CN107778317B - Methoxyl group fluorescein derivative and preparation method thereof and carboxypeptidase y detection method and detection kit - Google Patents
Methoxyl group fluorescein derivative and preparation method thereof and carboxypeptidase y detection method and detection kit Download PDFInfo
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Abstract
The present invention relates to the detection fields of carboxypeptidase y, and in particular to methoxyl group fluorescein derivative and preparation method thereof and carboxypeptidase y detection method and detection kit.Methoxyl group fluorescein derivative and preparation method thereof is disclosed, methoxyl group fluorescein derivative has the structure of formula (1), and R is the alkyl or cycloalkyl of C1-C10；Method includes: that in organic solvent, formula (2) compound is contacted with one of acyl chlorides or acid anhydrides in the presence of necleophilic reaction condition and acid binding agent.Also disclose application and detection carboxypeptidase y active method of the methoxyl group fluorescein derivative in the activity of detection carboxypeptidase y.Methoxyl group fluorescein derivative of the invention is for related with carboxypeptidase y Activity determination in application, high sensitivity, good, fluorescence intensity the variation of selectivity are easy to detect greatly, and stability is higher, using more convenient, price is less expensive.
Description
Technical field
The present invention relates to the detection fields of carboxypeptidase y, and in particular, to a kind of methoxyl group fluorescein derivative and its preparation
Method and carboxypeptidase y detection method and detection kit.
Background technique
Carboxypeptidase y (No. EC: 3.4.16.1), all amino acid with carboxyl terminal of hydrolyzable, it has also become polypeptide
Common tool enzyme in the analysis of chain C-terminal.Furthermore carboxypeptidase y can also make other amino acid derivativges or nucleophilic object by transpeptidation reaction
Matter replaces the amino acid residue of peptide chain end, to form new peptide.The measurement active main method of carboxypeptidase y mainly has purple at present
Outer absorption spectrometry, substrate structure required for this method is simple, easy to operate, has been widely used at present, but purple
There are following both sides defects for outer method: firstly, the background interference of ultraviolet absorption spectroscopy causes greatly the sensitivity of analysis low；
Secondly, the substrate for analysis is mostly peptides, such substrate synthesis difficulty is big, higher cost.Second of common method is glimmering
Light spectroscopic, for this method mainly using polypeptide fluorescence probe as substrate, reaction sensitivity is higher than ultraviolet spectrometry spectroscopic methodology, but
Peptide substrate synthesis difficulty for analysis is big, at high cost, and mostly stability is poor under normal conditions, storage and enabled condition
Harshness, atom utilization are low.
There are no the active reports that non-peptides fluorescence probe is used to detect carboxypeptidase y both at home and abroad at present.Therefore, design is opened
Hair is based on non-peptide, and inexpensively, Small-molecule probe easy to operate and stable property improves the sensitivity and selection of detection carboxypeptidase y
Property be very urgent and needs.
Summary of the invention
It is an object of the invention to overcome defect present in existing carboxypeptidase y Activity determination technology, provide a kind of sensitive
Degree is high, property is stable, it is easy to operate, be easy to storage and transportation and low-cost non-peptides carboxypeptidase y Activity determination fluorescence probe and
Carboxypeptidase y activity test method.
In order to achieve the above objectives, on the one hand, the present invention provides a kind of methoxyl group fluorescein derivative, the methoxyl group fluorescence
Plain derivative has structure shown in formula (1), wherein R is the alkyl or cycloalkyl of C1-C10；
Second aspect, the present invention also provides the preparation method of methoxyl group fluorescein derivative as described above, this method
Include: in the presence of necleophilic reaction condition and acid binding agent, in organic solvent, by structural compounds shown in formula (2) and acyl chlorides or
The contact of one of acid anhydrides；
Wherein, the general formula of the acyl chlorides is R '-C (=O) Cl, R ' and R selection having the same；
The general formula of the acid anhydrides is R "-C (=O)-O- (O=C)-R ", R " and R selection having the same.
