Gary R. Gray

Proteins containing reductively aminated disaccharides: Synthesis and chemical characterization

Abstract Synthetic glycoproteins can be prepared by reductive amination of protein and reducing disaccharide in the presence of sodium cyanoborohydride. The reaction proceeds readily in aqueous solutions over a broad pH range to give high degrees of substitution. The degree of substitution can be determined by amino acid analysis, as the secondary amine linkage formed by reductive amination in stable to acid-catalyzed protein hydrolysis conditions. In order to demonstrate that coupling occurs to lysine residues, synthetic α- N -1-(1-deoxyglucitol)-lysine and ϵ- N -1-(1-deoxyglucitol)-lysine were prepared and compared with bovine serum albumin conjugates of maltose, cellobiose, lactose, and melibiose by amino acid analysis after acid hydrolysis. These studies demonstrate that the expected secondary amine linkages are formed with the ϵ-amino groups of lysine.

Analysis of the linkage positions in D-fructofuranosyl residues by the reductive-cleavage method.

The suitability of the reductive-cleavage method for analysis of the linkage positions in D-fructofuranosyl residues of D-fructans was examined by using sucrose, chicory-root inulin, and Aerobacter levanicum levan as models. Permethylation, and reductive cleavage with triethylsilane in the presence of either boron trifluoride etherate or trimethylsilyl trifluoromethanesulfonate, gave the expected methylated derivatives of 2,5-anhydro-D-mannitol and 2,5-anhydro-D-glucitol. With either catalyst, nonreducing (terminal) D-fructofuranosyl groups and D-fructofuranosyl residues linked at O-1 gave derivatives having the manno configuration as the major product, whereas D-fructofuranosyl residues linked at O-6, and at both O-1 and O-6, gave derivatives having the gluco configuration as the major product. The independent synthesis, and n.m.r.- and mass-spectral characterization, of the methylated 2,5-anhydro-D-mannitol and 2,5-anhydro-D-glucitol derivatives formed from these residues by reductive cleavage are reported.

A new catalyst for reductive cleavage of methylated glycans

Several per-O-methylated D-glucans and D-fructans were used as models in an attempt to identify new catalysts for carrying out reductive cleavage. Included in these model studies were several D-glucans that contained 4-linked D-glucopyranosyl residues as well as one having a 4-linked D-glucitol residue, as both types of residue had previously been found to give rise to substantial proportions of artifactual products. These studies led to the development of a new catalyst for carrying out reductive cleavage, namely, a mixture of 5 equivalents of trimethylsilyl methanesulfonate (Me3SiOSO2Me) and 1 equivalent of boron trifluoride etherate (BF3 . Et2O) per equivalent of acetal. This new catalyst was found to accomplish the reductive cleavage of per-O-methylated, 4-linked D-glucopyranosyl residues and 4-linked D-glucitol residues, to give only the expected derivatives of 1,5-anhydro-D-glucitol and D-glucitol, respectively. The mixture of Me3SiOSO2Me and BF3 . Et2O also catalyzed reductive cleavage of the D-fructofuranosyl residues of per-O-methylated sucrose and inulin, to give only the expected derivatives of 2,5-anhydro-D-mannitol and 2,5-anhydro-D-glucitol. Indeed, when used alone, Me3SiOSO2Me also rapidly catalyzed the reductive cleavage of D-fructofuranosyl residues, but, under the same conditions, D-glucopyranosyl residues were unaffected. The results of these and other model studies demonstrated that catalysis of reductive cleavage by the mixture of Me3SiOSO2Me and BF3 . Et2O occurs in a synergistic manner. Examination of the mixture of Me3SiOSO2Me and BF3 . Et2O by 1H-n.m.r. spectroscopy demonstrated that a reaction occurs to generate trimethylsily fluoride and species of the type F2BOSO2Me, FB(OSO2Me)2, or B(OSO2Me)3 via ligand exchange.

Analysis of linkage positions in saccharomyces cerevisiaed-mannans by the reductive-cleavage method☆

The positions of linkage in the D-mannans derived from Saccharomyces cerevisiae X2180 and its mutants, mnn1, mnn2, and mnn4, were established by perethylation and subsequent reductive cleavage with triethylsilane in the presence of boron trifluoride etherate (BF3 . Et2O) or trimethylsilyl trifluoromethanesulfonate. With the latter as the catalyst, all glycosidic carbon-oxygen bonds were cleaved, to produce a mixture of ethylated 1,5-anhydro-D-mannitol derivatives. With BF3 . Et2O as the catalyst, 2-, 3-, and 6-linked residues were incompletely cleaved, and residues linked at both O-2 and O-6 were not cleaved at all. It was concluded that reductive cleavage is an attractive method for determination of the structure of polysaccharides.

