CN102154003A - Novel carbazole-bridge-based fluorescent cyanine dye probe and preparation method thereof - Google Patents

Abstract

The present invention provides a carbazole bridging fluorescent cyanine dye probe, the cyanine dye probe structure includes benzothia(oxa)azole and 4-methyl carbazole bridging group respectively bonded to thiazole orange and oxazole yellow A cyanine dye probe formed between quinoline salts; one end of the 3-position side chain of the carbazole ring is connected to the 2-position carbon of benzothia (oxa) azole, and the 6-position formyl group at the other end of the carbazole is connected to the 4- Vinyl quinolate compound is connected. The invention also provides a preparation method of the carbazole bridging fluorescent cyanine dye probe. The effect of the present invention is that the preparation method of the fluorescent dye biological probe is simple, and the raw materials are easy to obtain, so that the maximum emission wavelength of the newly synthesized fluorescent dye probe is red-shifted, the fluorescence intensity increases, the Stocks shift increases, and the fluorescence quantum yield increases , enhanced photostability. The invention retains the advantages of thiazole orange and oxazole yellow embedded fluorescent dyes, and is beneficial to further improving sensitivity, reducing dosage and improving stability. It has the characteristics of wide spectral range, large molar extinction coefficient, high ratio of two assets and high sensitivity.

Description

Novel fluorescent cyanine dye probe of carbazole bridge group and preparation method thereof

technical field

The invention relates to a fluorescent dye technology starting from the chemical field and applied to the biological field, in particular to a novel fluorescent cyanine dye probe based on a carbazole bridge and a preparation method thereof.

Background technique

As we all know, cancer has become the number one killer that threatens human health, and how to effectively control its occurrence and development has become a top priority. Most scholars believe that in the prevention and treatment of tumors, secondary prevention of tumors, that is, early detection, early diagnosis, and early treatment will be the direction for greatly improving the curative effect of solid tumors for a long period of time in the future. Molecular biomarkers have attracted more and more attention in medical early diagnosis due to their advantages of cheapness, safety and accuracy. At present, this technology has been well applied in the identification of cancer and AIDS.

Compared with traditional isotope detection, the relevant detection methods and technologies of bioluminescent probes in the biological field have the characteristics of fast speed, good repeatability, less consumption and no radiation. Currently, bioluminescent pigment probes used for cell labeling mainly include acridine, phenanthridine, fluorescein, difluoroalkaneboron, stilbene, naphthalimide, and cyanine dyes. As a labeling dye for biomacromolecules, cyanine dyes have the characteristics of fast, sensitive and safe, and have become a new generation of fluorescent labeling dyes for biomolecules. As embedded fluorescent dyes, thiazole orange (TO) and oxazole yellow (YO) have become two important branches of cyanine dyes. There is almost no fluorescence in the state, but the fluorescence is significantly enhanced after being combined with nucleic acid, so that there is no fluorescence interference of the dye itself during fluorescence detection, which improves the detection sensitivity and reduces the complicated steps of removing free dye during the labeling process. However, due to the weak fluorescence intensity of thiazole orange (TO) and oxazole yellow (YO), the shortcomings of the maximum fluorescence emission wavelength and the small Stocks shift restrict the application of these two fluorescent dyes in biomarkers.

The fluorescence properties of fluorescent dye probes are related to factors such as the size of the conjugated system of the probe, the coplanarity and rigidity of the conjugated bond system, and carbazole and its derivatives are an electron-rich system. Conjugate system, strong intramolecular electron transfer ability, special rigid planar fused ring structure, strong fluorescence and easy structural modification of carbazole ring to introduce a variety of functional groups and other advantages are widely used in dyes, biology, medicine, optoelectronic materials and other fields . Therefore, the special rigid ring of carbazole ring is bonded in thiazole orange (TO) or oxazole yellow (YO), which increases the conjugated system and planar rigid structure of the original fluorescent dye, which will be beneficial to the development of fluorescent dyes. The maximum fluorescence emission wavelength is red-shifted, the fluorescence intensity increases, the Stocks shift increases, and the fluorescence quantum yield increases.

