CN108383832A - A kind of carbazoles stable isotope sulfhydryl compound labelled reagent and its synthetic method and application - Google Patents
A kind of carbazoles stable isotope sulfhydryl compound labelled reagent and its synthetic method and application Download PDFInfo
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Abstract
The invention belongs to organic molecule stable isotope labeling technical field, a kind of carbazoles stable isotope sulfhydryl compound labelled reagent and its synthetic method and application are provided.The stable isotope labeling reagent is N (4 (9 base of carbazole) phenyl) N [d0]/[d2] maleimide, its synthetic method is that carbazole and 4 acetobromanilides are carried out substitution reaction, obtain intermediate I 9 ((4 acetylamino) phenyl) carbazole, then intermediate I heats hydrolysis under alkaline condition, obtains intermediate II 9 ((4 amino) phenyl) carbazole；By intermediate II and [d0]/[d2] maleic anhydride carries out acylated condensation reaction, generate target product N (4 (9 base of carbazole) phenyl) N [d0]/[d2] maleimide.Simple synthetic method of the present invention, it is easily achieved, the deuterated sulfhydryl compound labelled reagent of obtained stable isotope, isotope labelling position is stablized, all kinds of sulfhydryl compounds can be selectively marked, has many advantages, such as that marked product yield is high, mark reaction rate fast and to sulfhydryl compound high selectivity.
Description
Technical field
The invention belongs to organic molecule stable isotope labeling technical applications, and in particular to a kind of carbazoles stabilization
Isotope sulfhydryl compound labelled reagent and its synthetic method and application.
Background technology
High Performance Liquid Chromatography/Mass Spectrometry serial connection technology (abbreviation HPLC-MS/MS) due to its it is highly sensitive and highly selective and by
It is widely used in the research fields such as life analysis, environment measuring, food evaluation.Using the Selective reaction monitoring of HPLC-MS/MS
(abbreviation SRM) or reaction of high order ion monitoring (abbreviation MRM) pattern can selectivity monitoring sample in targeting analyte with
And partly target analyte.However, the signal caused by sample mesostroma or co-elute substance weakens or the matrix of enhancing effect
The problem of answering makes the analytical performance of HPLC-MS/MS have a greatly reduced quality, and application is subject to certain restrictions, and the prior art is but not
Provide effective total solution.
In recent years, the isotope-coded technology stablized receives prodigious concern, and especially stable isotope coding derives
Technology encodes derivative reagent by light/heavy isotope, and the isotope that can get the analyte of the same race with same functional group derives
Object.It usually carries out HPLC-MS/MS quantitative analyses using heavy isotope derivatives as internal standard compound.However, existingization so far
Labeling method has many defects, and such as label reaction rate is slow, label low yield, marked product signal is weak, marks accuracy
It is poor etc. with sensitivity, and the synthesis of many labelled reagents is complicated, is not easy to obtain corresponding isotope product, therefore limit
Being widely used of such technology.
Invention content
The object of the present invention is to provide a kind of label reaction rates soon, marked product signal is high, Mass Spectrometer Method sulfydryl chemical combination
Object accuracy by force and sensitivity height and convieniently synthesized carbazoles stable isotope sulfhydryl compound labelled reagent；The present invention is simultaneously
Its specific synthetic method and application are provided.
Carbazoles stable isotope sulfhydryl compound labelled reagent provided by the present invention is using carbazole as parent ring, Malaysia
Acid imide is reactive group and isotope labelling group, its chemical name is：N- (4- (carbazole -9- bases)-phenyl)-N- [d0]/
[d2] maleimide, chemical structural formula is：
Wherein：It is N- (4- (carbazole -9- bases) -) phenyl as X=H)-N- [d0] maleimide, it is abbreviated as NCPM-
[d0]；It is N- (4- (carbazole -9- bases) -) phenyl as X=D)-N- [d2] maleimide, it is abbreviated as NCPM- [d2].At this
In invention, H refers to the isotope protium of hydrogen, and D refers to the isotope deuterium of hydrogen.
