CN108148057B - Prepare the method for detecting the fluorescence probe of cysteine - Google Patents
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Abstract
Method the present invention relates to preparation for detecting the fluorescence probe of cysteine.Probe prepared by the method for the present invention can be acted on cysteine, and have the characteristics that high sensitivity and selectivity, can be applied in cysteine high sensitivity and highly selective identification or its concentration that can measure cysteine in sample.
Description
Technical field
Method the present invention relates to preparation for detecting the fluorescence probe of cysteine.Probe prepared by the present invention can be with
It is acted on cysteine, and has the characteristics that high sensitivity and selectivity, it can be in cysteine high sensitivity and highly selective
It is applied in identification or its concentration that can measure cysteine in sample.
Background technique
There are many kinds of the types of mercaptan, but there are three types of small molecule biological thiols, respectively homocysteine, glutathione
And cysteine.And wherein cysteine is present in many small molecules and protein of biological tissue cell, is to form them
Important substance, and cysteine mammal regulating system adjust played in a variety of physiology and pathologic process it is quite important
Effect, mainly maintained in cellular environment by the substance that the reproducibility speciality of cysteine can be oxidized to oxidisability
Redox equilibrium.Homocysteine plays an important role in organism, it be methionine in some reactions
Intermediate product, rear once being formed there are two types of outlets, first is that methionine is re-converted into, second is that the shape under the catalysis of some substances
At cysteine.Glutathione is the most abundant non-protein sulfydryl in human body, in the cell there are many function, including removing toxic substances
Free radical and peroxide, adjust cell growth and protein function, and maintenance immune function, so, glutathione it is dense
Degree can be used as assessment redox state and the index in biologic detoxication state status in organism.But cysteine is in people
It is to play one of vital amino acid in body, it can change the disulfide bond between protein molecule to weaken protein sky
Between structure, so that protein be enable to extend.In addition cysteine also has removing toxic substances, anti-aging, changes inflammation and pre-
Anti- treatment radiation disease.A large amount of investigation show that the biological thiol beyond normal level has been demonstrated the generation with human diseases
It is related, it such as slowly grows, liver is impaired and skin damage.Cysteine deficiency will lead to many diseases, including retarded growth, liver damage
Bad, drowsiness, muscle weakness and fat loss.The homocysteine of human blood middle and high concentration is to cause nerve channel effect, bone
The risk factor of the diseases such as matter osteoporosis and inflammatory bowel disease.
Currently, the method for being used to quantitative detection cysteine is varied, main method includes：Titration method, ultraviolet suction
Receipts method, molecular fluorescent method, emission spectrometry method, mass spectrometry etc..Several method in contrast, utilizes molecular fluorescence
The fluorescence probe of analysis has an apparent advantage, including it is highly selective, have it is highly sensitive, can fast reaction, detection in time,
Cell imaging etc..The probe for being used to detect cysteine at present has very much, but the fluorescence probe of Ratio-type near-infrared and can
Carry out the seldom of bio-imaging.
On the other hand, Ratiometric fluorescent probe can mitigate ambient enviroment using the variable value between two adjacent peaks
Influence to cysteine detection, so having bigger practical value.
To sum up, it is very significant that preparation, which is able to detect the Ratio-type near infrared fluorescent probe of cysteine,.
Summary of the invention
A kind of method that this field urgent need prepares high sensitivity and highly selective Ratio-type measurement cysteine, so as to
Effectively detection can especially detect cysteine in the sample.For this purpose, the invention proposes prepare a kind of novel detection half
The method of cystine, the probe can be used directly, and not need to do further organic synthesis modification, and can be highly sensitive and high
Optional ratio's type identifies cysteine.Probe of the present invention can carry out high sensitivity to cysteine and highly selective
Measurement.
Specifically, the method the present invention provides preparation for detecting the fluorescence probe of cysteine, preparation method
It is as follows：
Wherein：R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12And R13For independently selected from by hydrogen atom, straight chain or
Branched alkyl, straight or branched alkoxyl, the group of sulfonic group, ester group and hydroxyl composition；And R therein1, R2, R3, R4, R5, R6,
R7, R8, R9, R10, R11, R12And R13It can be identical or different.
