CN103980886A - Fluorescent molecular probe, and preparation method and application thereof - Google Patents
Fluorescent molecular probe, and preparation method and application thereof Download PDFInfo
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- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 claims description 6
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 6
- 239000008117 stearic acid Substances 0.000 claims description 6
- PWYVVBKROXXHEB-UHFFFAOYSA-M trimethyl-[3-(1-methyl-2,3,4,5-tetraphenylsilol-1-yl)propyl]azanium;iodide Chemical compound [I-].C[N+](C)(C)CCC[Si]1(C)C(C=2C=CC=CC=2)=C(C=2C=CC=CC=2)C(C=2C=CC=CC=2)=C1C1=CC=CC=C1 PWYVVBKROXXHEB-UHFFFAOYSA-M 0.000 claims description 6
- 150000001413 amino acids Chemical class 0.000 claims description 5
- 125000004432 carbon atom Chemical group C* 0.000 claims description 5
- JLZUZNKTTIRERF-UHFFFAOYSA-N tetraphenylethylene Chemical group C1=CC=CC=C1C(C=1C=CC=CC=1)=C(C=1C=CC=CC=1)C1=CC=CC=C1 JLZUZNKTTIRERF-UHFFFAOYSA-N 0.000 claims description 5
- 125000003277 amino group Chemical group 0.000 claims description 4
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- 238000004020 luminiscence type Methods 0.000 claims description 4
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- 238000002474 experimental method Methods 0.000 description 5
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- YPTNAIDIXCOZAJ-LHEWISCISA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-6-[[(4-methylphenyl)-diphenylmethyl]amino]hexanoic acid Chemical compound C1=CC(C)=CC=C1C(C=1C=CC=CC=1)(C=1C=CC=CC=1)NCCCC[C@@H](C(O)=O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 YPTNAIDIXCOZAJ-LHEWISCISA-N 0.000 description 2
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- Peptides Or Proteins (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明涉及一种荧光分子探针及其制备方法和应用。所述荧光分子探针包括亲水多肽以及连接于所述亲水多肽同一端的疏水烷基链和受限发光基团。本发明的荧光分子探针中，亲水多肽能够提高荧光分子探针在水溶液中的溶解性和稳定性，疏水烷基链能够嵌入细胞膜，受限发光基团只在受限条件下发射荧光。该荧光分子探针稳定性强、特异性好，因此是一种良好的可用于细胞膜标记的荧光分子探针，可应用于生物或医学研究中。本发明所述荧光分子探针的制备方法，简单易行，不涉及复杂的操作和苛刻条件，并且成本较低，光学稳定性好。
The invention relates to a fluorescent molecular probe and its preparation method and application. The fluorescent molecular probe includes a hydrophilic polypeptide, a hydrophobic alkyl chain and a restricted luminescent group connected to the same end of the hydrophilic polypeptide. In the fluorescent molecular probe of the present invention, the hydrophilic polypeptide can improve the solubility and stability of the fluorescent molecular probe in aqueous solution, the hydrophobic alkyl chain can be embedded in the cell membrane, and the restricted luminescent group can only emit fluorescence under restricted conditions. The fluorescent molecular probe has strong stability and good specificity, so it is a good fluorescent molecular probe that can be used for cell membrane labeling and can be applied in biological or medical research. The preparation method of the fluorescent molecular probe of the present invention is simple and easy, does not involve complex operations and harsh conditions, and has low cost and good optical stability.
Description
Technical field
The present invention relates to fluorescent molecular probe field, relate in particular to a kind of fluorescent molecular probe for cytolemma mark and its preparation method and application.
