SYNTHESIS AND CHARACTERIZATION OF FUNCTIONALIZED RECOMBINANT THROMBOMODULIN FOR ANTITHROMBOTIC DRUG DEVELOPMENT LIN WANG Bachelor of Pharmaceutical Science Shenyang Pharmaceutical University, China July 2009 Submitted in partial fulfillment of requirements for the degree of DOCTOR OF PHILOSOPHY IN CLINICAL-BIOANALYTICAL CHEMISTRY at the CLEVELAND STATE UNIVERSITY AUGUST 2015 We hereby approve this dissertation for Lin Wang Candidate for the Doctor of Philosophy in Clinical-Bioanalytical Chemistry Degree for the Department of Chemistry and CLEVELAND STATE UNIVERSITY College of Graduate Studies _____________________________________________________________ Dissertation Chairperson, Xue-Long Sun, Ph.D. _____________________________________ Department & Date ______________________________________________________________ Anthony Berdis, Ph.D. ______________________________________ Department & Date _______________________________________________________________ Baochuan Guo, Ph.D. ________________________________________ Department & Date ______________________________________________________________ Nolan Holland, Ph.D. ______________________________________ Department & Date ________________________________________________________________ Sihe Wang, Ph.D. ___________________________________________ Department & Date _________________________________________________________________ Aimin Zhou, Ph.D. _____________________________________________ Department & Date Date of Defense: August 4th 2015 ACKNOWLEDGEMENTS I owe my gratitude to all those people who have helped me to make this dissertation. I am grateful for their time and efforts which have contributed to my professional growth and education throughout my years as a graduate student. First and foremost, I would like to give my deepest gratitude to my advisor, Dr. Xue- Long Sun for providing me the opportunity to work with him and for his persistent support and guidance throughout my PhD process. His enduring enthusiasm and rigorousness in scientific research will always inspire me throughout my life careers. Without his guidance and persistent help this dissertation would not have been possible. Next, I would like to thank my committee members, Dr. Anthony Berdis, Dr. Baochuan Guo, Dr. Nolan Holland, Dr. Sihe Wang and Dr. Aimin Zhou, for their insightful comments and suggestions and valuable support for my projects. I would also like to thank all past and current members in Dr. Sun lab for all their assistance and suggestions for my experiments. Particularly, I would like to thank Dr. Jiang for giving me the guidance and training in chemical biology. Last but not the least, I would like to give my sincere acknowledgement to my family members. Their continuous encouragement, support and love are always the driving forces for me to work hard. SYNTHESIS AND CHARACTERIZATION OF FUNCTIONALIZED RECOMBINANT THROMBOMODULIN FOR ANTITHROMBOTIC DRUG DEVELOPMENT LIN WANG ABSTRACT Thrombomodulin (TM), an endothelial cell surface membrane glycoprotein, is a cofactor for protein C activation via thrombin, and therefore a crucial regulator in protein C pathway and coagulation cascade. TM contains six epidermal growth factor (EGF)–like structures, in which the forth to sixth EGF-like region (TM456) is the minimum functional domain responsible for protein C activation activity and could serve as a pure anticoagulant agent. However, recombinant TM456 has a short half-life (6-9 min) that limits its therapeutic application. Modification with polymer such as PEG should be a choice to enhance the pharmacokinetics of recombinant TM456. In addition, incorporation of protein into liposome provides several benefits including prolonged half-life and targeted drug delivery as well. Therefore, this study aimed to develop TM456 conjugates as novel and potential antithrombotic drugs. Site-specific modification of protein is the key for protein conjugate preparation for either defined conjugate formation or unchanged protein activity. Firstly, a one-pot strategy for site-specific PEGylation through copper-free click chemistry (CFCC) and fluorescent labeling through sortase-mediated ligation (SML) of recombinant TM without prior chemical modification and without diminishing the protein activity has been iv developed. Modification with polymer PEG aims enhances the pharmacokinetics of recombinant TM456. In addition, incorporation of a tag to the recombinant TM456 for subsequent detection or affinity purification facilitates efficient biological evaluation for both in vitro and in vivo experiments. Secondly, in order to obtain the azide bearing TM456 for further protein modification via CFCC, four site-selective strategies to introduce azide functionality into TM456, via direct recombinant expression with unnatural amino acid, chemical, and enzymatic modification were fully investigated. Among them, SML of recombinant protein affords the highest overall yield for incorporating azide functionality into recombinant TM456 and retained the full TM anticoagulant activity as well. Finally, synthesis and characterization of liposome-TM456 conjugates were investigated by synthesizing the DSPE-PEG2000-TM456 from azido-TM456 and DSPE- PEG2000-DBCO via CFCC followed by direct liposome formation method. This liposomal formulation of TM456 retained the same protein C activation activity as the unmodified TM456, and turned out to be much more stable in plasma than azido-TM456. Further, liposome-TM456 showed a comparable anticoagulant effect in thrombin-induced thromboembolism mouse model by reducing the mortality from 80% to 20%. Moreover, liposome-TM456 enhanced the circulation time than free azido-TM456 in vivo. v TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iv LIST OF TABLES ............................................................................................................. xi LIST OF FIGURES .......................................................................................................... xii ABBREVIATIONS ......................................................................................................... xiv CHAPTER I. INTRODUCTION ........................................................................................................... 1 1.1 Thrombomodulin and its antithrombotic activities ................................................... 1 1.1.1 Structure of TM .................................................................................................. 2 1.1.2 Biological functions of TM ................................................................................ 5 1.1.3 Therapeutic application of recombinant TM ...................................................... 8 1.2 Bioconjugation strategies for protein functionalization .......................................... 11 1.2.1 Click chemistry ................................................................................................. 15 1.2.2 Sortase-mediated ligation ................................................................................. 17 1.3 Liposomes as drug delivery systems ....................................................................... 19 1.3.1 Conventional liposomes ............................................................................................20 1.3.2 Stealth liposomes .............................................................................................. 22 1.3.3 Targeted liposomes ........................................................................................... 23 1.3.4 Preparation methods of liposomes with ligand................................................. 26 1.3.4.1 Direct liposome formation with ligands .................................................... 26 1.3.4.2 Post-insertion approach .............................................................................. 26 vi 1.3.4.3 Post-functionalization approach ................................................................. 27 1.4 Research Design and Rational................................................................................. 28 1.5 References ............................................................................................................... 29 II. CHEMOENZYMATIC BIO-ORTHOGONAL CHEMISTRY FOR SITE-SPECIFIC DOUBLE MODIFICATION OF RECOMBINANT TM456AL ....................................... 47 2.1 Introduction ............................................................................................................. 47 2.2 Results and discussion ............................................................................................. 50 2.2.1 Expression and purification of TM456AL ......................................................... 50 2.2.2 Double-labeling of TM2 with DIBO-AF647 or/and Gly2-Bodipy ................... 51 2.2.3 Optimization of one-pot double-modification reaction conditions ................. 52 2.2.4 One-pot PEGylation and fluorescent labeling of TM456AL ............................. 56 2.2.5 PEGylation of TM456AL via sortase-mediated ligation with Gly2-PEG5000-OMe ................................................................................................................................... 58 2.2.6 Protein C activation activity of TM456AL and TM456AL conjugates ............... 61 2.3 Conclusion ............................................................................................................... 63 2.4 Materials and methods ...........................................................................................