CN104672300A - Double enzyme-sensitive fluorescent probe and preparation method and application thereof - Google Patents
Double enzyme-sensitive fluorescent probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a double enzyme-sensitive fluorescent probe, namely, a fluorescent probe capable of detecting matrix metalloproteinase and apoptotic enzyme at the same time. The double enzyme-sensitive fluorescent probe comprises an apoptotic enzyme specific recognition polypeptide sequence, a matrix metalloproteinase specific recognition polypeptide sequence and two molecular fluorescent pairs with resonance energy transfer. The fluorescent probe can selectively act with one enzyme in the coexistence of the matrix metalloproteinase and the apoptotic enzyme, and namely specifically recognize corresponding polypeptide sequences and cut the corresponding polypeptide sequences to restore quenched fluorescence so as to detect the two enzymes; the fluorescent probe can perform rapid non-invasive detection on a complex microenvironment system with the coexistence of the matrix metalloproteinase and the apoptotic enzyme, is good in selectivity and high in sensitivity, has a great significance for early detection of a tumor and evaluation of a therapeutic effect and has a wide application prospect.
Description
Technical field
The present invention relates to chemical analysis, bioanalysis, field of clinical medical detection, belong to organic molecule fluorescent probe technique field.
Background technology
Matrix metalloproteinase is the proteolytic enzyme of a class extracellular matrix components, in the pathological lesion of disease, play the part of vital role.The expression of matrix metalloproteinase and the active strict regulation and control being subject to such as cytokine, hormone etc., and be subject to the suppression of its natural inhibitor, the overexpression of matrix metalloproteinase can cause the such as disease such as tumour invasion and transfer, therefore, for detection and the active diagnosis and detection differentiating to be conducive to diseases such as infantile tumours thereof of matrix metalloproteinase.
Apoptosis enzyme is a kind of cysteine hydrolases, plays an important role in startup and execution antiapoptotic signals process.Apoptosis enzyme exists with inactive form in cell, after running into apoptotic stimulus signal, realizes it to the structural transformation with physiologically active by intracellular proteolyzing, thus regulating cell programmed cell death.Meanwhile, most anti-tumor medicine all has the relevant mechanism of apoptosis, and therefore, the detection of apoptosis enzyme is conducive to the evaluation of infantile tumour result for the treatment of, propelling and optimization cancer immunotherapies.
Resonance energy transfer be excited state to body fluorophor by dipole effect, give contiguous acceptor fluorophore with non-radiative processes by transmission ofenergy, be converted into other energy, when not releasing energy in the mode of fluorescent emission, fluorescent quenching.But the distance between fluorophor exceedes the scope of resonance energy transfer
time, resonance energy transfer phenomenon disappears, thus the fluorescence launched when recovery excited state gets back to ground state to body.Just due to the characteristic of this resonance energy transfer distance sensitive, be widely used in the monitoring of biomacromolecule, dynamic process etc.
Polypeptide is the material with particular organisms activity that a class biocompatibility is good, the structure consisted of according to certain sequence certain amino acid can by the specific identification of involved enzyme and cut-out, is widely used the transport of the detection of biomolecules, the diagnosis of disease and medicine.Adopt Solid-phase synthesis peptides technology, the effective synthesis to target molecule and separation can be realized, and diversified amido protecting can realize modifying the multifunction of polypeptide, thus meets the demands such as different determination and analysis.
At present, the fluorescent probe for apoptosis enzyme or matrix metalloproteinase is extensively designed and is utilized.But conventional fluorescent probe all only designs for a certain enzyme, due to the interference that living things system complicacy detects specific enzymes, the recognition capability of enzyme to specific polypeptide sequence is limited, and present stage is to the demand of the Multifunction fluorescent probe that diagnosing tumor and treatment effectiveness evaluation are integrated, for two enzyme sensitivity, the fluorescent probe of design has great using value, and, dual energy resonance transfer fluorescent quenching process can realize the effective cancellation to fluorescence molecule fluorescence, thus improve the signal to noise ratio of probe, greatly can increase specific detection ability and the range of application of fluorescent probe.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of detect matrix metalloproteinase and apoptosis enzyme fluorescent probe and synthetic method and application.
