University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES STRUCTURE DETERMINATION AND BIOACTIVITY OF NOVEL CHLORINATED PEPTIDES ISOLATED FROM RHODOCOCCUS SP. M1042 BY SAMUEL KWAIN (10341226) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF PHILOSOPHY DEGREE IN CHEMISTRY DEPARTMENT OF CHEMISTRY JULY, 2018 i University of Ghana http://ugspace.ug.edu.gh DECLARATION I, Samuel Kwain, declare that the work contained in this thesis was undertaken solely by me under supervision and has neither wholly nor partially been presented elsewhere for another degree. ………………………………………. Samuel Kwain 10341226 (Student) ………………………………………. Dr. Kwaku Kyeremeh (PhD) (PRINCIPAL SUPERVISOR) ………………………………………. Prof. Kwabena M. Bosompem (Co-Supervisor) ii University of Ghana http://ugspace.ug.edu.gh ABSTRACT The Rhodococcus sp. M1042 is a new microbial strain that was isolated from soil sediments collected from Munzur Valley in Tunceli, Turkey. Species in this genus have been found to harbour extensive secondary metabolic pathways that produce novel metabolites with bioactivity with industrial and environmental bio-applications. The complete genome sequence of this strain contains 5,948,800 mega base pairs with 230 NRPS, PKS and NRPS-PKS gene clusters, some of which have been identified to produce already known bioactive compounds. The total crude extract (TCE) obtained after harvesting the fermentation broth of the Rhodococcus sp. M1042 was analysed by spectroscopic and spectrometric techniques to reveal the presence of three chlorinated peptides. A Kupchan solvent partitioning process, gave four fractions amongst which the WB fraction was found to contain these chlorinated peptides. Using column chromatography by gravity, Sephadex LH20 size exclusion and normal phase thin layer chromatography followed by HPLC led to the isolation and purification of these three peptides. The structure of one of the three chlorinated peptides was determined by 1D and 2D NMR data interpretations and mass spectrometry. The structure determination of the remaining two peptides is currently in progress. Subsequently, a connection is being made to the gene clusters that are responsible for the biosynthesis of these peptides. One of the compounds was tested for antiparasitic activity against Leishmania donovani, Trypanosoma brucei, Trichomonas mobilensi, and Plasmodium falciparum but was found to show minimal activity with IC50 >100 µM. The antibacterial activity against Gram-positive S. aureus ATCC 25923, the Gram-negative E. coli ATCC 25922 and a panel of clinical isolates of methicillin-resistant S. aureus (MRSA) strains are currently in progress. iii University of Ghana http://ugspace.ug.edu.gh DEDICATION This thesis is dedicated to my parents Mr. and Mrs Kwain for their encouragement and financial support throughout my years in school. iv University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS To the Lord and sovereign God of my life, praise, worship and honour be given, for he is the one who granted me strength and knowledge to undertake this study. I am most grateful to my supervisors, Dr. Kwaku Kyeremeh and Professor Kwabena M. Bosompem for their support, mentorship, guidance, encouragement, sacrifice and immense contribution during this study. I thank Dr. Kwaku Kyeremeh for his constructive comments and criticism which helped to improve the quality of this thesis. I thank him so much for he has always being there just like a father trying so hard to impart some of his knowledge into my life. I am very thankful to Mr. Enoch Osei as a true friend and brother who has been there for me throughout my challenges and has always been supporting me morally, spiritually, and financially, God richly bless you. You are such an amazing and intelligent friend and I will forever miss your company. Appreciation also goes to Miss Adwoa Padiki, Mr. Kenedy Mawunya, Mr. Tetevi Gilbert Kwame Mawuli and Mr. David Bakomna for their friendship, encouragement and love. I am also grateful to Mr. Charles Fosu, Mr. Evans Osei Boakye, Mr. Emmanuel Lamptey and Mr. John Kupagme as true friends and brothers for their advice, encouragement and support when I needed it the most. My sincere gratitude goes to Professor Mustafa Camas of Tunceli University in Turkey, for making available the Rhodococcus species used for the study. My earnest appreciation also goes to Mr Baffour Awuah and his team at NMIMR for the bioactivity analysis. İ am also thankful to Professor Marcel Jaspars and researchers at the Marine Bio-discovery Center, Chemistry Department, University of Aberdeen, Scotland, for their invaluable assistance. Finally, and most importantly, I thank my beloved family for their continuous support, prayers and encouragement in the course of my work and studies. v University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION ........................................................................................................................ ii ABSTRACT ............................................................................................................................... iii DEDICATION ........................................................................................................................... iv ACKNOWLEDGEMENTS ........................................................................................................ v TABLE OF CONTENTS ........................................................................................................... vi LIST OF FIGURES .................................................................................................................. xiv LIST OF SCHEMES ............................................................................................................... xvii LIST OF TABLES ................................................................................................................. xviii LIST OF ABBREVIATIONS .................................................................................................. xix CHAPTER ONE ......................................................................................................................... 1 Introduction ................................................................................................................................. 2 1.0 Introduction .................................................................................................................................... 2 1.1 Current microbial natural product discovery ................................................................................. 5 1.2 Problem Statement ........................................................................................................................ 6 1.3 Overall Goal .................................................................................................................................... 7 1.4 Hypothesis ...................................................................................................................................... 7 1.5 Objectives ....................................................................................................................................... 7 CHAPTER TWO ......................................................................................................................... 9 Literature review ....................................................................................................................... 10 2.0 Biodiversity of the Munzur Valley in Turkey ................................................................................ 10 2.1 Rhodococcus as prospective source of future antibitotics .......................................................... 10 vi University of Ghana http://ugspace.ug.edu.gh 2.2 Industrial and environmental importance of Rhodococcus strains. ............................................ 11 2.2.1 Bioconversion of Indene to Indandiols, precursors to the HIV/AIDS drug Indinavir ............. 11 2.2.2 Biodegradation and bioremediation of pollutants. ............................................................... 13 2.2.3 Biosensors ............................................................................................................................. 14 2.2.4 Production of biosurfactants and bioflocculants. ................................................................. 15 2.2.5 Desulphurization of fossil fuels. ............................................................................................ 16 2.2.6 Industrial biotransformation and syntheses. ........................................................................ 16 2.3 Secondary metabolites isolated from Rhodococcus .................................................................... 17 2.3.1 Antibiotics from the genus Rhodococcus .............................................................................. 17 2.3.1.1 Lariatins .......................................................................................................................... 18 2.3.1.2 Aurachins ........................................................................................................................ 19 2.3.1.3 Rhodostreptomycins ...................................................................................................... 20 2.3.1.4 Indole-3-acetaldehyde and indole-3-acetic acid. ........................................................... 21 2.3.1.5 Saframycin AR1, AR2 and AR3 ........................................................................................ 22 2.3.2 Antifungals from the genus Rhodococcus ............................................................................. 23 2.3.2.1 Rhodopeptins ................................................................................................................. 23 2.3.3 Other important compounds produced by Rhodococcus species ........................................ 24 2.3.3.1 Siderophores .................................................................................................................. 24 2.3.3.1.1 Heterobactin A and B .............................................................................................. 25 2.3.3.1.2 Rhodobactin ............................................................................................................ 26 2.3.3.1.3 Rhodochelin ............................................................................................................. 26 2.3.3.2 Rhodococcus pigments ................................................................................................... 27 vii University of Ghana http://ugspace.ug.edu.gh 2.3.3.3 4,7,8-trihydroxyisoflavone 7-O-α-D-arabinofuranoside ................................................ 28 2.3.3.4 Mycothiols ...................................................................................................................... 29 2.4 Complete genome sequencing of microbes for natural product discovery ................................. 29 2.4.1 Biosynthesis of Rhodochelin ................................................................................................. 31 2.4.2 Known compounds expressed by the gene clusters in the complete genome sequence of Rhodococcus sp. strain M1042 ...................................................................................................... 33 2.5 Terminologies in bioactivity measurements ................................................................................ 34 2.5.1 Half maximal inhibitory concentration (IC50)......................................................................... 34 2.5.2 Half maximal effective concentration (EC50) ......................................................................... 35 2.5.3 Half maximal Effective dose (ED50) ...................................................................................... 36 2.5.4 Lethal dose for 50% (LD50) ................................................................................................... 36 2.5.5 Lethal concentration for 50% (LC50) ...................................................................................... 37 2.5.6 Minimum inhibitory concentration (MIC) ............................................................................. 37 2.5.7 Minimum bactericidal concentration (MBC) ......................................................................... 37 2.5.8 Growth inhibition concentration for 50% (GI50) .................................................................... 37 CHAPTER THREE ................................................................................................................... 38 3.0 Materials and methods ................................................................................................................ 39 3.1 Sampling ....................................................................................................................................... 39 3.1.1 Soil sample collection from Munzur Valley ........................................................................... 39 3.1.2 Sampling site ......................................................................................................................... 40 3.2 Bacteria Media Preparation ......................................................................................................... 41 3.2.1 Materials ................................................................................................................................ 41 viii University of Ghana http://ugspace.ug.edu.gh 3.2.2 Preparation of International Streptomyces Protocol (ISP) 2 liquid media ............................ 41 3.2.3 Preparation of International Streptomyces Protocol (ISP) 2 agar media .............................. 41 3.3 Culturing, small scale fermentation (seeding) and preservation of bacteria ............................... 43 3.3.1 Material ................................................................................................................................. 43 3.3.2 Isolation of Rhodococcus sp. strain M1042 from glycerol stock ........................................... 43 3.3.3 Small Scale Fermentation of Pure Rodococcus sp. strain M1042 ......................................... 44 3.3.4 Cryopreservation of bacteria species .................................................................................... 44 3.4 Screening of crude extracts from small scale culture .................................................................. 45 3.5 Large scale fermentation of Rhodococcus sp strain M1042......................................................... 45 3.6 Isolation of compounds from M1042 extracts ............................................................................. 46 3.7 Isolation of compounds from M1042 WB fraction ...................................................................... 48 3.7.1 Sephadex LH20 size exclusion purification of M1042-WB fraction....................................... 48 3.7.2 Gravity column chromatography purification of M1042-WB-SFC ........................................ 49 3.7.3 Isolation of peptides from M1042-WB-SFC-C6-MDe fraction using HPLC ............................ 51 3.7.4 1D and 2D NMR Analysis ....................................................................................................... 53 3.8 Biological activity test ................................................................................................................... 53 3.8.1 Chemicals and reagents ........................................................................................................ 53 3.8.2 Preparation of compounds for bioactivity testing ................................................................ 53 3.8.3 Anti-malaria activity study .................................................................................................... 54 3.8.3.1 Blood collection and erythrocytes preparation ............................................................. 54 3.8.3.2 Giemsa stained thin blood smear and parasitaemia determination ............................. 54 3.8.3.3 In vitro cultivation of Malaria Parasite ........................................................................... 55 ix University of Ghana http://ugspace.ug.edu.gh 3.8.3.4 Screening for anti-malaria activity by SYBR Green I assay ............................................. 55 3.8.4 Screening of compounds for Anti-trypanosomal activity...................................................... 56 3.8.4.1 Culturing of Trypanosome parasites. ............................................................................. 56 3.8.4.2 Trypanosome parasites in vitro viability test. ................................................................ 56 3.8.5 Screening of compounds for Anti- leishmania activity.......................................................... 57 3.8.5.1 Cuturing of Leishmania Parasites ................................................................................... 57 3.8.5.2 Leishmania parasites in vitro viability test. .................................................................... 57 3.8.6 In-vitro susceptibility testing of Trichomonas mobilensis ..................................................... 