Pilocarpine biosynthesis

The biosynthesis of pilocarpine in Pilocarpus pennatifolius was studied by the administration of radiolabeled precursors. Radioactive sodium acetate, histidine, histidinol, methionine, and threonine were administered by the cut-stem method. Histidine, methionine, and threonine were administered together by a wick inserted through the stem of an intact plant. Sodium acetate and histidine were fed to root cuttings by suspending the roots in aqueous solutions of the precursors. After 64 - 7 5 h, the roots were harvested and total alkaloid extracts made. These extracts were then fed to stem cuttings. 

The methyl group of methionine has been found to be the biological source of the /V-methyl group of pilocarpine. This portion of the biosynthesis occurred within the leaves. At present, it seems reasonable to suppose... 

Most A. are colorless (yellow colored e.g., chelido-nine), as fiee bases they are readily soluble in water, even more soluble in alcohols, ethers, and chloroform. With acids they often form nice crystalline salts that are well soluble in water because of their solubility the latter are used preferentially in medicine (e. g., mor-phinum hydrochloricum, pilocarpine hydrochloride). Up to the end of 1957 2233 A. from 3761 plant species of 156 families had been isolated, in 1976 the numbers had increased to 5000 A. from 7000 plant species, and in 1989 over 10000 alkaloids were known. In 1995 the number of known A. was estimated to be ca. 15 000. As early as in the 19th century, structure investigations on A. led to the development of useful degradation methods which provided a major improvement to the arsenal of organic chemistry. Investigations on biosynthesis of A. started much later. 

Pilocarpine possesses an imidazole nucleus and the biosynthesis of this alkaloid is regarded as beginning with histidine, although the details are lacking. 

Pilocarpine has been suggested to arise from condensation of an alcohol involved in the biosynthesis of histidine (as the phosphate) and acetoacetate (Fig. 37,2). A second path has also been suggested. There is little experimental evidence to establish either pathway (Maat and Beyerman, 1983). 

Imidazole alkaloids alkaloids of sporadic occurrence, possessing the imidazole ring system. Tlieir most important representative is Pilocarpine (see). The biosynthesis of the I. a. is coupled to histidine metabolism. 

The amino acid L-histidine is the assumed building block in the biosynthesis of pilocarpine (27) and other imidazolic alkaloids due to the presence of glyoxaline ring [8, 23]. First attempts to clarify this pathway were proposed by Boit and Leete that considered the phosphate derivative of 2-oxo-3-(5-imidazofyl)-propanol, also known as imidazole pyruvic acid, as imidazole ring precursor (Scheme 25.1-Pathway 1) [8]. This initial biosynthesis proposal was improved detailing the lactone ring formation by aldol condensation. Another biosynthetic approach suggested condensation of 2-oxobutyric acid (lactone moiety) with urocanic acid (imidazole moiety) (Scheme 25.1- Pathway 2) [8, 39]. 

Isotopic studies with labeled potential precursors such as sodium acetate- " C, threonine- C, histidine- " C, histidinol- " C, and L-methionine-(S-methyl- " C) were carried out with P. pennatifolius in an attempt to prove the proposed pathways [8, 39]. Only the methylation of pilocarpidine, last step in both mechanisms, was attested by significant incorporation of radioactivity in the methyl group attached to the imidazole nucleus. This data confirmed an important assumption, the one that considers methionine the biological source of the A -methyl group of pilocarpine. Also, one should consider that these radiolabeled studies were carried out with stems, no other parts of the plant were analyzed, and thus some other site involved in biosynthesis was not considered in this study. 

The presence of the enzyme histidine aminotransferase, (HT) (EC 2.6.1.38) was reported in P. pennatifolius roots [40], which could link L-histidine to imidazole alkaloids biosynthesis and reinforce the hypothesis of a specific site for pilocarpine production. The enzyme activity was estimated in 46.09 nKat.mg of protein and optimal reaction at pH 8-9. HPLC data from enzymatic reaction indicated the formation of a product with the same retention time of the imidazole pyruvic acid standard obtained by synthesis. Such an enzyme activity could not be detected in P. pennatifolius leaves, reinforcing these organs could be involved only in the later steps of the biosynthesis, such as iV-methylation, or could play a role as accumulation site and not in the productimi. 

A cell suspension study with P. microphyllus recorded after several subcultures a stable cell line in pilocarpine and other imidazole alkaloids production and the same alkaloid profile found in leaves [41], This study also characterized three new alkaloids with imidazole ring in this plant species for the first time, by means of ESI-MS. Posterior studies using the same technique in leaves of several samples of Pilocarpus suggested three different pathways may occur in imidazole alkaloids biosynthesis [27, 41],... 

Identification of the genes involved in imidazole alkaloids biosynthesis could be the next and final boundary in pilocarpine production in plants. Nevertheless, isolation fmm natural sources is still a feasible way to obtain this alkaloid. 

Brochmann-Hanssen E, Nunes MA, Olah CK (1975) On the biosynthesis of pilocarpine. Planta Med 28 1-5... 

Abreu IN, Andreazza NL, Sawaya ACHF, Eberlin MN, Mazzafr P (2007) Cell suspension as a tool to study the biosynthesis of pilocarpine in jabtHandi. Plant Biol 9 793-799. 

SCHEME 13J Potential starting materials in the biosynthesis of pilocarpine. 

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