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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2011-05-31.

Permanent Link: http://ufdc.ufl.edu/UFE0024375/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2011-05-31.
Physical Description: Book
Language: english
Creator: Leonard, James
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by James Leonard.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Wagener, Kenneth B.
Electronic Access: INACCESSIBLE UNTIL 2011-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024375:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024375/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2011-05-31.
Physical Description: Book
Language: english
Creator: Leonard, James
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by James Leonard.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Wagener, Kenneth B.
Electronic Access: INACCESSIBLE UNTIL 2011-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024375:00001


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1 PRECISION POLYETHYLENE DESIGNED FOR BIOLOGICAL APPLICATIONS By JAMES KLEIN LEONARD A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 James K. Leonard

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3 To my loving and supportive parents, Jim and Dee Leonard

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4 ACKNOWLEDGMENTS There are several people who have helped and inspired me to accomplish all that I have in my education so far. First and foremost, I must thank my parents Jim and Dee. They have always selflessly and endlessly given me their love and encouragement throughout my life that has allowed me to become the man I a m today. My father helped me purchase my first home in Gainesville and always gives me wise advise that motivates me to tackle any problem. Mom gives me emotional support that never hurts and she visited me in Florida more than anyone else from Michigan. Mom and Dad are my heroes and I am always grateful to have been their son. My sisters, Christina and Julia, continually keep me working. They give me competition to always try a little harder and pride to never let them down. I love all of them and I thank them for all they give me. Three people have made my graduate experience a truly exceptional one: Prof. Ken Wagener, Prof. Ken Sloan, and Dr. Khalil Abboud. Professor Wagener, my research advisor, is an amazingly kind and sincere man who always put s people first. His guidance, patience, friendship, and encouragement throughout the years have made me a better person and scientist. Under -commit and over -deliver will be a motto I will live by and I will always treat a person with respect and kindne ss no matter how busy or stressed I am. Prof. Wagener always had time for me and was always willing to talk about science or everyday life. If my career ever leads me to academics, Prof. Wagener will be the model I will always strive to imitate. I will be forever thankful to him for giving me the opportunity to be a Gator and to pursue all I have in graduate school. My endless thanks go to the Alumni Fellowship and Prof. Sloan, because of them I was able to pursue a Medicinal Chemistry Masters in Pharm acy degree during my time here. Prof. Sloans early morning group meetings during my time here was always a pleasure to attend and I

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5 will surely miss our enthusiastic banter over science, politics, and life. I must give credit to Prof. Sloan for helping me incorporate the ideas of drugs and sunscreen molecules into the type of polymers we make. These projects were my favorite to work on and his insightful ideas and suggestions made me think about Chemistry with different perspectives. Dr. Khalil Abbo ud took me under his tutelage my second semester here and taught me to become competent in single crystal X -ray crystallography. By the end of my second summer I was able to pick, cut, gather, and solve the data on most crystal samples submitted to the la b. With this background I have been able to help fellow researchers on the polymer floor with their X ray data and better understand this technique. Dr. Abboud has been a great friend of mine since the day I started working with him and I cherish the lunches and laughs we had together. During my time here, I have had the opportunity to study and do research with a number of amazing fellow scientists. The George and Josephine Butler Polymer Research Laboratories have given me the privilege to work in a n environment where teamwork, collaboration, and friendships are fostered forever. Prof. John Reynolds was continuously a positive and effective leader on the floor and I really appreciate how he treated me and more so what he taught me while I was here. I will never forget the bet I should have never won against him, Prof. Reynolds looks fantastic in Maize and Blue and makes a great Wolverine! I offer my sincere thanks to former Wagener research group members Dr. Timothy Hopkins, Dr. John Sworen, Dr. Tr avis Baughman, Dr. Piotr Matloka, Dr. Florence Courchay, Dr. Violeta Petkovska, Dr. Giovanni Rojas, Dr. Emine Boz, Dr. Erik Berda and my undergraduate researchers Briana Jackson and Tahnie Danastor along with current members Sam Popwell, Kate Opper, Yuying Wei, Paula Delgado, Brian Aitken, and Bora Inci. I must also thank Justin Gardner, Denise Sharbaugh, and Brian MacMillan for being my rent paying roommates to help me make my first home a

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6 worthwhile investment. My time at the University of Florida would not have been the same without Dr. Benoit Lauly, Dr. Nick Taylor, Dr. Eric Libra, Candace Zielenuk, and Chase Rainwater for their commitment to quarter -barrel Saturday and competing in intramural sports with me. Thank you to Emily Rasch. I have dated her during my entire time in Gainesville. She never knows what I am talking about when it comes to science but she always listens and she has been my best friend here. I could not imagine how much more difficult life would be without Emily and her love and support. Enough gratitude cannot be expressed here to Sara Klossner, Gina Borrero, and during my first year, Lorraine Williams. Without them, the normal everyday flow of research could never have gone as smoothly as they have the past five years. Sara a nd Gina give the polymer floor a family feel and I will always consider them both friends for life. Many thanks are extended to my committee members Prof. Eric Enholm and Prof. Nicole Horenstein. Finally, I would like to thank Professor George Butler. I was fortunate enough to meet him a handful of times and even sit with him one evening at dinner. His generosity and foresight to create the Butler Polymer Floor has made for an educational environment that I would recommend to anyone looking for an exceptional place to learn and conduct polymer research. I worked and studied hard while I was here and I pray that as I go out to compete and work in the real world I will always be able to represent the Butlers and our program here because I believe it to be the best polymer program in the country.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES .............................................................................................................................. 11 LIST OF FIGURES ............................................................................................................................ 12 ABSTRACT ........................................................................................................................................ 15 CHAPTER 1 INTRODUCTION ....................................................................................................................... 17 Synthesis of Biopolymers ........................................................................................................... 17 Techniques for Biopolymer Synthesis ................................................................................ 17 Metathesis Approach ........................................................................................................... 24 Synthetic Background of Diene Premonomers .................................................................. 28 Amino Acid Containing Materials ...................................................................................... 31 Polyethylene Pr odrugs ......................................................................................................... 35 Sunscreen Polymers ............................................................................................................. 37 Dissertation Purpose ................................................................................................................... 39 2 SYNTHE SIS AND THERMAL CHARCTERIZATION OF PRECISION ETHYLENE VINYL AMINE COPOLYMERS ............................................................................................. 40 Introduction ................................................................................................................................. 40 Experimental ................................................................................................................................ 42 Materials ............................................................................................................................... 42 Instrumentation and Analysis .............................................................................................. 42 Premonomer Amine Diene Synthesis ................................................................................. 43 1 -Undec 10-enyl dodec 11 -enylamine (3,3NH2). ...................................................... 44 Tricosa 1,22-dien 12amine (9,9NH2). ....................................................................... 44 1 Dec 9 -enyl undec 10 -enylamine (8,8NH2). ............................................................ 45 (9H -fluoren 9 -yl)methyl henicosa 1,20-dien 11 -ylcarbamate (9,9NHFmoc). ........ 45 General Boc Protection of the Amine Dienes .................................................................... 46 Tert butyl undeca 1,10-dien 6 ylcarbamate 3,3NHBoc (21). ................................. 46 Tert butyl heptadeca 1,16-dien 9 -ylcarbamate 6,6NHBoc (2 2). ............................. 46 Tert butyl henicosa 1,20 dien 11 ylcarbamate 8,8NHBoc (23). ............................. 47 Tert butyl tricosa 1,22 -dien 12 ylcarbamate 9,9NHBoc (2 4). ................................ 47 General ADMET Polymerization Procedure for Symmetrical Boc Amine Monomers ......................................................................................................................... 47

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8 Polymerization of tert -butyl undeca 1,10-dien 6 -ylcarbamate 3,3NHBoc (2 -5). ... 48 Polymerization of tert -butyl heptadeca 1,16-dien 9 -ylcarbamate 6,6NHBoc (2 6). ............................................................................................................................... 48 Polymerization of tert -butyl henicosa 1,20-dien 11-ylcarbamate 8,8NHBoc (27). ............................................................................................................................... 48 Polymerization of tert -butyl tricosa 1,22 dien 12 ylcarbamate 9,9NHB oc (2 8). ............................................................................................................................... 49 Hydrogenation of Unsaturated ADMET Polymers ........................................................... 49 Polysat3,3NHBoc (2 9). .............................................................................................. 49 Polysat6,6NHBoc (2 10). ............................................................................................ 50 Polysat8,8NHBoc (2 11). ............................................................................................ 50 Polysat9,9NHBoc (2 12). ............................................................................................ 50 Removal of the Boc Protection Group from the Polymers ............................................... 50 Results and Discussion ............................................................................................................... 51 Polyme r Design and Synthesis ............................................................................................ 51 Structural Analysis with 13C and 1H NMR and IR ............................................................. 52 Thermal Analysis ................................................................................................................. 55 Conclusions .......................................................................................................................... 61 Future Work ................................................................................................................................. 61 3 SEMICRYSTALLINE LYSINE FUNCTIONALIZED PRECISION POLYOLEFINS ....... 63 Introduction ................................................................................................................................. 63 Experimental Section .................................................................................................................. 65 Materials ............................................................................................................................... 65 Instrumentation and Analysis .............................................................................................. 65 General Monomer Synthesis ............................................................................................... 67 [(S )5 Benzyloxycarbonylamino5 (1 undec 10 -enyl -dodec 11 enylcarbamoyl) -pentyl] -carbamic acid benzyl ester (3 3). .................................... 67 (S )6 Benzyloxycarbonylamino2 [3 (1 undec 10 enyl -dodec 11enylcarbamoyl) -propionylamino ] -hexanoic acid methyl ester (3 4). ................... 68 (S )-6 Benzyloxycarbonylamino2 (2 undec 10 -enyl -tridec 12 -enoyl amino) hexanoic acid methyl ester (3 5). ............................................................................ 68 (S) Methyl 6 (((9H -fluoren 9 -yl)methoxy)carbonylamino) 2 (2 (undec 10enyl)tridec 12 -enamido)hexanoate (3 6). .............................................................. 69 Polymer Synthesis ................................ ............................................................................... 69 Polymerization of [( S ) 5 Benzyloxycarbonylamino 5 (1 undec 10 -enyl -dodec 11enylcarbamoyl) pentyl] -carbamic acid benzyl ester (3 3a). ............................ 70 Polymerization of ( S ) 6 Benzy loxycarbonylamino2 [3 (1 undec 10 -enyl dodec 11-enylcarbamoyl) -propionylamino] -hexanoic acid methyl ester (3 4a). ............................................................................................................................. 71 Polymerization ( S )-6 Benzyloxycarbonylamino2 (2 undec 10 -enyl -tridec 12 e noyl amino) hexanoic acid methyl ester (3 5a). ................................................... 71 Polymerization of (S) -methyl 6 (((9H -fluoren 9 yl)methoxy)carbonylamino) 2 (2 (undec 10 -enyl)tridec 12 enamido)hexanoate (3 6a). ................................... 71 Deprotection Chemistry of the 9-fluorenylmethyloxycarbonyl (Fmoc) Lysine Polymer ............................................................................................................................. 72

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9 Deprotection of (S) -methyl 6 (((9H -fluoren 9 -yl)methox y)carbonylamino) 2 (2 (undec 10-enyl)tridec 12 -enamido)hexanoate (3 6b). ..................................... 72 Results and Discussion ............................................................................................................... 72 Polymer Design and Synthe sis ............................................................................................ 72 Molecular Weight and Physical Properties ........................................................................ 74 Deprotection Chemistry ...................................................................................................... 75 Thermal and X Ray Characterization of Lysine Monomers and Polymers ..................... 77 Conclusions .......................................................................................................................... 81 Future Work ................................................................................................................................. 82 4 POLYETHYLENE PRODRUGS USING PRECISELY PLACED PHARMACEUTICAL AGENTS .............................................................................................. 83 Introduction. ................................................................................................................................ 83 Experimental ................................................................................................................................ 85 Materials ............................................................................................................................... 85 Instrumentation and Analysis .............................................................................................. 85 Method of Hydrolysis .......................................................................................................... 86 Monomer Synthesis ............................................................................................................. 87 2 (Undec 10-enyl)tridec 12-enoic acid (4 1). ............................................................ 87 2 (Undec 10-enyl)tridec 12-en 1 ol (4 2). ................................................................. 88 General Coupling of the 9,9 Primary Alcohol to Either the Ibuprofen or Naproxen Drug Molecules ................................................................................................................ 89 (S) 2 (undec 10 -enyl)tridec 12 enyl 2 (4 isobutylphenyl)propanoate (4 3). .......... 89 (S) 2 (undec 10 -enyl)tridec 12 enyl 2 (6 -methoxynapthalen 2 -yl)propanoate (4 4). .......................................................................................................................... 90 General Coupling of the Drug to Either Decanediol or Tetraethylene glycol ................. 90 (S) 2 (2 -(2 (2 -hydroxyethoxy)et hoxy)ethoxy)ethyl 2 -(4 isobutylphenyl)propanoate (4 5). ............................................................................ 91 (S) 10-hydroxydecyl 2 (4 -isobutylphenyl)propanoate (4 6). .................................... 91 (S) 2 (2 -(2 (2 -hydroxyethoxy)ethoxy)ethoxy)ethyl 2 (6 -methoxynaphthalen 2 yl)propanoate (4 7). .................................................................................................. 91 (S) 10-hydroxydecyl 2 (6 -methoxynaphthalen 2 -yl)propanoate (4 8). ................... 92 General Coupling of the 9,9 Acid Diene to Either Decanediol Ester Drugs or Tetraethylene Glycol Ester Drugs ................................................................................... 92 (S) 14(4 isobutylphenyl) 13 -oxo 3,6,9,12-tetroxap entadecyl 2 (undec 10 enyl)tridec 12 -enoate (4 9). ..................................................................................... 92 (S) 10(2 (4 isobutylphenyl)propanoyloxy)decyl 2 (undec 10 -enyl)tridec 12 enoate (4 10). ............................................................................................................ 93 (S) 14(6 -methoxynaphthalen 2 yl) 13-oxo 3,6,9,12 tetroxapentadecyl 2 (undec 10-enyl)tridec 12-enoate (4 11). ................................................................. 93 (S) 10(2 (6 -methoxynaphthalen 2 yl)propanoyloxy)decyl 2 (und ec10enyl)tridec 12 -enoate (4 12). ................................................................................... 94 General Polymer Synthesis. ................................................................................................ 94 Poly9,9TEGIbuprofen (4 13a). ................................................................................... 95 Poly9,9DecanediolIbuprofen (4 14a). ........................................................................ 95 Poly9,9OHIbuprofen (4 15a). ..................................................................................... 96

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10 Poly9,9TEGNaproxen (4 13b). ................................................................................... 96 Poly9,9DecanediolNaproxen (4 14b). ........................................................................ 96 Poly9,9OHNaproxen (4 15b). ..................................................................................... 97 Results and Discussion ............................................................................................................... 97 Polymer Design and Synthesis ............................................................................................ 97 Structural Analysis ............................................................................................................. 102 Thermal Analysis ............................................................................................................... 104 Hydrolysis of Polymers ..................................................................................................... 106 Conclusions ........................................................................................................................ 109 Future Work ............................................................................................................................... 110 5 PRECISION SUNSCREEN POLYMERS .............................................................................. 113 Introduction ............................................................................................................................... 113 Experimental .............................................................................................................................. 115 Materials ............................................................................................................................. 115 Instrumentation and Analysis ............................................................................................ 116 Monomer Synthesis ........................................................................................................... 117 Undeca 1,10-dien 6 ol (5 1). ..................................................................................... 117 Undeca 1,10-dien 6 amine (5 1a). ............................................................................ 117 Undeca 1,10-dien 6 yl 4 (dimethylamino)benzoate 3,3OHPABA (5 2). .............. 118 4 (Dimethylamino) N -(undeca 1,10 dien 6 yl)benzamide 3,3NHP ABA (5 3). ... 119 (E ) undeca 1,10 dien 6 yl 3 (4 -methoxyphenyl)acrylate 3,3OHMCA (5 4). ....... 120 General Procedure for Polymerization ............................................................................. 120 Polymerization of ( E ) undeca 1,10 dien 6 -yl 3 (4 -methoxyphenyl)acrylate (54a). ........................................................................................................................... 121 Results and Discussion ............................................................................................................. 121 Conclusions ........................................................................................................................ 131 Future Work ............................................................................................................................... 131 LIST OF REFERENCES ................................................................................................................. 133 BIOGRAPHICAL SKETCH ........................................................................................................... 141

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11 LIST OF TABLES Table page 2 1 Molecular weight and thermal data for unsaturated ethy lene vinyl amine copolymers ... 52 2 2 Thermal data for the protected and deprotected polymers. ................................................. 55 3 1 Molecular weights and therma l behavior of lysine monomers and corresponding polymers. ................................................................................................................................. 75 4 1 Molecular weight and thermal data for polymer prodrugs ................................................ 104

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12 LIST OF FIGURES Figure page 1 1 Telechelics, post polymerization modifications, and attaching a vinyl group as approaches to making biopolymers. ...................................................................................... 18 1 2 Synthesis of 5 -fluorouracil -terminated MeO PEG. ............................................................. 19 1 3 Post polymerization modification by addition of doxorubicin and antibodies. ................. 19 1 4 Radical copolymerizations of vinyl monomers containing NSAIDs and sunscreen chromophores. ........................................................................................................................ 20 1 5 Steps for a radical chain growth addition type polymerization ........................................... 21 1 6 Introduction of chain defects ................................................................................................. 23 1 7 The ADMET polycondensation reaction. ............................................................................. 24 1 8 Olefin metathesis reactions .................................................................................................... 25 1 9 Well defined metathesis catalysts ......................................................................................... 26 1 10 Monomer to precision polymer ............................................................................................. 26 1 11 ADMET Mechanism .............................................................................................................. 28 1 12 Synthesis of 9 spacer alkenyl halides. ................................................................................... 29 1 13 Synt hesis of 18 spacer alkenyl bromide. .............................................................................. 29 1 14 Synthesis of the acid diene. ................................................................................................... 30 1 15 Synthesis of amine diene ....................................................................................................... 31 1 16 Synthesis of 8,8 spacer amine diene. .................................................................................... 31 1 17 Synthesis of amino acid containing monomers with both a TEG spacer and no spacer with the amino a cid branched directly off of the backbone. ............................................... 32 1 18 Langmuir Blodgett isotherms with corresponding Brewster Angle Microscopy (BAM) images. ....................................................................................................................... 33 1 19 Representation of a peptide functionalized surface through a polymer backbone. ............ 34 1 20 General methodology developing a polymer prodrug through ADMET. .......................... 36 1 21 Synthetic design for an improved sunscreen through a precision polymer. ....................... 38

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13 2 1 Initial protection strategy for amine monomers. .................................................................. 51 2 2 Ethylene co -vinyl amine copolymer synthesis. .................................................................... 52 2 3 Progression from monomer to polymer monitored by 1H NMR. ........................................ 54 2 4 Progression of monomer to polymer monitored by 13C NMR. ........................................... 54 2 5 TGA traces for (left) unsaturated protected polymers (right) saturated protected polym ers. ................................................................................................................................. 56 2 6 DSC of ADMET (left) unsaturated protected polymers (right) saturated protected polymers. ................................................................................................................................. 57 2 7 1H NMR of protected and deprotected amine branch every 15th carbon. ........................... 59 2 8 TGA traces of (left) polysat6,6NHBoc and polysat6,6NH2 (right) polysat9,9NHBoc and polysat9,9NH2 polymers ................................................................................................. 59 2 9 TGA traces of polysat9,9NH2 polymers deprotected from heat and acid .......................... 60 2 10 (left) DSC of amine branch every 9th, 15th, and 21st carbon on polymer. (r ight) overlay of 1st heat of saturated protected polymer and 2nd heat of deprotected polymers. ................................................................................................................................. 61 2 11 Ethylene co -vinylTEGamine copolymer synthesis. ............................................................ 62 3 1 Lysine polymers synthesized w/ varied connectivity through a spacer or directionally through its C or N terminus ................................................................................................ 73 3 2 Deprotection mechanism of the Fmoc group using piperidine. .......................................... 77 3 3 DSC second heating scans for the CBz protected monomers. ............................................ 78 3 4 WAXS data for the precipitated CBz prot ected lysine monomers and resultant polymers.. ............................................................................................................................... 80 3 5 DSC second heating scans for the monomer, protected polymer, and deprotected polymer for the Fmoc lysine materials. ................................................................................ 81 4 1 Acid and alcohol diene premonomer synthesis .................................................................... 98 4 2 Synthesis of non-spaced Ibuprofen and Naproxen diene monomers .................................. 99 4 3 Synthesis of decanediol and tetraethylene glycol spaced Ibuprofen and Naproxen monomers ............................................................................................................................. 100 4 4 Polymerization of spaced and non-spaced monomers ....................................................... 101

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14 4 5 1H and 13C NMR of three representative monomers with varying spacers. ..................... 103 4 6 Thermal degradation for polymer prodrugs. The Ibuprofen family is displayed on left and the Naproxen family on the right. .......................................................................... 105 4 7 DSC of polymer produgs. The Ibuprofen family is displayed on left and the Naproxen family on the right. ............................................................................................. 106 4 8 Chemical hydrolysis rate profiles for the Ibuprofen Polymers in pH=2 and pH=8 phosphate buffers. ................................................................................................................ 109 4 9 Synthesis of ACOM and AOCOM spaced Ibuprofen monomers. .................................... 111 4 10 Proposed synthesis of 9,9 TEG Acid to make water -soluble polymers. ........................... 112 5 1 Three s unscreen chromophores used for this study. .......................................................... 122 5 2 Premonomer synthesis. ........................................................................................................ 122 5 3 Synthetic scheme for attaching sunscreens to di ene premonomers. ................................. 124 5 4 UV-Vis absorbance for MCA with respective monomer and polymer. ........................... 126 5 5 UV-Vis absorbance for PABA and i ts respective monomer. ............................................ 126 5 6 Photodimerization of MCA polymers (crosslinking) ......................................................... 127 5 7 Different structural isomers for Avobenz one. .................................................................... 128 5 8 Synthetic attempt to alkylate avobenzone .......................................................................... 129 5 9 Synthesis of avobenzone stabilizer polymer. ..................................................................... 130 5 10 Proposed synthesis of UVA/UVB monomer. ..................................................................... 132

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PRECISION POLYMERS TARGETED FOR BIOLOGICAL APPLICATIONS SYNTHESIZED BY ADMET By James K. Leonard May 2009 Chair: Kenneth B. Wagener Major: Chemistry The incorporation of amino acids, peptides, drugs, and sunscreens into polymers can result in materials with properties that blend the properties of both the small molecule incorporated and the polymer itself. A polymer can improve the ability of a drug to induce its pharmacological action, preven t a sunscreen chromophore from entering the skin, or give a peptide the ability to act as an immobile functional surface. All of these functional groups mentioned are covalently attached to the polymer backbone through a variety of both labile and unreact ive linkages depending on the purpose of the material. In each example of polymer made, the material is built upon a polyethylene backbone. These materials are not readily biodegradable but this gives them the advantage of being biologically nontoxic in addition to being used as functional coatings. Acyclic diene metathesis (ADMET) allows for the synthesis of perfectly linear polyolefins with precisely controlled distribution of branches either along or within the backbone. This approach to polymer synthesis allows absolute control of ethylene run length distribution, functional group identity, and branch concentration giving a pristine polymer microstructure. Materials synthesized in this manner possess properties that can be varied by

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16 changing the fu nctional group incorporated and the number of methylene units between the terminal alkenes; these effects are evident and shown by structural analysis by 1H and 13C nuclear magnetic resonance (NMR) and Fourier Transform Infrared (FT IR) absorbance, in addi tion to thermal analysis using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The research presented in this dissertation contributes to the understanding of structure property relationships and how the systematic study of t hese properties allow for the tuning of polymers for ideal material performance.

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17 CHAPTER 1 INTRODUCTION Synthesis of Biopolymers The combination of synthetic organic chemistry, medicinal chemistry, and biochemistry have allowed for the synthesis and deve lopment of biopolymers that mimic those that occur naturally.1,2 Nature has provided wonderfully complex and functional macromolecules that vary from the famous double helix of deoxyribonucleic acid to the less cele brated structure of cellulose. Proteins, DNA, and RNA work in tandem to synthesize and maintain each other with incredible precision in both chemical structure and tertiary conformation. These biopolymers have been tailored by evolution over a very long timescale to perform specific functions with unique structures that enable them. In contrast, manmade polymers lack this structural control with regards to polydispersity, conformation, and primary structure. New synthetic techniques and catalysts are co ntinually being developed that better control polymer structures. Depending on the type of synthetic macromolecule, the mechanism that is chosen to synthesize the polymer is critical. The principle that synthetic chemists should focus on to develop biomim etic polymers is understanding that primary sequence of the biopolymer predisposes the secondary structure that in turn leads to defined macromolecular architecture, functional group placement, and the desired morphological structure.1 The biopolymers presented in this document will include those materials that contain amino acids or peptides, drug molecules, amino groups, and sunscre ens. This introductory chapter will focus on some of the common techniques and mechanisms chemists use to make such biopolymers. Techniques for Biopolymer Synthesis The literature is rich with examples of incorporating biologically active substituents suc h as drugs, sunscreens, or peptides into polymers.3,4 Scientists from many areas of science

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18 approach the development of these materials differently. Engineers take a top -down approach and take existing systems and try to understand how to make them perform as desired. Chemists often make fundamental changes to polymer structure to determine the effects on properties relative to other synthetic polymers. Biochemists often have the best resources to add complex subs tituents to a polymer and then look for specific biological responses. The more interaction and collaboration there is between these three fields, the better this research area will grow outside of the individual fundamental ways of thinking about these s ystems. Figure 1 1. Telechelics, post polymerization modifications, and attaching a vinyl group as approaches to making biopolymers. Other than physically mixed or absorbed approaches,46 the three most common techniques used to covalently link biounits onto a polymer backbone are displayed in Figure 1 1. The first option comes from linking the desired biological unit to the polymers chain ends only; these molecules are called telechelic polymers. Telechelic p olymers are often comprised of a well characterized polymer backbone but lack a significant amount of drug or functionality at the polymer chain ends. This lack of functionality in addition to low drug percent weight is often a drawback of such polymers. Figure 1 2 shows an example of a telechelic polymer produced by Yuyama7 with plenty more examples discussed in the review by Zalipsky.8

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19 Figure 1 2. Synthesis of 5 -fluorouracil terminated MeO -PEG. The synthesis of a polymer with reactive groups along the polymer backbone allow for the post polymerization modification by the addition of bioactive units after the polymer is made. This technique is o ften used when complicated or reactive groups could not withstand the polymerization conditions. The review by Khandare and Minko9 discuss the selective attachment of doxorubicin, antibodies, and enzymes to polyethylene glycol grafted, N (2 hydroxypropyl)methacrylamide (HMPA) and poly(lact ide co -glycolide) (PLGA) copolymers. The specific synthetic approach of Pechar and Ulbrich is shown in Figure 1 -3 as taken from their research.10 Figure 1 3. Post polymerizatio n modification by addition of doxorubicin and antibodies.

