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8703560

Houck, David Renwick

STUDIES ON THE BIOSYNTHESIS OF THE MODIFIED-PEPTIDE ANTIBIOTIC, NOSIHEPTIDE

The Ohio State University Ph.D. 1986

University Microfilms

I nternetionsi!300 N. Zeeb R oad, Ann Arbor, Ml 48106

STUDIES ON THE BIOSYNTHESIS OF THE MODIFIED-PEPTIDE

ANTIBIOTIC, NOSIHEPTIDE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

David Renwick Houck, B.S., M.S. *****

The Ohio State University

1986

Dissertation Committee: Approved by

Heinz G. Floss f ^

Robert M. Mayer

David J. Hart Department of Chemistry To my family

ii ACKNOWLEDGEMENTS

I am very fortunate to have studied with an exceptional scientist such as Dr. Heinz G. Floss. As my academic advisor for five years. Dr. Floss has provided an excellent atmosphere for both my scientific training and my research.

Merck & Co. is acknowledged for granting me a paid, educa tion leave of absence. I would like to thank Paul Keller,

John Beale, and Jonathan Lee for their help with acquisition of the NMR data. Dr. Edith W. Miles was very generous with the gift of tryptophan synthase. Diego Belinzoni and

Rosangela Casati taught me techniques of organic synthesis.

Kay Kampsen was a very efficient typist and ensured that all the deadlines were met. My parents are responsible for starting my academic studies, and Dr. Lawrence Wittle, of

Alma College, first stimulated my interest in biochemistry.

My wife, Allyson, has been incredibly devoted and patient during my preoccupation with science. Allyson's literature searches were invaluable for the completion of this work, as was her moral support.

Ill VITA

April 10, 1956 ...... Born - Detroit, Michigan

May, 1978 ...... B.S., Alma College, Alma, Michigan

September, 1981 ...... M.S., Purdue University West Lafayette, Indiana

October, 1981 to Present ...... Research Biochemist Merck and Co. Inc., Rahway, New Jersey

December, 1984 to Present ...... Recipient of a Merck Grant for Ph.D. studies at the Department of Chemistry, The Ohio State University

PUBLICATIONS

1. Houck, D., Chen, L.-C., Keller, P., Beale, J., Floss, H.G. (1986) "Biosynthesis of Nosiheptide" J . Amer. Chem. Soc., in press. 2. Houck, D.R., Kobayashi, K., Williamson, J.M. and Floss, H.G. (1986) "Stereochemistry of Méthylation in Thienamycin Biosynthesis" J. Amer. Chem. Soc. 108, 5365. 3. Asano, Y., Woodard, R.W., Houck, D.R., Floss, H.G. (1984) Arch. Biochem. Biophvs. 231, 253. 4. Miles, E.W., Houck, D.R. and Floss, H.G. (1982) J. Biol. Chem. 257, 14203. 5. Houck, D.R. and Floss, H.G. (1981) J. Natural Products, 44, 759.

FIELDS OF STUDY

Biochemistry, enzymology and natural products

IV TABLE OF CONTENTS

Page ACKNOWLEDGEMENTS ...... Ü i

VITA ...... iv

LIST OF TABLES ...... V Ü

LIST OF FIGURES ...... v i ü

LIST OF ABBREVIATIONS ...... X Ü

INTRODUCTION ...... 1

RESULTS ...... 7

Fermentation ...... 7 High Performance Liquid Chromatography ...... 10 Nosiheptide Isolation ...... 14 Chloramphenicol Inhibition of Nosiheptide Biosynthesis ...... 16 Incorporation of ^“C-Labeled Amino Acids into Nosiheptide ...... 19 Incorporation of ^®C-Enriched Amino Acids into Nosiheptide ...... 22 Incorporation of [U-^®Ca]Glycerol into Nosi heptide ...... 44

DISCUSSION ...... 62

EXPERIMENTAL SECTION ...... 79

Materials ...... 79 Organism and Fermentation...... 80 Isolation of Nosiheptide ...... 81 Nosiheptide Assay and HPLC ...... 83 Feeding Experiments ...... 84 Preparation of 4-Methyl-[3 ' “C]tryptophan .... 87 Preparation of [2-^®C]Indole ...... 88 Preparation of L-[2,l'-i3C2]Tryptophan ...... 90 ^ ^C-NMR Spectroscopy...... 91

V Table of Contents (continued)

Page APPENDIX ...... 93

LIST OF REFERENCES ...... 99

VI LIST OF TABLES

TABLE Page 1. Effect of Medium Components on Nosi heptide Biosynthesis ...... 11

2. Incorporation of ^“C-labeled amino acids into nosiheptide ...... 20

3. couplings observed in the reverse INEPT spectrum of nosiheptide biosyn thesized from L-[3-i3C]serine...... 31

4. ^^C-NMR assignments and enrichments of nosiheptide derived from ^^C-labeled precursors ...... 43

5. The carbon connectivities observed in the 2D-INADEQUATE spectrum of nosiheptide biosynthesized from [ U - ^ ]glycerol ... 55

6. ^^C-NMR assignments of nosiheptide (40-70 mg/0.4 ml, DMSO-dg, 320°K)...... 61

VI 1 LIST OF FIGURES

FIGURE Page 1. Nosiheptide and the sequence of its residues ...... 2

2. Thiostrepton and the sequence of its residues (top) ...... 3

3. The HPLC profile of a crude extract (tetrahydrofuran) of streptomyces actuosus grown in synthetic medium 13

4. The effect of chloramphenicol on nosi heptide production in synthetic medium. Chloramphenicol was added (10 pg/ml) at 30 h(x) or 54 h (A) or not at all (oscontrol) ...... 18

5. The HPLC profiles of crude nosiheptide- precipitates from fermentations con taining [^“C]tryptophan; labeled at the CHg group (top) or ring-C7a (bottom traces). The fluorescence detector was in series with radioactive flow detector. 21

6. Natural abundance of the ^^C-NMR spectrum of nosiheptide ...... 24

7. An expansion of the ^^C-NMR spectrum of nosiheptide biosynthesized from DL-[3-^^C]cysteine ...... 25

8. The sites of ^^C-enrichemnt (•) in nosi heptide biosynthesized from DL-[3-^^C]cysteine ...... 26

9. An expansion of the ^ ^C-NMR spectrum of nosiheptide biosynthesized from L-[3-i3C]serine. Stars indicate enriched signals ...... 28

Vlll List of Figures (continued)

Figure Page 10. The ^H-NMR spectra of nosiheptide biosyn thesized from L-( 3 - serine. The reverse INEPT sequence was run with t =1.4 msec (top) or x=1.6 msec (middle trace). The ^^C-NMR satellites are bracketed immediately above the main proton signal (bottom trace) ...... 30

11. The coupling observed between pyridine C3 and C4 in the spectrum of nosiheptide biosynthesized from L - [3- i3g ]serine ...... 32

12. The transfer of carbon-13 (A) from L-[3-i3c]serine to nosiheptide ...... 33

13. An expansion of the ^^C-NMR spectrum of nosiheptide biosynthesized from L-[CHa-^^C]methionine. The intense signal at 65.9 ppm corresponds to indole C4'...... 34

14. The ^H-NMR (A) and reverse INEPT (B) spectra of nosiheptide biosynthesized from L-[CHa-^^cImethionine. With a delay of t =1.6 msec, the pulse sequence for B uncovers the ^^C-satellites of the protons at indole C4'...... 35

15. The downfield region of the ^^C-NMR spec trum of nosiheptide biosynthesized from DL-[1-^^C]serine. The stars denote enriched signals ...... 37

16. The transfer of (•) from DL-[1-^®C]- serine to nosiheptide ...... 38

17. The synthesis of L-[2,1'-^^Cj]tryptophan .. 41

18. The ^^C-NMR spectrum of nosiheptide biosynthesized from L-[2,l'-^^Cjl- tryptophan ...... 42

IX List of Figures (continued)

Figure Page 19. A portion of the ^®C-NMR spectrum of nosiheptide biosynthesized from [U-^’Cglglyeroi (top) and a natural abundance spectrum (bottom) ...... 47

19. Continued ...... 48

20. The 2D-INADEQUATE spectrum of nosiheptide biosynthesized from [U-^^Cglglycerol. Coupled signals appear at the same double-quantum frequency (FI, abscissa) and are listed in Table 5 ...... 53

21. An expansion of the 2D-INADEQUATE spectrum (Fig. 20). Coupled thiazole-carbons are bracketed. Note the weak signals for pyr C4-C5 at 4000 Hz in FI ...... 54

22. A schematic representation of intact transfer of neighboring carbons from [U-^^Cg]glycerol. The bars indicate intact transfer of contiguous i^C-labeled carbons ...... 59

23. The structure of Berninamycin and the product of acid hydrolysis, berninamycinic acid ...... 63

24. The assembly of a thiazole ring in nosi heptide. Both precursors are assumed to be part of a peptide chain before ring closure ...... 65

25. The precursors and construction of the pyridine ring in nosiheptide. The three amino-acid precursors are thought to be part of a peptide chain before final modification and "tail to tail" joining of two serine residues ...... 67 List of Figures (continued)

Figure Page 26. The modification of tryptophan to generate the indole moiety in nosiheptide. Activated methionine donates a methyl group at indole C4 after (modified?) tryptophan is incorporated into the structure. The dashed arrow illustrates excision of the a-carbon ...... 72

27. The putative peptide precursor (left) of nosiheptide (right). Tryptophan may originate at the C- or N- terminus of the peptide before modification...... 74

28. ID-INADEQUATE spectrum of nosiheptide biosynthesized from [U-^^C^ ]glycerol 94

29. The 2D-INADEQUATE spectrum of nosiheptide biosynthesized from [U-^^Cj]glycerol .... 95

30. The ^^C-NMR spectrum of nosiheptide biosynthesized from DL-[3-^®C]cysteine.. 96

31. The ^^C-NMR spectrum of nosiheptide biosynthesized from L-[ 3 - serine .... 97

32. The ^^C-NMR spectrum of nosiheptide biosynthesized from DL-[1-^®C3serine ... 98

XI LIST OF ABBREVIATIONS

Common Abbreviations

CPM counts per minuts DOC dicyclohexyIcarbodiimide 2D two-dimensional FVM frozen-vegetative mycelia FifFz frequency dimensions corresponding to ti and tg HPLC high performance liquid chromatography INADEQUATE incredible natural abundance double quantum transfer experiment J coupling constant NMR nuclear magnetic resonance length of the evolution time ^2 acquisition time THF tetrahydrofuran UCi micro-Curie UV ultraviolet

Shorthand designation of the peptide residues and their carbons : but dehydrobutyr ine but C2 carbon-2 of dehydrobutyrine cys cysteine deala dehydroalanine deala-CO carbonyl group of dehydroalanine glu 4-hydroxyglut amate glu C4-C0 two-carbon unit of hydroxyglutamate carbons-4 and -5 ind indolic acid residue ind-CO carbonyl group attached to indole carbon-2 ind C3a-C7a two-carbon unit of indole carbons-3a and -7a pyr pyridine ring pyr C2-C3 two-carbon unit of pyridine carbons-2 and -3 thr threonine thz(l) thiazole number 1 thz(l)-CO carbonyl group attached to carbon-4 of thiazole(l) thz(4)C5-C4-pyr 06 three carbon unit corresponding to carbons-5 and -4 of thiazole(4) and pyridine carbon-6

Xll INTRODUCTION

Nosiheptide (Fig. 1), a metabolite of Streptomyces actuosus, is a modified peptide antibiotic commercially produced by Rhone-Poulenc^ as a growth proraotant for chickens. This antibiotic^, and related compounds, such as thiostrepton*, thiopeptins^, and micrococcins®, form a class of sulfur rich peptides known to interfere with protein synthesis in gram-positive bacteria. In general, their mode of action appears to be inhibition of the EF-G dependent activity in the 508 ribosomal subui\it^. The gram negative bacteria and yeasts are resistant to these antibiotics®. A gene coding for thiostrepton resistance has been cloned and is used as a marker in cloning vectors of Streptomyces (for review see 9). Extensive background information on nosihep tide, including a review of the chemical properties, has recently been given by Chen^®.

