反应详情

Acceptor specificity of the β1-4GlcA transferase activity of PmHS2 was also explored in one-pot three-enzyme system, as shown in FIG. 19. The first enzyme was a glucose-1-phosphate uridylyltransferase (GalU) which catalyzes the reversible conversion of Glc-1-P in the presence of UTP to produce UDP-Glc and inorganic pyrophosphate. The second enzyme was a UDP-glucose dehydrogenase (Ugd) for oxidation of 6-OH in glucose residue of UDP-Glc to form the UDP-glucuronic acid (UDP-GlcA) in the presence of its coenzyme NAD+. The third enzyme is PmHS2 transferring GlcA from UDP-GlcA for the formation of β1-4 linkage. As shown in FIG. 20, trisaccharides GlcAβ1-4GlcNAcα1-4GlcAβ2AAMe F20-1, GlcAβ1-4GlcNAc6N3α1-4GlcAβ2AAMe F20-3, GlcAβ1-4GlcNAcN3α1-4GlcAβ2AAMe F20-5 were synthesis by small-scale reaction and analyzed by HPLC method in 100%, 100% and 95% yields, respectively. The relative low yield (72%) for the formation of GlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe F20-2 was due to the formation of byproduct GlcAβ1-4GlcNH2α1-4GlcAβ2AAMe in which the TFA group was removed. Disaccharide F18-4 with N-glycolyl group in C2 position of glucosamine residue acts as a good acceptor for PmHS2, leading to the formation of GlcAβ1-4GlcNGcα1-4GlcAβ2AAMe F20-4 in 75% yield, but the disaccharide F18-6 with N-glycolyl group in C6 position of GlcNAc was converted to trisaccharide GlcAβ1-4GlcNAc6NGcα1-4GlcAβ2AAMe F20-6 only in 14% yield. Taken together, these results indicate that the donor and acceptor substrate activity of PmHS2 can tolerate a limited number of modifications on C-2 and C-6 position of glucosamine residue.