Vol. 14, No. 4 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1978, p. 601-604 0066-4804/78/0014-0601$02.00/0 Copyright © 1978 American Society for Microbiology Printed in U.S.A. Microbial Transformations of Natural Antitumor Agents: 0- Demethylation of Vindoline by Sepedonium chrysospermum GENG-SHUEN WU, THOMAS NABIH, LEISA YOUEL, WANDA PECZYNSKA-CZOCH, AND JOHN P. ROSAZZA* Division of Medicinal Chemistry and Natural Products, College of Pharmacy, The University of Iowa, Iowa City, Iowa 52242 Received for publication 16 May 1978 Vindoline (la) was transformed to O-demethylvindoline (lb) by Sepedonium chrysospermum ATCC 13378 in 33% yield. Thin-layer and high-pressure liquid chromatographic methods distinguished lb from previously reported microbial metabolites of vindoline. The structural proof of lb was based on its mass spectral fragmentation pattern and on its chemical and nuclear magnetic resonance spectral properties. The Vinca alkaloids vincristine and vinblas- tine possess significant antitumor activity and are widely used clinically. They have resisted simple syntheses, and their structural complex- ity has hindered the preparation of other useful derivatives of the alkaloids by chemical meth- ods. Microbial transformations are being exam- ined for their potential to prepare potentially important Vinca alkaloid derivatives. Vindoline (la) is one of the monomeric Vinca alkaloids and one of the most abundant alkaloids in Vinca rosea L. Since it is more readily avail- able than vincristine and vinblastine, it was used in an effort to identify new types of biotransfor- mation products of this group of alkaloids. Pre- vious reports document the production of a va- riety of metabolites of vindoline by streptomy- cetes. Known vindoline metabolites include (Fig. 1): deacetylvindoline (Ic) and deacetyldihydro- vindoline ether (2c) (9); dihydrovindoline ether (2a); 3-acetonyldihydrovindoline ether (2b); a ring contraction product known as 16-dehy- droxy-14,15-epoxy-14-oxo-3-norvindoline (12); N-demethylvindoline (Id) (13); and a dimer con- sisting of two dihydrovindoline ether moieties linked through their 3 and 14 positions (11). This report describes selective cleavage of the methyl ether group of vindoline by Sependon- ium chrysospermum ATCC 13378. The reaction gives lb in 33% yields, and it is not complicated by the formation of other side products. MATERIALS AND METHODS Melting points were determined on a Thomas-Hoo- ver apparatus and are uncorrected. Infrared spectra were determined on a Perkin-Elmer 267 grating in- frared spectrophotometer. Nuclear magnetic reso- nance (NMR) spectra were determined on a Varian Associates model T-60 spectrometer using tetrame- thylsilane as an internal standard. Low-resolution mass spectra were obtained on a Finnigan model 3200 spectrometer, and high-resolution mass spectral data were provided by the Department of Chemistry, Mas- sachusetts Institute of Technology, Cambridge. UV spectra were obtained on a Beckman DK-2 instru- ment. Elemental analyses were determined by Integral Microanalytical Laboratories, Inc., Raleigh, N.C. Vindoline, deacetylvindoline, dihydrovindoline ether, N-demethylvindoline, 3-acetonyldihydrovindo- line ether, and 16-dehydroxy-14,15-epoxy-14-oxo-3- norvindoline were obtained as authentic reference standards from Eli Lilly and Co., Indianapolis, Ind. The purity of each compound was verified by thin- layer chromatography (TLC), high-pressure liquid chromatography, and mass spectral analysis. Vindo- line (7) dihydrovindoline ether and the dihydrovin- doline ether dimer (11) were characterized by melting points and UV, NMR, and mass spectra. Cultures and general growth procedures. S. chrysospermum ATCC 13378 was maintained on Sa- bouraud-maltose agar slants in sealed screw-cap tubes stored at 4°C. All cultures were grown in a soybean meal-glucose medium (1) for screening and prepara- tive work. They were incubated according to the two- stage procedure previously described (1). All media were sterilized in an autoclave at 121°C for 15 min at 15 lb/in2 before use. Small-scale fermentations were conducted in 25 ml of medium held in 125-ml cotton- plugged Erlenmeyer flasks, which were shaken at 250 rpm with a 1-inch stroke at 26°C. Larger-scale incu- bations were performed under the same conditions in 1-liter flasks containing 200 ml of medium. Vindoline was added to the 24-h second-stage cul- tures in dimethylformamide (125 mg of vindoline per ml) to a final concentration of 0.5 mg/ml of culture medium. Samples (4 ml) were withdrawn at various time intervals, adjusted to pH 9.0 with 10% NH40H, and extracted with 1 ml of ethyl acetate, and 30 p1 of the extracts was examined by TLC. Controls were used to verify that metabolites ob- 601 on April 23, 2020 by guest http://aac.asm.org/ Downloaded from