The third aspect, the present invention also provides methoxyl group fluorescein derivatives as described above in the work for detecting carboxypeptidase y
Application in property.
Fourth aspect, the present invention also provides the detection active methods of carboxypeptidase y, wherein this method comprises:
Sample to be tested containing carboxypeptidase y is contacted with methoxyl group fluorescein derivative as described above, after obtaining contact
Material；
The value added of the fluorescence intensity that material after detection contact issues under excitation light at any time, the fluorescence intensity
Value added indicates the activity of carboxypeptidase y in sample to be tested；The wavelength of the exciting light is 410-490nm, the hair of the fluorescence
The a length of 505-555nm of ejected wave.
5th aspect, the present invention also provides a kind of active detection kit of carboxypeptidase y, the detection kit packets
It includes: methoxyl group fluorescein derivative, carboxypeptidase y standard items and reaction buffer as described above.
Methoxyl group fluorescein derivative of the invention with carboxypeptidase y for having higher when detecting carboxypeptidase y activity
Affinity and higher selectivity, detection sensitivity are higher.
Methoxyl group fluorescein derivative fluorescence probe provided by the invention and is adopted in the activity for detecting carboxypeptidase y
It is compared with polypeptide fluorescence probe, there is higher sensitivity and selectivity；And synthetic method is simple and quick, fluorescence background value
It is low.
Therefore, methoxyl group fluorescein derivative fluorescence probe provided by the invention is for related with carboxypeptidase y Activity determination
In application, high sensitivity, good, fluorescence intensity the variation of selectivity are easy to detect greatly, and methoxyl group fluorescein of the invention
Derivative is the substrate type fluorescence probe of non-peptides, and non-peptides fluorescence probe is higher compared to peptides fluorescence probe stability, makes
With more convenient, price is less expensive.Substrate type fluorescence probe is lower compared to suppressive fluorescence probe background values, to cytotoxicity more
It is low.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
The schematic diagram of Fig. 1 carboxypeptidase y catalytic fluorometry probe hydrolysis reaction, wherein hv indicates photon energy.
Fig. 2 difference hydrolase or protein are (from left to right successively are as follows: carboxypeptidase y, protaminase, chymotrypsin, pancreas
Protease, acetylcholinesterase, butyrylcholine esterase, elastoser and bovine serum albumin) it is urged under 10 μ g/mL concentration conditions
Change the relative speed of 10 μM of methoxyl group fluorescein derivative micromolecular probe hydrolysis.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
On the one hand, the present invention provides a kind of methoxyl group fluorescein derivative, which has formula
(1) structure shown in, wherein R is the alkyl or cycloalkyl of C1-C10；
In the present invention, it is illustrate on the contrary in the case where, term " alkyl " and " naphthenic base " are two side by side general
It reads, there is no the relationships for including with being included between the two.
Preferably, R is the alkyl of C2-C6.
It is furthermore preferred that R is in ethyl, propyl, butyl, amyl, hexyl, cyclopropyl, cyclobutyl, cyclopenta and cyclohexyl
One kind.
It is further preferred that R is selected from one of ethyl, propyl, butyl and cyclohexyl.
Still further preferably, R is selected from one of ethyl, n-propyl, isopropyl, tert-butyl and cyclohexyl.
Highly preferred, R is tert-butyl or cyclohexyl.
Second aspect, the present invention also provides the preparation method of methoxyl group fluorescein derivative as described above, this method
Include: in the presence of necleophilic reaction condition and acid binding agent, in organic solvent, by structural compounds shown in formula (2) and acyl chlorides or
The contact of one of acid anhydrides；
Wherein, the general formula of the acyl chlorides is R '-C (=O) Cl, R ' and R selection having the same；That is, R ' is C1-C10
Alkyl or cycloalkyl；Preferably, R ' is the alkyl of C2-C6；It is furthermore preferred that R ' be selected from ethyl, propyl, butyl, amyl, oneself
One of base, cyclopropyl, cyclobutyl, cyclopenta and cyclohexyl；It is furthermore preferred that R ' is selected from ethyl, propyl, butyl and cyclohexyl
One of, still more preferably, R ' be selected from one of ethyl, n-propyl, isopropyl, tert-butyl and cyclohexyl (that is,
The acyl chlorides is selected from one of propionyl chloride, n-butyryl chloride, isobutyryl chloride, trimethyl-aceyl chloride and cyclohexyl formyl chloride).The most
Preferably, the acyl chlorides is trimethyl-aceyl chloride or cyclohexyl formyl chloride.
