The putative biosynthetic gene cluster for the ␣-glucosidase inhibitor acarbose was identified in the producer Actinoplanes sp. 50/110 by cloning a DNA segment containing the conserved gene for dTDP-D-glucose 4,6-dehydratase, acbB. The two flanking genes were acbA (dTDP-D-glucose synthase) and acbC, encoding a protein with significant similarity to 3-dehydroquinate synthases (AroB proteins). The acbC gene was overexpressed heterologously in Streptomyces lividans 66, and the product was shown to be a C 7 -cyclitol synthase using sedo-heptulose 7-phosphate, but not ido-heptulose 7-phosphate, as its substrate. The cyclization product, 2-epi-5-epi-valiolone ((2S,3S,4S,5R)-5-(hydroxymethyl)cy-

To study the biosynthesis of the pseudotrisaccharide antibiotic, validamycin A (1), a number of potential precursors of the antibiotic were synthesized in (2)H-, (3)H-, or (13)C-labeled form and fed to cultures of Streptomyces hygroscopicus var. limoneus. The resulting validamycin A from each of these feeding experiments was isolated, purified and analyzed by liquid scintillation counting, (2)H- or (13)C NMR or selective ion monitoring mass spectrometry (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 open-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 is efficiently incorporated into 1, followed by dehydration, although a low level of incorporation of 2-epi-valienone (15) is also observed. Reduction of 11 affords validone (12), which is also incorporated specifically into 1, but 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 not incorporated into 1, although each of these amines has been isolated from the fermentation, with 3 being most prevalent. Demonstration of in vivo formation of [7-(3)H]validamine ([7-(3)H]-3) from [7-(3)H]-12 suggests that 3 may be a pathway intermediate and that the nonincorporation of [7-(3)H]-3 into 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-glucose unit to the 4'-position of 13 then completes the biosynthesis of 1. Other possibilities for the mechanism of formation of the nitrogen bridge between the two pseudosaccharide units are also discussed.

The biosynthetic pathway leading to the mC7N cyclitol (valienamine) moiety of acarbose (1) in Actinoplanes sp. strain SN 223/29 has been studied using 3H-, 2H-, and 13C-labeled cyclitols. These precursors were synthesized from d-glucose or d-mannose as starting materials. The feeding experiments demonstrated that cyclitols having the same stereochemistry at C-2 as the valienamine moiety of acarbose; i.e., valienone, valienamine, valiolone, valiolamine, and 1-epi-valienol, were not incorporated and thus are not plausible intermediates in 1 biosynthesis. 2-epi-Valiolone (10b), which has the same stereochemistry as the presumed open-chain precursor, sedoheptulose 7-phosphate, was also not incorporated. However, its C-5 epimer (10a) was incorporated efficiently and specifically into the valienamine moiety of 1. Surprisingly, the dehydrated form of 2-epi-5-epi-valiolone, 2-epi-valienone, was not incorporated. This suggests that 2-epi-5-epi-valiolone must be converted directly into the pseudodisaccharide moiety of acarbose without the intervention of other free cyclitol intermediates. This may occur by linkage to the amino group of TDP-4-amino-4,6-dideoxyglucose to form the imine, epimerization at C-2 to the correct stereochemistry, dehydration between C-5 and C-6 aided by enamine formation, and finally reduction to the amine. It is proposed that these reaction steps all take place on a single enzyme without free intermediates. Alternative mechanistic possibilities are also discussed.

Tubulin polymerization inhibitors had emerged as one of promising anticancer therapeutics because of their dual mechanism of action, i.e. apoptosis by cell-cycle arrest and VDA, vascular disrupting agent. VDAs are believed to be more efficient, less toxic, and several of them are currently undergoing clinical trials. To identify novel tubulin inhibitors that possess potent cytotoxicity and strong inhibition of tubulin polymerization as well as potent in vivo antitumor efficacy, we have utilized benzophenone scaffold. Complete SAR analysis of newly synthesized analogues that were prepared by incorporation of small heterocycles (C2, C4, and C5 position) into B-ring along with the evaluation of their in vitro cytotoxicity, tubulin polymerization inhibition, and in vivo antitumor activity allowed us to identify 22 (S516). Compound 22 was found to have potent cytotoxicity against several cancer cells including P-gp overexpressing MDR positive cell line (HCT15). It also induced cell cycle arrest at G(2)/M phase, which is associated with strong inhibition of tubulin polymerization. Its in vivo efficacy was improved by preparing its (l)-valine prodrug, 65 (CKD-516), which together with greatly improved aqueous solubility has shown marked antitumor efficacy against both murine tumors (CT26 and 3LL) and human xenogratfs (HCT116 and HCT15) in mice.

Chemists, particularly those of the organic or biochemical persuasion, tend to be preoccupied with large molecules and complex structures, not with small and simple compounds. Yet, despite this general mindset, chiral (stereogenic) methyl groups, the smallest asymmetric objects ever synthesized, have for some reason managed to capture our imagination. Ever since the initial disclosure1•2 of methods to synthesize the enantiomers of a methyl group carrying one atom each of , 2H, and 3H and to distinguish the R from the S enantiomer of such an entity, there has been considerable fascination with chiral methyl groups and their use to analyze stereochemical problems in chemistry and particularly in biochemistry. In the following we review some applications of chiral methyl groups in work carried out by our laboratory.