7p-Hydroxyhyoscyamine

In this article, we demonstrate the establishment of the adventitious and the hairy root cultures of several solanaceous plant species including Datura, Duboisia, Hyoscyamus, and Scopolia etc. for the production and biosynthetic studies of tropane alkaloids. In addition, the isolation and structural elucidation of the new tropane alkaloid 7P-hydroxyhyoscyamine and the piperidone alkaloid hyalbidone from them is also presented. 

Synthesis ofHPLC reference substances (littorine and 7p-hydroxyhyoscyamine)... 

Acid hydrolysis of this new alkaloid with 10% HCl gave (-)-tropic acid and a hydrolysate whose spectral data, H-and C-NMR and electron-impact (El) MS, were completely identical with that of 3a, 6p-dihydroxytropane HCl. This evidence indicated the hydrolysate to be the enantiomer of 3a, 6P-dihydroxytropane HCl. Concomitant with the structural confirmation by preparing the alkaloid from (-)-scopolamine HBr using Raney Ni, this new tropane alkaloid was characterized as 7P-hydroxyhyoscyamine [12]. 

Table 3 C-NMR spectral data of 7P-hydroxyhyoscyamine, 6p-hydroxyhyoscyamine, 3a, 6P-dihydroxytropane HCl and tropic acid at 67.5 MHz (5 values, in CDCI3, in acetone-dg + D2O, " in acetone-dg).

The tropane alkaloids isolated together with hyalbidone were identified as hyoscyamine, 6p-hydroxyhyoscyamine, 7P-hydroxyhyoscyamine, scopolamine, littorine, tropine, P-tropine and tropinone (Fig.l). 

The adventitious roots of H. albus and H. niger showed the highest level of 6P- and 7p-hydroxyhyoscyamines. The amount of hyoscyamine in the adventitious roots of H. albus increased remarkably when the roots were cultured in MS or 1/2 MS medium and it became the main alkaloid. In contrast, scopolamine was the main constituent of the adventitious roots of H. albus in Gamborg B5 (B5) [16] and WP media. Thus, in Hyoscyamus species, the ratio of tropane alkaloids produced during the culture pteriod seemed to depend mainly on the culture conditions and not on the species, as described by Hashimoto et al. [17]. In adventitious roots of H. albus the production of 7P-hydroxyhyoscyamine was at its highest level when the roots were cultured in WP medium, whereas the adventitious roots of D. innoxia and S. tangutica did not produce any detectable level of 7P-hydroxyhyoscyamine. In the Duboisia hybrid a trace amount of 7p-hydroxyhyoscyamine was detected. In the adventitious roots of H. aureus, H. muticus and H. pusillus, traces of 7P-hydroxyhyoscyamine were detected (Table 4). 

In the hairy roots, transformed with A. rhizogenes 15834, hyoscyamine was the main alkaloid only for the first two weeks of culture in phytohormone-free WP liquid medium. After that time scopolamine became the major alkaloid. The production of scopolamine increased rapidly and it became almost double the amount of hyoscyamine at the end of the culture period. The yield of 6P-hydroxyhyoscyamine and 7P-hydroxyhyoscyamine produced in these hairy roots was comparable to that in the adventitious roots (Fig.7). 

To increase the growth of hairy roots, the influence of the sucrose concentration in the WP liquid medium was tested (Fig. 10). The fastest growth was obtained with 8% sucrose (8.91 g fresh weight after 19 days). A similar effect was observed to that described in the previous experiment, i. e. the total alkaloid content decreased with the higher sucrose concentration in the medium, whereas the amount of 7P-hydroxyhyoscyamine increased up to 0.012% dry weight in the media with 8% and 10% sucrose. The content of littorine produced by the hairy roots particularly decreased with higher sucrose concentrations in the medium. 

In the hairy roots cultured in WPN03 medium, over 30 mg of tropane alkaloids was produced in one 100 ml flask (50 ml medium) within 28 days of culture (Fig. 13). This was S to 6-fold largest amount to that produced in the hairy roots cultured in the other media. Hyoscyamine was the main alkaloid produced by the hairy roots during the entire culture period in all media tested. In the hairy roots cultured in BS medium the highest content of scopolamine was obtained, while the content of 7P-hydroxyhyoscyamine and littorine was comparably low during the entire culture period. On the other hand littorine was produced in larger amounts in all WP media. Until 18 days... 

In a few species scopolamine, normally the final member of the pathway, may be transformed to 7p-hydroxyhyoscyamine (presumably by cleavage of the 6p,7P-epoxide group of the aminoalkanol moiety) or anisodine (presumably by hydroxylation at C-2 of the tropic acid moiety) thus representing a branched... 

Table 2 H-NMR spectral data of 7P-hydroxyhyoscyamme and 6p-hydroxyhyoscyamine at 270 MHz (5 values, in CDClj).

Tropinone (20) is a key intermediate in the synthesis of many tropane alkaloids, for example, hyoscyamine (18) [an ester of tropine (21) and (5)-tropic acid (22)] and scopolamine (17) (Leete, 1990). Scopolamine is formed from 6p-hydroxyhyoscyamine by a direct attack of the 6(3-hydroxyl group at C-7, displacing the 7p-hydrogen. The requisite enzyme, 6 3-hydroxyhyoscyamine epoxidase, requires 2-keto-glutarate and oxygen as cofactors (Leete, 1990). 

The alkaloids traditionally named 6 3-hydroxyhyoscyamine and 7P-hydroxyhy-oscyamine represent two natural diastereoisomers, namely (+)-(3R,6R,2 S)- and (-)-(3S,dS,2 -6P-hydroxyhyoscyamine, respectively (Munoz et al. 2(X)6). This is again a consequence of the C-3 (pseudo)stereocenter of 3-hydroxytropane. 

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