CN112812141A - Preparation method of 3-fluoromethyl-substituted coumarin compound and fluorescent probe - Google Patents

Preparation method of 3-fluoromethyl-substituted coumarin compound and fluorescent probe Download PDF

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CN112812141A
CN112812141A CN201911129662.8A CN201911129662A CN112812141A CN 112812141 A CN112812141 A CN 112812141A CN 201911129662 A CN201911129662 A CN 201911129662A CN 112812141 A CN112812141 A CN 112812141A
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谢贺新
谭庆伟
江嘉玲
赵书轩
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East China University of Science and Technology
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    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract

The invention provides a preparation method of 3-bit fluoromethyl substituted coumarins and a coumarin self-anchoring enzyme detection fluorescent probe. Specifically, the preparation method introduces the fluorine-containing methyl group at the 3-position of the coumarin by a one-step method in the presence of Eosin Y, an oxidant, an alkali metal salt, an alkaline earth metal salt and the like at the same time. The invention also provides a coumarin self-anchoring fluorescent probe with strong self-anchoring capability and high fluorescence intensity.

Description

Preparation method of 3-fluoromethyl-substituted coumarin compound and fluorescent probe
Technical Field
The invention relates to the field of fluorescent probes, in particular to a preparation method of 3-bit fluoromethyl substituted coumarins and a coumarin self-anchoring enzyme detection fluorescent probe.
Background
The small molecular fluorescent probe has the characteristics of low background signal, high sensitivity, intuitive result, safety, no wound, convenience, economy and the like, and is practically applied to the fields of fluorescent imaging, flow cytometry, high-throughput screening, environmental monitoring and the like; but also widely used in studies in various biomedical related fields such as detection of enzymes, in vivo tracing, development of drugs, diagnosis of diseases, and guidance of surgical operations. However, the conventional small molecule fluorescent probe is easy to diffuse out of the detection site and even to be discharged out of the cell after being activated by the target enzyme, so that the fluorescent signal is weakened, and the detection sensitivity and resolution of the fluorescent probe are reduced.
The self-anchoring fluorescent probe can be specifically identified and hydrolyzed by enzyme to generate fluorescence, can form covalent labels with peripheral protein, and is not easy to diffuse and discharge, so that the detection sensitivity and the practicability of the fluorescent probe can be improved.
Therefore, it is necessary to provide more fluorescent probes with strong self-anchoring ability and high fluorescence intensity, and a preparation method with mild reaction conditions and high yield.
Disclosure of Invention
The invention aims to provide a preparation method of a 3-fluoromethyl substituted coumarin compound, which can introduce fluoromethyl into the 3-position of coumarin by only one-step reaction, and has the advantages of mild reaction conditions, high selectivity and high yield.
Another purpose of the invention is to provide a coumarin self-anchoring enzyme detection fluorescent probe with strong self-anchoring capability and high fluorescence intensity.
In a first aspect of the invention, there is provided a process for the preparation of a compound of formula I, comprising the steps of:
(a) in an inert solvent, in the presence of Eosin Y and an additive, a compound of a formula II and a compound of a formula III react under blue light illumination to obtain a compound of a formula I,
Figure BDA0002277933130000021
wherein the content of the first and second substances,
a is selected from the following group: CH (CH)2F or CHF2；
X is selected from the group consisting of: o, NH or S;
r is selected from the group consisting of: h or an enzyme recognition group;
the enzyme recognition group is a group capable of being recognized by an enzyme and interacting with a targeting recognition group of the enzyme, thereby generating-XH;
R1、R2、R3、R4each independently selected from: hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkenyl, substituted or unsubstituted C1-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, C1-C6 haloalkyl, substituted or unsubstituted C6-C10 aryl, C6-C10 haloaryl, or substituted or unsubstituted C3-C10 cycloalkyl;
unless otherwise specified, the term "substituted" means that one or more hydrogens on the group is replaced with a group selected from the group consisting of: H. -OH, -NH2Halogen, oxo (═ O), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C3 alkoxy, C1-C3 haloalkyl, -COOH, phenyl or benzyl;
m is a cation pair;
and the additive is selected from the group consisting of: an oxidizing agent, an alkali metal salt, an alkaline earth metal salt, or a combination thereof.
In another preferred embodiment, the halogen is selected from the group consisting of: fluorine, chlorine, bromine or iodine.
In another preferred embodiment, M is Na+Or K+。
In another preferred embodiment, said compound of formula II is subjected to step (a) after addition of a protecting group.
In another preferred embodiment, the protecting group is selected from the group consisting of: boc, Cbz or Fmoc.
In another preferred embodiment, the oxidizing agent is selected from the group consisting of: t-butyl peroxy-alcohol, hydrogen peroxide, sodium periodate, potassium periodate, or a combination thereof, preferably t-butyl peroxy-alcohol.
In another preferred embodiment, the alkali metal salt is a sodium or potassium salt, preferably, KHSO4。
In another preferred embodiment, the alkaline earth metal salt is a zinc salt, preferably ZnCl2Or Zn (OAc)2。
In another preferred embodiment, the compound of formula I has the structure of formula Ia:
Figure BDA0002277933130000022
wherein R, X, A is as defined above.
In another preferred embodiment, the compound of formula I is selected from the group consisting of:
Figure BDA0002277933130000031
in another preferred embodiment, the Eosin Y is 2 ', 4 ', 5 ', 7 ' -tetrabromo-3 ', 6 ' -dihydroxyspiro [ isophenylfuran-1 (3H),9 ' (9H) -xanthen ] -3-one, a salt thereof (e.g., disodium salt), or a combination thereof.
