BIOMIMETIC LIGNIN DEPOLYMERIZATION USING SMALL MOLECULE THIOLS : MIMICKING THE BETA - ARYL ETHER CLEAVAGE PATHWAY By Grace Elizabeth Klinger A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirement for the degree of Chemistry Doctor of Philosophy Biochemistry & Molecular Biology Dual Major 20 20 ii ABSTRACT BIOMIMETIC LIGNIN DEPOLYMERIZATION USING SMALL MOLECULE THIOLS: MIMICKING THE BETA - ARYL ETHER CLEAVAGE PATHWAY By Grace Elizabeth Klinger A key requirement in the effort to replace petroleum products with renewable - based products is an efficient and affordable method to depolymerize lignin from biomass for downstream valorization. Reductive processes main tain the energy content of the phenolic units of lignin but usually involve high pressures and temperatures. One mechanism for biological lignin depolymerization entails reduction of keto - aryl ether bonds through a series of reactions that include an S N 2 m - aryl ether cleavage pathway is the main catabolic pathway to cleave the most prevalent linkage in lignin, the - O - - hydroxy by a NAD + - dependent dehydrogena se is required. Next, nucleophilic substitution of the aryl ether is achieved with an etherase glutathione cofactor. The final step is reductive cleav age of the thioether intermediate with a lyase glutathione cofactor, liberating the aromatic fragment and formation of a glutathione disulfide. Mimicking this chemistry in a simple protein - free process, we surveyed small organic thiols for their ability to cleave aryl ether models found in lignin. High yields were achieved for both synthetic dimers and polymer s. We next extended our study to lignin obtained from biomass and found molecular weight reductions of up to 60%. In both models and biomass - derived lignin, oxidation was required, as seen in the enzymatic pathway. Our work provides mechanistic insight, su bstrate scope, and utility in polymeric systems. Furthermore, this thiol mediator has the potential to be electrochemically reactivated providing a mild, green approach to depolymerize lignin for bio - derived fuels and chemicals. iii ACKNOWLEDGEMENTS The work in this dissertation would not have been possible without the guidance and contributions of many individuals. First and foremost, I want to thank my advisors, Dr. Eric Hegg and Dr. Ned Jackson, for their willingness to trust me in starting a new r esearch topic and new collaborations. Their encouragement, feedback, and patience have allowed me to be the successful person I am today. I would also like to thank my committee members, Drs. John Frost, Bob Hausinger, and David Hodge for their assistance and contributions. I acknowledge with gratitude the Great Lakes Bioenergy Research Center for funding me. I am grateful for the collaborations in the center, Dr. Shannon Stahl, Dr. Yanbin Cui, Manar Alherech, and M ohammad Rafiee and the administration supp ort: Becca Blundell, Chri s lyn Pa r ticka, Linda Steinman , Brenda Boyce , Tina Nielson , and Sary n na Lopez Meza whose support have been priceless. I would also like to express my gratitude to those that assisted me with facility and research resources. Specific ally, Dr. Dan Holmes in the NMR facility; Cliff Foster, Lee Alexander, and Sam Deloy at the GLBRC Cell Wall Facility; Dr. Dan Jones and especially Dr. Tony Schilmiller in the Mass Spec Facility ; and Dr s . Ned Jackson and Leslie Kuhn for all their computatio nal assistance. I want to also thank the individuals that helped me with all my electrochemistry questions: Neda Rafat, Nathan Frantz, Sarah McFall - Boegeman, and Kathy Severin. In my time at Mic h igan State University, I have had the fortunate opportunity to be a part of two great labs (and an honorary member of another). I would like to thank the Hegg lab for a wonderful time and many conversations and mentorship s : Dr s . Aditya Bhalla , Namita Bansa l, iv Emily Herwaldt , Josh Haslun, Hui Yang , Julius Campecino, Ruchi Gaur, Saravanakumar Thiyagarajan, Zhaoyang Yuan, Zhen Fang, Thanaphong Phongpreecha and ; and Antoineen White, John Wright, Clarisse Finders, Elise Rivett, and Krystina Hird. M ost import antly , I want to thank Dr. Eric Hegg who really is the glue that hold us together. I would also like to thank the Jackson lab: , Greg Spahlinger, Tayeb Kakeshpour, Mikhail Redko, Pengchao Hao ; and Yuti ng Zhou, Bill Killian, Sophie Bedford, Monique Noel, Cesar Plascencia, Benjamin Appiagyei , and Darya Howell for the great company and of course Dr. Ned Jackson for making the lab a family. I would also like to thank the Bo r han group for welcoming me as an honorary member. I must acknowledge the support of the undergraduates that have assisted this project: Rachel Semaan, Jadan Norman, Gabriel Del Alamo Cardoso de Moraes, Alec Hegg, Dayton Buchanan, Megan Freds and most especially Andrew Kozel, Juliet Foot e, Caleb Geissler, and Abby Wester. I want to thank MSU - Technologies : Tom Herlache and coworkers and John G. Shedd Aquarium : Andy Kough , Kelsey Ryan , and others for allowing me to be a part of their team to learn and grow as a professional and a communicat or. Lastly, and most importantly, I would like to thank my husband Aritra Sarkar. If not for him, this PhD may have never been finished. He has been my support, my family , and my best friend. v TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ....................... viii LIST OF FIGURES ................................ ................................ ................................ ....................... ix KEY TO SYMBOLS AND ABBREVIATIONS ................................ ................................ ....... xvii Chapter 1. Introduction to Lignin Valorization ................................ ................................ .............. 1 1.1 Lignin Background ................................ ................................ ................................ ................ 2 1.1.1 Reductive Lignin Cleavage ................................ ................................ ............................ 6 1.1.2 Enzymatic - aryl Ether Cleavage Pathway ................................ ................................ .... 8 1.1.3 Sulfur in Biomass Conversion ................................ ................................ ..................... 10 1.1.3 - i Kraft Process ................................ ................................ ................................ ...... 10 1.1.3 - ii Sulfite Process ................................ ................................ ................................ ... 12 1.1.3 - iii Thioacidolysis ................................ ................................ ................................ .. 12 1.2 Research Objectives ................................ ................................ ................................ ............ 15 1.3 Disser tation Outline ................................ ................................ ................................ ............. 15 R EFERENCES ................................ ................................ ................................ .......................... 18 - O - 4 Keto Aryl Ether Cleavage of Dimers with Thiols ................................ ............ 24 2.1 Introduction ................................ ................................ ................................ ......................... 24 2.2 Computation ................................ ................................ ................................ ........................ 26 2.3 Me thodology: Parameter Screening ................................ ................................ .................... 27 2.4 Mechanism ................................ ................................ ................................ .......................... 2 9 2.5 Conclusion ................................ ................................ ................................ ........................... 34 2.6 Experimental D etail ................................ ................................ ................................ ............. 35 2.6.1 General Information ................................ ................................ ................................ ..... 35 2.6.2 Results of molecular simulations ................................ ................................ ................. 36 2.6.2 - i Docking of substrate models + glutathione in LigE and LigF enzymes ............ 36 2.6.2 - ii Activation barriers and reaction paths from quantum chemical modeling ....... 37 2.6.3 Sy nthesis of Model Lignin Dimers ................................ ................................ .............. 38 2.6.4 General Procedure for Reduction of Lignin Model Dimers ................................ ........ 51 2.6.4 - i Methodology Summerization of Thiol - Dependent 2 - Phenoxyacetophenone Cleavage ................................ ................................ ................................ ............................ 51 2.6.4 - ii Thiol Exploration on Cleavage of 2 - Phenoxyacetophenone ............................. 53 2.6.4 - iii Solvent and Temperature Exploration on Cleavage of 2 - Phenoxyacetophenone ................................ ................................ ................................ ................................ ........... 61 2.6.4 - iv Exploring the Effect of Base on the Cleavage of 2 - Phenoxyacetophenone ..... 70 2.6.4 - v Reaction Scope: Tolerance of Structural Diversity During Ether Cleavage ..... 71 2.6.4 - vi Hammett Stu dies ................................ ................................ .............................. 79 2.6.5 Control Experiments ................................ ................................ ................................ .... 82 vi 2.6.6 Proposed Electrochemical Cycle for the Thiol - - O - 4 Cleavage .. 88 2.6.7 Lignin Polymer Structural Model ................................ ................................ ................ 89 2.7 Spectra ................................ ................................ ................................ ................................ . 90 R EFERENCES ................................ ................................ ................................ ........................ 116 Chapter 3. Non - - O - 4 Ether Cleavage with O rganic Thiols ................................ ...................... 122 3.1 Introduction ................................ ................................ ................................ ....................... 122 3.2 4 - O - 5 Model Dimer Cleavage ................................ ................................ ........................... 125 3. - O - 4 / - 5 Model Dimer Cleavage ................................ ................................ .................... 126 3.4 Conclusion ................................ ................................ ................................ ......................... 127 3.5 Experimental Detail ................................ ................................ ................................ ........... 128 3.5.1 General Information ................................ ................................ ................................ ... 128 3.5.2 Synthesis of Model Lignin Dimers ................................ ................................ ............ 128 3.5.3 General Procedure for Cleavage Reactions and Analysis ................................ .......... 130 3.5.4 Cleavage of 4 - O - 5 Models ................................ ................................ ......................... 131 - O - - 5 Models ................................ ................................ .................. 133 3.5.6 Dimer Control Experiments ................................ ................................ ....................... 135 3.5.7 Proposed Dimer Cleavage Mechanism ................................ ................................ ...... 136 3.6 Spectra ................................ ................................ ................................ ............................... 137 R EFERENCES ................................ ................................ ................................ ........................ 144 Chapter 4. Lignin Polymer Cleavage with Nucleophilic Thiols ................................ ................. 148 4.1 Introduction ................................ ................................ ................................ ....................... 148 - O - 4 Linked Polymer Cleavage ................................ ................................ ...... 152 4.3 Cu - AHP Lignin Cleavage ................................ ................................ ................................ . 154 4.4 Other Lignin Cleavage ................................ ................................ ................................ ...... 158 4.5 Co nclusion ................................ ................................ ................................ ......................... 159 4.6 Experimental Detail ................................ ................................ ................................ ........... 160 4.6.1 General Information ................................ ................................ ................................ ... 160 4.6.2 Synthesis of Model Lignin Oligomers ................................ ................................ ....... 161 4.6.3 General Procedure for Cleavage Reactions and Analysis ................................ .......... 163 4.6.3 - i Synthetic Polymer ................................ ................................ ............................ 163 4.6.3 - ii Lignin ................................ ................................ ................................ .............. 163 4.6.4 Synthetic Polymer Cleavage ................................ ................................ ...................... 167 4.6.5 Lignin Polymer Model ................................ ................................ ............................... 169 4.6.6 Cu - AHP Lignin Cleavage ................................ ................................ .......................... 170 4.6.7 Cleavage of Oxidized Lignin ................................ ................................ ..................... 185 4.6.8 Clea vage of Other Lignin Polymers ................................ ................................ ........... 191 4.6.9 Lignin Control Experiments ................................ ................................ ....................... 195 4.6.10 Proposed Electrochemical Cycle for the Thiol - - O - 4 Cleavage 201 R EFERENCES ................................ ................................ ................................ ........................ 202 Chapter 5. Electrochemical Recyc ling of Disulfides ................................ ................................ .. 208 5.1 Introduction ................................ ................................ ................................ ....................... 208 5.2 Optimization ................................ ................................ ................................ ...................... 213 vii 5.2.1 Thiols ................................ ................................ ................................ ......................... 213 5.2.2 Choice of material ................................ ................................ ................................ ...... 216 5.3 Reversibility ................................ ................................ ................................ ...................... 219 5.3.1 Adsorption ................................ ................................ ................................ .................. 221 5.3.2 Chemical Reversibility ................................ ................................ ............................... 226 5.3. 3 New Electrolytes ................................ ................................ ................................ ........ 228 5.4 Challenges in Closing the Catalytic Cycle: ................................ ................................ ....... 228 5.5 Conclusion ................................ ................................ ................................ ......................... 232 5.6 Experimental Detail ................................ ................................ ................................ ........... 233 5.6.1 General Information ................................ ................................ ................................ ... 233 5.6.2 Control Experiments ................................ ................................ ................................ .. 234 R EFERENCES ................................ ................................ ................................ ........................ 238 Chapter 6. Conclusions and Future Directions ................................ ................................ ........... 240 viii LIST OF TABLES Table 2.1 Summary of dimer cleavage methodology. ................................ ................................ .. 31 Table 2.2 Summary table of 2 - phenoxyacetophenone cleavage and product yields methodology ................................ ................................ ................................ ................................ ....................... 53 Table 3.1 Cleavage yields of 4 - O - 5 dimers. ................................ ................................ ............... 131 - O - - 5 dimers. ................................ ................................ ......... 133 ix LIST OF FIGURES Figure 1.1 Carbon balance of a petro - barrel compared to a bio - barrel. ................................ .......... 1 Figure 1.2 Three phenylpropano ids that make up lignin. ................................ ............................... 2 Figure 1.3 Representation of lignin and its common linkages highlighted by color. ..................... 3 Figure 1.4 Lignin degradation under acidic conditions. ................................ ................................ . 4 Figure 1.5 Lignin degradation under alkaline conditions. ................................ .............................. 5 Figure 1.6 - keto lignin side reactions unde r alkaline conditions. ................................ ................. 6 Figure 1.7 - aryl ether cleavage pathway . ................................ ................................ .... 9 Figure 1.8 Mechanism outlined for the kraft process. ................................ ................................ .. 11 Figure 1.9 Mechanism outlined for the sulfite process. ................................ ................................ 12 Figure 1.10 Thioacidolysis mechanism. ................................ ................................ ....................... 14 Figure 2.1 - aryl ether cleavage scheme. ................................ ................................ ...................... 26 Figure 2.2 Summary of key cleavage reactions. ................................ ................................ ........... 29 Figure 2.3 Cleavage of non - - O - 4 aryl ethers and yields ................................ ............................. 32 Figure 2.4 Hammett plot for para - subsitution ................................ ................................ ............... 33 Figure 2.5 Molecular docking of glutathione with lignin substrates at the active site in Lig etherases 8 using Autodoc Vina. 12 ................................ ................................ ................................ .. 36 Figure 2.6 Calculated energetics for the 2 - step nucleophilic cleavage of 2 - phenoxyacetophenone with 2 - mercaptoethanol ................................ ................................ ................................ ................ 38 Figure 2.7 Cleavage of 2 - phenoxyacetophenone (10 mg, 2.36 mM) by reaction with glutathione in refluxing MeCN ................................ ................................ ................................ ............................ 53 Figure 2.8 Cysteine was probed as a mediator for the cleavage of 2 - phenoxyacetophenone ...... 54 Figure 2.9 2 - Phenoxyacetophenone cleavage with cysteine in MeCN/H 2 O mix. ........................ 55 x Figure 2.10 N - acetylcysteine cleavage of 2 - phenoxyacetophenone. ................................ ............ 56 Figure 2.11 2 - Phenoxyacetophenone cleavage varying BME equivalence. ................................ . 57 Figure 2.12 2 - Phenoxyacetophenon e cleavage varying DTT equivalence. ................................ .. 58 Figure 2.13 2 - Phenoxyacetophenone cleavage varying 1,3 - propanedithiol equivalence. ............ 59 Figure 2.14 2 - Phenoxyacetophenone cleavage varying thiophenol equivalence. ........................ 60 Figure 2.15 2 - Phenoxyacetophenone cleavage using a thiol salt. ................................ ................ 60 Figure 2.16 2 - Phenoxyacetophenone cleavage under neat thiol conditions. ................................ 61 Figure 2.17 2 - Phenoxyacetoph enone cleavage in dioxane. ................................ .......................... 61 Figure 2.18 2 - Phenoxyacetophenone cleavage with BME in MeCN at various temperatures . .... 62 Figure 2.19 2 - Phenoxyacetophenone cleavage with BME in DMF at various temperatures . ...... 63 Figure 2.20 2 - Phenoxyacetophenone cleavage with BME in THF at various temperatures . ....... 63 Figure 2.21 2 - Phenoxyacetophenone cleavage with BME in DMSO at various temperatures . ... 64 Figure 2.22 2 - Phenoxyacetophenone cleavage with BME in NMP at various temperatures . ...... 65 Figure 2.23 2 - Phenoxyacetophenone cleavage with BME in GVL at various temperatures . ...... 66 Figure 2.24 2 - Phenoxyacetophenone cleavage with BME in MeOH at various temperatures . ... 67 Figure 2.25 2 - Phenoxyacetophenone cleavage with BME in w ater at various temperatures . ...... 67 Figure 2.26 Comparison of 2 - phenoxyacetophenone (10 mg, 2.36 mM) cleavage in optimized solvent tempe ratures using DTT ................................ ................................ ................................ ... 68 Figure 2.27 Comparison of 2 - phenoxyacetophenone (10 mg, 2.36 mM) cleavage in optimized solvent temperatures using 1,3 - propanedithiol ................................ ................................ ............. 69 Figure 2.28 2 - Phenoxyacetophenone cleavage with BME using various bases. .......................... 70 Figure 2.29 Dimer (10 mg), modeling syringyl and guaiacyl monomers found in real lignin, cleavage in refluxing MeCN: ................................ ................................ ................................ ........ 71 Figure 2.30 - - aryl ether cleavage: .... 72 Figure 2.31 - - aryl ether cleavage: .... 73 xi Figure 2.32 Refluxing control reactions on 3 - hydroxy - 2 - phenoxy - 1 - phenylpropan - 1 - one. ........ 74 Figure 2.33 Room temperature control reactions on 3 - hydroxy - 2 - phenoxy - 1 - phenylpropan - 1 - one. ................................ ................................ ................................ ................................ ....................... 75 Figure 2.34 Control reactions to test reduced dimer cleavage when there is a hydroxy methyl - carbon. ................................ ................................ ................................ ................ 76 Figure 2.35 Investigation of the importance of the aryl - moiety in the ether cleavage reaction: .. 76 Figure 2.36 Lignin - like d imer cleavage using neat BME and neat DTT ................................ ...... 77 Figure 2.37 - O - 4 trimer cleavage in refluxing MeCN ................................ ................................ 78 Figure 2.38 Hammett studies with p - withdrawing groups on the leaving group of 2 - phenoxyacetophenone ................................ ................................ ................................ ................... 79 Figure 2.39 Hammett studies with p - donating groups on the leaving group of 2 - phenoxyacetophenone. ................................ ................................ ................................ .................. 80 Figure 2.40 Hammett studies with p - withdrawing group on the keto - side of 2 - phenoxyacetophenone. ................................ ................................ ................................ .................. 80 Figure 2.41 Hammett studies with p - donating groups on the keto - side of 2 - phenoxyacetophenone. ................................ ................................ ................................ ................................ ....................... 81 Figure 2.42 Control experiment to support ether cleavage via thiol - mediated nucleophilic attack over radical initiated cleavage. ................................ ................................ ................................ ..... 82 Figure 2.43 Control reactions testing if products formed increase reaction rate by increasing the active mediator. ................................ ................................ ................................ ............................. 82 Figure 2.44 Control experiments to test if stirring the reaction accelerated the aryl ether cleavage directly or indirectly (by gr inding the base, thereby creating more surface area). ....................... 83 Figure 2.45 Control experiment to test if the thiol mediator, BME, oxidizes to O - BME under reaction conditions ................................ ................................ ................................ ........................ 84 Figure 2.46 - O - 4 ether bond. ................................ ................................ ................................ ................................ ..... 84 Figure 2.47 Control experiments to test whether BME demethylates the methoxy groups on lignin monomers. ................................ ................................ ................................ ................................ ..... 85 Figure 2.48 Control reaction of 2 - ((2 - hydroxyethyl)thio) - 1 - phenylethan - 1 - one, the proposed intermediate of the 2 - phenoxyacetophenone cleavage: ................................ ................................ 85 xii Figure 2.49 Control experiment to test if O - BME can cleave 2 - phenoxyacetophenone, the simplest - O - 4 model lignin dimer. ................................ ................................ ................................ ............ 86 Figure 2.50 Control reactions to determine if the cleavage products undergo further side reactions. ................................ ................................ ................................ ................................ ....................... 87 Figure 2.51 Control reactions to determine if the oxidized thiol product reacts with the products of ether cleavage. ................................ ................................ ................................ ............................... 87 Figure 2.52 Organocatalytic cycle envisioned for the thiol - - aryl ether bonds followed by a 2e - reduction to recycle the mediator. ................................ ......... 88 Figure 2.53 Representation of lignin and its common linkages highlighted by color. ................. 89 Figure 2.54 2 - phenoxy - 1 - phenylethan - 1 - ol 29 (2 - 1) ................................ ................................ ...... 90 Figure 2.55 3 - hydroxy - 2 - phenoxy - 1 - phenylpropan - 1 - one 29 (2 - 3) ................................ ............... 91 Figure 2.56 2 - ((2 - hydroxyethyl)thio) - 1 - phenylethan - 1 - one (2 - 1 - a) ................................ ............. 92 Figure 2.57 1 - (4 - hydroxy - 3 - methoxyph enyl) - 2 - (2 - methoxyphenoxy)ethan - 1 - one (2 - 4) ............ 93 Figure 2.58 2 - (2,6 - dimethoxyphenoxy) - 1 - (4 - hydroxy - 3,5 - dimethoxyphenyl)ethan - 1 - one (2 - 5) 94 Figure 2.59 (1 - methoxy - 2 - phenoxyethyl)benzene 29 (2 - 14) ................................ .......................... 95 Figure 2.60 2 - methyl - 2 - phenoxy - 1 - phenylpropan - 1 - one 29 (2 - 7) ................................ ................. 96 Figure 2.61 2 - phenoxy - 1 - phenylpropan - 1 - one 29 (2 - 6) ................................ ................................ . 97 Figure 2.62 1 - phenyl - 2 - (p - tolyloxy)ethan - 1 - one 29 (2 - 18 - b) ................................ ......................... 98 Figure 2.63 2 - (4 - methoxyphenoxy) - 1 - phenyle than - 1 - one 29 (2 - 18 - a) ................................ ........... 99 Figure 2.64 1 - (4 - methoxyphenyl) - 2 - phenoxyethan - 1 - one (2 - 17 - b) ................................ ........... 100 Figure 2.65 1 - phenyl - 2 - (4 - (trifluoromethyl)phenoxy)ethan - 1 - one (2 - 18 - c) .............................. 101 Figure 2.66 2 - phenoxy - 1 - (4 - (trifluoromethyl)phenyl)ethan - 1 - one (2 - 17 - d) .............................. 102 Figure 2.67 4 - (2 - oxo - 2 - phe nylethoxy)benzonitrile (2 - 18 - d) ................................ ...................... 103 Figure 2.68 2 - (4 - acetylphenoxy) - 1 - phenylethan - 1 - one ................................ .............................. 104 Figure 2.69 2 - phenoxy - 1 - (p - tolyl)ethan - 1 - one (2 - 17 - c) ................................ ............................. 107 Figure 2.70 1 - (4 - (dimethylamino)phenyl) - 2 - phenoxyethan - 1 - one (2 - 17 - a) .............................. 108 xiii Figure 2.71 2,2' - (1,4 - phenylenebis(oxy))bis(1 - phenylethan - 1 - one) ................................ ........... 111 Figure 2.72 (1 - methoxyethyl)benzene 29 ................................ ................................ ..................... 112 Figure 2.73 1 - (3,4 - dimethoxyphenyl) - 2 - (2 - methoxyphenoxy)et han - 1 - one 29 (2 - 8) .................... 113 Figure 2.74 2 - phenoxy - 1 - phenylpropane - 1,3 - diol 29 ................................ ................................ ... 114 Figure 2.75 2 - bromo - 1 - (3,4 - dimethoxyphenyl)ethan - 1 - one 29 ................................ .................... 115 Figure 3.1 Representation of lignin and its common linkages highlighted by color. ................. 124 Figure 3.2 Cleavage of 4 - O - 5 model lignin dimers ................................ ................................ .... 125 - O - - 5 model lignin dimers ................................ .............................. 127 Figure 3.4 Cleavage of other ether dimers ................................ ................................ .................. 127 Figure 3.5 Time course of 4 - O - 5 dimer cleavage with BME. ................................ .................... 132 Figure 3.6 Time course of other aryl ether cleavage with BME. ................................ ................ 133 - O - - 5 dimer cleavage with BME. ................................ .............. 134 Figure 3.8 Control reactions to determine a non - radical mechanism for the cleavage of 4 - O - 5 and - O - - 5 dimers with p - directing aldehydes. ................................ ................................ ............ 135 Figure 3.9 Proposed mechanism of non - - O - 4 ether cleavage. ................................ .................. 136 Figure 3.10 4 - p henoxybenzonitrile ................................ ................................ ............................. 137 Figure 3.11 4 - t olyl phenyl ether (3 - c) ................................ ................................ ......................... 138 Figure 3.12 4 - p henoxybenzaldehyde (3 - g) ................................ ................................ ................. 139 Figure 3.13 1 - m ethoxy - 4 - phenoxybenzene (3 - b) ................................ ................................ ....... 140 Figure 3.14 1 - p henoxy - 4 - (trifluoromethyl)benzene (3 - e) ................................ .......................... 141 Figure 3.15 4 - phenoxybenzamide (3 - h) ................................ ................................ ...................... 142 Figure 3.16 1 - ( p henoxymethyl) - 4 - (trifluoromethyl)benzene (3 - m) ................................ ........... 143 - aryl ether cleavage via thiol nucleophilic attack. ................................ ................... 151 Figure 4.2 Cleavage of synthetic - O - 4 linked polymers. ................................ .......................... 154 xiv Figure 4.3 Cleavage of oxidized vs unoxidized lignin with thiol. ................................ .............. 158 Figure 4.4 Thiol - - O - 4 lignin. ................................ .................. 167 - O - 4 lignin. ............ 168 Figure 4.6 Representation of general hardwood lignin and its common linkages highlighted by color. ................................ ................................ ................................ ................................ ........... 169 Figure 4.7 Thiol - mediated Cu - AHP lignin depolymerization in water. ................................ ..... 170 Figure 4.8 Comparison of percent mass change variability during Cu - AHP lignin depolymerization with various solvents. ................................ ................................ ................................ .................. 172 Figure 4.9 Time course study to determine reaction length needed for sufficient lignin depolymerization with neat thiol. ................................ ................................ ............................... 173 Figure 4.10 Polymer cleavage characterization usin g pH 9 workup. ................................ ......... 174 Figure 4.11 Polymer cleavage characterization using pH 7 workup. ................................ ......... 175 Figure 4.12 Polymer cleavage characterization using pH 2 workup. ................................ ......... 176 Figure 4.13 Depolymerization of Cu - AHP lignin with neat thiol for 24 h worked up with water to pH 9. ................................ ................................ ................................ ................................ ............ 177 Figure 4.14 Analysis of Cu - AHP lignin depolymerization whole polymer and fragments. ....... 178 Figure 4 .15 Susceptibility of Cu - AHP lignin depolymerization with 1,3 - Propanedithiol over time. ................................ ................................ ................................ ................................ ..................... 179 Figure 4.16 Determination of percent lignin cleavage by measuring mass loss after thiol - treatment. ................................ ................................ ................................ ................................ ..................... 180 Figure 4.17 Analysis of sulfur incorporated into depolymer ized Cu - AHP lignin. ..................... 180 Figure 4.18 Measurement of - OH content to determine lignin cleavage. ................................ ... 181 Figure 4.19 HSQC NMR of Cu - AHP lignin demonstrating changes to the polymer structure after depolymerization with thiol. ................................ ................................ ................................ ....... 183 Figure 4.20 Thioacidolysis S/G ratio of monomer units in remaining lignin polymer after thiol - treatment. ................................ ................................ ................................ ................................ .... 183 Figure 4.21 Thioacidolysis % monomer in thiol - treated and non - treated lignin polymer. ......... 184 Figure 4.22 Susceptibility of thiol - mediated Cu - AHP lignin depolymerization when the lignin is prepared with double the amount of hydrogen peroxide and thus presumably more oxidized. . 186 xv Figure 4.23 AcNH - TEMPO oxidized lignin compared to unoxidized lignin and its susceptibility to thiol - mediated depolymerization. ................................ ................................ ........................... 187 Figure 4.24 Time course of AcNH - TEMPO oxidized Cu - AHP depolymerization using BME. 188 Figure 4.25 Comparison of Cu - AHP lignin depolymerization on improved AcNH - TEMPO oxidized vs non - oxidized lignin. ................................ ................................ ................................ . 190 Figure 4.26 Comparison of Cu - AHP lignin depolymerization vs industrially made lignin from MetGen. ................................ ................................ ................................ ................................ ...... 191 Figure 4.27 GVL Lignin Depolymerization. ................................ ................................ .............. 192 Figure 4.28 Comparison of various lignin depolymerization with 1,3 - propanedithiol vs BME. 193 Figure 4.29 1,3 - propanedithiol depolymerization of various lignin. ................................ .......... 19 4 Figure 4.30 Control experiment to test lignin work - up precipitation to obtain accurate mass loss yields . ................................ ................................ ................................ ................................ .......... 195 Figure 4.31 Control reactions to determine changes in molecular weight based on differences in Cu - AHP lignin extract ion procedure ................................ ................................ .......................... 196 Figure 4.32 Control study to determine if scale - up of Cu - AHP lignin extraction affects its thiol - mediated depolyme rization. ................................ ................................ ................................ ........ 199 Figure 4.33 Control experiment to determine the scalability of thiol - mediated lignin depolymerization. ................................ ................................ ................................ ........................ 200 Figure 4.34 Organocatalytic cycle envisioned for the thiol - - aryl ether bonds followed by a 2e - reduction to recycle the mediator. ................................ ....... 201 Figure 5.1 Schematic of electron transfer from an applied potential. ................................ ......... 209 Figure 5.2 Mechanism of disulfide reductions through a 2e - addition. ................................ ...... 210 Figure 5.3 Simplified catalytic cycle for the thiol - mediated keto - aryl ether cleavage reaction. 211 Figure 5.4 Schematic of the electrochemically driven catalyst turnover during lignin dimer cleavage. ................................ ................................ ................................ ................................ ...... 212 Figure 5.5 CV of O - DTT and O - BME using Ag/AgBr and TBAB. ................................ ........... 214 Figure 5.6 Chronoamperometry of O - BME at - 1.2 V. ................................ ............................... 215 Figure 5.7 Working electrode comparison for the CV of di phenyl disulfide. ............................ 216 Figure 5.8 Electrolyte comparison for the CV of diphenyl disulfide. ................................ ........ 217 xvi Figure 5.9 NMR detection from diphenyl disulfide reduction at 1.5 V. ................................ ..... 218 Figure 5.10 CV Quantification of diphenyl disulfide reduction. ................................ ................ 219 Figure 5.11 Diagram of the disulfide redox cycle in a reversible and irreversible system. ........ 220 Figure 5.12 CV of diphenyl disulfide with irreversible features. ................................ ............... 220 Figure 5.13 Peak current as a function of the v 1/2 to determine adsorption by the Randles - Sevcik equation. ................................ ................................ ................................ ................................ ...... 222 Figure 5.14 Peak current as a function of concentration to determine ads orption by the Randles - Sevcik equation. ................................ ................................ ................................ .......................... 222 Figure 5.15 Charge as a function of t 1/2 to determine adsorption charge using the integr ated Cottrell equation. ................................ ................................ ................................ ................................ ...... 224 Figure 5.16 Peak current fluctuation over multiple scans to determine adsorption. ................... 225 Figure 5.17 Clean vs used electrode adsorption test. ................................ ................................ .. 225 Figure 5.18 Chemical reversibility monitoring current ratios over time. ................................ ... 226 Figure 5.19 Concentration of dipheny l disulfide electrochemical reduction overtime. ............. 227 Figure 5.20 Proposed chemical degradation of the electrochemically reduced diphe nyl disulfide. ................................ ................................ ................................ ................................ ..................... 228 Figure 5.21 Reduction potential overlap of disulfide and lignin model dimer. .......................... 229 Figure 5.22 Chronoamperometry of 2 - phenoxyacetophenone. ................................ .................. 230 Figure 5.23 Applied current to 2 - phenoxyacetophenone cleavage reaction for one catalytic turn - over. ................................ ................................ ................................ ................................ ............ 232 Figure 5.24 CV of ferrocene used as a control. ................................ ................................ .......... 234 Figure 5.25 CV of O - DTT and O - BME using Ag/AgCl and NB u 4 ClO 4 in MeCN. ................... 235 Figure 5.26 Gatorade as an electrolyte/solvent for O - BME reduction. ................................ ...... 235 Figure 5.27 CV of O - BME using water and NaCl as the electrolyte. ................................ ........ 236 Figure 5.28 Lithium perchlorate as an electrolyte. ................................ ................................ ..... 236 xvii KEY TO SYMBOLS AND ABBREVIATIONS 2,6 - lutidine 2,6 - d imethylpyridine 4 - acetamido TEMPO 4 - a cetamido - 2,2,6,6 - tetramethylpiperidine 1 - oxyl 4 - O - 5 phenoxyphenol 5 - 5 biphenyl - - 1 diarylpropane - O - - 5 phenylcoumaran - - diarylpropane - - O - 4 phenylcoumaran - pinoresinol - O - 4 - aryl ether p differen ce between the anodic and cathodic peak potentials sum * surface coverage of absorbed species (mol/cm 2 ) A area of the electrode (cm 2 ) AcNH - TEMPO 4 - a mino - 2,2,6,6 - tetramethylpiperidine - 1 - oxyl AcOH acetic acid BHT b utylated hydroxytoluene BME 2 - m ercaptoethanol N - (2,2,6,6 - tetramethyl - 1 - oxopiperidin - 1 - ium - 4 - yl)acetamide tetrafluoroborate xviii C 0 analyte concentration (mol/cm 3 ) Cu - AHP copper catalyzed alkaline hydrogen peroxide pretreatment CV c yclic voltammetry D o diffusion coefficient of analyte (cm 2 /s) DBU 2,3,4,6,7,8,9,10 - octahydropyrimido[1,2 - a]azepine DCM d ichloromethane DDQ 2,3 - d ichloro - 5,6 - dicyano - 1,4 - benzoquinone tBuONO tert - b utyl nitrite DFRC derivatization followed by reductive cleavage DMF d imethylformamide DMSO d imethyl sulfoxide DTT d ithiothreitol ECH electrocatalytic hydrogenation EtOAc e thyl acetate F G3(MP2) Gaus sian Møller - Plesset computation theory GCMS g as chromatography mass spectrometry gHSQC - AD gradient HSQC adiabatic GLBRC Great Lakes Bioenergy Research Center GPC gel permeation chromatography GS glutathione GSSG glutathione disulfide GVL - valerolactone xix HOMO highest occupied molecular orbital HPLC h igh - performance liquid chromatography HSQC h eteronuclear single quantum coherence spectroscopy i p peak current i pa peak current at the anode i pc peak current at the ca thode LCMS l iquid chromatography mass spectrometry LUMO lowest unoccupied molecular orbital M i molecular weight of each polymer chain M n n umber average molecular weight M p m olecular weight at the peak M w w eight average molecular weight MeCN a cetonitrile MeOH methanol n number of electrons transferred in redox event N HPI N - h ydroxyphthalimide N i total number of different molecular weights chains NMR n uclear magnetic resonance spectroscopy NMP N - m ethyl - 2 - pyrrolidone O - BME - d ithiodiethanol O - DTT trans - 4,5 - d ihydroxy - 1,2 - dithiane PDI p olydispersity index Q total charge xx Q ads adsorbed charge Q d diffused charge Q dl double layer charge QTOF q uadrupole t ime - of - f light R ideal gas constant RCF r eductive catalytic fractionation RS generic thiol RS . generic thiyl radical RS - generic thiolate RSSR generic disulfide RT room temperature S/G ratio of syringyl to guaiacyl in lignin SM8 computation solvent model S N Ar n ucleophilic a romatic s ubstitution S N 2 b imolecular n ucleophilic s ubstitution T temperature TBAB t etrabutylammonium bromide TBA - HFP t etrabutylammonium hexafluorophosphate TBA - P t etrabutylammonium phosphate TBA - TFB t etrabutylammonium tetrafluoroborate TBA - TPB t etrabutylammonium Tetraphenylborate TCA tricarboxylic acid cycle TEMPO + (2,2,6,6 - t etramethylpiperidin - 1 - yl)oxyl xxi TH F t etrahydrofuran TLC thin layer chromatography TS transition state v sc an rate (V/s) 1 Chapter 1. Introduction to Lignin Valorization Fossil carbon is a finite resource used in everything from fuels and plastics to consumer goods and food additives. 