Enzymatic glycosylation of nonbenzoquinone geldanamycin analogs via Bacillus UDP-glycosyltransferase

Abstract

Geldanamycin (GM) is a naturally occurring anticancer agent isolated from several strains of Streptomyces hygroscopicus. However, its potential clinical utility is compromised by its severe toxicity and poor water solubility. For this reason, considerable efforts are under way to make new derivatives that have both good clinical efficacy and high water solubility. On the other hand, glycosylation is often a step that improves the water solubility and/or biological activity in many natural products of biosynthesis. Here, we report the facile production of glucose-conjugated nonbenzoquinone GM analogs using the Bacillus UDP-glycosyltransferase BL-C. Five aglycon substrates containing nonbenzoquinone aromatic rings were chosen to validate the in vitro glycosylation reaction. Putative glucoside compounds were determined through the presence of a product peak(s) and were also verified using LC/MS analyses. Further, the chemical structures of new glucoside compounds 6 and 7 were elucidated using spectroscopy data. These glucoside compounds showed a dramatic improvement in water solubility compared with that of the original aglycon, nonbenzoquinone GM.

Fig 1

Structures of nonbenzoquinone geldanamycin analogs (analogs 1 to 5), glucoside nonbenzoquinone geldanamycin analogs (analogs 6 to 10), and geldanamycin. Compound 1, 18-dehydroxy-17-demethoxyreblastatin; compound 2, 18-dehydroxy-17-O-demethylreblastatin; compound 3, 18-dehydroxy-17-O-demethyl-4,5-dehydroreblastatin; compound 4, 17-demethoxy-reblastatin; compound 5, 17-demethoxy-15-hydroxylreblastatin.

Fig 2

LC/MS analysis of the in vitro glycosylation reaction with 17-demethoxy-reblastatin. (A) Total ion chromatogram of in vitro glycosylation reaction mixture. The arrow indicates a new peak from the in vitro glycosylation reaction. The asterisk indicates 17-demethoxy-reblastatin. (B) MS spectra of selected ion peak at 4.92 min. The ion peak at m/z 679 [M-H]⁻ (m/z 725 [M+HCOOH]⁻) corresponds to a glucoside form of 17-demethoxy-reblastatin as expected. (C) MS spectra of 17-demethoxy-reblastatin (m/z 517 [M-H]⁻) at 6.57 min.

Fig 3

LC/MS analysis of the in vitro glycosylation reaction with 18-dehydroxy-17-O-demethylreblastatin. (A) Total ion chromatogram of the in vitro glycosylation reaction mixture. The arrows indicate new peaks from the in vitro glycosylation reaction; the asterisks indicate a nonbenzoquinone GM, 18-dehydroxy-17-O-demethylreblastatin. (B) MS spectra of 18-dehydroxy-17-O-demethylreblastatin (m/z 517 [M-H]⁻) at 6.50 min. (C) MS spectra of selected ion peak at 4.99 min. (D) MS spectra of selected ion peak at 4.58 min. (E) MS spectra of selected ion peak at 3.87 min.

Fig 4

Western blot analysis of the indicated Hsp90 client protein expression. Each compound, at various concentrations, was evaluated for its ability to regulate several client proteins as described in Materials and Methods. (2), compound 2; (7), compound 7.

Fig 5

Comparison of the HPLC data from analysis of the glycosylation reaction mixture. (A) Total enzymatic reaction mixture. The glycosylation conversion rate was approximately 40%. (B and C) The (B) solvent layer and (C) water layer after the ethylacetate extracted enzyme reaction mixture. The peaks at 7.1 min (S) represent nonbenzoquinone GM (17-demethoxy-reblastatin), and those at 5.5 min (P) represent glucoside product. The peaks at 1.77 min (A) and 1.80 min (C) represent water-soluble enzyme reaction material, i.e., UDP-glucose, enzyme, etc. mAU, milli-absorbance units.