CN106749517A - A kind of polysaccharide derivates containing polypeptide targeting factor and preparation method thereof - Google Patents

A kind of polysaccharide derivates containing polypeptide targeting factor and preparation method thereof Download PDF

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CN106749517A
CN106749517A CN201611073567.7A CN201611073567A CN106749517A CN 106749517 A CN106749517 A CN 106749517A CN 201611073567 A CN201611073567 A CN 201611073567A CN 106749517 A CN106749517 A CN 106749517A
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polysaccharide
dimapa
polypeptide
targeting factor
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吴燕琳
罗嘉浩
白冰
杨立群
张黎明
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National Sun Yat Sen University
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    • C07—ORGANIC CHEMISTRY
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    • 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
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    • C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

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Abstract

The invention discloses a kind of polysaccharide derivates containing polypeptide targeting factor and preparation method thereof, the polysaccharide derivates be the polysaccharide derivates with polypeptide targeting factor and containing 3 DIMAPA groups as raw material, at room temperature by sulfydryl alkene clicking chemistry react prepare.The preparation method reaction condition of the polysaccharide derivates containing polypeptide targeting factor of the present invention is gentle, avoid using strong acid and strong base reagent, can farthest keep the bioactivity of polypeptide and polysaccharide, and preparation process is simple, easy to operate, required equipment and raw material are cheap.The polysaccharide derivates containing polypeptide targeting factor that the present invention is prepared will can be used as the medicine or genophore for having targeting transport performance to lesions position, thus have certain application prospect in biomedical sector.

Description

A kind of polysaccharide derivates containing polypeptide targeting factor and preparation method thereof
Technical field
The invention belongs to biology medical material technical field, in particular it relates to a kind of polysaccharide containing polypeptide targeting factor spreads out Biology and preparation method thereof.
Background technology
Polysaccharide is the natural polymer that a class is formed by connecting by different sugar units by glycosidic bond, and it is universally present in certainly In right boundary, safety non-toxic, with extraordinary biocompatibility and biological degradability, so, polysaccharide is led in bio-medical material Domain receives much concern, and shows preferable application prospect and researching value.It is poly- including starch Portugal that relatively broad polysaccharide is studied at present Sugar, cellulose, shitosan, sodium alginate, hyaluronic acid etc..Contain hydroxyl, amino, carboxyl isoreactivity functional group on polysaccharide chain, The polysaccharide derivates of different structure and function can be synthesized by chemical reaction, lifting polysaccharide is so better able in bio-medical The application value of Material Field.For example can be used as carrier in medicine and gene delivery field polysaccharide derivates and control curative effect improving Really, can be used as the formation of timbering material promotion organization or organ in field of tissue engineering technology polysaccharide derivates.（Yifen W., et al, Macromolecular Rapid Communications, 2014, 35, 1819−1832）.
In recent years, the cation modifying of polysaccharide causes the extensive research interest of people.Because thin in organism Cellular surface carries negative electrical charge, and the polysaccharide after cationization is in positive charge, can enter cell by electrostatic interaction.According to this Can be transported into cell body for medicine or gene as carrier by one characteristic, cationic polysaccharide, so as to play a part of to treat disease（Such as Patent：A kind of cation hyperbranched polysaccharide derivates and its enhancing blood porphyrin sensitising agent to tumour cell phototoxicity in terms of Using publication number：CN105949344A）.In order to strengthen the targeting transport performance of medicine or genophore to lesions position, generally Targeting factor group is coupled on polysaccharide derivates carrier, is existed by acceptor in targeting factor group and lesion tissue cell special The recognition reaction of the opposite sex, and strengthen targeting transport performance of the polysaccharide derivates to lesions position.For example, being received in cancer cell Folic Acid Body is generally over-expressed, so, the glucan derivative containing the folate-targeted factor can be by folic acid group and cancer cell The specific binding of folacin receptor and embody to cancer cell targeting（Nakamura J., et al, Anticancer Research, 2010, 30, 903−910）.Further, since the albumen that there is overexpression in some lesion tissue cells is received Body, therefore, targeting factor of some polypeptides as these protein receptors can be found, it is coupled on polysaccharide chain, synthesis contains The cationic polysaccharide derivative of polypeptide targeting factor, to obtain more preferable lesions position targeting transport performance.