Wherein, the general formula of the acid anhydrides is R "-C (=O)-O- (O=C)-R ", R " and R selection having the same.That is,
R " is the alkyl or cycloalkyl of C1-C10；Preferably, R " is the alkyl of C2-C6；It is furthermore preferred that R " is selected from ethyl, propyl, fourth
One of base, amyl, hexyl, cyclopropyl, cyclobutyl, cyclopenta and cyclohexyl；It is furthermore preferred that R " is selected from ethyl, propyl, fourth
One of base and cyclohexyl, still more preferably, R " is in ethyl, n-propyl, isopropyl, tert-butyl and cyclohexyl
It is a kind of (that is, the acid anhydrides in propionic andydride, n butanoic anhydride, isobutyric anhydride, trimethyl acetic anhydride and cyclohexanecarboxylic acid acid anhydride one
Kind).Highly preferred, the acid anhydrides is trimethyl acetic anhydride or cyclohexanecarboxylic acid acid anhydride.
In the preparation method of methoxyl group fluorescein derivative of the present invention, the necleophilic reaction is in formula (2)
Hydroxyl is reacted with what various substituted acyl chlorides, acid anhydrides occurred, and the condition of the necleophilic reaction includes that temperature is subzero 5 to 30 DEG C,
Time is 10-200min, and under preferable case, the condition of the necleophilic reaction is that temperature is 0-25 DEG C, time 80-160min.
In the preparation method of methoxyl group fluorescein derivative of the present invention, the contact needs to exist in acid binding agent
Under conditions of carry out, the acid binding agent can be any acid that can be generated in absorbing reaction commonly used in the art, avoid contact with
The acid generated in reaction influences the substance of reaction or reaction balance, it is preferable that the acid binding agent is selected from triethylamine, diethylamine, two
Isopropylamine, pyridine, N, N- dimethylamino naphthyridine, 2,6- lutidines, potassium carbonate, cesium carbonate, sodium carbonate and sodium bicarbonate
At least one of, most preferably, the acid binding agent is triethylamine.
In order to improve the effect of haptoreaction mass transfer and heat transfer, haptoreaction of the present invention is in the environment of organic solvent
It carries out, the organic solvent can not occur instead to be any under contact conditions of the present invention with reactant of the invention
Ying Bingneng provides the organic solvent of suitable solution environmental for reaction, and the boiling point of the organic solvent is preferably 30-150 DEG C, more
For preferably, the organic solvent is methylene chloride, tetrahydrofuran, chloroform, carbon tetrachloride, acetonitrile, acetone, glycol dinitrate
Ether, n,N-Dimethylformamide and 1, at least one of 2- dichloroethanes, most preferably, the organic solvent is dichloromethane
Alkane.
In the preparation method of methoxyl group fluorescein derivative of the invention, change relative to shown in 1 mM of formula (2)
The dosage of conjunction object, the acyl chlorides or acid anhydrides is 1-2 mMs；The dosage of the acid binding agent is not particularly limited, in order to reach
The assimilation effect of the good acid generated to reaction, the dosage of the acid binding agent can be 1-3 mMs, described under preferable case
The dosage of acid binding agent is 2-3 mMs；Dosage for organic solvent of the invention is also not particularly limited, preferable case
Under, the dosage of the organic solvent is 20-30 milliliters.
