Biologically Active Secondary Metabolites from Freshwater and Terrestrial Cyanobacteria BY Hahk-Soo Kang B.S., Chung-Ang University, 2003 M.S., Chung-Ang University, 2005 DISSERTATION Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacognosy in the Graduate College of the University of Illinois at Chicago, 2012 Chicago, Illinois Defense Committee: Jimmy Orjala, Chair Alexander Mankin Brian Murphy Duncan Wardrop, Chemistry Steven Swanson DEDICATION My doctoral dissertation is dedicated to my parents, Kyoungdon Kang and Myoungjae Song. My doctoral dissertation is dedicated to my mother’s 60th birthday. My doctoral dissertation is also dedicated to my elder sister Joohyun Kang, brother-in-law Changil Shin and their two sons Woobin Shin and Dongbin Shin.     II EPIGRAPH I believe there is no philosophical high-road in science, with epistermological singposts. We are in a jungle and find our way by trial and error, building our road behind us as we proceed. Max Born (1882-1970), Nobel Prize, 1954 Wisdom is not a product of schooling but of the lifelong attempt to acquire it. Albert Einstein Try as hard as for perfection, the next result of our labors is an amazing variety of imperfectness. We are surprised at our own versatility in being able to fail in so many different ways. Samuel McChord Crothers Firmness of purpose is one of the most necessary sinews of character, and one of the best instru- ments of success. Without it, genius wastes its efforts in a maze of inconsistencies. Philip Dormer Chesterfield III ACKNOWLEDGEMENTS My graduate study and research here at UIC has not been always easy. I have faced lots of difficult prob- lems with regard to my research, preliminary exam and course work for the past four years, and it would not have been possible to overcome those problems without other people’s help. Therefore, I would like to thank many people who have made this dissertation possible. My family: I want to deeply thank my parents for their unfailing support, guidance, and love to me throughout my life. I feel fortunate to have such great parents. My parents have always been role models for me and taught me how to live a life without losing a passion and the importance of a life-long study. Also, every time I came back from vacation, they gave me books about the story of successful people to read on the planes that have influenced my life as a graduate student. I also would like to thank my elder sister and brother-in-law Changil Shin for their kindness and encouragement throughout my Ph.D. study. Dr. Orjala’s LAB: My deepest thank goes to my Ph.D advisor Dr. Jimmy Orjala. He has given me an opportunity to join his LAB, also always been supportive of my research and driven me to the right way throughout my Ph.D study, so that I have been able to concentrate on my research without any difficulty. I am also grateful to Dr. Alec Krunic for his meaningful discussions on my research projects and help with NMR experiments. Without him, my publications and this dissertation would not be possible. I also would like to thank Eddie Lee. He has been such a good friend of mine in the LAB and also taught me how to do micro-isolation and culture cyanobacteria. I really enjoyed the trip to Wisconsin we went to- gether to collect cyanobacterial samples. My thanks also go to Drs. Shunyan Mo and Goerge Chlipala. Shunyan helped me a lot to settle down in the LAB and taught me all the experiments while I was doing a LAB rotation. George set up the dereplication method in our LAB and trained me. I really enjoyed doing dereplication, and this dereplication shaped the early part of Ph.D research. In addition, He has given me a lot of meaningful dissection about cyanobacterial taxonomy. I also would like to thank other LAB IV ACKNOWLEDGEMENTS (continued) members including Dr. Megan Sturdy, Dr. Jiachen Zi, Hyunjung Kim and Shangwen Luo for having been good friends of mine and also good research colleagues, and also for their kindness and help on my jour- ney through a Ph.D. study. Other LAB: I deeply thank Dr. Steven M. Swanson for his consultation during my course work and writ- ing me a letter of recommendation in support of my postdoc application. I also would like to thank Qi Shen and Jilai Yang in Dr. Swanson’s LAB, who have produced most of the cell line assay data for ex- tracts, fractions and pure compounds described in this dissertation. I appreciate Dr. Bernard Santarsiero and Dr. Ben Ramirez for conducting an X-ray crystallography of merocyclophane A for providing me an access to 600 MHz NMR at Center for Structural Biology, respectively. My big thank also goes to Chaitanya Aggarwal in Dr. Federle’s LAB for helping me with molecular biology experiments including PCR and gel electrophoresis. I also appreciate Dr. Kirk Hevener and Henry Su in Dr. Jonson’s LAB for performing a molecular mechanic study of merocyclophane B. Preliminary examination and dissertation committee members: I am grateful to Dr. Brian Murphy, Dr. Chun-Tao Che, Dr. Michael Federle and Dr. Steven M. Swanson for being on my preliminary examina- tion committee. I also would like to deeply thank Dr. Alexander Mankin, Dr. Brian Murphy, Dr. Duncan Wardrop, Dr. Jimmy Orjala and Dr. Steven M. Swanson for being on my dissertation committee and also for meaningful comments on my dissertation. Korean community: Lastly, I would like to deeply thank people in Korean community at UIC College of pharmacy for their great kindness and encouragement throughout my Ph.D. study. Especially, Changwha Hwang, Hyunjung Kim and Jahye Myung have been really good friends of mine during my Ph.D. study, leaving me so many memories that I will never forget for the rest of my life. My deep appreciation goes V ACKNOWLEDGEMENTS (continued) to Dr. Hyunwoo Lee and Dr. Hyunyeong Jung for having been so kind to me and given me very meaning- ful consultation on my carrier as a natural products chemist throughout my Ph.D. study. All of the researches described in this dissertation were conducted as a part of PO1 anticancer project funded by NCI/NIH with the grant No. CA125066. HSK VI TABLE OF CONTENTS   Chapter 1. Introduction.................................................................................................................1 1.1 Natural products as a source for drug discovery ........................................................................... 