Dereplication of the Actinomycete Metabolome as a Source ...

Dereplication of the Actinomycete Metabolome as a Source ofBioactive Secondary Metabolites

Author

Romero Bonifaz, Christian Abraham

Published

2016

Thesis Type

Thesis (PhD Doctorate)

School

School of Natural Sciences

DOI

https://doi.org/10.25904/1912/3862

Copyright Statement

The author owns the copyright in this thesis, unless stated otherwise.

Downloaded from

http://hdl.handle.net/10072/365652

Griffith Research Online

https://research-repository.griffith.edu.au

Dereplication of the Actinomycete

Metabolome as a Source of Bioactive

Secondary Metabolites

Christian A. Romero BSc

School of Natural Sciences Griffith Sciences Griffith University

Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy

September 2015

ii

Abstract

Natural products (NPs) and their derivatives have historically played a vital role in drug

discovery by serving as an invaluable source of therapeutic agents and potential drug leads.

Among the established sources of NPs, microorganisms have proven to be promising

candidates for the production of novel scaffolds as well as marketable drugs. They have

yielded some of the most economically relevant leads for the pharmaceutical industry,

including penicillin G, cephalosporin C, tetracycline, mevastatin and rapamycin. At present,

approximately 32,000-34,000 bioactive microbial metabolites have been isolated and even

though it has been estimated that this amount represent less than 10% of the total number of

small molecules that these microorganisms can biosynthesise. Declining in productivity and

discovery of novel molecules over the past two decades has been one the reasons because

large pharmaceutical companies have closed their microbial drug discovery programs.

New approaches have been developed to address the problem of rediscovery of microbial

natural products. One of these strategies involves the isolation, characterisation and screening

of novel/rare actinomycete taxa sourced from unique and underexplored environments.

Hence, this investigations aims to access to the unique components of the drug-like natural

product metabolome of termite gut-associated actinomycetes using a new NMR-based

methodology. This approach was used to accelerate the identification of all the constituents

with unique spectral patterns comprising the lead-like enhanced fractions. The effectiveness

of the approach was demonstrated by the isolation and identification of nine new natural

products, namely, actinoglycosidines A (27) and B (28), actinopolymorphol D (29),

niveamycins A (36), B (37) and C (38), actinofuranosin A (41) and arglecins B (42) and C

(43).

iii

Table of Contents

Abstract ....................................................................................................................................................ii

Table of Contents .................................................................................................................................... iii

Statement of Originality ........................................................................................................................... v

Acknowledgements ................................................................................................................................. vi

List of Figures ........................................................................................................................................ vii

List of Tables ........................................................................................................................................... ix

List of Abbreviations ................................................................................................................................ x

Publications and Presentations Arising from this Thesis ...................................................................... xiii

Chapter 1: Introduction ........................................................................................................................ 1

1.1 Microbial resources ....................................................................................................................... 3

1.1.1 Microbial metabolites as pharmacological agents ................................................................. 3

1.2 Significance of actinomycetes....................................................................................................... 4

1.3 Mining for novel sources of actinomycete diversity ..................................................................... 7

1.4 Dereplication of bioactive natural products ................................................................................ 10

1.5 Aims of the thesis ........................................................................................................................ 12

Chapter 2: Biological investigations of termite-gut associated actinomycetes............................... 14

2.1 Introduction ................................................................................................................................. 14

2.2 Results and discussion ................................................................................................................ 17

Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592 ...................................................... 30

3.1 Introduction ................................................................................................................................. 30

3.2 Chemical studies of termite gut-associated actinomycetes ......................................................... 31


Chapter 4: 1H NMR Fingerprinting of Streptomyces sp. USC 593 ................................................. 47

4.1 Introduction ................................................................................................................................. 47


4.3 Computation of NMR chemical shifts ........................................................................................ 59

4.3.1. Conformational search ........................................................................................................ 61

4.3.2. Geometry optimisation and frequency calculation .............................................................. 62

iv

4.3.3. NMR shielding tensor calculations and conversion to chemical shift values ..................... 62

4.3.4. Boltzman analysis of DFT NMR data ................................................................................. 63

4.3.5. Comparison of experimental and computed chemical shifts ............................................... 64

4.4. Absolute configuration ................................................................................................................ 67

4.4.1. Absolute configuration of niveamycin B ............................................................................ 68

Chapter 5: Actinofuranosin A and arglecins B and C from a Streptomyces sp. USC 597 ............ 70

5.1 Introduction ................................................................................................................................. 70


Chapter 6: Summary........................................................................................................................... 82

Chapter 7: Experimental .................................................................................................................... 85

7.1 General experimental .................................................................................................................. 85

7.2 Culture conditions ....................................................................................................................... 85

7.3 Lead-like enhanced (LEE) fractions ........................................................................................... 86

7.4 Metabolic fingerprinting approach .............................................................................................. 87

7.5 Preliminary screening of isolates for production of antimicrobial compounds .......................... 87

7.6 Scale-up solid culture growth and isolation ................................................................................ 88

7.7 Anti-BCG assay .......................................................................................................................... 88

7.8 Phylogenetic characterisation of the actinomycetes strains ........................................................ 89

7.9 Chapter 3: Experimental ............................................................................................................. 91

7.10 Chapter 4: Experimental ........................................................................................................... 94

7.11 Chapter 5: Experimental ......................................................................................................... 966

Chapter 8: Conclusions ..................................................................................................................... 100

References .......................................................................................................................................... 101

Appendix I: CD NMR data list for thesis compounds ................................................................... 112

Appendix II: Journal manuscript .................................................................................................... 113

v

Statement of Originality

I declare that this work has not previously been submitted for a degree or diploma in any

university. To the best of my knowledge and belief, the thesis contains no material previously

published or written by another person except where due reference is made in the thesis itself

Christian A. Romero Date

vi

Acknowledgements

I would like to express my appreciation to my academic advisors Professor Ronald Quinn, Dr

Ipek Kurtböke and Dr Tanja Grkovic for their scientific insight and mentorship. Without their

expertise and full support, this dissertation would not have been possible.

I would like to acknowledge the former and present members of the drug discovery group at

the Eskitis Institute for their collaboration and friendship throughout these years. I would like

to thank the staff and students of the Genecology Research Centre from the University of the

Sunshine Coast who assisted me in completing the biological experiments. I would also like

to thank Dr Ken Wasmund, Division of Microbial Ecology (DOME), Department of

Microbiology and Ecosystem Science, University of Vienna, Austria for the construction of

the phylogenetic trees. I also acknowledge the support of the Griffith University eResearch

Services Team and the use of the High Performance Computing Cluster "Gowonda" to

complete this research.

I would specially like to acknowledge Escuela Superior Politécnica del Litoral (ESPOL),

Centro de Investigaciones Biotecnológicas del Ecuador (CIBE) and Secretaría Nacional de

Educación Superior Ciencia y Tecnología (SENESCYT) for the PhD scholarship provided.

My deepest gratitude goes to my family for their unconditional support and understanding.

vii

List of Figures

Figure 1. Snapshot of the antibiotic pipeline ........................................................................................ 16

Figure 2. Underexplored habitat of novel actinomycete species .......................................................... 17

Figure 3. Neighbour-joining phylogenetic tree based on partial 16S rDNA sequences for

Streptomyces. ......................................................................................................................................... 19


Micromonospora ................................................................................................................................... 20

Figure 5. Neighbour-joining phylogenetic tree based on partial 16S rDNA sequences showing the

relationships between the strains USC 6900 and 6908 with the most closely related type strains of

Microbispora. Numbers at the nodes indicate bootstrap values based on 1,000 replicates; only values

above 50% are shown. Bar 0.01 sequence divergence .......................................................................... 21

Figure 6. Phylogenetic placement based on partial 16S rDNA sequences ........................................... 22

Figure 7. Evaluation of the antibiotic activity of the actinomycete isolates in solid cultures .............. 24

Figure 8. Colony morphologies of Streptomyces sp. USC 592 ............................................................ 32

Figure 9. HPLC chromatogram depicting the drug-like/lead-like region ............................................. 35

Figure 10. 1H NMR fingerprint spectrum of LLE fraction 1 at 600 MHz in MeOD-d4 ....................... 35



Figure 13. Fragments found during the elucidation process and cruciall HMBC and NOESY

correlations for actinoglycosidines A and B ......................................................................................... 40

Figure 14. Acid Hydrolysis of actinoglycosidine A ............................................................................. 40

Figure 15. 1H NMR spectra comparison between compounds 27 and 28 ............................................ 41

Figure 16. 13C NMR spectra comparison between compounds 27 and 28 ........................................... 42

Figure 17. 1H NMR fingerprint spectra of actinoglycosidine B ........................................................... 42

Figure 18. Crucial COSY and HMBC correlations for actinopolymorphol D .................................... 44

Figure 19. 1H NMR fingerprint spectra of LLE fraction 4, BE-54017-derivative 4 ............................ 45

viii

Figure 20. Colony morphologies of Streptomyces sp. USC 593 .......................................................... 49




Figure 24. Fragments found during the elucidation process and crucial HMBC correlations for the

new natural product, niveamycin A....................................................................................................... 54

Figure 25. 1H NMR spectra comparison between compounds 36 and 37 ............................................ 55

Figure 26. 13C NMR spectra comparison between compounds 36 and 37 ........................................... 55

Figure 27. 1H NMR fingerprint spectra of niveamycin B .................................................................... 56

Figure 28. Crucial HMBC correlations for niveamycins B and C ....................................................... 58

Figure 29. 1H NMR fingerprint spectra of niveamycins A and C ........................................................ 58

Figure 30. 1H NMR fingerprint spectrum of LLE fraction 3, WS 5995 A ........................................... 59

Figure 31. Structural isomers of niveamycins A–C ............................................................................. 62

Figure 32. Calculated (9S) and experimental ECD spectra of niveamycin B....................................... 68

Figure 33. Comparison of calculated (9S) and experimental ECD spectra of niveamycin B ............... 69


Streptomyces .......................................................................................................................................... 72

Figure 35. 1H NMR spectrum of actinofuranosin A at 600 MHz in MeOD-d4 .................................... 74

Figure 36. Fragments found during the elucidation process and crucial HMBC and NOESY

correlations for actinofuranosin A ......................................................................................................... 75

Figure 37. 1H NMR spectrum of arglecin B at 900 MHz in MeOD-d4 ................................................ 77

Figure 38. 13C NMR spectrum of arglecin B at 225 MHz in MeOD-d4 ............................................... 77

Figure 39. Crucial HMBC and COSY correlations for arglecins B and C ........................................... 78

Figure 40. gCOSY spectrum of Arglecin B at 600 MHz in DMSO-d6 ................................................ 79

Figure 41. 1H NMR data comparison between arglecins B and C ....................................................... 80

Figure 42. Summary of the NP-drug discovery workflow followed on this thesis .............................. 84

ix

List of Tables

Table 1. Colony characteristics on OMA of the selected termite-gut associated actinomycetes ......... 23

Table 2. Antitubercular activity with MIC values for the 17 tested compounds .................................. 28

Table 3 Colony characteristics and chemical profiles of the selected actinomycete strains ................. 34

Table 4. 1H NMR and 13C NMR spectroscopic data for actinoglycosidines A and B in MeOD-d4. .... 38

Table 5. 1H NMR and 13C NMR spectroscopic data for actinopolymorphol D in Acetone-d5 ............. 44

Table 6. 1H NMR and 13C NMR spectroscopic data for niveamycins A and B in MeOD-d4 ............... 54

Table 7. 1H NMR and 13C NMR spectroscopic data for niveamycin C in MeOD-d4 ........................... 57

Table 8. Scaling factors used for DFT calculations .............................................................................. 63

Table 9. Experimental and calculated 1H NMR data for niveamycins A–C ......................................... 65

Table 10. Experimental and calculated 13C NMR data for niveamycins A–C...................................... 66

Table 11. 1H NMR and 13C NMR spectroscopic data for actinofuranosin A in MEOD-d4 ................. 76

Table 12. . 1H NMR and 13C NMR spectroscopic data for arglecins B and C in MeOD-d4 ................. 81

x

List of Abbreviations

[α]D Optical rotation at 589 nm

13C Carbon-13 nuclear magnetic resonance spectroscopy

1H Proton nuclear magnetic resonance spectroscopy

2D NMR Two dimensional nuclear magnetic resonance spectroscopy

AC Absolute configuration

Acetone-d5 Deuterated acetone

ATCC American type culture collection

br Broad

C18 Octadecyl-derivatized silica

C8 Octyl-derivatized silica

calcd. Calculated

CMAE Corrected absolute error

d Doublet

DBEs Double bond equivalents

dd Doublet of doublets

DFT Density functional theory

DMSO Dimethylsulfoxide

DMSO-d6 Deuterated dimethylsulfoxide

DNP Dictionary of natural products

ECD Electronic circular dichroism

ESIMS Electrospray ionization mass spectrometry

EtOAc Ethyl acetate

gCOSY Gradient correlation spectroscopy

xi

gHMBC Gradient heteronuclear multiple-bond correlation

gHSQC Gradient heteronuclear single-quantum correlation

GYES Glucose yeast extract agar

HPLC High-performance liquid chromatography

HPLC-DAD High-performance liquid chromatography with diode-array detection

HRESIMS High resolution electrospray ionization mass spectrometry

Hz Hertz

J Coupling constant

LC-MS Liquid chromatography-mass spectrometry

LFA Lupin flour agar

LLE Lead-like enhanced

m Multiplet

m/z Mass to charge ratio

MAE Mean absolute error

Me Methyl

MEOD-d4 Deuterated methanol

MeOH Methanol

MIC Minimum inhibitory concentration

MMCM Monte Carlo molecular mechanism

MS Mass spectrometry

Mtb Mycobacterium tuberculosis

NCEs New chemical entities

NMR Nuclear magnetic resonance

NOE Nuclear overhauser effect

NPs Natural products

xii

OMA Oatmeal agar

OPLS Optimised potential for liquid stimulations

PCA Principal component analysis

ppm Part per million

q Quartet

QM Quantum chemical methods

RFA Rye flour agar

RMSD Root mean square deviation

ROA Raman optical activity

ROESY Rotating frame overhauser enhancement spectroscopy

s Singlet

t Triplet

TB Tuberculosis

TDDFT Time dependant density functional theory

USC University of the Sunshine Coast

UV Ultra-violet

VCD Vibrational circular dichroism

δ ppm of the applied magnetic field

xiii

Publications and Presentations Arising from this Thesis

Journal Publication

C. A. Romero, T. Grkovic, J. Han, L. Zhang, J. R . J. French, D. İ. Kurtböke and R. J. Quinn.

NMR fingerprints, an integrated approach to uncover the unique components of the drug-like

natural product metabolome of termite gut-associated Streptomyces species. RSC Adv., 2015,

5, 104524.

Conference and Poster Presentations

C. A. Romero. Rapid identification of new drug-like natural products using 1H NMR

fingerprints. Brisbane Biological & Organic Chemistry Symposium. 2014. Brisbane-Australia.

Oral Presentation.

C. A. Romero. Rapid identification of new drug-like natural products from termite-associated

actinomycetes using NMR metabolic fingerprints. International Symposium on the Biology of

Actinomycetes. 2014. Kusadasi-Turkey. Poster Presentation.

C. A. Romero. Termite-associated actinomycetes as a source of bioactive secondary

metabolites. The Australian Society for Microbiology. 2013. Adelaide-Australia. Poster

Presentation.

