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
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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
3.3 Results and discussion ................................................................................................................ 33
Chapter 4: 1H NMR Fingerprinting of Streptomyces sp. USC 593 ................................................. 47
4.1 Introduction ................................................................................................................................. 47
4.2 Results and discussion ................................................................................................................ 49
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
5.2 Results and discussion ................................................................................................................ 71
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
Figure 4. Neighbour-joining phylogenetic tree based on partial 16S rDNA sequences for
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 11. 1H NMR fingerprint spectrum of LLE fraction 2 at 600 MHz in MeOD-d4 ....................... 36
Figure 12. 1H NMR fingerprint spectrum of LLE fraction 4 at 600 MHz in MeOD-d4 ....................... 36
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 21. 1H NMR fingerprint spectrum of LLE fraction 4 at 600 MHz in MeOD-d4 ....................... 50
Figure 22. 1H NMR fingerprint spectrum of LLE fraction 3 at 600 MHz in MeOD-d4 ....................... 50
Figure 23. 1H NMR fingerprint spectrum of LLE fraction 5 at 600 MHz in MeOD-d4 ....................... 51
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
Figure 34. Neighbour-joining phylogenetic tree based on partial 16S rDNA sequences for
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
Chapter: 1 Introduction
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]
Chapter: 1 Introduction
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]
Chapter: 1 Introduction
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,
Chapter: 1 Introduction
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
Chapter: 1 Introduction
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
Chapter: 1 Introduction
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
Chapter: 1 Introduction
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]
Chapter: 1 Introduction
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]
Chapter: 1 Introduction
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
Chapter: 1 Introduction
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
Chapter: 1 Introduction
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
Chapter: 1 Introduction
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
Chapter 2: Biological investigations of termite-gut associated actinomycetes
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]
Chapter 2: Biological investigations of termite-gut associated actinomycetes
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
Chapter 2: Biological investigations of termite-gut associated actinomycetes
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]
Chapter 2: Biological investigations of termite-gut associated actinomycetes
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]
Chapter 2: Biological investigations of termite-gut associated actinomycetes
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.
Chapter 2: Biological investigations of termite-gut associated actinomycetes
20
Figure 4. Neighbour-joining phylogenetic tree based on partial 16S rDNA sequences showing the
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
Chapter 2: Biological investigations of termite-gut associated actinomycetes
21
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
Chapter 2: Biological investigations of termite-gut associated actinomycetes
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.
Chapter 2: Biological investigations of termite-gut associated actinomycetes
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
Chapter 2: Biological investigations of termite-gut associated actinomycetes
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
Chapter 2: Biological investigations of termite-gut associated actinomycetes
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
Chapter 2: Biological investigations of termite-gut associated actinomycetes
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).
Chapter 2: Biological investigations of termite-gut associated actinomycetes
27
Chapter 2: Biological investigations of termite-gut associated actinomycetes
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
Chapter 2: Biological investigations of termite-gut associated actinomycetes
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
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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.
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
33
3.3 Results and discussion
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
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
36
Figure 11. 1H NMR fingerprint spectrum of LLE fraction 2 at 600 MHz in MeOD-d4
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.
Figure 12. 1H NMR fingerprint spectrum of LLE fraction 4 at 600 MHz in MeOD-d4
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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,
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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.
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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
Chapter 3: 1H NMR fingerprints of Streptomyces sp. USC 592
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.
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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]
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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
4.2 Results and discussion
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
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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.
Figure 21. 1H NMR fingerprint spectrum of LLE fraction 4 at 600 MHz in MeOD-d4
Figure 22. 1H NMR fingerprint spectrum of LLE fraction 3 at 600 MHz in MeOD-d4
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
51
Figure 23. 1H NMR fingerprint spectrum of LLE fraction 5 at 600 MHz in MeOD-d4
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).
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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),
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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)
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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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]
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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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]
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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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]
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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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
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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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
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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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.
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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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
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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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
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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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.
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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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)
Chapter 4: 1H NMR fingerprints of Streptomyces sp. USC 593
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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
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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
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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.
5.2 Results and discussion
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
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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.
Figure 34. 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
Chapter 5: Actinofuranosin A and arglecins B and C from a Streptomyces sp. USC 597
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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
Chapter 5: Actinofuranosin A and arglecins B and C from a Streptomyces sp. USC 597
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
Chapter 5: Actinofuranosin A and arglecins B and C from a Streptomyces sp. USC 597
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,
Chapter 5: Actinofuranosin A and arglecins B and C from a Streptomyces sp. USC 597
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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
Chapter 5: Actinofuranosin A and arglecins B and C from a Streptomyces sp. USC 597
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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
Chapter 5: Actinofuranosin A and arglecins B and C from a Streptomyces sp. USC 597
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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
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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
Chapter 5: Actinofuranosin A and arglecins B and C from a Streptomyces sp. USC 597
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]
Chapter 5: Actinofuranosin A and arglecins B and C from a Streptomyces sp. USC 597
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
Chapter 7: Experimental
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,
Chapter 7: Experimental
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).
Chapter 7: Experimental
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
Chapter 7: Experimental
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
Chapter 7: Experimental
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
Chapter 7: Experimental
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]
Chapter 7: Experimental
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)
Chapter 7: Experimental
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.
Chapter 7: Experimental
93
Actinoglycosidine B (28)
Amorphous colourless solid; HRESIMS m/z [M+H]+ 393.1513 (calcd. for C16H21N6O6,
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)
Chapter 7: Experimental
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).
7.10 Chapter 4: Experimental
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);
Chapter 7: Experimental
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
Chapter 7: Experimental
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').
7.11 Chapter 5: Experimental
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
Chapter 7: Experimental
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'),
Chapter 7: Experimental
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)
Amorphous colourless solid; HRESIMS m/z [M+H]+ 266.1867 (calcd. for C14H24N3O2,
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).
Chapter 7: Experimental
99
Arglecin C (43)
Amorphous colourless solid; HRESIMS m/z [M+H]+ 252.1715 (calcd. for C13H22N3O2,
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
comprising small molecules with unusual spectral patterns.
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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.
113
Appendix II: Journal manuscript
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