Rajan N. Shah

Control of glycoprotein synthesis. Characterization of (1 → 4)-N-acetyl-β-d-glucosaminyltransferases acting on the α-d-(1 → 3)- and α-d-(1 → 6)-linked arms of N-linked oligosaccharides

Abstract Hen oviduct membranes contain at least three N -acetyl-β- d -glucosaminyltransferases (GlcNAc-T) that attach a βGlcNAc residue in (1-4)-linkage to a d -Man p residue of the N -linked oligosaccharide core, i.e., (1 → 4)-β- d -GlcNAc-T III which adds a “bisecting” GlcNAc group to form the β- d -Glc p NAc-(1 → 4)-β- d -Man p -(1 → 4)- d -GlcNAc moiety; (1 → 2)-β- d -GlcNAc-T IV which adds a GlcNAc group to the (1 → 3)-α- d -Man arm to form the β- d -Glc p NAc-(1 → 4)-[β- d Glc p NAc-(1 → 2)]-α- d -Man p -(1 → 3)-β- d -Man p -(1 → 4)- d -Glc p NAc component; and (1 → 4)-β- d -GlcNAc-T VI which adds a GlcNAc group to the α- d -Man p residue of β- d -Glc p NAc-(1 → 6)-[β- d -Glc p NAc-(1 → 2)]-α- d -Man p -R to form β- d -Glc p NAc-(1 → 6)-[β- d -Glc p NAc-(1 → 4)]-[β- d -Glc p NAc-(1 → 2)]-α- d -Man p -R. We now report a novel (1 → 4)-(β- d -GlcNAc-T activity (GlcNAc-T VI′) in hen oviduct membranes that transfers GlcNAc to β- d -Glc p NAc-(1 → 2)-α- d -Man p -(1 → 6)-β- d -Man p -R to form β- d -Glc p NAc-(1 → 4)-[β- d -Glc p NAc-(1 → 2)]-α- d -Man p -(1 → 6)-β- d -Man p -R. The structure of the enzyme product was confirmed by 1 H NMR spectroscopy, FAB-mass spectrometry and methylation analysis. Previous work with GlcNAc-T IV was carried out with biantennary substrates; we now show that hen oviduct membrane GlcNAc-T IV can also transfer GlcNAc to monoantennary β- d -Glc p NAc-(1 → 2)-α- d -Man p -(1 → 3)-β- d -Man p -R to form β- d -Glc p NAc-(1 → 4)-[β- d -Glc p NAc-(1 → 2)]-α- d -Man p -(1 → 3)-β- d -Man p -R. The findings that GlcNAc-T VI′ and IV have similar kinetic characteristics and that hen oviduct membranes can convert methyl β- d -Glc p NAc-(1 → 2)-α- d -Man p to methyl β- d -Glc p NAc-(1 → 4)-[β- d -Glc p NAc-(1 → 2)]-α- d -Man p suggest that these two activities may be due to the same enzyme. The R-group of the β- d -Glc p NAc-(1 → 2)-α- d -Man p -(1 → 6)-β- d -Man p (or Glc p )-R substrate has an important influence on GlcNAc-T VI′ enzyme activity. When R is GlcNAc or βGlc-allyl, the activity is drastically reduced. This may be due to conformational factors and may explain why hen oviduct membranes add a GlcNAc residue in (1 → 4)-β-linkage mainly to the (1 → 3)-α- d -Man arm of the bi-antennary substrate β- d -Glc p NAc-(1 → 2)-α- d -Man p -(1 → 6)-[β- d -Glc p NAc-(1 → 2)-α- d -Man p -(1 → 3)]-β- d -Man p -R to form β- d -Glc p NAc-(1 → 2)-α- d -Man p -(1 → 6)-{β- d -Glc p NAc-(1 → 2)-[β- d -Glc p NAc-(1 → 4)]-α- d -Man p -(1 → 3)}-β- d -Man p -R.

Substrate specificity and inhibition of UDP-GlcNAc:GlcNAc beta 1-2Man alpha 1-6R beta 1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues.

UDP-GlcNAc:GlcNAc β1-2Manα1-6R (GlcNAc to Man) β1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAcβ1-6 branch to bi- and triantennaryN-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAcβ1-2Manα1-6Glc/Manβ-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Manα1-3 residue and the 4-hydroxyl of the Manβ- residue of the Manα1-6(Manα1-3)Manβ-RN-glycan core are not essential for catalysis but influence substrate binding. GlcNAcβ1-2(4,6-di-O-methyl-)Manα1-6Glcβ-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.

Flexibility in a tetrasaccharide fragment from the high mannose type of N-linked oligosaccharides

An analysis has been carried out of the three-dimensional structure of a tetrasaccharide, Man(alpha 1-3)Man(alpha 1-6)Man(beta 1-4)GlcN Ac beta 1-OCD3, which is a fragment from the high mannose type of N-linked oligosaccharides. Although earlier work had suggested that this fragment might adopt a stable three-dimensional structure, both n.m.r. and conformational energy calculations support the existence of an ensemble of structures. The conformational entropy calculated from the ensemble and the distribution of distances between the terminal Man(alpha 1-3) and GlcN Ac residues, however, suggests that a significant fraction of the ensemble has the two terminal residues in close proximity.

Accessibility of D-Mannopyranoside Glycosylating Synthons by Acetolysis for Preparations of Oligosaccharide Moieties of N-Linked Glycoproteins

Abstract Selective acetolysis of methyl 2, 3, 4, 6-tetra-O-benzyl-α-D-manno-pyranoside (2) allows for easy preparation of 1-acetates of 2, 3,4, 6-tetra-O-benzyl (5), 6-O-acetyl-2, 3, 4, tri-O-benzyl-(6), 4, 6-di-O-acetyl-2,3-di-O-benzyl-(7), 3, 4, 6-tri-O-acetyl-2-O-benzyl-(8), and 2, 4, 6-tri-O-acetyl-3-O-benzyl-D-mannopyranoside (9). 8 and 9 formed are separated by preparative HPLC in 30-60g scale. The time course of previously described acetolyses of 3, 4, 6-tri-O-benzyl- 1, 2-O-(1-methoxyethyidene)-β-D-mannopyranose (3), and methyl 2, 3-dt-O-benzyl-4, 6-O-benzylldene-α-D-mannopyranoside (4) giving 9, 1, 2, 6-tri-O-acetyl-3, 4-di-O-benzyl-(10), and 1, 2-di-O-acetyl-3, 4, 6-tri-O-benzyl-(11) α-D-mannopyranose as well 7 have been studied.

Insoluble Promoters of O-Glycosylation Reaction: Thallium, Cobalt, and Cadmium Zeolites 4A and 13X

Abstract TI+, Co2+ and Cd2+ salts of zeolites 4A and 13X were shown to be suitable solid-state promoters in a number of glycosylation reactions representing thus an alternative for Ag and Hg based promoters.

Synthesis of Trideuteriomethyl 5-Deuterium-β-D-Mannopyranoside

Abstract The title compound was prepared by first converting trideuteriomethyl 2,3,4-tri-O-benzyl-β-D-mannopyranoside to a 6-bromo-6-deoxy derivative which on elimination by using DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) or DBN (1,5-diazabi-cyclo[4.3.0]non-5-ene) gave a hex-5-enopyranoside derivative. The deuteroboration of the hex-5-enopyranoside followed by oxidation and subsequent deblocking produced trideuteriomethyl 5-deuterium-β-D-mannopyranoside.