CN109422758B - Near-infrared fluorescence ratio fluorescence probe and preparation method and application thereof - Google Patents
Near-infrared fluorescence ratio fluorescence probe and preparation method and application thereof Download PDFInfo
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0036—Porphyrins
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- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- 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
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- 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 near-infrared fluorescence ratio fluorescence probe and a preparation method and application thereof. The fluorescent probe provided by the invention is a ratio type fluorescent probe, and through double-wavelength ratio contrast detection of a red fluorescent molecule base and a near-infrared dye molecule base which have different fluorescent absorption peaks on probe molecules, data distortion of a fluorescent signal is avoided, the correctness and the accuracy of a measurement result are ensured, and the intracellular mercaptan level can be accurately detected; meanwhile, the fluorescent probe of the invention has low toxicity and is suitable for in vivo detection. Furthermore, the preparation method has simple and convenient steps and simple operation, does not need to use large or complex instruments, and is suitable for large-scale production and preparation.
Description
Technical Field
The invention relates to the field of organic probe molecules, in particular to a near-infrared fluorescence ratio fluorescent probe and a preparation method and application thereof.
Background
The application of fluorescent dyes or fluorescent reagents as molecular probes is one of the development directions that are currently attracting attention in the field of life sciences.
The fluorescence spectrometry has the advantages of high sensitivity, good selectivity, intuitive and accurate obtained information, scientific expression and explanation of the structure, distribution, content, physiological function and the like of a complex sample, so the fluorescence spectrometry is widely applied to the aspects of biological analysis and radiography.
However, many organisms and their tissues emit fluorescence themselves under excitation by visible light, which can seriously interfere with fluorescence detection and imaging of biological samples. For example, the fluorescence wavelength range of serum proteins in plasma is 325-350 nm, and the fluorescence wavelength ranges of reductive Nicotinamide Adenine Dinucleotide Phosphatase (NADPH) and bilirubin in human body are 430-470 nm, which is closer to the fluorescence wavelength of serum proteins, and this also greatly affects the sensitivity and accuracy of fluorescence analysis in visible light region.
Fluorescence detection in the near infrared spectral region is more suitable for biological tissue imaging analysis than fluorescence detection in the visible region. Optical imaging of living tissue is based on the penetration of light energy into the tissue, the depth of such penetration being closely related to the wavelength of the light. The wavelength of the light used for detection is larger than 600nm, and the detection light can penetrate into the tissues by several centimeters, so that some tissues with larger volumes can be imaged, and disease diagnosis can be carried out. The maximum absorption wavelength and the emission wavelength of the near-infrared fluorescent probe are 600-900 nm, so that background interference can be avoided. Therefore, the near-infrared fluorescence detection has obvious superiority in biological sample analysis.
Intracellular sulfhydryl compounds, such as cysteine (Cys), homocysteine (Hcy), Glutathione (GSH), etc., play a very important role in the redox processes of proteins, cells and organisms. If the content of the molecules in cells is abnormal, the health of human beings is adversely affected, diseases such as slow growth, liver injury and skin injury are related to the abnormal content of thiol in the cells, and therefore, the detection of the content of the molecules is of great medical reference value.
Currently, some fluorescent probes for detecting thiols have been reported in the literature. However, most thiol probes detect small molecule thiols by single wavelength fluorescence intensity increase or quenching. Such probes based on changes in fluorescence intensity are very susceptible to other factors such as sample environmental conditions, probe concentration, etc. At present, most thiol probes are short in wavelength, cannot avoid tissue self-absorption and self-fluorescence, are strong in background interference, large in ultraviolet absorption spectrum and fluorescence spectrum overlapping, or are single-wavelength detection, low in signal-to-noise ratio and high in toxicity, and cannot be used for detecting the level of cell thiol.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The fluorescent probe provided by the invention is a ratio type fluorescent probe, and through double-wavelength ratio comparison detection of a red fluorescent molecule base and a near-infrared dye molecule base which have different fluorescent absorption peaks on probe molecules, data distortion of a fluorescent signal is avoided, and the accuracy and the precision of a measurement result are ensured.
The second purpose of the invention is to provide a preparation method of the near-infrared fluorescence ratiometric fluorescent probe, which has simple and convenient steps and simple operation, does not need to use large or complex instruments, and is suitable for large-scale production and preparation.
The third purpose of the invention is to provide the application of the near-infrared fluorescence ratiometric fluorescent probe in the detection of mercaptan.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a near infrared fluorescence ratiometric fluorescent probe, which has the structure as follows: r1-X1-R3-S-S-R4-X2-R2(I)；
Wherein, in the compound (I), R1Is a red fluorescent molecular base, R2Is a near-infrared dye molecule group, and the emission peak of the red fluorescent molecule is overlapped with the absorption peak of the near-infrared dye molecule; r3、R4Respectively and independently C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 arylene or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; x1、X2Independently represent a chemical bond, a linear or branched chain alkylene group of C1-C30, a linear or linear chain substituted alkylene group of C0-C30, an arylene or substituted arylene group of C5-C30, an alkylene aryl, substituted alkylene aryl or arylene alkyl group of C6-C30, a substituted arylene alkyl group, an amide group, an ester group or an imino group; preferably, tetraphenylporphyrin or a derivative thereof; preferably, the near-infrared dye molecule is one of IR-755 and derivatives thereof, IR-780 and derivatives thereof, IR-783 and derivatives thereof, IR-797 and derivatives thereof, IR-806 and derivatives thereof, IR-808 and derivatives thereof, or IR-820 and derivatives thereof.