In another preferred embodiment, the step (a) has one or more of the following features:
(1) the light source of the illumination is a blue LED;
(2) the power of the light source for illumination is 10-50W, preferably 15-30W, more preferably 15-25W;
(3) the reaction time is 2-24h, preferably 12-24h, more preferably 14-18 h;
(4) the reaction temperature is 4-30 ℃, preferably 20 +/-5 ℃; and/or
(5) The inert solvent is selected from: dimethyl sulfoxide, acetonitrile, dichloromethane, water, preferably dimethyl sulfoxide.
In another preferred embodiment, the molar ratio of the compound of formula II to the compound of formula III is 1:1 to 10, preferably 1:2 to 8; more preferably, 1: 3-6.
In another preferred embodiment, the molar ratio of said compound of formula II to Eosin Y is 20 to 1:1, preferably, 10-2:1, more preferably, 8-4: 1.
In another preferred embodiment, the molar ratio of Eosin Y to additive is 1:0.1-50, preferably 1: 0.2-45.
In another preferred embodiment, the additive is an oxidizing agent, and the molar ratio of Eosin Y to the oxidizing agent is 1:1-100, preferably 1: 3-50, preferably 1: 20-45.
In another preferred embodiment, the additive is t-butyl peroxy-butanol, and the molar ratio of Eosin Y to t-butyl peroxy-butanol is 1:5-100, preferably 1: 20-50, preferably 1: 35-45.
In another preferred embodiment, the additive is an alkali metal, and the molar ratio of Eosin Y to alkali metal salt is 1:0.1-5, preferably 1: 0.2-3, preferably 1: 0.3-2.
In another preferred embodiment, the additive is an alkaline earth metal salt, and the molar ratio of Eosin Y to the alkaline earth metal salt is 1:0.1-5, preferably 1: 0.2-3, preferably 1: 0.3-2.
In a second aspect of the present invention, there is provided an enzyme detection fluorescent probe, wherein the fluorescent probe has a structure of a compound of formula I:
Figure BDA0002277933130000041
wherein the content of the first and second substances,
a is selected from the following group: CH (CH)2F or CHF2；
X is selected from the group consisting of O, NH or S;
r is selected from the group consisting of: a beta-galactosidase (beta-gal) recognition group, an alkaline phosphatase (ALP) recognition group, or a glutamyltranspeptidase (gamma-GT, GGT) recognition group,
R1、R2、R3、R4each independently selected from: hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkenyl, substituted or unsubstituted C1-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, C1-C6 haloalkyl, substituted or unsubstitutedSubstituted C6-C10 aryl, C6-C10 haloaryl, or substituted or unsubstituted C3-C10 cycloalkyl;
unless otherwise specified, the term "substituted" means that one or more hydrogens on the group is replaced with a group selected from the group consisting of: H. -OH, -NH2Halogen, oxo (═ O), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C3 alkoxy, C1-C3 haloalkyl, -COOH, phenyl or benzyl.
In another preferred embodiment, the halogen is selected from the group consisting of: fluorine, chlorine, bromine or iodine.
In another preferred embodiment, R-X-is selected from the group consisting of:
Figure BDA0002277933130000042
in another preferred embodiment, the compound of formula I has the structure of formula Ia:
Figure BDA0002277933130000043
wherein R, X, A is as defined above.
In another preferred embodiment, the compound of formula I is selected from the group consisting of:
Figure BDA0002277933130000044
in a third aspect of the present invention, a β -galactosidase assay kit is provided, comprising:
as fluorescent probes
Figure BDA0002277933130000045
And at least one container containing the fluorescent probe.
In a fourth aspect of the present invention, there is provided an alkaline phosphatase detection kit comprising:
as fluorescent probes
Figure BDA0002277933130000051
And accommodating the fluorescenceAt least one container of the probe.
In a fifth aspect of the present invention, there is provided a glutamyl transpeptidase assay kit comprising:
as fluorescent probes
Figure BDA0002277933130000052
And at least one container containing the fluorescent probe.
In a sixth aspect of the invention, there is provided a use of the fluorescent probe according to the second aspect of the invention for: (i) labeling, tracking and/or imaging cells that overexpress an enzyme; and/or (ii) preparing a composition for labeling, tracking and/or imaging cells overexpressing an enzyme; and/or (iii) preparing a pharmaceutical composition for treating a disease associated with enzyme overexpression.
In another preferred embodiment, the labeling, tracking and/or imaging is in vitro non-therapeutic non-diagnostic.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows fluorescence spectra of BGC-2 and its analogs.
FIG. 2 shows the detection of β -gal activity in living cells using fluorescent probes.
FIG. 3 shows the detection of beta-gal activity in living cells using flow cytometry
FIG. 4 shows the detection of β -gal activity in senescent cells using a fluorescent probe.
FIG. 5 shows the detection of ALP activity in living cells with a fluorescent probe "leave-on".
FIG. 6 shows the detection of GGT activity in living cells using a fluorescent probe.
