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'ltnv'.V‘O->\I 1‘ u\; G '0 ‘bl ‘II‘ 1-». 1" I It .. sun-11.1. ‘4. 3,-, I ‘ .J. l‘lo I 3")», .I I N . .I a ‘1 .‘9‘ \ I nllillhflplflv'illlclu. la I‘U‘. Illl III \ . . ‘1'“.Sl-Ioflun . no EH9...» 4. «6 luau? huh I LIERARY ' Michigan State University This is to certify that the dissertation entitled Synthesis and Properties of Heme in 11: And Sulfur-Containing Green Hemes presented by Chariklia Sotiriou has been accepted towards fulfillment of the requirements for Ph . D . degree in Chemistry Major professor Date October 29, 1987 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES .3- RETURNING MATERIALS: PIace in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped beiow. SYNTHESIS AND PMPRRTIKS OF m 4". d1" AND SULFUR‘CONTAINING GREEN MINES BY Chariklia Sotiriou AN ABSTRACT OF A DISSERTATION Submitted to Michigan State university in partial fulfillment of the require-ants . for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1987 ABSTRACT SYNTHESIS AND PROPERTIES or m 9—, 53;- AND SULFUR—CONTAINING GREEN ms By Chariklia Sotiriou The discovery of the presence of chlorin and isobacteriochlorin green heme prosthetic groups in a significant number of proteins and enzymes has generated much interest. In this work, syntheses of 9- hydroxy, alkyl, alkenyl, and oxo chlorins are described. The conditions and migratory aptitudes for the pinacol-pinacolone type rearrangements involved in the formation of oxo and dioxo compounds have been studied. These results have laid the foundation from which porphyrindiones such as heme gr can be synthesized. The vig—dihydroxychlorins in dilute acids can also undergo non-pinacolic rearrangement by which new functional groups can be introduced at the porphyrin side chain. This route has also been applied successfully to creating the acrylic acid group in the g;_-type porphyrindiones. group have a propensity to lactonize. Mild bases such as NaOAc cyclize the geminal groups without inversion of configuration while prolonged contact with silica gel, invariably gives the trans diastereomer. 1H Chariklia Sotiriou NMR spectra have provided important information on the conformation of the cis and trans spirolactones. This work supports the argument that the proposed Y—spirolactone structure of heme d in E. coli is most likely 12,l3-dihydroxyprotochlorin IX, the cis and trans isomers and the lactone forms of which have been synthesized. Sulfur-containing porphyrin thiones have been synthesized from the corresponding oxo—analogues and Lawesson’s reagent. These compounds exhibited "hyper" type absorption spectra. Moreover, their redox potentials obtained by cyclic voltmetry revealed a reduction of the HOMO-LUMO energy gap. Finally, the kinetic and equillibriun constants of C0 and 02 binding to myoglobin reconstituted with several synthetic green hemes were measured by using flash photolysis and spectrophotometric titrations. In comparison with the native myoglobin, the generally faster association rates observed in the green hemes might be attributed to their larger core size which facilitates the spin state change during ligand binding. To My Mother iv ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Professor C. K. Chang for his encouragement and support throughout the course of this "colorful" work. I would also like to thank Dr. K. Hallenga who taught me the "secrets" of the NOE experiments. Special thanks are also extended to the members of our group, particularly to Weishih Wu and Gladys Avilés for their friendship. Financial support from Michigan State University in the form of teaching assistantships, National Institute of Health in the form of research assistantships, and SOHIO Company in the form of one year industrial fellowship are gratefully acknowledged. My deepest appreciation is due to my mother for her constant encouragement and faith in me. Finally, I would like to thank Nick for his love, patience and understanding. TABLE OF CONTENTS Page LIST OF TABLES . ............ . ..... xiii LIST OF FIGURES. ..................... xiv LIST OF APPENDIX FIGURES . ..... . ..... xvi GENERAL INTRODUCTION . . . . ................ . 1 CHAPTER 1 SYNTHETIC METHODOLOGY FOR g—SUBSTITUTED CBLORINS AND OTHER PORPBYRINOIDS. . . . . ........... 4 I. INTRODUCTION . . ............. . 4 II. RESULTS AND DISCUSSION ........ . . . ..... 8 A. vjc~Dihydroxychlorins and Derivatives ...... 8 B. Migratory Aptitudes in Pinacol Rearrangement of vic~Dihydroxychlorins . . . . . . . . . . . . 17 Applications . . . . . . ........ . . . . 21 C. Differentiation of Bacteriochlorin and Isobacteriochlorin Formation by Mbtalation . . . 24 D. A Novel Method of Functionalizing the Ethyl Chain of Octaethylporphyrin. . . . . . . . . . 28 III. EXPERIMENTAL .................. . . . 32 General. . ...... . . . . . . . . . . . 32 Dimethyl cis-7,8-dihydroxy—3,7,8,12,l3, l7—hexamethylchlorin-2,lB-dipropionate (16a) . . 33 Dimethyl cis-2,3-dihydroxy~3,7,8,12,13, 17—hexamethylchlorin—2,lB-dipropionate (17a) . . 34 Methyl cis-3-hydroxy-3,7,8,12,13,17- hexamethyl— 2,Z-Trspirolactone—chlorin—lB— propionate (18a) ....... . . . . . . . . . 34 vi Methyl trans-3-hydroxy-3,7,8,12,13,17- hexamethyl-Z,Z-Trspirolactone-chlorin—IB- propionate (190) . . . . .......... Dimethyl cis-7,8,12,13-tetraethyl-7,8- dihydroxy-3,l7-dimethylchlorin-2,18- dipropionate (16b) .............. Dimethyl cis—7,8,12,13-tetraethyl*2,3- dihydroxy-3,l7—dimethylchlorin-2,18— dipropionate (17b) ............ Methyl cis-7,8,12,lS-tetraethyl-3-hydroxy— 3,l7-dimethyl-2,2-Tbspirolactone-chlorin- lB—propionate (18b) ........ . ..... Methyl trans-7,8,12,l3—tetraethyl-3-hydroxy~ 3,17-dimethyl-2,2-Yrspirolactone-chlorin- lB-propionate (19b) .............. Dimethyl trans-7,8,12,l3-tetraethyl- 2,3-dihydroxy-l3,l7-dimethy1chlorin- 2,18—dipropionate (20) ............ Dimethyl 3,8,8,12,13,17-hexamethyl-7- porphinone—Z,lB—dipropionate (21a) and 3,7,7,12,l3,l7-hexamethyl-8—porphinone—2,18- dipropionate (22a) .............. Dimethyl 8,8,12,l3-tetraethyl-3,17-dimethyl- 7-porphinone-2,lB—dipropionate (21b) and Dimethyl 7,7,12,l3-tetraethy1-3,l7-dimethyl —8-porphinone-2 , 18-dipropionate (22h) ..... . Dimethyl 3,7,7,12,13,17-hexamethyl-8— methylenechlorin-Z,18-dipropionate (23). . Dimethyl 3,7,7,8,12,13,l7-heptamethylchlorin- 2,18-dipropionate (24) .......... . General Procedure of Oxidation and Rearrangement of 15a, 29, 36 and 41 ...... Dimethyl 3,7,8,12,13,17-hexamethyl-18- porphinone-Z,17-dipropionate (28) ....... Dimethyl 12,18-diethy1-3,7,13,l7- tetramethylporphine-Z,8-diacetate (36) . . . . Dimethyl 12,lB-diethyl-IZ,l3-dihydroxy-3,7, 13,17-tetramethyl-chlorin-2,8-diacetate (37) . . vii Page 35 35 36 36 36 37 38 39 40 41 41 42 42 43 Dimethyl 12,lB-diethyl-Z,3—dihydroxy-3,7,13, l7-tetramethylchlorin-2,8—diacetate (38) . . . . Dimethyl 13,18-diethyl-3,7,13-17- tetramethyl-l2-porphinone-2,8—diacetate (39) . . Dimethyl 12,18-diethyl-2,7,13,17- tetramethyl-3-porphinane-2,8-diacetate (40). . . Dimethyl 7,8-dihydroxy—3,8,l3,18- tetramethyl-7,l7-dipentylchlorin-2,12- diacetate (42) ................. Dimethyl 2,3-dihydroxy~3,8,l3,18- tetramethyl-7,l7-dipcntylchlorin-2,12- diacetate (43) ................. 2,3-Dihydroxy~2,12-bis(2-hydroxyethyl)- 3, 8, l3, 18—tetramethyl-7, l7— dipentylchlorin (44) .............. Dimethyl 3,9,l3,l8—tetramethyl-8,17- dipentyl-7-porphinone-2,lZ-diacetate (45). . . . Dimethyl 2,8,13,18-tetramethyl-7,17- dipentyl-3-porphinone-2,lZ-diacetate (46). . . . 3,12-Bis(2~hydroxyethyl)-3,8,13,18- tetramethyl-7,17-dipentyl-2-porphinone (47). . . Tetramethyl 2,7,12,17-tetramethyl-3- porphinone-2,8,13,18-tetrapropionate (49). . . . t-Butyl 2-acetoxymethyl-3-ethyl-4-methyl- 5-pyrrole-carboxylate (55) ........... Ethyl 3’,4-diethyl—3,4’-dimethyl— 5’-t-butoxycarbonyl-dipyrromethane— 5-carboxylate (57) ............... Methyl 7,12-diethyl-3,8,13,17,18- pentamethylporphine—Z-propionate (50) ...... Methyl 7,12-diethyl-3,8,13,17,17-pentamethyl- l8-porphinone-2-propionate (51) and ’Methyl 7,12-diethyl-3,8,13,18,18-pentamethyl- l7—porphinone—Z-propionate (52) ........ Dimethyl 12,13-dihydroxy— 3,8,8,12,l3,l7-hexamethyl-7-porphinone- 2,18-dipropionate (68) ............. viii Page 44 44 44 45 45 46 46 47 47 48 49 49 49 51 52 Page Dimethyl 3,8,8,l3,13,17—hexamethyl-7,12- porphinedione-Z,18—dipropionate (69) ...... 53 Dimethyl l7,lB-dihydroxy-3,8,8,l3,13,17— hexamethyl-7,12-porphinedione-2,18- dipropionate (89) ................ 54 Methyl 18- [2- (methoxycarbonyl ) ethenyl ] - 3,8,8,l3,13,l7-hexamethyl-7,lZ-porphinedione ~2-propionate (70) ............... 54 Dimethyl 12,lS-dihydroxy-3,7,7,12,13,17— hexamethyl-8-porphinone—2,18—dipropionate (72) and Dimethyl 2,3-dihydroxy-3,7,7,12, 13,17-hexamethyl-8-porphinone—2,18- dipropionate (73) ................ 55 Dimethyl 3,7,7,l3,13,17-hexamethyl-8,12- porphinedione-Z,lB—dipropionate (74) ...... 55 Dimethyl 2,7,7,12,13,17-hexamethy1-3,8- porphinedione-Z,lB—dipropionate (75) . ..... 56 vjchihydroxyoctaethylchlorin (76) ....... 57 3,7,12,13,17,18-Heptaethyl-2- (l-hydroxyethy1)-porphine (77) ......... 57 3,7,8,12.13.17.18-Rsptaethyl- 2-(l-acetoxyethyl)-porphine (78) ........ 58 3,7,8,12,13,17,18-Reptaethyl- 2—(lemethoxyethyl)—porphine (79) ........ 58 3,7,8,12,13,17,18-Reptaethyl- 2-vinylporphine (80) .............. 59 2,3,7,8,12,13,l7-Heptaethylporphine (81) . . . . 59 Reactions of Vic-Dihydroxyetiochlorin I (82) . . 59 Dimethyl 7,8,12,13-tetraethy1- 3-(hydroxymethyl)-17-methyl-2,18- porphinedipropionate (87) ............ 60 CHAPTER 2 SYNTHESIS OF THE HEME Q PROSTHETIC GROUP OF BACTERIAL TERMINAL OXIDASE I. INTRODUCTION ............. . ........ 61 ix II. RESULTS AND DISCUSSION . A. Model Studies. ..... . . . . . ..... B. Synthesis of "Lactochlorin". . C. 1H NMR Spectra and Structure of the Chlorins . . D. Structure of Heme d: Lactone vs. Diol? ..... III. EXPERIMENTAL . . . . . . ...... . . ....... Dihydroxychlorins 94a, 94b, and 94d. . . . . ..... Methyl 7,8,12,13,17,lB—hexaethyl-12,13- dihydroxy-4-methyl-chlorin-2-propionate (94a). . . . . . . . . ......... Methyl 7,8,12,13,17,18—hexaethyl-7,8- dihydroxy-S-methyl-chlorin-2-propionate (94b). ......... . . . ..... . . Methyl 7,8,12,13,17,18-hexaethyl-l7,18- dihydroxy-3-methyl-chlorin—2-propionate (94d). . . . . . . . . . . . . . . . . . . . . . cis-7,8,12,13,17,18-Hexaethyl-3-hydroxy—3- methyl-2,2-Trspirolactone-chlorin (95) ........ trans-7,8,12,13,17,lB—hexaethyl-3-hydroxy-3- methyl-2,Z-Yrspirolactone-chlorin (96) ..... . . . 3,8-Bis(2,2-dimethoxyethyl)-deuteroporphyrin IX dimethyl ester (98) ................ 3,8-Bis(2-hydroxyethy1)-deuteroporphyrin IX dimethyl ester (100) ................. 3 ,8-Bis(2- chloroethyl) -deuteroporphyrin IX dimethyl ester (101) ..... . . Spirolactones 103a and 103b. . . . . . cis-3,8-Bis(2-chloroethyl)-12-hydr0xy-l3, 13~Y—spirolactone-deuterochlorin IX dimethyl ester (103a) . . . . cis-3,8-Bis(2-chloroethyl)-18-hydroxy—l7, 17-Y—spirolactone-deuterochlorin IX dimethyl ester (103b). . . . . . cis-12-Hydroxy-l3,lS—Y-spirolactone- protochlorin IX methyl ester (104) . . CHAPTER 3 II. III. cis-12,lS-dihydroxyprotochlorin IX dimethyl ester (106) ....... trans-lZ-Hydroxy-IS,l3-1rspirolactone- protochlorin IX methyl ester (92). . . . ..... cis-18-Hydroxy—l7,l7-Trspirolactone— protochlorin IX methyl ester (105a) ........ trans-18~Hydroxy—l7,l7-Yrspirolactone- protochlorin IX methyl ester (105b). . . ..... cis-l7,18-Dihydroxy-protochlorin IX dimethyl ester .................. trans-3,8—Bis(2-chloroethy1)-12-hydroxy- 13,l3-Tbspirolactone—deuterochlorin IX dimethyl ester (107) .............. trans-12,13—dihydroxyprotochlorin IX dimethyl ester (108) ....... . ....... SYNTHESIS AND PROPERTIES OF SULFUR-CONTAINING SATURATED OCTAETRYLPORPHYRINS ........... . INTRODUCTION ................... RESULTS AND DISCUSSION .............. Absorption Spectra ................ Cyclic Voltammetry ....... . . . . ..... Closing Remarks .................. EXPERIMENTAL . . .......... . ..... 3,3,7,8,12,13,17,18-Octaethy1- 2-porphinethione (111) ....... . . ..... 3,3,8,8,12,13,17,18-Octaethyl-2-thio- 7-prophinedione (112) and 3,3,8,8,12,13,17,18- Octaethyl~2,7-porphinedithione (113) ....... 2,2,8,8,12,13,17,lB-Octaethyl-3-thio- 7-porphinedione (114) and 2,2,8,8,12,13, 17,18-Octaethyl-3,7-porphinedithione (115) . . . . xi Page 89 89 90 91 91 92 92 93 100 106 108 109 3,3,8,8,13,13,17,lB-Octaethyl-Z-thio- 7,12—porphinetrione (117), 3,3,8,8,13,13, 17,18-Octaethyl-2,lZ-dithio-7-porphinetrione (118), 3,3,8,8,13,13,17,18~Octaethy1-2,7,dithio- lZ-porphinetrione (119), and 3,3,8,8,13,13, 17,18—Octaethy1-2,7,lZ—porphinetrithione (120) . . . . 3,3,7,8,12,13,l7,lB-Octaethyl-Z-methyl- 2~mercaptochlorin (121) ....... 3,3,7,8,12,13,17,18-Octaethy1-2- mercaptochlorin (122) ................. 2,2,7,8,12,13,17,18-Octaethylchlorin (123) ..... General Procedure for Zinc and Copper Insertion. . . . CHAPTER 4 KINETIC AND EQUILIBRIUM STUDIES 0? CO AND 02 BINDING TO HORSE HEART MYOGLOBIN RECONSTITUTED WITH SYNTHETIC GREEN HEMES . . . . I. INTRODUCTION . . . . ........ II. RESULTS AND DISCUSSION . . . . III. SMARY. ........... IV. MATERIALS AND METHODS ...... . . . . Preparation of Hemins ........ Preparation of Myoglobins. . . Kinetic and Equilibrium Measurements . . . REFERENCES AND NOTES . APPENDIX . . . . . . . . ............ xii ......... Page 110 111 112 112 113 114 114 118 125 126 126 126 127 132 Table Table Table Table Table Table LIST OF TABLES 1H NMR Assignments for the pyrroline substituents of the four forms of 12,13- dihydroxyprotochlorin ................ Couplings (Hz) and rotamer populations of the cis-dial 106 . . . . . . . . . . . . ...... 1H NMR Data of sulfur-containing saturated octaethylporphyrins in comparison with their oxo-analogues .................... Redox potentials of sulfur-containing saturated octaethylporphyrins and oxo-octaethylporphyrins. The latter are indicated in parenthesis. . . ... . . Kinetic and equilibrium constants for CO and 02 binding .................... Absorption spectral maxima of synthetic hemes and myoglobins ..................... xiii Page 78 79 99 104 119 122 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure name 10 11 12 LIST OF FIGURES Tautomeric forms of porphyrin ........... 1900-1500 or1 IR spectra of (A) porphyrin; (B) dihydroxychlorin; (C) Thapirolactone chlorin. . . . 250 MHz 18 NMR spectra of 21b and 22b. Irradiations of the methyl resonances as indicated resulted in NOE observable at the neighboring groups whose chemical shifts are marked by the pointers. . . ............ Arrangement of cytochromes in the respiratory chain of cells of E. 9911 in the late The Em’ values are indicated in parentheses. Cyt, cytochrome; Q, ubiquinone-8 ............ 1H NMR spectra (in CD013, 250 MR2) of trans- lactone 83 (A); of the hexaethyl trans-lactone 87 with simulation (B). . . . . . . . . . . . . . . . . 1H NMR spectra (in CDCla) of cis lactone 95 at 250 MHZ (A); at 500 MR2 with simulation (B) . . . . Suggested conformation of the isomeric spirolactones. The slope estimated for the trans isomer is 35° while for the cis isomer is less than 10° ................... 1H NMR spectra (in CDCla) of cis-diol 97 at 250 MHz (A); at 500 MR2 with simulation (B) . . . . 1!! INR spectra (in CDCla) of trans-diol 99 at 250 MHz (A); at 250 MHz with simulation (B). . . Sulfheme models .................. 1800-1300 cur1 IR spectra of: (A) porphyrinone 8; (B) porphyrinthione 111 ............. 250 MHz in NMR spectra of porphyrinone.8 and porphyrinthione 111 ................ xiv Page 6 11 62 71 72 76 77 93 96 97 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 13 14 15 16 17 18 19 20 21 22 Visible spectra (in CH2C12) of thione 111 (A); thiol 122 (B); Cu—thione (C); Cu-thiol (D). Visible spectra (in CHéC12) of 3,7-dithione 115(A); 2-7-dithione 113 (B); 2,7,12-trithione 120 (C) . Cyclic voltammograms of 2,7,12-trione 116 (A); 2,7,12-trithione 120 (B) .............. The low-resolution structure of Mb (A); the 02- binding site in Mb (8) ............. Structures of synthetic green hemes used for myoglobin reconstitutions ............. Optical spectra of 7-keto-heme myoglobin (A); 8— keto-heme myoglobin (B); Ferric (--), Deoxy (_')1 oxy (—"_')1 CO (...), in 10 ‘7 (pH 7'4) potassium phosphate buffer ......... . . . . Autoxidation of reconstituted myglobins at 22°C in lOmM (pH 7.4) potassium phosphate buffer, saturated with 02. Protoheme (native)C), slope = 8.6x10‘5, r = 0.939; P330, slope = 9.0x10‘5, = 0.984; methylchlorin-hemeA, slope = .7x10“5, r = 0.999; 7-keto-hsmeA, slope = .3x10'4, r = 0.999; PaALJ, slope = 1.9x10", r 0.989; 8-keto-heme II, slope = 2.5x10", r = u HID"! O u-wun for the recombination of PaA-Mb and CO after flash photolysis at 22°C in 10 mM (pH 7.4) potassium phosphate buffer. [CO] = 5.25x10'5M; sweeptime = 1,5,20 msec/div.; wavelength = 420 nm. (b) The recombination of PaA-Mb with 02 and CO at 422 nm. 3.49x10'5M. [CO] = 5.25x10’5, [02] = Upper trace; sweeptime = 0.1, 0.2 sec/div.; lower trace; sweeptime = 0.2, 1 msec/div. . Spectrophotometric titration of 7-keto-heme myoglobin in 10 mM (pH 7.4) potassium phosphate buffer, with CO at R.T.; [CO]xlO°M = 0.0, 2.63, 5.26, 9.65, 17.55 and 1760. Inset; plot of A- Ao/Am-A V8. [CO] at 615 nm ............. Spectrophotometric titration of dione-heme myglobin in 10 mM (pH 7.4) potassium phosphate buffer with CO at R.T.; [C0]x109M = 0.0, 2.66, 5.32, 9.31, 15.97, 1346. Inset: plot of A- Ao/Aau-A vs. [CO] at 628 nm ............. XV .996. . . ..................... Page . 101 102 105 . 115 117 121 124 . 129 . . 131 Figure A1 Figure A2 Figure A3 Figure A4 Figure A5 Figure A6 Figure A7 Figure A8 Figure A9 LIST OF APPENDIX FIGURES 250 MR2 ‘R NMR spectrum of dimethyl cis-7,8- dihydroxy-3,7,8,12,13,17-hexamethy1chlorin-2,18- dipropionate (16a) ........... . ..... 250 MHz 11! NMR spectrum of dimethyl cis- 7,8,12,l3-tetraethyl-7,8-dihydroxy-3,l7- dimethylchlorin-Z,18-dipropionate (165) ...... 250 MHz 18 NMR spectra of (a) dimethyl cis- 7,8,12,13-tetraethy1-2,3-dihydroxyb3,17- dimethylchlorin-Z,18-dipropionate (17b); (b) dimethyl trans-7,8,12,13-tetraethy1-2,3- dihydroxy—l3,l7-dimethylchlorin-2,18- dipropionate (20) . . . .. ............. 250 MR2 1H NMR spectra of (a) methyl cis- 7,8,12,l3-tetraethyl-3-hydroxyh3,17—dimethyl- 2 , 2- T-spirolactone-chlorin-18-propionate ( 1%) ; (b) methyl trans-7,8,12,l3-tetraethy1-3-hydroxy- 3,17-dimethy1-2,2-Ybspirolactone—chlorin-IB- propionate (195) .................. 250 MHz 1H NMR spectra of (a) dimethyl 3,8,8,12,13,l7-hexamethyl-7-porphinone-2,18- dipropionate (21a); (b) dimethyl 3,7,7,12,l3,17- hexamethyl-8-porphinone-2,lB—dipropionate (22a) . . . 250 MHz 1H NMR spectrum of dimethyl 3,7,7,12,l3,l7-hexamethy1-8emethylenechlorin- 2,18—dipropionate (23) ............... 250 MR2 1H NMR spectrum of dimethyl 3,7,7,8,12,13,17-heptamethy1chlorin-2,18- dipropionate (24) . . . ...... . ....... 250 MHz in NMR spectrum of dimethyl 3,7,8,12,13,l7-hexamethyl-18—porphinone-2,l7- dipropionate (28) ................. 250 MHz 1H NMR spectrum of dimethyl 12,18- diethyl—3,7,13,l7-tetramethylporphine—2,8- diacetate (36) ................... xvi Page 140 141 142 143 144 145 147 148 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure A10 A11 A12 A13 A14 ‘A15 A16 A17 A18 A19 250 MHz 1H NMR spectra of (a) dimethyl 12,18- diethyl-12,13—dihydroxy-3,7,13,17- tetramethylchlorin—Z,8~diacetate (37); (b) dimethyl 12,18—diethy1-2,3-dihydroxy-3,7,13,17- tetramethylchlorin-Z,8-diacetate (38) ....... 250 MHz 1H NMR spectra of (a) dimethyl 13,18- diethy1-3,7,13,l7-tetramethyl-lZ-porphinone-Z,8- diacetate (39); (b) dimethyl 12,18-diethy1- 2,7,13,17-tetramethy1-3-porphinone-2,8-diacetate (40) ........................ 250 MHz 1H NMR spectra of (a) dimethyl 7,8- dihydroxy—3,8,l3,lB—tetramethyl-7,l7- dipentylchlorin-Z,12-diacetate (42); (b) dimethyl 2,3-dihydroxy-3,8,l3,lB—tetramethyl- 7,17-dipentylchlorin-2,lZ-diacetate (43) ...... 250 MR2 1H NMR spectra of (a) dimethyl 3,9,13,lB-tetramethyl-B,17-dipentyl-7- porphinone-Z,12—diacetate (45); (b) dimethyl 2,8,13,lB-tetramethyl-7,l7-dipenty1-3- porphinone-Z,12-diacetate (46) ........... 250 M112 In. m spectrum of methyl 7,12-diethy1- 3,8,13,17,18-pentamethy1-porphine-Z-propionate (50) ........................ 250 MR2 1H NMR spectrum of dimethyl 12,13- dihydroxy-3,8,8,12,l3,17-hexamethyl-7- porphinone—Z,18-dipropionate (SI) ......... 250 MHz 1H NMR spectrum of dimethyl 3,8,8,13,13,17-hexamethyl-7,12—porphinedione— 2,18-dipropionate (69) ............... . 250 MR2 1H NMR spectrum of dimethyl 17,18- dihydroxy-3,8,8,13,13,17-hexamethyl-7,12- porphinedione—Z,18-dipropionate (SS) ........ 250 MHz 1H NMR spectrum of methyl 18-[2- (methoxycarbonyl)etheny1]-3,8,8,13,13,17- hexamethyl-7,12-porphinedione-Z-propionate (70) . . . 250 MHz 1H NMR spectrum of dimethyl 3,7,7,13,13,17-hexamethyl-8,12-porphinedione- 2,18-dipropionate (74) ............... xvii Page 149 150 , 151 152 153 154 155 156 157 158 Figure Figure A20 Figure A21 Figure A22 Figure A23 Figure A24 Figure A25 Figure A26 Figure A27 Figure A28 Figure A29 Figure A30 250 MHz 1H NMR spectra of (a) dimethyl 12,13- dihydroxy—3,7,7,12,13,17-hexamethy1-8- porphinone—Z,lB-dipropionate (72); (b) dimethyl 2,3-dihydroxy—3,7,7,12,13,17-hexamethyl-8- porphinone-Z,18—dipropionate (73) .......... 250 MR2 1H NMR spectrum of dimethyl 2,7,7,12,13,l7-hexamethy1-3,8-porphinedione- 2,18—dipropionate (75) ................ 250 MHz 1H NMR spectrum of vic- dihydroxyoctaethylchlorin (76) ............ 250 MHz 1H NMR spectra of (a) 3,7,8,12,l3,l7,18- heptaethyl-2-(l-acetoxyethyl)-porphine (78); (b) 3,7,12,13,l7,18-heptaethyl-2-(l-hydroxyethy1)- porphine (77); (c) 3,7,8,12,13,17,18-heptaethyl- 2-(1—methoxyethyl)-porphine (79) ......... ,. . 250 MHz 1H NMR spectra of (a) 3,7,8,12,13,17,18- heptaethyl-Z-vinylporphine (80); (b) 2,3,7,8,13,17-heptaethylporphine (81) ..... > . . . 250 MHz in NMR spectrum of dimethyl 7,8,12,13- tetraethyl-3-(hydroxymethyl)-l7-methy1-2,18- porphinedipropi onate (87 ) .............. 250 MHz in NMR spectrum of methyl 7,8,12,13,17,18-hexaethy1-4-methyl-porphine-2- propionate (93) ................... 250 MHz 1H NMR spectrum of methyl 7,8,12,13,17,lB-hexaethyl-lZ,l3-dihydroxy-4- methylchlorin-2-propionate (94a) ........... 250 MHz 18 NMR spectrum of methyl 7,8,12,13,17,18—hexaethy1-7,8-dihydroxy—3 methylchlorin-Z-propionate (94b) ........... 250 MR2 1H NMR spectrum of methyl 7,8,12,13,17,18-hexaethyl-l7,lB-dihydroxy-3- methylchlorin-2—propionate (94d) ........... 250 MHz IE NMR spectra of (a) cis- 7,8,12,l3,17,18-hexaethyl-3-hydroxy~3-methy1- 2,2-Trspirolactone-chlorin (95); (b) trans- 7,8,12,13,l7,18-hexaethyl-3-hydroxy-3-methyl- 2,2-Tespirolactone-chlorin (96) ........... xviii Page 159 160 161 162 163 164 165 166 167 168 169 Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure A31 A32 A33 A34 A35 A36 A37 A38 A39 A41 250 MHz 1H NMR spectrum of 3,8-bis(2-chloroethy1)— deuteroporphyrin IX dimethyl ester (101) ...... 250 MHz 1H NMR spectra of (a) cis-3,8-bis(2- chloroethyl)—12—hydroxy~13,13-Ybspirolactone- deuterochlorin IX dimethyl ester (103a); (b) cis-3,8-bis(2-chloroethy1)-18—hydroxyrl7,17-15 spirolactone-deuterochlorin IX dimethyl ester (103b) ....................... 250 MHz IH NMR spectrum of trans-3,8—bis(2- chloroethyl)-12-hydroxy~13,13—1bspirolactone- deuterochlorin IX dimethyl ester (107) ....... 250 MHz 1H NMR spectra of (a) 3.3.8.8,12,l3,l7,lB—octaethyl-Z,7-porphinedione (64); (b) 3, 3, 8, 8, 12, 13, 17, l8—octaethy1-27-thio- 7-porphinedione (112); (c) 3,3,8,8,12,13,17,18- octaethyl-Z,7-porphinedithione (113) ........ 