CN101039965A - Sialic acid derivatives - Google Patents

Abstract

An amine or hydrazide derivative of a sialic acid unit, e.g. in a polysaccharide, is reacted with a bifunctional reagent at least one of the functionalities of which is an ester of N-hydroxy succinimide, to form an amide or hydrazide product. The product has a useful functionality, which allows it to be conjugated, for instance to proteins, drugs, drug delivery systems or the like. The process is of particular utility for derivatising amine groups introduced in sialic acid terminal groups of polysialic acids.

Description

Sialic acid derivative

The present invention relates to the derivative of sialylated compound, be preferably and have the unitary polysaccharide of sialic acid in end or the chain.Preferably, polysaccharide only is made up of the sialic acid unit, for example by α-2,8,2,9 sialic acid connected to one another unit.This product can be used for puting together the connection substrate, for example peptide, protein, medicine, drug delivery system, virus, cell, microorganism, synthetic polymer etc.Reaction comprises that the reaction reagent that contains the NHS group is puted together with the sialic acid derivative with amino or hydrazides functional group to be connected.

Polysialic acid (PSA) is sialic naturally occurring non-branched chain polymer form, produces [Roth etc., 1993] by some bacterial isolates and mammiferous some cell.The PSA that produces can be the multiple polymerization degree: be low to moderate n=2 from n=about 80 or more sialic acid residueses, the polymkeric substance classification of the natural form that can come from cell or bacterium by the digestion or the origin of limited acid hydrolysis, neuraminidase and producing.The composition of different PSA also there are differences each other.Have homopolymer form, i.e. the PSA of α-2,8 connection comprises that coli strain K1 and B organize meningococcal capsular polysaccharide, and this form also is present in embryo's form of nerve cell adhesion molecule (N-CAM).Also there is the heteropolymer form, alternately α-2,8 α-2 of coli strain K92 for example, the C group polysaccharide of 9PSA and Neisseria meningitidis (N.meningitidis).In addition, sialic acid also can be present in to have and be different from sialic other monomeric alternating copolymer, for example the W135 of Neisseria meningitidis group or Y group.PSA has important biological function, comprise and make immunity system and complement system avoid malignant bacteria, and regulate the neuronic neuroglial tackyness of prematurity (wherein this polymkeric substance has the anti stickness effect) [Muhlenhoff etc., 1998 in the fetation process; Rutishauser, 1989; Troy, 1990,1992; Cho and Troy, 1994], but do not have known PSA acceptor in the mammalian body.The PSA that the α of coli strain K1-2,8 connects is also referred to as " colominic acid ", and is used for (for all lengths) exemplary illustration the present invention.

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 (n is up to 4), and thought that this shorter polymkeric substance can cause and keep the immunotolerance to PSA effectively.In the last few years, utilized the biological characteristics of the homopolymerization PSA of the biological characteristics of PSA, particularly α-2,8 connections, to change pharmacokinetics character [Gregoriadis, 2001 of protein and low-molecular-weight drug molecule; Jain etc., 2003; US-A-5,846,951; WO-A-0187922].The PSA derivative [Fernandes and the Gregoriadis that comprise some human cytokines of catalase and asparaginase, 1996 and 1997] make circulating half-life and stability thereof increase substantially, and make these albumen can be used for tackling preexisting antibody, described preexisting antibody owing to before be exposed to human cytokines and do not expect that (being inevitable sometimes) produces [Fernandes and Gregoriadis, 2001].Aspect a lot, the character of Polysialic acid protein improvement is with suitable with polyoxyethylene glycol (PEG) deutero-protein.For example all improved the transformation period, and protein and peptide are more stable in proteolytic digestion, but when adopting PSA bioactive confining force as if than PEG stronger [Hreczuk-Hirst etc., 2002].When needing the therapeutical agent of long term administration, employing use PEG also to have problems, because PEG biological degradation very lentamente [Beranova etc., 2000] and high molecular form and lower molecular weight form all are easy to accumulation [Bendele etc., 1998 in tissue; Convers etc., 1997].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 [Cheng etc., 1990] 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 [Hunter and Moghimi, 2002 to its immunotoxicology, pharmacology and metabolism; Brocchini, 2003].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.So α-2,8 PSA that connect become a kind of noticeable PEG surrogate, its biodegradable polymkeric substance as " invisible " in the immunity becomes the part of human body naturally, and can be degraded to nontoxic carbohydrate---sialic acid by organizing neuraminidase.

Our group has put down in writing purposes [Gregoriadis, 2001 in the pharmacokinetics character that natural PS A improving the protein therapeutic thing in before scientific paper and granted patent; Fernandes and Gregoriadis, 1996,1997,2001; Gregoriadis etc., 1993,1998,2000; Hreczuk-Hirst etc., 2002; Mital, 2004; Jain etc., 2003,2004; US-A-05846,951; WO-A-0187922].Here, we have put down in writing new PSA derivative, and this derivative produces new composition and makes the method for PSA derived protein (and therapeutical agent of other form).These novel materials and method are specially adapted to produce the PSA that intends being used for the human and animal therapeutical agent of deriving, because the safety requirements of medical ethics criterion and administration (for example FDA, EMEA), chemistry and the qualification on the molecule to the medicine body in this therapeutical agent are extremely important.

Before to polysaccharide is connected therapeutical agent for example the method on the protein report [Jennings and Lugowski, 1981 are arranged; US-A-5,846,951; WO-A-0187922].Some method in these methods relies on chemically derived to generate the activated aldehyde part of protein (Fig. 1) to " non-reduced " end of polymkeric substance.In puting together connection procedure, be the condition of chemical integrity the needs gentleness that keeps proteinic conformation and PSA, and PSA (and other polysaccharide) reducing end only have faint and activity proteins react under this mild conditions.The sialic acid unit of PSA non-reducing end contains the ortho position glycol, easily (with optionally) generated single aldehyde derivatives by periodate oxidation.This derivative is much higher to activity of proteins, and comprises and can be connected to the active part that is fit on the protein by reductive amination and other chemical actions.Before us at US-A-5,846,951 and WO-A-0187922 in this is put down in writing to some extent.This is reflected at and has carried out the example explanation among Fig. 1, wherein:

A) shown that use sodium periodate oxidation CA (PSA that connects from colibacillary α-2,8) generates the protein active aldehyde that responds with sialic non-reducing end endways, and

B) show the reaction of aldehyde and protein primary amine groups, used sodium cyanoborohydride (NaCNBH then ₃) carry out the selective reduction of Schiff (Schiff) alkali to form stable, irreversible covalent linkage with the protein amino group.

