Antibiotics Targeting Tuberculosis: Biosynthesis of A-102395 and Discovery of Novel Actinomycins

University of Kentucky UKnowledge

Theses and Dissertations--Pharmacy College of Pharmacy

2015

ANTIBIOTICS TARGETING TUBERCULOSIS: BIOSYNTHESIS OF A-102395 AND DISCOVERY OF NOVEL ACTINOMYCINS

Wenlong Cai University of Kentucky, [email protected]

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Recommended Citation Cai, Wenlong, "ANTIBIOTICS TARGETING TUBERCULOSIS: BIOSYNTHESIS OF A-102395 AND DISCOVERY OF NOVEL ACTINOMYCINS" (2015). Theses and Dissertations--Pharmacy. 52. https://uknowledge.uky.edu/pharmacy_etds/52

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REVIEW, APPROVAL AND ACCEPTANCE

The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above.

Wenlong Cai, Student

Dr. Steven Van Lanen, Major Professor

Dr. Jim Pauly, Director of Graduate Studies

ANTIBIOTICS TARGETING TUBERCULOSIS: BIOSYNTHESIS OF A- 102395 AND DISCOVERY OF NOVEL ACTINOMYCINS

DISSERTATION

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Pharmacy at the University of Kentucky

By Wenlong Cai

Lexington, Kentucky

Director: Dr. Steven Van Lanen，Professor of Pharmaceutical Science

Lexington, Kentucky

2015

ABSTRACT OF DISSERTATION

ANTIBIOTICS TARGETING TUBERCULOSIS: BIOSYNTHESIS OF A- 102395 AND DISCOVERY OF NOVEL ACTINOMYCINS

The increase in antibiotic resistance of many bacterial strains including multidrug- resistant tuberculosis (MDR-TB) due to over- and misuse of antibiotics is a serious medical and economical problem. Therefore discovery and development of new antibiotics are urgently needed. Two projects were undertaken to address the need for new anti- tuberculosis antibiotics. 1. Discovery of new chemical entities. A-102395, a new nucleoside inhibitor of bacterial MraY (translocase I, EC 2.7.8.13) that is essential for bacterial survival, was isolated from the culture broth of Amycolatopsis sp. SANK 60206 in 2007. Although A-102395 is a potent inhibitor of translocase I with IC50 of 11 nM, it contradictingly does not have any antibiotic activity. A-102395 is a derivative of capuramycin with a unique aromatic side chain. A semisynthetic derivative of capuramycin is currently in clinical trials as an anti- tuberculosis antibiotic, suggesting potential for using A-102395 as a starting point for antibiotic discovery. The biosynthetic gene cluster of A-102395 was identified, including 35 putative open reading frames responsible for biosynthesis and resistance. A series of gene inactivation abolished the A-102395 production, indicating those genes within the cluster are essential for A-102395 biosynthesis. Functional characterization of Cpr17, which has sequence similarity to aminoglycoside phosphotransferases, revealed that it functions as a phosphotransferase conferring self-resistance by using GTP as phosphate donor. Furthermore the enzyme is characterized by low substrate specificity, as Cpr17 was capable of modifying a large series of natural or semi-synthesized analogues of capuramycins. A series of organism-specific high-throughput screening models for potential antibacterial agents targeting on bacterial cell wall synthesis have been established, including Escherichia coli and Mycobacterium tuberculosis. For this screen ten enzymes were successfully used to reconstitute cell wall biosynthesis in vitro. This screening is expected to allow us to identify the targets of novel antibiotics rapidly and in a cost- efficient manner 2. Rediscovering old antibiotics. As part of our long term goal of discovering and developing novel anti-tuberculosis antibiotics, four novel actinomycins were isolated from the scale-up fermentation of Streptomyces sp. Gö-GS12, and their structures were characterized using mass spectrometry and 1D and 2D NMR. Their antibacterial activity against Gram-positive and Gram- negative strains were determined, as well as their cytotoxicity. Key words: capuramycins, peptidoglycan cell wall, phosphotransferase, actinomycins.

Wenlong Cai

Student’s Signiture

9/22/2015

Date

ANTIBIOTICS TARGETING TUBERCULOSIS: BIOSYNTHESIS OF A- 102395 AND DISCOVERY OF NOVEL ACTINOMYCINS

by Wenlong Cai

Steven Van Lanen

Director of Dissertation

Jim Pauly

Director of Graduate Studies

9/22/2015

To Ling and Lucy

ACKNOWLEDGMENTS

I would like to take this moment to express my gratitude to my mentor, Dr. Steven Van Lanen, for his guidance, support, and inspiration throughout my study in his group. His expertise and insightful discussions have benefited me greatly. I would also like to thank my committee members, Dr. Jürgen Rohr, Dr. Kyung-Bo Kim, and Dr. Joe Chappell, for their invaluable advice and comments over the years, and I would like to sincerely thank Dr. Aardra Kachroo for being the external- department examiner for my dissertation defense.

Special thanks to all current and previous members from the Van Lanen group: Dr. Zhaoyong Yang, Dr. Sandra Barnard, Dr. Xiuling Chi, Dr. Anwesha Goswami, Dr. Xiaodong Liu, Zheng Cui, Matthew McErlean, Ashley Arlinghaus, and Tyler Bucci. It was a great honor to work with and learn from you all!

And last but not least, I would like to acknowledge my family and friends for their constant support and encouragement.

iii

TABLE OF CONTENT

ACKNOWLEDGMENTS ...... iii TABLE OF CONTENTS ...... iv LIST OF FIGURES ...... vii LIST OF SCHEMES ...... x LIST OF TABLES ...... xi LIST OF ABBREVIATIONS ...... xii Chapter 1: Introduction and Background ...... 1 1.1 Need for new anti-tuberculosis antibiotics ...... 1 1.2 Significance of natural products in antibiotic discovery ...... 3 1.3 Current antibacterial targets ...... 4 1.4 Biosynthesis of peptidoglycan cell wall ...... 6 1.5 MraY: structure and inhibitors ...... 9 1.6 Targeting bacterial MraY: discovery of capuramycins ...... 13 1.7 Binding to DNA: a brief history of actinomycins ...... 16 1.8. Aims of this study ...... 20 Chapter 2: Characterization of the biosynthetic gene cluster for A-102395 and reconstituting bacterial cell wall biosynthesis ...... 21 2.1 Background ...... 21 2.2 Materials and methods ...... 27 2.2.1 Instrumentation, chemicals and reagents ...... 27 2.2.2 Identification and characterization of A-102395 biosynthetic gene cluster ...... 29 2.2.2.1 Isolation of A-102395, A-503083B and A-503083F ...... 29 2.2.2.2 Amplification of disruption cassette ...... 29 2.2.2.3 Introduction of pNCap02 and pNCap04 into E. coli BW25113/pIJ790 strain ...... 31 2.2.2.4 PCR targeting of supercosmids ...... 32 2.2.2.5 Transfer of mutant cosmids into Amycolatopsis sp. SANK 60206 ...... 34 2.2.2.6 Isolation of genomic DNA of wild-type and mutant Amycolatopsis sp. SANK 60206 35 2.2.2.7 PCR analysis of wild-type and mutant Amycolatopsis sp. SANK 60206 ...... 36 2.2.2.8 Preparation of the DIG-labeled probes ...... 36 2.2.2.9 Digestion of genomic DNA ...... 38 iv 2.2.2.10 DNA:DNA hybridization and detection ...... 38 2.2.2.11 LC-MS analysis of production of A-102395 in mutant strains ...... 38 2.2.2.12 Transcriptional analysis of Δcpr19, Δcpr25 and Δcpr51 strains ...... 3 9 2.2.2.13 Heterologous expression and functional assignment of Cpr19 and CapH ...... 41 2.2.3 Characterization of Cpr17 ...... 42 2.2.3.1 Heterologous production of Cpr17 and CapP in E. coli ...... 42 2.2.3.2 Functional characterization of Cpr17 and CapP ...... 44 2.2.3.3 Kinetic characterization of Cpr17 ...... 45 2.2.3.4 Chemical and semi-synthesis of unnatural substrates for Cpr17 ...... 46 2.2.3.5 Site-directed mutagenesis of Cpr17 ...... 48 2.2.3.6 Docking of A-503083E to MraY and development of in-silico models for Cpr17 ...... 49 2.2.4 Reconstitution of bacterial cell wall biosynthesis pathway ...... 49 2.2.4.1 Reconstitution of E. coli cell wall biosynthesis pathway ...... 49 2.2.4.2 Reconstitution of M. tuberculosis H37Rv cell wall biosynthesis pathway ...... 52 2.3 Results and discussion ...... 54 2.3.1 Identification and characterization of A-102395 gene cluster ...... 54 2.3.1.1 Isolation of capuramycin-type antibiotics ...... 54 2.3.1.2 Identification of A-102395 gene cluster ...... 56 2.3.1.3 Characterization of the gene cluster for 3 by inactivation of cpr19, cpr25 and cpr51. 61 2.3.1.4 Functional characterization of Cpr19 and CapH ...... 67 2.3.1.5 Proposed biosynthetic pathway of α-D-mannopyranuronate moiety and formation of polyamide side chain for 3 ...... 69 2.3.2 Functional characterization of Cpr17 as a phosphotransferase ...... 73 2.3.2.1 Functional characterization of Cpr17 ...... 73 2.3.2.2 Kinetic characterization of Cpr17...... 80 2.3.2.3 Promiscuity of Cpr17 ...... 82 2.3.2.4 Predicted structure of Cpr17 ...... 87 2.3.3. Reconstitution of bacterial cell wall biosynthesis in vitro...... 93 2.4 Conclusion ...... 101 Chapter 3: Discovery of novel actinomycins as potential anti-tuberculosis agents* ...... 104 3.1 Background ...... 104 3.2 Materials and methods ...... 106 3.2.1 Instrumentation, chemicals and bacterial strains ...... 106

v 3.2.2 Isolation of actinomycins ...... 107 3.2.3 Determination of amino acids configuration ...... 109 3.2.4 Synthesis of Fmoc-depsipeptide and 2-amino-3-hydroxy-4-methylbenzamide ...... 109 3.2.5 Cytotoxicity assay* ...... 114 3.2.6 Antibacterial activity assay**...... 115 3.3 Results and discussion ...... 117 3.3.1 Isolation of actinomycins from S. sp. Strain Gö-GS12 ...... 117

3.3.1.1 Isolation of actinomycin Y1, Y3 and Y4 ...... 117 3.3.1.2 Structural elucidation of 1 ...... 117 3.3.1.3 Structural elucidation of 2 ...... 128 3.3.1.4 Structural elucidation of 3 ...... 130 3.3.1.5 Structural elucidation of 4 ...... 132 3.3.1.6 Structural elucidation of 5 ...... 134 3.3.1.7 Summary of physicochemical properties of novel actinomycins ...... 136 3.3.2 Biological activity of actinomycins ...... 141 3.3 Conclusion ...... 144 Chapter 4: Summary ...... 147 References ...... 149 Appendix I...... I-1 Appendix II...... II-1 Vita...... A

vi LIST OF FIGURES

Figure 1.1. Structure of peptidoglycan cross-linking ...... 6 Figure 1.2. Summary of the peptidoglycan biosynthetic pathway ...... 8 Figure 1.3. MraY-catalyzed reaction...... 9

Figure 1.4. Structure of MraYAA ...... 10

Figure 1.5. The active site of MraYAA ...... 11 Figure 1.6. Representatives of different classes of MraY inhibitors ...... 13 Figure 1.7. Structures of capuramycin-type antibiotics discussed in this dissertation ...... 15 Figure 1.8. The structure (left) and stick model ...... 17 Figure 1.9 Stereoscopic drawings of the crystal structure of the actinomycin D-

(ATGCTGCAT)2 complex ...... 18 Figure 2.1.1. Isotopic enrichment studies using the indicated 13C-labeled precursors ...... 21 Figure 2.1.2. Structure and biosynthesis of A-90289, a GlyU-containing nucleoside antibiotic ...... 23 Figure 2.2.1. Map of pIJ773 plasmid...... 30 Figure 2.3.1. 1H and 13C NMR spectra of A-102395 in MeOD ...... 54 Figure 2.3.2. HRMS of purified A-503083F ...... 55 Figure 2.3.3. HRMS of purified A-503083B ...... 55 Figure 2.3.4 Cosmid map and genetic organization of the 3 biosynthetic gene cluster ...... 57 Figure 2.3.5. Comparative analysis of the sequenced region of chromosomal DNA from the 2- and 3-producing strains ...... 58 Figure 2.3.6. Inactivation of cpr19 in Amycolatopsis sp. SANK 60206...... 62 Figure 2.3.7. Inactivation of cpr25 in Amycolatopsis sp. SANK 60206...... 63 Figure 2.3.8. Inactivation of cpr51 in Amycolatopsis sp. SANK 60206...... 64 Figure 2.3.9. Analysis of gene inactivation in Amycolatopsis. Sp. SANK 60206 ...... 65 Figure 2.3.10. RT-PCR analysis of the indicated genes using mRNA isolated from the mutant strains or wild-type strain ...... 66 Figure 2.3.11. Biosynthesis of CarU of A-102395...... 68 vii Figure 2.3.13. The ATP-dependent phosphorylation reaction catalyzed by CapP ...... 73 Figure 2.3.14. Resistance conferred to Streptomyces albus upon heterologous expression of Cpr17 ...... 74 Figure 2.3.15. Expression of recombinant CPR17 and CapP ...... 75 Figure 2.3.16. HPLC analysis of the reaction catalyzed by Cpr17 and CapP ...... 77 Figure 2.3.17. HRMS analysis for the product of reactions catalyzed by Cpr17 using 2a as substrate...... 78 Figure 2.3.18. Mass spectrum for the products of reactions catalyzed by Cpr17...... 79 Figure 2.3.19. Plots for single-substrate kinetic analysis of Cpr17 ...... 81 Figure 2.3.20. Phosphorylation of unnatural capuramycin derivatives by Cpr17...... 82 Figure 2.3.21. HPLC analysis of reactions catalyzed by Cpr17 using unnatural capuramycins as substrates ...... 84 Figure 2.3.22. Mass spectrum for the products of reactions catalyzed by Cpr17 using unnatural capuramycins as substrates ...... 85 Figure 2.3.23. Docking 2d to the active site of MraY ...... 86 Figure 2.3.24. Sequence alignment of Cpr17, CapP to APHs using Clustal Omega...... 89 Figure 2.3.25. Structure comparison of APH and predicted Cpr17 ...... 90 Figure 2.3.26. Alignment of GTP binding site of APH(2’’)-IIIa (light blue) and Cpr17 (green)...... 91 Figure 2.3.27. Mutant Cpr17 exhibited altered activity ...... 91 Figure 2.3.28. Crystal of Cpr17 obtained from high throughput screening ...... 92 Figure 2.3.29. Expression of MurA-MurF and Ddl involed in E. coli cell wall synthesis ...... 94 Figure 2.3.30. NahK and GlmU convert GlcNAc to UDP-GlcNAc ...... 94 Figure 2.3.32. HPLC and HRMS analysis of UDP-MurNAc-pentapeptide ...... 95 Figure 2.3.33. Reconstituting E. coli bacterial cell wall biosynthesis ...... 97 Figure 2.3.34. Reconstituting M. tuberculosis bacterial cell wall biosynthesis ...... 99 Figure 2.3.35. HPLC analysis of the reaction catalyzed MurB ...... 100

Figure 3.1.1. Structure of actinomycin Y5 ...... 105 Figure 3.2.1. Isolation scheme for actinomycins from 5 L fermentation broth...... 108

Figure 3.3.1. Simultaneous conversion of 1 to Y3 under acidic condition...... 125 viii Figure 3.3.2. Chiral amino acid analysis of 1...... 126 Figure 3.3.3. HPLC profile of different actinomycins produced by Streptomyces sp. Strain Gö-GS12 ...... 137 Figure 3.3.4 UV/VIS spectra of actinomycins isolated from Streptomyces sp. Strain Gö- GS12...... 138 Figure 3.3.5. CD spectra of actinomycins isolated from Streptomyces sp. Strain Gö-GS12...... 139 Figure 3.3.6. Conformational changes of α- and β-ring of actinomycins ...... 146

ix LIST OF SCHEMES

Scheme 2.1. Synthesis of A-503083G ...... 46 Scheme 2.2. Synthesis of P1 – P4 catalyzed by CapW as N-acyltranferase ...... 47 Scheme 2.3. Synthesis of P5-P10 catalyzed by CapW as a transacylase...... 47 Scheme 3.1. Synthesis of Fmoc-depsipeptide ...... 110 Scheme 3.2. Synthesis of phenol-4...... 113

x LIST OF TABLES

Table 2.2.1. Primers for in-frame deletion...... 30 Table 2.2.2. Primers for amplification of cpr19, cpr25 and cpr51 ...... 32 Table 2.2.3. Primers for amplification of disruption cassette...... 33 Table 2.2.4. Primers for amplification of DNA probes for Southern blot analysis ...... 37 Table 2.2.5. Primers for transcriptional analysis of mutant and wild-type strains ...... 41 Table 2.2.6. Primers for amplification of cpr17 and capP ...... 42 Table 2.2.7. Substrates for enzymatic preparation of unnatural capuramycins* ...... 48 Table 2.2.8. Primers for mutagenesis of Cpr17...... 49 Table 2.2.9. Primers for amplification of NahK, GlmU and GDH...... 50 Table 2.2.10. Primers for amplification of Mur liagases from M. tuberculosis ...... 53 Table 2.3.1. Annotation of ORFs within the 3 gene cluster ...... 59 Table 2.3.2. Sequence comparison in % identity/% similarity among putative αKG:UMP dioxygenases ...... 61 Table 2.3.3. Michaelis-Menten kinetic parameters of CapP and Cpr17...... 80 Table 2.3.4. Semi-synthesized substrates for Cpr17 activity assay ...... 83 Table 2.3.5. Sequence comparison of MurA-MurF from different species in % identity/% similarity ...... 98

Table 3.2.1. NMR data of 6 in CDCl3 (1H: 400 MHz, 13C: 100 MHz, J in Hz) ...... 112

Table 3.3.1. NMR data of actinomycin Y6 (1) in CD3OD ...... 127

Table 3.3.2. NMR data of actinomycin Y7 (2) in CDCl3 ...... 129

Table 3.3.3. NMR data of actinomycin Y8 (3) in CDCl3 ...... 131

Table 3.3.4. NMR data of actinomycin Y9 (4) in CDCl3 ...... 133

Table 3.3.5. NMR data of actinomycin Zp (5) in CDCl3 ...... 135 Table 3.3.6: Overview on naturally occurring actinomycinsa ...... 140

Table 3.3.6. In Vitro antimicrobial activities of 1-5 and actinomycins Y1, Y3, Y4 and D. . 142 Table 3.3.7. Cytotoxic activity of actinomycins against different tumor cell lines ...... 143

xi LIST OF ABBREVIATIONS

2D-NMR two-dimensional nuclear magnetic resonance spectroscopy

7-AAD 7-aminoactinomycin

ACL aminocaprolactam

ADME absorption, distribution, metabolism and excretion amp ampicillin apr apramycin

ATP adenosine triphosphate

BLAST basic local alignment search tool

CarU uridine-5'-carboxamide chl chloramphenicol

COSY correlation spectroscopy

DAP diaminopimelic acid

DCM dichloromethane

Ddl D-Ala-D-Ala ligase

DIG digoxigenin

DMF dimethylformamide

DMSO dimethylsulfoxide

DNA deoxyribonucleic acid

DNA deoxyribonucleic acid E. coli Escherichia coli

EC50 half maximal effective concentration EDTA ethylenediaminetetraacetic acid

xii

FRT flippase recongition target

GDP guanosine diphosphate GlcNAc N-acetylglucosamine

GlyU (5′S,6′S)-5'-C-glycyluridine

GTP guanosine monophosphate

His6 hexahistidine HMBC heteronuclear multiple-bond correlation spectroscopy

HPLC high performance liquid chromatography hr hour

HRMS high-resolution mass spectrometry

HSQC heteronuclear single-quantum correlation spectroscopy

HTS high-throughput screening

IPTG isopropyl-β-D-thiogalactopyranoside k kilo kan kanamycin kb kilo base pairs kcat turnover rate kDa kilo Dalton

Km Michaelis-Menten constant LB Luria broth

LC-MS liquid chromatography–mass spectrometry

Lys lysine

MDR multiple drug resistant pathogens

MIC minimum inhibitory concentration min minute

MraY phospho-MurNAc-pentapeptide translocase

viii

MRM multiple reaction monitoring

MRSA methicillin-resistant Staphylococcus aureus MurNAc N-acetylmuramic acid

MW molecular weight

NADP+ nicotinamide adenine dinucleotide phosphate

NADPH reduced form of NADP+

NDP nucleoside diphosphate

NME new molecular entities

NMR nuclear magnetic resonance spectroscopy

NOESY nuclear overhauser effect spectroscopy

NTP nucleoside triphosphate

OD optical density

ORF open reading fram

OriT origin of transfer

PABA para-amino-benzoic acid

PAGE polyacrylamide gel electrophoresis

PBP penicillin binding protein

PCR polymerase chain reaction

PEP phosphoenolpyruvic acid or phosphoenolpyruvate

RNA ribonucleic acid rt room temperature

SAR structure-activity relationship

SDS sodium dodecyl sulfate

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

xiv

SHMT serine hydroxymethyltransferase sp. Species sp. Streptomyces

TB tuberculosis

TEA triethylamine

TFA trifluoroacetic acid

THF tetrahydrofolic acid

TOCSY total correlation spectroscopy

UA uridine 5'-aldehyde

UDP uridine-5′-diphosphate

UDP-GlcNAc5-diphospho-N-acetylglucosamine

UDP-MurNAcUDP-N-acetylmuramic acid

UMP uridine monophosphate

UTP uridine-5′-triphosphate

UV/Vis ultraviolet/visible

VRE vancomycin-resistant Enterococci

WT wildtype α-KG α -ketoglutarate

Chapter 1: Introduction and Background

1.1 Need for new anti-tuberculosis antibiotics

Despite the remarkable success of antibiotics, infectious diseases are still one of the highest causes of death worldwide leading to over 17 million people deaths each year

(1). This is largely due to the continually increasing number of bacteria that are resistant to antibiotics as a result of both intrinsic or acquired mechanisms (2). Bacterial resistance to every major classes of antibiotics has been developed shortly after their first clinical use (3), and the emergence of multiple drug resistant pathogens (MDR) such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant

Enterococci (VRE) has become serious hospital and community threat. It was estimated that MDR pathogens caused over 2 million infections and 23,000 of deaths in the USA in 2013 alone (4).

The rise of antibiotic resistance is typically associated with overuse and misuse of antibiotics. It was reported that the highest rates of antibiotic resistance were seen in those areas with the highest rate of antibiotic prescription (5). Along with increased drug resistance among pathogens, the discovery and development of new antibiotics, however, has dramatically declined since the “golden era” of antibiotics in 1950s when most classes of antibiotics were discovered. Over the past 40 years, only five new classes of antibiotics have been approved by FDA (6,7), and currently there are no new molecular entities (NME) in phase III clinical trials within the United States. Therefore discovery and development of new antibiotics remains a high priority.

1 Another major threat today is tuberculosis (TB) caused primarily by Mycobacterium tuberculosis, which is estimated to currently infect 2 billion people worldwide with 9 million new cases diagnosed each year (8). It is likewise estimated that TB is the cause

1.3 million deaths annually (9), making it the second largest infectious disease killer. The complicated mycolic acid-containing cell wall structure of M. tuberculosis makes it naturally resistant to most of the antibiotics such as penicillin. The cell-wall architecture is composed of peptidoglycan which is covalently linked to a linear galactofuran via an

L-Rha-D-GlcNAc-P linker, and several strands of highly branched arabinofuran which is further attached to mycolic acids (10). The thick layer of lipid on the outer part of the cell protects the tuberculosis bacillus from noxious chemicals and allows the bacterium to survive from host’s immune system by living inside macrophages (11). Thus TB is difficult to treat and it usually takes six months to cure an active TB infection with a combination of first-line drugs (isoniazid, rifampin, ethambutol and pyrazinamide) (12).

Like other pathogens, there has been a steady increase in reported cases of drug- resistant M. tuberculosis. Multidrug-resistant tuberculosis (MDR-TB) which is not treatable by isoniazid and rifampicin is becoming an increasing threat to public health in many developing countries, and extensively drug-resistant TB (XDR-TB) which is resistant to not only isoniazid and rifampicin but at least one additional anti-TB drug has been reported in more than 100 countries (9). The World Health Organization (WHO) estimated that 1.1 million (13%) of the TB patients worldwide were HIV-positive resulting

360,000 deaths from HIV-associated TB in 2013 (9). MDR-TB generally requires 20-24 months of treatment with additional five or six second-line drugs that are less effective and more toxic than first-line regimen (12).

2 1.2 Significance of natural products in antibiotic discovery

Natural products have played an important role in the discovery of antibacterial agent since the introduction of penicillin in the 1940s (13). Until 2012, natural products or derivatives of natural products contributed about 75% of total approved antibacterial agents (14). Although antibiotics have been discovered from many organisms ranging from unicellular bacteria to eukaryotic fungi, and from plants to marine invertebrates, nearly 80% of the antibiotics have been discovered from Actinomycetes (15). Many important classes of antibiotics can only be found in those producers including polyene macrolides, polyether antibiotics, cyclopolylactons, most aminoglycosides, actinomycins and quinoxaline-peptides (16).

In recent years, screening of natural products from microbial products has diminished because the discovery of novel natural products with useful antibiotic properties has become more expensive and laborious (17). However, it is increasingly clear that there is still large amount of natural products from diverse microorganisms from underexplored ecological niches that have yet to be identified. For example, it was estimated that 99% of the bacteria are “unculturable” (18) in conventional microbiology labs, and that 90-99% of the metagenome is not being sampled (19). Furthermore, the majority of bacteria operons for secondary metabolites are “silent” or cryptic under the typical growth conditions in the laboratory (20) while marine microbial communities have not yet been systematically collected and studied for bioactive secondary metabolites (17). Thus natural products are still promising sources for novel antibiotic discovery by applying new technologies including improved (21) or culture-independent molecular methods

(22), activating silent or cryptic biosynthetic gene clusters (23) and exploring microbial

3 natural products and their producing organisms discovered from unique environments such as deep oceans (24). In addition, many “old” antibiotics were abandoned for clinic use due to poor pharmacokinetics or lack of activity. Researchers who now access to new technologies such as whole-genome sequencing can identify biosynthetic pathways of antibiotics and possible resistant genes, genetic engineering and computer-assisted drug design will be able to overcome difficulties in development of natural products

(21,25,26).

1.3 Current antibacterial targets

There are over 200 conserved essential proteins in bacteria providing multiple targets for antibiotics to inhibit growth (3). Nonetheless, traditional screens used for antibiotic discovery have led to the identification of only a few metabolic pathways that utilize proteins that are able to be inhibited by small molecules leading to arrest of cell growth or cell death. The most successful of these pathways have been i) bacterial protein synthesis, ii) folic acid metabolism, iii) bacterial DNA replication and repair and iv) bacterial cell wall biosynthesis (27).

Bacterial ribosomes play a key role in protein synthesis in initiation, elongation and termination of protein assembly (28), and have several differences compared to eukaryotic ribosomes, making it an important target for antibacterial agents. A large number of clinically used antibiotics target the complex translational ribonucleoprotein machinery including erythromycin (interferes with aminoacyl translocation (29)), tetracyclines (30) and aminoglycosides (31) targeting on the ribosomal decoding site by blocking the attachment of aminoacyl-RNA (27). Tetrahydrofolic acid (THF) is essential

4 for bacteria as it serves as a precursor of thymidine synthesis. Antibiotics targeting on different steps of the biosynthetic pathway of THF such as trimethoprim (32) and sulfamethoxazole (33) are used to inhibit the formation of THF and thus bacterial DNA synthesis. Antibiotics that inhibit DNA replication, transcription, and repair can be divided into two categories: those that inhibit enzymes involved in this process and those that directly bind to DNA. Ciprofloxacin, a member of fluoroquinolones, inhibits DNA gyrase by forming a complex with the enzyme and double-cleaved DNA (34) and rifampin inhibits bacterial DNA-dependent RNA synthesis by binding to the RNA polymerase (35).

The antibiotics that directly interact with DNA by forming complexes with DNA or chemically modifying DNA usually show very low specificity, resulting in high toxicity.

Bleomycins bind to DNA and induce scission by abstracting the hydrogen atom from the base, resulting multiple DNA strand breaks (36), and mitomycins covalently and irreversibly bind to DNA (37). Actinomycins form complexes with DNA (also known as intercalators), and the high toxicity limits their clinical use as antibiotics (38). Many of these nonspecific antibiotics, however, have instead found utility in the clinic as anticancer agents.

The peptidoglycan layer of cell wall is an essential component of bacterial cell wall providing protection to bacterial cells from internal osmotic pressure, and thus is required for the survival of nearly all bacteria (39). The backbone of peptidoglycan consists of a

β-1,4-linked polysaccharide of alternating N-acetylglucosamine (GlcNAc) and N- acetylmuramic acid (MurNAc) units with a pentapeptide side chain attached to each lactyl group of MurNAc. The backbone is conserved in all bacteria, while primarily two different types of pentapeptide chain are utilized with the general structure of L-Ala-γ-

5 D-Glu-X-D-Ala-D-Ala, where X is the variable component. Most gram-positive bacteria have an L-lysine (L-Lys) as the third amino acid, and a meso-diaminopimelic acid (m-

DAP) is usually found in gram-negative bacteria (40). The peptide component of peptidoglycan is covalently cross-linked via transpeptidation between the ε-amino groups of the Lys/DAP residue and the D-Ala residue of a second strand, establishing the final structure of cell wall (fig 1.1). Multiple steps are required for the assembly of the peptidoglycan polymer that are unique to bacteria, making it an excellent target for many antibiotics. β-lactam-containing penicillins prevent the final transpeptidation step by covalently binding to transpeptidase (41,42), and the vancomycin family of glycopeptide antibiotics bind to the terminal D-Ala-D-Ala moiety of pentapeptide chain, preventing enzyme recognition of this substrate thus preventing peptidoglycan synthesis

(42).

Figure 1.1. Structure of peptidoglycan cross-linking (43).

1.4 Biosynthesis of peptidoglycan cell wall

The biosynthesis of peptidoglycan requires a minimum of 12 ubiquitous enzymatic reactions for both gram-positive and gram-negative bacteria (43). The earlier steps

6 proceed within the bacterial cytoplasm. The first committed step is the MurA-catalyzed transfer of the enolpyruvyl moiety from phosphoenolpyruvate (PEP) to uridine-5- diphospho-N-acetylglucosamine (UDP-GlcNAc), generating enolpyruvyl UDP-GlcNAc.