Preferably, the structure of the near-infrared fluorescence ratio fluorescence probe of the invention is as follows:
wherein, in the compound (IV), R5、R6Respectively and independently C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 aryl or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; x3、X4Independently represent a chemical bond, a linear or branched chain alkylene group of C1-C30, a linear or linear chain substituted alkylene group of C0-C30, an arylene or substituted arylene group of C5-C30, an alkylene aryl, substituted alkylene aryl or arylene alkyl group of C6-C30, a substituted arylene alkyl group, an amide group, an ester group or an imino group; r7、R11Respectively and independently C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 arylene or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; r8、R9、R10、R12、R13、R14、R15、R16、R17、R18、R19Respectively and independently C0-C30 linear chain or branched chain alkyl, C0-C30 linear chain or branched chain substituted alkyl, C5-C30 aryl or substituted aryl, C6-C30 alkylaryl or arylalkyl; m and n are respectively independent integers of 0-4; x5Is F, Cl, Br, or I.
Preferably, in the near infrared ratiometric fluorescent probe compound (IV) of the present invention, R5、R6Respectively and independently are C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene; x3、X4Independently an amide group, an ester group, or an imine group; r7Is arylene or substituted arylene of C5-C30, alkylenearyl, substituted alkylenearyl or arylene of C6-C30Arylalkyl, substituted arylenealkyl; r8、R9、R10Are respectively and independently C5-C30 aryl or substituted aryl, C6-C30 alkylaryl or arylalkyl; r11Is a linear or branched chain alkylene of C0-C30, a linear or branched chain substituted alkylene of C0-C30; r12、R13、R14、R15、R16、R17、R18、R19Respectively and independently C0-C30 linear chain or branched chain alkyl, C0-C30 linear chain or branched chain substituted alkyl; m and n are respectively independent integers of 0-4; x5Cl, Br, or I.
Preferably, the structure of the near-infrared fluorescence ratio fluorescence probe of the invention is as follows:
meanwhile, the invention also provides a preparation method of the near-infrared fluorescence ratio fluorescent probe, which comprises the following steps: mixing and reacting a disulfide compound, a red fluorescent molecule and a near-infrared dye molecule in a solvent to obtain the near-infrared fluorescence ratio fluorescent probe; wherein the disulfide compound has the structure: y is1-R5-S-S-R6-Y2(i)；
Wherein, in the compound (i), R5、R6Respectively and independently C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 arylene or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; y is1、Y2Independently are amino, amino hydrochloride, amino sulfate, hydroxyl, carboxyl, or acyl halide; and/or the structure of the red fluorescent molecule is as follows: r1-Y3(ii)；
Wherein, in the compound (ii), R1Is a red fluorescent molecular base, Y3Is amino, amino hydrochloride, amino sulfate, hydroxyl, carboxyl, or acyl halide; and/or, near infrared dyesThe molecular structure is as follows: r2-Y4(iii) (ii) a Wherein, in the compound (iii), R2Is a near-infrared dye molecule radical, Y4Is Cl, Br, I, amino hydrochloride, amino sulfate, hydroxyl, carboxyl or acyl halide.
Preferably, in the preparation method of the invention, the structure of the raw material disulfide compound is as follows:
Y1-R5-S-S-R6-Y2(i)；
wherein, in the compound (i), R5、R6Respectively and independently C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 arylene or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; y is1、Y2Independently are amino, amino hydrochloride, amino sulfate, hydroxyl, carboxyl, or acyl halide; and/or, the red fluorescent molecule has the following structure:
wherein, in the compound (iv), R7Is C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 arylene or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; y is3Is amino, amino hydrochloride, amino sulfate, hydroxyl, carboxyl, or acyl halide; r8、R9、R10Respectively and independently C0-C30 linear chain or branched chain alkyl, C0-C30 linear chain or branched chain substituted alkyl, C5-C30 aryl or substituted aryl, C6-C30 alkylaryl or arylalkyl;
and/or, the molecular structure of the near-infrared dye is as follows:
wherein, in the compound (v), R11Is C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 arylene or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; y is4Is Cl, Br, I, amino hydrochloride, amino sulfate, hydroxyl, carboxyl, or acyl halide; r12、R13、R14、R15、R16、R17、R18、R19Respectively and independently C0-C30 linear chain or branched chain alkyl, C0-C30 linear chain or branched chain substituted alkyl, C5-C30 aryl or substituted aryl, C6-C30 alkylaryl or arylalkyl; m and n are respectively independent integers of 0-4; x5Is F, Cl, Br, or I.