It, can be with thin-layer chromatography (TLC) to anti-in the preparation method of methoxyl group fluorescein derivative of the present invention
The carry out situation answered is monitored, and when testing result shows that raw material point disappears, shows that reaction terminates.
Preparation method of the invention further includes purifying to catalytic product, and the process of purification may include in 0-
Successively reaction system is washed with 20-60 milliliters of water and 20-60 milliliters of saturated common salt aqueous solutions at a temperature of 25 DEG C, is stood
Multi_layer extraction organic phase is simultaneously dried organic phase with anhydrous sodium sulfate, removes solvent by decompression method and obtains crude product,
Chromatography finally is carried out to crude product with silicagel column and obtains product.
The third aspect, the present invention provides methoxyl group fluorescein derivatives as described above of the invention in detection carboxypeptidase y
Activity in application.
The application can be related to the application of carboxypeptidase y (No. EC: 3.4.16.1) Activity determination for this field is any.Example
Such as, the application may include: the active detection of carboxypeptidase y, the screening of carboxypeptidase y inhibitor, carboxypeptidase y promotor sieve
Choosing, carboxypeptidase y inhibitor or the Kinetic Characterization of promotor etc..
Fourth aspect, the present invention also provides a kind of active detection methods of carboxypeptidase y, this method comprises:
Sample to be tested containing carboxypeptidase y is contacted with methoxyl group fluorescein derivative as described above, after obtaining contact
Material；
The value added of the fluorescence intensity that material after detection contact issues under excitation light at any time, the fluorescence intensity
Value added indicates the activity of carboxypeptidase y in sample to be tested；The wavelength of the exciting light is 410-490nm, the hair of the fluorescence
The a length of 505-555nm of ejected wave.
In the present invention, term " value added " refers within the scope of minute, the value added of fluorescence intensity and the ratio of time
Value, that is, fluorescence intensity with advance the speed.
In the active detection method of carboxypeptidase y of the present invention, the amount ranges of methoxyl group fluorescein derivative with
The concentration of carboxypeptidase y sample to be measured is related, it is generally the case that the dosage of methoxyl group fluorescein derivative can reach characterization carboxylic peptide
The activity of enzyme Y sample to be tested, under preferable case, relative to the carboxypeptidase y of 10 μ g/mL every in sample to be tested, the methoxy
The dosage of base fluorescein derivative can be 2-100 μM, and in order to reach optimal detection effect, the methoxyl group fluorescein is derivative
The dosage of object is preferably 2-10 μM.
In the active detection method of carboxypeptidase y provided by the invention, the carboxypeptidase y sample to be tested and methoxyl group fluorescence
It is 25-37 DEG C, pH value 7-9, time 5-40min that the condition of plain derivative contact (mixing is incubated for), which includes: temperature,.It is preferred that feelings
Under condition, the condition of the contact includes: that temperature is 30-37 DEG C, pH value 7.5-8.0, time 10-30min.
In detection method of the present invention, carboxypeptidase y sample to be measured and probe (methoxyl group fluorescein derivative) are preferred
Mixing is contacted under the conditions of being protected from light, and before contact mixing, preferably mixing buffer used and container are preheated,
Reaction vessel can be black plate, cuvette or ELISA Plate, the preferred cuvette of reaction vessel.It is excellent to preheat temperature to be achieved
It is selected as 25-37 DEG C.
The condition of the active detection method of carboxypeptidase y of the present invention further include 10mM Hepes (pH=7.5) or
It carries out in the phosphate buffer (pH=7.5) of 10mM, is preferably carried out in the Hepes of 10mM (pH=7.5) buffer.
The fluorescence intensity of material after contacting to the carboxypeptidase y sample to be tested with methoxyl group fluorescein derivative is at any time
The detection of value added can be carried out in microplate reader or sepectrophotofluorometer, measurement sample to be tested can be used and methoxyl group is glimmering
Fluorescence intensity changes with time value after the mixing of light element derivative, to obtain the enhancement value.Furthermore, it is possible to using being repeated several times
The mode for testing and being arranged several control groups guarantees the reliability of detection.