58 CHAPTER FOUR ..................................................................................................................... 59 4.0 Results and Discussion ........................................................................................................ 60 4.1 Discussion of the techniques used to isolate and study the chemistry of Rhodococcus sp. ....... 60 4.2 Taxonomy of Rhodococcus sp. strain M1042 ............................................................................... 61 4.3 Extraction, fractionation, isolation and structure elucidation of compounds from Rhodococcus sp. strain M1042 ...................................................................................................................................... 64 4.3.1 Structure elucidation of M1042-WB-SFC-C6-MDe-B (compound B) ..................................... 72 4.3.1.1 Tyrosine residues ............................................................................................................ 74 4.3.1.2 Proline residue ............................................................................................................... 77 4.3.1.2 Threonine residues ......................................................................................................... 78 4.3.1.3 Isoleucine residue ........................................................................................................... 80 4.3.1.4 Valine residue ................................................................................................................. 82 4.3.1.5 Asparagine residue ......................................................................................................... 83 4.3.1.6 2,8-Diaminooctanoic acid residue .................................................................................. 84 x University of Ghana http://ugspace.ug.edu.gh 4.3.1.7 Non-amino acid prenylation residues ............................................................................ 86 4.4 Sequencing of the genome of Rhodococcus sp. strain M1042 to identify the gene clusters of M1042-WB-SFC-C6-MDe-B, M1042-WB-SFC-C6-MDe-C, and M1042-WB-SFC-C6-MDe-D peptides 88 4.5 Cytotoxicity studies ...................................................................................................................... 88 CHAPTER FIVE ....................................................................................................................... 90 5.1 Conclusion .................................................................................................................................... 91 5.0 Recommendations ....................................................................................................................... 91 References ................................................................................................................................. 92 Appendices .............................................................................................................................. 121 Appendix 1: 13 C NMR spectrum of M1061-WB-SFC-C6-MDe-B ....................................................... 121 Appendix 2: 13 C NMR spectrum of M1061-WB-SFC-C6-MDe-B ....................................................... 122 Appendix 3: 13 C NMR spectrum of M1061-WB-SFC-C6-MDe-B ....................................................... 123 Appendix 4: 13 C NMR spectrum of M1061-WB-SFC-C6-MDe-B ....................................................... 124 Appendix 5: 13 C NMR spectrum of M1061-WB-SFC-C6-MDe-B ....................................................... 125 Appendix 6: 13 C NMR spectrum of M1061-WB-SFC-C6-MDe-B ....................................................... 126 Appendix 7: HSQC spectrum of M1061-WB-SFC-C6-MDe-B ............................................................ 127 Appendix 8: HSQC spectrum of M1061-WB-SFC-C6-MDe-B ............................................................ 128 Appendix 9: HSQC spectrum of M1061-WB-SFC-C6-MDe-B ............................................................ 129 Appendix 10: HSQC spectrum of M1061-WB-SFC-C6-MDe-B .......................................................... 130 Appendix 11: HSQC spectrum of M1061-WB-SFC-C6-MDe-B .......................................................... 131 Appendix 12: HSQC spectrum of M1061-WB-SFC-C6-MDe-B .......................................................... 132 Appendix 13: HSQC spectrum of M1061-WB-SFC-C6-MDe-B .......................................................... 133 xi University of Ghana http://ugspace.ug.edu.gh Appendix 14: HSQC spectrum of M1061-WB-SFC-C6-MDe-B .......................................................... 134 Appendix 15: HSQC spectrum of M1061-WB-SFC-C6-MDe-B .......................................................... 135 Appendix 16: TOCSY spectrum of M1061-WB-SFC-C6-MDe-B......................................................... 136 Appendix 17: TOCSY spectrum of M1061-WB-SFC-C6-MDe-B......................................................... 137 Appendix 18: TOCSY spectrum of M1061-WB-SFC-C6-MDe-B......................................................... 138 Appendix 19: TOCSY spectrum of M1061-WB-SFC-C6-MDe-B......................................................... 139 Appendix 20: TOCSY spectrum of M1061-WB-SFC-C6-MDe-B......................................................... 140 Appendix 21: TOCSY spectrum of M1061-WB-SFC-C6-MDe-B......................................................... 141 Appendix 22: HMBC spectrum of M1061-WB-SFC-C6-MDe-B ......................................................... 142 Appendix 23: HMBC spectrum of M1061-WB-SFC-C6-MDe-B ......................................................... 143 Appendix 24: HMBC spectrum of M1061-WB-SFC-C6-MDe-B ......................................................... 144 Appendix 25: HMBC spectrum of M1061-WB-SFC-C6-MDe-B ......................................................... 145 Appendix 26: HMBC spectrum of M1061-WB-SFC-C6-MDe-B ......................................................... 146 Appendix 27: HMBC spectrum of M1061-WB-SFC-C6-MDe-B ......................................................... 147 Appendix 28: HMBC spectrum of M1061-WB-SFC-C6-MDe-B ......................................................... 148 Appendix 29: HMBC spectrum of M1061-WB-SFC-C6-MDe-B ......................................................... 149 Appendix 30: HMBC spectrum of M1061-WB-SFC-C6-MDe-B ......................................................... 150 Appendix 31: HMBC spectrum of M1061-WB-SFC-C6-MDe-B ......................................................... 151 Appendix 32: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B ................................................. 152 Appendix 33: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B ................................................. 153 Appendix 34: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B ................................................. 154 Appendix 35: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B ................................................. 155 xii University of Ghana http://ugspace.ug.edu.gh Appendix 36: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B ................................................. 156 Appendix 37: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B ............................................... 157 Appendix 38: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B ................................................. 158 Appendix 39 Mass spectrum showing neutral loss of m/z 36 .......................................................... 159 xiii University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 2.1: Model of rhodochelin iron-coordination. ............................................................... 27 Figure 2.2: Proposed biosynthetic pathway for rhodochelin assembly. The two building blocks 2,3-DHB and L-fhOrn are synthesised through their respective pathways and linked together forming a tripeptide by the synthetase RhcB. The TE- catalysed an ester bond formation between the third building block L-fhOrn and the L-Thr side chain of the tripeptide resulting in rhodochelin. ............................................................................................................................... 32 Figure 2.3: Illustration of Half Inhibitory Concentration (IC50) ............................................... 35 Figure 2.4: Illustration of Half Maximal Effect Concentration (EC50) ..................................... 35 Figure 2.5: Illustration of Effective Dose (ED50). ..................................................................... 36 Figure 2.6: Illustration of Lethal Dose (LD50) .......................................................................... 36 Figure 3.1: A geographical may of Munzur Valley National Park showing the sampling sites. ................................................................................................................................................... 40 Figure 3.2: A picture showing preparation of the ISP 2 modified agar plates on which the microbes from the soil samples were grown ............................................................................. 42 Figure 3.3: A picture showing smearing of the glycerol stock containing the bacteria on the modified agar plates (left) and obtained pure strains of Rhodococcus species (right) ............. 43 Figure 3.4: Picture showing small scale culture of Rhodococcus strains. ................................ 44 Figure 3.5: Pictures showing preserved bacteria at preparation stage for storage at -80oC. ..... 45 Figure 3.6: A picture showing excerpt of kupchan solvent partitioning technique of the crude extract from M1042 ................................................................................................................... 48 Figure 3.7: A picture showing sephadex LH20 size exclusion purification of M1042-WB fraction ...................................................................................................................................... 49 Figure 3.8: A normal phase TLC plate of M1042-WB-SFC-C5 and M1042-WB-SFC-C6 fractions showing positive results for ninhydrin test. ............................................................... 50 xiv University of Ghana http://ugspace.ug.edu.gh Figure 4.1: Neighbour-joining tree based on almost complete 16 rDNA gene sequences (1462 nt) showing the position of Rhodococcus sp. M1042 amongst its phylogenetic neighbours. .. 62 Figure 4.2: HRESI/HPLC-DAD-MSn spectrum of M1042-FH fraction ................................... 65 Figure 4.3: 1H-NMR spectrum of M1042-FH fraction ............................................................. 65 Figure 4.4: HRESI/HPLC-DAD-MSn spectrum of M1042-FD fraction ................................... 66 Figure 4.5: 1H-NMR spectrum of M1042-FD fraction ............................................................. 67 Figure 4.6: HRESI/HPLC-DAD-MSn spectrum of M1042-FM fraction .................................. 68 Figure 4.7: 1H-NMR spectrum of M1042-FM fraction............................................................. 68 Figure 4.8: HRESI/HPLC-DAD-MSn spectrum of M1042-WB fraction .................................. 69 Figure 4.9: 1H-NMR spectrum of M1042-WB fraction ............................................................ 70 Figure 4.10: A normal phase TLC plate with M1042 WB fraction which shows positive results for ninhydrin test as an indication of either free amino acid or peptide present in the fraction. ................................................................................................................................................... 70 Figure 4.11: HPLC profile of M1042-WB-SFC-C6-MDe fraction showing the peaks that yielded the three peptides .......................................................................................................... 72 Figure 4.12: UV spectrum of M1042-WB-SFC-C6-MDe-B showing prominent absorption maxima at λmax of 275.9 ............................................................................................................ 73 Figure 4.13: HRESI/HPLC-DAD-MSn spectrum of pure M1042-WB-SFC-C6-MDe-B......... 74 Figure 4.14: Substructure for first tyrosine residue showing TOCSY, COSY, and HMBC correlations ................................................................................................................................ 75 Figure 4.15: Substructure for second tyrosine residue showing TOCSY, COSY, and HMBC correlations ................................................................................................................................ 76 Figure 4.16: Substructure for Proline residue showing TOCSY, COSY, and HMBC correlations ................................................................................................................................................... 77 xv University of Ghana http://ugspace.ug.edu.gh Figure 4.17: Substructure for first Threonine residue showing TOCSY, COSY, and HMBC correlations ................................................................................................................................ 79 Figure 4.18: Substructure for second Threonine residue showing TOCSY, COSY, and HMBC correlations ................................................................................................................................ 80 Figure 4.19: Substructure for Isoleucine residue showing TOCSY, COSY, and HMBC correlations ................................................................................................................................ 81 Figure 4.20: Substructure for Valine residue showing TOCSY, COSY, and HMBC correlations ................................................................................................................................................... 82 Figure 4.21:Substructure for Asparagine residue showing TOCSY, COSY, and HMBC correlations ................................................................................................................................ 83 Figure 4.22: Substructure for 2,8-Diaminooctanoic acid residue showing TOCSY, COSY, and HMBC correlations ................................................................................................................... 85 Figure 4.23: Substructure for Non-amino acid prenylation residue showing TOCSY, COSY, and HMBC correlations ................................................................................................................... 86 Figure 4.24: Substructure for Non-amino acid prenylation residue showing TOCSY, COSY, and HMBC correlations ................................................................................................................... 87 xvi University of Ghana http://ugspace.ug.edu.gh LIST OF SCHEMES Scheme 2.1: Proposed bioconversion pathway of indene to trans (1R, 2R) and cis (1S, 2R) indandiol by Rhodococcus MA 7205 strain. ............................................................................. 12 Scheme 2.2: Bioconversion of saframycin A to saframycin AR1, AR2 and AR3 by Rhodococcus nocadia following two main possible routes. ............................................................................ 22 Scheme 3.1: A flowchart of the modified Kupchan Solvent Partition technique. This technique separates compounds in crude extracts into four fractions according to their polarities. The butanol fraction (WB) being the most polar and hexane fraction (FH) being the least polar. The FD and FM fractions are of intermediate polarities. ................................................................. 47 Scheme 3.2: A flow chart of isolation of peptides from M1042-WB fraction of TCE of the fermentation broth of Rhodococcus sp ...................................................................................... 52 xvii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 4.1: A table showing other species of the genus Rhodococcus with their index similarity pairwise and completeness to the new strain M1042 ................................................................ 63 Table 4.2: 1H and 13C NMR data for first tyrosine residue ....................................................... 75 Table 4.3: 1H and 13C NMR data for second tyrosine residue .................................................. 76 Table 4.4: 1H and 13C NMR data for Proline residue ................................................................ 78 Table 4.5: 1H and 13C NMR data for first Threonine residue ................................................... 78 Table 4.6: 1H and 13C NMR data for second Threonine residue ............................................... 79 Table 4.7: 1H and 13C NMR data for Isoleucine residue ........................................................... 81 Table 4.8: 1H and 13C NMR data for Valine residue ................................................................. 83 Table 4.9: 1H and 13C NMR data for Asparagine residue ......................................................... 84 Table 4.10: 1H and 13C NMR data for 2,8-Diaminooctanoic acid ............................................ 