GREEN POLYMER CHEMISTRY: THE ROLE OF CANDIDA ANTARCTICA LIPASE
B IN POLYMER FUNCTIONALIZATION
A Dissertation
Presented to
The Graduate Faculty of The University of Akron
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
Yenni Marcela Castaño Gil May, 2014 i GREEN POLYMER CHEMISTRY: THE ROLE OF CANDIDA ANTARCTICA LIPASE
B IN POLYMER FUNCTIONALIZATION
Yenni Marcela Castaño Gil
Dissertation
Approved: Accepted:
Advisor Department Chair Dr. Judit E. Puskas Dr. Coleen Pugh
Advisor Dean of the College Dr. Matthew L. Becker Dr. Stephen Z. D. Cheng
Committee Member Dean of the Graduate School Dr. Abraham Joy Dr. George R. Newkome
Committee Member Date Dr. Toshikazu Miyoshi
Committee Member Dr. Chrys Wesdemiotis
Committee Member Dr. Nic D. Leipzig
ii ABSTRACT
The objective of this research was the precise functionalization of polymers using two methods: enzymatic functionalization by chain end functionalization and enzyme- catalyzed functionalization via polymerization. The polymers obtained through this method are intended to be used in cancer drug delivery systems.
The first method is described in Chapter IV. Enzymatic functionalization of poly(ethylene glycol)s (PEGs) with various functionalities (halo-esters, azide, and acrylate) using Candida antarctica lipase B (CALB) as a biocatalyst under solventless conditions is described. 2,2'-(ethane-1,2-diylbis(oxy))diethanamine was reacted with 3-
(acryloyloxy)-2-hydroxypropyl methacrylate in the presence of CALB as a model reaction for the selective addition of diamine polymers to the acrylate group. Exclusive conjugation in two cases, the model compound and the NH2-PEG-NH2, to the acrylate group was observed. This Chapter IV also discusses the functionalization of small molecules using CALB.
The second method is discussed in Chapter V. Well-defined poly(caprolactone)s were generated using alkyne-based initiating systems catalyzed by CALB. Propargyl alcohol and 4-dibenzocyclooctynol (DIBO) were shown to efficiently initiate the ring- opening polymerization (ROP) of -caprolactone (-CL) under metal free conditions. In addition, the CALB- catalyzed transesterification of divinyl adipate (DVA) with tetraethylene glycol (TEG) and PEGs under solvent-free conditions are also discussed.
iii During transesterification with TEG, polycondensation occurred. With the judicious selection of molar equivalencies and reaction times both symmetric and asymmetric telechelic TEGs can be produced with high efficiency. In the case of PEGs, quantitative vinyl chain-end functionalization of PEGs with DVA can be reached using DVA/PEG
20/1 molar ratio.
In the same chapter, the synthesis of poly(isobutylene-b--caprolactone) PIB-b-
PCL diblock and poly(-caprolactone-b-isobutylene-b--caprolactone) PCL-b-PIB-b-PCL triblock copolymers was described using a combination of living carbocationic polymerization of isobutylene (IB) with the CALB catalyzed ROP of -CL.
The polymers obtained through this method are intended to be used in cancer drug delivery systems. A modular synthetic approach for the synthesis of a vitamin-polymer conjugate using enzymatic catalysis was proposed. Chapter VI covers the chemo- enzymatic approach for biomolecules functionalization for this application. Precisely:
Targeting (Folic Acid/ Biotin), drug (Fluorouracil) and imaging (FITC) agents were functionalized using CALB-based methods.
In order to make a biodegradable PIB, Chapter VII describes the synthesis of low molecular mass telechelic PIBs. It began with the synthesis of IB oligomers. After that, it was of interest to determine whether and under what conditions would the tert-alcohols
(TMHDiOH), tert-chloride (TMHDiCl) and tert-ether (TMHDiOMe) diinitiator with
TiCl4 complex lead to well defined Cl-PIB-Cl. A new telechelic polyisobutylene diol,
HO-PIB-OH, carrying two terminal primary hydroxyl end groups was prepared.
iv DEDICATION
This dissertation is dedicated to my family: my role model, whom I admire and love, my mom, Maria Flor Gil; my father, Fabio Castaño; my siblings, Yazmin, Astrid y Brayan; my grandmother, Nena; and my husband, my eternal love, Mauricio Echeverri for all of their love and support.
v ACKNOWLEDGEMENTS
I would like to thank to my PhD advisors, Professors Judit Puskas and Matthew
Becker, for supporting me during my graduate studies. Dr Puskas is a great scientist. She is an inspiration for the role that women play as scientist. Dr Becker is someone you will instantly love and never forget once you meet him. He is one of the smartest people I know. I could not have asked for better role models, each inspirational, supportive, and patient. I also have to thank the members of my PhD committee, Professors Chrys
Wesdemiotis, Abraham Joy, Toshikazu Miyoshi, Abraham Joy and Nic D. Leipzig who provided encouraging and constructive feedback.
I thank all the past and present members of the Puskas and Becker’s group. Thank you very much for the helpful discussions and the wonderful memories we built together during these years. Special thanks to Dr Kwang Su Seo for his scientific advice and knowledge and many insightful discussions and suggestions as well for his friendship.
Finally, I would like to express my very great appreciation to all my friends: The ones who I left in Colombia and the ones that I met during my PhD and became my family here in Akron. Thanks to my family who believe in me. Thanks to my husband,
Mauricio, for his trust, love and wonderful sense of humor. You all are my inspiration!
vi TABLE OF CONTENTS
Page
LIST OF TABLES……………………………………………………………………….xv
LIST OF FIGURES……………………………………………………………………..xvi
CHAPTER
I. INTRODUCTION ...... 1
II. BACKGROUND ...... 9 2.1. Enzymes...... 9
2.1.1. Candida Antarctica Lipase B (CALB) ...... 11
2.1.2. CALB-catalyzed Transesterification ...... 12
2.1.3. CALB-catalyzed Michael Addition Reaction ...... 16
2.1.4. Enzymes in Polymer Chemistry...... 19
2.1.4.1. Enzyme-catalyzed Polymer Synthesis ...... 19 2.1.4.2. Enzyme-Catalyzed Post-Polymerization Functionalization of Polymers 33 ……………………………………………………………...33 2.2. Polymeric Conjugates for Cancer Treatment ...... 42
2.2.1. Vitamin-Based Drug-Delivery Systems ...... 42
2.2.1.1. Folic Acid ...... 43 2.2.1.2. Biotin ...... 50 2.2.1.3. Other Vitamins Commonly Used for Drug-Delivery Application 52 52 2.2.2. Modular Approach for the Synthesis of Polymer Drug Conjugates .. 54
vii 2.3. Synthesis of Polyisobutylene ...... 57
2.3.1. Cationic Polymerization...... 57
2.3.2. Living Cationic Polymerization of Isobutylene ...... 58
2.3.3. End-Functionalized PIBs ...... 59
2.3.3.1. Initiation from Functional Initiators ...... 59 2.3.3.2. End-Capping by Functional Terminators ...... 60 2.3.3.3. Functionalization by Post-Modification Processes ...... 62 2.3.4. Telechelic HO-PIB-OH ...... 62
2.3.4.1. Polyfunctional Initiator-Transfer Agents Inifer Technique ...... 63 2.3.4.2. Bifunctional Aliphatic Initiator System ...... 64 2.3.5. PIB-Based Biomaterials Tested for Biomedical Applications ...... 65
2.3.5.1. PIB-Based Thermoplastic Elastomers for Vascular Grafts ...... 66 2.3.5.2. PIB–Poly(Methyl methacrylates) (PMMA) for Toughening of Bone Cements ...... 67 2.3.5.3. PIB-Based Amphiphilic Networks (APNs) for Immunoisolatory Membranes ...... 67 2.3.5.4. New Biomaterial as a Promising Alternative to Silicone Breast Implants 68
III. EXPERIMENTAL ...... 69 3.1. Materials ...... 69
3.2. Procedures ...... 72
3.2.1. Enzyme-Catalyzed Functionalization of Polymer by Chain-End ...... 72
3.2.1.1. Candida antarctica lipase B-Catalyzed Halo-Ester Functionalization of PEGs ...... 72 3.2.1.2. Candida antarctica lipase B-Catalysis of Multifunctional PEGs 74 74 3.2.2. Enzyme-Catalyzed Functionalization Via Polymerization ...... 74
viii 3.2.2.1. Propargyl Alcohol Initiator for ROP of -CL ...... 74
3.2.2.2. Vinyl-PEG-Vinyl2000 (V-PEG-V2000) ...... 75 3.2.2.3. Synthesis of Poly(isobutylene-b--caprolactone) ...... 76 3.2.3. Chemo-Enzymatic Functionalization of Biomolecules ...... 76
3.2.3.1. Synthesis of the Biotin-PEG arm ...... 76 3.2.3.2. Synthesis of the Fluorouracil-TEG-OH arms...... 77 3.2.3.3. Synthesis of the Folic Acid linkers ...... 77 3.2.3.4. Synthesis of the FITC Linkers ...... 78 3.2.4. Preparation of Low Molecular Mass Telechelic Functionalized Polyisobutylenes ...... 79
3.2.4.1. Initiators ...... 79 3.2.4.2. Synthesis of Telechelic Oligomers of Isobutylene ...... 81 3.2.4.3. Preparation of Telechelic Functionalized Polyisobutylenes ..... 82 3.3. Characterization of Products ...... 84
3.3.1. Nuclear Magnetic Resonance (NMR) Spectroscopy ...... 84
3.3.2. Size Exclusion Chromatography (SEC)...... 85
3.3.3. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF MS) ...... 85
3.3.4. Electrospray Ionization Mass Spectrometry (ESI-MS) ...... 86
3.3.5. Thin Layer Chromatography (TLC) ...... 86
3.3.6. Column Chromatography...... 87
3.3.7. Differential Scanning Calorimetry (DSC) ...... 87
3.3.8. AFM ...... 87
3.3.9. Transmission Electron Microscopy (TEM) ...... 88
ix IV. ENZYME-CATALYZED FUNCTIONALIZATION OF POLYMERS BY CHAIN-END ...... 89 4.1. Introduction ...... 90
4.2. Results and Discussion ...... 91
4.2.1. Model Reactions: CALB-Catalyzed Halo-Ester Functionalization with BzTEG……………………………………………………………………….91
4.2.1.1. Transesterification of EBrA with BzTEG ...... 91 4.2.1.2. Transesterification of VClA with BzTEG ...... 93 4.2.1.3. Transesterification of VIA with BzTEG ...... 95 4.2.1.4. Transesterification of EBrV with BzTEG ...... 96 4.2.2. Candida antarctica lipase B-Catalyzed Halo-Ester Functionalization of PEGs ...... 98
4.2.2.1. Transesterification of EBrA with MeO-PEG-OH2000 ...... 98
4.2.2.2. Transesterification of VClA with MeO-PEG-OH2000 ...... 99
4.2.2.3. Transesterification of EIA with MeO-PEG-OH2000 ...... 101
4.2.2.4. Transesterification of EBrV with MeO-PEG-OH2000 ...... 101 4.2.3. Candida antarctica lipase B-Catalysis of PEGs for Click Chemistry ... 106 106
4.2.3.1. Synthesis of Ethyl 5-azidevalerate (EN3V) ...... 106
4.2.3.2. Synthesis of Azide-Functionalized poly(ethylene glycol)s (N3- PEG-N3) 108 4.2.4. Transesterification of Vinyl Acrylate with of Poly(ethylene glycol)s 111 111
4.2.4.1. Monofunctionalization of Poly(ethylene glycol)s by Enzyme- Catalyzed Transesterification of Vinyl Acrylate ...... 111 4.2.4.2. Transesterification of VA-DVA with Poly(ethylene glycol) .. 116 4.2.5. Candida antarctica lipase B-Catalysis of Multifunctional PEGs .... 118
4.2.5.1. Model Reaction ...... 119 4.2.5.2. Chemoselectivity in Enzyme-Catalyzed Michael Addition .... 120
x 4.2.6. CALB-Catalyzed Functionalization of Small Molecules ...... 122
4.2.6.1. Synthesis of bis(2-ethyl 5-bromopentanoate) disulfide ...... 122 4.2.6.2. Transesterification Reaction of Divinyladipate with 2- hydroxyethyl disulfide ...... 124 4.2.6.3. Disulfide Reduction of O,O'-(disulfanediylbis(ethane-2,1-diyl)) divinyl diadipate (DVA-S-S-DVA) ...... 128 4.2.7. Transesterification of Divinyl Adipate with 2-(Hydroxyethyl) Acrylate in Solventless Conditions ...... 130
V. ENZYMATIC FUNCTIONALIZATION BY POLYMERIZATION ...... 133 5.1. Enzyme-Catalyzed Ring-Opening Polymerization of -caprolactone Using Alkyne Functionalized Initiators...... 134
5.1.1. Introduction ...... 134
5.1.2. Propargyl Alcohol Initiator for ROP of -CL ...... 136
5.1.3. DIBO Alcohol Initiator for ROP of -CL ...... 141
5.1.4. Conclusions ...... 147
5.1.5. ROP of -Caprolactone Using Furfurylamine as Initiator ...... 147
5.2. Synthesis of Poly(isobutylene-b--caprolactone) and Poly(-caprolactone-b- isobutylene-b--caprolactone) Using Enzyme Catalysis ...... 150
5.2.1. Introduction ...... 150
5.2.2. Synthesis of Poly(isobutylene-b--caprolactone) Diblock ...... 152
5.2.3. Synthesis of Poly(isobutylene-b--caprolactone) Triblock ...... 155
5.2.4. Microphase Separation Analysis...... 157
5.2.4.1. Conclusion ...... 160 5.3. Enzyme-Catalyzed Quantitative Chain-End Functionalization of Poly(ethylene glycol)s Under Solventless Conditions ...... 161
xi 5.3.1. Synthesis of V-PEG-V2000...... 162
5.3.2. CALB-catalyzed polycondensation of DVA with Tetraethylenglycol 165 165
5.3.2.1. Effect of DVA Concentration ...... 167 5.3.2.2. Reaction Kinetics ...... 168 5.3.2.3. Conclusion ...... 174
VI. CHEMO-ENZYMATIC FUNCTIONALIZATION OF BIOMOLECULES .... 175 6.1. Exclusive γ-Conjugated Folic Acid Derivatives Via Chemo-Enzymatic Processes...... 175
6.1.1. Introduction ...... 175
6.1.2. Lithiation of FA ...... 178
6.1.3. Synthesis of FA-γ-Hexanol ...... 179
6.1.4. Synthesis of MPEG-FA via Chemo-enzymatic Procedures ...... 181
6.1.5. FA-γ- bis(2-ethyl 5-bromopentanoate) disulfide (FA-S-S-FA) ...... 188
6.1.5.1. FA-SH ...... 191 6.2. Synthesis of the Biotin-TEG-OH arms ...... 193
6.2.1. Synthesis of HO-TEG-allyl...... 193
6.2.2. Synthesis of Biotin-TEG-allyl ...... 195
6.2.3. Synthesis of Biotin-TEG-OH ...... 197
6.2.4. Synthesis of Biotin-PEG-OH ...... 198
6.3. Synthesis of the Fluorouracil Arms ...... 201
6.3.1. Michael Addition of Fluorouracil to Vinyl Acrylate ...... 201
6.3.2. Transesterification Reaction of Fluorouracil-Vinyl Ester with 2HEDS 203 203
6.4. Synthesis of the FITC linkers ...... 204
xii 6.4.1. Michael Addition of NH2-PEG-OH1000 to FITC-VA ...... 204
6.4.2. Transesterification of 2-(acryloyloxy)ethyl vinyl adipate (VA-DVA) with FITC-PEG-OH1000...... 208
6.5. Synthesis of the FITC-PEG1000- γ- FA ...... 210
6.5.1. Michael Addition of FA-SH to FITC-PEG-VA1000 ...... 210
6.5.2. Michael Addition of Diethanolamine to VA-FITC-VA ...... 215
VII. PREPARATION OF LOW MOLECULAR MASS TELECHELIC FUNCTIONALIZED POLYISOBUTYLENES ...... 218 7.1. Introduction ...... 218
7.2. Initiators ...... 219
7.2.1. Dimethyl 3,3-dimethylpentanedioate (DiMDiPD) ...... 219
7.2.2. 2,4,4,6-tetramethylheptane-2,6-diol (TMHDiOH) ...... 220
7.2.3. 2,6-dimethoxy-2,4,4,6-tetramethylheptane (TMHDiOMe) ...... 221
7.2.4. 2,6-dichloro-2,4,4,6-tetramethylheptane (TMHDiCl) ...... 223
7.3. Synthesis of Telechelic Oligomers of Isobutylene ...... 224
7.3.1. Introduction ...... 224
7.3.2. Model reaction for the Oligomerization of Isobutylene ...... 225
7.3.2.1. Synthesis of 2-chloro-2,4,4-trimethylpentane ( TMPCl) ...... 225 7.3.2.2. Synthesis of 2,4,4,6,6-pentamethyl-1-heptene (PMH) ...... 226 7.3.2.3. Synthesis of 2,4,4,6,6-pentanethylheptane-1-ol (PMH-OH) ... 227 7.3.2.4. Synthesis of 2,4,4,4,6,6-pentanethylheptyl-2-methylacrylate (PMH-MA) ...... 229 7.3.3. Oligomerization of Isobutylene ...... 231
7.3.3.1. Synthesis of 2,4,4,6,6,8-hexamethylnona-1,8-diene (HMND) 231
xiii 7.3.3.2. Synthesis of 2,4,4,6,6,8,8,10-octamethylundeca-1,10-diene (OMUD) ……………………………………………………………….233 7.3.3.3. Synthesis of 2,10-dichloro-2,4,4,6,6,8,8,10-octamethylundecane (DiClOMU) ...... 235 7.3.3.4. Synthesis of 4,4,6,6,8,8,10,10,12,12-decamethylpentadeca-1,14- diene (DMPDD) ...... 236 7.3.3.5. Synthesis of 4,4,6,6,8,8,10,10,12,12-decamethylpentadecane- 1,15-diol (DMPDDiOH) ...... 237 7.4. Preparation of Telechelic Functionalized Polyisobutylenes ...... 239
7.4.1. Introduction ...... 239
7.4.2. Screening Experiments ...... 242
7.4.3. In situ Chlorine Exchange ...... 245
7.4.3.1. Chlorine Exchange in CH2C12 ...... 245
7.4.3.2. Chlorine Exchange in CH3Cl ...... 247 7.4.4. Synthesis of allyl-PIB-allyl ...... 247
7.4.5. Synthesis of HO-PIB-OH ...... 248
VIII. SUMMARY AND RECOMMENDATIONS...... 251 8.1. Summary ...... 251
8.2. Recommendations ...... 256
8.2.1. Recyclability Study ...... 256
8.2.2. Biological Activity Test ...... 256
8.2.3. Multivalent Drug-Delivery System...... 257
8.2.4. Biodegradables Pressure Sensitive Adhesive Matrix Patches ...... 257
BIBLIOGRAPHY………………………………………………………………………258
APPENDIX……………………………………………………………………………..272
xiv LIST OF TABLES
Table Page
2.1. Aspects of enzyme catalysis relevant to green chemistry ...... 10
4.1. Reaction progress composition in the CALB-catalyzed transesterification of VA with
HO-PEG-OH1100...... 114 5.1. Characteristics of the alkyne-PCL polymers obtained under different conditions after 12h of reaction...... 140
5.2. Characteristics of the DIBO-PCL polymers obtained under different conditions ... 146
5.3. Effect of DVA/TEG molar ratio on the oligomerization of TEG ...... 168
5.4. Oligomer compositions with DVA/TEG 1/1 ...... 169
5.5. Oligomer compositions with DVA/TEG 1.5/1 and 3/1...... 170
5.6. Reaction kinetics in case of excess TEG...... 172
7.1. Screening experiments ...... 242
xv LIST OF FIGURES
Figure Page
2.1. Classification of enzymes...... 10
2.2. (Left) 3D structure of CALB [Image of PBD ID:1TCB created with Polyview 3D] and (right) schematic representation of the catalytic triad of CALB. Reprinted with permission from Dr Kwang Su Seo...... 12
2.3. Transesterification of esters with alcohols: a) Reversible with an alkyl ester or a halogenated alkyl ester, and b) Irreversible with a vinyl ester...... 13
2.4. Lipase-catalyzed transesterification vinyl acetate with n-octanol...... 14
2.5. Transesterification of vinyl acetate: comparison of CALB and a tin-based catalyst. 14
2.6. Illustration of the mechanism of CALB-catalyzed transesterification of vinyl acetate with 2-phenylpropane-1-ol. The different shading represents the two enzyme pockets. . 15
2.7. CALB-catalyzed aza-Michael addition of amine to acrylate ...... 16
2.8. Suggested mechanism for the CALB-catalyzed Michael addition of pyrrolidine to acrylonitrile...... 18
2.9. Chemoselectivity CALB-catalyzed Michael addition reaction...... 19
2.10. Major modes of lipase catalyzed polyester synthesis...... 20
2.11. CALB catalyzed polymerization of organosiloxane with alkenodiols in bulk at elevated temperature and reduced pressure...... 21
2.12. Lipase-catalyzed irreversible polytransesterification between divinyl adipate and 1,4-butanediol...... 21
2.13. CALB-based biocatalytic polymerization of free mercapto containing polyesters. 22
2.14. Synthesis of copolyesters containing L-malic acid units using lipase as catalyst. .. 23
2.15. CALB-catalyzed ROP of various monomers...... 24
xvi 2.16. Enzymatic ROP of DD-lactide (DLA) by CALB...... 25
2.17. Enzymatic ROP of trimethylene carbonate (TMC) by CALB...... 25
2.18. Enzymatic ROP of -CL by CALB...... 26
2.19. Enzymatic ROP of macrocyclic monomers by CALB...... 27
2.20. Enzymatic ROP of OC by CALB...... 27
2.21. Modeled Kinetic Reactions in Enzyme-Mediated Poly(ε-caprolactone) Synthesis. Note: Subscripts on kinetic rate parameters denote the reaction step number. Positive subscripts are reactions with enzyme-activated PCL chains; negative subscripts are reactions with free enzyme sites...... 28
2.22. Functionalization of -CL by initiator method...... 31
2.23. CALB-catalyzed polymerization of -CL in the presence of alkyl glucopyranosides...... 31
2.24. Synthesis of macromonomer by terminator method...... 32
2.25. CALB-catalyzed acylation of cellulose acetate...... 34
2.26. CALB-catalyzed functionalization of starch nanoparticles...... 34
2.27. CALB-catalyzed modification of pendant ester groups of a polystyrene derivative...... 35
2.28. CALB-catalyzed epoxidation of polybutadiene...... 36
2.29. Enzymatic grafting of copolymers with vinyl acetate...... 36
2.30. CALB-catalyzed acylation of comb-like methacrylate polymers...... 37
2.31. Telechelic carboxylic acid functionalized PDMSs in the presence of CALB...... 38
2.32. CALB-catalyzed methacrylation of PIB-OHs. PIB-OH (Mn=5,200 g/mol; ĐM =1.09), Glissopal-OH (Mn=3,600 g/mol; ĐM=1.34), and asymmetric telechelic HO-PIB- OH (Mn=7,200 g/mol; ĐM =1.04)...... 38
2.33. Functionalized PEGs vial CALB-catalyzed transesterification...... 39
2.34. (OH)2–PEG–(OH)2 as a core of novel dendrimers using sequential CALB...... 40
2.35. Vitamins commonly used for drug-delivery applications. The functional groups on folate and biotin amenable to payload attachment are shaded in blue.96,98 Payloads attached to the orange shaded groups on cobalamin have been shown to maintain full
xvii binding to all cobalamin-trafficking proteins, while attachment of the payload through the group shaded in green allows the generation of transcobalamin I selective constructs.4 . 43
2.36. Schematic presentation of tumor cellular uptake of a folate−drug conjugate by FR- mediated endocytosis. The folate−drug conjugate consists of a folic acid moiety (shown as a yellow oval) and the drug payload (shown in red). Reprinted with permission from Vlahov, I. R.; Leamon, C. P. Bioconjug. Chem. 2012, 23, 1357–1369. Copyright 2014 American Chemical Society...... 45
2.37. Folate−drug conjugates in human clinical trials. Comparison between a pH- sensitive linker and a disulfide linker...... 46
2.38. Folate−drug conjugates in human clinical trials...... 47
2.39. Folate-conjugated imaging agents in human clinical trials...... 49
2.40. Example of a 90Y-DOTA-biotin compound...... 51
2.41. Chemical structure of biotin-S-S-taxoid and biotin-S-S-taxoid-fluorescein...... 52
2.42. Modular approach for the synthesis of Vitamin−polymer conjugates for therapeutic applications...... 54
2.43. Modular approach for the synthesis of vitamin−polymer conjugates for diagnostics applications...... 55
2.44. Modular approach for the synthesis of vitamin−polymer conjugates for diagnostics applications...... 56
2.45. Functional initiators for end-functional PIBs...... 60
2.46. Functionalization of PIB through direct reaction of quasiliving PIB with a nucleophilic quenching or capping agent...... 61
2.47. Conversion of allyl end-functionalized PIB to the corresponding hydroxyl functional polymer...... 61
2.48. End-functional polyisobutylenes using nucleophilic substitution reactions...... 62
2.49. PIB diol and undesirable reactions obtained by the inifer technique: dicumyl ether/TiCl4 system...... 63
2.50. Reaction of tert-alcohol with BCl3 to produce Cl-PIB-Cl...... 65
2.51. PIB-based biomaterials tested for biomedical applications...... 66
4.1 Enzymatic transesterification of halo-esters with PEGs in the presence of CALB. ... 91
xviii 4.2. NMR spectra of the product of the enzymatic transesterification of EBrA with BzTEG: (Top) 1H NMR spectrum and (Bottom) 13C NMR spectrum (500 MHz, solvent: CDCl3)...... 93
4.3. NMR spectra of the product of the enzymatic transesterification of VClA with 1 13 BzTEG: (Top) H NMR spectrum and (Bottom) C NMR spectrum (solvent: CDCl3)... 94
4.4. NMR spectra of the product of the enzymatic transesterification of EIA with BzTEG: 1 13 (A) H NMR spectrum and (B) C NMR spectrum (solvent: CDCl3)...... 95
4.5. NMR spectra of the product of the enzymatic transesterification of EBrV with BzTEG: (Top) 1H NMR spectrum and (Bottom) 13C NMR spectrum (500 MHz, solvent: CDCl3)...... 97
4.6. NMR spectra of the product of the enzymatic transesterification of EBrA with MeO- 1 13 PEG-OH2000 (Mn = 2000 g/mol; ĐM= 1.2): (Top) H NMR spectrum and (Bottom) C NMR spectrum (500 MHz, solvent: DMSO-D6)...... 99
4.7. NMR spectra of the product of the enzymatic transesterification of VClA with MeO- 1 13 PEG-OH2000 (Mn = 2000 g/mol; ĐM= 1.2): (Top) H NMR spectrum and (Bottom) C NMR spectrum (solvent: DMSO-D6)...... 100
4.8. NMR spectra of the product of the enzymatic transesterification of EIA with MeO- 1 PEG-OH2000 (Mn = 2000 g/mol; ĐM= 1.2): (Top) H NMR spectrum (solvent: DMSO- 13 D6). and (Bottom) C NMR spectrum (solvent: CDCl3)...... 101
4.9. NMR spectra of the product of the enzymatic transesterification of EBrV with MeO- 1 13 PEG-OH2000 (Mn = 2000 g/mol; ĐM= 1.2): (Top) H NMR spectrum and (Bottom) C NMR spectrum (500 MHz, solvent: DMSO-D6)...... 102
4.10. MALDI- ToF mass spectrum of the MeO-PEG-OH2000 (top left) and the MeO-PEG- BrV2000 product (bottom left); zoom of the 39 and 40 mer fractions (right)...... 103
4.11. MALDI- ToF mass spectrum of the Br-PEG-Br3400 (left) and zoom of the 73 and 74 mer fractions (right). [HO-PEG-OH3400] = 0.77 mol/L, [EBrV] = 7.72 mol/L; [CALB] = 1.54×10-4 mol/L...... 105
4.12. Synthesis of ethyl 5-azidevalerate (EN3V). [EBrV]= 0.77 mol/L, [NaN3]= 1.14 mol/L...... 106
4.13. NMR spectra of ethyl 5-azidevalerate. (Top) 1H NMR spectrum and (bottom) 13C NMR spectrum (300 MHz, solvent: CDCl3)...... 107
4.14. Enzymatic transesterification of EN3V with HO-PEG-OH3400. [HO-PEG-OH3400]= -4 0.58 mol/L, [EN3V]= 5.77 mol/L, [CALB]= 1*10 mol/L...... 108
xix 4.15. NMR spectra of the product of the enzymatic transesterification of EN3V with HO- 1 13 PEG-OH3400 (Top) H NMR spectrum and (bottom) C NMR spectrum (500 MHz, solvent: DMSO)...... 109
4.16. MALDI- ToF mass spectrum of the N3-PEG-N3-3400 (left) and zoom of the 74 and 75 mer fractions (right)...... 110
4.17. Synthetic procedures of acrylate-PEG-OH. [HO-PEG-OH1100]= 0.053 mol/L, [VA]= 0.64 mol/L, [CALB]= 8.8*10-4 mol/L ...... 112
4.18. MALDI spectrum of the aliquot taken in the CALB-catalyzed transesterification of VA with HO-PEG-OH1100 at t = 45 min (top), and zoom of the 21 and 22 mer fractions (bottom)...... 113
4.19. NMR spectra of the product of the enzymatic transesterification of VA with HO- 1 13 PEG-OH1100 (Top) H NMR spectrum and (bottom) C NMR spectrum (500 MHz, solvent: DMSO-D6)...... 115
4.20. MALDI- ToF mass spectrum of the VA-PEG-OH1000 (left) and zoom of the 22 and 23 mer fractions (right)...... 116
13 4.21. C NMR spectra of (bottom) HO-PEG-OH8000 and (top) VA-PEG-VA8000 in DMSO-D6...... 117
4.22. MALDI- ToF mass spectrum of VA-PEG-VA8000 (left) and zoom of the 199 and 200 mer fractions (right). [HO-PEG-OH8000]= 0.37 mol/L, [VA-DVA]= 3.70 mol/L, [CALB]= 1.9*10-3 mol/L ...... 117
4.23. Stereospecificity in CALB-catalyzed Michael addition...... 118
4.24. Chemoselectivity in Enzyme-catalyzed Michael Addition. Model reaction: [2,2'- (ethane-1,2-diylbis(oxy))diethanamine]= 1*10-6 mol/L, [3-(acryloyloxy)-2- hydroxypropyl methacrylate]= 2*10-6 mol/L, [CALB]= 1*10-10 mol/L. PEG functionalization: [NH2-PEG-NH2-2000]= 0.3 mol/L, [3-(acryloyloxy)-2-hydroxypropyl methacrylate]= 0.6mol/L, [CALB]= 3*10-8 mol/L...... 118
4.25. 1H NMR spectrum of the product CALB catalyzed Michael Addition. [2,2'- (ethane-1,2-diylbis(oxy))diethanamine]= 1*10-6 mol/L, [3-(acryloyloxy)-2- hydroxypropyl methacrylate]= 2*10-6 mol/L, [CALB]= 1*10-10 mol/L. (300 MHz, solvent: CDCl3)...... 119
1 4.26. H NMR spectrum of the product CALB catalyzed Michael Addition. [NH2-PEG- NH2-2000]= 0.3 mol/L, [3-(acryloyloxy)-2-hydroxypropyl methacrylate]= 0.6 mol/L, -8 [CALB]= 3*10 mol/L. (500 MHz, solvent: CDCl3)...... 121
xx 4.27. Reaction scheme and TLC monitoring of the CALB-catalyzed transesterification of EBrV with 2HEDS in bulk. [EBrV] = 4.27 mol/L, [2HEDS] =1.90 mol/L; [CALB] = 7.1 × 10-5 mol/L...... 122
4.28. NMR spectra of the transesterification product of EBrV with 2HEDS in bulk: (top) 1 13 H NMR spectrum and (bottom) C NMR spectrum (300 MHz, solvent: CDCl3)...... 123
4.29. Reaction scheme and TLC monitoring of the CALB-catalyzed transesterification of DVA with 2HEDS. [DVA] = 4.81 mol/L, [2HEDS] = 0.24 mol/L; [CALB] = 7.1×10-5 mol/L...... 124
4.30. NMR spectra of the transesterification product of DVA with 2HEDS: (top) 1H 13 NMR spectrum and (bottom) C NMR spectrum (300 MHz, solvent: CDCl3)...... 126
4.31. ESI-MS spectrum of the transesterification product of DVA with 2HEDS (cationizing agent: NaTFA) ...... 127
4.32. Reaction scheme and TLC monitoring of the disulfide reduction of DVA-S-S-DVA. [DTT] = 8.6×10-3 mol/L, [DSDV] = 8.6×10-3 mol/L...... 128
4.33. NMR spectra of the of the disulfide reduction of DVA-S-S-DVA: (top) 1H NMR 13 spectrum and (bottom) C NMR spectrum (300 MHz, solvent: CDCl3)...... 129
4.34. Reaction scheme and TLC monitoring of the transesterification reaction of DVA with 2HEA. [DVA] = 8.6×10-3 mol/L, [2HEA] = 8.6×10-3 mol/L, [CALB] = 8.6×10-3 mol/L...... 130
4.35. NMR spectra of the transesterification product of DVA with 2HEA: (top) 1H NMR 13 spectrum and (bottom) C NMR spectrum (300 MHz, solvent: CDCl3)...... 131
5.1. CALB catalyzed ROP of -CL using as initiator propargyl alcohol. For the case [monomer]/ [initiator]= 20. [Propargyl-OH] = 0.26 mol/L, [-CL] = 5.2 mol/L; [CALB] = 1.31 × 10-4 mol/L...... 136
5.2. 1H NMR spectrum of the product CALB catalyzed ROP of -CL using as initiator propargyl alcohol [monomer]/ [initiator]= 20 (500 MHz, solvent: CDCl3)...... 137
5.4. (A) SEC eluograms for aliquots at 4 h, 8 h, 12 h and 24 h of CALB catalyzed ROP of -CL using as initiator propargyl alcohol. For the case [monomer]/ [initiator]= 50:1. [Propargyl-OH] = 0.1 mol/L, [-CL] = 5.2 mol/L; [CALB] = 1.3×10-4 mol/L. (B) Number-average molecular mass (Mn) and ĐM as a function of time at 70ºC in bulk and solution system. (C) SEC results of CALB catalyzed ROP of -CL using propargyl alcohol as the initiator for the reaction [monomer]/ [initiator]= 50:1 at 12 h. In both bulk and solution reaction conditions...... 139
xxi 5.5. (A) SEC results of CALB catalyzed ROP of -CL using propargyl alcohol as an initiator with different feed ratios. (B) Number-average molecular mass (Mn) and ĐM as a function of feed ratio [monomer] / [initiator] at 70 ºC...... 140
5.6. CALB catalyzed ROP of -CL using as initiator dibenzocyclooctynol. For the case [monomer]/ [initiator] = 500. [DIBO] = 7.8×10-3 mol/L, [-CL] = 3.9 mol/L; [CALB] = 7.9× 10-5 mol/L...... 142
5.7. 1H NMR spectrum: (Top) primary hydroxyl-derivatized DIBO, (Bottom) Aliquot at 4h of the reaction CALB catalyzed ROP of -CL using as initiator DIBO [monomer]/ [initiator]= 500 (500 MHz, solvent: CDCl3)...... 144
5.8. (A) SEC results of CALB catalyzed ROP of -CL using as initiator DIBO for the case [monomer]/ [initiator]= 500:1 for aliquots at 4 h, 8 h, 12 h and 24 h. (B) SEC chromatogram and number-average molecular mass (Mn) and ĐM as a function of time at 70ºC bulk and solution system. (C) SEC results of CALB catalyzed ROP of -CL using as initiator DIBO for the case [monomer]/ [initiator]= 50:1 at 12 h in bulk and solution conditions. (D) UV spectrum of the reaction aliquot at 4h. CALB catalyzed ROP of -CL using as initiator DIBO [monomer]/ [initiator]= 500 (solvent: THF)...... 145
5.9. CALB catalyzed ROP of -CL using as initiator furfurylamine. [monomer]/ [initiator] = 20. [Furfurylamine] = 0.26 mol/L, [-CL] = 4.61 mol/L; [CALB] = 2.23×10-3 mol/L...... 147
5.10. SEC results of CALB catalyzed ROP of -CL using as initiator furfurylamine. [monomer]/ [initiator] = 20. [Furfurylamine] = 0.26 mol/L, [-CL] = 4.61 mol/L; [CALB] = 2.23×10-3 mol/L...... 148
5.11. NMR spectra of the product of the CALB catalyzed ROP of -CL using as initiator furfurylamine. (Top) 1H NMR spectrum and (bottom) 13C NMR spectrum (500 MHz, solvent: CDCl3)...... 149
5.12. CALB catalyzed ROP of -CL using allyl-PIB-OH as macroinitiator. For the case [monomer]/ [macroinitiator]= 40. [HO-PIB-allyl] = 9.02x10-3 mol/L, [-CL] = 0.36 mol/L; [CALB] = 9.02 × 10-5 mol/L...... 152
5.13. Carbocationic polymerization of isobutylene initiated by propylene oxide/TiCl4 and its allylation by allyltrimethylsilane. [Propylene oxide]=0.015 M, [IB]=1.000 M, [TiCl4]=0.030 M, [DtBP]=0.007 M, [ATMS]=0.045 M, T = -80 °C, Hx/MeCl=60/40 v/v, 10 minutes...... 152
1 5.14. H spectrum of HO-PIB-allyl (solvent: CDCl3) clearly showing the presence of the allyl group at the polymer chain end...... 153
5.15. SEC traces of PIB-b-PCL:[monomer]/ [macroinitiator]= 40. [allyl-PIB-OH] = 0.0090 mol/L, [-CL] = 0.36 mol/L; [CALB] = 0.0001 mol/L...... 154
xxii 1 5.16. H NMR spectra of PIB-b-PCL (solvent: CDCl3)...... 155
5.17. CALB catalyzed ROP of -CL using as macroinitiator HO-PIB-OH. For the case [monomer]/ [initiator]= 20. [HO-PIB-OH] = 0.26 mol/L, [-CL] = 5.2 mol/L; [CALB] = 1.31 × 10-4 mol/L...... 155
5.18. SEC traces of poly(--caprolactone-b-isobutylene-b--caprolactone): [monomer]/ [initiator]= 20. [HO-PIB-OH] = 0.26 mol/L, [-CL] = 5.2 mol/L; [CALB] = 1.31 × 10-4 mol/L...... 156
5.19. 1H NMR spectra poly(--caprolactone-b-isobutylene-b--caprolactone) (solvent: CDCl3)...... 157
5.20. DSC thermograms at a heating rate of 10 ºC/min for PIB-b-PCL and PCL- b-PIB-b- PCL...... 158
5.21. TEM pictures for A-B: PIB-b-PCL diblock. C-D PCL-b-PIB-b-PCL Triblock. Staining: 1% OsO4 showing the microphase separation...... 158
5.22. A-B: Optical images of PIB-b-PCL diblock, reflected light. E-F: Optical images of PCL-b-PIB-b-PCL Triblock, reflected light. Optical Images for PCL reported in the 214 literature (I) PCL with Mn=1,900 g/mol and (J) PCL with Mn=6,700 g/mol...... 159
5.23. C: AFM Tapping mode height and amplitude images of sample 5 µm scan size for diblock. G-H: AFM Tapping mode height and amplitude images of sample 5 µm scan size and 10 µm scan size respectively for triblock showing the microphase separation. 160
5.24. Transesterification of vinyl ester-PEG with functionalized alcohols. The red ball represents functional groups derived from the alcohol...... 161
5.25. Reaction of DVA with HO-PEG-OH2000 in the presence of CALB under solventless -4 conditions. [DVA] = 5.29 mol/L, [HO-PEG-OH2000] = 0.26 mol/L; [CALB] = 1.6 × 10 mol/L...... 162
5.26. MALDI- ToF mass spectrum of the product of the reaction of DVA with HO-PEG- OH2000 at 4 hours...... 163
1 5.27. NMR spectra of the product of the reaction of DVA with HO-PEG-OH2000: H 13 NMR (top) and C NMR (bottom) (solvent: DMSO-D6)...... 164
5.28. CALB-catalyzed transesterification of Divinyladipate with Tetraethylenglycol. Polycondensation products...... 165
5.29. Transesterification of DVA with TEG: first cycle...... 166
5.30. Simplified scheme of the polycondensation reaction of the transesterification of Divinyladipate with Tetraethylenglycol...... 167
xxiii 5.31. End group composition (%) vs. reaction time (min) in the case of TEG excess ... 172
6.1. Chemical structure of FA...... 176
6.2. Retrosynthetic strategy for the preparation of α-protected FA with a free γ-site. ... 177
6.3. New method for the exclusive γ-conjugation of FA.162 ...... 178
6.4. Expanded region of 13C NMR spectra of its lithiated intermediate (top) and folic acid (bottom)...... 178
1 6.5. H NMR spectra of (top) FA and (bottom) FA-hexanol in DMSO-D6...... 179
6.6. Expanded region of 13C NMR spectrum of the carbonyl carbons in FA-hexanol (solvent: DMSO-D6)...... 180
6.7. FA conjugation with 5-bromovalerate. (i) [FA]= 0.05 mol/L, [n-BuLi]= 0.05 mol/L, [EBrV]= 0.04 mol/L. (ii) [FA-γ-valerate]= 0.11 mol/L, [MeO-PEG-OH2000]= 0.08 mol/L, [CALB]= 8.3*10-5 mol/L...... 181
6.8. NMR spectra of FA-valerate 13C NMR spectrum solvent: DMSO-D6. [FA]=0.049 mol/L, [5-EBrV]=0.044 mol/L, [n-BuLi]= 0.049 mol/L...... 182
6.9. Folic Acid conjugation with MeO-PEG-Br2000. [FA]=0.049 mol/L, [MeO-PEG2000- Br]=0.044 mol/L, [n-BuLi]= 0.049 mol/L...... 183
13 6.10. NMR spectra of FA reaction with MeO-PEG-BrV2000 C NMR spectrum (500 MHz, solvent: DMSO-D6)...... 184
6.11. FA conjugation with MeO-PEG-BrV2000 in the presence of modifier. [FA]= 0.069 mol/L, [n-BuLi]= 0.069 mol/L, [MeO-PEG-BrV2000]= 0.044 mol/L, [TMEDA]= 0.14 mol/L...... 186
1 13 6.12. NMR spectra of FA reaction with MeO-PEG-BrV2000 H and C NMR spectrum solvent: DMSO-D6...... 187
6.13. MALDI-ToF spectra of the reaction of FA with MeO-PEG-BrV2000 (left) and zoom of the 35 and 36 mer fractions (right). [FA]=0.069 mol/L, [MeO-PEG-BrV2000]=0.044 mol/L, [n-BuLi]= 0.069 mol/L, [TMEDA]= 0.138 mol/L...... 188
6.14. FA conjugation with bis(2-ethyl 5-bromopentanoate) disulfide and its reduction. (i) [FA]= 0.05 mol/L, [n-BuLi]= 0.05 mol/L, [bis(2-ethyl 5-bromopentanoate) disulfide]= 0.02 mol/L. (ii) [FA-S-S-FA]= 0.26 mol/L, [DTT]= 0.26 mol/L...... 189
6.15. 1H NMR spectra: bis (2-ethyl 5-bromopentanoate) disulfide (top) and FA-γ- bis(2- ethyl 5-bromopentanoate (bottom) (500 MHz, solvent: DMSO-D6)...... 190
xxiv 6.16. 13C NMR spectrum of FA-γ- bis(2-ethyl 5-bromopentanoate (125 MHz, solvent: DMSO-D6)...... 191
1 13 6.17. NMR spectra of FA-SH. H and C NMR spectrum solvent: DMSO-D6...... 192
6.18. Synthetic strategy for Biotin-TEG-OH...... 193
6.19. 1H NMR spectrum (top) and 13C NMR spectrum HO-TEG-allyl (300 MHz, solvent: CDCl3)...... 194
1 13 6.20. H NMR spectrum (top) (300 MHz, solvent: DMSO-D6) and C NMR spectrum Biotin-TEG-allyl (75 MHz, solvent: CDCl3)...... 196
1 6.21. H NMR spectrum of Biotin-TEG-allyl. (300 MHz, solvent: DMSO-D6)...... 198
6.22. Synthetic strategy for Biotin-PEG-OH3400. [NH2-PEG-OH3400]= 0.098 mol/L, [Et3N]= 0.088 mol/L, [NHS-Biotin]= 0.34 mol/L...... 198
1 13 6.23.NMR spectra of Biotin-PEG-OH3400: H NMR spectrum (top) C NMR spectrum (bottom) (500 MHz, solvent: DMSO-D6)...... 199
6.24. MALDI-ToF mass spectrum of Biotin-PEG-OH3400 (left) and zoom of the 77 and 78 mer fractions (right)...... 200
6.25. CALB catalyzed Michael addition of Fluorouracil to vinyl acrylate. [VA]= 1.58 mol/L, [Fluorouracil]= 0.79 mol/L, [CALB]= 8.0×10-5 mol/L...... 201
6.26. 1H NMR spectrum (Top) and 13C NMR spectrum (Bottom) fluorouracil vinyl ester product. (300 MHz, solvent: DMSO-D6)...... 202
6.27. Synthetic strategy to get a disulfide bond. [fluorouracil-vinylester]= 0.8 mol/L, [2HEDS]= 2.3 mol/L, [CALB]= 7.7×10-7 mol/L...... 203
6.28. 1H NMR spectrum (Top) and 13C NMR spectrum (Bottom) fluorouracil disulfide product. (500 MHz, solvent: DMSO-D6)...... 204
6.29. Conjugation of FITC to NH2-PEG-OH1000 (Mn=1000 g/mol; ĐM =1.04). [FITC-VA] -4 = 0.3 mol/L, [NH2-PEG-OH1000] = 0.3 mol/L, [CALB]= 3.6×10 mol/L...... 204
6.30. NMR spectra of FITC-VA: (Top) 1H NMR spectrum and (Bottom) 13C NMR spectrum (500 MHz, solvent: DMSO-D6)...... 205
1 6.31. H NMR spectra of (bottom) NH2-PEG-OH1000 and (top) FITC-PEG-OH1000 in DMSO-D6...... 206
13 6.32. C NMR spectra of (bottom) NH2-PEG-OH1000 and (top) FITC-PEG-OH1000 in DMSO-D6...... 207
xxv 6.33. Transesterification of 2-(acryloyloxy)ethyl vinyl adipate with FITC-PEG-OH1000 (Mn=1000 g/mol; ĐM =1.04). [FITC-PEG-OH1000] = 0.31 mol/L, [VA-DVA] = 0.46 mol/L, [CALB]= 3.1×10-4 mol/L...... 208
1 13 6.34. NMR spectra of FITC-PEG-VA1000: H NMR spectrum (top) C NMR spectrum (bottom) (500 MHz, solvent: DMSO-D6)...... 209
6.35. Michael Addition of FA-SH to FITC-PEG-VA1000 (Mn=1000 g/mol; ĐM =1.04). -5 [FITC-PEG-VA1000] = 0.078 mol/L, [FA-SH] = 0.094 mol/L, [CALB]= 1.4×10 mol/L...... 211
1 13 6.36. NMR spectra of FITC-PEG1000- γ- FA. H and C NMR spectrum solvent: DMSO- D6...... 212
6.37. Structure of the products present on the Michael Addition of FA-SH to FITC-PEG- VA1000...... 213
6.38. MALDI-ToF spectra of FITC-PEG1000- γ- FA (top) and zoom of the 20 and 21 mer fractions (bottom)...... 214
6.39. Conjugation of VA-FITC-VA to DEA. [FITC] = 0.075 mol/L, [DEA] = 0.15mol/L. [CALB]= 3.6×10-4 mol/L...... 215
6.40. NMR spectra of VA-FITC-VA: (Top) 1H NMR spectrum and (Bottom) 13C NMR spectrum (500 MHz, solvent: DMSO-D6)...... 216
6.41. 1H NMR spectrum of the product of the Michael addition of diethanolamine to VA- FITC-VA. (500 MHz, solvent: DMSO-D6) ...... 217
7.1. Synthesis of dinitiator for IB oligomerization and IB polymerization...... 219
7.2. NMR spectra of the product DiMDiPD: (Top) 1H NMR spectrum and (bottom) 13C NMR spectrum (300 MHz, solvent: CDCl3)...... 220
7.3. NMR spectra of TMHDiOH: (Top) 1H NMR spectrum and (bottom) 13C NMR spectrum (300 MHz, solvent: CDCl3)...... 221
7.4. NMR spectra of the product TMHDiOMe: (Top) 1H NMR spectrum and (bottom) 13 C NMR spectrum (300 MHz, solvent: CDCl3)...... 222
7.5. NMR spectra of the product TMHDiCl: (Top) 1H NMR spectrum and (bottom) 13C NMR spectrum (300 MHz, solvent: CDCl3)...... 223
7.6. Model reaction sequence for the IB oligomerization...... 225
7.7. 1H NMR spectrum of the TMP-1 (top) and 1H NMR spectrum of the chlorinated product TMPCl (bottom) (300 MHz, solvent: CDCl3)...... 226
xxvi 1 7.8. H NMR spectrum of the product PMH (300 MHz, solvent: CDCl3). [TMPCl]= 0.072 mol/L, [MAMS]= 0.16 mol/L, [TiCl4]= 0.13 mol/L, [DtBP]= 0.008 mol/L...... 227
7.9. NMR spectra of the product of the hydroboration/oxidation reaction of PMH-OH: 1 13 (Top) H NMR spectrum and (bottom) C NMR spectrum (300 MHz, solvent: CDCl3). [PMH]= 0.035 mol/L, [9-BBN]= 0.24mol/L, [KOH]= 0.84 mol/L, [H2O2]= 0.77 mol/L ...... 228
7.10. NMR spectra of the transesterification of vinyl methacrylate with PMH-OH using enzyme catalysis: (Top) 1H NMR spectrum and (bottom) 13C NMR spectrum PMH-MA (300 MHz, solvent: CDCl3). [PMH-OH]= 1.1 mol/L, [VMA]= 3.2 mol/L, [CALB]= 2.2 *10-4 mol/L...... 230
7.11. Synthetic processes for dihydroxy oligoisobutylene using carbocationic initiation...... 231
7.12. NMR spectra of the product HMND: 1H NMR spectrum (Top) and 13C NMR spectrum (bottom) (300 MHz, solvent: CDCl3). [DMPDiOH]= 0.061 mol/L, [MAMS]= 0.31 mol/L, [TiCl4]= 0.24 mol/L, [DtBP]= 0.005 mol/L, [DMA]= 0.061 mol/L...... 232
7.13. NMR spectra of the product OMUD: 1H NMR spectrum (Top) and 13C NMR spectrum (bottom) (300 MHz, solvent: CDCl3). [TMHDiOH]= 0.048 mol/L, [MAMS]= 0.21 mol/L, [TiCl4]= 0.20 mol/L, [DtBP]= 0.004 mol/L, [DMA]= 0.049 mol/L...... 234
1 7.14. H NMR spectrum of DiClOMU (300 MHz, solvent: CDCl3)...... 235
7.15. NMR spectra of the product DMPDD: 1H NMR spectrum (Top) and 13C NMR spectrum (bottom) (300 MHz, solvent: CDCl3). [DiClOMU]= 0.029 mol/L, [ATMS]= 0.14 mol/L, [TiCl4]= 0.11 mol/L, [DtBP]= 0.003 mol/L, [DMA]= 0.029 mol/L...... 237
1 7.16. H NMR spectrum of DMPDDiOH (300 MHz, solvent: CDCl3)...... 238
7.17. Synthesis of fully aliphatic HO-PIB-OH ...... 241
7.18. SEC chromatogram of Cl-PIB-Cl. [TMHDiCl]= 0.013 mol/L; [TiCl4]=0.053 mol/L; [DtBp]= 0.0007 mol/L; [DMA]= 0.013 mol/L; [IB]=0.32 mol/L; CH3Cl/ Hexane, 40/60, v/v, Vo=180 mL. -80 ºC Reaction time 40 min...... 243
1 7.19. H NMR spectrum of PIB obtained with system TMHDiCl/TiCl4. For conditions see. Figure 7.18...... 244
7.20. SEC traces of the PIB made by in situ chlorine exchange. First Step: [TMHDiOH]0= 0.021M; [TiCl4]0=0.084M; [DtBp]0= 0.0018M; [DMA]0= 0.021 M; CH2Cl2,V0 =500 mL -80ºC, 4 h. Second step: Addition of 737 mL of Hexane and 16 mL of IB [TMHDiCl]f= 0.008M; [TiCl4]f=0.034M; [IB]=0.28M; Vf =1253 mL. Reaction time: 40 minutes...... 246
xxvii 7.21. Synthesis of allyl-PIB-allyl. First Step: [TMHDiOH]0= 0.042M; [TiCl4]0=0.85M; [DtBp]0= 0.0018M; [DMA]0= 0.042 M; CH3Cl, V0 =94 mL; -80ºC, 1 h. Second step: Addition of 141 mL of Hexane and 5.6 mL of IB. [TMHDiCl]f= 0.016 M; [TiCl4]f=0.32 M, [IB]=0.29M; Vf =250 mL, after 10 min addition of [ATMS]=0.32 mol/L. Intermediate TMHDiCl is in equilibrium as shown in Figure 7.17...... 247
1 7.22. H NMR spectrum allyl-PIB-allyl. (300 MHz, solvent CDCl3) ...... 248
7.23. Synthetic route of HO-PIB-OH by thiol-ene reaction using mercaptoethanol: [allyl- PIB-allyl]=0.025 mol/L, [mercaptoethanol]= 0.61 mol/L, [Irgacure]= 0.24 mol/L ...... 249
1 7.24. H NMR spectrum of HO-PIB-OH. (300 MHz, solvent CDCl3)...... 249
7.25. SEC chromatogram of allyl-PIB-allyl and HO-PIB-OH...... 250
xxviii 1. CHAPTER I
INTRODUCTION
Chemo-enzymatic methods are an alternative strategy to increase the diversity of
functional groups in polymeric materials. Specifically, enzyme-catalyzed polymer
functionalization carried out under solventless conditions is a great advancement in the
design of green processes for biomedical applications, where the toxicity of solvents and
catalyst residues need to be considered. The objective of this research is the precise,
efficient and specific enzymatic functionalization of polymers using two methods:
enzyme-catalyzed functionalization via polymerization and enzymatic functionalization
by chain end functionalization. The polymers obtained through this method are free from
any metal catalyst, which often broadens the application of the products in particular for
medical use. Therefore, the functional polymers obtained during this research are
intended to be used in cancer drug delivery systems.