We have put down in writing a kind of polysaccharide derivates in PCT/GB04/03488, and it has the sulfydryl reaction active groups of introducing by terminal sialic acid unit.Derived by the sialic acid unit of the non-reducing end of polysaccharide usually and introduce in this unit.The sulfydryl reaction active groups is preferably maleimide base group.The reaction of introducing this group can comprise that aldehyde or amido that following assorted two senses (heterobifunctional) reaction reagent and the sialic acid of polysaccharide are derived on the terminal units react, described assorted two functional response's reagent at one end have the sulfydryl reaction active groups, have group such as hydrazides or ester at the other end.Product can be used for proteinic site-specific nature derives, and for example derives on the sulfydryl of halfcystine unit or introducing.

Although many put down in writing PSA is connected to method [US-A-5,846,951 on the therapeutical agent; WO-A-0187922] possible in theory, but the reaction of the non-reducing end (aldehyde form) by protein and PSA obtains to make the satisfied conjugate yield of people at high temperature to carry out the certain reaction time, and this is unfavorable for proteinic stable (for example Interferon Alpha-2b).Secondly, required reactant concn (being that polymkeric substance is excessive) may be that can't realize or uneconomic.

The invention provides a kind of novel method that generates the sialic acid compound derivatives, wherein make and a kind ofly contain the intermediate that the unitary initial compounds of terminal sialic acid carries out in advance and form step, in this step, form the group that is selected from primary amine group, secondary amine group and hydrazine on the sialic acid unit endways, carry out the reactions steps of this intermediate and following two functional response's reagent reacts then

Wherein R is H or alkylsulfonyl;

R ¹Be linking group; And

X is a functional group,

Ester group is cut apart in the reaction, and the amido of intermediate or diazanyl quilt-CO-R ¹-X acidylate forms derivative.

In first kind of embodiment, initial compounds has the terminal sialic acid unit that is connected to other parts by the 2-carbon atom, promptly as non-reduced terminal units, and preliminary step comprises sialic C-7, C-8 glycol radical oxidation is formed aldehyde radical in this embodiment, then by using H ₂NR ⁴Or its acid salt carries out reductive amination to form intermediate, described H ₂NR ⁴Middle R ⁴Be H or low alkyl group.This preliminary step as shown in Figure 3.

In this first kind of embodiment, initial compounds has following structural formula:

R wherein ²Be described other parts, be selected from monose, disaccharides, oligose or polysaccharide group, protein or peptide, lipid, medicine and drug delivery system (for example liposome), and wherein the acid amides derived products have following structural formula:

Wherein X, R ¹And R ⁴With identical in separately the initial compounds, R ³With R ²Identical or be R ²Oxidation, reductive amination and with the reactions steps of reagent I in product.According to this embodiment, the formation of compound as shown in Figure 6, wherein reagent I is two-NHS linking agent.

In second kind of embodiment, initial compounds has the reducing end under neutral sialic acid that is connected to other parts by the 8-carbon atom, and preliminary step comprises that ketal open loop reduction step has the group of ortho position glycol with formation in this embodiment, in the selective oxidation step, ortho position glycol group is oxidized to aldehyde radical then, then by using H ₂NR ⁴Or acid salt carries out reductive amination to form intermediate.

In this embodiment, initial compounds has following structural formula:

R wherein ⁵Is described other parts, is selected from carbohydrate group, oligose or polysaccharide group, alkyl, acyl group, lipid, drug delivery system, and wherein amide product has following structural formula:

R wherein ¹, X and R ⁴With identical in separately the initial compounds, R ⁶With R ⁵Identical or be R ⁵Reduction, oxidation, amination and with the reactions steps of reagent I in product.The formation of compound V as shown in Figure 2.

In the third embodiment, initial compounds has the terminal sialic acid unit (promptly as non-reduced terminal units) that is connected to other parts by the 2-carbon atom, and preliminary step comprises sialic C-7, C-8 glycol radical oxidation formation aldehyde radical in this embodiment, then by generating intermediate with hydrazine reaction and reduction.

In this embodiment, initial compounds has following structural formula:

R wherein ²Is described other parts, is selected from monose, disaccharides, oligose or polysaccharide group, protein or peptide, lipid, medicine or drug delivery system, and wherein derived products has following structural formula:

Wherein X and R ¹With identical in separately the initial compounds, R ³With R ²Identical or be R ²Oxidation, with hydrazine reaction, reduction and with the reactions steps of reagent I in product.

In the 4th kind of embodiment, initial compounds has the reducing end under neutral sialic acid that is connected to other parts by the 8-carbon atom, and preliminary step comprises that ketal open loop reduction step has the group of ortho position glycol with formation in this embodiment, in the selective oxidation step, ortho position glycol group is oxidized to aldehyde radical then, then by generating intermediate with hydrazine reaction and reduction.

In this embodiment, initial compounds has following structural formula:

R wherein ⁵Is described other parts, is selected from monose, disaccharides, oligose or polysaccharide group, alkyl, acyl group, lipid and drug delivery system, and wherein derived products has following structural formula:

Wherein X, R ¹With identical in separately the initial compounds, R ⁶With R ⁵Identical or be R ⁵Reduction, oxidation, with hydrazine reaction, reduction and with the reactions steps of reagent I in product.An example that has shown the reaction scheme of production IX compound among Fig. 5, wherein two sense reagent I are two-NHS.

In the method, it generally is very important before intermediate and reagent I contact it being separated with the mix products of preliminary step basically.This is because the reagent that uses in preliminary step may make formula I reagent inactivation.In addition, in preliminary step, comprise under the oxidation and the situation of reduction step or its backward process successively, should make oxygenant or reductive agent inactivation in the first step, and then add the reaction reagent of subsequent step.