Preferably, in the process of the present invention, in the compound (i), R5、R6Respectively and independently are C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, Y1、Y2Independently are amino, amino hydrochloride, or amino sulfate; and/or, in the compound (iv), R7Is C5-C30 arylene or substituted arylene, C6-C30 alkylenearyl, substituted alkylenearyl or arylenealkyl, substituted arylenealkyl; r8、R9、R10Are respectively and independently C5-C30 aryl or substituted aryl, C6-C30 alkylaryl or arylalkyl; y is3Is a carboxyl group; and/or, in the compound (v), R11Is a linear or branched chain alkylene of C0-C30, a linear or branched chain substituted alkylene of C0-C30; r12、R13、R14、R15、R16、R17、R18、R19Respectively and independently C0-C30 linear chain or branched chain alkyl, C0-C30 linear chain or branched chain substituted alkyl; y is4Is Cl, Br, or I; m and n are respectively independent integers of 0-4; x5Cl, Br, or I.
Preferably, in the preparation method of the present invention, the compound (i) is
And/or compound (iv) is
And/or compound (v) is
Preferably, the method of the present invention comprises the steps of: and (3) dissolving the compound (i), adding the compound (v) for reaction, adding the compound (iv) for continuous reaction, and purifying a reaction mixed system to obtain the near-infrared fluorescence ratio fluorescent probe.
Furthermore, the invention also provides an application of the near-infrared fluorescence ratio fluorescence probe in detection of mercaptan.
Compared with the prior art, the invention has the beneficial effects that:
(1) in the fluorescent probe provided by the invention, a red fluorescent molecular group is taken as a fluorescent donor, a near-infrared dye molecular group is taken as a fluorescent acceptor, and fluorescence resonance energy transfer is carried out through a disulfide bond; meanwhile, the disulfide bond is used as a reaction site and can be reduced by mercaptan molecules of an object to be detected, the fluorescence resonance energy transfer is stopped, so that the fluorescence spectrum is changed, effective fluorescence detection is realized through the change of the emission ratio under different wavelengths, and the problems of fluorescence signal data distortion and inaccuracy of detection data caused by factors such as probe concentration, excitation light intensity, detection efficiency, photobleaching, probe leakage, cell thickness and the like in single-wavelength fluorescence detection are avoided.
(2) The preparation method is simple, only once synthesis and once purification are needed, meanwhile, no complex reaction operation or higher-price instrument and equipment is needed in the preparation process, and the prepared fluorescent probe has low toxicity and can be used for in-vivo detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a mass spectrometric test chart of the product of example 1;
FIG. 2 shows the viability of cells at different concentrations of IR-TPP;
FIG. 3 is a graph of the effect of IR-TPP on GSH response; wherein a) the fluorescence spectra of IR-TPP at different GSH concentrations; b) fluorescence ratio (F) of IR-TPP at 648nm to 756nm648/F756) Linear relationship to GSH concentration;
FIG. 4 is a graph showing the effect of IR-TPP on GSH detection at the cellular level.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
One of the major problems often faced when fluorescent probes are used in practical applications for detecting analytes in environmental and biological systems is the accuracy of fluorescence measurement, because the fluorescence intensity of fluorescent probes is often affected by a series of factors, such as probe concentration, excitation light intensity, detection efficiency, photobleaching, probe leakage, cell thickness, etc., when detecting analytes. Therefore, when the fluorescence intensity is measured at a single wavelength, the fluorescence signal data is often distorted in practical applications, thereby causing errors in the measurement result. In order to solve the problem of fluorescence signal artifact faced by the fluorescence measurement method based on single-wavelength fluorescence intensity change, the invention provides a near-infrared fluorescence ratio fluorescence probe with a novel structure.
Specifically, the structure of the fluorescent probe provided by the invention is as follows:
R1-X1-R3-S-S-R4-X2-R2(I)；
wherein, in the compound (I), R1Is a red fluorescent molecular base, R2The fluorescent dye is a near-infrared dye molecule group, and the emission peak of the red fluorescent molecule is overlapped with the absorption peak of the near-infrared dye molecule, preferably, strong overlapping exists;
the design of the fluorescent probe is mainly based on a fluorescence resonance energy transfer mechanism, and the main structural segments comprise: a red fluorescent molecular group which can be used as a fluorescent donor, a near-infrared dye molecular group which can be used as a fluorescent acceptor, and a disulfide bond which is used for transferring energy and can react with mercaptan to be detected;
in the actual detection process, the energy of the red fluorescent molecular group is transferred to the red dye molecular group through the disulfide bond, and after the disulfide bond reacts with the mercaptan, the disulfide bond is broken due to the reduction reaction, so that the fluorescence resonance energy transfer is stopped, and the fluorescence spectrum of the fluorescent probe molecule is changed. Further, accurate detection of thiol concentration is achieved by ratiometric calculation of the emission ratios at different wavelengths.
Wherein, in the compound (I), R1Can be as follows: tetraphenyl porphyrin group or a derivative group thereof;
more preferably, the structure of the tetraphenylporphyrin may be:
Further preferably, R is1May be the above-mentioned substituted tetraphenylporphyrinyl group or the above-mentioned substituted tetraphenylporphyrin derivative group;
R2can be as follows: IR-755 and its derivative base, IR-780 and its derivative base, IR-783 and its derivative base, IR-797 and its derivative base, IR-806 and its derivative base, IR-808 and its derivative base, or one of IR-820 and its derivative base.
