POLY(ETHYLENE GLYCOL) TAILORED POLYMERS: NANOMICELLES WITH 

TUNABLE LOWER CRITICAL SOLUTION TEMPERATURE BEHAVIOR 

By 

Yu-Ling Lien 

 

 

 

 

 

 

 

A DISSERTATION 

Michigan State University  

in partial fulfillment of the requirements  

Submitted to  

for the degree of 

Chemistry – Doctor of Philosophy 

2018 

 

ABSTRACT 

POLY(ETHYLENE GLYCOL) TAILORED POLYMERS: NANOMICELLES WITH 

TUNABLE LOWER CRITICAL SOLUTION TEMPERATURE BEHAVIOR 

By 

Yu-Ling Lien 

Propargyl and 1,1-dimethyl propargyl substituted poly(ethyleneoxides) (propargyl 

substituted = poly(PGE), 1,1ʹ-dimethyl propargyl substituted = poly(MGE)) have been 

prepared  by  ring-opening  polymerization  of  epoxides,  which  were  synthesized  from 

epichlorohydrin  and  propargyl  or  1,1-dimethyl  propargyl  alcohol  via  Williamson  ether 

synthesis.  The  resulting  polymers  were  modified  by  Cu-catalyzed  azide  alkyne 

cycloaddition (CuAAC) of the polymer propargyl groups and organic azides. When these 

reactions were carried out with mixtures of azides, the ratios of azides incorporated in the 

polymer side chains were equal to the molar ratios of the organic azides reactants (± 2%).  

Mixtures  of  hydrophobic  (decyl  azide)  and  hydrophilic  (mDEG  azide)  azides 

result in amphiphilic polymers that exhibited a lower critical solution temperature (LCST) 

behavior.  The  polymer  LCSTs  scaled  from  48  to  97  ±  2  °C  (poly(PGE)  derived 

amphiphiles) and 4 to 46 ± 1 °C (poly(MGE) derived amphiphiles) in a roughly linear 

fashion with the mole fraction of hydrophilic side chains in the polymer. When charged 

azides, COOH azide and aminium azide, were used, the physical property as well as the 

LCST  behavior  oh  the  polymers  were  changed. The  LCSTs  of  polymers incorporating 

charged  azides  were  increased  and  the  LCSTs  were  decreased  by  adding  salts  in  the 

solutions. The hydrodynamic radii (RH) obtained from DLS measurements indicate that 

polymers form unimolecular micelles in water (Mn = 52,000 g/mol, PDI = 1.19, RH = 6 ± 

2 nm), and TEM data showed monodisperse domains (20 ± 4 nm, for Mn = 52,000) when 

water was evaporated at room temperature from solutions cast on TEM grids. This length 

scale is consistent with domains that consist of single polymer chains. When the TEM grid 

was heated during evaporation, the domain size increased to 74 ± 45 nm.  

In solution, the unimolecular micelles can solubilize hydrophobic small molecules, 

such as trans-azobenzene (trans-PhN=NPh) in water. DLS data suggested that polymer 

encapsulating  trans-PhN=NPh  (trans-PhN=NPh@poly(PGE)  or  poly(MGE))  derived 

amphiphiles) showed signs of aggregation in one case (RH = 12 ± 8 nm) and no signs of 

aggregation in another case (RH = 5 ± 2 nm). When the resulting solutions were raised 

above the polymer LCST the polymer and small molecule precipitated. When the mixture 

was cooled below the LCST, the polymer and hydrophobic small molecule re-dissolved. 

The  unimolecular  micelles  were  used  to  encapsulate  a  hydrophilic  macromolecule, 

Subtilisin  Carlsberg  (SC),  in  aqueous  solution  and  organic  media.  Poly(PGE)  or 

poly(MGE) derived amphiphiles with COOH pendant group slowed down SC aggregation 

in aqueous environment. Also, the activity of SC@poly(MGE) derived amphiphiles with 

COOH pendant group was assayed and the half-life of SC was increased to 10 h from 2 h 

at 50 °C. Initial studies of SC@poly(PGE) or poly(MGE) derived amphiphiles in organic 

media showed enzymatic activity in toluene after 16 h at 37 °C. 

 

 

 

 

 

 

 

 

 

 

 

 

 

For those who believed in me 

 

 

 

iv 

ACKNOWLEDGMENTS 

 

I would like to thank my advisor, Dr. Mitch Smith, for the guidance during my 

graduate study. I appreciate the freedom I had to explore my ideas for the project. I’m also 

grateful  for  the  critics  and  questions  from  him  so  I  can  build  the  project  on  a  sound 

scientific foundation. I would also like to thank my guidance committee, Dr. Xuefei Huang, 

Dr. James Geiger, and Dr. Rémi Beaulac, for their suggestions and help.  

Thanks  to  Dr.  Kathy  Severin,  Dr.  Dan  Holmes,  and  Dr.  Ardeshir  Azadnia  for 

teaching me various characterization techniques. Also, I would like to thank the previous 

and current group member, Budda, Dmitry, Sean, Behnaz, Kristin, Don, Tim, Olivia, Gina, 

Greg, Zhe, Salinda, Jack, Meisam, Zahra, Po-Jen, Reza, Mona, Ryan, Alex, Seokjoo, Jack, 

Chris, Aalyah, Suzi. Thank you for your support throughout my graduate study. Special 

thanks to the talented undergraduate students I worked with, Jack and Chris. Your work 

made great impacts on the project. 

I  would  like  to  thank  all  my  friends  from  MSU,  Tim,  Kristin,  Kristen,  Travis, 

Tanner, Michelle, Pengchao, Corey, Matt, Tyler, Ruwi, Cho-Ying, Feng-Kuo, Yen-Yao, 

Zhong-En, Tung-Wu, Yung-Hsiu, Jing-Ru, Lenore, Dana. You brought so much joys to 

my life in East Lansing. Jay, thank you for your support and encouragement! Finally, I 

would like to thank my family for their support. 

 

 

 

v 

TABLE OF CONTENTS 

 

Chapter 2. Synthesis, Characterization, and Post-Polymerization Modification of 

LIST OF TABLES ..................................................................................................... viii	
LIST OF FIGURES ..................................................................................................... ix	
LIST OF SCHEMES ................................................................................................. xxii	
KEY TO ABBREVIATIONS .................................................................................. xxiv	
Chapter 1. Introduction ................................................................................................ 1	
1.1. Overview of Polyether Synthesis .......................................................................... 1	
1.2. Overview of Post-Polymerization Modification .................................................... 6	
1.3. Overview of Lower Critical Solution Temperature (LCST) ................................. 10	
1.4. Overview of the Formation of Unimolecular Micelles (Unimicelles) .................. 12	
1.5. Overview of Nanocarriers ................................................................................... 15	
1.6. Overview of the Synthesis of Biocatalysts .......................................................... 18	
1.7. Protein Folding and Molecular Chaperones......................................................... 21	
1.8. Artificial Chaperones .......................................................................................... 25	
Propargyl-Substituted Poly(ethylene oxide)s ............................................................. 32	
2.1. Introduction ........................................................................................................ 32	
2.2. Synthesis and Characterization of Monomers...................................................... 33	
2.3. Living Anionic Ring-opening Polymerization ..................................................... 37	
2.4. Post-polymerization Modification (PPM) via “Click” Chemistry ........................ 44	
2.5. Summary ............................................................................................................ 58	
of Unimolecular Micelles............................................................................................. 59	
3.1. Lower Critical Solution Temperature (LCST) Behavior ...................................... 59	
3.2. Formations of Unimolecular Micelles ................................................................. 75	
3.3. Summary ............................................................................................................ 78	
Molecules ..................................................................................................................... 79	
4.1. Encapsulation of Hydrophobic Guest Molecules ................................................. 79	
4.2. Encapsulation of Hydrophilic Guest Biomacromoelcules .................................... 84	
4.3. Summary ............................................................................................................ 97	
Chapter 5. Experimental Section ................................................................................ 99	
5.1. Materials ............................................................................................................ 99	
5.2. Characterization.................................................................................................. 99	
5.3. Procedures ........................................................................................................ 101	

Chapter 3. Lower Critical Solution Temperature (LCST) Behavior and Formation 

Chapter 4. Unimicelles as Nanocarriers for Hydrophobic and Hydrophilic Guest 

 

vi 

Synthesis	of	1-azido	poly(ethylene	glycol)	monomethyl	ether,	550	(mPEG550	azide)229,230

Synthesis	of	propargyl	glycidyl	ether	(PGE)222	....................................................................................	101	
Synthesis	of	1,1´-dimethyl	propargyl	glycidyl	ether	(MGE)	...........................................................	101	
General	Procedure	for	Polymerization	...................................................................................................	102	
Synthesis	of	poly(propargyl	glycidyl	ether)	(poly(PGE))................................................................	103	
Synthesis	of	poly(1,1´-dimethyl	propargyl	glycidyl	ether)	(poly(MGE))	.................................	104	
General	Procedure	for	“Click”	Functionalization49,95......................................................................	105	
Synthesis	of	poly(PGE0.50)	..............................................................................................................................	105	
Synthesis	of	poly(MGE0.50)	.............................................................................................................................	106	
Safety	Information	for	Synthesizing,	Purifying,	and	Handling	Organic	Azides270	..............	107	
Synthesis	of	1-azidodecane	(decylazide)95,225,226.................................................................................	108	
Synthesis	of	1-(2-azidoethoxy)-2-(2-methoxyethoxy)ethane	(mDEG	azide)95	.....................	108	
	...................................................................................................................................................................................	110	
	...................................................................................................................................................................................	111	
azide)229,230	...........................................................................................................................................................	112	
Synthesis	of	6-azidophexanoic	acid	(COOH	azide)231	.......................................................................	113	
Synthesis	of	3-azido-N,N,N-trimethylpropan-1-aminium	iodide	(aminium	azide)232,233.	114	
General	Procedure	for	Azobenzene	(transPhN=NPh)	Encapsulation	......................................	115	
General	Procedure	for	Subtilisin	Carlsberg	(SC)	Encapsulation	in	Aqueous	Media	..........	115	
General	Procedure	for	Subtilisin	Carlsberg	(SC)	Encapsulation	in	Organic	Media	...........	115	
	...................................................................................................................................................................................	116	
APPENDIX ................................................................................................................ 117	
REFERENCES .......................................................................................................... 266	

General	Procedure	for	N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide	(Suc-AAPF-pNA)	Assay

Synthesis	of	1-azido	poly(ethylene	glycol)	monomethyl	ether,	750	(mPEG750	azide)229,230

Synthesis	of	1-azido	poly(ethylene	glycol)	monomethyl	ether,	2000	(mPEG2000	

 

 

 

 

vii 

LIST OF TABLES 

 

Table 1. Examples for cross-linking chemistry for the formation of SCNPs.115 ............. 14	

Table 2. Results of polymerization of PGE and MGE. ................................................. 40	

Table 3. GPC results of poly(PGE0.X). .......................................................................... 50	

Table 4. GPC results of poly(MGE0.X). ........................................................................ 51	

Table 5. Selective polymers and their LCSTs.92,93,234–238 ............................................... 65	

Table 6. Selective examples from literatures showing LCSTs at various 
concentrations.239,241–244 ................................................................................................. 71	

Table 7. Effects of SC concentration and temperatures on SC stabili`ty. ....................... 91	

Table 8. Results of t1/2 in the presence of various polymers. .......................................... 94	

 

 

 

 

 

 

viii 

LIST OF FIGURES 

 

Figure 5. Schematic illustration of the formation of polymeric micelles and unimicelles.

Figure 6. Timeline of nanotechnology-based biocatalysts. Reprinted from ref. 165. 

Figure 7. Energy landscape scheme of protein folding and aggregation. Reprinted from 

Figure 8. Protein fates in the proteostasis network. Reprinted from ref. 196. Copyright 

Figure 3. Historical timeline of clinical-stage nanoparticle technologies. Reprinted from 

Figure 1. Examples of functionalized epoxides. .............................................................. 1	
Figure 2. Models for intermolecular cross-linking of a polymer chain.115 ...................... 13	
ref. 143. Copyright 2011 American Chemical Society.143 .............................................. 16	
Figure 4. Structure of poly(lactic-co-glycolic acid) (PLGA).157..................................... 17	
...................................................................................................................................... 18	
Copyright 2012 Springer Science+Business Media New York.165 .................................. 19	
ref. 182. Copyright 2009 Nature America, Inc.182 .......................................................... 22	
2011 Macmillan Publishers Limited.196 ......................................................................... 23	
Francis Group, LLC.187 ................................................................................................. 25	
workers.204 .................................................................................................................... 26	
Chemistry.213 ................................................................................................................. 27	
Weinheim.214 ................................................................................................................. 29	
(poly(PGE)) in CDCl3................................................................................................... 34	
(poly(PGE)) in CDCl3................................................................................................... 35	

Figure 10. Artificial chaperone-assisted protein refolding (uppermost path) vs. dilution 
additive-assisted protein refolding (lowermost path) reported by Gellman and co-

Figure 12. Schematic representation mechanism of MSPMs-assisted protein refolding. 
Reprinted from ref 214. Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA, 

Figure 9. Schematic representation mechanism of GroEL/GroES chaperone-mediated 
protein folding. Reprinted from ref. 187. Copyright 2013 Garland Science, Taylor & 

Figure 13. 500 MHz 1H NMR spectra of a) PGE b) poly(propargyl glycidyl ether) 

Figure 14. 125 MHz 13C NMR spectra of a) PGE b) poly(propargyl glycidyl ether) 

Figure 11. Schematic illustration of the C12ESG nanogel-mediated one-step protein 
refolding system. Reprinted from ref 213. Copyright 2013 The Royal Society of 

 

ix 

Figure 22. 500 MHz 1H NMR spectra of a) poly(MGE) b) poly(MGE0) c) poly(MGE1.0) 

Figure 15. 500 MHz 1H NMR spectra of a) MGE b) poly(1,1(cid:1)-dimethyl propargyl 

Figure 16. 125 MHz 13C NMR spectra of a) MGE b) poly(1,1(cid:1)-dimethyl propargyl 

Figure 20. Conversion vs. time of the polymerization of MGE ([M]: [Al]: [I] = 200: 2: 

Figure 21. 500 MHz 1H NMR spectra of a) poly(PGE) b) poly(PGE0) c) poly(PGE1.0) 

Figure 17. Measurements of refractive index increment, dn/dc, of poly(PGE). Trial 1 

Figure 18. Measurements of refractive index increment, dn/dc, of poly(MGE). Trial 1 

glycidyl ether) (poly(MGE)) in CDCl3. ......................................................................... 36	
glycidyl ether) (poly(MGE)) in CDCl3 .......................................................................... 37	
(blue diamond), trial 2 (red square), and trial 3 (green triangle). .................................... 41	
(blue diamond), trial 2 (red square), and trial 3 (green triangle). .................................... 42	
square) with MGE conversion ([M]: [Al]: [I] = 200: 2: 1). ............................................ 43	
1). ................................................................................................................................. 44	
d) poly(PGE0.50) in CDCl3. ........................................................................................... 47	
d) poly(MGE0.50) in CDCl3. .......................................................................................... 48	
35 °C, at 1 mL/min flow rate. ........................................................................................ 50	
(green line), and poly(PGE0.57-Neg0.05) (blue line) in CDCl3 in aromatic region. ........... 54	
...................................................................................................................................... 56	
poly(MGE0.68-Neg0.10) (green line) in CDCl3 in aromatic region. .................................. 57	
(taken at 33 °C). ............................................................................................................ 61	
min. ............................................................................................................................... 62	

Figure 27. Photographs showing the lower critical solution temperature behavior of 
polymers p-MGE0.65 and trans-PhN=NPh@poly(MGE0.65) in Milli-Q water. a) 
poly(MGE0.65) (right) and trans-PhN=NPh@poly(MGE0.65) (left) below LCST (taken at 
25 °C) b) poly(MGE0.65) (right) and trans-PhN=NPh@poly(MGE0.65) (left) above LCST 

Figure 23. GPC traces of poly(MGE) (Mn = 21000, PDI = 1.20; black line) and 
poly(MGE0) (Mn = 49000, PDI = 1.20; red line). The polymers were analyzed in THF at 

Figure 24. 500 MHz 1H NMR spectra of poly(PGE0.60) (red line), poly(PGE0.57-Pos0.05) 

Figure 25. 500 MHz 1H NMR spectra of poly(MGE0.75) (red line), poly(MGE0.71-
Pos0.05) (green line), and poly(MGE0.71-Neg0.05) (blue line) in CDCl3 in aromatic region.

Figure 19. Variation of the number-average molar mass (black square) and PDI (red 

Figure 26. 500 MHz 1H NMR spectra of poly(MGE0.71-Neg0.05) (red line) and 

Figure 28. LCST determination via UV-vis spectrometer for poly(MGE0.70) (Mn = 
54,000, PDI = 1.21) in Milli-Q water (5 mg/mL) under various temperatures at 1 °C/3 

 

x 

Figure 33. LCST determination via DLS for poly(MGE0.70) (Mn = 54,000, PDI = 1.21) in 

Figure 34. Plots of LCSTs vs. the mole fraction of mDEG chains in poly(PGE0.X) by 
UV-Vis spectrometer (black circle) and by DLS (red triangle) and poly(MGE0.X) by UV-

Figure 35. LCSTs as a function of concentration of poly(MGE0.65) (blue circles) and 

Figure 31. LCST determination via UV-vis spectrometer at 450 nm for poly(MGE0.50) 
(Mn = 54,000, PDI = 1.19) in Milli-Q water (10 mg/mL) under various temperatures at 1 

Figure 32. LCST determination via UV-vis spectrometer at 450 nm for poly(MGE0.70) 
(Mn = 54,000, PDI = 1.21) in Milli-Q water (5 mg/mL) under various temperatures at 1 

Figure 29. Plots of LCSTs vs. the mole fraction of mDEG chains in poly(PGE0.X) and 
poly(MGE0.X) obtained by UV-Vis spectrometer under various temperatures at 1 °C/3 

Figure 30. Plots of LCSTs vs. the mole fraction of mDEG chains in poly(PGE0.X) and 

min except poly(MGE0.50) (at 1 °C/60 min). ................................................................. 64	
poly(MGE0.X), and poly(PGL0.X) obtained by UV-Vis spectrometer. ............................ 66	
°C/3 min (blue diamond) and 1 °C/60 min (red diamond). ............................................. 67	
°C/3 min........................................................................................................................ 68	
Milli-Q water (5 mg/mL) under various temperatures at 1 °C/3 min. ............................. 69	
Vis spectrometer (black diamond) and by DLS (red square). ......................................... 70	
polyphosphazene reported by Bi and co-workers (red circles)........................................ 73	
concentrations from 0 to 0.3 M. ..................................................................................... 75	
nm) in Milli-Q water at different concentrations. ........................................................... 76	
bar = 200 nm, 74 ± 45 nm) its LCST. ............................................................................ 77	
1.33), and trans-PhN=NPh@poly(PGE0.60) in Milli-Q water at room temperature. ....... 80	
nm) and trans-PhN=NPh@poly(MGE0.75) (red curve) in Milli-Q .................................. 82	

Figure 36. LCST values of poly(MGE0.75) (red square) measured in sodium bicarbonate 
solutions at concentrations from 0 to 0.3 M, poly(MGE0.71-Neg0.05) (blue circle) 
measured in sodium bicarbonate solutions at concentrations from 0 to 0.4 M, and 
poly(MGE0.71-Neg0.05) (orange circle) measured in sodium chloride solutions at 

Figure 40. a) DLS results for poly(MGE0.65) (blue curve, Mn = 52,000 g/mol, PDI = 
1.19, RH = 7 ± 2 nm) and trans-PhN=NPh@poly(MGE0.65) (red curve) in Milli-Q. b) 
DLS results for poly(MGE0.75) (blue curve, Mn = 52,000 g/mol, PDI = 1.22, RH = 6 ± 2 

Figure 38. TEM images of polyMGE0.65) (Mn = 52,000 g/mol, PDI = 1.19) a) below 
(taken at 100,000x, scale bar = 100 nm, 20 ± 4 nm) and b) above (taken at 40,000x, scale 

Figure 39. UV-vis spectra of trans-PhN=NPh, poly(PGE0.60) (Mn = 44,000 g/mol, PDI = 

Figure 37. DLS results for poly(MGE0.65) (Mn = 52,000 g/mol, PDI = 1.19, RH = 6 ± 2 

Figure 41. a) DLS results for poly(MGE0.65) (blue curve, Mn = 52,000 g/mol, PDI = 
1.19, RH = 7 ± 2 nm), trans-PhN=NPh@poly(MGE0.65) (orange curve, 50 mg/mL; green 

 

xi 

Figure 44. DLS results of SC (blue line), poly(MGE0.71-mPEG2000-Pos0.05) (orange 

Figure 45. DLS results of SC (dotted line) and SC@poly(MGE0.71-mPEG2000-Pos0.05) 

Figure 46. DLS results of SC (dotted line) and SC@poly(MGE0.71-mPEG2000-Neg0.05) 

Figure 47. UV-Vis spectra of the catalytic conversion of Suc-AAPF-pNA to p-

Figure 42. DLS results of SC (blue line), poly(MGE0.71-mPEG2000-Pos0.05) (orange 

Figure 43. DLS results of SC (blue line), poly(MGE0.71-mPEG2000-Pos0.05) (orange 

curve, dilute to 10 mg/mL from 50 mg/mL; red curve, 10 mg/mL) in Milli-Q. b) DLS 
results for poly(MGE0.75) (blue curve, Mn = 52,000 g/mol, PDI = 1.22, RH = 6 ± 2 nm) 
and trans-PhN=NPh@poly(MGE0.75) (orange curve, 50 mg/mL; green curve, dilute to 10 

mg/mL from 50 mg/mL; red curve, 10 mg/mL) in Milli-Q ............................................ 83	
line) and SC with 5 µL	of	poly(MGE0.71-mPEG2000-Pos0.05) added (green line) in Milli-
Q water. ........................................................................................................................ 85	
line) and SC with 50 µL	of	poly(MGE0.71-mPEG2000-Pos0.05) added (green line) in 
Milli-Q water. ............................................................................................................... 85	
line) and SC with	poly(MGE0.71-mPEG2000-Pos0.05) added (green line) in Milli-Q 
water. ............................................................................................................................ 86	
(solid line) in Milli-Q water over 48 h. .......................................................................... 88	
(solid line) in Milli-Q water over 48 h. .......................................................................... 88	
nitroaniline overtime. .................................................................................................... 90	
taken at 1 or 4 h after heated at 50 °C. ........................................................................... 93	
(solid line) in Tris-HCl buffer at 50 °C for 4 h. .............................................................. 95	
toluene (green line), and SC@ poly(MGE0.65) in toluene (red line). .............................. 97	
Figure A1. 500 MHz 1H NMR spectrum of PGE in CDCl3. ....................................... 118	
Figure A2. 500 MHz 1H NMR spectrum of PGE in CDCl3between 2.0-4.5 ppm. ....... 119	
Figure A3. 125 MHz 13C NMR spectrum of PGE in CDCl3. ...................................... 120	
Figure A4. 2D HSQC NMR spectrum of PGE in CDCl3. ........................................... 121	
Figure A5. 2D HSQC NMR spectrum of PGE in CDCl3. ........................................... 122	
Figure A6. 500 MHz 1H NMR spectrum of MGE in CDCl3. ...................................... 123	

Figure 48. UV-Vis spectra of the conversion of Suc-AAPF-pNA (blue solid line) to p-
nitroaniline (yellow solid line) by aliquots of SC (1 h, orange dotted line; 4 h green dotted 
line) or SC@poly(MGE0.71-mPEG750-Neg0.05) (1 h, purple solid line; 4 h, red solid line) 

Figure 49. DLS results of SC (dotted line) and SC@poly(MGE0.71-mPEG750-Neg0.05) 

Figure 50. UV-Vis spectra of SC suspension in toluene (blue line), poly(MGE0.65) in 

 

xii 

Figure A12. 125 MHz 13C NMR spectrum of poly(PGE) in CDCl3 between 25.0-35.0 

Figure A15. Mass spectrum of poly(PGE) (Mn = 2800, PDI = 1.14) via positive 

Figure A16. Mass spectrum of poly(MGE) (Mn = 3900, PDI = 1.16) via positive 

Figure A9. 2D HSQC NMR spectrum of MGE in CDCl3. Methyl region was omitted for 

Figure A7. 500 MHz 1H NMR spectrum of MGE in CDCl3 between 1.0-4.0 ppm. ..... 124	
Figure A8. 125 MHz 13C NMR spectrum of MGE in CDCl3. ..................................... 125	
clarity. ......................................................................................................................... 126	
Figure A10. 500 MHz 1H NMR spectrum of poly(PGE) in CDCl3. ............................ 127	
Figure A11. 125 MHz 13C NMR spectrum of poly(PGE) in CDCl3. ........................... 128	
ppm. ............................................................................................................................ 129	
Figure A13. 500 MHz 1H NMR spectrum of poly(MGE) in CDCl3. ........................... 130	
Figure A14. 125 MHz 13C NMR spectrum of poly(MGE) in CDCl3. .......................... 131	
electrospray ionization. ............................................................................................... 132	
electrospray ionization. ............................................................................................... 133	
square) with PGE conversion ([M]: [Al]: [I] = 200: 2: 1). ........................................... 134	
1). ............................................................................................................................... 134	
1 mL/min flow rate...................................................................................................... 135	
1 mL/min flow rate...................................................................................................... 135	
at 1 mL/min flow rate. ................................................................................................. 136	
at 1 mL/min flow rate. ................................................................................................. 136	
Figure A23. 500 MHz 1H NMR spectrum of poly(PGE0) in CDCl3. ........................... 137	
Figure A24. 125 MHz 13C NMR spectrum of poly(PGE0) in CDCl3. .......................... 138	
Figure A25. 500 MHz 1H NMR spectrum of poly(PGE0.20) in CDCl3. ........................ 139	
Figure A26. 125 MHz 13C NMR spectrum of poly(PGE0.20) in CDCl3. ....................... 140	

Figure A21. GPC traces of poly(PGE0.X). The polymers were analyzed in THF at 35 °C, 

Figure A17. Variation of the number-average molar mass (black square) and PDI (red 

Figure A18. Conversion vs. time of the polymerization of PGE ([M]: [Al]: [I] = 200: 2: 

Figure A19. GPC traces of poly(PGE). The polymers were analyzed in THF at 35 °C, at 

Figure A20. GPC traces of poly(MGE). The polymers were analyzed in THF at 35 °C, at 

Figure A22. GPC traces of poly(MGE0.X). The polymers were analyzed in THF at 35 °C, 

 

xiii 

Figure A27. 500 MHz 1H NMR spectrum of poly(PGE0.40) in CDCl3. ........................ 141	
Figure A28. 125 MHz 13C NMR spectrum of poly(PGE0.40) in CDCl3. ....................... 142	
Figure A29. 500 MHz 1H NMR spectrum of poly(PGE0.50) in CDCl3. ........................ 143	
Figure A30. 125 MHz 13C NMR spectrum of poly(PGE0.50) in CDCl3. ....................... 144	
Figure A31. 500 MHz 1H NMR spectrum of poly(PGE0.55) in CDCl3. ........................ 145	
Figure A32. 125 MHz 13C NMR spectrum of poly(PGE0.55) in CDCl3. ....................... 146	
Figure A33. 500 MHz 1H NMR spectrum of poly(PGE0.60) in CDCl3. ........................ 147	
Figure A34. 125 MHz 13C NMR spectrum of poly(PGE0.60) in CDCl3. ....................... 148	
Figure A35. 500 MHz 1H NMR spectrum of poly(PGE0.65) in CDCl3. ........................ 149	
Figure A36. 125 MHz 13C NMR spectrum of poly(PGE0.65) in CDCl3. ....................... 150	
Figure A37. 500 MHz 1H NMR spectrum of poly(PGE0.70) in CDCl3. ........................ 151	
Figure A38. 125 MHz 13C NMR spectrum of poly(PGE0.70) in CDCl3. ....................... 152	
Figure A39. 500 MHz 1H NMR spectrum of poly(PGE0.75) in CDCl3. ........................ 153	
Figure A40. 125 MHz 13C NMR spectrum of poly(PGE0.75) in CDCl3. ....................... 154	
Figure A41. 500 MHz 1H NMR spectrum of poly(PGE0.80) in CDCl3. ........................ 155	
Figure A42. 125 MHz 13C NMR spectrum of poly(PGE0.80) in CDCl3. ....................... 156	
Figure A43. 500 MHz 1H NMR spectrum of poly(PGE0.85) in CDCl3. ........................ 157	
Figure A44. 125 MHz 13C NMR spectrum of poly(PGE0.85) in CDCl3. ....................... 158	
Figure A45. 500 MHz 1H NMR spectrum of poly(PGE0.90) in CDCl3. ........................ 159	
Figure A46. 125 MHz 13C NMR spectrum of poly(PGE0.90) in CDCl3........................ 160	
Figure A47. 500 MHz 1H NMR spectrum of poly(PGE0.95) in CDCl3. ........................ 161	
Figure A48. 125 MHz 13C NMR spectrum of poly(PGE0.95) in CDCl3. ....................... 162	
Figure A49. 500 MHz 1H NMR spectrum of poly(PGE1.0) in CDCl3. ......................... 163	
Figure A50. 125 MHz 13C NMR spectrum of poly(PGE1.0) in CDCl3. ........................ 164	
Figure A51. 500 MHz 1H NMR spectrum of poly(MGE0) in CDCl3. .......................... 165	

 

xiv 

Figure A52. 125 MHz 13C NMR spectrum of poly(MGE0) in CDCl3.......................... 166	
Figure A53. 500 MHz 1H NMR spectrum of poly(MGE0.20) in CDCl3. ...................... 167	
Figure A54. 125 MHz 13C NMR spectrum of poly(MGE0.20) in CDCl3. ..................... 168	
Figure A55. 500 MHz 1H NMR spectrum of poly(MGE0.40) in CDCl3. ...................... 169	
Figure A56. 125 MHz 13C NMR spectrum of poly(MGE0.40) in CDCl3. ..................... 170	
Figure A57. 500 MHz 1H NMR spectrum of poly(MGE0.50) in CDCl3. ...................... 171	
Figure A58. 125 MHz 13C NMR spectrum of poly(MGE0.50). in CDCl3 ..................... 172	
Figure A59. 500 MHz 1H NMR spectrum of poly(MGE0.55) in CDCl3. ...................... 173	
Figure A60. 125 MHz 13C NMR spectrum of poly(MGE0.55) in CDCl3. ..................... 174	
Figure A61. 500 MHz 1H NMR spectrum of poly(MGE0.60) in CDCl3. ...................... 175	
Figure A62. 125 MHz 13C NMR spectrum of poly(MGE0.60) in CDCl3. ..................... 176	
Figure A63. 500 MHz 1H NMR spectrum of poly(MGE0.65) in CDCl3. ...................... 177	
Figure A64. 125 MHz 13C NMR spectrum of poly(MGE0.65) in CDCl3. ..................... 178	
Figure A65. 500 MHz 1H NMR spectrum of poly(MGE0.70) in CDCl3. ...................... 179	
Figure A66. 125 MHz 13C NMR spectrum of poly(MGE0.70) in CDCl3. ..................... 180	
Figure A67. 500 MHz 1H NMR spectrum of poly(MGE0.75) in CDCl3. ...................... 181	
Figure A68. 125 MHz 13C NMR spectrum of poly(MGE0.75) in CDCl3. ..................... 182	
Figure A69. 500 MHz 1H NMR spectrum of poly(MGE0.80) in CDCl3. ...................... 183	
Figure A70. 125 MHz 13C NMR spectrum of poly(MGE0.80) in CDCl3. ..................... 184	
Figure A71. 500 MHz 1H NMR spectrum of poly(MGE0.85) in CDCl3. ...................... 185	
Figure A72. 125 MHz 13C NMR spectrum of poly(MGE0.85) in CDCl3. ..................... 186	
Figure A73. 500 MHz 1H NMR spectrum of poly(MGE0.90) in CDCl3. ...................... 187	
Figure A74. 125 MHz 13C NMR spectrum of poly(MGE0.90) in CDCl3. ..................... 188	
Figure A75. 500 MHz 1H NMR spectrum of poly(MGE0.95) in CDCl3. ...................... 189	
Figure A76. 125 MHz 13C NMR spectrum of poly(MGE0.95) in CDCl3. ..................... 190	

 

xv 

Figure A81. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG550-Pos0.05) in CDCl3.

