FUNCTIONALIZABLE BIODEGRADABLE POLYESTERS FOR
DRUG DELIVERY APPLICATIONS
A Dissertation
Presented to
The Graduate Faculty of The University of Akron
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
Abhishek Banerjee
May, 2012
FUNCTIONALIZABLE BIODEGRADABLE POLYESTERS FOR DRUG DELIVERY
APPLICATIONS
Abhishek Banerjee
Dissertation
Approved: Accepted:
______Advisor Department Chair Dr. Coleen Pugh Dr. Ali Dhinojwala
______Committee Chair Dean of the College Dr. Li Jia Dr. Stephen Cheng
______Committee Member Dean of the Graduate School Dr. Abraham Joy Dr. George R. Newkome
______Committee Member Date Dr. William J. Landis
______Committee Member Dr. Yang H. Yun
ii
ABSTRACT
Current biodegradable polymers, like poly(lactic acid) (PLA), poly(glycolic acid)
(PGA) and their copolymers (PLGA) do not have functionalities on their backbones.
Such biodegradable polymer systems are therefore not able to covalently attach drugs or
other therapeutic molecules, which could be useful for making drug delivery devices.
Instead, the therapeutic molecules must be physically entrapped into these polymers,
either by forming micelles or by nano-encapsulation, thereby limiting their loading capacity.
Our research deals with the polyesterification of 2-bromo-3-hydroxypropanoic acid which is a halogenated isomer of lactic acid, yet has a primary alcohol group like glycolic acid. It is therefore an ideal co-monomer for incorporation into PLGA. Such co- polyesters are potentially biodegradable with halogen functionalities on the main chain.
We have synthesized brominated copolymers with LA and GA of number average molecular weights around 20,000 Da (PS standards), under bulk co-polymerization conditions. The number of functionalizable sites on the main chain of this polyester is controlled by varying the feed ratio of the halogen co-monomer. The biodegradability of
the polymer can also be tailored by varying the lactic acid and glycolic acid feed ratios.
Solution polymerization using carbodiimide chemistry at room temperature has also been
explored to prepare these co-polymers, with lesser success.
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The biodegradation behavior of these brominated polyesters were studied in the
form of compression molded tablets under physiological conditions, Phosphate buffer saline (pH 7.4), at 37 °C and these polymers were found to degrade to around 80% of their initial molecular weights.
iv
DEDICATION
This dissertation is dedicated to my grandfather, Subhash Chatterjee. I wish life allowed me to spend more time with him.
This dissertation is also dedicated to my parents, Kalpana and Tarun Banerjee. It is their hard work, sacrifices and exceptional parenting that is mostly responsible for my successes in past, present and in future.
This dissertation is also dedicated to my elder brother, Abhijeet Banerjee. He is a friend and mentor.
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ACKNOWLEDGEMENTS
I would like to begin by thanking my parents for supporting me, both financially and emotionally, throughout my academic career. Any success is a manifestation of their sacrifices, foresight and excellent parenting.
I am extremely grateful to my doctoral advisor, and mentor, Dr Coleen Pugh, for giving me the opportunity to perform research in her lab. I have been fortunate to learn and practice not only chemistry but also valuable life lessons. I wish that I had even half of her intelligence, energy and dedication towards everything she does.
I acknowledge my doctoral dissertation committee members, Dr. Li Jia, Dr.
Abraham Joy, Dr William Landis, and Dr Yang Yun who took the time to read my dissertation and give constructive feedback.
I thank my colleagues, both past and present, from Dr Pugh’s research group, Dr
Anirudha Singh, Dr Chau Tang, Dr James Baker, Lisa Collette, Gladys Montenegro,
William Storms, Colin Wright, Mina Garcia, Stephanie Vivod, Cesar Lopez, Xiang
Yan, Ajay Amrutkar, Brinda Shah and Nicole Swanson.
I would like to thank Dr Andrew Ditto, and Kush Shah from the Dr Yun research group who helped me formulate micro-particles and helping me with the dynamic light scattering experiments.
vi
I would like to thank Rajarshi Sarkar, for helping me with hydrogenation reactions, and wish him good luck the remaining of his doctoral research.
I thank Dr Sachin Gokhale for proving me insight into silicon deprotection chemistry.
I would like to thank Dr Boje Wang and Dr Sarang Bhawalkar who helped me with Scanning Electron Microscopy.
I would like to thank my graduate school buddies, Kurt Chiang and Andy
Heidenreich, who made graduate school fun.
On a personal note, I would like to thank Adam Pilz, whose friendship over the years made life in akron enjoyable, and his influence helped me broaden my horizons beyond science – into the world of economics, finance and business.
I would also like to acknowledge the friendship of Dr Abhimanyu Kumar, as an
‘almost roommate’, who was always motivating and great company. I am grateful to have known and been roommates with Sushil Sivaram, whose excellent cooking skills and conversations I still miss.
I would like to thank my oldest friend, Dr Sunny Sethi, whose friendship and advice has stuck with me, thick and thin.
Finally, I would like to acknowledge lady luck, who has always taken care of things beyond my control.
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TABLE OF CONTENTS
Page
LIST OF TABLES ...... xii
LIST OF FIGURES ...... xiv
LIST OF SCHEMES ...... xix
CHAPTER
I. INTRODUCTION ...... 1
II. LITERATURE REVIEW ...... 4
2.1 Application of Biodegradable Polymers ...... 6
2.1.1 Ecological Applications ...... 7
2.1.2 Medical Application ...... 8
2.2 Motivation and Scope ...... 13
2.3 Synthesis of Poly(hydroxy acids) ...... 21
2.3.1 Bulk ...... 21
2.3.2 Solution ...... 22
2.3.3 Sequence Controlled Copolymers ...... 26
2.4 Biodegradation ...... 27
2.4.1 Effect of Structure and Environment on Biodegradation ...... 33
2.4.1.1 Crystallinity ...... 33
2.4.1.2 Hydrophilicity ...... 35
2.4.1.3 Acid autocatalysis ...... 35
viii
2.4.2 Experimental aspects of Studying biodegradation ...... 37
2.4.2.1 Sample Preparation ...... 38
2.4.2.2 Biodegradation Markers ...... 39
2.4.2.2.1 Visual Examination ...... 39
2.4.2.2.2 Water Absorption ...... 40
2.4.2.2.3 Weight Loss ...... 41
2.4.2.2.4 Molecular Weight Loss ...... 42
2.4.2.2.5 Change in pH ...... 44
III. EXPERIMNETAL METHODS ...... 45
3.1. Materials ...... 45
3.2. General Techniques ...... 46
3.3. Synthesis of 2-bromo-3-hydroxypropionic acid ...... 49
3.4. Synthesis of poly(lactic acid-co-glycolic acid-co-2-bromo-3-hydroxypropionic acid) (PLGB801010) by bulk polycondensation ...... 50
3.5. Synthesis of poly(lactic acid-co-2-bromo-3-hydroxypropionic acid) (PLB8020) by solution polymerization ...... 51
3.6. Synthesis of benzyl (DL)-lactate (Bn-LA) ...... 52
3.7. Synthesis of benzyl glycolate (Bn-GA) ...... 53
3.8. Synthesis of methyl 2-(tert-butyldiphenylsilanyloxy) acetate ...... 54
3.9. Synthesis of (tert-butyldiphenylsilanyloxy) acetic acid (GA-SiTBDP) ...... 55
3.10. Synthesis of methyl 2-((tert-butyldiphenylsilyl)oxy)propanoate ...... 56
3.11. Synthesis of 2-((tert-butyldiphenylsilyl)oxy)propanoic acid (LA-SiTBDP) ..... 57
3.12. Synthesis of methyl 2-bromo-3-hydroxypropionate ...... 58
3.13. Synthesis of 2-bromo-3-((tert-butyldiphenylsilyl)oxy)propionate ...... 58
3.14. Synthesis of 2-bromo-3-((tert-butyldiphenylsilyl)oxy)propionic acid ...... 59
3.15. Synthesis of Bn-GL-SiTBDP (p-GL-p) ...... 60
ix
3.16. Synthesis of HOOC-GL-SiTBDP (HOOC-GL-p) ...... 61
3.17. Synthesis of Bn-LGL-SiTBDP (p-LGL-p) ...... 62
3.18. Synthesis of Bn-LB-SiTBDP (p-LB-p) ...... 63
3.19. Synthesis of Bn-LB-OH (p-LB-OH) ...... 64
3.20. Synthesis of Bn-LBrL-SiTBDP (p-LBL-p) ...... 65
IV. FUNCTIONALIZABLE BIODEGRADABLE POLYESTERS ...... 66
4.1. Synthesis of Functionalized Monomer ...... 66
4.2. Synthesis of PLGB polymers ...... 69
4.2.1. PLA and PLGA copolymers ...... 70
4.2.2. PLB copolymers ...... 76
4.2.3. PLGB copolymers ...... 83
4.3. Microparticle formulation with PLGB copolymers ...... 94
4.3.1. Microparticle formulation procedure ...... 94
4.3.2. Microparticles of PLGB601030 ...... 95
4.3.3. Microparticles of PLGB502030 ...... 97
4.4. Solution Polymerization ...... 99
V. SEQUENCE CONTROLLED FUNCTIONALIZED POLYESTERS ...... 107
5.1. Reactivity Difference ...... 107
5.2. General Strategy for Synthesizing the Trimers ...... 110
5.3. Synthesis of the Building Blocks ...... 115
5.3.1. Benzyl Protection of Hydroxy Acids ...... 115
5.3.2. Silyl Protection of the Hydroxy Acids ...... 120
5.4. Synthesis of the LGL trimer ...... 133
5.4.1. Synthesis of the p-GL-p Dimer ...... 133
5.4.2. Synthesis of HOOC-GL-p Dimer ...... 136
x
5.4.3. Synthesis of p-LGL-p Dimer ...... 138
5.5. Synthesis of the LBL trimer ...... 143
5.5.1. Synthesis of p-LB-p Dimer ...... 144
5.5.2. Synthesis of p-LB-OH Dimer ...... 148
5.5.3. Synthesis of the p-LBL-p Trimer ...... 151
VI. BIODEGRADATION OF FUNCTIONALIZABLE POLYESTERS - PLGB ...... 157
6.1. Degradation ...... 157
6.2. Sample Preparation ...... 157
6.3. Degradation Experiment ...... 158
6.4. Degradation ...... 159
6.4.1. Degradation by Visual Inspection ...... 160
6.4.2. Moisture Absorption ...... 163
6.4.3. Weight Loss ...... 166
6.4.4. Molecular Weight Loss ...... 169
REFERENCES ...... 177
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LIST OF TABLES
Table Page
2.1 Classification of biodegradable polymers...... 6
2.2 Monomers and structures of common products made from biodegradable polymers in biomedical applications...... 8
2.3 Reactivity of poly(methylacrylate)-Br towards primary amines ...... 16
2.4 Reactivity of bromine end functionalized polyacrylate model vs. chlorine end functionalized polyacrylate model towards primary and tertiary amines ...... 18
2.5 Comparison of the relative hydrolysis rates (at 25°C, pH 7) of different chemical bonds ...... 29
4.1. Molecular weight and glass transition temperature of PLA and PLGA synthesized by bulk polycondensation conditions ...... 71
4.2. Molecular weight and glass transition temperature of PLB copolymers synthesized by bulk polycondensation conditions ...... 77
4.3. Molecular weight and glass transition temperature of PLGB copolymers synthesized by bulk polycondensation conditions...... 85
4.4. PLGB copolymers used for microparticle formulation ...... 94
4.5. Increase in number average molecular weight by chain extension using DiPC, with and without the dehydration by solvent recycling ...... 101
4.6. Increase in number average molecular weight by chain extension using DiPC, after dehydration by solvent recycling ...... 103
5.1. Degree of of polymerization of PLGA compared with PLB copolymers, for 10% and 20% co-polymer ratios ...... 108
6.1. Percent moisture absorption for the PLGB613 and PLGB523 copolymers during their biodegradation in PBS (37 °C)...... 164
xii
6.2. Percent weight change for the PLGB601030 and PLGB502030 copolymers during their biodegradation in PBS (37 °C)...... 169
6.3. Loss in molecular weight data for the PLGB601030 copolymer...... 170
6.4. Loss in molecular weight data for the PLGB502030 copolymer...... 173
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LIST OF FIGURES
Figure Page
2.1 Biodegradable PLA cold drink cups and food trays ...... 7
2.2 Phusiline® Screws made from PLA, used to fix graft in (a) knee ligament reconstruction, and (b) foot surgery ...... 9
2.3 Schematic illustration of time course of changes in mass, molecular weights and mechanical strength during biodegradation of polymeric biomaterials undergoing either bulk degradation or surface erosion...... 32
2.4 Weight- average molecular weight (Mw) of high molecular weight (160,000 Da) PLLA mixed with 0%(●), 10%(■), 20%(▲), 30%(♦) oligomers (2,000 Da) ...... 34
2.5 SEM image of cross-section of a PLGA (75/25) specimen after 10 days of degradation in phosphate buffer (pH = 7.4) at 37 °C ...... 36
2.6 Percent of molecular weight remaining compared to day 0 values as a function of degradation time for (a) PLGA 75:25 (Mw = 67,700 Da), (□) thin and (◊) thick films (b) PLGA 50:50 (Mw = 43,900 Da), (○) thin and (∆) thick films. Thick films = 100 µm, thin films = 10 µm.39 ...... 36
2.7 Evolution of water absorption during the degradation of PLA50 (Mw = 153,100 Da, Mw/Mn = 1.8) in the shape of plates (1.5 mm), in phosphate buffer at 60°C and pH 7.4. 8 ...... 41
2.8 Evolution of weight loss during the degradation of PLA50 (Mw = 153,100 Da, Mw/Mn = 1.8) in the shape of plates (1.5 mm), in phosphate buffer at 60°C and pH 7.4.8 ...... 42
2.9 Gel permeation chromatograms of thin PLGA 50:50 (thickness = 10 µm, Mw = 43,900 Da, pdi = 4.1) films showing the changes in the molecular weight distribution (Mw) during degradation: at day 0 (1), after 21 days (2), and 56 days (3)...... 43
2.10 Evolution of pH during degradation of PDLLA (Mw = 65,000 Da, pdi = 1.6) in parallelepipedic shape (15 mm x 10 mm x 2 mm) in an unbuffered solution.24, 35 ...44
xiv
4.1. 1H NMR (300 MHz) spectrum of 2-bromo-3-hydroxypropionic acid synthesized by deaminohalogenation route; * = CHCl3...... 68
4.2. 13C NMR (75 MHz) spectrum of 2-bromo-3-hydroxypropionic acid synthesized by deaminohalogenation route; * = CHCl3...... 69
4.3. 1H NMR (300 MHz) spectra of PLA and PLGA synthesized by bulk polycondensation reaction conditions; * = CHCl3...... 72
4.4. 13C NMR (75 MHz) spectra of PLA and PLGA8020, synthesized by bulk polycondesantion; * = CHCl3...... 74
4.5. The gel permeation chromatographs of PLA and PLGA copolymers synthesized by bulk polycondensation...... 76
4.6. 1H NMR (300 MHz) spectra of PLB copolymers synthesized by bulk polycondensation; * = CHCl3...... 79
4.7. 13C NMR (75 MHz) spectrum of PLB5050 copolymer synthesized by bulk polycondensation; * = CHCl3...... 80
4.8. Expanded 13C NMR (75 MHz) spectra of PLA and PLB5050 copolymer, showing the methine (CH) and methylene (CH2) carbons...... 81
4.9. The gel permeation chromatographs of five PLB copolymers synthesized by bulk polycondensation...... 82
4.10. 1H NMR (300 MHz) spectra of PLGB co-polymers synthesized by bulk polycondensation reaction conditions; * = CHCl3...... 85
4.11. 1H NMR (300 MHz) spectra of PLGB co-polymers synthesized by bulk polycondensation reaction conditions; * = CHCl3...... 87
4.12. 13C NMR (75 MHz) spectra of PLB5050 and PLGB502030 co-polymers synthesized by bulk polycondensation; * = CHCl3...... 88
4.13. Expanded 13C NMR (75 MHz) spectra of PLA, PLGA8020, PLB5050 and PLGB502030, showing the methine (CH) and methylene (CH2) carbons...... 90
4.14. The gel permeation chromatographs of PLGB copolymers, with 10% glycolic acid content, synthesized by bulk polycondensation...... 91
4.15. The gel permeation chromatographs of PLGB copolymers, with 20% glycolic acid content, synthesized by bulk polycondensation...... 92
4.16. Particle sizes of PLGB601030 observed by dynamic light scattering ...... 95
4.17. Microparticles of PLGB601030, as seen under scanning electron microscopy (SEM)...... 96 xv
4.18. Particle sizes of PLGB502030 observed by dynamic light scattering ...... 97
4.19. Microparticles of PLGB502030, as seen under scanning electron microscopy (SEM) ...... 98
4.20. Procedure for dehydration of oligomers by solvent refluxing over molecular sieves ...... 100
4.21. Increase in Mn of PLA by chain extension using DiPC, with and without the dehydration by solvent recycling...... 101
4.22. Increase in Mn of PLB8020 by chain extension using DiPC, with and without the dehydration by solvent recycling ...... 102
4.23. Increase in Mn of PLA by chain extension using DiPC, after dehydration by solvent recycling ...... 104
4.24. Increase in Mn of PLB9010 by chain extension using DiPC, after dehydration by solvent recycling ...... 104
4.25. Increase in Mn of PLB8020 by chain extension using DiPC, after dehydration by solvent recycling ...... 105
4.26. Increase in Mn of PLB7030 by chain extension using DiPC, after dehydration by solvent recycling ...... 105
4.27. Increase in Mn of PLB6040 by chain extension using DiPC, after dehydration by solvent recycling ...... 106
5.1. 1H NMR (300 MHz) spectrum of benzyl lactate, synthesized by reacting lactic acid with benzyl bromide; * = CHCl3 ...... 117
5.2. 13C NMR (75 MHz) spectrum of benzyl lactate, synthesized from reacting lactic acid with benzyl bromide; * = CHCl3 ...... 118
5.3. 1H NMR (300 MHz) spectrum of benzyl glycolate synthesized by reacting glycolic acid with benzyl bromide...... 119
5.4. 13C NMR (75 MHz) spectrum of benzyl glycolate, synthesized from reacting glycolic acid with benzyl bromide; * = CHCl3 ...... 120
5.5. 1H NMR (300 MHz)spectra of TBDPSi-protected methyl glycolate and glycolic acid; * = CHCl3 ...... 122
5.6. 13C NMR (75 MHz) spectra of the TBDPSi-protected methyl glycolate and glycolic acid; * = CHCl3 ...... 123
5.7. 1H NMR (300 MHz) spectra of TBDPSi-protected methyl lactate and lactic acid; * = CHCl3 ...... 125 xvi
5.8. 13C NMR (75 MHz) spectrum of the TBDPSi protected methyl lactate and lactic acid; * = CHCl3 ...... 126
5.9. 1H NMR (300 MHz) spectra of methyl 2-bromo-3-hydroxypropionate, TBDPSi protected methyl 2-bromo-3-hydroxypropionate and 2-bromo-3-hydroxypropionic acid; * = CHCl3...... 129
5.10. Expanded 1H NMR spectrum of the methyl 2-bromo-3-hydroxypropionate and TBDPSi protected methyl 2-bromo-3-hydroxypropionate ...... 130
5.11. 13C NMR (75 MHz) spectra of the methyl 2-bromo-3-hydroxypropionate, TBDPSi- protected methyl 2-bromo-3-hydroxypropionate and TBDPSi-protected 2-bromo-3- hydroxypropionic acid; * = CHCl3...... 132
5.12. 1H NMR (300 MHz) spectrum of the p-GL-p dimer synthesized by coupling benzyl glycolate acid with TBDPSi-protected lactic acid; * = CHCl3...... 134
5.13. 13C NMR (75 MHz) spectrum of the p-GL-p dimer, synthesized by the coupling of benzyl glycolate with TBDPSi-protected lactic acid; * = CHCl3...... 135
5.14. 1H NMR (300 MHz) spectrum of the HOOC-GL-p dimer, synthesized by the deprotection of the bisprotected GL dimer, by a palladium-catalyzed hydrogenation; * = CHCl3...... 137
5.15. 13C NMR (75 MHz) spectrum of the HOOC-GL-p dimer, synthesized by the deprotection of the bisprotected GL dimer, by a palladium-catalyzed hydrogenation; * = CHCl3...... 138
5.16. 1H NMR (300 MHz) spectrum of the p-LGL-p trimer, synthesized by the esterification of benzyl lactate with HOOC-GL-p dimer ; * = CHCl3...... 140
5.17. 13C NMR (75 MHz)spectrum of the p-LGL-p trimer, synthesized by the esterification of benzyl lactate with HOOC-GL-p dimer; * = CHCl3...... 141
5.18. 2D HSQC plot of the p-LGL-p trimer expanded into the methyl region...... 142
5.19. 2D HSQC plot of the p-LGL-p trimer expanded into the methine and methylene regions...... 143
5.20. 1H NMR (300 MHz) spectra of the p-LB-p dimer, synthesized by the esterification of benzyl lactate with TBDPSi-protected 2-bromo-3-hydroxypropionic acid; * = CHCl3...... 146
5.21. 13C NMR (75 MHz) spectrum of the p-LB-p dimer, synthesized by the esterification of benzyl lactate with TBDPSi-protected 2-bromo-3-hydroxypropionic acid; * = CHCl3...... 147
xvii
5.22. 2D HSQC plot of the p-LB-p dimer, expanded into the methine and methylene regions...... 148
5.23. 1H NMR (300 MHz) spectrum of the p-LB-OH dimer, synthesized by the de- protection of TBDPSi group in p-LB-p, using reagent TBAF; * = CHCl3...... 150
5.24. 13C NMR (75 MHz) spectrum of the p-LB-OH dimer, synthesized by the de- protection of TBDPSi group in p-LB-p, using reagent TBAF; * = CHCl3...... 151
5.25. 1H NMR (300 MHz) spectrum of the p-LBL-p trimer, synthesized by the coupling of p-LB-OH with TBDPSi protected lactic acid; * = CHCl3...... 153
5.26. 13C NMR (75 MHz) spectrum of the p-LBL-p trimer, synthesized by the coupling of p-LB-OH with TBDPSi protected lactic acid; * = CHCl3...... 154
5.27. 2D HSQC plot of the p-LBL-p trimer expanded into the methyl region...... 155
5.28. 2D HSQC plot of the p-LBL-p trimer, expanded into the methine and methylene regions...... 156
6.1. Mold for preparing 100 mg compression molded samples for biodegradation study...... 158
6.2. (a) Mold resting on the bottom plunger, (b) 100 mg of the dry polymer was added in the cavity of the mold, (c) the top plunger was inserted, and (d) 600 PSI pressure was applied for 30 second to yield a 100 mg polymer tablet ...... 159
6.3. Degradation samples over days of degradation of PLGB601030, arranged chronologically ...... 161
6.4. Degradation samples over days of degradation of PLGB 502030, arranged chronologically...... 162
6.5. Percent moisture absorption by weight of the PLGB601030 and PLGB502030 copolymers...... 164
6.6. Percent weight loss of the PLGB601030 (grey) and PLGB502030 (blue) copolymers during their biodegradation in PBS (37 °C)...... 168
6.7. Stacked Gel Permeation Chromatographs for PLGB601030, showing the molecular weight loss behavior upon degradation. Peak molecular weights shown...... 171
6.8. Stacked Gel Permeation Chromatographs for PLGB502030, showing the molecular weight loss behavior upon degradation. Peak molecular weights shown ...... 174
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LIST OF SCHEMES
Scheme Page
1.1 Functionalized PLGA, poly (lacitc acid-co-glycolic acid-co-2-bromo-3- hydroxypropionic acid)...... 1
2.1 Degradation of poly(glycolic acid) to its monomer...... 4
2.2 Degradation products from the degradation of PLA and PGA ...... 6
2.3 (a) A representation of an amphiphilic molecule; (b) a common amphiphilic diblock co-polymer: poly(ethylene oxide)-b-poly(D,L-lactide) and (c) amphiphilic molecules arranging into a micelle in an aqueous medium ...... 11
2.4 Iodine-containing amphiphilic block copolymers forming micelles. Used as a carrier of contrast agent in computed tomography...... 12
2.5 Synthesized copolymers and their corresponding monomers ...... 15
2.6 Three Ag(I)-N-Heterocyclic carbene complexes derived from 4,5-dichloro-1H- imidazole...... 19
2.7 Synthesis of PEG-supported TEMPO ...... 20
2.8 Synthesis of PLLA from L-lactic acid, in bulk...... 22
2.9 Reaction mechanism of the polycondensation of a lactic acid using DiPC in the presence of DMAP and pTSA catalysts...... 25
2.10 Synthesis of poly(lactic-alt-glycolic acid), TBAF = tetra-n-butylammonium fluoride, AcOH = acetic acid, EtOAc = ethyl acetate...... 27
2.11 Schematic illustration of three main steps involved in polymer degradation and the effect of important structural and environmental parameters in each ...... 31
4.1 Synthesis of 2-bromo-3-hydroxypropionic acid ...... 66
4.2 Synthesis of PLGB from lactic acid using acid catalyst pTSA, and DPE as high boiling solvent/plasticizer ...... 70
xix
4.3 Synthesis of PLA from lactic acid and PLGA from lactic acid and glycolic acid monomers using acid catalyst pTSA, and DPE as high boiling solvent/plasticizer ....71
4.4 Synthesis of functionalizable PLA (PLB) from lactic acid and 2-bromo-3- hydroxypropionic acid using acid catalyst pTSA, and DPE as high boiling solvent/plasticizer ...... 77
4.5 Synthesis of functionalizable PLGA (PLGB) from lactic acid and 2-bromo-3- hydroxypropionic acid using acid catalyst pTSA, and DPE as high boiling solvent/plasticizer ...... 83
4.6 The oligomerization of lactic acid and 2-bromo-3-hydroxypropionic acid, and subsequent chain extension using N, N’-diisopropylcarbodiimide ...... 99
5.1 General reaction Scheme illustrating the difference between the reaction of PLGA and PLB co-polymers.Reaction 1: Lactic acid unit is connected to glycolic acid, Reaction 2: Lactic acid unit connected to a 2-bromo-3-hydroxypropinic acid unit. .109
5.2 The LGL and LBL trimers...... 110
5.3 Synthesis of bisprotected LGL trimer...... 111
5.4 Strategy for the synthesis of bisprotected LBL trimer...... 113
5.5 Synthesis of p-LBL-p trimer...... 114
5.6 Synthesis of sequence controlled LGL co-polymer...... 114
5.7 Synthesis of sequence controlled LBL co-polymer...... 115
5.8 Synthesis of benzyl lactate from lactic acid, using benzyl bromide as reagent...... 116
5.9 Synthesis of benzyl glycolate from glycolic acid using benzyl bromide...... 118
5.10 Synthesis of TBDPSi-protected glycolic acid ...... 121
5.11 Synthesis of TBDPSi-protected lactic acid ...... 124
5.12 Synthesis of TBDPSi-protected 2-bromo-3-hydroxypropionic acid...... 127
5.13 Synthesis of the p-GL-p dimer...... 134
5.14 Synthesis of the HOOC-GL-p dimer ...... 136
5.15 Synthesis of the p-LGL-p trimer, synthesized by esterifying HOOC-GL-p dimer with benzyl lactate ...... 139
5.16 Synthesis of the p-LB-p dimer...... 144
xx
5.17 Synthesis of the p-LB-OH dimer...... 149
5.18 Synthesis of the p-LBL-p trimer...... 151
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CHAPTER I
INTRODUCTION
The present work describes the synthesis of biodegradable polymers similar to poly (lactic acid) (PLA) or poly (lactic acid-co-glycolic acid) (PLGA) (Scheme 1.1)
copolymers that are functionalized with halogen groups on the main chain.1 The halogen group can be used for further functionalization of the polymer. The monomer I used in this study is 2-bromo-3-hydroxypropionic acid, which has been co-polyesterified with variable amounts of lactic acid and glycolic acid to create a library of brominated PLAs
and PLGAs (Chapter IV).
