CN106518846B - Stable isotope sulfhydryl compound labelled reagent and its synthetic method and application - Google Patents
Stable isotope sulfhydryl compound labelled reagent and its synthetic method and application Download PDFInfo
- Publication number
- CN106518846B CN106518846B CN201610828351.0A CN201610828351A CN106518846B CN 106518846 B CN106518846 B CN 106518846B CN 201610828351 A CN201610828351 A CN 201610828351A CN 106518846 B CN106518846 B CN 106518846B
- Authority
- CN
- China
- Prior art keywords
- reaction
- acridone
- ethyl
- solid
- synthetic method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/05—Isotopically modified compounds, e.g. labelled
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The present invention relates to small organic molecule isotope labelling techniques fields, and in particular to a kind of stable isotope sulfhydryl compound labelled reagent and its synthetic method and application.The isotope labeling reagent is [d0]/[d4]-acridone -10- ethyl-N- maleimide.The reagent is using acridone as isotope group, using maleimide as reactive group.Its synthetic method is to be made using [d0]/[d5]-bromobenzene as raw material through two step condensation reactions, hydroxylating, halogenating reaction, the reaction of drop cloth riel, acylation reaction.The stable isotope that the present invention obtains is deuterium-labeled, and after separating-purifying, isotope labelling position is stablized, and chemical purity is up to 99.0% or more, and isotope abundance is 99.0% or more.The reagent optionally labeling SH groups compound, has many advantages, such as to mark that reaction rate is fast, label yield is high, high to sulfhydryl-selective.
Description
Technical field
The present invention relates to small organic molecule isotope labelling techniques fields, and in particular to a kind of stable isotope sulfydryl chemical combination
Substance markers reagent and its synthetic method and application.
Background technique
High performance liquid chromatography-tandem mass technology (HPLC-MS/MS) is highly sensitive and highly selective and extensive due to it
In the research fields such as life analysis, environmental analysis, food analysis.Using the Selective reaction monitoring of HPLC-MS/MS
(SRM) or reaction of high order ion monitoring (MRM) the mode property of can choose monitoring sample in designated analysis object, can be effectively
Noise effect is reduced, the sensitivity of detection is improved.However, the signal due to caused by sample substrate or co-elute substance decaying or
The problem of matrix effect of enhancing, makes the analysis performance of HPLC-MS/MS have a greatly reduced quality, in Journal of
Chromatography A, 2010.1217 (25): is discussed in detail in 3929-3937.
In recent years, isotope-coded deriving technology receives very big concern, by light/derivative examination of heavy isotope coding
Agent can get the isotope derivatives of a kind of analyte with same functional group, carry out HPLC-MS/ in this, as internal standard compound
MS quantitative analysis, can be efficiently against the influence of matrix effect.However, chemical marker methods all so far has some lack
It falls into, such as label reaction rate is slow, marked product signal is weak, label accuracy and sensitivity are poor, and much label examinations
Agent is not easy to obtain, therefore limits being widely used for they.
Summary of the invention
The object of the present invention is to provide a kind of signals accelerated label reaction rate, improve marked product, enhancing mass spectrum inspection
Survey the stable isotope sulfhydryl compound labelled reagent of sulfhydryl compound accuracy and sensitivity；Present invention simultaneously provides its synthesis
Methods and applications.
Stable isotope sulfhydryl compound labelled reagent of the present invention is the Malaysia acyl using acridone as isotope group
Imines is reactive group, its chemical name is: [d0]/[d4]-acridone -10- ethyl-N- maleimide, chemical structure
Formula are as follows:
Wherein: as X=H, being [d0]-acridone -10- ethyl-N- maleimide, be abbreviated as [d0]-AENM；Work as X
When=D, it is [d4]-acridone -10- ethyl-N- maleimide, is abbreviated as [d4]-AENM.
