CN105733559B - The preparation method and application of oxamido- quinoline fluorescent molecular probe - Google Patents
The preparation method and application of oxamido- quinoline fluorescent molecular probe Download PDFInfo
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Abstract
The preparation method and application of oxamido- quinoline fluorescent molecular probe, it is related to the synthetic method of bioluminescence molecular probe and application.The present invention is to solve existing molecular probe synthesis material species is more, the technical problem of synthesis condition harshness.The general structure of oxamido- quinoline fluorescent molecular probe is：Wherein R is alkyl amino or arylamino.Preparation method one：8 aminoquinolines and diethy-aceto oxalate are reacted after preparing intermediate, then reacted with amine, obtain mono-substituted oxamido- quinoline fluorescent molecular probe；Preparation method two：8 aminoquinolines are added in dichloromethane with diethy-aceto oxalate and are reacted, after purification, obtain disubstituted oxamido- quinoline fluorescent molecular probe.Molecular probe can be used for detecting zinc ion, and zinc ion is the zinc ion in zinc ion or life entity in aqueous solution.
Description
Technical field
Synthetic method and application the present invention relates to bioluminescence molecular probe.
Background technology
Aminoquinoline and its derivative are due to having the advantages that stability is good, molecule is easily modified and good biocompatibility quilt
Fluorescent optical sensor is made and is widely used in the fields such as medicine, dyestuff, pesticide, for quantitative detection metal ion.Such as Zhang Yu, Guo Xiang
Peak etc. exists《Organic chemistry bulletin》The 473-476 pages article delivered of 2008 the 10th phases of (Organic Letters)《With alkane
Oxygen ethylamine chain as conjugated group carboxamide groups quinoline as fluorescent molecular probe in aqueous to zinc ion
Ratio identification》(Ratiometric and Water-Soluble Fluorescent Zinc of
Carboxamidoquinoline with an Alkoxyethylamino Chain as Receptor) disclose a kind of second
Amide groups quinoline fluorescent molecular probe, the detection zinc ion that such fluorescent molecular probe can be selective, the fluorescence point
The preparation of sub- probe be first 2- chloracetyl chlorides and 8- aminoquinolines reaction, it is purified to obtain intermediate, then by intermediate with
2- (2- amino ethoxies) ethanol, n,N-diisopropylethylamine and potassium iodide are added in acetonitrile, stir back in a nitrogen atmosphere
Flow 10 it is small when, obtain acetamido quinoline fluorescent molecular probe AQZ after purification.This kind of fluorescent molecular probe obtains afterwards
To extensive use.But the raw material type needed for this molecular probe synthesis is more, and synthesis will carry out in nitrogen atmosphere, close
Harsh into condition, present invention design has synthesized a kind of oxalyl amido quinoline fluorescent molecular probe.
The content of the invention
The present invention is to solve existing molecular probe synthesis material species is more, the technical problem of synthesis condition harshness, and
The preparation method and application of a kind of oxamido- quinoline fluorescent molecular probe are provided, have widened the spy of quinolines fluorescence molecule
The species of pin.
The general structure of oxamido- quinoline fluorescent molecular probe of the present invention is：
Wherein R is alkoxy, amino, alkyl amino, arylamino or hydroxy alkyl.
The preparation method of above-mentioned oxamido- quinoline fluorescent molecular probe is as follows：
First, it is 1 according to molar ratio by 8- aminoquinolines and diethy-aceto oxalate：(5~10) 150 are warming up to after mixing ,~
220 DEG C, 3~12h is reacted, reaction mixture is obtained, after reaction mixture is purified, obtains intermediate, represented with EOQ；
2nd, EOQ is mixed with amine according to 1 ﹕ of molar ratio (2~10), is warming up to 80~150 DEG C, react 2~12h, obtained thick
Product I；After obtained crude product I is purified, mono-substituted oxamido- quinoline fluorescent molecular probe is obtained；It is therein
Amine is ammonia, organic primary amine or secondary amine.
The preparation method of above-mentioned oxamido- quinoline fluorescent molecular probe can also carry out according to the following steps：
By 8- aminoquinolines and diethy-aceto oxalate according to molar ratio for (2~10) ﹕ 1 are mixed and added into dichloromethane, are risen
Temperature obtains crude product II, after crude product II is purified, obtains disubstituted oxamido- quinoline to 40~100 DEG C of 3~12h of reaction
Quinoline derivant fluorescent molecular probe.
The oxamido- quinoline fluorescent molecular probe of the present invention, can be used for detecting zinc ion, zinc ion is water
The zinc ion in zinc ion or life entity in solution.