250 MHz 1H NMR spectra of (a) 2,2,8,8,12,13,17,18-octaethy1-3,7-porphinedione (66); (b) 2,2,8,8,12,l3,l7,18-octaethy1-3,7- porphinedithione (115); (c) 2,2,8,8,12,13,l7,18— octaethy1-3-thio—7-porphinedione (114). . . . . . . . 250 MHz 1H NMR spectra of (a) 3,3,8,8,13,l3,l7,lB—octaethyl-Z-thio—7,12- porphinetrione (117); (b) 3,3,8,8,13,13,17,18— octaethyl-Z,12-dithio—7-porphinetrione (118); (c) 3,3,8,8,13,13,l7,18—octaethy1-2,7,dithio—12- porphinetrione (119) ............... 250 MHz 1H NMR spectra of (a) 3,3,8,8,l3,13,l7,18-octaethy1-2,7,12- porphinetrione (116); (b) 3,3,8,8,13,13,17,18— octaethy1-2,7,12-porphinetrithione (120) ..... 250 MHz 1H NMR spectrum of 3,3,7,8,12,13,17,18— octaethyl-2-methy1-2-mercaptochlorin (121) ..... 250 MHz 1H NMR spectrum of 3,3,7,8,12,13,17,18— octaethyl-2-mercaptochlorin (122) ...... . 250 MHz 1H NMR spectrum of 2,2,7,8,12,l3,17,18— octaethylchlorin (123) ............... Visible spectra (in CH2C12) of (a) 3, 3, 8, 8, 12, 13, 17, 18-octaethy1-2-thio—7-porphine- dione (112); (b) its Zn-complex .......... xix Page 170 171 172 173 174 175 176 177 178 179 180 Figure A42 Figure A43 Figure A44 Figure A45 Figure A46 Figure A47 Visible spectra (in CH2C12) of (a) 2,2,8,8,12,13,17,18—octaethylf3-thio-7-porphine— dione (114); (b) its Zn-complex . . . ...... Visible spectra (in CH2C12) of Zn-complexes of (a) 3,3,8,8,12,13,17,18-octaethy1-2,7- porphinedithione (113); (b) 2,2,8,8,12,13,17,18— octaethy1-3,7-porphinedithione (115). ....... Visible spectra (in CH2C12) of (a) 3,3,8,8,13,13,l7,18-octaethyl-2-thio—7,12- porphinetrione (117); (b) 3,3,8,8,13,13,17,18— octaethyl—Z,12-dithio—7-porphinetrione (118); (c) 3,3,8,8,13,13,l7,lB-octaethyl-Z,7-dithio—12- porphinetrione (119) ................ Visible spectra (in cuzc12) of (a) dihydro—OEP; (b) tetrahydro—OEP; (c) hexahydro—OEP (under argon). .'. . . . ................. Optical spectra of methylchlorin-heme myoglobin; Ferric ( ), Deoxy ( ), Oxy (-----), CO (--—) in 10 mM (pH 7.4) potassium posphate buffer ....................... Optical spectra of dione-heme myoglobin; Ferric (_)a Deoxy (-_)9 co (—-_)9 in 10 m (pH 7.4) potassium posphate buffer .......... XX Page 181 182 183 184 185 186 GENERAL INTRODUCTION The majority of heme-containing proteins and enzymes found in nature possess prosthetic groups in which an iron atom is bound to a fully unsaturated porphyrin macrocycle 1 and is further coordinated by one or two axial ligands from protein side chains. Familiar examples include myoglobin and hemoglobin (oxygen transport and storage), cytochromes b and c (electron transfer), cytochrome P450 (substrate. hydroxylation), and peroxidases (substrate halogenation and peroxidation).1 Porphyrins and certain of their metal complexes are subject to reduction leading to a variety of isolable products.2 Among these are dihydroporphyrins (Chlorins, 2) and tetrahydroporphyrins (bacterio- chlorins, 3; isobacteriochlorins, 4) resulting from saturation of peripheral double bonds of the parent porphyrin. Biological interest in N M porphyrin chlorin 1 2 C i 5 bocteriochbrin isobacteriochbrin 3 - ‘4 reduced porphyrins has centered principally on chlorine and bacterio- chlorins inasmuch as the magnesium complexes of these macrocycles are the essential chromophoric units of algae and plant chlorophylls and bacteriochlorophylls, respectively.3 However, a significant number of organisms have now been shown to contain pyrrolic macrocycles based on C-substituted Chlorins and isobacteriochlorins. Examples include bonellin (5), the sex- .a-u-o—nn‘. .... neon-....."nu 3.151.933” Factor I from the Biz—producing Clostridium tetanomorphum’, heme d (6) of Escherichia 99116 which catalyzes the reduction of 02 to H20, heme d1 (7) from Pseudomonas aeruginosa", which is involved in a no.1: co," aonemn Home 9 "em as process known as "denitrification"3 by reducing nitrite to nitrous oxide, and sirohydrochlorin of nitrite and sulfite reductases as well as a 31: intermediate.9 The principal difference between these macrocycles and the unsubstituted hydroporphyrins is that the C—substituted compounds can resist dehydrogenation (back to porphyrins) and therefore, are better suited for undertaking the redox processes with which they may be associated in vivo. As the structures of these unique molecules are being elucidated, it. has become timely to investigate the chemistry and to identify their functional roles in their respective host systems. To realize such goals, however, requires workable quantities of materials which are often difficult to obtain from natural sources. In this thesis, Chapter 1 is devoted to the development of short and reliable syntheses for functionalized C-substituted Chlorins and isobacteriochlorins for general reactivity and biomimetic studies. The work initially focused on vicinal dihyroxychlorins and progressed into other substituted ring systems as a consequence of the lability of the vicinal diol. Chapter 1 also describes a novel method by which alkyl groups of porphyrins can be functionalized. Chapter 2 then gives a detailed account of the total synthesis of heme g and the effort to‘ deduce its true structure.‘ In chapter 3 the synthesis and properties of several sulfur containing saturated porphyrins, derived from their oxo- analogues are reported for the first time. Finally, the kinetic study of CO and 02 binding to myoglobin reconstituted with some of the above synthetic green hence is presented in chapter 4, in an effort to cast some light on their structure-function relationship. CHAPTERI SYNTHETIC manpower FOR C"SUBSTITUTED CHLORINS AND OTHER PORPHYRIIDIDS I. INTRODUCTION Saturation of 3-3’ pyrrole double bonds in a porphyrin ring can be brought about by either reduction or oxidation. Fischer and his group pioneered the use of sodium in alcohol to reduce porphyrin to chlorinlO' and this method has been extented by others11 to Obtain reduction levels beyond the chlorin stage, e.g., to bacteriochlorin and isobacterio- chlorin. In the opposite direction, Fischer again was first to study the effect of oxidants on porphyrin. In the 1930s, he reacted porphyrin with hydrogen peroxide in concentrated sulfuric acid and obtained what he thought at first was the vino-dihydroxy adduct.12 This product was later determined to contain only one oxygen.13 It was not until the 1960s that the keto-gem—dialkylporphyrin (oxochlorin) structure was characterized.“-16 The acidic hydrogen peroxide oxidation, which yields not only oxochlorins but diketo—(dioxoisobacteriochlorins and dioxobacteriochlorins) and triketoporphyrins arising from pinacolic rearrangements, has prevented the isolation of the expected dihydroxy intermediate. Fischer, however, demonstrated that hydroxylation of type IX porphyrins can be achieved with osmium tetraoxide although the resultant trimeric dihydroxy Chlorins were not individually identified.13:17 The passage from a Vic—dihydroxy or epoxy chlorin to the keto porphyrin by means of pinacolic rearrangements could be a useful method to provide C-substituted derivatives. Chang has reported the synthesis of’ methyl octaethylchlorin as well as dimethyloctaethylisobacterio- chlorins via the keto porphyrins.18 However, precedence of this rearrangement in porphyrin so far has been limited to octaethylporphyrin (OEP) and etioporphyrin I with simple alkyl groups. A central question in the pinacolic rearrangement is the migratory aptitude of the side chains. While this question has been addressed amply in alicyclic systems,” the outcome when applied to porphyrin rings is not readily predictable. This information would be absolutely necessary if this. ' method is to be used for the synthesis of’ biologically relevant molecules whose side chain substituents often determine the function. Recently, the keto porphyrins themselves also became the center of interest because of the discovery made in our laboratory that the green colored g. heme prosthetic group present in cytochrome 9g. has a dioxo- isobacteriochlorin structure.7° The only method known to date for the synthesis of such compounds is the hydrogen peroxide-sulfuric acid oxidation of BPsubstituted porphyrins resulting in a complex mixture of isomeric products containing one, two, and three oxo groups on the ring with uniformly poor yields.1°'18 The oxochlorins (porphyrinones), such as 8 can be prepared with a significantly higher yield by an alternative 2-step reaction via 0904 oxidation and acid catalyzed pinacolic rearrangementfli"?O Unfortunately, further oxidation of 8 by 0304 invariably leads to the bacteriochlorin 9,16 which upon rearrangement gives two isomeric dioxobacteriochlorins, 10 and 11 (Scheme 1). The Scheme 1 preference of attacking the opposite pyrrole double bond may be prompted by the diagonal electron delocalization pathway in chlorin that bypass the outer fl-B’ bond of the pyrroline ring and its opposite partner, leading to the bacteriochlorin formation with minimum loss of I—energy. If this is the case, we reasoned, any disruption of such a locked-in tautmeric form should decrease the bacteriochlorin formation and at the —-> H {Igure l. Tautomeric forms of porphyrin. same time, promote the isobacteriochlorin formation. One simple way to accomplish this feat would be the use of metal ion in the ring so that the two diagonal NH protons would be removed and the overall 04 symmetry is enhanced. There is precedence that the reduction site of porphyrin can be altered by metalation. Whitlock and Oester observed that the diimide reduction of free base tetraphenylchlorin (TPC) produces only tetraphenylbacteriochlorin whereas anlTPC gives exclusively ZnU tetraphenylisobacteriochlorin.21 Similarly, reduction of the NiH pheophorbide family of chlorins by Haney nickel promotes the fOrmation of' isobacteriochlorins.22 However, the osmate reaction with metalloporphyrins has never been studied before. As it turned out, this hypothesis was a complete success and this method now becomes the foundation for the total synthesis of d; heme and its analogues.7°'23 In the course of our study of the gig-dihydroxy chlorins, it was' frequently observed that acid treatment of certain .diols also gave porphyrins that are very different from the expected oxo products. This phenomenon is particularly common if less concentrated acid was used to promote the rearrangement. The product is usually a mixture comprising red porphyrins and some purple porphyrinone derived from pinacolic rearrangement of the diol.1°'2° With OEP—diol, the major porphyrin component is OEP—alcohol, presumably derived via hydration of an ethenylhydroxychlorin intermediate. An analogous reaction has been observed previously in a vic-dihydroxybacteriochlorin (scheme 2).?) semi, CH,-CH, 11° H N T 601* / 13 H on I on H,c 3,0 .4” a -+ f1“ N N 12 ‘~. ‘L H,c cmom-CH, 37.1 Scheme 2 14 This reaction appeared to us as a potentially attractive method to introduce functional groups to the side chain of alkylporphyrins. For example, OEP has been a tremendously useful compound in porphyrin ch-istry but the lack of functional groups can hinder its application in studies wherein some manipulation of side chains would be required. In such cases, it is often a choice between total synthesis, which is usually lengthy and of low yield, and the natural protoporphyrin or its derivatives, which on the other hand may have too many functional groups all at once. The mild elimination-hydration reaction obtainable from OEP-dial gives a simple method to functionalize the ethyl ‘chain of OEP so that the broad range of chemical transformation-s2° ascribed for the vinyl group of protoporphyrin would become accessible to OEP. In the following sections the synthesis and reactivities of gig- dihydroxychlorins will be described first, followed by the pinacolic rearrangement migratory aptitudes and site specificity. Examples of Q- alkylated chlorins derived from the keto porphyrins are given and finally the functionalization of OEP and other porphyrin macrocycles are discussed. II. RESULTS AND DISCUSSION A . v1” c-D ihydroxychlorins and Derivat ives Osmium tetroxide was added to dimethyl 3,7,8,12,13,17- hexamethylporphine—Z,lB—dipropionate (15a)27 in CH2012. The reaction was quenched after 20 h to yield the two dihydroxychlorins 16a (37%) and 17a (88) plus the unreacted porphyrin (30%). Increasing the amount of 0804 and lengthening the reaction time invariably led to the formation of tetrahydroxybacteriochlorin and intractable piments at the expense of the dihydroxy product. A similar reaction was tested on dimethyl 9 Mao, C :2: R: E! CO,Me I + M"0":17» R= Et COnMe R o a a R 1’ Wow 'o Mao,c 212 R= Mu CO.Me M005 22' R "‘0 °°tM° Moo, c 18: R: Me 21!) R: it 221. R: £1 18!) R: St M00.C CO.Me MGO'C 193 R3M. 19b R=Et l l ' ’OH . ° N i“ E I Mao c CO,Me 20 SCHEME 3 10 7,8,12,13-tetraethy1-3,17—dimethylporphine-2,18-dipropionate (15b).28 With this porphyrin apparently fer steric reasons, the dihydroxylation occurred more favorably at the "southern" pyrroles (ring C or D reduced) affording nearly 1:1 ratio of 16a and 17a. During the separation of 16a and 17a on TLC plates with Cflaclz/CHSOH (pair 16a,l7a requires multiple developments, whereas pair 18>,l7b requires only one development for separation) we observed the gradual development of a third, fastest- moving green spot. The IR spectrum of this new pigment showed a strong new band at 1780 cm—1 that was characteristic of a ‘Y-spirolactone29 (Figure 2). Mass spectral analyses confirmed that, the south diol 17a had lost a methanol and had become a spirolactone. Further study” ' revealed that this intramolecular lactonization is a general base- catalyzed reaction that can be brought about by sodium acetate, pyridines, basic alumina, as well as silica gel. When a small amount of the above lactone chlorin (18a) was extensively chromatographed on TLC plate (silica gel, CH2012/CH30H), it was converted to an even faster moving green spot. IR and mass spectral analyses still showed the presence of a Y—spirolactone. This lactone chlorin model compound, 19a exhibits a 1H NMR spectrum almost indistinguishable from that of Timkovich’s lactochlorin (from heme £1)° for’ those structural elements that are directly comparable to one another. Particularly interesting is the region between 2.3 and 3.4 ppm where the methylene protons of the rigid spirolactone should appear. On the basis of the absence of a measurable NOE between the 3-Me group (2.0 ppm) and any lactone ring protons, as well as on analyses of the lactone methylene ,peaks, Timkovich et. al. concluded that the two oxygen substituents have a trans configuration.° In our model chlorin lactone, ll J J_ J I 1 1 _L 1000 1700 1600 FREQUENCY, cm-1 Figure 2 1100~1500 cm"1 IR spectra of (A) porphyrin; (B) dihydroxychlorin; (C) Yespirolactone chlorin. 12 19a we also could not detect any NOE between the 3-Me peak (1.92 ppm) and‘the lactone protons. The remarkable similarity of the overall pattern between the model complex and the heme d derived lactones would thus argue that the model should likewise have a trans configuration about the pyrroline substituents. If this is true, it means that an inversion of the diol configuration has taken place, because the osmium tetroxide oxidation only affords cis diols. In an effort to elucidate this possible inversion process, we have examined the lactonization of a number of south diol chlorins. The tetraethylchlorin complex 17b, by virtue of its superior chromatographic mobility on silica gel, proves to be a more informative system for' delineating the reactions involved. When diol 17b is heated briefly in MeOH with sodium acetate on a steam bath, TLC (silica gel, 10% ethyl acetate in CH2C12) indicates that the slow—moving diol (Rf 0.3) is cleanly converted into a fast-moving spot (Rf 0.8). Both mass spectral (diol 17b minus 32) and IR (1780 cm-1) analyses suggest that this compound is a lactone, but 1H NMR shows the lactone methylene protons merged together between 3.3 and 3.8 ppm, distinctively different from that of 11a or the lactochlorin methyl ester.° If this green compound is rechromatographed on TLC, an even faster moving spot (Rf 0.88) emerges when the major spot is halfway up on the plate. If the plate is sufficiently long or the chromatography is repeated, the new pigment will eventually replace the original one and become the major spot. This new compound, as shown by mass spectroscopy and IR after isolation is still a lactone and has 1H NMR features very similar to those of the trans lactones observed earlier. This compound can also be shown to be 13 identical with the lactone prepared by repeated chromatography of the diol 17b. On the basis of these observations, we have assigned the NaOAc- cyclized product to be the cis lactone, while the silica gel induced lactone is trans (the NMR assignments for the cis and trans chlorin lactones are discussed in detail in Chapter 2). The cis-trans isomerization is illustrated in Scheme 4, with the key step being a unimolecular alkyl-oxygen fission process. can ,u N CH, _ A ‘ «A 0H V 9 ' V CC 2 OH -3... Scheme 4 The north diol chlorins (16a and 1%), lacking the propensity to lactonize, apparently can resist inversion upon repeated chromatography on silica gel; we have not observed another isomeric diol during chromatography or base-catalyzed conditions. A possible indirect way then, for the cis-trans diol conversion might be through silica gel- promoted lactone opening. Indeed, heating the cis lactone 1% and trans lactone 1% under reflux in pyridine-303 aq. KOH for prolonged periods of time, resulted in their hydrolysis giving back the diols. The product from the cis lactone 1% was identical in all respects to the cis diol 17b, whereas the product from the trans lactone 1% was identified by spectral analyses (see Chapter 2) as being the trans diol 14 20. If repeatedly chromatographed on TLC plate, both diols would eventually convert to the trans lactone 19b. From all the above, the following general Scheme 5 for the'cis- trans diol and lactone transformations can be written. cis diol ..___-—* cis lactone trans diol :==trans lactone Scheme 5 Treating the dihydroxychlorin 16a in CHaClz with 708 H0104 cleanly produced the rearranged ketones 21b and 22b in equal amount. The two isomers were separated by chromatography and their structures were determined ‘by nuclear Overhauser enhancements (NOE) on the proton resonances. 'Selective irradiation of the methyl substituents resulted in NOEs (>58) at the adjacent positions; by determining the nearest meso protons it is possible to assign the structures unawiguously (see Figure 3). Similar reaction and characterization were applied successfully for the tetramethyl homologues 21a and 22a. It is interesting to note that the NH protons of 21a and 21b should appear as two peaks while they remain as singlet in 22a and 22b. It is not evident whether an alteration of the tautomeric patterns or structural distortions is a possible cause for the splitting of the NH resonance. The oxochlorin (porphyrinone) 22a reacted sluggishly with methylenetriphenylphosphorane. The excess Wittig reagent present in the reaction invariably converted the ester group into the irketo methylphosphonium salt.3° Thus the methyl 'ester 22s. was first hydrolized in aqueous KOH, and the carboxyl groups were protected as the lS e 1 I ‘4 In. L l’ YYYYY VIYYYYYYYYVIII YYYYYYY l YYYYYYYYY IYYIIYYYIIIVYWY'YYYI YYYYYYY TY]rlIYYYVIIIYVYUYYYTYIVYVVV'Y'VI'YT'YlIYVlIIYYY q I ;: r—i -l . :i b com. 21b CO."- '1 ': AL 5 I v -2 -4 I 10 8 6 4 2 0 PPM“ Figure 3 250 MH; ‘H NMR spectra of 21b and 22b. Irradiations of the methyl resonances as indicated resulted in NOE observable at the neighboring groups whose chemical shifts are marked by the pointers. 16 carboxylate ion during the Wittig reaction.31 The resultant methylenechlorin was esterified and then hydrogenated quantitatively to the methylchlorin 24 with PtOz in formic acid. The dihydroxychlorin isomers 16a/17a and 16b/17b have almost identical visible absorption spectra whose overall features are indistinguishable from that of the canon dihydroporphyrins or the methylchlorin 24. The dihydroxychlorins are inert toward quinone oxidation; at room temperature they are relatively stable in most acids (including concentrated HCl) and undergo the pinacolic rearrangement only' with >602 sulfuric acid or' perchloric acid. When left in concentrated HI/HOAc, the dihydroxychlorin slowly changes into porphyrin presumably via one of the sequences in Scheme 6. This conversion is ——»i:°i ——~i:31 we. ii~ii——/ Scheme 6 HO OH probably responsible for Barrett32 to propose that porphyrin d from Aerobacter aerogenes is a dihydroporphyrin. Reductive removal of OH group by III was also used previously by Chang to prepare sy-etric alkylated chlorins and isobacteriochlorins.m The present series of '9- methyl- and C-hydroxychlorindipropionic acids is particularly useful for hemoprotein reconstitution studies. 17 Several rational approaches toward the synthesis of C—alkyl chlorins, starting with alicyclic precursors, have been described very recently.33-35 Unfortunately these lengthy and demanding syntheses do not lend themselves as a more serviceable route than harvesting organisms for providing the compound. Our approach from the gig-dials would seem to be a highly attractive route at providing the C- substituted chlorins. There is one recent report3° that a C-alkyl chlorin (27) can be obtained from a hydroxy porphyrin (25) by a Claisen rearrangement followed by hydrogenation (Scheme 7). The generality of this approach, however, seems to be limited. COfiM, 25 Scheme 7. Reagents: (a) CHSC(OCHS)2N(CH3)2; (b) H2, Pd/C B. MigratorywAptitudes in Pinacol Rearrangement 23.--!!392?”