The synthetic method of the stable isotope sulfhydryl compound labelled reagent, comprising the following steps:
(1) first step condensation reaction: [d0]/[d5]-bromobenzene, ortho-aminobenzoic acid are dissolved into nitrobenzene, and copper is added
Powder and potassium carbonate back flow reaction 3 hours, remove solvent with steam distillation after having reacted, reaction solution are poured into water, adjusts pH value
To 5-6, solid and drying are recycled, intermediate I [d0]/[d5] -2- phenyl amino phenyl formic acid is obtained；
(2) second step condensation reaction: intermediate I is dissolved in the concentrated sulfuric acid that mass percent is 98wt.%, in 100
It is reacted 2 hours at DEG C, after cooling, is poured into water quenching reaction, solid and drying is recycled, with acetonitrile and n,N-Dimethylformamide
It is recrystallized according to volume ratio v:v=3:1 and intermediate II [d0]/[d4]-acridone is made；
(3) hydroxylating: intermediate II and ethylene carbonate are dissolved into dimethyl sulfoxide, and potassium carbonate conduct is added
Catalyst reacts 3 hours at 90 DEG C, pours into after cooling and recycles solid and drying in saturated brine, is made with recrystallized from acetonitrile
Mesosome III [d0]/[d4] -10- (2- ethoxy)-acridone；
(4) halogenating reaction: intermediate III is dissolved into methylene chloride, and triethylamine is added, dichloro is added dropwise under room temperature
Sulfoxide continues to be stirred to react 1 hour, has reacted back spin and solvent is evaporated off, and residue acetonitrile is redissolved in falling back, recycling
Intermediate IV [d0]/[d4] -10- (ethyl -2- chlorine)-acridone is made with recrystallized from acetonitrile in solid and drying；
(5) drop cloth riel synthetic reaction: intermediate IV and potassium phthalimide are dissolved in dimethyl sulfoxide, added
Heat to 100 DEG C react 3 hours, reaction solution is poured into water later, filters and dries obtained solid；Solid is dissolved in anhydrous
In ethyl alcohol, the hydrazine hydrate that mass percent is 80wt.% is added, back flow reaction 1.5 hours, is added dropwise thereto after the reaction was completed dense
Hydrochloric acid to no solid generates, and filters out solid and recycles filtrate, by filtrate concentrated by rotary evaporation, then pours into ice water, adjusts pH value to 9-
10, solid and drying are recycled, intermediate V [d0]/[d4] -10- (ethyl -2- ammonia)-acridone is obtained；
(6) acylation reaction: maleic anhydride is dissolved into acetonitrile, the acetonitrile solution dissolved with intermediate V is added dropwise, under room temperature
Reaction 1 hour, revolving removes solvent after the reaction was completed；Acetic anhydride is added and dissolves residue, anhydrous sodium acetate is added, in 100
It reacts 1 hour, is cooled down in falling back at DEG C, recycle solid and drying, obtain target product [d0]/[d4]-with recrystallized from acetonitrile
Acridone -10- ethyl-N- maleimide.
In step (1) first step condensation reaction, the molar ratio of [d0]/[d5]-bromobenzene and ortho-aminobenzoic acid is 1:
1.2, the additional amount of copper powder and potassium carbonate is respectively the 2.5% and 50% of [d0]/[d5]-bromobenzene quality.
In step (3) described hydroxylating, the molar ratio of [d0]/[d4]-acridone and ethylene carbonate is 1:1.3,
The additional amount of potassium carbonate is the 50% of [d0]/[d4]-acridone quality.
In step (4) described halogenating reaction, [d0]/[d4] -10- (2- ethoxy)-acridone, triethylamine, thionyl chloride
Molar ratio be 1:1.2:2.
In step (5) the drop cloth riel synthetic reaction, [d0]/[d4] -10- (ethyl -2- chlorine)-acridone and adjacent benzene two
The molar ratio of carboximide sylvite is 1:1.3, the additional amount of hydrazine hydrate and [d0]/[d4] -10- (ethyl -2- chlorine)-acridone
Mole is identical.
In step (6) described acylation reaction, mole of [d0]/[d4] -10- (ethyl -2- ammonia)-acridone, maleic anhydride
Than being the 10% of [d0]/[d4] -10- (ethyl -2- ammonia)-acridone quality for the additional amount of 1:1.2, sodium acetate.
