CN101475647A - Preparation and use of double branching polysialic acid active derivative - Google Patents

Abstract

The invention belongs to the technical field of high molecular materials, and in particular relates to a new polysialic acid active derivative with a double bond structure and a preparation method thereof and application of the derivative to the medicine preparation. The derivative can be widely applied to the modification of amino-contained biological functional molecules, in particular to macromolecules such as proteins and polypeptides, and the improvement of medicinal solubility, stability and immunogenicity, thereby prolonging the half life of the medicines and improving the efficacy.

Description

The preparation method of double branching polysialic acid active derivative and application

Technical field:

The invention belongs to technical field of polymer materials, be specifically related to a kind of polysialic acid active derivative and preparation method thereof and application in medication preparation of duplex structure.Described other molecule is macromole such as protein and polypeptide particularly, the invention still further relates to the pharmaceutical composition that comprises described binding substances.

Background technology:

At present, medicine of a great variety divided with regard to its molecular weight size, and generally can be divided into two big classes: the molecule amount is lower, usually below 1000, the overwhelming majority is common chemical synthetic drugs and some natural drugs, as mustargen, cis-platinum, 5 FU 5 fluorouracil, taxol etc. all belong to these row; Another kind of then molecular weight is bigger, most protein and polypeptide drugs of being produced by genetically engineered.But no matter small-molecule drug or macromolecular drug, shortcoming such as it is big all to exist toxicity, poorly soluble, and the transformation period is short.Also there are immunogenic problem in protein and polypeptide drugs.

The protein chemistry modification technique is fast-developing in recent years emerging technology.Polypeptide, the protein drug that utilizes gene engineering method to produce in clinical application, often exist in poor stability, active on the low side, the body transformation period short, cause problem such as immune response, can effectively improve above-mentioned weak point by chemically modified, simultaneously, the protein chemistry modification also is an important means of carrying out the protein structure functional study.

Proteinic chemically modified is meant by reacting with the active group as modifier and protein (polypeptide) such as some material such as polyoxyethylene glycol (PEG), dextran, heparin, improve protein properties thereby reach, as the purpose of removing foreign protein immunogenicity, toxic side effect and increasing the transformation period in activity, raising stability, the extension body.The polymkeric substance binding medicine that will have biocompatibility is an approach of dealing with problems.Polysialic acid (Polysialic acid) is the straight chain homopolymer of sialic acid (Sialic acid) monomer with α-2,8 or the connection of α-2,9 key, is the integral part of glycoprotein in some mammalian cells and the exocellular polysaccharide component of a few bacterium.α-2, the PSA of 8 types of attachment is unique non-immunogenic (not causing T cell or antibody response in the mammalian subject) in bacterial polysaccharides, even puting together when being connected with immunogenic carrier albumen also is that so this can show as it and exist as Mammals (and bacterium) polymkeric substance.In being distributed widely in intravital cell surface Sphingolipids,sialo, found short polymkeric substance (being up to 4), and thought that this shorter polymkeric substance can cause and keep the immunotolerance to PSA effectively.Utilize the biological characteristics of the homopolymerization PSA that the biological characteristics, particularly α-2,8 of PSA connect in the last few years, changed the pharmacokinetics character of protein and low-molecular-weight drug molecule.The PSA derivative that comprises some human cytokines of catalase, asparaginase and Regular Insulin makes circulating half-life and stability thereof increase substantially, and makes these albumen can be used for tackling preexisting antibody.Aspect a lot, the improved character of Polysialic acid protein is with suitable with polyoxyethylene glycol (PEG) deutero-protein.For example all improved the transformation period, and made protein and its more stable in proteolytic digestion, but as if bioactive confining force is stronger than PEG when adopting PSA.Use PEG also to have problems when employing needs the therapeutical agent of long term administration, because PEG biological degradation very lentamente, and high molecular form and lower molecular weight form all are easy to accumulate in tissue.Find that the albumen mass-energy of PEGization produces the antibody of anti-PEG, the antibody of this anti-PEG also can influence the residence time of conjugate in blood circulation.Although PEG has certain history as puting together the administered parenterally polymkeric substance that is connected with therapeutant, but still needs carry out more deep understanding to its immunotoxicology, pharmacology and metabolism.Equally also the application (therefore finally causing heavy dose of PEG) in the heavy dose of therapeutical agent that uses of needs is worried to some extent to PEG, but because the accumulation toxigenicity of PEG.The degradable controlled release agent that Polysialic acid is applied to protein and polypeptide drug has obtained good result.Being expected to becomes medicine construct of new generation and significantly improves polypeptide, proteic pharmaceutical property.Have vital role and function protein drug the Polysialic acid modification the field of biological pharmacy in the whole world cause increasing concern: the LipoXen company clinical trial of Britain the Polysialic acid Interferon, rabbit, find that its effect is longer than the transformation period of the Interferon, rabbit of PEGization; Other drug such as Regular Insulin, asparaginase and HRBC growth hormone etc. are also being tested by the said firm in addition.

