US-12625141-B2 - Fluorescent dye, preparation method and uses thereof

Abstract

A fluorescent dye, as well as a preparation method and uses thereof, wherein the fluorescent dye is sensitive and specific to viscosity and has low background fluorescence; it can also be used as a fluorescent activated and lighted probe used for fluorescent labeling, quantification or monitoring of protein, enzymes or nucleic acid.

Inventors

• Linyong ZHU
• Yi Yang
• Dasheng Zhang
• Xianjun Chen
• Qiuning LIN
• Ni SU

Assignees

• FLUORESCENCE DIAGNOSIS (SHANGHAI) BIOTECH COMPANY LTD.

Dates

Publication Date

20260512

Application Date

20200427

Priority Date

20190428

Claims (5)

1. 1 . A fluorescent dye, the structural formula of which is shown as Formula (I), wherein: D- is HO— or N(X 1 )(X 2 )—, X 1 and X 2 are respectively and independently selected from hydrogen, alkyl and modified alkyl; and X 1 and X 2 are optionally interconnected, and form a lipid heterocyclic ring with N atoms; R is selected from cyano group, carboxy, amide group, ester group, sulfoxide group, sulphone group, sulfonic ester group or sulfonamido group; Ar 1 is selected from the following Formulae (II-2) to (II-22) and Ar 2 is selected from the following Formulae (II-1) to (II-4): wherein hydrogen atoms in Ar 1 and Ar 2 being optionally, respectively and independently substituted by halogen atoms, hydroxyl group, aldehyde group, carboxyl group, ester group, amide group, cyano group, sulfonic acid group, phosphoric acid group, amino group, primary amino group, secondary amino group, alkyl or modified alkyl; X 1 and X 2 optionally and independently form a lipid heterocyclic ring with Ar 1 ; wherein: the “alkyl” is respectively and independently C 1 -C 10 straight or branched alkyl; optionally, the “alkyl group” is C 1 -C 7 , straight or branched alkyl: optionally, the “alkyl group” is C 1 -C 5 straight or branched alkyl; optionally, the “alkyl group” is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tertiary butyl, sec-butyl, n-amyl, 1-methyl butyl, 2-methyl butyl, 3-methyl butyl, isoamyl, 1-ethyl propyl, neoamyl, n-hexyl, 1-methyl amyl, 2-methyl amyl, 3-methyl amyl, isohexyl, 1,1-dimethyl butyl, 2,2-dimethyl butyl, 3,3-dimethyl butyl, 1,2-dimethyl butyl, 1,3-dimethyl butyl, 2,3-dimethyl butyl, 2-ethyl butyl, n-heptyl, 2-methyl hexyl, 3-methyl hexyl, 2,2-dimethyl amyl, 3,3 dimethyl amyl, 2,3-dimethyl amyl, 2,4-dimethyl amyl, 3-ethyl amyl or 2,2,3-methyl butyl; the “modified alkyl” is respectively and independently a group obtained by replacing any carbon atom in alkyl with one or more groups of halogen atom, —OH, —CO—, —O—, —CN—, —S—, —SO 2 —, —(S═O)—, azido, primary amino group, secondary amino group, tertiary amino group, and quaternary ammonium base, and the modified alkyl has 1-10 carbon atoms, wherein the carbon-carbon single bond is optionally and independently replaced by a carbon-carbon double bond or a carbon-carbon triple bond; the replacement of carbon atoms refers to that carbon atoms or the carbon atoms and hydrogen atoms thereon together are replaced by a corresponding group; the “halogen atom” is respectively and independently F, Cl, Br or I; the “lipid heterocyclic ring” is a saturated or unsaturated 4- to 15-membered monocyclic or polycyclic lipid heterocyclic ring containing one or more heteroatoms of N, O, S, or Si on the ring, and the lipid heterocyclic ring is —S—, —SO— or —SO 2 — when there are S atoms on the ring; the lipid heterocyclic ring is optionally substituted by a halogen atom, an alkyl, an aryl or a modified alkyl; the “arylene” is a 5- to 13-membered monocyclic or dicyclic or fused dicyclic or fused polycyclic subaromatic group; the “sub-heteroaryl” is a 5- to 13-membered monocyclic or dicyclic or fused dicyclic or fused polycyclic sub-heteroaromatic group containing one or more heteroatoms of N, O, S, or Si on the ring; the “ester group” is R′(C═O)OR″ group; the “amide group” is R′CONR″R′″ group; the “sulfonic acid group” is R′SO 3 H group; the “sulfonic ester group” is R′SO 2 OR″ group; the “sulfonamido group” is R′SO 2 NR″R′″ group; the “phosphoric acid group” is R′OP(═O)(OH) 2 group; the “sulphone group” is R′SO 2 R″ group; the “sulfoxide group” is R′SOR″ group; the “primary amino group” is R′NH 2 group; the “secondary amino group” is R′NHR″ group; the “tertiary amino group” is R′NR″R′″ group; the “quaternary ammonium base” is R′R″R′″R″″N + group; each R′, R″, R′″, R″″ respectively and independently being single bond, hydrogen, alkyl, alkylene, modified alkyl or modified alkylene; the “alkylene” is C 1 -C 10 straight or branched alkylene; optionally, it is C 1 -C 7 straight or branched alkylene: optionally, it is C 1 -C 5 straight or branched alkylene; and the “modified alkylene” is a group obtained by replacing any carbon atom in C 1 -C 10 (preferably C 1 -C 6 ) alkylene with a group selected from —O—, —OH, —CO—, —CS—, and —(S═O)—.
2. 2 . The fluorescent dye according to claim 1 , wherein the “modified alkylene” is a group containing one or more groups selected from —OH, —O—, ethylene glycol unit, monosaccharide unit, —O—CO—, —NH—CO—, —SO 2 —O—, —SO—, Me 2 N—, Et 2 N—, —S—S—, —CH═CH—, F, Cl, Br, I, and cyano group.
3. 3 . The fluorescent dye according to claim 1 , wherein the compound represented by Formula (I) is selected from the compounds below:
4. 4 . A method of preparing the fluorescent dye according to claim 1 , including a step of aldol condensation reaction between a compound of Formula (a) and a compound of Formula (b),
5. 5 . A fluorescent activated and lighted probe solution, comprising the fluorescent dye according to claim 1 .

