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Biomedical subjects

J Thiem

Publications and source records attributed to J Thiem.

At least 37 records · Page 2Linked to original sources
PubMed1997

The specificity of an alpha-(1-->2)-L-galactosyltransferase from albumen glands of the snail Helix pomatia.

The specificity of an L-galactosyltransferase (L-Gal-T) from albumen glands of the snail Helix pomatia has been studied. This enzyme transfers L-Gal from GDP-L-Gal to various disaccharides with beta-linked D-Gal in terminal non-reducing position, forming an alpha-(1-->2) linkage. The subterminal residue and the type of interglycosidie linkage proved to be of minor importance. However, the branched trisaccharide beta-D-Gal-(1-->3)-[beta-D-Gal-(1-->6)]-beta-D-Gal-(1-->O)Me is a very poor acceptor. The specificity of the L-Gal-T correlates well with the equimolar occurrence of L-Gal and the structural element-->2)-Gal- (1-->found in the storage polysaccharide of this snail. Since L-Fuc is also transferred from its GDP-activated form, the membrane preparations of the albumen glands can be used to synthesize fucosylated oligosaccharides.

Animals↗
PubMed1997

beta-Glucosylation of chitooligomers by galactosyltransferase.

Galactosyltransferase from bovine milk was found to be able to utilise UDP-Glc to transfer Glc onto GlcNAc and chitooligomers[-beta-GlcNAc-(1-->4)-]n, n = 2-4. beta-Glucosylated products were used in binding studies with NKR-P1A protein cloned from rat natural killer cells.

Acetylglucosamine↗
PubMed1997

Fucosyltransferase-catalyzed formation of L-galactosylated Lewis structures.

The Lewis (alpha 1-3/4) fucosyltransferase isolated from human milk could be used for preparative fucosylations of the disaccharide acceptors Gal(beta 1-3)GlcNAc(beta 1-O)R (at position OH-4) and Gal(beta 1-4)GlcNAc(beta 1-O)R (at position OH-3) [R = (CH2)8COOMe]. As donors GDP-L-Gal and deoxygenated derivatives were used to lead to a series of novel modified trisaccharides of the Lewis(a) and the Lewis(x) type, respectively.

Animals↗
PubMed1997

Further syntheses employing phosphorylase.

Maltopentaose was immobilized on silica gel and used as a recyclable primer in the reaction of glycogen phosphorylase with D-glucal. An improved method to obtain 2-deoxy-alpha-D-arabino-hexopyranosyl phosphate (4) by using this catalyst as well as the specific synthesis of low molecular weight, water-soluble 2-deoxy-maltooligosaccharides (12, 13, 14 and 15) are described. Further investigations with modified phosphorylase substrates showed that mannosyl phosphate (16) can be slowly transferred to the primer maltotetraose. alpha-1,4-Mannosyl-maltotetraose (17) and its degradation product alpha-1,4-mannosyl-maltose (18) were identified.

Carbohydrate Sequence↗
PubMed1997

Galactosylation with beta-galactosidase from bovine testes employing modified acceptor substrates.

In this study beta1-3 linked analogues of the T-antigen determinant were synthesized in preparative scale by transgalactosylation using beta-galactosidase from bovine testes to give synthetic antigens. Acceptors with modifications of the sugar residue such as alpha-glycosylated spacers, as well as GlcNAc-alphaOR- and 2dGal-alphaOR-substrates opened further possibilities for galactosylation.

Animals↗
PubMed1997

Inhibition of L-fucokinase from rat liver by L-fucose analogues in vitro.

By investigating the effects of more than 15 different L-fucose analogues on the activity of L-fucokinase (EC 2.7.1.52) from rat liver in vitro, certain structural requirements for potent inhibition of this enzyme were established. Of the novel compounds, 4,6-dideoxy-L-xylo-hexopyranose (4) and methyl 4,6-dideoxy-4-iodo-L-glucopyranose (9) were found to be competitive inhibitors with Ki-values of 0.5 mM and 5.0 mM respectively. Thus 4,6-dideoxy-L-xylo-hexopyranose is a better inhibitor of L-fucokinase than methyl-alpha-L-fucoside (1). Uptake of L-fucose into rat hepatoma cells is reduced by 52% in the presence of the deoxy derivative (4), leading to a decrease of 45% in the incorporation of L-fucose into total cellular glycoproteins.

Animals↗
PubMed1996

The myrosinase-glucosinolate interaction mechanism studied using some synthetic competitive inhibitors.

Using synthetic deoxy-glucotropaeolins (6d-GTL, 4d- GTL, 3d-GTL, 2d-GTL) as substrates, myrosinase activity was studied in comparison to that determined on native glucotropaeolin (GTL) isolated from ripe Lepidium sativum seeds. When the deoxy substrates were used, in addition to an overall strong reaction rate decline, a significant decrease in the reaction rate was observed in going from 6d- to 2d-GTL. This finding allows us to propose a mechanism of catalysis which appears to be similar in many respects to that established for beta-glucosidases. Finally, 2d-GTL was shown to be the first strong competitive inhibitor of myrosinase ever reported.