Scheme 1.1: Functionalized PLGA, poly (lacitc acid-co-glycolic acid-co-2-bromo-3-
hydroxypropionic acid).
1
2-bromo-3-hydroxypropionic acid has been previously used by our group to
develop hyperbranched polyacrylates, by self-condensing vinyl polymerization (SCVP)
of various ester substituted inimers.2 Both bulk polycondensation and solution
polymerization routes have been investigated for the synthesis of these brominated PLAs
and PLGAs. The bulk polycondensation route is the preferred route as it affords higher
GPCPSt number average molecular weights and ease of synthesis. Its one-pot synthesis makes it easy to commercialize.
Bisprotected sequence controlled monomers, using the lactic acid, glycolic acid and 2-bromo-3-hydroxyacid have also been prepared with the intention to prepare sequence controlled functionalized PLGAs (Chapter V). This will allow us to investigate the surprising trend in the decrease of GPCPSt number average molecular weights, upon
the introduction of glycolic acid and 2-bromo-3-hydroxyacid monomer to PLA, to form brominated PLA and PLGA. Preparing sequence controlled copolymers might also allow
us to achieve more efficient functionalizing reactions, due to optimum steric spacing
between the bromine functionalization sites.
The biodegradation profiles of two particular brominated PLGA copolymers have
also been studied in Phosphate Buffer Saline (PBS), at 37 °C (Chapter VI). The
polyesters studied were completely biodegraded in 2 months, under physiological
conditions.
Future work, regarding establishing functionalization reactions on the main chain
of these brominated PLGA is being current carried out by one of Dr Coleen Pugh’s
graduate student, Colin Wright, in our lab. These polymers are also going to be
functionalized with silicon, by Xiang Yan, another of Dr Coleen Pugh’s graduate
2
students, and formulated into salt leached sponge scaffolds, to study the effect of silicon on bone mineralization, in collaboration with Dr William Landis.
3
CHAPTER II
LITERATURE REVIEW
Biodegradable polymers have received a lot of attention in the last couple of
decades, and have found tremendous biomedical and ecological applications. A
biodegradable polymer is one that will depolymerize under the right conditions, into its oligomeric units, and then into its monomeric units.3 Depolymerization takes place by the
scission of covalent bonds that link the monomeric units together.3, 4 The simplest
example is the transformation of a widely used suture material, polyglycolide, into short
oligomers and eventually to single glycolic acid molecules (Scheme 2.1).3
O O O Hydrolysis Hydrolysis O m O n HO H nOH H n/mOH OH glycolic acid Polyglycolic acid
Scheme 2.1: Degradation of poly(glycolic acid) to its monomer.
In its broadest sense, biodegradation is a series of events that leads to the
breakdown of polymeric materials into smaller fragments and eventually to monomeric
4
materials and small molecules.3 In this sense, all natural and synthetic polymers are biodegradable, although degradation of fully hydrocarbon synthetic polymers like polyethylene or polystyrene is so slow that they are considered non-biodegradable under normal conditions.3 The timescale of degradation and the timescale of the application
differentiates degradable and non-degradable polymers. In the context of biomaterials
and human medicine, biodegradation means breakdown of polymer into resorbable
(metabolizable), absorbable or excretable fragments.3, 5 Polylactide (PLA), polyglycolide
(PGA), and their copolymers are perfect examples. After complete degradation of polylactide into D,L-lactate, it is converted into carbon dioxide and pyruvate, which enters the Krebs cycle.6 The majority of products from PLA degradation are eliminated as
carbon dioxide through respiration. PGA hydrolyzes into glycolate, some of which is
either eliminated via urine or is oxidized to glyoxylate which is converted into glycine,
serine and pyruvate (Scheme 2.2).6 The biodegradation products of PLA and PGA are
thus completely resorbed, absorbed or excreted by the body.
Biodegradable polymers have been classified into natural and synthetic polymers.
Table 2.1 presents a more comprehensive list. Natural polymers are mainly
polysaccharides, proteins or polyhydroxyalkanoates.3, 5, 6 Polysaccharides are
monosaccharides linked with glycosidic linkages with typical examples being starch and
chitin. Proteins include collagen and silk.3 Among synthetic biodegradable polymers,
polyhydroxy acids including polyglycolide, polylactides and polycaprolactone are the
most popular.3
5
Glycine Serine Pyruvate
Scheme 2.2: Degradation products from the degradation of PLA and PGA.
Table 2.1: Classification of biodegradable polymers5
Natural Polymers Synthetic Polymers Classes Examples Classes Examples Plant Origin Aliphatic polyesters Polysaccharides Cellulose, Starch Glycol and dicarbonicacid Poly(ethylene succinate) Alginate Polycondensates Poly(butyleneterephthalate) Polylactides Polyglycolide, polylactides Polylactones Poly(є-caprolactone) Miscellaneous Poly(butylene terephthalate) Animal Origin Polyols Poly(vinyl alcohol) Polysaccharides Chitosan, Hyaluronate Polycarbonates Poly(ester carbonate) Proteins Collagen, Albumin Microbe Origin Miscellaneous Polyanhydrides Polyesters Poly(3-hydroxyalkanoate) Poly(α-cyanoacrylates) Polysaccharides Hyaluronate Poly(orthoesters) Polyphosphazenes
2.1 Applications of Biodegradable Polymers
Biodegradable polymers currently have two major applications: one as ecological polymers, which are aimed at protecting the earth’s environment from plastic wastes; the other as biomedical polymers, which contribute to the medical care of the patients.5
6
2.1.1 Ecological Applications
Biodegradable plastics are able to replace non-biodegradablle polymers like polystyrene and poly(ethylene terephthalate) (PET) in a vaariety of applications. Cargill
Dow LLC, under the trade name Nature Workks, is using PLA to make biodegradable products like dairy containers, food trays, cold drink cups, products for packaging applications, bottles for fruit juices, sport drinks and jams and jellies (Figure 2.1).7
Poly(butylene succinate) is being used in agricultural applications in the form of mulch films, bags for seedlings and replanting pots.5 Poly(butylene succinate) is also being used for manufacturing packaging films, bags and ‘flushable’ feminine hygiene products because of its excellent mechanical properties.5
Figure 2.1: Biodegradable PLA cold drink cups and food trays.
7
2.1.2 Medical Applications
The use of polyglycolide and its copolymers with lactide as medical sutures is one of the oldest applications of biodegradable polymers. A number of materials are now on the market, such as Dexon®, Vicryl®, Maxon® and Monocryl® sutures.8 Table 2.2 shows the structures of some polymers commercialized for medical applications. Biodegradable polymers like poly(glycolide-co-lactide), poly(L-lactide) and poly(p-dioxanone) (PDS) are being used in the fixation of fractured bones. They have been commercialized in the form of osteosynthetic devices with the trade names
Lactomer®, Biofix® and Phusiline®, respectively (Figure 2.2).6
Table 2.2: Monomers and structures of common products made from biodegradable
polymers in biomedical applications.
Monomer Polymer
O O O O O O O n Glycolide O “DEXON” O O
O O O O Glycolide O H O HO O n O O O m O O
“VICRYL” O O
L Lactide
8
O O O O O n O “PDS”
p-Dioxanone
Figure 2.2: Phusiline® Screws made from PLA, used to fix graft in (a) knee ligament
reconstruction, and (b) foot surgery.9
These materials are replacing traditional metals in the form of plates, pins, screws and wires, which need to be removed after the re-union of fractured bones by further surgery. Biodegradable polymers such as collagen, poly(glycolide-co-lactide) (PLGA) copolymers and cross-linked polysaccharides aare being used for tissue engineering, where they are being used as the scaffolds for cell proliferation and differentiation, which help in tissue regeneration. They are also being used for a sustained release of growth factors at the location of tissue regeneration.5
9
One of the most interesting uses of biodegradable polymers is in the field of
controlled drug delivery. These drug carriers are made from amphiphilic block co-
polymers(Scheme 2.3). Usually, poly(ethylene glycol) (PEG) blocks with a molecular
weight from 1,000 to 15,000 Da are used as the hydrophilic part of the amphiphile.10 PEG has low toxicity, is inexpensive and has been approved by regulatory agencies for internal applications.11 Poly(N-vinyl-2-pyrrolidone) (PVP) has been used as an alternative to
PEG.10 Monomers like D,L-lactic acid, caprolactone and L-lysine are commonly used to
build the hydrophobic block. Scheme 2.3(b) shows an example of an amphiphilic diblock
copolymer. These amphiphilic copolymers assemble into spherical micelles in aqueous
solutions when their concentration is above the critical micelle concentration (CMC)
(Scheme 2.3 (c)).10, 11 In aqueous media, the hydrophobic segment of the amphiphile
forms the core of the micelle, and the hydrophilic segment forms the corona. These
micelles are able to solubilize poorly soluble, hydrophobic drugs and hence are used as
drug delivery systems,10, 11 in aqueous media thereby increasing their bioavailability in
pathological sites. They also help minimize premature drug degradation and other
undesirable side effects by protecting the drug from possible inactivation by the
biological surroundings.10, 11 Pharmaceutical companies use biodegradable polymers in
the form of micelle-forming amphiphilic copolymers to make formulations for drugs that are otherwise difficult to deliver due to their high hydrophobicity.10, 11 Decapeptyl® and
Zoladex® are examples of some commercialized drug delivery products that are based on
these polymers.8
10
Scheme 2.3: (a) A representation of an amphiphilic molecule; (b) a common
amphiphilic diblock co-polymer: poly(ethylene oxide)-b-poly(D,L-lactide) and (c)
amphiphilic molecules arranging into a micelle in an aqueous medium
Biodegradable polymers have also found applications in the field of diagnostic medical imaging. Medical diagnostic imaging requires that an area of interest to generate a signal of sufficient intensity to differentiate it from the surrounding tissues. Different organs and tissues are imaged with appropriate contrast agents for the early detection and localization of numerous pathologies.10, 11 For example, computed tomography (CT) uses substances containing X-ray absorbing heavy elements, such as iodine. Traditional contrast agents, like iodine-containing organic molecules have brief effectiveness due to rapid extravazation and clearance of contrast.10, 11 The circulation time of a micelle- forming biodegradable amphiphilic block copolymer of α-mmethoxy-poly(ethylene glycol)
11
(MPEG) and triiodobenzoic acid-substituted poly-L-lysine (Scheme 2.4) is longer that of traditional CT contrast agents, and provide strong signal intensity even 2 h after injection in rats and rabbits.10, 11
Scheme 2.4: Iodine-containing amphiphilic block copolymers forming micelles. Used as a carrier of contrast agent in coomputed tomography.
12
2.2 Motivation and Scope
The most widely used PLGA polymer systems have several limitations for both
ecological and medical applications. PLA has limitations like high crystallinity,
brittleness, lack of total absorption and thermal instability.12 But, most importantly, these
polymers have no functionalities12 on their backbones, and are therefore not able to
covalently attach drugs or other therapeutic molecules for drug delivery applications.
Instead, the therapeutic molecules must be physically entrapped into these polymers, either by forming micelles or by nano-encapsulation, thereby limiting their loading capacity.
We recently developed hyperbranched polyacrylates, by self-condensing vinyl polymerization (SCVP) of various ester substituted inimers. 2 Various inimers with
methyl, dodecyl, perfluoroalkyl, siloxane, oligooxyethylene, and mesogenic ester
substitutes were synthesized, which were polymerized by atom transfer radical
polymerization to yield soluble hyperbranched polyacrylates with different functional
substitutions. The key reagent is a 2-halo-3-hydroxypropionic acid, in which a free alkyl
ester is substituted, and then converted into an inimer. This 2-halo-3-hydroxypropionic
acid is a halogen constitutional isomer of lactic acid, and has a primary alcohol group,
similar to that of glycolic acid. The presence of the halogen group on the hydroxy acid
makes it a functional hydroxy acid monomer, which could be co-polymerized with lactic
acid and glycolic acid, to create halogenated PLGAs.
The present work is aimed at the synthesis of biodegradable polymers similar to
PLA or PLGA copolymers that are functionalized with halogen groups on the main chain.
The halogen group can be used for further functionalization of the polymer. The
13
monomer we used in this study is 2-bromo-3-hydroxypropionic acid, which can be homo-
polyesterified to form poly(bromo-hydroxypropionic acid), or co-polyesterified with
variable amounts of lactic acid and glycolic acid (Scheme 2.5) to create a library of
brominated PLGAs.
Monomer Copolymers
O O H HO OH O nOH Br Br
2-Bromo-3- Poly(2-bromo-3-hydroxypropionic acid) hydroxypropionic acid
O
HO OH
O O Lactic Acid O H O OH n m O Br
HO OH Poly(lactic acid-co-2-bromo-3-hydroxypropionic acid) Br
2-Bromo-3- hydroxypropionic acid
O
HO OH
Glycolic Acid O O O H O OH n m Br
poly(glycolic acid-co-2-bromo-3-hydroxypropionic acid)
14
O
HO OH Br
2-Bromo-3- hydroxypropionic acid
O
HO OH Glycolic Acid
O HO O O O OH O H O O OH x y z Lactic Acid Br
Poly(glycolic acid-co-lactic acid-co-2-bromo-3-hydroxypropionic acid) O
HO OH Br
2-Bromo-3- hydroxypropionic acid
Scheme 2.5: Synthesized copolymers and their corresponding monomers.
Bromine, alpha to the carbonyl group of an ester is a good leaving group and it
has been displaced with amines at the terminus of polymers by Matyjaszewski and
coworkers.13 Functionalization reactions were performed on bromine end-functionalized
15
polyacrylates synthesized by atom transfer radical polymerization. Substitution was
quantitative when poly (methyl acrylate)-Br was reacted with butylamine and 4-
aminobutanol as nucleophile (Table 2.3). Using ammonia as nucleophile gave two amino
groups by chain substitution and amidation. Reaction with 2-aminoethanol also gave
substitution and amidation products. The reactions were performed for 48 h at 25 °C in
DMSO as solvent, using 10-25 equivalents of the nucleophiles, in the presence of
triethylamine.
Table 2.3 Reactivity of poly(methylacrylate)-Br towards primary amines.
Br 10-25 equiv Nu Nu H n H n+1 25 °C, 48 h CO2Me CO2Me CO2Me
0.5 M in DMSO
Nucleophile Results 2 amino groups/chain NH3 (substitution and amidation)
quantitative substitution H2N (+ NEt3)
OH (+ NEt3) Susbtitution plus amidation H2N (via intramolecular transesterification
quantitative substitution OH (+ NEt3) H2N
In another study by the same group,14 the reactivities of bromine end
functionalized and chlorine end functionalized polyacrylates, towards nucleophilic
substitution, were compared. Methyl 2-chloropropionate (MClP) and methyl 2-
16
bromopropionate (MBrP), models for polyacrylates were reacted with one equivalent of
primary amine, n-butylamine. The reactivity of the primary amine towards nucleophilic
substitution of bromine end functionalized model, MBrP, was three orders of magnitude
higher than towards the chlorine end functionalized model, MClP (Table 2.4). MClP and
MBrP were also reacted with one equivalent of the following tertiary amines: pyridine,
2,2’-dipyridyl (bpy), triethylamine (TEA), dimethylethylamine (DMEA) and
N,N,N’,N’’,N’’-pentamethyldiethylenetriamine (PMDETA). Methyl 2-chloropropioniate
did not react, where as methyl 2-bromopropionate formed quaternary ammonium salts
with pyridine (28% conversion), DMEA (57% conversion) and PMDETA (74%
conversion). These reactions were performed in the presence of triethylamine at 25 °C in
DMSO. Based on this literature precedent, we decided to use the bromo-functionalized
hydroxyacid to create the bromo functionalized PLGAs.
17
Table 2.4: Reactivity of bromine end functionalized polyacrylate model vs.
chlorine end functionalized polyacrylate model towards primary and tertiary amines.
Our group is interested in using such functionalized PLGA coppolymers for the covalent attachment of antimicrobial silver N-heterocyclic carbene (NHC) complexes15, 16
(Scheme 2.6) developed by Youngs. The NHC-functionalized PLGA will then be formulated into microparticles for aerosolization. I will therefore develop the micro particle formulation technique using the halogenated PLGAs. This technique is an adoption from the work of Yang, et al.,17 where they formulated micro-particles of L-
18
tyrosine phosphate (LTP) copolymers of molecular weight of 8,000-10,000 using an oil-
in-water (o/w) emulsion formed by mechanical stirring and solvent evaporation.
Polyethylene glycol grafted to chitosan (PEG-g-CHN) and polyvinylpyrrolidone (PVP)
were used as surfactants.
Scheme 2.6: Three Ag(I)-N-Heterocyclic carbene complexes derived from 4,5-
dichloro-1H-imidazole.
The copolymer system (the pendant secondary bromine groups) will be
functionalized by quenching the radical generated from the secondary bromine with the
stable radical, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (4-hydroxy-TEMPO).
Molecules of interest such as drugs, etc will be attached at the pendent alcohol group of
the 4-hydroxy-TEMPO, converting this molecule of interest into a radical, which can be
conveniently used to quench the radical generated from the secondary bromine of the
functionalized PLGA. Various groups have successfully attached molecules like PEG to
4-hydroxy-TEMPO.18 In a typical reaction, the alcohol end group of the PEG is
mesylated, and then the mesyl group is replaced by the alcohol of the hydroxy TEMPO
(Scheme 2.7) to create a PEG-supported TEMPO. In this particular example, PEG6000-
(TEMPO)2 was synthesized from PEG6000 in a 70% yield.
19
1. MsCl, CH2Cl2, OH O N O O O n 2. OH ,NaH n
N n = 112 O DMF, 0 °C
Scheme 2.7: Synthesis of PEG-supported TEMPO.
The cytotoxicity of the nitroxide bond has been investigated by Chenal, et al.19
They exposed 3 cell lines, mouse fibroblast cells, human umbilical vein endothelial cells, and murine macrophage cells, all of which represent important mammalian cell types, and their sensitivity to synthetic materials is crucial to be investigated for all new bio-
materials. They found that the nitroxide did not affect the cell viability on the three cell lines.
The results were further corroborated by direct microscopic observation, which showed no
changes in the cell density and morphological appearance.
These functionalized PLGA copolymers are also being functionalized with silicon
and formulated into salt leached sponge scaffolds,20 to study the effect of silicon on bone
mineralization. The above research directions are current doctoral dissertations of two
graduate students in our lab.
This work also studies the degradation profile of the brominated PLGA (30%
bromine, 90/10 and 30% bromine, 80/20). Depending upon the degradation profile of this
new polymer, it will be copolymerized with different amounts of glycolic acid and lactic
acid to tailor its degradation behavior to suit its end application.
20
2.3 Synthesis of Poly(hydroxy acids).
Polymerization of hydroxy acids has been a popular area of study because of the many biological and ecological applications of the two most important members of its class, lactic acid and glycolic acid. The polymerization of lactic acid has been especially studied. There are numerous ways to polymerize lactic acid, depending upon the molecular weight targeted and the end application. Hydroxy acids have been polymerized both in bulk (Section 1.3.1) and in solution (Section 1.3.2).
2.3.1 Bulk
Hydroxy acids can be polymerized by condensation polymerization in bulk, catalyzed by strong protonic acids, such as p-toluenesulfonic acid (pTSA). Condensation polymerization of hydroxy acids is an equilibrium reaction, with water formed as a byproduct. The reaction is driven forward by removing the water generated using heat and by applying a vacuum. High molecular weight polymers of lactic acid are very difficult to prepare because it is difficult to remove water in the later stages of the reaction due to the increased viscosity of the system. Since this is a condensation polymerization, high molecular weights can only be achieved at very high conversion, which requires removal of more than 99.3% of the water.21
However, L-lactic acid has been polymerized to high molecular weights
(Mw≥100,000) by direct melt polycondensation using an equimolar catalyst system of
22 SnCl2 and pTSA (Scheme 2.8). The L-lactic acid was first dehydrated under reduced
pressure at 150°C, to a degree of oligomerization of 8, calculated by 1H NMR
21
spectroscopy. The oligomer was then mixed with 0.3wt% of both tin chloride dihydrate
and pTSA. Under mechanical stirring, the mixture was heated at 180°C in vacuo. After
the end of the reaction, the product was dissolved in chloroform and subsequently
precipitated into diethyl ether. According to the authors, the protonic acid activates the
Sn(II) catalyst, producing high molecular weight polymer.22
150 °C, 8 h SnCl + pTSA OH O 2 O m HO H HO H nm HO n 150 °C, 16 h nm O O O L-lactic acid Oligo(L-lactic acid) Poly(L-lactic acid), m>>n
Scheme 2.8: Synthesis of PLLA from L-lactic acid, in bulk.
