CN-121974830-A - Enzyme activated fluorescent molecular precursor, preparation method and application thereof

CN121974830ACN 121974830 ACN121974830 ACN 121974830ACN-121974830-A

Abstract

The invention discloses an enzyme activated fluorescent molecular precursor, a preparation method and application thereof, wherein the enzyme activated fluorescent molecular precursor has a chemical structure shown in the following formula I: . The invention develops an enzyme activated fluorescent molecular precursor, which can be used for tumor real-time imaging by targeting exogenous enzyme to tumor and specifically activating a probe at the tumor; according to the invention, exogenous enzyme is introduced as an activation trigger, and normal cells do not have the exogenous enzyme, so that the problem of co-expression of endogenous enzyme in non-tumor tissues can be avoided, the background fluorescence signal can be reduced to the greatest extent, and the imaging specificity and accuracy are obviously improved.

Inventors

CHEN ZHANG
LI LI
GUO YIFANG
DONG WENFEI

Assignees

中国科学院苏州生物医学工程技术研究所

Dates

Publication Date: 20260505
Application Date: 20260107

Claims (10)

1. An enzyme-activated fluorescent molecular precursor, characterized in that it has a chemical structure represented by the following formula I: 。
2. A method for preparing the enzyme-activated fluorescent molecular precursor according to claim 1, comprising the steps of: S1, synthesizing a compound III by adopting a compound II, wherein the chemical structures of the compound II and the compound III are respectively shown in the following formulas II and III: ; S2, synthesizing by adopting a compound III to obtain the enzyme activated fluorescent molecular precursor.
3. The method for preparing an enzyme-activated fluorescent molecular precursor according to claim 2, wherein step S1 specifically comprises: s1-1, mixing a compound II, dichloromethane, DIPEA and 4-nitrophenyl chloroformate, and stirring; s1-2, dispersing L-glutamic acid di-tert-butyl ester hydrochloride in dichloromethane, dropwise adding the obtained mixture into the product obtained in the step S1-1, and stirring for reaction; S1-3, pouring the product into saline water after the reaction is finished, extracting by using ethyl acetate, concentrating an organic phase in vacuum, purifying the obtained concentrate by using silica gel column chromatography, eluting an eluent, collecting the eluent, concentrating and drying to obtain the compound III.
4. The method for preparing an enzyme-activated fluorescent molecular precursor according to claim 3, wherein the eluent in step S1-3 is a mixture of petroleum ether and ethyl acetate.
5. The method for producing an enzyme-activated fluorescent molecule precursor according to claim 4, the method is characterized in that the step S1 specifically comprises the following steps: S1-1, mixing 0.3-1.2mmol of compound II, 2.5-10mL of dichloromethane, 1-4mmol of DIPEA and 0.4-1.5mmol of 4-nitrophenyl chloroformate, and stirring at room temperature for 0.5-2h; S1-2, dispersing 0.5-2mmol of L-glutamic acid di-tert-butyl ester hydrochloride in 2.5-10mL of dichloromethane, dropwise adding the obtained mixture into the product obtained in the step S1-1, and stirring at room temperature for reaction for 0.5-2h; s1-3, pouring the product into 50-200mL of saline water after the reaction is finished, extracting with ethyl acetate for 1-4 times, adopting 50-2000mL of ethyl acetate each time, combining organic phases, concentrating in vacuum, purifying the obtained concentrate by silica gel column chromatography, eluting by adopting a mixture of petroleum ether and ethyl acetate in a volume ratio of = 1.5-6:1 as eluent, collecting the eluent, concentrating and drying to obtain the compound III.
6. The method for preparing an enzyme-activated fluorescent molecular precursor according to claim 2, wherein step S2 specifically comprises: Dissolving the compound III in a mixed solvent consisting of trifluoroacetic acid and deionized water, stirring for reaction, then adding deionized water to stop the reaction, and separating and purifying the reaction product by high performance liquid chromatography to obtain the enzyme activated fluorescent molecular precursor.
7. The method for preparing an enzyme-activated fluorescent molecular precursor according to claim 2, wherein step S2 specifically comprises: dissolving 0.3-1.5 mmol of compound III in a mixed solvent of 0.3-1.2 mL, wherein the mixed solvent consists of trifluoroacetic acid and deionized water according to a volume ratio of 2-8:1, stirring at room temperature for reacting for 2-3 hours, then adding deionized water to stop the reaction, and separating and purifying a reaction product by using high performance liquid chromatography to obtain the enzyme activated fluorescent molecular precursor.
8. The method for preparing an enzyme-activated fluorescent molecular precursor according to claim 2, comprising the steps of: S1, synthesizing a compound III by adopting a compound II: S1-1, 0.673mmol of Compound II, 5 mL dichloromethane, 2.02mmol of DIPEA and 0.178 mmol of 4-nitrophenyl chloroformate are mixed and stirred at room temperature for 1 h; S1-2, dispersing 1.0 mmol L-di-tert-butyl glutamate hydrochloride in 5mL of dichloromethane, dropwise adding the obtained mixture into the product obtained in the step S1-2, and stirring at room temperature for reaction for 1h; S1-3, pouring the product into 100mL of saline water after the reaction is finished, extracting with ethyl acetate for 2 times, adopting 100mL of ethyl acetate each time, combining organic phases, concentrating in vacuum, purifying the obtained concentrate by silica gel column chromatography, eluting by adopting a mixture of petroleum ether and ethyl acetate in a volume ratio of = 3:1 as eluent, collecting the eluent, concentrating and drying to obtain a compound III; S2, dissolving 0.673mmol of compound III in 0.673mL of mixed solvent consisting of trifluoroacetic acid and deionized water according to the volume ratio of 4:1, stirring at room temperature for reaction for 3 hours, then adding deionized water to stop the reaction, and separating and purifying the reaction product by high performance liquid chromatography to obtain the enzyme-activated fluorescent molecular precursor.
9. Use of the enzyme-activated fluorescent molecular precursor according to claim 1 as or in the preparation of a tumor tissue-targeted fluorescent activation formulation.
10. Use of an enzyme-activated fluorescent molecular precursor according to claim 1 as or in the preparation of a tumor-specific fluorescent imaging formulation.