Binding Sites↗
PubMed1995

Conversion of aldonic acids to their corresponding 2-keto-3-deoxy-analogs by the non-carbohydrate enzyme dihydroxy acid dehydratase (DHAD).

Aldonic acids containing four to six carbon atoms were tested as potential substrates of dihydroxy acid dehydratase (DHAD), an enzyme from the biosynthetic pathway of branched chain amino acids. Novel assay systems for observing the course of DHAD catalysed reactions were developed in order to adapt the enzyme to extended practical applications. Kinetic studies for the new substrates (12/13) as well as inhibitor kinetics for the substrate analogue 3-deoxy-aldonic acids (25, 27 and 29) were performed. These gave indications for the restrictions of substrate modifications and contributed to the understanding of the individual effects. Finally L-threonic acid (12) and D-erythronic acid (13) could be successfully applied as substrates for DHAD and this led to the chemoenzymatic synthesis of their 2-keto-3-deoxy-analogue (20) in a preparative scale.

Carbohydrate Sequence↗
PubMed1995

Applications of enzymes in synthetic carbohydrate chemistry.

By photosynthesis 200 billion tons of glucose are formed per annum most of which remains in the sugar state. Thus, the well-known properties of saccharides as the basis for structural materials and energy stores is well established. Increasingly, focus is being addressed to their functions as recognition molecule. Therefore, carbohydrate chemistry became en vogue again as documented by classical syntheses of complex hetero-oligosaccharides employing protecting and activating group chemistry. In recent years enzymatic methods for acylation and deacylation in saccharide chemistry came into use. Novel saccharides could be approached following aldolase-catalyzed de novo syntheses. The very complex task of stereospecific glycosylation was addressed by use of enzymes. With glycohydrolases via transglycosylation as well as with glycosyltransferases including cofactor regenerating circles advances were made. Modified acceptor and also donor substrates allowed access to oligosaccharides in preparative quantities. This paper will discuss these modern developments and will focus on some selected examples from others' and our own laboratory.

Carbohydrate Sequence↗
PubMed1994

Analysis of oligosaccharides containing 2-deoxy-alpha-D-arabino-hexosyl residues by the reductive-cleavage method.

A mixture of oligosaccharides containing (1-->4)-linked 2-deoxy-alpha-D-arabino-hexosyl ("2-deoxyglucosyl") and (1-->4)-linked alpha-D-glucosyl residues (1) was analyzed by reduction, permethylation (perethylation), degradation to monomers, and GLC-MS. Degradation was performed either by hydrolysis with subsequent reduction, by methanolysis, or by reductive cleavage, always followed by acetylation. Reductive cleavage turned out to be the method of choice for the acid-labile 2-deoxy sugars. The main degradation product formed during acid hydrolysis of 2-deoxy-D-arabino-hexosyl residues yielded, after reduction and acetylation, (4R,S)-6-O-acetyl-2,3,5-trideoxyhexono-1,4-lactone (7). By methanolysis, in addition to the expected methyl glycosides, methyl 2,3,5-trideoxy-6-O-methyl-4-hexulosonate (12) is formed as a by-product. For determination of the distribution of chain lengths, the permethylated oligomers were separated by reversed-phase HPLC. For peak assignment, one isolated oligomer was investigated by FABMS and 1H NMR spectroscopy. The average degree of polymerization (dp) calculated from the HPLC chromatogram is in good agreement with the reductive-cleavage results.

Carbohydrate Conformation↗
PubMed1994

Synthesis of 2-deoxy-alpha-D-arabino-hexopyranosyl phosphate and 2-deoxy-maltooligosaccharides with phosphorylase.

A convenient one-step synthesis of 2-deoxy-alpha-D-arabino-hexopyranosyl phosphate on a millimolar scale is described by reaction of potato phosphorylase with D-glucal at equimolar phosphate concentration. Furthermore, in the presence of catalytic amounts of phosphate, a 2-deoxy-maltooligosaccharide is obtained from maltotetraose and D-glucal. The water-insoluble oligosaccharide was isolated and characterized by 1H and 13C NMR spectroscopy. An average dp of 20 was thus determined.

Carbohydrate Sequence↗
PubMed1994

One-pot-synthesis of alpha-linked deoxy sugar trisaccharides.

