阅读说明：本技术 一种硫酸酯酶的比率型双光子荧光探针及其合成方法和应用 (Ratio type two-photon fluorescent probe of sulfatase, synthetic method and application thereof ) 是由 袁林 李伟 张晓兵 尹姝璐 龚向阳 于 2019-08-27 设计创作，主要内容包括：本发明公开了一种硫酸酯酶的比率型双光子荧光探针及其合成方法和应用,所述荧光探针的结构式如下所示：<Image he="736" wi="429" file="DDA0002180995430000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明比率型双光子荧光探针以1,8-萘酰亚胺为双光子荧光基团,以硫酸酯作为酶切位点,构建比率型双光子硫酸酯荧光探针。该探针通过与硫酸酯酶的酶促反应诱导其硫酸酯基团的断裂及离去,分别通过探针及其反应产物的两个不同波长处荧光强度的比值(F<Sub>564</Sub>/F<Sub>451</Sub>)的变化,来检测硫酸酯酶的存在及其活性大小。该特异性探针为一类比率型的双光子荧光探针,其在活性检测过程不易受生物体背景及杂质的干扰,可用于各种活细胞及亚细胞水平及动物组织中硫酸酯酶活性的定量检测。(The invention discloses a ratio type two-photon fluorescent probe of sulfatase, a synthetic method and application thereof, wherein the structural formula of the fluorescent probe is as follows: The ratio type two-photon fluorescent probe of the invention is constructed by taking 1, 8-naphthalimide as a two-photon fluorescent group and taking sulfate as an enzyme cutting site. The probe induces the rupture and the leaving of the sulfate group of the probe through the enzymatic reaction with the sulfatase, and the rupture and the leaving are respectively determined by the ratio (F) of the fluorescence intensities of the probe and a reaction product thereof at two different wavelengths 564 /F 451 ) To detect the presence of sulfatase and the activity thereof. The specific probeThe two-photon fluorescent probe is a ratiometric type two-photon fluorescent probe, is not easily interfered by organism background and impurities in an activity detection process, and can be used for quantitative detection of the activity of the sulfate enzymes in various living cells and subcellular levels and animal tissues.)

1. A ratio type two-photon fluorescent probe of sulfatase is characterized in that the structural formula of the fluorescent probe is shown as (1):

2. A method for synthesizing a ratio type two-photon fluorescent probe of sulfatase is characterized by comprising the following steps:

(1) Heating and refluxing 4-bromo-1, 8-naphthalic anhydride and N- (2-aminoethyl) -4-methylbenzenesulfonamide in ethanol for 8-12 h, cooling, filtering, and drying to obtain a compound 1;

(2) dissolving the compound 1, N-hydroxysuccinamide and potassium carbonate in dimethyl sulfoxide, heating and refluxing for 4-6 h, cooling, pouring into ice water, adjusting the pH value to 5-8 with hydrochloric acid, precipitating a solid, filtering, and drying to obtain a compound 2;

(3) Dissolving the compound 2, triethylamine and dimethylaminopyridine in a dry mixed solution of dichloromethane and acetonitrile, adding trichloroethyl sulfuryl chloride under an ice bath condition, reacting at room temperature for 12-16 hours, and performing column chromatography separation by spin-drying a solvent to obtain a white compound 3;

(4) and dissolving the compound 3, zinc powder and ammonium formate in a mixed solution of dichloromethane and methanol, reacting at room temperature for 1-6 h, and performing column chromatography separation by spin-drying a solvent to obtain the ratio type two-photon fluorescence probe, which is named as probe ERNathS.

3. The method for synthesizing a ratiometric two-photon fluorescent probe for sulfatase according to claim 2, wherein in the step (1), the molar ratio of 4-bromo-1, 8-naphthalic anhydride to N- (2-aminoethyl) -4-methylbenzenesulfonamide is 1 (1-3); the molar volume ratio of the 4-bromo-1, 8-naphthalic anhydride to the ethanol is 1 (5-7) mmol/mL.