Specifically, covalent polymer-drug conjugates offer an exciting strategy for the
improved delivery of therapeutic agents, because not only alters the pharmacokinetics,
while retaining or preferably enhancing the bioavailability, but also for the improved
specificity, increase solubility and duration of action at the target site. Polymer-drug
1 conjugates are a promising strategy for drug delivery especially in the field of cancer therapy.
Frequently the anticancer drugs are nonselective antiproliferative agents that kill dividing cells by attacking their DNA process. The absence of selectivity results in significant toxicity to normal cells. These toxicities along with drug resistance are major therapy limiting factors that results into poor options for patients.1 Polymer-drug conjugates therapy provides an alternative approach to design more selective and less cytotoxic form of anticancer drugs.
Polymer-drug conjugates design involves the conjugation of the drug to a linker
(e.g. polymer, carbohydrate or small molecule) and to a targeting molecule.2,3 The targeting molecule is an entity like sugars, growth factors, vitamins, antibodies or peptides that can bind specifically to receptors overexpressed in the cancer cells and subsequently release it inside the tumor cells, giving the desired selectivity to the drug.4
From the current studies targeting molecules with vitamins will be the focus in this research. From the literature review it was found that several vitamin–drug conjugates are currently undergoing clinical evaluations, and some of them might become FDA- approved drugs in a few years. Especially Folic Acid (FA) as a targeting agent has been widely studied. FA has two conjugation sites: The α- and the γ-carboxylic acid group.
Folate- targeted drugs will be linked via the γ-carboxyl rather than -carboxyl because the former exhibits higher affinity for FR than the latter.5 Vintafolide (EC145, IC50 ≈
9Nm) is one representative example of such γ-carboxyl conjugated disulfide-linked cytotoxic drugs in current clinical trials (Phase III clinical trial).6,7 Additionally, folate-
2 conjugated metal chelates have been proposed as potential imaging agents for cancers.
For example, 99mTc-DTPA-folate (EC20, Phase III clinical trials).8
Optimization of synthetic procedure is a major challenge. Since the carboxylic acid groups at the and γ positions are in very similar chemical environment, their reactivity is very similar under the conditions normally used for conjugation.
Consequently the regioselectivity toward the desired γ-conjugated product is poor.
Equally important, despite the advantages of a polymer linker (increased aqueous solubility, biocompatibility, and prolonged plasma circulation half-life compared to small molecules); there has not been any clinical trial for vitamin-polymer conjugate. Even though there are several current clinical trials of targeted nanoparticles (NP) based polymeric systems using other targeting molecules (e.g. antigen, transferrin receptor).9
The established method of FA attachment or “conjugation” is the activated ester method. This method, however, yields FA esters and amides with both the α and the γ carboxylic acid groups, which later needs complex and costly separation techniques.
Therefore, a new method for the exclusive γ-conjugation of FA with high selectivity was explored. The first step involves the formation of the lithium salt of the γ-carboxyl group in the glutamic acid moiety of FA. We hypothesized that the γ-carboxyl will react preferentially because the pKa values (where Ka is the acid dissociation constant) of the two carboxylic groups are very different (pKa, α-carbon= 4.5; pKa, γ-carbon=2.5).
Subsequently the γ-lithiated FA is reacted effectively with a bromine compound.
Previously, Dr Kwang Su Seo in his PhD dissertation thesis demonstrated the method using small bromo derivatives. Initially this research started with the idea of building a poly(isobutylene) (PIB)-based drug system conjugate, but due to the ionization and
3 structure determination by MS of PIB-based polymers remain a challenge, we then choose to test a synthetic route with PEG that is easily characterized by MS techniques.
In this research an effective route to biomolecule conjugation with PEG is explored to be further used in targeting drug delivery applications.
To meet the demand for expedient and efficient regioselective synthesis of releasable vitamin-conjugates, this thesis is aimed to develop a modular synthetic approach for the synthesis of a vitamin-polymer conjugate using enzymatic catalysis where possible. We hypothesize that use of enzymatic catalysis will allow us to produce precise functional polymers with high efficiency.
There are three answers to the question “Why use enzymes?”: necessity, convenience and opportunity. New synthetic and catalytic methods are necessary to deal with the new classes of compounds that are becoming the key targets for covalent polymer-drug conjugates. The use of enzymatic catalysis is a convenient and attractive alternative strategy because it offers many advantages, such as high efficiency, recyclability, and the ability to react under mild and solvent-free conditions. For this reason, in this research the opportunity to use this methodology for the functionalization of polymers, especially those employed in biomedical applications was investigated.
Enzymes are nature’s catalysts that accelerate specific metabolic reactions in living cells. As an environmentally friendly alternative to conventional chemical catalysts, enzymes offer several advantages including high selectivity, high efficiency, ability to operate under mild conditions, catalyst recyclability and biocompatibility. The most useful lipases for organic synthesis are: Porcine pancreatic lipase (PPL), lipase from Pseudomoanas cepacia (Amano lipase PS), lipase from Candida rugosa (CRL), and
4 lipase B from Candida antarctica (CALB).10 This research illustrates how powerful
CALB is in chemical transformations, for this reason it will be discussed in more detail.
CALB is immobilized on a resin, therefore it can conveniently be separated from the product, yielding very pure compounds. This research demonstrates how useful CALB is in chemical transformations in small molecules with transesterification and Michael
Addition reactions and in polymer chemistry.
In a previous report the efficiency of CALB-catalyzed transesterification was demonstrated by comparison with tin octoate; this latter gave 95% conversion when reacting vinyl-acetate with 2-phenylpropane-1-ol in 12 hours, while CALB yielded 100% conversion of the vinyl-acetate in 2 hours.11 It was shown that commercial Novozyme
235 (CALB) can be recycled several times before losing activity. 12–14 In this research small molecules “building blocks” were synthesized using CALB as catalyst.
CALB has several applications in polymer chemistry. As a catalyst for polymerization, CALB has successfully been applied over the last two decades in two major polymerization strategies, namely the ring-opening polymerization (ROP) of lactones and polycondensation type reactions.15,16 Aliphatic polyesters have been synthesized by the use of diverse monomers. Generally, polymers of moderate molecular weight have been obtained. In this research we present the synthesis of alkyne functionalized poly(caprolactone) (PCL) through the CALB catalyzed ring-opening polymerization of -CL activated monomer using propargyl alcohol and primary hydroxy-derivatized 4-dibenzocyclooctynol (DIBO) as an initiator. Additionally the polycondensation reaction between tetraethylenglycol and divinyladipate in the presence of CALB under solventless conditions is studied.
5 Furthermore, CALB is also active for selective end-functionalization, as well as modification of polymers. Despite the advantages that the enzymatic catalysis offers, reports of enzyme-catalyzed polymer functionalization have been less than optimal.
Recently Dr Puskas’ group have shown that CALB catalyzed quantitative methacrylation and acrylation of poly(ethylene glycol)s (PEG)s, polyisobutylenes and poly(dimethyl siloxane)s without the use of solvent.11,17 Previously, Dr. Sen from Professor Puskas’ research group investigated the functionalization of PEGs with vinyl esters using CALB- catalyzed reactions in organic solvents.17 The methacrylation and acrylation of PEGs with various molecular weights and molecular weight distributions in THF were quantitative in 24 hours. After that, Dr Kwang Su Seo prepared a tetra-hydroxyl functionalized PEG by CALB-catalyzed reactions using transesterification and Michael addition reaction to create multifunctional chain ends that could be used as the building block for the targeted nanodevices and other biomedical applications. In this work a more diverse type of functionalization is investigated. For instance the enzyme-catalyzed halogen functionalization of PEGs under mild conditions is explored. The same methodology is extended for the production of azide functional PEG, that can be further reacted through click chemistry. Additionally, the preparation of monofunctional acrylate VA-PEG-OH is studied via enzymatic transesterification.
Moreover, it is encouraged that enzymatic catalysts are to be used not only for replacing the conventional catalysts for the production of existing polymeric materials but also for creating new processes to produce the value-added materials for various uses, such as biomedical, pharmacological, and drug- delivery applications.
6 A second very important part of this research is the synthesis of biodegradable
PIB-based polymer. Biodegradable PIBs are very desirable: The first generation of PIB- based block copolymers is currently in clinical use on the FDA-approved Taxus® drug eluting coronary stent. More than six million of such stents have been implanted since
2004, saving countless lives. The same polymer was licensed for pace maker lead applications. Boston Scientific Co. verified that the biocompatibility of this polymer is superior to all existing biomaterials. It is anticipated that the new biodegradable PIB- based materials will also have superior biocompatibility. Our concept is to make biodegradable polyesters from short HO-PIB-OH precursors (a: monodisperse oligomer with 5 repeat units, and b: PIB with Mn ~ 1000 g/mol) using enzymatic catalysis. Since the introduction of the living carbocationic polymerization technique a variety of linear
telechelic polyisobutylenes PIB carrying one and two -CH2C(CH3)2 Cl end groups have been synthesized and characterized. In earlier publications18,19 researchers have described that complexes of BCl3 with certain tert-ethers and tert-alcohols are efficient initiators for the living polymerization of IB. The clean synthesis of tert-chlorine capped polyisobutylenes Cl-PIB-Cl by living carbocationic polymerization using various initiating systems with BCl3 has been demonstrated. In the course of this investigation it is of interest to determine whether and under what conditions would the tert-alcohols
(TMHDiOH), tert-chloride (TMHDiCl) and tert-ether (TMHDiOMe) diinitiator with
TiCl4 complex lead to well defined Cl-PIB-Cl, which can be further functionalized by end-cappping with allyltrimethylsilane and post-functionalized through thiol-ene click chemistry to get HO-PIB-OH. Later, HO-PIB-OH is used as macroinitiator for the ROP of -CL using CALB as catalyst.
7 The necessary background (Chapter II) to understand this thesis will be described, followed by the details in the experimental part (Chapter III). Chapter IV will discuss the enzymatic functionalization of poly(ethylene glycol)s (PEGs) with diverse functionalities. Chapter V will discuss the functionalization of polymer via enzymatic polymerization. Chapter VI will cover the chemo-enzymatic approach for biomolecules functionalization. Followed by Chapter VII that will describe the synthesis of low molecular mass telechelic PIBs. Finally, Chapter VIII will summarize the work done and it will include some recommendations.
8 2. CHAPTER II
BACKGROUND
The synthesis of functional polymers with well-defined structure, end-group fidelity and physico-chemical properties useful for biomedical applications has proven challenging. Enzymatic methods are an alternative strategy to increase the diversity of functional groups in polymeric materials. The objective of this research was the precise functionalization of polymers using two methods: Enzyme-catalyzed functionalization via polymerization and enzymatic functionalization by chain end functionalization. The background necessary to understand this research is detailed in the sections below.
2.1. Enzymes
Green Chemistry is a relatively new emerging field that strives to work at the molecular level to achieve sustainability. The field has received widespread interest in the past decade due to its ability to harness chemical innovation to meet environmental and economic goals simultaneously.20 Biocatalysis has emerged as an elegant synthetic methodology that allows the development of eco-friendly processes within the 12 basic principles of sustainable chemistry.20 Enzymes are nature’s catalysts that accelerate specific metabolic reactions in living cells. As an environmentally friendly alternative to
9 conventional chemical catalysts, enzymes offer several advantages including high selectivity, high efficiency, and ability to operate under mild conditions, catalyst recyclability and biocompatibility.21 Table 2.1 summarizes the main reasons why enzymatic catalysis is considered a green chemistry alternative.
Table 2.1. Aspects of enzyme catalysis relevant to green chemistry
Property Green chemistry Relevance Fast reactions due to correct catalyst orientation Faster throughput Orientation of site gives high stereoespecificity Possibility for asymmetric synthesis High degree of substrate specificity due to limited High degree of selectivity flexibility of active site Water soluble Opportunity for aqueous phase reactions Natural occurring Non-toxic, low hazard catalysts Natural operating under condition found in body Energy efficient reactions under moderate conditions of pH, temperature, etc. Possibility for tandem reactions when using whole Possibility for carrying out organisms sequential one-pot syntheses
Enzymes can be classified based on the function and the type of transformation that
they catalyze.21 Figure 2.1 shows this classification.
Figure 2.1. Classification of enzymes.
10 Hydrolases are the enzymes most used in biocatalysis and lipases form an important part of this class. Lipases are used widely in esterification, transesterification, aminolysis, and Michael addition reactions in organic solvents.15,21 The most useful lipases for organic synthesis are: Porcine pancreatic lipase (PPL), lipase from
Pseudomoanas cepacia (Amano lipase PS), lipase from Candida rugosa (CRL), and lipase B from Candida antarctica (CALB).10 This research illustrates how powerful
CALB is in chemical transformations, for this reason it will be discussed in more detail.
2.1.1. Candida Antarctica Lipase B (CALB)
The structure of CALB was solved in 1994 and is shown in Figure 2.2. 22,23
CALB belongs to the α/β-hydrolase-fold superfamily,21 which contains enzymes that have evolved from a common ancestor to catalyze reactions as various as hydrolysis of esters, thioesters, peptides, epoxides, and alkyl halides or cleavage of carbon bonds in hydroxynitriles.10 CALB is built up of 317 amino acids and has a molecular weight of 33 kDa. The active site pocket of CALB is illustrated in Figure 2.2, which is approximately
10Å x 4Å wide and 12 Å deep.22 It contains the catalytic triad, Ser105-His224-Asp187, common to all serine hydrolases.23,24
The top pocket (gray dark shadow in the Figure 2.2) is the “carbonyl” pocket an oxyanion hole that stabilizes the transition state and the oxyanion in the reaction intermediate. This oxyanion hole is a spatial arrangement of three hydrogen-bond donors, one from the side chain of Thr40 and two from the back-bone amides of Thr40 and
Gln106. The bottom (gray light shadow in the Figure 2.2) is the “hydroxyl” pocket.
11
Figure 2.2. (Left) 3D structure of CALB [Image of PBD ID:1TCB created with Polyview 3D] and (right) schematic representation of the catalytic triad of CALB. Reprinted with permission from Dr Kwang Su Seo.
In most reported biotransformations CALB is used physically immobilized within a macroporous resin of poly(methyl methacrylate) and it is commercially available as
Novozyme 435. The product consists of bead-shaped particles of with a diameter in the range of 0.3-0.9 mm with a water content of 1-2 % (w/w) and a protein content of 20%
(w/w).25,26
One of the focuses of the research in this dissertation is the enzymatic transesterification and Michael addition reactions. These two reactions will thus be discussed further.
2.1.2. CALB-catalyzed Transesterification
Transesterification reactions are generally reversible. The equilibrium can be shifted towards the product if an “activated” ester is used. Thus is, the nucleophilicity of the leaving group of the acyl donor should be reduced by the introduction of electron- withdrawing groups (e.g., trihaloesters, enol esters, oxime esters, anhydrides, etc.) as described in Figure 2.3.27
12
Figure 2.3. Transesterification of esters with alcohols: a) Reversible with an alkyl ester or a halogenated alkyl ester, and b) Irreversible with a vinyl ester.
In the case of the use of enol esters28 such as vinyl or isopropenyl esters the reaction is irreversible when the unstable enols are liberated as by-products that rapidly tautomerize to give the corresponding aldehydes or ketones and evaporate. Acetaldehyde, which forms during the reactions with vinyl esters, is known to inactivate the lipases from Candida rugosa and Geotrichum candidum by forming a Schiff’s base with the lysine residues of the protein. In contrast, CALB has shown to be highly stable when exposed to the acetaldehyde formed from vinyl esters.29
Based on the structural information, CALB is expected to have broad specificity toward acyl donors and high selectivity towards alcohols. Yadav et al compared the catalytic activity of various commercially available lipases in transesterification of vinyl acetate with n-octanol (Figure 2.4). CALB was found to be the most active catalyst in heptane as a solvent.30
13
Figure 2.4. Lipase-catalyzed transesterification vinyl acetate with n-octanol.
On the other hand, comparison of the catalytic activities of CALB and distannoxane, a conventional tin-based catalyst revealed that the transesterification of vinyl acetate with 2-phenyl-1-propanol catalyzed by CALB was complete in 2 hours, while the traditional tin-based catalyst yielded 95% conversion in 12 hours (Figure 2.5).11
Figure 2.5. Transesterification of vinyl acetate: comparison of CALB and a tin-based catalyst.
The catalytic cycle of the CALB-mediated transesterification shown in Figure 2.6 can be visualized based on the mechanism described in the literature.31
14
Figure 2.6. Illustration of the mechanism of CALB-catalyzed transesterification of vinyl acetate with 2-phenylpropane-1-ol. The different shading represents the two enzyme pockets.
The different shadings in our rendition in Figure 2.6 represent the carbonyl and hydroxyl pockets of the enzyme. First, the nucleophilic serine (Ser105) residue interacts with the carbonyl group of the vinyl acetate, forming a tetrahedral intermediate which is stabilized by the oxyanion hole of the enzyme via three hydrogen bonds: one from glutamine (Gln106) and two from threonine (Thr40) units. In the second step, the ester bond is cleaved to form the acyl-enzyme complex (AEC) and the first product, vinyl alcohol. Vinyl alcohol will immediately tautomerize to acetaldehyde, rendering the reaction irreversible. In the third step, the alcohol reacts with the acyl-enzyme complex to
15 form a second tetrahedral intermediate which is again stabilized by the oxyanion hole. In the last step, the enzyme is deacylated to form the desired product. The nucleophilic attack by the Ser105 is mediated by the His224-Asp187 pair.
2.1.3. CALB-catalyzed Michael Addition Reaction
The Michael addition reaction, which is a conjugate addition type reaction, is a powerful and highly-used method for the formation of new carbon–carbon and carbon– heteroatom bonds. Michael-type reactions typically involve the use of either strongly basic or acidic conditions, leading to the generation of potentially hazardous waste by- products and/or undesired side products.32 Recent efforts geared to discovery of greener, more environmentally compatible catalysts have been directed towards the development of biocatalytic versions of the Michael reaction.33
Bhanage et al report an efficient enzymatic protocol for the synthesis of -amino esters via Michael addition of primary and secondary amines to acrylates using CALB as a biocatalyst (Figure 2.7). CALB was found to be the most efficient lipase to catalyze the reaction while other lipases catalyzed the reaction with a low yield ranging from 10% to
36% of the desired product. In the absence of CALB under the same reaction conditions only traces of the Michael addition product was obtained.34
Figure 2.7. CALB-catalyzed aza-Michael addition of amine to acrylate
16 Gotor et al demonstrated CALB-catalyzed Michael-type addition of secondary amines to acrylonitriles and proposed that the serine in the active site was not involved in the reaction.35 These additions take place when α,β-unsaturated systems are used as the electrophile moiety and amines as the nucleophile substrate. Some evidences of the mechanism of this promiscuous activity of CALB point out that the oxyanion hole (Thr40 and Gln106) of the active site stabilizes the negative charge of the transition state while the His224-Asp187 pair facilitates proton transfer during the catalysis. Consequently, it is proposed that solvents of low polarity may induce interaction between the oxyanion hole and the carbonyl oxygen in the catalytic intermediate complex, allowing the ability of
CALB to carry out this reaction. According to the suggested mechanism,35–37 the catalytic cycle of the CALB-catalyzed Michael addition pyrrolidine to acrylonitrile was visualized as shown in Figure 2.8.
First is the interaction with the Michael acceptor, the nitrile group (or carbonyl group in the case of α,β-unsaturated carbonyl compounds) of acrylonitrile is activated by the oxyanion hole of the enzyme. Then the conjugate addition of the incoming nucleophile, i.e. pyrrolidine, to α-carbon of the Michael-acceptor takes place resulting in an intermediate which is stabilized by both the histidine-aspartate pair and the oxyanion hole in the enzyme active site. In the last step, the histidine-aspartate pair catalyzes the proton transfer from the pyrrolidine to the α-carbon of acrylonitrile.
17
Figure 2.8. Suggested mechanism for the CALB-catalyzed Michael addition of pyrrolidine to acrylonitrile.
Castillo et al38 implemented a solvent engineering strategy in order to control the chemoselectivity in a CALB-catalyzed Michael addition reaction (Figure 2.9).
Chemoselectivity of the enzymatic process was elucidated in terms of polarity of the medium, hence, adduct 3 was preferentially accumulated in hydrophobic medium, whereas in polar solvents the amide 4 was preferentially formed.
18
Figure 2.9. Chemoselectivity CALB-catalyzed Michael addition reaction.
The examples given in the previous sections demonstrate the power of CALB as catalyst in the synthesis of small molecules. The next section will discuss the use of
CALB in polymer chemistry, particularly for the synthesis and functionalization of polymers.
2.1.4. Enzymes in Polymer Chemistry
CALB has several applications in polymer chemistry. As a catalyst for polymerization, it is enantioselective, regioselective and chemoselective, and can be used under mild reaction conditions. CALB is also active for selective end-functionalization, as well as modification of polymers. The following part will highlight these processes.
2.1.4.1. Enzyme-catalyzed Polymer Synthesis
The application of enzymes in biocatalytic polymerization reactions represents an alternative to classical chemical polymerization methods that employ either acid–base or transition metal catalyzed processes.31 Unlike chemical methods, enzymatic polymerization reactions are generally performed under mild reaction conditions
19 (ambient temperature, pressure, and at neutral pH) and possess a high degree of reaction control in terms of their chemo-, regio-, and stereoselectivities, generally providing polymers with highly regulated structure.33 Biocatalytic polymerization thus offers novel, greener routes for the preparation of polymeric materials and biocompatible products.
Biocatalytic polymerizations have successfully been applied over the last two decades in two major polymerization strategies, namely the ring-opening polymerization of lactones and polycondensation type reactions (Figure 2.10) catalyzed by lipases.15,16
Figure 2.10. Major modes of lipase catalyzed polyester synthesis.
While a number of lipases have been screened for their ability to perform biocatalytic polymerizations, to date the best studied enzyme used for polymerizations is
CALB lipase.16
2.1.4.1.1. Polycondensation
Comprehensive reviews15,39–44 have been written to describe the broad range of enzyme-catalyzed polyester syntheses performed since 1984.31 This section will highlight pioneer work over the past years that have significantly advanced the field in enzyme- catalyzed polyester condensation.
20 In a solvent-free system reported in 1998, CALB efficiently catalyzed the polycondensation of dicarboxylic acids and glycols under mild reaction conditions at 60
ºC, despite the initial heterogeneous mixture of the monomers and catalyst. Polymer with molecular weight higher than 10,000 g/mol was obtained by the reaction under reduced pressure.45
Important progress has been made in lipase-catalyzed copolymerizations of acid and alcohol building blocks using non-activated free acid. For example, CALB has been used to catalyze the condensation polymerization of 1,3-bis- (carboxypropyl)- tetramethyldisilane with a series of alkanediol units in bulk and under vacuum to produce organosiloxane polymers ranging from 6,100 to 11,000 g/mol. The molecular weight obtained was depending on the alkanediol used (Figure 2.11).46
Figure 2.11. CALB catalyzed polymerization of organosiloxane with alkenodiols in bulk at elevated temperature and reduced pressure.
Activated esters have been also investigated. Prominent examples of activated esters used include bis(2,2,2-trichloroethyl) and vinyl esters. For example, Russell and co-workers47 showed that, by using CALB, in bulk copolymerization of divinyl adipate and 1,4-butanediol gave the corresponding polyester with Mw=23,200 g/mol (Figure
2.12).47
Figure 2.12. Lipase-catalyzed irreversible polytransesterification between divinyl adipate and 1,4-butanediol.
21 Furthermore, reaction times have decreased from days to hours. For example,
CALB-catalyzed bulk polymerizations of 12- and 16-hydroxyacids have been performed
48 producing polyesters in only 4 h with DPavg= 90 (Mn up to 23,000 g/mol). Bulk condensation polymerizations of adipic acid and octanediol, also catalyzed by CALB,
49 generated polyesters with Mn=15,000 g/mol in 8 h.
The chemoselectivity of CALB was explored when was used to catalyze the direct polycondensation of hexane-1,6-diol and dimethyl-2-mercaptosuccinate in bulk to produce aliphatic polyesters containing free pendant mercapto groups with molecular weights of 14,000 g/mol (Figure 2.13).
Figure 2.13. CALB-based biocatalytic polymerization of free mercapto containing polyesters.
No formation of disulfide or thioester linkages was observed. The pendant thiol- containing polyesters were easily cross-linked to form gels by the air oxidation in
DMSO.50
Enzyme-regioselectivity offers the potential to develop simple and direct routes to prepare Malic acid (MA) copolymers. CALB-catalyzed copolymerization in bulk of L-
MA, adipic acid and 1,8- octanediol was recently investigated (Figure 2.14).51 By using
20 mol % L-MA in the monomer feed at 80 ºC, a copolyester was formed in 91% yield.
Most importantly, 1H NMR studies revealed CALB was strictly selective for
22 esterification of L-MA carboxylic groups while leaving hydroxyl pendant groups unchanged.51
Figure 2.14. Synthesis of copolyesters containing L-malic acid units using lipase as catalyst.
In conclusion, CALB-catalyzed synthesis of polyesters by polycondensation has been extensively investigated in the past decade. Aliphatic polyesters have been synthesized by the use a variety of monomers. Generally, polymers of moderate molecular weight have been obtained. The other method of polymerization where CALB has been extensively utilized is ring-opening polymerization.
2.1.4.1.2. Ring Opening Polymerization (ROP)
In the polymerization modes shown in Figure 2.10, ring opening polymerization
(ROP) has been very extensively studied and several scientifically new aspects specific to enzymatic catalysis have been developed.39,41,52–54
An alcohol can purposely be added to the reaction medium to initiate the polymerization instead of water. The polymerization can be carried out in bulk, in organic solvents, and in ionic liquids. Interestingly, Kobayashi and coworkers reported in
55 2001 the ROP of lactones by lipase CALB in supercritical CO2.
In this respect, it must be noted that water has influence on the course of the polymerization. Water can initiate the polymerization, and at the same time a minimum
23 amount of water has to be bound to the surface of the enzyme to maintain its conformational flexibility, which is essential for its catalytic activity.39 Lipase-mediated polymerization cannot therefore be achieved in strictly anhydrous conditions. CALB has been used in the ROP polymerization of lactones of a range of size. The following part will highlight specific examples of these polymerizations.
Figure 2.15. CALB-catalyzed ROP of various monomers.
ROP of a 4-membered lactone Poly(β-malic acid) was prepared by lipase- catalyzed ROP of benzyl β-malolactonate (BM) with subsequent debenzylation at 60 °C to yield poly(benzyl β-malate) (PMB) having a molecular weight greater than 7,000 g/mol (Figure 2.15).56
Due to its thermodynamic stability, ROP of the 5-membered lactone has proved difficult. Bonduelle et al have produced poly(l-lactide) with Mn up to 38,400 g/mol
24 (Figure 2.15) and low polydispersities (ĐM < 1.4) by the lipase-catalyzed ROP of lacOCA which is the O-carboxylic anhydride derived from lactic acid.57
Poly(1,4-dioxan-2-one) (PPDO) is an important biocompatible polymer with good flexibility and tensile strength, which has been widely used in biomedical applications. It
58 has been synthesized using CALB as catalyst with Mn of 41,000 g/mol and 58,000 g/mol (Figure 2.15).59 In contrast to L-lactide, D-lactide has been polymerized by CALB
60 catalysis, yielding poly(D-lactide) with Mn up to 12,000 g/mol (Figure 2.16).
Figure 2.16. Enzymatic ROP of DD-lactide (DLA) by CALB.
The 6-membered cyclic carbonate, trimethylene carbonate (TMC), is one of the most widely studied monomers to date (Figure 2.17). It has been enzymatically polymerized or copolymerized with other monomers using CALB.61–63 Elimination of carbon dioxide has not been detected in enzymatic ROP of cyclic carbonates, making them suitable for biomedical materials.
Figure 2.17. Enzymatic ROP of trimethylene carbonate (TMC) by CALB.
The most widely studied system is the enzymatic ROP of -CL. In this case, lipase CALB showed high catalytic activity toward its polymerization, less than 1 wt % of CALB was enough to induce the polymerization of -CL (Figure 2.18).64 Furthermore,
25 lipase CALB could be reused for the polymerization. In the range of five cycles, the catalytic activity hardly changed.65 Among the solvents examined, toluene was the best to produce high molecular weight poly(-CL) efficiently. Variation in the ratio of toluene to
-CL in the reaction at 70 °C showed that the monomer conversion and polymer molecular weight were the largest with a ratio of about 2:1.65
Figure 2.18. Enzymatic ROP of -CL by CALB.
In terms of the reactor design, microreactor technology is a good alternative.
Recently, Kundu et al constructed a microreactor for CALB-catalyzed ROP of ɛ- caprolactone, which was made of aluminum coated with Kapton film using a thermally cured epoxy and mounted on a uniform heating stage.66 Compared with traditional batch reactors, the microreactor containing immobilized CALB enabled faster polymerization and higher molecular mass polymers.
Due to the limitation of commercially available monomers and lack of unique properties of polymers, enzymatic polymerization of 8-membered lactone (7- heptanolactone), 9-membered lactone (8-octanolactone), 10-membered lactone (9- nonanolactone) and 11-membered lactone (10-decanolactone) has attracted relatively less attention. Using CALB as catalyst (Figure 2.19), ROP of these monomers generated corresponding polymers with Mn values of 23,600, 16,000, 16,000 and 20,000 g/mol, respectively.67
26
Figure 2.19. Enzymatic ROP of macrocyclic monomers by CALB.
A 12-membered lactone, 2-oxo-12-crown-4-ether (OC) underwent a highly reactive ROP with CALB (Figure 2.20), giving a poly(ester ether) with
68 Mn = 3500 g/mol.
Figure 2.20. Enzymatic ROP of OC by CALB.
The 16-membered lactone ω-pentadecanolide has also been widely used in enzymatic ROP. For example, ω-pentadecanolide was shown to be rapidly polymerized
69 in the presence of CALB affording poly(ω-pentadecanolide) with Mn = 34,00 g/mol.
In ring-opening polymerization of macrocyclic lactones, the lactones are typically strainless, and the reactions are considered to be the entropically driven. Manzini et al successfully carried out the enzymatic ring-opening polymerization of 24-, 26-, 28-, 36-,
39-, 56-, and 84-membered macrocycles.70
27 2.1.4.1.3. Mechanism
It is well-known that the catalytic site of lipase is a serine residue and that lipase- catalyzed reactions proceed via an acyl-enzyme intermediate. The mechanism of the enzymatic polymerization is shown in Figure 2.21 and can be decomposed into three main steps.39,41,71
Figure 2.21. Modeled Kinetic Reactions in Enzyme-Mediated Poly(ε-caprolactone) Synthesis. Note: Subscripts on kinetic rate parameters denote the reaction step number. Positive subscripts are reactions with enzyme-activated PCL chains; negative subscripts are reactions with free enzyme sites.
Step 1 the lactone is first activated by nucleophilic attack of the serine residue of the enzyme on the lactone carbonyl, forming an acyl-enzyme intermediate (“enzyme- activated monomer”, EM). An activated opened monomer is obtained, which is more reactive than lactone towards nucleophiles. In step 2 chain initiation proceeds by nucleophilic attack of a nucleophile like water (alcohol, thiol or an amine can also serves as nucleophile) on the acyl carbon of the intermediate, producing ω-hydroxycarboxylic acid (n = 1). During chain propagation (Step 3), the acyl-enzyme intermediate is nucleophilically attacked by the terminal hydroxyl group of the propagating chain end, to
28 produce a one-unit-more elongated polymer chain. Both the initiation and propagation steps involve deacylation of the lipase.71
Several studies indicate that the formation of the activated open monomer is the rate-limiting step. The kinetics of polymerization obey the usual Michaelis–Menten equation.67 Nevertheless, all experimental data cannot be accounted for by this theory.
