Comparative studies of differential expression of chitinolytic enzymes encoded by chiA, chiB, chiC and nagA genes in Aspergillus nidulans.

N-Acetyl-D-glucosamine, chito-oligomers and carbon starvation regulated chiA, chiB, and nagA gene expressions in Aspergillus nidulans cultures. The gene expression patterns of the main extracellular endochitinase ChiB and the N-acetyl-beta-D-glucosaminidase NagA were similar, and the ChiB-NagA enzyme system may play a morphological and/or nutritional role during autolysis. Alterations in the levels of reactive oxygen species or in the glutathione-glutathione disulfide redox balance, characteristic physiological changes developing in ageing and autolyzing fungal cultures, did not affect the regulation of either the growth-related chiA or the autolysis-coupled chiB genes although both of them were down-regulated under diamide stress. The transcription of the chiC gene with unknown physiological function was repressed by increased intracellular superoxide concentration.

Dominant antibody responses to Fucalpha1-3GalNAc and Fucalpha1-2Fucalpha1-3GlcNAc containing carbohydrate epitopes in Pan troglodytes vaccinated and infected with Schistosoma mansoni.

The development of the humoral anti-glycan immune response of chimpanzees, either or not vaccinated with radiation-attenuated Schistosoma mansoni cercariae, was followed during 1 year after infection with S. mansoni. During the acute phase of infection both the vaccinated and the control chimpanzees produce high levels of immunoglobulin G (IgG) antibodies against carbohydrate structures that are characteristic for schistosomes carrying the Fucalpha1-3GalNAc and Fucalpha1-2Fucalpha1-3GlcNAc motifs, but not to the more widespread occurring structures GalNAcbeta1-4GlcNAc, GalNAcbeta1-4(Fucalpha1-3)GlcNAc, and Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis(x)). In addition, high levels of IgM antibodies were found against the trimeric Lewis(x) epitope. Apparently, the schistosome-characteristic carbohydrate structures are dominant epitopes in the anti-glycan humoral immune response of the chimpanzees. All chimpanzees showed an increase in the level of antibodies against most of the carbohydrate structures tested directly after vaccination, peaking at challenge time and during the acute phase of infection. With the exception of anti-F-LDN antibody responses, the anti-carbohydrate antibody responses upon schistosome infection of the vaccinated animals were muted in comparison to the control animals.

Anomalous Zemplén deacylation reactions of alpha- and beta-D-mannopyranoside derivatives.

Reaction of mono-, di-, and trisaccharide derivatives of methyl beta-D- and octyl beta-D-mannopyranosides bearing ester groups at isolated and non-isolated positions on the same molecule, under Zemplén conditions (catalytic amount of sodium methoxide in methanol) gave partially deacylated compounds, in which the O-acyl groups were retained at isolated sites. In the case of one disaccharide, all the benzoyl groups remained intact at the reducing end, while all the acetyl functions were removable from the nonreducing end. In another case, both isolated ester groups at positions 2 and 4 were retained at the reducing end. The isolated 2-O-acyl groups on methyl alpha-D-mannopyranoside compounds were more labile than on the corresponding beta-mannosides under the same conditions. The mechanism of the reaction may be different for ester groups at isolated or non-isolated positions. In the latter case, acyl migration may take place and carry acyl groups into a less hindered position.

Chemical and chemo-enzymatic synthesis of the alpha-Neu p5Ac-(2-->6)- beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp element that is part of N-linked carbohydrate chains of human lutropin.

In the framework of a project aimed at the elucidation of the nature of the functional importance of the N-glycosylation of the alpha-subunit of the glycoprotein hormones human lutropin and human chorionic gonadotropin, the structural element alpha-Neu p5Ac-(2-->6)-beta-D-GalpNac-(1-->4)- beta-D-GlcpNAc-(1-->2)-alpha-D-Manp, which is part of the carbohydrate chains of human lutropin, has been prepared by chemical and chemo-enzymatic synthesis in the form of its propyl glycoside. Condensation of 4-O- acetyl-3,6-di-O-benzyl-2-deoxy-2-phthalimido-alpha/beta-D-glucopyranosyl trichloroacetimidate with allyl 3,4,6-tri-O-benzyl-alpha-D-mannopyranoside gave after deacetylation allyl (3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl) -(1-->2)-3,4,6-tri-O-benzyl-alpha-D-mannopyranoside. Ethyl 3-O-benzyl-2-deoxy-2-phthalimido-l-thio-beta-D-glucopyranoside was converted into the galacto-derivative ethyl 4,6-di-O-acetyl-3-O-benzyl-2-deoxy-2-phthalimido-1-thio-beta-D -galactopyranoside via an oxidation-reduction route, as well as via SN2-type substitution with acetate. The use of this galacto thioglycoside, after its conversion into the corresponding bromide, as GaIN donor for condensation with the mentioned disaccharide derivative yielded after deacetylation allyl (3-O-benzyl-2-deoxy-2-phthalimido-beta-D-galactopyranosyl)-(1-->4) -(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1-->2) -3,4,6-tri-O-benzyl-alpha-D-mannopyranoside. Methylsulfenyl bromide-silver triflate promoted sialylation of this trisaccharide derivative with O-ethyl S-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D -glycero-alpha-D-galacto-non-2-ulopyranosyl)onate] dithiocarbonate and subsequent deprotection resulted into the aimed tetrasaccharide structural element. Alternatively, this compound was prepared via a block synthesis, which, however, was not superior to the linear strategy. Finally, a stereose lective sialylation of synthetically prepared beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) CH2CH2CH3 with CMP-Neu5Ac and rat liver alpha-2,6-sialyltransferase was accomplished affording the same tetrasaccharide structural element.