The fluorescent probe of detection matrix metalloproteinase provided by the invention and apoptosis enzyme, i.e. two enzyme responsive type fluorescent probe, comprises the molecular fluorescence pair of apoptosis enzyme spcificity Recognition polypeptide sequence, matrix metalloproteinase specific recognition peptide sequence and two pairs of resonance energy transfers.
What two pairs of described resonance energy transfer molecular fluorescences were right is same as body to body.
Described molecular fluorescence is to being be same as body and the fluorescence quencher molecule fluorescence pair that is acceptor with different dimethyl amino-azo-benzene with fluorescein.
Described apoptosis enzyme spcificity Recognition polypeptide sequence is the peptide sequence comprising amino acid-amino acid-amino acid-aspartic acid; Described matrix metalloproteinase specific recognition peptide sequence is the one in proline-leucine-glycine-α-amino-isovaleric acid-arginine, proline-leucine-Gly-Leu-Ala-Gly, proline-leucine-glycine-halfcystine-Ala-Gly, Gly-Pro-proline(Pro)-glycine-α-amino-isovaleric acid-α-amino-isovaleric acid-glycine-glutaminic acid-LYS-GLY-Glu-Gln.
The described peptide sequence comprising amino acid-amino acid-amino acid-aspartic acid is Asp-Glu-valine aspartic acid or tryptophane-L-glutamic acid-histidine-asparate.
Further, described two enzyme responsive type fluorescent probes are preferably, chemistry dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine-Methionin (fluorescein)-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene by name, chemical structure is as shown in formula I:
The preparation method of two enzyme responsive type fluorescent probes of structure shown in formula I, comprises the following steps:
(A) dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine, fluorescence is synthesized
Element-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene;
(B) dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine and fluorescein-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene are carried out fragment condensation, namely obtain the fluorescent probe of the detection matrix metalloproteinase shown in formula I and apoptosis enzyme.
In described step (A), the synthesis of dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine and fluorescein-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene adopts Solid-phase synthesis peptides method;
The synthesis of described step (B) adopts the method for solid phase fragment condensation.
The resin adopted in Solid-phase synthesis peptides is the one in the chloro-trityl chloride resin of 2-, Rink Amide resin, mbha resin.
In described step (A), the synthesis of (i) dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine specifically comprises the following steps:
(1) by chloro-for 2-trityl chloride resin swelling 0.5 ~ 2h in heavy steamed DMF, solvent is extracted;
(2) glycine protected by FMOC, DIPEA are together dissolved in DMF, and room temperature reaction 1 ~ 2h, extracts solvent, then with DMF washing 2 ~ 4 times;
(3) by unreacted avtive spot end-blocking: by methyl alcohol, N, dinethylformamide, N, N-diisopropylethylamine is mixed with mixing solutions by the volume ratio of 0.5 ~ 2:9 ~ 11:0.5 ~ 2, room temperature reaction 0.5 ~ 1h, extract solvent, then with DMF washing 2 ~ 4 times;
(4) FMOC protecting group cut: add piperidines/DMF mixing solutions reaction 1 ~ 15min that volume fraction is 10 ~ 50%, repetitive operation twice, reaction terminates rear DMF washing 2 ~ 4 times;