Among them, IR-755
IR-780(
2- [2- [ 2-chloro-3- [ (1, 3-dihydro-3, 3-dimethyl-1-propyl-2H-indol-2-ylidene) ethylene]-1-cyclohexen-1-yl]Vinyl radical]3, 3-dimethyl-1-propylindolium iodide, CAS No.: 207399-07-3)
IR-783(
2- [2- [ 2-chloro-3- [2- [1, 3-dihydro-3, 3-dimethyl-1- (4-sulfobutyl) -2H-indol-2-ylidene]-ethylene radical]-1-cyclohexen-1-yl]-vinyl radical]3, 3-dimethyl-1- (4-sulfobutyl) -3H-indolium hydroxide inner salt sodium salt, CAS No.: 115970-66-6);
IR-797(
2- [2- [ 2-chloro-3- [2- (1, 3-dihydro-1, 3, 3-trimethyl-2H-indol-2-ylidene) -ethylidene]-1-cyclopenten-1-yl-vinyl]1,3, 3-trimethyl-3H-indolium chloride, CAS number: 110992-55-7);
IR-806(
2- [2- [ 2-chloro-3- [2- [1, 3-dihydro-3, 3-dimethyl-1- (4-sulfobutyl) -2H-indol-2-ylidene]-ethylene radical]-1-cyclopenten-1-yl]-vinyl radical]-3, 3-dimethyl-1- (4-sulfobutyl) -3H-indolium hydroxide inner salt sodium salt);
IR-808
IR-820(
2- [2- [ 2-chloro-3- [ [1, 3-dihydro-1, 1-dimethyl-3- (4-sulfobutyl) -2H-benzo [ e ]]Indol-2-ylidene]-ethylene radical]-1-cyclohexen-1-yl]-vinyl radical]-1, 1-dimethyl-3- (4-sulfobutyl) -1H-benzo [ e]Indolium hydroxide inner salt, sodium salt, CAS No.: 172616-80-7);
R3、R4respectively and independently C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 arylene or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; preferably, R3、R4Respectively and independently are C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene; more preferably, R3、R4Respectively and independently are C1-C12 linear chain or branched chain alkylene, C1-C12 linear chain or branched chain substituted alkylene; further preferably, R3、R4Each independently is a linear or branched alkylene group having from C1 to C6, or a linear or branched substituted alkylene group having from C1 to C6, and examples thereof include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, tert-butylene, pentylene, isopentylene, neopentylene, hexylene, and the like;
X1、X2each independently is a bond (i.e. R)1、R3Directly linked), a linear or branched alkylene group of C1 to C30, a linear or substituted alkylene group of C0 to C30, an arylene or substituted arylene group of C5 to C30, an alkylenearyl group of C6 to C30, a substituted alkylenearyl or arylenealkyl group, a substituted arylenealkyl group, an amide group, an ester group, or an imine group; preferably, X1、X2Are each independently an amide group
Ester group
Or an imine group.
Preferably, the structure of the fluorescent probe compound provided by the invention is as follows:
in the compound (I), R1 is a porphyrin derivative group, R2 is a heptamethine cyanine dye derivative group, namely, a fluorescent probe taking the porphyrin derivative as a fluorescence donor structure and the heptamethine cyanine dye derivative as a fluorescence acceptor structure;
wherein, in the compound (IV), R5、R6Respectively and independently C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 aryl or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; preferably, R5、R6Respectively and independently are C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene; more preferably, R5、R6Respectively and independently are C1-C12 linear chain or branched chain alkylene, C1-C12 linear chain or branched chain substituted alkylene; further preferably, R5、R6Each independently is a linear or branched alkylene group having from C1 to C6, or a linear or branched substituted alkylene group having from C1 to C6, and examples thereof include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, tert-butylene, pentylene, isopentylene, neopentylene, hexylene, and the like;
X3、X4independently represent a chemical bond, a linear or branched chain alkylene group of C1-C30, a linear or linear chain substituted alkylene group of C0-C30, an arylene or substituted arylene group of C5-C30, an alkylene aryl, substituted alkylene aryl or arylene alkyl group of C6-C30, a substituted arylene alkyl group, an amide group, an ester group or an imino group; preferably, X3、X4Are each independently an amide group
Ester group
Or an imino group;
R7is C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 arylene or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; preferably, R7Is C5-C30 arylene or substituted arylene, C6-C30 alkylenearyl, substituted alkylenearyl or arylenealkyl, substituted arylenealkyl; more preferably, R7Is phenylene or substituted phenylene of C6-C12, alkylene phenyl or phenylene alkyl of C7-C13;
R8、R9、R10is C0-C30 linear chain or branched chain alkyl, C0-C30 linear chain or branched chain substituted alkyl, C5-C30 aryl or substituted aryl, C6-C30 alkyl aryl or aryl alkyl; preferably, R8、R9、R10Are respectively and independently C5-C30 aryl or substituted aryl, C6-C30 alkylaryl or arylalkyl; more preferably, R8、R9、R10Is phenylene or substituted phenylene of C6-C12, alkylene phenyl or phenylene alkyl of C7-C13;
R11is C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 arylene or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; preferably, R11Is a linear or branched chain alkylene of C0-C30, a linear or branched chain substituted alkylene of C0-C30; more preferably, R11Is a linear or branched chain alkylene of C0-C12, a linear or branched chain substituted alkylene of C0-C12; further preferably, R11Is a straight chain or branched chain of C0-C6Alkylene radicals, straight-chain or branched substituted alkylene radicals of C0 to C6, e.g. R11May be a chemical bond (i.e. R)11Is C0 alkyl, X4Directly linked to the cyclohexene parent ring structure), methylene, ethylene, propylene, isopropylene, butylene, isobutylene, tert-butylene, pentylene, isopentylene, neopentylene, hexylene, and the like;
