CN101372482A - Synthesis and use of luciferin calcium ion indicator - Google Patents
Synthesis and use of luciferin calcium ion indicator Download PDFInfo
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- CN101372482A CN101372482A CNA2008102317909A CN200810231790A CN101372482A CN 101372482 A CN101372482 A CN 101372482A CN A2008102317909 A CNA2008102317909 A CN A2008102317909A CN 200810231790 A CN200810231790 A CN 200810231790A CN 101372482 A CN101372482 A CN 101372482A
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Abstract
The invention discloses a fluorescein calcium ion fluoresent indicator which is shown by structural general formula (1), wherein, R1 and R2 are selected from hydrogen, methyl, ethide, propyl or isopropyl. The synthesized crude product of the fluorescein calcium ion fluoresent indicator and absolute methanol are esterified to obtain methyl ester of the fluorescein calcium ion fluoresent indicator, and the esterified pure product is obtained by the separation of column chromatography; at last, the pure product of the fluorescein calcium ion fluoresent indicator is obtained by hydrolysis. The fluorescein calcium ion fluoresent indicator can be used for measuring the concentration of dissociative calcium ion in cells.
Description
Technical field
The invention belongs to the synthetic field of calcium ion fluorescent indicator, particularly have the synthetic and application of the indicator of high quantum production rate and fluorescent signal.
Background technology
Ca
2+As intracellular information transmitter substance, regulating cell from dividing, break up up to dead a series of different critical function, as substance metabolism, muscle is excited to be shunk, and neurotransmitter discharges, cytodifferentiation and propagation, vascular tone is regulated, nerve impulse conduction etc.As the second messenger, what calcium ion also participated in bio signal strides film transmission, the responsing reaction that mediated cell stimulates to external world etc.We can say the metabolism of calcium ion pair cell and all physiology and the biological process that the function regulating effect almost relates to cell.
Yet the distribution of intracellular calcium is extremely uneven, exists bigger concentration gradient between cell is inside and outside and between interior each organoid of born of the same parents and the kytoplasm.Total about 1mmol/L of calcium concn in the cell, intracellular Ca2+ is with combination (mainly combining with phosphate radical and intracellular protein) and free two kinds of uneven being distributed in nucleus, endochylema, cytolemma and other organoid of form, by how many arrangements that account for cell calcium content percentage ratio, its distribution situation is: nucleus 50%, plastosome 14%, cytolemma (being mainly skin) 5%, endochylema combining form 0.5%, free form 0.005%.Because plastosome and sarcoplasmic reticulum calcium contents exceed hundred times than endochylema content, so plastosome and sarcoplasmic reticulum be called this storage vault in the cell again, is called for short calcium storehouse (Calcium pool).Cytosol also has 0.5% calcium to exist with the complexing calcium form, mainly be with kytoplasm in some little organic anion complexings form complexing calcium (CaL).The organic anion that these are little and the binding ability of calcium are moderate, also can pair cell in the adjusting of free calcium concentration play a part certain.The unhomogeneity that distributes just because of calcium ion and the variation of space-time thereof have constituted the basis of metabolic defect in cellular calcium ion signal.When certain stimulation that arrives cell surface made that calcium ion enters cytosol a spot of born of the same parents outside, will cause increasing considerably of cytosol calcium ion concn this moment, thus generation calcium signal.Equally, when the cell calcium storehouse was upset, calcium ion just was released into cytosol in the calcium storehouse, and the calcium ion concn in the cytosol is raise, and this is another important channel that signal produces.Calcium ion concn increases considerably in the kytoplasm, can combine with affine protein or the enzyme of calcium ion height with some then, makes its activation, a series of biochemical reactions of trigger cell, thus play the effect of transmitting extracellular signal.Therefore, how to measure particularly important that the dynamic variation of calcium ion in the viable cell shows accurately and rapidly in the research of life science.
From the 1950's, successively set up and developed the method for multiple mensuration intracellular calcium, as calcium organicvisualization reagent method, microelectrode method, calcium ion Optochemical sensor method, nuclear magnetic resonance method and calcium ion fluorescent probe method, wherein calcium ion fluorescent probe method is present the most frequently used a kind of method.In fact, it is very difficult setting up the measuring method that reaches requirement fully and be fit to various test requirements documents.People usually will select different measuring methods according to the specific requirement that is experimentized according to the characteristics of the whole bag of tricks.As bioluminescent protein method, organicvisualization reagent method, ionization sensor method, microelectrode method, nuclear magnetic resonance method, fluorescent probe method etc.Wherein in the majority with the research of fluorescent probe method again.
Using the interior free calcium ion concentration of this specific specificity calcium ion fluorescent probe mensuration single living cell has the following advantages: (1) studies cell calcium down to viable cell not damaged or physiological condition, not interior physiology, biological process normally of interference cell; (2) highly sensitive, detection limit should reach 10
-7Mol/L; (3) selectivity is good, because interior some other mineral ion of cell such as Mg
2+, Na
+, K
+And Cl
-Deng concentration all 10
-3The mol/L level is so require the measuring method high specificity; (4) response speed is fast, in order to measure the variation of calcium concn, requires measuring method to Ca
2+The response of change in concentration is faster than Ca in the cell
2+Relevant physiological that signal causes and biochemical reaction; (5) spatial and temporal resolution height, the information that is mutually related again independently of one another that can provide interior each the subcellular area calcium signal pcl of born of the same parents to loose fluctuation in real time, in situ.
