Measurement of cyclomaltodextrin glucanotransferase activity by high-performance liquid chromatography using a fluorogenic substrate

A mixture of p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranoside (FG5P) and p-nitrophenyl alpha-D-glucoside (GP) was incubated with cyclomaltodextrin glucanotransferase (CGTase) [EC 2.4.1.19]. Analysis of the digest by HPLC showed that the products were p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P) and p-nitrophenyl alpha-D-maltoside (G2P), and no other product could be detected. Based on the reaction, a sensitive method to assay for CGTase was developed.     

Inspection of active sites of human salivary alpha-amylase isozymes by means of non-reducing-end substituted maltooligosaccharides with 2-pyridylamino residue

The modes of action of four alpha-amylase isozymes, which were purified from human saliva, on p-nitrophenyl alpha-maltopentaoside (G5P), maltohexaitol (G6R), and their 2-pyridylamino derivatives, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG5P) and O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O- alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D- glucitol (FG6R) were examined at various pH values. No differences in their modes of action on the substrates was found. Irrespective of which enzyme was used, the molar ratio of the hydrolysis products of G5P or G6R was almost constant at any pH examined. On the other hand, those of FG5P and FG6R varied with pH such that predominantly O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose (FG3) was formed at high pH ranges, while the formation of O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-gl ucose (FG4) increased at lower pH. The result indicates that the binding mode of FG5P or FG6R to the active sites of the enzymes changed with pH; namely, interactions between the 2-pyridylamino residue of the substrates and some amino acid residue(s) located in the active sites were influenced by pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation of human salivary alpha-amylase isozymes by high-performance liquid chromatography with a continuous monitor system of the activity

Human salivary alpha-amylase isozymes were rapidly separated from each other by high-performance liquid chromatography with a postcolumn assay. The eluate from the HPLC column was mixed continuously with an intramolecularly quenched fluorescent substrate, p-nitro-phenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside delivered by a pump. The mixture was incubated in a reaction coil, and the fluorescence intensity was continuously measured by a fluorescence detector. The assay was based on the marked increase in fluorescence with the enzymatic cleavage of the glycosidic bond of the substrate that links the fluorogenic and quenching moieties.     

Studies on the active site of Taka-amylase A: its action on phenyl maltooligosides with a charge at their non-reducing-ends

Five modified moltooligosaccharides, phenyl O-6-amino-6-deoxy-alpha-D- glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyransoide (AG4P), phenyl O-(alpha-D-glucopyranosyluronic acid)-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-d-glucopyran osy l- (1----4)-alpha-D-glucopyranoside (CG4P), phenyl O-6-amino-6-deoxy-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyra nos yl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyranoside (AG5P), phenyl O-(alpha-D-glucopyranosyluronic acid)-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)- alpha-D-glucopyranoside (CG5P), and phenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)- O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-a lph a-D- glucopyranoside (FG4P), were prepared to examine the active site of Taka-amylase A (TAA) [EC 3.2.1.1, Aspergillus oryzae]. Phenyl alpha-maltotetraoside (G4P) was predominantly hydrolyzed by TAA to maltose and phenyl alpha-maltoside (G2P). While G2P, phenyl alpha-glucoside (GP), and phenol were liberated from AG4P in the ratio of 7:63:30. G4P, phenyl alpha-maltotrioside (G3P), G2P, and GP were liberated from G5P in the ratio of 1:20:73:6, but AG5P was almost completely hydrolyzed to modified maltotriose and G2P. On the hydrolysis of CG4P and CG5P, no remarkable change was observed except for a decrease in the relative reaction rates compared with G4P and G5P, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Difference in transglycosylation between human pancreatic and salivary alpha-amylases

