CN109929548A - A kind of novel near infrared fluorescent probe for Carboxypeptidase A detection - Google Patents
A kind of novel near infrared fluorescent probe for Carboxypeptidase A detection Download PDFInfo
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- 102000000496 Carboxypeptidases A Human genes 0.000 title claims abstract description 21
- 108010080937 Carboxypeptidases A Proteins 0.000 title claims abstract description 21
- 238000001514 detection method Methods 0.000 title claims abstract description 20
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 6
- 239000000523 sample Substances 0.000 abstract description 34
- 210000001819 pancreatic juice Anatomy 0.000 abstract description 5
- 238000001727 in vivo Methods 0.000 abstract description 4
- 238000000799 fluorescence microscopy Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 201000010099 disease Diseases 0.000 abstract description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 2
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 238000003786 synthesis reaction Methods 0.000 abstract 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 21
- 239000000047 product Substances 0.000 description 16
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 13
- 102000005367 Carboxypeptidases Human genes 0.000 description 10
- 108010006303 Carboxypeptidases Proteins 0.000 description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 102000003670 Carboxypeptidase B Human genes 0.000 description 6
- 108090000087 Carboxypeptidase B Proteins 0.000 description 6
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000005160 1H NMR spectroscopy Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 239000012300 argon atmosphere Substances 0.000 description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 229910052938 sodium sulfate Inorganic materials 0.000 description 4
- 235000011152 sodium sulphate Nutrition 0.000 description 4
- NHGXDBSUJJNIRV-UHFFFAOYSA-M tetrabutylammonium chloride Chemical compound [Cl-].CCCC[N+](CCCC)(CCCC)CCCC NHGXDBSUJJNIRV-UHFFFAOYSA-M 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 229960000583 acetic acid Drugs 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 102000005572 Cathepsin A Human genes 0.000 description 2
- 108010059081 Cathepsin A Proteins 0.000 description 2
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- -1 butyl sulfonic acid lactone Chemical class 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- WBJINCZRORDGAQ-UHFFFAOYSA-N ethyl formate Chemical compound CCOC=O WBJINCZRORDGAQ-UHFFFAOYSA-N 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- CCERQOYLJJULMD-UHFFFAOYSA-M magnesium;carbanide;chloride Chemical compound [CH3-].[Mg+2].[Cl-] CCERQOYLJJULMD-UHFFFAOYSA-M 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N naphthalene-acid Natural products C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 239000012299 nitrogen atmosphere Substances 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- TXUICONDJPYNPY-UHFFFAOYSA-N (1,10,13-trimethyl-3-oxo-4,5,6,7,8,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-17-yl) heptanoate Chemical compound C1CC2CC(=O)C=C(C)C2(C)C2C1C1CCC(OC(=O)CCCCCC)C1(C)CC2 TXUICONDJPYNPY-UHFFFAOYSA-N 0.000 description 1
- GTACSIONMHMRPD-UHFFFAOYSA-N 2-[4-[2-(benzenesulfonamido)ethylsulfanyl]-2,6-difluorophenoxy]acetamide Chemical compound C1=C(F)C(OCC(=O)N)=C(F)C=C1SCCNS(=O)(=O)C1=CC=CC=C1 GTACSIONMHMRPD-UHFFFAOYSA-N 0.000 description 1
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 1
- RTZZCYNQPHTPPL-UHFFFAOYSA-N 3-nitrophenol Chemical compound OC1=CC=CC([N+]([O-])=O)=C1 RTZZCYNQPHTPPL-UHFFFAOYSA-N 0.000 description 1
- 101710130081 Aspergillopepsin-1 Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102100031007 Cytosolic non-specific dipeptidase Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229910021626 Tin(II) chloride Inorganic materials 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 229940126214 compound 3 Drugs 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- YDCHPLOFQATIDS-UHFFFAOYSA-N methyl 2-bromoacetate Chemical compound COC(=O)CBr YDCHPLOFQATIDS-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- WCVHUIPWSPEOIG-UHFFFAOYSA-N n,n-dimethylheptadecan-1-amine Chemical compound CCCCCCCCCCCCCCCCCN(C)C WCVHUIPWSPEOIG-UHFFFAOYSA-N 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000001119 stannous chloride Substances 0.000 description 1
- 235000011150 stannous chloride Nutrition 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of near infrared fluorescent probes for detecting Carboxypeptidase A, the sensing detection of the fluorescence probe Carboxypeptidase A interior, external for organism, and the synthesis of this probe is simple, it is cheap and easy to get, fluorescence imaging can be in vivo carried out, has good diagnostic function to pancreatic juice leakage disease.Fluorescence probe of the invention is selectively good, high sensitivity, it is often more important that launch wavelength in the near infrared region, makes it have excellent anti-autofluorescence ability.Probe of the invention has broad application prospects in analytical chemistry detection and bio-medical.