Syntheses of spacer-armed carbohydrate components of the Mycobacterium avium serocomplex serovar 8.

p-Nitrophenyl glycosides of 3-O-Me-beta-D-Glcp-(1-->3)-alpha-L-Rhap, alpha-L-Rhap-(1-->2)-6-deoxy-alpha-L-Talp, and 3-O-Me-beta-D-Glcp-(1-->3)-alpha-L-Rhap-(1-->2)-6-deoxy-alpha-L-++ +Talp have been prepared, related to Mycobacterium avium. Various glycosylation methods have been used for the formation of the interglycosidic linkages. The p-nitrophenyl derivatives were converted into p-isothiocyanatophenyl glycosides, capable of forming neoglycoproteins.

Synthesis of a fucosylated and a non-fucosylated core structure of xylose-containing carbohydrate chains from N-glycoproteins.

The synthesis is reported of methyl 2-acetamido-4-O-[2-acetamido-2-deoxy-O-(3,6-di-O-alpha-D- mannopyranosyl-2-O-beta-D-xylopyranosyl-beta-D-mannopyranosyl)-beta-D- glucopyranosyl]-2-deoxy-beta-D-glucopyranoside (4) and methyl 2-acetamido-4-O-[2-acetamido-2-deoxy-4-O- (3,6-di-O-alpha-D- mannopyranosyl-2-O-beta-D-xylopyranosyl-beta-D-mannopyranosyl)-beta-D- glucopyranosyl]-2-deoxy-6- O-alpha-L-fucopyranosyl-beta-D-glucopyranoside (5), which represent the invariant hexasaccharide core structure of the xylose-containing glycans of N-glycoproteins and its 6-O- fucosylated derivative. Ethyl 4-O-[3-O-allyl-4-O-benzoyl-6-O-tert-butyldimethylsilyl-2-O- (2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-1- thio-beta-D-glucopyranoside (9) was coupled with methyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranoside (11). Desilylation of the resulting tetrasaccharide derivative, followed by condensation with 2,3,4,6- tetra-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (7), gave methyl 4-O-(4-O-[3-O-allyl-4- O-benzoyl-6-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-2-O-(2,3,4 -tri-O- acetyl-beta-D-xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranosyl)- 3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside (14). Deallylation of 14, followed by condensation with 7 and deprotection, gave hexasaccharide 4. Ethyl 3,6-di-O- benzyl-2-deoxy-4-O- [4,6-di-O-acetyl-3-O-allyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl) - beta-D-mannopyranosyl]-2- phthalimido-1-thio-beta-D-glucopyranoside (17) was coupled with methyl 3-O- benzyl-2-deoxy-6-O- (4-methoxybenzyl)-2-phthalimido-beta-D-glucopyranoside. Demethoxybenzylation of the tetrasaccharide derivative thus obtained, followed by fucosylation using ethyl 2,3,4-tri-O- benzyl-1-thio- beta-L-fucopyranoside, gave methyl 3-O-benzyl-2-deoxy-4-O-[3,6-di-O-benzyl-2- deoxy-4-O-[4,6- di-O-acetyl-3-O-allyl-2-O-(2,3,4-tri-O-acetyl-beta-D-xylopyranosyl)-beta -D- mannopyranosyl]-2-phthalimido- beta-D-glucopyranosyl)-2-phthalimido-6-O-(2,3,4-tri-O-benzyl-alpha-L- fucopyranosyl)-beta-D-glucopyranoside (23). O-Deacetylation followed by tert-butyldimethylsilylation, benzoylation, and desilylation gave methyl 4-O-(4-O-[3-O-allyl-4-O-benzoyl-2-O-(2,3,4-tri-O-benzoyl-beta-D- xylopyranosyl)- beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta- D-glucopyranosyl)-3- O-benzyl-2-deoxy-2-phthalimido-6-O-(2,3,4-tri-O-benzyl-alpha-L-fucopy ran osyl)- beta-D-glucopyranoside (24). Mannosylation of 24 using 7, followed by deallylation, further mannosylation with 7, and deprotection, gave the heptasaccharide 5.