Background technology
Fluorescent molecular probe is to be based upon in spectrochemistry and optical waveguide and Foundation of Measurement Technology, optionally changes continuously the chemistry of analytic target and structural information into the analytical instrument easily molecule measuring apparatus of the fluorescent signal of measurement.Fluorescent molecular probe is subject to the impact of surrounding environment, shows different fluorescent characteristics under different condition.When in a certain specific environment, fluorescent molecular probe fluorescent characteristic changes, and as growing out of nothing of fluorescence, or the change of excitation wavelength, emission wavelength, thereby makes people know certain customizing messages existing in the feature of surrounding environment or environment.That fluorescent molecular probe has is highly sensitive, selectivity good, easy to use, cost is low, do not need pre-treatment, be not subject to that external electromagnetic field affects, the remote plurality of advantages such as luminous.Fluorescent molecular probe is comprised of recognition group and reporter group conventionally, and the former has determined selectivity and the specificity of probe molecule, and latter has determined the sensitivity of identification.
In biomedical research, people usually need labeled cell film with form and the size of observation of cell, or observe the position relationship of specific protein, vesica and cytolemma.Yet existing membrane molecule probe has the shortcomings such as poor specificity, easy photobleaching, has limited its application, cannot meet the demand of scientific research.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of fluorescent molecular probe for cytolemma mark, its cell membrane has specific marker ability, and the rear luminophore of being combined with cytolemma has fluorescence, therefore showed cell film location is a kind of fluorescent molecular probe that can be used in cytolemma mark.The present invention also provides the preparation method and application of described fluorescent molecular probe, and its preparation method is simple, does not relate to complicated operation and severe condition, and cost is lower, and optical stability is good.
For realizing object of the present invention, the invention provides following technical scheme:
In first aspect, the invention provides a kind of fluorescent molecular probe, comprise hydrophilic polypeptide and be connected in described hydrophilic polypeptide with hydrophobic alkyl chain and the limited luminophore of one end.
In fluorescent molecular probe of the present invention, described hydrophobic alkyl chain can embed cytolemma and cause luminophore limited, and emitting fluorescence under laser radiation is realized the specific marker of cell membrane.In fluorescent molecular probe of the present invention, hydrophilic polypeptide can improve stability and the targeting of probe in the aqueous solution, hydrophobic alkyl chain can embed cytolemma, and luminophore is emitting fluorescence under confined condition only, is therefore a kind of good fluorescent molecular probe that can be used for cytolemma mark.
In the present invention, covalently bound between described hydrophobic alkyl chain and limited luminophore and hydrophilic polypeptide can be stable covalent linkage arbitrarily, as long as can realize three's connection, such as amido linkage, ester bond or ehter bond etc.Certainly, also comprise the covalent linkage connection type of developing future.
As the preferred technical solution of the present invention, described hydrophobic alkyl chain is all connected with described hydrophilic polypeptid covalence by amido linkage with limited luminophore.
Verified, adopt amido linkage connection can realize hydrophobic alkyl chain and be connected with the efficient, stable of hydrophilic polypeptide with limited luminophore, it is all very single-minded and stable that two amido linkages connect, and can not produce interference to three partial functions.
As the preferred technical solution of the present invention, on the side chain R base of the n terminal amino acid of described hydrophilic polypeptide, there is amino group, described amino group and described hydrophobic alkyl chain formation amido linkage are covalently bound; It is covalently bound that the amino and described limited luminophore of α of the n terminal amino acid of described hydrophilic polypeptide forms amido linkage.The n terminal amino acid of described hydrophilic polypeptide is such as Methionin, arginine, l-asparagine and glutamine etc.
As the preferred technical solution of the present invention, the n terminal amino acid of described hydrophilic polypeptide is Methionin, and the side chain of described Methionin is amino covalently bound with described hydrophobic alkyl chain formation amido linkage; It is covalently bound that the amino and described limited luminophore of α of described Methionin forms amido linkage.
As the preferred technical solution of the present invention, the carbochain of described hydrophobic alkyl chain contains 8-20 carbon atom, for example 8,9,10,11,12,13,14,15,16,17,18,19 or 20 carbon atoms, are preferably 12-20 carbon atom, more preferably palmitinic acid, oleic acid or stearic acid.In above-mentioned scope, the ability that hydrophobic alkyl chain embeds cytolemma is stronger, and the molecular probe obtaining has good stability.