Concentration of the stable isotope sulfhydryl compound labelled reagent of the present invention for mercaptan in test sample, step
Are as follows:
(1) pH=7.4 is prepared, the PBS buffer solution that concentration is 0.01mol/L；Compound concentration is 1 × 10-6The sulphur of mol/L
Alcohol standard items acetonitrile solution；Compound concentration is 1 × 10-4The second of [d0]-acridone -10- ethyl-N- maleimide of mol/L
Nitrile solution is 1 × 10 as light-duty labelled reagent solution, concentration-4Mol/L [d4]-acridone -10- ethyl-N- maleimide
Acetonitrile solution as heavy labelled reagent solution；
(2) successively 50 μ L PBS buffer solutions, 100 μ L mercaptan standard solutions and the light-duty labelled reagent solution of 50 μ L are added
Peace to 2mL is cutd open in bottle, is reacted 10 minutes in 40 DEG C of water-bath；Successively by 50 μ L PBS buffer solutions, 100 μ L through column purification
The peace that sample and 50 μ L heavy type labelled reagent solution afterwards is added to 2mL is cutd open in bottle, is reacted 10 minutes in 40 DEG C of water-bath；Reaction
It after the completion, is by volume that 1:1 is mixed by above-mentioned two reaction solution, 1 μ L of sample introduction carries out HPLC-MS/MS analysis；
(3) peak area of the light-duty derivative of gained is expressed as A1 after light-duty labelled reagent is derivative, 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 is derivative, and the concentration of mercaptan is expressed as C2 in sample, then
According to formula C1/C2=A1/A2, the concentration of mercaptan in sample can be acquired.
Beneficial effects of the present invention are as follows:
The stable isotope that the present invention obtains is deuterium-labeled, and after separating-purifying, isotope labelling position is stablized, chemical purity
Up to 99.0% or more, isotope abundance is 99.0% or more.Isotope labeling reagent of the invention optionally labeling SH groups
Compound has the advantages that label reaction rate is fast, marked product signal is strong, label yield is high, high to sulfhydryl-selective, increasing
Strong Mass Spectrometer Method sulfhydryl compound accuracy and sensitivity can be applied to the researchs such as life analysis, environmental analysis, food analysis neck
In domain；Its synthetic method is simple, it is easy to accomplish；The reagent can mark rapidly mercaptan in the PBS solution that pH is 7.4, right
Mercaptan shows high sensitivity and selectivity, and detection mercaptan is easy, sensitive, quick, as a result accurately.
Detailed description of the invention
Fig. 1 is the synthetic route chart of labelled reagent prepared by embodiment 1.
Fig. 2 is the nuclear-magnetism 1HNMR map of labelled reagent prepared by embodiment 1.
Fig. 3 is the infrared spectrogram of labelled reagent prepared by embodiment 1.
Fig. 4 is the reaction route figure of labelled reagent prepared by embodiment 1 and sulfhydryl compound.
Fig. 5 is the MRM ion chromatography flow graph of the derivative 7 kinds of mercaptan standard items of 2 weight reagent of embodiment.
Fig. 6 is the second order ms figure of 3 furfuryl mercaptan of embodiment and the light-duty derivative of 3- sulfydryl-hexanol.
Specific embodiment
The present invention is described further with reference to embodiments.
Embodiment 1
As shown in Figure 1, the synthesis of stable isotope sulfhydryl compound label of the present invention shares 6 steps, synthetic route is such as
Fig. 1, for heavy labelled reagent using [d5]-bromobenzene as raw material, heavy labelled reagent synthetic operation is as follows:
1, the preparation of intermediate I [d5] -2- phenyl amino phenyl formic acid
22g ortho-aminobenzoic acid, 20g [d5]-bromobenzene, 10g potassium carbonate and 0.5g copper powder are added in 250mL three-necked flask,
150mL nitrobenzene is added as solvent, in being heated to reflux on electric jacket, is stirred to react 3 hours；It is steamed after the reaction was completed with vapor
Solvent is removed in distillation, mixed reaction solution is poured into 200 milliliters of water, is neutralized to pH value 5-6 with concentrated hydrochloric acid, is sufficiently stirred, will analyse
Solid out is filtered and is dried, and obtains intermediate I, is grey powder.Yield is 23.3g, yield 87.2%.
