阅读说明：本技术 基于2-苯乙烯基吲哚盐类衍生物长波发射可区分检测Cys/Hcy荧光探针及其应用 (Detection Cys/Hcy fluorescence probe and its application can be distinguished based on 2- styryl indole salts derivative longwave transmissions ) 是由 徐永霞 朱文慧 李秋莹 赵洪雷 周诗怡 钟克利 汤立军 于 2019-09-06 设计创作，主要内容包括：一种基于2-苯乙烯基吲哚盐类衍生物长波发射可区分检测Cys/Hcy荧光探针及其应用,该荧光探针L的结构式为：<Image he="434" wi="518" file="DDA0002193229560000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>该荧光探针L是以2-苯乙烯基吲哚盐为底物,与4-氯-7-硝基苯并-2-氧杂-1,3-二唑、三乙胺,通过温和简单的反应,获得具有长波发射的荧光探针L。该荧光探针L合成步骤简单,长波长发射,响应时间较短,在含水介质中只对Cys/Hcy的有识别,并可区分检测Cys/Hcy,具有高灵敏性,可应用在细胞中作为非疾病诊断和非疾病治疗目的的检测。(One kind can distinguish detection Cys/Hcy fluorescence probe and its application, the structural formula of fluorescence probe L based on 2- styryl indole salts derivative longwave transmissions are as follows: Fluorescence probe L is to obtain the fluorescence probe L with longwave transmissions by mild simple reaction with chloro- 7- nitro benzo -2- oxa- -1, the 3- diazole of 4-, triethylamine using 2- styryl indoles salt as substrate.Fluorescence probe L synthesis step is simple, long wavelength emission, and the response time is shorter, only there is identification to Cys/Hcy in water-bearing media, and detection Cys/Hcy can be distinguished, there is high sensitivity, can be applicable to the detection in cell as non-disease diagnosis and non-disease therapeutic purposes.)

1. a kind of longwave transmissions can distinguish detection Cys/Hcy fluorescence probe, it is characterized in that:

Fluorescence probe L is based on 2- styryl indole salts derivative, and structural formula is as follows:

2. longwave transmissions according to claim 1 can distinguish detection Cys/Hcy fluorescence probe, it is characterized in that:

The longwave transmissions can distinguish detection Cys/Hcy fluorescence probe, and specific synthesis step is as follows:

With CH₂Cl₂For solvent, by compound 1The chloro- 7- nitro benzo -2- oxa- -1,3- diazole of 4- (Cl-NBD), triethylamine is according to molar ratio 1:(1~2): (0.1~0.5) feeds intake, and stirs 6 hours~12 hours at room temperature, After reaction plus water, extraction drying obtain crude product, are purified with silica gel column chromatography, made with methanol and methylene chloride It is separated for eluant, eluent, obtains fluorescence probe L

3. longwave transmissions according to claim 1 can distinguish detection Cys/Hcy fluorescence probe, it is characterized in that: the methanol Volume ratio with methylene chloride is 1:50~1:100.

4. a kind of longwave transmissions as described in claim 1 can distinguish detection Cys/Hcy fluorescence probe distinguish detection Cys and Application in Hcy, it is characterized in that: Cys and Hcy are detected in the buffer solution that volume ratio is the THF and PBS of 2:8, it is described It is diagnosed and non-disease therapeutic purposes using for non-disease.

5. longwave transmissions according to claim 4 can distinguish detection Cys/Hcy fluorescence probe and distinguish detection Cys and Hcy In application, it is characterized in that: pH=7.4, volume ratio be 2:8 THF and PBS buffer solution in, with fluorescence probe L detection When, the fluorescence intensity in 30 seconds at 592nm is remarkably reinforced, and illustrates that there are Cys.

6. longwave transmissions according to claim 4 can distinguish detection Cys/Hcy fluorescence probe and distinguish detection Cys and Hcy In application, it is characterized in that: pH=10, volume ratio be 2:8 THF and PBS buffer solution in, with fluorescence probe L detection when, Fluorescence intensity at 592nm is remarkably reinforced, and illustrates that there are Hcy.

7. a kind of longwave transmissions as described in claim 1 can distinguish detection Cys/Hcy fluorescence probe distinguish detection Cys and Application in Hcy, it is characterized in that: detecting in cell to Cys and Hcy, the application is non-disease diagnosis and non-disease Therapeutic purposes.

Technical field

The present invention relates to one kind based on 2- styryl indole salts derivative longwave transmissions can distinguish detection Cys/Hcy it is glimmering Light probe and its application.

Background technique

Cysteine (Cys), homocysteine (Hcy) and reduced glutathione (GSH) are lifes common in organism Object mercaptan, and play an important role in various physiology and pathologic process, wherein Cys is the precursor of GSH, participates in protein and closes At removing toxic substances and metabolism.Hcy has been confirmed as the independent hazard factor of many diseases, and small raising can lead to (more than 15 μM) Serious homotype Homocysteine.GSH is aoxidized also as intracellular the most abundant mercaptan to anti-oxidation stress and maintenance It plays an important role in former stable state.Three kinds of common classical ways of biological thiol of detection include electrochemical process, colorimetric method, methylene at present Blue laws and gas chromatography.It is compared with the traditional method, fluorescence analysis is at low cost with its, easy to operate, high sensitivity, in real time The advantages that lossless bio-imaging, gets more and more people's extensive concerning.

