Pyochelin, a phenolic siderophore of Pseudomonas aeruginosa, was synthesized in three steps from salicylonitrile, L-cysteine, and L-N-methylcysteine. The synthetic product was determined to be identical to natural pyochelin by 1H nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, chromatographic analysis, and chemical reactivity with FeCl3 and ammoniacal silver nitrate reagent. Synthetic and natural pyochelin promoted bacterial growth in iron-depleted medium and were also found to mediate iron transport by P. aeruginosa to the same levels. Neopyochelin, a stereoisomeric by-product of the synthesis, showed less biological activity than did pyochelin in iron transport assays.

The synthesis of pyochelin has been much improved but gives four stereoisomers. The stereochemistry of the two naturally occurring pyochelins I and II has been assigned, based on (1) the similarity of the NMR spectra of pyochelin I methyl ester to those for 4-methylpyochelin I methyl ester, whose X-ray structure has been determined and relative stereochemistry assigned; (2) comparison of the rotation of the natural material to that of pyochelin I methyl ester synthesized from IV-methyl-L-cysteine methyl ester, which is not expected to epimerize during the synthesis and thus assigns pyochelin I methyl ester as 4'R,2''R,4"R; and (3) the known facile epimerization at C-2" of 2"-substituted thiazolidine-4-carboxylic acids, which assigns the other (unfavored) naturally occurring isomer, pyochelin II, as 4'R,2"S,4"R. The remaining two synthetic products, neopyochelin I methyl ester and neopyochelin II methyl ester, were assigned the stereochemistry 4'S,2"S,4"R and 4'S,2"R,4"R, respectively, based on (1) the use of IV-methyl-L-cysteine methyl ester in their synthesis, which establishes C-4" as R; (2) the known instability at C-2", which favors neopyochelin II (2"R) over neopyochelin I (2"S); and (3) the requirement of nonidentity with pyochelins I and II, which requires the S configuration at C-4'.Pyochelin (la, Scheme 1) is a unique iron-chelating siderophore isolated from Pseudomonas aeruginosa.2,3 As produced in nature pyochelin exists as a mixture of two interconvertible isomers, pyochelins I and II, whose absolute and relative configurations at the three chiral centers have not previously been assigned. The proposed structure of pyochelin has been corroborated by total synthesis4 and further spectroscopic studies,5 67and three mutasynthetic analogues were recently isolated from a salicylic acid idiotrophic mutant (Salphenotype).6,7 We have found that the free carboxylic acids cannot be isolated in pure form due to their rapid interconversion, but the methyl esters (lb) are more stable configurationally and can be easily purified. We report here the assignment of the absolute stereochemistry of the natural and synthetic isomers as well as the mutasynthetic analogues of pyochelin, based on spectral data and an X-ray crystal structure. We also report a significant improvement in the synthetic approach to the compounds, one that provides four of the eight possible stereoisomers with the ring system of pyochelin.1 ResultsSynthesis of Pyochelin. The previous synthetic approach to the pyochelins (Scheme 1) proceeded in 8% overall yield from salicylonitrile (2), with the critical step

[Ctfr&0xj;-14C]-3-ammo-5-hydroxybenzoic acid (AHBA) has been shown to be incorporated by Streptomyces spectabilis to the extent of greater than 0. 1 %(35 : 1 dilution) in the ansamycin antibiotic streptovaricin C, the major componentof the streptovaricin complex. When[carboxy-i3C]AHBA was similarly administered, C-21 (quinone methide carbonyl at 188.3ppm) of streptovaricin C was specifically labeled (at twenty one times natural abundance). In preparation for the 13C incorporation study the 13C NMR spectrum of streptovaricin C was investigated, making extensive use of short-and long-range HETCOR. These assignments revise some of those proposed earlier for streptovaricin C.The meta-C7N units in the aromatic chromophores of several ansamycin antibiotics have been shown to be biosynthetically derived from 3-amino-5-hydroxybenzoic acid (AHBA) as a discrete biological intermediate. Rifamycin,1) actamycin,2) geldanamycin,3) the ansamitocins,4) and the mitomycins5) have all been shown to incorporate AHBA as a precursor. Although the biosynthesis of AHBA from primary metabolic precursors has not yet been completely determined, there is evidence that the compoundcomes from a branch of the shikimic acid pathway.6) Wereport here the biosynthetic incorporation of 13C-and 14C-labeled AHBAinto the naphthoquinoid chromophore of streptovaricin C (1), thus providing additional evidence that AHBA is a commonprogenitor of the ansamycinantibiotics. In addition, we have revised some of the previously reported 13C NMRassignments of 1 based on new 2D heteronuclear correlation experiments.7) Our continued interest in the biosvnthesis of the streotovaricins is based in Dart on reports of their derivatives' antiviral activities,8) most recently against the HIV virus.9) Streptovaricin analogues with modified biological activities could conceivably be produced through mutasynthetic modifications based on a knowledge of the biosynthetic origin of these compounds. 10)

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