2.3.2 Solution
Ajioka23 and others from Mitsui Chemicals24 used solvent to obtain high
molecular weight PLA by direct polycondensation. The polycondensation reaction of the
hydrocarboxylic acid was performed in diphenylether, which forms an azeotrope with
water, and the azeotrope was distilled from the reaction system. Stannous chloride or
pTSA was used to catalyze the system. The average molecular weight of the PLA
depended on the moisture content of the organic solvent used. To further dehydrate the
solvent, the azeotropically distilled organic solvent was passed through a column packed
with molecular sieves before returning it to the reaction system. The amount of solvent
used was calculated such that the polymer concentration stayed between 10~80% by
weight. Under optimized conditions and long reaction times, PLAs with weight average
22
molecular weights higher than 300,000 were synthesized.23, 24 However, the use of solvents such as diphenyl ether complicates the process and increases the price of PLLA, since the solvent must be completely removed from the end product, which is difficult.23,
24
Hydroxy acids polymerize at room temperature in solution state suitable coupling
agents and catalyst. Steglich25 first used carbodiimides for esterification of small
molecule carboxylic acids. Self-condensation of monomer containing aliphatic acid and a
phenolic group was achieved in anhydrous dichloromethae using diisopropylcarbodiimide (DiPC) as a linking agent, catalyzed by an equimolar complex of the hyperacylation catalyst, 4-dimethylaminopyridine (DMAP) and protonic acid,
26 pTSA. High molecular weight polyesters with Mn≥15,000 (DPn>50) were obtained in good yield and high purity.26 Similarly, lactic acid was polymerized to high molecular weight using dicyclohexylcarbodiimide (DCC) as a linking agent, catalyzed by DMAP.27
The 90% lactic acid was first dried by azeotropic distillation with a Dean-Stark trap using dichloromethane as solvent. After removing water from the monomer, the carbodiimide and DMAP were added to yield high polymer. Under optimized conditions, PLA of Mn =
15,400 with 94% yield was achieved.27 In another example, carbodiimides were used to
28 synthesize poly(L-lactic acid) of Mn = 13,500 in supercritical carbon dioxide.
Scheme 2.9 illustrates the reaction mechanism for the reaction of lactic acid with
DiPC in the presence of pTSA and DMAP catalyst system. In this reaction, the
carbodiimide selectively activates the carboxylic acid by forming an O-acylurea, which is
more electrophilic and able to react with an alcohol to form an ester bond and an
insoluble urea, which precipitates out of the solution.29 The O-acylurea can rearrange into
23
inactive N-acylurea that does not propagate further and limits the formation of high
molecular weight polymer. The protonic acid catalyst, pTSA, protonates the O-acylurea,
which decreases its nucleophilicity and thereby hinders the formation of the N-acylurea
side product. DMAP then reacts with the protonated O-acylurea to form an
acylpyridinium cation and a urea by-product before it rearranges into the N-acylurea.29
The urea by-product simply precipitates out of solution. The acylpyridiniumcationis then activated towards nucleophilic attack by the alcohol group of another monomer molecule to form an ester dimer, regenerating DMAP.29 The dimer reacts with other monomers, dimers, trimers, etc. to eventually produce high molecular weight polyester.
24
Scheme 2.9: Reaction mechanism of the polycondensation of a lactic acid using
DiPC in the presence of DMAP and pTSA catalysts.
25
2.3.3 Sequence Controlled Copolymers
The copolymerization of hydroxy acids in bulk or in solution gives random
statistical copolymers. Meyers,30 and coworkers have used an innovative approach to
create copolymers of lactic acid and glycolic acid with a controlled sequence distribution
of perfectly alternating lactic acid and glycolic acid. The steps are illustrated in Scheme
2.10. The carboxyl end group of the lactic acid was protected by a benzyl group, and the
alcohol end group of the glycolic acid was protected as a t-butyl diphenyl silyl ether. Both of these mono-functional protected monomers were then coupled using DCC as the coupling agent, to form a segmer31 comprising an exact sequence of the monomers. This
di-protected segmer was then deprotected on both ends, to form a hydroxyacid which was
then polymerized in solution using DCC as a coupling agent and dichloromethane as the
solvent..The perfect alternating copolymer of lactic acid and glycolic acid had a number
average molecular weight of 2.88 x 104 and polydispersity index of 1.3. 32
26
Scheme 2.10: Synthesis of poly(lactic-alt-glycolic acid), TBAF = tetra-n-
butylammonium fluoride, AcOH = Acetic acid, EtOAc = ethyl acetate.
2.4 Biodegradation
For both biomedical and consumer applications, it is of major importance
to understand the degradation characteristics of biodegradable polymers. Polymers used for orthopedic fixtures in the form of pins or screws must not lose their strength before the function of fracture healing takes place. Similarly, for drug delivery applications, the
release of the drug is a direct consequence of the erosion process; release rates become
predictable if the erosion process of the polymer is understood. Biodegradation studies of
poly(hydroxy acid)s have been performed by various researchers and an extensive
amount of biodegradation data is available for polylactides8, 33, 34 and copolymers of lactide with glycolide.8, 35-39The biodegradation behavior of poly(D,L-lactic acid)-b-
poly(ethylene glycol) copolymers,40 blends of poly(DL-lactic-co-glycolic acid) with
27
polyethylene glycol41 and even poly(vinyl alcohol) grafted onto poly(lactic-co-glycolic acid)42 have also been studied.
There are different types of polymer degradation, such as photo-, thermal-,
mechanical and chemical degradation.6 The most important degradation mechanism for
polymeric biomaterials is chemical degradation via hydrolysis or enzyme-catalyzed
hydrolysis. Enzymatic degradation is effective only for naturally occurring biopolymers
such as polysaccharides, proteins (gelatin and collagen) and poly(β-hydroxy acid)s.3
Hence for most biodegradable materials from synthetic polymers, the most important mode of degradation is passive hydrolysis. Among all synthetic polymers, the degradation behavior of polyesters such as poly(lactic acid), poly(glycolic acid), and their copolymers are the most studied.
The rate of polymer degradation depends mainly on the type of bond within the polymer backbone. The biodegradation rates of common biodegradable polymers used for medical and pharmaceutical applications, were compared in a study.43 The time
required for 50% hydrolysis (half life) at pH=7 and 25°C was noted. The rate of
hydrolysis was normalized against that of the polyesters, to give us relative rates – useful
for making order of magnitude comparisons. The study showed, polymers with anhydride and ortho-esters backbones are the most reactive, followed by esters and ureas (Table
2.5). Reactivities, however, can change tremendously with catalysis or by altering the chemical neighborhood of the functional group through steric and electronic effects.3
28
Table 2.5: Comparison of the relative hydrolysis rates (at 25°C, pH 7) of different
chemical bonds1
Structure Polymer Class Relative Rate
Polyanhydride 2.9 x 105
Polyorthoester 7.2 x 103
Polyester 1
Polyurea 1.0 x 10-1
Polyamide 4.0 x 10-5
Polycarbonate 7.9 x 10-5
Polyurethane 7.9 x 10-5
Several multistep models describing the series of events involved in the
hydrolytic degradation of polymers have been proposed. The general tenets of the models
are summarized in Scheme 2.11.The first step involves the absorption of water by the
polymer, the rate and extent of which are governed by the hydrophilicity and porosity of
the polymer. In the second step, water is absorbed by the polymer, and hydrolysis starts.
The relative rates of water uptake and hydrolysis are governed by the hydrophilicity and
morphology of the polymer, and the type and concentration of hydrolyzable bonds within
the polymer backbone. These properties determine if degradation occurs primarily on the
29
surface or throughout the bulk. In the third stage the polymer loses mass until it
completely disappears. Mass loss occurs due to the dissolution of the resultant oligomers that have been rendered soluble, or the physical liberation of incompletely degraded
chains or pieces of crystalline domains whose surrounding regions have been
preferentially degraded. Mass loss continues until the polymer bulk is completely broken
down and disintegrated.3
30
Scheme 2.11: Schematic illustration of three main steps involved in polymer degradation and the effect of important structural and environmental parameters in each
step.
Biodegradable polymers degrade in two different modes: surface and bulk (Figure
2.3). Surface erosion occurs when the penetration of water into the bulk polymer is much
31
slower than hydrolysis, limiting the degradation to the outer surface of the polymer. For
example, polyanhydrides degrade by surface erosion.3 When the penetration of water is
much faster into the bulk polymer than the degradation reaction, degradation takes place
in nearly equal rates throughout the polymer surface and bulk. Polyesters generally
undergo bulk degradation.3, 6
Bulk Degradation Surface Erosion
strength Mw Property Property mass strength
Mw mass
Time Time
Figure 2.3: Schematic illustration of time course of changes in mass, molecular
weights and mechanical strength during biodegradation of polymeric biomaterials
undergoing either bulk degradation or surface erosion.
32
2.4.1 Effect of Structure and Environment on Biodegradation
The rate of polymer degradation also depends on the structural properties of the polymer involved, including crystallinity, glass transition temperature (Tg), molecular
weight, molecular weight distribution, wettability and its surrounding environment such
as temperature and pH.3
2.4.1.1 Crystallinity
Amorphous polymer regions are more susceptible to hydrolysis than crystalline
regions as they have higher free volume and greater chain mobility, allowing easier
access of water, enzymes, radicals and other catalyzing agents. Therefore, scanning electron microscopy (SEM) images of degrading polymers often show features at the
surface resembling crystallites.44
The critical degree of crystallinity affecting the biodegradation rate is 40%.3
However, studying the effect of crystallinity is complicated by its dependence on other polymer properties, such as chemical structure and molecular weight. The stereochemistry of a chiral polymer is one parameter that can give insight into the effect of crystallinity in the absence of other confounding factors. For example, amorphous polylactides of various D/L ratios degrade significantly more quickly than highly crystalline poly(L-lactide) (PLLA). In vitro degradation of solid cylinders of PLLA (Mw
= 160,000 Da) is 10-30 times slower than those of PLLA mixed with different amounts
-1 of lactic acid oligomers (Mw = 2,000 Da), i.e., 0.002 versus 0.058 day (for PLLA with
30% oligomers) (Figure 2.4).45 In another study, the authors found that adding only 5%
D-Lactide to PLLA disrupts the crystallinity to the extent that the degradation rate
33
increases substantially.34 Degradation rate of PLLA increases from 0.015 day-1 to more
than 0.10 day-1 for poly(LLA-DLA, 95/5).34
Figure 2.4: Weight-average molecular weight (Mw) of high molecular weight
(160,000 Da) PLLA mixed with 0%(●), 10%(■), 20%(▲), 30%(♦) oligomers (2,000
Da).45
34
2.4.1.2 Hydrophilicity
Polymer hydrophobicity is the measure of its compatibility with water, wetting and swelling in water, and hence has a pronounced effect on degradation rate. For a series
of essentially non-crystalline poly(D,L-lactic acid-co-glycolic acid) (PDLLGA)
copolymers, increasing the glycolic acidcomponent from 0% to 50%, increasedthe degradation rate from 0.022 day-1 to 0.054 day-1.45 As poly(glycolic acid) is more
hydrophilic than poly(lactic acid), increasing its content increases the
copolymers’hydrophilicity, which in turn increases its biodegradation rate.46
2.4.1.3 Acid autocatalysis
The hydrolysis of PLA, PGA and related copolymers releases oligomers, and eventually
monomeric lactic acid or glycolic acid as degradation products, which increases the
acidity of the degradation medium. This local acidity leads to faster degradation in the interior of molded PLGA devices (Figure 2.5), as the increases in the local acidity leads
to autocatalytic acceleration of the hydrolysis reaction.37 Because of this, thick films (100
µm) of PLGA (50/50 and 75/25) degrade two to three times faster than thin films (10 µm)
- 0.083 versus 0.043 day-1, and 0.050 versus 0.017 day-1, respectively (Figure 2.6).39
Porous foams are, however, not susceptible to acid autocatalysis. The acidity never builds up since the porous structure allows the transport of degradation products out of the foams, the degradation is therefore uniform.3
35
Figure 2.5: SEM image of cross-section of a PLGA (75/25) specimen after 10
days of degradation in phosphate buffer (pH = 7.4) at 37 °C. 37
Figure 2.6: Percent of molecular weight remaining compared to day 0 values as a
function of degradation time for (a) PLGA 75::25 (Mw = 67,700 Da), (□) thin and (◊) thick films (b) PLGA 50:50 (Mw = 43,900 Da), (○) thin and (∆) thick films. Thick films =
100 µm, thin films = 10 µm.39
36
2.4.2 Experimental aspects of studying biodegradation
Studying biodegradation begins with the sample preparation. The prepared
samples are then either implanted in animals for ‘in vivo’ testing or placed in a medium
to let it age, for ‘in vitro’ studies. ‘In vitro’ studies are usually done in a phosphorus
buffer saline, pH 7.4, at 37°C, which mimics the conditions of physiological body fluids.
This can be performed in either static or pseudodynamic conditions. A test is static if the buffer solution used for aging is not replaced over the testing period. This results in a dramatic drop in the pH of the solution over time owing to the release of the acid from the material or device. In pseudodynamic conditions, the pH of the buffer is monitored at regular intervals of time and kept constant at pH 7.4.34 This is a better simulation of
degradation, as acidic products are transported away from the implantation site in the
body, keeping the pH around the biodegrading material more or less constant. The
biodegradation profile is studied by tracking the change in specific properties of the
polymer, such as weight loss and molecular weight loss.
A variety of different devices have been used to study degradation. For example,
PLA-PGA degradation has been studied in the form of films,8 blocks or cylinders33, 34and
in the form of microspheres.47 The choice of the device depends on the end use of the
polymer whose degradation is being investigated. Groups have generally used blocks or
cylinders when investigating polymers for orthopaedic implants, and microspheres for
studying polymers for drug delivery systems. However, films are most commonly used to
study degradability, probably because of the ease of sample preparation, and the smaller amount of sample required, which is very useful for polymers that might be difficult or expensive to prepare.
37
2.4.2.1 Sample Preparation
A very popular and easy method to prepare samples is solvent casting. In their
studies, Mikos et al. prepared PLLA and PLGA films by casting polymer/chloroform
solutions of specific concentrations (12.5-150mg ml-1) onto smooth glass surfaces.38 The polymer solutions were prepared by dissolving a known mass of polymer in a specificvolume of chloroform. The concentrations ranged from 12.5 to 150 mg polymer per 1 ml of chloroform. The solutions were then cast onto glass coverslips of 12mm diameter, completely covering them. These samples were left in the hood for 20h to allow the solvent to evaporate and then further dried in vacuo for 24h. The coverslips were then soaked in distilled, deionized water for 90min, and then carefully lifted off of the glass with a razor blade. Once removed, the films were air and vacuum dried for 24h each, and subsequently stored in a desiccator over anhydrous CaSO4, under vacuum until use.
Films of two different thicknesses were prepared by altering the amount and
concentration of the solution cast, and were classified as thin (5 to 10µm) or thick (85 to
100µm).
38
2.4.2.2 Biodegradation Markers
The following properties are monitored to study the degradation profile of
polymers: Visual examination, water absorption, weight loss, molecular weight loss and
change in pH.
2.4.2.2.1 Visual examination
While observing the degradation of PLA50 shaped in round plates molded by
compression molding, Li found that the specimens became progressively whitish and
exhibited irregular deformations in spite of the absence of mechanical stress.35 This was
due to heterogeneous degradation, in which the inner part of the specimen appeared as a
very viscous liquid after week 5, whereas the outer layer was still rigid. At 12 months,
most of the inner viscous liquid disappeared, whereas the outer layer remained and
appeared as a flattened empty shell. In another work by Li and McCarthy, they studied
the degradation of amorphous PLA50 at 60°C and pH7.8In the degradation medium at
60°C, the initially transparent plates rapidly became white at the surface. After two days,
heterogeneous degradation with surface/interior differentiation was already detectable.
After 21 days, only traces of degradation residue in the form of a white powder remained.
No hollow structures were obtained, which was related to the rapid degradation at
elevated temperature and the increase in the diffusion coefficient of water and water
soluble oligomers above the Tg of PLA.
39
2.4.2.2.2 Water Absorption
Water absorption is the first event that occurs when a PLA specimen is in contact with an aqueous medium. It both causes and reflects the hydrolytic degradation. The percentage of water absorption(WA%) was calculated according to the following equation,8
%WA= (Ww-Wi)/Wi x 100
where Ww= the wet weight of the specimen after wiping and Wi= the initial dry weight of
the sample.8
Water absorption for PLA50 (compression molded circular plates, thickness = 1.5
mm, Mw = 153,100 Da, Mw/Mn = 1.8) in phosphate buffer solution (PBS) at 37°C
increased linearly for 7 weeks, and became 100% by the 9th week.34 In the case of PLA50
in PBS at 60°C, water absorption increased linearly to 20% for the first 7days. Between
the 7 and 10 days, the water absorption increased rapidly up to 90% (Figure 2.7).8
40
Figure 2.7: Evolution of water absorption during the degradation of PLA50 (Mw =
153,100 Da, Mw/Mn = 1.8) in the shape of plates (1.5 mm), in phosphate buffer at 60°C
and pH 7.4. 8
2.4.2.2.3Weight Loss
In the case of biodegradable polymers, weight loss occurs due to the release of
water-soluble oligomers whenever such oligomers are formed within the degraded
matrices. The percentage of weight loss (%WL) was calculated according to the
following equation,18
%WL= (Wi-Wr)/Wix 100
where Wi= initial weight of the specimen and Wr= remaining weight of the specimens.
The dynamics of weight loss for a number of PLGA films of different composition were
similar to each other.39 The thick PLGA 50:50 films lost mass faster than the
corresponding thin films. The weight loss of poly(DL-lactide) (PDLLA)was compared
between temperatures 37°C and 60°C at pH7.4.8 At 60°C only 1.5% weight loss was
41
detected in 7 days, and it increased to 10% at the end of 10 days. Between 10 and 21
days, the weight loss increased to 90% (Figure 2.8).The weight loss data confirmed that
PDLLA degrades rapidly at 60°C and pH7.4.
Figure 2.8: Evolution of weight loss during the degradation of PLA50 (Mw = 153,100 Da,
8 Mw/Mn = 1.8) in the shape of plates (1.5 mm), in phosphate buffer at 60°C and pH 7.4.
2.4.2.2.4 Molecular Weight Loss
The degradation of poly(hydroxyacid)s in the aqueous media proceeds via a random, bulk hydrolysis of ester bonds in the polymer chain.6 The products of
degradation, carboxylic acids, catalyze the degradation process. Absorption of water is
accompanied by ester bond cleavage and molecular weight decrease. The degradation
products in the bulk are not able to diffuse out of the polymer, and thereby decrease the
pH of the bulk, which begins to catalyze the degradation. This is the autocatalytic effect.
As a result, degradation is heterogeneous with the bulk degrading into oligomers that
diffuse from the surface once their molecular weight is low enough, leaving behind a
hollow shell.10, 21
42
The molecular weight of the PLGA films, of various co-polymer compositions,
decreased immediately after introducing to the phosphate buffer saline and continued to
decrease throughout the degradation. The decrease in molecular weight of both PLGA
75:25 and PLGA 50:50 was faster with thick films than thin ones. The gel permeation
chromatograms of the thin films of PLGA had a single broad peak before degradation and
showed bimodal peaks after 21 days of degradation, indicating the presence of fast and
slowly degrading domains, corresponding to the inner and surface layers(Figure 2.9).39
Figure 2.9: Gel permeation chromatograms of thin PLGA 50:50 (thickness = 10 µm, Mw
= 43,900 Da, pdi = 4.1) films showing the changes in the molecular weight distribution
(Mw) during degradation: at day 0 (1), after 21 days (2), and 56 days (3).
43
2.4.2.2.5 Change in pH
To monitor the release of the acidic components, the degradation of PDLLA (Mw
= 65,000 Da, pdi = 1.6) was performed in an unbuffered saline. The PDLLA were machined in parallelepipedic shape (15 mm x 10 mm x 2 mm), out of compression molded round plates of 2mm thickness. For the ffiirst 5 weeks, the pH remained constant at
6.8, but then dropped dramatically to 2.6 from week 8 to week 10, reflecting the release of oligomeric and monomeric lactic acid (Figure 2.10).35 Similarly, the pH of the medium containing poly(L-lactide), in the shape of cylindrical bars (outer diameter = 3.2 mm),
(Mw = 60,100, pdi = 2.1) decreased to 6.9 after 40 weeks and reached a value of 5.4 after
1 year. For poly(L/D-lactide) (Mw = 57,800, pdi = 2.3) and poly(L/DL-lactide) (Mw =
43,700, pdi = 2.2), the pH of the buffer sollution decreased throughout the entire experiment, reaching a value of 2.5-3 after 52 weeks.34
Figure 2.10: Evolution of pH during degradation of PDLLA (Mw = 65,000 Da, pdi = 1.6)
in parallelepipedic shape (15 mm x 10 mm x 2 mm) in an unbuffered solution.24, 35
44
CHAPTER III
EXPERIMENTAL METHODS
3.1 Materials
N,N’-diisopropyl carbodiimide (DiPC, TCI America, 97%), 1,8-
Diazabicycloundec-7-ene (DBU, Acros Organics, 98%), 4-dimethyl-aminopyridine
(DMAP, Acros, 99%), acetic acid, glacial (AcOH, EMD Chemicals, 99.7 %), benzyl bromide (Alfa Aesar, 99%), diphenyl ether (DPE, Sigma Aldrich, 99%), glycolic acid
(TCI America, 98%), hydrochloric acid (HCl, EMD Chemicals, 36.5% – 38.0%), hydrogen bromide (Acros Organics, 48% aqueous solution), DL-Lactic acid (Acros
Organics, 85%), lithium hydroxide (LiOH, Sigma Aldrich, 98%), methanol (Fisher
Scientific, 99.5%), methyl glycolate (TCI America, 99%), methyl lactate (TCI America,
98%), palladium (Pd/C, 10% wt on activated carbon), potassium bromide (Alfa Aesar,
99%), DL-serine (Alfa Aesar, 99%), sodium bicarbonate (Mallinckrodt), sodium nitrite
(Fisher Scientific, 97%), tert-butyldiphenylsilylchloride (TBDPSCl, Aldrich, 98%), tetra- n-butylammoniumfluoride (TBAF, Aldrich, 1.0M in THF), p-toluenesulfonic acid
(pTSA, Fisher Scientific) were used as received.
45
Reagent grade tetrahydrofuran (THF) was dried by distillation from purple
sodium benzophenone ketyl under N2. Reagent grade dichloromethane was washed with
concentrated H2SO4 (95-98%) and water, dried over MgSO4 and further dried by distillation over CaH under N2. Triethylamine (TEA, Alfa Aesar, 99%) was distilled from
KOH and stored over KOH. All other reagents and solvents were commercially available and used as received.
3.2 General Techniques
All reactions were performed under a N2 atmosphere using a Schlenk line unless
noted otherwise. 1H and 13C NMR spectra (δ, ppm) were recorded on a Varian Mercury
300 (300 MHz and 75 MHz, respectively) spectrometer, and the 2D-gHSQC on the
Varian Mercury 500 spectrometer. All spectra were recorded in CDCl3 or a mixture of
CDCl3 and DMSO-d6 unless mentioned otherwise; and the resonances were measured
relative to the residual solvent resonances and referenced to tetramethylsilane (0.00 ppm).
Two-dimensional 1H-13C gradient Heteronuclear Single Quantum Correlation
Spectroscopy (2D-gHSQC, from Varian pulse sequence library) data were acquired using
a relaxation delay (d1) 1 s and an acquisition time (at) 0.12 s with simultaneous 13C
Globally-optimized Alternating-phase Rectangular Pulses (GARP) decoupling. Spectral
widths of 5.1 KHz in the 1H dimension and 21.4 KHz in the 13C dimension were used.
The π/2 pulse widths for 1H and 13C were 8.1 and 16.2 µsec, respectively. The data
acquired consisted of 2 x 128 data points for the 1H and 512 data points for 13C
dimensions, with 4 transients for each increment.