Chemical synthesis of the pyruvic acetal-containing trisaccharide unit of the species-specific glycopeptidolipid from Mycobacterium avium serovariant 8.

The functionalized, pyruvic acetal-containing haptenic trisaccharide, p-trifluoroacetamidophenyl 6-deoxy-2-O-(3-O-[4,6-O-(S)-(1-methoxycarbonylethylidene)-3-O-meth yl- beta-D-glucopyranosyl]-alpha-L-rhamnopyranosyl)-alpha-L-talopyranosid e (19), a component of the glycolipid from Mycobacterium avium serovar 8 was synthesized. For the preparation of the terminal pyruvic acetal-containing unit, benzyl 2-O-benzyl-3-O-methyl-beta-D-glucopyranoside (6) was condensed with methyl 2,2-di(ethylthio)propionate (1) in the presence of SO2Cl2-CF3SO3H catalyst to yield benzyl 2-O-benzyl-4,6-O-(S)-(1-methoxycarbonylethylidene)-3-O-methyl-beta -D- glucopyranoside (7S), which was then converted into the suitably substituted glycosyl donor 2-O-acetyl-4,6-O-(S)-(1-methoxycarbonylethylidene)-3-O-methyl-alph a-D- glucopyranosyl trichloroacetimidate (11). The disaccharide glycosyl acceptor p-nitrophenyl endo-3,4-O-benzylidene-6-deoxy-2-O-(2,4-di-O-benzyl-alpha-L-rhamnopyrano syl)- alpha-L-talopyranoside (15) was glycosylated with 11 in the presence of trimethyl trifluoromethanesulfonate to furnish the protected trisaccharide p-nitrophenyl 2-O-(3-O-[2-O-acetyl-4,6-O-(S)-(1-methoxycarbonylethylidene)-3-O-m ethyl-beta- D-glucopyranosyl]-2,4-di-O-benzyl-alpha-L-rhamnopyranosyl)-endo-3,4- O-benzylidene-6-deoxy-alpha-L-talopyranoside (16). After deprotection, this gave the spacer-armed unprotected haptenic trisaccharide 19.

Synthesis of p-trifluoroacetamidophenyl 6-deoxy-2-O-(3-O-[2-O-methyl-3-O- (2-O-methyl-alpha-D-rhamnopyranosyl)-alpha-L-fucopyranosyl]-alpha-L- rhamnopyranosyl)-alpha-L-talopyranoside: a spacer armed tetrasaccharide glycopeptidolipid antigen of Mycobacterium avium serovar 20.

The synthesis of the title tetrasaccharide glycoside 38 is reported. p-Nitrophenyl endo-3,4-O-benzylidene-6-deoxy-alpha-L-talopyranoside (4), 3-O-acetyl-2,4-di-O-benzyl-alpha-L-rhamnopyranosyl trichloroacetimidate (7), methyl 3-O-acetyl-4-O-benzyl-2-O-methyl-1-thio-beta-L-fucopyranoside (15), 3-O-acetyl-4-O-benzyl-2-O-methyl-alpha-L-fucopyranosyl bromide (16), and ethyl 3-O-acetyl-4-O-benzyl-2-O-methyl-1-thio-alpha-D-rhamnopyranoside (33) were prepared as intermediates. Compound 4 was glycosylated with imidate 7 as well as with methyl 3-O-acetyl-2,4-di-O-benzyl-1-thio-alpha-L-rhamnopyranoside (9), affording the same disaccharide derivative 8. Deacetylation of 8 gave crystalline 17. Condensation of 17 with both fucosyl donors 15 and 16 yielded the same trisaccharide derivative 18 stereoselectively. Compound 18 was also prepared by the coupling of 4 with disaccharide glycosyl donor 20. After deacetylation of 18 (-->34), methyl triflate-promoted glycosylation with compound 33 resulted in tetrasaccharide 35. Conversion of the p-nitrophenyl group of 35 into the p-trifluoroacetamidophenyl group (-->36) and removal of the protecting groups gave the title tetrasaccharide glycoside 38.

