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CN107011324A - DPP IV enzyme near infrared fluorescent probe substrate and preparation method and application - Google Patents

DPP IV enzyme near infrared fluorescent probe substrate and preparation method and application Download PDF

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CN107011324A
CN107011324A CN201610056580.5A CN201610056580A CN107011324A CN 107011324 A CN107011324 A CN 107011324A CN 201610056580 A CN201610056580 A CN 201610056580A CN 107011324 A CN107011324 A CN 107011324A
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dpp
probe substrate
substrate
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alkylidene
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杨凌
钱星凯
葛广波
邹立伟
王平
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Dalian Institute of Chemical Physics of CAS
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Dalian Institute of Chemical Physics of CAS
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Abstract

The invention provides near infrared fluorescent probe substrate of a kind of DPP IV and preparation method and application, belong to biomedicine technical field.The probe substrate is the glycinylproline derivative GP-R of substitution 4- vinyl aniline, and it can be used for the enzyme activity for determining DPP-IV in different biosystems.The flow of DPP-IV enzyme activity determinations is as follows：It is probe reaction to select GP-R amide hydrolysis, and quantitatively GP-R takes off the growing amount of dipeptides based products to determine the activity of DPP-IV in different kind organism sample in the detection unit interval in physiological conditions.Quantitative determination of the present invention available for DPP-IV enzyme activity in the body fluid, cell and tissue preparation thing of separate sources, DPP-IV enzyme inhibitors or derivant screening.

Description

DPP IV enzyme near infrared fluorescent probe substrate and preparation method and application
Technical field
The invention belongs to biomedicine technical field, and in particular to DPP IV enzyme near infrared fluorescent probe substrate and Its preparation method and application.
Background technology
Dipeptidyl peptidase-IV (Dipeptidyl peptidase IV, DPP-IV, CD26, EC 3.4.14.5) is with two The II type transmembrane protein enzyme that dimer form is present, is distributed widely in the epithelial cell and vascular endothelial cell of mammal internal organs, Highest is wherein expressed in small intestine and kidney, can be also present in dissolved form in body fluid.It is used as important I phase metabolic enzymes, DPP- IV can specifically the amino acid residue proline (Pro) of catalytic polypeptide chain N-terminal the 2nd or alanine (Ala) peptide bond hydrolysis Fracture, so as to participate in the activation of internal multiple biological activities polypeptide, and makes various active polypeptide portion or complete deactivation, such as intestines Pancreotropic hormone, neuropeptide, gastrin releasing peptide, growth hormone releasing hormone etc..DPP-IV plays the part of important during glycometabolism Role, has become treatment diabetes B novel targets, while being also approved as important drug target by FDA.In addition it Or cell-membrane receptor and costimulatory molecules, so as to participate in the immunological regulation of body, cell migration, cell adhesion and natural death of cerebral cells Process, thus it is closely related with the generation development of a variety of diseases.Research shows that DPP-IV high expression in some tumour cells (is tied Intestinal cancer, primary lung cancer, prostate cancer, oophoroma, thyroid cancer, esophagus malignant adenoma etc.), utilize DPP-IV metabolism activation machines System, using DPP-IV as potential target spot, can design the anticancer prodrug of targeting.Clinical research shows that many diseases can cause patient Blood/urine DPP-IV contents are significantly changed, and such as hepatopathy cancer blood samples of patients/serum DPP-IV contents are significantly higher than normal person, sugar The sick nephrotic's urine DPP-IV contents of urine substantially increase.It has been reported that the DPP-IV in detection blood/serum or urine can make For some medicals diagnosis on disease and the mark assessed.Therefore, the Activity determination of DPP-IV enzymes has wide potential applicability in clinical practice, right Early diagnosis, assessment and the prognosis treatment of disease, and high frequency zone DPP-IV inhibitor are used as candidate's medicine for the treatment of diabetes Thing etc..