Various alpha-linked 2,6-dideoxy-ribo-trisaccharides, models for part of the antibiotic kijanimicin, were synthesised by the N-iodosuccinimide method employing different pathways. The efficiency of a sequential synthesis suffered from side reactions of the axial HO-3, which are typical of digitoxosides. These problems did not arise in a straightforward polymerisation, performed as a one-pot-procedure. It afforded the trisaccharide directly from the monosaccharide precursor in 30% yield. A combination of the oligomerisation pathway and the sequential synthesis led to trisaccharides with different protecting group patterns. In these reactions different glycal and alcohol components were used and allowed to define the optimal partners in a sequential synthesis: the two components should ideally be of comparable reactivity.

Carbohydrate Conformation↗
PubMed1994

Synthesis and structural studies of asparagine-modified 2-deoxy-alpha-N-glycopeptides associated with the renin-angiotensin system.

Following addition of N-iodosuccinimide to glycals, reductive hydrogenolysis and ring opening gave 2-deoxy-alpha-N-glycopeptides carrying a deaminated asparagine unit. This reaction could be performed employing glucal, galactal, L-rhamnal, L-fucal and lactal to give the corresponding glycoconjugate building blocks 11, 12, 17, 22, 27 and 32. Further NIS-mediated glycosylation of the rhamno derivative 21 led to simple trisaccharide peptide adducts 45. Peptide synthesis of the gluco building unit with different preassembled oligopeptides afforded glycoconjugates 36, 39, 41 and 42 assumed to be of interest as potential inhibitors of the renin-angiotensin system.

Amino Acid Sequence↗
PubMed1993

Synthetic approaches to 2-deoxyglycosyl phosphates.

By the use of the N-iodosuccinimide (NIS)-procedure, various glycals could be converted into 2-deoxy-2-iodoglycosyl phosphates. Treatment of glycals 1 and 7 with NIS and dibenzyl phosphate gave the corresponding alpha-1,2-trans-diaxial 2-deoxy-2-iodoglycosyl phosphates 2 and 8 as the main products. The beta-1,2-trans-diequatorial compounds 3 and 9 were isolated as by-products. Analogous reaction of glycals 4 and 10 gave the corresponding 2-deoxy-2-iodoglycosyl phosphates 5, 6, 11, and 12 as crude products, which were characterized by 1H NMR spectroscopy. Classical phosphorylation of 2-deoxy-glycosyl chlorides 14 and 16 with silver dibenzyl phosphate gave the corresponding dibenzyl 2-deoxy-alpha-glycosyl phosphates 15 and 17. Alternatively, glycosylation of tri-O-acetyl-D-glucal using dibenzyl phosphate and triphenylphosphine hydrobromide afforded 15 in lower yield. The application of S-(2-deoxyglycosyl) phosphorodithioates as glycosyl donors provided the most convenient way to dibenzyl 2-deoxyglycosyl phosphates. The alpha-glycosyl phosphates 15, 20, and 22 could be synthesized by reaction of the 2-deoxyglycosyl dithiophosphates 18, 19, and 21 with dibenzyl phosphate and activation by iodonium di-sym-collidine perchlorate. Similarly, the 2,6-dideoxyglycosyl dithiophosphates 23 and 25 gave the 2,6-dideoxy phosphates 24 and 26; however, the isolation of these labile compounds could not be effected.

Carbohydrate Sequence↗
PubMed1993

Chemoenzymatic galactosialylation with integrated cofactor regeneration.

As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac alpha (2-8)Gal beta (1-4)GlcNAc beta (1-O)-pent-4-ene was synthesized starting from GlcNAc beta (1-O)-pent-4-ene, UDP-glucose and N-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and alpha (2-6)sialyltransferase in a complete cofactor regeneration system.

Carbohydrate Sequence↗
PubMed1993

Partial purification and characterization of cytidine-5'-monophosphosialate synthase from rainbow trout liver.

Trout liver is a rich source of sialate cytidylyltransferase activity. Three procedures are described by which the enzyme was enriched between 67- and 647-fold with high specific activities varying between 0.67 and 1.88 U/mg protein. In the simplest procedure studied, 100,000 x g supernatant of liver homogenate was chromatographed on Q-Sepharose and beta-[3-(2-aminoethylthio)propyl]-N- acetylneuraminic acid as affinity matrix, leading to an enzyme preparation (0.67 U/mg protein) well suited for the synthesis of CMP-N-acetylneuraminic acid. The synthase has a molecular mass of 160 kDa, a temperature optimum of 28 degrees C, a pH-optimum of 9.3 and exhibits Km-values for CTP, N-acetylneuraminic acid and N-glycoloylneuraminic acid of 1.7 mM, 2.1 mM and 2.9 mM, respectively. It is inactive with N-acetyl-9-O-acetylneuraminic acid. The enzyme is inhibited by CMP, CDP and 2'-deoxy-CTP. The sialic acid fraction of trout liver after hydrolysis is composed by N-acetylneuraminic acid (86%), N-acetyl-9-O-acetylneuraminic acid (12%) and N-acetyl-9-O-lactoylneuraminic acid (2%).

Animals↗