Detailed Description
The present inventors have conducted extensive and intensive studies and, as a result, have provided a method for introducing a fluoromethyl group (i.e., a group) at the 3-position of coumarin by a large number of screenings and tests. The method adopts a photocatalysis method, can selectively introduce the fluorine-containing methyl group at the 3-position of the coumarin in one step, has very mild reaction conditions, is surprising, has greatly improved reaction yield after the photocatalyst Eosin Y, an oxidant and other additives are added in a reaction system, is suitable for substrates with different target recognition groups, and synthesizes the coumarin self-anchoring fluorescent probe with greatly enhanced fluorescence effect by utilizing the method. The present invention has been completed based on this finding.
Term(s) for
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
Unless otherwise indicated, the term "alkyl" by itself or as part of another substituent refers to a straight or branched chain hydrocarbon group having the indicated number of carbon atoms (i.e., C1-6 represents 1-6 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, and the like.
Unless otherwise indicated, the term "alkenyl" refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl group having one or more triple bonds. Examples of such unsaturated alkyl groups include ethenyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and higher homologs and isomers.
Unless otherwise indicated, the term "cycloalkyl group"means having the indicated number of ring atoms (e.g., C)3-10Cycloalkyl) and a hydrocarbon ring that is fully saturated or has no more than one double bond between ring vertices. "cycloalkyl" also refers to bicyclic and polycyclic hydrocarbon rings, e.g. bicyclo [2.2.1]Heptane, bicyclo [2.2.2]Octane, and the like.
Unless otherwise indicated, the term "aryl" denotes a polyunsaturated (usually aromatic) hydrocarbon group which may be a single ring or multiple rings (up to three rings) which are fused together or linked covalently.
Unless otherwise specified, all occurrences of a compound in the present invention are intended to include all possible optical isomers, such as a single chiral compound, or a mixture of various chiral compounds (i.e., a racemate). In all compounds of the present invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.
Herein, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on a group are replaced with a substituent selected from the group consisting of: H. -OH, -NH2Halogen, oxo (═ O), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C3 alkoxy, C1-C3 haloalkyl, -COOH, phenyl or benzyl
As used herein, the term "a" or "an" refers to,
Figure BDA0002277933130000072
indicating the attachment site to other groups.
As used herein, the site numbering on the coumarin ring is as indicated in the formula I compound structural formula.
As used herein, room temperature means a temperature of 4-35 deg.C, preferably, 20 + -5 deg.C.
As used herein, the term "alkali metal" includes lithium, sodium, potassium, rubidium, cesium, francium; "alkaline earth metals" include beryllium, magnesium, calcium, strontium, barium, radium.
Preparation method
The invention provides a preparation method of a compound shown in formula I, which comprises the following steps:
(a) in an inert solvent, in the presence of Eosin Y and an additive, a compound of a formula II and a compound of a formula III react under blue light illumination to obtain a compound of a formula I,
Figure BDA0002277933130000071
wherein the additive is selected from the group consisting of: an oxidizing agent, an alkali metal salt, an alkaline earth metal salt, or a combination thereof;
R1、R2、R3、R4x, A, M are as defined above.
In another preferred embodiment, the step (a) has one or more of the following features:
(1) the light source of the illumination is a blue LED;
(2) the power of the light source for illumination is 10-50W, preferably 15-30W, more preferably 15-25W;
(3) the reaction time is 2-24h, preferably 12-24h, more preferably 14-18 h;
(4) the reaction temperature is 4-30 ℃, preferably 20 +/-5 ℃; and/or
(5) The inert solvent is selected from: dimethyl sulfoxide, acetonitrile, dichloromethane, water, preferably dimethyl sulfoxide.
Preferably, when a labile group such as an amino group is present in the compound of formula II, the group can be subjected to step (a) after addition of a protecting group, which protecting group and method are known in the art.
In another preferred embodiment, the protecting group is selected from the group consisting of: boc, Cbz or Fmoc.
In another preferred embodiment, the oxidizing agent is selected from the group consisting of: t-butyl peroxy-alcohol, hydrogen peroxide, sodium periodate, potassium periodate, or a combination thereof, preferably t-butyl peroxy-alcohol.
In another preferred embodiment, the alkali metal salt is a sodium or potassium salt, preferably, KHSO4。
In another preferred embodiment, the alkaline earth metal salt is a zinc salt, preferably ZnCl2Or Zn (OAc)2。
In another preferred embodiment, the Eosin Y is 2 ', 4 ', 5 ', 7 ' -tetrabromo-3 ', 6 ' -dihydroxyspiro [ isophenylfuran-1 (3H),9 ' (9H) -xanthen ] -3-one, a salt thereof (e.g., disodium salt), or a combination thereof.
In another preferred embodiment, the molar ratio of the compound of formula II to the compound of formula III is 1:1 to 10, preferably 1:2 to 8; more preferably, 1: 3-6.
In another preferred embodiment, the molar ratio of said compound of formula II to Eosin Y is 20 to 1:1, preferably, 10-2:1, more preferably, 8-4: 1.
In another preferred embodiment, the molar ratio of Eosin Y to additive is 1:0.1-50, preferably 1: 0.2-45.
In another preferred embodiment, the additive is an oxidizing agent, and the molar ratio of Eosin Y to the oxidizing agent is 1:1-100, preferably 1: 3-50, preferably 1: 20-45.
In another preferred embodiment, the additive is t-butyl peroxy-butanol, and the molar ratio of Eosin Y to t-butyl peroxy-butanol is 1:5-100, preferably 1: 20-50, preferably 1: 35-45.
In another preferred embodiment, the additive is an alkali metal, and the molar ratio of Eosin Y to alkali metal salt is 1:0.1-5, preferably 1: 0.2-3, preferably 1: 0.3-2.
