Nobuyuki Yamaji

Syntheses of modified 2-chloro-4-nitrophenyl β-maltopentaosides as useful substrates for assay of human alpha amylase

Twenty-three novel 2-chloro-4-nitrophenyl beta-D-maltopentaosides modified at the 6(5) and/or 4(5) position were synthesized as substrates for human alpha amylase. Two human alpha amylases hydrolyzed 6(5)-deoxy-6(5)-, 6(5)-O-, and 4(5),6(5)-di-O-substituted derivatives at essentially a single D-glucosidic linkage, but 4(5),6(5)-O-bridged and 4(5)-O-substituted derivatives were hydrolyzed at two or more linkages. The amylases displayed smaller Km values for the compounds having hydrophobic modifications. In these derivatives, 2-chloro-4-nitrophenyl O-(6-bromo-6-deoxy-alpha-D-glucopyranosyl)-(1-->4)-tris[O-alpha-D- glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (10), 2-chloro-4-nitrophenyl O-(6-azido-6-deoxy-alpha-D-glucopyranosyl)-(1-->4)- tris[O-alpha-D-glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (19), and 2-chloro-4-nitrophenyl O-[6-O-(N-isopropyl)carbamoyl-alpha-D-glucopyranosyl]-(1-->4)- tris[O-alpha-D-glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (30), which were rapidly hydrolyzed by the two amylases at a limited position at an approximately equal rate, were shown to be very useful blocked-type substrates for assay of human alpha amylase.

Syntheses of subtractively modified 2-chloro-4-nitrophenyl β-maltopentaosides and their application to the differential assay of human alpha-amylases

Three novel maltopentaosides, 2-chloro-4-nitrophenyl O-(6-deoxy-alpha-D-xylo-hex-5-enopyranosyl)-(1-->4)-tris[O-alpha-D - glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (3), 2-chloro-4-nitrophenyl O-(6-deoxy-alpha-D-glucopyranosyl)-(1-->4)-tris[O- alpha-D-glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (10), and 2-chloro-4-nitrophenyl O-(3,6-anhydro-alpha-D-glucopyranosyl)-(1-->4)-tris[O-alpha-D-glucopyran osyl- (1-->4)]-beta-D-glucopyranoside (26) were synthesized by chemical and enzymatic reactions. Two human alpha-amylases, salivary alpha-amylase (HSA) and pancreatic alpha-amylase (HPA), hydrolyzed 3 and 10 with the same specificity, almost entirely at a single D-glucosidic linkage, but had no hydrolytic activity for 26. Compound 3 was hydrolyzed by each of these amylases at an approximately equal rate, while 10 was hydrolyzed by HSA 4-fold faster than by HPA. Taking advantage of the difference in the hydrolytic rate of 10, we developed a new method for the differential assay of these two human alpha-amylases.

New enzymatic synthesis of 63-modified maltooligosaccharides and their inhibitory activities for human α-amylases

Ten new 6(3)-modified maltopentaoses and tetraoses were synthesized by enzymatic reactions utilizing cyclodextrin glycosyltransferase (EC 2.4.1.19) and subsequent human salivary alpha-amylase (HSA) (EC 3.2.1.1). Among these compounds, alpha-D-glucopyranosyl-(1-->4)- alpha-D-glucopyranosyl-(1-->4)-(6-deoxy-alpha-D-glucopyranosyl)-(1-->4)- alpha-D-glucopyranosyl-(1-->4)-D-glucopyranose (11) and alpha-D-glucopyranosyl-(1-->4)-(6-deoxy-alpha-D-glucopyranosyl)-(1-->4)- alpha-D-glucopyranosyl-(1-->4)-D-glucopyranose (12) showed strong inhibitory activities for human pancreatic alpha-amylase (HPA) and HSA. The IC50 of 6(3)-deoxymaltopentaose 11 (8.0 x 10(-5) M for HPA, 1.0 x 10(-4) M for HSA) and 6(3)-deoxymaltotetraose 12 (2.0 x 10(-3) M for HPA, 2.0 x 10(-3) M for HSA) were lower than that of 6(3)-deoxymaltotriose [(6-deoxy-alpha-D-glucopyranosyl)-(1-->4)-alpha-D- glucopyranosyl-(1-->4)-D-glucopyranose 13; 2.0 x 10(-3) M for HPA, 4.2 x 10(-2) M for HSA].