°¥X9919£19§ . Four porphyrins 15a, 29, 36, 41 were chosen for investigation.37 These porphyrins were converted into the dihydroxychlorins using osmium tetroxide as reported.38 The porphyrins were allowed to react with 1.2 equivalents of osmium tetroxide in methylene chloride and the reaction was quenched after 20 hours with the osmium esters being decomposed by hydrogen sulfide. Analyses (tlc, silica gel, methylene chloride- SCHEME 8s 19 SCHEME 8;: 20 methanol) indicated that the reaction mixtures contained variable amounts of chlorins plus the unreacted porphyrin. Isolation of individual components by chromatography and crystallization afforded the chlorin isomers as well as the starting porphyrin with yields shown in Scheme 8. The formation of the osmium esters is highly dependent on the porphyrin substituents. The relative yields of the chlorins thus produced a crude reactivity scale for the osmium tetroxide addition to porphyrin flkp’ double bonds: as would be expected, barring electronic effects, the larger the side chain, the slower the rate. The chlorin structures were determined by 1H NMR and by mass spectra. In the case of deuteroporphyrin dimethyl ester 29, the separation of A-ring and B-. ring chlorins was difficult; the mixture was employed for the subsequent rearrangement study. ' The acid-catalyzed pinacol-pinacolone rearrangement required different acid strength depending on whether the substituents are electron releasing or withdrawing. For example, while diol 1611 was converted smoothly into equal amounts of the two ketones by one drop of 70% perchloric acid in methylene chloride, the rearrangement of 38 required dissolution in 98* sulfuric acid for 2 hours. Except for 15a, each diol gave only one oxochlorin (porphyrinone) with a yield generally greater than BOX (only 40 and 46 were obtained with ~30¥ yield). The structure of the porphyrinones was established by nuclear Overhauser enhancements (NOE). Selective irradiation of methyl or methylene protons resulted in enhancement (24%) at the nearest meso protons (see Scheme 8). Since the mesa proton adjacent to the reduced ring, but not next to the keto group, invariably appears as a singlet near 9.0-9.1 ppm and the other three meso protons are around 9.5-9.9 ppm, it is possible 21 to assign the structures unambiguously. In the case of the mixture of 34 and 35, irradiation of the 3 and B—pyrrole protons resulted in a strong enhancement of both the 9.12 and 9.50 ppm meso protons; had the rearrangement gone the other direction, NOE should occur only at the two downfield meso protons, not at the 9.12 ppm peak. These experiments thus established the migratory aptitudes of the substituents; hydrogen, ethyl, alkyl groups including propionate side chains will migrate over methyl group. The only group that has a lower mobility than methyl is acetate; this is confirmed in two compounds 40 and 46. It seems that the electron-withdrawing nature of the acetate plays a determinant role. If the acetate group is first reduced with LiAlI-h, the pinacol. ‘ rearrangement of the 2-hydroxyethy1 group has been found to migrate over the methyl group. Applications. The knowledge of the migratory aptitudes, besides being useful in discerning possible biosynthetic precursors, is i-ediately applicable in planning new chlorin syntheses. For example, starting with coproporphyrin I (48), the above hydroxylation- rearrangement sequence gave a type III coprochlorin 49. Alkylation of the keto groups by Wittig reagents as described before20 would afford all-alkyl chlorins (Scheme 9). Similarly, porphyrin 50, prepared by stepwise assembling of the a,c-biladiene dihydrobromide 61 followed by Cu(II)-catalyzed cyclization38 (Scheme 10), produced two easily with the appropriate Wittig reagent and hydrogenation, would provide and easy entry into the family of the exotic echurian pigment bonellin 53. Previously, (t) bonellin has been made by a rather long synthesis.34b P 53 P SCHEME 9 chazcupozm 23 1. Cu’YDMF “1,50, 50 P SCHEME IO 24 Finally, the total synthesis of the green heme prosthetic group in cytochrome Cd]. achieved recently," was based on the knowledge of the .. ....... migratory aptitudes. C. Eifferentiation of Bacteriochlorin and Isobacteriochlorin Formation by Metalation. The 0s04 addition preference can be altered dramatically in favor of the isobacteriochlorin formation simply by metalation of the ring. The zinc complex 62 was found to react with 0s04 (1.5 equiv.) in CH2012 containing 1% pyridine to give predominantly 63 (>608 yield) which can be treated with sulphuric acid to give 64. A small amount of the ring D diol 65 was also obtained which rearranged to yield about equally 64 and 66. If the synthetic goal is 64, the crude dihydroxylation product can be used directly in the pinacol rearrangement as the ratio of 64:66 is usually greater than 30. That the osmate addition mainly occurred at ring 8 (63) is possibly due to the electron withdrawing effect of the carbonyl group rendering the adjacent ring D double bond less reactive. It is also noteworthy that during the pinacol rearrangement of 63 or its free ‘base, none of the possible porphyrin-2,8-dione. was observed. Insertion of other metal ions such as CuIt and Nil! had the same effect of switching the osmate addition pattern but the yields of osmate esters were less satisfactory.’9 The remarkable alteration of site of attack by metalation in the chlorin system appears to be a general phenomenon. The previously observed diimide reduction of Zn”TPP21 as well as the reduction .of NiH pheophorbides22 all serve to attest the significance of the metalation effect. The tautomerization patterns were thought to be more equalizing in a metal chlorin than in a free base chlorin to promote isobacteriochlorin formation. This hypothesis ZS SCHEME l l 64 66 26 did not explain why the double bond saturation occurs exclusively at the adjacent ring in the metal complex since one would expect that the absence of a preferred r—delocalizing pattern only favors a more random attack. Neither did previous M0 calculations of ZnTPC show a significant difference in r-electron density between the opposite and the adjacent B—B’ double bonds.“0 Presently, extended Huckel calculations are being undertaken on the metalloporphyrinone system using the newly acquired crystal structure parameters of Ni” OEP- porphinone.‘1 It is hoped that the refined calculations may uncover clues to explain this phenomenon. The selective saturation of the porphyrinone double bonds has made.- possible the synthesis of a variety of porphyrin-2,7-diones with side chains at specific positions. For example, the stereochemically uncomplicated dione 69 and its acrylic derivative 70, either in solution or in a reconstituted protein environment, proved to be accurate model compounds and spectral probes for heme d1 . The dione 69 could be prepared by the H202-H2804 oxidation of 15a, with a <23 yield after tedious separations from a mixture of no less than 10 oxo products.“ With the zinc method, 69 was prepared from 15640 cleanly with a high yield (Sch-e 12), and the unreacted starting material in the 0304 oxidation of 15a and 67 could always be recovered for recycling. The intermediacy of the porphyrinone 67 seems necessary. Attempts to react the Zn complex of 15a or ZnH octaethylporphyrin directly with an excess of man have only resulted in intractable pigments. The two-stage oxidation via isolated porphyrinone has also imparted a higher degree of regi oselect ivi t y for the isobacteriochlorin-type porphyrindione formation. In the present study, if the isomeric 7130 is used, the 27 8.60 N. MEN—mom 2. 0.0... 1.3 2. .0... 28 major product is 73 under reaction times > 36 h, with osmate selectivity of ring D vs. B 4:3. However, for shorter reaction times (24 h) the osmate selectivity is not obvious (ring D vs. ring D 1.2:1). In any case, the attack of ring D is much more favorable for porphyrinone 71 than for porphyrin 15a. The pinacolic rearrangement of 72 gave 69 and 74 in equal amounts. 0n the other had, the porphyrindione derived from the pinacolic rearrangement of 73 is exclusively 75, apparently reversing the migratory aptitude of methyl < propionate observed in simple gig-dihydroxychlorins“ but fully agreeing with the above observation of porphyrinone diols. Further reaction of free base 69 with 1.8 equiv. of mo. resulted in exclusively a ring C-diol, presumably for the same reason cited above for U: to avoid the carbonyl group next to ring D. Beating the dial in HCl-dioxane-Hzo or HCl-benzene, triggered an elimination of 820 and yielded a p-hydroxy propionate which eliminates further to give the acrylate 70 in 80" yield using the first solvent system or quantitatively using the second one. D. A Novel Method of "Eunctionalizingwthe Ethyl Chain ”of -—~.u.-a—.«- "no... fiteashiilpqrphifliag_ When OEP-diol 76, is heated in aqueous HCl/dioxane, the major porphyrin component is OEP-alcohol 77 with yields never greater than 508. This reaction seemed highly sensitive to the acid concentration: dilute acid gave insufficient reaction while too strong an acid only let to pinacol rearrangement. To improve the reaction, other nucleophilic media were tested. When 76 was dissolved in glacial acetic acid and heated at 90°C, the acetoxy 78 was obtained within 10 min in 85k yield. Likewise, if 76 in methanol was heated in the presence of HCl, the 76 29 on F 80 a =cw=cw, 31R=H SCHEME 13 OH 77a=ow 78R=0Ac 79mm» 30 methoxy 79 can be isolated in >75% yields. Both 78 and 79, of course, can serve as starting points for further derivatizations. Vinylation may also be achieved in a single step (SO-95% yield) by heating 76 in benzene containing HCl. As 76 can be prepared readily from OEP by OsOn oxidation, the simple preparation of 80 therefore offers expeditious synthetic routes to a wide range of monosubstituted heptaethylporphyrins having for instance, Br,“ CN,‘5 CH0,2°v4° C0083,“ CH=CHCOzR,‘5"" 011208,“ CHzCHzOH,“ 01201126028,“ plus other moieties attached via these groups. The Experimental Section includes a procedure for making 81 by devinylating 80. It was also observed that the preparation of 77- 81 can be scaled up without lowering the yield. When the above procedures are applied to other lip-dials with dissimilar alkyl chains, two products are expected. .Indeed, dihydroxy- etiochlorin 82 has been found to yield the two alcohols fl! and 84 or the two acetoxyetioporphyrins 85 and 86 under appropriate conditions. The same is true with porphyrins bearing methyl and propionate substituents (e.g., 87 and 88). While the ratio of the two possible products, varies frm case to case, the methyl group appears to be the more favorable site of attack, at least with common dihydroxychlorins. Such a result would suggest that the reaction is subject more to kinetic control. With porphyrinones, the course of the reaction can be dependent upon the symmetry of the molecule as well as the reaction conditions employed.” However, in our recently reported synthesis of heme 9: analogue, 89 gave exclusively 90 before it was dehydrated to the acrylate.23 In this case, the stability of the exocyclic alkene intermediate seems to be the determinant factor. The specificity associated with these molecules further suggests that the .mild 31 32 elimination—hydration of vic-diol (or an epoxide precursor) may have some biosynthetic significance. We speculate that the acrylate group of heme g; is indeed produced biosynthetically from a propionate side chain by this route. The demonstrated conversion” from chlorophyll a to chlorophyll b via a yic-diol could be a viable biosynthetic pathway. As for the heme a moiety of cytochrome oxidase,50 the lB—carboxaldehyde group could come from a CH3, not by harsh direct oxidation but by way of 082011 resulted from the gig-dial. These hypotheses possibly can be tested by future experiments. III. EXPERIMENTAL NMR spectra were obtained at 250 MHz on a Bruker WM—250 instrument. Spectra were recorded in CD013; the residue CHCla was used as the internal standard set at 7.24 ppm. Nuclear Overhauser enhance- ments (NOE) were measured by difference between a spectrum with preirra- diation on a target peak minus a spectrum with equivalent pre- irradiation at a dummy position. Typical parameters: D1 (relaxation delay) 8 5Ti=l.5 sec, D2 (NOE generation time) = 0.03 or 0.095 sec, D3 (pulse interval) = 0.1-0.3 sec, DP (decoupling power) = 26 or 36 L (depending on D2), PW = 90-degree pulse. Magnitudes of NOEs were calcu- lated as the area of the enhanced resonance in difference spectra divided by the area in the control spectrum with no enhancement. Mass spectra were obtained using a Finnigan 4021 GC-MS (direct insertion probe, 70ev, 200—30000), or a JEOL HX 110—HF spectrometer equipped with a fast atom bombardment (FAB) gun. A matrix of thioglycerol- dithioerythreitol—dithiothreitol (2:1:1) containing 0.1x trifluoroacetic 33 acid was used for the FAB—MS. Elementary analyses were performed by MicAnal. Visible absorption spectra (in CHzclz) were measured with a Cary 219 or a Shimadzu 160 spectrophotometer. IR spectra were obtained from KBr pellets on a Perkin-Elmer 283B spectrophotometer. Melting points were obtained on an electrothermal melting point apparatus and are uncorrected. Preparative TLC plates were from Analtech (silica gel 6, 1000 or 1500 um). Methylene chloride and pyridine were distilled from CaHz, THF from LiAlHa, and methanol from sodium before use. Dimethyl cis-7,8-dihydroxyj3,7,8,l2,13,17-hexamethy1chlorin-2,18- dipropionate (169) Osmium tetroxide (300 mg, 1.2 mol) in anhydrous ether (3 ml) was- added to a methylene chloride (200 ml) solution of dimethyl 3,7,8,12,l3,l7-hexamethylporphine—2,lB—dipropionate (15a)27 (566 mg, 1 mol). Dry pyridine (0.2 ml) was added subsequently, and the mixture was allowed to stir at room temperature, under nitrogen, in the dark for 20 h. It was then diluted with methanol (100 ml) and bubbled with H28 for 10 min in order to decompose the osmium ester. The precipitated osmium sulfide was removed by filtration, and the crude product in the filtrate was chromatographed on a silica gel column. Unreacted porphyrin was eluted first with CH2012/3X MeOH. The slower moving major isomer which turned out to be 16a was crystallized from CH2012-hexane. The mother liquor combined with. the faster moving component was chromatographed once again by preparative TLC (CH2C12/22 MeOH) to give pure 16a yields: unreacted porphyrin, 164 mg, 30%; 16a, 220 mg, 37%; 17a, 50 mg, 896. 16a: NMR 6 2.10, 2.12 (3H each, s, 7,8-Me), 3.11, 3.13 (2H each, t, cuzcgzcoz), 3.40 (an, s, 2xMe), 3.43, 3.45 (3H each, 8, Me), 3.63, 34 3.66 (3H each, s, COzMe), 4.15, 4.22 (2H each, t, CH2CH2C02), 9.07, 9.09 (1H each, s, 5,10-H), 9.68 (2H, 5, 15,20- H), -2.78 (2H, br s, NH); UV- vis hex (an) 642 nm (44 700), 614 (3 800), 588 (4 700), 522 (3 400), 495 (13 300), 490 (13 300), 392 (179 000); MS, m/e 600.2936 (calcd for CadhoNqOe 600.2950). Dimethyl cis-2,3-dihydroxy—3,7,8,12,l3,17-hexamethylchlorin-2,18- dipropionate (178) mm a 2.12 (3H, 2, 3-Me), 2.47-2.83 (4H, m, 2-CH2CH2002), 3.16, 3.18 (1H each, t, 18—cnzcnecoz), 3.36, 3.37, 3.39, 3.45, 3.46 (3H each, s, ring Me), 3.50 (3H, s, 2-CCC02Me), 3.68 (3H, s, lB—CCCOzMe), 4.16, 4.20 (1H each, t, 18-CH2CH2002), 9.08, 9.10 (18 each, s, 5,20—H), 9.58,. 9.67 (111 each, s, 10,15-1-1), -2.91 (2H, br s, NH); UV-vis has (an) 643 nm (43 400), 614 (3 400), 589 (3 900), 521 (2 500), 495 (13 400), 490 (13 400), 392 (188 000). Methyl cis-3-hydroxy-3,7,8,12,13,l7-hexamethyl-2,2-1~spirolactone- chlorin—18-propionate (18a) Prepared either by heating the dihydroxychlorin, 17a, in methanol with sodium acetate under reflux for 20 min, or by repeated chroma- tography on preparative TLC plates. NMR 6 1.80 (3H, s, 3-Me), 3.05 (2H, t, 18—CHzClj12C02), 3.21-3.83 (4H, m, 2-CHzCH2002), 3.30 (6H, s, 2xMe), 3.34, 3.40, 3.43 (3H each, 6, ring Me), 3.48 (3H, s, C02Me), 3.87 (br s, 0H), 4.17 (28, m, 18-C32CH2002), 9.02, 9.08 (1H each, s, 5,20-H), 9.59, 9.71 (111 each, s, 10,15-H), —2.65 (2H, br s, NH); UV—vis hex (ca) 641 nm (41 600), 614 (3 500), 588 (4 000), 520 (3 100), 493 (12 900), 488 (13 100), 389 (176 000); MS, found: m/e 569.2753 for (M+H)*, C33H37N405 requires m/e 569.2766. 35 Methyl trans- 3-hydroxy-3, 7, 8,12,13,17-hexamethyl-2, 2-Y-sp1rolactone- chlor1n-18—propionate (19a) Obtained from the cis—lactone 18a by repetitive chromatography. MIR 6 1.92 (3H, s, 3-Me), 2.38 (1H, sext, 284), 2.98 (1H, oct, 2b2), 3.16 (2H, t, 18—CHeC82C02), 3.21 (1H, 2b3), 3.35 (6H, s, 2xMe), 3.39, 3.40, 3.50 (3H each, 3, Me), 3.47 (1H, 281), 3.66 (3H, s, C02Me), 4.18 (2H, t, 18-0880H3C02), 8.86, 8.91 (1H each, s, 5,20-H), 9.63, 9.71 (1H each, s, 10,15-H), -2.58 (2H, br 8, NH); UV-vis Max (In) 643 nm (34 800), 590 (3 400), 520 (2 500), 492 (11 400), 488 (11 100), 388 (150 000); MS, found: m/e 569.2761 for (M+H)*, 033H37N405 requires m/e 569.2766. Dimethyl cis-7,8, 12, l3-tetraethyl-—7,8-dihydroxy-3,17-dimethylchlorin- 2,18—d1propionate (16b) Osmium tetroxide (90 mg, 0.35 .01) in ether (1 ml) was added to dimethyl 7,8,12,lB—tetraethy1-3,l7-dimethy1porphine-2,18-dipropionate 15b28 (170 mg, 0.27 mmol) in CH2C12 (50 m1), followed by dry pyridine (0.1 ml). The reaction was allowed to proceed in the dark for 20 h and worked up in the same manner as described above. The products isolated according to their elution pattern from the silica gel column were the unreacted porphyrin (20 mg, 11.8%), isomer 16b (50 mg, 28%), and isomer 17b (47 mg, 2896). (Notice that the isomer 18) is the faster moving chlorin in this case). 16b: NMR 6 0.90, 0.98 (3H each, t, 7,8-CH2C113), 1.74, 1.76 (3H each, t, 12,13—CH2CH3), 2.50, 2.58 (2H each, q, 7,8—C82CH3), 2.93, 3.06 (2H each, t, CHzCH2C02), 3.27, 3.37 (3H each, 8, Me), 3.59, 3.66 (3H each, s, COzMe), 3.85 (4H, q, 12,13-08261-13), 3.94, 4.06 (2H each, t, C82CH2002), 8.92, 9.00 (18 each, s, 5,10-H), 9.46, 9.67 (1H each, s, 15,20-H), -2.62 (2H, br 5, NH); UV-vis Max (an) 643 nm (46 900), 614 (4 36 000), 590 (4 200), 5:22 (2 900), 494 (14 200), 490 (14 200), 392 (198 000). Dimethyl cis-7,8,12,13—tetraethy1—2,3 dihydroxy—3 17-d1methy1chlor1n— 2, 18-d1propionate (17b) MIR 6 1.79 (12H, m, 4xCHzCI13), 2.18 (3H, s, 3-Me), 2.52-2.92 (4H, m, 2-CHzCHzC02), 3.15 (2H, t, 18-CH2CH2C02), 3.48 (3H, s, 17-Me), 3.54 (3H, s, 2-CCC02Me), 3.68 (3H, s, 18—CCC02Me), 3.86, 3.90, 3.92, 3.96 (2H each, q, C112CH3), 4.19 (2H, t, 18-C1120H2002), 9.04, 9.08 (1H each, s, 5,20-H), 9.71, 9.73 (1H each, s, 10,15-H), -2.57 (211, br s, NH); UV-vis Lu (8:) 643 nm (44 000), 615 (3 600), 590 (3 900), 521 (2 600), 494 (13 700), 490 (13 700), 392 (193 000). Methyl cis- -7, 8, 12, 13 tetraethyl -3- hydroxy—3 l7—dimethyl- 2, 2-1: spirolactone-chlorin 18- propionate (18b) Prepared the same way as 188. NMR 6 1.79, 1.80, 1.81, 1.84 (3H each, t, CH2CHa), 1.86 (3H, s, 3-Me), 3.10, 3.11 (111 each, t, 18- CHzCEzCOz), 3.26-3.85 (41!, m, 2-CHzCI-12C02). 3.46 (3H, s, 17-Me), 3.52 (3H, s, COzMe), 3.87 (4H, q, 2xCH20H3), 3.93, 4.00 (2H each, q, cgzcfla), 4.17, 4.20 (IH each, t, 18-082CH2002), 9.10, 9.21 (1H each, s, 5,20-H), 9.76 (2H, s, 10,15-H), —2.62 (2H, br s, NH). IR 1780 cm-1, 1736; UV-vis he: (an) 640 nm (42 600), 614 (3 600), 586 (3 600), 521 (2 700), 492 (12 300), 488 (12 400), 389 (179 000); MS, found: m/e 625.3401 for (M+H)*, CS7H45N405 requires m/e 625.3392. Methyl trans-7, 8, 12,13- tetraethyl -3- hydroxy-3 17 dimethyl 2, 2 T- spirolactone-chlorin 18 propionate (19b) Prepared the same way as 198. NMR 6 1.79 (12H, m, 4xCH2C113), 1.96 (3H, s, 3-Me), 2.43 (1H, sext, 284), 3.04 (1H, oct, 2132), 3.22 (1H, quint, 2b3), 3.50 «.lH, sept, 281) [J(281,2b2 = 4.1 Hz, J(28.1,2b3) = 9.6, 37 J(281,284) = -12.8, J(2b2,2b3) = ~17.9, J (2b2,284 = 9.6, J(2b3,284) = 9.6], 3.15 (2H, t, 18—CH2CH2C02), 3.52 (311, s, 17—Me), 3.66 (3H, s, C02Me), 3.85 (2H, q, 2xCHzCH3), 3.88, 3.98 (2H each, q, CHzCHa), 4.20 (2H, t, 18-C82CH2C02), 8.89, 9.04 (1H each, s, 5,20-H), 9.75 (2H, s, 10,15-H), -2.54 (2H, br 3, NH). IR 1780 cm-1, 1734, 1714; UV—vis hax (an) 643 nm (34 800), 591 (3 300), 521 (2 800), 489 (10 900), 484 (10 800), 391 (162 000). MS, found: m/e 625.3387 for (M+H)*, C37H45N405 requires m/e 625.3392. Dimethyl trans-7,8,12,13-tetraethyl-2,3-dihydroxy—13,17- dimethylchlorinf2l18jd1propionate (20) To a refluxing solution of trans-lactone 19) (22 mg, 0.035 mmol). in pyridine (20 m1) under argon, KOH (0.48 g) in water (1.6 ml) was added and the heating was continued for 7 h before the mixture was evaporated to dryness under reduced pressure. The residue was redissolved in cold dry methanol saturated with HCl gas. The mixture was allowed to stir in an ice-water bath for 10 min before being partitioned between CH2012 and water. The organic layer was separated, washed three times with water, dried (N82 804) and evaporated to dryness. The product was chromatographed rapidly on TLC (10% EtOAc/CHzClz) to separate the slower moving trans diol 20 (8 mg, 36.4%) from the faster moving trans lactone 19) (10 mg, 43.5%). 20: MIR 6 1.76, 1.78, 1.79, 1.81 (3H each, t, CH2C113), 1.86 (3H, s, 3-Me), 1.88, 2.06, 2.47, 2.66 (111 each, quint, 2-CH2CH2C02) [J(281, 2b: = 7.3Hz, J(281,2b3) = 7.3, J(281,284) = -l4.6, J(2b2,2b3) = -14.6, J(2b2,284) = 7.3, J(2b3,284) = 7.3], 3.17 (2H, t, 18-CH2C112C02), 3.34 (3H, s, l7-Me), 3.54 (3H, s, 2—CCC02Me), 3.67 (3H, s, lB-CCCOzMe), 3.84 (6H, q, 3240112013). 3.97 (211, q, 01120113), 4.22 (211, t, 18—C1120H2C02), 38 8.97, 9.03 (If! each, s, 5,20-H), 9.70, 9.72 (1H each, s, 10,15~H), —2.34 (2H, br 3, NH); UV—vis )max (an) 647 nm (35 900), 592 (3 800), 522 (2 700), 495 (10 900), 490 (11 100), 391 (150 000); MS, found: m/e 657.3649 for (Mi-HY, C38H49N406 requires m/e 657.3655. Dimethyl 3, 8, 8, 12, 13, l7~ hexamethyl 7-porph1none-2 18-d1propionate (21a) and Dimethyl 3, 7, 7, 12,13,17-hexamethy1 8-porph1none-2 18-d1propionate (23.9.2, Perchloric acid (70%, 1 ml) was added to a methylene chloride solution (60 ll) of 16a (150 mg, 0.25 mol). The mixture was allowed to stir at room temperature for one-half hour before being extracted with water (3x, 60 ml each). The CHzClz layer contained the mixture of 21a and 22a, which were separated on preparative TLC plates (silica gel, CHéClz/l% MeOH), yielding 60 mg (42%) each of the porphyrinones. 21a (slower component on TLC): MIR 6 2.09 (6H, s, 8,8-Me), 3.12, 3.15 (211 each, t, CHzCHzCOz), 3.37, 3.43, 3.46, 3.49 (31! each, s, ring Me), 3.57,.3.59 (3H each, s, C02Me), 4.15, 4.30 (211 each, t, C§2CH2C02). 9.02, (13, s, lO-H), 9.70 (1H, s, 5-H), 9.75 (111, s, 15-H), 9.84 (18, 3, 20-11), -3.06, -2.86 (1H each, br 3, NH); MS, m/e 582.2838 (calcd for 034113911405); m.p. 265-2660C; UV-vis 7.3:: (an) 642 nm (32 400), 585 (6 000), 546 (12 000), 508 (9 500), 490 (6 200), 404 (169 000). 22a (faster component on TLC): NMR 6 2.00 (SH, 3, 7,7-Me), 3.13, 3.18 (211 each, t, CH2CHzC02), 3.39, 3.44, 3.46, 3.50 (38 each, s, ring Me), 3.59 (6H, s, 2xC02Me), 4.16 (2H, t, 18—0112CH2002), 4.32 (2H, t, 2- 01120112002), 9.07 (1H, s, 5—H), 9.74 (1H, s, lO-H), 9.79 (111, s, 20-1-1), 9.80 (1H, s, 15-H), *3.12 (2H, br 3, NH). Irradiating the triplet at 6 4.32 caused the singlets at 6 9.79 and 3.50 to increase in intensity. Moreover, irradiating the singlet at 6 3.50 caused the singlet at 6 9.07 (most upfield meso proton) to increase in intensity. MS, m/e 582 (M’); *’~ 39 m.p. 266-26800; UV—vis )nax (an) 642 nm (32 300), 585 (5 500), 546 (11 300), 508 (8 500), 490 (5 600), 404 (151 000). Anal. calcd: C, 70.07; H, 6.58; N, 9.62. Found C, 70.18; H, 6.66; N, 9.57. Dimethyl 8,8,12,13-tetraethyl-3,l7-dimethyl-7-porphinone-2,18— dipropionate (21b)and Dimethyl "7,7,12,l3-tetraethyl-3,l7-dimethyl-8- POFPhinone‘Zs13"d12592199§£2m£§391 The dihydroxychlorin 16b (50 mg, 0.076 mmol) in 082012 was treated with perchloric acid (70%, 1 ml), and the reaction was worked up in the same manner as described above to afford 19 mg of each (40%) of the isomeric porphyrinones. The structure assignment for the slower moving 21b and the faster moving 22b was achieved via NOE measurments ‘(see Figure 3). 21b: NMR 6 0.38 (6H, t, 8,8-Cflécfls), 1.86 (6H, t, 12.13—082033), 2.75 (48, q, 8,8-CHzCHa), 3.20, 3.24 (3H each, t, 082CH2C02). 3.48, 3.59 (3H each, 3, Me), 3.65, 3.66 (3H each, s, COzMe), 4.00, 4.06 (28 each, q, 12,13—cgzcna), 4.25 (23, t, ls—cgzcuzcoz), 4.40 (23, t, 2-ogsc32002), 9.13 (1H, s, lO—H), 9.84 (1H, s, 5-H), 9.85 (18, s, l5-H), 9.95 (1H, s, 20—11), -2.91, -2.78 (1H each, br 3, NH); UV-vis but (an) 642 nm (34 700), 586 (5 900), 546 (11 800), 508 (9 600), 490 (6 300), 406 (173 000). 22b: NMR 6 0.38 (6H, t, 7,7-CHzCH3), 1.85 (6H, t, 12,13-CH2CH3), 2.76 (4H, q,'7,7—cnzcaa), 3.21, 3.28 (3H each, t, cuscg2c02), 3.48, 3.58 (3H, 3, Me), 3.67, 3.68 (3H each, s, COaMe), 4.06 (4H, q, 12,13—cnacgb), 4.25 (23, t, 18-CH20H2C02), 4.41 (28, t, 2-CfléCH2C02), 9.13 (1H, s, 5- H), 9.84 (1H, s,’ lO-H), 9.88 (1H, 3, 20-11), 9.93 (1H, s, 15-H), 42.90 (211, br s, NH); UV—vis hex (m) 642 nm (35 000), 586 (5 700), 546 (11 800), 508 (9 000), 490 (6 000), 406 (162 000). 