As the preferred technical solution of the present invention, described hydrophilic polypeptide is with positive charge, is preferably to take the polypeptide that positively charged amino acid is main composition, more preferably contains arginic polypeptide.Except arginine, can also contain Methionin or Histidine etc.The positively charged hydrophilic polypeptide that above-mentioned amino acid forms can promote itself and cell to be in contact with one another when improving fluorescent molecular probe stability, thereby improves specificity.
As the preferred technical solution of the present invention, described limited luminophore is limited induced luminescence molecule, is preferably tetraphenyl ethylene molecule or Silole.Above-mentioned limited induced luminescence molecule has the limited characteristics of luminescence of molecular rotation, and has the stable advantages such as cancellation, luminous efficiency height that are difficult for of fluorescence.
In second aspect, the invention provides a kind of method of preparing the fluorescent molecular probe described in first aspect, comprising: same one end of hydrophobic alkyl chain and limited luminophore and hydrophilic polypeptide is connected to form to described fluorescent molecular probe.
As the preferred technical solution of the present invention, described method comprises: under the existence of catalyzer, make respectively to contact with the amino of hydrophilic polypeptide with limited luminophore with the hydrophobic alkyl chain of carboxyl, form amido linkage covalently bound, obtain described fluorescent molecular probe.
Preferably, described catalyzer is 2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester and DIPEA.
In the method for the invention, the characteristic of hydrophilic polypeptide, hydrophobic alkyl chain and limited luminophore and selection, as described in first aspect, do not repeat them here.
In the third aspect, the invention provides the application in cytolemma mark of fluorescent molecular probe as described in first aspect.
Compared to existing technology, beneficial effect of the present invention is: in fluorescent molecular probe of the present invention, hydrophilic polypeptide can improve solvability and the stability of fluorescent molecular probe in the aqueous solution, and hydrophobic alkyl chain can embed cytolemma, and limited luminophore is emitting fluorescence under confined condition only.This fluorescent molecular probe stability is strong, specificity good, is therefore a kind of good fluorescent molecular probe that can be used for cytolemma mark, can be applicable in biology or medical research.The preparation method of fluorescent molecular probe of the present invention, simple, do not relate to complicated operation and severe condition, and cost is lower, optical stability is good.
Accompanying drawing explanation
Fig. 1 is the fluorescent molecular probe of the embodiment of the present invention 1 preparation mark effect of cell membrane (shooting of single-photon laser Laser Scanning Confocal Microscope) after incubated cell 30min under 50 μ M concentration.
Fig. 2 is the fluorescent molecular probe of the embodiment of the present invention 1 preparation mark effect of cell membrane (shooting of two-photon laser Laser Scanning Confocal Microscope) after incubated cell 30min under 50 μ M concentration.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that following examples are only the preferred embodiments of the present invention, so that understand better the present invention, thereby should not be considered as limiting scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method; Experiment material used, if no special instructions, is and is purchased available from routine biochemistry chemical reagent work.
Amino acid (Fmoc-amino acids), 2-(7-azo the benzotriazole)-N of the source of material therefor: Fmoc protection in embodiment, N, N', N'-tetramethyl-urea phosphofluoric acid ester (O-(7-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronilum hexafluorophosphate, HATU) and DIPEA (diisopropylethylamine, DIEA) all purchased from Beijing Bo Maijie Science and Technology Ltd.; Palmitinic acid (>=99%, palmitic acid), oleic acid (>=99%, oleic acid), stearic acid (>=99%, stearic acid) are all purchased from Sigma-Aldrich (St Louis, the U.S.) company; Carboxylated tetraphenyl ethene (TPE-COOH) (people such as reference Chunqiu Zhang, Imaging Intracellular Anti-Cancer Drug Delivery by Self-Assembly Micelles with Aggregation-Induced Emission (AIE Micelles) .ACS Applied Materials & Interfaces.2014.9; 6 (7): 5212-5220.) and Silole (silole) (reference Silole-Containing Polyacetylenes.Synthesis, Thermal Stability, Light Emission, Nanodimensional Aggregation, and Restricted Intramolecular Rotation.Macromolecules, 2003,36 (4), pp1108 – 1117) be that oneself is synthetic; All chemical reagent are not with all processing through other before, and the ultrapure water that whole experiment is used adopts the Milli-Q tri-distilled water (18.2M Ω cm, Millipore System Inc.) of Millipore Corp..