At present, qualitative assessment DPP-IV method mainly includes p-nitrophenyl amine derivative GP-pNA substrates development process and 4- Methyl -7- aminocoumarin derivative GP-AMC fluorescence probe methods.Suitable for the DPP-IV's containing single component more than GP-pNA systems Detect (such as screening of single enzyme DPP-IV inhibitor), it is numerous with analysis process to there is sample preparation in actual biology sample detection The trivial, defect such as test flux is low, cost is high, bio-matrix interference is big.And to be applied to biological sample (body fluid, thin for GP-AMC systems Born of the same parents, tissue etc.) detection when, by absorb and launch wavelength limit (λex=360/ λem=460nm) easily carried on the back by bio-matrix Scape absorb and background fluorescence interference so that the sensitivity of fluorescence analysis and accuracy receive very big influence, using when hold The result being easy to get to false positive or false negative.In recent years, as people are to DPP-IV and the depth of disease development relation research Enter, the performance of existing DPP-IV fluorescence probes can not meet demand of the people to DPP-IV accurate quantification appraisal procedures, Especially for the quick detection or functional imaging of the complex biological system such as blood, urine, cancerous cells/tissue.Therefore, it is badly in need of out It is simple to operate to send out a kind of, strong antijamming capability, can highly sensitive quantitative detection of complex biosystem DPP-IV activity fluorescence probe Substrate.
Near-infrared (NIR) fluorescence probe can avoid environmental and biological samples reasons for its use from making an uproar because it excites and detected Sound, can be effectively prevented from the self-absorption of biological sample and the interference of autofluorescence.Meanwhile, in Photobiology window (600m- In the range of 900nm), the ultraviolet-visible light injury that life system can not be born is avoided, photobleaching and light injury is reduced. In addition, the tissue that near-infrared fluorescence imaging can be penetrated, compensate for the low defect of penetration into tissue in optical imagery.Therefore, closely Infrared fluorescence probes have obvious superiority in complex biological sample analysis detection.Develop the DPP-IV near-infrareds of high selectivity Fluorescence probe reacts and its supporting high-flux detection method has important practical value.
The content of the invention
It is an object of the invention to provide a kind of specific and infrared near-infrared for determining DPP IV (DPP-IV) Fluorescence probe substrate and its application, the fluorescence probe substrate and go two peptidyl products fluorescence emission wavelengths have substantially it is poor It is different, and product fluorescence quantum yield it is higher be more easy to detection.Can be to DPP-IV in a variety of biosystems using the probe reaction Distribution and function carry out quantitative assessment.
The invention provides the specificity fluorescent probe substrate of DPP-IV a kind of, the probe substrate can be by DPP-IV specificity Product of the catalysis generation with different fluorescence properties simultaneously generates corresponding substituted aromatic amines, and the substrate structure formula is as follows：
Wherein, X is selected from O, N；R is selected from C1-C10Alkyl ,-(C1-C8Alkylidene)-carboxyl ,-(C1-C8Alkylidene) -ester base ,- (C1-C8Alkylidene)-amino ,-(C1-C8Alkylidene)-cyano group ,-(C1-C8Alkylidene)-nitro ,-(C1-C3Alkylidene)-O- (C1- C3Alkyl), carbocylic radical ,-(C1-C3Alkylidene)-carbocylic radical, aryl ,-(C1-C3Alkylidene)-aryl, heteroaryl ,-(C1-C3It is sub- Alkyl)-heteroaryl, heterocyclic radical or-(C1-C3Alkylidene)-heterocyclic radical.
The invention provides a kind of preparation method of DPP IV near infrared fluorescent probe substrate, it is characterised in that：
1) using 2- acetyl phenols as initiation material, 2- acetoacetos phenol 2 is combined to by base catalysis acetyl, acid is urged Change Cyclization flavones 3；
2) acid catalysis flavones 3 and malononitrile reaction synthesis dicyan chromene 4, or flavones 3 and substitution amine reaction synthesis acid Property/neutrality/alkalescence, polar/non-polar, the N- substd quinolines ketone 5 of hydrophilic/hydrophobic and serial substituted radical not of uniform size, With malononitrile reaction synthesis dicyan N- substd quinolines 6；
3) dicyan chromene 4 or dicyan N- substd quinolines 6 and para aminotenzaldehyde reaction synthesis of trans substitution 4- ethene Base aniline 7；
4) trans substitution 4- vinyl aniline 7 (1.0eq) N- hydroxy benzo triazoles (0.5-2.0eq), 1- ethyls- (3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (0.5-2.0eq), 2- (7- azos BTA)-N, N, N', N'- In the presence of tetramethylurea hexafluorophosphoric acid ester (1.0-3.0eq), contracted with t-butoxycarbonyl glycine base proline (1.0-3.0eq) Close, 20-30 DEG C of stirring reaction 24-48 hours, the product of generation is purified through column chromatography, then through trifluoroacetic acid acid catalysis, 20-30 DEG C 4-10 hours removing tertbutyloxycarbonyls of stirring reaction generate the near infrared fluorescent probe substrate GP-R of DPP IV, after purification Yield is 30-80%.
