CN1696093A - Compound of di-butanedione, complexes of europium, combination of containing the complexes and application - Google Patents
Compound of di-butanedione, complexes of europium, combination of containing the complexes and application Download PDFInfo
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- CN1696093A CN1696093A CN 200510023820 CN200510023820A CN1696093A CN 1696093 A CN1696093 A CN 1696093A CN 200510023820 CN200510023820 CN 200510023820 CN 200510023820 A CN200510023820 A CN 200510023820A CN 1696093 A CN1696093 A CN 1696093A
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Abstract
A chemically luminous composition contains the Eu matches and enyl-bond contained compound. It can be used for immonoanalysis.
Description
Technical field
The present invention relates to a kind of novel compound of di-butanedione, contain this compound as the europium complex of ligand and the composition that contains this europium complex.The invention still further relates to the purposes of described composition in chemiluminescence immune assay.
Background technology
Up to now, the method for immunity of micro-biologically active substance has experienced by radioimmunoassay (RIA), fluoroimmunoassay (FIA), enzyme immunoassay (EIA) in the organism, and all stages such as chemiluminescence immune assay.The improving constantly of demand that this development process mainly is based on susceptibility, accuracy and property simple to operation to detection method determines.
Then from inventing to the course of using of weeding out the old and bring forth the new, chemiluminescence immunoassay detection by quantitative method becomes strong analysis means and the technology that replaces radioimmunoassay now as radioimmunity (RIA) analytical technology.In the international scope, chemiluminescence immunoassay detection by quantitative method has become the very important new technology and the detection means of Clinical Laboratory, and will extend into the every field of Clinical Laboratory, and this also is that the consistent of Clinical Laboratory circle known together.
Chemiluminescence immunoassay is the method that a kind of light wave that utilizes chemiluminescent substance to launch detects.Chemiluminescent substance serves as a mark and is applied in detection of nucleic acids and the immunodetection.For example, can certain a part of specific combination centering be combined with luminophore by number of ways and form light-emitting composite.This mixture can react with detected material in the sample (another molecule of specific combination centering), be allocated in solid phase and the liquid phase, and allocation proportion is relevant with the amount that detects thing.By measuring luminous quantity in solid phase or the liquid phase, can draw the respective concentration that detects thing in the sample.
Specifically, under homogeneous phase condition, will be coated with bioactive molecule and inner have the sensitization microballoon (nano level) of dyestuff and be coated with bioactive molecule and the mixture of the inner luminous microballoon (nano level) that has a luminophor mixes as the reagent and the test sample that might contain target molecule.The nano luminescent microballoon that is coated with the nanometer sensitization microballoon of bioactive molecule this moment and is coated with bioactive molecule is the captured target molecule quickly and effectively, and nanometer sensitization microballoon, nano luminescent microballoon and target molecule form in-plant sandwich complex together.Through excitation light irradiation, the dyestuff in the nanometer sensitization microballoon is induced the active oxygen ion (singlet oxygen) that activates and discharge high-energy state.The active oxygen ion of this high-energy state is captured by in-plant nano luminescent microballoon, thereby transmits energy to activate the luminophor in the described luminous microballoon.Luminophor in the luminous microballoon can be in discharging high level ruddiness after the number microsecond under the zero glitch-free situation of background.Measure these high level photons and photon numbers is scaled concentration of target molecules with photon counter, the concentration that how much has promptly accurately reflected target molecule of photon numbers by computer.
United States Patent (USP) 5; 709; 994 disclose a kind of method of measuring target molecule; it comprises the step of the composition that may contain target molecule being carried out photoirradiation; described composition comprises non-particulate matrix or particle matrix, described matrix be coated with through photoirradiation can produce singlet oxygen photosensitizers and can be by described singlet oxygen activatory chemiluminescence compound.It also specifically discloses described chemiluminescence compound and has been selected from 9-alkylidene group-N-methylacridine, enol ether and enamine, and described photosensitizers is selected from methylene blue, rose-red, porphyrin, phthalocyanine and chlorophyll.But the specific combination that this photosensitizers and chemiluminescence compound form is to having the low shortcoming of luminous efficiency.
United States Patent (USP) 5,780,646 disclose a kind of composition, and it comprises the metal chelate of (a) a kind of hexa-coordinate at least and (b) compound of the two keys of a kind of band with two aryl.It also mentions described compound (b) can have following general formula:
Wherein, X ' is S; D and D ' are selected from CH separately
3(CH
2)
9CH
3, R ' is (CH
2)
6CH
3Perhaps (CH
2)
7CH
3
Described metal chelate (a) can have following general formula:
It also discloses at following formula (3a) and a TTA (3b) can be replaced by a following molecule:
Perhaps, following formula (3a) and (3b) in two TTA group that can be selected from following molecule separately replace:
(3n)
(3o)
(3r)
Perhaps, three TTA group that can be selected from following molecule separately replaces:
Compared with prior art, United States Patent (USP) 5,780,646 have greatly improved luminous efficiency by adding europium complex, thereby have improved detection sensitivity.But also there is following shortcoming in this method:
Because existing europium complex huge legendary turtle is right not enough, metal europium ion meeting huge legendary turtle is combined in the surface of Nano microsphere, causes Nano microsphere pan coating capacity to descend.And reduced luminous efficiency.After this composition that contains the compound (b) of described metal chelate (a) and the two keys of described band with two aryl was placed the luminous microballoon of nano level, the bag of finding luminous microballoon was descended by capacity and is up to 40%; The luminous efficiency 30%-40% that descended.
