Structural Characterization of Secondary Metabolites
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STRUCTURAL CHARACTERIZATION OF SECONDARY METABOLITES PRODUCED BY FUNGI OBTAINED FROM DAMP CANADIAN BUILDINGS David Roderick McMullin B.Sc. Carleton University, 2008 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Chemistry with a Specialization in Chemical and Environmental Toxicology Ottawa-Carleton Institute of Chemistry Carleton University Ottawa, ON, Canada © Copyright 2014. David R. McMullin ABSTRACT A comprehensive investigation of the secondary metabolites produced by Chaetomium globosum, Wallemia sebi, Penicillium corylophilum and four Trichoderma species obtained from Canadian buildings is presented. Atopic and non-atopic individuals occupying damp, moldy buildings are at increased risk of both allergic and non-allergic adverse health effects. There is now strong toxicological evidence showing that secondary metabolites, including mold specific glucan, present on spores and mycelial fragments are in part responsible for these effects. At the low doses that could be experienced by the human lung indoors, metabolites from fungi have been demonstrated to alter the expression of genes involved with asthma in vivo and in vitro. These genetic alterations are accompanied by histological disruptions and inflammatory responses. The primary focus of this study was to identify and isolate the dominant toxins produced by the mentioned fungi obtained from Canadian buildings. Isolates were grown in liquid culture and screened for metabolite production. Metabolites were purified by various chromatographic methods and their structures were unambiguously determined by mass spectrometry and detailed analysis of spectroscopic data. In this work, C. globosum primarily produced chaetoglobosin A, C and F, chaetomugilin D and chaetoviridin A. Other chaetoglobosins and azaphilones were produced in minor amounts including three new azaphilones. The meroterpenoids, andrastin A and citreohybridinol, koninginins A, E and G, the eremophilane sesquiterpene phomenone and seven new metabolites, four isochromans and three α-pyrones, were isolated from P. corylophilum. This was the first 2 report of meroterpenoids and α-pyrones from this species as well as koninginins from any genus outside of Trichoderma. The xerophile W. sebi produced walleminone, a new alkaloid, wallimidione, tryptophol and other amino acid derived compounds. T. citrinoviride produced many sorbicillin derived compounds including spirosorbicillinol A, B and C, trichotetronine, bisvertinol and a new metabolite, the (R)- stereoisomer of vertinolide. Koninginins and α- pyrones were isolated from T. koningiopsis and T. atroviride, respectively. To determine the adverse health effects caused by fungi indoors, isolates obtained directly from built environments must be studied which was achieved here. Some of the dominant toxins isolated, chaetoglobosin A, chaetomugilin D, phomenone, andrastin A, walleminone, tryptophol, koninginin A and trichotetronine, are being utilized as analytical standards to continue toxicological experiments aimed at determining how fungal metabolites alter human lung biology. These secondary metabolites encompass each the genera investigated, have known bioactivities and exhibit the diversity of fungal natural products. Information from these experiments will contribute to a more comprehensive understanding of the mechanism for non- atopic asthma associated with exposure to mold and dampness indoors. 3 ACKNOWLEDGMENTS The funding that made this project possible was provided by the Natural Sciences and Engineering Council of Canada (NSERC). Most importantly, I want to express my gratitude to my supervisor Dr. J. David Miller for presenting me with the opportunity of working within his group and continued mentorship throughout my graduate studies. I appreciate that you allowed me to work through this fascinating project on my own but were always available to answer my questions or provide advice without hesitation. These experiences provided me with an understanding of how to do things the right way. I apologize for the so called “McMullin induced delays”. Secondly, I would like to thank Dr. Mark Sumarah who was tasked with teaching me about natural product chemistry but taught me more life lessons instead. I have not forgotten the important ones. Thank you for your continued advice, good times and all of the conversations, particularly the ones calming me down when I get worked up. I need to thank Dr. Tienabe Nsiama for imparting his chemistry knowledge, convincing me to perform organic reactions on our precious compounds and always willing to offer his assistance; it was a pleasure working with you. I sincerely thank and appreciate the efforts