2, intermediate II [d4]-acridone preparation
20g intermediate I and the 100mL concentrated sulfuric acid (98wt.%) are sufficiently mixed, and are transferred in 150mL round-bottomed flask, oil
Bath heats and controls reaction temperature at 100 DEG C, is stirred to react 2 hours；Reaction solution is slowly poured into along walls of beaker after cooling
In 300mL water, and stir 5 minutes；The solid of precipitation is filtered, and is washed twice with 5% solution of potassium carbonate, uses second after dry
Nitrile and n,N-Dimethylformamide (v:v=3:1) recrystallization, obtain intermediate II, are yellow flat crystal.Yield is 15.2g,
Yield is 83%.
3, the preparation of intermediate III [d4] -10- (2- ethoxy)-acridone
15g intermediate II, 8.5g ethylene carbonate, 7.5g potassium carbonate, with 150mL diformazan are added in 250mL round-bottomed flask
The dissolution of base sulfoxide, oil bath heating are stirred to react 3 hours to 90 DEG C；Reaction solution is poured into 300mL saturated sodium-chloride after the reaction was completed
It in aqueous solution and is stirred continuously, solid is collected by filtration, wash twice, use recrystallized from acetonitrile after dry, obtain light yellow crystal,
That is intermediate III.Yield is 15.4g, yield 83.6%.
4, the preparation of intermediate IV [d4] -10- (ethyl -2- chlorine)-acridone
It weighs 15g intermediate III to be put into 250mL round-bottomed flask, adds 7.5g triethylamine, filled with 100mL methylene chloride
Divide dissolution；9mL thionyl chloride is slowly added dropwise into flask, is added in 30 minutes, continues to be stirred to react 1 hour under room temperature；Reaction
After completely, rotary evaporation removes solvent, and 100mL acetonitrile is added, and stirring is completely dissolved to above-mentioned product, and liquid is poured into 200mL
In water, there is solid to be precipitated and filter, after solid is dry twice with recrystallized from acetonitrile, obtains brownish red crystal, i.e. intermediate IV.It produces
Amount is 13.5g, yield 84.4%.
5, the preparation of intermediate V [d4] -10- (ethyl -2- ammonia)-acridone
10g intermediate IV and 9g potassium phthalimide are added in 500mL round-bottomed flask, 250mL bis- is added
Methyl sulfoxide as solvent, stirred by reactant oil bath heating to 100 DEG C react 3 hours, after reaction by hybrid reaction
Liquid pours into 300mL saturated sodium-chloride water solution, and the solid of precipitation is filtered and dried；By the solid after drying with 200mL without
Water-ethanol dissolution, is added 80wt.% hydrazine hydrate 2mL, is heated to reflux and continues to stir, have white solid appearance after about 30 minutes,
Continue to be stirred at reflux 1 hour；Concentrated hydrochloric acid is added dropwise after cooling into reaction solution until not having Precipitation, white precipitate is crossed and is filtered out
It goes and washs filter cake twice with dehydrated alcohol, cleaning solution and mother liquor are merged, concentrated by rotary evaporation to about 20mL adds water 40mL in remnants
In liquid, any insoluble substance, continuation concentrated liquid to 30mL or so are filtered out, residual liquid is poured into 100mL ice water,
Liquid is neutralized to pH value 9-10 with sodium hydroxide solution, solid is collected and is dried in vacuo, obtain yellowish solid i.e. intermediate V.
Yield is 5.6g, yield 59%.