The synthetic method of the carbazoles stable isotope sulfhydryl compound labelled reagent provided, includes the following steps：
1) substitution reaction：Carbazole and 4- acetobromanilides are dissolved into dimethyl sulfoxide, and with cuprous iodide, two tertiary valeryls
Methane and potassium carbonate mixing, 130 DEG C of reaction 12h of oil bath, reaction terminate to filter after reaction solution is cooled to room temperature, and obtain smoke filtrate,
Then smoke filtrate with the NaCl of (10-30) wt% is mixed to precipitation precipitation, recycle precipitation solid and drying, obtain intermediate I 9-
((4- acetylaminos)-phenyl)-carbazole；
2) hydrolysis：Intermediate I is dissolved in the dimethyl sulfoxide solution dissolved with potassium hydroxide aqueous solution, in 100 DEG C
Reaction 2 hours mixes reaction solution with water after cooling, and solid and drying is precipitated in recycling, and intermediate II is made with ethyl alcohol recrystallization
9- ((4- amino)-phenyl)-carbazole；
3) acylation reaction：By [d0]/[d2] maleic anhydride is dissolved into acetone, the acetone soln dissolved with intermediate II is added dropwise,
It reacts 1 hour in 25 DEG C, is after reaction evaporated reaction solution solvent, recycle solid and drying；Acetic acid is added after solid drying
Acid anhydride dissolves, and adds anhydrous sodium acetate, reacts 1 hour at 85 DEG C, slowly poured into ice water after reaction solution is cooled to 25 DEG C,
Solid and drying are recycled, target product N- (4- (carbazole -9- bases)-phenyl)-N- [d are at least obtained three times with recrystallized from acetonitrile0]/
[d2] maleimide.
Further, in the substitution reaction of step 1), carbazole is 1 with 4- acetobromanilide molar ratios:1.5, iodate
The addition of cuprous, dipivaloylmethane and potassium carbonate is respectively 25%, 22% and the 50% of carbazole quality.
Further, in the hydrolysis of step 2), the addition of potassium hydroxide is 9- ((4- amino)-phenyl)-
The 30% of carbazole quality.
Further, in the acylation reaction of step 3), 9- ((4- amino)-phenyl)-carbazoles and [d0]/[d2] Malaysia
The molar ratio of acid anhydrides is 1:1.1, the addition of sodium acetate is the 10% of 9- ((4- amino)-phenyl)-carbazole quality.
The present invention also provides a kind of applications of above-mentioned carbazoles stable isotope sulfhydryl compound labelled reagent, for detecting
The concentration of mercaptan, step are in sample：
A. ammonium acetate-sodium hydroxide buffer solution and mercaptan standard items acetonitrile solution are prepared；Compound concentration is 1 × 10-
The acetonitrile solution of N- (4- (carbazole -9- bases)-phenyl)-N- [d0] maleimide of 4mol/L is molten as light-duty labelled reagent
Liquid；Compound concentration is the acetonitrile solution of N- (4- (carbazole -9- bases)-phenyl)-N- [d2] maleimide of 1 × 10-4mol/L
As heavy labelled reagent solution；
B. successively by volume ratio 1:2:1 ammonium acetate-sodium hydroxide buffer solution, mercaptan standard solution and light-duty label
Reagent solution mixes, and reacts 10min in 40 DEG C of water-bath；Successively by volume ratio 1:2:1 ammonium acetate-sodium hydroxide buffer is molten
Liquid, the sample after column purification/membrane filtration and heavy labelled reagent solution mixing, react 10min in 40 DEG C of water-bath；Reaction
After the completion, it is 1 by volume by above-mentioned two reaction solution:1 is mixed, and sample introduction carries out High Performance Liquid Chromatography/Mass Spectrometry series connection skill
Art is analyzed；
C. the peak area of the light-duty derivative of gained is expressed as A1 after light-duty labelled reagent derives, and the concentration of mercaptan is expressed as
C1；The peak area of the heavy derivative of gained is denoted as A2 after heavy labelled reagent derives, and the concentration of mercaptan is expressed as C2 in sample, then
According to formula C1/C2=A1/A2, you can acquire the concentration of mercaptan in sample.
Preferably, in step a, the pH=7.4 of ammonium acetate-sodium hydroxide buffer solution, a concentration of 0.01mol/L, sulphur
A concentration of 1 × 10-6mol/L of alcohol standard items acetonitrile solution.
