Indole alkaloids, separation using

The Pictet-Spengler route to tetrahydro-P-carbolines is frequently used in indole alkaloid synthesis, and much attention has been devoted in recent years to the development of enantiospecific Pictet-Spengler reactions and the factors which influence the diastereochemistry at C-l and C-3 (c.f 1). The diester la was prepared from 2 as 1 1 mixture of diastereomers and heated in 2% ethanolic hydrogen chloride for 3 hours in an attempt to effect epimerisation at C-l and increase the amount of trans isomer. The product, however, was 2, which was isolated in 76% yield. Heating of the cis and trans diesters 1 separately in ethanolic hydrogen chloride for 3 hours also gave 2. By comparison, when a cis, trans mixture (39 61) of lb was stirred in a mixture of methylene chloride and trifluoroacetic acid at room temperature for 90 minutes the trans diester was obtained in 96% yield. 

In 1978, Harada et al. [17] used polymerized CD with gel support for the chiral resolution of mandelic acid and its derivatives. Later Zsadon et al. [18-21] used cyclodextrin-based CSPs for the chiral resolution of indole alkaloids, with aqueous buffers as the mobile phases. Today CD-based CSPs have a good reputation. In separate studies, Fujimura [22] and Kawaguchi [23] and their colleagues resolved the enantiomers of aromatic compounds in the reversed-phase mode. Armstrong et al. [29,30,33,34,41,44 46,48,54-63] carried out extensive and remarkable work on the chiral resolution of various racemic compounds using CD-based CSPs. 

Since the adsorption of plant metabolite onto adsorbent is a strong function of pH, the effect of pH upon the adsorption capadty of a plant metabolite should be investigated to understand and quantify product adsorption. The pH dependence of indole alkaloids, yohimbine and ajmalicine, and berberine onto XAD-7 has been reported [ 16,20]. As a model system, XAD-7 was examined to quantify berberine adsorption, especially in terms of pH and concentration dependence, which were used for the in situ berberine separation in Thalictrum ru-gosum cultures [19, 20]. Dependence of berberine adsorption on pH and concentration was investigated as shown in Fig. 4. Equilibrium ratio (Q/C), an affinity of berberine, could be calculated by Eq. (1) ... 

To remove the feedback regulation mechanism and to avoid product degradation various adsorbents have been used for the in situ separation of plant cell cultures as shown in Table 1. In situ removal with polymeric adsorbents stimulated anthraquinone production more than the adsorbent-free control in Cinchona ledgeriana cells [35]. It was found that nonionic polymeric resins such as Amberlite XAD-2 and XAD-4 without specific functional groups are suitable for the adsorption of plant metabolite [36]. The use of the natural polymeric resin XAD-4 for the recovery of indole alkaloids showed that this resin could concentrate the alkaloids ajmalicine by two orders of magnitude over solvent extraction [37] but the adsorption by this resin proved to be relatively nonspecific. A more specific selectivity would be beneficial because plant cells produce a large number of biosynthetically related products and the purification of a several chemically similar solutes mixture is difficult [16]. 

It has also been proposed that the presence of endophytes can alter the usual suite of secondary metabolites of plants. For example, whilst Murraya spp. have been reported to produce indole and carbazole alkaloids, the Brazilian M. paniculata did not [25]. An endophytic Eupenicillium sp. was isolated from surface-sterilized leaf material of the Brazilian M. paniculata and subsequently cultured on white corn. The Eupenicillium sp. produced hydrophobic spiroquinazoline alkaloids that were separated using silica gel column chromatography and preparative gel-filtration... 

HPLC has been extensively applied in the analysis of indole alkaloids - for quite different purposes. A series of papers on the analysis of drugs of abuse includes the analysis of one or more indole alkaloids, particularly strychnine, a common adulterant of heroin (Table 8.16). Moreover strychnine and brucine have often been used as test compounds for a number of separation systems 2, 1 2 32,4 43 as well as internal standards1 33. 