6, target product [d4]-acridone -10- ethyl-N- maleimide preparation
2.5g maleic anhydride and 50mL acetonitrile are added in the round-bottomed flask of 100mL, stirring to maleic anhydride is completely dissolved
Afterwards, the acetonitrile solution that about 25mL contains 5g intermediate V is added dropwise, after continuing stirring 1 hour, revolving removes solvent；With 60mL acetic acid
Residue is dissolved, 0.5g anhydrous sodium acetate, which is added, then with oil bath heating 30 minutes of 100 DEG C becomes dun solution,
After the reaction was continued 30 minutes, reaction solution is poured into 150mL water, the solid of precipitation is filtered and dried, with recrystallized from acetonitrile two
It is secondary, [d4]-acridone -10- ethyl-N- maleimide sterling is obtained, is yellowish-brown crystal.Yield is 5.2g, and yield is
65%.
Characterization of The Products:
1H NMR:(CDCl3,400MHz,ppm)δ8.52(dd,2H,J1=8.0, J2=4.0Hz), 7.74-7.70 (m,
2H), 7.30-7.27 (m, 2H), 4.66 (t, 2H, J=8.0Hz), 3.87 (t, 2H, J=8.0Hz)；(Fig. 2)
MS:m/z:322.2[M+H]+；IR (KBr): 3318 (Ar-H), 2962 (- CH2), 1644 (C=O), 1546 (C=
C),1246(Ar-H)；(Fig. 3)
Found:C 70.77,H 5.52,N 8.74,O 14.97；Calculated:C 70.79,H 5.63,N 8.69,
O 14.89.
For light-duty labelled reagent using [d0]-bromobenzene as raw material, remaining synthesis step is identical as heavy labelled reagent synthesis step,
Characterization of The Products:
1H NMR:(CDCl3,400MHz,ppm)δ8.53(dd,2H,J1=8.0, J2=4.0Hz), 7.72 (S, 2H) 7.69
(t, 2H), 7.32 (S, 2H), 7.28 (t, 2H), 4.66 (t, 2H, J=8.0Hz), 3.89 (t, 2H, J=8.0Hz)；
MS:m/z:318.2[M+H]+；IR (KBr): 3332 (Ar-H), 2948 (- CH2), 1634 (C=O), 1544 (C=
C),1251(Ar-H)；
Found:C 71.71,H 4.50,N 8.76,O 15.03；Calculated:C 71.69,H 4.43,N 8.80,
O 15.08.
Embodiment 2
Prepare pH=7.4, the PBS buffer solution that concentration is 0.01mol/L；Compound concentration is 1 × 10 respectively-4Mol/L's
The acetonitrile solution of [d0]-AENM and [d4]-AENM；Compound concentration is 1 × 10-6A series of mercaptan standard items (3- mercaptos of mol/L
Base-hexanol, hexyl mercaptan, furfuryl mercaptan, 2- methyl -3- furanthiol, benzene ethyl mercaptan, 3- mercapto-acetic acid-own ester, 4- methyl -4-
Sulfydryl -2 pentanone) acetonitrile solution.
Successively by 50 μ L PBS buffer solutions, 100 μ L mercaptan standard solutions and the 50 light-duty labelled reagent of μ L [d0]-AENM
Acetonitrile solution be added to the peace of 2mL and cut open in bottle, reacted 10 minutes in 40 DEG C of water-bath.Meanwhile with weight under the conditions of same
Type reagent [d4]-AENM marks the grape wine actual sample after column purification.It is mixed by volume for 1:1 after the reaction was completed
It closes, then carries out HPLC-MS/MS analysis.
The peak area of the light-duty derivative of gained is expressed as A1 after light-duty labelled reagent is derivative, 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 is derivative, and the concentration of mercaptan indicates in grape wine actual sample
The concentration of mercaptan in grape wine actual sample can be acquired then according to formula C1/C2=A1/A2 for C2.
Reagent and thiol reaction are Michael addition reaction, reaction equation such as Fig. 4.The ion chromatography stream of 7 kinds of mercaptan standard items
Figure is shown in Fig. 5, wherein (light: weight=3:1), being kept completely separate for 7 kinds of thiol analytes to be realized in 15 minutes, light/weight derivative
Retention time difference complied fully with the quantitative requirement of mass spectrum less than 0.05 minute.