In another preferred embodiment, the additive is an alkaline earth metal salt, and the molar ratio of Eosin Y to the alkaline earth metal salt is 1:0.1-5, preferably 1: 0.2-3, preferably 1: 0.3-2.
In another preferred embodiment, the compound of formula I is selected from the group consisting of:
Figure BDA0002277933130000081
fluorescent probe, kit and application
The invention also provides a class of enzyme detection fluorescent probes, which have a structure of a compound shown in formula I:
Figure BDA0002277933130000091
wherein the content of the first and second substances,
r is selected from the group consisting of: a beta-galactosidase (beta-gal) recognition group, an alkaline phosphatase (ALP) recognition group, or a glutamyltranspeptidase (gamma-GT, GGT) recognition group,
R1、R2、R3、R4x, A are as defined above.
In another preferred embodiment, R-X-is selected from the group consisting of:
Figure BDA0002277933130000092
in another preferred embodiment, the compound of formula I is selected from the group consisting of:
Figure BDA0002277933130000093
the invention also provides an enzyme detection kit comprising the fluorescent probe of the invention. According to different enzyme recognition groups, the method can be used for preparing a detection kit of the corresponding enzyme.
Typically, when R-X-is
Figure BDA0002277933130000094
In this case, the compound I can be used in a beta-galactosidase assay kit.
Such "enzymatic assays" include (but are not limited to): enzyme activity detection, enzyme location detection, identification, labeling, tracking, and/or imaging of enzyme-overexpressing cells, and the like.
The fluorescent probes of the present invention can be used for: (i) labeling, tracking and/or imaging cells that overexpress an enzyme; and/or (ii) preparing a composition for labeling, tracking and/or imaging cells overexpressing an enzyme; and/or (iii) preparing a pharmaceutical composition for treating a disease associated with enzyme overexpression.
The main advantages of the invention include:
(1) the invention provides a synthetic method which can introduce the fluoromethyl group at the 3-position of the coumarin by only one-step reaction, and has the advantages of mild reaction conditions, simple operation, high selectivity, high yield, good reproducibility and cheap and easily-obtained raw materials.
(2) The method of the invention can be applied to the 3-position fluoromethylation of coumarin fluorescent probes with different target recognition groups.
(3) The invention further provides a self-anchoring enzyme detection fluorescent probe, and experiments prove that the fluorescent probe has strong self-anchoring capability and high fluorescence intensity, the two properties jointly reduce background signals, improve detection sensitivity and imaging definition, and can clearly mark, image and identify cells.
The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
Detection method
In the practice of the present invention, it is,1H-NMR、13C-NMR was measured with a Bruker 400Mz or 600Mz type instrument using deuterated dimethyl sulfoxide (DMSO-d)6) And deuterated methanol (MeOD), with Tetramethylsilane (TMS) as an internal standard; all solvents were chromatographically, analytically or chemically pure. The method further comprises the step of isolating and/or purifying the detection probe; preferably, the step of isolating and/or purifying the detection probe is purification using a reverse phase C18 preparative column, freeze-drying.
Reagent
Eosin Y, formula: 2 ', 4 ', 5 ', 7 ' -tetrabromo-3 ', 6 ' -dihydroxyspiro [ isohydrocinnamofuran-1 (3H),9 ' (9H) -xanthene ] -3-one, adalimusbeta (shanghai) chemical reagent ltd, purity: 95 percent.
The compounds BGC-1, CP-1, GC-1, BGC-3 and the like can be prepared by common synthetic methods.
Example 1
Screening of reaction conditions[a]
The conditions for the reaction conditions were selected as shown in Table 1,[a]unless otherwise stated, the reaction was carried out using BGC-1(9.25mol) and Eosin Y (0.45mol) in DMSO (0.3mL) and irradiation with a blue LED (20W) for 16h at room temperature.[b]The yield was analyzed by HPLC and the internal standard was 7-methoxycoumarin.[c]Blue LED illumination is not used.[d]3.0 equivalents of NaSO2CF2H。[e]Isolated yield. TBHP tert-butyl hydroperoxide (tert-butyl hydroperoxide).
Figure BDA0002277933130000101
TABLE 1
Figure BDA0002277933130000102
Figure BDA0002277933130000111
As shown in Table 1, TBHP alone did not promote product formation, Eosin Y alone resulted in low yields, and surprisingly, when Eosin Y was used with an oxidant, ZnCl2、KHSO4When the additives are used in combination, the reaction yield is greatly improved compared with that when the additives are used independently, and the selectivity on 3-position substitution on a coumarin ring is high.
When the column is used for purifying the product, metal salts may precipitate in the organic phase to damage the equipment. TBHP is liquid, does not clog columns and equipment lines, facilitates purification, and is highest in yield when Eosin Y is used with TBHP.
Example 2
Figure BDA0002277933130000112
BGC-1(0.03mmol, 10.0mg), sodium difluoromethylsulfinate (0.15mmol, 20.7mg) and Eosin Y (4.5. mu. mol, 2.9mg) were placed in a reaction flask with 0.5mL of dimethyl sulfoxide and t-butanol peroxide (70% strength in water, 170. mu.L, 1.2 mmol);
the reaction was allowed to react at room temperature for 16 hours in a 20W blue LED and after completion of the reaction, purification was performed using reverse phase C18 preparative column, which was lyophilized to give the compound BGC-2(7.8mg) in 70% yield as a yellow color.