Therefore, need exploitation a kind of new, have the metal chelate that very strong huge legendary turtle is made a concerted effort, it can avoid the reaction of metal europium ion and Nano microsphere surface group, thereby has avoided the decline of Nano microsphere pan coating capacity and luminous efficiency.
The content of invention
In view of the above-mentioned defective of prior art, an object of the present invention is to provide a kind of novel light emitting composition, the bag that it can not influence light-emitting particles is by capacity and significantly improved the luminous efficiency of luminescent ball, has especially improved detected signal value.
Another object of the present invention provides a kind of compound of di-butanedione that is used for immunoassay.
Therefore, the invention provides a kind of compound of di-butanedione with following general formula:
Wherein: X is the aryl that has 1-5 carbon atom straight chain alkyl or have following formula, and wherein Y is the straight or branched alkyl substituent of 1-5 carbon atom:
Perhaps
The present invention also provides a kind of chemical luminous composite, and it comprises:
1) has the europium complex of compound of di-butanedione of the present invention as ligand;
What 2) have a following general formula contains the ethylene linkage compound:
Wherein, X ' is S or NR, and R has the alkyl of 1-10 carbon atom or the aryl of 6-18 carbon atom;
R ' is hydrogen atom or the alkyl with 1-10 carbon atom;
Respectively do for oneself hydrogen atom or have the alkyl of 1-20 carbon atom of D and D '.
Description of drawings
Fig. 1 is that light-induced chemiluminescent immunoassay system (LICA) detects schematic diagram, wherein
Fig. 1 a is that bag is by first antibody and contain the synoptic diagram of the photosensitive microballoon of photosensitive reagents;
Fig. 1 b is detected antigen and a first antibody bonded synoptic diagram after the adding test samples;
Fig. 1 c adds the synoptic diagram that is coated with second antibody and contains the microballoon of light emitting composition;
Fig. 1 d is the synoptic diagram of photoluminescence.
Embodiment
In the present invention, except as otherwise noted, otherwise term " alkyl " be meant have 1-10 carbon atom, better 1-6 carbon atom, the straight or branched alkyl of better 1-4 carbon atom, it comprises methyl, ethyl, propyl group, sec.-propyl, normal-butyl, the tertiary butyl, isobutyl-, n-pentyl, isopentyl, n-hexyl, nonyl, decyl etc.
In the present invention, term " aryl " is meant the aromatic group with 6-18 ring carbon atom, and it can be unsubstituted, and the alkyl that is perhaps had 1-10 carbon atom replaces.
Fig. 1 is that light-induced chemiluminescent immunoassay system of the present invention (LICA) detects schematic diagram.As shown in Figure 1a, have photosensitizers in the nano level microballoon 10, this microsphere surface is coated with first antibody 1.Shown in Fig. 1 b, after the Nano microsphere that will be coated with first antibody 1 and contain photosensitizers added in the sample contain detected antigen 2, first antibody 1 can combine with antigen 2.Shown in Fig. 1 c, luminous microballoon 20 is the Nano microspheres that contain light emitting composition, and its pan coating has second antibody 3.After adding described luminous microballoon 20 in the said sample, on this luminous microballoon the second antibody 3 of bag quilt also can with combine with first antibody 1 bonded antigen 2, form the clamping body.Shown in Fig. 1 d, after with the photosensitive microballoon of laser irradiation, the photosensitizers in the photosensitive microballoon can be emitted singlet oxygen, after this singlet oxygen is captured by the light emitting composition in the luminous microballoon, understand the luminophor in the stimulated luminescence composition, send the light of specific wavelength.Just can measure antigenic amount by the light that detects this wavelength.
Illustrate in greater detail the present invention below in conjunction with accompanying drawing.
One. photosensitive microballoon
Nano level microballoon matrix as the photosensitive microballoon 10 of the present invention is microballoon matrix known in the art, and for example, it can be for example carboxy-modified latex particle.This particulate details can be referring to United States Patent (USP) 5,709,994 and 5,780,646 (document this draw in full be with reference to).
The photosensitizers that is used for photosensitive microballoon is without particular limitation, and it can be a photosensitizers known in the art, and its indefiniteness example for example has United States Patent (USP) 5,709,994 disclosed methylene blues, rose-red, porphyrin, phthalocyanine and chlorophyll.
The carrier band amount of photosensitizers is without particular limitation in photosensitive microballoon, and it can be this area amount commonly used.The method for coating that is applicable to photosensitive microballoon of the present invention and first antibody can be for example referring to United States Patent (USP) 5,709, and 994 and 5,780,646.
Being used for photosensitizers is added the method for Nano microsphere and wraps by the method for this photosensitive microballoon can be this area any method commonly used.This method can be for example referring to United States Patent (USP) 5,709, and 994 and 5,780,646.
Two. luminous microballoon
1. matrix
Nano level microballoon matrix as the luminous microballoon 20 of the present invention is microballoon matrix known in the art, and it can be same or inequality with described photosensitive microballoon matrix phase.For example, it can be for example carboxy-modified latex particle.This particulate details can be referring to United States Patent (USP) 5,780,646 and 5,709,994 (document this draw in full be with reference to).