At US05846951 and WO0187922 the pharmacokinetic property that utilizes natural PSA to improve the protein therapeutic agent has been described.Having described rely on the polymkeric substance non-reducing end chemically derived produces the active aldehyde radical of proteins react polysaccharide is connected to method on the therapeutical agent.In WO2005016973, the polysaccharide derivates of being introduced the sulfydryl reaction active groups by terminal sialic acid unit has been described.Product can be used for proteinic location and derives.Disclose a kind of new PSA derivative at CN101160326, wherein the terminal sialic acid of reduction and/or non-reducing end is converted into N-hydroxy-succinamide ester (NHS) gene.Can with contain amido or diazanyl the group substrate reactions.

Although the various methods of having described that PSA is connected on the healing potion are feasible in theory, but for the conjugate that the reaction of albumen and PSA non-reducing end (form of aldehyde) is generated reaches acceptable amount, the desired reaction times will be unfavorable for proteinic stability under comparatively high temps.Secondly, required reactant concn may be difficult to realize or be economical inadequately.

As everyone knows, structure, molecular weight, molecular weight distribution and the high molecular configuration of performance behind the medicine high molecular and used macromolecular material are relevant.Same structure, the polymer of different molecular weight can produce different character behind modified medicaments; Same structure, the same molecular amount, but the different polymer of configuration also can produce very big difference behind modified medicaments.What use when protein or other medicines are carried out Polysialic acid at present all is the strand Polysialic acid, and several problems of existence make the advantage of binding substances of prepared Polysialic acid and protein or medicine be difficult to realization.The combination of first PSA reduces the binding substances biological activity of protein or medicine.In addition, the part hydrolytic cleavage can take place in PSA when forming binding substances, and binding substances has been lost by the advantage of bringing in conjunction with PSA.It is found that under study for action PSA is relevant to the molecular weight of the modification effect of biologically active substance and the molecule of employed PSA and specific modification degree.In general, the molecular weight of used PSA is big more, and modification degree is high more, and the antigenicity of PSA-protein conjugate is more little, and the eliminating time is long more.In order to address this problem, we have synthesized double-stranded Polysialic acid.

Summary of the invention:

The purpose of this invention is to provide a kind of polysialic acid active derivative and preparation method thereof and the application in medicine of active duplex structure.Compare with the line style PSA of same molecular amount, the static viscosity of the double-stranded PSA of the type is little, and hydrodynamic volume is big, can be to more efficiently physiological actions of generation such as pharmaceutical grade proteins.

The active double-stranded PSA that the present invention proposes is to be combined and obtain through chemical reaction and PSA by the trifunctional micromolecular compound.This active double-stranded PSA can be designated as (R₂-PSA)₂-X-F.Wherein, R₂Be hydrogen or a kind of single, double, oligomerization or polysialic acids group, a kind of albumen, a kind of peptide, a kind of lipid, a kind of medicine, a kind of cytolemma or cell-wall component or a kind of drug delivery system; 2 represent two strands; The X tie point is the trifunctional micromolecular compound; F represents activity functional groups; PSA is connected with covalent linkage with the trifunctional micromolecular compound, and linking group is selected from amino, amide group, inferior amide group, carbamate, ester group, epoxy group(ing), carboxyl, hydroxyl, sulfydryl or carbohydrate, or several combinations a kind of wherein; The structural formula of used trifunctional micromolecular compound is following a kind of:

Here, n is the integer of 1-9, and m is the integer of 0-6;

For example wherein trifunctional micromolecular compound X is H₂N (CH₂) nCH (NH₂) CO₂H (n is the integer of 1-9), when linking group is amide group or carbamate, its structural formula such as following general formula:

Wherein A is NR₃, NR₃NR₄, O or S, wherein R₃And R₄Be independently selected from H, C_1-4Alkyl and aryl;

SylO is a sialic acids groups;

N is 1-9; Q is 1-100; P is 0 or 1;

W is one of O, S or NH;

F is one of following functional group:

H，OH，NH₂，

R₁Be linking group;

R₂Be hydrogen or a kind of single, double, oligomerization or polysialic acids group, a kind of albumen, a kind of peptide, a kind of lipid, a kind of medicine, a kind of cytolemma or cell-wall component or a kind of drug delivery system;

The preparation method of the polysialic acid active derivative of above-mentioned duplex structure is as once: earlier by active Polysialic acid and the reaction of trifunctional micromolecular compound, and then with the compound reaction that has active group F, promptly obtain the polysialic acid active derivative of active duplex structure.