Description
Technical field
The present invention design belongs to analytical chemistry detection technique field, and in particular to it is a kind of identification pancreatic juice in Carboxypeptidase A it is new
The preparation method and its application external in vivo of type near-infrared probe.
Background technique
Carboxypeptidase is that (CPs) is a kind of protease that one or more amino acid are divided out from the end c of polypeptide or protein,
Common includes Carboxypeptidase A, protaminase, cathepsin A and four kinds of carboxypeptidase y, all has weight in physiology course and various diseases
It acts on.Carboxypeptidase A and protaminase occur mainly in pancreas, play a role in the degradation process of protein, and carboxylic peptide
Expression of the enzyme in pancreatitis and cancer of pancreas is different, this just provides possibility to detect the leakage of pancreatic juice.Currently, right
In the detection of carboxypeptidase, high performance liquid chromatography, absorption spectrometry and coupling analysis method, but these types of detection method are mainly used
All it is the detection for being defined in external carboxypeptidase, carboxypeptidase can't be imaged in real time.In recent years, the method for fluorescence imaging because
Its distinctive advantage causes the extensive concern of people.The fluorescence probe of near infrared region has selectivity good, high sensitivity, group
The advantages that knitting strong penetrating power and the interference of anti-autofluorescence, is often used for histocyte imaging research external in vivo.
Nevertheless, cut-off is till now, the fluorescence probe for carboxypeptidase detection is fresh to be reported, and that reports detects for carboxypeptidase
Fluorescence probe all there is short launch wavelength.It is well known that long launch wavelength to cell fluorescence imaging be it is extremely beneficial, because
The light scattering that environmental induction can be reduced for the photon with long wavelength, to reduce the shadow of interior raw chromophore in detection process
Ring, and the light injury of living cells can be reduced, thus to launch wavelength in the near infrared region for the glimmering of carboxypeptidase detection
The design of light probe is just very urgent.
Summary of the invention
For the status of current carboxypeptidase detection, the present invention is synthesized a kind of with long launch wavelength by MOLECULE DESIGN
For Carboxypeptidase A detection near infrared fluorescent probe, and by probe be used for pancreas pancreatic juice leakage detection.
