A kind of β-glucuronidase precipitation type fluorescent substrate synthesis method

Abstract

The invention discloses a method for synthesizing a β-glucuronidase precipitation-type fluorescent substrate based on 2-(benzothiazole-2'-yl)-4-bromophenol. The synthesis method comprises the following three-step reactions: 1) glycosylation reaction; 2) aromatic ring reaction; 3) deprotection group reaction. In the synthesis method, the reaction yield of each step can reach above the middle level, the highest is more than 90%, the reaction conversion rate of the material with relatively high price or preparation cost is relatively high, and the total yield of the three steps can reach 37%, and the reaction conditions are mild. Easy to implement. At the same time, the glycosylation reaction step and the deprotection group reaction step in the synthesis method can be applied to the synthesis of common fluorescent substrates 4-methylumbelliferyl-β-D with 4-methylumbelliferone as a glycosyl acceptor ‑Glucuronide, the two-step reaction yields can reach 51%, 89%, respectively, and the total yield can reach about 45%.

Description

technical field [0001] The invention relates to the technical field of organic synthesis and enzyme analysis, in particular to a method for synthesizing a beta-glucuronidase precipitation type fluorescent substrate. Background technique [0002] Escherichia coli (E.coli) is generally regarded as an indicator bacteria of fecal contamination that is closely related to the hygienic quality, cleanliness or risk of pathogenicity of food, water, feed, clinical and environmental samples. Since most (96-98%) E.coli have β-glucuronidase (β-glucuronidase) activity, and non-E.coli bacteria with this enzyme activity are quite few, β-glucuronidase Has been used as a biomarker to detect E.coli. Although E.coli O157, the most dominant pathogenic serotype in enterohemorrhagic (or Shiga toxin-producing) E.coli, does not have β-glucuronidase activity, it can also be compared with non-O157E.coli to distinguish. Therefore, visual detection technology based on the specific reaction of synthet...

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Problems solved by technology

However, compared with the corresponding β-glucuronide, etc., β-glucuronide is more difficult to synthesize (Stachulski et al., Natural Product Reports, 1998, 15:173-186; Wei et al., Molecules, 2015 ,20:21681–21699.), the above-mentioned glycoside synthesis method based on 2-(benzothiazol-2'-yl)phenol derivatives may not be feasible for the corresponding β-glucuronide synthesis

For the synthesis of β-glucuronide based on 2-(benzothiazol-2'-yl)-4-bromophenol, the team of the present invention also tried to use 2-(benzothiazol-2'-yl)-4- Bromophenol was used as the sugarsyl acceptor, triacetyl-α-D-methyl bromoglucuronide was used as the sugarsyl donor, and the corresponding β-glucoside synthesis method reported—solid-liquid phase transfer catalytic method ( Wei et al., Chemical Communications, 2017, 53:103-106.) carried out research on glycosylation reaction, but found that 1,2-elimination HBr side reaction mainly occurred in the glycosyl donor, and the yield of the target product was very low (<20 %); while adopting silver oxide (or cesium carbonate) / acetonitrile condition to carry out glycosylation reaction, target yield is also still very low (<30%)

In addition, for the obtained protective group β-glucuronide, because it contains two different protective groups, methoxy and acetyl, and the side reaction of dehydration at the 4,5-position of the sugar ring may occur during the deprotection reaction. , resulting in relatively more troublesome deprotection reaction treatment (Stachulski et al., Natural Product Reports, 2013, 30:806-848.); in order to reduce the production of by-products that are difficult to separate, some researchers even use highly toxic KCN as a deprotection Protecting group reaction catalyst, but it is also relatively easy to produce impurities that are difficult to separate, resulting in the need for column purification before use in NMR analysis (Perry et al., Journal of Applied Microbiology, 2010, 101:977-985; Diwu et al ., Tetrahedron, 1997, 53:7159-7164; Haugland et al., 1994, U.S. Patent No. 5316906.)

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