CN110878094A - Novel coumarin two-photon sulfhydryl amino acid fluorescent probe - Google Patents
Novel coumarin two-photon sulfhydryl amino acid fluorescent probe Download PDFInfo
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- CN110878094A CN110878094A CN201911214372.3A CN201911214372A CN110878094A CN 110878094 A CN110878094 A CN 110878094A CN 201911214372 A CN201911214372 A CN 201911214372A CN 110878094 A CN110878094 A CN 110878094A
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- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
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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 discloses a two-photon fluorescent probe for detecting sulfhydryl amino acid, belonging to the technical field of analytical chemistry. The probe is prepared from the prepared novel coumarin dye and 2, 4-dinitrobenzenesulfonyl chloride in dichloromethane at room temperature. The fluorescent probe provided by the invention has the advantages of easily available preparation raw materials, simple synthesis method, high selectivity, high sensitivity, low detection limit and the like on the sulfhydryl amino acid. The fluorescent probe can be applied to detection of sulfhydryl amino acid in environment or cells.
Description
Technical Field
The invention provides a two-photon fluorescent probe for detecting sulfhydryl amino acid, belonging to the technical field of fluorescent probes.
Technical Field
Cysteine (Cys), homocysteine (Hcy) and Glutathione (GSH) are three sulfhydryl amino acids of great importance in cells, also known as biological thiols. They are involved in the redox reaction process in the organism to protect cells and the like from oxidative stress damage, and can also regulate the cellular homeostasis and cellular metabolism in the organism. First, during the growth and development of an organism, the deficiency of cysteine (Cys) leads to growth retardation, hair depigmentation, liver damage. Secondly, the increase of homocysteine (Hcy) content in human plasma is closely related to diseases such as Alzheimer disease, cardiovascular diseases and osteoporosis. Glutathione (GSH) plays an important role in maintaining the reducing environment of cells, including keeping cysteine (Cys) in proteins in a reduced state and protecting cells from oxidative stress by trapping free radicals that easily damage DNA and RNA. Due to the important physiological role of biological thiols, the development of a rapid, sensitive, simple method for detecting biological thiols is very important for academic research and potential disease diagnosis.
At present, high performance liquid chromatography, mass spectrometry, gas chromatography, electrochemical methods and the like can be used for detecting the biological thiol. However, these methods have disadvantages of complicated operation steps, expensive instrument, long response time, short excitation wavelength, and the like. In comparison, the fluorescent probe has the advantages of high sensitivity, simple operation, short detection time and low cost, and can realize real-time visual tracing of cells and living bodies, and can exactly make up the defects of the traditional detection method. Aiming at the defects, the invention develops the two-photon fluorescent probe capable of being used for detecting the sulfhydryl amino acid in the organism, which provides a theoretical basis for the synergistic development of the relevant fields of environment or chemical biology, medicine and the like.
Disclosure of Invention
The invention relates to a two-photon fluorescent probe for detecting sulfhydryl amino acid, which is characterized in that the chemical structural formula of the fluorescent probe
Shown in the figure:
the fluorescent probe for detecting the sulfhydryl amino acid is prepared by the following method:
under the protection of nitrogen and ice bath, 0.31 g of compound II and 0.23 g of 2, 4-dinitrobenzenesulfonyl chloride are dissolved in dry dichloromethane, triethylamine is added, the mixture is stirred at room temperature overnight, the crude product is obtained by vacuum concentration, and the compound I is obtained by column chromatography analysis, wherein the yield is 20%.
The fluorescent probe can be used for simultaneously detecting the sulfhydryl amino acid in the aqueous solution.
Further preferably, the fluorescent probe is used for rapid detection of biological thiol in a mixture of DMSO and PBS buffer (volume ratio of 2: 98) at pH 7.4. The detection limits of the probe relative to cysteine, homocysteine and glutathione are 2.5,1.7 and 0.84 nM respectively.
The fluorescent probe provided by the invention has a rapid response performance to sulfhydryl amino acid in a mixed solution of DMSO and PBS buffer solution (the volume ratio is 2: 98, and the volume ratio is 1 mMCTAB) with a pH value of 7.4. The invention verifies through experiments that the fluorescent probe takes a light source with the wavelength of 480nm as exciting light in a mixed solution of DMSO and PBS buffer solution (the volume ratio is 2: 98,1mM CTAB) with the pH value of 7.4, and the solution hardly emits light. With the addition of the biological thiol, the solution emits strong orange fluorescence at 617nm when excited by 480nm light, and the fluorescence enhancement factor is up to 20 times. The addition of other non-mercaptoamino acids had no effect on the fluorescence of the mercaptoamino acids. Therefore, the fluorescent probe compound of the present invention has high selectivity to biological thiol. In addition, the response of the compound I to the sulfhydryl amino acid can be completed within 2 seconds, and the characteristic of quick response enables the compound I to be used for tracking the sulfhydryl amino acid in cells.
