Regioselective Synthesis of Curdlan Derivatives Ruoran Zhang Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Macromolecular Science and Engineering Kevin J. Edgar, Chair S. Richard Turner Judy S. Riffle Maren Roman Abby R. Whittington Nov. 10, 2015 Blacksburg, VA Keywords: curdlan, curdlan derivatives, regioselective synthesis, amination at C-6, tight junction opening, water-soluble Copyright © 2015, Ruoran Zhang Regioselective Synthesis of Curdlan Derivatives Ruoran Zhang Abstract Curdlan, a (1→3) linked linear homopolysaccharide composed of β-D-glucan, is produced by the bacterium Alcaligenes faecalis var. myxogenes. Several strategies to synthesize chemically modified curdlan derivatives have been reported, but there have been few reports of regioselective functionalization at specific positions of the curdlan backbone, especially of aminated curdlan derivatives which have remarkable potential in biomedical and pharmaceutical applications. We demonstrate herein the design, synthesis and characterization of a family of regioselectively aminated curdlan derivatives including 6-deoxy-6-(bromo/azido/amino/amido/ammonium) curdlans starting from 6- bromo/azido-6-deoxycurdlan. A key reaction that enabled the whole synthesis of new curdlan derivatives at C-6 described in this dissertation was the highly selective bromination of curdlan. The resultant 6-bromo-6-deoxycurdlan, prepared with high regioselectivity, was treated with trialkylamines or heterocyclic amines to produce a range of water-soluble curdlan ammonium salts. The bromide was then nucleophilically displaced by sodium azide to produce the versatile precursor 6-azido-6-deoxycurdlan. Its water solubility was enhanced either by the incorporation of hydrophilic trioxadecanoate esters into O-2/4 positions or by the borohydride reduction to afford 6-amino-6-deoxycurdlan. The iminophosphorane intermediate generated during Staudinger reactions was further investigated for subsequent syntheses: i) 6-amino or 6-amido-6-deoxycurdlan by in situ reaction with water or excess carboxylic anhydride, ii) 6-monoalkylamino curdlan by reductive amination using aldehydes and sodium cyanoborohydride, and iii) 6- dialkylamino-/tri-alkylammoniocurdlans by reacting with methyl iodide. Such derivatives could have properties useful for a range of biomedical applications, including interactions with proteins, encapsulation of drugs, and formulation with genes or other biological compounds. Acknowledgements The completion of my dissertation and subsequent Ph.D. has been a long journey. It is true that “Life is like a box of chocolates, you never know what you are gonna get” when I am completing my dissertation. I never thought of coming aboard to pursue a Ph.D. when I was a kid but this is happening right now. At any rate, I have finished, but not alone, and am elated. I could not have succeeded without the support from my advisor and committee members, my friends and my family. First and foremost I want to express my gratitude to my advisor Dr. Kevin Edgar for his contributions of time, knowledge, patience, genuine caring and concern. He has been motivating, encouraging and enlightening. The joy and enthusiasm he has for the research was motivational for me especially during tough times. His expertise in polysaccharide and a variety of subjects are the major reasons that I could complete a productive work in the past four years. My gratitude is also extended to my committee board, Dr. Riffle, Dr. Turner, Dr. Roman and Dr. Whittington for guiding my research and helping me to learn new knowledge in organic chemistry, macromolecular chemistry and biological chemistry. No research is possible without lab mates, the source of friendship, support and insights. I wish to give big thanks to every member in our polysaccharide research group: Dr. Daiqiang Xu, Dr. Sidd Pawar, Dr. Junia Pereira, Dr. Haoyu Liu, Dr. Xueyan Zheng, Dr. Joyann Marks, Xiangtao Meng, Cigdem Arca, Yifan Dong, Shu Liu, Ashlee Lambert, Brittany Nichols and Chengzhe Gao. It is always a lovely and joyful lab! Special thanks to my dog Simba, the best dog in the world! He is always on my side not only for my happiness but also for my sadness. Lastly, I would show my greatest thanks to my beloved parents in China for their support, strength, encouragement and faith for me. They are best parents! iii Attribution One colleague aided with sample analysis and another two colleagues aided with writing and research behind two of my chapters presented as part of this dissertation. A brief description of their contributions is included here. Chapter 5: S. Liu, currently a PhD candidate of Chemistry at Virginia Tech, served as a co-author on this paper and helped to characterize the samples with 2D NMR spectroscopy. Chapter 7: Dr. Zheng served as a co-author on this paper for the pullulan and glucomannan experimental data. J. Kuang, currently a senior of Bioscience at Virginia Tech helped to run some curdlan experiments. iv Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iii Attribution .......................................................................................................................... iv Table of Contents ................................................................................................................ v Chapter 1. Dissertation overview ........................................................................................ 1 Chapter 2. Literature Review: Properties, Chemistry, and Applications of the Bioactive Polysaccharide Curdlan ...................................................................................................... 3 2.1 Introduction ............................................................................................................................ 3 2.2 Curdlan Background .............................................................................................................. 5 2.2.1 Chemical structure determination ................................................................................... 5 2.2.2 Resources and biosynthesis ............................................................................................ 6 2.2.3 Solution behavior ............................................................................................................ 8 2.2.4 Gel structure and mechanism .......................................................................................... 9 2.2.5 Degradation ................................................................................................................... 11 2.2.6 Application .................................................................................................................... 13 2.3 Curdlan derivatization .......................................................................................................... 15 2.3.1 Esterification of curdlan ................................................................................................ 16 2.3.2 Carboxymethylation of curdlan .................................................................................... 20 2.3.3 Phosphorylation of curdlan ........................................................................................... 23 2.3.4 Regioselective reaction at C-6 of curdlan ..................................................................... 25 2.3.4.1 Curdlan C-6 oxidation by TEMPO ..................................................................................... 26 2.3.4.2 Curdlan C-6 bromination and azidation .............................................................................. 28 2.3.4.3 Chemoselective coupling of 6-azido-6-deoxy-curdlan and alkyne-terminated functional modules by “click chemistry” ......................................................................................................... 29 2.3.5 Sulfation of curdlan ...................................................................................................... 31 2.3.6 Synthesis of branched curdlan derivatives .................................................................... 34 2.4 Conclusions and perspectives .............................................................................................. 38 2.5 Acknowledgements .............................................................................................................. 39 2.6 References ............................................................................................................................ 39 Chapter 3. Synthesis of Curdlan Derivatives Regioselectively Modified at C-6: O-(N)- Acylated 6-Amino-6-Deoxycurdlan ................................................................................. 48 3.1 Abstract ................................................................................................................................ 48 3.2 Introduction .......................................................................................................................... 48 3.3 Experimental ........................................................................................................................ 50 3.3.1 Materials ......................................................................................................................