4O Dimethylm§lll7.12.13.17thexamethyI-BfmethxlenechIQFin+21187 dipropionatsWLZ§1 The methyl ester groups of porphyrinone 22a (100 mg, 0.18 mmol) were hydrolyzed in a mixture of equal volume of THF and 2N aqueous KOH. The mixture was stirred for 12 h before the THF solvent was removed in a rotorvap. The remainder of the aqueous solution was acidified with H01, and the precipitated porphyrinone diacid was collected by filtration, washed with water and dried. To a suspension of PhaPCHaBr (614 mg, 1.72 mmol) in dry THF (20 ml) was added an equivalent amount of n-butyllithium (1.6M solution in hexane) under nitrogen. The resultant orange suspension was allowed to stir at room temperature for 30 min before being added to a solution of the porphyrinone diacid (95 mg, 0.172 mol) in dry THF (25 m1) at 0°C. The mixture was allowed to stir at room temperature for 12 h, after which time the reaction was quenched with water. The solvent was evaporated, and the residue was esterified in dry methanol (50 m1), saturated with HCl gas, and left overnight. The solvent was again evaporated, and the residue was taken 'in CH2C12, washed with water, and chromatographed on silica gel (CH2012). The methylenechlorin 23 (68 mg, 71% yield), migrating in front of the unreacted 22a (20 mg), was further purified by crystalization from CHzClz/hexane: m.p. 229—231°C; NMR 6 2.03 (6H, s, gem-Me), 3.17, 3.20 (2H each, t, CH2CH2C02), 3.41 (6H, s, 2xMe), 3.45, 3.49 (3H each, 3, Me), 3.66, 3.67 (3H each, s, C02Me), 4.19, 4.33 (2H each, t, CH2CHzCOz), 5.81, 6.78 (1H each, s, =CH2), 8.86, 9.38 (1H each, s, 5,10-H), 9.65, 9.71 (1H each, s, 15,20—H), -2.54 (211 br 3, NH); MS, m/e 580.3049 (calcd for 0351-140qu 580.3052); UV-vis ha»: (ha) 656 nm (36 000), 600 (4 400), 534 (13 000), 506 (9 600), 498 (9 600), 400 (136 000). 41 Dimethylm317z7.8zl3.13¢l7-heptamethY1chlorinfiZzIdeiPrOPipnatem(24) The above chlorin 23 (10 mg) was dissolved in formic acid (88%, 8 ml), to which a small amount of Adams catalyst (Pt02, 5 mg) was added. A gentle stream of hydrogen was passed into the mixture for 5 min. A distinct color change was observed. The hydrogenated product was obtained almost quantitatively by evaporating the formic acid and purified by passing through a short silica gel pad with CH2C12: m.p. 215-218°C; NMR 6 1.83, 2.01 (3H each, s, gem-Me), 1.98 (3H, d, tertiary Me), 3.17, 3.20 (211 each, t, CH2CH2C02), 3.41, 3.42, 3.47, 3.50 (3H each, s, ring Me), 3.67 (6H, s, 2xC92Me), 4.20, 4.33 (2H, t, C§2C32C02), 4.55 (in, q, tertiary n), 8.81, 8.85 (1H each, s, 5,10-H), 9.68, 9.70 (1H each, s, 15,20~H), -2.42 (2H, br 5, NH); MS, m/e 582.3200 (calcd for 035842N404 582.3208; UV-vis 7.3x (an) 643 nm (36 900), 614 (3 700), 589 (4 200), 524 (4 000), 497 (9 900), 490 (9 800), 392 (141 000). General Procedureof 0x1dationand Rearrangement of 15a, 29, 36 and 41 Osmium tetroxide (1.2 mmol) in ether (3 ml) was added to a dichloromethane solution (200 ml) of porphyrin (1 mol) containing pyridine (0.2 ml). The mixture was allowed to stir at room temperature under nitrogen for 20 hours. The reaction was quenched by addition of methanol (100 ml) and followed by bubbling hydrogen sulfide into the solution. The precipitated osmium sulfide was filtered, the filtrate was evaporated, and the residue was chromatographed on silica gel using dichloromethane/ l 3% methanol as eluent. The pinacolic rearrangement of the dihydroxychlorins was brought about by three different acid treatments: (1) dichloromethane with a couple drops of 70% perchloric acid (16a, 37, 42); (2) chlorin in 42 dichloromethane, shaking with concentrated sulfuric acid (17a, 30, 31, 44); (3) neat concentrated sulfuric acid for several hours, followed by esterification (38, 43). Pinethyl 3:7.8.12.13.17-hexamethyl-1§t2952919999r2111: dipropionate (28) Yield (from 17a): 85%; m.p. 191-1930C, MS (direct probe, 70 ev) m/e 582 (Mt). NMR 5 1.51 (25, m, 17-cgacuecoz), 2.08 (3H, s, 17-Me), 3.05 (2H, t, 17—0820112002), 3.23 (211, t, 241820112002), ring Me: 3.28 (311, s), 3.40 (511, s), 3.51 (311, s), 3.55 (311, s), 3.50 (3H, s, 17- CCCOeMc), 3.74 (an, s, 2-CCC02Me), 4.35 (2H, t, 2-cnecaeCOa), 9.08 (15, s, l5-H), 9.77 (2H, s, 10,20-H), 9.87 (18, s, 5-H), -2.94, -2.81 (13 each, br s, NH); UV-vis 7.... (an) 643 nm (35 400), 586 (5 500), 548 (10 800), 508 (8 500), 490 (5 300), 405 (150 000). Dimethyl 12,18—diethy1:3,7,l3,17—tetramethylporphine-2,8- diacetate (36) 4,4’-Dimethoxycarbonylmethyl-3,3’,5,5’—tetramethyl-2,2’-dipyrro- methenium bromide” (4.25 g, 10 mmol) and 5,5’-dibromo-4,4’-diethyl— 3,3’-dimethyl-2,2’-dipyrromethenium bromides1 (4.67 g, 10 mmol) were suspended in formic acid (50 m1, 98-100%) and treated with bromine (0.5 ml). The mixture, protected from moisture, was refluxed in an oil bath maintained at 130-13500 for 2.5 h. The condenser was then removed and the solvent was boiled off under air. The black residue was dissolved in methanol (100 ml), the solvent was boiled off again, and the residue redissolved in 100 ml of methanol. Trimethyl orthoformate (20 m1) and sulfuric acid (concentrated, 2 ml) were added and the mixture was allowed to stand at room temperature, protected from moisture in the dark, for a day. Crystalline porphyrin often separated from the liquid 43 by this time. If so the crystals were collected by filtration, the filtrate was evaporated and the residue was loaded onto a silica gel column. A black nonfluorescent band was eluted first by using dichloromethane and discarded. The porphyrin methyl ester came off with 1% CHaOH/CHzClz but often required 2% CH30H/CH2C12 to be eluted completely. The main porphyrin fractions were combined and evaporated while the heavily contaminated fractions required a second column. The porphyrin 36 was further purified by recrystallization from CH2012/CH30H. Yield (1.8 g, 32 %); m.p. 310-312°C; MS found: m/e 567.2887 for (M+H)*, Ca4H38N404 requires m/e 567.2895; NMR 6 (6H,t, 2xCHzC113), 3.58, 3.61 (6H, 3, ring Me), 3.74 (6H, s, 2x0002Me), 4.05 (4H, q, 2xCHzCIh), 5.01 (4H, s, 2xCH2C02), 10.01, 10.02 (1H each, s, 5,15-H), 10.06 (2H, s, 10,20-H), -3.82 (2H, br s, NH); UV-vis has (an) 620.5 nm (4 300), 567 (6 600), 531.5 (9 400), 498.5 (14 000), 399 (169 000). Pissfihxl,l21lB-diethyl+12.13:dih24r0327317113.lzétetramethyl-chlorin: Z1§:91299§§£sm$37) Slower moving component on TLC: yield 48%; MS found: m/e 601.3018 for (M+H)*, C34H41N405 requires m/e 601.3028; NMR 6 0.67 (3H, t, lZ-CHzCHa), 1.75 (3H, t, l8-CH2CH3), 2.12 (3H, s, l3-Me), 2.20, 2.37 (1H each, m, 12-CH2CH3), 3.22, 3.33, 3.37 (3H each, 3, ring Me), 3.66, 3.68 (3H each, s, C02Me), 3.89 (2H, q, 01120113), 4.55, 4.65 (lH'each, AB, JAB = 15.8 Hz, 9-CH2002), 4.72 (2H, s, l-CHzCOz), 8.88, 8.89 (1H each, s, 10,15-H), 9.50, 9.66 (1H each, s, 5,20-H), -2.61 (2H, br s, NH); UV- vis 7.3:: (an) 642 nm (41 000), 612 (3 200), 588 (3 900), 526 (3 000), 498 (12 800), 494 (12 500), 394 (173 000). 44 Dimethyl112.18:diethylfzi3fidih2459¥¥r317.13xl7ttstrsmsthylshlprin? Zifirsiasstats-(38) Faster moving component on TLC: yield 6%; MS found: m/e 601.3022 for (M+H)*, C34H41N405 requires m/e 601.3028; NMR 6 1.74, 1.75 (3H each, t, CH2CH3), 1.93 (3H, s, 3—Me), 3.33, 3.38, 3.47 (3H each, 3, ring Me), 3.67 (3H, s, 2—CC02Me), 3.62, 4.07 (1H each, AB, Jxa = 16.5 Hz, 2- 0112002), 3.85 (4H, m, CH2CH3), 3.92 (3H, s, 8—CCOzMe), 4.72, 4.82 (1H each, AB, JAB = 15.3 Hz, 8-CH2C02), 9.02, 9.14 (1H each, s, 5,20-H), 9.67, 9.73 (1H each, s, 10,15-H), -2.80 (2H, br 3, NH); UVf-vis )max (01) 642 nm (47 400), 614 (3 500), 590 (3 800), 538 (1 600), 520 (2 200), 492 (13 600), 488 (13 300), 389 (186 000). Dimethyl 13,18-diethy1-3,7,l3-l7-tetramethyl-lZ-porphinone-Z,8- diacetate (39')... Yield (from 37): 90%; MS found: m/e 583.2925 for (M+H)*, C34839N405 requires m/e 583.2923; MIR 6 0.44 (3H, t, l3-CH2C113), 1.82 (3H, t, 18-CH2CH3), 2.06 (3H, s, l3-CH3), 2.75 (2H, q, 139CH2CH3), 3.40 3.47, 3.51 (3H each, s, ring Me), 3.71, 3.80 (3H each, s, 2xCC02Me), 4.00 (2H, q, 18-CH2CH3), 4.78, 4.94 (2H each, s, CH2C02), 9.08 (1H, s, 15-H), 9.74, 9.80 (1H each, s, 10,20—H), 9.79 (1H, s, 5—H), -2.88, -2.73 (111 each, br s, NH); UV-vis Lax (m) 638 nm (26 400), 582 (5 000), 546 (10 600), 508 (8 200), 490 (5 500), 406 (151 000). Dimethyl 12,18-diethyl-2,7,13,17-tetramethyl-S—porphinone—Z,8- diacetate (40) Yield (from 38): 30%; MS found: ‘ m/e 583.2930 for (M+H)*, C341b9N405 requires m/e 583.2923; mu 6 1.79 (6H, t, 2xCthHs), 1.96 (3H, s, 2-Me), 2.90 (3H, s, 2-CC02Me), 3.44, 3.58 (3H each, s, 13,17- Me), 3.62 (3H, s, 7—Me), 3.74 (3H, s, 8—CC02Me), 3.94 (6H,‘m, 2xC1§zCHa and 2-C§2C02), 5.06 (2H, s, 8-CH2002), 9.07 (1H, s, 20-H), 9.82 (1H, s, 4S 15-H), 9.93 (1H, s, 5-H), 9.95 (1H, s, 10-H), -2.96, -2.73 (111 each, br 3, NH); UV-vis Max (In) 642 nm (35 300), 586 (5 400), 541 (9 700), 504 (9 900), 490 (6 400), 402 (162 000). Dimethyl 7,8-dihydroxyf3,8,13,18-tetramethy1-7,17-dipenty1chlorin-2,12- diacetate (42) Slower moving component on TLC: yield 11%; MS found: m/e 685.3961 for (M+H)*, C40HsaN405 requires m/e 685.3968; MIR 6 0.64 (3H, t, 7-CaHsCE), 0.95 (3H, t, l7-C4HsCHa), 1.03 (4H, m, 7—CzH«C§20&CHa), 1.52 (4H, m, l7-CaHsCHzCHa) and 7-CH20H2C3H7), 1.66 (2H, quint, 17- CzHaCHzCsz), 2.16 (2H, m, CH20H203H7), 2.24 (3H, s, 8-Me), 2.38 (2H, m, 7-C_HzC4H9), 3.38, 3.41, 3.49 (3H each, 3, ring Me), 3.73, 3.75 (3H each, s, C02Me), 3.81 (2H, t, l7-CH2C4H9), 4.84 (4H, s, 2xCH2C02), 9.01, 9.06 (18 each, s, 5,10-H), 9.58, 9.70 (1H each, s, 15,20-H), -2.50 (28, br 3, NH); UV-vis in»: (m) 648 nm (46 800), 622 (3 700), 594 (3 700), 546 (1 700), 520 (2 700), 494 (13 400), 490 (13 400), 392 (178 000). Dimethyl 2,3—dihydroxy-3,8,13,lB-tetramethyl-7,17—dipenty1chlorin-2,12- diacetate (43) Faster moving component on TLC: yield 19%; MS found: m/e 685.3959 for (M+H)*, C40H53N405 requires m/e 685.3968; NMR 60.94, 0.95 (3H each, t, CcHeCHa), 1.53 (4H, sext, 2x0:;HsC&CHa), 1.64 (4H, quint, 2140211401120285), 1.95 (3H, s, 3—Me), 2.19 (4H, quint, 2xCHzC1-1203H7), 3.44, 3.47, 3.52 (3H each, 3, ring Me), 3.68, 4.17 (1H each, AB, JAB = 16.5 Hz 2-CH2002), 3.73 (3H, s, 2—CC02Me), 3.89, 3.96 (2H each, t, C§2C4H9), 3.98 (3H, s, l2—CC02Me), 4.86 (2H, s, 12-CH2C02), 9.01, 9.18 (1H each, s, 5,20-H), 9.78, 9.80 (18 each, s, 10,15-H), -2.57, -2.59 (1H each, br 3, NH); UV-vis hex (m) 638 nm (39 900), 610 (2 800), 584 (3 700), 526 (3 600), 498 (12 700), 494 (12 300), 394 (170 000). 46 ZH§:Pihxérpxx:2112-b15(2éhydroxyeth71)f3.8.13.lBttetramethylé7.17a dipentylchlorinw(44) u -.- The dihydroxychlorin 43 (75 mg, 0.11 mol) was dissolved in dry THF (50 m1) and LiAlH4 (21 mg, 0.55 mol) was added carefully. The mixture was stirred at room temperature under argon for 1 h (the progress of the reaction can be monitored by TLC as the product’s Rf value is much smaller than that of the starting material), the reaction was then quenched by addition of 5 ml EtOAc, followed by addition of 5 m1 of water after 5 min. The solvent was removed in vacuo and the residue was redissolved in CH2012, washed with 5% NaOH, than water and dried (anhydrous N82 804). The tetrahydroxychlorin 44 was purified by passing through a short silica gel pad (CH2612/3% MeOH). Yield (62 mg, 90%); MS found: m/e 629.4059 for (M+H)*, 0381153qu requires m/e 629.4070; NMR 6 0.97, 0.98 (3H each, t, C4H8CHQ), 1.56 (4H, m, 2x03150H20H3), 1.68 (4H, m, 2szHaCH202Hs), 1.71 (3H, s, 3-Me), 2.23 (4H, m, 2xCH2CH203H-r), 2.28, 2.38 (H each, m, 2-CH20H20), 3.12, 3.18 (IH each, m, 2-CHzCH20), 2.79, 3.36, 3.43 (3H each, 8, ring Me), 3.70, 3.85 (2H each, t, 12—CH2CH20), 3.95 (4H, t, 2xCHzC4H9), 8.47, 8.93 (1H each, s, 5,20-H), 9.13, 9.45 (1H each, s, 10,15-H), -2.86 (2H, br s, NH); UV- vis hax (an) 640 nm (34 600), 614 (3 100), 588 (3 200), 524 (2 700), 496 (10 600), 492 (10 400), 392 (151 000). Dimethyl 3.9,13.18“:etramet8817811124i298Eyl-7-porphinone: 2.12-diacetater(45) Yield (from 42) 90%; MS found: m/e 667.3871 for (M+H)*, CcoHanOs requires m/e 667.3862; NMR 6 0.50 (3H, t, 8-CqHsC113), 0.86 (211, m, 8-C3H5CH2CH3), 0.95 (3H, t, 17-CqfiaCH3), 1.67 (4H, m, 17- Cal-IsCljoI-Ia and 8--CH2CI{203H'7), 1.70 (2H, quint, l7-C2340H202Hs), 2.05 47 (3H, s, 8-Me), 2.18 (2H, quint, l7-CH2CH2C3H7), 2.71 (2H, t, 8-CH204H9), 3.41, 3.56, 3.62 (3H each, 8, ring Me), 3.75 (6H, s, 2xC02Me), 3.82 (2H, t, 17—CH2C4H9), 4.98, 5.00 (2H each, s, CH2C02), 9.18 (1H, s, lO-H), 9.80, 9.84 (1H each, s, 5,15-H), 9.89 (1H, s, 20-H), -2.79, -1.84 (1H each, br 8, NH); UV-vis hex (an) 646 nm (39 000), 588 (5 100), 540 (9 700), 504 (10 200), 486 (6 100), 402 (172 000). Dimethyl 2,8,13,lB—tefiramethy1-7,17—dipentyl-3-porphinone— 2,12-diacetate (46) Yield (from 43) 31%; MS found: m/e 667.3867 for (M+H)*, 640115114405 requires m/e 667.3862; NMR 6 0.97 (6H, t, 2x04115039), 1.53 (4H, m, 2xCaHsC§CH3), 1.70 (4H, m, 2x02H40H2C2H5.), 1.96 (3H, s, 2-Me), 2.23 (4H, m 2xCH2CH203H7), 2.93 (3H, s, 2-CC02Me), 3.51, 3.52, 3.59 (3H each, s, ring Me), 3.75 (3H, s, 12—CC02Me), 4.01 (4H, t, 2xC_HzCaH9), 3.89, 3.98 (1H each, AB, Jxa = 16.8 Hz, 2-CH2C02), 4.92 (2H, s, 12- CH2C02), 9.08 (1H, s, 20-H), 9.87 (2H, s, 5,15-H), 9.98 (1H, s, 10-H), -3.00, ~2.92 (1H each, br s, NH); UV-vis hax (5x) 640 nm (30 500), 584 (5 500), 548 (10 800), 510 (8 400), 490 (5 400), 406 (160 000). 3,12-Bis(2-hydroxyethy1)—3,8,13,18—tetramethyl~7,17-dipenty1- 2-porphinone (47) Yield (from 44) 80%; MS found: m/e 611.3973 for (M+H)*, C38851N403 requires m/e 611.3964; MIR 6 0.93, 0.95 (3H each, t, 04110033), 1.56 (4H, m, 2x03H5CH20H3), 1.65 (4H, m, 2szH4C_H202Hs), 2.09 (3H, s, 3-Me), 2.22 (4H, m, 2xCH20112C3H-7), 2.98, 3.10, 3.32, 3.48 (1H each, m, 3-CH2CH20), 3.51, 3.56, 3.59 (3H each, 8, ring Me), 3.97, 4.03, 4.19, 4.42 (2H each, t, 2xCH2C4H9 and 12—CH2CH20), —2.98, -2.89 (1H each, br s, NH); UV—vis hex (m) 640 nm (30 900), 584 (5 600), 548 (9 900), 508 (8 200), 490 (6 000), 406 (152 000). 48 T9§F§W§§h¥1m227112:17*tgtramethY1T3’P9FPh190995278:13:18f 19£E§B§9219H§Pee(491 Osmium tetroxide (52 mg, 0.21 mmol) in ether (0.5 ml) was added to coproporphyrin I tetramethyl ester, 48 (100 mg, 0.14 mmol) in dry CH2C12 (50 m1) followed by dry pyridine 0.1 ml. The reaction was allowed to proceed in the dark for 26 h and worked up in the same manner as described before. Yield: unreacted porphyrin (31 mg, 31%), vip- dihydroxycoprochlorin I tetramethylester (42 mg, 40%). The latter was dissolved in 20 m1 CH2012 containing 0.5 ml of concentrated sulfuric acid. The mixture was allowed to stir at room temperature for 30 min before being extracted with water (3 x 20 ml each). The CH2C12 layer was dried (N82 804) and the solvent was removed under reduced pressure. Recrystallization from CH2C12/CH30H gave pure 49 (35 mg, 85%) as purple crystals. MS found: m/e 727.3338 for (M+H)*, C40387N409 requires m/e 727.3345; NMR 6 1.50 (2H, m, 2-CH2CH2002), 2.09 (3H, s, 2-Me), 3.10 (2H, m, 2—CH20H2002), 3.22 (6H, m, 3xcnzcgzcoa), 3.25, 3.49, 3.58 (3H each, 3, ring Me), 3.63, 3.65, 3.67, 3.69 (3H each, s, COzMe), 4.23, 4.34, 4.38 (2H each, t, CH2CH2002), 9.18 (1H, s, 20-H), 9.83 (1H,. s, 5-H), 9.85, 9.94 (1H each, s, 10,15—H), -2.88, -2.98 (1H each, br s, NH). Irradiation at 6 4.23 caused the signal at 6 9.85 to increase in intensity by 5%, while irradiation at 6 4.36 caused the signals at 6 9.18 and 6 9.94 to increase in intensity by 8%; had the rearrangement gone the other direction NOE should never occur at 6 9.18. UV-vis )nax (an) 642 nm (31 900), 614 (2 200), 584 (5 800), 544 (11 800), 508 (9 700), 490 (6 400), 406 (165 000). 49 t-Butyl Z-acetOXYmePhY1:379thxltfifPfithyli§72¥§§91§t9§§99¥¥l§§91(55) t-Butyl 3--ethyl--2,4-dimethyl-5-pyrrole—carboxy1ate$2 (54) (20 g, 0.09 mol) was dissolved in glacial acetic acid (100 ml) and acetic anhydride (10 ml). This solution was added to lead tetraacetate (47.68 g, 0.11 mol) and heated with stirring under nitrogen at 70°C for 15 min. The solution was then diluted with water until a precipitate formed. The product was isolated by filtration and purified by recrystallization from methanol. Yield (23 g, 91%); m.p. 96—98°C; MS m/e 281 (M‘); [MR 6 1.1 (3H, t, CHzCHs), 1.6 (911, s, t-Bu), 2.0, 2.2 (3H each, s, arom. Me and OAc), 2.4 (2H, q, CHcha), 5.0 (2H, s, CH20), 8.9 (1H, br 3, NH). Ethyl 3’,4—diethyl—3,4’-dimethyl-5’-t-butoxycarbonyl-dipyrromethane-5: carboxylate (57) A suspension of ethyl 3-ethyl-4---methyl-2-pyrrol'e-carboxylate53 (56) (7.73 g, 43 mmol) and the foregoing pyrrole (55) (12 g, 43 mmol) in methanol (300 ml) was treated with toluene-p-sulfonic acid. hydrate (350 mg) and heated with stirring under argon at 40°C for 5 h. The homogenized mixture was then partitioned between water (300 ml) and methylene chloride (400 ml) and the organic layer was dried (NeaSO4) and evaporated to dryness. The product was used in the next steps without further purification; MS m/e 403 (M*); NMR 6 1.0, 1.1 (3H each, t, CH:C1_-_b), 1.3. (3H, t, C02CH20H3), 1.6 (9H, s, t-Bu), 1.9, 2.2 (3H each, s, Me), 2.4, 2.7 (2H each, q, 01120113), 3.8 (2H, s, methane CH2), 4.3 (211, q, CO:CH2CH:1), 8.5 (2H, br 3, NH). Methyl 7,12—diethy173,8,l3,l7,18—pentamethylporphine-Z-propionate (50) Dipyrromethane 57 (3g, 7.5 mmol) was treated with trifluoro-acetic acid (20 ml) under argon atmosphere at ambient temperature for 5 min. A solution of formyl pyrrole 58!M (1.03 g, 7.5-01) in methanol (100 m1) 50 was then added all at once and the dark red solution was stirred for an additional 90 min, followed by addition of a 30% HBr-CHsCOOH solution (1 m1) and ether (200 ml). Continued stirring for 15 min resulted in the formation of reddish-orange crystals which were collected by filtration and washed thoroughly with ether (1.5 g). The mother liquor was concentrated to approximately 100 ml, and ether (200 ml) was added to give a second crop of the product (0.5 g). Overall yield 53%; MS m/e 421 (M* - HBr). This tripyrrin 59 (2 g, 4 mmol) was stirred in a mixture of 48% HBr (30 m1), acetic acid (20 ml) and trifluoroacetic acid (30 m1) under N2, at 65-70°C for 6 h. A solution of formylpyrrole 50“ (0.84 g, 4 mmol) in methanol (100 ml) was then added all at once and stirring was continued for 1 h at room temperature. The reaction mixture was evaporated under reduced pressure and redissolved in dry DMF (100 ml) containing copper (II) chloride (10 g). The solution was stirred for 8 h at room temperature under argon. The reaction mixture was then poured into water and extracted with methylene chloride. The organic layer was then dried (N82 804) and evaporated to dryness, followed by column chromatography (silica gel - CHzClz). The red eluants were evaporated to dryness and the residue was treated with concentrated H2804 (30 ml) in order to demetalate the copper-porphyrin. The mixture was then partitioned carefully between CH2012 and water. The organic layer was washed with water (2 x 50 ml), saturated NaHC03 (2 x 50 m1), dried (N82804) and evaporated to dryness. This crude product was further purified by column chromatography (silica gel-1% CH30H/CHzClz) and recrystallized from CH2012/CH30H to give purple crystals. Overall yield (from dipyrromethane 5.7): 324 mg, 8%; m.p. 234-236°C; MS found m/e 523.3085 for (M+H)+ C33H39N402 requires m/e Sl 523.2076; NMR 61.84 (6H, t, 2xCH2CH3), 3.24 (2H, t, 01120112002), ring Me: 3.55 (3H, s), 3.56 (6H, s), 3.57 (3H, s), 3.60 (3H, s), 3.67 (3H, s, 002Me), 4.03, 4.06 (2H each, q, CH2CH3), 4.35 (2H, t, CH2CH2002), 9.96, 9.98 (1H each, s, meso), 10.03 (2H, s, meso), -3.85 (2H, br 3, NH); UV-vis hax (an) 619.5 nm (5 600), 566 (7 700), 530.5 (11 000), 497 (14 700), 396.5 (166 000). Methyl 7,12-diethyle3,8,13,17,17-pentamethy1-18-porphinone-2-propionate (51) and Methyl 57:,ji'1'2f4d'iieitihy,l-3.8,713,18,lB—pentamethyl-l7-porphinone—2- propionate (52) Osmium tetroxide (90 mg, 0.35 mmol) in ether (0.9 ml) was added to porphyrin 50 (150 mg, 0.29 mol) in dry CH2012 (50 m1) followed by dry pyridine 0.1 ml. The reaction was allowed to proceed in the dark for 18 h and worked up in the same manner as described before. The reaction mixture was chromatographed on TLC plates, developed with CH2C12/1% CHaOH. There were four distinct bands: the recovered red porphyrin 50 moving at the front (30 mg, 20%), followed by ring C dihydroxychlorin (6 mg, 4%), mixture of ring A and B dihydroxychlorins (46 mg, 29%) and another green band containing ring D dihydroxychlorin (21 mg, 13%). The structure assignments were based on 1H NMR data: the mixture of A and B dihydroxychlorins had 6 0.61 (3H, t, pyrroline Et), ring C dihydroxy— chlorin had 6 2.05-2.58 (4H, m, pyrroline CH2082002) and ring D dihydroxychlorin had 2.13 (6H, s, 2 x pyrroline Me). The letter (20 mg, 36 mol) in CH2C12 was treated with perchloric acid (70%, 0.5 m1) and the reaction was worked up in the same manner as described for 21a, 22a to afford 8 mg of each (41%) of the isomeric porphyrinones. 7 51 (most polar band on TLC): MIR 6 1.78, 1.82 (3H each, t, CH:C_H3), 2.08 (6H, s, 17,17—Me), 3.23 (2H, t, CH20H2C02), 3.44, 3.54, 3.60 (3H each, 3, ring Me), 3.74 (3H, s, C02Me), 3.88, 4.04 (2H each, q, 52 CH2CH3), 4.35 (2H, t, CH20H2COz), 9.12 (1H, s, 15-H), 9.78 (1H, s, 10- H), 9.81 (1H, s, 20—H), 9.87 (1H, s, 5—H); -2.81, —2.96 (1H each, br 3, NH). Irradiating the triplet at 6 4.35 caused the singlet at 6 9.81 to increase in intensity by 12%. 52 (faster moving component on TLC): NMR 6 1.78, 1.81 (3H each, t, 01120113), 2.09 (6H, s, 18,18-Me), 3.21 (2H, t, CH20§2002), 3.44, 3.55, 3.61 (3H each, s, ring Me), 3.65 (3H, s, COzMe), 3.88, 4.03 (2H each, q, CH2CHb), 4.34 (2H, t, CH2CH2CO2), 9.15 (1H, s, 20-H), 9.81, 9.84, 9.89 (1H, s, 5,10,15—H), -2.93, -3.00 (1H each, br s, NH). Irradiating the triplet at 6 4.34 caused the singlet at 6 9.15 to increase in intensity by 5%. Dimethyl l2,13-dihydroxy—3,8,8,12,13,17-hexamethy1-7-porphinone- 2,18-dipropionate (68) To the porphyrinone 21a (500 mg, 0.86 mmol) in CHCls (200 ml) is added a saturated solution of zinc acetate in methanol (3 ml). After 15 min refluxing (the reaction can be monitored by TTC as the Rf value of the green-colored product is smaller than that of, the purple-colored starting material), the mixture was concentrated, diluted with a little methanol, and after cooling the zinc complex is filtered off .in virtually quantitative yield. To a solution of 68 (500 mg, 0.77 mmol) in CH2012 (250 m1), osmium tetroxide (295 mg, 1.16 mmol) and pyridine (0.3 ml) were added and the reaction was allowed to proceed in the dark, under argon for 36 h at 23°C. The reaction mixture was worked up in the same manner as described for 16a to give after separation on silica gel column (CH2012/1% MeOH, then CH2C12/2% MeOH): unreacted starting material, 200 mg, 40% and a green zinc dihydroxy-porphyrinone, 250 mg, 47%. A S3 methylene chloride solution of the latter was completely demetalated, by shaking with 10% HCl in a separtory funnel, to give the violet 68 as the exclusive isomer. M.p. 216-219°C; NMR 6 1.68, 1.71, 1.76, 1.99 (3H each, s, pyrroline Me), 2.84, 2.93 (3H each, 9, ring Me), 2.87 (4H, t, 2xCHzCHzCOz), 3.59, 3.62 (3H each, s, C02Me), 3.67, 3.73 (2H each, t, CH201h002), 7.42 (1H, s, lO-H), 7.78 (1H, s, l5-H), 8.56 (1H, s, 5-H), 8.66 (1H, s, ZO-H); UV-vis hex (an) 642 nm (7 500), 589 (13 100), 583 (10 000), 547 (10 000), 512 (6 600), 429 (18 100), 386 (57 800). Dimethyl 3,8,8,l3,13,17fhexamethyl—7,lZ-porphinedione-Z,18- dipropionate (69) The foregoing dihydroxyporphyrinone 68 (200 mg, 0.32 mol) was dissolved in 5 ml of concentrated H2804 and the reaction mixture was allowed to stir at room temperature for 5 min before the careful addition of CHaOH (20 ml). The solution was then partitioned between CH2012 (100 ml) and water (100 ml). The organic layer was separated, washed with saturated NaHCO:3 (100 ml), water (100 ml), dried (N82804) and evaporated to dryness, to give after recrystallization from CH2012/CH30H the green colored 69 in almost quantitative yield. M.p. 293—295°C; MIR 6 1.93, 1.97 (6H each, s, gem—Me), 3.12, 3.13 (2 H each, t, 011201112002), 3.28, 3.33, (3H each, 3, ring Me), 3.59, 3.62 (3H each, s, 002Me), 4.17, 4.18 (2H each, t, CH2CH2002), 8.51 (1H, s, 15-H), 8.74 (1H, s, lO-H), 9.33 (1H, s, 5-H), 9.58 (1H, s, 20-H), -0.32 (2H, br s, NH); UV—vis hex (an) 638 nm (15 500), 592 (14 400), 584 (14 800), 540 (9 700), 436 (91 300), 415 (88 600), 400 (74 900); MS found: m/e 599.2865 for (M+H)*, C34H39N405 requires m/e 599.2872. 