The complete structures of nosiheptide^^"^^ and thiostrepton^^ have been determined by degradation studies and X-ray crystallographic analysis. These cyclic peptides are composed of two loops of highly modified amino acid residues including thiazole rings, dehydroalanine, and "W HN NHz Nosiheptide OH

HO HN HN H3O

deala OH thz(5) pyridine

ll(4, thr I but thz(3) indole thz(2)

Figure 1. Nosiheptide and the sequence of its residues. ^0«clo(2ltHO«om»>i

*10121

TIW ») t Atotni Tftflll

T l> j(2 l|

Cl* H Thsin

0*ola(^ I OioiolSI H, W,,

HN' T m ( 4 l

IT h l(l) Aloni

A l0 (2 | I I N III I CM* MO “ M . HO^ iNM

HN, ThflZI CHj

HO'

[SI

Thsin = Thiostreptine residue Q = Quinaldic acid precursor Cys = Thiazoline Thz = Thiazole ring Pip = Piperidine ring

Figure 2. Thiostrepton and the sequence of its residues (top). 4 several oxidized or dehydrogenated amino acids. Nosiheptide is broken down into a number of well-characterized fragments by acid hydrolysis, and an indolic acid fragment under alka line conditons. Although some of the physiochemical proper ties are similar, nosiheptide has several structural fea tures distinct from thiostrepton (Fig. 2) and the thiopeptins. First of all nosiheptide has a 2,3,4-trisubstituted indole moiety in place of the quinaldic acid residue of thiostrepton. The piperidine ring of thiostrepton seems to be the start and finish of both peptide loops, whereas a pyridine ring completes a single loop in nosiheptide; a short loop containing the indole ring extends to a hydroxy glutamate residue of the larger loop and a D-cysteine residue anchors the indolic acid via a thioester bond. The research presented here deals with the biosynthesis of these interesting structures.

To date, the biogenesis of this class of antibiotics has received little attention. This is somewhat surprising because such information would be very useful for the directed biosynthesis of modified structures. The primary objective of this work is to determine the precursors of the individual residues in nosiheptide. This is accomplished by feeding ^**0- and ^®C-labeled substrates to Streptomyces actuosus and analyzing the resulting nosiheptide for isotope 5 incorporation. The information gained from these experi ments sheds light on the construction of the thiostrepton- type antibiotics. In addition, double-quantum NMR analysis of nosiheptide biosynthesized from [U-^®Ca]glycerol is used 16 17 to determined biochemical connectivities ' and make ^^C-

MMR assignments.

There are essentially three technical prerequisites for all biosynthesis studies; they might be termed product yield, product isolation and product analysis. First of all, a system, in this case a fermentation, must be developed for production of the natural product. The system must yield enough compound for the final analysis and must be amenable to the feeding of isotopically labeled sub

strates. Antibiotic fermentations are many times, good examples of nature's cryptic ways, because there exist no exact rules for choosing the growth and fermentation condi tions. Much effort is spent to find the delicate balance of substrates, usually carbon and nitrogen, necessary for

induction of antibiotic production.

Product isolation is the second phase of biosynthesis

studies; the compound of interest must be separated from the

fermentation broth in essentially pure form. The amount and purity of the material required depends on the final method 6 spectrometry and radioactive tracer experiments, chromato graphy (GC or HPLC) of small amounts of crude material can yield enough pure compound (less than 1 umole) for exact analysis. More elaborate purification schemes must be developed for the quantities (more than 10 ymole) of product needed for ^®C-NMR spectroscopy. In the end, there must be a reproducible method for unambiguously determining the sites of precursor incorporation. In the case of nosihep tide, the ^^C-NMR signals needed to be strictly assigned for the analysis of the antibiotic derived from ^®C-enriched precursors.

The technical aspects of this study were not trivial, and much of the time required to complete the work was spent developing the three primary methods. Just recently Chen^^ completed preliminary experiments on the fermentation, bio synthesis, and NMR of nosiheptide. In fact, with only a few exceptions, the ^^C-NMR spectrum was rigorously assigned.

Yet attempts by this author to reproduce the fermentation and isolation procedures failed to yield nosiheptide. Thus, in additon to the biosynthesis experiments, here will be described the development of a defined fermentation medium and an isolation scheme. RESULTS

Fermentation —

Fermentation development was performed on two parallel tracks; reisolating of S. actuosus (ATCC 25421) and optimizing fermentation conditions, such as medium, scale, and temperature. A synthetic, or defined, production medium was desired since it would simplify the feeding experiments as well as the isolation. Crude media, composed of corn steep liquor and soybean meal, contain a high and unknown background of many substrates. This background will dilute isotopically labeled precursors and usually contaminate initial extracts or column fractions. The production medium originally used to screen reisolated colonies of S. actuosus was the progenitor of the medium finally used for feeding experiments (medium C). It contained 0.25% (NH^)gSO,,, but

NagSO^, arginine, and aspartate were not present. With the original culture this medium would support titers of 1 to 10 mg nosiheptide per liter of fermentation broth. The results were never reproducible. The ^^C-NMR analysis required at least 40 mg of pure material in order to obtain inter pretable signal to noise after 10 hours of accumulation.

This fact and the expense of i^C-enriched compounds. 8 especially amino acids, dictated there be a 5- to 10-fold increase in titer.

Culture reisolation of apt antibiotic producing strain is usually a first step towards improving fermentation yield, because the population is many times heterogeneous with respect to antibiotic production. This can be the result of poor storage conditions and/or the techniques used for culture propagation. For instance, the culture of S. actuosus used in early studies had been stored for long periods (months) on solid medium in a refrigerator; the culture probably degenerated after only a few weeks.

Moreover, prior to storage, the culture was transferred several times from one set of agar slants to another; this was a good method for selecting against antibiotic production.

A lyophile tube of ATCC 25421 was used to generate 10 isolated colonies on each of five different solid media

(petri dish). The fifty colonies were picked and trans ferred to the seed medium (B) used for inoculating the production medium described above. After four days in the production phase, the cultures were assayed for nosiheptide

(HPLC). Nine cultures produced no detectable antibiotic, whereas the remaining cultures produced within a range of 9

0.1 to 12 mg/L. The replicate.flasks gave identical results. Therefore, the organism obtained directly from the culture source (ATCC) was heterogeneous for nosiheptide production. Overall, the solid medium A gave the greatest number (three) of "high producers". To prevent degenera tion, the "most productive" culture - E3 - was grown in the seed medium for 36 h and then stored at -60“C in 3 ml aliquots (frozen vegetative mycelia, or FVM). The FVM was used to inoculate the first stage seed medium for all experiments and could be stored (-60*C) for up to one year without noticeable degeneration.

Culture E3 was used to further optimize the fermenta tion conditions. The seed medium described by Chen^® supported very poor growth regardless of the culture source or incubation conditions. After testing several seed media, medium B was found to provide an extremely rich and repro ducible inoculum for the production phase. Improving the defined medium involved the tasks of substituting carbon and nitrogen sources, adding or deleting components and finally testing various concentrations of the components. The experiments were performed in duplicate and the nosiheptide titer in each flask was determined (HPLC) after 3 and 4 days of incubation. Usually, 20 to 25 experiments were run in parallel. Sucrose, maltose, lactose, and glycerol all supported growth, but nosiheptide production was low. At 10 supported growth, but nosiheptide production was low. At levels above 2 g/L, glycerol completely inhibited antibiotic biosynthesis. The twenty (common) amino acids were added individually at 1 to 2 g/liter; most had no effect, yet tryptophan and cysteine both inhibited (repressed?) produc tion. Aspartate and arginine increased the titer by 15%.

The most dramatic effect was observed with ammonium ions.

When the sulfate levels were kept constant, deletion of

(NH^) gSO,, increased production 20-fold to over 200 mg/L.

Raising the phosphate levels also decreases the titer.

Regulation of antibiotic production by ammonia and phosphate 18 is well documented . Table 1 reviews the effect of various medium components on titers. This information is critical for the design of feeding experiments with labeled amino acids.

High Performance Liquid Chromatography

The HPLC method used in this work is the first reported chromatographic assay for nosiheptide. Figure 3 shows the chromatogram of an extract (tetrahydrofuran/hexane) of a cell pellet from 2 ml of whole broth. Initial attempts to separate nosiheptide from broth components by HPLC were \ unsuccessful. Extreme peak tailing, or no retention at all, was observed with conventional mobile phases, such as aqueous mixtures of acetonitrile, methanol, and isopropanol. 11

Table 1. Effect of Medium Components on Nosiheptide Biosynthesis.

concentration nosiheptide Medium component (mMolar) titer (%)

Control* - 100 +(NH„)jSO^ 7.5 25 +Na2HP0^ 7.0 75 +L-serine 10.0 100 +L-cysteine 8.3 15 +L-methionine 12.4 100 +L-tryptophan 1.0 100 +L-tryptophan 10.0 20

* The control is medium C.

Without dioxane or acetic acid present as solvent modifiers, reverse phase chromatography of crude extracts produced an unresolved envelope. The dioxane could be replaced by tetrahydrofuran without loss of resolution, but the peak shape became non-Gaussian. In addition, it was necessary for the sample to be dissolved in mixtures of acetic acid and an organic solvent, usually tetrahydrofuran. For analy tical work a 1:1 mixture of acetic acid and tetrahydrofuran was used, but for semi-preparative separation, 100% acetic acid gave better resolution and peak shape. The separation could be achieved with octadecyl-silane or octylsilane packings only if the bonded phase was end-capped and the nominal particle size was 3-5 ym. 12

The most successful and reproducible H P L C system utilized a Hamilton P R P -1 column and a mobile phase consisting of 40% CH3CN, 0.2% C H 3 C O O H and 20% p-dio%ane in water. The choice of the solvent system was based on the solubility properties of nosiheptide. Injections into methanol/water or acetonitrile/water probably resulted in partial precipitation of the antibiotic on the column; this would lead to peak tailing. The acetic acid increases the solubility of nosiheptide in organic solvents and dioxane is one of the few solvents that dissolves nosiheptide.

At the given detector sensitivity (one relative fluorescence unit = 1 R.F.U.), the detector response is a linear function of nosihetpide concentration within the range of 2 to 300 mg/liter. In theory, if the detector is set to maximum sensitivity (0.001 R.F.U.), then the limit of detection would be on the order of 2 ppb. This is only an estimate and does not take into account that the emitted light has been attenuated by reducing the diameter of the exit aperture. For the nosiheptide peak, the capacity and asymmetry factors are k'=2.3 and a=0.7 respectively. The coefficient of variation for the entire assay (extraction and injection) is equal to 3.5%. Using an ultraviolet- absorbance detector, the column and mobile phase are also 13

c 01 o N 0) WT3

Minutes

ù c o o. 0) m Cl Qi

lO o 01r V ». ü

Minutes

Figure 3. The HPLC profile of a crude extract (tetrahydro furan) of Streptomvces actuosus grown in the synthetic medium. 14 useful for the assay of thiostrepton, actinorhodin and its derivatives^^.

Nosiheptide Isolation

The isolation of nosiheptide, as described by Chen^^, was attempted twice and the yield was determined at each step. Nosiheptide is not detected in clarified broth, but is tenaciously associated with the mycelia. For separation of the cells from the broth, filtration of one liter was a long process (3 hours) whereas centrifugation achieved the same result within 15 min. Extraction of the cells with methylene chloride/isopropanol (2:1), or methylene chloride/ ethanol (4:1), or tetrahydrofuran/hexane (4:1) appears to be very effient, because 85% of the antibiotic is recovered in one step if the cells are treated with 3 volumes of solvent.

The second two solvent mixtures can be used to extract aqueous cell suspensions whereas the first solvent forms a thick emulsion with water. Any attempt to dry the organic extract with NajSO^ or MgSO„ results in a 50% loss of product. The salts may hydrolyze or absorb nosiheptide.

Silica gel chromatography, column or TLC, yields very pure nosiheptide (HPLC, NMR) from crude fermentation extracts. Even so, when essentially pure nosiheptide is 1 5 chromatographed by previously described’methods^®, two compounds are collected and as little as 30% of the total charge is recovered. In addition the silica gel retains fluorescent compounds even after washing with several volumes of ethanol. Possibly, the silica gel partially hydrolyzes nosiheptide.

The solubility properties (or more accurately, insolubility properties) of this peptide antibiotic were, at first, the major problem of most isolation procedures.

