Design and synthesis of 4′-O-alkyl-chitobiosyl-4-methylumbelliferone as human chitinase fluorogenic substrates

Duivenvoorden, Boudewijn A.; Ghauharali, Karen; Scheij, Saskia; Boot, Rolf G.; Aerts, Johannes M. F. G.; Marel, Gijsbert A. van der; Overkleeft, Herman S.; Codée, Jeroen D. C.

doi:10.1016/j.carres.2014.07.007

Cited by 2 publications

(2 citation statements)

References 22 publications

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“…Both colorimetric and fluorometric monosaccharides have been used to test the activity of DspB; however, these monosaccharide analogues are poor substrates and are unable to distinguish between the activity of functionally related glycosidase enzymes that differ in their glyosidic linkage specificity. The use of fluorometric or colorimetric oligosaccharides has been successfully employed to characterize the activity of chitinolytic enzymes that specifically recognize poly‐β‐ d ‐GlcNAc‐(1→4)‐linked substrates . Here, we envisioned using a similar approach in the design of the PNAG oligosaccharide analogue 1 .…”

Section: Figurementioning

confidence: 99%

A Colorimetric Assay to Enable High‐Throughput Identification of Biofilm Exopolysaccharide‐Hydrolyzing Enzymes

Wang

Breslawec

et al. 2020

Chemistry A European J

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Glycosidase enzymes that hydrolyze the biofilm exopolysaccharide poly‐β‐(1→6)‐N‐acetylglucosamine (PNAG) are critical tools to study biofilm and potential therapeutic biofilm dispersal agents. Function‐driven metagenomic screening is a powerful approach for the discovery of new glycosidase but requires sensitive assays capable of distinguishing between the desired enzyme and functionally related enzymes. Herein, we report the synthesis of a colorimetric PNAG disaccharide analogue whose hydrolysis by PNAG glycosidases results in production of para‐nitroaniline that can be continuously monitored at 410 nm. The assay is specific for enzymes capable of hydrolyzing PNAG and not related β‐hexosaminidase enzymes with alternative glycosidic linkage specificities. This analogue enabled development of a continuous colorimetric assay for detection of PNAG hydrolyzing enzyme activity in crude E. coli cell lysates and suggests that this disaccharide probe will be critical for establishing the functional screening of metagenomic DNA libraries.

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Section: Figurementioning

confidence: 99%

A Colorimetric Assay to Enable High‐Throughput Identification of Biofilm Exopolysaccharide‐Hydrolyzing Enzymes

Wang

Breslawec

et al. 2020

Chemistry A European J

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“…几丁质酶是糖基水解酶的一种, 其活性检测方法主要包括比色法 [14] 、黏度测定法 [15] 、放射化学法 [16] 和 HPLC 法 [17] 等. 其中比色法最为常用, 其原理之一是利用 4-硝基苯基-β-D-N-乙酰氨基葡萄糖苷(pNP-Glc-NAc)、4-硝基苯基-β-D-N,N'-二乙酰壳二糖苷(pNP-(Glc-NAc) 2 )或 4-甲基伞形基-β-D-N-乙酰氨基葡萄糖苷 (Mu-Glc-NAc)等作为相关酶的底物分子, 通过糖基水解释放出硝基苯酚或甲基伞形酮来检测酶的活性 [18,19] . 我们课题组 [20] 在前期研究中, 设计报道了一种新型的糖基化萘酰亚胺基荧光探针, 具有较好的水溶性和荧光特性, 在氨基糖苷酶的活性检测中发现其比 Mu-GlcNAc 更有效.…”

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Synthesis and Enzyme Substrate Activity of p-Nitrophenyl N,N'-Diacetyl-4-thio-β-chitobioside

Guo¹,

Shen²,

Jin³

et al. 2019

Chin. J. Org. Chem.

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Based on the resistance of glycosidase to the hydrolysis of glycosidase, the probe molecule p-nitrophenyl N,N'diacetyl-4-thio-β-chitobioside (pNP-TCB) was designed and synthesized. The effects of pNP-TCB on the degradation of glycoside hydrolase family 18 (GH18) chitinase and 20 (GH20) β-N-acetylhexosaminidase were studied. The results showed that when pNP-TCB was combined with β-N-acetylhexosaminidase, the disaccharide substrate was hardly degraded. But when it reacted with chitinase, the pNP-TCB was degraded by enzyme to release p-nitrophenol group, and the corresponding ultraviolet absorption value can be detected by enzyme-labeled instrument at 405 nm. It indicates that the substrate molecule exhibits good specificity, which can be used to study the properties of GH18 chitinase at the cellular level without interference of β-N-acetylhexosaminidase. The pNP-TCB can also provide a good detection method for screening and isolation between the GH18 chitinase and the GH20 β-N-acetyl hexose.

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Design and synthesis of 4′-O-alkyl-chitobiosyl-4-methylumbelliferone as human chitinase fluorogenic substrates

Cited by 2 publications

References 22 publications

A Colorimetric Assay to Enable High‐Throughput Identification of Biofilm Exopolysaccharide‐Hydrolyzing Enzymes

A Colorimetric Assay to Enable High‐Throughput Identification of Biofilm Exopolysaccharide‐Hydrolyzing Enzymes

Synthesis and Enzyme Substrate Activity of p-Nitrophenyl N,N'-Diacetyl-4-thio-β-chitobioside

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