1 The need to replace this non - renewably sourced carbon with carbon from renewabl e source s becomes even more apparent when looking at the ir carbon flux es . While renewable carbon from plants is involved in a closed loop ( Figure 1. 1 ) non - renewable carbon from fossil sources has an open loop l eading to a net increase in CO 2 emissions. 2 W hile exploiting plants to replace heavy fuels and chemicals does not result in increases in atmospheric CO 2 , it does provide its own set of challenges due to the recalcitrant nature of biomass. Figure 1. 1 Carbon balance of a petro - barrel compared to a bio - barrel. The net CO 2 remains the same when using fuels and chemicals from biomass. There is an influx of CO 2 into the atmosphere when using fuels and chemicals from fossil carbon . This figure was adapted from an unpublished graphic created by J. Runde from the Great Lakes Bioenergy Research Center communications department. Plants are primarily composed of three polymers: cellulose, polymers of 6 - carbon sugar s that provide the skeletal structure of plants; hemicellulose, poly mers of 5 - and 6 - carbon sugar s that 2 strengthen the cell wall through interactions with cellulose and lignin ; and lignin, phenolic polymers that enhance cell wall rigidity and act as a barrier to enzymatic degradatio n. 3 While the sugar polymers can be converted into plastics or fermented into alcohols for further valorization (conversion to high value products) , lignin has not been widely used for fuels or chemicals despite contributing up to 50% of the energy content of plants. 4 Recently, however, significant emphasis has been placed on utilizing the lignin polymer in bio - based products, and a new strategy for depolymerizing t his complex structure will be the topic of discussion in the following chapters. 1.1 Lignin Background Lignin is an energy - and carbon - rich aromatic polymer, historically treated as a waste by - product of the pulp and paper industry. Depolymerization of th is polymer could produce bio - substitutes for many petro - based chemicals and fuels. However, lignin depolymerization is energy intensive and costly due to the chemical recalcitrance of the linkages that bind the aromatic phenylpropanoid subunits together. A cheap and efficient method for cleaving the polymer is the first step of valorization in to bio - products. Figure 1. 2 Three phenylpropanoids that make up lignin . Lignin is synthesized through radical coupling of H, G, and S phenylpropanoids to produce a heterogenous polymer. The numbering of these lignin units is shown for the p - coumaryl subunit. The polymer is assembled through radical coupling of three phenylpropanoids, p - coumaryl, coniferyl, and sinapyl units ( Figure 1. 2 ), - O - - - O - - - - 5 (biphenyl), - - 1 (diarylpropane), and 4 - O - 5 (phenoxyphenol) , shown in Figure 1. 3 . The most prevalent linkage is 3 - O - 4 bond (orange), representing up to 60% of the linkages i n lignin. It is this ether linkage that is the easiest to cleave and thus the focus of much work on lignin depolymerization. Figure 1. 3 Representation of lignin and its common linkages highligh ted by color. The most - O - 4 linkage (highlighted in orange). The percent of the various linkages observed is based on lignin from hardwoods. 5 The simplest methods of lignin extraction and depolymerization have employed acids, bases, heat, pressure, or a combination of these approaches . While these simple methods may initially fragment the lignin, the challenge lies in preventing degradation from side reactions and repolymerization through carbon - carbon bond formation . This crosslinking and repolymerization can also occur in more complex depolymerization methods and must be understood when evaluating new cleavage techniques . Under aqueous acid ic conditions ( Figure 1. 4 ) , lignin can be dehydrated through the formation of a quinone methide ( structure 1 - 3 ) . These quinone methides 4 can degrade into enols ( structure 1 - 10 and 1 - 12 ) or enol ethers ( structures 1 - 4 ) and undergo repolymerization through aldol condensations. One potential pathway for this acid - mediated degradation to aldol reactants is depicted below in Figure 1. 4 . Figure 1. 4 Lignin degradation under acidic conditions. Under aqueous acid ic conditions, lignin forms enols and enol ethers . These products can subsequently lead to repolymerization via aldol chemistries . 6 In aqueous alkaline conditions ( Figure 1. 5 ) , similar , albeit slower, processes can also result in crosslinking and repolymerizatio n. Base initiated formation of quinone methide ( structure 1 - 3 below ) can enable elimination to form eithe r , - unsaturated aldehydes ( structure 1 - 14 ) or enol ethers ( structure 1 - 15 ) that are open for a 1,2 or 1,4 attack. Furthermore, phenolates ( structure 1 - 7 below ) can attack these systems leading to formation of additional C - C bond s that are much more diffi cult to cleave than the original ether bonds. 5 Figure 1. 5 Lignin degradation under alkaline conditions. Under aqueous alkaline conditions, lignin undergoes degradation to reactive products. These products can crosslink and repolymeriz e in subsequent reactions . 6 Oxidation of the - position decreases the activation energy required to cleave the - O - 4 ether bond by 9 - 15 kcal/mol depending on the ring substituents. 7 Under alkaline conditions, the oxidized lignin can lose a - hydroxymethyl through a retro - aldol reaction ( Figure 1. 6 ) , result ing in a reactive enol ether ( structure 1 - 18 ) that could lead to repolymerization ra ther than protonation ( structure 1 - 19 ) . Likewise, a competing elimination of the - hydroxy group could form a different enol ether ( structures 1 - 20 and 1 - 21 ) that can further undergo aldol - type reaction s . In these case s , oxidation of the - hydroxy allows for additional lignin degradation challenges . 6 Figure 1. 6 - K eto lignin side reactions under alkaline conditions. Under alkaline conditions, a competition between retro - aldol and elimination is observed for the - hydroxymethyl of oxidized lignin. 8 Th ese reactions can lead to the formation of enol ethers that can subsequently repolymerize to generate new C - C crosslinks in the lignin. To mitigate some of these side reactions, w hich are mostly due to the primary and secondary - OH groups on the - and - carbons, the Luterbacher group devised a protective strategy that uses aldehydes ( e.g. , formaldehyde ) to - OH sites by form ing a cyclic acetal from the two neighboring - OH groups. 9 - 12 This process has been succes sful in protecting both models and real lignin , and it has been used both during lignin extraction of biomass and depolymerization. By understanding the mechanism of the pitfalls associated with lignin depolymerization, insight and guidance can be gained for develop ment of more efficient strategies to turn lignin into commodit y chemicals. 1.1.1 Reductive Lignin Cleavage To overcome the challenges associated with simple acid/base lignin depolymerization, much work has been developed using methods such as cr acking, catalytic oxidation or reduction, electrolysis, photolysis, and enzymatic catalysis. These methods employ heterogenous and homogeneous metal, organic, or biomimetic catalysts to achieve cheap, scalable, and efficient 7 process es for lignin cleavage. Oxidation is usually the easiest method for cleavage, but because this process decreases the energy content of the products through incorporation of oxygen or loss of electrons , oxidation is often considered to be an unfavorable depolymerization strategy for the production of fuels and chemicals. Therefore, this thesis focuses on non - oxidative processes to cleave the - O - 4 bond in lignin. Hydrogenolysis is one of the most common reductive technique s used to cleave bonds. This technique employs t ransition or precious metal catalysts with heat and/or pressurized hydrogen . Many different catalysts, solvents, temperatures, and pressures have been optimized to produce the highest yield of lignin monomers , and a number of different reviews summarize this field . 5, 13 - 15 Another popular reductive technique that utilizes metal catalysts, high temperatures, and pressurized solvents is reductive catalytic fractionation (RCF) . 16 In this process, lignin is extracted directly from biomass, depolymerized into fragments through solvolysis or hydrolysis, and stabilized through redox active metals to produce stable small lignin fragments. RCF can be performed with or without hydrogen in batch or in flow - through systems. 17 A metal - free reductive lignin depolymerization strategy was achieved using B(C 6 F 5 ) 3 /Et 3 SiH at room temp erature to produce high yields of aromatic products with efficiencies up to 85% . 18 In this strategy, a Lewis acid, B(C 6 F 5 ) 3 , promotes reduction by hydrosilanes. E ffective cleavage can also be achieved through a two - step, net redox neutral, process. The first step is oxidation of the secondary alcohol of the - O - 4 linkage to decrease the bond dissociation energy of the C - O bond. This oxidation is followed by C - O c leavage through some sort of reductant. For example, the Westwood group has had success in cleaving model dimers , polymers , and re al lignin using DDQ/ t BuONO ( 2,3 - d ichloro - 5,6 - dicyano - 1,4 - benzoquinone / tert - b utyl nitrite ) with O 2 followed by Zn in the presence of NH 4 Cl. Similarly, the Stahl group has 8 achieved efficient lignin depolymerization using 4 - acetamido - TEMPO ( 4 - a cetamido - 2,2,6,6 - tetramethylpiperidine 1 - oxyl ) with HNO 3 /HCl followed by a redox neutral process invo lving sodium formate in aqueous formic acid to cleave the ether bonds . 19 The Stephenson group has used electrocatalytic oxidation with NHPI ( N - h ydroxyphthalimide ) and 2,6 - lutidine to achieve benzylic oxidation followed by photocatalytic reductive cleavage with iridium complexes. 20 While t hese two - step cleavage examples focus on - O - 4 cleavage , this method can also be extended to - O - 4 cleavage when the oxidants, seen above, are paired with catalytic hydrogenation. 15 The redox - neutral cleavage of - O - 4 bonds has proven to be a successful technique through the use of specialized catalysts , and th is technique has also been exploited by enzymes , as discussed below . 1.1.2 Enzymatic - aryl Ether Cleavage Pathway In nature, polymeric lignin is degraded by extracellular fungal and some bacterial enzymes that oxidatively cleave the polymer into fragments . These fragments can then be taken - up by bacteria and some fungi 21 and subsequently broken - down through a number of pathways , eventually le ading to vanillate and syringate products. 22 These monomers are then converted into pyruvate - and oxaloacetate - type molecules through multiple demethylases and ring - opening steps, for incorpo ration into the TCA cycle ( tricarboxylic acid cycle ) . 22 Nearly all 23 enzymatic cleavage of the - O - 4 linkage is achieved through a series of stereospecific glutathione - S - transferase enzymes th at first oxidize the fragment and then reductively cleave the ether bond ( Figure 1. 7 ) . 24 - 27 I n this process, a - O - 4 fragment crosses the cell membrane and is oxidized at the - carbon by a NAD + - dependent dehydrogenase, LigD/O or LigL/N (depending on the enantiomer). 27, 28 After oxidation of the - hydroxy to a ketone, the etherases, LigE, P, and BaeAB 29 or LigF (depe nding on the enantiomer), use a glutathione cofactor to nucleophilically displace the phenolic C - O bond 9 at the - carbon. 26 This displacement results in the formation of a glutathione thioether and a phenolic fragment. The thioether is then red uced by a lyase, stereospecific LigG, or nonstereospecific GST3 or NaGST Nu , 30 with a glutathione cofactor to form the glutathione disulfide and release the second lignin fragment. 27 This enzymatic process can be seen in Figure 1. 7 . Figure 1. 7 - aryl ether cleav age pathway . Mechanism for the Lig enzymes in the - aryl ether cleavage pathway to cleave the - O - 4 bond in lignin fragments. The first step involves oxidation of the - hydroxy to a ketone using the NAD + - dependent dehydrogenase. The next step uses glutathione dependent etherases to nucleophilically displace the phenolic monomer. The last step is the reductive cleavage of the thioether intermediate with a glutathione - depende nt lyase. This lyase reaction re sults in release of the second aryl - ketone monomer and formation of a disulfide (GSSG) b y product. The NADH and GSSG are recycled using other enzymatic processes outside the cleavage cascade. Enzymes that cleave the - O - 4 linkage were first reported in the late 19 70s from soil bacteria. 31 In the following decade, the bacteri um Sphingobium sp. SYK - 6 was isolated from the waste stream of a kraft pulp mill and found to grow only on lignin - derived systems by utilizing specific bacterial etherase pathways . 22 T he Masai lab and others have characterized the activity , 28, 31, 32 mapped the genes involved in this process , 33 - 38 and proposed reaction mechanisms 25, 39 throu gh crystallographic analysis 26, 27, 30, 40 and computational modeling. 41 Additional work has been performed on Sphingobium and other bacterial systems that exhibit - O - 4 etherase activity 10 to probe the in vitro enzymatic depolymerization of both lignin m odels 42 - 44 and real lignin 45, 46 while simultaneously recycling the NAD + and glutathione cofactors . 29, 30, 47 48 - 50 1.1.3 Sulfur in Biomass Conversion Sulfur - based chemistries have been employed in biomass conversions for the last 150 years. 5 1 The pulp and paper industry devised two main methods to remov e lignin that involve sulfur, the kraft process and the sulfite process. These processes incorporate multiple sulfonate s or thiol groups into the lignin, making strategies for depolymerizing this industrial lignin challenging due to catalyst poisoning and structural changes to the lignin that increase the energy barrier to cleavage. Sulfuric acid has also been used to separate the sugars from the lignin in biomass by hydrolyz ing the carbohydrates , a process that result s in an acid insoluble fraction termed Klason lignin. 52 Due to the strongly acidic conditions utilized, Klas on lignin is highly c rosslinked 14 via the same degradation/condensation reactions observed in the acid catalyzed depolymerization ( Figure 1. 4 ). 1.1.3 - i Kraft Process The k raft process is the method of choice for most pulp and paper mills to isolate cellulose from wood. In this process, base, sodium sulfide, and heat are used to cleave the ester and ether bonds connecting lignin to cellulose/hemicellulose. 53 D uring this process, however, the ether - O - 4 linkage are also susceptible to cleavage that produce highly reactive species that can undergo crosslinking and repolymerization. Under these conditions , the base can initiate formation of a quinone methide that is well suited for attack by sodium sulfide , replacing the - OR at the - position. As seen in the base initiated degradation above, elimination results in the formation of enol ethers ( structure 1 - 23 ), - unsaturated aldehyde s ( structure 1 - 14 below ), and phenolates ( structure 1 - 7 below ), precursors to aldol chemistries . This mechanism is outlined in 11 Figure 1. 8 , which varies from the literature mechanism involving a thiol - epoxide initiated dimer cleavage. 5, 54, 55 Based on the reactivity of - hydroxymethyl groups in base , 8 the mechanism presented here seems more realistic to the degradation observed under alkaline conditions. Figure 1. 8 M echanism outlined for the k raft process. The k raft , or sulfate, process uses base and sodium sulfide to remove lignin from wood. Under alkaline aqueous conditions, the easiest lignin bond to break ( - O - 4) can undergo eliminat ion s to quinone methide s, poised for attack by sodium sulfides. Once thiol - substituted, t he - hyd r oxy can eliminate to form an enol ether ( structure 1 - 23 ) , or - phenolate elimination can generate an , - unsaturated aldehyde ( structure 1 - 14 ). Kraft lignin, produced at an industrial scale for the last century, has been well characterized. This lignin includes the crosslinks mentioned previously under alkaline conditions as well as removal of most of the - aryl ethers and - aryl ethers. Roughly 1.5 - 3% sulfur from the sodium sulfide is incorporated in the lignin. 15 This dark powder has been shown to have molecular weights of M n ~1000 g/mol and M w of ~3 - 6,000 g/mol depending on whether it is derived from hardwood or sapwood. 53 12 1.1.3 - ii Sulfite Process The second most utilized process for delignification in the pulp and paper industry is the sulfite , or lignosulfonate, process. This method uses calcium or magnesium sulfite under acidic pH s to separate lignin from cellulose, produ cing a soluble lignin fraction. 52 The mechanism ( Figure 1. 9 ) involves the formation of a carbocation intermediate ( structure 1 - 25 ) at the - carbon at which a sulfite can attack to form an - substituted sulfonate ( structure 1 - 26 ) . Alternatively, the carbocation can react with an electron rich group , such as a ph enolic monomer , resulting in C - C bond formation. Figure 1. 9 M echanism outlined for the sulfite process. The sulfite process uses sulfite salt under acidic conditions to remove lignin from wood. In this method, a carbocation is formed at the - carbon of lignin that can either result in sulfona tion or crosslinking through C - C bond formation . The lignosulfonat e process generates slightly larger lignin than the kraft process due to the addition of the sulfonate groups, where the sulfur accounts for 4 - 8% of the lignin, 15 with lignin molecular weights ranging from 4,000 to 40,000 g/mol. 51, 52 Both the kraft and sulfite process es were developed as a means to isolate cellulose with little concern for the accompanying changes to the lignin structure . As the focus of lignin utilization has shifted from burning for energy to adding value by upgrading to commodity chemicals, a process that does not lead to lignin condensation or a process that allows for cleavage of these crosslinked polymers is sought after. 1.1.3 - iii Thioacidolysis Thioacidolysis is an a - O - 4 linkages as well as the S:G:H monomer ratio s in lignin . As an improvement to acidolysis that uses acid, heat, and 13 dioxane, 56 - 58 this acidic solvolysis uses the Lewis acid boron trifluoride etherate and ethanethiol in dioxane to cleave aryl ether bonds, a process that results in thiolated monomers that can be reduced by Raney ® nickel. 51 In the literature mechanism outlined ( Figure 1. 10 ) , the BF 3 - - hydroxyl groups as well as the phenolic ether. While the order of attack is unknown, the ethanethiol can be assumed to attack the partially positive carbon ne xt to the activated - OH. In the - OH, this attack leads to release of BF 3 coordinated with the - OH and formation of a - position . Next, BF 3 - carbon through coordination with the phenolic ether. This activation - - position, releasing the phenolic fragment and forming a n episulfonium ion ( thiol - epoxide ) . Addition of a second - ethaneth iol substituted chain. Additional BF 3 can a lso - carbon and induce a third attack by ethanethiol, releasing BF 3 OH. The final product is a trisubstituted ethanethiol monomer/fragment in which the thio ether groups can be removed through reaction with Raney ® nickel. 51 This mechanism , in which the substrate is activated by a very potent Lewis acid, is much more likely to go through the proposed th iol - epoxide intermediate for ether cleavage compared to the base catalyzed sulfate process above . 14 Figure 1. 10 Thioacidolysis mechanism . Thioacidolysis utilizes a Lewis acid, ethanethiol, and acid in dioxane to cleave the - O - 4 bond of lignin. Through BF 3 activation of C hydroxyl , ethanethiol can attack both - and - sites, resulting in fragmentation, as well as the - site which leads to removal of the - hydroxyl. Each of these simple, non - metal - O - 4 chemical cleavage processes (acid, base, kraft, lignosulfonate, and thioacidolysis) utilize different mechanisms for ether cleavage : (a) pure acid - and base - promoted cleavage result s in quinone methides that produce reactive enol ethers, (b) kraft utilizes base with sulfide salts to enhance this alkaline cleavage, (c) lignosulfonate - carbocations, and (d) thioacidolysis employs Lewis acids - site nucleophilic attack and cleavage through formation of thiol - epoxides. The - O - 4 dimers with glutathione - S - transferases , however, present s a new mechanism for lignin depolymerization, a thiol - mediated direct S N 2 cleavage of aryl ethers. This enzymatic mechanism leads to the question of whether direct S N 2 chemical cleavage of aryl ethers by thiols or thiolates is possible in the absence of ac tivation by Lewis acids, proteins, metals, scaffold, or reactive intermediates. Th us the main topic of discussion for this thesis is an exploration of thiols - O - 4 bonds, mimicking the mechanism seen in the 15 enzymatic process, in search o f a simple , efficient, and cost - effective method for lignin depolymerization. 1.2 Research Objectives The project developed in this dissertation aims to chemically - aryl ether cleavage pathway , enabling lignin depolymerization into fra gments for further valorization into fuels and chemicals by downstream processes. The enzymatic process uses several key enzymes with glutathione cofactors to cleave aryl ethers through nucleophilic substitution and reductive elimination. To mimic this pat hway, we first optimize reaction conditions - aryl ether lignin dimers. Next, we apply the se optimized parameters to larger model systems and other aryl ethers with structural relevance to lignin linkages. Finally, we assess the ability of small o rganic thiols to penetrate complex polymer matrices, access the polymer backbone, and act as redox mediators to reductively cleave keto - aryl ether bonds in biomass - derived polymeric lignin . In addition, p reliminary studies are conducted to parameterize the conditions necessary for electrochemical reduction of the disulfide b y product to regenerate the thiolate . Ultimately, the objective of this work is to develop a lignin depolymerization process that avoids expensive catalysts and extreme co nditions, minimizes condensation, and allows the thiol mediator to be recycled for a cost - effective , green, and scalable process. 1.3 Dissertation Outline The remainder of this document describes our work to achieve this objective. Chapter 2 describes the biomimetic - aryl ether cleavage of model - O - 4 dimers. Optimization of the thiol mediator, solvent system, base, mixing, and reaction temperature are used to analyze substrate scope, probe the mechanism, and perform structure - reactivity analys es . Tools us ed include synthesis, Hammett analysis, computation, and control studies. In Chapter 3, non - - O - 4 aryl ether 16 model dimers are evaluated for thiol - mediated cleavage. Any successful, albeit modest, cleavage would contribute to a more fully depolymerized lign in . In C hapter 4 , synthetic - O - 4 linked polymers are used to assess the ability of thiols to penetrate bulky systems . Efforts are then extended to depolymerization of biomass - derived lignin. L ignin obtained from different extraction processes are characterized before and after depolymerization using various analytical tools. Information detailing fragment formation is also provided . Depolymerization of oxidized lignin is assessed in parallel wi th unoxidized lignin to determine if the heterogenous natural polymer follows the reactivity of the model systems. Finally, Chapter 5 dives into the parameters needed to recycle the thiol redox mediator and enable multiple turnovers of ether cleavage. This process involves e lectrochemical 2 - electron/2 - proton reduction of the disulfide byproduct and enable s net electrocatalytic lignin depolymerization . The tools used include cyclic voltammetry, chronoamperometry, and other analytical characterization tools . In the final chapter, Chapter 6, the main conclusions of the project are summarized and future work is outlined. 17 REFERENCES 18 R EFERENCES 1. Service, R. F., Can the world make the chemicals it needs without oil? Science 2019, September (News). 2. Dale, B. E.; Anderson, J. E.; Brown, R. C.; Csonka, S.; Dale, V. H.; Herwick, G.; Jackson, R. D.; Jordan, N.; Kaffka, S.; Kline, K. L.; Lynd , L. R.; Malmstrom, C.; Ong, R. G.; Richard, T. L.; Taylor, C.; Wang, M. Q., Take a Closer Look: Biofuels Can Support Environmental, Economic and Social Goals. Environmental Science & Technology 2014, 48 (13), 7200 - 7203. 3. Goldstein, I. S., Organic Chemicals from Biomass . CRC Press, Inc.: Boca Raton, FL, 1981. 4. Perlack, R. D.; Wright, L. L.; Turhollow, A. F.; Graham, R. L.; Stokes, B. J.; Erbach, D. C. Biomass as Feedstock for a Bioenergy and Bioproducts Industry: The Technical Feasibility of a Billion - Ton Annual Supply ; Oak Ridge National Laboratory: 2005. 5. Rinaldi, R.; Jastrzebski, R.; Clough, M. T.; Ralph, J.; Kennema, M.; Bruijnincx, P. C. A.; Weckhuysen, B. M., Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining and Catalysis. Angewandte Chemie - International Edition 2016, 55 (29), 8164 - 8215. 6. Li, C.; Zhao, X.; Wang, A.; Huber, G. W.; Zhang, T., Catalytic Transformation of Lignin for the Production of Chemicals and Fuels . Chemical Reviews 2015, 115 (21), 11559 - 11624. 7. Kim, S.; Chmely, S. C.; Nimos, M. R.; Bomble, Y. J.; Foust, T. D.; Paton, R. S.; Beckham, G. T., Computational Study of Bond Dissociation Enthalpies for a Large Range of Native and Modified Lignins. J ournal of Physical Chemistry Letters 2011, 2 (22), 2846 - 2852. 8. Klinger, G. E.; Zhou, Y.; Hao, P.; Robbins, J.; Aquilina, J. M.; Jackson, J. E.; Hegg, E. L., Biomimetic Reductive Cleavage of Keto Aryl Ether Bonds by Small - Molecule Thiols. Chemsuschem 2019, 12 (21), 4775 - 4779. 9. Shuai, L.; Amiri, M. T.; Questell - Santiago, Y. M.; Heroguel, F.; Li, Y.; Kim, H.; Meilan, R.; Chapple, C.; Ralph, J.; Luterbacher, J. S., Formaldehyde stabilization facilitates lignin monomer production during biomass depolymerization. Science 2016, 354 (6310), 329 - 333. 10. Lan, W.; Amiri, M. T.; Hunston, C. M.; Luterbacher, J. S., Protection Group Effects During alpha,gamma - Diol Lignin Stabilization Promote High - Selectivity Monomer Production. Angewandte Chemie - Inter national Edition 2018, 57 (5), 1356 - 1360. 11. Amiri, M. T.; Dick, G. R.; Questell - Santiago, Y. M.; Luterbacher, J. S., Fractionation of lignocellulosic biomass to produce uncondensed aldehyde - stabilized lignin. Nature Protocols 2019, 14 (3), 921 - 954. 19 12. Lan, W.; de Bueren, J. B.; Luterbacher, J. S., Highly Selective Oxidation and Depolymerization of alpha,gamma - Diol - Protected Lignin. Angewandte Chemie - International Edition 2019, 58 (9), 2649 - 2654. 13. Zakzeski, J.; Bruijnincx, P. C. A.; Jongerius, A. L.; Weckhuysen, B. M., The Catalytic Valorization of Lignin for the Production of Renewable Chemicals. Chemical Reviews 2010, 110 (6), 3552 - 3599. 14. Schutyser, W.; Renders, T.; Van den Bosch, S.; Koelewijn, S. F.; Beckham, G. T.; Sels, B. F., Chemical s from lignin: an interplay of lignocellulose fractionation, depolymerisation, and upgrading. Chemical Society Reviews 2018, 47 (3), 852 - 908. 15. Sun, Z. H.; Fridrich, B.; de Santi, A.; Elangovan, S.; Barta, K., Bright Side of Lignin Depolymerization: T oward New Platform Chemicals. Chemical Reviews 2018, 118 (2), 614 - 678. 16. Renders, T.; Van den Bossche, G.; Vangeel, T.; Van Aelst, K.; Sels, B., Reductive catalytic fractionation: state of the art of the lignin - first biorefinery. Current Opinion in Biotechnology 2019, 56 , 193 - 201. 17. Anderson, E. M.; Stone, M. L.; Hulsey, M. J.; Beckham, G. T.; Roman - Leshkov, Y., Kinetic Studies of Lignin Solvolysis and Reduction by Reductive Catalytic Fractionation Decoupled in Flow - Through Reactors. Acs Sustainable Chemistry & Engineering 2018, 6 (6), 7951 - 7959. 18. Feghali, E.; Carrot, G.; Thuery, P.; Genre, C.; Cantat, T., Convergent reductive depolymerization of wood lignin to isolated phenol derivatives by metal - free catalytic hydrosilylation. Energy & Environmental Science 2015, 8 (9), 2734 - 2743. 19. Rahimi, A.; Ulbrich, A.; Coon, J. J.; Stahl, S. S., Formic - acid - induced depolymerization of oxidized lignin to aromatics. Nature 2014, 515 (7526), 249 - 252. 20. Nguyen, J. D.; Matsuura, B. S.; Stephenson, C. R. J., A Photochemical Strategy for Lignin Degradation at Room Temperature. Journal of the American Chemical Society 2014, 136 (4), 1218 - 1221. 21. Marinovic, M.; Nousiainen, P.; Dilokpimol, A.; Kontro, J.; Moore, R.; Sipila, J.; de Vries, R. P.; Makela, M. R.; Hilden, K., Selective C leavage of Lignin beta - O - 4 Aryl Ether Bond by beta - Etherase of the White - Rot Fungus Dichomitus squalens. Acs Sustainable Chemistry & Engineering 2018, 6 (3), 2878 - 2882. 22. Kamimura, N.; Takahashi, K.; Mori, K.; Araki, T.; Fujita, M.; Higuchi, Y.; Mas ai, E., Bacterial catabolism of lignin - derived aromatics: New findings in a recent decade: Update on bacterial lignin catabolism. Environmental Microbiology Reports 2017, 9 (6), 679 - 705. 23. Otsuka, Y.; Sonoki, T.; Ikeda, S.; Kajita, S.; Nakamura, M.; Katayama, Y., Detection and characterization of a novel extracellular fungal enzyme that catalyzes the specific and hydrolytic cleavage of lignin guaiacylglycerol beta - aryl ether linkages. European Journal of Biochemistry 2003, 270 (11), 2353 - 2362. 20 24. Gal l, D. L.; Kim, H.; Lu, F. C.; Donohue, T. J.; Noguera, D. R.; Ralph, J., Stereochemical Features of Glutathione - dependent Enzymes in the Sphingobium sp Strain SYK - 6 beta - Aryl Etherase Pathway. Journal of Biological Chemistry 2014, 289 (12), 8656 - 8667. 25. Gall, D. L.; Ralph, J.; Donohue, T. J.; Noguera, D. R., A Group of Sequence - Related Sphingomonad Enzymes Catalyzes Cleavage of beta - Aryl Ether Linkages in Lignin beta - Guaiacyl and beta - Syringyl Ether Dimers. Environmental Science & Technology 2014, 4 8 (20), 12454 - 12463. 26. Helmich, K. E.; Pereira, J. H.; Gall, D. L.; Heins, R. A.; McAndrew, R. P.; Bingman, C.; Deng, K.; Holland, K. C.; Noguera, D. R.; Simmons, B. A.; Sale, K. L.; Ralph, J.; Donohue, T. J.; Adams, P. D.; Phillips, G. N., Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of beta - Aryl Ether Bonds in Lignin. Journal of Biological Chemistry 2016, 291 (10), 5234 - 5246. 27. Pereira, J. H.; Heins, R. A.; Gall, D. L.; McAndrew, R. P.; Deng, K.; Holland, K. C.; Donohue, T. J.; Noguera, D. R.; Simmons, B. A.; Sale, K. L.; Ralph, J.; Adams, P. D., Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin beta - Aryl Ether Cleavage Pathway from Sphingobium sp.SYK - 6. Journal o f Biological Chemistry 2016, 291 (19), 10228 - 10238. 28. Masai, E.; Ichimura, A.; Sato, Y.; Miyauchi, K.; Katayama, Y.; Fukuda, M., Roles of the enantioselective glutathione S - transferases in cleavage of beta - aryl ether. Journal of Bacteriology 2003, 185 (6), 1768 - 1775. 29. Kontur, W. S.; Olmsted, C. N.; Yusko, L. M.; Niles, A. V.; Walters, K. A.; Beebe, E. T.; Vander Meulen, K. A.; Karlen, S. D.; Gall, D. L.; Noguera, D. R.; Donohue, T. J., A heterodimeric glutathione S - transferase that stereospecifically breaks lignin's (R) - aryl ether bond reveals the diversity of bacterial - etherases. Journal of Biological Chemistry 2019, 294 (6), 1877 - 1890. 30. Kontur, W. S.; Bingman, C. A.; Olmsted, C. N.; Wassarman, D. R.; Ulbrich, A.; Gall, D. L.; Smith, R. W.; Yusko, L. M.; Fox, B. G.; Noguera, D. R.; Coon, J. J.; Donohue, T. J., Novosphingobium aromaticivorans uses a Nu - class glutathione S - transferase as a glutathione lyase in breaking the beta - aryl ether bond of lignin. Journal of Biolo gical Chemistry 2018, 293 (14), 4955 - 4968. 31. Masai, E.; Katayama, Y.; Nishikawa, S.; Yamasaki, M.; Morohoshi, N.; Haraguchi, T., D etection and L ocalization of a N ew E nzyme C atalyzing the B eta - A ryl E ther C leavage in the S oil B acterium (P seudomonas - P au cimobilis SYK - 6). Febs Letters 1989, 249 (2), 348 - 352. 32. Sonoki, T.; Iimura, Y.; Masai, E.; Kajita, S.; Katayama, Y., Specific degradation of beta - aryl ether linkage in synthetic lignin (dehydrogenative polymerizate) by bacterial enzymes of Sphingomon as paucimobilis SYK - 6 produced in recombinant Escherichia coli. Journal of Wood Science 2002, 48 (5), 429 - 433. 21 33. Masai, E.; Katayama, Y.; Kubota, S.; Kawai, S.; Yamasaki, M.; Morohoshi, N., A B acterial E nzyme D egrading the M odel L ignin C ompound B eta - E therase is a M ember of the Glutathione - S - T ransferase S uperfamily . Febs Letters 1993, 323 (1 - 2), 135 - 140. 34. Masai, E.; Kubota, S.; Katayama, Y.; Kawai, S.; Yamasaki, M.; Morohoshi, N., C haracterization of the C - A laph - D ehydrogenase G ene I nvolved i n the C leavage of B eta - A ryl E ther by P seudomonas - Paucimobilis . Bioscience Biotechnology and Biochemistry 1993, 57 (10), 1655 - 1659. 35. Masai, E.; Katayama, Y.; Nishikawa, S.; Fukuda, M., Characterization of Sphingomonas Paucimobilis SYK - 6 Genes Involved in Degradation of Lignin - Related Compounds. J. Ind. Microbiol. Biot. 1999, 23 (4 - 5), 364 - 373. 36. Masai, E.; Katayama, Y.; Fukuda, M., Genetic and biochemical investigations on bacterial catabolic pathways for lignin - derived aromatic compounds. Bioscience Biotechnology and Biochemistry 2007, 71 (1), 1 - 15. 37. Tanamura, K.; Abe, T.; Kamimura, N.; Kasai, D.; Hishiyama, S.; Otsuka, Y.; Nakamura, M.; Kajita, S.; Katayama, Y.; Fukuda, M.; Masai, E., Characterization of the Third Glutathione S - Transfera se Gene Involved in Enantioselective Cleavage of the beta - Aryl Ether by Sphingobium sp Strain SYK - 6. Bioscience Biotechnology and Biochemistry 2011, 75 (12), 2404 - 2407. 38. Sato, Y.; Moriuchi, H.; Hishiyama, S.; Otsuka, Y.; Oshima, K.; Kasai, D.; Nak amura, M.; Ohara, S.; Katayama, Y.; Fukuda, M.; Masai, E., Identification of Three Alcohol Dehydrogenase Genes Involved in the Stereospecific Catabolism of Arylglycerol - beta - Aryl Ether by Sphingobium sp Strain SYK - 6. Applied and Environmental Microbiolo gy 2009, 75 (16), 5195 - 5201. 39. Gall, D. L.; Kim, H.; Lu, F.; Donohue, T. J.; Noguera, D. R.; Ralph, J., Stereochemical Features of Glutathione - dependent Enzymes in the Sphingobium sp Strain SYK - 6 beta - Aryl Etherase Pathway. Journal of Biological Chem istry 2014, 289 (12), 8656 - 8667. 40. Meux, E.; Prosper, P.; Masai, E.; Mulliert, G.; Dumarcay, S.; Morel, M.; Didierjean, C.; Gelhaye, E.; Favier, F., Sphingobium sp SYK - 6 LigG Involved in Lignin Degradation is Structurally and Biochemically Related to the Glutathione Transferase Omega Class. Febs Lett. 2012, 586 (22), 3944 - 3950. 41. Prates, E. T.; Crowley, M. F.; Skaf, M. S.; Beckham, G. T., Catalytic Mechanism of Aryl - Ether Bond Cleavage in Lignin by LigF and LigG. Journal of Physical Chemistry B 2019, 123 (48), 10142 - 10151. 42. Pic art, P.; Muller, C.; Mottweiler, J.; Wiermans, L.; Bolm, C.; de Maria, P. D.; Schallmey, A., From Gene Towards Selective Biomass Valorization: Bacterial beta - Etherases with Catalytic Activity on Lignin - Like Polymers. Chemsuschem 2014, 7 (11), 3164 - 317 1. 43. Picart, P.; Sevenich, M.; de Maria, P. D.; Schallmey, A., Exploring glutathione lyases as biocatalysts: paving the way for enzymatic lignin depolymerization and future stereoselective applications. Green Chemistry 2015, 17 (11), 4931 - 4940. 22 44. Hig uchi, Y.; Aoki, S.; Takenami, H.; Kamimura, N.; Takahashi, K.; Hishiyama, S.; Lancefield, C. S.; Ojo, O. S.; Katayama, Y.; Westwood, N. J.; Masai, E., Bacterial Catabolism of beta - Hydroxypropiovanillone and beta - Hydroxypropiosyringone Produced in the Reductive Cleavage of Arylglycerol - beta - Aryl Ether in Lignin. Applied and Environmental Microbiology 2018, 84 (7), 21. 45. Picart, P.; Liu, H.; Grande, P. M.; Anders, N.; Zhu, L.; Klankermayer, J.; Leitner, W.; de Maria, P. D.; Schwaneberg, U.; Schallmey, A., Multi - step biocatalytic depolymerization of lignin. Applied Microbiology and Biotechnology 2017, 101 (15), 6277 - 6287. 46. Wang, C.; Ouyang, X. H.; Su, S. S.; Liang, X.; Zhang, C.; Wang, W. Y.; Yuan, Q. P.; Li, Q., Effect of sulfonated lignin on enzymatic activity of the ligninolytic enzymes C alpha - dehydrogenase LigD and beta - etherase LigF. Enzyme and Microbial Technolo gy 2016, 93 - 94 , 59 - 69. 47. Gall, D. L.; Kontur, W. S.; Lan, W.; Kim, H.; Li, Y.; Ralph, J.; Donohue, T. J.; Noguera, D. R., In Vitro Enzymatic Depolymerization of Lignin with Release of Syringyl, Guaiacyl, and Tricin Units. Applied and Environmental Microbiology 2018, 84 (3). 48. Husarcikova, J.; Schallmey, A., Whole - cell cascade for the preparation of enantiopure beta - O - 4 aryl ether compounds with glutathione recycling. Journal of Biotechnology 2019, 293 , 1 - 7. 49. Reiter, J.; Strittmatter, H.; Wiem ann, L. O.; Schieder, D.; Sieber, V., Enzymatic cleavage of lignin beta - O - 4 aryl ether bonds via net internal hydrogen transfer. Green Chemistry 2013, 15 (5), 1373 - 1381. 50. Rosini, E.; Allegretti, C.; Melis, R.; Cerioli, L.; Conti, G.; Pollegioni, L .; D'Arrigo, P., Cascade enzymatic cleavage of the beta - O - 4 linkage in a lignin model compound. Catalysis Science & Technology 2016, 6 (7), 2195 - 2205. 51. Rolando, C.; Monties, B.; Lapierre, C., Methods in Lignin Chemistry. In Wood Science , Timell, T. E., Ed. Springer - Verlag: Berlin Heidelberg, 1992; pp 334 - 349. 52. Pearl, I. A., The Chemistry of Lignin . Marcel Dekker, Inc.: New York, 1967. 53. Sameni, J.; Krigstin, S.; Sain, M., Characterization of Lignins Isolated from Industrial Residues and their Beneficial Uses. Bioresources 2016, 11 (4), 8435 - 8456. 54. Gierer, J.; Smedman, L. A., R eactions of Lignin During Sulfate Cooking .8. M echanism of S plitting of B eta - A rylether B onds in P henolic U nits by W hite L iquor . Acta Chemica Scandinavica 1965, 19 (5), 1103. 55. Miles, L. W. C.; Owen, L. N., D ithiols .12. The Alkaline Hydrolysis of A cetylated H ydroxy - T hiols - A N ew R eaction for the F ormation of C y clic S ulphides . Journal of the Chemical Society 1952, (MAR), 817 - 826. 56. Lapierre, C.; Monties, B.; Rolando, C., T hioacidolysis of Lignin - C omparison with A cidolysis . Journal of Wood Chemistry and Technology 1985, 5 (2), 277 - 292. 23 57. Lundquist, K.; Kirk, T. K., A cid Degradation of Lignin .4. A nalysis of Lignin Acidolysis Products by G as C hromatography , U sing T rimethylsilyl D erivatives . Acta Chemica Scandinavica 1971, 25 (3), 889 - 894. 58. Oae, S., Organic sulfur chemistry: Structure and mechanisms . CRC Press: Boca Raton, Florida, 1991. 24 Chapter 2. - O - 4 Keto Aryl Ether Cleavage of Dimers with Thiols The nucleophilic and reductive properties of thiolates and thiols make them ideal candidates as redox mediators via the thiol/disulfide couple. One mechanism for biological lignin depolymerization entails reduction of keto aryl ether bonds via an S N 2 mechanism with the thiol redox mediator glutathione. In an attempt to mimic this chemis try in a simple protein - and metal - free process, we surveyed several small organic thiols for their ability to cleave aryl keto ethers - O - - mercaptoethanol and dithio threitol yielded up to 100% formation of phenol and acetophenone products from 2 - phenoxyacetophenone, but not from its reduced alcohol congener. We assessed the effects of reaction conditions and of substituents on the aryl rings and the keto ether linkage . These results, together with activation barriers computed via quantum chemical simulations and direct observation of the expected intermediate thioether, point to an S N 2 mechanism. This study confirms that small organic thiols can reductively break down lignin - relevant keto aryl ether linkages. 2.1 Introduction A key need in the effort to replace fossil - with renewable carbon - based products is an efficient and affordable method for reductive cleavage of alkyl aryl ethers. These substructures are the most common linkage in lignin, an underutilized source of renewable carbon found in the cell walls of plants ( Figure 2. 53 ). 1, 2 Ideally, redox mediators would be small and diffusible to access the complex interior of the lignin polymer and would be readily recovered and recycled. Small This chapter is adapted from Klinger, G. E.; Zhou, Y.; Hao, P.; Robbins, J.; Aquilina, J. M.; Jackson, J. E.; Hegg, E. L. Biomimetic Reductive Cleavage of Keto Aryl Ether Bonds by Small Molecule Thiols. ChemSusChem 2019 , 12, 4775 - Y. Zhou synthesized several dimers, P. Hao synthesized one dimer, J. Robbins and J. M. Aquilina assisted Y. Zhou in synthesis. 25 thiol and dithiol rea gents fit these criteria, as the disulfides formed should be amenable to electrochemical recycling, as is known for cystine reduction to cysteine. 3, 4 This atom efficient organocatalytic cycle, summarized in EQN 2. 1 and EQN 2. 2 , offers a path for green, sustainable biomass deconstruction and reductive upgrading. We rep ort here that small thiols do, in fact, reductively cleave the ether bonds in lignin - relevant model substrates via an S N 2 - type mechanism ( EQN 2. 1 ) , liberating phenols, phenylpropanoids, and the corresponding disulfide byproducts of thiol oxidation. EQN 2. 1 EQN 2. 2 Having proposed the above strategy on chemical grounds, we were pleased to note recent 5 - 7 Aryl ether cleavage by thiols is see n in wood - digesting bacteria such as Sphingobium sp. strain SYK 6. 6, 8 - 10 Specifically, the Lig enzymes E, P, F (etherases), and G (lyase) use the c ysteine - containing tripeptide glutathione as the redox active cofactor. 6, 8 - 10 This thiol performs a protein - assisted S N 2 reaction on a keto aryl ether lignin dimer, forming a thioether ( Figure 2. 1 : green). A sec ond glutathione from a lyase then attacks the thioether, forming a disulfide bond and liberating an enolate that is protonated to form a ketone. 6, 8 - 10 - O - 4' - aryl ether cleavage, reductively fragmenting the oxidized lignin dimer. Enzymatic reduction of the glutathione disulfide co - product allows for further turnovers of this reductive aryl ether cleavage. Inspired by the chemistry exploited by Lig enzymes to cleave aryl ethers, we examined the nucleophilic and reductive capabilities of small, diffusible organic thiols to cleave aryl alkyl ether linkages of phenylpropanoid dimers ( Figure 2. 1 : orange). Compared to the Lig enzymes, whose substrates are generally limited to soluble dimers and oligomers, small thiols 26 have the potential to penetrate and cleave the polymer network. 11 As detailed below, this bio - inspired approach has proven surprisingly effective, despite the absence of protein or metal co - catalysts. Figure 2. 1 - aryl ether cleavage sc heme. - aryl ether cleavage pathway in Sphingobium sp. strain SYK - - carbon hydroxy site (green reaction), displayed in parallel with the biomimetic pathway using simple organic thiols for nucleophilic aryl ether cleavage (orange reaction). 