(5) by amino acid, the benzotriazole-N of FMOC protection, N, N ' N '-tetramethyl-urea hexafluorophosphate, 1-hydroxy benzo triazole, N, N-diisopropylethylamine is together dissolved in N, in dinethylformamide, room temperature reaction 1 ~ 2h, extracts solvent, then with DMF washing 2 ~ 4 times;
(6) test with the triketohydrindene hydrate/methanol solution of 10mg/mL, if reaction not exclusively, then repeating step (5); If react completely, then carry out the reaction of step (7); Other amino acid carries out according to step (4) (5) (6);
(7) by dimethyl amino-azo-benzene formic acid, benzotriazole-N, N, N ' N '-tetramethyl-urea hexafluorophosphate, 1-hydroxy benzo triazole, N, N-diisopropylethylamine is dissolved in N together, in dinethylformamide, room temperature reaction 1 ~ 2h, extracts solvent, then with DMF washing 2 ~ 4 times;
(8) methanol wash 2 ~ 4 times, washed with dichloromethane 2 ~ 4 times;
(9) cut: 0.5 ~ 4% trifluoroacetic acid/96 ~ 99.5% methylene dichloride, 1 ~ 10 minute once, cuts 4 ~ 10 times;
(10) collection cuts liquid, revolves steaming, vacuum-drying;
(ii) synthesis of fluorescein-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene specifically comprises the following steps:
(1) by Rink Amide resin swelling 0.5 ~ 1h in heavy steamed DMF that FMOC protects, solvent is extracted;
(2) FMOC protecting group cut: add piperidines/DMF mixing solutions reaction 1 ~ 15min that volume fraction is 10 ~ 50%, repetitive operation twice, reaction terminates rear DMF washing 2 ~ 4 times;
(3) by FMOC protect Methionin, benzotriazole-N, N, N ' N '-tetramethyl-urea hexafluorophosphate, 1-hydroxy benzo triazole, N, N-diisopropylethylamine is dissolved in DMF, room temperature reaction 1 ~ 2h, extract solvent, DMF washing 2 ~ 4 times;
(4) end-blocking: unreacted amino with being 2 ~ 6%2,6-lutidine/2 ~ 5% diacetyl oxide/DMF mixing solutions reaction 10 ~ 60min containing volume fraction, reaction terminates rear DMF washing 2 ~ 4 times;
(5) FMOC remove according to reactions steps (2), to remove and after washing and terminating, by FMOC protect Methionin, benzotriazole-N, N, N ' N '-tetramethyl-urea hexafluorophosphate, 1-hydroxy benzo triazole, N, N-diisopropylethylamine is dissolved in N, in dinethylformamide, room temperature reaction 1 ~ 2h, extracts solvent, DMF washing 2 ~ 4 times;
(6) that verifies previous step reaction process carries out degree, tests with the triketohydrindene hydrate/methanol solution 0.3 ~ 1mL of 10mg/mL, if reaction not exclusively, will repeat previous step, and react completely, and carries out next step reaction;
(7) all the other amino acid whose reactions are carried out according to step (5), step (6);
(8) by 5 (6)-Fluoresceincarboxylic acids, 2-(7-azo benzotriazole)-N, N, N ' N '-tetramethyl-urea phosphofluoric acid ester, 1-hydroxy benzo triazole, N-methylmorpholine are dissolved in N together, in dinethylformamide, room temperature reaction 2 ~ 12h, extract solvent, DMF washing 2 ~ 4 times;
(9) by dimethyl amino-azo-benzene, 2-(7-azo benzotriazole)-N, N, N ' N '-tetramethyl-urea phosphofluoric acid ester, 1-hydroxy benzo triazole, N-methylmorpholine are dissolved in N together, in dinethylformamide, room temperature reaction 2 ~ 12h, extract solvent, DMF washing 2 ~ 4 times;
(10) with DMF washing 2 ~ 4 times, methanol wash 2 ~ 4 times, washed with dichloromethane 2 ~ 4 times, dry.