Wherein, the method for qualitative detection aqueous zinc is specifically：
First, the pH value of oxamido- quinoline fluorescent molecular probe solution is adjusted between 6~9, and tests and be somebody's turn to do
The fluorescence spectrum of solution, reads the wavelength value A of fluorescence emission peak position1And probe is in wavelength value A1The fluorescence intensity at place
Value B1；
2nd, aqueous solution to be measured is added in oxamido- quinoline fluorescent molecular probe solution, test again should
The fluorescence spectrum of solution, reads the wavelength value A of fluorescence emission peak position2And its in A2The fluorescence intensity level B at place2；
If the 3rd, A2＞ A1And B2＞ 10B1, then contain zinc ion in aqueous solution to be measured, otherwise do not have in aqueous solution to be measured
Zinc ion.
The method for quantitatively detecting aqueous zinc is calibration curve method, specific as follows：
First, the standard series sample containing zinc ion of various concentrations is prepared, it is glimmering to be separately added into oxamido- quinoline
Optical molecule probe solution, and adjust between pH value 6~9, and the fluorescence spectrum of solution is tested, read the glimmering of maximum fluorescence emission
Light intensity value, marks using the concentration of zinc ion to be horizontal, is denoted as figure using the fluorescence intensity level of maximum fluorescence emission to be vertical, draw mark
Directrix curve；
2nd, to will in the aqueous solution to be measured containing zinc ion add oxamido- quinoline fluorescent molecular probe solution,
Under the same conditions, the fluorescence spectrum of the solution is being tested with step 1, the fluorescence intensity level of maximum fluorescence emission is being read, from mark
The aqueous zinc concentration to be measured containing zinc ion is found on directrix curve.
The detection method of zinc ion in life entity is as follows：
Life entity to be measured is placed into culture 1~6h, Ran Houyong in the aqueous solution containing oxalyl amido quinoline
Fluorescence microscope detects the living cells of life entity, if fluorescence phenomenon, can determine that and contains zinc ion in life entity.
A kind of oxamido- quinoline fluorescent molecular probe of the present invention has the ability of stronger complexing zinc ion,
And after being complexed, cause its fluorescence spectrum red shift, and fluorescence significantly increases, can be not only used for zinc in detection aqueous solution from
Son, and quantitative reckoning is carried out using standard working curve method, but also can be detected by cell imaging in life entity
Zinc ion.As Zn2+Identify probe, various common metal ion (Na can contained+、K+、Mg2+、Ca2+、Cr3+、Fe2+、Co2 +、Ni2+、Cu2+、Ag+、Cd2+、Hg2+、Pb2+、Al3+) single identification Zn in solution2+.This new fluorescence molecule of the present invention is visited
Pin --- -- oxamido- quinoline has the advantages that high selectivity, high sensitivity and easy to operate, and oxalyl amido quinoline
The raw material of quinoline derivant is easy to get, and condition is not harsh, and preparation method is simple.Available for detection aqueous solution or life entity in zinc from
Son.
Brief description of the drawings
Fig. 1 is common metal ion and its and Zn in the experiment of embodiment 112+Influence figure during competition to fluorescence intensity.
Fig. 2 is influence figure of the pH value to fluorescence intensity in the experiment of embodiment 12.
Fig. 3 is the Zn in the experiment of embodiment 132+Influence figure of the concentration to fluorescence intensity at fluorescence spectrum and 490nm.
Fig. 4 is common metal ion and its and Zn in the experiment of embodiment 142+Influence figure during competition to fluorescence intensity.
Embodiment
Embodiment one：The general structure of the oxamido- quinoline fluorescent molecular probe of present embodiment
For：
Wherein R is alkoxy, amino, alkyl amino, arylamino or hydroxy alkyl.
Embodiment two：The system of oxamido- quinoline fluorescent molecular probe described in embodiment one
Preparation Method is as follows：
First, it is 1 according to molar ratio by 8- aminoquinolines and diethy-aceto oxalate：(5~10) 150 are warming up to after mixing ,~
220 DEG C, 3~12h is reacted, reaction mixture is obtained, after reaction mixture is purified, obtains intermediate, represented with EOQ；
2nd, EOQ is mixed with amine according to 1 ﹕ of molar ratio (2~10), is warming up to 80~150 DEG C, react 2~12h, obtained thick
Product I；After obtained crude product I is purified, mono-substituted oxamido- quinoline fluorescent molecular probe is obtained；It is therein
Amine is ammonia, organic primary amine or secondary amine.
Embodiment three：Present embodiment and 8- aminoquinolines unlike embodiment two and oxalic acid diethyl
The molar ratio of ester is 1: 8.It is other identical with embodiment two.
Embodiment four：Reaction temperature in present embodiment step 1 unlike embodiment two or three
For 180~200 DEG C, the reaction time is 8~10h.It is other identical with embodiment two or three.
Embodiment five：EOQ in step 2 unlike one of present embodiment and embodiment two to four
With 1 ﹕ of molar ratio (3~5) of amine.It is other identical with one of embodiment two to four.