85 Table 4.11:1H and 13C NMR data for Non-amino acid prenylation residue ............................. 86 Table 4.12: 1H and 13C NMR data for Non-amino acid prenylation residue ............................ 87 Table 4.13: Cell growth-inhibitory potencies of pure compounds expressed as IC50 values .... 89 xviii University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS BLAST Basic Local Alignment Search Tool COSY Correlation Spectroscopy DCM Dichloromethane DHB 2,3-dihydroxybenzoic acid DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid EC50 Median Effective Concentration EtOAc Ethyl acetate ESMS Electrospray Mass Spectrometry FD Dichloromethane Fraction FH Hexane Fraction fhOrn δ-N-formyl-δ-N-hydroxyornithine fOrn δ-N-formylornithine FM 50% aqueous methanol fraction GI50 Median Growth Inhibition HMBC Heteronuclear Multiple Bond Correlation hOrn δ-N-hydroxyornithine HPLC High Performance Liquid Chromatography HSQC Heteronuclear Single Quantum Coherence HTS High throughput screening ICPMS Inductively Coupled Plasma Mass Spectrometry IC50 Median Inhibition Concentration LC-MS Liquid Chromatography-Mass spectrometry LD50 Median Lethal Dose xix University of Ghana http://ugspace.ug.edu.gh MeOH Methanol MIC Minimum Inhibition Concentration MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide bromide NMR Nuclear Magnetic Resonance Spectroscopy NRP Non-ribosomal peptide NRPS Non-ribosomal peptide synthetase Orn Ornithine PCP peptidyl-carrier-protein PK Polyketide PKS Polyketide synthase rDNA Ribosomal Deoxyribonucleic acid TE Thioesterase domain TCE Total Crude Extract TOCSY Total Correlation Spectroscopy TROESY Transverse Rotating Frame Overhauser Enhancement WB Water/Sec-butanol fraction xx University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1 University of Ghana http://ugspace.ug.edu.gh Introduction 1.0 Introduction Microbial natural products have made tremendous contributions to the health and general well- being of people throughout the world. Historically, microbial natural products have been one of the most prolific sources of new leads in modern drug discovery especially in the case of antibiotics.1 Despite the huge microbial biodiversity available, Streptomyces have proved to be an unlimited source of novel compounds some of which have become very important for the treatment of infections.2,3 The discovery of new antibiotics from Streptomyces began with the discovery of streptothricin in 1942 and the subsequent discovery of streptomycin in 1946.4,5 As a result of these two major discoveries, scientists intensified their research within the genus and this lead to the discovery of many of our current antibiotics. Currently, more than 80% of the antibiotics in our clinics are sourced from the genus Streptomyces with many more molecules from the genus, tracked at some stage of the drug discovery pipeline.6 The recognition that natural products of microbial origin held clinical 2 University of Ghana http://ugspace.ug.edu.gh potential spurred the discovery of myriad antibacterial natural products from readily cultured bacteria species.1,7 Despite the accomplishment of the discovery of antibiotics and advances in the techniques for their production, infectious diseases still remain the second leading cause of death worldwide and bacterial infections cause roughly 17 million deaths yearly with tuberculosis, now the leading cause of death among infectious diseases.8 This worldwide problem is mainly attributed to antimicrobial resistance which is gradually on the rise. The number of resistant organisms, the geographic areas influenced by drug resistance, and the breadth of resistance in single organisms are currently unprecedented. Diseases and disease agents that were once thought to be controlled by antibiotics are returning with increased virulence.9 The increasing resistance of pathogenic organisms, prompting extreme types of diseases that are hard to treat, has additionally complicated the situation, as on the account of Staphylococcus aureus and other microorganisms.10 Infections caused by resistant bacteria do not respond to treatment, resulting in prolonged ailment and greater risk of death. Most worrisome is that, resistance to virtually all antibiotics has increased. Philosophically, the tremendous increase in antibiotics resistance can be attributed to the fact that, most microbial antibiotics in the clinic today are predominantly isolated from the genus Streptomyces. The huge chemical diversity represented by all microbes is still yet untapped and it is possible that the structural diversity represented by molecules from Streptomyces alone is effectively a very small portion of this diversity. Therefore, pathogenic bacteria could have developed enough genetic variability to adapt to the offensive antibiotics delivered from Streptomyces. Henceforth, detailed research into antibiotics produced by other bacteria genus other than Streptomyces is important in order to extend the range of chemical diversity offered by 3 University of Ghana http://ugspace.ug.edu.gh antibiotics in the clinic. New biodiversity always provides unique biology which leads to genomes that possess the capability of producing chemically diverse molecules. For example, chemical investigation of one of the less studied genus Kocuria palustris led to the isolation of a new thiazolyl peptide antibiotic known as kocurin with activity against methicillin-resistant Staphylococcus aureus with MIC value of 0.25 μg/mL.11 There is therefore the need to discover new scaffolds from less studied bacteria strains such as, the genus Rhodococcus among others to provide a solid platform for the development of new antibiotics. The genus Rhodococcus is one of the most interesting bacteria species with many environmental and industrial applications. These applications range from bioremediation and biodegradation of pollutants12,13, fossil fuel bio-desulfurization14,15, production of acrylamide and acrylic acid16, production of bio-surfactants and biosensors17 to applications as microbial biocatalyst in industry18. The biotechnological importance of Rhodococcus is as a result of their lifestyles and highly evolved bio-genomes which enables them to degrade a wide range of organic compounds in the environment.19 Rhodococcus have been identified as having a simpler developmental lifecycle than Streptomyces. Therefore, this bacteria species is a great source of 4 University of Ghana http://ugspace.ug.edu.gh chemical diversity which can provide structurally intriguing molecules whose biosynthesis apparatus can be tracked to the genome level.20 1.1 Current microbial natural product discovery Currently, the rejuvenated philosophy in microbial natural product drug discovery is to conduct whole genome sequencing of talented bacteria strains. Whole genome sequencing normally results in data that is processed through bioinformatics to reveal not only the gene clusters responsible for the compounds which are expressed by the microbe under laboratory conditions but, also those molecules that are not expressed. Advances in the technology of this new area, coupled with increased access to DNA sequencing data, provides a wealth of important information about how natural products are assembled, mechanisms by which natural product gene clusters can be manipulated to yield new chemical diversity, and the genetic potential of individual organisms.21 Henceforth, another strategy to improve the discovery of new antibiotics is to study into detail the genomes of novel natural product producing bacteria. Comparing the gene clusters of metabolites that are expressed under laboratory conditions to the clusters of metabolites that are not expressed provides a holistic knowledge of the biosynthesis of interesting molecules and their possible expression in heterologous host. Such biosynthesis experiments enable the preparation of different derivatives and analogues of compounds that sometimes prove more effective than their naturally occurring counterparts (combinatorial biosynthesis). This approach used to study bacteria has turned out to be a particularly appealing method for the search for novel antibiotics because, the genes that encode the biosynthesis of individual secondary metabolites are generally clustered on bacterial chromosomes.22 Traditional fermentation-based activation strategies involved principally in changing culture media conditions also helps to activate orphan or cryptic gene clusters.23 5 University of Ghana http://ugspace.ug.edu.gh In this project, a Rhodococcus sp strain M1042 isolated from Munzur Valley Soil from 39° 7'43.73", 39°30'18.80" in Tunceli was obtained from Professor Mustafa Camas of Tunceli University in Turkey and studied into detail for its novel antibiotic producing capabilities. This strain was found to have a number of very important industrial and environmental applications including the biosynthesis of gold nanoparticles. Nonetheless, the antibiotic producing capability of this strain has not been studied eventhough the biosynthesized gold nanoparticles produced excellent cytotoxicity results against HUVEC (human umbilical vein endothelial cells) and HeLa (Henrietta Lacks) cells. The overall goal of this research is to emphasize the importance of intensifying the search for antibiotics from bacterial genus other than Streptomyces and also the importance of connecting expressed secondary metabolites to their respective clusters. This should pave the way towards the discovery of novel antibiotics with novel and promiscuos mechanisms of action. The complete genome sequence of this strain was obtained and three large peptides isolated and characterized from its extracts. Subsequently, a connection was made to the clusters that are responsible for these peptides and their biosynthesis proposed. 1.2 Problem Statement Rhodococcus strains isolated from soils and sediments around the world have shown tremendous biosynthetic capabilities which has proved useful in a number of industrial and environmental applications. However, this genus compared to Streptomyces has been less studied for the possibility of obtaining new antibiotics. With genomes that encode interesting biosynthetic clusters, Rhodococcus strains must be investigated further to discover their antibiotic producing capabilities. 6 University of Ghana http://ugspace.ug.edu.gh 1.3 Overall Goal The aim of this project is to isolate the novel secondary metabolites produced by Rhodococcus sp. strain M1042, determine their structures using a combination of mass spectrometry and UV, IR, 1D and 2D-NMR spectroscopic data interpretation. The metabolites whose structures are obtained will be connected to their respective biosynthesis gene clusters and possible biosynthesis pathways proposed. 1.4 Hypothesis The Rhodococcus sp strain M1042 is a bacteria strain with the capability to biosynthesise structurally intriguing bioactive molecules that have potential as future antibiotics. 1.5 Objectives a) To prepare a pure culture plate of Rhodococcus sp M1042 from -80 ℃ glycerol stock. b) To small scale culture and extract the secondary metabolites of Rhodococcus sp M1042. c) To perform detailed chemical profile study by subjecting the extract to a high resolution HPLC-Mass spectrometry with subsequent analysis of the data. d) To do a large scale culture in order to obtain isolable quantities of secondary metabolites from the crude extract of the strain. e) To subsequently purify and isolate compounds from the crude extract using column chromatograph and HPLC. f) To obtain 1H, 13C, HSQC, 1H-1H COSY, HMBC, HSQC-TOCSY, HRESI/HPLC-DAD- MSn data for isolated compounds. g) To determine the structures of isolated compounds using the spectroscopic and spectrometric data obtained. h) To do a complete genome sequence of the Rhodococcus sp. strain M1042 7 University of Ghana http://ugspace.ug.edu.gh i) To do a BLAST on the complete genome sequence of the Rhodococcus sp. strain M1042 and identify the gene clusters that code for the biosynthesis of secondary metabolites. j) To screen pure compounds for antibiotic and antiparasitic activity. 8 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 9 University of Ghana http://ugspace.ug.edu.gh Literature review 2.0 Biodiversity of the Munzur Valley in Turkey Turkey lies at the nexus of Europe, Central Asia, Middle East, and Africa.24 It is the only country that has 3 out of the 34 biodiversity rich hotspots in the world and these are the Mediterranean, Caucasus, and Irano-Anatolian.25,26 Turkey’s geographical location and landscape have brought about its fresh water, high terrestrial, and marine biodiversity.27-29 The Munzur Valley is the largest and most biodiverse national park in Turkey and it is located at the Munzur Mountain Range within the Tunceli Province of eastern Anatolia.30 It was fully established as a national park on December 21, 1971. Currently, the Munzur Valley is the home of about 1500 registered species of plants and it has a unique natural environment that offers adequate habitat for wildlife.31-33 This highly biodiverse hotspot provides several unique un-or underexplored habitats capable of providing novel microorganisms living under extreme conditions with the potential of biosynthesising new and novel bioactive molecules with potential as future antibiotics. 2.1 Rhodococcus as prospective source of future antibitotics Kingdom: Bacteria Phylum: Actinobacteria Order: Actinomycetales Suborder: Corynebacterineae Family: Norcardiaceae Genus: Rhodococcus Species: Many known important species Although there are millions of bacteria present here on earth, most of them can be comfortably classified into taxonomic groups that aid their study and investigation.34-36 From the bacterial phylogenetic tree for the order Actinomycetales, the family Norcadiaceae, which incorporates 10 University of Ghana http://ugspace.ug.edu.gh Rhodococcus, is identified with the family Corynebacterineae which has been distinguished as a drug-producing family.37-39 From this observation, the family Norcardiaceae, more particularly the genus Rhodococcus, will be a decent space of focus with expectations of potential bioactive compounds. Rhodococcus is a genus of aerobic, nonsporulating, Gram-positive, non-motile, mycolate-containing, and nocardioform actinomycetes.40,41 Rhodococcus is the only genera of actinomycetes to have mycolic acid in their cell wall consequently the term mycolate- containers.42,43 Rhodococci have been isolated from various sources including marine sediments, soils, boreholes, rocks, groundwater, the guts of insects, animal dung and from diseased animals and plants.44-46 The genus Rhodococcus was initially investigated for its environmental and industrial bio-applications until 2006, where a complete genome of the Rhodococcus strain RHA1 isolated from soil was sequenced by McLeod and colleagues.20 RHA1 was found to contain twenty four nonribosomal peptide synthase genes, six of which surpass 25kbp, and seven polyketide synthase genes, giving confirmation that Rhodococcus harbour a broad secondary metabolic pathways20. Notwithstanding their potent secondary metabolism, Rhodococcus offer benefits as experimental systems for the discovery of potential biologically active molecules due to their simpler developmental life cycles compared to the well-studied genus Streptomycetes.47 This therefore opens the door for the potential of the genus Rhodococcus to influence other areas of science especially drug discovery. 2.2 Industrial and environmental importance of Rhodococcus strains. 2.2.1 Bioconversion of Indene to Indandiols, precursors to the HIV/AIDS drug Indinavir The treatment of AIDS is a worldwide priority and selective inhibition of the proteolytic post- translational modifying activity of the human immunodeficiency virus (HIV) has emerged as a high priority target.50 Indanivir an orally active HIV/AIDS protease inhibitor possess five chiral centres however, just one of the thirty two possible stereoisomers is believed to have the 11 University of Ghana http://ugspace.ug.edu.gh specified activity.51 The right hand segment of Indanivir consist of (-)-cis-(1S,2R)-1- aminoindan-2-ol, which was obtained initially by a reaction involving acetonitrile and racemic indanoxide followed by recrystallization.51,52 This synthetic approach was very challenging due to the fact that a complicated mixture of isomers was the result. Alternatively, this right hand portion of Indanivir is now synthesized by a reaction using either trans (1R,2R) or cis (1S,2R) indandiol in enantiomerically pure form.52,53 One important application is the use of Rhodococcus MA 7205 strain as a microbial biocatalyst in the bioconversion of indene to trans (1R,2R) and cis (1S,2R) indandiols.48 The Rhodococcus MA 7205 strain is capable of metabolizing toluene and naphthalene as sole carbon sources. Scheme 2.1: Proposed bioconversion pathway of indene to trans (1R, 2R) and cis (1S, 2R) indandiol by Rhodococcus MA 7205 strain. 