Among the present invention, the active group of described trifunctional micromolecular compound (X) is carboxyl and two amino, perhaps is each two hydroxyl of carboxyl.Be specifically as follows a kind of of aforementioned structural formula.

The synthetic method of the active PSA of strand is existing in the art to be described, for example, and at US05846951, WO0187922, WO2005016973 and CN101160326.In the present invention, these all are introduced into as a reference.

The preparation method of double-stranded PSA is similar, when X is Methionin (Lysine), two chain PSA are the active CA-NHS class of non-reducing end and reducing end deutero-, W is NH, n is 4, P is 1, when F is NHS, can prepare polysialic acid active derivative (I the type) (R that active end group is the different duplex structures of NHS by following reaction scheme₂-PSA)₂-Lys-NHS. wherein active the PSA of used strand can prepare according to methods such as above-mentioned US05846951, WO0187922, WO2005016973, CN101160326.

Polysialic acid active derivative (II the type) (R of different duplex structures₂-PSA)₂-Lys-NHS.Can use method for preparing equally, as long as change the order that adds active strand Polysialic acid.

The preparation method of identical double-stranded PSA is similar, if with above-mentioned two strands change into identical PSA reactive derivative get final product the PSA reactive derivative of identical two strands.The PSA reactive derivative of identical two strands also can prepare by following method: when X is Methionin (Lysine), active PSA is a non-reducing end deutero-CA-NHS class, W is NH, n is 4, P is 1, when F is NHS, can prepare the polysialic acid active derivative (R that active end group is the NHS duplex structure by following reaction scheme₂-PSA)₂-Lys-NHS, wherein the active PSA of used strand can prepare according to methods such as above-mentioned US05846951, WO0187922, WO2005016973 and CN101160326.

When active PSA is non-reducing end deutero-CA-DSG, X is Methionin (Lysine), and W is NH, and n is 4, and P is 1, and the preparation method was as follows when F was succinimide:

When X is Methionin (Lysine), active PSA is a reducing end deutero-CA-NHS class, and W is NH, and n is 4, and P is 1, when F is NHS, can prepare the polysialic acid active derivative (R that active end group is the NHS duplex structure by following reaction scheme₂-PSA)₂-Lys-NHS, wherein the active PSA of used strand can prepare according to above-mentioned CN 101160326 methods.

When X is Methionin (Lysine), active PSA is reducing end deutero-CA-BS³, W is NH, n is 4, when F is NHS, can prepare the polysialic acid active derivative that active end group is the NHS duplex structure (CA) by following reaction scheme₂-Lys-NHS, the wherein active CA-BS of used strand³Can prepare according to above-mentioned CN101160326 method.

When X is Methionin (Lysine), active PSA is non-reducing end deutero-CA-CHO, and W is NH, and n is 4, and P is 0, when F is NHS, can prepare the polysialic acid active derivative that active end group is the NHS duplex structure (CA) by following reaction scheme₂-Lys-NHS. wherein active the CA-CHO of used strand can prepare according to above-mentioned CN 101160326 methods.

Come activated carboxyl by the N-hydroxy-succinamide ester in the above-mentioned route, also can come carboxyl is activated, as the p-nitrophenyl phenolic ester etc. with the method that generates other active ester.

Obviously,, only need in preparation process, select different active PSA as required for use, just can prepare above-mentioned I, II, III, the active various length of IV formula, double-stranded PSA that combination is different with comparalive ease according to top route.In conjunction with of the present invention open, point is conspicuous for the person skilled in the art of this area on this.