Embodiment 1
Polypeptide (the RRRR that the present embodiment forms with 4 arginine, R4, SEQ ID NO:1) as hydrophilic polypeptide, carboxylated tetraphenyl ethene (TPE-COOH), as limited luminophore, palmitinic acid, as hydrophobic alkyl chain, be example, the preparation of fluorescent molecular probe is described.By this fluorescent molecular probe called after TR4, hereinafter TR4 all refers to this probe.Wherein arginine positively charged, therefore R4 polypeptide presents positive polarity, has affinity to cell.Methionin N end of the follow-up connection of arginine and side chain end two are amino for being connected TPE-COOH and palmitinic acid.The carboxyl of palmitinic acid is easy to reaction, and hydrophobic alkyl chain can embed in cytolemma, and fixing whole fluorescent molecular probe.TPE molecule is not luminous under free state, once probe is incorporated into cytolemma, and phenyl ring limited in rotation, TPE emitting fluorescence.Its optimum excitation wavelength is 330nm, and emission wavelength is 466nm.Following formula is the molecular structure of synthetic TR4.
The specific experiment step of synthetic TR4 is as follows:
Adopt CLEAR Amide polypeptide synthetic resins, utilize the solid-phase peptide synthetic method of Fmoc chemistry, connect successively arginine, arginine, arginine, arginine, Methionin (Fmoc-Lys (MTT)-OH).Under 3% trifluoroacetic acid condition, slough the Side chain protective group MTT of Methionin (Fmoc-Lys (MTT)-OH), adopt condensation reagent HATU and DIEA palmitinic acid to be connected to the side chain of Methionin.Under 20% piperidines condition, deviate from Fmoc group, adopt condensation reagent HATU and DIEA TPE-COOH to be connected to the N-terminal of Methionin.With trifluoroacetic acid, cut polypeptide, cold diethyl ether precipitation, collects TR4 crude product high performance liquid chromatography purifying afterwards, and freeze-drying, obtains final product probe.
Finally, cell membrane is marked as picture, and specific experiment step is as follows:
The TR4 making with the present embodiment is hatched breast cancer cell MCF-730min under 50 μ M concentration, imaging under single photon or two-photon laser Laser Scanning Confocal Microscope, by observing TPE fluorescence, determine cytolemma position and form, as depicted in figs. 1 and 2, Fig. 1 and Fig. 2 all show the fluorescent signal along cytolemma to result.
Result shows: the cytolemma of TR4 mark is continuous, complete, does not occur TPE fluorescence in endochylema, and TR4 mark has been described cytolemma has good mark specificity.Cellular form is good, and TR4 itself and labeling process can not produce toxicity to cell, are applicable to viable cell labelling.
Embodiment 2 (changing amino acid forms)
The difference of the present embodiment and embodiment 1 is to change 4 arginine in embodiment 1 into 2 arginine and 2 Methionins (RKRK, SEQ ID NO:2), prepares to take the fluorescent molecular probe that RKRK is hydrophilic polypeptide.
The cytolemma mark result that the fluorescent molecular probe making with the present embodiment carries out is similar to embodiment's 1.
Embodiment 3 (changing polypeptide length)
The difference of the present embodiment and embodiment 1 is to change 4 arginine in embodiment 1 into 8 arginine, prepares with RRRRRRRR SEQ ID NO:3) be the fluorescent molecular probe of hydrophilic polypeptide.
The cytolemma mark result that the fluorescent molecular probe making with the present embodiment carries out is similar to embodiment's 1.