12 University of Ghana http://ugspace.ug.edu.gh This strain has a toluene-inducible dioxygenase, a naphthalene-inducible dioxygenase, and a naphthalene-inducible monooxygenase which facilitate oxygenation of indene to the conceivable idandiols.48 Both trans (1R,2R) and cis (1S,2R) are potential antecedents to (-)-cis (1S,2R)-1-aminoindan-2-ol, a key chiral synthon for Indinavir.49 2.2.2 Biodegradation and bioremediation of pollutants. The ability of Rhodococci to metabolise substituted hydrocarbons and other chemicals suggests that, they may play a vital role in both natural degradation and bioremediation of these compounds.54 The resistance of rhodococci to metabolite constraints, starvation and their environmental persistence make them incredible candidates for bioremediation treatments.55,56 Furthermore, the hydrophobicity of Rhodococcus cells are attributed to the mycolic acids of aliphatic chains in their cell wall. Hence, these features easily permit degradation of hydrophobic chemical pollutants by enabling the cells to hold fast to the water and oil interphases.13,57 Rhodococcus species can degrade a wide variety of chemical pollutants ranging from simple hydrocarbons to complex aromatic hydrocarbons and this includes other pollutants like chlorinated hydrocarbons, nitroaromatics and chlorinated polycyclic aromatics e.g. polychlorinated biphenyls.12,58 Polychlorinated biphenyls are class of chemical pollutants that poses great concern because, they are obstinate to degradation and have high tenacity as hazardous pollutants in groundwater and soils.59,60 Polychlorinated biphenyls have been utilised industrially for a range of functions owing to their stability. A number of studies have revealed that Rhodococcus species such as Rhodococcus chlorophenolicus, Rhodococcus rhodochrous, Rhodococcus globerulus, and Rhodococcus erythropolis are good candidates for the degradation of polychlorinated biphenyls.61,62 13 University of Ghana http://ugspace.ug.edu.gh Sulphonated azo dyes e.g. s-triazines, metamitron, and pesticides such as n-methyl carbamates are other toxic recalcitrant chemical pollutants which can be degraded by the genus Rhodococcus.63-66 Rhodococcus species offer great advantages in bioremediation processes by accumulation of significant heavy metal particles including those that are radioactive in their cells. This bioaccumulation by Rhodococcus species offer potential alternative to existing strategies for the recuperation of radioactive compounds or decontamination of both aquatic environment and wastewater or nuclear facilities. An example of the potential of Rhodococcus species as bioremediation facilitators is the ability of Rhodococcus sp. CS402 and Rhodococcus erythropolis CS98 to accumulate significant levels of caesium ions after 24 hours of incubation in the presence of caesium.67 2.2.3 Biosensors Artificial biosensors are routinely incorporated with Rhodococcus cells for identification of compounds like phenols, chlorophenols and other hydrocarbons.68,69 Biosensors can permit fast recognition of target compounds and evaluate their bioavailability which is important for environmental toxicity testing.70 The genus Rhodococcus offers a rich source of enzymes that can be utilised as part of biosensors. Currently, biosensors made up of Rhodococcus cells and a transducer is used in the detection of toxic xenobiotic such as herbicides, fungicides or general biocides for environmental protection.69 The heroin esterase produced from Rhodococcus sp. H1 strain has potential application as heroin biosensors.71 The heroin esterase was found to possess the ability to hydrolyse both acetylester groups in the structure of heroin to give morphine which could be acted upon by an illicit-drug biosensor made up of specific NADP+ -dependent morphine dehydrogenase.71,72 14 University of Ghana http://ugspace.ug.edu.gh Scheme 2.1: Coupled enzyme (Rhodococcus strain H1) assay for the detection of heroin 2.2.4 Production of biosurfactants and bioflocculants. Apart from the biodegradation capacities of Rhodococcus species, this group of microbes also have the capacity to produce biosurfactants.19 Biosurfactant compounds contain both hydrophobic and hydrophilic groups and in this way exist between the interfaces of aqueous and oil phases. Biosurfactants isolated from Rhodococcus species are more efficient, less toxic, and effective in biodegradation of chemical pollutants than their synthetic counterparts.73 Biosurfactants isolated from Rhodococcus ruber incredibly improved oil expulsion from soil washing tests in a laboratory analysis.74 Bioflocculant material produced by Rhodococcus erythropolis has the ability to cause flocculation of extensive variety of suspended solids.75 The flocculant consists of assemblies of lipids, especially mycolate-containing glycolipids and polypeptides.75 Such materials could help expulsion of suspended solids in squander water or emanating treatment.76 15 University of Ghana http://ugspace.ug.edu.gh 2.2.5 Desulphurization of fossil fuels. Desulphurization of petroleum and coal by microbial species has been recommended as a method for preventing sulphurous discharge after combustion into the atmosphere thereby minimizing issues with acidic rain and improving the quality of fuel.78Advancements in the technologies to prevent the release of sulphur containing compounds from crude oil and coal have been made but, the technology is still expensive and able to address only post-burning sulphur removal.77 Many reports have shown that inorganic sulphur can be removed from coal and crude oil yet the organic sulphur continues to pose a problem. Coal and unrefined petroleum contain significant amounts of organic sulphur structures, which include sulphoxides, mercaptans, sulphides, thiols, sulphones, disulphides and thiophenes.79 Dibenzothiophene (DBT) is a model compound illustrative of thiophenic structures found in petroleum and coal. Rhodococcus rhodochrous has been observed to possess the ability to selectively cleave carbon- sulphur bonds in dibenzothiophene, yielding 2-hydroxybiphenyl (2-HBP) as the sole end product.79 This selectivity of Rhodococcus rhodochrous is noteworthy on the grounds that fuel could be desulphurized to remove sulphur without influencing the carbon and calorific value.79,80 2.2.6 Industrial biotransformation and syntheses. The genus Rhodococcus has been identified to produce commercially intriguing and potentially valuable products. The Nitto Chemistry Industry Company in Japan uses Rhocococcus rhodochrous J1 to produce more than 30000 tons of acrylamide yearly.16 This is the first principal case of effective modern industrial production of a chemical commodity utilising a microorganism. Acrylamide is one of the most crucial chemical commodities and is used in adhesives, soil conditioners, paint, stock additives for paper treatment, coagulators and petroleum recovering agents.81 Conventional chemical synthesis of acrylamide includes the use 16 University of Ghana http://ugspace.ug.edu.gh of copper salts as a sole catalyst for the hydration of acrylonitrile. However, this approach has various complications including:16,81  The formation of acrylic acid is more pronounced than that of acrylamide.  The alkene functionality in both product and substrate causes the formation of nitrylotrispropionamide and ethylene cyanohydrin as by-products.  Polymerization takes place on the double bond of both product and substrate. With the biotransformation of the acrylonitrile to acrylamide using Rhodococcus rhodochrous J1, pure acrylamide is obtained in higher percentage without the formation of any by-product or polymerization. This approach is less expensive and simpler.16,81 Furthermore, nitrilases produced from Rhodococcus species have been used in industrial production of a variety of chemicals consisting of para-aminobenzoic acid, acrylic acid, nicotinamide, isonicotinic acid, hydrazide and pyrazinamide.37,82 These industrial biotransformation capabilities of Rhodococcus species offer high yields and specificity. 2.3 Secondary metabolites isolated from Rhodococcus 2.3.1 Antibiotics from the genus Rhodococcus Microbes capable of producing a wide variety of structurally different antibiotics normally have the ability to grow effectively in many different habitats by competing out other microbes. The production of antibiotics can be stimulated by other external factors such as the symbiotic relationships between microbes and plants or invertebrates either terrestrial or marine. In such 17 University of Ghana http://ugspace.ug.edu.gh instances, the microbe derives its nutrients from the plant or invertebrate and in turn protects its host through the biosynthesis of strong antibiotics.83 Since their discovery in the twentieth century, antibiotics have considerably decreased the risk of infectious diseases. The use of antibiotics coupled with enhancement in sanitation, food, housing and the arrival of mass immunization programs has led to a sensational drop in deaths from ailments that were once widespread and deadly. 2.3.1.1 Lariatins Rhodococcus jostii K01-B0171 strain was isolated from soil sediments collected from China.84 Lariatins A and B were produced from the culture broth of Rhodococcus jostii K01-B0171. Structure determination employing spectral analysis coupled with advanced protein chemical approaches revealed lariatins A and B to be unique cyclic peptides. They comprised of eighteen and twenty L-amino acid residues with an internal linkage between the α-amino group of Gly1 and γ-carboxyl group of Glu8.85 Lariatins A and B demonstrated growth inhibition against Mycobacterim smegmatis with MIC estimations of 3.13 and 6.25 μg/ml in agar dilution method, respectively. In addition, lariatin A showed growth inhibition against Mycobacterium tuberculosis with MIC value of 0.39 μg/ml.85 18 University of Ghana http://ugspace.ug.edu.gh 2.3.1.2 Aurachins Rhodococcus erythropolis JCM 6824 strain was isolated from marine sediments. Aurachin RE was produced from the culture broth of Rhodococcus erythropolis JCM 6824 strain. Biological activity test of Aurachin RE showed wide and strong antimicrobial inhibition against both low and high G+C Gram-positive bacteria.86 Furthermore, aurachins Q and R were also produced from the cultured broth of Rhodococcus acta 2259 strain isolated from activated sludge foam collected at Milcote Pilot Sewage Treatment Plant in UK.87 Structural characterisations of aurachin Q and R revealed an isoprenoid chain that is connected to a bicyclic quinoline core system at various positions. Aurachins Q and R exhibited significant growth inhibitory activity against broad range of Gram-positive bacteria in biological assay testing.87 19 University of Ghana http://ugspace.ug.edu.gh 2.3.1.3 Rhodostreptomycins A strain of Streptomyces padamus which is known to be an exceptionally stable actinomycin producer was competitively co-cultured with a multi-anbibiotic resistant strain of Rhodococcus fascians. This competitive co-culture resulted in a unique Rhodococcus 307CO strain. This strain emerged with concomitant elimination of the Streptomyces padamus. Subsequent genomic examination revealed that the new Rhodococcus 307CO strain harbours an extensive segment of DNA derived from the Streptomyces strain. Periodic investigation of the culture broth of Rhodococcus 307CO strain yielded a new set of antibiotics known as Rhodostreptomycin A and B. Rhodostreptomycins showed great antibiotic activities against broad scope of Gram-positive and Gram-negative bacteria such as Bacillus subtilis, Helicobacter pylori, Escherichia coli, Streptomyces padamus, and Staphylococcus aureus.88 20 University of Ghana http://ugspace.ug.edu.gh 2.3.1.4 Indole-3-acetaldehyde and indole-3-acetic acid. Indole-3-acetaldehyde and indole-3-acetic acid are biofilm inhibitors isolated from the spent media of the plant pathogen Rhodococcus BFI332 strain.116 A close examination of the activities of indole-3-acetic acid and indole-3-acetaldehyde revealed an extensive inhibition against the biofilm formation of Staphylococcus aureus, Escherichia coli, and Staphylococcus epidermidis. Pathogenic biofilms have been related to constant infections because of their high imperviousness to antimicrobial agents. Indole derivatives derived from Rhodococcus BFI332 strain have proven to be a great source of biofilm inhibitors against resistant pathogenic biofilm bacteria. 21 University of Ghana http://ugspace.ug.edu.gh 2.3.1.5 Saframycin AR1, AR2 and AR3 Saframycins are a class of antibiotics that belong to the tetrahydroisoquinoline family. Saframycin A was initially isolated from Streptomyces lavendulae 314 strain.112 Saframycin A exhibited an intense antiproliferative activity against wide range of different tumor cell lines at low doses and thus it is one of the most potent member of this group of natural products.112-114 Scheme 2.2: Bioconversion of saframycin A to saframycin AR1, AR2 and AR3 by Rhodococcus nocadia following two main possible routes. 22 University of Ghana http://ugspace.ug.edu.gh During a microbial conversion study, it was found out that, Rhodococcus nocardia converts saframycin A to three products, saframycin AR1 (25-dihydrosaframycin A), AR2 (21- decyanosaframycin A), and AR (25-dihydro-21-decyanosaframycin A).1153 This bioconversion follows two main possible routes. The first route is a reduction of the C-25 ketone of the pyruvoylamine side chain to a carbinol giving saframycin AR1, followed by a reductive elimination of the C-21 cyanide giving saframycin AR3. The second possible route is a reductive elimination of C-21 cyanide giving saframycin AR2, followed by reduction of the C-25 ketone to give saframycin AR3.115 2.3.2 Antifungals from the genus Rhodococcus Fungal infection is accompanied by great number of therapeutic issues than bacterial infections. This is due to the fact that fungal infection is mainly caused by eukaryotic organism.89 The cell wall of fungi contains chitin making it tougher to disrupt coupled with the fact that agents used against fungal cells can potentially affect human cells due to their close similarity. In spite of increased improvement in diagnostic, preventive, and therapeutic mediations, fungal infections still remain significant in terms of morbidity and mortality in patients with compromised immune systems such as HIV/AIDS or cancer patients.90 Resistance from most commercially available antifungal drugs on the market is becoming a major concern.90 Hence there is the need to search for more bioactive compounds that may serve as prospective antifungal drugs. 2.3.2.1 Rhodopeptins Rhodopeptins are novel cyclic tetrapeptides produced from the Rhodococcus Mer-N1033 strain isolated from soil sediments collected from Mt. Haychine in Japan.91 Five rhodopeptins namely rhodopeptin C1, C2, C3, C4 and B5 were isolated from the bacterial pellet, which forms the cell cake of the Rhodococcus strain. They are novel cyclic tetrapeptides, composed of a lipohilic β- 23 University of Ghana http://ugspace.ug.edu.gh amino acid and three α-amino acids. Rhodopeptins showed high in vitro antifungal activity against Candida albicans and Cryptococcus neoforman.91 2.3.3 Other important compounds produced by Rhodococcus species 2.3.3.1 Siderophores Siderophores are mainly low molecular weight organic chelators. Microorganisms under strict iron deficient conditions produce and secret siderophore compounds as an effective strategy for iron-scavenging to support their lifestyle.92,93 Siderophores are natural products with unique structural diversity. Their intense chemical diversity for iron coordination is as a result of their biosynthetic assembly.94 The chemical nature of the fundamental moieties in siderophore compounds mainly involved in iron coordination are generally classified into three groups namely; hydroxamates, catecholates, and carboxylates. Siderophores coordinate iron with extremely high affinity through six donor atoms mainly as an octahedral complex.95 The presence of 2,3-dihydroxybenzoic acid and the structurally modified ornithine residues are common iron-coordinating approach among the siderophores isolated from different 24 University of Ghana http://ugspace.ug.edu.gh Rhodococcus species so far.99,100 Siderophores after complexing with iron in the extracellular space, selectively and actively release the iron from the chelator-complex into the intracellular space of the organism.96,97 In addition to the biological importance of siderophores, rodoccocal producing siderophores are routinely employed in bioremediation processes.98 Outlined below are some examples of siderophores produced by Rhodococcus species. 