The second step is catalyzed by MurB, a NADPH-dependent reductase that converts enolpyruvyl UDP-GlcNAc to UDP-N-acetylmuramic acid (UDP-MurNAc). Subsequently, a series of highly-conserved ATP-dependent amino acid ligases (MurC-MurF) catalyze the sequential addition of L-Ala, D-Glu, L-Lys (gram-positive species) or meso-DAP

(gram-negative bacteria), and a dipeptide D-Ala-D-Ala which is formed by the ATP- dependent D-Ala:D-Ala ligase (Ddl), generating UDP-MurNAc-pentapeptide. A few inhibitors have been discovered targeting the early stages of peptidoglycan synthesis including fosfomycin, which selectively inactivates MurA (44),(45), and D-cycloserine, which targets Ddl (46) and is effective against M. tuberculosis(47). Following the formation of UDP-MurNAc-pentapeptide is the lipid cycle of cell wall biosynthesis, which is initiated by the membrane protein MraY (phospho-MurNAc-pentapeptide translocase), also known as translocase I. The reaction catalyzed by MraY involves the transfer of the

MurNAC-pentapeptide unit onto a lipid carrier (undecaprenyl phosphate) to generate lipid I, which is then converted to lipid II by incorporating a GlcNAc sugar onto the 4’- hydroxyl group of MurNAc catalyzed by a glycotransferase MurG. In certain gram- positive strains, additional amino acids may be added to lipid II from an aminoacyl-tRNA donor (48). The final cytoplasmic step in peptidoglycan synthesis following disaccharide formation is to flip lipid II to the outer leaflet of the cytoplasmic membrane by a membrane protein called flippase, but the mechanism of this step is still obscure (49). On the cell surface, lipid II undergoes polymerization into nascent peptidoglycan strand via

7 transglycosylation, which is then cross-linked by transpeptidation, catalyzed by a penicillin binding protein (PBP) (50) (fig 1.2) The undecaprenyl phosphate is released and recycled via dephosphorylation (48). Unlike most bacteria, the mycobacterial cell wall peptidoglycan has unique features including incorporation of N-glycolyl-muramic acid in polysaccharide chains and heavily cross-linked DAP-DAP bridge (51) and the

ATP-dependent MurE ligase in M. tuberculosis is highly specific to m-DAP.

Figure 1.2. Summary of the peptidoglycan biosynthetic pathway. L-Lys is found in Gram-positive bacteria while meso-DAP is found in Gram-negative strains. Antibiotics inhibit specific enzyme are highlighted in red (e.g. vancomycin inhibits transglycosylase or transpeptidase by binding to the terminal unit of peptidoglycan).

8 1.5 MraY: structure and inhibitors

The transferase activity of MraY was first characterized by Neuhaus in 1965 (52). The integral memberane enzyme catalyzes the transfer of MurNAc-pentapeptide from UDP-

MurNAc-pentapeptide to undecaprenyl phosphate, yielding undecaprenyl- pyrophosphoryl-MurNAc-pentapeptide, also known as lipid I, and UMP (fig 1.3).

Figure 1.3. MraY-catalyzed reaction.

MraY belongs to a subfamily of polyprenyl-phosphate N-acetyl hexosamine 1-phosphate transferase (PNPY) superfamily (53). Early studies on MraY led to functional characterization of recombinant MraY from several organisms (52), and the catalytic

9 mechanism has been proposed to proceed via either a one-step or a two-step mechanism. The isotopic aided experiment suggested that a two-step mechanism was more likely (52). Very recently the first crystal structure of MraY from Aquifex aeolicus

(MraYAA) was reported (54). The MraYAA crystalized as a dimer with an oval-shaped tunnel that is surrounded by hydrophobic amino acids to accommodate lipids. Each subunit consists of 10 transmembrane helices (TM), an interfacial helix, an interfacial helix (IH), a periplasmic β hairpin (PB), a periplasmic helix (PH), and five cytoplasmic loops (fig 1.4).

Figure 1.4. Structure of MraYAA (54).

10 The structure of MraY crystalize as a dimer in the asymmetric unit (A) with 10 transmembrane helix (TP) highlighted in different colors (B).

Consistent with previous mutagenesis studies of MraY from Bacillus subtilis, most of the

14 essential amino acids are localized in the “cleft” region suggesting that this region serves as the active site. Mutational inactivation of three conserved aspartate residues as well as one histidine residue (Asp117, Asp118, Asp265 and His324) resulted in

2+ complete loss of activity of MraYAA, and Asp265 is possibly involved in Mg binding while Asp117 interacts with and deprotonates the undecaprenyl phosphate. Additionally, an inverted-U-shaped groove within the membrane region of the enzyme could serve as the binding site (fig 1.5). Unfortunately crystals were only obtained in the apo-form.

Figure 1.5. The active site of MraYAA (54). Amino acids important for catalysis are colored in red and residues essential for structural maintenance are in cyan (figure was redrawn using published data, PDB ID: 4J72).

11 MraY is a promising target for development of new antibiotics because the Mg2+- dependent reaction is essential for bacterial viability of both gram-positive and gram- negative bacteria and there appears to be only one copy of the gene per bacterial genome (55). The difficulty in assaying the MraY activity and obtaining the recombinant protein for characterization has hindered the identification of inhibitors. Nonetheless, after years of screening for inhibitors targeting MraY, there are three classes of structurally distinct compounds that have been discovered, which includes lysis protein

E from bacteriophage φX174 (56), lipopeptides, and nucleoside antibiotics (43).

The integral membrane protein E is encoded by the DNA phage φX174, and the overexpression of protein E leads to inhibition of MraY by forming a stable complex with

MraY (57). It has been shown that an 18-residue region of a conserved transmembrane helix of protein E is required for MraY inhibitory activity, and site-directed mutagenesis has revealed specific residues of the helix that are important. The second class of MraY inhibitor consists of lipopeptides containing a lipid connected to a peptide. The representative member of lipopeptides is amphomycin which has been shown to form a complex with undecaprenyl phosphate in the presence of Ca2+ (58). The largest class of

MraY inhibitors is formed by the nucleoside antibiotics which can be further divided into four structural groups: peptidyl nucleosides represented by pacidamycins (59) and mureidomycins (60), lipodisaccharyl nucleosides represented by tunicamycins (61) and streptoniridins(62), lipopeptidyl nucleosides represented by liposidomycins (63) and A-

90289 (24), and glycosyl-peptidyl nucleosides represented by the capuramycins (64) (fig

1.6). All nucleoside antibiotics contain an uridine or dihydrouridine moiety, and the variable structural component of a different group leads to slight differences in the

12 mechanism of inhibition: peptidyl, lipodisaccharyl and lipopeptidyl nucleosides are competitive MraY inhibitors (65) while capuramycins show a mixed-type inhibition (66).

Figure 1.6. Representatives of different classes of MraY inhibitors.

1.6 Targeting bacterial MraY: discovery of capuramycins

Capuramycin, initially named 446-S3 in reference to the name of its producer strain

Streptomyces griseus 446-S3, was discovered in screening programs for new antibiotics in 1980s (67). The structure of capuramycin contains three distinct moieties: uridine-5'- carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam (L-

ACL) (fig 1.7). Despite having good inhibitory activity against Streptococcus pneumoniae (MIC 12.5µg/ml) and Mycobacterium smegmatis (MIC 3.13µg/ml) (67), its

13 actual target, bacterial tranlocase I (MraY), was not elucidated until a series of capuramycin derivatives, known as A-500359s (1), were isolated from Streptomyces griseus SANK 60196 in 2003 (64). The in vitro activity (68) assay demonstrated that capuramycin and its methylated analogue A-500359 A (1b) showed potent inhibitory effect on MraY (IC50 18nM and 17nM, respectively), and the mode of action of A-500359s was demonstrated to be mixed type inhibition to UDP-MurNAc-pentapeptide and noncompetitive inhibition to undecaprenyl-phosphate. Interestingly, A-500359 E (1d) which lacks the L-ACL showed a slightly reduced inhibitory activity (IC50 27ng/ml), indicating the lack of importance of this moiety for activity (69). Shortly after that, A-

503083s, a new series of antibiotics that are structurally related to capuramycin were discovered from Streptomyces sp. SANK 62799(69). The structures of A-503083s (2) differ from 1 in the CarU moiety, which is modified with a 2’-O-carbamoyl group. Not surprisingly, 2 showed excellent inhibition of MraY (e.g. IC50 38 nM for A-503083 B, 2a), except A-503083F (2c) that lacks the L-ACL but has a carboxylic acid in its place (IC50

17.9 µM). A-102395 (3), the latest member of the capuramycin family, was isolated from

Amycolatopsis sp. SANK 60206 in 2007 (70). Unlike other capuramycin-like antibiotics,

3 has a unique arylamine-containing polyamide instead of an ACL. Although having potent inhibition against MraY with IC50 11nM, 3 lacks antimicrobial activity against various strains due to poor permeability of bacterial membrane.

-D-manno- NH

pyranuronate H2N O N O O 5' O 5'' O O 2' X Uridine-5'- MeO OR1 carboxamide 4'' 3'' OH (CarU) OH

- L- amino-caprolactam A-500359 B (capuramycin, 1a) R1 = H, R2 = H

R2 A-503083 B (2a) R1 = CONH2, R2 = H = X HN N A-500359 A (1b) R1 = H, R2 = Me O H A-503083 A (2b) R1 = CONH2, R2 = Me

A-500359 F (1c) R1 = R3 = H

A-503083 F (2c) R1 = CONH2, R3 = H X = OR3 A-500359 E (1d) R1 = H, R3 = Me

A-503083 E (2d) R1 = CONH2, R3 = Me

O O OH

= X HOOC N N A-102395 (3) R1 = H H H OH N H

Figure 1.7. Structures of capuramycin-type antibiotics discussed in this dissertation.

In addition to capuramycins having excellent inhibitory activity to MraY, they also have very low toxicity in mice (68). Due to their selective inhibitory activity against

Mycobacterium tuberculosis, capuramycins are considered important leads for the development of anti-TB antibiotics. A series of semisynthetic analogues, SQ641 and

SQ922 for example, have shown comparable MraY inhibitory activity yet better anti-TB activity and broader antimicrobial spectrum (71-74). Notably, not only are capuramycins

15 different from any of the current anti-TB drugs, the analogues have been shown to cause unique phenotypic changes in M. tuberculosis causing bacterial disintegration and loss of culture turbidity (75).

1.7 Binding to DNA: a brief history of actinomycins

Actinomycins are pseudo-symmetric molecules consisting of two cyclic peptides (termed

α- and β-ring) linked to a phenoxazinone chromophore core (fig 1.8). Since the discovery of the first member of actinomycin by Waksman from Streptomyces antibioticus (76), many other actinomycins have been isolated from a variety of Streptomyces strains as overviewed by Bitzer et al (77). Actinomycin D, the most prominent member of the family, was the first natural product shown to have potent anticancer activity and it is currently used in the clinic as a chemotherapeutic agent for the treatment of a variety of cancers, including infantile kidney tumors (78), Ewing’s sarcoma (79) and gestational trophoblastic neoplasia (80) under the name dactinomycin since its FDA approval in

1964. Actinomycins have also been proposed as potential anti-AIDS agents because of its potency of inhibiting HIV-1 minus-strand transfer (81).

Figure 1.8. The structure (left) and stick model (right, PDB ID: 1OVF (82)) of actinomycin D (right).

The mechanism of action of actinomycins is associated with the inhibition of DNA- directed RNA synthesis (83,84), including interactions with single (85) and double- stranded (86) DNA as well as RNA-DNA hybrids (87), leading to inhibition of transcription in both prokaryotes and eukaryotes (fig 1.9). This type of non-selective action is also responsible for the high toxicity of actinomycins. Numerous studies have been carried out to elucidate the interaction of actinomycins with DNA. The actinomycin D-DNA complex was shown to be formed through insertion of the planar phenoxazone ring between the 5’-GpC-3’ sequence while pentapeptide side chains served to shield and stabilize the hydrogen bonds formed by the threonine units on both rings with the NH and CO groups to the nitrogen atoms of neighboring guanine bases (88).

Figure 1.9 Stereoscopic drawings of the crystal structure of the actinomycin D-

(ATGCTGCAT)2 complex (PDB ID: 1MNV (89)).

Due to the strong affinity for DNA, 7-aminoactinomycin D (7-AAD), a derivative of actinomycin, is widely used as a fluorescent marker for DNA in fluorescence microscopy and flow cytometry for cell cycle analysis and chromosome banding studies (90).

There are about 30 naturally occurring actinomycins that have been reported, and they are grouped largely according to the organism from which they were first discovered, including C-type, F-type, X-type, Z-type, G-type and Y-type (77). The overall structures of the actinomycins are similar. The phenoxazinone core is essentially identical while the variations in actinomycins are mostly restricted to modifications of amino acids in the peptide rings. The L-Pro is the most variable position, which may undergo hydroxylation at the 2 position (HPro), oxidation at the 3 position (OPro), and methylation at the 4 position (MPro). In a few cases, L-Thr at the 4 position of the β-ring is modified by chlorination (ClThr) and hydroxylation (HThr), and can undergo cyclization with the

18 phenoxazinone to form an addition ring (cTHr). Other than naturally occurred actinomycins, about 400 analogues of actinomycins have been prepared by semi- or total-synthesis. Extensive modifications of actinomycins have been carried out and resulted in altered bioactivity. The peptide ring opening resulted in complete inactivation of actinomycins, and removal of methyl groups and amino group from phenoxazone also yielded limited activity. Thus the integrity of peptide ring and the 2-amino substituent are essential for biological activity (91). Structure-activity studies suggest that the L-Thr plays an important role in DNA binding activity since among all the naturally occurred actinomycins the Thr at position 1 was present, and modification of Thr resulted in altered bioactivity. For example, chlorine-containing actinomycins are more toxic than

HThr-containing actinomycins (92,93). Interestingly, protactin that was structurally identified as a depsihexapeptide corresponding to a single ring of actinomycins was found from Streptomyces cucumerosporus stran L703-4, and was stated to have both antibiotic activity and toxicity (94).

Actinomycins have been shown with potent antibacterial activity, and Actinomycins X2 and D was reported to have potent anti-tuberculosis activity (95), but their high cytotoxicity hindered their development and use as anti-TB drugs. Actinomycin Y5 containing a unique bridging macrocycle formed between an L-Thr and phenoxazinone core showed excellent antibacterial activity but no observable cytotoxicity (77), and suggested it is possible to achieve a safe antibiotic-selective actinomycin by modulating the chemistry at this position.

19 1.8. Aims of this study

The first specific aim of this study was the identification and characterization of the biosynthetic gene cluster for A-102395. We will:

1. Isolation of A-102395 and other capuramycin-type antibiotics.

2. Generation of in-frame deletion of genes that putatively involved in the biosynthesis of A-102395.

3. Cloning and heterologous expression of Cpr19 and Cpr25 that responsible for the formation of CarU core of capuramycins.

4. Functional characterization of the enzyme conferring self-resistance.

The second aim of this study was to discover novel actinomycins from Streptomyces sp. Strain Gö-GS12. This includes:

1. Thorough investigation of metabolic profile of the strain.

2. Isolation, purification and of structural elucidation of novel actinomycins.

3. Determine the anti-microbial activity and cytotoxicity of novel actinomycins.

20 Chapter 2: Characterization of the biosynthetic gene cluster for A-102395 and reconstituting bacterial cell wall biosynthesis

2.1 Background

Shortly after the discovery of 1, initial evidences of building blocks for each component were interrogated using isotopic feeding experiments. The observed incorporation suggested that L-ACL originates from L-Lys, the unsaturated hexuronic acid is derived from D-mannose, and CarU originates from D-ribose and pyruvate. Feeding experiments done by the Van Lanen group have revealed that L-Thr but not pyruvate is a precursor of CarU (fig 2.1.1) (96-98).

OH O L-Lys L-Thr O OH

H N NH 2 OH 2

NH2 OH O OH O NH H N H 2 N HN O N O O O O O

1 H3CO OR

O OH HO O OH HO OH HO

OH HO OH D-mannose D-ribose

Figure 2.1.1. Isotopic enrichment studies using the indicated 13C-labeled precursors.

A series of studies have been carried out to identify the biosynthetic gene clusters of capuramycin-type nucleoside antibiotics from different producer strains. In 2009,

Funabashi et. al.(99) identified the gene cluster for 1 in Streptomyces griseus

SANK60196. In this study, an abundantly transcribed NDP-glucose dehydratase (NGDH)

21 gene in a high producing strain was first identified from the total RNA isolated from the mycelia by RT-PCR, which was then used as a probe to give two contiguous cosmids containing minimal 21 open reading frames (orfs, orf7-orf28) covering approximately 65 kb region. Based on the sequences of orf8 and orf26, digoxigenin-labeled probes were designed to identify the genetic locus for 2 from Streptomyces sp. SANK 62799 and two genes, capB (encoding a carbamoylgtransferase) and capU (encoding a nonribosomal peptide synthetase) showing high similarities to the probes, were identified leading to the discovery of the entire gene cluster that includes 21 orfs (capA-capW). Additional evidence that the correct gene clusters were identified were provided by the functional characterization of several genes involved in 2 biosynthesis including an L-ACL:2d transacylase (capW), two methyltransferases (capS and capK) and a 2’-carbamoyl transferase (capB) (97). Unexpectedly, two orfs encoding proteins with high amino acid sequence similarity to a non-heme Fe(II)-dependent α-ketoglutarate:uridine-5’-UMP dioxygenase (LipL) (100) and a pyridoxal-5-phosphate-dependent L-Thr:uridine-5’- aldehyde (UA) transaldolase (LipK) (101) were also located in both gene clusters for 1

(orf7 and orf14) and 2 (capA and capH) (97,98).

LipL and LipK sequentially convert UMP to (5′S,6′S)-5'-C-glycyluridine (GlyU) during the biosynthesis of A-90289, a member of a distinct group of MraY inhibitors that contain a GlyU component in the final product (fig. 2.1.2) (24,102). Homologs of LipL and LipK were also identified from the biosynthetic gene clusters for other GlyU-containing nucleoside antibiotics include liposidomycins ( 103 ) , caprazamycins( 104 ) , muraymycin ( 105 , 106 ) , muraminomicin (76 ) , and the recently discovered sphaerimicins (107). Thus, the uncovering of these two genes within the clusters for 1

22 and 2 suggested that the biosynthesis of CarU proceeds via condensation of L-Thr with

UA to generate GlyU as a shared pathway intermediate among nucleotide antibiotics.

Figure 2.1.2. Structure and biosynthesis of A-90289, a GlyU-containing nucleoside antibiotic. The GlyU was converted from UMP by LipL and LipK.

The biosynthesis of the hexuronic acid moiety is unknown. Among the shared proteins encoded in the capuramycin-type nucleotide antibiotics, it was proposed that a putative

NDP-hexose 2,3-dehydratase, a putative NDP-hexose 4-epimerase/dehydratase, a 3- ketoreductase, a 2,3-dehytrase and a 4-epimerase are responsible for the biotransformation. A glycosyltransferase (ORF13 in 1 gene cluster and CapG in 2 gene cluster) presumably transfers the activated hexuronic acid to CarU.

The adenylation domain of CapU in 2 biosynthetic gene cluster was tested and shown to activate several amino acids with a clear preference for L-Lys while a stand-alone C domain of NRPS CapV is also likely involved in ACL formation(97). The cyclized ACL is subsequently linked onto the carboxyl group on sugar moiety via an amide bond which occurs through an unusual ATP-independent transacylation mechanism: CapS, a methyltransferase, first forms a methyl ester and followed by an ester-amide exchange reaction catalyzed by CapW, an ACL-acyltransferase. Homologues of capS and capW

23 were uncovered in the gene clusters of 1 (orf24 and orf28), suggesting that the amide bond is generated in the same manner. Additionally, two genes encoding for tailoring enzymes, a 2’-carbamoyl transferase (orf8 and capB) and O-methyltransferase (cpr29 and capK) were identified. The orf8 was predicted to encode for a truncated and hence unfunctional carbamoyltransferase, hence explaining the observed difference in the chemical structures of the capuramycin-type antibiotic 1 and 2.

Actinomycetes, like other antibiotic producing microorganisms, have developed highly effective self-protection mechanisms, also known as self-resistance, to prevent suicide from their own metabolic products. There are three commonly observed mechanisms of self-protection including i) chemical modification of the antibiotic by an enzyme, ii) physical removal from the cells by an efflux pump therefore preventing it from reaching a toxic concentration, and iii) modification of the normally sensitive target to render it insensitive (108,109). The genes conferring self-resistance are not only critical for protecting the antibiotic producers, but they may also potentially be acquired by other bacteria by horizontal gene transfer (HGT) via transduction (110), transformation (111) or conjugation (112). HGT is the major cause of widespread development of multi-drug resistance beyond spontaneous mutation. For example, the first demonstrated enzymes that inactivate the aminoglycoside class of antibiotics were aminoglycoside phosphtransferases (APHs) (113), which regio-specifically add a phosphate group to an aminoglycoside. Genes encoding APH have not only been found encoded within the biosynthetic gene cluster of aminoglycoside producing strains, but homologous genes have also been found within many bacterial pathogens such as Enterobacteria and

Staphylococcus (99). An understanding of the self-resistance mechanism of an

24 antibiotic-producer is thus important for developing novel antibiotics for clinical use. It provides valuable insights on the structure-activity relationship (SAR) of antibiotics (114), helps in predicting the potential resistance that could develop after the drug is introduced for clinical use (115), and offers impetus for developing derivatives of existing antibiotics to prolong their clinical use.

For the capuramycins, bioinformatic analysis of the biosynthetic gene cluster for 1 revealed a gene product (ORF21) within the locus that has sequence similarity to APH.

The heterologous expression of orf21 provided resistance to capuramycin in two normally sensitive strains of Streptomyces albus and E. coli ΔtolC (99). This was the first study on the self-resistance of capuramycin producers. Later in 2010, CapP (114), a gene product encoded within the biosynthetic gene cluster for 2 with high sequence similarity to ORF21, was functionally characterized as an ATP-dependent phosphotransferase which regio-specifically transfers the γ-phosphate of ATP to the 3’’- hydroxyl of the unsaturated hexuronic acid moiety of 2. The 3’’-phospho-capuramycins lost their antibacterial activity and the IC50 in the bacterial MraY reaction increased more than 200-fold (98,99,114)

To overcome the rise of antibiotic resistance and hence diminished utility of the current antibiotic repertoire, it is critical to keep discovering antibiotics, particularly those that can be considered new chemical entities. Whole cell based high-throughput screening

(HTS) has played a vital role in the discovery of antibiotics in the past and resulted in a large number of bioactive compounds. This method is sometimes complicated by the complexity of cells, the subsequent difficulty in determining their targets, and the bioactive fractions can often be toxic to mammalian cells (116). Perhaps most limiting is

25 the realization that traditional whole cell based HTS has reached the point of diminishing returns such that the same antibiotics or family of antibiotics routinely rediscovered with this method. Thus a target-specific HTS model for in vitro screening offers an important alternative for finding novel antibiotic candidates as well as specific targets that they bind.

The resulting hit-target pairs (116) can be further advanced by structure-based drug design to improve their pharmacological properties such as absorption, distribution, metabolism and excretion (ADME) without compromising their target-specificity. Other than target-specificity, species-specificity is an equally important consideration for a HTS model. Subtle differences in targets among different species may lead to false-positive screening results. Bedaquiline (117), for example, selectively inhibits the growth of mycobacteria but shows no activity against other bacteria because of minor differences in the target ATP synthase of mycobacteria and other species (3,118,119). Nevertheless, species-specific antibiotics are important as they typically minimize the possibility of adverse effects on human gut microbes, several of which are now known to be beneficial for human health (3).

In this chapter, we will discuss the identification of biosynthetic gene cluster for A-102395, the latest member of capuramycin-type antibiotics, by bioinformatic analysis and gene inactivation, and the functional characterization of a phosphotransferase that confers self-resistance mechanism. At the end of the chapter, a validated species-specific HTS model of peptidoglycan cell wall biosynthesis will be reported which is expected to allow us to identify the novel antibiotics targeting on early stages of bacterial cell wall biosynthesis rapidly and in a cost-efficient manner.

26 2.2 Materials and methods

2.2.1 Instrumentation, chemicals and reagents

UV/Vis spectroscopy was performed with a Bio-Tek μQuant microplate reader using

Microtest™ 96-well plates (BD Biosciences) or a Shimadzu UV/Vis-1800

Spectrophotometer equipped with a TCC-240A thermoelectrically temperature controlled cell holder. PCR was performed with a Veriti® 96-well thermal cycler from

Applied Biosystems. Analytic HPLC was performed with one of two systems: a Waters

Alliance 2695 separation module (Milford, MA) equipped with a Waters 2998 diode array detector and an analytical Apollo C-18 column (250 mm x 4.6 mm, 5 µm) purchased from Grace (Deerfield, IL) or a Dionex Ultimate 3000 Focused separation module

(Bannockburn, IL) equipped with a DAD-3000(RS) and MWD-3000(RS) diode array detector and an Acclaim 120 C-18 column (4.6 mm x 100 mm, 3 µm). Semipreparative

HPLC was performed with a Waters 600 controller and pump (Milford, MA) equipped with a 996 diode array detector, 717plus autosampler, and an Apollo C-18 column (250 mm x 10 mm, 5 µm) purchased from Grace (Deerfield, IL). LC-electrospray ionization

(ESI)-mass spectroscopy (MS) was performed using an Agilent 6120 Quadrupole MSD mass spectrometer (Agilent Technologies, Santa Clara, CA) equipped with an Agilent

1200 Series Quaternary LC system and an Eclipse XDB-C18 column (150mm x 4.6 mm,

5 µm, 80Å). LC-MS-MS analysis of 3 was carried out using a Shimadzu UFLC coupled with an AB Sciex 4000-Qtrap hybrid linear ion trap triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode. NMR data were collected using a Varian

Unity Inova 400 or 500 MHz spectrometer (Varian, Inc., Palo Alto, CA) or a Bruker

AVANCE 500 MHz spectrometer (Bruker Biospin Corporation, Billerica, MA). Nanodrop 27 2000 UV-Vis spectrophotometer (Thermo Scientific, Waltham, MA) was used to measure concentration and purity of DNA, RNA and proteins.

Synthetic oligonucleotides were purchased from Integrated DNA Technologies

(Coralville, IA). Wizard® Plus SV Minipreps DNA Purification Systems,Wizard® SV Gel and PCR Clean-Up System were purchased from Promega (Madison, WI, USA). pET-

30 Xa/LIC Vector Kit was purchased from Calbiochem (San Diego. CA, USA). InstaGene

Matrix was purchased from Bio-Rad (Hercules, CA). Ni-NTA agarose was purchased from Qiagen (Valencia, CA). Amicon Ultra 10000 MWCO centrifugal filter was purchased from Millipore (Billerica, MA). PD-10 desalting column was purchased from GE

Healthcare (Pittsburgh, PA). DNA sequencing was performed using the

BigDye™Terminator version 3.1 Cycle Sequencing kit from Applied Biosystems, Inc.

(Foster City, CA) and analyzed at the University of Kentucky Advanced Genetic

Technologies Center.

Streptomyces sp. SANK 62799 and Amycolatopsis sp. SANK 60206 were obtained from

Daiichi Sankyo Co., Ltd (Japan). E. coli BW25113/pIJ790 and E. coli ET12567/pUZ8002 strains were purchased from E. coli Genetic Resources at Yale University. NovaBlue

GigaSingles Competent cells, BL21 (DE3) Competent cells were purchased from

Invitrogen (Camarillo, CA). Takara LA taq DNA polymerase was purchased from Takara

Bio Inc (Otsu, Shiga, Japan). Restriction enzymes were purchased from New England

Biolabs (Ipswich, MA). Expand long template PCR system was purchased from Roche

Applied Science (Indianapolis, IN). Phusion Green Hot Start II High-Fidelity DNA

Polymerase was purchased from Thermo Scientific (Waltham, MA).

28 All chemicals and reagents were purchased from Sigma Aldrich expect indicated otherwise.

2.2.2 Identification and characterization of A-102395 biosynthetic gene cluster

2.2.2.1 Isolation of A-102395, A-503083B and A-503083F

A-102395 standard was a gift from Dr. Koichi Nonaka, Daiichi-Sankyo. A-102395, A-

503083B and A-503083F were isolated from the producing strains and purified as described previously (69,70).

2.2.2.2 Amplification of disruption cassette

For amplification of the disruption cassette for each gene of interest, two long primers were used as listed in table 2.2.1. At the 5’ end, each primer has 39 nt of homologous sequences matching the gene to be inactivated, and at 3’ end, each primer has a 19 nt or 20 nt matching the disruption cassette containing an apramycin resistant marker

(aac(3)IV) and an origin of transfer (OriT) from pIJ773 vector (fig 2.2.1).

29 Table 2.2.1. Primers for in-frame deletion.

Name Sequence Cpr19_inact 5’ GGCTCTCTATCGGTACGGAGAGACCAAGGACTACTC _forward CGCATTCCGGGGATCCGTCGACC 3’ Cpr19_ inact 5’ ATCGTCGGTCGCGGTGCGCAGGAAGTCCAGCGCTC _reverse GGCGTGTAGGCTGGAGCTGCTTC 3’ Cpr25_ inact 5’ TGGCAGTTTCGGGGCGGCCAGGACGTCGCGGAGAT _forward GGAGATTCCGGGGATCCGTCGACC 3’ Cpr25_ inact 5’ TGGCATGCCGGGAAGATCTACCTGGACGTTGACCCG _reverse GATTGTAGGCTGGAGCTGCTTC 3’ Cpr51_ inact 5’ ACGCTGGTGGAGGACCACACTCTCCGCCTCAACGAC _forward CCGATTCCGGGGATCCGTCGACC 3’ Cpr51_ inact 5’ CGGCGGCCAGACGAATTTGGAGTACGCCTTCTGCC _reverse GCGGTGTAGGCTGGAGCTGCTTC 3’

Figure 2.2.1. Map of pIJ773 plasmid. Within the disruption cassette, it contains an apramycin resistance gene aac(3)IV (aprr, accession number: X99313) and an origin of transfer of plasmid RP4 (oriT, accession number: L27758). The plasmid is resistant to both ampicillin and apramycin.

30 Disruption cassettes were amplified by PCR using Expand Long Template PCR System from Roche (Indianapolis, IN) with supplied Buffer 2, 200 µM dNTPs, 5% DMSO, 10 ng template, 5 U DNA polymerase, and 200 nM each of the primer pairs in table 2.2.1. DNA template for PCR was pIJ773 plasmid. The PCR program begins at 94 °C for 2 min, followed by 10 cycles of 94 °C for 45 s, 50°C for 45 s and 72°C for 90 s, and then 15 cycles of 94 °C for 45 s, 55°C for 45 s and 72°C for 90 s, and a final extension at 72°C for 5 min. The resulting linear PCR products were named Δcpr19-cassette, Δcpr25- cassette and Δcpr51-cassette.

PCR products were purified by electrophoresis on 0.5% agarose gel, and the desired

DNA fragment was extracted.

2.2.2.3 Introduction of pNCap02 and pNCap04 into E. coli BW25113/pIJ790 strain

The supercosmids pNCap02 and pNCap04, a gift from Dr. Koichi Nonaka at Daiichi

Sankyo, were transformed into E. coli BW25113/pIJ790 by electroporation as described below. E. coli BW25113/pIJ790 was cultured in 10 ml LB containing chloramphenicol

(CHL, 25 µg/ml) overnight at 30 °C, and 100 µl was used to inoculate 10 ml SOB containing 20 mM MgSO4 and 25 µg/ml chloramphenicol (CHL). The strain was collected by centrifuge at 4000 rpm for 5 min at 4 °C, and resuspended by gentle mixing in 10 ml ice-cold sterile 10% glycerol. Following a second centrifugation at 4000 rpm for 5 min at

4 °C, the cell pellet was resuspended in 100 µl of 10% glycerol. 100 ng of pNCap02 or pNCap04 was added and mixed by pipette a few times. Electroporation was carried out in a 0.2 cm pre-chilled electroporation cuvette using a BioRad GenePulsor II under following condition: 200 Ω, 25 µF and 2.5 kV. The time constant was 4.5 ms. 1 ml ice

31 cold SOB was immediately added after electroporation, and cells were incubated shaking for 1h at 30 °C. The cells were spread onto LB agar containing 50 µg/ml APR and 25 µg/ml CHL and incubated overnight at 30 °C to yield E. coli BW- pNCap02 and

E. coli BW- pNCap04. To confirm that the resulting single colonies harbored the correct cosmid, colony PCR was conducted using primers for amplification of cpr19 and cpr25 with E. coli BW- pNCap02 as the template, and primers for amplification of cpr51 with E. coli BW- pNCap04 as the template (table 2.2.2).

Table 2.2.2. Primers for amplification of cpr19, cpr25 and cpr51.