However, because polypeptide and polysaccharide are the material with bioactivity, and polypeptide and polysaccharide nature difference, synthesis After polysaccharide derivates containing polypeptide targeting factor, the activity of polypeptide and polysaccharide is often destroyed, it is impossible to realize target, because This, seeks a kind of special suitable polysaccharide derivates of the method synthesis containing polypeptide targeting factor so that the work of polypeptide and polysaccharide Property is not destroyed, and has great importance.And correlative study report is there are no in currently available technology.
The content of the invention
The technical problems to be solved by the invention are to overcome the existing polysaccharide derivates synthesis skill containing polypeptide targeting factor The defect and deficiency of art, there is provided a kind of preparation method of the polysaccharide derivates containing polypeptide targeting factor, it is preparation-obtained to contain The polysaccharide derivates of polypeptide targeting factor can be as medicine or gene transmission vector, to strengthen polysaccharide derivates to lesions position Targeting transport performance.
It is an object of the invention to provide a kind of polysaccharide derivates containing polypeptide targeting factor.
It is a further object of the present invention to provide the preparation method of the above-mentioned polysaccharide derivates containing polypeptide targeting factor.
Above-mentioned purpose of the invention is that used following technical scheme gives realization：
A kind of preparation method of the polysaccharide derivates containing polypeptide targeting factor is with polypeptide targeting factor and contains 3- dimethylamino The polysaccharide derivates of propylamine group are raw material, reacted by sulfydryl-alkene clicking chemistry prepare containing polypeptide targeting at room temperature The polysaccharide derivates of the factor.
Preferably, the polypeptide targeting factor is arginine-glycine-aspartic acid polypeptide（RGD）, penetratin （TAT、PTD-4）Or growth hormone release inhibiting hormone（SST-14）.
Preferably, the polysaccharide is glucan, Propiram, glycogen or shitosan.
Preferably, the substitution value of the polypeptide targeting factor group is 0.1~0.5.
Preferably, the substitution value of the 3- DIMAPAs group is 0.5~1.5.
Specifically, the preparation method of the above-mentioned polysaccharide derivates containing polypeptide targeting factor, comprises the following steps：
S1. according to 3- DIMAPA polysaccharide derivatives：Water removal organic solvent=0.1~1.0g：The ratio of 5~20mL, to Water removal organic solvent is added in 3- DIMAPA polysaccharide derivatives, dissolving is stirred at room temperature under inert gas shielding, obtained To 3- DIMAPA polysaccharide derivative solution；
S2. according to 3- DIMAPA polysaccharide derivatives：Weak base catalyst=0.1~1.0g：The ratio of 0.1~1mL, to Weak base catalyst is slowly added dropwise in step S1 resulting solutions, under inert gas shielding, room temperature priming reaction；
S3. to carbon-carbon double bonds group reagent is slowly added dropwise in the solution of step S2, under inert gas shielding, it is stirred at room temperature anti- Should；The carbon-carbon double bonds group reagent is isometric with weak base catalyst；
S4. the reaction solution of step S3 obtains 3- DIMAPAs Quito of carbon-carbon double bonds group by dialysis, freeze-drying Sugar derivatives；
S5. according to the 3- DIMAPA polysaccharide derivatives of carbon-carbon double bonds group：Pure water=0.1~1.0g：5~20mL Ratio, to pure water is added in polysaccharide derivates, dissolving is stirred at room temperature, obtain the 3- DIMAPAs of carbon-carbon double bonds group Polysaccharide derivative solution；
S6. according to the 3- DIMAPA polysaccharide derivatives of carbon-carbon double bonds group：Polypeptide reagent mass ratio containing sulfydryl It is 0.1~1：0.1~1 ratio, is slowly added dropwise the polypeptide reagent solution containing sulfydryl in the solution to step S5, be stirred at room temperature anti- Should；
S7. the reaction solution of step S6 obtains the group of targeting factor containing polypeptide and 3- dimethylamino third by dialysis, freeze-drying The polysaccharide derivates of amine groups.