When the active detection method of carboxypeptidase y of the present invention is used for the screening of carboxypeptidase y inhibitor or promotor,
It can carry out, respectively be dissolved in certain density carboxypeptidase y, certain density inhibitor or promotor above-mentioned by the following method
In buffer, it is placed in thermostatic water bath and is preheated to preference temperature (25-37 DEG C)；Reaction vessel is placed in incubator in advance simultaneously
Heat is to preference temperature.Then, suitable buffer solution having been warmed up, a certain amount of carboxypeptidase y are inhibited in the reaction vessel
Agent or promotor are mixed with suitable methoxyl group fluorescein derivative.Carboxypeptidase y starting reaction is added under the conditions of being protected from light, and uses enzyme
Mark instrument or sepectrophotofluorometer are monitored the situation of change of fluorescent intensity.
The present invention also provides a kind of active detection kit of carboxypeptidase y, which includes of the present invention
Methoxyl group fluorescein derivative, carboxypeptidase y standard items and reaction buffer, the carboxypeptidase y standard items when detecting may be used
To function as control group or draw standard curve.Wherein, the buffer is buffer as described above.
Kit provided by the present invention can be used for detecting carboxypeptidase y in sample to be tested activity (including screening carboxylic peptide
Enzyme Y inhibitor or promotor and other necks relevant to carboxypeptidase y Activity determination or carboxypeptidase y inhibitor or accelerator function
Domain), the preservation condition of this kit is to be kept in dark place at subzero 20 to 4 DEG C.The particular condition in use user of the kit
Method can be carried out according to following staged an uprising example, by taking buffer is the Hepes (pH=7.5) of 10mM as an example.
(1) DMSO (dimethyl sulfoxide) solution of 10mM methoxyl group fluorescein derivative is configured, and at subzero 20 DEG C to 4
It is kept in dark place at DEG C, when use carries out gradient dilution.
(2) prepare 4mg/mL carboxypeptidase y liquid storage, solvent be 10mM Hepes (pH=7.5) and containing 50 weight % it is sweet
Oil carries out gradient dilution in subzero 20 DEG C or so preservations, use.
(3) sample to be tested containing carboxypeptidase y is prepared, solvent is the Hepes (pH=7.5) of 10mM.
(4) respectively by the buffer of the Hepes of 10mM (pH=7.5), sample to be tested and standard items containing carboxypeptidase y add
In cuvette, carboxylic in test sample and carboxypeptidase y standard items is measured using the detection method of carboxypeptidase y described in the invention
The activity of peptase Y.
(5) standard curve is drawn with the changing value of the fluorescence intensity of the carboxypeptidase y standard jar of the gradient dilution detected, led to
Cross the activity that comparison determines carboxypeptidase y in examined sample.
When kit of the invention is used for the screening of carboxypeptidase y inhibitor or promotor, the inhibitor can be existed
Before detection in appropriate proportions or viscosity makes an addition in reaction mixture.Suitable carboxypeptidase y standard items starting reaction is then added,
The condition of reaction is 25-37 DEG C, time 5-40min.
The present invention will be described in detail by way of examples below.In following embodiment and comparative example,
Carboxypeptidase y is purchased from Sigma-Aldrich Rate activity >=170U/mg
Protaminase is purchased from Sigma-Aldrich Rate activity >=50U/mg
Acetylcholinesterase is purchased from Sigma-Aldrich Rate activity >=1500U/mg
Butyrylcholine esterase is purchased from Sigma-Aldrich Rate activity >=900U/mg
Elastoser is purchased from Mayan company Rate activity >=30U/mg
Trypsase is purchased from Mayan company Rate activity >=250U/mg
Chymotrypsin is purchased from Mayan company Rate activity >=1500U/mg
Bovine serum albumin is 98% purchased from lark prestige scientific & technical corporation's purity
The change rate (△ F/ △ t) of fluorescence intensity is calculated according to following formula in following embodiment:
Wherein, F1 indicates the fluorescence intensity of t1 moment sample, and F2 indicates the fluorescence intensity of t2 moment sample.