2.2 Computation To determine whether active site constraints were essential to catalysis in the protein - directed S N 2 reaction, we conducted docking calculations on LigE and LigF etherases. Using AutoDock Vina, 12 - aryl ether. Consistent with the work of Helmich et al., 8 we found no unusual geometrical orientations or distortions ( Figure 2. 5 ). To probe the bond - breaking and - forming processes in the proposed reaction path, we then performed quantum chemical calculations on 2 - - mercaptoethanol at the G3(MP2) 13 level, with solvent model SM8 14 to simulate the acetonitrile medium. These studies found barriers of 21 kcal/mol for the S N 2 cleavage of the C - O bond, and 11 kcal/mol for attack on the thioether to form the disulfide ( Figure 2. 6 ) and ketone enolate. Both 27 reaction steps were also found to be energetically dow nhill, by 11 and 7 kcal/mol, respectively. These docking and detailed reaction path studies point to experimentally accessible reaction barriers, with TS geometries analogous to those in the enzymatic reaction. 2.3 Methodology : Parameter Screening Glutath ione is the redox mediator in the etherase enzyme. 6, 8, 10, 15 - 17 We therefore tested its capacity to cleave the simplest keto aryl ether lignin model dimer, 2 - phenoxyacetophenone, in the absence of proteins. When ten equivalents of glutathione we re stirred in the presence of solid K 2 CO 3 in refluxing acetonitrile (MeCN), relatively low yields of the phenol were obtained, indicating inefficient S N 2 attack on the phenolic dimer ( Figure 2. 2 - b). Furthermore, as indicated by the low acetophenone yield, glutathione was unable to complete the second step, the thioether reduction. Similar results were seen when using 2 equiv. of gluta thione ( Figure 2. 7 and Table 2. 2 ). We speculate that steric bulk impeded the reac tion of the thioether with a second glutathione. Neither cysteine (the thiol in glutathione) nor N - acetylcysteine, both poorly soluble under the reaction conditions, were able to cleave the ether bond of the dimer ( Figure 2. 8 , Figure 2. 9 , and Figure 2. 10 ), suggesting that the thiol mediator must be solubilized to be effective. Size, acidity, and the ability to form intramolecular disulfide bonds were considered in choosing additional small organic thiols to complete the two - step cleavage process. As evidenced by good to excellent yields of both phenol and acetophenone, efficient reductive ether cleavage was obtained using BME (p K a ~9.6) 18, 19 , DTT (p K a 9.3 and 10.3) 20 , 1,3 - propanedithiol (calculated p K a 9.2, 10.7), and thiophenol (p K a 6.6) 18 - 20 ( Figure 2. 2 - a, Figure 2. 11 , Figure 2. 12 , Figure 2. 13 , Figure 2. 14 , Figure 2. 16 , and Table 2. 2 ). Testing with various solvents, temperatures, bases, and mediator equivalent amounts ( Figure 2. 2 - c, Figure 2. 11 , Figure 2. 12 , Figure 2. 13 , Figure 2. 14 , Figure 2. 16 , Figure 2. 17 , Figure 2. 18 , Figure 2. 19 , Figure 2. 20 , Figure 2. 21 , Figure 2. 22 , Figure 28 2. 23 , Figure 2. 24 , Figure 2. 25 , Figure 2. 26 , Figure 2. 27 , and Figure 2. 28 ; Table 2. 2 ) revealed that DTT and BME achieved the highest yields with the lowest mediator loadings. Though both have p K a s similar to that of glutathione (p K a ~9.6), 21 their success may reflect their small size compared to the sterically bulky glutathione. Soluble bases such as DBU (p K a 13.5), 22 triethylamine (p K a 10.8), 22 or pyridine (p K a 5.2) 22 ( Figure 2. 28 ) in various proportions were ineffective, while the most successful base was solid K 2 CO 3 (p K a 10.3). 22 Increasing the surface area by grinding the solid K 2 CO 3 offered modest acceleration of the reaction ( Figure 2. 44 ). The optimal media were polar aprotic solvents with high dielectric constants, such as MeCN, where the base was largely insoluble. Reactions in water or methanol ( Figure 2. 24 , Figure 2. 25 , and Table 2. 2 ), where K 2 CO 3 is soluble, were less successful. Heat was required for cleavage of the aryl ether dimer except in the solvent DMF, where the cleavage occurred, albeit slowly, even at ambient temperature ( Figure 2. 19 and Table 2. 2 ). Typical S N 2 reactions are favored under polar aprotic conditions, so it wa s not unexpected that polar aprotic solvents like DMF and MeCN worked well. THF, another polar aprotic solvent, was ineffective ( Figure 2. 20 and Table 2. 2 ), likely due to its low boiling point (66 °C) and lower dielectric constant 22 22 Though radical mechanisms can be envisioned, they wer e deemed unlikely, in part because the radical scavenger BHT had essentially no effect on the reaction rate ( Figure 2. 42 ). 29 Figure 2. 2 Summary of key cleavage reactions. (a) Small molecule thiols explored in this study. Reaction proceeded in refluxing MeCN with 10 equivalents of thiol for (b) 2 - 1 and glutathione, (c) 2 - 1 and BME, (d) 2 - 5 and BME, and (e) 2 - hy droxyethyl thioether intermediate ( 2 - 1 - a ) and BME. For compound numbering see Table 2. 1 . 2.4 Mechanism Encouraged by the ability of thiol s to cleav e 2 - 1 (2 - phenoxyaceto phenone, the simplest lignin - relevant keto aryl ether dimer), we explored their reactivity with other ethers ( Table 2. 1 , 0 20 40 60 80 100 0 10 20 30 40 50 % Yield Time (hrs) Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield Time (hrs) Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 10 20 30 40 50 % Yield Time (hrs) Syringol Acetosyringone SS Dimer 0 20 40 60 80 100 0 5 10 15 20 25 % Yield Time (hrs) Acetophenone Thioether b) c) d) e) a) 30 Figure 2. 29 , Figure 2. 30 , Figure 2. 31 , Figure 2. 32 , Figure 2. 33 , Figure 2. 34 , Figure 2. 35 , Figure 2. 36 , and Figure 2. 37 ) - carbon hydroxy group must be oxidized to the keto form for nucleophilic thiolate to displace the phenoxide leaving group ( Table 2. 1 , entries a - c ; Figure 2. 30 ), analogous to the enzymatic Lig pathway. 6, 8 - 10 Thiol treatment gave no reaction with compounds containing sp 3 hybridized - - - methoxy analogues 2 - 2 and 2 - 14 , and also 2 - 13 , which - position. The ability of a carbonyl group to activate a neighbouring carbon for nucleophilic attack is well known, and variously attributed to dipolar or conjugative stabilization of the transition state. 23, 24 In fact, the reactivity of substrates 2 - 10 and 2 - 11 revealed that the carbonyl moiety without other sub stituents is sufficient to enable ether cleavage ( Figure 2. 35 ). However, an aryl substituent on the ether oxygen is needed to make it a viable leaving group, as shown b y the poor reactivity of substrate 2 - 9 . Phenoxyacetic acid 2 - 12 also gave no cleavage products, likely due to its rapid deprotonation and salt precipitation. Nucleophilic attack at sp 3 carbons becomes more difficult with increasing substitution. As expected, 2 - 6 and 2 - 7 showed the expected decrease in reactivity ( Table 2. 1 , entry g and h ; Figure 2. 31 ). Interestingly, BME and DTT cleaved 2 - 3 in moderate yields despite its seco - carbon; the pendant hydroxymethyl group ( Table 2. 1 , entry d ; Figure 2. 31 , Figure 2. 32 , and Figure 2. 33 ) is lost via a retro - aldol cleavage to form 2 - 1 followed by formation of the S N 2 cleavage products. Meanwhile, compounds 2 - 15 through 2 - 16 ( Figure 2. 30 ) showed no reactivity, further confirming the importance of the carbonyl moiety for successful cleavage. Thus, small organic thiols can cleave primary and secondary aryl ether bonds next to a ketone. 31 Entry Dimer Time I II Conv. a *b 2 - 1 22 h 3 h 45% 73% 51% 75% 61% 85% c 2 - 2 48 h 0% 0% 2% d 2 - 3 24 h **33% 45% 72% *e 2 - 4 48 h 13% 25% 99% f 2 - 5 48 h 29% 23% 97% g 2 - 6 24 h 1% 6% 10% h 2 - 7 24 h 1% 2% 2% i 2 - 8 24 h ~98% # 95% *Entries b and e were performed under neat conditions at 100 °C. **The product quantified was acetophenone. # Guaiacol was not able to be quantified. Table 2. 1 Summary of dimer cleavage methodology. Evaluation of ether cleavage of simple lignin - relevant dimers at a 1:10 mole ratio of substrate to thiol. 32 Figure 2. 3 Cleavage of non - - O - 4 aryl ethers and yields under refluxing MeCN, 10 equiv. of BME, and stirring K 2 CO 3 . - aryl ethers bearing lignin - relevant substituents. Methoxy groups on one or both positions ortho to the - OH of the phenol ring are found in lignin subunits. Deme thylation of such methoxy groups by thiols has been reported in studies aimed at production of catechol - type resins. 25, 26 In both cases, some ether cleavage was also seen. In the present work, demethylation by the thiol redox mediator should form a methyl thioether by - product. To test for demethylation, guaiacol, syringol, and acetovanill one monomers were each incubated with BME and K 2 CO 3 in refluxing MeCN and examined by GC - MS and HPLC. Neither 2 - methylthioethanol nor catechol species were observed, and the mass balance of the monomers was retained ( Figure 2. 47 ), indicating no demethylation side reactions. The successful reductive ether cleavage of 2 - 4 ( Table 2. 1 , entry e ; Figure 2. 29 and Figure 2. 36 ), 2 - 5 ( Figure 2. 2 - d; Table 2. 1 , entry f ; Figure 2. 29 and Figure 2. 36 ), and 2 - 8 ( Table 2. 1 , entry i ; Figure 2. 29 ) confirmed that the proposed S N 2 mechanism still occurs regardless of methoxy group steric hindrance and the methoxy groups were not demethylated under our reaction conditions. 33 Figure 2. 4 Hammett plot for para - substitution of the ( t op) aryl ketone and ( b ottom) aryl ether with electron donating and withdrawing groups. Hammett analyses were used to quantify the effect of para substituents on both the aryl ether and the aryl ketone sides on the S N 2 cleavage mechanism. A substantial effect was expected e aryl ether due to changes in the electronics of the leaving group: electron withdrawing substituents make aryl ethers better leaving groups while electron donating groups do the reverse. This finding supports the proposed S N 2 mechanism. Our results ( Figure 2. 4 , Figure 2. 38 , Figure 2. 39 , Figure 2. 40 , and Figure 2. 41 ) indicate that the aryl ether functionalizat ion 2 - 2 - 2 - 2 - 2 - 2 - 2 - 2 - 2 - 34 larger effects, it might have suggested a more complex mechanis m, such as one with direct thiol attack on the ketone carbonyl. To examine the second step of the proposed 2 - step reductive cleavage, the thioether intermediate 2 - 1 - a expected from BME cleavage of 2 - 1 was independently synthesized. 27 Under reaction conditions (2 - 4 h), 2 - 1 - a reacted quickly yielding the expected disulfide and acetophenone products with an excellent mass balance and 100% yield without any product decay over time ( Figure 2. 2 - e and Figure 2. 48 ). Importantly, with authentic material in hand, the thioether could then be identified in trace amounts in the ether cleavage reaction mixtures, verif ying its intermediacy. These results are consistent with our quantum chemical studies indicating that the - keto aryl ether cleavage is the rate - limiting step. Placing para electron withdrawing groups on the aryl ether accelerated the fir st step, thereby allowing the thioether intermediate to be observed in larger quantities ( Figure 2. 38 ). To test if the disulfide itself could contribute to aryl ether c leavage, 2 - 1 was combined with - dithiodiethanol in refluxing MeCN. When the disulfide was present in large excess, 2 - 1 was cleaved resulting in up to 30% phenol yields ( Figure 2. 49 ). This may result from disulfide oxidation and cleavage under our basic conditions, 28 forming sulfonate and thiolate that could enable nucleophilic cleavage. 2.5 Conclusion In summary, these studies lay the groundwork for the biomimetic reductive cleavage of lignin - relevant aryl ether bonds by small - molecule redox - active thiols. Such thiols readily cleave simple keto aryl ether dimers with near complete mass balance. Reaction of more complex aryl ether dimers exhibited up to 100% reductive cleavage yields using 100 equivalents of thiol. While this study focused on single turn - over reactions of the thiol redox mediator, we envision free thiol 35 regeneration via 2 - electron/2 - proto n reduction of the disulfide in an electrochemical cell, enabling net electro - catalytic aryl ether cleavage ( Figure 2. 52 ). Future studies will extend this work to aryl e ther polymers, including lignin, and analyze the feasibility of direct integration with electrochemical disulfide recycling. 2.6 Experimental Detail 2.6.1 General Information Chemicals were tested for purity using 1 H NMR prior to use. All water used was pu rified and deionized using Thermo Scientific four - holder Barnstead E - pure water purification 120 V systems. All reactions were performed under a nitrogen atmosphere using a nitrogen - filled balloon unless otherwise specified. Column purification was accompl ished using Silicycle (Quebec City, Canada) SiliaFlash P60 silica gel (40 - 63 µm). Thin layer chromatography (TLC) was performed on Sigma Aldrich (St. Louis, MO) plastic silica gel 60 F - 254 plates and the bands were visualized using either short wave UV lig ht (254 nm) or development by treatment with iodine or potassium permanganate solution followed by heating. HPLC analysis was performed using an Agilent 1260 Infinity equipped with an Agilent G1315D 1260 diode array detector VL, monitoring at 280 nm and re cording 190 - 400 nm. For dimer and monomer analysis, a Supelco Ascentis Express C18 injections included phenanthrene as an internal standard; external standards were also rechecked during each sequence of analyses. Instrumental control, data acquisition, and data processing for the HPLC were performed with Agilent ChemStation. GC - MS anal yses employed a 6890 Agilent GC equipped with a VF - - Guard column and a 5975b single quadrupole MS detector. 1 H NMR spectra were recorded on 500 MHz Varian spectrometers and 36 referenced to residual solvent peaks. Yuting Zhou synthesized various dimers for methodology and mechanism studies used in this work. 29 2.6.2 Results of molecular simulations 2.6.2 - i Docking of substrate models + glutathione in LigE and LigF enzymes Figure 2. 5 Molecular docking of glutathione with lignin substrates at the active site in Lig etherases 8 using Autodoc Vina. 12 Exhaustiveness was set to 100 and all bonds in the ligand were a) b) c) d) e) f) g) h) 37 ( Figure 2. 5 ) set to rotatable. LigE and LigF are the enzymes that catalyze the enzymatic n - aryl ether bond of lignin dimers. (a) Crystal structure of LigE (PDB ID 4YAN) shown in surface mode with ligands and selected side chains shown in stick view including the cofactor glutathione (green) overlaid with the glutath ion e - O - 4 dimer transition state (red) shown in panel (c) with residues <4 Å away displayed in tan. (b) LigE binding pocket with glutathione (pink) and residue s of <4 Å away (green). Crystallographically observed water molecules are depicted as blue spher es. (c) ChemDraw structure of LigE transition state. (d) Crystal structure of LigF (PDB ID 4XTO) shown in surface mode with ligands and selected sidechains shown in stick mode including the cofactor glutathione (green) overlaid with the thioether intermedi ate (red) shown in panel (f) with residues <4 Å away displayed in tan. (e) LigF binding pocket with glutathione (pink) and residue s of <4 Å away (green). Crystallographically observed water molecules are depicted as blue spheres. (f) ChemDraw structure of the LigF thioether intermediate. The predicted binding modes, affinities, and best fit is ranked for (g) LigE dimer docking and (h) LigF monomer docking. Neither LigE + thioether intermediate nor LigF + glutathion e - O - 4 dimer transition state are shown, b ut both were found to be essentially identical to the corresponding docked ligand as shown above. No distortive interactions were found that would aid in nucleophilic cleavage. The results indicate that neither stereospecific enzyme requires geometric dist ortions for the glutathione thiol to cleave the ether bond. 2.6.2 - ii Activation barriers and reaction paths from quantum chemical modeling Figure 2. 6 (continue onto next page) 38 ( Figure 2. 6 ) Figure 2. 6 Calculated energetics for the 2 - step nucleophilic cleavage of 2 - phenoxyacetophenone with 2 - mercaptoethanol in the gas phase at the G3(MP2) level, and in acetonitrile, simulated with solvent model SM8. Both steps are net downhill. Computed activation energies are consistent with the relative rates observed experimentally, where the first step is rate determining. Final calculations were performed by Ned Jackson. 2.6.3 Sy nthesis of Model Lignin Dimers 2 - phenoxy - 1 - phenylethan - 1 - ol ( 2 - 1) 29 : This compound was prepared following a literature procedure. 30 2 - phenoxyacetophenone (2.25 g, 10.6 mmol) was added to MeOH (80 mL). To this mixture, NaBH 4 (0.6 g, 15.9 mmol) was added in small portions and stirred overnight at R T. After the completion of the reaction, saturated NH 4 Cl (100 mL) solution was added and the mixture was extracted with CH 2 Cl 2 and washed with deionized water. The organic layer was dried over anhydrous Na 2 SO 4 and concentrated under vacuum, giving 97% yiel d of the pure product (2.2 g). 1 H NMR (500 MHz, CDCl 3 4.06 (m, 1H), 4.12 (dd, J = 9.6, 3.1 Hz, 1H), 5.14 (dd, J = 8.9, 3.1 Hz, 1H), 6.93 39 (d, J = 8.7 Hz, 2H), 6.98 (t, J = 7.4 Hz, 1H), 7.29 (dd, J = 8.7, 7.4 Hz, 2H), 7.34 (t, J = 7.3 Hz, 1H), 7.40 (t, J = 7.4 Hz, 2H), 7.47 (d, J = 7.0 Hz, 2H). Spectral data are consistent with those reported in the literature. 31 (1 - methoxy - 2 - phenoxyethyl)benzene ( 2 - 14) 29 : This compound was prepared following a literature procedure. 32 To a stirring solution of sodium hydride (0.14 g, 3.5 mmol, 60 % in mineral oil) in anhydrous THF (10 mL), 2 - phenoxy - 1 - phenylethanol (0.5 g, 2.33 mmol) in anyhydrous THF (10 mL) was added in two portions at 0 °C, under argon. The resulting suspension was stirred at 0 °C for 1 h. Then iodomethane (0.174 mL, 2.80 mmol) was added dropwise. The reaction mixture was monitored by TLC. The reaction was quenched with saturated ammonium chloride solution. The aqueous layer was extracted with diethylether (2×30 mL). The combined organic extracts were washed with brine, dried ove r Na 2 SO 4 and concentrated in vacuo. The crude product was purified by column chromatography, first with pentane to remove the mineral oil followed by (pent/DCM 4:1) to give (1 - methoxy - 2 - phenoxyethyl)benzene as an oil (0.39 g) in 75% yield after solvent rem oval. 1 H NMR (500 MHz, CDCl 3 J = 4.4 Hz, 4H), 7.38 7.33 (m, 1H), 7.30 7.24 (m, 2H), 6.97 6.89 (m, 3H), 4.60 (dd, J = 8.0, 3.6 Hz, 1H), 4.18 (dd, J = 10.2, 8.0 Hz, 1H), 4.01 (dd, J = 10.3, 3.6 Hz, 1H), 3.37 (s, 3H). Spectral data are in ac cordance with those previously reported. 32 2 - phenoxy - 1 - phenylpropan - 1 - one ( 2 - 6) 29 : This compound was prepared following a literature procedure. 33 To a stirring solution of sodium hydride (0.226 g, 5.6 mmol, 60% in mineral oil) in anhydrous THF (20 mL), 2 - phenoxyacetophenone (1.0 g, 4.71 mmol ) in anyhydrous THF (20 mL) was added in two portions at 0 °C, under argon. The reaction mixture was stirred for 20 min. Methyl iodide (0.32 mL, 5.6 mmol) was added dropwise at 0 °C, and the solution was warmed to room temperature. The reaction mixture was 40 monitored by TLC. The mixture was quenched by slow addition of water (30 mL) and extracted with diethylether (2×30 mL). The combined organic extracts were washed with brine and dried over anhydrous Na 2 SO 4 , filtered and evaporated to give a crude oil. The oil was purified by column chromatography (pent/DCM, 1:1) to give 2 - phenoxy - 1 - phenyl - 1 - propanone as white solid (0.57 g) in 54% yield. 1 H NMR (500 MHz, CDCl 3 8.04 (m, 2H), 7.68 7.55 (m, 1H), 7.48 (t, J = 7.8 Hz, 2H), 7.26 7.22 (m, 2H), 6.94 ( t, J = 7.4, 1.0 Hz, 1H), 6.88 (d, J = 7.3, 1.0 Hz, 2H), 5.49 (q, J = 6.9 Hz, 1H), 1.72 (d, J = 6.9, 0.7 Hz, 3H). Spectral data are in accordance with those previously reported. 33 2 - methyl - 2 - phenoxy - 1 - phenylpropan - 1 - one ( 2 - 7) 29 : This compound was prepared following a literature procedure. 32 To a stirring solution of sodium hydride (0.47 g, 11.7 mmol, 60% in mineral oil) i n anhydrous THF (20 mL), 2 - phenoxyacetophenone (1.0 g, 4.71 mmol) in anhydrous THF (20 mL) was added in two portions at 0 °C, under argon. The reaction mixture was stirred for 20 min. Methyl iodide (0.63 mL, 9.9 mmol) was added dropwise at 0 °C, and the so lution was warmed to room temperature. The reaction mixture was monitored by TLC. The mixture was quenched by slow addition of water (30 mL) and extracted with diethyl ether (2×30 mL). The combined organic extracts were washed with brine and dried over anh ydrous Na 2 SO 4 , filtered, and evaporated to give a crude oil. The oil was purified by column chromatography (pent/DCM, 4:1) to give 2 - methyl - 2 - phenoxy - 1 - phenylpropan - 1 - one as white solid (0.5 g) in 44% yield. 1 H NMR (500 MHz, CDCl 3 8.24 (m, 2H), 7 .49 (t, J = 7.2, 1.3 Hz, 1H), 7.42 7.35 (m, 2H), 7.18 7.11 (m, 2H), 6.89 (t, J = 7.3, 1.1 Hz, 1H), 6.79 6.74 (m, 2H), 1.70 (s, 6H). Spectral data are in accordance with those previously reported. 32 41 3 - hydroxy - 2 - phenoxy - 1 - phenylpropan - 1 - one ( 2 - 3) 29 : This compound was prepared following a literature procedure. 34 2 - phe n oxyacetopheone (2.12 g, 10.0 mmol) and formaldehyde (1.15 mL, 15.0 mmol) were added to an acetone/ethanol mixture (1:1, 100 mL). K 2 CO 3 (1.38 g, 10.0 mmol) was added into the solution mixture and stirred for 1.5 h. The mixture was then washed w ith water (250 mL) and extracted with CH 2 Cl 2 . The organic layer was dried over Na 2 SO 4 , concentrated, and purified by column chromatography with hexanes/EtOAc (9:1 to 1:1) to give the pure product (1.72 g) in 71% yield. 1 H NMR (500 MHz, CDCl 3 J = 12.1, 6.2 Hz, 1H), 4.18 (dd, J = 12.1, 3.9 Hz, 1H), 5.57 (dd, J = 6.2, 4.0 Hz, 1H), 6.90 (d, J = 7.8 Hz, 2H), 6.97 (t, J = 7.4 Hz, 1H), 7.28 7.22 (m, 2H), 7.49 (t, J = 7.8 Hz, 2H), 7.61 (t, J = 7.4 Hz, 1H), 8.05 (d, J = 8.3 Hz, 2H). Spectral data are in accordance with those previously reported. 34 2 - phenoxy - 1 - phenylpropane - 1,3 - d iol 29 : This compound was prepared following a literature procedure. 35, 36 To a stirring solution of 3 - hydroxy - 2 - phenoxy - 1 - phenyl - 1 - propanone (0.56 g, 2.31 mmol) in methanol (20 mL), NaBH 4 (0.13 g, 3.46 mmol) in anyhydrous THF (20 mL) was added in portions at 0 °C, under argon. The reaction mixture was monitored by TLC. The mixture was quenched by the slow addition of saturated NH 4 Cl (20 mL) and then extracted with EtOAc (2×30 mL). The combined organic extracts were washed with brine and dried over anhydrou s Na 2 SO 4 , filtered, and evaporated to give 2 - phenoxy - 1 - phenyl - 1,3 - propanediol as white solid (0.423 g) in 75% yield. 1 H NMR (500 MHz, CDCl 3 7.46 (m, 2H), 7.39 (dd, J = 8.1, 6.5 Hz, 2H), 7.33 7.28 (m, 3H), 7.04 6.98 (m, 3H), 5.08 (dd, J = 6.8, 3.2 Hz, 1H), 4.45 (ddt, J = 7.9, 6.9, 4.1 Hz, 1H), 3.84 (ddd, J = 12.1, 5.3, 4.0 Hz, 1H), 3.59 (ddd, J = 12.1, 7.3, 4.0 Hz, 1H), 2.81 (d, J = 3.2 Hz, 1H), 42 1.77 (dd, J = 7.4, 5.3 Hz, 1H). No attempt was made to separate the diastereomers. Spectral data are in accordance with those previously reported. 35, 36 1 - (4 - hydroxy - 3 - methoxyphenyl) - 2 - (2 - methoxyphenoxy)ethan - 1 - one ( 2 - 4): This compound was prepared following a literature procedure. 31, 37 - 39 Benzyl chloride (0.075 mL) was added to a stirring solution of acetovanillone (83 mg) and pyridine (1 mL) in an ice bath. The mixture was taken out of the ice bath and stirred at room temperature for 15 minutes. Hexane (5 mL) was added to the solution producing w hite crystals. The product was recrystallized using EtOAc/hexane and dried to form the protected monomer. The product (1.08 g) was dissolved in CHCl 3 (12 mL) and HBr was pipetted into the mixture and cooled in an ice bath. Br 2 ring until the solution became colorless. The solution was extracted with water and CH 2 Cl 2 producing the protected bromoacetovanillone. Guaiacol (0.223 g, 1.8 mmol) was dissolved in DMF (6.7 mL) and then added to the protected bromoacetovanillone (0.6067 g , 1.67 mmol). To this solution, K 2 CO 3 (3 eq.) was added and stirred for 2 h. The product was recrystallized from methanol forming the protected G - G dimer. The protected dimer (1 g) was added to THF (40 mL) and cooled in an ice bath to 0 °C. LiOH (1 M solut ion, 40 mL) was added dropwise to the solution. The reaction was removed from the ice bath, stirred overnight at RT, quenched with HCl to pH 4 and extracted with 3 x 20 mL of diethyl ether. The ether layer was washed with brine (100 mL), dried over anhydro us Na 2 SO 4 , concentrated, and purified through column chromatography with hexanes/EtOAc (1:1) to give the purified product. 1 H NMR (500 MHz, DMSO - d 6 J = 8.6 Hz, 1H), 6.75 6.82 (m, 2H), 6.84 (ddd, J = 7 .8, 6.3, 2.7 Hz, 1H), 6.91 6.99 (m, 1H), 7.06 (d, J = 2.4 Hz, 43 1H), 7.34 (dd, J = 8.7, 2.4 Hz, 1H). Spectral data are in accordance with those previously reported. 31, 37 - 39 2 - (2,6 - dimethoxyphenoxy) - 1 - (4 - hydroxy - 3,5 - dimethoxyphenyl)ethan - 1 - one ( 2 - 5): This compound was prepared following a literature procedure. 31, 37 - 39 Benzyl chloride (0.075 mL) was added to a stirring solution of acetosyringone (83 mg) and pyridine (1 mL) in an ice bath. The mixture was taken out of the ice bath and stirred at room temperature for 15 minutes. Hexane (5 mL) was added to the solution producing white crystals. The product was recrystallized using EtOAc/hexane and dried to form the protected monomer. The product (1.08 g) was dissolved in CHCl 3 (12 mL) and HBr was pipetted into the mixture and cooled in an ice bath. Br 2 was extracted with water and CH 2 Cl 2 producing the protected bromoacetosyringone. Syringol (0.223 g, 1.80 mmol) was dissolved in DMF (6.7 mL) and then added to the protected bromoacetosyringone (0.607 g, 1.67 mmol). To this solution, K 2 CO 3 (3 eq.) was added and stirred for 2 h. The pr oduct was recrystallized from methanol forming the protected S - S dimer. The protected dimer (1.00 g) was added to THF (40 mL) and cooled in an ice bath to 0 °C. LiOH (1.0 M solution, 40 mL) was added dropwise to the solution. The reaction was removed from the ice bath and stirred overnight at RT. The reaction was quenched with HCl to pH 4 and extracted with 3 x 20 mL of diethyl ether. The ether layer was washed with brine (100 mL), dried over anhydrous Na 2 SO 4 , concentrated, and purified through column chrom atography with hexanes/EtOAc (1:1) to give a yellow powder after solvent removal. 1 H NMR (500 MHz, CDCl 3 6H), 5.11 (s, 1H), 5.95 (s, 1H), 6.60 (d, J = 8.4 Hz, 2H), 7.03 (t, J = 8.4 Hz, 1H), 7.44 (s, 2H). Spectral data are in accor dance with those previously reported. 31, 37 - 39 44 1 - (3,4 - dimethoxyphenyl) - 2 - (2 - methoxyphenoxy)ethan - 1 - one ( 2 - 8) 29 : This compound was prepared following a literature procedure. 40 A 250 mL round bottom flask connected with a dropping funnel was charged with guaiacol (0.9 g, 7.27 mmol) and K 2 CO 3 (1.26 g, 9.09 mmol) in acetone (40 mL) and stirred at room temperature. To this solution, 2 - bromo - 1 - (3,4 - dimethoxyphenyl)ethanone (1.57 g, 6.06 mmol) in acetone (40 mL) was added dropwise at room temperature. The resulting suspension was stirred at room temperature overnight; the suspension was filtered and concentrated in vacuo . The crude product was purified by column chromatography using EtOAc/hexane (3:7) to give 1 - (3,4 - dimethoxyphenyl) - 2 - (2 - methoxyphenoxy)ethan - 1 - one as a white solid (1.31 g) in 72% yield after solvent removal. 1 H NMR (500 MHz, CDCl 3 J = 8.4, 2.0 Hz, 1H), 7.60 (d, J = 2.0 Hz, 1H), 6.96 (ddd, J = 8.0, 5.9, 3.0 Hz, 1H), 6.94 6.88 (m, 2H), 6.87 6.81 (m, 2H), 5.30 (s, 2H), 3.96 (s, 3H), 3.94 (s, 3H), 3.89 (s, 3H). Spectral data are in accordance with those previously reported. 40 2 - bromo - 1 - (3,4 - dimethoxyphenyl)ethanone 29 : This compound was prepared following a literature procedure. 32 To a stirring solution of 1 - (3,4 - dimethoxyphenyl)ethanone (4.0 g, 22.2 mmol) in 100 mL chloroform, Br 2 (3.7 g, 23.3 mmol) solu tion in 100 mL of chloroform was added dropwise over 2 h at room temperature. The reaction mixture was washed with saturated Na 2 S 2 O 3 water solution and brine. The organic layer was collected and dried over anhydrous MgSO4. The solvent was evaporated in vac uo and the crude product was purified by column chromatography (100% DCM) to give 2 - bromo - 1 - (3,4 - dimethoxyphenyl)ethanone as a white solid (4.71 g) in 82% yield after solvent removal. 1 H NMR (500 MHz CDCl 3 J = 8.4, 2.1, 0.9 Hz, 1H), 7.55 (d, J = 45 1.7 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 4.42 (d, J = 1.0 Hz, 2H), 3.96 (dd, J = 10.8, 0.9 Hz, 6H). Spectral data are in accordance with those previously reported. 32 2 - ((2 - hydroxyethyl)thio) - 1 - phenylethan - 1 - one ( 2 - 1 - a): This compound was prepared according to a literature procedure. 27 2 - Bromoacetophenone (500 mg) was added to a mixture of triethylamine (280 mg, 2.76 mM) and 2 - mercaptoethanol (196 mg, 2.51 mM) in CH 2 Cl 2 (8 mL). The mixture was stirred at room temperature for 3 0 minutes. The solution was diluted with CH 2 Cl 2 (60 mL) and extracted with 3 x 100 mL of deionized water. The organic layer was dried with sodium sulfate, concentrated, and purified by column chromatography with CH 2 Cl 2 /MeOH (95:5) resulting in a pale yello wish oil after solvent removal under vacuum. 1 H NMR (500 MHz, Acetonitrile - d 3 2.65 (t, J = 6.4 Hz, 2H), 2.96 (t, J = 5.9 Hz, 1H), 3.63 (q, J = 6.4 Hz, 2H), 3.94 (s, 2H), 7.51 (t, J = 7.8 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.97 (d, J = 7.1 Hz, 2H). Spectral data are in accordance with those previously reported. 27 2 - phenoxy - 1 - (p - tolyl)ethan - 1 - one ( 2 - 17 - c) : This compound was prepared according to literature procedure. 41 2 - bromo - 1 - ( p - tolyl)etha n - 1 - one (2.15 g, 0.67 M) was added to a mixture of potassium carbonate (2.07 g, 1 M) and phenol (0.95 g, 0.67 M) in acetone (15 mL). The mixture was stirred at reflux overnight. The solvent was removed by rotary evaporation and the resulting solid was re - d issolved in EtOAc /diethyl ether and washed with brine. The organic layer was dried with Na 2 SO 4 , rotary evaporated, and then recrystallized from 2 - propanol forming a white crystal (0.59 g) in 26% yield. 1 H NMR (500 MHz, CDCl 3 6.95 (d, J = 7.8 Hz, 2H), 6.98 (t, J = 7.4 Hz, 1H), 7.27 (d, J = 14.3 Hz, 4H), 7.90 (d, J = 8.2 Hz, 2H). This compound was prepared according to a literature procedure. 41 46 1 - phenyl - 2 - (p - toly loxy)ethan - 1 - one ( 2 - 18 - b) 29 : This compound was prepared following a literature procedure. 42 A 250 mL round bottom flask connected with a dropping funnel was charged with p - cresol (6.5 g, 60 mmol) and K 2 CO 3 (11 g, 79 mmol) in acetone (50 mL) and stirred at room temperature. To this solution, 2 - bromoacet ophenone (7.9 g, 40 mmol) in acetone (50 mL) was added dropwise at room temperature. The resulting suspension was stirred at room temperature overnight; the suspension was filtered and concentrated in vacuo . The crude product was purified by column chromat ography using EtOAc /hexane (1:9) to give 1 - phenyl - 2 - ( p - tolyloxy)ethan - 1 - one as a white solid (4.8 g) in 53% yield after solvent removal. 1 H NMR (500 MHz, CDCl 3 7.91 (m, 2H), 7.70 7.57 (m, 1H), 7.51 (t, J = 7.8 Hz, 2H), 7.15 7.05 (m, 2H), 6.9 2 6.78 (m, 2H), 5.26 (s, 2H), 2.29 (s, 3H). Spectral data are in accordance with those previously reported. 42 1 - (4 - methoxyphenyl) - 2 - phenoxyethan - 1 - one ( 2 - 17 - b) : This compound was prepared according to literature procedure. 43 Potassium carbonate (5.2 g, 0. 46 M) and phenol (3.5 g, 0.46 M) were stirred in acetone (75 mL) under nitrogen at room temperature for 30 minutes. 2 - bromo - 1 - (4 - methoxyphenyl)ethan - 1 - one (7 g, 0. 46 M) was added to the mixture and stirred overnight at room temperature. The reaction was filtered with What man paper, the solvent removed by rotary evaporation, and the resulting solid dissolved in EtOAc. The EtOAc was washed with water (20 mL) and brine (20 mL), and it was then dried with Na 2 SO 4 . The remaining EtOAc was rotary evaporated and pumped on overnigh t. The solids were recrystallized from ethanol producing a white crystal (7.77 g) in 93% yield. 1 H NMR (500 MHz, CDCl 3 J = 8.8 Hz, 2H), 7.32 7.27 (m, 2H), 7.01 6.91 (m, 5H), 5.22 (s, 2H), 3.89 (s, 3H). Spectral data are in accordance with t hose previously reported. 43 47 2 - (4 - methoxyphenoxy) - 1 - phenylethan - 1 - one ( 2 - 18 - a) 29 : This compound was prepared following a literature procedure. 42 A 250 mL round bottom flask connected with a dropping funnel was charged with 4 - methoxyphenol (0.3 g, 2.25 mmol) and K 2 CO 3 (0.5 g, 3.6 mmol) in acetone (20 mL) and stirred at room t emperature. To this solution, 2 - bromoacetophenone (0.3 g, 1.5 mmol) in acetone (20 mL) was added dropwise at room temperature. The resulting suspension was stirred at room temperature overnight; the suspension was filtered and concentrated in vacuo . The cr ude product was purified by column chromatography using 100% dichloromethane to give 2 - (4 - methoxyphenoxy) - 1 - phenylethan - 1 - one as a white solid (0.24 g) in 66% yield after solvent removal. 1 H NMR (500 MHz, CDCl 3 7.92 (m, 2H), 7.66 7.58 (m, 1H), 7.50 (t, J = 7.8 Hz, 2H), 6.93 6.87 (m, 2H), 6.86 6.80 (m, 2H), 5.23 (s, 2H), 3.76 (s, 3H). Spectral data are in accordance with those previously reported. 42 1 - phenyl - 2 - (4 - (trifluoromethyl)phenoxy)ethan - 1 - one ( 2 - 18 - c) : This compound was prepared according to literature procedure. 43 Potassium carbonat e (2.6 g, 0. 46 M) and 4 - (trifluoromethyl)phenol (3.0 g, 0. 46 M) were stirred in acetone (40 mL) under N 2 at room temperature for 30 minutes. 2 - bromoacetophenone (3.5 g, 0. 46 M) was added to the mixture and stirred overnight at room temperature. The reaction was filtered with Whatman paper, the solvent removed by rotary evaporation, and the resulting solid d issolved in EtOAc. The EtOAc was washed with water (20 mL) and brine (20 mL), and it was then dried with Na 2 SO 4 . The remaining EtOAc was rotary evaporated and pumped on overnight. The solids were recrystallized from ethanol producing white crystals (3.51 g ) in 68% yield. Spectral data are consistent with those reported in the literature. 1 H NMR (500 MHz, CDCl 3 J = 7.1 Hz, 48 2H), 7.66 (t, J = 8.5 Hz, 1H), 7.55 (m, 4H), 7.02 (d, J = 8.5 Hz, 2H), 5.36 (s, 2H). Spectral data are in accordance with tho se previously reported. 44 2 - phenoxy - 1 - (4 - (trifluoromethyl)phenyl)ethan - 1 - one ( 2 - 17 - d) : This compound was prepared according to literature procedure. 43 Potassium carbonate (2.6 g, 0. 46 M) and phenol (1.75 g, 0. 46 M) were stirred in acetone (40 mL) under N 2 at room temperature for 30 minutes. 2 - bromo - 1 - (4 - (trifluoromethyl)phenyl)ethan - 1 - one (4.65 g, 0. 46 M) was added to the mixture a nd stirred overnight at room temperature resulting in a bright pink solution. The reaction was filtered with Whatman paper, the solvent removed by rotary evaporation, and the resulting solid dissolved in EtOAc. The EtOAc was washed with water (20 mL) and b rine (20 mL), and it was then dried with NaSO 4 . The remaining EtOAc was rotary evaporated and pumped on overnight. The solids were recrystallized from ethanol producing a white crystal (0.088 g) in 17% yield. 1 H NMR (500 MHz, CDCl 3 J = 8.1 Hz, 2H), 7.78 (d, J = 8.2 Hz, 2H), 7.31 (dd, J = 8.8, 7.4 Hz, 2H), 7.02 (t, J = 7.4 Hz, 1H), 6.95 (d, J = 7.8 Hz, 2H). Spectral data are in accordance with those previously reported. 45 4 - (2 - oxo - 2 - phenylethoxy)benzonitrile ( 2 - 18 - d) : This compound was prepared according to literature procedure. 43 Potassium carbonate (5.2 g, 0. 46 M) and 4 - hydroxybenzonitrile (4.35 g, 0.46 M) were stirred in acetone (75 mL) under nitrogen at room temperature for 30 minutes. 2 - bromoacet ophenone (7 g, 0. 46 M) was added to the mixture and stirred overnight at room temperature. The reaction was filtered with Whatman paper, the solvent removed by rotary evaporation, and the resulting crude product was redissolved in EtOAc. The EtOAc was was hed with water (20 mL) and brine (20 mL) and dried with Na 2 SO 4 . The remaining EtOAc was rotary evaporated and pumped on overnight. The solids were recrystallized from ethanol producing a white crystal (1.705 g) in 21% yield. Spectral data 49 are consistent wi th those reported in the literature. 1 H NMR (500 MHz, CDCl 3 J = 8.1 Hz, 2H), 7.66 (t, J = 8.1 Hz, 1H), 7.59 (d, J = 9.0 Hz, 2H), 7.53 (t, J = 7.8 Hz, 2H), 6.98 (d, J = 8.9 Hz, 2H), 5.38 (s, 2H). Spectral data are in accordance with those previo usly reported. 44 1 - (4 - (dimethylamino)phenyl) - 2 - phenoxyethan - 1 - one ( 2 - 17 - a) : This compound was prepared according to literature procedure. 43 Potassium carbonate (160 mg, 0.46 M) and phenol (0.11 g, 0.46 M) were stirred in acetone (2.5 mL) under nitrogen at room temperature for 30 minutes. 2 - bromo - 1 - (4 - (dimethylami no)phenyl)ethan - 1 - one (0.28 g, 0.46 M) was added to the mixture and stirred overnight at room temperature. The reaction was filtered with Whatman paper, the solvent removed by rotary evaporation, and the resulting crude product dissolved in EtOAc. The EtOA c was washed with water (20 mL) and brine (20 mL) and dried with Na 2 SO 4 . The remaining EtOAc was rotary evaporated and pumped on overnight. The solids were recrystallized from ethanol producing a light brown crystal (0.085 g) in 29% yield. Spectral data ar e consistent with those reported in the literature. 1 H NMR (500 MHz, CDCl 3 J = 5.3 Hz, 2H), 7.37 7.24 (m, 2H), 7.06 6.86 (m, 3H), 6.69 ((d, J = 8.8 Hz, 2H), 5.19 (s, 2H), 3.08 (s, 6H). 13 C NMR (126 MHz, CDCl 3 44, 129.49, 122.55, 121.31, 114.81, 110.80, 70.55, 40.07. MS (ESI+, m/z ): 256.1363. 2 - (4 - acetylphenoxy) - 1 - phenylethan - 1 - one: This compound was prepared according to literature procedure. 41 2 - bromoacetophenone (2.01 g, 0.67 M) was added to a mixture of potassium carbonate (2.07 g, 1 M) and 1 - (4 - hydroxyphenyl)ethan - 1 - one (1 .38 g, 0.67 M) in acetone (15 mL). The mixture was stirred at reflux overnight. The solution was rotary evaporated and re - dissolved in EtOAc /diethyl ether and washed with brine. The organic layer was dried with Na 2 SO 4 , rotary 50 evaporated, and then recrysta llized from 2 - propanol forming a light yellow crystal (1.73 g) in 68% yield. Spectral data are consistent with those reported in the literature. 1 H NMR (500 MHz, CDCl 3 ) J = 8.3 Hz, 2H), 7.94 (d, J = 9.0 Hz, 2H), 7.65 (t, J = 7.5 Hz, 1H), 7.53 (t , J = 7.7 Hz, 2H), 6.97 (d, J = 9.0 Hz, 2H), 5.37 (s, 2H), 2.55 (s, 3H). 13 C NMR (126 MHz, CDCl 3 193.52, 161.75, 134.25, 134.16, 131.09, 130.64, 128.97, 128.06, 114.42, 70.44, 26.40. MS (ESI+, m/z ): 255.1032. (1 - methoxyethyl)benzene 29 : This compound was prepared following a literature procedure. 46 To a stirring solution of NaOH (2 g, 49.2 mmol, 60% in mineral oil) in anhydrous THF (40 mL), 2 - p henoxy - 1 - phenylethanol (3 g, 24.6 mmol) in anyhydrous THF (40 mL) was added under argon in two portions at 0 °C. The resulting suspension was stirred at 0 °C for 1 h. Then iodomethane (2.3 mL, 36.9 mmol) was added dropwise, and the reaction mixture was mon itored by TLC. The reaction was quenched with saturated NH 4 Cl solution. The aqueous layer was extracted with diethylether (2×80 mL). The combined organic extracts were washed with brine, dried over Na 2 SO 4 and concentrated in vacuo . The crude product was pu rified by column chromatography (100% pentane) and dried via rotary evaporation to give 2 - methoxy - 1 - phenoxy - 1 - phenylethane as an oil (2.36 g) in 71% yield. 1 H NMR (500 MHz, CDCl 3 7.35 (m, 2H), 7.34 7.27 (m, 3H), 4.31 (q, J = 6.5 Hz, 1H), 3.24 (s, 3H), 1.45 (d, J = 6.5 Hz, 3H). Spectral data are in accordance with those previously reported. 46 2,2' - (1,4 - phenylenebis(oxy))bis(1 - phenylethan - 1 - one): This compound was prepared according to literature procedure. 43 Potassium carbonate (5.2 g, 0. 46 M) and hydroquinone (4.0 g, 0. 46 M) were stirr ed in acetone (75 mL) under N 2 at room temperature for 30 minutes. 2 - bromoacetophenone (7 g, 0. 46 M) was added to the mixture and stirred overnight at 51 room temperature. The reaction was filtered with Whatman paper, the solvent removed by rotary evaporatio n, and the resulting solid was redissolved in EtOAc. The EtOAc was washed with water (20 mL) and brine (20 mL) and dried with sodium sulfate. The remaining EtOAc was rotary evaporated and pumped on overnight. The solids were recrystallized from ethanol pro ducing a tan powder (1.52 g) in 25% yield. 1 H NMR (500 MHz, CDCl 3 J = 7.8 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H), 7.50 (t, J = 7.7 Hz, 2H), 6.89 (d, J = 0.9 Hz, 2H), 5.22 (s, 2H). Spectral data are in accordance with those previously reported. 47 2.6.4 General Procedure for Reduction of Lignin Model Dimers Lignin model dimers (10 mg) and solid base (100 mg) were added to a round - bottom flask (50 mL) equipped with a stir bar, condenser, septum, and nitrogen - filled balloon, and the entire apparatus was purged with N 2 . Solvent (20 mL) and thiol (1:1, 1:2, 1:10, and 1:100 dimer to thiol mole ratio) were added through a septum, and the reaction was heated to the desired temperature with periodic sampling to monitor progress over 24 48 h. 2.6.4 - i Methodology Summarization of Thiol - Dependent 2 - Phenoxyacetophenone Cleavage Thiol Solvent Mole Ratio Temp (°C) Time (hrs) % Conv. I II BME Neat - 100 3 85 73 75 BME MeCN 1:1 Reflux 24 14 8 6 BME MeCN 1:2 Reflux 24 34 23 27 BME MeCN 1:10 Reflux 22 61 45 51 BME MeCN 1:100 Reflux 24 94 75 85 BME MeCN 1:10 50 24 7 2 1 BME MeCN 1:10 RT 24 1 0 3 BME MeOH 1:10 Reflux 24 30 1 16 Ta ble 2.2 Summary (C ont inue onto next page) 52 Table 2. 2 BME MeOH 1:10 40 12 11 1 4 BME MeOH 1:10 RT 24 1 0 2 BME DMF 1:10 100 24 97 70 56 BME DMF 1:10 50 24 94 53 61 BME DMF 1:10 RT 24 54 18 15 BME NMP 1:10 100 12 94 66 87 BME NMP 1:10 50 12 40 19 22 BME NMP 1:10 RT 24 6 1 1 BME DMSO 1:10 100 4 99 81 99 BME DMSO 1:10 50 12 80 36 25 BME DMSO 1:10 RT 24 38 16 7 BME GVL 1:10 100 24 29 0 0 BME GVL 1:10 50 24 1 0 0 BME GVL 1:10 RT 24 0 0 0 BME 1,4 - Dioxane 1:2 Reflux 24 15 0 0 BME 1,4 - Dioxane 1:10 Reflux 24 4 0 0 BME THF 1:10 Reflux 24 9 0 0 BME THF 1:10 RT 24 1 0 0 BME Water 1:10 Reflux 24 - 5 35 BME Water 1:10 50 24 - 1 31 BME Water 1:10 RT 24 - 0 4 DTT Neat 100 3 85 48 62 DTT MeCN 1:1 Reflux 24 18 6 13 DTT MeCN 1:2 Reflux 24 69 47 53 DTT MeCN 1:10 Reflux 24 55 25 34 DTT MeCN 1:100 Reflux 24 34 29 31 DTT MeOH 1:2 Reflux 24 3 2 0 DTT DMF 1:2 100 24 57 15 56 DTT NMP 1:2 100 24 94 5 71 DTT DMSO 1:2 100 2 98 73 78 Thiophenol Neat 100 6 - - 5 MeCN 1:1 Reflux 24 30 4 16 MeCN 1:2 Reflux 24 95 5 69 MeCN 1:10 Reflux 24 100 26 46 MeCN 1:100 Reflux - - - - Glutathione MeCN 1:1 Reflux 48 45 3 38 MeCN 1:2 Reflux 48 39 0 33 MeCN 1:10 Reflux 48 25 0 10 MeCN 1:100 Reflux 48 9 1 3 1,3 - Propanedithiol neat 100 3 23 15 15 MeCN 1:1 Reflux 24 8 2 7 53 Table 2. 2 Summary table of 2 - phenoxyacetophenone cleavage and product yields methodology under various solvents, temperatures, thiol mediators, and substrate to thiol ratios. 2.6.4 - ii Thiol Exploration on Cleavage of 2 - Phenoxyacetophenon e Figure 2. 7 Cleavage of 2 - phenoxyacetophenone (10 mg, 2.36 mM) by reaction with glutathione in refluxing MeCN at (a) 1:1 mole ratio of substrate to thiol, (b) 1:2 mole ratio of substrate to thiol, (c) 1:10 mole ratio of substrate to thiol, and (d) 1:100 mole ratio of substrate to thiol. (a) and (b) conditions gave the highest dimer cleavage yield of ~40%. Acetophenone formation is negligible in each case, suggesting that this rea ction may stop at the thioether adduct. Glutathione is largely insoluble under our reaction conditions, forming a milky - white emulsion. This could explain the counterintuitive trend of decreasing reactivity with increasing thiol concentrations. 