Carbazoles: A Tricyclic Nucleus of Benzo[b]indole Chemistry

Carbazoles (C12H9N) refers to a group of organic compounds made up of a tricyclic nucleus and two benzene rings surrounded d by a pyrrole ring. Carbazole secondary metabolites exist in higher plants and fungi. The carbazole is tricyclic heterocycle has a 14p electron ring system while the benzene rings are fused to the pyrrole ring at the 2,3 and 4,5 sites (Peng et al., 2018). Being a benzo[b]indole, it has similar chemistry to the indole despite having less basic structure. It has a pKa of only 19.9. Its acidity is increased by the electron-withdrawing substituents. plans such as Murraya koenigii of the family Rutaceae mainly found in India have medicinal phytocompounds like koenimbine, koenine, mahanimbine, murrayazolidine, murrayazoline, murrayacine, girinimbine, and mukoeic acid. The carbazoles have similar characteristics to the imino compounds. However, their NH band is dependent on the phase as a result of their strong intermolecular association in the solid phase (Meena et al., 2017). The carbazoles have two key bands at nmax = 856 and 915 cm1. On the other hand, the formylcarbazoles tend to have a unique spectrum because the formyl carbonyl is 1680 cm1 while the chelation tends to shift to a higher wavelength. The organic compounds mainly appear in the form of white crystals, plates, and leaflets. In can sublime readily and exhibits a strong fluorescence. When exposed to ultraviolet light, it forms a long phosphorescence

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Biosynthetic Pathway and Bioactivities.

M. koenigii has aromatic importance but its source of bioactive carbazole alkaloids makes it more important like the murrayafoline, and pyrafoline-D. It has several pharmacological activities because it has anticancer, antidiabetic, antimicrobials as well as antioxidant properties. While the biosynthesis of plant carbazole is not properly documented, there are proposals that the plant carbazole is derived from the shikimic acid trough the chorismic and isochronism acid to produce anthranilic acid which is later converted to quinolone 3. The quinolone is prenylated with prenyl pyrophosphate (pp) extracted from an MVA or MEP pathway to form 3-prenylquinolone and 3-methylcarbazole (Wu et al., 1996). The 3-methylcarbazole is later modified in the biosynthetic pathway to yield carbazole alkaloids. Other methods include through Reductive cyclization of 2-nitrobiphenyl with triethylphosphite and Graebe-Ullman Reaction.

Figure1, Structure of typical plant carbazole NPs

Figure 2. Structure of typical plant carbazole NPs

2-Bacteria Carbazole NPs

-Introduction of bacteria carbazoles and bioactivities

Bacterial carbazole is a natural product with strong inhibitory activity against a wide range of free radical-induced lipid peroxidation. Over the last decade, researchers have focused on how to develop bacterial carbazole into radical scavengers and they also can inhibit potential cell protecting activity (Huang et al., 2015). The free radicals initiate various diseases such as cerebral and myocardial ischemia, arteriosclerosis, inflammation, rheumatism, and cancer (Yamasaki et al., 1983).

While the carbazole alkaloids in higher plant and funding are much more common, the bacterial carbazole metabolizers are not very common. For example, the indole[2,3-a] pyrrolo[3,4-c] carbazoles are the first class of bacterial carbazole metabolites with staurosporine and rebeccamycin (Wang, Tabudravu, Jaspars, &, Deng, 2013). T#he second class of bacterial carbazoles include the indolosesequiterpenes comprising of oridamycins, and xiamycins. On the other hand, the third class of bacterial carbazoles is made up of simple tricyclic ring systems with aliphatic side chains (Huang et al., 2019). The third class includes carquinostatins, carazostatin, carbazoquinocins, carbazomycin, (S)-streptoverticillin, and neocarazostatins (Xu, Baunach, Ding &, Hertweck, 2012). For example, neocarazostatin A is a bacterial carbazole alkaloid that has a lot of potential because of its isoprene moiety found at the C-6 of the ring (Xu, Baunach, Ding & Hertweck, 2012). The alkaloid was isolated from Streptomyces sp. GP38 culture (Deng, Cross, McGlinchey, Hamilton & O'Hagan, 2008). In the biosynthesis cluster of 1, ten genes encode the putative anthranilate phosphosyltransferanse, isopentenyl diphosphate delta-isomerase, phenylalanine aminotransferase, and an acyl carrier protein (genes nzsB, C, D, and E). Through the gene knockout experiments, gene products nzsH, I, and J were found to play an important role in the biosynthesis of 1 which indicates that the genes help in the assembly of the carbazole skeleton.