54 94891821117.lfitéihydr952731818yl§r131lYtbsramethx1:1112:89§8919991909: 2.18-dipropi°n§t§-(39l o. ... .....- Osmimm tetroxide (115 mg, 0.45 mmol) and pyridine (0.4 ml) were added to a dichloromethane solution of 69 (150 mg, 0.25 mmol) and the reaction was allowed to proceed under argon at room temperature for 36 h. The reaction mixture was worked up in the same manner as described for 168. The products isolated from the silica gel column were the unreacted 69 (65 mg, 43%) followed by the gray 89 (55 mg, 35%). MIR 6 1.57 (6H, s, pyrroline Me), 1.59, 1.71, 1.72 (3H each, s, pyrroline Me), 2.52 - 3.01 (4H, m, 18-CH2CH2002), 2.80 (3H, s, ring Me), 3.52 (2H, t, 2—CH26H2002), 3.62 (3H, s, 18-CCC02Me), 3.74 (3H, s, 2-CCCOzMe), 3.88 (28, t, 2-CH2CH2CO2), 7.12 (1H, s, 15-H), 7.50 (18, s, lO-H), 7.55 (1H, s, 20—H), 8.42 (1H, s, 5-H). Irradiating the singlet at,6 2.80 caused the singlet at 6 8.42 to increase in intensity by 4.4%. UV-vis hex (In) 684 nm (7 200), 656 (8 700), 602 (6 600), 539 (8 100), 505 (6 300), 410 (39 700), 382 (47 800). Methyl 18-[2-(methoxycarbonyl) ethenyl]-3,8,8,13,l3,l7-hexamethyl-7,12- porphinedione-2-propionate (70) Compound 89 (50 mg, 0.08 mmol) was heated to reflux in benzene (25 ml), followed by the gradual addition of 5 drops of concentrated 1101. After 20 min, water (20 ml) was added, the organic layer was separated, washed two -more times with water (20 ml each), dried (NeaSOu), evaporated to dryness and recrystallized from CH2012/MeOH to give the green 70 in quantitative yield. M.p. 337-340°C, NMR 6 1.87, 1.94 (6H each, s, gem-Me), 3.04 (2H, t, CH20§2C02), 3.22, 3.32 (3H each, 3, ring Me), 3.62, 4.00 (3H each, s, C02Me), 4.03 (3H, t, CH20H2C02), 6.88, 8.94 (1H each, d, acrylic, Jen = 16.1 Hz), 8.31 (1H, s, 15-H), 8.48 (1H, s, lO-H), 9.15 (1H, s, 5-H), 9.39 (1H, s, 20-H), 0.67 (2H, br 3, NH); UV- SS vis (CHCla) hex (an) 661 nm (15 400), 611 (25 000), 568 (14 000), 446 (66 000), 423 (93 000); MS found: m/e 597.2721 for (M+H)* C34H37N405 requires m/e 597.2715. Dimethyl 12,l3-dihydroxy-3,7,7,12,13,l7-hexamethyl-8—porphinone-2,18- dipropionate (72) and Dimeghy1“2,3-dihydroxy-3,7,7,12,13,17-hexaEEthyl- 8-porphinone-2,18-dipropionate (73) Prepared in the same way as described for yield: unreacted starting material 71 (40%), green 72 (15%), and violet 73 (20%). 72 (slower moving component on TLC): NMR 6 1.86, 1.90, 2.02, 2.05 (3H each, s, pyrroline Me), 2.94, 3.03 (2H each, t, CH20§2C02), 3.13, 3.23 (3H each, s, ring Me), 3.57, 3.58 (3H each, s, 002Me), 3.93, 4.04 (2H each, t, CH2CHzCOz), 8.45 (1H, s, 5-H), 8.60 (1H, s, l5-H), 8.74 (1H, s, lO-H), 9.28 (1H, s, 20-H); UV-vis hex (an) 634 nm (15 700), 593 (14 000), 520 (6 200), 431 (92 000), 406 (104 000). 64 (Faster moving component on TLC): NMR 6 1.70, 1.71, 1.86 (3H each, s, pyrroline Me), 1.78 (2H, t, 24112012002), 2.64 (2H, t, 2- CthHzCOz), 2.86 (2H, t, 18-CH20H2002), 2.92, 2.94, 2.98 (3H each, 8, ring Me), 3.59 (2H, t, 18—CH20HzCOz), “3.64, 3.68 (3H each, s, 002Me), 7.43 (1H, s, 5-H), 7.78 (1H, s, 20-H), 8.59 (1H, s, lO-H), 8.67 (1H, s, l5-H); UV-vis hex (m) 640 nm (8 800), 589 (21 000), 546 (13 200), 412 (31 300), 385 (71 000), 376 (73 000). Dimethyl 3,7,7,13,13,l7—hexamethyl-8,12-porphinedione-2,18: dipropionate (74) Obtained from the pinacolic rearrangement of 72, the same way as described for 69, and separated from the faster moving isomer 69 by TLC plates using l-2% caeon/cnzclz. The yield of each isomer was 45%. The yellow-green porphyrindione 74 was further purified by recrystallization from CH2C12/MeOH. M.p. 295—297.5oc; mm a 1.99 (12H, 3, gem-Me), 3.19 S6 (4H, t, 2XCH2CH2002), 3.45 (6H, s, ring Me), 3.59 (6H, s, 2xC02Me), 4.33 (4H, t, 2xC_H2CH2C02), 8.91 (1H each, s, 5,15-H), 9.65 (1H, s, lO-H), 9.89 (1H, s, 20-H), -1.73 (2H, br 3, NH). Irradiating the triplet at 6 4.33 caused the singlet at 6 9.89 to increase in intensity by 9.4%. UV" vis hex (as) 634 nm (15 300), 620 (21 400), 590 (9 400), 577 (8 200), 432 (116 000), 411 (136 000); MS found: m/e 599.2881 for (M+H)*, CseksNaOe requires m/e 599.2872. Dimethyl 2,7,7,12,13,l7—hexamethy1-3,8-porphinedione-2,18- dipropionate (75) Obtained from the pinacolic rearrangement of 73 in the same manner as described for 69, except that the reaction time was 30 min. The products isolated from the TLC plates, (CH2C12/1-2% MeOH), were the faster moving porphyrinone 228 (42%) and the slower moving green porphyrindione 75 (42%). [Note: if the spirolactone of 73 is employed for the pinacolic rearrangement, porphyrinone 22a is the only product obtained.] MAR 6 1.95 (3H, s, 2-Me), 1.96 (6H, s, 7,7-Me), 1.74, 2.15 (1H each, m, 2—CH2_CH2002), 2.94 (2H, t, 2-CH20H2C02), 3.08 (2H, t, 18- 0120112002), 3.32, 3.37, 3.40 (3H each, 3, ring Me), 3.44 (3H, s, 2- CCCOzMe), 3.62 (3H, s, 18—C0002Me), 4.09 (2H, t, lHHzCHzCOz), 8.64 (1H, s, 20-H), 8.83 (1H, s, 5-H), 9.40 (1H, s, lO-H), 9.55 (1H, s, 15- H), -O.69 (2H, br 8, NH); UV-vis hex (ad) 634 nm (15 600), 591 (13 000), 582 (12 200), 542 (7 800), 435 (76 500), 4-13 (83 200), 400 (72 500); MS found: m/e 599.2863 for (M+H)*, C34H39N405 requires m/e 599. 2872. 57 .819:9}!1898935899t99thY,19h19§.i.8......( .792). To.a solution of OEP55 (1.168 g, 2.2 mmol) in CH2C12 (250 ml) and pyridine (1 ml) was added osmium tetroxide (1.0 g, 3.9 mmol) in diethyl ether (10 ml). The mixture was allowed to stir at room temperature in the dark for two days. This mixture was diluted with methanol (50 ml) and was bubbled with H28 for 15 min. The precipitated osmium sulfide was recovered by filtration, and the solvent was evaporated. The residue was triturated with methanol, which dissolved most of the diol chlorin from unreacted OEP. The solution was filtered, and the product was further purified on a silica gel column, eluting: with. CH2Clz containing 0.5% of methanol: yield 827 mg (66.6%), plus unreacted OEP [201 mg (17.1%)]; m.p. 213-214°C; NMR 6 0.96 (6H, t, yrroline Et), 1.74 (18H, t, St), 2.55 (4H, q, pyrroline Et), 3.38 (2H, s, OH), 3.79, 3.82, 3.91 (12H, q, Et), 9.00 (2H, s, 5,20—H), 9.68 (2H, s, 10,15-H), -2.68 (28, br s, NH); UV—vis 2.... (or) 543 a. (54 000), 590.5 (9 700), 523.5 (8 700), 496 (19 900), 392 (206 000); MS, found m/e 569.3887 for (M+H)*, CaeroNh02 requires m/e 569.3858. The dihydroxylation of OEP with OsOe was found to proceed poorly under catalytic conditions, e.g., using Ne methylmorpholine oxide.5° 3,7,12,13,17,18-Heptaethyl-2-(1—hydroxyethyl)-porphine (77) Biol 76 (20 mg, 0.035 mmol) was heated in a mixture consisting of dioxane (6 ml), water (3.5 ml), and concentrated HCl (0.5 ml) on a steam bath for 30 min. The mixture was partitioned in CH2C12 and water; the organic layer was evaporated and the residue was separated on TLC plate with CH2C12 as solvent. The major product 77 was further crystallized from CfleClz/hexane: yield 9.7 mg (49%); NMR 6 1.90, 1.91 (21H, t, Et), 2.34 (3H, d, CH(0H)CH3), 2.78 (1H, br s, OH), 4.06, 4.12 (14H, q, 8t), 58 6.56 (1H, q, CH(OH)CH3), 10.08 (2H, s, meso), 10.10, 10.62 (1H each, s, meso), -3.73 (2H, br 3, NH); UV-vis hex (04) 620.5 nm, 566.5, 533, 500, 400; MS, found m/e 551.3726 for (M-O-H)*, C35H47N40 requires m/e 551.3753. 317.8. 12. 13, 17. 13""??1391!!!1T2:(-1..7993?9533.9}.13.2.1.)32951?hi§3-fl§1 Biol 76 (20 mg, 0.035 mmol) was heated in glacial acetic acid (5 m1) at 90°C for 10 min. The reaction mixture was partitioned in 082012 and water; the organic layer was separated and evaporated. The residue was recrystallized from CH2012/MeOH: yield 17.8 mg (85%) [note: upon purification on TLC plates using CH2C12/MeOH, 78 was largely converted to the methoxide 79. Silica gel promoted solvolysis is a facile reaction for acetylated hematoporphyrins.57]; MIR 6 1.95 (21H, t, St), 2.30 (3H, s, acetyl), 2.41 (3H, d, CHCHa), 4.09, 4.16,, 4.20 (14H, 9, St), 7.53 (1H, q, CHCHa), 10.12 (2H, s, meso), 10.16, 10.52 (1H each, s, meso), -3.70 (2H, br 3, NH); UV-vis hex (an) 620 nm (9 400), 567 (12 000), 536 (15 300), 500 (17 300), 401 (156 000); 16, found m/e 593.3873 for (Md-HP, C38H49N402 requires m/e 593.3858. 3,7,8,12,l3,17,lB-Heptaethyl-Z-(l-methoxyethyl)-porphine (79) Biol 76 (20 mg) was heated to reflux for 1 h in methanol (20 ml) with one drop of concentrated HCl. The solvent was then evaporated, and the residue was crystallized from CH2012/MeOH: yield 15.4 mg (77.5%); m.p. 243-245°C; NMR 6 1.93 (21H, t, Et), 2.32 (3H, d, CHCHa), 3.62 (3H, s, We), 4.08, 4.14 (14H, q, Et), 5.96 (1H, q, CHCHa), 10.10 (2H, s, meso), 10.12, 10.66 (1H each, s, meso), -3.70 (2H, br s, NH); UV-vis hex (on) 620.5 nm (9 200), 566.5 (11 500), 533 (14 700), 499 (18 300), 399.5 (172 000); MS, found m/e 565.3888 for (M+H)*, 037H49N40 requires 565.3909. 59 3.7.8.12.13.17.18-Heptaethy172:839812952h1991(39) Biol 76 (100 mg, 0.175 mmol) was heated to reflux in benzene (25 ml) containing five drops of concentrated HCl for 3 h. The solvent was evaporated, and the residue was crystallized from CH2C12/MeOH: yield 85 mg (90%); m.p. > 300°C; BMR 6 1.91, 1.93 (21H, t, St), 4.09, 4.16, 4.22 (14H, q, 8t), 6.16, 6.38 (2H, dd, CH=C.H2), 8.25, 8.31 (1H, dd, CH=CH2), 10.12 (2H, s, meso), 10.16, 10.28 (1H each, s, meso), -3.68 (2H, br s, NH); UV—vis hex (on) 623.5 nm (10 000), 569.5 (14 700), 539 (19 300), 503 (20 000), 402.5 (179 000); MS, found m/e 533.3641 for (M+H)*, CasHesNe requires m/e 533.3647. 2, 3 , 7, 8, 12, 13, l7-Heptaethylporphine (81) Solid vinylporphyrin 80 (50 mg, 0.113 mmol) was mixed and ground with resorcinol (240 mg, 2.18 mmol) in a mortar. The powder placed in a test tube was swirled over a flame until boiling. The mixture was cooled momentarily and then heated again to boiling. This cycle was r‘epeated three times, and the residue, after cooling, was extracted with 0112012 and water. The porphyrin in the organic phase was purified by cr‘ystallization from CH2012/MeOH: yield 51 mg (89%); m.p. 236-23900; M48 5 1.98 (15H, t, St), 2.10 (58, t, Et), 4.15 (108, q, St), 4.30 (48, 9. Et), 9.16 (1H, s, 8-H), 10.11, 10.16, 10.18, 10.20 (1H each, s, lieso); UV—vis hex (In) 618.5 nm (9 700), 565 (13 000), 531.5 (16 300), 498 (21 000), 398.5 (215 000); MS, found m/e 507.3457 for (M+H)*, Caeneane requires m/e 507.3491. gsggtions of vjcjpjhydroxyetiochlorin I (82) Biol 82 was heated in dioxane/aqueous HCl in the same manner as described above. The overall yield for the two etioporphyrin alcohols 60 was about 50%, with the ratio of 83 to 84 being near 2:1. If the diol 82 was heated in acetic acid, the acetoxy porphyrin 85 was obtained in 67% yield and 86 in 11% yield (6:1 ratio). These ratios can be observed directly by using NMR peaks of CH20R vs. CH(OR)Me: 6 6.10 (s, 83):6.52 (q, 84) = 4:1; 6.61 (s, 85):7.55 (q, 86) = 12:1. Dimethyl 7,8,12,13-tetraethyl-3:(hydroxymethyl)-17~methyl-2,18- porphinedipropionate (87) Biol 17b20 (40 mg) was heated in dioxane (10 m1)/aqueous HCl (10%, 4 m1) on a steam bath overnight. The solvent was evaporated, and the residue was esterified in MeOH/HzSOe. The principal product isolated from TLC plates, was 87: 9.8 mg (~25% yield); m.p. 196-198°C; MR 6 _ 1.90 (12H, t, St), 3.26 (4H, t, 0820112002), 3.49 (3H, s, CH3), 3.61, 3.55 (3H each, s, 002Me), 4.05, 4.11 (8H, q, 8t), 4.42, 4.45 (28 each, t, 082082002), 5.11 (28, s, CHzOH), 10.05 (2H, s, meso), 10.08, 10.28 (1H each, s, meso), -3.78 (2H, br s, NH); UV-vis hex (an) 621.5 I, 567, 535.5, 499.5, 402; MS, found m/e 639.3521 for (M+H)*, 038114711405 requires m/e 639.3549. The alternative alcohol 88 was not detected, but a small amount of acrylic porphyrin [~5% yield; NMR 6 7.05, 7.11, 9.28, 9.36, acrylic] was isolated, which can only result from 88. CHAPTERZ SYNTHESIS OF THE m _d PMSTHBTIC GmUP 0F BACTERIAL TEMINAL OXIDASE I. INTRODUCTION achegggg 9011, like many other microbes, have a branched respiratory system with two terminal oxidases, cytochrome g and cytochrome g, which- catalyze the reduction of 02 to H20.“ Both cytochrome d and 9 complexes contain two polypeptides. Moreover, the cytochrome d complex contains two cytochrome 51 centers (h-e £1), one cytochrome 9595 center (high-spin protoheme IX), and one cytochrome hsss center (low-spin protoheme IX); whereas the cytochrome 9 complex contains one cytochrome bssz and one cytochrome g, which is again a 13- type cytochrome. Cytochrome g prevails in the early exponential phase during aerobic growth of culture while cytochrome d becomes important in the late exponential phase, or when cells are grown under limiting oxygen supply.°°'61 The Kn value for oxygen of cytochrome d is about eight times lower than that of cytochrome 9.59 The more efficient utilization of oxygen by cytochrome d presumably allows the microbes to maintain efficient oxidative energy conservation over a wide range of oxygen pressures by changing the relative ratio of the two oxidases (Figure 4). 61 62 Cyt b562 ——> CYt O —-> 02 (Hioh oz) (125 mV) c3" b555 —> 0 (-45 mV) . \ CYt bsss —>Cyt d —> 02 (Low 02) (10 mV) (240 mV) Figure 4. Arrangement of cytochromes in the respiratory .......n..e ..... aerobic growth. The 8111’ values are- indicated in parentheses. Cyt, cytochrome; Q, ubiquinone-B. The oxygen binding site in the membrane-bound cytochrome (1 complex is a green-colored heme prosthetic group displaying a prominent m—band near 630 nm. Keilin62 originally designated the name ”g2” for this cytochrome absorbing in the red region; a; was later changed to d to avoid confusion with age hemes of mammalian cytochrome oxidase. It should be made clear that cytochrome d does not reduce nitrite; it is ..... different from the soluble Edi—type oxidases74°° from Pseudomonas aeruginosa and faracoccug denitrificans, which primarily function as nitrite reductase and are not components of the aerobic respiratory chain. The green heme moiety of cytochrome 51 was first studied by Barrett (1956) who extracted cells of Aerobacter aerogenes and identified an iron chlorin core structure 91.32 The site of saturation and the nature of the side chains on the chlorin macrocycle could not be determined at that time. Barrett suggested the possible presence of vinyl, 63 hydroxyethyl, and propanoic acid substituents in an arrangement similar to a hydrated protoporphyrin IX. The tentatively formulated structure of Barrett remained unchallenged in the literature for almost 30 years and has served as the de facto model for many other green hemes“ subsequently found in various bacterial cytochromes. Recently, Timkovich and collaborators° isolated sufficient amounts of the heme g prosthetic group from purified E 9911 oxidase and characterized the structure of the metal-free, esterified chromophore by means of 1H NMR, IR, UV-vis, and mass spectroscopy. The pr0posed structure comprises an unusual chlorin core with a spiro—‘Y—lactone group at the saturated pyrrole ring C (92). This "lactochlorin" structure is certainly unique but as the authors noted it is not clear whether the lactone ring found in the metal-free macrocycle is an authentic feature of the heme, or whether it is an artifact formed during isolation. The evidence of the Thlactone in the metal-free heme g was largely based on an intense IR absorption at ~1782 cm-l. In a recent paper, however, the Timkovich group reported that this IR peak was not present in the extracted heme d,°5 thus supporting structure 6. ou __, I I H OH H OH CO H Ho,c CO,H Meo,c H026 2 91 92 5 64 Considering the limitation imposed by the scarcity of natural material, we believe that a full-scale study of heme d as well as the confirmation of the proposed structure must rely upon organic syntheses. In this chapter, we report the total synthesis of Timkovich’s "lactochlorin", its diastereomer, and the non—lactonized forms. The chemical reactivities inherent in these structures are also addressed. II. RESULTS AND DISCUSSION A. H.999..1.--§£H§i§3 In the first chapter, we examined several gig-dihydroxychlorins and the principal results can be summarized here.2°:43n°°'°7 Dihydroxylation of the porphyrin fl-fl’ double bond can be routinely accomplished by using osmium tetroxide. If the diol chlorin bears a g-inal propionate ester side chain, It is unstable during chromatography, especially on TLC plate. Given enough time, the initially slow moving diol can completely change into a fast moving 7- spirolactone as evidenced by the characteristic strong IR band near 1780 cm-1. It turns out that hydroxychlorins with an angular propionate ester always have a tendency to lactonize into the 5-membered ring under the influence of general base catalysis.“ A variety of bases including silica gel and sodium acetate are effective to bring about this cyclization.' However, preliminary results indicate that lactones resulting from different reagents may not be spectroscopically identical, a difference perhaps attributable to diastereomers.°7 The above 'description can be exemplified by the reaction of a model compound 93 in Scheme 15; the reason for choosing this particular porphyrin will become evident in later discussion. Treatment of 93 with 0304 - H28 yielded a mixture of four isomeric chlorins, 94a - 94d, which HO OH ‘8’ 948 + comm. 94b+ com. HO . -. 'OH HO K5: OH 94d C0361. 94c COgM. 96 trans 95 cis SCHEME l5 66 can be separated by preparative TLC into three green bands. The two faster moving chlorins were characterized by 1H NMR to be the two north diols, 94a and 94b; the site of saturation was unambiguously identified by nuclear Overhauser enhancements (NOE’s), as indicated in Scheme 15. The two south diols, contained in the slow moving TLC band, was difficult to separate directly. Consequently, we recovered the mixture and heated it in CHzClz/MeOH with sodium acetate, and the cyclized 95 was then separated easily from 94d. Lactone 95, which has a strong IR peak at 1780 cm—1, was found to undergo further changes during developing on TLC plates; it slowly converted to another green compound possessing unchanged mass spectral and IR peaks. However, 1H MGR of the . - two (% and 95) were different, which eventually allowed us to deduce the configuration of the two lactone forms (vide infra). The more stable % could also be obtained directly by lactonizing diol 94c under prolonged contact with silica gel. B- exhihee1e-of_"Lactochlorin" The synthetic strategy, which was guided by the results of model compounds, is shown in Schemes 16 and 17. We began with protoporphyrin (97), a probable precursor also in the biosynthesis of heme d. The reactive vinyl groups were protected as the chloroethyl side chains by oxidation with T1(N03)3 to the aldehyde 99,49 followed by reduction to 100 with NaBHe, and then by chlorination with PhCOCl/DMF‘38 to 101, each step giving essentially quantitative yield (literature procedures have been modified). Porphyrin 101 was treated with 1.3 equivalents of 0504 fora day before being; quenched with H28 to effect dihydroxylation at the four possible sites: pyrrole ring A (6.8%), ring B (6.8%), ring C (22%), and ring D (26%). The separation of the north vjcdiols from the M0010 OH Meozc 97 100 67 CO: M9 M00: 0 \ 0H N83 He f C OzMe M2 WOO/0MP Cl 01 M0020 101 02”. SCHEME l6 l H,o+/THF 99 002.40 10.0,: 101 con"! 1 NORTH DIOLS 111.0,: 105 CO.M¢ SCHEME 1'? 69 south was easily accomplished by chromatography. The two south regioisomers were separated by TLC via the lactones. Structural assignments of 1038 and 103b were based on NOE connectivities (key measurements are indicated by structures in Scheme 17). Regeneration of the vinyl side chains was brought about by heating 103a in pyridine-30% KOH,°° during which most of the lactone chlorin was hydrolized but some survived. The hydrolyzed chlorin was methylated with diazomethane to give the cis-diol 106. The dehydrated lactone 104 has all the structural elements of Timkovich’s "lactochlorin" yet it exhibited a 1H NMR spectrum showing discrepancies in the lactone proton region as compared with that of the natural compound. An obvious explanation is . that this compound does not have the correct configuration. Indeed, when this lactone was chromatographed on silica gel repeatedly, the expected isomeric chlorin emerged which was shown by IR and mass spectra to be a Y-lactone; more significant, it exhibited 1H NMR features basically indistinguishable from that of "lactochlorin." Furthermore, the synthetic lactone and the natural compound have identical retention time in HPLC analyses. Based on their NMR spectra discussed below, the lactones 103 and 104 were assigned a cis configuration (with respect to the two oxygen position) while the "lactochlorin" should have a trans Configuration, as suggested by Timkovich. When 107, obtained from 103a by repetitive chromatography on silica gel, was subjected to hydrolysis Under the same reaction conditions as 103a, a different diol form was Obtained. This dial, 108, assigned as trans (based on 1H NMR data) can ‘De lactonized to give once again the trans "lactochlorin" 92. When the regioisomer 103b was subjected to the same reaction Conditions described above, the cis and trans lactones, 105a and 105b, 70 were obtained. These unnatural compounds not only have provided us more variety of this class of chromophores, but may be used for probing the structure-function relationship in the heme protein. C. 1H NMR Spectra and Structure of the Chlorins The two synthetic lactones 104 and 92 have distinctive 1H MHR signatures, particularly in the 6 2.4-3.4 ppm region where the methylene protons of the Yblactone appear. As shown in Figure 5A, at 250 MHz 92 has a spectrum almost identical to the published spectrum of "lactochlorin."° Timkovich concluded the trans configuration based on the assumption that the single proton peaks around 2.4 ppm are from H4 (refer to Figure 7) which points away from the OH group and should have ‘ a chemical shift similar to those of tr-methylene protons of normal pyrroline alkyl substituents. The peaks of H1, because of the large deshielding imparted by the nearby OH oxygen, should occur relatively downfield. The four protons were thus labelled as the following: H1 3.159, 82 3.032, 83 3.231, and 84 2.431. However, these peak assignments and the reported simulation had a large degree of uncertainty due to the presence of overlapping peaks in this region. In fact, on close examination of Figure 5A and the spectra of the natural molecule, as well as several other synthetic analogues,” it becomes evident that there are peaks near 3.5 ppm (at the shoulder of the dominant 18~methyl singlet) which can be nothing but part of the lactone methylenes. This is illustrated by the spectrum of the model lactone 96 whose 4-spin system can be readily assigned and simulated (Figure 5B). Going back to the "lactochlorin" spectrum, we now assign H1 at 3.490 ppm and other parameters as tabulated on Table 1, 'which fit the natural spectrum more closely in terms of relative heights of some of the 71 .A TITTTTI'TTT—YIYTTTIIIIII FITflYITVTVITij1j l... 9.3 3 O I S I I :7 S 7 I 6,5 6 I Tfl fiTTTfir TTTjjTTT 1171 I I I I I‘TWH, 5.5 9.13 3.5 3.0 . 2.5 2.0 rm 8» WM Simulation TWIrTrrvTfiIrTv Figure 5 1H NMR spectra (in CDCla, 250 MHz) of trans- lactone 83 (A); of the hexaethyl trans-lactone 87 with simulation (B). 72 M ... It i111117j7lTj111Tj71] T‘Ujj leTWITTIWWIjT I... 3.8 I.- 1.0 6.- I 250 MHz 888‘8‘88888 888888888 888888818 8 1 l 1 1 5.0 9.fl 3.0 . 2.0 8 Simulation I 1111le 500 MHz Figure 6 1H MIR spectra (in CD013) of cis lactone 95 at 250 MHz (A); at 500 MHz with simulation (B). .oOA caga mmoH mm.uvacmfl ago on» gem flaws: own m« pmaomw manna ma» ham vmumawamm macaw 0:9 .mmcoaowaouflaw owhoaqu may we newumaL0%:oo veamawwzm «:9... 