Synthesis of a selectively protected trisaccharide building block that is part of xylose-containing carbohydrate chains from N-glycoproteins.

The synthesis is reported of ethyl 4-O-[3-O-allyl-4,6-O-isopropylidene-2-O-(2,3,4-tri-O-acetyl-beta-D- xylopyranosyl)-beta-D-mannopyranosyl]-3,6-di-O-benzyl-2-deoxy-2-phthalim ido-1 - thio-beta-D-glucopyranoside (16), a key intermediate in the synthesis of xylose-containing carbohydrate chains from N-glycoproteins. Condensation of ethyl 3,6-di-O-benzyl-2-deoxy-2-phthalimido-1-thio-beta-D- glucopyranoside (5) with 2,4,6-tri-O-acetyl-3-O-allyl-alpha-D-glucopyranosyl bromide, using silver triflate as a promoter, gave the beta-linked disaccharide derivative 8 (84%). O-Deacetylation of 8 and then isopropylidenation afforded 10, which was converted via oxidation-reduction into ethyl 4-O-(3-O-allyl-4,6-O-isopropylidene-beta-D-mannopyranosyl)-3,6-di-O-benz yl-2- deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (12). Silver triflate-promoted condensation of 12 with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl bromide gave 16 (71%). The Xylp unit in 16 and in de-isopropylidenated 16 (17) existed in the 1C4(D) conformation, but that in O-deacetylated 17 (18) existed in the 4C1(D) conformation.

ROESY studies and HSEA calculations on xylose-containing oligosaccharides related to N-glycoproteins.

Conformational studies on beta-D-Xyl-(1----2)-[alpha-D-Man-(1----6)]-beta-D-OMe, beta-D-Xyl-(1----2)-[alpha-D-Man-(1----3)]-beta-D-Man-OMe, and beta-D-Xyl-(1----2)-[alpha-D-Man-(1----3)]-[alpha-D-Man-(1----6)]-beta-D -Man-Ome have been carried out using rotating-frame n.O.e. experiments in combination with HSEA calculations. The estimated time-averaged torsional angles phi and psi about the various glycosidic linkages and the estimated time-averaged rotamer population around the C-5-C-6-linkage of beta-Man in the tetrasaccharide are similar to the corresponding parameters for both trisaccharides.

1H- and 13C-n.m.r. assignments for structural elements of xylose-containing N-linked oligosaccharides, using 1D-and 2D-n.m.r. experiments.

1H-N.m.r. and 13C-n.m.r. spectral assignments for synthetic beta-D-Xyl-(1----2)-beta-D-Man-OMe, beta-D-Xyl-(1----2)-[alpha-D-Man-(1----3)]-beta-D-Man-OMe, beta-D-Xyl-(1----2)-[alpha-D-Man-(1----6)]-beta-D-Man-OMe, and beta-D-Xyl-(1----2)-[alpha-D-Man-(1----3)]-[alpha-D-Man-(1----6)]- beta-D-Man-OMe, which are structural elements of xylose-containing carbohydrate chains from N-glycoproteins, have been made on the basis of 1D and 2D (DQF 1H-1H COSY, HOHAHA) 500-MHz 1H-n.m.r. spectroscopy and 1D 50-MHz 13C-n.m.r. spectroscopy, respectively.

Synthesis of four structural elements of xylose-containing carbohydrate chains from N-glycoproteins.

The synthesis of the oligosaccharides beta-D-Xylp-(1----2)-beta-D-Manp-OMe (12), beta-D-Xylp-(1----2)-[alpha-D-Manp-(1----6)]-beta-D-Manp+ ++-OMe (17), beta-D-Xylp-(1----2)-[alpha-D-Manp-(1----3)]-beta-D-Manp+ ++-OMe (21), and beta-D-Xylp-(1----2)-[alpha-D-Manp-(1----3)] [alpha-D-Manp-(1----6)]-beta-D-Manp-OMe (25) is described. Methyl 3-O-benzyl-4,6-O-isopropylidene-beta-D-mannopyranoside (6) was prepared from the corresponding glucoepimer (4) by oxidation, followed by stereoselective reduction. Condensation of 6 with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl bromide in the presence of mercuric cyanide gave a 1:9 mixture of methyl 3-O-benzyl-4,6-O-isopropylidene-2-O-(2,3,4- tri-O-acetyl-alpha- (7a) and -beta-D-xylopyranosyl)-beta-D-mannopyranoside (7), and then 7 was converted into the acetylated disaccharide-glycoside 11. Regioselective mannosylation, with 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl bromide, at position 6 of deisopropylidenated 7 (8), using mercuric bromide as a promoter, afforded the trisaccharide-glycoside derivative 13, which was transformed into the acetylated trisaccharide-glycoside 16. The disaccharide derivative 10, obtained from 8, and the trisaccharide derivative 15, obtained from 13, were glycosylated at position 3 with O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)trichloroacetimidate (19), using trimethylsilyl triflate as a promoter, giving rise to acetylated tri- (20) and tetra-saccharide (24) derivatives, respectively. O-Deacetylation of 11, 16, 20, and 24 gave 12, 17, 21, and 25, respectively.