Described step (B) specifically comprises the following steps:
(1) the Rink Amide resin comprising fluorescein-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene sequence is placed in the swelling 0.5 ~ 2h of DMF;
(2) methyltrityl side base is removed with the trifluoroacetic acid/dichloromethane mixed solution that volume ratio is 0.5 ~ 4:96 ~ 99.5, after removing end, washed with dichloromethane 2 ~ 4 times, DMF washing 2 ~ 4 times;
(3) by dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine, 2-(7-azo benzotriazole)-N, N, N ' N '-tetramethyl-urea phosphofluoric acid ester, 1-hydroxy benzo triazole, N-methylmorpholine are together dissolved in N, in dinethylformamide, room temperature reaction 2 ~ 10h, then solvent is extracted, DMF washing 2 ~ 4 times;
(4) cut: cut 1 ~ 3h with trifluoroacetic acid/water mixed liquid that volume ratio is 83 ~ 95:5 ~ 17, decompress filter also collects filtrate, by filtrate decompression concentrated by rotary evaporation; Then concentrated solution is dropwise instilled in freezing anhydrous diethyl ether, centrifugal, supernatant liquor is drained, dried in vacuo overnight.
The application of described two enzyme responsive type fluorescent probes in the screening of chemical system, living things system matrix metalloproteinase and apoptosis Enzyme assay and apoptosis enzyme inhibitors and matrix metallo-proteinase inhibitor.
Fluorescent probe of the present invention has maximum fluorescence and recovers under apoptosis enzyme and matrix metalloproteinase acting in conjunction, and the recovery of fluorescein fluorescence realizes the detection to apoptosis enzyme and matrix metalloproteinase, sees Fig. 2.
Fluorescent probe of the present invention formerly acts on identical effect with matrix metalloproteinase and apoptosis enzyme with having with the effect of apoptosis enzyme after matrix metalloproteinase effect simultaneously again, sees Fig. 3.
Fluorescent probe of the present invention has comparatively hypofluorescence and recovers under the effect of apoptosis enzyme inhibitors, sees Fig. 4.
Fluorescent probe of the present invention has comparatively hypofluorescence and recovers under matrix metallo-proteinase inhibitor effect, sees Fig. 5.
The present invention has the following advantages and beneficial effect:
1) whole building-up process adopts solid phase synthesis technique and fragment condensation mode, and productive rate height is purified simple.
2) this fluorescent probe has the ability of apoptosis enzyme and the identification of matrix metalloproteinase dual specificity.
3) only having in apoptosis enzyme and the coefficient situation of matrix metalloproteinase, the fluorescence of this probe has very big recovery.
4) this probe has the practical value for the complicated microenvironment of tumour and cancer therapy effect assessment.
Accompanying drawing explanation
Fig. 1: the Matrix-assisted laser desorption ionization figure (MALDI-TOF-MS) of fluorescent probe.
Fig. 2: the fluorescence of fluorescent probe under apoptosis enzyme and matrix metalloproteinase acting in conjunction recovers figure.
Fig. 3: fluorescent probe and matrix metalloproteinase and apoptosis enzyme acting in conjunction fluorescence recovers in time and fluorescent probe and matrix metalloproteinase and apoptosis enzyme successively act on fluorescence recovery figure in time.
Fig. 4: fluorescent probe fluorescence under the effect of apoptosis enzyme inhibitors recovers figure.
Fig. 5: fluorescent probe fluorescence under matrix metallo-proteinase inhibitor effect recovers figure.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
The synthesis of dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine:
(1) take the chloro-trityl chloride resin of 0.5g 2-(1.05mmol/g), swelling 0.5 ~ 2h in heavy steamed DMF (DMF), extracts DMF;
(2) first the amino acid-glycine of FMOC protection, DIPEA (DIEA) are dissolved in DMF, add room temperature reaction 1 ~ 2h in reactor, extract solvent, then with DMF washing 2 ~ 4 times;
(3) by unreacted avtive spot end-blocking: methyl alcohol, DMF, DIEA are mixed with mixing solutions by the volume ratio of 0.5 ~ 2:9 ~ 11:0.5 ~ 2 and add in reactor, and room temperature reaction 0.5 ~ 1h, extracts solvent, then with DMF washing 2 ~ 4 times.
