## 5 Biosynthesis of Indole Alkaloids from the Stigonematales
These studies, therefore, support an alternative route which does not utilized cyanide. Specifically, in this proposed pathway, the nitrogen of the isonitrile is derived, instead, from tryptophan, and the carbon from a donor (_i.e._, ribulose-5-phosphate in feeding studies); and subsequent oxidative decarboxylation, in turn, yields the indole-isonitrile intermediate (Figure 13). The origin of the isothiamate found in many of the variants, on the other hand, has yet to be investigated. Several feeding studies of isonitrile- and isothianate-containing terpenoids produced by marine sponges generally suggest inter-conversion between cyanide and thiocyanate, and subsequent incorporation to produce the corresponding isonitriles and isothiances, respectively [53]. However, the aforementioned genomic studies [54] which argue against incorporation of cyanide, and rather donation of carbon to the amine of tryptophan, would, in general, conflict with this proposal. An alternative hypothesis proposes the subsequent incorporation of sulfur into the isonitrile [49; 53]; and, most recently, it was suggested, based on a the lack of a dedicated gene within biosynthetic gene clusters, that either a \"pathway independent sulfur carrier protein,\" or alternative enzymes, such as a cysteine desulfurase, could be responsible for isothiances found in several haplindole-type alkaloids [49]. Likewise, as to the rare nitrile variants, specifically as found in ambiguines and fischerindoles, no studies have investigated the biosynthetic origin of this group, and previous studies simply propose an otherwise unexplained \"rearrangement\" of the isonitrile [40]. Figure 12: Originally proposed biosynthesis of a tricyclic haplindole, as an intermediate for other haplindole-type alkaloids (see Figure 14), via chloronium or H\({}^{+}\) induced condensation [45]. Recent molecular studies, however, suggest an alternative biosynthetic route [55]. Figure 13: Biosynthesis of the indole-isonitrile precursor as inferred based on the identification of the WellII-3 and AmblI-3 genes as homologs of previously characterized isonitrile synthase genes, and subsequent _in vitro_ studies of gene products [49; 54]. According to the originally proposed scheme (Figure 12), therefore, the first step in the common biosynthetic pathway might yield one of four of the tricyclic haplandoles (_i.e._, C-F), depending on whether an isonitrile or isothicarate indole precursor, was involved (_i.e._, C/E or D/F, respectively), and whether a hydrogen or chloronium ion was utilized in the condensation (_i.e._, C/D or E/F, respectively). That said, recent molecular studies cast doubt on the role of chloronium ion-induced condensation, and suggest instead an enzymatically driven condensation reaction followed by subsequent enzymatic halogenation of the resulting haplindole [18; 55]. In this case, either haplindole C or D (_i.e._, isonitrile and isothicar variants, respectively) would be the expected tricyclic precursor, but if subsequent introduction of sulfur to the isonitrile (to produce the isothicarate) is assumed, and which is supported by recent data, then haplindole C is the most likely tricyclic intermediate. However, if stereochemistry of the indole-isonitrile/isothiocyanate or monoterpenoid precursors does explain the existence of various stereoisomers (_i.e._, 12-epimers, C10/11/15 isomers), as has been propose, it is likely that other, yet to be identified, intermediates might exist. After the formation of a tricyclic intermediate, and thus Group 2 alkaloids, the proposed biosynthetic scheme diverges into three paths as shown in Figure 14. The first path suggests the cyclization between the C-4 carbon in the indole ring, and the C-16 carbon in the isopropylidene group [45; 56]. The cyclization would, thereby, produce the tetracyclic haplindoles of Group 1. The formation of oxidoles and formamides (Figure 6) are proposed to occur through oxidation of these tetracyclic haplindoles.