CN115611882A - Fluorescent compound and preparation method and application thereof - Google Patents

Fluorescent compound and preparation method and application thereof Download PDF

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CN115611882A
CN115611882A CN202110789962.XA CN202110789962A CN115611882A CN 115611882 A CN115611882 A CN 115611882A CN 202110789962 A CN202110789962 A CN 202110789962A CN 115611882 A CN115611882 A CN 115611882A
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compound
fluorescent compound
fluorescent
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quadruplex
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王波林
宋军
彭晓
屈军乐
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Shenzhen University
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Abstract

The invention provides a fluorescent compound and a preparation method and application thereof, wherein the structure of the fluorescent compound is shown as a formula I; wherein A is selected from O,NH or S, R 1 、R 2 Independently selected from H, CH 3 、C 2 H 5 Any one of F, cl, br, carboxyl or sulfonic group, R 3 Selected from any one of H, C1-C11 alkyl, C1-C11 alkoxy, carboxyl, carboxylate, sulfonic group, sulfonate, hydroxyl or C6-C12 aryl, n is an integer of 0-5, X ‑ Selected from any one of iodide ion, bromide ion, chloride ion, tetrafluoroborate ion or perchlorate ion; r when n is 0 3 Is directly connected to N, and R 3 Is- (CH) 2 CH 2 O) m CH 2 CH 2 And (5) OH. The fluorescent compound provided by the invention has high maximum emission wavelength which is more than 900nm, can perform fluorescence enhancement detection on G-quadruplexes, and can be suitable for detection of various samples.

Description

Fluorescent compound and preparation method and application thereof
Technical Field
The invention belongs to the field of fluorescence detection, particularly relates to a fluorescent compound and a preparation method and application thereof, and particularly relates to a fluorescent compound with a large maximum emission wavelength and a preparation method and application thereof.
Background
In recent years, researchers have conducted extensive research around the development of G-quadruplex fluorescent probes, and the G-quadruplex fluorescent probes have also made remarkable progress. However, the reported G-quadruplex organic fluorescent probes are mainly located in a visible region, and the fluorescent probes cannot image deep samples in practical application, are interfered by biological background fluorescent signals and have the problems of high phototoxicity. Fluorescent probes with long wavelengths, particularly those with wavelengths in excess of 900nm, have low phototoxicity, are capable of imaging deeper samples, and are energy efficient in avoiding interference of background fluorescent signals. In addition, the G-quadruplex fluorescent probe with the wavelength of more than 900nm can expand the dimensionality based on a G-quadruplex signal transduction element, and has important value for constructing a nucleic acid sensor for simultaneously detecting multiple analytes. However, there is no report of G-quadruplex fluorescent probes with maximum emission wavelength over 900 nm.
CN108191752A discloses a fluorescent probe for selectively detecting RNA-G-quadruplex in cells, a preparation method and an application thereof. The structure of the fluorescent probe is shown as a formula (I); (ii) a Wherein Ar is aromatic ring or aromatic heterocycle; r1 is fluorine, amino or amine substituent; a is N methylated anion, iodide ion, p-toluenesulfonic acid ion or trifluoromethanesulfonic acid ion. The fluorescent probe provided by the invention can specifically detect and identify the RNA-G-quadruplex in the cell, the detection process is not interfered by other components, and the real-time tracking of the movement process and the folding and unfolding processes of the RNA-G-quadruplex in the living cell is realized; meanwhile, the fluorescent probe is simple in preparation process, low in cost, stable in structure and convenient to store, and has wide application space in research of biological functions of RNA-G-quadruplexes.