(4) FMOC protecting group cut: add 10 ~ 50% piperidines/DMF solution, reaction 1 ~ 15min, so react twice, reaction terminate rear DMF washing resin 2 ~ 4 times;
(5) by amino acid, the benzotriazole-N of FMOC protection; N; N ' N '-tetramethyl-urea hexafluorophosphate (HBTU); 1-hydroxy benzo triazole (HoBt); DIEA is dissolved in DMF; add room temperature reaction 1 ~ 2h in reactor, extract solvent, then with DMF washing 2 ~ 4 times.
(6) that verifies previous step reaction process carries out degree, tests with the triketohydrindene hydrate/methanol solution 0.3 ~ 1mL of 10mg/mL, if reaction not exclusively, then and repeating step (5); If react completely, then carry out the reaction of step (7).
Other amino acid carries out according to step (4) (5) (6).
(7) dimethyl amino-azo-benzene formic acid, HBTU, HoBt, DIEA are dissolved in DMF together, add room temperature reaction 1 ~ 2h in reactor, extract solvent, then with DMF washing 2 ~ 4 times.
(8) methanol wash 2 ~ 4 times, washed with dichloromethane 2 ~ 4 times.
(9) cut: 0.5 ~ 4% trifluoroacetic acid/96 ~ 99.5% methylene dichloride, 1 ~ 10 minute once, cuts 4 ~ 10 times.
(10) collection cuts liquid, and revolve steaming, vacuum-drying ,-20 DEG C keep in Dark Place.
Embodiment 2
The synthesis of fluorescein-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene:
(1) take the Rink Amide resin (0.7mmol/g) that 0.5g FMOC protects, swelling 0.5 ~ 1h in heavy steamed DMF, extracts DMF;
(2) FMOC protecting group cut, with 10 ~ 50% piperidines/DMF solution, reaction 1 ~ 15min, so react twice, reaction terminate rear DMF washing resin 2 ~ 4 times;
(3) Methionin of FMOC protection, HBTU, HoBt, DIEA are dissolved in DMF, add room temperature reaction 1 ~ 2h in reactor, extract solvent, DMF washing 2 ~ 4 times;
(4) end-blocking, unreacted amino with 2 ~ 6%2,6-lutidine/2 ~ 5% diacetyl oxide/DMF solution reaction 10 ~ 60min, reaction terminates rear DMF and washs 2 ~ 4 times;
(5) the removing according to reactions steps (2) of FMOC, removes and after washing and terminating, by the amino acid that FMOC protects, HBTU, HoBt, DIEA are dissolved in DMF, add room temperature reaction 1 ~ 2h in reactor, extract solvent, DMF washing 2 ~ 4 times;
(6) that verifies previous step reaction process carries out degree, tests with the triketohydrindene hydrate/methanol solution 0.3 ~ 1mL of 10mg/mL, if reaction not exclusively, will repeat previous step, and react completely, and carries out next step reaction;
(7) other amino acid whose reactions are carried out according to step (5) (6);
(8) 5 (6)-Fluoresceincarboxylic acids (FAM), 2-(7-azo benzotriazole)-N, N, N ' N '-tetramethyl-urea phosphofluoric acid ester (HATU), HoBt, N-methylmorpholine, be dissolved in DMF, add room temperature reaction 2 ~ 12h in reactor, extract solvent, DMF washing 2 ~ 4 times;
(9) dimethyl amino-azo-benzene (Dabcyl) carries out according to the method for condensing of step (8);
(10) with DMF washing 2 ~ 4 times, methanol wash 2 ~ 4 times, washed with dichloromethane 2 ~ 4 times, dry ,-20 DEG C keep in Dark Place.