Name Sequence cpr19_forward 5'- GGTATTGAGGGTCGCATGCAGCAGCTGCAA GCCG-3' cpr19_reverse 5'- AGAGGAGAGTTAGAGCCTCAATTGGAGGCG CGGGG-3' cpr25_forward 5'-GGTATTGAGGGTCGCATGACTGACACTAAC GAG-3’ cpr25_reverse 5'- AGAGGAGAGTTAGAGCCTCATGGGTAATCG ACGA-3’ cpr51_forward 5'- GGTATTGAGGGTCGCATGGCCAGAGGTGG CCTCT-3’ cpr51_reverse 5'- AGAGGAGAGTTAGAGCCTCAGTCGTCGATC GCCTG-3’

2.2.2.4 PCR targeting of supercosmids

E. coli BW- pNCap02 and E. coli BW- pNCap04 were used to inoculate 10 ml of LB containing 50 µg/ml APR and 25 µg/ml CHL for overnight at 30 °C. Then 1 ml of each was transferred into 100 ml SOB containing 50 µg/ml APR, 25 µg/ml CHL and 10 mM L- arabinose. After growing for 3-4 h at 30 °C until OD600 reached 0.4-0.6, the cells were recovered by centrifuge at 4000 rpm for 5 min at 4 °C. Cell pellets were washed twice

32 with 10 ml ice-cold 10% glycerol and resuspended in 100 µl of 10% glycerol. Then 100 ng of the disruption cassette Δcpr19-cassette or Δcpr25-cassette was added and mixed with E. coli BW-pNCap02, and the Δcpr51-cassette was mixed with E. coli BW- pNCap04.

Electroporation was carried out in a 0.2 cm pre-chilled electroporation cuvette using a

BioRad GenePulsor II under following condition: 200 Ω, 25 µF and 2.5 kV. The time constant was 4.5 ms. 1 ml ice cold SOB was immediately added after electroporation, and cells were incubated with shaking for 1h at 37 °C before spreading onto LB agar containing 50 µg/ml APR and 50 µg/ml AMP. The LB agar plates were incubated at 37 °C for overnight.

The double crossover gene disruption of the resulting single colonies yielded E. coli BW- pNCap02-Δcpr19, E. coli BW- pNCap02-cprΔ25 and E. coli BW- pNCap04-cprΔ51. The gene deletion and presence of disruption cassette were confirmed by PCR. The universal primers listed in table 2.2.3 were used to amplify the disruption cassette in mutant cosmids, and the primers listed in table 2.2.1 were used to compare the difference in sizes between wildtype (WT) and mutant genes. Mutant cosmids pNCap02-

Δcpr19, pNCap02-Δcpr25 and pNCap04-Δcpr51 were isolated using Wizard Plus

Minipreps DNA purification system (Promega).

Table 2.2.3. Primers for amplification of disruption cassette.

Name Sequence

Disruption cassette_forward 5' ATTCCGGGGATCCGTCGACC 3' Disruption cassette_reverse 5' TGTAGGCTGGAGCTGCTTC 3'

33 2.2.2.5 Transfer of mutant cosmids into Amycolatopsis sp. SANK 60206

Mutant cosmids pNCap02-Δcpr19, pNCap02-Δcpr25 and pNCap04-Δcpr51 were transformed into E. coli ET12567/pUZ8002 competent cells by heat shock. The resulting colonies harboring the mutant cosmids were confirmed by PCR yielding E. coli ET- pNCap02-Δcpr19, E. coli ET-pNCap02-Δcpr25 and E. coli ET-pNCap04-Δcpr51.

Strains harboring the mutant cosmids were used to inoculate 50 ml LB containing 50

µg/ml APR after overnight an overnight 3 mL culture and grown for ~4 h at 37 °C to an

OD600 of 0.4 – 0.6. The cells were washed twice with fresh LB to remove APR, and resuspended in 1 ml of LB.

Wild type Amycolatopsis sp. SANK 60206 was grown on ISP4 agar for 2 weeks at 28 °C to allow sporulation before conjugation. The spores of were collected from ISP4 agar and added into 50 ml of 2×YT broth. After heat shock at 50 °C for 10 min, the broth was allowed to cool to room temperature and centrifuged at 4000 rpm for 10 min at 4 °C. The pellet was resuspended in 1 ml of LB containing E. coli ET-pNCap02-Δcpr19, E. coli ET- pNCap02-Δcpr25 and E. coli ET-pNCap04-Δcpr51, respectively. The resuspended cultures were transferred onto ISP4 agar plates containing 10 mM MgCl2 without antibiotics. The plates were incubated at 36 °C for 16 h prior to overlaying the cells with

1 ml sterile water containing 0.5 mg nalidixic acid and 1.25 mg APR for selection.

Successful double crossover events were confirmed by PCR using the primers listed in table 2.2.2, yielding Amycolatopsis-Δcpr19, Amycolatopsis-Δcpr25 and Amycolatopsis-

Δcpr51.

34 2.2.2.6 Isolation of genomic DNA of wild-type and mutant Amycolatopsis sp. SANK 60206

Amycolatopsis sp. SANK 60206, Amycolatopsis-Δcpr19, Amycolatopsis-Δcpr25 and

Amycolatopsis-Δcpr51 were grown on ISP4 plates for 2 weeks until sporulation. A single colony of each cell line was used to inoculate 50 ml of TSB and incubated at 28

°C for 48 h in a rotary shaker. 1 ml of the cultured strain was used to inoculate 50 ml of

TSB and allowed to grow for 2 additional days. Cell cultures were transferred into

Eppendorf tubes and recovered by centrifuge at 4,000rpm for 10min, and cell pellets were washed twice with TE buffer. Washed cells were resuspended in 5 ml TE buffer and 10 mg lysosome was added and mixed gently. During incubation at 30 °C, the cells were checked every 15 min until 1:1 mixture of cells with 10% SDS became clear.

1.2 ml of 0.5 M EDTA was added and mixed gently, and proteinase K was immediately added to a final concentration of 0.2 mg/ml. After incubation at 37 °C for 30 min, 0.7 ml of 10% SDS was added followed by an additional 2 h incubation at 37 °C. After returning the mixture to room temperature, 7 ml phenol:chloroform:isoamyl alcohol

(25:24:1, v/v) was added and mixed by shaking at room temperature (rt) for 10 min.

Chloroform (7 ml) was added and mixed by shaking at room temperature for 10 min.

The supernatant was transferred into a new tube after centrifuge at 4000 rpm for 5 min.

The phenol/chloroform extraction was repeated twice. RNase was added to a final concentration of 4µg/ml, and after 1 h incubation at 37 °C, 1.75 ml of 5 M NaCl was added and mixed by inverting the tubes. PEG6000 (4.9 ml of 30%) was added to final concentration of 10% and mixed by inverting the tube. DNA was precipitated and carefully transferred into a new tube. After dissolved in 5 ml TE buffer, 0.6 ml 3 M

NaAc and 12 ml cold EtOH were added and mixed gently. The precipitated DNA was 35 washed with 70% EtOH, and allowed to air dry. The remaining solid was dissolved in

5-10 ml TE buffer, and stored at 4 °C until use.

The quality of genomic DNA was estimated by agarose gel and the ratio of absorbance at 260 nm and 280 nm (A260/A280) using a NanoDrop 2000C.

2.2.2.7 PCR analysis of wild-type and mutant Amycolatopsis sp. SANK 60206

PCR analysis of the genotype of wild-type and mutant Amycolatopsis sp. SANK 60206 was carried out using LA taq DNA polymerase purchased from Takara (Mountain View,

CA) with supplied buffer 2, 200 , 200 µM dNTPs, 5% DMSO, 10 ng template, 5 U DNA polymerase, and 200 nM each of the primer pairs in table 2.2.2. DNA template for WT strain was the genomic DNA of Amycolatopsis sp. SANK 60206, and DNA templates for mutant strains were genomic DNA of Amycolatopsis-Δ19, Amycolatopsis-Δ25 and

Amycolatopsis-Δ51, respectively. The PCR program began at 97 °C for 2 min, followed by 20 cycles of 97 °C for 45 s, 50°C for 45 s and 72°C for 90 s, and final extension at

72 °C for 7 min. The resulting linear PCR products were analyzed by agarose gel electrophoresis.

2.2.2.8 Preparation of the DIG-labeled probes

Primers for amplification were designed according to the sequences of probes for detection of cpr19, cpr25 and cpr51 are listed in table 2.2.4. The first amplification was carried out using Expand Long Template PCR System from Roche (Indianapolis, IN) with supplied Buffer 2, 200 µM dNTPs, 5% DMSO, 10 ng template, 5 U DNA polymerase,

36 and 200 nM each of the primer pairs. DNA template was genomic DNA of WT or the appropriate mutant Amycolatopsis sp. SANK 60206. The PCR program begins at 94 °C for 2 min, followed by 10 cycles of 94 °C for 45 s, 50°C for 45 s and 72°C for 90 s, and then 15 cycles of 94 °C for 45 s, 55°C for 45 s and 72°C for 90 s, and final extension at

72 °C for 5 min. The resulting linear PCR products were purified by agarose gel electrophoresis.

Table 2.2.4. Primers for amplification of DNA probes for Southern blot analysis.

Name Sequence

cpr19-probe-forward 5` ATTCCGGGGATCCGTCGA CC 3` cpr19-probe-reverse 5` TGTAGGCTGGAGCTGCTT C 3` cpr25-probe-forward 5` ATTCCGGGGATCCGTCGA CC 3` cpr25-probe-reverse 5` TGTAGGCTGGAGCTGCTT C 3` cpr51-probe-forward 5` ATTCCGGGGATCCGTCGA CC 3` cpr51-probe-reverse 5` TGTAGGCTGGAGCTGCTT C 3`

The second amplification for DIG labeled probes was carried out using PCR Dig

Synthesis Kit (Roche) following the provider’s protocol, and purified DNA from first PCR were used as templates. The PCR program was same as first amplification to yielding probe-Δ19, probe-Δ25 and probe-Δ51.

37 2.2.2.9 Digestion of genomic DNA For Southern blot analysis of Amycolatopsis-Δ19 and Amycolatopsis-Δ51, ~10 µg genomic DNA of WT Amycolatopsis sp. SANK 60206 and Amycolatopsis-Δ19 or

Amycolatopsis-Δ51 were individually digested with ApaI at 37 °C overnight. For

Southern blot analysis of Amycolatopsis-Δ25, ~10 µg genomic DNA of WT

Amycolatopsis sp. SANK 60206 and Amycolatopsis-Δ25 were digested with BamHI at

37 °C overnight. The digested genomic DNA was separated by 0.8% agarose gel in

1×TAE buffer.

2.2.2.10 DNA:DNA hybridization and detection Gels were soaked with denaturation solution for 30 min, then transferred into neutralization buffer and kept at room temperature for 30 min. Then gels were then soaked in 2×SSC buffer for an additional 15 min. After transferring DNA onto Nylon

Membranes (Roche), DNA:DNA hybridization (DIG Easy Hyb Granules, Roche) and detection were carried out following manufacturer’s instructions.

2.2.2.11 LC-MS analysis of production of A-102395 in mutant strains Amycolatopsis-Δ19, Amycolatopsis-Δ25 and Amycolatopsis-Δ51 were grown as described in 2.2.2.1, and MeOH was added to the supernatant to a final concentration of 50%. The organic solvent was evaporated and the remaining liquid was lyophilized to yield crude extract. Crude extracts were weighed and dissolved in 1,600 µL of 1:1 water: methanol and the pH was adjusted to 3-4 with concentrated HCl if necessary. 6,400 µL of cold acetonitrile was then added followed by sonication for a minute and centrifuged at 4,000 rpm for 10 min. The supernatant was then dried under nitrogen. Dried extract

38 was resuspended in 50 µL of methanol. Analysis of 1 compound was carried out in multiple reaction monitoring (MRM) mode. 1 was analyzed using an Apollo C18 250 ×

4.6 mm, 5 um column (Grace) under a series of linear gradients from A (10 mM ammonium formate with 0.1% formic acid in H2O) to B (0.1% formic in acetonitrile) in the following manner (beginning time and ending time with linear increase to % B): 0-8 min, 2% B; 8-19 min, 90% B; 19-23 min, 2% B. The flow rate was kept constant at 0.5 mL/min with a column temperature of 30 ⁰C. The mass spectrometer was operated in the negative electrospray ionization mode with optimal ion source settings determined by a standard of 1 with a declustering potential of -135 V, entrance potential of -10 V, collision energy of -60 V, collision cell exit potential of -7 V, curtain gas of 20 psi, ion spray voltage of -4200 V, ion source gas1/gas2 of 40 psi and temperature of 550 ⁰C. MRM transitions monitored were as follows: 763.316/189.9 and 763.316/120. Negative ESI mode was used to analyze samples as it was more sensitive compared to positive ESI mode for 1 compound.

2.2.2.12 Transcriptional analysis of Δcpr19, Δcpr25 and Δcpr51 strains

To isolate total mRNA of WT and mutant Amycolatopsis, strains were grown in TSB medium at 37 ⁰C for 2 days. Culture medium (1.5 mL) was centrifuge and washed twice with sterile water. The isolation procedure was carried out using TRIzol MAX

Bacterial RNA Isolation Kit (life technologies) following the provided manual. The quality of total RNA was estimated by agarose gel and the ratio of absorbance at 260 nm and 280 nm (A260/A280) using a NanoDrop 2000C. The isolated total RNA was immediately used for RT-PCR.

39 The first-strand cDNA of WT and mutant Amycolatopsis was synthesized using

SuperScript III First-Strand Synthesis System (life technologies) following the provided manual. The quality of total RNA was estimated by agarose gel and the ratio of absorbance at 260 nm and 280 nm (A260/A280) using a NanoDrop 2000. The first- strand cDNA were kept at -20 ⁰C until use. The expression of inactivated and flanking genes for each mutant was examined by

PCR using primers listed in table 2.2.5. For Amycolatopsis-Δ19, expression levels of cpr18, cpr19 and cpr20 were compared with WT. For Amycolatopsis-Δ25, expression levels of cpr24, cpr25 and cpr26 were compared with WT. For Amycolatopsis-Δ51, expression levels of cpr50, cpr51 and cpr52 were compared with WT.

40 Table 2.2.5. Primers for transcriptional analysis of mutant and wild-type strains.

Name Sequence cpr18_forward 5' ATGCAGCAGCTGCAAGCC 3' cpr18_reverse 5’ TCAATTGGAGGCGCGGGG 3' cpr19_forward 5' GTGCAGCAGCTGCAAGCC 3' cpr19_reverse 5’ TCAATTGGAGGCGCGGGG 3’ cpr20_forward 5’ ATGGAACTAC TCCTGATC 3' cpr20_reverse 5’ CTAGTTGTCCGCCGATGC 3' cpr24_forward 5’ ATGAGCAACC GACCAGTC 3' cpr24_reverse 5’ TCATCCACCGTGTAATTC 3' cpr25_forward 5’ ATGACTGACA CTAACGAG 3' cpr25_reverse 5’ TCATGGGTAATCGACGAA 3' cpr26_forward 5’ GTGGACATCG GTTCACTC 3' cpr26_reverse 5’ TCATGCGGGATTCGACGC 3' cpr50_forward 5’ GTGGAGGGCG CTGGGACC 3' cpr50_reverse 5’ CAACCAGGAAGCTCGGC 3' cpr51_forward 5’ ATGGCCAGAG GTGGCCTC 3' cpr51_reverse 5’ TCAGTCGTCGATCGCCTG 3' cpr52_forward 5’ ATGGGGAACT TGATTGAG 3' cpr52_reverse 5’ TCAGCTGAGCCAGCAGTTC 3'

2.2.2.13 Heterologous expression and functional assignment of Cpr19 and CapH

Cpr19 and CapH were heterologously expressed in E. coli BL21 (DE3) and

Streptomyces lividans TK64, respectively, following the protocol of previous work in the

Van Lanen lab (98).

Native-PAGE analysis was carried out using Novex 4-12% Tris-Glycine Gel purchased from Life technologies.

41 2.2.3 Characterization of Cpr17

2.2.3.1 Heterologous production of Cpr17 and CapP in E. coli. cpr17 and capP were amplified by PCR using Phusion Green Hot Start II High-Fidelity

DNA Polymerase (Thermo Scientific) with supplied HF buffer, DMSO, 10mM dNTPs,

DMSO, 10ng DNA template, 2U DNA polymerase, and 100nM each of the following primers:

Table 2.2.6. Primers for amplification of cpr17 and capP.

Name Sequence 5' GGTATTGAGGGTCGCATGACCGAGACCGA cpr17_forward GATCAC 3' 5’ AGAGGAGAGTTAGAGCCCTATCCGTGGAA cpr17_reverse TCGGGC 3' 5' GGTATTGAGGGTCGCGTGACGGACACCGA capP_forward AAAT 3' 5’ AGAGGAGAGTTAGAGCCTCAGGAGTGAGT capP_reverse CGCGGT 3'

DNA templates for PCR were cosmid pNCap03 for cpr17 and cosmid N-1 for capP.

The PCR program began at 94 °C for 30s, followed by 30 cycles of 94 °C for 10s, 56°C for 20s and 72°C for 20s, and final extension at 68°C for 7min. The PCR product was purified by electrophoresis on 0.5% agarose gel, and the desired DNA fragment was extracted with Wizard V Gel and PCR Clean-Up System (Promega). The gel-purified

PCR product was inserted into pET-30 Xa/LIC vector (Invitrogen) following the manufacture’s protocol to yield pET30-cpr17 and pET30-capP. Genes ligated in pET-30 vector were sequenced to confirm fidelity.

42 Plasmids pET30-cpr17 and pET30-capP were transformed into E. coli BL21 (DE3) by heat-shock, and the transformed strains were grown in SOC medium for 30 min and then selected on LB agar supplemented with 50 µg/mL kanamycin to yield E. coli-cpr17 and

E. Coli-capP strains.

Following an overnight incubation of culture in 10 ml of LB supplemented with 50 µg/mL kanamycin, the recombinant strains were grown in 500ml of LB with 50 µg/ml kanamycin at 37 °C until the cell density reached an OD600=0.4-0.6, then 0.1 mM 1-thio-β-D- galactopyranoside (IPTG) was added to induce expression. After overnight incubation at 18 °C, cells were collected by centrifuge at 4°C for 10min then resuspended in lysis buffer containing 100 mM Tris-HCl (pH=7.5) and 250 mM NaCl. Lysis of cells was performed using sonication for 10 rounds while the culture was kept on ice. Immediately after lysis, cells debris were separated from lysate by centrifuge at 10,000 rpm for 40min.

The supernatant containing His6-tagged protein was loaded onto Ni-NTA agarose

(Qiagen) and washed with 50 ml of washing buffer containing 100 mM Tris-HCl (pH=7.5),

250 mM NaCl and 20 mM imidazole. The recombinant proteins were eluted with 15 ml of elution buffer containing 100 mM Tris-HCl (pH=7.5), 250 mM NaCl and 200 mM imidazole, and then concentrated with an Amicon Ultra 10,000 MWCO centrifugal filter

(Millipore), followed by desalting of the proteins using desalting buffer containing 50mM

Tris-HCl (pH=7.5) and 100mM NaCl. The proteins were stored in 30% glycerol at -20 °C until use. Size and purity of recombinant proteins were assessed by 12% acrylamide

SDS-PAGE, and the concentration was determined by Nano Drop according to the predicted extinction coefficient of each protein.

43 2.2.3.2 Functional characterization of Cpr17 and CapP

Reactions consisted of 50 mM Tris-HCl (pH=7.5), 10 mM MgCl2, 1 mM ATP, 1 mM substrates (3, 2a, 2d or 1c) and 0.3 mg/ml Cpr17 or CapP (total volume of 100 µl).

Individual reactions were prepared and incubated at 30 °C for 12 hr and quenched by adding 300 µl of chloroform. Followed by centrifuge at top speed, the aqueous layers were used for HPLC analysis using a C-18 reversed-phase column (Grace) under a series of linear gradients from 0.1% TFA in 2.5% acetonitrile (A) to 0.1% TFA in 90% acetonitrile (B) in the following manner (beginning time and ending time with linear increase to % B): 0-8 min, 100% B; 8-18 min, 60% B; 18-25 min, 95%B; 25-32 min,

95%B; and 32-35 min, 0%B. The flow rate was kept constant at 1 ml/min and elution was monitored at 260 nm. LC-MS analysis was performed using an Eclipse XDB-C18 reversed-phase column (Agilent) under a series of linear gradients from 0.1% formic acid in H2O to 0.1% formic acid in acetonitrile over 20 min. The flow rate was kept constant at 0.4 ml/min and elution was monitored at 254 nm.

The substrate specificity of Cpr17 was characterized using the identical condition as described above except that unnatural substrates were used (A-503083G, P1-P10 and

P11).

44 2.2.3.3 Kinetic characterization of Cpr17

The kinetic parameters (Km and kcat) for Cpr17 were determined using an enzyme- coupled assay monitoring the production of ADP generated by the phosphotransferase reaction as previously reported. The reaction (100 µL) used in determining the kinetic parameters for the substrate 2a contained 50 mM Tris (pH 7.5), 10 mM MgCl2, 50 mM

KCl, 2.5 mM phosphoenol pyruvate, 0.5 mg/ml NADH, 0.5 µM Cpr17, 2 mM ATP, and 2 mL of a pyruvate kinase/lactic dehydrogenase mixture (Sigma-Aldrich). Reactions were initiated with 2a at various concentrations (0, 50, 100, 250, 1000, and 2000 µM). ATP and GTP kinetics were determined identical to the conditions described above except reactions contained either varying concentrations of ATP (0, 50, 100, 250, 500, 2000 µM) or GTP (0, 10, 50, 75, and 100 µM) and included a constant concentration of 2a (500

µM for ATP kinetics; 100 µM for GTP kinetics). All experiments were performed in triplicate in 96-well plates and monitored at 340 nm on a SpectraMax M5 microplate reader, taking measurements every 30s for 10 min. The initial rates were determined and the resulting data were fitted to the Michaelis-Menten equation:

where v is the initial velocity, Vmax is the maximum reaction velocity, [S] is the substrate concentration, and KM is the Michaelis constant of the varied substrate.

45 2.2.3.4 Chemical and semi-synthesis of unnatural substrates for Cpr17

HO O MeO O

O NH O NH O 2 O O 2 O NH NH TMSCN2 O HO O O MeO O O O MeOH N OH N OH O O O O O O

NH2 NH2 A-503083F A-503083G

Scheme 2.1. Synthesis of A-503083G.

To a stirred solution of the A-503083 F (100.4mg, 0.2 mmol) in anhydrous methanol (0.4 mL) under a nitrogen atmosphere, 220 uL of 2.0 M trimethylsilyl diazomethane (0.48 mmol) in hexanes was added slowly at 0 ℃. After stirring for 30 minutes, the reaction was quenched with 0.1 mL distilled water and the solvent was removed to yield A-

503083 G.

To prepare unnatural capuramycin derivatives, the previously established method was followed using a variety of alkyamines as substrates (table 2.2.7). Briefly, a 1 mL reaction consisted of 50 mM potassium phosphate (pH = 8.0), 2 mM 2d, 10 mM alkylamines (S1-S4) and 37.5 μM CapW. After incubation at 30 °C for 12 hr, reactions were stopped by ultracentrifuge to remove protein and analyzed by LC-MS using an

Eclipse XDB-C18 reversed-phase column (Agilent) under a series of linear gradients from 0.1% formic acid in H2O to 0.1% formic acid in acetonitrile over 20 min. The flow rate was kept constant at 0.4 ml/min and elution was monitored at 254 nm. Purification of products was carried out by semi-preparative HPLC using a C-18 reversed-phase column. A series of linear gradients was developed from A (2.5% ACN with 0.1% TFA) to B (90% ACN with 0.1% TFA) in the following manner (beginning time and ending time

46 with linear increase to % B): 0-2 min, 0% B; 2-18 min, 90% B; 18-22 min, 90% B; 22-24 min, 0% B; and 24-26 min, 0% B. The flow rate was kept constant at 3.5 mL/min, and elution was monitored at 260 nm. The purified products were lyophilized to yield P1-P4 as white powder.

OH OH S1-S4 MeOH OH

OH R MeO O CarU O CarU CapW O O A-503083E P1-P4

Scheme 2.2. Synthesis of P1 – P4 catalyzed by CapW as N-acyltranferase.

Similar 1 mL reactions consisting of 50 mM potassium phosphate (pH = 8.0), 2 mM A-

503083B, 10 mM alkylamines (S5–S9) and 37.5 μM CapW were carried out to yield P5-

P10.

OH OH S5-S9 R-NH2 OH OH O H N R HN O CarU CapW O CarU O O

A-503083B P5-P9

Scheme 2.3. Synthesis of P5-P10 catalyzed by CapW as a transacylase.

Table 2.2.7. Substrates for enzymatic preparation of unnatural capuramycins*.

Name Structures Name Structures Name Structures

O O O S1 NH2 NH2 NH S2 EtN S3 HN 2 HN

O NH O 2 S4 NH2 S5 S6 HN NH2

e S7 M O NH2 S8 NH2 NH2 MeO S9 O Ph O O O

* CapW was a gift from Dr. Xiaodong Liu of the Van Lanen lab.

2.2.3.5 Site-directed mutagenesis of Cpr17

Point mutations of Cpr17 were generated by PCR using Phusion Green Hot Start II High-

Fidelity DNA Polymerase (Thermo Scientific) and primers listed in table 2.2.8. pET30- cpr17 was used as a template. Reactions were conducted according to the manufacturer’s protocol with supplied buffer 2, DMSO and each pair of primer. The PCR program began at 94 °C for 30 s, followed by 30 cycles of 94 °C for 10 s, 56°C for 20 s and 60 °C for 7.5 min, and final extension at 72 °C for 7 min. The template DNA was digested with 10 units of DpnI for 1 h at 37 °C, and the remaining PCR product was

48 transformed into E. coli Nova Blue competent cells by heat-shock. The introduction of a

point mutation was confirmed DNA sequencing to yield pET30-cpr17_D212A.

Table 2.2.8. Primers for mutagenesis of Cpr17.

Name Sequence

5’ ACGCTGGCGGGCGTCGTCGCCTTCGGCGCTCT cpr17_ D212A_forward GTTCGCCG 5’ CGGCGAACAGAGCGCCGAAGGCGACGACGCC cpr17_ D212A_reverse CGCCAGCGT 3’

2.2.3.6 Docking of A-503083E to MraY and development of in-silico models for Cpr17 A-503083E was manually positioned onto the reported binding pocket of MraY (128, 129) using AutoDock Vina (130).

The in-silico model of Cpr17 was generated using the SWISS-MODEL (120) using APH(2’’)-

IIIa (PDB ID: 4TDW) and APH(2’’)-IIa (PDB ID: 4ORK) as templates. The predicted Cpr17 secondary structure was aligned with the crystal structures of templates using PyMOL.

2.2.4 Reconstitution of bacterial cell wall biosynthesis pathway

2.2.4.1 Reconstitution of E. coli cell wall biosynthesis pathway

N-acetylhexosamine 1-kinase (NahK) was cloned into pET-30 Xa/LIC vector (Invitrogen)

using genomic DNA of Bifidobacterium infantis as template yielding pET30-nahK.

GlcNAc-1-P uridyltransferase (GlmU) was amplified from E. coli genomic DNA and

glutamate dehydrogenase (GDH) was subcloned from Bacillus subtilis genomic DNA.

49 MurA, MurB, MurC, MurD, MurE, MurF and Ddl in vector pCA24N were purchased from

National Bioresource Project (Tokyo, Japan).The primers used for amplification were listed in table 2.2.9.

Table 2.2.9. Primers for amplification of NahK, GlmU and GDH.

Name Sequence NahK_forward 5' GGTATTGAGGGTCGCATGAACAACACCAAT 3’ NahK _reverse 5' AGAGGAGAGTTAGAGCCTCACTTGGTCGTCT 3’ GlmU_forward 5' GGTATTGAGGGTCGC ATGTTGAATA ATGCT 3’ GlmU _reverse 5' AGAGGAGAGTTAGAGCCTCACTTTTTCTTTAC 3’ GDH _forward 5' GGTATTGAGGGTCGCATGTTGAATAATGCT 3’ GDH _reverse 5' AGAGGAGAGTTAGAGCCTCACTTTTTCTTTAG 3’

The PCR program began at 94 °C for 30 s, followed by 25-30 cycles of 94 °C for 10 s,

56 °C for 20 s and 72 °C for 20 s per kb DNA, and final extension at 72 °C for 7 min.

Electrophoresis with a 0.5% agarose gel was used for product purification, and the desired DNA fragment was extracted. The PCR products were inserted into pET-30

Xa/LIC vector (Invitrogen) to yield pET30-nahK, pET30-glmU, and pET30-gdh. All plasmids were transformed into E. coli BL21 (DE3) competent cells. Induction and purification of recombinant proteins were carried out as described in 2.3.2.1.

To prepare UDP-MurNAc-pentapeptide, a 10 mL solution of 50 mM Tris-HCl (pH=8.0) and 10 mM MgCl2, 0.1 mmol GlcNAc, 0.12 mmol ATP, 0.12 mol UTP, 0.5 mmol PEP,

0.1 mmol NADPH, 2 mmol glucose was prepared; subsequently 5mg NahK, 5mg GlmU,

5mg MurA, 5mg MurB, 100U GDH were added. After stirring at room temperature for 2

50 h under N2, (NH4)2SO4 (final concentration at 2 mM), 0.25 mmol L-alanine, 0.25 mmol D- glutamic acid, 0.25 mmol diaminopemilic acid, 0.25 mmol D-alanine, 1 mmol ATP, 1 mmol PEP, 5 mg MurB, 5 mg MurC, 5 mg MurD, 8 mg MurE, 5 mg MurF, 8 mg Ddl, 8 mg DAP-Epi and 50U PK (pyruvate kinase) were added. The reaction was stirred at room temperature overnight and quenched by heating at 80 °C for 20 min. After centrifuge at 14, 000 rpm for 10min, the supernatant was absorbed onto pre-washed

SEPABEADS SP207 resin (Sigma-Aldrich, USA). The resin was then washed with 1L

0.01N HCl followed by 1.5L H2O, and UDP-Mur-Pentapeptide was eluted with 1L 20% methanol. The eluent was concentrated in vacuo to remove methanol and lyophilized, yielding 12mg of white powder.

Further purification was carried out by semi-preparative HPLC. A series of linear gradients was developed from 5% methanol in 40mM NaH2PO4 (pH adjusted to 6.5 by

TEA, A) to 20% methanol in 40mM NaH2PO4 (pH adjusted to 6.5 by TEA, B) in the following manner (beginning time and ending time with linear increase to % B): 0-7 min,

0% B; 7-25 min, 30% B. The follow rate was kept constant at 3.5 ml/min, and elution was monitored at 365 nm. Molecular weight of purified UDP-Mur-pentapeptide was determined by HRMS, and purity was determined by HPLC.

For HTS, to a 100 µL solution of 50 mM Tris-HCl (pH=8.0) and 10 mM MgCl2, 10 µM

UDP-GlcNAc, 10 mM ATP, 20 µM PEP, 0.1 mM NADPH, 10 mM of each amino acids,

10 mM glucose and 10mM inhibitor were added. The reaction was initiated by adding

1U GDH, 0.5 mg MurA-MurF and 0.5 mg Ddl. The reaction was incubated at 30 °C for 3 hours and quenched by adding 100 µL DCM. The aqueous phase was applied for HPLC analysis. A series of linear gradients was developed from 5% methanol in 40mM

51 NaH2PO4 (pH adjusted to 6.5 by TEA, A) to 20% methanol in 40mM NaH2PO4 (pH adjusted to 6.5 by TEA, B) in the following manner (beginning time and ending time with linear increase to % B): 0-7 min, 0% B; 7-25 min, 30% B. The follow rate was kept constant at 0.5 ml/min, and elution was monitored at 365 nm.