R12、R13、R14、R15、R16、R17、R18、R19respectively and independently C0-C30 linear chain or branched chain alkyl, C0-C30 linear chain or branched chain substituted alkyl, C5-C30 aryl or substituted aryl, C6-C30 alkylaryl or arylalkyl; preferably, R12、R13、R14、R15、R16、R17、R18、R19Respectively and independently C0-C30 linear chain or branched chain alkyl, C0-C30 linear chain or branched chain substituted alkyl; more preferably, R12、R13、R14、R15、R16、R17、R18、R19Respectively and independently are C1-C12 linear chain or branched chain alkylene, C1-C12 linear chain or branched chain substituted alkylene; further preferably, R12、R13、R14、R15、R16、R17、R18、R19Each independently is a linear or branched alkylene group having from C1 to C6, or a linear or branched substituted alkylene group having from C1 to C6, and examples thereof include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, tert-butylene, pentylene, isopentylene, neopentylene, hexylene, and the like;
m and n are respectively independent integers of 0-4;
X5is F, Cl, Br, or I.
m and n are respectively independent integers of 0-4;
X5cl, Br, or I.
Still more preferably, the probe compound provided by the present invention has the following structure:
The probe compound is shown as a compound (V), and the most preferable probe compound provided by the invention is composed of three structural fragments, namely porphyrin (TPP), cystamine (containing disulfide bonds), heptamethine cyanine dye (IR 783) and the like;
wherein, the TPP emission peak and the absorption peak of the heptamethine cyanine dye (IR 783) have strong overlap. In the probe IR-TPP molecule, TPP is used as an energy donor, heptamethine cyanine dye (IR 783) is used as an energy acceptor, and cystamine is used as a small molecule and used as a connector, so that energy is transferred from the TPP donor to the heptamethine cyanine dye (IR 783) acceptor to generate Fluorescence Resonance Energy Transfer (FRET). And the disulfide bond is used as a reaction site, and the thiol exchange reaction with the disulfide bond in the presence of thiol molecules causes the termination of FRET process, thereby changing the fluorescence spectrum of the probe IR-TPP molecules. The detection of the intracellular thiol molecule level is realized;
by using the fluorescent probe provided by the invention and adopting a dual-wavelength ratio detection method, the problem that the fluorescence intensity of the fluorescent probe is often influenced by factors such as probe concentration, excitation light intensity, detection efficiency, photobleaching, probe leakage, cell thickness and the like when the fluorescent probe detects an analyte can be solved, the fluorescence signal data distortion generated in practical application is avoided, and the accuracy and the correctness of a measurement result are ensured.
Meanwhile, the invention also provides a preparation method of the probe compound, the method has simpler operation steps, only needs to mix and react the raw materials, and can obtain a product after one-time purification after the reaction.
Preferably, in the present invention, the raw materials for synthesizing the probe compound are: y is1-R5-S-S-R6-Y2(i) I.e., disulfide compounds that provide disulfide bonds;
wherein, in the compound (i), R5、R6Respectively and independently C0-C30 straight chain or branched chain alkyleneLinear or branched substituted alkylene of C0-C30, arylene or substituted arylene of C5-C30, alkylenearyl, substituted alkylenearyl or arylenealkyl, substituted arylenealkyl of C6-C30; preferably, R5、R6Respectively and independently are C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene; more preferably, R5、R6Respectively and independently are C1-C12 linear chain or branched chain alkylene, C1-C12 linear chain or branched chain substituted alkylene; further preferably, R5、R6Each independently is a linear or branched alkylene group having from C1 to C6, or a linear or branched substituted alkylene group having from C1 to C6, and examples thereof include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, tert-butylene, pentylene, isopentylene, neopentylene, hexylene, and the like;
Y1、Y2independently are amino, amino hydrochloride, amino sulfate, hydroxyl, carboxyl, or acyl halide; preferably, Y is1、Y2Independently are amino, amino hydrochloride, or amino sulfate;
and/or, the red fluorescent molecule has the following structure:
wherein, in the compound (iv), R7Is C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 arylene or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; preferably, R7Is C5-C30 arylene or substituted arylene, C6-C30 alkylenearyl, substituted alkylenearyl or arylenealkyl, substituted arylenealkyl; more preferably, R7Is phenylene or substituted phenylene of C6-C12, alkylene phenyl or phenylene alkyl of C7-C13;
Y3is amino, amino hydrochloride, amino sulfate, hydroxyl, carboxylOr an acid halide group; preferably, Y is3Is a carboxyl group;