Embodiment 3
The synthesis of dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine-Methionin (fluorescein)-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene (such as formula I):
(1) the Rink Amide resin of fluorescein-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene sequence is contained in Example 2, swelling 0.5 ~ 2h in DMF;
(2) methyltrityl side base is removed with the trifluoroacetic acid/dichloromethane mixed solution that volume ratio is 0.5 ~ 4:96 ~ 99.5, after removing end, washed with dichloromethane 2 ~ 4 times, DMF washing 2 ~ 4 times;
(3) dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine, HATU, HoBt, N-methylmorpholine (NMM) are dissolved in DMF, add room temperature reaction 2 ~ 10h in reactor, extract solvent, DMF washing 2 ~ 4 times;
(4) cut: volume fraction is that the trifluoroacetic acid/water mixed liquid of 83 ~ 95%:5 ~ 17% cuts 1 ~ 3h, decompress filter also collects filtrate, by filtrate decompression concentrated by rotary evaporation, concentrated solution is dropwise instilled in freezing anhydrous diethyl ether, centrifugal, supernatant liquor is drained, dried in vacuo overnight ,-20 DEG C of preservations.
(5) detection of target molecule: take a morsel sample dissolution in methyl alcohol, detected by Matrix-assisted laser desorption ionization.
The chemical structure application MALDI-TOF-MS of target molecule characterizes, as shown in Figure 1.
Embodiment 4: the fluorescence spectrum before and after fluorescent probe and matrix metalloproteinase and apoptosis enzyme act on simultaneously
Fluorescent probe being dissolved in pH is in the phosphoric acid buffer (PBS) of 7.4, be mixed with the storing solution of 1 μM, add 200pM apoptosis enzyme (Caspase-3) and 200ng matrix metalloproteinase (MMP-2) and corresponding PBS damping fluid, the concentration of fluorescent probe in working fluid is made to be 0.5 μM, react under 37 DEG C of conditions, fluorescence emission spectrum is detected over time by fluorescence spectrophotometer, excitation wavelength 465nm, slit width 5nm, as shown in Figure 2, fluorescent probe before cutting after to have fluorescence intensity change more than 18.5 times at 520nm place.
Embodiment 5
After fluorescent probe and matrix metalloproteinase effect with apoptosis enzyme effect fluorescence spectrum and with fluorescent probe with matrix metalloproteinase and apoptosis enzyme effect contrast simultaneously
Prepare fluorescence according to the method that embodiment 4 is identical and recover comparative group, simultaneously, 200ngMMP-2 will be added in the storing solution be mixed with and with the PBS damping fluid of pH 7.4, fluorescent probe concentration dilution is prepared into working fluid to 0.5 μM, react under 37 DEG C of conditions, by fluorescent emission in fluorescence spectrophotometer testing liquid over time, excitation wavelength 465nm, slit width 5nm.Treat that working fluid fluorescence intensity changes in time to tend to balance, two parts are divided into working fluid, 200pM caspase-3 enzyme is added in portion, another part adds same volume PBS, the PBS damping fluid simultaneously adding same volume in comparative group is identical to maintain three's concentration and probe concentration, reacts under 37 DEG C of conditions, by the emissive porwer of fluorescence spectrophotometer record fluorescence at 520nm place over time, excitation wavelength 465nm, slit width 5nm.As shown in Figure 3,200ng MMP-2 and 200pM Caspase-3 acts under simultaneously, there is the enhancing of 17.7 times of fluorescence after 10.5h, and under the condition only having 200ng MMP-2 effect, only have the Fluorescence Increasing of 5.9 times.And, under the condition only having 200ng MMP-2 effect, reaction times is increased to 25.1h, fluorescence intensity is compared with the MMP-2 enzyme effect 10.5h of same concentration, do not strengthen further, but, after 200ng MMP-2 effect 10.5h, again with the effect of 200pM caspase-3 enzyme to 25.1h, the 200ng MMP-2 of fluorescence intensity and effect same time and do not add compared with 200pM caspase-3 enzyme, have further enhancing, and its fluorescence intensity is identical with from the fluorescence intensity initially adding MMP-2 with the Caspase-3 effect of same concentrations simultaneously.