46
Number average (Mn) and weight average (Mw) molecular weights and
polydispersities (PDI = Mn / Mw) were determined by gel permeation chromatography
relative to linear polystyrene (GPCPSt) calibration curves of log Mn vs. elution volume at
35 °C using tetrahydrofuran (THF) as solvent (1.0 mL/min), a set of 50 Å, 100 Å, 500 Å,
104 Å and linear (50-104 Å) Styragel 5 µm columns, a Waters 486 tunable UV/Vis
detector set at 254 nm, a Waters 410 differential refractometer, and Millenium Empower
2 software. Thermal analysis was performed using a Perkin Elmer Pyris 1 differential
scanning calorimeter. Glass transition temperatures (Tg) were determined as the middle of
the heat capacity change. Heating and cooling rates were 10 °C/min. Transition
temperatures were calibrated using indium and tin standards; enthalpy was calibrated
using an indium standard.
Scanning electron microscopy (SEM) was performed using a JEOL 6300f
fieldemission scanning electron microscope. The samples were gold sputter-coated for
imaging.
Dynamic light scattering experiments were conducted using a Brookhaven
Instrument coupled with a BI-200SM goniometer, BI-9000AT correlator, and an EMI-
9863 photomultiplier tube for photon counting. A Meller Griot 35 mW He-Ne laser was
used as light source (632.8 nm). A cylindrical glass scattering cell with a diameter of 12
mm was placed at the center of a thermostated bath with decahydronaphthalene used for
refractive index matching. The measurements were carried out at 90° scattering angle at
25 °C.
47
The following procedure was followed for microparticle formulation.
1. Dissolved 3 mg of poly(ethylene glycol)-grafted-to-chitosan (PEG-g-CHN) in 0.9
ml of 0.1 acetic acid at 37 °C for 48 hours to prepare 3.33 mg/ml solution.
2. Dissolved 9.0 g of polyvinylpyrrolidone (PVP) in 90 ml of autoclaved and
deionized H2O to prepare 90 ml of 10% (w/v) PVP solution.
3. Dissolved 297 mg of PLB or PLGB copolymer in 2.97 ml of chloroform for 1
minute to prepare 100 mg/ml polymer solution in chloroform.
4. Water-in-oil: 2.97 ml of 100mg/ml polymer solution, 0.9 ml of 3.33 mg/ml PEG-
g-CHN, and 0.6 ml of autoclaved and deionized H2O, vortexed with impeller at
2000 rpm for 3 minutes.
5. Water-in-oil-in-water: Added 90 ml of 10% PVP to water-in-oil emulsion and
vortexed with impeller at 1600 rpm for 3 minutes.
6. Evaporated chloroform from the microspheres for 5 hours in a ventilated hood,
while mixing gently by a stir bar. Foil with a small ventilation hole was also
placed over the beaker.
7. Next, the microspheres were transferred to a 50 ml centrifuge tube and were
centrifuged at 12,000 x g for 20 minutes at 4 °C.
8. A pellet was formed and the supernatant was decanted and discarded. The pellets
were resuspended in autoclaved and deionized H2O and were centrifuged at
12,000 x g for 20 minutes.
9. A pellet was formed and the supernatant was decanted and discarded. The pellets
were resuspended in 1% PVP and were centrifuged at 12,000 x g for 20 minutes.
48
10. A pellet was formed and the supernatant was decanted and discarded. The pellets
were resuspended in 10 ml autoclaved and deionized H2O.
11. Using a bucket of dry ice, the microspheres were shell frozen in a 50 ml
centrifuge tube. The tube was constantly rotated for 10 minutes while lying nearly
horizontal and buried in the dry ice.
12. The shell frozen microspheres were connected to the lyophilizer and lyophilized
for 72 hours.
13. After the microspheres had been freeze-dried by the lyophilizer, they were placed
in a desiccator.
3.3 Synthesis of 2-bromo-3-hydroxypropionic acid
In a 3-neck, 1000 mL, round-bottom flask, sodium nitrite (12 g, 0.17 mol) was added in portions over 270 min to a solution of DL-serine (10 g, 0.10 mol), HBr (26 mL,
48% aq. w/w, 0.23 mol) and potassium bromide (40 g, 0.33 mol) in water (88 mL) at approximately -10 °C. After stirring at room temperature for 16 h, the light-greenish solution was saturated with NaCl and extracted five times with ethyl acetate (50 mL each). The combined organic extracts were washed five times with satd. aq. NaCl (50 mL each), and dried over anhyd. MgSO4. After filtration and removing the solvent by trap-to-
trap distillation, the residue was recrystallized from CH2Cl2 to obtain 2-bromo-3-
hydroxypropionic acid as a white solid. (10 g, 63%); 1H NMR δ 3.87 (dd, CHHOH, 3J =
49
5.5 Hz, 2J = 11.9 Hz), 3.98 (dd, CHHOH, 3J = 7.5 Hz, 2J = 11.9 Hz), 4.28 (dd, CHBr, 3J
= 5.4 Hz, 3J = 7.5 Hz), 8.0 (broad s, COOH and OH). 13C NMR δ: 45.6 (CBr), 64.0
(COH), 171.0 (C=O).
3.4 Synthesis of poly(lactic acid-co-glycolic acid-co-2-bromo-3-hydroxypropionic acid)
(PLGB801010) by bulk polycondensation
DL-Lactic acid (0.93 g, 8.98 mmol), glycolic acid (0.08 g, 1.11 mmol), 2-bromo-
3-hydroxypropionic acid (0.19 g, 1.11 mmol), diphenyl ether (1.0ml, 6.30 mmol) and catalyst pTSA (0.05 g, 0.29 mm) were added to a schlenk tube equipped with a stir bar.
The schlenk tube was then introduced to an oil bath at 95 °C, and the pressure was reduced to around 1-3 mm Hg. The reaction was stopped after 48 hrs by removing the
Schlenk tube from the oil bath, and bringing it to atmospheric pressure. The contents of the schlenk tube were dissolved in 10 ml dichloromethane. The diphenyl ether is removed by cooling the Schlenk tube and filtering the solution by gravity filtration. The polymer is isolated by precipitating into rapidly stirring methanol (50ml) five times, and drying in
1 vacuo. (0.53 g, 53%). H NMR: 1.4-1.8 (m, 3H, CH3), 4.4–4.7 (m, 3H, CHBrCH2), 4.7-
13 4.9 (m, 2H, CH2COO), 5.10-5.30 (m, H, CH(CH3)COO). C NMR: 16.60 (CH3), 39.36
(CBr), 60.75 (OCH2COO), 64.49 (OCH2CHBr), 69.05 (CH(CH3)COO), 166.46
(CH2COO), 166.52 (CHBrCOO), 169.30 (C(CH3)COO). GPC P st standards: Mn=2.99 x
4 4 10 , Mw=7.49 x 10 , pdi=2.51.
50
3.5 Synthesis of poly(lactic acid-co-2-bromo-3-hydroxypropionic acid) (PLB8020) by solution polymerization
A mixture of DL-lactic acid (0.69 g, 6.7 mmol), 2-bromo-3-hydroxypropionic
acid (0.28 g, 1.7 mmol), pTSA (0.050 g, 0.29 mmol) in a Schlenk tube was stirred at 110
°C under reduced pressure (~1-3 mm Hg) for 14 h to create a prepolymer. The Schlenk tube was removed from the oil bath, opened to the atmosphere, and its contents were dissolved in CH2Cl2 (60 mL) and half of the contents were transferred to a 50ml round
bottom flask. The solvent was refluxed over oven dried 4Å molecular sieves for 18 h, to
completely dehydrate the polymer solution. After cooling the solution to room
temperature, DPTS (0.10 g, 0.34 mmol) was added, and the solution was further cooled
to 0 - 5 °C using an ice bath and DiPC (0.20 g, 1.6 mmol) was added dropwise. The
reaction was performed for 7 h at RT. The polymer was precipitated into methanol
(50mL) stirring in a 100ml round bottom flask. The methanol was carefully decanted
away, and the polymer dried inside the round bottom flask under vacuum. The polymer was reprecipitated four times from CH2Cl2 (10 mL) into methanol (50 mL) to yield
3 halogenated PLA as a white powder. (0.23 g, 52%). GPCPst Prepolymer: Mn = 8.81 x 10 ,
4 1 pdi=2.15. GPCPst Polymer: Mn=1.53 x 10 , pdi=1.71. H NMR: 1.4-1.8 (m, CH3), 4.4–4.7
13 (m, CHBrCH2), 5.10-5.30 (m, CHCH3). C NMR: 16.62 (CH3), 39.78 (CBr), 64.49
(CH2CHBr), 68.95 (CHCH3), 166.63 (CHBrCO2), 169.33 (C(CH3)COO).
51
3.6 Synthesis of benzyl (DL)-lactate (Bn-LA)
1,8-Diazabicyclo[5.4.0]undec-7-ene (7.35 g, 48.27 mmol) was added slowly,
with stirring, to a solution of 85% DL-Lactic acid (5.00 g, 48.27 mmol) in methanol (25
ml). The solvent was removed under reduced pressure at 70-80 °C, and the resulting oil,
in dimethylformamide (35 ml), was cooled to 0 °C. Benzyl bromide (7.43 g, 43.44 mmol)
was added dropwise, and the reaction mixture was stirred at room temperature for 48 h.
After removal of most of the solvent by vacuum distillation, ethyl acetate (35 ml) was
added, followed by water (10 ml). The aqueous layer was washed with ethyl acetate (30
ml), and the combined organic extracts were washed successively with water (20 ml), 5%
citric acid (20 ml), saturated sodium bicarbonate solution (3 x 20 ml) and saturated sodium chloride solution (2 x 15 ml), dried over anhydrous MgSO4, and the solvent was
removed by rotary evaporation. The crude product was distilled (95 – 100 °C, 1-2mm
1 3 Hg), and obtained as a colorless oil. (3.78 g, 44%). H NMR: 1.44 (d, 3H, CH3, J = 7.0
3 Hz), 4.33 (q, H, CH(CH3)COO, J = 7.0Hz), 5.22 (s, 2H, C5H5CH2COO), 7.36 (m, 5H,
13 C5H5). C NMR: 20.35 (C, CH3), 66.86 (C, CH), 67.21 (C, CH2), 128.23, 128.51, 128.65,
135.29 (5C, Ar), 175.53 (C, COO).
52
3.7 Synthesis of benzyl glycolate (Bn-GA)
1,8-Diazabicyclo[5.4.0]undec-7-ene (8.01 g, 52.60mmol) was added slowly, with
stirring, to a solution of glycolic acid (4.00 g, 52.60mmol) in methanol (25 ml). The solvent was removed under reduced pressure at 70-80 °C, and the resulting oil, in dimethylformamide (50 ml), was cooled to 0 °C. Benzyl bromide (8.10 g, 47.34mmol) was added dropwise, and the reaction mixture was stirred at room temperature for 24 h.
After removal of most of the solvent by vacuum distillation, ethyl acetate (50 ml) was added, followed by water (25 ml). The organic layer was washed with water (50 ml), 5% citric acid (50 ml), twice with saturated sodium bicarbonate solution (2 x 50 ml) and twice with saturated sodium chloride solution (2 x 50 ml), dried over anhydrous MgSO4, and the solvent was removed by rotary evaporation. The crude product was distilled (95 –
100 °C, 1-2mm Hg), and obtained as a colorless oil. (4.51 g, 67%). 1H NMR: 3.08 (s, H,
13 OH), 4.20 (s, H, CH2COO), 5.22 (s, 2H, C5H5CH2COO), 7.36 (m, 5H, C5H5). C NMR:
60.68 (C, CH2OH), 67.09 (C, CH2C5H5), 128.42, 128.56, 128.65, 135.19 (C, 5H, Ar),
173.24 (C, COO).
53
3.8 Synthesis of methyl 2-(tert-butyldiphenylsilanyloxy) acetate
Methyl glycolate (1.00 g, 11.10mmol), triethylamine(2.25 g, 22.20mmol), DMAP
(0.68 g, 5.50 mmol), and anhydrous methylene chloride (50 mL) were added under
nitrogen to a 100 mL oven-dried 3 neck round bottom flask. After cooling the reaction
mixture to 0 °C, TBDPSCl (3.36 g, 12.21mmol) was added. The ice bath was removed
and the reaction was stirred at room temperature overnight (18 h). The reaction mixture was filtered, and the filtrate was washed with 10%HCl (2 x 30 mL), H2O (2 x 30 mL),
and dried over MgSO4. Solvent was removed under vacuo to obtain colorless oil. (3.34 g,
1 97%). H NMR: 1.09 (s, 9H, C(CH3)3), 3.68 (s, 3H, CH3), 4.23 (s, 2H, CH2), 7.44(m, 6H,
13 Ar), 7.64 (m, 4H, Ar). C NMR: 19.30 (C, C(CH3)3), 26.73 (3C, C(CH3)3), 51.61 (C,
CH3), 62.17 (C, CH2), 127.82, 129.93, 132.85, 135.61 (6C, Ar), 171.62 (C, COO).
54
3.9 Synthesis of (tert-butyldiphenylsilanyloxy) acetic acid (GA-SiTBDP)
Methyl 2-(tert-butyldiphenylsilanyloxy) acetate (1.0 g, 3.04 mmol) was dissolved in THF (50 mL) and cooled in an icebath. 0.2M LiOH (33ml, 6.09 mmol of LiOH) was added dropwise over 15 min. The ice bath was removed, and the reaction was stirred for
10min. Water (30 mL) was added and the THF was removed under vacuum. The aqueous phase was extracted with diethyl ether (2 x 20 mL). This ether phase is washed with saturated NaHCO3 solution (40 mL). Both aqueous layers are combined together and
carefully acidified to a pH of 4.0 by adding HCl dropwise, and then extracted with ether
(2 x 50 mL). The second ethereal phase was dried with MgSO4 and the solvent was
removed in vacuo to obtain colorless oil. The crude oil was chromatographed over silica
gel (5% ethyl acetate in hexanes) to obtain product as colorless oil. (0.38 g, 40%). 1H
13 NMR: 1.11 (s, 9H, C(CH3)3), 4.23 (s, 2H, CH2), 7.44 (m, 6H, Ar), 7.64 (m, 4H, Ar). C
NMR: 19.20 (C, C(CH3)3), 26.69 (C, C(CH3)3), 61.90 (C, CH2), 127.99, 130.20, 132.06,
135.47 (10C, Ar), 174.90 (C, COO).
55
3.10 Synthesis of methyl 2-((tert-butyldiphenylsilyl)oxy)propanoate
Methyl lactate (3.00 g, 28.8mmol), triethylamine (5.83 g, 57.6mmol), DMAP
(0.50 g, 14.4 mmol), and anhydrous methylene chloride (100 mL) were added under
nitrogen to a 250 mL oven-dried 3 neck round bottom flask. After cooling the reaction
mixture to 0 °C, TBDPSCl (8.71 g, 31.7mmol) was added. The ice bath was removed and
the reaction was stirred at room temperature overnight (18 h). The reaction mixture was
filtered, and the filtrate was washed with 10%HCl (2 x75 mL), H2O (2 x50 mL), and
dried over MgSO4.Removal of the solvent under vacuum gave colorless oil. (8.2 g, 83%).
1 3 H NMR: 1.09 (s, 9H, C(CH3)3), 1.37 (d, 3H, CH3, J = 6.0 Hz), 3.56 (s, 3H, CH3), 4.28
3 13 (q, H, CH, J = 6.0 Hz), 7.41(m, 6H, Ar), 7.66 (m, 4H, Ar). C NMR: 19.18 (C, CH3),
21.01 (C, C(CH3)3), 26.85 (3C, C(CH3)3), 65.83 (3C, CH3O), 69.07 (C, CH2), 127.86,
130.10, 132.22, 132.86, 135.72 (12C, Ar), 176.94 (C, COO).
56
3.11 Synthesis of 2-((tert-butyldiphenylsilyl)oxy)propanoic acid (LA-SiTBDP)
Methyl 2-((tert-butyldiphenylsilyl)oxy)propanoate (8.2 g, 24 mmol) was
dissolved in THF (100 mL) and cooled in an icebath. 0.2M LiOH (240 mL, 48mmol of
LiOH) was added dropwise for over 20 min. The ice bath was removed, and the reaction
was stirred for 30 min at 30 °C. THF was removed under vacuum. The aqueous phase
was extracted with diethyl ether (100 mL). This aqueous phase is then acidified to a pH
of 2 by adding HCl dropwise, and then extracted with diethyl ether (2 x 100 mL). The
ethereal phase was dried over MgSO4 and the solvent was removed to obtain colorless
oil. The crude oil was chromatographed over silica gel (5% ethyl acetate in hexanes) to
1 3 obtain colorless oil (4.7g, 49%). H NMR: 1.14 (s, 9H, C(CH3)3), 1.39 (d, 3H, CH3, J = 6
Hz), 4.35 (q, H, CH, 3J = 6 Hz), 7.43 (m, 6H, Ar), 7.75 (m, 4H, Ar). 13C NMR: 19.21 (C,
CH3), 21.03 (C, C(CH3)3), 26.82 (3C, C(CH3)3), 69.00 (C, CH2), 127.84, 130.11, 132.35,
132.98, 135.80 (12C, Ar), 177.63 (C, COO).
57
3.12 Synthesis of methyl 2-bromo-3-hydroxypropionate
A solution of 2-bromo-3-hydroxypropionic acid (13.87 g, 82.1 mmol) and a
catalytic amount of HBr (0.5 mL, 48% aq. w/w) in methanol (100 ml) was stirred at 65
°C for 21 h. The unreacted excess methanol was removed by rotary evaporation. The
brownish liquid residue was dissolved in CH2Cl2 (100 ml) and was washed twice with
dil. aq. NaHCO3 (100 ml), and once with satd. aq. NaCl (100 ml), and then dried over
MgSO4. After filtration and removing the solvent by rotary evaporation, methyl 2-bromo-
3-hydroxypropionate was obtained as a light yellow oil (8.40 g, 55.9 %) 1H NMR: 2.58
3 2 3 (t, H, OH, J = 6 Hz), 3.81 (s, 3H, CH3), 3.86 (dd, H, CHHOH, J = 5.5 Hz, J = 12.2
Hz), 3.98 (dd, H, CHHOH, 2J = 7.7 Hz, 3J = 11.9 Hz), 4.29 (dd, H, CHBr, 3J = 5.5 Hz, 3J
13 = 7.5 Hz). C NMR: 44.25 (C, CHBr), 53.20 (C, CH3), 63.64 (C, CH2), 169.43 (C, COO).
3.13 Synthesis of 2-bromo-3-((tert-butyldiphenylsilyl)oxy)propionate
Methyl 2-bromo-3-hydroxypropionate (8.30 g, 45.4mmol), triethylamine (9.18 g,
90.7mmol), DMAP (2.77 g, 22.7mmol), and anhydrous methylene chloride (100 mL) were added under nitrogen to a 250 mL oven-dried 3 neck round bottom flask. After
58
cooling the reaction mixture to 0 °C, TBDPSCl (13.71 g, 49.9mmol) was added. The ice
bath was removed and the reaction was stirred at room temperature overnight (18 h). The
reaction mixture was filtered, and the filtrate was washed with 10%HCl (2 x100 mL),
water (2 x100 mL), and dried over MgSO4. Solvent was removed under vacuo to obtain a
1 colorless oil.(18.79 g, 98%). H NMR: 1.08 (s, 3H, C(CH3)3), 3.81 (s, H, CH3), 3.96 (dd,
3 3 2 3 H, CHBr, J = 5.6 Hz, J = 8.5 Hz), 4.16 (dd, H, CH2, J = 10.3 Hz, J = 8.5 Hz), 4.31
2 3 13 (dd, H, CH2, J = 10.3 Hz, J = 5.6 Hz), 7.44 (m, 6H,Ar), 7.72 (m, 4H, Ar). C NMR:
19.24 (C, C(CH3)3), 26.67 (C, C(CH3)3), 43.95 (C, CHBr), 52.91 (C, CH3), 65.10 (C,
CH2), 127.85, 129.99, 132.83, 134.82, 135.56 (10C, Ar), 169.10 (C, COO).
3.14 Synthesis of 2-bromo-3-((tert-butyldiphenylsilyl)oxy)propionic acid (B-SiTBDP)
2-bromo-3-((tert-butyldiphenylsilyl)oxy)propionate (5.0 g, 10.4mmol) was
dissolved in THF (50 mL) and cooled in an icebath. 0.2M LiOH (105 mL, 20.9mmol of
LiOH) was added dropwise for over 15 min. The ice bath was removed, and the reaction was stirred at 45 °C for 1 h. THF was removed using a rotary evaporator. The aqueous phase was extracted with diethyl ether (100 mL). The aqueous layer was then acidified to a pH of 2 by adding HCl dropwise, and then extracted with diethyl ether (2 x 100 mL).
The second ethereal phase was dried over MgSO4 and the solvent was removed to obtain
1 3 colorless oil (3.29 g, 60%). H NMR: 1.06 (s, 3H, C(CH3)3), 3.96 (dd, H, CHBr, J = 5.5
59
3 2 3 2 Hz, J = 8.0 Hz), 4.12 (dd, H, CH2, J = 10.4 Hz, J = 8.3 Hz), 4.30 (dd, H, CH2, J =
3 13 10.6 Hz, J = 5.6 Hz), 7.65 (m, 4H, Ar), 7.41 (m, 6H,Ar). C NMR: 19.23 (C, C(CH3)3),
26.66 (C, C(CH3)3), 43.67 (C, CHBr), 64.89 (C, CH2), 127.87, 130.02, 132.66, 135.56
(10C, Ar), 174.01 (C, COO).
3.15 Synthesis of Bn-GL-SiTBDP (p-GL-p)
O O Si O O O
Benzyl glycolate (1.99 g, 12 mmol) and LA-SiTBDP (3.93 g, 12 mmol) were
dissolved with 4-dimethylaminopyridine (0.73 g, 5.9 mmol) in anhydrous
dichloromethane (25 ml). After cooling the solution to 0 °C using an ice-bath, N, N’- diisopropylcarbodiimide (2.27 g, 18mmol) was added dropwise and the reaction was stirred for 18 h at room temperature. White precipitate formed was filtered away using a filter paper, and the filtrate was concentrated under vacuum. The crude oil was chromatographed over silica gel (5% ethyl acetate in hexanes) to give a colorless oil. (2.9
1 3 g, 50%). H NMR: 1.09 (s, 9H, C(CH3)3), 1.39 (d, 3H, CH3, J = 6.7 Hz), 4.38 (q, H, CH,
3J = 6.73 Hz), 4.45 (d, H, CHH, 2J = 15.8 Hz), 4.61 (d, H, CHH, 2J = 15.8 Hz), 5.17 (s,
13 H, CH2), 7.36 (m, 11H, Ar), 7.68 (m, 4H, Ar). C NMR: 19.23 (C, CH3), 21.28
(C(CH3)3), 26.79 (C(CH3)3, 60.59 (C, CH2), 67.07 (C, CH2Bn), 68.66 (C, CH), 127.62,
60
127.66, 128.43, 128.62, 132.95, 135.01, 135.73, 135.90 (12C, Ar), 167.32 (C, COOBn),
173.10 (C, COOCH(CH3)).
3.16 Synthesis of HOOC-GL-SiTBDP (HOOC-GL-p)
Bn-GL-SiTBDP (4.50 g, 9.4 mmol) and Pd/C (0.8 g, 5% w/w) were dissolved in
ethyl acetate (25ml), and reacted in a Parr Hydrogenation Reactor at 50 PSI pressure of
H2 and 50 °C for 24 h. The solution was passed through a plug of celite to remove the
Pd/C catalyst and the solvent removed by rotary evaporation to give a white solid. (3.07
1 3 g, 83%). H NMR: 1.11 (s, 9H, C(CH3)3), 1.43 (d, 3H, CH3, J = 6.7 Hz), 4.41 (q, H, CH,
3J = 6.73 Hz), 4.48 (d, H, CHH, 2J = 15.8 Hz), 4.60 (d, H, CHH, 2J = 15.8 Hz), 7.39 (m,
13 11H, Ar), 7.68 (m, 4H, Ar). C NMR: 19.21 (C, CH3), 21.22 (C(CH3)3), 26.78 (C(CH3)3,
60.09 (C, CH2), 68.63 (C, CH), 127.60, 127.66, 129.84, 132.92, 135.72, 135.88 (12C,
Ar), 172.96 (C, COO), 173.04 (C, COOCH(CH3)).