It is highly preferred that the preparation method of the above-mentioned polysaccharide derivates containing polypeptide targeting factor, specifically includes following steps：
S1. according to 3- DIMAPA polysaccharide derivatives：Water removal organic solvent=0.1~1.0g：5~20mL（It is preferred that 0.1 ~1.0g：5~15mL）Ratio, in 3- DIMAPA polysaccharide derivatives add water removal organic solvent, in indifferent gas Lower 100~the 500r/min of body protection（It is preferred that 200~500r/min）Dissolving in 2~24 hours is stirred at room temperature, 3- dimethylamino is obtained Propylamine polysaccharide derivative solution；
S2. according to 3- DIMAPA polysaccharide derivatives：Weak base catalyst=0.1~1.0g：The ratio of 0.1~1mL, to Weak base catalyst is slowly added dropwise in step S1 resulting solutions, under inert gas shielding, 100~500r/min（200~500r/ min）Room temperature priming reaction 10~60 minutes；
S3. to the carbon-carbon double bonds group reagent isometric with weak base catalyst is slowly added dropwise in the solution of step S2, in inertia Under gas shield, 100~500r/min（It is preferred that 200~500r/min）Reaction is stirred at room temperature 1~24 hour；
S4. the reaction solution of step S3 is dialysed 1~5 day with water, and freeze-drying obtains the 3- dimethylamino third of carbon-carbon double bonds group Aminated polysaccharide derivative.
S5. according to the 3- DIMAPA polysaccharide derivatives of carbon-carbon double bonds group：Pure water=0.1~1.0g：5~ 20mL（It is preferred that 0.1~0.5g：5~15mL）Ratio, to adding pure water, 100~500r/min in polysaccharide derivates（It is preferred that 200~500r/min）Dissolving in 1~24 hour is stirred at room temperature, 3- DIMAPAs Quito sugar of carbon-carbon double bonds group is obtained Derivative solution；
S6. according to the 3- DIMAPA polysaccharide derivatives of carbon-carbon double bonds group：Polypeptide reagent mass ratio containing sulfydryl It is 0.1~0.5：0.1~1 ratio, is slowly added dropwise the polypeptide reagent solution containing sulfydryl in the solution to step S5,100~ 500r/min（It is preferred that 200~500r/min）Reaction is stirred at room temperature 1~24 hour；The group of the polypeptide reagent solution containing sulfydryl It is divided into the polypeptide reagent containing sulfydryl：Pure water=0.1~0.5g：1~10mL；
S7. the reaction solution of step S6 is dialysed 1~5 day with water, and freeze-drying obtains the group of targeting factor containing polypeptide and 3- diformazans The polysaccharide derivates of amido propylamine group.
Preferably, 3- DIMAPAs polysaccharide derivative described in step S1 is the Portugal of the group of DIMAPA containing 3- Polysaccharid derivative, the Propiram derivative of the group of DIMAPA containing 3-, the glycogen of the group of DIMAPA containing 3- derive The chitosan derivatives of thing, the group of DIMAPA containing 3-, its preparation method can refer to patent（It is a kind of cation hyperbranched many Sugar derivatives and its enhancing blood porphyrin sensitising agent to the application in terms of tumour cell phototoxicity, publication number： CN105949344A）.
Preferably, it is water removal dimethylformamide, water removal ethyl acetate, water removal dichloro that organic reagent is removed water described in step S1 Methane or water removal dimethyl sulfoxide (DMSO).
Preferably, step S1, S2 or inert gas described in S3 are nitrogen, argon gas or helium.
It is highly preferred that step S1, S2 or S3 simultaneous selections nitrogen, argon gas or helium.
Preferably, weak base catalyst described in step S2 is pyridine, piperidines, triethylamine or sodium acetate.
Preferably, carbon-carbon double bonds group reagent described in step S3 is acrylic acid, acrylic anhydride, methacrylic acid, methyl Acrylic acid acid anhydrides or methacrylic chloride.
Preferably, polypeptide reagent described in step S6 is arginine-glycine-aspartic acid polypeptide（RGD）, cell membrane penetrates Peptide（TAT or PTD-4）Or growth hormone release inhibiting hormone（SST-14）.
In addition, the polysaccharide derivates containing polypeptide targeting factor that the above method is prepared are also in protection scope of the present invention Within.
Application of the above-mentioned polysaccharide derivates containing polypeptide targeting factor on as medicine or gene transmission vector, Yi Ji The application on medicine or gene transmission vector is prepared, also all within protection scope of the present invention.
