Hydrolytic and transglycosylation reactions of N-acyl modified substrates catalysed by β-N-acetylhexosaminidases

Fialová, Pavla; Weignerová, Lenka; Rauvolfová, Jana; Přikrylová, Věra; Pišvejcová, Andrea; Ettrich, Rüdiger; Kuzma, Marek; Sedmera, Petr; Křen, Vladimı́r

doi:10.1016/j.tet.2003.10.111

Cited by 48 publications

(34 citation statements)

References 38 publications

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“…The strains were cultivated in submerse media as described previously. [33] Enzymes were obtained by (NH 4 ) 2 SO 4 precipitation (80 % sat.) of the cultivation media, and the precipitates were directly used for their respective reactions.…”

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confidence: 99%

Synthesis of Sulfated Glucosaminides for Profiling Substrate Specificities of Sulfatases and Fungal β‐N‐Acetylhexosaminidases

et al. 2009

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Systematic sulfation: Sulfated glycoconjugates are degraded either by desulfation followed by glycoside cleavage, or by glycoside cleavage followed by desulfation. To study these processes, here we report the synthesis of four regioisomerically sulfated p-nitrophenyl glucosaminides from the common precursor p-nitrophenyl N-acetyl-beta-D-glucosaminide. These substrates allowed the rapid analysis of the substrate preferences of a set of four sulfatases and 24 hexosaminidases.Sulfated carbohydrates are components of many glycoconjugates, and are degraded by two major processes: cleavage of the sulfate ester by a sulfatase, or en bloc removal of a sulfated monosaccharide by a glycoside hydrolase. However, these processes have proved difficult to study owing to a lack of homogeneous, defined substrates. We describe here the synthesis of a series of p-nitrophenyl beta-D-glucosaminides bearing sulfate esters at the 2-, 3-, 4- or 6-positions, by divergent routes starting with p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside. The sulfated p-nitrophenyl beta-D-glucosaminides were used to study the substrate specificity of four sulfatases (from Helix pomatia, Patella vulgata, abalone, and Pseudomonas aeruginosa), and revealed significant differences in the preference of each of these enzymes for desulfation at different positions around the sugar ring. The 3-, 4- and 6-sulfated p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucosaminides were screened against a panel of 24 fungal beta-N-acetylhexosaminidases to assess their substrate specificity. While the 4- and 6-sulfates were substrates for many of the fungal enzymes investigated, only a single beta-N-acetylhexosaminidase, that from Penicillium chrysogenum, could hydrolyze the 3-sulfated p-nitrophenyl glycoside. Together these results demonstrate the utility of sulfated p-nitrophenyl beta-D-glucosaminides for the study of both sulfatases and glycoside hydrolases.

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confidence: 99%

Synthesis of Sulfated Glucosaminides for Profiling Substrate Specificities of Sulfatases and Fungal β‐N‐Acetylhexosaminidases

et al. 2009

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“…[26] In the second step the actual docking was performed using the version of Autodock 4.0 [27] implemented in Yasara applying local Search Algorithm, grid space 0.375 nm, to determine the exact docked position of the standard substrate pNP-GlcNAc (1) as well as of individual C-6-modified substrates 2-5 in the active site. For molecular docking, the pyranosyl rings of the substrates were in a slightly distorted 1,4 B/ 1 S 3 conformation to 2416 asc.wiley-vch.de resemble the reactive conformations in the Michaelis structures reported by Vocadlo and co-workers. [28] The molecular dynamics simulations of the substrateenzyme complexes were run to further improve the docked positions, including the solvation effect, and to assess the stability of the docked position in the active site.…”

Section: Molecular Modelingmentioning

confidence: 70%

“…The strains were cultivated in submersed media as described previously. [4] Enzymes were obtained by (NH 4 ) 2 SO 4 precipitation (80% saturation) of the cultivation media, and the precipitates were directly used in the enzymatic screening. The crude enzyme preparations were tested for contaminating sulfatase and phosphatase activities using the respective pnitrophenyl substrate (p-nitrophenyl sulfate, p-nitrophenyl phosphate, Sigma) under the same conditions as the b-Nacetylhexosaminidase assay.…”

