Study On Synthesis Of RGD Tri-peptide And Recycling Of Coupling Reagent

Recently with the rapid progress of gene, protein and enzyme technology,discovery and synthesis of new bio-activity peptides are greatly promoted by themodernization of separation and analysis. Research and development of bio-activityor pharmaceutical peptides, including synthesis of precursor peptides, have becomeone of important aspects of international high technique fields. RGD , which has been put into high attention in recent years, is one of thebio-activity peptides composed of three amino acids: arginine(Arg),glycine(Gly) andaspartic acid(Asp). RGD is proved to be a recognition site for cellular adhesion,spreading and motility of cells. As the competitive and reversible inhibitors for fibbinding, a great prospect of application RGD in clinic has been illustrated. In this paper, the technique and scheme of synthesis RGD were studied. At thesame time, several factors which affected the synthesis of peptides were alsoinvestigated. And all these provided necessary technique parameters for large scaleproduction of RGD. Furthermore, recycling of the most widely used couplingreagents, such as DCC/HOBt, was firstly studied in this paper. The DCC and mixed anhydride methods were applied to synthesizing theprecursor of RGD and protected tri-peptide, respectively. All protected groups used inRGD synthesis were finally de-protected, and the crude de-protected RGD productwas obtained. The influence factors of synthesis such as reaction time, solvent andratio of substrates were further investigated. Based on above experimental results and analysis, the following conclusions aredrawn:(1) THF and DMF are favorable solvents in the synthesis of RGD.(2) The precursor of RGD, Boc-Gly-Asp-(oBzl)2 , is synthesized by both DCC and mixed anhydrides methods. To remove the Boc- group, HCl/EtoAc and TFA solution are used. However, only by using HCl/EtoAc, precursor crystals of HCl.Gly-Asp-(oBzl)2 are available. While by using TFA, it is difficult to get precursor crystals. So HCl/EtoAc solution is used to remove protection group. i(3) Using DCC method to synthesize protected tri-peptide (Boc-Arg(NO2)-Gly- Asp(oBzl)2) with THF and DMF solvents, the yield of protected tri-peptide is 76.1%. To avoid the product loss, TFA is used as de-protected reagent to remove Boc- group of the protected tri-peptide for 3h at room temperature, and the yield is 70.1% ( based on Boc-Arg(NO2)-OH). Using mixed anhydrides method to synthesize protected tri-peptide, Boc-Arg(NO2)-Gly-Asp-(oBzl)2, with THF and DMF solvents, the protected tri-peptide yield is 52.2%, TFA is also used as de-protected reagent to remove Boc- group of the protected tri-peptide for 3h at room temperature, and the yield is 83.8% ( based on Boc-Arg(NO2)-OH). However, the later product is not as dry as former synthesized by DCC method.(4) Arg(NO2)-Gly-Asp-(oBzl)2 is hydrogenated in methanol-acetic acid solution with Pd/C catalyst to get RGD, the yield is 97.4%.(5) Recycling coupling reagent DCC/HOBt is firstly investigated. HOBt is recycled by acidifying the step-disposal solution to recycle HOBt, and the recycle yield is 75.4%. DCC was recycled by dehydrating DCU by Phosphorus oxychloride, and the recycle yield is 64%.(6) On the 1kg RGD scale base, the cost for RGD production is reduced greatly from 35559.65RMB/kg to 14467.61RMB /kg through recycling coupling reagents and synthesizing protected amino acids. Among them, only recycling coupling reagents can reduce 11% of total cost of RGD production.