of Don Belisle, Paracel Inc., for providing indoor isolates, Dr. Dan Sørensen, McMaster University, for acquiring high quality NMR data that made many structural elucidations possible and for the invitation to Hamilton, Dr. Keith Seifert 4 and Dr. Tharcisse Barasubiye, Agriculture and Agri-Food Canada, for providing fungal identifications and depositing many of the studied strains, Dr. Barbara Blackwell, Agriculture and Agri-Food Canada, for taking the time to help me decipher NMR spectra, Dr. Jeffery Manthorpe for his guidance with organic reactions and all the invitations to play hockey. A special thank you to Dr. Sue Twine, National Research Council, for not only facilitating mass spectrometry analysis but for writing me so many recommendation letters and convincing me to go to graduate school in the first place. I not only thank but appreciate all of the past and present Miller group members that have helped me along the way and made this an enjoyable experience: Samantha Frasz, Blake Green, Natacha Provost, Natasha Prince, Gina Parker, Luke Johnson, Susan Richardson, Tamara Desroches, Shari Levac, Greg Slack and Aaron Wilson. I suppose I must apologize to a couple of you who know who you are for your retention within the group. The many friendships made while working in the Steacie building made the whole process a great personal experience. Particularly true of anyone who wanted to go for “a” drink. I finally want to thank my family who has supported me throughout this endeavor. I think we can all agree, no one would have seen me here a decade ago, but I´m happy I am. 5 TABLE OF CONTENTS ABSTRACT 2 ACKNOWLEGMENTS 4 LIST OF FIGURES 7 LIST OF TABLES 11 SCENTIFIC CONTRIBUTIONS 13 CHAPTER I – GENERAL INTRODUCTION 15 CHAPTER II – ISOLATION, CHARACTERIZATION AND 40 QUANTIFICATION OF CHAETOGLOBOSINS AND AZAPHILONES FROM CHAETOMIUM GLOBOSUM CHAPTER III – ISOLATION AND STRUCTURAL ELUCIDATION OF 79 SECONDARY METABOLITES FROM PENICILLIUM CORYLOPHILUM CHAPTER IV – CHARACTERIZATION OF METABOLITES FROM 125 WALLEMIA SEBI CHAPTER V – ISOLATION AND STRUCTURAL ELUCIDATION OF 146 SECONDARY METABOLITES FROM TRICHODERMA CITRINOVIRIDE, T. KONINGIOPSIS AND T. ATROVIRIDE CHAPTER VI – GENERAL DISCUSSION 182 LITERATURE CITED 195 APPENDIX I – 1H AND 13C NMR SPECTRA FOR ISOLATED 219 METABOLITES APPENDIX II – CHARACTERIZATION OF A W.SEBI ANTIGENIC 260 CELLULASE 6 LIST OF FIGURES Figure Title Page Figure 1.1: Structure of the polyketides aflatoxin B1, zearalenone and griseofulvin 22 Figure 1.2: Structure of the sesquiterpenes deoxynivalenol and PR toxin 23 Figure 1.3 Structure of cinnaminc acid and roquefortine C 24 Figure 1.4: Scheme of secondary metabolism 25 Figure 1.5: Structures of secalonic acid D, meleagrin and chrysogine isolated from 35 either P. chrysogenum or P. rubens Figure 1.6: Structures of the carcinogenic metabolites sterigmatocystin and 5- 37 methoxysterigmatocystin from A. versicolor and the trichothecene trichodermol from S. chartarum chemotype A and Tricoderma spp. Figure 2.1: C. globosum grown on 2% MEA 41 Figure 2.2: Structures of the toxin chetomin and purple pigment cochliodinol 42 Figure 2.3: Structures of chaetoglobosin A and C 43 Figure 2.4: Structures of chaetoviridin A-D 44 Figure 2.5: Structures of chaetomugilin A, D, I and M 45 Figure 2.6: Structures of chaetoglobosin U and chaetoglobin A 46 Figure 2.7: Structure of chaetoglobosin A 48 Figure 2.8: Structure of chaetoglobosin F 48 Figure 2.9: Structure of chaetoglobosin C 49 Figure 2.10: Structure of chaetomugilin D 51 Figure 2.11: Structure of chaetoviridin A 52 Figure 2.12: HRESIMS spectra of chaetoglobosin A and chaetomugilin D 53 Figure 2.13: HPLC chromatogram of C. globosum DAOM 240349 filtrate extract at 56 254 nm and 400 nm with associated UV spectra (200-600nm) of major metabolites 7 Figure 2.14: Calibration plots for chaetoglobosin A and chaetomugilin D 56 Figure 2.15: Structure of 4’-epi-N-2-hydroxyethyl-azachaetoviridin A 59 Figure 2.16: Observed COSY and key HMBC correlations for compound 4’-epi-N- 60 2-hydroxyethyl-azachaetoviridin A Figure 2.17: Structure of N-2-butyric-azochaetoviridin E 63 Figure 2.18: Structure of isochromophilone XIII 65 Figure 3.1: P. corylophilum growing on 2% MEA 80 Figure 3.2: Structure of the iscohroman DHMI isolated and its synthetic derivative 81 8-methoxy-DHMI Figure 3.3: Structure of the sesquiterpene phomenone and the isocoumarins (+)- 82 orthosporin and citreoisocoumarinol Figure 3.4: Structures of citrinin, epoxyagroclavine-I and decarestrictine D 83 Figure 3.5: Structure of (1S,3S)-1,6,8-trihydroxy-3-(7-hydroxyheptyl) isochroman- 85 7-carboxylic acid Figure 3.6: Methylation of compound 3.1 to its tetramethyl derivative 3.5 with 87 diazomethane Figure 3.7: Structure of CJ- 12,373 from an unidentified Penicillium