2.2.4.2 Reconstitution of M. tuberculosis H37Rv cell wall biosynthesis pathway

MurA-tb, MurB-tb, MurC-tb, MurD-tb, MurE-tb, MurF-tb and Ddl-tb were amplified using

M. tuberculosis H37Rv genomic DNA as template. Primers used for cloning were listed in table 2.2.10. The PCR amplification, purification, protein purification and reconstitution of M. tuberculosis cell wall biosynthesis pathway were carried out as describe in 2.2.4.1.

Table 2.2.10. Primers for amplification of Mur liagases from M. tuberculosis.

Name Sequence MurA-tb_forward 5' GGTATTGAGGGTCGCGTGGCCGAGCGT 3’ MurA-tb _reverse 5' AGAGGAGAGTTAGAGCCCTAACAGCATAC 3’ MurB-tb _forward 5' GGTATTGAGGGTCGCATGAAACGGAGC 3’ MurB-tb _reverse 5' AGAGGAGAGTTAGAGCCCTACAACATGCA 3’ MurC-tb _forward 5' GGTATTGAGGGTCGCGTGAGCACCGAG 3’ MurC-tb _reverse 5' AGAGGAGAGTTAGAGCCTCATCCCAGCAC 3’ MurD-tb _forward 5' GGTATTGAGGGTCGCGTGCTTGACCCT 3’ MurD-tb _reverse 5' AGAGGAGAGTTAGAGCCCTACCGGATCAC 3’ MurE-tb _forward 5' GGTATTGAGGGTCGCATGATCGAGCTG 3’ MurE-tb _reverse 5' AGAGGAGAGTTAGAGCCTCATGGGCGCAC 3’ MurF-tb _forward 5' GGTATTGAGGGTCGCATGATCGAGCTG 3’ MurF-tb _reverse 5' AGAGGAGAGTTAGAGCCTCATGGGCGCAC 3’ Ddl-tb _forward 5' GGTATTGAGGGTCGCGTGAGTGCTAAC 3’ Ddl-tb _reverse 5' AGAGGAGAGTTAGAGCCCTAGTGCAGGCC 3’

53 2.3 Results and discussion

2.3.1 Identification and characterization of A-102395 gene cluster

2.3.1.1 Isolation of capuramycin-type antibiotics

The structure of A-102395 was confirmed using 1H-NMR and 13C-NMR data (70) (fig

2.3.1). The HRMS data of A-503083B and A-503083F were consistent with calculated molecular weight. (fig 2.3.2 and fig 2.3.3).

Figure 2.3.1. 1H and 13C NMR spectra of A-102395 in MeOD.

Figure 2.3.2. HRMS of purified A-503083F. HRMS (ESI+) calcd. for [M + H]+ 503.1261; found 503.1247. Calcd. For [M+Na+] 525.1081; found 525.1068.

Figure 2.3.3. HRMS of purified A-503083B. HRMS (ESI+) calcd. for [M + H]+ 613.2105; found 613.2066.

55 2.3.1.2 Identification of A-102395 gene cluster

An orf encoding a protein with high amino acid sequence similarity to the transaldolase

LipK is located within the gene clusters that have been identified for the CarU- containing nucleoside antibiotics: 1 (ORF14, 55/81 % identity/% similarity) and 2 (CapH,

47/79) (107). Similarly, an orf encoding a protein with moderate sequence similarity to the dioxygenase LipL is found within the 1 (ORF7, 36/48) and 2 (CapA, 33/47) gene clusters. Previous work in the Van Lanen lab has demonstrated the direct incorporation of L-Thr via a transladol-like mechanism by undertaking feeding experiments with isotopically enriched Gly and L-Thr using the 2 producing strain. Using the previously designed degenerate primers based on the distinctive sequence blocks found within the functionally assigned L-Thr:UA transaldolases (107) as probes, we were able to identify three overlapping cosmids (pNCap01-03) harboring 49 complete ORFs. A 10-kb DNA fragment from pNCap03 was then used as another probe to identify a fourth cosmid pNCap04. Following DNA sequencing, bioinformatics analysis was carried out using

Frame-plot version 4.0 and database comparison for sequence homology was performed using the National Center for Biotechnology Information (Bethesda, MD). A total of ~85 kb of contiguous DNA was identified, including 70 complete ORFs (fig 2.3.4).

The closest homologs and putative functions of the ORFs are listed in table 2.3.1; ORFs predicted to be involved in the biosynthesis of 3 were annotated as cpr17-cpr51, for capuramycin- related nucleoside.

Figure 2.3.4 Cosmid map and genetic organization of the 3 biosynthetic gene cluster. The cpr25 gene highlighted in red encodes the putative L-Thr:uridine-5'- aldehyde transaldolase that was targeted for inactivation to confirm the identify the genetic locus.

Not surprisingly, the genetic architecture of the clusters for 1 and 2 are virtually identical due to the similarity of their structures, excluding the aforementioned orf encoding a truncated 2'-O-carbamoyltransferase within the 1 gene cluster that leads to the sole structural variation between these two groups of capuramycin-type antibiotics.

Conversely, the 16 conserved orfs within the 3 gene cluster have a different genetic organization than found in 1 and 2 (fig 2.3.5). Nonetheless, individual comparison of the gene products revealed a relatively high amino acid sequence identity, including the transaldolase (Cpr 25; 79% identity with CapH) and t he non-heme Fe(II)- dependent αKG:UMP dioxygenase (Cpr19; 67% identity with CapA). Additional shared gene products of note include a phosphotransferase Cpr17, likely involved in self- resistance (53% identity with CapP), a carboxymethyltransferase (Cpr27; 57% identity 57 with CapS), and a putative amide bond- forming N-transacylase (Cpr51; 65% identity

with CapW). CapS and CapW have been shown to function sequentially to activate the

carboxylic acid of 2c in the form of the methyl ester 2d followed by transacylation to

incorporate the L-ACL to generate 2a( 121 ) . The uncovering of genes for Cpr27 and

Cpr51 suggests the unusual arylamine-containing polyamide of 3 is incorporated using

a comparable mechanism, i.e., carboxylic acid 1c → methyl ester 1d → amide 1a (Fig.

6C), which was unexpected given the unique chemical nature of the acyl acceptors.

Figure 2.3.5. Comparative analysis of the sequenced region of chromosomal DNA from the 2- and 3-producing strains. Highlighted in blue are the minimal orfs predicted to be essential for 3 biosynthesis, while the two orfs in black, cpr19 and cpr25, are predicted to encode for a non-heme Fe(II)-dependent αKG:UMP dioxygenase and L- ThrA-:uridine-5'-aldehyde transaldolase, respectively.

58 Table 2.3.1. Annotation of ORFs within the 3 gene cluster

a b c Protein Size Proposed function Homolog (accession no.) Identity / Similarityd (%) ORF1 319 Cell-wall associated hydrolase Svir_07310 (YP_003132627) 44/58 ORF2 363 Hypothetical protein Francci3_2715 (YP_481805) 49/59 ORF3 855 Hypothetical protein Franean1_2549 (YP_001506885) 58/69 ORF4 639 Type IV secretory pathway VirB4 Franean1_2548 (YP_001506884) 60/73 ORF5 398 Hypothetical protein Franean1_2547 (YP_001506883) 57/66 ORF6 774 Hypothetical protein Caci_4083 (YP_003114789) 46/64 ORF7 107 Hypothetical protein Franean1_2741 (YP_001507073) 58/77 ORF8 322 Hypothetical protein Franean1_2740 (YP_001507072) 45/56 ORF9 247 Hypothetical protein SACE_2308 (YP_001104534) 47/56 ORF10 350 Hypothetical protein SACE_2307 (YP_001104533) 70/80 ORF11 233 Putative cellulase SCAB_16431 (CBG68778) 24/41 Cpr12 259 Aminotransferase class IV Caci_2606 (YP_003113364) 45/56 ORF13 162 Putative short chain dehydrogenase MAB_3919c (YP_001704647) 48/62 ORF14 373 Transposase RHA1_ro08502 (YP_707704) 76/85 ORF15 122 Transposase Svir_17380 (YP_003133592) 75/85 ORF16 123 Transposase SSAG_01038 (ZP_04996736) 68/77

e Cpr17 305 Capuramycin 3''-phosphotransferase CapP 53/64 Cpr18 331 Fe2+-dependent, KG dioxygenase CapD 63/74 Cpr19 275 Fe2+-dependent, KG:UMP dioxygenase CapA 76/88 Cpr20 317 putative 3-ketoreductase CapC 73/81 Cpr21 457 putative 2,3-dehydratase CapE 73/83 Cpr22 313 putative 4-epimerase CapF 70/79 Cpr23 378 SelA-related PLP-dependent enzyme ORF15 47/65 Cpr24 384 Putative glycosyltransferase CapG 77/86 Cpr25 412 L-Thr:uridine-5'-aldehyde transaldolase CapH 79/90 Cpr26 87 Putative pyrophosphatase CapI 67/81 Cpr27 261 Putative carboxyl methyltransferase CapS 65/79 Cpr28 774 Putative CO dehydrogenase CapJ 75/84 Cpr29 244 Putative O-methyltransferase CapK 71/84 Cpr30 165 Putative CO dehydrogenase CapL 76/89 Cpr31 289 Putative CO dehydrogenase CapM 60/72 Cpr32 350 Luciferase-like monooxygenase LLB_0745 (ZP_06185947) 43/56 Cpr33 234 3-oxoacyl-[acyl-carrier-protein] reductase Noca_2571 (YP_923762) 57/71 Cpr34 391 -Ketoacyl synthase ShygA5_010100009994 (ZP_05513707) 53/63 Cpr35 257 Hypothetical protein SCAB_69811 (CBG73975) 44/63 Cpr36 84 Aryl carrier protein AcmD (AAD30112) 35/55 Cpr37 466 Actinomycin synthetase I AcmA (AAD30111) 49/62 Cpr38 663 4-amino-4-deoxychorismate synthase PabAB (P32483) 55/64 Cpr39 521 AMP-dependent synthetase and ligase SrosN15_010100025847 (ZP_04696386) 50/61

Table 2.3.1 continued.

Cpr40 311 Dehydrogenase E1, TPP-dependent Caci_5069 (YP_003115770) 55/70 Cpr41 338 Pyruvate dehydrog. (acetyl-transferring) Sros_2206 (YP_003337932) 55/69

Cpr42 365 Dehydrogenase Npun_F31621 (YP_001866563) 38/57

Cpr43 433 Glycosyl transferase family 2 MicauDRAFT 1168 34/49 Cpr44 612 Acetolactate synthase, large subunit Vapar_2048 (YP_002943959) 26/42 Cpr45 227 Phosphatase (HAD superfamily) Yaldo0001_22190 (ZP_04621838) 39/56 Cpr46 599 TPP-dependent synthase/transketolase PlaT6 (ABB69756) 45/57 Cpr47 448 Condensation domain (NRPS) MicauDRAFT_1165 (ZP_06216171) 29/42 Cpr48 97 Acyl carrier protein SrosN15_010100019301 (ZP_04695086) 46/58 Cpr49 314 MitI transglutaminase MitI (AAD28463) 29/48 Cpr50 395 MitI transglutaminase MitI (AAD28463) 33/44 Cpr51 395 Putative transacylase CapW 65/75 Cpr52 191 Hypothetical protein SrosN15_010100025822 (ZP_04696381) 38/51

Cpr53 381 Acyl-CoA dehydrogenase SrosN15_010100025827 (ZP_04696382) 45/57 Cpr54 515 Long-chain-fatty-acid-CoA ligase MCAG_01785 (ZP_04605528) 75/82 Cpr55 84 Acyl carrier protein VinL (BAD08369) 44/63 Cpr56 424 Orn/DAP/Arg decarboxylase 2 MCAG_01775 (ZP_04605518) 74/83 Cpr57 293 MitI transglutaminase MitI (AAD28463) 29/45 ORF58 596 ABC transporter ATP-binding protein MCAG_02642 (ZP_04606385) 37/52 ORF59 608 ABC transporter ATP-binding protein SghaA1_010100034323 (ZP_04690305) 53/65 ORF60 594 ABC transporter related protein Cfla_3590 (YP_003638660) 45/62 ORF61 663 ABC transporter related protein Cfla_3591 (YP_003638661) 44/58 ORF62 441 MATE efflux family protein Amir_4280 (YP_003101980) 57/71 ORF63 391 AstB/chuR/nirj-related protein SghaA1_010100036797 (ZP_04690774) 74/83 ORF64 85 Hypothetical protein SghaA1_010100036802 (ZP_04690775) 55/72 ORF65 352 Hypothetical protein StAA4_010100023552 (ZP_05481114) 55/63 ORF66 320 Oxidoreductase domain protein Amir_4534 (YP_003102223) 51/63 ORF67 219 Putative phosphotransferase StAA4_010100023547 (ZP_05481113) 50/61 ORF68 358 Hypothetical protein StAA4_010100023542 (ZP_05481112) 53/67 ORF69 420 Hypothetical protein Sros_6837 (YP_003342286) 29/43 ORF70 179 Hypothetical protein StAA4_010100029435 (ZP_05482273) 57/75

a Sequences deposited at NCBI (accession no. KP995196). b Numbers are in amino acids. c Given in brackets are accession numbers. d % sequence identity/similarity for the entire protein. e Putative proteins within shaded rows have homologues within the A-503083 gene cluster (accession no. AB538860) and A-500359 gene cluster (accession no. AB476988)

60 2.3.1.3 Characterization of the gene cluster for 3 by inactivation of cpr19, cpr25 and cpr51

The genes cpr19 and cpr25 are proposed to be responsible for the initiation of the

biosynthesis of CarU core of 3, while cpr51 was proposed to form the amide bond

between hexuronic acid and the polyamide chain. To investigate the involvement of

those genes in biosynthetic pathway of 3, both cpr19 and cpr25 were individually

targeted for inactivation by double crossover homologous recombination, and the

successful inactivation of each was confirmed by PCR and Southern blot analysis

(fig 2.3.6 and fig 2.3.7). To determine if cpr51—encoding a putative N-transacylase

with a proposed functional role comparable to CapW—was involved in the

biosynthesis of 3, the gene was inactivated by double crossover homologous

recombination and also confirmed by PCR and Southern blot analysis (fig 2.3.8).

Table 2.3.2. Sequence comparison in % identity/% similarity among putative αKG:UMP dioxygenases.

a b Gene Product LiPL Cpz15 LpmM Mur16 SphE Mra24 ORF7 CapA Cpr19 c GlyU LipL ------86/90 81/87 39/51 25/35 26/36 36/48 33/47 39/53 Cpz15 ------82/89 38/51 25/34 25/36 34/48 33/48 39/54 LpmM ------37/50 24/35 27/38 36/50 33/47 37/53 Mur16 ------25/36 29/41 43/56 42/53 47/62 SphE ------21/32 28/40 28/40 27/39 Mra24 ------27/37 26/35 27/41 d CarU ORF7 ------79/86 70/82 CapA ------67/78 Cpr19 ------

a LipL is encoded within the biosynthetic gene cluster for A-90289, Cpz15 for caprazamycin, LpmM for liposidomycin, Mur16 for muraymycin, SphE for sphaerimicin, Mra24 for muraminomicin, ORF7 for A-500359, CapA for A-503083, and Cpr19 for A- 102395. b Mra24 appears truncated and inactive, missing 57 amino acids at the C-terminus when aligned with LipL. c GlyU, 5'-C-glycyluridine-containing nucleoside antibiotics. d CarU, uridine-5'-carboxamide-containing nucleoside antibiotics.

B WT 

M WT 

2 kb 1.5 kb 5265 bp 1 kb 4294 bp

Figure 2.3.6. Inactivation of cpr19 in Amycolatopsis sp. SANK 60206. (A) Gene location of cpr19 in the biosynthetic gene cluster of A-102395. (B) PCR analysis of genomic DNA isolated from the wild-type (WT; expected 0.9 kb) and Δcpr19 mutant (Δ; expected 1.8 kb) strains. (C) Strategy utilized for gene inactivation of cpr19. (D) Southern blot analysis of ApaI-digested genomic DNA from wild-type (WT) and Δcpr19 mutant (Δ) strains using the indicated probe.

M WT   WT

2 kb 1.5 kb 2854 bp 1 kb

1161 bp

Figure 2.3.7. Inactivation of cpr25 in Amycolatopsis sp. SANK 60206. (A) Gene location of cpr25 in the biosynthetic gene cluster of A-102395. (B) PCR analysis of genomic DNA isolated from the wild-type (WT; expected 1.2 kb) and Δcpr25 mutant (Δ; expected 1.8 kb) strains. (C) Strategy utilized for gene inactivation of cpr25. (D) Southern blot analysis of BamHI-digested genomic DNA from wild-type (WT) and Δcpr25 mutant (Δ) strains using the indicated probe.

WT  M WT  

2 kb 1.5 kb 3529 bp 1 kb 3335 bp 

       Figure 2.3.8. Inactivation of cpr51 in Amycolatopsis sp. SANK 60206. (A) Gene location of cpr51 in the biosynthetic gene cluster of A-102395. (B) PCR analysis of genomic DNA isolated from the wild-type (WT; expected 1.1 kb) and Δcpr51 mutant (Δ; expected 1.8 kb) strains. (C) Strategy utilized for gene inactivation of cpr51. (D) Southern blot analysis of PstI-digested genomic DNA from wild-type (WT) and Δcpr51 mutant (Δ) strains using the indicated probe.

64 For comparative analysis of 3 production in the wild-type and mutant strains, extracts from 2 L of culture broth were used for LC-MS-MS analysis with comparison to authentic 3. Multiple-reaction-monitoring transitions were monitored at 763.316/189.9 and 763.316/120. The resulting cpr19 and cpr25 mutant strains were unable to produce 3 (Figure 2.3.9), consistent with an essential role in the biosynthesis of 3.

As expected, the cpr51 mutant strain was also unable to produce 3, consistent with an essential role in 3 biosynthesis.

A-

Wild

Δcpr19

Δcpr25

Δcpr51

Figure 2.3.9. Analysis of gene inactivation in Amycolatopsis. Sp. SANK 60206. LC-MS-MS analysis of authentic 3 (i) and production from wild-type (ii), Δcpr19 (iii), Δcpr25 (iv), and Δcpr51 (v) strains.

Gene complementation has yet to be successful in this strain, and thus to exclude a possible polar effect, the expression levels of the upstream and downstream genes

65 were analyzed by RT-PCR. Using mRNA extracted from the Δcpr25 mutant strain at the onset of 3 production, expression of cpr25 was not detected while genes flanking cpr25 were clearly expressed (fig 2.3.10). In contrast, all three genes were detected within the wild-type strain, suggesting there is no polar effect due to cpr25 inactivation.

Ambiguous results were obtained with expression analysis of the Δcpr19 mutant strain. Utilizing mRNA extracted from the Δcpr51 mutant strain at the onset of 3 production, expression of cpr51 was not detected while genes flanking cpr51 were clearly expressed (fig 2.3.10).

Figure 2.3.10. RT-PCR analysis of the indicated genes using mRNA isolated from the mutant strains or wild-type strain. M, DNA ladder.

Ambiguous results were obtained for expression analysis of the cpr19 mutant strain, and hence we turned our attention to in vitro characterization to establish direct evidence of the involvement of cpr19 in the biosynthesis of 3.

66 2.3.1.4 Functional characterization of Cpr19 and CapH

The gene encoding cpr19 was heterologously expressed in E. coli to obtain pure, recombinant protein (fig 2.3.11 B). Based on prior characterization of LipL, Cpr19 was expected to convert UMP to UA in a reaction that is dependent upon Fe(II), O2, and αKG. HPLC analysis of reactions with all these components revealed a new peak that co-eluted with authentic UA (fig 2.3.11 C).

After demonstrating that UA is a biosynthetic intermediate of CarU-containing nucleosides, the probable L-Thr:UA transaldolase, Cpr25, was targeted for characterization; however, soluble protein could not be obtained using multiple hosts and a variety of expression conditions. Instead Cpr25 was substituted with CapH that is encoded within the 2 biosynthetic gene cluster (69). Similarly to LipK (101), soluble

CapH was only produced in S. lividans TK64 (fig 2.3.11 B). Enzyme assays monitored by HPLC revealed identical traces to LipK (fig 2.3.11 D), and the product peak was compared to synthetic standard (101) to demonstrate CapH is an L-Thr:UA transaldolase.

The detailed characterizations of Cpr19 and CapH were carried out by Dr. Anwesha

Goswami and Dr. Sandra Barnard-Briston.

Interestingly, Cpr19 was a monomer instead of a dimer as TauD (fig 2.3.11C, PDB

ID: 3SWT (110)), its closest homology, suggesting that this enzyme has a distinct three dimension structure compared with other αKG:taurine dioxygenase (111-113).

Structural studies of this enzyme are ongoing.

67 A O O O O NH NH NH O OH NH 2 NH N O HO P N O Cpr25 H2N ? H2N HO O O Cpr19 O O O N O O N O O O HO HO HO OH HO OH Acet- aKG Succ L-a-Thr aldehyde HO OH HO OH O2 CO2

B C Cpr19 Cpr19 CapH

66kD

45kD 50kD

D E

Figure 2.3.11. Biosynthesis of CarU of A-102395. (A) The pathway starts with UMP that is converted to GlyU by the sequential reactions catalyzed by Cpr19 and Cpr25. (B) SDS analysis of recombinant Cpr19 and CapH. (C) Native SDS-PAGE analysis indicated that Cpr19 was a monomer. (D) HPLC analysis of reaction catylized by Cpr19 using UMP as the prime substrate with all components (i) or the omission of

68 ascorbic acid (ii). (E) HPLC analysis of reaction catylized by CapH PLC analysis of the reaction without enzyme (i) or with CapH (ii) or LipK (iii). A260, absorbance at 260 nm.

2.3.1.5 Proposed biosynthetic pathway of α-D-mannopyranuronate moiety and formation of polyamide side chain for 3

A glycosyltransferase, Cpr24 encoded in the 3 cluster, is proposed to attach the sugar moiety to the CarU core, but the formation of the unsaturated uronic acid moiety is still not clear. The isopotic enrichment experiments suggested the sugar unit was derived from mannose, thus a GDP-D-mannuronic acid is expected to be an intermediate. GDP-D-mannuronic acid is a building block of alginate found in other bacterial strains, and therefore is a reasonable precursor (122). GDP-D-mannuronic acid is formed from GDP-D-mannose by a GDP-mannose-6-dehydrogenase in alginate biosynthesis while two gene products (Cpr21, a putative NDP-hexose 2,3- dehydratese and Cpr22, a putative NDP-hexose 4-epimerase/dehydrodates) that are shared in the 1-3 gene clusters are putatively involved in this chemistry. Lastly, 4,5- unsaturated double bond is putatively introduced by a dehydratase reaction, possibly by Cpr20, a putative ketoreductase, or by Cpr21/22. Cpr29 encoded a methyl transferase that is required for the 3’-O-methylation is found in all capuramycin-type antibiotics, although the timing of methylation is still unclear. The genetic system established in this study should help in defining the involvement and functions of those gene products.

69 The arylamine-containing polyamide moiety is exclusively found in 3. Bioinformatics analysis of 3 gene cluster revealed that a minimum 8 ORFs are required for the side chain biosynthesis, including Cpr12 and Cpr32-38. Cpr38 encodes a bidomain protein containing an L-Gln aminotransferase and a 4-amino-4-deoxychorismate

(ADC) synthase(123), and thus it is proposed to catalyze a two-step reaction involving amidohydrolysis of L-Gln and incorporation of the free ammonia into chorismic acid to generate ADC. The following step is putatively catalyzed by Cpr12, which is annotated as an ADC lyase, to form para-amino-benzoic acid (PABA) through elimination of pyruvate. Similar to the initial activation and loading of a 4-methyl-3- hydroxyanthranilic acid to a stand-alone carrier protein in biosynthesis of actinomycins(124), Cpr37 and Cpr36 together catalyze the formation of thioester- linked PABA. Adjacent Cpr34 (a ketosynthase) and Cpr35 (a chain length factor) catalyze the decarboxylative condensation to form a β-ketothioester via a similar mechanism that was proposed in hygromycin B biosynthesis (125). The following stereoselective reduction is proposed to be catalyzed by Cpr33, and Cpr32 likely catalyzes hydroxylation of C-2. The formation of the remaining two amide bond is proposed to involve ORFs between cpr47-cpr57, which encodes proteins with similarity to an adenylation domain protein (Cpr54), two carrier proteins (Cpr48 and

Cpr55), a condensation domain protein (Cpr47) and three transglutaminase-like proteins. The lack of precedence for this component in metabolites makes it difficult to propose how the amide bonds are generated; however, the genetic system that was developed can now minimally be used to assess the requirement of these ORFs.

70 The final step for 3 biosynthesis is the formation of an amide bond between the polyamide side chain and the unsaturated hexuronic acid. Two genes cpr29 and cpr51 show high homology to capS and capW which are responsible for loading of

ACL during the biosynthesis of 2, and gene inactivation suggested that cpr51 is essential for 3 biosynthesis. Unfortunately we were unable to isolate any precursors of 3. Nonetheless Cpr29 is proposed to activate the carboxylic acid by methylation to the ester and Cpr51 is proposed to catalyze the final coupling of the polyamide.

The biosynthetic gene cluster for 3 identified in this study has enabled a thorough comparative analysis with gene clusters for other capuramycin-type antibiotics, resulting in the proposed shared biosynthetic pathway as summarized in fig 2.3.12.

72 2.3.2 Functional characterization of Cpr17 as a phosphotransferase

2.3.2.1 Functional characterization of Cpr17

Previous study in the Van Lanen group demonstrated that: 1) within the biosynthetic gene cluster for 1 exists a gene encoding for a putative aminoglycoside 3- phosphotransferase (Orf21) that confers selective resistance to 1 when heterologously expressed in Streptomyces albus (99), and 2) a similar gene, capP, was identified within the biosynthetic gene cluster for 2 and was functionally characterized in vitro as a phosphotransferase that regiospecifically transfers the - phosphate of ATP to the 3′′-hydroxyl of the hexuronic acid moiety of 2a leading to

272-fold increase in the IC50 in the MraY inhibitory activity (fig 2.3.13) (114).

HO O P OH O O HO O OH OH NH NH O O H H2N CapP H H2N HN N O N N O HN O N O O O O O O O O O ATP ADP H CO O 3 O H3CO O O

Figure 2.3.13. The ATP-dependent phosphorylation reaction catalyzed by

CapP (114).

Figure 2.3.14. Resistance conferred to Streptomyces albus upon heterologous expression of Cpr17. The concentration of 1a is 0 µg/mL, column 1; 100 µg/mL, column 2; and 500 µg/mL, column 3. The upper two rows (boxed in blue) consist of mycelia diluted 1/10 following homogenization of a liquid culture of S. albus while the bottom two rows (boxed in red) consist of mycelia diluted 1/100 prior to spotting on ISP2 agar plates.

Within the biosynthetic gene cluster of 3, a gene encoding Cpr17 was identified that showed high sequence similarity to Orf21 and CapP (52% identity with Orf21 and 53% identity with CapP). It is thus proposed that Cpr17, as same as Orf21 and CapP, functions as a phosphotransferase conferring self-resistance to Amycolatopsis sp.

SANK 60206. In order to test the hypothesized function of Cpr17, the cpr17 gene was cloned from Cosmid pNCap01 and heterologously expressed in S. albus. Strains harboring the cpr17 gene were resistant to 2a at 500 µg/mL, while growth of the strains containing empty vector were susceptible at 100 µg/mL (fig 2.3.14).

74 The cpr17 gene was then cloned and expressed in E. coli to yield purified protein with expected size (fig 2.3.15, His-tag included). As a control, CapP was also produced.

A B

55 KD 37 KD

25 KD

Figure 2.3.15. Expression of recombinant CPR17 and CapP. (A) SDS-PAGE analysis of His6-Cpr17 (expected MW of 37.9 kD), and (B) SDS-PAGE analysis of

His6-CapP (expected MW of 37.8 kD).

In order to test the activity of Cpr17, the reaction was initially assessed by HPLC using

2a as a potential substrate, and a new peak eluting before 2a was observed. The retention time of the new peak was identical when Cpr17 was substituted by CapP, suggesting that Cpr17 catalyzes regiospecific phosphorylation at the 3′′-OH of 2a.

The HRMS analysis of the product revealed an [M-H]- ion at m/z = 691.1536 which is consistent to the expected 3′′-phospho-A-503083B (calculated [M-H]- ion at m/z =

691.1607) (fig. 2.3.17).

The authentic substrate for Cpr17, 3, was then used for the reaction as described above. A new peak appeared using either Cpr17 or CapP, and the analysis of the product revealed an [M-H]- ion at m/z = 843.0, which was consistent to the calculated

- MW of mono-phospho-3 (C31H37N6O20P, expected [M-H] ion at m/z = 843.2) (fig.

75 2.3.18). Although the limited availability of 3 precluded structural elucidation and product bioactivity analysis at the current time, the results suggested Cpr17, like

CapP, functions as a regiospecific phosphotransferase. The shared precursor of the capuramycins, 2d and 2c (fig 1.7) were used as substrates. Although both Cpr17 and

CapP were able to convert 2d into 3’’-phospho-2d, no appreciable activity was detected with 2c. The resulted was consistent to the previous study showing that

CapP utilized 2c with a very low turnover of 0.2 min-1 (99) (fig 2.3.16).

A B

C D

iii

Figure 2.3.16. HPLC analysis of the reaction catalyzed by Cpr17 and CapP. 2a (A), 3 (B), 2d (C) 1c (D) were used as substrates and incubated without enzyme (i), with Cpr17 (ii), and with CapP (iii). ▼, 3''-phospho-2a; ♦, 3''-phospho-2d; and ●, 3''- phospho-3. A260, absorbance at 260 nm. The asterisk (*) denotes an unidentified contamination peak.

A 693.1742

B 691.1536

Figure 2.3.17. HRMS analysis for the product of reactions catalyzed by Cpr17 using 2a as substrate. (A) HRMS (ESI+) calcd. for [M + H]+ 693.1769; found 693.1742; (B) HRMS (ESI-) calcd. for [M - H]- 691.1612; found 691.1535.

A B

Figure 2.3.18. Mass spectrum for the products of reactions catalyzed by Cpr17. (A) Mass spectrum for the peak eluting at 7.7 min yielding [M-H]- = 763.3 consistent with the calculated MW of authentic 3. (B) Mass spectrum for the peak eluting at 7.6 min of the reaction using 3 as substrate yielding [M-H]- = 843.0 consistent with the calculated MW of 3’’-phospho-3. (C) Mass spectrum for the peak eluting at 14.6 min of the reaction using 2d as substrate yielding [M-H]- = 595.2 consistent with the calculated MW of 3’’-phospho-2d.

79 2.3.2.2 Kinetic characterization of Cpr17.

After identifying the function of Cpr17, single-substrate kinetic experiments were

performed using 2a as substrate. Using near saturating ATP and variable 2a,

standard Michaelis-Menten kinetics were observed yielding a KM = 146 ± 13 µM and -1 kcat = 5.8 ± 0.2 min (fig. 2.3.19 A), the former constant similar to and the latter 5-

fold lower than that reported for CapP (table 2.3.3). This result was expected since

2a was not the authentic substrate. Single-substrate kinetic experiments with

saturating 2a and variable ATP yielded a KM = 1.4 ± 0.1 mM and kcat = 10.1 ± 0.4

-1 min (fig. 2.3.19 B). The unexpected high observed KM for ATP indicated that

ATP is not a preferred source of phosphate. This result prompted us to test GTP as

an alternative phosphate donor, which served as an excellent substrate for Cpr17

-1 yielding a KM = 9.2 ± 1.7 µM and kcat = 3.3 ± 0.1 min (fig. 2.3.19 C), a 50-fold

improvement in catalytic efficiency, suggesting GTP is the in vivo substrate for Cpr17.