Transglycosylation reactions of alpha-amylases from human pancreatic juice and saliva were examined by using O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose as a substrate and O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-1-deoxy-1-[(2-pyridyl)amino]-D-glucitol as an acceptor. The transfer reaction was estimated by quantitation of O-alpha-D-glucopyranosyl-(1 leads to 4)-1-deoxy-1-[(2-pyridyl)amino]-D-glucitol produced by the enzymes from the transfer products, because the acceptor was not hydrolyzed. The amount of O-alpha-D-glucopyranosyl-(1 leads to 4)-1-deoxy-1-[(2-pyridyl)amino]-D-glucitol in the digest with pancreatic alpha-amylase was six times that in the digest with salivary alpha-amylase at the stage when the substrate was completely consumed, and the difference increased gradually on further incubation. The phenomenon can be applied to differentiate the two alpha-amylases in human serum.     

Preparation of a new fluorogenic substrate of alpha-amylases and a simple alpha-amylase assay by HPLC

A new substrate of alpha-amylases, O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, was prepared using dextrin as a starting material. Compared with other substrates so far reported, the fluorogenic substrate is unique in that it is resistant to exo-alpha-glucosidases due to the blocking group introduced into the non-reducing end glucose residue. The product of alpha-amylase digestion was rapidly separated from the substrate and was detected very sensitively by HPLC and a fluorescence detector. This method for alpha-amylase assay was also applied for determination of alpha-amylase in human serum.     

Examination of aglycone-binding site of human salivary alpha-amylase by means of transglycosylation reactions

The active site of human salivary alpha-amylase is composed of tandem subsites (S3, S2, S1, S1',S2', etc.) geometrically complementary to several glucose residues, and the glycosidic linkage of the substrate is split between S1 and S1'. As a matter of convenience, the subsites to which the non-reducing-end part (glycone) and the reducing-end part (aglycone) of the substrate being hydrolyzed are bound are named the glycone-binding site (S3, S2, S1) and the aglycone-binding site (S1', S2'), respectively. The features of the aglycone-binding site of human salivary alpha-amylase were examined by means of transglycosylation reaction using phenyl alpha-maltoside (GG phi: G-G-phi) and its derivatives (GAG phi: G-AG-phi, GCG phi: G-CG-phi, AGG phi: AG-G-phi, and CGG phi: CG-G-phi) in which one of the glucose residues (G) has been converted to 6-amino-6-deoxy-glucose (AG) or glucuronic acid (CG) residue as the acceptor. A fluorogenic derivative of maltotetraose, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D -glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P, FG-G-G-G-P), was used as the substrate. HSA catalyzed both hydrolysis of FG4P to FG3 (FG-G-G) and p-nitrophenyl alpha-glucoside (G-P) and transfer of the FG3 residue of FG4P to the acceptors. Transfer to GAG phi occurred more effectively than to GG phi. Transfers to GCG phi and CGG phi were less than to GG phi and very little transfer to AGG phi occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of Aspergillus oryzae alpha-amylase, potato phosphorylase, and rabbit muscle phosphorylase a and b on phosphorylated (1----4)-alpha-D-glucan

Aspergillus oryzae alpha-amylase [(1----4)-alpha-D-glucan glucanohydrolase, EC 3.2.1.1] produced O-(6-phosphoryl-alpha-D-glucopyranosyl)-(1----4)-O-alpha-D-glucopyran osy l-(1----4)-D-glucopyranose (6(3)-phosphorylmaltotriose) and O-alpha-D-glucopyranosyl-(1----4)-O-(3-phosphoryl-alpha-D-glucopyranosyl )- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucopyranose (3(3)-phosphorylmaltotetraose) from potato starch upon exhaustive hydrolysis. These products indicate that the enzyme hydrolyses the same linkages in the vicinity of the 6-phosphorylated residue as porcine-pancreatic alpha-amylase, but hydrolyses different linkages in the vicinity of the 3-phosphorylated residue when compared with B. subtilis and pancreatic alpha-amylases. Potato phosphorylase [(1----4)-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1] and rabbit muscle phosphorylase a and b were unable to by-pass the phosphorylated D-glucosyl residue of 6-phosphorylated (1----4)-alpha-D-glucan, leaving three D-glucosyl residues attached to the 6-phosphorylated residue on the non-reducing side.     