Preferably, the medicine or gene transmission vector can target transport toward lesions position.
Polypeptide and polysaccharide in the present invention are the materials with BA, it is necessary to anti-under the conditions of comparatively gentle The loss of activity that will not just should make.Therefore the present invention is from sulfydryl-alkene clicking chemistry method polysaccharide of the synthesis containing polypeptide targeting factor Derivative, the synthetic method does not use metallic catalyst, and reaction condition is gentle（Such as room temperature）, can farthest keep polypeptide and many The bioactivity of sugar, the preparation-obtained polysaccharide derivates containing polypeptide targeting factor can be used as medicine or the transmission of gene is carried Body, to improve the effect for lesions position magnetic target therapy disease.
Compared with prior art, the invention has the advantages that：
（1）Process is simple of the invention, easy to operate, required equipment and raw material are cheap.
（2）Chemical reaction of the present invention is simple, can be reacted in room temperature, it is to avoid use strong acid and strong base reagent, gently Reaction condition can ensure that the bioactivity of polysaccharide and polypeptide in product is unaffected.
（3）The polysaccharide derivates containing polypeptide targeting factor that the present invention is prepared can be used as medicine or genophore, have Beneficial to medicine or gene target arrival lesion tissue is carried, to reduce the toxic and side effect to normal cell.
Brief description of the drawings
Fig. 1 is the process chart that the present invention prepares the polysaccharide derivates containing polypeptide targeting factor.
Fig. 2 is the synthetic route chart of polysaccharide derivates of the present invention containing polypeptide targeting factor.
Fig. 3 is 3- DIMAPAs base glycogen of the present invention（DMAPA-Gly）And product RGD containing peptides targeting factor group Glycogen derivative（RGD-DMAPA-Gly）Infrared spectrogram（FTIR）.
Fig. 4 is 3- DIMAPAs base glycogen of the present invention（DMAPA-Gly）And product RGD containing peptides targeting factor group Glycogen derivative（RGD-DMAPA-Gly）Nuclear magnetic spectrogram（1H NMR）.
Fig. 5 be fluorescence marker groups containing FITC, TAT peptide groups and the targeting factor group of polypeptide containing FITC-TAT and The glucan derivative of 3- DIMAPA groups（FITC-TAT-DMAPA-DEX）Targeting enters bone marrow interstital stem cell Copolymerization Jiao's photo（20 μm of scale）：（a）It is distribution of the FITC-TAT-DMAPA-DEX derivatives in bone marrow interstital stem cell, （b）Wie passes through the fluorometric reagents of Hoechst 33258 by the bone marrow interstital stem cell of nuclei dyeing au bleu,（c）It is by Lyso Tracker Red DND-99 fluorometric reagents are dyed the bone marrow interstital stem cell of red by the organelles such as cell membrane, lysosome,（d） For（a）、（b）With（c）Merging photo.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited In this.Unless stated otherwise, reagent involved in embodiment, method are reagent and method commonly used in the art.
Embodiment 1
1. preparation method
The present invention prepares the process chart and synthetic route chart of the polysaccharide derivates containing polypeptide targeting factor respectively such as Fig. 1 and Tu Shown in 2.
Specific preparation method is as follows：
（1）Weigh 0.1g 3- DIMAPA base glycogen derivatives（DMAPA-Gly）Add into dry flask, add 10mL removes water dimethylformamide, and 200r/min is stirred at room temperature dissolving in 12 hours under nitrogen protection.
（2）0.5 mL pyridines to step are slowly added dropwise with syringe（1）In resulting solution, 200r/min is stirred at room temperature 10 points Clock, is then slowly added dropwise 0.5 mL acrylic acid with syringe, and under nitrogen protection, 200r/min is stirred at room temperature reaction 24 hours.
（3）Deionized water hemodialysis reaction liquid 1 day, freeze-drying obtains the 3- DIMAPA bases containing acrylic acid groups Glycogen derivative（MAA-DMAPA-Gly）.
（4）3- DIMAPA base glycogen derivatives of the 0.5g containing acrylic acid groups is weighed to add to dry flask In, add 5mL pure water, 200r/min that dissolving in 24 hours is stirred at room temperature.