R8、R9、R10respectively and independently C0-C30 linear chain or branched chain alkyl, C0-C30 linear chain or branched chain substituted alkyl, C5-C30 aryl or substituted aryl, C6-C30 alkylaryl or arylalkyl; preferably, R8、R9、R10Are respectively and independently C5-C30 aryl or substituted aryl, C6-C30 alkylaryl or arylalkyl; more preferably, R8、R9、R10Is phenylene or substituted phenylene of C6-C12, alkylene phenyl or phenylene alkyl of C7-C13;
and/or, the molecular structure of the near-infrared dye is as follows:
wherein, in the compound (v), R11Is C0-C30 linear chain or branched chain alkylene, C0-C30 linear chain or branched chain substituted alkylene, C5-C30 arylene or substituted arylene, C6-C30 alkylene aryl, substituted alkylene aryl or arylene alkyl, substituted arylene alkyl; preferably, R11Is a linear or branched chain alkylene of C0-C30, a linear or branched chain substituted alkylene of C0-C30; more preferably, R11Is a linear or branched chain alkylene of C0-C12, a linear or branched chain substituted alkylene of C0-C12; further preferably, R11Is a linear or branched alkylene group of C0-C6, a linear or branched substituted alkylene group of C0-C6, e.g. R11May be a chemical bond (i.e. R)11Is C0 alkyl, X4Directly linked to the cyclohexene parent ring structure), methylene, ethylene, propylene, isopropylene, butylene, isobutylene, tert-butylene, pentylene, isopentylene, neopentylene, hexylene, and the like;
Y4is Cl, Br, I, amino hydrochloride, amino sulfate, hydroxyl, carboxyl, or acyl halide; preferably, Y is4Is Cl, Br, or I;
R12、R13、R14、R15、R16、R17、R18、R19respectively and independently C0-C30 linear chain or branched chain alkyl, C0-C30 linear chain or branched chain substituted alkyl, C5-C30 aryl or substituted aryl, C6-C30 alkylaryl or arylalkyl; preferably, R12、R13、R14、R15、R16、R17、R18、R19Respectively and independently C0-C30 linear chain or branched chain alkyl, C0-C30 linear chain or branched chain substituted alkyl; more preferably, R12、R13、R14、R15、R16、R17、R18、R19Respectively and independently are C1-C12 linear chain or branched chain alkylene, C1-C12 linear chain or branched chain substituted alkylene; further preferably, R12、R13、R14、R15、R16、R17、R18、R19Each independently is a linear or branched alkylene group having from C1 to C6, or a linear or branched substituted alkylene group having from C1 to C6, and examples thereof include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, tert-butylene, pentylene, isopentylene, neopentylene, hexylene, and the like;
m and n are respectively independent integers of 0-4, for example, m and n are respectively independent 0, 1, 2, 3 or 4;
X5is F, Cl, Br, or I.
Further preferably, the preparation method disclosed by the invention comprises the following raw materials:
Further, the preparation method of the present invention may preferably include the steps of: dissolving the compound (i), adding the compound (v) for reaction, adding the compound (iv) for continuous reaction, and purifying a reaction mixed system to obtain the near-infrared fluorescence ratio fluorescent probe;
more preferably, the compound (v) may be dissolved first and then reacted with the compound (i), and further preferably, the compound (v) may be added dropwise to a solution in which the compound (i) is dissolved to carry out the reaction, preferably under nitrogen protection; then adding the compound (iv) into the reaction system, and continuing the reaction;
the purification steps of the mixed system after the reaction can be preferably referred to as follows: performing rotary evaporation on the mixed system to remove the solvent, and then performing column chromatography purification; more preferably, the eluent is a mixture of dichloromethane and methanol, gradient elution is carried out on the eluent with different concentration ratios, and the obtained eluent is subjected to rotary evaporation to remove the solvent, so that the product is obtained.
Still more preferably, the preparation method of the present invention can be referred to as follows:
adding cystamine dihydrochloride, methanol and triethylamine into a round-bottom flask at room temperature (preferably at the temperature of 20-30 ℃), adding acetonitrile, quickly stirring, and then enabling the solution to become transparent and the cystamine dihydrochloride to be completely dissolved;
then, dissolving IR783 in acetonitrile, dropwise adding the solution into cystamine dihydrochloride solution by using a dropping funnel, heating the solution under the protection of nitrogen, adding TPP (thermoplastic vulcanizate), and continuing to react;
spin off solvent, purify on silica gel column, sample dissolved with dichloromethane, wet load, eluent with dichloromethane: gradient elution is carried out on methanol from 200:1 to 20:1, solvent is removed by rotation, and indigo powdery solid is obtained after concentration, namely the product;
among them, it is preferable that the molar ratio of cystine hydrochloride, TPP and IR783 is 1:1: 1.
Meanwhile, the probe compound provided by the invention has good corresponding capacity to a compound containing a thiol structure, so that the probe compound can be used for detecting the compound. Furthermore, the fluorescent compound of the invention has low toxicity, so the fluorescent compound can be further used for in vivo detection, and the practical application is more convenient.