Nosiheptide is soluble in dimethylsulfoxide, dimethyl' formamide, and mixtures of CHjClj or CH3CI with ethanol or isopropanol. It is slightly soluble in tetrahydrofuran and dioxane. Microcystalline precipitates are formed when excess petroleum ether (or other hydrocarbons) is added to the tetrahydrofuran extracts from mycelia. When the preci pitate is collected by centrifugation and then dissolved in acetic acid, HPLC shows that the major component is nosi heptide. Precipitation with polar solvents has also been tested: including methanol, acetone, ethyl acetate, ethyl ether, and acetonitrile. The antibiotic is very slightly soluble in ethyl acetate and acetonitrile, so these solvents do not give adequate recoveries (<85%). Methanol is not selective and precipitates a major contaminant found in cell extracts. Three solvent systems have been developed for 16 recovery and purification of nosiheptide. Combining at least one volume of petroleum ether with tetrahydrofuran quantitatively precipitates nosiheptide. Similar results are obtained when one or more volumes of ethyl ether are mixed with solutions of nosiheptide in CHjClj/CHaCHjOH (4:1 or 3:1) or CH3CI/CH3CH2OH (4:1). Recovery of nosiheptide from the NMR solvent, DMSO-dg, can be accomplished by dropping the NMR sample in 100 ml of ethyl ether/acetone

(1:1); in some cases it is necessary to cool the solution

(4“C) overnight. Note that nosiheptide forms a thin film on flasks and tends to bind to the glass. This film can be dissolved in tetrahydrofuran. The solvent partitioning methods appear to provide a faster and more quantitative means of purification than any type of chromatography.

Chloramphenicol Inhibition of Nosiheptide Biosynthesis

Since nosiheptide is a modified peptide, it would be of interest to know how the amino acids are polymerized. Many peptide antibiotics, such as bacitracin and gramicidin S, are known to be biosynthesized on enzyme templates, and not 20 via ribosomal protein synthesis . The two systems for peptide synthesis can be experimentaly distinguished because of the sensitivity of the ribosomal process to numerous antibiotics: chloramphenicol, puromycin, streptomycin, and the tetracyclines. Template systems are not influenced by 17 these antibiotics. Chloramphenicol inhibits the transpep tidase reaction and is inhibitory to gram-positive (10 21 ug/ml) and gram-negative (0.2-5 ug/ml) bacteria . When S. actuosus is exposed to 10 or 20 g/ml chloramphenicol during the first 20 h of fermentation, growth stops and nosiheptide is not produced. After growth is complete (24 to 96 h ) ,

20 ug/ml causes cell lysis. Figure 4 shows the effect of 10 ug/ml chloramphenicol on nosiheptide biosynthesis when the antibiotic is added at 30 or 50 h after inoculation. In both experiments, biosynthesis is halted for approximately

24 h and then recovers at a rate comparable to the control.

Note that chloramphenicol can be inactivated by acetyl 22 23 transferases and hydrolases found in numerous Strepto- myces species. Therefore, these results indicate that nosiheptide biosynthesis is sensitive to the ribosome directed antibiotic, chloramphenicol, and that actuosus can slowly inactivate the antibiotic. The results are not complete, because of the long time periods between data points. Thus, the effect could be indirect; e.g., altering the level of a critical enzyme in the pathway. 150-, 18

130-

110-

4-> —4 30- e (U TD 7 0 - Q. 0) £ o c 5 0 -

10_

20 40 60 80 100 h o u r s

Figure 4. The effect of chloramphenicol on nosiheptide production in synthetic medium. Chloramphenicol was added (10 ug/ml) at 30 h(x) or 54 h (A) or not at all (o=control). 19

Incorporation of ^‘*C-Labeled Amino Acids into Nosiheptide

If the ^^C-NMR spectrum of a natural product has been unambiguously assigned, labeling will give the most specific data on the biosynthesis of a molecule. Yet as an initial probe, ^“C-labeled precursors provide preliminary information to guide the experiments with stable isotopes.

The exact position of radioactive incorporation can be determined by degradation of the molecule into well charac terized fragments, but the labor involved is not rewarded with the pinpoint accuracy of ^®C-NMR spectrosocpy.

Prior work on nosiheptide biosynthesis^® has demon strated L-[U-^'*C] threonine and L-[U-^“C] cysteine to be efficiently incorporated into the antibitoic. Inspection of the structure leads one to conclude that the dehydrobutyrine and the threonine residues are derived from threonine and that cysteine is the direct precursor of the thiazole rings.

This has yet to be confirmed.

Table 2 reviews the recent data obtained from feeding radioactive amino acids to actuosus. Serine is expected to be an effective precursor since it is the progenitor of cysteine and also feeds one-carbon metabolism. One of the possible precursors of the dehydroalanine residue, alanine, does not label the molecule to a significant degree. Table 2. Incorporation of ^“C-labeled amino acids into nosiheptide.

amino acid nosiheptide ÙCi/mmol mMolar UCi/mmol molar specific incorporation(%)

L-1 u - ^“C ]alanine 165 1.0 2 1.4 L-[methyl-Imethionine 130 0.9 24 18.5 L-[U-^"*C] serine 38 1.5 14 37.0 L-phenyl [1-^""C] alanine 1200 0.5 4 0.3 L- [ 2 ' -1 “C ] tryptophan 60 0.5 5 9.0 DL- [ 7a- ] tryptophan 50 0.5 5 9.0 DL-4-methyl-[3'-] 53 1.0 NSNS tryptophan

NS indicates no significant radioactivity above background.

K> o 21

280

210

Oi Id 140 I c o I a . 70 ^

0) w »- 0 4-» u «->0) 01 T3

Minutes

250

k200 , I 150 ' Oi £ lA C o -100 ^ o. lA (U 50 k. k- 0 o

dl TO

Figure 5. The HPLC profiles of crude nosiheptide-precipitates from fermentations containing [ ^**0]tryptophan: labeled at the CHj group (top) or ring-C7a (bottom traces). The fluoresence detector was in series with a radioactive flow detector. 22

Dehydration of serine or cysteine could also form dehydro alanine. The high incorporation of L- [ methyl-i"C] methionine

into nosiheptide suggests that ind C3' and/or ind C4* may arise from méthylations. If both of these substituents are derived from methionine, then the indole moiety can be envisioned as a cyclized phenylalanine residue. This hypothesis is not borne out by the present data; whereas very little of the radioactivity of L-phenyl [l-i"C] alanine ends up in nosiheptide, both ring- and side chain-labeled tryptophan are efficiently incorporated into the structure.

Figure 5 shows the HPLC profile of the nosiheptide isolated from the fermentations containing L-[3'-^“C]- and

DL-[7a-tryptophan. Thus, tryptophan appears to be the precursor to the indolic acid residue.

Incorporation of ^^C-Enriched Amino Acids into Nosiheptide

Streptomvces actuosus was fed a number of i^C-labeled

amino acids to determine unequivocally the origin of the

residues that make up nosiheptide; the experiments included

L-[3-i3c]serine, DL-[l-^^C]serine, L-[methyl-Imethionine,

DL-[3-^^C]cysteine, and L-[2,1'-^^C2]tryptophan. Relative

enrichments were calculated by normalizing the peak

integrals within specific regions of the spectra of natural

abundance and labeled samples. The integrals used as

standards depended on the region and the sample under study; 23 peaks were always referenced to signals of the same carbon type (hybridization, number of protons). The ratio of a normalized integral in the labeled sample to that in the natural abundance sample provided à value for enrich ment. Table 4 reviews the data from the labeling studies and Figure 6 shows the natural abundance ^^C-NMR spectrum of nosiheptide. The complete spectra of the nosiheptide samples from these feeding studies are shown in the Appendix.

From inspection of the structure, cysteine would appear to be the precursor to at least six residues, including the thiazoles, D-cysteine, and possibly dehydroalanine. Cys teine inhibits nosiheptide biosynthesis, so DL-[3-^®C]- cysteine was gradually added to the fermentation over a period of 24 h. Despite this precaution, the titer dropped from a control of 195 mg/1 to about 70 mg/1. Even so, only a brief perusal of the spectrum (Fig. 7) is necessary to observe that all the thiazole rings are enriched at carbon-5. As anticipated, carbon-3 of D-cysteine is also labeled (Fig. 8 and 30). The lack of enrichment at deala C3 indicates that serine is the precursor of dehydro alanine . i 8

60 It

3 5 s ë. 3 9

omi I, k, W w>»ftn> UmA^

I n I rr I rf I r i I I I I I I I I T I I I I I rT I I I I I I I I I I I I I I r[ I n I |-r r n [ iTi I -p I r I I I I I I I I I I I I i i n [ i t t i | i i i i lai I7S 17* I6S 161 ISS 151 MS HI IIS 131 IIS I» IIS III IIS III

Figure 6. Natural abundance ^^C-NMR spectrum of nosiheptide. to 25

T h z (3 ) C5 T h z (2 ) C5 T h z ( l) C5 T h z (4 ) C5

T hz(S ) C5^

In d C5 Ind C3

130 122 120 118 ppm

Figure 7. An expansion of the ^^C-NMR spectrum of nosihep tide biosynthesized from DL-t3-“ C]cysteine. 26

HN NHz HSCH2-CHCO2H OH I NH2

Nosiheptide

HO HN HN NH

Figure 8. The sites of “ C-enrichemnt (•) in nosiheptide biosynthesized from DL-[3-“ C]cysteine. 27

L-Serine, enriched with at carbon-3, is incor porated into nosiheptide as shown in Figure 12. The greatest enrichment is found in deala C3; therefore, dehy dration of serine must form the dehydroalanine residue.

Since cysteine is biosynthesized from serine one would expect to observe labeling of all the residues derived from cysteine. Integration of the signals between 115 and 130 ppm (Fig. 9) provides evidence for some enrichment at thiazole(1-5) C5. Yet, the normalized integral of cysteine

C3 is unchanged in the Icdseled sample. In addition, lines at 127.1 and 151.5 ppm have peak areas close to that of deala C3. These facts, and the complexity of the downfield spectrum, make peak integrals unconvincing criteria for enrichment; the possibility still exists for NMR misassign- ments or contamination of the isolated nosiheptide.

A NMR technique known as reverse INEPT^^ can unequi vocally determine enrichments if the ^H-NMR spectrum has been rigorously assigned. The method basically allows observation of the satellites of proton signals with

suppression of the intense center line. In the usual 25 proton-carbon INEPT experiment , a polarization transfer

from to enhances the sensitivity of the respective carbon lines in the ^^C-NMR spectrum. The spin populations 28

DealaCS *

PyrC3 PyrC4

* T h z (l)C 5 I *Thz(3)C5 A * T h z(2 )C 5

150 145 140 135 130 125 120 115 110 105 ppm

Figuré 9. An expansion of the ^®C-NMR spectrum of nosihep tide biosynthesized from L-[3-^®C]serine. Stars indicate enriched signals. 29 of the protons and the attached carbons are interchanged and since is the more sensitive nucleus, an enhanced absorp tion or emission is observed for the proton satellites of the carbon signal. As the term implies, the roles of the two nuclei are switched in reverse INEPT; antiphased satellites are seen in the proton spectrum and the main proton line is suppressed. For small quantities of large molecules, like nosiheptide, the polarization transfer from carbon to proton would usually be unobservable due to the low natural abundance of Therefore, only protons attached to a i^C-enriched carbons will be detected when this technique is applied to nosiheptide.

The results of the reverse INEPT experiment with the nosiheptide biosynthesized from L-[ 3 - serine (Fig. 10 and

Table 3) clearly reveals the enrichment of deala, C3, pyr C4, and C5 of all the thiazoles. The success of this NMR experiment is very dependent on the value chosen for t , the time that the vectors are allowed to process between pulses. If T is not set close to l/(4J^jj), then the signals may not be observed. Thus, the experiment cannot prove that a particular carbon is not enriched. With the two t values chosen in Figure 10, the satellites for the protons on indole C3 and C4 were not detected. Even so, the integrals of those firmly-assigned signals show Thiazole H5

H3t Deala H3c

8 6 ppm

Figure 10. The *^H-NMR spectra of nosiheptide biosynthesized from L-[3-i3C]serine. The reverse INEPT sequence was run with x=1.4 msec (top) or x=1.6 msec (middle trace). The ^^C-NMR satellites are bracketed immediately above the main proton signal (bottom trace). 31

Table 3. couplings observed in the reverse INEPT spectrum of nosiheptide biosynthesized from L_[3_i3c]serine.

