Isolation and chemical structure of aklanonic acid, an early intermediate in the biosynthesis of anthracyclines.

Abstract

The fermentation, isolation and structure elucidation of aklanonic acid are described. The compound was isolated from fermentations of Streptomyces strain ZIMET 43,717. Aklanonic acid is a yellow-orange crystalline substance, melting at 203-204'C (dec), having the molecular formula C:1H1sOs, and possessing UV maxima at 258, 282 (sh) and 438 nm (CHCI3). In dimethyl sulfoxide or pyridine aklanonic acid is unstable and a new compound (aklanone) is formed as a conversion product.The elucidation of the structures has shown that aklanonic acid and aklanone are derivatives of 1,8-dihydroxyanthraquinone.During our antibiotic screening program, a Streptomyces strain (designated as Streptomyces sp.ZIMET 43,717) was found to produce a new anthracycline-related pigment. The compound, which has been named aklanonic acid, is not active as an antibiotic. However, aklanonic acid is of particular interest because we have found that this compound was biotransformed to structurally different anthracyclinones in feeding experiments with several pigment-negative mutants"".Its role in the biosynthetic pathway of anthracyclines is now under study. The present paper is concerned with the physico-chemical properties of aklanonic acid and the results of the structural elucidation. The biological characteristics of the producing strain, which is unable to form aerial mycelia and spores, will be the subject of a separate communication. Results Isolation and Physico-chemical PropertiesAklanonic acid was obtained mainly from the mycelium by extraction with acetone. After purification by column chromatography and recrystallization from ethyl alcohol the compound was obtained as pure yellow-orange needles with a melting point of 203' 204°C (dec). Aklanonic acid is only slightly soluble in water, moderately soluble in ethyl alcohol, methanol, acetone and chloroform, and soluble in dimethyl sulfoxide and a solution of sodium bicarbonate in water. In concentrated sulfuric acid and sodium hydroxide the compound dissolves with a red color. When dissolved in dimethyl sulfoxide or pyridine aklanonic acid was rapidly converted to a new compound which we have named aklanone. The UV and visible spectrum of aklanonic acid in chloroform contains maxima at 258, 282 (sh), and 438 nm (s 23,300, 19,300, and 10,300). The IR spectrum in potassium bromide had characteristic absorptions at 1622, 1670 and 1700 cm-1.Aklanonic acid has the molecular formula C21H16O6. Attempts to derive the exact molecular formula based upon accurate molecular weight measurements by mass spectrometry and microanaly-

Minimal Streptomyces sp. strain C5 daunorubicin polyketide biosynthesis genes required for aklanonic acid biosynthesis

“…For extraction, the pH of each culture broth was adjusted to 1.5 with 6 N HCl, and the acidified broths were extracted with an equal volume of chloroform, reduced to dryness, and reconstituted in 100 l of chloroform. Aklanonic acid produced by the recombinant strains was compared with authentic aklanonic acid obtained from K. Eckardt (9) in parallel and by cochromatography, using TLC as described previously (4). Aklanonic acid also was analyzed by HPLC as previously described (4, 7), using a C 18 reverse-phase column and a solvent system of methanol:water:glacial acetic acid (65:30:5) (4).…”

“…strain C5 and Streptomyces peucetius ATCC 29050, are synthesized by condensation of nine extender units derived from malonyl coenzyme A (CoA) onto a propionyl moiety to make a theoretical C 21 polyketide intermediate (16,(33)(34)(35) which is reduced at C-9 (34), cyclized, and aromatized to form aklanonic acid ( Fig. 1), the first characterized chromophore in these pathways (9,16,(33)(34)(35). Aklanonic acid is converted in four enzymatic steps to ε-rhodomycinone (1,4,(33)(34)(35), which is then glycosylated and modified by a series of reactions to form daunorubicin and doxorubicin (6,16,33,35).…”

Biosynthesis of anthracyclines: analysis of mutants of Streptomyces sp. strain C5 blocked in daunomycin biosynthesis

“…C5 dauA mutants. Eckardt et al (1985) isolated aklanonic acid, an anthraquinone metabolite accumulated by Streptomyces sp. ZIMET 437 17, and showed that this compound and its chemically produced methyl ester could be converted in vivo by blocked mutants of the daunomycin-producing strain, S. griseus IMET JA 5142, to aklavinone, an intermediate in daunomycin biosynthesis .…”

“…This suggests that aklanonic acid or analogous anthraquinones may be universal intermediates in the formation of various naphthacenequinone polyketides. AAME and aklaviketone have previously been converted to anthracyclines in vivo Eckardt et al, 1985;Schumann et al, 1986). When spores of the maggiemycin-accumulating dauE mutant, SC5-24, were treated with NTG, doubly-mutated strains that accumulated aklanonic acid (dauC, E), AAME (dauD, E ) or aklaviketone (dauE, F ) were isolated.…”

In vivo and in vitro bioconversion of epsilon-rhodomycinone glycoside to doxorubicin: functions of DauP, DauK, and DoxA

“…1) are clinically important anthracycline chemotherapeutic agents (2, 11, 17), which are synthesized by a type II polyketide synthase (23,(42)(43)(44). The aglycone is formed by condensing nine extender units derived from malonyl coenzyme A onto a propionyl moiety to make a C 21 polyketide intermediate (21,23,(42)(43)(44)(45)50), which is reduced at C-9 from the carboxy terminus (43, 44), cyclized, and aromatized to form aklanonic acid (18,19,23,40,(42)(43)(44). Aklanonic acid is converted in four steps to ε-rhodomycinone (3,7,15,16,30,40,42,44), an intermediate that is accumulated in large quantities by most daunorubicin-producing strains (27,42,44,46).…”