类肌肽4(5)-丙氨酰胺-5(4)-羧酸咪唑的酶促合成及表征

肌肽(β-Ala-L-His)是一种高效抗氧化剂,广泛应用于生物、化工、医药等领域。应用微水相酶促合成类肌肽,效率高价格低,且具有相似性质,开发前景广阔。本研究以L-丙氨酸和4,5-二羧酸咪唑制备类肌肽4(5)-丙氨酰胺-5(4)-羧酸咪唑,正交实验下的最佳合成条件为:四氢呋喃:pH8磷酸缓冲溶液=10:1.6(V/V),L-丙氨酸:4,5-二羧酸咪唑=1:3(m/m),α-胰凝乳蛋白酶:底物=1:200(m/m),35oC下磁力搅拌1.5h。硅胶G60薄层色谱(TLC)分离反应产物,Rf=0.81处出现新斑点;刮下该点纯化后进行紫外扫描,高效液相色谱(HPLC)和核磁共振,紫外光谱253nm处吸收明显增强,310nm处出现新吸收峰;253nm、310nm、330nm高效液相色谱保留时间均为4.0min;13C核磁共振显示8组碳原子。结合胰凝乳蛋白酶的催化机理,得出产物结构为4(5)-丙氨酰胺-5(4)-羧酸咪唑。

Enzymetic synthesis and characterization of a carnosine analogue in non-aqueous solvent

Carnosine (β-Ala-L-His) has high antioxidant activity, and it is widely used in biology, chemical engineering, medicine and other fields. Its analogue syntheised in non-aqueous solvent and catalyzed by enzymes is high-effective but low-price, so it has great prospect. Here, we synthesized a carnosine analogue imidazole 4(5)-alanylamide-5(4)-carboxylic acid with imidazole-4,5-dicarboxylic acid and L-Alanine as substrates, α-chymotrypsin as catalyst in tetrahydrofuran (THF) solvent. Based on the orthogonal experiments, the optimized synthetic conditions are 4,5-dicarboxylic acid : L-alanine = 1:3 (m/m), a-chymotrypsin : substrates (4,5-dicarboxyl acid and L-alanine )=l:200 (m/m), pH 8 phosphate bufferTHF = 1.6:10 (V/V), reaction temperature 35°C, time 1.5 h. We separated the product with silica gel G60 thin-layer chromatography (TLC), and a new spot appeared at R/(ratio to front) = 0.81; then the new spot was purified and characterized with UV spectra, high performance liquid chromatogram (HPLC) and ~(13)C NMR (~(13)C nuclear magnetic resonance). The UV spectra shows a new absorption peak at 310 nm, and the peak in 253 nm is largely strengthened; HPLC reserve times are all 4.5 min at 253 nm, 310 nm, 330 nm;~(13)C NMR shows 8 carbons. Combing with the catalytic mechanism of α-chymotrypsin, structure of the analogue is confirmed, i.e. imidazole 4(5)-alanylamide-5(4)-carboxylic acid.