Proton Assignment PPM Jc h (h z )

Deala H3c 5.76 165 Deala H3t 6.37 165 Pyr H4 7.82 163 Thz(4) H5 7.88 194 Thz(2) H5 8.16 198 Thz(3) H5 8.30 193 Thz(5) H5 8.55 191 Thz(l) H5 8.65 191

significant enrichment (Fig. 31 and Table 4). Rather surprisingly, pyridine carbons 3 and 4 are enriched to an extent that they show coupling (Fig. 11). The satellites represent approximately 16% of the total peak areas. Prior to this experiment the signal at 172.6 ppm was assigned to pyr C3; the coupling observed in this experiment proves that the line at 151 ppm is pyr C3. The 6-carbon of serine can be channeled into many metabolic pathways, so these data alone do not point to any specific precursor(s) of the pyridine ring.

The incorporation of L-[3-^^C]serine into the methyl

(C3') and methylene (C4') groups attached to the indole ring suggests that they may be derived from the one-carbon pool.

Yet, L-[methyl"Imethionine only labels the CHj-O- group attached to C4- (Fig. 13). Using cys C3 as the reference 32

Pyr C3 Pyr C4

66Hz> 66Hz>

J h z ( 5 ) C5

T h z ( l) C5 T h z ( l) C4

T h z(5 ) C4

“T" 151 150 127 126 ppm ppm

Figure 11. The coupling observed between pyridine C3 and C4 in the spectrum of nosiheptide biosynthesized from L-[ 3-“ C] serine. 33

HN NHz HOCHz-CHCOzH I lilHz OH slA

Nosiheptide

HsC

HO HN HN NH HsC HaC

Figure 12. The transfer of carbon-13(A) from L_[3_ i3C]serine to nosiheptide. 34

J L ' I ' I 1-J-. -j-T )■ 1 -j-ry-j 1-1 1. 1 j 1 1 < 66 64 62 €0 56 56 54 52 50 46 46 PPM

Figure 13. An expansion of the ^^C-NMR spectrum of nosihep tide biosynthesized from L-[CH3~^®CImethionine. The intense signal at 65.9 ppm corresponds to indole C4*. 35

156 Hz

156Hz

Indole H4*

Indole H4'

X X ' I ' XXX X X X XXX j— L 6 0 0 5.80 5.60 5.40 5.20 5.00 ppm

Figure 14. The ^H-NMR (A) and reverse INEPT (B) spectra of nosiheptide biosynthesized from L-[CH,-^]methionine. With a delay of x=1.6 msec, the pulse sequence for B uncovers the i*C-satellites of the protons at indole C4'. 36 peak, the normalized Integral of the 65.9 ppm signal shows an enrichment of 28%. The reverse INEPT spectrum (Fig. 14) of the sample confirms the site of enrichment to be the indole-CHg-O- group. At this point, there still is a small possibility that the indole-CHg group is derived from the tetrahydrofolate pool of one-carbon units. Later results of this study rule out this hypothesis.

To establish the significance of the results with

L-[3-^®C]serine, S. actuosus was fed DL-[ 1 -^^C]serine. The

^®C-NMR spectrum of the isolated nosiheptide shows enrich ment of ten signals between 136 and 182 ppm (Figs. 15 and

16). Again the greatest enrichment from serine is found in the dehydroalanine residue. All the carbons corresponding to the carboxyl group of a cysteine are also labeled; thiazole-CO, thz(4) C2, and pyrC6. In contrast with L-[3-

^®C]serine, L-[l-^®C]serine is incorporated into the D- cysteine residue. The biosynthesis of D-cysteine would be expected to involve a racemase because of the known pathway for converting L-serine to L-cysteine. Contrary to a biochemist's intuition, the results from feeding DL- and L- serine suggest that D-cysteine is effectively formed from

D- but not L-serine (i.e.. not via L-cysteine). Such a process would require an additional set of enzymes catalyzing the formation of these D- amino acids. 37

D eala-CO *

Thz-CO T h z ( l) * Ind-CO C2 * T h z (5 ) C2 * T h z (4 ) C2 *

Pyr C6 *

Glu-CO £ I- Pyr C3 J 11 UJILJ n I I I I I I I r I I " [ ■ i- r i r | n " I I I I I I I I 1 I 1 j I I I I j I I I 180 175 170 165 160 155 150 145 ppm

Figure 15. The downfield region of the ^®C-NMR spectrum of nosiheptide biosynthesized from DL-[l-^®c]serine. The stars denote enriched signals. 38

HN NH2 HOCH2CH-CO2H OH NH2 I

Nosiheptide

H3C

HO HN NH H3O H3C

Figure 16. The transfer of ^®C(*) from DL-[1-^®C]serine to nosiheptide. 39

DL-[1-^®C]Serine is effectively incorporated into C2 of both thiazole 1 and 5. This result, together with the labeling of pyr C3 and pyr C4 of L-[3-^®C]serine, indicate that two serine units form carbon atoms 2, 3, 4, and 5 of the pyridine ring. The carboxyl of a dehydrocysteine (thz no. 4) residue contributes to form pyr C6.

A very interesting result is the labeling of the carbonyl group attached to indole C2 by L-[1-^®C]serine.

This is also an important result because it adds to the evidence that tryptophan is the direct progenitor of the indolic acid moiety. At this point the following is known:

Both [7a-^“C]- and [CHj-^^C 3 tryptophan are efficiently incorporated into nosiheptide. Moreover, the methyl group at indole C3 is not derived from an activated methionine, yet it is labeled by L-[ 3 - serine. Thus, since the side 26 27 chain of tryptophan is derived from L-serine ' one could postulate that a cyclization of tryptophan forms the 2,3 substituted indole ring; the carboxyl becomes connected to

C2 of indole, followed by excision of the a-carbon and the attached amino group.

To test the above hypothesis L-[2,l'-^^Cjltryptophan was synthesized for feeding experiments, and some comment should be made on the route used to make this isotopically 40 enriched amino acid (Fig. 17). The strategy was to condense

[2-^®C]indole and L-[1-^®C]serine to make tryptophan in a reaction catalyzed by tryptophan synthase. This route has the advantage of producing and using only the L isomers of product and substrate respectively. The enzyme reaction proved to be a very efficient method for making labeled tryptophan; over 110 mg was produced and this represented an isolated yield of 88%. With the amount of enzyme used for the reaction, at least another 110 mg could have been made, because the enzyme remains active for 2-3 days under the given conditions.

The production of nosiheptide over the course of the feeding experiment with L-r2,1'-^^Cj]tryptophan was on the order of 48 mg/L. The material (35 mg) isolated from the fermentation had to be combined with authentic nosiheptide prior to final precipitation. The reason for the four-fold drop in titer during this critical experiment remains unclear. Previous studies have shown that 1 mM tryptophan had no effect on nosiheptide production.

Despite the technical difficulties, the ^®C-NMR (Fig.

18) of nosiheptide biosynthesized from L-[2 ,1 '-^®C2]- tryptophan clearly shows a pair of coupled triplets: one centered at the signal of indole-C2 (130.40 ppm) and one DL-[l-'^C] DCC * NaH/HCOOK HCOOH f y a: atNCHO o-toluidine •N Tryptophon " 305® Synthase

N H g

Figure 17. The synthesis of L-[2,1'-“ C2]tryptophan. Ind—CO

75.5Hz > Ind C2

75.5H z>

CHS

182 ppm 130 ppm

I I I j I T T I I f I I I I I I I I I I I I I [ I I I I I I I I { I I r r I I I I I I T T - f 180 170 160 150 140 130 120 110 ppm

Figure 18. The ^®C-NMR spectrum of nosiheptide biosynthe sized from L-[2,l'-i3cjtryptophan. •b. to Table 4. ^^C-NMR assignments and enrichments in nosiheptide derived from i^C-labeled precursors. enrichments*, % (J^^) derived from

chem assignment DL-C3-13C]- L-[3-13c ]- DL-E1-13C]- shift cysteine serine serine ppm

12.23 Indole-CH3(C-3 ') 2.8 29.49 CysC3 7.2 65.90 Indole-CH2-0 (C-4 ') 3.4 103.60 DealaC3 6.7 119.98 Thz(4)C5 9.5 2.2 124.45 Thz(2)C5 9.9 2.9 125.25 Thz(3)C5 9.9 2.8 125.98 Thz(l)C5 8.1 2.4 126.80 Thz(5)C5 8.3 2.8 127.12 PyrC4 3.3 (66 Hz) 150.80 PyrC3 3.1 (66 Hz) 142.52 PyrC6 3.6 158.20 Thz-CO 3.0 159.45 Thz-CO 2.4 159.60 Thz-CO 4.0 159.80 Thz-CO 3.9 163.85 Thz(l)C2 6.3 165.00 Deala-CO 5.4 167.10 Thz(5)C2 4.2 168.98 Thz(4)C2 3.4 181.60 Indole-C0(C-2') 6.2

Signals of the other thirty carbons were not significantly enriched. W 44 at the resonance of the thioester carbonyl (181.6 ppm).

Most interestingly, the areas of the satellites vs the total signal areas indicate that the relative enrichment of ind-CO and ind C2 is the same as the starting material, L-

[ 2 , 1 ' ] tryptophan. The satellites are 90% of the ind C2 signal and about 50% for ind-CO; thus 90% of the species enriched at ind 02 are also enriched at ind-CO and 50% of the species enriched at ind-CO are simultaneously enriched at ind C2. This experiment proves that a curious intra molecular rearrangement of tryptophan forms the indole moiety of nosiheptide. Note, 4-methyl-[CHj-^^C]tryptophan is not incorporated into nosiheptide (Table 2). Therefore, the méthylation at indole C4 must occur after typtophan is rearranged and/or attached to the peptide.

Incorporation of [U-^^C,3Glycerol into Nosiheptide

When two adjacent carbons are both enriched with i^C, the coupling of the NMR signals can serve as an assignment critérium, and at the same time solve a biosynthetic problen if the enrichment is the result of a biochemical process.

Labeling of a single carbon in a natural product, from a ^®C enriched precursor, cannot be used to assign the ^®C signal without making assumptions on the biosynthesis of the molecule; that is unless the biosynthetic pathway has been proved beyond a shadow of a doubt. Thus the ^^C-NMR 45 assignments must be unequivocal before biochemical informa tion can be gleaned from single enrichments. The coupling information provided by intact incorporation of di- or trilabeled carbon units is independent of the biochemistry.

Yet, the level of intact incorporation, relative to random labeling from metabolic scrambling, is indicative of the pathways leading to the natural product. For these reasons,

[l,2-^^Cz]acetate^^, [U-^^CgJglycerol^®, and [U-^^Cg]- glucose^,17,30,51,52,53 ^ave become important tools for solving complex biosynthesis problems. Since nosiheptide is in part, constructed of eight amino acids of the serine family, [U - 3]glycerol has been fed to S. actuosus. and the ^^C-NMR analysis of the enriched antibiotic is presented here (Figs. 19-22, 28, and 29).

Before addressing the nosiheptide problem specifically, it is pertinent at this point to review the coupling patterns that can result from feeding [U-^^Calglycerol.

First of all, the center uncoupled line of a carbon resonance will always be observed if the enrichment of the substrate, in this case glycerol, is not 100% at each site and/or as long as some of the bonds are broken by intervening metabolic processes. Thus incor poration of a two carbon unit will yield a pair of triplets with outerlines separated by If three contiguous 46 the expected coupling of the center carbon (B) would be a triplet with the outer lines separated by when

Jj^-JgC. The signals for the flanking carbons (A and C) will again be triplets ( and Jg^). If does not approximate Jg^, then the central carbon will be observed as a quintet: superimposed over Jg^. The pattern for the middle carbon can be even more complex when the population of molecules contains some trilabed (A-B-C) and dilabeled species (A-B and/or B-C). In such cases as many as nine 30 lines could be observed . In a trilabeled species, two bonding coupling can split the outerlines of the A and C triplets by about 5 Hz, but many times this is not resolvable. Second order effects of strongly coupled system can yield an almost uninterpretable set of lines^®.

Not surprisingly, nosiheptide biosynthesized from

[ U - ^ ]glycerol yields a very complex spectrum, containing a series of overlapping multiplets in the downfield region

(Fig. 19). Unfortunately, since glycerol is a central meta bolite, many products of the fermentation other than nosi heptide will be enriched with carbon-13; this is observed as impurities in the sample at 30, 34, 60 to 62, and 70 ppm.