The double-stranded PSA of activity provided by the invention can be used for the preparation of medicine.Above-mentioned reactive derivative forms binding substances by F group and other molecule.The polysialic acid active derivative of above-mentioned duplex structure can combine with the molecule with free amino group at the physiological condition of gentleness, as being widely used in the modification of small-molecule drug, protein and polypeptide drugs, i.e. the bioactive molecules that contains primary amine groups of activity functional groups F and small-molecule drug, polypeptide or pharmaceutical grade protein etc. by two strands.Small-molecule drug is preferred but be not limited to: Chlorambucil, cis-platinum, 5-fluor-uracil, taxol, Zorubicin or Rheumatrex.Described pharmaceutical grade protein is preferred but be not limited to: Interferon, rabbit, interleukin, tumour necrosis factor, somatomedin, G CFS, erythropoietin or superoxide-dismutase.Use the medicine of macromolecular material modification of the present invention, can improve solvability, stability and the immunogenicity of medicine,, improve curative effect with the transformation period of prolong drug.

Except above-mentioned, other molecules can also be to have the molecule that has free amino group in the drug molecules such as amino amino acid, carbohydrate, organic acid, alkaloid, flavonoid, glycoside, quinones, terpene.

Another aspect of the present invention provides a kind of pharmaceutical composition that comprises above-mentioned double-stranded Polysialic acid and pharmacology acceptable carrier or vehicle, and described pharmaceutical composition can be the formulation of tablet, suppository, pill, soft hard-gelatin capsules, powder, solution, suspensoid or aerosol.

Experiment shows that the present invention has following advantage: one, because the hydrodynamic volume of active double-stranded PSA is big, when it with after certain position on the protein combines, because sterically hindered effect, other positions are difficult to and another double-stranded PSA reaction, thereby improved selectivity to combining site.And the biological activity retention value of such protein drug is also higher; They are two years old, can control its hydrodynamic volume by the molecular weight of controlling two PSA, make its inaccessibility activity of proteins position, the Polysialic acid binding substances of gained can keep higher biological activity like this, they are three years old, because the increase of chain number will more effectively stop macromole or cell near medical surfaces than linear PSA, thereby further improves binding substances circulation time in vivo, reduces immunoreactive generation.

Embodiment:

The present invention further specifies in following embodiment.These embodiment are for illustrative purposes, rather than are used for limiting the scope of the invention.For convenience of explanation, in following embodiment, polysialic acids all adopts colominic acid CA.

Embodiment 1 different double-stranded colominic acid CA₂-Lys-NHS preparation

With Methionin (439mg, 3mmol) be dissolved in 20ml pH value in the water of 8.0-8.3, in 2.5 hours, activate the active strand colominic acid CA-DSG (1mmol) (preparing) of deutero-then, come the pH value of maintenance system 8.3 with 0.2mol/L NaOH simultaneously by CN 101160326 methods to wherein add freshly prepd non-reducing end in batches.After stirred overnight at room temperature, reactant is cooled to 0 ℃.Now extract impurity with ether from water, extract three times continuously with chloroform, extracting solution is added dropwise in the anhydrous diethyl ether after concentrating, obtain white precipitate, the gained precipitation obtains the mono-substituted Methionin of CA (CA-Lys-COOH) behind twice recrystallization of ethanol, its functional end-group is a carboxyl.

In the anhydrous methylene chloride that is dissolved with the said products (0.9mmol) (30mL), add triethylamine (TEA) and reach 8 up to the pH value.Within three hours, add reducing end activation deutero-strand Polysialic acid CA-BS in batches³(1.1mmol) to reaction solution, use the pH value of TEA maintenance system simultaneously about 8.After reactant refluxed 72 hours, after concentrating, filter, use ether sedimentation.Use the small amount of ethanol recrystallization then.Excessive strand Polysialic acid is the Na of 9-10 at pH₂CO₃Be hydrolyzed after stirring is spent the night in the damping fluid, solution is chilled to 0 ℃.With the impurity in the ether extraction solution.Extract three times continuously with chloroform, the extracting solution drying concentrates the back with the anhydrous diethyl ether precipitation, uses the dehydrated alcohol recrystallization then again, and products therefrom is further purified with QSESephadexA50 post (5 * 80cm, the borate buffer solution of leacheate: pH=8.7), obtains CA₂-Lys-COOH.

At 0 ℃, to being dissolved with above-mentioned CA₂Add N-hydroxy-succinamide (1.6mmol) and DCC (1.6mmol) in the anhydrous methylene chloride (20mL) of-Lys-COOH (0.6mmol), after the stirred overnight at room temperature, filter, precipitate with anhydrous diethyl ether after the concentrating filter liquor, through re-crystallizing in ethyl acetate, obtain carboxyl again by N-hydroxy-succinamide activatory CA₂-Lys-NHS.

