Phenazine 1-carboxylic acid

Phenazines — The phenazines are biosynthesized by the shikimic acid pathway, through the intermediate chorismic acid. The process was studied using different strains of Pseudomonas species, the major producers of phenazines. The best-known phenazine, pyocyanine, seems to be produced from the intermediate phenazine-1-carboxylic acid (PCA). Although intensive biochemical studies were done, not all the details and the intermediates of conversion of chorismic acid to PCA are known. In the first step, PCA is N-methylated by a SAM-dependent methyltransferase. The second step is a hydroxylative decarboxylation catalyzed by a flavoprotein monooxygenase dependent on NADH. PCA is also the precursor of phenazine-1-carboxamide and 1-hydroxyphenazine from Pseudomonas species. - - ... 

Phenazines — This large class of compounds includes more than 6,000 natural and synthetic representatives. Natural phenazines are secondary metabolites of certain soil and marine microorganisms. The main phenazine producers are Pseudomonas and Streptomyces species. Pseudomonas strains produce the most simple phenazines tubermycin B (phenazine-1-carboxylic acid), chlororaphine, pyocyanin, and iodinine. Pyocyanin is a blue pigment while chlororaphine is green both are produced by Pseudomonas aeruginosa. They can be seen in infected wounds of animal and human skins. Iodinine is a purple phenazine produced by Pseudomonas aureofaciens. 

Figure 10 Predicted roles of gene products involved in the synthesis of phenazine-1-carboxylic acid by P. jluorescens 2-79. (From Ref. 48.)...

Slininger, P.J., Jackson, M.A. Nutridonal factors regulating growth and accumulation of phenazine 1-carboxylic acid by Pseudomonas fluorescens 2-79. Appl Microbiol Biotechnol 1992 37 388-392. 

Timms Wilson, T.M., Ellis, R.J., Renwick, A., Rhodes, D.J., Mavrodi, D.V., Weller, D.M., Thomashow, L.S., Bailey, M J. Chromosomal insertion of phenazine-1 -carboxylic acid biosynthetic pathway enhances efficacy of damping-off disease control by Pseudomonas fluorescens. Mol Plant-Microbe Interact 2000 13 1293-300. 

Using ether-treated cells of P. aureofaciens, dicarbonyl-14C2 phenazme-l,6-dicarboxylic acid las (121)1 was found to be an efficient and specific precursor for phenazine-1-carboxylic acid (123), and also for 2-hydroxyphenazine-l-carboxylic acid (124). The rate of growth of the organism appeared to be important, because an incorporation was also recorded of the labelled (121) into (123), albeit at a lower level, with cultures that had been grown rapidly. The position of phenazine-1,6-dicarboxylic acid (121) as a universal intermediate in the biosynthesis of phenazines now seems secure. The previously reported failure of dimethyl phenazine-1,6-dicarboxylate (122) to act as a precursor of phenazines cf. Vol. 10, p. 28 Vol. 9, p. 29) has been confirmed with ether-treated cells of P. aureofaciens. Efficient hydrolysis of (125) to (123) did, however, occur.101... 

Reductive cyclization of substituted jV-(2 -fluorophenyl)-3-nitroanthranilic acids 10 provides a reliable synthetic route by reductive ring closure exclusively to the fluoro-substituted carbon to yield 6-, 8-, and 9-substituted phenazine-1-carboxylic acids 11 in good yield.This ring closure occurs via S Ar displacement of fluoride. 

Several phenazine derivatives, e.g. phenazin-2-ol, phenazine-l-carboxylic acid, 2-hydroxy-phenazine-1-carboxylic acid, phenazine-2,3-diol, and 2,3-dihydroxyphenazine-l-carboxylic acid have been isolated from Pseudomonas aureofaciens. 

Further support for the conclusion that two molecules of shikimic acid are involved in phenazine biosynthesis comes from the incorporation of >-[1,6,7,- CaJshikimic acid [as (134)] into phenazine-1-carboxylic acid (138) in Pseudomonas aureofaciens with close to a fifth of the activity present in the carboxy-group, as required if two molecules of shikimic acid are involved [however, the same result would have been obtained if only one molecule of shikimic add was implicated provided that a symmetrical intermediate of type (139) was also involved in the elaboration of (138)]. 

Phenazines and Phenoxazlnones.— A preliminary report (cf. Vol. 9, p.28) concerning the incorporation of phenazine-1,6-di-carboxylic acid (113) and its methyl ester (114) into phenazine-1-carboxylic acid (115) in Pseudomonas aureofaciens, and of (113) into lomofungin (112) in Streptomyces lomodensis, has appeared in full (cf. Vol. 10, p.28 Vol. 12, p.29). In addition,(111) has been isolated from an extract of S, lomodensis cultures after the extract had been treated with excess diazomethane. The (111) incorporated label from radioactive (113). It is reasonable to conclude from this that (110) (or a methyl ester ) is produced from (113) as an intermediate in the biosynthesis of lomofungin (112). 

Phenazine-1-carboxylic acid (115) was identified as a normal intermediate in the formation of (117). It was not significantly... 