Chapter: 1 Introduction

1

Chapter 1: Introduction

Natural products (NPs) represent the richest source of inspiration for the identification

of novel scaffold structures that can serve as the basis for drugs.[1] In an extensive

review performed by Cragg and Newman[2] of the new drugs introduced between 1981

and 2010, it was estimated that from the 1073 new chemical entities reported (NCEs),

up to 34% were either directly derived from a NP or were inspired by a NP.[2-3] A

further 30% of these entities were synthetic compounds based on a NP

pharmacophore.[2, 4] Thus, from the 1,073 NCEs, 686 (64%) were classified either as an

unmodified NP or synthetic molecule modelled on a NP scaffold.[2] As of 2013, at least

100 NPs and NP-derived compounds were being either evaluated in clinical trials or in

registration.[5] Thirty eight of these compounds were being investigated as potential

oncology treatments, twenty six as anti-infectives, nineteen for the treatment of

cardiovascular and metabolic diseases, eleven for inflammatory and related diseases,

and six for neurological disorders.[5]

NP-based drug discovery is a complex, interdisciplinary pursuit of chemistry,

pharmacology, and clinical sciences.[6] Despite the significant number of NP-derived

drugs being ranked in the top 35 worldwide selling ethical drugs in 2000, 2001, and

2002, the majority of big pharmaceutical industries have terminated or significantly

scaled down their operations.[6-7] This decline was due in part to the costs behind high

rates of rediscovery in the late stages of the isolation process and developments in the

field of combinatorial chemistry and molecular biology.[7b, 8] Drug companies are now

predominantly relying upon the screening of large synthetic compound libraries over

NP libraries to identify novel lead compounds.[5, 8] The screening of NP extract libraries


2

is generally more problematic than synthetic libraries as the former contain complex

mixtures of mostly uncharacterised compounds, some of which have undesirable

properties.[6] An additional complication that may be present in the extracts is the

presence of interfering compounds which may mask their biological effect.[6]

Nevertheless, the major advantage of NPs screening in biological essays is their

inherently large structural diversity which is unsurpassable by any synthetic

compound.[7a, 9] NPs comprise larger numbers of chiral centres and increased steric

complexity than either synthetic drugs or combinatorial libraries.[7a] Furthermore, it has

been shown that 83% of the core ring scaffolds present in NPs were absent from both

commercially available molecules and screening libraries.[10] The unique and vast

chemical diversity of NPs has been optimised through evolutionary selection to bind to

multiple, unrelated classes of protein receptors as high affinity ligands.[11] This means

that these compounds are not only biologically active but also likely to be substrates for

one or more of the many transporter systems that can deliver them to their intracellular

site of action.[12]. Consequently, by including molecules with a NP-product-like scaffold

into a screening library, the number of hit rates can be increased.[10a]

With the emergence of novel high-content phenotypic cell-based screening systems, the

need to rapidly identify effective, novel chemical structures and bioactive lead

molecules is a vital necessity.[2, 13] As only a small fraction of the world’s biodiversity

has been evaluated for biological activity, it can be assumed that NPs will continue to

be a major source of lead molecules for clinical development.[14] Among the established

sources of NPs, microorganisms have proven to be promising candidates for the

production of novel scaffolds as well as marketable drugs.[2, 15]


3

1.1 Microbial resources

1.1.1 Microbial metabolites as pharmacological agents

Microorganisms have yielded some of the most economically relevant leads for the

pharmaceutical industry. These include: antibacterial agents, such as penicillin G (1)

(sourced from the fungus Penicillum); cephalosporin C (2) (sourced from the fungus

Cephalosporium acremonium); tetracycline (3), aminoglycosides, and other polyketides

of many structural types (sourced from different Streptomyces species); cholesterol

lowering agents, such as mevastatin (4) (sourced from the fungus Penicillum

brevicompactum) and lovastatin (5) (sourced from the fungus Aspergillus terreus);

immunosuppressive agents, such as rapamycin (sirolimus) (6) (sourced from the

actinomycete Streptomyces hygroscopicus) and ciclosporin A (7) (sourced from the

fungus Tolypocladium inflatum); as well as anthelmintics and antiparasitic drugs such as

invermectin (8) (sourced from the actinomycete Streptomyces avermintilis).[2, 16]


4

A significant number of chemotherapeutic agents isolated from microbes have been

used to treat bacterial infections and have greatly contributed to the improvement of

human health during the past century.[17] At present, between 32,000-34,000 bioactive

microbial metabolites have been described.[18] However, it has been estimated that only

10% of the total number of small molecules that these microorganisms have the

potential to biosynthesise have been discovered.[18-19]

1.2 Significance of actinomycetes

Actinomycetes are the most widely distributed group of bacteria in nature forming a

large part of the microbial population of soil and aquatic ecosystems such as rivers,


5

lakes, streams, marine environments and salt marshes.[20]. They belong to the Domain

Bacteria, Phylum Actinobacteria, order Actinomycetales and are composed of a mass of

thread resembling branched filaments which frequently give rise to a mycelium.[21] The

most abundant and easy to isolate genus is Streptomyces which is ubiquitous in soil.[22]

The next most common actinomycete genera are in descending order, Micromonospora

(up to 104-105 colony forming units/g of dry soil), Actinoplanes, Actinomadura and

Nocardia.[23]

Actinomycetes are prolific producers of a variety of bioactive secondary metabolites

with diverse chemical structures and biological activities.[18, 24] These small molecules

are biosynthesised during the aerial hyphae formation from the substrate mycelium and

often hold complex structures which result from long enzymatic pathways.[25]

Consequently, it seems that the genes involved in secondary metabolite production may

be subjected to some of the regulatory mechanisms that control aerial mycelium

formation.[26] Under laboratory conditions, the biosynthesis of these metabolites is

believed to be triggered by fermentation-dependent events such as the depletion of

nutrients, the biosynthesis of an inducer or a decrease in growth rate. In response to

these conditions, actinomycetes generate signals which trigger a cascade of regulatory

events resulting in chemical differentiation (secondary metabolism) and morphological

differentiation (morphogenesis).[25-26]

Bull et al., have pointed out that actinomycetes are the richest source of small molecules

(mainly antibiotics) and lead compounds as they have provided approximately 12,000

of all described bioactive metabolites.[18, 27] The genus Streptomyces has been identified

as the largest producer, accounting for approximately 80% of the total amount.[18] The


6

remaining 20% have been isolated from rare actinomycete genera, including

Salinispora, Actinoplanes, Micromonospora, Actinomadura, and Streptoverticillium.[28]

Diverse and unique compounds exhibiting high biological activity and low toxicity have

been identified from rare actinomycetes.[24] The discovery of the aminoglycoside

gentamicin (9) (sourced from the actinomycete Micromonospora purpurea) in 1963, an

antibiotic that inhibits bacterial protein synthesis, greatly increased the interest in rare

actinomycetes.[29] Micromonospora is the second most important producer of bioactive

compounds (more than 740 bioactive secondary metabolites have been described) after

Streptomyces.9 Further commercially relevant antibiotics from rare actinomycetes

include rifamycin SV (10) (sourced from the actinomycete Amycolatopsis

rifamycinica); erythromycin (11) (sourced from the actinomycete Saccharopolyspora

erythrea) and vancomycin (12) (sourced from the actinomycete Amycolatopsis

orientalis).20

https://en.wikipedia.org/wiki/Amycolatopsis_rifamycinica


7

1.3 Mining for novel sources of actinomycete diversity

During the last decades intensive screening programs were carried out worldwide in

order to access to the actinomycete biodiversity.[30] Large numbers of samples from a

wide range of geographical locations and habitats were processed and millions of strains

were isolated and screened in industrial laboratories and research centres.[30] As a

consequence, the rate of discovering commercially relevant bioactive small molecules

from common actinomycete sources has decreased as this practice frequently conducts

to the re-isolation of known compounds.[31] New approaches have been developed to

address the problem of rediscovery of microbial compounds.[30, 31b] One of these


8

strategies involves the isolation, characterisation and screening of novel/rare

actinomycete taxa sourced from unique and underexplored environments.[30] Novel

actinomycete strains producing new structurally diverse bioactive natural products have

been discovered from desert biomes, marine ecosystems, deep-sea sediments and insect-

associated actinomycetes.[32]

Goodfellow and Fiedler[31b] have recently report on the isolation and characterisation of

sediment actinomycetes collected from geographically diverse areas using taxon

specific isolation procedures.[31b] Sediment samples comprising novel actinomycete

genera, including Demequina, Iamia, Marinactinospora, Marisediminicola,

Paraoerskovia, Verrucosispora were collected from the Canary Basin, the Japan

Trench, the Norwegian fjords and the Challenger Deep of the Mariana Trench in the

western Pacific Ocean.[31b]

A series of unique polycyclic polyketide synthase type 1-antibiotics, namely,

abyssomicins B (13) and C (14) and atrop-abyssomicins C (15), D (16), G (17) and H

(18) were isolated from Verrucosispora maris using a combination of a targeted assay

and HPLC-DAD monitoring. Atrop-abyssomicin C (13) exhibited antibiotic activity

against two multi-drug resistance clinical isolates of Staphylococcus aureus (N315 and

Mu50). The minimum inhibitory concentration (MIC) values of atrop-abyssomicin C

against S aureus N315 and S. aureus Mu50 were in the range of 4 μg/mL and 13 μg/mL,

respectively.[31b, 33]


9

Lately, interest has been paid in studying actinomycetes associated to eusocial insects

such as termites, beewolves and beetles, which have hardly been exploited. Examples of

novel small molecules discovered from these sources include microtermolides A (19)

and B (20) isolated from a Streptomyces sp. strain associated with fungus-growing

termites,[32a] sceliphrolactam (21) a previously unreported 26-membered polyene

macrocyclic lactam displaying antifungal activity against amphotericin B-resistant

Candida albicans (MIC= 4 μg/mL),[32b] and mycangimycin (22) a polyene peroxide

with pronounce antifungal activity against the antagonistic ascomycetes, Ophiostoma

minus (MIC= 1.2 μg/mL), Saccharomyces cerevisiae (MIC= 0.4 μg/mL) and Candida

albicans ATCC 10231 (MIC= 0.2 μg/mL), of the pine beetle-associated fungus

Dendroctonus frontalis.[32c, 32d]


10

1.4 Dereplication of bioactive natural products

Dereplication refers to the rapid and competent identification of secondary metabolites

and their quantification in fractionated or unfractionated crude extracts in order to

eliminate from consideration compounds that have already been studied.[32d, 34]

Dereplication approaches can vary, but typically combine chromatographic and

spectroscopic methods with database searching which allow the comparison of known

metabolites with internal and external databases.[35] Some of the most comprehensive

databases include Chapman & Hall’s Dictionary of Natural Products (DNP) containing

over 270,000 natural products,[36] AntiBase with more than 42,950 entries[37] and

MarinLit comprising ~24,000 marine natural products.[38]

A number of authors have proposed different early stage dereplication strategies to

identify novel bioactive small molecules from a variety of microbial strains. Hou et al.,

have developed an untargeted method to support drug discovery efforts and evaluate

rapidly and efficiently marine-derived bacterial natural products using a LC-MS-PCA


11

(principal component analysis) based metabolomic approach.[39] This method was

effective at prioritising strains and significantly increased the efficiency to discover new

natural products compared with traditional LC-MS trace analyses.[39] Similarly, Carr et

al., have reported an early stage dereplication approach to rapidly identify novel

compounds from eusocial insect-associated actinomycetes using HPLC-HRMS based

metabolomics.[32a] This approach relied on careful processing of bacterial extracts

employing PCA of pre-processed samples to promptly identify unique actinomycete

producers from similar ecological niches.[32a] Using this strategy, two new compounds,

namely, microtermolides A (19) and B (20) and more likely produced by hybrid

nonribosomal-polyketide (NRPS-PKS) pathways were identified.[32a]

Although LC-MS-based dereplication approaches offer the major advantage of

detecting the accurate mass of the analytes (usually in the fentomolar-attomolar range)

present in the extracts or fractions. It can also be problematic for some compound

classes, especially if they have molecular masses lower than 300 Da as they may

generate a mix of fragment ion adducts and dimeric and double charged ions hence,

complicating the task of identifying the elemental composition of the desired

compounds.[40] 1H NMR spectroscopy on the other hand is a quantitative, non-selective

and non-destructive technique that allows the rapid, high-throughput and automated

analysis of all molecules containing hydrogen nuclei including compounds that are less

tractable to LC-MS analysis, such as sugars, amines, volatile ketones and relatively non-

reactive compounds.[41]

Over the past ten years, metabolomics-type NMR spectroscopy has been mostly used to

analyse the small molecule composition of tissue or biofluid samples in order to


12

determine changes in the organism’s metabolic status as a consequence of disease,

genetic manipulation or environmental stress.[42] The incorporation of NMR-based

metabolomics to explore the microbial drug-like metabolome in a broader sense than

just dereplicating the known secondary metabolites of complex mixtures or fractions is

not very common in natural products-based literature.[12] Recently, Lang at al.,

described a HPLC-NMR-ESMS/UV based dereplication methodology for the rapid

identification of known compounds from fungal and bacterial extracts.[43] Having access

to 1H NMR data at the initial steps of the dereplication proved to be highly

discriminating for the recognition of a wide range of known compounds, as the

structural information of the small molecules comprising the extracts could be obtained

and interpreted in a relatively short period of time.[43]

In a different study, Grkovic et al., reported a strategy to uncover and reveal unique

spectral patterns of the drug-like natural product metabolome of marine sponges from

the family Poecilosclerida.[44] A small subset of twenty sponges was studied using an

NMR-based metabolomic method focused on the analysis of 1H NMR fingerprints. This

innovative methodology allowed the identification of four new natural products and one

novel compound, named iotrochotazine A which may become a useful tool to

investigate the mechanisms underlying Parkinson’s disease.[44]

1.5 Aims of the thesis

This thesis aims to identify new microbial natural products from actinomycete

symbionts of the Australian wood-feeding termite Coptotermes lacteous (Froggatt).

Fifty actinomycete strains previously isolated from the gut of C. lacteous and held at the

Microbial Library of the University of the Sunshine Coast were selected to perform


13

chemical investigations. The strains were grown in small-scale using four different solid

culture conditions and examined to determine if the variation of media components

could induce the production of new compounds. Culture extracts were fractionated

following an in-house methodology and in order to access to the unique components of

the drug-like natural product metabolome of actinomycetes, a NMR metabolic

fingerprinting approach was established. It was found that the analysis of the 1H NMR

fingerprints from a pre-fractionated library provided well resolved and less complex

NMR spectra with minimum overlapping that allowed spectral comparison between

samples. The NMR-guided metabolic fingerprinting approach enabled a non-targeted

interrogation of the drug-like natural product metabolome and consequently was shown

to simplify and accelerate the identification of new natural products.

Chapter 2: Biological investigations of termite-gut associated actinomycetes

14

Chapter 2: Biological investigations of termite-gut

associated actinomycetes

2.1 Introduction

Actinomycetes are the most widely distributed group of bacteria in nature forming a

large part of the microbial population of soil and aquatic ecosystems such as rivers,

lakes, streams, marine environments and salt marshes.[45] These Gram-positive bacteria

exhibit varied morphologies, physiologies, and metabolic properties that allow them to

degrade and recycle organic materials.[46] Actinomycetes possess large genomes (>8

Mb), contain many biosynthetic gene clusters (i.e., Streptomyces coelicolor and

Streptomyces avermitilis comprise more than 20 biosynthetic genes) and devote over 5%

of their coding capacity to the production of a variety of chemically diverse and

biologically active secondary metabolites.[46-47] Secondary metabolites are small

molecules generally with a low molecular weight (< 2000 atomic mass units) which are

not directly involved in the normal growth, development or reproduction of the

producing organism.[19b]

The discovery of streptothricin (23) (sourced from the actinomycete Streptomyces

lavendulae) in 1942, the first microbial natural product with broad antimicrobial

spectrum, and streptomycin (24) (sourced from the actinomycete Streptomyces griseus)

two years later, triggered the systematic screening of the genus Streptomyces for the

identification of novel antimicrobial compounds.[19b, 48] For the next 17 years, the

discovery of novel antibiotics increased almost exponentially and then continued to rise


15

at a lesser linear rate, reaching its peak in the 1970s. The following 20 years, however,

were marked by a rapid decline in the identification of new antibiotics, reaching the

lowest peak in 1997.[19b]

The re-emergence of multi-drug resistant bacteria and fungi over the last decades has

presented a threat to public health and consequently has made the search for new drug

treatments a priority.[49] According to the World Health Organization (WHO; Geneva),

more than 95% of Staphylococcus aureus strains worldwide are now resistant to

penicillin, and up to 60% are resistant to its derivative, methicillin.[50] The development

of resistance is inevitable following the introduction of a new antibiotic (Figure 1) as

bacteria have evolved a plethora of resistance mechanisms to foil antibiotics, including

vertical and horizontal gene transfer, enzymatic inactivation of the antibiotic, alteration

of antibiotic target to reduce binding, reduced drug uptake into the cell, active efflux of

the drug from the cell, sequestration of antibiotic by protein binding, metabolic bypass

of the inhibited reaction, binding of specific immunity protein to the antibiotic and

overproduction of the antibiotic target.[50-51]


16

Figure 1. Snapshot of the antibiotic pipeline showing the dates when commercial antibiotics were

introduced and antibiotic resistance was first described Högberg et al.[49]

An additional problem is posed by Mycobacterium tuberculosis (Mtb), a contagious

airborne bacterial species and the causative agent of most cases of tuberculosis (TB)

which is showing an increasing trend towards multi-resistant variants.[52] This growing

public health problem underscores an increasingly desperate need to discover the next

generation of antibacterial agents with mechanisms of action radically different from the

existing drugs and therefore capable of combating the spread of multi-drug resistant

pathogens.[50-51]

Hence, here we examined the potential of an actinomycete library isolated from the gut

of the wood-feeding termite Coptotermes lacteus (Froggatt)[53] (Figure 2) to

biosynthesise new natural products. From this collection, fifty strains previously


17

characterised up to family level on the basis of their cultural and morphological

characteristics were selected to perform biological investigations, including

phylogenetic studies and antibacterial susceptibility testing.

Figure 2. Underexplored habitat of novel actinomycete species. A) Shows a C. lacteus nest. B) Members

of C. lacteus collected from a termite nest. C) Close-up view of C. lacteus (courtesy of Ken Harris)

2.2 Results and discussion

For this study, a subset of fifty actinomycete strains formerly isolated from the gut of

the subterranean termite Coptotermes lacteous (Froggatt) using a phage battery and held

at the Microbial Library of the University of the Sunshine Coast were subjected to

chemical and biological investigations.[53] A phylogenetic analysis conducted on the

selected strains using the 16s rDNA gene marker showed that 74.0% of the isolates

belonged to the genus Streptomyces (Figure 3), 8.0% to Micromonospora (Figure 4),

4.0% to Microbispora (Figure 5), 2.0% to Saccharopolyspora (Figure 6) and 12% got

either contaminated or were not successfully amplified using this gene marker. Thus,

these 13 isolates were not contemplated for further analysis. The distribution of rare

actinomycetes was lower than Streptomyces confirming previous reports conducted on

different environments such as tropical rainforest, beehives, desserts, marine sediments

and wasp mud nests.[54]


18

The Streptomyces isolates did not form monophyletic groups but instead they were

distributed across much of the phylogeny of the genus. Most of the isolates were closely

related to species more commonly found in soil and decaying vegetation.[55] This may

be explained either by C. lacteus ability to use soil to build their mounds thus

incorporating soil actinomycetes to their guts or by feeding on surrounding soil and

forage material where actinomycetes are ubiquitous.[53b, 56]


19

Figure 3. Neighbour-joining phylogenetic tree based on partial 16S rDNA sequences showing the relationships

between the strains USC 6922, 6921, 596, 6916, 595, 6901, 6903, 594, 6918, 6909, 6910, 6930, 597, 6923, 593, 6905,

6927, 6907, 6919, 6904, 592, 590, 6929, 6928, 6931, 6911, 6920, 6934, 6926, 6933 with the most closely related type

strains of Streptomyces. Numbers at the nodes indicate bootstrap values based on 1,000 replicates; only values above

50% are shown. Bar 0.05 sequence divergence.