Contents of the invention

The purpose of the present invention is to provide a carbazole bridging fluorescent cyanine dye probe and a preparation method thereof, to increase the conjugated system and planar rigid structure of the original fluorescent dye probe, and to facilitate the red shift of the maximum fluorescent emission wavelength of the fluorescent dye , the fluorescence intensity increases, the Stocks shift increases, and the fluorescence quantum yield and photostability are improved.

In order to achieve the above object, the technical solution adopted in the present invention is to provide a carbazole bridging fluorescent cyanine dye probe, wherein: the cyanine dye probe structure includes a carbazole bridging group bonded to thiazole orange (TO) and The cyanine dye probe that forms between the benzothia (ox) azole of azole yellow (YO) and 4-methyl quinoline salt; The 2-carbon of the carbazole is connected, and the 6-formyl group at the other end of the carbazole is connected with the 4-vinyl quinolate compound.

The invention also provides a preparation method of the carbazole bridging fluorescent cyanine dye probe.

Effect of the present invention is:

1. The preparation method of the fluorescent dye biological probe of the present invention is simple, and the raw materials are easily available.

2. The introduction of the carbazole bridging group makes the maximum emission wavelength of the newly synthesized fluorescent dye probe red-shift, the fluorescence intensity increases, the Stocks shift increases, the fluorescence quantum yield increases, and the photostability is enhanced.

3. The present invention retains the advantages of thiazole orange (TO) and oxazole yellow (YO) embedded fluorescent dyes, which will help to further improve sensitivity, reduce dosage and improve stability.

4. It has the characteristics of wide spectral range, large molar extinction coefficient, high dual asset ratio and high sensitivity.

Description of drawings

Fig. 1 is the design structural roadmap of carbazole bridging fluorescent cyanine dye probe of the present invention;

Fig. 2 is the experimental roadmap of the carbazole bridging fluorescent cyanine dye probe of the present invention.

Detailed ways

The novel fluorescent cyanine dye probe of the present invention and its preparation method will be further described in conjunction with the accompanying drawings and examples.

As shown in Figure 1, the novel fluorescent cyanine dye probe based on carbazole bridging group of the present invention is based on carbazole and its derivatives, which is an electron-rich system with a large conjugated system, strong intramolecular electron transfer ability, and special rigidity. Planar fused ring structure, strong fluorescence and easy structural modification to introduce various functional groups and other advantages. Therefore, the special rigid ring of carbazole ring is bonded in the molecular structure of thiazole orange (TO) or oxazole yellow (YO), which increases the conjugated system and planar rigid structure of the original two types of fluorescent dye probes, It will be conducive to the red shift of the maximum fluorescence emission wavelength of the fluorescent dye, the increase of fluorescence intensity, the increase of Stocks shift, and the improvement of fluorescence quantum yield and photostability.

The carbazole bridging fluorescent cyanine dye probe of the present invention, the cyanine dye probe structure includes benzothia (oxa)azole and carbazole bridging group respectively bonded to thiazole orange (TO) and oxazole yellow (YO) A cyanine dye probe formed between 4-methyl quinoline salts; one end of the 3-position side chain of the carbazole ring is connected to the 2-position carbon of benzothia (oxa) azole, and the 6-position formazan at the other end of the carbazole The acyl group is attached to the 4-vinylquinolate compound.

The 4-methylquinoline salt is a 4-methylquinoline salt with ^Br- as an anion, and the groups at the N-position of the 4-methylquinoline salt are -CH ₂ CH ₂ COOH, -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ Br,

The characteristics of the carbazole bridging fluorescent cyanine dye probe:

Fluorescence emission wavelength: 630nm-660nm;

Fluorescence intensity: 30-55;

Stokes shift (Stocks): 130-151nm;

Fluorescence quantum yield: 0.02-0.6.

The preparation method of the carbazole bridging fluorescent cyanine dye probe of the present invention comprises the following steps:

1) 3-benzothiazolyl-6-formyl-N-ethylcarbazole, 3-benzoxazolyl-6-formyl-N-ethylcarbazole, 3-benzoxazolyl-6 Preparation of -formyl-N-benzylcarbazole, 3-benzothiazolyl-6-formyl-N-benzylcarbazole

Add N,N-dimethylformamide (DMF) and phosphorus oxychloride at a ratio of 1:1 to four 100mL round-bottomed flasks respectively, after dropping, stir at room temperature until the reaction system turns reddish. , the amount of substance added dropwise is 3-benzothiazolyl-N-ethylcarbazole, 3-benzoxazolyl-N-ethylcarbazole, 3-benzothiazolyl-N-ethylcarbazole, 3-benzoxazolyl- N-benzylcarbazole, 3-benzothiazolyl-N-benzylcarbazole in 1,2-dichloroethane solution, the total volume is 40-70mL, reflux and stir at the temperature of 79-85°C for 6h -12h, cooled to room temperature, poured into ice water, extracted with dichloromethane, separated and purified by column chromatography to obtain 3-benzothiazolyl-6-formyl-N-ethylcarbazole, 3-benzoxa Azolyl-6-formyl-N-ethylcarbazole, 3-benzoxazolyl-6-formyl-N-benzylcarbazole, 3-benzothiazolyl-6-formyl-N-benzyl base carbazole;

2) Preparation of carbazole bridging fluorescent cyanine dye probes

1 mole of 3-benzothiazolyl-6-formyl-N-ethylcarbazole, 3-benzoxazolyl-6-formyl-N-ethylcarbazole, 3-Benzoxazolyl-6-formyl-N-benzylcarbazole, 3-benzothiazolyl-6-formyl-N-benzylcarbazole and four 1.5 molar N-positions with different Dissolve the 4-methylquinoline salt of the substituent in ethanol, add piperidine equivalent to the 4-methylquinoline salt dropwise, reflux and stir at a temperature of 75-80°C for 8-16 hours, and cool to room temperature , and then add diethyl ether to precipitate a red solid, and filter with suction to obtain a filter cake that is separated by column chromatography to obtain the carbazole bridging fluorescent cyanine dye probe.

The quinolate in the structure of the carbazole bridging fluorescent cyanine dye probe of the present invention is 4 ^- methylquinolate with Br- as the anion, and the groups at the N position are -CH ₂ CH ₂ COOH, - _{CH2CH2CH2CH2CH2COOH , -CH2CH3 , -CH2CH2CH2Br , _ _ _ _}

The preparation method of the carbazole bridging fluorescent cyanine dye probe of the present invention is further illustrated by the following examples, as shown in FIG. 2 , but not limited thereto.

The target compound ae represents that R ₁ is ethyl, X is S, and R ₂ are -CH ₂ CH ₂ COOH, -CH _{2 CH} 2 CH ₂ Br, -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH N-ethylcarbazole bridging thiazole orange dye probes with different substituent groups; fj means that R ₁ is ethyl, X is O, and R ₂ is -CH ₂ CH ₂ COOH, -CH2CH ₂ CH ₂ Br , -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH and other N-ethylcarbazole bridging oxazole yellow cyanine dye probes with different substituent groups; ko means that R ₁ is benzyl, X is S, R ₂ is N-benzyl with different substituent groups such as -CH ₂ CH ₂ COOH, -CH ₂ CH 2 CH ₂ Br, -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH Carbazole bridging thiazole orange dye probe; pt means that R ₁ is benzyl, X is O, and R ₂ are -CH ₂ CH ₂ COOH, -CH2CH ₂ CH ₂ Br, -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH and other N-benzylcarbazole bridging oxazole yellow cyanine dye probes with different substituent groups, the experimental route and target compounds are shown in Table 1:

Table 1 Target compounds a~t

The preparation of embodiment 1N-ethylcarbazole bridging thiazole orange dye probe comprises the following steps:

1.3-Benzothiazolyl-6-formyl-N-ethylcarbazole

Add DMF 7.7mL (100mmol) in a 100mL round-bottomed flask, dropwise add phosphorus oxychloride 9.5mL (15.2g, 100mmol) under an ice-water bath, dropwise, stir at room temperature until the reaction system is reddish, slowly add dropwise 3- A solution of 2 g (6 mmol) of benzothiazolyl-N-ethylcarbazole in 30 mL of 1,2-dichloroethane was added and reacted under reflux at 83° C. for 6 h. After cooling to room temperature, it was poured into ice water, extracted with dichloromethane, and separated by column chromatography to obtain 3-benzothiazolyl-6-formyl-N-ethylcarbazole.

2. Preparation of N-ethylcarbazole bridging thiazole orange dye probe a

Dissolve 0.1g (0.28mmol) of 3-benzothiazolyl-6-formyl-N-ethylcarbazole in 30mL of ethanol, then dissolve 0.13g (0.42mmol) of carboxyquinoline salt in 20mL of ethanol, and Mix the two in a flask, add 0.04mL (0.42mmol) piperidine dropwise, reflux at 78°C for 12h, cool to room temperature, add diethyl ether to precipitate a red solid, filter with suction, and separate by filter cake column chromatography to obtain the red target product a.

取代基的喹啉盐代替上述0.13g(0.42mmol)羧基喹啉盐，重复上述步骤2的化学反应，得本发明中的目标产物b～e。Take 0.42mmol of N-position with -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ Br,

The quinoline salt of the substituent replaced the above 0.13 g (0.42 mmol) carboxyquinoline salt, and the chemical reaction of the above step 2 was repeated to obtain the target products b-e of the present invention.

The preparation of embodiment 2N-benzylcarbazole bridge thiazole orange dye probe comprises the following steps:

1. Preparation of 3-benzothiazolyl-6-formyl-N-benzylcarbazole

Add DMF 7.7mL (100mmol) to the flask, add phosphorus oxychloride 9.5mL (15.2g, 100mmol) dropwise under ice-water bath, after dropping, stir at room temperature until the solution is reddish, slowly add 3-benzothiazolyl - A solution of 2.34g (6mmol) of N-benzylcarbazole in 30mL of 1,2-dichloroethane, after dropping, reflux at 83°C for 16h. After cooling to room temperature, it was poured into ice water, extracted with dichloromethane, and separated by column chromatography to obtain 3-benzothiazolyl-6-formyl-N-benzylcarbazole.

2. Preparation of N-benzylcarbazole bridging thiazole orange dye probe k

Dissolve 0.1g (0.28mmol) of 3-benzothiazolyl-6-formyl-N-benzylcarbazole in 30mL of ethanol, then dissolve 0.13g (0.42mmol) of carboxyquinoline salt in 20mL of ethanol, and The two were mixed and placed in a flask, 0.04mL (0.42mmol) piperidine was added dropwise, refluxed at 78°C for 12h, cooled to room temperature, ether was added to precipitate a red solid, filtered by suction, and filtered by filter cake column chromatography to obtain the red target product k.

取代基的喹啉盐代替上述0.13g(0.42mmol)羧基喹啉盐，重复上述步骤2的化学反应，得本发明中的目标产物l～o。Take 0.42mmol of N-position with -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ Br,

The quinoline salt of the substituent replaces the above-mentioned 0.13g (0.42mmol) carboxyquinoline salt, and repeats the chemical reaction of the above step 2 to obtain the target products 1-o of the present invention.

The preparation of embodiment 3N-ethylcarbazole oxazole yellow cyanine dye probe comprises the following steps:

1. Preparation of 3-benzoxazolyl-6 formyl-N-ethylcarbazole

Add DMF 7.7mL (100mmol) in the flask, dropwise add phosphorus oxychloride 9.5mL (15.2g, 100mmol) under ice-water bath, dropwise, stir at room temperature until the solution is reddish, slowly add 3-benzoxazole dropwise A solution of 2.25g (6mmol) of N-ethylcarbazole in 30mL of 1,2-dichloroethane was added, and the solution was refluxed at 83°C for 16h. After cooling to room temperature, it was poured into ice water, extracted with dichloromethane, and separated by column chromatography to obtain 3-benzoxazolyl-6-formyl-N-ethylcarbazole.

2. Preparation of N-ethylcarbazole oxazole yellow cyanine dye probe f

Dissolve 0.11g (0.28mmol) of 3-benzoxazolyl-6-formyl-N-ethylcarbazole in 30mL of ethanol, and then dissolve 0.13g (0.42mmol) of carboxyquinoline salt in 20mL of ethanol, Mix the two in a flask, add 0.04mL (0.42mmol) piperidine dropwise, reflux at 78°C for 12h, cool to room temperature, add diethyl ether to precipitate a red solid, suction filter, filter cake column chromatography to obtain the red target product f .

Take 0.42mmol of N-position with -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ Br, The quinoline salt of the substituent replaces the above 0.13 g (0.42 mmol) carboxyquinoline salt, and repeats the chemical reaction of the above step 2 to obtain the target products g~j of the present invention.

Embodiment 4N-benzylcarbazole bridging base oxazole yellow cyanine dye probe comprises the following steps:

1. Preparation of 3-benzoxazolyl-6 formyl-N-benzylcarbazole

Add DMF 7.7mL (100mmol) in the flask, dropwise add phosphorus oxychloride 9.5mL (15.2g, 100mmol) under ice-water bath, dropwise, stir at room temperature until the solution is reddish, slowly add 3-benzoxazole dropwise Base-N-benzylcarbazole 1.71g (6mmol) in 1,2-dichloroethane 30mL solution, dropwise, reflux at 83°C for 16h. After cooling to room temperature, it was poured into ice water, extracted with dichloromethane, and separated by column chromatography to obtain 3-benzoxazolyl-6-formyl-N-benzylcarbazole.

2. Preparation of N-benzylcarbazole bridging oxazole yellow cyanine dye probe p

Dissolve 0.11g (0.28mmol) 3-benzoxazolyl-6-formyl-N-benzylcarbazole in 30mL ethanol, then dissolve 0.13g (0.42mmol) carboxyquinoline salt in 20mL ethanol, Mix the two in a flask, add 0.04mL (0.42mmol) piperidine dropwise, reflux at 78°C for 12h, cool to room temperature, add diethyl ether to precipitate a red solid, suction filter, filter cake column chromatography to obtain the red target product p .

取代基的喹啉盐代替上述0.13g(0.42mmol)羧基喹啉盐，重复上述步骤2的化学反应，得本发明中的目标产物q～t。Take 0.42mmol of N-position with -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ Br,

The quinoline salt of the substituent replaces the above-mentioned 0.13 g (0.42 mmol) carboxyquinoline salt, and repeats the chemical reaction of the above step 2 to obtain the target products q-t in the present invention.

The properties shown by the carbazole bridging fluorescent cyanine dye probe of the present invention are fluorescence emission wavelength: 630nm-660nm; compared with TO emission wavelength 547nm, red shift 80nm-110nm; fluorescence intensity: 30-55; Compared with 0.77, it increased by 39-71 times, so the fluorescence intensity was significantly enhanced; Stocks displacement (Stocks): 130-151mm, TO was 67, the displacement increased by 2-3, and the fluorescence quantum yield: 0.02-0.6, compared with Compared with TO, it is 2-5 times larger, and the photostability is 1-6 times larger. At the same time, the large planar molecular structure of the conjugated system is retained, and the embedded fluorescent dyes of thiazole orange (TO) and oxazole yellow (YO) are retained. Structural features, especially the methylene quinoline structure that can be embedded in NDA or RNA, so it has the advantages of embedded cyanine dyes, such as weak fluorescence background, greatly enhanced fluorescence after combining with DNA or RNA, non-toxic, high sensitivity, etc. .

Claims (4)

1. A carbazole bridging base fluorescent cyanine dye probe, characterized in that: the cyanine dye probe structure includes a benzothiazide that is bonded to thiazole orange (TO) and oxazole yellow (YO) respectively with a carbazole bridging base The cyanine dye probe formed between (ox) azole and 4-methyl quinoline salt; one end of the 3-position side chain of the carbazole ring is connected with the 2-position carbon of benzothia (ox) azole, and the carbazole is another The 6-formyl group at one end is connected with the 4-vinyl quinolate compound. 2. according to the described carbazole bridging fluorescent cyanine dye probe of claim 1, it is characterized in that: described 4-methyl quinoline salt is to be the 4-methyl quinoline salt of anion with ^Br- , in 4-methyl quinoline salt The N-position groups of quinolinate are -CH ₂ CH ₂ COOH, -CH ₂ CH 2 CH ₂ CH ₂ CH ₂ COOH, -CH _{2 CH 3 ,} -CH ₂ CH ₂ CH ₂ Br, 3. the carbazole bridging fluorescent cyanine dye probe according to claim 1, characterized in that: the characteristics of the carbazole bridging fluorescent cyanine dye probe: Fluorescence emission wavelength: 630nm-660nm; Fluorescence intensity: 30-55; Stokes shift (Stocks): 130-151nm; Fluorescence quantum yield: 0.02-0.6. 4. according to the preparation method of the described carbazole bridging fluorescent cyanine dye probe of claim 1, this preparation method comprises the following steps: 1) 3-benzothiazolyl-6-formyl-N-ethylcarbazole, 3-benzoxazolyl-6-formyl-N-ethylcarbazole, 3-benzoxazolyl-6 Preparation of -formyl-N-benzylcarbazole, 3-benzothiazolyl-6-formyl-N-benzylcarbazole Add N,N-dimethylformamide (DMF) and phosphorus oxychloride at a ratio of 1:1 to four 100mL round-bottomed flasks respectively, after dropping, stir at room temperature until the reaction system turns reddish. , the amount of substance added dropwise is 3-benzothiazolyl-N-ethylcarbazole, 3-benzoxazolyl-N-ethylcarbazole, 3-benzothiazolyl-N-ethylcarbazole, 3-benzoxazolyl- N-benzylcarbazole, 3-benzothiazolyl-N-benzylcarbazole in 1,2-dichloroethane solution, the total volume is 40-70mL, reflux and stir at the temperature of 79-80℃ for 6h -12h, cooled to room temperature, poured into ice water, extracted with dichloromethane, separated and purified by column chromatography to obtain 3-benzothiazolyl-6-formyl-N-ethylcarbazole, 3-benzoxa Azolyl-6-formyl-N-ethylcarbazole, 3-benzoxazolyl-6-formyl-N-benzylcarbazole, 3-benzothiazolyl-6-formyl-N-benzyl base carbazole; 2) Preparation of carbazole bridging fluorescent cyanine dye probes 1 mole of 3-benzothiazolyl-6-formyl-N-ethylcarbazole, 3-benzoxazolyl-6-formyl-N-ethylcarbazole, 3-Benzoxazolyl-6-formyl-N-benzylcarbazole, 3-benzothiazolyl-6-formyl-N-benzylcarbazole and four 1.5 molar N-positions with different Dissolve the 4-methylquinoline salt of the substituent in ethanol, add piperidine equivalent to the 4-methylquinoline salt dropwise, reflux and stir at a temperature of 75-80°C for 8-16 hours, and cool to room temperature , and then add diethyl ether to precipitate a red solid, which is suction filtered, and the filter cake is separated by column chromatography to obtain the carbazole bridging fluorescent cyanine dye probe.

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