CN111995621A discloses a benzindole derivative for a G-quadruplex RNA fluorescent probe and a preparation method and application thereof. The molecular structure of the benzindole derivative is shown as the formula (I): wherein R is selected from hydroxyl, amido, carboxyl or phosphate, n is the number of carbon atoms, and n =1-6. The benzindole derivative can be used as a fluorescent probe to dye G-quadruplex RNA in cells, is not interfered by other components in the detection process, has the characteristics of high fluorescence intensity and strong anti-interference capability, and can specifically identify the G-quadruplex RNA in vivo and in vitro; meanwhile, the benzindole derivative has the advantages of simple preparation process, low cost, stable structure and convenient storage, and has wide application prospect in research of biological functions of G-quadruplex RNA.
As no G-quadruplex fluorescent probe with the maximum emission wavelength of more than 900nm exists at present, the G-quadruplex fluorescent probe with the maximum emission wavelength of more than 900nm has important value for constructing a nucleic acid sensor for simultaneously detecting multiple analytes. Therefore, how to provide a fluorescent probe with a large maximum emission wavelength is a problem to be solved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a fluorescent compound and a preparation method and application thereof, and particularly provides a fluorescent compound with a large maximum emission wavelength and a preparation method and application thereof. The fluorescent compound provided by the invention has high maximum emission wavelength which is more than 900nm, can perform fluorescence enhancement detection on the G-quadruplex, can perform imaging on a deep sample G-quadruplex, and can be suitable for detection of various samples.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a fluorescent compound, wherein the structure of the fluorescent compound is represented by formula I:
Figure BDA0003160699930000021
Figure BDA0003160699930000031
wherein A is selected from any one of O, NH or S, R 1 、R 2 Independently selected from H, CH 3 、C 2 H 5 Any one of F, cl, br, carboxyl or sulfonic group, R 3 Selected from any one of H, C1-C11 alkyl, C1-C11 alkoxy, carboxyl, carboxylate, sulfonic group, sulfonate, hydroxyl or C6-C12 aryl, n is an integer of 0-5, such as any one of 0, 1, 2, 3, 4 or 5, X - Selected from any one of iodide ion, bromide ion, chloride ion, tetrafluoroborate ion or perchlorate ion.
R when n is 0 3 Is directly linked to N, and R 3 Is- (CH) 2 CH 2 O) m CH 2 CH 2 OH and m is selected from any one of 1, 2 or 3.
Wherein C1-C11 respectively mean that the structure contains one carbon atom, two carbon atoms, three carbon atoms and the like, and the description is omitted for the sake of brevity.
The fluorescent compound with the specific structure has the maximum emission wavelength which is higher than 900nm, can perform fluorescence enhancement detection on the G-quadruplex, and can image the deep sample G-quadruplex; the method can be used for constructing a nucleic acid fluorescence sensor with long-wave signal output, and is suitable for detection of various samples; the fluorescent probe can be used together with other fluorescent probes with the maximum emission wavelength below 900nm to detect a plurality of samples simultaneously, so that the time is saved.
Preferably, the structure of the fluorescent compound comprises
Figure BDA0003160699930000032
In a second aspect, the present invention provides a method for preparing a fluorescent compound as described above, comprising the steps of: and mixing the compound A and the compound B with alkali for reaction to obtain the fluorescent compound.
The reaction formula is shown as formula II:
Figure BDA0003160699930000041
wherein A and R 1 、R 2 、R 3 、n、X - Having the same limitations as described above.
Preferably, the molar ratio of compound a to compound B is 1 to 5, preferably 1.2.
Preferably, the base comprises an organic base and/or an inorganic base.
Preferably, the organic base comprises any one of triethylamine, diisopropylethylamine, pyridine, N-dimethylaminopyridine, N-dimethylaminoaniline, 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU), or 1, 4-diazabicyclo [2.2.2] octane (DABCO), or a combination of at least two thereof.
Preferably, the inorganic base comprises any one of sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydride or potassium fluoride or a combination of at least two thereof.
Preferably, the molar ratio of compound a to base is 1.
Preferably, the reaction temperature is 40-200 ℃ and the reaction time is 0.5-18h.
Wherein the molar ratio of compound a to compound B can be 1, 1.2.