A similar nucleotide preference has been recently reported for certain aminoglycoside

phosphotransferases (126-128)

Table 2.3.3. Michaelis-Menten kinetic parameters of CapP and Cpr17.

-1 Enzyme Substrate KM (µM) kcat (min ) kcat/KM Relative efficiency a CapP 2a 170 ± 40 34 ± 4 2.0 x 10-1 Cpr17 2a 146 ± 13 5.8 ± 0.2 4.0 x 10-2 ATP (1.4 ± 0.1) x 103 10.1 ± 0.4 7.2 x 10-3 1 GTP 9.2 ± 1.7 3.3 ± 0.1 3.6 x 10-1 50 a Data taken from Ref (114).

A B

Figure 2.3.19. Plots for single-substrate kinetic analysis of Cpr17. (A) Single- substrate kinetic analysis with variable 2a. (B) Single-substrate kinetic analysis with variable ATP. (C) Single-substrate kinetic analysis with variable GTP.

81 2.3.2.3 Promiscuity of Cpr17

Cpr17 phosphorylates not only its authentic substrate 3 but also other capuramycin-

type antibiotics including 2a and 2d. This suggested a low specificity of Cpr17 for

different substrate sidechains (fig 2.3.20). the substrate specificity of Cpr17 were

further tested by using semi-synthetically prepared capuramycin derivatives P1 – P10

and A-503083G as substrates (table 2.3.4). After overnight incubation with Cpr17

along with ATP, all tested substrates were converted into their corresponding mono-

phosphorylated products with the only exception of A-503083 G (fig 2.3.21). The lack

of activity with A-503083G, which contains a 3’’-methyl ether instead of the hydroxyl,

provided further evidence that Cpr17, as well as other capuramycin

phosphotransferase, regiospecifically transfers γ–phosphate to this position. MS

analysis of the product peaks was consistent with each product being

monophosphorylated (fig 2.3.22). It is of worth to note that all the capuramycin

derivatives tested here showed potent inhibitory activity against M. smegmatis MC2

155* except A-503083 G.

HO O P R2 O O O HO O OH OH NH NH H N Cpr17 H N 2 R 2 R1 O N O 1 O N O O O O O O O O O ATP ADP

H3CO O O H3CO O O

Figure 2.3.20. Phosphorylation of unnatural capuramycin derivatives by Cpr17.

* Data not yet published.

82 Table 2.3.4. Semi-synthesized substrates for Cpr17 activity assay*. Absolute stereo chemistry was shown.

R 2 O O OH NH H2N R1 O N O O O O O

H3CO O O

Name R1 R2 Name R1 R2

P1 O H P2 O H H H N N EtN HN

P3 O H H P4 O H N NH HN HN

P5 H H P6 H H N N O

P7 MeO HN P8 H HN H MeO O

P9 H A-503083G O CH3 HN MeO

* Structures of unnatural capuramycins were compared with the standards provided by Dr. Xiaodong Liu except for P5 (Appendix I).

P1 P2 P3

P4 P5 P6

P7 P8 P9 A-503083G

Figure 2.3.21. HPLC analysis of reactions catalyzed by Cpr17 using unnatural capuramycins as substrates without enzyme (upper black traces) and with Cpr17

(lower red traces). A260, absorbance at 260 nm. All substrates were monophosphorylated except for A-503083 G.

Mono-phospho-P1 Mono-phospho-P2 Mono-phospho-P3

Mono-phospho-P4 Mono-phospho-P5 Mono-phospho-P6

Mono-phospho-P7 Mono-phospho-P8 Mono-phospho-P9

Figure 2.3.22. Mass spectrum for the products of reactions catalyzed by Cpr17 using unnatural capuramycins as substrates. Mass spectrum for the peak was consistent with calculated MW of mono-phospho P1-P9.

85 Functional assignment of Cpr17 as a regiospecific and substrate-promiscuis phosphotransferase suggests that the 3′′-OH is critical for the inhibitory activity of capuramycins against MraY. To elucidate the importance of 3′′-OH, I docked 2d, the structurally simplest but active member of capuramycins, into the proposed substrate binding site of MraY using the reported structure using autodock vina (129,130) (fig

2.3.23). It is of interest to notice that 3′′-OH is interacting through a hydrogen bond with D117, which is one of the three catalytically important residues in the active site of MraY (54). A similar interaction was not observed for A-503083G on which the 3′′-

OH was chemically protected by a methyl ether.

MraY D117 O

D117 O OH O OH NH H2N O O O O N O O O

H3CO O O

A-503083E

Figure 2.3.23. Docking 2d to the active site of MraY. The 3’’-OH of 2d formed a putative hydrogen bond to D117, one of three catalytic residues (highlighted in red and depicted as sticks, left). The structure used for binding and the possible interaction was highlighted (right).

86 2.3.2.4 Predicted structure of Cpr17

The protein kinase (PK) superfamily which transfers the γ-phophate group from nucleotide to the target substrates contain a number of highly conserved catalytic residues related to nucleotide binding and phophoryl transfer (131). Cpr17 and CapP, showing highest sequence similarity to aminoglycoside phosphotransferase (APH), were aligned to a series of well-studied APHs including APH(2′′)-IIIa (accession number 3TDW_A, PDB ID: 3tdw), APH(2′′)- Ia (accession number 4ORK_D, PDB ID:

4ORK), APH(2′′)-IIa (accession number ), APH(2′′)-IVa (accession number 3N4V_B,

PDB ID: 3N4V), and APH(2′′)-Iva (accession number 4N57_B, PDB ID: 4N57) (fig

2.3.24). The presence of the conserved triphosphate binding residue (highlighted in red), and the essential DXXXXN catalytic loop that is conserved (Asp-194 in Cpr17 and highlighted in blue) was confirmed. The acidic glutamic acid (Glu-63, highlighted in green) is essential for maintaining the protein structure, and nucleotide binding sites (highlighted in dark green) are also conserved. The alignment indicated that

Cpr17 and CapP share the conserved nucleotide binding site with other members of the APH superfamily as expected resulting in high overall sequence identity. However, it was quite unexpected that neither Cpr17 nor CapP contain a “gatekeeper” tyrosine as seen with other GTP specific APHs as reported previously (126) but instead contain a Val and Thr in Cpr17 and CapP, respectively, thus suggesting that the capuramycin phosphotransferease are nucleoside unspecific, which is inconsistent with the kinetics data.

87 10 20 30 Cpr17 ------VTETEITAGLIRDLLRDQHPDLADLPLREVEGGWG capP ------MTDTENEITADLITGLLREQHPDLADLPLTFGAHGWD 3tdv ------MKQNKLHYTTMIMTQFPDISIQSVESLGEGFR 4ork ------MEYRYDDNATNVKAMKYLIEHYFDNFKVDSIEIIGSGYD 4dca ------MVNLDAEIYEHLNKQIKINELRYLSSGDD 3n4v ------MRTYTF------DQVEKAIEQLYPDFTINTIEISGEGND 4N57: MGSSHHHHHHSSGLVPRGSHMRTYTF------DQVEKAIEQLYPDFTINTIQISGKGND

40 50 60 70 80 90 Cpr17 NQMWRLGDELAVRIQRMDND-PQFQLRERQWLPLLAPR--LPLPIPVPVRNGAPSERFPK capP NQLWRLGDDLAVRLPWATEDADDLLLKEHAFLPAMASR--LPLPVPVPQRLGRPSERFPR 3tdw NYAILVNGDWVFRFPKSQQG-ADELNKEIQLLPLLVGC--VKVNIPQYVYIGKR--SDGN 4ork SVAYLVNNEYIFKTKFSTNK-KKGYAKEKAIYNFLNTNLETNVKIPNIEYSYI---SDEL 4dca SDTFLCNEQYVVKVPKRDSV-RISQKREFELYRFLEN-CKLSYQIPAVVYQSD-----R- 3n4v CIAYEINRDFIFKFPKHSRG-STNLFNEVNILKRIHNK--LPLPIPEVVFTGMPSETYQM 4N57: CIAYEINRDFIFKFPKHSRG-STNLFNEVNILKRIHNK--LPLPIPEVVFTGMPSETYQM

100 110 120 130 140 Cpr17 LWTVVTWVPGLPLDQG-----SITRGDDAADTLAAFLRALHVAAPADAPV------DNDR capP PWIVTTWVPGEPADRA-----PATRGVEAADALSAFLKALHQPAPDDAPG------GRNR 3tdw PFVGYRKVQGQILGEDGMAVLPDDAKDRLALQLAEFMNELSAFPVETAISAG-VPVTNLK 4ork SILGYKEIKGTFLTPEIYSTMSEEEQNLLKRDIASFLRQMHGLDYTDISE---CTI-DNK 4dca -FNIMKYIKGERITYEQYHKLSEKEKDALAYDEATFLKELHSIEIDCSVSLFSDALVNKK 3n4v SFAGFTKIKGVPLTPLLLNNLPKQSQNQAAKDLARFLSELHSINISGFKS---NLVLDFR 4N57: SFAGFTKIKGVPLTPLLLNNLPKQSQNQAAKDLARFLSELHSINISGFKS---NLVLDFR

150 160 170 180 190 200 Cpr17 GAHPKDSTNGFNYFLNSVDADAIGHDAADVRAVWDDAVAAPA-WEGPAMWVHGDLHPANV capP GGPLADADEGFEHFLKETTNRGLIPEPDTVREVWKDALAAPV-WTGPSLWLHADLHPANL 3tdw NKILLLSEAVEDQVFP----LLDESLRDYLTLRFQSYMTHPVYTRYTPRLIHGDLSPDHF 4ork QNVLEEYILLRETIYN----DLTDIEKDYIES-FMERLNATTVFEGKKCLCHNDFSCNHL 4dca DKFLQDKKLLISILEKEQ--LLTDEMLEHIETIYENILNNAVLFKYTPCLVHNDFSANNM 3n4v EKINEDNKKIKKLLSR----ELKGPQMKAVDDFYRDILENEIYFKYYPCLIHNDFSSDHI 4N57: EKINEDNKKIKKLLSR----ELKGPQMKKVDDFYRDILENEIYFKYYPCLIHNDFSSDHI

210 220 230 240 250 Cpr17 VVA----DGTLAGVVDFGALFAGDPALDLSSAWVLLPAGAAA----RFFAAYADA-DEAT capP LTT----DGTFCGVVDFGDLCAGDPACDLAAGWHVLPDGAID----RFHQSYSSAADAAT 3tdw LTNLNSRQTPLTGIIDFGDAAISDPDYDYVYLLEDC----GELFTRQVMAYRGEVDLDTH 4ork LLD--G-NNRLTGIIDFGDSGIIDEYCDFIYLLEDSEEEIGTNFGEDILRMYGNIDIEKA 4dca IFR----NNRLFGVIDFGDFNVGDPDNDFLCLLDCSTDDFGKEFGRKVLKYYQHKAPEVA 3n4v LFD--TEKNTICGIIDFGDAAISDPDNDFISLMEDD-EEYGMEFVSKILNHYKHKDIPTV 4N57: LFD--TEKNTICGIIDFGDAAISDPDNDFISLMEDD-EEYGMEFVSKILNHYKHKDIPTV

260 270 280 290 300 Cpr17 VRRARGLAALKSLFLMLMGQNGDRGLPGGKPAWGPAGRSALDRVMGERLARFHG capP LRRARGWAVLKALACLLIGDNGVHGRTGGKATWGPPADAALRRLTATHS----- 3tdw IRKVSLFVTFDQVSYLL------EGLRARDQDWISEGLELLEEDKANNFGANSA 4ork KEYQDIVEEYYPIETIV------YGIKNIKQEFIENGRKEIYKRTYKD------4dca ERKAELNDVYWSIDQII------YGYERKDREMLIKGVSELLQTQAEMFIF--- 3n4v LEKYRMKEKYWSFEKII------YGKEYGYMDWYEEGLNEIRSAKIK------4N57: LEKYRMKEKYWSFEKII------YGKEYGYMDWYEEGLNEIRSIKIK------

88 Figure 2.3.24. Sequence alignment of Cpr17, CapP to APHs (126) using Clustal Omega. The conserved residues involved in triphosphate binding were highlighted in red and the catalytic loop HGDXXXN was in blue. The glutamic acid in green is responsible for electrostatic interaction with surrounding basic residues. The “gatekeeper” residues in black, together with nucleoside binding residues highlighted in dark green, determines ATP or GTP is preferred (125).

The structure of Cpr17 was predicted using SWISS MODEL based on a GTP specific

APH(2′′)-IIIa (PBD ID 3TDW) and a nucleotide unspecific APH(2′′)-IIa (PBD ID 4DCA), and showed considerable similarity to the template (fig 2.3.25). The conserved residues involved in nucleoside binding and the catalytic residues superimposed perfectly between models and templates (fig 2.3.26 while the “gatekeeper” tyrosine residue was not found in the putative nucleoside binding site of Cpr17. Based on this model, a D212A mutant were prepared and expressed in E. coli BL21 strains to yield soluble proteins. HPLC analysis results indicated that D212A_Cpr17 showed no activity with 2a, which was consistent to the proposed function of D212 (fig 2.3.27).

Figure 2.3.25. Structure comparison of APH and predicted Cpr17. (A) comparison of the structures of APH(2’’)-IIIa, APH(2’’)-IIa and the predicted structure of Cpr17. (A) The predicted structure of Cpr17 (blue, left) was imposed onto the template APH(2’’)-IIIa. (B) The predicted structure of Cpr17 (blue, left) was imposed onto the template APH(2’’)-IIa.

Tyr 92/ Val 96 Arg 45/ Arg 48

Glu 59/ Glu 62

Gly 97/ Asp 218/ Gly 102 GDP Asp 212

Asp 196/ His 201/ Asp 195 Asn 199

Figure 2.3.26. Alignment of GTP binding site of APH(2’’)-IIIa (light blue) and Cpr17 (green).

3’’-phospho-2a ii

2a i

Figure 2.3.27. Mutant Cpr17 exhibited altered activity. CPR17_D212A resulted complete loss of activity (i) compared to wild-type Cpr17 (ii).

91 Even though several APHs of the phosphotransferase/kinase family have been crystalized, it is still difficult to precisely predict the capuramycin binding site in Cpr17 because of the significant difference in structures between capuramycins and aminoglycosides. We subsequently attempted to crystalize Cpr17 and co-crystalize

Cpr17 along with its substrates capuramycin, AMPPNP and magnesium ion.

Although crystals of Cpr17 have been obtained (fig 2.3.28), further optimization of the conditions for crystallization in order to acquire decent structural information by

X-ray diffraction is still ongoing.

Figure 2.3.28. Crystal of Cpr17 obtained from high throughput screening.

92 2.3.3. Reconstitution of bacterial cell wall biosynthesis in vitro.

The genes encoded for MurA-MurF and Ddl that are involved in E. coli cell wall biosynthesis were cloned and expressed in E. coli (BL21) DE3 strains to yield soluble proteins (fig 2.3.29). Two additional genes were added to the reaction to avoid using expensive UDP-GlcNAc (132): N-acetylhexosamine 1-kinase (NahK (133), subcloned from Bifidobacterium infantis ATCC 15697) that converts GlcNAc to N- acetyl-glucosamine 1-phosphate (GIcNAc-1P) by using ATP as the phosphate donor; and a bifunctional enzyme GlmU (134) (subcloned from E. coli BL21) that converts

GlcNAc-1P into UDP-GlcNAc using UMP as the co-substrate (fig 2.3.30). When preparing the UDP-MurNAc-pentapeptide (12), another two enzymes including a glutamate dehydrogenase (GDH, subcloned from Bacillus subtilis strain 168) that recycles the NAPDH while oxidizing glucose into glucono-1,5-lactone (135), and a pyruvate kinase (purchased from Sigma-Aldrich) that regenerates nucleotides were added to the reaction. The one pot reaction containing all of the enzymes and supplemented with the appropriate substrates (fig 2.3.31) was incubated at rt for overnight, and the product was purified as described in Methods section. The production of UDP-MurNAc-pentapeptide (12), which is the final product of the Mur ligases, was purified using SEPABEADS SP207 resin and semi-preparative HPLC.

The purity of 12 was 95% based on HPLC and the molecular weight was confirmed by HRMS (fig 2.3.32)

75k 50k 37k

Figure 2.3.29. Expression of MurA-MurF and Ddl involed in E. coli cell wall synthesis.

HO HO HO O HO O NahK HO O HO O GlmU HO NH HO HO AcHN O O O O O O AcHN ATP ADP AcHN UTP P P N OH O OH PPi O O P OH OH OH HO OH

ATP UTP

iii

UDP-GlcNAc ii

Figure 2.3.30. NahK and GlmU convert GlcNAc to UDP-GlcNAc. (i) Reaction with NahK and GlmU, (ii) without NahK and (iii) without GlmU.

HO HO O HO HO O HO O NahK HO O GlmU HO NH MurA HO O HO O HO AcHN O O O NH HO AcHN O O P O P O N AcHN O O O O O N AcHN O P OH O O P P O OH UTP PPi OH OH HO O O ATP ADP PEP OH OH Pi OH HO OH HO OH 4 5 7 6

HO O O HO O O HO O MurC NH MurD MurB O NH AcHN O O O O O O O AcHN O O O P P N O P P N O HN O O O OH OH ATP ADP HO O OH OH ATP ADP NADPH NAD+ L-Ala HO OH D-Glu HO OH O OH GDH 8 9 Gluconolactone Glucose

HO HO HO O O O HO O HO O HOO O O O NH NH MurF AcHN O O NH AcHN O O O O O O O O O O MurE AcHN O O O P P N O O P P N HN O N O P P O OH OH HN O O HN O O OH OH OH OH ATP ADP D-Ala-Ala HO OH HO OH ATP ADP O NH HO OH H O NH HO COOH meso-DAP O NH ADP N HO NH OH HO H COOH O 2 N O Ddl O O O NH NH O 2 ATP H O O N O OH D-Ala OH 10 11 O 12

Figure 2.3.31. Enzymatic synthesis of UDP-MurNAc-pentapeptide.

A B

Figure 2.3.32. HPLC and HRMS analysis of UDP-MurNAc-pentapeptide (12). (A) HPLC analysis of purified 12. (B) HRMS analysis of purified 12, calcd. for [M - H]- 1192.3414; found 1192.3337.

A similar one pot reaction was used to establish a rapid and cost-efficient HTS model for discovery of antibiotics targeting on the Mur liagases. In short, candidate inhibitors would be expected to stop the flux when a Mur ligase is inhibited. Firstly, the reactions were carried out individually for each enzymatic step, and the HPLC traces revealed that the reactions can be monitored stepwise (fig 2.3.33). To validate this model, a known inhibitor targeting Ddl (cycloserine, purchased from Sigma-Aldrich) was added to reactions at a concentration of 10 mM. After the reaction was quenched after 3 h, the HPCL results indicated that the reactions containing cycloserine stopped at the expected step leading to the accumulation of 11 (fig 2.3.33).

7 8 9 11 10 12

UDP-GlcNAc

MurA i

MurB ii

MurC iii

MurD iv

MurE v

Ddl and MurF vi

MurA-F, 10 mM cycloserine vii

Figure 2.3.33. Reconstituting E. coli bacterial cell wall biosynthesis. HPLC analysis of reactions carried out using individual enzymes (MurA-MurF and Ddl) with appropriate substrates (traces i - vi). When 10 mM cycloserine was added, the reaction stopped at MurE step (last vii).

97 The MurA-MurF and Ddl in M. tuberculosis (Mur_tb and Ddl_tb) showed only moderate identity/similarity to those in E. coli, suggesting possible structural differences between isoenzymes from different species (table 2.3.5). Thus a species- specific HTS model is necessary to avoid false-negative results. To expand the system beyond detecting inhibitors for the E. coli recombinant enzymes, the homologous genes from M. tuberculosis R37v were cloned and expressed. All proteins from M. tuberculosis were soluble excluding MurD_tb, which failed to express in E. coli BL21 (DE3) and therefore E. coli MurD was purified and used instead (fig 2.3.33). HPLC traces of the reactions catalyzed by Mur ligases from M. tuberculosis were compared with the ones from E. coli. The final product 12 was purified using SEPABEADS SP207 resin. The product after purification was confirmed as authentic 12 by LC-MS (fig 2.3.34).

Table 2.3.5. Sequence comparison of MurA-MurF from different species in % identity/% similarity.

Protein Protein Identity/ E. coli BL21(DE3) M. tuberculosis Similarity (%) H37Rv MurA MurA-tb 45/60 MurB MurB-tb 34/50 MurC MurC-tb 36/51 MurD MurD-tb 31/44 MurE MurE-tb 39/53 MurF MurF-tb 31/46 DdlA DdlA-tb 37/54

75k 50k 37k

B 12

ii ii D

i i i

Figure 2.3.34. Reconstituting M. tuberculosis bacterial cell wall biosynthesis. (A) Expression of MurA-MurF and Ddl involed in M. tuberculosis cell wall synthesis. (B) One pot reaction of MurA-MurF and Ddl from M. tuberculosis (i) and E. coli (ii). (C Mass spectrum for the peak eluting at 13.5 min yielding [M-H]- = 1192.3 consistent with the calculated MW of authentic 12. (D) Mass spectrum for the peak eluting at 13.5 min yielding [M+H]+ = 1194.4 consistent with the calculated MW of authentic 12. The asterisk (*) denotes an unidentified contamination peak.

99 MurB_tb has not been functionally characterized from this Mycobacteria before, and the HPLC results suggest this enzyme is indeed an UDP-N- acetylenolpyruvoylglucosamine reductase (fig 2.3.35), the same activity of the E. coli

MurB.

Figure 2.3.35. HPLC analysis of the reaction catalyzed MurB. (A) Reactions were carried out using authentic 7 (iii) incubacted with MurB from M. tuberculosis (i) and E. coli (ii).

100 2.4 Conclusion

In summary, the biosynthetic gene cluster of 3 was identified by using a pair of degenerate primers based on the possible involvement of an L-Thr:UA transaldolase in CarU biosynthesis, and a total of 70 ORFs were identified to be responsible for the biosynthesis of 3. Unlike the producing strains for 1 and 2, which have not been amenable to genetic manipulation, gene inactivation was possible with the 3 producing strain, thus enabling the first in vivo analysis of the gene clusters for the capuramycin-type antibiotics to validate our proposed gene cluster. A series of gene inactivations were carried out. Three orfs cpr19, cpr25 and cpr51 were chosen and individually replaced by a resistant marker on the genome of 3 producer, and no mutant strains were able to produce 3 as determined by LC-MS-MS analysis.

Functional characterization of CapH and Cpr19 indicated that they catalyze identical reactions as LipK and LipL, suggesting a shared biosynthetic paradigm for CarU in capuramycin-type antibiotics and (5′S,6′S)-5'-C-glycyluridine (GlyU) in uridyl lipopeptide antibiotics (136,137): uridine-5'-monophophate (UMP) is oxidized to uridine-5'-aldehyde (UA) in a reaction catalyzed by a a non-heme, Fe(II)-dependent

α-ketoglutarate:UMP dioxygenase and UA is subsequently converted to GlyU through an aldol-type condensation in a reaction catalyzed by an L-threonine:uridine-5'- aldehyde transaldolase. The final step(s) to furnish CarU from GlyU, however, remains unclear.

We have functionally and kinetically characterized Cpr17 as a phosphotransferase conferring self-resistance to 3, and explored its substrate promiscuity toward capuramycins. Cpr17 has a similar amino acid sequence to ORF21 and CapP from

101 the biosynthetic gene clusters for 1 and 2, respectively, and was demonstrated to function as a phosphotransferase that converts 3 to 3′′-monophosphate-3 using a γ- phosphate group from a nucleotide donor. The kinetic characterization revealed

Cpr17 has similar kinetic parameters as CapP including an unexpectedly high apparent KM for ATP indicated that Cpr17 probably utilizes a different nucleotide as the phosphate donor. GTP was therefore tested with Cpr17, resulting in a 50-fold increase of catalytic efficiency suggested that GTP is the authentic substrate in vivo.

Cpr17 was also shown to phosphorylate other capuramycins including 2a and 2d.

The only exception among the natural capuramycins was 2c, which neither Cap17 nor CapP was able to modify under the conditions employed here. This substrate promiscuity of Cpr17 caught our interest and led us to explore the substrate flexibility in more detail. We chemically and chemo-enzymatically synthesized a series of unnatural capuramycins using CapW as a biocatalyst. As expected, A-503083 G, the

3′′-methoxy capuramycin was not phosphorylated. On the other hand, however, all the unnatural capuramycins were phosphorylated by Cpr17 regardless of the side chain structures. Finally, the conserved amino acid residues in Cpr17 involving in nucleotide triphosphate binding and catalytic residues were confirmed by global sequence alignment with representative aminoglycoside phosphotransferase, homologous modeling and small molecule docking. The abolishment of activity of

D212A_Cpr17 testified that the aspartate is a key residue involved in triphosphate binding. Interestingly, the “gatekeeper” tyrosine residue which determines the preference of nucleotide in aminoglycoside phosphotransferase was absent, but

102 Cpr17 is selective to GTP over ATP. Future crystal structure of Cpr17 may reveal the mechanism of nucleoside selectivity.

Lastly, a target- and species-specific HTS model was established and validated by using cycloserine, a known inhibitor targeting on Ddl which is one of the key enzymes in cell wall biosynthesis. The one pot reaction also allowed us to accumulate expensive intermediates that occur in the pathway. In the future, this model will be optimized to determine the most suitable concentration of starting material, best incubation time and so on. It is envisioned that this HTS model can eventually be utilized to screen natural products and their derivatives and synthetic libraries to identify new antibacterial therapeutics.

103 Chapter 3: Discovery of novel actinomycins as potential anti-tuberculosis agents*

3.1 Background

Previous studies revealed that the mechanism of action of actinomycin involves intercalation of the phenoxazinone between a GC base pair of double-stranded DNA with each peptide ring orienting in opposite directions to bind within the minor groove

(138). While the phenoxazinone moiety has been demonstrated to be essential for the activity of actinomycins, the strength and specificity of binding is highly dependent on amino acid composition of the peptides (77,92,93,139). In 2009, several new actinomycin congeners were identified (termed Y-type actinomycins) from the fermentation broth of Streptomyces sp. Strain Gö-GS12. Actinomycin Y5, which contains a unique bridging macrocycle between L-Thr on the β-ring and the phenoxazinone core, had potent antibacterial activity against Staphylococcus aureus and Bacillus subtilis while no apparent cytotoxicity against tested human cancer cell lines HM02 (gastric adenocarcinoma), HepG2 (hepatocellular carcinoma), and MCF7

(breast adenocarcinoma) (77) (fig 3.1.1). The improved selectivity of the fascinating structures of the newly discovered Y-type actinomycins suggested a possible antibiotic-selective actinomycin. Our group initiated studies aimed at identifying additional novel actinomycins with potential anti-tuberculosis activity by thoroughly investigating the metabolite profile of the producer strain for Y-type actinomycins.

Consequently, this would allow us to isolate more actinomycin Y5 for further characterization.

104 *This chapter was carried out in collaboration with Dr. Xiachang Wang (structural elucidation), Drs. Larissa.Ponomareva and Sherif Elshahawi (bioactivity assays), Center for Pharmaceutical Research and Innovation (CPRI).

HO O O O O N N O N O N N O N

NH O O NH O O

O O HN O O NH N NH

O O

Figure 3.1.1. Structure of actinomycin Y5.

In this chapter, structures of four novel actinomycins isolated from Streptomyces sp.

Strain Gö-GS12 (actinomycins Y6-Y9, 1-4) and a known semisynthetic compound actinomycin Zp (5) which for the first time, isolated from Streptomyces as a natural product will be discussed. The new members of actinomycins provided more details into structure-activity relationship. The unstable actinomycin Y6 (1) containing an additional ring structure between β-ring and chromophore underwent ring opening under acidic conditions, suggesting that biosynthesis of the bridging macrocycle is potentially due to nucleophilic attack of the amino group of the chromophore to C-4 of HThr.

105 3.2 Materials and methods

3.2.1 Instrumentation, chemicals and bacterial strains

UV spectra were recorded on an Ultrospec 8000 spectrometer (GE, Pittsburgh, PA,

USA). CD spectra were obtained on a Jasco J-810 spectropolarimeter (Jasco, Easton,

MD). All NMR data was recorded at 500 MHz or 400 MHz for 1H and 100 MHz for 13C with Varian Inova NMR spectrometers (Agilent, Santa Clara, CA). LC-MS was conducted with an Agilent 6120 Quadrupole MSD mass spectrometer (Agilent

Technologies, Santa Clara, CA) equipped with an Agilent 1200 Series Quaternary LC system and an Eclipse XDB-C18 column (150 × 4.6 mm, 5 µm). HR-ESI-MS spectra were recorded on an AB SCIEX Triple TOF 5600 System (AB Sciex, Framingham,

MA, USA). Analytic HPLC was performed with one of two systems: a Waters Alliance

2695 separation module (Milford, MA) equipped with a Waters 2998 diode array detector and an analytical Apollo C-18 column(250 mm x 4.6 mm, 5 µm).

Semipreparative HPLC was performed with a Waters 600 controller and pump

(Milford, MA) equipped with a 996 diode array detector, 717plus autosampler, and an

Apollo C-18 column (250 × 10 mm, 5 µm) purchased from Grace (Deerfield, IL). All solvents used were of ACS grade and purchased from Pharmco-AAPER (Brookfield,

CT). Sephadex LH-20 (25 ~ 100 μm) was purchased from GE Healthcare (Little

Chalfont, United Kingdom). TLC silica gel plates (60 F254) were purchased from EMD

Chemicals Inc. (Darmstadt, Germany). Celite (TM) was purchased from Fisher

Scientific (Pittsburgh, PA). Amino acids and marfey’s reagent were purchased from

Sigma Aldrich (St. Louis, MO).

106 Streptomyces sp. Strain Gö-GS12 was provided by Dr. Stephanie Grond (University of Göttingen).

3.2.2 Isolation of actinomycins

Streptomyces sp. Strain Gö-GS12 was cultivated in three 250 mL Erlenmeyer flasks, each containing 50 mL of oatmeal medium (20 g/L) enhanced with trace elements solution (CaCl2·2 H2O 3 g/L , Fe(III)-citrate 1 g/L, MnSO4 0.2 g/L, ZnCl2 0.1 g/L,

CuSO4·5 H2O 25 mg/L, Na2B4O7 ·10 H2O 20 mg/L, CoCl2 4 mg/L, Na2MoO4·2 H2O

10 mg/L). After three days of incubation at 28°C with 200 rpm agitation, the cultures were used to inoculate 50 flasks (250 mL), each containing 100 mL of oatmeal medium (77). The fermentation was continued for seven days at 28°C with 200 rpm agitation.

All culture flasks were combined, mixed with 300 g of celite follwed by filteration under vacum to seperate the mycelium and culutre broth. The mycelial cake-celite portion was extracted with acetone (3 × 500 mL) by sonnication, filltered off and the organic phase was evaporated to afford 2g of a dark red crude extract. The supernatant was extracted with CH2Cl2 (3 × 1 L). The organic phase was dried in vacuo to afford 3 g of dark red crude extract.

Crude extract from mycelium and supernatant was combined and subjected to a silica gel colunm chromatography using a gradient of CHCl3-MeOH (100:0 ~ 90:10) to yield four fractions, I-IV. Fraction I was applied to Sephadex LH-20 column chromatography and then semi-preparative HPLC (CH3CN/H2O, 0.01% TFA; flow rate: 3.5 mL/min) to offer 5 (10 mg). Compounds Y1 (30 mg), 3 (10 mg) and 4 (15 mg)

107 were isolated from fractions II, III by applying Sephadex LH-20 and then semi- preparative HPLC (CH3CN/H2O, 0.1% TFA; flow rate: 3.5 mL/min). Similarly, 1 (2 mg),

2 (10 mg) and Y4 (10 mg) were purified from fraction IV. Since 1 was unstable under acidic condition, 0.1‰ THF was used during HPLC purification for fraction IV, and solvent was rapidly dried in vacuo (fig 3.2.1).