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs 2 - Phenoxyacetophenone : Glutathione 1:1 in Refluxing MeCN Phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs 2 - Phenoxyacetophenone : Glutathione 1:2 in Refluxing MeCN Phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs 2 - Phenoxyacetophenone : Glutathione 1:10 in Refluxing MeCN Phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs 2 - Phenoxyacetophenone : Glutathione 1:100 in Refluxing MeCN Phenol Acetophenone 2-phenoxyacetophenone Table 2. 2 MeCN 1:2 Reflux 24 12 4 10 MeCN 1:10 Reflux 24 71 63 67 MeCN 1:100 Reflux 8 98 90 94 MeOH 1:100 Reflux 24 15 12 0 DMF 1:100 100 2 100 84 86 NMP 1:100 100 4 100 80 91 DMSO 1:100 100 2 100 93 98 ( a ) (b) (c) (d) 54 Figure 2. 8 Cysteine was probed as a mediator for the cleavage of 2 - phenoxyacetophenone d ue to the bulkiness of the glutathione tripeptide. The dimer (10 mg, 2.36 mM) was refluxed in MeCN with cysteine at (a) 1:1 mole ratio of substrate to thiol, (b) 1:2 mole ratio of substrate to thiol, (c) 1:10 mole ratio of substrate to thiol, and (d) 1:100 mole ratio of substrate to thiol. Low reactivity was seen in all cases with (b) and (c) conditions givi ng the highest product yield of ~10%. The low reactivity was presumably due to the poor solubility of cysteine in MeCN. 0 20 40 60 80 100 0 10 20 30 40 % yield hrs 2 - Phenoxyacetophenone : Cysteine 1:1 in Refluxing MeCN phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 10 20 30 40 % yield hrs 2 - Phenoxyacetophenone : Cysteine 1:2 in Refluxing MeCN phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 10 20 30 40 % yield hrs 2 - Phenoxyacetophenone : Cysteine 1:10 in Refluxing MeCN phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 10 20 30 40 % yield hrs 2 - Phenoxyacetophenone : Cysteine 1:100 in Refluxing MeCN phenol acetophenone 2-phenoxyacetophenone ( a ) (b) (c) (d) 55 Figure 2. 9 2 - Phenoxyacetophenone cleavage with cysteine in MeCN/H 2 O mix. Presumably due to its low solubility in MeCN, 2 - with the addition of 5% water to MeCN to combat the solubility issue, due to the low reactivity of cysteine. The dimer (10 mg, 2.36 mM) was refluxed at (a) 1:1 mole ratio of substrate to thiol, (b) 1:2 mole ratio of substrate to thiol, (c) 1:10 mole ratio of substrate to thiol, (d) 1:100 mole ratio of substrate to thiol, and (e) same conditions as in (d) except cysteine was pre - equilibrate d with 1 mL H 2 O and base prior to addition of MeCN and substrate. Little product formation was observed, suggesting that the base may inhibit the reaction when solubilized, see Figure 2. 23 and Figure 2. 28 . 0 20 40 60 80 100 0 5 10 15 20 25 % yield hrs 2 - phenoxyacetophenone : cysteine 1:1 in Refluxing ACN (1 mL H 2 O) phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % yield hrs 2 - phenoxyacetophenone : cysteine 1:2 in Refluxing ACN ( 1 mL H 2 O ) phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % yield hrs 2 - phenoxyacetophenone : cysteine 1:10 in Refluxing ACN ( 1 mL H 2 O ) phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % yield hrs 2 - phenoxyacetophenone : cysteine 1:100 in Refluxing ACN ( 1 mL H 2 O ) phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 2 4 6 8 10 % yield hrs 2 - phenoxyacetophenone : cysteine 1:100 in Refluxing ACN (1 mL H 2 O) Phenol Acetophenone 2-Phenoxyacetophenone ( a ) (b) (c) (d) (e) 56 Figure 2. 10 N - acetylcysteine cleavage of 2 - phenoxyacetophenone. N - acetylcysteine was probed as a mediator in the same reaction conditions above to overcome the solubility issues of cysteine. 2 - Phenoxyacetophenone (10 mg, 2.36 mM) was refluxed in MeCN with N - acetylcysteine at (a) 1:1 mole ratio of substrate to thiol, (b) 1:2 mole ratio of substrate to thiol, (c) 1:10 mole ratio of substrate to thiol, and (d) 1:100 mole ratio of substrate to th iol. N - acetylcysteine was also largely insoluble in the non - aqueous reaction conditions and therefore ineffective at cleaving 2 - phenoxyacetophenone. 0 20 40 60 80 100 0 5 10 15 % Yield hrs 2 - phenoxyacetophenone : N - acetyl cysteine 1:1 in Refluxing MeCN phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 % Yield hrs 2 - phenoxyacetophenone : N - acetyl cysteine 1:2 in Refluxing MeCN phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 % Yield hrs 2 - phenoxyacetophenone : N - acetyl cysteine 1:10 in Refluxing MeCN phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 % Yield hrs 2 - phenoxyacetophenone : N - acetyl cysteine 1:100 in Refluxing MeCN phenol acetophenone 2-phenoxyacetophenone ( a ) (b) (c) (d) 57 Figure 2. 11 2 - Phenoxyacetophenone cleavage va rying BME equivalence. 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in refluxing MeCN using BME at a (a) 1:1 mole ratio of substrate to thiol, (b) 1:2 mole ratio of substrate to thiol, (c) 1:10 mole ratio of substrate to thiol, and (d) 1:100 mole rati o of substrate to thiol. The conditions that utilized the least amount of thiol for the largest percent conversion (c) were used for the subsequent reactions with BME. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : BME 1:1 in refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : BME 1:2 in refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 Refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 % Yield hrs 2 - phenoxyacetophenone : BME 1:100 in refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone ( a ) (b) (c) (d) 58 Figure 2. 12 2 - Phenoxyacetophenone cleavage varying DTT equivalence. 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in refluxing MeCN using DTT at a (a) 1:1 mole ratio of substrate to thiol, (b) 1:2 mole ratio of substrate to thiol , (c ) 1:10 mole ratio of substrate to thiol, and (d) 1:100 mole ratio of substrate to thiol. The conditions that utilized the least amount of thiol for the largest percent conversion (b) were used for the subsequent reactions with DTT. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : DTT 1:1 refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : DTT 1:2 refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : DTT 1:10 refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : DTT 1:100 refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone ( a ) (b) (c) (d) 59 Figure 2. 13 2 - Phenoxyacetophenone cleavage varying 1,3 - propanedithiol equivalence. 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in refluxing MeCN using 1,3 - propanedithiol at a (a) 1:1 mole ratio of substrate to thiol, (b) 1:2 mole ratio of substrate to thiol, (c) 1:10 mole ratio of substrate to thiol, and (d) 1:100 mole ratio of substrate to thiol. Condition (d) gave the best yield, and the 1:100 mole ratio was used for su bsequent reactions with 1,3 - propanedithiol. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : 1,3 - propanedithiol 1:1 in refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : 1,3 - propanedithiol 1:2 in refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : 1,3 - propanedithiol 1:10 in refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : 1,3 - propanedithiol 1:100 in refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone ( a ) (b) (c) (d) 60 Figure 2. 14 2 - Phenoxyacetophenone cleavage varying thiophenol equivalence. 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in refluxing MeCN using thiophenol at a (a) 1:1 mole ratio of substrate to thiol, (b) 1:2 mole ratio of substrate to thiol, and (c) 1:10 mole ratio of substrate to thiol. The thioether is observed but does no t complete the 2 nd half of the proposed reaction as seen by the low acetophenone yields. Under 1:100 mole ratio of substrate to thiol, the chromatograms are too convoluted to determine yields. Due to the low acetophenone yields, this thiol was not used for further studies. Figure 2. 15 2 - Phenoxyacetophenone cleavage using a thiol salt. 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in refluxing MeCN using 2 - (diethylamino)ethane - 1 - thiol·HCl at a (a) 1:10 mole ratio of substr ate to thiol. Little reactivity was observed for this thiol, possibly due to solubility in MeCN, and it was therefore not used in further studies. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : Thiophenol 1:1 in Refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : Thiophenol 1:2 in Refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : Thiophenol 1:10 in Refluxing MeCN Phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol Acetophenone 2-phenoxyacetophenone ( a ) (b) (c) 61 Figure 2. 16 2 - Phenoxyacetophenone cleavage under neat thiol conditions. Yields of reaction of 2 - phenoxyacetophenone (10 mg) with neat thiol 2 CO 3 at 100 °C stopped at 3 h, 6 h, or 15 h (3 separate reactions per set of conditions). (a) dimer + BME, (b) d imer + DTT, (c) dimer + thiophenol, and (d) dimer + 1,3 - propanedithiol. Under neat conditions, 3 hours is sufficient for cleavage of the ether bond and formation of phenol and acetophenone. 2.6.4 - iii Solvent and Temperature Exploration on Cleavage of 2 - Ph enoxyacetophenone Figure 2. 17 2 - Phenoxyacetophenone cleavage in dioxane. Reaction of 2 - phenoxyacetophenone (10 mg, 2.36 mM) with BME at (a) 1:2 and (b) 1:10 mole ratio of substrate to thiol in refluxing 0 20 40 60 80 100 0 5 10 15 20 25 % yield hrs 2 - phenoxyacetophenone : BME 1:2 in Refluxing Dioxane phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % yield hrs 2 - phenoxyacetophenone : BME 1:10 in Refluxing Dioxane phenol acetophenone 2-phenoxyacetophenone ( a ) (b) 62 dioxane, the conventional medium for thioacidolysis. Very little dimer cleavage occurs using this solvent, distinguishing our cleavage from that of thioacidolysis . Figure 2. 18 2 - Phenoxyacetophenone cleavage with BME in MeCN at various temperatures . 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in MeCN using BME at a 1:10 mole ratio of substrate to thiol under (a) refluxing temperatures, (b) 50 °C, and (c) room temperature. For the reaction to proceed in MeCN, the reaction must be refluxed; under cooler temperatures, minimal to no reaction was observed. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 Refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 MeCN 50 C Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 MeCN RT Phenol Acetophenone 2-phenoxyacetophenone ( a ) (b) (c) 63 Figure 2. 19 2 - Phenoxyacetophenone cleavage with BME in DMF at various temperatures . 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in dimethylformamide (DMF) using BME at a 1:10 mole ratio of substrate to thiol at (a) 100 °C, (b) 50 ° C, and (c) room temperature. Heat was not required to cleave the dimer using DMF, but heat increased the rate of product formation and substrate conversion. Temperatures above 100 °C give 100% conversion and 100% yield of phenol and acetophenone within 2 h (not shown); however, solvent decomposition at high temperatures caused side reactions with cleavage products over time. Figure 2. 20 2 - Phenoxyacetophenone cleavage with BME in THF at various temperatures . 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in tetrahydrofuran (THF) using BME at a 1:10 mole ratio of substrate to thiol at (a) refluxing temperatures and (b) room temperature. Although THF is a polar aprotic solvent, makin g it ideal for nucleophilic reactions, no reaction was observed. (Figure 2.20 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : BME 1:10 DMF 100 C phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 DMF 50 C Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % yield hrs 2 - Phenoxyacetophenone : BME 1:10 DMF RT Phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : BME 1:10 in refluxing THF phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : BME 1:10 in RT THF Phenol Acetophenone 2-phenoxyacetophenone ( a ) (b) (c) ( a ) (b) 64 cle avage products under the same conditions. Figure 2. 21 2 - Phenoxyacetophenone cleavage with BME in DMSO at various temperatures . 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in dimethylsulfoxide (DMSO) using BME at a 1:10 mole ratio of substrate to thiol at (a) 100 °C, (b) 50 °C, and (c) room temperature. Increasing the temperature increased product formation. Temperatures above 100 °C increased side reactions, and the experiment s were not quantifiable (not shown). 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 DMSO 100 C Phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 DMSO 50 ° C Phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 DMSO RT Phenol Acetophenone 2-phenoxyacetophenone ( a ) (b) (c) 65 Figure 2. 22 2 - Phenoxyacetophenone cleavage with BME in NMP at various temperatures . 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in N - methylpyrrolidone (NMP) using BME at a 1:10 mole ratio of substrate to thiol at (a) 100 °C, (b) 50 °C, and (c) room temperature. Temperatures above 100 °C gave 100% conversion and nearly 100% yield of phenol and acetophenone within 2 h (not shown). 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : BME 1:10 NMP 100 C phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : BME 1:10 NMP 50 ° C Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % yield hrs 2 - phenoxyacetophenone : BME 1:10 NMP RT Phenol Acetophenone 2-Phenoxyacetophenone ( a ) (b) (c) 66 Figure 2. 23 2 - Phenoxyacetophenone cleavage with BME in GVL at various temperatures . 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in gamma - valerolactone (GVL) using BME at a 1:10 mole ratio of substrate to thiol at (a) 100 °C, (b) 50 °C, and (c) room temperature. Essentially no cleavage was observed using GVL as a solvent. Temperatures above 100 °C gave chromatograms too convoluted to determine yields. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 GVL 100 ° C Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 GVL 50 ° C Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 GVL RT Phenol Acetophenone 2-Phenoxyacetophenone ( a ) (b) ( c) 67 Figure 2. 24 2 - Phenoxyacetophenone cleavage with BME in Me OH at various temperatures . 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in methanol (MeOH) using BME at a 1:10 mole ratio of substrate to thiol at (a) refluxing temperatures, (b) 40 °C, and (c) room temperature. Even with heating, very little reaction was observed. Figure 2. 25 2 - Phenoxyacetophenone cleavage with BME in water at various temperatures . 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage yields after 24 h in water using 10 equiv. of BME at refluxing, 50 °C, and room temperature. Some cleavage can be observed with heating. Low acetophenone yields were observed regardless of whether or not the samples were heated. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 in Refluxing MeOH phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 MeOH 40 C Phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 MeOH RT phenol acetophenone 2-phenoxyacetophenone ( a ) (b) (c) 68 Figure 2. 26 Comparison of 2 - phenoxyacetophenone (10 mg, 2.36 mM) cleavage in optimized solvent temperatures using D TT at a 1:2 mole ratio of substrate to thiol. (a) 100 °C DMF, (b) 100 °C DMSO, (c) refluxing MeOH, and (d) 100 °C NMP. The reaction in MeOH had very li ttle conversion. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : DTT 1:2 DMF 100 C Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : DTT 1:2 DMSO 100 C Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : DTT 1:2 in Refluxing MeOH Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : DTT 1:2 NMP 100 C Phenol Acetophenone 2-Phenoxyacetophenone ( a ) (b) (c) (d) 69 Figure 2. 27 Comparison of 2 - phenoxyacetophenone (10 mg, 2.36 mM) cleavage in optimized solvent temperatures using 1,3 - propanedithiol at a 1:100 mole ratio of substrate to thiol. (a) 100 °C DMF, (b) 100 °C DMSO, (c) refluxing MeOH, and (d) 100 °C NMP. The reaction in MeOH had very little conversion. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : 1,3 - Propanedithiol 1:100 DMF 100 C Phenol Acetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : 1,3 - Propanedithiol 1:100 DMSO 100 ° C Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : 1,3 - Propanedithiol 1:100 in Refluxing MeOH Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : 1,3 - Propanedithiol 1:100 NMP 100 C phenol acetophenone 2-phenoxyacetophenone ( a ) (b) (c) (d) 70 2.6.4 - iv Exploring the Effect of Base on the Cleavage of 2 - Phenoxyacetophenone Figure 2. 28 2 - Phenoxyacetophenone cleavage with BME using various bases. 2 - Phenoxyacetophenone (10 mg, 2.36 mM) cleavage in refluxing MeCN (20 mL) using BME at a 1:10 mole ratio of substrate to thiol with: (a) n o base, (b) excess triethylamine, (c) 5 equiv. of diazabicycloundecene ( DBU ), (d) excess DBU, (e) excess pyridine, and (f) excess potassium carbonate (K 2 CO 3 ). Solid K 2 CO 3 (f) gave the highest yields and was used in all subsequent reactions. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : BME 1:10 in refluxing MeCN (no base) phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : BME 1:10 in refluxing MeCN (Triethylamine) Phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 in refluxing MeCN (DBU 5 equiv.) Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 in refluxing MeCN (DBU) Phenol Acetophenone 2-Phenoxyacetophenone OBME 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - phenoxyacetophenone : BME 1:10 in refluxing MeCN (Pyridine) Phenol Acetophenone Dimer 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 in Refluxing MeCN (potassium carbonate) Phenol Acetophenone 2-Phenoxyacetophenone ( a ) (b) (c) (d) (e) (f) 71 2.6.4 - v Reaction Scope: Tolerance of Structural Diversity During Ether Cleavage Figure 2. 29 Dimer (10 mg), modeling syringyl and guaiacyl monomers found in real lignin, cleavage in refluxing MeCN: (a) (2 - (2,6 - dimethoxyphenoxy) - 1 - (4 - hydroxy - 3,5 - dimethoxyphenyl)ethan - 1 - one) and 10 equiv. of BME; (b) (2 - (2,6 - dimethoxyphenoxy) - 1 - (4 - hydroxy - 3,5 - dimethoxyphenyl)ethan - 1 - one) and 2 equiv. of DTT; (c) (1 - (4 - hydroxy - 3 - methoxyphenyl) - 2 - (2 - methoxyphenoxy)ethan one) and 10 equiv. of BME; (d) (1 - (4 - hydroxy - 3 - methoxyphenyl) - 2 - (2 - methoxyphenoxy)ethanone) and 2 equiv. of DTT; and (e) 1 - (3,4 - dimethoxyphenyl) - 2 - (2 - methoxyphenoxy)ethenone and 10 equiv. of BME. Lower yields were observed for substrates with a para - hydrox y group (a - d). BME out - performed DTT in dimer cleavage. The reaction (e) resulted in 100% cleavage and yields in only 27 h relative to only 60% cleavage and <20% yields after 48 h in (c), indicating that it is the para - OH group that slows the reaction down and not the steric bulk of the methoxy groups. 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs Syringol Acetosyringone S-S Dimer 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs Syringol Acetosyringone S-S Dimer 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs Guaiacol Acetovanillone G-G Dimer 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs Guaiacol Acetovanillone G-G Dimer 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 3',4'-Dimethoxyacetophenone 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)ethanone (a) (b) (c) (d) (e) 72 Figure 2. 30 - - aryl ether cleavage: dimer (10 mg) in refluxing MeCN at a 1:10 mole ratio of substrate to BME. (a) 2 - phenoxyacetophenone, (b) phenethoxybenzene, (c) 2 - phenoxy - 1 - phenylethan - 1 - ol, (d) 2 - methoxy - 1 - phenoxy - 2 - phenylethane, (e) (benzyloxy)benzene, and (f) diphenoxymethane. Adding an electron donati ng group (c - d) abolishes the reactivity, while removing functionality to the alpha position (b) halts cleavage as well. Aromatic ethers cannot be cleaved without a ketone (e - f). 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenethoxybenzene 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs Phenol 1-Phenylethanol 1-phenyl-2-phenoxyethanol 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol 1-Methoxyethylbenzene 2-Methoxy-1-phenoxy-2-phenylethane 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol Toluene Benzyl phenyl ether 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol Anisole (Phenoxymethoxy)benzene (a) (b) (c) (d) (e) (f) 73 Figure 2. 31 - - aryl ether cleavage: d imer cleavage (10 mg) in refluxing MeCN at a 1:10 mole ratio of substrate to BME. (a) 2 - phenoxyacetophenone, (b) 2 - phenoxypropiophenone, (c) 2 - methyl - 2 - phenoxy - 1 - phenylpropan - 1 - one, and (d) 3 - hydroxy - 2 - phenoxy - 1 - phenylpropan - 1 - one. Low reactivity is seen for (b - c) while (d) resulted in the retro - aldol product, 2 - phenoxyacetophenone, which w as followed by the thiol - mediated aryl ether cleavage seen in (a). Nucleophilic reactions prefer attack on primary sites (a), followed by secondary sites (b and d)), and usually do not attack tertiary sites (c). The reactions above follow S N 2 reactivity wi th additional autocatalytic formation of the parent molecule in (d) followed by primary site cleavage. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol Propiophenone 2-Phenoxypropiophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol Isobutyrylbenzene 2-methyl-2-phenoxy-1-phenylpropan-1-one 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol Acetophenone 2-Phenoxyacetophenone 3-Hydroxy-2-phenoxy-1-phenyl-1-propanone (a ) (b) (c) (d) 74 Figure 2. 32 Refluxing control reactions on 3 - hydroxy - 2 - phenoxy - 1 - phenylpropan - 1 - one. Dimer cleavage (10 mg, 2.07 mM) in refluxing MeCN (a) no thiol and no base, (b) no thiol and excess K 2 CO 3 , (c) 10 equiv. of BME and no base, (d) 10 equiv. of BME and excess K 2 CO 3 , (e) 2 equiv. of DTT and excess K 2 CO 3 , and (f) 100 equiv. of 1,3 - propanedithiol and excess K 2 CO 3 . The dimer is heat stable (a), but with heat and base (b), the parent dimer (red) is observed, presumably formed via a retro aldol reaction, which then undergoes nucleophilic cleavage resultin g in phenol formation. Similarly, with thiol present but no base (c), the parent dimer is also observed (red). The presence of both base and thiol (d) additionally produce the acetophenone product. Without thiol and base, the second monomer is never seen. DTT does not cleave the dimer as efficiently (e). In the presence of a 1,3 - propanedithiol (f), near mass balance is obtained with several acetophenone adducts observed. This study indicates that the lignin - relevant hydroxy - methyl on - carbon may first be cleaved via a retro - aldol process into the parent dimer under basic conditions before the thiol initiates the S N 2 cleavage reaction. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs B-Hydroxymethyl 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol 2-Phenoxyacetophenone B-Hydroxymethyl 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol 2-phenoxyacetophenone B-Hydroxymethyl 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol Acetophenone 2-Phenoxyacetophenone 3-Hydroxy-2-phenoxy-1-phenyl-1-propanone 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs Phenol 3-Hydroxypropiophenone B-methylhydroxy 2-phenoxyacetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol Acetophenone 2-Phenoxyacetophenone B-Hydroxymethyl 2-phenoxyacetophenone Propiophenone (a) (b) (c) (d) (e) (f) 75 Figure 2. 33 Room temperature control reactions on 3 - hydroxy - 2 - phenoxy - 1 - phenylpropan - 1 - one. Dimer cleavage (10 mg, 2.07 mM) in room temperature MeCN (a) no thiol and no base, (b) no thiol and excess K 2 CO 3 , (c) 10 equiv. of BME and no base, and (d) 10 equiv. of BME and excess K 2 CO 3 . The dime r is stable in MeCN over time (a) but with base (b) the dimer is converted to something else that is neither parent dimer nor monomers. When thiol is present but no base (c), the dimer is unreactive and stable. The presence of both base and thiol (d) show an unreactive dimer suggesting that the thiol prohibits the base catalyzed polymerization. Purple lines show that over time the thiol mediator is not oxidized in the presence (c) or absence (d) of base supporting the fact that no reaction took place. This study demonstrates that the lignin - relevant hydroxy - carbon is stable at room temperature under our reaction conditions and the thiol stops any base - catalyzed condensation. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 3-Hydroxy-2-phenoxy-1-phenyl-1-propanone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 3-Hydroxy-2-phenoxy-1-phenyl-1-propanone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs OBME 3-Hydroxy-2-phenoxy-1-phenyl-1-propanone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol OBME 3-Hydroxy-2-phenoxy-1-phenyl-1-propanone (a) (b) (c) (d) 76 Figure 2. 34 Control reactions to test reduced dimer cleavage when there is a hydroxy methyl - carbon. Dimer cleavage (10 mg) in refluxing MeCN using 10 equiv. of BME and excess K 2 CO 3 for (a) 2 - phenoxy - 1 - phenylpropane - 1,3 - diol, and (b) 1 - (4 - hydroxy - 3 - methoxyphenyl) - 2 - (2 - methoxyphenoxy)propane - 1,3 - diol (guaiacylglycerol - beta - guaiacyl ether). Low amounts of guaiacol can be seen in (b) while no phenol formation occurs in (a). No evidence of elimination or retro - aldol reaction to aid in cleavage was observed in these instances where the - - carbon to be oxidized, mimicking the enzymatic pathway. Figure 2. 35 Investigation of the importance of the aryl - moiety in the ether cleavage reaction: dimer cleavage (10 mg) in refluxing MeCN at a 1:10 mole ratio of substrate to BME. (a) 1 - phenoxypropan - 2 - one, (b) 2 - methoxy - 1 - phenylethan - 1 - one, and (c) 2 - phenoxyacetaldehyde. The aromatic ring next to the ether is necessary for reactivity, as evidenced in reaction (b) by the low 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2-Phenoxy-1-phenylpropane-1,3-diol 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs Guaiacol Guaiacylglycerol-beta-guaiacyl ether 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs 1 - Phenoxypropan - 2 - one : BME 1:10 in Refluxing MeCN phenol Phenoxy-2-propanone 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs 2 - methoxy - 1 - phenylethan - 1 - one : BME 1:10 in Refluxing MeCN acetophenone Methoxyacetophenone 0 20 40 60 80 100 0 10 20 30 40 50 % Yield hrs 2 - Phenoxyacetaldehyde : BME 1:10 in Refluxing MeCN phenol ( a ) (b) (c) (a) (b) 77 reactivity. This is most likely due to the electron withdrawing effect of the aryl group, ma king phenoxy a better leaving group relative to methoxy. The aromatic ring next to the ketone is not necessary for ether cleavage as evidenced by (a) and (c). Figure 2. 36 Lignin - like d imer cleavage using neat BME and neat DTT Lignin dimer (10 mg) cleavage at 100 ° C using neat BME and neat DTT - (2,6 - dimethoxyphenoxy) - 1 - (4 - hydroxy - 3,5 - dimethoxyphenyl)ethan - 1 - one), (b) (1 - (4 - Hydroxy - 3 - methoxyphenyl) - 2 - (2 - methoxyphenoxy)ethanone), (c) 3 - hydroxy - 2 - phenoxy - 1 - phenylpropan - 1 - one, and (d) 2 - phenoxy - 1 - phenylethan - 1 - ol. Very little conversion is seen for (d), suggesting that the alpha hydroxy group must be oxidized for nucleophilic cl eavage. Attack on a secondary site decreases the cleavage yields compared to attack on a primary site. The addition of methoxy groups to the aromatic ring also decrease s product formation. (a) (b) (c) (d) 78 Figure 2. 37 - O - 4 trimer cleavage in refluxing MeCN - O - 4 trimer cleavage of 2,2' - (1,4 - phenylenebis(oxy))bis(1 - phenylethan - 1 - one) (10 mg, 1.45 mM) in refluxing MeCN using 10 equiv. of BME. Keto - aryl ether cleavage is observed with mode l lignin trimers. Additional equivalents of thiol may be required to cleave both ether bonds. Note that the response factor for 1 - phenyl - 2 - ( p - tolyloxy)ethan - 1 - one was used for the 2 - (4 - hydroxyphenoxy) - 1 - phenylethan - 1 - one response factor in determining yiel ds. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs acetophenone 2-(4-Hydroxyphenoxy)-1-phenylethanone p-hydroxy dimer 79 2.6.4 - vi Hammett Studies Figure 2. 38 Hammett studies with p - withdrawing groups on the leaving group of 2 - phenoxyacetophenone Dimer cleavage (10 mg) in refluxing MeCN at a 1:10 mole ratio of substrate to BME. (a) 1 - Phenyl - 2 - [4 - (trifluoromethyl)phenoxy]ethanone, (b) 4 - (2 - oxo - 2 - phenylethoxy) - Benzonitrile, (c) 4 - (2 - oxo - 2 - phenylethoxy)benzaldehyde, and (d) 4 - (phenacyloxy)acetophenone. Adding electron withdrawing groups to the para position of the aryl ether leaving group increased reaction rates. The thioether intermediate (red) is also readily observed, compared to 2 - phenoxyacetophenone cleavage. This is likely due to the increase in rate of the first step (rate limiting) that allows the intermediate to be seen before the fast second half of the reaction. A comparison to the electron withdrawing groups on the aromatic next to the ketone suggests that the electronic effects of the leaving group have a greater influence on the ov erall rate - carbon as - carbon and possible cleavage via a different mechanism. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 4-Hydroxybenzotrifluoride Acetophenone Thioether 1-Phenyl-2-[4-(trifluoromethyl)phenoxy]ethanone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 4-Hydroxybenzonitrile Acetophenone Thioether 4 - - - - - Benzonitrile 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 4-hydroxybenzaldehyde Acetophenone Thioether 4-(2-Oxo-2-phenylethoxy)benzaldehyde 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs p-Acetophenol Acetophenone Thioether 4-(Phenacyloxy)acetophenone ( a ) (b) (c) (d) 80 Figure 2. 39 Ha mmett studies with p - donating groups on the leaving group of 2 - phenoxyacetophenone. Dimer cleavage (10 mg) in refluxing MeCN at a 1:10 mole ratio of substrate to BME. (a) 1 - Phenyl - 2 - ( p - tolyloxy)ethan - 1 - one, and (b) 2 - (4 - methoxyphenoxy) - 1 - phenylethanone. Addition of electron donating groups to the para position of the aryl ether leaving group decreases reaction rates. A comparison to the electron donating groups on the aromatic next to the ketone suggests that the electronic effects have a greater influenc e on the leaving group. - carbon as opposed to thiol - carbon and possible cleavage via a different mechanism. Figure 2. 40 Hammett studies with p - withdrawing group on the keto - side of 2 - phenoxyacetophenone . Dimer cleavage (10 mg) in refluxing MeCN at a 1:10 mole ratio of substrate to BME. (a) 2 - Phenoxy - 1 - [4 - ( trifluoromethyl )phenyl]ethanone. Addition of an electron wtihdrawin g group to the para position of the aromatic ring next to the ketone increases reaction rates. A comparison to the electron withdrawing groups on the aryl ether suggests that the electronic effects have a greater influence on the leaving group. This suppor ts a mechanism - - carbon and cleavage via a different mechanism. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs p-cresol acetophenone 1-Phenyl-2-(p-tolyloxy)ethan-1-one 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 4-Methoxyphenol Acetophenone 2-(4-Methoxyphenoxy)-1-phenylethanone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol 4-Acetylbenzotrifluoride 2-Phenoxy-1-[4-(trifluoromethyl)phenyl]ethanone (a) (b) 81 Figure 2. 41 Hammett studies with p - donating groups on the keto - side of 2 - phenoxyacetophenone . Dimer cleavage (10 mg) in refluxing MeCN at a 1:10 mole ratio of substrate to BME. (a) 1 - (4 - Methylphenyl) - 2 - - methoxy - 2 - phenoxyacetophenone, and (c) 1 - [4 - ( dimethylamino )phenyl] - 2 - phenoxy - ethanone. Addition of electron donating groups to the para position of the aromatic ring next to the ketone decreases reaction rates. A comparison to the electron donating groups on the aryl ether suggests that the elec tronic effects have a greater influence on the leaving group. This supports a mechanism - - carbon and cleavage via a different mechanism. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol p-Acetyltoluene 1-(4-Methylphenyl)-2-phenoxyethanone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol 4'-Methoxyacetophenone 4'-Methoxy-2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol 4'-(Dimethylamino)acetophenone 1 - - - - - ethanone ( a ) (b) (c) 82 2.6.5 Control Experiments Figure 2. 42 Control experiment to support ether cleavage via thiol - mediated nucleophilic attack over radical initiated cleavage. 2 - Phenoxyacetophenone (10 mg, 2. 36 mM) in refluxing MeCN (20 mL) with 10 equiv. of BME and (a) no radical trap, and (b) 1 equiv. of butylated hydroxytoluene (BHT). BHT is a radical scavenger and would slow or stop the cleavage reaction if it went through a radical mechanism. The time cou rse shows that reaction yields are unaffected by the addition of BHT. Figure 2. 43 Control reactions testing if products formed increase reaction rate by increasing the active mediator. 2 - Phenoxyacetophenone cleavage (10 mg, 2.36 mM) in refluxing MeCN with 10 equiv. of BME and (a) ~2 equiv. of phenol or (b) 5 equiv. of diethanol disulfide. Phenol was added to the stirring base first, producing phenoxide to act as active base and aid in th e cleavage reaction. The yield did not improve with the phenoxide present, suggesting that product 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 Refluxing MeCN Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 in Refluxing MeCN and 1 equiv. of BHT Phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol Acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Phenol Acetophenone 2-Phenoxyacetophenone (a) (b) ( a ) (b) 83 formation does not aid in cleavage. Diethanol disulfide was added to promote thiolate formation. The yield did not increase with extra disulfide present, suggesting that formation of a disulfide does not promote an increase in the active mediator (thiolate). Figure 2. 44 Control experiments to test if stirring the reaction accelerated the aryl ether cleavage directly or indirectly (by grinding the base, thereby creating more surface area). 2 - phenoxyacetophenone (10 mg, 2.36 mM) and 10 equiv. of BME were refluxed with (a) stirring and powdered K 2 CO 3 , (b) stirring a nd granular K 2 CO 3 , (c) no stirring and powdered K 2 CO 3 , and (d) no stirring and granular K 2 CO 3 . The results indicate that the role of stirring is to grind the base and facilitate deprotonation of the thiol to the active thiolate. Pre - grinding the base resul ted in a 20% larger yield in aryl ether cleavage over 24 h. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 in Refluxing MeCN (Stirring and powdered K 2 CO 3 ) Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 in Refluxing MeCN (Stirring and granular K 2 CO 3 ) Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 in Refluxing MeCN (No stirring and powdered K 2 CO 3 ) Phenol Acetophenone 2-Phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Phenoxyacetophenone : BME 1:10 in Refluxing MeCN (No stirring and granular K 2 CO 3 ) Phenol Acetophenone 2-Phenoxyacetophenone (a) (b) (d) (c) 84 Figure 2. 45 Control experiment to test if the thiol mediator, BME, oxidizes to O - BME under reaction conditions (excess K 2 CO 3 in refluxing MeCN but in the absence of substrate). The lack of disulfide formation indicates that little to no oxygen was present in our reaction vessel. Figure 2. 46 Control - O - 4 ether bond. Under the conditions shown above at a 1:10 and 1:100 substrate to thiol mole ratio over 24 hours, 15 - 30% conversion of the dimer is found with a phenol yield of <10%. While it is unclear whether the proposed 1 st half of the reaction occurred or if phenol formation was derived some other way, there is no evidence of the 2 nd half of the proposed mechanism occurring with 0% yield of acetophenone. 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs 2 - Mercaptoethanol in Refluxing MeCN OBME 85 Figure 2. 47 Control experiments to test whether BME demethylates the methoxy groups on lignin monomers. Within error, no demethylation occurred under the reaction conditions shown with 10 mg of substrate, 20 mL solv ent, and 100 mg of base; GC - MS analysis did not detect either the catechol or 2 - (methylthio)ethan - 1 - ol. Figure 2. 48 Control reaction of 2 - ((2 - hydroxyethyl)thio) - 1 - phenylethan - 1 - one, the proposed intermediate of the 2 - phenoxy acetophenone cleavage: (left) under refluxing MeCN with K 2 CO 3 monitored by HPLC, (right) ~70 °C MeCN with K 2 CO 3 monitored by NMR over 17 hours. Left: Slow degradation of the thioether occurs which does not produce the acetophenone monomer. This side reaction occurred considerably more slowly than the thioether conversion to acetophenone when it reacted with BME, as seen in Figure 2. 2 - e . Right: No obvious formation of other products was observed from heating the thioether over time. The stability of 2 - ((2 - hydroxyet hyl)thio) - 1 - phenylethan - 1 - one was also tested at RT in MeCN in the presence of K 2 CO 3 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hrs Control Rxn: Thioether in Refluxing MeCN Thioether Acetophenone 86 and monitored over 24 hours by NMR. No change occurred (not shown), indicating the thioether is stable at room temperature under our basic reaction conditions. Figure 2. 49 Control experiment to test if O - BME can cleave 2 - phenoxyacetophenone, the - O - 4 model lignin dimer. Yield is given for 2 - phenoxyacetophenone (10 mg, 2.36 mM) cleavage. Standard reaction conditions: refluxing MeCN (20 mL) in the presence of K 2 CO 3 (100 mg) for 48 h. (a) 1 equiv. of disulfide, (b) 2 equiv. of disulfide, (c) 10 equiv. of disulfide, and (d) 100 equiv. of disulf ide. Conditions (b) and (c) gave the highest dimer cleavage yields, 20 - 30%. In each case phenol was produced in varying amounts but acetophenone was not produced, suggesting that the missing monomer may be a thioether adduct. The acetophenone thioether is present but in low (< 2%) yield for (a) - (d). 0 20 40 60 80 100 0 10 20 30 40 50 % yield hrs 2 - phenoxyacetophenone: Dithiodiethanol 1:1 in Refluxing MeCN phenol 2-phenoxyacetophenone 0 20 40 60 80 100 0 10 20 30 40 50 % yield hrs 2 - phenoxyacetophenone: Dithiodiethanol 1:2 in Refluxing MeCN phenol 2-phenoxyacetophenone 0 20 40 60 80 100 0 10 20 30 40 50 % yield hrs 2 - phenoxyacetophenone: Dithiodiethanol 1:10 in Refluxing MeCN phenol 2-phenoxyacetophenone 0 20 40 60 80 100 0 10 20 30 40 50 % yield hrs 2 - phenoxyacetophenone: Dithiodiethanol 1:100 in Refluxing MeCN phenol 2-phenoxyacetophenone ( a ) (b) (c) (d) 87 Figure 2. 50 Control reactions to determine if the cleavage products undergo further side reactions. Standard reaction conditions: refluxing MeCN (20 mL) in the presence of K 2 CO 3 (100 mg) for 24 h. (a) Percent remaining of acetophenone treated with 2 equiv. of BME, (b) percent remaining of phenol and acetophenone (1:1) treated with 2 equiv. of BME, and (c) percent remaining of acetophenone, phenol, and 2 - phenoxyacetophenone (1:1:1) when refluxed with MeCN and K 2 CO 3 over 24 hours. Based on these results, we conclude that no significant side reactions occurred among products nor products and mediator (BME). Figure 2. 51 Control reactions to determine if the oxidized thiol product reacts with the products of ether cleavage. Standard reaction conditions: refluxing MeCN (20 mL) in the presence of K 2 CO 3 (100 mg) for 24 h. (a) Percent remaining of acetophenone treated with 10 equiv. of dithiodiethanol. (b) Percent remaining of phenol and acetophenone treated with 10 equiv. of dithiodiethanol (oxidized BME). No reaction occurs b etween the oxidized BME and ether cleavage products. This suggests the general lower yield of acetophenone in thiol ether cleavage is not due 0 20 40 60 80 100 2 hrs 6 hrs 24 hrs % Remaining Control Rxn: Acetophenone + BME acetophenone 0 20 40 60 80 100 2 hrs 6 hrs 24 hrs % Remaining Control Rxn: Phenol + Acetophenone + BME phenol acetophenone 0 20 40 60 80 100 2 hrs 6 hrs 24 hrs % Remaining Control Rxn: 2 - phenoxyacetophenone + Phenol + Acetophenone phenol acetophenone 2-phenoxyacetophenone 0 20 40 60 80 100 0 5 10 15 20 25 % Remaining hrs Control Rxn: Disulfide + Acetophenone acetophenone dithiodiethanol 0 20 40 60 80 100 0 5 10 15 20 25 % Remaining hrs Control Rxn: Disulfide + Products phenol acetophenone dithiodiethanol ( a ) (b) ( a ) (b) (c) 88 to acetophenone side reactions. Oxidized BME does decrease in yield over time, suggesting a side reaction with itself. 2.6.6 Proposed Electrochemical Cycle for the Thiol - - O - 4 Cleavage Figure 2. 52 Organocatalytic cycle envisioned for the thiol - mediated nucleophilic cleavage of - aryl ether bonds followed by a 2e - reduction to recycle the mediator. Step 1: Nucleophilic organic thiol ate - aryl ether bond of lignin displaces a phenolic unit. Step 2: A second thiol ate attacks the thio ether intermediate, forming a disulfide, and releasing the arylpropanone fragment. Step 3: The thiol can be regenerated via a 2 - electron electrochemical reduction of the disulfide bond, potentially enabling a net electrocatalytic lignin cleavage process. 89 2.6.7 Lignin Polymer Structural Model Figure 2. 53 Representation of lignin and its common linkages highlighted by color. The most - O - 4 linkage (highlighted in orange). The percent of the various linkages observed is based on lignin from hardwoods. 1 90 2. 7 Spectra Figure 2. 54 2 - phenoxy - 1 - phenylethan - 1 - ol 29 ( 2 - 1) 3 1 2 4 6 7 8 5 91 Figure 2. 55 3 - hydroxy - 2 - phenoxy - 1 - phenylpropan - 1 - one 29 ( 2 - 3) 3 1 2 8 92 Figure 2. 56 2 - ((2 - hydroxyethyl)thio) - 1 - phenylethan - 1 - one ( 2 - 1 - a) 3 1 4 6 7 5 93 Figure 2. 57 1 - (4 - hydroxy - 3 - methoxyphenyl) - 2 - (2 - methoxyphenoxy)ethan - 1 - one ( 2 - 4) 7 6 4 1 5 2 3 8 94 Figure 2. 58 2 - (2,6 - dimethoxyphenoxy) - 1 - (4 - hydroxy - 3,5 - dimethoxyphenyl)ethan - 1 - one ( 2 - 5) 5 3 1 6 4 7 95 Figure 2. 59 (1 - methoxy - 2 - phenoxyethyl)benzene 29 ( 2 - 14) 5 6 4 7,9 5 1 2 96 Figure 2. 60 2 - methyl - 2 - phenoxy - 1 - phenylpropan - 1 - one 29 ( 2 - 7) 5 3 2 6 1 7 97 Figure 2. 61 2 - phenoxy - 1 - phenylpropan - 1 - one 29 ( 2 - 6) 6 3 2 7 1 8 4 98 Figure 2. 62 1 - phenyl - 2 - (p - tolyloxy)ethan - 1 - one 29 ( 2 - 18 - b) 2 99 Figure 2. 63 2 - (4 - methoxyphenoxy) - 1 - phenylethan - 1 - one 29 ( 2 - 18 - a) 100 Figure 2. 64 1 - (4 - methoxyphenyl) - 2 - phenoxyethan - 1 - one ( 2 - 17 - b) 3 2,5,7 4 1 101 Figure 2. 65 1 - phenyl - 2 - (4 - (trifluoromethyl)phenoxy)ethan - 1 - one ( 2 - 18 - c) 3 2,6 5 1 102 Figure 2. 66 2 - phenoxy - 1 - (4 - (trifluoromethyl)phenyl)ethan - 1 - one ( 2 - 17 - d) 1 5 2 3 4 103 Figure 2. 67 4 - (2 - oxo - 2 - phenylethoxy)benzonitrile ( 2 - 18 - d) 3 5 1 6 2 104 Figure 2. 68 2 - (4 - acetylphenoxy) - 1 - phenylethan - 1 - one 3 6 1 2 105 106 107 Figure 2. 69 2 - phenoxy - 1 - (p - tolyl)ethan - 1 - one ( 2 - 17 - c) 3 5,6 1 7,2 4 108 Figure 2. 70 1 - (4 - (dimethylamino)phenyl) - 2 - phenoxyethan - 1 - one ( 2 - 17 - a) 5, 7 2 6 109 110 111 Figure 2. 71 2,2' - (1,4 - phenylenebis(oxy))bis(1 - phenylethan - 1 - one) 3 1 5 2 112 Figure 2. 72 (1 - methoxyethyl)benzene 29 1,2,3 4 6 5 113 Figure 2. 73 1 - (3,4 - dimethoxyphenyl) - 2 - (2 - methoxyphenoxy)ethan - 1 - one 29 ( 2 - 8) 5 7 6 4 9,10 114 Figure 2. 74 2 - phenoxy - 1 - phenylpropane - 1,3 - diol 29 115 Figure 2. 75 2 - bromo - 1 - (3,4 - dimethoxyphenyl)ethan - 1 - one 29 116 REFERENCES 117 R EFERENCES 1. Rinaldi, R.; Jastrzebski, R.; Clough, M. T.; Ralph, J.; Kennema, M.; Bruijnincx, P. C. A.; Weckhuysen, B. M., Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining and Catalysis. Angewa ndte Chemie - International Edition 2016, 55 (29), 8164 - 8215. 2. Sun, Z. H.; Fridrich, B.; de Santi, A.; Elangovan, S.; Barta, K., Bright Side of Lignin Depolymerization: Toward New Platform Chemicals. Chemical Reviews 2018, 118 (2), 614 - 678. 3. Ralph, T. R.; Hitchman, M. L.; Millington, J. P.; Walsh, F. C., T he Electrochemistry of L - C ystine and L - C ysteine .2. E lectrosynthesis of L - C ysteine at S olid Electrodes . Journal of Electroanalytical Chemistry 1994, 375 (1 - 2), 17 - 27. 4. Botte, G. G., Electrochemical Manufacturing in the Chemical Industry. Electrochemical Society Interface 2014, 23 (3), 49 - 55. 5. Gall, D. L.; Ralph, J.; Donohue, T. J.; Noguera, D. R., A Group of Sequence - Related Sphingomonad Enzymes Catalyzes Cleavage of beta - Aryl Ether Linkages in Lignin beta - Guaiacyl and beta - Syringyl Ether Dimers. Environmental Science & Technology 2014, 48 (20), 12454 - 12463. 