Embodiment 6
For Ac-DEVD-CHO, use probe in detecting apoptosis enzyme inhibitors to the restraining effect of apoptosis enzymic activity
By 200pM caspase-3 enzyme and 50 μMs of apoptosis enzyme inhibitors Ac-DEVD-CHO effect 2h, add fluorescent probe again and be mixed with working fluid, and make the concentration of fluorescent probe in working fluid be 0.5 μM, react under 37 DEG C of conditions, by the change of fluorescence spectrophotometer record fluorescence intensity at 520nm place, excitation wavelength 465nm, slit width 5nm.As shown in Figure 4, add the effect of apoptosis enzyme inhibitors, after fluorescent probe and apoptosis enzyme reaction 6.6h, only have the enhancing of 1.1 times in the fluorescence intensity at 520nm place, show that this inhibitor is to the identification of apoptosis enzyme spcificity with cut off specific peptide sequence in probe and have restraining effect significantly.Otherwise, this probe can by fluorescence to change in fluorescence before and after desired inhibitor effect in order to screen corresponding enzyme inhibitors.
Embodiment 7
With 1,10-phenanthroline for example, use probe in detecting matrix metallo-proteinase inhibitor to the restraining effect of matrix metal proteinase activity
By 200ng MMP-2 enzyme and 1,10-phenanthroline effect 2h, add fluorescent probe again and be mixed with working fluid, and make the concentration of fluorescent probe in working fluid be 0.5 μM, react under 37 DEG C of conditions, by the change of fluorescence spectrophotometer record fluorescence intensity at 520nm place, excitation wavelength 465nm, slit width 5nm.As shown in Figure 5, add matrix metallo-proteinase inhibitor effect, after fluorescent probe and matrix metalloproteinase react 4.6h, only have the enhancing of 1.8 times in the fluorescence intensity at 520nm place, show that this inhibitor has restraining effect significantly to peptide sequence in the specific identification of matrix metalloproteinase and cut-out probe.Otherwise, this probe can by fluorescence to change in fluorescence before and after desired inhibitor effect in order to screen corresponding enzyme inhibitors.
Claims (10)
1. a two enzyme responsive type fluorescent probe, is characterized in that: the molecular fluorescence pair comprising apoptosis enzyme spcificity Recognition polypeptide sequence, matrix metalloproteinase specific recognition peptide sequence and two pairs of resonance energy transfers.
2. according to claim 1 pair of enzyme responsive type fluorescent probe, is characterized in that: what two pairs of described resonance energy transfer molecular fluorescences were right is same as body to body.
3. according to claim 2 pair of enzyme responsive type fluorescent probe, is characterized in that: described molecular fluorescence is to being be same as body and the fluorescence quencher molecule fluorescence pair that is acceptor with different dimethyl amino-azo-benzene with fluorescein.
4. the two enzyme responsive type fluorescent probes according to claim 1 or 3, is characterized in that: described apoptosis enzyme spcificity Recognition polypeptide sequence is the peptide sequence comprising amino acid-amino acid-amino acid-aspartic acid; Described matrix metalloproteinase specific recognition peptide sequence is the one in proline-leucine-glycine-α-amino-isovaleric acid-arginine, proline-leucine-Gly-Leu-Ala-Gly, proline-leucine-glycine-halfcystine-Ala-Gly, Gly-Pro-proline(Pro)-glycine-α-amino-isovaleric acid-α-amino-isovaleric acid-glycine-glutaminic acid-LYS-GLY-Glu-Gln.
5. according to claim 4 pair of enzyme responsive type fluorescent probe, is characterized in that: the described peptide sequence comprising amino acid-amino acid-amino acid-aspartic acid is Asp-Glu-valine aspartic acid or tryptophane-L-glutamic acid-histidine-asparate.