20


relationships between the strains USC 591, 599, 6917 and 6912 with the most closely related type strains

of Micromonospora. Numbers at the nodes indicate bootstrap values based on 1,000 replicates; only

values above 50% are shown. Bar 0.01 sequence divergence


21


relationships between the strains USC 6900 and 6908 with the most closely related type strains of

Microbispora. Numbers at the nodes indicate bootstrap values based on 1,000 replicates; only values

above 50% are shown. Bar 0.01 sequence divergence


22

Figure 6. Phylogenetic placement based on partial 16S rDNA sequences obtained from the termite gut

associate bacterium USC 6906, compared against sequences from public databases. Numbers at the nodes

indicate bootstrap values based on 1,000 replicates; only values above 50% are shown. Bar 0.01 sequence

divergence

The thirty seven isolates were grown in small-scale (four Petri dishes, 100 x 15 mm) for

15 days using four different culture conditions: oatmeal agar (OMA), lupin flour agar

(LFA), rye flour agar (RFA) and glucose yeast extract agar (GYES) and analysed to

determine if the variation of media components could induce the production of new

natural products. Previous studies have shown that the manipulation of nutritional

factors and growth conditions can produce substantial impacts on the quantity and

diversity of the secondary metabolites that are being biosynthesised.[57] The cultures

were visually examined to annotate the differences or similarities in their aerial

mycelium, substrate mycelium and diffusible pigments. The isolates showed differences

in the colours of their aerial and substrate mycelia in the four culture conditions.

However, diffusible pigments were only produced on OMA and GYES media, these

results are summarised in Table 1.


23

Table 1. Colony characteristics on OMA of the selected termite-gut associated actinomycetes

USC

Code AM [a] SM [b] DP [c] IG [d]

USC 590 White Cherry Cherry-red Streptomyces

USC 591 None Brown Light brown Micromonospora

USC 592 Lime Yellow Light yellow Streptomyces

USC 593 Yellow Orange Orange Streptomyces

USC 594 Beige-pink Brown Light-yellow Streptomyces

USC 595 Pink-grey Cherry Cherry Streptomyces

USC 596 Pink white Light orange Light orange Streptomyces

USC 597 Beige-light pink Dark brown Light brown Streptomyces

USC 599 None Black Light black-brown Micromonospora

USC 6900 White Cherry Light brown Microbispora

USC 6901 None Dark reddish Light brown Streptomyces

USC 6903 Purple

Cherry Cherry Streptomyces

USC 6904 Beige brown Cherry Cherry Streptomyces

USC 6905 Yellow Orange Orange Streptomyces

USC 6907 Pink-grey Cherry Cherry Streptomyces

USC 6906 None Brown Brown Saccharopolyspora

USC 6908 White Cherry Light brown Microbispora

USC 6909 Yellow grey Orange Orange Streptomyces

USC 6910 Grey Brown Light Brown Streptomyces

USC 6911 Grey Brown Light brown Streptomyces

USC 6912 Yellow Orange Light brown Micromonospora

USC 6914 Grey Cherry Cherry Streptomyces

USC 6916 Grey

Brown Brown Streptomyces

USC 6917 Grey-brown Red Orange-red Micromonospora

USC 6918 Powdery pink Orange Light orange Streptomyces

USC 6919 Pink Cherry Cherry Streptomyces

USC 6920

Grey with white puffs

Brown Light brown Streptomyces

USC 6921 Chalky-white White Light yellow Streptomyces

USC 6922 Lime white Orange Light orange Streptomyces

USC 6923 Grey Grey Grey Streptomyces

USC 6926 Beige pink Pink-brown Light pink Streptomyces


24

Preliminary screening for antimicrobial activity of the isolates against four multidrug

resistant reference strains obtained from the American Type Culture collection (ATCC)

namely, Escherichia coli (ATCC BAA-196), Kleibsiella pneumoniae (ATCC BAA-

1705), Staphylococcus aureus (ATCC 29247) and Staphylococcus aureus (ATCC

51575) was done using the agar plug method (Figure 7).

Figure 7. Evaluation of the antibiotic activity of the actinomycete isolates in solid cultures. This bar chart

shows the recorded inhibition zones in mm (for some of the actinomycete isolates) obtained after

performing the antimicrobial activity test against four multi-drug resistant reference strains using the agar

plug method

USC 6927

Pink Cherry Cherry Streptomyces

USC 6928 Grey Grey Light grey

Streptomyces

USC 6929 Chalky white-grey

Yellow

Light yellow Streptomyces

USC 6931 Grey Grey Beige Streptomyces

USC 6933 Chalky-pink Pink Light pink Streptomyces

USC 6934 Beige-grey Brown Beige

Streptomyces

[a] Aerial mycelium. [b] Substrate mycelium. [c] Diffusible pigment. [d] Identified genera


25

A total of 37 isolates were tested out of which 10.8% (n = 4) showed activity

exclusively against the Gram-negative bacteria Escherichia coli (ATCC BAA-196) and

Kleibsiella pneumoniae (ATCC BAA-1705); 35.1% (n = 13) displayed activity only

against the Gram-positive bacteria S. aureus (ATCC 29247) and S. aureus (ATCC

51575, 10.8% (n = 4) exhibited antimicrobial activity against both Gram-positive and

negative bacteria, suggesting that antibacterial compounds able to penetrate into the

bacterial cell to exert inhibitory effects are being produced. Similar results have been

reported on different studies conducted on actinomycetes isolated from conventional

and underexplored environments which have shown that actinomycete strains usually

exhibit stronger antimicrobial activity against Gram-positive rather than Gram-negative

bacteria.[58] Sixteen of the actinomycete strains (43.2%) did not show activity against

any of the ATCC multidrug resistant bacteria. Follow up work was carried out only on

the 21 bioactive isolates.

Solid cultures containing the 21 bioactive isolates were cut into small squares,

transferred to a 1L Erlenmeyers and flooded with EtOAc. The EtOAc extracts were

concentrated to dryness in vacuo to afford between 10 to 15 mg for each culture

condition, which were subsequently subjected to NMR metabolic fingerprinting analysis

in order to identify the most promising strains for natural product discovery. The

metabolic fingerprinting approach consisted of the generation, through RP-HPLC, of

five LLE fractions for each of the eighty four crude extracts (21 strains/4 crude extracts:

OMA, LFA, RFA, and GYES) using parameters such as logP < 5 that permitted the

retention of molecules with lead and drug-like properties.[59] Visual examination of the

resulting chromatograms was used as the first step for prioritization and allowed us to

reduce the number of samples to be further analysed. Only those chromatograms

containing constituents within the drug-like region and showing non-redundant


26

retention times were selected to be characterised by LC-MS spectrometry and high-field

NMR spectroscopy where only strains with well resolved NMR fingerprints were

selected in order to enable rapid, NMR-guided isolation follow-up work. . Based on this

analysis, we determined that the metabolic profile of a given strain was medium

dependant as variations in its chemical profile were observed.

Five promising actinomycete strains worth of pursuing solid fermentation in larger

volumes (40-60 Petri dishes) were grown on OMA, GYES or RFA media. Large-scale

NMR-guided isolation of three of these isolates led to the identification of nine new

natural products, including actinoglycosidines A (27) and B (28), actinopolymorphol D

(29) (from Streptomyces sp. USC 592), niveamycins A (36) B (37) and C (38) (from

Streptomyces sp. USC 593), actinofuranosin A (41) and arglecins B (42) and C (43)

(from Streptomyces sp. USC 597), together with six known co-occurring compounds,

namely, BE-54017-derivative 4 (30), BE-54017 (31), 2-amino-6-methoxy9H-

pyrrolo[2,3-d]pyrimidine-7-carbonitrile (32) (from Streptomyces sp. USC 592), WS-

5995 A (39), and B (40), 3H-Pyrrolo[2,3-d]pyrimidine-5-carboxylic acid, 2-amino-4,7-

dihydro-4-oxo-, methyl ester (44) (from Streptomyces sp. USC 593). The two other

strains were found to only biosynthesised the known major metabolites, namely,

phenazine-1-carboxamide (25)[60] (from Microbispora sp. USC 6900) and TMC-66

(26)[61] (from Streptomyces sp. USC 590).


27


28

Table 2. Antitubercular activity with MIC

values for the 17 tested compounds

Compounds tested

M. bovis BCG Pasteur 1173P2

MIC (µg/mL)

25 >100

26 3,12

27 >100

28 >100

29 100

30 100

31 >100

32 >100

36 50

37 100

38 100

39 >100

40 100

41 >100

42 >100

43 >100

44 >100


29

Pure compounds were recovered in small quantities ranging from 0.7 to 2.4 mg. Thus,

they were only tested for their antibacterial activity against one culture of

Staphylococcus aureus (ATCC 29247) and one of Escherichia coli (ATCC BAA-196)

using the agar plug method. No inhibitory activity was detected for any of the tested

compounds at concentrations as high as 100 μg/mL. All the isolated compounds were

also tested for their antitubercular activity against Mycobacterium bovis bacillus

Calmette-Guérin (BCG) Pasteur 1173P2 strain transformed with green fluorescent

protein (GFP) constitutive expression plasmid pUV3583c with direct readout of

fluorescence as a measure of bacterial growth.[62] Table 2 showed the MIC values

obtained for all the tested natural products. Compound 26 showed selective

antitubercular activity against M. bovis BCG 1173P2 with a MIC value of 3,12 µg/mL.

Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592

30

Chapter 3: 1H NMR fingerprints of Streptomyces sp.

USC 592

3.1 Introduction

Natural products (NP) and their derivatives have historically played a vital role in drug

discovery by serving as an invaluable source of therapeutic agents and potential drug

leads.[63] NP structures usually exhibit a wide range of pharmacophores, high degree of

stereochemistry, have advantages over synthetic compounds of being chemically

diverse within biologically relevant ‘chemical space’ and therefore are likely to be

substrates for many of the transporter systems that can deliver the compounds to their

intracellular site of action.[10a, 12, 64] However, due to technological challenges and the

emergence of combinatorial chemistry, NP-based drug discovery has diminished in the

last two decades and has been shifted from Nature to synthetic libraries.[65] In order to

improve natural product research competitiveness, more innovative and productive

strategies are needed to rapidly identify novel lead structures from natural sources.[2, 66]

A strategy to front-load NP extracts with lead-and drug-like molecules to facilitate the

drug discovery process has been recently reported.[59] This approach consisted of the

generation of lead-like enhanced (LLE) fractions containing components with desirable

physicochemical properties.[59] A subset of 18,453 biota samples, sourced from the in-

house Nature Bank biota repository was used to generate a drug-like natural product

library comprising 20,2983 LLE fractions. The filter used to maximize the recovery of

the desired molecules was partition coefficient (log P < 5). This optimised method

facilitated the isolation of NP occupying mid-polarity physicochemical space, an


31

essential property for oral bioavailability and cell permeability.[59] Subsequently, a 1H

NMR metabolic fingerprinting approach was developed to uncover and reveal unique

spectral patterns of the drug-like natural product metabolome of an Australian marine

sponge and allowed the identification of one novel compound, iotrochotazine A which

may become a useful tool to investigate the mechanisms underlying Parkinson’s

disease.[44]

3.2 Chemical studies of termite gut-associated actinomycetes

It has been argued that the likelihood of finding new structurally diverse small-

molecules from underexplored environments such as desert biomes, marine ecosystems,

deep-sea sediments and insect-associated actinomycetes is relatively high as they may

be valuable sources of novel Streptomyces species and other rare actinomycetes with the

capacity to produce complex molecules with a variety of biological activities.[18, 24, 32a-c,

32e, 67] Hence, the usefulness of the 1H NMR metabolic fingerprinting approach for the

discovery of new drug-like natural products in cultures of termite gut-associated

actinomycetes was evaluated. Twenty one actinomycete strains, isolated from the gut of

the wood-feeding termite Coptotermes lacteus (Froggatt),[53] were grown in solid media

(four Petri dishes, 100 x 15 mm) using four different solid culture conditions: OMA,

LFA, RFA and GYES and analysed to determine if the variation of media components

could induce the production of new natural products (Figure 8). The actinomycete

cultures were incubated at 28oC for 15 days, and then the agar containing the cells and

mycelia was cut into small squares and soaked overnight in Ethyl acetate (EtOAc). The

EtOAc extracts were dried under reduced pressure to yield between 10 to 15 mg for


32

each culture condition and were subsequently subjected to metabolic fingerprinting

analysis. Each extract was fractionated onto five lead-like enhanced (LLE) fractions

following published methodology.[59] A data set of 420 LLE fractions was manually

examined for the occurrence of unique chemical profiles (i.e., non-repetitive or unique

NMR resonances and distinctive ESIMS ion peaks). Based on this analysis, five strains

(Streptomyces sp. USC 590, Streptomyces sp. USC 592, Streptomyces sp. USC 593,

Streptomyces sp. USC 597 and Microbispora sp. USC 6900) showing unique

chemotypes were selected to be grown on 40-60 Petri dishes (100 x 15 mm) containing

RFA, OMA, or GYES solid media.

Figure 8. Colony morphologies of Streptomyces sp. USC 592 on four different solid culture conditions

This chapter describes in detail the 1H NMR fingerprint method used to isolate three

new drug-like natural products, namely, actinoglycosidines A (27) and B (28) and

actinopolymorphol D (29) together with three co-occurring compounds, namely, BE-

54017-derivative 4 (30), BE-54017 (31) and 7H-Pyrrolo[2,3-d]pyrimidine-5-carbonitrile,

2-amino-4-methoxy (32) from one of the selected strains, Streptomyces sp. USC 592.


33


The metabolic fingerprinting approach consisted of the generation, through reverse

phase-high performance liquid chromatography (RP-HPLC), of five LLE fractions

(Figure 9) for each of the eighty four crude extracts (21 strains/4 crude extracts: OMA,

LFA, RFA and GYES) using parameters such as log P < 5 that permitted the retention

of molecules with lead and drug-like properties.[1, 59]

The resulting HPLC chromatograms were examined one by one for the presence of

constituents within the drug-like region showing non-redundant retention times.

Performing this analysis before characterising the LLE fractions by high-field NMR

spectroscopy and LC-MS spectrometry allowed us to obtain not only a higher degree of

chemical diversity but also focused on the fractions that were more likely to contain

new microbial natural products. Therefore, the number of LLE fractions selected for


34

further investigations was reduced from 420 to 150 (10 strains/3 solid cultures). Table 3

summarises the chemical profiles of five strains belonging to two different genera,

Streptomyces and Microbispora, which were selected to be grown in larger quantities on

solid media.

For instance, the actinomycete strain, Streptomyces sp. USC 592, mostly showed unique

1H NMR spectral fingerprints in the LLE fractions generated from the GYES crude

extract. The GYES-extract sourced LLE fraction 1 showed unique proton signals (in

MeOD-d4) in the aromatic region at δH 7.61 (s), 7.56 (s) and 1.96 (s); as well as

resonances at δH 5.28 (d, J = 9.6 Hz), 4.07 (s), 4.06 (s), 3.85 (m), 3.69 (m), 3.58 (m),

Table 3 Colony characteristics and chemical profiles of the selected actinomycete strains

Actinomycete

species AM[a] SM[b] DP[c]

RT

(min)[d]

ESIMS [e]

NMR

Fingerprints[f]

Streptomyces sp.

USC 590 White Cherry Cherry red

7.0 529.12 LLE 5. 13.38,

12.53, 7.84, 7.81,

7.76, 7.41, 6.87,

4.72, 4.56, 1.43.

Streptomyces sp.

USC 592 Lime Yellow Light yellow

3.0

5.8

393.14

401.61

LLE 1. 12.25,

7.99, 7.89, 6.80,

5.01, 4.46, 3.98.

LLE 4. 8.54,

4.26, 4.13, 2.62.

Streptomyces sp.

USC 593 Yellow Orange Orange

5.2

5.6

408.15

369.14

LLE 4. 7.66,

7.60, 7.48, 7.55,

7.42, 7.33, 7.25,

7.06, 7.02, 6.87,

6.12, 5.68, 3.73,

3.65, 3.63, 2.39,

2.28.

Streptomyces sp.

USC 597

Beige

light pink

Dark

brown Light brown

2.9

3.1

266.18

252.17

LLE 1. 8.26,

7.90, 7.16, 2.37,

2.11, 2.01, 1.46,

1.00, 0.96.

LLE 2. 8.28,

8.21, 7.24, 5.91,

3.70, 3.58, 1.77,

0.96.

Microbispora sp.

USC 6900 White Cherry Light brown

4.9 225.5 LLE 3. 10.26,

8.93, 8.44, 8.31,

8.09, 8.05.

[a] Aerial mycelium. [b] Substrate mycelium. [c] Diffusible pigment. [d] Retention times. [e] Positive ionization mode [M+H]+. [f] Unusual/interesting resonances in ppm. All samples were acquired in DMSO-d6 at 600 MHz


35

3.41 (m) suggesting the presence of a sugar moiety (Figure 10). Similar proton

resonances as those described in LLE 1 were found on LLE 2, except for the downfield

shift of the sp2-hybridized methine from δH 7.56 (s) to 7.59 (s) and a very low intensity

proton signal at δH 6.01 (d, J = 5.0 Hz) which suggested the presence of an additional

anomeric proton (Figure 11). LC-MS data of both LLE 1 and 2 indicated the presence of

one molecular ion at 393.14 [M+H]+ which after exhaustive searching of the DNP

database did not show any hits containing neither the molecular ion nor the distinctive

NMR resonances.