Preferably, the method the present invention provides preparation for detecting the fluorescence probe of cysteine, preparation method is such as
Under：
Ratiometric fluorescent probe prepared by the present invention can be acted on cysteine, lead to the variation of fluorescence spectrum,
To realize the Ratio-type quantitative detection to cysteine.
Specifically, cysteine Ratiometric fluorescent probe prepared by the present invention respectively with Cys, Gly, Pro, Leu,
Val, Thr and Ala, which are acted on, cannot lead to substantially changeing for fluorescence spectrum, to realize the selectivity to cysteine
Identification, and then can be optionally used for excluding the interference of the quantitative determination of the presence of these amino acid to cysteine.
The stability of cysteine colorimetric probe prepared by the present invention is good, being capable of long-term preservation use.
Further, the cysteine probe prepared by the present invention being capable of half Guang of highly sensitive and highly selective Ratio-type measurement
Propylhomoserin, and do not need to do further organic synthesis modification, be conducive to commercialized popularization and application.
Detailed description of the invention
Fig. 1：Probe has good response for the cysteine of various concentration.Probe (5 μM) and cysteine (0,
0.1,0.2,0.3 0.4,0.5,0.6,0.7,0.8,0.9,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,
7.5,8 μM) it is put into sepectrophotofluorometer and is detected after reaction 10min.
Fig. 2：Have when concentration is 0 to 8 μM linear well, detection is limited to 1 μM.Probe (5 μM) and cysteine (0,
0.1,0.2,0.3 0.4,0.5,0.6,0.7,0.8,0.9,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,
7.5,8 μM) it is put into sepectrophotofluorometer and is detected after reaction 10min.
Fig. 3：When other amino acid (Cys, Gly, Pro, Leu, Val, Thr, Ala) concentration is 100 μM, probe (5 μM)
To their fluorescence response value, and the response of probe after cysteine is inside added again.Detecting solution system is second
Alcohol：Water=5：5 (v/v) contain PBS=5mM, pH=7.4, and test temperature is 25 DEG C.
Specific embodiment
The synthetic route and method of cysteine Ratio-type probe of the invention are as follows：
Wherein：R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12And R13For independently selected from by hydrogen atom, straight chain or
Branched alkyl, straight or branched alkoxyl, the group of sulfonic group, ester group and hydroxyl composition；And R therein1, R2, R3, R4, R5, R6,
R7, R8, R9, R10, R11, R12And R13It can be identical or different.
Preferably, heretofore described linear or branched alkyl group, straight or branched alkoxyl are the alkyl or alcoxyl of C1-10
Base.
Preferably, heretofore described linear or branched alkyl group, straight or branched alkoxyl are the alkyl or alcoxyl of C1-5
Base.
Preferably, heretofore described linear or branched alkyl group, straight or branched alkoxyl are the alkyl or alcoxyl of C1-3
Base.
Preferably, heretofore described linear or branched alkyl group be methyl, ethyl, propyl, n-pentyl, 2- methyl butyl,
Isobutyl group or 4- methylheptyl.
Preferably, heretofore described straight or branched alkoxyl is methoxyl group, ethyoxyl or propoxyl group.
Preferably, R in the present invention1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12And R13For independently selected from hydrogen original
Son, methyl, ethyl, propyl, methoxyl group, ethyoxyl or propoxyl group.
Preferably, R in the present invention1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12And R13Selected from hydrogen, methyl, second
Base or propyl；Most preferably, R1, R2, R3, R4, R5,
R6, R7, R8, R9, R10, R11, R12And R13It is H.
It is further preferred that the method for fluorescence probe of the preparation for detecting cysteine is in dehydrated alcohol in the present invention
As what is carried out under conditions of solvent.
It is further preferred that the method for fluorescence probe of the preparation for detecting cysteine is to react 12 at normal temperature in the present invention
It carries out within a hour.
Preferably still, in the present invention in the method for fluorescence probe of the preparation for detecting cysteine, 2- formoxyl -6-
The molar ratio of benzothiazole phenol and N- methyl -2- methylquinoline salt is 1:3-3:1, preferably 1:1.
Probe prepared by the present invention can highly sensitive and highly selective Ratio-type measure cysteine, and/or in amino
Quantitative analysis accurately can be carried out to cysteine in the presence of acid.