6. Gall, D. L.; Kim, H.; Lu, F.; Donohue, T. J.; Noguera, D. R.; Ralph, J., Stereochemical Features of Glut athione - dependent Enzymes in the Sphingobium sp Strain SYK - 6 beta - Aryl Etherase Pathway. Journal of Biological Chemistry 2014, 289 (12), 8656 - 8667. 7. Picart, P.; Mueller, C.; Mottweiler, J.; Wiermans, L.; Bolm, C.; Dominguez de Maria, P.; Schallmey, A., From Gene Towards Selective Biomass Valorization: Bacterial beta - Etherases with Catalytic Activity on Lignin - Like Polymers. Chem S us C hem 2014, 7 (11), 3164 - 3171. 8. Helmich, K. E.; Pereira, J. H.; Gall, D. L.; Heins, R. A.; McAndrew, R. P.; Bingman , C.; Deng, K.; Holland, K. C.; Noguera, D. R.; Simmons, B. A.; Sale, K. L.; Ralph, J.; Donohue, T. J.; Adams, P. D.; Phillips, G. N., Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of beta - Aryl Ether Bonds in Lignin. Jou rnal of Biological Chemistry 2016, 291 (10), 5234 - 5246. 9. Kamimura, N.; Takahashi, K.; Mori, K.; Araki, T.; Fujita, M.; Higuchi, Y.; Masai, E., Bacterial catabolism of lignin - derived aromatics: New findings in a recent decade: Update on bacterial lignin catabolism. Environmental Microbiology Reports 2017, 9 (6), 679 - 705. 10. Pereira, J. H.; Heins, R. A.; Gall, D. L.; McAndrew, R. P.; Deng, K.; Holland, K. C.; Donohue, T. J.; Noguera, D. R.; Simmons, B. A.; Sale, K. L.; Ralph, J.; Adams, P. D., Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin beta - Aryl Ether 118 Cleavage Pathway from Sphingobium sp.SYK - 6. Journal of Biological Chemistry 2016, 291 (19), 10228 - 10238. 11. Gall, D. L.; Kontur, W. S.; Lan, W.; Kim, H.; Li, Y.; Ralph, J.; Donohue, T. J.; Noguera, D. R., In Vitro Enzymatic Depolymerization of Lignin with Release of Syringyl, Guaiacyl, and Tricin Units. Applied and Environmental Microbiology 2018, 84 (3). 12. Trott, O.; Olson, A. J., Software News and Update AutoDock Vina: Improving the Speed and Accura cy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading. Journal of Computational Chemistry 2010, 31 (2), 455 - 461. 13. Curtiss, L. A.; Redfern, P. C.; Raghavachari, K.; Rassolov, V.; Pople, J. A., Gaussian - 3 theory using re duced Moller - Plesset order. Journal of Chemical Physics 1999, 110 (10), 4703 - 4709. 14. Marenich, A. V.; Olson, R. M.; Kelly, C. P.; Cramer, C. J.; Truhlar, D. G., Self - consistent reaction field model for aqueous and nonaqueous solutions based on accurat e polarized partial charges. Journal of Chemical Theory and Computation 2007, 3 (6), 2011 - 2033. 15. Kontur, W. S.; Bingman, C. A.; Olmsted, C. N.; Wassarman, D. R.; Ulbrich, A.; Gall, D. L.; Smith, R. W.; Yusko, L. M.; Fox, B. G.; Noguera, D. R.; Coon, J. J.; Donohue, T. J., Novosphingobium aromaticivorans uses a Nu - class glutathione S - transferase as a glutathione lyase in breaking the beta - aryl ether bond of lignin. Journal of Biological Chemistry 2018, 293 (14), 4955 - 4968. 16. Kontur, W. S.; Ol msted, C. N.; Yusko, L. M.; Niles, A. V.; Walters, K. A.; Beebe, E. T.; Vander Meulen, K. A.; Karlen, S. D.; Gall, D. L.; Noguera, D. R.; Donohue, T. J., A heterodimeric glutathione S - transferase that stereospecifically breaks lignin's (R) - aryl eth er bond reveals the diversity of bacterial - etherases. Journal of Biological Chemistry 2019, 294 (6), 1877 - 1890. 17. Meux, E.; Prosper, P.; Masai, E.; Mulliert, G.; Dumarcay, S.; Morel, M.; Didierjean, C.; Gelhaye, E.; Favier, F., Sphingobium sp SYK - 6 LigG Involved in Lignin Degradation is Structurally and Biochemically Related to the Glutathione Transferase Omega Class. Febs Lett. 2012, 586 (22), 3944 - 3950. 18. Jencks, W. P.; Salvesen, K., E quilibrium D euterium I soptope E ffects on I onization of Thiol Acids . Journal of the American Chemical Society 1971, 93 (18), 4433. 19. Thapa, B.; Schlegel, H. B., Density Functional Theory Calculation of pK(a)'s of Thiols in Aqueous Solution Using Explicit Water Molecules and the Polarizable Continuum Model. Jo urnal of Physical Chemistry A 2016, 120 (28), 5726 - 5735. 20. Whitesides, G. M.; Lilburn, J. E.; Szajewski, R. P., R ates of Thiol - Disulfide Interchange R eactions Between Mono - and D ithiols and E llmans R eagent . Journal of Organic Chemistry 1977, 42 (2), 332 - 338. 119 21. Dawson, R. M. C.; Elliott, D. C.; Elliott, W. H.; Jones, K. M., D ata for Biochemical Research . Oxford University Press: New York, N.Y. 1986 , 3 Ed., 580 . 22. Weast, R. C., CRC Handbook Of Chemistry And Physics . 1980 ; Vol. 60 ed. 23. Bordwell, F. G.; Brannen, W. T., E ffect of Carbonyl + R elated Groups on R eactivity of H alides in S(N)2 Reactions . Journal of the American Chemical Society 1964, 86 (21), 4645. 24. Rablen, P. R.; McLarney, B. D.; Karlow, B. J.; Schneider, J. E., How Alkyl Halide Stru cture Affects E2 and S(N)2 Reaction Barriers: E2 Reactions Are as Sensitive as S(N)2 Reactions. Journal of Organic Chemistry 2014, 79 (3), 867 - 879. 25. Hu, L.; Pan, H.; Zhou, Y.; Hse, C. - Y.; Liu, C.; Zhang, B.; Xu, B., Chemical Groups and Structural C haracterization of Lignin via Thiol - Mediated Demethylation. Journal of Wood Chemistry and Technology 2014, 34 (2), 122 - 134. 26. Sawamura, K.; Tobimatsu, Y.; Kamitakahara, H.; Takano, T., Lignin Functionalization through Chemical Demethylation: Preparatio n and Tannin - Like Properties of Demethylated Guaiacyl - Type Synthetic Lignins. Acs Sustainable Chemistry & Engineering 2017, 5 (6), 5424 - 5431. 27. Fascione, M. A.; Adshead, S. J.; Mandal, P. K.; Kilner, C. A.; Leach, A. G.; Turnbull, W. B., Mechanistic Studies on a Sulfoxide Transfer Reaction Mediated by Diphenyl Sulfoxide/Triflic Anhydride. Chemistry - A European Journal 2012, 18 (10), 2987 - 2997. 28. Koval, I. V., C hemistry of Disulfides . Uspekhi Khimii 1994, 63 (9), 776 - 792. 29. Zhou, Y.; Klinger, G. E.; Hegg, E. L.; Saffron, C. M.; Jackson, J. E., Multiple Mechanisms Mapped in Aryl Alkyl Ether Cleavage via Aqueous Electrocatalytic Hydrogenation over Skeletal Nickel. Journal of the American Chemical Society 2020, 142 (8), 4037 - 4050. 30. Hu, Z.; Zhang, S.; Zhou, W.; Ma, X.; Xiang, G., Synthesis and antibacterial activity of 3 - benzylamide derivatives as FtsZ inhibitors. Bioorganic & Medicinal Chemistry Letters 2017, 27 (8), 1854 - 1858. 31. Crestini, C.; Dauria, M., Singlet oxyge n in the photodegradation of lignin models. Tetrahedron 1997, 53 (23), 7877 - 7888. 32. Galkin, M. V.; Dahlstrand, C.; Samec, J. S. M., Mild and Robust Redox - Neutral Pd/C - Catalyzed Lignol - O - 4 Bond Cleavage Through a Low - Energy - Barrier Pathway. Chem S us C hem 2015, 8 (13), 2187 - 2192. 33. Fabbri, C.; Bietti, M.; Lanzalunga, O., Generation and reactivity of ketyl radicals with lignin related structures. On the importance of the ketyl pathway in the photoyellowing of lignin containing pulps and papers. Journal of Organic Chemistry 2005, 70 (7), 2720 - 2728. 120 34. Zhang, J.; Liu, Y.; Chiba, S.; Loh, T. P., Chemical conversion of beta - O - 4 lignin linkage models through Cu - catalyzed aerobic amide bond formation. Chemical Communications 2013, 49 (97), 11439 - 11441. 35. Kr uger, J. S.; Cleveland, N. S.; Zhang, S. T.; Katahira, R.; Black, B. A.; Chupka, G. M.; Lammens, T.; Hamilton, P. G.; Biddy, M. J.; Beckham, G. T., Lignin Depolymerization with Nitrate - Intercalated Hydrotalcite Catalysts. ACS Catalysis 2016, 6 (2), 1316 - 1328. 36. Choi, Y. S.; Singh, R.; Zhang, J.; Balasubramanian, G.; Sturgeon, M. R.; Katahira, R.; Chupka, G.; Beckham, G. T.; Shanks, B. H., Pyrolysis reaction networks for lignin model compounds: unraveling thermal deconstruction of beta - O - 4 a nd alpha - O - 4 compounds. Green Chemistry 2016, 18 (6), 1762 - 1773. 37. Dawange, M.; Galkin, M. V.; Samec, J. S. M., Selective Aerobic Benzylic Alcohol Oxidation of Lignin Model Compounds: Route to Aryl Ketones. Chemcatchem 2015, 7 (3), 401 - 404. 38. Badamali, S. K.; Luque, R.; Clark, J. H.; Breeden, S. W., Co(salen)/SBA - 15 catalysed oxidation of a beta - O - 4 phenolic dimer under microwave irradiation. Catalysis Communications 2011, 12 (11), 993 - 995. 39. Luo, J.; Zhang, X.; Lu, J. Z.; Zhang, J., Fine Tuning the Redox Potentials of Carbazolic Porous Organic Frameworks for Visi ble - Light Photoredox Catalytic Degradation of Lignin beta - O - 4 Models. ACS Catalysis 2017, 7 (8), 5062 - 5070. 40. Magallanes, G.; Karkas, M. D.; Bosque, I.; Lee, S.; Maldonado, S.; Stephenson, C. R. J., Selective C - O Bond Cleavage of Lignin Systems and P olymers Enabled by Sequential Palladium - Catalyzed Aerobic Oxidation and Visible - Light Photoredox Catalysis. A CS Catalysis 2019, 9 (3), 2252 - 2260. 41. Enthaler, S.; Spilker, B.; Erre, G.; Junge, K.; Tse, M. K.; Beller, M., Biomimetic transfer hydrogenat ion of 2 - alkoxy - and 2 - aryloxyketones with iron - porphyrin catalysts. Tetrahedron 2008, 64 (17), 3867 - 3876. 42. Cao, Y.; Wang, N.; He, X.; Li, H. R.; He, L. N., Photocatalytic Oxidation and Subsequent Hydrogenolysis of Lignin beta - O - 4 Models to Aromatics Promoted by In Situ Carbonic Acid. Acs Sustainable Chemistry & Engineering 2018, 6 (11), 15032 - 15039. 43. Wang, M.; Li, L. H.; Lu, J. M.; Li, H. J.; Zhang, X. C.; Liu, H. F.; Luo, N. C.; Wang, F., Acid promoted C - C bond oxidative cleavage of beta - O - 4 and beta - 1 lignin models to esters over a copper catalyst. Green Chemistry 2017, 19 (3), 702 - 706. 44. Andersen, M. L.; Mathivanan, N.; Wayner, D. D. M., Electrochemistry of electron - transfer probes. The role of the leaving group in the cleavage of radic al anions of alpha - aryloxyacetophenones. Journal of the American Chemical Society 1996, 118 (20), 4871 - 4879. 45. Jin, K. B.; Kim, H. E.; Hyun, M. H., Liquid Chromatographic Resolution of Mexiletine and Its Analogs on Crown Ether - Based Chiral Stationary Ph ases. Chirality 2014, 26 (5), 272 - 278. 121 46. Shah, S. T. A.; Singh, S.; Guiry, P. J., A Novel, Chemoselective and Efficient Microwave - Assisted Deprotection of Silyl Ethers with Selectfluor. Journal of Organic Chemistry 2009, 74 (5), 2179 - 2182. 47. Mohamed, M. I. F.; Devi, G. K. D., Micellar mediated reactions: Synthesis of substituted phenacyl phenolic ethers. Der Chemica Sinica 2011, 2 (1), 136 - 141. 122 Chapter 3. Non - - O - 4 Ether Cleavage with Organic Thiols Ether bonds represent up to 75% of the linkages found in lignin. 1 Our previous successful work focused on the most common ether linkage, the - O - 4 bond, which accounts for roughly 60% of ligni n bonds ( Figure 3. 1 ). In this work, we focused on the remaining ~15% of the ether bonds to see if they may be successfully cleaved using the potent nucleophilicity of small organic thiols and thiolates . We subjected 4 - O - 5 and - O - 4/ - 5 linkages to thiol - mediated cleavage under previously optimized conditions and found little reactivity. Only the addition of p - substituted aldehydes on the ring allowed successful cleavage of both 4 - O - 5 and - O - 4/ - 5 model lignin dimers with yields ranging from 5% to 78%. These results suggest that other ether cleavage reactions, apart from those using the - O - 4 S N 2 cleavage mecha nism, may occur when depolymerizing real heterogenous lignin and allow additional cleavage through activation with a carbonyl. 3.1 Introduction Lignin is an aromatic polymer found in the cell wall of plants with a high energy density that would make it use ful in the replacement of petroleum derived fuels and chemicals. We have previously found that organic thiols can mimic the chemistry found in the enzymatic - aryl ether cleavage pathway of lignin. 2 - aryl ether linkages account for up to ~60% of the bonds that link the aromatic polymer . This cleavage process, both in the enzymatic pathway and our mimetic pathway, involves a nucleophilic attack by a thiol on the - carbon of an oxidized - O - 4 bond. This This chapter is adapted from Klinger, G. E.; Zhou, Y.; Foote, J. A.; Wester, A. M.; Cui, Y.; Alherech, M.; Stahl, S. S.; Jackson, J. E.; Hegg, E. L. Nucleophilic Thiols Reductively Cleave Ether Linkages in Lignin Model Polymers and Lignin. ChemSusChem 2020 , accepted. (invited Special Collections: Lignin Valorization from Theory to Practice) Y. Zhou synthesized all model dimers, J. A. Foote assisted G. E. Klinger in lignin analysis, A. M. Wester assisted G. E. Klinger in analytical quantification, Y. Cui and M. Alherech oxidized Cu - AHP lignin, S. Stahl oversaw oxidation of Cu - AHP lignin. 123 first step releases a phenoxide and forms a thioether. The second and final step invol ves another nucleophilic attack by a thiol on the thioether to form a disulfide and release of the second polymer fragment. This process has been successful using lignin dimer models and has the potential to cleave real lignin. The successful depolymerizat ion of lignin, which comprises five other linkages in addition - O - 4 bond ( Figure 3. 1 ), requires a multiple number of its linkages to be cleaved i n order to - O - - 5 and the 4 - O - 5 linkages are the other two ether bonds found in lignin. These two ether linkages are resistant to thermal degradation 3 , even under certain pyrolytic conditions, and thus a great target for mild cleavage processes. For decades the most common m ethod of - O - - 5 and 4 - O - 5 linkages has been catalytic hydrogenolysis. This process typically involves various metal catalysts 4 at high temperatures with pressurized hydrogen to break the dimers bonds. Some of these successful catalysts include cobalt molybdenum complexes 5 or other metal - Mo complexes 6 to break the 4 - O - 5 bond. Ni/SiO 2 7 , Pd/C 8 , Ni/C 9 , and composites of titanium nitride with nickel 10 have also proven successful. The Zhang group was able to cleave both - O - 4 and 4 - O - 5 dimers using a Ni/Al 2 O 3 catalyst with alcoholic solvents as the hydrogen donor rather than H 2 gas. 11 Others have been able to decrease the hydrogenolysis temperatures to RT using a Pd/C catalyst to cleave - O - 4 bonds. 12 Each of these methods usually require H 2 pressurized systems. Electrocatalytic h ydrogenolysis (ECH) is an alternative process to catalytic hydrogenolysis where the hydrogen needed for cleavage is formed in - situ and there is no need for pressurized H 2 . 13 ECH cleavage of 4 - O - 5 and - O - 4/ - 5 models have been effective with catalyst s such as RANEY® nickel 14 , Ru/ACC 15 , and transition or precious metal particles on RCV 16 . The Huang 124 group has even developed a metal - free ECH process that uses NaBH 4 converted to TBABH 4 by the electrolyte to cleave both 4 - O - 5 and - O - 4/ - 5 dimers 17 . Other mild methods of cl eaving 4 - O - 5 and - O - 4/ - 5 ether bonds have included Co(salen) complexes with O 2 at RT to oxidatively cleave the - O - 4 bond 18 , hydrosilanes with B(C 6 F 5 ) 3 at mild temperatures and ambient pressures to reduce the - O - 4 bond 19 , and the use of chloro - substituted metalloporphyrins at RT and ambient pressures to oxidize the - O - 4 bond 20 . The Yao group was able to cleave 4 - O - 5 bonds using just heat, LiAlH 4 and Fe(acac) 3 . 21 The challenge for many of these methods involves balancing severity, cost, toxicity, efficiency, and recyclability ; thus, there remain openings for new milder processes that are cost effective and green. Due to the challenges listed above, coupled with a need for mechanisti c insight on how our biomimetic thiol system would be able to successfully cleave real lignin, we sought to evaluate - O - - 5 and 4 - O - 5 cleavage using thiolates that had previously proven successful in the - O - 4 ether cleavage. Figure 3. 1 Representation of lignin and its common linkages highlighted by color. The most - O - 4 bond (highlighted in orange) with other ether bonds highlighted in red (4 - O - - O - - 5). The percent of the various linkages in lignin is based on hardwoods. 1 125 3.2 4 - O - 5 Model Dimer Cleavage To probe possible 4 - O - 5 lignin linkage cleavage by thiols, simple diaryl ethers were used as model dimers (dimers defined as one linkage between two aromatic rings) under the optimized conditions 2 of 2 - mercaptoethanol in refluxing acetonitrile with K 2 CO 3 . Consistent with leaving group ability and previous Hammett studies, only the p - ketone and p - aldehyde - substituted diaryl ethers were cleaved ( Figure 3. 2 , Figure 3. 5 , and Table 3. 1 ) wit h yields of 7% and 78%, respectively . Addition of 1 equiv. of butylated hydroxytoluene (BHT), a known radical scavenger, did not affect the yield of cleavage ( Figure 3. 8 ), suggesting that the reaction mechanism does not involve a free radical. These reactions presumably proceed via a nucleophilic aromatic substitution mechanism ( Figure 3. 9 - a ), yielding the easily detected free phenol product. Other diaryl ethers with p - substituted electron withdrawing or donating groups were unreactive to thiol - mediated cleavage. These results s uggest thiolate side reactions in lignin may be minimal for 4 - O - 5 ether linkages unless prior pretreatment methods produce activating carbonyls. Figure 3. 2 Cleavage of 4 - O - 5 model lignin dimers Yields of phenolic monomer after 24 h are indicated in red. 126 3.3 - O - 4 / - 5 Model Dimer Cleavage - - O - 4 linkages, simple benzyl phenyl ethers were used as model dimers. Like the 4 - O - - - O - 4 model di mers were generally unreactive. Again, carbonyl moieties enabled a small amount of reaction; only substrates with p - aldehyde groups either on the benzyl or on the phenoxyl sides of the dimer released the phenolic products upon thiol treatment, doing so in 25% and 5% yields, respectively. ( Figure 3. 3 , Figure 3. 7 , and Table 3. 2 ). Likewise, a ddition of 1 equiv. of butylated hydroxytoluene (BHT) did not affect the yield of cleavage in - O - 4/ - 5 model dimers ( Figure 3. 8 ), also suggesting that the reaction mechanism does not involve a free radical. Presumably, when activated by an aldehyde on the benzyl side, the alpha position may be poised for nucleophilic attack ( Figure 3. 9 - b) . The decrease d yield seen when the aldehyde is placed on - position are more important for ether cleavage than the leaving group. Based on these yields, - O - 4 / - 5 linkages are even less reactive than 4 - O - 5 linkages to thiol - mediated cleavage, even when activated by a carbonyl. Therefore, it can be concluded that little, if any, side reactions of thiolates with - O - 4 / - 5 ethers will occur in real lignin. Despite representing an unhindered primary site for potential S N 2 attack with two phenoxide leaving groups, the acetal diphenoxymethane, with no carbonyls to activate it, was unreactive to cleavage by thiol ( Figure 3. 4 and Figure 3. 6 ). 127 Figure 3. 3 Cleavage of - O - - 5 model lignin dimers Yields of phenolic monomer are indicated in red. Figure 3. 4 Cleavage of other ether dimers Diphenoxymethane , an acetal , cleavage yields no phenolic monomers (red) 3.4 Conclusion In summary , this study illustrat e s that there are at least two other routes for thiol mediated lignin depolymerization, - aryl ether S N 2 cleavage . Furthermore, these results give additional evidence that 4 - O - - O - - 5 , and - O - 4 (previous work) ether linkages are no t cleave d through a thiol assisted radical mechanism. These thiol - mediated cleavage reactions may allow further depolymerization in extracted lignin that has been partially or fully oxidized. The oxidation that activates the - O - 4 cleavage may also produce carbonyls that promote other ether 128 cleavages. While these thiol - mediated side reactions may be less efficient than the published S N 2 cleavage of lignin - like compounds , they may contribute to a more fully depolymerized lignin. 3.5 Experimental Detail 3.5.1 General Information Chemicals were tested for purity using 1 H NMR prior to use. All water used was filtered with a Millipore SAS Milli - Q ® Reference Water Purification system. All reactions were performed under a nitrogen atmosphere using a ba lloon unless otherwise specified. Column purification was accomplished using Silicycle SiliaFlash P60 silica gel (40 - (TLC) was performed on aluminum silica gel 60 F - 254 plates and the bands were visualized using short wave UV light (254 nm). HPLC analysis was performed using an Agilent 1260 Infinity equipped with a G1315D 1260 diode array detector VL, monitoring at 280 nm and recording from 190 - 400 nm and a G1362 refractive index detector. For dimer and monomer analysis, a Supelco with a mobile phase of 70:30 acetonitrile:water, adjusted to 60:40 for more difficult separations; 5 an internal standard; external standards were run during each sequence of analysis. 1 H and NMR spectra were recorded using Agilent DDR2 500 MHz NMR spectrometers equipped with 7600AS 96 sample autosamplers running VnmrJ 3.2A and referenced to residual sol vent peaks. 3.5.2 Synthesis of Model Lignin Dimers General Protocol for 4 - O - 5 Synthesis: Yuting Zhou synthesized 4 - O - - O - 4 dimers. The following 4 - O - 5 dimers were prepared using a literature procedure 22 unless otherwise noted. A mixture of aryl bromide (2 mmol), phenol (3 mmol), Cs 2 CO 3 (4 mmol), copper iodide (0.2 mmol), N,N - dimethylglycine hydrochloride salt (0.6 mmol), and dioxane (100 mL) were sealed in a round 129 bottom flask and heated at 90 °C under a nitrogen atmosphere, monitoring for completion by TLC. The reaction was cooled and extracted using EtOAc and water. The organic layer was separated, and the aqueous layer was extracted with additio nal EtOAc. The combined organic layers were washed with brine, dried over Na 2 SO 4 , and concentrated in vacuo . The residual oil was purified by column chromatography using EtOAc/hexane (1:30) to afford the product. 4 - p henoxybenzonitrile: Spectral data are c onsistent with those reported in the literature. 23 1 H NMR (500 MHz, CDCl 3 7.56 (m, 2H), 7.45 7.37 (m, 2H), 7.25 7.20 (m, 1H), 7.11 7.04 (m, 2H), 7.04 6.96 (m, 2H). 4 - t olyl phenyl ether (3 - c) : Spectral data are consistent with those reported in the literature. 23 The product was obtained with 63% yield. 1 H NMR (500 MHz, CDCl 3 7.28 (m, 2H), 7.14 (d, J = 8.1 Hz, 2H), 7.07 (tt, J = 7.4, 1.1 Hz, 1H), 7.02 6.96 (m, 2H), 6.96 6.88 (m, 2H), 2.34 (s, 3H). 4 - p henoxybenzalde hyde (3 - g) : Spectral data are consistent with those reported in the literature. 24 The product was obtained with 38% yield. 1 H NMR (500 MHz, CDCl 3 9.92 (s, 1H), 7.88 7.79 (m, 2H), 7.45 7.37 (m, 2H), 7.23 (ddt, J = 8.6, 7.4, 1.2 Hz, 1H), 7.12 7.03 (m, 4H). 1 - m ethoxy - 4 - phenoxybenzen e (3 - b) : Spectral data are consistent with those reported in the literature. 24 The product was obtained with 66% yield. 1 H NMR (500 MHz, CDCl 3 7.28 (m, 2H), 7.08 7.02 (m, 1H), 7.02 6.97 (m, 2H), 6.97 6.93 (m, 2H), 6.93 6.86 (m, 2H), 3.82 (s, 3H). 1 - p henoxy - 4 - (trifluoromethyl)benzene (3 - e) : Spectral data are consistent with those reported in the literature. 23 The product was obtained with 67% yield. 1 H NMR (500 130 MHz, CDCl 3 7.53 (m, 2H), 7.43 7.36 (m, 2H), 7.19 (ddd, J = 8.5, 6.8, 1.1 Hz, 1H), 7.11 6.99 (m, 4H). 4 - phenoxybe nzamide (3 - h) : This compound was prepared following a literature procedure. 25 A 100 mL round bottom flask was charged with 1.06 g of 4 - phenoxybenzonitrile (5.4 mmol), 0.112 g of K 2 CO 3 (15 mol%), 3.8 mL of aqueous 30% H 2 O 2 solution and 22 mL of DMSO. The mixture was stirred at 0 °C for 2 h and monitored by TLC. Upon completion, the mixture was diluted slowly with DI water, the precipitation was filtered and washed by DI water. The crude product was recrystallized by EtOH/H 2 O (1:1) to afford a white solid product in 55 % yield. Spectral data are consistent with those reported in the literature. 25 1 H NMR (500 MHz, CDCl 3 7.76 (m, 2H), 7.44 7.34 (m, 2H), 7.23 7.15 (m, 1H), 7.10 7.05 (m, 2H), 7.05 6.99 (m, 2H), 6.23 5.35 (m, 2H). 1 - ( p henoxymethyl) - 4 - (trifluoromethyl)benzene (3 - m) : This compound was prepared according to a literature procedure. 26 4 - (trifluoromethyl)benzyl bromide (2 g, 8.37 mmol) was added to a mixture of phenol (0.866 g, 8.37 mmol; 1.1 equiv.) and K 2 CO 3 (1.39 g, 10.06 mmol) in acetone (60 mL). The mixture was stirred at room temperature for 24 h. The reaction was filtered with Whatman paper, the solvent removed by rotary evaporation, and the resulting residue was purified by column chromatography with D CM/pentane (4:1 and then 3:7) to afford the product (1.69 g) in 76% yield. Spectral data are consistent with those reported in the literature. 27 1 H NMR (500 MHz, CDCl 3 7.61 (m, 2H), 7.61 7.53 (m, 2H), 7.36 7.29 (m, 2H), 7.04 6.92 (m, 3H), 5.14 (d, J = 1.2 Hz, 2H). 3.5.3 General Procedure for Cleavage Reactions and Analysis Lignin model dimers (10 mg) and dried powdered K 2 CO 3 (~100 mg) were added to an oven dried round - bottom flask (50 mL) equipped with a stir bar, condenser, septum, and nitrogen - 131 filled balloon, and the entire apparatus was purged with N 2 . Acetonitrile (20 mL) and thiol (10 equiv. to dimer) were added through a septum, and t he reaction was refluxed with periodic sampling for HPLC analysis to monitor progress over 24 h. 3.5.4 Cleavage of 4 - O - 5 Models R - Group Conversion Yield Yield Yield R1 = H 0% 0% - 0% R1 = OMe 1% 0% 0% 0% R1 = Me 1% 0% - 0% R1 = OH 4% 0% - 3% R1 = CF3 0% 0% 0% 0% R1 = COCH3 9% 7% 0% 7% R1 = CN 21% 0% - - R1 =CHO 85% 2% 0% 78% R1 =CONH2 5% - - 0% D iphenoxymethane Table 3. 1 Cleavage yields of 4 - O - 5 dimers. Reactions were carried out using 10 equiv. of BME in refluxing MeCN with stirring K 2 CO 3 for 24 h. Conversion is shown for substrate and yields are given for products. Yields were assessed by phenolic product. Benzene (1) and toluene (3) wer e not quantified due to insolubility in direct injection HPLC. (7) was cleaved but quantification of phenol and benzonitrile where unsuccessful due to chromatography separation. (9) No phenol product was produced with no external standards for 4 - hydroxyben zamide or benzamide. Only the aldehyde (8) and ketone (6) substituted 4 - O - 5 dimers were cleaved successfully suggesting that a carbonyl is needed to activate a nucleophilic aromatic substitution using thiols. No cleavage was observed for diphenoxymethane. 132 Figure 3. 5 Time course of 4 - O - 5 dimer cleavage with BME. 4 - O - 5 dimer cleavage using 10 equiv. of BME and K 2 CO 3 while stirring with refluxing MeCN for 24 h. Very little cleavage is 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Phenol Diphenyl ether 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Phenol 4-Phenoxyphenol 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Phenol 4-phenoxybenzamide 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Phenol 1-Methoxy-4-phenoxybenzene 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Phenol 1-methyl-4-phenoxybenzene 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Phenol 4-(trifluoromethyl)diphenylether 0 20 40 60 80 100 0 10 20 30 % Yield hr Phenol 4-hydroxyacetophenone 4-Phenoxyacetophenone 0 20 40 60 80 100 0 10 20 30 % Yield hr Phenol 4-hydroxybenzaldehyde 4-phenoxybenzaldehyde (a) (b) (d) (c) (e) (f) (g) (h) 133 seen except in the case of (h) where the p - directing aldehyde promotes nucleophilic aromatic substitution, rel easing phenol. The corresponding implied thioether product was not quantified. Figure 3. 6 Time course of other aryl ether cleavage with BME . Diphenoxymethane was subjected to 10 equiv. of BME and K 2 CO 3 under refluxing MeCN for 24 h. but did not undergo cleavage. This control reaction supports the need of a carbonyl to promote ether cleavage using small thiols. 3.5.5 - O - - 5 Models R - Group Conversion Yield Yield Yield R 1 = H; R 2 = H 0% 0% 0% 0% * R 1 = NH 2 ; R 2 = H 2% - 0% - R 1 = OH; R 2 = H 6% 0% 0% 0% R 1 = CN; R 2 = H 12% 2% 0% 0% R 1 = ring; R 2 = H 0% 0% 0% 0% R 1 = CF 3 ; R 2 = H 0% 0% 0% 0% R 1 = H; R 2 = CF3 1% 0% 0% 0% R 1 = H; R 2 = CHO 49% 25% 0% 0% R 1 = CHO; R 2 = H 13% 2% 0% 0% Table 3. 2 - O - - 5 dimers. Reactions were carried out using 10 equiv. of BME in refluxing MeCN with stirring K 2 CO 3 for 24 h. Conversion is shown for substrate and yields are given for products. Yields were assessed by phenolic product. Benzene and toluene were not quantified due to insolubility in direct injection HPLC. ( * ) No phenol product was observed or quantified . Similar to the 4 - O - - O - - 5 dimers activated with an aldehyde enable cleavage through a possible S N 2 mechanism . 0 20 40 60 80 100 0 10 20 30 % Yield hr Diphenoxymethane 134 Figure 3. 7 Time course - O - - 5 dimer cleavage with BME . - O - - 5 dimer cleavage using 10 equiv. of BME and stirring K 2 CO 3 with refluxing MeCN for 24 h. Very little cleavage is 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Phenol Benzyloxybenzene 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr 2-Naphthol 2-Benzyloxynaphthalene 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Hydroquinone 4-Benzyloxyphenol 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr 4-(benzyloxy)aniline 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Phenol 4-Methylbenzaldehyde 4-Phenoxymethylbenzaldehyde 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr 4-Hydroxybenzaldehyde 4-Benzyloxybenzaldehyde 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr 4-Trifluoromethylphenol 4-Benzyloxybenzotrifluoride 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Phenol 4-(Trifluoromethyl)benzylphenyl ether (a) (b) (d) (c) (e) (f) (g) (h) 135 seen except (e) where the p - direc ting aldehyde promotes nucleophilic attack on the C - position, releasing a phenol product. (f) resulted in <5% cleavage suggesting that the - position are more important for cleavage. 3.5.6 Dimer Control Experiments Figure 3. 8 Control reactions to test for radical mechanism s of 4 - O - - O - - 5 dimer cleavage using p - directing aldehyde dimers . (a), (c), and (e) on the left side were reacted with 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Phenol 4-hydroxybenzaldehyde 4-phenoxybenzaldehyde 0 20 40 60 80 100 0 10 20 30 % Yield hr Phenol 4-hydroxybenzaldehyde 4-phenoxybenzaldehyde 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Phenol p-tolualdehyde 4-(phenoxymethyl)benzaldehyde 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr Phenol 4-Methylbenzaldehyde 4-Phenoxymethylbenzaldehyde 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr 4-hydroxybenzaldehyde Toluene 4-(benzyloxy)benzaldehyde 0 20 40 60 80 100 0 5 10 15 20 25 % Yield hr 4-Hydroxybenzaldehyde 4-Benzyloxybenzaldehyde (a) (b) (d) (c) (e) (f) 136 10 equiv. of BME in stirring K 2 CO 3 with refluxing MeCN and the addition of 1 equiv. of BHT, a known radical scavenger. (b), (d), and (f) on the right side were subjected to the same conditions but without BHT. Very little difference in yield is seen between the 3 separate substrates and their corresponding BHT control reaction. This suggests that the cleavage mechanism does not involve a free radical. 3.5.7 Proposed Dimer Cleavage Me chanism Figure 3. 9 Proposed mechanism of non - - O - 4 ether cleavage. A) Proposed 4 - O - 5 ether - O - - 5 ether cleavage via an S N 2 mechanism activated by the p - aldehyde. 137 3. 6 Spectra Figure 3. 10 4 - p henoxybenzonitrile 1 4 5 2 138 Figure 3. 11 4 - t olyl phenyl ether (3 - c) 1 4 3 5 2 139 Figure 3. 12 4 - p henoxybenzaldehyde (3 - g) 1 4 5 2 3 140 Figure 3. 13 1 - m ethoxy - 4 - phenoxybenzene (3 - b) 1 4 5 2 3 141 Figure 3. 14 1 - p henoxy - 4 - (trifluoromethyl)benzene (3 - e) 1 4 5 142 Figure 3. 15 4 - phenoxybenzamide (3 - h) 1 4 5 2 3 143 Figure 3. 16 1 - ( p henoxymethyl) - 4 - (trifluoromethyl)benzene (3 - m) 1 4 5 2 3 6 144 REFERENCES 145 R EFERENCES 1. Rinaldi, R.; Jastrzebski, R.; Clough, M. T.; Ralph, J.; Kennema, M.; Bruijnincx, P. C. A.; Weckhuysen, B. M., Paving the Way for Lignin Valorisation: Recent Advances in Bioengineering, Biorefining and Catalysis. Angewandte Chemie - International Edition 2016, 55 (29), 8164 - 8215. 2. Klinger, G. E.; Zhou, Y.; Hao, P.; Robbins, J.; Aquilina, J. M.; Jackson, J. E.; Hegg, E. L., Biomimetic Reductive Cleavage of Keto Aryl Ether Bonds by Small - Molecule Thiols. Chem S us C hem 2019, 12 (21), 4775 - 4779. 3. Parth asarathi, R.; Romero, R. A.; Redondo, A.; Gnanakaran, S., Theoretical Study of the Remarkably Diverse Linkages in Lignin. Journal of Physical Chemistry Letters 2011, 2 (20), 2660 - 2666. 4. Koyama, M., H ydrocracking of Lignin - Related Model Dimers . Bioresou rce Technology 1993, 44 (3), 209 - 215. 5. Petrocelli, F. P.; Klein, M. T., C hemical Modeling Analysis of the Yields of S ingle - R ing P henolics from Lignin L iquefaction . Industrial & Engineering Chemistry Product Research and Development 1985, 24 (4), 635 - 641. 6. Shabtai, J.; Nag, N. K.; Massoth, F. E., C atalytic Functionalities of S upoorted S ulfides .4. C - O H ydrogenolysis S electivity as a Function of P romotor Type . Journal of Catalysis 1987, 104 (2), 413 - 423. 7. He, J.; Zhao, C.; Lercher, J. A., Ni - Catalyzed Cleavage of Aryl Ethers in the Aqueous Phase. Journal of the American Chemical Society 2012, 134 (51), 20768 - 20775. 8. Li, Y.; Karlen, S. D.; Demir, B.; Kim, H.; Luterbacher, J.; Dumesic, J. A.; Stahl, S. S.; Ralph, J., Mechanistic Study of Diaryl Ether Bond Cleavage during Palladium - Catalyzed Lignin Hydrogenolysis. Chem S us C hem 2020 . Preprint. 9. Gao, F.; Webb, J. D .; Hartwig, J. F., Chemo - and Regioselective Hydrogenolysis of Diaryl Ether C - O Bonds by a Robust Heterogeneous Ni/C Catalyst: Applications to the Cleavage of Complex Lignin - Related Fragments. Angewandte Chemie - International Edition 2016, 55 (4), 1474 - 1478 . 10. Molinari, V.; Giordano, C.; Antonietti, M.; Esposito, D., Titanium Nitride - Nickel Nanocomposite as Heterogeneous Catalyst for the Hydrogenolysis of Aryl Ethers. Journal of the American Chemical Society 2014, 136 (5), 1758 - 1761. 146 11. Jiang, L.; Guo, H.; Li, C.; Zhou, P.; Zhang, Z., Selective cleavage of lignin and lignin model compounds without external hydrogen, catalyzed by heterogeneous nickel catalysts. Chemical Science 2019, 10 (16), 4458 - 4468. 12. Xie, T.; Cao, J. - P.; Zhu, C.; Zhao, X. - Y.; Zhao, M.; Zhao, Y. - P.; Wei, X. - Y., Selective cleavage of C - O bond in benzyl phenyl ether over Pd/AC at room temperature. Fuel Processing Technology 2019, 188 , 190 - 196. 13. Garedew, M.; Lin, F.; DeWinter, T. M.; Song, B.; Saffron, C. M.; Jackson, J. E.; Lam, C. H.; Anastas, P., Greener Routes to Biomass Waste Valorization: Lignin Transformation Through Electrocatalysis for Renewable Chemicals and Fuels Production. ChemSusChem 2020 . Preprint. 14. Mahdavi, B.; Lafrance, A.; Martel, A.; Lessard, J.; Menard, H.; Brossard, L., Electrocatalytic hydrogenolysis of lignin model dimers at Raney nickel electrodes. Journal of Applied Electrochemistry 1997, 27 (5), 605 - 611. 15. Garedew, M.; Young - Farhat, D.; Bhatia, S.; Hao, P.; Jackson, J. E.; Saffron, C . M., Electrocatalytic cleavage of lignin model dimers using ruthenium supported on activated carbon cloth. Sustainable Energy & Fuels 2020, 4 (3), 1340 - 1350. 16. Dabo, P.; Cyr, A.; Lessard, J.; Brossard, L.; Menard, H., Electrocatalytic hydrogenation o f 4 - phenoxyphenol on active powders highly dispersed in a reticulated vitreous carbon electrode. Canadian Journal of Chemistry - Revue Canadienne De Chimie 1999, 77 (7), 1225 - 1229. 17. Wu, W. - B.; Huang, J. - M., Electrochemical Cleavage of Aryl Ethers Promoted by Sodium Borohydride. Journal of Organic Chemistry 2014, 79 (21), 10189 - 10195. 18. Canevali, C.; Orlandi, M.; Pardi, L.; Rindone, B.; Scotti, R.; Sipila, J.; Morazzoni, F., Oxidative degradation of monomeric and dimeric phenylpropanoids: reactivity and mechanistic investigation. Journal of the Chemical Society - Dalton Transactions 2002, (15), 3007 - 3014. 19. Zhang, J.; Chen, Y.; Brook, M. A., Reductive Degradation of Lignin and Model Compounds by Hydrosilanes. Acs Sustainable Chemistry & Engineering 2014, 2 (8), 1983 - 1991. 20. Cui, F. T.; Wijesekera, T.; Dolphin, D.; Farrell, R.; Skerker, P., B iomimetic Degradation of Lignin . Journal of Biotechnology 1993, 30 (1), 15 - 26. 21. Ren, Y.; Yan, M.; Wang, J.; Zhang, Z. C.; Yao, K., Selective Reductive Cleavage of Inert Aryl C - O Bonds by an Iron Catalyst. Angewandte Chemie - International Edition 2013, 52 (48), 12674 - 12678. 22. Ma, D. W.; Cai, Q., N,N - Dimethyl glycine - promoted Ullmann coupling reaction of phenols and aryl halides. Organic Letters 2003, 5 ( 21), 3799 - 3802. 23. Tlili, A.; Monnier, F.; Taillefer, M., Selective One - Pot Access to Symmetrical or Unsymmetrical Diaryl Ethers by Copper - Catalyzed Double Arylation of a Simple Oxygen Source. Chemistry - A European Journal 2010, 16 (41), 12299 - 12302. 147 24. Hu, T.; Schulz, T.; Torborg, C.; Chen, X.; Wang, J.; Beller, M.; Huang, J., Efficient palladium - catalyzed coupling reactions of aryl bromides and chlorides with phenols. Chemical Communications 2009, (47), 7330 - 7332. 25. Murth y, S.; Desantis, J.; Verheugd, P.; Maksimainen, M. M.; Venkannagari, H.; Massari, S.; Ashok, Y.; Obaji, E.; Nkizinkinko, Y.; Luescher, B.; Tabarrini, O.; Lehtio, L., 4 - (Phenoxy) and 4 - (benzyloxy)benzamides as potent and selective inhibitors of mo no - ADP - ribosyltransferase PARP10/ARTD10. European Journal of Medicinal Chemistry 2018, 156 , 93 - 102. 26. Wang, M.; Li, L. H.; Lu, J. M.; Li, H. J.; Zhang, X. C.; Liu, H. F.; Luo, N. C.; Wang, F., Acid promoted C - C bond oxidative cleavage of beta - O - 4 a nd beta - 1 lignin models to esters over a copper catalyst. Green Chemistry 2017, 19 (3), 702 - 706. 27. Kuwano, R.; Kusano, H., Benzyl protection of phenols under neutral conditions: Palladium - catalyzed benzylations of phenols. Organic Letters 2008, 10 (10), 1979 - 1982. 148 Chapter 4. Lignin Polymer Cleavage with Nucleophilic Thiols Lignin may serve as a renewable feedstock for the production of chemicals and fuels if mild, scalable processes for its depolymerization can be devised. The use of small organic thiols represents a bioinspired - O - 4 bond, the most common linkage in lignin. In the present study, s - O - 4 linked polymers were treated with organic thiols, yielding up to 90% cleaved monomer products . L ignin extracted from poplar was also treated with o rganic thiols resulting in molecular weight reduction s as high as 65 % number average molecular weight (M n ) in oxidized lignin . Lignin extracted from other processes was also successfully cleaved to varying degrees. The success of thiol - mediated cleavage o f model polymers, as well as biomass - derived lignin, illustrate s the potential utility of small redox - active molecules to penetrate complex polymer matrices for depolymerization and subsequent valorization of lignin into fuels and chemicals. 4.1 Introductio n The need to replace fossil fuels with renewable feedstocks for the manufacture of chemicals and fuels is critical due to finite petroleum resources and rising greenhouse gas emissions. 1 - 3 Most efforts to make transportation fuels and chemicals from lignocellulosic biomass have focused on depolymerization of cellulose and fermentation of the result ing sugars to alcohols. 4 However, the carbon - rich lignin fraction could also displace substantial fossil petrol eum if practical methods were available for its depolymerization into chemically tractable fragments. 4 This chapter is adapted from Klinger, G. E.; Z hou, Y.; Foote, J. A.; Wester, A. M.; Cui, Y.; Alherech, M.; Stahl, S. S.; Jackson, J. E.; Hegg, E. L. Nucleophilic Thiols Reductively Cleave Ether Linkages in Lignin Model Polymers and Lignin. ChemSusChem 2020 , accepted. (invited Special Collections: Lign in Valorization from Theory to Practice) Y. Zhou synthesized all model dimers, J. A. Foote assisted G. E. Klinger in lignin analysis, A. M. Wester assisted G. E. Klinger in analytical quantification, Y. Cui and M. Alherech oxidized Cu - AHP lignin, S. Stahl oversaw oxidation of Cu - AHP lignin. 149 Lignin i s an energy - and carbon - rich aromatic polymer found in plant cell walls. Historically treated as a waste byproduct of the pulp and paper industry, it has recently gained attention as a possible bio - based substitute for petroleum feedstocks in fuel and chem ical production. 4 Lignin depolymerization, however, is energy intensive and costly due to the chemical recalcit rance of the linkages between the aromatic propylphenol subunits. Additionally, simple oxidative cleavage methods decrease the energy density of the products and often lead to the formation of new undesired covalent crosslinks. A number of non - oxidative li gnin depolymerization methods have been developed, many of which focus on the cleavage of aryl ethers from - O - 4 bonds, the most prevalent linkages in lignin. Reductive methods for ether cleavage often begin with a nucleophilic halide paired with a Lewis a cid. 5 Derivatization followed by reductive cleavage (DFRC) has been developed as a lignin analysis procedure, cleaving ether bonds via treatment with acetyl bromide and subsequent reduction with zinc metal. 6 In the acidolysis method, hydrochloric acid, heat, and dioxane are used to cleave aryl ethers, 7 - 9 but low cleavage yields led researchers to develop an improved variation called thioacidolysis. This acidic solvolysis uses boron trifluoride etherate, a potent Lewis acid, together with ethanethiol in dioxane to cleave aryl ether bonds. The resulting thiolated monomers can then be reduced with Raney nickel. 10 Like DFRC, thioacidolysis i s mainly used to analyze lignin. Catalytic hydrogenolysis, another reductive lignin cleavage technique, utilizes pressurized hydrogen and heat in the presence of catalysts such as Raney® nickel, 11 - 13 ruthenium, platinum, or palladium. 12 - 14 Condensation of the lignin fragments after depolymerization, a common problem of hydrogenolysis and other high temperature lignin depolymerization reactions, has been minimized by the Luterbacher gro up by using aldehydes as stabilizers. 15 - 18 The above meta l 150 catalysts are also key in electrocatalytic hydrogenation (ECH), a reductive technique where lignin linkages are cleaved in an electrochemical cell. 19 - 25 Oxidation of the C - OH in - O - 4 lignin models, followed by reductive cleavage, an overall redox - neutral process, has met with some success. Common oxidants such as TEMPO + or DDQ readily oxidize the HC - OH to a C =O carbonyl. This oxidation lowers the C - O ether bond dissociation energy, allowing reductants such as zinc used by the Westwood group 26 or photo - reductive i ridium complexes used by the Stephenson group, 27 - oxidized model - O - 4 dimers. Another effective redox neutral process for the cleavage of - - O - 4 linkages uses formic acid and water, and has successfully been applied to lignin from various sources . 28 Despite these advances, new simple and inexpensive processes remain of interest to diversif y lignin depolymerization strategies. Thiols are both strong nucleophiles and reductants, and they have been employed in applications ranging from protein denaturation to thiol - click chemistry. 29, 30 Thiols also play key roles in biochemical pathways, including one that cleaves ether bonds vicinal to carbonyl groups - oxidized lignin ( Figure 4. 1 - a). A relatively unexplored avenue for ether cleavage is a biomimetic approach, mimicking etherases such as the glutathione - dependent enzymes from wood - digesting bacteria like Sphingobium sp . strain SYK - 6. 31 - 34 Glutathione (GS) is a tripeptide used by the Lig enzymes E, P, F, and BaeAB (e therases), and Lig enzyme G and GST Nu (lyase). 31 - 35 This cofactor contains a cysteine (thiol) residue that performs a protein - assisted S N 2 reaction on an - keto lignin dimer, forming a thioether. The thioether is then reduced by another glutathione resulting in the formation of a disulfide bond and liberation of an enolate that is protonated to form the ketone. 31 - 34 - O - 4' - aryl ether C - O bond, reductively fragmenting the alpha - k eto - O - 4 linkage, as summarized in Figure 4. 1 - a. 151 Figure 4. 1 - aryl ether cleavage via thiol nucleophilic a ttack. A) - aryl ether cleavage of C - oxidized lignin dimer with glutathione (GS). B) - O - 4 model dimers cleavage with organic thiols (RS). C) This work focusing on lignin and lignin - like polymer cleavage using orga nic thiols to form lignin fragments that can be fed into other processes for subsequent valorization. 