2.3.3.1.1 Heterobactin A and B Heterobactin A and B are unique siderophores isolated from the culture broth of Rhodococcus erythropolis IGTS8 strain. Heterobactins are foremost the first set of structurally characterized siderophore compounds isolated from any known species of Rhodococcus.99 These compounds consists of a liner assembly of tripeptide sequence of (N-OH)-L-Orn-Gly-D-Orn-(δ-N- dihydroyxbenzoate). The alpha amino group of the D-Orn remains free in heterobactin B or is derivatized as a 2-hydroxybenzoxazolate in heterobactin A.99 The two new compounds are true siderophores because of their ability to relieve iron limited growth in the producing strain. Heterobactins are also transported by other non-producing bacteria as a source of iron for their developmental circle.99 These structurally intriguing siderophore compounds produced by Rhodococcus erythropolis makes this particular strain a better candidate for environmental bioremediation processes.94,99 25 University of Ghana http://ugspace.ug.edu.gh 2.3.3.1.2 Rhodobactin Rhodobactin is a siderophore compound isolated from iron-deficient culture of Rhodococcus rhodochrous. Rhodococcus rhodochrous is capable of internalizing variety of iron complexes as a result of its intense iron scavenging activity from various sources. Uptake studies revealed that, Rhodococcus rhodochrous takes up iron from its siderophore through a well-structured energy-dependent process.100 The structural characterization of rhodobactin revealed that, it is a complexed hexadentate ligand siderophore with one hydroxamate and two catecholates moieties for iron chelation.100 This makes Rhodococcus rhodocrous a better scavenger for iron and therefore a prospective candidate for environmental bioremediation processes.100 2.3.3.1.3 Rhodochelin Rhodochelin is a unique tetrapeptidic siderophore isolated from Rhodococcus jostii RHA1 strain cultured in iron deficient medium conditions. Rhodochelin is known to be the first natural product isolated from this strain. Structural characterization and genetic analysis of the biosynthesis of Rhodochelin revealed that, it is a branched tetrapeptide composed of 2,3- dihydroxybenzoic acid, threonine, and 2 moieties of δ-N-formyl-δ-N-hydroxyornithine.101 26 University of Ghana http://ugspace.ug.edu.gh The 2,3-dihydroxybenzoic acid part of the molecule forms the catecholate layer and the two moieties of δ-N-formyl-δ-N-hydroxyornithine form the hydroxymate layers. The catecholate and the hydroxymate functionalities are all involved in the iron coordination process.101 Figure 2.1: Model of rhodochelin iron-coordination. 2.3.3.2 Rhodococcus pigments Biological pigments such as carotenoids and their metabolites produced by different types of organisms play major role in protecting organisms from oxidative damage posed by active singlet oxygen species (1O2) during photochemical reaction.118 Carotenoids are also essential machinery in photosynthesis, nutrition, vision and cellular differentiation.117 Rhodococcus species have been associated as one of the non-photosynthetic bacterium producing carotenoids. 27 University of Ghana http://ugspace.ug.edu.gh OH-chlorobactene glucoside, OH-γ-carotene glucoside, OH-4-keto-γ-carotene glucoside hexadecanoate and OH-chlorobactene glucoside hexadecanoate are novel antioxidative carotenoids isolated from the culture broth of Rhodococcus species CIP strain.118 The singlet oxygen (1O2) quenching model of these carotenoids showed potent antioxidative activities of IC50 6.5µM, 9.9µM, 7.3µM and 14.6µM respectively for the aforementioned carotenoids.118 2.3.3.3 4,7,8-trihydroxyisoflavone 7-O-α-D-arabinofuranoside The compound 4,7,8-trihydroxyisoflavone 7-O-α-D-arabinofuranoside is an α-glucosidase inhibitor isolated from Rhodococcus sp.119 α-glucosidase inhibitors are anti-diabetic drugs that work by inhibiting the digestion of carbohydrates and subsequently decreasing the effect of carbohydrates on blood sugar.119 This compound showed a potent inhibitory activity against the α-glucosidases of rat liver microsome with an IC50 value of 0.46ng/ml.119 28 University of Ghana http://ugspace.ug.edu.gh 2.3.3.4 Mycothiols The pseudodisaccharide mycothiol is a dominant thiol produced by most bacteria species including Rhodococcus. Thiol compounds including glutathione and mycothiol principally serve as cofactors in detoxification reactions for free radicals, alkylating agents and xenobiotics by acting as electron acceptor/donor during the reaction.120 For this reason, mycothiol serves as a major biological adaptation that is crucial for the subsistence of organisms under various toxic conditions.121 2.4 Complete genome sequencing of microbes for natural product discovery Complete genome sequencing of microbes offers exceptional access to the genes involved in secondary metabolism; the means of their biosynthetic assembly; and, in some instances, what products they may yield.102,103 This makes it viable to examine the compounds expressed through the usage of traditional fermentation techniques with those anticipated from gene sequences, and also design fermentation methods which could prompt or enhance the production 29 University of Ghana http://ugspace.ug.edu.gh of expected compounds of individual gene clusters from the genome.104 Traditional fermentation-based activation strategies involved changing culture media conditions to activate orphan or cryptic metabolism for biosynthesis of metabolites. However, complete genome sequence data provides a more efficient way to carry out more directed gene cluster induction and molecule detection studies.105 The ready accessibility of genome sequence data has prompted the advancement of easy to use bioinformatics tools, like ClustScan, NP.searcher, and AntiSMASH, which detect biosynthetic gene clusters in assembled genomes. This predict structural elements of molecules based upon the characteristics of key enzymes involved in the polyketide snynthases (PKS) and nonribosomal peptide synthetases (NRPS) assembly and tailoring of natural products.106-108 PKS and NRPS have for quite some time been used by bacteria for cell defence and offense against predators. Genome sequencing analysis is currently applied to newly assembled genomes, allowing researchers to prioritize cryptic or orphan gene clusters for molecule discovery studies. For example, the complete genome sequence analysis of Rhodococcus jostii RHA1 strain revealed twenty-three secondary metabolite gene clusters all considered to be orphan with respect to their products.101 Bioinformatics investigation of the genome of this strain revealed three distant NRPS gene clusters responsible for rhodochelin biosynthesis. The first gene cluster (cluster A) involved RhcA, RhcB, RhcC, RhcD, RhcE, and RhcF genes with a size of approximately 12 kbp. It encodes a typical modular NRPS, composed of two complete modules and a terminal thioesterase (TE) domain. The first module has a substrate specificity preference for L-Threonine which is incorporated during the assembly of rhodochelin.101 Cluster B is composed of eight genes covering a DNA size of about 25 kbp. This cluster contains enzymes ornithine monooxygenase (RMO) and formyltransferase (Rft) that encodes for the L-Ornithine assemblage which is incorporated during the assembly of rhodochelin. Cluster C (DhbE) involves genes associated with 2,3-dihydroxybenzoate dehydrogenase (DhbA) and 30 University of Ghana http://ugspace.ug.edu.gh isochorismate synthase (DhbC) which encodes for the assemblage of 2,3-dihydroxybenzoic acid (2,3-DHB) incorporated during the assembly of rhodochelin.101 2.4.1 Biosynthesis of Rhodochelin Rhodochelin assembly is initiated by a stand-alone adenylating enzyme DhbE which activates 2,3-dihydroxybenzoic acid (2,3-DHB) by adenylation of the carboxyl group and then subsequently bounding to its cognate stand-alone aryl carrier protein RhcE.101 The RhcB protein catalyzes the nucleophilic attack of the L-Threonine (L-Thr) α-amino acid group onto the RhcE- bound 2,3-DHB resulting in a peptidyl carrier protein (PCP1) bound dipeptide known as 2,3- dihydroxybenzoic-L-Threonine (2,3-DHB-L-Thr). On the other hand, L-Ornithine (L-Orn) side chain amino group is hydroxylated by a N-hydroxylating flavoprotein monooxygenase enzyme RMO, resulting in L-ẟ-N-hydroxyornithine (L-hOrn). The L-hOrn is further modified by formylation leading to the generation of L-ẟ-N-formyl- ẟ-N-hydroxyornithine (L-fhOrn). The RhcB protein catalyzed bond formation between the L-fhOrn and the PCP1-bound 2,3-DHB-L-Thr dipeptide resulting in a formation of 2,3- dihydroxybenzoic-L-Threonine-L- ẟ-N-formyl- ẟ-N-hydroxyornithine (2,3-DHB-L-Thr-L- fhOrn) tripeptide bounded to PCP2. The newly assembled tripeptide is transferred to the thioesterase (TE) domain of the RhcB-PCP3. Subsequently, a second L-fhOrn is activated and tethered to RhcB-PCP3 in a similar mechanism described earlier. The hydroxyl group of the L- Thr side chain of the tripeptide initiates a nucleophilic attack on the adjacent L-fhOrn thioester resulting in the fully assembled rhodochelin. 31 University of Ghana http://ugspace.ug.edu.gh Figure 2.2: Proposed biosynthetic pathway for rhodochelin assembly. The two building blocks 2,3-DHB and L-fhOrn are synthesised through their respective pathways and linked together forming a tripeptide by the synthetase RhcB. The TE- catalysed an ester bond formation between the third building block L-fhOrn and the L-Thr side chain of the tripeptide resulting in rhodochelin. 32 University of Ghana http://ugspace.ug.edu.gh 2.4.2 Known compounds expressed by the gene clusters in the complete genome sequence of Rhodococcus sp. strain M1042 The complete genome sequence of Rhodococcus sp. strain M1042 is composed of 5,948,800 mega base pairs with 230 NRPS, PKS and NRPS-PKS genes most of which are non-functional. AntiSMASH database identified 19 out of the 230 gene clusters to produced already known bioactive compounds ranging from antibiotics, antifungals to anticancer agents. Some of these compounds include: Bleomycin, Fuscachelin, Laspartomycin, Ectoine, SF2575, Isorenieratene, Streptomycin and Rifamycin. 33 University of Ghana http://ugspace.ug.edu.gh 2.5 Terminologies in bioactivity measurements 2.5.1 Half maximal inhibitory concentration (IC50) This is a numerical measure in vitro that demonstrates the amount of a specific substance or drug expected to inhibit half a particular biological or biochemical process or a component of that process occurring within a cell.125-131 34 University of Ghana http://ugspace.ug.edu.gh Figure 2.3: Illustration of Half Inhibitory Concentration (IC50) 2.5.2 Half maximal effective concentration (EC50) The term EC50 is commonly used to depict or measure the potency of a drug. It represents the concentration of a compound, drug, antibody or toxicant at which 50% of its maximal effect is observed after a specified exposure time.132-136 Figure 2.4: Illustration of Half Maximal Effect Concentration (EC50) 35 University of Ghana http://ugspace.ug.edu.gh 2.5.3 Half maximal Effective dose (ED50) This is the dose or amount of drug that produces a specific effect or therapeutic response in some fraction (50%) of the subjects or test sample that have taken it. It is a measure of average effectiveness of the drug or the tolerance level of toxin in a sampled set of individuals.137-142 Figure 2.5: Illustration of Effective Dose (ED50). 2.5.4 Lethal dose for 50% (LD50) This is the quantity of a substance (toxin, drug, pesticide etc) which is enough to cause the death of 50% of a group of test animals in a specified period. It is commonly used in bioassay assessment to measure the acute toxicity of a chemically active agent.143-146 Figure 2.6: Illustration of Lethal Dose (LD50) 36 University of Ghana http://ugspace.ug.edu.gh 2.5.5 Lethal concentration for 50% (LC50) This is the quantity of a substance (toxin, drug, pesticide etc) present in the air or water which is enough to cause the death of 50% of a group of test animals in a specified period.146,147 2.5.6 Minimum inhibitory concentration (MIC) A minimum concentration of a particular drug or substance required to inhibit the visible growth of an organism after incubation overnight. MICs play important role in diagnostic assessments of the potency a new antimicrobial agents or to detect resistance of microbes to already existing antimicrobial agents.148-151 2.5.7 Minimum bactericidal concentration (MBC) A minimum concentration of drug or a substance required to kill and stop the rate of growth in a microorganism after incubation overnight.152-156 2.5.8 Growth inhibition concentration for 50% (GI50) This is the concentration of a substance or drug required to inhibits the growth cells by 50%. For an anticancer agent, it is the concentration of the drug that inhibits the growth rate of cancer cells by 50% or produces a 50% reduction in cell proliferation. 157,158 37 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE 38 University of Ghana http://ugspace.ug.edu.gh 3.0 Materials and methods 3.1 Sampling Turkey is the only country that has 3 out of the 34 biodiversity rich hotspots in the world still unexplored or underexplored. In this project, sampling was done in Munzur Valley located in the Tunceli Province of Turkey. The Munzur Valley is the largest and most biodiverse national park in Turkey. Soil samples were collected from different parts of the entire Menzur Valley national park. 3.1.1 Soil sample collection from Munzur Valley Soils samples were collected from different parts of the Munzur Valley in Tunceli, a forest reserve which is home to about 1500 registered species of plants with unique variety of organisms. The selection of sampling sites was based on the level of human activities that existed in the prospective area to be sampled. The areas chosen for sampling were noted for the predominance of many undisturbed habitats which represents the most bio-diverse environments in the Munzur Valley. Many soils were sampled for sediments and water collected at locations which were situated 100m apart. The GPS coordinates of sampling locations were recorded and stored in Google Earth. Soils were collected by clearing the humus or topsoil to a depth of between 30 and 40 cm after which the sediments were scooped directly into 50 mL sterile centrifuge tubes and covered. The samples were collected from different sections of the sampling sites at about 100m apart and labelled. The GPS coordinates of the sample site that gave the microbes described in this thesis was (coordinates: 39° 7'43.73"N, 39°30'18.80"W). A geographical map of the sampling sites in the Munzur Valley is shown in Figure 3.1. Fifty (50) samples were collected and labelled including the sample studied in this project, M1042. 39 University of Ghana http://ugspace.ug.edu.gh 3.1.2 Sampling site Figure 3.1: A geographical map of Munzur Valley National Park showing the sampling sites. 40 University of Ghana http://ugspace.ug.edu.gh 3.2 Bacteria Media Preparation 3.2.1 Materials Agar powder (Oxoid), cycloheximide 25 mg/ml (Fisher Bioreagent), nalidixic acid at 25 mg/mL (Fisher Bioreagent), nystatin at 25 mg/mL (Fisher Bioreagent), Yeast extract, Malt extract, D- glucose, water, 1 L autoclave bottle with cap (Pyrex). 3.2.2 Preparation of International Streptomyces Protocol (ISP) 2 liquid media Ten grams of malt extract, 4 g each of yeast extract and D-glucose were placed in a 2 L beaker and filled with 0.9-1 L of tap water. With the help of a magnetic stirrer, the content of the beaker was thoroughly mixed for 30 minutes and pH adjusted to 7.2 after which it was transferred into a 1 L autoclave flask and autoclaved using a set program. The media was then stored at 4 oC for future use. 3.2.3 Preparation of International Streptomyces Protocol (ISP) 2 agar media 10 g of malt extract, 4 g each of yeast extract and d-glucose and 15 g of agar powder (Oxoid) were placed in a 1 L beaker and filled with 0.9-1 L of tap water. Stirring and autoclaving was done as described in 3.2.2. After autoclaving, 1 ml each of 25 mg/ml cycloheximide, nystatin and nalidixic was added to the autoclaved agar medium and swirled gently. The agar was then emptied into sterilized Petri dishes in the clean bench to fill about two-thirds the volume of the Petri dishes whiles still hot. The agar was then allowed to set under sterile conditions. The agar plates were sealed and stored at 4 oC for future use. 41 University of Ghana http://ugspace.ug.edu.gh Figure 3.2: A picture showing preparation of the ISP 2 modified agar plates on which the microbes from the soil samples were grown 42 University of Ghana http://ugspace.ug.edu.gh 3.3 Culturing, small scale fermentation (seeding) and preservation of bacteria The Rhodococcus sp. strain M1042 was isolated from soil sediments collected from Munzur Valley in Tunceli. About 600 µg of 60% glycerol stock containing 1ml of pure colony of Rhodococcus sp. strain M1042 was obtained from Professor Mustafa Camas of Tunceli University in Turkey. 3.3.1 Material ISP2 agar media, ISP2 liquid media, sterile inoculation loops (Sigma Aldrich), sterile 60 % glycerol stock solution, 50 ml falcon tubes (Greiner), sterile Eppendorf tubes (Fischer), 250 ml and 500 ml Erlenmeyer flasks (Fischer), 2 ml screw capped cryo-preserved tubes (Fischer). 