Biomedical subjects
Publications and source records attributed to P Mischnick.
A new mathematical model is presented for the analysis of the substituent distribution in the polymer chain of polysaccharide derivatives. For the first time, the influence of substitution on the reactivity of neighbored monomer units is taken into account. The model was applied to various cellulose and amylose ethers and esters and excellently fits the experimental data.
The distribution of substituents in hydroxypropylated potato amylopectin starch (amylose deficient) modified in a slurry of granular starch (HPPAPg) or in a polymer 'solution' of dissolved starch (HPPAPs), was investigated. The molar substitution (MS) was determined by three different methods: proton nuclear magnetic resonance (1H NMR) spectroscopy, gas-liquid chromatography (GLC) with mass spectrometry, and a colourimetric method. The MS values obtained by 1H NMR spectroscopy were higher than those obtained by GLC-mass spectrometry analysis and colourimetry. The relative ratio of 2-, 3-, and 6-substitution, as well as un-, mono-, and disubstitution in the anhydroglucose unit (AGU) were determined by GLC-mass spectrometry analysis. Results obtained showed no significant difference in molar distribution of hydroxypropyl groups in the AGU between the two derivatives. For analysis of the distribution pattern along the polymer chain, the starch derivatives were hydrolysed by enzymes with different selectivities. Debranching of the polymers indicated that more substituents were located in close vicinity to branching points in HPPAPg than in HPPAPs. Simultaneous alpha-amylase and amyloglucosidase hydrolysis of HPPAPg liberated more unsubstituted glucose units than the hydrolysis of HPPAPs, indicating a more heterogeneous distribution of substituents in HPPAPg.
A method has been developed to determine the distribution of substituents in the glucose units of sulfonylbutylethers of cyclomaltoheptaose (beta-cyclodextrin). This method involves hydrolysis of the glucosidic linkages, permethylation, formation of sulfonylchlorides and subsequent transformation to the permethylated sulfonylfluoride derivatives. The latter were thermostable and could be analyzed by GLC and identified by EI and CIMS. For confirmation, the 2-, 3-, and 6-O-substituted standard compounds were independently synthesized and characterized by NMR and GLC-MS.
The distribution of substituents in sulfonylbutylethers of cyclomaltoheptaose (beta-cyclodextrin) formed in aqueous medium has been determined by gas chromatography after hydrolysis and formation of the permethylated sulfonylfluoride derivatives. In contrast to other etherification reactions of beta-cyclodextrin, preferred substitution in position 3 of the glucose units has been detected. From 1H NMR and microcalorimetric experiments, the formation of host-guest complexes by beta-cyclodextrin and the reagent 1,4-butane sultone in water became evident. This spatial preorganization presumably favors the reaction with the O-3. In contrast, in methyl sulfoxide preferred 2-O-alkylation was obtained, indicating that host-guest interaction does not influence regioselectivity in this solvent.
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Adult snails synthesize in their albumen glands a polysaccharide which is composed exclusively of D- or D- and L-galactose (Gal) residues which are interglycosidically linked by 1 --> 3 and 1 --> 6 bonds. It is the only carbohydrate source for embryos and freshly hatched snails. Two galactosyltransferases are described in this study which are most likely involved in the biosynthesis of this polysaccharide. One identified in Helix pomatia acts on oligosaccharides and could be used to synthesize a tetrasaccharide when the branched trisaccharide D-Gal-beta-(1 --> 3)-[D-Galbeta-(1 --> 6)]-D-Galbeta-1 --> OMe was offered as acceptor. This enzyme, requiring Mg++-and Mn++-ions for activity, introduced a linear beta-(1 --> 6) linkage at the terminal non-reducing ends and was not detected in Biomphalaria glabrata. The other enzyme, which introduced beta-(1 --> 6) linkages at subterminal D-Gal residues, thus forming branching points in the polysaccharide, was found in H. pomatia, Arianta arbustorum and B. glabrata with comparable activities. With the enzyme preparation of H. pomatia, up to four D-Gal residues were introduced into vicinal positions, forming single-membered side chains, if a hexasaccharide with five linearly beta-(1 --> 3)-linked D-Gal residues was offered as a acceptor. The multiple-branched structure formed is typical for snail galactans, making this enzyme a prime candidate for the branching enzyme in galactan synthesis. The enzyme activity could be solubilized and purified by affinity chromatography. In SDS-polyacrylamide electrophoresis, the Helix-derived eluate displayed two bands (68, 37 kDa) and that of Biomphalaria five bands (68, 63, 17.5; 15; 13 kDa). The purified material showed only 8% of the total activity of the crude extracts, but it could be shown that a phosphatase present in the crude extract can degrade UDP formed in the transfer reaction and thus drive the reaction to completion.
When 2,6-di-O-tert-butyldimethylsilylated cyclomalto-oligosaccharides (cyclodextrins) are treated with allyl or methyl iodide and NaH in dry tetrahydrofuran, O-2-->O-3 migration of the secondary 2-O-tert-butyldimethylsilyl groups occurs, leading to 2-O-alk(en)yl-3,6-di-O-tert-butyldimethylsilyl-cyclodextrin derivatives. The detection and identification of the reaction step during which migration occurred is described and possible mechanisms of migration are discussed.