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20 Perhaps the most common and straightforward manner to synthesize a biopolymer is to attach a vinyl group to the desired substituent that may induce some sort of biological response. By attaching a vinyl group through a myriad of possible linkers such as esters, amides, carbonates, and carbamates, one can synthesize high molecular weight polymer by ATRP, RAFT, or radicals through chain growth addition type polymerizations. Two examples are shown in Figure 1 4 of attaching a vinyl group to (a) non-steroidal anti inflammatory drugs11 and (b) sunscreen chromophores.12 Figure 1 4. Radical copolymerizations of vinyl monomers containing NSAIDs and sunscreen chromophores. The radical polymerization mechanism of such vinyl polymerizations is congruous across all polymer chemistry and is carried out in three distinct steps: initiation, propagation, and termination These three steps are shown in Figure 1 5 for a styrene polymerization.

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21 Figure 1 5. Steps for a radical chain growth addition type polymerization Initiation begins with either the thermal or photolytic homolytic bond cleavage of a peroxy or azobis in itiator species. In the azobis species, nitrogen gas is evolved and in both cases one initiator molecule generates two equivalents of radical initiator that then has the ability to add to two monomer units. This initiated monomer completes the initiation step of the polymerization mechanism. It is important to note that the reactive center on the growing chain is uncharged but lacks its full valence octet.

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22 Once the monomer undergoes initiation, the radical propagates itself by adding monomer one unit a t a time. This reaction transfers the radical down the propagating backbone. The driving force of a vinyl polymerization is the thermodynamic stability of an alkane to an alkene. Assuming a perfect head to tail addition of monomer during the propagation phase of this reaction, the resultant polymers backbone structure can be denoted with brackets around the styrenyl repeat unit. Termination can happen in a variety of ways but the most common ways are coupling and disproportionation reactions. Coupling quenches two propagating chains with each other and disproportionation occurs when a growing chain extracts a hydrogen atom from the carbon of another growing chain. This mechanism results in a terminal alkene on the hydrogen donating chain and a satur ated methyl group on the chain that extracted the hydrogen atom. Radicals also have the ability to quench themselves with chain transfer reactions with the flask, solvent, initiator, and polymer. One very important characteristic of radical polymerization s is the introduction of chain defects into the polymers structure. For a vinyl polymerization to yield a perfectly known microstructure the addition of monomer needs to always be head to tail and the termination mechanism can only arise form disproporti onation or coupling. Figure 1 6 demonstrates the effect of head to head or tail to tail additions of monomer, the resulting polymer backbone is distorted from the polymer denoted from any single set of repeat unit brackets. Another type of chain defect t hat can be introduced to the polymer backbone is by backbiting or transfer to polymer. In this case the propagating chain transfers the radical to either itself or another polymer through hydrogen abstraction. This transfer of radicals to other positions along a polymer backbone results in branching that can dramatically change the polymers structure and

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23 as a consequence the final applicable use. Low density polyethylene (LDPE) is a textbook example of a polyolefin produced by this method. Figure 1 6 Introduction of chain d efects Nonuniform addition of monomer and backbiting. It is often the researchers intention to vary the amount of the drug or bioactive unit in the polymer. For a vinyl polymerization, this is often accomplished by loading a s eparate vinyl monomer into the polymerization with the bio -monomer. Although this technique does work in reducing the weight percent of the biological unit desired in the polymer, it also has its negative effects on polymer structure as well. The dispari ty in reactivity ratios between two different vinyl monomers is often significant so that the corresponding polymer is often block like due to the fact that once a particular monomer is initiated it will only react with more of the same monomer. Once this monomer reacts with monomer B, one would then expect monomer B to then react with itself preferentially due to the reactivity ratio discrepancy of the two monomers. It would be wrong to assume that two vinyl monomers would react with each other randomly if the appropriate care were not given to matching the reactivity of the two monomers. The polymers in Figure 1 4 would be expected to have a multitude of chain defects including main chain branching and blocks of the two monomers.

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24 Metathesis Approach In order to circumvent this reactivity discre pancy and to minimize th e side reactions that introduce chain defects into the polymer structure, the polymers discussed in this dissertation are made via acyclic diene metathesis (ADMET). This technique is a step growth condensation type polymerization that is driven to comple tion by the removal of ethylene Figure 1-7. Figure 1-7. The ADMET polycondensation reaction. The advancements in ADMET reflect the mechanistic understanding and advancements in olefin metathesis chemistry.13 This mild carbon-carbon double bond scrambling reaction was first discovered by accident in th e 1960s at Goodyear when resear chers exposed alpha olefins to a combination of tungsten hexachloride and a le wis acid in hopes of developing a new catalyst for the polymerization of vinyl olefins.14,15 Instead of producing high polymer, a complex mixture of scrambled olefin products was observed. The mechanism of metathesis, first proposed by Chauvin16 in 1971 and later confirmed by Katz17 in 1975 led to the development of metal carbene catalysts that invol ve the 2+2 cyclo addition of an olefin to the metal carbene to form a metallocyclobutane, followed by a 2+2 cyclo reversion to yield a ne w olefin and a metal carbene. This metathesis reaction earned the 2005 Nobel Prize in Chemistry for Robert H. Grubbs, Richard R. Schrock, and Yves Chauvin for their collective effort in the field.18 The development of these metal carbene catalysts to function more effi ciently with a greater variety of functional groups has been established as an indispensable tool to the synthetic organic chemist: Figure 1-8.

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25 Figure 1-8. Olefin metathesis reactions Numerous reviews have been published examini ng the recent synthetic advances in olefin metathesis.19,20 In addition there are reviews that in clude acyclic diene metathesis (ADMET),21,22 ring-opening metathesis polymerization (ROMP),23 small molecule chemistry using ring-closing metathesis (RCM),24,25 cross metathesis (CM),26,27 and ring-opening metathesis (ROM) reacions.28 The primary purpose of this research is the synthesis of ethylene copolymers with a focus on the morphological consequences of ethyle ne run length distributi ons and the type of branch. Branch frequency effects are show n by spectroscopy and differential scanning calorimetry. Over the past decade, the Wagener group had dedicated its research efforts to the synthesis of a variety of functionalized polymers using ADMET. Symmetr ical diene monomers and their respective polymers with varieties of alkyl,29-31 halide,32-36 alkoxy silanes,37 amino acid,38-40 carboxylic acid,41 amine, drugs, and sunscreen branch es have all been synthesized and

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26 characterized. There are a variety of metathesis catalysts at the disposal of this polymerization technique: Figure 1-9. Implementing the ADM ET reaction with these catalysts under many different varieties of functional branches perm its the development of the olefin metathesis application to create new functional polymers. Figure 1-9. Well defined metathesis catalysts Perhaps the biggest advantage of this polymerization technique is the built in precision of the polymer obtained from the monomer design. The monomers are synthesized possessing the branch of interest within a symmetrical di ene monomer and this monomer symmetry is transferred to the polymer repeat unit. This symmetry in the polymers primary structure is conveyed to higher orders of polymer struct ure, ultimately changing conventional morphology Figure 1-10. Figure 1-10. Monomer to precision polymer The mechanism of ADMET is similar to all olefin metathesis reactions, but exploiting the equilibrium involved to afford high molecular weight product is key to this methodology. Figure

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27 1 11 displays the ADMET mechanism illustrating the need to remove the ethylene condensate, thereby driving the equilibrium reaction to high molecular weight polymer. The a ctive catalyst species, the free methylidene, is first generated after the initial metathesis cycle. The benzilidene catalyst is able to react with the monomer upon disassociation of the tricyclohexylphosphine catalyst. Upon monomer addition the reaction releases styrene while producing a new metal alkylidine species. Upon reaction with another monomer unit and the subsequent formation of the metallocyclobutane, retro 2+2 addition releases the methylidene catalyst and one dimer molecule. The newly creat ed methylidene then reacts with another monomer, releasing ethylene and reforming the metal alkylidene. This cycle continues as polymer is created through a stepwise addition of monomer to dimer, trimer, etc. This type of equilibrium step growth condensa tion type polymerization is typical of all step growth processes yielding polydispersities near 2.0 in addition to requiring monomer conversions of over 95% to achieve high molecular weight products.42 The applicability of metathesis catalysts to perform ADMET depends on monomer purity, catalyst lifetime, and the functional groups incorporated into the monomer. In contrast with the high oxidation state metal metathesis catalysts such as Schrocks cat alyst,21 ruthenium catalysts are used for the myriad of functional polymers used in this document.

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28 Figure 1 11. ADMET Mechanism Synthetic Background of Diene Premonomers The Wagener groups ability to make precisely branched polyethylene hinges on the availability and synthetic capacity to obta -olefins for the synthesis -dienes. Isomerization of the monomers terminal alkenes is not tolerated because even minimal amounts lead to inexact spacing of the functional group within the polymer. Since one of ADMETs greatest features is precise structure, large and high yielding -olefins has been developed in the group.43 The reduction of zinc undecylenate and its subsequent halogenation is a two step transformation from a readily available starting material: Figure 1 12. Bromination of the alcohol ( 1 -1 ) is accomplished with carbon tetrabromide and triphenyl phosphine while chlorination is performed with thionyl chloride. Both alkenyl halides can be purified via

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29 columns and vacuum distillation. A strong vacuum will reduce distillation temperatures that often are the leading cause of isomerizati on. O O 8 2 Z n2+ L i A l H4T H F O H 9 C B r4, P P h3S O C l2 B r 9 C l 9 11 12 13 Figure 1 12. Synthesis of 9 spacer alkenyl halides. The coupling of shorter alkenyl halides with subsequent hydrogenation and elimination is a straightforward process for synthesizing longer alkenyl halides but isnt as efficient as the two step re duction/halogenation reactions. Figure 1 13 displays the self cross metathesis of 1 -2 with Grubbs first generation catalyst to afford 1 -4 Exhaustive hydrogenation of the backbone alkene is done in a Parr bomb with Wilkinsons Rh catalyst under hydrogen pressure. The saturated 20-carbon dibromide 1 -5 -olefin 1 -6 using a potassium t -butoxide elimination in a tetrahydrofuran/toluene mixture. Compound 1 -6 can be purified from the 20 -diene and 1 -5 with column chromatography. B r 9 B r B r 9 9 R u (P P h3)2Cl2H2, t o l u e n eB r B r 1 8 1s t G e n G ru b b s t o l u e n e T H F t o l u e n e B r 1 8 1 -4 1 5 1 6 K O t Bu Figure 1 13. Synthesis of 18 spacer alkenyl b romide. All of the functional monomers in this study are synthesized by attaching the drug, amino acid, or sunscreen to the ethylene backbone through either an amine or carboxylic acid as initially developed by Hopkins38 40,4447 or an alcohol as demonstrated by Baughman.48 These functional requirements necessitate the development of symmetric dienes with a functional handle Compounds 1 -7, 1 -8, and 1 -9 have a carboxylic acid group symmetrically placed between the terminal alkenes Figure 1 14. The synthesis of these acids is accomplished via two

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30 consecutive Sn2 reactions between the deprotonated diethyl malonate and the cor responding alkenyl bromide or alkenyl tosylate to form the diester. This alkylation reaction is monitored closely by TLC and it is common that excess NaH and alkenyl bromide is added to optimize the malonate di -coupling. The diester is then saponified wi th KOH in ethanol and water. The product diacid mixture is concentrated and then mixed with a minimal amount of Decalin along with a catalytic amount of DMAP. The Decalin mixture is heated to 190 and vigorous bubbling is observed representing the intram olecular decarboxylation reaction. The large difference in polarity between product and Decalin results in easy separation of the two on a pad of silica. It should be noted that additional purification after saponification is required for the synthesis o f the 18,18 acid diene. Recrystallization combined with column chromatography allows for suitable purification of this set of materials. B r n D i e t hyl M a l ona t e T H F N a H n n O E t O O E t O E t ha nol N a O H n n O H O O H O D e c a l i n O O H n n 2 1 -7 ) n = 3 1 -8 ) n = 9 1 -9 ) n = 1 8 Figure 1 14. Synthesis of the acid diene. The amine branched premonomer used for coupling functionality to the die ne monomer is prepared through one of two possible methodologies. The first method of synthesizing amine dienes consists of the three steps shown in Figure 1 15. The diene alcohol is made by the Grignard reaction with the appropriate alkenyl halide with ethyl formate. The alcohol is oxidized to the ketone with pyridinium chlorochromate (PCC) and then reductively aminated in methanol with ammonium acetate and sodium cyanoborohydride to yield amine diene. These premonomer amines are purified with column c hromatography but have been found to quickly

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31 oxidized if exposed to the atmosphere. It is best to use the near -colorless oil immediately upon production and if needed, should be stored in a dark place under vacuum. C l n 2 E t hyl F or m a t e O H O E t O H n n P C C O n n N H2 n n N H4O A c N a C N B H3 M g, e t he r1 -1 0 ) n = 3 1 -1 1 ) n = 9 1 -1 2 ) n = 1 8 Figure 1 15. Synthesis of amine diene Another option of producing a symmetrical amine diene is shown is Figure 116. Compound 1 -13 is synthesized in two steps with excellent yields using Zantours method.39 The starting material, 10 undecenoyl chloride, is reacted with 1.8 equivalents of triethylamine to form a ketene. The ketenes then couple to form the i lactone intermediate in a sodium hydroxide solution opens the lactone, allowing decarboxylation to occur which in turn forms the symmetric ketone. The ketone is easily purified with recrystallization in acetone and is reductively aminated using the same methodology as used with the other amine monomers. O C l 2 C O O O O O O O N H2 N H4 +( O A c )-N a C N B H3M e O H 4A S i e ve s 8 8 8 7 7 7 7 7 8 8 2 T E A N a N a O H H2O C O2 1 -1 3 Figure 1 16. Synthesis of 8,8 spacer amine diene. Amino Acid Containing Materials Amino acid containing polymers are a subject of considerable research effort sinc e these materials lend themselves to applications such as drug delivery agents, chiral recognition

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32 stationary phases, asymmetric catalysts, metal ion absorbents, and biocompatible materials.49,50 The ADMET method ology has been proven a useful tool in modeling structure property relationships in bio -olefins and developing new functionalized materials.5154 Amino acids/peptides can be incorporated into a polyethylene polymer via ADMET with two methodologies. The first e ntails the incorporation of the amino acid/peptides directly into the polymer backbone while the second method is branching the amino acid/peptide moiety off of the backbone. The polymers possessing amino acid units in the backbone will likely be biodegra dable,44 while the polymers with the amino acid units pendant to the main chain will be hydrolytically stable. This leads to the synthesis of semicrystalline, chiral polyme rs having considerable tensile strength at moderate molecular weights. Figure 1 17. Synthesis of amino acid containing monomers with both a TEG spacer and no spacer with the amino acid branched directly off of the backbone.

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33 With the recently acquired ab ility to deprotect amine functionality in these polymers,55 work in this project is now focused on synthesizing materials that can be proven to induce biological activity. Figure 1 17 shows the t wo variables that we are interested in controlling for these materials : the choice of peptide attached and the type of linkage between this peptide and the polyethyle ne backbone. A hydrophilic tetra ethylene glycol (TEG) spacer is presented here as a possible alternative to previously published work with the amino acid connected directly to the ethylene backbone. 3840 Figure 1 18. Langmuir Blodgett isotherms with corresponding Brewster Angle Microscopy (BAM) images. The se protected monomers were polym erized with second generation Grubbs catalyst to afford polymers with number average molecular weights ranging from 15,000 to 40,000 Da relative to polystyrene. The Fmoc protected amino acid polymers, both non -spaced and spaced, were quantitatively deprot ected by adding piperidine to a solution of THF and polymer. The polymers were characterized with NMR and IR to confirm complete de protection of the amine. The protected polymers in Figure 1 17 were tested for their thermal behavior and their abilities

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34 t o cast films both through Langmuir Blodgett (Figure 1 18) and spin casting techniques. Contact angles for both the protected a nd deprotected polymer films were also measured and compared. ADMET has proven to be effective in synthesizing polymers containi ng amino acids connected though a TEG spacer or directly to the polyethylene backbone. The material properties of the spaced versus non-spaced polymers are significantly different in solubility, mechanical strength, and film forming ability. One example o f such a difference in the Fmoc protected glycine polymers is in the nonTEG spaced monomer, only insoluble oligomers are formed with ADMET with this monomer. The same glycine monomer with a TEG spacer incorporated forms a polymer with a Mn of 20,000. Th is TEG spaced polymer along with every other polymer with a TEG spacer is readily dissolved in numerous solvents and doesnt precipitate out of solution upon polymerization. The best option to use in making a film where nearly all the peptide branches are pointing out away from the surface is a Langmuir Blodgett trough where one transfers a monolayer to a silanized glass slide. Depending on the type of application, these materials can be modified to suit ones needs. Further development in the chemistry associated with larger peptides will be crucial in demonstrating optimal biological activity. Figure 1 19. Representation of a peptide functionalized surface through a polymer backbone.

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35 Polyethylene Prodrug s The combination of synthetic organic polymer chemistry, medicinal chemistry, and biochemistry have allowed for the engineering of polymer based therapeutics designed for controlled degradation along with specific cellular uptake and response. A polymer prodrug is a low molecular weight pharmaceutical immobilized on a polymeric carrier that in itself has no biological activity, thus any therapeutic effect.56,57 Polymer prodrugs require transformation of itself to the active drug in order to elicit the desired therapeutic action. One of the most important characteristics of a polymer prodrug is the stability of the drug-polymer linkage.58,59 Drugs and polymers can be conjugated using a variety of different functional chemical linkages: esters, carbonates, carbamates, methoxy esters or amides. Our research in this area has been focused on the synthesis of drug branched polyethylene using ADMET. This method has been proven to be a useful synthetic technique in modeling structure -property relat ionships in linear low -density polyethylene (LLPE),52,54,60,61 in addition to developing new functionalized materials such as bio -olefins.47 The three variables that we are most interested in controlling for these materials are the choice of drug, the frequency at which this pharmaceutical is attached, and lastly the type of spacer and its linkage between the drug and polyethylene backbone. Figure 1 20 shows the general outline of how polyethylene can be synthesized and used as a film whose partial degradation generates the active drug molecule.

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36 Figure 1 20. G eneral methodology developing a polymer prodrug through ADMET. The non -steroidal anti -inflammatory drugs (NSAID) Ibuprofen and Naproxen were involves the drug atta ched directly off of t he backbone the second uses a hydrophilic tetraethylene glycol spacer, and lastly a hydrophobic decanediol spacer is implemented to connect the two. Each of these three cases presents a different environment for the drug and its cleavable ester group, which will yield considerably different rates of drug release through hydrolysis. Two different types of hydrolysis, enzymatic and chemical, are studied here since both are in competition with each other in the body. The amount of dru g loaded on the polymer can be varied through monomer design. For high drug loading one can use smaller spaced diene monomers such as the 3,3 or 6,6 dienes, while lower loading can be achieved with the 9,9 diene monomer. These materials are most likely to be used as biocoatings due to their minimal water solubility. Once the drug is cleaved from the polyethylene backbone and imparts it therapeutic

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37 effect, the polyethylene would remain as a nontoxic coating on the implant. The TEG or decanediol spacers that would also be cleaved from the film are relatively non -toxic and can be easily eliminated from the body. accomplished as p reviously mentioned here The alcohol br anched diene was coupled directly to the NSAIDs with carbodiimide coupling chemistry. The prodrug monomers were polymerized with second generation Grubbs catalyst to afford polymers with number average molecular weights ranging from 15,000 to 40,000 Da re lative to polystyrene. The polymer prodrugs, both non-spaced and spaced were fully characterized with NMR, DSC and IR to determine structure and their thermal behavior. Each material was then subjected to enzymatic and chemical hydrolysis to determine ra te of drug release under both sets of conditions. The amount of cleaved drug versus time was determined over a 48 hr period, monitored by HPLC at 1 hr increments. Roughly 40% of the total drug was released from these polymers, which corresponds to other papers published with NSAIDs linked through ester bonds.62,63 ADMET has proven to be effective in synthesizing polymers containing drug molecules connected through a variety of spacers or directly to the polyethylen e backbone. The material properties and hydrolytic stability of the spaced versus non -spaced polymers are significantly different. Depending on the type of pharmaceutical response desired and the amount of drug required to be loaded onto the polymer, thes e materials can be modified to suit ones needs. Further development in the chemistry associated with hydrolysis and more chemically complex drugs will be crucial in demonstrating optimal pharmaceutical potential. Sunscreen Polymers Skin cancer has becom e one of the leading forms of cancer in the United States with nearly 1.21.5 million newly diagnosed cases annually. As a consequence the need for better UV

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38 protection has made the sun care market the fast -growing personal care sector with over 5 billion dollars in global sales in 2005. As the demand for more convenient and effective products rise, sunscreen producers have to address such factors such as higher SPF values, better water resistance while maintaining film integrity, and resistance to rubbin g off. One factor commonly overlooked with sunscreens is the safety of the chromophore itself. It had recently come to the attention of researchers that many common sunscreens have the ability to absorb into the skin and create cell damage much like the UV damage itself was intended to alleviate.64 67 Recent papers have published work considering the possibility of immobilizing a sunscreen chromophore onto a macromolecule.12 The considerable molecular weight of a polymer chain containing covalently attached sunscreens would prevent it from passing into the skin to cause any possible damage: Figure 1 21. Our research group has begun work on taking commonly used sunscreen molecules such as para aminobenzoic acid, para-methoxycinnamic acid, and avobenzone and attaching them to diene monomers that can then be converted to polymers through ADMET. The absorbance of the sunscreen remains intact through this chemistry and the resultant polymer offers the same UV absorbance as the initial sunscreen. Figure 1 21. Synthetic design for an improved sunscreen through a precision polymer.

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39 Dissertation Purpose This dissertation describes the incorporation of bioactive units onto a polyethylene backbone with both covalent and precise attachment. The appli cability of these materials for bio applications is touched upon but the synthesis of new materials with this potential is the focus. Chapter 2 discusses ethylene co -vinyl amine polymers and their potential for drug delivery. Polyethylene containing amino acids and peptides is discussed in Chapter 3 with respect to the crystallinity of the monomers and their corresponding polymers. Chapter 4 discusses the development of new polyethylene prodrugs with the ability to be tuned to suit ones pharmacological needs. Lastly, the synthetic incorporation of sunscreens onto a polymer backbone while maintaining their regular absorbance values is detailed in Chapter 5. NMR, IR, DSC, TGA, and GPC are used on all polymers synthesized for analysis to confirm that the s tructure of these materials mimic those previously made in the Wagener Group but with the potential for applications never before targeted in this group.

