类肌肽4(5)-丙氨酰胺-5(4)-羧酸咪唑的酶促合成及表征

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

摘要:

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:pH 8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1.5 h。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253 nm处吸收明显增强, 310 nm处出现新吸收峰; 253 nm、310 nm、330 nm高效液相色谱保留时间均为4.5 min; 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。

Abstract:

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), α-chymotrypsin : substrates (4,5-dicarboxyl acid and L-alanine )=1:200 (m/m), pH 8 phosphate buffer:THF = 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 Rf (ratio to front) = 0.81; then the new spot was purified and characterized with UV spectra, high performance liquid chromatogram (HPLC) and 13C NMR (13C nuclear magnetic resonance). The UV spectra shows two new absorption peaks 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; 13C 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.