Thiazoline Ring Formation Research Articles

Monitoring the in Vitro Thiazolidine Ring Formation of Antioxidant Drug N-Acetyl-l-cysteine at Basic pH and Detection of Reaction Intermediates: A Raman Spectroscopic and Ab Initio Study.

The important cyclization reaction of antioxidant drug N-acetyl-l-cysteine (NAC) has been monitored in vitro at basic pH with the help of time series Raman spectroscopy. The thiazoline ring formation of NAC at acidic pH is a well-known reaction and has been studied extensively. However, the formation of a thiazolidine ring from NAC at basic pH has not been investigated precisely till date. The effect of basicity of the medium on the rate of cyclization has been investigated by studying the reaction at five different basic pH values. Raman signatures of cyclization have been observed with the passage of time and are found to appear faster as the basicity of the medium increases. Ab initio calculations have been done to understand the plausible mechanism of the reaction at basic pH. It is observed that formation of a thiazolidine ring from NAC occurs primarily in four steps, which involve proton abstraction from the thiol (SH) group of NAC and subsequent formation of an S-C bond by a nucleophilic attack of the C-S group on the protonated C-O-H group in NAC. Correlation of the theoretically calculated results with experimental Raman spectral analysis has led to a detailed and proper understanding of this important biochemical reaction.

Improved Total Synthesis and Biological Evaluation of Potent Apratoxin S4 Based Anticancer Agents with Differential Stability and Further Enhanced Activity

Apratoxins are cytotoxic natural products originally isolated from marine cyanobacteria that act by preventing cotranslational translocation early in the secretory pathway to downregulate receptor levels and inhibit growth factor secretion, leading to potent antiproliferative activity. Through rational design and total synthesis of an apratoxin A/E hybrid, apratoxin S4 (1a), we have previously improved the antitumor activity and tolerability in vivo. Compound 1a and newly designed analogues apratoxins S7–S9 (1b–d), with various degrees of methylation at C34 (1b,c) or epimeric configuration at C30 (1d), were efficiently synthesized utilizing improved procedures. Optimizations have been applied to the synthesis of key intermediate aldehyde 7 and further include the application of Leighton’s silanes and modifications of Kelly’s methods to induce thiazoline ring formation in other crucial steps of the apratoxin synthesis. Apratoxin S9 (1d) exhibited increased activity with subnanomolar potency. Apratoxin S8 (1c) lacks the propensity to be deactivated by dehydration and showed efficacy in a human HCT116 xenograft mouse model.

Synthesis and Biological Activity of Kalkitoxin and its Analogues

Total syntheses of kalkitoxin, isolated from the Caribbean Lyngbya majuscula, and its analogues, 3-epi-, 7-epi-, 8-epi-, 10-epi-, 10-nor-, and 16-nor-kalkitoxin, were achieved via oxazolidinone-based diastereoselective 1,4-addition reaction of a methyl group and efficient TiCl(4)-mediated thiazoline ring formation as the key steps. The biological activities of synthetic kalkitoxin and its analogues were evaluated with brine shrimp.

Iron acquisition with the natural siderophore enantiomers pyochelin and enantio-pyochelin in Pseudomonas species

The bacterial siderophore pyochelin is composed of salicylate and two cysteine-derived heterocycles, the second of which is modified by reduction and N-methylation during biosynthesis. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation, whereas the second cysteine remains in its L-configuration. Stereochemistry is opposite in the Pseudomonas fluorescens siderophore enantio-pyochelin, in which the first ring originates from L-cysteine and the second ring from D-cysteine. Both siderophores promote growth of the producer organism during iron limitation and induce the expression of their biosynthesis genes by activating the transcriptional AraC-type regulator PchR. However, neither siderophore is functional as an iron carrier or as a transcriptional inducer in the other species, demonstrating that both processes are highly stereospecific. Stereospecificity of pyochelin/enantio-pyochelin-mediated iron uptake is ensured at two levels: (i) by the outer membrane siderophore receptors and (ii) by the cytosolic PchR regulators.