Detection of human urinary alpha-amylase encoded by the AMY2B gene using a fluorogenic substrate, FG5P.

The existence of alpha-amylase (HXA) encoded by alpha-amylase gene AMY2B in healthy humans was examined using a fluorogenic substrate, FG5P (FG-G-G-G-G-P: FG, 6-deoxy-6-[(2-pyridyl)amino]-D-glucose residue; G, glucose residue; P, p-nitrophenyl residue; -, alpha-1,4-glycosidic bond). Chromatofocusing of urine from a healthy human was carried out. FG5P was digested with the fractions exhibiting alpha-amylase activity and each digest at an early stage was analyzed by HPLC. FG5P was hydrolyzed to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P), and to FG4 (FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P). The molar ratios of FG4 to FG3 (FG4/FG3) in the digests with basic fractions were larger than those in the digests of human pancreatic alpha-amylase (HPA, 1.11) and human salivary alpha-amylase (HSA, 0.51). Considering that the value for the AMY2B gene product with yeast (yHXA) is 1.88, a value of more than 1.11 implies that HXA exists. The amount of HXA was determined after removal of HSA on an anti-human salivary alpha-amylase antibody bound column. The FG4/FG3 values for six urine samples free from HSA were 1.23-1.26. Assuming that the FG4/FG3 value for HXA is the same as that for yHXA, the ratios of HXA and HPA were estimated to be 1:5.4-4.1. The results obtained showed that the AMY2B gene is usually expressed as HXA in healthy humans.     

Synthesis of p-nitrophenyl 6(5)-O-benzyl-alpha-maltopentaoside, a substrate for alpha amylases

p-Nitrophenyl alpha-maltopentaoside, having a benzyl group on O-6 of the terminal (nonreducing) D-glucosyl group was prepared by use of a reductive ring-opening reaction. Highly regioselective reduction of p-nitrophenyl O-(2,3-di-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl)-(1----4)- tris[O-(2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl)-(1----4)]-2,3,6-tri- O- benzoyl-alpha-D-glucopyranoside by dimethylamine-borane and p-toluenesulfonic acid, followed by debenzoylation, gave p-nitrophenyl O-(6-O-benzyl-alpha-D-glucopyranosyl)-(1----4)-tris[O-alpha-D-glucopyran osyl- (1----4)]-alpha-D-glucopyranoside. An experiment was done on the mode of action of human pancreatic and salivary alpha amylases on this derivative. The compound is suitable as a substrate for the assay of alpha amylase when used with glucoamylase and alpha-D-glucosidase as coupling enzymes.     

Scammonins I and II, the resin glycosides of radix scammoniae from Convolvulus scammonia.

The ether-soluble resin glycoside ('jalapin') fraction obtained from scammony roots, on alkaline hydrolysis, gave a glycosidic acid, scammonic acid A, together with isobutyric, 2S-methylbutyric and tiglic acids. In addition, two kinds of resin glycosides, named scammonin I and II, were isolated and characterized, respectively, as (11S)-hydroxyhexadecanoic acid, 11-[( O-6-deoxy-4-O-(2(E)-methyl-1-oxo-2- butenyl)-beta-D-glucopyranosyl-(1----4)-O-6-deoxy-2-O-(2-methyl-1-oxobut yl)- alpha-L-mannopyranosyl-(1----2)-O-beta-D-glucopyranosyl-(1----2)-6-deoxy -beta- D-glucopyranosyl]oxy)-, intramol. 1,3"'-ester and (11S)-hydroxyhexadecanoic acid, 11-[( O-beta-D-glucopyranosyl-(1----4)-O-6-deoxy-2-O-(2-methyl-1-oxobutyl)- alpha-L-mannopyranosyl-(1----2)-O-beta-D-glucopyranosyl-(1----2)-6-deoxy -beta-D - glucopyranosyl]oxy)-, intramol. 1,3"'-ester.     