Embodiment 3
The quantitative of mercaptan uses 1290 type Ultra Performance Liquid Chromatography instrument of Agilent, the triple quadrupole rods tandem mass spectrometries of 6460 types
Instrument is equipped with electrospray ionisation source (ESI).Chromatographic condition: mobile phase A is+95% water of 5% acetonitrile, and Mobile phase B is 100% acetonitrile,
Ionization Efficiency of the formic acid that addition concentration is 0.1% in mobile phase to enhance analyte；Using gradient elution, mobile phase is set
B Initial Gradient is 30%, becomes 100% after ten minutes, and kept for 5 minutes；Sample volume is 1 μ L, flow velocity 0.2mL/min.Matter
Spectral condition: ESI positive ion mode, dry 300 DEG C, flow velocity 10L/min, atomizing pressure 40psi of temperature degree, 250 DEG C of sheath temperature degree,
Flow velocity 8L/min, capillary voltage 3.5KV.Collision voltage (Fragmentor) and cracking energy (CE) parameter are as follows:
Derivative shows stronger [M+H]+Signal, light-duty derivative mainly generate m/z's 353.1 and m/z 319.1
Fragment ion, heavy derivative mainly generate the fragment ion of m/z 357.1 and m/z 323.1, with furfuryl mercaptan and 3- sulfydryl-
The light-duty derivative of hexanol is that the cleavage of mass spectrum figure of representative is shown in Fig. 6.
Claims (6)
1. a kind of synthetic method of stable isotope sulfhydryl compound labelled reagent, it is characterised in that: using acridone as isotope
Group, maleimide are reactive group, its chemical name is: [d0]/[d4]-acridone -10- ethyl-N- maleimide,
Its chemical structural formula are as follows:
Wherein: being [d0]-acridone -10- ethyl-N- maleimide as X=H；It is [d4]-acridine as X=D
Ketone -10- ethyl-N- maleimide；
The synthetic method, comprising the following steps:
(1) first step condensation reaction: [d0]/[d5]-bromobenzene, ortho-aminobenzoic acid are dissolved into nitrobenzene, be added copper powder and
Potassium carbonate back flow reaction 3 hours, removes solvent with steam distillation after having reacted, reaction solution is poured into water, adjusts pH value
To 5-6, solid and drying are recycled, intermediate I [d0]/[d5] -2- phenyl amino phenyl formic acid is obtained；
(2) second step condensation reaction: intermediate I is dissolved in the concentrated sulfuric acid that mass percent is 98wt.%, at 100 DEG C
Reaction 2 hours after cooling, is poured into water quenching reaction, solid and drying is recycled, with acetonitrile and n,N-Dimethylformamide
It is recrystallized according to volume ratio v:v=3:1 and intermediate II [d0]/[d4]-acridone is made；
(3) hydroxylating: intermediate II and ethylene carbonate are dissolved into dimethyl sulfoxide, and potassium carbonate is added as catalysis
Agent is reacted 3 hours at 90 DEG C, is poured into after cooling and is recycled solid and drying in saturated brine, is made intermediate with recrystallized from acetonitrile
Body III [d0]/[d4] -10- (2- ethoxy)-acridone；
(4) halogenating reaction: intermediate III is dissolved into methylene chloride, and triethylamine is added, thionyl chloride is added dropwise under room temperature,
Continue to be stirred to react 1 hour, reacted back spin and solvent is evaporated off, residue acetonitrile is redissolved in falling back, recycling is solid
Intermediate IV [d0]/[d4] -10- (ethyl -2- chlorine)-acridone is made with recrystallized from acetonitrile in body and drying；
(5) drop cloth riel synthetic reaction: intermediate IV and potassium phthalimide are dissolved in dimethyl sulfoxide, are heated to
100 DEG C are reacted 3 hours, are later poured into water reaction solution, are filtered and dry obtained solid；Solid is dissolved in anhydrous second
In alcohol, the hydrazine hydrate that mass percent is 80wt.% is added, back flow reaction 1.5 hours, is added dropwise thereto after the reaction was completed dense
Hydrochloric acid to no solid generates, and filters out solid and recycles filtrate, by filtrate concentrated by rotary evaporation, then pours into ice water, adjusts pH value extremely
9-10 recycles solid and drying, obtains intermediate V [d0]/[d4] -10- (ethyl -2- ammonia)-acridone；
(6) acylation reaction: maleic anhydride is dissolved into acetonitrile, and the acetonitrile solution dissolved with intermediate V is added dropwise, reacts 1 under room temperature
Hour, revolving removes solvent after the reaction was completed；Acetic anhydride is added and dissolves residue, anhydrous sodium acetate is added, at 100 DEG C
Reaction 1 hour cools down in falling back, and recycles solid and drying, obtains target product [d0]/[d4]-a word used for translation with recrystallized from acetonitrile
Pyridine ketone -10- ethyl-N- maleimide.