Figure 3.2.1. Isolation scheme for actinomycins from 5 L fermentation broth.

108 3.2.3. Determination of amino acids configuration

The absolute configuration of each amino acid residue was determined following

Marfey’s method (140). 2.0 mg of 1 were hydrolyzed in 6 N HCl (1 ml) at 110 °C for

4 h. After drying under nitrogen, the corresponding hydrolysate was dissolved in 2 ml of EtOAc-H2O (1:1). The aqueous layer was dried in vacuo, to which a solution of 1%

Marfey’s reagent in acetone (200 ml) was subsequently added, followed by 1 M

NaHCO3 (50 ml). The reaction was incubated at 40 °C for 1 h, then allowed to cool.

It was then acidified with 25 ml of 2 N HCl. The reaction mixture was diluted with

MeOH (0.5 ml) and analyzed by HPLC using the following gradient: 0–55 min, linear gradient from 10–55% CH3CN in 50 mM triethylammonium phosphate buffer

(Phenomenex C18 column, 250*4.6 mm). The flow rate was kept constant at 1 ml/min and elution was monitored at 430 nm. Derivatized standards were prepared from the authentic D- and L-valine, L-sarcosine, L-threonine and L-methyl valine (50 ml of a 50 mM stock) following an identical procedure.

3.2.4. ynthesis of Fmoc-depsipeptide and 2-amino-3-hydroxy-4- methylbenzamide

The Fmoc-depsipeptide (6) was synthesized through a combination of solid- and liquid-phase peptide synthesis (Scheme 3.1).

109

O FmocHN OH O H O 1) 20% Pip/DMF 1) 20% Pip/DMF N Cl NHFmoc O NHFmoc O DIPEA, DCM 2) O 2-Cl Trityl resin 2) O FmocHN O OH N OH HATU, DIPEA Fmoc C1 C2 HATU, DIPEA

O O O O H H 1) 20% Pip/DMF N 1) 20% Pip/DMF N O N O N O O H N 2) H N 2) O FmocHN O FmocHN Fmoc (R) O OH NHFmoc OH

OH HATU, DIPEA HATU, DIPEA C3 C4

O O H O H O N N O O N 40% TFA/DCM HO N N H DIC, H (2.5% TIPS) O N H O N HOAt, NH N O O DMAP O O HN HN O O O O NH HO HO NHFmoc NHFmoc NHFmoc

C5 C6 6

Scheme 3.1. Synthesis of Fmoc-depsipeptide (6).

Under a nitrogen atmosphere, a filter column was charged with 2-chlorotrityl chloride

resin (1 equiv., 100 mg, 0.15 mmol, 100-200 mesh). The resin was washed with DCM

(3 × 10 mL) and swollen with DMF/CH2Cl2 (5:1, 10mL) for 20 min. susequently, a

solution of Fmoc-N-Me-Val-OH (2 equiv., 102 mg, 0.3 mmol) in DMF/ DCM (5:1, 3

mL) was added, followed by DIPEA (6 equiv., 158 µl, 0.9 mmol). The suspended resin

was stirred at rt for 1 h. The resin was drained and washed with DCM (3 × 20 mL),

DMF (3 × 20 mL), IPA (3 × 20 mL),and DCM (3 × 20 mL). The first loading and

washing was performed twice. 5 mL of 20% piperidine in DCM was added and

110 resuspended resin was stirred for 20 min to remove Fmoc protectin group, and same washing procedure was followed to afford resin-bond C1.

Then a solution of Fmoc-Sar-OH (2 equiv., 90 mg, 0.3 mmol), HATU (2 equiv., 115 mg, 0.3 mmol) and DIPEA (4 equiv., 105 µl, 0.6 mmol) in DMF/DCM (5:1, 1.5 mL) was added. The reaction was stirred at rt for 1 h, and washed as described above.

The reaction was repeated twice, and resin was drained to yield resin-bond C2.

Successive condensation with Fmoc-Pro-OH, Fmoc-D-Val and Fmoc-Thr(tBu)-OH was carried out as described above to give resin-bound C5.

The resin-bound C5 was released by adding 4 mL of 40% TFA in DCM containing

2.5% TIPS, and the resin was drained and washed with DCM (3 × 20 mL). The filtrate was dried in vacuo and purified by flash silica-gel chromatography purification

(DCM/MeOH) to afford 103 mg of C6 as white powder.

C6 (1 equiv., 103 mg, 0.15 mmol) was dissolved in DMF/DCM (1:1, 10 mL) and HOAt

(6 equiv., 123 mg, 0.9 mmol), DIPEA (6 equiv., 161 µl, 0.9 mmol) was added and stirred until solution was clear. Then DIC (2 equiv., 48 µl, 0.3 mmol) and DMAP (0.1 equiv., 1.8mg, 0.015 mmol) was added and stirred at rt for 3 h. The reaction was susequently washed with saturated NH4Cl, NaHCO3 and borine. The orgainc solvent was dried in vacuo, and remaining substances were purified by flash silica-gel chromatography purification (DCM/MeOH) to afford 33 mg of 6 as oily liquid.

The overall yield was 16%.

111

1 13 Table 3.2.1. NMR data of 6 in CDCl3 ( H: 400 MHz, C: 100 MHz, J in Hz)

Pentapeptidolactones α-ring positions δC δH β-ring positions δC δH Thr 1 170.0 Gly 1 171.5 2 57.2 4.54 (dd, 9.3, 2 42.7 4.44, 3.64 (d, 4.2) 2.2) 3 71.9 5.28 (m) NH 7.00 (brs) 4 15.9 1.24 (d, 6.2) L-Val 1 167.4 NH 5.64 (d, 9.3) 2 56.2 4.61 (dd, 9.9, 5.2) D-Val 1 169.3 3 28.8 2.28 (m) 2 58.1 4.06 (dd, 7.3, 4 17.5 0.93 (d, 7.0) 3.1) 3 29.9 1.96 (m) 5 19.6 0.95 (d, 6.8) 4 18.9 0.97 (d, 6.9) NH 7.36 (d, 8.2) 5 18.7 1.01 (d, 6.6) NH 7.02 (brs) Pro 1 171.6 2 61.4 4.44 (m) 3 29.3 2.17 (m), 1.22 (m) 4 24.5 2.03 (m) 5 47.9 3.96, 3.55 (m) Chromophore δH 7.74 (d, 7.5, 2H, 4/5-H), 7.59 (t, 7.2, 2H, 1/8-H), 7.37 (t, 7.6, 2H, 3/6-H), 7.29 (t, 7.4, 2H, 2/7-H), 4.37 (d, 7.6, 2H, 10-H), 4.22 (m, 1H, 9-H). δC 125.4 (CH, C-1/8), 127.6 (CH, C-2/7), 127.7 (CH, C-3/6), 119.9 (CH, C-4/5), 47.0 (CH, C-9), 67.5 (CH2, C-10), 143.7 (qC, C-11/14), 141.2 (qC, C-12/13).

112

2-amino-3-hydroxy-4-methylbenzamide (Phenol-4) was synthesized as described below:

COOH CONH2 CONH2 NO2 o NO2 NH2 1. (COCl)2, 60 C H2/Pd/EtOH

2. OH NH4OH OH OH

Phenol-3 Phenol-4

Scheme 3.2. Synthesis of phenol-4.

A solution of 3-hydroxy-4-methyl-2-nitrobenzoic acid (1.97 g, 10 mmol) and oxalyl chloride (10 mL) was refluxed at 60 °C until clear. After cooling to rt, solvent was evaporated to give 3-hydroxy-4-methyl-2-nitrobenzoyl chloride. Ammonium hydroxide (20 mL) was added dropwise within 5 min at 0 °C. The mixture was stirred at 0 °C for 30 min, and at room temperature for an additional 30 min. 30 mL of water was added to the mixture. After the white precipitate was filtered, the aqueous solution was adjusted to pH 3 and then extracted with ethyl acetate. The organic solution was dried with Na2SO4, and evaporated to give 1.4 g of 3-hydroxy-

4-methyl-2-nitrobenzamide (Phenol-3) as a white solid.

To a solution of 3-hydroxy-4-methyl-2-nitrobenzamide (1.96 g, 10 mmol) in 20 mL methanol, Pd/C (0.19 g) was added and reaction was stirred under hydrogen

113 atmosphere at room temperature for 4 h. The insoluble particles were filtered, and organic solution was concentrated to give 1.56 g of 2-amino-3-hydroxy-4- methylbenzamide (Phenol-4) as brown powder.

The overall yield was 93%.

3.2.5 Cytotoxicity assay*

A resazurin-based cytotoxicity assay, also known as AlamarBlue assay, was used to assess the cytotoxicity of agents against the human lung non-small cell carcinoma cell line A549 and human prostate cancer cell lines PC3 where the degree of cytotoxicity was based upon residual metabolic activity as assessed via reduction of resazurin (7-hydroxy-10-oxido-phenoxazin-10-ium-3-one) to its fluorescent product resorufin. A549 and PC3 cells, purchased from ATCC (Manassas, VA, USA), were grown in DMEM/F-12 Kaighn’s modification and MEM/EBSS media, respectively

(Thermo scientific HyClone, Logan, UT, USA), with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine. Cells were seeded at a density 2 × 103 cells per well onto 96-well culture plates with a clear bottom (Corning, NY, USA), incubated 24 hrs at 37 oC in a humidified atmosphere containing 5% CO2 and were exposed to test agents for 2 days (positive controls: 1.5 mM hydrogen peroxide, 10 µg/ml actinomycin D). Resazurin (150 µM final concentration) was subsequently added to each well and the plates were shaken briefly for 10 seconds and were incubated for another 3 h at 37 oC to allow viable cells to convert resazurin into resorufin. The fluorescence intensity for resorufin was detected on a scanning microplate spectrofluorometer FLUOstar Omega (BMG

LABTECH GmbH, Ortenberg, Germany) using an excitation wavelength of 560 nm

114 and an emission wavelength of 590 nm. The assay was repeated in 3 independent experimental replications. In each replication, the emission of fluorescence of resorufin values in treated cells were normalized to, and expressed as a percent of, the mean resorufin emission values of untreated control (metabolically active cells;

100%, all cells are viable).

3.2.6 Antibacterial activity assay**

The protocol used for the determination of the minimum inhibitory concentration (MIC) was as that described previously with minor modifications (141,142). The bacterial strains Staphylococcus aureus ATCC 6538, Micrococcus luteus ATCC 15307,

Bacillus subtilis ATCC 6633, Salmonella enterica ATCC 10708, Escherichia coli

ATCC 12435, Mycobacterium aurum ATCC 23366, in addition to the fungal strain

Saccharomyces cerevisiae ATCC 204508 were used as model strains for antimicrobial susceptibility assays. All strains were grown in appropriate liquid or on agar plates using tryptic soy broth (BD211825) for S. aureus and M. luteus, LB medium (BD244620) for B. subtilis and E. coli, nutrient broth (BD 234000) for S. enterica, Middlebrook 7H9 with OADC enrichment (Sigma-Aldrich M0178-500G) for

M. aurum, and YAPD (ATCC medium number 1069) for S. cerevisiae. Individual strains were grown in 5 mL of medium for 16 h at 37 °C with shaking (250 rpm). An aliquot of a fully grown culture (100 μL) was diluted to using sterile liquid medium to

OD600 ~ 0.1. Aliquots (160 μL) of each diluted culture were then transferred into the individual wells of a 96-well plate supplied with 2 μL of the tested compound. Various concentrations with maximum concentration 120 μM were maintained to assess the

115 antimicrobial activities and compared to the negative control containing vehicle

(DMSO) alone and positive controls (rifampicin and ciprofloxacin for M. aurum,

amphotericin B for S. cerevisiae, and kanamycin for the other microorganisms. The

culture plates was incubated at 28-37 °C for 16-48 h with shaking (160 rpm), and then

the OD600 of each well measured using a FLUOstar Omega scanning microplate

spectrofluorometer (BMG Labtech). The acquired OD600 values were normalized to

the negative control wells (100% viability). The minimal concentration of the tested

compound that caused growth inhibition was recorded as the MIC.

* Cytotoxicity assay was carried out by Dr. Larissa.Ponomareva, CPRI.

116 **Antibacterial activity assay was carried out by Dr. Sherif Elshahawi, CPRI.

3.3 Results and discussion

3.3.1 Isolation of actinomycins from S. sp. Strain Gö-GS12

3.3.1.1 Isolation of actinomycin Y1, Y3 and Y4

Our initial goal was to isolate and accumulate known Y-type actinomycins for

additional bioactivity studies, and Y1, Y3 and Y4 were successfully identified from the

culture medium. Their 1H and 13C NMR spectrum was identical to reported data (77).

Because the yield of Y2 and Y5 was very low, it was difficult to purify sufficient amounts

for NMR analysis, even though the HRMS results suggested the presence Y2 and Y5.

During these studies, several additional, novel actinomycins were identified; analysis

of these new compounds is described below.

3.3.1.2 Structural elucidation of 1

Compound 1 was obtained as red amorphous powder. The molecular formula of 1

was established as C61H80N12O18 on the basis of (+)-HRESIMS at m/z 1269.5816 [M

+ + H] (calcd for C61H81N12O18, 1269.5792). It was recognized as a member of

actinomycin family due to the high molecular weight around 1300, the presence of

twelve nitrogen atoms, and its UV spectrum (λmax 238 and 429 nm) that indicated

typical features of actinomycins containing an aminophenoxazinone chromophore.

An additional absorption max at 309 nm was observed, which is also found in

actinomycin Y5, suggesting that 1 may contain a third ring. Initially we thought that it

was actinomycin Y5 because of the same molecular weight and UV absorption profile.

However, the NMR spectrum of 1 in CD3OD was not consistent with Y5. Furthermore,

117 the molecular weight of 1 did not correspond to any other chomopeptide, indicating that 1 was a new member of actinomycins.

The 1H NMR spectrum (table 3.3.1) of 1 showed characteristic features of actinomcyins, including four N-methyl groups resonating at δH 2.84 (s), 2.88 (s), 2.92

(s) and 3.05 (s), two phenylmethyl groups at δH 2.25 (s) and 2.55 (s), and two adjacent protons attached to a phenyl group with resonances at δH 7.45 (d, J=7.8) and δH 7.50

(d, J=7.9). The 13C-NMR also showed typical features with actinomycins, including

12 aromatic carbons on the phenoxazinone chromophore, and 12 amide/acid carbonyl carbons at δC 165.5, 167.8, 168.5 (two overlapping signals), 169.1, 169.7,

170.6, 171.8, 172.5, 174.9, 175.6 and 177.2, which were consistent with the identify as a peptide-containing compound.

Correlations obtained from COSY, HMBC and HMQC and TOCSY experiments of 1 were used to establish the structure. The methyl group (δC 17.2 and δH 1.26, d, J=5.9) was chosen as a start point from TOCSY analysis, which showed correlations with two methines at δH 5.34 (m) and at δH 4.80. The COSY experiments revealed correlations between the methyl (δH 1.26) and the CH at δH 5.34, and between δH

5.34 and δH 4.80. Additionally, HMBC correlation was observed from δH 4.80 to a quaternary carbon resonating at δC 171.8. The independent spin system of

XCHCHCH3 indicated the presence of the first threonine and it was named Thrα.

Similarly, the second fragment was identified. In contrast to Thrα, the chemical shift at the β-position (δC 64.0, δH 5.68) and the γ-position (δC 71.5, δH 4.94, d, J=12.6, and

5.22, d, J=12.6) suggested that there was a significant conformational change including a C-N bond at C-3 and an oxygen-substituted C-4 whereby comparison of

118 carbon chemical shift with actinomycin G3 and G5 (77). The second fragment was thereby a 4-hydroxylthreonine, and named as HThrβ.

O . . . HN 171 8 1 74 7 5 34 17 . 165.5 64.0 5.68 2 1 2 3 O 4.24 254.6 4.94, 5.22 O 4 .80 56 6 . 3 . 4 471.5 1 26 . HN HN O 8 23

Thrα HThrβ

An active hydrogen resonating at δH 7.88 (d, J=5.5) showed TOCSY correlations between δH 3.77 (m), δH 2.04 (m), δH 0.92 (d, J=6.7), and δH 1.10 (d, J=6.7) revealed that they were clustered together. The COSY experiment indicated that the two methyl groups (δH 0.92, d, J=7.1, and 1.10, d, J=6.7) were linked via a CH at δH 2.04.

Additional COSY correlations were observed between δH 2.04 and δH 3.77, and between δH 3.77 and δH 7.88. The HMBC correlations further confirmed the structure with an independent spin system of XCHCH(CH3)2, which indicated the presence of a valine and it was named Valα. Similarly, the other active hydrogen resonating at δH

8.78 (d, J=5.3) resulted in another valine (Valβ) moiety from 2D NMR analysis.

177.2 175.6 O O

1.10 3.77 1.19 4.05 19.9 2.04 61.0 7.88 19.2 58.2 33.5 NH NH 8.78 2.25 32.9

0.92 19.5 0.89 19.8

Valα Valβ

119 A methyl group resonating at δH 1.48 (d, J=6.0) was chosen as a start point for the next fragment. It showed TOCSY correlations between δH 4.23 (m), δH 1.97/2.13 (m) and δH 6.29 (s), and a COSY correlation between δH 4.23. Further COSY correlations were observed between δH 4.23 and δH 1.97/2.12, between δH 1.97/2.12 and δH 4.27.

The methyl group also showed HMBC correlations to a methine at δC 40.4 (δH 1.97, m, 2.12, m) and a methine at δC 55.4 (δH 4.23, m), while other key HMBC correlations were observed from δH 6.29 to methines at δC 40.4 and δC 74.8, and to a quaternary carbon at δC 172.5, which further confirmed a XCHCHCH2CHX’CH3 spin system and suggested the presence of a methyl proline. The chemical shift of C-3 (δC 74.8) indicated that it was attached to a hydroxyl group. Thus the structure of this fragment was a 3-hydoxyl-5-methyl proline (HMPro). The relative conformation of HMPro was determined as 3-OH trans because 2-H at δH 6.29 was a singlet (indicating a 90° dihedral angle to 3-H at δH 4.27) and there was no COSY correlation between 2-H and 3-H. HO O 4.27 172.5 74.8 6.29 1.97 69.4 2.12 40.4 N 4.23 55.4 1.48 18.9

HMPro

13 13 A diagnostic C NMR signal at δC 209.5 was observed in the C NMR spectrum, to which five key HMBC correlations were observed from δH 3.94 (d, J=19.0), δH 4.40

(d, J=19.0), δH 2.38 (m), δH 3.42 (m) and δH 6.68 (d, J=7.2). Additional HMBC correlations from δH 2.38 to δC 174.9 revealed an independent spin system of

120 XCHCH2CCH2X’ as a 4-oxoproline (OPro) which was also present in Z and G type actinomycins.

O 2.38 3.42 174.9 6.68 209.5 56.2 O 42.4 54.2 N 3.94 4.40

OPro

Two remaining CH2 groups resonating at δC 52.9 (δH 4.09, d, J=17.8 and δH 4.80, d,

J=17.8), and δC 52.8 (δH 4.09, d, J=17.8 and δH 4.66, J=17.8) did not have COSY nor TOCSY correlations to other protons, suggested the presence of glycine moieties. The CH2 groups showed HMBC correlations to carbonyl carbons at δC

168.53 and δC 168.52, respectively. The correlations from CH2 groups to N-methyl groups at δC 35.7 (δH 2.84, s) and at δC 35.4 (δH 2.88, s) indicated two sarcosines

(Sarα and Sarβ) instead of glycines. Those sarcosines were named Sarα, and Sarβ.

2.84 35.7 2.88 35.4 4.09, 4.80 O 4.09, 4.66 O N 52.9 N 52.8 168.53 168.52

Sarα Sarβ

Similar to the Val discussed earlier, an extra valine was identified using the methyl group resonating at δC 19.6 (δH 0.82, d, J=6.9) as a starting point. This valine, different from the other two, has an additional HMBC correlation from the methine resonating

121 at δH 3.07 to a methyl group resonating at δC 39.6 (δH 3.05, s) indicating that this valine contained a N-methyl group. This fragment was named MeVal.

1.01 21.8

2.60 28.4 39.6 N 0.8 2 72.1 3.05 3.07 19.5

169.7 O

MeVal

The HMBC and COSY correlations observed from the very last methyl group resonating at δC 13.7 (δH 1.43, d, J=6.8) revealed the presence of an alanine with a spin system of XCHCH3. The HMBC correlation from the CH resonating at δH 3.76

(m) to a N-methyl group revealed the structure of a methyl alanine, and this fragment was named MeAla.

1.43 2.92 N 13.7 37.3 3.76 61.1

170.6 O

MeAla

The chromophore core of 1 was identified by 2D NMR and the comparison of the

13 measured C data with that obtained for known actinomycins, where actinomcyin Y5 was especially helpful.

169.1 O O 167.8 NH 127.1 133.0 N 88.4

7.45 128.7 147.2 153.4

7.50 149.0 177.4 132.6 129.9142.4 O 116.6 O

2.55 15.1 2.25 8.3 Chromophore of 1

122 The remaining analysis was aimed at connecting the fragments to give an overall structure of 1. For the α-ring, Thrα could be connected to Valα through the amide hydrogen at δH 7.87 which showed a HMBC correlation to C-1 of Thrα at δC 171.8.

The carbonyl carbon of Val at δC 177.2 contained a HMBC correlation from the methine at δH 6.29, thus the HMPro was attached to Valα. The Sarα was connected to HMPro due to the HMBC correlation from the N-methyl group at δH 2.84 (s) to the carbonyl of HMPro at δC 172.5. The last amino acid, MeVal was joined to Sarα according to the HMBC correlation from the N-methyl group at δH 3.05 (s) to the carbonyl of Sarα at δC 168.53. The key HMBC correlation from the CH at Thrα (δH

5.34) to the carbonyl at δC 169.7 of MeVal helped to establish the first pentapeptide ring (α-ring) as cyclo-(-Thr-Val-HMPro-Sar-MeVal-). The β-ring was established as cyclo-(-HThr-Val-OPro-Sar-MeAla-) by a similar manner as the α-ring. The major difference between α- and β-ring was that the Thr in the β-ring was connected to

MeAla via an amide bond instead of an ester. This type of “rearrangement” was also observed in other members of actinomycins including Y3, Y4 and G6. The α-ring was connected to the chromophore through an amide bond since carbonyl group at δC

169.1 contained HMBC correlations from δH 4.80 (C-2 of Thrα) and δH 7.45 (C-8 of chromophore). The β-ring was connected to the chromophore through an ester bond as a correlation was observed from the C-4 of Thrβ at δH 4.94 and 5.22 to C-14 on the chromophore resonating at δC 167.8. The 18 amu difference between compound

1 and actinomycin Y3 indicated a loss of H2O in 1, and the significant low field shift of

H-3 of HThrβ at δH 5.68 compared to actinomycin Y3 at δH 4.57 and Y4 at δH 4.33 suggested an additional ring structure similar to actinomycin G5 in which chemical

123 shift of H-4 at δH 4.64 and 4.86 shifted to low field significantly compared to G3 at δH

3.23 and 3.76 (77).

Interestingly, while purifying 1, we noticed that this compound was unstable under acidic condition. Similar phenomenon was also observed with Y5 (77). Thus the degradation product was collected and structurally characterized. 1 underwent hydrolysis, and the chromophore-β-peptide ring was opened under 0.1% of TFA

(pH≈4) to generate actinomycin Y3 (fig 3.3.1). This result further confirmed the presence of the bridging macrocycle in 1, and also provided some clue to the biosynthetic origin of the third ring: the ring closure is potentially formed by nucleophilic attack of the amino group of the chromophore to C-4 of HTHr concomitant with dehydration. This observation also provided important information for future biochemical characterization of the cyclization reaction, as all acidic conditions should be avoided during analysis and reactions can be monitored in real- time using difference spectrum analysis.

124

O A HO O O O O O N N N O O N O N N O N N O N

Acidic condition NH O O NH HN O NH HN O

O O O

HN O O O O O OH 13 10 14 NH 10 14 9a N 10a 10a 8 9 N NH 1 2 2 1 2 7 5a 6 O 4a O 4a 4 3 O 3 O 5 4 5 11 12 12

1 Y3 B 1

1 Y3

Figure 3.3.1. Simultaneous conversion of 1 to Y3 under acidic condition. (A)

Conversion of 1 to Y3 under 0.1% TFA. (B) 15% of 1 was converted to Y3 within 1 h. Each compound had a signature UV profile.

The CD spectra of 1 (fig 3.3.4) showed a strong cotton effect at about 210 nm, plus the origin from same strain, indicating the absolute configurations of the amino acids in 1 were supposed to be identical with actinomycin Y3. Thus, the structure of compound 1 was established as a new member of the Y-type actinomycins and subsequently named as actinomycin Y6.

125 The predicted amino acid stereochemical configuration was confirmed by Marfey’s method. The retention times for Marfey’s derivatives of authentic L-Val, D-Val, Sar, L-

Thr and L-MeVal were 34.2, 39.4, 24.9, 22.9, 38.7 min, respectively. Those for the

Marfey’s derivatives of D-Val, L-Val, L-Sar, L-Thr and L-MeVal in the hydrolysate of 1 were 22. (L-Thr), 24.9 (Sar), 38.7 (L-MeVal) and 39.4 (D-Val) min, respectively (fig

3.3.2).

Figure 3.3.2. Chiral amino acid analysis of 1.

126 1 13 Table 3.3.1. NMR data of actinomycin Y6 (1) in CD3OD ( H: 500 MHz, C: 100 MHz, J in Hz)

Pentapeptidolactones α-ring positions δC δH β-ring positions δC δH Thr 1 171.8 crHThr 1 165.5 2 56.6 4.80 (m) 2 54.6 4.27 (d, 3.5) 3 74.7 5.34 (m) 3 64.0 5.68 (m) 4 17.2 1.26 (d, 5.9) 4 71.5 4.94 (d, 12.6) NH 8.23 (d, 7.7) 5.22 (d, 12.6) D-Val 1 177.2 D-Val 1 175.6 2 61.0 3.77 (m) 2 58.3 4.05 (m) 3 33.5 2.04 (m) 3 32.9 2.25 (m) 4 19.9 1.10 (d, 6.7) 4 19.8 0.89 (d, 6.2) 5 19.5 0.92 (d, 7.1) 5 19.2 1.19 (d, 6.6) NH 7.88 (d, 5.5) NH 8.78 (d, 5.3) HMPro 1 172.5 OPro 1 174.9 2 69.4 6.29 (s) 2 56.2 6.68 (d, 7.2) 3 74.8 4.27 (d, 3.4) 3 42.4 2.38 (m) 4 40.4 1.97 (m) 3.42 (m) 2.12 (m) 4 209.5 5 55.4 4.23 (m) 5 54.2 3.94 (d, 19.0) 6 18.9 1.48 (d, 6.0) 4.40 (d, 19.0) Sar 1 168.5 Sar 1 168.5 2 52.9 4.09 (d, 17.8) 2 52.8 4.09 (d, 17.8) 4.80 (d, 17.8) 4.66 (d, 17.8) NMe 35.7 2.84 (s) NMe 35.4 2.88 (s) MeVal 1 169.7 MeAla 1 170.6 2 72.1 3.07 (d, 9.4) 2 61.1 3.76 (m) 3 28.4 2.60 (m) 3 13.7 1.43 (d, 6.8) 4 21.8 1.01 (d, 6.4) NMe 37.3 2.92 (s) 5 19.5 0.82 (d, 6.9) NMe 39.6 3.05 (s) Chromophore δH 2.25 (s, 3H, 12-H3), 2.55 (s, 3H, 11-H3), 7.45 (d, 7.8, 1H, 8-H), 7.50 (d, 7.9, 1H, 7-H). δC 8.3 (CH3, C-12), 15.1 (CH3, C-11), 88.4 (qC, C-1), 116.6 (qC, C-4), 127.1 (CH, C-8), 128.7 (qC, C-9a), 129.9 (qC, C-6), 132.6 (CH, C-7), 133.0 (qC, C-9), 142.4 (qC, C-5a), 147.2 (qC, C-10a), 148.9 (qC, C-4a), 153.4 (qC, C-2), 167.8 (qC, C-14), 169.1 (qC, C-13), 177.4 (qC, C- 3).

127 3.3.1.3 Structural elucidation of 2

The molecular formula of compound 2 (obtained as red amorphous powder), was

+ determined as C61H82N12O19 from the (+)-HRESIMS (m/z 1287.5857 [M + H] ). The

UV and NMR data of 2 revealed actinomycin-characteristic features, which were quite

similar to that of actinomycin Y1 (77). Compared to Y1, the two high-field shifted proton

signals (δH 3.31 and 3.70, H2-4) of HThr in the β-ring, a much higher chemical shift

for the C-4 (δC 59.6), and the absence of a chloride atom in 2, indicated a hydroxyl

substitution at C-4. Further COSY, TOCSY and HMBC correlations were in full

agreement with compound 2 as a new analog of the actinomycin series and 2 was

thereby designated as actinomycin Y7.

HMPro O O O HO Sar O ar HO OPro S O O O O N N O N O MeAla N N O N N O N N O N N O N MeVal NH O O NH O O - - NH O O NH O O D Val r D Val Th O O O HThr O NH HN O O OH HN O O NH OH

N NH2 N NH2

O O O O

COSY HMBC (H C)

128 1 13 Table 3.3.2. NMR data of actinomycin Y7 (2) in CDCl3 ( H: 500 MHz, C: 100 MHz, J in Hz)

Pentapeptidolactones α-ring positions δC δH β-ring positions δC δH Thr 1 169.0 HThr 1 168.2 2 55.0 4.61 (m) 2 52.2 4.96 (m) 3 74.6 5.23 (m) 3 77.1 5.15 (m) 4 17.0 1.15 (d, 6.0) 4 59.6 3.31 (m) NH 7.17 (brs) 3.70 (m) NH 8.15 (d, 6.1) D-Val 1 174.2 D-Val 1 173.8 2 59.1 3.43 (m) 2 57.0 3.84 (m) 3 31.9 2.10 (m) 3 31.9 2.22 (m) 4 19.3 1.14 (d, 6.6) 4 19.3 0.94 (d, 6.2) 5 18.9 0.94 (d, 6.4) 5 18.8 1.18 (d, 6.5) NH 7.82 (d, 4.6) NH 8.35 (d, 5.1) HMPro 1 170.7 OPro 1 172.6 2 68.3 5.87 (s) 2 54.6 6.54 (d, 11.0) 3 75.5 4.19 (m) 3 41.8 2.33 (d, 17.7) 4 41.0 2.12 (m) 3.93 (m) 2.19 (m) 4 208.6 5 53.4 4.76 (m) 5 52.8 4.00 (d, 19.0) 6 18.8 1.52 (d, 6.0) 4.55 (d, 19.1) Sar 1 166.0 Sar 1 165.8 2 51.4 3.65 (d, 18.0) 2 51.1 3.65 (d, 17.9) 4.72 (d, 18.0) 4.49 (d, 17.9) NMe 35.0 2.90 (s) NMe 34.7 2.88 (s) MeVal 1 167.4 MeAla 1 169.0 2 71.2 2.69 2 60.0 3.26 (m) 3 27.0 2.67 (m) 3 13.4 1.36 (d, 6.8) 4 21.6 0.96 (d, 6.4) NMe 37.0 2.92 (s) 5 19.0 0.75 (d, 6.9) NMe 39.3 2.91 (s) Chromophore δH 2.12 (s, 3H, 12-H3), 2.53 (s, 3H, 11-H3), 7.36 (d, 7.6, 1H, 7-H), 7.55 (d, 7.8, 1H, 8-H). δC 7.7 (CH3, C-12), 14.9 (CH3, C-11), 98.9 (qC, C-1), 113.5 (qC, C-4), 125.9 (CH, C-8), 127.6 (qC, C-6), 128.7 (qC, C-9a), 130.4 (CH, C-7), 132.7 (qC, C-9), 140.4 (qC, C-5a), 145.2 (qC, C-4a), 146.1 (qC, C-10a), 148.3 (qC, C-2), 166.0 (qC, C-13), 168.6 (qC, C-14), 178.2 (qC, C- 3).