Total synthesis and cytotoxicity of bisebromoamide and its analogues

A highly convergent route for assembling bisebromoamide, its stereoisomers and a simplified analogue has been accomplished, which features connecting its left part and right part via thiazoline ring formation at the final stage. Preliminary biological studies revealed that compounds with both proposed and revised structures, and a simplified analogue have similar potency against the proliferation of HeLa S 3 cell, indicating that the stereochemistry of methylthiazoline part, and methyl group at the 4-methylproline residue in bisebromoamide, have limited influence on its cytotoxicity.

Interaction Studies and Alanine Scanning Analysis of a Semi-synthetic Split Intein Reveal Thiazoline Ring Formation from an Intermediate of the Protein Splicing Reaction

We recently reported an artificially split intein based on the Ssp DnaB mini-intein that consists of a synthetic N-terminal intein fragment (Int(N)) and a recombinant C-terminal part (Int(C)), which are 11 and 143 amino acids in length, respectively. This intein holds great promise for the preparation of semi-synthetic proteins by protein trans-splicing. In this work we synthesized a set of Int(N) peptide variants to investigate their structure-function relationship with regard to fragment association and promotion of protein trans-splicing. A further truncation of the Int(N) sequence below 11 amino acids resulted in loss of activity, whereas C-terminal extensions were tolerated. Alanine scanning analysis identified three essential hydrophobic residues, whereas substitutions at other positions were tolerated. We developed assays to monitor association of Int(N) with an Int(C) mutant blocked in protein splicing by native PAGE and fluorescence anisotropy. The kinetic parameters of intein complex formation were K(d) = 1.1 mum, k(on) = 16.8 m(-1) s(-1), and k(off) = 1.8 x 10(-5) s(-1) for the native Int(N11) sequence. Intriguingly, a G(-1)A substitution, previously known to significantly impair protein splicing, was revealed to result in thiazoline ring formation involving the catalytic Cys-1, likely by aberrant dehydration of a oxythiazolidine intermediate. This finding provides experimental evidence for the postulated intermediate during the initial N/S acyl shift and underlines the delicate spatial and temporal alignment required in the intein active site to prevent side reactions of the protein-splicing pathway.

Pseudomonas fluorescens CHA0 Produces Enantio-pyochelin, the Optical Antipode of the Pseudomonas aeruginosa Siderophore Pyochelin

The siderophore pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation whereas the second cysteine remains in its L-configuration, thus determining the stereochemistry of the two interconvertible pyochelin diastereoisomers as 4'R, 2''R, 4''R (pyochelin I) and 4'R, 2''S, 4''R (pyochelin II). Pseudomonas fluorescens CHA0 was found to make a different stereoisomeric mixture, which promoted growth under iron limitation in strain CHA0 and induced the expression of its biosynthetic genes, but was not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promoted growth and induced pyochelin gene expression in P. aeruginosa, but was not functional in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of the P. aeruginosa siderophore pyochelin. Enantio-pyochelin was chemically synthesized and confirmed to be active in CHA0. Its potential biosynthetic pathway in CHA0 is discussed.

Epimerization of an L-cysteinyl to a D-cysteinyl residue during thiazoline ring formation in siderophore chain elongation by pyochelin synthetase from Pseudomonas aeruginosa.

The thiazoline-containing siderophores pyochelin, yersiniabactin, and Micacocidin A all have D-thiazoline rings, participating in high-affinity chelation of ferric iron. However, studies with pyochelin (Pch) synthetase and yersiniabactin (Ybt) synthetase reconstituted from pure protein components have shown that only L-cysteine is activated and tethered as a covalent aminoacyl-S-enzyme intermediate. Nor are any of the canonical epimerase domains of nonribosomal peptide synthetase (NRPS) assembly lines found in the Ybt or Pch synthetase modules. Here, we report that the PchE subunit of the Pch synthetase exchanges solvent deuterium into the C(2) center of the thiazoline moieties during siderophore chain elongation. Both PchE and HMWP2, from Ybt synthetase, subunits have a 310-360-residue insert in their amino acid activation domains that look like defective methyltransferase (MT) domains. We suggest these inserts are noncanonical epimerase domains, reversibly deprotonating and reprotonating acyl-S-enzyme intermediates at the C(2) locus. The PchE subunit does not epimerize the Cys-S-enzyme intermediate, but once amide bond formation from a benzoyl-S-PchE donor is catalyzed by the cyclization (Cy) domain of PchE, the N-benzoyl-Cys-S-PchE intermediate is present as a D,L-mixture. The subsequent phenylthiazolinyl-S-PchE intermediate, arising from cyclodehydration of the N-benzoyl-Cys-S-PchE intermediate, is likewise a D,L-mixture on hydrolytic release and enantiomer analysis. These results suggest a default role for MT domains of NRPS assembly lines in generating alpha-carbanionic species from thioester intermediates during siderophore chain elongation.