3-Biosynthesis of Bacteria Carbazole NPs

Introduction of Streptomyces sp MA37 and related NPs isolated from this strain.

The biosynthesis pathways of bacterial carbazole studies were proposed after the feeding experiment in which the L-tryptophan, pyruvate, and acetate produce the carbazole nucleus. When neocarazonstatin A1 is isolated from the soil bacterium Streptomyces sp (Deng et al., 2014). MA37 strain (Yamasaki et al., 1983). The actinobacterial biosynthetic gene cluster was found to be responsible for the biosynthesis of the NZS. Enzymes NzsA and NzsG were the only enzymes biomechanically characterized (Deng, Cross, McGlinchey, Hamilton & O'Hagan, 2008). While NzsA is a P450 hydroxylase, the enzymes NzsG is a phytoene-synthase-like (PSL) prenyltransferase that catalyzes the prenylation of the carbazole. The novel fluorinates from the strain include Nocardia brasiliensis and an Actinoplanes sp. These two have high homology (80-87 % identity) compared to the original S. cattleya enzyme (Deng et al., 2014). The Streptomyces sp. MA37 strain exhibited the fluorometabolites, fluoroacetate, and 4-fluorothreonine which are very similar to the S. cattleya (Tanaka, Shin-Ya, Furihata & Seto, 1995).

Figure3, the structure of NPs from MA37

4- biosynthesis pathway of neocarastain A

Neocarazostatin A (NZS) one of the key bacterial alkaloids that have presented promising bioactivities against a wide range of free radicals. Neocaratostatin A has a tricyclic carbazole nucleus, a prenyl moiety, and C4 alkyl side chain attached to ring "A". Its biosynthetic pathways are through genome mining and gene activation. The biosynthesis of a gene cluster of n's involves several steps and enzymes (Huang et al., 2015). Three enzymes are involved. MA37-FabG from the primary fatty acid metabolism, and NzsE and NzsF enzymes that are pathways specific are involved in the formation of the side-chain precursor (Deng et al., 2014). It is important to note that the NzsE is a free-standing acyl carrier protein (ACP) while the NzsF is a homolog of the protein synthase III KAS III that caries v-ketoacyl-acyl. The enzymes catalyze decarboxylative commendation between the acetyl-CoA and the NzsE bound malonyl thioester to generate acetoacetyl-NzsE (Su, Zhang, Kyeremeh, Deng, Deng, &Yu, 2017). The NADPH-dependent reductase homologs from then fatty acid biosynthesis pathways can significantly reduce the 3-keto groups of the acetoacetyl- NzsE to produce the 3-hydroxybutyl-NzsE product for producing the neocarazostatin analogs (Su, et al, 2017).

The mechanism mediated by the enzymes involves the decarboxylation of the a-keto acid and succeeded by the carbonization of the active ThDP-bound enolate intermediate with the acyl acceptor to create a-hydroxyl ketone (an acyloin). The YerE enzyme can catalyze the decarboxylation of pyruvate and transfer the activated acetaldehyde onto the 3-keto sugar to form hydroxyethyl branched chain of yersiniosis A.

Biosynthesis model of bacteria carbazole NPs with the introduction of acyl-SNACs.

The proposed model for the biosynthesis of NZS is as follows, in the scheme (A) is the Proposed model for indole-3-pyruvate. (B) is the Biosynthesis of DMAPP, (C) is the Biosynthesis of 3-hydroxy-butyryl-ACP while (D) is the Proposed biosynthetic pathway of 1? Bracket: 9 was observed to be present in the extract of the mutant DnzsG based on HR-ESIMS and MSn analyses (Su, et al. 2016).

Key:

The dashed line is the proposed reaction.

Solid line: reactions were confirmed biochemically.

Figure4, biosynthesis model of bacteria carbazole NPs.

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Xu, Z., Baunach, M., Ding, L., &...