4. The method for synthesizing a ratio type two-photon fluorescent probe for sulfatase according to claim 2, wherein in the step (2), the molar ratio of the compound 1 to the N-hydroxysuccinamide is 1 (1-3); the molar volume ratio of the compound 1 to the dimethyl sulfoxide is 1 (2-5) mmol/mL.

5. The method for synthesizing the ratio type two-photon fluorescent probe of the sulfatase according to claim 2, wherein in the step (3), the molar ratio of the compound 2 to the triethylamine is 1 (1-3); the molar ratio of the compound 2 to the dimethylaminopyridine is 1 (0.5-2); the molar volume ratio of the compound 2 to dichloromethane is 1 (2-5) mmol/mL; the molar volume ratio of the compound 2 to the acetonitrile is 1 (1-3) mmol/mL.

6. The method for synthesizing the ratio type two-photon fluorescent probe of the sulfatase according to claim 2, wherein in the step (4), the molar ratio of the compound 3 to the zinc powder is 1 (2-6); the molar ratio of the compound 3 to the ammonium formate is 1 (0.5-3); the molar volume ratio of the compound 3 to dichloromethane is 1 (1-5) mmol/mL; the molar volume ratio of the compound 3 to the methanol is 1 (2-6) mmol/mL.

7. The use of the ratiometric two-photon fluorescent probe of claim 1 for detecting sulfatase.

8. The use of the ratiometric two-photon fluorescent probe of claim 7 for detecting sulfatase, wherein ERNathS is used as a substrate specific for sulfataseThe probe itself is strong blue fluorescence, and emits yellow fluorescence after inducing the leaving of the sulfate group by the enzymatic reaction with the sulfatase, and the ratio (F) of the fluorescence intensity of the reaction product in unit time to the fluorescence intensity of the probe is quantitatively detected₅₆₄/F₄₅₁) To quantitatively determine the activity of sulfatase in different biological systems.

9. The application of the ratio type two-photon fluorescent probe in detecting sulfatase according to claim 7 or 8, wherein the specific method for detecting sulfatase is as follows:

in the system, 1, 8-naphthalimide sulfated derivatives are used as a ratio two-photon probe substrate, the reaction temperature is 20-45 ℃ in a Tri-HCl buffer solution, the incubation pH environment is 3-10, and the reaction time is 0-120 min, so that the corresponding desulfated ester products of the substrates reach the quantitative limit, and the rapid and sensitive detection of the substrates and the products can be realized by adopting an ultraviolet spectrophotometer and a fluorescence detector; fluorescence detection conditions: the excitation wavelength was 405nm and the maximum emission wavelengths were 451nm and 564nm, respectively.

10. The use of the ratiometric two-photon fluorescent probe for detecting sulfatase according to claim 9, wherein the reaction temperature is preferably 37 ℃; the incubation pH environment is preferably 5.

Technical Field

The invention belongs to the technical field of fluorescent probes, and relates to a ratio type two-photon fluorescent probe of sulfatase, a synthetic method and application thereof.

Background

Sulfatase is a hydrolase belonging to the sulfatase family, mainly involved in the catalytic hydrolysis process of aryl sulfate, and can regulate the sulfation level of various biological molecules in human body and bacteria. The regulation process is closely related to hormone regulation in cells, degradation of cell components, regulation of signal pathways, bacterial infection, inflammation and the like. One class of sulfatases is localized to the endoplasmic reticulum, and because of their ability to regulate steroids, this enzyme is also known as steroid sulfatase (STS). STS hydrolyzes to release steroid precursors, affecting hormone-related signaling pathways, and thus is closely associated with the growth and migration of common hormone-dependent tumors (breast, ovarian, endometrial, and prostate). Inactive estrogen sulfate can only be converted to active estrogen by STS-mediated desulfurization and can only bind to estrogen receptors to initiate a series of enhancements in cancer signaling pathways. STS has been widely focused as a target for cancer therapy in recent years due to its high activity in hormone-dependent tumor tissues, and studies on the treatment of hormone-related tumors by STS small molecule inhibitors have been applied to phase I clinics. However, it is difficult to understand the physiological and pathological effects of STS due to the lack of in situ detection tools. Therefore, there is still a need to develop a direct and efficient method for in situ detection of STS in living cells and tissues, which is of great significance in early diagnosis and treatment of tumors.