The spectrogram is characterized in that:1H NMR(400MHz,DMSO-d6)δ8.41(s,1H),7.82(d,J＝8.7Hz,1H),7.13(d,J＝2.2Hz,1H),7.09(dd,J＝8.6,2.3Hz,1H),6.90(t,J＝54.5Hz,1H),5.25(bs,1H),5.03(d,J＝7.7Hz,1H),4.65(bs,3H),3.75–3.40(m,6H).13C NMR(101MHz,DMSO-d6)δ162.07,158.52(t,J＝4.0),155.81,142.81(t,J＝6.0),131.39,117.73(t,J＝23.0),114.81,112.47,112.09(t,J＝237),103.44,100.96,76.21,73.65,70.49,68.57,60.83.19F NMR(376MHz,DMSO-d6)δ-121.14(d,J＝52.6Hz)；HRMS(ESI)m/z calcd for C16H15F2O8(M-H)-373.0735,found 373.0731.
this example demonstrates that the preferred embodiment of example 1 is very reproducible.
Example 3
Preparation of fluorescent Probe (CP-2)
Figure BDA0002277933130000121
CP-1(0.04mmol, 9.7mg), sodium difluoromethylsulfinate (0.20mmol, 27.6mg) and Eosin Y (6.0. mu. mol, 3.9mg) were placed in a reaction flask, 0.5mL of dimethyl sulfoxide and t-butanol peroxide (70% strength in water, 170. mu.L, 1.2 mmol);
the reaction was allowed to react at room temperature for 16h with a 20W blue LED and after completion of the reaction, purification was performed using a reverse phase C18 preparative column, which was lyophilized to give the compound in yellow color, i.e., 52% yield of the compound CP-2(6.2 mg). Namely the fluorescent probe (CP-2).
The spectrogram is characterized in that:1H NMR(400MHz,DMSO-d6)δ8.44(s,1H),7.88(d,J＝8.5Hz,1H),7.29–7.22(m,2H),6.92(t,J＝54.3Hz,1H).13C NMR(101MHz,DMSO-d6)δ156.59(t,J＝4.0Hz),154.56(d,J＝6.0Hz),153.50,140.89(t,J＝6.0Hz),129.86,117.48(t,J＝23.0Hz),116.17(d,J＝6.0Hz),112.60,110.33(t,J＝237.4Hz,1H),106.17(d,J＝5.0Hz).19F NMR(376MHz,DMSO-d6)δ-120.30(d,J＝56.4Hz)；HRMS(ESI)m/z calcd for C10H6F2O6P(M-H)-290.9876,found 290.9888.
example 4
Preparation of fluorescent Probe (GC-2)
Figure BDA0002277933130000122
GC-0(0.04mmol, 9.7mg), sodium difluoromethylsulfinate (0.20mmol, 27.6mg) and Eosin Y (6.0. mu. mol, 3.9mg) were placed in a reaction flask with 0.5mL of dimethyl sulfoxide and t-butanol peroxide (70% strength in water, 170. mu.L, 1.2 mmol);
the reaction system was reacted at room temperature for 16 hours in a 20W blue LED, and after the reaction was completed, it was purified by reverse phase C18 preparative column, and lyophilized to obtain a yellow compound, i.e., the compound GC-1'.
GC-1' (0.01mmol, 5.0mg), anhydrous dichloromethane (1mL and trifluoroacetic acid (1mL in a reaction flask, ice bath conditions for 4 hours), after the reaction, reverse phase C18 preparative column purification was performed, and freeze drying was performed to obtain a yellow compound, namely the fluorescent probe GC-2(3.5mg), with a yield of 28% in two steps.
The spectrogram is characterized in that:1H NMR(600MHz,CD3OD)δ8.21(d,J＝4.2Hz,1H),7.92(dd,J＝4.8,1.8Hz,1H),7.68(t,J＝8.4Hz,1H),7.44(dd,J＝7.8,5.4Hz,1H),6.75(t,J＝56.4Hz,1H),3.35(s,1H),2.72(m,2H),2.23(m,2H).13C NMR(151MHz,CD3OD)δ173.57,172.67,169.41,160.44,156.46,144.98,142.76,131.21,117.44(d,J＝13.6Hz,),115.09(d,J＝7.6Hz),112.41(t,J＝236Hz),107.46,53.90,33.04,26.60.19F NMR(565MHz,MeOD)δ-120.51(d,J＝51.0Hz)。
example 5
BGC-2 and its analogs fluorescence spectra
The experimental conditions are as follows: fluorescent probes BGC-2 and BGC-3(5 mu M) and beta-gal (4U/mL) were added to PBS (pH:7.4), incubated in a water bath at 37 ℃ for 1h, and then excited with 416nm (BGC-2) and 360nm (BGC-3), respectively, to determine their fluorescence spectra.
Figure BDA0002277933130000131
The experimental results are as follows: as shown in FIG. 1, the fluorescence spectrum of fluorescent probe BGC-2 under the activation of beta-gal is shown on the left side of FIG. 1, and the fluorescence spectrum of fluorescent probe BGC-3 under the activation of beta-gal is shown on the right side. When the fluorescent probe BGC-2 and BGC-3 are hydrolyzed by beta-gal, the fluorescence intensity of BGC-2 is much higher than that of BGC-3.