In the method for the invention, being reflected in the aprotic solvent between intermediate and the formula I reagent is favourable, preferably contains a spot of protonic solvent in the described solvent.Reduce the early stage inactivation of NHS group that the protonic solvent that exists in the reaction can be avoided formula I reagent as far as possible.It has been generally acknowledged that aprotic solvent can destroy biomolecules.Surprisingly, use dimethyl sulfoxide (DMSO) DMSO, particularly use DMSO to make the PSA dissolving, can obtain to be connected degree with good the puting together of NHS reagent, and can not make the passivation exceedingly before reaction of NHS group, and make and from mix products, to reclaim derivative.Therefore preferred aprotic solvent is DMSO.

Formula I reagent dosage is common than excessive with the stoichiometric amount of intermediate reaction, and is preferably the twice at least of the stoichiometric amount of reacting with intermediate, more preferably at least five times.

In a kind of embodiment of formula I reagent, X is following group

Wherein the R definition as above.

X is selected from following group in an alternative embodiment: vinyl sulphone, N-dimaleoyl imino, N-iodo kharophen, ortho position pyridyl disulfide group, hydroxyl and protected, shielded amino and azido-.

Formula I reagent is preferably selected from:

N-(α-dimaleoyl imino acetoxyl) succinimide ester (AMAS),

N-(β-dimaleoyl imino propoxy-) succinimide ester (BMPS),

N-(the sad base of ξ-dimaleoyl imino) succinimide ester (EMCS) or its sulfo group analogue,

N-(γ-dimaleoyl imino butyric acid base) succinimide ester (GMBS) or its sulfo group analogue,

Succinimido-4-(N-maleimide ylmethyl)-hexanaphthene-1-carboxyl-(6-aminocaprolc acid ester) (LC-SMCC),

Between dimaleoyl imino benzoyl-N-hydroxysuccinimide eater (MBS) or its sulfo group analogue,

Succinimido-4-(N-maleimide ylmethyl)-hexanaphthene-1-carboxylicesters (SMCC) or its sulfo group analogue,

Succinimido-4-(to the dimaleoyl imino phenyl) butyric ester (SMPB) or its sulfo group analogue,

Succinimido-6-(β-dimaleoyl imino-propionamido) capronate (SMPH),

N-(k-dimaleoyl imino undecanoyl oxygen base) sulfosuccinimide ester (sulfo-KMUS),

Succinimido 6-[3-2 (2-pyridyl disulfide group)-propionamido] capronate (LC-SPDP) or its sulfo group analogue,

4-succinimido oxygen base carbonyl-methyl-α-(2-pyridyl disulfide group) toluene (SMPT) or its sulfo group-LC analogue,

N-succinimido-3-(2-pyridyl disulfide group) propionic ester (SPDP),

N-succinimido [4-vinylsulfonyl] benzoic ether (SVSB),

Succinimido 3-(bromo kharophen) propionic ester (SBAP) and

N-succinimido iodo acetic ester (SIA) and

N-succinimido (4-iodo ethanoyl) Aminobenzoate (SIAB) or its sulfo group analogue.

The assorted two sense reagent of another kind of formula I have optical active group X, for example azido group.The example of this type of reaction reagent has:

N-5-azido--2-nitro benzoyl oxygen base succinimide (water insoluble) (ANB-NOS),

N-hydroxy-succinamide base-4-azido-Whitfield's ointment (water insoluble, indivisible) (NHS-ASA),

N-succinimido (4-azido-phenyl)-1,3 '-dithio propionic ester (SADP),

Sulfosuccinimide base 2-(7-azido--4-methyl-tonka bean camphor-3-kharophen) ethyl-1,3 '-dithio propionic ester (SAED),

Sulfosuccinimide base 2-(-azido--neighbour-nitro-benzamido) ethyl-1,3 '-dithio propionic ester (SAND),

N-succinimido 6-(4 '-azido--2 '-nitro-phenyl amino) capronate (SANPAH),

Sulfosuccinimide base 2-(right-azido--neighbour-salicylyl amino) ethyl-1,3 '-dithio propionic ester (SASD),

Sulfosuccinimide base-(perfluor triazobenzene formamido group) ethyl-1,3 '-dithio propionic ester (SFAD) and

N-hydroxysulphosuccinimide base-4-triazobenzene manthanoate (Sulfo-HSAB).

Formula I reagent can be selected from two [2-succinimido oxygen base carbonyl-oxygen base) ethyl] sulfone (BSOCOES) and sulfo group analogue thereof,

Two (sulfosuccinimide base) suberate (BS ³),

Two succinimido glutarates (DSG),

Dithio two (succinyl phosphorons amino propyl acid ester) (DSP),

Two succinimido suberates (DSS),

Two succinimido tartrates (DST) or its sulfo group analogue,

3,3 '-dithio two (sulfosuccinimide base propionic ester) (DTSSP) and

Ethylene glycol bisthioglycolate (succinimido succinate) (EGS) and the sulfo group analogue.

Radicals R ¹Be two sense organic groups.Preferably, R ¹Be selected from alkane two bases, arylidene, alkarylene, inferior heteroaryl and alkylene heteroaryl, above-mentioned group all can be replaced and/or be interrupted by carbonyl, ester, sulphur, ether, acid amides and/or amine.Preferred especially R ¹Be C ₃-C ₆Alkane two bases.Most preferably, R ¹Suitable part among one of corresponding above-mentioned preferred reagent I.Substituting group can be selected from R listed above ¹Substituting group, or can be amino acid side chain.

Derived products is preferred in present method fully is separated with excessive reaction reagent basically.

Usually the reaction conditions of the above-mentioned reaction of adopting also can adopt in the present invention, for example referring to Hermanson, (1995).

More preferably, the product amide derivatives fully is separated with mix products basically.This separation and recovery can comprise drying step, and described drying step preferably under reduced pressure carries out, and most preferably is step of freeze drying.

Can obtain so stable, can be used for follow-up and biological available compound reactive activity sialic acid derivative.

In conjunction with embodiment that encloses and accompanying drawing the present invention is carried out further example explanation.