Other studies suggest that the nature of the rate-limiting step depends upon the structure of the lactone.72 Indeed, the reaction of nucleophilic hydroxyl-functionalized compounds with activated opened monomers can become the rate limiting step, especially if sterically hindered nucleophilic species are involved. Unlike chemical ROP, enzymatic
ROP is not governed by the ring strain. Unexpectedly, several studies showed that macrolides polymerize faster than smaller ring lactones when lipases are used as catalysts.73 It was shown that macrolides exhibit a higher dipole moment and are thus more hydrophobic than smaller ring lactones.73 Indeed, macrolides have a chemical structure closer to the glycerol fatty acids esters hydrolyzed by lipases in nature.72
Lipase-catalyzed ROP of lactones accompanies the formation of cyclic oligomers
(step 4) in addition to major products of linear polyesters.74 This behavior is well known as a ring-chain (cyclic-linear structure) equilibrium generally involved in the all polymerization reaction, including enzymatic ROP75 and enzymatic polycondensation.76
Reaction behaviors of the cyclic oligomer formation in the ROP of -CL have been investigated in detail. The polymerization was carried out using CALB as catalyst at 60
ºC in bulk or in an organic solvent. In bulk, cyclic dimers of -CL and linear poly(-CL) were formed (2 and 98%, respectively). Among the organic solvents examined
29 acetonitrile gave a major portion of cyclic oligomers, total 70% and a minor portion of linear poly(-CL) (30%).75
2.1.4.1.4. Functionalization of Polymers by ROP Polymerization
For the synthesis of end-functionalized polymers, typically macromonomers and telechelics, the polymer terminal structure is to be precisely controlled. Lipase catalysis provided new methodologies for single-step functionalization of polymer terminal. The enzyme-catalyzed polymer synthesis gives product polymers free from any metal catalyst, which often broadens the application of the products in particular for medical use.52
Initiator Method
According to the speculated mechanism of lipase catalysis, a nucleophile, like water, an alcohol, an amine or a thiol can act as an initiating species in the ROP of lactones. To obtain highly functionalized polyesters, the enzyme and the reaction system are to be dried to reduce the water content. At the same time, it is also true that a completely dry reaction system cannot be realized because the enzyme requires traces of water to retain its activity.39 Thus is, control of the water content is very important for the functionalization. For example, benzyl alcohol was used as initiator for the ROP of -CL, functionalization was found to vary from 20%77 to 73%78 depending of the water content in the media.
As shown in Figure 2.21, an alcohol acted as an initiating species in the ring- opening polymerization of lactones by lipase CALB catalyst to introduce the alcohol
30 moiety at the polymer terminal (“initiator method”).79 Cordova and co-workers75 prepared end-functionalized polycaprolactone (PCL) macromers using CALB.
Caprolactone (-CL) was polymerized in the presence of functional initiators that included 9-decenol (A), 2-(3-hydroxyphenyl)- ethanol (B), 2-(4-hydroxyphenyl)ethanol
(C), and cinnamyl alcohol (D) (Figure 2.22). This methodology was expanded to synthesis of ö-alkenyl and alkynyl-type macromonomers by using 5-hexen-1-ol and 5- hexyn-1-ol as initiator.
Figure 2.22. Functionalization of -CL by initiator method.
Polyesters bearing a sugar moiety at the polymer terminal were synthesized by
CALB-catalyzed polymerization of -CL in the presence of alkyl glucopyranosides
(Figure 2.23).80,81 In the initiation step, the regioselective acylation at the 6-position of the glucopyranoside took place.
Figure 2.23. CALB-catalyzed polymerization of -CL in the presence of alkyl glucopyranosides.
31 Terminator Method
The terminator method involves a lipase-catalyzed single-step acylation of a polyester alcohol end-group with a vinyl ester. Cordova explored this concept (Figure
2.24), first, -CL ROP was initiated by lipase catalysis from OH-containing initiators, such as a benzyl ester of 2,2-bis(hydroxymethyl)propionic acid or ethyl glucopyranoside; then, the selective end group functionalization was introduced to the terminal OH of the
-CL chain by end-capping with 10-undecenoic acid, vinyl acrylate, and vinyl methacrylate as acyl donors. Polymers were obtained with and average molecular weight up to Mw= 3,100 g/mol.82
Figure 2.24. Synthesis of macromonomer by terminator method.
In summary, CALB was broadly used as catalyst for the polymerization of a variety of monomers. Enzyme catalyzed polymerization offer several benefits such as mild reaction conditions, selective reactions to give well defined structures and
32 substitution of toxic metal catalysts with natural enzymes. The control of polymer architecture is possible because of enantio and region selectivity of enzyme.
Another area of polymer science where the use of enzymes has potential advantages over conventional catalysts is the selective end-functionalization, as well as modification of polymers. This area will be discussed in the next section.
2.1.4.2. Enzyme-Catalyzed Post-Polymerization Functionalization of Polymers
Synthesis of new polymeric materials can be achieved not only by polymerization reactions but also by modification reactions of the existing polymers. Despite all the advantages that the enzymatic catalysis offers, the area in the end-functionalization of preformed synthetic symmetric or asymmetric telechelic polymers modification has not been fully developed. Enzymes can catalyze the modification of a polymer through functional groups located at the polymer terminal, in the main chain or in the side chain in a specific reaction manner.
Natural Polymers
Site specific chemical modification of hydroxy groups in polysaccharide chains is hardly possible via conventional organic synthetic method. Preparation of various esters was performed via CALB-catalyzed acylation of cellulose acetate83 and hydroxypropyl cellulose.84 In the case of the acylation of cellulose acetate with lauric and oleic acids
(Figure 2.25), the final conversion of both fatty acids was about 35% after 96 h of incubation at 50 °C. In the case of hydroxypropyl the final ester content was about 11% after 6-day incubation at 50°C.
33
Figure 2.25. CALB-catalyzed acylation of cellulose acetate.
Starch nanoparticles in microemulsions were reacted with vinyl stearate, - caprolactone, and maleic anhydride in the presence of CALB at 40 °C for 48 h to give starch esters with degrees of substitution (DS) of 0.8, 0.6, and 0.4, respectively.
Substitution occurred regioselectively at the C-6 position of the glucose repeat units
(Figure 2.26).85
Figure 2.26. CALB-catalyzed functionalization of starch nanoparticles.
Another example is a novel regioselective strategy for the transesterification of
Konjac glucomannan (KGM) with vinyl acetate using CALB in a solvent-free system.
KGM is an abundant, naturally occurring polysaccharide isolated from the tubers of
Amorphophallus konjac plant. It consists of -1,4-linked D-glucose and D-mannose
34 units, and the molar ratio of glucose to mannose has been reported to be around 1 to 1.60.
Degree of substitution (DS) depend of temperature of reaction 0.34 to 0.58 at 30 and
60ºC respectively. It has also been found that the DS (from 0.67 to 0.16) of modified
KGM sample decreases with increase in KGM molecular weight (from 114,000 to
980,000 g/mol).86
Synthetic Polymers
Lipase (CALB)-catalyzed modification of pendant ester groups of a polystyrene derivative provided with a clear-cut regioselective transesterification reaction.87 In the pendant two ester groups are present, only the ester group distant from the polymer backbone was involved in the reaction. It is possible that due to the proximity of the acyl
(ester) group (A) to the bulk of the polymer backbone, the enzyme is incapable of coordinating to the acyl group, but only for the distant group (B) (Figure 2.27).
Figure 2.27. CALB-catalyzed modification of pendant ester groups of a polystyrene derivative.
35 Jarvie et al showed that CALB was able to catalyze the selective epoxidation of polybutadiene (Mn=1,300 g/mol) (35% trans, 20% cis, 45% vinyl) in organic solvents in the presence of hydrogen peroxide and catalytic quantities of acetic acid (Figure 2.28).
The cis and trans alkene bonds of the backbone were epoxidised in yields of up to 60% while the pendant vinyl groups are untouched.88
Figure 2.28. CALB-catalyzed epoxidation of polybutadiene.
Enantioselective enzymatic transesterification by CALB was investigated (Figure
2.29).89 First, copolymers of styrene and p-vinlyphenylethanol (R-isomers and S-isomers) were prepared from these monomers over the whole range of compositions from 100% R to 100% S. When a backbone containing 100% S groups was used for the enzymatic transesterification with vinyl acetate, no acetylation was detected. By contrast, when a backbone containing 100% R groups was used, the enzymatic transesterification of vinyl acetate occurred from 75% of the alcohol groups within 24 h.
Figure 2.29. Enzymatic grafting of copolymers with vinyl acetate.
36 In another study, CALB-catalyzed acylation of comb-like methacrylate polymers was induced through OH groups in the side chains using vinyl acetate, phenyl acetate, 4- fluorophenyl acetate, and phenyl stearate, as acylating agents (Figure 2.30). The OH groups in the side chains of the methacrylate/ styrene copolymer were acylated in THF at an ambient temperature for 6 days (Figure 2.30). Conversions vary from 20% to 93% depending of the acylating agent.90
Figure 2.30. CALB-catalyzed acylation of comb-like methacrylate polymers.
Telechelic carboxylic acid functionalized PDMSs were reacted with α,β- ethylglucoside at 70 °C under vacuum for 34 hours in the presence of CALB, but the product was a mixture of mono- and difunctional esters (Figure 2.31).91 In other studies, the synthesis of telechelic methacrylate-functionalized oligoesters by the CALB- catalyzed polycondensation of ethylene glycol, hydroxyethyl methacrylate and divinyl adipate (DVA) was reported.92 However, 5-9% of the product was not difunctional methacrylate.
37
Figure 2.31. Telechelic carboxylic acid functionalized PDMSs in the presence of CALB.
Puskas et al reported the first examples of quantitative functionalization of synthetic polymer using CALB catalyzed reactions with and without organic solvents.93
Sen Mustafa94 in his doctoral thesis shows different chain end conjugations catalyzed by
CALB.
For example, Figure 2.32 shows the quantitative methacrylation by transesterification of vinyl methacrylate with the primary hydroxyl groups of hydroxy- functionalized polyisobutylenes (PIBs) in the presence of CALB within the 24 h in hexane and 2 h in bulk, respectively.
Figure 2.32. CALB-catalyzed methacrylation of PIB-OHs. PIB-OH (Mn=5,200 g/mol; ĐM =1.09), Glissopal-OH (Mn=3,600 g/mol; ĐM=1.34), and asymmetric telechelic HO- PIB-OH (Mn=7,200 g/mol; ĐM =1.04).
38 Additionally, asymmetric methacrylation of α, -hydroxy functionalized PIB was achieved by the regioselective transesterification of vinyl methacrylate using CALB in hexane within 24h, leaving the sterically hindered hydroxyl group intact. The reaction was irreversible because the vinyl alcohol product instantaneously tautomerized into acetaldehyde, which was easily removed from the system due to its low volatility.
Recently, functionalized PEGs under solvent free conditions within 4 h was achieved, by dissolving low molecular weight HO–PEG–OH (Mn = 1,050 and 2,000 g/mol) in the corresponding acyl donors (vinyl methacrylate, vinyl acrylate and vinyl crotonate) at 50 ºC (Figure 2.33). 1H and 13C NMR along with MALDI-ToF confirmed quantitative conversion with the expected structures.
Figure 2.33. Functionalized PEGs vial CALB-catalyzed transesterification.
Additionally other polymers are also functionalized with CALB. Commercially available polydimethylsiloxanes (PDMS), PDMS-monocarbinol and PDMS-dicarbinols were also methacrylated with vinyl methacrylate under solventless conditions within 2 hours in the presence of CALB.17,94 Primary hydroxy-functionalized polystyrene (PS-
39 (CH3)2Si-CH2-OH, Mn=2,600 g/mol; ĐM =1.06) was quantitatively methacrylated by transesterification of vinyl methacrylate within 48 hours.
CALB in Dendrimer Synthesis
Puskas et al synthesized (HO)2–TEG–(OH)2 and (OH)2–PEG–(OH)2 as a core of novel dendrimers using sequential CALB-catalyzed transesterification and Michael addition of diethanolamine to the acrylate double bonds (Figure 2.34).11 Both transesterification and Michael addition reactions were successful as quantitative conversions were reached within 24 h and 2 h, respectively.
Figure 2.34. (OH)2–PEG–(OH)2 as a core of novel dendrimers using sequential CALB.
In summary, CALB was shown to be an effective catalyst for selective end- functionalization, as well as modification of polymers. Despite the advantages that the enzymatic catalysis offers, the end functionalization is still an ongoing work where there
40 is much room for new applications. Therefore, it is encouraged that lipase catalysts are to be used not only for replacing the conventional catalysts for the production of existing polymeric materials but also for creating new processes to produce the value-added materials for various uses, such as biomedical, pharmacological, and drug delivery applications.
Specifically, covalent polymer-drug conjugates offer an exciting strategy for the improved delivery of therapeutic agents, because not only alters the pharmacokinetics, while retaining or preferably enhancing the bioavailability, but also for the improved specificity, increase solubility and duration of action at the target site. Polymer-drug conjugates are a promising strategy for drug delivery especially in the field of cancer therapy. Research is ongoing concerning the conjugation of the therapeutic agent to the polymer.
There are three answers to the question “Why use enzymes?”: necessity, convenience and opportunity. New synthetic and catalytic methods are necessary to deal with the new classes of compounds that are becoming the key targets for covalent polymer-drug conjugates. The use of enzymatic catalysis is convenient because endorse the concept of green chemistry and sustainability as highlighted in previous section. For this reason, in this research the opportunity to use this methodology for the functionalization of polymers, especially those employed in biomedical applications was investigated.
41 2.2. Polymeric Conjugates for Cancer Treatment
Frequently the anticancer drugs are nonselective antiproliferative agents that kill dividing cells by attacking their DNA process. The absence of selectivity results in significant toxicity to normal cells. These toxicities along with drug resistance are major therapy limiting factors that results into poor options for patients.1 Polymer-drug conjugates therapy provides an alternative approach to design more selective and less cytotoxic form of anticancer drugs.
Polymer-drug conjugates design involves the conjugation of the drug to a linker
(e.g. polymer, carbohydrate or small molecule) and to a targeting molecule.2,3 The targeting molecule is an entity like sugars, growth factors, vitamins, antibodies or peptides that can bind specifically to receptors overexpressed in the cancer cells and subsequently release it inside the tumor cells, giving the desired selectivity to the drug.
Thus is, the targeting often display high affinities to their associated receptors and undergo a rapid receptor-mediated endocytosis after binding. The binding of the ligand to the target cell results in the ligand–receptor complex folding into a vesicle, which subsequently fuses with the early endosome (Figure 3).4 From the current studies targeting molecules with vitamins will be the focus in this research. Therefore vitamin- based conjugates and clinical trials will be discussed in the following section.
2.2.1. Vitamin-Based Drug-Delivery Systems
Cancer cells have an increased requirement for vitamins essential for biosynthesis and nutrient metabolism such as folate, biotin, riboflavin and cobalamin (Figure 2.35), to sustain the rapid cell-division cycles.95 The corresponding cell-surface uptake-transporter
42 proteins are thus often overexpressed on tumor cells and have been exploited for the targeted delivery of various therapeutic effectors.4,96,97
Figure 2.35. Vitamins commonly used for drug-delivery applications. The functional groups on folate and biotin amenable to payload attachment are shaded in blue.96,98 Payloads attached to the orange shaded groups on cobalamin have been shown to maintain full binding to all cobalamin-trafficking proteins, while attachment of the payload through the group shaded in green allows the generation of transcobalamin I selective constructs.4
2.2.1.1. Folic Acid
The most extensively studied example of vitamin-based drug delivery is the folate/folate receptor (FR) system,99 largely because its receptor (FR) is overexpressed in many, but not all, variety of tumors (e.g., breast, lung, kidney, ovary, colon, brain, and hematologic malignancies)95,100 yet absent in most normal tissues.100 For example in serous carcinoma (ovarian cancer) FR= 34 pM/mg membrane protein compare to a normal ovarian cell FR= 1 pM/mg membrane protein.100
43 Folates are needed within the cell to carry out carbon methylation reactions as well as de novo synthesis of nucleotide bases (most notably thymine, but also purine bases).101 Therefore the reason for this overexpression is that folates are essential to the viability of normal, but especially proliferating cells.96 Folic Acid can bind to the FR with
−9 97,102 high affinity (Kd ∼ 10 M). Folate can be conjugated to low- or high-molecular weight therapeutic or imaging agents and the folate-conjugates preserve the binding property.103 More importantly, FR undergoes internalization via endocytosis, delivering folate (or its therapeutic conjugates) into the cell interior. As shown in Figure 2.36, Step
1, a folate−drug conjugate extravasates from circulation to approach an FR-expressing tumor cell. From there, it readily binds to a FR (Step 2) and is brought inside the cell via an endocytosis process. After that, a biocleavable linkers placed within the folate−drug conjugate construct is broken inside this endosomal compartment (Step 3) to release the drug that diffuses out of the endosome to interact with its biological target (Step 4). The
FR protein recycles back to the cellular membrane.101 The activity of folate−drug conjugates is closely dependent on an efficient cleavable linker system that ensure release of the attached drug only after internalization by the target cell.6
44
Figure 2.36. Schematic presentation of tumor cellular uptake of a folate−drug conjugate by FR-mediated endocytosis. The folate−drug conjugate consists of a folic acid moiety (shown as a yellow oval) and the drug payload (shown in red). Reprinted with permission from Vlahov, I. R.; Leamon, C. P. Bioconjug. Chem. 2012, 23, 1357–1369. Copyright 2014 American Chemical Society.
Two different trigger systems have been utilized to promote the programmed break of the linker system within the endosome: a disulfide linker or a pH-sensitive linker.101 The most effective approach, a disulfide- bond-based linker system, takes advantage of the reducing power within the endosomal milieu.104 The second approach, a pH- sensitive linker system, releases the active parent drug from its folate−drug conjugate as the result of the drop in the pH in the endosome (pH5-6).105 After release occurs by either of these two methods, highly lipophilic drugs cross the endosomal membrane and find their pharmacological target within the cytosol.
45 Leamon et al6 compared the effect of the trigger system based on folate- desacetylvinblastine monohydrazide conjugates: EC140 with an acylhydrazone (pH- sensitive) linker and EC145 (commercial name Vintafolide) with a disulfide linker
(Figure 2.37). As anticipated, in vivo tests have demonstrated EC145 to be dramatically more potent than EC140.6
Figure 2.37. Folate−drug conjugates in human clinical trials. Comparison between a pH- sensitive linker and a disulfide linker.
Moreover, the folic acid has two conjugation sites: the α- and the γ-carboxylic acid group (Figure 2.35). Folate- targeted drugs will be linked via the γ-carboxyl rather than -carboxyl because the former exhibits higher affinity for FR than the latter.5
46
Figure 2.38. Folate−drug conjugates in human clinical trials.
Representative examples of such γ-carboxyl conjugated disulfide-linked cytotoxic drugs (Figure 2.38) include Vintafolide (EC145, IC50 ≈ 9nM),6,7 folate-maytansine DM1
(EC131, IC50 ≈ 16-25 nM),106 folate-tubulysin B (EC0305, IC50 ≈ 1-10 nM),107 and
47 folate-mitomycin C (EC72, IC50 ≈ 5nM).108 Each of the aforementioned folate-targeted cytotoxic drugs has demonstrated remarkable activity in animal tumor models with greatly reduced off-target toxicity to healthy cells and they were in current human clinical trials. Vintafolide is being evaluated in a global Phase III study named PROCEED for women with platinum-resistant ovarian cancer. Endocyte and Merck entered into a $1 billion partnership for vintafolide in April 2012.109Additionally, vintafolide will be evaluated in women with triple-negative breast cancer in a Phase IIb study scheduled to begin in 2014,96 whereas EC1456 is currently being evaluated in a Phase I study in patients with advanced solid tumors.
Several folate-diagnostic conjugates have recently been covered in a number of excellent reviews.96,97,101 Folate-conjugated metal chelates have been proposed as potential imaging agents for cancers. 99mTc-DTPA-folate [EC20] and 111In-DTPA-folate were synthesized and introduced into the clinic trials for imaging of cancer patients.
8,102,110,111 Uptake of 111In–DTPA–folate in ovarian cancer patients was seen in both the cancer cells and kidneys, whereas in healthy patients only kidney uptake was observed
Figure 2.39. Currently Etarfolatide (EC20) imaging has been a component of more than
13 clinical studies, in patients with different cancer; and has been administered to more than 1000 patients. It is being co-developed as the companion imaging agent to vintafolide (EC145, PROCEED Phase III) and EC1456 (folate-tubulysin).109
48
Figure 2.39. Folate-conjugated imaging agents in human clinical trials.
Another folate-targeted therapeutic agent to enter the human clinical trial is EC17,
112 a folate-linked fluorescent hapten that was designed to enhance the immunogenicity of
FR-positive tumors (Figure 2.39). In humans, the therapy has recently completed phase II clinical studies in kidney cancer. Phase I trial has started for the intraoperative imagery of triple negative breast cancer with Folate-FITC (EC17).
Assuming that the observations from the clinical trials results of the folate- conjugates can be extrapolated, one general lesson can be shared: Folate targeting can revive other useful drugs that have proven to be too toxic for human use in their normal non-targeted forms. In general, in all the current human clinical trials the drugs are tethered to folate via a hydrophilic spacer (to confer water solubility on the entire conjugate) and a disulfide bond (for facile release of the attached drug following uptake
49 into reducing endosomes). And as with EC145, each of the targeted drugs displays significantly reduced off-site toxicity compared to its non-targeted counterpart and this improved toxicity profile is seen without loss in anti-tumor activity.
2.2.1.2. Biotin
Biotin is a cofactor responsible for carbon dioxide transfer in several carboxylase enzymes, and as a result, is important in fatty acid synthesis, branched-chain catabolism, and gluconeogenesis, and an essential micronutrient for normal cell growth and development.113 In the area of targeted cancer therapy, biotin has been also studied.
Biotin uptake in tumor cells is greater than in normal cells.114 In fact, biotin receptors are overexpressed more than FA or Vitamin B12 receptors on several cancer cell lines, including leukemia (L1210FR), lung (M109), renal (RENCA, RD0995), and breast (4T1,
JC, MMT06056).95 The structure(s) of the biotin receptor(s) are not known; however, the sodium dependent multivitamin transporter is believed to be involved in biotin uptake.115
Biotin has been found to bind with the proteins avidin and streptavidin with a dissociation constant of the order of 10-15 M. This is one of the strongest known-protein ligand interactions.116 Several recent examples of the use of biotin-avidin technology in cancer research can be found in the literature.4,9,114,117 A 90Y-DOTA-biotin conjugate entered a phase II trial for the treatment of metastic colon cancer (Figure 2.40).118
50
Figure 2.40. Example of a 90Y-DOTA-biotin compound.
The research group of Ojima described several conjugates of biotin to fluorescein, coumarin, a taxoid-fluorescein derivative, and the improved taxoid SB-T-1214 (Figure
2.41).98 The authors were able to show that L1210FR leukemia cells could take up biotin–fluorescein conjugates efficiently. Furthermore, a self-immolative disulfide linker could be cleaved intracellularly, as demonstrated by activation of quenched coumarin fluorescence. The biotin–linker–taxoid–fluorescein conjugate was also cleaved efficiently, thereby resulting in the green fluorescence labeling of microtubule bundles.
Finally, while the free taxoid exhibited low nanomolar cytotoxicity (IC50 =9.5–10.7 nM) against biotin receptor positive and negative cell lines, the targeted cytotoxic selectively killed biotin receptor positive cells with a potency equal to that of the free taxoid (IC50
=8.8 nM).98
51
Figure 2.41. Chemical structure of biotin-S-S-taxoid and biotin-S-S-taxoid-fluorescein.
2.2.1.3. Other Vitamins Commonly Used for Drug-Delivery Application
Besides folate, and biotin cobalamin (vitamin B12), which is essential for thymidine biosynthesis, has also been actively investigated as a ligand for the delivery of cytotoxic drugs into solid tumors.95 Indeed, co-vitamin B12 was tested for imaging applications in murine models of cancer. The translation of this approach into the clinic was hampered by the high uptake in the liver, pancreas, and kidneys.119 Nevertheless, cobalamin conjugates with chelators for radiometals such as 99m Tc and 111 In, which are more suitable for imaging in humans, were prepared,120 and showed improved targeting properties in vivo.
Multi targeting systems (MTS) generally describe surfaces decorated with two or more targeting ligands that recognize different receptors on the same or different cells.
This type of targeting has gained a substantial amount of interest as a way to increase prodrug uptake by cancer cells using a ‘‘two-punch’’ (or ‘‘three-punch’’) approach. Patil
52 et al121 also prepared dual-targeted PLA-PEG NPs using folic acid and biotin as targeting agents and investigated the tumor accumulation and efficacy of this construct versus folate-targeted and biotin-targeted mono-targeted NPs. The targeting strategy resulted in improved efficacy and higher tumor accumulation when compared to controls.
In conclusion, several vitamin–drug conjugates are currently undergoing clinical evaluations, and some of them might become FDA-approved drugs in a few years.
Polymer−drug conjugates design involves the conjugation of the drug to a linker (e.g. polymer, carbohydrate or small molecule) and to a targeting molecule (e.g. vitamins).
Optimization of synthetic procedure is a major challenge as the chemistries associated with drug molecule and the carrier may not always be compatible. Also, despite the advantages that a polymer linker can offer (increased aqueous solubility, biocompatibility, and prolonged plasma circulation half-life compared to small molecules); to my knowledge there has not been any clinical trial for vitamin-polymer conjugate. Even though there are several current clinical trials of targeted nanoparticles
(NP) based polymeric systems using other targeting molecules (e.g. antigen, transferrin receptor).9
To meet the demand for expedient and efficient regioselective synthesis of releasable vitamin-conjugates, and considering the advantages of CALB-catalyzed modifications detailed above, this thesis is aimed to develop a modular synthetic approach for the synthesis of a vitamin-polymer conjugate using enzymatic catalysis where possible. See Figure 2.42.
53 2.2.2. Modular Approach for the Synthesis of Polymer Drug Conjugates
The vitamin (FA or Biotin) ligand serves as Module 1, while the active drug is represented as Module 4. The role of the polymer (Module 2) can be summarize in three main parts: (i) to properly position the ligand and drug cargo thereby preserving FR binding potential, (ii) to provide the desired hydrophilicity to the overall construct and
(iii) to prolong plasma circulation half-life. Between the polymer and the drug
(Fluorouracil) modules there is a cleavable linker system (pH-sensitive or disulfide linker), represented as Module 3.
Figure 2.42. Modular approach for the synthesis of Vitamin−polymer conjugates for therapeutic applications.
The presented retro-synthetic analysis is a roadmap for expedient assembly of vitamin−polymer conjugates (Figure 2.42). As indicated by the double lined arrows,
54 strategic disconnection transformed the final construct into two key synthon units: a
Vitamin-polymer linker and the linker−drug derivative.
Additionally in order to build a vitamin−polymer conjugate for diagnostics the
Figure 2.43 shows the strategy. Module 5 represents fluorescein (FITC). As indicated by the retrosynthetic analysis, strategic disconnection transformed the final construct into two key synthon units: a Vitamin and the polymer−FITC derivative.
Figure 2.43. Modular approach for the synthesis of vitamin−polymer conjugates for diagnostics applications.
The same strategy can be used in the case of a difunctional FITC Module that is placed in the middle (Figure 2.44). In this case, 2 targeting units are attached to the conjugate.
55
Figure 2.44. Modular approach for the synthesis of vitamin−polymer conjugates for diagnostics applications.
Biodegradable elastomers have a number of potential applications in the biomedical area, especially in the emerging field of soft-tissue engineering where the mechanical properties of the polymer scaffold should match those of the tissue to be grown.
PIB triblock copolymers with end segments formed from cyclic esters are expected to be novel materials with semicrystalline, biodegradable end blocks.
56 2.3. Synthesis of Polyisobutylene
In this dissertation the synthesis of a poly(isobutylene-b--caprolactone) diblock and poly(-caprolactone-b-isobutylene-b--caprolactone) triblock copolymers was accomplished by the combination of living carbocationic polymerization of isobutylene
(IB) with the ring-opening polymerization (ROP) of -caprolactone (-CL) using CALB as catalyst. The following section will discuss in detail the carbocationic polymerization of PIB and its biomedical applications.
2.3.1. Cationic Polymerization
In the 1980s living cationic polymerization was first reported with vinyl ethers, and then with isobutylene (IB), and styrenic monomers.122,123 The first break- through was made by Higashimura and Sawamoto with living polymerization of alkyl vinyl ethers,124,125 followed by Kennedy and Faust with living polymerization of isobutylene.126,127
Cationic polymerization is known as chain polymerization in which propagating chains, namely active species are positively charged carbenium ions (trivalent
+ carbocations of the type R3C ), or onium ions in vinyl polymerization or ring opening polymerization, respectively. 123
Various types of the initiation processes involving addition of Brønsted acids,
Lewis acids, Lewis acids in conjunction with a proton or carbocation source, photochemical reactions,128 and treatment with high electric field have been developed.
The initiation process is followed by propagation in which a nucleophilic attack of the monomer onto the active growing centers paired with non-nucleophilic counterions
57 occurs. Nucleophilicity of counterion of the cationic growing species is crucial to achieve the living character of the polymerization. Therefore, counterions differ with respect to type of the monomer including vinyl ethers; styrenic monomers; substituted alkenes; and cyclic monomers, and mode of the polymerization (conventional or living cationic polymerization).129
2.3.2. Living Cationic Polymerization of Isobutylene
Isobutylene (IB), being polymerizable only cationically, has been investigated extensively, since PIB is of interest as a commercial material. PIB has unique properties: very low permeability, good thermal and oxidative stability, ozone resistance, chemical resistance, high hysteresis (mechanical dampening), and tack. Low- and medium- molecular-weight PIBs are used as viscosity modifiers, fuel and lubricating oil additives, tack improvers in adhesive formulations, and sealing compounds.122
After the discovery of its living polymerization, initiating systems for IB were vigorously investigated by many groups.122 The first living system gave relatively broad
126,127 ĐM polymers. Later, combinations of BCl3 or TiCl4 with a small amount of strong
Lewis bases [also called as “electron pair donors (ED)”], such as DMSO and N,N- dimethylacetamide (DMA), were found to be effective for the better control of the reaction.130 For example, living polymerization was achieved using dicumyl chloride/BCl3/DMSO, dicumyl alcohol/BCl3/DMSO, or 2-chloro-2,4,4-trimethylpentane
131 (TMP-Cl)/TiCl4/DMA, yielding polymers with narrow ĐM. Using certain EDs various undesirable side reactions, i.e., uncontrolled initiation, chain transfer, irreversible termination, indanyl end-group formation, etc., can be eliminated and well- defined
58 narrow ĐM products can be obtained. Without the ED, only ill-defined reactions occurred with these three initiating systems.
A proton trap also improved the molecular weight distribution of PIBs. With
132 BCl3 or TiCl4, narrowly distributed PIB was obtained in the presence of 2,6-di-tert- butylpyridine (DtBP). DtBP had only interaction with the scavenged protic impurities.
This statement was confirmed by the fact that the polymerization rate was not influenced by the excess of DtBP and the polymerization was of first order with respect to both monomer and the Lewis acid.
2.3.3. End-Functionalized PIBs
End-functionalized polymers are relevant due to their potential applications in many areas, and a variety of end- functionalized polymers have been successfully prepared by use of various living/controlled polymerizations. Efficient methods for the synthesis of PIB carrying functional end groups have long been sought to facilitate the creation of new PIB-based materials. In the living cationic polymerizations reported at this moment,133 there are three major methods for the synthesis of end-functionalized
PIBs: (i) Functionalization by using functional initiators, (ii) End-capping by functional terminators, and (iii) Derivatization of functional groups by post-modification processes of PIBs created using method ii.
2.3.3.1. Initiation from Functional Initiators
Several end-functionalized PIBs have been obtained from functional initiators (A-
D, Figure 2.45). For example, the PIB-methacrylate macromonomer was obtained by
59 living cationic polymerization of IB using 3,3,5-trimethyl-5-chloro- 1-hexyl methacrylate
134 (A). The living cationic polymerization of IB with B/TiCl4 in hexane/CH3Cl at -80 °C gave well- defined PIBs having mono-, di-, and trichlorosilyl head- groups.135 Puskas et al have employed a class of unique epoxide initiators C in the living cationic
136 polymerization of IB using TiCl4 with DtBP. On quenching with methanol, PIB with a primary hydroxy head group and a tert-alkyl chloride end group was obtained.
137 Additionally the polymerization of D/ TiCl4 yielded cylopentene functionalized PIB.
Figure 2.45. Functional initiators for end-functional PIBs.
2.3.3.2. End-Capping by Functional Terminators
Functionalization by end-capping by functional terminators is a convenient alternative to functionalize PIB through direct reaction of quasiliving PIB with a nucleophilic quenching or capping agent. For example, Kennedy et al138 reported the direct end-capping on a PIB living end with allyltrimethylsilane, to give an allyl-capped polymer (Figure 2.46).138 The report also includes the conversion of allyl end- functionalized PIB to the corresponding hydroxyl functional polymer by regioselective hydroboration of the double bond with 9-borabicyclo[3.3.l]nonane followed by oxidation reaction. PIB-Br can be the starting material for subsequent transformation into the primary amine, hydroxy and methacrylate group (Figure 2.47).139
60 Various classes of compounds have demonstrated the effectiveness of this technique (Figure 2.46). They include other olefins such as butadiene,140 hindered nucleophiles,141,142 and activated aromatic compounds such as 2-alkylfurans,143 thiophene,144 N-substituted pyrroles,145 and alkoxy benzenes.146
Figure 2.46. Functionalization of PIB through direct reaction of quasiliving PIB with a nucleophilic quenching or capping agent.
Figure 2.47. Conversion of allyl end-functionalized PIB to the corresponding hydroxyl functional polymer.
61 2.3.3.3. Functionalization by Post-Modification Processes
End-functionalized PIBs can be transformed further by typical organic reactions including nucleophilic substitution, addition, elimination and esterification reactions
(Figure 2.48). Faust et al147 reported the syntheses of end-functional polyisobutylenes
(PIBs) including hydroxy, amino, carboxy, azide, propargyl, methoxy, and thymine end groups using nucleophilic substitution reactions, SN2 reactions on PIB-Allyl-X (X= Cl or
Br).
Figure 2.48. End-functional polyisobutylenes using nucleophilic substitution reactions.
2.3.4. Telechelic HO-PIB-OH
New methodologies to get precise hydroxyl functional polymers are of great interest in terms of their versatility for further chemical modification. It is important to consider the significant reactivity difference between tertiary and primary alcohols for subsequent chain-extension and functionalization reactions.
62 2.3.4.1. Polyfunctional Initiator-Transfer Agents Inifer Technique
The first well-defined PIB diol was obtained by the inifer technique.148 In the inifer method, the polymerization of isobutylene is mediated by an initiating system
149 consisting of a benzylic dichloride (e.g., dicumyl chloride) initiator plus BCl3 or
150 TiCl4 as coinitiator. The dichloride fulfills two functions: it is an initiator and a chain transfer agent, hence the term inifer. Despite their impressive application in the preparation of linear ,-di ( tert-chloro)polyisobutylenes, cumyl initiators often suffer cyloalkylation by the nucleophilic attack of the initiator with the active-end group in isobutylene polymerization. Figure 2.49 shows the mechanism of this reaction in the case
150 of dicumyl ether/TiCl4 system.
Figure 2.49. PIB diol and undesirable reactions obtained by the inifer technique: dicumyl ether/TiCl4 system.
This side reaction hampers the clean synthesis of perfect telechelic polyisobutylenes by the formation of undesirable indanyl and diindane end group.150 To
63 circumvent these difficulties several methods have been developed to prevent the undesirable cycloalkylation but an aliphatic initiator is an alternate choice for an aromatic initiator that I will investigate in this research and it will be discussed.
Due to the importance of getting HO-PIB-OH, several investigations have been conducted in order to reduce the cost of its production. It was discovered that living isobutylene polymerization could be induced with TiCl4, a significantly less expensive
150 coinitiator than BCl3, and that it yields the same tCl-PIB-Clt as the inifer method.
Important further cost-reducing discoveries that drove developments were as follows: (1) one-pot terminal functionalization with the relatively inexpensive allyltrimethylsilane
148 (ATMS) that yielded CH2-CH=CH2 end groups, (2) low-cost synthesis for the preparation of the ‘‘blocked’’ initiator,151 and, importantly, (3) new end-functionalization strategies that eliminated the use of hydroboration oxidation. Storey et al152 described a simple, rapid synthesis of primary halogen, amine, carboxylic acid, and hydroxyl- functional PIBs (mono- and difunctional) via thiol–ene click chemistry. The method produces functional PIBs with 98% conversion within 10 min without difficult purification or reaction conditions.152
2.3.4.2. Bifunctional Aliphatic Initiator System
Since the introduction of the living carbocationic polymerization technique a
126 124 variety of linear telechelic polyisobutylenes PIB carrying one and two CH2C(CH3)2-
Cl end groups have been synthesized and characterized. In earlier publications researchers have described that complexes of BCl3 with certain aliphatic initiators like tert-ethers and tert-alcohols are efficient initiators for the living polymerization of IB.149
64 The clean synthesis of tert-chlorine capped polyisobutylenes Cl-PIB-Cl by living carbocationic polymerization using various initiating systems with BCl3 has been demonstrated.149 In this case aliphatic bifunctional initiators are very convenient because the structure of the specie expected to form from this diol mimicks that of the growing
~PIB~ structure (Figure 2.50). Even at low initiator efficiency the structure of the growing PIB chain is symmetrical.
Figure 2.50. Reaction of tert-alcohol with BCl3 to produce Cl-PIB-Cl.
In the course of this investigation it was of interest to determine whether and under what conditions would the tert-alcohols, tert-chloride and tert-ether aliphatic bifunctional initiator with TiCl4 complex lead to well defined Cl-PIB-Cl. These initiating system were of particular interest because of its relatively low cost, high stability, and advantageous solubility characteristics.
2.3.5. PIB-Based Biomaterials Tested for Biomedical Applications
PIB is known to possess superior biocompatibility, and noninflammatory property as many segmented polymers based on PIB have been studied as potential biomaterials
(Figure 2.51).153,154 PIB has been combined with materials widely used for biomedical
65 applications [polyacrylates and -methacrylates, polysiloxanes, polylactones, polyurethanes, poly(ethylene oxide), and poly(vinyl alcohol) (PVA)]. Some applications that use PIB-based materials are approved by the Food and Drug Administration (FDA).
The following section will highlight some of these applications.
Figure 2.51. PIB-based biomaterials tested for biomedical applications.
2.3.5.1. PIB-Based Thermoplastic Elastomers for Vascular Grafts
Drug eluting stents are known to have polymeric coatings over the stent to release a drug to counteract the effects of in-stent restenosis.155 One of the most relevant coatings is the poly(styrene-block-isobutylene-block-styrene) (SIBS). SIBS is a very soft, transparent biomaterial resembling silicone rubber, with superior mechanical properties.
66 It was U.S. FDA approved the use of these triblocks as drug-eluting coatings of coronary stents, and Boston Scientific Co. started marketing these devices in the United States under the trade name Taxus Express Paclitaxel-Eluting Stents (Figure 2.51).153
2.3.5.2. PIB–Poly(Methyl methacrylates) (PMMA) for Toughening of Bone
Cements
Bone cement is composed mainly by the glassy (brittle) PMMA material. To toughen bone cements, Kennedy et al156 developed a series of new polyisobutylene
(PIB)-toughened poly(methyl methacrylate) (PMMA) networks in which the PIB domains are covalently bonded to a PMMA matrix (Figure 2.51). The synthesis involves free-radical solution copolymerization of methyl methacrylate (MMA) and methacrylate tritelechelic PIB.156 The effect PIB chain length and weight percent (wt %) on the mechanical properties of the novel cements ((PIB)-toughened poly(methyl methacrylate) was investigated. It was found that the bone cements containing 20% PIB (Mn= 18,000 g/mol) showed the greatest toughness enhancement and the highest work of rupture in comparison with commercial bone cements.156
2.3.5.3. PIB-Based Amphiphilic Networks (APNs) for Immunoisolatory
Membranes
Immunoisolatory membranes are used to encapsulate and transplant living tissue from a donor to a host organism (xeno-transplantation). Systematic experimentation showed that amphiphilic membranes containing approximately 50/50 poly(N,N-dimethyl acrylamide)/PIB had semipermeability and diffusion rates suitable for the
67 immunoisolation of pancreatic islets.157 These membranes allowed the countercurrent diffusion of glucose and insulin (Mn=180 and 5,700 g/mol, respectively) but prevented the diffusion of albumin (Mn= 66,000 g/mol), and the diffusion rates (fluxes) of glucose and insulin were deemed appropriate for islet immunoisolation. The device was also implanted subcutaneously in rat.153
2.3.5.4. New Biomaterial as a Promising Alternative to Silicone Breast Implants
Despite the desirable properties of the first generation SIBS. Puskas et al continued investigating structure–property relationships in PIB–PS block copolymers, and developed new carbocationic initiators, and novel PIB-based structures (Figure 2.51).
A new generation of SIBS polymers with a branched (arborescent) structure polyisobutylene (arbIBS) was developed. Puskas et al showed that this new material has a promising potential as a biomaterial alternative to silicone rubber with a pre-clinical research study using a rabbit implantation model.158 The objective was to evaluate in vivo tissue and material interactions of a third generation polymer (arbIBS) compared to commercial SIBS (SIBSTAR 103T) and silicone controls. The data presented show that arbIBS is a very promising biocompatible candidate for breast implants.159
68 3. CHAPTER III
EXPERIMENTAL
3.1. Materials
General solvents: tetrahydrofuran (THF, 99%, bp: 66℃, Fisher Scientific), ethyl
acetate (≥ 99.5%, bp: 77.1℃, Fisher Scientific), hexanes ( ≥ 98.5%, bp: 68℃, Fisher
Scientific), n-pentane (≥ 98%, bp: 36℃, EMD Chemicals), benzene (≥ 99.0%, bp: 80.1℃,
EMD Chemicals), methanol (bp: 65℃, Fisher Scientific), acetone (≥ 99.8%, bp: 56℃,
Sigma-Aldrich), N,N-Dimethylformamide (DMF, anhydrous, 99.8%, Aldrich) and
Ethanol (≥ 98.5%, bp: 78℃, Fisher Scientific) were used as received.
When used in a reaction, tetrahydrofuran (THF, bp: 66℃, Fisher Scientific), and
hexane (Hexane, bp: 68℃, Fisher Scientific) were purified with the MBraun, MB-SPS
purification system. Diethyl ether ethylether (Et2O, ≥ 99.0%, bp: 34.6℃, Aldrich) was
first distilled over sodium (≥ 99.9%, Aldrich) and benzophenone (99%, Sigma-Aldrich)
prior to use. Toluene (99%, EMD Chemicals) and methylene chloride (CH2Cl2, ≥ 99.8%,
bp: 39.6℃, EMD Chemicals) were distilled from CaH2 (95%, Aldrich). Dimethyl
sulfoxide (DMSO, anhydrous, ≥99.9%, Aldrich), deuterated chloroform (CDCl3, 99.8%,
bp: 60.9℃, Chemical Isotope Laboratories Inc.) dimethyl sulfoxide – D6 (DMSO-D6,
99.9%, bp: 189℃, Cambridge Isotope Laboratories Inc.).