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40 CHAPTER 2 SYNTHESIS AND THERMAL CHARCTERIZATION OF PRECISION ETHYLENE -VINYL AMINE COPOLYMERS Introduction Precise ethylene co -vinyl amine (EVAm) polymers have never been made. The copolymerization of ethylene with vinyl amine type monomers is a difficult task due to both the large reactivity ratio disparity of the two vinyl monomers and the ability of t he vinyl amine to act as an efficient chain -transfer agent during radical and cationic polymerization. Several properties make EVAm interesting for polymer chemists: the high reactivity of the polymers primary amine functionality in derivatization and cr osslinking, the cationic charge density of the ammonium ion makes it a candidate for ionomer applications,68 and the ability for amines to form chelating complexes with various metal ions,69,70 and support for enzymes.71 The synthesis of pol yvinylamine (PVAm) and its copolymers have been a challenge for generations. The simplest precursor monomer to synthesize PVAm with, vinyl amine, doesnt exist in the free state because the monomer tautomerizes to the acetaldehyde imine. This monomer lab ility necessitates the synthesis of PVAm indirectly from a precursor material; a similar synthetic approach is used with polyvinylalcohol (PVA).72 Although PVAm has been synthesized in numerous ways,7375 the main two precursors used to make PVAm via radical chemistry with minimal structural defects and reasonably high molecular weights st em from the tert -butyl N -vinylcarbamate (TBNVC)76 and vinylformamide (VFA) monomers.77,78 BASF is the lone commercial producer of PVAm and it is synthesized from vinylformamide (VFA) and its subsequent hydrolys is to PVAm. It has been discovered and recognized that copolymers of ethylene and vinylamine are of particular interest, especially at ethylene:vinylamine molar ratios of at least 1:1. Such materials are ideal to be used as flocculants for water clarifica tion. The molar ratio of these copolymers is

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41 modified by monomer addition under high-pressure reactors via radical chemistry. The ideal vinylamine ratio in these materials is 2:1 to 4:1 because within this range the desired physical and chemical properti es imparted by the amine units are preserved while the cost of the polymers is markedly lowered by the presence of the more economical ethylene units.79 It our groups desire to make perfectly modeled polymers by avoiding the random nature of addition in addition polymerizations combined with bypassing unwanted side reactions often seen in radical polymerizations of viny l monomers. Accomplishing this has been possible by taking advantage of a clean, step polymerization chemistry offered by acyclic diene metathesis (ADMET). This mild chemistry avoids the defects usually imparted by catalysts during chain propagation proc esses. These defects, in either small or large quantities, can have a profound effect on the macromolecules material behavior in addition to its thermal response. Defects encountered with these PVAm materials, other than the usual backbone defects elicit ed by the radical chain polymerization of these materials, comes from both the acid and basic hydrolysis of the poly(N vinylformamide). Acid hydrolysis is unable to surpass the 80% level due to mutual repelling of positive charges; it is interesting to po int out that this mechanism can proceed via transiently formed amidine rings. Basic hydrolysis can be carried out completely, but Spange and Bortel have both discovered the elimination of ammonia as noted by elemental analysis and the perceptible smell du ring the hydrolysis.77,78 The utility of metathesis chemistr y lends itself to the challenge of synthesizing polymers with a wide range of functionality. One such example, ring opening metathesis polymerization (ROMP), has been used to make amine -functionalized polymers.80,81 Herein, we report the synthesis and thermal characterization of four linear EVAm copolymers. These materials have the amine branches precisely spaced along the polyethylene backbone at intervals of every 9th,

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42 15th, 19th, and 21st carbon. This paper reports the preparation of precise, amine branched polyolefins that resemble a modified precise copolymer of vinyl amine and ethylene. Their preparation has been accomplished using ADMET chemistry, which assures that the branches are set at specific, not ran dom, intervals along the backbone, generating polymers incapable of being made by other methodologies.36,39,43,82 84 Primary structural analysis of all polymers is discussed by using NMR and FT IR techniques. Detai led calorimetry data is presented to demonstrate the morphological differences between the regular distributions of amine branches combined with branch frequency. Experimental Materials Reagents and chemicals were used as received from Aldrich chemical c ompany unless otherwise noted. Diethyl ether and THF were used as dry solvents from the Aldrich keg system and dried ove r 4 bis(2,4,6 trimethylphenyl) 4,5 dihydroimidazol 2 ylidene] [benzylidene]ruthenium (IV) dichloride) was exclusively used and synthesized as previously described by Grubbs et al.85 Instrumentation and Analysis 1H NMR and 13C NMR spectra were recorded on a Varian Associates Mercury 300 spectrometer. Chemical shifts for 1H and 13C NMR were referenced to residual signals from CDCl3 (1H = 7.24 ppm and 13C = 77.23 ppm) with 0. 03% v/v TMS as an internal reference. High resolution mass spectra (HRMS) data were obtained on a Finnegan 4500 gas chromatograph/mass spectrometer using the chemical ionization mode. Gel permeation chromatography (GPC) of polymers was performed at 40C using a Waters Associates GPCV2000 liquid chromatography system with an internal differential index detector (DRI) and two Waters Styragel HR

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43 HPLC grade tetrahydrofuran as the mobile phase at a flow rate of 1.0 mL/min. Injection 0.07 w/v sample concentration. Retention times were calibrated against a minimum of nine narrow molecular weight polystyrene standards purchased from Polymer Laboratories (Amherst, MA). Diffe rential scanning calorimetry (DSC) was performed on a TA Instruments Q1000 equipped with a liquid nitrogen cooling accessory calibrated using sapphire and high purity indium metal. All samples were prepared in hermetically sealed pans (4 7 mg/sample) and w ere referenced to an empty pan. A scan rate of 10 C per minute was used. Melting temperatures were taken as the peak of the melting transition, glass transition temperatures as the mid point of a step change in heat capacity. Thermal experiments were con ducted as follows: samples were heated through the melt to erase thermal history, followed by cooling at 10 C per minute to 150 C, and then heated through the melt at 10 C per minute. Data reported reflects this second heating scan. Premonomer Amine Di ene Synthesis Magnesium ( 4.89 g, 0.20 mol ) was added to a 500 mL three neck flask equipped with a reflux c ondenser and an addition funnel. T he reaction vessel was backfilled three times with Ar and flamed dried after each backfill. Dry THF (100 mL) was a dded, followed by the addition of 5 bromo1 pentene (25.0 g, 0.17 mol ) via dropwise addition by syringe. The solution was refluxed for 2 h to completely form the Grignard. Ethyl formate ( 5.64 g, 0.076 mol ) in 30 mL THF was added dropwise to the cooled mi xture (0 C), and the solution was allowed to warm slowly to room temperature and refluxed for 21 h r. Hydrochloric acid (1M, 100 mL) was added, and the solution was extracted with ether (3 x 25 mL), washed with 1M HCl (1 x 30 mL), and washed with brine (3 x 20 mL). The solution was dried over MgSO4, followed by evaporation of the solvent to yield 14.18 g of the crude alcohol.

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44 To a 500 mL round -bottom flask equipped with an addition funnel was added pyridinium chlorochromate (PCC) (26.0 g, 0.12 mol ), cel ite ( equal weight to crude alcohol ), and methylene chloride (100 mL) followed by the addition of the crude alcohol ( 1 equiv. ) The reaction was stirred for 4 h r diethyl ether (200 mL) was added and the mixture was filtered t hrough a pad of silica gel. S olvent evaporation yielded 13.0 g of the crude ketone. To a 500 mL round -bottom fl ask was added the crude ketone dry methanol (225 mL), ammonium acetate ( 60g, 0.78 mol ), NaCNBH3 (25g, 0.40 mol ), and a spatula tip of crushed 4 molecular sieves and the mi xture was refluxed for 48 hr under N2. The crushed molecular sieves were filtered via Bchner filtration and deionized water (200 mL) was added to the filtrate, followed by extraction with diethyl ether (3 x 50 mL). The organic layer was washed with 1 M NaOH (2 x 50 mL) and brine (2 x 30 mL) and dried over MgSO4. The solution was concentrated to a brown viscous oil, which was purified by flash column chromatography using a 3:1:1 (hexane:ethyl acetate:methanol) mobile phase yielding 9.28 g of the desired 3,3NH2 product for an overall yield of 73 %. 1 -U ndec -10-enyl dodec -11-enylamine (3,3NH2). 1H NMR (300 MHz, CDCl3 1.70 (m, 8H), 2.01 2.15 (br,4H), 2.652.76 (br, 1H), 4.905.10 (m, 4H), 5.75 5.90 (m, 2H). 13C NMR (75 MHz, CDCl3 37.84, 51.45, 114.95, 139.19. Tricosa -1,22-dien-12-amine (9,9NH2). The 9,9NH2 was synthesized as described above using the 11 -bromo 1 undecene (25.0 g, 0.106 mol) instead of the 5 -bromo 1 -pentene. After purification a final yield of 48% (13.0 g) was obta ined. 1H NMR (300 MHz, CDCl3 1.45 (br, 32H), 2.05 (q, 4H), 2.652.75 (br, 1H), 4.885.05 (m, 4H), 5.725.38 (m, 2H). 13C NMR (75 MHz, CDCl3 29.76, 29.84, 29.90, 30.07, 34.08, 38.02, 51.50, 114.36, 139.50.

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45 1 -D e c -9 -enyl -undec -10-enylamine (8,8NH2). S ynthesis was performed using a modified procedure by Zantour and co-workers.39 To a 500 mL three neck roundbottom flask equipped with a reflux condenser and an addition funnel was added 10 undecenoyl chloride (20.27 g, 100 mmol) and dry diethyl ether (150 mL). The solution was cooled to 0 C, and triethylamine (18.21 g, 180 mmol) was added dropwise, instantly forming white triethylamonium chloride salts. The reaction mixture was warmed to room temperature and stirred for 24 h r, followed by Bchner filtra tion of the salts and evaporation to yield the liquid intermediate -lactone. Deionized water (100 mL) and NaOH (8.80 g, 2.10 mol) were added, and the mixture was refluxed for 12 h r. The solution was acidified with 1 M HCl and extracted with diethyl ethe r (3 x 50 mL). T he combined organic layers were washed with 1 M HCl (2 x 20 mL), and brine (2 x 20 mL). After drying over MgSO4 and recrystallizing from MeOH, 13.15 g of the pure ketone was obtained. The ketone was converted to the amine using the same methodology as described with the 3,3NH2 and 9,9NH2 synthesis. The overall yield for the two steps was 58%. 1H NMR (300 MHz, CDCl3 1.181.62 (br, 28H), 2.04 (q, 4H), 2.80 2.94 (m, br, 1H), 4.034.54 (br, 2H), 4.885.07 (m, 4H), 5.715.91 (m, 2H). 13C NMR (75 MHz, CDCl3 34.21, 38.18, 41.61, 114.46, 139.50. (9H -fluoren -9 -yl)methyl henicosa-1,20-dien-11-ylcarbamate (9,9NHFmoc). To a dry 500 mL round bottom flask was added 150 mL dry THF, 50 mL dry pyridine, and Fmoc Cl (1.0 g, 3.90 mmol) under argon. 9,9NH2 (1 g, 3.25 mmol) was slowly added over .5 hr and the reaction was allowed to stir at room temperature for an additional 2 hr. After 2 hr, 100 mL of ether was added to the reaction and it was extracted wi th 1M HCl (2 x 50 mL) and brine (2 x 50 mL). The protected amine solution was dried over MgSO4 followed by rotary evaoporation to yield the 9,9NHFmoc product. The 9,9NHFmoc was purified via column

PAGE 46

46 chromatography using ethyl acetate:hexane (3:2). 1H NMR (300 MHz, CDCl3 7.61 (d, 2H), 7.40 (t, 2H), 7.28 (t, 2H), 5.82 (m, 2H), 4.94 (m, 4H), 4.40 (d, 2H), 4.23 (t, 1H), 2.04 (m, 4H), 1.151.45 (br, 28H). EI/HRMS [M + 1]: Calcd. for C36H51NO2: 530.3998. Found: 530.4006. General Boc Protec tion of the Amine Dienes To a dry 500 mL round bottom flask was charged 150 mL dry THF and the appropriate amine (2.5 g) under argon. A syringe was used to add the Boc anhydride (1M in THF, 1 equiv.) over 15 min at room temperature. The reaction was allo wed to stir for 24 hr and was monitored by TLC (ethyl acetate: hexane, 1:19) for disappearance of the starting material amine. At the end of the 24 hr, 100 mL of ether was added and the solution was extracted with water (1 x 50 mL), NaHCO3 (2 x 50 mL), and brine (2 x 50 mL). The washed solution was dried over MgSO4 followed by rotary evaporation to yield Boc protected product. Monomer was purified via column chromatography using ethyl acetate:hexane (1:19). T ert -butyl undeca -1,10-dien -6 -ylcarbamate 3,3NH Boc (2 -1). 1H NMR (300 MHz, CDCl3 1.46 (br, m, 8H), 1.48 (s, 9H), 2.01 (m, 4H), 3.52 (br, 1H), 4.22 (br, d, 1H), 4.93 (m, 4H), 5.76 (m, 2H). 13C NMR (75 MHz, CDCl3 28.62, 33.79, 35.25, 50.57, 79.00, 114.78, 138.83, 155.89. FT IR (KBr pellet): 3348, 3077, 2978, 2934, 2860, 1814, 1692, 1641, 1522, 1457, 1443, 1416, 1391, 1366, 1284, 1249, 1174, 1120, 1056, 1026, 944, 910, 868, 773, 637. E S I/HRMS [2M +1]: calcd for C37H62O2, 535.4469; found, 535.4463. Anal. Calcd for CHNO: C, 71.86; H, 10.93; N, 5.24. Found: C, 71.91; H, 11.06; N, 5.23. T ert -butyl heptadeca -1,16-dien -9 -ylcarbamate 6,6NHBoc (2 -2). 1H NMR (300 MHz, CDCl3 1.40 (br, m, 8H), 1.41 (s, 9H), 2.01 (q, 4H), 3.50 (br, 1H), 4.20 (br, d, 1H), 4.91 (m, 4H), 5.78 (m, 2H). 13C NMR (75 MHz, CDCl3

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47 29.05, 29.27, 29.63, 33.97, 35.79, 50.83, 79.00, 114.37, 139.35, 155.92. FT IR (KBr pellet): 3444, 3346, 3077, 2977, 2928, 2856, 1821, 1703, 1693, 1641, 1522, 1455, 1441, 1415, 1390, 1365, 1248, 1174, 1092, 1057, 993, 909, 869, 778, 750, 725, 636, 555. EI/HRMS [2M +1]: calcd for C37H62O2, 703.6347; found, 703.6327. Anal. Calcd for CHNO: C, 75.16 ; H, 11.75 ; N, 3.98. Found: C, 75.26 ; H, 11.99 ; N, 3.93. T ert -butyl henicosa -1,20-dien-11-ylcarbamate 8,8NHBoc (2 -3). 1H NMR (300 MHz, CDCl3 1.41 (br, m, 28H), 1.42 (s, 9H), 2.01 (q, 4H), 3.50 (br, 1H), 4.20 (br, d, 1H), 4.93 (m, 4H), 5.80 (m, 2H). 13C NMR (75 MHz, CDCl3 29.14, 29.33, 29.64, 29.76, 29.80, 34.02, 35.82, 50.87, 78.95, 114.31, 139.42, 155.94. FT IR (K Br pellet): 3348, 3077, 2977, 2926, 2855, 1701, 1641, 1503, 1456, 1390, 1365, 1245, 1173, 1046, 993, 909, 865, 723, 640. EI/HRMS [2M +1]: calcd for C37H62O2, 815.7599; found, 815.7466. Anal. Calcd for CHNO: C, 76.60; H, 12.11; N, 3.44. Found: C, 76.58; H, 12.26; N, 3.47. T ert -butyl tricosa -1,22-dien-12-ylcarbamate 9,9NHBoc (2 -4). 1H NMR (300 MHz, CDCl3 1.40 (br, m, 32H), 1.41 (s, 9H), 2.01 (q, 4H), 3.49 (br, 1H), 4.20 (br, d, 1H), 4.91 (m, 4H), 5.78 (m, 2H). 13C NMR (75 MHz, CDCl3 28.66, 29.15, 29.34, 29.68, 29.74, 29.80, 34.02, 35.84, 50.90, 78.95, 114.30, 139.43, 155.94. F T IR (KBr pellet): 3446, 3349, 3077, 2977, 2927, 2855, 1820, 1705, 1641, 1503, 1456, 1415, 1390, 1365, 1247, 1174, 1049, 992, 909, 867, 780, 750, 722, 636, 552, 464. EI/HRMS [2M +1]: calcd for C37H62O2, 871.8225; found, 871.8195. Anal. Calcd for CHNO: C, 77.18; H, 12.26; N, 3.21. Found: C, 77.24; H, 12.27; N, 3.25. General ADMET Polymerization Procedure for Symmetrical Boc Amine Monomers Monomer was transferred into a dry 25 mL Schlenk tube equipped with a stir bar and glass stopcock and dried by heatin g the vessel in an oil bath at 50 C under full vacuum (103

PAGE 48

48 mmHg) for 24 hr. After 24 hr the reaction vessel was backfilled with Argon and first -generation Grubbs Ru catalyst (200:1/monomer:catalyst) was added. The full vacuum was placed back on the po lymerization reaction after .5 hr. Additional catalyst was added 60 hr into the polymerization to ensure maximum possible couplings. The polymerization reaction was monitored closely by 1H NMR to confirm that no remaining terminal olefin could be detecte d. Upon completion the reaction was quenched by opening the flask and adding 25 mL of toluene and 1 mL of ethyl vinyl ether. The polymer was purified by precipitation of the polymer solution into 1.5 L of cold methanol. The polymer was then filtered and dried for characterization. Polymerization of tertbutyl undeca -1,10-dien-6 ylcarbamate 3,3NHBoc (2 -5). 1H NMR (300 MHz, CDCl3 1.48 (br, 17H), 1.92 (br, 4H), 3.48 (br, 1H), 4.26 (br, 1H), 5.245.40 (br, 2H). 13C NMR (75 MHz, CDCl3 35.31, 50.63, 78.92, 129.95, 130.43, 155.88. FT IR (KBr pellet): 3443, 3341, 2977, 2931, 2857, 2248, 1691, 1523, 1456, 1391, 1365, 1248, 1174, 1056, 968, 912, 867, 779, 734, 647, 462. Polymerization of tertbutyl heptadeca -1,16-dien -9 -ylcarbamate 6,6NHBoc (2 -6). 1H NMR (300 MHz, CDCl3 4.21 (br, 1H), 5.285.5.37 (br, 2H). 13C NMR (75 MHz, CDCl3 26.04, 27.39, 28.64, 29.03, 29.25, 29.33, 29.47, 29.67, 29.90, 32.76, 33.95, 35.79, 50.85, 78.91, 130.03, 130.49, 155.93. FT IR (KBr pellet): 3445, 3344, 2977, 2927, 2855, 1692, 1525, 1456, 1390, 1365, 1248, 1174, 1090, 1013, 967, 909, 867, 778, 728, 646, 463. Polymerization of tertbutyl henicosa -1,20-dien-11-ylcarbamate 8,8NHBoc (2 -7). 1H NMR (300 MHz, CDCl3 1.40 (br, m, 28H), 1.41 (s, 9H), 1.91 1.99 (br, 4H), 3.49 (br, 1H), 4.22 (b r, d, 1H), 5.29 5.39 (br, 2H). 13C NMR (75 MHz, CDCl3 28.67, 29.37, 29.70, 29.81, 29.88, 29.98, 32.82, 35.85, 50.93, 78.95, 130.53, 155.93. FT IR

PAGE 49

49 (KBr pellet): 3443, 3344, 2975, 2927, 2854, 1691, 1523, 1456, 1390, 1365, 1247, 1174, 1093, 1019, 967, 914, 864, 778, 724, 645, 464. Polymerization of tertbutyl tricosa -1,22-dien-12-ylcarbamate 9,9NHBoc (2 -8). 1H NMR (300 MHz, CDCl3 1.41 (br, m, 32H), 1.42 (s, 9H), 2.01 (br, 4H), 3.49 (br, 1H), 4.21 (br, d, 1H), 5.305.38 (br, 2H). 13C NMR (75 MHz, CDCl3 29.42, 29.55, 29.74, 29.81, 29.84, 29.90, 30.00, 32.84, 35.84, 50.92, 78.93, 130.10, 130.56, 155.94. FT IR (KBr pellet): 3445, 3346, 3136, 2975, 2925, 2854, 2248, 1693, 1523, 1456, 1390, 1365, 1247, 1174, 1097, 1048, 1024, 966, 910, 865, 778, 723, 647, 463. Hydrogenation of Unsaturated ADMET Polymers The crude polymer solution was transferred to a Parr Bomb glass sleeve and diluted to 200 mL with toluene. Argon was bubbled through the solution for 30 minute s after which a spatula tip of Wilkinsons catalyst (RhCl(PPh3)3) was added to the solution and the sleeve was sealed inside a Parr Bomb equipped with a mechanical stirrer and temperature control. The vessel was purged three times with 600 psi hydrogen ga s, then filled to 600 psi with hydrogen gas and was left for 4 days at room temperature. Upon depressurization, Argon was bubbled through the crude reaction mixture for 30 minutes. The solution was concentrated to 50 mL and slowly dripped into 1 L of co ld methanol. The precipitated polymer was filtered and dried for characterization. P olysat3,3NHBoc (2 -9). 1H NMR (300 MHz, CDCl313C NMR (75 MHz, CDCl3 35.85, 50.90, 78.95, 155.95. FT IR (KBr pellet): 3446, 3342, 3134, 2928, 2855, 2248, 1692, 1524, 1456, 1390, 1365, 1248, 1175, 1098, 1048, 1020, 909, 865, 802, 733, 667, 647, 556, 463.

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50 P olysat6,6NHBoc (2 -10). 1H NMR (300 MHz, CDCl3 H), 1.41 ( br, s, 13H), 3.49 (br, 1H), 4.21 (br, d, 1H). 13C NMR (75 MHz, CDCl3 28.67.29.48, 29.86, 29.91, 32.10, 35.84, 50.92, 78.94, 155.95. FT IR (KBr pellet): 3446, 3346, 3137, 2977, 2924, 2854, 2249, 1695, 1525, 1456, 1390, 1365, 1247, 1175, 1060, 1013, 909, 866, 778, 734, 646, 465. P olysat8,8NHBoc (2 -11). 1H NMR (300 MHz, CDCl3 1.41 (br, m, 36H), 1.42 (s, 9H), 3.49 (br, 1H), 4.21 (br, d, 1H). 13C NMR (75 MHz, CDCl3 35.80, 50.87, 78.92, 155.93. FT IR (KBr pellet): 3445, 3348, 3135, 2925, 2854, 2248, 1693, 1526, 1457, 1390, 1365, 1248, 1175, 1096, 1019, 909, 865, 801, 732, 647, 464. P olysat9,9NHBoc (2 -12). 1H NMR (300 MHz, CDCl3 1.34 (br, 40H), 1.41 (s, 9H), 3.49 (br, 1H), 4.21 (br, d, 1H). 13C NMR (75 MHz, CDCl3 29.94, 32.13, 35.82, 50.88, 78.92, 155.93. FT IR (KBr pellet): 3446, 3346, 3136, 2925, 2854, 1696, 1523, 1465, 1457, 1390, 1365, 1248, 1175, 1058, 1019, 866, 783, 753, 721, 650, 462. Removal of the Boc Protection Group from the Polymers Each saturated, p rotected polymer was readily dissolved in THF and transferred to a 10 mL screwcap vial. The solution was rotovapped with fast spinning to create as thin a film as possible along the walls of the vials. The vial was attached to a vacuum vial adapter and p laced under high vacuum (103 mmHg) for one day to dry. After the polymer was dry the vial was submerged in 275 C sand in an aluminum foil lined heating mantle and left under heat and vacuum for 2 hr. Each polymer went from a light beige color to a dark brown color within the first 5 minutes of heating. Bubbling of each sample was initially observed and the every 9th material melted to form a viscous liquid upon initial heating that solidified upon cooling.

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51 Results and Discussion Polymer Design and Synt hesis The synthesis of linear ethylene co vinyl amine (EVAm) polymers requires the synthesis of a symmetrically branched diene monomers for the ADMET reaction. An initial attempt at polymerizing the free amine diene was performed due to the recent su ccess of other groups at performing ROMP on the free amine but the attempt failed due to its ability to facilitate catalyst decomposition. As a result a protection strategy was required to polymerize the amine. A 9 fluorenyl carbamate (Fmoc) amine deprot ection strategy had already been developed in our group by Leonard et al55 on amino acid containing polyolefins and was attempted for these materials. 9,9NHFmoc monomer synthesis was readily accomplished by protecting the corresponding amine with 9 -fluorenylmethyl chloroformate (Fmoc -Cl) to yield a high melting white fluffy powder. Polymerization of this monomer in THF led to the formation of dimers and trimers almost immediately upon addition of catalyst as observed by the precipitati on of these oligomers out of solution (Figure 2 1). Attempts at characterizing these oligomers failed due to no observed solubility in any known solvent. N H2 9 9 H N 9 9 O O H N 9 9 O O n i i i9 9 N H F m o c i n s o l u b l e o l i g o m e r s 9 9 N H2 Figure 2 1. Initial protection strategy for amine monomers : (i) FmocCl, DMAP, THF; (ii) first gene ration Grubbs catalyst As an alternative protection strategy, the t -butyl oxycarbonyl (Boc) group was employed due to its ability to be readily removed under acid or heat with some literature examples of strong sterically hindered Bronsted base. Figure 2 2 details the synthetic approach employed to conduct the metathesis polymerization, hydrogenation, and deprotection. Monomer 2 -1 is a

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52 colorless viscous oil while monomers 2 -2, 2 -3, and 2 -4 are white crystalline solids that melt at 38 43 and 46 C r espectively. As a result, all four monomers can be polymerized neat in the melt with no solvent. The ADMET polymerization of the Boc protected monomers proceeded normally under vacuum with no noticeable effect on the reaction. Hydrogenation of the mater ials was performed in toluene with Wilkinsons catalyst under hydrogen pressure in a Parr reactor. These hydrogenations were done at room temperature and there was no evidence that any Boc protection groups were removed during this step. N H O O x x N H O O x x n N H2 N H O O 2 x + 2 n N H2 N H2 N H2 x = 3 ( 2 1 ) 6 ( 2 2 ) 8 ( 2 3 ) 9 ( 2 4 ) x = 3 ( 2 5 ) 6 ( 2 6 ) 8 ( 2 7 ) 9 ( 2 8 ) x = 3 ( 2 9 ) 6 ( 2 1 0 ) 8 ( 2 1 1 ) 9 ( 2 1 2 ) 2 1 6 ( 9 5 m o l % V A m ) 2 1 5 ( 1 1 m o l % V A m ) 2 1 4 ( 1 3 m o l % V A m ) 2 1 3 ( 2 2 m o l % V A m ) n n n n i i i i i i Figure 2 2. E thylene co -vinyl amine copolymer synthesis: (i) first generation Grubbs catalyst; (ii) H2 (500 psi), RhCl(PPh3)3, toluene, RT; (iii) 250 C. Table 2 1. Molecular weight and thermal data for unsaturated ethylene vinyl amine copolymers: a calculated from theoretical repeat unit, confirmed by NMR. b 10 C/min scan rate, values determined from second scan data. c reported in kg/mol and performed in THF at 40 C with calibration against polystyrene standards. p olymer mol % vinyl amine a T g b M w c PDI c D p c 2 5 22 19 7.4 1.45 21 2 6 13 5 11.4 1.43 25 2 7 10.5 8 14.7 1.99 20 2 8 9.5 24 13.3 1.67 20 Structural Analysis with 13C and 1H NMR and IR Primary structure analysis by 1H NMR revealed the kind of pristine primary structure that can be obtained when using sequenced ADMET materials produced by clean metathesis and

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53 hydrogenation reactions. These proton and carbon spectra are the best techniques to determine primary structure of monomer, unsaturated polymer, and saturated polymer. Figures 2 3 and 2 4 show the overlapped spectra of the 9,9 and 6,6 molecules, respectively, and display the structural purity of these materials as the chemistry progresses from monomer through ADMET and hydrogenation reactions to yield fully saturated, protected polymer. Co nfirmation of the precision of branches and knowledge of methylene run lengths between these branches confidently allows for the determination of molar ratios of ethylene and vinyl amine. For the proton spectra in Figure 2 3, one can see the resonances fr om terminal olefin at roughly 4.8 and 5.8 ppm of the monomer condense to one peak at 5.3 ppm while other peaks shifts are maintained and slightly broadened. From the unsaturated to saturated polymer, it can be seen that the internal olefin and its alpha proton peak at 2.0 ppm are completely removed from the sample through the hydrogenation step. The hydrogenation of the unsaturated polymer also leads to the formation of methyl end groups as seen appear at 0.9 ppm. The same trends are seen in the carbon spectra (Figure 2 4), the terminal olefin resonances at 130 and 156 ppm are condensed to an internal olefin peak that shows the cis/trans isomers. This internal olefin peak is then eliminated from the sample upon hydrogenation in addition to the protons a t 2 ppm on the alpha methylene carbons to the alkene as verified from the spectra. The final hydrogenated polymer shows five proton shifts relating to the polymer: (1) methyl end groups at 0.8 ppm; (2) backbone protons at 1.23 ppm; (3) nine Boc group prot ons at 1.41 ppm; (4) proton on the carbon with the amine branched at 3.48 ppm; (5) carbamate nitrogens proton at 4.21 ppm.