Below be brief description of drawings:

Fig. 1 a is the reaction scheme of prior art activation irreducibility sialic acid terminal units;

Fig. 1 b is that prior art uses protein amido part with the aminating reaction scheme of aldehyde partial reduction among the reaction scheme 1a;

Fig. 2 has shown the preparation (when non-reducing end does not have the ortho position glycol) of reducing end deutero-NHS colominic acid;

Fig. 3 has shown reducing end deutero-NH ₂The preparation of-CA colominic acid (removing the ortho position glycol) at non-reducing end;

Fig. 4 has shown the general route of preparation CA-NHS-protein conjugate;

Fig. 5 has shown by NHS and has prepared the CA-protein conjugate at reducing end;

Fig. 6 has shown the preparation of non-reducing end deutero-CA;

Fig. 7 has shown use two (sulfosuccinimide base) suberate (BS ³) prepare the CA-protein conjugate at non-reducing end;

Fig. 8 has shown that use linking agent DSG carries out the representative schematic diagram that CA-protein is puted together;

Fig. 9 has shown that CA-GH puts together the HPLC of ligation;

Figure 10 has shown sodium lauryl sulphate (the SDS)-polyacrylamide gel electrophoresis (PAGE) of CA-NHS-GH conjugate (CA 35kDa);

Figure 11 has shown the non-denaturing polyacrylamide gel chromatogram (native-PAGE) of unreacted CA;

Figure 12 has shown the SDS-PAGE of CAM-β-gal and CAI-β-gal conjugate;

Figure 13 has shown that the SDS-PAGE of CAH-NHS reaction analyzes;

Figure 14 has shown the Size Exclusion Chromatograph SEC analysis of CAH-NHS reaction.

Embodiment

Material

Obtain sodium metaperiodate and molecular weight marker by Britain Sigma Chemical Laboratory.Used CA is intestinal bacteria K1 PSA (molecular-weight average 22.7kDa, the polydispersity coefficient (p.d.) 1.34 of linear α-(2,8)-connection; 39kDa, p.d.1.4; 11kDa, p.d.1.27), from Camida, Ireland.Other materials comprises 2,4 dinitrophenyl hydrazines (Aldrich Chemical Company, Britain), (3.5kDa and 10kDa hold back scope to dialysis tubing; Medicell International Limited, Britain), agarose SP HiTrap, PD-10 post (Pharmacia, Britain), XK50 post (Amersham Biosciences, Britain), agarose Q FF (Amersham Biosciences), Tris-glycine polyacrylamide gel (4-20% and 16%), Tris-glycine sodium lauryl sulphate running buffer and sample loading buffer (Novex, Britain).Deionized water obtains from Elgastat Option 4 water correction plants (Elga Limited, Britain).Used total overall reaction reagent is AG.Plate reader (plate reader) (Dynex Technologies, Britain) is used for the spectrometry of protein or CA detection.

Method

The mensuration of protein and CA

CA passes through other places [Gregoriadis etc., 1993 as sialic quantitative assay; Fernandes and Gregoriadis, 1996,1997] described Resorcinol method [Svennerholm 1957] is carried out.(bicinchoninic acid, BCA) colorimetry detects GH by dicinchonine acid.

Comparative examples 1: by IEC to CA carry out classification (CA, 22.7kDa, pd1.34)

Fill the XK50 post and with buffering washing fluid (the 20mM trolamine of 3 column volumes with 900ml agarose Q FF; PH7.4) flow velocity with 50ml/min carries out balance.With the flow velocity of 50ml/min CA (25 grams are dissolved in 200ml buffering washing fluid) is gone up sample to post by syringe bore.Use the buffering washing fluid coupled columns of 1.5 column volumes (1350ml) to wash then.

Different buffer elution liquid (trolamine damping fluids with 1.5 column volumes, 20mM, pH7.4, step-length with 25mM NaCl between the level changes to 475mM NaCl from 0mM) with bonded CA wash-out, the final same buffer wash-out that is dissolved with 1000mM NaCl of using is to shift out all residual CA and other residues (if existence).

Use the film (Vivascience, Britain) of 5kDa that sample concentration is arrived 20ml by the high pressure ultrafiltration.Under 4 ℃, these samples are exchanged in the deionized water from damping fluid by repeating ultrafiltration.Analyze molecular-weight average and other parameters of these samples by GP and native-PAGE (with liking alizarin blue dyeing).The narrow dispersion CA fraction sodium periodate oxidation that will obtain by aforesaid method, and with the overall variation of GPC and native-PAGE analyzing polymers.

The activation of comparative examples 2:CA

Under 20 ℃ with freshly prepd 0.02M sodium metaperiodate (NaIO ₄With respect to the excessive 6 times of mole numbers of CA) solution mixes with CA, with reaction mixture lucifuge magnetic agitation 15 minutes (shown in the first step among Fig. 3).With the ethanol of 70% (ultimate density) with the CA precipitation of oxidation and with mixture under 3000g centrifugal 20 minutes.Remove supernatant liquor, with the deionized water dissolving of particulate matter with minimum.With 70% ethanol CA is precipitated again, then 12, centrifugal under the 000g.With the water dissolution of particulate matter with minimum, freeze-drying also stores until next step use under-20 ℃.

The mensuration of the oxidation state of comparative examples 3:CA and derivative

With 2,4 dinitrophenyl hydrazines (2,4-DNPH) carry out the quantitative measurment of CA degree of oxidation, 2,4-DNPH obtains 2,4 dinitrophenylhydrazones of indissoluble by interacting with carbonyl compound.The CA (CAO) (every kind of 5mg) of non-oxide attitude (CA) and oxidation state is added to 2, and in the 4-DNPH reaction reagent (1.0ml), vibration solution leaves standstill it then until observing crystal settling [Shriner etc., 1980] under 37 ℃.The degree of oxidation of CA (quantitatively) is measured based on the method [Park and Johnson, 1949] that the hexacyanoferrate ion in the alkaline solution is reduced to yellow prussiate (Persian indigo plant) by its principle, measures under 630nm then.In this case, glucose is used as standard substance.