Preferably, in stepb, mercaptan standard solution is 100 μ L, and the sample after column purification/membrane filtration is 100 μ
L。
Synthesis caused by the present invention has the beneficial effect that：
1. the deuterium-labeled reagent of stable isotope that the present invention obtains, after separating-purifying, isotope labelling position is stablized, and is changed
Purity is learned up to 99.5% or more, isotope abundance is 99.0% or more.The light-duty labeled derivative object of the present invention and heavy isotope derivative method
Biology chromatography retention behavior having the same in mass spectral analysis, the matrix effect between sample and standard items is also identical, can have
The influence of effect ground calibration matrix effect.2. the present invention isotope labeling reagent in maleimide labelling groups, by with
The Michael addition reaction of contained sulfydryl in sulfhydryl compound, optionally labeling SH groups compound, has synthetic method letter
Just, it is easy to accomplish；Label reaction rate is fast, marked product signal is strong, label yield is high, the advantage high to sulfhydryl-selective,
The advantages that enhancing Mass Spectrometer Method sulfhydryl compound accuracy and sensitivity.3. the reagent can selectively mark all kinds of sulfydryl chemical combination
Object is widely used in the research fields such as life analysis, environmental analysis and food analysis, for sulfydryl chemical combination in a variety of actual samples
The analysis of object detects.
Specifically, reagent of the present invention being capable of rapid labeling SH groups in ammonium acetate-sodium hydroxide buffer solution that pH is 7.4
Compound shows sulfhydryl compound high sensitivity and selectivity, and detection sulfhydryl compound is easy, sensitive, quick, as a result
Accurately.
Description of the drawings
Fig. 1 is the synthetic reaction route map of labelled reagent prepared by embodiment 1.
Fig. 2 is the nuclear-magnetism of heavy labelled reagent prepared by embodiment 11H NMR spectras.
Fig. 3 is the nuclear-magnetism of light-duty labelled reagent prepared by embodiment 11H NMR spectras.
Fig. 4 is the reaction route figure of labelled reagent and sulfhydryl compound prepared by embodiment 1.
Fig. 5 is that the mass spectrum that the light-duty labelled reagent of 2 present invention of embodiment and heavy labelled reagent derive 5 kinds of mercaptan standard items is more
Reaction monitoring (abbreviation MRM) ion chromatography flow graph.
Fig. 6 is light-duty derivative of the embodiment 3 using the n-propyl mercaptan and n-butyl mercaptan of labelled reagent of the present invention label
Second order ms figure.
Specific implementation mode
The present invention is described further with reference to embodiments.
Embodiment 1
As shown in Figure 1, the synthesis of carbazoles stable isotope sulfhydryl compound labelled reagent of the present invention shares 3 steps.
Heavy labelled reagent is with [d2]-maleic anhydride is basic raw material, and steps are as follows for specific synthetic operation：
1, the preparation of intermediate I 9- ((4- acetylaminos)-phenyl)-carbazole
10g carbazoles and 16.5g4- acetobromanilides are added in 250mL three-necked flasks, 150mL dimethyl sulfoxides are added as molten
Agent, is added 5g potassium carbonate, 2.5g cuprous iodides and 3mL dipivaloylmethanes, and 130 DEG C of reaction 12h of oil bath are stayed overnight；Reaction terminates to wait for
Reaction solution filters after being cooled to room temperature 25 DEG C, then pours into smoke filtrate and precipitation is precipitated in the NaCl of 600mL 25wt%, recycling
Precipitation solid and drying obtain intermediate I 9- ((4- acetylaminos)-phenyl)-carbazole, yield 85wt%.
2, the preparation of intermediate II 9- ((4- amino)-phenyl)-carbazole
10g intermediate Is are added in 250mL three-necked flasks, the potassium hydroxide of 100mL dimethyl sulfoxides and 20mL 50% is added
Solution, oil bath heating and controlling reaction temperature are stirred to react 2 hours at 100 DEG C；After cooling by reaction solution along walls of beaker slowly
It pours into 600mL water, and stirs 5 minutes；The solid of precipitation is filtered and dried, intermediate II 9- is made with ethyl alcohol recrystallization
((4- amino)-phenyl)-carbazole, yield 90wt%.