In recent years, due to the advantages of high spatial and temporal resolution, high sensitivity, high selectivity, non-invasiveness and the like, fluorescence analysis and detection technology has been widely applied to various fields such as biomolecular labeling, enzyme analysis, environmental monitoring, cell staining and clinical test and diagnosis. Based on the probe for commercial sulfatase: modification of 4-methylumbelliferone sulfate, several new fluorescent probes were reported for in vitro detection of sulfatase [ j.s. rush et al, chem biochem,2010,11, 2096-2099; c.h. taiet al, chem.commun.,2014,50, 6116-6119; k.e. beatty et al, proc.natl.acad.sci.u.s.a.,2013,110,12911-12916 ]. However, these probes lack the rate features and subcellular organelle targeting and are therefore difficult to use for quantitative detection of sulfatases (e.g., endoplasmic reticulum sulfatase STS) in living cells or subcellular organelles.

disclosure of Invention

Aiming at the technical problems that the existing probe lacks ratio characteristics and subcellular organelle targeting and is difficult to be used for quantitative detection of sulfatase in living cells or subcellular organelles, the invention aims to provide a ratio type two-photon fluorescent probe of sulfatase, a synthesis method and application thereof. The probe can be used for quantitatively evaluating the activity and distribution of endoplasmic reticulum sulfatase in living cells, and imaging the ratio of deep tumor tissues by using the advantages of two photons, and is particularly suitable for bioanalysis and medical research.

the invention provides a ratio type two-photon fluorescent probe of the sulfatase, which has a structural formula shown as (1):

the fluorescent probe was named: 2- (2- ((4-methylphenyl) sulfonylamino) ethyl) -1, 3-dioxo-2, 3-dihydro-1H-benzo [ de ] isoquinolin-6-yl hydrogen sulfate has the characteristics of easy observation before and after identification, high selectivity and high sensitivity.

The invention provides a synthetic method of a ratio type two-photon fluorescent probe of sulfatase, which comprises the following steps:

(1) Heating and refluxing 4-bromo-1, 8-naphthalic anhydride and N- (2-aminoethyl) -4-methylbenzenesulfonamide in ethanol for 8-12 h, cooling, filtering, and drying to obtain a compound 1;

(2) Dissolving the compound 1, N-hydroxysuccinamide and potassium carbonate in dimethyl sulfoxide, heating and refluxing for 4-6 h, cooling, pouring into ice water, adjusting the pH value to 5-8 with hydrochloric acid, precipitating a solid, filtering, and drying to obtain a compound 2;

(3) Dissolving the compound 2, triethylamine and dimethylaminopyridine in a dry mixed solution of dichloromethane and acetonitrile, adding trichloroethyl sulfuryl chloride under an ice bath condition, reacting at room temperature for 12-16 hours, and performing column chromatography separation by spin-drying a solvent to obtain a white compound 3;

(4) And dissolving the compound 3, zinc powder and ammonium formate in a mixed solution of dichloromethane and methanol, reacting at room temperature for 1-6 h, and performing column chromatography separation by spin-drying a solvent to obtain the ratio type two-photon fluorescence probe, which is named as probe ERNathS.

in the step (1), the molar ratio of 4-bromo-1, 8-naphthalic anhydride to N- (2-aminoethyl) -4-methylbenzenesulfonamide is 1 (1-3); the molar volume ratio of the 4-bromo-1, 8-naphthalic anhydride to the ethanol is 1 (5-7) mmol/mL.

In the step (2), the molar ratio of the compound 1 to the N-hydroxysuccinamide is 1 (1-3); the molar volume ratio of the compound 1 to the dimethyl sulfoxide is 1 (2-5) mmol/mL.

