Preparation Of The Caspase-3/7Substrate And A New Systhesis Method Of Fluorescent Molecule Used In The Substrate

As the principal peptidic substrate of caspase-3, Ac-DEVD-pNA and Ac-DEVD-AMC are widely used in cell biology, chemical biology and medicinal chemistry. So far, Ac-DEVD-pNA has been mainly used to monitor the activity of caspase-3/7in cell culture, although it has also been used for the in vitro identification and assessment of inhibitors of caspase-3/7and small molecule activators of procaspase-3/7:Caspase-3or caspase-7catalyzes the hydrolysis of Ac-DEVD-pNA and release the chromopHoric group p-nitroaniline; the activity of caspase-3/7is determined through the test of chromopHoric strength ofp-nitroaniline.This paper summarized the advantages and drawbacks of the current synthesis routes of polypeptides. Through balanced consideration of cost, atom economy and green chemistry, we choosed solution-pHase route and synthesized the caspase-3/7substrate Ac-DEVD-pNA in the gram-scale.(1)The synthesis of Ac-DEVD-pNA:the strong activator POCl3was used because of the relatively low nucleopHilicity of p-nitroaniline and the resulting Fmoc-Asp(Ot-Bu)-pNA was purified by column chromatograpHy and isolated in77%yield;(2)Peptide condensation:PyBOP was choosed as the condensing agent with quick reaction rate and yield(90%or more) at room temperature in each step;(3)Deprotection of amine:the Fmoc group was rapidly removed by diethylamine-DCM for2hours at room temperature, with quick reaction rate and yield(90%or more) at room temperature in each step;(4) Deprotection of the carboxyl group:The protected product was deprotected with trifluoroacetic acid to obtain the target product. In this paper, the overall yield is32%.This paper also made preliminary research into the synthesis of Ac-DEVD-AMC. Based on the work in our lab,7-amine-4-methylcoumarin was synthesized in a new ionic liquid,4-ethyl-2-butyl-5-benzyltetrazolium ethyl sulfate, both as the solvent and the catalysis.(1) as a1,3-Dipolar Cycloaddition reaction, zinc bromide was choosed as the catalyste in aqueous environment with yield up to78%;(2)1-bromobutane was used as the butyl agent and DBU as the base catalyst. The yield of the2-substituted product is up to61%;(3)2-butyl-5-benzyltetrazole reacted with diethyl sulfate under reflux in toluene to obtain the target ionic liquid, yielded up to74%.The reaction mechanism was investigated and the structure of all the intermediates and final product were confirmed by H’-NMR and MS.