Scopolamine, biosynthesis

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. 

Biosynthesis of tropan alkaloids hyosciamine and scopolamine by isomerization of alkaloid littorine in Datura. stramonium and related species 98CSR207. 

Keto add-dependent enzymes are involved in numerous reactions and are found in prokaryotes as well as eukaryotes. Apart from scopolamine and clavulanic add biosynthesis, a-keto add-dependent enzymes are also found in the biosynthetic... 

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... 

Simple Pyrrolidine Alkaloids.—It is well established that ornithine (1) is a key precursor in the biosynthesis of pyrrolidine alkaloids. Notably, the amino-acid (1) is utilized for the biosynthesis of nicotine (5) via the symmetrical intermediate putrescine (3), whereas the biosynthesis of tropane alkaloids, e.g. scopolamine (6), avoids any symmetrical intermediate1,2 (cf. Vol. 11. p. 1). 

Secondary metabolites can accumulate in the same cell and tissue in which they are formed, but intermediates and end-products can also be transported to other locations for further elaboration or accumulation. For example, TAs and nicotine are typically produced near the root apex, but mostly accumulate within leaf cell vacuoles. Even TA biosynthesis itself involves intercellular transport of several pathway intermediates (Fig.7.9A). P-Glucuronidase (GUS) localization in A. belladonna roots transformed with a PMT promoter-GUS fusion showed that PMT expression is restricted to the pericycle.144 Immunolocalization and in situ RNA hybridization also demonstrated the pericycle-specific expression of H6H.145,146 In contrast, TR-I was immunolocalized to the endodermis and outer root cortex, whereas TR-II was found in the pericycle, endodermis, and outer cortex.85 The localization of TR-I to a different cell type than PMT and H6H implies that an intermediate between PMT and TR-I moves from the pericycle to the endodermis (Fig.7.9A). Similarly, an intermediate between TR-I and H6H must move back to the pericycle. The occurrence of PMT in the pericycle provides the enzyme with efficient access to putrescine, ornithine, and arginine unloaded from the phloem. In the same way, scopolamine produced in the pericycle can be readily translocated to the leaves via the adjacent xylem. 

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]. 

There are considerable literatures on the production of tropane alkaloids in tissue and cell cultures derived firom various parts of intact plants [4]. In a number of cases, root differentiation is required for enhanced tropane alkaloid biosynthesis [3, 5]. The production of the economically valuable tropane alkaloids, scopolamine and hyoscyamine, by these cultures has not been commercially successful, however, root cultures are so far the best system to investigate the production and biosynthesis of tropane alkaloids. 

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]. 

Based on knowledge of a biosynthetic pathway one can select certain steps which could be of interest for bioconversion of (a) readily available precursor(s). This could, for example, be stereospecific reactions, like the reduction of quinidinone in quinine or quinidine and the epoxidation of atropine to scopolamine. For the bioconversion one can consider using plant cells [e.g., the production of L-dopa from tyrosine by immobilized cells of Mucuna pruriens (10)] or isolated enzymes from the plant itself. An interesting example of the latter is the (5)-tetrahydroprotoberberine oxidase (STOX) enzyme, which oxidizes (5)-reticuline but not its stereoisomer (11). This feature can be used in the production of (i )-reticuline from a racemic mixture (see below). Immobilized strictosidine synthase has been successfully used to couple secologanin and tryptamine. The gene for this enzyme has been isolated from Rauvolfia (6) and cloned in Escherichia coli, in which it is expressed, resulting in the biosynthesis of active enzyme (7). The cultured bacteria produced 20 times more enzyme... 

Comprehensive reviews have recently appeared on the tropane alkaloids of the Solanaceae and on the biosynthesis and metabolism of the same type of alkaloids. The stereospecific hydroxylation of tropine to 6/3-hydroxytropine and the conversion of its tropic acid ester into 6,7-dehydrohyoscyamine and further into scopolamine have been conclusively proven. Hence, the total synthesis of scopolamine from 6-tropen-3a-yl esters is indeed a biomimetic synthesis. 

Scheme 3. Biosynthesis of the tropane alkaloids hyoscyamine and scopolamine, the ca-lystegines, and nicotine. Molecular clones have been isolated for the enzymes shown. Abbreviations CYP82E4, nicotine A-demethylase H6H, hyoscyamine 6jS-hydroxylase ODC, ornithine decarboxylase PMT, putrescine A-methyltransferase TR-I, tropinone reductase-I TR-II, tropinone reductase-II.

PMT and H6H, which catalyze the first and last steps, respectively, in the biosynthesis of the tropane alkaloid scopolamine (Scheme 3), were localized to the pericycle in the roots of A. belladonna and Hyoscyamus muticus (Fig. 2A) 132,145). PMT also catalyzes the first step in nicotine biosynthesis (Scheme 3) and has been localized to the endodermis, outer cortex, and xylem in N. sylvestris (244,245). In contrast, TR-I, an intermediate enzyme in the tropane alkaloid pathway, resides in the endodermis and nearby cortical cells (Fig. 2A) (135) thus, intermediates of tropane alkaloid metabolism must also be transportai between cell types. The biosynthesis and storage of acridone alkaloids were also associated with endodermis in Ruta graveolens (246). 

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 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]. 

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