Thomson reported the reversed-phase ion-pair separation of psilocybin and psilocin. Because both alkaloids exist as zwitter-ions, cationic and anionic pairing ions can be used. Alkyl sulfonates (Cg-Cg) and tetraalkyl ammonium (C3-Cg) ions were found unsatisfactory for psilocybin. Good results were obtained with a long chain quaternary ammonium ion, cetrimonium. Optimal conditions for quantitative analysis on an octadecyl stationary phase were 0.15% pairing ion in methanol - 0.4% aqueous phosphate buffer (pH 7.Z). Some other quaternary indole alkaloids have also been separated by means of ion-pair HPLC. Parkin6 analyzed the bisquater-nary alkaloid alcuronium in biological fluids. After an ion-pair extraction, the alkaloid was analyzed on an octadecyl column with the mobile phase methanol - water (4 1) containing O.Z5% acetic acid and 0.005 M dodecylsulfate. 

Determination of the indole alkaloids psilocin and psilocybin in fungi has been the most popular application of EC in the analysis of alkaloids in a plant matrix. This research has primarily been done by two groups Christiansen and co-workers in Norway, and Kysilka and Wurst in the Czech Republic. Especially psilocin is well suited for EC, due to its hydroxy substituent on the indole ring, essentially a phenol, whereas in psilocybin this group is phosphorylated. Since the pH of the mobile phase is not critical to the oxidation of either the phenolic group or indole nucleus, it has been possible to use different separation techniques. 

Table 4 lists GC systems used for the analysis of terpenoid indole alkaloids and Fig. 4 illustrates a GC separation of a mixture of reference compounds. 

Six indole alkaloids (tryptamine, bufotenin, serotonin, tryptophan, 5-hydroxy-and 5-hydroxy-A -methyltryptophan) were separated in 15 min using a 10/90 ethanol/ water (0.1 M citrate/phosphate buffer at pH 2.8) mobile phase on a Cig column (/I = 267 nm). Excellent chromatographic results were obtained and detection limits down to tens of nanograms were obtained [305]. 

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. 

An aqueous electrolyte solution was initially used to separate the indole alkaloids mentioned in the experimental. The electrolyte tested was potassium hydrogenphosphate at different pH values where poor resolution was observed. Efforts to improve the separation by adding different organic solvents such as acetonitrile and methanol were found to be invalid. The use of nonaqueous solvents... 

The following remarks, although written specifically with indole alkaloids in mind, are good for alkaloids in general and basically true for any isolation problem. At some time during the workup of a plant extract advantage is taken of that property of the looked for substance(s) which will enable its separation from the other products. For an alkaloid one normally takes advantage of its basicity unless some other incidental property can be effectively used. In its simplest form an extract of the plant in a water immiscible solvent is washed with aqueous acid which upon subsequent basification affords the alkaloids. 

Ergot alkaloids are easily oxidized in the indole moiety by a two-electron transfer process affording a deep purple highly conjugated dimer as the principal product (Dankhazi et al., 1993). This feature can be used, e.g., for the electrochemical detection of EA after their HPLC separation (Pianezzola et al.. 

Brucine is a highly toxic alkaloid resembling strychnine and is also used as a central nervous system stimulant. In addition, it is used in analytical chemistry for separating racemic mixtures. These alkaloids possess an additional 2-carbon unit between the indolic Ng and C17. It has been shown that the 2-carbon unit comes from acetate at some stage of the biogenesis (123). 

Two years later, thiourea 21, functionalized with a cinchona alkaloid, was efficiently used in the synthesis of 3-indolylmethanamines in good yields and selec-tivities (up to 96% ee) [25]. The transformation takes place in the presence of aromatic, aliphatic, and heteroaromatic imine analogs. Chiral phosphoric acids 26a, mf-26d, and ent-26f were also identified as efficient catalysts for the F-C alkylation between indoles and imines [35, 38, 40]. In these cases, in contrast with thiourea 21, the processes were only successful in the presence of aromatic imines. More recently, the fmitful use of a thiourea supported on mesoporous silica (heterogeneous bifunctional catalyst) was reported [61]. The catalyst can be easily separated from the reaction mixture by simple filtration and reused several times without erasing the enantioselectivity. 

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