Xue-Zhong Zhang

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.

Synthesis of graphene-like g-C3N4/Fe3O4 nanocomposites with high photocatalytic activity and applications in drug delivery

Graphene-like g-C3N4 nanosheet (GCN)/Fe3O4 quantum dot (QD) nanocomposites were successfully synthesized by a facile electrostatic self-assembly method. Characterization shows that the GCN is at least several micrometers in size. The GCN/Fe3O4 nanocomposites were used as photocatalysts for degradation of Rhodamine B (RhB) under visible light irradiation. After irradiation for 1.5 h, the degradation efficiency was 72.5% for pure g-C3N4, 81% for GCN-1 wt% Fe3O4, 95% for GCN-2 wt% Fe3O4, 60.46% for GCN-3 wt% Fe3O4 and 57.2% for GCN-4 wt% Fe3O4, indicating that GCN-2 wt% Fe3O4 nanocomposites had the highest photocatalytic activity. We deduce that the efficient separation of the photogenerated electron–hole pairs and the high specific surface area of GCN play important roles in the photocatalytic activity of the nanocomposites. In addition, the nanocomposites can be loaded with a model drug (Rhodamine B) and the loading capacity was as high as 108.6 mg g−1, making it a potential candidate for photocatalysis and controlled magnetically targeted drug delivery.

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%.

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.

Protease-Catalyzed Synthesis of a Precursor Dipeptide, Z-Asp-Val-NH2 of Thymopentin, in Organic Solvents

Abstract The protease-catalyzed, kinetically controlled synthesis of a precursor dipeptide, Z-Asp-Val-NH2 of thymopentin (TP-5), in organic solvents was studied. Z-Asp-OMe and Val-NH2 were used as the acyl donor and the nucleophile, respectively. An industrial alkaline protease alcalase was 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-Asp-Val-NH2. The optimum conditions using alcalase as the catalyst are pH 10.0, 35°C, in acetonitrile/Na2CO3-NaHCO3 buffer system (9:1, V/V), reaction time 5 h, with a yield of 63%. The dipeptde product was confirmed by LC- MS.

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%.

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%.

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%.