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54 Figure 2 3. Progression from monomer to polymer monitored by 1H NMR. Figure 2 4. Progression of monomer to polymer monitored by 13C NMR.

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55 Thermal Analysis Table 2 2. Thermal data for the protected and deprotected polymers: a calculated from theoretical repeat unit, confirmed by NMR. b 10 C/min scan rate, values determined from second scan data. Polymer Mol % vinyl aminea T g b T m b 2 9 22 4 N/A 2 10 13 2 N/A 2 11 10.5 8 N/A 2 12 9.5 2 N/A 2 13 22 10 N/A 2 14 13 N/A 49 2 16 9.5 N/A 44 Thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC) was performed on each of the three groups of polymer sampl es: the unsaturated protected, the saturated protected, and the saturated, deprotected polymers. Table 2 2 summarizes the thermal properties and Figures 2 5, 2 6, 28, 29, and 2 10 detail the decomposition, glass transition, and melting points of these m aterials with TGA and DSC. It is obvious from this thermal data that the molar ratio of incorporated vinyl amine has a dramatic effect on the material behavior. The decomposition traces of both the unsaturated and saturated protected amine polymers are s hown in Figure 2 5 and are predictable. Yamamoto and Ahn have shown in their research that the Boc group can be removed thermally at about 175 C.86,87 Although the mechanism could proceed through a variety of mechanisms at this elevated temperature, a six membered ring transition state involving the amide nitrogens lone pair deprotonating a methyls hydrogen on the tert butyl group is our best guess at this point. We have no evidence that this deprotection could not also be occurring with through the formation of radicals. A sharp loss in weight is seen at this temperature for each of the eight protected polymers. It is assumed that a stable deprotected amine polymer exists at this plateau area of each curve. T he percent weight losses observed

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56 experimentally were 45, 32, 29, and 25%. These values match those calculated by the loss of carbon dioxide and isobutene from each repeat unit 42, 31, 26, and 25% respectively. The addition of hydrogen to the internal al kene has a minimal net effect on the overall weight percentage of the polymer as the unsaturated graph is mirrored in shape by the saturated polymers TGA figure. It can be assumed by these weight loss profiles that a stable polymer exists over a 100 win dow that could make these materials excellent candidates for a simple thermal deprotection step. Figure 2 5. TGA traces for (left) unsaturated protected polymers (right) saturated protected polymers Figure 2 6 shows the DSC data (second heating scan) for the series of unsaturated and saturated protected polymers. All four unsaturated polymers are amorphous but the Tgs are consistent for ADMET sequenced materials. Amorphous sequenced copolymers are a consequence of too much steric congestion along the polymer backbone preventing the ethylene run lengths between branches to crystallize. The decreasing Tg with decreasing branch frequency is explained by the t -butyl carbamate protection group. This group allows for hydrogen bonding with other such prote cting groups. This hydrogen bonding ability allows branches to become associated with each other and form a physical cross linked structure. The increasingly less crosslinked structure of polymers 2 -5 2 -6 2 -7 and 2 -8 show decreasing Tg

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57 values. The f ewer physical cross -links a polymer chain has, the greater chain flexibility and lower Tg. Figure 2 5 also shows the second heating scan for the series of saturated protected polymers. These DSC traces show complicated transitions that could resemble the start of a Tg but then look like they lead directly into a melt. The general trend noticed in these precision polymers is that increasing the pendant branch frequency (increasing the molar concentration of vinyl amine) yields less and less crystalline ma terials until a critical threshold is met and the material becomes amorphous. This threshold isnt obviously clear yet since a melt is observed with the every 9th amine polymer ( 2 -9 ). Figure 2 6. DSC of ADMET (left) unsaturated protected polymers (ri ght) saturated protected polymers Initial attempts at deprotecting these polymers were carried out in solution with hydrochloric acid. The protected polymer was readily dissolved into a minimum amount of a 1:1 THF/dioxane solvent mixture. Ten volume equ ivalents of 4 M HCl in dioxane was then added at room temperature. Within minutes a dark brown polymer started to precipitate out of solution onto the sides of the round bottom flask. After one hour the solution was extracted with ether but no polymer wa s obtained upon rotavapory evaporation of the organic layer. Multiple attempts were made to remove the polymer remaining on the walls of the flask but no solvent

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58 could dissolve the polymer. The polymer was finally removed from the flask by physically scr aping it from the sides and placing the black scrapings into a vial and dried. It is thought that the minimally soluble polymer that crashed out of solution was the ammonium chloride salt of the amine. These salt branches could cluster together in a poly mer coil and prevent any further solvation of the material. To prevent this potentially strong ionic clustering it was decided to use the information gathered from the TGA thermograms shown in Figure 2 5 to help in this deprotection. For this deprotectio n technique, the polymer was cast as a thin film in a 10 mL vial and placed in a 275 C sand bath under vacuum. The tan colored, protected polymer films of all these materials turned black within minutes upon heating and one could even see the samples bub bling with the removal of carbon dioxide and isobutene. Since this deprotection is done under vacuum, there is no need for further purification since the isobutene is removed upon elimination. Figure 28 shows the overlapping TGA traces of the saturated protected 6,6 polymer and 9,9 polymer with their respective deprotected counterparts. It can be seen that the corresponding weight loss from the removal of the Boc group no longer exists when the polymer recovered from the vial is run on the TGA. These t hermally deprotected polymers, like the chemically deprotected ones, have neglible solubility in all solvents tested. Small amounts of the 3,3 and 6,6 deprotected polymers dissolved in CDCl3 and the corresponding polysat66NH2 NMR is shown in Figure 2 7. Figure 2 9 shows one of the advantages of the thermal over the acid deprotection strategies. One can see the weight loss on the acid deprotected polymer begin before 100 C and this weight loss continues until the main chain decomposition at 400 C. The thermally deprotected polymer is immediately ready for analysis upon removal of heat and shows no solvent contamination or nonpolymer associated weight loss upon TGA analysis.

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59 Figure 2 7. 1H NMR of protected and deprotected amine branch every 15th carb on. Figure 2 8. TGA traces of (left) polysat6,6NHBoc and polysat6,6NH2 (right) polysat9,9NHBoc and polysat9,9NH2 polymers

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60 Figure 2 9. TGA traces of polysat9,9NH2 polymers deprotected from heat and acid The final deprotected amine on every 15th, and 21st carbon polymers exhibit crystallinity. Figure 2 10 shows the every 15th polymer has a sharp melt at 49 C and the every 21st polymer has a broader melt at 44 C. A trend is usually seen where most every 21st carbon family of precision polymers the Wagener group has synthesized has a higher melting point than the 15th materials. The every 9th polymer is expected to be amorphous compared to other materials with this precision and the trend is upheld. One very interesting point noticed in the thermal data for all of these materials is the 1st heat of the saturated protected materials and the 2nd heat of the deprotected materials (Figure 2 10). It can be seen that the melts associated with this first heat are strikingly similar to the melts of the dep rotected polymers. One can imagine a competition in the deprotected polymers between the ethylene run lengths trying to form lamellae and the amine groups trying to form hydrogen bonding to disrupt that interaction.

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61 Figure 2 10. (left) DSC of amine bra nch every 9th, 15th, and 21st carbon on polymer. (right) overlay of 1st heat of saturated protected polymer and 2nd heat of deprotected polymers. Conclusions Reported for the first time, a family of six linear copolymers of ethylene and vinyl amine has b een prepared with no branching defects with a predesigned target for comonomer ratios. These materials possess exact ethylene run lengths between pendant amine branches and the resultant materials have an amine branch on every 9th, 15th, 19th, and 21st ca rbon along the backbone. Spectroscopic analysis via NMR and FT -IR reveals the perfect microstructure control capable through metathesis polymerization by eliminating side reactions during polymerization or hydrogenation. The pristine nature of the copolymers primary structure imparts marked effects on thermal behavior. Although it is believed that the deprotection went to completion, the deprotected amine branched polymers on every 21st, 19th, and 15th show minimal solubility. Two deprotection approach es were used to make the final polymer but both yielded the same minimally soluble product. IR and thermal analysis show a marked difference from the saturated protected polymer and it is believe these materials are what we say they are. Future Work Solubility appears to be the biggest obstacle encountered with these potentially useful amine -branched materials. Synthesis of polyethylene with tetraethylene glycol (TEG) spaced

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62 amines could maintain the molar amount of amine branches on the polymer chain in addition to imparting solubility. Figure 2 11 is a suggested synthetic scheme that could be implemented to make these materials. Sam Popwell has already begun the synthesis of these materials for his dissertation and will work in developing these polymer s as antibacterial coatings. This TEG amine branched polymer could also be cast as a film and then functionalized on the surface with peptides or enzymes or even attached chemically or physically to drugs. Amine branched surfaces like these would be idea l for a very wide range of applications. 9 9 O T s T r O O H 4 9 9 T r O 4 O 9 9 H O 4 O 9 9 N H2 4 O 9 9 N H B o c 4 O n O O O O N H2 +i i i i i i i v v Figure 2 11. Ethylene co vinylTEG amine copolymer synthesis: (i) NaH, THF; (ii) 4M HCl; (iii) PCC followed with NH4OAc and NaCNBH3; (iv) (Boc)2O, THF; (v) fi rst -generation Grubbs catalyst, then H2 (500 psi) with RhCl(PPh3)3 in toluene followed by subsequent deprotection with heat.

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63 CHAPTER 3 SEMICRYSTALLINE LYSINE FUNCTIONALIZED PRECISION POLYOLEFINS Introduction In the early years of implantable materials, device function was the primary consideration with issues associated with biostability and biocompatibility being of secondary concern.88 As the understanding of interactions between the body and the implanted materials advanced, materials have been better engineered to improve their biostability and biocompatibility.89 The current frontier in biomaterials is focusing on engineering the surface of a material for a favorable interaction by mimicking the surface of a known biological surface.90,91 One example of this sort of surface modification is the covalent attachment of heparin to prevent blood clot formation in blood oxygenation devices.92,93 Such modifications represent successful approach es to modifying metals, ceramics, and crystalline or high glass transition temperature (Tg) polymers. Low Tg, flexible biomaterials that are tough and biostable, such as in the case of polyurethanes and silicones, are a different matter.9496 The challenge associated with surface modification of such low Tg materials relates to their surface having significant molecular mobility. As a consequence, the molecular sequences presented on the surface of these polymers in the ex vivo atmospheric environment are different than those presented in the in vitro wet environment. These molecular sequences fluctuate again in the in vivo environment where the surface is likely to rearrange, perhaps burying the attached moieties in response to the variety of local chemistry changes that are known to occur as the tissue near the implant surface continues along the healing cascade. These dynamics were elegantly delineated for polyurethane materials by Ratner.97 The utility of metathesis chemistry lends itself to the challenge of synthesizing polymers with a wide range of functionality and numerous potential biological applications. One such

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64 example, ring opening metathesis polymerization (ROMP), has been use d to make a variety of amino acid and peptide branched polynorbornenes with such properties.98100 This synthetic strategy has also proven useful for preparing polymers capable of inhibiting cell binding to fibrone ctin.101 ROMP of cyclooctene m onomers functionalized with either lysine or pentalysine groups has been shown to be useful in electrolyte applications by varying charge density.102 In addition, the radical polymerization of amino acid and peptide functionalized acrylic acid and methacrylic acid derivatives, some even biologically active, has proven viable to prepare many new highly functional polymers.103106 This paper reports the preparat ion of precise, semicrystalline amino acid branched polyolefins, termed bio -olefins, that resemble a modified precise copolymer of either acrylamide, or N -vinyl amide and ethylene.3840,44 Their preparation has bee n accomplished using ADMET chemistry, which assures that the branches are set at specific, not random, intervals along the backbone, generating polymers incapable of being made by other methodologies.21,2931,54 Th is well defined structure results in materials with high tensile strength and semicrystallinity, physical properties not seen in typical amino acid and peptide branched polyacrylates, polynorbornenes, and polymethacrylates.38,44 Discussed herein is the synthesis and structural characterization of these bio olefins using differential scanning calorimetry (DSC), wide angle x ray scattering (WAXS), and small angle x ray scattering (SAXS). The re sults suggest that the amino acid protecting groups pack such that certain length scales of the monomer crystalline unit cells are preserved after polymerization. Although these polymeric materials are semicrystalline in nature, the polyethylene backbone itself is amorphous, with the protected amino acid groups packed in a crystalline manner similarly ordered to that of the monomer. The viability of this last statement is amplified by one example reported herein, where the amino acid protecting group is

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65 co mpletely removed, thereby rendering the material amorphous. These phenomena are unprecedented in the literature. Experimental Section Materials Reagents and chemicals were used as received from Aldrich chemical company unless otherwise noted. Diethyl eth er and THF were used as dry solvents from the Aldrich keg system and dried over 4 Bachem. The second generation Grubbs catalyst (tricyclohexylphosphine[1,3bis(2,4,6 trimethylphenyl) 4,5 -dihydroimidazol 2 -ylidene] [benzylidene]ruthenium (IV) dichloride ) was exclusively used and synthesized as previously described by Grubbs et al .85 Instrumentation and Analysis 1H NMR and 13C NMR spectra were recorded on a Varian Associates Mercury 300 spectrometer. Chemical shifts for 1H and 13C NMR were referen ced to residual signals from CDCl3 (1H = 7.24 ppm and 13C = 77.23 ppm) with 0.03% v/v TMS as an internal reference. High resolution mass spectra (HRMS) data were obtained on a Finnegan 4500 gas chromatograph/mass spectrometer using the chemical ionization mode. Gel permeation chromatography (GPC) of polymers 3a -6a was performed at 40C using a Waters Associates GPCV2000 liquid chromatography system with an internal differential index detector (DRI) and two Waters Styragel HR 300 mm length) in HPLC grade tetrahydrofuran as the mobile phase at a flow rate of 1.0 mL/min. Injection 0.07 w/v sample concentration. Retention times were calibrated against a minimum of nine narrow polystyrene st andards purchased from Polymer Laboratories (Amherst, MA).

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66 Both monomers and their polymers were evaluated with wide angle x -ray scattering (WAXS) and small angle x ray scattering (SAXS). All x ray analysis was performed in the Institute of Technology Cha racterization Facility at the University of Minnesota. For the WAXS experiments, the data was collected using a Bruker -AXS Microdiffractometer equipped with CuK radiation ( =1.54). For the SAXS experiments, CuK radiation ( = 1.54) was generated by a Rigaku Ru 200BVH rotating anode. Small angle data was collected at a sample to detector distance of 58cm and 230 cm. Wide angle data was collected over a range from 0 2 32. In both the small and wide angle experiments, two dimensional diffraction data were collected with a Siemens HI STAR multi -wire area detector. All data were corrected for background and detector response characteristics. Azimuthal averages produced one dimensional plots. By convention, the WAXS data is presented as intensity (arbitrary units) vs. 2 while the SAXS data is presented as intensity (arbitrary units) vs. q (the magnitude of the scattering wave vector). The magnitude of the scattering vector, q, is related to the scatting angle by q=4 / sin /2, where is equal to t he radiation wavelength. Using Braggs Law ( =2dsin ), q can be related to the characteristic domain spacing, d =2 /q. All x ray experiments were performed at room temperature. The polymers were solvent -cast on a Teflon plate from chloroform or tetrahydr ofuran and allowed to dry slowly. Differential scanning calorimetry (DSC) was performed on a TA Instruments Q1000 equipped with a liquid nitrogen cooling accessory calibrated using sapphire and high purity indium metal. All samples were prepared in hermet ically sealed pans (4 7 mg/sample) and were referenced to an empty pan. A scan rate of 10 C per minute was used. Melting temperatures were taken as the peak of the melting transition, glass transition temperatures as the mid point of a step change in hea t capacity. Thermal experiments were

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67 conducted as follows: samples were heated through the melt to erase thermal history, followed by cooling at 10 C per minute to 150 C, and then heated through the melt at 10 C per minute. Data reported reflects this second heating scan. General Monomer Synthesis Synthesis of the monomers 3 -3 to 3 -5 was performed as described in previous publications and the characterization matched that previously reported with additional IR characterization provided here.39,40 Monomer 3 -6 was made as follows: to a 100 mL round bottom flask wa s added the hydrochloride salt of the Fmoc and methyl ester protected lysine (1 equiv), HOBt (3 equiv) and the 9,9 diene acid premonomer ( 3 -2 ). The flask was then equipped with a septum and purged with argon, followed by the addition of THF and DIC (1.25 equiv). Triethylamine (1 equiv) was added to neutralize the hydrochloride salt of the amino acid. The reaction was allowed to stir at room temperature for 2 hrs then the reaction was refluxed for 48 hrs. D.I. water (30 mL) was added followed by extracti on with diethyl ether (3x30 mL). The organic layer was collected and washed with 1 M HCl (2x20 mL) and brine (2x20 mL). After drying with MgSO4, the solution was evaporated yielding the crude diene, which was purified using column chromatography using hex ane:ethyl acetate as the eluent. [(S )-5 -Benzyloxycarbonylamino -5 -(1 -undec -10-enyl -dodec -11-enylcarbamoyl) -pentyl] carbamic acid benzyl ester (3 -3). The pure product 3 -3 was obtained in 47 % yield as a white solid after purification by three recrystalli zations from CH3CH2OH/H2O (mp = 99 104 C). 1H NMR (300 MHz, CDCl3, ppm): 1.91 (br, 37H), 1.952.13 (br, 1H), 2.23 (q, br, 4H), 3.253.44 (br, 2H), 3.964.12 (br, 1H), 4.184.33 (br, 1H), 4.91 5.07 (br, 1H), 5.08 5.24 (br, 4H), 5.30 (s, br, 4H), 5.565.71 (br, 1H), 5.89 (d, br, 1H), 5.936.09 (m, 2H), 7.49 7.60 (b r, 10H). 13C NMR (75 MHz, CDCl3 22.66, 25.97, 29(m), 33.91, 34.12, 35.37, 40.41, 49.59, 55.00, 66.71, 114.29, 128(m), 138.63,

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68 156.49, 171.11. FTIR (KBr pellet, cm1): 3316, 3278, 2926, 2854, 1691, 1650, 1535, 1467, 1266, 1247, 1153, 1080, 1054, 1024, 911, 696. Anal. Calcd. for C45H69N3O5: C 73.83, H 9.50, N 5.74. Found C 73.48, H 9.59, N 5.65. EI/HRMS [M + 1]: Calcd. for C34H64N2O3: 731.5237; Found: 731.5315. (S )-6 -Benzyloxycarbonylamino -2 -[3 -(1 -undec -10-enyl dodec -11-enylcarbamoyl) propionylamin o] hexanoic acid methyl ester (3 -4). Product 3 -4 was obtained pure in 72 % yield as a white solid after purification by three recrystallizations using CH3CH2OH/H2O (mp = 129 132 C). 1H NMR (300 MHz, CDCl3, ppm): 1.75 (br, 38H), 1.751.88 (br, 1H), 1.901.96 (br, 1H), 2.02 (q, 4H), 2.362.61 (br, 4H), 3.063.27 (br, 2H), 3.71 (s, 3H), 3.753.90 (br, 1H), 4.46 4.61 (br, 1H), 4.86 5.04 (m, 4H), 5.065.22 (br, 3H), 5.58 (d, br, 1H), 5.715.91 (m, 2H), 6.68 (d, br, 1H), 7.297.39 (br, 5H). 13C NMR (75 MHz, CDCl3 34.03, 35.30, 35.39, 40.62, 49.69, 52.19, 52.55, 66.77, 114.33, 128.28, 128.73, 136.91, 139.45, 156.83, 171.67, 172.46, 172.96. FTIR (KBr pellet, cm1): 3316, 3077, 2924, 2852, 1748, 1687, 1637, 1540, 1467, 1435, 1365, 1290, 1264, 1211, 1145, 993, 910, 739, 696, 668, 466. Anal. Calcd. for C42H69N3O6: C 70.85, H 9.77, N 5.90; Found: C 70.65, H 9.90, N 5.74. EI/HR MS [M + 1]: Calcd. for C42H69N3O6: 711.5186; Found: 711.5252. (S )-6 -Benzyloxycarbonylamino -2 -(2 -undec -10-enyl tridec -12-enoyl amino) -hexanoic acid methyl ester (3 -5). The pure product 3 -5 was obtained in 78 % yield as a white solid after purification by three recrystallizations from CH3CH2OH/H2O (mp = 102110 C). 1H NMR (300 MHz, CDCl3, ppm): 1.45 (br, 32H), 1.471.94 (br, 6H), 1.962.11 (br, 5H), 3.17 (q, br, 2H), 3.73 (s, 3H), 4.564.66 (m, 1H), 4.774.88 (br, 1H), 4.895.84 (m, 4H), 5.06 5. 16 (br, 2H), 5.685.86 (m, 2H), 6.03 (d, br, 1H), 7.29 7.41 (br, 5H). 13C NMR (75 MHz, CDCl3

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69 29.37, 29.57, 29.70, 29.73, 29.78, 29.86, 29.92, 32.38, 33.17, 33.27, 34.04, 40.75, 48.11, 51.76, 52.57, 66.85, 114.33, 128.3 2, 128.74, 136.79, 139.46, 156.73, 173.25, 176.19. FTIR (KBr pellet, cm1): 3318, 3079, 2924, 2851, 1739, 1686, 1638, 1466, 1342, 1263, 1213, 1179, 1129, 1027, 910, 782, 725, 695, 636. Anal. Calcd. for C39H64N2O5: C 73.08, H 10.06, N 4.37; Found: C 7 3.01, H 10.07, N 4.32. EI/HRMS [M + 1]: Calcd. for C39H64N2O5: 640.4815; Found: 640.4831. (S) -Methyl 6 -(((9H -fluoren -9 -yl)methoxy)carbonylamino) -2 -(2 -(undec -10-enyl)tridec -12enamido)hexanoate (3-6). The pure product 3 -6 was obtained in 76 % yield a s a white solid after purification by column chromatography using 4:1 hexane:ethyl acetate as the eluent (mp = 136 142 C). 1H NMR (300 MHz, CDCl3 1.35 (br, 28H), 1.57 (m, 4H), 1.83 (b, 4H), 2.01 (q, 4H), 3.20 (q, 2H), 3.73 (s, 3H), 4.19 (t 1H), 4.38 (d, 2H), 4.62 (t, 1H), 4.94 (m, 4H), 5.79 (m, 2H), 5.99 (t, 1H), 6.91 (m, 1H), 7.34 (t, 2H), 7.40 (t, 2H), 7.58 (d, 2H), 7.75 (d, 2H).13C NMR (75 MHz, CDCl3 32.37, 33.16, 33.27, 34.02, 40.74, 47.49, 48.13, 51.72, 52.76, 66.82, 114.30, 120.18, 125.22, 127.24, 127.88, 139.43, 141.52, 144.17, 156.74, 173.24, 176.21. FTIR (KBr pellet, cm1): 3322, 3075, 2926, 2852, 1944, 1905, 1731, 1688, 1637, 1536, 1466, 1345, 1261, 1025, 912, 801, 758, 740. Anal. Calcd. for C46H68N2O5: C 75.78, H 9.40, N 3.84; Found: C 73.41, H 9.40, N 3.72. EI/HRMS [M + 1]: Calcd. for C46H68N2O5: 729.5201; Found: 729.5131. Polymer Synthesis Polymerization of monomers 3 -3 to 3 -5 was performe d as described in previous publications and the characterization matched that previously reported with additional IR characterization provided here.39,40 Polymerization of 3 -6 and the other lysine monomers differs from a typical bulk ADMET polymerization due to the fact that all four monomers are solids at

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70 and abov e room temperature. This technique requires adding just enough tetrahydrofuran to solubilize both the monomers and catalyst so that the terminal alkene is at its maximum concentration. The general procedure is described as follows. Monomer is placed in a dry, argon filled, 50 mL Schlenk tube equipped with a stir bar and septum followed by addition of second generation Grubbs Ru catalyst (100:1/monomer:catalyst); all additions being done while maintaining a positive Ar flow throughout the system. A mini mal amount of dry, degassed THF is then added producing a homogeneous solution, which is then stirred at 50 C for 144 hr. Dry THF was added daily to replace that which had evaporated out. The reaction was then sampled and assessed for complete conversio n of terminal olefin to internal olefin by 1NMR. Upon completion the reaction mixture is mixed with 30 mL of chloroform and transferred to an addition funnel where a few drops of tris(hydroxymethyl)phosphine (THP, 1M in isopropanol, 30 equiv to catalyst) is added and extracted with D.I. water (1 x 20 mL) and brine (1 x 20 mL). The remaining polymer solution is dried over MgSO4 followed by rotary evaporation to yield the purified polymer. Polymerization of [( S )-5 -Benzyloxycarbonylamino -5 -(1 -undec -10-enyl -d odec -11enylcarbamoyl) pentyl] -carbamic acid benzyl ester ( 3 -3 a). 1H NMR (300 MHz, CDCl3 1.71 (br, 37H), 1.732.05 (br, 5H), 3.013.23 (br, 2H), 3.72 3.93 (br, 1H), 3.99 4.19 (br, 1H), 4.79 5.17 (br, 5H), 5.22 5.44 (br, 2H), 5.475.75 (br, 1H), 5.78 6.15 (br, 1H), 7.31 (s, 10H). 13C NMR (75 MHz, CDCl3 29(m), 32.38, 32.82, 35.30, 40.53, 49.67, 55.16, 66.84, 67.20, 128.27, 128.72, 130.57, 136.41, 156.84, 171.33. FTIR (KBr pellet, cm1): 3423, 3314, 3057, 2989, 2930, 2856, 1719, 1685, 1648, 1519, 1456, 1423, 1266, 1137, 1028, 973, 897, 742, 704.