6. according to claim 5 pair of enzyme responsive type fluorescent probe, it is characterized in that: chemistry dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine-Methionin (fluorescein)-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene by name, chemical structure is as shown in formula I:
7. a preparation method for according to claim 6 pair of enzyme responsive type fluorescent probe, is characterized in that: comprise the following steps:
(A) dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine, fluorescein-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene is synthesized;
(B) dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine and fluorescein-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene are carried out condensation, namely obtain the fluorescent probe of the detection matrix metalloproteinase shown in formula I and apoptosis enzyme.
8. the preparation method of according to claim 7 pair of enzyme responsive type fluorescent probe, is characterized in that:
In described step (A), the synthesis of dimethyl amino-azo-benzene-Gly-Pro-leucine-glycine-α-amino-isovaleric acid-arginine-glycine and fluorescein-Methionin-serine-aspartate-glutamic acid-valine-asparate-serine-Methionin-dimethyl amino-azo-benzene adopts Solid-phase synthesis peptides method;
Described step (B) adopts the method for solid phase fragment condensation.
9. the preparation method of according to claim 8 pair of enzyme responsive type fluorescent probe, is characterized in that: the resin adopted in Solid-phase synthesis peptides is the one in the chloro-trityl chloride resin of 2-, Rink Amide resin, mbha resin.
10. the application of the two enzyme responsive type fluorescent probes described in any one of claim 1-6 in the screening of chemical system, living things system matrix metalloproteinase and apoptosis Enzyme assay and apoptosis enzyme inhibitors and matrix metallo-proteinase inhibitor.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105694851A (en) * | 2016-03-08 | 2016-06-22 | 武汉大学 | Tumor-targeted diagnosis and treatment integrated fluorescent probe |
| CN105949281A (en) * | 2016-05-04 | 2016-09-21 | 新乡医学院 | Fluorescence resonance energy transfer probe, preparation method and application thereof |
| CN106753342A (en) * | 2017-01-09 | 2017-05-31 | 武汉大学 | A kind of double-bang firecracker answers ring-type fluorescence probe and its preparation method and application |
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| CN110590801A (en) * | 2019-09-05 | 2019-12-20 | 中国药科大学 | Glutaminase small-molecule fluorescent probe and preparation method and application thereof |
| CN115925803A (en) * | 2022-07-01 | 2023-04-07 | 华中科技大学 | Lighting type GPx4 fluorescent molecular probe and application thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105694851A (en) * | 2016-03-08 | 2016-06-22 | 武汉大学 | Tumor-targeted diagnosis and treatment integrated fluorescent probe |
| CN105694851B (en) * | 2016-03-08 | 2018-07-27 | 武汉大学 | A kind of cancer target diagnosis and treatment fluorescence probe |
| CN105949281A (en) * | 2016-05-04 | 2016-09-21 | 新乡医学院 | Fluorescence resonance energy transfer probe, preparation method and application thereof |
| CN106753342A (en) * | 2017-01-09 | 2017-05-31 | 武汉大学 | A kind of double-bang firecracker answers ring-type fluorescence probe and its preparation method and application |
| CN106753342B (en) * | 2017-01-09 | 2019-05-24 | 武汉大学 | A kind of double-bang firecracker answers cyclic annular fluorescence probe and its preparation method and application |
| CN108707184A (en) * | 2018-05-21 | 2018-10-26 | 浙江大学 | A kind of MS probe and its preparation method and application for thrombin activity detection |
| CN108707184B (en) * | 2018-05-21 | 2021-01-29 | 浙江大学 | Mass spectrum probe for thrombin activity detection and preparation method and application thereof |
| CN110590801A (en) * | 2019-09-05 | 2019-12-20 | 中国药科大学 | Glutaminase small-molecule fluorescent probe and preparation method and application thereof |
| CN110590801B (en) * | 2019-09-05 | 2020-11-03 | 中国药科大学 | Glutaminase small-molecule fluorescent probe and preparation method and application thereof |
| CN115925803A (en) * | 2022-07-01 | 2023-04-07 | 华中科技大学 | Lighting type GPx4 fluorescent molecular probe and application thereof |
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