2. light emitting composition
Contained light emitting composition comprises a kind of with europium complex and a kind of compound that contain unsaturated ethylene linkage of compound of di-butanedione as ligand in the luminous microballoon of the present invention.These two kinds of components contents are without particular limitation, as long as can make the luminous efficiency of said composition satisfy final immunoassay.In a better example of the present invention, described europium complex accounts for the 10-400mg/g microballoon, better accounts for the 50-200mg/g microballoon, better accounts for the 100mg/g microballoon; The described compound that contains unsaturated ethylene linkage better accounts for the 50-500mg/g microballoon, better accounts for the 100-300mg/g microballoon, preferably accounts for the 200mg/g microballoon.
A. the compound that contains ethylene linkage
The compound that contains ethylene linkage that is applicable to the present composition has following general formula:
Wherein, X ' is S or NR, and R has the alkyl of 1-10 carbon atom or the aryl of 6-18 carbon atom;
R ' is hydrogen atom or the alkyl with 1-10 carbon atom;
Respectively do for oneself hydrogen atom or have the alkyl of 1-20 carbon atom of D and D '.
In the present invention, X ' can be NR, and wherein R has the straight or branched alkyl of 1-10 carbon atom or the replacement or the unsubstituted aryl of 6-18 carbon atom.In a better example of the present invention, described R group is the straight or branched alkyl with 1-6 carbon atom, is preferably the straight or branched alkyl of 1-4 carbon atom.Of the present invention another preferably in the example, described R group is the aryl with 6-18 carbon atom, it is selected from phenyl for example, had the phenyl that the alkyl of 1-10 carbon atom replaces; The example of described alkyl substituent has methyl, ethyl, propyl group, butyl, isobutyl-, the tertiary butyl, n-pentyl, isopentyl, hexyl, heptyl, octyl group, iso-octyl etc.
In a better example of the present invention, the described compound that contains ethylene linkage has following general formula:
Wherein, D and D ' are selected from hydrogen atom separately or have the straight or branched alkyl of 1-20 carbon atom,
Good for having 6-20 carbon atom straight chain or branched-chain alkyl, more preferably having the straight or branched alkyl of 6-15 carbon atom, be preferably (CH
2)
5CH
3Perhaps (CH
2)
13CH
3
R ' is for hydrogen atom or have the straight or branched alkyl of 1-10 carbon atom, is preferably the straight or branched alkyl that has 1-6 carbon atom straight chain or branched-chain alkyl, more preferably has 1-4 carbon atom, is preferably (CH
2)
6CH
3Perhaps (CH
2)
7CH
3
In another better example of the present invention, the described compound that contains ethylene linkage has following general formula:
Wherein, D and D ' are selected from (CH separately
2)
5CH
3Perhaps (CH
2)
13CH
3
R ' is hydrogen atom, (CH
2)
6CH
3Perhaps (CH
2)
7CH
3
The present invention another preferably in the example, the described compound that contains ethylene linkage is following compound:
Each substituent connotation A that sees the following form wherein:
Table A
Compounds X Ar Ar '
1????????????????O?????????
2????????????????S?????????
3????????????????S?????????
4????????????????S?????????
5????????????????S?????????????????????
6?????????????
7?????????????
The above-mentioned synthetic method that contains the compound of ethylene linkage of the present invention is well-known in the art.For example, it can adopt United States Patent (USP) 5,780, and 646 described methods are synthetic.
B. di-butanedione ligand
The di-butanedione ligand that is applicable to europium complex of the present invention has following general formula:
Wherein, X is the aryl that has 1-5 carbon atom straight chain alkyl or have following formula, and wherein Y is hydrogen atom or the straight or branched alkyl substituent with 1-10 carbon atom:
Perhaps
In a better example of the present invention, described X group is selected from methylene radical, ethylidene, propylidene, butylidene, adjacent phenylene, the 3-methyl isophthalic acid, the 2-phenylene, the 4-methyl isophthalic acid, the 2-phenyl, 3-ethyl-1, the 2-phenylene, 4-ethyl-1, the 2-phenyl, 2, the 3-naphthylidene, 1, the 8-naphthylidene, the 4-methyl isophthalic acid, the 8-naphthylidene, 3-amyl group-1, the 2-phenylene, 4-hexyl-1, the 2-phenyl, 5-heptyl-2, the 3-naphthylidene, 5-(2 '-the methyl hexyl)-2, the 3-naphthylidene, 4-ethyl-1, the 8-naphthylidene, the 5-methyl isophthalic acid, the 8-naphthylidene, 4-propyl group-1, the 8-naphthylidene, 4-methyl-2, the 3-naphthylidene, 4-ethyl-2, the 3-naphthylidene, 5-methyl-2, the 3-naphthylidene, 4-propyl group-2, the 3-naphthylidene.
The indefiniteness example that is applicable to compound of di-butanedione of the present invention has, for example following compounds:
1) 1,1-two (to (1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) methane;
2) 1,2-two (to (1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) ethane;
3) 1,3-two (to (1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) propane;
4) 1,6-two (to (1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) butane;
5) 4,4 '-two (1 ", 1 ", 1 " three fluoro-2 ", the adjacent terphenyl of 4 " dimethyl diketone-4 "-yl);
6) 3-methyl isophthalic acid, 2-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) benzene;
7) 4-methyl isophthalic acid, 2-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) benzene;
8) 3-ethyl-1,2-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) benzene;
9) 4-ethyl-1,2-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) benzene;
10) 2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
11) 5-heptyl-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
12) 5-(2 " methyl hexyl)-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
13) 4-methyl-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
14) 4-ethyl-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
15) 5-methyl-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
16) 4-propyl group-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene.