3.3.2 Isolation of Rhodococcus sp. strain M1042 from glycerol stock Sterilized inoculation loops were used to collect small portion of the glycerol stock containing the Rhodococcus sp strain M1042 and smeared evenly on the ISP2 agar plate. The agar plate was then labelled, para-filmed and incubated at 28 ◦C for 7 days to allow the growth of the bacteria. Figure 3.3: A picture showing smearing of the glycerol stock containing the bacteria on the modified agar plates (left) and obtained pure strains of Rhodococcus species (right) 43 University of Ghana http://ugspace.ug.edu.gh 3.3.3 Small Scale Fermentation of Pure Rodococcus sp. strain M1042 Single colony of the strain was transferred from the pure culture plate and inoculated into 250 ml Erlenmeyer flask filled with about 100 ml of ISP2 liquid media. The inoculated flask was labelled and placed in an incubator-shaker at 28oC for 14 days at 165 rpm. After the 14 days, the culture broth of the sample was filtered by suction filtration to separate bacteria mycelia from the broth. The broth or the filtrate of the sample was extracted with about 100 ml ethyl acetate and the mycelium was extracted repeatedly and alternatively with methanol and dichloromethane. The ethyl acetate, methanol and dichloromethane extracts were dried under vacuum and subsequently combined to give a total crude extract or TCE. Figure 3.4: Picture showing small scale culture of Rhodococcus strains. 3.3.4 Cryopreservation of bacteria species After small scale culture of bacteria as described in 3.3.3, 1 ml of the bacteria suspension was pipetted into a 2 ml cryo-screwed tube and 400 μl of 60 % glycerol stock solution was added to it and shaken. They were then labelled before storage in the -80oC freezer. The glycerol solution 44 University of Ghana http://ugspace.ug.edu.gh was supposed to form a hibernation wall around the bacteria in order to enhance their preservation. Triplicates of the bacteria species were preserved at -80 oC Figure 3.5: Pictures showing preserved bacteria at preparation stage for storage at - 80oC. 3.4 Screening of crude extracts from small scale culture About 1ml of TCE solution of concentration 1.0 mg/ml was then prepared using 100 % CH3OH as solvent and placed in an LCMS vial and submitted to our collaborators at the University of Aberdeen, Scotland, UK for HRESI/HPLC-DAD-MSn analysis. 3.5 Large scale fermentation of Rhodococcus sp strain M1042 A seed culture was prepared by taking a small portion of the pure bacteria strain and inoculating it into a sterilized 1 L Erlenmeyer flask containing 250 ml of ISP2 liquid media. The Erlenmeyer flask was labelled, plugged with non-adsorbent cotton wool and placed in an incubated shaker at 28 °C for 7 days with continuous agitation to allow the bacteria to grow. After 7 days, the 250 ml seed culture was used to inoculate nine sterilized 1 L flasks each containing 250 ml ISP2 liquid media. The flasks were labelled, plugged with non-adsorbent cotton wool and placed in an incubated shaker at 28°C for 21 days with continuous agitation to allow the bacteria to grow. 3 days prior to harvesting, Diaion HP-20 resin (15 g) were added under sterile conditions using 45 University of Ghana http://ugspace.ug.edu.gh a serological pipette to each of the culture broth and allowed to shake again this time for an additional 3 days. After three weeks of incubation, the nine 1 L cultures were harvested and filtered under pressure using a piece of glass wool placed in a Buchner funnel. The filtrates were combined and extracted with ethyl acetate after which the ethyl acetate portion was rotary evaporated and concentrated into a sterile vial. The mycelia residues consisting mainly of the Diaion HP-20 resin with adsorbed organics were repeatedly and alternatively extracted with methanol and dichloromethane as described earlier. The methanol and dichloromethane extracts were combined and rotary evaporated at 60 0C to give a total crude extract of TCE (1865.2254 mg). 3.6 Isolation of compounds from M1042 extracts The modified Kupchan solvent partition scheme was adopted as a pre-clean up procedure to separate the crude extract into fractions of well-defined polarities. This was done by dissolving the crude extract (1865.2254 g) in about 200 ml of de-ionised water and placing this in a 1 L separating funnel. The 200 ml water layer was then extracted 3 times with 200 ml portions of CH2Cl2 (1:1 ratio per extraction). The 3 CH2Cl2 fractions were combined and concentrated under reduced pressure. The remaining aqueous layer was extracted once with 200 ml sec-butanol. The sec-butanol layer was dried under reduced pressure to give a water-butanol fraction labelled WB (589.6452 mg) while the remaining water layer was discarded. The CH2Cl2 extract was dissolved in 200 mL CH3OH: H2O (90:10 v/v) mixture. This mixture was then placed in a 1 L separating funnel and extracted three times with 200 ml of hexane. The hexane layer was dried under reduced pressure to afford the hexane fraction FH (245.7523mg). The CH3OH: H2O (90:10 v/v) mixture was phase adjusted to CH3OH: H2O (50:50 v/v) by adding 160 ml of de- ionised water. The CH3OH: H2O (50:50 v/v) mixture was then extracted three times with 200 ml of DCM. The DCM layer was dried under reduced pressure to obtain the fraction FD 46 University of Ghana http://ugspace.ug.edu.gh (653.1256 mg). The remaining CH3OH: H2O (50:50 v/v) layer left in the separating funnel was also dried under reduced pressure to obtain the water-methanol fraction labelled FM (112.3231 mg). About 0.5 mg/ml of all 4 fractions were submitted to the University of Aberdeen for HRESI/HPLC-DAD-MSn analysis. 1H -NMR was also obtained for all the 4 fractions where CD3OD was used as solvent for the more polar fractions WB, FM and FD and CDCl3 was used as solvent for the less polar FH fraction. The LC-MS and 1H NMR for the 4 fractions were analysed and the compounds of interest were found in both WB and FD fractions. However, the most interesting compounds were seen to be more concentrated in WB fraction. Scheme 3.1: A flowchart of the modified Kupchan Solvent Partition technique. This technique separates compounds in crude extracts into four fractions according to their polarities. The butanol fraction (WB) being the most polar and hexane fraction (FH) being the least polar. The FD and FM fractions are of intermediate polarities. 47 University of Ghana http://ugspace.ug.edu.gh Figure 3.6: A picture showing excerpt of kupchan solvent partitioning technique of the crude extract from M1042 3.7 Isolation of compounds from M1042 WB fraction 3.7.1 Sephadex LH20 size exclusion purification of M1042-WB fraction The TCE obtained from the solvent extraction as described in section 3.6 was taken through the modified Kupchan solvent partitioning process which is described in section 3.7 above. From the 1H NMR and LC-MS data analysis, the compounds of interest were in the WB fraction. The WB fraction was subjected to column chromatography by size exclusion using sephadex LH20 as stationary phase and CH3OH:CH3CN (50:50 v/v) mixture as mobile phase. A total of 4 fractions were collected and concentrated under reduced pressure. These semi-pure fractions were then transferred into vials and labelled M1042-WB-SFA (57.1245 mg), M1042-WB-SFB (63.4213 mg), M1042-WB-SFC (93.2411 mg) and M1042-WB-SFD (42.3126 mg). 1H NMR and LC-MS data were acquired for each of the samples and analysed. TLC was done on both the crude M1042-WB and the sub-fractions using aluminium foil coated with silica as stationary 48 University of Ghana http://ugspace.ug.edu.gh phase and a mixture of methanol and ethyl acetate (50:50 solvent ratio) as mobile phase to ascertain the level of purity and metabolite pattern. From the 1H NMR, LC-MS and TLC analysis, the compounds of interest were in the M1042-WB-SFC fraction. The semi-purified M1042-WB-SFC fraction was subjected to further purification processes. Figure 3.7: A picture showing sephadex LH20 size exclusion purification of M1042-WB fraction 3.7.2 Gravity column chromatography purification of M1042-WB-SFC The M1042-WB-SFC fraction was further subjected to column chromatography by gravity using silica gel as stationary phase and varying mixtures of hexane, ethyl acetate and methanol of increasing polarity as the mobile phase. A total of 7 fractions were collected from the column. The first fraction was eluted with 200 ml hexane:ethyl acetate (90:10) mixture. The second fraction was eluted with 100 ml hexane:ethyl acetate (70:30) mixture. The third fraction was eluted with 300 ml hexane:ethyl acetate (40:60) mixture. The fourth fraction was eluted with 200 ml ethyl acetate:methanol (90:10) mixture. The fifth fraction was eluted with 300 ml ethyl acetate:methanol (70:20) mixture. The sixth fraction was eluted with 450 ml ethyl 49 University of Ghana http://ugspace.ug.edu.gh acetate:methanol (30:70) mixture. The seventh fraction was also eluted with 300 ml 100 % methanol. All the fractions were concentrated under reduced pressure and transferred into vials and labelled M1042-WB-SFC-C1-7. 1H NMR and LC-MS data were acquired for each fraction and analysed. In addition, the fractions were subjected to TLC and phytochemical screening using ninhydrin reagent to detect the presence of nitrogenous compounds. Two fractions M1042-WB-SFC-C5 (5.2142 mg) and M1042-WB-SFC-C6 (19.1425 mg) each showed a purple colour on the TLC plate after staining with ninhydrin reagent with the most intense one observed in M1042-WB-SFC-C6. This is an indication of either free amino acid or peptide present in the fraction. The LC-MS analysis further confirmed the presence of three peptides in the M106- WB-SFC-C6 fraction. The M1042-WB-SFC-C6 fraction was subjected to vacuum flash chromatography to afford M1042-WB-SFC-C6-MDe (14.3216 mg) fraction which was subsequently subjected to HPLC for isolation of the three peptides. Figure 3.8: A normal phase TLC plate of M1042-WB-SFC-C5 and M1042-WB-SFC-C6 fractions showing positive results for ninhydrin test. 50 University of Ghana http://ugspace.ug.edu.gh 3.7.3 Isolation of peptides from M1042-WB-SFC-C6-MDe fraction using HPLC The HPLC separation and purification was performed with Waters 1525 series binary pump and waters 2998 photodiode array detector with column heater and in-line degasser. A Phenomenex Luna reversed-phase C18 column (C18 250 × 10 mm, L × i.d.) was used with solvents systems of gradients 15:75 (CH3CN: H2O) as solvent A and 100 % CH3CN as solvent B used as eluent with column flow rates set at 1.5 ml/min. The first compound, M1042-WB-SFC-C6-MDe-B (5.1231 mg) was eluted at a retention time of 21.03 minutes to afford a yellow oily compound. The second compound, M1042-WB-SFC-C6-MDe-C (3.1321 mg) was eluted at a retention time of 26.31 minutes to afford a white crystalline compound. The third compound, M1042-WB- SFC-C6-MDe-D (2.5121 mg) was eluted at a retention time of 36.23 minutes to afford also a white crystalline compound. These three pure compounds were dried under reduced pressure and transferred into vials. Concentration of 1.0 mg/ml of each compound was prepared using 100 % methanol and submitted to the University of Aberdeen, Scotland, UK for HRESI/HPLC- DAD-MSn. 51 University of Ghana http://ugspace.ug.edu.gh Scheme 3.2: A flow chart of isolation of peptides from M1042-WB fraction of TCE of the fermentation broth of Rhodococcus sp 52 University of Ghana http://ugspace.ug.edu.gh 3.7.4 1D and 2D NMR Analysis 1D and 2D NMR data were acquired on a Bruker 500 MHz spectrometer with either DMSO or CD3OD as solvents. Data acquired included 1H, 13C, DEPT-135°, 90° and 45°, 1H-1H-COSY, HSQC, HMBC, gHSQC-TOCSY, 1D and 2D-TOCSY and TROESY. 3.8 Biological activity test 3.8.1 Chemicals and reagents RPMI-1640, IMDM, M-199, HEPES, YI-S, Foetal Bovine Serum (FBS), Adult Bovine Serum (ABS) Gentamycin, Penicillin-Streptomycin-L-Glutamine (PSG), 2-[4-(2- hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), Artesunate, Alamar dye, Dimethyl sulphoxide (DMSO), Sodium citrate, Adenine, Sodium bicarbonate (NaHCO3), AlbuMax II, Sodium chloride (NaCl), Sodium Phosphate Dibasic (Na2HPO4), Potassium chloride (KCl), Sodium Phosphate Monobasic (KH2PO4), Sodium hydroxide (NaOH), and Sodium bicarbonate (NaHCO3). All these chemicals and reagents were purchased from Sigma-Aldrich, USA. 3.8.2 Preparation of compounds for bioactivity testing A stock solution of 10 mM concentration of M1042-WB-SFC-C6-MDe-B was prepared. The compound was first dried using nitrogen gas after which its mass was obtained by means of a mass balance. The compound (1 mg) was then dissolved in an appropriate volume of dimethyl sulfoxide (DMSO) to obtain the 10 mM concentration. This was followed by vortexing of solution and filter sterilization into vials through 0.45 µm millipore filters under sterile conditions. The solution was then stored and at -20 °C until used. 53 University of Ghana http://ugspace.ug.edu.gh 3.8.3 Anti-malaria activity study 3.8.3.1 Blood collection and erythrocytes preparation Erythrocytes were obtained from blood of consented volunteers (Blood group O+). Venous blood was drawn and collected into containers containing citrate phosphate dextrose (CPD) solution and kept at 4 oC overnight. It was centrifuged at 2,000 rpm for 10 minutes to separate the serum and buffy coat. Packed erythrocytes were washed three times with parasite washing medium (RPMI 1640, buffered with, 2 mM L-glutamine and 50 µg/mL gentamicin) Each washing step involved addition of wash medium, pipetting up and down thrice, centrifuging at 2,000 rpm for 10 minutes and then discarding suspended medium. After washing, wash medium was added to the packed erythrocytesand stored at 4 oC until ready for use. Washed RBCs were stored and used for up to 2 weeks’ maximum after which new blood was collected. 3.8.3.2 Giemsa stained thin blood smear and parasitaemia determination A drop of infected erythrocytes cultured medium was placed on a microscope slide and spread with the aid of another slide to prepare a thin blood film. Slides were air dried, dipped into absolute methanol for some seconds to fix and air dry again. A 10 % Giemsa stain was added to cover completely the surface of the fixed slides for at least 10 minutes and then gently rinsed off with running water. Stained slides were air dried and then viewed under a light microscope (source) (with immersion oil at 100x objective) to determine parasitaemia. Percent parasitaemia was estimated by counting the number of infected cells in a total of 500 erythrocytes in the Giemsa-stained thin blood smear. Parasitaemia was determined as a percentage of the number of infected erythrocytes to the total number of erythrocytes counted. 54 University of Ghana http://ugspace.ug.edu.gh 3.8.3.3 In vitro cultivation of Malaria Parasite Erythrocytic stages of malaria parasite (Plasmodium falciparum-chloroquine sensitive strain 3D7) were cultured in 25 cm2 flasks using the method of Trager and Jensen (1976) with modifications. Erythrocytes were maintained at 2% haematocrit (v/v) cell suspension in complete malaria parasite medium (buffered with 25 mM HEPES, RPMI 1640, supplemented with 7.5% NaHCO3, 25 ug/ml gentamycin, 5 % heat-inactivated human O + serum and 5 mg/mlAlbuMax II) and incubated at 37 oC under gas condition of 2 % O2, 5 % CO2 and 93 % N2. Parasite growth and development were monitored with Giemsa stained thin blood smear. Parasite culture was purified by using 5 % sorbitol to obtain matured erythrocyte parasitic stages (late trophozoites and schizonts) from uninfected cells. The matured erythrocyte parasitic stages (purity > 90 %) obtained were used to screen the compounds for anti-malaria activity. 3.8.3.4 Screening for anti-malaria activity by SYBR Green I assay The compound was screened for anti-malaria activity by using the SYBR Green I fluorescence assay as established by Smilktein.161 Serial dilution of the standard (artesunate) which served as an experimental positive control and stock solution of the compound to yield final concentrations ranging from 100 ng/ml to 400 ng/ml and 3.13 µg/ml to 25 µg/ml respectively were prepared. The matured erythrocyte parasitic stages were treated with the compound and washed erythrocytes in 96 well plates (Nunc) and incubated with complete malaria parasite medium until it is harvested after 24 hrs. Slides were then prepared and percent parasitaemia was estimated by counting the number of infected cells in a total of 500 erythrocytes in the Giemsa-stained thin blood smear. Briefly an aliquot of 5 µL per each concentration of the standard drug and the compound was dispensed into test wells. 95 µL of complete malaria parasite medium with washed erythrocytes at 2 % 55 University of Ghana http://ugspace.ug.edu.gh haematocrit and the purified matured erythrocyte parasitic stages (1 % parasitaemia) were added, and incubated at 37 oC under gaseous conditions as stated above and untreated erythrocytes were used as control. Wells containing erythrocytes at 2 % haematocrit, infected erythrocytes at 2 % haematocrit and complete parasite medium alone served as negative controls, positive controls and blank controls respectively. Furthermore, wells containing infected parasites and 0.1 % DMSO served as reference controls. Final volume per well was 100 µL. Plates were then incubated for 24 hr as described above in the cultivation of malaria parasites. 100 µL aliquot of 2.5x buffered SYBR Green I (0.25 µL of SYBR Green I/mL of phosphate buffer saline) was added to each well after the incubation period and incubated in the dark place for 30 min at 37 oC. Fluorescence was detected by Guava EasyCyte HT FACS machine (Millipore, USA). 