152 depolymerization of lignin dimers, we recently reported that organi c thiols, under alkaline polar aprotic solvent conditions, can cleave keto aryl ether bonds in model lignin systems with nearly 100% yields ( Figure 4. 1 - b). 36 In this study, we examined the nucleophilic and reductive capabilities of free organic thiols to cleave ether bonds in polymeric lignin ( Figure 4. 1 - c). The ability of small, diffusible redox medi ators to penetrate and chemically attack the lignin network is a potential advantage relative to the Lig enzymes which are mostly limited to small lignin fragments. 35, 37, 38 Furthermore, these diffusible thiol - based redox carriers can th eoretically be recycled using 2 - electron/2 - proton reduction of the disulfide product in an electrochemical cell ( Figure 4. 34 ), analogous to the electrochemical oxidative mediators employed by the Stahl group. 39, 40 Herein, we first explore alkaline thiol - - aryl ether polymers into monomeric p roducts, and then extend our studies to biomass - extracted lignin. 4.2 - O - 4 Linked Polymer Cleavage The ability of small organic thiols to penetrate polymeric networks should enable nucleophilic cleavage of lignin from cellulosic biomass. To test sites in bulky polymeric systems, three synthetic - O - 4 - linked polymers were constructed. Degrees of polymerization were ~10, as analyzed through elemental analysis (described in the experimental detail section) . These i nsoluble 10 - mers 41, 42 were subjected to depolymerization using neat 1,3 - propanedithiol as shown in Figure 4. 2 . The dithiol, 1,3 - propanedithiol, was chosen for its reducing abilities due to stabilization of the oxidized 5 - membered ring product via an intramolecular disulfide bond. Synthetic polymer A ( - O - 4 linked polyphenol), a polyphenol made entirely of - O - 4 linkages, was depolymerized resulting in soluble monomer yields as high as 90%. Synthetic polymer B ( - O - 4 linked polyguaiacol), a methoxylated analog of polymer A, was also 153 cleaved with up to 80% yields. This lower yield may be due either to the electroni c effects of the methoxy groups that decrease the partial positive charge on the - position or to steric effects as observed previously in our - O - 4 model studies. 36 Reaction of Polymers A and B with BME was slightly less successful than 1,3 - propanedithiol but still resulted in monomer yields of up to 70% ( Figure 4. 4 ). This trend was also seen in previous work 36 on dimer cleavage when using neat or high concentrations of BME and 1,3 - propanedithiol. The low sulfur content, < 2%, of the remaining polymer ( Figure 4. 5 ) indicates that the thiol - dependent depolymerization reaction completed both the S N 2 ether cleavage and the reductive elimination of t he thioether intermediate, - hydroxymethylated analog of polymer B, a more representative model of - O - 4 linkages in lignin, would be expected to yield the same products a s those from polymer B . This is due to loss of the hydroxymethyl moiety on the - position via the retro - aldol side reaction seen in our previous work. 36 This process would form polymer B, which would be cleaved as seen above. Importantly, synthetic polymer C (reduced - O - 4 linked polyphenol), the - hydroxy analog of polymer A, was unreactive to thiol mediated depolymerization, as expected. Oxidation of th e - position is required for nucleophilic attack by the thiol, as seen both in our earlier work 36 and in the enzymatic pathway being mimicked. 43, 44 154 Figure 4. 2 Cleavage of synthetic - O - 4 linked polymers . Top: Synthetic model - O - 4 polymer cleavage reaction (3 h) with 1,3 - propanedithiol to yield monomer cleavage products. A) - O - 4 linked polyphenol synthetic polymer, B) - O - 4 linked polyguaiacol syn thetic polymer, C) reduced - O - 4 linked polyphenol. Bottom: Yields of monomers cleaved from top reactions in triplicate. No cleavage products were seen from polymer C. Yields reported are monomer yields performed without thiol subtracted from monomer yield s of reactions performed with thiol. 4.3 Cu - AHP Lignin Cleavage With the above promising results on lignin polymer model systems, efforts were extended to an exploration o f biomass - derived lignin oxidized at the - keto position as represented by Figure 4. 6 . All lignin used in this section, unless otherwise stated, was isolated via the copper - catalyzed 0 20 40 60 80 100 Polymer A + Thiol Polymer B + Thiol Polymer C + Thiol Monomer Yield (%) 155 alkaline hydrogen peroxide (Cu - AHP) pretreatm ent of poplar. 45 This lignin is slightly oxidized at the alpha position. 46 The synthetic polymer reactions, above, were performed under neat conditions due to low solubility. Biomass - derived lignin, which has more polar groups to aid e in solubility, was tested using various solvents, reaction times, thiols, and work - up procedures to optimize parameters for depolymerization and analysis. Lignin depolymerization with BME under aqueous conditions ( Figure 4. 7 ) was relatively unsuccessful as was found in the previous dimer studies. 36 In fact, GPC of products from treatment under aqueous conditions showed molecular weight increases at long reaction times, possibly due to condensation r eactions. Similar results were observed with DMSO as the solvent ( Figure 4. 8 ). The lignin could be solubilized by other polar aprotic solvents, such as DMF or NMP, but s howed little molecular weight change upon treatment with BME or DTT ( Figure 4. 8 ) in these media. Neat thiol conditions achieved the largest molecular weight reductions, with DTT and BME as the most successful thiols ( Figure 4. 8 ) under a 24 h reaction time ( Figure 4. 9 ). To analyze lignin cleavage under neat conditions, work - up proc edures had to be thoughtfully carried out to minimize condensation reactions of the polymer. Lignin molecular weight changes were examined for neat reactions with BME and DTT, worked up with water at pH 9 ( Figure 4. 10 ), neutral ( Figu re 4. 11 ), and pH 2 ( Figure 4. 12 ). Various acids were compared to determine recoverability of unreacted lignin ( Figure 4. 22 ). Sulfuric acid gave complete recovery of solubilized lignin a nd was therefore used for all work - up acidification procedures. Complete analysis of total polymer was obtained with an alkaline work - up ( Figure 4. 10 , Figure 4. 13 , and Figure 4. 14 ), whereas acidification, even to a neutral pH, precipitated the lignin and made depolymerization comparisons inaccurate. With the above reaction parameters optimized, Cu - AHP lignin was stirred in the presence of powdered K 2 CO 3 with either neat BME ( Figure 4. 13 ), neat DTT ( Figure 4. 13 ), or neat 1,3 - 156 propanedithiol ( Figure 4. 3 - a, Figure 4. 25 - a, c) at 100 °C for 24 h. For molecular weight analysis, mixtures were diluted with water to solubilize th e base and lignin. Large decreases in molecular weight were observed when Cu - AHP lignin was depolymerized with BME and DTT ( Figure 4. 13 and Figure 4. 14 ) despite these thiols being less successful in dimer and synthetic oligomer cleavage. This cleavage resulted in 60 - 80% polymer mass loss, presumably through formation of small fragment s that are acid soluble ( Figure 4. 16 ). Furthermore, sulfur incorporation ( Figure 4. 17 ) into the remaining polymer (roughly 15 - 20%) suggests that the thiolate can access the polymer backbone and cleave the ethers but may not completely proceed through the second step of the reaction involving disulfide bond formation and release of the seco nd fragment. This inability to complete the reaction may be due to the bulky nature of the polymer that may decrease accessibility to the polymer backbone. Analysis of the - OH content showed a doubling of guaiacyl - OH sites, confirming that ether cleavage had occurred ( Figure 4. 18 ). Similarly, in the remaining lignin, the oxidized - O - 4 dimer NMR signal decreased after treatment with DTT and BME, indicating that these ox idized fragments had been removed from the polymer ( Figure 4. 19 ). Finally, lignin analysis via thioacidolysis, an analytical technique to quantify lignin - O - 4 content, also showed that the - O - 4 content decreased, presumably due to the thiolate - mediated ether cleavage ( Figure 4. 20 and Figure 4. 21 ). Interestingly, while BME and DTT were moderately successful at Cu - AHP lignin depolymerization, 1,3 - propanedithiol under neat conditions and over various reaction times ( Figure 4. 15 ) was unsuccessful, with little to no decrease in the Cu - AHP lignin molecular weight. These results contrast with the high cleavage yields achieved with 1,3 - propanedithiol treatment of model dimers or synthetic oligomers ( Figure 4. 3 ). 157 Noting the importance of the - keto moiety in activating the cleavage, we explored further oxidation of the lignin with Bob 49 which resulted in an ~5 - fold increase in oxidation of the - OH groups ( Figure 4. 25 - e - f ). Incubation of BME with this oxidized Cu - AHP lignin resulted in the M n decreasing by ~65% compared to ~40% for BME treatment of the unoxidized Cu - AHP lignin ( Figure 4. 3 - a - c ). The use of the less reactive thiol, 1,3 - propanedithiol, on the oxidized Cu - AHP lignin resulted in an M n decrease of 49% ( Figure 4. 25 - a - c ). Thus, oxidation enhances thiol - mediated depolymerization. Furthermore, this degree of depolymerization cannot be accounted for by only end group cleavage. Other oxidization techniques were less successf ul resulting in increases in molecular weight of unreacted lignin and little molecular weight reductions when treated with thiol ( Figure 4. 22 , Figure 4. 23 , and Figure 4. 24 ). Importantly, performing the Cu - AHP lignin depolymerization reaction on a 1 g scale ( Figure 4. 33 ) proved to be just as efficient as the base case 100 mg scale, indicating that the lignin depolymerization reaction can be successfully scale d up. F urthermore, scale - up and variations in procedural details of the Cu - AHP lignin isolation did not alter the lignin properties ( e.g. , NMR chemical shift, molecular weight, solubility) or affect depolymerization ( Figure 4. 31 and Figure 4. 32 ), demonstrating the robustness of the process. While the exact reaction mechanism remain s to be verified, our results clearly demonstrate that diffusible organic thiols are able to penetrat e and cleav e the polymer ic network not only of synthetic linear polymers, but also of oxidized poplar lignin . 158 Figure 4. 3 Cleavage of oxidized vs unoxidized lignin with thiol . Gel permeation chromatograms of A) Cu - AHP lignin before and after treatment with BME , B) Cu - AHP lignin before and after treatment with BME . C) Molecular weight distributions for M n in blue , M w in orange , M p in green, and PDI in purple . * EQN 3. 1 , EQN 3. 2 , and EQN 3. 3 were used to analyze the molecular weight values. 4.4 Other Lignin Cleavage Lignin extracted through oxidative processes o r subjected to oxidation has proven to be a useful substrate for thiol - mediated depolymerization. To test the nucleophilicity of thiolates to other biomass - derived lignins, we obtained lignin from various extraction processes and subjected them to neat thi ol in 100 °C with stirring K 2 CO 3 for 24 h. GVL ( - valerolactone) lignin, obtained from thermalcatalytic saccharification of biomass using GVL, water, H 2 SO 4 , and heat to solubilize 0 1 2 3 4 5 6 7 0 5000 10000 15000 20000 25000 30000 Cu-AHP Lignin Thiol + Cu-AHP Lignin Oxidized Cu-AHP Lignin Thiol + Oxidized Cu-AHP Lignin PDI Molecular Weight (g/mol) Mn Mw Mp PDI A) B) C) 159 and separate the sugars from lignin 50 , was subjected to BME ( Figure 4. 27 and Figure 4. 28 - a) and 1,3 - propandithiol ( Figure 4. 28 - b) and analyzed for molecular weight reductions using GPC. Both thiols cleaved the lignin with decreases in M n and M w around 20% and little change to the M p or PDI. Decreases in lignin molecular weight were compared to unreacted lignin controls due to only 68% of the GVL lignin soluble under work - up conditions (aqueous base). MetGen crude lignin, obtained from an industrial biorefinery before oxidative enzymatic treatment, was also reacted with 1,3 - propanedithiol and mo nitored for molecular weight changes. This poorly reactive thiol mediator was able to decrease M n by up to 59% and M w by 76% ( Figure 4. 26 ). Similarly, sodium lignosulfo nate lignin had molecular weight decreases of 41% for M n and 18% for M w ( Figure 4. 29 ) when treated with 1,3 - propanedithiol. Alkaline and dealkaline lignin were unreactiv e to 1,3 - propanedithiol mediated depolymerization ( Figure 4. 29 ). This inactivity is most likely due to cross links from its previous extraction process, making it poorly suitable for keto aryl ether cleavage. Among the various lignins tested, BME mediated depolymerization is the most promising method ( Figure 4. 28 ) for cleavage with mos t lignin showing some amount of reduction in molecular weight. 4.5 Conclusion In summary , building on our prior model - based studies, we have now demonstrated that small, diffusible thiols can penetrate complex matrices, access the polymer backbone, and act as redox mediators to reductively cleave ether linkages in polymeric substrates, including biomass - derived lig nin, that bear the - keto functionality . S mall organic thiols can readily cleave simple model lignin oligomers containing - keto - O - 4 linkages with near complete mass balance. The penetration and depolymerization of oxidized natural lignin suggests this s trategy could be a viable technique for lignin fragmentation for further downstream valorization. Furthermore, the 160 success ful, albeit modest, cleavage of non - - O - 4 linkages (Chapter 3) potentially expands the utility of this thiol - mediated depolymerization process. Th ese findings open the door for future studies in which electrochemical 2 - electron/2 - proton reduction s of the disulfide byproducts would regenerate the thiols , enabling net electrocatalytic lignin depolymerization. 4.6 Experimental Detail 4.6. 1 General Information Chemicals were tested for purity using 1 H NMR prior to use. All water used was filtered with a Millipore SAS Milli - Q ® Reference Water Purification system . All reactions were performed under a nitrogen atmosphere using a balloon unless otherwise specified. Column purification was accomplished using Silicycle SiliaFlash P60 silica gel (40 - (TLC) was performed on aluminum silica gel 60 F - 254 plates and the bands were visualized using short wave UV light (2 54 nm). HPLC analysis was performed using an Agilent 1260 Infinity equipped with a G1315D 1260 diode array detector VL, monitoring at 280 nm and recording from 190 - 400 nm and a G1362 refractive index detector. For dimer and monomer analysis, a Supelco at 0.4 mL/min with a mobile phase of 70:30 acetonitrile:water , adjusted to 60:40 for more difficult separations ; 5 (0.28 mM) as an internal standard; external standards were run during each sequence of analysis. Lignin analysis was performed using a Waters Ultrahydrogel 250 7.8 x 300 mm gel permeation chromatography (GPC) column attached to a Waters Ultrahydrogel 6 x 40 mm guard column held at a constant 40 °C temperature in isocratic mode at 0.7 mL/min with a mobile phase of 0.005 M NaOH in 80/20 0.1 M aqueous sodium (8.6 mM) as an internal standard with pol ystyrene sulfonic acid external standards run during each sequence. Instrumental control, data 161 acquisition, and data processing for the HPLC were performed with Agilent ChemStation software. LC - MS analyses employed a Waters Xevo G2 - XS UPLC/MS/MS equipped w ith a Quadrupole/Time - of - Flight (QTOF) system . 1 H and 31 P NMR spectra were recorded using Agilent DDR2 500 MHz NMR spectrometers equipped with 7600AS 96 sample autosamplers running VnmrJ 3.2A and referenced to residual solvent peaks. 2D - NMR spectra were r ecorded using a Varian 600 MHz superconducting NMR s pectrometer operating at 599.892 MHz interfaced with a Dell Precision T3500, running CentOS 5.6 with VnmrJ 3.2A and a Bruker Avance 900 MHz superconducting NMR s pectrometer. 4.6.2 Synthesis of Model Ligni n Oligomers Synthetic Polymer Protocol: The following polymers were prepared using a literature procedure 41, 42, 51 . Br - acetophenone monomer (500 mg, 2.0 mmol) (2 - bromo - acetophenone or 2 - bromo - acetovanillone) was stirred in anhydrous DMF (2.5 mL) under a N 2 atmosphere. Dry powdered K 2 CO 3 (422 mg, 3.0 mmol) was added to the mixture and the reaction was stirred at 50 °C overnight. The mixture was poured into ice water (100 mL), filtered, and washed with water and methanol. The remaining solids were frozen and lyophilized to afford an insolu ble dry powder. Further reduction of the synthetic polymer was achieved as follows: The polymer (200 mg) was stirred in DMSO (10 mL). To the suspension, NaBH 4 (230 mg) was added and the reaction was heated to 50 °C under N 2 atmosphere overnight. The reacti on mixture was poured into ice water (200 mL) and acidified to pH 3 with HCl. The slurry was filtered, washed with water, frozen, and lyophilized. The dry powder was stirred with dioxane and poured into diethyl ether to remove low molecular weight compound s. The mixture was filtered, and the solids were frozen and lyophilized to afford a dry powder. The bromine content of the polymers was analyzed by Midwest Microlab 162 (Indianapolis, IN) and used to calculate the approximate polymer molecular weight of the in soluble polymers. Lignin Extraction Protocol: Lignin was prepared from 18 - year - old debarked ( Populus nigra var. charkoviensis x caudina cv. NE - 19) hybrid poplar (harvested in 2011 from University of Wisconsin - Madison, Arlington plots) that was chipped and hammer - milled to 5 mm grind size. Lignin was isolated according to previous procedures using copper - catalyzed alkaline hydrogen peroxid e (Cu - AHP pretreatment). 9 Briefly, poplar (100 g) was incubated with aqueous NaOH (270 mM) at 10% solid loadings (1 liter) at 30 °C shaking for 1 h. The biomass was washed with deionized water (500 mL) and pretreated for an additional 23 h at 10% solid loa dings (1 liter) with additional NaOH (270 mM), copper (1 mM), bipyridine (2 mM), and hydrogen peroxide (100 mg H 2 O 2 per g of biomass added over the course of 10 h). The mixture was then filtered to remove the solid biomass, and acid insoluble lignin was pr ecipitated from the filtrate by lowering the pH to 2 with H 2 SO 4 . The precipitated lignin was centrifuged and washed 3 times with pH 2 water, frozen, and lyophilized yielding a light - yellow powder. This powder was then used for depolymerization studies. Polymer A Polymer B Polymer C 163 4.6 .3 General Procedure for Cleavage Reactions and Analysis 4.6.3 - i Synthetic Polymer Synthetic polymer (10 mg), dried powdered K 2 CO 3 (100 mg), and thiol (0.5 mL) were added to a glass vial (~5 mL) and closed with an aluminum cap. The mixture was stirred at 1 00 °C in an oil bath for 3 h. The solution was dissolved in 20 mL of water and analyzed by LC - MS according to the specifications written out in the General Information section above. For sulfur analysis, the reaction was scaled up 5 - fold and the depolymeri zed thiol - treated solution was centrifuged, decanted, and the remaining solid was washed 3 times with alkaline water. This washed solid was frozen, lyophilized, and the dry powdered was sent to A&L Great Lakes (Fort Wayne, IN) for sulfur analysis. 4.6.3 - ii Lignin Thiol - treated lignin was treated and processed as follows unless otherwise noted : Lignin (100 mg) and dried powdered K 2 CO 3 (100 mg) was added to a glass vial (~10 mL) with a stir bar, capped with a septum, and purged with nitrogen. Thiol (1 mL) was added to the reaction and capped with an aluminum covered cap and stirred at 100 °C in an oil bath for 1 - 24 h. HPLC: For molecular weight analysis, the resulting mixture was cooled to room temp, dissolved in 20 mL of water, centrifuged, and injected dir ectly to the HPLC - GPC using the conditions stated in the G eneral I nformation section. Lignin , unreacted with thiol , was worked up in the same manner (100 mg of lignin dissolved in 20 mL of water with 100 mg of K 2 CO 3 ) and injected directly on to the HPLC as a control comparison . Retention times were compared to a sodium polystyrene sulfonate kit from Scientific Polymer Products (M n 1,440 - 85,600 g/mol). The molecular weight at the highest peak (M p ) was calculated directly from external standards while the number average 164 molecular weight (M n ) (average of all the molecular weights of the polymer) , weight average molecular weight (M w ) (molecular weights of the polymer weighted for the larger polymeric fraction) , and polydispersity (PDI) (width of the molecular range) were calculated from the following equations (1 - 3): EQN 3. 1 EQN 3. 2 EQN 3. 3 where N i represents the total number of different molecular weights chains and M i is the molecular weight of each polymer chain. For elemental analysis, mass balance, thioacidolysis, and NMR characterization, the aqueous solubiliz ed thiol - treated lignin was precipitated with H 2 SO 4 (HCl for sulfur analysis) to pH 2, centrifuged and washed with acidic water, frozen, and lyophilized. The resulting dry depolymerized lignin was then characterized by the following: NMR: For 2 - D NMR anal ysis on lignin before and after depolymerization, g HSQC - AD NMR was used according to literature procedure. 52, 53 Briefly, dry lignin (40 mg) was dissolved in 500 - d 6 . The solution was added to an NMR tube and r u n with either a Varian 600 MHz or a Bruker 900 MHz NMR. The Bruker BioSpin Avance GmbH 900 MHz superconducting NMR s pectrometer equipped with a TCI triple resonance inverse detection Cryoprobe , 5 mm CPTCI 1H - 13C/ 15N/ D Z - GRD Z44910/ 0007 used the following parameters: DMSO - d 6 solvent peak was used as an internal reference ( C 39.5, H 2.49 ppm); The 13 C - 1 H correlation experiment was adiabatic 165 g HSQC - AD using the pulse sequence hsqcedetgpsisp2.2 with spectra acquired from 13 to - 3 ppm in F2 ( 1 H) using a pulse width of 9.25 sec , a relaxation delay of 1.5 sec , acquisition time 0.0713 sec, F2 ( 1 H) spectral width of 14367.8, spectral and acquired size of 1024 ; 200 to 0 ppm in F1 ( 13 C) using a spectral width of 45196.2, acquired size 400, and a spectral size of 1024; 32 scans , with a total acquisition time of 16 h. The Varian Inova 600 MHz superconducting NMR s pectrometer oper ating at 599.892 MHz, running CentOS 5.6 with VnmrJ 3.2A and equipped with an HCN probe used the following parameters: DMSO - d 6 solvent peak was used as an internal reference (39.5, 2.49 ppm); The 13 C - 1 H correlation experiment was adiabatic gHSQC - AD using t he standard gHSQC - AD pulse sequence with spectra acquired from 14 to - 2 ppm in F2 ( 1 H) using a pulse width of 8.5 sec, a relaxation delay of 1.0 sec , acquisition time 0.15 sec, F2 ( 1 H) spectral width of 9595.8, spectral size of 2048, and an acquired size of 1439 ; 190 to - 10 ppm in F1 ( 13 C) using a spectral width of 20155.9, acquired size 512, and a spectral size 2048; 72 scans, with a total acquisition time of 2 4 h. For - OH content of lignin before and after depolymerization, 31 P NMR was us ed according to literature procedure. 54 pyridine/deuterated chl - chloro - 4,4,5,5, - tetramethyl 1,3,2 - dioxaphosph olane were added and vortexed until fully dissolved. The solution was added to an NMR tube and r u n with a 500 MHz Agilent DDR2 NMR equipped with a phosphorous probe using a 45° pulse angle, 5 sec pulse delay, and 512 scans. Peaks were integrated using the internal standard, cyclohexanol. 166 Mass balance: The dry solids remaining were weighed and compared to the weight of lignin un - reacted with thiol, that was worked up in the same manner (dissolved in base and water and acidified, centrifuged, washed with aci dic water, frozen, and lyophilized). Elemental analysis: Samples were analyzed by A & L Great Lakes (Fort Wayne, IN) for sulfur content. Thioacidolysis: Samples were analyzed by the GLBRC - Core F acility (Michigan State University - East Lansing, MI) for S:G ratio. 55 167 4.6.4 Synthetic Polymer Cleavage Figure 4. 4 Thiol - - O - 4 lignin . Monomer cleavage yields are - O - 4 polymers (top) using neat 1,3 - propanedithiol or neat BME in stirring K 2 CO 3 at 100 °C for 3 h. These monomer yields are compared to yields without addition 0 10 20 30 40 50 60 70 80 90 100 Monomer Yield (%) Synthetic - O - 4 Polymer Cleavage 168 of thiol. Polymer A (purple) and B (blue) have high cleavage yields , >70%, when treated with 1,3 - propanedithiol . However, no monomer products are seen from the reaction with out the addition of thiol. Furthermore, Polymer C, the reduced version of Polymer A, did not yield any monomer products whether subjected to thiol treatment or not. These results support the ability of thiols such as BME and 1,3 - propanedithiol to diffuse t hrough bulky polymeric systems - O - 4 bonds. T hese results also give additional - position must be oxidized, as seen in previous dimer studies 36 and in the enzymatic pathway 56 , in order for thiols to - aryl ether site. Reactions were performed in triplic ate. Figure 4. 5 Analysis of sulfur incorporated into depolymerized synthetic - O - 4 lignin . - O - 4 polymer after depolymerization with 1,3 - propanedithiol and K 2 CO 3 at 100 °C for 24 h. Roughly 22% of polymer A remained uncleaved after thiol mediated depolymerization and from that 22%, there was 1.69% sulfur content (purple) or 1.86 mg of sulfur remaining. Roughly 13.6% of polymer B remaine d uncleaved after thiol mediated depolymerization and from that 13.6%, there was 0.51% sulfur content (blue) or 0.350 mg of sulfur remaining. The low percent of sulfur suggests that the remaining unreacted polymer successfully completed the first step of t he S N 2 reaction, forming a thioether, and continued to the second step, formation of the disulfide and releas e of the second monomer , to complete the cleavage reaction . 0 10 20 30 40 50 60 70 80 90 100 Polymer A Polymer B % Sulfur by weight % Sulfur in Thiol - Treated Synthetic - O - 4 Polymer 169 4.6.5 Lignin Polymer Model Figure 4. 6 Representation of general hardwood lignin and its common linkages highlighted by color. - O - 4 bond (highlighted in orange) with other ether bonds highlighted in red (4 - O - - O - - 5). The percent of the various linkages in lignin is based on hardwoods. 57 In poplar lignin, a small amount of p - hydroxybenzoic acid is present on the gamma hydroxyl groups of S - units, but they are largely removed during the Cu - AHP lignin isolation process. 170 4.6.6 Cu - AHP Lignin Cleavage Figure 4. 7 Thiol - mediated Cu - AHP lignin depolymerization in water . Molecular weight comparisons for Cu - AHP lignin treated with 10 equiv. of BME and stirring K 2 CO 3 in refluxing water for 0, 6, 12, and 24 h. (a) Gel permeation chromatogram of molecular weight distributions of Cu - AHP lignin at different reaction times (0 h, blue , 6 h, red , 12 h, green , and 24 h, purple h). (b) Calculated molecular weight comparisons f or M n in blue , M w in orange , and M p in green for 0, 6, 12, and 24 h. M n and M p axis is shown on the left and M w axis is shown on the right. Visually, from the chromatogram and the calculated values, lignin depolymerization does not work well in aqueous con ditions, similar to what was found in our dimer studies 36 . Reactions were performed in duplicate. 0 250000 500000 750000 1000000 1250000 0 2500 5000 7500 10000 12500 15000 BME + Lignin water initial BME + Lignin water 6 hrs BME + Lignin water 12 hrs BME + Lignin water 24 hrs M w Molecular Weight (g/mol) M n & M p Molecular Weight (g/mol) Cu - AHP Lignin Depolymerization in Water with 10 equiv. BME Mn Mp Mw (a) (b) (280 nm) 171 Cu - AHP Lignin Depolymerization Conditions Mass Change (%) Conditions Mass Change (%) DTT in DMF - 65.2 ± 5.09 BME in DMF - 82.5 ± 10.61 DTT in DMSO +62.15 ± 15.34 BME in DMSO +116.25 ± 68.66 DTT in NMP - 75.5 ± 1.13 BME in NMP - 86.35 ± 1.77 DTT in H2O - 47.75 ± 8.56 BME in H2O +111.6 ± 77.22 DTT in H2O and NaOH +46.1 ± 58.55 BME in H2O and NaOH +59.3 ± 53.60 Cu - AHP Lignin Depolymerization Conditions M p Conditions M p DTT in DMF 6,929 BME in DMF 6,182 DTT in DMSO 1,772 BME in DMSO 1,767 DTT in NMP 7,333 BME in NMP 6,726 DTT in H 2 O 6,041 BME in H 2 O 5,364 DTT in H 2 O (NaOH) 927 BME in H 2 O (NaOH) 933 Cu - AHP Lignin 5,311 Figure 4.8 continued onto next page 172 Cu - AHP Lignin Depolymerization Conditions Mass Change (%) Neat DTT - 54.2 Neat thiophenol + 52.2 Neat BME - 64.4 Neat 1,3 - propanedithiol + 62.8 Figure 4. 8 Comparison of percent mass change variability during Cu - AHP lignin depolymerization with various solvents. (+) indicates mass gained due to thiol incorporation and/or crosslinking of polymer chains and ( - ) indicates mass loss due to possible solubilization of clea ved fragments. (a) Mass change (%) of Cu - AHP lignin reaction with DTT or BME with stirring Na 2 CO 3 at 100 ° C in DMF, DMSO, NMP, H 2 O, or H 2 O (with NaOH instead of Na 2 CO 3 ) . The reactions were quenched with acid to pH 2, centrifu g ed, frozen, lyophilized, and the dry un cleaved /acid insoluble polymer was weighed. Standard deviation reported for 3 replicates. (b) M p from GPC analysis of (a) compared with Cu - AHP lignin M p . (c) Mass change (%) of Cu - AHP lignin reacted with neat thiol (DTT, BME, Thi ophenol, or 1,3 - propanedithiol) and stirring K 2 CO 3 at 100 °C for 24 h. From the above tables it is clear that the lignin reacted under polar aprotic solvents (and water) is not an accurate representation of the lignin depolymerization due to the variabilit y in the mass changes and the molecular weight changes compared to unreacted Cu - AHP lignin. Furthermore, mass loss change is evident in most cases but structural changes in lignin in solvating conditions resulted in solubility/precipitation problems after the reaction. Thiophenol and 1,3 - propanedithiol increased the mass of the uncleaved polymer. Therefore, neat reaction conditions using BME or DTT appear to be ideal for lignin depolymerization. 173 Cu - AHP Lignin 0 h. Cu - AHP Lignin + BME 6 h. Cu - AHP Lignin + BME 24 h. M n 6778 2294 1954 M w 55740 41726 34357 PDI 8.22 18.19 17.58 M p 1624 1060 1051 Figure 4. 9 Time course study to determine reaction length needed for lignin depolymerization with neat thiol . Molecular weight comparison of Cu - AHP lignin either unreacted or reacted with neat BME for 6 or 24 h. (Top) GPC chromatogram of molecular weight distribution between Cu - AHP lignin (grey), Cu - AHP lignin treated with BME for 6 h. (green), and Cu - AHP lignin treated with BME for 24 h. (purple) . (Bottom) Molecular weight comparisons for M n , M w , M p , and PDI for the 3 time points . Cleavage of lignin after 6 h. is similar to c leavage after 24 h. , suggesting that the lignin does not need to be treated with BME for as long as 24 h. to produce ether cleavage fragments. 174 Figure 4. 10 Polymer cleavage characterization using pH 9 workup. Molecular weight comparisons for Cu - AHP lignin treated with neat BME or neat DTT with stirring K 2 CO 3 at 100 °C for 24 h. compared to a Cu - AHP lignin control, worked up in the same manner but without the addition of thiol. Reactions were quenched with water (20 mL) with a final pH of 9 (due to the dissolved K 2 CO 3 ). (a) GPC chromatogram of molecular weight distributions of Cu - AHP lignin (grey) , BME - treated Cu - AHP lignin (orange) , and DTT - treated Cu - AHP lignin (blue). (b) Calculated molecular weight comparisons for M n in blue, M w in orange, and M p in green for Cu - AHP lignin, BME - treated Cu - AHP lignin, and DTT - treated Cu - AHP lig nin. M n and M p axis is shown on the left and M w is shown on the right. Visually, from the chromatogram and the calculated values, both BME and DTT decrease the polymer molecular weight significantly. Reactions were run in duplicate. 0 10000 20000 30000 40000 50000 60000 0 2000 4000 6000 8000 10000 DTT pH9 BME pH9 Lignin pH9 M w Molecular Weight (g/mol) M n & M p Molecular Weight (g/mol) Cu - AHP Lignin Depolymerization in Neat Thiol (pH 9) Mn Mp Mw (a) (b) 175 Figu re 4. 11 Polymer cleavage characterization using pH 7 workup. Molecular weight comparisons for Cu - AHP lignin treated with neat BME or neat DTT with stirring K 2 CO 3 at 100 °C for 24 h. compared to Cu - AHP lignin worked up the same way (minus the thiol and heat) as a control. Reactions were quenched with water (20 mL) and acidified with 72% H 2 SO 4 to pH 7. (a) Gel permeation chromatogram of molecular weight distributions of Cu - AHP lignin ( grey) , BME - treated Cu - AHP lignin (orange) , and DTT - treated Cu - AHP lignin (blue). (b) Calculated molecular weight comparisons for M n in blue, M w in orange, and M p in green for Cu - AHP lignin, BME - treated Cu - AHP lignin, and DTT - treated Cu - AHP lignin. M n and M p axis is shown on the left and M w is shown on the right. Visually, from the chromatogram and the calculated values, both BME and DTT decrease the polymer molecular weight significantly but have larger variations than the reactions analyzed directly (pH 9) . Reactions were run in duplicate. 0 10000 20000 30000 40000 50000 60000 0 2500 5000 7500 10000 DTT pH7 BME pH7 Lignin pH7 M w Mol. Wt. (g/mol) M n & M p Mol. Wt. (g/mol) Cu - AHP Lignin Depolymerization in Neat Thiol (pH 7) Mn Mp Mw (a) (b) 176 Figure 4. 12 Polymer cleavage characterization using pH 2 workup. Molecular weight comparisons for Cu - AHP lignin treated with neat BME or neat DTT with stirring K 2 CO 3 at 100 °C for 24 h. compared to Cu - AHP lignin worked up the same way (minus the thiol and heat) as a control. Reactions were quenched with water (20 mL) and acidified with 72% H 2 SO 4 to pH 2. (a) Gel permeation chromatogram of molecular weight distributions of Cu - AHP lignin (grey) , BME - treated Cu - AHP lignin (orange) , and DTT - treated Cu - AHP lignin (blue). (b) Calculated molecular weight comparisons for M n in blue, M w in orange, and M p in green for Cu - AHP lignin, BME - treated Cu - AHP lignin, and DTT - treated Cu - AHP lignin. M n and M p axis is shown on the left and M w is shown on the right. Visually, from the chromatogram and the calculated values, both BME and DTT polymer molecular weight values vary significantly with little differe nce between the thiol depolymerized lignin and the lignin control. From these quenching reactions, addition of acid appears to cause large variations in molecular weight with possible precipitation. Moving forward, direct analysis of the reaction (pH 9) ap pears to be the most informative work - up owing to the fact that the entire polymer/polymer products are dissolved and are observable and quantifiable using GPC with very little variability in replicates. Reactions were run in duplicate. 0 10000 20000 30000 40000 50000 60000 0 200 400 600 800 1000 1200 1400 1600 DTT pH2 BME pH2 Lignin pH2 M w Molecular Weight (g/mol) M n & M p Molecular Weight (g/mol) Cu - AHP Lignin Depolymerization in Neat Thiol (pH 2) Mn Mp Mw (a) (b) 177 Figure 4. 13 Depolymerization of Cu - AHP lignin with neat thiol for 24 h worked up with water to pH 9 . (a) Molecular weight distributions of untreated Cu - AHP lignin (grey) , Cu - AHP lignin treated with neat DTT (orange) , a nd Cu - AHP lignin treated with neat BME (blue) . (b) Calculated molecular weights of M n in blue , M w in orange , and M p in green from the top chromatogram. M n and M p axis is shown on the left and M w is shown on the right. BME - treated lignin has similar molecular weight decreases in M n , M w , and M p compared to DTT - treated lignin. Reactions were run in triplicate. 0 20000 40000 60000 80000 0 2000 4000 6000 8000 10000 Lignin pH9 DTT pH9 BME pH9 M w Molecular Weight (g/mol) M n & M p Molecular Weight (g/mol) Average Molecular Weights of Neat Thiol - Treated Lignin (24 h.) Mn Mp Mw 178 Cu - AHP Lignin Cu - AHP Lignin + DTT Cu - AHP Lignin + BME M n 8009 2583 3208 M w 57702 49786 25832 M p 1749 703 1150 PDI 7.20 19.27 8.05 Figure 4. 14 Analysis of Cu - AHP lignin depolymerization whole polymer and fragments . Cu - AHP lignin reactions were run with neat thiol and K 2 CO 3 at 100 °C for 24 h. and worked up with water at pH 9 . (a) GPC chromatogram of Cu - AHP lignin (grey), DTT - treated Cu - AHP lignin (orange), and BME - treated Cu - AHP lignin (blue). The lignin polymer is highlighted from retention volume 5 - 10 mL while the lignin fragments small er than 1,000 g/mol are highlighted from retention volume 10 - 30 mL. After 1,000 g/mol, molecules are separated by polarity with the disulfide products seen at ~13 mL. (b) Calculated molecular weights of M n , M w , and M p , and P DI from the top chromatogram of the lignin polymer. (c) Relative percent ages of monomers, dimer, trimers, tetramers, and pentamers - decamers of the lignin fragments less than 1,000 g/mol for (a). Percentage is based on relative intensity of molecular weight ranges from 84 - 1068 g/mol. Percent Monomer 22% Dimers 7% Trimers 28% Tetramers 14% Pentamer - decamer 29% Cu - AHP Lignin Monomer 38% Dimers 14% Trimers 20% Tetramers 8% Pentamer - decamer 20% BME + Cu - AHP Lignin Monomer 38% Dimers 12% Trimers 20% Tetramers 6% Pentamer - decamer 24% DTT + Cu - AHP Lignin Lignin Polymer Lignin Fragments <1,000 g/mol 179 polymers is broken down as follows: monomers 84 - 199 g/mol, dimers 200 - 299 g/mol, trimers 300 - 399 g/mol, tetramers 400 - 499 g/mol, and polymers 500 - 1068 g/mol. Values were obtained using a high - resol ution LCMS - QTOF with all molecules with sulfur mass defects removed from analysis. GPC analysis shows a 60 - 73% molecular weight reduction in lignin. Soluble lignin fragment analysis suggests that there is significant cleavage by the thiol due to the larger % monomer compared to the lignin control. Figure 4. 15 Susceptibility of Cu - AHP lignin depolymerization with 1,3 - p ropanedithiol over time. Cu - AHP lignin was reacted with neat 1,3 - propanedithiol and K 2 CO 3 at 100 °C for 1, 3, 6, 12, and 24 h. as well as 7 days. Calculated molecular weight M n in blue, M w in orange, M p in green, and PDI in purple was found for each time and compared to non - thiol treated Cu - AHP lignin. While 1,3 - propanedithiol is very effective at cleaving dimers and synthetic polymers at high concentrations, this specific thiol appears slower in cleaving real lignin. 0.00 1.00 2.00 3.00 4.00 5.00 6.00 0 10000 20000 30000 40000 50000 60000 70000 80000 Cu-AHP Lignin 1hr average 3hr average 6hr average 12hr average 24hr average 7 days average PDI Molecular Weight (g/mol) Cu - AHP Lignin Depolymerization with 1,3 - propanedithiol (Timecourse) Mn Mw Mp PDI 180 Figure 4. 16 Determination of percent lignin cleavage by measuring mass loss after thiol - treatment. Mass loss (measured in triplicate) of Cu - AHP lignin precipitated using H 2 SO 4 to pH 2 following reaction with either neat DTT in orange or neat BME in blue in stirring K 2 CO 3 at 100 °C for 24 h. Control precipitation reactions for this work - up method are shown in Figure 4. 30 , demonstrating that without thiol mediated depolymerization, all Cu - AHP lignin mass is retained. This m ass loss of 60 - 80% is presumably due to the formation of small fragments that are no longer insoluble at low pH demonstrating the success of thiol - mediated cleavage of lignin. Figure 4. 17 Analysis of sulfur incorporated into depolymerized Cu - AHP lignin. Elemental analysis of Cu - AH P lignin (grey) compared to thiol treated Cu - AHP lignin with neat BME in blue or neat DTT in orange and K 2 CO 3 at 100 °C for 24 h. for percent sulfur left in the polymer after cleavage as a way to determine catalyst recovery. The rise in sulfur content impl ies incorporation - site of lignin, releasing a lignin fragment, but no subsequent disulfide bond formation and release of the second fragment. Each bar graph represents duplicates. 0 20 40 60 80 100 Neat DTT Neat BME Mass Loss (%) Mass Loss of Thio - Treated Lignin 0 5 10 15 20 25 30 Lignin BME DTT % Sulfer by Mass Percent Sulfur in Lignin Samples 181 Figure 4. 18 Measurement of - OH content to determine lignin cleavage. 31 P - NMR demonstrates relative concentration of hydroxy ( - OH) groups via tagging. Changes in - OH content in lignin that has been depolymerized would suggest ether bond cleavage and - OH formation. Top: 31 P - NMR chromatograms of Cu - AHP lignin (red) and (a) Cu - AH P lignin reacted with neat BME and K 2 CO 3 at 100° C in triplicate (purple, blue, and green) and (b) Cu - AHP lignin reacted with DTT and K 2 CO 3 at 100° C in triplicate (purple, blue, and green) . Bottom: (c) Calculated values of - OH content from the spectra (top) integrated to an internal standard (cyclohexanol) with Cu - AHP lignin in grey, BME - treated Cu - AHP lignin in blue, and DTT - treated Cu - AHP lignin in orange . A significant difference between the guaiacyl - OH content of lignin and thiol treated li gnin is observed suggesting that the guaiacyl phenolic monomer is readily cleaved through thiol - mediated lignin depolymerization. 0 2 4 6 8 10 12 14 Relative 31P - NMR Integration Cu - AHP Lignin - OH Content Cu-AHP Lignin Cu-AHP Lignin + BME Cu-AHP Lignin + DTT - BME Treated Lignin 1 - BME Treated Lignin 2 - BME Treated Lignin 3 Aliphatic Syringyl Guaiacyl Hydroxy Phenol - DTT Treated Lignin 1 - DTT Treated Lignin 2 - DTT Treated Lignin 3 Aliphatic Syringyl Guaiacyl Carboxylic Acid Hydroxy Phenol Carboxylic Acid 182 C - AHP Lignin Cu - AHP Lignin + BME Cu - AHP Lignin + DTT Methoxy Methoxy Methoxy A: 74 % 16 % B: 3 % C: 8 % A: 84 % 9 % B: 2 % C: 5 % A: 77 % 13 % B: 3 % C: 8 % A A (S/G - S) A (S/G - G) A A A A (S/G - S) A (S/G - G) A A (S/G - S) A (S/G - G) A B B B B B B C C C C C C C C C C C C - S) A A - S) - S) A 183 Figure 4. 19 HSQC NMR of Cu - AHP lignin demonstrat ing changes to the polymer structure after depolymerization with thiol . Cu - AHP lignin was reacted with neat BME or neat DTT with stirring K 2 CO 3 in 100 °C for 24 h. The remaining polymer was precipitated and worked up according to Section II. (a) Cu - AHP lignin, (b) BME - treated Cu - AHP lignin, and (c) DTT - treated Cu - AHP lignin. The relative volume integrals of characteristic peaks are given. Cross peaks are color coded according each linkage assigned. Cu - AHP lignin is slightly oxidized as shown in (a) - treatment of lignin (b - - O - 4 linkage, assigned to the red cross peaks, is the only expected integrals to decrease by thiol depolymerization due to the carbonyl requirement to activate nucleophilic cleavage. Figure 4. 20 Thioacidolysis S/G ratio of monomer units in remaining lignin polymer after thiol - treatment. Cu - AHP lignin was subjected to neat thiol: BME, DTT, 1,3 - propanedithiol, or thiophenol and K 2 CO 3 at 100 °C for 24 h. The mixture was worked up with water, pH adjust ed to 2 with H 2 SO 4 , washe d, frozen, lyophilized, and submitted for thioacidolysis analysis. Y - axis: lignin is Cu - AHP lignin powder, lignin control is Cu - AHP lignin worked up in the same manner as thiol - treated lignin, BME is BME - treated Cu - AHP lignin, DTT is DTT - treated Cu - AHP lig nin, 1,3 - propanedithiol is 1,3 - propanedithiol - treated Cu - AHP lignin, and thiophenol is thiophenol - treated Cu - AHP lignin. The little change in S/G ratio indicates no preference in lignin unit cleavage for syringyl versus guaiacyl monomers. Very little diffe rence is seen between lignin and lignin control suggesting that the work - up does not change the thioacidolysis results. 0 0.5 1 1.5 2 2.5 Thiophenol 1,3-Propanedithiol DTT BME Lignin Control Lignin S/G S/G Ratio of Thiol - Treated Lignin 184 Figure 4. 