Summary of the invention
One of purpose of the present invention provides a kind of fluoresceins calcium ion indicator of excited by visible light, and this indicator has stronger selectivity to calcium ion.
A further object of the present invention provides the synthetic method of above-mentioned calcium ion indicator, advantage such as that this method has is easy and simple to handle, yield is high, the product separation and purification is easy.
Fluoresceins calcium ion indicator provided by the invention has following structure:
R
1, R
2Be selected from hydrogen, methyl, ethyl, propyl group or sec.-propyl.
The main synthesis step of above-mentioned fluoresceins calcium ion fluorescent indicator is as follows:
(1) R
1, R
21-chloro-2-methyl-5-nitro-4-(2-(2-nitro-phenoxy) oxyethyl group) benzene that replaces obtains R through reduction
1, R
22 (2-(2-amino-4-chlorine 5-methylphenoxy) oxyethyl group) aniline that replaces, but hydrogenating reduction or use iron powder reducing;
(2) R
1, R
22 (2-(2-amino-4-chlorine 5-methylphenoxy) oxyethyl group) aniline that replace and methyl bromoacetate be the synthetic R that obtains in diisopropyl ethyl amine
1, R
2The 2-(2-(2-amino-4-chloro-5-methylphenoxy) phenetole-N, N, N ', the N '-tetraacethyl methyl esters that replace;
(3) R
1, R
2(N ' obtains fluoresceins calcium ion indicator with the condensation under the methylsulphonic acid effect of 4-chloro resorcinol after N '-tetraacethyl methyl esters formylation to the 2-that replaces for 2-(2-amino-4-chloro-5-methylphenoxy) phenetole-N, N;
Step (1)～(3) described R
1, R
2Be selected from hydrogen, methyl, ethyl, propyl group or sec.-propyl.
Described R
1, R
21-chloro-2-methyl-5-nitro-4-(2-(2-nitro-phenoxy) oxyethyl group) the benzene structure that replaces is as follows:
Described R
1, R
22 (2-(2-amino-4-chlorine 5-methylphenoxy) oxyethyl group) the aniline structure that replaces is as follows:
Described R
1, R
2The 2-that replaces (2-(2-amino-4-chloro-5-methylphenoxy) phenetole-N, N, N ', N '-tetraacethyl methyl esters structure is as follows:
Obtain class BAPTA compound after the above-mentioned product formylation, its structure is as follows:
The above-mentioned synthetic fluoresceins calcium ion indicator crude product method of purification that obtains is as follows: fluoresceins calcium ion indicator crude product that condensation obtains and anhydrous methanol esterification obtain fluoresceins calcium ion indicator methyl esters, obtain the pure product of esterification through column chromatography for separation, the post separated flow is used the mixed solvent of methyl alcohol and ethyl acetate mutually, stationary phase is the column chromatography silica gel of 53-75 μ m, and last hydrolysis obtains the pure product of fluoresceins calcium ion indicator.
Above-mentioned fluoresceins calcium ion indicator can be used for free calcium ion concentration mensuration in the born of the same parents.
Advantage of the present invention and positively effect: 1. The compounds of this invention has been introduced the alkyl or the chlorine substituent of different numbers, wherein with on the phenyl ring that fluorescein base group directly links to each other introduces donor residues---and alkyl can strengthen the binding ability of compound and calcium ion; With on the phenyl ring that fluorescein base group directly links to each other do not introduce the chlorine atom, can strengthen the hydrophobicity of compound, thereby obtain to stride preferably the film effect.2. the invention provides a class fluoresceins calcium ion indicator, the excitation spectrum of such indicator is compared with the Fluo-3 of present widespread use with absorption spectrum and is changed not quite, so the key instrument of the interior free calcium ion concentration of used mensuration born of the same parents all is applicable to the fluoresceins calcium ion indicator that this class is brand-new on the market.
Description of drawings
Fig. 1 is the synthetic Ca that obtains of the present invention
2+The fluorescence emission spectrum of fluorescent indicator Fluo-3MClM;
Fig. 2 is the synthetic Ca that obtains of the present invention
2+The fluorescence emission spectrum of fluorescent indicator Fluo-3ClMM ';
Fig. 3 is the synthetic Ca that obtains of the present invention
2+The fluorescence emission spectrum of fluorescent indicator Fluo-3ClM.
Embodiment
Further describe the synthetic method of the fluoresceins calcium ion indicator of invention below in conjunction with example.
The concrete synthesis step of fluoresceins calcium ion indicator of the present invention is as follows:
(1) R
1, R
2Synthesizing of 1-(2-the chloroethoxy)-2-oil of mirbane (intermediate product 2) that replaces
To N, add the o-NP, 1 that replaces, 2-ethylene dichloride and Anhydrous potassium carbonate in the dinethylformamide, o-NP, 1 wherein, the mol ratio of 2-ethylene dichloride and Anhydrous potassium carbonate is 1:3:1.5, is heated to 120 ℃ of reaction 2h, TLC detection reaction process.Pour in the ethyl acetate, the water extraction merges, concentrates organic phase, the dry intermediate product 2 that gets;
(2) R
1, R
2Synthesizing of 1-chloro-2-methyl-5-nitro-4-(2-(2-nitro-phenoxy) oxyethyl group) benzene (intermediate product 3) that replaces
In the intermediate product 2 that step (1) obtains, add 4-chloro-3-methyl-2-nitrophenols and the Anhydrous potassium carbonate that replaces, wherein intermediate product 2, the 4-chloro-3-methyl-2-nitrophenols that replaces and the mol ratio of Anhydrous potassium carbonate are 1:1.2:2, use N, dinethylformamide is as solvent.Mixture is in 140 ℃ of-150 ℃ of following backflow 4-5h, TLC detection reaction process.Pour in the frozen water suction filtration, the dry intermediate product 3 that gets into;
(3) R
1, R
2Synthesizing of 2 (2-(2-amino-4-chlorine 5-methylphenoxy) oxyethyl group) aniline (intermediate product 4) that replaces
Add intermediate product 3 and iron powder in dehydrated alcohol, the mol ratio of the two is 1:6, adds a small amount of concentrated hydrochloric acid simultaneously and makes catalyzer.Back flow reaction 4-6h, suction filtration, filtrate is used KOH solution respectively, the H of 6moL/L
2SO
4, and saturated NaOH solution is handled suction filtration, the dry intermediate product 4 that gets successively;
(4) R
1, R
2The 2-that replaces (2-(2-amino-4-chloro-5-methylphenoxy) phenetole-N, N, N ', N's '-tetraacethyl methyl esters (intermediate product 5) is synthetic
Add methyl bromoacetate and diisopropyl ethyl amine in the acetonitrile solution of intermediate product 4, wherein the mol ratio of intermediate product 4, methyl bromoacetate and diisopropyl ethyl amine is 1:5:5.After carrying out 24-36h under reaction refluxes, TLC detection reaction process.Pour in the ethyl acetate, concentrated filtrate obtains intermediate product 5;
(5) class BAPTA compound (intermediate product 6) is synthetic
To N, slowly drip POCl in the dinethylformamide
3, temperature is controlled at 5 ℃-10 ℃.Subsequently intermediate product 5 is dissolved in N, slowly drips in above-mentioned mixed solution in the dinethylformamide.Dropwise, reaction solution is incubated 12-24h, TLC detection reaction process down at 40 ℃-45 ℃.Pour in the frozen water suction filtration, the dry intermediate product 6 that gets into;
(6) fluoresceins calcium ion indicator crude product is synthetic
Add intermediate product 6 and 4-chloro resorcinol in methylsulphonic acid, wherein the mol ratio of intermediate product 6 and 4-chloro resorcinol is 1:2.5.Reaction is earlier at N
2Protection in 50 ℃-60 ℃ reaction 30min, then feeds O down
2, in 120 ℃ of reaction 3-6h.Cooling, TLC detection reaction process.Pour in the frozen water suction filtration, the dry crude product that gets target compound into.
(7) fluoresceins calcium ion indicator methyl esters is synthetic
Add fluoresceins calcium ion indicator crude product in exsiccant methyl alcohol, wherein the mol ratio of exsiccant methyl alcohol and crude acid is 10:1.Add an amount of vitriol oil as catalyzer, stir backflow 36h down.Cooling is poured in the frozen water, and suction filtration, product carry out post to be separated.Elution requirement is, sherwood oil dress post carries out gradient elution with the mixed solvent of trichloromethane and methyl alcohol different ratios, collects pure product orange part.
(8) the pure product of fluoresceins calcium ion indicator is synthetic
Add the pure product of fluoresceins calcium ion indicator methyl esters in the mixing solutions of methyl alcohol and water, and add the LiOH solid in batches, wherein the mol ratio of esterified prod and LiOH is 1:4.After stirring 24h under the reaction solution room temperature, transfer pH=2, collect and separate out solid, dry pure fluoresceins calcium ion indicator with concentrated hydrochloric acid.
In above-mentioned synthetic method, described TLC monitoring solvent for use is a normal hexane, ethyl acetate, methyl alcohol, trichloromethane or their mixed solvent.
In above-mentioned synthetic method, the used mobile phase solvent of described column chromatography is the mixed solvent of ethyl acetate and methyl alcohol, and used stationary phase is the column chromatography silica gel of 53-75 μ m.
Synthetic (the R of embodiment 1:Fluo-3MClM
1Be CH
3, R
2Be H)
(1) compound 1-(2-chloroethoxy) 4-methyl-2-oil of mirbane (intermediate product 2) is synthetic, and synthesis step is as follows:
Reflux condensing tube is being housed, add 7.65g (50mmol) 4-methyl-2-nitrophenols in the 100mL there-necked flask of drying tube, 40mL N, dinethylformamide, after treating dissolving fully, stir adding 12.4mL (0.15mol) 1 down, the 2-ethylene dichloride, stirring at room 5 minutes adds 10.35g (0.1mol) K
2CO
3, be warming up to 120 ℃ of reaction 2h, be cooled to room temperature, add the dilution of 70mL ethyl acetate, water (50mL * 3) washing merges organic phase, and vacuum is steamed and is desolventized suction filtration, drying.The crude product ethyl alcohol recrystallization, productive rate 96%.m.p.33～34℃
Nuclear-magnetism
1H NMR (CDCl
3) δ (ppm): 7.657 (s, 1H), 7.339 (d, 1H), 6.993 (d, 1H), 4.388-4.332 (t, 2H), 3.848-3.665 (t, 2H), 2.360 (s, 3H);
Infrared FT-IR (KBr), υ/cm
-1: 3420.9,2930.3,1626.9,1530.3,1357.4,1258.1,1157.8,1030.6,810.7.
(2) compound 1-chloro-2-methyl-4-(2-(4-methyl-2-nitro-phenoxy) oxyethyl group) 5-oil of mirbane (intermediate product 3) is synthetic, and synthesis step is as follows:
In the three-necked bottle that mechanical stirrer, reflux condensing tube and thermometer are housed, add 21.5g (0.1mol) intermediate product 2,18.4g (0.12mol) 4-chloro-5-methyl-2-nitrophenols, 100mL N, dinethylformamide stirs and makes it dissolving.In above-mentioned reaction solution, add 27.6g (0.2mol) K
2CO
3Heat up, make temperature maintenance between 140～160 ℃, stirring and refluxing 4-5h.Question response is chilled to room temperature after finishing, and vacuum concentration is scattered in residue in the water, and suction filtration is collected the solid that forms.Crude product 95% ethyl alcohol recrystallization, productive rate 88.30%.m.p.126～128℃
Nuclear-magnetism
1H NMR (CDCl
3) δ (ppm): 7.882 (s, 1H), 7.636 (s, 1H), 7.355 (s, 1H), 7.141-7.109 (d, 2H), 4.497 (s, 4H), 2.458 (s, 3H), 2.353 (s, 3H);
Infrared FT-IR (KBr), υ/cm
-1: 3441.7,2924.9,1614.9,1526.9,1355.9,1258.7,1112.4,1038.4,811.3.
(3) compound 2-(2-(2-amino-4-chloro-5-methylphenoxy) oxyethyl group-5-methyl-aniline (intermediate product 4) synthetic, synthesis step is as follows:
In the 100mL three-necked bottle of reflux condensing tube and agitator is housed, add 17g (0.3mol) reduced iron powder, 20mL dehydrated alcohol and 1.04mL (0.012mol) concentrated hydrochloric acid.Stir down, add 18.3g (0.05mol) intermediate product 3, afterwards reflux 2-4h in batches.Question response fully back adds potassium hydroxide solution in hot mixt, remove iron powder while hot, and with hot absolute ethanol washing reaction flask and filter cake.Merging filtrate, washing lotion add 6mol/L sulfuric acid, separate out vitriol, and cooling is filtered.Solid is through dry air.The above-mentioned vitriol that makes is dissolved in 60 ℃ of hot water, but fully the dissolving after, be chilled to 40 ℃, add saturated sodium hydroxide solution make be alkalescence, the cooling, the solid that suction filtration is separated out.Productive rate 85.2%.m.p.152～154℃
Nuclear-magnetism
1H NMR (CDCl
3) δ (ppm): 6.749-6.729 (d, 1H), 6.704 (s, 1H), 6.680 (s, 1H), 6.556 (s, 1H), 6.525-6.505 (d, 1H), 4.306 (s, 4H), 2.251 (s, 3H), 2.222 (s, 3H);
Infrared FT-IR (KBr), υ/cm
-1: 3439.5,3358.7,1617.1,1512.5,1218.3,1169.2,1071.9,791.7.
(4) compound 2-(2-(2-amino-4-chloro-5-methylphenoxy) oxyethyl group-5-methylbenzene-N, N, N ', N's '-tetraacethyl methyl esters (intermediate product 5) is synthetic, synthesis step is as follows:
In the 100mL three-necked bottle of reflux condensing tube is housed, add the hot acetonitrile of 25mL, stir down, add 9.17g (0.025mol) intermediate product 4, after treating to dissolve fully, add 21.75mL (0.125mol) (iPr)
2NEt, 11.75mL (0.125mol) methyl bromoacetate is with the reaction solution 24-36h that refluxes.Add 0.7mL methyl bromoacetate and 2.2mL (iPr) again behind the reaction 12h
2NEt.After question response is finished, reaction solution is chilled to 40-60 ℃, in the impouring 50mL ethyl acetate, suction filtration, washing, merging filtrate, washing lotion, solvent removed in vacuo adds 28mL methyl alcohol, the solid that stirring at room 4-6h, suction filtration separate out.Productive rate 81.7%.m.p.108～110℃
Nuclear-magnetism
1H NMR (CDCl
3) δ (ppm): 6.795 (s, 1H), 6.756-6.695 (m, 3H), 6.637 (s, 1H), 4.228 (s, 4H), 4.146-4.110 (d, 8H), 3.647-3.590 (s, 12H), 2.270 (s, 3H), 2.241 (s, 3H);
Infrared FT-IR (KBr), υ/cm
-1: 2954.7,1754.8,1512.2,1173.1,1008.4,713.2.
(5) class BAPTA compd B APTA-MClM-CHO's (intermediate product 6) is synthetic, and synthesis step is as follows:
In the 250mL there-necked flask of reflux condensing tube, thermometer is housed, add 50mL N, dinethylformamide is chilled to 5～10 ℃ with this reaction solution, drips the 9.6mL phosphorus oxychloride in 40～45min, after dripping, 11.9g (0.02mol) intermediate product 5 is dissolved in 50mL N, in the dinethylformamide, in 40～45min, drops in the above-mentioned reaction solution, after adding, reaction mixture is heated to 40～45 ℃ of stirrings spends the night.After question response is finished, be chilled to room temperature, pour in the 200Kg ice/water-bath, suction filtration is collected the solid that generates, and is washed with water to pH=4～5.Productive rate 77.9%.m.p.150～152℃
Nuclear-magnetism
1H NMR (CDCl
3) δ (ppm): 10.130 (s, 1H), 7.305 (s, 1H), 6.807 (s, 1H), 6.685 (s, 1H), 6.451 (s, 1H), 4.272-4.261 (s, 4H), 4.220 (s, 4H), 4.104 (s, 4H), 3.619-3.603 (s, 12H), 2.553 (s, 3H), 2.273 (s, 3H);
Infrared FT-IR (KBr), υ/cm
-1: 3444.6,2952.2,1744.2,1679.4,1514.8,1252.8,1204.9,1172.2,1005.5,709.1.
MALDI-TOF?MS，m/z：622.58(calcd.622.19)。
(6) the Fluo-3MClM crude product is synthetic, and synthesis step is as follows:
In the 50mL there-necked flask of reflux condensing tube, thermometer is housed, stir adding 1.25g (2mmol) intermediate product 6 and 0.61g (4mmol) 4-chloro resorcinol down, wherein the mol ratio of intermediate product 6 and 4-chloro resorcinol is 1:2.Reaction is earlier at N
2Protection in 50 ℃-60 ℃ reaction 30min, then feeds O down
2, in 120 ℃ of reaction 3-6h.Cooling is slowly poured into (150mL) in the frozen water, and the brownish black precipitation is arranged, suction filtration, and washing becomes red-brown until the solid color.Crude product does not separate, and dry air 2-3 days, is directly used in next step esterification.
(7) the Fluo-3MClM methyl esters is synthetic, and synthesis step is as follows:
In the 100mL single port bottle of reflux condensing tube is housed, stir the vitriol oil that adds 4.08g Fluo-3MClM ' crude product and 50mL anhydrous methanol and catalytic amount down.With the reaction solution 36h that refluxes, cooling is poured in the 100mL frozen water, treats that solid separates out suction filtration, drying fully.Crude product carries out post to be separated, sherwood oil dress post, and eluent employing trichloromethane and methyl alcohol carry out gradient elution by the mixed solvent of different ratios.Collect pure product orange part.
Infrared FT-IR (KBr), υ/cm
-1: 3423.1,2950.3,1740.7,1590.1,1509.3,1250.1,1204.8,1173.9,998.1,536.9.
MALDI-TOF?MS，m/z：874.92(calcd.874.11)。
(8) the pure product of Fluo-3MClM is synthetic, and synthesis step is as follows:
In 50mL single port bottle, add 5mL methyl alcohol and 15mL water, stir adding 1.75g (2mmol) Fluo-3MClM ' methyl esters down, add 0.19g (8mmol) LiOH solid subsequently in batches.Reaction solution is transferred pH=2 with concentrated hydrochloric acid in ice-water bath after stirring 24h under the room temperature, collect and separate out solid, dry pure fluoresceins calcium ion indicator Fluo-3MClM.
The fluorescence emission spectrum of Fluo-3MClM as shown in Figure 1.
Synthetic (the R of embodiment 2:Fluo-3ClMM '
1Be H, R
2Be CH
3)
(1) compound 1-(2-chloroethoxy) 2-oil of mirbane (intermediate product 2) is synthetic, and synthesis step is as follows:
Reflux condensing tube is being housed, is adding 6.95g (50mmol) 2-nitrophenols in the 100mL there-necked flask of drying tube, 40mL N, dinethylformamide, treat dissolving fully after, stir and add 12.4mL (0.15mol) 1 down, the 2-ethylene dichloride, stirring at room 5 minutes adds 10.35g (0.1mol) K
2CO
3, be warming up to 120 ℃ of reaction 2h, be cooled to room temperature, add the dilution of 70mL ethyl acetate, water (50mL * 3) washing merges organic phase, and vacuum is steamed and is desolventized suction filtration, drying.The crude product ethyl alcohol recrystallization, productive rate 95.0%.m.p.44～45℃
Nuclear-magnetism
1H NMR (CDCl
3) δ (ppm): 7.855-7.835 (d, 1H), 7.574-7.529 (t, 1H), 7.111-7.074 (d, 2H), 4.434-4.304 (t, 2H), 3.917-3.851 (t, 2H);
Infrared FT-IR (KBr), υ/cm
-1: 3443.1,2928.4,1607.9,1521.6,1345.9,1247.2,1159.7,1025.3,745.9.
(2) compound 2-chloro-1,3-dimethyl-4-nitro 5-(2-(2-nitro-phenoxy) oxyethyl group) benzene (intermediate product 3) synthetic, synthesis step is as follows:
In the three-necked bottle that mechanical stirrer, reflux condensing tube and thermometer are housed, add 20.1g (0.1mol) intermediate product 2,24.12g (0.12mol) 4-chloro-3,5-dimethyl-2-nitrophenols, 100mLN, dinethylformamide stirs and makes it dissolving.In above-mentioned reaction solution, add 27.6g (0.2mol) K
2CO
3Heat up, make temperature maintenance between 140～160 ℃, stirring and refluxing 4-5h.Question response is chilled to room temperature after finishing, and vacuum concentration is scattered in residue in the water, and suction filtration is collected the solid that forms.Crude product 95% ethyl alcohol recrystallization, productive rate 80.1%.m.p.158～160℃
Nuclear-magnetism
1H NMR (CDCl
3) δ (ppm): 7.845-7.821 (d, 1H), 7.575-7.535 (t, 1H), 7.138-7.117 (d, 1H), 7.100-7.062 (t, 1H), 6.966 (s, 1H), 4.471-4.427 (s, 4H), 2.443 (s, 3H), 2.300 (s, 3H);
Infrared FT-IR (KBr), υ/cm
-1: 3438.4,1605.4,1520.2,1367.7,1278.0,1094.9,847.9,735.7.
(3) compound 2-(2-(2-amino-4-chloro-3,5-dimethyl phenoxy) oxyethyl group) aniline (intermediate product 4) is synthetic, and synthesis step is as follows:
In the 100mL three-necked bottle of reflux condensing tube and agitator is housed, add 17g (0.3mol) reduced iron powder, 20mL dehydrated alcohol and 1.04mL (0.012mol) concentrated hydrochloric acid.Stir down, add 20.1g (0.05mol) intermediate product 3, afterwards reflux 2-4h in batches.Question response fully back adds potassium hydroxide solution in hot mixt, remove iron powder while hot, and with hot absolute ethanol washing reaction flask and filter cake.Merging filtrate, washing lotion add 6mol/L sulfuric acid, separate out vitriol, and cooling is filtered.Solid is through dry air.The above-mentioned vitriol that makes is dissolved in 60 ℃ of hot water, but fully the dissolving after, be chilled to 40 ℃, add saturated sodium hydroxide solution make be alkalescence, the cooling, the solid that suction filtration is separated out.Productive rate 81.0%.m.p.105～107℃
Nuclear-magnetism
1H NMR (CDCl
3) δ (ppm): 6.862-6.813 (m, 2H), 6.742-6.722 (d, 2H), 6.639 (s, 1H), 4.370-4.348 (s, 4H), 3.801 (s, 4H), 2.298 (s, 3H), 2.241 (s, 3H);
Infrared FT-IR (KBr), υ/cm
-1: 3434.7,3354.7,1615.6,1505.7,1216.6,1154.5,1084.8,746.8.
(4) compound 2-(2-(2-amino-4-chloro-3,5-dimethyl phenoxy) oxyethyl group) benzene-N, N, N ', N's '-tetraacethyl methyl esters (intermediate product 5) is synthetic, and synthesis step is as follows:
In the 100mL three-necked bottle of reflux condensing tube is housed, add the hot acetonitrile of 25mL, stir down, add 7.65g (0.025mol) intermediate product 4, after treating to dissolve fully, add 21.75mL (0.125mol) (iPr)
2NEt, 11.75mL (0.125mol) methyl bromoacetate is with the reaction solution 24-36h that refluxes.Add 0.7mL methyl bromoacetate and 2.2mL (iPr) again behind the reaction 12h
2NEt.After question response is finished, reaction solution is chilled to 40-60 ℃, in the impouring 50mL ethyl acetate, suction filtration, washing, merging filtrate, washing lotion, solvent removed in vacuo adds 28mL methyl alcohol, the solid that stirring at room 4-6h, suction filtration separate out.Productive rate 77.8%.m.p.102～103℃
Nuclear-magnetism
1H NMR (CDCl
3) δ (ppm): 6.926-6.909 (s, 1H), 6.868 (s, 1H), 6.641 (s, 1H), 4.365-4.355 (s, 4H), 4.262-4.251 (s, 4H), 4.149 (s, 4H), 3.874-3.845 (d, 8H), 3.642-3.605 (d, 12H), 2.521 (s, 3H), 2.333 (s, 3H);
Infrared FT-IR (KBr), υ/cm
-1: 2953.9,1753.3,1509.1,1253.2,1203.7,1175.5,1003.5,747.3.
(5) class BAPTA compd B APTA-ClMM '-CHO's (intermediate product 6) is synthetic, and synthesis step is as follows:
In the 250mL there-necked flask of reflux condensing tube, thermometer is housed, add 50mL N, dinethylformamide is chilled to 5～10 ℃ with this reaction solution, drips the 9.6mL phosphorus oxychloride in 40～45min, after dripping, 11.9g (0.02mol) intermediate product 5 is dissolved in 50mL N, in the dinethylformamide, in 40～45min, drops in the above-mentioned reaction solution, after adding, reaction mixture is heated to 40～45 ℃ of stirrings spends the night.After question response is finished, be chilled to room temperature, pour in the 200Kg ice/water-bath, suction filtration is collected the solid that generates, and is washed with water to pH=4～5.Productive rate 72.5%.m.p.103～105℃
Nuclear-magnetism
1H NMR (CDCl
3) δ (ppm): 9.814 (s, 1H), 7.438-7.435 (s, 1H), 7.415-7.268 (d, 1H), 6.630 (s, 1H), 4.427-4.404 (t, 2H), 4.262-4.239 (t, 2H), 4.220 (s, 4H), 3.863-3.849 (d, 4H), 3.642-3.626 (s, 6H), 3.617-3.602 (s, 6H), 2.522 (s, 3H), 2.339 (s, 3H);
Infrared FT-IR (KBr), υ/cm
-1: 3476.9,2953.1,1748.2,1687.9,1595.3,1519.5,1259.5,1203.9,1168.5,1005.1,893.3.
MALDI-TOF?MS，m/z：622.50(calcd.622.19)。
(6) Fluo-3ClMM ' crude product is synthetic, and synthesis step is as follows:
In the 50mL there-necked flask of reflux condensing tube, thermometer is housed, stir adding 1.25g (2mmol) intermediate product 6 and 0.61g (4mmol) 4-chloro resorcinol down, wherein the mol ratio of intermediate product 6 and 4-chloro resorcinol is 1:2.Reaction is earlier at N
2Protection in 50 ℃-60 ℃ reaction 30min, then feeds O down
2, in 120 ℃ of reaction 3-6h.Cooling is slowly poured into (150mL) in the frozen water, and the brownish black precipitation is arranged, suction filtration, and washing becomes red-brown until the solid color.Crude product does not separate, and dry air 2-3 days, is directly used in next step esterification.
(7) Fluo-3ClMM ' methyl esters is synthetic, and synthesis step is as follows:
In the 100mL single port bottle of reflux condensing tube is housed, stir the vitriol oil that adds 4.08g Fluo-3ClMM ' crude product and 50mL anhydrous methanol and catalytic amount down.With the reaction solution 36h that refluxes, cooling is poured in the 100mL frozen water, treats that solid separates out suction filtration, drying fully.Crude product carries out post to be separated, sherwood oil dress post, and eluent employing trichloromethane and methyl alcohol carry out gradient elution by the mixed solvent of different ratios.Collect pure product orange part.
Infrared FT-IR (KBr), υ/cm
-1: 3408.9,2951.0,1740.1,1588.1,1494.3,1249.7,1204.4,1165.3,946.9,532.3.
MALDI-TOF?MS，m/z：874.70(calcd.874.11)。
(8) the pure product of Fluo-3ClMM ' is synthetic, and synthesis step is as follows:
In 50mL single port bottle, add 5mL methyl alcohol and 15mL water, stir adding 1.75g (2mmol) Fluo-3MClM ' methyl esters down, add 0.19g (8mmol) LiOH solid subsequently in batches.Reaction solution is transferred pH=2 with concentrated hydrochloric acid in ice-water bath after stirring 24h under the room temperature, collect and separate out solid, dry pure fluoresceins calcium ion indicator Fluo-3ClMM '.
The fluorescence emission spectrum of Fluo-3ClMM ' as shown in Figure 2.
Embodiment 3: the synthetic (R of compound F 17-hydroxy-corticosterone luo-3ClM
1Be H, R
2Be H)
(1) compound 1-(2-chloroethoxy) 2-oil of mirbane is identical with embodiment 2 steps (1), productive rate 95.0%.
(2) compound 1-chloro-2-methyl-5-nitro-4-(2-(2-nitro-phenoxy) oxyethyl group) benzene is synthetic similar with embodiment 1 step (2), and different is that 4-chloro-5-methyl-2-nitrophenols replaces 4-chloro-3,5-dimethyl-2-nitrophenols, productive rate 89.6%.
(3) compound 2-(2-(2-amino-4-chloro-5-methylphenoxy) oxyethyl group) aniline is synthetic similar with embodiment 1 step (3), productive rate 85.2%.
(4) compound 2-(2-(2-amino-4-chloro-5-methylphenoxy) oxyethyl group) benzene-N, N, N ', synthetic and embodiment 1 step (4) of N '-tetraacethyl methyl esters is similar, productive rate 91.6%.
(5) class BAPTA compd B APTA-ClM-CHO's is synthetic similar with embodiment 1 step (5), productive rate 72.6%.
(6) the Fluo-3ClM crude product is synthetic similar with embodiment 1 step (6).
(7) the synthetic and separation of Fluo-3ClM methyl esters is similar with embodiment 1 step (7).
(8) the pure product of Fluo-3ClM is synthetic similar with embodiment 1 step (8).
