CN108997476A - A kind of novel polypeptide fluorescence nano structural material and preparation method thereof - Google Patents
A kind of novel polypeptide fluorescence nano structural material and preparation method thereof Download PDFInfo
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- CN108997476A CN108997476A CN201810880643.8A CN201810880643A CN108997476A CN 108997476 A CN108997476 A CN 108997476A CN 201810880643 A CN201810880643 A CN 201810880643A CN 108997476 A CN108997476 A CN 108997476A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/14—Macromolecular compounds
- C09K2211/1408—Carbocyclic compounds
- C09K2211/1425—Non-condensed systems
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Abstract
The present invention provides a kind of novel polypeptide fluorescence nano structural material and preparation method thereof, feature is crosslinked by way of aoxidizing with amino addition by the polypeptide containing tyrosine and lysine.Its preparation process are as follows: (1) polypeptide of the sequence containing GYK in right amount is dissolved in the alkaline solution that pH is 8, its solution concentration is made to be greater than 1 mg/mL；(2) hydrogen peroxide oxidant of 0.05%~0.1% volume is added, 25~80 DEG C of 2~10h of heating water bath are cooled to room temperature, ultrafiltration remove unreacted polypeptide monomer to get.Preparation of the invention does not need additionally to add any cross-linking reagent, easy to operate, and product fluorescence property is stablized, and facilitates connection and modification identification sequence to carry out total assembling, has the potentiality for being widely used in biomarker, detection and imaging field.
Description
Technical field
The present invention relates to biochemical synthesis technical fields, more particularly, to a kind of novel polypeptide fluorescence nano structural material
And preparation method thereof.
Background technique
In recent years, aggregation-induced emission (AIE) technology is widely used in biomarker and imaging field, and cardinal principle is base
Assemble limitation aromatic group rotation in organic molecule, reduces energy dissipation to improve fluorescence property, with traditional organic dyestuff phase
Than with higher stability, the functional exploitation that it forms conjugate in conjunction with polypeptide has also obtained good in medical mark
Development.Nevertheless, the luminous original part of these materials still relies on inanimate molecule.
Have found green fluorescent protein (GFP) the 1960s, excellent fluorescence property becomes most to be made extensively
One of protein.Wherein, yellow fluorescence protein is in all GFP variant medium wavelength longests, relative to the spectral red shift of GFP,
Its wavelength length is caused by being replaced as 203 threonines by tyrosine, and exists between the phenols anion of the latter's chromophore
Pi-pi accumulation effect.This active force reduces excited energy, promotes also to mention while GFP spectral red shift for self-assembling polypeptide
For driving force.Artificial reconstructed, to be formed by beta sheet in fractionation/combination GFP molecule tubbiness is further carried out to this structure
Structure adjusts the characteristics of luminescence of fluorescin, assembles molecular switch, and utilize the mould of the artificial constructed fluorescin of its chromophore
Quasi- object, is used for biomarker and detection.
By the improvement for fluorescence protein amino acid sequence, novel photoactivation fluorescin is produced, i.e., is swashed specific
Spectral characteristic changes under luminous irradiation, including photoactivation (fluorescence intensity changes from weak to strong) and light conversion (fluorescence color by
It is green to redden) two kinds of forms.Fluorescin is converted derived from the green feux rouges of coral, it is highly conserved in sequence and structure, wherein representing
The chromophore of property fluorescin is all by the Sequence composition containing tyrosine.Although fluorescin be current optical property well at
As material, but its structure is complex and at high price, also not simple in surface modification and connection, thus in further assembling and
There are still many limitations in.Need that development structure is simpler, the better nano-luminescent material of versatility.
Polypeptide has many advantages as the macromolecular for constituting organism basic structure, utilizes amino acid sequence design construction
Self assembly module forms the new direction that orderly functionalized nano structure is medical material development.Small peptide self-assembled structures by
Prove that there is good semiconductor optical performance, their band gap can compare favourably with traditional material, and stringent self assembly can make
Potential cytotoxicity minimizes, and the biocompatibility of supramolecular structure also meets application demand.In various polypeptide nano structures,
Hot spot, compared with the inorganic fluorescent material largely used at present, the biology of peptide nanoparticles are increasingly become to optical property research
Degradability is undoubtedly great advantage.However, the intrinsic spectral region of polypeptide chromophore is predominantly located at ultra-violet (UV) band, itself optics
The limitation of performance limits its application as effective probe.Up to the present, existing research be related to control self-assembling polypeptide obtain
To required pattern and structure, but changes intrinsic optical property polypeptide nano structure by self assembly and be still in exploration rank
Section.
