Chemical Synthesis And Evaluation Of A Chromogenic Peptide Substrate

Objective:To study the main conditions of liquid-phase synthesis of chromogenic peptide substrate H-D-Chg-Ala-Arg-PNA?2HCl（CAAP）,synthesize chromogenic peptide substrate and evaluate its activity,providing a basis for efficient and economical preparation of chromogenic peptide substrates.Methods:（1）Synthesis of chromogenic peptide substrate:the condensation of arginine with p-nitroaniline is carried out by the activated ester method using pyridine as solvent and phosphorus oxychloride as condensing agent;the 9-fluorenylmethoxycarbonyl is removed with 50%piperidine/dichloromethane;the condensation of alanine and glycine is carried out by carbodiimide method using dichloromethane as solvent and EDCI-HOBt as condensing agent;the Tert-butoxycarbonyl is removed with HCl in acetone.（2）HPLC analysis of chromogenic peptide substrate:the imported chromogenic peptide substrate is used as the standard to select the mobile phase,trifluoroacetic acid content,mobile phase volume and wavelength in the HPLC analysis method to determine the optimal analysis conditions.The synthetic chromogenic peptide substrate,the imported chromogenic peptide substrate,and the domestic chromogenic peptide substrate are detected under the determined HPLC analysis conditions,and the three are qualitatively analyzed according to the retention time,the purity of the synthetic chromogenic peptide substrate is obtained according to the area normalization integration method.The yield of the synthetic chromogenic peptide substrate is calculated by drawing a standard curve on the imported chromogenic peptide substrate.（3）Evaluation of chromogenic peptide substrates:the chromogenic peptide substrates are added to an enzymatic reaction system,and the change in absorbance per unit time（ΔA/t）is detected by ultraviolet-visible spectrophotometer at a wavelength of 405 nm,thereby evaluating the reactivity of the chromogenic peptide substrates.The greater theΔA/t,the higher the activity of the chromogenic peptide substrate.Results:（1）A chromogenic peptide substrate is obtained by the activated ester method and the carbodiimide method,which is a pale yellow water-soluble solid.The main synthesis conditions are:1)The condensation of Fmoc-Arg（Boc）₂-OH with p-nitroaniline adopted activated ester method using phosphorus oxychloride as condensing agent and pyridine as solvent:the ratio of pyridine（mL）to reactant（mmol）was 3:1,and the reaction was carried out at-15^-10℃for 40 min;2)The9-fluorenylmethoxycarbonyl was removed with 50%piperidine/dichloromethane for 30min;3)The condensation of Fmoc-Ala-OH and Boc-D-Chg-OH adopted carbodiimide methodusingEDCI-HOBtascondensingagent,dichloromethaneand N,N-dimethylformamide as solvent:the amino acid substrate was activated at-5⁰℃for 30 min and then condensed with EDCI-HOBt with a total of 8 hours of reaction.The molar ratio of carboxyl component,amino component,EDCI-HOBt was 1.5:1:1.5.The volume ratio of dichloromethane to N,N-dimethylformamide is 7:1.4)The Tert-butoxycarbonyl was removed with HCl in acetone.（2）HPLC analysis of chromogenic peptide substrates:the retention time of the synthetic chromogenic peptide substrate,the imported chromogenic peptide substrate,and the domestic chromogenic peptide substrate were 9.357 min,9.316 min,and 9.415 min,respectively.The retention time of the three is relatively consistent.The target peak of the synthetic chromogenic peptide substrate was relatively clear,and the purity of the synthesized chromogenic peptide substrate was 34.70⁴8.95%by the area-integrated integration method,and the yield of the synthetic chromogenic peptide substrate calculated by the standard curve was 15²0%.（3）The evaluation results of the chromogenic peptide substrate:the change in absorbance per unit time of the synthetic chromogenic peptide substrate,the imported chromogenic peptide substrate,and the domestic chromogenic peptide substrate was 0.0045 s^-1,0.0052 s^-1,and 0.0031 s^-11 within 30¹50 s,respectively.Conclusion:（1）The water-soluble chromogenic polypeptide substrate obtained by the liquid-phase synthesis method was identified as thrombin chromogenic peptide substrate by HPLC and enzymatic reaction,and the purity was 34.70⁴8.95%,and the yield was 15²0%.（2）The initial synthetic method was:1)The condensation of Fmoc-Arg（Boc）₂-OH with p-nitroaniline used phosphorus oxychloride as condensing agent and pyridine as solvent;2)The removal of the 9-fluorenylmethoxycarbonyl was carried out with 50%piperidine/dichloromethane;3)The condensation of Fmoc-Ala-OH and Boc-D-Chg-OH used EDCI-HOBt as condensing agent,dichloromethane and N,N-dimethylformamide as solvent;4)The removal of the Tert-butoxycarbonyl is carried out with a solution of HCl in acetone.（3）In response to thrombin,the activity of the synthetic chromogenic peptide substrate is higher than that of the domestic chromogenic peptide substrate,but less than the imported chromogenic peptide substrate.