Automated Glycan Assembly of Oligomannose Glycans for Sensing Applications Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry, and Pharmacy of Freie Universität Berlin by Priya Mohanrao Bharate 2017 This work was performed between November 2013 and February 2017 under the guidance of Prof. Dr. Peter H. Seeberger in the Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces Potsdam, and the Institute of Chemistry and Biochemistry, Freie Universität Berlin. 1st reviewer: Prof. Dr. Peter H. Seeberger 2nd reviewer: Prof. Dr. Rainer Haag Date of oral defense: 22.11.2017 Declaration This is to certify that the entire work in this thesis has been carried out by Ms. Priya Bharate. The assistance and help received during the course of investigation have been fully acknowledged. ----------------------- ------------------------ (Date, Place) (Signature) Acknowledgements I dedicate my special thanks to Prof. Dr. Peter H. Seeberger for providing me the opportunity and support in realizing this thesis. Prof. Dr. Rainer Haag for generously agreeing to review this thesis. My colleagues from the Carbohydrate Materials group especially Ms. Silvia Varela- Aramburu and Dr. Martina Delbianco for proof reading this thesis, Dr. Chian-Hui Lai, Ms. Zhou Ye and Dr. Guillermo Orts-Gil for friendly atmosphere. Dr. Claney Lebev Pereira for proof reading the first chapter and for his suggestions for third chapter of this thesis. Prof. Dr. Rainer Haag, Dr. Zhenhui Qi, and Mr. Benjamin Ziem for their extensive efforts to make a close collaboration and for providing valuable graphene material. Dr. Christoph Böttcher, Ms. Andrea Schulz for transmission electron and atomic force microscopy experiments as well as Mr. Benjamin Hatting for Raman spectroscopy experiment. Prof. Virgil Percec, Dr. Sebastian Krauss for their extensive efforts to make a close collaboration. Dr. Sharavathi Parameshwarappa and Dr. Claney Lebev Pereira for their advice and moral support which was key in finishing this dissertation. Dr. Bopanna Monnanda Ponnappa for proof reading and for the biological experimental suggestions for second chapter of this thesis. Ms. Dorothee Böhme, Ms. Katharina Tebel and Ms. Lydia Alnajjar for all the back office help. Especially Ankita, Silvia, Maria, Zhou, Martina, and Monika for the last few months of my PhD and for helping me to recover faster. and my esteemed colleagues and friends of the Biomolecular Systems department Anish, Andrew, Alonso, Annette, Andreas, Anika, Andreia A., Ben, Bart, Chandradhish, Camille, Deborah, Eva, Fei-Fei, Felix B., Fabian, Frank, Hahm, Madhu, Maria B., Marilda, Matt, Max, Naresh, Naeem, Uwe V., Oren, Olaf, Pietro, Renee, Ramesh, Rich, Somesh, Sahid, Sandip, Sourav, Kathir, Malte, Martín, Advik, and many others for wonderful time in Berlin. I also like to thank Dr. Raghavendra Kikkeri for his support, and to all my friends back home in India. Finally, I express my deepest gratitude to my family members and in-laws for their love and support. And big thank you to Balaji for his unfailingly support and without him I would not have come so far! Dedicated To My beloved Parents List of Publications A) Scientific Publication and Reviews 1. Multivalency at Interfaces: Supramolecular Carbohydrate-Functionalized Graphene Derivatives for Bacterial Capture, Release, and Disinfection. Zhenhui, Q.* Bharate, P.* Lai, C. Ziem, B. Böttcher, C. Schulz, A. Beckert, F. Hatting, B. Mülhaupt, R. Seeberger, P.H. and Haag, R. Nano Lett., 2015, 15, 6051−6057. 2. Carbohydrates in supramolecular chemistry. Delbianco, M. Bharate, P. Varela- Aramburu, S. Seeberger, P.H. Chem. Rev. 2016, 116, 1693−1752. 3. Tumor-targeted drug delivery with mannose-functionalized nanoparticles self-assembled from amphiphilic β-cyclodextrins. Ye, Z. Zhang, Q. Wang, S. Bharate, P. Varela- Aramburu, S. Lu, M. Seeberger, P.H. Yin, J. Chem. Eur. J. 2016, 22, 15216 – 15221. * equal contribution B) Scientific Conferences and Symposia 1. International Carbohydrate Symposium, New Orleans, USA (2016): “Self-Assembling Carbohydrate-functionalized Graphene for Capturing and Killing E. coli Bacteria” (Poster). 2. International Symposium on Macrocyclic and Supramolecular Chemistry, Strasbourg, France (2015): “Self-Assembling Carbohydrate-functionalized Graphene for Capturing and Killing E. coli Bacteria” (Poster). 3. COST Multiglyconano, Bangor, UK (2015): “Self-Assembling Carbohydrate- functionalized Graphene for Capturing and Killing E. coli Bacteria” (Oral presentation + Poster). 4. MPIKG Ringberg Conference, Rottach-Egern, Germany (2014): “Self-Assembling Carbohydrate-functionalized Graphene for Capturing and Killing E. coli Bacteria” (Oral presentation). Table of Contents Table of contents……….