Example 1
Adding cystamine dihydrochloride (135mg, 0.6mmol), 1.5mL of methanol, 416 mu L of triethylamine and 2mL of acetonitrile into a 25mL round-bottom flask at room temperature, and quickly stirring for about 0.5 hour until the solution becomes transparent and the cystamine dihydrochloride is completely dissolved;
then IR783(
383.4mg, 0.6mmol) was dissolved in 4mL acetonitrile, added dropwise to cystamine dihydrochloride solution using a dropping funnel, heated, reacted at 35 ℃ under nitrogen for 4 hours, and then added with TPP
(
395.4mg,0.6mmol), and the reaction was continued for 4 hours.
The solvent was removed by rotary evaporation, purified on a silica gel column, the sample was dissolved in dichloromethane, wet loaded, eluent dichloromethane: eluting with methanol in a gradient manner from 200:1 to 20:1, removing the solvent by spinning, and concentrating to obtain solid IR-TPP (I-TPP) of indigo powder
79.6mg, 0.057mmol), yield: 9.5 percent.
Example 1 Mass Spectrometry of the product the profile of HRMS (EI) m/z C is shown in FIG. 183H79N8OS2 +(M+):1267.5813，Found 1267.5803。
The reaction step scheme of example 1 is referenced below:
example 2
At room temperature, adding cystamine dihydrochloride (1350mg,6mmol), 10mL methanol, 4160 μ L triethylamine and 15mL acetonitrile into a 25mL round-bottom flask, rapidly stirring for about 2 hours until the solution becomes transparent and the cystamine dihydrochloride is completely dissolved;
IR783(3834mg,6mmol) was then dissolved in 4mL acetonitrile and added dropwise to cystamine dihydrochloride solution using a dropping funnel, reacted at room temperature for 6 hours under nitrogen, then TPP (3954mg,06mmol) was added and the reaction was continued for 6 hours.
The solvent was removed by rotary evaporation, purified on a silica gel column, the sample was dissolved in dichloromethane, wet loaded, eluent dichloromethane: methanol was gradient eluted from 200:1 to 20:1 and concentrated by spin off the solvent to give solid IR-TPP as indigo powder (670.0mg, 0.48mmol), yield: 8.0 percent.
Example 3
At room temperature, adding cystamine dihydrochloride (270mg,1.2mmol), 2mL of methanol, 800. mu.L of triethylamine and 3mL of acetonitrile into a 25mL round-bottom flask, and rapidly stirring for about 0.5 hour until the solution becomes transparent and the cystamine dihydrochloride is completely dissolved;
IR783(776.8mg,1.2mmol) was then dissolved in 4mL acetonitrile and added dropwise to cystamine dihydrochloride solution using a dropping funnel, heated, reacted at 45 ℃ under nitrogen for 4 hours, then TPP (790.8mg,1.2mmol) was added and the reaction was continued for 4 hours.
Rotary evaporation to remove solvent, silica gel column purification, sample dissolution with chloroform, wet loading, eluent chloroform: ethanol was gradient eluted from 100:1 to 5:1 and concentrated by spin off the solvent to give indigo as a powder IR-TPP (226.15mg, 0.114mmol) in yield: 13.5 percent.
Example 4
Adding cystamine dihydrochloride (2700mg, 12mmol), 5mL methanol, 1000 mu L triethylamine and 20mL acetonitrile into a 25mL round-bottom flask at room temperature, quickly stirring for about 1 hour, and then enabling the solution to become transparent and the cystamine dihydrochloride to be completely dissolved;
IR783 (7768mg,12mmol) was then dissolved in 30mL acetonitrile and added dropwise to cystamine dihydrochloride solution using a dropping funnel, heated and reacted at 40 ℃ for 6 hours under nitrogen.
Removing solvent by rotary evaporation, purifying with silica gel column, dissolving sample with chloroform, loading by wet method, eluting with chloroform: methanol was eluted in a gradient from 100:1 to 8:1, and after concentration with spin-off of the solvent, indigo was obtained as a powdery solid (1787mg, 1.28mmol), yield: 10.7 percent.
Experimental example 1
(1) IR-TPP cytotoxicity assay:
taking MCF-7 cells growing in logarithmic phase, preparing single cell suspension with culture solution containing 10% fetal calf serum, laying 6 × 7 rows in 96-well plate, adding 100 μ L culture solution into each well, cell concentration is 5000/well (sterile PBS is filled in the residual marginal well), and culturing at 37 deg.C and 5% CO2Culturing in an incubator until cell monolayers are paved on the bottom of the hole;
then, 6 groups of IR-TPP with different concentrations are respectively added, the concentrations are respectively 0.1, 5, 10, 30, 60 and 90 mu M, each group is provided with 5 multiple holes, after the hole plate is coated by the tinfoil paper, the culture is continued for about 1d in an incubator until the cells grow out adherent to the wall;
then, taking out the pore plate, then sucking out the supernatant, adding a PBS solution for washing, and then sucking out the supernatant;
then, 180. mu.L of fresh RPMI 1640 medium was added to each well of the well plate, and 20. mu.L of 5mg/L MTT solution was added thereto at 37 ℃ with 5% CO2Culturing for 4h in the incubator, and then sucking liquid in the holes;
then, adding 150 mu L of DMSO into each hole, and oscillating on a shaking table at a low speed for 5-15 min to serve as an experimental test group;
meanwhile, a control well (adding cells, culture medium, MTT and dimethyl sulfoxide) and a withering well (adding culture medium, MTT and dimethyl sulfoxide) are respectively arranged;
then, the absorbance of each well was measured at 490nm in an enzyme linked immunosorbent assay (ELISA) monitor, the detection results of each well were recorded, the cell viability was calculated, and the toxicity of the probe was observed by plotting a graph with the concentration of IR-TPP molecules as the horizontal axis and the cell viability as the vertical axis, and the detection results are shown in FIG. 2.