Figure A82. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG750-Pos0.05) in CDCl3.

Figure A83. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG2000-Pos0.05) in 

Figure A84. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG2000-Pos0.05) in 

Figure A77. 500 MHz 1H NMR spectrum of poly(MGE1.0) in CDCl3. ........................ 191	
Figure A78. 125 MHz 13C NMR spectrum of poly(MGE1.0) in CDCl3. ....................... 192	
Figure A79. 500 MHz 1H NMR spectrum of poly(PGE0.57-Pos0.05) in CDCl3. ............ 193	
Figure A80. 125 MHz 13C NMR spectrum of poly(PGE0.57-Pos0.05) in CDCl3. ........... 194	
.................................................................................................................................... 195	
.................................................................................................................................... 196	
CDCl3. ........................................................................................................................ 197	
CDCl3. Peak height was increased for clarity. .............................................................. 198	
Figure A85. 500 MHz 1H NMR spectrum of poly(PGE0.57-Neg0.05) in CDCl3. ........... 199	
Figure A86. 125 MHz 13C NMR spectrum of poly(PGE0.57-Neg0.05) in CDCl3. .......... 200	
.................................................................................................................................... 201	
.................................................................................................................................... 202	
CDCl3. ........................................................................................................................ 203	
CDCl3. Peak height was increased for clarity. .............................................................. 204	
.................................................................................................................................... 205	
Figure A92. 500 MHz 1H NMR spectrum of poly(MGE0.71-Pos0.05) in CDCl3. ........... 206	
Figure A93. 125 MHz 13C NMR spectrum of poly(MGE0.71-Pos0.05) in CDCl3. .......... 207	
CDCl3. ........................................................................................................................ 208	
CDCl3 ......................................................................................................................... 209	

Figure A87. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG550-Neg0.05) in CDCl3.

Figure A88. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG750-Neg0.05) in CDCl3.

Figure A89. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG2000-Neg0.05) in 

Figure A90. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG2000-Neg0.05) in 

Figure A94. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG550-Pos0.05) in 

Figure A95. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG750-Pos0.05). in 

Figure A91. 500 MHz 1H NMR spectrum of poly(PGE0.57-Neg0.025-Pos0.025) in CDCl3.

 

xvi 

Figure A102. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG2000-Neg0.05) in 

Figure A103. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG2000-Neg0.05) in 

Figure A100. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG550-Neg0.05) in 

Figure A101. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG750-Neg0.05) in 

Figure A96. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG2000-Pos0.05) in 

Figure A97. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG2000-Pos0.05) in 

CDCl3. ........................................................................................................................ 210	
CDCl3. Peak height was increased for clarity. .............................................................. 211	
Figure A98. 500 MHz 1H NMR spectrum of poly(MGE0.71-Neg0.05) in CDCl3. .......... 212	
Figure A99. 125 MHz 13C NMR spectrum of poly(MGE0.71-Neg0.05) in CDCl3. ......... 213	
CDCl3. ........................................................................................................................ 214	
CDCl3. ........................................................................................................................ 215	
CDCl3. ........................................................................................................................ 216	
CDCl3. Peak height was increased for clarity. .............................................................. 217	
.................................................................................................................................... 218	
Figure A105. 500 MHz 1H NMR spectrum of poly(MGE0.68-Neg0.10) in CDCl3. ........ 219	
(green), and poly(PGE0.57-Neg0.05) (blue) in CDCl3. .................................................... 220	
(green), and poly(PGE0.57-Neg0.05) (blue) in CDCl3 in aliphatic region. ....................... 221	
(green), and poly(MGE0.71-Neg0.05) (blue) in CDCl3. .................................................. 222	
(green), and poly(MGE0.71-Neg0.05) (blue) in CDCl3 in aliphatic region....................... 223	
Figure A110. 500 MHz 1H NMR spectrum of mDEG azide in CDCl3. ...................... 224	
Figure A111. 500 MHz 1H NMR spectrum of decyl azide in CDCl3. ......................... 225	
Figure A112. 500 MHz 1H NMR spectrum of mPEG550 OTs in CDCl3. ................... 226	
Figure A113. 125 MHz 13C NMR spectrum of mPEG550 OTs in CDCl3................... 227	
Figure A114. 500 MHz 1H NMR spectrum of mPEG550 azide in CDCl3. ................. 228	

Figure A109. 500 MHz 1H NMR spectra of poly(MGE0.75) (red), poly(MGE0.71-Pos0.05) 

Figure A108. 500 MHz 1H NMR spectra of poly(MGE0.75) (red), poly(MGE0.71-Pos0.05) 

Figure A104. 500 MHz 1H NMR spectrum of poly(MGE0.71-Neg0.025-Pos0.025) in CDCl3.

Figure A106. 500 MHz 1H NMR spectra of poly(PGE0.60) (red), poly(PGE0.57-Pos0.05) 

Figure A107. 500 MHz 1H NMR spectra of poly(PGE0.60) (red), poly(PGE0.57-Pos0.05) 

 

xvii 

Figure A115. 125 MHz 13C NMR spectrum of mPEG550 azide in CDCl3. ................ 229	
Figure A116. 500 MHz 1H NMR spectrum of mPEG750 OTs in CDCl3. ................... 230	
Figure A117. 500 MHz 1H NMR spectrum of mPEG750 azide in CDCl3. ................. 231	
Figure A118. 125 MHz 13C NMR spectrum of mPEG750 azide in CDCl3. ................ 232	
Figure A119. 500 MHz 1H NMR spectrum mPEG2000 OTs in CDCl3. ..................... 233	
Figure A120. 500 MHz 1H NMR spectrum of mPEG2000 azide in CDCl3. ............... 234	
Figure A121. 125 MHz 13C NMR spectrum of mPEG2000 azide in CDCl3. .............. 235	
Figure A122. 500 MHz 1H NMR spectrum of COOH azide in CDCl3. ...................... 236	
Figure A123. 125 MHz 13C NMR spectrum of COOH azide in CDCl3. ..................... 237	
Figure A124. 500 MHz 1H NMR spectrum of aminium azide in D2O. ....................... 238	
Figure A125. 125 MHz 13C NMR spectrum of aminium azide in D2O. ...................... 239	
Figure A126. Study of LCST behavior of poly(PGE0.65) via UV-vis. .......................... 240	
Figure A127. Study of LCST behavior of poly(PGE0.70) via UV-vis. ......................... 240	
Figure A128. Study of LCST behavior of poly(PGE0.75) via UV-vis. ......................... 241	
Figure A129. Study of LCST behavior of poly(PGE0.80) via UV-vis. ......................... 241	
Figure A130. Study of LCST behavior of poly(PGE0.85) via UV-vis. ......................... 242	
Figure A131. Study of LCST behavior of poly(PGE0.90) via UV-vis. ......................... 242	
Figure A132. Study of LCST behavior of poly(PGE0.95) via UV-vis. ......................... 243	
Figure A133. Study of LCST behavior of poly(PGE1.0) via UV-vis. ........................... 243	
Figure A134. Study of LCST behavior of poly(MGE0.55) via UV-vis. ........................ 244	
Figure A135. Study of LCST behavior of poly(MGE0.50) via UV-vis. ........................ 244	
Figure A136. Study of LCST behavior of poly(MGE0.65) via UV-vis. ........................ 245	
Figure A137. Study of LCST behavior of poly(MGE0.75) via UV-vis. ........................ 245	
Figure A138. Study of LCST behavior of poly(MGE0.80) via UV-vis. ........................ 246	
Figure A139. Study of LCST behavior of poly(MGE0.85) via UV-vis. ........................ 246	

 

xviii 

Figure A140. Study of LCST behavior of poly(MGE0.90) via UV-vis. ........................ 247	
Figure A141. Study of LCST behavior of poly(MGE0.95) via UV-vis. ........................ 247	
Figure A142. Hydrodynamic diameter of poly(PGE0.55) determined by DLS.............. 248	
Figure A143. Hydrodynamic diameter of poly(MGE0.60) determined by DLS. ........... 248	
Figure A144. Study of LCST behavior of poly(PGE0.60) via DLS............................... 249	
Figure A145. Study of LCST behavior of poly(PGE0.65) via DLS............................... 249	
Figure A146. Study of LCST behavior of poly(PGE0.70) via DLS............................... 250	
Figure A147. Study of LCST behavior of poly(PGE0.75) via DLS............................... 250	
Figure A148. Study of LCST behavior of poly(PGE0.80) via DLS............................... 251	
Figure A149. Study of LCST behavior of poly(PGE0.85) via DLS............................... 251	
Figure A150. Study of LCST behavior of poly(PGE0.90) via DLS............................... 252	
Figure A151. Study of LCST behavior of poly(PGE0.95) via DLS............................... 252	
Figure A152. Study of LCST behavior of poly(MGE0.60) via DLS. ............................ 253	
Figure A153. Study of LCST behavior of poly(MGE0.65) via DLS. ............................ 253	
Figure A154. Study of LCST behavior of poly(MGE0.70) via DLS. ............................ 254	
Figure A155. Study of LCST behavior of poly(MGE0.75) via DLS. ............................ 254	
Figure A156. Study of LCST behavior of poly(MGE0.80) via DLS. ............................ 255	
Figure A157. Study of LCST behavior of poly(MGE0.85) via DLS. ............................ 255	
Figure A158. Study of LCST behavior of poly(MGE0.90) via DLS. ............................ 256	
Figure A159. Study of LCST behavior of poly(MGE0.95) via DLS. ............................ 256	
Figure A160. Study of LCST behavior of poly(MGE1.0) via DLS............................... 257	
line) and SC with 5 µL	of	poly(PGE0.57-mPEG2000-Pos0.05) added (green line) in Milli-
Q water. ...................................................................................................................... 257	
line) and SC with 50 µL	of	poly(PGE0.57-mPEG2000-Pos0.05) added (green line) in 
Milli-Q water. ............................................................................................................. 258	

Figure A161. DLS results of SC (blue line), poly(PGE0.57-mPEG2000-Pos0.05) (orange 

Figure A162. DLS results of SC (blue line), poly(PGE0.57-mPEG2000-Pos0.05) (orange 

 

xix 

Figure A163. DLS results of SC (blue line), poly(PGE0.57-mPEG2000-Pos0.05) (orange 

Figure A164. DLS results of SC (blue line), poly(PGE0.57-mPEG2000-Pos0.05) (orange 

Figure A165. DLS results of SC (blue line), poly(PGE0.57-mPEG2000-Pos0.05) (orange 

Figure A166. DLS results of SC (blue line), poly(PGE0.57-mPEG2000-Pos0.05) (orange 

Figure A167. DLS results of SC (blue line), poly(MGE0.71-mPEG2000-Pos0.05) (orange 

Figure A168. DLS results of SC (blue line), poly(MGE0.71-mPEG2000-Pos0.05) (orange 

line) and SC with	poly(PGE0.57-mPEG2000-Pos0.05) added (green line) in Milli-Q water.
.................................................................................................................................... 258	
line) and SC with 5 µL	of	poly(PGE0.57-mPEG2000-Pos0.05) added (green line) in PBS.
.................................................................................................................................... 259	
line) and SC with 50 µL	of	poly(PGE0.57-mPEG2000-Pos0.05) added (green line) in PBS.
.................................................................................................................................... 259	
line) and SC with poly(PGE0.57-mPEG2000-Pos0.05) added (green line) in PBS.......... 260	
line) and SC with 5 µL	of	poly(MGE0.71-mPEG2000-Pos0.05) added (green line) in PBS.
.................................................................................................................................... 260	
line) and SC with 50 µL	of	poly(MGE0.71-mPEG2000-Pos0.05) added (green line) in 
PBS. ............................................................................................................................ 261	
line) and SC with	poly(MGE0.71-mPEG2000-Pos0.05) added (green line) in PBS. ....... 261	
SC@poly(MGE0.71-mPEG2000-Pos0.05) (solid line) in Milli-Q water over 48 h. ........ 262	
SC@poly(MGE0.71-mPEG2000-Neg0.05) (solid line) in Milli-Q water over 48 h. ........ 262	
temperature. ................................................................................................................ 263	
mg/mL; red square, 0.1 mg/mL; orange triangle, 1.0 mg/mL) over time at 40 °C. ........ 263	
mg/mL; red square, 0.1 mg/mL) over time at 50 °C. .................................................... 264	
50 °C. .......................................................................................................................... 264	
Neg0.05) (solid line) in Tris-HCl buffer at 50 °C for 4 h. ............................................... 265	

Figure A172. Concentrations of p-nitroaniline converted by SC (blue diamond, 0.04 
mg/mL; red square, 0.1 mg/mL; orange triangle, 1.0 mg/mL) over time at room 

Figure A170. DLS results of control experiments for SC (dotted line) and 

Figure A171. DLS results of control experiments for SC (dotted line) and 

Figure A173. Concentrations of p-nitroaniline converted by SC (blue diamond, 0.04 

Figure A174. Concentrations of p-nitroaniline converted by SC (blue diamond, 0.04 

Figure A175. Concentrations of p-nitroaniline converted by SC (1 mg/mL) over time at 

Figure A176. DLS results of SC (dotted line) and SC@poly(MGE0.71-mPEG750-

Figure A169. DLS results of SC (blue line), poly(MGE0.71-mPEG2000-Pos0.05) (orange 

 

xx 

 

 

 

 

xxi 

LIST OF SCHEMES 

 

Scheme 7. Proposed mechanism of copper(I)-catalyzed 1,3-dipolar cycloaddition via 

Scheme 10. Schematic representation of the properties of poly(PGL0.X) and 

Scheme 11. Synthesis and post-polymerization modification poly(PGL) and 

Scheme 12. Synthesis and polymerization of propargyl glycidyl ether (PGE) and 1,1´-

Scheme 1. Anionic ring-opening polymerization (AROP) of epoxides initiated by alkali 

Scheme 3. The “activated monomer” reaction mechanism for epoxide ROP, exemplified 

metal alkoxides.4 ............................................................................................................. 2	
Scheme 2. Chain transfer reaction in AROP.5 ................................................................. 3	
for the polymerization of propylene oxide (PO).1 ............................................................ 4	
Scheme 4. Epoxide polymerization with bimetallic cobalt catalyst and [PPN][OAc].12 ... 5	
Scheme 5. Post-polymerization modification of polymers ............................................... 6	
Scheme 6. Schematic representations of “click” reactions used as PPM. ......................... 8	
dinuclear copper intermediate.53 ...................................................................................... 9	
Scheme 8. Synthesis and PPM of poly(PGL) and poly(PGL0.X). .................................. 10	
Scheme 9. Schematic representation of LCST behavior. ............................................... 11	
poly(PGL0.X) as nanocarriers for hydrophobic and the hydrophilic guest molecules. ..... 30	
poly(PGL0.X).49 ............................................................................................................. 32	
dimethyl propargyl glycidyl ether (MGE). .................................................................... 33	
(Oct4NBr).8 ................................................................................................................... 38	
with mDEG azide and decyl azide. .............................................................................. 44	
azide. ............................................................................................................................ 51	
Scheme 16. Synthesis of mPEG550 azide, mPEG750 azide, and mPEG2000 azide. .. 52	
Scheme 17. Synthesis of COOH azide. ........................................................................ 52	

Scheme 15. PPM of poly(PGE) and poly(MGE) with mDEG azide, decyl azide, 
mPEG550 azide, mPEG750 azide, mPEG2000 azide, COOH azide, and aminium 

Scheme 13. Proposed mechanism of ring-opening polymerization of functionalized 
epoxides using triisobutylaluminum (i-Bu3Al) and tetraoctylammonium bromide 

Scheme 14. Post-polymerization modification (PPM) of poly(PGE) and poly(MGE) 

 

xxii 

Scheme 18. Synthesis of aminium azide. ..................................................................... 53	
Scheme 19. Schematic representation of LCST behavior............................................... 59	
Scheme 20. Schematic representative of azobenzene encapsulation............................... 79	
Scheme 21. Schematic representation of Subtilisin Carlsberg (SC) encapsulation. ........ 87	
SC@polymer................................................................................................................. 90	
Scheme 23. Schematic representation of SC encapsulation in toluene. .......................... 95	

Scheme 22. Catalytic hydrolysis of Suc-AAPF-pNA to p-nitroaniline by SC or 

 

 

 

 

 

xxiii 

KEY TO ABBREVIATIONS 

 

Allyl glycidyl ether 

Anionic ring-opening polymerization 

Atom transfer radical polymerization 

Boiling point 

Carbonic anhydrase B 

Copper-catalyzed alkyne-azide cycloaddition 

β-Cyclodextrin 

Cholesteryl group-bearing pullulan 

Critical micelle concentration 

Cetyltrimethylammonium bromide 

Doublet 

Chemical shift 

Dalton 

Doublet of doublets 

Dynamic light scattering 

N,N-Dimethylformamide 

Epichlorohydrin 

Ethoxy ethyl glycidyl ether 

Enzymatically synthesized glycogen 

Functional group 

Glycidyl methacrylate 

xxiv 

AGE 

AROP 

ATRP 

b.p. 

CAB 

CuAAC 

β-CD 

CHP 

cmc 

CTAB 

d 

δ 

Da 

dd 

DLS 

DMF 

ECH 

EEGA 

ESG 

FG 

GMA 

 

GPC 

h 

HSQC 

Hz 

IR 

J 

LC 

LCST 

m 

M 

m/z 

MALS 

mDEG 

mg 

MGE 

MHz 

min 

mL 

Mn 

mPGE 

MS 

MSPM 

Mw 

 

Gel permeation chromatography 

Hour 

Heteronuclear single quantum coherence 

Hertz 

Infrared spectroscopy 

Coupling constant 

Liquid chromatography 

Lower critical solution temperature 

Multiplet 

Molar 

Mass of ion (atomic units)/its charge number 

Multiangle light scattering detector 

Triethylene glycol monomethyl ether 

Milligram 

1,1´-Dimethyl propargyl glycidyl ether 

MegaHertz 

Minute 

Milliliter 

Number-average molecular weight 

Poly(ethylene glycol) monomethyl ether 

Mass spectrometry 

Mixed-shell polymeric micelles 

Weight-average molecular weight 

xxv 

MWCO 

NMR 

PBS 

PCL 

PDI 

PEG 

PEO 

PGE 

Poly(MGE) 

PMMA 

PNIPAM 

PO 

Poly(PGE) 

Poly(PGL) 

ppm 

PPM 

PPO 

PTFE 

RAFT 

rac 

RH 

ROP 

rt 

 

Molecular weight cutoff 

Nuclear magnetic resonance 

Phosphate buffered saline 

Poly(ε-caprolactone) 

Polydispersity index 

Poly(ethylene glycol) 

Poly(ethylene oxide) 

Propargyl glycidyl ether 

Poly(1,1´-dimethyl propargyl glycidyl ether) 

Poly(methyl methacrylate) 

Poly(N-isopropylacrylamide) 

Propylene oxide 

Poly(propargyl glycidyl ether) 

Poly(propargyl glycolide) 

Part per million 

Post-polymerization modification 

Poly(propylene oxide) 

Poly(tetrafluoroethylene) 

Reversible-addition fragmentation chain transfer 

Racemic 

Hydrodynamic radius 

Ring-opening polymerization 

Room temperature 

xxvi 

s 

SC 

SCNPs 

SEC 

Singlet 

Subtilisin Carlsberg 

Single-chain nanoparticles 

Size exclusion chromatography 

Suc-AAPF-pNA 

N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide 

t 

TEM 

tert 

Tg 

THF 

TOF 

Tris-HCl 

UV-Vis 

 

 

Triplet 

Transmission electron microscopy 

Tertiary 

Glass transition temperature 

Tetrahydrofuran 

Turnover frequency 

Tris(hydroxymethyl)aminomethane hydrochloride 

Ultraviolet-visible 

xxvii 

Chapter 1. Introduction 

1.1. Overview of Polyether Synthesis 

Polyethers represent an important class of polymers whose unique properties, in 

particular the high flexibility, low glass transition temperatures (Tg), and hydrophilicity, 

arise  from  their  C−O−C  backbones.  Polyethers  are  typically  synthesized  via  the  ring-

opening  polymerization  (ROP)  of  epoxide  monomers.1  Among 

the  polyethers, 

poly(ethylene oxide) (PEO, molecular weight > 30,000 g/mol)/poly(ethylene glycol) (PEG, 

molecular weight < 30,000 g/mol) is widely used in pharmaceutical, cosmetic, and medical 

applications  as  a  result  of  its  biocompatibility,  non-toxicity  and  non-immunogenic 

nature.1,2 More recent research has explored syntheses and applications of polyethers where 

the polyether backbone has pendant chemical functionality. These materials are most often 

prepared from epoxides bearing functional groups, like those in Figure 1.1,3,4  

 

O

Cl

O

O

O

O

epichlorohydrin (ECH)

allyl glycidyl ether (AGE)

propargyl glycidyl ether (PEG)

O

O

O

O

O

O

O

O

ethoxy ethyl glycidyl ether (EEGE)

glycidyl methacrylate (GMA)

1,1´-dimethyl propargyl glycidyl ether (MGE)

 

Figure 1. Examples of functionalized epoxides. 

 

 

 

1 

Scheme 1. Anionic ring-opening polymerization (AROP) of epoxides initiated by alkali 
metal alkoxides.4 

Initiation Step
O

R′

RO−Mt+
Mt = Na, K, Cs
R = H, alkyl, polymer…etc.

Propagation Step

RO

O

Mt

R′

O

n

R′

RO

O

Mt

R′

R′

O

Mt

RO

O

n

R′

Termination Step
R′

O

n

R′

O

Mt

RO

 

HR″

RO

R′
R″ = HO−, R2N−, RCOO−…etc.

R′

O

n

OH

+ R″−Mt+

 

PEO/PEG is often obtained by the anionic ring-opening polymerization (AROP) of 

ethylene oxide in the presence of metal hydroxide or alkoxide initiator (Scheme 1).1,4–6 

Alkali  metal  derivatives  (hydrides,  alkyls,  aryls, amides,  and  alkoxides)  are  commonly 

used initiators and the polymerizations are carried out in polar aprotic solvents when high 

molecular weights are desired. In AROP, the epoxide monomer undergoes nucleophilic 

attack by the anionic initiator to generate a new alkoxide species. Subsequent attack by this 

species  on  the  monomer  propagates  the  chain  in  an  iterative  fashion.  The  addition  of 

alcohols  or  water  terminates  AROP.  AROP  is  an  efficient  method  for  synthesizing 

PEO/PEG from ethylene oxide, but AROP of mono-substituted epoxides is hampered by 

chain transfer side reactions (Scheme 2),5 which broaden molecular weight distributions 

 

2 

for  the  polyethers.  Also,  the  resulting  polyethers  synthesized  from  mono-substituted 

epoxides by AROP are atactic in nature; therefore, the physical properties are different 

from  their  stereoregular  counterparts.  The  efforts  to  overcome  the  shortcomings  have 

resulted in two main approaches: the activation of the monomers and the formation of 

stereospecific reactive molecules. 

 

Scheme 2. Chain transfer reaction in AROP.5 

H
C ONa

CH2

+

O
H2C C
H

CH3

chain
transfer

CH2

H
C OH
CH3

+

O
H2C C
H

CH2Na

O

CH2

H3C C
H

O

CH2

H3C C
H

NaO

H2C
H
C
CH3

O

CH2

NaO

H2C
H
C
CH3

O

CH

CH2

CH3

C
H

C
H

O

CH2

H3C C
H

O

CH2

H3C C
H

NaO

CH2

NaO

CH

C
H

C
H

CH2

CH3

 

CH2

CH3

C
H

C
H

H2C
H
C
CH3

O

CH2

O

CH

H2C
H
C
CH3

 

The major breakthrough of the activation of epoxide monomers came from Carlotti, 

Deffieux, and co-workers in 2004 (Scheme 3).7 In the study, a “controlled” high speed 

AROP of propylene oxide (PO) was achieved and high molecular weight poly(propylene 

oxide) (PPO) (Mn up to 20,000 g/mol) was obtained. PO was activated by the addition of a 

Lewis acid to reduce the electron density on propylene oxide. Also, the initiator, alkali 

metal  alkoxides  and  the  Lewis  acid  catalyst  formed  an  initiating  complex  during  the 

initiation step. Hence, it is crucial to ensure the ratio between the catalyst and the initiator 

is greater than 1:1. To prevent the reaction between the catalyst and solvents, aprotic and 

non-coordinating 

solvents  are  preferred 

such  as  cyclohexane, 

toluene,  and 

 

3 

dichloromethane. To date, this method, using specifically triisobutylaluminum (i-Bu3Al) 

as  the  catalyst  and  tetraoctylammonium  bromide  (Oct4NBr)  as  the  initiator,  has  been 

applied to the synthesis of various mono-functionalized epoxides including the work will 

be presented in the following chapter.4,8 

 

Scheme 3. The “activated monomer” reaction mechanism for epoxide ROP, exemplified 
for the polymerization of propylene oxide (PO).1 

Epoxide activation

O

Al(i-Bu)3

Al(i-Bu)3
O

δ+

Initiating complex formation

+

Al(i-Bu)3
X−Y+
X = i-PrO, Cl, Br, N3
Y = Na, NBu4, NOCl4, PBu4

X

Al(i-Bu)3

−Y+

Initiation and propagation

X

Al(i-Bu)3

−Y+

Al(i-Bu)3
O

O

n

X

O

n

O Al(i-Bu)3

−Y+

Termination

X

O

n

 

O Al(i-Bu)3

−Y+

EtOH
− Al(OH)3
− YOH
− Isoprene

X

O

n

OH

 

The polymer stereochemistry, known as tacticity, greatly influences the physical 

property  of  the  polymers.9,10  For  instance,  PPO  synthesized  by  traditional  AROP  is  a 

flexible  material  with  a  low  Tg  due  to  its  atactic  (random  stereochemical)  backbone. 

However, isotactic PPO, where all methyl substituents are located on the same side of the 

zigzag  plane  of  the  extended  polyether  backbone,  is  semicrystalline.  Although  the 

discovery  of  isotactic  PPO  was  reported  in  1949,  further  advance  in  the  synthesis  of 

 

4 

stereoregular PPO was not made until recent years.10 Among the various catalysts, a cobalt-

based system developed by Coates and co-workers is especially attractive.11,12 In 2005, 

Coates and co-workers published the first catalyst to generate regioregular rac-isotactic 

PPO from rac-PO.11 As shown in Scheme 4, in 2008, a homogeneous bimetallic catalyst, 

which  is  activated  by  an  ionic  cocatalyst  salt,  bis(triphenylphosphine)iminium  acetate 

([PPN][OAc]),  was  used  for  the  polymerization  of  a  variety  of  mono-substituted 

epoxides.12  This  bimetallic  catalyst  and  cocatalyst  system  is  capable  of  kinetically 

resolving racemic epoxides into valuable enantiopure epoxides and isotactic polyethers 

with high turnover frequencies (TOFs) up to 30,000 h-1.12–14 This bimetallic cobalt catalyst 

system remains one of the most efficient system for synthesizing isotactic PPO but the 

synthesis of the ligand for the catalyst is non-trivial with low total yield. In 2018, Coates 

and  co-workers  reported  a  new  bimetallic  chromium  system  to  overcome  the  high 

polydispersity indices (PDIs) of the isotactic PPOs obtained by the cobalt system.15  

 

Scheme 4. Epoxide polymerization with bimetallic cobalt catalyst and [PPN][OAc].12 

tBu

(R)
N

O

(R)
N

O

Co
Cl

tBu

(S)

tBu

O

N
(R)

Cl
Co

O

N
(R)

tBu

O

(S)
R

O

(R)
R

+

 

[PPN][OAc]

O

(R)
R

+

(S)

O

R

n

 

 

5 

1.2. Overview of Post-Polymerization Modification 

Post-polymerization modification (PPM) of polymers is a valuable approach for 

synthesizing a range of polymers with diverse properties from a single polymer synthon.16–

23  PPM  not  only  overcomes  the  limited  functional  group  tolerance  of  many  common 

polymerization techniques, but it also reduces the number of synthetic steps when new 

polymers  are  synthesized.  Scheme  5  illustrates  the  PPM  concept.  Monomers  bearing 

certain  functional  groups  (FG1),  which  are  inert  toward  polymerization  conditions,  are 

polymerized. Subsequent reactions with modifying groups (FG2) allows to transform the 

polymer synthon into a variety of different polymers. 

 

Scheme 5. Post-polymerization modification of polymers 

monomer with site (FG1) for 
subsequent modification

M

polymerization

M

n

modifying group with 
“click” functionality (FG2)

R

“click” reaction

M

n

R

 

 

For PPM to be effective, the polymer synthon must possess a functional group that 

reacts with molecular or macromolecular entities with high fidelity and broad compatibility 

with other functionalities. In the most synthetically-flexible examples, reactions can be 

carried out in a wide range of solvents. It is possible in this way to prepare polymers that 

are  otherwise  difficult—even  impossible—to  prepare  by  direct  polymerization  of 

functional  co-monomers.  Among  the  various  synthetic  strategies  for  PPM,  “click” 

chemistry is a powerful and an effective way for fabricating polymer architectures.24–27  

 

6 

The term “click” chemistry was first introduced by Sharpless and co-workers in 

2001.28 It refers to reactions that are “modular, wide in scope, high yields, generating only 

inoffensive  byproducts  that  can  be  removed  by  nonchromatographic  methods,  and 

stereospecific.”  Copper(I)-catalyzed  1,3-dipolar  cycloaddition  of  azides  and  alkynes 

(CuAAC), developed simultaneously by Fokin, Sharpless and co-workers29 and Meldal and 

co-workers30,31 is one of the most used “click” reactions. Several other types of reaction 

have also been identified to fulfill these criteria such as thiol-ene addition,32–38 thiol-yne 

addition,39–41  Diels-Alder  cycloaddition,42,43  and  Michael  addition  (Scheme  6).38,44  

CuAAC24,45–51 for PPM will be discussed further since it is the “click” reaction used used 

for PPM in this thesis. 

 

 

 

 

7 

Scheme 6. Schematic representations of “click” reactions used as PPM. 

CuAAC

R

Thiol-ene
R
SH

Thiol-yne

R

SH

Diels-Alder

O

R

+

+

+

+

Michael addition

R

SH

+

N N N

R′

R′

R′

O

N

R′

O

O

R
N N

N

R′

R

R

S

R
S

R′

R′

S

O

R

O

N

R′

O

O

R

S

 

 

The classic thermal Huisgen 1,3-dipolar cycloaddition of azides and alkynes often 

yields a mixture of regioisomers hence it fails to meet the criteria of “click” chemistry.52 

However,  CuAAC  affords  the  1,4-disubstituted  regioisomer  with  high  fidelity  and  has 

broad functional group tolerance under a wide range of reaction conditions. Additionally, 

the products can usually be purified by nonchromatographic methods. This makes it a great 

fit  as  a  “click”  reaction.  Fokin  and  co-workers  conducted  extensive  research  on  the 

mechanism of CuAAC and found direct evidence for a dinuclear copper intermediate in 

CuAAC.53  Scheme  7  shows  the  proposed  mechanisms  of  CuAAC  from Fokin  and  co-

 

8 

workers. The reaction begins with the π-complexation of the acetylene to the first copper 

([Cu]a). Then coordination of the second copper ([Cu]b), is accompanied by proton loss and 

migration of [Cu]a to generate a s-bound acetylide.Then, the reversible coordination of the 

organic  azide  to  the  active  complex  results  in  the  formation  of  the  dinuclear  copper 

intermediate. After the generation of C-N bond and the removal of one copper, a triazolyl-

copper derivative is formed. Protonation of Cu-C bond releases the triazole product and 

reforms the initial copper catalyst. 

