Synthesis and utility of sulfated chromogenic carbohydrate model substrates for measuring activities of mucin-desulfating enzymes

Clinch, Keith; Evans, Gary B.; Furneaux, Richard H.; Rendle, Phillip M.; Rhodes, P.; Roberton, Anthony M.; Rosendale, Douglas; Tyler, Peter C.; Wright, Damian P.

doi:10.1016/s0008-6215(02)00104-0

Cited by 21 publications

(17 citation statements)

References 48 publications

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“…Acetylation of 11 under standard conditions gave 12, which was subsequently treated with hydrogen fluoride-pyridine complex to give the 6-hydroxy derivative 13 in good yield (75 %). Initially, we attempted sulfation of 13 using SO 3 ·Me 3 N in pyridine; [14] in our hands SO 3 ·pyr in pyridine/DMF gave better results. Purification was achieved by evaporation of the solvent and sequential size-exclusion and cation-exchange chromatographies of the residue to provide 14 as the sodium salt.…”

Section: Synthesis Of Sulfated Substratesmentioning

confidence: 88%

“…[14] Here, we adopted the latter approach. Thus, 5 was deacetylated by using NaOMe in MeOH to afford 10 in quantitative yield (Scheme 3).…”

Section: Synthesis Of Sulfated Substratesmentioning

confidence: 99%

“…A similar assay was developed by Clinch et al for the detection of bacterial carbohydrate sulfatases using chromogenic p-nitrophenol glycosides but using an exogenous glycoside hydrolase. [14] The sulfatases from snail (Helix pomatia), limpet (Patella vulgata) and abalone (unspecified species) are commercially available, are widely used in analytical applications, and have been utilized for the preparation of monosulfated carbohydrates through the desulfation of readily accessible di-or trisulfated precursors. [23][24][25] For these reasons they were chosen for study.…”

Section: Synthesis Of Sulfated Substratesmentioning

confidence: 99%

“…A similar approach using sulfated chromogenic glycosides has been reported. [13,14] These approaches have allowed the monitoring of sulfatase activity in several systems, but the limited number of suitable substrates prevents its more widespread use.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Synthesis Of Sulfated Substratesmentioning

confidence: 88%

“…[14] Here, we adopted the latter approach. Thus, 5 was deacetylated by using NaOMe in MeOH to afford 10 in quantitative yield (Scheme 3).…”

Section: Synthesis Of Sulfated Substratesmentioning

confidence: 99%

Section: Synthesis Of Sulfated Substratesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2009

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“…The enzyme assay involves the desulfation of SO 3 -6-GlcNAc-1-PNP by the glycosulfatase, followed by removal of 4-nitrophenol (PNP) from the 4-nitrophenyl 2-acetamido-2-deoxy-␤-D-glucopyranoside (GlcNAc-1-PNP) by the hexosaminidase auxiliary enzyme, leaving 2-acetamido-2-deoxy-␤-D-glucopyranoside (GlcNAc), and finally adjustment of the pH to 9.6 to measure PNP (2).…”

Section: Methodsmentioning

confidence: 99%

A Novel Bacterial Mucinase, Glycosulfatase, Is Associated with Bacterial Vaginosis