74 resolved transitions and in explaining the weak signals overlapping with the lB-methyl. The spectrum of the lactone isomer cyclized by NaOAc has very different features in the spectral region discussed above (Figure 6A). The most evident difference is that theme are no lactone peaks lower than 3.3 ppm. The complex splitting pattern of the methylene protons is not discernible at 250 MHz but reduced to first-order at 500 MHz (Figure 68). The chemical shifts and parameters are listed in Table l. The lack of large differentiation between H1 and H4 or, for that matter, any two protons in this group suggests that the shielding and deshielding. effect of the OH group is absent or diminished. 'The fact that the chemical shifts are all located at a much downfield region indicates that the four methylene protons must be experiencing uniformly a greater deshielding than in the trans isomer. Using the known patter of isoshielding lines of porphyrin ring current,7° our NMR data can best be fit into a cis-lactone in which the methylene protons are near the horizontal plane of the macrocycle (Figure 7). In the trans isomer, the relatively small deshielding effect experienced by the methylene protons, particularly H4, is an indication that they are located higher above the plane, near the "blank region" interfacing the opposite isotropic and anisotropic ring current effects. The presence of the adjacent on oxygen to H1 could add up to 0.8 ppm deshielding to Hl but the shielding effect on H4 should not exceed 0.2 ppm. These arguments can be applied to the 12—methyl equally well. In the cis isomer (1.823 ppm), it is axial and has no nearby deshielding oxygen whereas in the trans form (1.992 ppm) it is more equatorial and also closer to the ester oxygen. 75 The broad peak at 3.924 ppm in Figure BB is the 12-0H proton. NOE experiments revealed that irradiation of the 12-Me singlet enhances the lactone H1 peaks as well as the lO-H proton (9.272 ppm), and irradiation of the lZ-OH peak enhances the same meso proton. This observation ruled out the possibility that the NaOAc-cyclized product may be a 6-membered lactone fused across the 12,13-position. The M!!! spectra of the diols 106 and 108 are shown in Figures 8 and 9. The methylene protons of the angular propionate side chain were anaylzed at 500 and 250 MHz, respectively (Figure BB and SB). The 4- spin systems were simulated to give the chemical shifts and J values tabulated in Table 1. Their configuration was deduced from several considerations. Firstly, cis—dials were frequently obtained in model compounds following the osmate cleavage. In these cis--.diols,2° the MIR peaks of the methylene protons of the pyrroline propionic acid side chain usually gave a densely packed pattern which resembled the ABCM pattern of the diol 106. Second, when either the cis or trans diol was lactonized in the presence of NaOAc, which should not epimerize the lactone, each gave only the corresponding cis or trans lactone. The symetric pattern of diol 108 suggests there is a greater rotational freedom of the pyrroline methylene protons in the trans configuration. The lack of a large differentiation between 111 and H4, in contrast to the case of the trans lactone 92 (Figure 7), is again a consequence of this freedom of rotation. In both diols, no NOE could be detected between the lZ-Me or any of the methylene protons. The vicinal couplings obtained from the cis-diol 106 can be used to provide information about the conformation of the pyrroline propionic group. These measured numbers are average values of the component 76 1'1“!" 811888] 7.. L TjjTjITWIWjjjTjjjjljjjwjjj‘ljltfil' I'r'n 3.:a z.ra 250MHz 1.0 500 MHz I I r8 I I I I I 81' I I1 I r"I— I ‘1 r I l r"r 2.80 2.70 2.60 2.50 Figure W 1H NMR spectra (in CDCl:\ of cis"diol 97 at I 250 MHz (A); at 500 MHz with simulation (8). 44.4» bdfll TlWlTllTllTITfi lellflTTTTTlTTTlTflTTI 1% 0 S 0 8.0 7 m 814 JW L I Ii TjTIWjjTjjjj T11jTfifiTWTjj ‘LB 3B 20 k. Figure 9 'H VMR spvctra (in CD013) of trans~diol 99 i? 380 MHz (A); at 250 MHz with simulation (8). 78 Table l. 1H NMR Assignments for the Pyrroline Substituents of the Four Forms of 12,13-Dihydroxyprotochlorin. Form H 6(ppm) Coupling Constant (Hz) Lactone trans 1 3.490 J1,2 = 4.1, J2,4 = 9.6 3 3.213 31.3 = 9.6, 33.4 = 9.6 2 3.032 J1,4 = -13.3 4 2.430 J2,3 = -l7.9 lZ-Me 1.992 cis 4 3.846 J1,2 = 7.4, J2,4 = 9.8 2 3.731 J1,3 = 9.8, J3,4 = 5.2 1 3.443 J1,4 = -13.3 3 3.345 32.3 = -17.8 12*Me 1.823 Diol cis 1 2.816 J1,2 = 5.0, J2,4 = 9.4 4 2.614 J1,3 = 9.2, J3,4 = 5.0 2 2.562 J1,4 = -14.4 3 2.508 J2,3 = -16.2 12-Me 2.110 trans 2 3 2.660 J1,2 = 7.3, 32.4 = 7.3 ' 2.417 J1,3 = 7.3, J3,4 = 7.3 1 4 2.116 J1,4 = -14.6 ’ 1.882 J2,3 = -14.6 12~Me 1.847 79 coupling constants in rotamers I to III weighted by their fractional populations.71 The component coupling constants of I - III have previously"been estimated for chlorophyll derivatives72 and can be applied directly in this case. These J values and our calculated populations for 106 are given in Table 2. While this anaylsis may have substantial error margins (110%), the results are entirely consistent with what a molecular model would qualitatively predict: the anti conformer I is most favorable while the sterically congested rotamer II may be neglected. In the trans-dial 108, because of the greater rotational freedom, there should be no significant difference in the population of the rotamers. Table 2. Couplings (Hz)3 and Rotamer Populations of the cis- Diol 106. co,cn, H, Ha H' c" H‘ H! H2 w, ”J ' cu,o,c "’ c ”O O" HO OH I II III J1.2 ' 4.4 13.2 2.8 J1.3 13.2 3.6 3.6 J2,4 13.2 3.6 3.6 J3,4 . 4.4 2.8 13.2 Population 0.60 0.12 0.28 3From reference 72. 80 D. Structure of Heme dAMWLactoas versus.Diol? Before commenting on this quesiton, we would like to recapitulate the experimental observation. cis,Vic-Dihydroxychlorins carrying a pyrroline propionate ester chain would readily cyclize into either a cis or trans lactone by reagents encountered in common heme extraction protocols. Mild bases such as sodium acetate, sodium bicarbonate, and pyridine cleanly lactonize the ester without inversion of configuration. Silica gel acts upon the diol in two steps: first it lactonizes the geminal groups and then promotes the inversion of lactone to give the more stable trans diastereomer. In an ideal case, all three compounds can be seen on TLC plate during developing, arising from a pure diol. Under alkaline hydroytic conditions, the cis or trans lactone, if not. hydrolyzed, apparently do not epimerize, and the hydrolysis of which yields the dial with complete retention of configuration, undoubtedly the result of a common Bac2 mechanism.73 Given these intrinsic labilities, we may quite safely conjecture that the lactone ring found in "lactochlorin" is produced during the chromatographic purification of the demetalated and esterified chromophore. The observed trans configuration is also irrelevant as far as the true structure of the in give heme d is concerned. That issue, unfortunately, remains unanswered. From a biosynthetic point of view, saturation of the protoporphyrin ring is most likely brought about by an epoxidation. There have been efforts to prepare epoxychlorin but thus far such a compound has not been observed, presumably it is too labile to have a stable existence. If the epoxy ring is opened in aqueous medium, most likely a trans diol will result. However, one cannot be certain about the configuration as the propionate group may participate 81 in epoxide opening even though the resulting lactone may not be the final form of heme d. To date, the strongest argument against the lactone being present in the in vivo heme d is the absence of IR peak in the extracted, but unesterified, heme d.55 We noticed that the lactones are uniformly resistant to acid or base hydrolysis and the extraction procedure employed by Timkovich et.al. is too mild to open the lactone ring. In any case, the true structure of heme g awaits further confirmation. Our finding that the various forms of dihydroxychlorin have slightly different absorption and resonance Raman (RR) spectra" indicates that this is a problem solvable by RH studies of the heme enzyme . III. EXPERIMENTAL 1MB spectra were routinely obtained at 250 MHz on a Bruker 104-250 instrument. Occasionally we managed to obtain spectra recorded at 360, 400, or 500 MHz on spectrometers (all Bruker make) located at other institutions. Spectra were recorded in CDCla; the residue CHCla was used as the internal standard set at 7.240 ppm. The concentration of chlorin samples were maintained at 2-3 M to avoid concentration effects. All NOE’s were positive and are expressed as the area of the enhanced resonance in difference spectra divided by the area in the control spectrum. Simulations were carried out first on an IBM-PC with the PMH software and then on Aspect 2000 which outputs to the NMR plotter. Dihydroxychlorins 94a, 94b,” and94d Methyl 7,8,12,13,17,l8—hexaethy1-4-methylporphine—Z-propionate (93) was synthesized by condens ing 5 , 5 ’-dibromo-3 , 3 ’ , 4 ’ , 4 ’-tetraethy1- 82 2,2’-dipyrrylmethene hydrobromide74 and 4’-(2—carboxyethyl)—3,4-diethyl- 3’,5,5’-trimethyl-2,2’-dipyrrylmethene hydrobromide74 in formic acid and worked up in the usual manner;51 yield: 32X; m.p. 193-195°C; 1H NMR: 1.94 (98 each, t, Et), 3.29 (28, t, CH2CH2C02), 3.65, 3.68 (38 each, 3, ring’ Me and C02Me), 4.09, 4.12 (6H each, q, Et), 4.42 (2H, t, CH2C82C02), 10.09, 10.10 (1H each, s, 5,20-H), 10.12 (2H, s, 10,15-H), - 3.73 (211, br s, NH); UV-vis he): (an) 619 nm (8 800), 566.5 (10 400), 533 (13 100), 499 (15 900), 399 (142 000). To a solution of this porphyrin (200 mg, 0.35 mmol) in CH2C12 (60 m1) and pyridine (0.25 ml) was added osmium tetraoxide (114 mg. 0.45 -01). The mixture was stirred at room temperature in the dark for 36 h; it was then diluted with methanol (20 ml) and bubbled with 3:3 for 10 min. The precipitated osmium sulfide was removed by filtration and the filtrate was concentrated and chromatographed on preparative TLC plates, developed with. 082012/5X EtOAc. There 'were four distinct bands: the red porphyrin moving at the front followed by 94a, 94b, and another green band containing 94c and 94d. Unambiguous structure assiments were achieved by NOE (see Scheme 15). The two south diols were dissolved in a mixture of methanol (20 ml) and CH2C12 (5 ml) and was brought to reflux in the presence of anhydrous NaOAc (l g). The reflux was continued for 20 min before the mixture was washed with water and evaporated. The residue was chromatographed on TLC to separate the lactonized 95 (higher Rf) and 94d; overall yields: unchanged 93, 22 mg (11%); 94a, 34 mg (16%); 94b, 29 mg (13.73); 94d, 32 mg (153); and 95, 28 mg (14%). Methyl] 8. 12. 13 . 1.7 .187hexsstthy1-12. 13-dihydroxy-4-methyl-chlorin- 2-propionate (94a). M.p. 138°C (dec); NMR 6 1.00 (6H, t, pyrroline Et), 83 1.79, 1.81, 1.82, 1.85 (3H each, t, Et), 2.59, 2.61 (2H each, q, pyrroline Et), 3.11 (2H, t, CHzCHzCOz), 3.40 (3H, S, Me), 3.68 (3H, S, C02Me), 3.91 (8H, q, Et), 4.14 (2H, t, CH2CH2002), 9.01, 9.02 (1H each, s, 10,15-H), 9.66 (1H, s, 20-H), 9.69 (H, s, 5-H), —2.53 (2H, br 3, NH): UV-vis 7.3x (as) 642 nm, 590.5, 525, 496, 393; MS, found m/e 613.4152 for (M+H)*, C37849N4O4 requires m/e 613.4189. Methyl 7.8,12.13.17,1thexssthxI:7ifitéihy§§9§¥2§rwsthyl-ch1Grin-2- propionate (94b). M.p. 180-182°C (dec); NMR 6 0.90, 1.00 (38 each, t, pyrroline Et), 1.80, 1.83 (6H each, t, Et), 2.54, 2.64 (2H each, q, pyrroline Et), 3.08 (2H, t, CH2CH2002), 3.36 (3H, 3, Me), 3.64 (3H, s, 002Me), 3.88, 3.89 (4H each, q, Et), 4.15 (2H, t, CH20H2002), 8.96 (1H, . s, 5-H), 9.02 (1H, s, lO-H), 9.66, 9.73 (In each, s, 15,20-H), -2.57 (2H, br 8, NH): UV-vis Max (m) 644 nm, 590.5, 523, 494, 392. Mfifihllmzi§11§il3i17’13‘hexaeth21317213"dihydPOXY“3‘methyl-Ch1Grin" 2-propionate (94d). NMR 6 0.92, 1.02 (3H each, t, pyrroline Et), 1.80, 1.83 (6H each, t, Et), 2.58, 2.64 (2H each, q, pyrroline Et), 3.10 (2H, t, CH2CH2002), 3.41 (3H, 3, Me), 3.65 (3H, s, C02Me), 3.86, 3.92 (4H each, q, Et), 4.11 (2H, t, CH2CHzC02), 8.94 (1H, s, 20—H), 9.04 (1H, s, 15-H), 9.65, 9.72 (1H each, s, 5,10—H): UV-vis Max (on) 643.5 nm, 591, 523, 497, 391.5. 013'7:8:12213:17i1B‘Hexa?tthTQIhYdFQXXfigiygfihX}$212217 spirolactone-chlorin ( 95) M.p. 239-2410C; NMR 6 1.82, 1.85 (9H each, t, Et), 1.88 (3H, 3, Me), 3.29-3.54 (2H, m 2a2 and 2b4), 3.75-4.14 (14H, m, 6xEt, 2a1,2b3), 9.02, 9.20 (1H each, s, 5,20-H), 9.78, 9.85 (1H each, s, 10,15-H), -2.65, -2.57 (1H each, br 3, NH); UV-vis has: (an) 640.5 nm (42 000), 84 588 (5 900), 522 (5 000), 492 (14 000), 390 (161 000); MS, found m/e 581.3480 for (M+H)+, C35845N403 requires m/e 581.3494. EF§9§:21811g213117i187H3399thy173ih¥9593Y7§IWch¥1T?1277: spirolactonstshl9rin-($61 The cis-lactone 95 (20 mg, 0.034 mmol) was loaded on a 1500 um TLC plate and left in the dark overnight. The plate was developed with CHzClz/3% EtOAc to give the faster moving % in about 20% and the unchanged 95 (75%). If the plate was developed repetitively, 95 was completely converted to %; however, some minor degradation was also observed. M.p. 253—2550c; NMR 5 1.79, 1.83 (9H each, t, Et), 1.97 (an, 3, Me), 2.38 (1H, sext, 2a4), 2.97 (1H, oct, 2b2), 3.21‘(lH, quint, 2b3), 3.48 (1H, sept, 2al-) [J(2a1,2b2) = 3.7 Hz, J(2a1,2b3) = 9.6,. J(2a1,2a4) = -13.3, J(2b2,2b3) = -17.9, _J(2b2,2a4) = 9.6, J(2b3,2a4 = 9.6], 3.87, 3.90, 4.00 (4H each, q, Et), 8.87, 9.00 (1H each, 3, 5,20- H), 9.76, 9.77 (1H each, s, 10,15-H), -2.51 (2H, br 8, NH); UV-vis Max (In) 643 nm (31 500), 590 (2 600), 522 (4 800), 493 (11 000), 390 (153 000); MS, found m/e 581.3473 for (M+H)*, CaefiasNaOB requires m/e 581.3494. 3,8-8is(2,2-dimethoxyethy1)-deuteroporphyrin IX dimethyl ester (98) To a solution of 1 1t of dichloromethane and 170 ml of methanol containing 4 g of protoporphyrin IX dimethyl ester” was added 10.5 g (3.3 mol equiv) of thallium (III) trinitrate trihydrate dissolved in 340 m1 of methanol. The mixture was stirred under argon for 10 min at room temperature. Hydrogen sulfide was then bubbled through the mixture for 10 min, followed by the addition of 17 m1 of concentrated hydrochloric acid. The mixture was stirred for 5 min before the supernatant was decanted and the precipitated thallium (I) salts were washed with 85 methylene chloride. The combined organic solutions were washed three times with 1 1t of water and evaporated under vacuum, to give porphyrin 98 in quantitative yield. NMR 6 3.29, 3.32 (2H each, t, CH2CHzC02), ring methyl: 3.45 (3H, s), 3.53 (6H, s), 3.60 (3H, s), 3.46, 3.51 (6H, each, s, 00113), 4.09, 4.20 (2H each, d, C112CH(0CH3)2), 4.34, 4.39 (2H each, 1:, C1120HzC02), 5.08, 5.14 (1H each, t, CH2CH(OCHs)2), 9.84, 9.87, 9.95, 9.97 (1H each, s, meso), -4.05 (2H, br 3, NH). 3.8-Bis<2-hydroxyethy.l.).-.slsatemporphyrin IX dimethyl ss..t.93:-.._..(l992. The foregoing porphyrin 98 (2 g, 2.8 mol) was dissolved in 500 ml of tetrahydrofuran and the solution was brought to reflux, followed by the addition of 5 m1 conc. HCl in 15 ml of H20. After 5 min, the“ solution was cooled immediately, diluted with 500 ml 082C12, and washed three times with water. The solvent was evaporated under vacuum and the residue was dissolved in an arbitrary mixture of THF/MeOH. This solution (mixture of dialdehyde diester 99 and dialdehyde monoesters) was treated at 0°C with 6 g of Nam-14 in ice-cold methanol. It was stirred for 10 min before ~20 ml of acetic acid was carefully added to quench excess borohydride. The solvent was evaporated under vacuum and the residue was stirred for 12 h in 500 m1 of dry methanol containing 25 m1 of concentrated sulfuric acid. The acidic solution was then diluted with 500 ml 0112012 and washed with 3 x 500 ml of water. The product was chromatographed on neutral alumina (Brochmann Grade III) to separate a small amount of the faster moving mixture of 3-(2-hydroxyethyl)-8-(2,2’- dimethoxyethyl ) ~deuteroporphyrin IX dimethyl ester and 8- ( 2- hydroxyethyl)-3-—(2,2’—dimethoxyethyl)-deuteroporphyrin IX dimethyl ester (elution with crime) from the desired compound 100 (elution with CH013/2% CH30H). Yield 71%; NMR (CDC13 at 315°K) 6 3.25 (4H,' t, 86 C82C1712002), 3.57 (12H, 5, ring Me), 3.65 (611, s, C02Me), 4.23 (4H, t, CH2CH2C02), 4.37 (8H, m, CHzCHzO), 9.98, 10.00 (2H each, s, meso), -3.80 ..... (2H, br 3, NH). 3,8-Bis(2-ch1oroethyl)jdeuteroporphyrin IX dimethyl ester (101) To a solution of the foregoing porphyrin 100 (1 g, 1.59 mmol) in dry DMF (200 ml) was added benzoyl chloride (20 ml). The mixture was heated as 98°C for l h under nitrogen and allowed to cool. Water (500 m1) and triethyl amine (30 ml) were then added and the precipitated compound was filtered, washed with water and purified by passing through a short silica gel column using CH2012/l% CH30H as eluent. The product was further purified by recrystallization from CH2012/CHaOH. Yield 91%; m.p. 216-217°C (lit.53° m.p. 216—217°C); NMR 6 3.24, 3.26 (2H each, 1:, CH20H2002), 3.46, 3.50, 3.53, 3.56 (3H each, 3, ring Me), 3.66, 3.67 (3H each, s, C02Me), 4.19-4.38 (12H, m, CH2CH201 and CH2CH2002), 9.75, 9.82, 9.87, 9.96 (1H each, s, meso), -4.06 (2H, br 8, NH); MS, m/e 663.1049 (calcd for C36841N404012 663.2508). Spirolactones.1039naQQMIQ3b To a solution of 3,8—bis(2-chloroethy1)deuteroporphyrin IX dimethyl ester 101 (850 mg, 1.28 mmol) in CH2C12, osmium tetraoxide (423 mg, 1.66 mmol) was added, followed by addition of pyridine (0.2 ml). The reaction was stirred in the dark for 26 h before being diluted with methanol (50 m1) and quenched by 1128. The chlorin products were chromatographed on a silica gel column. Porphyrin 101 (180 mg, 21% recovered) was eluted first with CH2012 while the mixture of the four dihydroxychlorins was washed out with CH2C12/2% MeOH. This mixture was then chromatographed on TLC plates (CH2C12/10% EtOAc) to separate the 87 two faster moving north diols (120 mg, 1:1 ratio) from the two south diols 102a and 102b (430 mg, 1.2:1 ratio). The distinction of the north and south diols was based on NMR of the methyl ester singlets: 6 3.64, 3.63, 3.60, 3.59 (north diols) versus 3.67, 3.65, 3.55, 3.52 (south dials). The mixture of 102a and 102b in MeOH (100 ml) was refluxed with anhydrous NaOAc (5 g) for 30 min. the solution was evaporated and the residue was dissolved in CH2012 and chromatographed on TLC (CH2C12/10% EtOAc) without interruption, in a single path, to yield the faster moving 103a (160 mg) and the slower moving 103b (180 mg), the structure assignment of which had been based on NOE (see Scheme 17). cis-3,8-Bis(2-chloroethyl)-12—hydroxy~13,13-Tbspirolactone- deuterochlorin IX dimethyl ester (103a). M.p. 226—228°C; MIR 6 1.84 (3H, s, 12-Me), 3.06 (28, t, 17— CH20§2C02), 3.38, 3.41 (3H each, s, 2.18—Me), 3.51 (3H, s, 7—Me), 3.52 (3H, s, C02Me), 3.26-3.90 (48, m, 13- CHzCHz), 4.06 (1H, br s, OH), 4.12, 4.20, 4.28 (10H, t, 2x0HzCHzCl and l7-CH20H2C02) 9.08, 9.12 (111 each, s, 10,15-H), 9.64 (1H, s, 20-H), 9.71 (1H, s, 5-H), —2.67 (2H, br 3, NH): UV-vis hex (m) 641 nm (57 000), 588.5 (10 000), 522.5 (9 000), 497 (21 900), 392 (226 000); MS, found m/e 665.2345 for (M+H)*, CssllseNdOsClz requires m/e 665.2300. cis-3,8-Bis(Zechloroethyl)-18-hydroxy-l7,17-Thspirolactone- deuterochlorin IX dime$112.1...-...s§.§s_r_:.._.-,5.19.392.- M.p. 239—240°C; NMR 6 1.85 (3H, s, 18-Me), 3.12 (2H, t, 13- cmcgecoz), 3.41, 3.49 (311 each, s, 7,12-Me), 3.44 (3H, s, 2-Me), 3.52 (an, s, C02Me), 3.27-3.86 (4H, m, 17- CIhCHz), 3.98 (1H, br s, on), 4.19, 4.22, 4.30 (10H, t, 2xCH2CH2C1 and l3-CHzCHzC02), 9.14 (1H, s, 15-H), 9.19 (1H, s, 20-1-1), 9.66, 9.70 (111 88 each, s, 5,10-H), —2.70 (2H, br 5, NH); UV-vis Max (an) 641 nm (55 000), 588 (11 900), 524.5 (12 800), 494 (22 000), 395.5 (255 000). QE§7127HY9§9¥Yfl3p13*T'SPiF01§CFQPGTPFQF°9919F1911XiflfithYL ester (104) To a refluxing solution of cis-lactone 103a (100 mg, 0.15 mmol) in pyridine (50 m1) under argon, KOH (1.2 g) in water (4 ml) was added and the heating was continued for 6 h before the mixture was evaporated to dryness under reduced pressure. The residue dissolved in ice-water was treated with 10% HCl whereupon the product precipitated. The solid was filtered, washed with water and esterified in MeOH with diazomethane. The product was chromatographed rapidly on TLC to separate the faster moving lactone 104 (8 mg) and the slower moving dial 106 (12 mg). The non-mobile material, after eluting with CH:C12/2% MeOH was believed to be degradation products due to the prolonged heating with strong base. However, if more dilute solution of ROI! or less time ((3 h) was allowed for the reaction, the mono-vinyl compound: 3-(2-chloroethyl)-12,13- dihydroxy—8-vinyldeuterochlorin IX dimethyl ester was mainly resulted (structure determined by NOE). The condition for this elimination has not been optimized. NMR of.104: ring methyl (3H each, 3): 3.433 (18- Me), 3.490 (2), 3.527 (7); propionate: 4.263 (2H, t, 17a), 3.087 (2H, t, 17b) [J(17a,17b) = 7.4 Hz], 3.558 (3H, s, COzMe); vinyl (1H each): 8.073 (Xi), 8.073 (X3), 6.281 (At), 6.101 (Bi), 6.165 (lb), 6.022 (83) [J(Xi,Ai) = 17.8, J(Xi,Bi) = 11.5, J(Ai,Bi) = 1.3, J(AJ,BJ) = 1.7]; meso (1H each, 3): 9.856 (5), 9.272 (10), 9.048 (15), 9.737 (20); NH: -2.618 (2H, br s): pyrroline substituents: see Table 1. IR 1780 cm-1, 1735; UV-vis Max (01) 650 nm (37 200), 594 (7 500), 530 (8 000), 500 89 (14 200), 401 (147 000). MS, found m/e 593.2742 for (M+H)", Casi-137N405 requires m/e 593.2766. cis-'12...13-dihydr98ypmteehlorin.......IX dimethyl ester .,...<.....1.9§l NMR 6 2.11 (3H, s, 12—Me), 3.06 (2H, t, 17b), 4.04 (2H, t, 17a) [J(l7a,l7b) = 7.4], 3.32, 3.34, 3.44 (3H each, a, ring Me), 3.49 (3H, s, l3-CCC02Me), 3.66 (3H, s, 17-CCCOzMe), 5.99, 6.05, 6.13, 6.22 (1H each, dd, vinyl), 8.01 (2H, m, vinyl), 8.84 (1H, s, 15-H), 9.03 (1H, s, 10- H), 9.57, 9.72 (1H each, s, 5,20—H), -2.41 (2H, br 3, NH), l3-CHzCHzCOz: see Table l; UV-vis hax (an) 650 nm (45 000), 594 (4 600), 532 (4 400), 498 (13 800), 401 (179 000). MS found m/e 625.3039 for (M+H)*, Gael-141N405 requires m/e 625.3028. trans-lZ-Hydroxy—l3,13fTbspirolactone-protdchlorin IX methyl ester (92) The cis lactone 104 (20 mg) was loaded on a TLC plate and was developed with CH2C12/3% EtOAc) in the dark for at least, 4 times to convert 104 into the slightly faster moving 92. NMR 6 ring methyl (3H each, s): 3.484 (18-Me), 3.514 (2), 3.584 (7); propionate: 3.132 (2H, t, 17b), 4.189 (2H, t, 17b) [J(l7a,l7b) = 7.4 Hz]; 3.662 (38, s, COzMe), vinyl (1H each): 8.069 (Xi), 8.069 (Xi), 6.286 (Ai), 6.091 (Bi), 6.157 (M). 6.010 (85) [J(Xi,Ai) = 17.8, J(Xi,8i) = 11.5, J(Ai,Bi) = 1.3, J(~,BJ) = 1.7]; meso (1H each, 3): 9.889 (5), 9.113 (10), 8.856 (15), 9.752 (20); NH: -2.254 (2H, br s); pyrroline substituents: see Table 1. IR 1780 cm-1, 1738, 1718; UV-vis hax (ea) 653 nm (40 500), 596 (6 300), 532 (6 600),, 501 (14 500), 401 (156 000). MS, found m/e 593.2736 for (Mi-H)’, CssHs'rNdOs requires m/e 593.2766. 90 QiS'IB‘HXdFOXXI17i177TISPEEQlQQFQR§IRKQIQ9thE19m1me§Fthm§§§E!