 

Scheme  7.  Proposed  mechanism  of  copper(I)-catalyzed  1,3-dipolar  cycloaddition  via 
dinuclear copper intermediate.53 

NN

R1

H+

N

R2

R1

H

H

[Cu]

[Cu]a

R1

H

NN

N

R2

[Cu]

R1

[Cu]

 

R2

[Cu]
[Cu]

N N
N

R1

R2
N

N
N
R1

[Cu]b

[Cu]a

[Cu]b

H+

[Cu]b

R1

[Cu]a

N3 R2

 

In previous research done in our group, “clickable” polyglycolides, poly(propargyl 

glycolide)  (poly(PGL))  were  synthesized  and  applied  CuAAC  as  PPM  to  modify  the 

properties  of  poly(PGL)s  (Scheme  8).49  Hydrophilic  azide,  1-(2-azidoethoxy)-2-(2-

methoxyethoxy)-ethane  (mDGE  azide),  and  hydrophobic  azide  (decyl  azide),  1-

 

9 

azidodecane, were used to synthesize the amphiphilic polymers, poly(PGL0.X), where X = 

the mole fraction of hydrophilic azide incorporated in the polymer. poly(PGL0.X) presented 

several properties that will be reviewed in the following sections. 

 

Scheme 8. Synthesis and PPM of poly(PGL) and poly(PGL0.X). 

 

O

x

O

O

+

O

RO

O

O

+

1-x

N3

H
n

O

N3

O

O

CuSO4 (5 mol %)
sodium ascorbate 

(12 mol %)
DMF, 2 h, rt

RO

3

O

N

N

N

1-x

O

N

N N
poly(PGL0.X)

9

O

x

H
n

 

O

BBA, SnII

O

O

O

 

1.3. Overview of Lower Critical Solution Temperature (LCST) 

PPM  is  also  a  convenient  tool  for  designing  stimuli-responsive  polymers.54–57 

Stimuli-responsive polymers, which are capable of chemical or conformational changes in 

response  to  external  stimuli,  have  attracted  attention  in  diverse  applications  such  as 

biomedical  and  drug  delivery,58–63  sensing,64–67  smart  surfaces,68  and  biological 

interfaces.69 Temperature,20,70–76 pH,77,78 and light,79–81 are a few examples of stimuli that 

can induce a change in polymer properties. Thermoresponsive polymers are an interesting 

class  of  stimuli-responsive  materials.  A  common  temperature  response  is  a  change  in 

polymer solubility. Certain polymers precipitate from solution, typically aqueous, when 

the temperature is increased. This phenomenon is referred to as lower solution temperature 

(LCST) behavior (Scheme 9).71,75,82–85 

 

 

10 

Scheme 9. Schematic representation of LCST behavior. 

H

bound water
H
OH
HO
H
O
H
O
H
H
H O H
H O
H
H
H
soluble coil

H

H
O
H
H O

H
HO
H
O

H
HO
O H
H
H
O H

O
H

T > LCST
T < LCST

H
O
H
H O H

H
HO

H

HO

H
OH

insoluble globule

H O
H

O H
H
O H
released water

H

Entropically unfavorable

 

Entropically favorable

 

An early challenge in polymer chemistry was understanding the deviation of phase 

behaviors of polymer solutions from the law for ideal solutions of ions and small molecules. 

Independently, Flory86,87 and Huggins88,89 proposed theories for these phase behaviors of 

polymer solutions, but explaining phase separation of polymer solutions at temperatures 

above the LCST remained a great challenge. Although LCST was known to exist in other 

systems,  the  first  experimental  results  of  LCST  phenomenon  in  non-polar  polymer 

solutions was observed in 1960 by Freeman and Rowlinson.90 In many polymer systems, 

the critical temperature was close to the boiling point of the solvent. Hence, the difficulty 

for observations was the result of the increasing system pressures at critical temperatures.91  

LCST behavior of polymer solutions can be understood in terms of the entropy of 

solvation, ΔSsolv. For some water-soluble polymers, the water molecules in the solvation 

sphere are highly ordered and ΔSsolv < 0. As the temperature is increased, the polymer 

precipitates when |TΔSsolv| > |ΔHsolv| (i.e., ΔGsolv > 0). One of the most studied polymers 

exhibiting LCST behavior in water are poly(N-isopropylacrylamide) (PNIPAM) and its 

copolymers for their potential in biomedical applications.60,65,66,75,92–94 Previous research 

done  in  our  group  on  amphiphilic  degradable  mDEG/alkyl-grafted  poly(propargyl 

 

11 

glycolide) polymers, p-PGL0.X, where X is the mole fraction of mDEG side chains, showed 

tunable LCST behavior by adjusting the ratio between hydrophilic and hydrophobic side 

chains.49,95 

 

1.4. Overview of the Formation of Unimolecular Micelles (Unimicelles) 

The term unimolecular micelles, first described by Newkome and co-workers,96,97 

was  used  to  refer  to  depicting  spherical  single molecular  polymers  that  are  capable  of 

molecule inclusion. These cascade polymers, now known as dendrimers, are one of the 

earliest examples of unimicelles.97,98 Since then, dendrimers have been attractive materials 

for various applications.99–106 Two major strategies have been developed for dendrimer 

synthesis,  the  so-called  divergent  and  convergent  synthesis.100,101,103,107  The  divergent 

method, also known as “inside-out” strategy, is the growth of a dendron from the core of 

the dendrimer to the molecular surface. The convergent method, also known as “outside-

in” strategy, starts the synthesis from the molecular surface inward to the focal point at the 

core.  Dendrimers  are  important  examples  of  unimicelles,  but  their  syntheses  require 

multiple steps and the number of branches from the core limits their size, which seldom 

exceeds 10 nm diameter. 

In the past two decades, interest in the formation of the single-chain polymeric 

nanoparticles  by  chain-collapse  has  increased.108–115  These  single-chain  nanoparticles 

(SCNPs)  also  exhibit  properties  associated  with  unimicelles.  Three  models  of  chain-

collapsing were proposed for the construction of SCNPs: homofunctional, heterofunctional, 

and cross-linker mediated collapse (Figure 2).115 Homofunctional collapse is where the 

polymer  chain  is  functionalized  with  one  type  of  reactive  pendent  groups  that  react 

 

12 

intramolecularly 

through  covalent  bonding  or  non-covalent 

interactions.  In 

the 

heterofunctional  collapse  model,  the  polymer  chain  carries  two  functional  side  chains, 

which undergo intramolecular reaction to form SCNPs. The cross-linker mediated collapse 

involves the reaction between a monofunctional polymer and bifunctional cross-linkers.  

 

Homofunctional collapse

Heterofunctional collapse

Cross-linker mediated collapse

 

Figure 2. Models for intermolecular cross-linking of a polymer chain.115 

 

Three synthetic approaches for intramolecular cross-linking have been employed 

to  the  formation  of  SCNPs:  irreversible  covalent,  reversible  covalent  (dynamic),  and 

noncovalent  cross-linking  (Table  1).115  “Click”  reactions,112  free  radical  coupling  of 

alkenes,116 and amine quaternization117 are few of the examples that have been used for 

irreversible  covalent  cross-linking.  Dynamic  covalent  cross-linking  is  achievable  by 

disulfide,118 hydrazone,119 and enamine chemistry.120 Hydrogen bonding,121 hydrophobic 

interaction,122 and metal complexation123 are often applied in noncovalent cross-linking. 

The  synthesis  of  SCNPs  allows for  precise  size control  and  tailored functionality. The 

 

13 

increasing  interest  in  SCNPs  also  prompted  further  research  on  their  potential 

applications.19,113,124–126 Although much progress has been made, challenges remain.114 

 

Table 1. Examples for cross-linking chemistry for the formation of SCNPs.115 

Cross-linking chemistry

(Model)

CuAAC “click” chemistry

(Hetero)

Free radical coupling

(Homo)

Amine quaternization

(Cross-linker)

Disulfide chemistry

(Homo)

Hydrazone chemistry

(Hetero)

Enamine chemistry

(Hetero)

Upy dimerization

(Homo)

 

FG precursors

Cross-linked structure

N3

O

O

+

or

+ X

N

X = I or Br

X

SH

O

+ O

O

+

O

NH2

N
H

NH2

O

N

N
H

N
H

N

O

O2N

N

N N

O

O

or

n

X−N

n

X−

N

S S

N

N
H

O

O

H
N

O

N
H
O

HN
N
H
N

N
H
N
NH

O
H
N

O

n

 

 

14 

1.5. Overview of Nanocarriers 

Nanoparticles, which can be prepared from metals, ceramics, polymeric materials, 

and composite materials, are defined as particles with at least one dimension sized between 

1  to  100  nm.  With  unique  size-dependent  properties  different  from  the  bulk  materials, 

extensive research has invested in exploring applications in electronics,127 energy,128,129 

and medical fields.130–139 The development of functional nanoparticles is a relatively new 

avenue  toward  advancement  in  biological  and  medical  fields  for  applications  such  as 

imaging, sensing, and divery.63,130,132,134,140,141 It has been shown that engineered functional 

nanoparticles can improve the solubility of poorly water soluble drugs, enhance drug half-

life  by  reducing  immunogenicity,  and  achieve  controlled  releasing  and  targeted 

delivery.130,131,139,142–144  To  date,  some  therapeutic  nanoparticle  platforms  have  been 

approved and commercialized, and many others have entered clinical trials (Figure 3).143 

In  the  following,  nanocarriers  will  be  used  to  describe  these  therapeutic  nanoparticle 

platforms.  

 

 

15 

Figure 3. Historical timeline of clinical-stage nanoparticle technologies. Reprinted from 
ref. 143. Copyright 2011 American Chemical Society.143 

 

 

Liposomes, first reported in 1960s,145,146 are spherical vesicles formed by one or 

more phospholipid bilayers with size between 50 to 100 nm.134 Liposomes are attractive 

nanocarriers  because  they  are  biocompatible,  biodegradable,  non-toxic,  and  non-

immunogenic. In addition, liposomes can encapsulate both hydrophobic and hydrophilic 

guest  molecules.131,134,139,141  The  similarity  between  liposomes  and  cellular  membranes 

assists in delivering the guest molecules into the cell. In 1995, liposomes became the first 

nanocarrier to reach commercialization with the FDA approval of DOXIL (doxorubicin-

liposome).143  Since  then,  liposomal  formulations  have  been  widely  utilized  for  in  vivo 

delivery.147 Despite their significant impact as nanocarriers, liposomes have limitations. 

For example, the common synthesis of liposomes first generates multilamellar vesicles, 

which have several bilayers, and the single vesicles that are obtained via sonication often 

have  broad  size  distributions.148  Further  processing  is  required  for  reducing  their 

 

16 

polydispersity. Moreover, burst release of guest molecules from liposomes is common and 

often undesirable.131,137,149  

Polymeric micelles, generated by self-assembly of amphiphilic block copolymers 

in aqueous solutions, have also been used as nanocarriers.130,131,139,141,144,150–153 Polymeric 

micelles  consist  of  hydrophobic  core  and  hydrophilic  corona,  and  are  thus  similar  to 

micelles constructed from conventional detergents, but are usually more stable owing to 

low critical micelle concentrations (cmc).133 One advantage of polymeric nanocarriers is 

that they can be prepared in a simple and reproducible manner with precise control over 

morphology and architecture.133,154–156 The tunability of the designs makes it possible to 

produce  stimuli-responsive  or 

targeted  delivery  vehicles.  Moreover,  polymeric 

nanocarriers are often made with biocompatible and biodegradable materials, which are 

important criteria for biomedical applications.  

 

HO

O

O

O

O

O

x

O
PGA-b-PLA (PLGA)

x = 0, PLA
y = 0, PGA

H

O

y

O

 

Figure 4. Structure of poly(lactic-co-glycolic acid) (PLGA).157 

 

Aliphatic  polyesters  derived  from  lactide,  glycolide,  and  ε-caprolactone  are 

especially attractive materials for delivery application.158 Delivery devices composed by 

US Food and Drug Administration(FDA)-approved poly(lactic-co-glycolic acid) (PLGA) 

polymers  have  been  an  important  component  in  some  commercialized  drugs  such  as 

Lupron Depot and Decapeptyl (Figure 4).157 PEG/PEO is one of the most studied non-

 

17 

degradable biocompatible polyethers and is widely used in composing nanocarriers. PEG 

is  often  used  with  PLA  in  block  copolymers  because  PLA  is  water  insoluble.  PEG’s 

hydrophilicity makes PLA-b-PEG polymers water soluble.159,160 Although there have been 

many  successes,  micelle  formation  is  still  concentration-dependent,  which  can  pose 

challenges for these polymeric nanocarriers (Figure 5).131,161,162 As mentioned in previous 

section, SCNPs, which can function as unimicelles in solution, have unique features as 

nanocarriers for delivery applications.19,113,124–126   

 

below cmc

above cmc

Block copolymers

Polymeric micelles

H2O

Amphiphilic polymers

Unimolecular micelles

 

Figure 5. Schematic illustration of the formation of polymeric micelles and unimicelles. 

 

1.6. Overview of the Synthesis of Biocatalysts 

As interest in nanocarriers as delivery vehicles has grown, research on coupling 

nanomaterials  and  biology,  dubbed  bionanotechnology,  has  also  blossomed.163,164  One 

particular  area  drawing  significant  attention  is  advancing  biocatalysis  by  conjugating 

 

18 

proteins  with  synthetic  polymers.165  As  shown  in  Figure  6,  the  first  polymer-protein 

conjugates, specifically, PEG-enzyme conjugates, were approved by FDA in 1990s, but 

the concept of PEG-protein conjugation, now known as PEGylation, was first envisioned 

and  pioneered  by  Davis  and  co-workers.166–168  PEGylation  is  now  a  well-established 

method that is used to enhance the solubility and stability of enzymes and to decrease the 

immunogenicity. Since then, many researches have been focused on developing various 

types of biocatalysts to expand applications of enzyme on different fronts.165,168–171 

 

Figure  6.  Timeline  of  nanotechnology-based  biocatalysts.  Reprinted  from  ref.  165. 
Copyright 2012 Springer Science+Business Media New York.165 

 

 

Enzyme  immobilization  can  be  an  effective  method  and  is  used  routinely  in 

industry.172–176  Enzyme 

immobilization 

facilitates 

their  handling, 

reusing,  and 

isolation …etc., therefore minimizing the protein contamination to other products during 

the process. It was also shown that immobilized enzymes have increased stability and/or 

 

19 

improved  activity  or  selectivity.174,176  Enzyme  immobilization  is  categorized  into  three 

types:  support  binding,  entrapment,  and  cross-linking.  Support  binding  can  be 

accomplished by hydrophobic or van der Waals interactions, electrostatic interactions, or 

covalent binding. Depending on the strength of the interactions, leaching or reusability are 

potential issues. Entrapment can be used to confine enzymes in a polymer matrix, but extra 

treatment  may  be  needed  to  prevent  enzyme  leaching.  Cross-linking  of  enzymes  is  a 

support-free approach that uses bifunctional reagents to make intermolecular connections 

between enzymes. Reaction rates can increase significantly because the concentration of 

active sites is higher than when enzymes are attached to an inert support.  

Polymer-enzyme  conjugates  offer  the  potential  of  merging  the  advantages  from 

both worlds.165,168–170,177,178 The implement of polymers renders the conjugates solubile in 

aqueous  or  organic  environments,  which  is  beneficial  for  substrates  with  poor  water 

solubility. By using stimuli-responsive polymers, recyclable polymer-enzyme conjugates 

can be designed that preserve the benefits of immobilization. In addition, the properties of 

polymer-enzyme conjugates can be modified by changing the polymer architecture and 

composition. Polymer-enzyme conjugates can be synthesized by two methods: “grafting 

to”  or  “grafting  from”.  “Grafting  to”  starts  with  presynthesized,  end-functionalized 

polymers whose functionality reacts with accessible amino acid side chains or end termini 

on the enzymes. A wide variety of monomers and polymers that can be used in the “grafting 

to”  method,  but  excess  polymer  reactants  is  often  required  to  fully  convert  the  native 

enzyme to the conjugate form. The separation of unreacted polymer from the conjugates 

can be difficult. In the “grafting from” method, the polymerization is initiated directly from 

the  surface  of  enzymes  via  controlled  radical  polymerization.  Atom  transfer  radical 

 

20 

polymerization (ATRP) and reversible-addition fragmentation chain transfer (RAFT) are 

often employed since both polymerizations can be performed under biological relevant 

conditions.  Separation  of  unreacted  monomer  is  easier  in  “grafting  from”  method  and 

obtaining high polymer density conjugates is achievable. 

 

1.7. Protein Folding and Molecular Chaperones 

Protein  folding  converts  newly  synthesized  polypeptide  chains  into  their  native 

three-dimensional structures.179–184 In the early 1960s, Anfinsen and co-workers conducted 

groundbreaking  protein  folding  experiments  with  ribonuclease  A  (RNase  A),  which 

catalyzes  the  cleavage  of  RNA  into  ribonucleotides.185–188  By  adding  and  removing  a 

denaturant and a reducing agent, the catalytic activity of RNase A was suppressed and 

restored, respectively. Assuming the catalytic activity of RNase A only exists when it folds 

into its proper three-dimensional structure, Anfinsen and co-workers concluded that RNase 

A was unfolded and refolded reversibly in vitro. They also demonstrated that the structural 

information of RNase A is encrypted in its amino acid sequence.185–188 These conclusions 

provided  the  foundation  for  the  thermodynamic  hypothesis  for  protein  folding.  The 

thermodynamic hypothesis states that the native structure of a protein is determined solely 

by the intrinsic properties of its amino acid sequence and is not the result of an external 

template. 

Thermodynamics is considered to be the governing factor for protein folding.187–189 

In the free-energy landscape of protein folding, shown in Figure 7, the native state of the 

protein resides at a local energy minimum and unfolded conformations occupy at higher 

energies,  where  each  of  the  conformations  folds  along  a  unique  pathway.  The  folding 

 

21 

energy landscape allows proteins to fold into their native structure within a reasonable 

amount of time. However, the unfolded conformation can also follow a reaction coordinate 

to undesired minima like amorphous aggregates and amyloid fibrils. 

 

Figure 7. Energy landscape scheme of protein folding and aggregation. Reprinted from ref. 
182. Copyright 2009 Nature America, Inc.182 

 

 

Hydrophobic  interactions  between  hydrophobic  protein  residues,  driven  by  the 

entropic  gains  as  water  molecules  are  expelled  from  the  solvation  sphere,  is  the  main 

driving force  for  protein folding.184,190–192  In  addition,  noncovalent  interactions  such  as 

hydrogen bonding,184 salt-bridge interaction,193 and aromatic-aromatic interaction194 can 

 

22 

further stabilize the native structure. Hydrophobic interactions are not only the key driving 

force for protein folding but also the cause of protein aggregation at high concentration of 

unfolded or partially folded proteins. When high concentration of unfolded or partially 

folded proteins are present in solution, the intermolecular hydrophobic interaction between 

the exposed hydrophobic residues can lead to amorphous aggregates or amyloid fibrils at 

or  near  the  global  minimum  of  reaction  coordinate.  Amyloid  fibrils,  the  irreversibly 

ordered  fibrils,  are related  to  neurodegenerative diseases,  such  as  Alzheimer’s  disease, 

Parkinson’s disease and spongiform neuro encephalopathies.187,195 Although the formation 

of amorphous aggregates or amyloid fibrils can be thermodynamically favored, cells have 

developed  strategies  to  avoid  or  reduce  the  formation  of  aggregates  in  the  early  stage 

(Figure 8). 

Figure 8. Protein fates in the proteostasis network. Reprinted from ref. 196. Copyright 
2011 Macmillan Publishers Limited.196 

 

 

23 

Molecular  chaperones,  whose  functions  include  preventing  protein  aggregation, 

assisting proper protein folding and assembly, and disassembling aggregates or misfolded 

proteins, are functionally related families of proteins.181,182,187,196–201 Molecular chaperones 

often convert between two states, driven by adenosine triphosphate (ATP) binding and 

hydrolysis. In one state, chaperones have the ability to bind unfolded or partially folded 

polypeptides via hydrophobic interactions to prevent aggregation. In the other state, the 

binding affinity decreases to release the protein. Molecular chaperones bind to the unfolded 

or  partially  folded  polypeptides  to  impede  protein  aggregation.  They  can  also  induce 

conformational changes in misfolded proteins to assist correct protein folding. 

One of the most studied molecular chaperone families is a bacterial chaperonin 

system, known as GroEL and GroES.182,187,197–199,202 The term “GroE” is from the name of 

a bacterial gene that was discovered and the “L” and “S” in GroEL and GroES indicates 

the larger and the smaller of the two subunits. Three structural domains can be found in 

GroEL, the apical domain containing binding sites for unfolded proteins, intermediate, and 

equatorial domain containing the ATP-binding site. The internal structure of GroEL is a 

hollow cylinder divided by the equatorial domain to form two cage-like chambers, known 

as Anfinsen cages. The co-chaperone GroES is a dome-like structure acts as a cap while 

binding to GroEL. GroES and ATP are bound to GroEL after the unfolded polypeptide 

chain enters GroEL. This isolated environment allows polypeptide to fold into the native 

state without aggregation, with the hydrolysis of ATP functioning as a timer to open the 

chambers and to release the folded protein (Figure 9).187 

 

 

24 

Figure  9.  Schematic  representation  mechanism  of  GroEL/GroES  chaperone-mediated 
protein  folding.  Reprinted  from  ref.  187.  Copyright  2013  Garland  Science,  Taylor  & 
Francis Group, LLC.187 

 

 

1.8. Artificial Chaperones 

Aggregation during protein folding can be eliminated in the presence of molecular 

chaperones. However, in the absence of molecular chaperones, protein aggregation is a 

common occurrence in vitro. The idea of artificial chaperons was first reported by Gellman 

and co-workers in 1995.203 Inspired by molecular chaperones, they used small molecules, 

 

25 

detergents  or  cyclodextrins,  to  discourage  the  aggregation  and  to  guide  the  folding 

process.203–205 Similar to molecular chaperones, this artificial chaperone system consists of 

two  consecutive  steps.  In  the  first  step,  a  cationic  detergent  (cetyltrimethylammonium 

bromide, CTAB) was added to the denatured protein solution to create protein-detergent 

complexes  and  to  prevent  denatured  proteins  from  aggregation.  In  the  second  step,  β-

cyclodextrin (β-CD) was introduced to the solution and to interact with the detergents and 

to allow proper refolding of the proteins. Figure 10 shows the schematic view of artificial 

chaperone-assisted  protein  refolding  vs.  additive-assisted  protein  refolding  reported  by 

Gellman and co-workers.204 

 

Dilute + detergent
(“capture step”)

Cyclodextrin

(“stripping step”)

Protein-detergent
complex (inactive)

Native Protein

(+ some aggregate)

Gdm-denatured

Protein

Dilute

Aggregate
(inactive)

HO

O

O
OH
HO

Dilute + additive

Native Protein

(+ some aggregate)

HO

N Br

CTAB

HO
O
O OH

OH
O
OH
O

HO

OH
O
OH

O

!-CD

OH

O
OH
O
HO

OH

O

OH
O
OH

OH

OH
OH
O

O

OH

 

Figure 10. Artificial chaperone-assisted protein refolding (uppermost path) vs. dilution 
additive-assisted  protein  refolding  (lowermost  path)  reported  by  Gellman  and  co-
workers.204 

 

 

26 

Since  2003,  Akiyoshi  and  co-workers  developed  series  of  polysaccharide-based 

self-assembled  nanogels,  cholesteryl  group-bearing  pullulan  (CHP)206–212  and  dodecyl 

group-bearing enzymatically synthesized glycogen (C12ESG),213 as artificial chaperones. 

Similar to the work by Gellman,203–205 CHPs showed the ability to complex with denatured 

proteins and to prevent the aggregation. Upon the addition of cyclodextrin to disassemble 

CHPs,  the  refolded  proteins  were  recovered  retaining  their  activity.  C12ESGs  also 

demonstrated the ability to capture denatured proteins. The release of the refolded proteins 

relied on the fine balance of hydrophobic interaction between the nanogels and the proteins; 

therefore, the addition of cyclodextrin was no longer required (Figure 11). 

 

Figure  11.  Schematic  illustration  of  the  C12ESG  nanogel-mediated  one-step  protein 
refolding  system.  Reprinted  from  ref  213.  Copyright  2013  The  Royal  Society  of 
Chemistry.213 

 

 

 

27 

Shi  and  co-workers  designed  temperature-responsive  mixed-shell  polymeric 

micelles (MSPMs) as a one-step artificial chaperone system that mimics the GroEL/GroES 

molecular  chaperones.214–219  The  MSPMs  usually  consisted  of  two  types  of  block 

copolymers. One is composed by a mixture of hydrophobic segment, poly(lactide) (PLA) 

or  poly(ε-caprolactone)  (PCL),  and  hydrophilic  segment,  PEG.  The  other  one  is 

constructed  with  the  same  hydrophobic  portion  and  a  temperature-responsive  portion, 

usually  PNIPAM.  Poly(2-(2-methoxyethoxy)  ethyl  methacrylate)  was  also  used  as  the 

temperature-responsive  section  with  a  LCST  around  26  °C.  After  self-assembly  of  the 

polymeric micelles, the MSPMs evolved into core-shell-corona micelles above the LCST 

of  the  temperature-responsive  polymer.  The  collapse  of  the  temperature-responsive 

polymer  in  the  mixed  shell  left  behind  cavities  surrounded  by  PEG  inner  walls.  The 

denatured  proteins  enter  these  cavities  and  refold.  Figure  12  shows  the  first  MSPMs-

assisted protein refolding published by Shi and co-workers. In this case, MSPMs were 

generated by PLA100-b-PEG45 and PLA125-b-PNIPAM180 and it effectively prevented the 

aggregation and assist the refolding of denatured carbonic anhydrase B (CAB). In more 

recent  reports,  Shi  and  co-workers  studied  the  effects  of  surface  charges  or  surface 

hydrophobic/hydrophilic ratio on the properties of MSPMs for designing more efficient 

artificial chaperones.217,218 

 

 

28 

Figure  12.  Schematic  representation  mechanism  of  MSPMs-assisted  protein  refolding. 
Reprinted  from  ref  214.  Copyright  2013  Wiley-VCH  Verlag  GmbH  &  Co.  KGaA, 
Weinheim.214 

 

 

As mentioned earlier, the formation of polymeric micelles is limited by their cmc. 

Hence, we explored the possibility of using SCNPs as artificial chaperones. In previous 

research,  our  group  examined  the  properties  of  poly(PGL0.X)  and  observed  that 

poly(PGL0.X)  formed  unimicelles  and  exhibited  tunable  LCSTs.49,95,220  To  survey  the 

potential  of  using  poly(PGL0.X)  as  nanocarriers, the  encapsulation  of  hydrophobic  and 

hydrophilic small guess molecules was investigated. Furthermore, poly(PGL0.X) was used 

 

29 

to  encapsulated  10  kDa  azobenzene-initiated  poly(methyl  methacrylate)  (azobenzene-

PMMA)  (Scheme  10).95  The  success  of  macromolecule  encapsulation  opens  up  the 

possibility  of  utilizing  poly(PGL0.X)  in  biomacromolecule-related  study  such  as 

nanocarriers for protein delivery and artificial chaperones for assisting protein refolding. 

The biocompatibility and biodegradability poly(PGL0.X) is desirable for some applications, 

degradability can pose challenges in synthesizing unimicelles and studying their properties. 

 

Scheme 10. Schematic representation of the properties of poly(PGL0.X) and poly(PGL0.X) 
as nanocarriers for hydrophobic and the hydrophilic guest molecules. 

Unimolecular

micelles

no cmc
H2O

Azobenzene
N N

p-PGL0.X

N

> LCST

Insoluble
globule

< LCST

with azobenzene 

10 kD PMMA
head group

OH

O

N

O
Cl

Rhodamine B

 

 

In  order  to  evaluate  the  effect  of  the  degradability,  we  have  designed  non-

degradable  “clickable”  polyether  analogs  of  poly(PGL),  poly(propargyl  glycidyl  ether) 

(poly(PGE)) and poly(1,1′-dimethyl propargyl glycidyl ether) (poly(MGE)), which were 

prepared  via  ROP  of  alkyne-functionalized  epoxides.  The  ROP  method  developed  by 

 

30 

Carlotti, Deffieux, and co-workers was employed considering the catalyst and the initiator 

are commercially available and controlled polymerization can be achieved. By applying 

PPM  vai  “ckick”  chemistry,  we  were  able  to  adjust  the  properties  of  the  “clickable” 

polyethers and the resulting amphiphilic polymers formed SCNPs as nanocarriers. Further 

modifications enabled us to install charge pendant groups on the polymer backbone and to 

increase  the  interaction  between  polymers  and  biomacromolecules.  The  results  will  be 

presented in the following chapters. 

 

 

 

 

31 

Chapter 2. Synthesis, Characterization, and Post-Polymerization 

Modification of Propargyl-Substituted Poly(ethylene oxide)s 

2.1. Introduction 

In  previous  research,  our  group  synthesized  a  “clickable”  polyglycolides  and 

applied  PPM  to  generate  degradable  mDEG/alkyl-grafted  poly(propargyl  glycolide) 

polymers, poly(PGL0.X), where X is the mole fraction of mDEG side chains (Scheme 11).49 

It was showed that LCSTs are between 25 ºC to 65 ºC for poly(PGL0.X) and it could be 

tuned by modifying ratios between hydrophilic and hydrophobic pendant groups. It was 

also  demonstrated  that  poly(PGL0.X)  forms  unimolecular  micelles  with  31  nm 

hydrodynamic  radius  in  Milli-Q  water.95  These  unimicelles  were  further  used  to 

encapsulate  hydrophobic  and  hydrophilic  guest  molecules  as  a  proof  of  concept  for 

delivery vehicles.95  

 

Scheme  11.  Synthesis  and  post-polymerization  modification  poly(PGL)  and 
poly(PGL0.X).49 

x

O

O

O

+

N3

O

BBA, SnII

O

RO

1-x

O

O

+

H
n

O

N3

O

O

CuSO4 (5 mol %)
sodium ascorbate 

(12 mol %)
DMF, 2 h, rt

RO

3

O

N

N

N

1-x

O

N

N N
poly(PGL0.X)

9

O

x

H
n

 

O

O

O

 

To further extend this concept and assess the effects of degradability, we designed 

and synthesized “clickable” poly(ethylene glycol)s (PEGs) polymers as non-degradable 

analogs of poly(propargyl glycolide). We envisioned the synthesis of these materials using 

 

32 

the  ring-opening  polymerization  scheme  developed  by  Carlotti,  Deffieux,  and  co-

workers8,221  to  polymerize  propargyl  glycidyl  ether  (PGE)  and  1,1′-dimethyl  propargyl 

glycidyl  ether  (MGE).  The  resulting  polymers  could  then  be  modified  via  CuAAC 

reactions of the polymers with azides. The polymer side chains significantly impact the 

LCST  behavior.  In  addition,  experiments  show  that  these  polymers  function  as 

unimolecular micelles in aqueous solution and do not aggregate when films are cast on 

surfaces below the LCST. 

 

2.2. Synthesis and Characterization of Monomers 

The preparation of PGE and MGE is outlined in Scheme 12. The synthesis of PGE 

was  modified  from  a  literature  report  from  the  reaction  of  (±)-epichlorohydrin  and 

propargyl  alcohol 

in  a  Williamson  ether  synthesis  using 

tetrabutylammonium 

hydrogensulfate (TBAHS) as a phase transfer reagent.222 Pure, colorless PGE was isolated 

as liquid in 76% yield upon distillation of the reaction mixture. The dimethyl analog, MGE, 

was synthesized in similar fashion from the reaction of (±)-epichlorohydrin and 2-methyl-

3-butyn-2-ol as a colorless oil in 50% yield.  

 

Scheme 12. Synthesis and polymerization of propargyl glycidyl ether (PGE) and 1,1´-
dimethyl propargyl glycidyl ether (MGE). 

O

Cl

+

HO

R R

 

 

O

NaOH (aq) 
TBAHS
0 °C to rt

O
R R

i-Bu3Al/Oct4NBr
toluene, -37 °C to rt

PGE, R = H
MGE, R = CH3

33 

H

O

Br

n

R

O

R

poly(PGE), R = H
poly(MGE), R = CH3

 

The 1H NMR spectrum of PGE is shown in Figure 13a. The acetylene proton (He) 

appears as a triplet centered at 2.44 ppm. This splitting pattern arises from 4JHH coupling 

with the diastereotopic propargyl methylene protons, Hd and Hd′. The two doublets centered 

at 4.19 ppm and 4.20 ppm correspond to Hd and Hd′. The two doublets of doublets centered 

at 3.46 ppm and 3.81 ppm correspond the diastereotopic methylene protons Hc, Hc′ that are 

alpha to the epoxide ring. Resonances for Ha, Ha′, and Hb are assigned based on data from 

related  structures  and  2D  HSQC.222 The  13C{1H}  NMR  spectrum  of PGE  is  shown  in 

Figure 14a, which shows six distinct signals. The epoxide carbons, C1 and C2, appear at 

44.18 and 50.40 ppm, respectively, and the acetylene carbons (C5 and C6) are observed at 

79.18 and 74.78 ppm, respectively. 

