Xiao-Xia Wu

Protease-catalyzed small peptide synthesis in organic media

Abstract Four kinds of supports were used to prepare immobilized papain using different methods: simple absorption on Celite, ionic absorption on CM-cellulose and QAE-Sephadex, and covalent cross-linking on egg white protein. The direct effects of the support on the catalytic properties of the enzyme were examined by studying the dependence of water content, pH, ionic strength, and reaction temperature on the yields of a model dipeptide Boc-Phe-ValOMe in ethyl acetate under thermodynamic control. It was found that the supports with low hydrophilicity demonstrated much higher catalytic activity by the enzyme than those with high hydrophilicity. Egg white protein was the best support with a good yield (94.5%) of the dipeptide. Papain and α-chymotrypsin immobilized on egg white protein were used to synthesize a number of dipeptides with Boc-Phe-XOMe showing different substrate specificity. Alcalase was used in the synthesis of some dipeptides containing hydrophilic amino acids and d -amino acids with reasonable yields.

Lipase-catalyzed synthesis of RGD diamide in aqueous water-miscible organic solvents

Abstract 1 RGD tripeptide as the cellular adhesion factor was synthesized by a combination of chemical and enzymatic methods in this study. First of all, Gly-Asp diamide was synthesized by a novel chemical method in three steps including preparation of L-aspartic acid dimethyl ester, chloroacetylation of L-aspartic acid dimethyl ester and ammonolysis of chloroacetyl L-aspartic acid dimethyl ester. Secondly, Porcine Pancreas Lipase (PPL) was used to catalyze the synthesis of Benzyl-Arg-Gly-Asp diamide in water-organic cosolvents systems. The reaction condition was optimized by examining the main factors affecting the yield of the tripeptide. The optimal reaction condition was set up as pH7.7, 15°C in 50% DMF for 8 h with the maximum yield of 76.4%. It was also found that 50% DMSO was another alternative with the tripeptide yield of 71.7%.

Protease-catalyzed synthesis of precursor dipeptides of RGD with reverse micelles

Abstract The precursor dipeptides of RGD, Boc-Arg-Gly-OEt and Ac-Gly-Asp-diOMe, were synthesized in three reverse micellar systems using different proteases. The reaction conditions for the Boc-Arg-Gly-OEt synthesis catalyzed by alcalase and for Ac-Gly-Asp-diOMe synthesis catalyzed by trypsin in AOT/isooctane reverse micelles were optimized by examining the effects of several factors including water content, temperature, pH, reaction time, and the enzymes on the dipeptide yields. The best yields for alcalase-catalyzed synthesis of Boc-Arg-Gly-OEt and trypsin-catalyzed synthesis of Ac-Gly-Asp-diOMe were 62.7% and 78.8%, respectively. Two other reverse micelle systems, Triton/ethyl acetate and HTAB/heptane/hexanol were also tested for the syntheses of the same dipeptides.

Kinetically controlled syntheses catalyzed by proteases in reverse micelles and separation of precursor dipeptides of RGD

Abstract The precursor dipeptides of RGD (Arg-Gly-Asp), N-CBZ-Arg-Gly-NH2 and CBZ-Gly-Asp-NH2 were synthesized in reverse micellar system and organic solvent using several proteases in different forms under kinetical control. The synthesis reaction conditions for CBZ-Arg-Gly-NH2 and CBZ-Gly-Asp-NH2 catalyzed by chymotrypsin in AOT/isooctane reverse micelles were optimized by examining the effects of several factors including water content, temperature, pH, and reaction time on the dipeptide yields. The best yields for the syntheses of CBZ-Arg-Gly-NH2 and CBZ-Gly-Asp-NH2 were 87.3% and 80.8%, respectively. The dipeptide products were separated from the reverse micelles by using a silica gel column.

Purification and characterization of thermostable α-amylase II from Bacillus sp-JF2 strain

Abstract Thermostable α-amylase from Bacillus sp - JF 2 strain was found to have three active components (names α-amylase I, II, and III) with molecular weights of 110,000, 140,000, and 300,000, respectively. α-Amylase II was isolated and purified in the current work by different procedures from that for α-amylase I. α-Amylase II consists of two identical subunits (MW 70,000). The isoelectric point is 4.7. The temperature optimum is at 90°C and the pH optimum is 5.5. for the enzyme activity. The half-life of the enzyme at 90°C is 30 min, and the enzyme is stable over a pH range of 7.0–9.0. The K m value of the enzyme was estimated to be 3.3 mg ml −1 . A considerable difference in amino acid composition was observed between α-amylase I and α-amylase II. The α-helix content of α-amylase II was calculated to be 51% from the circular dichroism spectrum. The number of Ca 2+ binding to each molecule of α-amylase II was determined to be 10 by atomic adsorption.

Lipase-catalyzed synthesis of precursor dipeptides of RGD in aqueous water-miscible organic solvents.

Abstract PPL-catalyzed synthesis of the precursor dipeptides of RGD as a cellular adhesion factor, Benzyl-Arg-Gly-NH2 and CBZ-Gly-Asp-NH2, was conducted in water-organic cosolvents systems. Five water-miscible organic solvents, which have some advantage over the water-immiscible organic solvent systems or the anhydrous organic solvent systems used often in protease-catalyzed synthesis of a peptide bond, were tested. The reaction condition of PPL-catalyzed synthesis of the dipeptides was optimized by examining the main factors affecting the product yield. The optimal reaction condition for the synthesis of Benzyl-Arg-Gly-NH2 was set up as pH 8.0, 15°C in 40% MeOH for 10 h with the maximum yield of 73.6%. The optimum condition for the synthesis of CBZ-Gly-Asp-NH2 was pH 7.0, 15°C in 50% MeOH for 10 h with the maximum yield of 67.0%.

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.