Embodiment 1
The present embodiment is used to illustrate the preparation method of methoxyl group fluorescein derivative of the present invention.
It, will be shown in formula (2) in the presence of acid binding agent (triethylamine, 2mmol) in organic solvent (methylene chloride, 20mL)
Compound (methoxyl group fluorescein, 1mmol) and acyl chlorides (trimethyl-aceyl chloride, 2mmol) at 5 DEG C, maintain connecing for 120min
Touching, the material after being contacted.Then the material after successively washing contact with water (50mL) and saturated common salt aqueous solution (50mL),
Material after being washed.The organic phase in material after separating, washing is simultaneously dried with anhydrous sodium sulfate, and decompression removal is molten
Agent obtains crude product, and crude product is obtained white solid product 0.27g through silica gel column purification.
The white solid product is characterized with NMR and HRMS detection, data are as follows.1H NMR(600MHz,DMSO-d6):δ
8.02 (d, J=11.4Hz, 1H), 7.80 (t, J=11.1Hz, 1H), 7.74 (t, J=10.8Hz, 1H), 7.32 (d, J=
11.4Hz, 1H), 7.23 (s, 1H), 6.94 (s, 1H), 6.91-6.83 (m, 2H), 6.72 (q, J=12.2Hz, 2H), 3.84 (s,
3H),1.36(s,9H).HRMS calcd for[M+H]+:430.1153.Found:430.1144.。
The data are consistent completely with the theoretical value of formula (1) compound represented, it was demonstrated that the product is to change as shown in formula (3)
Close object.
Embodiment 2
The present embodiment is used to illustrate the preparation method of methoxyl group fluorescein derivative of the present invention.
The preparation of methoxyl group fluorescein derivative is carried out according to the method for embodiment 1, unlike, acyl chlorides uses ring
Hexyl formyl chloride, it is final to obtain 0.31g white solid.
The white solid product is characterized with NMR and HRMS detection, data:1H NMR(600MHz,DMSO-d6):δ8.05(d,
J=7.2Hz, 1H), 7.81 (t, J=7.2Hz, 1H), 7.75 (t, J=7.2Hz, 1H), 7.33 (d, J=7.2Hz, 1H), 7.23
(s, 1H), 6.96 (s, 1H), 6.92-6.86 (m, 2H), 6.74 (q, J=8.8Hz, 2H), 3.82 (s, 3H), 2.61
(quintuplet, J=9.6Hz, 1H), 1.99-1.97 (m, 2H), 1.73-1.71 (m, 2H), 1.51-1.46 (m, 2H),
1.36-1.30(m,2H),1.25-1.22(m,2H).HRMS calcd for[M+H]+:457.1646.Found:
457.1648.。
The data are consistent completely with the theoretical value of formula (1) compound represented, it was demonstrated that the product is to change as shown in formula (4)
Close object.
Embodiment 3
The present embodiment is used to illustrate the preparation method of methoxyl group fluorescein derivative of the present invention.
The preparation of methoxyl group fluorescein derivative is carried out according to the method for embodiment 1, unlike, acyl chlorides uses third
Acyl chlorides, it is final to obtain 0.35g white solid.
Characterizing the white solid product with NMR and HRMS detection is target product.
Test case 1
This test case is used to illustrate the activity test method of carboxypeptidase y provided by the invention.