17) 1,8-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
18) 5-heptyl-1,8-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
12) 5-(2 " methyl hexyl)-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
13) 4-methyl isophthalic acid, 8-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
14) 4-ethyl-1,8-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
15) 5-methyl isophthalic acid, 8-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
16) 4-propyl group-1,8-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene.
With X is that phenyl is an example, in a better example of the present invention, described ligand is X compound and the acetyl halide reaction that phenylbenzene is replaced, form the X compound that two (to the methyl carbonyl phenyls) replace, make the X compound and the Trifluoroacetic Acid Ethyl Ester reaction of two (to the methyl carbonyl phenyls) replacement that obtains subsequently again, generate compound of di-butanedione of the present invention.The synthesis path of compound of di-butanedione of the present invention is as follows:
C. with the europium complex of di-butanedione ligand of the present invention
Europium complex of the present invention can be used general formula E u
2(X ")
3(DYE)
2Expression, wherein, DYE is this area dyestuff commonly used, its indefiniteness example has for example TOPO (trioctylphosphine oxide (TOPO)) or DPP.Better be DPP (4,7-phenylbenzene-1,10-phenanthroline), it is a class dyestuff commonly used, and it has following array structure:
Ligand X in this title complex " be compound of di-butanedione of the present invention, and dyestuff DYE can adopt this area method commonly used to make, or buy from chemical company.
3. the manufacture method of luminescent ball
Method and the bag of described europium complex being put into the nano luminescent ball can be this area any methods commonly used by the method for this luminescent ball.In a better example of the present invention, adopt United States Patent (USP) 5,780,646 disclosed luminescent ball manufacture method, just the europium complex with band di-butanedione ligand of the present invention replaces its metallo-chelate.
In another better example of the present invention, the bag that the novel light-emitting composition that uses europium complex of the present invention to form can not influence light-emitting particles is by capacity and significantly improved the luminous efficiency of luminescent ball, especially improve detected signal value, thereby reduced measuring error.
Below, further specify the present invention in conjunction with the embodiments:
Testing method
1. bag is by capacity
The dextran standardized solution of getting one group of different concns adds the 1mL anthrone respectively, hatches 30 minutes at 85 ℃.After being cooled to room temperature, measuring the absorption photometric value (Hitachi U-3000 type spectrophotometer available from HIT) of different solutions and make typical curve at the 636nm place.
Bag is added the 1ml anthrone subsequently by dextran on sample nano luminescent ball, at 85 ℃, hatches 30 minutes.After being cooled to room temperature, measure its absorption photometric value, calculate the bag of microsphere surface by capacity according to typical curve at the 636nm place.
2. luminous efficiency
Get the sensitization ball that is coated with antibody (50 μ g) and luminescent ball (6.25 μ g) of equal-volume 500 μ l, add 50 μ l antigens and mix as the 50mM phosphate buffered saline buffer (available from Sigma) that detects damping fluid with 100 μ l.37 degree incubations utilized PMT photon counter (available from Pengyuantai Biotech Co., Ltd., Shanghai) to read number of photons after 15 minutes.
Synthesis example 1
The synthetic compound (A) that contains unsaturated ethylene linkage: 34.4g (0.2mol) bromobenzene amine solvent in 125ml DMF, is added 0.6mol bromotetradecane and 0.6mol diisopropylethylamine, and reaction soln is heated to 100 ℃, reacts then 12 hours.The solution of reaction adds the 300ml methylene dichloride, washes and removes DMF three times.Remove methylene dichloride, the product ethyl alcohol recrystallization.Obtain N, the dibasic para-bromoaniline of N-(formula I compound), the productive rate of reaction is 70%
In an I ml three-necked round bottom flask that dropping funnel, thermometer and reflux condensing tube be housed, add 100ml exsiccant THF, add 3g metal magnesium rod subsequently, under refluxad add the N that obtains above on a small quantity, the dibasic para-bromoaniline product of N-(formula I compound) reacts to start, and adds a spot of iodine to help the reaction startup.With the above-mentioned N of 0.11mol, the dibasic para-bromoaniline product of N-(formula I compound) is dissolved among the 50ml exsiccant THF, and places addition funnel, gently drips this solution and keeps backflow.Finish relief reaction of liquid feeding refluxed 1 hour.Use the ice bath cooling reaction system, ℃ the time gently drip the 50ml THF solution of 0.1mol following formula (II) compound (available from Aldrich/Sigma).Allow be reflected at 0 ℃ and carried out 30 minutes, be positioned over room temperature then 2 hours.Gently add the magnesium salts that 0.1N HCl solution generates with hydrolysis.Add 200mlCH
2Cl
2, wash organic layer 2-3 time then with water.The organic layer anhydrous sodium sulfate drying.Separate desired product with silicagel column.With 20% ethyl acetate, 80% hexane solution separating product, the Rf value of product is about 0.5.(product (formula III compound) productive rate behind the purifying of 20% ethyl acetate/80% hexane/TLC) is 35%.