Embodiment six：Reacted in step 2 unlike one of present embodiment and embodiment two to five
Temperature is 100~120 DEG C, and the reaction time is 8~10h.It is other identical with one of embodiment two to five.
Embodiment seven：The system of oxamido- quinoline fluorescent molecular probe described in embodiment one
Preparation Method is as follows：By 8- aminoquinolines and diethy-aceto oxalate according to molar ratio for (2~10) ﹕ 1 are mixed and added into dichloromethane,
40~100 DEG C of 3~12h of reaction are warming up to, crude product II is obtained, after crude product II is purified, obtains disubstituted oxamido-
Quinoline fluorescent molecular probe.
Embodiment eight：Present embodiment and 8- aminoquinolines unlike embodiment seven and oxalic acid diethyl
The molar ratio of ester is (5~8) ﹕ 1.It is other identical with embodiment seven.
Embodiment nine：Present embodiment reaction temperature 50~80 unlike embodiment seven or eight
DEG C, the reaction time is 8~10h.It is other identical with embodiment seven or eight.
Embodiment ten：Oxamido- quinoline fluorescent molecular probe described in embodiment one should
With being to be used to oxamido- quinoline fluorescent molecular probe detect zinc ion, zinc ion is the zinc ion in aqueous solution
Or the zinc ion in life entity.
Embodiment 11：Present embodiment utilizes the qualitative inspection of oxamido- quinoline fluorescent molecular probe
The method for surveying aqueous zinc, carries out according to the following steps：
First, the pH value of oxamido- quinoline fluorescent molecular probe solution is adjusted between 6~9, and tests and be somebody's turn to do
The fluorescence spectrum of solution, reads the wavelength value A of fluorescence emission peak position1And probe is in wavelength value A1The fluorescence intensity at place
Value B1；
2nd, aqueous solution to be measured is added in oxamido- quinoline fluorescent molecular probe solution, test again should
The fluorescence spectrum of solution, reads the wavelength value A of fluorescence emission peak position2And its in A2The fluorescence intensity level B at place2；
If the 3rd, A2＞ A1And B2＞ 10B1, then contain zinc ion in aqueous solution to be measured, otherwise do not have in aqueous solution to be measured
Zinc ion.
Embodiment 12：Present embodiment and A in step 3 unlike embodiment 112-A1>
10nm.It is other identical with embodiment 11.
Embodiment 13：Present embodiment is quantitatively examined using oxamido- quinoline fluorescent molecular probe
The method for surveying aqueous zinc is calibration curve method, is specifically carried out according to the following steps：
First, the standard series sample containing zinc ion of various concentrations is prepared, it is glimmering to be separately added into oxamido- quinoline
Optical molecule probe solution, and adjust between pH value 6~9, and the fluorescence spectrum of solution is tested, read the glimmering of maximum fluorescence emission
Light intensity value, marks using the concentration of zinc ion to be horizontal, is denoted as figure using the fluorescence intensity level of maximum fluorescence emission to be vertical, draw mark
Directrix curve；
2nd, to will in the aqueous solution to be measured containing zinc ion add oxamido- quinoline fluorescent molecular probe solution,
Under the same conditions, the fluorescence spectrum of the solution is being tested with step 1, the fluorescence intensity level of maximum fluorescence emission is being read, from mark
The aqueous zinc concentration to be measured containing zinc ion is found on directrix curve.
Embodiment 14：Present embodiment utilizes the qualitative inspection of oxamido- quinoline fluorescent molecular probe
The detection method of the zinc ion in life entity is surveyed, is carried out according to the following steps：
Life entity to be measured is placed into culture (1-6h), Ran Houyong in the aqueous solution containing oxalyl amido quinoline
Fluorescence microscope detects the living cells of life entity, if fluorescence phenomenon, judges to contain zinc ion in life entity.
Further verified to beneficial effects of the present invention with reference to specific embodiment.
Embodiment 1：The fluorescent molecular probe of the present embodiment, is synthesized according to the following steps：
First, the 8- aminoquinolines of 1.00g and 12mL diethy-aceto oxalates are mixed, is heated to 180 DEG C, stir 3h, up to anti-
Answer mixture；Using dichloromethane as eluant, eluent, which is subjected to pillar layer separation, obtained solution is depressurized
Solvent removed by evaporation, obtains white solid, which is represented with EOQ；
2nd, EOQ, 0.357g hydroxy ethoxy ethamine of 0.550g and 10mL ethanol are mixed, are heated to 85 DEG C, stir 2h,
Up to crude product I；Using ethyl acetate as eluant, eluent, crude product I is subjected to pillar layer separation, rotary evaporation removes solvent, obtains
White solid, the product are fluorescent molecular probe, are represented with OAQ.
Using FT-IR,1The EOQ that H-NMR prepares step 2 is characterized.