（5）Weigh 0.5g arginine-glycine-aspartic acids（RGD）Polypeptide is added into drying flask, adds 1 mL Pure water dissolves, to step（4）Solution in be slowly added dropwise RGD solution 1 mL, 200r/min is stirred at room temperature reaction 8 hours；
（6）Deionized water hemodialysis reaction liquid 1 day, freeze-drying obtains RGD containing peptides targeting factor group and 3- dimethylamino The glycogen derivative of propylamine group（RGD-DMAPA-Gly）.
2. result
（1）Fig. 3 is 3- DIMAPA base glycogen raw materials（DMAPA-Gly）And the FTIR spectrograms of derivative, can from Fig. 3 b Find out and be coupled acrylic acid 3- DIMAPAs base glycogen derivative（MAA-DMAPA-Gly）In 1630cm-1Occur in that new Absworption peak, is attributed to the stretching vibration peak of carbon-carbon double bond, shows that acrylic acid is coupled with 3- DIMAPA base glycogens, together When 1709 cm-1Locate the carbonyl of carbamate（C=O）Characteristic peak is also present, and shows the 3- DIMAPA groups of derivative Property and uninfluenced.
（2）Fig. 4 is 3- DIMAPA base glycogen raw materials（DMAPA-Gly）And derivative1H NMR spectras, with 3- DIMAPA base glycogens1H NMR spectras（Fig. 4 a）Compare, MAA-DMAPA-Gly derivatives1H NMR spectras（Figure 4b）In occur in that 2 characteristic resonances peaks of acrylic acid groups：（e）Peak（5.6 ppm）、（e’）Peak（6.1 ppm）, further demonstrate,prove Real acrylic acid is coupled with 3- DIMAPA base glycogens, synthesizes MAA-DMAPA-Gly derivatives.
（3）Pass through1The Proton integration method of H H NMR spectroscopies, i.e., respectively to different head in 3- DIMAPA base glycogen derivatives H1（5.3-4.8 ppm）Formant and 3- DIMAPA group methylenes proton [（a）Peak] it is integrated, according to formulaThe substitution value DS of 3- DIMAPA groups in DMAPA-Gly derivatives can be calculated1（It is coupled i.e. in each sugar unit Ligand groups number）.By elemental microanalysis method, the weight ratio of N/S in RGD-DMAPA-Gly is drawn, according to formulaCan calculate Go out the substitution value DS of rgd peptide group in RGD-DMAPA-Gly derivatives2。
In formula,N(DMAPA)It is the number of 3- DIMAPA groups in DMAPA-Glyp derivatives,N(Glc)It is The number of glucose unit in DMAPA-Glyp derivatives,I(>CH2)It is 3- DIMAPA group methylene protons The integral area at [(a) peak],I(H1)It is the integral area of different head H1 formants in DMAPA-Glyp derivatives.
In formulaN/SIt is the weight ratio of nitrogen and element sulphur in RGD-DMAPA-Gly,M N WithM S Respectively nitrogen and sulphur two The atomic mass of element is planted,DS 1 WithDS 2 Respectively 3- DIMAPAs group and polypeptide targeting factor substitution value.
The substitution value of 3- DIMAPA groups is 1.7, RGD in the present embodiment gained RGD-DMAPA-Gly derivatives The substitution value of polypeptide is 0.1.
Embodiment 2
1. preparation method
（1）Weigh 0.4g 3- DIMAPA base enclosure polysaccharid derivatives to add into dry flask, add 5mL water removals two Chloromethanes, under nitrogen protection 300r/min dissolving in 24 hours is stirred at room temperature.
（2）0.1 mL triethylamines to step are slowly added dropwise with syringe（1）In resulting solution, 300r/min is stirred at room temperature 20 Minute, 0.1 mL acrylic anhydrides then are slowly added dropwise with syringe, under nitrogen protection, it is small that 300r/min is stirred at room temperature reaction 1 When.
（3）Deionized water hemodialysis reaction liquid 2 days, freeze-drying obtains the 3- DIMAPA bases containing acrylic acid groups Chitosan derivatives.
（4）3- DIMAPA base enclosure polysaccharid derivatives of the 0.1g containing acrylic acid groups is weighed to add to dry flask In, add 10mL pure water, 300r/min that dissolving in 1 hour is stirred at room temperature.
