Yi-Bing Huang

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%.

Synthesis of a precursor dipeptide of thymopentin in organic solvents by an enzymatic method.

Abstract The protease‐catalyzed, kinetically controlled synthesis of a precursor dipeptide of thymopentin(TP‐5), Z‐Arg‐Lys‐NH2 in organic solvents was studied. Z‐Arg‐OMe was used as the acyl donor and Lys‐NH2 was used as the nucleophile. An industrial alkaline protease alcalase and trypsin were used to catalyze the synthesis of the target dipeptide in water‐organic cosolvent systems. The conditions of the synthesis reaction were optimized by examining the effects of several factors, including organic solvents, water content, temperature, pH, and reaction time on the yield of Z‐Arg‐Lys‐NH2. The optimum conditions using alcalase as the catalyst are pH 10.0, 35°C, in acetonitrile/DMF/Na2CO3‐NaHCO3 buffer system (80∶10∶10, V/V), 6 h, with the dipeptide yield of 71.1%. Compared with alcalase, the optimum conditions for trypsin are pH 8.0, 35°C, in ethanol/Tris‐HCl buffer system (80∶20, V/V), 4 h, with the dipeptide yield of 76.1%.

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.

Trypsin‐Catalyzed Kinetically Controlled Synthesis of a Precursor Dipeptide of Thymopentin in Organic Solvents, Using a Free Amino Acid as Nucleophile

Abstract Trypsin‐catalyzed, kinetically controlled synthesis of a precursor, dipeptide of thymopentin (TP‐5), Bz–Arg‐Lys–OH (or–OEt) in organic solvents was studied. Bz–Arg–OEt was used as the acyl donor and Lys–OH and Lys–OEt were used as the nucleophiles. Ethanol was selected as the organic solvent from ethanol, methanol, acetonitrile, and ethyl acetate tested under the experimental conditions. As expected, Lys–OEt is not a suitable nucleophile in trypsin‐catalyzed reaction, due to its competition with the protective Arg–OEt as acyl donor for the active site of trypsin, while Lys–OH does not have this problem. The optimal reaction condition for the synthesis of Bz–Arg‐Lys–OH was set up as 20% Tris‐HCl buffer, pH 8.0, 35°C for 6 h with the yield of 52.5%, or for 18–24 h with the yield of about 60%.

Alcalase®-catalysed synthesis of the precursor tetrapeptide N-benzoylarginylglycylaspartylserinamide (Bz-RGDS-NH2) of the cell-adhesion peptide arginylglycylaspartylserine (RGDS)

In the present study, a precursor tetrapeptide Bz‐RGDS‐NH2 (N‐benzoylarginylglycylaspartylserinamide) of cell‐adhesion peptide RGDS (arginylglycylaspartylserine) was synthesized by a novel route. First of all, the precursor tripeptide GDS‐NH2 (glycylaspartylserinamide) was synthesized by a chemical method only using aspartic acid and serine at gram scale in four steps. The linkage of the fourth amino acid Bz‐Arg‐OEt (N‐benzoyl‐ L‐arginine ethyl ester) to GDS‐NH2 was completed by an enzymatic method under kinetic control in water‐miscible organic media. An industrial alkaline protease, Alcalase®, with a wide substrate specificity, was used as the catalyst. The effects of organic solvents, pH value, reaction temperature, water content and molar ratio of substrates on the yield of Bz‐RGDS‐NH2 synthesis were examined. The optimum reaction conditions were found to be pH 10.0, 35 °C, 8 h, in an acetonitrile/(Na2CO3/NaHCO3) buffer system (93:7, v/v) with a maximal yield of 65.2%. We found that secondary hydrolysis of the peptide product did not take place in these water‐miscible organic solvents.

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.

Inhibition of Growth of Human Ileocecal Adenocarcinoma Cells HCT-8 and Inducing Apoptosis by Different RGD-containing Peptides

Abstract Human ileocecal adenocarcinoma cells HCT-8 were treated with RGD-containing cellular adhesion peptides including RGD, RGD(NH 2 ) 2 ( i.e. , RGE-NH 2 ), RGDS, and RGDS-NH 2 . MTT assay was prepared to examine their inhibiting effects on HCT-8 cells after treatment. The methods including Haematoxylin and Eosin(HE) staining, transmission electron microscopy(TEM), immunohistochemistry, flow cytometry, and Reverse Transcription-Polymerase Chain Reaction(RT-PCR) were used to observe the morphology of the apoptotic cells and analyze the mechanism of apoptosis. The experimental results indicate that RGD-containing cellular adhesion peptides can inhibit the growth and proliferation of tumor HCT-8 cells in a dose-dependent manner and induce the apoptosis of HCT-8 cells. At the same time, the high conservative property of RGD was confirmed again.

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%.