Summary of the invention
Idea of the invention is that providing a kind of novel polypeptide fluorescence nano structural material and preparation method thereof, it is based primarily upon
Polypeptide of the one kind containing tyrosine and lysine is cross-linked to form polymer by way of aoxidizing with amino addition and realizes, principle
See Fig. 1.Crosslinking between small peptide monomer is mainly due to the effect of the pi-pi accumulation between tyrosine and tyrosine and another monomer
Lysine side chain amino groups form intermolecular coupling caused by imines under alkaline condition, the interaction between this monomer is formed
Polypeptide nano particulate polymers, i.e. polypeptide fluorescent nano material.
The purpose of the present invention is what is be achieved through the following technical solutions:
A kind of novel polypeptide fluorescence nano structural material, the polypeptide containing tyrosine and lysine are handed over by oxidation and amino addition
The mode of connection forms polymer.Specifically polypeptide sequence (is abbreviated as Lys comprising tyrosine (being abbreviated as Tyr or Y), lysine
Or K), glycine (being abbreviated as Gly or G), therefore sequence include containing this 3 kinds of amino acid tripeptides (GYK, GKY, YGK, YKG,
KGY, KYG) and repetitive sequence of the tripeptides as a unit, such as GYK, GYKGYK, GYKGYKGYK ..., (GYK) n,
N is the integer greater than zero.
The synthesis step of novel polypeptide fluorescence nano structural material are as follows: (1) polypeptide of the sequence containing GYK in right amount is dissolved in pH
For in 8 alkaline solution, as phosphate buffer (PBS), borax (sodium tetraborate) buffer, Tris buffer, MES buffer or
In HEPES buffer solution, its solution concentration is made to be greater than 1 mg/mL；(2) hydrogen peroxide oxidation of the volume of 0.05 %~0.1% is added
Agent, 25~80 DEG C of 2~10h of heating water bath, is cooled to room temperature, ultrafiltration remove unreacted polypeptide monomer to get.
Further, the peptide C end can select identification sequence by carrying out sequence design to it and carrying out functionalized modification
RGD peptide but it is not limited to RGD peptide, the accurate modification for specific cells is reinforced with this.
Compared with the prior art, the advantages of the present invention are as follows:
(1) the present invention provides a kind of novelties, stablize, easy polypeptide fluorescence nano structural material synthesis mode, sequence containing GYK
Polypeptide pH be 8 alkaline solution under the conditions of carry out oxidation cross-linked reaction, the side chain phenolic hydroxyl group of tyrosine is by oxygen in the reaction
It is melted into quinone, meanwhile, the addition reaction of the amino and tyrosine side chain phenyl ring of lysine forms covalent bond, and peptide molecule relies on junket ammonia
The addition of crosslinking and tyrosine and lysine between acid connects, and this double action forms polymer, compared with monomer, point
Son amount increases by 10 times or more, and has the good fluorescence property of visible region under ultraviolet light.Therefore, it can use and contain junket ammonia
The polypeptide of the GYK sequence of acid and lysine prepares fluorescence nano structural material, and reaction condition is mild, and reaction process is easy quickly,
It can connect and modify identification sequence and carry out total assembling, be with a wide range of applications in biomarker, detection and imaging field.
(2) synthetic material of the invention is pure polypeptide molecule, and the organic or inorganic ingredient without any abiotic source property is raw
Object compatibility is good, is expected to be used for living imaging and treatment, and synthesis condition is mild, it is thus only necessary in common buffer
Oxidation cross-linked reaction is completed, does not need additionally to add any cross-linking reagent, it is easy to operate, it is suitable for batch production, product morphology is steady
It is fixed.Other than there is stable fluorescence property in physiological pH range, remain to show under the conditions of high temperature, with high salt, Strong oxdiative
Good optical property.
(3) present invention can form nano-scale particle (nano structural material), easily enter histocyte, convenient for marking in biology
Remember research field application, and since polypeptide sequence has editability, facilitates a variety of function such as modified cells identification peptide, cell-penetrating peptide
Sequence can be changed, meet different biomarker demands, be with a wide range of applications.
Detailed description of the invention
Polypeptide fluorescence nano structural material composition principle figure of the Fig. 1 containing Trp-Lys.
Fig. 2 polypeptide fluorescence nano structural material SDS-PAGE electrophoresis result.
Fig. 3 polypeptide fluorescence nano structural material dynamic light scattering particle diameter distribution testing result.