3, target product N- (4- (carbazole -9- bases)-phenyl)-N- [d2] maleimide preparation
2.5g [d are added in the round-bottomed flask of 100mL2]-maleic anhydride and 20mL acetone, stirring to [d2]-maleic anhydride
After being completely dissolved, the acetone soln that about 25mL contains 4g intermediate IIs is added dropwise and is evaporated solvent after continuing stirring 1 hour, recycles
Solid and drying are added solution of acetic anhydride dissolving, 0.5g anhydrous sodium acetates are added, then with 85 DEG C of oil bath after solid drying
Heating reaction 1 hour slowly pours into 500mL ice water along walls of beaker after reaction solution cooling, the solid of precipitation is filtered and done
It is dry, three times with recrystallized from acetonitrile, obtain N- (4- (carbazole -9- bases)-phenyl)-N- [d2] maleimide sterling, yield is
60wt%.
Characterization of The Products：
1H NM R (500MHz, DMSO, ppm) δ 8.39 (dd, J=8.0,1.5Hz, 2H), 7.78 (d, J=8.6Hz,
2H), 7.73-7.66 (m, 4H), 7.36 (dd, J=11.3,4.2Hz, 2H), 6.76 (d, J=8.6Hz, 2H)；(such as Fig. 2 institutes
Show)
Found:C 75.0,H 4.34,N 7.61,O 13.04；Calculated:C 74.99,H 4.38,N 7.60,
O13.03.
MS:m/z:[M+H]+=368.1.
Light-duty labelled reagent is with [d0]-maleic anhydride is basic raw material, and synthesis step synthesizes step with heavy labelled reagent
Rapid identical, Characterization of The Products：
Nuclear-magnetism1H NMR (500MHz, DMSO, ppm) δ 8.39 (d, J=8.0Hz, 2H), 7.78 (d, J=8.2Hz, 2H),
7.73-7.66 (m, 4H), 7.36 (t, J=7.5Hz, 2H), 7.30 (s, 2H), 6.76 (d, J=8.6Hz, 2H)；(such as Fig. 3 institutes
Show)
Found:C 75.40,H 3.82,N 7.65,O 13.10；Calculated:C 75.40,H 3.85,N 7.65,
O13.11.
Mass spectrum MS:m/z:[M+H]+=366.1.
Embodiment 2
The present embodiment synthesizes carbazoles stable isotope sulfhydryl compound label examination of the present invention using 1 the method for embodiment
Agent, while being applied to sulfydryl sample detection.
Prepare ammonium acetate-sodium hydroxide buffer solution of pH=7.4, a concentration of 0.01mol/L；Compound concentration is 1 respectively
×10-4NCPM- [the d of mol/L0] and NCPM- [d2] acetonitrile solution；Compound concentration is 1 × 10-65 kinds of mercaptan standards of mol/L
Product, 2 mercapto ethanol, n-propyl mercaptan, n-butyl mercaptan, the acetonitrile solution of 3- sulfydryls hexanols and benzyl mercaptan.
50 μ L ammonium acetates-sodium hydroxide buffer solution, 100 μ L mercaptan standard solutions and the light-duty labels of 50 μ L are tried successively
Agent NCPM- [d0] acetonitrile solution be added to the peace of 2mL and cut open in bottle, react 10min in 40 DEG C of water-bath.Meanwhile in same
Under the conditions of use heavy type labelled reagent NCPM- [d2] onion actual sample of the label after column purification/membrane filtration.It will after the completion of reaction
Above-mentioned two is light/heavy label after reaction solution be 1 by volume:1 is mixed, and HPLC-MS/MS analyses are then carried out.This hair
Bright column purification can be used general purification splitter and handle or use existing 0.22 μm of organic membrane filtration, in the present embodiment
In specifically isolated and purified using LiChrolut-EN solid phase pillars.
The peak area of the light-duty derivative of gained is expressed as A1 after light-duty labelled reagent derives, and the concentration of mercaptan is expressed as C1；
The peak area of the heavy derivative of gained is denoted as A2 after heavy labelled reagent derives, and the concentration of mercaptan is expressed as in onion actual sample
C2, then according to formula C1/C2=A1/A2, you can acquire the concentration of mercaptan in onion actual sample.
Labelled reagent of the present invention is Michael addition reaction with thiol reaction, and selectivity is strong, and reaction speed is fast, tool
Body chemical equation is as shown in Figure 4.
Fig. 5 shows the ion chromatography flow graph of 5 kinds of mercaptan standard items.Wherein light-duty labeled derivative object and heavy labeled derivative
Object concentration ratio is 4:1, being kept completely separate for 5 kinds of mercaptan standard items derivatives of analysis is realized in 10 minutes, light/weight derivative
Retention time difference is less than 0.05 minute, complies fully with the quantitative requirement of mass spectrum.