Section: Enzymesmentioning

confidence: 99%

Charged Hexosaminides as New Substrates for β‐N‐Acetylhexosaminidase‐Catalyzed Synthesis of Immunomodulatory Disaccharides

et al. 2011

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This work is a structure-activity relationship study that investigates the influence of the nature and amount of negative charge in carbohydrate substrates on the affinity of b-N-acetylhexos-A C H T U N G T R E N N U N G aminidases, and on the stimulation of natural killer cells. It describes synthetic procedures yielding novel glycosides that are useful in immunoactivation. Specifically, we present a thorough study on the ability of six C-6 modified b-N-acetylhexosaminides (aldehyde, uronate, 6-O-sulfate, 6-O-phosphate) to serve as substrates for cleavage and glycosylation by a library of b-N-acetylhexosaminidases from various sources. Four novel disaccharides with one or two (negatively) charged groups were prepared in synthetic reactions in good yields. Surprisingly, the 6-Ophosphorylated substrate, although cleaved by a number of enzymes from the series, worked neither as a donor nor as an acceptor in transglycosylation reactions. The results of wet experiments were supported by molecular modeling of substrates in the active site of two representative enzymes from the screening. All ten prepared compounds were examined in terms of their immunoactivity, namely as ligands of two activation receptors of natural killer (NK) cells, NKR-P1 and CD69, both with isolated proteins and whole cells. Sulfated disaccharides in particular acted as very efficient protectants of NK cells against activation-induced apoptosis, and as stimulants of the natural killing of resistant tumor cells, which makes them good candidates for potential clinical use in cancer treatment.

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“…On the contrary, azide 1 was cleaved by none of the a-galactosidases tested (< 1 % referred to 5). In summary, the respective azides were cleaved by most tested b-galactosidases, b-glucosidases and a-mannosidases (related to common rates for modified substrates), [19] contrary to a-galactosidases.…”

Section: Introductionmentioning

confidence: 94%

Glycosyl Azides – An Alternative Way to Disaccharides

et al. 2007

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This paper is dedicated to the memory of Prof. Zoltµn Gyçrgydeàk, who greatly contributed to the chemistry of glycosyl azides.Abstract: Glycosyl azides are shown to be efficient donors for b-galactosidases, b-glucosidases and amannosidases. Only a-galactosidases do not cleave the respective glycosyl azide 1 and, moreover, they exhibit competitive inhibition (especially a-galactosidase from Talaromyces flavus). High water solubility and ready synthesis of glycosyl azides enable transglycosylation reactions even with difficult acceptors like N-acetyl-d-mannosamine in good yields. The versatility of glycosyl azides was demonstrated in the synthesis of five disaccharides -two of them are described for the first time. All the reactions were highly regioselective, yielding bA C H T U N G T R E N N U N G (1!6) isomers. bGalactosidase from E. coli proved to have the best synthetic capabilities. The present study shows that glycosyl azides are a valuable alternative to common p-nitrophenyl glycoside donors and in many synthetic reactions.

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Hydrolytic and transglycosylation reactions of N-acyl modified substrates catalysed by β-N-acetylhexosaminidases

Cited by 48 publications

References 38 publications

Synthesis of Sulfated Glucosaminides for Profiling Substrate Specificities of Sulfatases and Fungal β‐N‐Acetylhexosaminidases

Synthesis of Sulfated Glucosaminides for Profiling Substrate Specificities of Sulfatases and Fungal β‐N‐Acetylhexosaminidases

Charged Hexosaminides as New Substrates for β‐N‐Acetylhexosaminidase‐Catalyzed Synthesis of Immunomodulatory Disaccharides

Glycosyl Azides – An Alternative Way to Disaccharides

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