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Dioxetane compounds and their use for the detection of microorganisms

A technology of compounds and heterocycles, applied in the field of dioxetane compounds and their use in the detection of microorganisms, can solve the problems of limited shelf life, complexity, and high cost of use

Active Publication Date: 2021-01-08
尼米斯技术公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, despite being the current industry standard, this system has a number of disadvantages
For example, the system is a complex multi-component system (standard composition: 1. pro-luciferin enzyme substrate (for example, fluorescein-β-D-galactopyranoside), 2. fluorescent Sulfase, 3. Bovine serum albumin, 4.ATP, 5.EDTA, 6.D / L-cysteine, 7.MgSO 4 , 8. Sodium pyrophosphate), which are usually more complex than one-component systems
Furthermore, the luciferase-luciferin system requires the use of luciferase, which makes its use very costly and limits shelf life due to the notorious instability of commercially available luciferase
Furthermore, the sensitivity of the luciferase-luciferin system is limited, thus leaving room for improvement

Method used

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  • Dioxetane compounds and their use for the detection of microorganisms
  • Dioxetane compounds and their use for the detection of microorganisms
  • Dioxetane compounds and their use for the detection of microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0352] General method:

[0353] All reactions were performed at room temperature unless otherwise stated. Chemicals and solvents were A.R. grade or purified by standard techniques. Thin Layer Chromatography (TLC): Silica gel plates Merck 60F254: Compounds were visualized by irradiation with UV light. Column chromatography (FC): silica gel Merck 60 (particle size 0.040-0.063 mm), eluents are given in parentheses. Reverse phase high pressure liquid chromatography (RP-HPLC): C18 5 μm, 250×4.6 mm, eluents are given in brackets. Preparative RP-HPLC: C18 5 μm, 250×21 mm, eluents are given in brackets. Fluorescence and chemiluminescence were recorded on a Molecular Devices Spectramax i3x.

[0354] All chemicals were purchased from Merck and Biosynth AG and used as received if not stated otherwise.

[0355] Abbreviation. AcOH-acetic acid, MeCN-acetonitrile, DCM-dichloromethane, DMF-N,N'-dimethylformamide, EtOAc-ethyl acetate, Hex-hexane, MeOH-methanol, TFA-trifluoroacetic acid, ...

Synthetic example 1

[0356] Synthesis Example 1: Synthesis of Compound IIa

[0357]

[0358] DCC (457 mg, 2.21 mmol, 1.1 eq) was added to a mixture of octanoic acid (350 μl, 2.21 mmol, 1.1 eq) and 4-hydroxybenzyl alcohol (250 mg, 2.01 mmol, 1 eq) in DCM (2 ml). The reaction mixture was stirred at room temperature and monitored by TLC (40:60 EtOAc:Hex). Upon completion, DCC was filtered off and the crude product was purified by column chromatography on silica gel (30:70 EtOAc:Hex) to give compound 1a (246 mg, 49% yield) as a pale yellow solid. 1 H NMR (400MHz, CDCl 3 )δ7.35(d, J=8.5Hz, 2H), 7.05(d, J=8.5Hz, 2H), 4.63(s, 2H), 2.55(t, J=7.5Hz, 2H), 1.76(dt, J=15.1, 7.5Hz, 2H), 1.50-1.18(m, 8H), 0.97-0.82(m, 3H). 13 C NMR (101MHz, CDCl 3 )δ 173.75, 150.68, 133.71, 129.49, 121.78, 65.50, 34.48, 31.72, 29.16, 29.14, 29.11, 28.98, 25.01, 22.67, 14.13.

[0359]

[0360] Compound la (200 mg, 0.8 mmol, 1 eq) was dissolved in 4 mL of ACN and cooled to 0 °C. Sodium iodide (360 mg, 2.4 mmol, 3 eq) ...

Synthetic example 2

[0365] Synthesis Example 2: Synthesis of Compound IIIa

[0366]

[0367] 1,2:4,5-Di-O-isopropylidene-inositol (250 mg, 0.96 mmol, 1 eq) and imidazole (98 mg, 1.44 mmol, 1.5 eq) were dissolved in dry pyridine (3 ml) and cooled to -10°C. Tert-butyldiphenylchlorosilane (275 μl, 1.06 mmol, 1.1 eq) was added slowly via syringe. The reaction was allowed to warm to room temperature and monitored by TLC (50:50 EtOAc:Hex). Upon completion, the reaction mixture was diluted with EtOAc and washed with saturated NH 4 Cl wash. The organic layer was separated, washed with Na 2 SO 4 Dry, filter and evaporate the solvent under reduced pressure. The crude product was purified by column chromatography on silica gel (50:50 EtOAc:Hex) to afford compound la (350 mg, 73% yield) as a white foam. MS (ES+): C 28 h 38 o 6 m / z calculated for Si: 498.24; found: 499.4 [M+H] + .

[0368]

[0369] Compound 1a (170 mg, 0.341 mmol, 1 eq) was dissolved in DCM and ethyl vinyl ether (652 μl, 6...

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PUM

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Abstract

The present invention relates to dioxetane compounds, their use in the detection of presence or absence, quantification and identification of microorganisms including bacteria, bacterial fragments (e.g., LPS, endotoxin), viruses, fungi as well as other pathogens by means of chemiluminescence and to corresponding methods.

Description

technical field [0001] The present invention relates to dioxetane compounds and their use for detecting the presence, quantification and identification of microorganisms including bacteria, bacterial fragments (eg, LPS, endotoxins), viruses, fungi and other pathogens by means of, Said means are chemiluminescence indicating the action of metabolic enzymes, reagent enzymes or reference enzymes on appropriate molecular probes, by means of reagent enzymes indicating the process produced by the enzymatic oxidation of microbial metabolites or nutrients. Hydrogen oxide, or inorganic phosphates that function as nutrients, substrates, metabolites, or by-products are detected by reagent enzymes. Background technique [0002] Bacterial contamination of, for example, food, water, and blood stocks poses major health problems. According to the World Health Organization, waterborne diseases, which are diseases caused by pathogenic microorganisms that travel in water, are associated with a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D321/00
CPCC07D321/00C07F9/302C07H15/203C07F5/025C12Q1/025C12Q1/44C07D407/04C12Q1/04C12Q1/10C12Q1/14C12Q1/22C12Q1/54G01N21/76
Inventor D·沙巴特M·E·罗斯康福特N·哈纳尼亚O·格林U·斯皮茨L·威克J·伊森R·沃伯格R·柯瑞彪Z·巴贾科娃姚春妍
Owner 尼米斯技术公司
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