 

a) 

d, d¢ 

b) 

d, d¢ 

He

Ha
Ha′

O

Hc

Hb

O
Hd
Hc′

Hd′

c¢ 

c 

b 

a 

a¢ 

a-c 

a, a'

H

O

b

c, c'

O
d, d'

Br

n

e

e 

e 

Figure  13.  500  MHz  1H  NMR  spectra  of  a)  PGE  b)  poly(propargyl  glycidyl  ether) 
(poly(PGE)) in CDCl3. 

 

 

34 

 

a) 

b) 

6 

5 

2, 3 

1 

5 

6 

3 

O

1

2

3

4 

6

1 

O

4

5

2 

4 

H

O

1

3

Br

n

2

O
4
5

6

Figure  14.  125  MHz  13C  NMR  spectra  of  a)  PGE  b)  poly(propargyl  glycidyl  ether) 
(poly(PGE)) in CDCl3. 

 

 

Figure  15a  shows  the  1H  NMR  spectrum  of  MGE.  The  acetylene  proton,  He, 

appears as a singlet at 2.42 ppm and protons for the diastereotopic methyl groups, Hd and 

Hd′, appear as resolved singlets at 1.45 ppm and 1.46 ppm. The diastereotopic methylene 

protons Hc and Hc′ appear as doublets of doublets centered at 3.55 ppm and 3.75 ppm. The 

epoxide protons are assigned as above. The 13C{1H} NMR spectrum of MGE is shown in 

Figure 16a. Carbons for the diastereotopic methyl groups (C7 and C7′) are observed at 28.41 

and 28.66 ppm, respectively. Two peaks represent epoxide carbons, C1 and C2, appear at 

44.84 and 50.86 ppm, similar to the ones seen in PGE. The acetylene carbons, C5 and C6, 

are shown at 85.52 and 72.35 ppm, respectively.  

 

35 

a) 

b) 

He

Ha
Ha′

O

Hc

Hb

O
Hc′

d, d′

e 

c¢ 

c 

b 

a 

a¢ 

a, a'

O

Br

n

H

O

b

c, c'

d

d'

e

e 

a-c 

d, d¢ 

d, d¢ 

Figure 15. 500 MHz 1H NMR spectra of a) MGE b) poly(1,1′-dimethyl propargyl glycidyl 
ether) (poly(MGE)) in CDCl3. 

 

 

 

36 

a) 
O

1

2

3

6

O

4

7

5

7'

5 
b) 
H

O

5 

1

3
7

2

Br

n

O
4
5

7′

6

1 

3 

1 

2 

6 

4 

4 

6 

2, 3 

7, 7′ 

7, 7′ 

 

Figure 16. 125 MHz 13C NMR spectra of a) MGE b) poly(1,1′-dimethyl propargyl glycidyl 
ether) (poly(MGE)) in CDCl3 

 

2.3. Living Anionic Ring-opening Polymerization 

Anionic ring-opening polymerization of epoxides is one of the most studied and 

important methods for epoxide polymerization. In 2008, Carlotti, Deffieux and co-workers 

reported a new living/controlled polymerization of functionalized epoxides, which operates 

via an anionic coordination mechanism.8,221 In the reports, triisobutylaluminum (i-Bu3Al) 

was  used  as  the  catalyst  and  tetraoctylammonium  bromide  (Oct4NBr)  was  used  as  the 

initiator,  which  resulted  in  controlled  molecular  weight  polymers  under  moderate 

conditions. Scheme 13 shows the proposed mechanism of the ring-opening polymerization 

of functionalized epoxides. Two events were proposed including the formation of initiating 

complex and the activation of the monomer in the initiation step. When the catalyst and 

 

37 

initiator ratio is greater than one, the initiating complex will be formed, and the excess 

catalyst will activate the corresponding monomer by coordinating with the oxygen atom 

on  the  epoxide.  Nonetheless,  to  the  best  of  our  knowledge,  there  is  no  spectroscopic 

evidence for the bromide end group in these polymers. 

 

Scheme  13.  Proposed  mechanism  of  ring-opening  polymerization  of  functionalized 
epoxides  using 
tetraoctylammonium  bromide 
(Oct4NBr).8 

triisobutylaluminum  (i-Bu3Al)  and 

Oct4NBr

O

R

+ Al(i-Bu)3

 

Br - Oct4N+
(i-Bu)3Al
initiating complex formation

+

δ+

O

Al(i-Bu)3

R
monomer activation

Br

-

Oct4N+

O Al(i-Bu)3
R

propagation

termination

+ Al(i-Bu)3

Br

H

n

O

R

 

Polymerizations of PGE and MGE were performed in toluene using i-Bu3Al as the 

catalyst and Oct4NBr as the initiator. The reagents were combined at -37 °C and the stirred 

reaction mixtures were allowed to warm to room temperature, after which stirring was 

continued from 2.5 to 24 h. Poly(propargyl glycidyl ether) (poly(PGE)) and poly(1,1′-

dimethyl propargyl glycidyl ether) (poly(MGE)) were obtained in good yields (Table 2). 

As shown in Table 2, the [i-Bu3Al]/[Oct4NBr] ratio was 2:1 for all polymerizations of PGE. 

Polymerization conversions were calculated by comparing the 1H NMR integrations of the 

monomer epoxide peaks at 2.66 ppm with those on the polymer backbone at 3.52-3.63 ppm 

(Figure 13b). Resonances from the pendant propargyl groups of poly(PGE) were observed 

in 1H NMR spectra at 2.46 ppm (He) and the alkyne carbon (C5 and C6) resonances appear 

 

38 

in  13C  NMR  spectra  at  79.82  and  74.56  ppm  (Figure  14b),  respectively,  similar  to  the 

resonances observed in PGE (79.18 and 74.78 ppm). It is worth noting that carbons on the 

polymer backbone (C1, C2, and C3) showed multiple peaks compared to PGE. This can be 

attributed  to  variations  in  the  poly(PGE)  backbone  stereochemistry  and/or  the 

regioselectivity of ROP for the epoxide. 

The MGE monomer was polymerized in analogous fashion except for entry 10 of 

Table 2, where the [i-Bu3Al]/[Oct4NBr] ratio was increased to 3:1 from 2:1. Polymerization 

conversions  were  determined  from  1H  NMR  spectra  as  described  above  for  PGE  by 

comparing the 1H NMR integration of the residual epoxide peak at 2.64 ppm with those on 

polymer  backbone  at  3.56-3.63  ppm  (Figure  15b).  The  pendant  alkyne  group  was 

confirmed by 1H NMR spectrum at 2.43 ppm and 13C NMR spectrum at 72.2 and 86.1 ppm 

(Figure 15b and Figure 16b). The acetylene proton (He) is observed at 2.43 ppm and the 

alkyne carbons appear as multiple peaks at 72.17-72.27 ppm (C6) and 86.06 and 86.08 ppm 

(C5). More resonances were also observed that the tertiary carbon (C4) and carbons on 

polymer  backbone  (C1,  C2,  and  C3)  compared  to  poly(PGE).  It  is  proposed  that  the 

dimethyl  groups  impede  the  rotation  of  the  side  chains  and  cause  the  differences  in 

chemical environment. 

It is worth mentioning that, in  13C NMR spectrum of poly(PGE), a peak at 29.6 

ppm is observed, which could be assigned to the carbon attached to the bromine end group. 

In  the  MS  spectra  of  low  molecular  weight  poly(PGE)  (Mn  =  2800,  PDI  =  1.14)  and 

poly(MGE) (Mn = 3900, PDI = 1.16) (Figure A15 and Figure A16, Appendix), the distinct 

isotope peaks for bromine are also detected. 

 

 

 

39 

Table 2. Results of polymerization of PGE and MGE. 
Conversion 

Entry  Monomer 

Mntheory 
(g mol-1)b 

Mnexp 
(g mol-1)c  PDI 
2800 
1.14 
1.30 
5500 
1.19 
10000 
1.26 
19000 
3900 
1.16 
1.15 
7000 
1.17 
12000 
24000 
1.24 
1.10 
22000 
31000 
1.33 
aDetermined by 1H NMR. b Calculated from the monomer to initiator ratio and corrected 
for conversion. c Measured by GPC in THF via light scattering. 

[M]: 
[Al]: [I] 
25: 2: 1 
50: 2: 1 
100: 2: 1 
200: 2: 1 
25: 2: 1 
50: 2: 1 
100: 2: 1 
200: 2: 1 
500: 2: 1 
500: 3: 1 

Time 
(h) 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
24 
24 

2800 
5500 
11000 
22000 
3500 
7000 
14000 
28000 
34000 
51000 

PGE 
PGE 
PGE 
PGE 
MGE 
MGE 
MGE 
MGE 
MGE 
MGE 

(%)a 
> 99 
98 
> 99 
97 
> 99 
> 99 
> 99 
> 99 
48 
73 

1 
2 
3 
4 
5 
6 
7 
8 
9 
10 

Typical results for the polymerization of PGE and MGE are listed in Table 2. The 

molecular weights of poly(PGE) and poly(MEG), measured by GPC in THF via light 

scattering, were in close agreement with the theoretical values with narrow to moderate 

polydispersities. The refractive index increment (dn/dc) is the change in refractive index 

of  a  solution  at  a  given  increment  in  concentration.  The  dn/dc  values  for  poly(PGE) 

(0.0939 mL/g, Figure 17) and poly(MGE) (0.0750 mL/g, Figure 18) were measured and 

applied  during  data  processing.  Weight-average  molecular  weight  (Mw)  and  dn/dc  are 

connected through Zimm equation. A simplified version of the Zimm equation is shown 

below223,224 

where Rθ	is the	Rayleigh ratio, c	is	the sample concentration, θ	is	the measurement angle, 

A2 is the second Virial coefficient, and K is defined as 

$%&'=)1+,+2/0%112' 
$= 4;0
<=>?@)A=BAB%10

 

 

40 

where λ0 is the laser wavelength in a vacuum, NA is Avogadro’s number, and n0 is the 

refractive index of the solvent.   

Pq is the single chain form factor, whose reciprocal is given by 

12'=1+16;0A=0&D0
3<=0

sin0)F21 

where Rg is the molecule’s radius of gyration. 

 

Figure 17. Measurements of refractive index increment, dn/dc, of poly(PGE). Trial 1 (blue 
diamond), trial 2 (red square), and trial 3 (green triangle). 

 

 

41 

Figure 18. Measurements of refractive index increment, dn/dc, of poly(MGE). Trial 1 
(blue diamond), trial 2 (red square), and trial 3 (green triangle). 

 

 

In the attempts to synthesize polymers with higher molecular weights in entries 9 

and 10, a higher [i-Bu3Al]/[Oct4NBr] ratio was necessary to increase the conversion and 

molecular weight. However, full conversion was not achieved with 3:1 [i-Bu3Al]/[Oct4NBr] 

ratio. A higher ratio may be needed to complete the polymerization. 

The  progression  of  poly(MGE)  number-average  molecular  weight  (Mn)  with 

monomer  conversion  is  shown  in  Figure  19.  A  linear  increase  of  the  Mn  with  percent 

conversion is observed. The linear relationship between Mn and percent conversion and 

low  PDIs  indicate  a  controlled  polymerization  mechanism  without  any  noticeable 

undesired  reactions.  A  kinetic  study  of  poly(MGE)  consumption  with  time  at  room 

temperature, following initiation at -37 °C, was also performed (Figure 20). It revealed two 

 

42 

distinct polymerization regimes. The polymerization is rapid up to ~85% conversion, after 

which  the  polymerization  slows  down.  These  results  are  consistent  with  the  previous 

reports by Carlotti and co-workers.8,221 

 

25000

20000

15000

10000

)
l
o
m
g
(

/

n

M

5000

0

0

20

40
Conversion (%)

60

80

2
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
100

I

D
P

 

Figure 19. Variation of the number-average molar mass (black square) and PDI (red square) 
with MGE conversion ([M]: [Al]: [I] = 200: 2: 1). 

 

43 

100

80

60

40

20

)

%

i

(
 
n
o
s
r
e
v
o
C

0

0

50

100

Time (min)

150

200

 

Figure 20. Conversion vs. time of the polymerization of MGE ([M]: [Al]: [I] = 200: 2: 1). 

 

2.4. Post-polymerization Modification (PPM) via “Click” Chemistry 

Scheme 14. Post-polymerization modification (PPM) of poly(PGE) and poly(MGE) with 
mDEG azide and decyl azide. 

x

O

O

N3

O

+

CuSO4 (5 mol %)
sodium ascorbate

(12 mol %)
DMF, rt, 3.5 h

H

O

Br

n

R

O

R

poly(PGE), R = H
poly(MGE), R = CH3

+

N3

 

1-x

 

O

O

O

N

N

N

R

O

R

H

O

O

1-x

Br

x n

R

O

R

N

N

N

poly(PGE0.X), R = H
poly(MGE0.X), R = CH3

 

44 

To  further  extend  the  scope  of  the  polymer  properties,  “click”  chemistry, 

specifically copper(I)-catalyzed 1,3-dipolar cycloaddition of azides and alkynes (CuAAC), 

was used for PPM. The synthesis of “click” modified polymers is illustrated in Scheme 14. 

The “click” reaction was performed in the presence of CuSO4•5H2O and sodium ascorbate 

in  DMF  at  room  temperature.  1-(2-Azidoethoxy)-2-(2-methoxyethoxy)ethane  (mDEG 

azide) was synthesized from triethylene glycol monomethyl ether and sodium azide.95 Also, 

1-azidodecane  (decyl  azide)  was  synthesized  from  1-bromodecane  and  sodium 

azide.95,225,226 To synthesize polymer poly(PGE0.X) the desired amount of mDEG azide 

and/or decyl azide, and 12 mol % sodium ascorbate were dissolved in DMF (all equivalents 

and mole percentages are with respect to acetylene units in poly(PGE) (Mn = 18000, PDI 

= 1.35 or Mn = 19000, PDI = 1.26). Then, 5 mol % of CuSO4•5H2O in DMF was added 

via syringe. After the desired reaction time, the residual copper was removed by stirring 

the polymer solution with ion exchange resin beads (Amberlite® IRC-748 ion exchange 

resin) for 16-24 hours. The polymers were purified by dialysis (molecular weight cutoff 

(MWCO) = 6-8 kDa).49,95 Similar procedures were followed to synthesize poly(MGE0.X) 

from poly(MGE) (Mn = 22000, PDI = 1.13, Mn = 21000, PDI = 1.20). By varying the ratio 

of mDEG azide and decyl azide, a series of polymers poly(PGE0.X) and poly(MGE0.X) 

(X is mole percent of mDEG azide) were obtained. 

In  1H  NMR  spectra,  the  absence  of  acetylene  protons  at  2.44  ppm  poly(PGE) 

(Figure  21)  and  2.43  ppm  poly(MGE)  (Figure  22)  confirmed  that  the  reactions  were 

complete after 3.5 hours. As shown in Figure 21b, c, and d, triazole proton resonances at 

7.66 ppm and/or 7.76 ppm are observed for poly(PGE0.X). The spectrum of poly(PGE0) 

shows that the triazole proton for alkyl side chain is the upfield resonance, 7.66 ppm, and 

 

45 

in the spectrum of poly(PGE1.0), the triazole proton for mDEG side chain is the down field 

resonance, 7.76 ppm. Likewise, new peak(s) for the proton on the triazole ring appear(s) at 

7.59 ppm and/or 7.66 ppm for poly(MGE0.X) (Figure 22b, c, and d), where the proton for 

the triazole for alkyl side chain is upfield and for alkyl side chain is downfield. The ratio 

of the incorporated mDEG azide and decyl azide in polymer side chain was calculated by 

the integration of the triazole protons and it was within 2% error of the molar ratio used in 

the reaction. 

 

 

 

46 

a) 

b) 

c) 

d) 

H

O

Br

n

O

poly(PGE)

CDCl3 

O

H

Br

n

O

N

N

N
poly(PGE0)

9

O

H

Br

n

O

N

N

N

poly(PGE1.0)

O

3

O

N

N

N

3

O

Br

0.50 n

H

O

O

0.50

O

N

N

N

9

poly(PGE0.50)

H2O 

Acetone 

H2O 

Figure 21. 500 MHz 1H NMR spectra of a) poly(PGE) b) poly(PGE0) c) poly(PGE1.0) d) 
poly(PGE0.50) in CDCl3. 

  

 

 

47 

a) 

b) 

c) 

d) 

H

O

Br

n

O

poly(MGE)

CDCl3 

O

H

Br

n

O

N

N

N
poly(MGE0)

9

O

H

Br

n

O

N

N

N

poly(MGE1.0)

O

3

O

N

N

N

3

O

Br

0.50 n

H

O

O

0.50

O

N

N

N

9

poly(MGE0.50)

Acetone 

  

Figure 22. 500 MHz 1H NMR spectra of a) poly(MGE) b) poly(MGE0) c) poly(MGE1.0) 
d) poly(MGE0.50) in CDCl3. 

 

48 

The increase in the molecular weight of the resulting polymer poly(PGE0.X) and 

poly(MGE0.X) was confirmed by size exclusion chromatography coupled with a multiangle 

light scattering detector (SEC-MALS) (Table 3 and Table 4). The experimental values of 

poly(PGE0.X) and poly(MGE0.X) are in good agreement of calculated values with slight to 

no change in polydispersity, which is consistent with high fidelity in the “click” reacion. 

In Figure 5, the GPC traces for poly(MGE) (Mn = 21000, PDI = 1.20) and the product from 

its “click” reaction with decyl azide, poly(MGE0) (Mncalc. = 50000, Mnexpt. = 49000, PDI = 

1.20),  have  similar  retention  times  with  no  noticeable  change  in  peak  shape  and 

polydispersity. The small difference in retention times despite a 2.3-fold increase in Mn is 

likely  due  to  similar  hydrodynamic  volumes  for  poly(MGE)  and  highly  branched 

poly(MGE0), as has been observed for other branched polymers.227,228  

 

 

49 

poly(GHI)
poly(GHI₀)

16

16.5

17

 

13.5

14

14.5

15

15.5

Retention Time (min)

Figure  23.  GPC  traces  of  poly(MGE)  (Mn  =  21000,  PDI  =  1.20;  black  line)  and 
poly(MGE0) (Mn = 49000, PDI = 1.20; red line). The polymers were analyzed in THF at 
35 °C, at 1 mL/min flow rate. 

 

Table 3. GPC results of poly(PGE0.X). 
mol % 
mDEG 

 (g mol-1)a 

Mnorig 

aMeasured by GPC in THF via light scattering. 

 

50 

18000 
18000 
20000 
18000 
20000 
18000 
20000 
20000 
20000 
18000 
20000 
20000 
20000 
18000 

0 
20.3 
40.7 
48.2 
54.4 
60.0 
62.9 
71.9 
72.5 
78.1 
82.4 
90.3 
95.4 
100 

Mntheory 
(g mol-1) 
49000 
49000 
49000 
49000 
49000 
49000 
49000 
49000 
49000 
49000 
49000 
50000 
50000 
50000 

Mnexp 

(g mol-1)a 

27000 
35000 
41000 
46000 
44000 
44000 
45000 
50000 
53000 
47000 
51000 
55000 
52000 
53000 

Mwexp 

(g mol-1)a 

35000 
45000 
48000 
54000 
51000 
59000 
54000 
58000 
60000 
53000 
57000 
61000 
59000 
58000 

PDI 
1.30 
1.29 
1.17 
1.17 
1.16 
1.34 
1.20 
1.16 
1.13 
1.13 
1.12 
1.11 
1.13 
1.09 

Polymer 
poly(PGE0) 
poly(PGE0.20) 
poly(PGE0.40) 
poly(PGE0.50) 
poly(PGE0.55) 
poly(PGE0.60) 
poly(PGE0.65) 
poly(PGE0.70) 
poly(PGE0.75) 
poly(PGE0.80) 
poly(PGE0.85) 
poly(PGE0.90) 
poly(PGE0.95) 
poly(PGE1.0) 

 

Table 4. GPC results of poly(MGE0.X). 
mol % 
mDEG 

(g mol-1)a 

Polymer 

Mnorig 

poly(MGE0) 
poly(MGE0.20) 
poly(MGE0.40) 
poly(MGE0.50) 
poly(MGE0.55) 
poly(MGE0.60) 
poly(MGE0.65) 
poly(MGE0.70) 
poly(MGE0.75) 
poly(MGE0.80) 
poly(MGE0.85) 
poly(MGE0.90) 
poly(MGE0.95) 
poly(MGE1.0) 

 

22000 
22000 
21000 
22000 
21000 
21000 
21000 
21000 
21000 
22000 
21000 
21000 
24000 
22000 

Mntheory 
(g mol-1) 
49000 
50000 
50000 
50000 
50000 
50000 
50000 
50000 
50000 
50000 
50000 
50000 
50000 
50000 

Mnexp 

(g mol-1)a 

49000 
55000 
48000 
54000 
54000 
55000 
52000 
54000 
52000 
59000 
55000 
53000 
59000 
61000 

Mwexp 

(g mol-1)a 

60000 
63000 
60000 
64000 
65000 
64000 
62000 
65000 
64000 
69000 
65000 
62000 
69000 
71000 

PDI 
1.22 
1.15 
1.25 
1.19 
1.20 
1.16 
1.19 
1.20 
1.23 
1.17 
1.18 
1.17 
1.17 
1.16 

0 
20.5 
39.9 
46.4 
55.5 
57.8 
63.3 
68.7 
74.4 
79.7 
85.0 
89.3 
93.9 
100 

aMeasured by GPC in THF via light scattering. 

 

Scheme  15.  PPM  of  poly(PGE)  and  poly(MGE)  with  mDEG  azide,  decyl  azide, 
mPEG550 azide, mPEG750 azide, mPEG2000 azide, COOH azide, and aminium azide. 

O

N3
m
m = 3, 12, 16, 45

N3

N3

N3

N

I
OH

O

H

O

Br

n

+

R

O

R

a

b

c

d

poly(PGE), R = H
poly(MGE), R = CH3

CuSO4 (5 mol %)
sodium ascorbate

(12 mol %)
DMF, 3.5 h, rt

O

N

N

m

N

R

O

HO

O

N N

5

N

R

O

R

O

O

c

Br
n

d

H

O

R

O

a

O

R

b

O

R
R

N I

R

N

9

N

N

N

N N
poly(PGE0.a-Neg0.d-Pos0.c), R = H, m = 3
poly(PGE0.a-mPEG550-Neg0.d-Pos0.c), R = H, m = 12
poly(PGE0.a-mPEG750-Neg0.d-Pos0.c), R = H, m = 16
poly(PGE0.a-mPEG2000-Neg0.d-Pos0.c), R = H, m = 45
poly(MGE0.a-Neg0.d-Pos0.c), R = CH3, m = 3
poly(MGE0.a-mPEG550-Neg0.d-Pos0.c), R = CH3, m = 12
poly(MGE0.a-mPEG550-Neg0.d-Pos0.c), R = CH3, m = 16
poly(MGE0.a-mPEG550-Neg0.d-Pos0.c), R = CH3, m = 45

 

 

To further examine side chain effects on polymer properties, azides with longer 

hydrophilic  segments,  and  others  bearing  ammonium  or  carboxylic  acid  groups,  were 

 

51 

synthesized and PPMs of poly(PGE) and poly(MGE) were performed with these azides, as 

shown in  

Scheme  15.  The  syntheses  of  more  hydrophilic  azides  were  modified  from 

published  procedures.229,230  1-azido  poly(ethylene  glycol)  monomethyl  ether,  550 

(mPEG550  azide),  1-azido  poly(ethylene  glycol)  monomethyl  ether,  750  (mPEG750 

azide), and 1-azido poly(ethylene glycol) monomethyl ether, 2000 (mPEG2000 azide), 

were synthesized from the corresponding poly(ethylene glycol) monomethyl ethers and 

sodium  azide  as  a  liquid,  viscous  liquid,  and  solid,  respectively  (Scheme  16).  6-

azidophexanoic acid (COOH azide) is used as a short chain negatively charged azide under 

physiological condition and was obtained from the reaction of 6-bromohexanoic acid and 

sodium azide as a clear liquid (Scheme 17).231 For a short chain positively charged azide, 

3-azido-N,N,N-trimethylpropan-1-aminium  iodide  (aminium  azide)  was  chosen.  The 

synthesis was carried out with 3-dimethylaminopropyl chloride hydrochloride and sodium 

azide followed by the addition of methyliodide to acquire aminium azide as a pale-yellow 

solid (Scheme 18).232,233 

 

Scheme 16. Synthesis of mPEG550 azide, mPEG750 azide, and mPEG2000 azide. 

TsCl

KOH, THF/water
0 °C to rt, 16 h

HO

O

m

m = 12, mPEG550
m = 16, mPEG750
m = 45, mPEG2000

TsO

O

m

NaN3

DMF, rt, 16 h

N3

O

m

m = 12, mPEG550 OTs
m = 16, mPEG750 OTs
m = 45, mPEG2000 OTs

m = 12, mPEG550 azide
m = 16, mPEG750 azide
m = 45, mPEG2000 azide

 

Scheme 17. Synthesis of COOH azide. 

Br

OH

O

NaN3
DMF

90 °C, 16 h

N3

O
COOH azide

OH

 

 

52 

Scheme 18. Synthesis of aminium azide. 

Cl

N (cid:380)(cid:3)HCl

NaN3
water

80 °C, 24 h

N3

N

MeI
MeOH

reflux, 20 h

N

N3
aminium azide

I

 

 

PPM  of  poly(PGE)  and  poly(MGE)  with  various  azides  were  carried  out. 

Specifically,  poly(PGE)  (Mn  =  18000,  PDI  =  1.35  or  Mn  =  19000,  PDI  =  1.26)  or 

poly(MGE) (Mn = 22000, PDI = 1.13, Mn = 21000, PDI = 1.20), the desired amount of 

hydrophilic azide (mDEG azide or mPEG550 azide or mPEG750 azide or mPEG2000 

azide), hydrophobic azide (decyl azide), charged azide (COOH azide and/or aminium 

azide), and 12 mol % sodium ascorbate were dissolved in DMF (all equivalents and mole 

percentages are with respect to acetylene units in poly(PGE) and poly(MGE)). Then, 5 

mol % of CuSO4•5H2O in DMF was added via syringe. After the desired reaction time, the 

residual  copper  was removed  by  stirring  the  polymer  solution  with  ion  exchange resin 

beads for 16-24 hours and were purified via dialysis. A series of polymers poly(PGE0.a-

(mPEG#)-Neg0.d-Pos0.c) and poly(MGE0.a-(mPEG#)-Neg0.d-Pos0.c) (a is the mole percent 

of hydrophilic azide, d is the mole percent of COOH azide, c is mole percent of aminium 

azide, if longer hydrophilic azide was used, # is the molecular weight of mPEG) were 

obtained. 

 

53 

Figure 24. 500 MHz 1H NMR spectra of poly(PGE0.60) (red line), poly(PGE0.57-Pos0.05) 
(green line), and poly(PGE0.57-Neg0.05) (blue line) in CDCl3 in aromatic region. 

 

 

The  1H  NMR  spectra  of  the  aromatic  region  of  poly(PGE0.60),  poly(PGE0.57-

Pos0.05), and poly(PGE0.57-Neg0.05) are shown in Figure 24. Similar chemical shifts of the 

triazole  protons  at  7.66  and  7.76  ppm  were  observed  for  poly(PGE0.57-Pos0.05)  and 

poly(PGE0.57-Neg0.05). However, peak broadening was seen for poly(PGE0.57-Pos0.05). The 

 

54 

new peak for the aminium methyl group on poly(PGE0.57-Pos0.05) appears at 3.23 ppm and 

the methylene protons on hexanoic acid group on poly(PGE0.57-Neg0.05) emerge at 1.60 

and 2.22 ppm (Figure A106 and Figure A107, Appendix) Similarly, the protons on the 

triazole  ring  were  seen  at  7.59  ppm  and  7.66  ppm  for  poly(MGE0.75),  poly(MGE0.71-

Pos0.05),  and  poly(MGE0.71-Neg0.05)  and  the  peak  broadening  was  observed  for 

poly(MGE0.71-Pos0.05) (Figure 25). It is worth noting that a shoulder peak appears at around 

7.72 ppm for poly(MGE0.71-Neg0.05) and it is proposed to be the proton on the triazole ring 

bonded  to  hexanoic  acid. This  was  confirmed  by  comparing  the  1H  NMR  spectrum  of 

poly(MGE0.71-Neg0.05) and poly(MGE0.68-Neg0.10), shown in Figure 26, the integration the 

peak at 7.75 ppm in poly(MGE0.68-Neg0.10) was twice of the value in poly(MGE0.71-Neg0.05) 

(Figure A98 and Figure A105, Appendix). The new peak for the aminium methyl group on 

poly(MGE0.71-Pos0.05) appears at 2.45 ppm and the methylene protons on hexanoic acid 

group  on  poly(PGE0.57-Neg0.05)  emerge  at  2.27  ppm  (Figure  A108  and  Figure  A109, 

Appendix). 

 

 

55 

Figure 25. 500 MHz 1H NMR spectra of poly(MGE0.75) (red line), poly(MGE0.71-Pos0.05) 
(green line), and poly(MGE0.71-Neg0.05) (blue line) in CDCl3 in aromatic region. 

 

 

56 

Figure  26.  500  MHz  1H  NMR  spectra  of  poly(MGE0.71-Neg0.05)  (red  line)  and 
poly(MGE0.68-Neg0.10) (green line) in CDCl3 in aromatic region. 

 

 

The physical appearance of polymers with charged side chains are different than 

the  neutral  polymers.  For  instance,  positively  charged  polymer,  poly(PGE0.57-Pos0.05), 

poly(PGE0.57-mPEG550-Pos0.05), poly(MGE0.71-Pos0.05), and poly(MGE0.71-mPEG550-

Pos0.05),  are  red  transparent  viscous  liquids  due  to  oxidation  of  the  iodide  counterions 

associated with the aminium side chains. The color can be removed by ion exchange with 

chloride  to  generate  off-white  transparent  viscous  liquids.  Furthermore,  polymers  with 

mPEG750  side  chain,  poly(PGE0.57-mPEG750-Pos0.05),  poly(PGE0.57-mPEG750-

Neg0.05), poly(MGE0.71-mPEG750-Pos0.05), and poly(MGE0.71-mPEG750-Neg0.05) were 

low-melting  point  solids.  Polymers  with  mPEG2000  side  chain,  poly(PGE0.57-

mPEG2000-Pos0.05),  poly(PGE0.57-mPEG2000-Neg0.05),  poly(MGE0.71-mPEG2000-

 

57 

Pos0.05), and poly(MGE0.71-mPEG2000-Neg0.05) were obtained as solids. The charged side 

chains not only change the physical appearance of the polymers, but it also alters the lower 

critical  solution  temperature  behavior.  This  will  be  further  discussed  in  the  following 

chapter. 

 

2.5. Summary 

Two  alkyne-functionalized  epoxides,  PGE  and  MGE,  were  synthesized  and 

characterized. The polymerization of PGE and MGE were carried out using i-Bu3Al as 

catalyst and Oct4NBr as initiator. Poly(PGE)s with molecular weight between 2,800 to 

19,000 g/mol and poly(MGE)s with molecular weight between 3,900 to 31,000 g/mol were 

obtained with good to moderate PDI. PPM of poly(PGE) and poly(MGE) were performed 

with mEDG azide and decyl azide via CuAAC “click” reaction and a series of amphiphilic 

poly(PGE0.X)  and  poly(MGE0.X)  were  synthesized  and  characterized.  Furthermore, 

charged side chains were placed via CuAAC with various hydrophilic azides (mPEG550 

azide,  mPEG750  azide,  mPEG2000  azide)  and  charged  azides  (COOH  azide  and 

aminium azide). This modification further expanded the chemical properties of these non-

degradable polymers.  

