Pseudotropine alkaloids

The gas chromatography of cocaine has mainly been dealt with in connection with investigations to develop selective and sensitive methods for its detection and quantitative determination in pharmacological and toxicological analysis. One study has so far been done on naturally occurring alkaloidal mixtures present in the coca plant (Erythroxyion coca Lam.).  

The gas chromatography of cocaine has mostly been carried out on packed columns with non- 

1-2 m long packed columns with 1-2 % stationary phase and column temperatures of about 175-240°C were used to obtain reasonable retention times for cocaine. 

For a gas chromatographic identification of alkaloids, Brochmann-Hanssen and Fontan recommended a combination of two columns, one non-polar (SE-30) and one semi-polar (XE-60). Ham-12 

Scaringelli separated cocaine and a number of other caines (Procaine, Tetracaine, Benzocaine) on a 2 feet long silicone rubber column (10 %) at 220-225°C, and with temperature programming, starting at 75°C. He collected the separated compounds and identified them by their special crystal tests. Estimation of cocaine was obtained by using benzocaine as an internal standard. 

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The analysis of the pseudotropine alkaloids has been given considerable attention because of the abuse of cocaine. Cocaine diastereoisomers have been separated by GC using a packed column (Allen et al. 1981). Apolar columns separated the stereoisomers better than columns of medium... 

The product of the reaction in Entry 8 was used in the synthesis of the alkaloid pseudotropine. The proper stereochemical orientation of the hydroxy group is determined by the structure of the oxazoline ring formed in the cycloaddition. Entry 9 portrays the early stages of synthesis of the biologically important molecule biotin. The reaction in Entry 10 was used to establish the carbocyclic skeleton and stereochemistry of a group of toxic indolizidine alkaloids found in dart poisons from frogs. Entry 11 involves generation of a nitrile oxide. Three other stereoisomers are possible. The observed isomer corresponds to approach from the less hindered convex face of the molecule. 

Tropinone reductases, which catalyse the stereospecific reduction of the keto group of tropinone to 3a- and 3 3-hydroxy groups (Drager, 2005), were analysed in detail by dissection of the peptides and construction of chimeric enzymes. The opposite stereospecificity of the two reductases was ascribed to the carboxy-half of the proteins, to which the substrate tropinone is assumed to bind with the reverse orientation in the two enz)mries (Nakajima et al, 1993, 1994 Hashimoto and Yamada, 1994). Only tropinone with the 3a-hydroxy group is used to produce hyoscyamine (Leete, 1990). Tropinone with the 3 3-hydroxy group forms esters with other acids, but these occur only as minor alkaloids. When tril was overexpressed in A. belladonna root cultures, a substantial amount of pseudotropine and related alkaloids was observed (Richter et al, 2005). 

Another enzyme involved in the production of minor alkaloids, tigloyl-CoA pseudotropine acylfransferase, has been purified from roots of D. stramonium (Rabot et al, 1995). If cafalyses fhe fransfer to pseudotropine of an acyl group from a range of acyl-CoA fhioesfers. Esters of pseudotropine do not accumulate significantly in D. stramonium roots, although they do appear under abnormal metabolic conditions (Drager et al, 1992). 

Rabot, S., Peerless, A.C.J. and Robins, R.J. (1995) Tigloyl-CoA pseudotropine acyl transferase a novel enzyme of tropane alkaloid biosynthesis. Phytochemistry, 39, 315-22. 

Both alkaloids have (+) and (-) forms but only the (-) hyoscyamine and (-) scopolamine are active. The biosynthetic pathway of tropane alkaloids, Fig. (1) is not totally understood, especially at the enzymatic level. Edward Leete has pioneered the biosynthetic studies of tropane alkaloid since 1950"s using whole plants and isotope labels [85-86]. The tropane alkaloid hyoscyamine is bioconverted by the enzyme H6H (hyoscyamine 6p-hydroxylase, EC 1.14.11.11) to scopolamine via 6p-hydroxyhyoscyamine. Hyoscyamine is the ester of tropine and (S)-tropic acid. The (S)-tropic acid moiety derives from the amino acid L-phenylalanine, while the bicyclic tropane ring derives from L-omithine primarily or L-arginine via tropinone. Tropinone is stereospecifically reduced to form either, tropine which is incorporated into hyoscyamine, or on the other hand into pseudotropine which proceeds to calystegines, a group of nortropane derivates that were first found in the Convolvulaceae family [87]. 