In the step (3), the molar ratio of the compound 2 to triethylamine is 1 (1-3); the molar ratio of the compound 2 to the dimethylaminopyridine is 1 (0.5-2); the molar volume ratio of the compound 2 to dichloromethane is 1 (2-5) mmol/mL; the molar volume ratio of the compound 2 to the acetonitrile is 1 (1-3) mmol/mL.

In the step (4), the molar ratio of the compound 3 to the zinc powder is 1 (2-6); the molar ratio of the compound 3 to the ammonium formate is 1 (0.5-3); the molar volume ratio of the compound 3 to dichloromethane is 1 (1-5) mmol/mL; the molar volume ratio of the compound 3 to the methanol is 1 (2-6) mmol/mL.

In addition, the inventors respectively perform characterization by means of nuclear magnetic resonance hydrogen spectrum, carbon spectrum, mass spectrum, ultraviolet spectrum and the like, and show that the ratiometric two-photon fluorescence probe ERNathS is successfully synthesized.

the invention also provides application of the ratio type two-photon fluorescent probe in detecting sulfatase.

The application of the ratio type two-photon fluorescent probe in detecting the sulfatase adopts the probe ERNathS as a specific substrate of the sulfatase, and the probe is strong blueFluorescence, which is induced by enzymatic reaction with sulfatase to leave the sulfate group and then emits yellow fluorescence, and the ratio of the fluorescence intensity of the reaction product per unit time to the fluorescence intensity of the probe (F)₅₆₄/F₄₅₁) To quantitatively determine the activity of sulfatase in different biological systems.

the specific method for detecting the sulfatase comprises the following steps:

in the system, 1, 8-naphthalimide sulfated derivatives are used as a ratio two-photon probe substrate, the reaction temperature is 20-45 ℃ in a Tri-HCl buffer solution, the incubation pH environment is 3-10, and the reaction time is 0-120 min, so that the corresponding desulfated ester products of the substrates reach the quantitative limit, and the rapid and sensitive detection of the substrates and the products can be realized by adopting an ultraviolet spectrophotometer and a fluorescence detector; fluorescence detection conditions: the excitation wavelength was 405nm and the maximum emission wavelengths were 451nm and 564nm, respectively.

further, the reaction temperature is preferably 37 ℃; the incubation pH environment is preferably 5.

The procedure for the synthesis of the ERNathS probe is as follows:

According to the ERNathS probe, when the sulfatase is not added, the maximum absorption of the probe is 353nm and the fluorescence emission wavelength is 451nm, when the sulfatase is added, the absorption at the 353nm is weakened, the fluorescence at the 451nm is weakened, and the maximum absorption and the maximum emission are red-shifted to 450nm and 564 nm.

as a two-photon ratio type fluorescent probe of high-specificity sulfatase, the compound can be used for detecting the activity of the sulfatase in various living cells, can be quickly positioned in endoplasmic reticulum by virtue of endoplasmic reticulum targeting, and is used for detecting the activity of the endoplasmic reticulum sulfatase.

According to the activity of the endogenous sulfatase in different types of cells, not only normal cells and tumor cells can be distinguished, but also different types of tumor cells can be distinguished, in particular hormone-dependent tumors.

The specific probe is a ratiometric two-photon fluorescent probe, is not easily interfered by organism background and impurities in the activity detection process, and can be used for quantitative detection of the activity of the sulfate enzymes in various living cells and subcellular levels and animal tissues. The fluorescence ratio detection method of the probe substrate and the desulfate metabolite can also be used for rapid screening of sulfatase inhibitors and quantitative evaluation of inhibition capability.

the ratio type two-photon fluorescent probe provided by the invention takes 1, 8-naphthalimide as a two-photon fluorescent group, takes sulfate as an enzyme cutting site, and utilizes the difference that blue fluorescence is changed into yellow fluorescence before and after probe reaction through two-photon excitation in a near infrared region (700-1000 nm) with strong penetrability to construct the ratio type two-photon sulfate fluorescent probe. The probe induces the rupture and the leaving of the sulfate group of the probe through the enzymatic reaction with the sulfatase, and the rupture and the leaving are respectively determined by the ratio (F) of the fluorescence intensities of the probe and a reaction product thereof at two different wavelengths₅₆₄/F₄₅₁) To detect the presence of sulfatase and the activity thereof.