The identical double-stranded colominic acid CA of embodiment 2 non-reducing ends activation deutero-₂-Lys-NHS preparation

Methionin (0.2mmol) is dissolved in 40ml pH value in the borate buffer of 8.0-8.3, to wherein adding the active strand colominic acid CA-DSG (0.6mmol) (preparing) of freshly prepd non-reducing end activation deutero-, come the pH value of maintenance system 8.3 with 0.2mol/Lr NaOH simultaneously then by CN 101160326 methods.After stirred overnight at room temperature, reactant is cooled to 0 ℃.Now extract impurity with ether from water, extract three times continuously with chloroform, extracting solution is added dropwise in the anhydrous diethyl ether after concentrating, and obtains white precipitate, and gained precipitates behind twice recrystallization of ethanol, obtains the disubstituted Methionin (CA of CA₂-Lys-COOH), its functional end-group is a carboxyl.

At 0 ℃, to being dissolved with above-mentioned CA₂Add N-hydroxy-succinamide (0.6mmol) and DCC (0.6mmol) in the anhydrous methylene chloride (20mL) of-Lys-COOH (0.2mmol), after the stirred overnight at room temperature, filter, precipitate with anhydrous diethyl ether after the concentrating filter liquor, through re-crystallizing in ethyl acetate, obtain carboxyl again by N-hydroxy-succinamide activatory CA₂-Lys-NHS.

The identical double-stranded colominic acid CA of embodiment 3 reducing ends activation deutero-₂-Lys-NHS preparation

The active strand colominic acid of non-reducing end in the above-mentioned example 2 activation deutero-CA-DSG is changed (preparing by CN 101160326 methods) into the active strand colominic acid of the reducing end activation deutero-CA-BS of equivalent³Get final product.

Embodiment 4 is initiated with the identical double-stranded colominic acid CA of CA-CHO₂-Lys-NHS preparation

With the 2mL deionized water of the Methionin that contains 2mmol, dissolving molar equivalent 2-6 CA-CHO doubly, and place the 50mL container, add NaCNBH then₄(to concentration be 5mg/mL).Mixture at room temperature reacted 2-5 days.Add the freezing ethanol of 5mL, under the double-stranded colominic acid Acid precipitation of product.Reclaimed precipitation in centrifugal 30 minutes with desk centrifuge 4000r/min.In the resuspended precipitation of 2mL deionization, precipitate again at the freezing ethanol of the ultracentrifugation Guan Zhongyong of 10mLr 5mL then.Reclaimed precipitation in centrifugal 30 minutes with 30000r/min.Precipitation resuspended and freeze-drying in the 2mL deionized water once more, standby.

At 0 ℃, to being dissolved with above-mentioned CA₂Add N-hydroxy-succinamide (1.6mmol) and DCC (1.6mmol) in the anhydrous methylene chloride (20mL) of-Lys-COOH (0.8mmol), after the stirred overnight at room temperature, filter, precipitate with anhydrous diethyl ether after the concentrating filter liquor, through re-crystallizing in ethyl acetate, obtain carboxyl again by N-hydroxy-succinamide activatory CA₂-Lys-NHS.

Embodiment 5 CA₂The preparation of-Lys-p-NP

The p-NP that N-hydroxy-succinamide in above-mentioned example 1,2,3,4 examples is changed into equivalent gets final product.

The polysialic acid active derivative CA of embodiment 6 duplex structures₂The preparation of-Lys-haFGF

(10-50mmol, PH7.2) human acid fibroblast growth factor in mixes with mol ratio 1:5-1:50 with the double-stranded colominic acid of example 1,2,3,4,5 preparations, is positioned over 4-37 ℃, reacts 2-72 hour will to be dissolved in the PBS damping fluid.After reacting completely, after reaction solution crossed Sephedax G-25 post desalination, last CL-6B heparin affinity chromatography post separates, separation condition is: CL-6B prepares type affinity column (20cm * 1.5cm), adopt 10-50mmol/L, PBS damping fluid balance columns, sample on the 1mL/min flow velocity is used 10-50mmol/L, the PBS damping fluid wash post to baseline steadily after, 10-50mmol/L, PBS damping fluid+0.1-1.2M NaCl buffer solution elution, eluent flow rate is 1mL/min, collects elution peak, SDS-PAGE checks, and obtains the pure product of polysialic acid active derivative-haFGF of the duplex structure of different modifying degree.