Nevertheless, genetic engineering of a key enzyme in a chosen pathway does not always result in the enhancement of the end product thus, overexpression of pmt gene carried out in A. belladonna resulted in a 5-fold increased in pmt transcript level but an unchanged tropane alkaloid profile (hyoscyamine and scopolamine), as well as the tropane alkaloids biosynthetic precursors tropine, pseudotropine and tropinone which were not affected [134-135], Similarly, the overexpression of tobacco pmt gene in Dubosia hybrid hairy root cultures produced amounts of tropane alkaloid similar to control roots [131, 136]. All these results seem to indicate the presence of different points of control in the tropane alkaloid metabolic pathway. 

Tropinone is stereospecifically reduced to yield both tropine (3a-hydroxytropine), which led to the formation of tropane alkaloids, and pseudotropine (3p-hydroxytropine), the precursor of calystegines. These stereospecific reductions are catalyzed by two different tropinone reductases, tropinone reductase I and II (TRI and TRII). Both enzymes have been isolated from many Solanaceous species. Thus, TR I and TR II were isolated from D. innoxia roots and the crude extract favoured the production of pseudotropine over tropine [151]. Also, from transformed root cultures of D. stramonium two different tropinone reductases were obtained. In this species, TRI showed about 5-fold larger activity than TRII, and TRI displayed a pronounced pH-dependency, while TRII was more tolerant to different pH values [152]. Moreover, two tropinone reductases were also isolated from H. niger root cultures. TRI-reduction was reversible, whereas TRII-reduction was essentially irreversible [153]. In subsequent studies it was found that the accumulation of both TRs was the highest in the lateral roots of H. niger throughout development, with different cell-specific patterns [154]. 

The second group comprises one alkaloid, tropacocaine, which is a benzoyl-pseudotropein. This alkaloid is important not merely because of its use in medicine but also because the base pseudotropine is isomeric with the base tropine found in atropine. 

A number of difficulties exist with this postulate. Stermitz1 group has shown that fresh aerial parts of field bindweed (Convolvulus arvensis) contain pseudotropine as the major alkaloid, together with trace amounts of tropine and tropinone, as well as the pyrrolidine alkaloids hygrine and... 

Although the alkaloid content of the aerial parts and roots of Datura sanguinea has been examined by various workers the seeds of this species have not previously been investigated, with the exception of a paper chromatographic determination by Drey and Foster,2 in which the presence of hyoscine was established. Leary3 has now confirmed that the most abundant alkaloid present in the seeds is hyoscine, and that it is accompanied by hyoscyamine apohyoscine, tropine, pseudotropine and two other, unidentified, alkaloids are also present. Datura suaveolens contains4 meteloidine in addition to the... 

Plants in the Solanaceae family produce a variety of alkaloids, some of them having a considerable therapeutic importance. One such group of alkaloids possesses a tropane nucleus. Tropane alkaloids are structurally related natural products having in common the azabicyclo[3.2.1]octane structure and therefore the systematic name for tropane is 8-methyl-8 azabicyclo[3.2.1]octane (Fig. 1). The majority of these alkaloids are esters between organic acids and hydroxytropanes. 3a-Hydroxytropane, called tropine, is the amino alcohol most frequently encountered. In addition, its 3 (3-isomer (pseudotropine), the di- (3,6- 3,7- or 6,7-) and trihydroxylated... 

Scheme 10.7 gives some other examples of 1,3-DPCA reactions. Entries 1 to 3 are typical intermolecular 1,3-DPCA. The 1,3-dipoles in each instance are isolatable compounds. Entries 4 and 5 are intramolecular nitrone cycloadditions. The product from Entry 5 was used in the synthesis of the alkaloid pseudotropine. The proper stereochemical orientation of the hydroxyl group is ensured by the structure of the isoxazoline from which it is formed. 

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