The ratio type two-photon fluorescent probe can image the sulfatase in the deep tumor tissue by utilizing two-photon excitation.

Two-photon fluorescent probes are a technique that uses near-infrared laser pulses for imaging, which has many advantages over single-photon probes: deeper tissue penetration depth, higher spatial resolution, higher signal to magnification ratio, and less photodamage. Therefore, the introduction of the advantages of two photons overcomes the defects of the existing sulfatase probe, and the development of the fluorescent probe of the two-photon ratio type endoplasmic reticulum sulfatase for detecting and monitoring living cells and tissues has great significance.

Compared with the prior art, the beneficial technical effects that have are:

1) the method is cheap and easy to obtain: the compound can be obtained by simple chemical synthesis, the synthetic raw materials are cheap and easy to obtain, the synthetic process is simple and easy to implement, the cost is low, and the popularization is easy;

2) Two-channel ratio detection: the probe itself is strong blue fluorescence and reacts with sulfataseChange to yellow fluorescence, qualitative detection by fluorescence color change, and ratio of fluorescence intensities of two maximum emission wavelengths (F)₅₆₄/F₄₅₁) Can be quantitatively detected;

3) high specificity and high sensitivity: the probe can be specifically hydrolyzed into a yellow fluorescent hydroxynaphthalene amide fluorophore by sulfatase, and the lower limit of detection for quantitatively detecting the sulfatase through a standard curve is 0.002U/mL;

4) Detection of living cells and tissues: the probe can be used for detecting the activity of sulfatase in living cells, and carrying out ratio imaging and three-dimensional imaging on deep tissues of tumor tissues by utilizing a two-photon microscopic imaging technology.

Drawings

FIG. 1 is a drawing of Compound 3¹H NMR spectrum;

FIG. 2 is a drawing of Compound 3¹³A C NMR spectrum;

FIG. 3 shows the fluorescent probe ERNathS¹h NMR spectrum;

FIG. 4 shows the fluorescent probe ERNathS¹³A C NMR spectrum;

FIG. 5 is a high resolution mass spectrum of the fluorescent probe ERNathS;

FIG. 6 is a UV-Vis spectrum of the response of fluorescent probe ERNathS to sulfatase in example 3 (a);

FIG. 7 is a graph showing fluorescence spectra of the response of the fluorescent probe ERNathS to various concentrations of sulfatase in example 3 (a); (b) fluorescence intensity ratio (F) of the fluorescent probe ERNathS in the examples₅₆₄/F₄₅₁) A graph relating to sulfatase at different concentrations;

FIG. 8 is a bar graph showing the selection test of ERNathS for the fluorescent probe in the example, i.e., the fluorescence intensity ratio (F) of the probe₅₆₄/F₄₅₁) The relationship between the protein, enzyme, biomolecule, etc. (1. blank, 2. sulfatase, 3. alkaline phosphatase, 4. apyrase, 5. glutamyl transpeptidase, 6. leucine aminopeptidase, 7. matrix metalloproteinase-2, 8. lipolytic enzyme, 9. tyrosinase, 10. beta-galactosidase, 11. reduced glutathione, 12. cysteine, 13. hydrogen sulfide, 14. vitamin C,15. bovine serum albumin, 16. ferrous ion, 17. peroxideHydrogen, 18 sodium hypochlorite, 19 potassium superoxide, 20 potassium phosphate);

FIG. 9 is an image of the cellular co-localization of the fluorescent probe ERNathS with a commercial subcellular organelle localization agent in an example;

FIG. 10 is a graphic image of cells in which the fluorescent probe ERNathS of the example detects sulfatase in various living cells;

FIG. 11 is a two-photon imaging chart of the fluorescent probe ERNathS in the example for detecting sulfatase in tumor tissues.

Detailed Description