69 Isobutylene (IB, 99 %, ExxonMobil) and methyl chloride (CH3Cl, 99.9 %,
ExxonMobil) were dried by passing through a column containing BaO and CaCl2 before condensing them from the gas phase inside the dry box at the polymerization temperature.
Candida antarctica lipase B (CALB, ≥ 10,000 U/g, 33273 Da, 20 wt% immobilized on macroporous acrylic resin, Novozyme® 435, Sigma-Aldrich), vinyl methacrylate (VMA, 98%, bp: 112 ºC, TCI America), vinyl acrylate (VA, >90%, bp: 92
ºC, Monomer-Polymer& Dajac Labs), divinyl adipate (DVA, >98%, TCI America),
2,4,4-trimethyl-1-pentene (TMP-1, 99%, Aldrich), phosphomolybdic acid in 30% ethanol
(Aldrich), sodium chloride (NaCl, Fisher Scientific), sulfuric acid (EMD Chemicals), potassium hydroxide (Fisher Scientific), sodium hydroxide (NaOH, ≥ 98.0%, J.T. Baker), sodium bicarbonate (NaHCO3, Fisher Scientific), magnesium sulfate (MgSO4, EMD
Chemicals), ammonium chloride (NH4Cl, 99%, Aldrich) titanium tetrachloride (TiCl4,
99.9%, Aldrich), 2,6-di-tert-butyl pyridine (DtBP, >97 %, TCI America), allyltrimethylsilane (ATMS, 97.7 %, Gelest Inc.), methylallyltrimethylsilane (MTMS,
97.7 %, Gelest Inc.), 9-Borabicyclo[3.3.1]nonane (9-BBN, 0.5 M in THF, Aldrich), methanol (Fisher Scientific) and hydrogen peroxide (30% w/w in H2O, Aldrich) were used as received. Celite® 545 (Aldrich), 3,3-dimethyl glutaric acid (>97%, Aldrich), methyl magnesium bromide (>97%, solution in ethyl ether 3M, Aldrich), N,N-dimethyl acetamide (DMA, 99.8%, Aldrich) were used as received. TMHDiOH was synthesized using the method described by Kennedy et al.18 Tetraethylene glycol (TEG, 99 %,
Aldrich), tetraethylene glycol monobenzyl ether (BzTEG, ≥ 95%, TCI America),
70 poly(ethylene glycol) monomethyl ether (MeO-PEG-OH1100, Mn=1,100 g/mol, ĐM =1.09,
Polymer Source, Inc. MeO-PEG-OH2000, Mn=2,000 g/mol, ĐM =1.2, Aldrich), poly(ethylene glycol)s (HO-PEG-OH1000, Mn=1,000 g/mol, ĐM =1.08, Polymer Source,
HO-PEG-OH2000, Mn=2,000 g/mol, ĐM =1.20, Alfa Aesar, HO-PEG-OH3400, Mn=3,400 g/mol, Polymer Source, HO-PEG-OH8000, Mn=8,000 g/mol, ĐM =1.2, Aldrich) and heterofunctional poly(ethylene glycol)s (NH2-PEG-OH1000, Mn=1,000 g/mol; ĐM =1.04,
Laysan BioInc, NH2-PEG-OH3400, Mn=3,400 g/mol; ĐM =1.04, Laysan BioInc), poly(ethylene glycol) diamine (NH2-PEG-NH2-2000, Mn=2,000 g/mol, ĐM =1.08,
Scientific Polymer Products, Inc., 95 % content amine). Ethyl bromo acetate (EBrA,
95%, Aldrich), ethyl iodoacetate (EIA, 95%, Aldrich), ethyl 5-bromovalerate (EBrV,
98%, Aldrich), vinyl chloroacetate (VClA, 98%, Monomer Polymer & Dajac Labs). - caprolactone (-CL, 97%, Aldrich) was dried over CaH2 and distilled under vacuum, propargyl alcohol (99%, Aldrich) was distilled under vacuum. Hydroxy-derivatized 4- dibenzocyclooctynol (DIBO) was synthesized by Jukuan Zheng.160 2-hydroxyethyl disulfide ( 2HEDS, 85%, Aldrich). Dithiodiethanol (DTT, Sigma-Aldrich ~90%). α-
(allyl) ω-(hydroxy)- polyisobutylene ( OH-PIB-allyl, Mn=4,306 g/mol, ĐM =1.21) was prepared by Alejandra Alvarez via the propylene oxide (PPEx)/ TiCl4 initiated carbocationic polymerization of isobutylene.161 2,2'-(ethane-1,2- diylbis(oxy))diethanamine (≥96%, Aldrich), 3-(acryloyloxy)-2-hydroxypropyl methacrylate (≥96%, Aldrich). Allyl bromide (≥ 99%, Aldrich), biotin N- hydroxysuccinimide ester (NHS-Biotin, ≥ 98%, AK Scientific Inc), biotin (≥ 99%, AK
Scientific Inc), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC,
≥98%, Aldrich), 4-(Dimethylamino)pyridine (DMAP, ≥98.0%, Aldrich), 2-
71 mercaptoethanol (≥ 99.0%, Aldrich), 5-Fluorouracil (99.0%, Aldrich), n-butyllithium (n-
BuLi, 1 M in cyclohexane, Aldrich), sodium hydride (NaH, 95%, Aldrich), methyl iodide
(MeI, ≥99.5%, Aldrich), sodium azide (NaN3, ≥99.5%, Aldrich), 2,2-dimethoxy-2- phenylacetophenone (Irgacure® 651, 99%, Sigma-Aldrich), fluorescein o-acrylate (FITC-
VA, 95%, Aldrich), fluorescein O,O′-diacrylate (VA-FITC-VA, 98%, Aldrich), diethanolamine (DEA , ≥98.0%, Aldrich), folic acid (FA, ≥97%, Aldrich). Triethylamine
(Et3N, ≥99%, Aldrich), N,N,N’,N’-tetramethylethylenediamine (TMEDA, ≥99.5%,
Aldrich), 12-4 Crown ether (98%, Alfa Aesar).
3.2. Procedures
In this research, precise functionalization of polymers was achieved using two methods: enzyme-catalyzed functionalization of polymers by chain-end and enzyme- catalyzed functionalization via polymerization.
3.2.1. Enzyme-Catalyzed Functionalization of Polymer by Chain-End
3.2.1.1. Candida antarctica lipase B-Catalyzed Halo-Ester Functionalization of
PEGs
3.2.1.1.1. Model Reactions with BzTEG
Transesterification of EBrA with BzTEG. BzTEG (0.50 g, 1.76 mmol, 1.0 eq.) was reacted with EBrA (0.97 mL, 8.79 mmol, 5.0 eq.) in the presence of CALB (0.029 g resin @ 20 wt% enzyme, 1.76 × 10-4 mmol, 0.00010 eq.) in bulk at 65 °C under vacuum
(70 millitorr). The solid CALB was removed by a 0.45 µm PTFE filter and the excess
EBrA was removed by vacuum distillation.
72 Transesterification of EBrV with BzTEG. BzTEG (0.50 g, 1.76 mmol, 1.0 eq.) was reacted with EBrV (1.39 mL, 8.79 mmol, 5.0 eq.) in the presence of CALB (0.029 g resin @ 20 wt% enzyme, 1.76 × 10-4 mmol, 0.00010 eq.) in bulk at 65 °C under vacuum
(70 millitorr). The solid CALB was removed by a 0.45 µm PTFE filter and the excess
EBrV was removed by vacuum distillation.
The progress of both reactions was monitored with Thin Layer Chromatography
(TLC) using alumina plates with hexane/THF (2/1; vol/vol) as an eluent and phosphomolibdic acid as a staining agent.
3.2.1.1.2. Transesterification of EBrV with MeO-PEG-OH2000
MeO-PEG-OH2000 (0.50 g, 0.25 mmol) was reacted with EBrV (0.20 mL, 1.25 mmol, 5.0 eq. per OH in PEG) in the presence of CALB (0.0083 g resin @ 20 wt% enzyme, 5.0×10-5 mmol, 0.00020 eq.) in bulk at 65 °C for 4 h under vacuum
(70 millitorr). After the reaction, 5 mL of dried THF was added to the mixture and the solid CALB was removed by a 0.45 µm PTFE filter. The polymer was precipitated twice into hexane to remove the excess of EBrV and dried in a vacuum oven at room temperature.
73 3.2.1.2. Candida antarctica lipase B-Catalysis of Multifunctional PEGs
3.2.1.2.1. Chemoselectivity in Enzyme-Catalyzed Michael Addition:
Michael Addition of NH2-PEG1000-NH2 to 3-(acryloyloxy)-2-
hydroxypropyl methacrylate
NH2-PEG1000-NH2 (0.20 g, 0.20 mmol, 1.0 eq.) in 0.7 mL of anhydrous DMSO was reacted with 3-(acryloyloxy)-2-hydroxypropyl methacrylate (0.028 g, 0.44 mmol, 2.2 eq.) in the presence of CALB (3.3 mg resin @ 20 wt% enzyme, 2.0×10-5 mol, 0.00010 eq.) at 50 °C. After 24h of reaction the solid CALB was removed by a 0.45 µm PTFE filter. The product was precipitated and washed in 200 mL of diethyl ether.
3.2.2. Enzyme-Catalyzed Functionalization Via Polymerization
Well-defined poly(caprolactone)s were generated using alkyne-based initiating systems catalyzed by CALB. Propargyl alcohol and 4-dibenzocyclooctynol (DIBO) were shown to efficiently initiate the ring opening polymerization of -caprolactone under metal free conditions. A model procedure for CALB-catalyzed functionalization via polymerization is included. Specific concentrations are given in the Chapter V.
3.2.2.1. Propargyl Alcohol Initiator for ROP of -CL
Dry toluene (2.0 mL) and propargyl alcohol (0.078 mL) were transferred via syringe under dry N2(g) into a flask containing CALB (100 mg). This suspension, as well as a separate flask containing ε-CL, was equilibrated for 15 min to the reaction temperature (70 °C). Thereafter, ε-CL (3.0 mL) was transferred to the reaction flask via syringe under dry N2 (g) to start the polymerization. Aliquots were taken at intervals of 4
74 h, 8 h, and 12 h and precipitated in methanol, dried under vacuum and analyzed by size exclusion chromatography (SEC). The polymerization continued until being stopped at
24 h. The polymer was precipitated in methanol, and washed 3 times with methanol, filtered and dried under vacuum for 24 h at room temperature. The ratio of initiator/monomer was varied from 20:1 to 500:1. Each reaction condition was repeated 3 times.
In the case of bulk reaction the same amounts were taken but in this case not toluene was added.
3.2.2.2. Vinyl-PEG-Vinyl2000 (V-PEG-V2000)
HO-PEG-OH2000 (2.0 g, 1.00 mmol) was reacted with DVA (3.96 g, 20.00 mmol,
20.0 eq.) in the presence of CALB (100 mg, 6.0 × 10-4 mmol) for 4 hours at 50 °C. A sample was taken for MALDI-ToF analysis at 2 hours reaction time. After 4 hours, 5 mL of dried THF was added to the mixture and the solid CALB was removed by a syringe fitted with a 0.45 µm PTFE filter. The mixture was cooled to room temperature, yielding a solid polymer with excess liquid DVA. The excess DVA was removed by washing with hexane twice and recovered by distilling off the hexane using a rotary evaporator. The polymer was dried in a vacuum oven at room temperature.
Additionally the synthesis of a poly(isobutylene-b--caprolactone) block copolymer and poly(-caprolactone-b-isobutylene-b--caprolactone) triblock copolymers
75 was accomplished by the combination of living carbocationic polymerization of isobutylene (IB) with the ring-opening polymerization (ROP) of -caprolactone (-CL).
3.2.2.3. Synthesis of Poly(isobutylene-b--caprolactone)
A solution of HO-PIB-allyl (0.18 g, 9.0x10-3 mol/L) and dry toluene (5.0 mL) were transferred via syringe under dry N2(g) into a flask containing CALB (75 mg,
9.0×10-5 mol/L). This suspension, as well as a separate flask containing ε-CL, was equilibrated for 15 min to the reaction temperature (70 °C). Thereafter, ε-CL (0.2 mL,
0.36 mol/L) was transferred to the reaction flask via syringe under dry N2 (g) to start the polymerization. The polymerization continued until being stopped for 24 h. The polymer was precipitated in methanol, filtered and dried under vacuum for 24 h at room temperature. (yield 0.235 g, ε-CL conversion 28 %).
3.2.3. Chemo-Enzymatic Functionalization of Biomolecules
3.2.3.1. Synthesis of the Biotin-PEG arm
In a 10mL round bottom flask, 0.1g of NH2-PEG-OH3400 (Mn=3,400 g/mol, ĐM
=1.01, 0.10 g, 2.94*10-5 mol, 1.0 eq) was dissolved in 0.2 mL of anhydrous acetonitrile at room temperature. Triethylamine (0.0088 mL, 2.65*10-5 mol, 0.9 eq.) and 0.1 mL of anhydrous CH2Cl2 was added. The flask was covered with aluminum foil. After 5 min of stirring, 0.026 g of NHS-Biotin (1.03*10-5 mol, 3.5 eq) was added and stirring was continued overnight under nitrogen (the mixture is not completely soluble).
The mixture was first filtered; the supernatant was precipitated in 120mL of Et2O with 3mL THF to rinse the flask. The precipitate was then washed with 100 mL Et2O,
76 and then twice more with 1:1 ratio of 100mL Et2O: hexane solution. After that the solid was re-dissolve in 10 mL of isopropanol, heated at 70 ºC and then cool it down until precipitation. The product was filtered and dry under vacuum at room temperature.
3.2.3.2. Synthesis of the Fluorouracil-TEG-OH arms
Vinyl ester of fluorouracil was prepared by Michael addition of fluorouracil to vinyl acrylate. Specifically, vinyl acrylate (0.75 mL, 7.6 mmol, 2.0 eq.) was added to a flask containing fluorouracil (0.50 g, 3.8 mmol, 1.0 eq.) in 4.8 mL of anhydrous DMSO and CALB (0.064 g, 3.84x10-4 mol) under an inert atmosphere. The mixture was stirred at 300 rpm for 24 hours at 50 °C. After the reaction, the solid CALB was removed by a
0.45 µm PTFE filter and the solvent was removed by vacuum distillation.
3.2.3.3. Synthesis of the Folic Acid linkers
3.2.3.3.1. FA-γ-valerate
FA (0.46 g, 1.05 mmol, 0.049 mol/L) was dissolved in 21 mL of anhydrous
DMSO overnight under N2 gas. n-BuLi (0.53 mL, 1.1 mmol, 1.0 eq.) was added dropwise into the FA solution. The progression of the reaction was indicated by the evolution of butane which bubbled out of the solution. The solution was stirred for 20 min at 20°C.
Subsequently 5-bromo-valerate (0.20 g, 0.90 mmol, 1.0 eq) was added into the reaction mixture. The solution was stirred for 24 h at room temperature. The dark yellow product was precipitated in 400 mL diethyl ether and washed with hexane and THF. The solid product was dried in a vacuum oven for further analysis.
77 3.2.3.3.2. MeO-PEG2000 γ-FA
FA (0.069g, 0.15 mmol, 0.049 mol/L) was dissolved in 3.1 mL of anhydrous
DMSO overnight under N2 gas. 0.078 mL of n-BuLi (0.15 mmol, 1 eq) was added dropwise into the folate solution. The progression of the reaction was indicated by the evolution of butane which bubbled out of the solution. The solution was stirred for 20 min at 20°C. Subsequently 0.3 g of MeO-PEG-Br2000 (0.142 mmol, 1 eq) was added into the reaction mixture. The solution was stirred for 120 h at room temperature. Aliquots were taken at 24 h, 48 h, 72 h and 120 h and analyzed by NMR. The dark yellow product was precipitated in 400 mL diethyl ether and washed with hexane and THF. The solid product was dried in a vacuum oven for further analysis.
Note: When using modifier (THF or TMEDA) it was added before the addition of
MeO-PEG-Br2000 and let it to homogenize for 5 min.
3.2.3.4. Synthesis of the FITC Linkers
3.2.3.4.1. Michael Addition of NH2-PEG-OH1000 to FITC-VA
NH2-PEG-OH1000 (0.15 g, 0.15 mmol, 1.0 eq per acrylate group of FITC-VA, 0.3 mol/L, Mn=1,000 g/mol; ĐM =1.04) in 0.5 mL of anhydrous DMSO was reacted with
FITC acrylate (0.058 g, 0.15 mmol, 0.3 mol/L) in the presence of CALB (120 mg resin @
20wt% enzyme, 7.4 × 10-4 mmol, 3.6×10-4 mol/L) under nitrogen. The reaction was stirred at 300 rpm for 5 hours at 50 oC. The CALB was removed using a 0.45 μm syringe filter. The product was precipitated in 200 mL of diethyl ether, and then washed with 100 mL of hexane. The yellow solid product was dried in a vacuum oven at room temperature
78 (0.1051 g). Aliquots were taken at 1 h, 1.5 h and 5 h. The reaction is complete at 5 h. The product was purified by precipitation.
3.2.4. Preparation of Low Molecular Mass Telechelic Functionalized
Polyisobutylenes
3.2.4.1. Initiators
Three different initiators were employed for the synthesis of telechelic PIBs,
2,4,4,6-tetramethylheptane-2,6-diol (TMHDiOH), 2,6-dimethoxy-2,4,4,6- tetramethylheptane (TMHDiOMe) and 2,6-dichloro-2,4,4,6-tetramethylheptane
(TMHDiCl), in the presence of TiCl4.
3.2.4.1.1. Dimethyl 3,3-dimethylpentanedioate (DiMDiPD)
DiMDiPD was prepared by refluxing a mixture of 3,3-dimethyl glutaric acid (100 g, 0.62 mole) with anhydrous methanol (2.5 L, 78 mol) and 125 ml concentrated sulfuric acid under reflux for 48 h. The mixture was cooled to room temperature and the methanol was removed by rotovaporation. Re-dissolved with 500 mL of hexane and washed with
NaHCO3 solution (100mL * 5) and then with water several times until the waste water was acid free. The solution was dried with anhydrous magnesium sulfate, filtered, and the solvent was removed. After that slightly yellow oil was recovered (yield 89g, 76% conversion). The product was passed through an alumina column (30g), clear oil was obtained. The ester product was dried using 3 freeze pump-thaw cycles.
79 3.2.4.1.2. 2,4,4,6-tetramethylheptane-2,6-diol (TMHDiOH)
To a solution of DiMDiPD (60 g, 0.24 mol) of in 200 ml ethyl ether under a blanket of N2 were added dropwise methylmagnesium bromide in ethyl ether (237 mL,
0.71 mol, 3M) and stirred for 15 h at < 5 ºC. Then the charge was slowly added to a 35 g
NH4Cl-400 g ice mixture under stirring and leave to react for 5h. The system was extracted with ethyl ether while the pH of the aqueous phase is around 7. The extract was washed with water, dried over anhydrous magnesium sulfate overnight. The product was passed through a column packed with carbon black (40g). After that the ether is removed by roto-evaporation and crystallization with ethyl acetate help to recover the product
(yield 33 g, 53 % conversion).
3.2.4.1.3. 2,6-dichloro-2,4,4,6-tetramethylheptane (TMHDiCl)
NaCl (13 g, 0.44 mol) was placed into a 100 mL, three-necked, round-bottom flask. Concentrated sulfuric acid (12 mL, 0.44 mol) was added to the NaCl very slowly from a dropping funnel to form HCl gas. The HCl gas was bubbled into a solution of
TMHDiOH (2.0 g, 0.011 mol, 0.7 M) with 15mL of CH2Cl2 in a 50 mL tube which was kept in an ice-water bath. The reaction flask was connected to a trap and another flask containing sodium hydroxide solution to neutralize the unreacted acid. The reaction was conducted at 0ºC for 5 hours. Thin Layer chromatography was checked to follow the reaction progress, in a system of hexane: ethyl acetate (5:1 v/v). After completion of the reaction, TMHDiCl was neutralized by the slow addition of sodium bicarbonate. The solution was filtered and the solvent was removed under vacuum (not heating). The
80 product was purified using a column packed on alumina and a system of hexane: ethyl acetate (5:1). (yield 2.267 g, 100% conversion).
3.2.4.2. Synthesis of Telechelic Oligomers of Isobutylene
3.2.4.2.1. Hydrochlorination of the Oligoisobutylenes General
Procedure.
In a two-necked flask, a solution of the oligoisobutylene in methylene chloride was cooled in an ice bath. After passing gaseous HCl through the solution for 4 h, the organic layer was neutralized with NaHCO3. The organic fraction was dried over MgSO4, the solvent was evaporated in vacuum, and the residue was distilled. Specific concentrations are given in the Chapter VII.
3.2.4.2.2. Reaction of the Alkyl Chlorides with the
Allylsilanes/methylallylsilane General Procedure.
The reaction was carried out under a dry nitrogen atmosphere (H2O < 1 ppm and
O2 < 3ppm) in a MBraun LabMaster 130 glove box equipped with a bath filled with hexane cooled by an FTS Flexi Cool Immersion Cooler and liquid nitrogen. The temperature was kept constant during the reaction at -80 °C. The chloro compound and the allylsilane/methylallylsilane were dissolved in dry methylene chloride. After cooling in a hexane bath at -80 ºC, the TiCl4 stock solution was added slowly and the reaction mixture was stirred for 4-5 h. Aqueous ammonia (30 mL) was added and the mixture was filtered over Celite. The aqueous layer was extracted with CH2Cl2, the combined organic
81 fractions were dried over MgSO4, after filtration the solvent was removed, and the reaction products were purified. Specific concentrations are given in the Chapter VII.
3.2.4.3. Preparation of Telechelic Functionalized Polyisobutylenes
Polyisobutylenes (PIB)s were synthesized by carbocationic polymerization of isobutylene. The polymerizations were carried out under a dry nitrogen atmosphere (H2O
< 1 ppm and O2 <3ppm) in a MBraun LabMaster 130 glovebox at The University of
Akron. The drybox was equipped with a bath filled with hexane cooled by a coil cooled with liquid nitrogen. The temperature was controlled by regulating the liquid N2 flow with a control valve regulated with a PID controller (Omron E5AK).
3.2.4.3.1. IB Polymerization
One general description is given below. Specific conditions are listed in the
Chapter VII.
In a typical reaction, a 500 mL, 3-necked, round-bottom flask equipped with an overhead stirrer was charged with the co-solvents (CH3Cl, CH2Cl2, or CH3Cl/ Hexane,
40/60, v/v) and the other chemicals (initiator, DtBP, DMA), the flask was allowed to stir for 15 min. IB was added and the polymerization started with the rapid introduction of a prechilled stock solution of TiCl4 in CH2Cl2 or Hexane into the reactor. The solution color became faint yellow. Samples were taken at specified times. The mixture was allowed to react for a specific time, and then terminated by the addition of a prechilled solution of 10% NaOH in methanol. The reaction flask was then removed from the glove box, and MeCl and IB were allowed to evaporate. After filtration, the polymer was
82 precipitated from the organic phase into cold methanol (5 °C). The precipitated polymer was dried under vacuum and further purified by freeze drying and vacuum heating to remove traces of solvents. Polymer yield was determined by gravimetry.
Chlorine exchange experiments with TMHDiOH and TMHDiOMe were carried out in CH3Cl or CH2Cl2 in the absence of IB. Aliquots were taken for NMR to determine the reaction time. After the determination of the reaction time necessary for quantitative chlorine exchange, the polymerization was carried out in one pot. In these experiments the addition sequences of the ingredients were: CH3Cl or CH2Cl2, initiator and TiCl4.
After chlorine exchange, hexane was added to keep the system in solution [CH3Cl or
CH2Cl2/ Hexane (40:60 v/v)] and lastly the IB was added. After 40 min of reaction,
ATMS was added, a color change from yellow to red was observed. The reaction mixture was neutralized after 30 min with a saturated solution of NaHCO3 at -5°C. After filtration, the polymer was precipitated from the organic phase in cold methanol (5°C) and dried under vacuum. The polymer was further purified by freeze drying and vacuum heating to remove traces of solvents. Conversions and Ieff = Mntheo/Mnexp are reported for each case.
3.2.4.3.2. Conversion of allyl-PIB-allyl to HO-PIB-OH
Allyl-PIB-allyl (0.17 g, 0.025 mol/L) was dissolved in 1.5 mL CHCl3, then mercaptoethanol (0.071 mL, 0.61 mol/L) and Irgacure (0.094 g, 0.24 mol/L) were added to the solution. The contents were shaken in darkness for 20 min. The sample was then irradiated using a UV lamp (354 nm) and stirred for 10 min at 0 ºC. The product was
83 precipitated in cold methanol (-10 °C) and freeze-dried under vacuum. (yield 0.16 g, conversion 100%).
3.3. Characterization of Products
Nuclear magnetic resonance (NMR) spectroscopy, size exclusion chromatography
(SEC), matrix assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-ToF MS), thin layer chromatography (TLC), column chromatography, differential scanning calorimetry (DSC), AFM, and transmission electron microscopy
(TEM) were utilized in this research.
3.3.1. Nuclear Magnetic Resonance (NMR) Spectroscopy
1H and 13C NMR spectra were recorded on Varian Mercury 300 or Varian NMRS
500 spectrometer using deuterated chloroform (Chemical Isotope Laboratories, 99.8%
CDCl3) or deuterated dimethylsulfoxide (Chemical Isotope Laboratories, 99.9% DMSO-
D6) as solvent. The resonances of non-deuterated chloroform at δ=7.27 ppm and δ=77.23 ppm, and that of non-deuterated dimethylsulfoxide at δ=2.5 ppm and δ=39.51 ppm were used as internal references for the 1H and 13C NMR spectra, respectively. 1H NMR samples were prepared in 5 mm NMR tubes with 25-50 mg of polymer dissolved in 0.7 mL of NMR solvent and the spectra were acquired after 64-128 transients with a relaxation time of 1-5 sec. 13C NMR samples were prepared in 5 mm NMR tubes with
100-150 mg of polymer dissolved in 0.7 mL of NMR solvent and the spectra were acquired after 5000-20000 transients with a relaxation time of 1-5 sec.
84 3.3.2. Size Exclusion Chromatography (SEC)
Size exclusion chromatography (SEC) measurements were conducted using a
HPLC pump (Waters 515 HPLC Pump), a Waters 2487 Dual Absorbance UV Detector
(UV), a Wyatt OPTILAB DSP Interferometric Refractometer (RI), a Wyatt DAWN EOS multi-angle light scattering detector (LS), a Wyatt ViscoStar viscometer (VIS), a Wyatt
QELS quasi-elastic light scattering instrument (QELS), a Waters 717 plus autosampler and 6 Styragel® columns (HR6, HR5, HR4, HR3, HR1 and H0.5). The columns were thermostated at 35 °C and THF, freshly distilled from CaH2, was used as the mobile phase at a flow rate of 1 mL/min. The results were analyzed by using the ASTRA software (Wyatt Technology). The molecular mass calculations were carried out using a refractive index increment (dn/dc) value of 0.108 for polyisobutylenes; the results agreed with data obtained assuming 100% mass recovery. Copolymer dn/dc was calculated based on the weight fraction and dn/dc of the individual components; PCL = 0.053 and
PIB = 0.108. Copolymer dn/dc was calculated based on the weight fraction and dn/dc of the individual components (PCL and PIB).
3.3.3. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass
Spectrometry (MALDI-ToF MS)
MALDI-ToF mass spectra were acquired with a Bruker UltraFlex-III time-of- flight (ToF) mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with a
Nd:YAG laser (355 nm), a two-stage gridless reflector, and a single stage pulsed ion extraction source. Separate THF (anhydrous, 99.9%, Aldrich) solutions of polymer (10 mg/mL), 1,8,9-anthracenetriol (dithranol, 20 mg/mL, >97%; Alfa Aesar), sodium
85 trifluoroacetate (10 mg/mL, >98%; Aldrich) or silver trifluoroacetate (10 mg/mL, 98%,
Aldrich) were mixed in a ratio of 10:1:2 or 14:1:4 (matrix:cationizing salt:polymer), and
0.5 μL of the resulting mixture was introduced on to the MALDI target plate and allowed to dry. The spectra were obtained in the reflectron mode. The attenuation of the nitrogen laser was adjusted to minimize unwanted polymer fragmentation and to maximize the sensitivity. The calibration of mass scale was carried out externally using poly(methyl methacrylate) or polystyrene standard having similar molecular weight as the sample.
3.3.4. Electrospray Ionization Mass Spectrometry (ESI-MS)
ESI mass spectra were acquired with a Bruker Daltonics HCTultra ETD II ion trap mass spectrometer for the identification of mass. The sample was dissolved in anhydrous THF (99.5%, Aldrich) at 1μg/μl and mixed with a 1μg/μl solution of sodium trifluoroacetate cationizing agent (98%, Aldrich) in MeOH in the ratio 100:1(sample:salt)
(v/v). Experimental conditions: positive mode; drying gas, nitrogen (8 L/min); drying temperature, 300 °C; nebulizer gas, nitrogen (10 psi). The sodiated solutions were introduced into the ESI source by direct infusion using a syringe pump at a flow rate of
250 μl/h.
3.3.5. Thin Layer Chromatography (TLC)
Thin layer chromatography analyses were carried out by spotting and developing the samples on aluminum-backed silica gel/ or alumina plates with fluorescent indicator
(Dynamic Adsorbents Inc.) using various eluents. The plates were then analyzed using either phosphomolybdic acid staining reagent or UV light (λ=254 nm).
86 3.3.6. Column Chromatography
Silica gel column chromatography was employed for the purification of some of the products. The silica gel (Dynamic Adsorbents Inc., Silica Gel Classic Column 60A) was added to a beaker containing the appropriate eluent; and the resulting slurry was poured to a column which was half filled with the eluent. After all of the silica gel was added, the solvent was allowed to drain through the column until the solvent level was just above the surface of silica gel. The crude product which was dissolved in the minimum amount of solvent was loaded onto the column and the solution was allowed to drain until it was again just above the level of silica gel. The eluent solvent was then run through the column and fractions were analyzed by TLC. The fractions having the pure product were then combined and the solvent was removed under reduced pressure.
3.3.7. Differential Scanning Calorimetry (DSC)
DSC was carried out on a TA Q2000 DSC using a heat-cool-heat thermal cycle.
The samples were collected in a DSC pan with a mass of typically 5 mg. The samples were then subjected to heating/cooling cycles of 10C/min in the temperature range from
-100 C to 200 C. Transition temperatures Tg and Tm were calculated as the mean value between the onset and end point temperatures. A nitrogen atmosphere was used to minimize thermal degradation of the polymers.
3.3.8. AFM
The polymer samples were provided as films deposited on silicon wafers for AFM analysis. The samples were analyzed using a Veeco Instruments Multimode AFM with a
87 Nanoscope IV controller, operated in tapping-mode with height and phase images collected simultaneously. Silicon cantilevers with a nominal resonance frequency of 170 kHz (Aspire CT170R) were used, with medium-light tapping forces as characterized by a
2.0 V free amplitude and a 1.6 V setpoint amplitude. Images were processed using flattening and plane fitting routines in the Nanoscope (v. 5.30) software. Optical images were collected with an Olympus BX51 optical microscope using reflected light.
3.3.9. Transmission Electron Microscopy (TEM)
Transmission electron microscopy (TEM, Philips Tecnai 12 at an accelerating voltage of 120 kV) experiments, the films were prepared on a carbon coated glass surface by spin coating. One drop of a solution 1% of poly(isobutylene-b--caprolactone) block copolymer or poly(-caprolactone-b-isobutylene-b--caprolactone) triblock copolymers solution in THF was used in the spin coating procedure. By inserting the specimen into a distilled water bath, the polymeric film along with the beneath carbon layer was floated on the water surface, and then picked up by a clean TEM copper grids (400 mesh,
SPI). Before TEM observation, samples on the grids were annealed for 15 h at 40 ºC.
.
88 4. CHAPTER IV
ENZYME-CATALYZED FUNCTIONALIZATION OF POLYMERS BY CHAIN-END
Parts of this chapter have been published:
M Castano, KS Seo, EH Kim, ML Becker, JE Puskas* “Synthesis of Halo-ester
Functionalized Poly(ethylene glycol)s via Enzymatic Catalysis” Macromolecular Rapid
Communications, 2013, 34, 1375-1380. Reproduced with permission of © 2013 WILEY-
VCH Verlag GmbH & Co. KGaA, Weinheim
In this research, precise functionalization of polymers was achieved using two
methods: enzyme-catalyzed functionalization of polymers by chain-end and enzyme-
catalyzed functionalization via polymerization.
This chapter will discuss the first method. Previously, Dr. Sen from Professor
Puskas’ research group investigated the functionalization of PEGs with vinyl esters using
CALB-catalyzed reactions in organic solvents.17 The methacrylation and acrylation of
PEGs with various molecular weights and molecular weight distributions in THF were
quantitative in 24 hours. After that, Dr Kwang Su Seo prepared a tetra-hydroxyl
functionalized PEG by CALB-catalyzed reactions using transesterification and Michael
addition reaction in the presence of CALB to create multifunctional chain ends that could
be used as the building blocks for the targeted nanodevices and other biomedical
89 applications.162 This chapter will discuss the functionalization of PEGs with other functionalities, which include halo-esters, azide, mono and difunctionalization with acrylate. All reactions were run under bulk conditions.
4.1. Introduction
Poly(ethylene glycol) (PEG) is a non-toxic, hydrophilic polymer that is used widely for biomedical applications .163,164 One application is to enhance the circulation time and blood half-life for cell imaging, drug delivery, and antibody-based therapy.165–
167 However, the HO- end groups that are available for chemical derivatization are only a small fraction of the molecular mass of the polymer, and chemistries utilized for end- group modification must be high fidelity in nature and leave few or ideally no residuals.168–170 Because halogens, especially bromine make a great leaving group, PEG-
Br is often used as an intermediate for further functionalization. PEG is usually brominated using thionyl bromide or phosphorous tribromide in toluene.171,172 An attractive alternative strategy is the use of enzymatic reactions. This “green” polymer chemistry approach offers many advantages, such as high efficiency, recyclability, and the ability to react under mild and solvent-free conditions.21 Before our group had started working on the end-functionalization of preformed synthetic telechelic polymers, enzymatic catalysis has not extensively been utilized in this area despite the advantages enzymes offer. Recently our group have shown that Candida antarctica lipase B (CALB) catalyzed quantitative methacrylation of poly(ethylene glycol)s (PEG)s, polyisobutylenes and poly(dimethyl siloxane)s without the use of solvents. 17,173 Since CALB is immobilized on a resin, it can conveniently be separated from the product, yielding very
90 pure compounds for potential pre-clinical and clinical applications. 174–176 In this work we investigated the enzyme-catalyzed halogen functionalization of PEGs under mild conditions.
4.2. Results and Discussion
Figure 4.1 Enzymatic transesterification of halo-esters with PEGs in the presence of CALB.
4.2.1. Model Reactions: CALB-Catalyzed Halo-Ester Functionalization with
BzTEG
4.2.1.1. Transesterification of EBrA with BzTEG
TLC showed that the transesterification of EBrA with BzTEG (Figure 4.1) was complete after 2h under solventless conditions. The 1H NMR spectrum of the product is shown in Figure 4.2. The resonance at δ=4.53 ppm, corresponding to the -OH proton of
BzTEG disappeared and the peak of the methylene protons adjacent to hydroxyl group shifted downfield to δ=4.20 ppm (a’) after the reaction. The new peak corresponding to
91 the methylene protons adjacent to the bromine was observed at δ = 3.76 ppm (f). The relative integrals of the methylene protons of the ester group (a’) and the methylene protons next to the benzyl group in the TEG moiety at δ = 4.48 ppm (d) demonstrate successful functionalization with the integration ration of 2:2.05. The 13C NMR spectrum
(Figure 4.2) showed that the carbons connected to the hydroxyl groups at δ=60.1 ppm in
2013_05_02_Bz_TEG-E-2_Br_va_1H_300M.esp the BzTEG shifted downfield to δ=64.77 ppm (A’) and the carbon signals associated with the bromo ester group at δ = 25.46 ppm (F) and δ = 166.63ppm (G) appeared.
(c) (b)
O (f) (c)(d) (a') O Br (e) O O (b) 3 (f) (d)
(e)
(a')
4.21 14.591.962.052.00
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 Chemical Shift (ppm)
92 2013_05_02_Bz_TEG-E-2_Br_va_13C_300M.esp
O (C)(D) (A') (E') O Br (E) O O(G) (B) 3 (F) (E) (C) (E) (B)
(E) (A')
(D) (F)
(G) (E')
180 160 140 120 100 80 60 40 20 Chemical Shift (ppm)
Figure 4.2. NMR spectra of the product of the enzymatic transesterification of EBrA with BzTEG: (Top) 1H NMR spectrum and (Bottom) 13C NMR spectrum (500 MHz, solvent: CDCl3).
4.2.1.2. Transesterification of VClA with BzTEG
Vinyl chloroacetate and ethyl iodoacetate were also successfully transesterified to produce BzTEG-Cl (Figure 4.1B) and BzTEG-I (Figure 4.1C), respectively. Quantitative functionalization was reached after 2 h (Figure 4.3 and Figure 4.4). From our results, the presence of halogens did not affect the enzyme.
93
Figure 4.3. NMR spectra of the product of the enzymatic transesterification of VClA with 1 13 BzTEG: (Top) H NMR spectrum and (Bottom) C NMR spectrum (solvent: CDCl3).
1 H NMR (300 MHz, CDCl3, δ): 3.57 - 3.73 (b,c) (m, 14 H) 4.05 (f)(s, 2 H) 4.30
13 (a’) (m, 2 H) 4.53(d) (s, 2 H) 7.30 (e) (m, 5 H). C NMR (75 MHz, CDCl3, δ): 40 (F)
(CH2), 64.95 (A’) (CH2), 68.51(B) (CH2), 69.16 (B) (CH2), 70.30 (B) (CH2), 70.36 (C)
(CH2), 72.97 (D) (CH2), 127.40 (E) (CH), 127.53 (E) (CH), 137.93 ((E’) (C4), 167.15 (G)
(C=O). ESI m/z: [M+Na] calculated for C17H25IO6, 383.12; found 383.0.
94 4.2.1.3. Transesterification of VIA with BzTEG
Figure 4.4. NMR spectra of the product of the enzymatic transesterification of EIA with 1 13 BzTEG: (A) H NMR spectrum and (B) C NMR spectrum (solvent: CDCl3).
1 H NMR (300 MHz, CDCl3, δ): 3.60 - 3.69 (b,c,f) (m, 16 H) 4.20 - 4.28 (a’) (m,
2 H) 4.53 (d) (s, 2 H) 7.18 - 7.44 (e) (m, 4 H)
95 13 C NMR (75 MHz, CDCl3, δ): -5.66(F) (CH2), 64.91 (A’) (CH2), 68.48 (B)
(CH2), 69.24 (B) (CH2), 70.37 (B) (CH2), 70.43 (C) (CH2), 72.96 (D) (CH2), 127.32 (E)
(CH), 127.43 (E) (CH), 138.07 (E’)(C4), 168.45 (G) (C=O).
4.2.1.4. Transesterification of EBrV with BzTEG
The presence of a longer alkyl chain between the Br and the carbonyl group could relieve steric hindrance in further reactions, so we also transesterified EBrV with BzTEG
(Figure 4.1D).
TLC showed that the reaction was complete after 2.5 h under solventless conditions. The 1H NMR spectrum is shown in the Figure 4.5. The resonance at δ=4.53 ppm, corresponding to the -OH proton of BzTEG disappeared and the peak of the methylene protons adjacent to hydroxyl group shifted downfield to δ=4.12 ppm (a’) after the reaction. The new peak corresponding to the methylene protons adjacent to bromine were observed at δ = 3.32 ppm (j). The new peaks corresponding to the methylene
[δ=2.24 ppm (g), δ=1.80ppm (i) and δ=1.67ppm (h)] protons of the bromo valerate group were observed at the expected positions. The relative integrals of the methylene protons of the ester group (a’) and the methylene protons next to the benzyl group in the TEG moiety at δ = 4.47 ppm (d) demonstrate successful functionalization with the integration ration of 2:2.05. The 13C NMR spectrum (Figure 4.5) showed that the carbons connected to the hydroxyl groups at δ=60.1 ppm in the BzTEG shifted downfield to δ=63.13 ppm
(A’) and the carbon signals associated with the bromo valerate group at δ = 23.08 ppm (I)
δ = 31.56 ppm (H), δ = 32.69 ppm (G), δ = 32.74 ppm (J) and δ = 172.54 ppm (F)
96 appeared. ESI confirmed the product with a mass of 468.9 (ESI m/z: [M+Na]+ calculated for C20H31BrNaO6 469.12).
Figure 4.5. NMR spectra of the product of the enzymatic transesterification of EBrV with BzTEG: (Top) 1H NMR spectrum and (Bottom) 13C NMR spectrum (500 MHz, solvent: CDCl3).
Following the model reactions, we turned our attention to polymer functionalization.
97 4.2.2. Candida antarctica lipase B-Catalyzed Halo-Ester Functionalization of
PEGs
4.2.2.1. Transesterification of EBrA with MeO-PEG-OH2000
MeO-PEG-OH2000 was liquefied at the reaction temperature so the transesterification of EBrA could be carried out without solvents (Figure 4.1A’). The 1H
NMR spectrum of a sample taken at 2.5 hours still showed the presence of unreacted
1 MeO-PEG-OH2000. The H NMR spectrum of the product after 4h reaction time is shown in Figure 4.6. The resonance at δ=4.50 ppm, corresponding to the -OH proton of MeO-
PEG-OH2000 disappeared and the peak of the methylene protons adjacent to hydroxyl group shifted downfield to δ=4.13 ppm (a’) after the reaction. The new peak corresponding to the methylene protons adjacent to the bromine was observed at δ = 3.76 ppm (f). The 13C NMR spectrum (Figure 4.6) showed that the carbons connected to the hydroxyl groups at δ=60.1 ppm in the MeO-PEG-OH2000 shifted downfield to δ=64.90 ppm (E) and the carbon signals associated with the bromo ester group at δ = 26.97 ppm
(F) and δ = 167.15ppm (G) appeared after 4 h reaction time. The chloro- and iodo-esters were also successfully produced (Figure 4.1B’ and Figure 4.1C’).
98
Figure 4.6. NMR spectra of the product of the enzymatic transesterification of EBrA with 1 MeO-PEG-OH2000 (Mn = 2000 g/mol; ĐM= 1.2): (Top) H NMR spectrum and (Bottom) 13 C NMR spectrum (500 MHz, solvent: DMSO-D6).
4.2.2.2. Transesterification of VClA with MeO-PEG-OH2000
1 H NMR (500 MHz, DMSO-D6, ): 3.25(e) (s, 3 H), 3.46 - 3.56 (d) (m, 160 H)
3.61 - 3.68 (c) (m, 2 H), 4.21 - 4.26 (b) (m, 1 H), 4.39 (a) (s, 1 H).13C NMR (126 MHz,
DMSO-D6, ): 40.98 (F) (CH2), 57.98 (F) (CH3), 64.79 (D) (CH2) 68.00 (C) (CH2), 69.76
(CH2) 71.25 (B) (CH2), 167.26 (A) (C=O). MALDI-ToF: m/z = 2200.20 (calculated
2200.918).