Probe prepared by the method for the present invention has the following advantages that：
1, there is very good selectivity to cysteine, and for other amino acid then without apparent response；
2, the probe response time is 20min, therefore can be used as the means of quickly detection cysteine；And/or
3, the detection of the probe is limited to 1 μM, 1 to 8 μM of detection of the cysteine that can be quantified, and sensitivity is relatively high.
The probe has the blue shift of a 170nm after reacting with cysteine, this is closely red in the Ratio-type of detection cysteine
It is fewer in the probe of outer class.The present invention is also to synthesize a kind of Ratio-type probe to propose a new method.
It below will be by the way that the present invention be described in more detail by following embodiment.Following embodiment is merely illustrative,
It should be understood that the present invention is not limited by following embodiments.
The preparation of 1 probe of embodiment
The synthesis of 2,6- diformyl phenol and purification
It weighs phenol 940.5mg in the balance and hexamethylenetetramine 2.80g is put into the glass reaction bottle of 100mL, be added
The trifluoroacetic acid of 30mL is put into the small magneton an of wash clean, is heated to reflux later using 84-1 magnetic agitation control-temperature electric heating set,
Recording reacting time tracks process using tlc silica gel plate in reaction process, about needs reaction 6h, after fully reacting, turns off
Reaction.Synthetic reaction is shown in Formulas I：
The beaker for preparing a 100mL, in the distilled water for wherein pouring 50mL or so into.Stop in reaction, it, will be anti-after cooling
Solution is answered to be added dropwise in preprepared beaker,
It is stirred evenly with dropper, the liquid in beaker is poured into separatory funnel, then wherein adding 20mL's or so
Methylene chloride, fastening plug rock with strength it is several under, separatory funnel is placed on brandreth stands a few minutes later, make liquid point
Layer.Since the density ratio water of methylene chloride is big, so dichloromethane solution is below, our product is organic matter, generally not
It is dissolved in water, to be completely dissolved in methylene chloride during extraction.The piston for opening separatory funnel flows methylene chloride from lower part
Out.Then the methylene chloride extraction of 20mL or so is added in remaining liquid in separatory funnel again, repeatedly repeatedly 3 to 5
It is secondary, make product completely into methylene chloride.
After having extracted, we are extracted obtained two with this flask by the glass reaction bottle of one 25mL of weighing
Chloromethanes liquid is spin-dried for Rotary Evaporators.Then it takes the substance being spin-dried for a little in the centrifuge tube of 4mL, adds a small amount of two
Chloromethanes makes it dissolve, and pipettes a little solution with capillary glass tube, the contact plate on tlc silica gel plate, later by thin-layer chromatography
Silica gel plate is put into the glass jar containing about 1mL methylene chloride, about waits for 3min quietly, until methylene chloride liquid is fastly by thin layer
Tlc silica gel plate is taken out with tweezers when chromatographic silica gel plate all soaks, with the 365nm's in portable uv analyzer
Fluorescent lamp excitation a, it can be observed that phosphor dot, without other points, illustrates that extraction is spin-dried in the fluorescent lamp excitation of 254nm
The solid obtained afterwards is purer, can directly cast single step reaction.It weighs again after equal flasks are cooling, the weight for obtaining product is
976.5mg, yield 65.1%.
The synthesis and purification of 2- formoxyl -6- benzothiazole phenol
Product 2,6- diformyl phenol obtained above is weighed into 450.0mg and near amino thiophenols weigh 375.6mg
It is put into the glass reaction bottle of 100mL, adds 25mL ethanol solution, be completely dissolved two kinds of drugs, be put into wash clean
Small magneton, glass reaction bottle is placed on the strong magnetic stirring apparatus of 84-B is stirred at room temperature later.It is added during stirring
37% hydrochloric acid solution of 9mmol is eventually adding 30% hydrogenperoxide steam generator of 18mmol.Continue to be stirred at room temperature
30min or so.Synthetic reaction is shown in Formula II.
Reaction is completed, and solid matter is sucked out using dropper, then filters, is rinsed with dehydrated alcohol.
