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HPLC purification technique: synthesis of unsymmetrical thiobarbituric acids.

ABSTRACT: Synthesis of thiobarbituric acids by the reaction of 1,3-disubstituted thioureas and malonic acid in acetyl chloride-acetic acid medium and synthesis of cyclized pyrimidin-7-one by the interaction of 1-(2-hydroxyethyl)-aryl thioureas, with malonic acid in p-tolyl sulphonic acid and acetyl chloride-acetic acid medium at room temperature stirring has been reported. The present protocol is highly eco-friendly alternative to existing methods, reduces the excess use of acetyl chloride and purity of all synthesized molecules checked with the help of reverse phase high performance liquid chromatography with photo diode array (PDA) detection at 254 nm with spectral characterization by 1H, 13C NMR, and MS spectra.

SUBMITTER: Deotale VD

PROVIDER: S-EPMC6664147 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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HPLC purification technique: synthesis of unsymmetrical thiobarbituric acids.

Deotale Vinod D VD Katiya Manish M MM Dhonde Madhukar G MG

Heliyon 20190725 7

Synthesis of thiobarbituric acids by the reaction of 1,3-disubstituted thioureas and malonic acid in acetyl chloride-acetic acid medium and synthesis of cyclized pyrimidin-7-one by the interaction of 1-(2-hydroxyethyl)-aryl thioureas, with malonic acid in <i>p</i>-tolyl sulphonic acid and acetyl chloride-acetic acid medium at room temperature stirring has been reported. The present protocol is highly eco-friendly alternative to existing methods, reduces the excess use of acetyl chloride and puri ...[more]

PMID: 31384677

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Project description:The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2-6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins which were obtained in 8-33% overall yield with 90-98% purity despite the omission of HPLC purification.

Project description:Using a robust and quantitative assay, we have identified a novel class of DNA polymerase inhibitors that exhibits some specificity against an enzyme involved in resistance to anti-cancer drugs, namely, human DNA polymerase eta (hpol η). In our initial screen, we identified the indole thiobarbituric acid (ITBA) derivative 5-((1-(2-bromobenzoyl)-5-chloro-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (ITBA-12) as an inhibitor of the Y-family DNA member hpol η, an enzyme that has been associated with increased resistance to cisplatin and doxorubicin treatments. An additional seven DNA polymerases from different subfamilies were tested for inhibition by ITBA-12. Hpol η was the most potently inhibited enzyme (30 ± 3 μM), with hpol β, hpol γ, and hpol κ exhibiting comparable but higher IC50 values of 41 ± 24, 49 ± 6, and 59 ± 11 μM, respectively. The other polymerases tested had IC50 values closer to 80 μM. Steady-state kinetic analysis was used to investigate the mechanism of polymerase inhibition by ITBA-12. Based on changes in the Michaelis constant, it was determined that ITBA-12 acts as an allosteric (or partial) competitive inhibitor of dNTP binding. The parent ITBA scaffold was modified to produce 20 derivatives and establish structure-activity relationships by testing for inhibition of hpol η. Two compounds with N-naphthoyl Ar-substituents, ITBA-16 and ITBA-19, were both found to have improved potency against hpol η with IC50 values of 16 ± 3 μM and 17 ± 3 μM, respectively. Moreover, the specificity of ITBA-16 was improved relative to that of ITBA-12. The presence of a chloro substituent at position 5 on the indole ring appears to be crucial for effective inhibition of hpol η, with the indole N-1-naphthoyl and N-2-naphthoyl analogues being the most potent inhibitors of hpol η. These results provide a framework from which second-generation ITBA derivatives may be developed against specialized polymerases that are involved in mechanisms of radio- and chemo-resistance.

Dataset Information

HPLC purification technique: synthesis of unsymmetrical thiobarbituric acids.

Publications

HPLC purification technique: synthesis of unsymmetrical thiobarbituric acids.

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