99
Figure 4.7. NMR spectra of the product of the enzymatic transesterification of VClA with 1 MeO-PEG-OH2000 (Mn = 2000 g/mol; ĐM= 1.2): (Top) H NMR spectrum and (Bottom) 13 C NMR spectrum (solvent: DMSO-D6).
100 4.2.2.3. Transesterification of EIA with MeO-PEG-OH2000
Figure 4.8. NMR spectra of the product of the enzymatic transesterification of EIA with 1 MeO-PEG-OH2000 (Mn = 2000 g/mol; ĐM= 1.2): (Top) H NMR spectrum (solvent: 13 DMSO-D6). and (Bottom) C NMR spectrum (solvent: CDCl3).
1 H NMR (300 MHz, DMSO-D6, δ): 3.23 (d) (s, 3H), 3.47 - 3.60 (c) (m, 160H)
13 3.81(e) (s, 1 H), 4.18 (a’) (m, 2 H). C NMR (75 MHz, CDCl3, δ): -5.73 (E) (CH2), 59
(D) (CH3), 64.98 (A’) (CH2), 68.98 (B) (CH2), 70 (C) (CH2), 71.77 (G)(CH2), 168.55
(F)(C=O).
4.2.2.4. Transesterification of EBrV with MeO-PEG-OH2000
The transesterification of EBrV with MeO-PEG-OH2000 (Figure 4.1D’) was also carried out under solventless conditions. The 1H NMR spectrum of a sample taken at 2.5
101 10_08_2012_MC_MeO_PEG_Br_2.5h_1H_500M.esp
1 hours still showed the presence of unreacted MeO-PEG-OH2000. The H NMR spectrum of the product after 4 h reaction time is shown in Figure 4.9.
10_08_2012_MC_MeO_PEG_Br_2.5h_13C_500M.esp (c) (a') O (h) (j) (b) (d) (c) O O O Br (b) n (g) (i) (d)
(a') (g) (j) (i) (h)
2.01 2.902.042.49 2.15 2.142.08
6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Chemical Shift (ppm)
O (D) (C) (A') (H) (J) O O O (F) Br (C) (B) n (G) (I)
(B) (G) (A')(D) (H) (I) (F) (J)
180 160 140 120 100 80 60 40 20 Chemical Shift (ppm)
Figure 4.9. NMR spectra of the product of the enzymatic transesterification of EBrV with 1 MeO-PEG-OH2000 (Mn = 2000 g/mol; ĐM= 1.2): (Top) H NMR spectrum and (Bottom) 13 C NMR spectrum (500 MHz, solvent: DMSO-D6).
The resonance at δ=4.50 ppm, corresponding to the -OH proton of the MeO-PEG-
OH2000 disappeared and the peak of the methylene protons adjacent to hydroxyl group shifted downfield to δ=4.13 ppm (a’) after the reaction. The new peak corresponding to the methylene protons adjacent to bromine were observed at δ = 3.37 ppm (j). The new peaks corresponding to the methylene [δ=2.36 ppm (g), δ=1.83ppm (i) and δ=1.65ppm
(h)] protons of the bromo valerate group were observed at the expected positions. The 13C
102 NMR spectrum (Figure 4.9) showed that the carbons connected to the hydroxyl groups at
δ=60.10 ppm in the MeO-PEG-OH2000 shifted downfield to δ=63.06 ppm (A’) and the carbon signals associated with the bromo valerate group at δ = 23.05 ppm (I) δ = 31.42 ppm (H), δ = 32.40 ppm (G), δ = 34.51 ppm (J) and δ = 172.51 ppm (F) appeared after 4 hours reaction.
Figure 4.10. MALDI- ToF mass spectrum of the MeO-PEG-OH2000 (top left) and the MeO-PEG-BrV2000 product (bottom left); zoom of the 39 and 40 mer fractions (right).
MALDI-ToF mass spectrometry was utilized for further confirmation of the chain end structure. Figure 4.10 shows the MALDI-ToF spectrum of the starting MeO-PEG-
OH2000 (top left) and the product (bottom left). The peak at m/z 1978.19 corresponds to the sodium complex of the 39-mer fraction of MeO-PEG-BrV2000. The calculated monoisotopic mass for this ion is 1978.18 [39 × 44.03 (C2H4O repeat unit) + 238.02 (-
+ C8H15O2Br end group) + 22.99 (Na )]. Within the distribution, the peaks were separated by 44 Da, corresponding to an ethylene glycol repeat unit (Figure 4.10 inset where the
103 peaks in each cluster represent isotope distributions). This confirms that each oligomer in the MeO-PEG-BrV2000 carries the bromo-valerate end group. Therefore, MALDI-ToF MS analysis confirmed that the conversion of MeO-PEG-OH2000 was quantitative within 4 hours of reaction time under solventless conditions.
The same methodology can be used to synthesize difunctional Br-PEG-Br3400.
Figure 4.11 shows the MALDI-ToF spectrum of the product. There are one major and a minor distribution with peaks of 44 m/z units, corresponding to Na+ and K+ Br-PEG-
Br3400, respectively. The peak at m/z 3623.16 corresponds to the sodium complex of the
73-mer fraction of Br-PEG-Br3400. The calculated monoisotopic mass for this ion is
3623.152 [73 × 44.03 (C2H4O repeat unit) + 385.97(-C12H20O4Br2 end group) + 22.99
(Na+)]. Within the distribution, the peaks were separated by 44 Da, corresponding to an ethylene glycol repeat unit (Figure 4.11 inset where the peaks in each cluster represent isotope distributions). The minor peaks in the same region, which differ from the main
+ series by 15.16 m/z units, are attributed to the K cationized Br-PEG-Br3400 (the mass difference between Na+ and K+ is 15.974 Da).
104
Figure 4.11. MALDI- ToF mass spectrum of the Br-PEG-Br3400 (left) and zoom of the 73 and 74 mer fractions (right). [HO-PEG-OH3400] = 0.77 mol/L, [EBrV] = 7.72 mol/L; [CALB] = 1.54×10-4 mol/L.
In conclusion, we successfully synthesized halo-ester functionalized PEGs using
CALB catalyzed transesterification in the absence of solvents. The resin-supported enzyme can easily be separated from the reaction mixture, producing very pure products.
According to the literature, the resin-supported CALB can be recycled at least 4 times without loss of activity.12–14 Both the absence of solvents and the recyclability of the catalyst are endorsing green chemistry concepts. In this work a low molecular weight
MeO-PEG-OH2000 was used to aid structure characterization, but CALB was shown to catalyze transesterifications with PEGs with molecular weights up to Mn ~ 10,000 g/mol.173
In order for chemical reactions to be most effective in adapting to a wide range of processes and demands, they should be simple, efficient, regioselective, stereoselective
105 where applicable, yield a single product, and be amendable to occurring at ambient temperature and atmospheric conditions. Specifically, alkyne–azide click chemistry is one powerful strategy to produce distinctive, highly functional materials. In the next section a difunctional azide PEG is synthesized using CALB-catalysis. This functional
PEG can be used as a building block for other functional materials taking the advantage of click chemistry.
4.2.3. Candida antarctica lipase B-Catalysis of PEGs for Click Chemistry
4.2.3.1. Synthesis of Ethyl 5-azidevalerate (EN3V)
Figure 4.12. Synthesis of ethyl 5-azidevalerate (EN3V). [EBrV]= 0.77 mol/L, [NaN3]= 1.14 mol/L.
The synthesis of ethyl 5-azidevalerate (EN3V) is described in Figure 4.12. EBrV
(0.70 g, 3.3 mmol, 1.0 eq) was dissolved in DMSO (4.3 mL) and stirred under N2 (g) overnight at 55°C with sodium azide (0.36 g, 4.9 mmol, 1.5 eq). The 1H NMR spectrum of the product after 12 h reaction time is shown in Figure 4.13.
106
Figure 4.13. NMR spectra of ethyl 5-azidevalerate. (Top) 1H NMR spectrum and 13 (bottom) C NMR spectrum (300 MHz, solvent: CDCl3).
The peak corresponding to the methylene protons adjacent to bromide group disappear δ = 3.37 ppm and the methylene protons adjacent to the azide group were observed at δ = 2.18 ppm (c). The peaks corresponding to valerate group were observed at the expected positions [δ=3.99ppm (a), δ=3.15 ppm (b), δ=1.51 ppm (d) and δ=1.10
107 ppm (e)]. The 13C NMR spectrum (Figure 4.13) showed that the carbons connected to azide group appeared at δ=50.59 ppm and the carbon signals associated with the valerate group at δ = 172.45 ppm (A), δ = 33.114 ppm (D) δ = 27.82 ppm (E), δ = 21.68 ppm (F) and δ = 13.71 ppm (G) appeared at the expected positions.
ESI confirmed the product with a mass of 194.1 (ESI m/z: [M+Na]+ calculated for
C7H13N3NaO2 194.09).
4.2.3.2. Synthesis of Azide-Functionalized poly(ethylene glycol)s (N3-PEG-N3)
Figure 4.14. Enzymatic transesterification of EN3V with HO-PEG-OH3400. [HO-PEG- -4 OH3400]= 0.58 mol/L, [EN3V]= 5.77 mol/L, [CALB]= 1*10 mol/L.
The transesterification of EN3V with HO-PEG-OH3400 (Figure 4.14) was also carried out under solventless conditions. HO-PEG-OH3400 (Mn=1000 g/mol; ĐM =1.04,
0.26 g, 0.15 mmol, 1.0 eq.) was reacted with EN3V (0.26 g, 1.5 mmol, 5.0 eq. per OH in
-5 HO-PEG-OH3400) in the presence of CALB (0.014 g resin @ 20 wt% enzyme, 2.8 × 10 mmol, 0.00020 eq.) in bulk at 65 °C for 4 h under vacuum (70 millitorr). The 1H NMR spectrum of the product after 4 h reaction time is shown in Figure 4.15.
108
Figure 4.15. NMR spectra of the product of the enzymatic transesterification of EN3V 1 13 with HO-PEG-OH3400 (Top) H NMR spectrum and (bottom) C NMR spectrum (500 MHz, solvent: DMSO).
The resonance at δ=4.50 ppm, corresponding to the -OH proton of the HO-PEG-OH3400 disappeared and the peak of the methylene protons adjacent to hydroxyl group shifted downfield to δ=4.13 ppm (a) after the reaction. The new peak corresponding to the methylene protons adjacent to azide was observed. The new peaks corresponding to the methylene protons of the azide valerate group were observed at the expected positions.
The 13C NMR spectrum (Figure 4.15) showed that the carbons connected to the hydroxyl
109 groups at δ=60.1 ppm in the HO-PEG-OH3400 shifted downfield to δ=63.06 ppm (C) and the carbon signals associated with the azide valerate group appeared after 4 hours reaction. MALDI-ToF mass spectrometry was utilized for further confirmation of the chain end structure (Figure 4.16).
Figure 4.16. MALDI- ToF mass spectrum of the N3-PEG-N3-3400 (left) and zoom of the 74 and 75 mer fractions (right).
Figure 4.16 shows the MALDI-ToF spectrum of the product. There are one major
+ + and a minor distribution with peaks of 44 m/z units, corresponding to Na and NH4 N3-
PEG-N3-3400, respectively. The peak at m/z 3623.16 corresponds to the sodium complex of the 75-mer fraction of N3-PEG-N3-3400. The calculated monoisotopic mass for this ion is 3637.48 [75 × 44.03 (C2H4O repeat unit) + 312.15(-C12H20O4N6 end group) + 22.99
(Na+)]. Within the distribution, the peaks were separated by 44 Da, corresponding to an ethylene glycol repeat unit (Figure 4.16 inset where the peaks in each cluster represent isotope distributions). The minor peaks in the same region, which differ from the main
110 + series by 4.79 m/z units, are attributed to the NH4 cationized N3-PEG-N3-3400. The calculated monoisotopic mass for this ion is 3632.40 [75 × 44.03 (C2H4O repeat unit) +
+ 312.15(-C12H20O4N6 end group) + 18 (NH4 )].
4.2.4. Transesterification of Vinyl Acrylate with of Poly(ethylene glycol)s
4.2.4.1. Monofunctionalization of Poly(ethylene glycol)s by Enzyme-Catalyzed
Transesterification of Vinyl Acrylate
One of the uses of PEG is in drug delivery, where it is used as a linker between a drug and a carrying agent; and generally two different functional groups on each end are required for PEG to be used as a linker. However, the synthesis of a monofunctionalized
PEG is a challenge.
The preparation of mono-functionalized VA-PEG-OH1100 might be possible via enzymatic transesterification of vinyl acrylate (0.063 g, 0.64 mmol, 0.66 eq) with HO-
PEG-OH1100 (Mn=1100 g/mol, ĐM=1.1, 0.99 g, 1.1 mmol, 1.0 eq.) in the presence of
CALB (0.16 g resin @ 20 wt% enzyme, 8.8 x 10-4 mmol, 0.001eq). Aliquots were taken from t = 15 min to 4 h. The concept was evaluated using MALDI-ToF MS which is a powerful tool for the characterization of functional polymers.
111
Figure 4.17. Synthetic procedures of acrylate-PEG-OH. [HO-PEG-OH1100]= 0.053 mol/L, [VA]= 0.64 mol/L, [CALB]= 8.8*10-4 mol/L
At t = 45 min of reaction, there was a mixture of unreacted HO-PEG-OH1000, monosubstituted VA-PEG-OH1000 and disubstituted VA-PEG-VA1000 with the unreacted
HO-PEG-OH1000 being the major distribution (Figure 4.18). Within each distribution the peaks were separated by the C2H4O repeat unit (m/z = 44). The representative peak at m/z
= 965.3 in the major distribution, designated as “A” in Figure 4.18, corresponds to the sodiated 21-mer of the HO-PEG-OH1100. The calculated monoisotopic mass for this peak
+ [m/z = 22x44.03 (C2H4O repeat unit) + 18.01 (OH2 end groups) + 22.99 (Na )] is 1009.68
Da. The representative peak of one of the minor distributions, designated as “B” in Figure
4.18, at m/z = 975.7 corresponds to the sodiated 22-mer of VA-PEG-OH1100. [m/z =
+ 21x44.03 (C2H4O repeat unit) + 86.04 (C4H6O2 end groups) + 22.99 (Na )] is 1033.68
Da. The peak at m/z = 985.3 which belongs to the other minor distribution, designated as
“C” in Figure 4.18, corresponds to the sodiated 19-mer of VA-PEG-VA1000. The calculated monoisotopic mass for this peak [m/z = 19x44.03 (C2H4O repeat unit) +
+ 154.08 (C8H10O3 end groups) + 22.99 (Na )] is 1013.64 Da.
112 965.3 921.3 1009.3
500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z 965.3 1009.3
44.03 975.73
44.03 985.3
960 970 980 990 1000 1010 1020 1030 1040 m/z
Figure 4.18. MALDI spectrum of the aliquot taken in the CALB-catalyzed transesterification of VA with HO-PEG-OH1100 at t = 45 min (top), and zoom of the 21 and 22 mer fractions (bottom).
Table 4.1 is built based on the MALDI analysis of every aliquot taken to compare the percent of monofunctional and difunctional product at specific time.
113 Table 4.1. Reaction progress composition in the CALB-catalyzed transesterification of VA with HO-PEG-OH1100.
Time (h) HO-PEG-OH1000 % VA-PEG-OH1000 % VA-PEG-VA1000 0.28 82.20 13.41 4.29 0.5 82.70 13.60 3.70 0.75 87.10 14.43 2.85 1 72.80 24.47 2.71 1.5 74.80 21.22 3.91 2 71.30 26.03 2.60 4 66.00 29.32 4.42
The analysis indicated that at the very beginning of the reaction both, the monofunctional and difunctional products are present. Other conditions were tried: change in the CALB concentration and ratio of the alcohol to the vinyl ester, bulk and solvent. In all cases the difunctional product doesn’t overcome the 5% of the mixture, but in solution the monofunctional product is produced in a significant amount compare with bulk conditions.
The purification process is necessary to remove VA-PEG-VA1100 and HO-PEG-
OH1100. The obtained crude product was purified by column chromatography alumina
(CH2Cl2: THF: MeOH = 6:3:0.3). TLC Rf= 0.23.
1 Figure 4.19 shows the H NMR spectrum of the VA-PEG-OH1100. The peak corresponding to the methylene protons next to hydroxyl group can be observed at
=3.50 ppm and the new methylene protons from the ester group appeared at =4.42 ppm
(c). New resonances attributed to the vinylidene [=6.20 ppm (b)] and vinyl [=6.32 ppm
(a) and =5.97 ppm (a’)] protons appeared with the integration ratios of
(c):(a’):(a):(b):OH as 2:1:1:1:1.
114
Figure 4.19. NMR spectra of the product of the enzymatic transesterification of VA with 1 13 HO-PEG-OH1100 (Top) H NMR spectrum and (bottom) C NMR spectrum (500 MHz, solvent: DMSO-D6).
The 13C NMR spectrum of the transesterification product also confirmed the structure of the VA-PEG-OH1000. The carbon connected to the hydroxyl group can be observed at =60.13 ppm (E) and the new methylene carbon from the ester group appeared at =63.41 ppm (F) and the carbon signals associated with the acrylate group at
= 166.02 ppm (A), = 131.86 ppm (B) and = 128.55 ppm (C) appeared after the acrylation.
115 Figure 4.20 shows MALDI-ToF mass spectrum of the transesterification product.
The distribution corresponds to VA-PEG-OH1000. In the expanded spectrum, the representative peak at m/z 1107.77 corresponds to the sodium complex of the 22-mer of
VA-PEG-OH1000; its calculated monoisotopic mass is 1107.70 Da [22 × 44.03 (C2H4O repeat unit) + 99.054 (C5H7O2 acrylate group) + HO (15.99) + 22.99 (Na+)].
Figure 4.20. MALDI- ToF mass spectrum of the VA-PEG-OH1000 (left) and zoom of the 22 and 23 mer fractions (right).
4.2.4.2. Transesterification of VA-DVA with Poly(ethylene glycol)
VA-DVA (0.3376 g, 1.25 mmol, 5 eq. per OH) was reacted with HO-PEG-OH8000
(Mn=8,000 g/mol, ĐM=1.2, 1 g, 0.13 mmol, 1.0 eq.) in the presence of CALB (0.1 mg
-4 -3 resin @ 20 wt% enzyme, 6.3 × 10 mmol, 4.8 × 10 eq.) at 50 ºC for 4 hours.
116
13 Figure 4.21. C NMR spectra of (bottom) HO-PEG-OH8000 and (top) VA-PEG-VA8000 in DMSO-D6.
Figure 4.21 shows the 13C NMR spectrum of the starting material and the product. The
The signals appeared at the expected position, but some excess of DVA-VA is still
present.
Figure 4.22 shows the MALDI, the series corresponding to VA-PEG-VA8000 was
observed (
Figure 4.22)
9288.988 9332.272 9376.839 9376.839 9288.988
7500 8000 8500 9000 9500 10000 10500 11000 m/z 9280 9300 9320 9340 m/z 9380 9360
117 Figure 4.22. MALDI- ToF mass spectrum of VA-PEG-VA8000 (left) and zoom of the 199 and 200 mer fractions (right). [HO-PEG-OH8000]= 0.37 mol/L, [VA-DVA]= 3.70 mol/L, [CALB]= 1.9*10-3 mol/L. 4.2.5. Candida antarctica lipase B-Catalysis of Multifunctional PEGs
Dr. Kwang Su Seo in his doctoral dissertation studied the stereoselectivity of the
CALB-catalyzed Michael addition. Diethylamine was reacted with 2-(acryloyloxy)ethyl crotonate (Figure 4.23). It was found that diethylamine exclusively reacted with the acrylate group.162
Figure 4.23. Stereospecificity in CALB-catalyzed Michael addition.
Figure 4.24. Chemoselectivity in Enzyme-catalyzed Michael Addition. Model reaction: [2,2'-(ethane-1,2-diylbis(oxy))diethanamine]= 1*10-6 mol/L, [3-(acryloyloxy)-2- hydroxypropyl methacrylate]= 2*10-6 mol/L, [CALB]= 1*10-10 mol/L. PEG functionalization: [NH2-PEG-NH2-2000]= 0.3 mol/L, [3-(acryloyloxy)-2-hydroxypropyl methacrylate]= 0.6mol/L, [CALB]= 3*10-8 mol/L.
118
4.2.5.1. Model Reaction
The Michael Addition of 2,2'-(ethane-1,2-diylbis(oxy))diethanamine to 3-
(acryloyloxy)-2-hydroxypropyl methacrylate in the presence of CALB is described in
Figure 4.24. This serves a model reaction of the PEG functionalization.
2,2'-(ethane-1,2-diylbis(oxy))diethanamine (1.54 g, 0.010 mol, 1.0 eq.) in 10 mL of anhydrous DMSO was reacted with 3-(acryloyloxy)-2-hydroxypropyl methacrylate
(4.50 g, 0.020 mol, 2.0 eq.) in the presence of CALB (0.17 g resin @ 20 wt% enzyme,
1.0 × 10-6 mol, 0.00010 eq.) at 50 °C. Only the acrylate group reacted, leaving the methacrylate group intact.
Figure 4.25. 1H NMR spectrum of the product CALB catalyzed Michael Addition. [2,2'- (ethane-1,2-diylbis(oxy))diethanamine]= 1*10-6 mol/L, [3-(acryloyloxy)-2- hydroxypropyl methacrylate]= 2*10-6 mol/L, [CALB]= 1*10-10 mol/L. (300 MHz, solvent: CDCl3).
119 Figure 4.25 shows the NMR spectra of the product at 24 h reaction time. The vinylidene [δ=6.18 ppm] and vinyl [δ=6.32 ppm and δ=5.97 ppm] protons of the acrylate group disappeared while the proton resonances of the methacrylate group at δ = 6.06 ppm
(a), δ = 5.68 ppm (b) and δ = 0.9 ppm (c) remained intact.
The 1H NMR spectrum confirmed that the Michael addition to the acrylate group was complete within 30 min, while the methacrylate group remained intact. Based on these results polymer functionalization was carried out successfully.
4.2.5.2. Chemoselectivity in Enzyme-Catalyzed Michael Addition
The Michael Addition of NH2-PEG-NH2-2000 to 3-(acryloyloxy)-2-hydroxypropyl methacrylate in the presence of CALB is described in Figure 4.24. NH2-PEG-NH2-2000
(Mn=2,000 g/mol; ĐM=1.04, 0.2 g) in 0.7 mL of anhydrous DMSO was reacted with 3-
(acryloyloxy)-2-hydroxypropyl methacrylate (0.028 g) in the presence of CALB (3.3 mg resin @ 20 wt% enzyme) at 50 °C. Only the acrylate group reacted, leaving the methacrylate group intact.
120
1 Figure 4.26. H NMR spectrum of the product CALB catalyzed Michael Addition. [NH2- PEG-NH2-2000]= 0.3 mol/L, [3-(acryloyloxy)-2-hydroxypropyl methacrylate]= 0.6 mol/L, -8 [CALB]= 3*10 mol/L. (500 MHz, solvent: CDCl3).
Figure 4.26 shows the NMR spectra of the product at 24 h reaction time. The vinylidene [δ=6.18 ppm] and vinyl [δ=6.32 ppm and δ=5.97 ppm] protons of the acrylate group disappeared while the proton resonances of the methacrylate group at δ = 6.06 ppm
(a), δ = 5.68 ppm (b) and δ = 1.88 ppm (c) remained intact. In the 13C NMR spectrum, the carbons resonances corresponding to the acrylate group shifted upfield to δ=42.60 ppm
(H) and δ=34.97 ppm (G) after the reaction and the carbon resonances of the methacrylate group at δ = 166.82 ppm (M), δ = 135.67 ppm (L), δ = 125.72 ppm (A) and
δ = 172.06 ppm (N) did not change.
121 4.2.6. CALB-Catalyzed Functionalization of Small Molecules
4.2.6.1. Synthesis of bis(2-ethyl 5-bromopentanoate) disulfide
Figure 4.27. Reaction scheme and TLC monitoring of the CALB-catalyzed transesterification of EBrV with 2HEDS in bulk. [EBrV] = 4.27 mol/L, [2HEDS] =1.90 mol/L; [CALB] = 7.1 × 10-5 mol/L.
Bis (2-ethyl 5-bromopentanoate) disulfide was synthesized following the Figure
4.27. 2HEDS (1.0 g, 6.5 mmol, 1.0 eq.) was reacted with EBrV (8.1 mL, 38.9 mmol, 6.0 eq.) in the presence of CALB (0.1 g resin @ 20 wt% enzyme, 6.5 × 10-4 mmol, 0.00010 eq.) in bulk at 65°C under vacuum. The progress of the reaction was monitored with TLC using hexane/THF (1/1; vol/vol) as the eluent mixture. TLC showed that the reaction was complete after 2 h under solventless conditions. The excess of the EBrV was removed by vacuum distillation at 85ºC. But there was a 4% remaining in the product.
The product was analyzed by 1H and 13C NMR spectroscopy (Figure 4.28). The
1H NMR spectrum of the product is shown in Figure 4.28.
122 cdcl3_01
cdcl3_02 (f) (b) (a) (c) (f) (d) (a)
(e) (c) (b) (d) (e)
2.00 2.00 1.98 2.11 2.05
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 Chemical Shift (ppm)
(D)(F) (E) (B) (G) (A) (G) (E) (B) (C) (F) (D) (A)
(C)
170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 Chemical Shift (ppm)
Figure 4.28. NMR spectra of the transesterification product of EBrV with 2HEDS in bulk: 1 13 (top) H NMR spectrum and (bottom) C NMR spectrum (300 MHz, solvent: CDCl3).
The resonance at δ=4.53 ppm, corresponding to the -OH proton of 2HEDS disappeared and the peak of the methylene protons adjacent to hydroxyl group shifted downfield to δ=4.48 ppm (a) after the reaction. The new peak corresponding to the methylene protons adjacent to the bromine was observed at δ = 3.47 ppm (f). The relative integrals of the methylene protons of the ester group (a) and the methylene protons next to the bromine moiety at δ = 3.48 ppm (f) demonstrate successful functionalization with the integration ration of 2:2. The 13C NMR spectrum (Figure 4.28) showed that the carbons connected to the hydroxyl groups at δ=60.1 ppm in the 2HEDS shifted downfield
123 to δ=64.77 ppm (A) and the carbon signals associated with the bromo ester group at δ =
25.46 ppm (F) and δ = 166.63ppm (G) appeared.
4.2.6.2. Transesterification Reaction of Divinyladipate with 2-hydroxyethyl
disulfide
In this section, a linker carrying a mercapto and vinyl ester was synthesized in two steps. The first step involved the transesterification reaction of divinyladipate with 2- hydroxyethyl disulfide using CALB to yield DVA-S-S-DVA product. The second step involved the disulfide bond reduction using DTT to yield HS-DVA.
O,O'-(disulfanediylbis(ethane-2,1-diyl)) divinyl diadipate (DVA-S-S-DVA) was synthesized following the Figure 4.29. 2HEDS (1.0 g, 6.5 mmol, 1.0 eq.) was reacted with divinyladipate (25.6 mL, 128 mmol, 20 eq.) in 3.3 mL of anhydrous THF and CALB
(0.5 g resin @ 20 wt% enzyme, 8.7x10-6 mmol, 1.3x10-6 eq.) under an inert atmosphere.
Figure 4.29. Reaction scheme and TLC monitoring of the CALB-catalyzed transesterification of DVA with 2HEDS. [DVA] = 4.81 mol/L, [2HEDS] = 0.24 mol/L; [CALB] = 7.1×10-5 mol/L.
124 The progress of the reaction was monitored with TLC using hexane/THF (1/1; vol/vol) as the eluent mixture. TLC showed that the reaction was complete after 24 h.
The product was purified by chromatography column in silica.
The product was analyzed by 1H and 13C NMR spectroscopy (Figure 4.30). The resonance at δ=4.53 ppm corresponding to the -OH (a) protons of 2HEDS disappeared and the peak of the methylene protons adjacent to the hydroxyl groups shifted downfield from δ=3.50 to δ=4.22 ppm (b’). New resonances attributed to the vinylidene [δ=4.87 ppm (e), δ= 4.65 (e’)], and methine [δ=7.24 ppm (f)] protons appeared. In the product
(b):(c+d):(e):(e’):(f) = 2.00:6.44:0.95:0.97:0.94.
In the 13C NMR spectrum, the carbons connected to the hydroxyl group at
δ=60.13 ppm in the 2HEDS shifted downfield to δ=62.10 ppm (B) and new carbon resonances of the vinyl groups [δ=141.00 ppm (B) and δ=97.60 ppm (A)] and carbonyl carbons resonances of adipic ester groups [δ=172.92 ppm (K) and δ=170.25 ppm (J)] appeared at the expected positions.
125
Figure 4.30. NMR spectra of the transesterification product of DVA with 2HEDS: (top) 1H
13 NMR spectrum and (bottom) C NMR spectrum (300 MHz, solvent: CDCl3).
The product was also analyzed by electrospray ionization mass spectrometry
(ESI-MS) using sodium trifluoroacetate (NaTFA) as a cationizing agent. The ESI-MS confirmed the formation of the desired product with a peak at m/z 484.7 [M+Na+], which
126 is in good agreement with the calculated monoisotopic mass of the expected product:
462.14 (C20H30O8S2) (Figure 4.31). 484.7
ESI shows the product is pure 500.7
200 300 400 500 600 700 800 m/z
Figure 4.31. ESI-MS spectrum of the transesterification product of DVA with 2HEDS (cationizing agent: NaTFA)
127 4.2.6.3. Disulfide Reduction of O,O'-(disulfanediylbis(ethane-2,1-diyl)) divinyl
diadipate (DVA-S-S-DVA)
Figure 4.32. Reaction scheme and TLC monitoring of the disulfide reduction of DVA-S- S-DVA. [DTT] = 8.6×10-3 mol/L, [DSDV] = 8.6×10-3 mol/L.
In dark condition, a mixture of DVA-S-S-DVA (0.25 g, 0.43 mmol, 1.0 eq.) and
DTT (0.068 g, 0.43 mmol, 1.0 eq.) was dissolved in 50 mL of ethyl acetate under a nitrogen atmosphere. The progress of the reaction was monitored with TLC using hexane/THF (1/1; vol/vol) as the eluent mixture. TLC showed that the reaction was complete after 3 h (Figure 4.32). The product was purified by chromatography column in silica. The 1H and 13C spectra confirmed the purity of the product (Figure 4.33).
128
Figure 4.33. NMR spectra of the of the disulfide reduction of DVA-S-S-DVA: (top) 1H 13 NMR spectrum and (bottom) C NMR spectrum (300 MHz, solvent: CDCl3).
129 4.2.7. Transesterification of Divinyl Adipate with 2-(Hydroxyethyl) Acrylate in
Solventless Conditions
Figure 4.34. Reaction scheme and TLC monitoring of the transesterification reaction of DVA with 2HEA. [DVA] = 8.6×10-3 mol/L, [2HEA] = 8.6×10-3 mol/L, [CALB] = 8.6×10-3 mol/L.
DVA (2.0 g, 10.1 mmol, 1.0 eq.) was reacted with 2HEA (1.2 g, 10.1 mmol, 1.0 eq.) in the presence of CALB (200 mg resin @ 20 wt% enzyme, 1.2×10-3 mmol, 1.2×10-4 eq.) in bulk conditions at 50 ºC. The progress of the reaction was monitored with TLC using hexane/THF (3/1; vol/vol) as the eluent mixture (Figure 4.34). The reaction was complete after 3 hours, as the spot at Rf = 0.36 corresponding to 2HEA disappeared and new spots appeared at Rf = 0.57 and 0.75. The product was purified by silica gel column chromatography using hexane and THF mixture (5/1; vol/vol). The liquid product was dried using a vacuum oven (2.1 g yield, 100% conversion). The product was analyzed by
1H and 13C NMR spectroscopy (Figure 4.35).
The hydroxyl protons at δ=4.76 ppm from 2HEA disappeared and the signals of the methylene protons adjacent to hydroxyl group shifted downfield to δ=4.35 ppm (g) after the reaction. The new peaks corresponding to the vinylidene [δ=4.87 ppm (a) and
130 δ=4.65 ppm (a)] and methine [δ=7.26 ppm (b)] protons of the vinyl ester group appeared with the integration ratios of (g):(a):(a):(b) as 4:1:1:1.
(e, d) (h, g)
(f) (c) (j) (i) (a) (a) (b) (j)
(I) (F) (J) (D) (G)(H) (C) (E)
(B) (K) (M) (A) (L)
Figure 4.35. NMR spectra of the transesterification product of DVA with 2HEA: (top) 1H 13 NMR spectrum and (bottom) C NMR spectrum (300 MHz, solvent: CDCl3).
The 13C NMR spectrum of the transesterification product also confirmed the structure of 2-(acryloyloxy)ethyl vinyl adipate (VA-DVA). The carbons connected to the hydroxyl group in the starting material at δ=59.47 ppm shifted downfield to δ=62.17 ppm
(H,G) after the reaction and the carbon resonances of the vinyl ester group appeared at
131 δ=97.70 ppm (A), δ=141.15 ppm (B) and δ=165.26 ppm (K). The carbons corresponding to the acrylate group appeared at δ=127.95 ppm (I), δ=131.68 ppm (J) and δ=170.10 ppm
(M), confirming the structure of the VA-DVA product.
132 5. CHAPTER V
ENZYMATIC FUNCTIONALIZATION BY POLYMERIZATION
Parts of this chapter have been published:
M Castano, J Zheng, JE Puskas*, ML Becker* “Enzyme-Catalyzed Ring-Opening
Polymerization of -caprolactone using Alkyne Functionalized Initiators” Polymer
Chemistry. 2014, 5, 1891-1896. Reproduced with permission from The Royal Society of
Chemistry.
KS Seo, M Castano, M Casiano, C Wesdemiotis, ML Becker, JE Puskas*
“Functionalization of Poly(ethylene glycol)s by Enzyme-catalyzed Transesterification of
Divinyl Adipate under Solventless Conditions” RSC Advances, 2014, 4, 1683-1688.
Reproduced with permission from The Royal Society of Chemistry.
In this research, precise functionalization of polymers was achieved using two methods: enzyme-catalyzed functionalization of polymers by chain-end and enzyme- catalyzed functionalization via polymerization. This chapter will discuss the second method.
133 5.1. Enzyme-Catalyzed Ring-Opening Polymerization of -caprolactone Using
Alkyne Functionalized Initiators.
5.1.1. Introduction
Chemo-enzymatic methods are an alternative strategy to increase the diversity of functional groups in polymeric materials. In the past two decades, enzymatic catalysis has been applied to polymer synthesis177–181 and functionalization11,17,182,183 due to several distinct advantages, including high efficiency, catalyst recyclability, and mild reaction conditions.178,184 Lipases belong to the group of hydrolyses and are the most widely utilized enzyme-based catalysts. Lipases are used widely in esterification, transesterification, aminolysis, and Michael addition reactions in organic solvents.180,181,185 The most useful lipases for organic synthesis are: Porcine pancreatic lipase (PPL), lipase from Pseudomoanas cepacia (Amano lipase PS), lipase from
Candida rugosa (CRL), and lipase B from Candida antarctica (CALB).31 In particular,
CALB has been used in a range of polymer forming reactions, including the ring-opening polymerization of cyclic monomers (e.g., lactones, carbonates) and polycondensation reactions.186–188
The synthesis of functional, degradable polymers with well-defined structure, end-group fidelity and physico-chemical properties useful for biomedical applications has proven somewhat challenging. In particular, aliphatic polyesters prepared by ring- opening polymerization (ROP) of lactones and lactides have received significant attention due to their mechanical properties, degradation behavior and biocompatibility. To achieve defined structures and properties, the techniques for ROP of lactones, lactides
134 and cyclic carbonates has been continuously refined in recent years.52,189–196 Multiple combinations of initiators and catalysts have been evaluated.77,78,160,188,197–200
Enzyme-catalyzed ROP is one of the most promising tools and avoids the use of organo-metallic catalysts (Zn, Al, Sn or Ge) which are known to be cytotoxic to cellular systems are often difficult to remove from polymeric products.200 Additionally, other reports include the combination of chemoenzymatic monomer transformation with other polymerization mechanism like ATRP201 and RAFT.202 As the CALB enzyme is immobilized on a resin, purification is achieved via filtration and characterization of the resulting products proceeds using established protocols, which is convenient if the product is intended to be used in clinical applications or devices. The seven-membered unsubstituted lactone ε-CL is one of the most studied systems with respect to lipase- catalyzed ring-opening polymerization initiated by water.
End-functionalized -caprolactone (-CL) is attractive in that biologically active molecules can be tethered in a manner that preserves the biological activity and does not change significantly the semi-crystalline nature of the polymer backbone. Similar to the metal mediated ROP of CL, a nucleophile can be considered as initiator of the polymerization. This initiator can be water, alcohol, amine, or thiol.79,203,204 For example, benzyl alcohol was used as initiator for the ROP of -CL, functionalization was found to vary from 20%77 to 73%78 depending of the water content in the media.
We are interested in developing polymerization methods that use functional alkynes as initiating systems as the -CL can be functionalized post-polymerization using click chemistry. In this paper we present the synthesis of alkyne functionalized poly(caprolactone) (PCL) through the CALB catalyzed ring-opening polymerization of -
135 CL activated monomer using propargyl alcohol and primary hydroxy-derivatized 4- dibenzocyclooctynol (DIBO) as an initiator. The alkyne-PCL functionalized material obtained through the ROP of -CL initiated by propargyl alcohol can be further functionalized using conventional copper-catalyzed click chemistry. On the other hand, the -CL initiated by DIBO yields alkyne-PCL functionalized that can subsequently be derivatized though an strain-promoted azide alkyne cycloaddition, which avoid the use of copper or other metal catalysts in the reactions.
5.1.2. Propargyl Alcohol Initiator for ROP of -CL
-unsaturated polymers are useful as building block for synthesis of functional materials. For the preparation of end-functionalized polymer, propargyl alcohol was used as the initiator of the enzymatic ring opening polymerization of -CL Figure 5.1.
-unsaturated polymers are useful as building block for synthesis of functional materials. For the preparation of end-functionalized polymer, propargyl alcohol was used as the initiator of the enzymatic ring opening polymerization of -CL Figure 5.1.
Figure 5.1. CALB catalyzed ROP of -CL using as initiator propargyl alcohol. For the case [monomer]/ [initiator]= 20. [Propargyl-OH] = 0.26 mol/L, [-CL] = 5.2 mol/L; [CALB] = 1.31 × 10-4 mol/L.
The number-average molecular mass (Mn) of the polymer was determined by SEC
1 as Mn =4,800 g/mol (ĐM =1.3). Figure 5.2 shows the H spectrum of the product. Signals for structural analysis included those at 3.68 ppm (t, J 6.5 Hz,-[C-O]-(CH2)4CH2-OH),
136 due to methylene protons of PCL chain-end units; resonances at 3.99-4.11 ppm (m, -[C-
O]-(CH2)4CH2-O), due to PCL repeat units along chains. The signal at =4.7 ppm and
=2.452013_02_13_MC_Propargyl_CL_30h_1H_300M.esp ppm was assigned to the methylene (CH2) protons and methyne protons (CH) of polycaprolactone (PCL) end-capped with propargyl alcohol. The Mn of the polymer was calculated from the ratio of the relative integral of methylene protons of the repeat unit to the methylene protons of PCL to chain-end units at = 3.68 ppm (c) to that of the backbone methylene protons at = 3.99-4.11 ppm (b).
O (a) (c) (d) O O OH (b) nO
(b)
(a) (c)
(d)
5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)
Figure 5.2. 1H NMR spectrum of the product CALB catalyzed ROP of -CL using as initiator propargyl alcohol [monomer]/ [initiator]= 20 (500 MHz, solvent: CDCl3).
The functionalization was determined by the ratio of the signals at =3.68 ppm (c) and =2.45 ppm (d), in this case 99%. Mn calculated by the NMR is 4,373 g/mol. The Mn obtained from 1H NMR was in good agreement with SEC results.
Figure 5.3 shows MALDI-ToF spectrum two distributions of the product obtained by the ROP polymerization reaction of propargyl alcohol with -CL. There is one major
137 and one minor distribution, corresponding to the sodium complex of linear alkyne-PCL
(98%), and cyclic polymer (2%), which is formed by the back-biting reaction of a polymer acyl-enzyme intermediate. Within the distributions, the peaks are separated by
114.1 Da, corresponding to a PCL repeat units.
Figure 5.3. MALDI- ToF mass spectrum of the product from the CALB catalyzed ROP of -CL using as initiator propargyl alcohol; [monomer]/ [initiator]= 20.
Aliquots were taken at defined intervals for the reaction posessing a monomer/ initiator ratio equal to 50. TheFigure 5.4A-B shows the SEC eluograms and the plot of
Mn vs time. The sequencial increase in the molecular mass and the polydispersity is observed with time. At 24 h there is a decrease in the molecular mass. This is due with long reaction time the enzyme start to catalize transesterification reactions.
Additionally, polymerizations were carried using different feed ratios. The various feed ratio conditions and the resulting molecular mass and molecular mass
138 distributions of the polymers were measured using SEC (THF as eluent) and are shown in
Figure 5.5. Monomodal distributions in all polymerizations were observed.
Figure 5.4. (A) SEC eluograms for aliquots at 4 h, 8 h, 12 h and 24 h of CALB catalyzed ROP of -CL using as initiator propargyl alcohol. For the case [monomer]/ [initiator]= 50:1. [Propargyl-OH] = 0.1 mol/L, [-CL] = 5.2 mol/L; [CALB] = 1.3×10-4 mol/L. (B) Number-average molecular mass (Mn) and ĐM as a function of time at 70ºC in bulk and solution system. (C) SEC results of CALB catalyzed ROP of -CL using propargyl alcohol as the initiator for the reaction [monomer]/ [initiator]= 50:1 at 12 h. In both bulk and solution reaction conditions.
The polymerization was also performed in dioxane and in bulk conditions. Using dioxane as solvent, no polymerization was observed at 24 h. Figure 5.4C shows the chromatogram for the alkyne-PCL product in bulk and toluene conditions and the plot of
Mn vs time. In the case of bulk conditions, lower molecular mass is obtained. The reason is because there is an increase in the viscosity, there are differences in the matrix swelling
139 where the enzyme is immobilized and therefore there is a difference in the enzyme activity.
(A) (B) 25000 2.0
20000 1.8 ) 15000 1.6 M mol Đ (g/
n 10000 1.4 M
5000 1.2
0 1.0 0 200 400 600 Feed Ratio
Series1Mn Mw/MnĐM
Figure 5.5. (A) SEC results of CALB catalyzed ROP of -CL using propargyl alcohol as an initiator with different feed ratios. (B) Number-average molecular mass (Mn) and ĐM as a function of feed ratio [monomer] / [initiator] at 70 ºC.
Table 5.1 summarizes the conditions and results for the propargyl initiate ROP of
-CL carried out at 70ºC. CALB was used as catalyst. Well-defined PCL in the Mn range of 8,000 to 24,000 g/mol and dispersity indices (ĐM = 1.1-1.6) were obtained.