The following technical solution is employed by the present invention:
A kind of novel near infrared fluorescent probe for Carboxypeptidase A detection, has following molecular structural formula:
The synthetic route of the fluorescence probe is as follows:
Details as Follows for preparation method: (1) take 1,8- naphthalene lactim (0.85g, 5mmol), potassium hydroxide (0.56g, 10mmol),
Be dissolved in n-methyl-2-pyrrolidone (10ml), 30min be stirred at room temperature, later be added butyl sulfonic acid lactone (0.75g,
5.5mmol), 90 DEG C of reaction 10h are again heated to, are cooled down later, then are handled with acetone (35ml), product 1a is obtained；(2) 1a is taken
(1.50g, 5mmol), tetrabutylammonium chloride (1.51g, 5.5mmol) are dissolved in acetic acid (8ml), 0.5h are stirred at 90 DEG C, so
After ethyl acetate is added dropwise, refilter, rotate, obtain the product 2 of viscous oil shape；(3) product 2(2.73g, 5mmol are taken) and
Methyl-magnesium-chloride is dissolved in anhydrous tetrahydro furan (20ml), in a nitrogen atmosphere, is heated to 60 DEG C and is stirred 1h, then cold
But, 1M hydrochloric acid is added to be neutralized, and is diluted with ethyl alcohol (15ml) and ether (20ml), be cooled back to 0 DEG C, overnight, obtain
To product 3；(4) product 3(606mg, 2mmol are taken), substance 3a(360mg, 1mmol), sodium acetate (164g, 2mmol) is dissolved in vinegar
Acid anhydrides (40ml), is stirred at room temperature 2h, is handled later with ether (60ml), finally crosses chromatographic column, obtains product 4;(5) 3- is taken
Nitrophenol (347mg, 2.5mmol), potassium carbonate (345mg, 2.5mmol) are dissolved in acetonitrile (15ml), later in argon atmosphere
Under 10min is stirred at room temperature, take IR-780(765mg, 1mmol) be dissolved in acetonitrile, then add it in said mixture, room temperature
Lower stirring 4h, mixture is evaporated under reduced pressure later, and obtained concentrate is dissolved in methylene chloride, is then washed with water three times,
Dry with sodium sulphate again, the residue obtained after evaporating is dispersed in methanol (30ml) for future use；Take stannous chloride (4g,
It 20mmol) is dissolved in concentrated hydrochloric acid, is added in aforementioned methanol solution under an argon atmosphere later, reaction is heated to 70 DEG C, and stirs
It mixes overnight, is then neutralized with the sodium carbonate liquor of saturation, sediment is filtered out later, and washed with methylene chloride, by collection
Filtrate water is handled three times, then dry with sodium sulphate, is evaporated under reduced pressure later, is crossed chromatographic column and is obtained product 5；(6) bromoacetic acid first is taken
Ester and product 5 react in acetonitrile, add sodium hydroxide, are washed three times later, are being rotated, dissolving the residue in second
Nitrile adds phenylalanine, is heated to 120 DEG C, flows back, and overnight, after extracting liquid separation, is filtered, obtains required probe FD-
CPA。
That Details as Follows is described for the detection mechanism of fluorescence probe of the invention to Carboxypeptidase A, with bromoacetic acid by dye molecule with
Phenylalanine connects to form fluorescence probe, and the electronics transfer (ICT) of intramolecular is blocked, Carboxypeptidase A (CPA) there are the case where
Under, the phenylalanine of specific identification carbon teminal, and cut down, restore ICT process, under excitation light, has launched
Fluorescence signal.Response process is as follows:
In the near infrared region, itself does not emit fluorescence to fluorescence probe launch wavelength of the invention, after reacting with CPA, issues very
Strong fluorescence, maximum emission wavelength 826nm.
The Stokes shift of fluorescence probe of the invention reaches 166nm, maximum absorption wave a length of 660nm, anti-with CPA
Ying Hou, maximum emission wavelength 826nm.
Fluorescence probe of the invention has good selectivity to Carboxypeptidase A, in the buffer solution of PBS of the PH equal to 7.4,
Probe solution does not emit fluorescence, and after the pancreatic juice of 1.25 μ g is added, fluorescence intensity is gradually increased, and fluorescence intensity increases to after 30 minutes
42 times of 0 minute, are separately added into other chemical substances that may interfere with, including protaminase under similarity condition, HSH, Hcy, Cys,
Na+,K+,Mg2+Deng the fluorescence intensity of probe is without significant change.
Fluorescence probe of the invention have very strong anti-interference ability, it is other may interfere with chemical substance (protaminase,
HSH,Hcy,Cys,Na+,K+,Mg2+) presence do not influence response of the probe molecule to Carboxypeptidase A.
