6p-Hydroxyhyoscyamine

H Hyosoyamine, 6P 6p-hydroxyhyoscyamine, S Scopolamine, T.A Total yield referred to biomass formed, Yp/si Alkaloid yield referred to substrate 

The percentage values of 6p-hydroxyhyoscyamine and scopolamine are the means of three independent determinations which differs in no more than 10%. 

Hyoscyamine, anisodamine (6p-hydroxyhyoscyamine) and scopolamine are the most important tropane alkaloids from a therapeutical point of view. These alkaloids are well known anticholinergic agents and were historically used in medicine. 

In all Argentinean clones, the 6P-hydroxyhyoscyamine content predominates over hyoscyamine and scopolamine, while in Colombian clones the main alkaloid was hyoscyaniine. In addition, the Argentinean clones produced the highest 6P-hydroxyhyoscyamine concentration. In these cultures, scopolamine constituted about 4% of the three alkaloids measured and 6P-hydroxyhyoscyamine about 68%. 

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).

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

It was estimated that (—)-scopolamine was biosynthesized from 6P-hydroxyhyoscyamine formed via (—)-hyoscyamine. In this biosynthetic pathway, the two routes (a) and (b) could be considered. When [6- 0]6P-hydroxyhyoscyamine was fed to the cultured stems and leaves of Duboisia myoporoides, it was found that (—)-scopolamine retained 100% of the [9], The experimental result indicated that biosynthetic route (b) was taken in the pathway from the hydroxy precursor to the epoxy product [10]. 

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

When the reaetion was incubated for 2hs, the tagged protein was able to produce a 15% of 6P-hydroxyhyoscyamine. The other 85% remained as hyoscyamine. On the other hand, the untagged protein produced a 53.7% of the intermediate in the same incubation time. 

A simple modification introduced in a stirred tank bioreactor allowed us the establishment of B. Candida hairy root cultures for scopolamine and 6P-hydroxyhyoscyamine production with increased alkaloid concentration compared to the Erlenmeyer flask cultures [28], It is worth pointing out that these results are potentially applicable to perform the rational scale-up of the process [28]. 

Furthermore, the fast atom bombardment (FAB) mass spectrum (MS) with the prominent [M+H]" " peak at m/z 306 indicated the same molecular mass as that of 6P-hydroxyhyoscyamine. From these spectral data, this new tropane alkaloid was presumed to be the structural isomer of 6P-hydroxyhyoscyamine. 

Hyoscyamine (duboisine) and the racemate atropine are mACh-R antagonists and a number of atropine derivatives also have this property, namely anisodamine (6P-hydroxyhyoscyamine), 7 (3-hydroxyhyoscyamine, hyoscine (6,7-epoxyhyoscyamine or scopolamine), benzoyltropein (tropine benzoate), littorine (tropine a-hydroxyphenylpropionate), tigloidine (pseudotropane tiglate) and tropacocaine (pseudotropine benzoate). The further derivatives apoatropine (a-dehydrohyoscyamine) and tropine are very toxic. 

According to the features mentioned above, the fact that scopolamine has a 10-times higher commercial demand than that of hyoscyamine [17] and the increased interest in 6P-hydroxyhyoscyamine for medical applications, we constructed transgenic S. cerevisiae strains which are able to produce the H6H enzyme from B. Candida [29]. 

Kursinszki et al. [36] have found that D. innoxia and A. belladonna hairy root cultures behaved in a similar way to Argentinean B. Candida hairy roots. In these cultures 6P-hydroxyhyoscyamine was also the predominant alkaloid detected [27, 36]. 

It is frequently mentioned that there is a decrease in productivities when processes are scale-up from Erlenmeyer flasks to bioreactors [66]. However, our studies show that 6P-hydroxyhyoscyamine volumetric productivity was 3-fold higher in the bioreactor system [28]. 

The biotechnological processes based on undifferentiated cell culture s were not successful according to the low productivity. For this reason, the investigations in order to improve 6P-hydroxyhyoscyamine and scopolamine yields are based mainly on hairy root culture systems [6,43], 

The results reported by Zarate et al. [62] about the clone 19 of baetica hairy roots were coincident to the results that we found for Colombian hairy root clones. In this case, the scopolamine content predominated over the 6P-hydroxyhyoscyamine one [27]. 

Table 2 shows the kinetic parameters calculated for the bioreactor and the Erlenmeyer system [28], It is interesting to note that the growth index found in bioreactor cultures was a 36% higher than that reached in Erlenmeyer flask processes showing an improvement in the liquid-phase culture environment by the application of the modified bioreactor [47]. In this case, 6P-hydroxyhyoscyamine was also the predominant alkaloid (Table 2). 

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). 

Both alkaloids have (+) and (-) forms but only the (-) hyoscyamine and (-) scopolamine are active. The biosynthetic pathway of tropane alkaloids, Fig. (1) is not totally understood, especially at the enzymatic level. Edward Leete has pioneered the biosynthetic studies of tropane alkaloid since 1950"s using whole plants and isotope labels [85-86]. The tropane alkaloid hyoscyamine is bioconverted by the enzyme H6H (hyoscyamine 6p-hydroxylase, EC 1.14.11.11) to scopolamine via 6p-hydroxyhyoscyamine. Hyoscyamine is the ester of tropine and (S)-tropic acid. The (S)-tropic acid moiety derives from the amino acid L-phenylalanine, while the bicyclic tropane ring derives from L-omithine primarily or L-arginine via tropinone. Tropinone is stereospecifically reduced to form either, tropine which is incorporated into hyoscyamine, or on the other hand into pseudotropine which proceeds to calystegines, a group of nortropane derivates that were first found in the Convolvulaceae family [87]. 

Through the incorporation studies using D. innoxia, it was found that a P-ketothioester was the intermediate in the biosynthesis of (-)-hyoscyamine and (-)-scopolamine [3-7].Tropinone is formed from the P-ketothioester, and is stereoselectively reduced by a NADPH-dependent oxidoreductase TR-1 to give tropine.Then littorine is formed by adding a phenyllactic acid moiety derived from phenylalanine via phenylpyruvic acid. Regarding the incorporation of phenyllactic acid into tropine and its transformation, it was found that [1,3A C2](—)-hyoscyamine was obtained when [1,3- C2] phenyllactic acid was incorporated into D. innoxia [8]. A transformation therefore occurred on littorine to form (—)-hyoscyamine, and scopolamine was biosynthesized from 6P-hydroxyhyoscyamine by oxidation as described below. 

---