-(19991 The chlorin-bearing lactone 1035 was treated with KOH in refluxing pyridine and worked up in the same manner as described for 104. NMR 6 1.84 (3H, s, 18-Me), 3.06 (2H, t, 13b), 3.27—3.88 (4H, ABMN, lactone), 3.32, 3.39, 3.43 (3H each, 3, ring Me), 3.54, (3H, s, 002Me), 4.05 (1H, br s, OH), 4.10 (2H, t, 138), 5.99, 6.07, 6.13 6.25 (1H each, dd, vinyl), 8.00 (2H, m, vinyl), 9.04, 9.15 (1H each, s, 15,20-H), 9.71, 9.72 (111 each, s, 5,10-H), -2.70 (2H, br 3, NH); UV-vis hax (an) 650 nm (40 000), 596 (7 300), 533 (7 400), 501 (15 800), 402 (164 000). trans-lB—Hydroxy-17,17-Y—spirolactone-protochlorin IX methyl ester (105b) Obtained from the cis-lactone 105a by a repetitive chromatography. MIR 6 2.01 (2H, s, 18—Me), 2.43 (1H, sext, l7a4), 3.04: (1H, oct, 17b2), 3.22 (1H, quint, 17b3), 3.48 (1H, sept, l7al) [J(l7a1,17b2) = 3.7 Hz, J(17al,l7b3) = 9.6, J(17a1,l7a4) = -13.3, J(l7b2,l7b3) = -17.9, J(17b2,l7a4) = 9.6, J(l7b3,17a4) = 9.6], 3.13 (2H, t, 13b), 3.66 (SH, s, COzMe), 4.19 (2H, t, 13a), 6.02, 6.13, 6.19, 6.35 (1H each, dd, vinyl), 8.11 (23, m, vinyl), 8.88, 9.05 (In each, s, 15,20-3), 9.86, 9.93 (1H each, s, 5,10-H), -2.34 (2H, br s, NH); UV-vis )max (ea) 652 nm (41 800), 597 (8 200), 533 (9 000), 500 (16 800), 401 (166 000); MS, found m/e 593.2730 for (M+H)*, CasfiaquOs requires m/e 593.2766. cis-17,18-Dihydroxyjprotochlorin IX dimethyl ester Obtained by pyridine/K011 treatment of 105a. MIR 6 2.09 (3H, s, 12-Me), 2.32-2.78 (4H, ABCM, l7-CHzCH2002), 3.01 (2H, t, 13b), 3.24, 3.40, 3.41 (311 each, 3, ring Me), 3.50 (3H, s, 17-CCCOzMe), 3.65 (3H, s, l3~CCCOzMe), 3.97 (2H, t, 13a), 5.99, 6.07, 6.11, 6.24 (1H each, dd, vinyl), 7.98 (2H, m, vinyl), 8.82, 8.91 (111 each, s, 15,20-H), 9.62, 91 9.71 (1H each, s, 5,10—H), —2.45 (2H, br 3, NH); UV-vis 2.3:: (an) 651 nm (41 000), 597 (8 000), 533 (8 600), 501 (15 800), 402 (159 000). tr§9§:31§:§i§32:9h1orpethxl171%:hx§§951:1§i13:1:§21:91993999: dsgtssgshlsxiyhixméimstbylwesterMIIQZi Obtained from the cis-lactone 103a by repetitive chromatography. MIR 6 1.99 (3H, s, 12-Me), 2.41 (1H, sext, 1384), 3.03 (1H, oct, 13b2), 3.23 (1H, quint, 13b3), 3.45 (1H, sept, 13ai) [J(13ai,l3b2 = 4.1 Hz, J(13ai,l3ba) = 9.6, J(13ai,l3a4) = -l3.3, J(l3b2,l3b3) = ~17.9, J(13b2,l3a4) = 9.6, J(13b3,l3a4) = 9.6], 3.14 (2H, t, 17b), 3.42, 3.52, 3.54 (3H each, 3, ring Me), 3.66 (3H, s, C02Me), 4.16, 4.20, 4.21, 4.24, 4.30 (2H each, t, 2x0112CHzCl and 17a), 8.98, 8.90 (1H each, s, 10,15-H), 9.75, 9.66 (1H each, s, 5,20-H), -2.46 (2H, br s, NH); UV-vis 7mm): (at) 641 nm (42 200), 588 (6 300), 520 (4 600), 495.5 (18 700), 392 (184 000); MS, found m/e 665.2335 for (M+H)*, CssH39N¢OsC12 requires m/e 665.2300. trans-12,l3-Dihydroxyprotochlorin IX dimethyl ester (108) Obtained by pyridine/XOH treatment of 107. MIR 6 1.85 (3H, s, 12- Me), 3.15 (2H, t, 17b), 3.39, 3.49, 3.52 (3H each, s, ring Me), 3.59 (3H, s, l3-CCC02Me), 3.66 (3H, s, l7—CC002Me), 4.19 (2H, t, 17a), 6.00, 6.07, 6.15, 6.29 (1H each, dd, vinyl), 8.08 (2H, m, vinyl), 8.93, 9.11 (1H each, s, 10,15-H), 9.72, 9.86 (1H each, s, 5,20-H), -2.08 (2H, br s, NH), l3-CHzCI-12C02: see Table 1; UV-vis has: (m) 653 nm (37 200), 598 (4 600), 533 (4 800), 499.5 (12 800), 401 (154 000); MS found m/e 625.3014 for (M+H)*, C36H41N406 requires m/e 625.3028. MR3 SYNTHESIS AND MISS OF SWNTAINIM SATURATBD MTABTHYIPORPHYRINS I. INTRODUCTION Our interest in sulfur-containing saturated porphyrins stems from sulfmyoglobin (SW) and sulfhemoglobin (SHb). These sulfglobins are unusual green derivatives of either myoglobin or hemoglobin produced in 31.559 according to the following scheme: Fe(III)Mb + H202 + Fe(IV)M>-Peroxo Fe(IV)Mb-Peroxo + H28 -> Fe(III)SM> The existence of sulfglobins is more than a laboratory curiosity. conditions or in the presence of high dosages of some drugs related to cmon analgesics such as phenacetin." Increased levels of Shb have been correlated with exposure to chemical pollutants.77 Over the years, several groups have made contributions to the available structural knowledge of sulfglobins.7°i7° Today, it is generally accepted that these abnormal globins contain a chlorin prosthetic group with a sulfur moiety on the pyrroline ring, however, both the final sulfur modification and the site of ring reduction are still questions under investigation by a number of laboratories. Recently, La Mar79 and Timkovich“ have independently shown that it is possible to isolate a stable sulfchlorin from Sn; and suggested a 92 93 thiophene—like cyclic structure (III) based on 1H NMR data. They further proposed than an episulfide (I) or a thio-substituted chlorin (II) is most likely to be the initial product during sulfheme formation (Figure 10). S s\ s B/ N” N Figure 10. Sulfheme Models Since such a sulfchlorin structure has not been know in the literature, we decided to develop the synthesis and to study the properties of sulfur-containing chlorins. Moreover, our curiosity let us proceed further and explore the chemistry of even higher saturated porphyrin systems containing up to three sulfur atoms. II. RESULTS AND DISCUSSION Our approach towards the synthesis of sulfur-containing saturated porphyrins starts with the transformation of a carbonyl into a thiocarbonyl group, using 2,4—bis(p~methoxy—l,3-dithiadiphosphetane-2,4- disulfide) 109 now popularly referred to as Lawesson’s reagent. This reagent has been used in the literature for the high yield thionation of a variety of aliphatic and aromatic carbonyls.5°’°1 It has been suggested that a highly reactive dithiophosphine ylide 110, rather than Lawesson’s reagent itself, might be the active thionating agent‘32 and two possible mechanisms might be envisioned, both involving Wittig-type intermediates83 (Scheme 18). 94 sf 7? 5° , Arluks/PAr A‘— 2 Aug- + R-C_R s o 109 110 i e i 7°: 9 Ar- P- S (__ Ar—P—S—?_R' l l O_C_R R R! Scheme 18 In an effort to synthesize OEP-thione (111), we reacted OEP- monoketone18 (8) with two equivalents of Lawesson’s reagent in refluxing THF for 24 h, to yield the thione 111 (60%), plus the unreacted starting material (35%). Lengthening the reaction time had no effect on the product’s yield. On the other hand, increasing the amount of Lawesson’s reagent led to the formation of polymeric material at the expense of the desired product. Thione lll exhibited a characteristic IR band at 1230 cm‘1 3‘ (Figure 11) and a 1H NMR spectrum shown in Figure 12 in comparison with its oxo-analogue. Notable features of this spectrum are: (l) the deshielding effect of the sulfur on the adjacent meso proton; (2) the multiplication of the geminal ethyl groups, suggesting that these groups have less freedom of rotation in comparison with the oxo-analogue 8; and (3) the slight downfield shift of the N-H protons. The thione structure is stable in acids and on silica gel (during purification), however, in the presence of oxidizing agents, e.g., OsOu, 115 114 118 117 SCHEME l9 96 I 708(C-O) B. I 230(0-8) I I I I I I I 700 I 600 I 500 I 400 l 300 IZOO FREQUENCY, cm-I Figure 11 1800—1300 cm‘1 IR spectra of: (A) porphyrinone 8; 'B) porphyrinthione lll. A: ecoflnacwnhnmsom can m omosflshnmaom he masons—m $2 as was. 93 NH scam: I hi I.NI I.I ... I. N I. "ELI I... I.“ I.“ I... a... I!“ I In a J 31, 1 j: AWIII a a new] 111 98 MOLI Rsnoy NI ’ W2 SCHEME 20 0304 HS 21 121 122 123 99 Table 3. 1H NMR Data of Sulfur-Containing Saturated Octaethyl- porphyrins in Comparison with Their Oxo-Analogues. Meso Protons, 6 Compound 5-H lO-H 15-H 20-H Ketone (8) 9.13 9.86 9.94 9.84 Thione (111) 9.10 9.69 9.78 10.32 Methyl-thiol (121) 8.81 (9.75) (9.77) 9.32 Thiol (122) 8.79 9.76 9.76 9.35 2,7-dione (64) 8.61 8.42 9.41 9.26 2-thiol-7-dione (112) 8.65 8.37 ' 9.25 i 9.72 2,7—dithione (113) 9.04 8.37 9.19 9.69 3,7-dione (66) 9.58 “8.87 9.77 8.87 3-thio—7—dione (114) 10.01 (8.87) 9.61 (8.80) 3,7-dithione (115) 10.40 8.74 9.40 , 8.74 2,7,12-trione (116) 8.05 7.81 8.12 8.92 2-thio-7,12-trione (117) 8.03 7.70 7.98 9.38 2,12-dithio-7-trione (118) 8.04 8.26 8.10 9.34 2,7-dithio-12-trione (119) 8.59 7.92 8.08 9.53 2,7,12-trithione (120) 8.60 8.46 8.19 9.48 Note: The chemical shifts in parentheses are tentative assignments. 100 it reverts to the starting ketone. Moreover, thione lll reacts readily with alkyllithium reagents, e.g., CH3Li in a manner similar to porphyrinone 813, giving rise to methylthiol 121. In contrast to porphyrinone 8 which requires LiAlHo for complete reduction,85 thione 111 can be reduced cleanly to 122 with NaBHs. Finally, desulfuration of 111 with Haney Nickel (Vii-2)88 affords gem—OEC (octaethylchlorin) (123), quantitatively. Dithiones 113, 115 and trithione 120 can also be Obtained from the reaction of their corresponding oxo-analogues“:18 with 4eq and 6eq of Lawesson’s reagent, respectively (Scheme 19). The low yields ((12%) associated with these molecules can be attributed-to both steric and electronic reasons. The 1H NMR data for the meso protons of all the above sulfur-containing saturated octaethylporphyrins are listed in Table 3 for a general comparison.v Desulfurations by Ra-Ni have also been carried out with dithione 113 and trithione 120, providing an easy access to the tetrahydro- isobacteriochlorins11b and hexahydro-pyrrocorphinsa7, respectively. Absorption Spectra. The visible spectra of thione lll, thiol 121 and their Cu—complexes as well as those of dithiones 113, 115 and trithione 120 are shown in Figures 13 and 14. Thione 111 gives rise to a "hyper" type spectrum, presumably as a consequence of the mixing of n- I“ and r1“ transitions, where n are the nonbonding sulfur orbitals, ‘l’ are the highest occupied molecular orbitals (HOMO) and 1'" are the lowest unoccupied molecular orbitals (LIMO) of the porphyrin ring. Moreover, . the absorption bands are shifted to longer wavelengths due to the conjugation of the thione with the ring.88 On the other hand, thiol 121 101 .Aav Homes so ”see oceans -se ”Ame «we Hones “Ase Ham scones co Awesome one ossooan seasons s... ...-.9335! my madman 4 aim-03 oo— n00 who IQNIJOIQI 9N. «X .0» r 1 a” . l e m b P p p p b q a I d u q J1 r i T. 102 IOO - A 460 80 r ‘ 60 r " U 4101- ‘ 734 20 I- 794 '1 070 1 l l 1 l l l l l l 1 l l 460 560 600 gravelongth, on Figure 14 Visible spectra (in CHeClz) of 3,7—dithione 115(A); 2-7-dithione 113 (B); 2,7,12—trithione 120 (C). 103 exhibits a typical chlorin spectrum since the sulfur atom is no longer in conjucation with the aromatic ring. Introduction of a second and third sulfur atom into the porphyrin ring (dithiones 113, 115 and trithione 120) shifts the absorption bands further to the red and the overall pattern depends on the relative position of the sulfur atoms, as well as the saturation degree of the porphyrin ring. Cyclic Voltammetry. The redox properties of free-base and metal complexes of' porphyrins,99i9° hydroporphyrins°°'97 and oxo~ porphyrins“,98 have been extensively investigated and the principal resutls, for the OEP series, can be summarized here. Porphyrinones exhibit ring oxidation potentials very similar to those of the parent porphyrin, in sharp contrast to those of chlorins, isobacteriochlorins and bacteriochlorins, which are significantly easier'to oxidieze than the porphyrin (by as much as 0.6 V). Again, in contrast to the behavior of the isobacteriochlorin derivatives, which are harder to reduce than porphyrins by ~O.3 V, the dioxo—isobacteriochlorins are easier to reduce by ~0.2 V for a net change of ~0.5 V in reduction potential upon introduction of the dioxo—functions onto the isobacteriochlorin skeleton. The redox properties of sulfur containing saturated octaethyl- porphyrins are being examined here, for the first time. Potentials are listed in Table 4 in comparison with those of oxoporphyrins. These measurements reveal that substitution of 0 with S renders the porphyrin ring easier to reduce (by as much as 0.53 V in trithione 120, see Figure 15) and easier to oxidize (by as much as 0.3 V in 2n—3,7-dithione 115. The potentials of the redox processes of the Cu and Zn complexes of 104 Table 4. Redox Potentialsa of Sulfur-Containing Saturated Octaethyl- porphyrins and Oxo—Octaethylporphyrins. The latter are Indicated in Parenthesis. Compound Ring Oxidation Ring Reduction 0/1+ 0/1‘ l'/2' 2’/3‘ Thione (111) He 0.76(0.84) -l.03(-l.36) Cu 0.61(0.68) -l.08(-l.37) Zn 0.50(0.56) -l.lO(-l.46) Thiol (122) Hz 0.68 -l.49 Cu 0.47 -l.49 Zn 0.52 -l.51 2-thio—7-dione (112) He 0.72b -1.01 Zn 0.40 -l.20 2,7-dithione (113) He 0.70°(0.82) -0.85(-l.29) -l.20 Zn 0.44(0.70) ~0.97 -1.40 3-thio-7-dione (114) He 0.70b -0.86 -l.4l Zn 0.38 —0.94 -l.50 3,7-dithione (115) H: 0.70b(0.79) -0.77(-l.l4) -l.24 Zn 0.40(0.70) —0.85 —l.38 2,7,12-trithione (120) H: 0.70(0.79) -0.7l(-l.24) -l.02 -l.43 (-l.50) --__ ———-- °El/2 vs. SCE obtained by cyclic voltammetry at 8 Pt electrode in CH2C12 containing 0.1 M tetrabutylammonium perchlorate. bQuasi-reversible. Scan rate: 20 mv/s. 105 .2. 8: osowzuwsusm~.b.m .A 0q~o>0 m. sesame wow 3 23> m. . I 0.... 0.0.. 0 0.0 0.. 0.. fidqfiq—-fi+—q_fiqlfi—d-qadd-uddflfiqq—l 106 these sulfur-containing saturated porphyrins are shifted negatively relative to the potentials of their free bases in a fashion parallel to the shifts observed upon metalation of oxoporphyrins.41“V7 Thus, the metal exerts an inductive influence (via the signs framework) on the porphyrin Ihlevels. To a first approximation, the redox span for formation of the monocation and monoanion radicals corresponds to the HOMO-LUMO energy gap which can be related further to the wavelength of the first absorption band.9°‘99 Since the inductive substituent effect is expected to shift the whole stack of I and 1* orbital energies up or down without altering the HOMO-LING gap while the resonance effect should narrow the gap with increasing delocalization, it is obvious from the electrochemical data that sulfur atoms conjugated to the porphyrinoid ring have strong rinteractions with it. The redox potentials of thiol 122 which is not in conjugation with the ring, are very similar to those of any typical chlorin. An intriguing point is that the redox potentials are also sensitive to the position of the sulfur atoms at the prophyrinoid ring, e.g., 114 and 115 are easier to reduce than 112 and 113, respectively. Sisaliggwfiemarks The above work presented the synthesis and properties of the very first model sulfchlorins. In addition, the sulfur effects were examined in model compounds with different number and position of thio groups. Further efforts will be aimed at the synthesis of episulfides and vicinal -SH and ~OH (ring opened products) which are closer mimics of the alleged biological sulfhemes. 107 The chemical events that lead to the sulfheme formation in proteins remain to be the most intriguing question. Judging from the obligatory presence of ferryl (Fe‘V) species in the proteins prior to the sulfheme formation, it may be reasonable to consider the involvement of certain electron-deficient forms of sulfur (RS+ or Fe(IV)-OS) as the reactive species. This hypothesis awaits to be tested by future model experiments (for example, reactions of Fe(IV)OEP with (8114 )2S or other appropriate sulfur reagent). III. EXPERIMENTAL Visible spectra were recorded on a Cary 219 spectrophotometer interfaced to a Bascom-Turner recorder. Spectra shown here were plotted direclty from data stored on floppy diskettes. Cyclic voltametry was performed using a Bioanalytical System CV-IA unit. All measuraents were carried out in CH2012 containing 0.1 M tetrabutylamonium perchlorate at a scan rate of 200 mV/sec. 3,3,7,8,12,l3,17,l8—Octaethyl-Z-porphinethione (lll) Lawmson’s reagent (40 mg, 0.1 mmol) was added to a solution of porphinone 8 (55 mg, 0.1 mol) in dry THF (50 ml) and the reaction mixture was refluxed under N2 for 24 h or until no more product was formed (monitored by TLC). The solvent was evaporated in vacuo and the residue was chromatographed on preparative TLC plate (silica gel, CHzClz/hexane) to separate the faster moving yellow—green thione 111 (34 mg, 60%) from the purple starting material 8 (20 mg, 35%). Thione 111 was further purified by recrystallization from CH2C12/CHsOH. M.p. 234- 236°C; MIR 6 0.07 (6H, t, CHzClls sat), 1.79, 1.83, 1.86 (6H each, t, canons), 2.70, 2.97 (23 each, m, cgecus sat), 3.86, 4.00, 4.03 (43 each, 108 q, CH2CH3), 9.10 (1H, s, 5-H), 9.69 (1H, s, lO-H), 9.78 (1H, s, 15-H), 10.32 (18, s, 20-H), -2.38, -2.42 (1H each, br 5, NH); IR 1230 cm‘l; UV— vis haw (m) 680 nm (24 200), 624 (20 500), 458 (63 700), 442 (63 700), 404 (54 800), 382 (48 400), 344 (32 200), 312 (24 200); MS (direct probe, 70 eV) m/e 566(W). Anal. Calcd for CasHMiNds: C, 76.28; H, 8.18; N, 9.88. Found: C, 76.17; H, 8.28; N, 9.79. 3,3,8,8 3,3 8,8 I I ! 12,13,17,lB—Octaethylj2-thio-7-prophinedione (112) and 12,l3,l7,18jOctaethyl-2,7-porphinedithione (113) 56 mg (0.1 mmol) of dione 64 and 81 mg (0.2 mmol) of Lawesson’s reagent were reacted as described for 111. Separation on TLC plate gave the fastest moving brown-orange dithione 113 (5.6 mg, 9.4%) followed by the green 112 (31.7 mg, 53%) and the dark-green starting material 64 (10.1 mg, 17.8%). 112: MIR 6 0.17, 0.58 (6H each, t, CH2CH3 sat), 1.69, 1.70, 1.72, 1.73 (3H each, t, CH2CH3), 2.60 (6H, m, CH2CHs sat), 2.87 (28, m, CH20112 sat), 3.73 (8H, m, CHzCHa), 8.37 (1H, s, lO-H), 8.65 (1H, s, 5-H), 9.25 (1H, s, 15-H), 9.72 (1H, s, 20-H), 0.47 (2H, br 3, NH). Irradiating the multiplets at 6 2.60 and 2.80 caused the respective singlets at 6 8.37 and 8.65 to increase in intensity. IR 1716 cm”, 1248, 1216; UV-vis has: (In) 681 nm (24 000), 641 (16 900), 612 (12 900), 476.5 (34 900), 453 (37 400), 419.5 (59 800). MS (direct probe, 70 eV), m/e 582 (M‘). 113: NMR 6 0.15, 0.26 (6H each, t, CH2CH3 sat), 1.66, 1.70 (6H each, t, 01120113), 2.56, 2.79 (411 each, m, CHzCHs sat), 3.63 (2H, q, CH20H3), 3.73 (6H, m, 01120113), 8.37 (1H, s, lO-H), 9.04 (1H, s, 5-H), 9.19 (1H, s, 15-H), 9.69 (18, s, 20-H). Irradiating the multiplets at 6 2.56 and 2.79 caused the respective singlets at 6 8.37 and 9.04 to increase in intensity. IR 1073 cm”; UV—vis hax (an) 769 nm (5 100), 109 716 (8 400), 681 (12 700), 523 (25 800), 489 (18 200), 450 (28 500), 428 (19 600); MS (direct probe, 70 eV) m/e 598 (W). .2. . ....i 21.2.; Prepared as above. Separation on TLC plate gave three green bands. The least polar band 115 (4.1%) followed by 114 (22.3%) and the recovered starting material 66 (50.2%). 114: NMR 6 0.19, 0.52 (6H each, t, 08201713 sat), 1.74, 1.76, 1.78, 1.81 (3H each, t, CH20H3), 2.62, 2.85 (6H,2H each, m, 01120113 sat), 3.83, 3.86, 3.94, 3.95 (2H each, q, CH20H3), 8.80, 8.87 (18 each, s, 10,20—H), 9.61 (1H, s, 15—H)_, 10.01 (1H, s, 5-H), -l.06 (28, br s, NH). Irradiating at 6 3.84 caused the singlets at 6 8.80 and 8.87 to increase 1 in intensity. Moreover, irradiating at 6 3.94 caused the singlet at 6 9.61 to increase in intensity. IR 1700 cm”, 1108, 1200; UV-vis 7.3::(01) 701 nm (34 100), 631 (11 200), 455 (124 000), 429 (68 500); MS (direct probe, 70 eV) m/e 582 (W). 115: NMR 6 0.20 (12H, t, CH20H3 sat), 1.71, 1.76 (611 each, t, 0820113), 2.60, 2.81- (4H each, m, CH20H3 sat), 3.76, 3.85(4H each, q, 01120113), 8.74 (2H, s, 10,20-H), 9.40 (1H, s, 15-H), 10.40 (1H, s, 5-H), -0.45 (2H, br 5, NH). Irradiating at 6 3.76 and 3.85 caused the respective singlets at 6 8.74 and 9.40 to increase in intensity. IR 1072 cm”, UV-vis )nsx(ei) 794 nm (17 300), 734 (27 900), 670 (9 900), 466 (82 900); Ms (direct probe, 70 eV) m/e 598 (Mt).. 110 3,3,8,8,13,13, l7, 18-Octaethyl-2-thio-7,12-porph1netr10ne (117), 8,2,8,8,13 13, 17, 18- Octaethyl- -2, 12- d1th10-7-porphlnetrione (118), 3,3,8,8,13, 13, 17, 18- Octaethyl 2, 7, dithio—12- porphinetrione (119), and 3,3,8,8,13,13,17,1887-Octaethyl 2 7, 12 porphinetrithlone (120) 58 mg (0.1 mmol) of trione 116 and 121 mg (0.3 mmol) of Lawesson’s reagent were reacted as described for 111. Separation on TLC plate gave the fastest moving rosy trithione 120 (7.1 mg, 11.3%) fellowed by the brown 119 (16.2 mg, 26.5%), the green 118 (7.1 mg, 11.6%) and the yellow-green 117 (21 mg, 35.2%). 117: NMR 6 0.17, 0.54, 0.57 (6H each, t, CH2083 sat), 1.54, 1.61 (3H each, t, CH2CHa), 2.27-2.60 (12H, m, CHzCHs sat), 3.48, 3.56 (2H each, q, CH2CH3), 7.70 (1H, s, lO-H), 7.98 (1H, s, l5-H), 8.03 (1H, s, 5-H), 9.38 (1H, s, 20-H). Irradiating at 6 3.48‘and 3.56 caused the respective singlets at 6 7.98 and 9.38 to increase in intensity. UV-vis luax(:u) 692 nm (30 100), 670 (35 200), 636 (24 300), 448 (65 400), 430 (67 400). MS found: m/e 599.3411 for (M+H)*, C36H47N4028 requires m/e 599.3423. 118: NMR 6 0.18, 0.26, 0.60 (6H each, t, CH2C§3 sat), 1.57, 1.63 (6H, t, CH2083), 2.30 (1H, br 3, NH), 2.34-2.64 (12H, m, CH2CH3 sat), 3.51, 3.58 (2H each, q, 082083), 8.04 (1H, s, 5-H), 8.10 (1H, s, 15-H), 8.26 (1H, s, lO-H), 9.34 (1H, s, 20-H). Irradiating at 6 3.51 and 3.58 caused the respective singlets at 6 8.10 and 9.34 to increase in intensity. UV-vis Maiden) 767 nm (50 400), 698 (26 300), 652 (9 400), 486 (67 600), 454 (47 300), 445 (45 500), 422 (37 900), 388 (21 700), 376 (20 400); MS found: m/e 615.3200 for (M+H)+, 036H87N4082 requires m/e 615.3195. 119: NMR 6 0.18, 0.28, 0.54 (6H each, t, CH2CH3 sat), 1.57, 1.64 (3H each, t, CH20H3), 1.92, 2.22 (1H each, br 8, NH), 2.32-2.81 (12H, m, 082083 sat), 3. 53, 3. 61 (2H each, q, 082083), 7. 92 (1H, s, lO-H), 8.08 111 (1H, s, 15-H), 8.59 (1H, s, 5-H), 9.53 (1H, s, 20—H). Irradiating at 6 3.53 and 3.61 caused the respectiv‘e singlets at 6 8.08 and 9.53 to increase in intensity. UV-vis Max (an) 794 nm (22 700), 692 (32 700), 646 (15 500), 556 (9 100), 498 (31 700), 468 (75 700), 443 (34 400), 396 (21 000); MS found: m/e 615.3180 for (M+H)*, 0351147N40S2 requires m/e 615.3195. 120: NMR 6 0.19, 0.26, 0.30 (6H each, t, 01120113 sat), 1.60, 1.65, (38 each, t, 01120113 sat), 3.56, 3.62 (2}! each, q, 01712083), 8.19 (11!, s, l5-H), 8.46 (111, s, 10-H), 8.60 (1H, s, 5-H), 9.48 (111, s, 20-H). Irradiating at 6 3.56 and 3.62 caused the respective singlets at 6 8.19 and 9.48 to increase in intensity. UV-vis flux (at) 784 nm (50 500), 718' (26 100), 595 (10 600), 505 (65 800), 423 (20 000); MS found: m/e - 631.2975 for (M+H)+, 035114711453 requires m/e 631.2967. 313.7,8:12.13.17118799tasthy1-2-metbylfztnstgaptochlorin.(121) Thione 111 (10 mg, 0.018 mmol) was dissolved in dry THF (10 m1) and treated with a two-fold excess of CHaLi (1.4 M in ether) at R.T. under argon. After a few minutes the reaction was quenched with water (2 ml). The organic layer was separated, dried and evaporated to dryness. The residue was redissolved in 082012 and passed through a short silica gel pad (0112012 as eluant) to afford 121 in almost quantitative yield. NMR 6 0.73, 0.99 (311 each, t, 01-12083 sat), 1.62 (3H, s, Me), 1.81, 1.86 (12H, 61! each, t, 01120113), 2.15, 2.49, 2.89 (18, 211,111 each, m, CHzCH—a sat), 3.90, 3.93, 4.03 (21! each, q, 0820113), 5.65 (IR, 3, SH), 8.81 (1H, s, 5-H), 9.32 (1H, s, ZO-H), 9.75, 9.77 (IH each, 10,15-H), -2.53 (2H, br 3, NH). Irradiating the singlet at 6 1.62 caused the singlet at 6 9.32 to increase in intensity. UV-vis Max 644 '112 nm, 615.5, 590.5, 524.5, 496, 393; MS (direct probe, 70 eV) m/e 582 (M*). 813,728,12J13A17,18f99taethyl-2-mercaptochlorin (132) Thione 111 (10 mg, 0.018 mol) was dissolved in dry THF (10 m1) and a three-fold excess of Nam-14 in 0113011 was added. After 10 min at R.T. , the reaction was worked up in the same manner as described for 121 to give (122), quantitatively. NMR 6 0.51, 0.94 (311 each, t, 01120113 sat), 1.79, 1.84 (9H each, t, 011201713), 2.34 (111, d, SH), 2.34, 2.58, 2.70 (2H,1H,1H each, m, 01120113 sat), 3.88, 3.95, 4.01 (411 each, q, 01120113), 5.92 (1H, d, 2-11), 8.79 (1H, s, 5-H), 9.35 (1H, s, 20-H), 9.76 (2H, s, 10,15-H), -2.55 (2H, br s, NH). Irradiating the doublet at 6 < 5.92 caused the singlet at 6 9.35 to increase in intensity. UV—vis 7.331(1)!) 644 nm (38 600), 614 (4 300), 591(4 000), 524 (3 600), 496 (11 700), 489 (11 700), 392 (143 000); MS (direct probe, 70 eV) m/e 568 (M'*). 