Synthesis of an appropriately protected core glycotetraoside, a key intermediate for the synthesis of "bisected" complex-type glycans of a glycoprotein

A stereocontrolled synthetic route to a glycotetraoside, allyl O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-O- (3,6-di-O-allyl-2-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-3, 6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1----4)-3-O- benzyl- 2-deoxy-6-O-p-methoxy-phenyl-2-phthalimido-beta-D-glucopyranoside, an important intermediate for the synthesis of "bisected" complex type glycans of glycoproteins has been established by employing two glycosyl donors, 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate and 4-O-acetyl-3,6-di-O-allyl-2-O-benzyl-alpha-D-mannopyranosyl bromide, and a glycosyl acceptor, allyl O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1----4) -3-O- benzyl-2-deoxy-6-O-p-methoxyphenyl-2-phthalimido-beta-D-glucopyranoside.     

Synthesis of an octasaccharide fragment of high-mannose-type glycans of glycoproteins.

O-(alpha-D-Mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----3)- O- [(alpha-D-mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----6)]- O- (alpha-D-mannopyranosyl)-(1----6)-O-(beta-D-mannopyranosyl)-(1----4)-O-( 2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy- glucopyranose, an octasaccharide fragment of high-mannose type glycan of glycoproteins, was synthesized. Crucial glycosylation of trisaccharide intermediate, benzyl O-(2,4-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(2-acetamido-3,6-di -O- benzyl-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-3,6-di-O-benz yl-2- deoxy-beta-D-glucopyranoside, was successful only with a di-O-acetyltetradeca-O-benzyl-D-mannopentaosyl chloride. The use of the corresponding hexadeca-O-acetyl-D-mannopentaosyl bromide did not give the desired product.     

Synthesis of p-nitrophenyl beta-glycosides of (1----6)-beta-D-galactopyranosyl-oligosaccharides

Sequential tritylation, benzoylation, and detritylation of p-nitrophenyl beta-D-galactopyranoside gave p-nitrophenyl 2,3,4-tri-O-benzoyl-beta-D-galactopyranoside (2). Reaction of 2 with 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl bromide gave p-nitrophenyl O-(2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl)-(1----6) -2,3,4-tri-O-benzoyl-beta-D-galactopyranoside (4) in 94% yield. Deprotection with sodium methoxide then gave the crystalline p-nitrophenyl O-(beta-D-galactopyranosyl)-(1----6)-beta-D-galactopyranoside (5). Condensation of 2 with 2,3,4-tri-O-benzoyl-6-O-bromoacetyl-alpha-D-galactopyranosyl bromide (3) readily yielded the protected disaccharide p-nitrophenyl O-(2,3,4-tri-O-benzoyl-6-O-bromoacetyl-beta-D-galactopyranosyl)-(1----6) -2,3,4-tri-O-benzoyl-beta-D-galactopyranoside (6) from which the bromoacetyl groups could be selectively removed. Condensation of the resulting material with tetra-O-benzoyl-alpha-D-galactopyranosyl bromide then yielded p-nitrophenyl O-(2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl)-(1----6)-O-(2,3,4 -tri-O-benzoyl-beta-D-galactopyranosyl)-(1----6)-2,3,4-tri-O-benzoyl-bet a-D -galactopyranoside, (8), which was converted into the crystalline trisaccharide p-nitrophenyl O-(beta-D-galactopyranosyl)-(1----6)-O-beta-D-galactopyranosyl)-(1----6) -beta -D-galactopyranoside (9) by treatment with sodium methoxide. Preliminary experiments on the interaction of p-(bromoacetamido)phenyl and p-isothiocyanatophenyl glycoside derivatives of some of these galacto-saccharides with monoclonal anti-(1----6)-beta-D-galactopyranan antibodies have been conducted.     

