Studies On The Synthesis Of Three Kinds Of Protected Amino Acids

With the further research on the biological action of peptide, protein in life science and the development of modern medical and biochemistry technology,many peptides with activity and their analogues have been developed and widely used in medical,health, health care,food,cosmetics,etc.Research and development of peptide drug also become an active field in biotechnology industry in the 21st century.In order to avoid side reaction,the amino which will lead to side reaction should be protected before forming a new peptide bond.So the primary question of peptide synthesis is how to protect amino acid.In order to ensure the reaction successfully,not only the amino,but also other active groups such as side amino,hydroxyl,mercapto,carboxyl, guanidine need to be protected.In this paper,the research progress in peptide drugs,peptide synthesis and the protection of amino acids are briefly reviewed.From the industrial point of view,the synthesis of three protected amino acids, Fmoc-L-lys(Boc)-OH,Fmoc-L-Val-OH and Fmoc-L-Asp(OtBu)-OH, were investigated in detail and the procedures were listed as follow:1.Fmoc-L-lys(Boc)-OHUsing H₂O-acetone as solvent,sodium bicarbonate as acid binder and catalyst,N^ε-tert-butoxycarbony-L-lysine copper(â…¡) complex was synthesized successfully by N^ε-tert-butoxycarbonylation of L-lysine copper(â…¡) complex which was furnished by protection of theα-amino and carbonyl of L-lysine with copper(â…¡)sulfate pentahydrate as complex agent.The freeα-amino was released from N^ε-tert-butoxycarbonylation of L-lysine copper(â…¡) complex promoted by copper catching regent,and followed by N^α-9-fluorenylmethyloxycarbonylation catalysed by sodium carbonate with H₂O-acetone as solvent to form N^α-9-fluorenylmethyloxycarbonyl-N^ε-tert-butyloxycarbonyl-1-lysine (Fmoc-L-Lys(Boc)-OH).The effects of four kinds of copper catching reagents on the N^α-9-fluorenylmethyloxycarbonylation were studied,and the results showed the excellent copper catching reagent were sodium sulfide and 8-hydroxyquinoline.The reaction conditions of N^α-9-fluorenylmethyloxycarbonylation such as ratio of N^ε-tert-butoxycarbony-L-lysine copper(â…¡) complex to Fmoc-Osu, reaction time,reaction temperature were investigated in detail.The optimum reaction conditions were obtained as follow:the mole ratio of N^ε-tert-butoxycarbony-L-lysine copper(â…¡) complex to Fmoc-Osu is 1.00:0.98,the reaction temperature is room temperature and the reaction time is 3 h.Under those conditions,Fmoc-L-Lys(Boc)-OH was reached in 91.7%yield with 99.1%purity.2.Fmoc-L-Val-OHUsing H₂O-acetone as solvent,sodium carbonate as acid binder and catalyst,Fmoc-Osu was used as the N-terminal protecting reagent to block the L-valine.Systemic studies on synthetic process of Fmoc-L-valine-OH was investigated.Some synthetic conditions for synthesis Fmoc-L-valine-OH,such as reaction temperature,reaction time,ratio of start materials,were optimized.The recrystallization conditions of crude products were also studied in detail The results showed that the optimal synthetic conditions were as follow:The ratio of Fmoc-Osu to L-valine was 0.98:1,the reaction time was 4 h(1.5 h at -3℃followed by 2.5 h at room temperature),and the best recrystallization solvent system was petroleum ether/ethyl acetate in 2:1 ratio.Under the optimal reaction conditions and recrystallization system, Fmoc-L-valine-OH was obtained in 94.5%yield with 99.4%purity.3.Fmoc-L-Asp(OtBu)-OHFmoc-L-Asp(OtBu)-OH was synthesized by the regioselective esteration of L-Aspartic acid with isobutene using sulfuric acid as catalyst,followed by neutralization with 20%sodium hydroxide(pH= 7) and sodium carbonate(pH=9～10),followed by introduce reaction of Fmoc with the solution of Fmoc-Osu in 1,4-dioxane at lower temperature.The optimal synthesis conditions were as follow:the regioselective esteration was carried out at 9℃for 72 h,the introduce reaction of Fmoc was runned -L-Asp(OtBu)-OH was run at 5℃for 4.5 h with the ratio of Fmoc-Osu to L-aspartic acid=0.95:1.The reaction mixture was filtered,the filter was washed with ethyl ether,the water phase was neutralized to pH=5.5 with 2 mol / L HC1 and extracted with ethyl acetate,the organic phase was concentrated to give white solid. After recrystallization with DCM/petroleum to give Fmoc-L-Asp(OtBu)-OH in 39%yield with 95.7%purity.