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71 Polymerizat ion of ( S )-6 -Benzyloxycarbonylamino -2 -[3 -(1 -undec -10-enyl -dodec -11enylcarbamoyl) propionylamino] hexanoic acid methyl ester (3 -4a). 1H NMR (300 MHz, CDCl3 1.76 (br, 38H), 1.902.16 (br, 5H), 3.023.23 (br, 2H), 3.71 (s, 3H), 4.524.66 (br, 1H), 4.995.25 (br, 3H), 5.28 5.45 (br, 2H), 6.266.44 (br, 1H), 7.247.40 (br, 5H). 13C NMR (75 MHz, CDCl3 29.53, 29.76, 29.86, 31.98, 32.82, 33.17, 33.29, 40.65, 47.89, 51.81, 52.43, 66.69, 128.18, 128.22, 128.66, 130.52, 136.85, 156.82, 173.21, 176.29. FTIR (KBr pellet, cm1): 3317, 2924, 2852, 1738, 1687, 1638, 1536, 1463, 1346, 1262, 1216, 1095, 1028, 967, 805, 731, 697. Polymerization ( S )-6 -Benzyloxycarbonylamino-2 -(2 -undec -10-enyl -tridec -12-enoyl -amino) hexanoic acid methyl ester ( 3 -5 a ). 1H NMR (300 MHz, CDCl3 1.77 (br, 36H), 1.772.06 (br, 6H), 2.402.63 (br, 4H), 3.06 326 (br, 2H), 3.72 (br, s, 3H), 3.79 3.90 (br, 1H), 4.47 4.61 (br, 1H), 5.04 5.17 (br, 2H), 5.225.47 (br, 3H), 5.696.13 (br, 1H), 6.697.02 (br, 1H) 7.20 7.40 (br, 5H). 13C NMR (75 MHz, CDCl3 52.44, 52.69, 66.94, 128.43, 128.88, 130.76, 137.14, 157.00, 171.86, 172.66, 173.10. FTIR (KBr pellet, cm1): 3318, 3070, 2963, 2924, 2852, 1746, 1686, 1637, 1542, 1442, 1364, 1262, 1207, 1097, 1024, 871, 801, 697, 465. Polymerization of (S) methyl 6 -(((9H -fluoren -9 -yl)methoxy)carbonylamino) -2 -(2 -(undec 10-enyl)tridec-12-enamido)hexanoate (3-6a). 1H NMR (300 MHz, CDCl3 1.78 (br, 36H), 1.782.20 (br, 7H), 3.023.32 (br, 2H), 3.69 (br, s, 3H), 4.15 4.34 (br, 1H), 4.33 4.46 (br, 2H), 4.544.67 (br, 1H), 4.845.10 (br, 1H), 5.26 5.53 (br, 2H), (br, 1H) 5.97 6.25 (br, 1H), 7.27 7.36 (br, 2H), 7.367.44 (br, 2H), 7.487.68 (br, 2H), 7.70 7.89 (br, 2H ). 13C NMR (75 MHz, CDCl3 27.90, 29.41, 29.70, 29.86, 32.24, 32.81, 33.18, 33.28, 40.68, 47.44, 48.08, 51.70, 52.50, 66.78, 120.14, 125.20, 127.20, 127.84, 130.50, 141.47, 144.13, 156.74, 173.20, 176.21. FTIR (KBr

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72 pellet, cm1): 3319, 2923, 2851, 1943, 1902, 1731, 1688, 1638, 1536, 1449, 1346, 1261, 1025, 802, 758, 734. Deprotection Chemistry of the 9 -fluorenylmethyloxycarbonyl (Fmoc) Lysine Polymer Removal of the Fmoc protection group from the side chain amine of lysine was performed by charging a 250mL round-bottom flask with the dry polymer 3 6a and dissolving it into THF. Upon complete dissolution of the polymer, piperidine (2.0 equiv) was pipetted into the reaction mixture and allowed to stir for 2 hr at room temperature with monitoring by TLC The solution was then concentrated by rotary evaporation and the polymer was precipitated in diethyl ether. The resultant polymer was analyzed by 1H NMR and IR. Deprotection of (S)methyl 6 -(((9H -fluoren -9 yl)methoxy)carbonylamino) -2 -(2 -(undec -10enyl) tridec -12-enamido)hexanoate (3-6b). 1H NMR (300 MHz, CDCl3, ppm): 1.78 (br, 36H), 1.782.20 (br, 7H), 3.023.32 (br, 2H), 3.73 (br, s, 3H), 4.56 4.77 (br, 1H), 4.84 5.10 (br, 1H), 5.265.53 (br, 2H), (br, 1H) 5.976.25 (br, 1H). FTIR (KBr pellet, cm1): 3308, 2927, 2852, 1744, 1638, 1541, 1464, 1262, 1062, 803. Results and Discussion Polymer Design and Synthesis All four lysine monomers are synthesized from either the 9,9 amine diene ( 3 -1 ) or the 9,9 acid diene ( 3 -2 ) premonomers as shown in Figure 3 1. Premonomer 3 -1 lends itself to the attachment of amino acid s or peptides through there C termini whereas premonomer 3 -2 is opposite, with attachment to amino acids or peptides through their N termini. Adding succinic anhydride to premonomer 3 -1 transforms the reactive functionality of the diene to that of a car boxylic acid while also introducing a spacing group between the amino acid and the diene. M onomer 3 -6 was synthesized using a similar method as monomers 3 -5 by using standard

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73 peptide coupling chemistry with the 9,9 acid diene ( 3 -2 ) and the hydrochloride s alt of H Lys(Fmoc) OMe. N H2 9 9 O O H 9 9 H N 9 9 N H H N O O O O O 3 3 H N 9 9 O O N H H N O O O O C H3 3 4 9 9 N H H N O O O O O C H3 3 5 9 9 N H H N O O O O O C H3 3 6 H N O N H H N O O O O C H3 n H N N H H N O O O O O n N H H N O O O O O C H3 n N H H N O O O O O C H3 n O 3 3 a 3 4 a 3 5 a 3 6 a i i i i v v vi vi vi vi 3 1 3 2 i i i Figure 3 1. Lysine polymers synthesized w/ varied connectivity through a spacer or directionally through its C or N terminus: (i) HOLysNHCBz, HOBt, DIC, THF (ii) succinic anhydride (iii) HLysCBzOMe HCl HOBt, DIC, TEA, THF (iv) HLysCBzOMe HCl, HOBt, DIC, TEA, THF (v) HLysFmocOMe HCl, HOBt, DIC, TEA, THF (vi) 2nd Generation Grubbs Catalyst, THF Coupling conditions included using the reagents 1 -hydroxybenzatriazole (HOBt), 1,3 diisopropylcarb odiimide (DIC), and 1 equivalent of triethylamine (TEA) to neutralize the amino acid salt (Figure 3 1). The reaction was carried out in THF due to the increased solubility of HOBt and amino acid in this solvent. Purification was accomplished by column chr omatography

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74 using hexane:ethyl acetate. Pure monomers 3 -3 to 3 -6 were obtained as white solids in a 47% 80% yield. All four monomers were polymerized under the same conditions including a 120 hr polymerization period traditionally used by our group to m aximize couplings for high molecular weights. At the end of this 120 hr reaction period, each polymer was analyzed for confirmation that no residual terminal olefin remained with either 1H or 13C NMR. Each polymer was readily soluble in either chloroform or tetrahydrofuran making them easily manageable for post polymerization analysis or manipulation. Polymers 3 -3a and 3 -5a could be cast as flexible films on a Teflon plate, while polymers 3 -4a and 3 -6a were too brittle for successful film casting. The syn thesis of precision polyolefins using a step growth, condensation type polymerization with symmetry built directly into the monomer allows the preparation of materials with pendant amino acid or peptide groups evenly spaced along the polyethylene backbone. The four polymers ( 3 -3a to 3 -6a in Figure 3 1) have the amino acid lysine branched off of the backbone. Polymer 3 -3a has lysine connected through its C terminus with both its N terminus and amine side chain capped with benzyloxycarbonyl (CBz) protecting groups. Polymer 3 -4a has a succinic acid spacer between the lysine and backbone and otherwise is similar to 3 -5 a Polymer 3 -5a has the amino acid connected through its N terminus with the C terminus protected with a methyl ester and the amine side chain protected with CBz. Polymer 3 6a has analogous connectivity as polymer 3 -5a but rather employs the 9 fluorenylmethyloxycarbonyl (Fmoc) protecting group rather than the CBz group. Molecular Weight and Physical Properties Each of these lysine monomers a nd their respective unsaturated polymers were examined for structural purity using 1H NMR, 13C NMR, and IR spectroscopy, elemental analysis, and mass spectrometry. Molecular weights and thermal behavior were measured using GPC, TGA,

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75 and DSC (Table 3 1). Each polymerization yielded a high melting, high molecular weight polymer with a polydispersity close to 2 which is predicted by its step growth polymerization mechanism. Table 3 1 Molecular weights and thermal behavior of lysine monomers and correspondi ng polymers. Monomer Monomer Tm (C) Monomer Hmelt (J/g) Polymer Mw (g/mol1) PDI Polymer Tm (C) Polymer Hmelt (J/g) 3 102 21.6 3a 49,000 2.07 101 13.9 4 130 88.0 4a 17,000 1.78 108 3.5 5 106 67.0 5a 56,000 2.10 85 18.6 6 141 101.0 6a 57,000 1.82 118 23.1 Deprotection Chemistry Free amine or carboxylic acid functional groups must be protected to avoid metathesis catalyst poisoning during polymerization; consequently, all ADMET bio-olefins synthesized to date bear protecting groups on the ami no acid or peptide moieties prior to and during polymerization. The initial protection group used in the synthesis of these polymers was the CBz group, with the thought that hydrogenation of the olefin in each repeating unit and deprotection of the amino acid could be performed simultaneously in one step. Surprisingly, hydrogenation driven deprotection proved not to be feasible for polymers 3 -3a to 3 -5a Attempts to remove the CBz group with 72 hours of hydrogenation using Pd/C in toluene with 300 PSI H2 resulted in only hydrogenation of the backbone. The protecting group itself remained untouched. A variety of harsher hydrogenation conditions were employed, e.g. higher temperatures (>100 C), higher pressures (600 PSI H2), the use of Pearlmans catalyst which is well known for the removal of benzyl groups, and the use of formic acid/Pd(OH) hydrogen transfer type hydrogenation/deprotection; all methodologies proved to be ineffective in removing the protecting group.

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76 Inaccessibility of the protection gr oup to the conditions designed to remove it could be the result of the amino acid branches collapsing on themselves to form micelles, which in effect screen them from hydrogenation regardless of the rigor associated with attempting to do so. The core of t hese micelles would be made up of the aromatic protection groups and the amino acid connecting them to the polyethylene outer shell. A variety of alcohols were used along with toluene in the hydrogenation step in an attempt to break up possible micelle fo rmation, but these added solvents had no effect in deprotecting the amino acids themselves. In order to bypass the difficulty associated with deprotection due to the apparent formation of micelles in the hydrogenation/deprotection step, an alternate prote ction strategy was employed, one that utilized a more labile amine protecting group. The 9fluorenylmethyloxycarbonyl (Fmoc) amine protecting group, originally published by Carpino107,108 and quickly implemented into the synthesis of peptides via the Merrifield technique,109 was chosen due to its ease of removal under mild basic conditions which could be performed prior to the hydrogenation step. In fact, deprotection of the Fmoc protected amine polymer 3 -6a pro ved possible at room temperature in THF using 2 equivalents of piperidine. Figure 2 shows the general base mechanism for piperidine to deprotect the Fmoc group. Evolution of carbon dioxide was immediately observed, as was fulvene via TLC assessment. The reaction was stirred for two hours, concentrated on the rotary evaporator, and then added to cold diethyl ether to precipitate the deprotected polymer overnight. Spectroscopic measurements (1H NMR and IR) confirmed complete deprotection of the polymer; in particular, the lack of aromatic proton peaks in the NMR and the absence of aromatic overtones at 1943cm1 and 1902cm1 in the IR, unequivocal evidence for the success of this reaction.

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77 O N H O O H N O O 9 9 H H N P i p e r i d i n e O N H O O H2N 9 9 C O2 T H F + 36a 36b Figure 3 2. Deprotection mechanism of the Fmoc group using piperidi ne. Thermal and X -Ray Characterization of Lysine Monomers and Polymers Data in Table 3 1 shows that all the protected monomers and polymers are semicrystalline materials with reasonably high melting points. The differential scanning calorimetry curves in Figures 3 and 5 amplify this point. Such crystallinity in amino acid modified polyolefins is unusual, since heretofore polymers of this nature have lacked sufficient structural regularity to allow crystallization to occur. This crystallization phenomenon is observed in spite of the fact that the stereocenters along the polymer backbone are atactic in nature. The DSC of polymer 3 -4a has substantially more complicated thermal transitions than the sharper melts observed in polymers 3 -3a and 3 -5 a The succini c acid spacer included in 3 -4a allows for better branch separation from the backbone. This promoted phase separation of branch and backbone allows for a small endotherm at 35 C, which could correspond to the unsaturated backbone melting, and a broad mel t (25 110 C) corresponding to the protection group that can not form as well a defined crystal as without the spacer. Each polymer shows a decreased melt temperature but is still relatively proximal to the monomers melting temperature. The enthalpies of melting for the polymers are all less than their respective monomers; this is

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78 most likely due to the crystalline regions not being as well defined from the polymers added conformational constrictions. Figure 3 3. DSC second heating scans for the CBz protected monomers 3 -5 and respective polymers 3a -5a Wide angle (WAXS) and small angle (SAXS) data allow for more definitive structural analysis. The SAXS data demonstrate that structural order present is not representative of long range order. Lon g sample to -detector distances (230 cm) display only amorphous behavior. At

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79 WAXS. None of the polymers have any evidence of longrange order that might be ind icative of crystalline spacing typical for folded chain lamella that stack alternately with amorphous regions, ie, which would be the structured feature expected for bulk crystallization of polymer chains. The WAXS data for both the monomers and their respective polymers is shown in Figure 3 4, where two observations are of value. First, the crystalline structure is dissimilar to high density polyethylene, (HDPE); compare with the WAXS data for HDPE, also shown in Figure 3 4. This result suggests that if the polymer backbone were involved in crystallization, then the corresponding structure is significantly distorted from the thermodynamically favorable conditions of polyethylene. More than likely, the bulky amino acid branches precisely placed at every 21st carbon prevent polyethylene -like crystallization, meaning the semicrystalline nature of the polymer is directly (and only) a result of the presence of the protected amino acid branch itself. Second and equally as interesting, the crystalline nature of t he polymer resembles the crystalline structure of the monomers, albeit with some WAXS peaks slightly shifted. This result suggests that the crystalline packing of the monomer is somewhat preserved after polymerization, further supporting the notion that t he protected amino acid branch, and not the polymer backbone itself, drives the crystallization process.

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80 Figure 3 4. WAXS data for the precipitated CBz protected lysine monomers and resultant polymers. The intensity units are arbitrary. As such, th e peak height is a function of the amount of polymer in the xray beam and the x-ray flux. The intent of these experiments was to assess the crystalline structure, not the degree of crystallinity. While future research is needed to elucidate these eff ects, the protecting group itself appears to be responsible for these crystallization phenomena. In the case of monomer 3 -6 and polymer 3 -6 a removal of the Fmoc protecting group renders the polymer amorphous (Figure 3 5). It should be noted that even wit h the side chain amine on polymer 3 -6a deprotected, the C terminus is still capped as a methyl ester. Additional hydrolysis of this C terminal ester could impart enough additional hydrogen bonding, to the already available amide and amine hydrogen bonding to reintroduce sufficient order for some crystallinity.

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81 Figure 3 5. DSC second heating scans for the monomer, protected polymer, and deprotected polymer for the Fmoc lysine materials. Conclusions A family of four ethylene based polymers with the a mino acid lysine on every 21st carbon along the backbone has been prepared and characterized. Thermal and X ray analysis reveal that these polymers have a crystalline structure similar to that of their respective monomers. Crystallinity is driven by the pr ecision placement of the branch (and perhaps just the protecting group itself), rather than the polymer chain. Deprotection of the amino acid functionality although initially found to be difficult, perhaps due to self organization of the amino acid

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82 branch es into micellar structures, can be readily performed using the Fmoc protection strategy. These resulting deprotected amorphous materials could prove to be potentially beneficial in engineering a bioactive surface that could readily respond to changes in its in vivo environment. Developing an understanding of the factors influencing the self organization and crystallization behavior of these bio -olefins will become important in applying bio -olefins as biomimetic materials. Future Work This project has lim itless possibilities for future studies. From a synthetic viewpoint, the next person on this project needs to focus on specific applications. For instance, cell binding is one application that is of considerable interest for coatings. The tripeptide RGD (arginine, glycine, aspartic acid) is the smallest active peptide isolated from fibronectin that elicits cell binding. With a peptide that has a specific cellular response like cell binding, one could team up with a biochemist who knows how to culture ce lls and try to grow these cells on a surface and monitor cell adhesion. Targeting diseases could be studies by working with antibodies know for binding to specific diseases. Making a surface that could be used as a sensor for diagnosing certain diseases would be of incredible importance. Just as a Wang resin is used for the solid phase synthesis of peptides, one could use a film made by the polymers discussed here to act as a surface template to grow peptides off of. The possibilities capable with this project are only limited by the imagination.

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83 CHAPTER 4 POLYETHYLENE PRODRUGS USING PRECISELY PLACED PHARMACEUTICAL AGENTS Introduction. The combination of synthetic organic polymer chemistry, medicinal chemistry, and biochemistry have allowed for the en gineering of polymer based therapeutics designed for controlled degradation along with specific cellular uptake and response. Prof. Helmut Ringsdorf first recognized in 1975 the potential for polymer chemists and biologists to work together to make pharma cologically active polymers.110 This proposed model suggests five main components of a polymer therapeutic: the polymeric backbone, the drug, the spacer, the targeting group, and the solubilizing agent. All five components are examined in our research presented here along with their effects on these polyethylene polymer prodrugs. A polymer prodrug is a low molecular weight pharmaceutical immobilized on a polymeric carrier that in itself has no biological activity, thus no therapeutic effect.56,57,59 Polymer prodrugs require transformation to the active drug in order to illicit the desired therapeutic action. The ben efits of using such polymer systems include: (1) protect the drug from metabolism thus preserving its activity during circulation while improving transport to target organs or tissues; (2) improve the water solubility of an otherwise poorly soluble pharmac eutical; therefore, enhancing its bioavailability; (3) improve the drugs pharmacokinetics; (4) reduce the antigenic activity of the drug which leads to a less pronounced immunological response; (5) provide passive or active targeting of the drug to specif ic sites of action; (6) form advanced targeting delivery systems in which the polymer system targets the site where it will then release its drug. Any combination of these six factors makes polymer therapeutics a desirable field of research, since all cou ld potentially improve dosage regimes for a patient.9,57,111

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84 Nonsteroidal anti -inflammatory drugs (NSAIDs) are a class of therapeutic agents with analgesic, antipyretic, and anti -inflammatory effects, but are oft en limited by their poor water solubility and relatively short plasma half -lives. Excessive use of NSAIDs often leads to irritation of the gastro enteric mucosal membranes and in some cases can lead to ulcers. Several polymer -drug approaches have been studied to solve these problems. Cecchi et al synthesized PEG derivatives of Ibuprofen with molecular weights ranging from 400 to 2400 Da/mol, these materials showed better anti inflammatory activity than the molar equivalent of free drug in the carrageen an induced rat paw assay.112 Davaran et al prepared acryloyl and methacryloylethyl ester and amide monomers of ibuprofen and indomethacin.62 It was shown that these prodrugs could be readily cleaved between the drug and spacer with either ester or amide linkages. Several other examples of various polymer backbones being attached to ibuprofen,62,113,114 indomethacin,62,115 naproxen,116 and ketoprofen113 have been studied for their hydrolytic behavior. Babazadeh has recently shown the cleavage of ibuprofen, ketoprofen, and naproxen from amide linkages on a poly2 (1 -propene)oxyethylamine backbone.117 119 The literature cited above focuses largely on improving the anti inflammatory behavior, or reducing the unwanted side effects, of NSAID polymer prodrugs. Although the polymer prodrugs presented in this manuscript also aim to achieve the same overall improvements in a new delivery system, our goal is to prove that our delivery system is tailorable to obtain any desirable release rate of drug. One of the most important components of a polymer prodrug is the stability of the drug -polymer linkage.58 Drugs and polymers can be conjugated using a variety of diffe rent functional chemical linkages: esters, carbonates, carbamates, methoxy esters or amides.

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85 Our research has been focused on the synthesis of drug branched polyethylene using acyclic diene metathesis (ADMET), a step growth, condensation type polymerization driven to high conversion by the removal of ethylene. This method has proven to be a useful synthetic technique in modeling structure -property relationships in linear low -density polyethylene (LLPE),30,43 in addi tion to developing new functionalized materials such as bioolefins.3840,44,54 The three variables that we are most interested in controlling for these materials are the choice of drug, the frequency at which this pharmaceutical is attached, and the type of spacer and its linkage between the drug and polyethylene backbone. Experimental Materials Reagents and chemicals were used as received from Aldrich chemical company unless otherwise noted. Diethyl ether and THF were used as dry solvents from the Aldrich keg system and dried over 4 bis(2,4,6 trimethylphenyl) 4,5 dihydroimidazol 2 ylidene] [benzylidene]ruthenium (IV) dichloride) was exclusi vely used and synthesized as previously described by Grubbs et al.85 Instrumentation and Analysis 1H NMR and 13C NMR spectra were recorded on a Varian Associates Mercury 300 spectrometer. Chemical shifts for 1H and 13C NMR were referenced to residual signals from CDCl3 (1H = 7.24 ppm and 13C = 77.23 ppm) with 0.03% v/v TMS as an internal reference. High resolution mass spectra (HRMS) data were obtained on a Finnegan 4500 gas chromatograph/mass spectrometer using the chemical ionization mode. Gel permeation chromatography (GPC) of polymers was performed at 40C using a Waters Associates GPCV2000 liquid chromatography system with an internal differential index detector (DRI) and two Waters Styragel HR

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86 HPLC grade tetrahydrofuran as the mobile pha se at a flow rate of 1.0 mL/min. Injection 0.07 w/v sample concentration. Retention times were calibrated against a minimum of nine narrow polystyrene standards purchased from Polymer Laboratories (Amherst, MA). The polymers were solvent -cast on a Teflon plate from chloroform or tetrahydrofuran and allowed to dry slowly. Differential scanning calorimetry (DSC) was performed on a TA Instruments Q1000 equipped with a liquid nitrogen cooling accessory calibrated using s apphire and high purity indium metal. All samples were prepared in hermetically sealed pans (4 7 mg/sample) and were referenced to an empty pan. A scan rate of 10 C per minute was used. Melting temperatures were taken as the peak of the melting transitio n, glass transition temperatures as the mid point of a step change in heat capacity. Thermal experiments were conducted as follows: samples were heated through the melt to erase thermal history, followed by cooling at 10 C per minute to 150 C, and then heated through the melt at 10 C per minute. Data reported reflects this second heating scan. Method of Hydrolysis Two 10 mmol monobasic and dibasic phosphate solutions were made in 1 L volumetric flasks with 50% (v/v) water and 50% (v/v) THF. The monobas ic solution was then added to the dibasic solution until a pH of 8 was obtained. This buffer was sealed in a pyrex bio -jar and stored for no more than three days before being used. The pH=2 buffer was made by adding phosphoric acid to a 50% (v/v) water a nd 50% (v/v) THF solution until a pH of 2 was obtained. Extinction coefficients ( ) were obtained for both Ibuprofen and Naproxen at each pH using five different drug concentrations in the corresponding buffer to determine

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87 For the actual hydrolysis st udy, polymer was dissolved in 3 5 mL of THF and then added to 5 mL of the corresponding phosphate buffer. This slightly cloudy solution was then added to dialysis tubing and placed in exactly 250 mL of the buffer. Aliquots were taken every hour on the ho ur for 33 hours. Each aliquot was analyzed at the max of the corresponding drug and the concentration was determined by using the calculated extinction coefficient. A blank was run for reference by dissolving each polymer in THF and running it like a no rmal hydrolysis experiment but with dry THF instead of an aqueous buffer. No Ibuprofen was observed in solution, which proves that all drug observed during hydrolysis experiments is actually drug being freshly cleaved from the backbone. Monomer Synthesis 2 -(Undec -10-enyl)tridec -12-enoic acid (4 -1). To a 500 mL three neck round-bottom flask equipped with a stir bar and addition funnel was added diethyl malonate (7.16 g, 45 mmol), 11 -bromoundec 1 ene (23.0 g, 99 mmol), and 150 mL THF. NaH (3.22 g, 134 mmo l) was added through a powder funnel over 10 minutes and then allowed to stir at room temperature for 1 h and at reflux for an additional day. The reaction was monitored by TLC using a 3:1 hexanes:ethyl acetate mobile phase. More NaH or alkenyl bromide i s added if monoalkylated product exists. The mixture was cooled to room temperature and water was added slowly to neutralize the remaining NaH. Then 100 mL of water (total ~ 125 mL) and ethanol (100 mL) were added along with KOH (15 g, 375 mmol) and the reaction was refluxed for 24 hr. The reaction was monitored for the disappearance of the diester using TLC (3:1 hexanes:ethyl acetate mobile phase). The solution was neutralized with concentrated HCl and extracted with diethyl ether. Following the evaporation of the diethyl ether, Decalin (40 mL), and a catalytic amount of DMAP were added to a 250 mL round -bottom flask equipped with a stir bar and a condenser. The flask was lowered into a 190C oil bath and decarboxylation

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88 was observed by excessive frot hing caused by the decarboxylation. Once the frothing ceased the reaction was stirred for an additional 2 h and the reaction was allowed to cool to room temperature. The crude mixture was flashed through a plug of silica gel using hexane as the eluent to remove the Decalin the Decalin could be observed as a clear ring moving through the plug. Once the Decalin was removed, the eluent was switched to ethyl acetate to remove the desired product. 1H NMR (300 MHz, CDCl3 1.55 (br, 28H), 1.63 (m, 4H), 2.05 (q, 4H), 2.06 (q, 4H), 2.37 (m, 1H), 4.98 (m, 4H), 5.82 (m, 2H). 13C NMR (75 MHz, CDCl3 29.15, 29.34, 29.68, 29.75, 29.77, 32.37, 34.03, 45.75, 114.31, 139.44, 183.14. 2 -(U ndec -10-enyl)tridec -12-en -1 -ol (4 -2). To a 250 mL three neck round-bottom flask equipped with a stir bar was added 125 mL of dry THF upon which LiAlH4 powder (4.6g 109 mmol) was stirred in slowly. To this slurry was added impure 9,9 acid in the form of a colorless oil (10 g, 27 mmo l) over 15 minutes via syringe. The reaction was placed under Ar and left to stir at room temperature for 18 hr. The reaction was quenched with water and acidified with concentrated HCl. A small amount of diethyl ether was added and the solution was ext racted with 1M HCl (2 x 50mL) and brine (2 x 50mL). The organic layer was dried over MgSO4 and concentrated down to a light yellow oil. This product was purified by flash chromatography using 9:1 (hexane:ethyl acetate) mobile phase yielding 4.1 g of the desired primary alcohol. 1H NMR (300 MHz, CDCl3 2.03 (q, 4H), 3.54 (d, 2H), 4.96 (m, 4H), 5.81 (m, 2H). 13C NMR (75 MHz, CDCl3 26.94, 28.99, 29.20, 29.57, 29.67, 30.14, 30.98, 33.88, 40.57, 65.61, 114.13, 139.21.27.58, 29.15, 29.34, 29.68, 29.75, 29.77, 32.37, 34.03, 45.75, 114.31, 139.44, 183.14.