3.8.4 Screening of compounds for Anti-trypanosomal activity. 3.8.4.1 Culturing of Trypanosome parasites. The GUT at 3.1 strain of the bloodstream form of the parasites T. brucei was used in this work. Parasites were cultured in vitro as described previously. Parasites were used when they reached a confluent concentration of 1X106 parasites/ml. The Neubauer counting chamber was used to estimate the parasitemia. Concentration of 3X105 parasites/ml was prepared by dilution of the parasites with IMDM medium and was used for the drug assay. 3.8.4.2 Trypanosome parasites in vitro viability test. The viability of the treated or untreated trypanosome parasites were ascertained by the alamar Blue assay test. The assay was carried out in a 96-well plate through the instructions of the manufacturer with slight modifications. 1.5X104 parasites were seeded with varied concentrations 56 University of Ghana http://ugspace.ug.edu.gh of the compound ranging from 0 µM to 100 µM. Final concentrations (DMSO) were maintained at 0.1 %, respectively. After incubation of parasites with or without the compound for 24 h at 37 °C in 5 % CO2, 10 % alamar Blue dye was added, and the parasites were incubated for another 24 h in darkness. The Tecan Sunrise Wako spectrophotometer was used to read the absorbance at 540 nm for the plate after 48 hours. A trend curve was drawn to estimate IC50 of the compound. 3.8.5 Screening of compounds for Anti- leishmania activity 3.8.5.1 Cuturing of Leishmania Parasites The log-phase promastigotes of L. donovani (D10) and L. major (NR48815) were cultured in M-119 growth medium with a working concentration of 6 × 106 cells/ml. At a confluent concentration of 1X106 parasite/ml, the parasites were used in the test study. The Neubauer counting chamber was used in the estimation of parasitemia. A concentration of 3X105 parasites/ml by diluting the parasites with M199 medium and used for the drug assay test. 3.8.5.2 Leishmania parasites in vitro viability test. The viability of the treated or untreated trypanosome parasites were ascertained by the alamar Blue assay test. The assay was carried out in a 96-well plate through the instructions of the manufacturer with slight modifications. 1.5X104 parasites were seeded with varied concentrations of the compound ranging from 0 µM to 100 µM. Final concentrations (DMSO) were maintained at 0.1 %, respectively. After incubation of parasites with or without the compound for 24 h at 37 °C in 5 % CO2, 10 % alamar Blue dye was added, and the parasites were incubated for another 24 h in the dark. The Tecan Sunrise Wako spectrophotometer was used to read the absorbance at 540 nm for the plate after 48 hours. A trend curve was drawn to estimate IC50 of the compound. 57 University of Ghana http://ugspace.ug.edu.gh 3.8.6 In-vitro susceptibility testing of Trichomonas mobilensis M1042-WB-SFC-C6-MDe-B was prepared at a concentration of 0 - 100 µM in 96 well plates. Trophozoites of Trichomonas mobilensis were seeded in the well plates at concentration of 2X106 cells/ml. the plates were sealed with seal stickers to provide anaerobic conditions for parasites then the plates were transferred into an Anaero Pack jar with gas generators. The jar was incubated at 28°C for 48 hours. Parasites were transferred into black well plates, 100 µl ATP bioluminescent were added and plates were incubated for 20 minutes before luminescence reading. 58 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 59 University of Ghana http://ugspace.ug.edu.gh 4.0 Results and Discussion 4.1 Discussion of the techniques used to isolate and study the chemistry of Rhodococcus sp. Rhodococcus sp. M1042 is a talented strain and capable of biosynthesizing novel secondary metabolites. The initial stages of the secondary metabolite isolation and purification process involved an appropriate and efficient de-replication techniques. These techniques were conducted on the kupchan solvent partitioning sub-fractions (FH, FD, WB, and FM) obtained from the total crude extracts (TCE) of Rhodococcus sp. strain M1042 as described in section 3.7. These FH, FD, WB, and FM sub fractions were subjected to HRESI/HPLC-DAD-MSn analysis which provided data on all the secondary metabolites present in these fractions with regard to their accurate masses, fragmentation patterns and UV profiles. The data obtained from these analyses provided good search criteria which were entered into two natural product databases AntiMarin and Marin-Lit as a means of de-replication. Accurate masses and UV profiles entered into the two databases were checked against all compounds published in all journals to date that were of microbial origin. Compounds (peaks or MSn = m/z) which did not provide relevant hits when checked from the two databases became high priority targets for further fractionation and purification. Henceforth, HRESI/HPLC-DAD-MSn data acquired gave clues on which sub-fraction contained the compounds of interest. As part of the de-replication strategies, 1H -NMR was also obtained for all the 4 fractions where CD3OD was used as solvent for the more polar fractions WB, FM and FD and CDCl3 was used as solvent for the less polar FH fraction. In addition, these sub fractions were subjected to phytochemical screening on TLC plates using ninhydrin reagent to detect the presence of nitrogenous compounds. Various purification techniques including size-exclusion chromatography, gravity column chromatography and HPLC were employed in isolation, in various strategic combinations or in succession until the pure secondary metabolites were obtained. 60 University of Ghana http://ugspace.ug.edu.gh 4.2 Taxonomy of Rhodococcus sp. strain M1042 The taxonomy of Rhodococcus sp. strain M1042 was studied and the neighbour joining tree constructed as shown in Figure 4.1. A table containing the closest neighbour of this strain was also constructed (Table 4.1) to give an idea of the possible biological and chemical behaviour of the strain. The new strain Rhodococcus sp. M1042 exhibited a range of chemotaxonomic and phenotypic properties typical of members of the genus Rhodococcus. An almost complete 16S rDNA gene sequence (1,455 nt) was determined for the organism. Primary sequence analysis with the sequences of representatives of the family Rhodococcus confirmed that the M1042 was closely related to the species of the genus Rhodococcus. The phylogenetic tree based on the neighbour-joining algorithm showed that the strain M1042 formed a cluster with R. coprophilus DSM 43347T/X80626 among members of the genus Rhodococcus (Fig. 4.1). Strain M1042 shared 16S rDNA gene sequence similarities of 98.97 % (8 nt differences at 1462 locations), 98.43 % (23 nt differences at 1462 locations) and 98.29 % (25 nt differences at 1461 locations) with R. coprophilus DSM 43347T, R. phenolicus DSM 44812T and R. zopfii DSM 44108T respectively. Sequence similarities with all other members of the genus Rhodococcus were <98.29 %. 61 University of Ghana http://ugspace.ug.edu.gh 66 Rhodococcus koreensis DNP505T/AF124343 Rhodococcus jostii IFO 16295T/AB046357 Rhodococcus marinonascens DSM 43752T/X80617 Rhodococcus maanshanensis M712T/AF416566 99 Rhodococcus erythropolis DSM 43066T/X79289 Rhodococcus erythropolis NBRC 100887/AP008957 76 100 Rhodococcus qingshengii djl-6T/DQ090961 99 Rhodococcus jialingiae djl-6-2T/DQ185597 Rhodococcus nanhaiensis SCSIO 10187T/JN582175 Rhodococcus wratislaviensis NBRC 100605T/BAWF01000105 51 96 Rhodococcus opacus DSM 43205T/X80630 99 Rhodococcus corynebacterioides DSM 20151T/AF430066 79 Rhodococcus trifolii T8T/FR714843 Rhodococcus triatomae IMMIB RIV-085T/AJ854055 Rhodococcus canchipurensis MBRL 353T/JN164649 Rhodococcus agglutinans CFH S0262T/KP232908 93 Rhodococcus defluvii Ca11T/JPOC01000058 58 Rhodococcus equi NBRC 101255T/APJC01000042 88 Rhodococcus phenolicus DSM 44812T/AM933579 Rhodococcus zopfii DSM 44108T/AF191343 97 M1042 57 Rhodococcus coprophilus DSM 43347T/X80626 Rhodococcus artemisiae YIM 65754T/GU367155 Rhodococcus rhodochrous DSM 43241T/X79288 98 Rhodococcus gordoniae W4937T/AY233201 62 97 Rhodococcus pyridinivorans PDB9T/AF173005 59 Rhodococcus biphenylivorans TG9T/KJ546454 Rhodococcus ruber DSM 43338T/X80625 100 Rhodococcus aetherivorans 10bc312T/AF447391 Rhodococcus rhodnii DSM 43336T/X80621 Corynebacterium diphtheriae 0.01 Figure 4.1: Neighbour-joining tree based on almost complete 16 rDNA gene sequences (1462 nt) showing the position of Rhodococcus sp. M1042 amongst its phylogenetic neighbours. 62 University of Ghana http://ugspace.ug.edu.gh Table 4.1: A table showing other species of the genus Rhodococcus with their index similarity pairwise and completeness to the new strain M1042 Rank Nam/Title Accession Pairwise Diff/Total Completeness Similarity nt (%) (%) 1 Rhodococcus coprophilus X80626 98.97 15/1462 100 DSM 43347(T) 2 Rhodococcus phenolicus AM93357 98.43 23/1462 100 DSM 44812(T) 9 3 Rhodococcus zopfii DSM AF191343 98.29 25/1461 100 44108(T) 4 Rhodococcus rhodochrous X79288 97.95 30/1461 100 DSM 43241(T) 5 Rhodococcus ruber DSM X80625 97.88 31/1462 100 43338(T) 6 Rhodococcus pyridinivorans AF173005 97.88 31/1460 100 PDB9(T) 7 Rhodococcus KJ546454 97.47 37/1461 100 biphenylivorans TG9(T) 8 Rhodococcus artemisiae GU36715 97.46 37/1458 100 YIM 65754(T) 5 9 Rhodococcus aetherivorans AF447391 97.24 38/1375 94.18 10bc312(T) 10 Rhodococcus gordoniae AY23320 97.19 39/1386 94.93 W4937(T) 1 63 University of Ghana http://ugspace.ug.edu.gh 11 Rhodococcus X80617 97.12 42/1456 100 marinonascens DSM 43752(T) 4.3 Extraction, fractionation, isolation and structure elucidation of compounds from Rhodococcus sp. strain M1042 Rhodococcus sp. strain M1042 was cultured on large scale (2.5 L) and metabolites from the culture extracted/adsorbed by adding Diaion HP-20 resin 50 g/L. The Diaion HP-20 resin was decanted by filtration through a piece of glass wool from the culture medium. Subsequently, the Diaion HP-20 resin with adsorbed organics was repeatedly and alternatively extracted using equal volumes of methanol and dichloromethane to afford the total crude extract (TCE). The TCE was taken through a modified Kupchan solvent partitioning process as described in section 3.7. This solvent portioning process resulted into four fractions WB, FM, FD, and FH listed in order of decreasing polarity of metabolite contained in each fraction. Analysis of the HRESI/HPLC-DAD-MSn data (Fig 4.2) for the FH did not identify any interesting metabolites which could be targeted for isolation and characterization. This could be as a result of the fact that most metabolites in this extract are too polar for the FH fraction or metabolites within this fraction are too aliphatic for the LCMS system in which case they will not fly very well in the orbitrap. Most of the peaks seen in this spectrum were probably contaminants that were picked up during the sample processing. Hence, a 1H-NMR was acquired for this fraction (Figure 4.3) but, analysis of this data still did not yield any interesting results. 64 University of Ghana http://ugspace.ug.edu.gh Figure 4.2: HRESI/HPLC-DAD-MSn spectrum of M1042-FH fraction Figure 4.3: 1H-NMR spectrum of M1042-FH fraction 65 University of Ghana http://ugspace.ug.edu.gh Figure 4.4: HRESI/HPLC-DAD-MSn spectrum of M1042-FD fraction The HRESI/HPLC-DAD-MSn data (Fig 4.4) for the FD fraction was also analysed and a series of huge masses were seen to emerge from the HPLC column at the end of the chromatogram as shown in Figure 4.4 above but, upon careful analysis of these peaks, it was obvious that they were not natural products but mere contaminants of the sample processing. Analysis of the 1H- NMR for the FD fraction (Figure 4.5) still did not show any interesting natural product metabolites. 66 University of Ghana http://ugspace.ug.edu.gh Figure 4.5: 1H-NMR spectrum of M1042-FD fraction Results from analysing both the LCMS (Figure 4.6) and 1H-NMR (Figure 4.7) data for the FM fraction showed metabolites that were worthy of isolation and subsequent characterization but, the amount of sample obtained after Kupchan for FM did not permit this study. 67 University of Ghana http://ugspace.ug.edu.gh Figure 4.6: HRESI/HPLC-DAD-MSn spectrum of M1042-FM fraction Figure 4.7: 1H-NMR spectrum of M1042-FM fraction 68 University of Ghana http://ugspace.ug.edu.gh It appears that, most of the secondary metabolites produced by Rhodococcus sp. M1042 are very polar and concentrate in the WB fraction during Kupchan. Hence, the WB fraction seemed to contain quite a number of metabolites notable amongst which were the three peptides shown in Figure 4.8 The 1H-NMR for the WB fraction (Figure 4.9) further confirmed these peptides and subsequent phytochemical screening with ninhydrin detection provided more evidence of the presence of these interesting natural products. Figure 4.8: HRESI/HPLC-DAD-MSn spectrum of M1042-WB fraction 69 University of Ghana http://ugspace.ug.edu.gh Figure 4.9: 1H-NMR spectrum of M1042-WB fraction Figure 4.10: A normal phase TLC plate with M1042 WB fraction which shows positive results for ninhydrin test as an indication of either free amino acid or peptide present in the fraction. 70 University of Ghana http://ugspace.ug.edu.gh Hence, the WB fraction was subjected to column chromatography by size exclusion using sephadex LH20 as stationary phase and CH3OH:CH3CN (50:50 v/v) mixture as mobile phase to afford four main fractions; M1042-WB-SFA (57.1245 mg), M1042-WB-SFB (63.4213 mg), M1042-WB-SFC (93.2411 mg) and M1042-WB-SFD (42.3126 mg) as described in section 3.8.1. 1H-NMR, HRESI/HPLC-DAD-MSn and phytochemical screening analysis showed the compounds of interest to be in the M1042-WB-SFC (93.2411 mg) fraction and hence this became a priority sample for further purification. The M1042-WB-SFC fraction was further subjected to column chromatography by gravity using silica gel as stationary phase and varying mixtures of hexane, ethyl acetate and methanol of increasing polarity as the mobile phase to afford a total of seven fractions M1042-WB-SFC-C1-7. Subsequent investigation using phytochemical screening, 1H –NMR, and HRESI/HPLC-DAD-MSn showed the presence of three peptides in the M1042-WB-SFC-C6 fraction and this was further subjected to vacuum flash chromatography to afford M1042-WB-SFC-C6-MDe (14.3216 mg) fraction as described in section 3.8.2. The M1042-WB-SFC-C6-MDe fraction was subjected to a repeated HPLC procedure involving the use of a Phenomenex Luna reversed-phase C18 column (C18 250 × 10 mm, L × i.d.) at a flow rate of 1.5 ml/min. The gradient involved the use of solvent mixtures A: 75/15 (H2O/CH3CN) and B: 100 % CH3CN with the gradient set to run from 100 % A to 100 % B in 30 minutes and maintained at 100 % B for 30 minutes. This led to the isolation of M1042-WB- SFC-C6-MDe-B (5.1231 mg) as yellow oily compound eluted at a retention time of 21.03 minutes, M1042-WB-SFC-C6-MDe-C (3.1321 mg) as white crystalline compound eluted at a retention time of 26.31 minutes, and M1042-WB-SFC-C6-MDe-D (2.5121 mg) also as white crystalline compound eluted at a retention time of 36.23 minutes within 60 minutes elution period after 24 injections. 71 University of Ghana http://ugspace.ug.edu.gh 3.00 2.80 2.60 2.40 2.20 2.00 1.80 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Minutes Figure 4.11: HPLC profile of M1042-WB-SFC-C6-MDe fraction showing the peaks that yielded the three peptides 4.3.1 Structure elucidation of M1042-WB-SFC-C6-MDe-B (compound B) The compound B was isolated from Rhodococcus sp. M1042 as deep yellowish oil with slight pungent smell. The UV spectrum of this compound showed prominent absorption maxima at λmax of 275.9 (Figure 4.12). 72 AU University of Ghana http://ugspace.ug.edu.gh 2.60 2.40 2.20 2.00 1.80 1.60 1.40 1.20 1.00 0.80 0.60 275.9 0.40 0.20 0.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00 nm 20.918 Extracted Figure 4.12: UV spectrum of M1042-WB-SFC-C6-MDe-B showing prominent absorption maxima at λmax of 275.9 The high resolution mass spectrometry data gave m/z 1537.8738 [M+H]+. Due to the rather large size of this molecule, a different approach to the analysis of the 1D and 2D-NMR was adopted and this involved solving the structure of the component amino acids one at a time and using the HRLCMS data to verify proposed structures. 