21 Thioacidolysis % monomer in thiol - treated and non - treated lignin polymer. Cu - AHP lignin was subjected to neat thiol: BME, DTT, 1,3 - propanedithiol, or thiophenol and K 2 CO 3 at 100 °C for 24 h. The mixture was worked up with water, pH adjust ed to 2 with H 2 SO 4 , washed, frozen, lyophilized, and submitted for thioacidolysis analysis. Labels are as foll ows: lignin is Cu - AHP lignin powder, lignin control is Cu - AHP lignin worked up in the same manner as thiol - treated lignin, BME is BME - treated Cu - AHP lignin, DTT is DTT - treated Cu - AHP lignin, 1,3 - propanedithiol is 1,3 - propanedithiol - treated Cu - AHP lignin, a nd thiophenol is thiophenol - treated Cu - AHP lignin. The results indicate that BME - and DTT - treated lignin have decreased thioacidolysis monomer yields, potentially indicating that those monomers may have already been released during thiol - mediated depolymer - O - 4 linkages remaining to react during the analysis. No hydroxy phenol units were seen because the Cu - AHP lignin used was made from poplar which has a low H - content. 0 5 10 15 20 25 Syringyl avg Guaiacyl avg HydroxyPhenyl avg Unit in each sample (%) % Monomer Released from Lignin Polymer following Thioacidolysis Lignin Control Lignin BME DTT 1,3-propanedithiol thiophenol 185 4.6.7 Cleavage of Oxidized Lignin 0 10000 20000 30000 40000 50000 60000 70000 80000 0 2000 4000 6000 8000 10000 12000 Poplar Cu-AHP Lignin Thiol + Poplar Cu- AHP Lignin Poplar Cu-AHP Lignin (double H2O2) Thiol + Poplar Cu- AHP Lignin (double H2O2) Switchgrass Cu- AHP Lignin (double H2O2) Thiol + Switchgrass Cu-AHP Lignin (double H2O2) M w Molecular Weight (g/mol) M n & M p Molecular Weight (g/mol) Lignin Depolymerization with 2 - mercaptoethanol Mn Mp Mw (a) (b) (c) (d) (e) (f) G: 32% : 2% pBA:23% S: 36% 7% pBA 2/6 G 5/6 G 2 G 5/ 6 pBA 3/5 S 2/6 2/6 2 6 pBA 2/6 G 5/6 G 5/6 G 2 pBA 3/5 S 2/6 2/6 2 6 pBA 2/ 6 G 5/6 G 2 G 5/6 pBA 3/5 S 2/6 2/6 2 6 Poplar Cu - AHP Lignin: 1 0% H 2 O 2 Poplar Cu - AHP Lignin: 2 0% H 2 O 2 G: 3 6 % : 2% pBA: 19 % S: 3 8 % 5 % Switchgrass Cu - AHP Lignin: 20% H 2 O 2 G: 51 % : 6 % pBA: 15 % S: 4 % 25 % 186 Figure 4. 22 Susceptibility of thiol - mediated Cu - AHP lignin depolymerization when the lignin is prepared with double the amount of hydrogen peroxide and thus presumably more oxidized. Cu - AHP lignin was prepared using the standard protocols from Section II for poplar biomass and was also prepared using poplar and switchgrass with double the amount of hydrogen peroxide (20% H 2 O 2 Cu - AHP l ignin). (a) GPC chromatogram of poplar Cu - AHP lignin that was prepared with 20% H 2 O 2 (blue) overlaid with poplar Cu - AHP lignin (20% H 2 O 2 ) treated with BME in red . (b) GPC chromatogram of switchgrass Cu - AHP lignin prepared with 20% H 2 O 2 (blue) overlaid with switchgrass Cu - AHP lignin (20% H 2 O 2 ) treated with BME (red) . Visually, the molecular weight slightly decreases when treated with BME. (c) Calculated molecular weight M n in blue, M w in orange, and M p in green for (a) and (b) compared with BME - treated and non - treated poplar Cu - AHP lignin (standard method : 10% H 2 O 2 ). The M n and M p axis is on the left and the M w axis on the right for clarity. (d - f) HSQC NMR for (d) Cu - AHP poplar lignin made with the standard method (10% H 2 O 2 ), (e) Cu - AHP poplar lignin made with double the H 2 O 2 (20%), and (f) Cu - AHP switchgrass lignin made with double the H 2 O 2 (20%). The relative volume integrals of characteristic peaks are given. Cross peaks are color coded according each linkage assigned. These NMR illustrate the changes in oxidation of Cu - AHP lignin that have been prepared with extra H 2 O 2 . Little change is observed between G (bl cross peaks that represent the non - oxidized and oxidized version of guaiacyl or syringyl linkages, respectively. If oxidation were enhanced, one would expect the integrals of G (blue) and S (burgundy) to decre unoxidized linkages and slight increases in the oxidized linkages are observed suggesting that the 20% H 2 O 2 - made Cu - AHP lignin may be slightly more oxidized than when made with 10% H 2 O 2 , which in turn may slightly increase thiol - mediated depolymerization. (a) 187 Figure 4. 23 AcNH - TEMPO o xidized lignin compared to unoxidized lignin and its susceptibility to thiol - mediated depolymerization. (a) Difference in color and solubility in alkaline water of Cu - AHP lignin and oxidized Cu - AHP lignin through a 4 - acetamido - TEMPO (AcNH - TEMPO) oxidation . 49 (b - c) Calculated molecular weight M n in blue, M w in orange, M p in green, and PDI in purple for (b) Cu - AHP ligni n and AcNH - TEMPO oxidized Cu - AHP lignin compared to the BME - treated lignins; and for (c) the AcNH - TEMPO oxidized and non - oxidized Cu - AHP lignin compared to the 1,3 - propanedithiol treated lignins. 0 2 4 6 8 0 10000 20000 30000 40000 50000 60000 Cu-AHP Lignin Cu-AHP Lignin + BME Oxidized Cu-AHP Lignin Oxidized Cu-AHP Lignin + BME PDI Molecular Weight (g/mol) Mn Mw Mp PDI 0 2 4 6 8 0 20000 40000 60000 80000 Cu-AHP Lignin Cu-AHP Lignin + 1,3- propanedithiol Oxidized Cu-AHP Lignin Oxidized Cu-AHP Lignin + 1,3- propanedithiol PDI Molecular Weight (g/mol) Mn Mw Mp PDI Cu - AHP Lignin Cu - AHP Lignin Oxidized 100% soluble 70% soluble (a) (b) (c) 188 Figure 4. 24 Time course of AcNH - TEMPO oxidized Cu - AHP depolymerization using BME. AcNH - TEMPO o xidized lignin was treated with neat BME and st irring K 2 CO 3 in 100 °C for 6, 12, and 24 h. (a) GPC chromatogram of Cu - AHP lignin (blue) compared with AcNH - TEMPO oxidized Cu - AHP lignin (red) and oxidized Cu - AHP lignin treated with BME for 6 (green) , 12 (purple) , and 24 h (turquoise) . (b) Calculated molecular weight M n in blue, M w in orange, and M p in green for (a). The M n and M p axis is on the left and the M w axis on the right for clarity. Oxidation increases molecular weights but after BME - treatment, the oxidized Cu - AHP lignin decreases i n size with the ideal reaction time as 24 h. 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 0 5000 10000 15000 20000 25000 30000 35000 Cu-AHP Lignin Oxidized Cu- AHP Lignin Oxidized Cu- AHP Lignin + BME 6 hrs Oxidized Cu- AHP Lignin + BME 12 hrs Oxidized Cu- AHP Lignin + BME 24 hrs M w (g/mol) M n & M p (g/mol) Oxidized Cu - AHP Lignin Depolymerization Time Course Mn Mp Mw (a) (b) 189 0 10000 20000 30000 40000 50000 60000 0 2000 4000 6000 8000 10000 12000 Cu-AHP Lignin Thiol + Cu-AHP Lignin Oxidized Cu-AHP Lignin Thiol + Oxidized Cu-AHP Lignin M w Mol Wt. (g/mol) M n & M p Mol Wt (g/mol) Mn Mp Mw Oxidized Cu - AHP Lignin ( Cu - AHP Lignin (a) (b) (c) (d) 190 Figure 4. 25 Comparison of Cu - AHP lignin depolymerization on improved AcNH - TEMPO oxidized vs non - oxidized lignin. Cu - AHP lignin was reacted with neat 1,3 - propanedithiol and stirring K 2 CO 3 in 100 °C for 24 h. (a) GPC chromatogram of Cu - AHP lignin (blue) and thiol treated Cu - AHP lignin (red). (b) GPC chromatogram of Cu - Salt 49 (blue) , using a new and improved method than previous AcNH - TEMPO oxidation ( Figure 4. 23 and Figure 4. 24 ), and thiol treated oxidized Cu - AHP lignin (red). Visually the oxidized lignin appears t o undergo a larger decrease in molecular weight than the non - oxidized lignin. (c) Calculated molecular weights M n in blue, M w in orange, and M p in green for (a) and (b) with the M n and M p axis on the left and the M w axis on the right. The oxidized lignin h as a larger decrease in molecular weight when subjected to thiol than the unoxidized lignin. After depolymerization oxidized Cu - n , M w , and M p decreased by 49%, 36%, and 40%, respectively. (d) Both unoxidized (left) and oxidized (right) Cu - AHP lignin is completely soluble in alkaline water. a light yellow and the unoxidized lignin is a tan color. (e - f) HSQC - NMR of (e) Cu - AHP lignin and (f) Bobbitt - AHP lignin. The relative volume integrals of characteristic peaks (e) Cu - AHP Lignin (f) Oxidized Cu - AHP Lignin G: 19 % : 3 % pBA: 36 % S: 36 % 6 % G: 4 % : 17 % pBA: 27 % S: 13 % 39 % pBA 2/6 G 5/6 G 2 G 5/6 S 2/6 2/6 2 6 pBA 2/6 G 5/6 G 2 G 5/6 S 2/6 2/6 2 6 pBA 2/6 pBA 2/6 191 are given. Cross peaks are color coded according to each linkage assigned. The Cu - AHP lignin exhibits about 14% oxidation of both S and G units due to the oxidative extraction process. After oxidation of the Cu - exhibit approximately 74% and 81% oxidation levels, respectively. From these results it is clear that oxidized lignin is highly susceptible to thiol - mediated depolymerization. 4.6.8 Cleavage of Other Lignin Polymers Figure 4. 26 Comparison of Cu - AHP lignin depolymerization vs industrially made lignin from MetGen . Lignin was reacted with neat 1,3 - propanedithiol for 24 h in stirring K 2 CO 3 at 100 ° C. The Cu - AHP lignin molecular weight increased after 24 h. The MetGen crude lignin decreases in molecular weight after 24 h by 59% in M n (blue), 76% in M w (orange), 41% in PDI, and 8% in M p fractionation. This lignin is darker in color and has lower molecular weights than Cu - AHP lignin. 1,3 - propanedithiol has been shown to work well with oxidized lignin but not well with Cu - AHP lignin. This suggests that the MetGen crude biorefinery lignin may have properties such as oxidized alpha carbons that allow for successful depolymerization by a less reactive thiol. 0 10000 20000 30000 40000 50000 60000 0 2000 4000 6000 8000 10000 12000 Cu-AHP Lignin Thiol + Cu-AHP Lignin Metgen Crude Lignin Thiol + Metgen Crude Lignin M w (g/mol) M n & M p (g/mol) Mn Mp Mw 192 Figure 4. 27 GVL Lignin Depolymerization . Lignin obtained from the GVL pretreatment process 50 was reacted with neat BME at 100 ° C in stirring K 2 CO 3 for 24 h. (a) Slight molecular weight decreases were seen for M n (blue), M w (orange), M p (green), and PDI (purple). (b) GPC chromatogram of molecular weight distributions of (a). (c) GVL lignin dissolved in water and K 2 CO 3 . The undissolved p ortion seen on the bottom of the tube represents 32% of the polymer and thus, only the dissolved portion is represented in the chromatogram (b) and the calculated molecular weight values (a). These results suggest that the GVL is not an ideal substrate for thiol - mediated depolymerization. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0 500 1000 1500 2000 2500 3000 3500 GVL Lignin BME + GVL Lignin PDI Molecular Weight (g/mol) Mn Mw Mp Polydispersity (c) (b) (a) GVL Lignin 68% dissolved 193 Figure 4. 28 Comparison of various lignin depolymerization with 1,3 - propanedithiol vs BME . Lignin was reacted with neat BME (a) or neat 1,3 - propanedithiol (b) in stirring K 2 CO 3 at 100 ° C for 24 h and analyzed for molecular weight changes in M n (blue), M w (orange), M p (green), and PDI (purple). In general, Cu - AHP lignin depolymerization was scalable for both thiols with molecular weight changes of similar magnitude when reaction were run at the base case 100 mg scale compared to the 1 g scale. BME appears to be a more suitable mediator for Cu - AHP lignin depolymerization than 1,3 - propanedithiol with large molecular weight decreases for BME treated 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 10000 20000 30000 40000 50000 60000 70000 80000 BME + Cu- AHP Lignin Scale Up Cu-AHP Lignin repeat BME + Cu- AHP Lignin GVL Lignin BME + GVL Lignin Oxidized Cu-AHP Lignin BME + Oxidized Cu-AHP Lignin PDI Molecular Weight (g/mol) Lignin Depolymerization with 2 - Mercaptoethanol Mn Mw Mp Polydispersity 1 gram 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0 10000 20000 30000 40000 50000 60000 70000 80000 Prop + Cu- AHP Lignin Scale-Up Cu-AHP Lignin Prop + Cu- AHP Lignin GVL Lignin Prop + GVL Lignin Oxidized Cu-AHP Lignin Prop + Oxidized Cu-AHP Lignin PDI Molecular Weight (g/mol) Lignin Depolymerization with 1,3 - Propanedithiol Mn Mw Mp Polydispersity 1 gram ( a ) (b) 194 lignin com pared to molecular weight increases for 1,3 - propanedithiol treated lignin. GVL lignin appeared to have the same reactivity to both thiols. Oxidized Cu - AHP lignin made from the old AcNH - TEMPO method was unreactive to 1,3 - propanedithiol cleavage but showed m olecular weight decreases when reacted with BME. In general, BME allowed for larger molecular weight decreases amongst various lignin sources. Figure 4. 29 1,3 - propanedithiol depolymerization of various lignin . Lignin was reacted with neat 1,3 - propanedithiol in stirring K 2 CO 3 at 100 ° C for 24 h and analyzed for molecular weight changes in M n (blue), M w (orange), M p (green), and PDI (purple). All reactions showed molecular weight decreases at various levels except dealkaline lignin and alkaline lignin. These two lignins showed molecular weight increases which may be due to crosslinking or condensation reactions. ignosulfonate, GVL lignin, and Cu - AHP lignin show the variation in molecular weight before depolymerization and the degree of cleavage after treatment with thiol. Lignin molecular decreases are seen across multiple lignin sources, even with the use of the poorly reactive thiolate, 1,3 - propanedithiol. 0.00 2.00 4.00 6.00 8.00 10.00 0 20000 40000 60000 80000 100000 PDI Mol. Wt. (g/mol) Lignin Depolymerization with 1,3 - propanedithiol Mn Mw Mp PDI 195 4.6.9 Lignin Control Experiments Figure 4. 30 Control experiment to test lignin work - up precipitation to obtain accurate mass loss yields . Cu - AHP lignin was solubilized in NMP or water and K 2 CO 3 , filtered or not filtered, and then precipitated with different acids ( hydrochloric acid (HCl) , formic acid (FA) , acidic acid (AcOH) , or sulfuric acid (H 2 SO 4 )) to determine the recoverability of the lignin during a work - up procedure to obtain accurate mass loss yields . Sulfuric acid precipitation in water with no filtering gave quantitative recovery of lignin (0% mass loss) as indicated by no bar shown for mass loss. 0 10 20 30 40 50 60 70 80 90 100 Mass loss (%) Lignin Reprecipitation Control NMP/Filtered NMP/Unfiltered Water/Filtered Water/Unfiltered 196 Figure 4. 31 Control reactions to determine changes in molecular weight based on differences in Cu - AHP lignin extraction procedure : grind size of wood and work - up procedure of extracting the acid insoluble lignin. Cu - AHP lignin was made with either 1mM or 5mm wood gri nd size and combined with different precipitation procedures to extract the lignin: wash with cold water 3x, wash with acid 2x and cold water 1x, or the usual procedure of wash with acidic water 3x (labeled as original). These different Cu - AHP lignins were then subjected to depolymerization with neat BME and K 2 CO 3 at 100 °C for 24 h. and compared with the original procedure mentioned in Section II: Lignin Extraction Protocol. Calculated molecular weights M n in blue , M w in orange , M p in green , and PDI in pur ple between the samples indicate that wood mesh size and work - up do not effect depolymerization results. Samples were run in triplicate. 0 1 2 3 4 5 6 7 8 9 10 0 5000 10000 15000 20000 25000 1mm Poplar Cold Wash BME + 1mm Poplar Cold Wash 1mm Poplar Acid + Cold Wash BME + 1mm Poplar Acid + Cold Wash 5mm Poplar Cold Wash BME + 5mm Poplar Cold Wash 5mm Poplar Acid + Cold Wash BME + 5mm Poplar Acid + Cold Wash Original 5mm Poplar Acid Wash BME + Original 5mm Poplar Acid Wash PDI Molecular Weight (g/mol) Various Worked - Up Cu - AHP Lignin Depolymerization with BME Mn Mw Mp PDI 197 (d) (a) (c) G: 44 % : 2 % pBA: 9 % S: 39 % 6 % G: 41 % : 2 % pBA: 14 % S: 36 % 6 % pBA 2/ 6 G 5/6 G 2 G 5/6 S 2/6 2/6 2 6 pBA 2/6 G 5/6 G 2 G 5/6 S 2/6 2/6 2 6 (b) 198 0 2 4 6 8 10 12 14 16 Relative 31P - NMR Integration Cu - AHP Lignin - OH Content Cu-AHP Lignin (Standard Scale) Cu-AHP Lignin (Bucket Scale) 0 2 4 6 8 10 12 14 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 Cu-AHP Lignin (Standard) Cu-AHP Lignin (Bucket) PDI Molecular Weight (g/mol) Mn Mw Mp PDI 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 Cu-AHP Lignin BME + Cu-AHP Lignin PDI Molecular Weight (g/mol) Mn Mw Mp Polydispersity PDI Cu - AHP Lignin (Bucket Prep) Depolymerization (e) (f) (g) (h) (i) (Bucket Prep) (Bucket Prep) (Bucket Prep) (Bucket Prep) 199 Figure 4. 32 Control study to determine if scale - up of Cu - AHP lignin extraction affects its thiol - mediated depolymerization. (a) Cu - AHP reaction in a 5 - gallon bucket for production of lignin. (b) Lignin liquor from bucket scale Cu - AHP pretreatment before acid precipitation. (c - d ) HSQC 2D - NMR of (c) Cu - AHP lignin made at standard scale, (d) Cu - AHP lignin made at bucket scale. The relative volume integrals of characteristic peaks are given. Cross peaks are color coded according each linkage assigned. ( e ) - OH content calculated from 31 P - NMR of Cu - AHP lignin made at standard scale and duplicates made at bucket scale. ( f ) GPC chromatogram comparison of Cu - AHP lignin made at standard scale and bucket scale in duplicate. ( g ) Calculated molecular weight distributions of M n in blue , M w in orange , M p in green , and PDI in purple of ( f ). ( h ) GPC chromatogram of Cu - AHP lignin (bucket scale) compared to the BME - treated Cu - AHP lignin (bucket scale) reacted in neat conditions at 100 °C for 24 h. ( i ) Calculated molecular weight distributions of M n in blue , M w in orange , M p in green , and PDI in purple of ( h ). Lignin prepared in a large bucket scale had similar molecular weights as the standard scale Cu - AHP lignin but had less phenolic and aliphatic - OH s which may be due to increase s in oxidation. NMR - analysis determined very little structural changes between standard and bucket scale Cu - AHP lignin. The scaled - up Cu - AHP lignin was depolymerized using BME with decreases in M n , M w , and PDI similar to results seen with standard - scale Cu - AHP lignin . 200 Figure 4. 33 Control experiment to determine the scalability of thiol - mediated lignin depolymerization. Cu - AHP lignin reacted with neat BME or neat 1,3 - propanedithiol and K 2 CO 3 at 100 °C for 24 h. (a) GPC chromatogram of BME - treated Cu - AHP lignin at a 1 - gram scale (blue) and a 100 - milligram scale (red). (b) GPC chromatogram of 1,3 - propanedithiol - treated Cu - AHP lignin at a 1 - gram scale (blue) and a 100 - milligram scale (red). Calculated molecular wei ghts M n in blue , M w in orange , M p in green ( seen at 7.75 mL above), and PDI in purple of Cu - AHP lignin compared to (c) the 1 - g and 100 - mg scale of BME - treated lignin and (d) the 1 - g and 100 - mg scale of the 1, 3 - propanedithiol treated lignin. From these resu lts it can be stated that the thiol depolymerization strategy is scalable from 100 mg to 1 g. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 0 10000 20000 30000 40000 50000 60000 70000 Cu-AHP Lignin 1 g Cu-AHP Lignin + BME 100 mg Cu- AHP Lignin + BME PDI Molecular Weight (g/mol) Mn Mw Mp PDI 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 0 10000 20000 30000 40000 50000 60000 70000 Cu-AHP Lignin 1 g Cu-AHP Lignin + 1,3- propanedithiol 100 mg Cu-AHP Lignin + 1,3- propanedithiol PDI Molecular Weight (g/mol) Mn Mw Mp PDI (a) (b) (c) (d) 201 4.6.10 Proposed Electrochemical Cycle for the Thiol - - O - 4 Cleavage Figure 4. 34 Organocatalytic cycle envisioned for the thiol - mediated nucleophilic cleavage of - aryl ether bonds followed by a 2e - reduction to recycle the mediator. Step 1: Nucleophilic - aryl ether bond of lignin displaces a phenolic unit. Calculated activation energy in MeCN: 21.3 k cal/mol. Step 2: A second thiol attacks the thioether intermed iate, forming a disulfide, and releasing the aryl propanone fragment. Calculated activation energy in MeCN: 10.7 k cal/mol. Step 3: The thiol can be regenerated via a 2 - electron electrochemical reduction of the disulfide bond, potentially enabling a net ele ctrocatalytic lignin cleavage process. Electrochemical cell illustrated inside the catalytic cycle. E a = 21.3 E a = k cal/mol G3(MP2), MeCN E a = 10.7 C arbon P latinum e - - + e - 202 REFERENCES 203 R EFERENCES 1. Administration, U. S. E. I., International Energy Outlook 2019. 2019; Vol. IEO2019. 2. Agency, U. S. E. P., Inventory of U.S. Greenhouse Gas Emissions and Sinks: 1990 - 2017, Executive Summary. April 2019. 3. Administration, U. S. E. I., Monthly Energy Review: Environment. May 2019. 4. Zakzeski, J.; Bruijnincx, P. C. A.; Jongerius, A. L.; Weckhuysen, B. M., The Catalytic Valorization of Lignin for the Production of Renewable Chemicals. Chemical Reviews 2010, 110 (6), 3552 - 35 99. 5. Long, L. H.; Freeguar , G . F. L ow - Temperature Cleavage of Ethers . Nature 1965, 207 (4995), 403. 6. Lu, F. C.; Ralph, J., DFRC method for lignin analysys. 1. New method for beta aryl ether cleavage: Lignin model studies. Journal of Agricultural and Food Chemistry 1997, 45 (12), 4655 - 4660. 7. Lapierre, C.; Monties, B.; Rolando, C., T hioacidolysis of Ligni n - C omparison with Acidolysis . Journal of Wood Chemistry and Technology 1985, 5 (2), 277 - 292. 8. Lundquist, K.; Kirk, T. K., A cid Degradation of Lignin .4. A nalysis of Lignin Acidolysis P roducts by Gas Chromatography , U sing T rimethylsilyl D erivatives . Act a Chemica Scandinavica 1971, 25 (3), 889. 9. Oae, S., Organic sulfur chemistry: Structure and mechanisms . CRC Press: Boca Raton, Florida, 1991. 10. Rolando, C.; Monties, B.; Lapierre, C., Methods in Lignin Chemistry. In Wood Science , Timell, T. E., Ed. Spr inger - Verlag: Berlin, 1992 , 334 - 349. 11. Wang, X.; Rinaldi, R., Solvent Effects on the Hydrogenolysis of Diphenyl Ether with Raney Nickel and their Implications for the Conversion of Lignin. Chem S us C hem 2012, 5 (8), 1455 - 1466. 12. Li, C.; Zhao, X.; Wang, A.; Huber, G. W.; Zhang, T., Catalytic Transformation of Lignin for the Production of Chemicals and Fuels. Chemical Reviews 2015, 115 (21), 11559 - 11624. 13. Sun, Z. H.; Fridrich, B.; de Santi, A.; Elangovan, S.; Barta, K., Bright Side of Lignin Depoly merization: Toward New Platform Chemicals. Chemical Reviews 2018, 118 (2), 614 - 678. 204 14. Laskar, D. D.; Tucker, M. P.; Chen, X.; Helms, G. L.; Yang, B., Noble - metal catalyzed hydrodeoxygenation of biomass - derived lignin to aromatic hydrocarbons. Green Ch emistry 2014, 16 (2), 897 - 910. 15. Shuai, L.; Amiri, M. T.; Questell - Santiago, Y. M.; Heroguel, F.; Li, Y.; Kim, H.; Meilan, R.; Chapple, C.; Ralph, J.; Luterbacher, J. S., Formaldehyde stabilization facilitates lignin monomer production during bio mass depolymerization. Science 2016, 354 (6310), 329 - 333. 16. Amiri, M. T.; Dick, G. R.; Questell - Santiago, Y. M.; Luterbacher, J. S., Fractionation of lignocellulosic biomass to produce uncondensed aldehyde - stabilized lignin. Nature Protocols 2019, 14 ( 3), 921 - 954. 17. Lan, W.; Amiri, M. T.; Hunston, C. M.; Luterbacher, J. S., Protection Group Effects During alpha,gamma - Diol Lignin Stabilization Promote High - Selectivity Monomer Production. Angewandte Chemie - International Edition 2018, 57 (5), 1356 - 1360. 18. Lan, W.; d e Bueren, J. B.; Luterbacher, J. S., Highly Selective Oxidation and Depolymerization of alpha,gamma - Diol - Protected Lignin. Angewandte Chemie - International Edition 2019, 58 (9), 2649 - 2654. 19. Cyr, A.; Chiltz, F.; Jeanson, P.; Martel, A.; Brossard, L.; Lessard, J.; Menard, H., Electrocatalytic hydrogenation of lignin models at Raney nickel and palladium - based electrodes. Canadian Journal of Chemistry 2000, 78 (3), 307 - 315. 20. Lam, C. H.; Lowe, C. B.; Li, Z.; Longe, K. N.; Rayburn, J. T.; Caldwell, M. A.; Houdek, C. E.; Maguire, J. B.; Saffron, C. M.; Miller, D. J.; Jackson, J. E., Electrocatalytic upgrading of model lignin monomers with earth abundant metal electrodes. Green Chemis try 2015, 17 (1), 601 - 609. 21. Li, H.; Fang, Z.; Smith, R. L., Jr.; Yang, S., Efficient valorization of biomass to biofuels with bifunctional solid catalytic materials. Progress in Energy and Combustion Science 2016, 55 , 98 - 194. 22. Garedew, M.; Young - F arhat, D.; Jackson, J. E.; Saffron, C. M., Electrocatalytic Upgrading of Phenolic Compounds Observed after Lignin Pyrolysis. A CS Sustainable Chemistry & Engineering 2019, 7 (9), 8375 - 8386. 23. Garedew, M.; Young - Farhat, D.; Bhatia, S.; Hao, P.; Jackso n, J. E.; Saffron, C. M., Electrocatalytic cleavage of lignin model dimers using ruthenium supported on activated carbon cloth. Sustainable Energy & Fuels 2020, 4 (3), 1340 - 1350. 24. Lam, C. H.; Das, S.; Erickson, N. C.; Hyzer, C. D.; Garedew, M.; And erson, J. E.; Wallington, T. J.; Tamor, M. A.; Jackson, J. E.; Saffron, C. M., Towards sustainable hydrocarbon fuels with biomass fast pyrolysis oil and electrocatalytic upgrading. Sustainable Energy & Fuels 2017, 1 (2), 258 - 266. 205 25. Zhou, Y.; Klinger, G. E.; Hegg, E. L.; Saffron, C. M.; Jackson, J. E., Multiple Mechanisms Mapped in Aryl Alkyl Ether Cleavage via Aqueous Electrocatalytic Hydrogenation over Skeletal Nickel. Journal of the American Chemical Society 2020, 142 (8), 4037 - 4050. 26. Lancefiel d, C. S.; Ojo, O. S.; Tran, F.; Westwood, N. J., Isolation of Functionalized Phenolic Monomers through Selective Oxidation and C - O Bond Cleavage of the beta - O - 4 Linkages in Lignin. Angewandte Chemie - International Edition 2015, 54 (1), 258 - 262. 27. Nguyen , J. D.; Matsuura, B. S.; Stephenson, C. R. J., A Photochemical Strategy for Lignin Degradation at Room Temperature. Journal of the American Chemical Society 2014, 136 (4), 1218 - 1221. 28. Rahimi, A.; Ulbrich, A.; Coon, J. J.; Stahl, S. S., Formic - acid - i nduced depolymerization of oxidized lignin to aromatics. Nature 2014, 515 (7526), 249 - 252. 29. Hoyle, C. E.; Lowe, A. B.; Bowman, C. N., Thiol - click chemistry: a multifaceted toolbox for small molecule and polymer synthesis. Chemical Society Reviews 2010, 39 (4), 1355 - 1387. 30. Cleland, W. W., D ithiothreitol N ew Protective Reagent for SH G roups . Biochemistry 1964, 3 (4), 480. 31. Gall, D. L.; Kim, H.; Lu, F.; Donohue, T. J.; Noguera, D. R.; Ralph, J., Stereochemical Features of Glutathione - dependent En zymes in the Sphingobium sp Strain SYK - 6 beta - Aryl Etherase Pathway. J. Biol. Chem. 2014, 289 (12), 8656 - 8667. 32. Kamimura, N.; Takahashi, K.; Mori, K.; Araki, T.; Fujita, M.; Higuchi, Y.; Masai, E., Bacterial catabolism of lignin - derived aromatics: New findings in a recent decade: Update on bacterial lignin catabolism. Environmental Microbiology Reports 2017, 9 (6), 679 - 705. 33. Pereira, J. H.; Heins, R. A.; Gall, D. L.; McAndrew, R. P.; Deng, K.; Holland, K. C.; Donohue, T. J.; Noguera, D. R. ; Simmons, B. A.; Sale, K. L.; Ralph, J.; Adams, P. D., Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin beta - Aryl Ether Cleavage Pathway from Sphingobium sp.SYK - 6. Journal of Biological Chemistry 2016, 291 (19), 1 0228 - 10238. 34. Helmich, K. E.; Pereira, J. H.; Gall, D. L.; Heins, R. A.; McAndrew, R. P.; Bingman, C.; Deng, K.; Holland, K. C.; Noguera, D. R.; Simmons, B. A.; Sale, K. L.; Ralph, J.; Donohue, T. J.; Adams, P. D.; Phillips, G. N., Jr., Stru ctural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of beta - Aryl Ether Bonds in Lignin. Journal of Biological Chemistry 2016, 291 (10), 5234 - 5246. 35. Kontur, W. S.; Olmsted, C. N.; Yusko, L. M.; Niles, A. V.; Walters, K. A.; Beebe, E. T.; Vander Meulen, K. A.; Karlen, S. D.; Gall, D. L.; Noguera, D. R.; Donohue, T. J., A heterodimeric glutathione S - transferase that stereospecifically breaks lig nin's (R) - aryl ether bond reveals the diversity of bacterial - etherases. Journal of Biological Chemistry 2019, 294 (6), 1877 - 1890. 206 36. Klinger, G. E.; Zhou, Y.; Hao, P.; Robbins, J.; Aquilina, J. M.; Jackson, J. E.; Hegg, E. L., Biomimetic Reductive C leavage of Keto Aryl Ether Bonds by Small - Molecule Thiols. Chem S us C hem 2019, 12 (21), 4775 - 4779. 37. Gall, D. L.; Kontur, W. S.; Lan, W.; Kim, H.; Li, Y.; Ralph, J.; Donohue, T. J.; Noguera, D. R., In Vitro Enzymatic Depolymerization of Lignin with R elease of Syringyl, Guaiacyl, and Tricin Units. Applied and Environmental Microbiology 2018, 84 (3). 38. Kontur, W. S.; Bingman, C. A.; Olmsted, C. N.; Wassarman, D. R.; Ulbrich, A.; Gall, D. L.; Smith, R. W.; Yusko, L. M.; Fox, B. G.; Noguera, D. R.; Coon, J. J.; Donohue, T. J., Novosphingobium aromaticivorans uses a Nu - class glutathione S - transferase as a glutathione lyase in breaking the beta - aryl ether bond of lignin. Journal of Biological Chemistry 2018, 293 (14), 4955 - 4968. 39. Badalyan, A.; Stahl, S. S., Cooperative electrocatalytic alcohol oxidation with electron - proton - transfer mediators. Nature 2016, 535 (7612), 406 - 410. 40. Rafiee, M.; Alherech, M.; Karlen, S. D.; Stahl, S. S., Electrochemical Aminoxyl - Mediated Oxidation of Primary Alc ohols in Lignin to Carboxylic Acids: Polymer Modification and Depolymerization. Journal of the American Chemical Society 2019, 141 (38), 15266 - 15276. 41. Kishimoto, T.; Uraki, Y.; Ubukata, M., Synthesis of beta - O - 4 - type artificial lignin polymers and thei r analysis by NMR spectroscopy. Organic & Biomolecular Chemistry 2008, 6 (16), 2982 - 2987. 42. Kishimoto, T.; Uraki, Y.; Ubukata, M., Easy synthesis of beta - O - 4 type lignin related polymers. Organic & Biomolecular Chemistry 2005, 3 (6), 1067 - 1073. 43. Gall , D. L.; Ralph, J.; Donohue, T. J.; Noguera, D. R., A Group of Sequence - Related Sphingomonad Enzymes Catalyzes Cleavage of beta - Aryl Ether Linkages in Lignin beta - Guaiacyl and beta - Syringyl Ether Dimers. Environmental Science & Technology 2014, 48 (20), 12454 - 12463. 44. Gall, D. L.; Kim, H.; Lu, F.; Donohue, T. J.; Noguera, D. R.; Ralph, J., Stereochemical Features of Glutathione - dependent Enzymes in the Sphingobium sp Strain SYK - 6 beta - Aryl Etherase Pathway. Journal of Biological Chemistry 2014, 289 (12), 8656 - 8667. 45. Li, Z. L.; Bansal, N.; Azarpira, A.; Bhalla, A.; Chen, C. H.; Ralph, J.; Hegg, E. L.; Hodge, D. B., Chemical and structural changes associated with Cu - catalyzed alkaline - oxidative delignification of hybrid poplar. Biotechnology f or Biofuels 2015, 8 , 12. 46. Bhalla, A.; Bansal, N.; Stoklosa, R. J.; Fountain, M.; Ralph, J.; Hodge, D. B.; Hegg, E. L., Effective alkaline metal - catalyzed oxidative delignification of hybrid poplar. Biotechnology for Biofuels 2016, 9 , 10. 47. Li, Z. L.; Bansal, N.; Azarpira, A.; Bhalla, A.; Chen, C. H.; Ralph, J.; Hegg, E. L.; Hodge, D. B., Chemical and structural changes associated with Cu - catalyzed alkaline - oxidative delignification of hybrid poplar. Biotechnology for Biofuels 2015, 8 , 12. 207 48. Bhalla, A.; Bansal, N.; S toklosa, R. J.; Fountain, M.; Ralph, J.; Hodge, D. B.; Hegg, E. L., Effective alkaline metal - catalyzed oxidative delignification of hybrid poplar. Biotechnology for Biofuels 2016, 9 . 49. Rahimi, A.; Azarpira, A.; Kim, H.; Ralph, J.; Stahl, S. S., Che moselective Metal - Free Aerobic Alcohol Oxidation in Lignin. Journal of the American Chemical Society 2013, 135 (17), 6415 - 6418. 50. Luterbacher, J. S.; Rand, J. M.; Alonso, D. M.; Han, J.; Youngquist, J. T.; Maravelias, C. T.; Pfleger, B. F.; Dumesic , J. A., Nonenzymatic Sugar Production from Biomass Using Biomass - Derived gamma - Valerolactone. Science 2014, 343 (6168), 277 - 280. 51. Kishimoto, T.; Uraki, Y.; Ubukata, M., Chemical synthesis of beta - O - 4 type artificial lignin. Organic & Biomolecular Chem istry 2006, 4 (7), 1343 - 1347. 52. Kim, H.; Ralph, J., Solution - state 2D NMR of ball - milled plant cell wall gels in DMSO - d(6)/pyridine - d(5). Organic & Biomolecular Chemistry 2010, 8 (3), 576 - 591. 53. Mansfield, S. D.; Kim, H.; Lu, F.; Ralph, J., Whole plant cell wall characterization using solution - state 2D NMR. Nature Protocols 2012, 7 (9), 1579 - 1589. 54. Kalami, S.; Arefmanesh, M.; Master, E.; Nejad, M., Replacing 100% of phenol in phenolic adhes ive formulations with lignin. Journal of Applied Polymer Science 2017, 134 (30). 55. Harman - Ware, A. E.; Foster, C.; Happs, R. M.; Doeppke, C.; Meunier, K.; Gehan, J.; Yue, F.; Lu, F.; Davis, M. F., A thioacidolysis method tailored for higher - throug hput quantitative analysis of lignin monomers. Biotechnology Journal 2016, 11 (10), 1268 - 1273. 56. Gall, D. L.; Ralph, J.; Donohue, T. J.; Noguera, D. R., A Group of Sequence - Related Sphingomonad Enzymes Catalyzes Cleavage of beta - Aryl Ether Linkages in Lignin beta - Guaiacyl and beta - Syringyl Ether Dimers. Environ. Sci. Technol. 2014, 48 (20), 12454 - 12463. 57. Rinaldi, R.; Jastrzebski, R.; Clough, M. T.; Ralph, J.; Kennema, M.; Bruijnincx, P. C. A.; Weckhuysen, B. M., Paving the Way for Lignin Valoris ation: Recent Advances in Bioengineering, Biorefining and Catalysis. Angewandte Chemie - International Edition 2016, 55 (29), 8164 - 8215. 208 Chapter 5. Electrochemical Recycling of Disulfides The use of diffusible r edox mediators for lignin depolymer ization offers the advantage of a closed system where the mediator can be recycled using electrochemistry. Using this approach, - effective organocatalytic method t o produce small molecules for renewable chemicals and fuels. We have previously developed a redox mediator process that uses small molecule thiols to cleave the keto - aryl ether bond in polymeric lignin resulting in a disulfide b y product. To turn over the m ediator, parameters were needed to optimize the reduction of the disulfide in an electrochemical cell. Through the use of cyclic voltammetry and chronoamperometry studies we have found that the disulfide reduction is electrochemically irreversible due to c hemical irreversibility under our reaction conditions. Furthermore, we have obtained substantial evidence that this chemical irreversibility is due to background reactions from thiolate/thiol radical products and not through adsorption of any thiol species on the cathode. These studies lay the groundwork for electrochemical parameters needed to make thiol - mediated lignin cleavage a sustainable and cost - effective technology. 5.1 Introduction The reductive cleavage of disulfides is a well - documented process t hat can be achieved in numerous ways. Most reducing agents are capable of this reduction including metal hydrides ( i.e. , LiAlH 4 , NaBH 4 ), tricoordinate phosphorus compounds ( i.e. , Ph 3 P), formamidinesulfinic acid, or other thiols or selenols under basic conditions ( i.e. , NaSH, Na 2 S 2 O 4 , HSe - ). Electron transfer processes can also reduce disulfides through photolysis (aided with a radical initiator), SET mediators under acidic conditions ( i.e. , Na - Sn and HCl or Zn - HCl), or electrochemistry. 1 209 Electrochemical reductions have several advantages over chemical reduction methods with the most obvious being that the activation energy is supplied thr ough a n applied potential rather than thermally or through high energy reagents . 2 Adjustments in applied potential can efficiently help control the selectivity and the rate of the reaction . 2 For instance, in the reduction of disulfides, potentials applied at or below (less negative) the potential energy of the disulfide LUMO will not allow for electron tr ansfer. Increasing the negative potential of the electrode to a higher energy (more negative) than the disulfide LUMO allows an electron to be transferred. This is demonstrated in Figure 5. 1 - top. Similarly, thiolate oxidation will not occur when the electrode potential is at or below (less positive) the potential energy of the thiolate HOMO. Increasing the positive potential of the electrode to a higher energy (more posit ive) than the thiolate HOMO allows electron transfer from the thiolate ( Figure 5. 1 - bottom). Figure 5. 1 Schematic of electron transfer from an applied potential. Electrochemical reduction requires the potential applied by an electrode be more negative (higher in energy) than the potential 210 energy of the LUMO of the analyte. Similarly, electrochemical oxidation requires that the potential applied by an electrode be more positive (higher in energy) than the potential energy of the HOMO of the analyte. This scheme was adapted from Dempsey et al. 3 Electrochemical disulfide reduction involves a 2 - electron transfer. 4 There is much debate on the mechanism of the first step. Addition of the first electron to the disulfide m ay proceed through a concerted mechanism, whereby fragmentation occurs producing a thiyl radical and a thiolate, or a stepwise mechanism, where the electron is transferred to the disulfide to form a radical anion before fragmenting to the thiyl radical and thiolate. 5 Regardless of this mechani stic detail , the second step involves the addition of a second electron to the thiyl radical to form another thiolate. The overall process results in the production of two thiolates as shown in Figure 5. 2 . Figure 5. 2 Mechanism of disulfide reductions through a 2e - addition. The additional of one electron to a disulfide results in the production of a thiolate and thiyl radical. This step may be concerted or stepwise. Addition of a second electron to the thiyl radical produces a second thiolate. This scheme is adapted from Gla ss et al 4 and Antonello et al 5 . When choosing a process for disu lfide reduction, scale - up, separations, and side reactions with the reducing agent must be considered. Electrochemical reductions may circumvent many of these limitations due to operations under mild conditions ( i.e. , room temperature and ambient pressure), heterogeneous electrodes, and optimization of potentials that avoid reduction potentials that overlap with other substrates. Electrochemical reduction of disulfide has even been the method of choice for many industrial processes including the re duction of cystine to cysteine for precursors in the food, pharmaceutical, and personal - care industries. 6 Thus, disulfide/thiolate redox mediators 211 have an advantage to the chemistries they perform in that they ca n be turned over electrochemically allowing for an efficient organocatalytic system that can be applied to real world applications. It is for these reasons that we have developed a thiolate redox catalyst for the production of fuels and chemicals from lig nin. Thiolates are potent nucleophiles but have previously been known to only participate in S N 2 reactions with alkyl halides and sulfonates. 7 We have fou nd in our work that thiolates can nucleophilically displace phenols when activated by a neighboring ketone under alkaline polar aprotic conditions. 8 This is advantageous to the bioenergy area where energy dense polymeric lignin from plants can be converted to fuels and chemicals if mean s to break the phenolic linkages that make up the lignin are addressed. The b y product from this thiol - mediated keto - aryl ether reductive cleavage (phenolic displacement) is a disulfide formed from two separate nucleophilic displacements. Additional of 2e - to the disulfide would allow for catalytic turnover as illustrated in Figure 5. 3 . Figure 5. 3 Simplified cat alytic cycle for the thiol - mediated keto - aryl ether cleavage reaction. Two thiolates are used in the nucleophilic substitution and reductive elimination reactions to cleave keto - aryl ether bonds. The oxidized catalyst is then reduced through a 2 - electron transfer to form two thiolate molecules. We hypothesized that electrochemistry could provide these two electrons for disulfide cleavage, as described above. To test this hypothesis, preliminary studies were done to probe the 212 electrochemical behavior of dis ulfides in the reaction conditions needed to perform the keto - aryl ether cleavage. This electrochemical reduction would require several parameters: 1) Potentials lower than that of direct electrolysis o f the lignin dimer substrate, which would otherwise reduc e the ketone to an alcohol and inhibit thiol - mediated S N 2 cleavage. 2) An electrolytic system in polar aprotic solvents with the presence of insoluble base and temperature ~80 - 100 ° C. 3) An electrolyte that does not interact with the lignin substrate or thiol mediator. 4) Electrodes that do not adsorb the thiol. This electrolytic system is shown in Figure 5. 4 as a 1 - compartment cell but could also be envisioned as a 2 - compartment system. Figure 5. 4 Schematic of the electrochemically driven catalyst turnover during lignin dimer cleavage. Thiolates cleave keto - aryl ethers resulting in disulfide formation and the production of two monom ers. One electron addition to the disulfide results in the formation of a thiolate and a thiyl radical. Another one electron addition to the thiyl radical forms a second thiolate which then proceeds again through the keto - aryl ether cleavage reaction. 213 5.2 Optimization 5.2.1 Thiols Electrochemical disulfide reductions have been reported since the 1930s. 9 In many cases, these processes were p erformed under acidic aqueous conditions. Our previous work 8 indicates that thiol - mediated ether cleavage is best suited in polar aprotic solvents such as MeCN with an insoluble pow d ered base. Furthermore, optimization of the ether cleavage suggests small thiols with p K as of ~9, such as BME and DTT, are more suitable than bulkier thiols with lower p K as, like thiophenol. However, the current literature on disulfide electrochemical reduction in polar aprotic solvents is dominated by the thiophenol/diphenyl disulfide pair. 10 We sought to replicate these thiophenol experiments using the less bulky thiols, DTT and BME, to obtain redox potentials under our reaction conditions. The redox potential for DTT is - 0.33 V at pH 7 under aqueous conditions 11 and that for BME is - 0.26 V at pH 7 12 under aqueous conditions . There is much debate on the accuracy of these values and what the best method is to obtain accurate values. 11, 13, 14 For example, Corwin et al , suggests that the use of Pt wire as an electrode results in sluggish potentials (for cysteine) which may be due to formation of Pt thiol compounds on the surface of the electrode. 13 Both gold and mercury electrodes also tend to strongly adsorb thiol s to their surface, causing fluctuation s in thiol/disulfide potentials. 4, 13 Similarly, thiophene was found to stick to glassy carbon electrodes in acetonitrile within four CV scans. 15 Not only can thiol adsorption to the electrode alter the potential but potentials may also be shifted by solvent, electrode material, and electrolyte. 3 Due to these key points, it is important to experimentally determine a reduction potential that is relev ant to the ether cleavage conditions of interest for lignin upgrading . 214 Figure 5. 5 CV of O - DTT and O - BME using Ag/AgBr and TBAB. Cyclic voltammetry for O - BME and O - DTT performed in MeCN result s in differences in electrochemical reversibility with large E p . The CV of O - BME has been labeled with the reduction peak of interest. Electrolyte reduction potential is labeled. The CV of the disulfides, O - DTT (oxidized DTT) and O - BME (oxidized BME) , were obtained using a glassy carbon working electrode and Ag/AgBr in MeCN as shown in ( Figure 5. 5 ). The reduction peak of O - BME at ~1.9 V is not symmetrical to the oxidatio n peak at ~0.6 indicating that there are additional processes occu r ring during reduction at the electrode. The O - DTT CV exhibited a similar trend. Generally, for 2 - e lectron transfer steps, if the 2 nd electron transfer is more thermodynamically favored, the n the voltammogram will show an overlap of the 2 - step process where there is only 1 reduction potential. If the 1 st step becomes more thermodynamically favored, then the 2 electrochemical steps will separate into their own reduction peaks. 3 This e ffect will also be observed on the anodic wave. In the case of O - BME and O - DTT, the reduction peak appears to -0.2 0.0 0.2 0.4 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 Current (mA) E (V) vs. Ag/AgBr O - BME -0.2 0.0 0.2 0.4 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 Current (mA) E (V) vs. Ag/AgBr O - DTT Analyte Concentration: 10 mM Electrolyte conc always 50 mM Electrolyte: NBu 4 Br Working: Glassy Carbon Counter: Platinum Reference: Ag/AgBr in MeCN Scan rate: 100 mV/s Solvent: MeCN Disulfide Reduction Electrolyte Reduction 215 have multiple species and/or electron transfer processes taking place but the lack of symmetry in the anodic wave suggests that this is not due to its 2 - electro n transfer steps , but rather a b y product of another species. The v oltammogram also exhibits a wide peak to peak separation ( E p ) between the anodic and cathodic peak potentials, for both O - BME and O - DTT. When this difference is greater than 57 mV at RT it indicates electrochemical irreversibility. 3 Furthermore, after - 1.2 V was applied to O - BME for 2 h, no reduction products were observed b y HPLC or NMR analysis ( Figure 5. 