FT-IR(KBr pellet)v cm-1:3320,2980,2922,1709,1530,1290,595-824；
1H NMR(600MHz,CDCl3):δ ppm 11.39 (s, 1H), 8.90 (t, J=1.8Hz, 1H), 8.80 (d, J=
7.2Hz, 1H), 8.20 (d, J=8.4Hz, 1H), 7.57-7.63 (m, 2H), 7.50 (d, J=4.2Hz, 1H), 4.49 (q, J=
7.2Hz, 2H), 1.48 (t, J=7.2Hz, 3H)
By analyze EOQ FT-IR and1H-NMR spectrum, it is known that the structural formula of EOQ is：
Using FT-IR and1H-NMR characterizes the OAQ that step 3 obtains.
FT-IR(KBr pellet)v cm-1:3477,3309,2919,2888,1658,1328,1126,599-840；
1H NMR(600MHz,CDCl3):δ ppm 11.64 (s, 1H), 8.90 (dd, J=1.8,4.2Hz, 1H), 8.73
(dd, J=1.8,7.2Hz, 1H), 8.17 (dd, J=1.8,8.4Hz, 1H), 7.93 (s, 1H), 7.60 (m, 2H), 7.48 (q, J
=4.2Hz, 1H), 3.79-3.62 (m, 8H), 2.11 (br, 1H)
By analyze OAQ FT-IR and1H-NMR spectrum, the structural formula for determining OAQ are：
The chemical name of OAQ：N- quinolyl-N ' -2- (2- hydroxy ethoxies) ethyl Oxalic acid diamides.
The fluorescent molecular probe OAQ prepared using the present embodiment 1 carries out tests below.
The luciferase assay reagent of the present embodiment is by OAQ and Tris-HClO4Buffer solution forms, and wherein buffer solution is with water
It is formulated for solvent, the pH value of buffer solution is 7.24, and the concentration of buffer solution is 1.0 × 10-2The concentration of mol/L, OAQ
For 1.0 × 10-5mol/L.Preparation method is：OAQ is added to Tris-HClO4Buffer solution, adding the methanol of certain volume makes
V in the system of obtainingMethanol:VWater=1:9 formation.
Experiment 1：Be separately added into the fluorescent molecular probe OAQ solution described in the embodiment various common metals from
Son (Na+、K+、Mg2+、Ca2+、Cr3+、Fe2+、Co2+、Ni2+、Cu2+、Ag+、Cd2+、Hg2+、Pb2+、Al3+), measure its fluorescence spectrum
(concentration of common metal ion is 5 times of OAQ concentration).Zn2+Addition cause the launch wavelength of OAQ from 410nm red shifts to
490nm, red shift 80nm.Influence figure of the obtained common metal ion to fluorescence intensity is as shown in white column in Fig. 1, from Fig. 1
As can be seen that only Zn2+The fluorescence of OAQ is caused to significantly increase (B2=32B1), and the addition of other common metal ions cannot be led
Cause the enhancing of OAQ fluorescence.Fluorescent molecular probe OAQ i.e. manufactured in the present embodiment can the single identification in common metal ion
Zn2+.Fig. 1 wherein abscissas represent different metal ions, and (1 is Na+, 2 be K+, 3 be Mg2+, 4 be Ag+, 5 be Cd2+, 6 be Hg2+, 7
For Pb2+, 8 be Al3+, 9 be Ca2+, 10 be Cr3+, 11 be Fe2+, 12 be Co2+, 13 be Ni2+, 14 be Cu2+, 15 be Zn2+), indulge and sit
It is designated as the change of OAQ fluorescence intensities.
Experiment 2：The concentration for preparing fluorescent molecular probe OAQ is 1.0 × 10-5The solution (duplicate) of mol/L, thereto
Zn is added in portion2+Make wherein Zn2+Concentration be 5.0 × 10-5Mol/L, uses HClO4Or NaOH adjusts its pH (pH is from 2 increases
To 12), the fluorescence intensity of two parts of solution is measured with the change of pH.Influence figure of the obtained pH value to fluorescence intensity as shown in Fig. 2,
Wherein a is the curve that the fluorescence intensity of OAQ solution changes with pH value, and b is containing Zn2+The fluorescence intensity of OAQ solution become with pH value
The curve of change, compares the curve that two fluorescence intensities change with pH value, it is known that from pH value since 4.6, OAQ is gradually and Zn2+Knot
Conjunction forms compound so that and the fluorescence intensity of OAQ gradually increases, and when pH value is between 6-9, Zn2+It can make the fluorescence of OAQ
Significantly increase (B2＞ 10B1).Fig. 2 wherein abscissas represent pH value, and ordinate represents fluorescence intensity.
Experiment 3：The Zn of 5 times of equivalents of OAQ is separately added into the OAQ solution of the present embodiment2+With various common metal ions
(Na+、K+、Mg2+、Ca2+、Cr3+、Fe2+、Co2+、Ni2+、Cu2+、Ag+、Cd2+、Hg2+、Pb2+、Al3+), measure its fluorescence spectrum.