Embodiment 3
The present embodiment is marked and is detected using 2 the method for embodiment, and is further carried out mercaptan to sample and determined
Amount analysis.
The quantitative analysis of the present embodiment mercaptan uses 1290 type ultra performance liquid chromatography system of Agilent, 6460 types triple four
Grade bar tandem mass spectrometer, is equipped with electron spray ionisation source (ESI).
Chromatographic condition：Mobile phase A is+95% water of 5% acetonitrile, and Mobile phase B is 100% acetonitrile, and concentration is added in mobile phase
It is 0.1% formic acid to enhance the Ionization Efficiency of analyte；Using gradient elution, setting Mobile phase B Initial Gradient is 65%,
Become 80% after 6 minutes, and is kept for 2 minutes；Sample size is 1 μ L, flow velocity 0.2mL/min.Mass Spectrometry Conditions：ESI：Cation mould
Formula, dry temperature degree：300 DEG C, flow velocity：10L/min, atomizing pressure：40psi, sheath temperature degree：250 DEG C, flow velocity：8L/min, hair
Tubule voltage：3.5KV.Percentage is percent by volume in conditions above.
Collision voltage (Fragmentor) and cracking energy (CE) parameter are as shown in the table：
Derivative is shown stronger [M+H]+Signal, light-duty derivative mainly generate m/z's 338.2 and m/z 372.3
Fragment ion, heavy derivative mainly generate the fragment ion of m/z 340.2 and m/z 374.3, and Fig. 6 is shown with n-propyl mercaptan
Light-duty derivative with n-butyl mercaptan is the cleavage of mass spectrum figure of representative.
Claims (10)
1. a kind of carbazoles stable isotope sulfhydryl compound labelled reagent, it is characterised in that：Using carbazole as parent ring, Malaysia acyl
Imines is reactive group and isotope labelling group, its chemical name is：N- (4- (carbazole -9- bases)-phenyl)-N- [d0]/[d2]
Maleimide, chemical structural formula are：
Wherein：It is N- (4- (carbazole -9- bases) -) phenyl as X=H)-N- [d0] maleimide；It is N- (4- as X=D
(carbazole -9- bases) -) phenyl)-N- [d2] maleimide.
2. a kind of synthetic method of carbazoles stable isotope sulfhydryl compound labelled reagent as described in claim 1, feature
It is, carbazole and 4- acetobromanilides is subjected to substitution reaction, obtain intermediate I 9- ((4- acetylaminos)-phenyl)-carbazole,
Then intermediate I heats hydrolysis under alkaline condition, obtains intermediate II 9- ((4- amino)-phenyl)-carbazole；By intermediate II
With [d0]/[d2] maleic anhydride carries out acylated condensation reaction, generate target product N- (4- (carbazole -9- bases)-phenyl)-N- [d0]/
[d2] maleimide.
3. synthetic method according to claim 2, which is characterized in that the synthetic method specifically includes following steps：
1) substitution reaction：Carbazole and 4- acetobromanilides are dissolved into dimethyl sulfoxide, and with cuprous iodide, dipivaloylmethane
It is mixed with potassium carbonate, 130 DEG C of reaction 12h of oil bath, reaction terminates to filter after reaction solution is cooled to room temperature, and obtains smoke filtrate, then
Smoke filtrate is mixed to precipitation precipitation with the NaCl of (10-30) wt%, precipitation solid and drying is recycled, obtains intermediate I 9- ((4-
Acetylamino)-phenyl)-carbazole；
2) hydrolysis：Intermediate I is dissolved in the dimethyl sulfoxide solution dissolved with potassium hydroxide aqueous solution, reacts 2 in 100 DEG C
Hour, after cooling, reaction solution is mixed with water, recycles solid and drying, intermediate II 9- ((4- ammonia is made with ethyl alcohol recrystallization
Base)-phenyl)-carbazole；
3) acylation reaction：By [d0]/[d2] maleic anhydride is dissolved into acetone, the acetone soln dissolved with intermediate II is added dropwise, in 25
Reaction solution solvent, is evaporated, recycles solid and drying by DEG C reaction 1 hour after reaction；It is molten that acetic anhydride is added after solid drying
Solution, adds anhydrous sodium acetate, reacts 1 hour at 85 DEG C, be slowly poured into water after reaction solution is cooled to 25 DEG C, and recycling is solid
Body and drying at least obtain target product N- (4- (carbazole -9- bases)-phenyl)-N- [d three times with recrystallized from acetonitrile0]/[d2] Malaysia
Acid imide.