Table 5.1. Characteristics of the alkyne-PCL polymers obtained under different conditions after 12h of reaction.
a a System Ratio Mn ĐM %Functionality %Conversion [monomer] / (g/mol) [initiator] 1c 50 14,900 1.46 96 53 1'd 50 8,000 1.12 97 69 2 100 16,700 1.38 93.0 76 3 200 21,200 1.55 94.0 83 4 300 22,800 1.58 99.0 72 5 500 21,900 1.57 88.0 64 a Determined by SEC; b Determined by 1H NMR; c Polymerization of -CL in toluene; d Polymerization of -CL in bulk
140 This indicates these polymerizations are well controlled and exhibit linear growth of the average number molecular weight (Mn) respect to the feed ratio. The end- functionality is clearly evidenced and quantified by the characteristic peak of the alkyne proton at 2.45 ppm.
The alkyne-PCL functionalized obtained through the ROP of -CL initiated by propargyl alcohol can be further functionalized using copper-catalyzed click chemistry.
However, using conventional methodology for the polymer post-functionalization can restrict its application due to the copper catalyst cytotoxicity. Recently, Bertozzi et al has introduced a copper-free click reaction to overcome this limitation, the strain-promoted azide-alkyne [3+2] cycloaddition, that exploits the spontaneous reactivity of cyclooctynes toward an azide originating from its ring strain.205–208 In a previous report, we describe a polymerization method utilizing 4-dibenzocyclooctynol (DIBO compound 4) as an initiator for the ring-opening polymerization of -caprolactone catalysed by stannous octoate.30 Using this as motivation, we investigated the ROP of -CL using as initiator
DIBO and CALB as catalyst.
5.1.3. DIBO Alcohol Initiator for ROP of -CL
When using the DIBO compound 4 as initiator not polymerization occur. The proposed mechanisms for the lipase catalysed polymerization of lactones proceeded via an acyl-enzyme intermediate (enzyme-activated monomer, EM) at a serine residue of the catalytic site of lipase as the principal reaction course. The key step is the reaction of the lactone with lipase involving the ring opening of the lactone to produce the acyl-enzyme intermediate (EM).11,42 The initiation is the nucleophilic attack by the alkyne alcohol on
141 the acyl carbon of the intermediate, which is regarded as the basic propagation species.
That is, the lipase-catalyzed polymerization proceeds via a monomer-activated mechanism. In this case, the initiator 4 is a bulky secondary alcohol, due to steric effects the alcohol cannot access to the EM and therefore not initiation is achieved. For that reason we proceed to the synthesis of the initiator DIBO compound 6 that contains a primary alcohol. The DIBO-PCL was synthesized as described in Figure 5.6. The strained hydroxy-derivatized DIBO precursor was synthesized according to previously described methods.40,41 The DIBO was derivatized with p-nitrophenyl chloroformate and further reacted with excess 3-aminopropan-1-ol to yield the primary hydroxy-derivatized
DIBO compound 6.
Figure 5.6. CALB catalyzed ROP of -CL using as initiator dibenzocyclooctynol. For the case [monomer]/ [initiator] = 500. [DIBO] = 7.8×10-3 mol/L, [-CL] = 3.9 mol/L; [CALB] = 7.9× 10-5 mol/L.
Figure 5.7 shows the 1H NMR spectrum of the DIBO initiator (top) and resulting
PCL polymer (bottom). The retention of the phenyl resonances (f, g and h) (=7.46-7.18 ppm), the methylene (CH2) from the strained ring (=2.9 ppm and 3.1 ppm), and the methane proton (d) at =5.45 ppm shows that DIBO successfully initiated the polymerization of PCL, and survived intact during the polymerization process. The
142 functionalization was determined by the ratio of the methylene protons of PCL to chain- end units at δ = 3.62 ppm and =5.45 ppm (d), in this case 39%. The incomplete functionalization is due to some contaminants in the initiator and water initiation. When comparing the two functional nucleophiles, propargyl alcohol and DIBO 6, the incorporation of the initiator is observed to be different. A non-bulky functional nucleophile like propargyl alcohol yields 98% incorporation into the PCL polymer. On the other hand, the bulky alcohol 6 is incorporated very slowly, giving only 36% of incorporation, while the 64% of the chains are water initiated. It is possible that the hydroxy-derivatized DIBO linker is not long enough and there is still some steric hindrance of the initiator to access the active-pocket of the enzyme
143
Figure 5.7. 1H NMR spectrum: (Top) primary hydroxyl-derivatized DIBO, (Bottom) Aliquot at 4h of the reaction CALB catalyzed ROP of -CL using as initiator DIBO [monomer]/ [initiator]= 500 (500 MHz, solvent: CDCl3).
144
Figure 5.8. (A) SEC results of CALB catalyzed ROP of -CL using as initiator DIBO for the case [monomer]/ [initiator]= 500:1 for aliquots at 4 h, 8 h, 12 h and 24 h. (B) SEC chromatogram and number-average molecular mass (Mn) and ĐM as a function of time at 70ºC bulk and solution system. (C) SEC results of CALB catalyzed ROP of -CL using as initiator DIBO for the case [monomer]/ [initiator]= 50:1 at 12 h in bulk and solution conditions. (D) UV spectrum of the reaction aliquot at 4h. CALB catalyzed ROP of -CL using as initiator DIBO [monomer]/ [initiator]= 500 (solvent: THF).
Aliquots were taken at certain intervals for the reaction with a monomer/ initiator ratio equal to 500. Figure 5.8A-B shows the chromatogram for the DIBO-PCL aliquots and the plot of Mn vs time. Molecular mass and molecular mass distribution of PCL initiated with DIBO show nearly linear growth kinetics with increasing polymerization time while maintaining relatively narrow molecular mass distribution. SEC chromatogram as a function of polymerization time exhibit mono-modal molecular mass distributions.
145 To confirm the survival of the strained DIBO group following the polymerization process, the polymer was dissolved in THF, and UV-vis spectrum of the solutions showed the π–π* optical transition at 306nm, which correspond to the alkyne group in
DIBO (Figure 5.8D).
The polymerization was also performed in bulk conditions. Figure 5.8C shows the
SEC eluogram for the DIBO-PCL product in bulk and toluene conditions and the plot of
Mn vs time. Using bulk conditions, lower molecular mass is obtained. The lower molecular mass tendency was similar to that observed in the case of the ROP initiated by propargyl alcohol.
Table 5.2 summarizes the conditions and results for DIBO initiate ROP of -CL carried out at 70 ºC. CALB was used as catalyst. Well-defined DIBO-PCL in the Mn range of 5,000 to 14,000 g/mol and dispersity indices (ĐM= 1.19-1.54) were obtained.
This indicates these polymerizations are well controlled and exhibit linear growth of the average number molecular mass (Mn) with respect to time.
Table 5.2. Characteristics of the DIBO-PCL polymers obtained under different conditions
Bulk Toluene a a b a a b t h Mn ĐM %Conversion Mn ĐM %Conversion (g/mol) 4 5,500 1.19 37 6,100 1.41 26 8 7,200 1.32 55 11,000 1.31 31 12 8,400 1.41 60 12,000 1.48 34 24 9,200 1.32 74 14,000 1.54 55 a Determined by SEC; b Determined by gravimetric measurements.
146 5.1.4. Conclusions
Alkyne functionalized poly(caprolactone) (PCL) was synthesized by CALB- catalyzed Ring-Opening Polymerization of -CL using propargyl alcohol and 4- dibenzocyclooctynol (DIBO) as initiators. A very high extent of the alkyne functionality is retained post-polymerization. The combination of enzymatic polymerization and functional initiating systems resulted in the metal free synthesis of poly(caprolactone) which normally requires the use of tin or other transition metal which is difficult to remove. This methodology can be expanded to synthesis of additional cyclic monomers to yield functional biomaterials containing the degradable polyester unit.
The alkyne-PCL functionalized materials can be further functionalized using conventional copper-catalyzed or strain-promoted azide alkyne cycloaddition.
Functionalization of PCL with a furan group was also explored. In this case the polymer can be post-functionalized using Diels Alder reaction.
5.1.5. ROP of -Caprolactone Using Furfurylamine as Initiator
Figure 5.9. CALB catalyzed ROP of -CL using as initiator furfurylamine. [monomer]/ [initiator] = 20. [Furfurylamine] = 0.26 mol/L, [-CL] = 4.61 mol/L; [CALB] = 2.23×10-3 mol/L.
Dry toluene (3.0 mL) and furfurylamine (0.15 g) were transferred via syringe under dry N2(g) into a flask containing CALB (150 mg) (Figure 5.9). This suspension, as well as a separate flask containing ε-CL, was equilibrated for 15 min to the reaction
147 temperature (70°C). Thereafter, ε-CL (3.0 mL) was transferred to the reaction flask via syringe under dry N2(g) to start the polymerization. The polymerization proceeds for 24 h.
The number-average molecular weight of the polymer was determined by SEC
(Figure 5.10) as Mn =4,153 g/mol (ĐM = 1.23).
Figure 5.10. SEC results of CALB catalyzed ROP of -CL using as initiator furfurylamine. [monomer]/ [initiator] = 20. [Furfurylamine] = 0.26 mol/L, [-CL] = 4.61 mol/L; [CALB] = 2.23×10-3 mol/L.
Figure 5.11 shows the 1H NMR spectrum of the product. Signals for structural analysis included those at 3.68 ppm (t, J 6.5 Hz,−[C-O]−OCHH2CH2−OH), due to methylene protons of ε-CL to chain-end units; resonances at 3.99 ppm (t, 6.5 Hz, −[C-
O]−OCHH2CH2−), due to PCL repeat units along chains. The ratios of signals at 3.99 ppm to 3.68 ppm were used to calculate the Mn, respectively. The signals of the furfural moiety appear at the expected positions, confirming a polycaprolactone (PCL) end- capped with furfurylamine. The Mn of the polymer was calculated from the ratio of the relative integral of methylene protons of the repeat unit to the methylene protons of ε-CL
148 to chain-end units at δ = 3.68 ppm (c) to that of the backbone methylene protons at δ =
1 3.99 ppm (b). Mn calculated by the NMR is 4,618 g/mol. The Mn obtained from H NMR was in good agreement with SEC results.
Figure 5.11. NMR spectra of the product of the CALB catalyzed ROP of -CL using as initiator furfurylamine. (Top) 1H NMR spectrum and (bottom) 13C NMR spectrum (500 MHz, solvent: CDCl3).
Additionally the synthesis of a poly(isobutylene-b--caprolactone) block copolymer and poly(-caprolactone-b-isobutylene-b--caprolactone) triblock copolymers
149 was accomplished by the combination of living carbocationic polymerization of isobutylene (IB) with the ring-opening polymerization (ROP) of -caprolactone (-CL).
5.2. Synthesis of Poly(isobutylene-b--caprolactone) and Poly(-caprolactone-b-
isobutylene-b--caprolactone) Using Enzyme Catalysis
5.2.1. Introduction
The design of novel macromolecular architectures is a continuous focus in polymer science. Many of these architectures, such as block copolymers, possess unique properties which make them interesting candidates for special applications in nanotechnology and biomedical materials. Controlled ring-opening polymerization
(ROP) of cyclic esters, such as lactide, glycolide, cyclic carbonate, and/or -caprolactone
(-CL), have received significant attention due to the good mechanical properties, degradation behavior and biocompatibility of the resulting polymers.194,209,190
Polyisobutylene (PIB) has commercial utility as a stabilizing fuel and motor oil additive, packaging elastomer, adhesive and sealant, and more recently, as a biomaterial.
This saturated hydrocarbon elastomer has excellent thermal and oxidative stability, gas barrier properties, and biocompatibility.154 Additionally there have been very successful materials developed that have harnessed the advantages and versatility of this polymer.
154,210,155 This is the case of poly(styrene-block-isobutylene-block-styrene) that has successfully been used as the drug-eluting coating on Boston Scientific’s TAXUSs stents.155 The same polymer is being investigated for ophthalmic implants to treat glaucoma, synthetic heart valves and other applications.210 PIB is not degradable under biological conditions, however, its copolymers can be. Biodegradable elastomers have a number of potential applications, especially in the emerging field of soft-tissue
150 engineering where the mechanical properties of the polymer scaffold should match those of the tissue to be grown. Block copolymers of PIB with L-lactide and pivalolactone have been synthesized from primary hydroxyl functionalized PIBs and metal-containing activators. It was found that the blocks had phase separated morphologies, and the crystallization behavior of the polylactide and polypivalolactone was influenced by the presence of the PIB blocks. However, we found only two papers discussing the synthesis of PIB-P(-CL) block copolymers. Both papers used telechelic HO-PIB-OH macroinitiators obtained by multistep processes, and triethyl aluminum or HCl·Et2O. The first paper concentrated on structural analysis. The second paper studied the microphase separation by DSC and found two transitions (Tg=-60 ºC for the PIB segment and Tm =
60 ºC for the PCL block).211 Both papers reported that the anionic ROP is -CL is plagued by backbiting and transesterification reactions
This section reports the synthesis of a poly(isobutylene-b--caprolactone) diblock and poly(-caprolactone-b-isobutylene-b--caprolactone) triblock copolymers using PIB-
OH and HO-PIB-OH. ROP of -CL catalyzed by CALB. Enzyme-catalyzed ROP is one of the most promising tools and avoids the use of organo-metallic catalysts (Zn, Al, Sn or
Ge) which are known to be cytotoxic to cellular systems and are often difficult to remove from polymeric products.209 However, when Gross and Hillmyer used anionically synthesized hydroxyl-functional polybutadiene of various molecular weights (Mn ~ 2,600
– 19,000 g/mol) to initiate the polymerization of caprolactone (-CL) and pentadecalactone (PDL) by CALB, water initiation led to homoPCL that needed to be separated from the block copolymer. We report conditions leading to pure di- and triblock copolymers.
151 5.2.2. Synthesis of Poly(isobutylene-b--caprolactone) Diblock
The enzyme-catalyzed ROP of -CL was initiated by the HO-PIB-allyl macroinitiator as shown in Figure 5.12. In this case [monomer]/ [macroinitiator]= 40 was used based on Storey report. A higher ratio was used but in this case not clean diblock was obtained.
Figure 5.12. CALB catalyzed ROP of -CL using allyl-PIB-OH as macroinitiator. For the case [monomer]/ [macroinitiator]= 40. [HO-PIB-allyl] = 9.02x10-3 mol/L, [-CL] = 0.36 mol/L; [CALB] = 9.02 × 10-5 mol/L.
HO-PIB-allyl was synthesized by Alejandra Alvarez in one pot following the route shown in Figure 5.13. The resulting material had a number average molecular mass of Mn = 4,300 g/mol with ĐM = 1.21.
Figure 5.13. Carbocationic polymerization of isobutylene initiated by propylene oxide/TiCl4 and its allylation by allyltrimethylsilane. [Propylene oxide]=0.015 M, [IB]=1.000 M, [TiCl4]=0.030 M, [DtBP]=0.007 M, [ATMS]=0.045 M, T = -80 °C, Hx/MeCl=60/40 v/v, 10 minutes.
Figure 5.14 shows the 1H NMR spectrum of the HO-PIB-allyl. In that spectrum, the proton resonances at δ=1.13 ppm and δ=1.45 ppm correspond to the methyl and methylene protons respectively of the repeat units (HO-PIB-allyl). Resonances at δ=5.04
152 ppm (e) and δ=5.77 ppm (f) confirm the presence of allylic end groups in the polymer.
The proton resonances at δ=3.33 and δ=3.51 ppm were assigned to the protons next to primary hydroxyl group (-CH2-OH) (a and a’). The splitting pattern also supports the assignments because the (-CH2-OH) signal is split into doublet of quadruplets, which is in
212,136 agreement with other NMRs of PIBs synthesized by the epoxy initiators. The Mn =
4,236 g/mol of the polymer was calculated from the integral ratio of the protons of the allyl end groups [δ = 5.77 ppm (e)] to the backbone methylene protons [δ = 1.43ppm (b)],
13 which is in good agreement to Mn detected by SEC (Mn = 4,300 g/mol). The C NMR also verified the expected structure.
1 Figure 5.14. H spectrum of HO-PIB-allyl (solvent: CDCl3) clearly showing the presence of the allyl group at the polymer chain end.
Figure 5.15 shows the SEC traces for the block copolymer (PIB-b-PCL). Relative to the starting HO-PIB-allyl (Mn = 4,300 g/mol), the SEC RI traces of PIB-b-PCL diblock copolymers shifted to higher molecular mass and the molecular mass distribution remained narrow. The Mn =6,058 g/mol and ĐM= 1.26 were determined by SEC using
153 dn/dc= 0.093 (Copolymer dn/dc was calculated based on the weight fraction [%wt
PIB=0.7] and dn/dc of the individual components). This would correspond to a PIB4300-
1 b-PCL1758 structure. Figure 5.16 shows the H NMR spectrum of PIB-b-PCL. The resonances at δ=5.04 ppm (e) and δ=5.77 ppm (f) belong to the allylic protons of HO-
PIB-allyl. Signals for structural analysis included those at 3.68 (t, J 6.5 Hz,-[C-O]-
(CH2)4CH2-OH), due to methylene protons of PCL chain-end units (h); resonances at
3.99-4.11 ppm (g) (m, -[C-O]-(CH2)4CH2-O)), due to PCL repeat unit. The Mn of the poly(-caprolactone) block was calculated from the ratio of the relative integral of methylene protons of the repeat unit of PCL (g) at =3.99-4.11 ppm to the allyl-end units of HO-PIB-allyl (f) at = 5.77 ppm (c) Mn calculated by the NMR is. The Mn = 1,590 g/mol obtained from 1H NMR is in good agreement with the SEC results.
1.2
1.0
PIB-PCL 0.8 allyl-PIB-OH
0.6
0.4 Relative Relative scale RI
0.2
0.0 30 40 50 60 70 Time (min)
Figure 5.15. SEC traces of PIB-b-PCL:[monomer]/ [macroinitiator]= 40. [allyl-PIB-OH] = 0.0090 mol/L, [-CL] = 0.36 mol/L; [CALB] = 0.0001 mol/L.
154
1 Figure 5.16. H NMR spectra of PIB-b-PCL (solvent: CDCl3).
5.2.3. Synthesis of Poly(isobutylene-b--caprolactone) Triblock
Figure 5.17 shows the triblock synthesis. HO-PIB-OH was synthesized from allyl-
PIB-allyl by click reaction with mercaptoethanol as reported in Chapter VII.
Figure 5.17. CALB catalyzed ROP of -CL using as macroinitiator HO-PIB-OH. For the case [monomer]/ [initiator]= 20. [HO-PIB-OH] = 0.26 mol/L, [-CL] = 5.2 mol/L; [CALB] = 1.31 × 10-4 mol/L.
Figure 5.18 shows the SEC trace of the triblock copolymer (PCL-b-PIB-b-PCL).
The SEC RI traces of PCL-b-PIB-b-PCL triblock copolymer shifted to higher molecular weight and the molecular weight distribution is broad. The number average molecular mass (Mn) of the polymer was determined by SEC as Mn =8,398 g/mol (PCL2156-b-
155 PIB4085-b-PCL2156) and ĐM =1.48. Using dn/dc= 0.073 (Copolymer dn/dc was calculated based on the weight fraction [%wt PIB=0.37] and dn/dc of the individual components).
Figure 5.18. SEC traces of poly(--caprolactone-b-isobutylene-b--caprolactone): [monomer]/ [initiator]= 20. [HO-PIB-OH] = 0.26 mol/L, [-CL] = 5.2 mol/L; [CALB] = 1.31 × 10-4 mol/L.
Figure 5.19 shows the 1H NMR spectrum of PCL-PIB-PCL. The signal of methylene protons (g) (-CH2-OH) at 3.77 ppm disappeared. Signals for structural analysis included those at 3.65 (t, J 6.5 Hz,-[C-O]-OCHCH2CH2-OH), due to methylene protons of PCL chain-end units; resonances at 3.99-4.11ppm (h) (m, -[C-O]-
OCHCH2CH2-), due to PCL repeat unit. The proton resonances at δ=1.13 ppm (e) and
δ=1.45 ppm (d) correspond to the methyl and methylene protons respectively of the repeat unit of PIB. Knowing the number average molar mass of the starting HO-
PIBOH by SEC analysis, from the ratio of the relative integral of methylene protons of the repeat unit of PCL (h) at =3.99-4.11 ppm to the methyl protons of the repeat unit of
PIB (e), the DP of PCL segment of the triblock copolymer can be calculated. From these data the number average molar mass of PCL blocks calculated by the 1H NMR
156 1 is 2,204 g/mol. The Mn obtained from H NMR was in good agreement with SEC results.
This indicates that on average, the 4,000 g/mol PIB center block is flanked on either side by 2,200 g/mol PCl outer blocks (PCL2200-b-PIB4085-b-PCL2200).
Figure 5.19. 1H NMR spectra poly(--caprolactone-b-isobutylene-b--caprolactone) (solvent: CDCl3).
5.2.4. Microphase Separation Analysis
DSC thermograms of samples of PCL homopolymer, allyl-PIB-OH homopolymer and PIB-b-PCL diblock copolymers are presented in Figure 5.20. In the heating scan the diblock copolymer exhibited the Tg of amorphous rubbery PIB segment around -70 °C and the Tm of PCL segment around 45 °C. The shape of the curve around the Tg can be explained based on the cooling rate used, this shape corresponds to the fastest cooling in the second cycle of DSC.213 This clearly indicated microphase separation in the block copolymers. This microphase separation is also visualized in the transmission electron micrograph (Figure 5.21).
157 PIB-b-PCL
PCL-b-PIB-b-PCL
Figure 5.20. DSC thermograms at a heating rate of 10 ºC/min for PIB-b-PCL and PCL- b- PIB-b-PCL.
Figure 5.21. TEM pictures for A-B: PIB-b-PCL diblock. C-D PCL-b-PIB-b-PCL Triblock. Staining: 1% OsO4 showing the microphase separation.
Figure 5.22 shows the Optical images of PIB-b-PCL diblock, reflected light and the AFM Tapping mode height and amplitude images (Figure 5.23). It can be observed from the optical images that there is a change in the crystallization behavior of PCL block due to the presence of the PIB block.
In the case of the triblock microphase separation is clearly demonstrated in the transmission electron micrograph (Figure 5.21). The film was stained with
OsO4, therefore the black dots belong to the poly(caprolactone) domains, i. e., the
158 OsO4, accumulates in greater amount in the polar than in the apolar phase. The
DSC measurement (Figure 5.20) shows two glass transitions (Tg= -67 ºC characteristic of polyisobutylene segment and Tm = 52 ºC (characteristic of poly(caprolactone) segment), and this is a strong support of microphase separation.
500 m A 500 m C E F
50 m B 50 m D
Figure 5.22. A-B: Optical images of PIB-b-PCL diblock, reflected light. C-D: Optical images of PCL-b-PIB-b-PCL Triblock, reflected light. Optical Images for PCL reported 214 in the literature (E) PCL with Mn=1,900 g/mol and (F) PCL with Mn=6,700 g/mol.
Figure 5.22 shows the Optical images of PCL-b-PIB-b-PCL Triblock, reflected light and the AFM Tapping mode height and amplitude images (Figure 5.23). It can be observed from the optical images that there is a change in the crystallization behavior of
PCL block due to the presence of the PIB block.
159 AA C
D B
0 5 m 5 m 500 nm 2 m
E F
G
5 m 0 500 nm 2 m
Figure 5.23. A-D: AFM Tapping mode height for diblock. E-F: AFM Tapping mode height triblock showing the microphase separation.
5.2.4.1. Conclusion
The combination of enzymatic polymerization and functional initiating systems resulted in the metal free synthesis of poly(caprolactone) which normally requires the use of tin or other transition metal which is difficult to remove. This methodology can be
160 expanded to synthesis of additional cyclic monomers to yield functional biomaterials containing the degradable polyester unit.
5.3. Enzyme-Catalyzed Quantitative Chain-End Functionalization of Poly(ethylene
glycol)s Under Solventless Conditions
Vinyl ester functionalized telechelic PEG building blocks are very attractive for further transformations. Transesterification of vinyl ester-PEGs with functionalized alcohols would lead to new functional groups with high efficiency: the vinyl alcohol product immediately tautomerizes into acetaldehyde, rendering the reaction irreversible as shown in Figure 5.24.
Figure 5.24. Transesterification of vinyl ester-PEG with functionalized alcohols. The red ball represents functional groups derived from the alcohol.
We set out to investigate the transesterification of divinyl adipate (DVA) with
PEGs without the use of solvents. The polycondensation of DVA with various diols via enzyme catalysis in various solvents to produce polyesters has extensively been investigated and reviewed.39,41,47,179 Based on experimental data and a mathematical model it was concluded that the polymerizations occurred by a step-condensation mechanism and the rate of polymerization decreased with increasing alcohol molecular weight.179 The transesterification of DVA with 1,4-butanediol in bulk catalyzed by
CALB yielded the corresponding polyester with Mn=8,273 g/mol and ĐM = 2.78.
161 Hydrolysis of the DVA invariably occurred so the product had a mixture of vinyl- and
HO- end groups. The cause of the hydrolysis was believed to be the release of water attached to the enzyme. The presence of a solvent such as tetrahydrofuran THF was found to promote this process, leading to more hydrolysis.179
5.3.1. Synthesis of V-PEG-V2000
Figure 5.25. Reaction of DVA with HO-PEG-OH2000 in the presence of CALB under solventless conditions. [DVA] = 5.29 mol/L, [HO-PEG-OH2000] = 0.26 mol/L; [CALB] = 1.6 × 10-4 mol/L.
HO-PEG-OH2000 was reacted with 20 molar equivalent of DVA in the presence of
CALB under solventless conditions (Figure 5.25). In this case, no polycondensation was observed, this is because the large excess of DVA and the molecular weight of the diol.
In the MALDI-ToF mass spectrum of the product (Figure 5.26), the representative peak at m/z 2242.44 corresponds to the sodium complex of the 43-mer of telechelic V-PEG-
V2000. The calculated monoisotopic mass for this peak [m/z = 43 × 44.03 (C2H4O repeat
+ unit) + 326.14 (C16H22O7 end groups) + 22.99 (Na )] is 2242.42 Da. Traces of the potassium complex can also be seen in the spectra.
162
Figure 5.26. MALDI- ToF mass spectrum of the product of the reaction of DVA with HO-PEG-OH2000 at 4 hours.
The 1H NMR spectrum (Figure 5.27) shows the vinyl protons of the end groups
[δ=4.87 (e) and δ=4.65 (e’) ppm, and δ=7.26 ppm (f)], the adipic ester groups [δ=2.32 (i)
163 and δ=2.43 (g) ppm, and δ=1.56 ppm (h)], and the HO-PEG-OH2000 repeat units [δ=4.11 ppm (b), δ=3.59 ppm (c), and δ=3.50 ppm (d)].
Figure 5.27. NMR spectra of the product of the reaction of DVA with HO-PEG-OH2000: 1 13 H NMR (top) and C NMR (bottom) (solvent: DMSO-D6).
The 13C NMR spectrum (Figure 5.27) shows the vinyl groups [δ=141.17 ppm (F) and δ=97.97 ppm (E)], the carbonyl carbons resonances of adipic ester groups [δ=172.59 ppm (K) and δ=170.17 ppm (G)] and the HO-PEG-OH2000 units [δ=63.05 ppm (B),
δ=68.26 ppm (C), and δ=69.98 ppm (D)]. The resonance of the carbons connected to the hydroxyl groups in the starting HO-PEG-OH2000 shifted downfield to δ=63.05 ppm (B)
164 and new carbon resonances of the vinyl groups (F and E) and carbonyl groups (G and K) appeared at the expected positions, confirming the structure of the product.
5.3.2. CALB-catalyzed polycondensation of DVA with Tetraethylenglycol
Figure 5.28. CALB-catalyzed transesterification of Divinyladipate with Tetraethylenglycol. Polycondensation products.
Figure 5.28 shows the polycondensation products obtained when DVA is reacted with TEG at two different conditions: when DVA is in excess (condition 1) and when
TEG is in excess (condition 2). The percent of these oligomers will depend on the conditions employed.
165
Figure 5.29. Transesterification of DVA with TEG: first cycle.
The catalytic cycle of the CALB-catalyzed transesterification of DVA with TEG is visualized in Figure 5.29, based on the mechanism described in the literature for monofunctional vinyl esters with alcohols.31,136,215 The different shadings in our rendition represent the carbonyl (top) and hydroxyl (bottom) pockets of the enzyme. First, the nucleophilic serine (Ser105) residue interacts with one of the carbonyl groups of the
166 DVA, forming a tetrahedral intermediate which is stabilized by the oxyanion hole of the enzyme via three hydrogen bonds: one from glutamine (Gln106) and two from threonine
(Thr40) units. In the second step, the ester bond is cleaved to form the first product, vinyl alcohol which will tautomerize to acetaldehyde, and the acyl-enzyme complex. In the third step, one of the HO- groups of TEG reacts with the acyl-enzyme complex to form a second tetrahedral intermediate which is again stabilized by the oxyanion hole. In the last step, the enzyme is deacylated to form the desired product. The HO-TEG-V can react with an activated DVA, yielding difunctional V-TEG-V. The vinyl ester group of HO-
TEG-V can also be activated by the enzyme. This complex then can react with another
HO-TEG-OH, yielding a dimer with OH end groups. The simplified scheme is shown in
Figure 5.30. Higher oligomers can also be formed in this stepwise manner. We investigated the TEG – DVA reaction using MALDI-ToF.
Figure 5.30. Simplified scheme of the polycondensation reaction of the transesterification of Divinyladipate with Tetraethylenglycol.
5.3.2.1. Effect of DVA Concentration
TEG was reacted with 1, 1.5 and 3 molar equivalent of DVA for 30 minutes and
Table 5.3 summarizes the results.
167 Table 5.3. Effect of DVA/TEG molar ratio on the oligomerization of TEG
Composition (%) by MALDI-ToF DVA/TEG molar ratio 1 1.5 3.0
V-TEG-V, 0
n=0 VO Dimer, n=1 0 Vinyl-[TEG-DVA] -TEG- n Trimer, n=2 6.11 OH Tetramer, n=3 7.89 Higher 22.13 Total 36.13 V-TEG-V, 0 0 42.5 n=0 VV Dimer, n=1 0 37.59 35.5 Vinyl-[TEG-DVA] -TEG- n Trimer, n=2 14.20 27.57 17.7 Vinyl Tetramer, n=3 14.29 14.29 3.8 Higher 0.5 20.55 0.5 Total 63.88 100 100 Grand total 100 100 100 -4 -4 -4 [CALB] = 2.77 × 10 mol/L1.0 eq., 2.19 × 10 mol/L1.5 eq., 1.35 × 10 mol/L3.0 eq.
VO stands for Vinyl-(TEG)n-OH and VV stands for Vinyl-(TEG)n-Vinyl structures. Dihydroxy telechelics HO-(TEG)n-OH (OO) were not detected in any of the reactions, indicating that this intermediate disappears very quickly. VO oligomers
(36.13%) were detected only at 1/1 molar ratio. The ratio of VV/VO = 2 at 1/1 molar ratio indicates that the rate of reaction between VV and VO structures is twice as fast than between VO structures. At excess DVA (1.5 and 3 molar equivalent) only VV structures were detected.
5.3.2.2. Reaction Kinetics
It can be seen from the data in Table 5.3 that in the case of DVA excess only vinyl end groups were detected in all oligomers. To get a better understanding of the mechanism, we investigated the time-dependence of five reactions: one at equimolar
168 DVA/TEG ratio, two with excess DVA (1.5 and 3 molar equivalent), and two with excess
TEG (1.5 and 3 molar equivalent). Samples were taken at the specified times and the composition and end group structures were obtained by MALDI-ToF. After 5 minutes the starting TEG was not detected in any of the reactions, indicating very quick conversion of the TEG. Table 5.4 summarizes the data at DVA/TEG 1/1 molar ratio.
Table 5.4. Oligomer compositions with DVA/TEG 1/1
DVA/TEG = 1/1 Composition (%) by MALDI-ToF Time (min)
5.0 10 15 20 25 30 V-TEG-O 0 0 0 0 0 0 Dimer, n=1 5.86 7.45 5.57 2.55 3.68 0 Trimer, n=2 8.56 12.38 10.68 9.18 10.29 6.11 Tetramer, n=3 8.26 9.42 10.57 7.14 10.66 7.89 Pentamer, n=4 4.35 6.33 6.7 3.57 6.25 7.62 VO Higher 5.98 3.66 3.98 3.06 3.68 5.72 Vinyl-[TEG- 2.11 2.16 1.02 2.57 3.98 DVA]n-TEG-OH 1.27 1.25 0.51 1.84 2.61 0.56 0.8 0.51 0.74 1.31 0.28 0.23 - 0.74 0.53 - - - - 0.36 Total 33.03 43.46 41.94 27.54 40.45 36.13 OO Not detected V-TEG-V, n=0 10.06 7.74 5.68 3.57 2.57 0 VV Dimer, n=1 16.82 14.91 16.25 17.86 12.5 0 Vinyl-[TEG- Trimer, n=2 16.52 15.19 17.16 21.43 19.12 14.2 DVA]n-TEG- Tetramer, n=3 9.76 9.14 9.32 13.78 11.03 14.29 Vinyl Higher 6.16 4.78 5 7.65 6.99 12.55 3.9 2.39 2.61 4.08 3.68 9.7 2.55 1.41 1.25 2.55 2.21 6.49 0.75 0.7 0.57 1.02 1.1 2.08 0.3 0.28 0.23 0.51 0.37 2.4 0.15 - - - - 1.47 - - - - - 0.43 - - - - - 0.17 Total 66.97 56.54 58.07 72.45 59.57 63.88 Total 100 100 100 100 100 100
169 At 5 minutes no V-TEG-O was detected, indicating a very fast first cycle (large k1 in Figure 5.30). Oligomers up to undecamer were found. The amount of oligomers with
VV end groups was twice as much than those of VO end groups, thus the reaction of VV oligomers with VO oligomers is twice as faster than the reaction between VO oligomers.
Table 5.5. Oligomer compositions with DVA/TEG 1.5/1 and 3/1.
DVA/TEG = 1.5/1 Composition (%) by MALDI-ToF Time (min)
5.0 10 15 20 25 30 Trimer, n=2 1.86 4.71 0.876 - - - VO Tetramer, n=3 0.63 2.23 0.292 - - - Vinyl-[TEG- Higher - 0.992 - - - - DVA]n-TEG-OH - 0.248 - - - - Total 2.49 8.18 1.168 0 0 0 OO Not detected Dimer, n=1 46.27 37.47 43.73 40.65 38.26 37.59 Trimer, n=2 26.4 24.57 26.82 23.57 23.47 27.57 VV Tetramer, n=3 13.97 12.66 14.577 15.71 14.8 14.29 Vinyl-[TEG- Higher 6.83 7.94 6.12 8.4 9.18 9.02 DVA]n-TEG- 2.8 4.96 4.37 5.96 6.12 5.76 Vinyl 1.24 2.73 2.33 3.53 4.34 3.51 - 1.24 0.875 1.64 2.55 1.75 - 0.248 - 0.54 1.276 0.51 Total 97.51 91.818 98.822 100 100 100 Total 100 100 100 100 100 100
Composition (%) by MALDI-ToF DVA/TEG = 3/1 Time (min) 5 10 15 20 25 30 VO Not detected OO Not detected V-TEG-V, n=0 42.86 51.47 49.5 44.19 42.6 38.61 VV Dimer, n=1 27.27 28.30 29.1 31.29 29.56 29.17 Vinyl-[TEG- Trimer, n=2 18.51 13.97 14.04 17.10 17.97 18.89 DVA]n-TEG- Tetramer, n=3 8.10 5.15 5.69 6.13 7.54 9.17 Vinyl Higher 2.60 1.10 1.34 1.29 2.03 3.33 0.65 - 0.33 - 0.29 0.83 Total 100 100 100 100 100 100
170 Table 5.5 shows the oligomer compositions with DVA/TEG 1.5/1 and 3/1. In the case of 1.5 DVA excess OO groups were not detected at all, and no VO end groups were detected after 20 minutes. In the case of the 3 eq DVA the composition of the 5 minute sample is very similar to that of the 30 min sample. Oligomers up to hexamer were detected, with the V-TEG-V species being the most abundant. All oligomers had vinyl end groups.
Table 4 shows the product compositions in case of excess TEG. The oligomer composition now is much more complex and changes with time. At 5 minutes reaction time no TEG starting material was detected, and ~60% of the oligomers had only OH end groups (OO), while ~40% had VO end groups. At 30 minutes reaction time less than
12% of the oligomers had a vinyl group and 35.4% of the product was a TEG dimer connected with DVA. Figure 5.31 shows the change of end group composition with time.
At 1.5 eq excess TEG at 5 minutes 40% VO and 60% OO was detected. At 10 minutes
70% VO, 10% OO and 20% VV were seen.
At 30 minutes no VV is detected, with 90% OO and 15% VO. At 3 excess of
TEG the composition is very similar with ~12% VO and 88% OO.
171
Figure 5.31. End group composition (%) vs. reaction time (min) in the case of TEG excess
Table 5.6. Reaction kinetics in case of excess TEG. TEG /DVA= 1.5/1 Composition (%) by MALDI-ToF Time (min)
5.0 10 15 20 25 30 VO Trimer, n=2 20.73 27.42 11.1 Vinyl-[TEG- Tetramer, n=3 10.46 16.09 7.64 3.95 3.16 3.36 DVA]n-TEG-OH Pentamer, n=4 4.59 10.42 4.37 2.07 2.16 2.18
Table 5.6. Reaction kinetics in case of excess TEG (continued)
Higher 2.02 6.21 2.64 1.318 1.38 1.39 1.1 4.388 2 1.13 1.18 1.188 0.73 2.742 1.24 0.753 0.788 0.792
- 1.462 0.62 0.3766 0.39 0.396 - 0.914 0.31 9.5976 9.058 9.306 Total 39.643 69.644 29.92 9.611 9.42 9.364
172 Table 5.6. Reaction kinetics in case of excess TEG (continued)
Trimer, n=2 27.52 10.6 23.4 28.249 29.58 29.7 Tetramer, n=3 18.35 - 17.2 22.599 19.9 20.2 Pentamer, n=4 7.52 - 9.98 12.05 12.62 12.67 Higher 3.67 - 7.33 8.851 9.07 9.3 OO 1.83 - 4.37 6.967 7.1 6.14 HO-[TEG-DVA]n- 0.92 - 3.43 5.085 5.3 5.3 TEG-OH 0.55 - 2.8 3.578 3.47 3.76 - - 1.1 1.88 1.97 1.98 - - 0.468 1.13 1.18 1.19 - - - - 0.39 0.396 Total 60.357 0 70.078 90.389 90.58 90.636 Trimer, n=2 - 8.23 - - - - Tetramer, n=3 - 4.75 - - - - Pentamer, n=4 - 2.74 - - - - VV Higher - 1.645 - - - - Vinyl-[TEG- - 1.097 - - - - DVA]n-TEG-Vinyl - 0.548 - - - - - 0.366 - - - - - 0.366 - - - - Total 0 19.742 0 0 0 0 Total 100 100 100 100 100 100 Composition (%) by MALDI-ToF TEG/DVA = 3/1 Time (min) 5.0 10 15 20 25 30 Dimer, n=1 24.5 28.52 29.75 26.8 29.13 35.32 Trimer, n=2 19.2 20.8 22.1 23.87 25.59 24.82 OO Tetramer, n=3 13 12.9 14.37 18.8 19.88 15.75 HO-[TEG-DVA]n- Pentamer, n=4 6.17 6.94 7.49 9.94 10.83 8.35 TEG-OH Higher 3.17 3.3 3.24 4.34 4.33 3.1 0.82 1.16 1 1.63 1.38 0.95 0.3 0.38 0.4 0.35 0.2 0 VO V-TEG-OH, n=0 17.5 12.5 8.7 3.43 1.18 4.06 Vinyl-[TEG- Dimer, n=1 8.17 6.94 6.48 4.16 2.95 3.82 DVA]n-TEG-OH Trimer, n=2 4.67 3.67 3.64 3.61 2.75 2.15 Tetramer, n=3 2 1.93 2.02 2.17 1.18 1.43 Pentamer, n=4 0.5 0.77 0.61 0.9 0.59 0.24 Higher - 0.19 0.2 - - - Total 100 100 100 100 100 100
173 At DVA/TEG = 10, after 30 minutes 73 % of the product was V-TEG-V, with 23
% VV dimers and 4% vv trimers. At DVA/TEG = 20, 81% of the product was V-TEG-V along with 17% dimers and 2 trimers. At TEG/DVA =10, after 30 minutes 5% VO dimer,
86% OO dimer and 9% OO tetramer and 5% VO was detected and VV was not detected at all. At TEG/DVA =20 the composition was very similar (3% VO dimer, 93% OO dimer and 4% OO tetramer).
In conclusion, investigation of the kinetics of the enzyme-catalyzed reaction of
DVA with TEG allowed us to draw some mechanistic conclusions. With the judicious selection of molar equivalencies and reaction times both symmetric and asymmetric telechelic TEGs can be produced with high efficiency.
5.3.2.3. Conclusion
The data presented in this work demonstrate that enzyme catalysis is very effective in end-functionalizing PEGs in the absence of solvents and under dry nitrogen, producing very pure products as demonstrated by quantitative MALDI-ToF analysis. The excess DVA was recovered for reuse, according to green chemistry concepts. The vinyl groups enable further effective end-functionalization of PEGs and/or the attachment of drugs and proteins.
174 6. CHAPTER VI
CHEMO-ENZYMATIC FUNCTIONALIZATION OF BIOMOLECULES
Parts of this chapter were the preliminary results for a SBIR-Phase II proposal that was recently accepted.
6.1. Exclusive γ-Conjugated Folic Acid Derivatives Via Chemo-Enzymatic
Processes.
6.1.1. Introduction
Figure 6.1 shows the chemical structure of FA, consisting of a glutamic acid and a pteroate residue. The glutamic acid residue has two conjugation sites: the α- and the γ- carboxylic acid groups. Folate- targeted drugs will be linked via the γ-carboxyl rather than -carboxyl because the former exhibits higher affinity for FR than the latter.5
Furthermore, successful endocytosis of pteroate-conjugated drugs (without the glutamic acid moiety) has been reported.216,217 In any case, regioselective conjugation is a desirable objective.
175
Figure 6.1. Chemical structure of FA.
In most cases the conjugation of FA with drugs, imaging and bioactive agents is carried out by the so-called “activated ester” method using condensing agents such as
N,N'-dicyclohexylcarbodiimide (DCC),111,218 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)219 and (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP).220 However, since the carboxylic acid groups at the α and
γ positions are in very similar chemical environment, their reactivity is very similar under the conditions normally used for conjugation. Consequently the regioselectivity toward the often desired γ-conjugated product is quite poor. If only the γ functionalized FA is bioactive in cancer treatment, it needs to be isolated which poses considerable purification challenges.