2. the synthetic method of stable isotope sulfhydryl compound labelled reagent according to claim 1, it is characterised in that:
In step (1) first step condensation reaction, the molar ratio of [d0]/[d5]-bromobenzene and ortho-aminobenzoic acid is 1:1.2, copper powder
Additional amount with potassium carbonate is respectively the 2.5% and 50% of [d0]/[d5]-bromobenzene quality.
3. the synthetic method of stable isotope sulfhydryl compound labelled reagent according to claim 1, it is characterised in that:
In step (3) described hydroxylating, the molar ratio of [d0]/[d4]-acridone and ethylene carbonate is 1:1.3, potassium carbonate
Additional amount is the 50% of [d0]/[d4]-acridone quality.
4. the synthetic method of stable isotope sulfhydryl compound labelled reagent according to claim 1, it is characterised in that:
In step (4) described halogenating reaction, the molar ratio of [d0]/[d4] -10- (2- ethoxy)-acridone, triethylamine, thionyl chloride
For 1:1.2:2.
5. the synthetic method of stable isotope sulfhydryl compound labelled reagent according to claim 1, it is characterised in that:
In step (5) the drop cloth riel synthetic reaction, [d0]/[d4] -10- (ethyl -2- chlorine)-acridone and phthalimide
The molar ratio of sylvite is 1:1.3, the mole of the additional amount of hydrazine hydrate and [d0]/[d4] -10- (ethyl -2- chlorine)-acridone
It is identical.
6. the synthetic method of the stable isotope sulfhydryl compound labelled reagent according to claim 1, it is characterised in that:
In step (6) described acylation reaction, [d0]/[d4] -10- (ethyl -2- ammonia)-acridone, maleic anhydride molar ratio be 1:
1.2, the additional amount of sodium acetate is the 10% of [d0]/[d4] -10- (ethyl -2- ammonia)-acridone quality.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610828351.0A CN106518846B (en) | 2016-09-18 | 2016-09-18 | Stable isotope sulfhydryl compound labelled reagent and its synthetic method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610828351.0A CN106518846B (en) | 2016-09-18 | 2016-09-18 | Stable isotope sulfhydryl compound labelled reagent and its synthetic method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106518846A CN106518846A (en) | 2017-03-22 |
| CN106518846B true CN106518846B (en) | 2019-02-22 |
Family
ID=58343834
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610828351.0A Expired - Fee Related CN106518846B (en) | 2016-09-18 | 2016-09-18 | Stable isotope sulfhydryl compound labelled reagent and its synthetic method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106518846B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108383830B (en) * | 2018-03-29 | 2020-01-10 | 曲阜师范大学 | Acridone stable isotope sulfhydryl compound marking reagent and synthesis method and application thereof |
| CN108383832A (en) * | 2018-03-29 | 2018-08-10 | 曲阜师范大学 | A kind of carbazoles stable isotope sulfhydryl compound labelled reagent and its synthetic method and application |
| CN113402352A (en) * | 2021-06-17 | 2021-09-17 | 曲阜师范大学 | Stable isotope amino compound labeling reagent and synthetic method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1357542A (en) * | 2000-12-13 | 2002-07-10 | 中国科学院大连化学物理研究所 | Fluorescent reagent 2-(N-acridone)-chlorethyl formate |
-
2016
- 2016-09-18 CN CN201610828351.0A patent/CN106518846B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1357542A (en) * | 2000-12-13 | 2002-07-10 | 中国科学院大连化学物理研究所 | Fluorescent reagent 2-(N-acridone)-chlorethyl formate |
Non-Patent Citations (2)
| Title |
|---|
| N-取代马来酰亚胺巯基荧光探针的研究进展;梁淑彩，等，;《化学通报》;20011231(第8期);第478-782页 |