The reaction scheme of the reaction is as follows：
The present invention also provides a kind of application of measure DPP-IV specificity fluorescent probe substrate, special using the DPP-IV Property substrate, enzymatic reaction is carried out after being mixed with the biological sample containing DPP-IV, by it is quantitative detection the unit interval in substrate disappear Except rate or its go the production rate of two peptidyl products to quantitative determine the activity of DPP-IV in different biosystems, specific measure side Method and condition are as follows：
A. Ratio-type probe substrate is used as using GP-R in system；Concentration of substrate selects 1/10~10Km；
B. in PBS, reaction temperature is between 20~60 DEG C；Incubation system pH is between 5.5~10.5；
C. the reaction time be 5~120 minutes, it is ensured that the corresponding N- of above substrate go two peptidyl products reach quantitative limit and Terminating reaction when substrate conversion efficiency is no more than 20%；
D. substrate decrement or N- go two peptidyl product formations commenting as DPP-IV activity in the analytical unit time Valency index.
In specific assay method and condition, concentration of substrate preferred K during single point assaym。
In specific assay method and condition, preferably 37 DEG C of reaction temperature, the preferred pH7.4 of incubation system pH.
Described biosystem is the mono- enzymes of recombination expression DPP-IV, human or animal's blood/serum, urine, tissue preparation liquid Or any one in mammalian species histocyte and its prepared product.
The probe substrate and its go dipeptides based products that there is different UV absorption and fluorescence emission spectrum, available for ultraviolet And fluoroscopic examination.The probe substrate can be additionally used in the quick screening of DPP-IV inhibitor and the quantitative assessment of rejection ability.
The Specific probe is near infrared fluorescent probe, and it is difficult by biosystem in DPP-IV Activity determination processes The interference of matrix and impurity, available for various restructuring DPP-IV, people and animal tissue's preparation solution, blood/serum, urine and all kinds of The quantitative determination of DPP-IV enzyme activity in histocyte；It can also be commented simultaneously as the probe substrate in body and animal entirety DPP-IV Estimate metabolic enzyme DPP-IV individual and species variation.The probe substrate and N- remove the fluorescence detection method of two peptidyl metabolites It can be additionally used in the quick screening of DPP-IV inhibitor and the quantitative assessment of rejection ability.
As the fluorescence probe substrate of the mono- enzymes of the DPP-IV of high specific, the compound can be for detecting DPP-IV work Property, it is especially suitable for the DPP-IV produced to bacterium, insect cell, mammalian cell and saccharomycete clonal expression system Enzyme activity determination, and in the prepared product such as microsome, S-9 of a variety of mammalian tissues organ origins DPP-IV activity mark It is fixed.
From the mono- enzyme external activity tools of near infrared fluorescent probe reaction detection DPP-IV of the mono- enzymes of DPP-IV of the present invention There is advantage following prominent：
(1) high specific：GP-R can be metabolized to a metabolite with high specificity by DPP-IV, i.e. N- goes dipeptidesization to produce Thing.
(2) it is cheap and easy to get：GP-R can be obtained through chemical synthesis, and synthesis technique is simple and easy to apply.
(3) easily high flux detection：It can be determined on the common all kinds of fluorescence microplate readers in laboratory and clinical big biochemical instruments, can profit Batch detection is carried out with 96 or 386 microwell plates.
(4) it is anti-interference：, can due to environmental and biological samples reasons for its use noise can be avoided in near-infrared (NIR) detection To be effectively prevented from the self-absorption of biological sample and the interference of autofluorescence.
Brief description of the drawings
The general structure of Fig. 1 4- (Gly-Pro)-substituted ethylene base amino benzenes compounds；
The synthetic route of Fig. 2 .N- (Gly-Pro)-(E- chromenes-ethene) aniline (GP-ACM)；
Fig. 3 .N- (Gly-Pro)-(E- chromenes-ethene) aniline (GP-ACM)1H-NMR spectrum；
The synthetic route of Fig. 4 .N- (Gly-Pro)-(E- quinoline-ethene) aniline (GP-ABP)；
The selectivity of Fig. 5 .N- (Gly-Pro)-(E- chromenes-ethene) aniline (GP-ACM)；
Fig. 6 .DPP-IV quantitation curves；
The enzyme kinetic analysis of Fig. 7 .DPP-IV catalysis GP-ACM hydrolysis；
Fig. 8 Chemical Inhibitions are tested；
Fig. 9 human normal plasma DPP-IV determinations of activity；
Embodiment
The following examples will be further described to the present invention, but not thereby limiting the invention.
