Rui-Zhen Hou

Synthesis of a precursor dipeptide of RGDS (Arg‐Gly‐Asp‐Ser) catalysed by the industrial protease alcalase

Synthesis of Bz‐Arg‐Gly‐NH2 (N‐benzoylargininylglycinamide) [a precursor dipeptide of RGDS (Arg‐Gly‐Asp‐Ser)] catalysed by protease in water/organic co‐solvent systems was studied. Starting substrates were N‐benzoyl‐l‐arginine ethyl ester hydrochloride (acyl donor) and glycinamide (nucleophile). Acetonitrile was selected as the organic solvent. Alcalase, an industrial alkaline protease, was applied to the synthesis of the target dipeptide. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including water content, temperature, pH, molar ratio of the substrates and reaction time, on the yield of Bz‐Arg‐Gly‐NH2. The optimum conditions were established to be pH 10.0, 45 °C, in acetonitrile/0.1 M Na2CO3/NaHCO3 buffer system (90:10, v/v) for 1 h with a dipeptide yield of 82.9%.

Alcalase‐Catalyzed, Kinetically Controlled Synthesis of a Precursor Dipeptide of RGDS in Organic Solvents

Abstract The protease‐catalyzed, kinetically controlled synthesis of a precursor dipeptide of RGDS, Z‐Asp‐Ser‐NH2 in organic solvents was studied. Alcalase, an industrial alkaline protease, was used to catalyze the synthesis of the target dipeptide in water‐organic cosolvents systems with Z‐Asp‐OMe as the acyl donor and Ser‐NH2 as the nucleophile. Acetonitrile was selected as the organic solvent from acetonitrile, ethanol, methanol, DMF, DMSO, ethyl acetate, 2‐methyl‐2‐propanol, and chloroform tested under the experimental conditions. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including water content, temperature, pH, and reaction time on the Z‐Asp‐Ser‐NH2 yields. The optimum conditions are pH 10.0, 35°C, in acetonitrile/Na2CO3‐NaHCO3 buffer system (85:15, v/v), 6 h, with a dipeptide yield of 75.5%.

New synthetic route for RGD tripeptide.

Abstract A new route was employed to synthesize RGD. First, Gly‐Asp dipeptide was synthesized by a novel chemical method in two steps, including chloroacetylation of L‐aspartic acid and ammonolysis of chloroacetyl L‐aspartic acid. Second, Nα‐Z‐ L‐Arginine was reacted with Gly‐Asp to synthesize RGD by the N‐carboxyanhydride method. Less protected amino acids were used in this synthesis. This method possessed advantages of low cost, simplicity, and rapidity with a reasonable yield of 62% calculated from arginine. In addition, compared with the above method, a conventional solid phase method was also used to synthesize RGD, the yield was 75% calculated from the first amino acid anchored to resin.

Synthesis of Cell Adhesive Motif RGD Tripeptide by a Novel Chemical Method and Its Purification

The cell adhesive motif RGD tripeptide was synthesized by using a novel chemical method. First, Gly-Asp(GD) was synthesized in two steps including the chloroacetylation of free L-aspartic acid and the ammonolysis of the chloro-acetylated L-aspartic acid. The yield of chloroacetylated L-aspartic acid was 83. 0%. For the ammonolysis of chloro-acetylated L-aspartic acid, the yield of the ammonolyzed product was 92. 3%. Second, the coupling between Arg and Gly-Asp was carried out by using the NCA method. The maximum yield of RGD was about 50% at 0 °C and pH = 9. 5. The prepared RGD tripeptide was confirmed by using amino acid component analysis and mass spectrographic analysis.

Protease-catalyzed Synthesis of Bz-Arg-Gly-Asp-OMe in Full Aqueous Medium

Synthesis of N-benzoyl-argininylglycylasparagine methyl ester (Bz-Arg-Gly-Asp-OMe), a precursor tripeptide of Arg-Gly-Asp) was catalyzed by papain under kinetic control, at alkaline pH, in a full aqueous medium. The substrates were N-benzoyl-argininylglycine ethyl ester and asparagine dimethyl ester. An aqueous solution of 0.1 mol/L KCl/NaOH containing 8 mmol/L EDTA and 2 mmol/L DTT was selected as the reaction medium. The synthesized hydrophilic tripeptide was soluble in the reaction medium during the reaction process, however, the secondary hydrolysis of the tripeptide product was not considerable. The effects of different factors, including water content, temperature, reaction time, and molar ratio of the substrates, on the yield of Bz-Arg-Gly-Asp-OMe were examined. The optimal reaction conditions were 0.05 mol/L Bz-Arg-Gly-OEt and 0.15 mol/L Asp(-OMe)2 . HCl in 0.1 mol/L KCl/NaOH solution (pH 8.5), at 40°C, and a reaction time of 60 min, with a maximum conversion yield of 62.4%.

Cellular Adhesion Tripeptide RGD Inhibits Growth of Human Ileocecal Adenocarcinoma Cells HCT-8 and Induces Apoptosis

Abstract The tripeptide, Arg-Gly-Asp(RGD) motif is an integrin-recognition site found in adhesive proteins present in extracellular matrices (ECM) and in the blood. HCT-8 cells were treated with cellular adhesion tripeptide RGD at various concentrations. MTT assay was performed to examine the growth and proliferation of HCT-8 cells after treatment with RGD for 48 h. Haematoxylin and Eosin (HE) staining and electromicroscope were used to observe the morphology of apoptotic cells. Survivin and flow cytometry were also used to analyze the HCT-8 apoptosis. Cellular adhesion tripeptide RGD significantly inhibits the growth and proliferation of HCT-8 cells in a dose-dependent manner and induces apoptosis of HCT-8. These results indicate that cellular adhesion tripeptide RGD inhibits the growth and proliferation of tumor HCT-8 cell, probably by the aid of inducing apoptosis of HCT-8 cell.

Chemoenzymatic Synthesis of Cellular Adhesion Tripeptide RGD Precursor in Organic Media

Abstract Chemoenzymatic synthesis of tripeptide Bz-RGD-(OEt) 2 was conducted in this study. First, the free dipeptide Gly-Asp was synthesized via a novel chemical method, wherein only L -aspartic acid was used and was followed by the esterification of Gly-Asp. The formation of the linkage between the third amino acid Bz-Arg-OEt and Gly-Asp-(OEt) 2 was completed by using the enzymatic method in organic media. The effects of several factors such as pH, the water content, triethylamine(TEA), the molar ratio of the substrates, and the reaction time on the yield of Bz-RGD-(OEt) 2 were examined. It was obtained that the optimum conditions for Bz-RGD-(OEt) 2 synthesis in an ethanol/ Tris-HC1 buffer system (volume ratio 93: 7) were as follows; pH=8.0; temperature, 30 °C; reaction time, 7 h. The tripeptide yield was 75. 2%.