Exclusive γ-conjugation was achieved by a “retrosynthetic” approach.221,222 This method is based on the cleavage of the FA group into pteroate and glutamate derivatives, followed by selective protection and reconstruction of the γ-FA involving an imidazole pteroate derivative and a final deprotection step. For example, FA was cleaved into pteroic and glutamic acids using the carboxylpeptidase G hydrolase enzyme, followed by protection of the α-carboxylic acid site in the glutamic acid using 2-trimethylsilylethanol
(TMSCH2CH2OH) and carbonyldiimidazole (CDI). The pteroate was also protected using
176 a similar chemistry. The product was then reconstructed from the protected pteroate and glutamate moieties, yielding the FA with a free γ-site for exclusive conjugation with nucleoside units (Figure 6.1).
Figure 6.2. Retrosynthetic strategy for the preparation of α-protected FA with a free γ- site.
While this method leads to exclusive γ-conjugation, it is a very tedious process.
In this chapter, we describe a new method for the exclusive γ-conjugation of FA with high selectivity. The first step involves the formation of the lithium salt of the γ- carboxyl group in the glutamic acid moiety of FA using 1 molar equivalent of an organometallic compound such as n-BuLi (Figure 6.3). We theorized that the γ-carboxyl will react preferentially because the pKa values (where Ka is the acid dissociation constant) of the two carboxylic groups are very different (pKa, α-carbon= 4.5; pKa, γ- carbon=2.5). Subsequently the γ-lithiated FA is reacted with 1 equivalent of a compound
177 that is able to react with the lithium salt effectively. Previously, Dr. Kwang Su Seo from
Professor Puskas’ research group demonstrated the method using short bromo derivatives.162
Figure 6.3. New method for the exclusive γ-conjugation of FA.162
6.1.2. Lithiation of FA
FA was reacted with 1 molar equivalent of n-BuLi in anhydrous DMSO. Upon lithiation, the peaks corresponding to the acid carbons (A) and (E) at δ=173.97 and
δ=173.78 ppm shifted downfield to δ=175.58 and δ=174.92 ppm, and became more separated (0.66 ppm) as shown in the expanded regions of the 13C NMRs in Figure 6.4.162
Figure 6.4. Expanded region of 13C NMR spectra of its lithiated intermediate (top) and folic acid (bottom).
178 6.1.3. Synthesis of FA-γ-Hexanol
The lithiated intermediate was then reacted with 6-bromo-1-hexanol (Figure 6.3).
1 The H NMR spectrum of the product in Figure 6.5 shows the -CH2- protons (x) of the conjugated hexanol group at δ=4.0 ppm.
1 Figure 6.5. H NMR spectra of (top) FA and (bottom) FA-hexanol in DMSO-D6.
179 There is no signal at δ=3.42 ppm, characteristic of the CH2 protons next to the Br group in 6-bromo-1-hexanol, indicating complete reaction. Figure 6.5 demonstrates the absence of the -folate and mixed product signals between 7.8 and 8.0 ppm, but shows the γ –substituted NH proton signal (r’) at 8.2 ppm. The NH proton signal (r) at 8.05-8.06 ppm indicates the presence of residual free FA. The ratio of (u) and (x) to (b) and (c) is
0.9/1, indicating 90% conversion. The m/z 564.2051 ([M+Na]+) observed by ESI MS agreed very well with the calculated m/z 564.5453.
Figure 6.6 shows an expanded region of the 13C NMR spectrum of FA-hexanol.
The disappearance of the peaks at 174.92 and 175.58 ppm indicate that the lithiated intermediate reacted completely. The small peak (173.97-173.99 ppm) near (E) indicates the presence of some free FA, in agreement with the 1H NMR spectrum. This is most likely due to base-catalyzed hydrolysis during the water-wash of the Li-residues. The peak (E) at δ=173.95 ppm is assigned to the carbon of the free -acid in a γ-substituted folate, based on the assignment by Mindt et al222 The peak at δ=172.29 ppm in Figure
4.27 is assigned to the substituted γ carbonyl group; the substitution by an alkyl group is expected to shift the carbonyl signal upfield.
Figure 6.6. Expanded region of 13C NMR spectrum of the carbonyl carbons in FA- hexanol (solvent: DMSO-D6).
180 6.1.4. Synthesis of MPEG-FA via Chemo-enzymatic Procedures
Figure 6.7. FA conjugation with 5-bromovalerate. (i) [FA]= 0.05 mol/L, [n-BuLi]= 0.05 mol/L, [EBrV]= 0.04 mol/L. (ii) [FA-γ-valerate]= 0.11 mol/L, [MeO-PEG-OH2000]= 0.08 mol/L, [CALB]= 8.3*10-5 mol/L.
1 The H NMR spectrum of FA-γ- valerate in DMSO-d6 is shown in Figure 6.8. The
CH2 protons (j) of the CH2 conjugated to the γ-carboxylic acid in FA appear at 4.02 ppm.
The signal of the γ-substituted product corresponds to the NH proton appeared at δ=7.95 ppm. The proton signals of FA at δ=6.64 ppm (r), δ=7.59 ppm (s), and δ=8.63 ppm (x) appeared at the expected positions. The proton resonances of the valerate group at δ=2.29 ppm (g), δ=1.90 ppm (i), δ=1.66 ppm (h) and δ=1.14 ppm (b) did not change after the reaction. The 13C NMR spectra of the product is shown in Figure 6.8, the peaks corresponding to the two carboxyl groups in the glutamic moiety of the conjugated FA appeared at δ = 174.97 ppm (O) and δ = 172.32 ppm(K). The ester peak from the valerate group appears at δ = 174.74 ppm (F). The 13C NMR spectrum showed that the carbon connected to the bromine group at δ=34.5 ppm shifted downfield to δ=63.67 ppm (J), confirming the conjugation.
181
Figure 6.8. NMR spectra of FA-valerate 13C NMR spectrum solvent: DMSO-D6. [FA]=0.049 mol/L, [5-EBrV]=0.044 mol/L, [n-BuLi]= 0.049 mol/L.
Next, the transesterification of FA-γ-valerate with MeO-PEG-OH2000 was performed. But in this case no reaction was observed even after 72 h Figure 6.7. DMSO was used because of the insolubility of FA-γ-valerate in solvents with low polarity.
However, it is known that CALB shows higher efficiency when used in hydrophobic solvents.31 In this case, the fact that we were using a polar solvent affected the enzyme reactivity and therefore the reaction did not proceed.
Then we proceed to direct conjugation of MeO-PEG-BrV2000 to FA as shown in
Figure 6.9.
182
Figure 6.9. Folic Acid conjugation with MeO-PEG-Br2000. [FA]=0.049 mol/L, [MeO- PEG2000-Br]=0.044 mol/L, [n-BuLi]= 0.049 mol/L.
Aliquots were taken to follow the formation of MeO-PEG2000-γ- FA at different times (24h, 48h, 72h, 120h) and analyzed by 1H and 13C NMR (Figure 6.10). In the 1H
NMR the signals chosen to follow this reaction was the disappearance of the methylene protons adjacent to bromine at δ = 3.37 ppm, the integral ratio of this signal was compared with the methylene protons adjacent to the ester group at δ=4.13 ppm.
Conversions were 5%, 24%, 30% and 88% at 24 h, 48 h, 72 h and 120 h respectively. In the 13C NMR the signals chosen to follow this reaction were the disappearance of the carbon adjacent to the bromine group at δ=34.5 ppm and the appearance of the new signal at δ=63.77 ppm corresponding to the carbon connected to the new formed ester bond. After 24h the reaction is not complete, the carbon connected to the bromine group at δ=34.5 ppm is still there and there is the appearance of a new peak downfield to
δ=63.77 ppm. The signal at δ=34.5 ppm disappeared after 120h of reaction.
183
13 Figure 6.10. NMR spectra of FA reaction with MeO-PEG-BrV2000 C NMR spectrum (500 MHz, solvent: DMSO-D6).
The fact the reaction takes considerate longer time (24h vs 120h) compare to the shorter bromine molecules, is that the PEG long chain contains several ether groups and due to the different helical conformation changes that the polymer chain can has, it will be like pseudo-crown ethers. PEGs certainly have the ability to form complexes with metal cations. Compared with crown ether complexation, the same PEG can coordinate
184 different sizes of metal cations such as Sr2+ (1.12 ) and Li+ (0.76 ) , 223 thus, PEG presents a more flexible structure for metal cation complexation. The formation of these aggregates makes the lithiated compound less reactive and therefore the reaction is longer.
Lithium chemistry has been widely studied in anionic polymerizations. The intermolecular association of alkyllithium in solid state as well as in solution is well known.224 The extent of aggregation depends strongly on the polarity of the solvent. As alkyllithiums are known to exist in different forms with different degree of intermolecular aggregation, the aggregation affects the efficiency of initiation of hydrocarbon monomers. Studies related to the kinetics of polymerization of styrene in benzene using n-BuLi as initiator began with the work of Worsfold and Bywater in 1960.225 Owing to their ability to solvate lithium ions, electron donors, such as ethers or amines, have a dramatic effect on the polymerization of styrene in nonpolar solvents. The addition of
THF to the polymerization of styrene in benzene strongly increases the rate of polymerization. Depending on the concentration, tertiary amines such as N,N,N,N_- tetramethylethylenediamine (TMEDA) can lead to an increase or decrease in the rate of polymerization. 224In this case, we proceed to use these two modifiers as shown in Figure
6.11. In the case of THF not difference in the reaction time was found. When using
TMEDA the reaction was complete after 24h.
185
Figure 6.11. FA conjugation with MeO-PEG-BrV2000 in the presence of modifier. [FA]= 0.069 mol/L, [n-BuLi]= 0.069 mol/L, [MeO-PEG-BrV2000]= 0.044 mol/L, [TMEDA]= 0.14 mol/L.
1 The H NMR spectrum of MeO-PEG2000-γ- FA in DMSO-D6 is shown in Figure
6.12. There is no proton resonance signal at δ=3.37 ppm, characteristic of the CH2 protons next to the -Br group in MeO-PEG-BrV2000. The CH2 protons (j) of the MeO-
PEG-BrV2000 conjugated to the γ-carboxylic acid in FA appear at 4.09 ppm. The signal of the γ-substituted product corresponds to the NH proton appeared at δ=7.77 ppm. The proton signals of PEG repeat units at δ=3.50 ppm (b and c) and FA at δ=6.59 ppm (r),
δ=7.55 ppm (s), and δ=8.57 ppm (x) appeared at the expected positions. The proton resonances of the valerate group at δ=2.34 ppm (g), δ=1.65 ppm (i) and δ=1.49 ppm (h) did not change after the reaction. The 13C NMR spectrum showed that the carbon connected to the bromine group at δ=35.10 ppm (J) in Figure 4.42 shifted downfield to
δ=63.20 ppm (J), confirming the conjugation. Most of the carbon signals match the expected structure of MeO-PEG2000-γ- FA and the carbon signal associated with the methyl in the methoxy end group at δ = 58.02 ppm (D) did not change after the reaction.
The carbons attributed to the two carboxyl groups in the glutamic moiety of the conjugated FA appeared at δ = 175.28 ppm (O) and δ = 172.42 ppm (K). The ester peak from the valerate group appears at δ = 174.57 ppm (F).
186
1 13 Figure 6.12. NMR spectra of FA reaction with MeO-PEG-BrV2000 H and C NMR spectrum solvent: DMSO-D6.
Figure 6.13 shows the MALDI-ToF spectrum of the FA reaction with MeO-PEG-
BrV2000 product. There is only a single distribution of peaks, separated by 44 m/z units, corresponding to TMED complex with MeO-PEG2000-γ- FA. In the expanded spectrum, the peak at m/z 2256.69 corresponds to the TMED complex of the 36-mer of MeO-
PEG2000-γ- FA. The calculated monoisotopic mass for this peak is 2256.45 Da [36 ×
187 44.03 (C2H4O repeat unit) + 555.27 (end groups) + 116.20 (TMEDA)]. There were no peaks at higher molecular weights. Quaternary salts can be detected in the MS analysis.226 2256.688 2212.651 2256.688 2168.627
TMEDA (116.20) NO Cationizing agent: Monoisotopic mass for 36-mer (Na+) m/z = 36 x 44.03 440.1 + 84.17 31 116.20 2256.45 Da
2160 2180 2200 2220 2240 2260 1250 1500 1750 2000 2250 2500 2750 3000 3250 m/z
Figure 6.13. MALDI-ToF spectra of the reaction of FA with MeO-PEG-BrV2000 (left) and zoom of the 35 and 36 mer fractions (right). [FA]=0.069 mol/L, [MeO-PEG- BrV2000]=0.044 mol/L, [n-BuLi]= 0.069 mol/L, [TMEDA]= 0.138 mol/L.
6.1.5. FA-γ- bis(2-ethyl 5-bromopentanoate) disulfide (FA-S-S-FA)
FA-γ- bis(2-ethyl 5-bromopentanoate) disulfide was synthesized as shown in
Figure 6.14.
188
Figure 6.14. FA conjugation with bis(2-ethyl 5-bromopentanoate) disulfide and its reduction. (i) [FA]= 0.05 mol/L, [n-BuLi]= 0.05 mol/L, [bis(2-ethyl 5-bromopentanoate) disulfide]= 0.02 mol/L. (ii) [FA-S-S-FA]= 0.26 mol/L, [DTT]= 0.26 mol/L.
The 1H NMR spectrum of bis (2-ethyl 5-bromopentanoate) disulfide (top) and
FA-γ- bis(2-ethyl 5-bromopentanoate (bottom) is shown in Figure 6.15. The proton resonance signal at δ=3.45 ppm (f), characteristic of the CH2 protons next to the -Br group in bis (2-ethyl 5-bromopentanoate) disulfide shift to δ=3.45 ppm e CH2 protons (j) of the CH2 conjugated to the γ-carboxylic acid in FA appear at 3.98 ppm (g). The signal of the γ-substituted product corresponds to the NH proton appeared at δ=7.90 ppm. The proton signals of FA at δ=6.61 ppm (r), δ=7.59 ppm (s), and δ=8.59 ppm (x) appeared at the expected positions. The proton resonances of the valerate group at δ=2.26 ppm (i), and δ=1.53 ppm (h) did not change after the reaction.
189
Figure 6.15. 1H NMR spectra: bis (2-ethyl 5-bromopentanoate) disulfide (top) and FA-γ- bis(2-ethyl 5-bromopentanoate (bottom) (500 MHz, solvent: DMSO-D6).
The 13C NMR spectra of the product is shown in Figure 6.16, the peaks corresponding to the two carboxyl groups in the glutamic moiety of the conjugated FA appeared at δ = 174.94 ppm (I) and δ = 172.53 ppm (H). The ester peak from the valerate group appears at δ = 172.53 ppm (C). The 13C NMR spectrum of the product also confirmed the structure.
190
Figure 6.16. 13C NMR spectrum of FA-γ- bis(2-ethyl 5-bromopentanoate (125 MHz, solvent: DMSO-D6).
From Chapter IV in section 4.2.6.1 it was point out that there was 4% of EBrV in the product that couldn’t be removed. In this case the EBrV can react as well with the 0.1 excess of the FA-Li. This mean there is a few percent of this EBrV that will react with
FA-Li and form the FA-γ-valerate. By the 1H NMR this product doesn’t appear, by the
13 C NMR there is a small signal at δ = 14.1 ppm that may correspond to the CH3 from the valerate group. This impurity will be present in the subsequent reactions performed with bis (2-ethyl 5-bromopentanoate) disulfide.
6.1.5.1. FA-SH
FA-SH was synthesized as shown in Figure 6.14. In dark condition, a mixture of
FA-S-S-FA (0.50 g, 0.42 mmol, 1.0 eq.) and DTT (0.064 g, 0.42 mmol, 1.0 eq.) was dissolved in 1.6 mL of DMF under a nitrogen atmosphere. After stirring at room temperature for 20 h, the dark yellow product was precipitated in 50 mL diethyl ether and washed with hexane and THF. The solid product was dried in a vacuum oven for further analysis.
191
1 13 Figure 6.17. NMR spectra of FA-SH. H and C NMR spectrum solvent: DMSO-D6.
Figure 6.17 displays the NMR spectra of the product. The 1H NMR spectrum shows signals of FA at =7.59 ppm (s), =6.61 ppm (r) and =8.58ppm (x) appeared at the expected positions. The proton resonances of the valerate group at =2.27 ppm (j) and
=1.52 ppm (i) did not change after the reaction. A new signal at =2.69 ppm that corresponds to the CH2 (b’) attached to the SH group appeared. Some water is present in the spectrum. The 13C NMR spectrum also confirmed the conjugation. Most of the carbon signals match the expected structure of FA-SH and the carbon signal associated with the
192 valerate carbons did not change after the reaction. The carbons attributed to the two carboxyl groups in the glutamic moiety of the conjugated FA appeared at δ = 174.83 ppm
(I) and δ = 172.65 ppm (H).
It has to be also pointed out that in the 1H NMR spectrum of the FA conjugates the signal of the carboxylic acid proton (COOH) at =12 ppm did not appear. This is because the acidic proton is easily exchangeable with water that can be present in
DMSO-D6. This agrees with the reports in the literature. In order to see the COOH proton one may use a water-signal suppressing program as reported.
6.2. Synthesis of the Biotin-TEG-OH arms
This strategy involves the synthesis of a monofunctional allyl tetraethylenglycol.
Then this product can be esterified with the biotin. At the end we will have a biotin-TEG- allyl that can be coupled with a mercapoethanol through thiol-ene click reaction (Figure
6.18).
Figure 6.18. Synthetic strategy for Biotin-TEG-OH.
6.2.1. Synthesis of HO-TEG-allyl
The synthesis of HO-TEG-allyl is shown in Figure 6.18. TEG (5.0 g, 25 mmol,
1.0 eq.), was dissolved in a 50% aqueous solution of NaOH(0.20 mL, 4.4 mmol, 0.17 eq.)
193 under vigorous stirring at 50 ºC for 4 h. Allyl bromide (0.50 g, 4.4 mmol, 0.17 eq.), was added slowly over 30 min, the mixture was stirred at 60 ºC for 2h and at 85 ºC for 22h.
The crude was purified by column chromatography on silica, using CH2Cl2/ ethyl acetate/ethanol 2/1/0.3 as eluent to give HO-TEG-allyl (1 g, 39%) as colorless oil.
Disubstituted product was also obtained in 14% yield.
Figure 6.19. 1H NMR spectrum (top) and 13C NMR spectrum HO-TEG-allyl (300 MHz, solvent: CDCl3)
The 1H NMR spectrum of the product is shown in Figure 6.19. The resonance at
δ=3.16 ppm, corresponding to the -OH proton of HO-TEG-allyl is present. The peak of the methylene protons adjacent to hydroxyl group and now close to the allyl group shifted downfield to δ=3.83 ppm (a) after the reaction. The new peak corresponding to the allyl protons were observed at δ = 5.70 ppm (a), δ = 5.07 ppm (b) and δ = 4.97 ppm (b’). The
194 relative integrals of the methylene protons of the allyl group and the methylene protons next to the OH group in the TEG moiety at δ = 3.83 ppm (c) demonstrated successful functionalization with the integration ratio of 1:2:2.
The 13C NMR spectrum (Figure 6.19) showed that the carbons connected to the hydroxyl groups at δ=60.1 ppm (F) in the HO-TEG-allyl is present. The signal at δ=72.10 ppm (C) corresponds to the methylene carbon close to the allyl group and the carbon signals associated with the allyl group at δ = 134.29 ppm (A) and δ = 116.33 ppm (B) appeared. ESI confirmed the product with a mass of 257.1 (ESI m/z: [M+Na]+ calculated for C11H22NaO5 257.135).
6.2.2. Synthesis of Biotin-TEG-allyl
The synthesis of HO-TEG-allyl is shown in Figure 6.18. In dark condition, a mixture of HO-TEG-allyl (1.0 g, 4.2 mmol, 1.0 eq.), biotin (1.0 g, 4.2 mmol, 1.0 eq.),
EDC (1.1 g, 5.9 mmol, 1.4 eq.), and DMAP (0.51 g, 4.2 mmol, 1.0 eq.) was dissolved in
212 mL anhydrous CH2Cl2 (0.02 mol/L) under a nitrogen atmosphere. The obtained crude product was purified by column chromatography alumina (CH2Cl2: EtOAc: MeOH
= 6:3:0.3) to give 2.1 g of the product as a yellowish solid. TLC Rf=0.23.
195
1 13 Figure 6.20. H NMR spectrum (top) (300 MHz, solvent: DMSO-D6) and C NMR spectrum Biotin-TEG-allyl (75 MHz, solvent: CDCl3).
The 1H NMR spectrum of the product is shown in Figure 6.20. The resonance at
δ=3.16 ppm, corresponding to the -OH proton of HO-TEG-allyl disappeared. The peak of the methylene protons adjacent to hydroxyl group and now part of the ester group shifted downfield to δ=4.13 ppm (g) after the reaction. The new peaks corresponding to biotin moiety were observed at δ = 1.18 ppm (j), δ = 1.33 ppm (h), δ = 1.53 ppm (i), δ = 4.13
196 ppm (m) and δ = 6.40 ppm (p). The relative integrals of the protons of the allyl group at δ
= 5.70 ppm (a), δ = 5.07 ppm (b), δ = 4.97 ppm (b’) and the methylene protons next to the ester group at δ = 4.13 ppm (g) demonstrates successful functionalization with the integration ratio of 1:1:1:2. The 13C NMR spectrum (Figure 6.20) showed that the carbons connected to the hydroxyl group shifted from δ=60.1 ppm to δ=62.39 ppm. The carbonyl part of the biotin moiety and the ester group are observed at δ=172.43 ppm (I) and δ=163.13 ppm (H) respectively. The carbon signals associated with the allyl group at
δ = 134.29 ppm (A) and δ = 116.33ppm (B) are intact. ESI confirmed the product with a
+ mass of 483.1 (ESI m/z: [M+Na] calculated for C21H36N2S1NaO7 483.2141).
6.2.3. Synthesis of Biotin-TEG-OH
The synthesis of Biotin-TEG-OH is shown in Figure 6.18. Biotin-TEG-allyl (0.50 g, 1.0 mmol, 1.0 eq.) were dissolved in 3mL CH2Cl2, and then mercapto ethanol (0.11 mL, 1.6 mmol, 1.5 eq. ) and Irgacure (0.14 g, 0.52 mmol, 0.5 eq.) of were added to the solution. All the mixture was homogenized during 1min. The reaction started with a UV lamp (364 nm) and stirred during 20 min. The reaction was followed by TLC.
The 1H NMR spectrum of the product is shown in Figure 6.23. The resonance of the allyl group at δ = 5.70 ppm (a), δ = 5.07 ppm (b), δ = 4.97 ppm (b’) disappeared. The peak of the methylene protons adjacent to hydroxyl group appeared at δ=4.13 ppm (g) after the reaction. ESI confirmed the product with a mass of 561.1 (ESI m/z: [M+Na]+ calculated for C23H42N2S2NaO8 561.23).
197
1 Figure 6.21. H NMR spectrum of Biotin-TEG-allyl. (300 MHz, solvent: DMSO-D6).
6.2.4. Synthesis of Biotin-PEG-OH
Figure 6.22. Synthetic strategy for Biotin-PEG-OH3400. [NH2-PEG-OH3400]= 0.098 mol/L, [Et3N]= 0.088 mol/L, [NHS-Biotin]= 0.34 mol/L.
Biotin was reacted with exclusively amine group of NH2-PEG-OH3400 in the presence of triethylamine via nucleophilic addition (Figure 6.22). The product was analyzed by 1H and 13C NMR (Figure 6.23). The proton resonances corresponding to the biotin residue [δ = 7.79 ppm (NH), δ = 6.37 ppm (NH), δ = 6.31 ppm (NH), δ = 4.53 ppm
(l), δ = 4.30 ppm (k), δ = 1.59 ppm (h), δ = 1.48 ppm (j), and δ = 1.31 ppm (i)] were observed at the expected positions. The proton resonance next to amine at δ = 2.71 ppm
(a) shifted downfield to δ = 3.50 ppm, overlapping with the protons of the PEG repeat units.
198 2013_03_09_MC_Biotin_PEG_OH_13C_300M.esp 69.76 39.51
(E) (C) (B’) (H) (J) (F) (G) (I) (K) (D) (A’) (L)
(M)
(B’) (L) (D) (E) (A’) (I)(G) (H,J) 60.19 69.14 61.00 (F) (M) 72.31 59.17 55.37 28.00 28.15 35.06 25.22 172.05 162.64
160 140 120 100 80 60 40 20 Chemical Shift (ppm)
1 13 Figure 6.23.NMR spectra of Biotin-PEG-OH3400: H NMR spectrum (top) C NMR spectrum (bottom) (500 MHz, solvent: DMSO-D6).
13 Figure 6.23 shows C NMR spectrum of Biotin-PEG-OH3400 in DMSO-D6. The carbons next to the amine group of NH2-PEG-OH3400 shifted from δ=40.75 ppm (A) and
δ=71.69 ppm (B) to δ=55.37 ppm (A’) and δ=69.14 ppm (B’), respectively, indicating quantitative conversion. Also, the signal of the carbon next to the OH in the NH2-PEG-
OH3400 at δ=60.19 ppm (E) did not shift, indicating that the OH group remained intact.
199 Furthermore, the carbon of the carboxyl group of Biotin shifted from δ=174.45 ppm to
δ=172.05 ppm (F).
Figure 6.24. MALDI-ToF mass spectrum of Biotin-PEG-OH3400 (left) and zoom of the 77 and 78 mer fractions (right).
Figure 6.24 shows MALDI-ToF spectrum two distributions of the product obtained by the reaction of biotin with NH2-PEG-OH3400 (Mn=3,400 g/mol). There is one major and one minor distribution, corresponding to the sodium and the potassium complex of Biotin-PEG-OH3400, respectively. Within the distributions, the peaks are separated by 44 Da, corresponding to an ethylene glycol repeating unit. In the expanded spectrum, the peak at m/z 3744.599 corresponds to the sodium complex of the Biotin-
PEG-OH3400 (78 mer). The calculated monoisotopic mass for this peak is 3744.45 Da [78
× 44.03 (C2H4O repeat unit) + 287.12 (C12H21N3O3S end groups) + 22.99 (Na+)]. The
200 minor peaks in the same region differ from the main series by 16 m/z units, attributed to the potassium complex of the Biotin-PEG-OH3400.
6.3. Synthesis of the Fluorouracil Arms
6.3.1. Michael Addition of Fluorouracil to Vinyl Acrylate
Figure 6.25. CALB catalyzed Michael addition of Fluorouracil to vinyl acrylate. [VA]= 1.58 mol/L, [Fluorouracil]= 0.79 mol/L, [CALB]= 8.0×10-5 mol/L.
Previously, Dr. Sen demonstrated quantitative Michael addition of thymine to VA in DMSO in the presence of CALB. Thus, Michael addition was also performed in
DMSO. Vinyl ester of fluorouracil was prepared by Michael addition of fluorouracil to vinyl acrylate Figure 6.25.
Figure 6.26 shows NMR spectra of the Michael addition product of fluorouracil to vinyl acrylate in DMSO-D6.
201
Figure 6.26. 1H NMR spectrum (Top) and 13C NMR spectrum (Bottom) fluorouracil vinyl ester product. (300 MHz, solvent: DMSO-D6).
The resonances of the vinylidene [δ=6.18 ppm] and vinyl [δ=6.32 ppm and
δ=5.97 ppm] protons in the acrylate group in vinyl acrylate shifted to at δ = 3.86 ppm (e) and δ = 2.80 ppm (f), respectively. The new peaks corresponding to the fluorouracil moiety appeared at δ = 11.69 ppm (a) and δ = 7.00 ppm (b). The relative intensities
[(a):(b):(c):(e):(f)] 1:1:1:2:2 indicated quantitative functionalization. The 13C NMR spectrum of the Michael addition product also confirmed the structure. The carbons connected to fluorouracil moiety at δ=162.62 ppm (B), δ=157.30 ppm (C) and δ=138.43 ppm (E) appeared after the reaction. The carbon resonances of the acrylate group shifted upfield to δ=43.77 ppm (H) and δ= 32.11 ppm (I), respectively.
202 6.3.2. Transesterification Reaction of Fluorouracil-Vinyl Ester with 2HEDS
Figure 6.27. Synthetic strategy to get a disulfide bond. [fluorouracil-vinylester]= 0.8 mol/L, [2HEDS]= 2.3 mol/L, [CALB]= 7.7×10-7 mol/L.
Figure 6.27 shows the synthetic strategy to get a fluorouracil disulfide linker.
2HEDS (0.8 mL, 6.9 mmol, 3.0 eq.) was added to a flask containing fluorouracil- vinylester (0.3 g, 2.3 mmol, 1.0 eq.) in 3mL of anhydrous THF and CALB (0.3 g,
2.31x10-6 mmol, 1.0x10-6 eq.) under an inert atmosphere. The mixture was stirred at 300 rpm for 48 hours at 50 °C. After the reaction the solid CALB was removed by a 0.45 µm
PTFE filter and the solvent was removed by vacuum distillation.
After purification by column chromatography, the product was analysed with 1H and 13C NMR spectroscopy (Figure 6.28). In the 1H NMR spectrum two different peaks were observed at δ=3.61 (f) and 4.27 (d) ppm corresponding to the methylene protons next to the hydroxyl group and the ester linkage, respectively. There is an excess of
2HEDS that couldn’t be removed by column. The vinyl ester protons disappeared. In the
13C NMR spectrum, two separate carbon signals corresponding to the carbons next to the hydroxyl group and the ester linkage were observed at δ=62.20 ppm (F) and 59.40 (G) ppm, respectively.
203
Figure 6.28. 1H NMR spectrum (Top) and 13C NMR spectrum (Bottom) fluorouracil disulfide product. (500 MHz, solvent: DMSO-D6).
6.4. Synthesis of the FITC linkers
The following part described the synthesis of FITC linkers using enzymatic catalysis. Previously in our group was followed the conventional methodology that involves using FITC isothyocyanate and triethylamine. Using CALB gives the advantage of being a green methodology for this functionalization.
6.4.1. Michael Addition of NH2-PEG-OH1000 to FITC-VA
Figure 6.29. Conjugation of FITC to NH2-PEG-OH1000 (Mn=1000 g/mol; ĐM =1.04). -4 [FITC-VA] = 0.3 mol/L, [NH2-PEG-OH1000] = 0.3 mol/L, [CALB]= 3.6×10 mol/L.
204 FITC-PEG-OH1000 was prepared by Michael addition of NH2-PEG-OH1000 to
FITC-VA Figure 6.29. Figure 6.30 shows the 1H and 13C NMR spectra of FITC-VA starting material, verifying the structure. All proton and carbon resonances corresponded to the expected structure.
Figure 6.30. NMR spectra of FITC-VA: (Top) 1H NMR spectrum and (Bottom) 13C NMR spectrum (500 MHz, solvent: DMSO-D6).
205
1 Figure 6.31. H NMR spectra of (bottom) NH2-PEG-OH1000 and (top) FITC-PEG-OH1000 in DMSO-D6.
1 Figure 6.31 shows H NMR spectra of NH2-PEG-OH1000 and FITC-PEG-OH1000.
The proton resonances corresponding to the FITC residue [δ = 10.09 ppm (m), δ = 7.95 ppm (g), δ = 7.74 ppm (h), δ = 6.67 ppm (i), δ = 6.59 ppm (l) and δ = 6.57 ppm (k)] were observed at the expected positions. After the Michael Addition the acrylate protons from the FITC moiety shifted high field from δ = 6.19 ppm (l), δ = 6.42 ppm k) and δ = 6.61 ppm (m) to δ = 2.69 ppm (j’) and δ = 2.35 ppm (o’). The proton resonance next to amine in the of NH2-PEG-OH1000 moiety at δ = 2.71 ppm (a) shifted downfield to δ = 3.15 ppm.
Resonances attributed to NH2-PEG-OH1000 repeat units are also observed.
206
13 Figure 6.32. C NMR spectra of (bottom) NH2-PEG-OH1000 and (top) FITC-PEG-OH1000 in DMSO-D6.
13 Figure 6.32 shows C NMR spectra of NH2-PEG-OH1000 and FITC-PEG-OH1000 in DMSO-D6. The carbons next to the amine group of NH2-PEG-OH1000 shifted from
δ=40.75 ppm (A) and δ=71.69 ppm (B) to δ=43.66ppm (A’) and δ=68.40 ppm (B’), respectively, indicating quantitative conversion. Also, the signal of the carbon next to the
OH in the NH2-PEG-OH1000 at δ=60.17 (F) did not shift, indicating that the OH group remained intact. The carbon resonances corresponding to the FITC residue [δ = 168.43 ppm (H), δ = 162.24 ppm (G), δ = 159.40 ppm (M), δ = 151.42 ppm (P, Q), δ = 128.92 ppm (U, V)] were observed at the expected positions. After the Michael Addition the acrylate carbons shifted high field from δ = 127.30 ppm (O) and δ = 135.74 ppm to δ =
35.72ppm (O’) and δ = 45.67 ppm (J’).
207 6.4.2. Transesterification of 2-(acryloyloxy)ethyl vinyl adipate (VA-DVA) with
FITC-PEG-OH1000.
2-(acryloyloxy)ethyl vinyl adipate (VA-DVA, 0.087 g, 0.32 mmol, 1.5 eq. per
OH) was reacted with FITC-PEG-OH1000 (0.3 g, 0.21 mmol) in dried THF (0.7 mL) in the presence of CALB (35 mg resin @ 20wt% enzyme, 2.1 × 10-4 mmol) for 24 hours
(Figure 6.33).
Figure 6.33. Transesterification of 2-(acryloyloxy)ethyl vinyl adipate with FITC-PEG- OH1000 (Mn=1000 g/mol; ĐM =1.04). [FITC-PEG-OH1000] = 0.31 mol/L, [VA-DVA] = 0.46 mol/L, [CALB]= 3.1×10-4 mol/L.
The product was precipitated in diethyl ether and washed with hexane to remove the excess VA-DVA. The yellowish precipitate was analyzed by 1H and 13C NMR spectroscopy. Figure 6.34 displays the NMR spectra of the product. The 1H NMR spectrum shows the signal of the hydroxyl proton of FITC-PEG-OH1000 at δ = 4.50 ppm disappeared after the reaction and the signals assigned to methylene proton resonances next to the oxygen at δ = 4.12 ppm (f’) appeared.
208
1 13 Figure 6.34. NMR spectra of FITC-PEG-VA1000: H NMR spectrum (top) C NMR spectrum (bottom) (500 MHz, solvent: DMSO-D6).
The vinylidene [δ=6.17 ppm (u)] and vinyl [δ=6.31 ppm (v’) and δ=5.96 ppm (v’)] protons of the acrylate group appeared. The proton resonances corresponding to the FITC residue [δ = 7.96 ppm (t), δ = 7.78 ppm (v), δ = 7.71 ppm (s), δ = 6.68 ppm (y) and δ =
6.53 ppm (z’)] were observed at the expected positions. The relative integrals of the protons of the newly formed acrylate chain end [v’), (v’), (u)] and the new methylene proton
209 resonances next to the oxygen from the FITC-PEG-VA1000 moiety (f’): (v’) : (v’) : (u) = 1.7 :
1: 0.96 : 0.76 demonstrate successful functionalization.
The 13C NMR spectrum showed that the carbon connected to the hydroxyl group at δ=60.12 ppm in the FITC-PEG-OH shifted downfield to δ=63.14 ppm (F) and the carbon signals corresponding to the acrylate group appeared at δ=131.90 ppm (U’) and at
δ=128.02 ppm (V’), respectively. The carbonyl carbons of the acrylate linker appear at δ
= 174.88 ppm (Q’, K’) and δ = 165.37 ppm (T’). The carbon resonances corresponding to the FITC residue were observed at the expected positions.
6.5. Synthesis of the FITC-PEG1000- γ- FA
As established in the Chapter II this thesis was aimed to develop a modular synthetic approach for the synthesis of a vitamin-polymer conjugate using enzymatic catalysis where possible. The next challenge was synthesize a polymer conjugate that has the folic acid as targeting agent and fluorescence as imaging agent at the same time.
6.5.1. Michael Addition of FA-SH to FITC-PEG-VA1000
FITC-PEG1000- γ- FA was prepared by Michael addition of FA-SH to FITC-PEG-
VA1000 (Figure 6.35). FA-SH (23 mg, 0.38 mmol, 1.2 eq. per acrylate group of FITC-
PEG-VA-1000) in 0.4 mL of anhydrous DMSO was reacted with FITC-PEG-VA1000
(Mn=1000 g/mol; ĐM=1.04, 50 mg, 0.31 mmol, 1.0 eq.) in the presence of CALB (9.4 mg, 5.6×10-4 mmol) under nitrogen. The reaction was stirred at 300 rpm for 20 hours at
50oC.
210
Figure 6.35. Michael Addition of FA-SH to FITC-PEG-VA1000 (Mn=1000 g/mol; ĐM -5 =1.04). [FITC-PEG-VA1000] = 0.078 mol/L, [FA-SH] = 0.094 mol/L, [CALB]= 1.4×10 mol/L.
Figure 6.36 displays the NMR spectra of the product. The vinylidene [δ=6.17 ppm (u)] and vinyl [δ=6.31 ppm (v’) and δ=5.96 ppm (v’)] protons of the acrylate group shifted upfield to δ = 2.73 ppm (v’) and δ = 2.66 ppm (u’). The proton resonances corresponding to the FITC residue [δ = 8.03 ppm (v), δ = 7.89 ppm (s), δ = 7.71 ppm (s),
δ = 6.90 ppm (y)] were observed at the expected positions. The signals of FA moiety at
=7.65 ppm (i), =6.61 ppm (r) and =8.64ppm (x’) were observed at the expected positions. The signal at =2.69 ppm that corresponds to the CH2 (a) attached to the thioether group shifted upfield to =2.40 ppm (a). The 13C NMR spectrum showed the carbons resonances corresponding to the acrylate group shifted upfield to δ=33.14 ppm
(U’) and δ=27.46 ppm (V’) after the reaction and the carbon resonances of the FITC moiety did not change. The carbon connected to the thiol group at =18.40 ppm (A’)
211 shifted downfield to δ=31.74 ppm (A’), confirming the conjugation. The carbons attributed to the FA moiety appeared at the expected positions.
1 13 Figure 6.36. NMR spectra of FITC-PEG1000- γ- FA. H and C NMR spectrum solvent: DMSO-D6.
From the section 6.1.5 it was discuss the presence of FA-γ-valerate as an impurity. The amino group (NH2) from the FA moiety can also react though Michael addition with the acrylate group of FITC-PEG-VA1000 to form the compound B (Figure
6.37). Previously Dr Kwang Su Seo studied the Michael-Addition reaction of FA to vinyl
212 acrylate (data not published). He found that the reaction proceeds after 72 h. With this knowledge the presence of FA-γ-valerate in the system was not a concern. Thus is, the reaction was run for 20h and the structure of FA-γ-valerate does not contain the SH as a
Michael acceptor. Based on the NMR the product FITC-PEG1000- γ- FA is pure. MALDI confirmed the presence of A and B.
Figure 6.37. Structure of the products present on the Michael Addition of FA-SH to FITC-PEG-VA1000.
213
Figure 6.38. MALDI-ToF spectra of FITC-PEG1000- γ- FA (top) and zoom of the 20 and 21 mer fractions (bottom).
214 Figure 6.38 shows the MALDI-ToF spectrum of the FITC-PEG1000- γ- FA. Two set of peaks were observed. The main series (96%) corresponds to the [M+H] of A and the minor series (4%) corresponds to the product B. In the expanded spectrum, the peak at m/z 2202.47 corresponds to the M+H of the 20-mer of FITC-PEG1000- γ- FA. The calculated monoisotopic mass for this peak is 2202.63 Da [20 × 44.03 (C2H4O repeat unit) + 448.124 (FITC moiety) + 872.91 (FA moiety) + 1 (H+)]. There were no peaks at lower molecular weights. The peak at m/z 2212.49 corresponds to the M+ of the 21-mer of FITC-PEG1000- γ- FA. The calculated monoisotopic mass for this peak is 2213.09 Da
[21 × 44.03 (C2H4O repeat unit) + 448.124 (FITC moiety) + 840.34 (FA moiety)].
6.5.2. Michael Addition of Diethanolamine to VA-FITC-VA
Figure 6.39. Conjugation of VA-FITC-VA to DEA. [FITC] = 0.075 mol/L, [DEA] = 0.15mol/L. [CALB]= 3.6×10-4 mol/L.
2(OH)-FITC-(OH)2 was prepared by Michael addition of DEA to VA-FITC-VA
Figure 6.39. VA-FITC-VA (0.050 g, 0.11 mmol, 1.0 eq.) in 0.15 mL of anhydrous
DMSO was reacted with diethanolamine (DEA) (0.024 g, 0.23 mmol, 1.01 eq per acrylate group of VA-FITC-VA) in the presence of CALB (3.8 mg, 2.29×10-8 mmol,
215 2×10-7 eq.) under nitrogen. The reaction was stirred at 300 rpm for 6 hours at 50 oC. The progress of the reaction was monitored with silica gel TLC using CH2Cl2/Ethanol (30/1; vol/vol) as the eluent.
Figure 6.40. NMR spectra of VA-FITC-VA: (Top) 1H NMR spectrum and (Bottom) 13C NMR spectrum (500 MHz, solvent: DMSO-D6).
Figure 6.40 shows the 1H and 13C NMR spectra of VA-FITC-VA starting material, verifying the structure. All proton and carbon resonances corresponded to the expected structure. Figure 6.41 displays the 1H NMR spectrum of the conjugated product.
216
Figure 6.41. 1H NMR spectrum of the product of the Michael addition of diethanolamine to VA-FITC-VA. (500 MHz, solvent: DMSO-D6)
217 7. CHAPTER VII
PREPARATION OF LOW MOLECULAR MASS TELECHELIC FUNCTIONALIZED
POLYISOBUTYLENES
7.1. Introduction
Telechelic polymers, defined as macromolecules that contain two reactive end groups, are used as cross-linkers, chain extenders, and important building blocks for various macromolecular structures, including block and graft copolymers, star, hyperbranched or dendritic polymers. Cationic polymerization is known as chain polymerization in which propagating chains, namely active species are positively charged carbon centered ions or onium ions in vinyl polymerization.122 Various types of the initiation processes involving addition of Brønsted acids, Lewis acids, Lewis acids in conjunction with a proton or carbocation source have been described. The initiation process is followed by propagation in which a nucleophilic attack of the monomer onto the active growing centers paired with non-nucleophilic counterions occurs.
Nucleophilicty of counterion of the cationic growing species is crucial to achieve the living character of the polymerization.122
Living mode of cationic polymerization provides to introduce functional groups at polymer chain termini. The method involves the use of a functional initiator or a
218 termination agent. Moreover, transformation of the primary end-capping groups to any other functional groups is an alternative method to obtain functional polymers. This section, will describe the synthesis of low molecular mass telechelic PIBs.
7.2. Initiators
Three different initiators were employed for the synthesis of telechelic PIBs,
2,4,4,6-tetramethylheptane-2,6-diol (TMHDiOH),18 2,6-dimethoxy-2,4,4,6- tetramethylheptane (TMHDiOMe) and 2,6-dichloro-2,4,4,6-tetramethylheptane
(TMHDiCl), in the presence of TiCl4 coinitiator Figure 7.1.
Figure 7.1. Synthesis of dinitiator for IB oligomerization and IB polymerization.