Figure 9. HPLC chromatogram depicting the drug-like/lead-like region containing the desired

constituents of one of the selected crude extracts. Adapted from Camp et al.[59]

Figure 10. 1H NMR fingerprint spectrum of LLE fraction 1 at 600 MHz in MeOD-d4


36


LLE fraction 4 showed resonances at δH 2.62 (d, J = 4.7 Hz) and 8.54 (q, J = 4.7 Hz),

indicative of the presence of a methyl group attached to a secondary amine (Figure 12).

In addition, this fraction exhibited resonances characteristic of an aromatic ring system

at δH 8.17 (s), 8.00 (d, J = 2.3 Hz), 7.80 (d, J = 8.2 Hz), 7.73 (d, J = 8.7 Hz), 7.59 (d, J =

8.2 Hz), 7.39 (dd, J = 8.7, 2.3 Hz), 7.25 (dd, J = 8.2, 1.1 Hz), 7.12 (dd, J = 8.2, 1.1 Hz).

LC-MS data analysis revealed a quasimolecular ion at 401.61 [M+H]+ which did not

correspond to any of the known compounds reported in the DNP from the genus

Streptomyces.



37

Although the NMR fingerprint of LLE fraction 5 presented background noise that

interfered with the recognition of its spectral patterns, it was possible to identify NMR

resonances at δ 8.49 (s, 1H), 8.11 (d, J = 8.5 Hz) , 7.93 (d, J = 2.0 Hz), 7.70 (d, J = 8.0

Hz), 7.57 (d, J = 8.7 Hz), 7.28-7.14 (m, 6H) indicating that one of the compounds in this

fraction has a similar scaffold to the LLE fraction 4 constituents. Furthermore, NMR

resonances in the aromatic region showed that at least one more molecule was present in

this fraction. This was further supported by LC-MS analysis that showed two molecular

ion peaks at 261.13 [M+H]+ and 452.09 [M+H]+.

Due to the occurrence of interesting NMR fingerprint patterns in the LLE fractions

generated from the GYES extract, the producing strain Streptomyces sp. USC 592 was

grown in 60 Petri dishes (100 x 15 mm) containing GYES medium. The plates were

incubated for 15 days at 28ºC and then extracted overnight with EtOAc to yield 190.0

mg of the crude extract. A portion of the crude extract (43.0 mg) was separated on a

reversed-phase C18 HPLC column, 60 fractions were collected and analysed by 1H

NMR spectroscopy. NMR-guided isolation led to the identification of 3 new natural

products, namely, actinoglycosidines A (27) and B (28) and actinopolymorphol D (29)

together with 3 co-occurring compounds, namely, BE-54017-derivative 4 (30), BE-

54017 (31) and 2-amino-6-methoxy-9H-pyrrolo[2,3-d]pyrimidine-7-carbonitrile (32).

Actinoglycosidine A (27) was isolated as a stable amorphous solid. The HRESIMS of

27 contained the protonated molecular peak at 393.1513 [M+H]+ consistent with a

molecular formula of C16H21N6O6 (calcd. for C16H21N6O6, 393.1414) requiring 10

double-bond equivalents (DBEs). The 1H NMR spectrum of 27 (Table 4) in MeOD-d4

displayed ten resonances attributable to two sp3-hybridized methyls at δH 4.06 (3H, s,


38

H-7), 1.96 (3H, s, H-9'), seven sp3-hybridized methines at δH 5.29 (1H, d, J = 10.0 Hz,

H-1'), 3.88 (1H, dd, J = 10.0, 3.3 Hz, H-2'), 3.56 (1H, ddd, 10.0, 8.5, 3.3 Hz, H-3'), 3.40

(1H, m, H-4'), 3.41 (1H, m, H-5'), 3.85 (1H, dd, 12.0, 2.8 Hz, H-6a'), 3.70 (H, dt, J =

12.0, 5.1 Hz, H-6b') and one sp2-hybridized methine at δH 7.58 (1H, s, H-9).

The 13C NMR spectrum of 27 (Table 4) exhibited 16 resonances comprised of two sp3-

hybridized methyls at δC 54.3 (C-7), 22.8 (C-9'), six sp3-hybridized methines at δC 83.7

(C-1'), 56.2 (C-2'), 76.6 (C-3'), 72.3 (C-4'), 79.4 (C-5'), and 62.9 (C-6'), one sp2-

Table 4. 1H NMR and 13C NMR spectroscopic data for actinoglycosidines A (27) and B (28) in MeOD-

d4.

27[a] 28[a]

No δC [ppm] δH [ppm]

(J in Hz) HMBC δC [ppm]

δH [ppm]

(J in Hz) HMBC

2 160.5 160.3

3

4 155.4 155.0

5 98.7 98.8

6 164.7 164.7

7 54.3 4.06 (s) C-6 54.6 4.08 (s) C-6

8 84.8 84.3

9 131.2 7.58 (s) C-4, C-5, C-8,

C-10 131.7 7.60 (s)

C-4, C-5, C-8,

C-10

10 116.5 116.6

1' 83.7 5.29 (d, 10.0)

C-2, C-2´, C-3´, C-5´

78.7 6.02 (d, 5.1) C-2, C-2´, C-3´

2' 56.2 3.88

(dd, 10.0, 3.3) C-1´, C-3´, C-8´ 54.4

4.11

(dd, 11.2, 5.1) C-1´, C-3´, C-8´

3' 76.6

3.56

(ddd, 10.0, 8.5, 3.3)

C-2´, C-4´ 73.8

3.62

(ddd, 9.6, 5.1, 2.6)

C-6´

4' 72.3 3.40 (m)[b] C-3´, C-5´, C-6´ 71.9 3.79

(dd, 8.8, 6.7) C-2´, C-3´, C-5´

5' 79.4 3.41 (m)[b] C-3´, C-4´, C-6´ 72.1 3.44

(dd, 9.6, 8.8) C-3´, C-4´, C-6´

6' 62.9 3.85

(dd, 12.0, 2.8) C-4´, C-5´ 62.3

3.76

(dd, 9.6, 2.6) C-2´, C-3´

3.70

(dt, 12.0, 5.1)

3.72

(dd, 12.0, 5.1)

7'

8' 174.6 174.3

9' 22.8 1.96 (s) C-8´ 22.9 1.93 (s) C-8´

[a] Proton and carbon resonances were acquired at 600 MHz and 150 MHz, respectively. [b] Coupling constants of these resonances are unclear due to overlapping


39

hybridized methine at δC 131.2 (C-9), and seven quaternary resonances including one

carbonyl at δC 174.6 (C-8), one oxygen-bearing aromatic carbon δC 164.7 (C-6), four

olefinic carbons at δC 160.5 (C-2), 155.4 (C-4), 98.7 (C-5), and 84.8 (C-8), and one

unsaturated carbon at δC 116.5 (C-10) suggesting that either an acetylene or a nitrile

were attached to it.[68] Interpretation of 1D and 2D NMR data allowed for the

identification of 2 partial structures, fragments A and B depicted in Figure 13.

Fragment A, showed characteristic resonances of an N-acetylglucosamine moiety, with

the configuration of the anomeric proton assigned to be β based on a trans diaxial

relationship of H-1' and H-2' coupling constant at δH 5.29 (1H, d, J = 10.2 Hz, H-1').

The absolute configuration of the sugar was determined to be D via hydrolysis (Figure

14) and subsequent comparison of its optical rotation value with the commercial N-β-D-

acetylglucosamine (Sigma-Aldrich). The structural assignment of fragment B was

challenging due to the absence of proton resonances and a large number of heteroatoms.

The only proton peak observed was a singlet at δH 7.58 (H-9) showing HMBC

correlations to the quaternary carbons at δC 155.4 (C-4), 98.7 (C-5), 84.8 (C-8), 116.5

(C-10). These correlations were consistent with the presence of a purine residue which

was further confirmed by NMR data comparison with related natural products

containing the same residue such as dapiramicins A and B.[69] Crucial HMBC

correlations from the anomeric proton (H-1') to the quaternary carbon (C-2) indicated

that fragment A and B are connected through a nitrogen atom. Actinoglycosidine A (27)

was therefore concluded to be N-β-D-acetylglucosamine 2-amino-6-methoxy-9H-

pyrrolo[2,3- d]pyrimidine-7-carbonitrile.


40

Figure 13. Fragments found during the elucidation process and crucial HMBC and NOESY correlations

for actinoglycosidines A (27) and B (28)

Figure 14. Acid Hydrolysis of actinoglycosidine A (27)

The molecular formula of actinoglycosidine B (28) was established to be C16H21N6O6 by

HRESIMS at m/z 393.1513 [M+H+], isomeric to the natural product 28. The 1H NMR


41

and 13C NMR spectra of 28 (Table 4) resembled to those of 27 and in particular the

spectral data attributable to the purine residue which were almost superimposable

(Figures 15 and 16). The most significant NMR spectral differences were observed in

the sugar moiety, especially the resonances of the anomeric carbon C-1' and C-5' had

shifted upfield to δC 78.7 and 72.1, respectively. The stereochemistry of the anomeric

proton of the sugar residue were consistent with the presence of an α sugar, based on the

magnitude of the 1H-1H coupling constant [δH 6.02 (1H, d, J = 5.0 Hz, H-1')]. This was

further confirmed by strong NOESY correlations between H-1´ and H-2´ which were

indicative of the presence of the α anomer.

Figure 15. 1H NMR spectra comparison between compounds 27 and 28


42

Figure 16. 13C NMR spectra comparison between compounds 27 and 28

Figure 17. 1H NMR fingerprint spectra. Top spectrum depicts characteristic NMR resonances of a sugar

moiety. The spectrum at the bottom shows the pure natural product, actinoglycosidine B (28) which was

identified by large-scale NMR-guided isolation


43

Based on the co-occurrence of actinoglycosidines A (27) and B (28) in the same

organism and in agreement with the NMR spectral data, optical rotation and ECD

values, the structure of 28 was determined to be N-α-D-acetylglucosamine 2-amino-6-

methoxy-9H-pyrrolo[2,3-d]pyrimidine-7-carbonitrile (Figure 17).

Actinopolymorphol D (29) was isolated as a stable amorphous solid and gave a

molecular formula of C18H16N2 deduced from the HRESIMS peak at m/z 261.1380

[M+H+] (calcd. for C18H16N2, 261.1313), indicating that this molecule required 12

degrees of unsaturation. The 1H NMR spectrum of 29 (Table 5) displayed eight

resonances attributable to two sp3-hybridized methylenes at δH 4.12 (2H, s, H-7) and six

sp2-hybridized methines at δH 7.31 (2H, dd, J = 7.9, 5.6 Hz, H-2), 7.19 (2H, dd, J = 7.3,

5.6 Hz, H-3), 7.28 (1H, dd, J = 7.9, 5.6 Hz, H-4), 8.46 (1H, s, H-9). The 13C NMR

spectrum of 29 (Table 5) exhibited 9 resonances comprised of one sp3-hybridized

methylene at δC 41.8 (C-7), six sp2-hybridized methines at δC 129.8 (C-2), 127.2 (C-3),

129.4 (C-4), 127.2 (C-5), 129.8 (C-6), 144.4 (C-9), and two quaternary resonances at δC

140.1 (C-1) and 154.8 (C-8). From the molecular formula of 29 it was observed that the

remaining atoms required by the molecular formula C18H16N2 were C9H8N2 and

consequently six additional degrees of unsaturation were needed to be established.

On the basis of the analysis of the proton and carbon resonances, the structure of

actinopolymorphol D (29) was concluded to be a symmetric dimer. Moreover,

interpretation of the proton resonances in the aromatic region allowed for the

identification of two monosubstitued benzene rings. Characteristic proton and carbon

resonances at δH 8.46 (H-9) and δC 144.4 (C-9) indicated the presence of a pyrazine

molecule substituted at C-8 and C-8'.[70] COSY correlations observed between the


44

methylene at H-7 to the olefinic protons at H-2 and H-9 suggested that the two parts of

the molecule were connected through a sp3-hybridized methylene.

This was further confirmed by HMBC correlations from H-7 to the quaternary carbons

at C-1 and C-8 and to the olefinic carbons at C-2 and C-9 (Figure 18). Thus, the

structure of 29 was assigned to be 2,5-dibenzyl pyrazine. While the synthesis of 29 has

been reported elsewhere,[71] this is the first report of the structure as a naturally-

occurring 2,5-dibenzyl pyrazine.

Figure 18. Crucial COSY and HMBC correlations for actinopolymorphol D (29)

Large-scale NMR-guided isolation accelerated the identification of compound 30

(Figure 19). After extensive interpretation of 1H and 13C and 2D NMR spectroscopic

Table 5. 1H NMR and 13C NMR spectroscopic data for actinopolymorphol

D (29) in Acetone-d5

29[a]

No δC [ppm] δH [ppm] (J in Hz) HMBC

1; 1' 140.1

2; 2' 129.8 7.31 (dd, 7.9, 5.6) C-3, C-4, C-7

3; 3 127.2 7.19 (dd, 7.3, 5.6) C-2

4; 4' 129.4 7.28 (dd, 7.9, 5.6) C-1

5; 5' 127.2 7.19 (dd, 7.3, 5.6) C-2

6; 6' 129.8 7.31 (dd, 7.9, 5.6) C-3, C-4, C-7

7; 7' 41.8 4.12 (s) C-1, C-2, C-8, C-9

8; 8' 154.8

9; 9' 144.4 8.46 (s)

[a] Proton and carbon resonances were acquired at 600 MHz and 150 MHz, respectively


45

data of 30, it was concluded that the spectral data of this compound was in accordance

with the described literature values for the known natural product BE-54017-derivative

4.[72] The BE-54017 and its derivatives are bis-indole alkaloids closely related to

cladoniamides and are characterised by having an unusual indenotryptoline structure

rarely observed among bis-indole alkaloids.[72b]

Figure 19. 1H NMR fingerprint spectra. Top spectrum exhibits the distinctive proton resonances of LLE

fraction 4. The bottom spectrum displays the proton NMR resonances of compound 30, after comparison

of its spectral values with those of the literature led to the rapid identification of 30 as the known natural

product BE-54017-derivative 4


46

Similarly, compounds 31 and 32 were identified upon comparison of their 1H NMR

spectroscopic and LC-MS spectrometric data with those of the known natural products

BE-54017[72] and 2-amino-6-methoxy-9H-pyrrolo[2,3-d]pyrimidine-7carbonitrile,[69]

respectively.


47

Chapter 4: 1H NMR Fingerprinting of Streptomyces sp.

USC 593

4.1 Introduction

Over the last 80 years several thousand microbial natural products displaying a

remarkable and diverse array of biological activities have been isolated.[24a, 73] Many of

these molecules were discovered from actinomycetes, including some clinically useful

antibiotics with unique mechanism of action, such as daptomycin (33), an antibiotic that

kills Gram-positive bacteria by disrupting multiple aspects of the bacterial membrane.[74]

Actinomycetes remain to be one of the most prolific sources of bioactive compounds, it

has been estimated that at present, only 10% of the total number of small molecules that

these microorganisms can biosynthesised have been found.[18, 19b] However, discovering


48

novel natural products from traditional actinomycete sources has become more difficult

as this practice generally leads to the rediscovery of known secondary metabolites.[75]

Thus, new approaches have been proposed to identify promising untapped chemical

scaffolds. One of which is the exploration of unique environments such as insect-

associated actinomycetes which may represent a particularly promising source of new

structurally diverse natural products.[32a-c, 32e, 76] Recently, this was exemplified by the

discovery of novel compounds, including microtermolides A (19) and B (20) from a

termite-associated Streptomyces sp.,[32a] one sceliphrolactam (21) and one

mycangimycin (22) (both with pronounced antifungal activity) from a wasp-associated

Streptomyces sp. and a pine beetle-associated Streptomyces sp., respectively.[32b, 32c, 76]

The implementation of effective dereplication strategies is essential to increase the

number of new/novel natural products that can be identified.[39] Dereplication

approaches can vary but usually utilize liquid chromatography combined with UV and

mass spectrometry techniques for the identification of lead compounds from crude

extracts. Bioactivity-directed fractionation methodologies have been largely used for the

isolation of promising drug candidates such as platensimycin (34) and platencin (35).[77]


49

However, it is often criticized for resulting in the reisolation and recharacterization of

known compounds.[40c] Hence, for this study we evaluated the usefulness of the NMR-

guided metabolic fingerprinting approach to uncover new drug-like natural products in

solid cultures of a termite-associated Streptomyces sp. USC 593.[53] The producing

strain was grown in small-scale (four Petri dishes, 100 x 15 mm) in OMA, LFA, RFA

and GYES (Figure 20). Cultures containing the cells and mycelia were cut in small

squares and soaked into EtOAc overnight, the EtOAc extracts were used to dereplicate

the Streptomyces sp. USC 593 metabolome.

Figure 20. Colony morphologies of Streptomyces sp. USC 593 in four different solid culture conditions


The metabolic fingerprinting approach comprised the generation of LLE fractions using

parameters such as log P > 5 that allowed the retention of molecules with lead and drug-

like properties.[59] Five LLE fractions were collected for each extract and subsequently

analysed by high-field NMR spectroscopy and LC-MS spectrometry. Unique 1H NMR

spectral fingerprints were observed only in the LLE fractions generated from the GYES

crude extract. LLE fraction 4 showed distinctive proton resonances at δH 6.16 (s) and

5.64 (s) which suggested the presence of an exocyclic methylene (Figure 21). In


50

addition, this fraction displayed resonances characteristics of fused aromatic rings at δH

7.66 (dd, J = 8.2, 7.6 Hz), 7.60 (d, J = 7.2 Hz), 7.55, (dd, J = 8.2, 7.6 Hz), 7.48 (d, J =

7.2 Hz), 7.26 (d, J = 8.2 Hz) and 7.06 (d, J = 8.2 Hz), and trisubstituted benzene rings at

δH 7.42 (s), 7.33 (s), 7.02 (s) and 6.87 (s). Likewise, LLE fractions 3 (Figure 22) and 5

(Figure 23) revealed NMR resonances at δH 7.68 (dd, J = 8.2, 7.6 Hz), 7.58 (dd, J = 8.2,

7.6 Hz), 7.21 (dd, J = 8.2, 1.0 Hz), 7.50 (d, J = 0.8 Hz), 7.12 (s), and at δH 7.69 (dd, J =

7.6, 8.2 Hz), 7.61 (dd, J = 8.2, 7.6 Hz), 7.29 (dd, J = 8.2, 1.0 Hz), 7.55 (s), 7.12 (s),

respectively, thus indicating the occurrence of other analogues.