2 1.2 Cyanobacteria ................................................................................................................................ 7 1.2.1 General characteristics ......................................................................................................... 7 1.2.2 Taxonomic classification ..................................................................................................... 7 1.3 Ecotypes of freshwater and terrestrial cyanobacteria and their secondary metabolites .............. 10 1.3.1 Mat-forming cyanobacteria ............................................................................................... 10 1.3.2 Bloom-forming cyanobacteria ........................................................................................... 12 1.3.3 Symbiotic cyanobacteria .................................................................................................... 22 1.3.4 Other non-bloom forming cyanobacteria in freshwater and terrestrial environments ....... 25 1.4 Rationalization and objective of the study .................................................................................. 33 Chapter 2. Antiproliferative lipodecapeptides from two cultured cyanobacteria, Anabaena minutissima (UTEX 1613) and Cf. Anabaena sp. (UIC 10035) .............................35 2.1 Introduction ................................................................................................................................. 36 2.2 Minutissamides A-D, cyclic lipodecapeptides from the cultured cyanobacterium Anabaena minutissima (UTEX 1613) ............................................................................................. 37 2.2.1 Isolation of minutissamides A-D ....................................................................................... 37 2.2.2 Structure determination of minutissamides A-D ............................................................... 38 2.3 Homesteadamides A-H, cyclic lipodecapeptides from the cultured cyanobacterium cf. Anabae- na sp. (UIC 10035) ........................................................................................................ 51 2.3.1 Isolation of homesteadamides A-H ................................................................................... 51 2.3.2 Structure determination of homesteadamides A-H ............................................................ 51 2.4 Antiproliferative activity of minutissamides A-D and homesteadamides A-H against cancer cells ................................................................................................................................ 63 2.5 Morphological and phylogenetic analyses for taxonomic identification of the strain UIC 10035 ....................................................................................................................................... 64 2.6 Experimental ............................................................................................................................... 67 2.6.1 General experimental procedures ...................................................................................... 67 2.6.2 Biological material ............................................................................................................ 67 2.6.3 Morphological and phylogenetic analyses for taxonomic identification ........................... 68 2.6.4 Extraction and isolation ..................................................................................................... 68 2.6.5 Marfey’s analysis ............................................................................................................... 71 2.6.6 Advanced Marfey’s analysis .............................................................................................. 72 2.6.7 Preparation of OMeThr standards of the homesteadamides .............................................. 73 2.6.8 Antiproliferative assay ....................................................................................................... 74 Chapter 3. Merocyclophanes A and B, antiproliferative cyclophanes from the cultured ter- restrial cyanoabacterium Nostoc sp. (UIC 10062) .................................................75 3.1 Introduction ................................................................................................................................. 76 3.2 Merocyclophanes A and B, antiproliferative cyclophanes from the cultured terrestrial cyanobac- terium Nostoc sp. (UIC 10062) ...................................................................................... 77 3.2.1 Isolation of merocyclophanes A and B .............................................................................. 77 3.2.2 Structure determination of merocyclophanes A and B ...................................................... 78 3.3 Antiproliferative activity of merocyclphanes A and B ............................................................... 85 3.4 Taxonomic identification of the strain UIC 10062 ..................................................................... 85 3.5 Experimental ............................................................................................................................... 88 3.5.1 General experimental procedures ...................................................................................... 88 3.5.2 Biological material ............................................................................................................ 88 VII TABLE OF CONTENTS (continued) 3.5.3 Strain identification ........................................................................................................... 88 3.5.4 DNA extraction, PCR amplification and sequencing ........................................................ 89 3.5.5 Phylogenetic analysis......................................................................................................... 89 3.5.6 Extraction and isolation ..................................................................................................... 90 3.5.7 X-ray crystallographic analysis of merocyclophane A ...................................................... 91 3.5.8 HT-29 antiproliferative assay ............................................................................................ 