Phoiazines.— DL-[l,6- " C2]Shikimic acid [as (175)] is a specific precursor for pyocyanin (176) and phenazine-1-carboxylic acid (177) in Pseudomonas species. The activity was found to be approximately equally divided between the two groups of atoms C-1, C-4, C-6, and C-9 and C-4a, C-5a, C-9a, and C-lOa [see (177)]. Additional, more definitive, results were subsequently obtained with d-[6- C] shikimic acid activity in the derived phenazine-1-carboxylic acid was confined to C-4a, C-5a, C-9a, and C-lOa. 

Administration of the same precursor to Brevibacterium iodinum gave radioactive iodinin (178). This material was subjected to the usual degradation (Scheme 19). In accord with the labelling pattern observed in phenazine-1-carboxylic acid (177), 100% of the activity appeared in the pyrazine (180) but strangely 12% also appeared in the carbon dioxide. 

The results obtained for iodinin (178) with and D-[l,6,7- C3]shikimic acids and for phenazine-1-carboxylic acid (177) with and DL-[l,6- C2]shikimic acids are now in agreement with... 

Phenazines.—Consideration of structural relationships between the various microbial phenazines, in association with evidence on the way in which these metabolites are formed from two molecules of shikimic acid (151), leads to phenazine-l,6-dicarboxylic acid (152) as a likely common intermediate (Scheme 8), but it has not been found to act as a precursor for various phenazines, exemplified by phenazine-1-carboxylic acid (153). Although the dimethyl ester of (152) has been reported to be a precursor for (153) in Pseudomonas aureo-faciens, it could not be confirmed as such under various conditions. The dihydro-derivative (156) was likewise found not to be a precursor for (153) and (154) in P. aureofaciens. ... 

The above negative results with (152) and its dimethyl ester were obtained in Pseudomonas and closely related species for metabolites bearing a single aryl-Ci substituent or none. It was still an attractive possibility that (152) could be a precursor for phenazines bearing two aryl-Ci substituents, e.g. lomofungin (155), and indeed it was found to be ineorporated into this metabolite in Streptomyces lomodensis with an efficiency which indicated that it is an intermediate in lomofungin biosynthesis. It is not yet clear whether this positive result was obtained because (152) is transported across the cell walls of Streptomyces but not Pseudomonas species or because (152) is only a precursor for phenazines with two aryl-Ci substituents. Experiments with another Streptomyces species (5. luteoreticuli) which produces the methyl ester of phenazine-1-carboxylic acid (153) should allow resolution of the problem. 

Hou, C.T., L.K. Nakamura, D. Weisleder, R.E. Peterson, and M.O. Bagby, Identification of NRRL Strain B-18602 (PR3) as Pseudomonas aeruginosa and Effect of Phenazine-1 -carboxylic Acid Formation on 7,10-Dihydroxy-8( )-octadecenoic Acid Accumulation, World J. Microbiol. Biotechnol. 9 570-573 (1993). 

Scheme 1. Proposed General Biosynthetic Pathway of Common Phenazine Precursors, Phenazine-1-carboxylic Acid and Phenazine-1,6-dicarboxylic Acid ...

Phenazine natural products are believed to be secondary metabolites derived from a mutual primary metabolite. Most of the investigation of phenazine biosynthetic pathways has been carried out in Pseudomonas strains, but results from studies of Streptomyces support the belief that these species have similar biosynthetic pathways. Accordingly, it has been shown that phenazine-1,6-dicarboxylic acid (Iq) and phenazine-1-carboxylic acid (Ih) are precursors for more complex phenazine metabolites. 

Studies of pyocyanin (3) production in Pseudomonas strains have shown that the biosynthetic transformations from phenazine-1-carboxylic acid (Ih) to pyocyanin (3) are highly consistent in fluorescent Pseudomonas sp. First, the 5-methylphenazine-1-carboxylic acid betaine (59) is formed by methylation of the 5-position on phenazine-1-carboxylic acid, catalyzed by SAM-dependent (5-adenosyl-L-methion-ine) methyltranferase, and subsequently an NADPH-dependent flavoprotein monooxygenase catalyzes the hydroxylative decarboxylation (Scheme 3). ° 5-Meth-ylphenazine-1-carboxylic acid betaine (59) is also... 

Hydroxyphenazine-l-carboxylic acid (li) and 2-hy-droxyphenazine (lb) are believed to be derived sequentially from phenazine-1-carboxylic acid (Ih) via known biotransformation reactions, presumably via an arene oxide intermediate (Scheme 3). In Pseudomonas aureofaciens, an NADPH-dependent reductase is responsible for the hydroxylation to form li, whereas the subsequent decarboxylation to give lb occurs spontaneously and nonenzymatically. ... 

Phenazine-1-carboxylic acid, 2-hydroxyphenazine, and 2-hydroxyphenazine-l-carboxylic acid were extracted from Pseudamonas aureofaciens cultures and analyzed on a Cig column (A = 257 nm) using a 25-min 65/35/0.1 ->0/100/0.1 water/acetonitrile/TFA gradient [1001]. Elution of the components is complete in 15 min and the two carboxylic acids are not resolved using this gradient system. Therefore, it seems prudent to change to a shallower gradient or have an initial isocratic hold period prior to a gradient in order to force a separation. 

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