 

 

 

 

58 

Chapter 3. Lower Critical Solution Temperature (LCST) Behavior and 

Formation of Unimolecular Micelles 

3.1. Lower Critical Solution Temperature (LCST) Behavior 

Materials  that  have  lower  critical  solution  temperature  (LCST)  behavior  are 

interesting  and  have  biomedical  applications.71,75,83,84  At  the  LCST,  these  materials 

undergo a solution to gel transition that is driven by entropic gain as water molecules are 

expelled from the polymer solvation sphere (Scheme 19).82,85 In 1968, the phase diagram 

of  poly(N-isopropylacrylamide)  (PNIPAM)  was  first  studied  systematically  by  Heskins 

and Guillet.92,93 They showed that the LCST of PNIPAM (Mn = 290,000, PDI = 3.50) is 31 

°C in water (Table 5). Since then, PNIPAM and its copolymers have been the most studied 

thermoresponsive polymers.65,66,75,93,94  

 

Scheme 19. Schematic representation of LCST behavior. 

H

bound water
H
OH
HO
H
O
H
O
H
H
H O H
H O
H
H
H
soluble coil

H

H
O
H
H O

H
HO
H
O

H
HO
O H
H
H
O H

O
H

T > LCST
T < LCST

H
O
H
H O H

H
HO

H

HO

H
OH

O H
H
O H
released water

H

insoluble globule

H O
H

Entropically unfavorable

 

Entropically favorable

 

Initial solubility tests of poly(PGE0.X) and poly(MGE0.X) showed that poly(PGE0.X) 

containing more than 55% mDEG and poly(MGE0.X) containing more than 60% mDEG 

were soluble in water at room temperature. When aqueous solutions of poly(PGE0.55) were 

cooled in an ice bath, the polymer solubility was not affected. However, poly(MGE0.50), 

 

59 

poly(MGE0.55), which are water insoluble at room temperature, dissolved when samples 

were cooled in an ice bath. To better quantify the LCST behavior of poly(PGE0.X) and 

poly(MGE0.X), cloud points were determined. First, aqueous solutions of poly(PGE0.X) 

and  poly(MGE0.X)  were  prepared  and  filtered  through  0.2  µm  Whatman  PTFE 

(poly(tetrafluoro-ethylene)) filter prior to analysis. Then the solutions were slowly heated 

in  a  temperature  controlled  bath  until  the  transparent  solution  began  to  cloud.  A 

representative picture of cloud point determination is shown in Figure 27. In the course of 

heating, the solution of poly(MGE0.65), which was transparent below 32 °C (Figure 27a), 

rapidly became cloudy at 33 °C (Figure 27b). 

 

 

 

 

60 

a)

b)

Figure  27.  Photographs  showing  the  lower  critical  solution  temperature  behavior  of 
polymers  p-MGE0.65  and 
in  Milli-Q  water.  a) 
poly(MGE0.65) (right) and trans-PhN=NPh@poly(MGE0.65) (left) below LCST (taken at 
25 °C) b) poly(MGE0.65) (right) and trans-PhN=NPh@poly(MGE0.65) (left) above LCST 
(taken at 33 °C). 

trans-PhN=NPh@poly(MGE0.65) 

 

 

To  better  LCST  behavior  for  the  large  number  of  materials  synthesized  in  this 

project, polymers were dissolved in Milli-Q water and solution turbidity was monitored 

via  variable  temperature  UV-vis  spectrometer  (Figure  28).  LCSTs  were  determined  by 

dissolving  each  polymer  in  Milli-Q  water  (5  mg/mL  except  poly(PGE1.0)  and 

poly(MGE0.50),  for  which  10  mg/mL  was  used),  and  then  monitoring  the  solution 

absorbance at 450 nm as a function of temperature. Specifically, the first spectrum of the 

clear solution was recorded several degrees below the LCST. Then the temperature was 

increased  by  1  °C  increments  and  successive  spectra  were  taken  with  3  minutes 

 

61 

equilibration times between temperature jumps (except for poly(MGE0.50)). As the solution 

approaches  the  LCST,  the  solution  becomes  cloudy  and  the  absorbance  increases.  The 

temperature  was  raised  until  the  absorbance  plateaued.  The  LCST  is  defined  as  the 

temperature at which the absorbance is half of the maximum value. Plots of the LCSTs as 

a function of the mole fraction of mDEG chains present in poly(PGE0.X) and poly(MGE0.X) 

are shown in Figure 29. 

 

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
350

e
c
n
a
b
r
o
s
b
A

28 °C
32 °C

29 °C
33 °C

30 °C
34 °C

31 °C

450

550

Wavelength (nm)

650

750

 

Figure 28. LCST determination via UV-vis spectrometer for poly(MGE0.70) (Mn = 54,000, 
PDI = 1.21) in Milli-Q water (5 mg/mL) under various temperatures at 1 °C/3 min. 

 

Similar to the previously reported click-modified glycolide analogs,49 the LCSTs 

for poly(PGE0.X) and poly(MGE0.X) have an approximately linear relationship with mole 

fraction of mDEG side chains between 18.4 to 70.8 °C. The linear portions of the plots in 

 

62 

Figure  29  have  similar  slopes  (0.5  for  poly(PGE0.X)  and  0.7  for  poly(MGE0.X)). 

Substitution at the propargyl C1 position significantly impacts the LCST with the dimethyl 

propargyl p-MGE0.X polymers exhibiting LCSTs ~ 20 °C lower than poly(PGE0.X) analogs. 

As shown in Figure 29, a solution of poly(PGE0.60) has an LCST at 48.2 °C. Interestingly, 

solution of poly(PGE1.0) gave an LCST near the boiling point of water of 96.8 °C. Near 

lower  the  freezing  point  of  water,  poly(MGE0.50)  gave  an  LCST  at  3.6  °C,  while 

poly(MGE1.0)  set  the  upper  LCST  limit  for  the  poly(MGE0.X)  family  at  55.5  °C. 

Combining the temperature ranges of poly(PGE0.X) and poly(MGE0.X), the LCSTs of these 

PEG-based polymers can extend to the full range of liquid phase water. It is worth noting 

that poly(MGE0.50) has one of the lowest LCSTs measured in the literature (Table 5). 

Table  5  compares  previously  reported  polymers  with  low  LCST  values  to 

poly(MGE0.50).92,93,234–238 A low molecular weight oligomer of poly(propylene glycol) (2, 

Mn = 1,025), reported by Firman and Kahlweit in 1986, has the lowest LCST at 1.0 °C at 

55 wt % concentration in water.234 In 2006, Laschewsky and co-workers published series 

of water soluble poly(acrylamide)s and poly(acrylate)s.235 Among these polymers, it was 

demonstrated that the LCST of poly(2-(2-ethoxyethoxy)ethyl acrylate) (3, Mn = 17,500, 

PDI = 1.66) is 9.0 °C in water. Satoh and co-workers synthesized a series of aliphatic 

glycidyl ethers and their homopolymers with LCSTs ranging from 10.3 °C (4, poly(ethoxy 

glycidyl ether) (poly(EtGE), Mn = 5,290, PDI = 1.04) to 91.6 °C (poly(2-methoxyethyl 

glycidyl ether) poly(MeEOGE), Mn = 4,860, PDI = 1.05) in 2017.236 In 2005, Mori, Endo 

and co-workers reported the synthesis of N-acryloyl-L-proline methyl ester (A-Pro-OMe) 

and poly(A-Pro-OMe) (5, Mn = 5,500, PDI = 1.15) with LCST at 15.0 °C.237 Kobayashi 

and co-workers presented the synthesis of a series of poly(vinyl ether)s with oxyethylene 

 

63 

pendant  groups  and  the  phase  separation  behavior  in  water.238  For  example,  poly(2-

ethoxyethylvinylether) (6, Mn = 22,000, PDI = 1.13) exhibited a LCST at 20.0 °C. At 3.6 

°C, the LCST for poly(MGE0.50) lies between entries 2 and 3 in Table 5, but differs from 

those polymers by having a significantly higher Mn, and the LCST was measured at a much 

lower concentration than in entry 2.   

 

Figure 29. Plots of LCSTs vs. the mole fraction of mDEG chains in poly(PGE0.X) and 
poly(MGE0.X) obtained by UV-Vis spectrometer under various temperatures at 1 °C/3 min 
except poly(MGE0.50) (at 1 °C/60 min). 

 

 

 

64 

Table 5. Selective polymers and their LCSTs.92,93,234–238 

Polymer Structure

Mn (g/mol)

PDI

LCST (℃)

Concentration

(wt %)

Ref.

n
N
H

n
O

18

O

O

O

O

2

H O

OnBu
51

O

1

2

3

4

O

5

MeO2C

O

6

n
N

n

O

O

N

N

N

3

O

Br

0.50 n

H

O

O

0.50

O

N
poly(MGE0.50)

N

N

9

 

290,000

3.50

31

-

92,93

1,025

-

17,500

1.66

1

9

5,290

1.04

10

55.0

234

-

1

235

236

5,500

1.15

15

0.1

237

22,000

1.13

20

1.0

238

54,000

1.19

4

0.5

-

 

 

65 

Figure 30. Plots of LCSTs vs. the mole fraction of mDEG chains in poly(PGE0.X) and 
poly(MGE0.X), and poly(PGL0.X) obtained by UV-Vis spectrometer. 

 

 

The comparison of degradable poly(PGL0.X) and the non-degradable poly(PGE0.X) 

and poly(MGE0.X)is shown in Figure 30. The slope for p-PGL0.X is 1.3, which is almost 

double the slope value for poly(PGE0.X) and poly(MGE0.X), 0.5 and 0.7, respectively. This 

result reveals that the backbone of the polymers influences the LCST behavior significantly. 

Not only can one use side chains to control the LCSTs of polymers, one can also use the 

backbone structure to adjust the LCSTs. This design allows a precise way to adjust the 

LCST in future applications. In addition, the result is in good agreement with our previous 

research on “click” modified poly(PGL0.X).  

 

 

66 

Figure 31. LCST determination via UV-vis spectrometer at 450 nm for poly(MGE0.50) (Mn 
= 54,000, PDI = 1.19) in Milli-Q water (10 mg/mL) under various temperatures at 1 °C/3 
min (blue diamond) and 1 °C/60 min (red diamond). 

 

 

As shown in representative graph, Figure 31, the polymer exhibits the lowest LCST, 

poly(MGE0.50)  (Mn  =  54,000,  PDI  =  1.19,  10  mg/mL),  shows  very  broad  change  in 

absorbance at 1 °C/3 min heating rate over temperature range of 10 °C. On the contrary, 

the absorbance of poly(MGE0.70) (Mn = 54,000, PDI = 1.21, 5 mg/mL) at 450 nm increases 

sharply during gel formation upon heating over temperature range of ~ 3 °C (Figure 32). 

When the heating rate was changed to 1 °C/60 min for poly(MGE0.50), the absorbance 

increases sharply over temperature range of ~ 3 °C. For the less concentrated (5 mg/mL) 

sample of poly(MGE0.50), it required more than 150 min to reach equilibrium.  

 

 

67 

Figure 32. LCST determination via UV-vis spectrometer at 450 nm for poly(MGE0.70) (Mn 
= 54,000, PDI = 1.21) in Milli-Q water (5 mg/mL) under various temperatures at 1 °C/3 
min. 

 

 

Variable temperature dynamic light scattering (DLS) can also be used to survey the 

LCST  behavior  by  measuring  the  average  hydrodynamic  radius  (Rh)  as  a  function  of 

temperature. The average hydrodynamic radius of poly(MGE0.70) stays constant at ~7 nm 

before  heating  to  its  LCST.  While  heating  to  and  above  its  LCST,  the  average 

hydrodynamic radius changed rapidly during gel formation (Figure 33). A similar roughly 

linear relationship between the LCST and the mole fraction of mDEG side chains in the 

polymers was observed in both UV-vis and DLS. Figure 34 shows the overlay of LCST vs. 

mole fraction of mDEG in polymer obtained from UV-Vis spectrometer and DLS. It is 

concluded that both methods gave comparable data. 

 

68 

)

%

(
 
y
t
i
s
n
e
t
n

I

40

35

30

25

20

15

10

5

0

 

30 °C
31 °C
32 °C
33 °C
34 °C

0.4

4

Hydrodynamic Radius (nm)

40

400

4000

 

Figure 33. LCST determination via DLS for poly(MGE0.70) (Mn = 54,000, PDI = 1.21) in 
Milli-Q water (5 mg/mL) under various temperatures at 1 °C/3 min. 

 

 

69 

Figure 34. Plots of LCSTs vs. the mole fraction of mDEG chains in poly(PGE0.X) by UV-
Vis spectrometer (black circle) and by DLS (red triangle) and poly(MGE0.X) by UV-Vis 
spectrometer (black diamond) and by DLS (red square). 

 

 

As is the case for small molecules and salts, polymer solubilities are concentration 

and temperature dependent.82 Likewise, the LCSTs for most polymers that exhibit LCST 

behavior  are  concentration  dependent.  Nevertheless,  examples  where  the  LCST 

concentration  dependence  is  small  or  undetected  are  documented.239,240  Generally,  a 

polymer’s LCST is concentration independent over wide concentration ranges but rises at 

low concentrations. The increase in LCST at low concentration can vary significantly with 

chemical  composition  as  illustrated  for  selected  examples  from  the  literature  (Table 

6).239,241–244 

 

70 

 

Table 6. Selective examples from literatures showing LCSTs at various 
concentrations.239,241–244 

Polymer Structure

Mn (g/mol)

Concentration

(wt %)

LCST Range

(℃)

LCST (℃)

Ref.

O
O

O
O

H

n
N

O

7

O

N P

n

O

8

O
N P
HN

O

n

9

O

O

O

O
O

O

O

O

7

O
N P
O

O

n

O

2

R

O

R = Et (10), n-Pr (11), n-Bu (12)

O

N

N

N

3

O

Br

0.65 n

H

O

O

0.35

O

N
poly(MGE0.65)

N

N

9

 

130,000

0.01-1.0

31.6-29.9

29.9

239

100,000

0.1-30.0

43.0-38.0

38.0

241

22,400

0.1-30.0

76.0-65.5

65.5

242

Et = 6,187
n-Pr = 4,711
n-Bu = 4,891

0.13-15.0

Et = 77.0-52.0
n-Pr = 73.0-41.0
n-Bu = 69.0-33.0

Et = 52.0
n-Pr = 41.0
n-Bu = 33.0

243,
244

52,000

0.1-10.0

27.3-30.8

27.5

-

 

Freitag  and  co-workers  synthesized  and  examined  the  LCSTs  of  poly-N,N-

diethylacrylamide (polymer 7, Mn = 130,000 g/mol) in 1994.239 The change in LCST at 

 

71 

various concentrations was minimal. The LCST was 29.9 °C at 1.0 wt % and rose to 31.6 

°C  at  0.01  wt  %.  Allcock  and  co-workers  reported  a  series  of  alkyl  ether  based 

polyphosphazenes in 1996.241 The LCSTs of these polyphosphazenes were investigated in 

the concentration range of 0.1-30 wt %. The LCSTs were concentration independent in the 

range of 2-30 wt %. For example, the LCST of polymer 8 (Mn = 100,000 g/mol) was 38.0 

°C above 2 wt % and increased to 43.0 °C at 0.1 wt %. In 1999, Shon and co-workers 

published a series of polyphosphazenes with methoxy-PEGs and amino acid esters as side 

chains.242 Majority of these polyphosphazenes exhibited LCSTs from 25.2 to 98.5 °C and 

the LCSTs were concentration independent between 3 to 30 wt %. For instance, polymer 

9 (Mn = 22,400 g/mol) exhibited LCST at 65.5 °C above 3 wt % and the LCST rose sharply 

to 76.0 °C at 0.1 wt %. Bi and co-workers reported a series of polyphosphazenes containing 

lactate esters and 2-(2-methoxyethoxy)ethoxy side chains in 2010 and 2011.243,244 These 

polyphosphazenes exhibited LCSTs from 33 to 52 °C in water dependending on the ester 

group (Et = 52 °C, 10, Mn = 6,187 g/mol, n-Pr = 41 °C, 11, Mn = 4,711 g/mol, n-Bu = 33 

°C, 12, Mn = 4,891 g/mol). The LCSTs were concentration independent in the range of 1.5-

15 wt %.  Below 1.5 wt %, the LCSTs increased sharply. For R = Et, the LCST at 0.13 wt 

% rose to 77 °C–an increase of 25 °C. The LCST for propyl and butyl analogs at at 0.13 

wt % rose by 32 and 36 °C, respectively. 

 

 

72 

Figure  35.  LCSTs  as  a  function  of  concentration  of  poly(MGE0.65)  (blue  circles)  and 
polyphosphazene reported by Bi and co-workers (red circles). 

 

 

The influence of polymer concentration on LCSTs for poly(MGE0.65) (Mn = 52,000 

g/mol, PDI = 1.19) was examined and is shown in Figure 35. Notably, the concentrations 

of poly(MGE0.65) showed little impact on cloud points and only a ~ 3 °C difference was 

observed within the studied range, 0.1-10 wt %. The small variation of cloud points of 

poly(MGE0.65) suggests a concentration independent LCST behavior in comparison with 

polyphosphazenes reported by Bi and co-workers even at low polymer concentration. 

We have also found that addition of the charged side chains significantly altered 

the LCST behavior of the polymers. For example, the LCST for poly(MGE0.75) is 36.9 °C; 

however, the LCST of poly(MGE0.71-Neg0.05) is 49.5 °C, with only 5% of the side chains 

were COOH azide incorporated. Moreover, the LCST for poly(MGE0.75) was obtained at 

 

73 

5 mg/mL concentration but it requires 15 mg/mL in concentration to measure the LCST of 

poly(MGE0.71-Neg0.05). In addition, polymers containing 5% of aminium azide such as 

poly(MGE0.71-Pos0.05) showed no LCST behavior. 

The interactions of salts with macromolecules in aqueous solution can influence 

their behaviors.245–254 In 1888, the ability of salts to precipitate proteins from an aqueous 

solution with a recurring trend was first systematically studied by Hofmeister.255,256 Now 

known as the Hofmeister series, the ionic sequence refers to the effectiveness of ions on a 

range of phenomena. In 2005, Bergbreiter, Cremer, and co-workers reported specific ion 

effects  on  the  LCST  behavior  of  PNIPAM.257  Herein,  we  have  studied  the  effects  of 

Na2CO3 and NaCl on the LCSTs of poly(MGE0.75) and poly(MGE0.71-Neg0.05) at various 

concentrations, shown in Figure 36. In the Bergbreiter, Cremer, and co-workers’ study, the 

changes in LCSTs of PNIPAM in Na2CO3 solution were the sharpest and the LCST was 

lowered to 21.0 °C with 0.33 M Na2CO3 in the solution.257 However, in our study, the 

LCST of poly(MGE0.71-Neg0.05) was first increased in 0.1 M Na2CO3 solution to 54.5 °C 

then was decreased to 27.1 °C when the concentration of Na2CO3 was increased to 0.4 M. 

On  the  contrary,  the raise  of  the  LCST  of  poly(MGE0.75)  in  Na2CO3  solution  was  not 

observed. The LCST of poly(MGE0.75) was lowered to 21.8 °C from 36.9 °C at 0.3 M 

Na2CO3 concentration. The effects of NaCl on the LCST behavior of poly(MGE0.71-Neg0.05) 

was also investigated and its LCST was reduced to 32.7 °C from 49.5 °C in 0.3 M NaCl 

solution.  More  studies  should  be  conducted  to  further  understand  the  effects  of  the 

Hofmeister series on the “click” derivatives of poly(PGE0.X) and poly(MGE0.X). This can 

provide another way to adjust LCSTs for future applications.  

 

 

74 

Figure 36. LCST values of poly(MGE0.75) (red square) measured in sodium bicarbonate 
solutions at concentrations from 0 to 0.3 M, poly(MGE0.71-Neg0.05) (blue circle) measured 
in sodium bicarbonate solutions at concentrations from 0 to 0.4 M, and poly(MGE0.71-
Neg0.05) (orange circle) measured in sodium chloride solutions at concentrations from 0 to 
0.3 M. 

 

 

3.2. Formations of Unimolecular Micelles 

Micelles are dynamic aggregates of surfactant molecules above the critical micelle 

concentration (cmc). DLS is often used to determine the cmc. When traditional micelles 

reach  the  cmc,  there  is  a  dramatic  change  in  the  hydrodynamic  radius.  However,  our 

previous  work  on  “clickable”  polyglycolides  showed  that  hydrodynamic  radius  of  the 

poly(propargyl  glycolide)-graft-poly(ethylene  glycol)  monoethyl  ether  (poly(PGL1.0))95 

polymers  was  unaffected  by  polymer  concentration,  which  suggested  the  formation  of 

unimolecular micelles.97,109,110,113,114,122 To perform the unimolecular micelle study on the 

amphiphilic  poly(PGE0.X)  and  poly(MGE0.X),  the  polymer  samples  were  dissolved  in 

 

75 

Milli-Q water and filtered through 0.2 µm Whatman PTFE filter prior to analysis. All the 

analysis was done at a temperature below the LCST of the sample. Similar to poly(PGL0.X) 

system,  DLS  data  shows  that  the  hydrodynamic  radius  of  the  particles  at  different 

concentrations  was  relatively  similar,  which  indicates  the  formation  of  unimolecular 

micelles. As shown in Figure 37, the hydrodynamic radius of poly(MGE0.65) (Mn = 52,000 

g/mol, PDI = 1.19) is 7 ± 3 nm at 50 mg/mL and is 6 ± 2 nm at 10, 5, 1 mg/mL. 

 

18
16
14
12
10
8
6
4
2
0

0.2

)

%

(
 
y
t
i
s
n
e
t
n

I

50 mg/mL
10 mg/mL
5 mg/mL
1 mg/mL

200

 

2

Hydrodynamic Radius (nm)

20

Figure 37. DLS results for poly(MGE0.65) (Mn = 52,000 g/mol, PDI = 1.19, RH = 6 ± 2 nm) 
in Milli-Q water at different concentrations. 

 

The formation of unimolecular micelles and the aggregation of polymer above its 

LCST can also be observed by TEM (Figure 38). Poly(MGE0.65) was dissolved in Milli-Q 

water and filtered through 0.2 µm Whatman PTFE (poly(tetrafluoro-ethylene)) filter prior 

 

76 

to analysis. The solution was dropped on Formvar-coated copper grids below LCST for the 

preparation of TEM samples. One sample was kept below its LCST before removing the 

excess water and the other sample was heated on a hot plate above the LCST for 2 minutes 

before  removing  the  excess  water.  Both  samples  were  stained  with  2%  potassium 

phosphotungstate (PTA) solution to generate a dark background. As shown in Figure 38a, 

which was taken at 100,000x, the size of polymer particles is 20 ± 4 nm. This is slightly 

bigger than the hydrodynamic diameter of poly(MGE0.65) measured by DLS (~13 nm). 

This is caused by the surface tension between the solution and the grid. After heating above 

its LCST (Figure 38b, taken at 40,000x), the size of the particles increased to 74 ± 45 nm. 

 

a)

b)

Figure 38. TEM images of polyMGE0.65) (Mn = 52,000 g/mol, PDI = 1.19) a) below (taken 
at 100,000x, scale bar = 100 nm, 20 ± 4 nm) and b) above (taken at 40,000x, scale bar = 
200 nm, 74 ± 45 nm) its LCST. 

 

 

 

77 

3.3. Summary 

“Click”  modified  amphiphilic  poly(PGE0.X)  and  poly(MGE0.X)  showed  water 

solubility and LCST behavior when X = 55 in poly(PGE0.X) and X = 50 in poly(MGE0.X). 

The LCST behavior was controlled through varying the ration between mDEG azide and 

decyl azide. In our study, both UV-Vis spectrometer and DLS gave consistent results for 

LCSTs.  A  close  to  linear  relationship  was  obtained  between  18.4  to  70.8  °C  for  both 

poly(PGE0.X) and poly(MGE0.X) with similar slopes but with nearly 20 °C differences. 

These results indicated the backbone and the pendant groups of the polymers both play an 

important role for tuning the LCSTs. It is worth highlighting that poly(MGE0.50) with 3.6 

°C for LCST and poly(PGE1.0) with 96.8 °C for LCST. This allows us to manipulate the 

LCSTs of these polymers across the whole temperature span of liquid phase water. We also 

demonstrated  that  the  LCSTs  of  poly(PGE0.X)  and  poly(MGE0.X)  are  concentration 

independent. Furthermore, it was found that the LCST of poly(MGE0.71-Neg0.05) is 49.5 

°C, 12.6 °C higher than poly(MGE0.75), with only 5% of side chains were acid derivatives. 

The effects of Na2CO3 and NaCl on poly(MGE0.75) and poly(MGE0.71-Neg0.05) were also 

investigated  and  this  provided  another  factor  to  alter  the  LCST  behavior  of  polymers. 

Moreover, poly(PGE0.X) and poly(MGE0.X) formed unimolecular micelles, which can be 

characterized by DLS and TEM. The potential applications for unimolecular micelles will 

be further discussed in the following chapter.   

 

 

 

 

78 

Chapter 4. Unimicelles as Nanocarriers for Hydrophobic and 

Hydrophilic Guest Molecules 

4.1. Encapsulation of Hydrophobic Guest Molecules 

Scheme 20. Schematic representative of azobenzene encapsulation. 

O

N

N

N

3

O

Br

0.60 n

=

H

O

O

0.40

O

N

N

N

9

poly(PGE0.60)

=

N N

Azobenzene
H2O

 

 

Polymers that form unimolecular micelles are promising as drug carriers because 

they can be prepared by controlled polymerization methods and are not subject to cmc. As 

a proof of concept, azobenzene (trans-PhN=NPh), which is a water insoluble, UV-active 

model  for  hydrophobic  drugs,  was  chosen  as  a  hydrophobic  guest  molecule  to  be 

encapsulated in the unimolecular micelles (Scheme 20). Azobenzene and poly(PGE0.60) 

(Mn = 44,000 g/mol, PDI = 1.33), poly(MGE0.65) (Mn = 52,000 g/mol, PDI = 1.19), and 

poly(MGE0.75) (Mn = 52,000 g/mol, PDI = 1.22) were dissolved in Milli-Q water. The 

solution was stirred for 24 hours followed by filtered through 0.2 µm Whatman PTFE filter 

 

79 

to  remove  unencapsulated  azobenzene.  The  resulting  solution  appeared  yellow  which 

indicates the encapsulation of azobenzene. Also, as shown in Figure 39, the characteristic 

absorbance at 425 nm in UV spectrum confirmed that azobenzene was encapsulated in the 

micelles. With increasing concentration of poly(PGE0.60), an increase in absorbance was 

also observed. Furthermore, when the resulting solution was heated above the LCST of 

poly(MGE0.65), turbidity of solution was observed (Figure 27b). After the mixture was 

cooled down below the LCST, the precipitates re-dissolved and the color of the solution 

returned to yellow again (Figure 27a). 

 

Figure 39. UV-vis spectra of trans-PhN=NPh, poly(PGE0.60) (Mn = 44,000 g/mol, PDI = 
1.33), and trans-PhN=NPh@poly(PGE0.60) in Milli-Q water at room temperature. 

 

 

 

80 

The DLS results of poly(MGE0.65) and poly(MGE0.75) encapsulated azobenzene 

are  shown  in  Figure  40  and  Figure  41.  Interestingly,  when  10  mg/mL  polymer 

concentration  was  used  during  the  encapsulation  process,  the  hydrodynamic  radii  of 

poly(MGE0.65) encapsulated azobenzene (trans-PhN=NPh@ poly(MGE0.65)) were almost 

two times larger (RH = 12 ± 8 nm) than those of poly(MGE0.65) (RH = 7 ± 2 nm) (Figure 

40a). When 50 mg/mL polymer concentration was used, the hydrodynamic radii of trans-

PhN=NPh@ poly(MGE0.65) increased to 13 ± 6 nm and decreased to 9 ± 4 nm upon five-

fold dilution (Figure 41a). A different trend was observed when poly(MGE0.75) was used. 

The hydrodynamic radii of poly(MGE0.75) and poly(MGE0.75) encapsulated azobenzene 

(trans-PhN=NPh@poly(MGE0.75))  were  practically  identical,  6  ±  2  nm  and  5  ±  2  nm, 

respectively (Figure 40b). The hydrodynamic radii of trans-PhN=NPh@ poly(MGE0.75) 

increased only to 6 ± 3 nm when 50 mg/mL polymer concentration was used and decreased 

to 5 ± 2 nm upon five-fold dilution. Our hypothesis is that the polymer composition and its 

hydrophilicity are the potential reasons for the change in hydrodynamic radii, but more 

studies need to be conducted to explore this phenomenon further. 

 

 

81 

Figure 40. a) DLS results for poly(MGE0.65) (blue curve, Mn = 52,000 g/mol, PDI = 1.19, 
RH = 7 ± 2 nm) and trans-PhN=NPh@poly(MGE0.65) (red curve) in Milli-Q. b) DLS results 
for poly(MGE0.75) (blue curve, Mn = 52,000 g/mol, PDI = 1.22, RH = 6 ± 2 nm) and trans-
PhN=NPh@poly(MGE0.75) (red curve) in Milli-Q 

 

 

82 

Figure 41. a) DLS results for poly(MGE0.65) (blue curve, Mn = 52,000 g/mol, PDI = 1.19, 
RH = 7 ± 2 nm), trans-PhN=NPh@poly(MGE0.65) (orange curve, 50 mg/mL; green curve, 
dilute to 10 mg/mL from 50 mg/mL; red curve, 10 mg/mL) in Milli-Q. b) DLS results for 
poly(MGE0.75) (blue curve, Mn = 52,000 g/mol, PDI = 1.22, RH = 6 ± 2 nm) and trans-
PhN=NPh@poly(MGE0.75) (orange curve, 50 mg/mL; green curve, dilute to 10 mg/mL 
from 50 mg/mL; red curve, 10 mg/mL) in Milli-Q 

 

 

83 

4.2. Encapsulation of Hydrophilic Guest Biomacromoelcules 

Subtilisin Carlsberg (SC) was chosen as the hydrophilic guest biomacromolecule. 

Subtilisin  is  a  non-specific  protease  and  a  serine  protease  originated  from  Bacillus 

subtilis.258–260  SC  has  been  widely  studied  and  used  in  enzyme-containing  cleaning 

products.261  Also,  it  is  conveniently  purchased  from  commercial  sources.  We  have 

therefore chosen it as a model protein. To study the interaction between SC and polymers, 

DLS was utilized to monitor the particle sizes as the polymer solution was titrated in the 

SC  solution.  Poly(MGE0.71-mPEG2000-Pos0.05)  (25  mg/mL)  and  SC  (1  mg/mL)  were 

dissolved in Milli-Q water to make the final polymer/SC mixture with a 2:1 molar ratio. 

As shown in Figure 42, the hydrodynamic radius of SC in Milli-Q water is 5 ± 2 nm (blue 

line)  and  poly(MGE0.71-mPEG2000-Pos0.05) is  9  ±  3  nm  (orange  line).  When  5 µL	of	
hydrodynamic  radii  were  6  ±  3  nm  and  55  ±  30  nm  (green  line).  When  50  µL	of	

poly(MGE0.71-mPEG2000-Pos0.05)  was  added,  a  new  peak  was  observed  and  the 

poly(MGE0.71-mPEG2000-Pos0.05) was added, the smaller size peak was shifted to 13 ± 

12 nm with a shoulder and two larger size peaks at 198 ± 70 nm and 2603 ± 236 nm 

appeared (Figure 43). It is proposed that the large peaks are the results of the interaction 

between  SC  and  poly(MGE0.71-mPEG2000-Pos0.05)  forming  large  particles  that  are 

observable by DLS. When equal volumes of SC and poly(MGE0.71-mPEG2000-Pos0.05) 

were  combined,  a  single  peak  appeared  at  9  ±  4  nm  (green  line),  similar  to  the 

hydrodynamic radius of poly(MGE0.71-mPEG2000-Pos0.05) (Figure 44). However, this is 

insufficient to prove that SC was encapsulated by poly(MGE0.71-mPEG2000-Pos0.05) due 

to the limitation in resolution of DLS. More studies are required to further investigate the 

interaction between SC and polymers.	