91 Chapter 4. Sanctolide A, a 14-membered PK-NRP hybrid macrolide from the cultured cy- anobacterium Oscillatoria sancta (SAG 74.79) ......................................................92 4.1 Introduction ................................................................................................................................. 93 4.2 Isolation of sanctolide A ............................................................................................................. 93 4.3 Structure determination of sanctolide A ...................................................................................... 95 4.4 Biological activity of sanctolide A ............................................................................................ 100 4.5 Experimental ............................................................................................................................. 104 4.5.1 General experimental procedures .................................................................................... 104 4.5.2 Biological material .......................................................................................................... 104 4.5.3 Extraction and isolation ................................................................................................... 104 4.5.4 Methanolysis of sanctolide A .......................................................................................... 105 4.5.5 Mosher ester analysis of 2 ............................................................................................... 105 4.5.6 Chiral HPLC analysis of 2-hydroxyisovaleric acid (3) ................................................... 106 Chapter 5. Stigonemapeptin, an Ahp-containing depsipeptide with elastase-inhibitory ac- tivity from the bloom-forming freshwater cyanobacterium Stigonema sp.. .....107 5.1 Introduction ............................................................................................................................... 108 5.2 Sample collection and isolation of stigonemapeptin ................................................................. 109 5.3 Structure determination of stigonemapeptin ............................................................................. 110 5.4 Serine protease activity of stigonemapeptin ............................................................................. 114 5.5 Taxonomic identification of Stigonema sp. WI53 .................................................................... 115 5.6 Experimental ............................................................................................................................. 118 5.6.1 General experimental procedures .................................................................................... 118 5.6.2 Sample collection and morphological study .................................................................... 118 5.6.3 DNA extraction and phylogenetic analysis of 16S rRNA gene sequence ....................... 118 5.6.4 Extraction and isolation ................................................................................................... 119 5.6.5 Absolute configuration of amino acids and Ahp by the advanced Marfey’s method ...... 119 5.6.6 Protease inhibition assays ................................................................................................ 120 Chapter 6. Conclusion and Perspective ...................................................................................122 6.1 Conclusion & Perspective ......................................................................................................... 123 6.1.1 Chemical investigation of the cultured freshwater and terrestrial cyanobacteria ............ 123 6.1.2 Chemical investigation of the bloom-forming freshwater cyanobacteria ........................ 127 References ...................................................................................................................................129 Appendices ..................................................................................................................................141 Curriculum Vitae .......................................................................................................................170 VIII LIST of FIGURES   Chapter 1. .......................................................................................................................................1 Figure 1.1 Some of the landmark drugs discovered from natural sources ......................................... 2 Figure 1.2 Structures, sources and targets of natural products discovered since 1970 that led to an approved drug in 1981-2006 ............................................................................................ 6 Figure 1.3 Phylogenetic tree constructed using 16S rRNA gene sequences of reference cyanobacte- ria retrieved from Bergey’s manual of systematic bacteriology ...................................... 8 Figure 1.4 Two types of cyanobacterial cells where carbon and nitrogen fixation are performed .. 10 Figure 1.5 Typical laminated structures of cyanobacteria-dominated microbial mats..................... 11 Figure 1.6 The structure and biosynthesis of the cyanobacterial sunscreen pigment scytonemin ... 12 Figure 1.7 The structures of microcystins and nodularin ................................................................. 13 Figure 1.8 The schematic description of the microcystin-LR biosynthetic pathway ....................... 15 Figure 1.9 The originally proposed and revised structures of cylindrospermopsin ......................... 16 Figure 1.10 The structure and biosynthetic pathway of cylindrospermopsin ................................... 16 Figure 1.11 The structure and biosynthetic pathway of anatoxin-a .................................................. 18 Figure 1.12 The structure and biosynthetic pathway of saxitoxin .................................................... 19 Figure 1.13 The structures of cyanobacterial Ahp-containing depsipeptides isolated from bloom samples of freshwater cyanobacteria ............................................................................. 21 Figure 1.14 The structure of cryptophycin 1 and its biosynthetic pathway ...................................... 