61
3.17 Synthesis of Bn-LGL-SiTBDP (p-LGL-p)
Benzyl lactate (1.43 g, 7.9 mmol) and HOOC-GL-SiTBDP (3.07 g, 7.9mmol)
were dissolved with 4-dimethylaminopyridine (0.10 g,0.8mmol) in anhydrous
dichloromethane (10 ml). After cooling the solution to 0 °C using an ice-bath, N, N’- diisopropylcarbodiimide (1.15 g, 9.1mmol) was added dropwise and the reaction was stirred for 18 h at room temperature. White precipitate formed was filtered away using a filter paper, and the filtrate was concentrated under vacuum. The crude oil was chromatographed over silica gel (5% ethyl acetate in hexanes) to give a colorless oil.
1 3 3 (2.20g, 50%). H NMR: 1.10 (s, 9H, C(CH3)3), 1.41 (dd, 3H, CH3, J = 2.6 Hz, J = 6.7
3 3 Hz), 1.49 (dd, 3H, CH3, J = 2.8 Hz, J = 7.2 Hz), 4.39 (m, H, CH), 4.56 (m, 2H, CH2),
3 3 5.17 (s, 2H, CH2), 5.20 (dd, H, CH, J = 7.0 Hz, J = 1.0 Hz), 7.39 (m, 11H, Ar), 7.67
13 (m, 4H, Ar). C NMR: 16.81 (C, CH3), 19.23 (C, C(CH3)3), 21.28 (C, CH3), 26.81 (C,
C(CH3)3), 60.31 (C, CH2), 67.17 (C, CH2Bn), 68.69 (C, CH(CH3)OSiTBDP), 69.30 (C,
CH(CH3)COOBn), 127.66, 128.15, 128.44, 128.61, 129.82, 132.99, 133.48, 135.19,
135.73, 135.90 (18C, Ar), 168.84 (C, COOCH2), 169.87 (C, COOBn), 172.96 (C,
COOCHCH3OSiTBDP).
62
3.18 Synthesis of Bn-LB-SiTBDP (p-LB-p)
Benzyl lactate (5.57 g. 30.9 mmol) and B-SiTBDP (12.58 g, 30.9 mmol) were
dissolved with dimethylaminopyridine (0.38 g, 3.1 mmol) in anhydrous dichloromethane
(25 ml). After cooling the solution to 0 °C using an ice-bath, N, N’-
diisopropylcarbodiimide (4.48 g, 35.2 mmol) was added dropwise and the reaction was
stirred for 18 h at room temperature. White precipitate formed was filtered away using a
filter paper, and the filtrate was concentrated under vacuum. The crude oil was
chromatographed over silica gel (5% ethyl acetate in hexanes) to give a colorless oil. (9.2
1 3 g, 52%). H NMR: 1.05 (s, 9H, C(CH3)3), 1.56 (d, 3H, CH3, J = 7.0 Hz), 3.96 (dd, H,
CHH, 3J = 5.6 Hz, 2J = 10.4 Hz), 4.18 (dd, H, CHH, 3J = 8.5 Hz, 2J = 10.4 Hz), 4.35 (dd,
3 3 3 H, CHBr, J = 5.7 Hz, J = 8.3 Hz), 5.21 (d, 2H, CH2, J = 2.9 Hz), 5.26 (d, H, CH, J =
13 7.0 Hz), 7.35 (s, 5H, (C5H5)CH2)), 7.68 (m, 4H, Ar), 7.40 (m, 6H, Ar). C NMR: 16.85
(C, CH3), 19.24 (C, C(CH3)3), 26.65 (C(CH3)3), 43.69 (C, CHBr), 64.95 (C, CH2), 67.20
(C, CH2), 69.87 (C, CH), 127.85, 128.29, 128.60, 130.00, 132.58, 132.76, 135.52 (18C,
Ar), 168.10 (C, COOCHBr), 169.78 (C, COOCHCH3).
63
3.19 Synthesis of Bn-LB-OH (p-LB-OH)
Bn-LB-SiTBDP (4.19 g, 7.4 mmol) and acetic acid (0.66 g, 11 mmol) were
combined under nitrogen with 10 ml of THF, in an ice bath. TBAF (1M in THF, 8.8 ml,
8.8 mmol) was added, and the reaction mixture was stirred for 2 h in an ice bath. THF
was removed by rotary evaporation, and product extracted with diethyl ether (50 ml). The
ethereal phase was washed brine (50 ml x 2) and dried over MgSO4. The solvent was
removed in vacuo and product was chromatographed over silica gel (25% ethyl acetate in
1 3 hexanes) to give a colorless oil. (0.90 g, 37 %). H NMR: 1.57 (d, 3H, CH3, J = 7 Hz),
3.16 (s, broad, H, OH), 3.91 (dd, H, CHH, 3J = 5.3 Hz, 2J = 11.9 Hz), 4.10 (dd, H, CHH,
3J = 8.6 Hz, 2J = 11.9 Hz), 4.44 (dd, H, CHBr, 3J = 5.4 Hz, 3J = 8.6 Hz), 5.20 (d, 2H,
3 13 CH2, J = 6.4 Hz), 5.30 (q, H, CH, J = 7.1 Hz), 7.36 (s, 5H, (C5H5)CH2)). C NMR:
16.56 (C, CH3), 43.38 (C, CHBr), 64.03 (C, CH2CHBr), 67.66 (C, CH2), 69.68 (C, CH),
128.32, 128.66, 134.83, (5C, Ar), 167.88 (C, COOCHBr), 170.61 (C, COOCHCH3).
64
3.20 Synthesis of Bn-LBrL-SiTBDP (p-LBL-p)
Bn-LB-OH (1.54 g, 6.7 mmol) and LA-SiTBDP (1.84 g, 5.6 mmol) were
dissolved with dimethylaminopyridine (0.06 g, 0.5 mmol) in anhydrous dichloromethane
(10 ml). After cooling the solution to 0 °C using an ice-bath, N, N’-
diisopropylcarbodiimide (0.67 g, 5.4 mmol) was added dropwise and the reaction was
stirred for 18 h at room temperature. White precipitate formed was filtered away using a
filter paper, and the filtrate was concentrated under vacuum. The crude oil was
chromatographed over silica gel (5% ethyl acetate in hexanes) to give a colorless oil.
1 3 (1.56 g, 52%). H NMR: 1.11 (s, 9H, C(CH3)3), 1.38 (d, 3H, CH3, J = 6.7 Hz), 1.52(m,
3H, CH3COOBn), 4.29 (m, H, CHBr), 4.32 (m, H, CH(CH3)SiTBDP), 4.38 (m, 2H,
3 CH2CHBr), 5.16 (d, 2H, CH2, J = 3.5 Hz), 5.18 (d, H, CH, J = 2.3 Hz), 7.46-7.34 (m,
13 11H, Ar), 7.71 – 7.66 (m, 4H, Ar). C NMR: 16.64 (C, CH3COOBn), 19.22 (C,
C(CH3)3), 21.24 (C, CH3CHOSiTBDP), 26.82 (3C, C(CH3)3), 40.05 (C, CHBr), 63.88 (C,
CH2CHBr), 67.21 (C, CH2Bn), 68.67 (C, CHOSiTBDP), 70.17 (C, CHCOOBn), 127.64,
128.26, 128.60, 130.04, 135.09, 135.72, 135.87(18C, Ar),166.81 (C, COOCHBr), 169.44
(C, COOBn), 172.83 (C, COOCH(CH3)OSiTBDP).
65
CHAPTER IV
FUNCTIONALIZABLE BIODEGRADABLE POLYESTERS
4.1 Synthesis of Functionalized Monomer
The functionalizable monomer, 2-bromo-3-hydroxypropionic acid was prepared
through the diazotization of the secondary amine on DL-Serine, and then a halogenation
of the diazo intermediate. This route can produce both chloro and bromo derivatives, but
throughout my work, I only used the bromo derivative.2
Scheme 4.1: Synthesis of 2-bromo-3-hydroxypropionic acid
The serine was dissolved in acidic water and sodium nitrite was added in small
fractions at sub-zero temperatures. After reacting for 24 hours, the product was salted out
and extracted with ethyl acetate. After recrystallizing from CH2Cl2, the halohydrin was
obtained as white crystals. For example, the 2-bromo-3-hydroxypropionic acid
(bromohydrin) was synthesized in ~60% yield from acidic aqueous solution (HBr) of
66
(DL)-2-amino-3-hydroxypropionic acid (DL-Serine) in the presence of potassium
bromide (KBr). Figures 4.1 and 4.2 present the 1H and 13C NMR spectra, respectively, of
2-bromo-3-hydroxypropionic acid in CDCl3/DMSO-d6 at room temperature. Both
methylene (CH2) protons appear as doublets of doublets since the two protons are
chemically inequivalent due to the α asymmetric center (CHBr). The two resonances
appear at 3.87 ppm and 3.98 ppm, with coupling constants of 2J = 5.5 Hz and 3J = 11.8
Hz, and 2J = 7.5 Hz and 3J = 11.9 Hz. The methine (CH) at 4.28 ppm also appears as a
doublet of doublets with coupling constants 3J = 5.4 Hz and 3J = 7.5 Hz. In the 13C NMR
spectrum, the carbon attached to the bromine resonates at 45.13 ppm. In all of the
polymers presented in this dissertation, the presence of the bromine functionality can be
verified by a carbon (CHBr) in the range of 40-50 ppm on the 13C NMR spectrum.
67
22’ 1 dd dd dd
3 J = 11.8 Hz 3J = 11.9 Hz
2J = 7.5 Hz 2 3J = 7.5 Hz J = 5.5 Hz 3.97 3.90 3 3.89 4.29 J = 5.4 Hz 3.94 4.28 4.26 4.30 4.01 3.87 3.85 3.98
1.00 1.06 1.05
4.30 4.25 4.20 4.15 4.10 4.05 4.00 3.95 3.90 3.85 3.80 Chemical Shift (ppm)
12, 2’
3.97 1 3.90 3.89
4.29 2, 2’ 3.94 4.28 4.30 4.01 3.87 3.85
*
1.66 1.051.00
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)
Figure 4.1: 1H NMR (300 MHz) spectrum of 2-bromo-3-hydroxypropionic acid
synthesized by deaminohalogenation route; * = CHCl3.
68
1 2 45.13 63.71
3 1 2 3 170.93
*
180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)
Figure 4.2: 13C NMR (75 MHz) spectrum of 2-bromo-3-hydroxypropionic acid
synthesized by deaminohalogenation route; * = CHCl3.
4.2 Synthesis of the PLGB polymers
Biodegradable polymers and copolymers of lactic acid and glycolic acid have
been prepared before using acid catalysts in bulk polycondensation conditions, driven by
heat and low pressure, as discussed in section 2.3.1 of this dissertation. Adapting from the
literature, I optimized the reaction conditions for synthesizing PLA, PLGA and PLGB as
depicted in Scheme 4.2.
69
Scheme 4.2: Synthesis of PLGB from lactic acid using acid catalyst pTSA, and DPE as
high boiling solvent/plasticizer.
The polycondensation reaction of hydroxyacids is a reversible reaction, in which water is
released as a byproduct of the condensation of the alcohol and carboxylic acid. Removing
the water byproduct using heat and low pressure drives the reaction forward, by Le
Chatelier’s principle. At higher conversions, it becomes increasingly difficult to remove
the water byproduct due to the high viscosity of the bulk system which limits the conversion. I found that introducing a small amount (1ml/gm of feed monomers) of high
boiling solvent diphenylether (DPE, bp = 121 °C at 10.05 mm Hg) plasticizes the bulk
and eases the removal of water resulting in higher conversion.
4.2.1 PLA and PLGA polymers
For comparison PLA and PLGA copolymers were made on a 1 gram scale
(Scheme 4.3). Their molecular weights are listed in Table 4.1.
70
Scheme 4.3: Synthesis of PLA from lactic acid and PLGA from lactic acid and glycolic
acid monomers using acid catalyst pTSA, and DPE as high boiling solvent/plasticizer.
Table 4.1: Molecular weight and glass transition temperature of PLA and PLGA
synthesized by bulk polycondensation conditions.
Feed Molecular Weight Co‐polymer Glass Ratio Polystyrene Standards Composition Transition 4 4 Sample LA GA Mn x 10 Mw x 10 PDI DP LA GA Tg (°C) Yields (%) PLA 100 0 3.16 5.12 1.62 439 100 0 51 42.4 PLGA9010 90 10 2.29 3.49 1.53 324 89.3 10.7 50 40.9 PLGA8020 80 20 2.01 3.04 1.51 291 78.4 21.6 47 51.0
71
1
* 2
2 1
PLA
1.00 3.68
3 2 1
PLGA‐9010 3
1.00 0.24 3.13
PLGA‐8020
1.00 0.55 3.12
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)
Figure 4.3: 1H NMR (300 MHz) spectra of PLA and PLGA synthesized by bulk
polycondensation reaction conditions; * = CHCl3.
The 1H NMR traces of the polymers are shown in Figure 4.3. In the 1H NMR
spectra, the methine (CH) proton in the backbone of the PLA is observed at 5.2 ppm, and the methyl (CH3) protons are observed at 1.5 ppm. In the PLGA copolymers, the
methylene (CH2) protons from the glycolic acid units are observed at 4.75 ppm. These resonances agree to the literature.48
Since the methylene protons of the glycolic acid units and the methine proton of the lactic acid units are well resolved we can calculate the copolymer composition by
72
comparing the contribution of the integrals under the methine proton from lactic acid
units and the methylene protons from the glycolic acid units. For example, in the case of
the PLGA8020 (Figure 4.3, red spectrum):
where x is the percent of lactic acid units, and y is the percent of glycolic acid units. ICHL is the integrated peak area at 5.2 ppm, and ICH2G is the integrated peak area at 4.75 ppm.
Solving for x and y, x = 78.4% and y = 21.6%. This is close to the feed ratio of 80 mol%
lactic acid and 20 mol% glycolic acid. The copolymer composition of PLGA9010 was
calculated similarly, and reported in the Table 4.1.
Figure 4.4 shows the 13C NMR spectra of both PLA and PLGA. The methyl
(CH3) carbon of the lactic acid unit is observed at 16.62 ppm for the PLA, and at 16.68
ppm for PLGA. The methine (CH) carbon of the lactic acid unit is observed at 68.95 ppm
for the PLA and 68.98 ppm for the PLGA. The carbonyl (C=O) carbon of the lactic acid
unit appears at 169.22 pm, for the PLA and at 169.25 ppm for the PLGA copolymer. In
the PLGA 13C NMR spectrum, the methine (CH) carbon of the glycolic acid unit is
observed at 60.75 ppm and the carbonyl (C=O) carbon resonates at 166.44 ppm.
73
1 2 16.62 68.95
2 3 3 * 1 169.22
PLA
1 4 2 5 3 2 16.68 1 3
68.98 4 5 60.75 169.25 166.44 PLGA8020
180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)
Figure 4.4: 13C NMR (75 MHz) spectra of PLA and PLGA8020, synthesized by bulk
polycondesantion; * = CHCl3.
The glass transition temperature (Tg) of polymers are also reported in Table 4.1.
The Tg of the copolymers decrease with increasing glycolic acid content. This behavior is
expected, glycolic acid (Tg of PGA = 35 – 40 °C) is less rigid than lactic acid, and its
introduction increases the copolymer chain mobility.
74
The gel permeation chromatography (GPC) traces showing the molecular weight
and polydispersity indices of the PLA and PLGA copolymers are shown in Figure 4.5.
Increasing the glycolic acid content in the PLGA copolymers from 0% to 10%, and then
to 20% decreased in the polystyrene-equivalent number average molecular weights (Mn).
However, because the repeat unit of glycolic acid (58 a.m.u.) is smaller than that of the lactic acid (72 a.m.u.), it is important to compare the degree of polymerizations (DP) of the two copolymers of PLGA (PLGA9010 and PLGB8020) to that of the PLA.
Increasing the glycolic acid content in the PLGA copolymers from 0% to 10%, and then to 20% shows a decrease in the degree of polymerization as well. This implies that the reactivity of glycolic acid is lower than that of lactic acid. This is surprising considering that glycolic acid has a primary alcohol and lactic acid has a secondary alcohol. We therefore believe that the decrease in number average polystyrene-equivalent molecular weight and degree of polymerization may not be real, and instead are due to changes in the hydrodynamic volume and errors in GPCPSt. The polydispersity index (PDI) of the
copolymers also seemed to decreases with increasing glycolic acid content. The % of
glycolic acid was limited to 20% only, as beyond 30% glycolic acid content, the PLGA
copolymers become insoluble in common solvents (like chloroform and tetrahydrofuran)
and need highly-fluorinated solvent for dissolution,48 making workup and analysis by
NMR spectroscopy and GPC difficult.
75
Figure 4.5: The gel permeation chromatographs of PLA and PLGA copolymers
synthesized by bulk polycondensation.
4.2.2 PLB copolymers
The functional poly(lactic acid) was prepared by copolymerization of lactic acid with 2- brromo-3-hydroxypropionic acid, in various ratioss, ranging from 10% functionalization to
50% functionalization (Scheme 4.4).
76
Scheme 4.4: Synthesis of functionalizable PLA (PLB) from lactic acid and 2-bromo-3-
hydroxypropionic acid using acid catalyst pTSA, and DPE as high boiling
solvent/plasticizer
The polymers were synthesized on a 1 gm scale, and their molecular weights are tabulated in Table 4.2.
Table 4.2: Molecular weight and glass transition temperature of PLB copoylmers
synthesized by bulk polycondensation.
Feed Molecular Weight Co‐polymer Glass Ratio Polystyrene Standards Composition Transition 4 4 Sample LA GA Mn x 10 Mw x 10 PDI DP LA GA Tg (°C) Yields (%) PLB9010 90 10 2.08 4.13 1.99 260 88.8 11.2 43 49.7 PLB8020 80 20 1.8 3.96 2.2 205 71.2 21.8 41 38.5 PLB7030 70 30 1.68 3.67 2.18 176 67.2 32.8 41 48.9 PLB6040 60 40 1.71 3.44 2.01 165 56.1 43.9 37 48.4 PLB5050 50 50 2.03 3.91 1.93 182 54.9 45.1 35 36.4
The 1H NMR traces of the functionalized PLA are shown in Figure 4.6. The methine
(CH) proton in the backbone of the lactic acid units resonate at around 5.2 ppm, the
methyl (CH3) protons resonate at 1.5 ppm. The overlapping methine (CHBr) and
methylene (CH2CHBr) protons from the 2-bromo-3-hydroxypropionic acid are observed
at 4.5 ppm. Comparing the contribution of the integrals due to the methine (CH) proton
of the lactic acid units and the three protons from the methylene (CH2) and methine (CH)
units of the 2-bromo-3-hydroxypropionic acid units allows us to estimate the copolymer
77
composition. For example, in the case of the PLB7030 (Figure 4.6 1H NMR spectrum in
Red):
where x is the percent of lactic acid units, and z is the percent of 2-bromo-3-
hydroxypropionic acid units. ICHL is the integrated peak area at 5.2 ppm, and ICH2B+CHB is
the integrated peak area at 4.5 ppm. This equation was used to calculate the copolymer
compositions for all six of the PLB copolymers in Table 4.2. Solving for x and z, for
PLB7030 (red spectrum in Figure 4.6), x = 67.2% and z = 32.8%. This is close to the feed
ratio of 70 mol% lactic acid and 30 mol% 2-bromo-3-hydroxypropionic acid.
Figure 4.7 shows the 13C NMR spectrum of the PLB5050 copolymer. The methyl
(CH3) carbon of the lactic acid unit appears at 16.57 ppm, and the methine (CHBr)
carbon of the 2-bromo-3-hydroxypropionic acid unit resonates at 39.29 ppm. The
methine (CH(CH3)) carbon of the lactic acid unit resonates at 69.11 ppm and 70.04 ppm.
These resonances are shown in detail in Figure 4.8. Because of the random statistical
nature of the copolymers, there are two kinds of methines of lactic acid in the polymer.
The resonance at 69.11 ppm is assigned to the methine (CH) carbon of a lactic acid
homodyad (~LL~), as this particular methine in the PLA resonates at 68.95 ppm. The
resonance at 70.04 ppm is assigned to the methine (CH) carbon of a hetrodyad (~ BL~).
78
1
*
4 2 5 2 1
5 4 PLB9010
7.97 3.00 28.20
PLB8020
3.58 3.00 13.00
PLB7030
2.05 3.00 7.44
PLB6040
1.28 3.00 4.66
PLB5050
0.82 3.00 2.88
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)
Figure 4.6: 1H NMR (300 MHz) spectra of PLB copolymers synthesized by bulk
polycondensation; * = CHCl3.
79
Similarly, there are two resonances (64.47 and 65.27 ppm) corresponding to the
methylene (CH2CHBr) carbon of the 2-bromo-3-hydroxypropionic acid repeat unit
(Figure 4.8). The methylene (CH2CHBr) carbon for a 2-bromo-3-hydroxypropionic acid
unit of the heterodyad (~LB~) resonates at 64.47 ppm while the methylene (CH2CHBr)
carbon of the homodyad (~BB~) resonates at 65.27 ppm. The carbonyl (C=O) carbon
from the lactic acid unit is observed at 169.26 ppm and that of the 2-bromo-3- hydroxypropionic repeat unit resonates at 166.41 ppm.
*
1
6 6 16.57 2 7 3 8 2 7 8 1 65.27 70.04 3 69.11 64.51 39.29 166.41 169.26
180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)
Figure 4.7: 13C NMR (75 MHz) spectrum of PLB5050 copolymer synthesized by bulk
polycondensation; * = CHCl3.
80
2 68.95
2 3 69.14 1 69.27 69.37
6’ 2’ 6 2 6 2 7 3 8 65.27 1 70.04 69.11 64.47
70 68 66 64 62 60 Chemical Shift (ppm)
Figure 4.8: Expanded 13C NMR (75 MHz) spectra of PLA and PLB5050 copolymer,
showing the methine (CH) and methylene (CH2) carbons.
Figure 4.9 shows the gel permeation chromatographs of the functionalizable
4 4 PLA’s synthesized. The Mn of the copolymers fall in the range of 1.6 x 10 to 2 x 10 Da.
With increase concentration of the functionalization groups, 2-bromo-3-
81 hydroxypropionic acid, the molecular weight and DP of the copolymers decrease.
Although this indicates that the reactivity of the 2-bromo-3-hydroxypropionic acid is lower than that of the lactic acid, it may be an aartifact of the GPCPSt. The PDI of these copolymers are approximately 2 as expected for step polymerrizations.
Figure 4.9: The gel permeation chromatographs of five PLB copolymers synthesized by
bulk polycondensation.
82
4.2.3 PLGB Copolymers
Functionalized PLGAs were prepared by copolymerization of lactic acid and
glycolic acid with 2-bromo-3-hydroxypropionic acid in various ratios, ranging from 10
mol% to 30 mol% functionalized monomer (Scheme 4.5).
Scheme 4.5: Synthesis of functionalizable PLGA (PLGB) from lactic acid and 2-bromo-
3-hydroxypropionic acid using acid catalyst pTSA, and DPE as high boiling
solvent/plasticizer.
The 1H NMR traces of the functionalized PLGA with 10% glycolic acid is shown
in Figure 4.10 and that of the functionalized PLGA with 20% glycolic acid content is
shown in Figure 4.11. The methine (CH) proton in the backbone of the polylactic acid is
seen observed at 5.2 ppm and the methyl (CH3) protons are observed at 1.5 ppm. The two
methylene (CH2) protons of the glycolic acid units resonate at 4.8 ppm, and the
overlapping methine and methylene protons from the 2-bromo-3-hydroxypropionic acid
resonate at 4.5 ppm. Comparing the contribution of the integrals under the one methine
proton from lactic acid units, two methylene protons from the glycolic acid units and a total of three methylene and methine protons from the 2-bromo-3-hydroxypropionic acid units allows us to estimate the copolymer composition. For example, in the case of the
PLB701020 (red spectrum in Figure 4.10):
83
where x is the percent of lactic acid units, y is the percent of glycolic acid units and z is
the percent of 2-bromo-3-hydroxypropionic acid units. Solving for x,y and z using the
above two equations formulated by looking at the integrals due to the methine (CH) of
lactic acid, the methylene of glycolic acid, and overlapping methine (CHBr) and
methylene (CH2CHBr) peaks: x = 68.1%, y = 7.0% and z = 24.9%. This is very close to
the feed ratio of 70 mol% lactic acid, 10 mol% glycolic acid and 20 mol% of 2-bromo-3-
hydroxypropionic acid. The glycolic acid content estimation is lower because of the
slight overlapping of glycolic acid resonances at 4.6 ppm with the 2-bromo-3-
hydroxypropionic acid resonances, leaving the 2-bromo-3-hydroxypropionic acid content
estimation on the higher end. The other copolymer compositions are calculated by using
the same three formulae, and are listed in the Table 4.3.
