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Bicyclo[4.1.0]heptane nitrosourea derivative for biological orthogonal reaction, and preparation method and application thereof

A nitrosourea and bio-orthogonal technology, applied in the field of chemical biology, can solve the problems of inactivation, achieve high activity, avoid instability, and fast reaction rate

Active Publication Date: 2020-05-12
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to their own high tension, TCO and its related analogs are prone to inactivation due to cis-trans isomerization of double bonds in complex biological systems, making this type of reaction unable to maintain good biological stability while maintaining a high reaction rate. compatibility

Method used

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  • Bicyclo[4.1.0]heptane nitrosourea derivative for biological orthogonal reaction, and preparation method and application thereof
  • Bicyclo[4.1.0]heptane nitrosourea derivative for biological orthogonal reaction, and preparation method and application thereof
  • Bicyclo[4.1.0]heptane nitrosourea derivative for biological orthogonal reaction, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1, the hydrolysis rate constant (pH 6.0-8.3) of pH triggering compound 1 (bicyclo[4.1.0]heptane nitrosourea derivatives) measured by UV-Vis

[0076] The evoked rate constant of compound 1 was obtained by measuring the decrease of the absorbance of compound 1 at a wavelength of 296 nm under different pH reaction conditions. The prepared stock solution of Compound 1 was stored in extremely dry methanol. Moreover, these stock solutions are best prepared for immediate use (25mM). A certain volume of stock solution (0.2ml) was measured and placed in 1.8ml of different buffer solutions (pH=6.0, 7.0, 7.2, 8.0, 8.3). The concentration of compound 1 after dilution was 2.5 mM. The absorbance of Compound 1 was measured every 5 seconds from 10 minutes to 12 hours. All reactions were run in triplicate. The first-order rate constant of eliciting compound 1 was determined by Originlab9.0.

[0077] Pseudo-first-order rate constant k obs : The absorbance of compound 1 in d...

Embodiment 2

[0082] Example 2, the model reaction between compound 1 and compound 2 (3,6-dipyridine-1,2,4,5-tetrazine)

[0083] The mechanism for the IEDDA reaction of compounds 1 and 2 is as follows:

[0084] Wherein each reaction condition is as follows:

[0085] Sequence 1: At room temperature, NaHCO 3 (24.4 mg, 0.291 mmol) powder was added to a solution of compound 1 (26.7 mg, 0.145 mmol) and compound 2 (23.4 mg, 0.097 mmol) in methanol (5 mL). NaHCO 3 Once added in the solution, nitrogen will be generated, and the color of the reaction solution will change from rose red to light yellow after 30min, which will indicate that the reaction has been completed. Afterwards, the reaction solution was spin-dried, and the reaction solution was purified by column chromatography. A mixed system of methanol and dichloromethane (5:95) was used as the eluent of column chromatography to obtain compound 5' (35.0 mg, 0.095 mmol, 98%) as a yellow solid.

[0086] Sequence 2: At room temperature, NaH...

Embodiment 3

[0095] Embodiment 3, preparation of bicyclo[4.1.0]heptane nitrosourea derivatives shown in formula I

[0096] 1) Preparation of ethyl 3-((benzyloxy)-methyl)bicyclo[4.1.0]heptane-7-carboxylate (compound 8):

[0097] The reaction equation is as follows:

[0098]

[0099] Compound 7 can be prepared according to existing methods.

[0100] Ethyl diazoacetate (2.72ml, 25.9mmol) was added dropwise to anhydrous dichloromethanesulfonate catalyst (936.7mg, 2.59mmol) containing compound 7 (10.487g, 51.8mmol) using a flow pump. in methane solution. After the reaction was completed, the reaction solution was spin-dried in vacuo, and then column chromatography was carried out under the mixed eluent of petroleum ether-dichloromethane (3:1) to obtain a colorless oily mixture 8 of a pair of diastereoisomers (4.725 g, 32%, dr=1.5:1) 1 H NMR (400MHz, CDCl 3 ) δ7.36–7.27(m,5H),4.48–4.45(m,2H),4.10(q,J=7.2Hz,2H),3.30–3.15(m,2H), 2.23–2.04(m,1H) ,2.00(d,J=9.4Hz,1H),1.80(ddd,J=18.2,9.2,4.7H...

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Abstract

The invention discloses a bicyclo[4.1.0]heptane nitrosourea derivative for a biological orthogonal reaction, and a preparation method and an application thereof. The structural formula of the bicyclo[4.1.0]heptane nitrosourea derivative is represented by formula I; and in the formula I, R represents an alkyl group with the carbon atom number of 1-15, an aryl group with the carbon atom number of 6-15 or H, X represents O or N, and n is a natural number between 1 and 10. A pH-initiated IEDDA reaction of trans-cycloheptene and tetrazine is provided for the first time, and can be used as a biological orthogonal reaction. A bicyclonitrosourea derivative (BNU)-bicyclo[4.1.0]heptane nitrosourea derivative is introduced as a precursor for storing and releasing trans-cycloheptene, so that the highactivity of the trans-cycloheptene is utilized, and the instability of the trans-cycloheptene is avoided as much as possible. The pH-initiated biological orthogonal reaction can be applied to in-vitroprotein labeling, living cell imaging and tissue pre-labeling imaging, and can also be applied to living animal tumor pre-labeling imaging. The novel biological orthogonal reaction has a wide application prospect.

Description

technical field [0001] The invention relates to a bicyclo[4.1.0]heptane nitrosourea derivative used in a bioorthogonal reaction and a preparation method and application thereof, belonging to the technical field of chemical biology. Background technique [0002] Since Bertozzi first applied the Staudinger reaction to chemical biology in 2000, many new bioorthogonal reactions have been gradually developed. From the perspective of reaction mode, the existing bioorthogonal reactions can be roughly divided into two categories: one is "one-stop reaction" ( figure 1 A)), most of the currently known bioorthogonal reactions are of this type, such as the Staudinger reaction, copper-catalyzed alkyne / azide [3+2] cycloaddition and ring strain-driven alkenes / Tetrazine [4+2] cycloaddition reaction. In contrast, another type of response uses the "elicit-release-bind" model, that is, a potential bioorthogon is released when induced by some factor, and then reacts with another orthogon. T...

Claims

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Application Information

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IPC IPC(8): C07D207/46G01N21/64A61K49/00
CPCA61K49/0021A61K49/0036A61K49/0052C07D207/46G01N21/6428G01N21/6486G01N2021/6439
Inventor 唐叶峰杜娟娟朱珠贾向前吴云飞杨鸿智曹玉
Owner TSINGHUA UNIV
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