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

Abstract

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.

Characterization of Glycosaminoglycan (GAG) Sulfatases from the Human Gut Symbiont Bacteroides thetaiotaomicron Reveals the First GAG-specific Bacterial Endosulfatase

“…After stirring at 0°C for 0.5 h and at room temperature for 1 h, a 0.15 M aqueous solution of sodium hydrogen carbonate (2.6 ml) was added at 0°C and stirred for 16 h. The reaction mixture was then concentrated under reduced pressure and purified by reverse phase column chromatography (H 2 O/MeOH) to finally give pure GlcNAc6S-O-pNP (21 mg, 34%) after lyophilization. 1 H and 13 C NMR data were in agreement with previously reported data (25).…”

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

“…After 11 days of cultivation, the mycelium was filtered off and the enzyme was purified from the culture medium by cation exchange chromatography [Fractogel EMD SO 3 − (Merck, Germany), 10 mM sodium citrate/phosphate buffer pH 3.5, linear gradient 0–1 M NaCl] and gel permeation chromatography [Superdex 200 (Amersham Biosciences, SW), 50 mM sodium citrate/phosphate buffer pH 5 and 150 mM NaCl] using an Äkta Purifier protein chromatography system (Amersham Biosciences, SW). The β‐ N ‐acetylhexosaminidase activity was determined in an end‐point assay as described previously,5 with a 2 mM starting concentration of p NP‐GlcNAc standard substrate 1 or substrates 2 – 6 and 8 and with 11–15 mU mL −1 or 220–300 mU mL −1 of β‐ N ‐acetylhexosaminidase, respectively.…”

“…β‐ N ‐Acetylhexosaminidases1 (EC 3.2.1.52, CAZy GH20; http://www.cazy.org) belong to the group of glycosidases with a well‐documented broad synthetic potential;2 at the same time, they are also extensively studied from the viewpoint of their structure and their involvement in human physiology and disease 3. Previously, we have demonstrated that fungal β‐ N ‐acetylhexosaminidases possess a wide tolerance to substrates bearing various modifications, including N ‐acyls,4 sulfates,5 and an absent C‐4 hydroxy 6. Moreover, GlcNAc substrates with two unusual CN linked aglycones, namely the azido group7 and the aromatic triazole moiety,8 were cleaved by some fungal β‐ N ‐acetylhexosaminidases; the azide even worked as an efficient glycosyl donor in transglycosylation reactions 7…”

Glycosidases in Carbohydrate Synthesis: When Organic Chemistry Falls Short

“…[41] Glycosylations with 6-O-sulfo-N-acetyl-d-glucosaminyl moiety was demonstrated in several recent works. [45] Loft and Williams [46] published challenging syntheses of regioselectively sulfated p-nitrophenyl 2-acetomido-2deoxy-b-d-glucopyranosides (6-O-sulfate, cosynthase [65] mutants with altered substrate specificity have been obtained. An example is the xylosynthase derived from Agrobacterium sp.…”