Description

Enzyme activated fluorescent molecular precursor, preparation method and application thereof Technical Field The invention relates to the field of ‌ biomedical engineering, in particular to an enzyme activated fluorescent molecular precursor, a preparation method and application thereof. Background Malignant tumors are serious diseases threatening human health, and their therapeutic effects are highly dependent on early accurate diagnosis and complete excision. Real-time high-specificity imaging is a key for realizing accurate pathological diagnosis in malignant tumor operation. However, current clinical imaging approaches and histopathological methods have difficulty achieving intraoperative real-time tumor diagnosis. In addition, tissue biopsies rely on post-operative paraffin section and immunohistochemistry, which are time-consuming procedures and cannot be used for in vivo on-demand diagnosis. Although the traditional fluorescence imaging technology improves the imaging signal to noise ratio to a certain extent, the problem of overhigh background fluorescence exists in the tumor imaging process of the normally bright (always-on) fluorescent molecules, and the clinical application of the fluorescent molecules is severely limited. To improve imaging specificity and diagnostic accuracy, activatable probes (activatable probes) have been developed that typically quench fluorescence in an initial state, but are activated and emit intense fluorescence only after specific recognition of specific biomarkers within the tumor microenvironment, thereby greatly improving imaging contrast and detection signal-to-noise ratio. Currently, activatable fluorescent probes have been reported to rely on chemical component activation or endogenous enzyme activation mechanisms, such as hydrogen peroxide, glutathione activation, endogenous enzymes such as Nitroreductase (NTR), aminopeptidase N (APN), and various Matrix Metalloproteinase (MMPs) activation, and the like. Wherein the endogenous enzymes are often overexpressed in various malignant tumors, and can efficiently catalyze the cleavage of specific chemical bonds (such as amide bonds) in probe molecules, thereby causing the release or conformational change of fluorophores and realizing fluorescent "on" response. Based on the tumor specificity of endogenous enzyme activated probes, the application of the endogenous enzyme activated probes in the field of tumor imaging is limited by the problem that enzymes are expressed in non-tumor tissues. Probes that rely on endogenous enzyme activation have difficulty completely avoiding off-target activation in normal tissues, may lead to false positive signals, and reduce diagnostic specificity. Although endogenous enzyme-activated fluorescent probes have made significant progress in improving tumor/normal tissue (T/N) contrast, their inherent off-target risks still limit the clinical transformation prospects. The development of new generation probes with higher tumor specificity, faster activation kinetics and lower non-specific background signals remains a key challenge in achieving accurate intraoperative real-time pathological diagnosis. Disclosure of Invention The technical problem to be solved by the invention is to provide an enzyme activated fluorescent molecular precursor, a preparation method and application thereof, aiming at the defects in the prior art. The invention focuses on the design and application ‌ of a ‌ molecular probe in ‌ biomedical engineering ‌ and simultaneously relates to enzymatic reaction in ‌ biochemistry ‌, and the core of the invention is to develop a novel fluorescent molecular precursor which can be activated and change fluorescent signals under the action of specific biological enzymes, is mainly used for diagnosing and imaging diseases at the cellular and living body level, and aims to provide a fluorescent detection tool with high sensitivity and low background interference. In order to achieve the above purpose, the invention adopts the technical scheme that in the first aspect of the invention, an enzyme activated fluorescent molecular precursor is provided, which is marked as a compound I and has a chemical structure shown as the following formula I: 。 The invention designs and synthesizes a fluorescent molecular precursor which is applied to tumor tissue targeted fluorescent activation, such as but not limited to tumor fluorescent imaging, and the fluorescent molecular precursor can be specifically activated at a tumor part by CPG2 to release an active fluorescent molecule, thereby reducing background fluorescence. The compound I can be used as a substrate of carboxypeptidase G2 (CPG 2), and is hydrolyzed to remove glutamic acid protecting groups and spontaneously remove carbon dioxide through enzymatic reaction to release active fluorescent molecules (compound II), so that the fluorescent signal enhancement effect is achieved, and the action process is as follows: In a second aspect of the