Hygrine, biosynthesis

A/ -Methoxycarbonyl-2-pyrroline undergoes Vilsmeier formylation and Friedel-Crafts acylation in the 3-position (82TL1201). In an attempt to prepare a chloropyrroline by chlorination of 2-pyrrolidone, the product (234) was obtained in 62% yield (8UOC4076). At pH 7, two molecules of 2,3-dihydropyrrole add together to give (235), thus exemplifying the dual characteristics of 2,3-dihydropyrroles as imines and enamines. The ability of pyrrolines to react with nucleophiles is central to their biosynthetic role. For example, addition of acetoacetic acid (possibly as its coenzyme A ester) to pyrroline is a key step in the biosynthesis of the alkaloid hygrine (236). 

The mechanism of acetoacetate coupling in the biosynthesis of hygrine (209) has been studied by feeding sodium [3-14C]- and [4-14C] acetoacetate to Nicandra physaloides 134). 

The pivotal role of hygrine in the biosynthesis of cuscohygrine and of the tropane alkaloids has justified numerous investigations. The following scheme represents the now generally admitted pattern of formation of these bases. Ornithine (119) is first converted to putrescine or V-methylputrescine (120)... 

Hygrine.—The alkaloid hygrine (3) is an intermediate in the formation of tropane bases. Biosynthesis is from acetic acid, plausibly via acetoacetic acid (cf. Vol. 10, p. 12), and from ornithine (1), very reasonably in the manner shown in Scheme l.1,2 Acetoacetate has been confirmed as an intact precursor for hygrine in experiments with [3-14C]- and [4-14C]-acetoacetic acid in Nicandra phys-aloides. Labelling in (3) was, respectively, of C-2 and C-3, which confirms the suspected orientation of acetoacetate in hygrine (see Scheme l).3... 

The first committed step in TA and nicotine biosynthesis is catalyzed by putrescine JV-methyltransferase (PMT) (Fig.7.4).82 A PMT cDNA isolated from tobacco showed extensive homology to spermidine synthase from mammalian and bacterial sources.83 A-Methylputrescine is oxidatively deaminated to 4-aminobutanal, which undergoes spontaneous cyclization to form the reactive A-methyl-A1-pyrrolinium cation. Although the enzymes involved are unknown, the A-methyl-A1-pyrrolinium cation is thought to condense either with acetoacetic acid to yield hygrine as a precursor to the tropane ring, or with nicotinic acid to form nicotine. 

The biosynthesis of hygrine (28) and its possible role as a precursor for cusco-hygrine (29) and the tropane alkaloids have been investigated. Both [2- " C]-ornithine and acetate were specifically incorporated into (28, the acetate... 

The presence of some enamine, at equilibrium, is demonstrated by the conversion of piperideine into a dimer, indeed, the ability of these two systems to serve as both imines and enamines in such condensations is at the basis of their roles in alkaloid biosynthesis. Formed in nature by the oxidative deamination and decarboxylation of ornithine and lysine, they become incorporated into alkaloid structures by condensation with other precursor units. Hygrine is a simple example in which the pyrroline has condensed with ace-toacetate, or its equivalent. 

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... 

Scheme 5. The classical proposal for the biosynthesis of tropine 1 via hygrine 16 and a bio-mimetic reaction which supports it...

The most straightforward interpretation of this result invokes the formation of the tropane nucleus via both Path C and Path D, the former possibly occurring nonenzymatically as discussed previously. It should be noted that this rationalization of the observed labeling pattern still invokes a role for hygrine (16) in the biosynthesis of (1) as outlined in Scheme 10. In accordance with experimental observations, two labeling patterns in (18) and, after reduction, in (1) would result if (18) is formed by both pathways. 

The question of whether (22) is produced by Path D1 or D2 really amounts to the question of whether an enzyme can form an enol or enolate of a methyl ketone, e.g. at C-4 of acetoacetate or at C-3 of hygrine (16). Current thinking on the role of hygrine (16) in tropane alkaloid biosynthesis would suggest that this is not possible. If one considers all four pathways of Scheme 8 and the subsequent steps required to convert (16) or (22) into tropinone (18), shown in Scheme 10, one can see that pathways Cl, C2 and Dl, respectively, involve enolization of a methyl ketone, which is not nearly as easy as enolization of a -dicarbonyl compound. Only Path D2, which invokes (21) and (22) as intermediates, postulates that all of the carbon-carbon bond-forming reactions receive the benefit of stabilization of the nucleophile via a -dicarbonyl system (assiuning the acetate imits are intro duced stepwise via malonate or its CoA ester). The only experimental fact not in accord with this last proposal is the failure so far to achieve incorporation of (21) into (1) or (19). A new proposal for the formation of (22) which circiun-vents this problem will be introduced in Section 5 of this contribution. 

ScHWARTiNG et al. [150 b] have employed TLC in qualitative and quantitative studies of the biosynthesis of hygrine, cuscohygrine, ana-hygrine, anapherine and isopelletierine. 

Tropane alkaloid Biosynthesis Scopolamine, nicotine, hygrine, calystegines Bl, calystegines B2, spermidine, spermine, cocaine, tropinone, littorine, tropine... 

Leete E (1983) Biosynthesis and metabolism of the tobacco alkaloids. In Pelletier SW (ed) Alkaloids - chemical and biological perspectives, vol 1. Wiley, New York, pp 85-152 Leete E (1985) Biosynthesis of hygrine from [5- C]omithine via a symmetrical intermediate in Nicandra physaloides. Phytochemistry 24 953-955 Leete E (1990) Recent developments in the biosynthesis of the tropane alkaloids. Planta Med 56 339-352... 

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... 

Economy in the use of a few primary metabolites has been of importance in allowing the development of useful ideas [3] about alkaloid biosynthesis, e.g. tropine 6.3) and hygrine 6.2) could be thought of simply as deriving from ornithine and acetate (see Scheme 6.1). Two further things have been important in useful speculation... 

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