The PH scope of application of fluorescence probe of the invention is wider, and probe molecule is not affected in the range of 7 to 13 to carboxylic
The detection of PEPA A.
Advantages of the present invention: probe molecule raw material is cheap and easy to get, and synthetic yield is higher, and detection carboxypeptidase is selectively good, spirit
Sensitivity is high, has long launch wavelength, small to the light injury of cell in vivo in imaging process, has in chemical analysis field
Wide application prospect.
Example is embodied
Embodiment 1: the preparation of sylvite 1a
1,8- naphthalene lactim (0.85g, 5mmol) is taken, potassium hydroxide (0.56g, 10mmol) is dissolved in N- methyl -2- pyrrolidines
Ketone (10ml), is stirred at room temperature 30min, and butyl sulfonic acid lactone (0.75g, 5.5mmol) is added later, is again heated to 90 DEG C instead
10h is answered, is cooled down later, then is handled with acetone (35ml), product 1a (1.65 g, 96%) are obtained. 1H NMR (400 MHz,
DMSO-d6): 8.18 (d,J = 8.1 Hz, 1H), 8.07 (d, J = 8.3 Hz, 1H), 7.81 (t, J = 8.7
Hz, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.55 (t, J = 8.7 Hz, 1H), 7.23 (d, J =7.5
Hz, 1H), 3.91 (t, J = 7.2 Hz, 2H), 2.46 (m, 2H), 1.79 (m, 2H), 1.62 (t, J =
7.2 Hz, 2H)。
Embodiment 2: the preparation of intermediate product 2
It takes 1a (1.50g, 5mmol), tetrabutylammonium chloride (1.51g, 5.5mmol) is dissolved in acetic acid (8ml), stirs at 90 DEG C
0.5h is mixed, ethyl acetate is then added dropwise, is refiltered, rotates, obtains the product 2 (2.5 g, 94%) of viscous oil shape.1H NMR
(400 MHz, DMSO-d6): 8.01 (t, J = 7.4 Hz, 1H) ,7.66 (t, J = 7.4 Hz, 1H), 7.51
(m, 2H) 7.24 (d, J = 7.2 Hz, 1H), 7.17 (d, J = 7.1 Hz, 1H), 6.97 (d, J = 7.1
Hz, 1H), 3.93 (t, J = 7.4Hz, 1H), 3.26 (t, J = 8.6 Hz, 8H), 2.90 (m, 2H),
1.94 (m, 4H), 1.64 (m, 8H), 1.42 (m, 8H), 0.98 (t, J = 8.6 Hz, 12H)。
Embodiment 3: the preparation of compound 3
Take product 2(2.73g, 5mmol) and methyl-magnesium-chloride, it is dissolved in anhydrous tetrahydro furan (20ml), in a nitrogen atmosphere,
It is heated to 60 DEG C and is stirred 1h, then cool down, 1M hydrochloric acid is added and is neutralized, and with ethyl alcohol (15ml) and ether (20ml)
It is diluted, is cooled back to 0 DEG C, overnight, obtain product 3 (2.58 g, 85%).1H NMR (D2O): 8.51 (d, J =
8.1 Hz, 1H),8.43 (d, J = 8.1 Hz, 1H), 8.09 (m, 2H), 7.85 (t, J = 8.5 Hz, 1H),
7.73 (t, J = 8.2 Hz, 1H), 4.51, (t, J = 8.5 Hz, 2H), 3.02 (s, 3H),2.85 (m,
2H), 2.04 (m, 2H), 1.78 (t, J = 8.2 Hz, 2H). MS (maldi) m/z: calcd. for
[C16H19NO3S]+, 304.10; found 304.10。
Embodiment 4: the preparation of flower cyanine type dye 4
Take product 3(606mg, 2mmol), substance 3a(360mg, 1mmol), sodium acetate (164g, 2mmol) is dissolved in acetic anhydride
2h is stirred at room temperature in (40ml), later with ether (60ml) handle, finally cross chromatographic column (CH2Cl2: CH3OH=2:
1) product 4 (612 mg, 81%), is obtained1H NMR(400 MHz, DMSO-d6): 8.37 (d, J = 13.1 Hz,
2H), 8.04 (d, J = 7.1 Hz, 2H), 7.84 (d, J = 7.4 Hz, 2H), 7.73 (t, J = 7.1 Hz,
2H),7.42 (t, J = 7.1 Hz, 2H), 7.29 (d, J = 7.4 Hz, 2H), 6.88 (d, J = 7.1 Hz,
2H), 6.20 (d, J = 13.1 Hz, 2H), 4.01 (t, J = 7.5 Hz, 4H),2.83 (t, J = 7.5 Hz,
4H), 1.82 (m, 14H). High-resolution MS (ESI, positive ion mode) m/z: calcd.
