David H. G. Crout

Glycosidase-catalysed oligosaccharide synthesis of di-, tri- and tetra-saccharides using the N-acetylhexosaminidase from Aspergillus oryzae and the β-galactosidase from Bacillus circulans

The N-acetylhexosaminidase from Aspergillus oryzae catalyse transfer of N-acetylglucosaminyl and N-acetylgalactosaminyl residues selectively on to the 6-OH group of N-acetylgalactosamine to give the corresponding (1→6)-linked disaccharides 3 and 6 in 26 and 38% yield, respectively. The disaccharide β-D-GlcpNAc-(1→6)-β-D-GalpNAc 3 thus synthesized acts as acceptor for transfer of a β-D-galactosyl residue from the corresponding p-nitrophenyl glycoside on to the 4-OH group of the non-reducing unit to give the ovarian cyst fluid mucin fragment β-D-Galp-(1→4)-β-GlcpNAc-(1→6)-D-GalpNAc 8 in 48% yield together with the tetrasaccharide β-D-Galp-(1→4)-β-D-Gal-(1→4)-β-D-GlcpNAc-(1→6)-D-GalpNAc 9 in 7% yield. With lactose as acceptor, the trisaccharide β-D-Galp-(1→4)-β-D-Galp-(1→4)-D-Glcp 12, a growth factor for human intestinal bifidobacteria, is produced in 52% yield.p

Glycosidase-catalysed synthesis of oligosaccharides: a two-step synthesis of the core trisaccharide of N-linked glycoproteins using the β-N-acetylhexosaminidase and the β-mannosidase from Aspergillus oryzae

Using a partially purified β-mannosidase from Aspergillus oryzae, a β-mannosyl unit is trasferred from p-nitrophenyl β-D-mannopyranoside 5 to di-N-acetylchitobiose 4 to give the core trisaccharide β-D-Manp-(1→4)-β-D-GlcpNAc-(1→4)-D-GlcpNAc 6 of the N-linked glycoproteins.

Glycosidase-catalysed synthesis of oligosaccharides: trisaccharides with the α-D-gal-(1 → 3)-D-gal terminus responsible for the hyperacute rejection response in cross-species transplant rejection from pigs to man

α-Galactosidases from Coffea arabica, Aspergillus niger and A. oryzae catalyse the transfer of α-galactosyl residues on to lactose thioglycosides to give 1 → 3 and 1 → 6 linked trisaccharides, including an unusual branched trisaccharide 11 and the isoglobotriose glycoside 4, which, as ‘linear B type 6’, has been recognised as of the type responsible for the hyperacute rejection response in pig-to-man xenotransplantation.

Acetoin metabolism: stereochemistry of the acetoin produced by the pyruvate decarboxylase of wheat germ and by the α-acetolactate decarboxylase of Klebsiella aerogenes

Acetoin (3-hydroxybutan-2-one)(1) produced from pyruvate and acetaldehyde by the pyruvate decarboxylase of wheat germ consists of a mixture of enantiomers with the (S)-(+)-isomer preponderating. With pyruvate alone as substrate, racemic acetoin is produced. Acetoin produced from pyruvate by the sequential action of the acetohydroxy acid synthetase and α-acetolactate decarboxylase of Klebsiella aerogenes consists solely of the (R)-(–)-isomer.

Pyrrolizidine alkaloid analogues. Preparation of semisynthetic esters of retronecine

Methods have been devised for the selective esterification of retronecine (I) to give analogues of the naturally occurring hepatotoxic pyrrolizidine alkaloids. Selective esterification at C-9 of retronecine (I) with simple acids was achieved by using NN′-dicyclohexylcarbodi-imide. Selective esterification and esterification with αβ-unsatur-ated and α-hydroxy-αα-dialkyl acids was achieved through the intermediacy of N-acylimidazoles.