7.2.1. Dimethyl 3,3-dimethylpentanedioate (DiMDiPD)
The esterification reaction of 3,3 dimethyl glutaric acid (Figure 7.1) was complete after 48h. The 1H NMR spectrum of the ester product is shown in Figure 7.2. There is a new resonance at δ=3.57 ppm that corresponds to the methyl protons (a’). The methylene protons peak (c) shifted upfield from δ=2.52 ppm to δ=2.34 ppm after the reaction. The
219 relative integrals of the methyl protons (c) and the methyl protons (d) demonstrate successful functionalization with the integration ratio of 6:6.3. In the 13C NMR spectrum is shown in Figure 7.2, two carbon peaks corresponding to the methyl carbon ( = 51.12 ppm) and ester carbon ( = 172.16 ppm) were observed.
Figure 7.2. NMR spectra of the product DiMDiPD: (Top) 1H NMR spectrum and 13 (bottom) C NMR spectrum (300 MHz, solvent: CDCl3).
7.2.2. 2,4,4,6-tetramethylheptane-2,6-diol (TMHDiOH)
DiMDiPD was converted to TMHDiOH through a Grignard reaction (Figure 7.1).
The 1H NMR of the initiator in Figure 7.3 shows the resonances at δ=1.07 ppm, δ=1.27 ppm, δ=1.73 ppm, and δ=3.54 ppm characteristic of methyl (d and c), methylene (b) and
OH (a) protons respectively. The 13C NMR shows the resonances at δ=72.36 ppm,
220 δ=51.46 ppm, δ=33.71 ppm, and δ=33.12 ppm characteristic of methyne (B), methylene
(C), methyl (B), methyl (D) and methyne (D’) carbons respectively.
Figure 7.3. NMR spectra of TMHDiOH: (Top) 1H NMR spectrum and (bottom) 13C NMR spectrum (300 MHz, solvent: CDCl3).
7.2.3. 2,6-dimethoxy-2,4,4,6-tetramethylheptane (TMHDiOMe)
The eterification reaction of TMHDiOH (Figure 7.1) yields the TMHDiOMe in
80% conversion.
221
Figure 7.4. NMR spectra of the product TMHDiOMe: (Top) 1H NMR spectrum and 13 (bottom) C NMR spectrum (300 MHz, solvent: CDCl3).
The 1H NMR spectrum of the ether product is shown in Figure 7.4. There is a new resonance at δ=3.16 ppm that corresponds to the methyl protons (a’). The methylene protons close to the alcohol disappeared (a). The methylene protons (c) shifted upfield from δ=1.72 ppm to δ=1.59 ppm after the reaction. The relative integrals of the methyl protons (a’) and the methyl protons (d) demonstrate successful functionalization with the integration ratio of 6.13:6. In the 13C NMR spectrum is shown in Figure 7.4, one new carbon peak corresponding to the methyl carbon from the ether group ( = 48.64 ppm) is observed. The methyne proton signal (B’) shifted downfield from = 72.36 ppm to =
75.98 ppm. Some residual solvent remains in the product.
222 7.2.4. 2,6-dichloro-2,4,4,6-tetramethylheptane (TMHDiCl)
The HCl gas was bubbled into a solution of TMHDiOH (Figure 7.1).
Figure 7.5. NMR spectra of the product TMHDiCl: (Top) 1H NMR spectrum and 13 (bottom) C NMR spectrum (300 MHz, solvent: CDCl3).
The 1H NMR spectrum of the chlorine product is shown in Figure 7.5. The methylene protons close to the alcohol disappeared (a). The methylene protons (c) shifted downfield from δ=1.72 ppm to δ=2.07 ppm after the reaction. The relative integrals of the methylene protons (c) and the methyl protons (d) demonstrate successful functionalization with the integration ratio of 4:6. In the 13C NMR spectrum is shown in
223 Figure 7.5, the methyne proton signal (B’) shifted upfield from = 72.36 ppm to =
71.74 ppm.
7.3. Synthesis of Telechelic Oligomers of Isobutylene
7.3.1. Introduction
Polyisobutylene (PIB) is an essential component used in the synthesis of gasoline and lubricant additives which is manufactured commercially by the oligomerization of isobutene in continuous-flow processes using aluminum chloride or boron trifluoride as catalysts. The products from these commercial processes can have high halide contents which interfere with their subsequent functionalization in the synthesis of additives. In this chapter is discussed how to synthesize functional oligomers of IB. First model reactions were run and after that the oligomerization process is explained.
224 7.3.2. Model reaction for the Oligomerization of Isobutylene
Figure 7.6. Model reaction sequence for the IB oligomerization.
7.3.2.1. Synthesis of 2-chloro-2,4,4-trimethylpentane ( TMPCl)
The chlorination reaction of TMP-1 (Figure 7.6) was achieved by the bubbling of
HCl gas into TMP-1 and it was complete after 24 h. The 1H NMR spectrum of the starting material and the product are shown in Figure 7.7.
The resonance at δ=4.83 ppm and δ=4.62 ppm, corresponding to the vinyl protons of TMP-1 (1H top) disappeared and the peak of the methyl protons adjacent to vinyl group shifted upfield from δ=1.77 ppm to δ=1.68 ppm (c) after the reaction. The relative integrals of the methylene protons (b) and the methyl protons (c) and (a) demonstrate successful functionalization with the integration ratio of 2:6.2: 9.
225
Figure 7.7. 1H NMR spectrum of the TMP-1 (top) and 1H NMR spectrum of the chlorinated product TMPCl (bottom) (300 MHz, solvent: CDCl3).
7.3.2.2. Synthesis of 2,4,4,6,6-pentamethyl-1-heptene (PMH)
The methyl allylation reaction of TMPCl yield the product PMH (Figure 7.6) and it was synthesized using the method of Roth and Mayr.227 The 1H NMR spectrum of the product is shown in Figure 7.8. Four new peaks appear after the reaction: the peaks at =
4.86-4.64 ppm corresponding to the vinylidene protons (g, g’), the methyl protons (f) at
δ=1.78 ppm and the methylene protons (e) at δ=2 ppm. The methylene protons (b) shifted
226 upfield to δ=1.30 ppm. The relative integrals of the vinyl protons (g, g’) and the methylene protons (b) demonstrate successful functionalization with the integration ratio of 1:1:2.
1 Figure 7.8. H NMR spectrum of the product PMH (300 MHz, solvent: CDCl3). [TMPCl]= 0.072 mol/L, [MAMS]= 0.16 mol/L, [TiCl4]= 0.13 mol/L, [DtBP]= 0.008 mol/L.
7.3.2.3. Synthesis of 2,4,4,6,6-pentanethylheptane-1-ol (PMH-OH)
PMH-OH was synthesized by hydroboration/oxidation of PMH (Figure 7.6) using the process reported by Kennedy and Ivan5. PMH-OH was obtained with 54% conversion.
227
Figure 7.9. NMR spectra of the product of the hydroboration/oxidation reaction of PMH- OH: (Top) 1H NMR spectrum and (bottom) 13C NMR spectrum (300 MHz, solvent: CDCl3). [PMH]= 0.035 mol/L, [9-BBN]= 0.24mol/L, [KOH]= 0.84 mol/L, [H2O2]= 0.77 mol/L.
Comparison of the 1H NMR spectra of PMH (Figure 7.8) and the PMH-OH intermediate (Figure 7.9) revealed that the vinyl protons ( = 4.86-4.64 ppm) had shifted upfield to = 3.48-3.32 ppm, corresponding to the methylene protons adjacent to the hydroxyl group of PMH-OH. A new signal corresponding to the methyne proton (h) at =
1.68 ppm appeared. The methylene protons (e) and (b) shifted upfield to = 1.27 ppm.
The relative integrals of the methylene protons (i, i’) and the methylene protons (b) and
(c) demonstrate successful functionalization with the integration ratio of 1:1:4.
228 The 13C NMR spectrum (Figure 7.9) showed that the carbon connected to the hydroxyl group appear at δ=69.6 ppm and the methyne carbon (H) appeared at δ=28.72 ppm.
7.3.2.4. Synthesis of 2,4,4,4,6,6-pentanethylheptyl-2-methylacrylate (PMH-
MA)
In the enzyme-catalyzed transesterification using CALB (Figure 7.6), 100% conversion was obtained. The 1H NMR (Figure 7.10) showed the methylene protons adjacent to the hydroxyl group of PMH-OH at = 3.48–3.32 ppm shifted downfield due to the methacrylation as expected. The integration of the vinylidene protons ( = 5.56 and
6.12 ppm) and methyl protons ( = 1.96 ppm) of the newly formed methacrylate-end group indicated quantitative functionalization. In the 13C NMR spectrum of PMH-MA, two carbon peaks corresponding to the carbons of double bond ( = 136-125 ppm) and ester carbon ( = 167 ppm) were observed.
229
Figure 7.10. NMR spectra of the transesterification of vinyl methacrylate with PMH-OH using enzyme catalysis: (Top) 1H NMR spectrum and (bottom) 13C NMR spectrum PMH- MA (300 MHz, solvent: CDCl3). [PMH-OH]= 1.1 mol/L, [VMA]= 3.2 mol/L, [CALB]= 2.2 *10-4 mol/L.
From this model reaction we can conclude that PMH-OH was successfully synthesized from TMP-1 using chlorination, methylallylation, and hydroboration/oxidation. The product structure was confirmed by 1H and 13C NMR spectroscopy. The PMH-OH was subsequently used to transesterify vinyl methacrylate
230 using enzymatic catalysis with 100% yield. Then we moved to the oligomerization of isobutylene using a difunctional initiator.
7.3.3. Oligomerization of Isobutylene
In this work two separate tertiary alcohol initiators were tested in conjunction with TiCl4 to synthesize monodisperse telechelic oligoIBs (Figure 7.11).
Figure 7.11. Synthetic processes for dihydroxy oligoisobutylene using carbocationic initiation.
7.3.3.1. Synthesis of 2,4,4,6,6,8-hexamethylnona-1,8-diene (HMND)
HMND was synthesized as shown in Figure 7.11.
231
Figure 7.12. NMR spectra of the product HMND: 1H NMR spectrum (Top) and 13C NMR spectrum (bottom) (300 MHz, solvent: CDCl3). [DMPDiOH]= 0.061 mol/L, [MAMS]= 0.31 mol/L, [TiCl4]= 0.24 mol/L, [DtBP]= 0.005 mol/L, [DMA]= 0.061 mol/L.
The 1H NMR of the crude 2,4,4,6,6,8-hexamethylnona-1,8-diene (HMND) product obtained by the methallylation of DMPDiOH showed the presence of OH groups, indicating incomplete and/or monodirectional initiation. The HO-containing compounds
232 were easily separated from the product by column chromatography. The 1H NMR spectrum of the product after chromatography, obtained with 24.9% conversion (yield
0.37 g), is shown in Figure 7.12. The signals corresponding to the unsaturated end group protons ( = 4.84-4.63 ppm) relative to the methyl (c) and ethyl (d) protons ( = 1.78 ppm and = 1.35 ppm respectively) indicate that the desired telechelic methallyl structure is the main product, with some contaminants. The 13C NMR spectrum (Figure 7.12) verifies the presence of the expected structure.
7.3.3.2. Synthesis of 2,4,4,6,6,8,8,10-octamethylundeca-1,10-diene (OMUD)
OMUD was synthesized as shown in Figure 7.11. The 1H NMR spectrum of the crude 2,4,4,6,6,8,8,10-octaamethylundeca-1,10 diene (OMUD) shows that the HO- proton signals at 3.4 ppm disappeared and two new signals corresponding to the unsaturated end group protons ( = 4.88-4.66 ppm) appeared. There are some impurities in the aliphatic region.
The crude was purified using column chromatography packed with silica gel and with hexane as the eluent. OMUD was obtained with 56% conversion (yield 1.05 g). The
1H NMR spectrum (Figure 7.13) of pure compound shows the signals corresponding to the unsaturated end group protons ( = 4.88-4.66 ppm). The integration of the saturated protons and methyl protons ( = 1.96 ppm) of the newly formed methallyl end groups indicated quantitative functionalization. The 13C NMR spectrum of OMUD (Figure 7.13) also verified the structure.
233
Figure 7.13. NMR spectra of the product OMUD: 1H NMR spectrum (Top) and 13C NMR spectrum (bottom) (300 MHz, solvent: CDCl3). [TMHDiOH]= 0.048 mol/L, [MAMS]= 0.21 mol/L, [TiCl4]= 0.20 mol/L, [DtBP]= 0.004 mol/L, [DMA]= 0.049 mol/L.
In conclusion, our results demonstrate that the structure of the initiator affects tremendously the initiator efficiency and conversion. When the reaction was initiated using DMPDiOH, only 24% conversion was obtained with 47% initiator efficiency. On
234 the other hand, with TMHDiOH as initiator the conversion was 56% with 94 % Initiator efficiency.
HMND and OMUD were successfully synthesized from DMPDiOH and
TMHDiOH in conjunction with TiCl4. The conversions were 24% and 56% respectively.
The product structures were confirmed by 1H and 13C NMR spectroscopy.
7.3.3.3. Synthesis of 2,10-dichloro-2,4,4,6,6,8,8,10-octamethylundecane
(DiClOMU)
The chlorination reaction of OMUD (Figure 7.11) was achieved by the bubbling of HCl gas into OMUD and it was complete after 1 h to yield the product 2,10-dichloro-
2,4,4,6,6,8,8,10-octamethylundecane (DiClOMU) in a 99% conversion (yield 1.7 g).
1 Figure 7.14. H NMR spectrum of DiClOMU (300 MHz, solvent: CDCl3).
Figure 7.14 shows the 1H NMR spectrum of the product, one new signal corresponding to the methylene protons ( = 1.97 ppm) appeared and the unsaturated end group protons ( = 4.88-4.66 ppm) disappeared. The integration of the mehylene and methyl protons ( = 1.96 ppm) indicate that the desired telechelic chlorinated structure is obtained. 235 7.3.3.4. Synthesis of 4,4,6,6,8,8,10,10,12,12-decamethylpentadeca-1,14-diene
(DMPDD)
The allylation reaction of DiClOMU (Figure 7.11) was complete after 5 h. The 1H
NMR spectrum of the product is shown in Figure 7.15. Four new peaks appear after the reaction: the peaks =5.85ppm (h) and =5.05 ppm (i, i’) corresponding to the allylic protons, the methylene protons (g) at δ=2.04 ppm and the methyl protons (f) at δ=1.11 ppm. The methylene protons (e) shifted upfield to δ=1.43 ppm. The relative integrals of the allyl protons (i, i’), (h) and the methylene protons (g) demonstrate successful functionalization with the integration ratio of 2:4:4. 13C NMR spectroscopy (Figure 7.15) also confirmed the presence of the product DMPDD. The resonances of the allyl carbons are observed at δ = 136.31 (H) and 117.0 ppm (I) and the carbon of the methylene group adjacent to the double bond is observed at δ = 50.47 ppm (G).
236
Figure 7.15. NMR spectra of the product DMPDD: 1H NMR spectrum (Top) and 13C NMR spectrum (bottom) (300 MHz, solvent: CDCl3). [DiClOMU]= 0.029 mol/L, [ATMS]= 0.14 mol/L, [TiCl4]= 0.11 mol/L, [DtBP]= 0.003 mol/L, [DMA]= 0.029 mol/L.
7.3.3.5. Synthesis of 4,4,6,6,8,8,10,10,12,12-decamethylpentadecane-1,15-diol
(DMPDDiOH)
In the next step, DMPDD was converted to the corresponding primary hydroxyl- functionalized oligoIB, via hydroboration followed by alkaline oxidation (Figure 7.11).
DMPDDiOH was obtained with 54% conversion. Comparison of the 1H NMR spectrum of DMPDD (Figure 7.15) and the DMPDDiOH product (Figure 7.16) revealed that the allyl protons at =5.85ppm (h) and =5.05 ppm (i, i’) had shifted upfield to = 3.62 ppm (i), that corresponds to the methylene protons adjacent to the hydroxyl group of
DMPDDiOH and the signal at = 1.54 ppm (h’) that corresponds to the methyne proton.
The relative integrals of the methylene protons (i) and the methylene protons (h’, g) and
(e, c) demonstrate successful functionalization with the integration ratio of 4:8:8
237
1 Figure 7.16. H NMR spectrum of DMPDDiOH (300 MHz, solvent: CDCl3).
In conclusion, we based the synthetic strategy shown in Figure 7.11 on the work of Mayr227 who produced monofunctional IB-Cl by ionizing 2-chloro-2,4,4-
º trimethylpentane with TiCl4 in CH2Cl2 at -80 C. We modified the procedure: 2,6-
dihydroxy-2,4,4,6-tetramethylheptane TMDDiOH was ionized by TiCl4 in the presence of 2,6-di-tert-butyl-pyridine DtBp as a proton trap to prevent initiation by adventitious
o moisture. The first reaction in Figure 7.11 was completed in CH2Cl2 at -80 C and yielded
OMUD with 56% conversion and 94 % initiator efficiency. Hydrochlorination of OMUD was successful. 1H and 13C NMR confirmed the structure of the pentamer dichloride
DiClOMU. Our preliminary results also demonstrated that the structure of the initiator affects tremendously the initiator efficiency and conversion: with the commercially available DMPDiOH (2,4-dimethyl-2,4-pentanediol), only 24% conversion was obtained with 47% initiator efficiency. The hydroboration/oxidation of DMPDD yielded
DMPDDiOH HO-MID-OH with 5 repeat units as shown in Figure 7.11
238 7.4. Preparation of Telechelic Functionalized Polyisobutylenes
7.4.1. Introduction
New methods to precision synthesize α,ω-primary-dihydroxy PIBs (HO-PIB-OH) are of great interest in terms of their versatility for further chemical modification and the synthesis of hydrolysis-resistant polyurethanes.228 It is important to consider the significant reactivity difference between tertiary and primary alcohols for subsequent chain-extension and functionalization reactions.
The first well-defined HO-PIB-OH was obtained by the inifer technique.148 In the inifer method, the polymerization of isobutylene (IB) is mediated by an initiating system
149 150 consisting of a benzylic dichloride (e.g., DiCumCl) initiator plus BCl3 or TiCl4 as coinitiator. The dichloride fulfills two functions: it is an initiator and a chain transfer agent, hence the term inifer. Despite their impressive application in the preparation of linear ,-di-tert-chloro-PIB, cumyl initiators often suffer cycloalkylation by the nucleophilic attack of the initiator of the active-end group in IB polymerization.150 This side reaction leads to the formation of undesirable indanyl and diindane end groups, preventing precise functionalization. Several methods have been developed to prevent the undesirable cycloalkylation, leading to the clean synthesis of tert-chlorine capped Cl-
148 PIB-Cl using various initiating systems with BCl3. However, BCl3 only works in polar solvents that do not dissolve PIBs with Mn > 3000 g/mol. Subsequently it was discovered that living IB polymerization could be induced with TiCl4 in a mixed solvent
150 (Hexane/MeCl 60/40 v/v), a significantly less expensive coinitiator than BCl3.
Important further developments were as follows: (1) one-pot terminal functionalization
239 with the relatively inexpensive allyltrimethylsilane (ATMS) that yielded -CH2-CH=CH2 end groups,148 followed by hydroboration-oxidation, (2) synthesis of ‘‘blocked’’ initiators that would not undergo indanyl ring formation,151 and, importantly, (3) new end- functionalization strategies that eliminated the use of hydroboration-oxidation. Storey et al152 described a simple, rapid synthesis of primary hydroxyl functionalized PIB via thiol–ene click chemistry from exo-olefin terminated PIB with 98% conversion. Puskas
136,161 reported direct functionalization using epoxide/TiCl4 initiating systems, Kennedy reported anti-Markovnikov HBr addition to olefin-functional PIBs followed by hydrolysis, yielding HO-PIB-OH.138 Faust reported the synthesis of hydroxyallyl telechelic PIBs by the hydrolysis of chloro- and bromoallyl PIBs derived from living
PIBs end-capped with 1,3-butadiene.147
This paper reports a new method for the synthesis of fully aliphatic backbone HO-
PIB-OH using aliphatic difunctional initiators in conjunction with TiCl4 coinitiator.
Figure 7.17 shows the synthetic strategy to produce fully aliphatic HO-PIB-OH
240
Figure 7.17. Synthesis of fully aliphatic HO-PIB-OH
The Kennedy school reported the synthesis of Cl-PIB-Cl from the
19 TMHDiOH/BCl3 initiating system. Since TMHDiOH had limited solubility in the
CH3Cl diluent, first the TMHDiOH was reacted with BCl3 in the presence of dimethyl acetamide (DMA) as a strong Electron Pair Donor (ED) to yield TMHDiCl that subsequently initiated IB polymerization. The diether TMHDiOMe with good solubility
18 was also used to produce Cl-PIB-Cl in conjunction with BCl3. These systems are heterogeneous since the forming PIB precipitates from the polar diluents used (CH3Cl
241 and CH2Cl2). No report was found to produce fully aliphatic Cl-PIB-Cl in solution using
TiCl4. In that system the living PIB chains can be turned into allyl-PIB-allyl by termination with allyltrimethylsilane (ATMS). Click chemistry with HS-CH2-CH2-OH would then yield α,ω-dihydroxy-PIB with primary hydroxy groups (HO-PIB-OH). First we screened three initiators, TMHDiOMe, TMHDiOH and TMHDiCl in conjunction with TiCl4 and DMA in Hexane/CH3Cl 60/40 v/v.
7.4.2. Screening Experiments
Table 7.1 summarizes the results of the screening experiments.
Table 7.1. Screening experiments
Initiator Diluent Conversion Mn ĐM Ieff End % g/mol % groups TMHDiOMe Hexane/CH3Cl <1 35,000 1.2 - - TMHDiOH Hexane/CH3Cl 86 16,000 1.6 4 trace CH3Cl 90 8,965 1.7 10 olefin olefin TMHDiCl Hexane/CH3Cl 60 2,166 1.5 34 trace CH3Cl 51 2,573 1.4 24 olefin CH2Cl2 38 2,910 1.8 16 tert-Cl 30% olefin
Tertiary ethers in conjunction with TiCl4 have been shown to be effective initiators in the living solution polymerization of IB in Hexane/CH3Cl. However, we found that TMHDiOMe/TiCl4 after 40 minutes reaction time yielded only traces of PIB with Mn = 35,000 g/mol and ĐM = 1.2. TMHDiOH/TiCl4 yielded 86% IB conversion in
1 40 minutes and a PIB with Mn = 16,400 g/mol with ĐM = 1.6 (Ieff = 4%). The H NMR spectrum showed no signals related to tert-Cl end. The other signals were all in the
242 aliphatic region but the exact end group structure could not be identified.
TMHDiCl/TiCl4 yielded Cl-PIB-Cl with Mn = 2,166 g/mol with ĐM = 1.5 at 60 % IB conversion after 40 minutes and Ieff = 34 %, with a very small high molecular weight shoulder in the SEC trace (Figure 7.18).
Figure 7.18. SEC chromatogram of Cl-PIB-Cl. [TMHDiCl]= 0.013 mol/L; [TiCl4]=0.053 mol/L; [DtBp]= 0.0007 mol/L; [DMA]= 0.013 mol/L; [IB]=0.32 mol/L; CH3Cl/ Hexane, 40/60, v/v, Vo=180 mL. -80 ºC Reaction time 40 min.
The 1H NMR spectrum (Figure 7.19) verifies the expected structure. The peaks designated as (a) and (b) at δ = 1.97 and 1.70 ppm correspond to the methylene (CH2) and methyl protons (CH3) next to the tert-Cl chain end, respectively. The peak at δ = 1.43 ppm (c) is assigned to the methylene protons of the repeat unit. The Mn of the polymer was calculated from the integral ratio of the protons of the methylene end group [δ =1.97 ppm (a)] to the backbone methylene protons [δ = 1.43ppm (c)] as Mn = 1,779 g/mol,
243 which is in good agreement with the Mn = 2,166 g/mol by SEC. However, the spectrum shows trace amount of exo/endo olefinic structures at δ =4.86 ppm, δ =4.83 ppm and δ
=4.65 ppm.
1 Figure 7.19. H NMR spectrum of PIB obtained with system TMHDiCl/TiCl4. For conditions see. Figure 7.18.
Next screening experiments were carried out in CH3Cl and CH2Cl2. PIB with Mn
> 3000 g/mol is not soluble in these systems so it would precipitate after the addition of
TiCl4. TMHDiOMe/TiCl4 yielded no polymer in CH3Cl, and only traces of polymer in
CH2Cl2. TMHDiOH/TiCl4 in CH3Cl produced polymer with Mn = 8.965 g/mol, ĐM =
1.71 at 90% IB conversion and Ieff = 10 %. However, the same system yielded no polymer in CH2Cl2. TMHDiCl/TiCl4 in CH3Cl yielded Cl-PIB-Cl Mn = 2,573 g/mol, ĐM
244 = 1.4 at 51% IB conversion and Ieff = 24 %. These data are quite similar to those obtained in the solution system (see Table 1). However, in this case no olefinic end groups were detected. The Mn = 2,540 g/mol calculated from the NMR spectrum is in good agreement with the SEC measurement. The same initiating system in CH2Cl2 yielded a polymer with
1 Mn = 2,910 g/mol and ĐM = 1.84 at 38 % IB conversion (Ieff = 16%). The H NMR of this polymer showed end groups with tert-Cl and exo/endo olefinic structures at δ =4.86 ppm,
δ =4.83 ppm and δ =4.65 ppm.
While TMHDiCl/TiCl4 gave clean Cl-PIB-Cl in CH3Cl, the Ieff values were low and TMHDiCl is very unstable so it is difficult to isolate and purify. Therefore we studied the in situ formation of TMHDiCl from TMHDiOH.
7.4.3. In situ Chlorine Exchange
7.4.3.1. Chlorine Exchange in CH2C12
The screening experiments yielded olefinic end groups in CH2Cl2, so first this solvent was studied. The chlorine exchange reaction with TMHDiOH /TiCl4 was performed by taking aliquots to determine the reaction time. In these experiments the addition sequences of the ingredients were: CH2Cl2, TMHDiOH, DMA, DtBP and TiCl4.
Aliquots were taken (18min, 55 min, 2h, 3h and 4h), quenched in NaOH/MeOH and analyzed by NMR. Based on the 1H NMR spectra (not shown) the chlorine exchange was complete after 4 h. In a separate reaction after 4 h chlorine exchange the necessary amount of hexane was added to keep the system in solution (Hexane/CH2Cl2 60/40 v/v),
245 followed by IB. The reaction was allowed to proceed for 40 minutes before termination with methanol.
Figure 7.20. SEC traces of the PIB made by in situ chlorine exchange. First Step: [TMHDiOH]0= 0.021M; [TiCl4]0=0.084M; [DtBp]0= 0.0018M; [DMA]0= 0.021 M; CH2Cl2,V0 =500 mL -80ºC, 4 h. Second step: Addition of 737 mL of Hexane and 16 mL of IB [TMHDiCl]f= 0.008M; [TiCl4]f=0.034M; [IB]=0.28M; Vf =1253 mL. Reaction time: 40 minutes.
The SEC traces of the polymer with light scattering (LS) and refractive index (RI) detectors are shown in Figure 7.20. A high MW shoulder was visible in the LS and RI traces, indicating the presence of a high MW polymer fraction (Mn=23,500 g/mol). Mn =
2,201 g/mol and ĐM = 1.97 was measured by SEC, assuming 100% mass recovery. The
1 IB conversion was 89 % and the initiator efficiency Ieff = 26%. The H spectrum showed the formation of Cl-PIB-Cl with no traces of olefinic end groups. The Mn = 2,266 g/mol calculated from NMR is in good agreement with the SEC measurement. 246 7.4.3.2. Chlorine Exchange in CH3Cl
The same reaction was performed in CH3Cl, but with higher TiCl4 concentration.
In this case chlorine exchange, monitored by NMR, was complete within 1 hour. In a separate reaction, after 1 h chlorine exchange the necessary amount of hexane was added to keep the system in solution (CH3Cl/Hexane (40:60 v/v) and polymerization was initiated by the addition of IB. After 10 min the reaction was stopped. The conversion was 97% and the initiator efficiency Ieff = 26%. SEC analysis gave Mn = 3,401 g/mol with
1 ĐM = 1.20. The H spectrum demonstrated the formation of clean Cl-PIB-Cl. The Mn=
3,941 g/mol calculated from the NMR is in good agreement with the SEC data.
7.4.4. Synthesis of allyl-PIB-allyl
The synthetic strategy is shown in Figure 7.21.
Figure 7.21. Synthesis of allyl-PIB-allyl. First Step: [TMHDiOH]0= 0.042M; [TiCl4]0=0.85M; [DtBp]0= 0.0018M; [DMA]0= 0.042 M; CH3Cl, V0 =94 mL; -80ºC, 1 h. Second step: Addition of 141 mL of Hexane and 5.6 mL of IB. [TMHDiCl]f= 0.016 M; [TiCl4]f=0.32 M, [IB]=0.29M; Vf =250 mL, after 10 min addition of [ATMS]=0.32 mol/L. Intermediate TMHDiCl is in equilibrium as shown in Figure 7.17.
After the chlorine exchange reaction (1 h, TMHDiOH/TiCl4) hexane and IB were added. After 10 min reaction time the polymerization was terminated by ATMS (Scheme
2). The resulting material had Mn = 3,797 g/mol with ĐM = 1.20. At 97% conversion Ieff =
26% was calculated. Figure 7.22 shows the 1H NMR spectrum of allyl-PIB-allyl. The
247 proton resonances at δ=1.13 ppm (e) and δ=1.45 ppm (d) correspond to the methyl and methylene protons of the repeat unit of PIB, respectively. The proton resonances at
δ=2.00 ppm correspond to the methylene protons(c) of the end group. Resonances at
δ=5.04 ppm (b) and δ=5.77 ppm (a) confirm the presence of allylic end groups in the polymer. The Mn of the polymer was calculated from the ratio of the relative integral of vinylidene protons at δ = 5.02 ppm (b) to that of the backbone methylene protons at δ =
1.43 ppm (d). The Mn = 4,046 g/mol, is in good agreement with the SEC data (Mn =
3,797 g/mol).
1 Figure 7.22. H NMR spectrum allyl-PIB-allyl. (300 MHz, solvent CDCl3)
7.4.5. Synthesis of HO-PIB-OH
HO-PIB-OH was synthesized by thiol-ene chemistry as reported by Storey
(Figure 7.23).152
248
Figure 7.23. Synthetic route of HO-PIB-OH by thiol-ene reaction using mercaptoethanol: [allyl-PIB-allyl]=0.025 mol/L, [mercaptoethanol]= 0.61 mol/L, [Irgacure]= 0.24 mol/L
Figure 7.24 shows the 1H NMR spectrum of the HO-PIB-OH. The peaks at δ =
1.97 and 1.70 ppm correspond to the methylene (CH2) and methyl protons (CH3) of the
PIB chain, respectively. The methylene protons adjacent to the hydroxyl head group are observed at δ = 3.73 ppm (g). The resonances at δ=2.74 ppm, δ=2.50 ppm and δ = 1.70 ppm corresponds to methylene protons (f), (b’) and (a’) respectively. Mn = 4,362 g/mol was calculated from the integral ratio of the protons of the methylene end group [δ = 3.73 ppm (g)] to the backbone methylene protons [δ = 1.43ppm (c)].
1 Figure 7.24. H NMR spectrum of HO-PIB-OH. (300 MHz, solvent CDCl3).
SEC analysis yielded Mn =4,085 g/mol and ĐM =1.2, with a very clean monomodal trace (Figure 7.25). 249
Figure 7.25. SEC chromatogram of allyl-PIB-allyl and HO-PIB-OH.
In summary, a new fully aliphatic HO-PIB-OH, carrying two terminal primary hydroxyl end groups has been prepared from allyl-PIB-allyl by thiol-ene click reaction with mercaptoethanol. The allyl-PIB-allyl was synthesized by a new method: first chlorine exchange reaction with TMHDiOH/TiCl4 for 1h , followed by the addition of hexane and IB and then termination of the polymerization with ATMS. 1H-NMR analysis verified the structure of the HO-PIB-OH.
250 8. CHAPTER VII
SUMMARY AND RECOMMENDATIONS
8.1. Summary
Precise enzymatic functionalization of polymers was achieved using two methods: enzyme-catalyzed functionalization via polymerization and enzymatic functionalization by chain end functionalization.
In Chapter IV halo-ester functionalized PEGs were successfully prepared by the transesterification of alkyl halo-esters with PEGs using Candida antarctica lipase B
(CALB) as a biocatalyst under solventless conditions. Transesterifications of chlorine, bromine and iodine esters with tetraethylene glycol monobenzyl ether (BzTEG) were quantitative in less than 2.5h. The transesterification of halo-esters with PEGs were complete in 4h. The same methodology was extended to produce N3-PEG-N3-3400.
Additionally, the preparation VA-PEG-OH1100 was possible via enzymatic transesterification of vinyl acrylate (VA) with HO-PEG-OH1100 in the presence of CALB.
It was found the presence of a mixture of unreacted HO-PEG-OH1000, monosubstituted
VA-PEG-OH1000 and disubstituted VA-PEG-VA1000 with the unreacted HO-PEG-OH1000 being the major distribution. Conditions were optimized where the difunctional product doesn’t overcome the 5% of the mixture, and the monofunctional product was produced
251 in a significant amount. Column chromatography was necessary for the purification process. VA-DVA was reacted with HO-PEG-OH8000 in the presence of CALB.
The chemoselectivity of CALB was also analyzed using first a model compound and later a polymer. The Michael Addition of 2,2'-(ethane-1,2-diylbis(oxy))diethanamine to 3-(acryloyloxy)-2-hydroxypropyl methacrylate in the presence of CALB was described. This served as a model reaction of the PEG functionalization. The 1H NMR spectrum confirmed that the Michael addition to the acrylate group was complete within
30 min, while the methacrylate group remained intact. Similar results were achieved in the Michael Addition of NH2-PEG2000-NH2 to 3-(acryloyloxy)-2-hydroxypropyl methacrylate in the presence of CALB. This Chapter IV also discussed the functionalization of small molecules using CALB. DVA was reacted with 2HEA in the presence of CALB in bulk conditions the reaction was complete after 3 hours. Bis (2- ethyl 5-bromopentanoate) disulfide was synthesized when 2HEDS was reacted with
EBrV in the presence of CALB. Additionally a SH-DVA linker was synthesized by two steps. The first step involved the transesterification reaction of DVA with 2-hydroxyethyl disulfide using CALB to yield DVA-S-S-DVA product. The second step involved the disulfide bond reduction using DTT to yield HS-DVA.
Chapter V discussed the functionalization of polymers via enzymatic polymerization: Ring Opening Polymerization (ROP) and polycondensation. The combination of enzymatic polymerization and functional initiating systems resulted in the metal free synthesis of poly(caprolactone) which normally requires the use of tin or other transition metal which is difficult to remove and not ideal for biomedical applications of
252 the material. Propargyl alcohol and 4-dibenzocyclooctynol (DIBO) were shown to efficiently initiate the polymerizations under metal free conditions and yielded 4 to 24
KDa polymers with relatively narrow molecular mass distribution. The alkyne bonds in the strained and unstrained initiating systems survived the polymerization conditions and the purification process. The resulting polymers can be further functionalized using metal free and copper catalyzed click chemistry conditions.
Chapter V also discussed the quantitative vinyl chain-end functionalization of
PEGs. DVA was transesterified with tetraethylene glycol (TEG) and PEGs using CALB as a biocatalyst at 50 oC under dry nitrogen and solventless conditions. During transesterification with TEG polycondensation occurred. With the judicious selection of molar equivalencies and reaction times both symmetric and asymmetric telechelic TEGs were produced with high efficiency. Additionally, at DVA/TEG 20/1 molar ratio ~82% of the product was the Vinyl-TEG-Vinyl, together with dimers and trimers. Vinyl-PEG-
Vinyl was obtained with HO-PEG-OH of Mn = 2,000 g/mol. with. After the reactions excess DVA was removed by hexane extraction and recovered by distilling off the hexane.
The synthesis of poly(isobutylene-b--caprolactone) PIB-b-PCL diblock and poly(-caprolactone-b-isobutylene-b--caprolactone) PCL-b-PIB-b-PCL triblock copolymers was accomplished using a combination of living carbocationic polymerization of isobutylene (IB) with the ring-opening polymerization (ROP) of - caprolactone (-CL). The synthesis involved the living carbocationic polymerization of
IB using 1,2-propylene oxide and 2,4,4,6-tetramethyl-heptane-2,6-diol /TiCl4 as the
253 initiating system to yield PIB-OH and HO-PIB-OH, respectively. Which were used as macroinitiators for the ROP of -CL using CALB, yielding PIB-b-PCL diblock and PCL- b-PIB-b-PCL triblock copolymers, respectively. DSC, TEM and AFM demonstrated that the amorphous PIB and the semicrystalline PCL block segments are phase separated, creating nanostructured phase morphology.
Chapter VI covered the chemo-enzymatic approach for biomolecules functionalization. Precisely: Folic Acid, Biotin, Fluoruracil and FITC. We disclose a new process for the preferential/exclusive γ-substitution of the glutamic acid moiety in folic acid and similar drugs, leaving the bioactive α-position available. Biotin was reacted with exclusively amine group of NH2-PEG-OH3400 in the presence of triethylamine via nucleophilic addition. Vinyl ester of fluorouracil was prepared by Michael addition of fluorouracil to vinyl acrylate. Additionally, a fluorouracil disulfide linker was also synthesized. Finally, the synthesis of FITC linkers using enzymatic catalysis is described.
As established in the Chapter II this thesis was aimed to develop a modular synthetic approach for the synthesis of a vitamin-polymer conjugate using enzymatic catalysis where possible. FITC-PEG-FA conjugate was synthesized using this approach. The use of CALB gives the advantage of being a green methodology for this functionalization.
1H and 13C NMR spectroscopy with MALDI-ToF and ESI mass spectrometry confirmed the structure and purity of the products.
Living mode of cationic polymerization provides to introduce functional groups at polymer chain termini. Chapter VII described the synthesis of low molecular mass telechelic PIBs. It started with the synthesis of IB oligomers. We based the synthetic
254 strategy on the work of Mayr45 who produced monofunctional IB-Cl by ionizing TMPCl with TiCl4. We then modified the procedure: 2,6-dihydroxy-2,4,4,6-tetramethylheptane
46 (TMDDiOH, synthesized as reported), was ionized by TiCl4 in the presence of DtBp to yield OMUD with 56% conversion and 94 % initiator efficiency. Hydrochlorination of
OMUD was successful to yield the pentamer dichloride DiClOMU. The hydroboration/oxidation of DMPDD yielded DMPDDiOH with 5 repeat units with 56% conversion.
In the course of this investigation it was of interest to determine whether and under what conditions would the tert-alcohols (TMHDiOH), tert-chloride (TMHDiCl) and tert-ether (TMHDiOMe) diinitiator with TiCl4 complex lead to well defined Cl-PIB-
Cl. A new telechelic polyisobutylene diol, HO-PIB-OH, carrying two terminal primary hydroxyl end groups has been prepared from α,ω-di(isobutenyl)polyisobutylene, allyl-
PIB-allyl, by thiol-ene click reaction of mercaptoethanol. The thiol-ene click reaction is more efficient and simpler than the often-used hydroboration-oxidation method – this was demonstrated with an allyl-functionalized oligomer PIB. 1H-NMR analysis of the polymers indicates quantitative functionalization to two hydroxy termini per polyisobutylene chain. The telechelic diol prepolymer opens new avenues to the synthesis of many new materials, e.g., polyurethanes, polyesters.
Advantages
The resin-supported enzyme can easily be separated from the reaction mixture, producing very pure products. According to previous works, the resin-supported CALB can be recycled at least 4 times without loss of activity from physical desorption or
255 leaching of the CALB from the acrylic resin. Both the absence of solvents and the recyclability of the catalyst are endorsing green chemistry concepts.
Limitations
In terms of CALB-based transformations in solution: Challenges surround issues of incompatibility between the solvents tolerate by the CALB and the substrates solubility. In the case of solventless transformations, the substrate must be liquid at the working temperature (50 ºC) and in the case of polymerization of polyesters the formation of highly viscous product limits the application. If the reaction proceeds for long periods of time, the CALB starts to catalyze the hydrolysis of the polyesters. As a result, it is still quite challenging to synthesize high molar mass polyesters. The control of polymerization remains less efficient with enzymes than with chemical initiators.
8.2. Recommendations
8.2.1. Recyclability Study
A recyclability study to reduce process costs showing the economic advantage of the use of CALB in biotransformation will help to commercialize these new methods.
8.2.2. Biological Activity Test
In vitro screening needs be carried out. Three cell lines can be co-cultured with the FITC-PEG-FA synthesized in this work. The cell lines could be: MDA-MB-468, a folate negative breast cancer cell line; MDA-MB-231, a folate expressing breast cancer cell line; and MCF10, an unaltered breast cancer cell line.
256 8.2.3. Multivalent Drug-Delivery System
The controlled presentation of multiple targeting ligands by a single macromolecule is commonly referred to as multivalent targeting. The approach relies on presenting an optimized arrangement of targeting ligands to a set of expressed receptors on the cell surface. New ongoing work is aimed at demonstrating that a platform of folate-targeted Multivalent Polymer Diagnostic/Drug Conjugates (MPDC) has better efficiency than the competitive folate-targeted Small Molecule Diagnostic/Drug
Conjugates (SMDC) platform of Endocyte LLC. SMDCs are currently in Phase III clinical trials.
8.2.4. Biodegradables Pressure Sensitive Adhesive Matrix Patches
A method of transdermally or transmucosally delivering of a drug with a pressure sensitive hydrophobic adhesive matrix patch may be of interest. One reason that PIBs can be used for this application is the polymer’s critical surface tension. In order for an adhesive to wet a substrate, the surface energy of the adhesive must be equal or less than that of the adherend. For skin, the critical surface energy varies between 38 and 56 mN m-1 depending on the temperature and relative humidity of the skin. PIB has a critical surface tension of 30 to 32 mN m-1 so wetting is possible.
I propose to explore the adhesive properties of the new poly(isobutylene-b-- caprolactone) PIB-b-PCL diblock and study its degradation behavior.
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271 APPENDIX
1 Determination of Mn of PIB by H NMR spectroscopy:
SEC traces of PIB polymer using as initiator TMHDiCl/TiCl4 initiating complex at -80ºC in CH3Cl.
The peaks designated as (a) and (b) at δ = 1.97 and 1.70 ppm, respectively, in the
1 H NMR spectrum correspond to the methylene (CH2) and methyl protons (CH3) of the isobutenyl chain end. The peak at δ = 1.43 ppm (c) is assigned to the methylene protons 272 of the repeat unit. The Mn of the polymer was calculated from the integral ratio of the protons of the methylene end group [δ = 1.97 ppm (a)] to the backbone methylene protons [δ = 1.43ppm (c)]. According to 1H NMR the number average molecular weight
(Mn) was calculated as Mn = 2,341 g/mol, which is in good agreement to Mn detected by
SEC (Mn = 2,573 g/mol, ĐM = 1.37).
( )
( ) ( ) )
( ) ( )
273
1 H NMR spectra of Cl-PIB-Cl polymer using as initiator TMHDiCl-TiCl4 initiating complex at -80ºC.
274