84
Table 4.3: Molecular weight and glass transition temperature of PLGB
copolymers synthesized by bulk polycondensation conditions.
Feed Molecular Weight Co‐polymer Glass Ratio Polystyrene Standards Composition Transition 4 4 Sample LA GA BrH Mn x 10 Mw x 10 PDI DP LA GA BrH Tg (°C) Yields (%) PLGBr801010 80 10 10 2.99 7.49 2.51 381 79.4 6.8 13.8 48 53.0 PLGBr701020 70 10 20 2.37 9.45 3.99 274 68.1 7.0 24.9 45 52.2 PLGBr601030 60 10 30 2.3 10.0 4.35 244 61.4 7.7 30.9 43 54.9 PLGBr702010 70 20 10 2.32 5.87 2.52 301 69.5 16.2 14.3 47 56.8 PLGBr602020 60 20 20 2.20 9.10 4.13 259 60.3 16.7 23.0 41 51.8 PLGBr502030 50 20 30 1.89 7.28 3.86 203 49.7 16.6 33.9 39 67.3
1
3 2 5 4 1 *
2
4,5 PLGB801010 3
3.000.995.74 21.26
PLGB701020
3.000.562.73 10.93
PLGB601030
3.000.501.99 6.24
8 7 6 5 4 3 2 1 0 2 Chemical Shift (ppm)
Figure 4.10: The 1H NMR (300 MHz) spectra of PLGB co-polymers synthesized by bulk
polycondensation reaction conditions; * = CHCl3.
85
Figure 4.12 compares the 13C NMR spectra of PLGB502030 and PLB5050
copolymers. In the PLGB502030 copolymer spectrum, the methyl (CH3) of the lactic acid
unit appears at 16.70 ppm. The methine (CH(CH3)) carbons of the lactic acid appear at
69.14 ppm and 69.99 ppm. These resonances are shown in detail in Figure 4.13. Because
of the random statistical nature of the copolymers, there are two kinds of methine of
lactic acid in the polymer. The resonance at 69.14 ppm is assigned to the methine (CH) of
a lactic acid unit homodyad (~LL~) and a ~GL~ heterodyad, based on the observance of
this methine (CH) carbon at 68.95 ppm in the PLA homopolymer and at 68.98 ppm in the
PLGA8020 copolymer. The resonance at 69.99 ppm is assigned to the methine (CH) of
the ~BL~ heterodyad. I hypothesize that the bromine group through the ester bond shifts
the methine carbon downfield, from 69.14 ppm to 69.99 ppm, as there is no other group
that could cause such a downfield shift.
86
1
3 2 5 4 1
*
2
3 4,5 PLGB702010
4.85 2.263.00
PLGB602020
2.62 1.453.00 8.46
PLGB502030
3.000.981.47 4.89
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)
Figure 4.11: The 1H NMR (300 MHz) spectra of PLGB co-polymers synthesized by bulk
polycondensation reaction conditions; * = CHCl3.
Similarly, the methylene (CH2) carbon of the glycolic acid unit is observed at
60.86 ppm and 61.60 ppm. The methylene (CH2) at 60.86 ppm is assigned to ~LG~ and
~GG~ dyads since in the PLGA8020, the same methylene (CH2) carbon is observed at
60.75 ppm. I hypothesize that the other methylene (CH2) carbon at 61.60 ppm
87
corresponds to ~BG~ heterodyad, and the bromine group of the heterodyad shifts the
methylene (CH2) carbon downfield.
*
1
6 16.57 6 2 3 7 2 8 65.27 8 7 1 70.04 69.11 64.51
3 39.29 166.41 169.26
1
4 16.68 2 5 3 7 8 6 8 1 2 3 6 4 5 7 69.14 166.43 64.55 60.86 169.22 69.99 39.33 65.30
180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)
Figure 4.12: The 13C NMR (75 MHz) spectra of PLB5050 and PLGB502030 co-
polymers synthesized by bulk polycondensation; * = CHCl3.
The same is observed for the methylene (CH2CHBr) carbon of the 2-bromo-3-
hydroxypropionic acid unit. The methylene (CH2CHBr) carbon for a ~LB~ or ~GB~
88
heterodyad is observed at 64.55 ppm while the methylene (CH2CHBr) carbon from a 2-
bromo-3-hydroxypropionic acid unit ~BB~ homodyad resonates at 65.30 ppm. I
hypothesize that in the ~BB~ homodyad, the presence of the bromine group though the
ester bond shifts the methylene (CH2CHBr) carbon downfield. The methine (CHBr)
carbon of the 2-bromo-3-hydroxypropionic acid unit resonates at 39.33 ppm. The
carbonyl (C=O) carbon of the lactic acid unit is at 169.24 ppm, and those of the glycolic acid and 2-bromo-3-hydroxypropionic acid are at 166.68 ppm and 166.46 ppm, respectively.
89
2 68.95
69.14 2 3 69.27 69.37 1
PLA 68.98 2
69.15 4
4 60.75 2 5 3 69.39 1
PLGA8020
2’ 2 65.27 6 70.04 2 7
69.11 3 8 64.47 1 6’ 6 PLB5050
2 4 2 5 8 6 3 6 7 2’ 69.14 1 4
6’ 64.55
4’ 60.86 69.99 65.30 61.60
PLGB502030
70 68 66 64 62 60 Chemical Shift (ppm)
Figure 4.13: Expanded 13C NMR (75 MHz) spectra of PLA, PLGA8020,
PLB5050 and PLGB502030, showing the methine (CH) and methylene (CH2) carbons.
90
In the functionalized PLGA copolymers, with 10% glycolic acid content (Figure
4.14), increasing the bromohydrin content from 10% to 20% to 30%, decreases the polystyrene-equivalent number average molecular weight (Mn) and DP. This behavior is also surprising considering that glycolic acid and 2-bromo-3-hydroxypropionic acid both have primary alcohols and that lactic acid has a secondary alcohol. We therefore believe that the decrease in number average polystyrene-equivalent molecular weight and degree of polymerization may not be real, and instead aare due to changes in the hydrodynamic volume and errors in GPCPSt. The PDI of the copolymers also increases, and polymers with the higher bromination content demonstrate PDI’s as high as 4. This could be because increasing the comonomer concentrations, makes condensation polymerization have less control due to the different reactivities of each of the three monomers.
Figure 4.14: The gel permeation chromatographs of PLGB copolymers, with 10%
glycolic acid content, synthesized bby bulk polycondensation.
91
A similar trend is noticed in the PLGA copolymeers with 20% glycolic acid content (Figure 4.15). Upon increasing the 2-bromo-3-hydroxypropionic acid content from 10% to 20% to 30%, the polystyrene-equivalent number average molecular weight
(Mn) and DP decrease. We believe that the decrease in number average polystyrene- equivalent molecular weight and degree of polymerization also may not be reall, and instead are due to changes in the hydrodynamic volume and errors in GPCPSt. The co- polymers with higher bromination (20% & 30%) also show broader PDIs of around 4.
This could also be because increasing the comonomer concentrations, makes condensation polymerization have less control duue to the different reactivities of each of the three monomers.
Figure 4.15: The gel permeation chromatographs of PLGB copolymers, with 20%
glycolic acid content, synthesized bby bulk polycondensation.
92
The Tg of the PLGB copolymers is also listed Table 4.3. Keeping the glycolic acid
content constant at 10% and increasing the bromination content by 10%, from 10% to
30%, the Tg of the copolymers decreases from 48 °C (PLGB801010), to 45 °C
(PLGB701020) to finally 43 °C (PLGB601030). The same trend is also observed in the
’s three PLGB copolymers with 20% glycolic acid content. Their Tg decrease from 47 °C
(PLGB702010) to 41 °C (PLGB602020) to finally 39 °C (PLGB502030). The same trend
of decreasing Tg’s is observed at constant % bromination and increasing [glycolic acid].
Tg decreases from PLGB80101 to PLGB702010, 48 °C compared to 47 °C. Similarly, the
Tg decreases from 45 °C to 41 °C, for sample PLGB701020 vs. PLGB602020, and
consistently, Tg also decreases from 43 °C to 39 °C, for the samples PLGB601030 vs.
PLGB502030.This demonstrates that the glycolic acid units disrupts the chain packing,
increasing chain mobility and decreasing Tg. The decrease in Tg is much more
pronounced upon introduction of the 2-bromo-3-hydroxypropionic acid compared to the
introduction of glycolic acid comonomer. This trend is observed upon comparing the Tg’s
of PLGA9010 and PLB9010, with that of PLA, where the Tg changed only slightly from
51 °C to 50 °C vs. Tg changing from 51 °C to 43 °C. Similarly, comparing the change in
Tg of PLGA8020 to PLA (50 °C vs. 51 °C) vs. change in Tg of PLB8020 to PLA (41 °C
vs. 51 °C), it is also observed the change in Tg upon adding 20% 2-bromo-3- hydroxypropionic acid is much more than the change in Tg upon adding glycolic acid co-
monomer. This is not unexpected, as introducing the brominated comonomer, 2-bromo-3-
hydroxypropionic acid, introduces a bulky bromine group into the main chain of the copolymer, which creates void spaces inbetween the adjacent chains of the copolymers, decreasing its Tg by as much as 16-20%. Introducing glycolic acid also decreases the Tg,
93
but to a much lesser extent, 2-7%, as introduction of glycolic acid merely disrupts the packing, and does not have much of an effect on the void space creation inbetween
adjacent polymer chains. Hence, introduction of glycolic acid comonomer has a milder
effect on Tg.
4.3 Microparticle formulation with PLGB polymers
The PLGB polymers discussed in the previous sections were formulated into
microparticles, since many drug deliveries takes place in the form of microparticles.
4.3.1 Microparticle formulation procedure
The brominated polymers were formulated into microparticles following the
procedure by Yun, et al. The procedure is described in the experimental chapter of this
dissertation. In the above procedure, the PEG acts as an emulsifying agent. The
hydrophobic nature of the chitosan makes it stick to the polymer surface, and the
hydrophilic nature of the PEG chains make the chain extend out as arms in the aqueous
solution, keeping the particles suspended. The PVP solution further emulsifies the
solution stopping the particles from aggregating together.
Two different copolymers were made into microparticles. Their details are presented
in Table 4.4.
Table 4.4: PLGB copolymers used for microparticle formulation
Sample Co‐polymer Mn PDI 1 PLGB601030 2.1 x 104 4.8 4 2 PLGB502030 1.9 x 10 3.9
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The size distribution of the micro-particles obtained were measured by dynamic light scattering (DLS), and visualized by scanning electron microscopy (SEM).
4.3.2 Microparticles of PLGB601030
The DLS results (Figure 4.16) show that the microparticles have a broad range of sizes, from around 250 nm to 4 microns. The broad polydispersity in sizes is also seen in the SEM images (Figure 4.17), which clearly show particles ranging from the sub-micron range, to a few micrometers. Some aggregation is also seen in the SEM images, which could be reason for a higher percent of larger particles sizes seen on the DLS.
Figure 4.16: Particle sizes of PLGB601030 observed by dynamic light scattering.
95
Figure 4.17: Microparticles of PLGB601030, as seen under scanning electron microscopy
(SEM).
96
4.3.3 Microparticles of PLGB502030
The DLS (Figure 14.18) results show that almost all of the particles formulated are in the 5 to 10 micron ranges, with very few in the sub-miccron sizes. However, looking at these particles through SEM (Figure 4.19) shows that there is a lot of aggregation in these particles, as compared to PLGB601030. It should be noted that PLGB502030 has a slightly higher glycolic acid content, which might have made the PLGB polymer slightly less hydrophobic, causing the aggregation. Using higher amount of PEG-g-CHN might give us better separation between the particles. Aggregation aside, by examining the SEM images, we conclude that particles were formed in a very broad range of sizes, quite similar to that of PLGB 601030.
Figure 4.18: Particle sizes of PLGB502030 observed by dynamic light scattering.
97
Figure 4.19: Microparticles of PLGB502030, as seen under scanning electron microscopy
(SEM).
98
4.4 Solution Polymerization
I also investigated the solution polymerization of the lactic acid and 2-bromo-3-
hydroxypropionic acid comonomers. The monomers were not directly polymerized, but
converted to oligomers, and then chain extended with N, N’-diisopropylcarbodiimide
(DiPC). The oligomerization conditions were similar to the polymerization conditions
described in Section 4.1, except for the absence of the high boiling solvent, DPE. The
temperature and reaction times were also been varied slightly.
Scheme 4.6: The oligomerization of lactic acid and 2-bromo-3-hydroxypropionic acid,
and subsequent chain extension using N, N’-diisopropylcarbodiimide
The oligomers produced by bulk polycondesation were dissolved in dichloromethane and refluxed. The condensing dichloromethane was passed through
molecular sieves before returning to the flask containing the oligomers (Figure 4.20).
Dichloromethane forms an azeotrope with water and thereby removes moisture trapped in
the bulk oligomer. The drying agent (molecular sieves), then dries the azeotrope, and the
anhydrous solvent is recycled back into the reaction flask. After 18 hours of dehydration
of the oligomer, the contents of the flask were cooled to 0 °C, and DiPC was introduced
dropwise, and the reaction was stirred at room temperature. The DiPC activates the
99 carboxylic acid end of the oligomeric polyester, according to the mechanism described in section 2.9, leading to chain extension.
N2
condenser
Molecular Sieves
Hydroxy acid monomers or oligomers
in CH2Cl2 60 °C
Dehydration by Solvent Recycling
Figure 4.20: Procedure for dehydration of oligomers by solvent refluxing over molecular
sieves.
To ascertain the usefulness of the dehydration by solvent recycling step, one half of PLA and PLB8020 oligomers were chain extended using DiPC with the dehydration by solvent recycling, and the other half without. The molecular weight results are listed in
Table 4.5 and the GPCPSt traces are shown in Figure 4.21 and Figure 4.22. When the chain extension is performed without the dehydration by solvent recycling, there is 89%
100 increase in Mn for PLA, and 25% increase in Mn for PLB8020. When the same oligomers are first dehydrated by solvent recycling, and then chain extended by DiPC, there is a
224% increase and 70% increase their number average molecular weights.
Table 4.5: Increase in number average molecular weight by chain extension using
DiPC, with and without the dehydration by sollvent recycling.
Without Dehydration Percent Dehydration Percent Oligomer DIPC Coupling Mn DIPC Coupling Mn 4 4 Mn x 10 PDI Mn x 10 PDI Yield %Increase Mn PDI Increase Yield % PLA 7.4 x 103 1.6 1.4 x 104 2.3 60.2 89% 2.4 x 104 1.9 224% 56.3 3 4 4 PLB8020 8.8 x 10 2.1 1.1 x 10 1.8 46.4 25% 1.5 x 10 1.7 70% 38.4
Figure 4.21: Increase in Mn of PLA by chain extension using DiPC, with and without the
dehydration by solvent recycling.
101
Figure 4.22: Increase in Mn of PLB8020 by chain extension using DiPC, with and
without the dehydration by solvent recyycling.
Oligomerization and chain extension (Scheme 4.6) of various copolymers of PLB were performed, and their chain extension efficiencies and molecular weights are tabulated in Table 4.6. The GPC traces illustrating chain extension are shown in Figures
4.23-4.28.
102
Table 4.6: Increase in number average molecular weight by chain extension using
DiPC, after dehydration by solvent recycling
Dehydration Percent Oligomer DIPC Coupling Mn 3 4 Mn x 10 PDI Mn x 10 PDI Increase Yield % PLA 15.00 1.70 3.10 1.70 107 45.20 PLB9010 5.70 2.40 2.20 2.30 286 43.00 PLB8020 13.00 2.60 1.80 3.00 39 61.30 PLB7030 8.50 3.60 1.10 2.70 29 55.10 PLB6040 7.80 4.40 1.10 2.60 41 49.80
The highest molecular weight is observed for the PLA homopolymer. The highest
% increase of around 3 fold is seen for the PLB9010. The chain extensions of the other
PLB copolymers were less effective. The chain extension takes place by the activation of
the carboxylic acid group by the carbodiimide. The presence of the bromine group α to the carboxylic acid might be making the activation of the carboxylic acid group more difficult. The increase in molecular weight for the PLB copolymers for higher bromohydrin content were less, and the number average molecular weights achieved were either equal to or less than that obtained by using bulk conditions presented in
Section 4.1. The bulk conditions also have the advantage of being a one-pot process, which makes it possible to scale up for industrial applications. The solution polymerization route described in this section, however, could be useful for making high molecular weight polyesters of temperature-sensitive hydroxy acids, as the chain extension reaction is performed at room temperature.
103
Figure 4.23: Increase in Mn of PLA by chain extension using DiPC, after dehydration by
solvent recycling.
Figure 4.24: Increase in Mn of PLB9010 by chain extension using DiPC, after
dehydration by solvent recycling.
104
Figure 4.25: Increase in Mn of PLB8020 by chain extension using DiPC, after
dehydration by solvent recycling.
Figure 4.26: Increase in Mn of PLB7030 by chain extension using DiPC, after
dehydration by solvent recycling.
105
Figure 4.27: Increase in Mn of PLB6040 by chain extension using DiPC, after
dehydration by solvent recycling.
106
CHAPTER V
SEQUENCE CONTROLLED FUNCTIONALIZED POLYESTERS
5.1 Reactivity Difference
The previous chapter described the synthesis of functionalized PLGAs. Under identical conditions, the degree of polymerization of PLB and PLGA copolymers, measured by GPCPst was found to be lower than that of a PLA (Table 5.1). This suggests that both glycolic acid and 2-bromo-3-hydroxypropinic acid have lower reactivity than lactic acid. This is counter intuitive, as lactic acid possesses a secondary alcohol, which should be less reactive than the primary alcohol of both glycolic acid and 2-bromo-3- hydroxypropinic acid.
107
Table 5.1: Degree of polymerization of PLGA compared with PLB copolymers,
for 10% and 20% co-polymer ratios.
Degree of Polymer Polymerization PLA 439 PLGA9010 324 PLGA8020 291 PLB9010 260 PLB8020 205
The degree of polymerization of PLB with 10% bromohydrin units has a degree
of polymerization of 260, 20% lower than the DP of the analogous PLGA with 10%
glycolic acid units. A similar fact is observed in PLB with 20% 2-bromo-3-
hydroxypropinic acid units. The PLB with 20% 2-bromo-3-hydroxypropinic acid units has a DP of 205, 30% lower than PLGA with 20% glycolic acid units. The general
condensation reaction taking place in both of these cases is shown in Scheme 5.1. This
suggests that the reactivity of a lactic acid unit attached to a 2-bromo-3-hydroxypropinic
acid unit (LB), towards another lactic acid is lower than the reactivity of a lactic acid unit
attached to glycolic acid (LG) unit towards another lactic acid unit.
108
Reaction 1: LLBL + L LLBLL
Reaction 2: LLGL + L LLGLL
Scheme 5.1: General reaction Scheme illustrating the difference between the reaction of
PLGA and PLB co-polymers.Reaction 1: Lactic acid unit is connected to glycolic acid,
Reaction 2: Lactic acid unit connected to a 2-bromo-3-hydroxypropinic acid unit.
In order to compare the relative reactivities, it is proposed to synthesize two trimeric hydroxy acids, one in which a reactive lactic acid is flanked by two glycolic acid units, and another in which the reactive lactic acid is flanked by two 2-bromo-3- hydroxypropinic acid units. The two molecules are abbreviated as LGL and LBL
(Scheme 5.2).
The rest of this section describes the strategies and steps for the synthesis of a bisprotected LGL and LBL trimer.
109
LGL trimer: Hydroxy acid composed of a lactic acid unit connected to a glycolic acid unit.
LBL trimer: Hydroxy acid composed of a lactic acid unit connected to a bromohydrin unit.
Scheme 5.2: The LGL and LBL trimers.
5.2 General Strategy for Synthesizing the Trimers
The general strategy for synthesizing the dimers and trimers was adapted from the work of Dr. Tara Meyer and Dr. Ryan Stayshich.30-32 All three of the building blocks, lactic acid, glycolic acid and the 2-bromo-3-hydroxypropionic acid are hydroxy acids.
This allows us to use the same reaction conditions to protect and de-protect the alcohol group in all three, and the same reaction conditions to protect and de-protect the carboxylic acid group. The strategy
In the first step, a dimer was synthesized by coupling a protected alcohol with a protected carboxylic acid, yielding a hydroxy ester, in which both terminal alcohol and terminal carboxylic acid groups were protected. To make the trimer, one of the terminal groups needs to be de-protected and then coupled with the third building block. The
110 alcohol group in these hydroxy acids was protected with tert-butyldiphenylsilyl
(TBDPSi) protecting group forming a silyl ether protecting group. This protecting group was removed using tetrabutylammoniumfluoride (TBAF). The carboxylic acid group was protected with a benzyl protecting group, which was deprotected by palladium-catalyzed hydrogenation. Due to the higher yields and ease of benzyl deprotection, it is a much preferred route to create the trimer from the dimer as compared to deprotecting the dimer by the cleavage of the TBDPSi bond, and then esterifying with another TBDPSi- protected carboxylic acid. For creating the bisprotected LGL trimer (p-LGL-p) (Scheme
5.3), a bisprotected GL (p-GL-p) dimer was first synthezised, and the carboxylic acid was deprotected by hydrogenation. This carboxylic acid was then further reacted with benzyl lactate, creating the bisprotected LGL trimer.
p‐GL‐p
p‐LGL‐p Total yield = 21%
Scheme 5.3: Synthesis of bisprotected LGL trimer.
The same strategy was attempted to synthesize the p-LBL-p trimer. The dimer was first synthesized by coupling a benzyl-protected 2-bromo-3-hydroxypropionic acid
111
with TBDPSi-protected lactic acid. However, to my surprise, the hydrogenation reaction
to deprotection the benzyl protecting group did not work. Three different strategies were
tried to de-protect the benzyl protecting group, none of which were successful (Scheme
5.4). In the first case, it was attempted to deprotect the benzyl group by hydrogenation in
the presence of Pd/C catalyst. The reaction was carried out in ethyl acetate, in a Parr
Instruments hydrogenator for 24 h, under 50 psi hydrogen gas and 50 °C.30 These
conditions were unsuccessful for the deprotection. Deprotection of the benzyl group was
then attempted by hydrogenation in the presence of W-2 Raney Ni catalyst.49 The
reaction was carried out in ethyl alcohol, in a Parr Instruments hydrogenator for 24 h,
under 50 psi hydrogen gas and 50 °C. These conditions were unsuccessful for the
deprotection as well. The benzyl protecting group in p-BL-p was also attempted to
deprotect by catalytic hydrogenation employing hydrazinium monoformate and
magnesium (Mg/NH2-NH2●HCOOH). In this reaction, the Mg acts as a catalyst for the
removal of the hydrogenolysable protecting group, and the hydrazinium monoformate
acts as a hydrogen donor.50 The p-BL-p was reacted with Mg and hydrazinium
monoformate, in methanol at 60 °C overnight. This deprotection reaction was unsuccessful aswell.
112
Scheme 5.4: Strategy for the synthesis of bisprotected LBL trimer.
To overcome this problem, I decided to create the trimer by deprotecting the alcohol end of the dimer, and then further couple it with the TBDPSi-protected lactic acid. This meant synthesizing a new p-LB-p dimer (Scheme5.5). This strategy does allow me to make the bisprotected trimer, but the low yield (~10%) of the intermediates provides very little products to experiment with. The complete synthetic strategy for making the LBL copolymer is shown in Scheme 5.5. This chapter only discusses the synthesis of the bisprotected trimers, p-LGL-p and p-LBL-p. The deprotection of both ends of the trimers and their subsequent polymerizations (Scheme 5.6 and Scheme 5.7) will be subsequently added.