4. synthetic method according to claim 3, which is characterized in that in the substitution reaction of step 1), carbazole with
4- acetobromanilide molar ratios are 1:1.5, the addition of cuprous iodide, dipivaloylmethane and potassium carbonate is respectively carbazole quality
25%, 22% and 50%.
5. synthetic method according to claim 3, which is characterized in that in the hydrolysis of step 2), hydroxide
The addition of potassium is the 30% of 9- ((4- amino)-phenyl)-carbazole quality.
6. synthetic method according to claim 3, which is characterized in that in the acylation reaction of step 3), 9- ((4-
Amino)-phenyl)-carbazole and [d0]/[d2] maleic anhydride molar ratio be 1:1.1, the addition of sodium acetate is 9- ((4- ammonia
Base)-phenyl)-carbazole quality 10%.
7. a kind of application of carbazoles stable isotope sulfhydryl compound labelled reagent, it is characterised in that：The labelled reagent structure
As described in claim 1, or by the synthetic method as described in any one of claim 2-6 it synthesizes；
Wherein, which is used to detect the content of sulfhydryl compound in sample.
8. application according to claim 7, which is characterized in that include the following steps：
A. ammonium acetate-sodium hydroxide buffer solution and mercaptan standard items acetonitrile solution are prepared；Compound concentration is 1 × 10-4Mol/L's
The acetonitrile solution of N- (4- (carbazole -9- bases)-phenyl)-N- [d0] maleimide is as light-duty labelled reagent solution；It prepares dense
Degree is 1 × 10-4The acetonitrile solution of N- (4- (carbazole -9- bases)-phenyl)-N- [d2] maleimide of mol/L is marked as heavy
Remember reagent solution；
B. successively by volume ratio 1:2:1 ammonium acetate-sodium hydroxide buffer solution, mercaptan standard solution and light-duty labelled reagent
Solution mixes, and reacts 10min in 40 DEG C of water-bath；Successively by volume ratio 1:2:1 ammonium acetate-sodium hydroxide buffer solution,
Sample after column purification/membrane filtration and heavy labelled reagent solution mixing, react 10min in 40 DEG C of water-bath；It has reacted
Above-mentioned two reaction solution is 1 by volume by Cheng Hou:1 is mixed, and sample introduction carries out High Performance Liquid Chromatography/Mass Spectrometry serial connection technology
Analysis；
C. the peak area of the light-duty derivative of gained is expressed as A1 after light-duty labelled reagent derives, and the concentration of mercaptan is expressed as C1；Weight
The peak area of the heavy derivative of gained is denoted as A2 after phenotypic marker reagent derives, and the concentration of mercaptan is expressed as C2 in sample, then basis
Formula C1/C2=A1/A2, you can acquire the concentration of mercaptan in sample.
9. application according to claim 8, which is characterized in that in step a, ammonium acetate-sodium hydroxide buffer solution
PH=7.4, a concentration of 0.01mol/L, a concentration of the 1 × 10 of mercaptan standard items acetonitrile solution-6mol/L。
10. application according to claim 8, which is characterized in that in stepb, mercaptan standard solution is 100 μ L, warp
Sample after column purification/membrane filtration is 100 μ L.
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| CN108490093A (en) * | 2018-03-29 | 2018-09-04 | 曲阜师范大学 | A kind of fluoroscopic examination application of switching mode mercaptan fluorescent labeling reagent and its synthetic method |
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| CN106518846A (en) * | 2016-09-18 | 2017-03-22 | 曲阜师范大学 | Stable isotope mercapto compound labeling reagent, synthesis method and application thereof |
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| CN106518846A (en) * | 2016-09-18 | 2017-03-22 | 曲阜师范大学 | Stable isotope mercapto compound labeling reagent, synthesis method and application thereof |
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| CN108490093A (en) * | 2018-03-29 | 2018-09-04 | 曲阜师范大学 | A kind of fluoroscopic examination application of switching mode mercaptan fluorescent labeling reagent and its synthetic method |
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