Even so, these do not interfere with the interpretation of the data. There are a few observations I 'l ] I I I I I I I I I I r r I I I I T I T I I I I I I I I I I I I T 1 I I I I I I I I I I I I I I I I I I ITT I I I I I I I I I I I I I I I I I I I I I I te s 175 178 165 168 155 158 1*5 1*8 135 138 125 128 115 118 185 ppm

Figure 19. A portion of the ^^C-NMR spectrum of nosiheptide biosynthesized from [U-^^C]glycerol (top) and a natural abundance spectrum (bottom). ir1

■.‘-iyw>Vk*eV^ "««w4 r »M»«»w v . i L

Y I I I I I I I I I I I I I I I I I IT I I i-r If I I I I [-1-1 I I |-r-TT I I I n I I I II I I I I I I ( I I i~r 70 65 60 55 50 95 <0 35 30 25 20 IS ppm

•i^ Figure 19. Continued. 00 49

that can be made directly from inspection of this ^®C-NMR

spectrum. First of all, this experiment confirms the results obtained with doubly labeled tryptophan, because ind-CO (181.6 ppm) and ind 0 3 ’ (CH3 at 12.2 ppm) are sin glets. The proposed rearrangement of tryptophan would essentially carve out the central carbon of a unit, leaving two isolated enrichments at ind-CO and ind

C3'. No statistical coupling is observed at these sites.

Secondly, intact glycerol units appear to be transferred to residues derived from cysteine. The signal of cys C2 shows a five line pattern of a doubly coupled carbon. Moreover, the line widths (8 Hz) of the satellites of pyr C6 (142 pm) and thz(4) C5 (119 ppm) indicate two bond coupling of a trilabeled species. The satellites of the thiazole-carbonyl groups (159 ppm) are also split (7 Hz) due to two bond coupling. There is obviously much more information to be gained from this sample, but the complexity of the spectrum and some assignment ambiguities prevent further interpreta tion of the single quantum ^^C spectrum. In addition, coupling constants are of litle help in the assignment of specific coupled pairs, because many of the satellites are obscured by overlapping and moreover the magnitudes of are very similar for the various coupled systems. For instance, the region between 164 and 172 ppm contains seven triplets with splittings of 6 8 , 65, 78, 67, 63, 57, and 62 50

Hz, respectively. Therefore, a double-quantum NMR experi- 32 33 ment, know as INADEQUATE ' , has been used to determine the carbon-carbon connectivities in nosiheptide biosynthe sized from [ U - ^ ]glycerol.

32 The INADEQUATE experiment, developed by Bax et. al is based on the phenomenon of double quantum coherence. It essentially allows detection of coupled species while masking the strong signals of noncoupled carbons. Since only the coupled carbon spins can generate double-quantum signals, the pulse sequence produces a ^^C-NMR spectrum exclusively composed of satellites. Thus, the technique has become very important for the detailed analysis of natural products biosynthesized from multiply ^^C-enriched IG 17 31 precur-sors ' ' . h number of antiphased doublets in the

INADEQUATE spectrum of nosiheptide (Fig. 28, Appendix), immediately demonstrate a few connectivities. For instance the signals at 172 and 66 ppm are coupled (J^g = 62 Hz) as are 170 and 45 ppm (57 Hz). Yet the complexity in the regions of thiazole C5 (125 ppm), C4 (148 ppm), and the attached carbonyls (159 ppm) prevents direct analysis of their connectivities. Moreover, not all the satellites are detected in this single experiment because the time allowed for coherence development, t, is based on an estimated value for the average (t = 1/(4 • 51

The 2D-INADEQUATE technique^^ can detect the signals at

all the double quantum frequencies by incrementing the delay

time following the generation of double quantum coherence.

The information thus obtained is a function of both a delay

time, ti, and the acquisition time, tj. After Fourier

transformation, signals will appear on a two-dimensional

contour map at points determined by the chemical shift (F2,

ordinate) and the double quantum frequency of a coupled pair

(FI, abscissa). Carbons sharing the same FI frequency at

points equidistant from the center of gravity (FI = 2 x F2)

are necessarily connected; and carbons giving a signal at

more than one FI must be connected to more than one carbon.

In addition, the coupled carbons will give signals at a

double quantum frequency equal to the sum of their chemical

shifts, relative to the transmitter. Since the frequency of

double quantum coherence (FI) provides the proof, the actual

value of is not required to determine the connec

tivities. The INADEQUATE technique is inherently insensi

tive and for the nosiheptide case, only ^^C-enriched pairs will be detected. Since the carbon-13 enrichment of nosi

heptide is a result of a biochemical transfer of carbon from

[U-^^CjJglycerol, the technique determines both structural

and biochemical connectivities^®. Therefore the

2D-INADEQUATE experiment can prove assignments in the 52

^^C-NMR spectrum as well as uncover the pathways involved in the construction of a natural product.

The results of the 2D-INADEQUATE experiment with nosiheptide biosynthesized from [U-^^C^ ]glycerol is pre sented as a contour plot (Figs. 20, 21 and 29) and in tabular form (Table 5). Only one half of the FI axis is shown in Figure 20. All but one of the coupled pairs in

Table 5 are observed in the positive half of FI; cys C2 (49 ppm) and cys C3 (29.5 ppm) are detected at -9.5 KHz (Fig.

29).

The ^^C-NMR assignments of nosiheptide, made by Depaire 34 et al. , were largely based on multiplicity and chemical shift theory. A more rigorous study by Chen^® utilized two- dimensional NMR techniques to assign unequivocally all the proton bearing carbons and many of the quaternary carbons.

Some of the assignments have been corrected here as a result of the coupling information obtained from feeding L-

[3-i3C]serine, L-[2,l'-^®Cj]tryptophan, and [U-^^CjJgly- cerol; these include pyr C3, glu-CO, ind C4, ind C3a, ind-

CO, and thr-CO. Since the assignments for protonated carbons are firm, they can be used to identify neighboring carbons in the 2D-INADEQUATE spectrum. For instance, glu C4

(66.4 ppm) is connected to a carbon resonating at 172.6 ppm, there can be no doubt that this downfield signal corresponds 53

-2

- 4

-6

-8

-10

T r n — '— r T T T 1 -- 160 140 120 100 80 60 40 ppm

Figure 20. The 2D-INADEQUATE spectrum of nosiheptide biosynthesized from [U-^Cj]glycerol. Coupled signals appear at the same double-quantum frequency (FI, abscissa) and are listed in Table 5. ■4000

■5000

■ 6000

•7000

8000 Hz

170 160 150 140 130 120 ppm

Figure 21. An expansion of the 2D-INADEQUATE spectrum (Fig. 20). Coupled thiazole-carbons are bracketed. Note the weak signals for pyr C4-C5 at 4000 Hz in FI.

oi ■t^ 55

Table 5. The carbon connectivities observed in the 2D-INADEQUATE spectrum of nosiheptide biosynthe sized from [U-^®C3]glycerol. coupled pair Jcc'Hz F2,ppm<— >ppm FI,kHz glu-CO<— >glu C4 62 172.6<-->66.4 2.400 thz (3)C2<— >glu C2 57 170.0<— >45.1 0.800 thr-CO<— >thr C2 57 167.7<— >56.6 1.500 thz(2) C2<— >but C2 78 166.3<— >129.3 6.800 thz-CO<— >thz C4* 74 159<-““>148 7.800 thz C4<— >thz C5* 58 148<— ->125 5.200 thz(4) C2<— >cys C2 63 168.9<— >49.0 1.000 cys C2<— >cys C3 56 49.0<— >29.5 9.500 deala-CO<— >deala C2 65 165.0<— >134.3 7.200 deala C2<— >deala C3 75 134.3<— >103.6 2.500 thz(5) C2<— >pyr C2 67 167. K- - > 1 3 5 .0 7.300 thz(l) C2<— >pyr C5 68 163.9<— >129.9 6.600 pyr C4<— >pyr C5 ND 127.1<— >129.9 3.900 ind C7a<— >ind C3a 55 137.6<— >124.7 4.300

* For simplicity, the connectivities within the individual thiazoles are not specified. The signals appear in clusters at the chemical shifts listed above. ND, not determined. 56 to the glutamate carbonyl group. Originally this signal was assigned to hydroxyl-bearing pyr C4^^. The double quantum spectrum also yielded the assignment of thr-CO, because of the connectivity with thr C2. The assignment of ind C3a

(124.7 ppm) is based on its connectivity to ind C7a (137.6 ppm). As for ind C7a, its assignment can be accepted because it shows a long range correlation with the proton at indole 06^® and it agrees with the extensive ^ ^C-NMR data on indole alkaloids^^.

The strongest signals in the 2D-INADEQUATE spectrum are the glu C4-C0 and thz(3) C2-glu 02 pairs; these correspond to glutamic acid C1-C2 and 04-05, respectively, because degradation of [U-^^0 3 ]glycerol, via glycolysis, to a

[1,2-^®0 2 Jacetate labels a-ketoglutarate at 04-05 after a half turn of the Krebs cycle, and 01-02 of a-ketoglutarate after a complete turn. Incorporation of two-carbon units are also observed at thr 02-00 and but 02-thz(2) 02. The connectivity of carbons 1 and 2 of threonine is expected since threonine is biosynthesized from aspartate (via oxal- acetate). The same labeling pattern at but 02-thz(2) 02 provides evidence that dehydration of threonine forms the dehydrobutyrine residue. Another doubly labeled piece is indole 03a-07a. This can be explained by the fact that carbons 2 and 3 of phosphoenol pyruvate are converted, via the shikiraate pathway, to carbons 3a and 7a of tryptophan. 57

Only a few biochemical reactions are required to convert glycerol to the precursor of serine and cysteine,

3-phosphoglyceric acid. Thus it is no surprise to find several triply labeled species in the nosiheptide isolated from the fermentation of [ U - ^ ]glycerol. Three-carbon units have been incorporated, without disruption of the i3c_i3c-i3c bonds, into the thiazole rings, and the D- cysteine residue. The thiazoles show two-bond coupling

(Fig. 19, 7Hz) in the single quantum ^^C-NMR spectrum. The two-bond coupling of thz-CO and 05 is also apparent in the

2D-INADEQUATE spectrum; the satellites for thz-CO appear at double quantum frequencies with thz-C5 and thz-C4. In addition, carbon-2 of D-cysteine is doubly coupled as is indicated by the appearance of a four-line pattern at two double quantum frequencies: with thz(4) 02 and cys 03. The corresponding connectivities for thiazole(4) 05-04-pyr 06 are not observed in the 2D-INADEQUATE spectrum because their peak heights are below the minimum intensity of the contour plot. As mentioned previously, the single quantum ^^0 spectrum shows there is two-bond coupling between thz(4) 05 and pyr 06, and this provides the proof of an intact three- carbon unit at thiazole-4.

Interestingly, residues derived from cysteine show a greater amount of intact incorporation of units 58

than a residue formed directly from serine, namely dehydro

alanine. This is made obvious by the relative signal inten

sities in the 2D-INADEQUATE spectrum and by the areas of the

satellites versus the total signal areas. For instance, the

satellites for deala C3 (103 ppm) are 30% of the total area

while for thz(4) C5 (119 ppm) the satellites are 67% of the

total signal area; the relative peak areas (total) for deala

C3 versus thz(4) C5 are 1:1.2 in the natural abundance

spectrum and 1:1.3 in the labeled sample. Therefore, the

total carbon-13 enrichment is the same at both sites, but in

terms of intact units, the thiazole rings show

twice the enrichment of dehydroalanine. Keeping in mind .

that serine is the progenitor of cysteine, just the opposite

is anticipated because the dehydroalanine residue shows the

greatest amount of enrichment from ^^C-labeled serines

(Table 4). In addition, the signals for deala C2-C3 are very weak in the 2D-INADEQUATE spectrum. The incorporation of three-carbons units into the corresponding pieces of the pyridine ring is also lower than expected; from the results with L-[3-^^C]- and DL-El-'-^C]serine, three-carbon connec

tivities for pyr C3-C2-thz(5) C2 and pyr C4-C5-thz(l) C2 are

anticipated. The coupling of thz(5) C2 to pyr C2 is readily

apparent at FI = 7.3 KHz but the connectivity of pyr C2-C3 59

"WHN NHe ÇHeOH CHzOH OH CHzOH

Nosiheptide HsC

HO HN NH HsC HsC

Figure 22. A schematic representation of intact transfer of neighboring carbons from [U-^®Ca]glycerol. The bars indicate intact transfer of contiguous i*C-labeled carbons. 60

is not observed in the contour plot. Signals for the pyr

C4-C5 and pyr-thz(l) C2 pairs are present but only one

satellite per signal is detected. Slice plots at 3.9, 6 .6 ,

7.3, and 6.1 KHz in FI may show more detail of the enrich ment and couplings in the pyridine ring. This has yet to be

completed. 61

Table 6 . ^^C-NMR assignments o f .nosiheptide (40-70 mg/0.4 ml, DMSO-dg, 320“K).