113
p‐LB‐p
Total yield = 10% p‐LBL‐p
Scheme 5.5: Synthesis of p-LBL-p trimer.
p‐LGL‐p
Poly(LGL) or PLGA66‐33 LGL
Scheme 5.6: Synthesis of sequence controlled LGL copolymer.
114
p‐LBL‐p
LBL Poly(LBL) or PLB66‐33
Scheme 5.7: Synthesis of sequence controlled LBL copolymer.
5.3 Synthesis of the Building Blocks
Two kinds of building blocks were synthesized. The carboxylic acid ends of the hydroxy acids were protected using the benzyl ester protecting group and the alcohol end was protected using silyl ether bonds.
5.3.1 Benzyl Protection of Hydroxy acids
The carboxylic acid groups of the 3- hydroxy acids were protected using benzyl bromide, in the presence of the non-nucleophilic base, 1,8-diazabicycloundec-7-ene
(DBU). Methanol was used as a solvent to dissolve the hydroxy acid before adding the base. After the base was added, methanol was removed, and the reaction was carried out in dimethylformamide (DMF).The product was extracted using ethyl acetate, washed
115
with dilute acid, and aqueous sodium bicarbonate, and then purified by vacuum
distillation (100 °C, 1 - 2 mm Hg). These reactions typically yield about 50%. The
reaction Scheme for benzyl protecting lactic acid is shown in Scheme 5.8.
Scheme 5.8: Synthesis of benzyl lactate from lactic acid, using benzyl bromide as
reagent.
Figure 5.1 shows the 1H NMR spectrum of the purified product. The methylene
(CH2) of the benzyl protecting group is seen at 5.22 ppm. The methine (CH) of the lactic
acid is a quartet at 4.33 ppm with a coupling constant of 3J = 7.0 Hz, and the methyl
3 (CH3) group appears as a doublet at 1.44 ppm with a coupling constant of J = 7.0 Hz.
The alcohol group of the lactic acid is seen at 2.85 ppm and the five benzylic protons
resonate as a singlet at 7.36 ppm. The resonances match with the reported values in the
literature.30
116
4 1 3 4 5 3 2 4
4 4 1 4
* 2 5
3.87 2.00 0.75 0.60 3.49
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)
Figure 5.1:1H NMR (300 MHz) spectrum of benzyl lactate, synthesized by reacting lactic
acid with benzyl bromide; * = CHCl3.
In the 13C NMR spectrum (Figure 5.2), the methyl carbon is seen at 20.35 ppm. The
methine (CH) of the lactic acid and the methylene (CH2) of the benzyl protecting group
are seen very close together at 66.86 ppm, and 67.21 ppm respectively. The quaternary
carbon of the benzyl group is seen at 135.29 ppm and the five other carbons of the benzyl
group are seen at 128.23, 128.51 ppm and 128.65 ppm. The carbonyl group appears at
175.53ppm.
117
6
4 6 1 6 3 5 6 6 2 6
4
1 2 3 5
*
180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)
Figure 5.2: 13C NMR (75 MHz) spectrum of benzyl lactate, synthesized from reacting
lactic acid with benzyl bromide; * = CHCl3.
Similarly, the benzyl protected glycolic acid was synthesized in 50-60% yield
(Scheme 5.8), by reacting glycolic acid with benzyl bromide in the presence of a non- nucleophilic base, DBU. Methanol was used to dissolve the glycolic acid, and after adding the base, DBU, methanol was removed and the reaction was carried on in DMF at room temperature for 24 h. The product was extracted using ethyl acetate, washed with dilute acid, and aqueous sodium bicarbonate, and then purified by vacuum distillation
(60-70 °C, 1 - 2 mm Hg).
O O DBU, Methanol OH OH O HO BnBr, DMF RT, 24 h
Scheme 5.9: Synthesis of benzyl glycolate from glycolic acid using benzyl bromide.
118
Figure 5.3 shows the 1H NMR spectrum of the purified compound. The two methylenes (CH2) of the glycolic acid and benzyl protecting group are seen as two singlets at 4.2 ppm and 5.22 ppm, respectively. The OH of the alcohol shows up at
3.08ppm and the five protons of the benzyl group are seen as a singlet at 7.36 ppm. In the
13 C NMR spectrum (Figure 5.4), the methylene (CH2) carbon of the glycolic acid unit is seen at 60.68 ppm, and the methylene (CH2) carbon of the benzyl protecting group appears at 67.09 ppm. The ipso carbon of the phenyl ring appears at 135.19 ppm, and the ester (COO) carbon is downfield at 173.24 ppm.
3
3 2 4 3 2 1 3 3 3 1
4
4.93 2.00 2.02 0.95
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)
Figure 5.3: 1H NMR (300 MHz) spectrum of benzyl glycolate synthesized by reacting
glycolic acid with benzyl bromide.
119
5 5 3 5 4 2 1 5 5 5
1
3 2 4 *
180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)
Figure 5.4: 13C NMR (75 MHz) spectrum of benzyl glycolate, synthesized from reacting
glycolic acid with benzyl bromide; * = CHCl3.
5.3.2: Silyl Protection of the Hydroxy Acids
Tert-butyldiphenylsilylchloride (TBDPSCl) is used to protect the alcohol group.
The starting points of the reactions were not the hydroxyl acids themselves, but their methy esters. In the case of glycolic acid and lactic acid, the methyl esters were
purchased, but in the case of the bromohydrin, it was synthesized.
Scheme 5.10 shows the two step reaction Scheme for protecting the alcohol group
of the glycolic acid. Methyl glycolate was reacted with TBDPSCl, in the presence of triethylamine (TEA), forming triethylammonium hydrochloride, a white salt, that
precipitates out in the dichloromethane reaction solvent. The silyl protected methyl
glycolate was hydrolyzed into silyl protected glycolic acid, by deprotection with 0.2 M
120
lithium hydroxide (LiOH) in tetrahydrofuran (THF) at room temperature. The LiOH32 selectively cleaves the methyl group, giving a silyl-protected glycolic carboxylic acid.
Scheme 5.10: Synthesis of TBDPSi-protected glycolic acid.
The methyl (CH3) of the protected methyl glycolate appears at 3.68 ppm, and the
methylene (CH2) from the glycolate at 4.23 ppm (Figure 5.5). Both peaks are singlets.
The tert-butyl ((CH3)3) group has nine protons which show up at 1.09 as a singlet, and the
ten protons of the diphenyl group appear at 7.41-7.48 ppm and 7.63-7.66 ppm. After the
cleavage of the methyl ester to yield TBDPSi protected glycolic acid, the 1H NMR spectrum shows the disappearance of the methyl (CH3) at 3.68 ppm. The methylene
(CH2) group from the glycolic acid remains at 4.23 ppm as a singlet, and the protons
from the TBDPSi protecting group remain virtually unchanged.
121
2 1.12
4 4 4 3 3 5 2 1 5 3.69 1 1.11
4 4.27
3
*
7.124.55 2.00 3.05 10.26
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm) 4 1 4 4
4.22 3 O 3 O 2 4 HO Si 3 * 1
4.12 6.37 2.00 9.61
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)
Figure 5.5: 1H NMR (300 MHz)spectra of TBDPSi-protected methyl glycolate and
glycolic acid; * = CHCl3.
Figure 5.6 shows the stacked 13C NMR spectra of TBDPSi-protected methyl glycolate and glycolic acid. The methylene (CH2) carbon of the methyl glycolate appears at 62.17 ppm and the methyl (CH3) carbon of the methyl ester is at 51.61 ppm. Upon cleavage of the methyl group with LiOH, the resonance is no longer present in the bottom
122
spectrum, and the methylene shifts slightly upfield from 62.17 ppm to 61.90 ppm. The
cleavage of the methyl ester leads to the downfield shift of the carbonyl (COO) carbon,
from 171.62 ppm to 174.90 ppm. The quaternary (C(CH3)3)carbon and the 3 methyl
(C(CH3)3) groups of the tert-butyldiphenylsilyl protecting group appear at 19.30 ppm
and 26.73 ppm, respectively, and shift slightly upfield to 19.20 and 26.69 ppm,
respectively after cleavage of the methyl ester.
4 3 5 3 1 2 26.73
1 2 5 4 62.17 19.30 51.61 171.62 *
3
3 26.69 5 2 1
1 2 5 19.20 * 61.90 174.90
180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)
Figure 5.6: 13C NMR (75 MHz) spectra of the TBDPSi-protected methyl glycolate and
glycolic acid; * = CHCl3.
123
The alcohol group of the lactic acid was also similarly protected (Scheme 5.11).
Methyl lactate was reacted with TBDPSCl in the presence of TEA, forming the TBDPSi-
protected methyl lactate. The TEA reacts with the by-product HCl, forming a white salt
that precipitates out in the dichloromethane reaction solvent, which is later filtered away.
The silyl protected methyl lactate is converted into silyl protected lactic acid, by reacting
with 0.2 M LiOH in THF at room temperature. The LiOH cleaves the methyl group,
giving a silyl protected lactic acid.
Scheme 5.11: Synthesis of TBDPSi-protected lactic acid.
Figure 5.7 shows the stacked 1H NMR spectra of TBDPSi-protected methyl
lactate and lactic acid stacked. The methyl (CH3) of the protected methyl lactate appears
as a singlet at 3.56 ppm. The methyl (CH3) of the lactic acid backbone appears as a
doublet at 1.37 ppm with a coupling constant of 6 Hz, and the methine (CH) from the
lactic acid is a quartet at 4.28 ppm is a quartet with a coupling constant of 6 Hz. The tert-
butyl ((CH3)3) group has nine protons that show up at 1.09 as a singlet, and the ten
protons of the diphenyl group appear at 7.33-7.48 ppm and 7.63 - 7.70 ppm. After the cleavage of the methyl ester with LiOH, in 1H NMR spectrum of the TBDPSi-protected
lactic acid, the resonance of the methyl (CH3) at 3.56 ppm disappears. The methyl (CH3) of the lactic acid shifts to 1.39 ppm and methine (CH) shifts to 4.35 ppm. The splitting
124 patterns remain the same, with the same coupling constant of 6 Hz. The nine protons from the tert-butyl ((CH3)3) also shift to 1.14 ppm.
5 1.09
6 3 2 5 3 6 1 3 4
3.56 4 4 1 1.38
2 1.36 4 3 4.30 * 4.27 4.32 4.25
7.11 1.00 3.14 10.53 1.14
2 5 3 1 3 4 1 4 4 2 4 1.39 3 4.37 4.34 4.32 4.39
6.93 1.00 9.09
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)
Figure 5.7: 1H NMR (300 MHz) spectra of TBDPSi-protected methyl lactate and lactic acid; * = CHCl3.
Figure 5.8 shows the 13C NMR spectrum of TBDPSi-protected lactic acid. The methine (CH) and methyl (CH3) carbons of the lactic acid appear at 68.96 ppm and 21.09 ppm, respectively. The quaternary (C(CH3)3) carbon and the 3 methyl (C(CH3)3) groups 125 of the tert-butyldiphenylsilyl protecting group appear at 19.22 ppm and 26.87 ppm, respectively. The carbonyl (COO) carbon of the lactic acid is observed at 177.92 ppm.
5 26.87
2 5 3 4 1 4 19.22
2 1 21.09 3 68.96 * 177.92
180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)
13 Figure 5.8 C NMR (75 MHz) spectrum of the TBDPSi protected lactic acid; * = CHCl3.
126
The protection of the alcohol group of 2-bromo-3-hydroxypropionic acid is shown
in Scheme 5.12.
Scheme 5.12: Synthesis of TBDPSi-protected 2-bromo-3-hydroxypropionic acid
2-bromo-3-hydroxypropionic acid was esterified by reacting it with methanol using
methanol as solvent in the presence of hydrobromic acid as a catalyst. The methyl ester of
2-bromo-3-hydroxypropionic acid is then converted into a TBDPSi-protected methyl
ester of 2-bromo-3-hydroxypropionic acid, using TBDPSCl as with the case of lactic and
glycolic acid protections, and then converted into TBDPSi protected 2-bromo-3-
hydroxypropionic acid, by LiOH cleavage of the methyl ester. Figure 5.9 shows three 1H
NMR spectra leading up to the synthesis of TBDPSi protected 2-bromo-3-
hydroxypropionic acid. The first spectrum on the stack is that of methyl 2-bromo-3- hydroxypropionate. The methyl (CH3) of the methyl ester is a singlet at 3.81 ppm. The
methine (CHBr) appears at 4.29 ppm as a doublet of doublets (3J = 5.5 Hz, 3J = 7.5 Hz),
and the two methylene (CH2) protons alpha to the bromine split into two sets of doublets
of doublets at 3.86 ppm and 3.98 ppm with coupling constants of 2J = 5.5 Hz and 3J =
12.2 Hz and 2J = 7.7 Hz and 3J = 11.9 Hz, respectively. The alcohol of the methyl 2-
127
bromo-3-hydroxypropionate appears as a triplet at 2.58 ppm, with a coupling constant of
7.5 Hz. Upon protection with TBDPSi, the methylene (CH2Br) of the bromohydrin shifts downfield, and the doublet of doublets of the protons of the methylene spread apart. The splitting pattern and coupling constants, however, remain the same (Figure 5.10). The methyl (CH3) of the methyl ester of the bromohydrin also shifts downfield from 3.76
ppm to 3.81 ppm. The methyl (CH3) of the methyl 2-bromo-3-hydroxypropionate
resonates at the same frequency on protecting with TBDPSi. Each of the two protons of
the methylene (CH2CHBr) appear as a doublet of doublets at 4.16 ppm and 4.31 ppm,
with coupling constants, 3J = 8.5 Hz,2J = 10.3 Hz and 3J = 5.6 Hz, 2J = 10.3 Hz,
respectively. The methine (CHBr) connected to the bromine also appears as a doublet of
doublets at 3.96 ppm with coupling constants of 3J = 5.6 Hz and 3J = 8.5 Hz. The three
methyls of the tert-butyl appear at 1.08 ppm as a singlet, and the ten protons from the
diphenyl groups belonging to the TBDPSi-protecting group are seen at 7.39 – 7.51 ppm
and 7.67 – 7.77 ppm.
128
3 1.08
4 1 3 2,2’ 1 2,2’ 4 2.58 4.33 4.35 4.35 4.37 2.60 2.55 *
1.00 3.26 1.09 7 3 6
3.81 6 6 5 5 1 7 3 2,2’ 1.06
6
5 1 2,2’ 4.16 3.99 4.31 4.29 4.32 4.13 4.34 *
8.155.37 3.231.09 12.46 6 6 7 6 5 5 1 7 2,2’ 6 5 2,2’ 1 4.14 4.32 4.00 4.31 4.15 3.98 4.29 4.17 4.11 4.34 *
4.226.41 0.941.00 9.26
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)
Figure 5.9: 1H NMR (300 MHz) spectra of methyl 2-bromo-3-hydroxypropionate,
TBDPSi protected methyl 2-bromo-3-hydroxypropionate and 2-bromo-3-
hydroxypropionic acid; * = CHCl3.
129
On reacting the protected methyl 2-bromo-3-hydroxypropionate with LiOH to hydrolyze the ester group, the methyl (CH3) protons at 3.81 ppm disappear, proving the cleavage of the methyl ester generating a carboxylic acid. The nine protons of the tert- butyl from the TBDPSi-protecting group are a singlet at 1.08 ppm and the ten protons from the two phenyl groups appear as complex multiplets in the regions 7.41-7.51 ppm and 7.67-7.77 ppm. The rest of the protons resonate at the same positions with the same splitting patterns. 3.76
1 3 2,2’ 2’ 1 2 3.97 3.90 3.88 3.95 3 4.29 4.27 4.29 4.31 4.01 3.86 3.84 3.99
1.00 1.08 1.07 3.08
1 3 2,2’ 3.81 2 2’ 1 4.16 3.99 3 3.97 4.31 4.29 4.19 4.32 3.96 4.13 4.17 4.34 3.94
1.00 1.05 1.10 3.21
4.3 4.2 4.1 4.0 3.9 3.8 Chemical Shift (ppm)
Figure 5.10: Expanded 1H NMR (300 MHz) spectrum of the methyl 2-bromo-3-
hydroxypropionate and TBDPSi protected methyl 2-bromo-3-hydroxypropionate
130
Figure 5.11 shows three stacked 13C NMR spectra, illustrating the protection of
the alcohol group of bromohydrin with TBDPSi. After protection of methyl 2-bromo-3- hydroxypropionic acid with TBDPSi, the methylene (CH2CHBr) carbon shifts downfield
from 63.64 ppm to 65.10 ppm due to the formation of the new silyl ether bond. The
methine (CHBr) and methylester(CH3O) carbons appear at 43.95 ppm and 52.91 ppm, respectively, and are not significantly changed compared to methyl 2-bromo-3- hydroxypropionic acid. The ester carbon (COO) also doesnot change, and appears at
169.10 ppm. Two new carbon resonances belonging to the TBDPSi-protecting group appear at 19.24 and 26.67 ppm, belonging to the quaternary carbon (C(CH3)3) and three
overlapping methyls (C(CH3)3) of the tert-butyl group, respectively. After cleavage of the
methyl ester with LiOH, the carbon resonance corresponding to the methyl ester at 52.91
ppm is no longer present, and the carbonyl (COO) carbon shifts downfield to 174.01
ppm, for the carboxylic acid.
131
1 2
3 44.25
1 63.64 4 4 2 53.20 3 169.48
*
26.67 6 3 6 135.56 1 4 2 5 127.85 4 2 1 5 3 52.91 129.99 43.95 19.24 65.10 169.10 * 26.66 3 6 6 127.84 1 135.56 2 5 5 3 2 1
* 19.23 130.02 174.01 43.67 64.89
180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)
Figure 5.11: 13C NMR (75 MHz) spectra of the methyl 2-bromo-3-hydroxypropionate,
TBDPSi-protected methyl 2-bromo-3-hydroxypropionate and TBDPSi-protected 2-
bromo-3-hydroxypropionic acid; * = CHCl3.
132
5.4 Synthesis of the LGL trimer.
The LGL trimeric segmer was synthesized by first creating a bisprotected GL dimeric segmer. The benzyl protecting group (of the carboxylic acid) of the glycolic acid side was removed, and the dimer was coupled with a benzyl lactate, creating a bisprotected LGL trimeric segmer (Scheme 5.3).
5.4.1 Synthesis of p-GL-p Dimer
The GL dimer was synthesized in around 50% yields, by coupling benzyl glycolate and silyl-protected lactic acid building blocks using carbodiimides (Scheme
1 5.13). The H NMR spectrum in Figure 5.12 shows that the two methylene (CH2) protons
of the glycolic acid unit split into two sets of doublets at 4.45 ppm and 4.61 ppm, after
coupling, from 4.2 ppm in the benzyl glycolate. The two protons couple with each other
with 2J =15.8 Hz. This methylene shifts due to the change in its electronic environment
caused by the change from an alcohol (OH) to an ester (COO). The methine (CH) of the
lactic acid appears at 4.38 ppm as a quartet with a coupling constant of 3J = 6.7 Hz. The
methine couples with the methyl (CH3) of the lactic acid unit at 1.39 ppm, which appears
3 as a doublet with a coupling constant of J = 6.7 Hz. The methylene (CH2) of the benzyl
protecting group appears at 5.17 ppm as a singlet. The nine tert-butyl protons are seen at
1.09 ppm and the fifteen aryl protons are seen as multiplets between 7.31-7.42 ppm and
7.65-7.71 ppm.
133
Scheme 5.13: Synthesis of the p-GL-p dimer.
3 3’ 2 d d q
2J = 15.8 Hz 2J = 15.7 Hz 3J = 6.7 Hz 8 8 5 8 O 3, 3’ Si O 6 4.48 4.58 8 O O 8 4 2 7 O 1 6 7 7 4.37 4.39 4.43 4.63 4.41 4.35 5
2.06 1.00
4.6 4.5 4.4 Chemical Shift (ppm)
8 4 1 7 6 3 3’ * 2
4.2612.25 2.00 1.19 3.02 9.74
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)
Figure 5.12: 1H NMR (300 MHz) spectrum of the p-GL-p dimer synthesized by coupling
benzyl glycolate acid with TBDPSi-protected lactic acid; * = CHCl3.
Figure 5.13 shows the 13C NMR spectrum of the bisprotected GL dimer. The methylene carbon (CH2) and ester carbon (COO) of the glycolic acid unit resonate at
134
60.59 and 167.32 ppm, respectively. The methyl carbon (CH3) of the lactic acid unit appears at 19.23 ppm, and the methine carbon (CH) and the ester carbon appear at 68.66 ppm and 173.10 ppm, respectively. The quaternary carbon (C(CH3)3) of the tert-butyl protecting group is seen at 21.28 ppm, and the three carbons from the methyl groups
(3CH3) in the tert-butyl protecting group overlap at 26.79 ppm with a much stronger intensity, due to contributions from 3 methyl groups. The methylene carbon (CH2) of the benzyl protecting group is seen at 67.07 ppm; this resonance should disappear after the following benzyl deprotection reaction.
8 4 5 2 7 128.62 3 6 1
128.43 8 135.90 127.66 26.79 135.73 127.62
*
6 4 1
5 67.07 3 19.23
2 60.59 7 167.32 135.01 132.95 173.10 68.66 21.28
180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)
Figure 5.13: 13C NMR (75 MHz) spectrum of the p-GL-p dimer, synthesized by the
coupling of benzyl glycolate with TBDPSi-protected lactic acid; * = CHCl3.
135
5.4.2 Synthesis of HOOC-GL-p dimer
The benzyl group of the p-GL-p dimer was deprotected (Scheme 5.14) by
hydrogenation. The reaction was carried out in a Parr Instruments hydrogenator for 24
hours, under 50 psi hydrogen gas in the presence of a Pd/C catalyst. This reactor
maintains constant hydrogen pressure, heat and stirring.
Scheme 5.14: Synthesis of the HOOC-GL-p dimer
In the 1H NMR spectrum (Figure 5.14), after the benzyl deprotection, the benzyl
methylene (CH2) protons at 5.17 ppm are absent. There is no change in the appearance of
the methylene (CH2) resonances of the glycolic acid unit, and the methyl and methine
resonances of the lactic acid units. The splitting patterns of the multiplets, and their
coupling constants remain the same after the benzyl deprotection, since there is little
change in the electronic atmosphere around these protons.
136
5
5 3 6 2 7 1 6 7 7
7 1 6 3 * 2
7.134.10 1.13 9.233.33
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)
Figure 5.14: 1H NMR (300 MHz) spectrum of the HOOC-GL-p dimer, synthesized by the deprotection of the bisprotected GL dimer, by a palladium-catalyzed hydrogenation; * =
CHCl3.
The 13C NMR spectrum also shows similar results (Figure 5.15). The resonance at
67.07 ppm belonging to the benzyl protecting group is absent. The ester carbon (COO) of the lactic acid unit appears at 173.04, same as that in the bi-protected GL dimer.
However, the ester carbon of the glycolic acid units shift down-field from 167.32 to
172.96 ppm, because of the change from a benzyl protected ester bond to a carboxylic acid.
137
7 26.78 7 4 5 2 6 3 1 127.60 127.65
135.72 * 135.88 1 77.01 77.43 76.58 19.21 3,5
129.84 2 6 173.04 132.92
68.63 4 21.22 60.09
180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)
Figure 5.15: 13C NMR (75 MHz) spectrum of the HOOC-GL-p dimer, synthesized by the deprotection of the bisprotected GL dimer, by a palladium-catalyzed hydrogenation; * =
CHCl3.
5.4.3 Synthesis of p-LGL-p dimer
The trimeric segmer is made by coupling the silyl protected GL-carboxylic acid dimer with benzyl lactate, using carbodiimide coupling chemistry (Scheme 5.15). Figure
5.16 shows the 1H NMR spectrum of the bisprotected trimer. The methine (CH) proton of the benzyl lactate unit shifts from 4.3 ppm (Figure 5.1) to 5.20 ppm upon coupling. This is because this proton changes from being alpha to an alcohol to being alpha to an ester, proving the esterification. The methine (CH) is split into a doublet of doublets, with the
138 coupling constants of 3J = 7 Hz and 3J = 1 Hz (Figure 5.16 (b)), which is surprising, as that methine should split into a quartet by coupling with the 3 protons of the attached methyl group. The methyl (CH3) protons with which this methine couples, appears at
1.49 ppm, as a doublet of doublets, with 3J = 7.2 Hz and the 3J = 2.8 Hz (Figure 5.16 (a)).