Comparative examples 4a: amino colominic acid (CA-NH ₂) preparation

CAO and 300 times of mole number excessive N H with 2 preparations of 10-100mg/ml comparative examples ₄Cl is dissolved in the 2ml deionized water in the test tube of 50ml, then with NaCNBH ₄(mother liquor of 5M is dissolved among the 1N NaOH (aqueous solution)) adds (Fig. 4, the first step) with the ultimate density of 5mg/ml.At room temperature mixture was cultivated 5 days.AO carries out control reaction with the CA replaced C.By adding the ice-cold ethanol of 5ml product colominic acid sulfonamide derivatives is precipitated.Thereby at room temperature in desk centrifuge, under the rotating speed of 4000rpm, will precipitate recovery in centrifugal 30 minutes.Keep particulate matter and it is suspended in the 2ml deionized water again, in 10ml ultracentrifugation pipe, reuse the ice-cold ethanol of 5ml then and precipitate.At room temperature 30, thereby reclaimed throw out under the rotating speed of 000rpm in centrifugal 30 minutes.Again be suspended in the 2ml deionized water once more particulate matter and freeze-drying.

Comparative examples 4b: the detection of amine content

Adopt the quantity [Satake etc., 1960] of the amino group that exists in TNBS (picryl sulfonic acid, promptly 2,4,6-trinitro-benzene-sulfonic acid) the detection assay product.

In the hole of microtiter plate, TNBS (TNBS of 15mM, 0.5 μ l) is added to 90 μ lpH and is in 9.5 the 0.1M borate buffer solution.In said mixture, add the CA-amide solution of 10 μ l 50mg/ml, plate was at room temperature left standstill 20 minutes, under 405nm, read absorbancy then.Use glycine as standard substance, concentration range is 0.1 to 1mM.TNBS is with the primary amine group trinitrophenylization.Can detect the TNP adducts of amine.

Use the TNBS detection method that the cold ethanol precipitation purifying is crossed twice product and detect, demonstrate transformation efficiency near 90%.

The preparation of embodiment 1:CA-NHS

With synthetic CA-NH among the above-mentioned comparative examples 4a ₂(35kDa) (15-20mg) is dissolved in 0.15M PBS (350 μ L, pH7.2), what add 50 or 75 molar equivalents then is dissolved in PBS (150 μ L, pH7.2) BS in ³The mixture eddy current is stirred 5 seconds kinds, it was reacted 30 minutes down at 20 ℃.Shown situation about using prevailingly in second step in Fig. 4, more specifically shown use BS among Fig. 7 with two senses (homobifunctional) linking agent ³Situation.Use PBS by the PD-10 post CA-NHS product to be carried out purifying, and purified product is used for NH with protein and peptide at once as elutriant (pH7.2) ₂The site-specific nature of group is puted together connection.By Resorcinol check and analysis sialic acid content, thus the CA concentration in definite PD10 fraction.CA and NHS reaction soln are analyzed at the 260nm place by use UV spectrum, and under 254nm, develop by thin-layer chromatography, thus the NHS content in the measurement CA polymkeric substance.

With synthetic CA-NH in the foregoing description 1 ₂(35kDa) (15-20mg) is dissolved in the minimum water (50-65 μ L) and to wherein adding DMSO (300-285 μ L), or with described CA-NH ₂Be dissolved in＞95% DMSO (350 μ L) in and be aided with heating (100-125).The DSG that will be dissolved in 75 molar equivalents among the DMSO (150L) is added to CA-NH ₂In the solution, eddy current stirred for 5 seconds, made it react 30 minutes (Fig. 8) down at 20 ℃ then.By diox precipitation (* 2) or by the PD-10 post use PBS as elutriant (pH7.2) with the CA-NHS product purification, and purified product is used for NH with protein and peptide at once ₂The site-specific nature of group is puted together connection.As previously mentioned, detect the CA concentration of determining in the PD10 fraction by Resorcinol.By using the NHS content in UV spectrum (260nm) and thin-layer chromatography (254nm) the measurement CA polymkeric substance.

The preparation of embodiment 2:CA-NHS-protein conjugate (is used BS ³And DSG)

The GH that will be dissolved in sodium bicarbonate (pH7.4) is covalently bound to CA-NHS (35kDa), and described CA-NHS passes through to use excessive BS according to the method described in the comparative examples 4b ³And prepare.Using mol ratio is the CA-NHS of 25: 1 or 50: 1: GH, under 20 ℃ at 0.15MPBS (pH7.2; 1.5ml) the middle reaction 30 minutes.By SDS-PAGE the GH of Polysialic acidization is characterized, and put together the connection yield by FPLC-Size Exclusion Chromatograph SEC mensuration.Control experiment is included under the condition that does not have any CA-NHS and uses BS ³Make natural protein put together Connection Step.Same under the condition that does not have natural GH, use BS ³Make CA-NH ₂Put together Connection Step.

The GH that will be dissolved in sodium bicarbonate (pH7.4) is covalently bound to CA-NHS (35kDa), and described CA-NHS prepares by using excessive DSG according to the method described in the embodiment 4b.Using mol ratio is 50: 1 CA-NHS: GH, under 20 ℃ at 0.15M PBS (pH7.2; 1.5ml) the middle reaction 30 minutes.By SDS-PAGE the GH of Polysialic acidization is characterized, and put together the connection yield by HPLC-Size Exclusion Chromatograph SEC mensuration.Control experiment is included under the condition that does not have any CA-NHS and makes natural protein put together Connection Step with DSG.

The CA-GH conjugate is dissolved in ammonium bicarbonate buffers (0.2M; PH7) in, at the enterprising circumstances in which people get things ready for a trip spectrum analysis of superose6 post and use UV index to detect (Agilent, 10/50 system, Britain).Sample (1mg/ml) filters with 0.45 μ m nylon membrane, injects 175 μ l, moves (Fig. 9) as moving phase with the speed of 0.25cm/min with ammonium bicarbonate buffers.

(VGT 1 type, power supply are Consort E132 for MiniGel, Vertical Gel Unit to use SDS-PAGE; VWR, Britain) detect that the molecular size of GH changes after the Polysialic acidization.The SDS-PAGE of use the 4-20% polyacrylamide gel to carry out GH and conjugate (with CA-NHS) thereof---sample of the 0th minute (control sample) and the 30th minute in the reaction mixture---and method control sample (CA of non-oxide attitude).By the various molecular weights marker sample is demarcated (Figure 10 and 11).

The result

With CA and derivative (22.7kDa) thereof successfully be classified as polydispersity coefficient less than 1.1, molecular-weight average is up to 46kDa and has the various narrow dispersion kind of different % amounts.Table 2 has shown the separating resulting of this 22.7kDa material.