Above-mentioned formula III compound dissolution adds a small amount of ZnCl in 100ml toluene with 0.1mol in a 500ml round-bottomed flask that has a prolong
2And 0.11mol formula IV compound.Backflow is also removed the water that generates.Reacting reaction in 4 hours finishes substantially.Remove unnecessary toluene with Rotary Evaporators.Use the last product of silicagel column separation and purification (using 95 hexanes, 5% ethyl acetate) then, obtain compd A, productive rate is 60%.
1H NMR:7.0ppm-7.26ppm (multiplet, 7H), 6.42ppm-6.44ppm (multiplet, 2H), and 4.48ppm-4.50ppm (multiplet, 2H), 3.16ppm-3.32ppm (multiplet, 4H), and 1.52ppm-1.57ppm (multiplet, 4H), 1.3ppm (multiplet, 44H), and 0.86ppm (3 heavy peaks, 6H).
Synthesis example 2
The synthetic compound (B) that contains unsaturated ethylene linkage
34.4g (0.2mol) bromobenzene amine solvent in 125ml DMF, is added 0.6mol butyl bromide and 0.6mol diisopropylethylamine, and reaction soln is heated to 100 ℃, reacts then 12 hours.The solution of reaction adds the 300ml methylene dichloride, washes and removes DMF three times.Remove methylene dichloride, the product ethyl alcohol recrystallization.Obtain N, the dibasic para-bromoaniline of N-(formula 1 compound), the productive rate of reaction is 70%
In a 500ml three-necked round bottom flask that dropping funnel, thermometer and reflux condensing tube be housed, add 100ml exsiccant THF, add 3g metal magnesium rod subsequently, under refluxad add the N that obtains above on a small quantity, the dibasic para-bromoaniline product of N-(formula 1 compound) is to start reaction.With the above-mentioned N of 0.11mol, the dibasic para-bromoaniline product of N-(formula 1 compound) is dissolved among the 50ml exsiccant THF, and places addition funnel, gently drips this solution and keeps backflow.Finish relief reaction of liquid feeding refluxed 1 hour.Use the ice bath cooling reaction system, ℃ the time gently drip the 50ml THF solution of 0.1mol following formula (2) compound (available from Aldrich/Sigm).Allow be reflected at 0 ℃ and carried out 30 minutes, be positioned over room temperature then 2 hours.Gently add the magnesium salts that 0.1N HCl solution generates with hydrolysis.Add 200mlCH
2Cl
2, wash organic layer 2-3 time then with water.The organic layer anhydrous sodium sulfate drying.Separate desired product with silicagel column.With 20% ethyl acetate, 80% hexane solution separating product, the Rf value of product is about 0.5.(product (the formula 3 compounds) productive rate behind the purifying of 20% ethyl acetate/80% hexane/TLC) is 35%.
Above-mentioned formula 3 compound dissolutions add a small amount of ZnCl in 100ml toluene with 0.1mol in a 500ml round-bottomed flask that has a prolong
2And 0.11mol formula 4 compounds.Backflow is also removed the water that generates.Reacting reaction in 4 hours finishes substantially.Remove unnecessary toluene with Rotary Evaporators.Use the last product of silicagel column separation and purification (using 95 hexanes, 5% ethyl acetate) then, obtain compd B, productive rate is 60%.
1H NMR 7.0ppm-7.26ppm (multiplet, 6H), 6.42ppm-6.44ppm (multiplet, 2H), 4.48ppm-4.50ppm (multiplet, 2H), 3.16ppm-3.32ppm (multiplet, 4H), 2.2ppm (singlet, 3H) .1.52ppm-1.57ppm (multiplet, 4H), 0.86ppm (3 heavy peaks, 6H).1.3ppm (multiplet, 4H).
Synthesis example 3
(1) the adjacent phenyl biphenyl of 2.3g is dissolved in 20ml exsiccant CH
2Cl
2In the solution, add 2.94g AlCl
3, be cooled to 0 ℃ then.The CH that under violent stirring, adds 2g
3C (O) Cl (available from Aldrich/Sigma).Be added dropwise to complete relief and be reflected at room temperature reaction 1-2 hour.The organic solution water is given a baby a bath on the third day after its birth inferior, uses anhydrous sodium sulfate drying then.Obtain 2.6g product (81% productive rate) after removing solvent.
1H NMR:7.1-7.9ppm (multiplet, 12H), 2.3ppm (singlet, 6H)
(2) the above-mentioned product of 1.6g is dissolved in the anhydrous diethyl ether of 20ml, adds the CH of 1g
3ONa (sodium methylate) and 2g CF
3-C (O) OEt (Trifluoroacetic Acid Ethyl Ester).Allow reaction at room temperature stir 2-3 hour.Water is given a baby a bath on the third day after its birth inferior then.The organic layer anhydrous sodium sulfate drying obtains 1.6g product (productive rate 80%) after removing organic solution
1H NMR 7.1-7.9ppm (multiplet, 12H), 4.5ppm (unimodal, 4H)
Synthesis example 4
(1), the 1.7g ditane is dissolved in 20 exsiccant CH
2Cl
2In the solution, add 2.94g AlCl
3, be cooled to 0 ℃ then.The CH that under violent stirring, adds 2g
3C (O) Cl.Be added dropwise to complete afterreaction at room temperature reaction 1-2 hour.The organic solution water is given a baby a bath on the third day after its birth inferior, uses anhydrous sodium sulfate drying then.Obtain 1.4g product (70% productive rate) after removing solvent
1H NMR:7.6-7.9 (doublet, 4H), 3.1 (singlet, 2H), 2.3 (singlet, 6H)
(2) the above-mentioned product of above-mentioned 1.3g is dissolved in the anhydrous diethyl ether of 20ml, adds the CH of 1g
3ONa (methyl alcohol is received) and 2g CF
3-COOEt (Trifluoroacetic Acid Ethyl Ester).Allow reaction at room temperature stir 2-3 hour.Water is given a baby a bath on the third day after its birth inferior then.The organic layer anhydrous sodium sulfate drying obtains 1.2g product (productive rate 80%) after removing organic solution
1H NMR:7.6-7.9 (doublet, 4H), 4.5 (singlet, 4H), 3.1 (singlet, 2H).