（5）Weigh 0.2g penetratins（TAT）Add into drying flask, the dissolving of 2 mL pure water is added, to step （4）Solution in be slowly added dropwise TAT solution 2 mL, 300r/min is stirred at room temperature reaction 12 hours；
（6）Deionized water hemodialysis reaction liquid 2 days, freeze-drying obtains the targeting factor group of polypeptide containing TAT and 3- dimethylamino The chitosan derivatives of propylamine group.
2. result
It is demonstrated experimentally that the present embodiment successfully synthesizes the targeting factor group of polypeptide containing TAT and 3- DIMAPA groups Chitosan derivatives, the substitution value of 3- DIMAPA groups is in the present embodiment gained TAT-DMAPA-CHI derivatives The substitution value of 1.0, TAT polypeptides is 0.2.
Embodiment 3
1. preparation method
（1）Weigh 1g 3- DIMAPA base Propiram derivatives to add into dry flask, add 10mL water removal diformazans Base formamide, 500r/min is stirred at room temperature dissolving in 2 hours under argon gas protection.
（2）1.0 mL pyridines to step are slowly added dropwise with syringe（1）In resulting solution, 500r/min is stirred at room temperature 30 points Clock, is then slowly added dropwise 1.0 mL methacrylic acids with syringe, and under argon gas protection, it is small that 500r/min is stirred at room temperature reaction 12 When.
（3）Deionized water hemodialysis reaction liquid 3 days, freeze-drying obtains the 3- dimethylamino third containing methacrylic acid group Amido Propiram derivative.
（4）3- DIMAPA base Propiram derivatives of the 0.2g containing methacrylic acid group is weighed to add to dry In flask, add 15mL pure water, 500r/min that dissolving in 12 hours is stirred at room temperature.
（5）Weigh 1.0g penetratins（PTD-4）Add into drying flask, add the dissolving of 2 mL pure water, Xiang Bu Suddenly（4）Solution in be slowly added dropwise PTD-4 solution 2 mL, 500r/min is stirred at room temperature reaction 1 hour；
（6）Deionized water hemodialysis reaction liquid 3 days, freeze-drying obtains the targeting factor group of polypeptide containing PTD-4 and 3- dimethylamine The Propiram derivative of base propylamine group.
2. result
It is demonstrated experimentally that the present embodiment successfully synthesizes the targeting factor group of polypeptide containing PTD-4 and 3- DIMAPA groups Propiram derivative, the present embodiment gained PTD-4-DMAPA-Pul derivatives in 3- DIMAPA groups substitution value The substitution value for being 1.5, PTD-4 polypeptides is 0.1.
Embodiment 4
1. preparation method
（1）Weigh 1g 3- DIMAPA base glucan derivatives to add into dry flask, add 15mL water removal dichloros Methane, 200r/min is stirred at room temperature dissolving in 10 hours under helium protection.
（2）0.5mL sodium acetates to step are slowly added dropwise with syringe（1）In resulting solution, 200r/min is stirred at room temperature 50 Minute, 0.5 mL methacrylic chlorides then are slowly added dropwise with syringe, under helium protection, 200r/min is stirred at room temperature reaction 12 hours.
（3）Deionized water hemodialysis reaction liquid 5 days, freeze-drying obtains the 3- dimethylamino third containing methacrylic acid group Amido glucan derivative.
（4）3- DIMAPA base glucan derivatives of the 0.5g containing methacrylamide group is weighed to add to drying Flask in, add 5mL pure water, 200r/min is stirred at room temperature dissolving in 10 hours.
（5）Weigh 0.1g growth hormone release inhibiting hormones（SST-14）Add into drying flask, the dissolving of 10 mL pure water is added, to step （4）Solution in be slowly added dropwise SST-14 solution 10 mL, 200r/min is stirred at room temperature reaction 24 hours；
（6）Deionized water hemodialysis reaction liquid 5 days, freeze-drying obtains the targeting factor group of polypeptide containing SST-14 and 3- diformazans The glucan derivative of amido propylamine group.
2. result
It is demonstrated experimentally that the present embodiment successfully synthesizes the targeting factor group of polypeptide containing SST-14 and 3- DIMAPA groups Glucan derivative, the present embodiment gained SST-14-DMAPA-DEX derivatives in 3- DIMAPA groups substitution value The substitution value for being 1.2, SST-14 polypeptides is 0.1.