In a third aspect, the present invention provides the use of a fluorescent compound as described above for the detection of G-quadruplexes.
In a fourth aspect, the present invention provides a signalling platform comprising a fluorescent compound as described above and a G-quadruplex precursor.
In a fifth aspect, the present invention also provides an application of the signal transduction platform as described above in detecting an analyte Y, where the analyte Y includes any one of a metal ion, a small molecule compound, a large molecule compound or a nucleic acid sequence.
Preferably, the metal ions comprise Ag + 、Hg 2+ 、Pb 2+ 、K + 、Cu 2+ 、Tb 3+ 、UO 2 2+ 、Mg 2+ Or Sr 2+ Any one of them.
Preferably, the small molecule compound comprises any one of ATP, AMP, or cocaine.
Preferably, the macromolecular compound comprises any one of thrombin, nucleolin, HIV-1 integrase, ribonuclease, uracil DNA glycosylase, glucose oxidase or tyrosine kinase.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a fluorescent compound with a specific structure, the maximum emission wavelength of the fluorescent compound is high and exceeds 900nm, the fluorescent compound can carry out fluorescence enhancement detection on a G-quadruplex, and a deep sample G-quadruplex can be imaged; the method can be used for constructing a nucleic acid fluorescence sensor with long-wave signal output, and is suitable for detection of various samples; the fluorescent probe can be used together with other fluorescent probes with the maximum emission wavelength below 900nm to simultaneously detect a plurality of samples, so that the time is saved; the method can be used for simultaneously extracting various information in a complex biological system, and is favorable for deeply and accurately researching a life system; detection of multiple analytes can also improve the accuracy of disease diagnosis.
Drawings
FIG. 1 is a graph of the fluorescence titration spectrum of the fluorescent compound provided in example 1 for G-quadruplexes;
FIG. 2 is a graph of the fluorescence spectrum of the fluorescent compound 1 provided in example 1 for a single-stranded DNA FM2 sequence;
FIG. 3 is a graph of the fluorescence spectrum of the fluorescent compound 1 provided in example 1 for a double-stranded DNA D26 sequence;
FIG. 4 shows fluorescent Compound 1 provided in example 1 with 5mM K added to different concentrations of Oligo-3 + The ratio result of the fluorescence intensity before and after the fluorescence intensity is shown;
FIG. 5 is a schematic representation of the gradual addition of K to fluorescent compound 1 provided in example 1 in the presence of Oligo-3 + Fluorescence titration spectrogram of (1).
Detailed Description
The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
This example provides a fluorescent compound 1 prepared according to the following reaction scheme:
Figure BDA0003160699930000071
the method comprises the following specific steps:
N 2 under protection, a mixture of compound 1-A (88mg, 0.36mmol), compound 1-B (200mg, 0.30mmol), triethylamine (0.4mL, 2.9mmol) and N, N-dimethylformamide (20 mL) was reacted at 90 ℃ for 2.0h. DMF was removed under reduced pressure and purified by silica gel column separation (volume ratio dichloromethane: methanol =10 = 1) to obtain 127mg of a dark blue solid (yield 63.5%).
The characterization data are as follows: 1 H NMR(400MHz,DMSO-d 6 )δ:8.50(d,J＝13.9Hz,1H),8.20(d,J＝7.4Hz,1H),8.09(d,J＝8.0Hz,1H),,8.01(d,J＝7.88Hz,1H),7.93(d,J＝8.00Hz,1H),7.85(t,J＝7.6Hz,1H),7.66(s,1H),7.55(d,J＝7.8Hz,1H),7.51(d,J＝7.1Hz,1H),7.46(t,J＝5.9Hz,1H),7.42–7.28(m,2H),7.25(s,1H),7.05(s,1H),6.68(d,J＝14.0Hz,1H),4.27(q,J＝7.2Hz,2H),2.83(t,J＝6.2Hz,4H),1.93(t,J＝6.1Hz,2H),1.32(t,J＝7.2Hz,3H))。
the successful synthesis of fluorescent compound 1 was demonstrated.