And (4) experimental conclusion: BGC-2 is hydrolyzed by beta-gal to quickly form a methyl benzoquinone active intermediate, and products generated by reaction with water in a solution have higher fluorescence intensity (after hydrolysis, the maximum fluorescence intensity of BGC-2 is enhanced by about 14 times compared with BGC-3), which shows that the fluorescent probe has good application prospect in the aspects of reducing background interference, improving detection sensitivity and improving imaging effect.
Example 6
Cell fluorescence imaging of BGC-2 and analogs thereof
The experimental conditions are as follows: the fluorescent probes BGC-1, BGC-2 and BGC-3(5 μ M) were incubated with ct26.cl25(Lac Z +), ct26.cl25(Lac Z-) for 1h in a confocal dish (35mm), followed by discarding the probe-containing medium and washing three times with PBS (pH 7.4) to remove probe molecules that did not establish covalent linkage with the membrane surface; 1mL of sterile PBS (pH 7.4) was added and cell imaging was performed using a fluorescence microscope (λ)ex＝360±20nm,λem425 nm). And (5) performing fluorescence imaging photographing.
Figure BDA0002277933130000141
The experimental results are as follows: the experimental results are shown in FIG. 2, the left group 3 is the labeling experiment of the fluorescent probes BGC-1, BGC-2, BGC-3 and CT26.CL25(Lac Z +) cells on the cells under the activation of beta-gal; the right group 3 is a marking experiment of fluorescent probes BGC-1, BGC-2 and BGC-3 on CT26.WT (Lac Z-) cells with low expression of beta-gal. In the figure, "FL" is a blue fluorescence signal; BF is cell morphology under white light irradiation; scale bar 25 μm. As can be seen from FIG. 2, compared with the ordinary fluorescent probe BGC-1 without labeling ability, the fluorescent probes BGC-2 and BGC-3 with covalent bond "anchor point" can realize the recognition of beta-gal overexpression cells, obviously distinguish beta-gal low expression cell strains (CT26.WT) from high expression cell strains (CT26.CL25), and the labeling effect of BGC-2 is obviously better than that of BGC-3.
And (4) experimental conclusion: the fluorescent probes BGC-2 and BGC-3 can realize the identification and covalent labeling effects on the beta-gal over-expressed tumor cells at the level of in vitro living cells, so that the dispersion of the fluorescent probes in cell imaging experiments is reduced, background signals are reduced, the detection sensitivity is improved, and the BGC-2 has obviously higher fluorescence intensity than the BGC-3 and is clearer in imaging.
Example 7
Flow cytometric assay for BGC-2 and analogs thereof
The experimental conditions are as follows: the fluorescent probes BGC-1, BGC-2 and BGC-3 (50. mu.M) were respectively mixed with CT26.CL25(Lac Z +) CT26.CL25(Lac Z-) (5X 10)5One/tube) were incubated in a microcentrifuge tube for 1h at 37 ℃. Subsequently, the mixture was centrifuged at 800 Xg for 2min in a centrifuge, and the supernatant was discarded. Resuspending in 5mL PBS (pH 7.4), centrifuging 800 × g for 3min, carefully discarding the supernatant, resuspending in 200 μ L PBS (pH 7.4), and finally placing in a flow cytometer for assay (. lamda.) (λ. assay)ex/em＝405/450nm)。
Figure BDA0002277933130000151
The experimental results are as follows: the experimental results are shown in FIG. 3, and in FIG. 3, the left side is the flow detection experiment of the fluorescent probes BGC-1, BGC-2, BGC-3 and CT26.CL25(Lac Z +) cells under the activation of beta-gal; the right side is the flow detection experiment of fluorescent probes BGC-1, BGC-2 and BGC-3 on CT26.WT (Lac Z-) cells with low expression of beta-gal. As can be seen from FIG. 3, compared with the ordinary fluorescent probe BGC-1 without labeling ability, the fluorescent probes BGC-2 and BGC-3 with covalent bond "anchor point" can realize the recognition of beta-gal overexpression cells, obviously distinguish beta-gal low expression cell strains (CT26.WT) from high expression cell strains (CT26.CL25), and the labeling effect of BGC-2 is obviously better than that of BGC-3.
And (4) experimental conclusion: the flow cytometry experiment can detect the fluorescent labeling effect of a certain number of cells, the result has good statistical significance, and further demonstration shows that the fluorescent probes BGC-2 and BGC-3 can realize the identification and covalent labeling effect on the tumor cells over-expressed by beta-gal at the level of in vitro living cells, so that the dispersion of the fluorescent probes in the cells is reduced, background signals are reduced, the detection sensitivity is improved, and BGC-2 has higher fluorescent intensity than BGC-3.
Example 8
Fluorescence imaging of BGC-2 and analogs thereof in senescent cells
The experimental conditions are as follows: incubating fluorescent probes BGC-1, BGC-2 (5. mu.M) with aged HeLa cells (aged 5 days with camptothecin (15 nM)) and normal HeLa, respectively, in a confocal dish (35mm) for 2h, then discarding the medium containing the probes and washing three times with PBS (pH 7.4) to remove probe molecules that do not establish covalent links to the membrane surface; 1mL of sterile PBS (pH 7.4) was added and cell imaging was performed using a fluorescence microscope (λ)ex＝360±20nm,λem425 nm). And (5) performing fluorescence imaging photographing.