 

84 

and  SC  with  5  µL	of	 poly(MGE0.71-mPEG2000-Pos0.05)  added  (green line)  in  Milli-Q 

Figure 42. DLS results of SC (blue line), poly(MGE0.71-mPEG2000-Pos0.05) (orange line) 

 

water. 

and SC with 50 µL	of	poly(MGE0.71-mPEG2000-Pos0.05) added (green line) in Milli-Q 

Figure 43. DLS results of SC (blue line), poly(MGE0.71-mPEG2000-Pos0.05) (orange line) 

 

water. 

 

85 

and SC with	poly(MGE0.71-mPEG2000-Pos0.05) added (green line) in Milli-Q water. 

Figure 44. DLS results of SC (blue line), poly(MGE0.71-mPEG2000-Pos0.05) (orange line) 

 

 

To investigate the polymers’ ability to slow or stop the aggregation of proteins, SC 

was 

encapsulated 

by 

poly(MGE0.71-mPEG2000-Pos0.05) 

(SC@poly(MGE0.71-

mPEG2000-Pos0.05)) 

or 

poly(MGE0.71-mPEG2000-Neg0.05) 

(SC@poly(MGE0.71-

mPEG2000-Neg0.05))  in  the  following  manner  (Scheme  21).  SC  and  poly(MGE0.71-

mPEG2000-Pos0.05) or poly(MGE0.71-mPEG2000-Neg0.05) were dissolved in phosphate-

buffered saline (PBS) solution (pH = 7.4), and the resulting solution was placed in a dialysis 

bag (MWCO = 6-8 kDa) with Milli-Q water for 24 h to replace the counterions at the 

protein surface with polymers. The solution was then dried under vacuum and re-dissolved 

in  PBS.  A  set  of  control  was  prepared  by  dissolving  only  SC  and  poly(MGE0.71-

mPEG2000-Pos0.05) or poly(MGE0.71-mPEG2000-Neg0.05) without the dialysis process. 

 

86 

It is worth noting that the hydrodynamic radius of SC in PBS is 3 ± 1 nm, which is smaller 

than in Milli-Q water. 

 

Scheme 21. Schematic representation of Subtilisin Carlsberg (SC) encapsulation. 

+

dialysis

Subtilisin Carlsberg

(SC)

Charged amphiphilic polymers

 

Encapsulated SC
(SC@polymer)

 

The solutions were left at room temperature for 48 h and the DLS of the solutions 

were taken at 0, 24, and 48 h. As shown in Figure 45 and Figure 46, the peak corresponding 

to the hydrodynamic radius of SC decreased over time as a new peak for aggregates at 26 

± 9 nm increased. If the aggregation process can be slowed or stopped by polymer, little to 

no  aggregation  should  be  observed  overtime.  However,  as  shown  in  Figure  45,  the 

aggregation at 61 ± 47 nm was detected after 24 h for SC@poly(MGE0.71-mPEG2000-

Pos0.05), similar as SC alone in solution. On the contrary, no aggregation was seen at 24 h 

for SC@poly(MGE0.71-mPEG2000-Neg0.05) and only small amount of aggregations at 602 

± 194 nm was observed after 72 h (Figure 46). It is concluded that polymers with COOH 

pendant groups exhibit a better ability to encapsulate SC and to slow down SC aggregation 

in solution. It is necessary to perform activity assay to evaluate the activity of SC before 

and after aggregations. 

 

 

87 

Figure 45. DLS results of SC (dotted line) and SC@poly(MGE0.71-mPEG2000-Pos0.05) 
(solid line) in Milli-Q water over 48 h. 

 

Figure 46. DLS results of SC (dotted line) and SC@poly(MGE0.71-mPEG2000-Neg0.05) 
(solid line) in Milli-Q water over 48 h. 

 

 

88 

The  catalytic  hydrolysis  of  N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide  (Suc-

AAPF-pNA, <max = 315 nm, J = 14,000 M-1 cm-1) to amino acids and p-nitroaniline (<max 
= 410 nm, J = 8,800 M-1 cm-1) by protease has been frequently employed as the activity 

assay for protease (Scheme 22).262–264 As shown in Figure 47, 5 µL of SC (0.04 mg/mL, 

Tris-HCl buffer, pH = 8.0) was added to 2 mL of Suc-AAPF-pNA (0.02 mM, Tris-HCl 

buffer,  pH  =  8.0)  and  the  progression  of  the  hydrolysis  was  monitored  by  UV-Vis 

spectrometer.  Suc-AAPF-pNA  was  fully  converted  to  p-nitroaniline  in  35  min.  For 

consistency, the conversion of Suc-AAPF-pNA was measured 5 min after aliquots of SC 

or SC@polymer were added. 

It has been shown that the concentration of SC affects the rate of autolysis and 

denaturation.265–268 Therefore, a series of experiments were performed to investigate the 

effects  of  SC  concentration  at  various  temperatures  on  the  activity.  The  half-life  (t1/2), 

which  refers  to  the  time  required  for  the  enzyme  activity  to  reduce  to  half its  original 

activity, of SC at 0.01, 0.4, and 1.0 mg/mL at room temperature, 40 °C, and 50 °C are listed 

in  Table  7  (Figure  A172Figure  A175,  Appendix).  As  expected,  t1/2  decreases  as  the 

temperature is increased. Additionally, higher SC concentrations results in longer t1/2. We 

were puzzled by the fact that our results of the concentration effect were in contradicting 

with the results reported by Russell and co-workers.269 However, some studies have shown 

that SC aggregates remain active,265,267 which is consistent with what was observed in our 

studies. 

 

 

 

89 

Scheme  22.  Catalytic  hydrolysis  of  Suc-AAPF-pNA  to  p-nitroaniline  by  SC  or 
SC@polymer. 

O

HO

H
N

O

O

N
H

N

O

O

N
H

Substrate

(Suc-AAPF-pNA)

NO2

Subtilisin or
SC@polymer
Tris-HCl buffer
pH = 8.0, 50 °C

H2N

NO2

Nitroaniline

 

0 min

5 min

15 min

20 min

25 min

35 min

 

0.25

0.2

0.15

0.1

0.05

e
c
n
a
b
r
o
s
b
A

0
260

310

360

Wavelength (nm)

410

460

 

Figure 47. UV-Vis spectra of the catalytic conversion of Suc-AAPF-pNA to p-nitroaniline 
overtime. 

 

 

 

90 

Table 7. Effects of SC concentration and temperatures on SC stability. 

SC Concentration 

(mg/mL) 

0.04 

0.1 

1 

 

 

Temperature (°C) 

25 
40 
50 
25 
40 
50 
25 
40 
50 

t1/2 (h) 
94.1 
5.9 
1.6 
244.5 
11.7 
2.0 
390.4 
59.7 
14.8 

To assess the ability of “click” modified poly(MGE)s to stabilize SC in solution, 

the activity of solutions of SC@polymer over 24 h was tested. As mentioned in previous 

section, polymers with COOH pendant groups display a better ability to slow down the 

aggregation of SC. Therefore, poly(MGE0.71-Neg0.05), poly(MGE0.71-mPEG550-Neg0.05), 

poly(MGE0.71-mPEG750-Neg0.05),  and  poly(MGE0.71-mPEG2000-Neg0.05)  were  first 

used in the study. The molar ratio of SC to polymer was kept at 1 to 2 and the solution were 

prepared by dissolving SC and polymer in Tris-HCl (pH = 8.0) buffer at room temperature. 

The dialysis process described above was eliminated for simplicity. The concentration of 

SC in SC@polymer solution was fixed at 0.1 mg/mL and solutions of SC@polymer were 

heated at 50 °C to accelerate the degradation of SC. Aliquots (10 µL) of SC@polymer 

solutions were taken out at 0, 1, 2, 3, 4, and 24 h. Each aliquot was then added to a freshly 

prepared solution of Suc-AAPF-pNA in Tris-HCl buffer (2 mL, 0.02 mM, pH = 8.0). The 

UV-Vis spectrum was obtained after 5 min and the absorbance at 412 nm (J = 8,480 M-1 

cm-1) was used to calculate the concentration of p-nitroaniline. The control experiments 

were performed following the same procedure on solutions containing only SC. 

 

91 

Representative UV-Vis spectra of the activity study are presented in Figure 48. The 

blue solid line represents the absorbance for Suc-AAPF-pNA and the absorbance for p-

nitroaniline is shown in yellow solid line. At 0 h, when SC@poly(MGE0.71-mPEG750-

Neg0.05)  solutions  were  freshly  made,  Suc-AAPF-pNA  was  fully  converted  to  p-

nitroaniline  within  5  min.  After  SC  and  SC@poly(MGE0.71-mPEG750-Neg0.05)  were 

heated at 50 °C for 1 h, the activity of SC decreased to 83% of its original activity (orange 

dotted line) and the activity of SC@poly(MGE0.71-mPEG750-Neg0.05) decreased to 87% 

of its original activity (purple solid line). The reduction of the activities after 1 h was similar 

but the differences became very noticeable after 2 h. The activity of SC decreased to 48% 

and the activity of SC@poly(MGE0.71-mPEG750-Neg0.05) remained at 72%. After 4 h, as 

shown in Figure 48, the activity of SC reduced to 33% (green dotted line) and the activity 

of  SC@poly(MGE0.71-mPEG750-Neg0.05)  only  lowered  to  52%  (red  solid  line).  Our 

results demostrate that the activity of SC increases in the presence of polymers. 

 

 

92 

Figure 48. UV-Vis spectra of the conversion of Suc-AAPF-pNA (blue solid line) to p-
nitroaniline (yellow solid line) by aliquots of SC (1 h, orange dotted line; 4 h green dotted 
line) or SC@poly(MGE0.71-mPEG750-Neg0.05) (1 h, purple solid line; 4 h, red solid line) 
taken at 1 or 4 h after heated at 50 °C. 

 

 

t1/2 values in the presence of various polymers are listed in Table 8. Poly(MGE0.71-

Neg0.05) 

and  poly(MGE0.71-mPEG750-Neg0.05)  promoted 

the 

largest 

activity 

enhancements of SC and extended the t1/2 to 9.8 h and 10.4 h, respectively. The neutral 

counterpart 

of 

poly(MGE0.71-Neg0.05) 

and 

poly(MGE0.71-mPEG750-Neg0.05), 

poly(MGE0.75) and poly(MGE0.75-mPEG750), also increased the t1/2 to 7.0 h and 7.1 h. 

By adding 5% of COOH side chains, we were able to lengthen the t1/2 by 3 h. Interestingly, 

polymers  with  mPEG750  outperformed  polymers  with  mPEG550  and  mPEG2000  as 

hydrophilic pendant groups. It is important to note that the studies were conducted without 

 

93 

the dialysis as encapsulation procedure; therefore, it will be necessary to carry out these 

experiments after dialysis to study the effects of dialysis procedure. 

 

Table 8. Results of t1/2 in the presence of various polymers. 

SC or SC@polymer 

SC 

t1/2 (h) 

2.0 
7.0 
7.1 
9.8 
6.7 
10.4 
8.2 

SC@poly(MGE0.75) 

SC@poly(MGE0.75-mPEG750) 
SC@ poly(MGE0.71-Neg0.05) 

SC@poly(MGE0.71-mPEG550-Neg0.05) 
SC@poly(MGE0.71-mPEG750-Neg0.05) 
SC@poly(MGE0.71-mPEG2000-Neg0.05) 

 

 

The hydrodynamic radii of SC and SC@poly(MGE0.71-mPEG750-Neg0.05) at 50 

°C were monitored by DLS over 4 h (Figure 49). Similar to SC in PBS, the hydrodynamic 

radius of SC in Tris-HCl buffer is 3 ± 1 nm (dotted lines). After 1 h of heating at 50 °C, 

only SC aggregates were detected, and the hydrodynamic radius of SC increased to 59 ± 

18 nm. As shown in Figure 49, a broad peak was observed and the hydrodynamic radius 

of SC@poly(MGE0.71-mPEG750-Neg0.05) is 13 ± 11 nm (solid lines). Small amount of 

large aggregates, RH = 187 ± 109 nm, were seen after 1 h of heating at 50 °C. Combining 

with the activity studies, we propose that slower SC aggregation contributes to enhancing 

the SC activity. However, our study also shows that SC remains active upon aggregation. 

In order to establish a clearer correlation between protein aggregation and activity, proteins 

that lose their activity as they aggregate will be needed for future studies. 

 

 

94 

Figure 49. DLS results of SC (dotted line) and SC@poly(MGE0.71-mPEG750-Neg0.05) 
(solid line) in Tris-HCl buffer at 50 °C for 4 h. 

 

Scheme 23. Schematic representation of SC encapsulation in toluene. 

 

+

toluene

Amphiphilic polymer

Subtilisin Carlsberg

(SC)

 

 

Aside from dissolving hydrophobic guest molecules in water, these amphiphilic 

polymers can also be utilized to solubilize hydrophilic guest molecules in organic media. 

As a proof of concept, poly(PGE0.60) and poly(MGE0.65) were used for SC encapsulation 

in organic media (Scheme 23). The encapsulation of SC was performed under two methods 

 

95 

which both led to similar results. The first method involved the addition of an aqueous 

solution of SC (1 mg/mL) and poly(PGE0.60) or poly(MGE0.65) (10 mg/mL), in which the 

molar  ratio  between  SC  and  poly(PGE0.60)  or  poly(MGE0.65)  was  1:2,  to  toluene.  The 

resulting two-phase mixture was then vigorously evaporated in vacuo. The resulting solid 

was re-dissolved in toluene. In the second method, SC (1 mg/mL) and poly(PGE0.60) or 

poly(MGE0.65) (10 mg/mL) were dissolved in PBS, and the solution was lyophilized. The 

resulting solid was re-dissolved with toluene. The encapsulation of SC, SC@poly(PGE0.60) 

or SC@poly(MGE0.65), was confirmed by UV-Vis spectrometer. As shown in Figure 50, 

absorbance  at  285  nm  was  detected  for  SC@poly(MGE0.65),  resulting  from  tyrosine, 

tryptophan, and phenylalanine. Control experiments were conducted following the same 

methods with SC in the absence of poly(PGE0.60) or poly(MGE0.65) and the resulting solid 

was insoluble in toluene. Therefore, no absorption was seen in the aromatic region of UV-

vis spectrum.  

The activity of SC@poly(MGE0.65) was assayed by transesterification of benzyl 

alcohol  and  vinylbutyrate  at  45  °C  in  toluene.  GC-MS  show  approximately  ~35% 

conversion after 16 h. Controls with lyophilized or as provided SC suspensions showed no 

activity in toluene. However, the generation of formaldehyde during the reaction would 

potentially compromise the activity of SC therefore a new approach should be taken to 

examine the enzymatic activity. These initial studies demonstrate that our “click” modified 

amphiphilic polymers enhance protein solubility as well as the activity in organic media.  

 

 

96 

Figure  50.  UV-Vis  spectra  of  SC  suspension  in  toluene  (blue  line),  poly(MGE0.65)  in 
toluene (green line), and SC@ poly(MGE0.65) in toluene (red line).  

 

 

4.3. Summary 

By employing the “click” modified amphiphilic polymers as unimicelles, we were 

able to solubilize a hydrophobic guest molecule, azobenzene, in water. Interestingly, when 

the solubilized azobenzene-loaded micelles were heated above the LCST of the polymer, 

the solution appeared cloudy and after it was cooled down below LCST, the precipitates 

re-dissolved  and 

the  color  of 

the  solution  returned 

to  yellow  again.  Trans-

PhN=NPh@poly(MGE0.65)  showed  different  hydrodynamic 

radius  compared 

to 

poly(MGE0.65) but the same hydrodynamic radius was observed for poly(MGE0.75) and 

trans-PhN=NPh@poly(MGE0.75). It is proposed that the differences were caused by the 

 

97 

compositions  and  hydrophilicities  of  polymers,  but  further  studies  are  required  to 

investigate our hypothesis. 

By  exchanging  surface  ions  on  SC  via  dialysis  in  aqueous  solutions,  we 

encapsulated  SC  in  poly(MGE0.71-mPEG2000-Pos0.05)  and  poly(MGE0.71-mPEG2000-

Neg0.05).  It  was  shown  that  only  little  aggregation  of  SC@poly(MGE0.71-mPEG2000-

Neg0.05)  was  observed  after  48  h.  Furthermore,  the  activity  of  SC  in  the  presence  of 

polymers was assayed via the hydrolysis of Suc-AAPF-pNA and monitored by UV-Vis 

spectrometer.  The  half-life  of  SC  was  enhanced  to  about  10  h  in  the  presence  of 

poly(MGE0.71-Neg0.05) and poly(MGE0.71-mPEG750-Neg0.05), 5 times longer than in the 

absence of polymers. It is worth noting that poly(MGE0.75) and poly(MGE0.75-mPEG750) 

increased the t1/2 of SC to ~7 h, 3 h shorter without the COOH pendant group. However, 

our studies indicated that SC aggregates remained active. In order to further investigate the 

correlation  between  reduced  aggregation  and  enhanced  activity  of  proteins,  a  different 

model should be selected. Preliminary studies also show that these amphiphilic polymers 

can not only solubilize SC but also stabilize SC in organic media. 

 

 

 

 

98 

Chapter 5. Experimental Section 

5.1. Materials 

(±)-Epichlorohydrin (≥98%, Fluka), propargyl alcohol (99%, Sigma-Aldrich), 2-

methyl-3-butyn-2-ol (98%, Aldrich), tetrabutylammonium hydrogensulfate (TBAHS, 97%, 

Aldrich), triethylene glycol monomethyl ether (95%, Sigma-Aldrich), polyethylene glycol 

monomethyl ether, 550 (Alfa Aesar), polyethylene glycol monomethyl ether, 750 (Acros), 

polyethylene  glycol  monomethyl  ether,  2000  (Aldrich),  p-toluenesulfonic  chloride 

(Spectrum), 6-bromohexanoic acid (98+%, Alfa Aesar), 3-dimethylaminopropyl chloride 

hydrochloride  (98%,  Alfa  Aesar),  sodium  L-ascorbate  (>98%,  TCI),  and  sodium  azide 

(Sigma-Aldrich) were used as received. Tetraoctylammonium bromide (Oct4NBr, 98%, 

Sigma-Aldrich)  and  triisobutylaluminum  (i-Bu3Al,  1  mol/L  in  toluene,  Sigma-Aldrich) 

were  stored  in  glovebox  under  nitrogen  atmosphere  and  were  used  without  further 

purification. Ion exchange resin beads (Amberlite® IRC-748) were purchased from Alfa 

Aesar and soaked in N,N-dimethylformamide (DMF) prior to use. Toluene was dried by 

refluxing  over  sodium  benzophenone  and  stored  under  nitrogen  atmosphere.  DMF, 

tetrahydrofuran (THF), dichloromethane (CH2Cl2), methanol, ethanol, diethyl ether, and 

ethyl acetate were used as received. 

 

5.2. Characterization 

The  molecular  weights  of  polymers  were  determined  by  gel  permeation 

chromatography  (GPC)  at  35  °C  using  two  PLgel  10µ  mixed-B  columns  in  series 

(manufacturer stated linear molecular weight range of 500-10,000,000 g/mol) with THF as 

the eluent solvent at a flow rate of 1 mL/min. A Waters 2410 differential refractometer was 

 

99 

used as the detector. An Optilab rEX (Wyatt Technology Co.) and a DAWN EOS 18-angle 

light scattering detector (Wyatt Technology Co.) with a laser wavelength of 684 nm were 

used to calculate absolute molecular weights. Monodisperse polystyrene standards (Mn = 

2727, 3680, 12860, 24150, 32660, 45730, 95800, 184200, 401340 g/mol) were used to 

calibrate the molecular weights. The concentration of polymer solutions used for GPC was 

1-4 mg/mL and all samples were filtered through a 0.2 µm Whatman PTFE syringe filter. 

1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were acquired using either a Varian 

Inova-500 spectrometer or Agilent DirectDrive2 500 spectrometer. Chemical shifts for 1H 

and  13C spectra were recorded in parts per million relative to the residual  1H and 13C of 

CDCl3 (δ 7.26, 77.0) and D2O (δ 4.79). Elemental analyses were determined using a Perkin-

Elmer 2400 CHNS/O analyzer. Mass spectral analyses were carried out on a Waters Xevo 

G2-S QTof UPLC/MS/MS. Dynamic light scattering (DLS) data were obtained using a 

Malvern Zetasizer Nano ZS. All samples were filtered through a 0.2 µm Whatman PTFE 

syringe filter and then equilibrated in the instrument for 2 minutes at each temperature 

before taking the data used to calculate the hydrodynamic radius (Rh). The particle size 

uniformity was determined by a monomodal curve fit, which assumes a single particle size 

with  a  gaussian  distribution.  TEM  micrographs  were  collected  at  40,000-100,000x 

magnifications  on  a  JEOL-100CX11  transmission  electron  microscope.  UV-vis  spectra 

were recorded with an ATI UNICAM UV2 UV/Vis spectrometer or a Shimadzu UV-2600 

UV-Vis spectrometer. 

 

 

100 

5.3. Procedures 

Synthesis of propargyl glycidyl ether (PGE)222 

O

O
PGE

 

In an Erlenmeyer flask, (±)-epichlorohydrin (98.53 g, 1.06 mol) and TBAHS (1.60 

g, 4.71 mmol) were added to a 40% NaOH aqueous solution (60 mL) at 0 °C. Propargyl 

alcohol (14.85 g, 0.27 mol) was then added dropwise to the flask at 0 °C while stirring, and 

the mixture was allowed to reach room temperature. After 3 h, water was added to the 

reaction mixture to quench the reaction. The aqueous phase was extracted with diethyl 

ether  (3×50  mL)  and  the  organic  phase  was  dried  over  Na2SO4.  Diethyl  ether  was 

evaporated under reduced pressure. The product was purified by vacuum distillation (b.p. 

= 50 °C, 10 torr) to obtain a colorless liquid of propargyl glycidyl ether (PGE, 22.57 g, 

0.20 mol, 76% yield). 1H NMR (CDCl3, 500 MHz): δ 2.44 (t, 1H, J = 2.5 Hz), 2.62 (dd, 

1H, J = 5.0 and 2.7 Hz), 2.79 (t, 1H, J = 4.4 Hz), 3.12-3.17 (m, 1H), 3.46 (dd, 1H, J = 5.9 

and 11.3 Hz), 3.81 (dd, 1H, J = 3.0 and 11.4 Hz), 4.19 (d, 1H, J = 2.4 Hz), 4.20 (d, 1H, J 

= 2.4 Hz). 13C NMR (CDCl3, 125 MHz): δ 44.18, 50.40, 58.36, 70.25, 74.78, 79.18. Anal. 

Calcd. for C6H8O2: C, 64.27; H, 7.19. Found: C, 64.48; H, 7.10. 

 

Synthesis of 1,1´-dimethyl propargyl glycidyl ether (MGE) 

O

O

MGE

 

In an Erlenmeyer flask, (±)-epichlorohydrin (98.04 g, 1.06 mol) and TBAHS (3.2 

g, 9.42 mmol) were added to 25% NaOH aqueous solution (240 mL) at 0 °C. 2-Methyl-3-

 

101 

butyn-2-ol (44.56 g, 0.53 mol) was then added dropwise to the flask at 0 °C while stirring, 

and the mixture was allowed to reach room temperature.. After 24 h, water was added to 

the reaction mixture to quench the reaction. The aqueous phase was extracted with diethyl 

ether  (3×100  mL)  and  the  organic  phase  was  dried  over  Na2SO4.  Diethyl  ether  was 

evaporated under reduced pressure. The product was purified by vacuum distillation (b.p. 

= 60 °C, 10 torr) to obtain a colorless liquid of 1,1´-dimethyl propargyl glycidyl ether 

(MGE, 37.54 g, 0.27 mol, 50% yield). 1H NMR (CDCl3, 500 MHz): δ 1.45 (s, 3H), 1.46 

(s, 3H), 2.42 (s, 1H), 2.62 (dd, 1H, J = 2.9 and 5.4 Hz), 2.79 (dd, 1H, J = 3.9 and 4.9 Hz), 

3.11-3.15 (m, 1H), 3.55 (dd, 1H, J = 5.6 and 10.7 Hz), 3.75 (dd, 1H, J = 3.6 and 10.7 Hz). 

13C NMR (CDCl3, 125 MHz): δ 28.41, 28.66, 44.84, 50.86, 65.22, 70.37, 72.35, 85.52. 

HRMS-ESI (m/z): [M + H]+ calcd. for C8H13O2, 141.0916; found, 141.0919. 

 

General Procedure for Polymerization  

All  polymerizations  were  performed  in  a  glovebox  or  using  standard  Schlenk 

techniques at 25 °C in an atmosphere of high-purity nitrogen. The Schlenk flasks were 

dried  in  an  oven  at  115  °C  overnight  and  allowed  to  cool  prior  to  use.  Monomer(s), 

tetraoctylammonium  bromide  (Oct4NBr,  initiator),  and  triisobutylaluminum  (i-Bu3Al, 

catalyst) were transferred into Schlenk flasks in a glovebox under nitrogen atmosphere, 

and then the catalyst solution in toluene was transferred into the monomer and initiator 

mixture in toluene at -30 °C. After the reaction mixture reached room temperature, it was 

stirred until the reaction was completed or the desired reaction time was reached. A small 

amount of ethanol was then added to stop the polymerization. Then, the toluene solvent 

 

102 

was  removed  in  vacuo.  Polymerization  conversions  were  determined  by  1H  NMR 

spectroscopy. 

 

Synthesis of poly(propargyl glycidyl ether) (poly(PGE))  

H

O

Br

n

O

poly(PGE)

 

Under a nitrogen atmosphere, propargyl glycidyl ether (PGE, 1.12 g, 10 mmol) and 

Oct4NBr (27.5 mg, 0.05 mmol) were dissolved in toluene (10 mL) and the solution was 

cooled to -30 °C. i-Bu3Al (0.1 mL, 0.1 mmol) was added via syringe while stirring. The 

reaction mixture was slowly warmed up to room temperature and stirred for 2.5 h. A small 

amount  of  ethanol (1-2  mL)  was  added  to  quench  the  polymerization  and  toluene  was 

removed  under  reduced  pressure.  Unreacted  propargyl  glycidyl  ether  was  removed  by 

washing with hexanes and then Oct4NBr and i-Bu3Al was removed by passing the crude 

product through silica plug with methanol/CH2Cl2 (1:19, v/v). The solvent was evaporated 

under  reduced  pressure  to  obtain  clear  viscous  gel  of  poly(propargyl  glycidyl  ether) 

(poly(PGE), 1.02 g, 92% yield, Mn = 19000, PDI = 1.26). Polymerization conversions were 

calculated by comparing the 1H NMR integrations of the monomer epoxide peaks at 2.66 

ppm with those on the polymer backbone at 3.52-3.63 ppm. 1H NMR (CDCl3, 500 MHz): 

δ 2.46 (m, 1H), 3.41-3.81 (m, 5H), 4.16 (d, 2H, J = 2.3 Hz). 13C NMR (CDCl3, 125 MHz): 

δ 58.47, 69.54, 69.64, 69.70, 69.80, 69.84, 74.56, 74.62, 74.64, 78.41, 78.51, 78.60, 79.81, 

79.82.  

 

 

103 

Synthesis of poly(1,1´-dimethyl propargyl glycidyl ether) (poly(MGE)) 

H

O

Br

n

O

poly(MGE)

 

Under a nitrogen atmosphere, 1,1´-dimethyl propargyl glycidyl ether (MGE, 2.80 

g, 20 mmol) and Oct4NBr (55.0 mg, 0.05 mmol) were dissolved in toluene (20 mL) and 

the solution was cooled to -30 °C. i-Bu3Al (0.2 mL, 0.2 mmol) was added via syringe. The 

reaction mixture was slowly warmed up to room temperature and stirred for 2.5 h. A small 

amount of ethanol (1-2 mL) was added to quench the reaction and toluene was removed 

under reduced pressure. Unreacted propargyl glycidyl ether was removed by washing with 

hexanes and then Oct4NBr and i-Bu3Al was removed by passing the crude product through 

silica plug with methanol/CH2Cl2 (1:19, v/v). The solvent was evaporated under reduced 

pressure  to  obtain  clear  viscous  gel  of  poly(1,1´-dimethyl  propargyl  glycidyl  ether) 

(poly(MGE), 2.38 g, 85% yield, Mn = 21000, PDI = 1.20). Polymerization conversions 

were determined by comparing the  1H NMR integration of the residual epoxide peak at 

2.64 ppm with those on polymer backbone at 3.56-3.63 ppm. 1H NMR (CDCl3, 500 MHz): 

δ 1.42 (s, 6H), 2.43 (s, 1H), 3.41-3.78 (m, 5H).  13C NMR (CDCl3, 125 MHz): δ 28.63, 

28.69, 28.75, 64.27, 64.30, 64.38, 64.44, 69.86, 69.88, 70.03, 70.06, 70.08, 70.12, 70.30, 

70.33, 72.17, 72.19, 72.22, 72.25, 72.27, 78.54, 78.56, 78.68, 78.70, 78.73, 78.76, 78.85, 

78.88, 86.06, 86.08.  

 

 

104 

General Procedure for “Click” Functionalization49,95  

The  desired  amount  of  acetylene-functionalized  polymer,  azide  (1  equiv  with 

respect to acetylene groups), and 12 mol % sodium ascorbate were dissolved in DMF. The 

resulting solution was transferred to a Schlenk flask and deoxygenated by three freeze-

pump-thaw cycles. After the solution had warmed to room temperature, a 0.1 M solution 

of CuSO4•5H2O in deoxygenated DMF (5 mol % with respect to the acetylene groups) was 

added via syringe under nitrogen and the reaction mixture was stirred at room temperature 

for desired time. At the end of the reaction, the solids in the reaction mixture were removed 

by filtration. Ion exchange resin beads (Amberlite® IRC-748 ion exchange resin) were 

added to the solution and the solution was stirred for 12-24 hours to remove residual copper. 

The ion exchange resin beads were removed by filtration. The polymer was purified via 

dialysis (MWCO = 6-8 kDa) in acetone/water mixture (4:1 v/v) for 24 h and then dried 

under vacuum. 

 

Synthesis of poly(PGE0.50) 

O

N

N

N

3

O

Br

0.50 n

H

O

O

0.50

O

N

N

N

9

poly(PGE0.50)

 

Poly(PGE) (0.20 g, Mn = 19000, PDI = 1.26), mDEG azide (0.17 g, 0.9 mmol, 50 

mol %), decyl azide (0.17 g, 0.9 mmol, 50 mol %), and sodium ascorbate (44.1 mg, 0.2 

 

105 

mmol, 12 mol %) were dissolved in DMF (6 mL). The resulting solution was transferred 

to a Schlenk flask and deoxygenated by three freeze-pump-thaw cycles. After the solution 

had warmed to room temperature, a 0.1 M solution of CuSO4•5H2O in deoxygenated DMF 

(0.9 mL, 5 mol %) was added via syringe under nitrogen and the reaction mixture was 

stirred at room temperature for 3.5 h. At the end of the reaction, the solids in the reaction 

mixture were removed by filtration. Ion exchange resin beads (Amberlite® IRC-748 ion 

exchange resin) were added to the filtrate and the solution was stirred for 18 h to remove 

residual copper. The ion exchange resin beads were removed by filtration. The polymer 

was purified via dialysis (MWCO = 6-8 kDa) in acetone/water mixture (4:1 v/v) for 24 h 

and the product was dried under vacuum to obtain poly(PGE0.50) as a viscous gel (0.45 g, 

84% yield). 1H NMR (CDCl3, 500 MHz) δ 0.86 (t, 3H, J = 6.9 Hz), 1.24-1.30 (m, 12H), 

1.64 (s, 2H), 1.87 (s, 2H), 3.35 (s, 3H), 3.39-3.71 (m, 14H), 3.84-3.86 (m, 2H), 4.29-4.32 

(m, 2H), 4.44-4.54 (m, 2H), 4.54-4.68 (m, 3H), 7.66 (s, 1H), 7.76 (s, 1H). 13C NMR (CDCl3, 

125 MHz) δ 14.10, 22.63, 26.51, 29.03, 29.24, 29.42, 29.48, 30.35, 31.83, 50.02, 50.22, 

58.97, 64.66, 64.84, 69.38, 70.41, 70.44, 71.83, 122.77, 123.87, 144.70, 144.81. 