The ion-exchange chromatography of table 2:CA22.7 (pd1.3)

Buffer elution liquid (trolamine damping fluid+mM NaCl of 20mM, pH7.4)	Molecular weight	Pd	The % amount
				325mM	12586	1.091	77.4％
350mM	20884	1.037	3.2％
				375mM	25542	1.014	5.0％
400mM	28408	1.024	4.4％
				425mM *			7.4％
450mM	43760	1.032	2.3％
				475mM	42921	1.096	0.2％

^*Do not implement

This method can be between 1ml to 900ml matrix convergent-divergent, and the grading curve of every kind of scale (does not show whole results) much at one.

[(classification pd1.4) produces the kind that is up to 90kDa for CA, 39kDa to big polymkeric substance.Present method can be successfully used to the more large batch of polymkeric substance of classification.The result shows that the fraction dispersion of ion-exchange gained is narrow.This and GPC data consistent.]

All narrow dispersive fractions are all successfully by the 20mM periodate oxidation, and to different steps sampling in the preparation process and carry out gpc analysis and native-PAGE analyzes, the result is presented on molecular weight and the polymolecularity and does not change.

Method [Park and Johnson, 1949] by the hexacyanoferrate ion in the alkaline solution being reduced to yellow prussiate (Persian indigo plant) also uses glucose as standard substance, and the oxidation state of CA has been carried out quantitative measurment.The CA that finds oxidation state compares the apparent aldehyde that has near 100 moles of % with natural polymer.The qualitative detection result that this use hexacyanoferrate carries out the result of detection by quantitative and uses 2,4 dinitrophenyl hydrazines to carry out CA intermediate in the oxidation step is consistent, obtains deep orange look precipitation after 10 minutes thereby at room temperature react; Generate faint yellow precipitation with natural CA when wherein using 2,4 dinitrophenyl hydrazines to detect, obtain the deep orange look with the CA reaction that contains the aldehyde polymer form and precipitate.

The amination degree that records polymkeric substance is 85%, and CA-NHS is the NHS positive.In addition, the content that also records mercaptan in the polymkeric substance is 60%.

By the integrity of the Neu5Ac residue of α-2,8 connection of inside after gpc analysis periodate and the hydroborate processing, with oxidation state (CAO), the amino CA (CA-NH that obtains ₂), the color atlas of CA-NHS material and the color atlas of natural CA compare.Find that (Fig. 9) all CA demonstrate elution curve much at one, do not have evidence to show that each step has significant fracture or crosslinked (for CA-NHS) effect to polymer chain.Small peak is represented buffering salt.

Analyze the generation of CA-GH conjugate by SEC-HPLC and SDS-PAGE.Put together ligation when adopting DSG, SDS-PAGE shows do not have residual free GH and put together ligation and finish.This has also obtained confirmation by SEC-HPLC, and wherein the CA-GH conjugate was washed out (peak of not observing free GH) before the free GH that is estimated washes out the time.On the other hand, to using BS ³The CA-NH that carries out ₂Have free GH with the SDS-PAGE analysis revealed of puting together ligation of GH, this is also confirmed by SEC-HPLC, has wherein occurred the elution peak of free protein in SEC-HPLC about 70 minutes.In addition, also to have measured the degree of puting together connection be 53% to SEC-HPLC.

Result (Figure 10) shows that bands of a spectrum have skew in the swimming lane of conjugate, and this illustrates the GH and GH specific mass increase mutually of Polysialic acidization usually.In addition, by SEC-HPLC the GH conjugate is separated into different kinds.

The preparation of the iodo acetic ester derivative (CAI) of embodiment 3:CA

3.1 it is synthetic

In the 40mg of the PBS that is dissolved in 1ml pH7.4 colominic acid amine (85mol% amine) (described in comparative examples 2), add 5mg N-succinimido iodo acetic ester (SIA).Make mixture 25 ℃ of following lucifuge reactions 1 hour, then by Hightrap at 5ml ^TMDesalting post (AP Bioscience) is gone up and is carried out gel-filtration with the PBS wash-out, thereby removes excessive SIA.Press 0.5ml from post and collect fraction, detect the content (Resorcinol detections) of colominic acid in the sample of each fraction, and use with the reactive behavior of halfcystine and indicate iodide (Ellman detection).The fraction that iodine and CA are positive merges.

3.2 CAI was connected with puting together of beta-galactosidase enzymes

Intestinal bacteria beta-galactosidase enzymes (5.0mg, 4.3 * 10 in 1ml PBS ^-8Mol) add 15mg CAI (6.59 * 10 in ^-7Mol, 15 molar equivalents).With the tinsel parcel, make reaction at room temperature carry out leniently mixing simultaneously in 1 hour the test tube sealing.By SDS-PAGE the gained conjugate is analyzed, carried out purifying to remove free CAI according to the method for generally acknowledging then.The polymkeric substance of test sample and protein content as stated above.

Carry out control reaction in contrast with CA.β-gal activity by the whole samples of methods analyst in following 3.3 joints.

3.3 the detection of enzymic activity

The standard substance of the fresh beta-galactosidase enzymes of preparation 60 μ g/ml to 3.75 μ g/ml in PBS.With same buffer with CAM-β-gal diluted sample to 60 μ g/ml.Measure the enzymic activity of conjugate by the following method:

In microtiter plate, in 100 μ l samples or standard substance, add 100 μ l All-in-One (All-in-One) β-gal substrates (Pierce).Plate was cultivated 30 minutes down at 37 ℃, read absorbancy at 405nm.Obtain calibration curve by standard substance, use the activity of the equation of linear regression calculation sample of curve.

3.4 conclusion

Fraction 3-6 all is positivity to polymkeric substance and iodo acetic ester, with its merging.SDS-PAGE (4-12%Bis/Tris gel; Figure 12) demonstrate the apparent molecular weight rising of the sample of cultivating with the iodo-acetamide derivative, and use comparison polymer not have this phenomenon.In detection, measure and put together than being 1.63CAI: 1 β-gal to protein and polymkeric substance.β-gal the activity that calculates the conjugate sample is in a ratio of 100.9% with resolvase.

