Chemo-enzymatic Synthesis Of Bz-RGD-(NH₂)₂, The Precursor Of Cell Adhesive Motif RGD Tripeptide In Organic Solvents

Biologically active peptides possess multiplebiological functions and they are beneficial for biologyorganism. In recent years, one hundred kinds of biologicallyactive peptides have been found in body. Indeed, numerousstudies have described their antithrombosis, antihypertension,immunomodulating, hormonal, and antibiotic properties. Almostall the diseases occurring, recrudescence, therapy andhealing are related with peptides. The prospect of utilizingat least some of these molecules and their structural analoguesas new drugs has provided a powerful impetus for intensiveresearch by biotechnological methods in this area.Peptides containing Arg-Gly-Asp widely exist in manymultiorganisms and different tissues of organism. SincePierschbacher and Ruoslahti firstly reported RGD sequencewithin fibronectin, which is a cell recognition site in 1984,the research on RGD has become a hot point. In 1987, Hynessummarized RGD receptor, ligand and other biologic characters.RGD receptor was named integrin, and then RGD sequence has beenfound in many extra-cellular adhesive proteins. The tripeptideRGD is a common cell recognition site of many adhesive proteinscontained in extracellular matrices and in the blood. Peptidecontaining RGD have been found to be useful in anti-metastasis,anti-thrombosis, and treating osteoporosis. Bz-Arg-Gly-Asp-(NH2)2 , a precursor tripeptide of RGD wassynthesized by a combination of chemical and enzymatic methodsin this study. Firstly,Gly-Asp-(NH2)2 was synthesized by a novelchemical method, compared with conventional methods, thismethod possesses higher efficiency , lower cost , larger scaleand so on. The target tripeptide was synthesized in ethanol/This-HCl buffer system with Bz-Arg-OEt as acyl donor andGly-Asp-(NH2)2 as the nucleophile. In this study, we foundtrypsin treated with Tris-HCl buffer was much better comparedwith trypsin powder. Furthermore, ethanol/ This-HCl buffersystem is suitable as the reaction medium for the synthesis ofhydrophilic amino acid-containing peptides because of bettersolubility of substrates. The peptide product was easier to bepurified. The reaction conditions were optimized by examiningthe effects of several factors including water content,temperature, pH, and reaction time on the Bz-Arg-Gly-Asp-NH2yields. The optimum conditions are pH 8.0, 30℃, in ethanol/This-HCl buffer system (85:15, V/V), 80 min with the tripeptideyield of 74.4%. Purification is more important in our study.Some methods were used including gel chromatography, rotaryvaporization and lyophilizing. The samples were analyzed bymeans of RP-HPLC, HPLC-MS. The price is 51.50$ per 10mg RGD in Sigma productcatalogue. We designed an efficient and low-cost peptide...