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MUTANTS OF droxy analog of thienamycin corresponding to CATTLEYA PRODUCING N-ACETYL NS-5 (2b). AND DESHYDROXY This is the first report on the use of mutants RELATED TO THIENAMYCIN of S. cattleya for the production of thienamycin analogues. These mutants should be useful in elucidating biosynthetic pathways leading to this DAVIDRosi, MARIONL. DROZD, interesting class of carbapenems. MICHAELF. KUHRT,LOUIS TERMINIELLO, S. cattleya NRRL 8057 was incubated on N-Z PAULE. CAMEand SOLJ. DAUM amine agar') for 7 days at 27°C. Spores were Sterling-Winthrop Research Institute, harvested in TX buffer') and subjected to the Rensselaer, New York 12144, U.S.A. mutagenic action of N-methyl-N'-nitro-N-nitro- soguanidine at 1.0 mg/ml for 2 hours. The (Received for publication October 24, 1980) spores were collected via filtration, suspended in 0.1 M phosphate-buffered saline and plated on Streptomyces cattleya NRRL 8057 produces N-Z amine agar and incubated as above. Iso- the jl-lactam antibiotic thienamycin (1a) and lated colonies were transferred to N-Z amine traces of N-acetylthienamycin (lb)1). A closely agar slants. These stock cultures served as a related , PS-5 (2a) is produced by source of seed for growth in flasks containing a Streptomyces cremetis subsp auratilis.2,11)* The thienamycin production medium (medium B)9' latter has recently been deacetylated with L- incubated on a rotary shaker at 210 rpm at 27°C amino acid acylase from porcine kidney and D- for 48 hours. Samples from each fermentation amino acid acylase from Streptomyces olivaceus were clarified via centrifugation and aliquots of affording a desacetyl compound NS-5 (2b).3) In the crude broths were subjected to chromato- addition, a European patent published in 1979 graphy on Whatman No. 1 paper developed in discloses the preparation of NS-5 (deshydroxy- ethanol-water, 70: 30 (v/v) using authentic thiena- thienamycin) via a lengthy chemical synthesis.') mycin as a reference.** The developed chro- These compounds display very potent antimic- matograms were subjected to bioautography robial activity against Gram-positive and nega- using S. aureus as the test organism. Using tive , as well as (3-lactamase inhibitory these methods we have found two mutants; S- properties.a, s) WRI-M459 producing a compound correspond- We would like to report the production of ing to N-acetylthienamycin (Rf 0.59) and S-WRI- carbapenems by two new mutants of S. cattleya M5301 producing a compound corresponding to NRRL 8057. One of these mutants, S-WRI- NS-5 (Rf 0.50). Thienamycin (Rf 0.36) was not M459 produces an N-acetyl carbapenem cor- observed in either case. responding to N-acetylthienamycin (Ib) while For isolation of these antibiotics, fermenta- the second, S-WRI-M5301 produces a deshy- tions were conducted in 14 liter jars containing

a RH; Thienamycin a RCOCH3; PS-5 b R=COCH,; N-Acetylthienarnycin b R=H; NS-5 * Since submission of this paper , carbapenems PS-6 and PS-7 have been reported.") ** Thienamycin was kindly supplied by Dr. J . BIRNBAUM of Merck Institute of Therapeutic Research, Rahway, NJ, U.S.A. 342 THE JOURNAL OF ANTIBIOTICS MAR. 1981