Embodiment 4: the preparation of colominic acid hydrazides (CAH)

4.1 it is synthetic

The colominic acid (19kDa) that makes the 50mg oxidation state under 25 ℃ and 2.6mg hydrazides (liquid state) are reaction 2 hours in 5.5 the 20mM sodium acetate buffer at 400 μ l pH.Use 70% ethanol that colominic acid is precipitated then.Precipitation is dissolved in the phosphate buffered saline (PBS) of 350 μ lpH7.4 again, and adds NaCNBH ₄To concentration be 5mg/ml.Mixture was reacted 4 hours, then freeze overnight down at 25 ℃.Be filled with PD10 post, the use 0.15M NH of Sephadex G25 by use ₄HCO ₃Carry out gel permeation chromatography as moving phase and separate, to remove NaCNBH ₄And byproduct of reaction.(amino had specificity by the TNBS detection; As previously mentioned) each fraction (each 0.5ml) is analyzed.Fraction 6,7,8 and 9 (void volume fraction) has the strong signal far above background signal.Owing to there is NH ₄ ⁺Ion, so background signal is higher.Fraction 6,7,8 and 9 also contains colominic acid.With these four kinds of fraction freeze-drying to reclaim CA-hydrazides (CAH).

4.2 the preparation of colominic acid NHS (CA-NHS) and colominic acid-protein conjugate

Make 19kDa CA-hydrazides and the 9mg BS of 10mg under the room temperature ³Reaction is 30 minutes in 400 μ l PBS (pH 7.4).Reaction mixture is added the PD10 post that is filled with Sephadex G25, press 0.5ml and collect fraction.In each fraction of 5 to 9, add 0.1mg BSA.Room temperature makes each fraction and BSA reaction after following 2 hours.Analyze these samples by SDS-PAGE and SEC-HPLC.

These fractions contain colominic acid hardly.The fraction (6 and 7) that is rich in colominic acid existing bands of a spectrum and the BSA bands of a spectrum, also has protein spectra in having other samples, this clearly proves to exist puts together (Figure 13).

The HPLC color atlas of fraction 6 shows that the retention time of conjugate compared very big skew with free protein, proves to exist to put together (Figure 14 a and b).

Employed BSA contains impurity.The BSA peak appears at 56 minutes locates that (Figure 14 a).

Except the peak of locating in 56 minutes, also there is bigger conjugate kind.A big peak, the NHS that this peak discharges when being CA-NHS and proteins react have been located at 80 minutes.This can not be free BS ³, because CAH process gel permeation chromatographic column, chromatographic column will be removed.This has effectively illustrated has the NHS ester group to generate (Figure 14 b) on the CA molecule.

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Claims (28)

1. method that forms the sialic acid compound derivatives, wherein make and a kind ofly contain the intermediate that the unitary initial compounds of terminal sialic acid carries out in advance and form step, in this step, form the group that is selected from primary amine group, secondary amine group and hydrazine on the sialic acid unit endways, carry out the reactions steps of this intermediate and formula I two functional response's reagent reacts then

Wherein R is H or alkylsulfonyl;

R ¹Be linking group; And

X is a functional group,

Ester group is cut apart in described reaction, and the amido of intermediate or diazanyl quilt-CO-R ¹-X acidylate forms derivative.

2. the process of claim 1 wherein that initial compounds has the terminal sialic acid unit that is connected to other parts by the 2-carbon atom, and wherein preliminary step comprises sialic 6,7-glycol radical oxidation is formed aldehyde radical, then by using H ₂NR ⁴Or its acid salt carries out reductive amination to form intermediate, described H ₂NR ⁴Middle R ⁴Be H or low alkyl group.

3. the method for claim 2, wherein initial compounds has following structural formula:

R wherein ²Is described other parts, is selected from monose, disaccharides, oligose or polysaccharide group, protein or peptide, lipid, medicine or drug delivery system, and wherein the acid amides derived products has following structural formula:

Wherein X, R ¹And R ⁴With identical in separately the initial compounds, R ³With R ²Identical or be R ²Oxidation, reductive amination and with the reactions steps of reagent I in product.

4. the method for claim 1, wherein initial compounds has the reducing end under neutral sialic acid that is connected to other parts by the 8-carbon atom, and wherein preliminary step comprises that ketal open loop reduction step has the group of ortho position glycol with formation, in the selective oxidation step, ortho position glycol group is oxidized to aldehyde radical then, then by using H ₂NR ⁴Or acid salt carries out reductive amination to form intermediate, described H ₂NR ⁴Middle R ⁴Be H or low alkyl group.

5. the method for claim 4, wherein initial compounds has following structural formula:

R wherein ⁵Is described other parts, is selected from carbohydrate group, oligose or polysaccharide group, alkyl, acyl group, lipid and drug delivery system, and wherein amide product has following structural formula:

R wherein ¹, X and R ⁴With identical in separately the initial compounds, R ⁶With R ⁵Identical or be R ⁵Reduction, oxidation, amination and with the reactions steps of reagent I in product.

6. the method for claim 1, wherein initial compounds has the terminal sialic acid unit that is connected to other parts by the 2-carbon atom, and wherein preliminary step comprises sialic 6,7-glycol radical oxidation is formed aldehyde radical, then by generating intermediate with hydrazine reaction and reduction.

7. the method for claim 6, wherein initial compounds has following structural formula:

R wherein ²Is described other parts, is selected from monose, disaccharides, oligose or polysaccharide group, protein or peptide, lipid, medicine or drug delivery system, and wherein derived products has following structural formula:

Wherein X and R ¹With identical in separately the initial compounds, R ³With R ²Identical or be R ²Oxidation, with hydrazine reaction, reduction and with the reactions steps of reagent I in product.

8. the method for claim 1, wherein initial compounds has the reducing end under neutral sialic acid that is connected to other parts by the 8-carbon atom, and wherein preliminary step comprises that ketal open loop reduction step has the group of ortho position glycol with formation, in the selective oxidation step, ortho position glycol group is oxidized to aldehyde radical then, then by generating intermediate with hydrazine reaction and reduction.