Other common metal ions and Zn arrived2+Figure of fluorescence intensity changes during competition is as shown in figure 3, from figure 3, it can be seen that remove Ni2 +, Cu2+Outside, other metal ions do not disturb OAQ-Zn2+The fluorescent emission of complex compound, fluorescence intensity and only Zn2+In the presence of class
Seemingly.Wherein, abscissa represents different metal ions (1 is Zn2++Na+, 2 be Zn2++K+, 3 be Zn2++Mg2+, 4 be Zn2++Ag+, 5
For Zn2++Cd2+, 6 be Zn2++Hg2+, 7 be Zn2++Pb2+, 8 be Zn2++Al3+, 9 be Zn2++Ca2+, 10 be Zn2++Cr3+, 11 be Zn2+
+Fe2+, 12 be Zn2++Co2+, 13 be Zn2++Ni2+, 14 be Zn2++Cu2+, 15 be Zn2++Zn2+), ordinate represents fluorescence intensity.
Experiment 4：Zn is gradually added into the OAQ solution of the present embodiment2+, Zn2+Concentration is 0~11.2 times of OAQ concentration
When, measure its fluorescence spectrum.Obtained Zn2+Influence figure of the concentration to fluorescence spectrum as shown in figure 4, from fig. 4, it can be seen that with
Zn2+Addition, fluorescent emission peak intensities of the OAQ at 490nm gradually increase.Work as Zn2+Concentration is 0~9.0 × 10-5Mol/L models
In enclosing, the fluorescence intensity and Zn of solution2+Good linear relationship (R is presented in concentration2=0.9916), its equation of linear regression is Y
=5.561+4.510X；20 parallel determinations are carried out to blank sample, (σ is the standard deviation of blank sample, and K is recurrence side by 3 σ/K
The slope of journey), calculate detection and be limited to 2.0 × 10-8mol/L.Show that the fluorometric reagent of the present embodiment highly sensitive can detect
Zn2+.Abscissa represents wavelength of fluorescence in Fig. 3, and ordinate represents fluorescence intensity, and the direction of arrow meaning refers to Zn2+Concentration increases
Direction.
Experiment 5：By yeast cells in OAQ solution (1 × 10-4Mol/L in), at 37 DEG C, 2h is cultivated, then in Zn2+Solution
In, 2h is cultivated at 37 DEG C, is observed respectively with fluorescence microscope.When yeast cells only uses OAQ hydroponics, cell is almost
There is no fluorescence；Cultivated when cell is first in OAQ solution, then in Zn2+After being cultivated in solution, the fluorescence of blueness is sent.Show this reality
The fluorescent molecular probe OAQ for applying example can be to the Zn in living cells2+It is detected.
Embodiment 2：The fluorescent molecular probe of the present embodiment, is synthesized according to the following steps：
First, the 8- aminoquinolines and 12mL oxalic acid diethyls of 1.00g is sequentially added into the three-necked flask with reflux
Ester, is heated to 180 DEG C, stirs 3h, obtains reaction mixture；Using dichloromethane as eluant, eluent, which is subjected to column
Chromatographic isolation, carries out reduction vaporization by obtained solution and removes solvent, obtain white solid, which is represented with EOQ.
2nd, the EOQ of 0.600g, 10mL ethanol and 0.649g1,4- are sequentially added into the three-necked flask with reflux
Butanediamine, is heated to 85 DEG C, 5h is stirred, up to crude product I；, will with methanol/chloroform (1/3, v/v) mixed solution for eluant, eluent
Crude product I carries out pillar layer separation, obtains colourless transparent solution；Again by the colourless transparent solution carry out reduction vaporization remove it is molten
Agent, obtains white solid, which is fluorescent molecular probe, is represented with PQOA.
Using FT-IR,1H-NMR characterizes PQOA.
FT-IR(KBr pellet)v cm-1:3311,2937,2853,1671,1526,1480,595-953；
1H NMR(600MHz,CDCl3):δ ppm 11.66 (s, 1H), 8.90 (t, J=2.1Hz, 1H), 8.73 (d, J=
7.2Hz, 1H), 8.17 (d, J=7.8Hz, 1H), 7.93 (s, 1H), 7.60 (m, 2H), 7.48 (q, J=4.2Hz, 1H), 3.47
(q, J=6.6Hz, 2H), 2.76 (t, J=4.2Hz, 2H), 1.69 (q, J=7.2Hz, 2H), 1.56 (br, 2H).
By analyze PQOA FT-IR and1H-NMR spectrum, it is known that the structural formula of PQOA is：
The chemical name of PQOA：To N- (3- aminopropyls)-N '-(quinolyl) oxamides.