Equipment and its model of the present invention：Fluorescent emission/excitation spectrum is by SynergyH1 global function micropores Board detector detection is completed；1H-NMR spectrum is to be detected to complete by nuclear magnetic resonance chemical analyser (Avance II 700MHz).
The general structure of 4- (Gly-Pro)-aromatic amine compounds is as shown in Figure 1.
Embodiment 1
The synthesis of N- (Gly-Pro)-(E- chromenes-ethene) aniline (GP-ACM)
Synthetic route is as shown in Figure 2.
(1) synthesis of compound 2
Room temperature, ethyl acetate (85mL) and tetrahydrofuran (15mL) are added by o-hydroxyacetophenone 1 (1.36g, 10mmol) Mixed solution in.It is stirred vigorously down and sodium block is added portionwise, is reacted at room temperature after adding.After completion of the reaction, frozen water (50mL) is added, Watery hydrochloric acid adjusts pH=5-6, be extracted with ethyl acetate (50mL × 3), washing (30mL × 1), saturated nacl aqueous solution wash (30mL × 1), anhydrous sodium sulfate drying.After drying fully, solvent is evaporated off, crude compound 2 is obtained, next step reaction is directly used in without purifying.
(2) synthesis of compound 3
Room temperature, the crude product of compound 2 obtained by previous step is added in glacial acetic acid (10mL) solution, slowly dripped after stirring Enriching sulfuric acid (1mL), adds room temperature reaction.Acetic acid is evaporated off after completion of the reaction, adds water (50mL), is extracted with ethyl acetate (50mL × 2), merge organic phase washing (30mL × 1), saturated nacl aqueous solution and wash (30mL × 1), anhydrous sodium sulfate drying.Steam Except solvent, crude product column chromatography (petrol ether/ethyl acetate=10/1) compound 3, faint yellow solid, yield 70-80%, ESI-MS m/z 161.0[M+H]+。
(3) synthesis of compound 4
Room temperature, compound 3 (480.5mg, 3mmol), malononitrile (792.7mg, 12mmol) addition acetic anhydride (3mL) is molten In liquid, after stirring and dissolving, back flow reaction.After completion of the reaction, frozen water (10mL) is added, saturated sodium bicarbonate solution adjusts pH=7-8, Ethyl acetate extracts (50mL × 2), merge organic phase washing (20mL × 2), saturated nacl aqueous solution wash (20mL × 1), it is anhydrous Sodium sulphate is dried.Be evaporated off solvent, crude product column chromatography (petrol ether/ethyl acetate=10/1) compound 4, faint yellow solid, Yield 50-60%, ESI-MS m/z 207.1 [M-H]-。
(4) synthesis of compound 7
Room temperature, successively by compound 4 (218.5mg, 1.05mmol), para aminotenzaldehyde (121.1mg, 1mmol), piperidines (0.5mL), acetic acid (0.5mL) are added in toluene (40mL) solution, after stirring and dissolving, back flow reaction.After completion of the reaction, it is cooled to Room temperature, is evaporated off ethanol and adds water (40mL), ethyl acetate extraction (50mL × 2) merges organic phase washing (20mL × 1), saturation Sodium chloride solution washes (20mL × 1), anhydrous sodium sulfate drying.Solvent, crude product column chromatography (petroleum ether/dichloromethane/second is evaporated off Acetoacetic ester=5/5/1) obtain compound 7, red brown solid, yield 40-50%, ESI-MS m/z 312.1.1 [M+H]+。
(5) synthesis of compound 8
