The Biosynthesis of Hyoscyamine

In D. stramonium there exists a close relationship between PMT activity, the morphological state of cultures and the biosynthesis of hyoscyamine. In fact, it was found that when de-differentiation was induced with a mixture of kinetin, 2,4-dichlorophenoxyacetic acid and a-naphthalene-acetic acid, the PMT activity and hyoscyamine biosynthesis were lost [127]. Furthermore, it seems that PMT... 

Hyoscyamine is considered to be biosynthesized from a C4 unit derived from two molecules of malonyl CoA attached to the N-methyl-A -pyrrolinium ion, followed by decarboxylation, cyclization, reduction, and esterification [1,2]. The biosynthetic route through which ornithine becomes putrescine by decarboxylation and is transformed to N-methyl-A -pyrrolinium during the biosynthesis of (—)-hyoscyamine was described in the section of the biosynthesis of nicotine. 

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. 

It has been confirmed that isoleucine but not 3-hydroxy-2-methylbutanoic acid is a precursor for the tiglic acid which is the esterifying acid in some tropane alkaloids [e.g., meteloidine (77) (735)]. In the biosynthesis of meteloidine (77) from 3a-hydroxytropane (1), the hydroxyl groups at C-6 and C-7 are most probably introduced after esterification at C-3 (5) (Scheme 23). In this connection we would point out that scopolamine (89) is a well-known 2,3) metabolite of hyoscyamine (27) and that the reaction proceeds via 6-hydroxyhyoscyamine [(—)-anisodamine (63)] and 6,7-dehydrohyoscyamine (211) (Scheme 26). 

Tropane Alkaloids.—It is known that tropine (26) is a precursor for meteloidine (27),42 and its close relative hyoscyamine (29) is a precursor for scopolamine (28).43 Experiments with samples of (26) labelled with /3-tritium at C-6 and C-7 show that entry of the two /S-hydroxy-groups in (27) must occur with normal retention of configuration since almost complete loss of tritium occurred.44 Tritium was again lost almost completely on formation of scopolamine (28). On the assumption that early conclusions45 on the sequential intermediacy of (30) and (31) in the biosynthesis of (28) are correct, formation of (30) involves normal retention of configuration (loss of half the tritium) and cis-dehydration then occurs to give (31) (loss of remaining tritium). [In these experiments the hyoscyamine (29) isolated showed appropriately no loss of tritium.]44... 

The usefulness of GC-MS analysis for biosynthetic studies was demonstrated by Patterson and O Hagan [74] in their investigation of the conversion of littorine to hyoscyamine after feeding transformed root cultures of Datura stramonium with deuterium-labeled phenyllactic adds. This study complements previous investigations on the biosynthesis of the tropate ester moiety of hyoscyamine and scopolamine [75], where GC-MS played a key role. It also has general relevance in the biosynthetic pathway of tropane alkaloids in the entire plant kingdom [76]. 

Chesters, N.C.J.E., O Hagan, D. and Robins, R.J. (1995a) The biosynthesis of tropic acid the (R)-d-phenyllactyl moiety is processed by the mutase involved in hyoscyamine biosynthesis in Datura stramonium.. Chem. Soc. Chem. Commun., 127-8. 

A correlation of configuration of (-t- )-a-methyltropidine (LXXXIV) therefore gave a clue to the absolute configuration of labeled hyoscyamine. This was achieved by converting it (82) into the V-dimethyl-glutaminol antimer XCa, via the tetraol LXXXVIII and with periodate. The reduction of A,A-dimethyl-L-(-f )-glutamic acid afforded its antimer XCb. Consequently, C-1 was radioactive in hyoscyamine, but, C-5 was not. This ultimate information concerning the biosynthesis of the tropane skeleton had not been anticipated. 

Independent results in Atropa belladonna confirm that it is the 5-amino-group of ornithine which is utilized in the biosynthesis of cuscohygrine (29) and hyoscyamine (30). " Whilst 5-iV-methyl-[ H]ornithine serves as a good precursor for these alkaloids with a major portion of the radioactivity confined to the... 

The biosynthesis of tropane alkaloids has been extensively studied over the last few decades. This is mainly due to the pharmacological importance of compounds such as (-)-hyoscyamine, (-)- scopolamine and (-)-cocaine. An excellent review has been published on that subject by Leete [26]. 