 

Synthesis of poly(MGE0.50) 

O

N

N

N

3

O

Br

0.50 n

H

O

O

0.50

O

N

N

N

9

poly(MGE0.50)

 

106 

 

Poly(MGE) (0.20 g, Mn = 22000, PDI = 1.13), mDEG azide (0.13 g, 0.7 mmol, 50 

mol %), decyl azide (0.13 g, 0.7 mmol, 50 mol %), and sodium ascorbate (35.6 mg, 0.2 

mmol, 12 mol %) were dissolved in DMF (6 mL). The resulting solution was transferred 

to a Schlenk flask and deoxygenated by three freeze-pump-thaw cycles. After the solution 

had warmed to room temperature, a 0.1 M solution of CuSO4•5H2O in deoxygenated DMF 

(0.7 mL, 5 mol %) was added via syringe under nitrogen and the reaction mixture was 

stirred at room temperature for 3.5 h. At the end of the reaction, the solids in the reaction 

mixture were removed by filtration. Ion exchange resin beads (Amberlite® IRC-748 ion 

exchange resin) were added to the filtrate and the solution was stirred for 18 h to remove 

residual copper. The ion exchange resin beads were removed by filtration. The polymer 

was purified via dialysis (MWCO = 6-8 kDa) in acetone/water mixture (4:1 v/v) for 24 h 

and the product was dried under vacuum to obtain poly(MGE0.50) as a viscous gel (0.39 g, 

82% yield). 1H NMR (CDCl3, 500 MHz) δ 0.86 (t, 3H, J = 6.8 Hz), 1.23-1.29 (m, 14H), 

1.52 (s, 12H), 1.85-1.88 (m, 3H), 3.19-3.59 (m, 18H), 3.85-3.87 (m, 2H), 4.27-4.30 (m, 

2H), 4.48-4.50 (m, 2H), 7.59 (s, 1H), 7.66 (s, 1H). 13C NMR (CDCl3, 125 MHz) δ 14.08, 

22.61, 26.55, 26.71, 26.82, 26.89, 27.0, 27.01, 27.07, 27.27, 27.50, 29.03, 29.24, 29.41, 

29.47, 30.40, 31.82, 49.94, 50.17, 58.95, 62.87, 63.03, 69.43, 69.61, 70.02, 70.41, 70.45, 

71.83, 72.43, 72.64, 120.93, 122.00, 152.14, 152.39. 

 

Safety Information for Synthesizing, Purifying, and Handling Organic Azides270 

Organic azides are potentially explosive materials that can decomposed violently 

which may result in injury if proper precautions are not taken. Read material safety data 

sheet before conducting experiments. NEVER use chlorinated solvents as reaction solvent 

 

107 

or  during  workup.  Chlorinated  solvents,  such  as  dichloromethane  and  chloroform,  and 

sodium azide can generate explosive and unstable di- or tri- azomethane. 

 

Synthesis of 1-azidodecane (decylazide)95,225,226 

N3

 

decyl azide

 

WARNING! NEVER use chlorinated solvents as reaction solvent or during workup! 

1-Bromodecane (10.00 g, 45.2 mmol) and sodium azide (14.70 g, 226.0 mmol) 

were dissolved in DMF (180 mL) and the solution was stirred and heated to 80 °C. After 

24 h, the solution was cooled to room temperature and was added to a 600 mL beaker 

containing DI water (100 mL). The solution was extracted with diethyl ether (4×100 mL). 

The  combined  organic  layers  were  washed  with  distilled  water  (3×100  mL),  saturated 

aqueous NaCl solution (2×100 mL), and dried over MgSO4. Diethyl ether was evaporated 

under reduced pressure and the product was purified via vacuum distillation (b.p. = 41.1 

°C, 5 torr) to obtain 1-azidodecane as a clear liquid (7.18 g, 39.2 mmol, 87% yield).  1H 

NMR (CDCl3, 500 MHz) δ 0.88 (t, 3H, J = 6.9 Hz), 1.25-1.36 (m, 14H), 1.55-1.61 (m, 2H), 

3.22-3.25 (t, 2H, J = 7.0 Hz). 13C NMR (CDCl3, 125 MHz) δ 14.08, 22.66, 26.71, 28.82, 

29.14, 29.27, 29.49, 31.86, 51.49. 

 

Synthesis of 1-(2-azidoethoxy)-2-(2-methoxyethoxy)ethane (mDEG azide)95 

N3

O
mDEG azide

O

O

 

Triethylene  glycol monomethyl  ether  (20.00  g,  0.12 mol)  in  THF (60  mL)  was 

added dropwise to a solution of NaOH (14.60g, 0.37 mol) in a water/THF mixture (6:4 v/v, 

 

108 

200 mL) at 0 °C while stirring. The mixture was stirred at 0 °C for 30 minutes and then p-

toluenesulfonic chloride (23.00 g, 0.12 mol) in THF (100 mL) was added dropwise. The 

mixture was stirred at 0 °C for 3 h and then at room temperature for 6 h. The mixture was 

then  poured  into  a  600  mL  beaker  containing  ice  water  (30  mL). The  water  layer  was 

extracted with diethyl ether (3×100 mL). The combined organic layers were washed with 

saturated  aqueous  NaCl  solution  and  dried  over MgSO4.  Diethyl  ether  was  evaporated 

under  reduced  pressure  and  the  tosylate  product  was  used  without  further  purification 

(32.86 g, 0.10 mol, 85% yield). 

WARNING! NEVER use chlorinated solvents as reaction solvent or during workup! 

The mDEG tosylate (32.86 g, 0.10 mol) and sodium azide (13.43 g, 0.20 mol) were 

dissolved in DMF (200 mL) and the solution was heated to 60 °C. After 15 h, the solution 

was cooled to room temperature and was added to a 600 mL beaker containing water (100 

mL). The solution was extracted with diethyl ether (4×100 mL). The combined organic 

layers  were  washed  with  saturated  aqueous  NaCl  solution  (2×100  mL)  and  dried  over 

MgSO4. Diethyl ether was evaporated, and the product was purified via vacuum distillation 

(b.p.  =  141.7  °C,  5  torr)  to  obtain  a  clear  liquid  of  1-(2-azidoethoxy)-2-(2-

methoxyethoxy)ethane (mDEG azide, 10.93g, 0.06 mol, 60% yield).  1H NMR (CDCl3, 

500 MHz) δ 3.25-3.28 (m, 5H), 3.42-3.45 (m, 2H), 3.52-3.57 (m, 8H). 13C NMR (CDCl3, 

125 MHz) δ 50.36, 58.62, 58.64, 69.71, 70.26, 70.27, 70.33, 70.34, 70.36, 70.37, 71.61. 

 

 

 

 

109 

Synthesis  of  1-azido  poly(ethylene  glycol)  monomethyl  ether,  550  (mPEG550 

azide)229,230 

O

N3
12
mPEG550 azide

 

Polyethylene glycol monomethyl ether, 550 (mPEG550, 20.00 g, 36.4 mmol) and 

p-toluenesulfonic chloride (10.40 g, 54.5 mmol) were dissolved in THF (80 mL) and the 

mixture was cooled to 0 °C. A solution of KOH (13.4g, 0.24 mol) in water (15 mL) was 

added dropwise to the reaction mixture at 0 °C while stirring. The solution was stirred at 0 

°C for 30 minutes then at room temperature for 16 h. A solid suspension was observed after 

16 h. The reaction was quenched with CH2Cl2/water mixture (9:1 v/v) until all the solid 

was dissolved. The water layer was extracted with CH2Cl2 (3×100 mL). The combined 

organic layers were washed with saturated aqueous NaCl solution and stirred with Na2SO4 

overnight. CH2Cl2 was evaporated under vacuum and the tosylate product was obtained as 

a colorless liquid and was used without further purification (22.37 g, 31.8 mmol, 87% 

yield). 

WARNING! NEVER use chlorinated solvents as reaction solvent or during workup! 

The tosyl mPEG550 (10.00 g, 14.2 mmol) and sodium azide (1.39 g, 21.3 mmol) 

were  dissolved  in  DMF  (100  mL)  and  the  solution  was  stirred  at  room  temperature 

overnight. DMF was evaporated under vacuum at 70 °C and the solid was redissolved in 

ethyl acetate. The solution was filtered through Celite and ethyl acetate was evaporated 

under reduced pressure to obtain 1-azido polyethylene glycol monomethyl ether, 550 as a 

clear liquid (mPEG550 azide ,7.76 g, 13.5 mmol, 95% yield). 1H NMR (CDCl3, 500 MHz) 

δ, 3.37-3.39 (m, 4H), 3.53-3.54 (m, 2H), 3.62-3.67 (m, 38H). 13C NMR (CDCl3, 125 MHz) 

 

110 

δ 50.62, 59.00, 69.98, 70.45, 70.49, 70.53, 70.57, 70.60, 70.64, 71.86. IR (dry film, cm-1): 

2106 (N=N=N) 

 

Synthesis  of  1-azido  poly(ethylene  glycol)  monomethyl  ether,  750  (mPEG750 

azide)229,230 

O

N3
16
mPEG750 azide

 

Polyethylene glycol monomethyl ether, 750 (mPEG750, 20.00 g, 26.7 mmol) and 

p-toluenesulfonic  chloride  (7.6  g,  40  mmol)  were  dissolved  in  THF  (80  mL)  and  the 

mixture was cooled to 0 °C. A solution of KOH (9.8g, 0.18 mol) in water (11 mL) was 

added dropwise to the reaction mixture at 0 °C while stirring. The solution was stirred at 0 

°C for 30 minutes then at room temperature for 16 h. A solid suspension was observed after 

16 h. The reaction was quenched with CH2Cl2/water mixture (9:1 v/v) until all the solid 

was dissolved. The water layer was extracted with CH2Cl2 (3×100 mL). The combined 

organic layers were washed with saturated aqueous NaCl solution and stirred with Na2SO4 

overnight. CH2Cl2 was evaporated under vacuum and the tosylate product was obtained as 

a clear low melting point solid, which was used without further purification (21.20 g, 23.4 

mmol, 88% yield). 

WARNING! NEVER use chlorinated solvents as reaction solvent or during workup! 

Tosyl mPEG750 (10.00 g, 11.1 mmol) and sodium azide (1.08 g, 16.6 mmol) were 

dissolved in DMF (100 mL) and the solution was stirred at room temperature overnight. 

DMF was evaporated under vacuum at 70 °C and the solid was redissolved in ethyl acetate. 

The solution was filtered through Celite and ethyl acetate was evaporated under reduced 

 

111 

pressure  to  obtain  1-azido  polyethylene  glycol  monomethyl  ether,  750  as  a  clear,  low-

melting point solid (mPEG750 azide, 7.16 g, 9.3 mmol, 84% yield). 1H NMR (CDCl3, 500 

MHz) δ, 3.33-3.35 (m, 4H), 3.49-3.51 (m, 2H), 3.58-3.64 (m, 50H). 13C NMR (CDCl3, 125 

MHz) δ 50.50, 58.88, 69.87, 70.34, 70.39, 70.43, 70.46, 70.49, 70.52, 71.75. IR (dry film, 

cm-1): 2106 (N=N=N). 

 

Synthesis  of  1-azido  poly(ethylene  glycol)  monomethyl  ether,  2000  (mPEG2000 

azide)229,230 

O

N3
45
mPEG2000 azide

 

Polyethylene glycol monomethyl ether, 2000 (mPEG2000, 10.00 g, 5.0 mmol) and 

p-toluenesulfonic chloride (1.43 g, 7.5 mmol) were dissolved in THF (40 mL) at 50 °C and 

the mixture was cooled to room temperature. A solution of KOH (1.84 g, 32.8 mmol) in 

water (2 mL) was added dropwise to the reaction mixture while stirring and the solution 

was stirred at room temperature for 16 h. A solid suspension was observed after 16 h. The 

reaction was quenched with CH2Cl2/water mixture (9:1 v/v) until all the solid was dissolved. 

The water layer was extracted with CH2Cl2 (3×100 mL). The combined organic layers were 

washed with saturated aqueous NaCl solution and stirred with Na2SO4 overnight. CH2Cl2 

was evaporated under reduced pressure and the tosylate product was obtained as an off-

white solid, which was used without further purification (9.43 g, 4.4 mmol, 88% yield). 

WARNING! NEVER use chlorinated solvents as reaction solvent or during workup! 

Tosyl mPEG2000 (8.73 g, 4.1 mmol) and sodium azide (0.40 g, 6.1 mmol) were 

dissolved in DMF (100 mL) and the solution was stirred at room temperature overnight. 

 

112 

DMF was evaporated under vacuum at 70 °C and the solid was redissolved in ethyl acetate. 

The solution was filtered through Celite and ethyl acetate was evaporated under reduced 

pressure to obtain 1-azido polyethylene glycol monomethyl ether, 2000 as a white solid 

(mPEG2000 azide, 7.83 g, 3.87 mmol, 95% yield). 1H NMR (CDCl3, 500 MHz) δ, 3.37 

(s, 3H), 3.53 – 3.55 (m, 2H), 3.62-3.65 (m, 125H). 13C NMR (CDCl3, 125 MHz) δ 50.63, 

59.01, 70.47, 70.52, 70.56, 70.59, 70.62, 71.88. IR (dry film, cm-1): 2105 (N=N=N). 

 

Synthesis of 6-azidophexanoic acid (COOH azide)231 

N3

O
COOH azide

OH

 

WARNING! NEVER use chlorinated solvents as reaction solvent or during workup! 

6-Bromohexanoic acid (5.00 g, 25.6 mmol) and sodium azide (3.33 g, 51.2 mmol) 

were dissolved in DMF (20 mL). The solution was heated to 85 °C and was stirred for 

overnight.  The  solution  was  cooled  to  room  temperature  and  was  diluted  with  0.1  M 

aqueous HCl solution (20 mL). The solution was extracted with ethyl acetate (3×50 mL). 

The combined organic layers were washed with 0.1 M aqueous HCl solution (5×20 mL) 

and dried over Na2SO4. Ethyl acetate was evaporated under reduced pressure to obtain 6-

azidophexanoic acid as a clear liquid (1.57 g, 10.0 mmol, 39% yield). 1H NMR (CDCl3, 

500 MHz) δ 1.39-1.47 (m, 2H), 1.60-1.68 (m, 4H), 2.37 (t, 2H, J = 7.4 Hz), 3.28 (t, 2H, J 

= 6.9 Hz). 13C NMR (CDCl3, 125 MHz) δ 24.11, 26.11, 28.50, 33.81, 51.15, 179.93. IR 

(dry film, cm-1): 2097 (N=N=N), 1708 (C=O). 

 

 

113 

Synthesis of 3-azido-N,N,N-trimethylpropan-1-aminium iodide (aminium azide)232,233 

N3

N

aminium azide

I

 

WARNING! NEVER use chlorinated solvents as reaction solvent or during workup! 

3-Dimethylaminopropyl  chloride  hydrochloride  (10.01  g,  63.3  mmol)  were 

dissolved in DI water (127 mL) and sodium azide (8.23 g, 126.6 mmol) was added. The 

solution was heated to 80 °C and was stirred for 24 h. The solution was cooled in ice bath, 

and KOH was added to adjust the pH to approximately 10. The solution was extracted with 

diethyl ether (3×50 mL). The combined organic layers were dried over MgSO4. Diethyl 

ether  was  evaporated  under  reduced  pressure  to  obtain  3-azido-N,N-dimethylpropan-1-

amine as a pale-yellow liquid, which was used without further purification (7.11 g, 55.5 

mmol, 88% yield). 

To a solution of methyl iodide (1.00 g, 7.1 mmol) in methanol (5 mL), a solution 

of 3-azido-N,N-dimethylpropan-1-amine (1.11 g, 8.7 mmol) in methanol (5 mL) was added. 

The reaction mixture was refluxed for 20 h and then was cooled to room temperature. 

Methanol and the unreacted 3-azido-N,N-dimethylpropan-1-amine were evaporated under 

reduced  pressure  to  obtain  3-azido-N,N,N-trimethylpropan-1-aminium  iodide  as  pale-

yellow solid (aminium azide, 1.60 g, 5.9 mmol, 84% yield). 1H NMR (D2O, 500 MHz) δ 

2.03-2.17 (m, 2H), 3.14 (s, 9H), 3.40-3.47 (m, 2H), 3.50 (t, 2H, J = 6.4 Hz).  13C NMR 

(D2O, 125 MHz) δ 22.25, 47.69, 47.71, 47.72, 52.95, 52.98, 53.01, 63.97, 64.00, 64.02 

 

 

114 

General Procedure for Azobenzene (transPhN=NPh) Encapsulation 

Two methods were used to encapsulate azobenzene, and the results obtained from 

both methods were identical. The first method was used as a direct comparison to previous 

work, where azobenzene and the “click” modified polymer were dissolved in acetone (<1 

mL). The resulting solution was slowly added dropwise to stirred ice-cold Milli-Q water 

(5  mL)  in  a  Schlenk flask.95  The  acetone  was  removed  in  vacuo  and  the  solution  was 

filtered to remove unencapsulated azobenzene. In the second azobenzene and the “click” 

modified polymer were dissolved in Milli-Q water. The solution was then stirred for 24 

hours followed by filtration to remove unencapsulated azobenzene. 

 

General Procedure for Subtilisin Carlsberg (SC) Encapsulation in Aqueous Media 

SC and the “click” modified polymer, in which the molar ratio between SC and 

polymer was 1:2, were dissolved in phosphate-buffered saline (PBS) solution (pH = 7.4). 

The solution was then placed in dialysis bag (MWCO = 6-8 kDa) with Milli-Q water for 

24 h to replace the counterions on protein surface with polymers. The solution was dried 

under vacuum and redissolved in Milli-Q water, PBS, or Tris-HCl. 

 

General Procedure for Subtilisin Carlsberg (SC) Encapsulation in Organic Media 

Two methods were used for the encapsulation of SC in organic media. The first 

method involved adding an aqueous solution of SC and the “click” modified polymer, in 

which the molar ratio between SC and polymer was 1:2, to toluene and the solvents in the 

vigorously  mixed  two-phase  mixture  were  removed  in  vacuo.  The  resulting  solid  was 

redissolved in toluene. In the second method, SC and the “click” modified polymer were 

 

115 

dissolved  in  PBS,  after  which  the  solution  was  lyophilized.  The  resulting  solid  was 

redissolved in toluene. 

 

General Procedure for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA) 

Assay 

Aliquots (10 µL) of SC or SC@polymer solution were taken at designated time 

points. Each aliquot was immediately added to a freshly prepared solution of Suc-AAPF-

pNA in Tris-HCl buffer (2 mL, 0.02 mM, pH = 8.0). The UV-Vis spectrum was obtained 

after 5 min and the absorbance at 412 nm (J = 8,480 M-1 cm-1) was used to calculate the 

concentration of p-nitroaniline. 

 

 

 

 

116 

 

 

 

 

 

 

 

 

 

 

 

 

 

APPENDIX 

117 

He

Ha
Ha′

O

Hc

Hb

O
Hd
Hc′

Hd′

Figure A1. 500 MHz 1H NMR spectrum of PGE in CDCl3. 

 

 

118 

e 

a 

a¢ 

d, d¢ 

c¢ 

c 

b 

Figure A2. 500 MHz 1H NMR spectrum of PGE in CDCl3between 2.0-4.5 ppm. 

 

 

119 

O

1

2

3

6

O

4

5

3 

4 

1 

2 

6 

5 

Figure A3. 125 MHz 13C NMR spectrum of PGE in CDCl3. 

 

 

120 

Figure A4. 2D HSQC NMR spectrum of PGE in CDCl3. 

 

 

121 

Figure A5. 2D HSQC NMR spectrum of PGE in CDCl3. 

 

 

122 

He

Ha
Ha′

O

Hc

Hb

O
Hc′

d, d′

Figure A6. 500 MHz 1H NMR spectrum of MGE in CDCl3. 

 

 

123 

d, d¢ 

e 

c¢ 

c 

b 

a 

a¢ 

Figure A7. 500 MHz 1H NMR spectrum of MGE in CDCl3 between 1.0-4.0 ppm. 

 

 

124 

O

1

2

3

6

O

4

7

5

7'

3 
 

6 
 

4 
 

5 
 

7, 7¢ 

 

1 
 

2 
 

Figure A8. 125 MHz 13C NMR spectrum of MGE in CDCl3. 

 

 

125 

Figure A9. 2D HSQC NMR spectrum of MGE in CDCl3. Methyl region was omitted for clarity.  

 

 

126 

a, a'

H

O

b

c, c'

O
d, d'

Br

n

e

d, d¢ 

a-c 

e 

Figure A10. 500 MHz 1H NMR spectrum of poly(PGE) in CDCl3. 

 

 

127 

H

O

1

3

Br

n

2

O
4
5

6

4 

2, 3 

6 

1 

5 

Figure A11. 125 MHz 13C NMR spectrum of poly(PGE) in CDCl3. 

 

 

128 

Figure A12. 125 MHz 13C NMR spectrum of poly(PGE) in CDCl3 between 25.0-35.0 ppm. 

 

 

129 

a, a'

O

Br

n

H

O

b

c, c'

d

d'

e

d, d¢ 

a-c 

e 

Figure A13. 500 MHz 1H NMR spectrum of poly(MGE) in CDCl3. 

 

 

130 

H

O

1

2

3
7

O
4
5

7′

Br

n

6

7, 7¢ 

 

4 
 

6 
 

2, 3 

 

5 
 

1 
 

Figure A14. 125 MHz 13C NMR spectrum of poly(MGE) in CDCl3. 

 

 

131 

Figure A15. Mass spectrum of poly(PGE) (Mn = 2800, PDI = 1.14) via positive electrospray ionization. 

 

 

132 

Figure A16. Mass spectrum of poly(MGE) (Mn = 3900, PDI = 1.16) via positive electrospray ionization.

 

 

133 

16000
14000
12000
10000
8000
6000
4000
2000
0

)
l
o
m
g
(

/

n

M

0

20

2
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
100

I

D
P

80

40
60
Conversion (%)

 
Figure A17. Variation of the number-average molar mass (black square) and PDI (red 
square) with PGE conversion ([M]: [Al]: [I] = 200: 2: 1). 

100
90
80
70
60
50
40
30
20
10
0

)

%

i

(
 
n
o
s
r
e
v
n
o
C

0

50

 
Figure A18. Conversion vs. time of the polymerization of PGE ([M]: [Al]: [I] = 200: 2: 
1). 

100

Time (min)

150

200

 

 

134 

25:	2:	1
50:	2:	1
100:	2:	1
200:	2:	1

13.5

14.5

25:	2:	1
50:	2:	1
100:	2:	1
200:	2:	1
500:	2:	1
500:	3:	1

15.5

16.5

17.5

 
Figure A19. GPC traces of poly(PGE). The polymers were analyzed in THF at 35 °C, at 
1 mL/min flow rate. 

Retention	Time	(min)

13.5

14.5

15.5

16.5

17.5

 
Figure A20. GPC traces of poly(MGE). The polymers were analyzed in THF at 35 °C, at 
1 mL/min flow rate. 

Retention	Time	(min)

 

135 

14

14.5

15

15.5

16

16.5

17

 
Figure A21. GPC traces of poly(PGE0.X). The polymers were analyzed in THF at 35 °C, 
at 1 mL/min flow rate. 

Retention	Time	(min)

p-PGE	0
p-PGE	0.20
p-PGE	0.40
p-PGE	0.50
p-PGE	0.55
p-PGE	0.60
p-PGE	0.65
p-PGE	0.70
p-PGE	0.75
p-PGE	0.80
p-PGE	0.85
p-PGE	0.90
p-PGE	0.95
p-PGE	1.0

p-MGE	0
p-MGE	0.20
p-MGE	0.40
p-MGE	0.50
p-MGE	0.55
p-MGE	0.60
p-MGE	0.65
p-MGE	0.70
p-MGE	0.75
p-MGE	0.80
p-MGE	0.85
p-MGE	0.90
p-MGE	0.95
p-MGE	1.0

13.5

14

14.5

15

15.5

16

16.5

17

 
Figure A22. GPC traces of poly(MGE0.X). The polymers were analyzed in THF at 35 °C, 
at 1 mL/min flow rate. 

Retention	Time	(min)

 

 

136 

O

H

Br

n

O

N

N

N
poly(PGE0)

9

Figure A23. 500 MHz 1H NMR spectrum of poly(PGE0) in CDCl3. 

 

 

137 

O

H

Br

n

O

N

N

N
poly(PGE0)

9

Figure A24. 125 MHz 13C NMR spectrum of poly(PGE0) in CDCl3. 

 

 

138 

O

N

N

N

3

O

Br

0.20 n

H

O

O

0.80

O

N

N

N

9
poly(PGE0.20)

Figure A25. 500 MHz 1H NMR spectrum of poly(PGE0.20) in CDCl3. 

 

 

139 

O

N

N

N

3

O

Br

0.20 n

H

O

O

0.80

O

N

N

N

9
poly(PGE0.20)

Figure A26. 125 MHz 13C NMR spectrum of poly(PGE0.20) in CDCl3. 

 

 

140 

O

N

N

N

3

O

Br

0.40 n

H

O

O

0.60

O

N

N

N

9

poly(PGE0.40)

Figure A27. 500 MHz 1H NMR spectrum of poly(PGE0.40) in CDCl3. 

 

 

141 

O

N

N

N

3

O

Br

0.40 n

H

O

O

0.60

O

N

N

N

9

poly(PGE0.40)

Figure A28. 125 MHz 13C NMR spectrum of poly(PGE0.40) in CDCl3. 

 

 

142 

O

N

N

N

3

O

Br

0.50 n

H

O

O

0.50

O

N

N

N

9

poly(PGE0.50)

Figure A29. 500 MHz 1H NMR spectrum of poly(PGE0.50) in CDCl3. 

 

 

143 

O

N

N

N

3

O

Br

0.50 n

H

O

O

0.50

O

N

N

N

9

poly(PGE0.50)

Figure A30. 125 MHz 13C NMR spectrum of poly(PGE0.50) in CDCl3. 

 

 

144 

O

N

N

N

3

O

Br

0.55 n

H

O

O

0.45

O

N

N

N

9

poly(PGE0.55)

Figure A31. 500 MHz 1H NMR spectrum of poly(PGE0.55) in CDCl3. 

 

 

145 

O

N

N

N

3

O

Br

0.55 n

H

O

O

0.45

O

N

N

N

9

poly(PGE0.55)

Figure A32. 125 MHz 13C NMR spectrum of poly(PGE0.55) in CDCl3. 

 

 

146 

O

N

N

N

3

O

Br

0.60 n

H

O

O

0.40

O

N

N

N

9

poly(PGE0.60)

Figure A33. 500 MHz 1H NMR spectrum of poly(PGE0.60) in CDCl3. 

 

 

147 

O

N

N

N

3

O

Br

0.60 n

H

O

O

0.40

O

N

N

N

9

poly(PGE0.60)

Figure A34. 125 MHz 13C NMR spectrum of poly(PGE0.60) in CDCl3. 

 

 

148 

O

N

N

N

3

O

Br

0.65 n

H

O

O

0.35

O

N

N

N

9

poly(PGE0.65)

Figure A35. 500 MHz 1H NMR spectrum of poly(PGE0.65) in CDCl3. 

 

 

149 

O

N

N

N

3

O

Br

0.65 n

H

O

O

0.35

O

N

N

N

9

poly(PGE0.65)

Figure A36. 125 MHz 13C NMR spectrum of poly(PGE0.65) in CDCl3. 

 

 

150 

O

N

N

N

3

O

Br

0.70 n

H

O

O

0.30

O

N

N

N

9

poly(PGE0.70)

Figure A37. 500 MHz 1H NMR spectrum of poly(PGE0.70) in CDCl3. 

 

 

151 

O

N

N

N

3

O

Br

0.70 n

H

O

O

0.30

O

N

N

N

9

poly(PGE0.70)

Figure A38. 125 MHz 13C NMR spectrum of poly(PGE0.70) in CDCl3. 

 

 

152 

O

N

N

N

3

O

Br

0.75 n

H

O

O

0.25

O

N

N

N

9

poly(PGE0.75)

Figure A39. 500 MHz 1H NMR spectrum of poly(PGE0.75) in CDCl3. 

 

 

153 

O

N

N

N

3

O

Br

0.75 n

H

O

O

0.25

O

N

N

N

9

poly(PGE0.75)

Figure A40. 125 MHz 13C NMR spectrum of poly(PGE0.75) in CDCl3. 

 

 

154 

O

N

N

N

3

O

Br

0.80 n

H

O

O

0.20

O

N

N

N

9

poly(PGE0.80)

Figure A41. 500 MHz 1H NMR spectrum of poly(PGE0.80) in CDCl3. 

 

 

155 

O

N

N

N

3

O

Br

0.80 n

H

O

O

0.20

O

N

N

N

9

poly(PGE0.80)

Figure A42. 125 MHz 13C NMR spectrum of poly(PGE0.80) in CDCl3. 

 

 

156 

O

N

N

N

3

O

Br

0.85 n

H

O

O

0.15

O

N

N

N

9

poly(PGE0.85)

Figure A43. 500 MHz 1H NMR spectrum of poly(PGE0.85) in CDCl3. 

 

 

157 

O

N

N

N

3

O

Br

0.85 n

H

O

O

0.15

O

N

N

N

9

poly(PGE0.85)

Figure A44. 125 MHz 13C NMR spectrum of poly(PGE0.85) in CDCl3. 

 

 

158 

O

N

N

N

3

O

Br

0.90 n

H

O

O

0.10

O

N

N

N

9

poly(PGE0.90)

Figure A45. 500 MHz 1H NMR spectrum of poly(PGE0.90) in CDCl3. 

 

 

159 

O

N

N

N

3

O

Br

0.90 n

H

O

O

0.10

O

N

N

N

9

poly(PGE0.90)

Figure A46. 125 MHz 13C NMR spectrum of poly(PGE0.90) in CDCl3 

 

 

160 

O

N

N

N

3

O

Br

0.95 n

H

O

O

0.05

O

N

N

N

9

poly(PGE0.95)

Figure A47. 500 MHz 1H NMR spectrum of poly(PGE0.95) in CDCl3. 

 

 

161 

O

N

N

N

3

O

Br

0.95 n

H

O

O

0.05

O

N

N

N

9

poly(PGE0.95)

Figure A48. 125 MHz 13C NMR spectrum of poly(PGE0.95) in CDCl3. 

 

 

162 

O

H

Br

n

O

N

N

N

poly(PGE1.0)

O

3

Figure A49. 500 MHz 1H NMR spectrum of poly(PGE1.0) in CDCl3. 

 

 

163 

O

H

Br

n

O

N

N

N

poly(PGE1.0)

O

3

Figure A50. 125 MHz 13C NMR spectrum of poly(PGE1.0) in CDCl3. 

 

 

164 

O

H

Br

n

O

N

N

N
poly(MGE0)

9

Figure A51. 500 MHz 1H NMR spectrum of poly(MGE0) in CDCl3. 

 

 

165 

O

H

Br

n

O

N

N

N
poly(MGE0)

9

Figure A52. 125 MHz 13C NMR spectrum of poly(MGE0) in CDCl3. 

 

 

166 

O

N

N

N

3

O

Br

0.20 n

H

O

O

0.80

O

N

N

N

9

poly(MGE0.20)

Figure A53. 500 MHz 1H NMR spectrum of poly(MGE0.20) in CDCl3. 

 

 

167 

O

N

N

N

3

O

Br

0.20 n

H

O

O

0.80

O

N

N

N

9

poly(MGE0.20)

Figure A54. 125 MHz 13C NMR spectrum of poly(MGE0.20) in CDCl3. 

 

 

168 

O

N

N

N

3

O

Br

0.40 n

H

O

O

0.60

O

N

N

N

9

poly(MGE0.40)

Figure A55. 500 MHz 1H NMR spectrum of poly(MGE0.40) in CDCl3. 

 

 

169 

O

N

N

N

3

O

Br

0.40 n

H

O

O

0.60

O

N

N

N

9

poly(MGE0.40)

Figure A56. 125 MHz 13C NMR spectrum of poly(MGE0.40) in CDCl3. 