6 ). Changes in solvent to aqueous conditions ( Figure 5. 26 and Figure 5. 27 ) or a change of electrolyte and reference electrode ( Figure 5. 25 ) were unsuccessful in decreasing the E p or producing a reversible process, and in fact contributed to additional anodic and cathodic peaks. Figure 5. 6 Chronoamperometry of O - BME at - 1.2 V. An applied voltage of - 1.2 V to O - BME did not reduce the substrate. Secondary analysis using NMR showed no reduced products. Due to the above challenges and the substantial literature surrounding oxidized thiophenol in MeCN, diphenyl disulfide was used in all further studies as a m odel for electrochemical optimization for the reduction of disulfides in MeCN. Cyclic voltammograms recorded with diphenyl disulfide in acetonitrile, unlike O - BME or O - DTT, showed clear anodic and cathodic peak s ( Figure 5. 7 - red trace). However, a large E p was still observed with large differences in 0 5 10 15 0 20 40 60 80 100 120 Concentration (mM) Time (min) O - BME Analyte Concentration: 10 mM Electrolyte conc always 50 mM Electrolyte: NBu 4 ClO 4 Working: Glassy Carbon Counter: Platinum Reference: Ag/AgCl in Water Voltage: - 1.2V Solvent: MeCN 216 anodic/cathodic peak current suggesting irreversible features associated with thiophenol/diphenyl disulfide that requires further optimization. 5.2.2 Choice of material The large E p is a consequence of inefficient electron shuttling that may be due to a number of reasons. To improve this efficiency and thereby decrease E p , several parameters can be optimized includ ing the electrolyte, electrode, and solvent. Our initial screening of diphenyl disulfide used glassy carbon as the working electrode (cathode). We screened several other working electrodes, shown in Figure 5. 7 , and found that the glassy carbon (red) was actually the best material to reduce the disulfide with clear reductions at - 1.2 V vs Ag/AgBr. However, the difference in anodic/cathodic peak current indicate some form of chemical irreversibility and would need to be addressed after further parameter optimizations. Figure 5. 7 Working electrode comparison for the CV of diphenyl disulfide. Glassy carbon was found to be the best electrode for the reduction of diphenyl disulfide at the cathode with large single anodic and cathodic peak currents. Literature reports document the most commonly used electrolyte in MeCN as tetrabutylammonium bromide (TBAB or NBu 4 Br ). We opted to change the anion to gauge any e ffects to the reduction potential. Figure 5. 8 illustrates that changes to the anion had a minimal -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 -2 -1.5 -1 -0.5 0 0.5 1 Current (mA) E (V) vs. Ag/AgBr Electrode Comparison Carbon Tow Platinum Disk Platinum Cylinder Glassy Carbon Analyte: Diphenyl Disulfide Analyte Concentration: 10 mM Electrolyte conc always 50 mM Electrolyte: NBu 4 Br Working: Change Counter: Platinum Reference: Ag/ AgBr in MeCN Scan rate: 100 mV/s Solvent: MeCN 217 effect on diphenyl disul fide redox potentials. For this reason, we moved forward with all further experiments using TBAB (light blue). Figure 5. 8 Electrolyte comparison for the CV of diphenyl disulfide. Tetrabutylammonium (NBu 4 or TBA ) is a common organic electrolyte cation for MeCN electroch emistry. The anion had minimal effect on diphenyl disulfide reduction/oxidation. Though the reduction peak current showed some irreversibility, we wanted to confirm if our optimized electrolyte and working electrode in MeCN could reduce diphenyl disulfide to thiophenol. We chose to apply - 1.5 V , slightly higher than the reduction potential, to make sure we had enough potential to cleave the disulfide. Using NMR to detect any thiophenol, we compared the reaction mixture after 5 h of chronoamperometry to pure thiophenol and diphenyl disulfide and saw no evidence of the reduced product ( Figure 5. 9 ). Furthermore, we used CV as a way to measure concentration of the analyte usi ng a concentration gradient and compared it to diphenyl disulfide after chronoamperometry. The reduction peak did increase slightly, and the oxidation peak nearly disappeared after applying - 1.5 V for 5 h. This equated to about a 1.3 mM concentration of th e reduced product, thiophenol. Figure 5. 10 illustrates the small changes from the reduced product compared to the starting disulfide with thiophenol as a reference. -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 -2 -1 0 1 Current (mA) E (V) vs. Ag/AgBr Electrolyte Comparison TBA-HFP TBA-TPB TBA-TFB TBA-P TBA-B Analyte: Diphenyl Disulfide Analyte Concentration: 10 mM Electrolyte: Change Electrolyte conc always 50 mM Working: Glassy Carbon Counter: Pla tinum Reference: Ag/AgBr in MeCN Scan rate: 100 mV/s Solvent: MeCN 218 Figure 5. 9 NMR detection from diphenyl disulfide reduction at 1.5 V. Diphenyl disulfide was analyzed for reduction after applying - 1.5 V for 5 h. No thiophenol peaks were observed through N MR analysis. The mediocre reductions seen from the disulfide chronoamperometry studies, the large anodic/cathodic peak current difference, and the large E p indicated that the irreversibility needed to be addressed in order to allow for an efficient disulf ide cleavage. A) Thiophenol B) Diphenyl disulfide Analyte Concentration: 10 mM Electrolyte conc always 50 mM Electrolyte: NBu 4 Cl Working: Carbon Tow Counter: Platinum Reference: Ag/AgCl in MeCN Voltage: - 1.5 V Solvent: MeCN C) Diphenyl disulfide after chronoamperometry 219 Figure 5. 10 CV Quantification of diphenyl disulfide reduction. A - 1.5 V potential was applied to diphenyl disulfide for 5 h and the peak current was compared to a concentration gradient of diphenyl disulfide as an external standard. The ox idation peak disappeared while the reduction peak increased by approximately 1.3 mM. 5.3 Reversibility Figure 5. 1 Figure 5. 11 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 -2 -1 0 1 Current (mA) E (V) vs. Ag/AgBr CV Quantification Diphenyl disulfide Thiophenol Diphenyl disulfide after chronoamperometry Analyte Concentration: 10 mM Electrolyte conc always 50 mM Electrolyte: NBu 4 Br Working: Glassy Carbon Counter: Platinum Reference: Ag/AgBr in MeCN Scan rate: 100 mV/s (positive scan first) Solvent: MeCN 220 Figure 5. 11 Diagram of the disulfide redox cycle in a reversible and irreversible system. A disulfide - thiolate redox schematic is shown with both reversible and irreversible processes outlined. In the case of diphenyl disulfide reduction, Figure 5. 12 , it is clear that the E p is > 57 mV indicating electrochemical irreversibility. 16 Furthermore, the anodic peak current is one sixth the cathodic peak current indicating either adsorption or side reactions. Due to the symmetry of the single peaks at the anodic and cathodic potential we decided that side reaction were less likely, with the absence of addition peaks, and therefore focused on any adsorption to the electrode that would account for the both the chemical/adsorption ir reversibility (y - axis) or the electrochemical irreversibility (x - axis). Figure 5. 12 CV of diphenyl disulfide with irreversible features. The reduction/oxidation of diphenyl disulfide shows both electrochemical irreversibility (large E p on the x - axis) and chemical irreversibility (large difference in anodic and cathodic peak currents on the y - axis). -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 -2 -1 0 1 Current (mA) E (V) vs. Ag/AgBr Glassy Carbon CV 1760mV (57mV is reversible) 0.37mA - 0.06mA Electrolyte: NBu 4 Br Analyte: Diphenyl Disulfide 221 5.3.1 Adsorption Determination of adsorption of an analyte on an electrode can be accomplished using several equations. T he Randles - Sevcik equation can be plotted in two ways to show linear dependence when there are adsorbed species versus when there are freely diffusing species. Using EQN 5. 1 , if a linear dependence is found whe n plotting peak current (i p ) with the scan rate ( v ) , this suggests the presence of adsorbed species. However, rearranging the Randles - Sevcik equation to EQN 5. 2 to plot i p with the square root of v will show a linear relationship when the reaction is an electrochemically reversible with freely diffusing redox species. 3 EQN 5. 1 EQN 5. 2 where n is the number of electrons for the disulfide reduction ( n is the area of electrode (cm 2 ), C 0 is the disulfide concentration (mol cm - 3 ), v is the scan rate ( V s - 1 ), D o is the diffusion constant of the disulfide (cm 2 s - 1 ), R is the ideal gas constant, T is the temperature , and * is the surface coverage of the adsorbed species (mol cm - 2 ) . From the graph, peak current increases linearly with v 1/2 ( Figure 5. 13 ) with diphenyl disulfide. This indicates that the redox species are freely diffusing in solution. A freely diffusing analyte suggests no adsorption. 222 Figure 5. 13 Peak current as a functio n of the v 1/2 to determine adsorption by the Randles - Sevcik e quation . The peak current increases linearly with v 1/2 suggesting that any diphenyl disulfide redox species freely diffuse through solution rather than adsorbed to the electrode surface. Using the Randles - Sevcik equation, we can also plot peak current with respect to concentration to give insight into electrode adsorption. A linear correlation, as seen in Figure 5. 14 , suggests mass transport of the analyte proceeds through normal diffusion as scan rate is increased and it can be assumed that there is no adsorption of the analyte of the electrode. Figure 5. 14 Peak current as a function of concentration to det ermine adsorption by the Randles - Sevcik e quation . The peak current increases linearly with concentration for diphenyl disulfide suggesting that diffusion of the analyte proceeds unimpeded by adsorption to the electrode. y = 1.025x + 0.0453 R² = 0.9936 0 0.2 0.4 0.6 0.1 0.15 0.2 0.25 0.3 0.35 Peak Current (mA) v 1/2 ((V/s) 1/2 )) Scan Rate versus Peak Current y = 0.0303x - 0.0312 R² = 0.9982 0 0.2 0.4 0.6 5 10 15 20 Peak current (mA) Concentration (mM) Concentration Dependence Analyte: Diphenyl Disulfide Analyte Concentration: 10 mM Electrolyte conc always 50 mM Electrolyte: NBu 4 Br Working: Glassy Carbon Counter: Platinum Reference: Ag/AgBr in MeCN Scan rate: Change Solvent: MeCN Analyte: Diphenyl Disulfide Analyte Concentrat ion: Change Electrolyte conc always 50 mM Electrolyte: NBu 4 Br Working: Glassy Carbon Counter: Platinum Reference: Ag/AgBr in MeCN Scan rate: 100 mV/s Solvent: MeCN 223 A separate equation, the integrated Cottrell equation, may also be used to determine if there is any analyte adsorption on the electrode. In this method, chronoamperometry instead of cyclic voltammetry is used to evaluate adsorption. The current is monitored as the potential is stepped up/d own over time and the equation can be separated into diffused and adsorbed charge, EQN 5. 3 where Q is the total charge, Q ads is the adsorbed charge, Q d is the diffused charge, and Q dl is the double layer charge. The total charge of the reduction is equal to the Q dl and Q d then a plot of Q as a function of t 1/2 will give a straight line where the intercept equals Q ads and the slope Q d . The Q dl is ignored because it is instantaneous. 17 Using this equation, a voltage of - 1.5 V was applied to diphenyl disulfide while monitoring the charge over time and plotted as shown below ( Figure 5. 15 ). This was compared to when no analyte was present. When the y - intercept < 0, this indicates there is no adsorbed charge, as seen in Figure 5. 15 . Q d Q ads 224 Figure 5. 15 Charge as a function of t 1/2 to determine adsorption charge using the integrated Cott rell equation. The disulfide total charge was plotted as a function of t 1/2 and compared to electrolyte only. The y - intercept equals the adsorption charge in the integrated Cottrell equation whereby intercepts < 0 indicate no adsorbed charge. Diphenyl disulfide (blue) would cross the intercept below 0 and thus, does not have any adsorbed charge suggesting no adsorbed species to the electrode. A third and more straightforward method to determine analyte adsorption is to monitor the peak current over the course of many sweeps during cyclic voltammetry. In this method, if the peak cu rrent changes over time it suggests that mass transport to the electrode changes possibly due to adsorption. Figure 5. 16 clearly shows that over the course of 18 CV swee ps , the peak current does not change. y = 0.0002x - 2E - 06 R² = 0.9991 y = 2E - 05x - 2E - 07 R² = 0.9958 0.0E+00 5.0E-06 1.0E-05 1.5E-05 2.0E-05 2.5E-05 0.00 0.05 0.10 0.15 Charge (C) t 1/2 (s 1/2 ) Adsorbed Charge Disulfide Electrolyte Analyte: Diphenyl Disulfide Analyte Concentration: 10 mM Electrolyte conc alwa ys 50 mM Electrolyte: NBu 4 Br Working: Glassy Carbon Counter: Platinum Reference: Ag/AgBr in MeCN Potential: - 1.5 V Solvent: MeCN 225 Figure 5. 16 Peak current fluctuation over multiple scans to determine adsorption. No decrease of peak current with repeated scans indicates no adsorption of diphenyl disulfide species. Similar to monitoring current over CV swe eps, another straightforward method to detect analyte adsorption to the electrode is to overlay the CV of a newly polished electrode to a used electrode in new electrolytic solution. As seen in Figure 5. 17 , no diphenyl disulfide is observed on the used electrode with only the electrolyte reduction/oxidation potential observed. To summarize, an abundance of evidence (5 separate methods) all support that no adsorbed analyt e species is present on the electrode that would result in electrochemical irreversibility. Figure 5. 17 Clean vs used electrode adsorption test. No diphenyl disulfide peaks were observed with a used electrode in fresh media. Both clean and used electrodes showed only electrolyte anodic/cathodic peaks. This indicates no thiol species were adsorbed to the electrode. 0.70 0.72 0.74 0.76 0.78 0.80 0 5 10 15 20 Peak Current (mA) Sweep Number Repeated CV Peak Current -0.02 0.00 0.02 0.04 0.06 -2 -1 0 1 Current (mA) E (V) vs. Ag/AgBr Electrode Immersion Immersed Electrode Clean Electrode Analyte: Diphenyl Disulfide Analyte Concentration: 10 mM Electrolyte conc always 50 mM Electrolyte: NBu 4 Br Working: Glassy Carbon Counter: Platinum Reference: Ag /AgBr in MeCN Scan rate: 100 mV/s Solvent: MeCN Analyte: Diphenyl Disulfide Analyte Concentration: 10 mM Electrolyte conc always 50 mM Electrolyte: NBu 4 Br Working: Glassy Carbon Counter: Platinum Reference: Ag/AgBr in MeCN Scan rate: 100 mV/s Solvent: MeCN 226 5.3.2 Chemical Reversibility Base d on the evidence provided that the irreversibility of diphenyl disulfide reduction is not caused by adsorption to the electrode, as much literature has warned against, 4, 13, 15 we moved on to determine if analyte degradation or background reactions may have caused the irreversibility seen in our system. 3 Figure 5. 18 Figure 5. 18 Chemical reversibility monitoring current ratios over tim e. Diphenyl disulfide peak ratio (i cp /i pa ) decreasing with scan rate, indicating a side reaction. To take a closer look at the chemical degradation of the analyte, we used GCMS to qualitatively determine side reactions. We found that diphenyl disulfide di d decrease, as we had seen from previous quantification studies above. We also found two products forming over the course of 20 h ( Figure 5. 19 ). 0 5 10 15 20 25 0 0.2 0.4 0.6 0.8 1 i pc /i pa v (V/s) Peak Current Ratios Analyte: Diphenyl disulfide Analyte Concentratio n: 10mM Electrolyte conc always 50mM Electrolyte: NBu 4 Br Working: Glassy Carbon Counter: Platinum Reference: Ag/AgBr in MeCN Scan rate: Change Solvent: MeCN 227 Figure 5. 19 Concentration of diphenyl disulfide electrochemical reduction overtime. Diphenyl disulfide decreases over the course of 20 h as two products form, qualitatively measured by GCMS. One product was identified as butylthiolbenzene , formed through reaction of the reduced product, thiophenolate, and the electrolyte, TBAB. This was confirmed with the addition of thiophenol and TBAB in acetonitrile, without an applied potential, that also produced the butylthiolbenzene. Interestingly, many articles that report disulfide reduction use tetrabutylammonium electrolytes in acetonitrile but have never addressed this side reaction. 10, 15 The second product from the electrochemical reduction was another thiol species that coul d not be identified but may be a product of a possible thiyl radical side reaction. No quantification with internal/external standards were performed. The proposed diphenyl disulfide chemical transformation leading to chemical irreversibility is shown belo w in Figure 5. 20 . 0 20 40 60 80 100 0 5 10 15 20 Percent of Peak Area Time (hr) Reduction of Diphenyl Disulfide Diphenyl Disulfide Product A Product B Analyte: Diphenyl disulfide Analyte Concentration: 10mM Electrolyte conc always 50mM Electrolyte: NBu 4 Br Working: Glassy Carbon Counter: Platinum Reference: Ag/AgBr in MeCN Potential: - 1.2V Solvent: MeCN 228 Figure 5. 20 Proposed chemical degradation of the electrochemically reduced diphenyl disulfide. Thiophenolate is produced but undergoes side reactions with the electrolyte to form butylthiolbenzene. Thiyl radical may also undergo side reactions, preventing the full r eduction of diphenyl disulfide to two thiophenolates. 5.3.3 New Electrolytes After finding that the electrolyte was the cause of the irreversibility in diphenyl disulfide, new electrolytes were screened for electrochemical reversibility. Perchlorates are u sed in the reference electrode but proved to be inefficient for the redox chemistry of disulfides ( Figure 5. 28 ). LiCl, LiOH, and Li 2 CO 3 were not soluble in acetonitrile. LiBr, LiBF 4 , and the bulky PPh 4 Cl were soluble in acetonitrile and did not react with the disulfide (as analyzed by GCMS). LiBr, over time, corroded the electrode and required cleaning between each scan making this electrolyte inefficient. Future work wil l detail finding the redox potential with the remaining potential electrolytes: LiBF 4 or PPh 4 Cl. 5.4 Challenges in Closing the Catalytic Cycle : The challenges that arose from finding parameters for electrochemical cycling of disulfides were unexpected. Ho wever, a well foreseen challenge was that of reducing the disulfide, our redox mediator, at low enough potentials to not reduce lignin substrates in our ether cleavage reaction mixture. Figure 5. 21 illustrates the reduction potential of diphenyl disulfide (green) in our TBAB 229 conditions from above compared to the reduction of 2 - phenoxyacetophenone (red), a simple lignin model dimer. Two clear electron transfers are seen f or the reduction of the ketone to an alcohol in the lignin model dimer. These cathodic peaks overlap with the reduction potential needed to reduce the disulfide in the same electrolytic system. This overlap makes it imperative to find electrolytic paramete rs to decrease the E p of the disulfide, thus decreasing the overlap of the two compounds potentials. Figure 5. 21 Reduction potential overlap of disulfide and lignin model dimer. 2 - phenoxyacetophenome (red) has overlapping reduction pe aks with diphenyl disulfide (green). Selective reduction of the disulfide may be difficult in cleavage reaction media. To test the overlap in reduction potentials and its effect on reducing both the disulfide and the lignin model dimer, chronoamperometry s tudies were performed using - 1.2 V for 3 h with HPLC analysis. The 2 - phenoxyacetophenone HPLC trace was compared before and after the applied voltage ( Figure 5. 22 a - b). S mall, more polar peaks were observed after applying a voltage indicating that at - 1.2 V, there is some, albeit small, electrolytic conversion occurring. When diphenyl disulfide and 2 - phenoxyacetophenone were mixed together and compared before and after chr onoamperometry ( Figure 5. 22 c - d), there was little difference seen from the HPLC trace compared to the dimer alone. -0.20 0.00 0.20 0.40 -2 -1 0 1 Current (mA) E (V) vs. Ag/AgBr Disulfide and Lignin Dimer CVs Disulfide Dimer Analyte: Diphenyl disulfide & 2 - phenoxyacetopheone Analyte Concentration: 10mM both Electrolyte conc always 50mM Electrolyte: NBu 4 Br Working: Glassy Carbon Counter: Platinum Reference: Ag/AgBr in MeCN Scan rate: 100mV/s Solvent: MeCN 230 Figure 5. 22 Chronoamperometry of 2 - phenoxyacetophenone. (A - B) HPLC of 2 - phenoxyacetophenone (B) compared to 2 - phenoxyacetophenone after a constant potential of - 1.2 V was applied for 3 h (A). Little reductive products are observed. (C - D) HPLC of 2 - phenoxyacetophenone with diphenyl disulfide (D) compared to 2 - phenoxyacetophenone and diphenyl disulfide after a constant potential of - 1.2 V was applied for 3 h (C). Little reductive produc ts are observed. The control reaction above was slightly promising in that little dimer reduction was seen. We decided to move forward and obtain preliminary data for our organocatalytic keto - aryl ether cleavage. In this system, as illustrated by the catal ytic cycle in Figure 5. 23 , the dimer cleavage reaction would take place over the course of 24 h, at which time the reaction would be cooled and a current would be appli ed to reduce the disulfide b y products from the reaction. (Current applied was calculated by how many electrons needed to be passed in a given time to cleave the disulfide). Dimer Dimer after chronoamperometry Dimer and disulfide Analyte Concentration: 10 mM Electrolyte conc alwa ys 50 mM Electrolyte: NBu 4 Br Working: Glassy Carbon Counter: Platinum Reference: Ag/AgBr in MeCN Potential: - 1.2 V Solvent: MeCN Dimer and disulfide after chronoamperometry A) B) C) D) 231 After 2 h, the current would be turned off and the reaction heated again overnight to obtain a rough estimate of newly cleaved dimer. It is established that at room temperature no thiol - medated dimer cleavage occurs. 8 Figure 5. 23 illustrates the quantitative change between substrat es, products, and b y products before a current is applied (blue) to after applied current and additional heating (orange). The bar graph indicates that the dimer is completely consumed, the disulfide b y product was produced in roughly the same amount as the first cleavage cycle, and that phenol is produced in near complete yields. Acetophenone yields decreased most likely due to the common electrolytic side reactions observed in literature, forming a pinacol dimer (not quantified). 18 - 22 These preliminary results indicate that, though electrochemical parameters must be optimized to stop chemical irreversibility, the turnover of the thiol - media tor in lignin ether cleavage reactions is a possible solution to decreasing costs and improving scale - up of this technology. 232 Figure 5. 23 Turn - over of 2 - phenoxyacetophenone cleavage reaction using an applied current . 2 - phenoxyacetophenone (2.3 mM) was treated with BME (22 mM) for 24 h in refluxing MeCN and stirring K 2 CO 3 . After 24 h, the reaction was cooled and ~50 mA of current was applied for 2 h to reduce the used catalyst. The reaction was heated back to refluxing overnight and the substrate, products, and oxidized catalyst were measured (orange) and compared to concentrations before electrolysis (blue). The dimer was completely consumed with near complete yields of phenol. 5.5 Conclusion In summary, thiol - mediated lignin ether cleavage can be improved to b e cost effective, more efficient, and sustainable if coupled to electrochemical reduction of the thiol redox mediator. We proposed that the disulfide b y product of the lignin ether cleavage reaction could be recycled through a 2e - reduction to allow for mul tiple turnovers of the ether cleavage reaction. Through cyclic voltammetry and chronoamperometry studies we have detailed the electrochemical behavior 0 0.5 1 1.5 2 Dimer Acetophenone Phenol Oxidized BME Concentration (mM) Turn - over of 2 - Phenoxyacetophenone Cleavage Using Current Before After Analyte Concentration: Start 22 mM BME Electrolyte is always 50 mM Electrolyte: NBu4ClO4 Working: Carbon Cloth Counter: Platinum wire Reference: Unknown Current: 48 - 46 mA (12.8 - 13.5 V) Solvent: MeCN 2 h 233 of disulfides in relevant reaction conditions. Furthermore, we have assessed electrochemical and chemical irreversibility and proposed further parameterizations. Lastly, we have found promising results in the regeneration of disulfide b y product for additional lignin dimer cleavage. Future work will expand on the parameters found here and allow for a fed - batch process for a net electrocatalytic depolymerization of lignin. 5.6 Experimental Detail 5.6.1 General Information All reagents were tested for purity by 1 H - NMR. All solvents used were distilled, purged with nitrogen, and stored over 3 Å molecular sieves. Cyclic voltammetry experiments were performed with a standard 3 - electrode setup, using a glassy carbon electrode that was polished with alumina after each experiment, a Ag/Ag + reference electrode, and a Pt wire counter electrode in a one cell system with sparging nitrogen. At the beginning of each experiment, a scan of ferrocene was taken to reference the Ag/Ag + potential to Fc +/0 , after which the electrodes were rinsed and transferred to a fresh solution. All cyclic voltammetry experiments were conducted at a scan rate of 100 mV/s in acetonitrile unless otherwise stated. N 2 gas was purged into solution before scans. Platinum electrodes were clean and stored in concentrated H 2 SO 4 . HPLC analysis was performed using an Agilent 1260 Infinity equipped with a G1315D 1260 diode array detector VL, monitoring at 280 nm and recording from 190 - 400 nm and a G1362 refractive index detector. A Supelco Ascentis Express C18 used in isocratic mode at 0.4 mL/min with a mobile phase of 70:30 acetonitrile:water, adjusted to an internal standard; extern al standards were run during each sequence of analysis. GC - MS analyses employed a 6890 Agilent GC equipped with a VF - 234 m EZ - Guard column and a 5975b single quadrupole MS detector. 1 H NMR spectra were recorded on 500 MHz Vari an spectrometers and referenced to residual solvent peaks. 5.6.2 Control Experiments Figure 5. 24 CV of ferrocene used as a control. A CV of ferrocene was run in fresh solution before every experiment to test the electrochemical system for problems or changes in potential. -0.10 -0.06 -0.02 0.02 0.06 0.10 0.5 0.7 0.9 1.1 1.3 1.5 Current (mA) E (V) vs. Ag/AgBr Ferrocene Analyte Concentration: 2.85 mM Electrolyte conc always 50 mM Electrolyte: NBu 4 Br Working: Glassy Carbon Counter: Platinum Reference: Ag/AgBr in MeCN Scan rate: 100 mV/ s Solvent: MeCN 235 Figure 5. 25 CV of O - DTT and O - BME using Ag/AgCl and NBu 4 ClO 4 in MeCN. BME was chosen for its performance in the cleavage reaction but appea rs electrochemically irreversible under NBu 4 ClO 4 electrolyte conditions. O - DTT shows similar results. Figure 5. 26 Gatorade as an electrolyte/solvent for O - BME reduction. G2 Low Calorie Glacier Freeze Gatorade, which has known electrolytes of sodium citrate, sodium chloride, and potassium phosphate, was used as a solvent/electrolyte combination for CV studies of O - BME. Large E p are seen suggesting electrochemical irreversibility. -0.10 -0.05 0.00 0.05 0.10 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 Current (mA) E (V) vs. Ag/AgCl O - DTT -0.10 -0.05 0.00 0.05 0.10 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 Current (mA) E (V) vs. Ag/AgCl O - BME -0.4 -0.2 0.0 0.2 -2.5 -1.5 -0.5 0.5 1.5 Current (mA) E (V) vs. Ag/AgCl Gatorade as Electrolyte Gatorade Disulfide Analyte Concentration: 10 mM Electrolyte conc always 50 mM Electrolyte: NBu 4 ClO 4 Working: Glassy Carbon Counter: Platinum Reference: Ag/AgCl in Water Scan rate: 100 mV/s Solvent: MeCN Analyte: O - BME Analyte Concentration: 10 mM Electrolyte: G atorade ions Working: Glassy Carbon Counter: Platinum Reference: Ag/AgCl in Water Scan rate: 100 mV/s Solvent: G atorade 236 Figure 5. 27 CV of O - BME using water and NaCl as the electrolyte. Under aqueous conditions with NaCl as the electrolyte, O - BME is not electrochemically reversible . Figure 5. 28 Lithium perchlorate as an electrolyte. CV of diphenyl disulfide using lithium perchlorate as the electrolyte. No oxidation peak of the electrolyte is observed. Repeated sweeps show decrease in peak height of the analyte. -0.70 -0.50 -0.30 -0.10 0.10 -2 -1 0 1 Current (mA) E (V) vs. Ag/AgCl Sodium Chloride as an Electrolyte NaCl Oxidized BME -0.02 0.00 0.02 0.04 0.06 -2 -1 0 1 Current (mA) E (V) vs. Ag/AgBr Lithium Perchlorate Electrolyte Background Disulfide Sweep 1 Disulfide Sweep 2 Disulfide Sweep 3 Disulfide Sweep 4 Analyte: O - BME Analyte Concentration: 10 mM Working: Glassy Carbon Counter: Platinum Reference: Ag/AgCl in Water Scan rate: 100 mV/s Solvent: water Analyte: Diphenyl Disulfide Analyte Concentration: 10 mM Electrolyte conc always 50 mM Electrolyte: Lithium perchlorate Working: Glassy Carbon Counter: Platinum Reference: Ag/AgBr in MeCN Scan rate: 100 mV/s Solvent: MeCN 237 REFERENCES 238 R EFERENCES 1. Oae, S., Organic sulfur chemistry: Structure and mechanisms . CRC Press: Boca Raton, Florida, 1991 . 2. Garedew, M.; Lin, F.; DeWinter, T. M.; Song, B.; Saffron, C. M.; Jackson, J. E.; Lam, C. H.; Anastas, P., Greener Routes to Biomass Waste Valorization: Lignin Transformation Through Electrocatalysis for Renewable Chemicals and Fuels Production. ChemS usChem 2020 . Preprint 3. Elgrishi, N.; Rountree, K. J.; McCarthy, B. D.; Rountree, E. S.; Eisenhart, T. T.; Dempsey, J. L., A Practical Beginner's Guide to Cyclic Voltammetry. Journal of Chemical Education 2018, 95 (2), 197 - 206. 4. Glass, R. S., Sulfur - , Selenium - , and Tellurium - Containing Compounds. In Organic Electrochemistry: Revised and Expanded , 5th ed.; Hammerich, O.; Speiser, B., Eds. CRC Press: Boca Raton, FL, 2016; pp 1035 - 1102. 5. Antonello, S.; Maran, F., Intramolecular dissociative electron t ransfer. Chemical Society Reviews 2005, 34 (5), 418 - 428. 6. Fuchigami, T.; Inagi, S.; Atobe, M., Fundamentals and Applications of Organic Electrochemistry: Synthesis, Materials, Devices. Fundamentals and Applications of Organic Electrochemistry: Synthesis , Materials, Devices 2015 , 1 - 226. 7. Organic Sulfur Chemistry: Theoretical and Experimental Advances . Elsevier Science Publishers: Amsterdam, The Netherlands, 1985 , ( 19 ) . 8. Klinger, G. E.; Zhou, Y.; Hao, P.; Robbins, J.; Aquilina, J. M.; Jackson, J. E.; Hegg, E. L., Biomimetic Reductive Cleavage of Keto Aryl Ether Bonds by Small - Molecule Thiols. Chem S us C hem 2019, 12 (21), 4775 - 4779. 9. Kadin, H., E lectrochemical Reduction of Disulfides . Methods in Enzymology 1987, 143 , 257 - 264. 10. Borsari, M.; Cannio, M.; Gavioli, G., Electrochemical behavior of diphenyl disulfide and thiophenol on glassy carbon and gold electrodes in aprotic media. Electroanalysis 2003, 15 (14), 1192 - 1197. 11. Cleland, W. W., D ithiothreitol New Protective Reagent for SH Groups . Biochemistry 1964, 3 (4), 480 - 482. 12. Aitken, C. E.; Marshall, R. A.; Puglisi, J. D., An oxygen scavenging system for improvement of dye stability in single - molecule fluorescence experiments. Biophysical Journal 2008, 94 (5), 1826 - 1835. 239 13. Freedman, L. D.; Corwin, A. H., O xidation - R eduction P otentials of T hiol - D isulfide Systems . Journal of Biological Chemistry 1949, 181 (2), 601 - 621. 14. Lees, W. J.; Whitesides, G. M., E quilibrium - C onstants for Thiol Disulfide I nterchang e R eactions - A C oherent , C orrected S et . Journal of Organic Chemistry 1993, 58 (3), 642 - 647. 15. Mostafavi, S. M.; Rouhollahi, A.; Adibi, M.; Mohajeri, A.; Pashaee, F.; Pyriaee, M., Electrochemical Investigation of Thiophene on Glassy Carbon Electrode and Quantitative Determination of it in Simulated Oil Solution by Differential Pulse Voltammetry and Amperometry Techniques. Asian Journal of Chemistry 2011, 23 (12), 5356 - 5360. 16. Saveant, J. M.; Costentin, C., Elements of Molecular and Biomolecular Elec trochemistry: an Electrochemical Approach to Electron Transfer Chemistry, 2nd Ed ., 2019 , 1 - 616. 17. Harvey, D., Modern Analytical Chemistry . 1 ed.; McGraw - Hill Companies: Boston, MA, 2000 . 18. Dorrestijn, E.; Hemmink, S.; Hulstman, G.; Monnier, L.; van Scheppingen, W.; Mulder, P., The reduction of alpha - X - acetophenones (X = PhO, Br, Cl) in hydrogen - donating solvents at elevated temperatures. European Journal of Organic Chemistry 1999, 1999 ( 3), 607 - 616. 19. Andersen, M. L.; Wayner, D. D. M., Electrochemistry of electron transfer probes. alpha - aryloxyacetoveratrones and implications for the mechanism of photo - yellowing of pulp. Acta Chemica Scandinavica 1999, 53 (10), 830 - 836. 20. Andersen, M. L.; Mathivanan, N.; Wayner, D. D. M., Electrochemistry of electron - transfer probes. The role of the leaving group in the cleavage of radical anions of alpha - aryloxyacetophenones. Journal of the American Chemical Society 1996, 118 (20), 4871 - 4879. 21. Net toferreira, J. C.; Avellar, I. G. J.; Scaiano, J. C., E ffect of Ring Substitution on the P hotochemistry of A lpha - (A ryloxy )A cetophenones . Journal of Organic Chemistry 1990, 55 (1), 89 - 92. 22. Nakahara, K.; Naba, K.; Saitoh, T.; Sugai, T.; Obata, R.; N ishiyama, S.; Einaga, Y.; Yamamoto, T., Electrochemical Pinacol Coupling of Acetophenone Using Boron - Doped Diamond Electrode. Chemelectrochem 2019, 6 (16), 4153 - 4157. 240 Chapter 6. Conclusions and Future Directions The objective of this project w as to develop a biomimetic and inherently green, scalable, and cost - effective means to reductively depolymerize lignin into fragments for downstream valorization. To this end, this document describes our attempt to model a Lig enzymatic pathway, the - aryl ether cleavage pathway, which uses thiol cofactors for nucleophilic substitution and reductive elimination that result in keto - aryl ether cleavage of small lignin fragments. We hypothesized we could mimic this pathway using small molecule thiols without t he assistance of proteins or metal co - catalysts. We explored this hypothesis in Chapter 2, where we used simple keto - aryl ethers to model the - O - 4 lignin linkage. From the exploration and optimization of this cleavage reaction, we found that polar aprotic solvents, which are also optimal solvents for S N 2 reactions, achieved the largest monomer yields. Temperatures ranging from 80 - 100 ° C overcame the calculated energy barrier of ~21 kcal/mol for nucleophilic attack. Insoluble bases, such as K 2 CO 3 with incre ased surface area through grinding, allowed for the highest yields. Soluble small thiols with p K a s ~ 9, such as BME and DTT, were chosen as the optimal redox mediator s . Once parameters were optimized , exploration on the mechanism of thiol - mediated cleavage was completed to determine if this mimetic system followed the S N 2 mechanism of the enzymatic pathway. Using synthesis, computations, Hammett analysis, structure - reactivity relationships, and many controls , we were able to expand the substrate scope and r each the following key conclusions: 1) The reaction most likely occurs in two steps. A thioether intermediate is observed after activation with p - electron withdrawing groups, suggesting a second step in the reaction. 241 2) The first step, nucleophilic attack, is the rate limiting step. With the parent keto - aryl ether dimer , 2 - phenoxyacetophenone , no thioether was observed. However, model dimers activat ed with p - electron withdrawing groups, which increased the rate of nucleophilic attack, lead to observable bui ldup of thioether. Thus, it can be concluded that the second step, the reductive elimination and disulfide formation, normally has a lower activation energy resulting in a faster reaction. This was verified by computational modeling. 3) The first step of t he reaction involves nucleophilic attack at the - carbon. Hammett analysis revealed that aryl ether functionalization impacts cleavage rates more than keto functionalization, consistent with a n attack on the - carbon. 4) There is most likely no free radica l involved in the reaction mechanism; no changes were observed in cleavage rates when BHT, a known radical trap, was added to the thiol - mediated cleavage reaction. 5) The - OH must be oxidized for successful ether cleavage. Substrates with only an - OH or no functionality on the alpha position were unreactive. 6) The leaving group must be an aryl ether for cleavage to occur. Other ethers were unreactive suggesting, once again, an S N 2 mechanism where an aryl ether is a much better leaving group than an alkyl ether. However, t he aromatic ring on the ketone side of the lignin model dimer was unnecessary for ether cleavage. 7) Under alkaline conditions, the hydroxymethyl moiety attached to t he - carbon of lignin model dimers can be eliminate d via a retro aldol reaction, resulting in an open primary site for thiol nucleophilic attack. 242 8) Ring substituents alter cleavage yields. Methoxy groups on the meta positions and alkoxy groups on the para position are seen in natural lignin. High cleavage yields were observed when the p - position was changed from a hydroxyl to a methoxy unit. The n on - - O - 4 ether model lignin dimer linkages, 4 - O - 5 and - O - 4/ - 5, were also examined for ether cleavage using small thiol redox mediators. Little activity was observed unless the dimer was activated by a p - aldehyde or p - ketone on the ring. The 4 - O - 5 model c leavage was presumed to undergo a nucleophilic aromatic substitution resulting in addition of the thiol to the aromatic ring and release of a phenol. The - O - 4/ - 5 models may be cleaved through a typical S N 2 mechanism, but this has not been confirmed. From these two ether studies, it can be concluded that some amount of side reactions may occur in thiol - mediated biomass - derived lignin cleavage and could contribute to additional depolymerization. We expanded our thiol - mediated cleavage to larger systems inc luding synthetic - O - 4 linked polymers and biomass - derived lignin in Chapter 4. We found that bulky synthetic polymers were successfully cleaved with up to 90% monomer yields. This finding suggests that thiols can diffuse through bulky systems and cleave k eto - aryl ether bonds. When applied to biomass - derived lignin, this strategy afforded molecular weight decreases of up to 50%. We also found that lignin depolymerization was enhanced by oxidation, presumably through the conversion of the - OH groups to keto nes that activate the ether bond. The reaction b y product, the disulfide, requires reduction to regenerate the thiol and achieve additional keto - aryl ether cleavage. In Chapter 5, parameters were assessed for electrochemical disulfide reduction in acetonit rile with several key findings: 1) TBAB, a common electrolyte for electrochemical analysis in MeCN, is unsuitable for our reactions. TBAB was found to react with thiols to form a butylated thio ether , with or without an 243 applied potential. Thiolates are nec essary for ether cleavage so butylation of thiolates would kill the lignin depolymerization reaction. 2) The disulfide reduction is electrochemically irreversible, most likely due to the chemical irreversibility from interaction with the electrolyte. 3) N either the disulfide, thiolate, nor any other reduction product absorbs to the electrodes surface. 4) The most promising electrolytes for disulfide cleavage includes LiBr, LiBF 4 , and PPh 4 Cl which are all soluble in acetonitrile and show no side reactions with thiols. LiBr , however, was found to corrode the electrode over time. Current electrochemical work (Zhen Fang) uses applied current, instead of voltage, with/without thiols to cleave lignin model dimers. Future studies of thiol - mediated lignin depolym erization include: 1) Characterizing and quantifying lignin depolymerization monomeric products is a priority . While characterization of unreacted polymer has been extensively described in Chapter 4, small molecule fragments, such as monomers, dimers, trimers, etc., need further characterization through spectroscopic methods. Quantifying the yields of these fragm ents will allow further development of this depolymerization technology. 2) A second key goal is to screen other biomass - derived lignin s to compar e and characteriz e the depolymerized polymer and monomer products. Current work has focused primarily on Cu - AH P lignin from poplar wood due to its more native lignin structure, intact - O - 4 linkages, solubility in alkaline conditions, and partial oxidation. Preliminary studies have evaluated lignin streams from large scale productions such as Kraft lignin, alkalin e/dealkaline lignin, sodium lignosulfonate lignin, and industrial produced MetGen lignin. These various lignins have different polymeric structures depending on their extraction process. Some of these structural differences include 244 crosslinking via C - C bon ds, decreases in - O - 4 content, addition of sulfonate groups, etc. To determine how feedstock - agnostic our thiol - mediated lignin depolymerization is, characterization of different extracted lignin s before and after thiol - mediated cleavage must be accomplis hed. 3) Oxidation of other biomass - derived lignins followed by thiol - mediated cleavage should be performed to provide further evidence as to how well oxidation enhanced depolymerization. Chapter s 2 - 4 outline the importance of - OH oxidation to the thiol - me diated reductive cleavage of both models and polymeric lignin. In Chapter 4, we found that additional oxidation of Cu - AHP lignin increase d depolymerization up to 40% with 1,3 - propanedithiol. Performing a pre - oxidation of other extracted lignins will give f urther evidence of the effectiveness of thiol - mediated cleavage. 4) Determination of disulfide redox potentials in lignin cleavage reaction media is needed . Parameters as well as pitfalls were established in Chapter 5 for the reduction of disulfides. Using this knowledge, better electrochemical conditions can be established to electrochemically characterize disulfides and determine the reduction potentials needed for electrocatalytic turnover in our reaction conditions. 5) Cycling the disulfide in the pres ence of lignin models will be essential to test for side redox reactions. A key challenge envisioned for electrochemical recycling of disulfides is the overlap in reduction potential needed for disulfide cleavage and the potential at which ketones are redu ced to alcohols in lignin - type structures. Under thiol - mediated lignin cleavage conditions, carbonyls on the - position are crucial for activation of the - carbon for nucleophilic attack. With aryl ketone reduction potentials close to those of disulfides, accurate values are needed as it is imperative that applied potentials remain low enough to avoid electrolysis of substrates other than the disulfide. 6) Multiple turnovers of disulfide b y product is important for complete lignin dimer cleavage. Chapter 2 o utlined the success of thiols in cleav ing model lignin dimers . However, t he resulting 245 disulfide b y product does not cleave additional dimers . Therefore, yields from these reactions only involve one cycle of cleavage. Future work will apply a potential, peri odically, to batch reduce the disulfide to the thiol redox mediator , allowing multiple turnovers and complete dimer cleavage. 7) Fed - batch model lignin dimer cleavage will be the next stage of process development . Once the dimer is fully cleaved into monomers, as described in 6), additional dimer may be added to the reaction mixture for cleavage by the cycling thiolate/disulfide. 8) Net electrocatalytic lignin depolymerization is the ultimate goal . Future work will utilize the parameters obtained above from 5) - 7) and apply each of these advances to biomass - derived lignin, rather than models. In this way, potential overlap, complete fragmentation, and fed - batch processing can be assessed for lignin. Once optimized, the thiol - mediated lignin depolymerizat ion could be scaled - up and utilized as a viable technology for industrial processing of biomass to fuels and chemicals.