Li Xu

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%.

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%.

Fabrication of a magnetite nanoparticle-loaded polymeric nanoplatform for magnetically guided drug delivery

We developed a magnetite nanoparticle-loaded polymeric nanoplatform for magnetically guided 10-hydroxycamptothecin(HCPT) delivery. The nanoplatform was fabricated by simultaneously incorporating magnetite nanoparticles(NPs) and HCPT into the polymer micelle self-assembled from methoxy polyethylene glycol-poly(D,L-lactide-co-glycolide)(MPEG-PLGA) copolymer. Successful loading of HCPT into the nanoplatform was confirmed by Fourier transform infrared(FTIR) spectroscopy. Subsequently, we examined the in vitro antitumor efficacy of free HCPT and nanoplatform against three different cancer cell lines—HeLa, A549 and HepG2. Flow cytometric analysis was conducted to reveal the cell apoptosis caused by free HCPT and nanoplatform. Finally, the magnetic targeting property of the nanoplatform was evaluated by a self-designed in vitro experiment.

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.

In vitro Anticancer Efficacy by Magnetic Targeted Nanocarrier with Local Delivery of Paclitaxel

A magnetic nanoparticles-loaded polymeric nanocarrier was developed. Amphiphilic copolymer, methoxy polyethylene glycol-poly(D,L-lactide-co-glycolide)(MPEG-PLGA) could self-assemble to form nanomicelle with the help of emulsion-solvent evaporation technique. This nanocarrier with core-shell structure was loaded with magnetic iron oxide nanoparticles(IONPs) and anticancer drug paclitaxel(PTX). The hydrodynamic diameter of IONPs-PTX-loaded nanocarrier showed an average size of 110 nm with a polydispersity index(PDI) of 0.136, and its zeta potential was (–4.76±0.36) mV. The drug-loading content and encapsulation efficiency were 4.47% and 31.28%, respectively. In vitro drug release experiment was performed and a sustained release profile was observed. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT) assay indicated that IONPs-PTX-loaded nanocarrier showed comparable cytotoxicity with free paclitaxel. When an external magnetic field was applied, the nanocarrier significantly localized at the target area, demonstrating that the nanocarrier could be used for potential magnetic targeted drug delivery.

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%.

Novel chemical strategy for the synthesis of RGDCySS tetrapeptide

A new route was described to synthesize Arg-Gly-Asp-X(RGDX, X=amino acid) tetrapeptide. To better understand the method, the tetrapeptide Arg-Gly-Asp-CySS(RGDCySS) was chosen as a model target for X. First, GDCySS was obtained in four steps, comprising the chloroacetylation of L-aspartic acid(ClCH2COAsp), synthesis of chloroacetyl L-aspartic acid anhydride[ClCH2COAsp(CO)2O], formation of ClCH2COAsp-CySS and ammonolysis of ClCH2COAsp-CySS. Second, preparation of Arg-NCA, which was coupled with GDCySS to synthesize RGDCySS by the NCA method(Leuchs’ anhydrides method, NCA: N-carboxy-α-amino acid anhydride). The purity of the product was analyzed by the high performance liquid chromatography(HPLC). Molecular weights of the peptide products were confirmed by mass spectroscopy. In the developed approach, less protected amino acids were used compared to conventional solid-phase synthesis. The new route offers advantages of low cost, simplicity and rapid synthesis with a reasonable yield of 63.0%(calculated according to arginine content).

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%.