Fluorescence photo under Fig. 4 polypeptide fluorescence nano structural material solution ultraviolet light.
The excitation of Fig. 5 polypeptide fluorescence nano structural material fluorescence and transmitting map.
Fig. 6 polypeptide fluorescence nano structural material difference pH fluorescence Spectra.
Fluorescence Spectra under Fig. 7 polypeptide fluorescence nano structural material difference salt concentration conditions.
Fig. 8 polypeptide fluorescence nano structural material marks cell imaging picture.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be described in detail, and embodiments of the present invention are not limited thereto.
It takes 3 mg polypeptide solutions to be dissolved in 1 mL borate buffer solution, adds 0.1 % volume hydrogen peroxide, 80 DEG C of water-bath 6h are cooling
To room temperature, ultrafiltration remove unreacted polypeptide monomer to get.Survey about 5 kDa(of its molecular weight and see Fig. 2), average grain diameter is 2 nm
(see figure 3), and ultraviolet irradiation.
In the present embodiment, the fluorescence excitation peak of polypeptide fluorescence nano structural material is located at 350nm, and fluorescence emission peak is located at
490nm, therefore solution shows dark green fluorescence under the blue light illumination of different gel imaging systems, sees Fig. 4 and Fig. 5.Separately
Outer fluorescence property of the invention is not limited to that the adjustment of the amino acid sequence sequence of GYK may be implemented to fluorescence color
Regulation, fluorescence emission peak can be prepared and be located in the section of 450 ~ 500 nm, it is seen that fluorescence color migrate from blue light to blueness
Color and green light can develop the functionalized nanostructure material for multi-color marking by design to polypeptide sequence and modification.
In the present embodiment, which can still keep good stability under the conditions of high temperature and Strong oxdiative, and it is long when
Between ultraviolet light also have no apparent photobleaching phenomenon.In addition, in physiological conditions, the fluorescence nano structural material optics
Performance is stablized, the smaller (see figure 6) of fluctuation of fluorescence spectrum at least in 7 ~ 9 range of pH.Under the conditions of high ionic strength, fluorescence
Stability is equally ideal, even if the also (see figure 7) within 20% is quenched in fluorescence intensity under 10 × PBS high salt concentration.
Cell marking imaging test
Polypeptide fluorescence nano structural material surface carries positive charge in the present invention, can also obviously see in common non denatured electrophoresis
Band is observed from anode to cathode swimming, is proven this material.In consideration of it, the positive electricity fluorescence nano structural material is easily
The cell surface mostly in negative electricity is adsorbed in by charge interaction.Specifically, by mouse glioma cell climbing sheet and this
Invention polypeptide fluorescence nano structural material solution is incubated overnight altogether, after PBS buffer solution rinses, glycerol mounting, in fluorescence microscopy
Label effect of the microscopic observation fluorescence nano structural material to cell.Experiments have shown that polypeptide fluorescence nano structural material of the present invention is not
Add any modification that can carry out good label, visible clearly cellular morphology (see figure 8) under burst of ultraviolel to cell.
In conclusion reaction condition of the present invention is mild, operation technological process is simple, and product fluorescence property is stablized, biofacies
Capacitive is good, also shows great advantage in terms of polypeptide sequence design and modification, has extensive biomarker and medicine
Imaging applications prospect.
Finally, it should be noted that the above examples are only used to illustrate the technical scheme of the present invention rather than protects to the present invention
The limitation of range, all local change or modification and derived from technical idea of the invention and be the personnel for being familiar with the field technology
It is easy to deduce, does not depart from patent right range of the invention all.
Claims (9)
1. a kind of novel polypeptide fluorescence nano structural material, which is characterized in that passed through by the polypeptide containing tyrosine and lysine
The mode of oxidation and amino addition is cross-linked to form.
2. a kind of novel polypeptide fluorescence nano structural material according to claim 1, which is characterized in that the polypeptide is packet
Containing tyrosine, lysine, glycine polypeptide.
3. a kind of novel polypeptide fluorescence nano structural material according to claim 2, which is characterized in that the polypeptide includes
Tripeptides, the combining form of the tripeptides include (GYK, GKY, YGK, YKG, KGY or KYG).
4. a kind of novel polypeptide fluorescence nano structural material according to claim 3, which is characterized in that the polypeptide includes
The repetitive sequence that tripeptides is formed as a unit, including GYK, GYKGYK, GYKGYKGYK ... (GYK) n, wherein n is big
In zero integer, i.e. the polypeptide polypeptide that is the sequence containing GYK.