Embodiment 4 (changing hydrophobic alkyl chain length)
The difference of the present embodiment and embodiment 1 is to change the palmitinic acid in embodiment 1 into oleic acid, prepares to take the fluorescent molecular probe of oleic acid as hydrophobic alkyl chain.
The cytolemma mark result that the fluorescent molecular probe making with the present embodiment carries out is similar to embodiment's 1.
Embodiment 5 (changing hydrophobic alkyl chain length)
The difference of the present embodiment and embodiment 1 is to change the palmitinic acid in embodiment 1 into stearic acid, prepares to take the fluorescent molecular probe of stearic acid as hydrophobic alkyl chain.
The cytolemma mark result that the fluorescent molecular probe making with the present embodiment carries out is similar to embodiment's 1.
Embodiment 6 (changing limited luminophore)
The difference of the present embodiment and embodiment 1 is to change the tetraphenyl ethene in embodiment 1 into Silole, prepares to take the fluorescent molecular probe that Silole is limited luminophore.
The cytolemma mark result that the fluorescent molecular probe making with the present embodiment carries out is similar to embodiment's 1.
Applicant's statement, the present invention illustrates detailed features of the present invention and detailed method by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and detailed method, do not mean that the present invention must rely on above-mentioned detailed features and detailed method could be implemented.Person of ordinary skill in the field should understand, any improvement in the present invention is selected the selection of the equivalence replacement of component and the interpolation of ancillary component, concrete mode etc., within all dropping on protection scope of the present invention and open scope to the present invention.
Claims (10)
1. a fluorescent molecular probe, comprises hydrophilic polypeptide and is connected in described hydrophilic polypeptide with hydrophobic alkyl chain and the limited luminophore of one end.
2. fluorescent molecular probe according to claim 1, is characterized in that, described hydrophobic alkyl chain is all connected with described hydrophilic polypeptid covalence by amido linkage with limited luminophore.
3. fluorescent molecular probe according to claim 1 and 2, is characterized in that, on the side chain R base of the n terminal amino acid of described hydrophilic polypeptide, has amino group, and described amino group and described hydrophobic alkyl chain formation amido linkage are covalently bound; It is covalently bound that the amino and described limited luminophore of α of the n terminal amino acid of described hydrophilic polypeptide forms amido linkage.
4. according to the fluorescent molecular probe described in claim 1-3 any one, it is characterized in that, the n terminal amino acid of described hydrophilic polypeptide is Methionin, and the side chain of described Methionin is amino covalently bound with described hydrophobic alkyl chain formation amido linkage; It is covalently bound that the amino and described limited luminophore of α of described Methionin forms amido linkage.
5. according to the fluorescent molecular probe described in claim 1-4 any one, it is characterized in that, the carbochain of described hydrophobic alkyl chain contains 8-20 carbon atom, is preferably 12-20 carbon atom, more preferably palmitinic acid, oleic acid or stearic acid.
6. according to the fluorescent molecular probe described in claim 1-5 any one, it is characterized in that, described hydrophilic polypeptide is with positive charge, is preferably to take the polypeptide that positively charged amino acid is main composition, more preferably contains arginic polypeptide.
7. according to the fluorescent molecular probe described in claim 1-6 any one, it is characterized in that, described limited luminophore is limited induced luminescence molecule, is preferably tetraphenyl ethylene molecule or Silole.
8. a method of preparing the fluorescent molecular probe described in claim 1-7 any one, comprising: same one end of hydrophobic alkyl chain and limited luminophore and hydrophilic polypeptide is connected to form to described fluorescent molecular probe.
9. method according to claim 8, it is characterized in that, described method comprises: under the existence of catalyzer, make respectively to contact with the amino of hydrophilic polypeptide with limited luminophore with the hydrophobic alkyl chain of carboxyl, formation amido linkage is covalently bound, obtains described fluorescent molecular probe;
Preferably, described catalyzer is 2-(7-azo benzotriazole)-N, N, N', N'-tetramethyl-urea phosphofluoric acid ester and DIPEA.
10. the application of the fluorescent molecular probe as described in claim 1-7 any one in cytolemma mark.
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