129 3.3.1.4 Structural elucidation of 3

Structurally related to 2, actinomycin Y8 (3) was obtained as red amorphous powder

and assigned a molecular formula C61H82N12O18 on the basis of (+)-HRESIMS,

suggestive of a deoxygenated derivative of 2. This deduction was corroborated by

the NMR data (table 3.3.3), in which the diagnostic methyl signal at C-4 of Thr in β-

ring (δC 17.4, δH 1.28 (d, J=6.2), showed a COSY correlation with H-3 and an HMBC

correlation with C-2 and C-3, which indicated the absence of 4-OH in the Thr residue.

3 was thereby designated as actinomycin Y8.

HO O O HMPro O O O O HO Sar Sar O N O OPro O N N N O N O N N O N MeAla N O N N O N MeVal NH O O NH O O NH O O NH O O D-Val Thr D-Val O O O O HN O NH Thr O HN O O NH

N NH2 N NH2

O O O O

COSY HMBC (H C)

130 1 13 Table 3.3.3. NMR data of actinomycin Y8 (3) in CDCl3 ( H: 400 MHz, C: 100 MHz, J in Hz)

Pentapeptidolactones α-ring positions δC δH β-ring positions δC δH Thr 1 168.4 HThr 1 167.8 2 54.5 4.49 (dd, 6.9, 3.1) 2 54.7 4.90 (dd, 6.6, 2.6) 3 74.5 5.20 (m) 3 75.2 5.22 (m) 4 17.0 1.12 (d, 6.1) 4 17.4 1.28 (d, 6.2) NH 6.92 (d, 7.0) NH 7.72 (d, 6.4) D-Val 1 173.9 D-Val 1 173.9 2 59.0 3.40 (dd, 10.7, 2 56.9 3.83 ( dd, 9.9, 6.6) 4.7) 3 31.9 2.05 (m) 3 31.9 2.17 (m) 4 19.3 1.12 (d, 6.0) 4 19.2 0.91 (d, 6.6) 5 19.2 0.91 (d, 6.0) 5 18.7 1.15 (d, 6.6) NH 7.64 (d, 5.0) NH 8.27 (d, 6.2) HMPro 1 171.0 OPro 1 172.7 2 68.5 5.92 (s) 2 54.4 6.60 (d, 10.4) 3 75.8 4.11 (m) 3 41.7 2.31 (d, 18.0) 4 41.5 2.10 (m) 3.97 (dd, 17.4, 11.1) 2.20 (m) 4 208.8 5 54.0 4.69 (m) 5 52.7 3.97 (d, 19.0) 6 18.9 1.51 (d, 5.9) 4.53* Sar 1 166.1 Sar 1 165.7 2 51.4 3.65 (d, 17.7) 2 51.2 3.61 (d, 17.6) 4.75 (d, 17.6) 4.53* NMe 35.0 2.88 (s) NMe 34.6 2.86 (s) MeVal 1 167.4 MeAla 1 168.9 2 71.2 2.66* 2 60.5 3.23 (m) 3 26.9 2.64 (m) 3 13.5 1.36 (d, 6.8) 4 21.6 0.94 (d, 5.9) NMe 37.2 2.94 (s) 5 19.0 0.72 (d, 6.1) NMe 39.4 2.92 (s) Chromophore δH 2.20 (s, 3H, 12-H3), 2.52 (s, 3H, 11-H3), 7.34 (d, 7.8, 1H, 7-H), 7.54 (d, 7.7, 1H, 8-H). δC 7.8 (CH3, C-12), 15.0 (CH3, C-11), 100.8 (qC, C-1), 113.8 (qC, C-4), 125.8 (CH, C-8), 127.6 (qC, C-6), 128.9 (qC, C-9a), 130.3 (CH, C-7), 132.7 (qC, C-9), 140.5 (qC, C-5a), 145.0 (qC, C- 4a), 146.0 (qC, C-10a), 147.3 (qC, C-2), 166.0 (qC, C-13), 167.8 (qC, C-14), 178.6 (qC, C-3).

* Overlapped signals.

131 3.3.1.5 Structural elucidation of 4

Compound 4, obtained as a red amorphous powder, was assigned to be the deoxygenated analogue of 3 by (+)-HRESIMS and NMR data analysis (tables 3.3.4).

1 The representative oxygenated methine (δH 5.92) in the H NMR of HMPro in the α- ring of 3 was absent, and a methylene (δH 1.75 and 2.74) appeared in 4. The

13 corresponding methylene signal (δC 29.4) in the C NMR of 4 was determined by an

HSQC experiment. Analysis of the key COSY and HMBC correlations and comparison of the data with that of 3 indicated that there was an MPro residue in α- ring. Thus, the structure of 4 was confirmed and named actinomycin Y9.

O O MPro O Sar O O OPro Sar O O O N N O N O N MeAla N O N N O N N O N N O N MeVal NH O O NH O O NH O O NH O O D-Val Thr D-Val O O O O HN O O NH Thr HN O O NH

N NH 2 N NH2

O O O O

COSY HMBC (H C)

132 1 13 Table 3.3.4. NMR data of actinomycin Y9 (4) in CDCl3 ( H: 400 MHz, C: 100 MHz, J in Hz)

Pentapeptidolactones α-ring positions δC δH β-ring positions δC δH Thr 1 168.7 HThr 1 168.7 2 54.6 4.59 (m) 2 54.9 4.48 (m) 3 74.7 5.14 (m) 3 75.4 5.15 (m) 4 17.1 1.11 (d, 6.2) 4 17.4 1.25 (d, 6.2) NH 7.14 (d, 7.1) NH 7.63 (d, 6.0) D-Val 1 173.9 D-Val 1 173.2 2 59.1 3.51 (dd, 9.6, 2 57.0 3.78 (m) 6.0) 3 32.0 2.07 (m) 3 32.3 2.08 (m) 4 19.3 1.08 (d, 6.6) 4 19.2 0.88 (d, 7.0) 5 19.0 0.91 (d, 6.8) 5 18.7 1.16 (d, 6.6) NH 7.61 NH 8.38 (d, 5.6) MPro 1 173.6 OPro 1 172.7 2 58.2 6.00 (d, 9.0) 2 54.3 6.57 (d, 10.7) 3 29.4 1.75 (m) 3 41.7 2.31 (d, 17.1) 2.74 (m) 3.79 (dd, 9.5, 18.0) 4 31.9 1.99 (m) 4 208.9 2.23 (m) 5 55.4 4.35 (m) 5 52.8 3.95 (d, 19.0) 6 18.8 1.47 (d, 6.0) 4.58 (m) Sar 1 166.3 Sar 1 165.6 2 51.4 3.62* 2 51.2 3.62* 4.64 (d, 16.1) 4.49 (d, 15.8) NMe 34.8 2.86 (s) NMe 34.6 2.86 (s) MeVal 1 167.4 MeAla 1 168.8 2 71.3 2.64 2 60.5 3.23 (m ) 3 26.8 2.65 (m) 3 13.4 1.36 (d, 6.8) 4 21.6 0.93 (d, 6.0) NMe 37.2 2.92 (s) 5 19.0 0.71 (d, 5.8) NMe 39.2 2.90 (s) Chromophore δH 2.21 (s, 3H, 12-H3), 2.53 (s, 3H, 11-H3), 7.34 (d, 7.7, 1H, 7-H), 7.61 (d, 7.7, 1H, 8-H). δC 7.7 (CH3, C-12), 15.0 (CH3, C-11), 101.8 (qC, C-1), 113.5 (qC, C-4), 126.2 (CH, C-8), 127.7 (qC, C-6), 129.1 (qC, C-9a), 130.1 (CH, C-7), 132.2 (qC, C-9), 140.4 (qC, C-5a), 144.9 (qC, C-4a), 145.9 (qC, C-10a), 147.3 (qC, C-2), 165.8 (qC, C-13), 168.8 (qC, C-14), 179.0 (qC, C-3).

* Overlapped signals.

133 3.3.1.6 Structural elucidation of 5

Compound 5 was obtained as red amorphous powder and had a molecular formula of C64H90N12O16 as determined by the pseudo-molecular ion peak at m/z 1283.6666

[M + H]+ (calcd for C64H91N12O16) by (+)-HRESIMS. Its NMR data showed the existence of representative actinoyl chromophore and two identical pentapeptidolactone residues which contained Val, MPro, Sar, MeVal and Thr moieties (table 3.3.5). Compound 5 differed from actinomycin D at the methylation at

C-3 of proline, which was confirmed through COSY, TOCSY, HMBC and NOESY experiments. By comparison with published structures of known actinomycins, 5 was determined to be identical to the known actinomcyin Zp shich was isolated as the monomeric protactin and was converted to Zp by ferricyanide oxidation (143).

However, 5 was, for the first time, isolated as a natural product.

O O O O Sar ro Sar MP O MPro O O O N N N N N N N O N N O N O N O N MeVal MeVal NH O O NH O O - NH O O - NH O O D Val D Val Thr O O O O Thr HN O NH HN O O NH O

N N NH2 NH2

O O O O 5 COSY HMBC (H C)

134 1 13 Table 3.3.5. NMR data of actinomycin Zp (5) in CDCl3 ( H: 400 MHz, C: 100 MHz, J in Hz)

Pentapeptidolactones α-ring positions δC δH β-ring positions δC δH Thr 1 168.4 Thr 1 168.9 2 55.1 4.51 (m) 2 54.7 4.65 (m) 3 75.0 5.20 (m) 3 75.0 5.20 (m) 4 17.4 1.24 (d, 5.3) 4 17.8 1.24 (d, 6.4) NH 7.05 (d, 5.3) NH 7.61 (d, 6.4) D-Val 1 173.4 D-Val 1 173.4 2 59.3 3.48* 2 59.2 3.48* 3 32.0 2.11 (m) 3 32.0 2.11 (m) 4 19.2 1.10* 4 19.2 1.10* 5 19.0 0.90 (d, 6.8) 5 19.0 0.88 (d, 6.6) NH 8.39 (d, 5.7) NH 8.20 (d, 6.0) MPro 1 173.4 MPro 1 173.4 2 58.0 6.10 (d, 9.0) 2 58.2 6.03 (d, 9.0) 3 29.4 1.27 (m) 3 29.6 1.27 (m) 1.73 (m) 1.73 (m) 4 31.8 1.92 (m) 4 31.8 1.92 (m) 2.23 (m) 2.23 (m) 5 55.4 4.29 (m) 5 55.2 4.40 (m) 6 18.8 1.46 (d, 6.0) 6 18.8 1.46 (d, 6.0) Sar 1 166.4 Sar 1 166.5 2 51.4 3.58 (d, 11.5) 2 51.4 3.60 (d, 11.5) 4.68* 4.68* NMe 34.8 2.87 (s) NMe 34.8 2.87 (s) MeVal 1 167.6 MeVal 1 167.5 2 71.3 2.65 (brs) 2 71.3 2.65 (brs) 3 26.7 2.68* 3 26.7 2.68* 4 21.5 0.95 (d, 5.8) 4 21.6 0.95 (d, 6.1) 5 19.1 0.72 (d, 5.8) 5 19.1 0.72 (d, 6.1) NMe 39.2 2.90 (s) NMe 39.3 2.91 (s) Chromophore δH 2.22 (s, 3H, 12-H3), 2.52 (s, 3H, 11-H3), 7.32 (d, 7.7, 1H, 7-H), 7.58 (d, 7.7, 1H, 8-H). δC 7.7 (CH3, C-12), 15.0 (CH3, C-11), 102.0 (qC, C-1), 113.3 (qC, C-4), 125.7 (CH, C-8), 127.4 (qC, C-6), 129.1 (qC, C-9a), 130.2 (CH, C-7), 132.9 (qC, C-9), 140.4 (qC, C-5a), 144.9 (qC, C-4a), 145.7 (qC, C-10a), 147.3 (qC, C-2), 166.1 (qC, C-13), 166.3 (qC, C-14), 179.1 (qC, C-3).

* Overlapped signals.

135 3.3.1.7 Summary of physicochemical properties of novel actinomycins

Actinomycin Y6 (1): red amorphous powder; UV (MeOH) λmax (log ε) 234 (sh, 9.13),

306 (1.22), 429 (4.30) nm; CD (MeOH) λmax ([θ]) 212 (‒103,949), 244 (7,851), 306

(14,000), 375 (1,261); 13C and 1H NMR data, see Tables 1 and 2; (+)-HR-ESI-MS:

+ m/z 1269.5770 [M + H] (calcd for C61H81N12O18, 1269.5792).

Actinomycin Y7 (2): red amorphous powder; UV (MeOH) λmax (log ε) 241 (sh, 7.91),

441 (3.73) nm; CD (MeOH) λmax ([θ]) 209 (‒94,694), 243 (9,034), 271 (16,047), 306

(1,664), 378 (5,637); 13C and 1H NMR data, see Tables 1 and 2; (+)-HR-ESI-MS:

+ m/z 1287.5918 [M + H] (calcd for C61H83N12O19, 1287.5897); m/z 1309.5748 [M +

+ Na] (calcd for C61H82N12O19Na, 1309.5717).

Actinomycin Y8 (3): red amorphous powder; UV (MeOH) λmax (log ε) 240 (sh, 9.37),

440 (4.34) nm; CD (MeOH) λmax ([θ]) 215 (‒125,568), 242 (26,967), 267 (56,168),

379 (20,196); 13C and 1H NMR data, see Tables 1 and 2; (+)-HR-ESI-MS: m/z

+ + 1271.5968 [M + H] (calcd for C61H83N12O18, 1271.5948); m/z 1293.5788 [M + Na]

(calcd for C61H82N12O18Na, 1293.5768).

Actinomycin Y9 (4): red amorphous powder; UV (MeOH) λmax (log ε) 238 (sh, 8.58),

440 (3.95) nm; CD (MeOH) λmax ([θ]) 210 (‒67,274), 242 (11,893), 272 (21,000), 381

(7,255); 13C and 1H NMR data, see Tables 1 and 2; (+)-HR-ESI-MS: m/z 1255.5935

+ + [M + H] (calcd for C61H83N12O17, 1255.5999), m/z 1277.5749 [M + Na] (calcd for

C61H82N12O17Na, 1277.5819).

136 Actinomycin Zp (5): red amorphous powder; UV (MeOH) λmax (log ε) 242 (sh,

9.44), 442 (4.13) nm; CD (MeOH) λmax ([θ]) 212 (‒52,199), 241 (11,713), 269

(16,359), 380 (5,068); 13C and 1H NMR data, see Tables 1 and 2; (+)-HR-ESI-

+ MS: m/z 1283.6575 [M + H] (calcd for C64H91N12O16, 1283.6676), m/z 1305.6400 [M

+ + Na] (calcd for C64H90N12O16Na, 1305.6495).

Figure 3.3.3. HPLC profile of different actinomycins produced by Streptomyces sp. Strain Gö-GS12.

137 The UV/VIS spectra (λmax 238, 429 and 441 nm) of the new actinomycins suggested the aminophenoxazinone chromophore was shared by these compounds. An additional absorption at 309 nm of 1 suggested the phenoxazinone-fused L-Thr congener. Also a red shift at 452 nm was observed for Y4 and Y3 indicating the rearrangement of the β-ring also results in a distinct UV-VIS spectrum that is diagnostic of this modification (fig 3.3.4).

Norm.

1750

Standard β-ring 1500 actinomycins rearrangement

1250

1000 Phenoxazinone- 750 fused L-Thr

500

250

250 300 350 400 450 500 550 nm

Figure 3.3.4 UV/VIS spectra of actinomycins isolated from Streptomyces sp. Strain Gö-GS12.

Actinomycins have a strong negative absorption at 210 nm in the CD spectra. The

β-ring rearranged actinomycins including 1 and Y4 had two positive absorptions around 275 nm and 360 nm, while the other members of actinomycins had a strong negative absorption at these wavelengths (fig. 3.3.5).

138

ECD spectra of actinomycins

150

100

0 200 250 300 350 400 450 500

-50

-100

Y6 Y7 Y8 Y9 Zp Y1 Y4 -150

Figure 3.3.5. CD spectra of actinomycins isolated from Streptomyces sp. Strain Gö-GS12.

139 a Table 3.3.6: Overview on naturally occurring actinomycins

α-ring β-ring (1) Actinomycin D as core structure Actinomycin D Thr-D-Val-Pro-Sar-MeVal Thr-D-Val-Pro-Sar-MeVal (2) N-demethyl actinomycins Actinomycin D0 Thr-D-Val-Pro-Sar-MeVal Thr-D-Val-Pro-Gly-MeVal N,N'-Didemethyl- Thr-D-Val-Pro-Gly-MeVal Thr-D-Val-Pro-Gly-MeVal actinomycin D (2) C-type actinomycins Actinomycin C2 Thr-D-Val-Pro-Sar-MeVal Thr-D-aIle-Pro-Sar-MeVal Actinomycin C2a Thr-d-aIle-Pro-Sar-MeVal Thr-D-Val-Pro-Sar-MeVal Actinomycin C3 Thr-D-aIle-Pro-Sar-MeVal Thr-D-aIle-Pro-Sar-MeVal (3) F-type actinomycins Actinomycin F8 Thr-D-Val-Sar-Sar-MeVal Thr-D-Val-Sar-Sar-MeVal Actinomycin F9 Thr-D-Val-Sar-Sar-MeVal Thr-D- Thr-D-Val-Pro-Sar-MeVal Val-Pro-Sar-MeVal Thr-D-Val-Sar-Sar-MeVal (4) X-type actinomycins

Actinomycin X0α Thr-D-Val-Sar-Sar-MeVal Thr-D-Val-Hyp-Sar-MeVal Actinomycin X0β Thr-D-Val-Pro-Sar-MeVal Thr-D-Val-Hyp-Sar-MeVal Actinomycin X0δ Thr-D-Val-Pro-Sar-MeVal Thr-D-Val-aHyp-Sar-MeVal Actinomycin X1a Thr-D-Val-Sar-Sar-MeVal Thr-D-Val-OPro-Sar-MeVal Actinomycin X2 Thr-D-Val-Pro-Sar-MeVal Thr-D-Val-OPro-Sar-MeVal (5) Z-type actinomycins Actinomycin Z1 Thr-D-Val-HMPro-Sar-MeVal HThr-D-Val-MOPro-Sar-MeAla Actinomycin Z2 Thr-D-Val-HMPro-Sar-MeVal Thr-D-Val-MOPro-Sar-MeAla Actinomycin Z3 Thr-D-Val-HMPro-Sar-MeVal ClThr-D-Val-MOPro-Sar-MeAla Actinomycin Z4 Thr-D-Val-MPro-Sar-MeVal Thr-D-Val-MOPro-Sar-MeAla Actinomycin Z5 Thr-D-Val-MPro-Sar-MeVal ClThr-D-Val-MOPro-Sar-MeAla Actinomycin Zp(5) Thr-D-Val-MPro-Sar-MeVal Thr-D-Val-MPro-Sar-MeVal (6) G-type actinomycins Actinomycin G1 Thr-D-Val-Pro-Sar-MeVal HThr-D-Val-HMPro-Sar-MeAla Actinomycin G2 Thr-D-Val-HMPro-Sar-MeVal ClThr-D-Val-Pro-Sar-MeAla Actinomycin G3 Thr-D-Val-HMPro-Sar-MeVal HThr-d-Val-Pro-Sar-MeAla Actinomycin G4 Thr-D-Val-HMPro-Sar-MeVal Thr-D-Val-Pro-Sar-MeAla Actinomycin G5 Thr-D-Val-HMPro-Sar-MeVal cHThr-D-Val-Pro-Sar-MeAla Actinomycin G6 Thr-D-Val-HMPro-Sar-MeVal rHThr-D-Val-Pro-Sar-MeAla (7) novel Y-type actinomycins Actinomycin Y1 Thr-D-Val-HMPro-Sar-MeVal ClThr-D-Val-OPro-Sar-MeAla Actinomycin Y2 Thr-D-Val-HMPro-Sar-MeVal ClThr-D-Val-Hyp-Sar-MeAla Actinomycin Y3 Thr-D-Val-HMPro-Sar-MeVal rHThr-D-Val-OPro-Sar-MeAla Actinomycin Y4 Thr-D-Val-HMPro-Sar-MeVal rHThr-D-Val-Hyp-Sar-MeAla Actinomycin Y5 Thr-D-Val-HMPro-Sar-MeVal cThr-D-Val-OPro-Sar-MeAla Actinomycin Y6 (1) Thr-D-Val-HMPro-Sar-MeVal crThr-D-Val-OPro-Sar-MeAla Actinomycin Y7 (2) Thr-D-Val-HMPro-Sar-MeVal HThr-D-Val-OPro-Sar-MeAla Actinomycin Y8 (3) Thr-D-Val-HMPro-Sar-MeVal Thr-D-Val-OPro-Sar-MeAla Actinomycin Y9 (4) Thr-D-Val-MPro-Sar-MeVal Thr-D-Val- OPro -Sar-MeAla

140 Differences to actinomycin D are shown in bold letters. All amino acids are L-configurated except when indicated otherwise. Abbreviations: MeVal=N-methylvaline, MeAla=N-methyl-L-alanine, aIle=allo-isoleucine, Sar=sarcosine, MPro=cis-5-methylproline, HMPro=trans-3-hydroxy-cis-5-methylproline, Hyp=trans-4-hydroxyproline, OPro=4- oxoproline, MOPro=cis-5-methyl-4-oxoproline, aHyp=cis-4-hydroxyproline, HThr=4-hydroxythreonine, ClThr=4- chlorothreonine, cThr/cHThr=cyclic Thr or HThr, i.e. forming an additional ring closure to the chromophore, rHThr= rearrangement of the HThr (β-ring) connectivities. a. This table was updated based on previous overview on naturally occurring actinomycins (77).

3.3.2 Biological activity of actinomycins

The biological activities of the novel Y-type actinomycins, along with known isolated actinomycins as controls, were evaluated against different Gram-positive and Gram- negative strains by using commercial actinomycin D as a clinical reference. Most actinomycins showed potent activity against Gram-positive strains except for Y3 and

Y4. 4 showed the most potent antibacterial activity, followed by 3 and 5. 2 had significant decreased activity while 1 was 50-fold less active than 2.

The results of the antibacterial screen revealed insight into structure-activity relationship (SAR) of actinomycins. 5 differed from actinomycin D only in the 5- methylproline moiety of the β-ring and showed a 2-fold decrease in activity, indicating that methylation of proline had a slight negative impact. When compared with 4, a decreased activity of 3 against S. aureus and B. subtilis indicated that introduction of an extra hydroxyl group to the methylproline of α-ring was also detrimental to the antibacterial activity. Modifications on the threonine moiety of β-ring also significantly lowered the activities, since 2 was at least 10-fold less active than 3 which only differed by the hydroxyl group on threonine. Another important factor that effected the activity was the nature of the cyclization of the β-peptide ring. The connection between threonine and alanine of β-ring is typically through an ester bond, and the chromophore was attached via an amide. This was not the case for 1, Y3, and Y4

141 which undergo an acyl rearrangement. 1 had significantly decreased activity, while

Y3 and Y4 showed no appreciable activity suggesting that the β-peptidolactone ring played an important role. On the other hand, the fusion of the peptidolactone to the phenoxazine had a positive impact on activity since 1 was at least 4-fold more potent than Y3.

As part of our program aimed at rapidly accessing peptide antibiotics, we implemented a solid phase synthetic strategy to obtain potential actinomycin precursors and derivatives. The synthesized partial 6 and phenol-4 showed no appreciable activity against any tested strains. This result indicated that neither the peptide chain with altered aromatic moiety nor 4-MHA can provide any antibacterial activity. Thus protactin (94) is the minimum bioactive unit (fig 3.3.6).

Table 3.3.6. In Vitro antimicrobial activities of 1-5 and actinomycins Y1, Y3, Y4 and D.

MIC ( μM) Compound S. M. B. M. S. aureus luteus subtilis aurum S. enterica E. coli cerevisae 1 30 7.5 7.5 12 >120 >120 120 2 6 1.5 1.5 12 >120 >120 >120 3 0.4 0.04 0.15 12 >120 >120 120 4 0.15 0.04 0.04 12 >120 >120 120 5 0.6 0.075 0.075 12 >120 120 >120

Y1 0.3 0.04 0.02 7.5 >120 >120 60

Y3 >120 60 >120 >120 >120 >120 >120 Y4 >120 60 >120 >120 >120 >120 >120 D 0.075 0.04 0.02 7.5 120 120 60 C7 >120 >120 >120 >120 >120 >120 >120 Phenol-4 >120 60 >120 >120 >120 >120 >120

142 Structural modification to actinomycins had parallel impact to antibacterial activity and cytotoxicity as noticed in this study. For most cases, actinomycins with higher antibacterial potency were more toxic to different cell lines (PC3 and A549 cells were used in this study). Importantly, actinomycins with iso-actinomycins such as 5 and actinomycin D showed highest toxicity against human cell lines, and this feature should be avoid for development of actinomycins as antibacterial agents.

Table 3.3.7. Cytotoxic activity of actinomycins against different tumor cell lines. EC (nM) Compound 50

PC3 Cells A549 Cells

1 76.70 71.48 2 51.18 119.30 3 2.77 7.67 4 0.47 1.54 5 0.16 0.74

Y1 0.70 1.10 Y3 2051 3392 Y4 1474 3272 D 0 .61 1.64

Importantly, even though structurally different actinomycins varied in most antibacterial activities, their anti-Mycobacteria activity was apparently independent from structural modifications. For example, 1 and 2 showed the least cytotoxicity against human cancer cell lines and are about 100-fold less toxic than actinomycin

D, and their activity against S. aureus, M. Iuteus and B. subtilis paralleled the decrease in cytotoxicity. Nonetheless, 1 and 2 were still shown to have moderate activity against M. aurum with MIC at 12 µM that is comparable to actionomycin D

(MIC of 7.5 µM). This divergence between toxicity and activity suggests a possible

143 distinct mechanism of inhibition in M. aurum. This unexpected discovery suggest that the actionmycin scaffold may be a good candidate for development of anti-

Mycobacteria agents, and further structural optimization may enhance this anti- mycobacteria activity without compromising drug safety. We are currently testing all

actinomycins against Mycobacterium tuberculosis, the primary causative agent of TB.

3.4 Conclusion

The high antibiotic potency and low toxicity of actinomycin Y5 suggested it is possible to be a lead compound to achieve an antibiotic-selective actinomycin through structural modification. With the goal of expanding the family of actinomycins by discovering novel members, we conducted a thorough characterization of the metabolic profile of Streptomyces sp. Strain Gö-GS12 strain by LC-MS, and several actinomycins were characterized by 1D and 2D NMR. We successfully identified four new members of actinomycin family Y6-Y9 (1-4) which differ in the asymmetric (aniso-) peptidolactones compared to other actinomycins isolated from Streptomyces sp.

Strain Gö-GS12, along with actinomycin Zp (5), which was for the first time isolated as a natural product. Similar to previously discovered actinomycins from this strain,

1-4 bear the rare OPro in β-ring and HMPro or MPro in α-ring. 1 also contains two important structural features including a two-fold acyl shift and an additional ring closure. 5 is the only symmetric (iso-) actinomycins produced by this strain, and this is also for the first time that iso- and aniso-actinomycins were isolated from the same strain. The new members of actinomycins were tested for activity to provide more detailed information on antimicrobial/toxicity structure-activity relationship (SAR) of

144 the actinomycins. The oxidation of proline or threonine moiety diminishes the bioactivity in terms of both antibacterial activity and cytotoxicity, and the rearrangement of β-ring has a similar negative impact. The additional ring closure of

1 and Y5 seemed to be the only plausible modification β-ring which leads to significant improved antibacterial activity. A structural model of compound 1 containing the rearranged β-ring and the additional third ring suggested the β-ring significantly changes conformation while the conformation of α-ring showed only minor changes

(fig. 3.3.6). The differences in conformation of the β-ring may contribute to the binding of actinomycins to DNA molecules resulting in various antibacterial activities and cytotoxicity. It was interesting to note that the two least toxic actinomycins against human cancer cell lines, 1 and 2, maintained potency against M. aurum that was comparable to actinomycin D, suggesting divergent activity may be achieved for developing an anti-TB agent.

β-ring

α-ring

Chromophore core

145 Figure 3.3.6. Conformational changes of α- and β-ring of actinomycins. The structure of actinomycin D (blue line, PDB ID: 4HIV) is shown in blue line. Actinomycin

Y3 containing a rearranged β-ring was shown in yellow, Y5 and 1 containing the additional ring structure were shown as red and green lines, respectively. Structures were superimposed by chromophore cores, and the structures of Y3, Y5 and 1 were established and optimized using Gaussian program*.

Streptomyces sp. Strain Gö-GS12 produces at least 10 different actinomycins, from actinomycin Y1-Y9 and Zp, with various on both peptide chains and includes almost all of the important structural features that have been discovered for different actinomycins. The ability of producing a variety of analogues makes this strain a perfect candidate to establish a model strain to study SAR and possibly the biosynthetic pathway of actinomycins.

* I am grateful to Prof. Dr. Zhaoyong Yang at Chinese Academy of Medical Science & Peking Union Medical College for giving me the remote access to the program in his group.

The discovery of novel antibiotics has dramatically decreased over the last few decades while tuberculosis (TB) remains a global major threat to public health. Thus, discovery and development of new antibiotics are urgently needed. Capuramycins, a class of nucleoside antibiotics, show potent anti-TB activity by inhibiting the bacterial

MraY, a key enzyme involved in the biosynthesis of peptidoglycan cell wall. The first part of this dissertation describes the identification and characterization of the biosynthetic gene cluster for A-102395, which is the latest member of capuramycins.

Bioinformatic analysis of the gene cluster led us to identify 35 putative open reading frames (ORF) which were responsible for the biosynthesis and resistance. A series of gene inactivations indicate those genes are essential for A-102395 biosynthesis.

Among the cluster were the genes Cpr19 and Cpr25, two enzymes involved in the assembly of the (5’S, 6’S)-5’-C-glycyluridine (GlyU). After this pair of genes was coexpressed and was functionally characterized, a shared biosynthesis paradigm for uridine-5'-carboxamide (CarU) in uridyl lipopeptide antibiotics was suggested. The enzyme conferring self-resistance, Cpr17, was characterized as a phosphotransferase, and its substrate promiscuity was explored. Cpr17 transfers the

γ-phosphate from a nucleoside triphosphate, and GTP was the preferred the phosphate donor. Additionally, a validated target- and species-specific high- throughput screening model was established, and this model will be used to screen natural products and their derivatives to identify new antibacterial therapeutics.

The second part of this dissertation describes the discovery of novel actinomycins via a thorough investigation of the metabolic profile from Streptomyces sp. Strain Gö-

147 GS12. Four new members of actinomycin family Y6-Y9 were isolated and structurally elucidated by LC-MS, 1D and 2D NMR. Their anti-TB activity and cytotoxicity were determined, and the unexpected divergence between them suggested that the actinomycin scaffold may be a good candidate for development of anti-TB agents, and further structural optimization may enhance their anti-microbial activity without sacrificing drug safety.

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157

Appendix I.