Embodiment 5
1. preparation method
（1）Weigh 0.2g 3- DIMAPA base Propiram derivatives to add into dry flask, add 5mL water removal second Acetoacetic ester, 400r/min is stirred at room temperature dissolving in 12 hours under helium protection.
（2）1.0 mL triethylamines to step are slowly added dropwise with syringe（1）In resulting solution, 400r/min is stirred at room temperature 60 Minute, 1.0 mL acrylic acid then are slowly added dropwise with syringe, under helium protection, it is small that 400r/min is stirred at room temperature reaction 24 When.
（3）Deionized water hemodialysis reaction liquid 5 days, freeze-drying obtains the 3- DIMAPA bases containing acrylic acid groups Propiram derivative.
（4）3- DIMAPA base Propiram derivatives of the 0.2g containing acrylic acid groups is weighed to add to dry flask In, add 10mL pure water, 400r/min that dissolving in 8 hours is stirred at room temperature.
（5）Weigh 0.8 g PTD-4 to add into drying flask, the dissolving of 5 mL pure water is added, to step（4）Solution in It is slowly added dropwise PTD-4 solution 5 mL, 400r/min and reaction 12 hours is stirred at room temperature；
（6）Deionized water hemodialysis reaction liquid 5 days, freeze-drying obtains the targeting factor group of polypeptide containing PTD-4 and 3- dimethylamine The Propiram derivative of base propylamine group.
2. result
It is demonstrated experimentally that the present embodiment successfully synthesizes the targeting factor group of polypeptide containing PTD-4 and 3- DIMAPA groups Propiram derivative, the present embodiment gained PTD-4-DMAPA-Pul derivatives in 3- DIMAPA groups substitution value The substitution value for being 1.0, PTD-4 polypeptides is 0.5.
Embodiment 6
1. preparation method
（1）Weigh 0.2g 3- DIMAPA base glucan derivatives to add into dry flask, add 5mL water removal second Acetoacetic ester, 400r/min is stirred at room temperature dissolving in 12 hours under helium protection.
（2）1.0 mL triethylamines to step are slowly added dropwise with syringe（1）In resulting solution, 400r/min is stirred at room temperature 60 Minute, 1.0 mL acrylic acid then are slowly added dropwise with syringe, under helium protection, it is small that 400r/min is stirred at room temperature reaction 24 When.
（3）Deionized water hemodialysis reaction liquid 5 days, freeze-drying obtains the 3- DIMAPA bases containing acrylic acid groups Glucan derivative.
（4）3- DIMAPA base glucan derivatives of the 0.2g containing acrylic acid groups is weighed to add to dry flask In, add 10mL pure water, 400r/min that dissolving in 8 hours is stirred at room temperature.
（5）Weigh 1.0 g FITC-TAT to add into drying flask, the dissolving of 5 mL pure water is added, to step（4）It is molten FITC-TAT solution 5 mL, 400r/min are slowly added dropwise in liquid reaction 12 hours is stirred at room temperature；
（6）Deionized water hemodialysis reaction liquid 5 days, freeze-drying obtains the targeting factor group of polypeptide containing FITC-TAT and 3- bis- The glucan derivative of methylamino propylamine group.
2. result
It is demonstrated experimentally that the present embodiment successfully synthesizes the targeting factor group of polypeptide containing FITC-TAT and 3- DIMAPA bases The glucan derivative of group, the substitution value of 3- DIMAPA groups in the present embodiment gained TAT-DMAPA-DEX derivatives The substitution value for being 2.0, TAT polypeptides is 0.1.
The application of the polysaccharide derivates of targeting factor containing polypeptide of embodiment 7
By verification experimental verification, the polysaccharide derivates of targeting factor containing polypeptide prepared by the method for the present invention can be protected farthest The bioactivity of polypeptide and polysaccharide is held, can be used as the medicine or genophore that there is targeting transport performance to lesions position.