Thiazoline Ring Formation from 2- Methylcysteines and 2-Halomethylalanines

A systematic survey of conditions and substrates for the formation of 2,4,4-trisubstituted thiazoline rings is presented. The substitution patterns of these thiazolines is particularly relevant for the synthesis of the tantazole, mirabazole, and thiangazole classes of natural products, which contain a linear array of these heterocycles. Methods for the formation of these thiazolines from 2-methyl cysteines and 2-halomethylalanines are discussed.

The bacitracin biosynthesis operon of Bacillus licheniformis ATCC 10716: molecular characterization of three multi-modular peptide synthetases.

The branched cyclic dodecylpeptide antibiotic bacitracin, produced by special strains of Bacillus, is synthesized nonribosomally by a large multienzyme complex composed of the three bacitracin synthetases BA1, BA2 and BA3. These enzymes activate and incorporate the constituent amino acids of bacitracin by a thiotemplate mechanism in a pathway driven by a protein template. The biochemical features of these enzymes have been studied intensively but little is known about the molecular organization of their genes. The entire bacitracin synthetase operon containing the genes bacA-bacC was cloned and sequenced, identifying a modular structure typical of peptide synthetases. The bacA gene product (BA1, 598kDa) contains five modules, with an internal epimerization domain attached to the fourth; bacB encodes BA2 (297kDa), and has two modules and a carboxy-terminal epimerization domain; bacC encodes BA3, five modules (723kDa) with additional internal epimerization domains attached to the second and fourth. A carboxy-terminal putative thioesterase domain was also detected in BA3. A putative cyclization domain was found in BA1 that may be involved in thiazoline ring formation. The adenylation/thioester-binding domains of the first two BA1 modules were overproduced and the detected amino-acid specificity coincides with the first two amino acids in bacitracin. Disruption of chromosomal bacB resulted in a bacitracin-deficient mutant. The genes encoding the bacitracin synthetases BA1, BA2 and BA3 are organized in an operon, the structure of which reflects the modular architecture expected of peptide synthetases. In addition, a putative thiazoline ring formation domain was identified in the BA1 gene.

Total Synthesis of Antibiotic Althiomycin

Abstract Total Synthesis of antibiotic althiomycin has been achieved starting from d-cysteine. The imide bond between thiazoline and the pyrrolinone part was constructed by coupling reaction of sodium salt of pyrrolinone with cysteine active ester or by photoreaction with diketene. The hydroxymethyl group attached on the carbon adjacent to C-2 of the thiazoline ring, was introduced by aldol condensation posterior to the thiazoline ring formation. The thiazole part was introduced in a final step in whole process of the total synthesis of the antibiotic. The synthetic althiomycin was identical with the natural antibiotic in all respects.

Synthetic Studies on Bacitracin. VII. Isomerization of Amino Acid Components of Thiazoline Peptides

Abstract Ethyl 2-(1-benzyloxycarbonylaminoethyl)-R-Δ2-thiazoline-4-carboxylate (III), ethyl 2-(1-benzyloxycarbonylamino-2-methyl-n-butyl)-R-Δ2-thiazoline-4-carboxylate (VI) and their acid amide derivatives (V, VII and VIII) were prepared by either the iminoether coupling method or the dehydration method. As a general character of thiazoline peptide, it was found that the amino acid residue connected to thiazoline ring at N-terminal side is completely racemized at the stage of thiazoline ring formation, and further epimerized, in the case of isoleucine, by treatment with the basic reagent.