CN102633788A - P-methylphenyl rhodamine oxadiazole as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses 2-p-methylphenyl-5-[rhodamine B-9'-(2'-benzoyl) yl]-1,3,4-oxadiazole with an asymmetrical structure, namely a compound I. According to the invention, the compound I is a derivative of rhodamine B fluorescent dye. In the compound I, one side of an oxadiazole ring is substituted by rhodamine B, and the other side of the oxadiazole ring is substituted by p-methylphenyl. The compound I has good water solubility and can be used as a cell fluorescence colouring agent.

Description

P-methylphenyl Luo Danming oxadiazole and preparation method thereof and application

The present invention carries out under the subsidy of Tianjin Normal University's development fund (fund number for 52XK1102).

Technical field

Ben Mingfa belongs to the chemical sensitisation technical applications, relates to 2-p-methylphenyl-5-[rhodamine B-9 '-(2 ' '-benzoyl) base]-1,3, the preparation method of 4-oxadiazole I, and as the purposes of the biological stain of cell dye.

Background technology

The luminescent dye molecule probe has in fields such as molecular biology, microbiology, biological chemistry, analytical chemistry, chemical industry and medicine industries widely to be used.Subjects such as its development and life science, chemistry, biotechnology, diagnostic medicine are closely related simultaneously.Along with development of technologies and universal, the application of luminescent dye molecule probe more and more widely, and is meanwhile also increasingly high to fluorescent probe dyestuff performance demands.Synthetic and the applied research of fluorescent probe dyestuff developed into the intercrossing forward position research field of life science and chemistry two big subjects.

Rhodamine class fluorescent probe dyestuff has the fluorescence quantum yield height, advantages such as excitation wavelength, value wide ranges.In addition can also be through the modification of parent being satisfied the needs of different working conditionss (for example: maximum absorption wavelength, maximum emission wavelength, quantum yield etc.).So such fluorescent probe dyestuff has been widely used in the fields such as research of pH value, ion, micromolecular detection and complex biological system in the living microbe.

Change rhodamine fluorochrome molecular probe in bioaffinity, water miscible performance in order to reach, we introduce the heterocycle structure of oxadiazole on the precursor structure of rhodamine, designed and synthesized novel rhodamine class biological fluorescent labeling.

Summary of the invention

First purpose of the present invention has provided the preparation method of the compound with general formula I.

Second purpose of the present invention provided the purposes that compound I is used for active somatic cell optical dye aspect.

For realizing the above-mentioned purpose following technology contents of having asked disclosure of the Invention:

2-p-methylphenyl-5-[rhodamine B-9 '-(2 ' '-benzoyl) base]-1,3 with structural formula I, 4-oxadiazole compound:

The present invention further discloses the preparation method of compound I, it is to be undertaken by following step:

The synthesis step of compound I:

(1) the rhodamine B hydrazides is synthetic

The rhodamine compounds is used dissolve with ethanol, then 85% Hydrazine Hydrate 80 is dropwise joined in the mixed system under the condition of room temperature, after dropwising with mixture heating up strong inverse flow 3-10 hour; Reaction is cooled to room temperature, removal of solvent under reduced pressure after accomplishing; Suction filtration, distilled water wash product 3-5 time; The mol ratio 1: 1.5 ~ 5 of rhodamine B and 85% Hydrazine Hydrate 80 wherein;

(2) target compound I's is synthetic

The rhodamine hydrazides of above-mentioned steps (1) preparation mixed with p-methylbenzoic acid be dissolved in the POCl3; Reflux, after reaction is accomplished with its cool to room temperature and pour in the frozen water dichloromethane extraction into; Anhydrous magnesium sulfate drying; Through volume ratio is methylene chloride 100 ~ 20:1, and column chromatography is separated, and obtains target compound I; Rhodamine hydrazides wherein: p-methylbenzoic acid: the mol ratio of POCl3 1: 1: 1.5 ~ 5.

The present invention further discloses the application of compound I as cell fluorescence dyestuff aspect.

The molecular characterization of the I compound that the present invention designed is: a side of the oxadiazole compound that this type of is asymmetric is replaced by rhodamine class material, and opposite side is replaced by p-methylphenyl.The present invention utilizes the carboxyl reaction in interior hydrazides structure of the active part in the raw material rhodamine hydrazides and the p-methylbenzoic acid, utilizes POCl3 to make its dehydration condensation simultaneously, generates a Xin De oxadiazole heterocycle.New compound I has good water-solubility, and has pair active somatic cell to carry out the purposes of fluorescent dye.