Room temperature, by compound 7 (155.7mg, 0.5mmol), Boc-Gly-Pro-OH (272.3mg, 1.0mmol), N- hydroxyls Base BTA (67.6mg, 0.5mmol), 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (95.9mg, 0.5mmol), 2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acids ester (380.2mg, 1.0mmol) it is added sequentially to react in DMF (5mL) solution, thin plate chromatography (TLC) monitoring reaction.Reaction After 36h, reaction system is cooled to 0-5 DEG C, and add water (25mL), and ethyl acetate extracts three times (30mL × 3), merges organic phase washing Three times (15mL × 3), 5% sodium bicarbonate solution is washed (20mL × 1), and washing (15mL × 1), saturated nacl aqueous solution wash (20mL × 1), solvent is evaporated off in anhydrous sodium sulfate drying, and crude product column chromatography (methylene chloride/methanol=50/1) obtains compound 8, yellow Grease, yield 65-75%, ESI-MS m/z 564.3 [M-H]-。
(6) compound GP-ACM synthesis
Room temperature, is added to dichloromethane (12mL) by compound 8 (200mg, 0.35mmol) and is mixed with trifluoroacetic acid (3mL) Reacted in solution, thin plate chromatography (TLC) monitoring reaction.React after 6h, reaction dissolvent be evaporated off, crude product is added to the water (15mL), Saturated sodium bicarbonate solution adjusts pH=7-8, and dichloromethane extracts three times (25mL × 3), merges organic phase washing (15mL × 1), Saturated nacl aqueous solution is washed (20mL × 1), anhydrous sodium sulfate drying, is evaporated off solvent, crude product column chromatography (methylene chloride/methanol/ Water=20/5/0.5) obtain compound GP-ACM, its nuclear magnetic spectrogram as shown in figure 3, the product is red brown solid, yield 50- 60%.
The NMR spectrum of the product of preparation is specific as follows：
1H NMR (700MHz, DMSO) δ 8.73 (d, J=8.4Hz, 1H), 8.22 (s, 1H), 7.93 (t, J=7.8Hz, 1H), 7.79 (d, J=8.4Hz, 1H), 7.77-7.68 (m, 5H), 7.62 (t, J=7.8Hz, 1H), 7.41 (d, J=16.0Hz, 1H), 7.00 (s, 1H), 4.51 (dd, J=8.4,3.3Hz, 1H), 3.78 (s, 2H), 3.64-3.59 (m, 1H), 3.53 (m, 1H),2.24-2.16(m,1H),2.06-1.85(m,3H).ESI-MS m/z 466.2[M+H]+。
Embodiment 2
The synthesis of N- (Gly-Pro)-(E- quinoline-ethene) aniline (GP-ABP)
Synthetic route is as shown in Figure 4.
(1) synthesis of compound 5
Room temperature, compound 3 (961mg, 6mmol) crude product obtained by previous step is added in glacial acetic acid (10mL) solution, stirred N-butylamine (2mL), back flow reaction is slowly added dropwise after mixing uniformly.Room temperature is down to after completion of the reaction, adds frozen water (100mL), filtering Obtain crude product.Crude product column chromatography (petrol ether/ethyl acetate=8/1) compound 5, red brown solid, yield 30-40%, ESI-MS m/z 216.1[M+H]+。
(2) synthesis of compound 6
Room temperature, acetic anhydride (3mL) solution is added by compound 5 (430mg, 1mmol), malononitrile (792.7mg, 12mmol) In, after stirring and dissolving, back flow reaction.After completion of the reaction, frozen water (10mL) is added, saturated sodium bicarbonate solution adjusts pH=7-8, second Acetoacetic ester extracts (50mL × 2), merges organic phase washing (20mL × 2), saturated nacl aqueous solution and washes (20mL × 1), anhydrous sulphur Sour sodium is dried.Solvent is evaporated off, crude product column chromatography (petrol ether/ethyl acetate=5/1) obtains compound 6, faint yellow solid, yield 50-60%, ESI-MS m/z 262.1 [M-H]-。
(3) compound 7' synthesis
Room temperature, successively by compound 6 (275.4mg, 1.05mmol), para aminotenzaldehyde (121.1mg, 1mmol), piperidines (0.5mL), acetic acid (0.5mL) are added in toluene (40mL) solution, after stirring and dissolving, back flow reaction.After completion of the reaction, it is cooled to Room temperature, is evaporated off ethanol and adds water (40mL), ethyl acetate extraction (50mL × 2) merges organic phase washing (20mL × 1), saturation Sodium chloride solution washes (20mL × 1), anhydrous sodium sulfate drying.Solvent, crude product column chromatography (petroleum ether/dichloromethane/second is evaporated off Acetoacetic ester=5/5/1) obtain compound 7', red brown solid, yield 40-50%, ESI-MS m/z 367.2 [M+H]+。