A regio- and stereo-controlled synthesis of beta-D-Glcp NAc6SO3-(1----3)-beta-D-Galp6SO3-(1----4)-beta-D-GlcpNAc 6SO3- (1----3)-D-Galp, a linear acidic glycan fragment of keratan sulfate I

A stereocontrolled synthesis of beta-D-GlcpNAc6SO3-(1----3)-beta-D-Galp6SO3-(1----4)-beta-D- GlcpNAc6SO3- (1----3)-D-Galp, was achieved by use of benzyl O-(2-acetamido-3,4 di-O-benzyl-2-deoxy-6-O-p-methoxyphenyl-beta-D- glucopyranosyl)-(1----3)-O-(2,4-di-O-tert-butyldiphenylsilyl-beta- D- galactopyranosyl-(1----4)-O-(2-acetamido-3-O-benzyl-2-deoxy-6-O-p-methox yphenyl - beta-D-glucopyranosyl)-(1----3)-2,4,6-tri-O-benzyl-beta-D-galactopyranos ide as a key intermediate, which was in turn prepared by employing two glycosyl donors, 3,4-di-O-benzyl-2-deoxy-6-O-p-methoxyphenyl-2-phthalimido-beta-D- glucopyranosyl trichloroacetimidate and O-(3,6-di-O-acetyl-2,4-di-O-benzyl-beta-D-galactopyranosyl)-(1----4)-3-O - benzyl-2-deoxy-6-O-p-methoxyphenyl-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate, and a glycosyl acceptor, benzyl 2,4,6-tri-O-benzyl-beta-D-galactopyranoside.     

Synthesis of methyl glycoside derivatives of tri- and penta-saccharides related to the antithrombin III-binding sequence of heparin, employing cellobiose as a key starting-material

Two key synthons for the title pentasaccharide derivative, methyl O-(methyl 2-O-benzoyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-acetyl- 2-azido - 3-O- benzyl-2-deoxy-beta-D-glucopyranoside and O-(methyl 2,3-di-O-benzyl-4-O- chloroacetyl-beta-D-glucopyranosyluronate)-(1----4)-3,6-di-O-acetyl-2-az ido-2- deoxy-alpha-D- glucopyranosyl bromide, were prepared from a common starting material, cellobiose. They were coupled to give a tetrasaccharide derivative that underwent O-dechloroacetylation to the corresponding glycosyl acceptor. Its condensation with the known 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl bromide afforded a 77% yield of suitably protected pentasaccharide, methyl O-(6-O- acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)- O- (methyl 2,3- di-O-benzyl-beta-D-glucopyranosyluronate)-(1----4)-O-(3,6-di-O-acetyl-2- azido-2 - deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-benzoyl-3-O-benzyl-alpha-L- idopyranosyluronate)- (1----4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranoside. Sequential deprotection and sulfation gave the decasodium salt of methyl O-(2- deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl)-(1----4)-O-(be ta-D- glucopyranosyl-uronic acid)-(1----4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-gluco pyranosyl)- (1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1----4)-2-deoxy-2- sulfamido-6-O- sulfo-beta-D-glucopyranoside (3). In a similar way, the trisaccharide derivative, the hexasodium salt of methyl O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D- glucopyranosyl)- (1----4)-O-(beta-D-glucopyranosyluronic acid)-(1----4)-2-deoxy-2-sulfamido-3,6- di-O- sulfo-alpha-D-glucopyranoside (4) was synthesized from methyl O-(6-O-acetyl-2- azido- 3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2,3-di-O- benzyl-beta- D-glucopyranosyluronate)-3,6-di-O-acetyl-2-azido-2-deoxy-alpha-D- glucopyranoside. The pentasaccharide 3 binds strongly to antithrombin III with an association constant almost equivalent to that of high-affinity heparin, but the trisaccharide 4 appears not to bind.     