Assignment ppm Assignment ppm

Ind-CHgtCS') 12.23 Ind 02 130.40 But-CHa 13.50 Deala 02 134.26 Thr-CHa 18.25 Pyr 02 135.00 Cys C3 29.49 Ind 07a 137.60 Glu C3 37.60 Pyr 06 142.52 Glu C2 45.15 Thz(2) 04 147.62 Cys 02 49.05 Thz(3) 04 148.70 Thr 02 56.57 Thz(5) 04 149.57 Ind-0H2O(04*) 65.90 Thz(l) 04 149.83 Glu 04 66.40 Pyr 03 150.80 Thr 03 66.50 Thz(4) 04 153.10 Deala 03 103.60 Thz-00 158.20 Ind 07 114.40 ThZ-00 159.45 Ind 03 118.35 Thz-00 159.60 Thz(4) 05 119.98 Thz-00 159.80 Ind 05 123.23 Thz(l) 02 163.85 Thz(2) 05 124.45 Deala-00 165.00 Ind 03a 124.70 Thz(2) 02 166.32 Ind 06 124.91 Thz(5) 02 167.10 Thz(3) 05 125.25 Thr-00 167.69 Thz (1) 05 125.98 Thz(4) 02 168.98 Thz (5) 05 126.80 Thz(3) 02 170.04 Pyr 04 127.12 Glu-00 172.62 But 03 128.85 Ind-00 181.80 Ind 04 129.20 But 02 129.27 Pyr 05 129.90 DISCUSSION

It is useful to consider the work of Pearce and Rine- 36 37 hart ' on the biosynthesis of a related antibiotic, berninamycin (Fig. 23). This cyclic peptide is composed of ten residues, including two 5-methyloxazole rings, five dehydroalanines, and a dehydrobutyrine residue. The producing organism, Streptomyces bernesis, was incubated with i*C-labeled amino acids and the resulting berninamycin was degraded to berninamycinic acid (Fig. 23) and to pyruvic acid, the isolated form of dehydroalanine residues. Serine was discovered to be the most effective precursor of berninamycin and pyruvic acid. This was the first evidence that dehydration of hydroxyl-bearing amino acids forms the unsaturated residues in these modified peptides. The incor poration of i^C-labeled serine into nosiheptide clearly demonstrates that the dehydroalanine is derived from serine.

The labeling patterns observed in the threonine and dehydro butyrine residues from [U-^^C^]glycerol supports the general hypothesis on the conversion of hydroxy-aunino acids to dehydro-amino acids.

62 63 Berninamycyl

0*ala NH ^CH Dtola

Htf Dtala ÇH, Thr I I I

i " N ^ O Ox-A H Ox-B C-NH NH-CO CHj H,C CO N a A ÇH' " C 0 - ( ^ " Cb "'CHi CH,-C-OH CH* CH,

Hyvol<---Deala<— Deala

6 /VHCI 110", 18 hr

^ V ti^ cooh OH

Figure 23. The structure of Berninamycin and the product of acid hydrolysis, berninamycinic acid. 64

When [U-^‘‘C]cysteine was fed to S. bernesis, the molar-

specific radioactivities of berninamycin and its degradation product, berninamycinic acid, were equivalent. This suggests that the thiazole part of the heterocyclic system is derived from cysteine. The efficient incorporation of

[ ^**C]cysteine into nosiheptide has been reported by Chen^®, and as shown here [3-^^C]cysteine specifically labels carbon-5 of the thiazole rings into nosiheptide. Therefore, cysteine contributes the sulfur, nitrogen, C5, C4, and the attached carbonyl group while another amino acid donates a carboxyl group to form thiazole C2 (Fig. 24). From this, one anticipates that the 5-methyloxazole rings in berninamycin are derived from threonine.

The mechanism of thiazole formation still needs to be examined. Thiostrepton (Fig. 2), produced by S. azureus. contains a 4,5-dihydrothiazole ring with R configuration at

C4. This could be either a direct product of a cyclization

(D-cys), or a product from reduction of a pre-formed thiazole ring. A feeding experiment with [3-^®C,*Hj]cysteine would eliminate one of the alternatives; for instance, if only one deuterium is retained at C-5, the cysteine must be dehydrogenated before formation of the 4,5-dihydeothia- zole. Also unknown is which enantiotopic hydrogen from carbon-3 of cysteine is eliminated to form a thiazole. HOzCv^NHz S . QCtUOSUS + CO2H I •ï. R Cysteine Nosiheptide

Figure 24. The assembly of a thiazole ring in nosiheptide. Both precursors are assumed to be part of a peptide chain before ring closure.

m U1 66

Assuming the system utilizes the L-isomer of cysteine, a trans elimination would remove the a-proton and the pro-S hydrogen at carbon-3. Serine, stereospecifically labeled with *H or at C-3 could be fed to S. actuosus to resolve this problem. The only basis for assuming the L-isomer of cysteine is the normal precursor to the thiazole rings is the finding that L-[ 3 - serine labeled the thiazoles but not D-cys. Considering this, and the fact that L-[3-i*C]- serine was not incorporated into carbon-3 of the D-cysteine residue, further studies are needed to determine whether

D- or L-amino acids are the natural precursors to these pep tides. A direct approach to this problem would be to feed a

1:1 mixture of L-[1-“ C]serine and D-[3 - serine to the producers of nosiheptide (S. actuosus) and thiostrepton (S. azureus ). The scime experiment with D- and L-t cysteine would unequivocally determine the stereoisomers that are used to make the thiazole rings.

From the experiments with i*C-labeled serines, the pyridine ring in nosiheptide appears to be formed by a

"tail-to-tail” joining of two serine units; the nitrogen and carbons-2, 3, 4, 5 are derived from serine, while carbon-6 is donated by a (modified) cysteine residue (Fig. 25). This transformation must require an interesting mechanism for connecting the two hydroxymethyl carbons of the serine resi dues. The results of the experiment with [U-^^CjJglycerol O H EEI I . • HzC^COzH OH A I ser HOCHz + NHa S. actuosus

• x k , H O a C ^ ^ N H 2 Nosiheptide CO2H I R [r h thiazole]

[r 5 cys]

Figure 25. The precursors and construction of the pyridine ring in nosiheptide. The three amino-acid precursors are thought to be part of a peptide chain before final modifica tion and "tail to tail" joining of two serine residues.

a\ 68 do not strongly support this scheme, because the pyr C4-C5 signals are weak and the pyr C2-C3 connectivity is not observed (Fig. 21). Yet as mentioned before, the [i*C]- serine and [U-^^Calglycerol experiments are inconsistent with respect to the relative labeling of the dehydroalanine versus the (modified) cysteine residues. Serine is incor porated to a greater extent into deala and the pyridine ring relative to the residues derived from cysteine (Table 4); just the opposite is observed for the transfer of intact

units from glycerol. If serine is the intermediate for the incorporation of ] glycerol then one expects similar results in both feeding experiments.

The discrepancy can be explained by the following: Keeping in mind that serine levels in the medium have little or no effect on the production of nosiheptide, and that cysteine and glycerol inhibit the biosynthesis, regulatory mechanisms can alter labeling patterns via changes in the relative flux of carbon through branches pathways. Somehow glycerol must influence the relative levels of serine versus cysteine that are available for biosynthesis. Thus, because the connec tivities of pyr C4-C5 and C2-C3 are observed, the lack of extensive incorporation of three-carbon units into the respective pieces of the pyridine ring does not argue against "tail-to-tail" serine hypothesis. The biochemical connectivities within the pyridine ring can be confirmed by 69 feeding [U-^^Ca]serine or [U-^^Cg]glucose; the carbon source of the fermentation is glucose and it does not appear to repress nosiheptide biosynthesis. There is an alternative route for the biosynthesis of the pyridine ring that can lead to the labeling patterns observed in the [^®C]serine experiments: Glycine, derived from carbons 1 and 2 of serine could be the precursor of the thz(l) C2-pyr C5 and thz(5) C2-pyr C2 pieces while methylene tetrahydrofolate provides pyr C3 and C4. In this case, any coupling between pyr C2 and C3, or pyr C4 and C5 would be observed only when the two adjacent sites are simultaneously enriched due to random processes: i.e.. statistical coupling. This pathway seems improbable, but it cannot be ruled out at this point.

Most likely, the piperidine ring in thiostrepton (Fig.

2) is also derived from two serine residues. The amino group of serine probably gives rise to the nitrogen attached to carbon-5 of the ring; this can be tested by feeding L-[2-

^®N]serine to s. actuosus. The origin of the protons at piperidine C3 and C4 can be determined by feeding L-[3-

serine, and 3R- and 3S-L-[3-^®C,“Hi]serines. In nosiheptide, it is possible that the hydroxyl group at pyr

C3 is derived from the corresponding group of serine. A very elegant method to show this would be to feed [3-

““C]serine enriched with oxygen-18 in the hydroxyl group. 70

Intact incorporation of the bond would be observed

as an isotope shift (approx. 0.01 ppm) of the “ C-NMR signal

of pyridine C3. This technique has been used for probing a 38 number of biosynthetic pathways . The origin of the proton

at pyr C4 can be explored in the manner suggested above for

the piperidine hydrogens in thiostrepton. Feeding multiply-

labeled amino acids is an expensive and time consuming

proposition, but such experiments are very rewarding because

they can reveal the detailed chemistry of complex trans

formations.

The biosynthesis of this pyridine ring is highly

unusual and there is no precedent for such a formation of

CgN rings. There are two textbook-pathways leading to pyri

dine rings both involving quinolinic acid as an inter

mediate. Nicotinic acid is biosynthesized from quinolinate

which arises either via tryptophan (animals and aerobic microorganisms) or by a condensation of aspartic acid with

glyceraldehyde-3-phosphate (plants and some microorganisms).

The latter route is utilized by Streptomvces pvridomvceticus 39 for the biosynthesis of pyridomycin . The penta-substi-

tuted pyridine ring of streptonigrin is formed in an unusual manner from tryptophan; the indole carbons 2 and 3 provide

pyridine C-4 and C-5 while the side chain (C-2' and C-3')

forms pyridine c-2 and C-3^®. The major route to pyridoxol

in E. coli involves the joining of three trioses*^'^^. From 71 the present study, it appears that the CjN rings of the thiostrepton-type antibiotics are assembled from three amino acids that are presumably part of a peptide chain; this requires the joining of two serine units through their hydroxymethyl carbons. This interesting transformation warrants further study.

The indole moiety of nosiheptide is the product of a novel rearrangement of tryptophan (Fig. 26). In general, the amino-acid tryptophan provides the indole ring found in a variety of natural products such as the indole alkaloids.

Many of the compounds are the result of chemical modifica tions at C-3 and C-2, and most of the reactions at the indole nucleus can be rationalized in terms of the poten tially nucleophilic character of C-3. For instance, harmine has incorporated into its structure a Cg-unit (pyruvate) between indole C-2 and what was the primary amino group of tryptamine; as an intermediate step in the biosynthesis, C-3 of the indole ring is believed to attack the Schiff's base of tryptamine and pyruvate (for review see 43, 43). Yet, there is no example for an internal cyclization of trypto phan that connects indole C-2 with the carboxyl group. This rearrangement, demonstrated by feeding double i^C-labeled tryptophan (Fig. 26), also requires excision of the a-carbon and its attached amino group. The mechanism of this (Met) CH3

ANHz C H 3 Streptomyces actuosus

L-tryptophon Nosiheptide

Figure 26, The modification of tryptophan to generate the indole moiety in nosiheptide. Activated methionine donates a methyl group at indole C4 after (modified?) tryptophan is incorporated into the structure. The dashed arrow illustrates excision of the a-carbon.

«vj N) 73 reaction deserves careful investigation and there are

several feeding experiments that could yield some informa tion. The ^®C-NMR spectrum of nosiheptide derived from a

fermentation containing L-ECHj-^^C,^Hj]- or L-ECHj-^Hjltryp- tophan, or L-[ 3 - serine would show how many protons of the CH; group (c-2* in tryptophan) are retained in the

formation of indole-CHg group. If, as one anticipates, both hydrogens are retained, then feeding experiments with 3R- and 3S-[3-^H,^H]serine would uncover the stereochemistry of the displacement at carbon-3* of tryptophan. Of course this would involve stereochemical analysis of the acetic acid produced by oxidative degradation of the labeled nosiheptide*^'^^. It is even possible that the a-proton of tryp

tophan is transferred to carbon-3* (indole-C3*) during the cleavage of the carbon 2*-3* bond. This somewhat unlikely possibility could be explored by feeding L-[2*-®H,l*-^‘*C]-

tryptophan. The conversion of DL-[2*,3*-®H]- plus L-I3’-

^“C]typtophan to nosiheptide takes place with 25% ^H-reten-

tion, but the details of the experiment are not described here^®.