The methyl (CH3) protons of the silyl-protected lactic acid unit are upfield at 1.42 ppm, and these proton are split in an identical doublet of doublets splitting pattern, with coupling constants 3J = 6.7 Hz and the 3J = 2.6 Hz (Figure 5.16 (a)). The methine (CH) to which this methyl group couples is the methine of the silyl-protected lactic acid, at 4.56 ppm, which splits into a complex multiplet pattern (Figure 5.16 (c)). The methylene
(CH2) of the benzyl protecting group appears as a singlet at a predictable 5.2 ppm.
Scheme 5.15: Synthesis of the p-LGL-p trimer, synthesized by esterifying HOOC-GL-p
dimer with benzyl lactate.
139
8
dd 9 3J = 7.0 Hz
M01(dd)
3 5.19
J = 1.0 Hz 5.19 5.21
5.21 3 4.54 4.59 2 4.47 4.64 2.00
(b) 4.42 4.39 4.41 4.40 5.225 5.200 5.175 5.150 4.69 4.38 Chemical Shift (ppm) 4.65 4.49
(c)
4 1 4.7 4.6 4.5 4.4 3J = 6.7 Hz dd dd Chemical Shift (ppm)
3J = 7.2 Hz
5 M02(dd) M03(dd) 3J = 2.8 Hz 10 9 3 3J = 2.6 Hz 10 8 6 1.50 1.42 1.51 1.48 1.43
1.40 2
1.41 7 1.47 10 10 4 1 6 7 10 7
5
(a) 3.16 3.00
1.50 1.45 1.40 * Chemical Shift (ppm) 10
7 9 41 6 3 2 8
4.2512.01 3.00 3.53 3.23 9.58
8 7 6 5 4 3 2 1 0 Chemical Shift (ppm)
Figure 5.16: 1H NMR (300 MHz) spectrum of the p-LGL-p trimer, synthesized by the
esterification of benzyl lactate with HOOC-GL-p dimer ; * = CHCl3.
140
The 13C spectrum (Figure 5.17) of the trimer is complicated, and its peak assignments were elucidated with the help of 2D 1H 13C correlation gHSQC experiment. This experiment identifies the directly bonded carbons and protons.
11 11 26.81 9 4 7 5 2 10 8 3 6 1 128.61 127.66 135.73
135.90 * 129.82 9 10 2 4
7 19.23 1 6 67.17 60.31 69.30 35 8 21.28 16.81 166.84 133.48 172.96 169.87
180 160 140 120 100 80 60 40 20 0 Chemical Shift (ppm)
Figure 5.17: 13C NMR (75 MHz)spectrum of the p-LGL-p trimer, synthesized by the
esterification of benzyl lactate with HOOC-GL-p dimer; * = CHCl3.
Figure 5.18 shows the 2D HSQC plot of the trimer, expanded in the methyl regions. Based on the 13C crosspeaks with the known 1H assignments, this plot helps to assign the resonance at 16.81 ppm (crosspeak to 1.52 ppm) to the methyl (CH3) carbon of the benzyl-protected lactic acid unit, and 21.28 ppm (crosspeak to 1.45 ppm) to the methyl (CH3) carbon of the silyl-protected lactic acid unit. The three overlapping methyl
(C(CH3)3) carbons of the tert-Bu group are at 26.81 ppm. The remaining resonance in this
141
t region, is that of the quaternary carbon (C(CH3)3) of the Buu group, which does not show a cross peak since it is not bonded to any protons.
Figure 5.18: 2D HSQC plot of the p-LGL-p trimer expanded into the methyl region.
The region between 60 ppm and 70 ppm on the 13C spectrum is also elucidated with the help of the 2D HSQC plot (Figure 5.19), by matching the crosspeaks with known 1H assignments from figure 5.16. The methylene (CH2) carbon of the glycolic acid unit is at
60.31 ppm, and the other methylene of the benzyl protecting group is at 67.17 ppm. The two methine carbons (CH) appear at 68.7 ppm and 69.3 ppm belong to the silyl-protected lactic acid unit and the benzyl-protected lactic acid unit respectively. All of the three ester
(COO) resonances appear in the region betweeen 160 to 180 ppm. The resonance at
142
172.96 ppm is that of the ester of the silyl-protected lactic acid, since the same ester appears at 173 ppm in the dimer. There is practically no change in the electronic environment around this carbon upon forming the trimer from the dimer. The ester of the glycolic acid is next, at 169.87 ppm, and the ester of to the benzyl protected lactic acid unit shifts upfield to 166.84 ppm, upon esterifying with the GL dimer.
Figure 5.19: 2D HSQC plot of the p-LGL-p trimer expanded into the methine and
methylene regions.
5.5 Synthesis of the LBL Trimer
The LBL trimeric segmer was synthesized by first creating a bisprotected LB dimeric segmer. The silyl-protected end of the dimer was deprotected to yield an alcohol,
143
CHAPTER VI
BIODEGRADATION OF FUNCTIONALIZABLE POLYESTERS - PLGB
6.1 Degradation
Polymer degradation studies were performed on PLGB601030 and PLGB502030, to investigate the biodegradation behavior of these novel functionalizable polyesters.
Both polymers of the polymers studied have 30 mol% brominated groups, but vary in their glycolic acid content (10 mol% and 20 mol%). The polymers were biodegraded in a phosphate buffer saline (PBS) (pH = 7.4) at 37 °C with constant stirring.
6.2 Sample Preparation
The two copolymers were compression molded into 100mg tablets using a custom machined steel mold (Figure 6.1). The copolymers were first finely ground into a powder.
To prepare each sample, 100 mg of this powder was added into the circular cavity of the mold, and the top plunger was pushed on it to begin shaping it into a circular tablet. This mold was then inserted in a Carver Press, and 5000 pounds of pressure (600 PSI) was applied for 30 seconds. The mold was then removed from the press, and the top plunger
157
was withdrawn to retrieve the polymer tablet (Figure 6.2). The exact weight of the polymer tablet obtained was recorded on a microbalance.
Top Plunger
Cylinder with cavity
Polymer
Bottom Plunger
Figure 6.1: Mold for preparing 100 mg compression molded samples for biodegradation
study.
6.3 Degradation Experiment
For each set of polymer, 16 tablets were compression molded and each added to a
20 ml drum vial, making a total of 32 vials for the two polymers. Each of the drums was filled with 15 ml of the PBS solution and introduced to an incubator maintained at 37 °C, stirring at the rate of 100 rpm. One vial of PLGB601030 and PLGB502030 was taken out of the incubator at an interval of 3-4 days. The PBS solution was changed at the interval of once every week to maintain the buffer solution. The study was continued for 54 days.
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Figure 6.2: (a) Mold resting on the bottom plunnger, (b) 100 mg of the dry polymer was added in the cavity of the mold, (c) the top plunger was inserted, and (d) 600 PSI pressure
was applied for 30 second to yield a 100 mg polymer tablet.
6.4 Degradation
The degradation of the two PLGB copolymers were evaluated by visual inspection (section 6.4.1), moisture absorption (section 6.4.2), weight loss (section 6.4.3) and loss in molecular weight (section 6.4.4)
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6.4.1 Degradation by Visual Inspection
Degradation can be evaluated visually, by looking first for signs of swelling,
which indicate water absorption. Water absorption is the first step for degradation by
hydrolysis. Swelling leads to the deformation of the polymers, and the onset of
degradation. Eventually, massive erosion is seen, leading eventually to complete
degradation.
Looking at both PLGB601030 (Figure 6.3) and PLGB502030 (Figure 6.4), there
is no visual difference in the degradation behaviors of the PLGB601030 and
PLGB502030. Visually, there is no physical change in the polymers till 9 days. Signs of water absorption and swelling begin to be seen around 12-17 days. Till 12-17 days, the tablets maintained their disk-like physical form and do not show any loss of material, but from 12-17 days onwards, the polymers begin deforming due to swelling from water
absorption. From 28 days onwards, the polymers begin showing significant material loss,
and by around 40 days, most of the polymer has eroded away. By the end of the study,
only a spatter of the original polymer remains.
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Figure 6.3: Degradation samples over days of degradation of PLGB601030, arranged
chronologically.
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Figure 6.4: Degradation samples over days of degradation oof PLGB 502030, arranged
chronologically.
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6.4.2 Water Absorption:
Water absorption is the very first step of the degradation process. To measure water absorption, the polymer was removed from vial and washed twice with distilled deionized water. The polymer disk was wiped dry with a kim-wipe to remove any water sticking to the surface of the polymer. The polymer disk was then weighed, and water absorption was calculated by the following formula. It is important to wipe the polymer dry before weighing it to ensure that only water absorbed in the bulk of the tablet is calculated.
Ww = weight of wetted polymer and Wi = initial weight of the polymer.
The data from the water absorption experiment is tabulated in Table 6.1. Figure
6.5 shows the water absorption curves for PLGB601030 (in blue) and PLGB502030 (in grey). The X axis on the curves show number of days in degradation media, and the Y axis shows the percent water absorbed for both polymers.
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Table 6.1: Percent water absorption for the PLGB613 and PLGB523 copolymers during
their biodegradation in PBS (37 °C).
Number PLGB613 PLGB523 of days% Water Absorption % Water Absorption 3 1.7 6.5 5 2.4 9.2 7 4.2 12.2 9 2.7 16.9 12 4.1 25.5 17 9.1 34.2 19 15.3 47.2 23 37.7 80.4 28 57.4 77.7
Figure 6.5: Percent watere absorption by weight of the PLGB601030 and PLGB502030
copolymers.
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Both the PLGB copolymers display similar water absorption behavior to PLGA
copolymers. The PLGB copolymers are hydrophilic and are able to absorb water almost
equal to their weight in about 4 weeks. The tablets used in this degradation study are non-
porous; hence the water absorption occurs first by wetting of the surface, swelling the
polymer (visualized in section 6.4.1), and finally leading to absorption of water in the
bulk. The wetting of the surface occurs in the first 17 days, where the polymers absorb
only about 10% (PLGB601030) and 35% (PLGB502030) of water. Once, the surface has
been wetted, the water is able to be absorbed in the bulk, and the rate of water absorption
increases, as seen by the increase in the slope of the water absorption curve after 19 days, seen in figure 6.5.
Water absorption in the bulk is important for these hydrophilic copolymers, as the rate of water absorption (rwa) is higher than the rate of ester bond hydrolysis (rh). This implies that the rate of degradation can be tailored by varying the rate of water absorption. The PLGB502030 shows faster absorption of water compared to the
PLGB601030. This is not unexpected as the PLGB502030 has a higher content of glycolic acid, which is slightly more hydrophilic than lactic acid units, and aids in the water absorption. Hence, increasing the ratio of glycolic acid units would afford us a polymer with faster water absorption properties and thereby faster hydrolytic degradation profile.
Even with 30% 2-bromo-3-hydroxy propionic acid units in the polymer, both polymers show good water absorption, which is the starting point of degradation. Once a critical amount of degradation medium has been absorbed in the bulk, chain hydrolysis starts, leading eventually to complete degradation. The water absorption was only
165
performed till 28 days, since it was not possible to pick up the polymer samples after that
to measure their wet weights.
6.4.3 Weight Loss
To measure the weight loss of the polymers, they were first dried in vacuum for a
couple of days, after measuring their weight for the moisture absorption data. The
polymers were dried in their respective vials, as after 23 days, the polymers were stuck to
the vials, and it would not have been possible to weigh them without losing polymer in
just transferring them to weigh. The polymers were hence dissolved in around 5 mL
CH2Cl2 and transferred into vials of known weights, and dried in vacuum for a couple of
days. The increase in weight of these new vials correspond to the weight of polymer left,
after degradation.
An analysis of the weight loss of the two copolymers also helps us understand
degradation behavior of these copolymers. Table 6.2 displays the weight loss data, and
the graph in Figure 6.6 compares the trends between the two copolymers. The weight loss
profile indicates that the degradation of the PLGB copolymers occurs by bulk
degradation and not by surface degradation.
Both polymers gained some weight in the first 23 days. This could be explained
by the fact that these polymers absorbed some PBS solution in their bulk, and upon drying the polymers, the salts from the absorbed PBS solution were trapped in the bulk.
The weights measured are thus not only the weight of the polymer, but also include some
salt from the PBS solution.
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After 23 days, there was a steady loss of weight, which continues to about 80% in
60 days. The weight loss behavior shows that the PLGB copolymers degrade by bulk degradation, rather than surface erosion. There is practically no weight loss till the first
23 days of the study, as there was water absorption and degradation in the bulk
(corroborated in section 6.4.4). However, there was no weight loss in the first 23 days, as hydrolysis of the ester bonds in the bulk was not leading to diffusible small molecules. A critical point was reached at 23 days, after which further scission of chain gave small monomeric or oligomeric species, leaching out of which led to loss in matter. This behavior is also observed visually in Figures 6.3 and 6.4. This behavior is typical of hydrophilic polymers like PLGA which degrade by bulk degradation and not by surface erosion.
Had these copolymers been degrading by surface erosion, we would have observed a steady weight loss over the degradation period, with no induction period.
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Figure 6.6: Percent weight loss of the PLGB601030 (greyy) and PLGB502030 (blue)
copolymers during their biodegradation in PBS (37 °C).
Comparison of the behavior of the PLGB502030 and PLGB601030 demonsttrates that PLGB502030 shows a higher and faster weiight loss. This can again be attributed to the fact that PLGB502030 has a higher glycolicc acid content, which makes the polymer slightly more hydrophilic, accelerating it degradation. By the end of the study, both polymers lost around 80% of their mass.
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Table 6.2: Percent weight change for the PLGB601030 and PLGB502030 copolymers
during their biodegradation in PBS (37 °C).
PLGB601030 PLGB502030 # days Wt after Wt Initial % Wt Loss Wt after Wt Initial % Wt Loss 3 110.4 100.46 ‐9.9 102.51 100.3 ‐2.2 5 106.9 98.89 ‐8.1 110.02 101.5 ‐8.4 7 94.95 101.49 6.4 96.19 99.48 3.3 9 101.77 102.61 0.8 102.36 99.4 ‐3.0 12 111.43 102.22 ‐9.0 99.55 99.12 ‐0.4 17 106.17 97.78 ‐8.6 106.46 100.85 ‐5.6 19 103.44 97.53 ‐6.1 94.05 99.33 5.3 23 104.84 100 ‐4.8 104.48 99.84 ‐4.6 28 88.13 98.86 10.9 84 97.97 14.3 32 89.65 101.24 11.4 79.78 100.1 20.3 36 54.89 100.4 45.3 44.56 103.47 56.9 40 70.65 96.67 26.9 60.8 100.96 39.8 44 52.26 101.28 48.4 39.43 101.18 61.0 49 34.72 99.83 65.2 23.6 99.4 76.3 54 24.06 102.26 76.5 17.72 101.44 82.5
6.4.4 Molecular Weight Loss
The loss in molecular weight for both the PLGB601030 and PLGB502030 are tabulated in Table 6.3 and Table 6.4. Their GPCPSt chromatographs are stacked in Figure
6.7 and Figure 6.8.
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Table 6.3: Loss in molecular weight data for the PLGB601030 copolymer.
Number Peak of Days Mn Mw PDI Molecular Weight 3 5,889 16,682 2.83 14,313 7 5,127 12,688 2.47 11,343 12 2,283 8,672 2.56 8,416 19 2,258 4,267 1.89 3,396 28 1,394 2,348 1.68 1,372 36 1,048 1,322 1.26 945 44 1,046 1,313 1.26 898 54 869 1,010 1.16 697
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Figure 6.7: Stacked Gel Permeation Chromatographs for PLGB601030, showing the
molecular weight loss behavior upon degradation. Peak molecular weights shown.
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Both PLGB601030 and PLGB502030 show a steady decrease in both number
average and weight average molecular weight. The peak molecular weight also shows a
steady decrease with increasing degradation time. The polydispersity indexes of both the polymers also decrease with degradation time. While for physical properties like weight
loss (section 6.4.3) and water absorption (section 6.4.2) an induction period is observed, loss in molecular weight is observed from the first day. This is because hydrolysis occurs in the parts of the polymer matrix exposed to the PBS solution. In the beginning, only the surface of the polymer absorbs water, and there is hydrolytic degradation on the surface.
This does not lead to any changes in the structural integrity of the polymer tablet, and there is no observed weight loss (section 6.4.3) as the chain scission still leaves behind high molecular weight chains. Once a critical amount of water has been absorbed, more and more chain scission occurs in the bulk, creating small monomeric and oligomeric species which are able to diffuse out of the bulk material, compromising its structural integrity. Visually, the tablet swells, and then begins to grow smaller in size. Physically, there is increased water absorption and a steady weight loss is observed. All this while, the molecular weight of the polymer decreases in a steady manner. This behavior is typical for hydrophilic polymers like PLGA.
By comparing the loss in molecular weight of PLGB601030 with PLGB502030, there seems to be no difference in the rate of decrease of the molecular weights. So, while the increase in glycolic acid content in PLGB502030 made it swell faster and lose more mass faster during the course of the biodegradation, the loss in molecular weight were similar.
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Table 6.4: Loss in molecular weight data for the PLGB502030 copolymer.
Number Peak of Days Mn Mw PDI Molecular Weight 3 5,705 16,388 2.87 17,461 7 4,699 13,582 2.89 14,983 12 3,224 7,834 2.43 8,239 19 2,726 5,662 2.08 4,969 28 1,799 3,585 1.99 2,766 36 1,082 1,330 1.22 1,103 44 1,082 1,366 1.26 850 54 913 1,035 1.13 789
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Figure 6.8: Stacked Gel Permeation Chromatographs for PLGB502030, showing the
molecular weight loss behavior upon degradation. Peak molecular weights shown.
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REFERENCES
1. Pugh, C.; Banerjee, A.; Storms, W.; Wright, C. U.S. Provisional Patent Application 61/368413, 2011.
2. Pugh, C.; Singh, A.; Samuel, R.; Bernal Ramos, K. M. Macromolecules 2010, 43, 5222.
3. Arshady, R., Biodegradable Polymers, Vol 2. Citus Books: London, 2003; Vol. 2.
4. Frazza, E. J.; Schmitt, E. E. J. Biomed. Mater. Res. 1971, 5, 43.
5. Ikada, Y.; Tsuji, H. Macromol. Rapid Commun. 2000, 21, 117.
6. Domb, A. J.; Kost, J.; Wiseman, D. M., Handbook of Biodegradable Polymers. Harwood Academic Publishers: Amsterdam, 1997; p Ch. 1, Ch. 2.
7. LLC, N. NatureWorks LLC. http://www.natureworksllc.com (10 June 2011).
8. Li, S.; McCarthy, S. Biomaterials 1999, 20, 35.
9. Phusiline® Resoscarf screw. http://www.phusis.fr/uk/produit/resoscarf.htm (25 April 2011)
10. Torchilin, V. P. J. Control Release 2001, 73, 137.
11. Torchilin, V. P. Pharm. Res. 2006, 24, 16.
12. Williams, C. K. Chem. Soc. Rev. 2007, 36, 157312.
13. Coessens, V.; Matyjaszewski, K. Macromol. Rapid Commun. 1999, 20, 127.
14. Coessens, V.; Matyjaszewski, K. J Macromol. Sci. A 1999, 36, 811.
15. Garrison, J. C.; Youngs, W. J. Chem. Rev. 2005, 105, 3978.
16. Medvetz, D. A.; Hindi, K. M.; Panzner, M. J.; Ditto, A. J.; Yun, Y. H.; Youngs, W. J. Met. Based Drugs 2008, 1-7.
17. Ditto, A. J.; Shah, P. N.; Lopina, S. T.; Yun, Y. H. Int. J. Pharm. 2009, 368, 199.
175
18. Ferreira, P.; Phillips, E.; Rippon, D.; Tsang, S. C.; Hayes, W. J. Org. Chem. 2004, 69, 6851.
19. Chenal, M.; Mura, S.; Marchal, C.; Gigmes, D.; Charleux, B.; Fattal, E.; Couvreur, P.; Nicolas, J. Macromolecules 2010, 43, 9291-9303.
20. Ratner, B. D.; Hoffman, A. S.; Schoen, F. J.; Lemons, J. E., Biomaterials Science: An Introduction to Materials in Medicine, 2nd ed. Elsevier Academic Press: New York, 2004.
21. Buchholz, B. US Patent 5,302,694, 1994.
22. Moon, S. I.; Lee, C. W.; Miyamoto, M.; Kimura, Y. J. Polym. Sci. Part A 2000, 38, 1673.
23. Ajioka, M.; Enomoto, K.; Suzuki, K.; Yamaguchi, A. J. Environ. Polym. Degr. 1995, 3, 225.
24. Enomoto, K.; Ajioka, M.; Yamaguchi, A. US Patent 5,310,865, 1994.
25. Neises, B.; Steglich, W. Angew. Chem. Int. Edit. 1978, 17, 522.
26. Moore, J. S.; Stupp, S. I. Macromolecules 1990, 23, 65.
27. Akutsu, F.; Inoki, M.; Uei, H.; Sueyoshi, M.; Kasashima, Y.; Naruchi, K.; Yamaguchi, Y.; Sunahara, M. Polym. J. 1998, 30, 421.
28. Yoda, S.; Bratton, D.; Howdle, S. M. Polymer 2004, 45, 7839.
29. Wagener, K. B.; Linert, J. G.; O'Gara, J. E. J.M.S. Pure Appl. Chem. 1994, A31, 775.
30. Stayshich, R. M.; Meyer, T. Y J. Polym. Sci. Part A 2008, 46, 4704.
31. Stayshich, R. M.; Weiss, R. M.; Li, J.; Meyer, T. Y. Macromol. Rapid Commun. 2011, 32, 220.
32. Stayshich, R. M.; Meyer, T. Y. J. Am. Chem. Soc. 2010, 132, 10920.
33. Mainil-Varlet, P.; Rahn, B.; Gogolewski, S. Biomaterials 1997, 18, 257.
34. Mainil-Varlet, P.; Curtis, R.; Gogolewski, S. J. Biomed Mater. Res. 1997, 36, 360.
35. Li, S. M.; Garreau, H.; Vert, M. J. Mater. Sci. 1990, 1, 123.
36. Li, S. M.; Garreau, H.; Vert, M. J. Mater. Sci. 1990, 1, 198.
176
37. Li, S. M.; Garreau, H.; Vert, M. J. Mater. Sci. 1990, 1, 131.
38. Lu, L.; Garcia, C. A.; Mikos, A. G. J. Biomat. Sci – Polym. E. 1998, 9, 1187.
39. Lu, L.; Garcia, C. A.; Mikos, A. G. J. Biomed. Mater. Res. 1999, 46, 236.
40. Shah, S. S.; Zhu, K. J.; Pitt, C. G. J. Biomed. Mater. Res. 1994, 5, 421.
41. Cleek, R. L.; Ting, K. C.; G. Eskin, S.; Mikos, A. G. J. Control. Release 1997, 48, 259.
42. Breitenbach, A.; Pistel, K. F.; Kissel, T. Polymer 2000, 41, 4781.
43. Pierre, T. S.; Chiellini, E. J. Bioact. Compat. Pol. 1986, 1, 32.
44. Mathiowitz, E.; Jacob, J.; Pekarek, K.; Chickering, D. Macromolecules 1993, 26, 6756.
45. Gautier, S. E.; Oudega, M.; Fragoso, M.; Chapon, P.; Plant, G. W.; Bunge, M. B.; Parel, J.-M. J. Biomed. Mater. Res. 1998, 42, 642.
46. Wu, X. S.; Wang, N. J. Biomat. Sci. - Polym. E. 2001, 12, 21.
47. Park, T. G. Biomaterials 1995, 16, 1123.
48. Gao, Q.; Lan, P.; Shao, H.; Hu, X. Polym. J. 2002, 34, 786.
49. Horita, K.; Yoshioka, T.; Tanaka, T.; Oikawa, Y.; Yonemitsu, O Tetrahedron 1986, 42, 3021.
50. Gowda, D. C Tetrahedron Lett. 2002, 43, 311.
177