51


LC-MS data of LLE fractions 4 and 5 consisted of molecular ion peaks at 407.113,

369.08 [M+H]+ and 410.12 [M+H]+, respectively, which did not correspond to any of

the known compounds reported on the DNP from the genus Streptomyces.

Herein, Streptomyces sp. USC 593 was grown in 60 Petri dishes (100 x 15 mm)

containing GYES medium. The plates were incubated for 15 days at 28ºC and then

extracted overnight with EtOAc to yield 140.0 mg of the crude extract. A portion of the

crude extract (~40.0 mg) was separated on a reversed-phase C18 HPLC column, 60

fractions were collected and subsequently analysed by 1H NMR spectroscopy. NMR-

guided isolation led to identification of three new 5-hydroxy-1,4-naphthoquinones

natural products, namely, niveamycins A (36), B (37) and C (38), together with two co-

occurring known compounds, namely, WS-5995 A (39) and B (40).


52

Niveamycin A (36) was isolated as a yellow amorphous solid. The molecular formula of

36 was established as C23H19O7 by HRESIMS at m/z 407.1115 [M+H]+ (calcd. for

C23H19O7, 407.1053). The 1H NMR spectrum of 36 (Table 6) in MeOD-d4 displayed 10

resonances attributable to three sp3-hybridized methyls at δH 2.27 (3H, s, H-12), 3.68

(3H, s, H-7'), 2.42 (3H, s, H-8'), five sp2-hybridized methines at δH 7.30 (1H, dd, J = 8.3,

1.1 Hz, H-6), 7.69 (1H, dd, J = 8.3, 7.6 Hz, H-7), 7.61 (1H, dd, J = 7.6, 1.1 Hz, H-8),

7.04 (1H, s, H-3'), 7.48 (1H, s, H-5'), and one sp2-hybridized methylene at δH 6.16 (1H,

s, H-10a), 5.64 (1H, s, H-10b). The 13C NMR spectrum of 36 exhibited 23 resonances

(Table 6) comprised of three sp3-hybridized methyls at δC 26.4 (C-12), 56.0 (C-7'), and

21.6 (C-8'), five sp2-hybridized methines at δC 124.7 (C-6), 137.4 (C-7), 120.0 (C-8),

116.3 (C-3'), and 123.9 (H-5'), one sp2-hybridized methylene at δC 130.1 (C-10), and

fourteen quaternary resonances including four carbonyls at δC 184.5 (C-1), 190.4 (C-4),

199.3 (C-11) and 170.9 (C-9'), two oxygen-bearing aromatic carbons δC 162.5 (C-5),


53

157.4 (C-2'), eight olefinic carbons at δC 149.5 (C-2), 143.4 (C-3), 116.1 (C-4a), 134.1

(C-8a), 144.5 (C-9), 122.1 (C-1'), 141.5 (C-4'), 128.5 (C-6').

Detailed analysis of the COSY and HMBC spectra allowed the identification of three

partial fragments, A, B and C (Figure 24). In fragment A, COSY correlations between

the olefinic protons at H-6, H-7 and H-8 to each other indicated the presence of an ortho,

meta-substituted aromatic spin system. Moreover, critical HMBC correlations from the

aromatic protons at H-6 to C-4a, C-5, C-8; H-7 to C-4a, C-5, C-8a, and H-8 to C-1, C-

4a, C-5, C-6 were consistent with the presence of a hydroxyl naphthoquinone moiety. In

fragment B, COSY correlations from H-8' to the singlets at δH 7.48 (H-5') and 7.04 (H-

3') suggested that a methyl group is attached to the olefinic carbon at δC 141.5 (C-4').

This was further confirmed by HMBC correlations from H-8' to C-3', C-4' and C-5'. The

position of the methoxy substituent was established based on HMBC correlations from

H-7' to the carbon at δC 157.4 (C-2') and ROESY correlations from the singlet at H-3' to

H-7'.

HMBC correlations from the aromatic proton at H-5' to C-6' and C-9' indicated that a

carboxylic acid was attached to C-6'. The methylene pair at δH 6.16 and 5.64 from

fragment C showed HMBC correlations to the quaternary carbons at C-3 (fragment A),

C-9 and C-11. Furthermore, HMBC correlations from H-12 to C-11, C-10, and C-9

indicated the presence of a terminal acetyl group. These correlations clearly indicated

that fragment A substituent is connected to C-3. These correlations clearly indicated that

fragment A substituent is connected to C-3. Based on the degrees of unsaturation

calculated from the molecular formula (C23H19O7) and comparison of the spectroscopic

data of 36 with those of the known natural products WS 5995 A, B, and C,[78] fragments


54

A-C were connected to complete the structure of 36 as 2-(5-hydroxy-1,4-dioxo-3-(3-

oxobut-1-en-2-yl)-1,4-dihydronapththalen-2-yl)-3-methoxy-5-methyl benzoic acid.

Figure 24. Fragments found during the elucidation process and crucial HMBC correlations for the new

natural product, niveamycin A (36)

Table 6. 1H NMR and 13C NMR spectroscopic data for niveamycins A (36) and B (37) in MeOD-d4

36[a] 37[a]

No δC

[ppm]

δH [ppm]

(J in Hz) HMBC

δC

[ppm]

δH [ppm]

(J in Hz) HMBC

1 184.5 184.4

2 149.5 150.7

3 143.4 145.6

4 190.4 191.0

4a 116.1 116.2

5 162.5 162.4

6 124.7 7.30 (dd, 8.3, 1.1 ) C-4a, C-5, C-8 124.7 7.30 (dd, 8.3, 1.0) C-4a, C-5, C-6, C-8

7 137.4 7.69 (dd, 8.3, 7.6) C-4a, C-5, C-8a 137.5 7.70 (dd, 8.3, 7.6) C-4a, C-5, C-8a

8 120.0 7.61 (dd, 7.6, 1.1) C-1, C-4a, C-5, C-6

120.0 7.61 (dd, 7.6, 0.9) C-1, C-3, C-4a, C6, C-7

8a 134.1 133.8

9 144.5 49.9 3.03 (q, 6.8) C-2, C-3, C-4, C-10, C-11

10 130.1 6.16 (s) C-3, C-9, C-11 14.3 1.27 (d, 6.8) C-3, C-9, C-11

5.64 (s) C-3, C-9, C-11

11 199.3 208.0

12 26.4 2.27(s) C-9, C-11 28.5 1.99 (s) C-11

1' 122.1 121.1


55

2' 157.4 157.9

3' 116.3 7.04 (s) C-2', C-5’, C-8' 116.3 7.15 (s) C-1', C-2', C-4', C-5', C-8'

4' 141.5 142.0

5' 123.9 7.48 (s) C-1', C-3', C-6', C-8', C-9'

124.3 7.58 (s) C-1', C-3', C-5', C-8', C-9'

6' 128.5 [b]

7' 56.0 3.68 (s) C-2' 56.2 3.78 (s) C-2'

8' 21.6 2.42 (s) C-3', C-4', C-5' 21.6 2.47 (s) C-3', C-4', C-5'

9' 170.9 169.8

[a] Proton and carbon resonances were acquired at 600 MHz and 150 MHz, respectively. [b] Signal not observed

Figure 25. 1H NMR spectra comparison between compounds 36 and 37

Figure 26. 13C NMR spectra comparison between compounds 36 and 37


56

Niveamycin B (37) was isolated as a yellow amorphous solid, the molecular formula of

37 was established as C23H21O7 by HRESIMS measurements at m/z 409.1279 [M+H]+

(calcd. for C23H21O7, 408.1209), indicating the presence of two additional protons

compared to 37. The 1H and 13C NMR spectroscopic data for 36 were superimposable

with those of 37 (Figures 25 and 26), except that a 3-methylbut-3-en-2-one group of 36

at C-9 was replaced by a 3-methylbutan-2-one group in 37. Consequently, the C-11

resonance was observed to shift downfield from δC 199.3 to δC 203.1. The planar

structure of 37 (Figure 27) was established on the basis of the 1H-1H COSY, HSQC, and

HMBC spectral analysis. The absolute configuration at C-9 was established using ECD

calculations (see Section 3.4).

Figure 27. 1H NMR fingerprint spectra. Top spectrum depicts characteristic NMR resonances of a fused

aromatic ring. The spectrum at the bottom shows the pure natural product, niveamycin B (37), identified

by large-scale NMR-guided isolation


57

Niveamycin C (38) was isolated as a yellow amorphous solid. The molecular formula of

38 was established as C20H17O7 from the HRESIMS at m/z 368.1509 [M+H]+ (calcd. for

C20H17O7, 369.0896), indicating the absence of three carbons and two protons compared

to 36 (Table 7). Comparison of 1D and 2D NMR spectroscopic data of 36 with those of

38 revealed that these compounds were virtually identical, except that the 3-methylbut-

3-en-2-one group at C-3 of 36 was substituted by a methoxy group in 38. As a

consequence, the oxygen-bearing aromatic carbon resonance has significantly shifted

downfield to δC 168.0. This side chain substitution was supported by a HMBC

Table 7. 1H NMR and 13C NMR spectroscopic data for niveamycin C (38) in MeOD-d4

38[a]

Position δC [ppm] δH [ppm] (J in Hz) HMBC

1 182.7

2 [b]

3 168.0

4 [b]

4a 114.1

5 162.5

6 122.8 7.23 (dd, 8.3, 1.0) C-4a, C-8

7 137.6 7.67 (dd, 8.3, 7.6) C-5, C-8a

8 119.2 7.56 (dd, 7.6, 1.0) C-1, C-4a, C-6, C-8a

8a 134.0

9 52.0 3.68 (s) C-3

11

12

1' 121.9

2' 157.7

3' 116.6 7.11 (s) C-2', C-5', C-8'

4' 139.1

5' 123.3 7.43 (s) C-1', C-3', C-6', C-8', C-9'

6' 128.3

7' 56.2 3.76 (s) C-2'

8' 21.2 2.44 (s) C-3', C-4', C-5'

9' 167.8

[a] Proton and carbon resonances were acquired at 600 MHz and 150 MHz, respectively. [b] Signal not observed


58

correlation from the methyl at δH 3.68 (H-8) to C-3 (Figure 28). Hence, the structure of

Niveamycin C (38) was established as 2-(5-hydroxy-3-methoxy-1,4-dioxo-1,4-

dihydronaphthalen -2-yl)-3-methoxy-5-methylbenzoic acid (Figure 22).

Figure 28. Crucial HMBC correlations for niveamycins B (37) and C (38)

Figure 29. Top spectrum depicts characteristic NMR resonances of fused aromatic rings and a tri-

substituted benzene ring. The spectrum in the middle shows the proton NMR of the new natural product,

niveamycin A (36). The bottom spectrum displays the new natural product, niveamycin C (38)


59

Based upon comparison of the 1H NMR and LC-MS spectral data of the co-occurring

compounds 39 and 40 with those reported on the literature, it was determined that

compound 39 corresponded to WS 5995 C and 40 (Figure 30) to WS 5995 A.[78]

Figure 30. Top spectrum depicts the 1H NMR fingerprint of LLE fraction 3. The bottom spectrum

displays the proton spectrum of the known compound named WS 5995 A (39)

4.3 Computation of NMR chemical shifts

The use of quantum chemical methods (QM) for predicting proton and carbon NMR

chemical shifts and determining the relative configuration of organic compounds, has

now evolved to the point where compounds of considerable (and ever-increasing)

complexity and size are amenable for study.[79] Over time, density functional theory

(DFT) has emerged as a successful method to elucidate important properties concerning

structure identification, confirmation and stereochemical reassignment of a number of

natural products.[79b, 80]


60

Although both 1H and 13C calculated chemical shifts have demonstrated to be useful for

the assessment of the relative configuration of unknown molecules.[79a] Calculated

carbon chemical shifts has shown to be more accurate to predict structural differences or

similarities for most of the compounds that have been analysed.[81] However, some

sources of magnetic inequivalence might have a more obvious effect on 1H NMR rather

than on 13C NMR spectra.[80] For instance, differences induced by the magnetic

anisotropy characteristic of aromatic systems, can produce an upfield effect in protons

which are in the vicinity of the face of a benzene ring.[82] As the experienced upfield

effect is in the same order of magnitude than that of the carbon atom, it will have a more

noticeable effect in 1H NMR rather than in 13C NMR spectra.[82]

Several methods can be used to compare the experimental and computed chemical shifts

of a candidate structure to determine the goodness of fit.[79, 83] It can be express in

different ways such as by the correlation coefficient R, the mean absolute error (MAE),

the corrected absolute error (CMAE), the root mean square deviation (RMSD) and other

methods.[79a] There is no universally accepted best practice for performing the

evaluation of the goodness of fit. Nonetheless, comparison of the MAE and CMAE are

the most commonly used criteria.[79b]

In the present work, we accounted on the use of 1H and 13C NMR chemical shifts

predictions for the determination of the correct structures for the niveamycins. We were

especially interested in confirming the position of the hydroxyl group at C-5 (fragment

A) and the position of the covalent bond that links fragment A and B at C-2 and C-1',

respectively. Calculations were performed according to the protocol described by

Willoughby et al., which consisted of five operations:[79b]


61

Conformational search

Geometry optimisation and frequency calculation

NMR shielding tensor calculations and conversion to chemical shift values

Boltzmann-weighting of shielding tensors and conversion to chemical shifts

Comparison of experimental and computed chemical shifts and assessment of

goodness of fit

4.3.1. Conformational search

To calculate the theoretical conformational analysis of the niveamycins, the

corresponding isomeric structures, 36–I and 36–II (niveamycin A), 37–I and 37–II

(niveamycin B) and 38–I and 38–II (niveamycin C) (Figure 31) were subjected to

molecular mechanics energy minimization and subsequent conformational search using

Monte Carlo molecular mechanics (MMCM) as implemented in MacroModel 9.9.[84],

the optimised potential for liquid stimulations was calculated by OPLS 2005. The value

of energy window for saving new structures was 5 kcal mol-1 with a maximum number

of steps of 30,000 and 1,000 steps per rotatable bond.[79b, 85]


62

Figure 31. Structural isomers of niveamycins A–C

4.3.2. Geometry optimisation and frequency calculation

Each minimum energy conformer was further optimised using DFT calculations,

(comprising geometry optimisation and frequency calculation). Geometry optimisation

was carried out in methanol solvation by using the default parameters implemented in

Gaussian 09 at the M06-2X functional with the 6-31+G(d,p) level of theory as it

provides more accurate geometries and energies.[86] Frequency calculations allow for

structure validation by ensuring that each optimised geometry is not a local saddle point

on the potential energy diagram, which, if present is indicated by the presence of a

negative (or imaginary) frequency.[79b]

4.3.3. NMR shielding tensor calculations and conversion to chemical shift values

These calculations were performed using the B3LYP functional with the 6-311+G(2d,p)

level of theory. The resulting set of tensor values were converted to chemical shifts by


63

applying scaling (slope) and referencing (intercept) factors (Table 8) which are derived

from linear regression analysis of a test set of molecules[81] to each of the computed

tensor values. This has the effect of reducing some of the systematic error inherent in

the theory used for the computation.[79b]

4.3.4. Boltzman analysis of DFT NMR data

Energetic data resulting from geometry optimisation and NMR shielding tensor

calculations were manipulated by the use of the following script.[79b]

nmr-data_compilation.py

Boltzmann weighting factors were calculated for each conformer at 25°C by using the

relative free energies obtained from the frequency calculations. The resulting weighting

factors were applied to the computed NMR shielding tensors for each nucleus of each

individual conformer. Summation of the weighted tensors across all conformers gave

the Boltzmann-weighted average NMR shielding tensors for the candidate structure.[79b]

Table 8. Scaling factors used for

DFT calculations

Slope Intercept

1H -1.0767 31.9477 13C -1.0522 181.2412


64

4.3.5. Comparison of experimental and computed chemical shifts

The MAE and CMAE methods were used to determine the goodness of fit of the

niveamycins. The experimental and calculated proton and carbon chemical shifts for the

niveamycins are showed in Tables 9 and 10, respectively. Based on this analysis, we

concluded that the correct configuration of niveamycins A–C (36–38) was given by the

structural isomers 36–I, 37–I and 38–I. Furthermore, in order to determine the

efficiency of the 1H and 13C nuclei to discriminate between the right and wrong

diastereomers, the match ratio between the CMAE for the right and wrong match was

calculated.[79a] The computed average match ratio for 1H and 13C data was 1.3 and 0.9,

respectively, and as larger match ratio values are indicative of a better ability to predict

the right structural isomer, we determined that 1H nucleus is more discriminating than

13C for performing stereochemical assignments by quantum chemical calculations.