for [C40H39ClN2NaO6S2]+,765.1830; found 765.1827。
Embodiment 5: the preparation of dyestuff 5
It takes 3- nitrophenol (347mg, 2.5mmol), potassium carbonate (345mg, 2.5mmol) is dissolved in acetonitrile (15ml), Zhi Hou
10min is stirred at room temperature under argon atmosphere, takes IR-780(765mg, 1mmol) it is dissolved in acetonitrile, then add it to above-mentioned mixing
In object, 4h being stirred at room temperature, mixture is evaporated under reduced pressure later, obtained concentrate is dissolved in methylene chloride, then uses water
Three times, then with sodium sulphate drying, the residue obtained after evaporating is dispersed in methanol (30ml) for future use for washing；Take chlorination
Stannous (4g, 20mmol) is dissolved in concentrated hydrochloric acid, is added in aforementioned methanol solution under an argon atmosphere later, and reaction is heated to 70
DEG C, and be stirred overnight, it is then neutralized with the sodium carbonate liquor of saturation, filters out sediment later, and washed with methylene chloride, it will
The filtrate water of collection is handled three times, then dry with sodium sulphate, is evaporated under reduced pressure later, is crossed chromatographic column and is obtained product 5.
Embodiment 6: the preparation of probe molecule
It takes methyl bromoacetate and product 5 to react in acetonitrile, adds sodium hydroxide, wash three times, rotated later, it will
Residue is dissolved in acetonitrile, adds phenylalanine, is heated to 120 DEG C, flows back, and overnight, after extracting liquid separation, is filtered, is obtained
To required probe FD-CPA.
Embodiment 7: the application of fluorescence probe of the present invention
Take probe molecule mother liquor (1.0 × 10-3Mol/L) in PBS buffer solution (10mM, PH=7.4,1.0Mm hexadecyl
Trimethylamine) in be made into solution to be measured, then be separately added into various substances (protaminase, HSH, Hcy, Cys, Na+,K+,Mg2+) 37 DEG C
Heat preservation measures after 1 hour, and the probe solution fluorescence of Carboxypeptidase A is only added substantially almost without significant change in solution fluorescence
Degree enhancing, it is seen that fluorescence probe can be to Carboxypeptidase A specific recognition.As other substances (protaminase, HSH, Hcy, Cys, Na+,K+,Mg2+) respectively and carboxypeptidase be added simultaneously, 37 DEG C heat preservation 1 hour after measure, probe solution fluorescence remain unchanged significantly increase,
The fluorescence probe of invention goes out very strong anti-interference ability to the detected representation of Carboxypeptidase A.PH does not affect probe molecule 7 to 13
Detection to Carboxypeptidase A, it is seen that fluorescence probe of the invention has good biocompatibility.
Claims (1)
1. a kind of novel near infrared fluorescent probe for Carboxypeptidase A detection, structure are as follows:
。
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