The luciferase assay reagent of the present embodiment is by PQOA and Tris-HClO4Buffer solution forms, and wherein buffer solution is
It is formulated using water as solvent, the pH value of buffer solution is 7.24, and the concentration of buffer solution is 1.0 × 10-2Mol/L, PQOA
Concentration be 1.0 × 10-5mol/L.Preparation method is：PQOA is added to Tris-HClO4Buffer solution, adds certain volume
Methanol cause V in systemMethanol:VWater=1:9 formation.
Experiment 1：Various common metals are separately added into the fluorescent molecular probe PQOA solution described in the embodiment
Ion (Na+、K+、Mg2+、Ca2+、Cr3+、Fe2+、Co2+、Ni2+、Cu2+、Ag+、Cd2+、Hg2+、Pb2+、Al3+), measure its fluorescence light
Spectrum (concentration of common metal ion is 5 times of PQOA concentration).The result shows that only Zn2+Cause the fluorescence spectrum red shift of PQOA (red
Move 68nm), fluorescence intensity significantly increases (B2=22B1), i.e., fluorescent molecular probe PQOA manufactured in the present embodiment can be normal
See single identification Zn in metal ion2+。
The luciferase assay reagent of PQOA in the present embodiment for pH value between 6-9 when can be used in Zn2+Detection, and
From the influence of other common metal ions.With Zn2+Concentration increase, PQOA solution fluorescence peak intensities gradually strengthen.
Experiment 2：By yeast cells in PQOA solution (1 × 10-4Mol/L 2h is cultivated at 37 DEG C in), then in Zn2+Solution
In cultivate 2h at 37 DEG C, observed respectively with fluorescence microscope.When yeast cells only uses PQOA hydroponics, cell is almost
There is no fluorescence；Cultivated when cell is first in PQOA solution, then in Zn2+After being cultivated in solution, the fluorescence of blueness is sent.Show this reality
The fluorescent molecular probe PQOA for applying example can be to the Zn in living cells2+It is detected.
Embodiment 3：The fluorescent molecular probe of the present embodiment, is synthesized according to the following steps：
First, the 8- aminoquinolines and 12mL oxalic acid diethyls of 1.00g is sequentially added into the three-necked flask with reflux
Ester, is heated to 180 DEG C, 3h is stirred, up to reaction mixture；Using dichloromethane as eluant, eluent, which is subjected to column
Chromatographic isolation, carries out reduction vaporization by obtained solution and removes solvent, obtain intermediate E OQ；
2nd, the EOQ of 0.550g, 10mL ethanol and 0.207g aminoethyle alcohols are sequentially added into three-necked flask, is heated to 85
DEG C, 3h is stirred, up to crude product I；Using ethyl acetate as eluant, eluent, crude product I is subjected to pillar layer separation, the solution that will be obtained
Carry out reduction vaporization and remove solvent, obtain white solid, i.e. purpose product fluorescent molecular probe, be denoted as HQOA.
Using FT-IR,1H-NMR characterizes HQOA.
FT-IR(KBr pellet)v cm-1:3462,3315,2879,1640,562-893；
1H NMR(600MHz,CDCl3):δ ppm 11.64 (s, 1H), 8.90 (dd, J=1.8,4.2Hz, 1H), 8.73
(dd, J=1.8,7.2Hz, 1H), 8.17 (dd, J=1.8,8.4Hz, 1H), 7.93 (s, 1H), 7.60 (m, 2H), 7.48 (q, J
=4.2Hz, 1H), 3.79 (q, J=4.2Hz, 2H), 3.15 (q, J=3.6Hz, 2H), 2.01 (br, 1H)
By analyze HQOA FT-IR and1H-NMR spectrum, it is known that the structural formula of HQOA is：
The chemical name of HQOA：N- (2- ethoxys)-N '-(quinolyl) oxamides.
The luciferase assay reagent of the present embodiment is by HQOA and Tris-HClO4Buffer solution form, wherein buffer solution be with
What water was formulated for solvent, the pH value of buffer solution is 7.26, and the concentration of buffer solution is 1.0 × 10-2Mol/L, HQOA's
Concentration is 1.0 × 10-5mol/L.Preparation method is：HQOA is added to Tris-HClO4Buffer solution, adds certain volume
Methanol causes V in systemMethanol:VWater=1:9 formation.
Experiment 1：Various common metals are separately added into the fluorescent molecular probe HQOA solution described in the embodiment
Ion (Na+、K+、Mg2+、Ca2+、Cr3+、Fe2+、Co2+、Ni2+、Cu2+、Ag+、Cd2+、Hg2+、Pb2+、Al3+), measure its fluorescence light
Spectrum (concentration of common metal ion is 5 times of HQOA concentration).The result shows that only Zn2+Cause the fluorescence spectrum red shift of HQOA (red
Move 72nm) fluorescence intensity significantly increases (B2=29B1), i.e., fluorescent molecular probe HQOA manufactured in the present embodiment can be common
Single identification Zn in metal ion2+。
The luciferase assay reagent of HQOA in the present embodiment can be used in Zn when being more than 5 for pH value2+Detection, and from
The influence of other common metal ions.With Zn2+Concentration increase, HQOA solution fluorescence peak intensities gradually strengthen.
