Characterization of 17β-estradiol 3-(β-D-glucopyranoside) and 17-(α-D-glucopyranoside) as the metabolites of 17β-estradiol in the cultured ovaries of the silkworm,Bombyx mori

Abstract

Summary. The structures of the metabolites formed upon incubation of 17fl-estradiol with the ovaries of silkworm, Bombyx mori, have been determined as 17fl-estradiol 3-(fl-D-glucopyranoside) (1) and 17-(c~-D-glucopyranoside) (2) by spectroscopic means.Key words. Silkworm; Bombyx mori ; 17fl-estradiol metabolites; ovaries.1H NMR data (400 MHz, recorded in CDC13) of the pentaacetates (3) and (4) 2), respectively. The obtain materials for structure elucidation, non-labeled substrate was incubated and metabolites were isolated according to procedures essentially the same as previously reported< 17/?-estradiol (100 gg) was incubated in Grace's insect medium (10 ml) in the presence of the ovarian tissues (40 heads, dissected from the pupae 60 h after larval-pupal ecdysis) of B. mori for 5 h.The incubation was repeated 5 times. Apolar and polar forms of steroids were separated by silicic acid column chromatography with benzene-methanol, 9 : 1, and methanol, respectively. For the isolation of apolar compounds (containing A and B), eluate from the silicic acid column was applied to TLC (aforementioned conditions). The bands corresponding to A and B were scraped from the TLC plate and eluted with methanol, and purified by HPLC using a reversed phase column Wakogel ODS-10K (4.0 mm i.d. x 50 cm) and solvent system of methanol-water, 3:2, flow rate being 1 ml/min. Fractions eluted at 12.2 rain and 17.0 rain 6 afforded the metabolite A (80 gg)7, UV (MeOH) 2max 278 rim, and B (20 gg)7, UV (MeOH) 2~ X 280 nm.At the outset of the work, we thought A and B might be a hydroxylated estradiol derivative such as estriol, judging from their mobility in HPLC. This, however, turned out to be incorrect after the tH NMR measurement of these metabolites. The 1H NMR data 8 (in CD3OD ) revealed that the two metaboIRes are mono-sugar substituted estradiols; A has the substitution at 3-position with 17-OH being intact; whereas B has the substitution at 17-position with 3-OH being intact, although the overlapped signals at 6 3.3-3.7 disturbed a detailed analysis.FAB mass spectra of (1) and (2) both exhibited a peak at m/z 457[presumably M + (C24H3707)+Na]. Further, the peracetylated compounds (3) and (4), obtained from (1) and (2), respectively, by treatment with Ac20-pyridine at room temperature, showed a peak at m/z 644 (M +, C34H44012 , corresponds to the pentaacetate of (1) and (2)) in their FD mass spectra. These mass spectral data indicated that the sugar moiety is a hexose. The analysis of the ~H NMR data of the penta-acetates (3) and (4) allowed the assignation of the hexose as glucose. All the hydrogen signals of the glucose moiety were unambiguously assigned as listed in the table by the aid of decoupling experiments, and the coupling constants between adjacent hydrogens were quite reasonable as for the J-values of glucose (acetate).Comparison of the tH NMR. data of (1) and (2)

Structures of the Pregnenediol Tri- and Di-Glucosides from Eggs of the Tobacco Hornworm, Manduca sexta

“…'H-nmr spectra were recorded on peracetylated derivatives. Peracetylation (6)(7)(8) simplified the spectra by avoiding the CHOH coupling on the glucose moieties, removing O-H signals, and improving solubility in CDC13 and C6D6. Spectra were recorded in both these solvents; CDC13 was generally more useful for literature comparisons, but, in several cases, superior resolution was realized with C6D6.…”