Epoxidation in Vivo of Hyoscyamine to Scopolamine Does Not Involve a Dehydration Step

Abstract

Hyoscyamine is epoxidized to scopolamine via 6,6-hydroxyhyoscyamine in several solanaceous plants. 6,7-Dehydrohyoscyamine has been proposed to be an intermediate in the conversion of 6#-hydroxyhyoscyamine to scopolamine on the basis of the observation that this unsaturated alkaloid is converted to scopolamine when fed to a Datura scion. To determine whether a dehydration step is involved in scopolamine biosynthesis, 16-'8016j0-hydroxyhyoscyamine was prepared from I-hyoscyamine and '02 using hyoscyamine 6f6-hydroxylase obtained from root cultures of Hyoscyamus niger L. When 16-'8016,-hydroxyhyoscyamine was fed to shoot cultures of Duboisia myoporoides R. BR., the labeled alkaloid was converted to scopolamine which retained "O in the epoxide oxygen. It is concluded that 6,6-hydroxyhyoscyamine is converted in vivo to scopolamine without a dehydration step.Scopolamine, the epoxide of hyoscyamine, is one of the major alkaloids which accumulate in the family Solanaceae (7). The formation of the epoxide bridge in scopolamine begins with the hydroxylation of L-hyoscyamine to Hyos-OH' by a 2-oxoglutarate-dependent dioxygenase, hyoscyamine 6#-hydroxylase (12 De-hyos (scheme 1 in Fig. 1); '"0 should be retained if dehydration is not involved (scheme 2 in Fig. 1). MATERIALS AND METHODSChemicals. '"02 (>99 atom %) was purchased from CEA, France. L-Hyoscyamine hydrobromide and scopolamine hydrobromide were obtained from Nakarai Chemicals, Kyoto. Hyos-OH hydrobromide was prepared using hyoscyamine 6#-hydroxylase from root cultures of Hyoscyamus niger L. (12). De-hyos was synthesized by the method of Sharpless et al. (23). Other chemicals were obtained as described previously (12).Root and Shoot Cultures. Culture conditions for root cultures ofH. niger L. have been reported elesewhere (12). Shoot cultures ofDuboisia myoporoides R. BR. were initiated according to Endo et al. (6) and were subcultured on a Gyrotory shaker (model G 10-21, New Brunswick Scientific, Edison, NJ) at 100 rpm and 25°C under light (2,700-4,500 lux) in 100-ml flasks containing 25 ml of liquid B5 medium (10) supplemented with 3% (w/v) sucrose and 10 Mm 6-BA. In the feeding experiments, alkaloid solutions were neutralized with HCI when necessary and were sterilized by passing through 0.22 Am membrane filters. These were added to 10 ml ofthe above culture medium in 50-ml flasks to a final concentration of0.2 mm. The shoot cultures containing young leaves were inoculated in each flask and cultured under the above conditions.Partial Purification of Hyoscyamine 6,8-Hydroxylase. Hyoscyamine 6t3-hydroxylase was partially purified from root cultures ofH. niger. The hydroxylase in the crude extract was precipitated between 60 and 80% saturation of (NH4)2SO4 and subsequently chromatographed on a DEAE-Toyopearl 650M column. The detailed purification procedure has been reported (13). The partially purified enzyme preparation (about 23-fold in specific activity) was concentrated using Amicon

Metabolic engineering of medicinal plants: transgenic Atropa belladonna with an improved alkaloid composition.

“…Tropane alkaloids were extracted from plant tissues and then purified and quantified by gas-liquid chromatography, as described (24). Alkaloids were identified by comparing their mass spectra with those of authentic samples (25). Next, four plant parts (leaf, stem, main root, and branch root) were analyzed by immunoblotting and by enzyme assays (Fig.…”

On the Transformation of (S)-2-Hydroxypropylphosphonic Acid into Fosfomycin in Streptomyces fradiae—A Unique Method of Epoxide Ring Formation

“…b-Hydroxyphosphonic acid 7 is oxidatively cyclised (loss of two hydrogen the biosynthesis of an oxirane is reminiscent of the biosynthesis of the plant alkaloid scopolamine, where 6b-hydroxyhyoscyamine is dehydrogenated to scopolamine, an epoxide. [12] Contrary to the biosynthesis of fosfomycin, the carbonÀoxygen bond is formed with retention of configuration in the scopolamine synthesis. Normally, epoxides are biosynthesised by monooxygenases from olefins.…”

Two‐step epoxidation of hyoscyamine to scopolamine is catalyzed by bifunctional hyoscyamine 6β‐hydroxylase

“…Previous feeding experiments with alkaloid precursors [1,2] have suggested that the 6,7-epoxide bridge of scopolamine is formed by hyoscyamine by way of B/?-hydroxyhyoscyamine {Fig. 1).…”