Experiment 2：By yeast cells in HQOA solution (1 × 10-4Mol/L 2h is cultivated at 37 DEG C in), then in Zn2+Solution
In cultivate 2h at 37 DEG C, observed respectively with fluorescence microscope.When yeast cells only uses HQOA hydroponics, cell is almost
There is no fluorescence；Cultivated when cell is first in HQOA solution, then in Zn2+After being cultivated in solution, the fluorescence of blueness is sent.Show this reality
The fluorescent molecular probe HQOA for applying example can be to the Zn in living cells2+It is detected.
Embodiment 4：The fluorescent molecular probe of the present embodiment, is synthesized according to the following steps：
First, the 8- aminoquinolines of 0.501g, 15mL dichloromethane and 0.257g oxalic acid two are sequentially added into three-necked flask
Methyl esters, is heated to 45 DEG C, 3h is stirred, up to crude product II；Crude product II is washed three times with ether, is filtered under diminished pressure, obtains palm fibre
Black solid；Using dichloromethane as eluant, eluent, brownish black solid is subjected to pillar layer separation, obtains colourless transparent solution；Will step
The solution obtained in rapid three carries out reduction vaporization and removes solvent, obtains white solid, which is represented with DQOA.
Using FT-IR,1H-NMR characterizes DQOA.
FT-IR(KBr pellet)v cm-1:3311,1613,1518,496-862；
1H NMR(600MHz,CDCl3):δ ppm 11.85 (s, 1H), 8.96 (dd, J=1.2,4.2Hz, 1H), 8.89
(dd, J=2.4,6.6Hz, 1H), 8.21 (dd, J=1.2,8.4Hz, 1H), 7.64 (s, 1H), 7.52 (q, J=4.2Hz, 1H).
By analyze DQOA FT-IR and1H-NMR spectrum, it is known that the structural formula of DQOA is：
The chemical name of DQOA：N, N '-two (quinolyl) Oxalic acid diamides.
The luciferase assay reagent of the present embodiment is by DQOA and Tris-HClO4Buffer solution forms, and wherein buffer solution is
It is formulated using water as solvent, the pH value of buffer solution is 8.66, and the concentration of buffer solution is 1.0 × 10-2Mol/L, DQOA
Concentration be 1.0 × 10-5mol/L.Preparation method is：DQOA is added to Tris-HClO4Buffer solution, adds certain volume
Methanol cause V in systemMethanol:VWater=9:1 formation.
Experiment 1：Various common metals are separately added into the fluorescent molecular probe DQOA solution described in the embodiment
Ion (Na+、K+、Mg2+、Ca2+、Cr3+、Fe2+、Co2+、Ni2+、Cu2+、Ag+、Cd2+、Hg2+、Pb2+、Al3+), measure its fluorescence light
Spectrum (concentration of common metal ion is 5 times of DQOA concentration).The result shows that only Zn2+Cause the launch wavelength of DQOA from 450nm
Red shift is to 536nm, and fluorescence significantly increases (B at red shift 86nm, 536nm2=27B1), i.e., fluorescence molecule manufactured in the present embodiment
Probe DQOA can in common metal ion single identification Zn2+。
The luciferase assay reagent of the present embodiment can be used in Zn when being more than 7 for pH value2+Detection, and from other common
The influence of metal ion.With Zn2+Concentration increase, DQOA solution fluorescence peak intensities gradually strengthen.Work as Zn2+Concentration 0~
1.5×10-5In the range of mol/L, the fluorescence intensity and Zn of solution2+Good linear relationship, linear equation Y=is presented in concentration
0.29276+0.059X, linearly dependent coefficient R2=0.9800；20 parallel determinations are carried out to blank sample, (σ is blank by 3 σ/K
The standard deviation of sample, K are the slope of regression equation), calculate detection and be limited to 2.37 × 10-6mol/L.Show the fluorescence examination of the present invention
Agent can highly sensitive detection Zn2+。
Experiment 2：By yeast cells in DQOA solution (1 × 10-4Mol/L 2h is cultivated at 37 DEG C in), then in Zn2+Solution
In cultivate 2h at 37 DEG C, observed respectively with fluorescence microscope.When yeast cells only uses DQOA hydroponics, cell is almost
There is no fluorescence；Cultivated when cell is first in DQOA solution, then in Zn2+After being cultivated in solution, the fluorescence of blueness is sent.Show this reality
The fluorescent molecular probe DQOA for applying example can be to the Zn in living cells2+It is detected.