9. the method for claim 8, wherein initial compounds has following structural formula:

R wherein ⁵Is described other parts, is selected from monose, disaccharides, oligose or polysaccharide group, alkyl, acyl group, lipid and drug delivery system, and wherein derived products has following structural formula:

Wherein X, R ¹With identical in separately the initial compounds, R ⁶With R ⁵Identical or be R ⁵Reduction, oxidation, with hydrazine reaction, reduction and with the reactions steps of reagent I in product.

10. any one method in the claim 1 to 9, wherein at intermediate with before formula I reagent contacts, intermediate is separated with the mix products of preliminary step basically.

11. any one method in the claim 1 to 10, wherein being reflected in the aprotic solvent between intermediate and the general formula I reagent carried out, and described aprotic solvent preferably contains a spot of protonic solvent.

12. the method for claim 11, wherein aprotic solvent is a dimethyl sulfoxide (DMSO), and protonic solvent is a water.

13. any one method in the claim 1 to 12, its Chinese style I reagent be than excessive with the stoichiometric amount of intermediate reaction, and be preferably the twice at least with the stoichiometric amount of intermediate reaction, more preferably at least five times.

14. the method for claim 12 or 13, wherein X is following group

Wherein R such as claim 1 definition.

15. the method for claim 14, its Chinese style I reagent is selected from

Two [2-succinimido oxygen base carbonyl-oxygen base) ethyl] sulfone (BSOCOES) and sulfo group analogue thereof,

Two (sulfosuccinimide base) suberate (BS ³),

Two succinimido glutarates (DSG),

Dithio two (succinyl phosphorons amino propyl acid ester) (DSP),

Two succinimido suberates (DSS),

Two succinimido tartrates (DST) or its sulfo group analogue,

3,3 '-dithio two (sulfosuccinimide base propionic ester) (DTSSP) and

Ethylene glycol bisthioglycolate (succinimido succinate) (EGS) and the sulfo group analogue.

16. the method for claim 12 or 13, wherein X is the functional group that is selected from following group: vinyl sulphone, N-dimaleoyl imino, N-iodo kharophen, ortho position pyridyl disulfide group, hydroxyl and protected, shielded amino and azido-.

17. the method for claim 16, wherein said reagent is selected from:

N-(α-dimaleoyl imino acetoxyl) succinimide ester (AMAS),

N-(β-dimaleoyl imino propoxy-) succinimide ester (BMPS),

N-(the sad base of ξ-dimaleoyl imino) succinimide ester (EMCS) or its sulfo group analogue,

N-(γ-dimaleoyl imino butyric acid base) succinimide ester (GMBS) or its sulfo group analogue,

Succinimido-4-(N-maleimide ylmethyl)-hexanaphthene-1-carboxyl-(6-aminocaprolc acid ester) (LC-SMCC),

Between dimaleoyl imino benzoyl-N-hydroxysuccinimide eater (MBS) or its sulfo group analogue,

Succinimido-4-(N-maleimide ylmethyl)-hexanaphthene-1-carboxylicesters (SMCC) or its sulfo group analogue,

Succinimido-4-(to the dimaleoyl imino phenyl) butyric ester (SMPB) or its sulfo group analogue,

Succinimido-6-(β-dimaleoyl imino propionamido) capronate (SMPH),

N-(k-dimaleoyl imino undecanoyl oxygen base) sulfosuccinimide ester (sulfo-KMUS),

Succinimido 6-[3-2 (2-pyridyl disulfide group)-propionamido] capronate (LC-SPDP) or its sulfo group analogue,

4-succinimido oxygen base carbonyl-methyl-α-(2-pyridyl disulfide group) toluene (SMPT) or its sulfo group-LC analogue,

N-succinimido-3-(2-pyridyl disulfide group) propionic ester (SPDP),

N-succinimido [4-vinylsulfonyl] benzoic ether (SVSB),

Succinimido 3-(bromo kharophen) propionic ester (SBAP) and

N-succinimido iodo acetic ester (SIA) and

N-succinimido (4-iodo ethanoyl) Aminobenzoate (SIAB) or its sulfo group analogue.

Any one method, wherein R during 18. aforesaid right requires ¹Be selected from alkane two bases, arylidene, alkarylene, inferior heteroaryl and alkylene heteroaryl, above-mentioned group all can be replaced and/or be interrupted by carbonyl, ester, sulphur, ether, acid amides and/or amine.

19. the method for claim 18, wherein R ¹Be C ₃-C ₆Alkane two bases.

20. any one method during aforesaid right requires wherein fully is separated derived products basically with excessive reaction reagent.

21. the method for claim 20 wherein fully is separated product acid amides or hydrazide derivatives basically with mix products.

22. the method for claim 21 is wherein finished product by the step of drying under reduced pressure removal solvent and reclaimed, described drying step is preferably freeze-drying.

23. formula III or formula VIII compound,

Wherein X is the functional group that is selected from following group: NHS-ester, vinyl sulphone, N-dimaleoyl imino, N-iodo kharophen, ortho position pyridyl disulfide group, hydroxyl, hydroxyl and protected, amino, shielded amino, carboxyl, shielded carboxyl or azido-;

R ¹Be linking group;

R ⁴Be hydrogen or C _1-4Alkyl; And

R ³Be monose, disaccharides, oligose or polysaccharide, protein, peptide, lipid, medicine or drug delivery system.

24. formula V or formula IX compound,

Wherein X is the functional group that is selected from following group: NHS-ester, vinyl sulphone, N-dimaleoyl imino, N-iodo kharophen, ortho position pyridyl disulfide group, hydroxyl, hydroxyl and protected, amino, shielded amino, carboxyl, shielded carboxyl or azido-;

R ¹Be linking group;

R ⁴Be hydrogen or C _1-4Alkyl; And

R ⁶Be monose, disaccharides, oligose or polysaccharide group.

25. the compound of claim 23 or 24, wherein R ¹Be selected from alkane two bases, arylidene, alkarylene, inferior heteroaryl and alkylene heteroaryl, above-mentioned group all can be replaced and/or be interrupted by carbonyl, ester, sulphur, ether, acid amides and/or amine.

26. the compound of claim 25, wherein R ¹Be C ₃-C ₆Alkane two bases.

27. any one compound in the claim 23 to 26, wherein R ³Or R ⁶Can be oligose or polysaccharide, be preferably low Polysialic acid or Polysialic acid.

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