By the carboxypeptidase y standard items liquid storage of 4mg/mL, with Hepes (pH=7.5) gradient dilution of 10mM to various concentration；
Carboxypeptidase y mark of the 3 liquid storage gradient dilution of structural formula that the embodiment 1 of 10mM is synthesized to 200 μM, after as above diluting
Quasi- product, buffer solution (Hepes (pH=7.5) of 10mM) and 200 μM of methoxyl group fluorescein derivatives are respectively at 30 DEG C of incubation 5-
10min.By the peptase Y solution after incubation, buffer solution and 200 μM of methoxyl group fluorescein derivatives are added in calculating ratio, most
Final concentration of 10 μM of methoxyl group fluorescein derivative are obtained eventually；The final concentration of 0.1-6 μ g/mL of carboxypeptidase y standard items is (such as 1 institute of table
Show).The solution of the mixed carboxypeptidase y standard items containing various concentration is added in cuvette and is surveyed under 455nm excitation wavelength
Determine fluorescence intensity in 5-10min to change with time at 515nm rate, is as a result listed in table 1.Wherein, carboxypeptidase y catalytic fluorometry is visited
The schematic diagram of needle hydrolysis is as shown in Figure 1.
Table 1
| Carboxypeptidase y standard items (μ g/mL) | The change rate (△ F/ △ t) of fluorescence intensity |
| 0.1 | 1.74 |
| 0.2 | 4.41 |
| 0.5 | 9.38 |
| 1 | 20.97 |
| 2 | 43.55 |
| 3 | 56.88 |
| 4 | 74.45 |
| 6 | 99.48 |
Contrast test example 1
The contrast test example is for illustrating the active detection method of carboxypeptidase y in the prior art.
The substrate hydrolysis of series of concentrations gradient is catalyzed using UV, visible light spectrophotometric method detection carboxypeptidase y standard items
Situation, unlike, substrate probe used is trimethylace tonitric p-nitrophenyl ester, and reaction density is 10 μM, buffer solution
(Hepes (pH=7.5) of 10mM) docks ultravioletvisible absorption value after touch in the case where wavelength is the exciting light of 400nm and carries out power
Scanning is learned, the change rate of the ultravioletvisible absorption value in 5-10 minutes is obtained, is as a result listed in table 2.
Table 2
| Carboxypeptidase y standard items (μ g/mL) | Change rate (the Δ A/ Δ t) of ultravioletvisible absorption value |
| 2 | 0.0003 |
| 5 | 0.0006 |
| 10 | 0.0012 |
| 15 | 0.0020 |
| 20 | 0.0029 |
Test case 2
This test case is used to illustrate the specificity of carboxypeptidase y activity test method provided by the invention.
With the buffer of the Hepes (pH=7.5) of 10mM by various enzymes (carboxypeptidase y, protaminase, chymotrypsin,
Trypsase, acetylcholinesterase, butyrylcholine esterase, elastoser and bovine serum albumin) sample preparation at 200 μ g/mL,
Respectively by buffer solution, 200 μ g/mL different enzyme samples, the methoxyl group fluorescein derivative of 200 μM of embodiments 1 preparation is in 30
DEG C be incubated for 5-10min.According to 200 μ g/mL each 40 μ L of different enzyme samples, 720 μ L of buffer solution, 200 μM of methoxyl group fluoresceins
40 μ L total volume of derivative, 800 μ L is added in cuvette after beating reaction system mixing, with microplate reader in 455nm excitation wavelength
Fluorescence intensity changes with time rate at 515nm in lower measurement 5-10min, the product hydrolysis in different enzymatic embodiments
Relative speed it is as shown in Figure 2.
It can be seen that the methoxy that structure shown in formula (1) can be made using method of the invention by the result of embodiment 1
Base fluorescein derivative.
It can be seen that compared with existing polypeptide Substrate fluorescence probe by the result of test case 1 and contrast test example 1,
Non-peptide methoxyl group fluorescein derivative fluorescence probe and carboxypeptidase y of the invention has higher sensitivity and affinity.
It is glimmering to methoxyl group fluorescein derivative provided by the present invention to can be seen that carboxypeptidase y by the result of test case 2
The hydrolysis ability of light probe is significantly higher than other kinds of enzyme, therefore, methoxyl group fluorescein derivative provided by the present invention
Specificity it is high.