Claims (9)
1. the preparation method of oxamido- quinoline fluorescent molecular probe, it is characterised in that this method according to the following steps into
OK：
First, it is 1 according to molar ratio by 8- aminoquinolines and diethy-aceto oxalate：(5~10) after mixing, 150~220 DEG C are warming up to,
3~12h is reacted, reaction mixture is obtained, after reaction mixture is purified, obtains intermediate, represented with EOQ；
2nd, EOQ is mixed with amine according to 1 ﹕ of molar ratio (2~10), is warming up to 80~150 DEG C, reacted 2~12h, obtain crude product
I；After obtained crude product I is purified, mono-substituted oxamido- quinoline fluorescent molecular probe is obtained；Amine therein is
Ammonia, organic primary amine or secondary amine；The general structure of the fluorescent molecular probe is：
Wherein R is alkyl amino or arylamino.
2. the preparation method of oxamido- quinoline fluorescent molecular probe according to claim 1, it is characterised in that
The molar ratio of 8- aminoquinolines and diethy-aceto oxalate is 1 in step 1：8.
3. the preparation method of oxamido- quinoline fluorescent molecular probe according to claim 1 or 2, its feature exist
Reaction temperature is 180~200 DEG C in step 1, and the reaction time is 8~10h.
4. the preparation method of oxamido- quinoline fluorescent molecular probe, it is characterised in that this method according to the following steps into
OK：By 8- aminoquinolines and diethy-aceto oxalate according to molar ratio for (2~10) ﹕ 1 are mixed and added into dichloromethane, are warming up to 40
~100 DEG C of 3~12h of reaction, obtain crude product II, after crude product II is purified, obtain disubstituted oxamido- quinoline derivatives
Thing fluorescent molecular probe；The general structure of the fluorescent molecular probe is：
Wherein R is alkyl amino or arylamino.
5. the preparation method of oxamido- quinoline fluorescent molecular probe according to claim 4, it is characterised in that
The molar ratio of 8- aminoquinolines and diethy-aceto oxalate is (5~8) ﹕ 1.
6. the application of oxamido- quinoline fluorescent molecular probe prepared by claim 1 or 4, it is characterised in that the application
It is to be used to oxamido- quinoline fluorescent molecular probe detect zinc ion, zinc ion is zinc ion or life in aqueous solution
Order the zinc ion in body.
7. the application of oxamido- quinoline fluorescent molecular probe according to claim 6, it is characterised in that utilize
The method of oxamido- quinoline fluorescent molecular probe qualitative detection aqueous zinc, carries out according to the following steps：
First, the pH value of oxamido- quinoline fluorescent molecular probe solution is adjusted between 6~9, and tests the solution
Fluorescence spectrum, read the wavelength value A of fluorescence emission peak position1And probe is in wavelength value A1The fluorescence intensity level B at place1；
2nd, aqueous solution to be measured is added in oxamido- quinoline fluorescent molecular probe solution, tests the solution again
Fluorescence spectrum, read the wavelength value A of fluorescence emission peak position2And its in A2The fluorescence intensity level B at place2；
If the 3rd, A2＞ A1And B2＞ 10B1, then contain zinc ion in aqueous solution to be measured, otherwise do not have in aqueous solution to be measured zinc from
Son.
8. the application of oxamido- quinoline fluorescent molecular probe according to claim 6, it is characterised in that utilize
The method that oxamido- quinoline fluorescent molecular probe quantitatively detects aqueous zinc carries out according to the following steps：
First, the standard series sample containing zinc ion of various concentrations is prepared, is separately added into oxamido- quinoline fluorescence point
Sub- probe solution, and adjust between pH value 6~9, and the fluorescence spectrum of solution is tested, the fluorescence for reading maximum fluorescence emission is strong
Angle value, marks using the concentration of zinc ion to be horizontal, is denoted as figure using the fluorescence intensity level of maximum fluorescence emission to be vertical, draw standard song
Line；
2nd, to will in the aqueous solution to be measured containing zinc ion add oxamido- quinoline fluorescent molecular probe solution, with
Step 1 under the same conditions, tests the fluorescence spectrum of the solution, reads the maximum fluorescence emission of the aqueous solution to be measured containing zinc ion
Fluorescence intensity level, the aqueous zinc concentration to be measured containing zinc ion is found from standard curve.
9. the application of oxamido- quinoline fluorescent molecular probe according to claim 6, it is characterised in that utilize
The detection method of zinc ion in oxamido- quinoline fluorescent molecular probe qualitative detection life entity, according to the following steps into
OK：Life entity to be measured is placed into 1~6h of culture in the aqueous solution containing oxalyl amido quinoline, is then shown with fluorescence
Micro mirror detects the living cells of life entity, if fluorescence phenomenon, judges to contain zinc ion in life entity.
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