Chemo-enzymatic Synthesis Of Bz-RGDS-NH₂, The Precursor Of Cellular Adhesion Motif RGDS Tetrapeptide

The tripeptide Arg-Gly-Asp(RGD) sequence, which present inextracellar matrix (ECM), including fibronectin, vitronectin, osteopntin,collagens, thrombospondin, fibrinogen, and von Willebrand factor, plays akey role in mediating integrin-matrix interaction. RGD peptides ascompetitive, reversible inhibitors for the binding of adhesive proteins havebeen used to study adhesive interaction between cells and inhibit tumormetastasis, tumor-induced angiogenesis and tumor-elicited plateletaggregations. And RGD peptides not only play a major role as anchoringmolecules but also are important in processes like embryogenesis, celldifferentiation, immune response. Rencengtly, X₁-RGD-X₂ (X₁ =Gly, Asp;X₂ =Ser, Cys, Val, PHe), which is the further extension of the RGD, havebeen reported to make these sequences be even more efficient adhesionmotifs to integrins of cell membrane. For example, RGDS peptide exhibitsadditional potent anti-chemotactic and pro-apoptotic effects independentlyfrom its anti-adhesive action, likely by entering the cells and directlyactivating caspase 8 and 9, and lately caspase 3, implying unexpectedintracellular actions of the RGD-motif.As we know, it is not available to isolate RGD or other biologicallyactive peptides from whole tissue directly. So many researchers tried tosynthesize RGD and RGD-containing peptides by chemical or enzymaticmethods. Chemical and enzymatic method has different characteristic andsuit different synthesis scale. Chemical method is suitable for synthesis themoderate length peptide and with large-scale. An enzymatic method issuitable for synthesis the short peptide and with small-scale. As compared tothe chemical method, the important benefits of enzymatic peptide synthesisare:a) the mild conditions of the reaction;b) the high regiospecifity ofenzyme allowing the use of minimally protected substrates;c) the reactionbeing stereospecificity without racemization. A number of hydropHobicsmall peptides were synthesized in high yields using proteases in organicmedia as largely reported. However, the synthesis of the hydropHilicpeptide generally faced some problems: such as substrates solubility,activity and stability of enzyme in organic solvents. In hydropHobic organicsolvents, the enzyme usually shows better activity and selectivity, but thesolubility of hydropHilic substrates was too low. The polar solvents could toincrease the solubility of hydropHilic substrates. However, they are oftenharmful to enzyme because it has a greater tendency to strip the tightlybound essential water from the enzyme molecules. The appropriate reactionsystem should be selected considering the balance of solubility of substratesand enzymatic activity. RGDS tetrapeptide is a good hydropHilic peptidemodel containing three charged or polar residues (Arg, Asp and Ser) and aneutral one (Gly). So it was selected as the model tetrapeptide in this study.For enzymatic method, a number of proteases have been used aspractical catalysts in peptide synthesis, particularly in synthesis of smallpeptides. For protease-catalyzed synthesis of peptides under the kineticcontrol in organic solvents, the enzymes are limited to serine and cysteineproteases which could form acyl-enzyme intermediate with the acyl donor.Acyl transfer reaction is completed by deacylation of the covalentacyl-enzyme intermediate via possible two pathways of aminolysis andhydrolysis. Although protease-catalyzed synthesis of peptides has severaladvantages over chemical methods, such as no racemization, no essentialrequirement of side chain protection, and good stereo-and regioselectivity,but its shortcomings including an undesirable hydrolysis of the growingpeptide chain due to an amidase activity, a narrow substrate specificity, anddeactivation due to organic solvents still hamper its use in peptide synthesiswith long sequence. So lipase, which has esterase activity no amidaseactivity, was aplly to formation between an N-protected amino acid esterand a nucleopHile such as an amino acid amide could be completed in asimilar manner with protease-catalyzed synthesis of peptides under thekinetic control. High water content, therefore, could be applied to keepactivity of enzyme and improve solubility of hydropHilic substrates but lessside-reaction.In this study, we succeed in synthesizing the tetrapeptideBz-RGDS-NH2 by combining a novel chemical method with enzymemethod successfully. First, the chemical method was used to prepare theprecursor tripeptide GDS-NH2 at large scale with low cost. The linkage ofthe fourth amino acid (Arg) to GDS-NH2 was completed by enzymaticmethod under kinetic control in organic media. Trypsin, Alcalase and lipase(PPL) were selected to the synthesis of the peptide bond with Bz-Arg-OEtas the acyl donor. The synthesis reaction conditions were optimized byexamining the effects of several factors including water content,temperature, pH, and reaction time on the Bz-Arg-Gly-Asp-NH2 yields.Accidentally, the secondary hydrolysis of the tetrapeptide product did nottake place not oly in PPL catalyse peptide synthesis, but also in the otherreaction conditions because of the low water organic media.In addition, Z-Asp-Ser-OMe was catalyzed synthesis by enzymaticmethod under kinetically controlled in aqueous buffer system, which is ableto dissolve of the hydropHilic amino acids substrates well and keep activityand specificity of the enzyme. Also, Chymopapain was be used as catalystbecause its broad P1 specificity and a preference for small, relativelyhydropHilic groups in the P1' site.