5. a kind of novel polypeptide fluorescence nano structural material as described in claim 1, which is characterized in that the peptide C end can
By carrying out sequence design and functionalized modification to it, selecting identification sequence RGD peptide but being not limited to RGD peptide.
6. a kind of novel polypeptide fluorescence nano structural material as described in claim 1, which is characterized in that the polypeptide fluorescence is received
Rice material surface carries positive charge and is easily attached and modifies identification sequence and carry out total assembling.
7. a kind of preparation method of novel polypeptide fluorescence nano structural material, which comprises the steps of: (1) will fit
The polypeptide of amount sequence containing GYK is dissolved in the alkaline solution that pH is 8, its solution concentration is made to be greater than 1 mg/mL；(2) 0.05 % is added
The oxidant of~0.1% volume, 25~80 DEG C of 2~10h of heating water bath, is cooled to room temperature, and ultrafiltration removes unreacted polypeptide list
Body to get.
8. a kind of preparation method of novel polypeptide fluorescence nano structural material as claimed in claim 7, which is characterized in that (1)
Described in alkaline solution can be selected but be not limited only to phosphate buffer (PBS), borax (sodium tetraborate) buffer, Tris buffering
Liquid, MES buffer or HEPES buffer solution.
9. a kind of preparation method of novel polypeptide fluorescence nano structural material as claimed in claim 7, which is characterized in that (2)
Described in oxidant be hydrogenperoxide steam generator.
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| CN201810880643.8A CN108997476A (en) | 2018-08-04 | 2018-08-04 | A kind of novel polypeptide fluorescence nano structural material and preparation method thereof |
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| CN201810880643.8A CN108997476A (en) | 2018-08-04 | 2018-08-04 | A kind of novel polypeptide fluorescence nano structural material and preparation method thereof |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015057820A2 (en) * | 2013-10-15 | 2015-04-23 | Roberts S Kenny | Peptide constructs and well-defined aggregates thereof |
| CN104749377A (en) * | 2015-03-12 | 2015-07-01 | 浙江大学 | Fluorescent probe with aggregation-induced luminescent property and preparation method and application of fluorescent probe |
| CN105061557A (en) * | 2015-08-18 | 2015-11-18 | 浙江大学 | Polypeptide for DPP-4 detection and fluorescence probe containing polypeptide |
| CN105949322A (en) * | 2016-05-12 | 2016-09-21 | 苏州大学附属第医院 | Biomimetic active peptide suitable for titanium-based medical material modified with one-step method |
| CN109554912A (en) * | 2018-11-23 | 2019-04-02 | 青岛大学 | A kind of preparation method of fluorescence falsification preventing camel wool fiber and products thereof and purposes |
-
2018
- 2018-08-04 CN CN201810880643.8A patent/CN108997476A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015057820A2 (en) * | 2013-10-15 | 2015-04-23 | Roberts S Kenny | Peptide constructs and well-defined aggregates thereof |
| CN104749377A (en) * | 2015-03-12 | 2015-07-01 | 浙江大学 | Fluorescent probe with aggregation-induced luminescent property and preparation method and application of fluorescent probe |
| CN105061557A (en) * | 2015-08-18 | 2015-11-18 | 浙江大学 | Polypeptide for DPP-4 detection and fluorescence probe containing polypeptide |
| CN105949322A (en) * | 2016-05-12 | 2016-09-21 | 苏州大学附属第医院 | Biomimetic active peptide suitable for titanium-based medical material modified with one-step method |
| CN109554912A (en) * | 2018-11-23 | 2019-04-02 | 青岛大学 | A kind of preparation method of fluorescence falsification preventing camel wool fiber and products thereof and purposes |
Non-Patent Citations (5)
| Title |
|---|
| A J GROSS 等: "The oxidation of tyramine, tyrosine, and related compounds by peroxidase", 《J BIOL CHEM》 * |
| CHRISTINE C WINTERBOURN 等: "Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides", 《BIOCHEM J》 * |
| GIDEON OUDGENOEG: "Peroxidase catalyzed conjugation of peptides, proteins and polysaccharides via endogenous and exogenous phenols", 《WAGENINGEN UNIVERSITY》 * |
| J W HEINECKE 等: "Tyrosyl radical generated by myeloperoxidase catalyzes the oxidative cross-linking of proteins", 《J CLIN INVEST》 * |
| RAIJA LANTTO: "Protein cross-linking with oxidative enzymes and transglutaminase: Effects in meat protein systems", 《ESPOO》 * |
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Application publication date: 20181214 |