In a mixed solution of DMSO and PBS buffer solution (the volume ratio is 2: 98,1mM CTAB), the concentration of the sulfhydryl amino acid in the fluorescent probe compound I has a linear relation with the fluorescence intensity. The working ranges of the concentration of the probe are 0-160nM, 0-140nM and 0-80nM for cysteine, homocysteine and glutathione, respectively, so that the content of mercaptoamino acid in the solution can be quantitatively determined.
The fluorescent probe can be used for detecting the sulfhydryl amino acid in the cell.
The fluorescent probe can be applied to the detection of the sulfhydryl amino acid in the cell. The specific detection method comprises the following steps: HeLa cells were incubated in compound I (10. mu.M) culture for 10 minutes at 37 ℃ and the cells emitted strong red fluorescence under excitation at 488nm and 880 nm. The compound I can detect endogenous sulfhydryl amino acid in single photon and two-photon modes, and has potential application value in biomedicine and other aspects.
The invention has the beneficial effects that:
the two-photon fluorescent probe for detecting the biological thiol has the following advantages: 1. the probe can rapidly react with biological thiol (completed in 2 seconds), and has strong fluorescence intensity emission (the fluorescence intensity is increased by 20 times); 2. the probe has higher selectivity in the process of detecting the biological thiol; 3. the probe detects endogenous biological thiol in living cells.
Drawings
FIG. 1 is a drawing of Compound I1H NMR spectrum.
FIG. 2 is a graph of the selective fluorescence spectrum of Compound I.
FIG. 3 is a fluorescence spectrum of compound I with cysteine concentration under 480nm excitation.
FIG. 4 is a photograph of the cellular image of Compound I with cysteine under 488nm laser excitation.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but is not limited thereto. The various materials in the examples were purchased from the market.
EXAMPLE 1 Synthesis of Compound I
Under nitrogen atmosphere and ice bath, adding 2, 4-dinitrobenzenesulfonyl chloride and compound II into 20mL of dry dichloromethane, and dropwise adding triethylamine. Stirring at room temperature overnight, after the reaction is finished, vacuum concentrating, and performing column chromatography separation to obtain a deep red solid compound I. FIG. 1 is a nuclear magnetic diagram of the probe.
EXAMPLE 2 Selective analysis of Compound I
The final concentration of the thiol amino acid was 50. mu.M by adding the thiol amino acid-containing DMSO solution to a 10. mu.M mixture of Compound I in DMSO and PBS buffer (volume ratio: 2: 98,1mM CTAB) at once, and the change in fluorescence intensity of the solution was detected after mixing, and the results are shown in FIG. 2. When the wavelength of the excitation light is 480nm, the probe compound I has strong orange fluorescence at 617nm to glutathione, cysteine and homocysteine, and the probe compound I has no important response to other non-sulfhydryl amino acids. The probe compound I is proved to have excellent selectivity on glutathione, cysteine and homocysteine.
EXAMPLE 3 Probe analysis of response of Compound I to changes in the concentration of mercaptoamino acids
Solutions containing different concentrations of glutathione, cysteine and homocysteine were added to 10. mu.M of a mixture of probe compound I in DMSO and PBS buffer (volume ratio 2: 98,1mM CTAB), and the fluorescence intensity at 617nm was changed regularly with the increase of the amounts of glutathione, cysteine and homocysteine, as shown in FIG. 3. The fluorescence intensity is plotted against the concentration, and the linear relation is shown, so that the concentration of the probe compound I on various sulfhydryl amino acids is detected.
EXAMPLE 4 Probe Compound I detection of Mercapto-amino acids in Living cells
Compound I was added to a culture of Hela cells at 20. mu.M, incubated at 37 ℃ for 10 minutes, and imaged under excitation with excitation light of 880nm, as shown in FIG. 4. Experiments show that the probe compound I can carry out single photon and two-photon imaging on the cell endogenous sulfhydryl amino acid.
Claims (1)
1. A coumarin-based two-photon fluorescent probe specifically responding to sulfhydryl amino acid is characterized in that the chemical structure of the fluorescent probe is shown as formula I
。
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560027A (en) * | 2015-01-13 | 2015-04-29 | 中国科学技术大学 | Fluorescent probe capable of distinguishing and detecting biological mercaptans and preparation method thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560027A (en) * | 2015-01-13 | 2015-04-29 | 中国科学技术大学 | Fluorescent probe capable of distinguishing and detecting biological mercaptans and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| CHENG DONG等: "A novel 3,6-diamino-1,8-naphthalimide derivative as a highly selective fluorescent "turn-on"probe for thiols", 《RSC ADV.》 * |
| MASAFUMI YOSHIDA等: "A highly sensitive,cell-membrane-permeable fluorescent probe for glutathione", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| YICHAO WAN等: "A new xanthene-based fluorescent probe with a red light emission for selectively detecting glutathione and imaging in living cells", 《SPECTROCHIMICA ACTA PART A: MOLECULAR AND BIOMOLECULAR SPECTROSCOPY》 * |
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Application publication date: 20200313 |