Keiko Sujino

GLYCOSYLTRANSFERASES IN OLIGOSACCHARIDE SYNTHESIS

Recent demonstrations that oligosaccharides play important roles in diverse biological events have resulted in renewed interest in the synthesis of oligosaccharides and their analogs. The availability of such molecules can facilitate studies on carbohydrate-protein recognition and help to elucidate molecular mechanisms of oligosaccharide-mediated biological processes that could eventually lead to rationally designed carbohydrate-based therapeutics. Despite many advances that have been made over the past decades, the chemical synthesis of oligosaccharides remains a challenge. This is attributed to the inherent chemical difficulties presented by this class of molecules. Each monosaccharide carries at least three hydroxyl groups that must be protected and deprotected during synthesis. Also, glycosylation generates a new stereocenter at the anomeric carbon, and there are no general methods for the introduction of all types of glycosidic linkage in a manner that is both stereo-controlled and high yielding. The chemical synthesis of oligosaccharides is therefore very timeconsuming and requires specialized expertise. The synthesis of oligosaccharide analogs containing modified sugars is an even more complex task than the preparation of natural structures. Almost twice as many steps are usually required for analog synthesis and the steps are more difficult, since most chemical protocols in the literature have been optimized for natural sugars. In nature, glycosyltransferase enzymes accomplish the ‘‘daunting’’ task of the construction of diverse and complex oligosaccharide. These enzymes catalyze

Stereocontrolled synthesis of pyrimidine 2′,3′-dideoxy-β-nucleosides by intramolecular glycosylation

Abstract β-2′,3′-Dideoxynucleosides and their 3′-azido and 3t-fluoro substituted derivatives were synthesized in a stereocontrolled manner by the dimethyl(methylthio)sulfonium tetrafluoroborate promoted intramolecular glycosylation of phenyl 2,3-dideoxy-5-O-(2-pyrimidyl)-1-thioglycosides followed by hydrolysis or ammonolysis.

Acceptor hydroxyl group mapping for calf thymus α-(1 → 3) - galactosyltransferase and enzymatic synthesis of α-d-Galp-(1 → 3)-β-d-Gal p-(1 → 4)-βd-GlcpNAc analogs

Abstract The epitope of the acceptor substrate for α -(1 → 3)-galactosyltransferase from calf thymus has been mapped by using a series of mono-deoxygenated and mono- O -alkylated Type II (β- d - Gal p-(1 → 4)-β- d - Glc p NAc ) disaccharides. The 4-OH group of the β- d -galactopyranosyl residue is a key polar group essential for glycosyl transfer, tolerating neither deoxygenation nor O -alkylation. Substitution at positions 6 and 6′ by a variety of polar alkyl substituents was readily tolerated, allowing the preparative enzymatic synthesis of a series of trisaccharide derivatives carrying polar substituents on each of these hydroxyl groups. These new analogs are potential inhibitors of Clostridium difficile toxin A and of a human anti-α-Gal antibody.

Stereocontrolled Synthesis of Pyridazine, Pyrazine, and Triazine 2′-DEOXY-β-Nucleosides by Means of Intramolecular Glycosylation

Abstract The stereocontrolled synthesis of 2′-deoxynucleoside analogs was explored by intramolecular glycosylation of pyridazine, pyrazines and triazines. These heterocycles were temporarily connected to the 5-O position in 1-thioglycoside via ethereal linkage.

Frontal Affinity Chromatography Coupled to Mass Spectrometry: An Effective Method for Kd Determination and Screening of α‐Gal Derivatives Binding to Anti‐Gal Antibodies (IgG)

Frontal affinity chromatography with mass spectrometric detection (FAC/MS) was developed as an effective method for rapid determination of Kd values for α‐Gal derivatives binding to human anti‐Gal IgG antibodies. Using this method, Kd values for 23 α‐Gal compounds were determined for the first time, including an α‐Gal terminated N‐linked oligosaccharide which mimics a single N‐glycoform present on the surface of animal cells. A mixture of eight α‐Gal derivatives, a model for an α‐Gal compound library, was successfully screened against this anti‐Gal IgG using FAC/MS. The analyte breakthrough sequence, indicated by the ion chromatogram, reflected the magnitude of the Kd values, confirming its potential application in the screening of new α‐Gal derivatives and mimetics. Ten α‐Gal derivatives were designed and synthesized chemically or enzymatically. Among the compounds analyzed, trivalent compound 26 demonstrated the strongest binding affinity to anti‐Gal IgG with a Kd value of 3.1 µM. The α‐Gal terminated N‐linked oligosaccharide 28 had a Kd value of 8.6 µM.

Facile synthesis of 2′,3′-unsaturated nucleosides from 2-deoxyribose

Abstract A straightforward approach for the synthesis of 2′,3′-unsaturated nucleosides starting from 2-deoxyribose is described. This novel route involves two new methods; (1) preparation of 2-deoxy-1-thioribofuranoside by direct condensation of 2-deoxyribose and thiophenol, (2) formation of the nucleoside skeleton by the direct coupling of 2,3-unsaturated 1-thiopentofuranoside with pyrimidine bases.

Stereocontrolled synthesis of 3′-isomeric β-nucleoside by intramolecular glycosylation

Intramolecular glycosylation of a pyrimidine derivative, which was temporarily connected to a 2,3-unsaturated 1-thiopentofuranoside through an ethereal linkage, led to an unusual 3′-isomeric nucleoside in a stereoselective fashion.

Effects of amine modifiers on the separation of tetramethylrhodamine-labeled mono- and oligosaccharides by capillary zone electrophoresis

In this work, nine tetramethylrhodamine (TMR) labeled isomeric oligosaccharide derivatives of betaGal(1 --> 4) betaGlcNAc-O-TMR were separated by capillary zone electrophoresis coupled with laser-induced fluorescence detection. Charged species were created in situ by complexation with borate and phenylborate. Micellar separation was achieved by addition of 10 mM sodium dodecylsulfate to the running buffer. We have investigated the effects of adding a homologous series of monoamine modifiers on the separation efficiency of these oligosaccharides. The separation was significantly improved in the presence of the organic modifiers methyl- and ethylamines, but worsened in the presence of propyl- and butylamines. Possible mechanisms of the amine additives are discussed.