(4) compound 8' synthesis
Room temperature, by compound 7'(183.6mg, 0.5mmol), Boc-Gly-Pro-OH (272.3mg, 1.0mmol), N- hydroxyls Base BTA (67.6mg, 0.5mmol), 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (95.9mg, 0.5mmol), 2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acids ester (380.2mg, 1.0mmol) it is added sequentially to react in DMF (5mL) solution, thin plate chromatography (TLC) monitoring reaction.Reaction After 36h, reaction system is cooled to 0-5 DEG C, and add water (25mL), and ethyl acetate extracts three times (30mL × 3), merges organic phase washing Three times (15mL × 3), 5% sodium bicarbonate solution is washed (20mL × 1), and washing (15mL × 1), saturated nacl aqueous solution wash (20mL × 1), solvent is evaporated off in anhydrous sodium sulfate drying, and crude product column chromatography (methylene chloride/methanol=50/1) obtains compound 8', brown Grease, yield 65-75%, ESI-MS m/z 619.3 [M-H]-。
(4) compound GP-ABP synthesis
Room temperature, by compound 8'(150mg, 0.24mmol) it is added to dichloromethane (10mL) and trifluoroacetic acid (2mL) and mixes Close in solution and react, thin plate chromatography (TLC) monitoring reaction.React after 6h, reaction dissolvent is evaporated off, crude product is added to the water (15mL), saturated sodium bicarbonate solution adjusts pH=7-8, and dichloromethane extracts three times (25mL × 3), merges organic phase washing (15mL × 1), saturated nacl aqueous solution is washed (20mL × 1), anhydrous sodium sulfate drying, and solvent, crude product column chromatography (dichloro is evaporated off Methane/methanol/water=20/5/0.5) obtain compound GP-ABP, the product is red brown solid, yield 40-50%, ESI-MS m/z521.2[M-H]-。
Embodiment 3.
External test people recombinates the selectivity of the mono- enzymes of DPP-IV
(1) 198 μ L DPP-IV metabolic response systems are prepared in advance, include pH 7.4 PBS (50mM), restructuring The mono- enzymes of people DPP-IV (1 μ g/mL), the mono- enzymes of recombined human DPP-IX (1 μ g/mL), the mono- enzymes of recombined human DPP-VIII (1 μ g/mL), restructuring Human fibroblasts activated protein list enzyme (FAP, 1 μ g/mL), carbonic anhydrase (CA, 10 μ g/mL), trypsase (trypsin, 10 μ g/mL), pepsin (pepsin, 10 μ g/mL), butyrylcholine esterase (BChE, 10 μ g/mL), acetylcholinesterase (AChE, 10 μ g/mL), carboxy-lesterase (hCE1, hCE2,10 μ g/mL), bovine serum albumin(BSA) (BSA, 10 μ g/mL), human serum albumins (HAS, 10 μ g/mL), Proteinase K (Proteinase K, 5 μ g/mL), lysozyme (Lysozyme, 10 μ g/mL), lipase (Lipase, 5 μ g/mL), acid seromucoid (α-acidoglycoprotein, 5 μ g/mL) shakes under the conditions of 37 DEG C incubates 3 in advance Minute；
(2) the GP-ACM initial actions that 2 μ L concentration are 10mM are added into reaction system；
After (3) 30 minutes, 200 μ L ice acetonitriles are added, acutely after concussion, terminating reaction；
(4) with high speed freezing centrifuge at 4 DEG C, under conditions of 20,000 × g, high speed centrifugation takes supernatant, entered after 20 minutes Row fluoroscopic examination (Ex=400nm, Em=535nm)；The selectivity of recombined human DPP-IV enzymes is a maximum of about of 50 times of other single enzymes Left and right (Fig. 5).
Embodiment 4.
External test DPP-IV Monitoring lower-cut is determined
Experiment is measured on ELIASA using 96 orifice plates, 100 μM of GP-ACM, mono- μ g/mL~1.2 of enzyme 0.5 of DPP-IV μ g/mL, pH 7.4 PBS 50mM, cumulative volume is to be analyzed after being incubated 1h at 200 μ L, 37 DEG C by ELIASA, every group Average value is compared with being not added with DPP-IV control group, and the Monitoring lower-cut for as a result showing DPP-IV is 160ng/mL (Fig. 6).