24 Figure 1.15 The structures of tolytoxin and scytophycins ................................................................ 26 Figure 1.16 The structures of cyanobacterial indole alkaloids .......................................................... 28 Figure 1.17 The structures of naturally occurring [7.7]paracyclophanes isolated from terrestrial cy- anobacteria ..................................................................................................................... 29 Figure 1.18 Proposed biosynthetic pathways for cylindrocyclophane D by 13C feeding experiments ....................................................................................................................................... 30 Figure 1.19 The structures of cyclic lipodecapeptides isolated from terrestrial Anabaena spp.. ...... 32 Chapter 2. .....................................................................................................................................35 Figure 2.1 Key 2D correlations used for the determination of the planar structure of minutissamide A (1) ............................................................................................................................ 40 Figure 2.2 Sequential NOE correlations for the determination of amino acid sequence of minutis- samide A (1) .................................................................................................................. 41 Figure 2.3 ESI-MS/MS data of minutissamide A (1) acquired using CID fragmentation method .. 41 Figure 2.4 Newman projections for (a) C-2/C-3 and (b) C-3/C-4 .................................................... 42 Figure 2.5 The advanced Marfey’s method used to determine the absolute configuration of the β- amino acid residue at C-3 .............................................................................................. 44 Figure 2.6 Key 2D correlations used for the determination of the planar structure of homesteadam- ide A (5) ......................................................................................................................... 52 Figure 2.7 ESI-MS/MS fragmentation of homesteadamides A (5) and E (9) .................................. 54 Figure 2.8 Morphological comparison between Anabaena minutissima (UTEX 1613) and Cf. Ana- baena sp. (UIC 10035) .................................................................................................. 65 Figure 2.9 Evolutionary distances were determined using the minimum evolution method with 1,000 replicate boots trap re-sampling to construct the phylogenetic tree .................... 66 Chapter 3. .....................................................................................................................................75 Figure 3.1 Key 2D correlations used for the determination of the planar structure of merocyclo- phane A ....................................................................................................................... 78 Figure 3.2 ORTEP drawing of merocyclophane A (1) .................................................................... 80 Figure 3.3 Torsion energy profile of the hydroxyquinone ring in merocyclophane B (2) ............... 81 IX LIST of FIGURES (continued) Figure 3.4 Comparison of carbon skeletons between the cylindrocyclophanes, nostocyclophanes and merocyclophane A .................................................................................................. 82 Figure 3.5 Morphological description of Nostoc sp. (UIC 10062) .................................................. 86 Figure 3.6 Phylogenetic relationships of 16S rRNA genes from cyanobacteria for the taxonomic identification of the strain UIC 10062 ........................................................................... 87 Chapter 4. .....................................................................................................................................92 Figure 4.1 HPLC-based activity profiling of the combined active fractions ................................... 94 Figure 4.2 Key 2D NMR correlations used for the determination of the planar structure of sanc- tolide A .......................................................................................................................... 96 Figure 4.3 Newman projections for A4: C5-C6 and B1: C6-C7 ...................................................... 98 Figure 4.4 Key nOe correlations and a possible conformation of the C5-C4 fragment ................... 99 Figure 4.5 Methanolysis and acid hydrolysis for the absolute configuration of sanctolide A at C-5 and C-15 ...................................................................................................................... 100 Figure 4.6 1H NMR spectra (600 MHz, CDCl ) of the partially and completely degraded products 3 of sanctolide A ............................................................................................................. 102 Figure 4.7 Proposed enamide hydrolysis of 1 in neutral pH that leads to ring-opening of the macro- lide ring structure ......................................................................................................... 102 Chapter 5. ...................................................................................................................................107 Figure 5.1 The location and photos of the collection site of the sample WI53 .............................. 108 Figure 5.2 Key 2D NMR correlations used for the determination of the planar structure of stigonemapeptin .......................................................................................................... 111 Figure 5.3 Advanced Marfey’s analysis of stigonemapeptin for the determination of amino acid configurations .............................................................................................................. 112 Figure 5.4 Elastase and chymotrypsin inhibitory activity of stigonemapeptin .............................. 114 Figure 5.5 Photomicrograph of Stigonema sp. (collection ID WI53) ............................................ 116 Figure 5.6 Phylogenetic relationships of 16S rRNA genes from cyanobacteria for taxonomic identi- fication of the sample WI53 ........................................................................................ 117 X