As can be seen from the detection results in FIG. 2, the survival rate of the cells can reach more than 90% at low IR-TPP concentration; meanwhile, the experimental cells can still maintain high survival rate even at high IR-TPP concentration. Therefore, the probe compound provided by the invention has low toxicity and is suitable for in vivo detection.
(2) GSH detection assay
(i) Solution titration experiment:
accurately measuring 1mL of mother liquor with the concentration of 500 mu M of the IR-TPP probe molecule, and preparing 100mL of DMSO/PBS (20mM, pH 7.4, V/V9: l) test solution with the concentration of 10 mu M of the IR-TPP;
taking 0.5mL of test solution each time, adding glutathione solutions with different concentrations, enabling the final concentration of IR-TPP molecules to be 5 mu M and the final concentration of glutathione to be 0-4 mu M in the test solution after glutathione is added, obtaining a plurality of groups of solutions to be tested with different glutathione concentrations, and carrying out fluorescence spectrum detection on the solutions to be tested, wherein the detection result is shown in figure 3.
As can be seen from the detection results in FIG. 3, as the concentration of GSH increases, the fluorescence intensity of the solution to be detected at 648nm decreases and the fluorescence intensity at 756nm increases;
meanwhile, the ratio of the fluorescence intensity of any group of solutions to be detected at 648nm to the fluorescence intensity of any group of solutions to be detected at 756nm is basically consistent with the final concentration of the GSH in the group of solutions to be detected.
Therefore, the IR-TPP probe molecule provided by the invention not only can accurately react with the GSH concentration level in the solution; meanwhile, the GSH concentration value of the solution can be further accurately obtained by measuring the fluorescence intensity ratios of different wavelengths.
(ii) Cell detection experiments:
culturing MCF-7 cells in a DMEM cell culture solution, dissolving probe IR-TPP molecules in DMSO, and preparing a stock solution with the concentration of 50 mM;
the stock solution was diluted to 5. mu.M with a cell culture solution for cell culture. Inoculating cells into a 24-well plate, culturing for 12 hours by using a cell culture solution, observing under a microscope, enabling HeLa cells to adhere to the wall, washing out from the culture solution, soaking cells of an experimental group in a prepared cell culture solution containing 5 mu M of a probe for 30min at 37 ℃, washing for 3 times by using a phosphate buffer solution, and finishing a fluorescence imaging experiment under a Leica TCS SP5 laser confocal microscope to serve as an experimental group;
meanwhile, a control group is set, cells of the control group are firstly soaked in NEM (N-ethylmaleimide, thiol inhibitor) with the concentration of 100mM for 2h, and then are soaked in probe IR-TPP molecular solution with the concentration of 5 mu M for 30min, and the detection result is shown in figure 4.
Wherein, fig. 4(a) and 4(e) are red light channels of the TPP; FIG. 4(b) and FIG. 4(f) show blue light channels of IR-783; FIGS. 4(c) and 4(g) show the states of cells observed in a bright field;
FIG. 4(d) is an overlay of FIGS. 4(a) and 4(c) and FIG. 4(h) is an overlay of FIGS. 4(e) and 4(g), whereby the overlay enables localization of intact cells;
further, as can be seen from the obvious difference between the fluorescence in fig. 4(d) and the fluorescence in fig. 4(h), the IR-TPP probe provided by the present invention has a good detection effect on thiol in Hela cells, and the change of the fluorescence signal is consistent with the result of the GSH titration experiment of IR-TPP in solution.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (4)
1. A near-infrared fluorescence ratiometric fluorescent probe is characterized in that,
the structure of the near-infrared fluorescence ratio fluorescent probe is as follows:
2. the method for preparing a near-infrared fluorescence ratiometric fluorescent probe of claim 1, comprising the steps of:
mixing and reacting a disulfide compound, a red fluorescent molecule and a near-infrared dye molecule in a solvent to obtain the near-infrared fluorescence ratio fluorescent probe;
wherein the disulfide compound has the structure:
the red fluorescent molecular structure is
The near infrared dye has a molecular structure of
3. The method for preparing according to claim 2, characterized in that it comprises the steps of:
and dissolving the disulfide compound, adding the red fluorescent molecule for reaction, continuing the reaction of the near-infrared dye molecule, and purifying a reaction mixed system to obtain the near-infrared fluorescence ratio fluorescent probe.
4. Use of the near infrared fluorescence ratiometric fluorescent probe described in claim 1 for the detection of thiols for non-disease diagnostic and therapeutic purposes.
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