The positively effect that compound I fluorescence probe dye compound disclosed by the invention is compared with prior art had is:

(1) optimizes rhodamine B fluorochrome performance, blocked the generation of the ubiquitous quenching of fluorescence phenomenon that causes because of the change of its lactone and carboxylic acid structure in the similar dyestuff;

(2) in the compound I, increase nitrogen-atoms, Sauerstoffatom, improved water-soluble;

(3) in the big pi-conjugated system of original dyestuff, synthesize oxadiazole heterocycle, be beneficial to when dyeing and produce weak interaction, improve dyeing and fluoroscopic examination effect, and be beneficial to fluorescence or bore hole identification and detection with object.

Description of drawings:

Fig. 1. be the structural formula of compound I;

Fig. 2. compound I fluorescence spectrum figure (excitation wavelength 530nm);

Fig. 3. the cell fluorescence dyeing imaging experiment one of compound I: viable cell group fluorescent dye imaging;

Fig. 4. the cell fluorescence dyeing imaging experiment two of compound I: fixed cell group fluorescent dye imaging.

Embodiment

In order to explain enforcement of the present invention more fully, following preparing method's embodiment is provided.These embodiments only are to explain rather than limit scope of the present invention.Be simple and purpose clearly, in below discussing the description of technique known method, the process for preparation of solution repeated no more.Wherein rhodamine B and p-methylbenzoic acid are that reagent company directly buys.Other organic solvents are through no water treatment.

Embodiment 1.

2-p-methylphenyl-5-[rhodamine B-9 '-(2 ' '-benzoyl) base]-1,3,4-oxadiazole synthetic:

The synthetic of compound I prepares through following principal reaction:

(1) the rhodamine B hydrazides is synthetic

With the rhodamine B of 5 g (10.4 mmol) dissolve with ethanol with 100 mL, put into the flask of 250 mL, the Hydrazine Hydrate 80 with 0.63 mL, 85 % (10.4mmol) dropwise joins in the mixed system under the condition of room temperature then; After dropwising with mixture heating up strong inverse flow 3 hours; When solution becomes clarifying bright orange by mulberry, react and finish, be cooled to room temperature, removal of solvent under reduced pressure; 1M HCl is added in the mixture of evaporate to dryness; Then 1M NaOH is regulated mixture pH to 9, suction filtration, 15 mL distilled water wash products 3 times.Get product 3.38 g, productive rate: 71.4%.

(2) target compound I's is synthetic

Synthetic hydrazides 2 g (4.4 mmol) in the above-mentioned steps (1) mixed with 0.6 g p-methylbenzoic acid (4.4 mmol) be dissolved in the 2 mL POCl3s, reflux 6 hours, after the reaction completion with its cool to room temperature and pour in the frozen water; Dichloromethane extraction; Anhydrous magnesium sulfate drying, removal of solvent under reduced pressure, the gained bullion is 100: 1 methylene chloride column chromatography separation through volume ratio; Obtain target compound (0.32 g, yield 13.1%). ¹H?NMR:?δ(CDCl ₃,?400?MHz)?1.319(12H,?t),?2.403(3H,?s),?3.627(8H,?t),?6.846(2H,?s),?6.894(2H,?d,? J=9.6Hz),?7.152(2H,?d,? J=9.2Hz),?7.269(2H,?d,? J=8.0Hz),?7.474(1H,?d,? J=4.4Hz),?7.728(2H,?d,? J=8.0Hz),?7.859(2H,?t,),?8.391(1H,?t)。The mass spectrum theoretical value: M/zM ⁺, 557.3, measured value, 557.6.

Embodiment 2.

2-p-methylphenyl-5-[rhodamine B-9 '-(2 ' '-benzoyl) base]-1,3,4-oxadiazole synthetic:

(1) the rhodamine B hydrazides is synthetic

With the rhodamine B of 5 g (10.4 mmol) dissolve with ethanol with 100 mL, put into the flask of 250 mL, the Hydrazine Hydrate 80 with 8 mL 85% (138.6 mmol) dropwise joins in the mixed system under the condition of room temperature then; After dropwising mixture heating up was refluxed 10 hours; When solution becomes clarifying bright orange by mulberry, react and finish, be cooled to room temperature, removal of solvent under reduced pressure; 1M HCl is added in the mixture of evaporate to dryness; Then 1M NaOH is regulated mixture pH to 9, suction filtration, 15 mL distilled water wash products 5 times.Get product 4.3 g, productive rate: 91.1 %.