Studies on the substrate specificity of Taka-amylase A1. XIV. Preparation of 6-deoxy-6-halogenomaltotrioses and their hydrolysis by Taka-amylase A.

1. O-6-Deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, O-6-chloro-6-deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, O-6-bromo-6-deoxy-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, and O-6-deoxy-6-iodo-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose were prepared, taking advantage of the substrate specificities of Taka-amylase A and glucoamylase, and the action of Taka-amylase A on these substrates was investigated. 2. The Michaelis constant Km and the molecular activity ko were determined at 37 degrees C and pH 5.2 using the modified maltotrioses. The values of Km and ko decreased upon modification of maltotriose and those of ko/Km were in agreement with the comparative initial rates for the corresponding derivatives of phenyl alpha-maltoside at low substrate concentrations. This result suggested that a subsite of the enzyme may have a specific interaction with halogen atoms in the substrate. 3. All halogenomaltotrioses examined showed substrate inhibition at high substrate concentrations.     

Synthesis of alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D-Manp+ ++-(1----6)] - beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp- (1----6)]-D- GlcpNAc, a core glycoheptaose of a "bisected" complex-type glycan of glycoproteins

A synthesis of alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D-Manp++ +-(1----6)]- beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp-( 1----6)]-D- GlcpNAc was achieved by employing benzyl O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-O- (2-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(3,6-di-O-benzyl-2-deoxy-2 - phthalimido-beta-D-glucopyranosyl)-(1----4)-3-O-benzyl-2-deoxy-6-O-p- methoxyphenyl-2-phthalimido-beta-D-glucopyranoside as a key glycosyl acceptor. Highly stereoselective mannosylation was performed by taking advantage of the 2-O-acetyl group in the mannosyl donors. The alpha-L-fucopyranosyl residue was also stereoselectively introduced by copper(II)-mediated activation of methyl 2,3,4-tri-O-benzyl-1-thio-beta-L-fucopyranoside.     

p-Trifluoroacetamidophenyl O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D-mannopyranosyl- (1----6)]-beta-D-mannopyranoside

p-Nitrophenyl 2-O-benzyl-4,5-O-cyclohexylidene-beta-D-mannopyranoside (4) was condensed with tetra-O-benzoyl-alpha-D-mannopyranosyl bromide. The resulting, protected disaccharide was converted into p-nitrophenyl O-(2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl)-(1----3)-4-O-benzoyl-2-O- benzyl-beta-D-mannopyranoside (8), which was condensed with tetra-O-benzoyl-alpha-D-mannopyranosyl bromide to give p-nitrophenyl O-(2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl)-(1----3)-O -[2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1----6)]-4-O-benzoyl-2-O -benzyl-beta-D-mannopyranoside (9) in 75% yield. Conversion of the p-nitrophenyl group followed by deprotection then yielded the title compound, whose structure was confirmed by 1H- and 13C-n.m.r. spectroscopy.     

Differentiation of human non-salivary, non-pancreatic alpha-amylase from salivary and pancreatic alpha-amylases by use of FG5P

Human non-salivary, non-pancreatic alpha-amylase (yHXA) is the gene product of a newly found human alpha-amylase gene expressed in yeast. Its mode of action on a fluorogenic derivative of p-nitrophenyl alpha-maltopentaoside, FG5P (FG-G-G-G-G-P), was examined at various pH values to elucidate the difference between yHXA and pancreatic or salivary alpha-amylase. The product analysis of the digests by HPLC showed that the enzyme hydrolyzed FG5P to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P) and to FG4(FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P), and the ratio of the two reactions changed with pH. The three enzymes differed from each other in the mode of action at pH 5.5. The molar ratio of FG4 to FG3 in the digest with yHXA was the largest. This suggested that the expression of the new gene in human can be detected by the use of FG5P as the substrate in the alpha-amylase assay.