Reductive Cleavage of Glycosides, Stereochemistry of Trapping of Cyclic Oxonium Ions

Abstract Reductive cleavage of the glycosidic carbon-oxygen bonds of methyl 2,3,4,6-tetra-O-methyl-β-d-glucopyranoside (1), methyl 2,3,4,6-tetra-O-methyl-α-d-glucopyranoside (2), permethylated cellulose (6) and permethylated cyclohexaamylose (7) was carried out in the presence of deuteriotriethylsilane, and the configuration of deuterium in the l-deuterio-1,5-anhydro-d-glucitol derivatives (4, 5 and 9, 10) that were produced was established by 1H- and 2H-NMR spectroscopy. All reductions were carried out with boron trifluoride etherate as the catalyst as originally reported [D. Rolf and G. R. Gray, J. Am. Chem. Soc., 104, 3539 (1982)], as well as with trimethylsilyl trifluoromethanesulfon-ate which we now report efficiently catalyzes the regiospecific reductive cleavage of glycosides. Spectroscopic studies revealed that the configuration of deuterium in the products was independent of the configuration of the starting glycoside. The predominant (∼95%) axial configuration observed leads us to propose that f...

Analysis of positions of substitution of O-methyl or O-ethyl groups in partially methylated or ethylated cellulose by the reductive-cleavage method☆

A method is described for the analysis of positions of substitution of O-methyl and O-ethyl groups in commercial samples of O-methylcellulose and O-ethylcellulose, respectively. The method requires perethylation of O-methyl-cellulose and permethylation of O-ethylcellulose. Subsequent reductive cleavage of both polymers gives the same eight products, which are analyzed as their O-acetyl derivatives by gas-liquid chromatography.

Proteins containing reductively aminated disaccharides: Immunochemical characterization

Abstract Bovine serum albumin (BSA) conjugates containing reductively aminated maltose, cellobiose, and lactose are effective antigens in rabbits. Antisera to the cellobiose-BSA conjugate were examined in detail for the specificity of antibodies formed against the hapten by determining the effects of various synthetic inhibitors on the agglutination reaction between the conjugate and homologous serum. These studies demonstrate that antibodies to the hapten recognize the terminal β-glucopyranosyl residue, the acyclic reduced glucose residue, and the secondary ammonium linkage. Antibodies specific for the hapten were predominant in this serum as analogs containing these features inhibited 80–90% of the precipitin reaction.

Production of the phytotoxins radicinin and radicinol by Alternaria chrysanthemi

Abstract A biologically active compound isolated from liquid cultures of Altemaria chrysanthemi in large yield has been identified as radicinin by X-ray cry

Analysis by the reductive-cleavage method of linkage positions in a polysaccharide containing 4-linked d-glucopyranosyluronic residues☆

The fate of 4-linked D-glucopyranosyluronic residues under reductive-cleavage conditions was investigated by using the Klebsiella aerogenes type 54 strain A3 capsular polysaccharide. Treatment of the fully methylated polysaccharide with triethylsilane and trimethylsilyl trifluoromethanesulfonate in dichloromethane, followed by in situ acetylation, yielded 1,5-anhydro-2,3,4,6-tetra-O-methyl-D-glucitol, 3,4-di-O-acetyl-1,5-anhydro-2,6-di-O-methyl-D-glucitol, and 3-O-acetyl-1,5-anhydro-2,4-di-O-methyl-L-fucitol, as expected, but the expected product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue, namely, methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-L-gulonate, was not observed. Instead, methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-L-gulonate (6) was identified as the sole product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue. That compound 6 arose as a result of rearrangement during reductive cleavage rather than by reductive cleavage of a 5-linked D-glucofuranosyluronic residue, was established by reductive cleavage of the fully methylated polysaccharide following reduction of its ester groups with either lithium aluminum hydride or lithium aluminum deuteride. The products of the latter reductive cleavage were the same as before, except for the absence of 6 and the presence of 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-D-glucitol, or its 6,6-dideuterio isomer. Although the reductive-cleavage technique is suitable for the direct analysis of polysaccharides containing 4-linked D-glucopyranosyluronic residues, it does not establish whether the uronic residue is a 4-linked pyranoside or a 5-linked furanoside. The expected product is, however, derived from the 4-linked D-glucopyranosyluronic residue after sequential methylation, reduction of its ester group and reductive cleavage.

Analysis of positions of substitution of O-carboxymethyl groups in partially O-carboxymethylated cellulose by the reductive-cleavage method☆

A method is described for the analysis of positions of substitution of O-carboxylated groups in commercial samples of O-carboxymethylcellulose. Sequential permethylation of the polymer and reductive cleavage gives eight products, which are analyzed as their O-acetyl derivatives by gas-liquid chromatography.