The polysialic acid active derivative CA of embodiment 7 duplex structures₂The preparation of-Lys-IFN

Get the Interferon, rabbit borate buffer of 10mg/ml, pH=8.5 adds the prepared double-stranded colominic acid dry powder 20-400mg of example 1,2,3,4,5, and reaction is 2-72 hour under the 4-37 ℃ of condition, through the anionresin column purification, can get the PSA-IFN sample.

Claims (10)

1, a kind of polysialic acid active derivative of duplex structure is characterized in that randomly being lumped together by junction through chemical reaction and activatory strand Polysialic acid by the trifunctional micromolecular compound and forms, and can be expressed as (R₂-PSA)₂-X-F, wherein, R₂Be hydrogen or a kind of single, double, oligomerization or polysialic acids group, a kind of albumen, a kind of peptide, a kind of lipid, a kind of medicine, a kind of cytolemma or cell-wall component or a kind of drug delivery system; 2 represent two strands; X is a tie point, promptly a kind of trifunctional micromolecular compound; F represents activity functional groups, can form covalent linkage with the amino on medicine or the matrix and be connected; PSA is a Polysialic acid.

2, the polysialic acid active derivative of the described duplex structure of claim 1, it is characterized in that activatory strand Polysialic acid is connected with covalent linkage with the trifunctional micromolecular compound, linking group is selected from amido, imido grpup, amide group, inferior amide group, carbamate, ester group, epoxy group(ing), carboxyl, hydroxyl, sulfydryl, carbohydrate, or several combinations a kind of wherein.

3, the polysialic acid active derivative of the described duplex structure of claim 1, the active group that it is characterized in that the trifunctional micromolecular compound is carboxyl and two amino or carboxyl and two hydroxyls, and the structural formula of used trifunctional micromolecular compound is preferably from following a kind of:

Here, n is the integer of 1-9, and m is the integer of 0-6.

4, the polysialic acid active derivative of the described duplex structure of claim 1 is characterized in that array structure under the trifunctional micromolecular compound is:

Wherein, n is the integer of 1-9, when linking group is amide group or carbamate, and described double-stranded Polysialic acid (R₂-PSA)₂-X-F has structural formula I, II, III or IV

Wherein A is NR₃, NR₃NR₄, O or S, wherein R₃And R₄Be independently selected from H, C_1-4Alkyl and aryl;

SylO is a sialic acids groups;

N is 1-9; Q is 1-100; P is 0 or 1;

W is one of O, S or NH;

One of preferred following functional group of F:

R₁Be linking group;

R₂Be hydrogen or a kind of single, double, oligomerization or polysialic acids group, a kind of albumen, a kind of peptide, a kind of lipid, a kind of medicine, a kind of cytolemma or cell-wall component or a kind of drug delivery system.

5, the polysialic acid active derivative of the described duplex structure of claim 4, wherein R₁Be selected from alkylidene group, arylidene, alkarylene, heteroarylidene, alkyl-heteroarylidene, any one can be connected by thioester, ester, amine or amido linkage in them, and wherein A is NR₃Or for passing through NR₃Group is connected to a linking group of molecule remaining part.

6, a kind of preparation method of polysialic acid active derivative of duplex structure as claimed in claim 1, it is characterized in that by activatory strand Polysialic acid and the reaction of trifunctional micromolecular compound, and then with the compound reaction that has active group F, obtain the polysialic acid active derivative of active duplex structure.

7, a kind of application of polysialic acid active derivative of duplex structure as claimed in claim 1 is characterized in that it combines with the bioactive molecules that contains primary amine groups that comprises small-molecule drug, polypeptide or pharmaceutical grade protein by activity functional groups F.

8, the application of the polysialic acid active derivative of the described duplex structure of claim 7 is characterized in that described small-molecule drug is preferred but is not limited to: Chlorambucil, cis-platinum, 5-fluor-uracil, taxol, Zorubicin or Rheumatrex.

9, the application of the polysialic acid active derivative of the described duplex structure of claim 7 is characterized in that described pharmaceutical grade protein is preferred but is not limited to: Interferon, rabbit, interleukin, tumour necrosis factor, somatomedin, G CFS, erythropoietin or superoxide-dismutase.

10, a kind of pharmaceutical composition, said composition comprise the polysialic acid active derivative of the described duplex structure of claim 1 and the binding substances and the treatment inert support of protein or polypeptide.