Since all the modified residues of nosiheptide can be

traced to natural amino acids, it is logical to presume that

thé structure is originally synthesized as a linear peptide,

composed of 12 or 13 residues (Fig. 27). Extensive 74

COzH

deala / thz(5) pyridine thz(l) thz (4)

thz (3) indok thz(2) / \ /

Figure 21. The putative peptide precursor (left) of nosi heptide (right). Tryptophan may originate at the C- or N- terminus of the peptide before modification. 75 oxidation, dehydrogenation, and dehydration of the consti tuent amino acids would give rise to the modified peptide.

Serine-10 and the N-terminal serine must be joined to form the pyridine ring, and cysteine residues numbers 2, 5, 7, 9 and 11 form thiazole rings. Tryptophan may actually be a terminal residue in the peptide precursor. Note that in thiostrepton (Fig. 2), a nitrogen at piperidine carbon-5 connects the two peptide loops: the hydroxyl group of a threonine residue is the second anchor. Thus, one can hypo thesize that tryptophan originates as the N-terminus of the precursor and is displaced at some point during the trans formations. An alternative is that tryptophan is the

C-terminal amino acid of the original peptide.

Nosiheptide and thiostrepton could each result from different modifications of similar peptide precursors. Con sider the sequence trp-ile-ala-ser-ala-ser-cys-thr-thr-cys-

(X)-cys-(Y)“cys-ser-cys-ser-ser where X and Y are variable residues. Assuming tryptophan is the precursor to the quinaldic acid moiety, the peptide precursor of thiostrepton would have Y=threonine and X=an unknown amino acid (precur sor to thiostreptine residue). Hypothetically, serine-6 and serine-15 are joined to form the piperidine ring, and thr-13 is connected to the modified tryptophan residue (quinaldic 76

acid). The precursor peptide of nosiheptide would contain

X=glu, Y=cys, and one less serine at the C-terminus;

processing would involve clipping out residues ile-2 through

ala-5, modification of tryptophan and formation of the ester

and thioester bonds. If these speculations have any

validity, the biosynthetic machinery for thiostrepton and

nosiheptide must be almost identical with respect to peptide

synthesis; they would differ mostly in the systems respon

sible for peptide modification. Testing this hypothesis

directly would be no trivial task because first of all it

would involve the chemical synthesis of the proposed peptide

intermediate(s). Secondly a cell free system for peptide

modification needs to be developed for both organisms.

Another approach would be to produce blocked mutants of both

organisms and attempt cosynthesis of the antibiotics (with

whole cells and homogenates). In any case the enzymology

deserves study, because a detailed understanding of these

complicated biochemical processes will facilitate the production of novel structures via directed biosynthesis or mutasynthesis (review, 47). Multienzyme systems for the

assembly of peptide antibiotics - such as gramicidin S and bacitracin - have been well characterized, and it is generally assumed that all microbial bioactive-peptides are 20 synthesized by non-ribosomal processes . These systems are not influenced by inhibitors of ribosomal protein synthesis. 77 yet the present work indicates that the biosynthesis of nosiheptide by S. actuosus is sensitive to chloramphenicol.

At this point it is important not to over emphasize this result because there is no evidence that the antibiotic has a direct effect on the biosynthesis of the peptide precur sor. Chloramphenicol could be halting ribosomal synthesis of a key enzyme that has a very short half-life. To deter mine which system - ribosomal or enzyme template - con structs the backbone of nosiheptide, several different antibiotics known to interfer with prokaryotic ribosomes should be administered in the presence of [U-^**C]serine (or threonine). Thus, if the nosiheptide precursor is biosyn thesized on ribosomes, incorporation of radioactivity into both protein and antibiotic should cease upon exposure to the inhibitors. On the other hand, if a multienzyme complex assembles the peptide, incorporation of [U-^‘‘C]serine into nosiheptide may be accelerated (on the short term) in the presence of the inhibitors because the major route for amino acid consumption is blocked. If chloramphenicol are directly effecting biosynthesis, then nosiheptide would be the only peptide antibiotic known to be constructed by ribosomal peptide synthesis. Of course mRNA directed synthesis can only incorporate the natural L-amino acids, so feeding studies with D- and L-[ amino acids must show the

L-isomers to be the preferred substrates; epimerization of 78

L-cysteine could only occur after completion of peptide synthesis. The gene coding for such a peptide would be readily accessible because synthetic DNA probes, with the appropriate sequence, could be used to screen for the gene in chromosomal digests or plasmid libraries of chromosomal

DNA.

In conclusion, this work has accomplished two goals.

First it has provided the most detailed information to date on the biosynthesis of nosiheptide and thiostrepton-type antibiotics; the peptide backbone of nosiheptide is now clearly identified. Many of the biochemical transformations revealed here have never been observed and further study may uncover novel reaction mechanisms for amino acid modifica tion. Secondly, this work should provide an example of the general approach to the study of antibiotic biosynthesis; from fermentation of ^®C-NMR analysis of labeled products.

Hopefully, at the very least, results presented here will help guide future research on the biosynthesis of modified- peptide antibiotics. EXPERIMENTAL SECTION

Materials. Solvents, buffers, and any reagents obtained from commercial sources were of the highest quality available. Authentic nosiheptide was a gift from Rhone-

Poulenc Co. (France). The CjPz complex of tryptophan synthase was the generous gift of Dr. Edith Wilson Miles of the NIH in Bethesda, Maryland. Radioactive precursors were obtained from the following sources: L-[U-^“C]Alanine (150 mCi/mmole), L-[methyl-^‘*C]methionine (46 mCi/mmole), and

L-[U-^“C]serine (160 mCi/mmole) from New England Nuclear;

L-pheny 1 [ 1-^"*C]alanine (56 mCi/mole), L-[methylene-^“C]tryp tophan (58 mCi/mmole), and [^“C]formaldehyde (11 mCi/mmole) from Amer sham; and DL-[ring-7a-^‘*C] tryptophan (3.5 mCi/mmole) from ICN. The following companies supplied i^C- enriched compounds (atom % ^®c): DL-[3-^^C]cysteine (97) and [i3C]formic acid (98) from Cambridge Isotopes; L-

[methyl-^^C]methionine (90) from ICN: and L-[3-i*C]serine

(90) and DL-[l-^®C]serine (99) from Merck and Co. The

[U^^C]glycerol was synthesized by Jonathan P. Lee of Ohio

State University by modifications of published procedures^®' .

79 80

Organism and Fermentation. Streptomyces actuosus ATCC

25421 was transferred from a lyophile-tube and grown for 7 days (27*0 on medium A. Isolated colonies were picked to inoculate the seed culture in medium B. The seed culture was incubated on a gyratory shaker (220 rpm with 5.1 cm, or

260 rpm with 2.5 cm throw) at 27*C for 36 to 48 h. After growth was complete, aliquots (5 ml) were stored aseptically with glycerol (20%) at -60*C (frozen vegetative mycelia or

FVM). The FVMs could be stored for at least one year and were used to start all fermentations (medium B) for the feeding experiments. Medium C was inoculated with 2 ml of the seed culture (24 to 36 h) and incubated on a gyratory shaker for 84 to 96 h. The production of nosiheptide in medium C was very dependent on the rate (rpm) and throw (cm) of gyratory shaker; at 220 rpm and 5.1 cm antibiotic produc tion began at 24 h and abruptly terminated at 84 h. Yet, at a greater rate (260 rpm) and shorter throw (2.5 cm) nosihep tide production occurred between 36 and 96 h. Under ideal conditions the titer was usually 175-250 mg/liter.

The following media were used for this study: Medium A was used for solid culture and consisted of (g/liter) yeast extract (0.15), casamino acids (0.3), malt extract (0.3), glycerol (10.0), sucrose (20.0), CaClj (1), MgClj (1), and agar (20.0). The pH was adjusted to 7.0 with NaOH before 81

sterilization. Medium B, the liquid seed medium, contained

(g/liter) glucose (30), MgSO„*7HjO (0.5), Proraosoy (20.0), corn steep liquor (20.0), (NH„)(3) and CaCO, (5). The pH was adjusted to 6.8 with NaOH prior to dispensing 45 ml in 250-ml flasks (three baffles). Medium C was the produc tion medium used for all the feeding studies and was composed of (g/liter) L-glutamate (5), L-arginine (1),

L-aspartate (1), KjHPO^-VHzO (0.5), MgSO^-THjO (1), NajSO^

(2), ZnSO^-THjO (0.01), FeSO^-THjO (0.02), CaCO, (3), and glucose (40). The pH was adjusted to 7.25 before dispensing

45 ml into 250-ml flasks (nonbaffled).

Isolation of Nosiheptide. The following procedure is an example of the routine isolation of nosiheptide from 500- ml of fermentation broth. Isolation on any scale can be accomplished providing the proportions of solvents are kept constant. This method was used for preparing nosiheptide samples for ^^C-NMR spectroscopy.

The mycelia were separated from the whole broth by centrifugation (15 min at 3000 rpm) in two 250-ml polypro pylene bottles. After decanting the supernatant into a waste container, the pellet in each bottle was agitated with tetrahydrofuran (THF, 125 ml) and 15 ml of a buffer composed of 1 M sodium citrate and 0.5 M sodium phosphate at pH 4.0. 82

The mixture was agitated 15 mih, combined with hexane or petroleum ether (30 ml), and agitated for another 15 min period. In some cases centrifugation was necessary for separation of the two phases. The organic phases were pooled in a 1 liter round-bottom flask and evaporated to dryness under vacuum (35“C). The extraction of the aqueous phase was repeated and the organics were combined in the flask and evaporated to dryness. The residue was dissolved in 100 ml of THF and the resulting yellow or pink solution was filtered through Whatman No. 2 paper. The filtrate was combined with 200 ml of hexanes (or petroleum ether), stir red for 30 min, and placed in a cold room for 2 to 8 h. The white precipitate was collected by centrifugation and then redissolved in 50 ml of CH2CI2/CH3CH2OH, 4:1. Nosiheptide was precipitated by the addition of 100 ml of ethyl ether and collected by centrifugation. Precipitation was repeated from THF/hexane (1:2) and the nosiheptide was dried under vacuum for at least 12 h. The mother liquors were always assayed for nosiheptide before discarding them. The purity of nosiheptide was estimated by comparing HPLC integration

(fluorescence and 254 nra) with the mass of the isolate. In some cases 10 mg of authentic nosiheptide was combined with a sample prior to the final precipitation from THF/hexane. 83

Nosiheptide Assay and HPLC. A method was developed for rapid assay of nosiheptide in fermentation broths. The cells from a 2 ml aliquot of whole broth were collected by centrifugation (10 min at 3000 rpm) and the supernatant was discarded. The pellet was mixed thoroughly for 10 min with

0.1 ml of buffer (1 M citrate and 0.5 M phosphate, pH 4.0) and 1.7 ml tetrahydrofuran (THF). The water and the cells were removed from the THF by mixing the sample with 0.3 ml hexane. The phases were separated by centrifugation (10 min, 3000 rpm). An aliquot of the organic layer was com bined with an equal volume of glacial acetic acid and the resulting solution was directly injected (10 ]il) onto an

HPLC column.

The HPLC system was composed of a Reodyne-1025 injec tor, a Waters M-40 pump, a Hamilton PRP-1 column (10 um, 4.6

X 250 mm), a Gilson-121 fluorometer and a Hewlett Packard-

3390 integrator. The isocratic system consisted of acetonitrile, p-dioxane, water, and acetic acid (40:20:40:0.2) at a flow rate of 1 ml/min. A Packard Trace-II was used to detect radioactive compounds separated by HPLC. Nosiheptide was detected by fluorescence at 450 to 600 nm with excita tion at 390 nm. 84

Feeding Experiments. In general, isotopically labeled

substrates were fed to cultures grown in the synthetic

medium (C). Before adding a precursor to the fermentation,

the nosiheptide titer was assayed at 24, 32, and 36 h post

inoculation; the labeled substrate was added when the

nosiheptide titer reached 5 to 10 rag/L, usually at 32 h.