Synthesis of the tetrasaccharide core region of antigenic lipo-oligosaccharides characteristic of Mycobacterium kansasii.

The oligosaccharide core region, beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-alpha-D-Glcp- 1----1)-alpha-D-Glcp (1), of the lipo-oligosaccharide-type antigens isolated from M. kansasii has been synthesised from 2,3,2',3',4',6'-hexa-O-benzyl-6-O-(1-phenylethyl)-alpha, alpha-trehalose (4). Compound 4 was obtained by LiAlH4-AlCl3-type hydrogenolysis of 2,3,2',3',4',6'-hexa-O-benzyl-4,6-O-(S)-(1-phenylethylidene)-alpha , alpha-trehalose. The beta-laminaribiosyl part of the molecule was built-up by sequential glycosylation steps using 2,4,6-tri-O-acetyl-3-O-allyl-alpha-D-glucopyranosyl bromide in the presence of HgBr2 and methyl 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranoside promoted by methyl triflate. The complete a priori 13C-n.m.r. spectrum assignment of 1 was achieved by applying 2D methods.

Glycosylated trehalose. Synthesis of the oligosaccharides of the glycolipid-type antigens from Mycobacterium smegmatis.

The oligosaccharide components, 3-O-Me-beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-beta-D-Glcp-(1----6)- alpha-D-Glcp-(1----1)-alpha-D-Glcp (1) and beta-D-Glcp-(1----4)-beta-D-Glcp-(1----6)-alpha-D-Glcp-(1----1)-alpha-D- Glcp, of the glycolipid-type antigens isolated from M. smegmatis have been synthesised from 2,3,4,2',3',4',6'-hepta-O-acetyl-alpha,alpha-trehalose and the appropriate glycosyl bromides under Helferich conditions with Hg(CN)2 as the promoter. Condensation of the trisaccharide glycosyl bromide 27 gave an orthoester derivative (28) which could be rearranged, using HgBr2 or boron trifluoride etherate, into the acetylated derivative (29) of 1. The model compound beta-D-Glcp-(1----6)-alpha-D-Glcp-(1----1)-alpha-D-Glcp has also been synthesised.

Synthesis of the tetrasaccharide repeating-unit of the lipopolysaccharide isolated from Pseudomonas maltophilia.

The title tetrasaccharide having the structure 3-O-Me-beta-L-Xylp-(1----4)- alpha-L-Rhap-(1----4)-alpha-L-Rhap-(1----2)-L-Rhap was obtained by reaction of the alpha-acetobromo derivative of 4-O-(3-O-methyl-beta-L-xylopyranosyl)-L-rhamnopyranose and benzyl 3,4-di-O-benzyl-2-O-(2,3-O-isopropylidene-alpha-L-rhamnopyranosyl)- alpha-L-rhamnopyranoside, followed by removal of the protecting groups. The synthesised compounds were characterised on the basis of n.m.r. data.

Intracellular hydrolysis of EGTA-esters.

It has been established that the hydrolysis of EGTA-acetoxymethylester (AME) by red blood cells is about the tenth of the hydrolysis of acetylthiocholine (Ac-S-Ch). This splitting of AME could be inhibited by about 50% by prostigmine at a concentration of 0.75 X 10(-5) mol/l, while the splitting of Ac-S-Ch was totally inhibited by the same prostigmine concentration. The hydrolysis of AME by the so-called white-ghost preparation was considerably inhibited by prostigmine (KI = 5 X 10(-8) mol/l), and this inhibition proved to be a competitive one. The splitting of AME by membrane-free cytosol fraction could not be inhibited by prostigmine. Human red blood cells do not hydrolyse EGTA-ethylester (EE). This compound decomposes spontaneously at room-temperature, its reaction-product formed in the Hestrin-reaction is unstable, the developed colour gradually turns pale. On the other hand, AME does not hydrolyse spontaneously at room-temperature and the colour-intensity of its Hestrin-reaction does not decrease with time. Using chelator-and dye-indicator esters to reach different concentrations of free chelators and dye-indicators intracellulary (IC), the extracellular hydrolysis of esters has to be taken into account or this external breakdown has to be inhibited.