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89 General Coupling of the 9,9 Primary Alcohol to Either the Ibuprofen or Naproxen Drug Molecules To a flame dried three neck round -bottom flask equipped with stir bar and was added the 9,9 alcohol (1.25 equiv to drug) under argon. To this oil was added 150 mL of dry THF followed by the addition of either the Naproxen or Ibuprofen drug molecule (usually 5 grams), DMAP (1.25 equiv to drug) and EDCI (1.25 equiv to drug) upon which the reaction turned cloudy. The re action was sealed and kept stirring at RT for 24 hr until the reaction mixture went clear and grayish sticky salts crashed out of solution. After monitoring completion by TLC, 100 mL of water was added to dissolve the salts along with 50 mL of diethyl eth er to separated organic from aqueous. The organic layer was washed with water (2 x 100 mL) and brine (2 x 100 mL) and then dried over MgSO4. The resultant monomers were purified via column chromatography to give colorless oils in 70 80% yields. (S) -2 -(un dec -10-enyl)tridec -12-enyl 2 -(4 -isobutylphenyl)propanoate (4 -3). The pure product was obtained in 78% yield after purification using flash chromatography using 14:1 (hexane:ethyl acetate) mobile phase. 1H NMR (300 MHz, CDCl3 1.141.40 (m b, 32H), 1.47 (d, 3H), 1.54 (b, 1H), 1.83 (m, 1H), 2.03 (q, 4H), 2.42 (d, 2H), 3.67 (q, 1H), 3.94 (o, 2H), 4.93 (m, 4H), 5.80 (m, 2H), 7.06 (d, 2H), 7.18 (d, 2H) 13C NMR (75 MHz, CDCl3 30.14, 30.39, 31.34, 31.44, 34.03, 37.53, 45.28, 45.52, 67.44, 114.31, 127.40, 129.42, 138.13, 139.41, 140.57, 175.03. FT IR (KBr pellet): 3077, 2926, 2854, 1736, 1641, 1513, 1465, 1383, 1330, 1242, 1201, 1166, 1094, 1072, 1022, 993, 909, 848, 799, 722, 636, 551. EI/HRMS [M +1]: calcd for C37H62O2, 539.4823; found, 539.4809. Anal. Calcd for CHNO: C, 82.47; H, 11.60. Found: C, 82.44; H, 11.73.

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90 (S) -2 -(undec -10-enyl)tridec -12-enyl 2 -(6 methoxynapthalen -2 -yl)propanoate (4 -4). The pure product was obtained i n 71% yield after purification using flash chromatography using 14:1 (hexane:ethyl acetate) mobile phase. 1H NMR (300 MHz, CDCl3 1.40 (br, 33H), 1.57 (d, 3H), 2.02 (m, 4H), 3.83 (q, 1H), 3.89 (s, 3H), 3.97 (m, 2H), 4.94 (m, 4H), 5.80 (m, 2H), 7.0 8 7.13 (m, 2H), 7.39 (m, 1H), 7.66 (m, 3H). 13C NMR (75 MHz, CDCl3 18.45, 26.81, 26.87, 29.15, 29.35, 29.68, 29.73, 29.77, 30.09, 31.38, 31.47, 34.02, 37.52, 45.82, 55.43, 60.55, 67.62, 105.76, 114.30, 119.09, 126.12, 126.50, 127.21, 129.15, 129.43, 133.88, 136.04, 139.40, 157.80, 174.92. FT IR (KBr pellet): 3075, 3062, 2975, 2926, 2854, 1905, 1734, 1638, 1607, 1506, 1484, 1464, 1417, 1393, 1376, 1349, 1325, 1264, 1231, 1218, 1178, 1122, 1092, 1059, 1035, 994, 958, 924, 909, 889, 851, 810, 747, 722, 668, 555, 475. EI/HRMS [M +1]: calcd for C38H58O3, 563.4459; found, 563.4466. Anal. Calcd for CHNO: C, 81.09; H, 10.39. Found: C, 81.34; H, 10.45. General Coupling of the Drug to Either Decanediol or Tetraethylene glycol To a flame dried 250 mL three neck round -bottom flask with a stir bar was added the drug (2g of either Ibuprofen or Naproxen). This material was dissolved in 100mL of dry chloroform and then the carbonyl diimidazole (1.1 equiv to drug) was slowly added over 5 minutes upon which lots of CO2 bubbles evolved for approximately 10 minutes. The solution was allowed to stir for 2 hr at RT to ensure complete formation of the activated acid. An excess of diol (4 equiv to drug of either decanediol or tetraethylene glycol) was added quick ly and allowed to stir at RT for 48 h. Upon completion of the reaction, 25 mL of chloroform was added and this organic layer was washed with water (2 x 75 mL) and brine (2 x 75 mL) and was dried over MgSO4. The organic layer was then concentrated to a se mi -viscous oil and was purified by flash chromatography using 1:1 (ethyl acetate:diethyl ether) for the tetraethylene glycol esters and 1:1 (hexane:THF) for the decanediol esters.

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91 (S) -2 -(2 -(2 -(2 -hydroxyethoxy)ethoxy)ethoxy)ethyl 2-(4 -isobutylphenyl)propano ate (4 -5). The pure product was obtained in 92% yield after purification using flash chromatography using 14:1 (hexane:ethyl acetate) mobile phase 1H NMR (300 MHz, CDCl3 (d, 3H), 1.84 (m, 1H), 2.43 (d, 2H), 2.66 (s, 1H), 3.50 3.80 (m, br, 15H), 4.22 (m, 2H), 7.08 (d, 2H), 7.20 (d, 2H) 13C NMR (75 MHz, CDCl3 64.01, 69.22, 70.49, 70.68, 70.69, 70.77, 72.65, 127.34, 129.42, 137.82, 140.62, 174.84. EI/HRMS [M +1]: calcd for C21H34O6, 383.2482; found, 383.2481. (S) -10-hydroxydecyl 2 -(4 -isobutylphenyl)propanoate (4 -6). The pure product was obtained in 62% yield after purification using flash chromatography using 14:1 (hexane:ethyl acetate) mobile phase 1H NMR (300 MHz, CDCl3 7 (d, 6H), 1.201.1.30 (br, 12H), 1.46 (d, 3H), 1.54 (m, 4H), 1.82 (m, 2H), 2.02 (s, 1H), 2.42 (d, 2H), 3.60 (t, 2H), 3.65 (q, 1H), 4.03 (t, 2H), 7.06 (d, 2H), 7.18 (d, 2H) 13C NMR (75 MHz, CDCl3 18.59, 22.52, 25.89, 28.66, 29.27, 29.54, 29.62, 30.31, 3 2.91, 45.19, 45.36, 63.08, 64.90, 127.29, 129.39, 138.05, 140.54, 174.99. (S) -2 -(2 -(2 -(2 -hydroxyethoxy)ethoxy)ethoxy)ethyl -2 -(6 -methoxynaphthalen -2 yl)propanoate (4 -7). The pure product was obtained in 68% yield after purification using flash chromatography using 14:1 (hexane:ethyl acetate) mobile phase.1H NMR (300 MHz, CDCl3 (s,br, 1H), 3.48 3.88 (m, br, 14H), 3.88 (q, 1H), 3.89 (s, 3H), 4.23 (t, 2H), 7.11 (d, 1H), 7.15 (d, 1H), 7.40 (d, 1H), 7.43 (d, 1H), 7.67 (s, 1H), 7.71 (s, 1H) 13C NMR (75 MHz, CDCl3 18.64, 45.45, 55.40, 61.81, 64.08, 69. 14, 70.40, 70.56, 70.60, 70.66, 72.59, 105.67, 119.07, 126.10, 126.40, 127.22, 129.03, 129.38, 133.80, 135.75, 157.74, 174.73. EI/HRMS [M +1]: calcd for C22H30O7, 407.2064; found, 407.2044.

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92 (S) -10-hydroxydecyl 2 -(6 methoxynaphthalen -2 yl)propanoate (4 -8). The pure product was obtained in 56% yield after purification using flash chromatography using 14:1 (hexane:ethyl acetate) mobile phase 1H NMR (300 MHz, CDCl3 1.30 (br, 12H), 1.42 (s, 1H), 1.52 (m, 4H), 1.55 (d, 3H), 3.61 (t, 2H), 3.82 (q, 1H), 3.89 (s, 3H), 4.04 (t, 2H), 7.1 (s, 1H), 7.13 (d, 1H), 7.38 (d, 1H), 7.65 (s, 1H), 7.66 (s, 1H), 7.69 (s, 1H) 13C NMR (75 MHz, CDCl3 28.75, 29.33, 29.57, 29.64, 33.01, 45.76, 55.52, 63.27, 65.08, 105.84, 119.11, 126.12, 126.50, 127.27, 129.16, 129.48, 133.88, 136.09. General Coupling of the 9,9 Acid Diene to Either Decanediol Ester Drugs or Tetraethylene Glycol Ester Drugs To a flame d ried three neck round -bottom flask equipped with stir bar and placed under Ar was charged the 9,9 acid diene. To this solid was added 150 mL of dry THF followed by the addition of either the decanediol ester drug or the tetraethylene glycol ester drug (1. 25 equiv to the 9,9 acid diene), DMAP (1.25 equiv to the 9,9 acid diene) and EDCI (1.25 equiv to the 9,9 acid diene) upon which the reaction turned cloudy. The reaction was sealed and kept stirring at RT for 24 h until the reaction mixture went clear and grayish sticky salts crashed out of solution. After monitoring completion by TLC, 100mL of water was added to dissolve the salts along with 50mL of diethyl ether to separate organic from aqueous. The organic layer was washed with water (2 x 100mL) and b rine (2 x 100mL) and then dried over MgSO4. The resultant monomers were all purified via column chromatography to give colorless oils in 75% to 90% yields. (S) -14-(4 -isobutylphenyl) -13-oxo -3,6,9,12-tetroxapentadecyl 2 -(undec -10-enyl)tridec -12enoate (4 -9 ). The pure product was obtained in 82% yield after purification using flash chromatography using 14:1 (hexane:ethyl acetate) mobile phase. 1H NMR (300 MHz, CDCl3 1.201.60 (br, m, 34H), 1.82 (m, 1H), 1.98 (q, 4H), 2.31 (br, 1H), 2.41 (d, 2H), 3.503.75 (br, m, 13H), 4.19 (m, 4H), 4.91 (m, 4H), 5.78 (m, 2H), 7.05 (d, 2H), 7.17 (d, 2H). 13C NMR (75 MHz,

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93 CDCl3 29.65, 29.73, 29.75, 30.34, 32.59, 33.98, 45.22, 45.81, 63.22, 64.03, 69.27, 69.48, 70.73, 70.76, 70.83, 114.29, 127.37, 129.45, 137.87, 139.37, 140.65, 174.82, 176.65. FT IR (KBr pellet): 3076, 2926, 2855, 1736, 1640, 1512, 1460, 1383, 1350, 1250, 1200, 1167, 1138, 994, 909, 850, 800, 723, 635, 551. EI/HRMS [M +1]: calcd for C45H76O7, 729.5664; found, 729.5689. Anal. Calcd for CHNO: C, 74.13; H, 10.51. Found: C, 74.27; H, 10.55. (S) -10-(2 -(4 -isobutylphenyl)propanoyloxy)decyl 2 -(undec -10-enyl)tridec -12-e noate (4 -10). The pure product was obtained in 80% yield after purification using flash chromatography using 14:1 (hexane:ethyl acetate) mobile phase. 1H NMR (300 MHz, CDCl3 1.201.65 (br, m, 51H), 1.82 (m, 1H), 2.01 (q, 4H), 2.28 (m, 1H ), 2.42 (d, 2H), 3.65 (q, 1H), 4.04 (q, 4H), 4.92 (m, 4H), 5.78 (m, 2H), 7.06 (d, 2H), 7.18 (d, 2H). 13C NMR (75 MHz, CDCl3 18.66, 22.57, 25.97, 26.18, 27.67, 28.75, 28.94, 29.14, 29.33, 29.36, 29.43, 29.67, 29.72, 29.76, 30.37, 32.75, 34.00, 45.25, 45.41, 46.05, 64.29, 64.91, 114.30, 127.34, 129.44, 138.12, 139.38, 140.58, 174.96, 176.82. FT IR (KBr pellet): 3450, 3076, 2926, 2855, 1900, 1735, 1640, 1512, 1465, 1384, 1367, 1331, 1244, 1200, 1165, 1094, 1072, 1022, 993, 909, 848, 800, 723, 636, 551. EI/HRMS [M +1]: calcd for C47H80O4, 709.6129; found, 709.6111. Anal. Calcd for CHNO: C, 79.60; H, 11.37. Found: C, 79.76; H, 11.43. (S) -14-(6 -methoxynaphthalen -2 -yl) -13-oxo -3,6,9,12-tetroxapentadecyl 2 (undec -10enyl)tridec -12-enoate (4 -11). The pure pro duct was obtained in 78% yield after purification using flash chromatography using 14:1 (hexane:ethyl acetate) mobile phase. 1H NMR (300 MHz, CDCl3 1.45 (br, m, 32H), 1.55 (d, 3H), 2.00 (q, 4H), 2.32 (m, 1H), 3.453.65 (m, br, 12H), 4.20 (q, 1H), 4.21 (s, 3H), 4.22 (m, 4H), 4.92 (m, 4H), 5.76 (m, 2H), 7.057.15 (m, 2H), 7.38 (d, 1H), 7.67 (d, 3H). 13C NMR (75 MHz, CDCl3

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94 45.50, 45.78, 55.43, 63.19, 64.09, 69.20, 69.42, 70.65, 70.69, 70.71, 105.74, 114.27, 119.10, 126.13, 126.41, 127.25, 129.08, 129.41, 133.85, 135.78, 139.33, 157.81, 174.70, 176.61. FT IR (KBr pellet): 3451, 3075, 2926, 2855, 1735, 1638, 1607, 1506, 1484, 1463, 1418, 1393, 1376, 1350, 1324, 1264, 1232, 1218, 1177, 1150, 1034, 994, 909, 852, 811, 747, 723, 669, 554, 476. EI/HRMS [M +1]: calcd for C46H72O8, 753.5300; found, 753.5305. Anal. Calcd for CHNO: C, 73.37; H, 9.64. Found: C, 73.46; H, 9.75. (S) -10-(2 -(6 -methoxynaphthalen -2 yl)propanoyloxy)decyl 2-(undec -10-enyl)tridec -12enoate (4 -12). The pure product was obtained in 76% yield after purification using flash chromatography using 14:1 (hexane:ethyl acetate) mobile phase. 1H NMR (300 MHz, CDCl3 1.65 (br, m, 50H), 1.99 (q, 4H), 2.29 (m, 1H), 3.82 (q, 1H), 3.89 (s, 3H), 4.04 (t, 4H), 4.94 (m, 4H), 5.79 (m, 2H), 7.05 7.15 (m, 2H), 7.39 (d, 1H), 7.66 (d, 3H) 13C NMR (75 MHz, CDCl3 25.98, 26.14, 27.66, 28.74, 28.92, 29.13, 29.32, 29.40, 29.60, 29.66, 29.71, 29.75, 32.74, 33.99, 45.72, 46.04, 55.45, 64.29, 65.03, 105.78, 114.29, 119.10, 126.09, 126.45, 127.24, 129.14, 129.44, 133.86, 136.05, 139.38, 157.81, 174.87, 176.83. FT IR (KBr pellet): 3447, 3075, 2926, 2854, 1733, 1638, 1607, 1506, 1484, 1464, 1418, 1393, 1374, 1350, 1325, 1264, 1231, 1218, 1176, 1122, 1092, 1070, 1035, 994, 909, 851, 810, 746, 722, 662, 522, 475. EI/HRMS [M +1]: calcd for C48H76O5, 733.5766; found, 733.5768. Anal. Calcd for CHNO: C, 78.64; H, 10.45. Found: C, 78.56; H, 10.47. General Polymer Synthesis. Monomer was pipetted into a dry 100 mL Schlenk tube equipped with a stir bar and glass stopcock and dried by heating the vessel in an oil bath at 50 C under full vacuum (103 mmHg) for 24 hr. After 24 hr the reaction vessel was backfilled with Argon and first -genera tion Grubbs Ru catalyst (100:1/monomer:catalyst) was added. The full vacuum was placed back on the

PAGE 95

95 polymerization reaction after .5 hr. Additional catalyst was added 60 h into the polymerization to ensure maximum possible couplings. Upon completion of 120 h of reaction time and confirmation of complete conversion of terminal to internal olefin by 1NMR, the polymerization was quenched in THF or CHCl3 with a few drops of ethyl vinyl ether added to the solvent. The ruthenium catalyst was removed via compl exation by extracting the organic layer with tris(hydroxymethyl)phosphine (THP) 1M solution. The resultant organic layer was washed concentrated NaHCO3 (1 x 30 mL) and brine (1 x 30 mL) and dried over MgSO4 followed by rotary evaporation to yield the pure polymer. P oly9,9TEGIbuprofen (4 -13a). 1H NMR (300 MHz, CDCl3 1.45 (br, 28H), 1.46 (d, 3H), 1.56 (br, 4H), 1.82 (m, 1H), 2.00 (q, 4H), 2.32 (m, 1H), 2.41 (d, 2H), 3.50 3.71 (br, m, 13H), 4.154 24 (b, m, 4H), 5.29 5.36 (br, 2H), 7.05 (d, 2H), 7.18 (d, 2H) 13C NMR (75 MHz, CDCl3 22.58, 25.79, 27.43, 27.63, 29.44, 29.54, 29.73, 29.81, 29.90, 29.99, 30.36, 32.64, 32.83, 45.18, 45.21, 45.85, 63.21, 64.04, 68.15, 69.26, 69.47, 70.72, 70.74, 70.81, 127.37, 129.46, 130.05, 130.50, 137.85, 140.66, 174.86, 176.70. FT IR (KBr pellet): 3624, 3530, 3451, 2927, 2854, 2252, 1948, 1734, 1640, 1613, 1512, 1464, 1383, 1366, 1349, 1321, 1250, 1200, 1167, 1140, 1074, 1022, 967, 849, 801, 723, 636, 550. P oly9,9DecanediolIbuprofen (4 -14a). 1H NMR (300 MHz, CDCl3 1.45 (br, 44H), 1.46 (d, 3H), 1.61 (br, 4H), 1.82 (m, 1H), 1.94 (br, 4H), 2.28 (m, 1H), 2.42 (d, 2H), 3.65 (q, 1H), 4.04 (q, 4H), 5.305.37 (br, 2H), 7.06 (d, 2H), 7.17 (d, 2H) 13C NMR (75 MHz, CDCl3 26.17, 27.44, 27.72, 28.74, 28.93, 29.37, 29.44, 29.56, 29.64, 29.66, 29.74, 29.81, 29.91, 30.01, 30.38, 32.80, 32.85, 45.24, 45.40, 46.10, 64.31, 64.94, 127.34, 129.44, 130.06, 130.52, 138.10, 140.60, 175.00, 176.89. FT IR (KBr pellet): 3451, 2926, 2854, 2686, 1899, 1734, 1512, 1465,

PAGE 96

96 1422, 1383, 1367, 1332, 1319, 1244, 1201, 1166, 1094, 1072, 1022, 967, 883, 848, 800, 779, 723, 635, 551. P oly9,9OHIbuprofen (4 -15a). 1H NMR (300 MHz, CDCl3 1.32 (br, 32H), 1.47 (d, 3H), 1.56 (br, 1H), 1.82 (m, 1H), 1.95 (q, 4H), 2.42 (d, 2H), 3.66 (q, 1H), 3.93 (m, 2H), 5.37 (br, m, 2H), 7.05 (d, 2H), 7.17 (d, 2H) 13C NMR (75 MHz, CDCl3 29.60, 29.79, 29.85, 29.89, 29.94, 30.04, 30.19, 30.40, 31.36, 31.46, 32.87, 37.53, 45.27, 45.51, 67.42, 127.39, 129.43, 130.09, 130.55, 138.10, 140.59, 175.07. FT IR (KBr pellet): 2924, 2853, 1735, 1512, 1462, 1383, 1320, 1242, 1201, 1165, 1093, 1070, 1021, 966, 846, 799, 721. P oly9,9TEGNaproxen (4 -13b). 1H NMR (300 MHz, CDCl3 1.45 (br, 32H), 1.55 (d, 3H), 1.92 (br, 4H), 2.30 (br, m, 1H), 3.47 3.66 (br, m, 12H), 3.81 3.89 (br, m, 4H), 4.10 4.26 (br, t, 4H), 5.305.39 (br, 2H), 7.08 (br, s, 1H), 7.10 (d, 1H), 7.37 (br, d, 1H), 7.40 (br, d, 1H), 7.65 (br, s, 1H), 7.68 (b r, s, 1H). 13C NMR (75 MHz, CDCl3 45.54, 45.89, 55.49, 63.23, 64.15, 69.25, 69.47, 70.69, 105.75, 119.17, 126.18, 126.47, 127.30, 129.11, 129.46, 130.07, 130.52, 133.89, 135.81, 157.83, 174.79, 176.73, 188.01. FT IR (KBr pellet): 3451, 2925, 2854, 1734, 1634, 1607, 1506, 1484, 1463, 1418, 1392, 1376, 1349, 1324, 1264, 1231, 1218, 1176, 1034, 967, 926, 887, 852, 810, 746, 722, 522, 476. P oly9,9DecanediolNaproxen (4 -14b). 1H NMR (300 MHz, C DCl3 1.50 (br, 48H), 1.55 (d, 3H), 1.94 (br, 4H), 2.27 (br, m, 1H), 3.82 (q, 1H), 3.88 (s, 3H), 4.04 (t, 4H), 5.315.40 (br, 2H), 7.09 (br, s, 1H), 7.13 (d, 1H), 7.38 (br, d, 1H), 7.40 (br, d, 1H), 7.65 (br, d, 1H), 7.69 (br, s, 1H). 13C NMR (75 MHz, CDCl3): 32.79, 32.83, 45.70, 46.08, 55.45, 64.30, 65.04, 105.73, 119.10, 126.08, 126.45, 127.25, 129.11,

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97 129.43, 130.05, 130.51, 133.84, 136.02, 157.78, 174.90, 176.87. FT IR (KBr pellet): 3446, 3059, 2926, 2854, 2685, 2319, 2058, 1905, 1734, 1635, 1607, 1507, 1484, 1465, 1419, 1393, 1374, 1350, 1326, 1264, 1231, 1218, 1177, 1122, 1092, 1070, 1035, 968, 925, 888, 851, 810, 746, 722, 687, 669, 523, 475. P oly 9,9OHNaproxen (4 -15b). 1H NMR (300 MHz, CDCl3 1.40 (br, 33H), 1.55 (d, 3H), 1.93 (br, 4H), 3.654.15 (br, m, 6H), 5.30 5.39 (br, 2H), 7.08 (br, s, 1H), 7.12 (d, 1H), 7.36 (br, s, 1H), 7.39 (br, s, 1H), 7.65 (br, s, 1H), 7.67 (br, s, 1H). 13C NMR (75 MHz, CDCl38, 29.81, 29.87, 30.16, 31.43, 32.88, 37.54, 45.83, 55.47, 67.63, 105.75, 119.11, 126.14, 126.53, 127.25, 129.15, 129.46, 130.57, 133.89, 136.04, 157.80, 175.01. FT IR (KBr pellet): 3444, 3059, 2925, 2853, 1905, 1734, 1634, 1607, 1506, 1483, 1464, 1418, 1392, 1375, 1348, 1324, 1263, 1231, 1217, 1176, 1121, 1091, 1060, 1034, 967, 925, 887, 850, 808, 746, 722, 668, 522, 474. Results and Discussion Polymer Design and Synthesis All prodrug monomers were made from the acid ( 4 -1 ) and alcohol (4 -2 ) diene premono mers. Similar to the prep used by Hopkins et al ,40 the synthesis begins with the 9 spacer alkenyl bromide double substitution reac tion on diethyl malonate using NaH as the base. Soponification of the diester is then performed using basic hydrolysis to yield the 9,9 diacid. Decarboxylation is carried out at 185 C in Decalin until evolution of carbon dioxide ceases as monitored visu ally. The pure white solid, 9,9 diene acid ( 4 -1 ), is obtained from recrystallization in pentane. Reduction of ( 4 -1 ) using lithium aluminum hydride (LAH) yields the diene alcohol (4 -2 ) in nearly a quantitative yield resulting in a colorless oil: Figure 4 1.