Figure BDA0002277933130000152
The experimental results are as follows: the experimental results are shown in FIG. 4, the left group 2 is the labeling experiment of the fluorescent probes BGC-1 and BGC-2 and aged HeLa cells on the cells under the activation of beta-gal; the right 2 group is fluorescent probe BGC-1, BGC-2 is used for labeling experiment of normal HeLa cells. In the figure, "FL" is a blue fluorescence signal; BF is cell morphology under white light irradiation; scale bar 50 μm. As can be seen from FIG. 4, the fluorescent probe BGC-2 with covalent bond "anchor point" can realize the detection of beta-gal in senescent cells and obviously distinguish senescent cells from normal tumor cells compared with the ordinary fluorescent probe BGC-1 without labeling capability.
And (4) experimental conclusion: the fluorescent probe BGC-2 can realize the functions of identification and covalent labeling of aged tumor cells at the level of in vitro living cells, thereby reducing the dispersion of the fluorescent probe in a cell imaging experiment, reducing background signals and improving the detection sensitivity.
Example 9
Wash-free fluorescence imaging of CP-2 and analogs thereof in living cells
The experimental conditions are as follows: performing real-time wash-free fluorescence imaging on fluorescent probes CP-1 and CP-2(5 μ M) and HeLa cells and HEK293 cells in a confocal culture dish (35mm), or pre-treating HeLa cells with p-BTO ((-) -p-bromotramisone oxalate, an ALP inhibitor) for 0.5h, incubating with CP-2, and performing wash-free fluorescence imaging (λ -fluorescence imaging) with fluorescence microscopeex＝360±20nm,λem＝425nm)。
Figure BDA0002277933130000161
The experimental results are as follows: the experimental results are shown in FIG. 5, the upper group 2 is the washing-free labeling experiment of fluorescent probe CP-1, CP-2 and HeLa cells on the cells under ALP activation; the left side of the lower part is a washing-free labeling experiment of the fluorescent probe CP-2 and the inhibitor pretreated HeLa cells; the lower right part is the washing-free labeling experiment of the fluorescent probe CP-2 and HEK293 cells with low expression of ALP. In the figure, "FL" is a blue fluorescence signal; "BF" is the morphology of cells under white light irradiation; scale bar 10 μm. As can be seen from FIG. 5, the fluorescent probe CP-2 with covalent bond "anchor" can gradually aggregate on HeLa cells overexpressing ALP with time to achieve the effect of wash-free real-time fluorescence detection, while the ordinary fluorescent probe CP-1 without labeling ability gradually disperses on the cells with time to present a higher fluorescence background, which cannot be accurately positioned on tumor cells. In addition, in the ALP inhibitor group experiment, the fluorescent probe CP-2 did not detect a significant fluorescent signal in the HeLa cells in which ALP was inhibited; in the negative control group, no significant fluorescence signal was detected for the fluorescent probe CP-2 in HEK293 cells with low ALP expression, indicating that initiation of covalent labeling with the fluorescent probe is dependent on ALP-specific hydrolysis to induce a process for producing QM intermediates. The CP-2 can effectively and accurately realize real-time fluorescence detection of positive cells in a wash-free mode on the in vitro living cell layer closer to the physiological environment.
And (4) experimental conclusion: the fluorescent probe CP-2 can realize the identification and washing-free real-time fluorescent detection of tumor cells at the level of in vitro living cells, thereby reducing the dispersion of the fluorescent probe in cell imaging experiments, reducing background signals and improving the detection sensitivity.
Example 10
Cellular fluorescence imaging of GC-2 and analogs thereof
The experimental conditions are as follows: incubating fluorescent probes GC-1 and GC-2(200 mu M) with U87MG cells over-expressing GGT enzyme and HEK293 cells of normal tissues with low expression of GGT enzyme in a confocal culture dish (35mm) for 1h respectively; or U87MG cells were pretreated with GGs Top (GGT enzyme inhibitor) for 0.5h in advance and incubated with different probes for 1 h. The probe-containing medium was then discarded and washed three times with PBS (pH:7.4) to remove probe molecules that did not establish covalent links to the cells; 1mL of sterile PBS (pH:7.4) was added, and cell imaging was performed using a fluorescence microscope (. lamda.)ex＝360±20nm,λem＝425nm)。
Figure BDA0002277933130000171
The experimental results are as follows: the results are shown in FIG. 6, the left column 3 shows the positive cell labeling experiment of fluorescent probes GC-1 and GC-2 on GGT positive cells (U87MG) and inhibitor pretreatment; the right column 2 is the labeling experiment of the fluorescent probe GC-1, GC-2 on HEK293 cells with low expression of GGT. "FL" in FIG. 6 is the blue fluorescence signal; "BF" is the morphology of cells under white light irradiation; GGs Top is a commercial GGT enzyme inhibitor-Scale bar 25 μm. Compared with the ordinary enhanced fluorescent probe GC-1 without the labeling capability, the enhanced fluorescent probe GC-2 with covalent bond 'anchor point' can realize the identification of GGT over-expression cells and obviously distinguish GGT low-expression cell strains (HEK293) from high-expression cell strains (U87 MG); and the QM active intermediate formed after the GGT hydrolyzes the probe can be used for realizing 'anchoring' on positive cells. In addition, in the experiment of GGT inhibitor group, the fluorescent signal of the fluorescent probe GC-2 to GGT inhibited U87MG cell marker is weakened, which shows that the initiation of the covalent labeling effect of the fluorescent probe depends on the specific hydrolysis of GGT to induce the process of producing QM intermediate. The GC-2 can effectively and accurately detect the target enzyme and mark positive cells at the in vitro living cell layer closer to the physiological environment.