| 同位素编码衍生高效液相色谱串联质谱法测定果蔬样品中的羧酸类植物生长调节剂;蔡轶平，等，;《分析化学》;20150331;第43卷(第3期);第419-423页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106518846A (en) | 2017-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hong et al. | A red to near-infrared fluorescent probe featuring a super large Stokes shift for light-up detection of endogenous H2S | |
| Yu et al. | A novel fluorescent probe for highly sensitive and selective detection of cysteine and its application in cell imaging | |
| CN106518846B (en) | Stable isotope sulfhydryl compound labelled reagent and its synthetic method and application | |
| Zhang et al. | A new colorimetric and fluorescent probe with a large stokes shift for rapid and specific detection of biothiols and its application in living cells | |
| CN105712964B (en) | Preparation method and application of thiol fluorescent probe based on coumaroyl hydrazide | |
| CN104447421A (en) | Preparation and application of novel cysteine and homocysteine fluorescence probe | |
| CN105131035B (en) | Amino group compound and sugar chain labeled mass spectrum derivatization reagent with positive charges | |
| CN114591633B (en) | Xanthene-hemicyanine near-infrared fluorescent dye, and synthetic method and application thereof | |
| Sun et al. | An ultrafast turn-on thiol probe for protein labeling and bioimaging | |
| CN107033131A (en) | It is a kind of to be used to detect fluorescence probe of cysteine and its preparation method and application | |
| Zhang et al. | Highly sensitive and selective detection of biothiols by a new low dose colorimetric and fluorescent probe | |
| Shen et al. | A mitochondria-oriented fluorescent probe for ultrafast and ratiometric detection of HSO 3− based on naphthalimide–hemicyanine | |
| Yang et al. | Constructing a FRET-based molecular chemodosimeter for cysteine over homocysteine and glutathione by naphthalimide and phenazine derivatives | |
| CN108658838A (en) | A kind of formaldehyde fluorescence probe based on seven methine indoles cyanines and preparation method thereof and application method | |
| Yan et al. | A label-free turn-on fluorescence probe for rapidly distinguishing cysteine over glutathione in water solution | |
| Yu et al. | A novel near-infrared fluorescent probe for cysteine in living cells based on a push-pull dicyanoisophorone system | |
| You et al. | Development of a sensitive reagent, 1, 2-benzo-3, 4-dihydrocarbazole-9-ethyl-p-toluenesulfonate, for determination of bile acids in serum by HPLC with fluorescence detection, and identification by mass spectrometry with an APCI source | |
| CN106008435B (en) | One kind is used for Au3+Fluorescence Increasing type fluorescence probe of detection and preparation method thereof | |
| JP5529020B2 (en) | Use of cyanocinnamic acid derivatives as matrix in MALDI mass spectrometry | |
| CN109206351A (en) | A kind of near infrared fluorescent probe, preparation method and application for surveying palladium ion based on flower cyanines structure | |
| CN101274909B (en) | Brominated1-[3-(4-maleimido phenoxyl)propyl] trimethylamin, preparation and application thereof | |
| Rong et al. | A naphthalimide-indole fused chromophore-based fluorescent probe for the detection of biothiol with red emission and a large Stokes shift | |
| CN107033879A (en) | It is a kind of to be used to detect fluorescence probe of glutathione and its preparation method and application | |
| CN113402352A (en) | Stable isotope amino compound labeling reagent and synthetic method and application thereof | |
| CN105985770A (en) | Preparation method and application of hydrogen sulfide fluorescent probe |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190222 Termination date: 20190918 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |