The Biosynthesis of Nicotine

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). 

Tryptophan quite clearly follows different metabolic routes. Our studies have been devoted to the pathway that, proceeding through the formation of kynurenine (an amino acid but no longer an indole) to the biosynthesis of nicotinic acid, explains the formation of the various intermediates. Kynurenine is the key substance in this process. 

Putrescine is an important intermediate in the biosynthesis of tropane alkaloids acting also as a precursor for polyamines, spermine and spermidine, and also for the biosynthesis of nicotine [111, 122]. 

Almost the same method as described by Quin and Pappas was used by Alworth et al. for investigations on the biosynthesis of nicotine in Nicotiana giutlnosa. The assay of nicotine was carried out on the aerial and root sections of the plant separately. Polybutylene glycol 10 % on Firebrick was used, at column temperature 169°C. 

Outstanding stereochemical problems associated with the biosynthesis of nicotine have been resolved by n.m.r. measurements. An unexpected... 

Dawson, R.F., D.R. Christman, A. D Adamo, M.L. Solt, and A.P. Wolf The biosynthesis of nicotine from isoto-pically labelled nicotinic acids J. Am. Chem. Soc. 82 (1960) 2628-2633. 

A8. Auricchio, S., Rigillo, N., and Di Toro, R., Researcbes on the biosynthesis of nicotinic acid from tryptophan during pregnancy, the foetal and the neonatal periods. I. Tryptophan pyrrolase and 3-hydrorgrantfaranilic oxidase activity of the rat liver. Minerva Pediat. 12, 1463-1470 (I960). 

The biosynthesis of nicotine and anabasine can reasonably proceed via (30), which will be electrophilic towards (31) and (3), which are intermediates for the other parts of the alkaloids, subsequent aromatization leading to loss of tritium. Coupling of two molecules of (30), decarboxylation, and aromatization would give anatabine, with 50% retention of tritium observed the stereochemistry of (30) follows from that determined in anatabine (34). [Direct coupling of two molecules of (30) makes use of the higher electrophilicity associated with this molecule cf. fatty acid biosynthesis), and so is preferred to the suggested coupling with two molecules of (35).]... 

Plants produce various pyridine alkaloids derived from nicotinic acid. Trigonelline, the major component in coffee seeds, and ricinine, the toxic alkaloid produced by Ricinus communis, are formed from nicotinic acid originating from the NAD catabolism [20, 25, 26], Quinolinic acid was found to be an efficient precursor in the biosynthesis of nicotine [27]. 

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. 

The mode of action of vitamin PP can be understood only if its site of action in metabolism is clarified. It is therefore necessary to review the biosynthesis of nicotinic acid, its role in the synthesis of NAD, and the breakdown mechanism of nicotinic acid and NAD. 

Kynurenine is hydroxylated to hydroxykynurenine by an enzyme (kynurenine-3-hydroxylase) found in rat liver mitochondria. The reaction requires NADPH and molecular oxygen. In the presence of pyridoxal phosphate, hydroxykynurenine is hydrolyzed by an enzyme (kynurenase) found in liver and kidney. The product of this reaction is 3-hydroxyanthranilic acid. The same enzyme catalyzes the cleavage of the side chain of kynurenine to yield alanine and anthranilic acid. Studies made with labeled 3-hydroxyanthranilic acid demonstrated its role as an intermediate of the biosynthesis of nicotinic acid. These studies established that the label of the carbon 3 of 3-hydroxyanthranilic acid is transferred to the a-carbon of quinolinic acid and is lost as C14O2 during the conversion of quinolinic to nicotinic acid. The details of the metabolic conversion of 3-hydroxyanthranilic acid to nicotinic acid are known. 

From the role of tryptophan in the biosynthesis of nicotinic acid, it is obvious that the nutritional studies on nicotinic acid deficiency must take tryptophan intake into account. Indeed, 60 mg of tryptophan in the diet is as effective as 1 mg of nicotinic acid. Since 70 g of protein yields 720 mg of tryptophan, the intake of such an amount of protein corresponds to 12 mg of nicotinic acid in preventing niacin deficiency. Since the requirements for niacin, like those of thiamine, depend essentially on the caloric intake, it is useful to express the requirements in niacin equivalents per 1000 calories. The optimum requirement is 4.4 mg niacin per 1000 calories. 

In the biosynthesis of nicotine 6.71) in Nicotiana species it has been found [45, 46] that both ornithine 6.60) and putrescine 6.63) are again involved in pyrrolidine-ring formation. For this alkaloid, however, incorporation of the amino acid is through at least one symmetrical intermediate, logically putrescine 6.63), because [2- " C]ornithine gave nicotine 6.71) with label equally spread over C-2 and C-5 additional results were obtained with [ N]ornithines. This symmetrical pathway is supported by experiments with C02, although a few results indicate an unsymmetrical route [47, 48]. 

The biosynthesis of nicotinic acid in animals and some microorganisms is well established to be by degradation of tryptophan, whereas in plants nicotinic acid has its origins in aspartic acid and glycerol (glyceraldehyde) via quinolinic acid [6.72) (Scheme 6.16). 

Several authors have studied nicotine production (i.e., biosynthesis) in callus tissue cultures (Speake et al., 1964 Benveniste et al., 1966 Furuya et al.y 1966, 1971 Tabata et aL, 1968, 1971 Shiio and Ohta, 1973 and Heinze, 1975). The biosynthesis of nicotine is dependent upon the formation of organized tissue within the callus. Nodule-like structures similar to roots were observed in our laboratories using tobacco variety Maryland-872, which produces 96% of its alkaloids as nicotine. Shoot formation stimulated nicotine production in the callus, and nicotine may have been transported from the callus to the shoot. Nicotine production and tissue differentiation were dependent upon concentrations and types of growth regulators in the culture medium (Tables 4.3 and 4.4). The vegetative buds and leaves (shoots) contained about live times as much nicotine as callus without buds or leaves, which is in agreement with the results of Tabata et al (1968). 

Tryptophan was first isolated from casein hydrolysates, prepared by hydrolysis using pancreatic enzymes, by Hopkins in 1902. It occurs in animal proteins in relatively low amounts (1-2%) and in even lower amounts in cereal proteins (about 1%). Tryptophan is exceptionally abundant in lysozyme (7.8%). It is completely destroyed during acidic hydrolysis of protein. Biologically, tryptophan is an important essential amino acid, primarily as a precursor in the biosynthesis of nicotinic acid. 

NicotioHa Species.—Some progress is being made in isolating the enzymes responsible for the biosynthesis of nicotine. The immediate precursor of the pyrrolidine ring of nicotine is the 1-methyl-l-pyrrolinium salt (42). - This is formed from putresdne [(38), the decarboxylation product of ornithine] by the route illustrated in Scheme 9. A putrescine-JV-methyltransferase was... 

Figure 9.4 The biosynthesis of nicotine from ornithine and nicotinic acid. Anabasine,...

Stereoselective additions to C=N bonds are of prime importance for the synthesis both of alkaloids and also of a host other nitrogen-containing natural products. Nature s own ability to synthesize complex alkaloids is awe-inspiring. A relatively simple example where the sequence of synthetic steps has been well elucidated is the biosynthesis of nicotine (8) from nicotinic acid (vitamin B,) [43]. As outlined in Figure 11.1, the enzyme-mediated... 

For several years it was assumed that the putrescine, which forms a part of nicotine and other pyrrolidine alkaloids, arises through the action of ODC (Mizusaki et a/., 1973) and, in fact, pyrrolidine alkaloids have been classified as ornithine-derived compounds (Leete, 1980). In contrast, our investigations with tobacco callus tissue have shown that (1) changes in the ADC activity parallel changes in alkaloid levels, while changes in ODC activity do not (2) DFMA effectively inhibits the biosynthesis of nicotine and nomicotine, while DFMO is less effective and (3) the flow of from uniformly labeled arginine... 

Furthermore, SAM not only plays a pivotal role in PA and ethylene biosynthesis, but also in the formation of putrescine-derived alkaloids. Thus, SAM acts as a methyl donor in the conversion of putrescine into A-methylputresdne, an intermediate in the biosynthesis of nicotine (Mizusaki et al., 197 lb). Therefore, it would be interesting to explore the auxin-ethylene-PAs interaction on PA conjugates and putrescine-derived alkaloids metabolism in the above-mentioned tobacco callus tissue system. Indeed, it has been reported that inhibition of SAM decarboxylase by MGBG resulted in an increase of nicotine production in tobacco root cell suspension cultures (Blume, 1985). Unfortunately, nothing about PA conjugates was indicated in this report. 

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