Embodiment 5.
DPP-IV enzyme kinetic analysis is determined
Experiment is measured on ELIASA using 96 orifice plates, 1-500 μM of substrate, the mono- enzyme 0.25mg/mL of DPP-IV or intestines Microsome 2.5mg/mL, pH 7.4 PBS 100mM, cumulative volume 200 μ L, are incubated by ELIASA analysis 1h at 37 DEG C, Survey once within every 1 minute.Testing conditions：Excitation wavelength 480nm, maximum emission wavelength is 670nm.Obtained fluorescence intensity is substituted into and marked The Vmax and Km (Fig. 7) of the mono- enzymes of DPP-IV and people's intestines microsome (HIM) to GP-ACM are respectively obtained after directrix curve.
Embodiment 6.
Chemical Inhibition is tested
Inhibitor concentration gradient：Working solution concentration (μm) 250,100,50,25,10,5,2.5,1,0.5,0.25,0.1,0； React final concentration (nm) 2500,1000,500,250,100,50,25,10,5,2.5,1,0.Determine：
(1)HIM:2 groups of parallel laboratory test.5 μ l HIM and 93 μ l buffer are added, the μ l of vildagliptin 2 are added, 4min is incubated； Add 100 μ l (100 μM) substrate initial action, 30min, 200 μ l acetonitrile terminating reactions, detection.
(2)DPP-Ⅳ：According to above-mentioned experimental method, in DPP-IV system, 2 groups of parallel laboratory test is repeated.
As shown in figure 8, people's intestines microsome hydrolysis GP-ACM activity can be by Vildagliptin (2.5 μM) institute strong inhibition (Fig. 8).
Embodiment 7.
Human normal plasma DPP-IV determinations of activity
196ul PBS solutions are added in 1.5ml EP pipes, 2ul blood plasma (or serum) sample is then added, pot is being incubated In incubate 5min in advance.It is then respectively adding and has prepared substrate GP-ACM 2ul, each example reaction 30min.Added after 30min 200ul acetonitrile terminating reactions.200ul systems are drawn in 96 hole elisa Plates, fluorescent value is measured.The ratio measured is brought into mark song Calculate enzymatic activity (Fig. 9).

Claims (9)

1. a kind of near infrared fluorescent probe substrate of DPP IV, it is characterised in that：The probe substrate can be special by DPP-IV Opposite sex catalysis occurs peptide bond hydrolysis and reacts and generate corresponding arylamine, and the structure of the probe substrate is as follows：
Wherein, X is selected from O, N；R is selected from C1-C10Alkyl ,-(C1-C8Alkylidene)-carboxyl ,-(C1-C8Alkylidene) -ester base ,-(C1- C8Alkylidene)-amino ,-(C1-C8Alkylidene)-cyano group ,-(C1-C8Alkylidene)-nitro ,-(C1-C3Alkylidene)-O- (C1-C3Alkane Base), carbocylic radical ,-(C1-C3Alkylidene)-carbocylic radical, aryl ,-(C1-C3Alkylidene)-aryl, heteroaryl ,-(C1-C3Alkylene Base)-heteroaryl, heterocyclic radical or-(C1-C3Alkylidene)-heterocyclic radical.
2. a kind of preparation method of DPP IV near infrared fluorescent probe substrate as claimed in claim 1, it is characterised in that：
1) using 2- acetyl phenols as initiation material, 2- acetoaceto phenol, acid catalysis ring are combined to by base catalysis acetyl It is combined to flavones；
2) acid catalysis flavones and malononitrile reaction synthesis dicyan chromene, or flavones and substitution amine reaction synthetic acidic/neutrality/ Alkalescence, polar/non-polar, the N- substd quinolines ketone of hydrophilic/hydrophobic and serial substituted radical not of uniform size, with malononitrile Reaction synthesis dicyan N- substd quinolines；
3) dicyan chromene or dicyan N- substd quinolines and para aminotenzaldehyde reaction synthesis of trans substitution 4- vinyl aniline；
4) trans substitution 4- vinyl aniline, in N- hydroxy benzo triazoles, 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne two In the presence of inferior amine salt hydrochlorate, 2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester, with tertiary butyloxycarbonyl Base glycinylproline is condensed, 20-30 DEG C of stirring reaction 24-48 hours, and the product of generation is purified through column chromatography, then through trifluoro Acetic acid acid catalysis, 4-10 hours removing tertbutyloxycarbonyls of 20-30 DEG C of stirring reaction generate the near-infrared fluorescent of DPP IV Probe substrate GP-R, yield is 30-80% after purification；
The trans substitution 4- vinyl aniline, N- hydroxy benzo triazoles, 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne two Inferior amine salt hydrochlorate, 2- (7- azos BTA)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester, with the sweet ammonia of tertbutyloxycarbonyl The mol ratio of acidic group proline is 1.0：0.5-2.0：0.5-2.0：1.0-3.0：1.0-3.0.