The targeting factor group of polypeptide containing FITC-TAT and 3- DIMAPA groups for being prepared with embodiment 6 below Glucan derivative（FITC-TAT-DMAPA-DEX）As a example by, experimental data is presented as follows：
Fig. 5 is the glucan derivative of the targeting factor group of polypeptide containing FITC-TAT and 3- DIMAPA groups（FITC- TAT-DMAPA-DEX）Into copolymerization Jiao's photo of bone marrow interstital stem cell.Can be observed the derivative have been enter into bone marrow interstital do Cell, is mainly distributed in cell membrane, cytoplasm and nuclear membrane, shows that the derivative has to bone marrow interstital stem cell preferable Targeting, this is relevant with the performance that tat peptide can penetrate bone marrow interstital stem cell film.
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment Limitation, it is other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

Claims (10)

1. a kind of preparation method of the polysaccharide derivates containing polypeptide targeting factor, it is characterised in that be with polypeptide targeting factor and Polysaccharide derivates containing 3- DIMAPA groups are raw material, reacted by sulfydryl-alkene clicking chemistry prepare at room temperature Obtain the polysaccharide derivates containing polypeptide targeting factor.
2. preparation method according to claim 1, it is characterised in that the polypeptide targeting factor be arginine-glycine- Aspartic acid polypeptide, penetratin or growth hormone release inhibiting hormone.
3. preparation method according to claim 1, it is characterised in that the polysaccharide is glucan, Propiram, glycogen or shell Glycan.
4. preparation method according to claim 1, it is characterised in that the substitution value of the polypeptide targeting factor group is The substitution value of 0.1~0.5,3- DIMAPA group is 0.5~1.5.
5. preparation method according to claim 1, it is characterised in that comprise the following steps：
S1. according to 3- DIMAPA polysaccharide derivatives：Water removal organic solvent=0.1~1.0g：The ratio of 5~20mL, to Water removal organic solvent is added in 3- DIMAPA polysaccharide derivatives, dissolving is stirred at room temperature under inert gas shielding, obtained To 3- DIMAPA polysaccharide derivative solution；
S2. according to 3- DIMAPA polysaccharide derivatives：Weak base catalyst=0.1~1.0g：The ratio of 0.1~1mL, to Weak base catalyst is slowly added dropwise in step S1 resulting solutions, under inert gas shielding, room temperature priming reaction；
S3. to carbon-carbon double bonds group reagent is slowly added dropwise in the solution of step S2, under inert gas shielding, it is stirred at room temperature anti- Should；The carbon-carbon double bonds group reagent is isometric with weak base catalyst；
S4. the reaction solution of step S3 obtains 3- DIMAPAs Quito of carbon-carbon double bonds group by dialysis, freeze-drying Sugar derivatives；
S5. according to the 3- DIMAPA polysaccharide derivatives of carbon-carbon double bonds group：Pure water=0.1~1.0g：5~20mL Ratio, to pure water is added in polysaccharide derivates, dissolving is stirred at room temperature, obtain the 3- DIMAPAs of carbon-carbon double bonds group Polysaccharide derivative solution；
S6. according to the 3- DIMAPA polysaccharide derivatives of carbon-carbon double bonds group：Polypeptide reagent mass ratio containing sulfydryl It is 0.1~1：0.1~1 ratio, is slowly added dropwise the polypeptide reagent solution containing sulfydryl in the solution to step S5, be stirred at room temperature anti- Should；
S7. the reaction solution of step S6 obtains the group of targeting factor containing polypeptide and 3- dimethylamino third by dialysis, freeze-drying The polysaccharide derivates of amine groups.
6. preparation method according to claim 5, it is characterised in that it is water removal diformazan that organic reagent is removed water described in step S1 Base formamide, water removal ethyl acetate, water removal dichloromethane or water removal dimethyl sulfoxide (DMSO).
7. preparation method according to claim 5, it is characterised in that weak base catalyst described in step S2 be pyridine, piperidines, Triethylamine or sodium acetate.
8. preparation method according to claim 5, it is characterised in that carbon-carbon double bonds group reagent described in step S3 is third Olefin(e) acid, acrylic anhydride, methacrylic acid, methacrylic acid acid anhydrides or methacrylic chloride；Polypeptide reagent described in step S6 is essence Propylhomoserin-Gly-Asp polypeptide, penetratin or growth hormone release inhibiting hormone.
9. the polysaccharide derivates containing polypeptide targeting factor that any methods described of claim 1~8 is prepared.
10. the polysaccharide derivates containing polypeptide targeting factor described in claim 9 as or prepare medicine or gene transmission vector On application.
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