The fluorescence emission spectrum of Fluo-3ClM as shown in Figure 3.
Embodiment 4: the synthetic (R of compound F 17-hydroxy-corticosterone luo-M3ClMEt
1Be methyl, R
2Be ethyl)
(1) compound 1-(2-chloroethoxy)-4-methyl-2-oil of mirbane is identical with embodiment 1 step (1), productive rate 95.0%.
(2) compound 1-(2-(4-chloro-3-ethyl-5-methyl-2-nitro-phenoxy) oxyethyl group)-4-methyl-2-oil of mirbane is synthetic similar with embodiment 1 step (2), different is that 4-chloro-3-ethyl-5-methyl-2-nitrophenols replaces 4-chloro-3,5-dimethyl-2-nitrophenols, productive rate 82.3%.
(3) compound 2-(2-(2-amino-4-chloro-3-ethyl-5-methylphenoxy) oxyethyl group)-5-monomethylaniline is synthetic similar with embodiment 1 step (3), productive rate 77.5%.
(4) compound 2-(2-(2-amino-4-chloro-3-ethyl-5-methylphenoxy) oxyethyl group) benzene-N, N, N ', synthetic and embodiment 1 step (4) of N '-tetraacethyl methyl esters is similar, productive rate 82.3%.
(5) class BAPTA compd B APTA-MClMEt-CHO's is synthetic similar with embodiment 1 step (5), productive rate 66.8%.
(6) the Fluo-M3ClMEt crude product is synthetic similar with embodiment 1 step (6).
(7) the synthetic and separation of Fluo-M3ClMEt methyl esters is similar with embodiment 1 step (7).
(8) the pure product of Fluo-M3Cl MEt is synthetic similar with embodiment 1 step (8).
Claims (6)
1. the fluoresceins calcium ion fluorescent indicator that has following general structure (1):
R
1, R
2Be selected from hydrogen, methyl, ethyl, propyl group or sec.-propyl.
2. the synthetic method of the described fluoresceins calcium ion of claim 1 fluorescent indicator, its main synthesis step is as follows:
(1) R
1, R
21-chloro-2-methyl-5-nitro-4-(2-(2-nitro-phenoxy) oxyethyl group) benzene that replaces obtains R through reduction
1, R
22 (2-(2-amino-4-chlorine 5-methylphenoxy) oxyethyl group) aniline that replaces;
(2) R
1, R
22 (2-(2-amino-4-chlorine 5-methylphenoxy) oxyethyl group) aniline that replace and methyl bromoacetate be the synthetic R that obtains in diisopropyl ethyl amine
1, R
2The 2-(2-(2-amino-4-chloro-5-methylphenoxy) phenetole-N, N, N ', the N '-tetraacethyl methyl esters that replace;
(3) R
1, R
2(N ' obtains fluoresceins calcium ion indicator with the condensation under the methylsulphonic acid effect of 4-chloro resorcinol after N '-tetraacethyl methyl esters formylation to the 2-that replaces for 2-(2-amino-4-chloro-5-methylphenoxy) phenetole-N, N;
Step (1)～(3) described R
1, R
2Be selected from hydrogen, methyl, ethyl, propyl group or sec.-propyl.
3. the synthetic method of fluoresceins calcium ion fluorescent indicator according to claim 2, it is characterized in that: fluoresceins calcium ion indicator crude product that condensation obtains and anhydrous methanol esterification obtain fluoresceins calcium ion indicator methyl esters, obtain the pure product of esterification through column chromatography for separation, last hydrolysis obtains the pure product of fluoresceins calcium ion indicator.
4. the synthetic method of fluoresceins calcium ion fluorescent indicator according to claim 2 is characterized in that: step (1) uses iron powder as reductive agent.
5. the synthetic method of fluoresceins calcium ion fluorescent indicator according to claim 3 is characterized in that: the post separated flow is used the mixed solvent of methyl alcohol and ethyl acetate mutually, and stationary phase is the column chromatography silica gel of 53-75 μ m.
6. the described fluoresceins calcium ion of claim 1 fluorescent indicator application in the free calcium ion concentration mensuration in born of the same parents.
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|---|---|---|---|
| CNA2008102317909A CN101372482A (en) | 2008-10-20 | 2008-10-20 | Synthesis and use of luciferin calcium ion indicator |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102770417A (en) * | 2009-10-17 | 2012-11-07 | 阿桑特研究公司 | Fluorescent calcium indicators that are ratiometric and emit in the red spectrum |
| CN112592279A (en) * | 2020-12-23 | 2021-04-02 | 淄博鸿润新材料有限公司 | Refining and purifying method of 2,2' -dinitroglycol diphenyl ether |
-
2008
- 2008-10-20 CN CNA2008102317909A patent/CN101372482A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102770417A (en) * | 2009-10-17 | 2012-11-07 | 阿桑特研究公司 | Fluorescent calcium indicators that are ratiometric and emit in the red spectrum |
| CN112592279A (en) * | 2020-12-23 | 2021-04-02 | 淄博鸿润新材料有限公司 | Refining and purifying method of 2,2' -dinitroglycol diphenyl ether |
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