A little upper layer product is pipetted in centrifuge tube, is dissolved in a small amount of methylene chloride, is pipetted a little with capillary glass tube
Solution, the contact plate on tlc silica gel plate put the silica gel plate put into glass wide-mouth containing about 1mL methylene chloride later
In bottle, about wait for 3min quietly, until methylene chloride liquid fastly all soaks tlc silica gel plate with tweezers by thin-layer chromatography
Silica gel plate takes out, and is excited with the fluorescent lamp of the 365nm in portable uv analyzer, it can be observed that a phosphor dot,
When the fluorescent lamp excitation of 254nm, there is the stain of a very little, the solid for illustrating that suction filtration obtains is purer, since this is not final
Product, purity, which reaches 80% or more, may be used for reacting in next step, so we do not purify further, be directly used in down
Single step reaction.
A period of time is filtered, until after solid parches completely, is pipetted solid from funnel using spoon small to 25mL
It in glass reaction bottle, weighs to small glass reaction bottle, weighs again after being put into product in advance, to obtain the weight of product
For 521.6mg, yield 68.2%.
The preparation and purification of N- methyl -2- methylquinoline salt
In the glass reaction bottle for weighing the 50mL that 300mg 2- methylquinoline is put into wash clean in the balance, the iodine of 20mL is poured into
Methane is put into magneton, is heated to reflux or so 4 hours with 84-1 magnetic agitation control-temperature electric heating set.Synthetic reaction is shown in formula III.
Flask is removed after having reacted, after cooling, has solid precipitation, is filtered with Suction filtration device, obtain solid,
About 285mg after weighing, yield 98%.
The synthesis and purification of 2- (N- methyl -2- vinyl) quinoline -6- benzothiazole phenol
Weigh product 2- formoxyl -6- benzothiazole phenol 255mg and product N- methyl -2- methylquinoline salt 285mg
It is put into the glass reaction bottle of the 50mL of wash clean, pours the dehydrated alcohol of 20mL or so into, be as far as possible completely dissolved 2 kinds of drugs,
It puts small magneton into later, is heated to reflux with 84-1 magnetic agitation control-temperature electric heating set, reacts 12 hours.
After reaction, reaction dissolvent dehydrated alcohol is removed by revolving, and the methylene chloride that 15mL is added makes it dissolve.
Then solution is added dropwise in chromatographic column, the weight for obtaining sterling is 108.9mg, yield 20.8%.
1The analysis of H-NMR result
Hydrogen modal data is as follows：1H-NMR(400MHz,DMSO-d6)δ(*10-6):4.23 (s, 3H), 7.16 (t, J=
7.6Hz, 1H), 7.25-7.27 (m, 4H), 7.52 (t, J=7.0Hz, 1H), 7.59 (t, J=7.0Hz, 1H), 7.64 (d, J=
16.9Hz, 1H), 7.90 (d, J=8.0Hz, 1H), 7.99 (d, J=8.0Hz, 1H), 8.10 (d, J=8.0Hz, 1H), 8.15
(d, J=16.9Hz, 1H), 8.20 (d, J=8.0Hz, 1H), 8.22 (d, J=7.6Hz, 1H), 8.83 (d, J=7.6Hz, 1H),
13.32(s,1H).Above data meets the structural formula feature of 2- (N- methyl -2- vinyl) quinoline -6- benzothiazole phenol.
13The analysis of C-NMR result
Carbon modal data is as follows：13C-NMR(100MHz,DMSO-d6)δ(*10-6):46.28,114.26,116.27,
117.19,118.64,119.16,121.32,121.76,123.18,123.53,123.97,124.38,125.47,126.29,
127.62,130.36,131.28,131.38,134.25,144.47,150.18,152.47,155.29,167.36.The above number
According to the structural formula feature for meeting 2- (N- methyl -2- vinyl) quinoline -6- benzothiazole phenol.
2 solution of embodiment is prepared
The final products for weighing 5.22mg are configured to the probe mother liquor of 1mM.The cysteine for weighing 12.1mg is configured to
Cysteine (Cys) mother liquor of 10mM.
50 μ L liquid dilutings are pipetted from probe mother liquor to 10mL, are made into 5 μM of probe in detecting liquid.By 10mL probe in detecting
Liquid is divided into two, and the cysteine mother liquor of 25 μ L is added in portion wherein, and concentration is 50 μM.