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.
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.
In this study, the structure of the immunodominant carbohydrate epitope of the extracellular polysaccharides from mold species belonging to the order Mucorales reactive with rabbit IgG antibodies was elucidated. An exo-alpha-D-mannanase which was able to abolish the antigenicity of these polysaccharides completely was purified and characterized, and the activity was compared with that of an alpha-D-mannosidase. Analysis of the monomeric reaction products after enzymatic treatment revealed the presence of 2-O-methyl-D-mannose residues. This compound is a constituent of the polysaccharides from the mold genera Mucor, Rhizopus, Rhizomucor, Absidia, Syncephalastrum, and Thamnidium, and its occurrence in fungi has not been reported until now. Two mannan fractions which are highly reactive with rabbit IgG were isolated from the extracellular polysaccharides of Mucor racemosus and characterized with ethylation analysis. The role of the newly found 2-O-methyl-D-mannose residues in the immunoreactivity was assessed by specific degradation of these mannans with the exo-alpha-D-mannanase and subsequent ethylation analysis. It was concluded that the immunodominant carbohydrates reactive with rabbit IgG are chains composed of a single terminal non-reducing 2-O-methyl-D-mannose residue, alpha (1-2)-linked to a short sequence of alpha(1-2)-linked D-mannose residues.
Agarose was structurally characterised by permethylation and subsequent reductive cleavage. Treatment of the fully methylated polysaccharide with triethylsilane and a mixture of trimethylsilyl methanesulfonate and boron trifluoride etherate followed by a newly developed, acid-catalysed, in situ acetylation yielded two main products: the expected 4-O-acetyl-1,5:3,6-dianhydro-2-O-methyl-L-galactitol and 3-O-acetyl-1,5-anhydro-2,4,6-tri-O-methyl-D-galactitol in the molar ratio 1:1, and traces of 1,5-anhydro-2,3,4,6-tetra-O-methyl-D-galactitol. Reductive cleavage using triethylsilane and trimethylsilyl trifluoromethanesulfonate as the catalyst yielded the same anhydroalditols as well as a smaller amount of 1,4,5-tri-O-acetyl-3,6-anhydro-2-O-methyl-L-galactitol due to ring-opening of 3,6-anhydrogalactopyranosyl residues during reductive cleavage. In this paper, results from reductive cleavage are compared with results using standard methylation analysis.
To study the structures of the epitopes of the extracellular polysaccharides from Penicillium and Aspergillus species, an exo-beta-D-galactofuranosidase was purified from a commercial crude enzyme preparation from Trichoderma harzianum. Analysis of ring size and linkage position of the galactose residues of the extracellular polysaccharide of Penicillium digitatum, before and after enzymatic treatment, was determined by the reductive-cleavage technique. In addition to terminal and beta (1-5)-linked galactofuranosides, beta (1-6)-linked and beta (1,5,6)-linked branched galactofuranose residues could be identified. After degradation with the purified exo-beta-D-galactofuranosidase, all initial linkages of the galactofuranose residues were still present, but the amount of beta (1-5)-linked galactofuranose residues had decreased considerably. Treatment of the extracellular polysaccharides of Penicillium and Aspergillus species with the purified exo-beta-D-galactofuranosidase resulted in complete disappearance of the enzyme-linked immunosorbent assay reactivity of these polysaccharides, using immunoglobulin G antibodies raised against P. digitatum. Therefore, with the use of this enzyme, it was proved that the beta (1-5)-linked galactofuranosyl residues only are responsible for the antigenicity of the extracellular polysaccharides of Penicillium and Aspergillus molds. A new structural model for the antigenic galactofuranose side chains of the galactomannan from P. digitatum is proposed.
A new family of bouquet-like molecules based on cyclodextrins is described. These compounds were obtained by polymerization of ethylene oxide. This reaction was initiated by primary and secondary hydroxyl groups of cyclodextrins, which constitute an organizing core. Analysis of structure and composition of conjugates based on alpha-CD and beta-CD was performed using the data of MALDI-MS, GC-MS, and 13C-NMR spectra. Glass transition behavior of the conjugates shows that the above compounds are amorphous. Complexation properties of the conjugates are described with respect to sodium 4-nitrophenolate and calcium acetylhomotaurinate, which are used as guest molecules. Binding interaction between cyclodextrins or their conjugates and sodium 4-nitrophenolate was studied using differential absorption spectra. Association constants Ka between CD hosts and calcium acetylhomotaurinate composed of two equal anionic moieties were studied using 1H-NMR spectroscopy. The values of binding constant for beta-CD were found to increase by more than 2 orders of magnitude than that of the corresponding system based on alpha-CD. alpha-CD was shown to form the inclusion complex with one anionic moiety, whereas beta-CD produces a ternary complex with two anionic moieties of CAHT. For PEO derivatives of CDs, Ka decreases as compared with that of parent CD for both guests. These conjugates may be used as potent drug-delivery systems.