65

Table 9. Experimental and calculated 1H NMR data for niveamycins A–C[a]

δC experimental [ppm] δC calculated [ppm]

Isomer 1

δC calculated [ppm]

Isomer 2

No 36 37 38 36–I 37–I 38–I 36–II 37–II 38–II

5 [b] [b] [b] 7.63 7.54

6 7.30 7.30 7.23 7.26 7.26 7.18 7.28 7.31 7.22

7 7.69 7.70 7.67 7.58 7.57 7.53 7.61 7.61 7.60

8 7.61 7.61 7.56 7.55 7.53 7.49 [b] [b] [b]

9 3.03 3.68 3.11 3.53 3.03 3.86

10 6.16 1.27 6.19 1.39 6.20 1.44

5.64 5.53 5.55

12 2.27 1.99 2.48 2.42

3' 7.04 7.15 7.11 7.03 7.19 7.07 7.07 7.07 7.15

5' 7.48 7.58 7.43 7.54 7.66 7.44 7.51 7.51 7.52

7' 3.68 3.78 3.76 3.54 3.70 3.75 3.56 3.58 3.75

8' 2.42 2.47 2.44 2.43 2.47 2.46 2.42 2.46 2.47

MAE 0.08 0.02 0.05 0.37 0.32 0.11

CMAE 0.20 0.15 0.18 0.16 0.09 0.16

[a] Experimental and calculated 1H NMR data were obtained in MeOD-d4, [b] Exchangeable signal, not calculated


66

Table 10. Experimental and calculated 13C NMR data for niveamycins A–C[a]

δC experimental [ppm]

δC calculated [ppm]

Isomer I

δC calculated [ppm][b]

Isomer 2

No 36 37 38 36–I 37–I 38–I 36–II 37–II 38–II

1 184.5 184.4 182.7 181.9 182.1 181.8 185.8 186.9 179.0

2 149.5 150.7 [c] 145.9 147.9 122.6 146.1 147.0 119.7

3 143.4 145.6 168.0 143.4 145.1 155.2 143.4 144.3 154.8

4 190.4 191.0 [c] 185.7 186.0 182.0 181.7 183.9 186.6

4a 116.1 116.2 114.1 112.8 113.1 112.1 130.1 114.7 113.5

5 162.5 162.4 162.5 158.2 158.6 158.7 116.5 157.0 158.4

6 124.7 124.7 122.8 123.0 122.7 121.9 135.1 121.1 123.5

7 137.4 137.5 137.6 134.7 134.8 135.3 122.8 136.3 134.2

8 120.0 120.0 119.2 117.3 117.0 116.8 158.7 119.4 116.7

8a 134.1 133.8 134.0 130.5 130.5 130.3 113.2 135.9 131.3

9 144.5 49.9 52.0 143.3 51.0 52.3 143.8 56.5 52.3

10 130.1 14.3 129.5 13.2 129.6 12.2

11 199.3 208.0 198.0 211.4 197.9 209.7

12 26.4 28.5 27.5 29.2 27.5 30.4

1' 122.1 121.1 121.9 120.1 121.6 117.6 119.5 120.6 117.0

2' 157.4 157.9 157.7 153.5 154.9 155.1 153.9 154.4 165.7

3' 116.3 116.3 116.6 114.5 114.3 112.6 114.5 114.4 112.5

4' 141.5 142.0 139.1 141.7 141.7 141.6 142.0 144.3 141.4

5' 123.9 124.3 123.3 120.1 120.6 119.2 120.0 120.3 129.5

6' 128.5 [b] 128.3 128.1 125.9 129.9 128.2 127.1 128.1

7' 56.0 56.2 56.2 51.4 52.1 57.2 51.6 51.9 58.9

8' 21.6 21.6 21.2 19.9 20.0 19.9 19.9 18.0 20.0

9' 170.9 169.8 167.8 165.4 163.8 166.2 165.4 164.6 155.8

MAE 2.15 2.22 2.06 2.12 2.68 3.63

CMAE 7.87 5.12 6.66 7.84 6.12 6.24

[a] Experimental and calculated 1H NMR data were obtained in MeOD-d4. [b] Carbon chemical shifts were organised to match

with those of niveamycins. [c] Exchangeable signal, not calculated


67

4.4. Absolute configuration

Determination of the absolute configuration (AC) of natural products often poses a

challenging problem in structure elucidation.[87] To address this, several methods such

as X-ray crystallography, chiroptical spectroscopy and NMR anisotropy methods, each

having its own limitations, have been developed over the last years.[87-88] The AC of

chiral natural products comprising chromophores has been most commonly elucidated

using chiroptical methods, including electronic circular dichroism (ECD), vibrational

circular dichroism (VCD) and Raman optical activity (ROA).[87, 89]

Among these approaches, ECD has been most widely used over the past decade.[90]

ECD measures the differential response of a chiral molecule to the modulation of

UV/Vis radiation between left- and right-circularly polarised states.[90] In general, AC

determination using ECD compares the spectrum of new compounds against analogous

molecules having a known AC. However, recently an alternative non-empirical method

involving ECD calculations of time-dependent density functional theory (TDDFT) has

become a rapid and reliable way to establish the AC of chiral compounds.[87, 91] ECD

calculations usually include two steps, a conformational search to obtain the candidate

conformers and their subsequent optimisation using TDDFT.[87] The accuracy of

TDDFT calculations depends mainly on the basis set and functional used for the

calculations. Thus, the larger the basis set, the more accurate the results will be.


68

4.4.1. Absolute configuration of niveamycin B

The absolute configuration of the side chain chiral carbon at C-9 of niveamycin B (37)

was determined using ECD calculations. The ECD spectra of the most stable

conformers for 37 were calculated at the B3LYP/6-31+G(d,p)//CAM-B3LYP/SVP

(Figure 32) and B3LYP/6-311 + G(d,p)// B3LYP/6-311 + G(2d,p) (Figure 33) level on

six stable conformers. The ECD spectra for the six conformers were Boltzman-averaged

to obtain the ECD spectrum of the isomers. Although the two calculation levels agreed

well with that of the experimental and led to the conclusion that the absolute

configuration at C-9 was S, the B3LYP/6-311 + G(d,p)// B3LYP/6-311 + G(2d,p)

computed ECD spectrum provide a more accurate result as it matched the experimental

ECD spectrum better.

Figure 32. Calculated (9S) and experimental ECD spectra of niveamycin B (37)


69

Figure 33. Comparison of calculated (9S) and experimental ECD spectra of niveamycin B (37)

Chapter 5: Actinofuranosin A and arglecins B and C from a Streptomyces sp. USC 597

70

Chapter 5: Actinofuranosin A and arglecins B and C

from a Streptomyces sp. USC 597

5.1 Introduction

Actinomycetes represent a rich source of bioactive small molecules and lead

compounds with diverse chemical structures and biological activities.[53a, 92] Over the

last 60 years, millions of actinomycete strains isolated from diverse geographical

locations and habitats have been extensively screened for new bioactive small

molecules.[30, 54a] These screening campaigns led to the discovery of more than 12,000

naturally-occurring compounds, including many with medical importance and high

commercial value.[5-6, 18] However, as a result of the extensive screening programs, the

probability of finding new/novel chemical entities is proving to be increasingly

difficult.[30]

Several strategies have been proposed to address the problem of rediscovery; one of the

most promising is the selective isolation of rare and uncommon actinomycetes from

extreme and understudied environments such as desert biomes, marine ecosystems,

deep-sea sediments and insect-associated symbionts.[31b, 32a, 93] Actinomycetes sourced

from these habitats represent a rich source of novel strains with the potential to

biosynthesise unique scaffolds which may be used as leads for the development of drug

candidates.[24b, 31b, 94]

As part of a continuing effort to discover new natural products, a termite gut-associated

actinomycete strain (USC 597) was selected from the University of the Sunshine Coast


71

Microbial Collection to perform chemical and biological investigations.[53] Herein, we

report the isolation and structure elucidation of three new natural products, namely,

actinofuranosin A (41) and arglecins B (42) and C (43) and one known compound

named, 3H-Pyrrolo[2,3-d]pyrimidine-5-carboxylic acid, 2-amino-4,7-dihydro-4-oxo-,

methyl ester (44). The structures of these compounds were determined by

comprehensive spectroscopic and spectrometric analysis.


Comparative 16S rRNA gene sequence analysis revealed that the isolate USC 597 was a

Streptomycete species occupying a distant phylogenetic position compared with the

previously described species Streptomyces cinnamonensis strain ZZ043KJ995740

(Figure 34). Cultures of the producing strain Streptomyces sp. USC 597 were first


72

grown on four Petri dishes (100 x 15 mm) containing OMA, LFA, RFA and GYES

agar. Potential new natural products were detected only in the crude extract obtained

from the GYES solid culture.


relationships between the strains USC 6922, 6921, 596, 6916, 595, 6901, 6903, 594, 6918, 6909, 6910,

6930, 597, 6923, 593, 6905, 6927, 6907, 6919, 6904, 592, 590, 6929, 6928, 6931, 6911, 6920, 6934, 6926,

6933 with the most closely related type strains of Streptomyces. Numbers at the nodes indicate bootstrap

values based on 1,000 replicates; only values above 50% are shown. Bar 0.05 sequence divergence


73

Thus, with the aim of obtaining larger amounts of the desired compounds to perform 2D

NMR spectroscopic analysis, Streptomyces sp. USC 597 was grown in 40 GYES agar

plates (100 x 15 mm) for 15 days at 28°C, after which cultures comprising the cells and

mycelium were cut into small squares and soaked in EtOAc. The EtOAc extract was

concentrated to dryness in vacuo, to yield 52.3 mg of the crude extract. A portion of this

extract (44.3 mg) was subjected to reverse-phase chromatography using a C18 semi-

preparative column and subsequently to C8 semi-preparative HPLC to afford three new

natural products, namely, actinofuranosin A (41), arglecins B (42) and C (43) and one

known compound named, 3H-Pyrrolo[2,3-d]pyrimidine-5-carboxylic acid, 2-amino-4,7-

dihydro-4-oxo-, methyl ester (44).

Actinofuranosin A (41) was isolated as an optically inactive colourless amorphous solid.

The molecular formula of C13H20N5O4, m/z 310.15098 [M+H]+ (calcd. for C13H20N5O4,

310.1437) was determined on the basis of the (+)-HRESIMS and NMR measurements.

The 1H NMR spectrum of 41 (Table 11) in MeOD-d4 (Figure 35) displayed eleven

resonances which correspond to three sp3-hybridized methyls at δH 3.44 (3H, s, H-6')

and 3.50 (6H, s, H-7 and H-8), six sp3-hybridized methines at δH 3.65 (1H, dd, J = 10.8,

3.8 Hz, H-5a'), 3.73 (1H, dd, J = 10.8, 2.9 Hz, H-5b'), 4.18 (1H, dd, J = 8.2, 3.5 Hz, H-

4'), 4.32 (1H, t, J = 4.7 Hz, H-3'), 4.52 (1H, t, J = 4.7 Hz, H-2') and 6.06 (1H, d, J = 4.7

Hz, H-1'), two sp2-hybridized methines at δH 8.21 (1H, s, H-2) and 8.26 (1H, s, H-10).

The 13C NMR spectrum of 41 (Table 11) exhibited thirteen resonances comprised of

three sp3-hybridized methyls at δC 59.5 (C-6') and 38.9 (C-7 and C-8), five sp3-

hybridized methines at δC 73.2 (C-5'), 85.0 (C-4'), 71.8 (C-3'), 76.2 (C-2') and 89.7 (C-

1'), two sp2-hybridized methines at δC 153.2 (C-2) and 138.8 (1H, s, H-10) and three

quaternary carbons at δC 154.8 (C-6), 149.8 (C-4) and 119.5 (C-5). Interpretation of 1D


74

and 2D NMR data allowed for the identification of two partial structures which are

depicted in Figure 36. Fragment A, showed characteristic proton resonances indicative

of a N-ribofuranose moiety, with the configuration of the anomeric proton assigned to

be β based on a cis relationship of H-1' and H-4' coupling constant at δH 6.06 (1H, d, J =

4.7, H-1').[95] Moreover, reciprocal HMBC correlations between the methyl at δH 3.44

(H-6') to the sp3-hybridized methine carbon at δC 73.2 (C-5') and from the methine pair

at δH 3.65 (H-5a') and 3.73 (H-5b') to the sp3-hybridized methyl at (C-6') suggested a

naturally-occurring methylation of the ribofuranose at C-5'.

Figure 35. 1H NMR spectrum of actinofuranosin A (41) at 600 MHz in MeOD-d4

Detailed analysis of the NMR spectroscopic data of fragment B, indicate the presence of

a purine ring system. HMBC correlations from the methine at δH 8.21 (1H, s, H-2) to the

olefinic carbons at δC 149.8 (C-4) and 119.5 (C-6) as well as HMBC correlations from

the methine at δH 8.26 (1H, s, H-10) to the quaternary carbons at δC 149.8 (C-4) and

154.8 (C-5) were consistent with the presence of the aglycone 9H-purin-6-amine, N,N-

dimethyl-. This was further confirmed by NMR data comparison with related synthetic


75

and natural product compounds containing the same residue such as puromycin.[96]

Additionally, crucial HMBC correlations from the anomeric proton at δH 6.06 (H-1') to

the olefinic and quaternary carbons at δC 138.8 (C-10) and 149.8 (C-4), respectively,

unequivocally positioned the furanose ring at N-11. The relative configuration of

actinofuranosin A (41) was concluded to be N-β-ribofuranosyl-9H-Purin-6-amine, N,N-

dimethyl-.

Figure 36. Fragments found during the elucidation process and crucial HMBC and NOESY correlations

for actinofuranosin A (41)

Arglecin B (42) was obtained as an optically inactive colourless amorphous solid.

Analysis of the HRESIMS spectrum showed a quasimolecular ion at 266.1867 [M+H]+,

corresponding to the molecular formula C14H24N3O2 (calcd. for C14H24N3O2, 266.1790).

The 1H NMR spectrum of 42 in MeOD-d4 (Figure 37) revealed 10 resonances which

corresponded to three sp3-hybridized methyls at δH 1.92 (3H, s, H-17), and 0.93 (6H, d,

J = 7.1 Hz, H-9 and H-10), one sp3-hybridized methine at δH 2.15 (1H, m, H-8), five

sp3-hybridized methylenes at δH 2.58 (2H, d, J = 7.1 Hz, H-7), 3.19 (2H, t, J = 6.8 Hz,


76

H-14), 2.52 (2H, t, J = 7.7 Hz, H-11), 1.66 (2H, m, H-12), and 1.54 (2H, m, H-13), and

one sp2-ybridized methine at δH 7.16 (1H, s, H-5) (Table 12).

The 13C NMR spectrum of 42 in MeOD-d4 (Figure 38) showed the presence of three

methyls at δC 22.5 (C-17), and 22.9 (C-9 and C-10), one methine at δC 28.1 (C-8), five

methylenes at δC 42.5 (C-7), 39.9 (C-14), 30.8 (C-11), 27.1 (C-12), and 29.8 (C-13),

three olefinic carbons at δC 122.6 (C-5), 158.3 (C-3) and 141.1 (C-6), and two carbonyl

carbons at δC 173.3 (C-16), 157.7 (C-2).

Table 11. 1H NMR and 13C NMR spectroscopic data for

actinofuranosin A (41) in MEOD-d4

41[a]

No δC [ppm]

δH [ppm] (J in Hz) HMBC

2 153.2 8.21 (s) C-4, C-6

3

4 149.8

5 119.5

6 154.8

7 38.9 3.50 (s)

8 38.9 3.50 (s)

9

10 138.8 8.26 (s) C-4, C-5

11

1' 89.7 6.06 (d, 4.7) C-2´, C-10

2' 76.2 4.52 (t, 4.7) C-4´

3' 71.8 4.32 (t,4.7) C-1´, C-5´

4' 85.0 4.18 (dd, 8.2, 3.5) C-3´

5' 73.2 3.73 (dd, 10.8, 2.9)

3.65 (dd, 3.5, 10.8) C-3´, C-4´, C-6´

6' 59.5 3.44 (s) C-5´

[a] Proton and carbon resonances were acquired at 600 MHz and 150 MHz,

respectively


77

The COSY spectrum showed two partial structures (Figure 39) which were comprised

of an isopropyl spin system and an n-butyl side chain attached to a secondary amine.

The presence of the secondary amine at δH 7.77 (NH, brt, J = 6.8 Hz, H-15) attached to

the methylene at δH 3.19 (t, J = 6.8 Hz, H-14) was further confirmed by strong COSY

correlations between these protons when 42 was recorded in DMSO-d6 (Figure 40).

Figure 37. 1H NMR spectrum of arglecin B (42) at 900 MHz in MeOD-d4

Figure 38. 13C NMR spectrum of arglecin B (42) at 225 MHz in MeOD-d4

Crucial HMBC correlations displayed in Figure 39 were used to complete the structure

of 42. The methylene pair at δH 2.58 (H-7) showed correlations to C-2, C-3, C-8, C-9,

and C-10. Moreover, HMBC correlations from the methylene pair at δH 2.52 (H-11) to


78

the olefinic carbons at δC 122.5 (C-5) and 141.1 (C-6) suggested that the butyl side

chain may be attached to a pyrimidine ring system. HMBC correlations from the methyl

at δH 1.92 (H-17) and from the methylene pair resonances at δH 1.54 (H-13) to the

carbonyl carbon at δC 173.3 (C-16) indicated the presence of a terminal acetyl group.

From the molecular formula of 42 (C14H24N3O2) it was determined that a core ring

comprising two degrees of unsaturation needed to be established. HMBC correlations

from the methines at δH 7.16 (H-5) and 2.15 (H-8) to the carbonyl carbon at δC 157.7 (C-

2) as well as correlations from the methylene pair at δH 2.52 (H-11) to the olefinic

carbons at δC 122.6 (C-5) and 141.1 (C-6) were indicative of a 2(1H)-pyrazinone core.