Each radioactive precursor was diluted with an aqueous

solution (pH 7.0) of unlabeled compound, sterilized by

filtration and then added to the production culture (45 ml).

All radioactive experiments were run in duplicate or

triplicate. A second addition of the labeled precursor (^^C or i^C) was made 24 h after the initial feeding, and 24 h

later the flasks were harvested by centrifugation. Nosihep

tide was purified by the usual method and radiochemical purity was confirmed by comparing the HPLC peak integration versus the mass of the isolated solids. In most cases, a radioactivity-flow monitor was placed in series with the fluorescense or UV detectors. Authentic, purified nosihep tide was used to calibrate the HPLC assay.

A solution (13.1 mmolar) of DL-[3-^®C]cysteine was pre pared in 75 mM MES buffer (Sigma) at pH 7.0. The labeled cysteine was gradually added to a production culture which was grown at 27*C (260 rpm at 2.5 cm throw); 2 ml of the solution was added to each of 16 flasks (45 ml) at 40, 52, 85

and 64 h {post-inoculation). At the time of the final

addition only 30 mg/liter of nosiheptide had been produced

so the flasks were transferred to a shaker set to 27"C and

300 rpm (2.5 cm). After a total incubation time of 80 h,

the cultures were combined in 250-ml centrifuge bottles.

The nosiheptide was isolated as usual, but before the final

precipitation from THF/hexane, 10 mg of authentic nosihep

tide was added to the solution of labeled material. The dried precipitate (60 mg) was dissolved in 0.4 ml DMSO-dg.

Before feeding L - [3-^®C]serine, the production culture was grown at 27®C and 220 rpm (5.1 cm throw) for 34 h. The precursor was added as a 40 m M solution in water; 1 ml was

added to 15 flasks (40 ml) at 34 h and 58 h. The cells were extracted 24 h later. This fermentation yielded 90 mg of

isolated nosiheptide.

DL-[1-^®C]Serine was added to a culture of S. actuosus grown at 27“C and 260 rpm (2.5 cm throw); 1 ml of 47.6 mM

serine in water was added to each of 15 flasks (45 ml) at 48 h and 62 h. The fermentation was terminated after 96 h and nosiheptide was isolated as usual except 10 mg of authentic material was added before the final precipitation. The total yield was 60 mg. 86

L”[Methyl-^®C]methionine was added to 25 production flasks, or a total of 1 liter of culture. To each flask, at

41 and 65 h, was added 1 ml of a 27.4 mM solution in water.

The fermentation was harvested at 89 h and yielded 145 mg of nosiheptide.

For the experiment with L-[2,1'-^^Cj3tryptophan, the production culture was grown for 32 h at 27“C (5.1 cm at 220 rpm). The tryptophan was added at 32 and 56 h; 1 ml of a 16 mM solution was added to 15 flasks (45 ml). The fermenta tion was terminated at 72 h and nosiheptide was isolated as usual. Prior to the final precipitation from THF/hexane, 10 mg of authentic material was added to bring the isolated yield up to 45 mg.

For the feeding experiment with [U-^^gC]glycerol, S. actuosus was grown for 44 h in synthetic production medium at 27°C (260 rpm, 2.5 cm throw). A solution of [U-^^Cglgly- cerol (800 mg/45 ml) in water was filter sterilized (0.22 vm) and 1 ml aliquots were added to each of 15 flasks (40 ml). A second addition of 2 ml per flask was made at 68 h.

The cells were extracted after an additional 24 h of incuba tion. In this experiment 10 mg of authentic nosiheptide was combined with the 30 mg of isolated material. 87

Preparation of 4-Methyl-[3'-^‘*C]trvptophan^^. In a

10 ml test tube, equipped with a magnetic stir bar, the

following compounds were combined in sequence: 0.26 ml

dimethylamine (40%, Aldrich), 0.1 ml 30% aqueous form

aldehyde, 50 uCi (50 u D [ ^“C]formaldehyde, 1 ml acetic

acid, and 0.11 ml 4-methylindole (Aldrich). The test tube

was capped and the reaction was stirred for 24 h at room

temperature. 4-Methylgramine was detected by TLC on silica

gel (toluene/ ethanol, 85:5, Rf 0.75), and precipitated by

the addition of NaOH (0.7 g/7ml Hj) at 4°C. The precipitate

was collected by filtration and washed with 50 ml HjO.

After drying the product overnight in a vacuum, 126 mg of

4-methyl-(methylene-^‘*C]gramine was mixed with 270 mg of

diethyl formamidomalonate in 2.5 ml of xylene (10 ml flask,

14/20 Rg). Argon was bubbled through the mixture for 1 h

(30"C) and then, after the addition of 9 mg pulverized NaOH,

the reaction was refluxed under argon for 10 h. The

resulting brown mixture was reduced to a thick oil by rotary

evaporation (45*C). Hydrolysis and decarboxylation were

accomplished directly by first refluxing this oil with

aqueous NaOH (2.3 g in 22 ml, 9 h) followed by refluxing with glacial acetic acid (0.6 ml, 2 h). The entire reaction mixture was placed in 20 ml water which was then adjusted to pH 6.0. A mixture of n-butanol and toluene (10:1) was used

to extract the product (three 40 ml portions). After 88

evaporating the solvent under vacuum (40"C), the residue was

dissolved in 10 ml water. Final purification of 4-methyl-

DL-[3 tryptophan was performed on a cation exchange

column (AG50X8, 200-400 mesh, 20x1 cm). The column was

washed with 200 ml 0.1 N HCl, 200 ml water, and 1 M NH„OH

which upon evaporation, afforded 20 mg (5 uCi) of the

labeled 4-methyltryptophan. The yield was 10% based on

formaldehyde. This material, used directly for feeding

studies, co-chromatographed with 4-methyltryptophan on

silica gel (n-butanol/HgO/CH3COOH, 70:25:5).

Preparation of [ 2 - Indole. In a 125-ml Erlenmeyer

flask, 3.5 g o-toluidine (Aldrich, redistilled), 6.7 g

dicyclohexylcarbodiimide, and 40 mg 4-dimethylaminopyridine

were dissolved in 30 ml dichloromethane. The mixture was

stirred at room temperature for 10 min and then a solution

of 1 g [ formic acid (contained 5% HjO), 1 ml CH3CN, and

5 ml CH2CI2 was added dropwise to the flask over a period of

15 min. This mixture was stirred for another 2 h under

nitrogen and then filtered to remove the dicyclohexylurea.

The filtrate was concentrated under reduced pressure and

applied directly onto silica gel (Baker, 40 um, 2x30 cm) which had been equilibrated with CH2CI2/ether (95:5). The

column was washed with three portions (250 ml) of the

solvent in ratios of 95:5, #5:15, and 70:30 in succession. 89

Fractions (50 ml) were monitored by TLC and N-formyl-o-

toluidine was found in fractions 12-14. Evaporation of the

solvent gave an oil and a slow stream of nitrogen blown over

this material finally yielded 2 g of a pink, low melting

(50*0 solid. The compound gave a single spot on TLC

(CHjClj/Ether, 1:1) and a single peak by GC-MS (molecular

ion 136/135=9:1).

Dry o-toluidine (184-185*C, 9 g) and 0.9 g NaH (80% in

oil, Aldrich) were placed in a 50 ml three-neck flask equipped with a gas inlet, reflux condenser, boiling chips, and a glass stopper. While under a slow stream of argon, the mixture was heated intermittantly over a metal bath (330“C) until the evolution of nitrogen was complete (- 20 min); excessive foaming was avoided by raising and lowering the metal bath until all the NaH was utilized. Freshly fused

H C O O K (4.2 g) and N - [ C H O - ^ ^ C Jformyl-o-toluidine (1 g) were added to the flask and the reaction was refluxed at 305 to

310"C for 45 min^^^. After the black material was cooled to room temperature it was covered with diethyl ether and then gradually neutralized with 1 N HCl. The water phase was extracted with 3 volumes of diethyl ether. The ether was removed under vacuum and the resulting residue was dissolved in a minimum of CHCI3. The solution was applied to silica gel (Baker, 40 um, 2x30 cm) which had been equilibrated with 90

petroleum ether/CHaClj, 7:3. After washing the column with

2 volumes of solvent, the indole was eluted with 100% CHCI3.

The 12 - indole (600 mg) was crystallized from water (mp.

50-51“C; MS molecular ion 118/117 = 7.2:6.3). Carbon-2 was enriched about 50% with

Preparation of L-Cl ' .2-^^C]Tryptophan. The reaction mixture was composed of 5xlO“^M pyridoxal phosphate, 10 mM mercaptoethanol, 1.5 mM [2-^^]indole, 67 mM DL-[1-^^C]- 27 serine, and 9 mg of O 2P 2 complex of tryptophan synthase in

250-ml buffer (10 mM KjHPO.. and 100 mM TRIS-HCl, pH 7.8).

The entire mixture, less enzyme, was sterilized by filtra tion (0.22 um) and placed in a sterile 500 ml Erlenmeyer flask. After addition of enzyme (9 mg/0.5 ml) the mixture was incubated for 8 h at room temperature; at that time no indole could be detected by TLC. Thus, 17 ml (55 mg) of an aqueous solution of indole was added along with 2 ml of a sterile DL-[1-^^C]serine solution (10.5 mg/ml). Only a trace of indole was detected after another 1 h of incuba tion, so the reaction was terminated by the addition of 10 ml glacial acetic acid. The aqueous mixture was extracted with 2 volumes of diethyl ether and then reduced to 15 ml by rotary evaporation (35°C). The pH of this solution was adjusted to 5.9. Tryptophan was isolated from the mixture by reversed phase chromatography on Diaion HP-20 (Mitsubishi 91

Chemicals). The sample was applied to a 1.5 x 90 cm column which had been equilibrated with water (the HP-20 was previously washed thoroughly with acetone). The column was washed with 500 ml water and the tryptophan was eluted with

20% aqueous ethanol. Evaporation of the solvent afforded white plates which were recrystallized from water/ ethanol

(125 mg). The proton and carbon-13 NMR were identical to those of authentic tryptophan; the ^^C-NMR spectrum showed two intense signals: the carboxyl group at 177.3 ppm and indole C-2 at 128.1 ppm. The i^C-enrichments at the car boxyl group (C-1') and ring carbon-2 (C-2) of tryptophan must be equal to that of the starting materials; DL-[1- i*C]serine (99 atom % ^®C) and [2-^®C]indole (50 atom % i^C), respectively.

^^C-NMR Spectroscopy. ^®C-NMR spectra were measured at

75.4 MHz (7.0 T) on a Bruker WM-300 FT-NMR spectrometer.

Samples were dissolved in 0.25 to 0.5 ml DMSO-dg (Sigma).

The measurements were made under the following conditions:

320“ K, 30“ pulse, repetition time = 1.1 sec, spectral width

= 16.13 KHz, 16K data sets, 2 Hz line broadening, and continuous broadband ^H decoupling. The ID-INADEQUATE spec trum was acquired using 16K data sets, an acquisition time of 0.5 sec, and x = 4 msec. The 2D-INADEQUATE was acquired over a 48 h period under the following conditions: 92 repetition time of 1 sec, x = 4 msec, sw2 = 11.6 KHz in 4K data sets, swl = 1 1 . 6 KHz sampled in 256 increments and zero-filled to 512 data points. The reverse INEPT experi ment was performed with 16K data sets, and a spectral width of 16.13 KHz. APPENDIX

93 94

SÛ bU 40

170 150 130 ppm

°* nosiheptide biosyn- -10

KHz

170 / / o 50

Figure 29. The 2D-INADEQUATE spectrum of nosiheptide biosynthesized from [U-^^Cglglyderol.

U1VO 96

80 60 40 20 ppm

180 160 140 120 100 ppm

Figure 30. The ^®C-NMR spectrum of nosiheptide biosynthesized from DL- [ 3- ^ ]cysteine. 97

80 60 40 20 ppm

180 160 140 120 100 ppm

Figure 31. The ^^C-NMR spectrum of nosiheptide biosynthesized from L-[3-^^C]serine. i - j j j WfwVW#

ppm

IU ii#wAm#W ##W ¥¥

180 170 160 150 130 120 110 100

Figure 32. The ^^C-NMR spectrum of nosiheptide VO biosynthesized from DL-[1-^®C]serine. 00 LIST OF REFERENCES

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