The biosynthesis of alkaloids containing a pyrrolidine ring such as hyoscyamine (18) and hygrine (19) is similar to the biosynthesis of those with a piperidine... 

Proline and ornithine would also appear to be the most probable amino acid precursors of 1-hyoscyamine, and, as such, the source of the nitrogen of the tropane nucleus. The biosynthesis of the tropic acid half of the molecule has not yet received attention. Such investigations as have been made up to the present have been based upon Robinson s (113) proposed tropinone synthesis from ornithine plus acetone, and have been principally concerned with the identification of the original nitrogenous precursor. 

The biosynthesis of cuscohygrine, one of the main alkaloids of belladonna, was examined using C-labeled compounds [11,12]. According to the results, the labeling pattern of C of cuscohygrine obtained 15 days after feeding Cj or C2 labeled sodium acetate to the cultivated belladonna is as shown in the Figure. Namely, the skeleton of this alkaloid is biosynthesized from N-methyl-A -pyrrolidine and acetoacetic acid, as in the case of hyoscyamine biosynthesis. 

It is considered that the biosynthesis of cocaine resembles that of (—)-hyoscyamine and (—)-scopolamine. It was shown, through the feeding experiments using E. coca [2—5], that the P-ketothioester was the biosynthetic intermediate of cocaine, as in the case of (—)-hyoscyamine and (—)-scopolamine. It was also demonstrated that a thioester of benzoic acid derived from phenylalanie was the precursor of the benzoyl moiety of cocaine [6]. 

Genomic and transcriptomic technologies have been used to rapidly identify biosynthetic steps. There are currently over 40,000 expressed enzyme tags (ESTs) generated fi om alkaloid-producing plants that have been used to isolate genes involved in the alkaloid pathway [7]. Some alkaloid biosynthetic steps occur as spontaneous chemical reactions without the use of enzymes, for example, conversion of the intermediate neopine into codeinone in the morphine biosynthetic pathway. Also, some enzymes may catalyze two or more separate reactions in the pathway, for example, hyoscyamine 6-hydroxylase, which carries out two consecutive steps in the scopolamine biosynthetic pathway. Alkaloid biosynthesis also involves compartmentalization. Tissue-specific localization studies have shown that sequential biosynthetic enzymes can occur in distinct cell types [8, 9]. During the biosynthesis of the indole alkaloids vinblastine and vincristine in Catharanthus roseus, different enzymatic steps are carried out in different cellular compartments (Fig. 8.5) [10]. Various steps in the pathway are carried out in different types of cell. This requires the intercellular transport of metabolic intermediates. Similarly, scopolamine biosynthesis also involves two different cell types. 

Second, it has to be pointed out that the co-occurrence with scopolamine (hyoscine) is a consistent trait in hyoscyamine-containing species. Furthermore, this fact involves that anisodamine (6P-hydroxyhyoscyamine) must also be synthesized in all these species because it is an intermediate representing the direct precursor in the biosynthesis of scopolamine (Fig. 3.14). Nevertheless, due to a certain accumulation this intermediate could be identified in many species, e.g., in the genera Anthocercis, Cyphanthera, Duboisia, Datura, Hyoscyamus, Physochlaina. The trivial name of this alkaloid has been given due to its occurrence in Anisodus acutangulus. 

O Donovan DG, Keogh MF (1968) Biosynthesis of piperidine alkaloids. Tetrahedron Lett 265-267 O Donovan DG, Keogh MF (1969) The role of hygrine in the biosynthesis of cuscohygrine and hyoscyamine. J Chem Soc C 223-226... 

Hyoscyamine 6j8-hydroxylase (H6pH) is a 2-ODD enzyme that catalyzes a critical step in the biosynthesis of the tropane alkaloid scopolamine (Fig. 3) in members of the Solanaceae [82],... 

Littorine is the natural plant precursor for biosynthesis of S-hyoscy amine representing its isobaric isomer (Fig. 1). Even though littorine exhibits high affinity to MR similar to hyoscyamine [35] it is not used as drug, most reasonably due to its low concentration in plants. 

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