Upon comparison of the NMR spectroscopic data, with that of known natural products

containing similar core structures,[97] the presence of a 3,5,6-trisubstitued 2(1H)-

pyrazinone core was confirmed. The structure of compound 42 was therefore concluded

to be N-[4-(3-isobutyl-2-oxo-pyrazin-2(1H)-one-6-yl)butyl]acetamide.

Figure 39. Crucial HMBC and COSY correlations for arglecins B and C

Arglecin C (43) was obtained as an optically inactive colourless amorphous solid. Its

molecular formula was determined to be C13H22N3O2, m/z 252.1715 [M+H]+, (calc. for

C13H22N3O2, 252.1634) based on HRESIMS measurements. Comparison of NMR

spectral data (Figure 41) of 42 with that of 43 in MeOD-d4 (Table 12) revealed that


79

compound 43 possessed a similar skeleton to 42 except that the n-butyl side chain in 42

was substituted for an n-propyl side chain in 43. The structure of arglecin C was further

confirmed by interpretation of the 2D NMR spectra to be N-[4-(3-isobutyl-2-oxo-

pyrazin-2(1H)-one-6-yl)propyl]acetamide. .

Figure 40. gCOSY spectrum of Arglecin B at 600 MHz in DMSO-d6


80

Figure 41. 1H NMR data comparison between arglecins B (42) (top spectrum) and C (43) (bottom

spectrum)

The GYES crude extract sourced from Streptomyces sp. USC 597 also yield the known

natural product 44, which was isolated from the more polar fractions. The molecular

formula of C8H8N4O3 was deduced on the basis of HRESIMS measurements. Following

the interpretation of 1 and 2D NMR spectroscopic data of 44, it was concluded that the

spectral data of this fragment were in accordance to the described literature values for

3H-Pyrrolo[2,3-d]pyrimidine-5-carboxylic acid, 2-amino-4,7-dihydro-4-oxo-, methyl

ester.[98]


81

Table 12. . 1H NMR and 13C NMR spectroscopic data for arglecins B (42) and C (43) in MeOD-d4

42[a] 43[b]

No δC

[ppm]

δH [ppm] (J in Hz)

HMBC δC

[ppm]

δH [ppm] (J in Hz)

HMBC

2 157.7 158.2

3 158.3 159.0

5 122.6 7.16 (s) C-2, C-6 122.5 7.20 (s) ND[c]

6 141.1 140.3

7 42.5 2.58 (d 7.1) C-2,C-3, C-8, C-9, C-10

42.1 2.60 d (7.0) C-2,C-3, C-8, C-9, C-10

8 28.1 2.15 (m) C-3, C-7, C-9, C-10

27.7 2.17 (m) C-3, C-7, C-9, C-10

9 22.9 0.93 (d, 7.1) C-7, C-8, C-9 22.6 0.95 (d,7.0) C-7, C-8, C-10

10 22.9 0.93 (d, 7.1) C-5, C-6, C-12, C-13

22.6 0.95 (d, 7.0) C-7, C-8, C-9

11 30.8 2.52 (t, 7.7) C-5, C-6, C-12, C-14

28.2 2.54 (t, 7.7) C-5, C-6, C-12, C-14

12 27.1 1.66 (m) C-11, C-14 29.2 1.84 (m) C-11, C-14

13 29.8 1.54 (m) C-12, C-13, C-14

14 39.9 3.19 (t, 6.8) C-12, C-13, C-16

39.2 3.23 (t, 6.8) C-12, C-16

16 173.3 173.7

17 22.5 1.92 (s) C-16 22.2 1.96 (s) C-16

[a] Proton and carbon resonances were acquired at 900 MHz and 225 MHz, respectively. [b] Proton and carbon resonances were acquired at 600 MHz and 150 MHz, respectively. [c] No signal detected

Chapter 6: Summary

82

Chapter 6: Summary

Natural products have undergone evolutionary selection over time to bind to multiple,

unrelated classes of protein receptors as high affinity ligands.[11a] And while these small-

molecules often exhibit highly potent and selective bioactivity against a wide range of

infectious diseases and cancer, they have not evolved to satisfy the pharmacokinetic

properties envisioned by humans for a clinically useful drug.[99] Large number of

promising drug leads have failed to advance to preclinical or clinical development due

mainly to their toxicity, harmful side effects or low bioactivity.[100] The emergence of

modern tools of chemistry and biology allowed advances in improving formulation and

drug delivery methodology to determine the exact nature of the bioactive natural

products to uncover possible synergies for the development of more effective therapies

against many devastating diseases.[99a, 100] The application of novel techniques and

methods to induce the expression of cryptic biosynthetic pathways has become an

attractive form of drug discovery particularly in the search for novel anti-infectives.

While many methods may be used to induce such expression, the identification of novel

metabolites in the complex mixtures has many challenges and this thesis examined the

use of NMR metabolomics to identify the production of new metabolites.

In this study, twenty one actinomycete strains were subjected to different small-scale

culture conditions, including comparison of two strains, co-culture and variation of the

media components in order to determine if these conditions could induce the

biosynthesis of new secondary metabolites. Differences in the production of small-

molecules (based on their chemical profiles) were found on all the performed

Chapter 6: Summary

83

experiments. However, due to the short time frame, further work to trigger the

production of new compounds was carried out only using different carbon and nitrogen

sources.

Crude extracts were fractionated (LLE fractions) and then analysed by a 1H NMR-

guided metabolic fingerprinting approach. Based on this analysis, five strains

(Streptomyces sp. USC 590, Streptomyces sp. USC 592, Streptomyces sp. USC 593,

Streptomyces sp. USC 597 and Microbispora sp. USC 6900) showing unique

chemotypes were selected to be grown on 40-60 Petri dishes (100 x 15 mm) containing

RFA (Rye flour 5.0 g, peptone 100.0 mg, glucose 1.0 g, agar bacteriological 20.0 g,

dH2O 1L), OMA (Oatmeal 20.0 g, yeast extract 3.0 g, agar bacteriological 20.0 g, dH2O

1L), or GYES (glucose 10.0 g, yeast extract 2.50 g, corn starch 2.50 g, sodium chloride

1.25 g, calcium carbonate 0.75 g, agar bacteriological 20.0 g, dH2O 1L) solid media.

Although the five strains showed distinctive chemical profiles on the OMA solid culture,

only when Streptomyces sp. USC 592, Streptomyces sp. USC 593 and Streptomyces sp.

USC 597 were grown on GYES media, potential new natural products were detected.

The effectiveness of the NMR-based methodology was demonstrated by the isolation

and identification of nine new natural, namely, actinoglycosidines A (27) and B (28),

actinopolymorphol D (29), niveamycins A (36), B (37) and C (38), actinofuranosin A

(41) and arglecins B (42) and C (43).

In the context of NP-based drug discovery, NMR fingerprinting is important as it

rapidly achieves a non-targeted interrogation of the drug-like natural product

metabolome and consequently can simplify and accelerate the identification of new

secondary metabolites.[12, 44, 101] The study of the biological activity was limited to

Chapter 6: Summary

84

finding new antibacterial compounds in particular against Mycobacterium bovis bacillus

Calmette-Guérin (BCG) Pasteur 1173P2 strain and produced only one active known

compound named, TMC-66 (26).[61] A future task will involve the evaluation of all

isolated compounds using a phenotypic assay (Figure 42) which in contrast to high-

throughput screening (HTS) offers the possibility to analyse whole cell models where

all targets and biological pathways can be interrogated.[102] Combining NMR

fingerprinting with phenotypic assay may identify new targets and probes to better

understand some biological processes that occur on a disease state.

Figure 42. Summary of the NP-drug discovery workflow followed on this thesis. The biological activity

of the compounds was evaluated using an Antitubercular assay.[62] A future task will involve the a

phenotypic screening[102] of all new and known natural products isolated.

Chapter 7: Experimental

85


7.1 General experimental

The UV spectra were recorded on a JASCO Varian-650 UV/Vis spectrophotometer.

NMR spectra were recorded at 30°C on either a Varian 500 or 600 MHz Unity INOVA

spectrometer. The 1H and 13C NMR chemical shifts were referenced to the solvent peak

at δH 2.50 and δC 39.52 for DMSO-d6. (+)-LR-ESIMS were recorded on a Waters ZQ

single quadropole ESI spectrometer or on an Agilent 6120 quadruple LCMS system. All

HR-ESIMS were recorded on an Agilent Q-TOF 6520 mass spectrometer. RP-HPLC

prefractionation was performed on a Waters 600 pump equipped with Waters 996 PDA

detector and Gilson 717 liquid handler. Semipreparative HPLC separations were carried

out either with a Phenomenex Onyx Monolitic (100 x 10 mm) C18 column or a Thermo

Scientific BDS Hypersil C8 column (250 X 10 mm). All solvents used for

chromatography, UV, and MS were Lab-Scan HPLC grade (RCI Lab-Scan, Bangkok,

Thailand), and the H2O was Millipore Milli-Q PF filtered. Chiroptical measurements

([α]D and CD) were acquired on a Jacso J-715 and P-1020 spectrometer, respectively.

7.2 Culture conditions

Twenty one actinomycete species isolated from the gut of the wood-feeding termite

Coptotermes lacteus (Froggatt) were grown in solid media (four Petri dishes, 100 x 15

mm) using four different solid culture conditions, OMA (Oatmeal 20.0 g, yeast extract

3.0 g, agar bacteriological 20.0 g, dH2O 1L), LFA (Lupin flour 5.0 g, peptone 100.0 mg,


86

glucose 1.0 g, agar bacteriological 20.0 g, dH2O 1L), RFA (Rye flour 5.0 g, peptone

100.0 mg, glucose 1.0 g, agar bacteriological 20.0 g, dH2O 1L) and GYES (glucose 10.0

g, yeast extract 2.50 g, corn starch 2.50 g, sodium chloride 1.25 g, calcium carbonate

0.75 g, agar bacteriological 20.0 g, dH2O 1L), media was adjusted to a pH of 7.2 before

autoclaving. The actinomycete cultures were incubated at 28°C for 15 days, and then

the agar containing the cells and mycelium was cut into small squares and soaked

overnight in EtOAc. The EtOAc extracts were dried under reduced pressure to yield

between 10 to 15 mg for each culture condition. The extracts were afterward subjected

to chemical investigations where based on the NMR-guided fingerprinting approach,

five strains and three culture conditions were selected to perform solid fermentations in

larger amounts (sixty Petri dishes, 100 x 15 mm).

7.3 Lead-like enhanced (LEE) fractions

A portion of the microbial extracts (1 mg) was reconstituted in DMSO (150 μL). HPLC

separations were performed on a Phenomenex C18 Monolithic HPLC column (100 x 4.6

mm) using conditions that consisted of a linear gradient from 90% H2O/10% MeOH to

50 H2O/50% MeOH in 3 min at a flow rate of 4mL/min; a convex gradient to MeOH in

3.50 min at a flow rate of 3 mL/min, held at MeOH for 0.50 min at a flow rate of 3

mL/min, held at MeOH for a further 1.0 at a flow rate of 4 mL/min; then a linear

gradient back to 90% H2O/10% MeOH in 1 min at a flow rate, then held at 90% H2O/10%

MeOH fro to min at a flow rate of 4 mL/min. The total run time for each crude extract

was 11 min and 5 fractions were collected between 2.0 and 7.0 min (5 x 1 min).


87

Because the intent of this separation step was to collect fractions most likely to contain

compounds with drug-like properties, the early eluting material consisting of media

components and highly polar compounds was not collected nor was the late-eluting

lipophilic portion.

7.4 Metabolic fingerprinting approach

Five LLE fractions replicates were combined accordingly and further analyzed by 1H

NMR spectroscopy. The fractions were dried down under reduced pressure and

subsequently dissolved in 220 μL of MeOD-d4, the samples were run under the

following parameters: pw = 45º, pl = 0 μs, d2 = 0 s, d1 = 1 s, at = 1.7 s, sw = 9615Hz, nt

= 256 scans.

7.5 Preliminary screening of isolates for production of antimicrobial compounds

All isolates were preliminary screened for their antibacterial activity by the agar plug

method following a modified protocol from that of (Xie et al. 2005). The isolates were

grown for 15 days at 28°C in small-scale (4 Petri dishes, 100 x 15 mm) using four

different solid culture conditions, OMA (Oatmeal 20.0 g, yeast extract 3.0 g, agar

bacteriological 20.0 g, dH2O 1L), LFA (Lupin flour 5.0 g, peptone 100.0 mg, glucose

1.0 g, agar bacteriological 20.0 g, dH2O 1L), RFA (Rye flour 5.0 g, peptone 100.0 mg,

glucose 1.0 g, agar bacteriological 20.0 g, dH2O 1L), GYES (glucose 10.0 g, L-

asparagine 500.0 mg, dipotassium phosphate 1.0 g, agar bacteriological 20.0 g, dH2O

1L), media was adjusted to a pH of 7.2 before autoclaving. After 15 days, four discs (6

mm in diameter) were cut and placed on Muller-Hinton agar plates seeded with the test


88

organisms namely, Escherichia coli (ATCC BAA-196), Kleibsiella pneumoniae (ATCC

BAA-1705), Staphylococcus aureus (ATCC 29247) and Staphylococcus aureus (ATCC

51575) and then incubated at 37°C overnight. The inhibition zone diameter was

measured by calipers.

7.6 Scale-up solid culture growth and isolation

The producing strains Streptomyces sp. USC 590, Streptomyces sp. USC 592,

Streptomyces sp. USC 593, Streptomyces sp. USC 597 and Microbispora sp. USC 6900

were grown for 15 days at 28°C in 60 agar plates (100 x 15 mm) containing GYES,

OMA or RFA media. A similar methodology as described above was followed to obtain

the crude extract. The EtOAc extracts were dried down to yield 120.0 mg, 190.0 mg

130.0 mg, 44.3 mg and 210.2 mg of solid crude extract, respectively. A portion of the

crude extract (~43.0 mg) was run down a Phenomenex Onyx Monolitic (100 x 10 mm)

C18 column. Isocratic HPLC conditions of H2O/MeOH (90%/10%) were initially

employed for 10 min, followed by a linear gradient to 100% MeOH over 40 min, then

an isocratic condition of 100% MeOH was run for 10 additional minutes, all at a flow

rate of 9mL/min. 60 fractions were collected from 0 to 60 min (60 x 1 min), and then

analysed by (+)-LR-ESIMS.

7.7 Anti-BCG assay

Identification of inhibitors was performed in an aerobic, logarithmic growth screen of

BCG as previously reported.[103] The BCG used was a M. bovis BCG 1173P2 strain

transformed with green fluorescent protein (GFP) constitutive expression plasmid


89

pUV3583c with direct readout of fluorescence as a measure of bacterial growth. BCG

was grown at 37°C to mid log phase in Middle brook 7H9 broth (Becton Dickinson)

supplemented with 10% OADC enrichment (Becton Dickinson) 0.05% tween-80 and

0.2% glycerol, which then adjusted to OD600=0.025 with culture medium as bacterial

suspension. Aliquots (80 μL) of the bacterial suspension were added to each well of the

96-well microplates (clear flat-bottom), followed by adding compounds (2 μL in

DMSO), which were serially twofold diluted. Isoniazid served as positive control and

DMSO as negative control. The plate was incubated at 37°C for 3 days, and GFP

fluorescence was measured with Multi-label Plate Reader using the bottom read mode,

with excitation at 485 nm and emission at 535 nm. MIC is defined as the minimum

concentration of drug that inhibits more than 90% of bacterial growth reflected by

fluorescence value.[62]

7.8 Phylogenetic characterisation of the actinomycetes strains

Total genomic DNA samples from 50 actinomycetes strains were extracted with the

DNeasy® Blood & Tissue Kit (Qiagen, Austin, TX) following a protocol modified from

that of the manufacturer. In brief, a fresh colony was added to 1.5 mL Eppendorf tubes

containing 200µL of sterile deionised water, cells were centrifuged for 10 min at 12,000

rpm. Pellet was resuspended in 180 µL enzymatic lysis buffer (20mM Tris-Cl pH 8,

2mM sodium EDTA, 1.2% Triton ® X-100, 20 mg/mL lysozyme) and incubated for 30

mins at 37°C. Subsequently, 25µL proteinase K and 200µL Buffer AL (supplied with kit)

were added and the sample was incubated again at 56°C for 30 mins. After that, 200 µL

of ethanol 100% were added and mixed thoroughly by vortexing. Afterwards, the


90

mixture was pipetted to a spin column within a 2 mL collection tube and centrifuged for

1 min at 8,000 rpm. The collection tube with flow-through was discarded and the spin

column was placed in another 2 mL collection tube. 500 µL of buffer AW1 were added,

the mixture was centrifuged for 1 min at 8,000 rpm. Following centrifugation, flow-

through was discarded and spin column was transferred to a new 2 mL collection tube,

to which 500 µL of buffer AW2 were added. Column was again centrifuged for 4 mins

at 10.500 rpm before transferring to sterile 1.5 mL Eppendorf tubes for elution with 200

µL buffer AE. DNA purity and integrity was assessed using agarose gels (1% w/v) and

stained with ethidium bromide. The 16S rRNA gene was amplified by PCR using

universal primers F27 and R1492. Reactions in a final volume of 20 µL were carried out

using 10 µL of reaction mixture containing HotstarTaq plus, 0.25 mM of primers

forward and reverse 7 µL of deionised water and 2 µL (~ 100 ng) of genomic DNA.

Amplifications were carried out in a T100TM thermal cycler (Bio-Rad, Hercules, CA)

using the following conditions: preheat activation at 95°C for 5 min, 35 cycles of 94°C

for 30 s, 55°C for 45 s, 72°C for 1 min and a final extension at 72°C for 10 min.