2,2,7,8,12,l3,17,18—Octaethy1chlorin (123) Thione 111 (10 mg, 0.018 mol) was dissolved in dioxane (10 m1) and an excess of freshly prepared Haney Nickel-11288 was added under argon. The reaction mixture was heated in an oil bath at 60°C for 10 min, followed by filtration through a glass wool. The filtrate was evaporated to dryness to give 123 quantitatively. NMR 6 0.68 (6H, t, 011201113 sat), 1.80 (181-I, m, 01120113), 2.41 (411, q, 011120113 sat), 3.87, 3.90, 4.00 (4H each, q, 01120113), 4.60 (2H, s, 3,3-H), 8.71 (1H, s, 5-H), 8.96 (1H, s, 20-H), 9.71, 9.74 (111 each, 10,15-1-1), —2.53 (1H, br 8, NH). Irradiating the singlet at 6 4.60 caused the singlet at 6 8.96 to 113 increase in intensity. UV—vis Max 643 nm, 613, 589, 521, 495, 488, 391; MS (direct probe, 70 eV) m/e 536 (M‘). General Procednrs for Zips 294 QQRPEEWJQ§§§1399 To a solution of the free-base porphyrinoid (10 mg) in boiling chloroform (10 ml) was added a saturated solution (1 m1) of the metal (II) acetate in methanol, followed by the addition of sodium acetate (~2 mg). After ~30 min refluxing and checking by TLC (metal-complex moves slower than the corresponding free-base), the solvents were evaporated in vacuo and the residue was redissolved in 0H2012/HzO (1:1). The organic phase was separated, washed twice with water, dried (Nastu) and evaporated to dryness to give the metal complex in quantitative yield. The 1H NMR peaks of the zinc complexes are generally shifted upfield in relation to their free bases. m4 KINETIC AND EOUILIBRIIH STUDIES OF 00 AND 02 BINDIm “1'0 mass HEART MYOGIDBIN WTITUTED WITH SYNTHETIC MS I. INTRODUCTION Reconstitution of heme proteins is often used as a method of probing the prosthetic group structure-protein function relationship. To date, heme proteins that have been successfully reconstituted with foreign hemes or recombined with the native prosthetic group include myoglobin, hemoglobin, peroxidases, catalase, cytochrome P-450, b-type, and c-type cytochromes as well as cytochrome 9511 .100 Among these, myoglobin is the simplest system for monitoring perturbations brought by heme structure changes. Myoglobin (Mb) is a monomeric protein of 160 amino acid residues (m 17,800) and one molecule of heme (Protoheme IX). M) is found in the skeletal muscles and stores diogygen, transported to it by hemoglobin (11b) for use in the mitochondria.101 A generic structure for M) is shown in Figure 16A. A closer examination of the region near the heme in the deoxy form reveals the iron atom in a square pyramidal structure, the equator defined by the porphyrin plane and an axial position occupied by the imidazole of histidine F8 which is called the ”proximal histidine” (Figure 168). The iron atom is approximately 0.5A out of the plane of the porphyrin and the Fe-N (imidazole) bond vector is 114 113 F-Mlix PROXIMAL HISTIDINE (F81 HEME Gaoup DISTAL VALINE (E II) DISTAL HISTIDINE (E 7) E-hoflx Figure 16 The low-resolution structure of Mb (A); the 02- binding site in Mb (8). 116 approximately 8° off the heme normal.”2 The iron atom is high-spin (8:2) Fe(II). Upon binding a sixth ligand (00 or 02), Mb undergoes a change in the tertiary structure that places the iron within 0.2A.of the porphyrin plane, resulting in a diamagnetic, low-spin (S=0) Fe(II) for carbon-monoxymyoglobin (WCO).1°3 M has been discussed as various spin-coupled system.104 The ligand binding properties of myoglobin as well as myoglobin reconstituted with a variety of porphyrin hemes modified at the side chains, have been extensively investigated.1°5‘115 Studies on myoglobin containing saturated macrocycles are limited to only few systems which include sulfmyoglobin79'»"79'11° chlorophyllide-Mb117 as well as iron and cobalt complexes of pheophorbide—g—bb, pyro-, meso—, and mesopyropheophorbide-g-Mb.118 In each of these cases however, greater structural perturbation than simple pyrrole ring reduction has been involved. As part of an effort to probe structure-function relationships of green heme enzymes , we report here the oxygen and carbon monoxide binding behavior of horse heart myoglobin reconstituted with several synthetic green heme (124—130) shown in Figure 17. We believe that these derivatives are more useful approximations of the iron-prosthetic groups found in naturally occuring chlorin and isobacteriochlorin containing enzymess»54'55 than the previously reported cases. Moreover, the dehydrogenation (oxidation of chlorin to porphyrin) problem often encountered with regular chlorin hemes119 is not present in our stable systems. Finally, the static and kinetic parameters measured from reconstituted myoglobins may be closer to the primary systems than those obtained with isolated hemes in organic solution.119:12° 11" Ho,c 124 60:11 PI Ho,c COM HON: cont-I HO c 126 127 ' 128 60'" Ho,c 129 cog-I Ho,c 13o co," Figure 1? Structures of synthetic green hemes used for myoglobin reconstitutions. 118 11. RESULTS AND DISCUSSION The kinetic and equilibrium constants for 00 and 02 binding to the reconstituted Mbs of this study are summarized in Table 5. The results indicate that substitution of the protoheme with saturated hemes, particularly 126-130 affects the ligand binding constants. Focusing on methylchlorin hwe-Mb 128, it can be seen that the carbon monoxide association rate 9.’ underwent an almost seven-fold increase in comparison with the natural protoheme-fl). This can arise from the fact that chlorins have intrinsically larger cores than porphyrins13°b and can more easily accommodate the low—spin six— coordinate Fe(II) atom which moves towards the plane of the macrocycle. Clearly, other explanations may be offered. It could. be argued that chlorins are weaker bases than porphyrins causing an Fe(II) chlorin to be a stronger Lewis acid than an Fe(II) porphyrin towards the substrate ligand such as 00.1 In fact, it is well known that free-base chlorins are weaker Bronsted bases than free-base porphyrins}, ”1 However, metal-ligand affinities could be governed not by the basicity of the free-bases, but by the total basicity of the macrocycle dianions, of which nothing quantitative has been reported. The oxygen association rate, k’, for the same myoglobin has experienced only a two-fold increase, in comparison with the native Mb. Perhaps, triplet oxygen can induce the S=0 state of iron even before the heme becomes planar, consequently, diminishing the core-size effect. In the case of 00, this singlet molecule must combine with a high spin Fe(II) giving a singlet product. To pair these spins, the necessity for spin inversion could retard the rate of reaction.122 119 .OoNNION .¢.b Ea .hflhhzn uugnaOfia i A: GHQ mes posxm.e ~m.~ meo.o ocsx~.m m~o.c “ease oaasiaaoseuossso< e.HH~ posxm.m an.“ mmc.o oosxm.m mHo.c Assay oamsnonowa ”.mm posxm.¢ «m.o mmo.c .e~x~.¢ m~o.o Amuse onuanamuosaosssuoz was pofixw.m mm.~ o-.o ocfixm.m emo.o Abuse oamazosmxum m.~m posxo.¢ as.c s-.o ocsxe.m meo.o Amuse onasuosomus ~.m~ bogus.fi me.c smo.o nosxo.m «mo.o Amugv a o-wsosouaosogm ¢.~N .onm.~ mm.c one.c sedan.“ mmo.c Aeuflv < o-«sososnosoam ms .cflx¢.~ oe.o «No.9 nofixv.m mmc.o mausososm ATE.— ATmTzv... 2.8335 A73... TTmTzvk 2.3335 2333»: No as“: cossoawm on as“: consume: ..uaseawm No ecu on you usaauuaoo nassnssflaom was assoc“: .u wanna 120 Focusing on a different chlorin heme, 8-keto heme-Mb 127 the electronic‘effect can be monitored on the oxygen dissociation rate constant, k. The presence of the e‘ withdrawing oxygen atom in conjugation with the ring causes k to increase while k’ remains fairly constant. Therefore, the Puz°2 is larger. This indicates that decreasing a" density in the iron d orbitals could lessen the Fe-Oz r- backbonding and effectively weaken the Fe to 02 bonding. For the CO ligand, the same trend in 9. value can be seen, but to a lesser extent. This suggests the predominant role of t bond formation in determining the iron-carbon monoxide binding while rbond break-up may be predominant for iron-oxygen binding.111 To probe the protein effect, 7-keto heme-M) 126 possessing the oxo group on the same pyrrole ring but on the other f-pyrrole position has been examined in comparison with B-keto heme-M) 12?. These two Mas show differences in their electronic spectra, particularly in their met (ferric) forms. Moreover, the absorption maxima of 7-keto heme-M) 126 are shifted slightly, bathochromically relative to 8-keto heme-M: 127 (Figure 18). In contrast, visible absorption peaks of pyridine hemochrome of both hemes are essentially identical (see Table 6). In addition, their ligand binding constants are quite different (see Table 5). This can only be attributed to modulations by local environment. To verify these results, PaA-M) 124 and Pan-M) 125 (photoprotochlorin Mbs saturated at either ring A or B) were tested and indeed showed differences in electronic spectra (Table 6) and binding. properties (Table 5). Although these chlorin Mbs behave differently from the previously discussed chlorin Mas (126-128), due to the conjugation of a formyl group with the macrocycle, they offer another sbsorbancs absotbanee ...... .. _ fib- {>- 560 660 700 wavelength, nm Figure 18 Optical spectra of 7-keto-heme myoglobin (A); 8- kcto—heme myoglobin (B); Ferric (--), Deoxy (—')o OXY (_—)a C0 (---)a in 10 "‘4 (PH 7'4) potassium phosphate buffer. Table 6. Absorption Myoglobins.a Spectral 122 Maxima of Synthetic Hemes and Mb/Heme Soret (nm) Photoprotoheme A (124) Visible Bands (nm) met Mb 408.5 500, 602 deoxy Mb 431 562 02 Mb 414 542, 578, 598 CO Mb 421 542, 576, 594 Pyridine hemochromeb 419.5 558 Photoprotoheme B (125) met Mb 406 599, 500 deoxy Mb 428 593, 556 02 Mb 409 594, 544 CO Mb 415 593, 538 Pyridine hemochromeb 418.5 556.5 7-keto heme (126) _ met Mb 411 624, 672 deoxy Mb 406, 425 607 02 Mb 419 625 CO Mb 420 615 Pyridine hemochromeb 414 589 8—keto heme (127) met Mb 404 676 deoxy Mb 400, 422 608 02 Mb 414 620 CO Mb 418 612 Pyridine hemochromeb 415 590 methylchlorin heme (128) met Mb 390 602 deoxy Mb 408 616 02 Mb 400 624 C0 Mb 401 614 Pyridine hemochromeb 415 598.5 Dione heme (129) met Mb 388, 424 634, 638 deoxy Mb 398, 436 616 CO Mb 394, 439 628 Pyridine hemochrome° 395, 319, 445 605 Acrylo-dione heme (130) met Mb 411 643 deoxy Mb 424 632 CO Mb 410, 434 644 Pyridine hemochromeb 376, 404, 450 626 aIn phosphate buffer, pH 7.4; bIn pyridine-1X NHéNHz. 123 example in which the Mb reaction parameters can be affected upon changing the position of the "northern" saturated pyrrole ring. A similar case reported by Sono at 31.105 noticed that Mbs reconstituted with spirographis and isospirographis hemes exhibit different absorption maxima even though the two hemes have the same spectrum outside the protein. The stability of the various chlorin-containing oxymyoglobins was examined and their autoxidation kinetics are presented in Figure 19 in comparison with the native myoglobin. As is apparent, isomers PaA-bez/ Pan-W02 and 7-keto heme—b02/8-keto heme-W02 exhibit different autoxidation rates. PaB—WOz and 7-keto heme-+1302 are about two times slower towards autoxidation than their corresponding isomers. In view of the fact that Pea—Mb and 7-keto heme-M) have slower CO association rate constants (11’) when compared with their corresponding isomers, it would appear that the heme pocket is tighter around the ligand binding site that stabilizes the oxyhme. This stabilization is also reflected in the k’ values which are smaller in the PaB-Mb and 7-keto heme-M) cases. On the other hand, the autoxidation rate of native myoglobin and 125, 126, 128, the are approximately within experimental error with one another. This suggests that the saturation of a porphyrin ring alone, does not perturb the protein native structure and produces stable myoglobins. However, both the position of the saturated pyrrole ring and the side chains are crucial in determining myoglobin’s stability. Myoglobin has also been reconstituted with two porphinedione-hemes (heme g; analogues). However, the soret/Azao ratios (1.1 for dione heme-M) .129 and 0.6 for acrylo—dione heme-m) 130 indicate that these hemes do not fit well in the myoglobin pocket, particularly heme 130 D ’32’ ‘t I I 5 0 >? m < I | I 2 V I I: A "'1 - " A A A; " 4. I A m ’A . ’ . A e .’.’ A}‘,m0 /e L L l 1 2 3 Time (hr) Figure 19 Autoxidation of reconstituted myglobins at 22°C in lOmM (pH 7.4) potassium phosphate buffer, saturated with 02. Protoheme (native)C), slope 8.6x10‘5, r = 0.989; 933., slope = 9.0x10-5, = 0.984; methylchlorin-hemefik, slope = .7x10‘5, r = 0.999; 7-keto-hemeA, slope = .3x1o-4, r = 0.999; PaAtj, slope = 1.9x10'4, r 0.989; 8—keto-hemezll, slope = 2.5x10“, r = .996. Our—om“! II 125 that possesses one acrylic propionic acid chain. The significance of the two propionic acid groups of the heme at positions 2 and 18, which constitute hydrogen bonds with Arg C03 and His F62, respectively, of the apoprotein, has been addressed by Ogoshi.113 129-Mb and 130+!) retained the ability to bind CO in the ferrous state but bleached rather quickly upon exposure to oxygen. Apparently the macrocyclic ring destruction is faster than iron autoxidation in these cases. It is quite possible that the quinone porphyrin ring structure of these heme g1 analogues reduces 02 and generates its own hydrogen peroxide equivalent, which, in turn, might attack the macrocycle ring, resulting in its oppening.123 The fact that the protein conformation around these hemes is significantly altered due to the introduction of a second pair of angular methyl- groups should contribute to the instability of the oxy-form. In any case, this is a point that merits further investigation. III. SWAIN The present studies establish that apomyoglobin can be reconstituted with chlorin hemes to produce stable forms of protein that retain the ability to bind dioxygen and carbon monoxide in the reduced state. In comparison with the native Mb, the generally faster association rates observed in the green hemes might be attributed to their larger core size which facilitates the spin state change during ligand binding. In an attempt to probe the effect of the degree of ring saturation on the protein function, we reconstituted H) with two isobacteriochlorins, having the core structure of heme 311. Although these synthetic isobacteriochlorin-hemes didn’t provide us with any further clue for possible relationship between protein function and the degree of ring saturation, an interesting phenomenon was observed: 126 their ferrous forms bleached upon exposure to oxygen. Further studies of this phenomenon are necessary. For example, it would be interesting to see if the heme destruction was due to the protein environment or to an intrinsic property of this heme. IV . MATERIALS AND METHODS Preparation of Hemins. Photoprotoporphyrin IX dimethyl ester A(P3A) and B(PaB) were synthesized according to a literature procedure.124 Dimethyl 3, 8, 8, 12 , 13 , 17-hexamethyl-7—porphinone-2, 18-dipropionate (21a), dimethyl 3,7,7,12,l3,l7—hexamethyl-8-porphinone-2,18-dipropionate (22a), dimethyl 3, 7, 7 , 8, 12 , l3 , l7-heptamethy1chlorin-2, lB—dipropionate (24) , dimethyl 3 , 8, 8, l3 , l3, l7-hexamethyl-7 , lZ-porphinedione-Z, lB-di- propionate (69) and methyl 18-[2—(methoxycarbonyl)ethenyl]-3,8,8,l3, 13,17-hexamethyl-7,lZ-porphinedione-Z-propionate (70) were synthesized as described in Chapter 1 of this thesis. Iron insertions were accom- plished by the ferrous bromide methodlzs (PaA, P38 and methylchlorin 24) and by the ferrous sulfate method12° (porphyrinones 21a, 22a and porphyrindiones 69, 70). The ester groups of these hemins were hydrolyzed in 1:1 THF/2N KOH at R.T. as described for 22s (Chapter 1) to afford the diacid hemins shown in Figure 17. Preparation of Myoglobins. Horse heart myoglobin (Sigma, Type III) was used as received. Apomyoglobin was prepared from myoglobin, by the acidified butanone procedure.1°°»127'12° The concentration of apoprotein was determined on the basis of its absorbance at 280 nm (£230 = 15.4 film-”.129 For reconstitution, a 1.2-fold excess of green hme to apoprotein was dissolved in a minimal volume of 1% KOH/MeOH and added to the 127 apoprotein solution (0.2—0.5 M) at 0°C. The resultant solution was gently stirred for 20 min at 0°C (frothing should be avoided as it denatures the protein). The mixture was then adjusted to pH 8.0 with 0.2 M Tris-HCl buffer (PH 5.0) and dialyzed (Spectrapor: 25 or 45 mm x 100’ membrane tubing) against 10 dd phosphate buffer (pH 7.0) and against distilled water twice. The deionized Mb solution was centrifuged (15 KG at 4°C), and loaded onto a DB 23 column that was equilibrated with 10 11M phosphate buffer (pH 7.4). The column was developed. with the equilibrating ‘buffer at 4°C. Excess heme was adsorbed on the top of the column, while the reconstituted myoglobin was easily eluted from the column (reconstituted myoglobins have bathochromically shifted absorption maxima relative to their free hemes. and possess a new absorption peak at ~280 nm). Reconstituted Mas had Asorot/Aaso ratios: ~3.0 (PaA, P38, porphyrinones 126, 127 and methylchlorin 128 Mbs), l.l (dione 129 5b) and 40.6 (acrylo—dione 130 m). In the last two eased the Aunt/A230 ratio was increased upon storage (-70°C freezer).' For kinetic and equilibrium measurements, solutions of met Mas (~10‘5M) in pH 7.4, 10 mM potassium phosphate buffer were degassed in a 120 ml tonometer by freeze-pump-thraw cycles at 10'5 Torr and were reduced with a minimum amount of aqueous sodium dithionite in an argon atmosphere. Kinetic andwlfgquilibrim Measurements. Kinetic rates were measured at 22°C by flash photolysislzz't 13° according to: 2’ Fe + CO F9 FeCO 9. (hv) 128 kl Fe+02 <=‘- Fe02 R Fe02 " FeCO Flash photolysis was carried out with either a Xenon photographic flash gun (Braun 2000) or a flash lamp pumped dye laser (Phase-R DL 2100) with rhodamine 66 dye. ‘With carbon monoxide present, 23 was measured directly as a pseudo-first order decay, R’[CO] = R’obs which included concentration of CO (Figure 20s). However, when oxygen was added, two decays were observed (Figure 20b). The fast decay to a certain absorbance Anoz is k’[02] = k’obs followed by a decay R from this. absorbance to Anco. CO association rates (11’) were calculated from plots of the observed pseudo-first order rate constants vs. CO concentration and 02 association rates (k’) were calculated from similar plots. Puz°2 (oxygen pressure at half saturation) were obtained from direct titration or calculated according to the Gibson equationzlaob 1/R = l/k + K[02]/!f[CO] where R’[C0] is the pseudo-first order rate constant measured before introducing'Oa, H is the displacement rate of oxyMb by CO, k is the 02 dissociation rate constant and K is the 02 equilibrium constant. Carbon monoxide affinities (L) were determined by direct titration of each Mb with a gas mixture containing 1% CO in nitrogen, at H.T., using a standard spectrophotometric procedure developed by Halpern and coworkers131 (Figures 21, 22). The dissociation rate constants R and k were calculated from L = 5179. and k = k’/k, respectively; where L = (Pm:co x 1.35x10"5 M/torr)‘l and K = (Pl/202 x 1.80x10'5 M/torr)'1. Optical spectra were recorded on a Cary 219 spectrophotometer. Figure 20 (a) Oscilloscope trace of absorbance vs. time for the recombination of PaA-Mb and CO after flash photolysis at 22°C in 10 mM (pH 7.4) potassium phosphate buffer. [CO] = 5.25x10'5M; sweeptime : 1,5,20 msec/div.; wavelength = 420 nm. (b) The recombination of PaA-Mb with 02 and CO at 422 nm. [C0] = 5.25x10'5, [02] = 3.49x10'5M. Upper trace; sweeptime = 0.1, 0.2 sec/div.; lower trace; sweeptime = 0.2, 1 msec/div. ABSORBANCE .0 a: 2.0 is 0.0 130 —> A-Ao/AQ-A (I p o s 10 15 :0 Mb [to] x 10' in C0,}! X4 L 1 1 1 1 300 400 500 600 700 }\ nm Figure 21 Spectrophotometric titration of 7-keto—heme myoglobin in 10 n“ (pH 7.4) potassium phosphate buffer, with CO at R.T.; [C0]x103M = 0.0, 2.63, 5.26, 9.65, 17.55 and 1760. Inset: plot of A- A./Aa-A vs. [CO] at 615 nm. ABSORBANCE 1 / o s to u [‘0] X 10' w 20 1 3C“) Figure 22 1 <4CH) 5CK) GCK) 71X) Anm Spectrophotometric titration of dione-heme: myglobin in 10 mM (pH 7.4) potassium phosphate buffer with CO at R.T.; [CO]x103M = 0.0, 2.66, 5.32, 9.31, 15.97, 1346. Inset: plot of A- Ao/AarA vs. [CO] at 628 nm. mmnnncss AND NOTES 10. 11.. REFERENCES AND NOTES An exhaustive compilation on the ch-istry, physics, and biology of porphyrins appeared as the multivolume set entitled "The Porphyrins," Dolphin, 0., ed., Academic Press, New York, 1978. Shceer, H. In ”The Porphyrins,” Vol. II, Part B, Dolphin, 0., Ed., Academic Press, New York, 1978, Chapter 1. (a) Katz, J.J. In "Inorganic Biochemistry,” Vol. 2, Eichhorn, G.L., Ed., Elsevier Publishing 00., Amsterdam, 1973, Chapter 29. (b) Clayton, R.K.; Sistrom, W.H. "The Photosynthetic Bacteria," Plenum Press, New York, 1978. ' Agius, I..; Ballantine, J.A.; Ferrito, V.; Jaccarini, V.; Murray-- Rust, P.; Pelter, A.; Psaila, A.F.; Schewri, P.J. Pure 8. Appl. 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(a) Tamure, M.; Asakura, T.; Yonetani, T. Biochim. Biophys. Acta 1973, 266, 467. (b) Tamure, M.; Woodrow, 6.7, III: Yonetani, T.; Biochim. Biophys. Acta 1973, 617, 34. (a) Traylor, T.G.; Chang, C.K.; Geibel, J.; Benzinis, A.; Mincey, T.; Cannon, J. J. Am. Chem. Soc. 1979, 161, 6716. (b) Antonini, 8.; Bruhori, M. In ifHemoglobin and Myoglobin in Their Reactions with Ligands," North-Holland, Amsterdam, 1971. Marzilli, G.L.; Marzilli, P.A.; Helpern, J. J. Am. Chem. Soc. 1971, 66, 1374. APPENDIX 140 .Asesv oossossosossims.musssossosssooasxosxss.m~.-.m.s.m -sxosossseum.susso assooaso so assoooos :22 e. was com s< senses to... . as... s.m.. as as a... e.m as as: —b:bbbhbb—bbbbbbbbb—bbbbbbbbbhthtbbfibil—Ibbhbbbhhb—bebbhhbb —bhhbbbLbbhhbbtbbt—bbhbbhbhb_hbhhbhE —hbbhbbbb as . ... 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I b 64! 1 1 1 Q 1 1 1 k 1 400 ~ 500 600 700 wavelength, nm Figure A41 Visible spectra (in CH2C12) of (a) 3,3,8,8,12,13,17,l8—octaethyl-Z-thio—7-porphine— dione (112); (b) its Zn—complex. absorbance absorbance 181 b p 758 468 4| I p 440 p p 1 1 1 l 1 1 L 1 l I I I I I r I h F a P 455 701 83' 746 1 l L l l L J J 400 500 600 700 wavelength, nm Figure A42 Visible spectra (in CH2C12) of (a) 2,2,8,8,12,13,17,18—octaethyl-3-thio-7-porphine— dione (114); (b) its Zn-complex. absomanco ODSOIDIHCO 182 I I I I I I T I r b 551 ,— ‘1 b '1 . 9 Q q 454 n32' q 720 499 ‘ 389 756 .- 1 P -l l 4 l l l l L l l r I I I I I I I f 779 a - -4 h '1 h d 431 q d .1 579 735 p l Figure A43 460 J 56’_"'—"'6—'——“60 60 70 L wavelength, nm Visible spectra (in CH:C12) of Zn-conplexea of (a) 3,3,8,8,12,13,17,lB-octaethy1-2,7- porphinedithione (113); (b) 2.2.8.8,12,13,17,18- octaethyl-S,7-porphinedithione (115). 183 I d -4 d d o g -1 Q a b o Cl a a o d d _..l_ I q .1 7.7 d . d 0 e g -1 b o . d g 0.. c1 .5 .4 .- d l I J l l 1 ¥ 1 l I I I I l I l I I >- «I '1 P . ~ 1 p- '1 O '- fl’ .1 o I: 3 d 3F 0 a Q. a " 4n '“ '4 b- 7.‘ I1 390 L- 4 54 35. q 1 l J l 400 500 : 0'0 0 o ; o o wavelength, nm Figure A44 ViS1ble spectra (in CH0012) of (a) 3,3,8,8,13,13,17,18- octaethyI—Z-thio—7,12-porphinetrione (117); (b) 3,3,8,8,13,13,17,18-octaethy1-2,12-dithio-7-porphinetrione (118); (c) 3,3,8,8,13,13,17,18-octaethy1-2,7-dithio-12- porphinetrione (119). 184 absorbance absorbance absorbance 1 1 500 600 700 Wavelength, um I 300 400 Figure A45 Visible spectra (in CH2C12) of (a) dihydro-OEP; (b) tetrahydro—OEP; (c) hexahydro-OEP (under argon). 185 absorbance 390 H 40! ‘408 ”b I | I 400 ”0.: 60‘” I . ' —- Fem: ’ I ‘ —- Deoxy -‘ M O" 6 l 4 \I co A I 0 \ ‘I I 1 ,g/ \ 660 700'— wavelength, nm 1 1 l 300 400 500 Figure A46 Optical spectra of methylchlorin-hele myoglobin; Ferric ( ), Deoxy ( ), Oxy (-——--——), CO (----) in 10 mM (pH 7.4) potassium posphate buffer. 186 absorbance l l I 166 Wavelength, nm Figure A47 Optical spectra of dione~heme myoglobin; Ferric (--). Deoxy (--—). CO (--). in 10 mM (pH 7.4) potassium posphate buffer.