It is above-mentioned for prepare the system of solution to be ethyl alcohol：Water=5：5 (v/v) contain PBS=5mM, pH=7.4.
Influence of 3 response time of embodiment to probe identification cysteine
5 μM of probe in detecting liquid is made into as described in embodiment 2, rapidly joining cysteine mother liquor later makes its concentration
Be 10 μM, rock it is several under, it is quickly put into sepectrophotofluorometer after keeping the dispensed materials in solution uniform, be arranged fluorescence
Thus spectrophotometer obtains the response spectrum of different time in every 30 seconds run-downs, recording light spectrogram simultaneously saves, when detection
Room temperature is 25 DEG C.Detection solution system remains as ethyl alcohol：Water=5：5 (v/v) contain PBS=5mM, pH=7.4.As a result such as Fig. 1 institute
Show.Find out that pure probe solution peak value at 700nm is relatively high from fluorescence spectra, there is no peak at 530nm, is added
After cysteine, spectrogram is that the peak value at 700nm slowly reduces, and the peak value at 530nm slowly increases.
Quantitative effect of 4 probe of embodiment to cysteine
The same probe in detecting liquid for preparing 5 μM, and probe in detecting liquid is added in colorimetric cylinder and is settled to 10mL.Pipette half Guang
Propylhomoserin solution sequentially adds in colorimetric cylinder, makes the concentration of cysteine finally be：0,0.1,0.2,0.3 0.4,0.5,0.6,
0.7,0.8,0.9,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8 μM and it is made to be evenly distributed, detected
Solution system is ethyl alcohol：Water=5：5 (v/v) contain PBS=5mM, pH=7.4, are existed after placing 10min with sepectrophotofluorometer
Excitation wavelength is to be detected under 430nm and keep spectrogram.Room temperature is 25 DEG C when detection.As a result as shown in Figure 2.Probe pair
There is good response in the cysteine of various concentration.Have when concentration is 0 to 8 μM linear well, detection is limited to 1 μM.
It can be seen that probe synthesized by the present invention can detect cysteine by comparing the method for the fluorescence intensity at two neighboring peak
And there is good sensitivity.
Anti-interference when 5 probe of embodiment identifies cysteine
Prepare 7 colorimetric cylinders and probe solution constant volumes are added to 10mL, only 10 μM of cysteine of addition in first is remaining
Cys, Gly, Pro, Leu, Val, Thr, Ala are sequentially added, concentration is 100 μM, then adds 10 μM of cysteine.It is same quiet
After setting 10min, since the colorimetric cylinder of first, left side, it is sequentially placed into sepectrophotofluorometer and detects, and keep data.Inspection
Survey solution system is ethyl alcohol：Water=5：5 (v/v) contain PBS=5mM, pH=7.4, and room temperature is 25 DEG C when detection.As a result such as Fig. 3 institute
Show.
It can be seen from the figure that other thiol solutions being added in probe solution after reaction a period of time, in solution
The fluorescence intensity of probe is not responded to without too big variation, explanation, but after cysteine inside is added again, probe is just
It will appear relatively high response.Therefore, probe has good selectivity, anti-interference to cysteine.
It can be seen that probe synthesized by the method for the present invention be one have to cysteine it is highly selective and anti-dry well
The probe of immunity.
Although with above embodiments describe the present invention, it should be appreciated that before without departing substantially from spirit of the invention
It puts, the present invention further can be modified and be changed, and these modifications and variation all belong to the scope of protection of the present invention it
It is interior.
Claims (4)
1. a kind of method for preparing the Ratio-type probe for detecting cysteine, synthetic route are as follows：
Wherein：R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12And R13It is H.
2. according to the method described in claim 1, wherein, the method is carried out under conditions of dehydrated alcohol is as solvent
's.
3. according to the method described in claim 1, wherein, 2- formoxyl -6- benzothiazole phenol and N- methyl -2- methylquinoline
The molar ratio of salt is 1:3-3:1.
4. according to the method described in claim 1, wherein, 2- formoxyl -6- benzothiazole phenol and N- methyl -2- methylquinoline
The molar ratio of salt is 1:1.
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