Synthesis example 5
(1), the adjacent phenyl biphenyl of 2.5g methyl is dissolved in 20 exsiccant CH
2Cl
2In the solution, add 2.94g AlCl
3, be cooled to 0 ℃ then.The CH that under violent stirring, adds 2g
3C (O) Cl.Be added dropwise to complete afterreaction at room temperature reaction 1-2 hour.The organic solution water is given a baby a bath on the third day after its birth inferior, uses anhydrous sodium sulfate drying then.Obtain 1.4g product (70% productive rate) after removing solvent
1H NMR:7.6-7.9 (multiplet, 7H), 2.1 (singlet, 3H), 2.3 (singlet, 6H)
(2) the above-mentioned product of above-mentioned 1.7g is dissolved in the anhydrous diethyl ether of 20ml, adds the CH of 1g
3ONa (methyl alcohol is received) and 2g CF
3-COOEt (Trifluoroacetic Acid Ethyl Ester).Allow reaction at room temperature stir 2-3 hour.Water is given a baby a bath on the third day after its birth inferior then.The organic layer anhydrous sodium sulfate drying obtains 1.6g product (productive rate 80%) after removing organic solution
1H NMR:7.6-7.9 (multiplet, 7H), 4.5 (singlet, 4H), 2.1 (singlet, 3H).
Synthesis example 6
Synthetic ligand compound 4
(1), the adjacent phenylbenzene naphthalene of 2.9g is dissolved in 20 exsiccant CH
2Cl
2In the solution, add 2.94gAlCl
3, be cooled to 0 ℃ then.The CH that under violent stirring, adds 2g
3C (O) Cl.Be added dropwise to complete afterreaction at room temperature reaction 1-2 hour.The organic solution water is given a baby a bath on the third day after its birth inferior, uses anhydrous sodium sulfate drying then.Obtain 1.4g product (70% productive rate) after removing solvent
1H NMR:7.3-7.9 (multiplet, 10H), 2.3 (singlet, 6H)
(2) the above-mentioned product of above-mentioned 1.9g is dissolved in the anhydrous diethyl ether of 20ml, adds the CH of 1g
3ONa (methyl alcohol is received) and 2g CF
3-COOEt (Trifluoroacetic Acid Ethyl Ester).Allow reaction at room temperature stir 2-3 hour.Water is given a baby a bath on the third day after its birth inferior then.The organic layer anhydrous sodium sulfate drying obtains 1.8g product (productive rate 80%) after removing organic solution
1H NMR:7.3-7.9 (multiplet, 10H), 4.5 (singlet, 4H).
The europium complex of the two two butanone ligands of anamorphic zone
With 2.932 gram EuCl
36H
2O mixes with the ligand compound 1 that 6.39 grams make above, adds the 40mL anhydrous alcohol solution.Stir and add 2mL NH
3H
2O, pH are about 8.Stirring at room 1 hour.Solution is joined in the 800mL deionized water, filtering solution, collecting precipitation is 10.3g.Obtain Eu (ligand compound 1) after the vacuum-drying
2H
2O, purity is about 90%.
Get 4.0 gram DPP and 120mL ethanol heating for dissolving, the recrystallization crystal is collected in the cooling back.Take by weighing 3.014 gram Eu (ligand compound 1)
2H
2O and 0.997 gram DPP add 300mL toluene, 62 ℃ of stirring reactions one hour.Cool to room temperature utilizes rotary evaporation to remove toluene.Europium complex 1 is collected in vacuum-drying.
Make luminescent ball
With United States Patent (USP) 5,780, the described method of 646 embodiment with 200mg synthesis example 1 europium complex 1 that ethylene linkage compd A and 100mg make above containing of making put into 200nm nano particle (available from U.S. BangsLaboratories, Inc) in, form luminescent ball Eu-A.
Coated antibody
Get the above-mentioned luminescent ball (20mg/mL that makes of 10mL, the 50mM phosphate buffered saline buffer, dye inside is to contain the europium complex 1 that ethylene linkage compd A and the present invention make), add 40mg Amino-Dextran (4%, buffer system 0.05Mes buffer), 20mg EDAC (4%, buffer system 50mM phosphate buffered saline buffer).The stirring at room reaction is spent the night.Centrifugal 1 hour of 4 ℃ of Sorvall SA-600 rotary heads, rotating speed is 15,000rpm.Abandon supernatant liquor after centrifugal, with 8mL 50mM phosphate buffered saline buffer resuspension.Repeated centrifugation is cleaned three times.