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98 B r 9 D i e t hyl M a l ona t e T H F N a H 9 9 O E t O O E t O E t ha nol N a O H 9 9 O H O O H O D e c a l i n O O H 9 9 2 O H 9 9 L A H T H F4 -2 4 -1 Figure 4 1. Acid and alcohol diene premonomer synthesis It was predicted that by introducing both hydrophobic and hydrophilic spacers between the drug and polyethylene backbone, the resulting drug-polymer spaced materials would have a profound effe ct on the rate of hydrolysis. As a control study to eliminate any effect of the spacer on hydrolysis rates, Ibuprofen and Naproxen were connected directly to the monomer backbone with no spacer introduced. The primary alcohol diene ( 4 -2 ) was coupled to t he carboxylic acid functionality of the NSAIDs with carbodiimide coupling chemistry. Ethyl diisopropyl carbodiimide (EDCI) was specifically used for this coupling due to its ability to effectively couple weaker nucleophiles such as alcohols in making rea ctive ester linkages. Both Ibuprofen (4 -3 ) and Naproxen ( 4 -4 ) diene couplings gave high yields and the urea salts produced from the EDCI precipitated from the reaction solution. Pure product was isolated at reactions end by filtering off the byproduct u rea salts followed by column chromatography: Figure 4 2.

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99 O H 9 9 O O O O O T H F, E D C I T H F, E D CI I b u p ro f en N a p r o x en 4 2 4 -3 4 -4 Figure 4 2. Synthesis of non-spaced Ibuprofen and Naproxen diene monomers Synthesis of the NSAIDs with the hydrophilic and hydrophobic spacers began with choosing the type and length of the appr opriate diol spacer. Decanediol and tetraethylene glycol (TEG) were chosen since they both exhibit minimal toxicity to living systems in addition to each maximizing its respective dislike or like of water. TEG is made up of a thirteen atom backbone and i s often referred to as a rogue stealth molecule in the body since this highly hydrophilic molecule induces no immunological response in addition to no site accumulation or toxicity.120 Decanediol has a twelve atom long ch ain and is minimally toxic but does potentially pose an accumulation risk in the body due to its hydrophobic properties. To eliminate decanediol from the body, enzymes are required to glucuronidate or sulphate the terminal alcohols of the aliphatic diol t o makes it sufficiently water soluble for elimination. Coupling either of these spacers to the drug can be accomplished in high yields using carbonyl diimidazole (CDI) as described in the Cecchi prep.112 For either case, the NSAID was dissolved in dry chloroform an d CDI is added. Upon addition of the CDI, the reaction bubbles fervently for approximately 45 min. The reaction was continually stirred for an additional hour to ensure the carboxylic acid of the drug was completely activated as the imidazole amide. An

PAGE 100

100 excess of either TEG or decanediol was then added to this activated acid to produce the drug TEG conjugates 4 -5 and 4 -7 or the drug -decanediol conjugates 4 -6 and 4 -8 (Figure 4 3) Both materials are purified via column chromatography to yield colorless oil s. The spacer linked drugs are ultimately connected to the acid diene premonomer ( 4 -1 ) via the same EDCI coupling chemistry implemented for the ester synthesized in Figure 4 3. All four prodrug monomers were purified with column chromatography to yield c olorless oils ( 4 -9 to 4 -12). O O 9 9 O 1 0 O O 9 9 O 4 O N N N N O H O O H O O O O O O O O O O O O O H O H O H O H 4 4 4 4 O O 9 9 O 1 0 O O 9 9 O 4 O O O O O O E D C I T H F E D C I T H F E D C I T H F E D C I T H F 41 41 41 41CD IC H C l3 t e t r a e t hyl e ne gl yc ol C H C l3 de c a ne di olCD I CD I CD IC H C l3 t e t r a e t hyl e ne gl yc ol C H C l3 de c a ne di o lI b u p r o f e n N a p r o x e n45 4 6 4 7 48 49 410 411 412 Figure 4 3. Synthesis of decanediol and tetraethylene glycol spaced Ibuprofen and Naproxen monomers Polymerization of all six prodrug monomers was performed by adding Grubbss 1st Generation Metathesis catalyst in a 1:100 ( catalyst to monomer ratio) to each of the six liquid monomers. The condensation reaction was carried out at 50 C under vacuum until NMR showed no trace of external olefin; the reaction usually required 96120 hours. Performing this reaction in the bulk gives a very high concentration of terminal olefin that promotes acyclic diene metathesis (ADMET) rather than ring closing metathesis (RCM), a side reaction product we try to avoid since macrocycles can greatly alter our desired material properties. Figur e 4 4

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101 shows the six polymerization products of the monomer reactions. The Ibuprofen materials are notated 13a-15a and the Naproxen materials are 13b -15b respectively. O O O O D r u g 9 9 O O 9 9 O O D r u g 10 4 O 9 9 O D r u g 1 s t G en era t i o n G ru b b s 1 s t G en era t i o n G ru b b s 1 s t G e n e rat i o n G ru b b s O O D r u g O O O O O O O O O O O O M e I bu p ro fe n N a p ro x e nD rugs : D r u g D r u g 4-13 4-14 4-15 a b Figure 4 4. Polymerization of spaced and non -spaced monomers Figure 4 4s reaction is a step growth, condensation type polymerization yielding high molecular weight polymer by using Le Chateliers principle to drive the reaction equilibrium to polymer by removal of the condensate -ethylene. The effect of this polymerization is a linear pol ymer with branches at exact locations along the backbone. For these materials the monomer has nine methylene units (n=9) between the branch and terminal alkene. This symmetrical monomer structure means that the resultant polymer will have 2n + 2 carbon a toms between the branches on the polyethylene backbone. For these 9,9 diene monomers, their respective polymers will have the drug branch on every 21st carbon along the polymer backbone.30,43 The polyethylene backbone of these precisely branched polymers makes them insoluble in water. Consequently, the target application for these materials is for functional bio -coatings and films, as in stents and other medical implants. Ideally, these specialized coatings would release their bound pharmaceutical at varied rates and the remaining polyethylene would continue to act as a robust, non toxic co ating. Changing the size of the alky diene side chains of the monomer could vary drug loading for this family of polymers. For a higher amount of drug

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102 loading, one would synthesize the 3,3 or 6,6 alcohol or acid diene monomers rather than the 9,9 shown i n Figure 4 1. Structural Analysis Examination of the 1H and 13C NMR spectra of the monomers and polymers indicates the complete transformation and control over primary structure afforded by ADMET. Figure 4.5 shows both the 1H and 13C NMR spectra of mono mers 4 -3 4 -11, and 4 -10 and their respective polymers. Analysis of the olefin region (5 6 ppm) in the 1H spectra shows the complete conversion of terminal olefin (4.9 and 5.8 ppm) to internal olefin (5.4 ppm) for all three monomers. Comparison of the 13C NMR spectra of each monomer and respective polymer shows the dissapearance of the signals corresponding to termial olefin shown by singlets at 114.3 and 139.4 ppm and formation of internal olefin through the polymerization (cis at 130.1 and trans at 130. 6 ppm). Neither the 1H nor the 13C NMR spectra show any detectable trace of terminal olefin, which is desired for maximization of molecular weight in the polymer.

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103 Figure 4 5. 1H and 13C NMR of three representative monomers with varying spacers

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104 Table 4 1. Molecular weight and thermal data: a reported in kg/mol and performed in THF at 40 C with calibration against polystyrene standards. b 10 C/min scan rate, values determined from second scan data. Polymer M n a PDI a T g b T m b 13a 15.1 2.29 52 41 14 a 56.9 1.78 49 31 15a 17.0 1.90 51 N/A 13b 30.7 1,84 34 N/A 14b 20.8 1.81 41 21 15b 13.4 1.77 28 N/A Thermal Analysis Each of the six polymers synthesized, regardless of the spacer or NSAID incorporated exhibits thermal stability under argon i n excess of 300 C as shown in Figure 4 6. No weight loss was observed before 350 C for each sample as shown by the straight line observed for weight loss respective to temperature. The poly9,9decanediolNaproxen ( 4 -12 ) polymer is the only material that shows minor weight loss before its decomposition temperature; this is a result from residual solvent evaporating from the sample. Residual solvent was difficult to remove from these polymers since each one was a tan, thick, viscous oil. Each polymer was spun at high speeds on the rotovap under strong vacuum to create a thin film with maximum surface area to evaporate solvent. Polymer samples were also placed in oil baths preset to 50 C under high vacuum and left to sit for up to five days. A polymer wa s considered dry when no residual peaks could be observed in the 1H NMR spectra. High thermal stability could prove to be an important property in processing such materials in the bulk. This stability also eliminates the possibility of heat being able to cleave drug from the polymer chain in storage or coating applications.

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105 Figure 4 6. Thermal degradation for polymer prodrugs. The Ibuprofen family is displayed on left and the Naproxen family on the right. Figure 4 7 displays the representative DSC s for the Ibuprofen and Naproxen polymers. For each Ibuprofen polymer the Tg is below 40 C and for the Naproxen materials the Tgs are at 40 C or just above. One explanation for these trends is that the Naproxen molecule is a little bigger than the Ibuprofen, which increases the size of the amorphous regions thus raising the Tg temperature. Previous ADMET studies have shown that both the melting temperature and melting enthalpy decrease as the size of the pendant moieties increases, it is believed thi s trend is supported for these materials as well. In both decanediol spaced polymers, the endothermic transitions observed are complicated. The transition appears to represent a melt just after the Tg. This transition is more pronounced in the decanedio l branched Ibuprofen material. In addition, a small melting transition is seen after the Tg in the tetraethylene glycol spaced Ibuprofen material as well. The large side chain branches pendant to the polyethylene backbone, in addition to the alkene poten tially kinking the ethylene run lengths out of an ideal packing orientation for lamellae, explain the lack of significant crystallinity in these polymers. It is expected that these materials would exhibit significant crystallinity once the backbone polyet hylene backbone was saturated. It was decided that the unsaturated family of polymer prodrugs would be

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106 characterized over the saturated since the unsaturated materials are usually significantly more soluble. Figure 4 7. DSC of polymer produgs. The I buprofen family is displayed on left and the Naproxen family on the right. Hydrolysis of Polymers Polymeric prodrugs are only useful if they are labile enough to release the covalently attached pharmaceutical. It was our goal to prove that these material s can hydrolyze and release the NSAID through chemical and enzymatic hydrolysis of ester linkages. Even though these materials are targeted for use as coatings, we decided to do both the enzymatic and chemical hydrolytic studies in organic solvents to bet ter test the reactivity of the esters as a consequence of the spacer. Figure 4 8 shows the chemical hydrolysis data for the Ibuprofen polymers in phosphate buffers at pH=2 and pH=8. All three Ibuprofen polymers hydrolyze to release roughly 40% of the tot al contained drug within 24 hr. Going into these studies, certain hypothesis were formulated that needed to be tested. The polymer expected to hydrolyze the fastest are the TEG based polymers with the slowest being the decanediol spaced materials, while an intermediate rate would probably be shown by the materials with no spacer. These expected results could be explained by examining the ability of water to come into contact with the ester. The more

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107 hydrophilic the local environment of the ester, the be tter chance water will have to permeate the sample and hydrolyze the ester. The TEG spacer is quite hydrophilic, thus its hydrolysis is expected to be the fastest. The opposite extreme found with the decanediol spacer could explain the slowest rate of hydrolysis. To determine a reference for the hydrolysis studies, each polymer prodrug was dissolved in 5 mL of dry THF and placed in dialysis tubing, which was then placed in a beaker of dry THF. The THF outside of the dialysis tubing was monitored for any drug released but none was observed for the first 24 hr. This experiment proves that the drug that is observed to be released from the polymer is not an artifact in addition it also supports that all of the drug is initially bound to the polymer backbon e and not physically absorbed or mixed in. A few interesting points can be deciphered from the drug release profiles displayed in Figure 4 8. The first is that up to 40% of the drug is released within 48 hr at pH=8. The fastest release profile at this pH was shown for the decanediol and non -spaced materials with the slowest rate of release displayed with the TEG spaced polymer. For the pH=2 phosphate buffer solution, the fastest release is given with the decanediol and the non -spaced material has the slo west release at this pH. Although these trends cannot be easily explained, we believe that the predicted spacer effects noted above would be more likely to observed in a non -organic, aqueous solvent environment as used with bio -coatings. It can be clearl y noted that more drug is released at pH=8. This demonstrates that the mechanism for this hydrolysis occurs better with base than acid. With increasing pH, more hydroxide ions, more total drug is cleaved faster than at pH=2. More hydrolysis studies are being run in order to place error bars on these curves so more reliable curves can be displayed.

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108 All of the release profiles show an initial burst of drug being released for the first 5 10 hrs and then the hydrolysis rate flattens out. This trend is bel ieved to occur as a consequence of availability of drug able to be cleaved. When each polymer is first placed in the buffer the solution in the dialysis tubing is cloudy. The solubility of these polymer prodrugs, even in a solution that is half THF, is m arginal. The burst of drug released at the beginning of these profiles is believed to be a consequence of the drug on the out edge of the polymer that is in contact with the buffer being cleaved off. As the drug is hydrolyzed from the polymer, the result ant polymer is believed to possibly become less soluble and collapse in on itself, thus trapping any uncleaved drug inside a hydrophobic shell. With minimal water being able to permeate this shell, the rate of drug hydrolysis quickly falls off. The enzym atic hydrolysis results have not been gathered yet due to the difficulty of finding an enzyme system that can be utilized in an organic medium. Several examples exist in the literature that use enzymes in organic media121,122 but availability and cost are factors in choosing a workable system for this work. One enzyme of particular interest is the Candida -Antarctica lipase. This enzyme is a non -specific esterase used in 10 mmol phosphate buffers that has demonstra ted activity in up to 40 volume percent toluene. Speculating what the hydrolysis data would look like for these polymers: only the two drug -spaced materials would be expected to show any enzymatic hydrolysis. The non-spaced polymer would be expected to s how no detectable enzymatic hydrolysis. The fastest polymer to hydrolyze via the enzyme would be expected to be the decanediol spaced polymer over the TEG spaced one. These results could be explained by examining the esterase performing the hydrolysis ac tion. In esterases, the active site on the enzyme is made up of a catalytic triad of amino acids that contains serine, histidine, and aspartic acid. The pocket within the enzyme in which these amino acids react is

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109 exceptionally hydrophobic. Remembering that like dissolves like, Ibuprofen and its hydrophobic spacer decanediol are the most likely to enter the hydrophobic pocket of the esterase and be enzymatically hydrolyzed. Although the hydrophilic spacer TEG also would allow for enzymatic hydrolysis, it would be at a decreased rate from the difficulty of such a hydrophilic group to enter a hydrophobic pocket. The complete lack of any esterase activity on the non-spaced polymers would be a direct consequence of the steric bulk the polymer chain has on the ability of the drugs ester to enter the reactive enzyme pocket to hydrolyze. Although the polymer backbone is very hydrophobic, it is far too large to be able to enter the enzyme for the drug to be cleaved and released. Figure 4 8. Chemical hyd rolysis rate profiles for the Ibuprofen Polymers in pH=2 and pH=8 phosphate buffers. Conclusions ADMET has proven to be effective in synthesizing polymers containing drug molecules connected through a variety of spacers or directly to the polyethylene bac kbone. In this work, Ibuprofen and Naproxen were linked to polyethylene at every 21st carbon along the backbone either directly or through a tetraethylene glycol or decanediol spacer. The material properties

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110 and hydrolytic stability of the spaced versus non -spaced polymers were significantly different. Esters were implemented as the linker for all drug -spacer and spacer -backbone connectivitys and these linkages were shown to be readily hydrolysable under chemical conditions. At pH=2, chemical hydrolysi s was fastest for the decanediol spaced and slowest for the non -spaced. For pH=8, the rate of hydrolysis was roughly equal for the decanediol spaced and non-spaced polymer with the TEG spaced being the slowest. Depending on the type of pharmaceutical res ponse desired and the amount of drug required to be loaded onto the polymer, these materials can be modified to suit ones needs. Further development in the chemistry associated with hydrolysis and more chemically complex drugs will be crucial in demonstr ating optimal pharmaceutical potential. Future Work The focus of this project needs to expand to three specific areas: (1) utilize alternate types of chemical linkers to vary hydrolysis rates; (2) make water soluble versions of the same type of materials; (3) expand the families of drugs used in these materials. Development of new linkers such as the alkylcarbonyloxymethyl (ACOM) and alkoxycarbonyloxymethyl (AOCOM) as developed in the Sloan research group to make the 9,9ACOMIbu and 9,9AOCOMIbu monomers ha s already been accomplished although complete purification is still a challenge: Figure 4 9. The 1H and 13C NMR spectra of these two monomers have been gathered but minor impurity peaks are observed in the spectra. Diane Turek has begun work on making mo re of both the Ibuprofen and Naproxen derivatives of the ACOM and AOCOM linkages. It is expected to see an increase in the rate of hydrolysis for these materials as the parent drug, formaldehyde, and resulting polymer are formed during this cleavage.

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111 O O H 9 9 O H 9 9 O O 9 9 O 9 9 O O O O O O 9 9 A C OM I bu 9 9 A OC OM I bu i i i Fi gure 4 9 Synthesis of ACOM and AOCOM spaced Ibuprofen monomers: (i) CO2Cl2 then trioxane and NaI then added to Ibuprofen. (ii) chloromethyl carbonochloridate and addition of Ibuprofen. As the hydrolysis rates are determined for the varieties of linkers possible for these materials, work can then be invested into making a family of water -soluble ADMET polymers. The advantages of such polymers would be a variety of new possible applications such as polymers that could be used intravenously rather than ins oluble coatings. Hydrolysis profiles of these water -soluble polymers would also be likely to show a higher percentage of total drug released since the hydrolyzed polymer would be less likely to collapse on itself and prevent drug from being released. A w ater soluble family of polymer prodrugs coupled with the polymers now synthesized for coating applications would further allow for more control in tuning a material for an optimal dosage regime for a patient. Figure 4 10 outlines the synthesis of the wate r -soluble premonomer that could be used to make such a family of polymer prodrugs. Each polymer mentioned in this chapter could be re -synthesized with this water -soluble premonomer shown in Figure 4 10. These polymers should be readily soluble in normal buffer solutions, this would be expected to lead to better release profiles. Perhaps the initial burst of released drug observed in Figure 4 8 for each polymer would not flatten out and hydrolysis

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1 12 would continue until 100% of the drug was released. Idea lly, a polymer prodrug should release its entire load of drug to be a feasible delivery device. H O O T r 4 B r 3 O O H 4 3 O O 3 3 O T s O O 3 3 O O 3 3 O O E t O E t O O O 3 3 O O 3 3 O O H +i i i i i i i v 9 9 TEG A c i d Figure 4 10. Proposed synthesis of 9,9 TEG Acid to make water -soluble polymers: (i) NaH in THF followed by deprotection with HCl; (ii) TsCl; (iii) NaH and et hyl malonate followed by soponification; (iv) heat in Decalin with a catalytic amount of DMAP.

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113 CHAPTER 5 PRECISION SUNSCREEN POLYMERS Introduction No one would question the importance of sunlight. Of the countless benefits the sun offers, it provide s the required energy needed for life on earth. With sunlight playing a part in everyones daily lives, the general public needs to become increasingly aware and more concerned about the effects of UV radiation on the skin. Skin cancer has become one of the leading forms of new cancers in the United States with 1.2 1.5 million new cases diagnosed annually. The solar radiation most dangerous to humans is ultraviolet, UVA (320 400 nm) and UVB (290 320 nm). UVB radiation can have a direct impact on c ell DNA and proteins123 and is responsible for acute damage such as sunburn in addition to longterm cellular damage including cancer. UVA radiation, although not directly absorbed by biological targets, has the ability to drastica lly impair cell and tissue function through a variety of pathways:66,124,125 (1) The longer wavelength UVA penetrates deeper into the skin than UVB where it can produce reactive oxygen species that have negative eff ects on connective tissue.126 (2) UVA is believed to be a contributor to photocarcinogenesis.127 (3) UVA is a strong inducer of immune suppression.128 (4) Exogeneous photosensitizaton has an action spectrum in the UVA range. (5) UVA is involved in idiopathic photodermatosis as most c ases of polymorphous light eruption.129 Consumers and the general public have been aware of the dangers that UV radiation cause for some time now but are under the impression that sunscreens give them the total protec tion they need when out in the sun. Sunscreens enable people to spend more time in the sun without getting sunburn. With more sunscreen products offering higher sun protection factors (SPF) and more convenient formulations such as nonaerosol sprays, people have assumed that they are completely safe from UV danger when wearing these sun blocks. This complete

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114 sense of safety is not warranted and research has been coming to light that proves that the sunscreen chromophores meant to protect one from UV radia tion can itself cause and promote oxidative damage to the skin.130 Hanson has shown that sunscreens enhance UV induced reactive oxygen species131 while Xu has demonstrated that sunscreens can delay the cell cycle and cause mitochondrial stre ss.67 Lip pold has published work demonstrating a variety of sunscreens that exhibit significant flux through the skin.64 Knowing that the cure to the problem can in fact be contributing to cause the disease its trying to prevent, creates a high demand research area that involves improving the UV protection systems we currently have. Sloan has been a pioneer in the area of dermal delivery of drugs. His work has focused on demonstrating the importance of three factors when considering whether a molecule will cross t he skin: (1) water solubility, Saq (2) lipid solubility, Slipid (3) molecular weight, MW.132 The relationship between these factors has been elegantly delineated with the equation: log J = x + y log Slipid + (1 -y) log Saq-z MW The variables x, y, and z are constants pertaining to the system under study and J is the flux. It is a common misconception that one only needs to improve one of these three properties to improve membra ne permeability but in reality there is a subtle link between all three factors. Preventing a sunscreen from passing into the skin is the problem that must be solved. It is our intention to solve this problem by attaching the sunscreen to a polymer bac kbone that will increase its molecular weight to the point of making it unable to enter the skin. In addition to preventing oxidative damage once the sunscreen enters the skin, locking the sunscreen at the skins surface would also decrease the chance of allergic reaction, which could lead to irritation. Although we are not the first group to attach sunscreens to a polymer,12,133 we intend to make polymers with a degree of control unable to be synthesized by other techniques. Our research

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115 has been focused on the synthesis of sunscreen branched polyethylene using acyclic diene metathesis (ADMET), a step growth, condensation type polymerization driven to high conversion by the removal of ethylene. This method has pr oven to be a useful synthetic technique in modeling structure -property relationships in linear low -density polyethylene (LLPE),30,43,54 in addition to developing new functionalized materials such as bio-olefins.39,40 The three variables that we are most interested in controlli ng for these materials are the choice of sunscreen, the frequency at which this chromophore is attached, and the type of spacer and linkage used for connecting the sunscreen and polyethylene backbone. Trends have emerged from consumers as to what they find important in their product: higher sun protection factors (SPF) values, broad spectrum of UV protection, and enhanced water and rub -off resistance. Film -forming polymers serve ideally as a great delivery material to spread a sunscreen over a surface in a uniform film. Depending on the backbone, the water and rub off resistance would be greatly improved in addition to potentially increasing the SPF. The mechanism by which a film is cast on the skin would vary with formulation, which is usually done by a lcohol -based systems that use alcohol soluble materials. If the polymer is completely dissolved in the formulation, the resultant polymer film could spread out as a thin layer whereas if the dissolved polymer was an emulsion, the discrete particles would pack together upon alcohol evaporation and form a different type of film consisting of a layer of these particles. Experimental Materials Reagents and chemicals were used as received from Aldrich chemical company unless otherwise noted. Diethyl ether a nd THF were used as dry solvents from the Aldrich keg system and dried over 4 -

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116 bis(2,4,6 trimethylphenyl) 4,5 dihydroimidazol 2 ylidene] [benzylidene]ruthenium (IV) dichloride) was exclusively used and synthesized as previously described by Grubbs et al.85 Instrumentation and Analysis 1H NMR and 13C NMR spectra were recorded on a Varian Associates Mercury 300 spectrometer. Chemical shifts for 1H and 13C NMR were referenced to residual signals from CDCl3 (1H = 7.24 ppm and 13C = 77.23 ppm) with 0.03% v/v TMS as an internal reference. High resolution mass spectra (HRMS) data were obtained on a Finnegan 4500 gas chromatograph/mass spectrometer using the chemical ionization mode. Gel permeation chromatography (GPC) of polymer 5 -3a was performed at 40C using a Waters Associates GPCV2000 liquid chromatography system with an internal differential index detector (DRI) and two Waters Styragel HR with HPLC grade tetrahydrofuran as the mobile phase at a flow rate of 1.0 mL/min. Injection 0.07 w/v sample concentrations. Retention times were calibrated against a minimum of nine narrow polystyrene standards purchased from Polymer Laboratories (Am herst, MA). The polymers were solvent -cast on a Teflon plate from chloroform or tetrahydrofuran and allowed to dry slowly. Differential scanning calorimetry (DSC) was performed on a TA Instruments Q1000 equipped with a liquid nitrogen cooling accessory ca librated using sapphire and high purity indium metal. All samples were prepared in hermetically sealed pans (4 7 mg/sample) and were referenced to an empty pan. A scan rate of 10 C per minute was used. Melting temperatures were taken as the peak of the m elting transition, glass transition temperatures as the mid point of a step change in heat capacity. Thermal experiments were conducted as follows: samples were heated through the melt to erase thermal history, followed