et al. 2005

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The modifications to the vaginal habitat accompanying a change to vaginal flora in bacterial vaginosis (BV) are poorly understood. In this study enzymes involved in mucin degradation were measured, including a novel glycosulfatase assay. Women attending an emergency walk-in sexually transmitted disease clinic were studied. One high vaginal swab (HVS) was used to prepare a gram-stained smear to determine BV status, using Ison and Hay's criteria, and a separate swab was used for the purposes of the assays. The median glycosulfatase activity was 8.5 (range, ؊1.2 to 31.9) nmol h ؊1 1.5 ml ؊1 of HVS suspension in patients with BV compared to 0.5 (range, ؊0.7 to 9.4) nmol h ؊1 1.5 ml ؊1 of HVS suspension in patients without BV (P ‫؍‬ <0.001). The median glycoprotein sialidase activity was 29.2 (range, ؊17 to 190) nmol h ؊1 1.5 ml ؊1 of HVS suspension in patients with BV compared to ؊1.1 (range, ؊41 to 48) nmol h ؊1 1.5 ml ؊1 of HVS suspension in patients without BV (P < 0.001). A rapid spot test for sialidase was positive in 22/24 patients with BV (sensitivity, 91.7%; 95% confidence interval [CI], 73 to 99%) and negative in 32/35 patients without BV (specificity, 91.4%; 95% CI, 76.9 to 98.2%) (P < 0.001). Glycosulfatase activity significantly correlated with both glycoprotein sialidase activity and the sialidase spot test (P ‫؍‬ 0.006 and P < 0.001, respectively). The results are consistent with the hypothesis that the consortium of bacteria present in BV requires the ability to break down mucins in order to colonize the vagina and replace the normal lactobacilli.Bacterial vaginosis (BV) is a condition in which commensal lactobacilli in the vagina are replaced by a profound overgrowth of a mixed microflora of anaerobes and Gardnerella vaginalis. It is strongly associated with preterm birth (6,7,14), endometritis, and pelvic inflammatory disease (12, 26), acquisition of Neisseria gonorrhoeae, Chlamydia trachomatis, and human immunodeficiency virus (3,21,30), and probably enhanced human immunodeficiency virus transmission (4,15,21). The causative link between BV colonization and associated pathology is not firmly established.Mucolytic enzymes (mucinases), including those which degrade terminal carbohydrate residues or those which disrupt the mucin apoprotein primary structure, are produced by some bacteria associated with BV. Previous work has shown that the presence of glycosidases, including sialidase, is frequently associated with BV (1,8,14,17,33). Certain organisms related to those found in BV have been shown to produce sialidase (reviewed in reference 32). The detection of a variety of enzymes with mucin-degrading potential from BV organisms (1,8,17,22,32,33) indicates that a complementary relationship may exist between the different types of bacteria and that synergistic-enzymatic activity is required to effect maximum control over the vaginal environment. Intensive production of hydrolytic enzymes in BV might lead to diminished mucosal barrier protection through disruption of secreted mucus viscoelasticity and d...

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Sulfatasen: Struktur, Mechanismus, biologische Aktivität, Inhibition, Anwendung in Synthesen

2004

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Sulfatasen – d. h. Sulfatester spaltende Enzyme – spielen eine entscheidende Rolle bei der Regulierung von Sulfatierungszuständen, die über die Funktion vieler physiologischer Moleküle entscheiden. Die Substrate der Sulfatasen reichen von kleinen cytosolischen Steroiden wie dem Östrogensulfat bis hin zu komplexen Kohlenhydraten auf Zelloberflächen, etwa den Glycosaminoglycanen. Die Umwandlung dieser Moleküle wurde mit wichtigen zellulären Funktionen in Verbindung gebracht, z. B. mit der Hormonregulation, dem zellulären Abbau und der Modulation von Signalisierungsprozessen. Sulfatasen hängen mit dem Ausbruch etlicher pathophysiologischer Erscheinungen zusammen, etwa mit hormonabhängigen Krebsformen. Bis heute sind zwar zahlreiche Sulfatasen identifiziert worden, die Aufklärung des weitreichenden Spektrums ihrer biologischen Aktivitäten befindet sich aber erst am Anfang. Dieser Aufsatz gibt einen Überblick über den derzeitigen Wissensstand zu den Strukturen, Mechanismen und der Inhibition von Sulfatasen.

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Synthesis and utility of sulfated chromogenic carbohydrate model substrates for measuring activities of mucin-desulfating enzymes

Cited by 21 publications

References 48 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

A Novel Bacterial Mucinase, Glycosulfatase, Is Associated with Bacterial Vaginosis

Sulfatasen: Struktur, Mechanismus, biologische Aktivität, Inhibition, Anwendung in Synthesen

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