Get the above-mentioned luminescent ball (25mg/mL for preparing, the 50mM phosphate buffered saline buffer, dye inside is to contain the europium complex 1 that ethylene linkage compd A and the present invention make, the outside is coated with Dextran) 0.4ml, add 8.16mgIgG1 and press United States Patent (USP) 5,780,646 embodiment, the 1 described luminescent ball of the present invention that makes.
Make the sensitization ball
With United States Patent (USP) 5,780, the described method of 646 embodiment with the 200mg Chlorophyll A put into 200nm nano particle (available from U.S. Bangs Laboratories, Inc) in, and with reference to the method bag of above-mentioned coated antibody by IgG2, form the bright sensitization ball of this law.
Bag is by volumetric determination
The bag that is recorded sample of the present invention by capacity test method with bag recited above be the results are shown in table 1 by capacity.
Record the luminous efficiency of sample with top luminous efficiency test method, the results are shown in table 2.
Make the luminescent ball of embodiment 2 with the method identical, but the ligand compound 2 that is to use synthesis example 2 synthetic band ethylenic unsaturation key compound B and synthesis example 4 to make makes luminescent ball Eu-B with embodiment 1.
Use bag that sensitization ball identical with embodiment 1 and bag recorded sample by the capacity test method by capacity, the results are shown in table 1
Record the luminous efficiency of sample with top luminous efficiency test method, the results are shown in table 2.
Make the luminescent ball of embodiment 3 with the method identical, but the ligand compound 3 that is to use synthesis example 1 synthetic band ethylenic unsaturation key compound A and synthesis example 5 to make makes luminescent ball Eu-C with embodiment 1.
Use bag that sensitization ball identical with embodiment 1 and bag recorded sample by the capacity test method by capacity, the results are shown in table 1
Record the luminous efficiency of sample with top luminous efficiency test method, the results are shown in table 2.
Embodiment 4
Make the luminescent ball of embodiment 4 with the method identical, but the ligand compound 4 that is to use synthesis example 2 synthetic band ethylenic unsaturation key compound B and synthesis example 6 to make makes luminescent ball Eu-D with embodiment 1.
Use bag that sensitization ball identical with embodiment 1 and bag recorded sample by the capacity test method by capacity, the results are shown in table 1
Record the luminous efficiency of sample with top luminous efficiency test method, the results are shown in table 2.
Comparative example 1
Make luminescent ball with the method identical, but be to use TTA (thiophene trifluoro dimethyl diketone) to replace with embodiment 1
Di-butanedione ligand of the present invention makes europium complex:
Embodiment 5
Make the luminescent ball of embodiment 5 with the method identical with embodiment 1, but be to use according to United States Patent (USP) 5,780, the ligand compound 4 that band ethylenic unsaturation key compound 2 that the last Table A that 646 embodiment 2 make is listed and synthesis example 6 make makes luminescent ball Eu-E.
Use bag that sensitization ball identical with embodiment 1 and bag recorded sample by the capacity test method by capacity, the results are shown in table 1
Record the luminous efficiency of sample with top luminous efficiency test method, the results are shown in table 2.
Embodiment 6
Make the luminescent ball of embodiment 6 with the method identical with embodiment 1, but be to use according to United States Patent (USP) 5,780, the ligand compound 3 that band ethylenic unsaturation key compound 3 that the last Table A that 646 embodiment 3 make is listed and synthesis example 5 make makes luminescent ball Eu-F.
Use bag that sensitization ball identical with embodiment 1 and bag recorded sample by the capacity test method by capacity, the results are shown in table 1
Record the luminous efficiency of sample with top luminous efficiency test method, the results are shown in table 2.
Embodiment 7
Make the luminescent ball of embodiment 7 with the method identical with embodiment 1, but be to use according to United States Patent (USP) 5,780, the ligand compound 2 that band ethylenic unsaturation key compound 5 that the last Table A that 646 embodiment 4 make is listed and synthesis example 4 make makes luminescent ball Eu-G.
Use bag that sensitization ball identical with embodiment 1 and bag recorded sample by the capacity test method by capacity, the results are shown in table 1
Record the luminous efficiency of sample with top luminous efficiency test method, the results are shown in table 2.
Embodiment 8
Make the luminescent ball of embodiment 8 with the method identical with embodiment 1, but be to use according to United States Patent (USP) 5,780, the ligand compound 1 that band ethylenic unsaturation key compound 6 that the last Table A that 646 embodiment 5 make is listed and synthesis example 3 make makes luminescent ball Eu-H.
Use bag that sensitization ball identical with embodiment 1 and bag recorded sample by the capacity test method by capacity, the results are shown in table 1
Record the luminous efficiency of sample with top luminous efficiency test method, the results are shown in table 2.
Embodiment 9
Make the luminescent ball of embodiment 9 with the method identical with embodiment 1, but be to use according to United States Patent (USP) 5,780, the ligand compound 1 that band ethylenic unsaturation key compound 7 that the last Table A that 646 embodiment 6 make is listed and synthesis example 3 make makes luminescent ball Eu-I.
Use bag that sensitization ball identical with embodiment 1 and bag recorded sample by the capacity test method by capacity, the results are shown in table 1
Record the luminous efficiency of sample with top luminous efficiency test method, the results are shown in table 2.
For comparing, table 1 gives the bag of blank microballoon (the 50mM phosphate buffered saline buffer, inner no dyestuff, the outside is coated with Dextran for 200nm, 25mg/mL) by capacity, the results are shown in table 1.