And (4) experimental conclusion: the fluorescent probe GC-2 can realize the identification and accurate positioning effects on tumor cells over-expressed by GGT at the level of in vitro living cells, thereby reducing the dispersion of the fluorescent probe in cell imaging experiments, reducing background signals and improving the detection sensitivity. By combining the results, the self-anchoring coumarin fluorescent probe GC-2 can more effectively realize covalent labeling of cell membrane surface protein at the cellular level.
In conclusion, the method for introducing the fluoromethyl group at the 3-position of the coumarin can perform fluoromethylation on substrates with different target recognition groups through one-step reaction, and has the advantages of very mild reaction conditions, high selectivity, high yield and good reproducibility.
The coumarin fluorescent probe with the 3-position introduced fluoromethyl group prepared by the preparation method has strong self-anchoring capability and high fluorescence intensity, and the two properties reduce background signals and improve detection sensitivity and imaging definition, so that the fluorescent probe can clearly mark, image and identify cells, and has more promising prospect and practical application value in the fields of biological marking and biological medicine.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (10)

1. A process for the preparation of a compound of formula I, comprising the steps of:
(a) in an inert solvent, in the presence of Eosin Y and an additive, a compound of a formula II and a compound of a formula III react under blue light illumination to obtain a compound of a formula I,
Figure FDA0002277933120000011
wherein the content of the first and second substances,
a is selected from the following group: CH (CH)2F or CHF2；
X is selected from the group consisting of: o, NH or S;
r is selected from the group consisting of: h or an enzyme recognition group;
the enzyme recognition group is a group capable of being recognized by an enzyme and interacting with a targeting recognition group of the enzyme, thereby generating-XH;
R1、R2、R3、R4each independently selected from: hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkenyl, substituted or unsubstituted C1-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, C1-C6 haloalkyl, substituted or unsubstituted C6-C10 aryl, C6-C10 haloaryl, or substituted or unsubstituted C3-C10 cycloalkyl;
unless otherwise specified, the term "substituted" means that one or more hydrogens on the group is replaced with a group selected from the group consisting of: H. -OH, -NH2Halogen, oxo (═ O), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C3 alkoxy, C1-C3 haloalkyl, -COOH, phenyl or benzyl;
m is a cation pair;
and the additive is selected from the group consisting of: an oxidizing agent, an alkali metal salt, an alkaline earth metal salt, or a combination thereof.
2. The method of claim 1, wherein the oxidizing agent is selected from the group consisting of: t-butyl peroxy-alcohol, hydrogen peroxide, sodium periodate, potassium periodate, or a combination thereof, preferably t-butyl peroxy-alcohol.
3. The method of claim 1, wherein the compound of formula I has the structure of formula Ia:
Figure FDA0002277933120000012
wherein R, X, A is as defined above.
4. The method of claim 1, wherein step (a) has one or more of the following characteristics:
(1) the light source of the illumination is a blue LED;
(2) the power of the light source for illumination is 10-50W, preferably 15-30W, more preferably 15-25W;
(3) the reaction time is 2-24h, preferably 12-24h, more preferably 14-18 h;
(4) the reaction temperature is 4-30 ℃, preferably 20 +/-5 ℃; and/or
(5) The inert solvent is selected from: dimethyl sulfoxide, acetonitrile, dichloromethane, water, preferably dimethyl sulfoxide.
5. The method of claim 1, wherein the additive is an oxidizing agent, and the molar ratio of Eosin Y to oxidizing agent is 1:1 to 100, preferably 1: 3-50, preferably 1: 20-45.
6. An enzyme detection fluorescent probe having the structure of a compound of formula I:
Figure FDA0002277933120000021
wherein the content of the first and second substances,
a is selected from the following group: CH (CH)2F or CHF2；
X is selected from the group consisting of O, NH or S;
r is selected from the group consisting of: a beta-galactosidase (beta-gal) recognition group, an alkaline phosphatase (ALP) recognition group, or a glutamyltranspeptidase (gamma-GT, GGT) recognition group,
R1、R2、R3、R4each independently selected from: hydrogen, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkenyl, substituted or unsubstituted C1-C6 alkynyl, substituted or unsubstituted C1-C6 alkoxy, C1-C6 haloalkyl, substituted or unsubstituted C6-C10 aryl, C6-C10 haloaryl, or substituted or unsubstituted C3-C10 cycloalkyl;
unless otherwise specified, the term "substituted" means that one or more hydrogens on the group is replaced with a group selected from the group consisting of: H. -OH, -NH2Halogen, oxo (═ O), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C3 alkoxy, C1-C3 haloalkyl, -COOH, phenyl or benzyl.
7. The fluorescent probe of claim 6, wherein the compound of formula I has the structure of formula Ia:
Figure FDA0002277933120000022
Figure FDA0002277933120000031
wherein R, X, A is as defined above.
8. The fluorescent probe of claim 6, wherein the compound of formula I is selected from the group consisting of:
Figure FDA0002277933120000032
9. a beta-galactosidase assay kit, comprising:
as fluorescent probes
Figure FDA0002277933120000033
And at least one container containing the fluorescent probe.
10. Use of a fluorescent probe according to claim 6 for: (i) labeling, tracking and/or imaging cells that overexpress an enzyme; and/or (ii) preparing a composition for labeling, tracking and/or imaging cells overexpressing an enzyme; and/or (iii) preparing a pharmaceutical composition for treating a disease associated with enzyme overexpression.
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