Description

RELATED APPLICATIONS The present application is a U.S. National Phase of International Application Number PCT/CN2020087311 filed Apr. 27, 2020 and claims priority to Chinese Application Number CN 201910352348.X filed Apr. 28, 2019. INCORPORATION BY REFERENCE The sequence listing provided in the file entitled 072-2110093US-SEQUENCE LISTING_revised_27.10.2021.txt, which is an ASCII text file that was created on Oct. 27, 2021, and which comprises 2,896 bytes, is hereby incorporated by reference in its entirety. TECHNICAL FIELD The present invention relates to the technical field of fluorescent dye, and particularly relates to a fluorescent dye with viscosity responsiveness and low background fluorescence, as well as a preparation method and uses thereof. BACKGROUND Molecular rotors are a kind of dyes the fluorescence intensity of which changes with microenvironment viscosity. After excitation of molecular rotors, conformation of molecules is twisted and TICT (twisted intramolecular charge transfer) is formed, wherein the excited energy are mainly released in a non-radiative form; when the molecules are in a microenvironment of comparatively large viscosity or rigidity, the twisted molecular conformation will be restricted for this kind of molecules, and the excited energy of dye will be mainly released in the form of radioluminescence, namely, the fluorescence property of molecules is activated. It is important that the fluorescence intensity of this kind of molecules changes with the microenvironment viscosity, so that the viscosity change of the microenvironment is displayed in real time, in situ and in a sensitive and visual manner. At present, besides the field of viscosity detection, the twisted conformation based on restrictions of the molecular rotors is also widely used for constructing a fluorescent activated probe, for example, after the combination of molecular rotors with BSA, the conformation of molecules is restricted by protein, and the fluorescence is lit up, but the excited energy of the dye that is not combined with protein is still dissipated in a non-radiative form, thereby detecting and quantifying the protein in real time. For another example, Thiazole Orange is in a state of fluorescence quenching before it is combined with DNA or RNA, and the molecular conformation is restricted after it is combined with DNA or RNA, as a result of which the fluorescence is activated, so Thiazole Orange is widely used for the detection and tracing of DNA and RNA; molecular rotors such as Malachite Green are coated with antibodies so as to limit the conformation changes of the molecules and are used for protein-activated fluorescence imaging; DHBI is combined with an adapter so as to construct fluorescent protein simulators for RNA tracing; for another example, the combination with amyloid protein can restrict the conformation changes of molecules, and can be used for the detection, research and so on of Alzheimer's disease. However, current molecular rotors generally have the disadvantage of high fluorescence background, namely, the fluorescent intensity of molecular rotors in a free state is comparatively high, and thus can hardly be used for the sample detection and labeling with a small sample size, complicated components and low abundance of objects to be measured, such as endogenous proteins, nucleic acid, metabolites and so on in biological samples, so the development of a kind of molecular rotors with low background fluorescence can further expand the use of current molecular rotors. SUMMARY OF THE INVENTION The object of the present invention is to provide a fluorescent dye with viscosity responsiveness and low background fluorescence. For one aspect, the present invention provides a fluorescent dye, wherein the fluorescent dye is shown as Formula (I), wherein:D- is HO— or N(X1)(X2)—, X1 and X2 are respectively and independently selected from hydrogen, alkyl and modified alkyl; and X1 and X2 are optionally interconnected, and form a lipid heterocyclic ring with N atoms;R is selected from cyano group, carboxy, amide group, ester group, sulfoxide group, sulphone group, sulfonic ester group or sulfonamido group; Ar1 and Ar2 are respectively and independently selected from arylene and sub-heteroaryl; wherein hydrogen atoms in Ar1 and Ar2 being optionally, respectively and independently substituted by halogen atoms, hydroxyl group, aldehyde group, carboxyl group, ester group, amide group, cyano group, sulfonic acid group, phosphoric acid group, amino group, primary amino group, secondary amino group, alkyl or modified alkyl;X1 and X2 optionally and independently form a lipid heterocyclic ring with Ar1;wherein: the “alkyl” is respectively and independently C-Cia straight or branched alkyl; optionally, the “alkyl group” is C1-C7 straight or branched alkyl; optionally, the “alkyl group” is C1-C5 straight or branched alkyl; optionally, the “alkyl group” is selected from methyl, ethyl, n-propyl, isopropyl,