Hydrolytic and transglycosylation reactions of N-acyl modified substrates catalysed by β-N-acetylhexosaminidases
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Introduction
The recent dynamic development of glycosciences has brought about the increasing need for new glycostructures. Novel carbohydrate compounds can effectively be synthesized using enzymatic methods.1 Glycosidases (EC 3.2), although they often exhibit a poor regioselectivity and sometimes low yields, are readily available from different sources. They tolerate environmental stress and thanks to their broad substrate specificity they are able to accept a wide range of donors with different aglycon moieties and acceptors.2., 3., 4., 5., 6.
It has been revealed only recently that besides their natural substrates (glycons), glycosidases are capable of accepting substrates bearing various structural modifications. These modified substrates, often exhibiting important biological properties,7 find applications in many areas, such as structure-activity relationship studies8., 9., 10. or treatment of glycosidase-induced pathogenic states.9
The first papers dealing with the enzymatic recognition of substrates modified at the glycon moiety were published in the early 70s, mostly investigating the substrate specificity of the β-N-acetylhexosaminidase from Aspergillus oryzae isolated from a digestive amylase preparation Takadiastase®. Substrates bearing 3-O-methyl, 6-O-methyl, 3,4-di-O-methyl and 3,4,6-tri-O-methyl groups,11 as well as a range of N-acyl modified substrates12., 13. were studied.
The affinity of various β-N-acetylhexosaminidases from mushrooms and marine vertebrates to substrates modified at C-2 amino group was thoroughly studied by Molodtsov and Vafina.14., 15., 16. They isolated and characterised the ‘β-N-glycylhexosaminidase’ from the marine helminth Chaetopterus variopedatus17 and the ‘β-N-benzoylhexosaminidase’ from the scallop Mizuhopecten yessoensis.18 Besides, they detected a high tolerance to N-acyl modifications with the β-N-acetylhexosaminidase from the fungus Hohenbuehelia serotina.19 A similar study was carried out by Leaback and Walker with the commercial pig epididymal β-N-acetylhexosaminidase.20
Probably the first use of modified substrates in a transglycosylation reaction was described by Wong and Takayama with p-nitrophenyl 6-oxo-β-d-galactopyranoside.21 C-6 modifications were also studied by MacManus et al.22 with a set of 10 p-nitrophenyl galactopyranosides bearing different functionalities at C-6, such as methyl, alkene, alkyne or fluorine. A β-N-acetylhexosaminidase was first used by Hušáková et al. in a reaction with a 6-O-acetylated glycosyl donor.23 To our best knowledge, no case of transglycosylation with N-acyl modified substrates catalysed by glycosidases has yet been published.
In the present paper, we report the affinity of a library of 35 fungal β-N-acetylhexosaminidases to various N-acyl modified substrates from the viewpoint of their hydrolytic and transglycosylation potentials. Four novel p-nitrophenyl disaccharides were prepared in transglycosylation reactions with these new substrates. The experimental results are discussed regarding molecular models of modified substrates docked in the active centre of the β-N-acetylhexosaminidase from A. oryzae Culture Collection of Fungi 1066.
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Results and discussion
Compounds 3, 5 and 6 were prepared from the corresponding 2-acylamido-2-deoxy-d-glucopyranoses24., 25., 26. using modified method27 (Scheme 1). The selective formylation with p-nitrophenyl formiate28 as well as those using HCOOH with Ac2O29., 30. did not lead to the desired product, and so the formylation with a mixed acetic formic anhydride was performed.24 Substrate 2 was synthesized using modified method31 (Scheme 2). In the synthesis of substrate 4 (Scheme 2), 1,3,4,6-tetra-O
Conclusion
The acetamido group is a crucial structural feature of substrates for β-N-acetylhexosaminidases, which are quite sensitive to its modification. This study clearly demonstrates for the first time that besides cleavage, fungal β-N-acetylhexosaminidases are able to catalyse synthetic transglycosylation reactions with N-acyl modified substrates. They tolerate certain sterical changes at C-2 (shorter or longer acyls, a hydroxyl instead of a hydrogen). Nevertheless, they do not accept highly
Materials
Citrate/phosphate buffer (Mc Ilvaine) pH 5.0 was prepared by mixing 0.1 M citric acid (24.3 mL) and 0.2 M Na2HPO4 (25.7 mL), by diluting with water to 100 mL and adjusting pH to 5.0. The fungal strains producing β-N-acetylhexosaminidases (EC 3.2.1.52) originated from the CCF, Department of Botany, Charles University, Prague, or from the Culture Collection of the Institute of Microbiology (CCIM), Prague. The strains were cultivated as described previously.37., 38. Flasks (500 mL) with medium
Acknowledgements
This work was supported by Czech Science Foundation GA CR No. 204/02/P096. The preparation of enzymes used in this work was done under the support of GA CR grant No. 203/01/1018. Molecular modelling was financed by Ministry of Education of the Czech Republic (MSMT 123100001). We thank Dr K. Bezouška (Fac. Sci., Charles University, Prague) for providing computing facilities for molecular modelling and Dr P. Halada (Inst. Microbiol., Prague) for the MS measurements.
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