And (3) testing the detection effect:
dissolving 4.3mg of a fluorescent compound in 1347 mu L of chromatographic pure DMSO to prepare a 5mM DMSO stock solution; add 2.0. Mu.L of DMSO stock solution to a fluorescent cuvette containing 2.0mL of 50mM Tris-HCl (pH =7.2, 50mM KCl) buffer and mix well; mu.M of the G-quadruplex solution (5. Mu.L each) was gradually added dropwise, and the fluorescence spectrum at 20 ℃ was measured under 938nm wavelength excitation with a slit width of 5nm and 5nm, respectively (Fluorolog-3, horiba Jobin Yvon, france).
The above test was performed on fluorescent compound 1 provided in example 1, and the results are shown in fig. 1.
It can be seen from the figure that the emission wavelength of the fluorescent compound provided by the invention is significantly higher than 900nm, the maximum emission wavelength reaches about 1000nm, and the fluorescence intensity is continuously enhanced along with the continuous increase of the concentration of the G-quadruplex, thereby showing the effective detection of the G-quadruplex.
And (3) selective testing:
dissolving 4.3mg of a fluorescent compound in 1347 mu L of chromatographic pure DMSO to prepare a 5mM DMSO stock solution; add 2.0. Mu.L of DMSO stock solution to a fluorescent cuvette containing 2.0mL of 50mM Tris-HCl (pH =7.2, 50mM KCl) buffer and mix well; 5.0. Mu.M of single-stranded DNA FM2 or double-stranded DNA D26 was added, fluorescence emission spectra before and after the reaction were measured, the excitation emission slit was 5nm, and the excitation wavelength was 938nm.
The fluorescent compound 1 provided in example 1 was subjected to the above-described test, and the results are shown in FIGS. 2 to 3.
Single-stranded DNA FM2 sequence: 5 'CTCCACACTCACACACTACTC-3';
double-stranded DNA D26 sequence: from the complement sequence 5 'CAATCGGATGAATTCGATTG-3'.
It can be seen from the figure that the fluorescent compound provided by the invention has no obvious detection effect on single-stranded and double-stranded DNA sequences, and the change of the concentration of the fluorescent compound hardly causes the change of the fluorescence intensity, which indicates that the fluorescent compound provided by the invention has better selectivity relative to the single-stranded and double-stranded DNA sequences.
Constructing and testing a signal transduction platform:
construction of Signal transduction platform for K Using fluorescent Compound 1 provided in example 1 as raw Material + And (6) detecting.
Oligo-3 sequence: 5 'TGAGGGGAGGG 3'.
Without K + In the presence of the enzyme, oligo-3 cannot form a G-quadruplex structure in Tris-HCl buffer solution with the pH value of 7.2; when K is + When present, two Oligo-3 sequences can assemble to form a G-quadruplex structure, resulting in enhanced fluorescence of Compound 3, and thus, based on this principle, binding of Oligo-3 to fluorescent Compound 1 can achieve K + Detection of (3).
The concentration of the immobilized fluorescent compound 1 was 3.0. Mu.M, and Oligo-3 was added to each of the different concentrations, and these solutions were measured at the time of addition of 5mM K + The fluorescence spectra before and after the addition were compared to each other to obtain data, and the results are shown in FIG. 4.
It can be seen from the figure that there is the greatest fluorescence enhancement factor when the concentration of Oligo-3 is 4.0. Mu.M, and therefore, the system of 3.0. Mu.M fluorescent compound 1 and 4.0. Mu.M Oligo-3 is used as K + A signaling platform.
To a buffer solution of 3.0. Mu.M fluorescent compound 1 and 4.0. Mu.M Oligo-3 in Tris-HCl (pH = 7.2), K was gradually added + And measuring the fluorescence spectrum. The excitation emission slits are respectively 5nm, and the excitation wavelength is 938nm. The results are shown in FIG. 5.
