Biosynthesis of the validamycins: Identification of intermediates in the biosynthesis of validamycin A by Streptomyces hygroscopicus var. limoneus

To study the biosynthesis of the pseudotrisaccharide antibiotic, validamyci n A (1), a number of potential precursors of the antibiotic were synthesize d in H-2-, H-3-, Or C-13-labeled form and fed to cultures of Streptomyces h ygroscopicus var, limoneus. The resulting validamycin A from each of these feeding experiments was isolated, purified and analyzed by liquid scintilla tion counting, H-2- or C-13 NMR Or selective;ion monitoring mass spectromet ry (SIM-MS) techniques. The results demonstrate that 2-epi-5-epi-valiolone (9) is specifically incorporated into 1 and labels both cyclitol moieties. This suggests that 9 is the initial cyclization product generated from an o pen-chain C-7 precursor, D-sedoheptulose 7-phosphate (5), by a DHQ synthase -like cyclization mechanism. A more proximate precursor of 1 is valienone ( 11), which is also incorporated into both cyclitol moieties. The conversion of 9 into 11 involves first epimerization to 5-epi-valiolone (10), which i s efficiently incorporated into 1, followed by dehydration, although a low level of incorporation of Zepi-valienone (15) is also observed. Reduction o f 11 affords validone (12), which is also incorporated specifically into, b ut labels only the reduced cyclitol moiety. The mode of introduction of the nitrogen atom linking the two pseudosaccharide moieties is not clear yet. 7-Tritiated valiolamine (8), valienamine (2), and validamine (3) were all n ot incorporated into 1, although each of these amines has been isolated fro m the fermentation, with 3 being most prevalent. Demonstration of in vivo f ormation of [7-H-3]validamine ([7-H-3]-3) from [7-H-3]-12 suggests that 3 m ay be a pathway intermediate and that the nonincorporation of [7-H-3]-3 int o 1 is due to a lack of cellular uptake. We thus propose that 3, formed by amination of 12, and 11 condense to form a Schiff base, which is reduced to the pseudodisaccharide unit, validoxylamine A (13). Transfer of a D-glucos e unit to the 4'-position of 13 then completes the biosynthesis of 1. Other possibilities for the mechanism of formation of the nitrogen bridge betwee n the two pseudosaccharide units are also discussed.