73 AU University of Ghana http://ugspace.ug.edu.gh Figure 4.13: HRESI/HPLC-DAD-MSn spectrum of pure M1042-WB-SFC-C6-MDe-B 4.3.1.1 Tyrosine residues Two tyrosine residues were detected in the 1D and 2D NMR data of this compound. Olefinic protons at 6.72 (2H, d, J = 8.3 Hz, H-P), 7.09 (4H, d, J = 8.3 Hz, H-M) and 6.72 (2H, d, J = 8.3 Hz, H-Q) corresponding to C C-P (C116.3), C-M (C131.5) and C-Q (C116.3) respectively were direct indication of the presence of two tyrosine residues whose NMR data are summarized in Figure 4.14 and 4.15. The full NMR data for the two tyrosine residues are as shown in Tables 4.2 and 4.3 74 University of Ghana http://ugspace.ug.edu.gh Figure 4.14: Substructure for first tyrosine residue showing TOCSY, COSY, and HMBC correlations Table 4.2: 1H and 13C NMR data for first tyrosine residue # δ 13C 13C δ 1H Mult 1H-1H 1H-1H HMBC (ppm) mult (ppm) (Hz) COSY TOCSY H 173.4 C b., p.' K 157.3 C m, p M 131.5 CH 7.09 d, 8.3 p p p.' O 129.4 C P 116.3 CH 6.72 d, 8.3 m m p, m B. 56.0 CH 4.67 ov. p., p.' p.' P. 38.4 CH2 3.01 ov. b. m 2.93 ov. 75 University of Ghana http://ugspace.ug.edu.gh Figure 4.15: Substructure for second tyrosine residue showing TOCSY, COSY, and HMBC correlations Table 4.3: 1H and 13C NMR data for second tyrosine residue # δ 13C 13C δ 1H Mult 1H-1H 1H-1H HMBC (ppm) mult (ppm) (Hz) COSY TOCSY H 173.4 C u.' L 157.3 C m M 131.5 CH 7.09 d, 8.3 q q m, u.' O 129.4 C Q 116.3 CH 6.72 d, 8.3 m m m W... 56.8 CH 4.62 ov. u. U. 37.4 CH2 3.17 ov. w... 2.91 ov. 76 University of Ghana http://ugspace.ug.edu.gh 4.3.1.2 Proline residue Analysis of the 1D and 2D-NMR data also showed the presence of one proline residue which is quite easy to see by the appearance of three diastereotopic protons at  2.21 (1H, ov., H-I..),  1.97 (1H, ov., H-I..'),  2.07 (1H, ov., H-V..),  1.98 (1H, ov., H-V..'),  3.94 (1H, ov., H-L.),  3.69 (1H, ov., H-L.') with corresponding carbon atoms C-I.. (C 30.6), C-V.. (C 25.8) and C-L. (C 49.2) respectively. The a-carbon for this residue could also be seen at  4.40 (1H, ov., H-T) with corresponding carbon atom at C-T (C 62.1). The full NMR correlations are as indicated in Figure 4.16 while Table 4.4 contains a summary of this data. Figure 4.16: Substructure for Proline residue showing TOCSY, COSY, and HMBC correlations 77 University of Ghana http://ugspace.ug.edu.gh Table 4.4: 1H and 13C NMR data for Proline residue # δ 13C 13C δ 1H Mult 1H-1H 1H-1H HMBC (ppm) mult (ppm) (Hz) COSY TOCSY A... 165.7 C i.. T 62.1 CH 4.39 ov. i.. l., l.', i.., i..' L. 49.2 CH2 3.94 ov. v.., v..', t, 3.69 ov. v..', i..' v.., i.., i..' I.. 30.6 CH2 2.21 ov. l., l.', t 1.97 ov. V.. 25.8 CH2 2.07 ov. i.. l., l.', t, 1.98 ov. i.., i..' 4.3.1.2 Threonine residues Two threonine residues were also identified in this peptide and their presence was indicated by the appearance of  1.18 (3H, ov., H- B...),  1.15 (3H, ov., H- C...),  3.98 (2H, ov., H-S),  4.21 (2H, ov., H-V) corresponding to carbons C-B... (C 20.2), C-C... (C 20.2), C- S (C 68.8) and C- (C 60.9) respectively. The full 2D-NMR correlations are summarized in Figure 4.17 and 4.18 but a summary of the data is given Table 4.5 and 4.6 Table 4.5: 1H and 13C NMR data for first Threonine residue # δ 13C 13C δ 1H Mult 1H-1H 1H-1H HMBC (ppm) mult (ppm) (Hz) COSY TOCSY I 172.6 C v S 68.8 CH 3.98 ov. v, c... v, c... c... V 60.9 CH 4.21 ov. s s, c... C... 20.2 CH3 1.16 ov. v, s v 78 University of Ghana http://ugspace.ug.edu.gh Figure 4.17: Substructure for first Threonine residue showing TOCSY, COSY, and HMBC correlations Table 4.6: 1H and 13C NMR data for second Threonine residue # δ 13C 13C δ 1H Mult 1H-1H 1H-1H HMBC (ppm) mult (ppm) (Hz) COSY TOCSY I 172.6 C v S 68.8 CH 3.98 ov. v v b... V 60.9 CH 4.21 ov. s, b... b... B... 20.3 CH3 1.18 ov. 79 University of Ghana http://ugspace.ug.edu.gh Figure 4.18: Substructure for second Threonine residue showing TOCSY, COSY, and HMBC correlations 4.3.1.3 Isoleucine residue An isoleucine residue could also been seen in the sequence of this novel Rhodococcus derived peptide. This substructure was easily identified through  0.89 (3H, ov., H-O...),  1.12 (2H, ov., H-U..),  1.80 (1H, ov., H-R.),  4.32 (1H, ov., H-Y) and  0.83 (3H, ov., H-K...) with corresponding carbons at C-O... (C 11.8), C-U.. (C 26.3), C-R. (C 38.2), C-Y (C 58.3) and C-K... (C 15.9) respectively. The 2D NMR correlations are shown in Figure 4.19 while the summary of all this data is given in Table 4.7 80 University of Ghana http://ugspace.ug.edu.gh Figure 4.19: Substructure for Isoleucine residue showing TOCSY, COSY, and HMBC correlations Table 4.7: 1H and 13C NMR data for Isoleucine residue # δ 13C 13C δ 1H Mult 1H-1H 1H-1H HMBC (ppm) mult (ppm) (Hz) COSY TOCSY H 173.4 C y Y 58.3 CH 4.32 ov. r., k... k... R. 38.2 CH 1.80 ov. u.., k... q, y, k... y U.. 26.3 CH2 1.12 ov. r. r., y, o... y, k... K... 15.9 CH3 0.83 ov. r. y, r., o... y O... 11.8 CH3 0.86 ov. y, r. 81 University of Ghana http://ugspace.ug.edu.gh 4.3.1.4 Valine residue One valine residue was also identified in this peptide sequence by analysis of the 1D and 2D NMR data. This substructure was evident from the  1.00 (3H, ov., H-F...),  1.01 (3H, ov., H-G...),  2.16 (1H, ov., H-B..) and  4.55 (1H, d, J = 8.8 Hz, H-A.) with corresponding carbons at C-F... (C 19.5), C-G... (C 19.2), C-B.. (C 31.3) and C-A. (C 58.0) respectively. The crucial correlations in this spin systems are illustrated in Figure 4.20 with a summary of the data given in Table 4.8 Figure 4.20: Substructure for Valine residue showing TOCSY, COSY, and HMBC correlations 82 University of Ghana http://ugspace.ug.edu.gh Table 4.8: 1H and 13C NMR data for Valine residue # δ 13C 13C δ 1H Mult 1H-1H 1H-1H HMBC (ppm) mult (ppm) (Hz) COSY TOCSY H 173.4 C a. A. 58.0 CH 4.55 d, 8.8 b.. g..., b.. g..., f... B.. 31.3 CH 2.16 ov. a., f... a., g... F... 19.5 CH3 1.00 ov. b.. a., b.. G... 19.2 CH3 1.01 ov. f... 4.3.1.5 Asparagine residue Furthermore, one asparagine residue was also identified in the structure of this novel peptide. The presence of this amino acid was evident by the observation of protons  2.98 (1H, ov., H- O.),  2.92 (1H, ov., H-O.') and  4.35 (1H, ov., H-E.) with corresponding carbons at C-O. (C 40.0) and C-E. (C 55.2) respectively. The characteristic correlations of this structure are shown in Figure 4.21 with a summary of the data listed in Table 4.9 Figure 4.21:Substructure for Asparagine residue showing TOCSY, COSY, and HMBC correlations 83 University of Ghana http://ugspace.ug.edu.gh Table 4.9: 1H and 13C NMR data for Asparagine residue # δ 13C 13C δ 1H Mult 1H-1H 1H-1H HMBC (ppm) mult (ppm) (Hz) COSY TOCSY G 173.8 C o.' E. 55.2 CH 4.35 ov. O. 40.0 CH2 2.98 ov. 2.92 Apart from the -amino acids whose structures were determined for this peptide, there were some post-translationally modified amino acids as well. The structures of these were determined by interpretation of the current experimental data in comparison to literature data for peptide- like compounds that have been previously isolated from Rhodococcus sp.91 4.3.1.6 2,8-Diaminooctanoic acid residue A 2,8-diaminooctanoic acid residue was found in the structure of peptide B. Incidentally, similar diaminoalkanoic acids have been seen previously in the structure of Rhodococcus-derived peptides like rhodopeptin B1-5, C1-4 and lariatin A and B. The observed 1D and 2D NMR data are illustrated in Figure 4.22 and Table 4.10 84 University of Ghana http://ugspace.ug.edu.gh Figure 4.22: Substructure for 2,8-Diaminooctanoic acid residue showing TOCSY, COSY, and HMBC correlations Table 4.10: 1H and 13C NMR data for 2,8-Diaminooctanoic acid # δ 13C 13C δ 1H Mult 1H-1H 1H-1H HMBC (ppm) mult (ppm) (Hz) COSY TOCSY F 174.4 C s... S... 55.0 CH 4.35 ov. j.. N. 40.2 CH2 1.19 ov. X. 35.1 CH2 2.30 ov. j.., m.., s... A.. 32.7 CH2 2.25 ov. y.., m.., s... J.. 29.5 CH2 2.05 ov. s... x., s... M.. 28.6 CH2 2.14 ov. y.. s... Y.. 23.4 CH2 1.85 ov. m.. 85 University of Ghana http://ugspace.ug.edu.gh 4.3.1.7 Non-amino acid prenylation residues Two non-amino acid prenylation substructures were also obvious from the analysis of the NMR data. Interestingly, these substructures were also seen in other Rhodococcus-derived metabolites. Their characteristic correlations are shown in Figures 4.23 and 4.24 but the data is also summarized in Tables 4.11 and 4.12 Figure 4.23: Substructure for Non-amino acid prenylation residue showing TOCSY, COSY, and HMBC correlations Table 4.11:1H and 13C NMR data for Non-amino acid prenylation residue # δ 13C 13C δ 1H Mult 1H-1H 1H-1H HMBC (ppm) mult (ppm) (Hz) COSY TOCSY G 173.8 C m. R 69.6 CH 4.00 ov. q., m. f.., m., m. q. M. 44.4 CH2 4.40 ov. r r, f.., q. Q. 38.3 CH2 1.48 ov. r r F.. 30.8 CH2 1.29 ov. R.. 26.8 CH2 1.43 ov. q, f.., r, m. 86 University of Ghana http://ugspace.ug.edu.gh Figure 4.24: Substructure for Non-amino acid prenylation residue showing TOCSY, COSY, and HMBC correlations Table 4.12: 1H and 13C NMR data for Non-amino acid prenylation residue # δ 13C 13C δ 1H Mult 1H-1H 1H-1H HMBC (ppm) mult (ppm) (Hz) COSY TOCSY N. 40.2 CH2 1.19 ov. k.. z.. z.. K.. 29.1 CH 1.54 ov. n., z.. z.. z.. O.. 28.4 CH2 1.30 ov. Z.. 23.1 CH3 0.90 ov. k.. n. k.., n. The calculated mass generated up to this point accounts for m/z = 1335.7690 out of the m/z = 1537.8738 that was obtained from the experimental data as the full mass of this peptide. This leaves a mass of about m/z = 202.1048 to account for in order to solve the full structure of this molecule. Analysis of the LCMS data shows that this peptide indeed loses a m/z = 35.9767 (Appendix 39) which corresponds to an HCl that in turn is direct indication of the presence of a chlorine atom. This leaves m/z = 166.1281 which points to the fact that there are indeed three 87 University of Ghana http://ugspace.ug.edu.gh tyrosine present in this peptide but, the NMR data (Section 4.3.1.1) only facilitates the identification of two tyrosines due to the presence of overlaps that is particularly characteristic of all the NMR data of this huge chlorinated peptide. In order to confirm whether or not this peptide is linear or cyclic, the exact position of the chlorine atom and the sequence in which all amino acids are joined more mass spectrometry and bioinformatics data is required. 4.4 Sequencing of the genome of Rhodococcus sp. strain M1042 to identify the gene clusters of M1042-WB-SFC-C6-MDe-B, M1042-WB-SFC-C6-MDe-C, and M1042-WB- SFC-C6-MDe-D peptides The Rhodococcus sp. strain M1042 was subjected to genome sequencing and the gene clusters responsible for the biosynthesis of these three peptides M1042-WB-SFC-C6-MDe-B, M1042- WB-SFC-C6-MDe-C, and M1042-WB-SFC-C6-MDe-D have been identified. Currently, the insertion of gene clusters into heterologous hosts, the subsequent expression of enzymes and gene knockout studies are underway. However, the data for all these studies are not included in this thesis since this work has not been published, we are unable to include the full details but we will soon publish this data for the general public good. 4.5 Cytotoxicity studies Notwithstanding their high molecular weights, biological peptides, including Ribosomally synthesized and post-translationally modified peptides (RiPPs), and Nonribosomal peptides (NRP) have contributed significantly (15 %, 202 drugs) to disease fighting drugs on the market.224 These structures have attracted a lot of attention lately due to their high molecular weights, interesting novel structures and potent biological activities including anticancer, 88 University of Ghana http://ugspace.ug.edu.gh antiviral, antimicrobial, antimalarial and anti-protease enzyme inhibiting activities. Kolossin is a remarkable example of the interesting bioactivity for members in this group notwithstanding the fact that it is relatively difficult for microbes, parasites and cancer cells to develop resistance to these huge molecules compared to their small molecular drug counterparts. In this instance, we managed to test the activity of only one of our three peptides due to the rather small quantities of the metabolites isolated in this project. The results show minimal activity for the five parasites tested but, it is possible that these peptides could have cytotoxicity activity and hence this study is still on-going. Table 4.13: Cell growth-inhibitory potencies of pure compounds expressed as IC50 values Parasites cell line Compound IC50 (µM) M1042-WB-SFC-C6-MDe-B T. brucei (Trypanosomiasis) >100 µM L. donovani (Leishmaniasis) >100 µM L. major (Leishmaniasis) >100 µM Trichomonas mobilensis (Trichomoniasis) >100 µM Plasmodium falciparum (Malaria) >100 µM 89 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 90 University of Ghana http://ugspace.ug.edu.gh 5.1 Conclusion The huge chemical diversity resulting in the isolation of medicinally important natural products from all microbes is still yet untapped. In this thesis, a new Rhodococcus sp. strain M1042 was investigated for its capability of producing structurally diverse molecules as a source of future antibiotics. A very efficient HRESI/HPLC-DAD-MSn measurement and appropriate dereplication technique was employed in identifying and isolating secondary metabolites from this bacterial species. The WB fraction of the TCE of the Rhodococcus sp. M1042 gave positive results to Ninhydrin phytochemical screening showing the possible presence of either free amino acid or peptide. Three novel peptides were isolated from the WB fraction of the TCE. The structure of M1042-WB-SFC-C6-MDe-B peptide was fully elucidated with the structural determination of the remaining two still in progress. Currently, a connectoin is being made to the gene clusters that are responsible for the biosynthesis of these peptides. M1042-WB-SFC-C6-MDe-B showed minimal cytotoxicity activity against Leishmania donovani, Trypanosoma brucei, Trichomonas mobilensi, and Plasmodium falciparum with IC50 >100 µM. 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PharManufacturing: The international peptide review, 2, 10-15. 119 University of Ghana http://ugspace.ug.edu.gh 120 University of Ghana http://ugspace.ug.edu.gh Appendices Appendix 1: 13 C NMR spectrum of M1061-WB-SFC-C6-MDe-B Appendix 2: 13 C NMR spectrum of M1061- WB-SFC- C6-MDe-B 121 University of Ghana http://ugspace.ug.edu.gh Appendix 2: 13 C NMR spectrum of M1061-WB-SFC-C6-MDe-B 122 University of Ghana http://ugspace.ug.edu.gh Appendix 3: 13 C NMR spectrum of M1061-WB-SFC-C6-MDe-B 123 University of Ghana http://ugspace.ug.edu.gh Appendix 4: 13 C NMR spectrum of M1061-WB-SFC-C6-MDe-B 124 University of Ghana http://ugspace.ug.edu.gh Appendix 5: 13 C NMR spectrum of M1061-WB-SFC-C6-MDe-B 125 University of Ghana http://ugspace.ug.edu.gh Appendix 6: 13 C NMR spectrum of M1061-WB-SFC-C6-MDe-B 126 University of Ghana http://ugspace.ug.edu.gh Appendix 7: HSQC spectrum of M1061-WB-SFC-C6-MDe-B 127 University of Ghana http://ugspace.ug.edu.gh Appendix 8: HSQC spectrum of M1061-WB-SFC-C6-MDe-B 128 University of Ghana http://ugspace.ug.edu.gh Appendix 9: HSQC spectrum of M1061-WB-SFC-C6-MDe-B 129 University of Ghana http://ugspace.ug.edu.gh Appendix 10: HSQC spectrum of M1061-WB-SFC-C6-MDe-B 130 University of Ghana http://ugspace.ug.edu.gh Appendix 11: HSQC spectrum of M1061-WB-SFC-C6-MDe-B 131 University of Ghana http://ugspace.ug.edu.gh Appendix 12: HSQC spectrum of M1061-WB-SFC-C6-MDe-B 132 University of Ghana http://ugspace.ug.edu.gh Appendix 13: HSQC spectrum of M1061-WB-SFC-C6-MDe-B 133 University of Ghana http://ugspace.ug.edu.gh Appendix 14: HSQC spectrum of M1061-WB-SFC-C6-MDe-B 134 University of Ghana http://ugspace.ug.edu.gh Appendix 15: HSQC spectrum of M1061-WB-SFC-C6-MDe-B 135 University of Ghana http://ugspace.ug.edu.gh Appendix 16: TOCSY spectrum of M1061-WB-SFC-C6-MDe-B 136 University of Ghana http://ugspace.ug.edu.gh Appendix 17: TOCSY spectrum of M1061-WB-SFC-C6-MDe-B 137 University of Ghana http://ugspace.ug.edu.gh Appendix 18: TOCSY spectrum of M1061-WB-SFC-C6-MDe-B 138 University of Ghana http://ugspace.ug.edu.gh Appendix 19: TOCSY spectrum of M1061-WB-SFC-C6-MDe-B 139 University of Ghana http://ugspace.ug.edu.gh Appendix 20: TOCSY spectrum of M1061-WB-SFC-C6-MDe-B 140 University of Ghana http://ugspace.ug.edu.gh Appendix 21: TOCSY spectrum of M1061-WB-SFC-C6-MDe-B 141 University of Ghana http://ugspace.ug.edu.gh Appendix 22: HMBC spectrum of M1061-WB-SFC-C6-MDe-B 142 University of Ghana http://ugspace.ug.edu.gh Appendix 23: HMBC spectrum of M1061-WB-SFC-C6-MDe-B 143 University of Ghana http://ugspace.ug.edu.gh Appendix 24: HMBC spectrum of M1061-WB-SFC-C6-MDe-B 144 University of Ghana http://ugspace.ug.edu.gh Appendix 25: HMBC spectrum of M1061-WB-SFC-C6-MDe-B 145 University of Ghana http://ugspace.ug.edu.gh Appendix 26: HMBC spectrum of M1061-WB-SFC-C6-MDe-B 146 University of Ghana http://ugspace.ug.edu.gh Appendix 27: HMBC spectrum of M1061-WB-SFC-C6-MDe-B 147 University of Ghana http://ugspace.ug.edu.gh Appendix 28: HMBC spectrum of M1061-WB-SFC-C6-MDe-B 148 University of Ghana http://ugspace.ug.edu.gh Appendix 29: HMBC spectrum of M1061-WB-SFC-C6-MDe-B 149 University of Ghana http://ugspace.ug.edu.gh Appendix 30: HMBC spectrum of M1061-WB-SFC-C6-MDe-B 150 University of Ghana http://ugspace.ug.edu.gh Appendix 31: HMBC spectrum of M1061-WB-SFC-C6-MDe-B 151 University of Ghana http://ugspace.ug.edu.gh Appendix 32: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B 152 University of Ghana http://ugspace.ug.edu.gh Appendix 33: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B 153 University of Ghana http://ugspace.ug.edu.gh Appendix 34: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B 154 University of Ghana http://ugspace.ug.edu.gh Appendix 35: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B 155 University of Ghana http://ugspace.ug.edu.gh Appendix 36: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B 156 University of Ghana http://ugspace.ug.edu.gh Appendix 37: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B 157 University of Ghana http://ugspace.ug.edu.gh Appendix 38: 1H-1H COSY spectrum of M1061-WB-SFC-C6-MDe-B 158 University of Ghana http://ugspace.ug.edu.gh Appendix 39 Mass spectrum showing neutral loss of m/z 36 159