Esterase-catalysed regioselective 6-deacylation of hexopyranose per-acetates, acid-catalysed rearrangement to the 4-deprotected products and conversions of these into hexose 4- and 6-sulfates

The esterase from Rhodosporidium toruloides has been used to catalyse the hydrolysis of a series of per-acetylated α-D-hexopyranoses and α-D-hexopyranosides. Per-acetylated glucose 4, mannose 6, N-acetylgalactosamine 8, galactose 10, methyl α-D-glucoside 12, methyl α-D-mannoside 14 and methyl α-D-galactoside 16 have been selectively cleaved at the C-6 position by the esterase to give the 6-OH derivatives 5, 7, 9, 11, 13, 15 and 17. Acid-catalysed rearrangement of acetates 5, 7, 13, 15, 11, 17 and 9 with 4→6 acetyl migration gives the corresponding 4-deprotected derivatives 22–28, respectively. Hydrolyses of β-D-glucose pentaacetate 20 and α-D-lactose octaacetate 21 have been attempted, but no hydrolyses have been observed. 1,2,3,6-Tetraacylated α-D-hexopyranoses 3 and 22, derivatives of N-acetylglucosamine and glucose respectively, and 2,3,6-triacetylated α-D-hexopyranosides 24 and 25, derivatives of glucose and mannose, respectively, have been hydrolysed by the esterase to the corresponding 4,6-dihydroxy acetates 29, 18, 30 and 31. Acylation of methyl α-D-glucopyranoside 32 catalysed by the esterase provides the C-6 monoacetate 33 and the C-3 monoacetate 34 in 4 and 5% yield, respectively. The sodium salts of N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose and mannose 6-sulfates 38–42, respectively, are prepared in two steps from the 6-deacetylated hexopyranoses 2, 5, 9, 11 and 7, respectively. The sodium salts of N-acetylglucosamine, glucose and mannose 4-sulfates 43–45, respectively, are prepared in two steps from the 4-deacetylated precursors 3, 22 and 26 which are obtained via acid catalysed 4→6 acyl migration of compounds 2, 5 and 7.

Conversion of 2-acetamido-2-deoxy-β-D-glucopyranose (N-acetylglucosamine) into 2-acetamido-2-deoxy-β-D-galactopyranose (N-acetylgalactosamine) using a biotransformation to generate a selectively deprotected substrate for SN2 inversion

A procedure is described for the conversion of N-acetylglucosamine 2 into N-acetyigalactosamine 1, in which use is made of a 4,6-acetyl migration in a precursor selectively deprotected by a biotransformation procedure.

Stereospecific formation of R-aromatic acyloins by Zymomonas mobilis pyruvate decarboxylase

Recombinant pyruvate decarboxylase from Zymomonas mobilis catalysed the formation of R-aromatic acyloins of high optical purity from aromatic aldehydes and either pyruvate or acetaldehyde. The results are contrasted with those obtained with aliphatic acyloins and compared with those obtained with the pyruvate decarboxylase of Saccharomyces sp.

Kinetic control of regioselectivity in glycosidase-catalysed disaccharide synthesis: preparation of 2-acetamido-4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-D-glucopyranose (N,n′-diacetylchitobiose) and 2-acetamido-6-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-D-glucopyranose

During transfer of the N-acetyl-β-D-glucosaminyl (2-acetamido-2-deoxy-β-D-glucopyranosyl) residue from p-nitrophenyl N-acetyl-β-D-glucosaminide (p-nitrophenyl N-2-deoxy-β-D-glucopyranoside) on to N-acetyl-D-glucosamine (2-acetamido-2-deoxy-D-glucopyranose) catalysed by the N-acetylhexosaminidase from Aspergillus oryzae, the major isomer formed was found to depend on the time course of the reaction, 1 → 4 transfer predominating while the p-nitrophenyl glycoside was available as donor, but 1 → 6 transfer when the initially-formed 1 → 4 product took over as donor, results that could be interpreted in terms of a constant regioselectivity modulated by selective hydrolysis of the products.

Biotransformations using clostridia: stereospecific reductions of a β-keto ester

The reduction of methyle 4-(4-chlorophenylthio)-3-oxobutanoate 1 by clostridia has been studied. Clostridium pasteurianum ATCC 6013, C. tyrobutyricum DSM 1460 and C. kluyveri NCIB 10680 gave the D-(3S) reduction product 2, whereas C. kluyveri DSM 555 gave the L-(3R) reduction product 3. The products could be obtained in higher optical purities than by yeast reductions.