 

 

170 

O

N

N

N

3

O

Br

0.50 n

H

O

O

0.50

O

N

N

N

9

poly(MGE0.50)

Figure A57. 500 MHz 1H NMR spectrum of poly(MGE0.50) in CDCl3. 

 

 

171 

O

N

N

N

3

O

Br

0.50 n

H

O

O

0.50

O

N

N

N

9

poly(MGE0.50)

Figure A58. 125 MHz 13C NMR spectrum of poly(MGE0.50). in CDCl3 

 

 

172 

O

N

N

N

3

O

Br

0.55 n

H

O

O

0.45

O

N

N

N

9

poly(MGE0.55)

Figure A59. 500 MHz 1H NMR spectrum of poly(MGE0.55) in CDCl3. 

 

 

173 

O

N

N

N

3

O

Br

0.55 n

H

O

O

0.45

O

N

N

N

9

poly(MGE0.55)

Figure A60. 125 MHz 13C NMR spectrum of poly(MGE0.55) in CDCl3. 

 

 

174 

O

N

N

N

3

O

Br

0.60 n

H

O

O

0.40

O

N

N

N

9

poly(MGE0.60)

Figure A61. 500 MHz 1H NMR spectrum of poly(MGE0.60) in CDCl3. 

 

 

175 

O

N

N

N

3

O

Br

0.60 n

H

O

O

0.40

O

N

N

N

9

poly(MGE0.60)

Figure A62. 125 MHz 13C NMR spectrum of poly(MGE0.60) in CDCl3. 

 

 

176 

O

N

N

N

3

O

Br

0.65 n

H

O

O

0.35

O

N

N

N

9

poly(MGE0.65)

Figure A63. 500 MHz 1H NMR spectrum of poly(MGE0.65) in CDCl3. 

 

 

177 

O

N

N

N

3

O

Br

0.65 n

H

O

O

0.35

O

N

N

N

9

poly(MGE0.65)

Figure A64. 125 MHz 13C NMR spectrum of poly(MGE0.65) in CDCl3. 

 

 

178 

O

N

N

N

3

O

Br

0.70 n

H

O

O

0.30

O

N

N

N

9

poly(MGE0.70)

Figure A65. 500 MHz 1H NMR spectrum of poly(MGE0.70) in CDCl3. 

 

 

179 

O

N

N

N

3

O

Br

0.70 n

H

O

O

0.30

O

N

N

N

9

poly(MGE0.70)

Figure A66. 125 MHz 13C NMR spectrum of poly(MGE0.70) in CDCl3. 

 

 

180 

O

N

N

N

3

O

Br

0.75 n

H

O

O

0.25

O

N

N

N

9

poly(MGE0.75)

Figure A67. 500 MHz 1H NMR spectrum of poly(MGE0.75) in CDCl3. 

 

 

181 

O

N

N

N

3

O

Br

0.75 n

H

O

O

0.25

O

N

N

N

9

poly(MGE0.75)

Figure A68. 125 MHz 13C NMR spectrum of poly(MGE0.75) in CDCl3. 

 

 

182 

O

N

N

N

3

O

Br

0.80 n

H

O

O

0.20

O

N

N

N

9

poly(MGE0.80)

Figure A69. 500 MHz 1H NMR spectrum of poly(MGE0.80) in CDCl3. 

 

 

183 

O

N

N

N

3

O

Br

0.80 n

H

O

O

0.20

O

N

N

N

9

poly(MGE0.80)

Figure A70. 125 MHz 13C NMR spectrum of poly(MGE0.80) in CDCl3. 

 

 

184 

O

N

N

N

3

O

Br

0.85 n

H

O

O

0.15

O

N

N

N

9

poly(MGE0.85)

Figure A71. 500 MHz 1H NMR spectrum of poly(MGE0.85) in CDCl3. 

 

 

185 

O

N

N

N

3

O

Br

0.85 n

H

O

O

0.15

O

N

N

N

9

poly(MGE0.85)

Figure A72. 125 MHz 13C NMR spectrum of poly(MGE0.85) in CDCl3. 

 

 

186 

O

N

N

N

3

O

Br

0.90 n

H

O

O

0.10

O

N

N

N

9

poly(MGE0.90)

Figure A73. 500 MHz 1H NMR spectrum of poly(MGE0.90) in CDCl3. 

 

 

187 

O

N

N

N

3

O

Br

0.90 n

H

O

O

0.10

O

N

N

N

9

poly(MGE0.90)

Figure A74. 125 MHz 13C NMR spectrum of poly(MGE0.90) in CDCl3. 

 

 

188 

O

N

N

N

3

O

Br

0.95 n

H

O

O

0.05

O

N

N

N

9

poly(MGE0.95)

Figure A75. 500 MHz 1H NMR spectrum of poly(MGE0.95) in CDCl3. 

 

 

189 

O

N

N

N

3

O

Br

0.95 n

H

O

O

0.05

O

N

N

N

9

poly(MGE0.95)

Figure A76. 125 MHz 13C NMR spectrum of poly(MGE0.95) in CDCl3. 

 

 

190 

O

H

Br

n

O

N

N

N

poly(MGE1.0)

O

3

Figure A77. 500 MHz 1H NMR spectrum of poly(MGE1.0) in CDCl3. 

 

 

191 

O

H

Br

n

O

N

N

N

poly(MGE1.0)

O

3

Figure A78. 125 MHz 13C NMR spectrum of poly(MGE1.0) in CDCl3. 

 

 

192 

O

N

N

N

3

O

O

0.57

H

O

O

0.38

O

N

N

N

9

Br

0.05 n

O

N

N N

N I

poly(PGE0.57-Pos0.05)

Figure A79. 500 MHz 1H NMR spectrum of poly(PGE0.57-Pos0.05) in CDCl3. 

 

 

193 

O

N

N

N

3

O

O

0.57

H

O

O

0.38

O

N

N

N

9

Br

0.05 n

O

N

N N

N I

poly(PGE0.57-Pos0.05)

 

Figure A80. 125 MHz 13C NMR spectrum of poly(PGE0.57-Pos0.05) in CDCl3. 

 

 

194 

O

N

N

N
12

H

O

O

0.38

O

N

N

O

O

0.57

Br

0.05 n

O

N

N N

9

N
poly(PGE0.57-mPEG550-Pos0.05)

N I

Figure A81. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG550-Pos0.05) in CDCl3. 

 

 

195 

O

N

N

N
16

H

O

O

0.38

O

N

N

O

O

0.57

Br

0.05 n

O

N

N N

9

N
poly(PGE0.57-mPEG750-Pos0.05)

N I

Figure A82. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG750-Pos0.05) in CDCl3. 

 

 

196 

O

N

N

N
45

H

O

O

0.38

O

N

N

O

O

0.57

Br

0.05 n

O

N

N N

9

N
poly(PGE0.57-mPEG2000-Pos0.05)

N I

Figure A83. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG2000-Pos0.05) in CDCl3. 

 

 

197 

O

N

N

N
45

H

O

O

0.38

O

N

N

O

O

0.57

Br

0.05 n

O

N

N N

9

N
poly(PGE0.57-mPEG2000-Pos0.05)

N I

 
Figure A84. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG2000-Pos0.05) in CDCl3. Peak height was increased for clarity. 

 

198 

O

N

N

N

3

H

O

O

0.38

O

O

O

0.57

Br

0.05 n

O

N

N

N
poly(PGE0.57-Neg0.05)

9

N

N N

O

5

OH

Figure A85. 500 MHz 1H NMR spectrum of poly(PGE0.57-Neg0.05) in CDCl3. 

 

 

199 

O

N

N

N

3

H

O

O

0.38

O

O

O

0.57

Br

0.05 n

O

N

N

N
poly(PGE0.57-Neg0.05)

9

N

N N

O

5

OH

Figure A86. 125 MHz 13C NMR spectrum of poly(PGE0.57-Neg0.05) in CDCl3. 

 

 

200 

O

N

N

N
12

H

O

O

0.38

O

O

O

0.57

Br

0.05 n

O

N

9

N

N N
N
poly(PGE0.57-mPEG550-Neg0.05)

N

O

5

OH

Figure A87. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG550-Neg0.05) in CDCl3. 

 

 

201 

O

N

N

N
16

H

O

O

0.38

O

O

O

0.57

Br

0.05 n

O

N

9

N

N N
N
poly(PGE0.57-mPEG750-Neg0.05)

N

O

5

OH

Figure A88. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG750-Neg0.05) in CDCl3. 

 

 

202 

O

N

N

N
45

H

O

O

0.38

O

O

O

0.57

Br

0.05 n

O

N

9

N

N N
N
poly(PGE0.57-mPEG2000-Neg0.05)

N

O

5

OH

Figure A89. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG2000-Neg0.05) in CDCl3. 

 

 

203 

O

N

N

N
45

H

O

O

0.38

O

O

O

0.57

Br

0.05 n

O

N

9

N

N N
N
poly(PGE0.57-mPEG2000-Neg0.05)

N

O

5

OH

 
Figure A90. 500 MHz 1H NMR spectrum of poly(PGE0.57-mPEG2000-Neg0.05) in CDCl3. Peak height was increased for clarity. 

 

204 

O

N

N

N

3

HO

O

N N

5

N

O

O

0.57

H

O

O

0.38

O

N

N

N

9

O

Br
n

0.025

N I

O

0.025

O

N

N N

poly(PGE0.57-Neg0.025-Pos0.025)

Figure A91. 500 MHz 1H NMR spectrum of poly(PGE0.57-Neg0.025-Pos0.025) in CDCl3. 

 

 

205 

O

N

N

N

3

H

O

O

0.24

O

N

N

N

O

O

0.71

Br

0.05 n

O

N

N I

N N

9
poly(MGE0.71-Pos0.05)

Figure A92. 500 MHz 1H NMR spectrum of poly(MGE0.71-Pos0.05) in CDCl3. 

 

 

206 

O

N

N

N

3

H

O

O

0.24

O

N

N

N

O

O

0.71

Br

0.05 n

O

N

N I

N N

9
poly(MGE0.71-Pos0.05)

Figure A93. 125 MHz 13C NMR spectrum of poly(MGE0.71-Pos0.05) in CDCl3. 

 

 

207 

O

N

N

N
12

O

O

0.71

H

O

O

0.24

O

N

N

N

9

Br

0.05 n

O

N

N N

N I

poly(MGE0.71-mPEG550-Pos0.05)

Figure A94. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG550-Pos0.05) in CDCl3. 

 

 

208 

O

N

N

N
16

H

O

O

0.24

O

N

N

O

O

0.71

Br

0.05 n

O

N

N N

9

N
poly(MGE0.71-mPEG750-Pos0.05)

N I

Figure A95. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG750-Pos0.05). in CDCl3 

 

 

209 

O

N

N

N
45

H

O

O

0.24

O

N

N

O

O

0.71

Br

0.05 n

O

N

N N

9

N
poly(MGE0.71-mPEG2000-Pos0.05)

N I

Figure A96. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG2000-Pos0.05) in CDCl3. 

 

 

210 

O

N

N

N
45

H

O

O

0.24

O

N

N

O

O

0.71

Br

0.05 n

O

N

N N

9

N
poly(MGE0.71-mPEG2000-Pos0.05)

N I

 
Figure A97. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG2000-Pos0.05) in CDCl3. Peak height was increased for clarity. 

 

211 

O

N

N

N

3

H

O

O

0.24

O

O

O

0.71

Br

0.05 n

O

N

N
N
poly(MGE0.71-Neg0.05)

9

N

N N

O

5

OH

Figure A98. 500 MHz 1H NMR spectrum of poly(MGE0.71-Neg0.05) in CDCl3. 

 

 

212 

O

N

N

N

3

H

O

O

0.24

O

O

O

0.71

Br

0.05 n

O

N

N
N
poly(MGE0.71-Neg0.05)

9

N

N N

O

5

OH

Figure A99. 125 MHz 13C NMR spectrum of poly(MGE0.71-Neg0.05) in CDCl3. 

 

 

213 

O

N

N

N
12

H

O

O

0.24

O

O

O

0.71

Br

0.05 n

O

N

N N
N
poly(MGE0.71-mPEG550-Neg0.05)

N

N

9

O

5

OH

Figure A100. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG550-Neg0.05) in CDCl3. 

 

 

214 

O

N

N

N
16

H

O

O

0.24

O

O

O

0.71

Br

0.05 n

O

N

9

N

N N
N
poly(MGE0.71-mPEG750-Neg0.05)

N

O

5

OH

Figure A101. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG750-Neg0.05) in CDCl3. 

 

 

215 

O

N

N

N
45

H

O

O

0.24

O

O

O

0.71

Br

0.05 n

O

O

5

OH

N

N

N

N

N N
poly(MGE0.71-mPEG2000-Neg0.05)

9

Figure A102. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG2000-Neg0.05) in CDCl3. 

 

 

216 

O

N

N

N
45

H

O

O

0.24

O

O

O

0.71

Br

0.05 n

O

O

5

OH

N

N

N

N

N N
poly(MGE0.71-mPEG2000-Neg0.05)

9

Figure A103. 500 MHz 1H NMR spectrum of poly(MGE0.71-mPEG2000-Neg0.05) in CDCl3. Peak height was increased for clarity. 

 

 

217 

O

N

N

N

3

HO

O

N N

5

N

O

O

0.71

O

0.025

O

O

Br
n

0.025

N I

H

O

O

0.24

O

N

N

N

N N

9

N
poly(MGE0.71-Neg0.025-Pos0.025)

Figure A104. 500 MHz 1H NMR spectrum of poly(MGE0.71-Neg0.025-Pos0.025) in CDCl3. 

 

 

218 

O

N

N

N

3

H

O

O

0.22

O

O

O

0.68

Br

0.10 n

O

N

N
N
poly(MGE0.68-Neg0.10)

9

N

N N

O

5

OH

Figure A105. 500 MHz 1H NMR spectrum of poly(MGE0.68-Neg0.10) in CDCl3. 

 

 

219 

Figure A106. 500 MHz  1H NMR spectra of poly(PGE0.60) (red), poly(PGE0.57-Pos0.05) (green), and poly(PGE0.57-Neg0.05) (blue) in 
CDCl3. 

 

 

220 

Figure A107. 500 MHz  1H NMR spectra of poly(PGE0.60) (red), poly(PGE0.57-Pos0.05) (green), and poly(PGE0.57-Neg0.05) (blue) in 
CDCl3 in aliphatic region. 

 

 

221 

Figure A108. 500 MHz 1H NMR spectra of poly(MGE0.75) (red), poly(MGE0.71-Pos0.05) (green), and poly(MGE0.71-Neg0.05) (blue) in 
CDCl3. 

 

 

222 

Figure A109. 500 MHz 1H NMR spectra of poly(MGE0.75) (red), poly(MGE0.71-Pos0.05) (green), and poly(MGE0.71-Neg0.05) (blue) in 
CDCl3 in aliphatic region. 

 

 

223 

N3

O
mDEG azide

O

O

 

Figure A110. 500 MHz 1H NMR spectrum of mDEG azide in CDCl3. 

 

 

224 

N3

decyl azide

 

Figure A111. 500 MHz 1H NMR spectrum of decyl azide in CDCl3. 

 

 

225 

O

TsO
12
mPEG550 OTs

Figure A112. 500 MHz 1H NMR spectrum of mPEG550 OTs in CDCl3. 

 

 

226 

O

TsO
12
mPEG550 OTs

Figure A113. 125 MHz 13C NMR spectrum of mPEG550 OTs in CDCl3. 

 

 

227 

O

N3
12
mPEG550 azide

Figure A114. 500 MHz 1H NMR spectrum of mPEG550 azide in CDCl3. 

 

 

228 

O

N3
12
mPEG550 azide

Figure A115. 125 MHz 13C NMR spectrum of mPEG550 azide in CDCl3. 

 

 

229 

O

TsO
16
mPEG750 OTs

Figure A116. 500 MHz 1H NMR spectrum of mPEG750 OTs in CDCl3. 

 

 

230 

O

N3
16
mPEG750 azide

Figure A117. 500 MHz 1H NMR spectrum of mPEG750 azide in CDCl3. 

 

 

231 

O

N3
16
mPEG750 azide

Figure A118. 125 MHz 13C NMR spectrum of mPEG750 azide in CDCl3. 

 

 

232 

O

TsO
45
mPEG2000 OTs

Figure A119. 500 MHz 1H NMR spectrum mPEG2000 OTs in CDCl3. 

 

 

233 

O

N3
45
mPEG2000 azide

Figure A120. 500 MHz 1H NMR spectrum of mPEG2000 azide in CDCl3. 

 

 

234 

O

N3
45
mPEG2000 azide

Figure A121. 125 MHz 13C NMR spectrum of mPEG2000 azide in CDCl3. 

 

 

235 

OH

N3

O
COOH azide

Figure A122. 500 MHz 1H NMR spectrum of COOH azide in CDCl3. 

 

 

236 

OH

N3

O
COOH azide

Figure A123. 125 MHz 13C NMR spectrum of COOH azide in CDCl3. 

 

 

237 

N3

N

I

aminium azide

Figure A124. 500 MHz 1H NMR spectrum of aminium azide in D2O. 

 

 

238 

N3

N

I

aminium azide

Figure A125. 125 MHz 13C NMR spectrum of aminium azide in D2O. 

 

 

239 

2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

40

50

60

70

80

90

100

Temperature	(°C)

Figure A126. Study of LCST behavior of poly(PGE0.65) via UV-vis. 

2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

 

40

50

60

70

80

90

100

110

Temperature	(°C)

Figure A127. Study of LCST behavior of poly(PGE0.70) via UV-vis. 

 

 

240 

 

 

2.9
2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

40

50

60

70
80
Temperature	(℃)

90

100

110

Figure A128. Study of LCST behavior of poly(PGE0.75) via UV-vis. 

2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

 

40

50

60

70
80
Temperature	(℃)

90

100

110

Figure A129. Study of LCST behavior of poly(PGE0.80) via UV-vis. 

 

 

241 

 

 

2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

40

50

60

70
80
Temperature	(℃)

90

100

110

Figure A130. Study of LCST behavior of poly(PGE0.85) via UV-vis. 

2.9
2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

 

40

50

60

70
80
Temperature	(℃)	

90

100

110

Figure A131. Study of LCST behavior of poly(PGE0.90) via UV-vis. 

 

 

242 

 

 

2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

40

50

60

70
80
Temperature	(℃)

90

100

110

Figure A132. Study of LCST behavior of poly(PGE0.95) via UV-vis. 

00.20.40.60.811.21.41.61.82

e
c
n
a
b
r
o
s
b
A

 

40

50

60

70

80

90

100

110

Temperature	(°C)

Figure A133. Study of LCST behavior of poly(PGE1.0) via UV-vis. 

 

 

243 

 

 

3.5
3
2.5
2
1.5
1
0.5
0

e
c
n
a
b
r
o
s
b
A

-5

5

15

25

Temperature	(℃)

35

45

55

Figure A134. Study of LCST behavior of poly(MGE0.55) via UV-vis. 

3.5
3
2.5
2
1.5
1
0.5
0

e
c
n
a
b
r
o
s
b
A

 

-5

5

15

25

Temperature	(℃)

35

45

55

Figure A135. Study of LCST behavior of poly(MGE0.50) via UV-vis. 

 

 

244 

 

 

3.4
2.9
2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

-5

5

15

25

Temperature	(℃)

35

45

55

Figure A136. Study of LCST behavior of poly(MGE0.65) via UV-vis. 

2.9
2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

 

-5

5

15

25

35

45

55

Temperature	(°C)

Figure A137. Study of LCST behavior of poly(MGE0.75) via UV-vis. 

 

 

245 

 

 

3.4
2.9
2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

-5

5

15

35
25
Temperature	(℃)

45

55

Figure A138. Study of LCST behavior of poly(MGE0.80) via UV-vis. 

2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

 

25

-5

5

15

35

45

55

Figure A139. Study of LCST behavior of poly(MGE0.85) via UV-vis. 

Temperature	(°C)

 

 

246 

 

 

2.9
2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

-5

5

15

35
25
Temperature	(℃)

45

55

Figure A140. Study of LCST behavior of poly(MGE0.90) via UV-vis. 

2.9
2.4
1.9
1.4
0.9
0.4
-0.1

e
c
n
a
b
r
o
s
b
A

 

-5

5

15

25
35
Temperature	(℃)

45

55

Figure A141. Study of LCST behavior of poly(MGE0.95) via UV-vis. 

 

 

247 

 

 

)

%

(
	
y
t
i
s
n
e
t
n
I

10	mg/mL
4	mg/mL
2	mg/mL
1	mg/mL

0.4

4

40

Hydrodynamic	Radius	(nm)

Figure A142. Hydrodynamic diameter of poly(PGE0.55) determined by DLS. 

0246810121416
02468101214

0.4

)

%

(
	
y
t
i
s
n
e
t
n
I

4	mg/mL
2	mg/mL
1	mg/mL

 

4

Hydrodynamic	Radius(nm)

40

 

 

Figure A143. Hydrodynamic diameter of poly(MGE0.60) determined by DLS. 

 

 

248 

25
20
15
10
5
0

)

%

(
	
y
t
i
s
n
e
t
n
I

0.4

4

40

Size	(nm)

400

4000

Figure A144. Study of LCST behavior of poly(PGE0.60) via DLS. 

40
35
30
25
20
15
10
5
0

)

%

(
	
y
t
i
s
n
e
t
n
I

 

0.4

4

40

Size	(nm)

400

4000

Figure A145. Study of LCST behavior of poly(PGE0.65) via DLS. 

 

 

249 

PPGE-60-40C
PPGE-60-41C
PPGE-60-42C
PPGE-60-43C
PPGE-60-44C
PPGE-60-45C
PPGE-60-46C
PPGE-60-47C
PPGE-60-48C

PPGE-65-40C
PPGE-65-41C
PPGE-65-42C
PPGE-65-43C
PPGE-65-44C
PPGE-65-45C
PPGE-65-46C
PPGE-65-47C
PPGE-65-48C
PPGE-65-49C
PPGE-65-50C
PPGE-65-51C

 

 

35
30
25
20
15
10
5
0

)

%

(
	
y
t
i
s
n
e
t
n
I

0.4

4

40

Size	(nm)

400

4000

Figure A146. Study of LCST behavior of poly(PGE0.70) via DLS. 

60
50
40
30
20
10
0

)

%

(
	
y
t
i
s
n
e
t
n
I

 

0.4

4

40

400

4000

Figure A147. Study of LCST behavior of poly(PGE0.75) via DLS. 

Size	(nm)

 

 

250 

PPGE-70-50C
PPGE-70-51C
PPGE-70-52C
PPGE-70-53C
PPGE-70-54C
PPGE-70-55C
PPGE-70-56C
PPGE-70-57C
PPGE-70-58C

PPGE-75-49C
PPGE-75-50C
PPGE-75-51C
PPGE-75-52C
PPGE-75-53C
PPGE-75-54C
PPGE-75-55C
PPGE-75-56C
PPGE-75-57C

 

 

051015202530354045

)

%

(
	
y
t
i
s
n
e
t
n
I

0.4
40
35
30
25
20
15
10
5
0

0.4

)

%

(
	
y
t
i
s
n
e
t
n
I

4

40

Size	(nm)

400

4000

Figure A148. Study of LCST behavior of poly(PGE0.80) via DLS. 

Figure A149. Study of LCST behavior of poly(PGE0.85) via DLS. 

 

 

251 

PPGE-80-55C
PPGE-80-54C
PPGE-80-56C
PPGE-80-57C
PPGE-80-58C
PPGE-80-59C
PPGE-80-60C

PPGE-85-55C
PPGE-85-56C
PPGE-85-57C
PPGE-85-58C
PPGE-85-59C
PPGE-85-60C

 

 

 

400

4000

4

40

Size	(nm)

40

Size	(nm)

400

4000

Figure A150. Study of LCST behavior of poly(PGE0.90) via DLS. 

051015202530354045

)

%

(
	
y
t
i
s
n
e
t
n
I

0.4

4

40
35
30
25
20
15
10
5
0

)

%

(
	
y
t
i
s
n
e
t
n
I

 

400

4000

0.4

4

40

Size	(nm)

Figure A151. Study of LCST behavior of poly(PGE0.95) via DLS. 

 

 

252 

PPGE-90-55C
PPGE-90-56C
PPGE-90-57C
PPGE-90-58C
PPGE-90-59C

PPGE-95-65C
PPGE-95-66C
PPGE-95-67C
PPGE-95-68C
PPGE-95-69C
PPGE-95-70C
PPGE-95-71C

 

 

25
20
15
10
5
0

)

%

(
	
y
t
i
s
n
e
t
n
I

0.4

4

40

Size	(nm)

400

4000

Figure A152. Study of LCST behavior of poly(MGE0.60) via DLS. 

80
70
60
50
40
30
20
10
0

)

%

(
	
y
t
i
s
n
e
t
n
I

 

0.4

4

40

Size	(nm)

400

4000

Figure A153. Study of LCST behavior of poly(MGE0.65) via DLS. 

 

 

253 

PMGE-60-15C
PMGE-60-16C
PMGE-60-17C
PMGE-60-18C
PMGE-60-19C
PMGE-60-20C
PMGE-60-21C
PMGE-60-22C
PMGE-60-23C
PMGE-60-24C
PMGE-60-25C

PMGE-65-20C
PMGE-65-21C
PMGE-65-22C
PMGE-65-23C
PMGE-65-24C
PMGE-65-25C
PMGE-65-26C
PMGE-65-27C
PMGE-65-28C
PMGE-65-29C
PMGE-65-30C
PMGE-65-31C
PMGE-65-32C
PMGE-65-33C

 

 

40
35
30
25
20
15
10
5
0

)

%

(
	
y
t
i
s
n
e
t
n
I

0.4

4

40

400

4000

Figure A154. Study of LCST behavior of poly(MGE0.70) via DLS. 

Size	(nm)

25
20
15
10
5
0

)

%

(
	
y
t
i
s
n
e
t
n
I

0.4

4

40

Size	(nm)

PMGE-70-30C
PMGE-70-31C
PMGE-70-32C
PMGE-70-33C
PMGE-70-34C

PMGE-75-35C
PMGE-75-36C
PMGE-75-37C
PMGE-75-38C

 

 

 

400

4000

Figure A155. Study of LCST behavior of poly(MGE0.75) via DLS. 

 

 

254 

40
35
30
25
20
15
10
5
0

)

%

(
	
y
t
i
s
n
e
t
n
I

0.4

4

40

Size	(nm)

400

4000

Figure A156. Study of LCST behavior of poly(MGE0.80) via DLS. 

051015202530354045

)

%

(
	
y
t
i
s
n
e
t
n
I

 

0.4

4

40

400

4000

Figure A157. Study of LCST behavior of poly(MGE0.85) via DLS. 

Size	(nm)

 

 

255 

PMGE-80-38C
PMGE-80-39C
PMGE-80-40C
PMGE-80-41C

PMGE-85-37C
PMGE-85-38C
PMGE-85-39C
PMGE-85-40C
PMGE-85-41C
PMGE-85-42C

 

 

051015202530354045

)

%

(
	
y
t
i
s
n
e
t
n
I

0.4

4

40

Size	(nm)

400

4000

Figure A158. Study of LCST behavior of poly(MGE0.90) via DLS. 

16
14
12
10
8
6
4
2
0

)

%

(
	
y
t
i
s
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I

0.4

4

40

Size	(nm)

PMGE-90-40C
PMGE-90-41C
PMGE-90-42C
PMGE-90-43C
PMGE-90-44C
PMGE-90-45C

PMGE-95-46C
PMGE-95-47C
PMGE-95-48C
PMGE-95-49C

 

 

 

400

4000

Figure A159. Study of LCST behavior of poly(MGE0.95) via DLS. 

 

 

256 

40
35
30
25
20
15
10
5
0

)

%

(
	
y
t
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I

0.4

4

40

Size	(nm)

400

4000

Figure A160. Study of LCST behavior of poly(MGE1.0) via DLS. 

 

PMGE-100-50C
PMGE-100-51C
PMGE-100-52C
PMGE-100-53C
PMGE-100-54C
PMGE-100-55C
PMGE-100-56C
PMGE-100-57C
PMGE-100-58C

 

line) and SC with 5 µL	of	poly(PGE0.57-mPEG2000-Pos0.05) added (green line) in Milli-Q 

Figure A161. DLS results of SC (blue line), poly(PGE0.57-mPEG2000-Pos0.05) (orange 

 

water. 

 

257 

line) and SC with 50 µL	of	poly(PGE0.57-mPEG2000-Pos0.05) added (green line) in Milli-

Figure A162. DLS results of SC (blue line), poly(PGE0.57-mPEG2000-Pos0.05) (orange 

 

Q water. 

line) and SC with	poly(PGE0.57-mPEG2000-Pos0.05) added (green line) in Milli-Q water. 

Figure A163. DLS results of SC (blue line), poly(PGE0.57-mPEG2000-Pos0.05) (orange 

 

 

258 

line) and SC with 5 µL	of	poly(PGE0.57-mPEG2000-Pos0.05) added (green line) in PBS. 

Figure A164. DLS results of SC (blue line), poly(PGE0.57-mPEG2000-Pos0.05) (orange 

 

line) and SC with 50 µL	of	poly(PGE0.57-mPEG2000-Pos0.05) added (green line) in PBS. 

Figure A165. DLS results of SC (blue line), poly(PGE0.57-mPEG2000-Pos0.05) (orange 

 

 

259 

Figure A166. DLS results of SC (blue line), poly(PGE0.57-mPEG2000-Pos0.05) (orange 
line) and SC with poly(PGE0.57-mPEG2000-Pos0.05) added (green line) in PBS. 

 

line) and SC with 5 µL	of	poly(MGE0.71-mPEG2000-Pos0.05) added (green line) in PBS. 

Figure A167. DLS results of SC (blue line), poly(MGE0.71-mPEG2000-Pos0.05) (orange 

 

 

260 

line) and SC with 50 µL	of	poly(MGE0.71-mPEG2000-Pos0.05) added (green line) in PBS. 

Figure A168. DLS results of SC (blue line), poly(MGE0.71-mPEG2000-Pos0.05) (orange 

 

line) and SC with	poly(MGE0.71-mPEG2000-Pos0.05) added (green line) in PBS. 

Figure A169. DLS results of SC (blue line), poly(MGE0.71-mPEG2000-Pos0.05) (orange 

 

 

261 

Figure  A170.  DLS  results  of  control  experiments  for  SC  (dotted 
SC@poly(MGE0.71-mPEG2000-Pos0.05) (solid line) in Milli-Q water over 48 h. 

 
line)  and 

Figure  A171.  DLS  results  of  control  experiments  for  SC  (dotted 
SC@poly(MGE0.71-mPEG2000-Neg0.05) (solid line) in Milli-Q water over 48 h. 

 
line)  and 

 

262 

0.04 mg/mL
0.1 mg/mL
1 mg/mL

0

1500
 
Figure  A172.  Concentrations  of  p-nitroaniline  converted  by  SC  (blue  diamond,  0.04 
mg/mL; red square, 0.1 mg/mL; orange triangle, 1.0 mg/mL) over time at room temperature. 

Time (h)

1000

500

 

0.04 mg/mL
0.1 mg/mL
1 mg/mL

0.00002
0.000018
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0.000014
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50

100

Time (h)

 
Figure  A173.  Concentrations  of  p-nitroaniline  converted  by  SC  (blue  diamond,  0.04 
mg/mL; red square, 0.1 mg/mL; orange triangle, 1.0 mg/mL) over time at 40 °C. 

150

200

 

263 

0.000018
0.000016
0.000014
0.000012
0.00001
0.000008
0.000006
0.000004
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10

20

0.04 mg/mL
0.1 mg/mL

6

8

1 mg/mL

40

50

60

 
Figure  A174.  Concentrations  of  p-nitroaniline  converted  by  SC  (blue  diamond,  0.04 
mg/mL; red square, 0.1 mg/mL) over time at 50 °C. 

4

Time (h)

 
Figure A175. Concentrations of p-nitroaniline converted by SC (1 mg/mL) over time at 50 
°C. 

30

Time (h)

 

264 

 
Figure A176. DLS results of SC (dotted line) and SC@poly(MGE0.71-mPEG750-Neg0.05) 
(solid line) in Tris-HCl buffer at 50 °C for 4 h. 

 

 

 

 
 

265 

 

 

 

 

 

 

 

 

 

 

 

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