3. a kind of application of DPP IV near infrared fluorescent probe substrate as claimed in claim 1, it is characterised in that：Using The specific substrate of the DPP IV, carries out enzymatic reaction after being mixed with the biological sample containing DPP IV, passes through Quantitatively detection the unit interval in substrate elimination factor or its production rate of dipeptides based products is removed to quantitative determine different biosystems Middle DPP-IV activity, specific assay method and condition are as follows：
A. probe substrate GP-R is added in phosphate buffer；Concentration of substrate is 1/10~10Km；
B. reaction temperature is between 20~60 DEG C, incubation system pH is between 5.5~10.5；
C. the reaction time is 5~120 minutes, it is ensured that the corresponding N- of above substrate goes two peptidyl products to reach quantitative limit and substrate Terminating reaction when conversion ratio is no more than 20%；
D. substrate decrement or N- go two peptidyl product formations to refer to as the evaluation of DPP-IV activity in the analytical unit time Mark.
4. the application of the specific near infrared fluorescent probe substrate according to the measure DPP IV described in claim 3, its It is characterised by：In specific assay method and condition：The preferred K of concentration of substratem。
5. the application of the specificity fluorescent probe substrate according to the measure DPP IV described in claim 3, its feature exists In：In specific assay method and condition：Preferably 37 DEG C of reaction temperature, incubation system pH preferably 7.4.
6. the application of the specific near infrared fluorescent probe substrate according to the measure DPP IV described in claim 3, its It is characterised by that described biosystem is thin for the restructuring list enzyme containing DPP-IV, people's tissue preparation liquid, mammalian species tissue Any one in born of the same parents and its prepared product.
7. according to the application for the specificity fluorescent probe substrate that DPP IV is determined described in claim 3, it is characterised in that： The probe substrate and its go dipeptides based products that there is different UV absorption and fluorescence emission spectrum, available for the inspection of ultraviolet and fluorescence Survey.
8. a kind of application of the specific near infrared fluorescent probe substrate as claimed in claim 1 for determining DPP IV, It is characterized in that：DPP-IV enzyme activity in tissue fluid that the probe substrate can also originate as different genera, cell, tissue samples Quantitative detection.
9. a kind of application of the specific near infrared fluorescent probe as claimed in claim 1 for determining DPP IV, its feature It is：The probe substrate can be additionally used in the quick screening of DPP-IV inhibitor or derivant, and its suppress or inducibility is determined Amount is assessed.
CN201610056580.5A 2016-01-28 2016-01-28 DPP IV enzyme near infrared fluorescent probe substrate and preparation method and application Pending CN107011324A (en)

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Cited By (8)

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WO2019117812A1 (en) * 2017-12-12 2019-06-20 Nanyang Technological University Near infra-red molecular probes for use in diagnosis of fibrotic conditions and screening of anti-fibrotic drugs
CN108558859B (en) * 2018-06-06 2021-08-24 湖北大学 Preparation and application of visible long-wave Hg2+ fluorescent probe based on benzopyran
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CN113234014A (en) * 2021-04-26 2021-08-10 济南大学 Aggregation-induced emission fluorescent probe for detecting aminopeptidase N and preparation thereof
CN113667328A (en) * 2021-07-09 2021-11-19 中国化学工业桂林工程有限公司 Regenerated carbon black and preparation method and application thereof
CN114656520A (en) * 2021-12-24 2022-06-24 上海中医药大学 Prolinamide endopeptidase near-infrared fluorescent probe substrate, and preparation method and application thereof
CN116478153A (en) * 2023-04-25 2023-07-25 吉林大学 Near infrared fluorescent probe for detecting dipeptidyl peptidase IV and preparation method and application thereof

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