…………………………………………………………………....1 List of Abbreviations……….……………………………………………………………….5 Summary………………….…………………………………………………………………9 Zusammenfassung………………………………………………………………………….11 1 Introduction………………………………………………………………………………14 1.1 Cell surface glycans…………………………………………………………………….14 1.2 Gram-positive and Gram-negative Bacteria........……………………………………16 1.3 Carbohydrate binding lectins………………………………………………………....18 1.3.1 Plant lectins………………………………………………………………………….19 1.3.2 Bacterial lectins……………………………………………………………..............19 1.3.3 Carbohydrate-protein and carbohydrate-carbohydrate interactions…………………20 1.4 Supramolecular chemistry………………………………………………………….....22 1.4.1 Carbohydrate materials……………………………………………………………...24 1.4.1.1 Cyclodextrins (CDs)…………………………………………………………….24 1.4.1.2 Graphene functionalized CD’s…………………………………………………..26 1.5 Automated glycan assembly…………………………………………………………...29 1.5.1 Solid support: Merrifield resin……………………………………………………....31 1.5.2 Linkers and recent AGA syntheses………………………………………………….32 1.6 Aim of the Thesis……………………………………………………………………….39 2 Multivalency at Interfaces: Supramolecular Carbohydrate-Functionalized Graphene Derivatives for Bacterial Capture, Release, and Disinfection 2.1 Introduction…………………………………………………………………………….42 2.1.1 Graphene and cyclodextrin………………………………………………………….43 2.1.1.1 Graphene………………………………………………………………………...43 2.1.1.2 Cyclodextrin……………………………………………………………………..43 2.1.2 Strategy……………………………………………………………………………...45 2.2 Results…………………………………………………………………………………..46 2.2.1 Synthesis of cyclodextrin functionalized with mannose-host moiety…………….…46 2.2.2 Synthesis of TRGO and adamantyl-functionalized TRGO sheets-guest moiety……47 1 Table of Contents 2.2.3 Physicochemical characterization of AG4…………………………………………..48 2.2.4 Assembly of the supramolecular ManCD@AG4 (49) derivative and its characterization……………………………………………………………………………...51 2.2.5 Characterization of carbohydrate-lectin binding study of ManCD@AG4 using microscopic techniques……………………………………………………………………...54 2.3 Conclusion and Outlook……………………………………………………………….62 2.4 Supplementary Figures………………………………………………………………..64 2.5 Experimental Section…………………………………………………………………..72 2.5.1 General methods…………………………………………………………………….72 2.5.2 Methods of synthetic chemistry……………………………………………………..77 3. Automated Solid Phase Synthesis of Lipoglycans from Mycobacterium tuberculosis 3.1 Introduction…………………………………………………………………………….85 3.1.1 Mycobacterium tuberculosis………………………………………………………...85 3.1.2 Cell wall of MTB……………………………………………………………………86 3.2 Results…………………………………………………………………………………..92 3.2.1 Synthesis of Linear LM fragment α-(1→6)-Oligomannoside by AGA…………….92 3.2.2 Optimization of automated glycosylation conditions……………………………….95 3.2.3 Synthesis of branched hexasaccharide LM fragment 89……………………………97 3.2.4 Synthesis of longer LM and LAM fragments……………………………………...102 3.2.5 Deprotection of the linear and branched LM and LAM oligosaccharides…………107 3.3 Conclusion and Outlook……………………………………………………………...109 3.4 Experimental Section…………………………………………………………………111 3.4.1 General methods………………………...…………………………………………111 3.4.2 Methods of synthetic chemistry……………………………………………………112 3.4.2.1 Synthesis of Photolabile Linker and Functionalization of Resin………………112 3.4.2.2 Synthesis of Building Block…………………………………………………...115 3.4.2.3 Pre-Automation Steps………………………………………………………….122 3.4.2.4 Modules for automated synthesis………………………………………………122 3.4.2.5 Automated Synthesis…………………………………………………………...127 3.4.2.6 Deprotection of LM and LAM fragments……………………………………...148 4. Applications of Automated glycan Assembly………………………………………...157 2 4.1 Introduction…………………………………………………………………………..157 4.1.1 Self –assembling structures………………………………………………………..158 4.1.2 Strategy…………………………………………………………………………….161 4.2 Results…………………………………………………………………………………162 4.2.1 Synthesis of a library of linear mannose fragments by AGA……………………..163 4.2.3 Deprotection of the Mannose Oligosaccharides…………………………………...164 4.2.4 Synthesis of Glycodendrimers……………………………………………………..164 4.2.5 Conclusion and Outlook……...……………………………………………………166 4.3 Experimental Section…………………………………...……………………………167 4.3.1 Automated Synthesis………………………………………………………………168 5. References………………………………………………………………………………177 3 4 List of Abbreviations Ac Acetyl Ac2O Acetic anhydride ACN/CH3CN Acetonitrile A.I Agglutination indexes AGA Automated glycan assembly Azmb 2-(azidomethyl)benzoyl BF3.Et2O Boron trifluoride diethyl etherate Bn Benzyl BnBr Benzyl bromide BzCl Benzoyl chloride PhCH(OMe)2 Benzaldehyde dimethyl acetal CAN Ceric ammonium nitrate CHCl3 Chloroform CsOAc Cesium acetate Cs2CO3 Cesium carbonate CSA Camphor sulfonic acid CLRs C-type lectin receptors ConA Concanavalin A CFU Colony forming units δ Chemical shift d doublet