Amplification products were analysed by gel electrophoresis in agarose gels (2%, w/v)

stained with ethidium bromide. PCR products were sent to Macrogen, Korea

(http://dna.macrogen.com/eng/) for PCR purification and DNA sequencing. DNA

sequences were assembled using the DNA Sequence Analysis Software Sequencher 5.1

(reference), and the resulting partial 16S rRNA gene sequences (average length, 1,310

bp) were compared to those available in the GenBank

(http://www.ncbi.nlm.nih.gov/GenBank/index.html) using nucleotide BLASTn.

Sequences were aligned, and a phylogenetic tree was constructed using the Molecular

Evolutionary Genetics Analysis (MEGA) software version 5.[104]

http://www.ncbi.nlm.nih.gov/GenBank/index.html


91

7.9 Chapter 3: Experimental

Isolation and hydrolysis

Actinoglycosidine A (27) was isolated from HPLC fraction 14 with a yielded of 2.4 mg

(5.5% dry wt). Fraction 16 yielded 1.1 mg of Actinoglycosidine B (28) (2.5% dry wt).

Combined fractions 37 and 38 were further purified by semi-preparative HPLC using a

BDS Hypersil C8 column (250 x 10 mm) eluting with a gradient from 60% MeOH/40%

H2O to 90% MeOH/10 H2O over 30 min to yield 1.1 mg of Actinopolymorphol D (29)

(2.5% dry wt) and 2.0 mg of BE-54017-derivative 4 (30) (4.6% dry wt). Fractions 32 to

34 were combined and subsequently purified using a BDS Hypersil C8 column (250 x

10 mm) eluting with a gradient from 40% MeOH/60% H2O to 90% MeOH/10 H2O over

30 min to yield 1.0 mg of BE-54017 (31) (2.3% dry wt). Combined fractions 17 to 21

yielded 2.0 mg of 7H-Pyrrolo[2,3-d]pyrimidine-5-carbonitrile, 2-amino-4-methoxy (32)

(4.6% dry wt).

Actinoglycosidine A (27)


92

Amorphous colourless solid; HRESIMS m/z [M+H]+ 393.1513 (calcd. for C16H21N6O6,

393.1414); [α]25D = +40 (c 0.1, MeOH); UV (MeOH) λmax nm (log ε): 207 (3.65), 228

(4.29), 261 sh (3.59), 284 (3.55), 307 (3.13); 1H NMR (MeOD-d4, 600 MHz) δH 7.58

(1H, s, H-9), 5.29 (1H, d, J = 10.0 Hz, H-1'), 4.06 (3H, s, H-7), 3.88 (1H, dd, J = 10.0,

3.3 Hz, H-2'), 3.85 (1H, dd, 5.0, 3.3 Hz, H-6a'), 3.70 (H, dt, J = 5.2, 2.6 Hz, H-6b'), 3.56

(1H, ddd, 10.0, 8.5, 3.3 Hz, H-3'), 3.41 (1H, m, H-5'), 3.40 (1H, m, H-4'), 1.96 (3H, s,

H-9'); 13C NMR (MeOD-d4, 150 MHz) δC 174.6 (C-8), 164.7 (C-6), 160.5 (C-2), 155.4

(C-4), 131.2 (C-9), 116.5 (C-10), 98.7 (C-5), 84.8 (C-8), 83.7 (C-1'), 79.4 (C-5'), 76.6

(C-3'), 72.3 (C-4'), 62.9 (C-6'), 56.2 (C-2'), 54.3 (C-7), 22.8 (C-9').

A solution of actinoglycosidine A (27) (2.4 mg) in MeOH (2.0 mL) was treated with 5%

HCl (200 μL) and the reaction was kept stirring at 60°C for 81 hrs. After hydrolysis, the

solution was evaporated and subjected to semi-preparative HPLC using a Thermo

Scientific BDS Hypersil C8 column (250 X 10 mm). Isocratic HPLC conditions of 90%

H2O/10% MeOH were initially employed for 10 min, then a linear gradient to 21%

MeOH was run over 15 min, followed by a gradient to 100% MeOH in 10 min and then

isocratic conditions of 100% MeOH were held for 5 min, all at a flow rate of 4 mL/min.

Sixty fractions (60 x 30 sec) were collected from time = 0 min, and then analyzed by

high resolution NMR spectroscopy and (+)-LR-ESIMS. Fraction 10 yielded 0.3 mg of

N-β-D-acetylglucosamine.


93

Actinoglycosidine B (28)


393.1414); [α]25D = +39 (c 0.07, MeOH); UV (MeOH) λmax nm (log ε): 203 (3.93), 225

(4.20), 253 sh (3.68), 269 (3.35), 2.92 (3.57); 1H NMR (MeOD-d4, 600 MHz) δH 7.60

(1H, s, H-9), 6.02 (1H, d, J = 5.1 Hz, H-1'), 4.11 (1H, dd, J = 11.2, 5.1 Hz, H-2'), 4.08

(3H, s, H-7), 3.79 (1H, dd, J = 8.8, 6.7 Hz, H-4'), 3.76 (1H, dd, 9.6, 2.6 Hz, H-6a'), 3.72

(1H, dd, 12.0, 5.1 Hz, H-6b'), 3.62 (1H, ddd, 9.6, 5.1, 2.6 Hz, H-3'), 3.44 (1H, dd, J =

9.6, 8.8 Hz, H-5'), 1.93 (3H, s, H-9'); 13C NMR (MeOD-d4, 150 MHz) δC 174.3 (C-8),

164.7 (C-6), 160.3 (C-2), 155.0 (C-4), 131.7 (C-9), 116.6 (C-10), 98.8 (C-5), 84.3 (C-8),

78.7 (C-1'), 73.8 (C-3'), 72.1 (C-5'), 71.9 (C-4'), 62.3 (C-6'), 54.6 (C-7), 54.4 (C-2'),

22.9 (C-9').

Actinopolymorphol D (29)


94

Amorphous colourless solid; HRESIMS m/z [M+H]+ 261.1380 (calcd. for C18H16N2,

261.1313); UV (MeOH) λmax nm (log ε): 205 (4.06), 250 sh (3.63), 281 (3.65), 3.03 sh

(3.44); 1H NMR (Acetone-d6, 600 MHz) δH 7.31 (2H, dd, J = 7.9, 5.6 Hz, H-2, H-6),

7.28 (1H, dd, J = 7.9, 5.6 Hz, H-4), 7.19 (2H, dd, J = 7.3, 5.6 Hz, H-3, H-5), 8.46 (1H, s,

H-9), 4.12 (2H, s, H-7); 13C NMR (Acetone-d6, 125 MHz) δC 154.8 (C-8), 144.4 (C-9),

140.1 (C-1), 129.8 (C-2), 129.4 (C-4), 127.2 (C-3), 41.8 (C-7).


HPLC fractions 29 to 31 were combined to afford 1.7 mg of niveamycin A (36) (3.9%

dry wt). Combined fractions 32 and 33 yield 0.9 mg of niveamycin B (37) (2.0% dry

wt). Niveamycin C (38) was isolated from fraction 28 in very slow yields, 0.7 mg (1.6%

dry wt). Fractions 24 and 25 afforded 0.8 mg (1.8% dry wet) of WS-5995 A (39) and

1.9 mg (4.4% dry wet) of WS-5995 A (40).

Niveamycin A (36)

Yellow amorphous solid; HRESIMS at m/z 407.1115 [M+H]+ (calcd. for C23H19O7,

407.1053); UV (MeOH) λmax (log ε): 208 (4.65), 249 sh (4.10), 287 (4.00), 4.12 (3.61);


95

1H NMR (MeOD-d4, 600 MHz) δH 7.69 (1H, dd, J = 8.3, 7.6 Hz, H-7), 7.61 (1H, dd, J =

7.6, 1.1 Hz, H-8), 7.48 (1H, s, H-5'), 7.30 (1H, dd, J = 8.3, 1.1 Hz, H-6), 7.04 (1H, s, H-

3'), 6.16 (1H, s, H-10a), 5.64 (1H, s, H-10b), 3.68 (3H, s, H-7'), 2.42 (3H, s, H-8'), 2.27

(3H, s, H-12); 13C NMR (MeOD-d4, 125 MHz) δC 199.3 (C-11), 190.4 (C-4), 184.5 (C-

1), 170.9 (C-9'), 162.5 (C-5), 157.4 (C-2'), 149.5 (C-2), 144.5 (C-9), 143.4 (C-3), 141.5

(C-4'), 137.4 (C-7), 134.1 (C-8a), 130.1 (C-10), 128.5 (C-6'), 124.7 (C-6), 123.9 (C-5'),

122.1 (C-1'), 120.0 (C-8), 116.3 (C-3'), 116.1 (C-4a), 56.0 (C-7'), 26.4 (C-12), 21.6 (C-

8').

Niveamycin B (37)

Yellow amorphous solid; [α]25D = -100 (c 0.05, MeOH); HRESIMS m/z 409.1279

[M+H]+ (calcd. for C23H21O7, 409.1209); UV (MeOH) λmax (log ε): 208 (4.61), 248

(3.96), 290 (3.83), 407 (3.53); 1H NMR (MeOD-d4, 600 MHz) δH 7.70 (1H, dd, J = 8.3,

7.6 Hz, H-7), 7.61 (1H, dd, J = 7.6, 0.9 Hz, H-8), 7.58 (1H, s, H-5'), 7.30 (1H, dd, J =

8.3, 1.0 Hz, H-6), 7.15 (1H, s, H-3'), 3.78 (3H, s, H-7'), 3.03 (1H, q, J = 6.8 Hz, H-9),

2.47 (3H, s, H-8'), 1.99 (3H, s, H-12), 1.27 (3H, d, J = 6.8); 13C NMR (MeOD-d4, 125

MHz) δC 208.0 (C-11), 191.0 (C-4), 184.4 (C-1), 169.8 (C-9'), 162.4 (C-5), 157.9 (C-2'),

150.7 (C-2), 145.6 (C-3), 142.0 (C-4'), 137.5 (C-7), 133.8 (C-8a), 124.7 (C-6), 124.3


96

(C-5'), 121.1 (C-1'), 120.0 (C-8), 116.3 (C-3'), 116.2 (C-4a), 56.2 (C-7'), 49.9 (C-9),

28.5 (C-12), 21.6 (C-8'), 14.3 (C-10).

Niveamycin C (38)

Yellow amorphous solid; HRESIMS m/z 369.1509 [M+H]+ (calcd. for C20H17O7,

369.0896); UV (MeOH) λmax (log ε): 206 (4.37), 283 (3.84), 398 (3.30); 1H NMR

(MeOD-d4, 600 MHz) 1H NMR (MeOD-d4, 600 MHz) δH 7.67 (1H, dd, J = 8.3, 7.6 Hz,

H-7), 7.56 (1H, dd, J = 7.6, 1.0 Hz, H-8), 7.43 (1H, s, H-5'), 7.23 (1H, dd, J = 8.3, 1.0

Hz, H-6), 7.11 (1H, s, H-3'), 3.76 (3H, s, H-7'), 3.68 (1H, s, H-9), 2.44 (3H, s, H-8'); 13C

NMR (MeOD-d4, 125 MHz) δC 182.7 (C-1), 168.0 (C-3), 167.8 (C-9'), 162.5 (C-5),

157.7 (C-2'), 139.1 (C-4'), 137.6 (C-7), 134.0 (C-8a), 128.3 (C-6') 123.3 (C-5'), 122.8

(C-6), 121.9 (C-1'), 119.2 (C-8), 116.6 (C-3'), 114.1 (C-4a), 56.2 (C-7'), 52.0 (C-9), 21.2

(C-8').


Actinofuranosin A (41) was isolated from HPLC fraction 21 and further purified by

semi-prep HPLC using a Thermo Scientific BDS Hypersil C8 column (250 x 10 mm).

Isocratic conditions of MeOH/H2O (27%/83%) were employed for 10 min, and then a


97

linear gradient to 40% MeOH was run over 20 min at a flow rate of 4 mL/min to afford

1.7 mg of 41 (3.8% dry wt). Arglecin B (42) was isolated from HPLC fraction 20 and

further purified by semi-prep HPLC using a Thermo Scientific BDS Hypersil C8

column (250 x 10 mm). Isocratic conditions of MeOH/H2O (26%/84%) were employed

for 10 min, and then a linear gradient to 33% MeOH was run over 20 min at a flow rate

of 4 mL/min to afford 1.0 mg of 42 (2.2% dry wt). HPLC fraction 18 comprising

arglecin C (39) was further purified by semi-prep HPLC using a Thermo Scientific BDS

Hypersil C8 column (250 x 10 mm). Isocratic conditions of MeOH/H2O (31%/79%)

were employed for 10 min, then a linear gradient to 36% MeOH was run over 20 min at

a flow rate of 4 mL/min to yield 1.2 mg of 43 (2.7% dry wt). HPLC fraction 4 afforded

2.4 mg of 3H-Pyrrolo[2,3-d]pyrimidine-5-carboxylic acid, 2-amino-4,7-dihydro-4-oxo-,

methyl ester (44) (5.4% dry wt).

Actinofuranosin A (41)

White amorphous solid; HRESIMS m/z 310.1509 [M+H]+ (calcd. for C13H20N5O4,

310.1437); UV (MeOH) λmax (log ε): 203 (4.08), 216 sh (3.93), 272 (3.59); 1H NMR

(MeOD-d4, 600 MHz) 1H NMR (MeOD-d4, 600 MHz) δH 8.26 (1H, s, H-10), 8.21 (1H,

s, H-2), 6.06 (1H, d, J = 4.7 Hz, H-1'), 4.52 (1H, t, J = 4.7 Hz, H-2'), 4.32 (1H, t, J = 4.7

Hz, H-3'), 4.18 (1H, dd, J = 8.2, 3.5 Hz, H-4'), 3.73 (1H, dd, J = 10.8, 2.9 Hz, H-5b'),


98

3.65 (1H, dd, J = 10.8, 3.8 Hz, H-5a'), 3.50 (6H, s, H-7 and H-8), 3.44 (3H, s, H-6'); 13C

NMR (MeOD-d4, 125 MHz) δC 154.8 (C-6), 153.2 (C-2), 149.8 (C-4), 138.8 (1H, s, H-

10), 119.5 (C-5), 89.7 (C-1'), 85.0 (C-4'), 76.2 (C-2'), 73.2 (C-5'), 71.8 (C-3'), 59.5 (C-

6'), 38.9 (C-7 and C-8).

Arglecin B (42)


266.1790) UV (MeOH) λmax nm (log ε): 204 (3.42), 231 (3.37), 321 (3.16). 1H NMR

(MeOD-d4, 900 MHz) δH 7.16 (1H, s, H-5), 3.19 (2H, t, J = 6.8 Hz, H-14), 2.58 (2H, d,

J = 7.1 Hz, H-7), 2.52 (2H, t, J = 7.7 Hz, H-11), 2.15 (1H, m, H-8), 1.92 (3H, s, H-17),

1.66 (2H, m, H-12), 1.54 (2H, m, H-13), 0.93 (6H, d, J = 6.8 Hz, H-9, H-10); 13C NMR

(MeOD-d4, 225 MHz) δC 173.3 (C-16), 158.3 (C-3), 157.7 (C-2), 141.1 (C-6), 122.6 (C-

5), 42.5 (C-7), 39.9 (C-14), 30.8 (C-11), 29.8 (C-13), 28.1 (C-8), 27.1 (C-12), 22.9 (C-9

and C-10), 22.5 (C-17).


99

Arglecin C (43)


252.1634) UV (MeOH) λmax nm (log ε): 276 (2.80), 369 (1.65), 396 (1.54); 1H NMR

(MeOD-d4, 600 MHz) δH 7.20 (1H, s, H-5), 3.23 (2H, t, J = 6.9, H-14), 2.60 (2H, d, J =

7.1 Hz, H-7), 2.54 (2H, t, J = 7.7 Hz, H-11), 2.17 (1H, m, H-8), 1.84 (2H, m, H-12),

1.96 (3H, s, H-17), 0.95 (6H, d, J = 6.8 Hz, H-9, H-10). 13C NMR (MeOD-d4, 150

MHz) δC 173.7 (C-16), 159.0 (C-3), 158.2 (C-2), 140.3 (C-6), 122.5 (C-5), 42.1 (C-7),

39.2 (C-14), 29.2 (C-12), 28.2 (C-11), 27.7 (C-8), 22.6 (C-9 and C-10), 22.2 (C-17).

Chapter 8: Conclusions

100

Chapter 8: Conclusions

The remarkable chemical diversity encompassed by marine and terrestrial

microorganisms continues to be of relevance to drug discovery programs. Efforts to

identify new and novel natural products from under-explored environments include

approaches that are based on the premise that adaptation to unique environments will

include the production of new microbial metabolites and that those environments will

contain novel microbial strains. However, the identification of phylogenetic uniqueness

does not guarantee that the organism has the necessary biosynthetic machinery to

produce new or novel metabolites or that the pathway is expressed rather than being

cryptic. NMR metabolomics fingerprints offer the advantage that it detects, in a

quantitative fashion, all drug-like natural products produced by a microorganism. This

research demonstrated that the NMR-guided metabolic fingerprinting approach is an

effective method which can be used to rapidly prioritise and select LLE fractions

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Appendix I: CD NMR data list for thesis compounds

NMR spectra were recorded on a Varian 500 or 600 MHz Unity INOVA and referenced

to residual proto-deutero solvent signals in DMSO-d6 (δH 2.49, δC 39.51 ppm), MEOD-

d4 (δH 3.31, δC 49.15 ppm) or Acetone-d6 (δH 2.05, δC 206.68 ppm). Electronic copies of

1H and 13C NMR spectra of the new natural products are given as a word document file

on a CD-ROM attached to the back cover page of the thesis.

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Appendix II: Journal manuscript

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