In recent years, scientific research personnel designs and develops many in vitro and in vivo detection biological thiol (including Hcy, Cys And GSH) fluorescence probe, there is preferable selectivity, such as Org.Biomol.Chem., (2019), 17,1436-1441； Tetrahedron, (2017), 73,6651-6656, but its synthetic route is relative complex；Dyes Pigm.,(2018),158, 151-156；New J.Chem., (2018), 42,18172-18181, but its launch wavelength is shorter；Biosensors and Bioelectronics,(2016),81,341-348；CN106588912；It cannot distinguish between identification Cys/Hcy；Talanta, (2016),146,41-48；Analyst,2019,144,3676-3684；It cannot quickly detect.As it can be seen that the probe of existing report There is certain defect, while they are distinguished and detected, still due to the similitude of structure and respond by Hcy, Cys and GSH It is a challenging task.

Summary of the invention

The technical problem to be solved in the present invention is to provide one kind to be based on 2- styryl indole salts derivative longwave transmissions Detection Cys/Hcy fluorescence probe and its application can be distinguished, the fluorescence probe synthesis step is simple, long wavelength emission, the response time It is shorter, the only identification that has to Cys/Hcy in water-bearing media, and detection Cys/Hcy can be distinguished, there is high sensitivity, can apply As non-disease diagnosis and the detection of non-disease therapeutic purposes in cell.

The technical scheme is that

A kind of longwave transmissions can distinguish detection Cys/Hcy fluorescence probe, and fluorescence probe L is based on 2- styryl indoles Salt derivative, structural formula are as follows:

Further, the longwave transmissions can distinguish detection Cys/Hcy fluorescence probe, and specific synthesis step is as follows:

With CH₂Cl₂For solvent, by compound 1The chloro- 7- nitro benzo -2- oxa- -1,3- of 4- Diazole (Cl-NBD), triethylamine are according to molar ratio 1:(1~2): (0.1~0.5) feeds intake, and stirs 6 hours~12 at room temperature Hour, after reaction plus water, extraction drying obtain crude product, are purified with silica gel column chromatography, with methanol and dichloromethane Alkane is separated as eluant, eluent, obtains fluorescence probe L

The volume ratio of the methanol and methylene chloride is 1:50~1:100.

A kind of longwave transmissions can distinguish detection Cys/Hcy fluorescence probe and distinguish the application in detection Cys and Hcy, special Different place is: detecting in the buffer solution that volume ratio is the THF and PBS of 2:8 to Cys and Hcy, the application is non-disease Disease diagnosis and non-disease therapeutic purposes.

A kind of longwave transmissions can distinguish detection Cys/Hcy fluorescence probe and distinguish the application in detection Cys and Hcy, special Different place is: in pH=7.4, THF the and PBS buffer solution that volume ratio is 2:8, when being detected with fluorescence probe L, in 30 seconds Fluorescence intensity at 592nm is remarkably reinforced, and illustrates that there are Cys.

A kind of longwave transmissions can distinguish detection Cys/Hcy fluorescence probe and distinguish the application in detection Cys and Hcy, special Different place is: in pH=10, THF the and PBS buffer solution that volume ratio is 2:8, when being detected with fluorescence probe L, in 592nm The fluorescence intensity at place is remarkably reinforced, and illustrates that there are Hcy.

A kind of longwave transmissions can distinguish detection Cys/Hcy fluorescence probe and distinguish the application in detection Cys and Hcy, special Different place is: detecting in cell to Cys/Hcy, the application is non-disease diagnosis and non-disease therapeutic purposes.

The beneficial effects of the present invention are:

Fluorescence probe synthesis process is simple, and separating-purifying is easy；Fluorescence probe long wavelength's fluorescence can increase in an aqueous medium Strong identification Cys/Hcy, selectivity and good sensitivity with height, detection limit reach 10^-6mol/L；In pH=6-9 range It is interior quickly to detect, only there is response to Cys in 30 seconds, only has response to Hcy in pH=10, therefore, detection can be distinguished Cys and Hcy；In addition, the fluorescence probe can be used as non-disease diagnosis and non-disease therapeutic purposes are applied in cell and detect In Cys/Hcy.

Detailed description of the invention

Fig. 1 is fluorescence probe L of the present invention¹H NMR spectra；

Fig. 2 is fluorescence probe L of the present invention¹³C NMR spectra；

Fig. 3 is fluorescence probe L and Br of the present invention^-, I^-, NO₂ ^-, CO₃ ^2-, Cys, HCO₃ ^-, Ac^-, HPO₄ ^2-, H₂PO₄ ^-, PO₄ ^3-, CN^-, SCN^-, Hcy, HS^-, SO₄ ^2-, SO₃ ^2-, HSO₃ ^-, HSO₄ ^-, N₃ ^-, S₂O₃ ^2-Act on the fluorescence emission spectrogram of compound of front and back；

Fig. 4 is the fluorescence emission spectrum variation diagram of fluorescence probe L of the present invention and different multiples Cys effect front and back；

Fig. 5 is the fluorescence emission spectrum variation diagram of fluorescence probe L of the present invention and different multiples Hcy effect front and back；

Fig. 6 is the change in fluorescence spectrogram after fluorescence probe L of the present invention adds different anions to add Hcy again；

Fig. 7 is the change in fluorescence spectrogram after fluorescence probe L of the present invention adds different anions to add Cys again；

Fig. 8 is the time response figure of fluorescence probe L identification Hcy of the present invention；

Fig. 9 is the time response figure of fluorescence probe L identification Cys of the present invention；

Figure 10 is the pH response diagram of fluorescence probe L identification Hcy of the present invention；

Figure 11 is the pH response diagram of fluorescence probe L identification Cys of the present invention；

Figure 12 is selection change in fluorescence figure of the fluorescence probe L of the present invention in pH=10；

Figure 13 is the change in fluorescence spectrogram after fluorescence probe L of the present invention adds different anions to add Hcy again in pH=10；

Figure 14 is the change in fluorescence figure that fluorescence probe L of the present invention detects Hcy in cell.

Specific embodiment

Technical solution of the present invention is described in more detail combined with specific embodiments below.