Table of Contents Page Figure S1. Chemical structure and 1H, 1H-COSY correlations of P5 I-2 1 Figure S2. H NMR spectrum (D2O, 400 MHz) of P5 I-3 13 Figure S3. C NMR spectrum (D2O, 100 MHz) of P5 I-3 1 1 Figure S4. H, H COSY spectrum (D2O, 400 MHz) of P5 I-4

I-1 H N O

O NH2 O O NH O HO O N O OH O O O

NH2

Figure S1. Chemical structure and 1H, 1H-COSY correlations of P5

1 H NMR (400 MHz, D2O) δ 7.71 (d, J = 8.1 Hz, 1H), 5.98 (dd, J = 2.9, 1.1 Hz, 1H), 5.90 (dd, J = 6.2, 4.3 Hz, 2H), 5.39 (d, J = 3.1 Hz, 1H), 5.24 – 5.20 (m, 1H), 4.71 (d, J = 2.3 Hz, 1H), 4.56 (dd, J = 4.5, 2.5 Hz, 1H), 4.49 (dd, J = 5.5, 2.3 Hz, 1H), 4.23 – 4.17 (m, 1H), 3.95 (t, J = 5.5 Hz, 1H), 3.47 – 3.42 (m, 2H), 3.36 (d, J = 0.6 Hz, 3H), 2.46 (td, J = 6.6, 13 2.6 Hz, 2H), 2.36 (td, J = 2.6, 0.8 Hz, 1H). C NMR (101 MHz, D2O) δ 172.71, 165.96, 162.81, 157.08, 151.08, 141.48, 141.32, 109.52, 102.28, 98.94, 88.46, 82.08, 81.70, 77.72, 75.07, 73.47, 70.47, 64.51, 61.77, 58.45, 37.91, 18.31. HRMS (ESI): + C22H27N5O12+H , Calc: 554.1729, Found: 554.1731.

I-2 1 Figure S2. H NMR spectrum (D2O, 400 MHz) of P5

13 Figure S3. C NMR spectrum (D2O, 100 MHz) of P5

I-3

1 1 Figure S4. H, H COSY spectrum (D2O, 400 MHz) of P5 Appendix II.

Table of Contents Page

Figure S1. Chemical structures of the compounds isolated from Streptomyces sp. II-3 Strain Gö-GS12 1 Figure S2. H NMR spectrum (CD3OD, 500 MHz) of actinomycinY6 (1) II-4 13 Figure S3. C NMR spectrum (CD3OD, 100 MHz) of actinomycin Y6 (1) II-5 Figure S4. HSQC spectrum (CD3OD, 500 MHz) of actinomycin Y6 (1) II-6 Figure S5. HMBC spectrum (CD3OD, 500 MHz) of actinomycin Y6 (1) II-7

1 1 Figure S6. H, H COSY spectrum (CD3OD, 500 MHz) of actinomycin Y6 (1) II-8

Figure S7. TOCSY spectrum (CD3OD, 500 MHz) of actinomycin Y6 (1) III-9

Figure S8. (+)-HRESI-MS (positive mode) of actinomycin Y6 (1) II-10 1 Figure S9. H NMR spectrum (CDCl3, 500 MHz) of actinomycin Y7 (2) II-11

13 Figure S10. C NMR spectrum (CDCl3, 100 MHz) of actinomycin Y7 (2) II-12

Figure S11. HSQC spectrum (CDCl3, 500 MHz) of actinomycin Y7 (2) II-13

Figure S12. HMBC spectrum (CDCl3, 500 MHz) of actinomycin Y7 (2) II-14

1 1 Figure S13. H, H COSY spectrum (CDCl3, 500 MHz) of actinomycin Y7 (2) II-15

Figure S14. TOCSY spectrum (CDCl3, 500 MHz) of actinomycin Y7 (2) II-16

Figure S15. (+)-HRESI-MS (positive mode) of actinomycin Y7 (2) II-17 1 Figure S16. H NMR spectrum (CDCl3, 400 MHz) of actinomycin Y8 (3) II-18

13 Figure S17. C NMR spectrum (CDCl3, 100 MHz) of actinomycin Y8 (3) II-19

Figure S18. HSQC spectrum (CDCl3, 500 MHz) of actinomycin Y8 (3) II-20

Figure S19. HMBC spectrum (CDCl3, 500 MHz) of actinomycin Y8 (3) II-21

1 1 Figure S20. H, H COSY spectrum (CDCl3, 500 MHz) of actinomycin Y8 (3) II-22

Figure S21. TOCSY spectrum (CDCl3, 500 MHz) of actinomycin Y8 (3) II-23

Figure S22. NOESY spectrum (CDCl3, 500 MHz) of actinomycin Y8 (3) II-24

Figure S23. (+)-HRESI-MS (positive mode) of actinomycin Y8 (3) II-25 1 Figure S24. H NMR spectrum (CDCl3, 400 MHz) of actinomycin Y9 (4) II-26

13 Figure S25. C NMR spectrum (CDCl3, 100 MHz) of actinomycin Y9 (4) II-27

Figure S26. HSQC spectrum (CDCl3, 500 MHz) of actinomycin Y9 (4) II-28

Figure S27. HMBC spectrum (CDCl3, 500 MHz) of actinomycin Y9 (4) II-29

1 1 Figure S28. H, H COSY spectrum (CDCl3, 500 MHz) of actinomycin Y9 (4) II-30

II-1 Figure S29. TOCSY spectrum (CDCl3, 500 MHz) of actinomycin Y9 (4) II-31

Figure S30. NOESY spectrum (CDCl3, 500 MHz) of actinomycin Y9 (4) II-32

Figure S31. (+)-HRESI-MS (positive mode) of actinomycin Y9 (4) II-33 1 Figure S32. H NMR spectrum (CDCl3, 400 MHz) of actinomycin Zp (5) II-34

13 Figure S33. C NMR spectrum (CDCl3, 100 MHz) of actinomycin Zp (5) II-35

Figure S34. HSQC spectrum (CDCl3, 500 MHz) of actinomycin Zp (5) II-36

Figure S35. HMBC spectrum (CDCl3, 500 MHz) of actinomycin Zp (5) II-37

1 1 Figure S36. H, H COSY spectrum (CDCl3, 500 MHz) of actinomycin Zp (5) II-38 Figure S37. (+)-HRESI-MS (positive mode) of actinomycin Zp (5) II-39 1 Figure S38. H NMR spectrum (CDCl3, 500 MHz) of actinomycin Y1 II-40

13 Figure S39. C NMR spectrum (CDCl3, 100 MHz) of actinomycin Y1 II-41

Figure S40. HSQC spectrum (CDCl3, 500 MHz) of actinomycin Y1 II-42

Figure S41. HMBC spectrum (CDCl3, 500 MHz) of actinomycin Y1 II-43

1 1 Figure S42. H, H COSY spectrum (CDCl3, 500 MHz) of actinomycin Y1 II-44

Figure S43. TOCSY spectrum (CDCl3, 500 MHz) of actinomycin Y1 II-45 Figure S44. (+)-HRESI-MS (positive mode) of actinomycin Y1 II-46

1 Figure S45. H NMR spectrum (CDCl3, 500 MHz) of actinomycin Y3 II-47

13 Figure S46. C NMR spectrum (CDCl3, 100 MHz) of actinomycin Y3 II-48

Figure S47. (+)-HRESI-MS (positive mode) of actinomycin Y3 II-49

1 Figure S48. H NMR spectrum (CDCl3, 500 MHz) of actinomycin Y4 II-50

13 Figure S49. C NMR spectrum (CDCl3, 100 MHz) of actinomycin Y4 II-51

Figure S50. (+)-HRESI-MS (positive mode) of actinomycin Y4 II-52

1 Figure S51. H NMR spectrum (CDCl3, 500 MHz) of 6 II-53

13 Figure S52. C NMR spectrum (CDCl3, 100 MHz) of 6 II-54

Figure S53. HSQC spectrum (CDCl3, 500 MHz) of 6 II-55

Figure S54. HMBC spectrum (CDCl3, 500 MHz) of 6 II-56

1 1 Figure S55. H, H COSY spectrum (CDCl3, 500 MHz) of 6 II-57

III--22 HMPro O MPro/HMPro O Sar O HO Sar OPro R O Sar Sar O N O 1 O OPro O N MeVal N O N O N N O MeAla N O N N O N N O N MeAla MeVal NH HN O NH O O D-Val NH O O NH O O D-Val D-Val D-Val Thr Thr O O crHThr O O HN O O HN O O NH R2 13 O 10 14 NH HThr/Thr/ClThr 9a N 10a N NH2 8 9 1 2 7 5a 4a 6 O 4 3 O O O 5 11 12 actinomycin Y7 (2): R1 = OH; R2 = OH actinomycin Y6 (1) actinomycin Y8 (3): R1 = OH; R2 = H actinomycin Y9 (4): R1 = H; R2 = H actinomycin Y1: R1 = OH; R2 = Cl

O O HMPro O Sar Sar HO O Hyp/OPro Sar MPro O MPro O Sar O N N O N N R MeAla N O N N O N N O N N O N MeVal MeVal MeVal NH O O NH O O NH O O NH HN O D-Val D-Val D-Val D-Val Thr O O O Thr Thr O OH NH HN O O HN O O O rHThr N NH 2 N NH2

O O O O

actinomycin Y3 : R = O actinomycin Zp (5) actinomycin Y4 : R = OH

Figure S1. Chemical structures of the compounds isolated from Streptomyces sp. Strain Gö-

GS12

II-3 11

. . 8 7916 0 70 . 8 7809 8

F . . 8 2406 i 0 43 .

gu 8 2212 8

0 .

re . 7 8888 0 46 . 7 8777

S . 7 5132

7 . . 2

0 83 7 4974 .

5 . . . 0 83 7 4571 . 1 7 4416 H 7 N

. Rs . 6 6925 0 94 . 6 6782 6

5 pe

. . 0 90 6 2990 c t 6 r

. u

m f

( .

(

ppm 0 91 5 6803 C 5

. 5 D

) 5 3591

. . 1 09 5 23343761 3 .

O 0 94 . . 0 73 5 1204 D 5

0 . . , 1 86 4 8632 .

50 . 2 78 4 7989 . . 4 6837 1 15 . 0 4 5978 4

5 . M . 1 11 4 3895 . H 11 . 5182 . 4 . 2394 4 1147

z .

4 2 58

) .

0 . . 3 9661 1 94 o . 3 8639

f . .

1 96 3 7602

a . 3 6442 3 c

5 . t

1 11 . ino . 3 3256 1 74 . 3 2974

m . 3 3 37 .

3 0590 .

0 . y

2 . 63

c 2 . 83 . 2 68 2 8484 inY . 0 79 . 2 2 64

. 6 5

. .

1 06 . ( 2 55603734

1 . . 3 11 2 2597

) . 1 06 . 2 1393

2 .

1 00

. .

0 . 2 1124 1 08 . 2 0418 . 1 971728

. . 1 3 75 1 2502

. .

5 . 1 2012 3 76 . . 1 1879 . 10 32 1 1596 . . 2 73 1 1137 . 1 . 3 24 1 1003

0 . .

3 23 1 0134 . . 8 14 1 0003 . . 2 98 0 9282 . 0 9140 0 .

. 0 8999

. 0 8809 . 0 8304 . 0 8165

II-4 210 . 209 5319

200

190

. 180 177 4394 . 177 2912 . 175 6449 . 174 9904 . 171 7886 170

F . 169 1618

. i 167 8895 gu . 165 5719 re 160

3 . 153 4923 .

150 13 . 149 0723

C . 147 2522

N . 142 4122 140 M

. 132 6656 pe 130 . 129 9058

c 128 7761 .

t 127 1935 r u 120 m

. ( 116 6384 C D f

1 110

3 (

ppm

O D )

100 10

0 M 90 H

. 88 4169 z)

o 80 f

a 78 3693

c . 74 7212 t

ino . 71 5334 70 . 69 4437

. y 64 0929 c . 60

i 60 9436 n

. 55 4476 Y . 54 2397

6 . 52 8859

( 50 1 . ) 47 7066

. 42 4245 . 40 4982 40 . 39 6756

. 35 7024 . 33 5692 . 32 9256 . 30 30 6178

. 28 . 4715 21 8878

. 19 5363 . 19 2212 20 . 18 9113

. 17 2891 . 15 1031

10 13 7277

. 8 3880

II-5 Figure S4. HSQCspectrum (CD 3 OD, 500 OD, MHz) actinomycinof Y 6 ( 1 )

II-6 Figure S5. HMBCspectrum (CD 3 OD, 500 OD, MHz) actinomycinof Y 6 ( 1 )

II-7 Figure S6. 1 H, 1 HCOSY spectrum (CD 3 OD, 500 OD, MHz)actinomycinof Y 6 ( 1 )

II-8 II -9

Figure S7. TOCSY spectrum (CD3OD, 500 MHz) of actinomycin Y6 (1) Figure S8. (+)- HRESI-MS(positive mode)of actinomycin Y 10 6 ( 1 )

II-10 11

F 9

0 i

gu re 8

5 . . 0 71 8 3902 S

9 . . 0 81 8 1509 . 8

1 0

H . . 0 74 7 8419

. N 7 5568 . . 7 0 93 7 5412 M

. . 7 3747

Rs 1 09 . 7 3594 . 7 2613 7 pe

t . r . 6 5494 6 u 0 62 .

6 5274 5 m

( CDC 6

0 f

1 . .

0 61 5 8753

(

ppm l

) 5

50 . . 5 2451 0 87 . . 5 1570

0 0 86 . 5 1154 5

. . . M

0 0 64 4 9674

. 4 7386 H . 2 02 . z 4 5735 . . 4 ) 2 90 4 5349

5 .

. 4 5092 o 1 27 . 4 4744 f

. .

a 1 36 4 1928 . 4 0237 4 c

0 . t

3 08

ino . 3 8805 . 3 22 . m 3 6258 3

5 .

y 1 02 . 3 3489 c . 2 10 . 3 2461 i

n . 0 79 .

3 3 0917

. Y

0 .

13 26 . 2 8816 7

. ( 1 61 . 2 6886 2

2 . 3 00 .

)

. 2 5380

. . 1 52 2 3163 . . 1 99 2 2218 . . 3 73 2 1248 2 .

. 2 96 .

0 1 9948

. 1 5265

. 1 4165

1 . 3 69

. .

5 1 3591

. 4 36 . 1 1940 . 1 1713 . . 12 87 1 1397 1

. . 8 50 0 9312 . . 4 54 0 7511 . 0 7384 0

II-11 220

210 . 208 6691

200

190

180 F .

178 2663 i gu . 172 6127 re 170 . 169 0180 . 168 6430 . S1 168 2570 . 167 4132 . 166 0575 160 0 . 166 0011

. .

165 8455 13 C 150 . N 148 3120 . 146 1148 M . 145 2865 Rs

140 . 140 4475

. 130 4779 130

c . 128 7235

t .

r 127 6335 . u 125 9802 m 120

( CDC f

(

ppm 110

) l

3 ,

10 100 . 98 9753

0 M H 90

z) . 77 2885 . 77 1735 o . 76 9707 80 f .

76 6529

a . 75 5805

c . 74 6382 t

ino . 71 2235 70

. 68 3215 m . 60 0785

y . 59 6518 60 c . 59 1366

i . n 57 . 0393 54 6218 Y . 52 2182 7 50 . 51 4443

. ( 51 1794 2 . ) 41 8538 . 41 0900

40 . 39 3289

37 005

. 34 7301 . 31 9453

30 . 29 3308

. 27 0338

. 1291 0601284 20

. 17 0001 . 14 9944 . 14 0918 .

10 13 4779

. 7 7049

II-12 Figure S11. HSQCspectrum (CDCl 3 , 500 MHz) , actinomycinof Y 7 ( 2 )

II-13 Figure S12. HMBCspectrum (CDCl 3 , 500 MHz) , actinomycinof Y 7 ( 2 )

II-14 5 II -1

1 1 Figure S13. H, H COSY spectrum (CDCl3, 500 MHz) of actinomycin Y7 (2) Figure S14. TOCSYspectrum (CDCl 3 , 500 MHz) , actinomycin of Y 7 ( 2 )

II-16 Figure S15. (+)- HRESI-MS(positive mode)of actinomycin Y 7 ( 2 )

[ M+ H] +

II-17 10

F i gu 8

re . . 8 2846 0 81 . 8 2691 S1 8

. 0 7 7120 . 6 . 7 6555 0 85 .

. 7 6430 . . 1 00 7 5460 1 .

7 . H 1 09 7 5267

. . N 1 17 7 2423 . 7 2399 M . 7 2387 7 R s

. .

6 9371 0 .

1 06 . 6 9196

pe . . 6 6106 1 15 . 6 6 5845

c 5

t r u m 6

0 . . 1 00 5 9212 ( CDC f

. (

5 2603 ppm 5

. .

5 5 2111

l 5 2016 )

3 . 1 15 .

, 5 1948 . . 2 13 5 1858 40 . 5 5 . 1788

. 4 9136

0 .

. 0 0 97 4 8967

. . M 2 24 4 7241 . H 4 0 95 .

. 4 4963

5 . .

2 13

z) 4 4889 . 4 4714 . 3 9949

o . . 1 16 3 9019 f 4 .

3 8581 .

. 0

a 2 08 . 3 8319 . . c 1 49 3 8070 t

ino . . 2 52 3 6128 3

5 . .

m 1 37 3 3997 . . 3 3740 1 03 . y 3 2481 . c . 23 9241325 3

. i . .

6 64 0 n 2 9283 . . 7 21 2 8883

Y . 2 8683 . . 8 2 20 2 6442

2 .

. ( 2 5203

. 3 39

5 3

. ) 1 32 . . 2 2003 4 20 . . 2 1392 4 14 . 2 2 0363

. . 1 66 1 8358

. 1 3756 . 1 3 35 .

1 2943 . .

5 3 40 . . 1 2324 . . 13 1350 1 1646 . . 3 51 1 1219 . . 7 14 1 1055 1

0 . . 12 52 0 9131 . . 3 63 0 7322 . 0 7168 0

II-18 220

210 .

208 8004 200

190

F 180 . i

178 6676 gu . 172 7498

. re 168 9627 170 . 168 9292

. 168 5525

S1 . 167 9327 . 167 4954 .

7 160 166 2303 .

. 166 1697

. 165 8060 13 C 150

. N 147 4376 . 146 0795 M . 145 0885 Rs

140 . 140 5963

pe . 132 7403

130 .

c 130 4373 .

t 129 0431

r . 127 7730 u . 125 8867 m 120

( CDC f

1 . 113 8998

(

ppm 110

) l

3 ,

100 . 100 8616

0 M 90 H . 77 3634

z) . 77 2483 . 77 0457 . o

80 76 7277 .

f 75 8705

. 75 3049 a . 74 6158 c . t

70 71 3429 ino

. 68 5515

m 60 5576

. 60 y 56 9778

c 54 8393 .

i 54 5721

n . 54 0805 . 52 8369 Y 50 . 51 5246

8 . 51 3012

( . 3 41 8123 .

) 41 5356 40 . 39 4986

. 37 2881 . 34 7483 . 31 9935 30

. 27 0722

. 21 6804 20 .

19 2666 . 19 0781 . 18 8478 . 17 1119 . 10 13 5441

. 7 8512

II-19 Figure S18. HSQCspectrum (CDCl 3 , 500 MHz) , actinomycinof Y 8 ( 3 )

II-20 Figure S19. HMBCspectrum (CDCl 3 , 500 MHz) , actinomycinof Y 8 ( 3 )

II-21 2 II -2

1 1 Figure S20. H, H COSY spectrum (CDCl3, 500 MHz) of actinomycin Y8 (3) Figure S21. TOCSYspectrum (CDCl 3 , 500 MHz) , actinomycin of Y 8 ( 3 )

II-23 Figure S22. NOESYspectrum (CDCl 3 , 500 MHz) , actinomycin of Y 8 ( 3 )

II-24 Figure S23. (+)- HRESI-MS(positive mode)of actinomycin Y 8 ( 3 )

[ M+ H] + [ M+ N a] +

II-25 11

0 F

i gu 8 re

5 .

. 8 3915 0 64 . 8 3773 S2 8 4

0 .

. 7 6401

. 7 6249 1 . H 7 6160 . . 2 14 7 5967 7 N

. .

5 7 3500 . M . 1 00 7 3306 . 7 2397 R s . . 0 60 7 2390 . 7 7 1502

0 .

7 1323 pe

c . . 6 5737 t 6 0 98 . r

. 6 5468

5 u

( . 6 CDC . 6 0153

. 0 86

f . 0

1 5 9926

(

ppm l

) 3 5

, .

0 . 2 03 5 1446 5

. M

. 4 5915

H . 4 5699 . 2 27 . z) 4 5449 . 1 20 .

4 4 5095

. . .

5 o 1 30 4 4664

f . 1 03 4 3297

a c

4 .

t 3 9793

. .

0 ino 1 34 .

3 9313 . . 2 23 3 7504

m . . 2 26 3 6300

3 . y

1 21 . .

5 . 3 4654 c 1 05

i . n . 3 2345 1 05 . 3 2172

Y . 2 9395 3 .

. . .

9 3 36

0 86 0 . 2 9021

3 . 11 .

( 6 38 2 8618

4 . 0 94 . . II-26 2 6445 ) 2 32 . . 2 6275 2 3 19

. .

5 2 5305 . . 2 2581 1 97 . . 2 2160 4 31 . 2 1922 .

2 . 1 71 2 0523

0 . . 2 04 1 9291

. . 6 12 1 7060 . 1 3741 1 .

. 3 67

. . 3 . 14 1 2312 6 58 . . 1 1452 2 89 . 1 1085 . 6 80 .

1 1 0768

0 . . 12 41 0 8970 . . 3 56 0 7211 . 0 7064 0

II-26 210 . 208 9487

200

190

180 . 179 0773

. 173 6887

F . 172 7449 170 i

gu . 168 8779

. 168 7164 re . 167 4025 .

160 166 3190

S2 . 165 8832 . 165 6566 5 .

150 13 . 147 3233 C . 145 9161 . 144 9732 N

140 .

M 140 4702

. 132 2413 .

130 130 1948 pe . 129 1783 . 127 7806 c . 126 2036 t r u 120 m

( . CDC 113 5125 f

1 110 (

ppm

) l

, .

101 8521 100 10

0 M 90 H

z) . 77 2855 . 77 1706

o . 76 9678 80 . f

76 6500 . a 75 4476 . c 74 7528 t

ino . 71 3575 70

. 60 5894 m . 58 2790 y .

c 57 0458 60 .

i 55 4595

n . 54 9447 . 54 6772 Y . 54 3643 9 . 50

52 8466

( . 51 4055 4 . 51 2200 )

. 41 7382 40

. 37 2749 . 34 6695 . 31 6875

30 . 29 4815

. 26 . 8133 21 6111

. 19 0333 20 . 18 7014 . 17 1942 . 15 0383 . 13 4849 10

. 7 7387

II-27 Figure S26. HSQCspectrum (CDCl 3 , 500 MHz) , actinomycinof Y 9 ( 4 )

II-28 Figure S27. HMBCspectrum (CDCl 3 , 500 MHz) , actinomycinof Y 9 ( 4 )

II-29 Figure S28. 1 H, 1 HCOSY spectrum (CDCl 3 , 500 , MHz)actinomycinof Y 9 ( 4 )

II-30 Figure S29. TOCSYspectrum (CDCl 3 , 500 MHz) , actinomycin of Y 9 ( 4 )

II-31 Figure S30. NOESYspectrum (CDCl 3 , 500 MHz) , actinomycin of Y 9 ( 4 )

II-32 Figure S31. (+)- HRESI-MS(positive mode)of actinomycin Y 9 ( 4 )

[ M+ H] + [ M+ N a] +

II-33 11

F i gu

8 .

. 8 3956

5 re

. . 0 99 8 3814 . . 8 2123 S3 1 01 . 8 1973 8

0 2

1 . 7 5999 H . . 2 00 7 5805 7

. .

5 7 3446 N . . 1 22 7 3252 M . 7 2410

R s . .

7 7 0604 0 85

. .

0 7 0442

pe c 6

. t

5 r

u . m 6 1202 . . 1 02 6 0977

6 . . (

6 0487

. 1 00

CDC . 6 0261 f

(

ppm 5

l .

3 5 )

. 40 . 5 2183 2 30 . 5 2043 5 0

. .

0 4 6876

M . 4 6737 . 4 6491 . H 2 95 . 4 6303 . z) 4 1 04

. . .

5 0 95 4

4022

o . . 1 39 4 2905 f

a . 4 3 6302

. c

t . 3 5703 ino . . 0 79 3 5165 . . 3 4914 3 83 3 m .

. 3 4760 .

5 2 45 . 3 4611 y . 3 4487 c

i . 3 0840 n . 3 3 0417

0 Z . 15 52 . 2 8658 p .

. 2 6566

( 4 53 5

2 . .

3 54 . 2 5272 )

. 2 3481 . . 5 76 2 2257 . . 1 47 2 1899 2

0 . . 2 88 1 9263 . 4 88 . 1 7225 . 1 5998 1 .

8 22 5 . 1 4568 . 2 09 . . 1 2348 11 08 . 1 1126 . .

1 7 75 1 0898

. . 16 80 0 8873 . . 7 29 0 7318 . 0 7164 0

II-34 210

200

190

180 . 179 1075

. 173 8947 . 173 4103 .

F 168 9473 170 i . gu 167 6934 . 167 5922 .

re 166 5753 .

160 166 4105 . 166 3340

S3 . 166 1273 3 150 .

13 . 147 3833 .

C 145 7925 . 144 9824 N

140 . 140 4226 M

Rs . 132 9539

130 . 130 2064 pe

. 129 1146 . 127 4042 c . 125 7298 t r 120 u m

( . 113 3657 CDC f 110

(

ppm

l )

3 .

, 102 0577 100

0 M 90

H z) . 77 2830

o 77 1685 80 .

f 76 9651

. 76 6473 a . 75 0842 c

t .

ino 71 5431 70 . 71 3808

m . 59 2388

y . 58 3996 c 60 . 58 2634

i .

n 58 0457

. Z 54 6558

p . 51 4696 50

( 5 )

. 39 3608 40 .

39 2875 . 34 8891 . 34 8473 . 32 1076 30 .

29 2690 . 26 7403 . 24 7641

. 21 5205 20

. 19 0695 . 15 0067 . 14 0717 10

. 7 7441

II-35 Figure S34. HSQCspectrum (CDCl 3 , 500 MHz) , actinomycinof Zp ( 5 )

II-36 Figure S35. HMBCspectrum (CDCl 3 , 500 MHz) , actinomycinof Zp ( 5 )

II-37 Figure S36. 1 H, 1 HCOSY spectrum (CDCl 3 , 500 , MHz)actinomycinof Zp ( 5 )

II-38 Figure S37. (+)- HRESI-MS(positive mode)of actinomycin Zp ( 5 ) [ M+ H] + [ M+ N a] +

II-39

-4 II

1 Figure S38. H NMR spectrum (CD3OD, 500 MHz) of actinomycinY1 Figure S39. 13 C NMR spectrum (CD 3 OD,500 MHz) of actinomycinY 1

II-41 2 II -4

Figure S40. HSQC spectrum (CD3OD, 500 MHz) of actinomycinY1 3 II -4

Figure S41. HMBC spectrum (CD3OD, 500 MHz) of actinomycinY1 Figure S42. 1 H, 1 HCOSY spectrum (CD 3 OD, 500 MHz) of actinomycinY OD,500 MHz) of 1

II-44 Figure S43. TOCSYspectrum (CD 3 OD,500 MHz) of actinomycinY 1

II-45 [M+Na]+

[M+H]+ 6 II -4

Figure S44. (+)-HRESI-MS (positive mode) of actinomycin Y1 7 II -4

1 Figure S45. H NMR spectrum (CDCl3, 500 MHz) of actinomycin Y3 8 II -4

13 Figure S46. C NMR spectrum (CDCl3, 100 MHz) of actinomycin Y3 9 II -4

Figure S47. (+)-HRESI-MS (positive mode) of actinomycin Y3

II-5

1 Figure S48. H NMR spectrum (CDCl3, 400 MHz) of actinomycin Y4 Figure S49. 13 C NMR spectrum (CDCl 3 , 100 MHz) , of actinomycin Y 4

II-51 [M+Na]+ 2 II -5

[M+H]+

Figure S50. (+)-HRESI-MS (positive mode) of actinomycin Y4 Figure S51. 1 H NMR spectrum (CDCl 3 , 400 MHz) , of 6

II-53 4 II -5

13 Figure S52. C NMR spectrum (CDCl3, 100 MHz) of 6 5 II -5

Figure S53. HSQC spectrum (CD3OD, 500 MHz) of 6 6 II -5

Figure S54. HMBC spectrum (CD3OD, 500 MHz) of 6 Figure S55. 1 H, 1 HCOSY spectrum (CD 3 OD,500 MHz) of

II-57 Vita

WENLONG CAI

Department of Pharmaceutical Sciences, University of Kentucky 789 S. Limestone St., Lexington, KY, 40503 (859)-797-4817 [email protected]

Education

 PhD Candidate, Department of Pharmaceutical Sciences (Aug 2010 – present), University of Kentucky, KY Cumulative GPA: 3.7/4.0  Bachelor of Sciences, College of Pharmacy (Aug 2008), Chengdu University of Traditional Chinese Medicine, China Cumulative GPA: 3.3/4.0

Professionals

 Research Assistant, University of Kentucky, KY (Aug 2010 - present)  Research Assistant, Chengdu University of Traditional Chinese Medicine (2008 - 2010)

II-58 Presentations

 Poster presentation, Natural Product Symposium, Department of Pharmaceutical Sciences, University of Kentucky, 2014

 Poster presentation, 3rd Annual Conference of the International Chemical Biology Society, San Francisco, 2014

A Pubilications 1. Wenlong Cai, Anwesha Goswami, Zhaoyong Yang, Xiaodong Liu, Keith D Green, Sandra Barnard-Britson, Satoshi Baba, Masanori Funabashi, Koichi Nonaka, Manjula Sunkara, Andrew J Morris, Anatol P Spork, Christian Ducho, Sylvie Garneau-Tsodikova, Jon S Thorson, and Steven G Van Lanen, “The biosynthesis of capuramycin-type antibiotics: uridine-5'- carboxamide formation and characterization of the A-102395 biosynthetic gene cluster”, J. Biol. Chem., in press

2. Anwesha Goswami, Wenlong Cai, Thomas P. Wyche, Maïa Meurillon, SuzannePeyrottes, Tim S. Bugni, Jurgen Rohr, Steven G. Van Lanen, “Mechanism of the non-heme Fe(II), α- ketoglutarate:uridine-5’-monophosphate dioxygenase involved in nucleoside antibiotic biosynthesis”, J. Am. Chem. Soc., Submitted

3. Xiaodong Liu, Juanjuan Liu, Wenlong Cai, Keith D. Green, Anwesha Goswami, Sylvie Garneau-Tsodikova, Koichi Nonaka, Zhaoyong Yang, Steven G Van Lanen “Dissecting the Mechanisms of Amide Bond Formation for Chemoenzymatic Structural Diversification of Capurmaycin Antibiotics”, Angew. Chem. Int. Ed. , submitted

4. Xiaodong Liu, Anwesha Goswami, Wenlong Cai, Bertolt Gust, and Steven G. Van Lanen, “Biosynthesis of Nucleoside Antibiotics: Characterization of Gene Clusters and Functional and Mechanistic Insight of Key Enzymes”, Nat. Prod. Rep., in preparation

5. Wenlong Cai, et. al. “Crystal Structure of Cpr17, a Promiscuous Phosphotransferase Conferring Self-resistance to Capuramycin Antibiotics” in preparation.

6. Wenlong Cai, et. al. “Identification and Characterization of the Novel V-type Actinomycins Gene Cluster in Streptomyces sp.” J. Nat. Prod., in preparation.