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117 by cooling at 10 C per minute to 1 50 C, and then heated through the melt at 10 C per minute. Data reported reflects this second heating scan. Monomer Synthesis U ndeca -1,10-dien -6 -ol ( 5 -1) Magnesium ( 4.89 g, 0.20 mol ) was added to a 500 mL three neck flask equipped with a reflux c ondense r and an addition funnel. T he reaction vessel was backfilled three times with Ar and flamed dried after each backfill. Dry THF (100 mL) was added, followed by the addition of 5 bromo1 pentene (25.0 g, 0.17 mol ) via dropwise addition by syringe. The sol ution was refluxed for 2 h to completely form the Grignard. Ethyl formate ( 5.64 g, 0.076 mol ) in 30 mL THF was added dropwise to the cooled mixture (0C), and the solution was allowed to warm slowly to room temperature and refluxed for 21 h. Hydrochloric acid (1M, 100 mL) was added, and the solution was extracted with ether (3 x 25 mL), washed with 1M HCl (1 x 30 mL), and washed with brine (3 x 20 mL). The solution was dried over MgSO4, followed by evaporation of the solvent to yield 14.18 g of the crude alcohol ( 5 -1 ). 1H NMR (300 MHz, CDCl3 2.15 (m, 10H), 2.012.15 (br, 4H), 3.57 (br, 1H), 4.885.04 (m, 4H), 5.705.85 (m, 2H). 13C NMR (75 MHz, CDCl3 U ndeca -1,10-dien -6 amine ( 5 -1a). To a 500 mL round -bottom flask equipped with an additi on funnel was added pyridinium chlorochromate (PCC) (26.0 g, 0.12 mol ), celite ( equal weight to crude alcohol ), and methylene chloride (100 mL) followed by the addition of the crude alcohol ( 5 -1 1 equiv. ). The reaction was stirred for 4 hr, diethyl ether (200 mL) was added and the mixture was filtered through a pad of silica gel. Solvent evaporation yielded 13.0 g of the crude ketone. To a 500 mL round -bottom fl ask was added the crude ketone dry methanol (225 mL), ammonium acetate ( 60g, 0.78 mol ), NaCNB H3 (25g, 0.40 mol ), and a spatula tip of crushed 4

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118 molecular sieves and the mixture was refluxed for 48 hr under N2. The crushed molecular sieves were filtered via Bchner filtration and deionized water (200 mL) was added to the filtrate, followed by ex traction with diethyl ether (3 x 50 mL). The organic layer was washed with 1 M NaOH (2 x 50 mL) and brine (2 x 30 mL) and dried over MgSO4. The solution was concentrated to a brown viscous oil, which was purified by flash column chromatography using a 3: 1:1 (hexane:ethyl acetate:methanol) mobile phase yielding 9.28 g of the desired 3,3NH2 (5 -1a ) product for an overall yield of 73 %. 1H NMR (300 MHz, CDCl3 1.70 (m, 8H), 2.01 2.15 (br,4H), 2.65 2.76 (br, 1H), 4.90 5.10 (m, 4H), 5.755.90 (m, 2H). 13C NMR (75 MHz, CDCl3 U ndeca -1,10-dien -6 yl 4 -(dimethylamino)benzoate 3,3OHPABA (5 -2). To a 100 mL single neck round bottom flask was charged 50 mL of keg dry CH2Cl2 and para -dimethylaminobenzoic acid (1.25 g, 7.5 mmol). To this slurry of undissolved PABA was added 1.75 mL of oxalyl chloride. The solution immediately started bubbling and the reaction was pl aced under heat and refluxed for 6 hr. After this time, all of the product acid chloride was dissolved to give dark forest green color. A shortpath distillation head was attached and the residual CH2Cl2 and oxalyl chloride was removed to leave a green po wder remaining in the flask. To this solid was added 50 mL of THF which dissolved the acid chloride to give a bluish green solution to which was added the alcohol 5 -1 No color change was observed upon addition of the alcohol but the solution immediately turned a brown green color upon addition of 1 mL of triethylamine (TEA). The reaction solution was refluxed for 12 hr and then extracted with diethyl ether (2 x 75 mL). The ether was washed with H2O (2 x 75 mL) and brine (2 x 75 mL) and then dried with MgSO4. The reaction was closely monitored by TLC with 9:1 (hexane:ethyl acetate). The product was purified with 3 successive recrystallizations using methanol/water: the product was dissolved in hot ethanol, and water was added till the solution became c loudy. The

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119 pure product 5 -2 (1.78 g, 78 %, mp = 117119 C) was then collected by filtration through a Kontes filtration apparatus and dried under vacuum (102 mmHg) for 48 hours. 1H NMR (300 MHz, CDCl3 4.95 (m, 4H), 5.08 (m, 1H), 6.66 (d, 2H), 7.90 (d, 2H) 13C NMR (75 MHz, CDCl324.85, 29.91, 33.85, 3401, 40.40, 73.74, 111.09, 114.85, 131.45, 138.84, 153.36. FT IR (KBr pellet): 3076, 2928, 2859, 2357, 1700, 1641, 1610, 1526, 1484, 1445, 1414, 1366, 1318, 1276, 1232, 1183, 1107, 1065, 995, 912, 829, 770, 748, 698, 635, 596, 508. EI/HRMS [M+]: calcd for C37H62O2, 315.2198 g/mo l; found, 315.2199 g/mol. 4 -(D imethylamino) -N -(undeca -1,10-dien-6 yl)benzamide 3,3NHPABA (5 -3). PABA (1.2 g, 7.2 mmol) and 1 -hydroxybenzotriazole (HOBt) (1.6 g, 7.5 mmol) were added to a 100 mL round bottom flask. The flask was equipped with a septum and, under argon, 1,3 diisopropylcarbodiimide (DIC) (1.14 g, 9.05 mmo l, 1.42 mL) and dry THF (50 mL) were added. The reaction vessel was equipped with a reflux condenser and stirred one hour at room temperature, followed by the addition of 5 -1a (1.00 g, 5.98 mmol) and 1 mL of TEA. Then the reaction was stirred for 12 hr un der reflux conditions. The insoluble urea was removed via gravity filtration, and THF was evaporated to yield a crude yellow solid. The product was purified with 3 successive recrystallizations using ethanol/water; the product was dissolved in hot ethanol, and water was added till the solution became cloudy. The pure product 5 -3 (1.78 g, 78 %, mp = 117119 C) was then collected by filtration through a Kontes filtration apparatus and dried under vacuum (102 mmHg) for 48 hours. 1H NMR (300 MHz, CDCl3 1.44 (m, 4H), 1.57 (m, 4H), 2.05 (m, 4H), 2.99 (s, 6H), 4.12 (br, 1H), 4.94 (m, 4H), 5.64 (d, 2H), 5.76 (m, 2H), 6.73 (d, 2H), 7.63 (d, 2H) 13C NMR (75 MHz, CDCl325.45, 33.87, 35.19, 40.26, 40.36, 49.36, 111.31, 114.87, 132.99, 138.85, 167.17.

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120 (E)-undeca -1,10-dien -6 yl 3 -(4 methoxyphenyl)acrylate 3,3OHMCA (5 4). To a flame dried three neck round -bottom flask equipped with stir bar and placed under Ar was charged the para -methoxycinnamic acid. To this solid was added 50 mL of dry THF followed by the addition of the alcohol 5 -1 (1.25 equiv to acid), DMAP (1.25 equiv) and EDCI (1.25 equiv) were added when the reaction turned cloudy. The reaction was sealed and kept stirring at RT for 24 hr until the reaction mixture went clear and grayish sticky sal ts crashed out of solution. After monitoring completion by TLC, 100 mL of water was added to dissolve the salts along with 50mL of diethyl ether to separate organic from aqueous. The organic layer was washed with water (2 x 100 mL) and brine (2 x 100 mL) and then dried over MgSO4. The resultant monomer 5 -4 was then purified via column chromatography with 9:1 hexane:ethyl acetate to yield a colorless oil in a 70% yield. 1H NMR (300 MHz, CDCl3 (m, 4H), 2.05 (m, 4H), 3.81 (s, 3H), 4.94 (br, m, 5H), 5.77 (m, 2H) 6.29 (d, 1H), 6.88 (d, 2H), 7.46 (d, 2H), 7.61 (d, 2H) 13C NMR (75 MHz, CDCl324.81, 33.77, 33.92, 55.54, 74.03, 114.49, 114.92, 116.29, 127.47, 129.86, 138. 70, 144.32, 161.50, 167.31. FT IR (KBr pellet): 3398, 3206, 3075, 3035, 3001, 2976, 2937, 2862, 2839, 2560, 2291, 2040, 1888, 1823, 1708, 1635, 1605, 1576, 1514, 1459, 1442, 1423, 1351, 1303, 1288, 1254, 1204, 1171, 1116, 1033, 986, 912, 865, 829, 810, 78 1, 741, 638, 554, 520, 437. EI/HRMS [2M +1]: calcd for C37H62O2, g/mol; found, g/mol. Anal. Calcd for C HNO: C, 76.79; H, 8.59. Fo und: C, 75.28; H, 8.56. General Procedure for Polymerization Monomer was transferred into a dry 25 mL Schlenk tube equippe d with a stir bar and glass stopcock and dried by heating the vessel in an oil bath at 50 C under full vacuum (103 mmHg) for 24 hr. After 24 hr the reaction vessel was backfilled with Argon and first -generation Grubbs Ru catalyst (250:1/monomer:catalys t) was added. The full vacuum was placed back on the polymerization reaction after .5 hr. Additional catalyst was added 60 hr into the polymerization

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121 to ensure maximum possible couplings. The polymerization reaction was monitored closely by 1H NMR to co nfirm that internal olefin was being produced. Upon completion of the reaction after 36 hr, the polymerization was quenched by opening the flask and adding 25 mL of toluene and 1 mL of ethyl vinyl ether. The polymer was purified by precipitation of the p olymer solution into 1.0 L of cold methanol. The polymer was then filtered and dried for characterization. Polymerization of (E) -undeca -1,10-dien -6 yl 3 -(4 methoxyphenyl)acrylate (5 -4a). 1H NMR (300 MHz, CDCl3 4.94 (br, 1.3 H), 5.34 (br, 1.8H), 5.73 (br, .2H), 6.25 (br, 1H), 6.85 (br, 2H), 7.43 (br, 2H), 7.58 (br, 1H). 13C NMR (75 MHz, CDCl324.81, 25.51, 25.61, 27.26, 32.66, 33.78, 34.08, 55. 54, 74.18, 114.47, 114.91, 116.32, 127.46, 129.86, 129.95, 130.43, 138.72, 144.24, 161.45, 167.32. Results and Discussion To begin the synthesis of sunscreens incorporated onto a polymer backbone, specific sunscreen moieties needed to be chosen as a start ing point. Para -dimethylaminobenzoic acid (PABA) and para -methoxy cinnamic acid (MCA) were chosen due to both their availability and limited functional groups on the molecule but also since both are currently being used in sunscreen products available on the market today (Figure 5 1). PABA is commonly used as a UV filter in sunscreen formulations but it has been determined that it increases the formation of particular DNA defects in human cells, thus increasing the risk of skin cancers.134 MCA is also used as a UV filter but it is also used to minimize the appearance of scars. MCA has also has raised safety concerns regarding its toxicity to mouse cells at concentratio ns lower than that found in any cosmetic formulation. There are conflicting reports on the ability of MCA to penetrate and enter the skin to cause damage.65 Both PABA and MCA are UV B absorbers and have respective -maxes at 304 and 291 nm in THF. It was our intention when we began this

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122 research that we could immobilize any sunscreen on a polymer backbone and preserve, if not improve, its ability to filter UV radiation. O H O N O H O O M e pa ra-dime thylam ino ben zoic acid ( P ABA) para-m ethoxy cinna mic a cid (M CA) Figure 5 1. Three sunscreen chromophores used for this study. To form a functional diene monomer that can be covalently linked to a sunscreen we made a symmetrical alcohol 5 -1 and amine diene 5 -1a (Figure 5 2). This synthesis of the alco hol was accomplished by doing a double Grignard on ethyl formate and then purifying the colorless oil by column chromatography. To convert the alcohol to the amine, the alcohol was first oxidized to the ketone using pyridinium chloroformate and then a red uctive amination was done by converting the ketone to an imine and then reducing it in methanol with sodium cyanoborohydride. This hydride compound is especially suited for reductions in traditionally polar protic solvents. The final amine product was pu rified via column chromatography to yield a light brown oil in reasonable yields. O H B r N H2 i i i5 1 5 1 a Figure 5 2. Premonomer synthesis: (i) Mg, diethyl ether, ethyl formate (ii) PCC, celite, CH2Cl2 followed with NH4OAc, NaCNBH3, dry MEOH. Leonard was able to clearly pr ove that ester linkages pendant to an ADMET synthesized polyethylene backbone are not especially stable to hydrolysis. Since it is the intention of these materials to prevent any sunscreen compounds from entering the skin, it is critical for the compound to stay attached to the backbone. Any hydrolysis at the skins surface or in the

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123 formulation would defeat the purpose of these materials so an amide linkage with less reactivity to hydrolysis was included as a group of materials to make. The coupling of the alcohol and amine diene premonomer structures to the sunscreen was accomplished as shown in Figure 53. Formation of the amide linkage in both the PABA and MCA cases was performed using simple carbodiimide coupling chemistry in THF. Products were pur ified by column chromatography. Formation of the ester linkages was not quite as easy to make as the amides. The MCA ester could be synthesized with a stronger carbodiimide reagent such as EDCI. This reaction is facilitated by the salt of a diimide that facilitates proton transfer during the coupling step that DIC is not able to do. Both EDCI and DIC were rendered useless in trying to form the ester between 5 -1 and PABA. Attempts were used with both reagents but no product was observed with DIC and onl y trace amounts were observed with EDCI. It is assumed the lack of reactivity is related to the steric effects of the phenyl ring on the urea forming intermediate of the carbodiimide. Ester synthesis was accomplished by converting PABA to the acid chlori de with oxalyl chloride followed with its coupling to the alcohol in the presence of TEA to form the final product and ammonium chloride salt.

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124 O H O N O H O O M e O O O M e N O O M e O O N N O N O O O M e N O O M e n n O O N N O N n nH H H H i ii iii iv 5 -2 5 -3 5 -4 5 -5 5-2a 5-3a 5-4a 5-5a v v v v Figure 5 3. Synthetic scheme for attaching sunscreens to diene premonomers: (i) CO2Cl2, CH2Cl2 and then TEA a n d 1 -1 ; (ii) DIC, THF, TEA, HOBt and 1 -1a ; (iii) EDCI, THF, DMAP and 1 -1 ; (iv) DIC, THF, TEA, HOBt and 1 -1a ; (v) first -generation Grubbs catalysts, vacuum (103 mmHg). Performing ADMET on these dienes was not necessarily considered to be a sure success. The alkene separating the phenyl ring and carboxylic acid on MCA was thought to be potentially problematic although allylic substituted alkenes have previously been shown to have difficulty in undergoing olefin metathesis.135 Regardless, metathesis was successfully carried out on the monomers. All of the monomers synthesized except 5 -4 were solids; thus, polymerizations had to be performed i n THF as previously reported in amino acid containing dienes designed for ADMET.39,40,55 We have assumed a molecular weight of over 5 kg/mol should be able to prevent the absorbance of any sunscreen materials into t he skin. As a consequence, metathesis

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125 polymerization need not be necessarily run for the complete removal of noticeable terminal alkene as done in other ADMET reaction where it is the goal to maximize MW. The ADMET reaction on these materials is usually run overnight to obtain MWs of 7 10 kg/mol. 1H NMR of these materials show residual terminal olefin dwarfed by the formation of internal olefin. Even with a polydispersity of 2 there will be minimal polymer chains under the 5 kg/mol threshold sought. I t should also be pointed out that it is unclear how small this MW can be to completely eliminate the possibility of any sunscreen from entering the skin. Perhaps monomer itself could reduce absorbance enough to make a positive effect although short alkyl and PEG chains have been shown to enter the skin as well. UV-Vis studies were performed on each sunscreen as purchased and compared to the absorbance of the monomer. All synthesized monomers have a slight red shift in absorbance for their max but are fo r the most part super imposable with the parent sunscreen (Figure 5 4 and 55). The corresponding polymers for each compound displayed no shift in max and was essentially unchanged with respect to the monomer (Figure 5 4). Concentrations were varied one hundred fold to note any concentration effects, which could be indicative of systems interacting with each other but no red or blue shifts were observed although THF is usually considered to be an excellent solvent for dissolving ADMET polymers. These absorbance trends are very encouraging since we would like to see the absorbance profile maintained throughout the functionalization of these sunscreens into polymers.

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126 Figure 5 4. UV -Vis absorbance for MCA with respective monomer and polymer. Figure 5 5. UV -Vis absorbance for PABA and its respective monomer. One interesting problem that was encountered with the MCA polymer 5 -4a and monomer 5 -5 was their tendency to undergo a 2+2 photodimerization. The first time monomer 5 -4 was polymerized the resul ting polymer went from a white fluffy solid upon rotovapory evaporation to a light brown insoluble film Figure 5 6. This film was peeled from the round bottom flask it crosslinked in and the film still has the shape of the flask since it was cured. Kathy Novak from the Wagener research group studied single -crystal -to -single -crystal photodimerization of MCA during her time in graduate school.136 It is now understood that although these materials make excellent UVB absorbers, although they are n ot especially stable since the resultant polymers

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127 crosslink upon exposure to 350 nm light. From a materials standpoint, these polymers could be well suited to applications requiring UV curable polymers. O O O O n nC ro s s l i n k e d M C A Po l y m e r O O O M e 5 -4a O O O M e 3 5 0 n m l i g h t n n Figure 5 6. Photodimerization of MCA polymers ( crosslinking) 4 tert -butyl 4 -methoxydibenzoylmethane (avobenzone or Parsol 1789) of the dibenzoylmethane group is a particularly interesting compound because it exhibits the strongest and widest absorption curve in the UVA range of all commercially availa ble UV filters. Avobenzone has a wavelength absorption maximum ( max) ranging from 350 nm to 365 nm depending on the solvent used.137 The structure is in constant equilibriu m between two tautomeric forms: the enol tautomer and the keto tautomer (Figure 5 7). The keto form exists as only one stereoisomer but the enol form can take on any of the four different geometric conformations when the alkene forms. The cis enols relat ive to the trans are substantially more stable due to the intramolecular hydrogen bond (chelated enol). These many tautomers of avobenzone give rise to the unique photochemical properties that give it advantages for use as a UV filter.

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128 O H O O O O H O ke t oe nol t a ut om e r i z a t i on O O O A vobe nz one En ol f orm K et o f o rm Figure 5 7 Diff erent structural isomers for Avobenzone. Upon initial inspection of avobenzone, one can immediately observe the two acidic protons on the methylene unit flanked by both benzoyl groups. Before much research was invested into understanding the factors tha t contribute to the UV absorbance of this molecule, we attempted to dialkylate the avobenzone to transform it into a functional diene monomer for ADMET. The reaction was expected to work like the dialkylation of diethyl malonate but the alkylation reactio ns here failed due to monoalkylation of both the carbon and oxygens (Figure 5 8). No dialkylation was observed, most likely due to the reduced nucleophilicity of the substituted enolate with the counter ion being able to be chelated by the 1,3 diketo moie ty. A pKa difference of at least 2 units is usually observed for the difference of acidity of a diketo versus a diester moiety, this increased acidity for the diketo molecule can be assumed to translate to reduced nucleophilicity. This reduced nucleophil icity of the diketo anion is why monosubstitution is the dominant product. It was assumed that one could preferentially O or C alkylate using conditions suggested in Houses book but a mixture was always obtained of the different alkylated products with the majority of the product being unreacted starting material.

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129 Separation of the two monoalkylated products has proven difficult but 1H NMR shows the shifts corresponding to all three molecules shown in Figure 5 8. O O O i O O O O O O + C alkyla tion O-a lkylati on A v oben zone Figure 5 8 Synthetic attempt to a lkylate avobenzone: (i) NaH, THF In order for a sunscreen to be useful as a functional material, it must be able to impart a high screening efficiency and be photostable. Light -induced decomposition quickly minimizes the photoprotective ability of the f ilm by forming other non-UV absorbing compounds. Irreversible photo degradation of avobenzone has been shown in a variety of solvents and in concentrated solutions.138140 The photo degradation of avobenzone arise from the excitation of an electron from a transition and then conversion from a singlet to triplet state. The triplet state allows for the avobenzone to fragment a bond homolytically and form either a benzoyl or phenacyl radical. The introduction of another molecule to quench the triplet state of avobenzone would greatly stabilize the formulation. One particular stabilizing molecule of interest is octocrylene. Vollhardt and Bonda performed a number of studies in this area to test the ability of n umerous possible avobenzone stabilizers.141,142 Vollharts study showed octocrylene maintains the stability of 90% of the avobenzone in the formulation compared to only 20% maintained with no stabilizer. Such stabilization is a necessity to make these avobenzone formulations useful for any duration of time. One added benefit of incorporating octocrylene into our fami ly of avobenzone polymers is that octocrylene also functions as a UVB

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130 absorber. A more inclusive range of UV absorbance would only impart more utility as a much more useful sunblock for the consumer. Figure 5 9 shows the proposed synthesis of an octocry lene diene monomer similar to the synthetic methodology already performed for the PABA and MCA monomers. The octocrylene monomer could be homopolymerized and the resultant polymer blended in an approximate 1:1 ratio of avobenzone polymer. Another option to get both molecules in the same polymer matrix would be to perform a copolymerization of both the octocrylene monomer and the avobenzone monomer to produce a random copolymerization. Either way, octocrylene should stabilize avobenzone and the polymer ca n stabilize the entire system by blocking both the radiation from the sun and preventing the sunscreen from entering the skin. One factor that could be considered a drawback for octocrylene is that the cyano functionality contained in the chromophore has been know for some time in the chemical community to be a metathesis killer. The ability of the cyano group to act as a chelater towards the ruthenium center of the catalyst kills all activity towards metathesis. It is hoped for octocrylene that the cyan o group is tied into the system of the chromophore and its chelating ability will be negated. If this molecule cannot undergo metathesis, a suitable electron -withdrawing substitute will be implemented such as an ester or amide functional group. O O H N O O N 3 3 O ct o cr y l e n e U V B A b so r b e r a n d S t a b i l i ze r O n O N i i i Figure 5 9 Synthesis of avobenzone stabilizer polymer: (i) EDCI, THF, DMAP and 5 -1 ; (ii) first generation Grubbs catalysts, vacuum (103 mmHg).

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131 Conclusions ADMET has proven to be effective in synthesizing polymers containing sunscreen moieties connected dir ectly to the polyethylene backbone through either an ester or amide linkage. In this work, para -dimethylaminobenzoic acid and para -methoxycinnamic acid are linked to polyethylene at every 9th carbon along the backbone. The resultant materials are amorphous with high thermal decomposition temperatures. The UV -Vis absorbance of these materials is minimally changed from the parent sunscreen chromophore from which each polymer was built. Depending on the range and amount of absorbance required, these materi als can be modified to suit ones needs by varying the chromophore used, stabilizer incorporated, and ethylene run lengths between sunscreen branches. Further development in the chemistry associated with avobenzone and its stabilizers will be crucial in d emonstrating and formulating sunscreen materials that enable more stable and uniform UV protection across both the UVA and UVB range. Future Work It is mentioned in this chapter that the copolymerization of a UVA and UVB absorbing monomer is important for both wide UV absorption but also for the stabilization of the Avobenzone -like absorber. The future work of this project revolves around making a useful material with structural precision to yield a welldefined polymer tuned to the environment it will be applied to. The synthesis of a diene monomer with both the UVA and UVB chromophores attached is desired to eliminate the random incorporation of the two monomers separately which could potentially cause proximity considerations for triplet transfer betwe en absorbers. Figure 5 10 proposes a general synthetic scheme that could be implemented to make such a monomer. It is important to note that this monomer is not symmetric so some precision would be lost in the resultant polymer but the step growth nature of the polymerization can guarantee that the UVA

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132 chromophore will always be adjacent to at least one UVB chromophore. The head to tail coupling of this A B monomer would lead to an A -A, A B, and B B connectivity in equal amounts along the polymer backbon e. n n x O O O O N C O O O O U V A A bs or be r U V B A bs or be r a nd U V A S t a bi l i z e r A v ob e n z on e D e r i vat i ve O c t ac r yl e n en = 3 x = 3 t o 8 B r n n O E t O O E t O n n x H O O H O O n n x H O O H i i i i i i i v Figure 5 10. Proposed synthesis of UVA/UVB monomer: (i) NaH, alkenyl bromide, and diethyl malonate; (ii) NaH and alkyl dibromide, soponification and decarboxylation; (iii) lithium aluminum hydride in THF; (iv) octacrylene coupling followed by avobenzone coupling.

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141 BIOGRAPHICAL SKETCH James Klein Leonard, son of Ji m and Dee Leonard, was born in Bad Axe, Michigan on May 28, 1979 where he resided for 18 years. Upon finishing high school in May 1997, James attended the University of Michigan where he double majored with B.S. degrees in Chemistry and Spanish. While in Ann Arbor, James conducted three years of undergraduate research on the synthesis and characterization of vinazene polymers with Professor Paul G. Rasmussen. This research experience and introduction to polymer chemistry encouraged James to pursue his gr aduate studies at the University of Florida (Gainesville, FL) in organic and polymer chemistry under the advisement of Prof. Ken Wagener in August 2003. Upon completion of the Ph.D. requirement s in Spring of 2009, Dr. James K. Leonard will be relocating t o Madison, WI to attend law school for his J.D. at University of Wisconsin. James will maintain his Chemistry knowledge at Wisconsin because he was offered a teaching position at Wisconsin for a full tuition waiver.