As can be found from the figure, the signal transduction platform constructed by the compound provided by the invention can realize the detection of the sample to be detected, and the fluorescence intensity is increased along with the increase of the concentration of the sample to be detected, thereby showing the correlation; and because the maximum emission wavelength is more than 900nm, the fluorescent probe can be used together with other fluorescent probes with the maximum emission wavelength less than 900nm to simultaneously detect various samples, thereby saving time.
The applicant states that the present invention is illustrated by the above examples to show the standard substance for ultraviolet and visible wavelength calibration of the present invention, and the preparation method and application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims (10)

1. A fluorescent compound having the structure of formula I:
Figure FDA0003160699920000011
wherein A is selected from any one of O, NH or S, R 1 、R 2 Independently selected from H, CH 3 、C 2 H 5 Any one of F, cl, br, carboxyl or sulfonic group, R 3 Selected from any one of H, C1-C11 alkyl, C1-C11 alkoxy, carboxyl, carboxylate, sulfonic group, sulfonate, hydroxyl or C6-C12 aryl, n is an integer of 0-5, X - Selected from any one of iodide ion, bromide ion, chloride ion, tetrafluoroborate ion or perchlorate ion;
r when n is 0 3 Is directly linked to N, and R 3 Is- (CH) 2 CH 2 O) m CH 2 CH 2 And OH, m is selected from any one of 1, 2 or 3.
2. The fluorescent compound of claim 1, wherein the structure of the fluorescent compound comprises
Figure FDA0003160699920000012
3. A method for preparing a fluorescent compound according to claim 1 or 2, comprising the steps of: mixing the compound A and the compound B with alkali for reaction to obtain the fluorescent compound;
the reaction formula is shown as formula II:
Figure FDA0003160699920000021
wherein A and R 1 、R 2 、R 3 、n、X - Having the same limits as in claim 1.
4. The method for preparing a fluorescent compound according to claim 3, wherein the molar ratio of compound A to compound B is 1.
5. The method of preparing a fluorescent compound according to claim 3 or 4, wherein the base comprises an organic base and/or an inorganic base;
preferably, the organic base comprises any one of triethylamine, diisopropylethylamine, pyridine, N-dimethylaminopyridine, N-dimethylaminoaniline, 1, 8-diazabicyclo [5.4.0] undec-7-ene or 1, 4-diazabicyclo [2.2.2] octane, or a combination of at least two thereof;
preferably, the inorganic base comprises any one of sodium bicarbonate, sodium carbonate, potassium carbonate, sodium hydride or potassium fluoride or a combination of at least two thereof;
preferably, the molar ratio of compound a to base is 1.
6. A method of preparing a fluorescent compound according to any of claims 3 to 5, where the reaction is carried out at a temperature of 40 to 200 ℃ for a period of 0.5 to 18h.
7. Use of a fluorescent compound according to any one of claims 1 to 3 for the detection of G-quadruplexes.
8. A signaling platform comprising the fluorescent compound of any one of claims 1-3 and a G-quadruplex precursor.
9. The use of the signal transduction platform of claim 8 for detecting an analyte Y, wherein the analyte Y comprises any one of a metal ion, a small molecule compound, a large molecule compound or a nucleic acid sequence.
10. Use according to claim 9, wherein the metal ions comprise Ag + 、Hg 2+ 、Pb 2+ 、K + 、Cu 2+ 、Tb 3+ 、UO 2 2+ 、Mg 2+ Or Sr 2+ Any one of them;
preferably, the small molecule compound comprises any one of ATP, AMP, or cocaine;
preferably, the macromolecular compound comprises any one of thrombin, nucleolin, HIV-1 integrase, ribonuclease, uracil DNA glycosylase, glucose oxidase or tyrosine kinase.
CN202110789962.XA 2021-07-13 2021-07-13 Fluorescent compound and preparation method and application thereof Pending CN115611882A (en)

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