Make the luminescent ball of embodiment 10 with the method identical, make luminescent ball Eu-J but be to use TOPO to replace DPP with embodiment 1.
Use bag that sensitization ball identical with embodiment 1 and bag recorded sample by the capacity test method by capacity, the results are shown in table 1
Record the luminous efficiency of sample with top luminous efficiency test method, the results are shown in table 2.
Table 1
| Sample | The package amount of amino-polysaccharide (ug/mg particulate) |
| Comparative example 1 | ??41.3 |
| | ??62.1 |
| | ??60.7 |
| | ??62.3 |
| Embodiment 4 | ??60.4 |
| Embodiment 5 | ??60.2 |
| Embodiment 6 | ??61.5 |
| Embodiment 7 | ??60.1 |
| Embodiment 8 | ??59.9 |
| Embodiment 9 | ??61.6 |
| | ??60.5 |
| Blank ball | ??61.2 |
Table 2
| Sample | Photon counting |
| Comparative example 1 | ??1,000,000 |
| | ??1,550,000 |
| | ??1,400,000 |
| | ??1,460,000 |
| Embodiment 4 | ??1,370,000 |
| Embodiment 5 | ??1,320,000 |
| Embodiment 6 | ??1,580,000 |
| Embodiment 7 | ??1,340,000 |
| Embodiment 8 | ??1,290,000 |
| Embodiment 9 | ??1,430,000 |
| | ??1,275,000 |
By test-results as seen, insert Nano microsphere with light emitting composition of the present invention after, the bag of this microballoon is suitable with blank ball by capacity, and after adopting existing europium complex bag by Nano microsphere, its bag is obviously descended by capacity.On the other hand, by table 2 as seen, if the luminous efficiency of existing luminescent ball is decided to be 1, then the luminous efficiency of luminescent ball of the present invention is respectively 1.5 and 1.4, apparently higher than the luminous efficiency of existing luminescent ball.Above-mentioned evidence, the present invention is by changing the coordination compound of europium complex, and the bag that has greatly improved Nano microsphere has been obtained beyond thought effect by capacity and luminous efficiency.
Under the situation that does not depart from invention thought of the present invention, those of ordinary skill in the art can easily carry out various changes and modifications to the present invention.Present invention resides in the various changes and modifications in the claims scope.
Claims (12)
1. compound of di-butanedione with following general formula:
Wherein: X has 1-5 carbon atom straight chain alkyl; Perhaps
Aryl with following formula, wherein Y is the straight or branched alkyl substituent of 1-5 carbon atom:
2. as claimed in claim 1 pair of butanone compound, it is characterized in that described X group be selected from straight chained alkyl with 1-3 carbon atom,
3. as claimed in claim 1 or 2 pair of butanone compound is characterized in that x is selected from methylene radical or adjacent phenylene.
4. as claimed in claim 1 pair of butanone compound is characterized in that it is selected from:
1) 1,1-two (to (1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) methane;
2) 1,2-two (to (1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) ethane;
3) 1,3-two (to (1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) propane;
4) 1,6-two (to (1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) butane;
5) 4,4 '-two (1 ", 1 ", 1 " three fluoro-2 ", the adjacent terphenyl of 4 " dimethyl diketone-4 "-yl);
6) 3-methyl isophthalic acid, 2-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) benzene;
7) 4-methyl isophthalic acid, 2-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) benzene;
8) 3-ethyl-1,2-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) benzene;
9) 4-ethyl-1,2-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) benzene;
10) 2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
11) 5-heptyl-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
12) 5-(2 " methyl hexyl)-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
13) 4-methyl-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
14) 4-ethyl-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
15) 5-methyl-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
16) 4-propyl group-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene.
17) 1,8-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
18) 5-heptyl-1,8-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
12) 5-(2 " methyl hexyl)-2,3-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
1 3) 4-methyl isophthalic acid, 8-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
14) 4-ethyl-1,8-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
15) 5-methyl isophthalic acid, 8-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene;
16) 4-propyl group-1,8-two (4-(1 ', 1 ', 1 '-three fluoro-2 ', 4 '-dimethyl diketone-4 '-yl) phenyl) naphthalene.
5. chemical luminous composite, it comprises:
A) europium complex, this europium complex have among the claim 1-4 each described compound of di-butanedione as ligand;
What 2) have a following general formula contains the ethylene linkage compound:
Wherein, X ' is S or NR, and R has the alkyl of 1-10 carbon atom or the aryl of 6-18 carbon atom;
R ' is hydrogen atom or the alkyl with 1-10 carbon atom;
Respectively do for oneself hydrogen atom or have the alkyl of 1-20 carbon atom of D and D '.
6. composition as claimed in claim 5 is characterized in that X ' is a sulphur atom.
7. as claim 5 or 6 described compositions, it is characterized in that R ' is the alkyl of 1-4 carbon atom.
8. as claim 5 or 6 described compositions, it is characterized in that D and D ' are selected from hydrogen atom separately or have the alkyl of 4-14 carbon atom.
9. composition as claimed in claim 7 is characterized in that D and D ' are selected from hydrogen atom, butyl or tetradecyl separately.
10. composition as claimed in claim 5, the compound that it is characterized in that containing ethylene linkage is the compound with following structure:
Wherein each substituting group is selected from:
11. an europium complex, it contains among the claim 1-4 each described compound as ligand.
12. the purposes of europium complex as claimed in claim 11 in immunoassay.
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