10 liters of medium COI. These were inoculated lite CG-400, Bio Gel P-2 and XAD-2 followed with 500 ml of a 48-hour culture grown in the by HPLC purification on the Lichrosorb NH, same medium. The jar fermentors were operated column eluted with 74% acetonitrile and 26 at 28°C with an agitation rate of 200 rpm and an 0.02 M ammonium acetate buffer at pH 6.5 (v/v). air flow of 5 liters per minute for 48 hours. Anti- In the second method, successive chromato- biotic production was followed using an agar graphy was carried out over CM-Sephadex C-25, diffusion assay with S. aureus as the assay XAD-2 and Sephadex LH-20. Both methods organism. afforded material with an extinguishable absor- Antibiotic from S-WRI-M459 bance of 90 % of 297 nm) after treatment The culture filtrate from this organism, after with 10 mm hydroxylamine; MH+ (chemical adjustment to pH 7.0, was passed through Dowex ionization) 257 (M++C2H2 at 285), M+ (elec- 1-X2 (Cl- form) anion exchange resin. The tron impact, direct inlet) 256; pmr (D2O, ex- adsorbed antibiotic was eluted with 1.5% NaCI ternal TMS) 1.46 (t, CH,), 2.2 ppm (m, CH2); in 40%o aqueous methanol. Fractions display- 13Cnmr (D20, 0.02 M phosphate buffer, pH 7.9, ing antibiotic activity were combined, concen- internal TMS) 11.7 (C8), 22.7 (C9), 30.1, 40.2, trated and adsorbed on Amberlite XAD-2 from 40.4 (C4,11,12), 56.2 (C2), 60.9 ppm (C6). No which elution was conducted with 20% aqueous peaks were observed for C2,3.7,10since the small methanol. The material was then subjected to sample size resulted in a low signal to noise ratio. column chromatography via Bio-Gel P-2 gel The above data are consistent with a structure filtration and Diaion HP-20. Final purification corresponding to NS-5 (2b). was effected via preparative HPLC using a Lichrosorb NH, (10p) column measuring 10 x Acknowledgments 250 min with a solvent composed by volume of 81 ° , acetonitrile and 19 % 0.02 M phosphate buf- The authors wish to acknowledge the excellent fer at pH 6.0 and a detector measuring uv ab- assistance of the following colleagues in the Sterling- Winthrop Research Institute Laboratories: B. SAGE sorbance at 280 nm. Antibiotic fractions were and R. BECKERfor technical assistance with the fer- combined and lyophilized. On the basis of mentations, M. FANCHERand L. RICCAfor the muta- paper chromatography using three solvent sys- tion work and S. CLEMANSfor interpretation of the tems and HPLC retention times, this material physical data. was indistinguishable from N-acetylthienamycin prepared chemically from pure thienamycin ac- References cording to published procedures"). The pmr spectrum obtained in D20 showed a sharp 1) KAHAN,J. S.; F. M. KAHAN,R. GOEGELMAN, singlet at 2.51 ppm (relative to H2O) attributed S. A. CURRIE,M. JACKSON,E. 0. STAPLEY, to the methyl of an acetyl group. This signal T. W. MILLER,A. K. MILLER,D. HENDLIN,S. was also present in our chemically prepared MOCHALES,S. HERNANDEZ,H. B. WOODRUFF& sample of N-acetylthienamycin. J. BIRNBAUM:Thienamycin, a new 9-lactam antibiotic. I. Discovery, , isolation Antibiotic from S-WRI-M5301 and physical properties. J. Antibiotics 32: 1 - A 48-hour seed culture of this organism in- 12, 1979 cubated as above was used to inoculate jar fer- 2) OKAMURA,K.; S. HIRATA,A. KOKI,K. HORI, mentors containing 9 liters each of production N. SHIBAMOTO,Y. OKUMURA,M. OKABE,M. medium having the following composition: dis- OKAMOTO,K. KOUNO, Y. FUKAGAWA,Y. tillers solubles, 1.0%; CaCO3, 0.4% (pH 6.5). SHIMAUCHI,T. ISHIKURA& J. LEIN: PS-5, a These fermentations were conducted as described new (3-lactam antibiotic. I. Taxonomy of the above for 65 hours. The culture filtrate was producing organism, isolation and physico- chemical properties. J. Antibiotics 32: 262- passed over Dowex 1-X2 (CI- form) anion ex- 271, 1979 change resin. The antibiotic in the effluent was 3) FUKAGAWA,Y.; K. KUBO, T. ISHIKURA& K. adsorbed on Amberlite XAD-2 and eluted with KouNO: Deacetylation of PS-5, a new P- 30% aqueous methanol. lactam compound. I. Microbial deacetylation From this step, pure material was obtained of PS-5. J. Antibiotics 33: 543-549, 1980 via two methods. In one, use was made of 4) Merck and Co. Inc.: EP Appln. 78101157.2, successive column chromatography on Amber- Pub. No. 0001 628 Al (1979) VOL. XXXIV NO. 3 THE JOURNAL OF ANTIBIOTICS 343

5) OKAMURA,K.; M. SAKAMOTO& T. ISHIKURA: KACZKA, R. E. RHODES, J. S. KAHAN, F. M. PS-5 inhibition of a t4-lactamase from Proteus KAHAN, R. W. RATCLIFFE, E. WALTON, L. J. vulgaris. J. Antibiotics 33: 293302, 1980 RUSWINKLE,R. B. MORIN & B. G. CHRISTENSEN: 6) KROPP, H.; J. S. KAHAN, F. M. KAHAN, J. Structure and absolute configuration of thiena- SUNDELOF,G. DARLAND& J. BIRNBAUM:Thiena- mycin. J. Amer. Chem. Soc. 100: 6491-6499, mycin, a new P-lactam antibiotic. II. In vitro 1978 and in vivo evaluation. 16th ICAAC, Abst. 11) YAMAMOTO,K.; T. YOSHIOKA, Y. KATO, N. No. 228, 1976 SHIBAMOTO,K. OKAMURA,Y. SHIMAUCHI& T. 7) American Type Culture Collection, Catalogue ISHIKURA: Structure and stereochemistry of of Strains I, Thirteenth Edition, 441, 1978 antibiotic PS-5. J. Antibiotics 33: 796803, 8) HIRSCH, C. F. & J. C. ENSIGN: Nutritionally 1980 defined conditions for germination of Strepto- 12) SHIBAMOTO,N.; A. KOKI, M. NISIIINO, K. myces viridochromogenes spores. J. Bacteriol. NAKAMURA,K. KIYOSHIMA,K. OKAMURA,M. 126: 13-.23, 1976 OKABE,R. OKAMOTO,Y. FUKAGAWA,Y. SHIMA- 9) Merck and Co. Inc.: Antibiotics. U. S. Pat. UCHI, T. ISHIKURA& J. LEIN: PS-6 and PS-7, 3,950,357, Apr. 13, 1976 new P-lactam antibiotics. Isolation, physico- 10) ALBERS-SCHONBERG,G.; B. H. ARISON, O. D. chemical properties and structures. J. Anti- HENSENS,J. HIRSHFIELD,K. HOOGSTEEN,E. A. biotics 33: 1128-.1137, 1980