4-Chloro-l-kynurenine as fluorescent amino acid in natural peptides

Abstract

4-Chloro-L-kynurenine (3-(4-chloroanthraniloyl)-L-alanine, L-4-ClKyn), an amino acid known as a prospective antidepressant, was recently for the first time found in nature in the lipopeptide antibiotic taromycin. Here, we report another instance of its identification in a natural product: 4-chloro-L-kynurenine was isolated from acidic hydrolysis of a new complex peptide antibiotic INA-5812. L-4-ClKyn is a fluorescent compound responsible for the fluorescence of the above antibiotic. Whereas fluorescence of 4-chlorokynurenine was not reported before, we synthesized the racemic compound and studied its emission in various solvents. Next, we prepared conjugates of DL-4-ClKyn with two suitable energy acceptors, BODIPY FL and 3-(phenylethynyl)perylene (PEPe), and studied fluorescence of the derivatives. 4-Chloro-DL-kynurenine emission is not detected in both conjugates, thus evidencing effective energy transfer. However, BODIPY FL emission in the conjugate is substantially reduced, probably due to collisional or photoinduced charge-transfer-mediated quenching. The intrinsic fluorescence of L-4-ClKyn amino acid in antibiotics paves the way for spectral studies of their mode of action.

A Novel Peptide Antibiotic Produced by Streptomyces roseoflavus Strain INA-Ac-5812 With Directed Activity Against Gram-Positive Bacteria

“…Figure 1 illustrates the complete spectrum of compounds isolated from the extract by their detection at two UV wavelengths (214/364 nm). It should be noted that detection at 214 nm is typical for peptide bonds, whereas 364 nm was selected based on the UV absorbance spectrum of 5812-A/C as recorded earlier ( Alferova et al, 2018 ). The relative diversity of the obtained compounds suggests that a simplified method of extraction would be acceptable for obtaining the INA-5812 fraction, as no admixtures were found to disturb the elution of the target peptide ( Figure 1A ).…”

“…The antibiotic contains an arabinose residue, nine amino acids (one residue of Asp/Asn, Ser, Pro, Ala, Leu, Tyr, and Orn, two residues of Gly), and three non-identified amino acid residues. Acidic hydrolysis of the molecule, followed by fluorescence excitation/emission values and high resolution mass spectrometry of INA-5812, revealed the presence of 4-chloro-Lkynurenine (Alferova et al, 2018).…”

Biosynthesis of l‐4‐Chlorokynurenine, an Antidepressant Prodrug and a Non‐Proteinogenic Amino Acid Found in Lipopeptide Antibiotics

“…Biologically, 1 was recently identified as an amino acid building block in the lipopeptide antibiotics taromycin A ( 2 ) and B ( 3 ; Figure A) and in the putative glycopeptide antibiotic complex INA‐5812 . With the concurrent discovery of the taromycin biosynthetic gene cluster (BGC) from the marine actinomycete Saccharomonospora sp.…”

“…[3,4] Biologically, 1 was recently identified as an amino acid building block in the lipopeptide antibiotics taromycin A(2) and B( 3;F igure 1A) [5,6] and in the putative glycopeptide antibiotic complex INA-5812. [7] With the concurrent discovery of the taromycin biosynthetic gene cluster (BGC) from the marine actinomycete Saccharomonospora sp.C NQ-490, we sought to establish the biosynthetic logic for the bacterial synthesis of l-4-Cl-Kyn. Herein, we report the concise threestep l-4-Cl-Kyn pathway originating from l-tryptophan (l-Tr p, 4)a nd present it as an orthogonal approach to produce this promising drug candidate.…”

“…Considering that taromycin contains asecond chlorinated amino acid residue, l-6-Cl-Trp (7), and that its BGC encodes only one halogenase,w eh ypothesized that the chlorination reaction takes place directly on l-Trp rather than on acarrier protein bound substrate [16] or post-assembly on ar eleased peptide substrate. [17] Phylogenetic analysis of Tar14 showed that it clades with FDHs acting on free l-Trp ( Figure S1 in the Supporting Information).…”