Meanwhile methoxyl group fluorescein derivative fluorescence probe provided by the present invention has higher Atom economy, this
Outside from the aspect of the easy degree for obtaining raw material, methoxyl group fluorescein derivative fluorescence probe provided by the present invention is easy to close
At, and polypeptide substrate used in comparative example 1 is then difficult to synthesize.Therefore, methoxyl group fluorescein provided by the present invention is derivative
There is provided fluorescence probe object implementation to be more easier compared to the prior art, the lower advantage of cost.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (11)
1. a kind of methoxyl group fluorescein derivative, which has structure shown in formula (1), wherein R is
Tert-butyl；
2. the preparation method of methoxyl group fluorescein derivative described in claim 1, this method comprises: in necleophilic reaction condition and
In the presence of acid binding agent, in organic solvent, structural compounds shown in formula (2) are contacted with one of acyl chlorides or acid anhydrides；
Wherein, the general formula of the acyl chlorides is R '-C (=O) Cl, R ' and R selection having the same；
The general formula of the acid anhydrides is R "-C (=O)-O- (O=C)-R ", R " and R selection having the same.
3. preparation method according to claim 2, wherein relative to structural compounds shown in 1 mM of formula (2), institute
The dosage for stating acyl chlorides or acid anhydrides is 1-2 mMs, and the dosage of the acid binding agent is 1-3 mMs, the dosage of the organic solvent
It is 20-30 milliliters.
4. preparation method according to claim 3, wherein the acid binding agent is selected from triethylamine, diethylamine, diisopropyl
Amine, pyridine, N, in N- dimethylamino naphthyridine, 2,6- lutidines, potassium carbonate, cesium carbonate, sodium carbonate and sodium bicarbonate extremely
Few one kind, the organic solvent are selected from methylene chloride, tetrahydrofuran, chloroform, carbon tetrachloride, acetonitrile, acetone, glycol dinitrate
At least one of ether, N,N-dimethylformamide and 1,2- dichloroethanes.
5. the preparation method according to any one of claim 2-4, wherein the condition of the necleophilic reaction includes: temperature
Degree is subzero 5 DEG C to 30 DEG C, time 10-120min.
6. application of the methoxyl group fluorescein derivative described in claim 1 in the activity of detection carboxypeptidase y.
7. a kind of active method of detection carboxypeptidase y, which is characterized in that this method comprises:
Sample to be tested containing carboxypeptidase y is contacted with methoxyl group fluorescein derivative described in claim 1, after obtaining contact
Material；
The value added of the fluorescence intensity that material after detection contact issues under excitation light at any time, the increase of the fluorescence intensity
Value indicates the activity of carboxypeptidase y in sample to be tested；The wavelength of the exciting light is 410-490nm, the transmitted wave of the fluorescence
A length of 505-555nm.
8. detection method as claimed in claim 7, wherein relative to the carboxypeptidase y of 10 μ g/mL, the methoxyl group fluorescein spreads out
The dosage of biology is 2-100 μM.
9. detection method according to any one of claims 8, wherein relative to the carboxypeptidase y of 10 μ g/mL, the methoxyl group fluorescein spreads out
The dosage of biology is 2-10 μM.
10. detection method described in any one of claim 7-9, wherein the condition of contact includes: that temperature is 25-37 DEG C,
PH value is 7-9, time 5-40min.
11. a kind of active detection kit of carboxypeptidase y, which includes: that methoxyl group described in claim 1 is glimmering
Light element derivative, carboxypeptidase y standard items and reaction buffer.
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| A Minimalist Substrate for Enzymatic Peptide and Protein Conjugation;James W. Wollack;《Chembiochem》;20091214;第10卷(第18期);2934-2943 * |
| Fluorogenic ester substrates to assess proteolytic activity;Laurent Mugherli;《Bioorganic & Medicinal Chemistry Letters》;20060627;第16卷;4488-4491 * |
| Graphene Oxide−Peptide Nanocomplex as a Versatile Fluorescence Probe of Protein Kinase Activity Based on Phosphorylation Protection against Carboxypeptidase Digestion;Jiang Zhou;《Analytical Chemistry》;20130523;第85卷;5746-5744 * |
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