(2) target compound I's is synthetic

Synthetic hydrazides 2 g (4.4 mmol) in the above-mentioned steps (1) mixed with 0.6 g p-methylbenzoic acid (4.4 mmol) be dissolved in the 5 mL POCl3s, reflux 10 hours, after the reaction completion with its cool to room temperature and pour in the frozen water; Dichloromethane extraction; Dried over mgso, removal of solvent under reduced pressure, the gained bullion is that 20: 1 column chromatographys of methylene chloride are separated through volume ratio; Obtaining target compound I is 0.94 g, yield 38.5%.

Embodiment 3.

The cell dyeing of compound I and fluorescence imaging experiment thereof:

1. cell dyeing and fluorescent staining imaging experiment materials and methods thereof:

1.1 cell and cultured method thereof:

(a) cell of test: select NIH-3T3 cell (l cell) for use;

(b) cultivation of cell: DMEM (high sugar) substratum; 10 % foetal calf serums;

(c) cell bed board process (24 hole): get the good NIH-3T3 cell of growth conditions, process 5x10 ⁴/ mL cell suspension; The every hole of 24 orifice plates adds 1.0 mL cell suspensions.37 ^oC, 5% CO ₂Cultivated 48 hours under the condition, carry out the cell dyeing and the fluorescence imaging of viable cell group, fixing back groups of cells respectively and observe.

1.2 test is prepared with optical dye sample compound I solution:

According to standard method, compound I is mixed with 4.0 mg/mL solution with 1% acetum and uses sample solution as test.

1.3. cell fluorescence is coloured to picture:

(1) viable cell group experiment:

(1) draws through cultivation each hole substratum after 48 hours;

(2) add the test of 200 μ L/ holes and use the compound I sample solution, at room temperature left standstill 15 minutes;

(3) draw sample solution, wash 5 times with 1% acetum;

(4) use the fluorescence inverted microscope to observe, take pictures fluorescence excitation optical wavelength 570 nm.

(2) fix the back groups of cells:

(1) draws through cultivation each hole substratum after 48 hours;

(2) acetum 0.5 mL in every hole adding 10% fixes;

(3) 4 ^oLeft standstill under the C condition 1 hour;

(4) use PBS solution to wash 5 times;

(5) add the test of 200 μ L/ holes and use the compound I sample solution, under room temperature, left standstill 15 minutes;

(6) draw sample solution, use 1% acetum to wash 5 times;

(7) under the fluorescence inverted microscope, observe, take pictures, the fluorescence excitation optical wavelength is 570 nm.

2. the cell dyeing of compound I and fluorescence imaging experimental result and analysis

2.1 fluorescence imaging test-results show sample compound I has the red fluorescence imaging effect; Referring to the fluorescence imaging photo accompanying drawing 3 that is converted into grayscale mode;

2.2 fluorescent dye is the result show, fixing back groups of cells fluorescent dye imaging effect is superior to the Color of viable cell group, referring to the fluorescence imaging photo accompanying drawing 4 that is converted into grayscale mode.

Claims (3)

1. have structural formula I compound:

。

2. the said 2-p-methylphenyl-5-of claim 1 [rhodamine B-9 '-(2 ' '-benzoyl) base]-1,3, the preparation method of 4-oxadiazole compound I is characterized in that being undertaken by following step:

Synthesizing of compound I:

(1) the rhodamine B hydrazides is synthetic

Rhodamine B is used dissolve with ethanol, then 85% Hydrazine Hydrate 80 is dropwise joined in the mixed system under the condition of room temperature, after dropwising with mixture heating up strong inverse flow 3-10 hour; Reaction is cooled to room temperature, removal of solvent under reduced pressure after accomplishing; Suction filtration, distilled water wash product 3-5 time; The mol ratio 1: 1.5 ~ 5 of rhodamine B and 85% Hydrazine Hydrate 80 wherein;

(2) target compound I's is synthetic

The rhodamine hydrazides of above-mentioned steps (1) preparation mixed with p-methylbenzoic acid be dissolved in the POCl3; Reflux, after reaction is accomplished with its cool to room temperature and pour in the frozen water dichloromethane extraction into; Anhydrous magnesium sulfate drying; Through volume ratio is methylene chloride 100 ~ 20:1, and column chromatography is separated, and obtains target compound I; Rhodamine hydrazides wherein: p-methylbenzoic acid: the mol ratio of POCl3 1: 1: 1.5 ~ 5.

The said compound I of claim 1 in preparation as the application aspect the cell fluorescence dyestuff.

Images