Biosynthesis of aklavinone and aclacinomycins.

Anthracycline antibiotics aclacinomycins (ACMs) are aklavinone glycosides with variation in the trisaccharide moiety and were produced by Streptomyces galilaeus MA144-M1. Their production" and structural elucidation') were previously reported. By the isolation of various mutants') and the conversion of hypothetical precursors fed in the cultural medium'), we found that ACMs were formed by a step-wise glycosidation of aklavinone followed by enzymatic modification of the terminal sugars). We report here the 11C NMR study of the biosynthesis of aklavinone by the incorporation of "Cacetate and "C-propionate and the radioisotopic analysis of ACM labeled with 14C-precursors. For the incorporation experiments, a blocked mutant strain 3AR-33, which accumulated aklavinone by the genetic loss of glycosidation ability'), and an aclacinomycin A (ACM-A) producing strain 6U-213) were inoculated into a 500-m1 Erlenmeyer flask containing 50 ml of productive medium consisting of 1.5 % potato starch, 1.0 % glucose, 3.0 % soybean meal (Meat, Ajinomoto Co.), 0.1 % yeast extract, 0.3 NaCI, 0.1 % KZHPO4, 0.1 % MgSO4.7H2O, 0.0007 ; CuSO4.5H2O, 0.0008 % MnCl2.4H,O, 0.0002 % ZnSO4.5HZO and 0.0001 % FeSO4 7HZO, pH 7.41). During a rapid productive phase of antibiotic (around 20 35-hour cultivation), each 13Cor "C-labeled compound was added to the culture and the cultivation at 28°C on a rotary shaker (220 rpm) was continued for one to 20 hours. The cultured broth was then harvested and centrifuged. The crude antibiotic was extracted from the mycelium with acetone and froth the supernatant fluid with chloroform. The isolation and purification of aklavinone and ACM-A from the extracts were performed by preparative layer chromatography using silica gel 60 PF25} (E. Merck) according to the method previously described 2). Solvent systems used were CHCI3 MeOH (50: 1) for aklavinone and CHCI, MeOH (20: 1) for ACM-A. 1"C NMR spectra of the antibiotics were recorded in CDCI3 on a Varian model XL-100 with tetramethyl silane as an internal standard, and the radioactivity was measured by a liquid scintillation spectrometer (Aloka LSC-653) or by a radiochromatoscanner (Aloka JTC-203). 1"C-Labeled aklavinone was obtained from the culture (1.5 liters) of strain 3AR-33 supplemented with [1-13C]acetate (75 mg), [2-13C]acetate (75 mg), [1,2-1"C]acetate (100 mg), or [1-13C]propionate (75 mg); all were purchased from Merck Sharp & Dohme Canada Ltd. The 13C-enriched carbons were determined by comparison of 13C NMR spectra between the labeled and unlabeled antibiotics. The results are shown in Table 1. The incorporation of [1-13C]acetate resulted in enrichment of C-2, 4, 5, 6, 7, 1Oa, Ila, 12a, and 15, while that of [2-13C]acetate enriched C-I, 3, 4a, 5a, 6a, 8, 10, 11 and 12. These indicated the consecutive incorporation of intact acetate in the aklavinone molecule, further confirmed by the fact that all resonances, except those of C-9, 13, 14 and 16, in 13C NMR spectrum of aklavinone enriched with [1,2-13C]acetate were accompanied by two satellite signals due to spin "C -13C coupling as shown in Table 2, while [113C]propionate enriched only one signal at C-9. These results show that ring carbon of aklavinone was made up of nine acetates (or acetylCoA) and one propionate (or propionyl-CoA)