Polysialic acid derivatives

FIELD: chemistry; pharmacology.

SUBSTANCE: polysaccharide, in form of polysialic acid, with at least, two sialic acid links, joined to each other at positions 2,8 and/or 2,9, and with a lateral part, linked to at least, one end link, obtained from a sialic acid link, which consists of a functional group chosen from N-maleimide groups, vinylsufonyl groups, N-iodoacetamide groups and orthopyridyl disulphide groups. Polysaccharide is reacted with a hetero bi-functional reagent, with a first functional group, chosen from N-maleimide groups, vinylsufonyl groups, N-iodoacetamide groups and orthopyridyl disulphide groups, and a second functional group, different from the first group, where the stated second functional group reacts with the stated end link, derivative of sialic acid, with formation of a covalent bond and functional polysaccharide, suitable for selective pairing with a thiol group.

EFFECT: reaction with a hetero bi-functional reagent allows for introduction of a side functional group for site-specific bonding to sulfhydryl groups, for example, to side chains of cysteic links in medicines, medication delivery systems, proteins or peptides.

29 cl, 3 dwg, 1 tbl, 4 ex

 

The present invention relates to a derivative polisialovoi acid, which are used for conjugation with drugs, proteins and peptides, or with systems deliver drugs to the site of action, such as liposomes, having a sulfhydryl group, and to methods of synthesis of the products of the merger, such as derivatives and conjugates, and new synthetic intermediates.

Enhanced presence of drugs or inside the vascular system, or application outside of the vessel is often a necessary condition for their optimal use. For example, many antibiotics and cytostatics, as well as a number of therapeutic peptides, proteins and liposomes are removed from the bloodstream before time and before can be achieved in an effective concentration in the target tissue. The elimination half-life of a number of short-lived proteins (e.g. enzymes, cytokines, etc.) increased by conjugation with polyethylene glycol. From this it is clear that the molecules of PEG prolong the circulation time of proteins and particles, covering their surface, thereby sterically hindering the interaction with the factors responsible for their clearance. However, PEG is not biodegradable and accumulation of PEG-isolation of proteins inside cells may be undesirable, especially when glutelin the m application [Bendel, A., Seely J., C. Richey, Senello G., G. Shopp Renal tubular vacuolation in animals treated with polyethylene-glycol conjugated proteins, Toxicologycal sciences, 42 (1998) 152-157; Convers C.D., L. Lejeune, K. Shum, Gilbert C., Shorr R.G.L, Physiological effect of polyethylene-glycol conjugated on stroma-free bovine hemoglobin in the conscious dog after partial exchange transfusion, Artifical organ, 21 (1977) 369-378].

The authors described the conjugation of polysaccharide with 2→or 8 and 2→9 (e.g., interleaved 2→8 and 2→9) related links sialic acid conjugated to proteins to increase their half-life, reducing their immunogenicity/antigenicity or increase the stability of a number of proteins. In WO92/22331 politologie acid is subjected to interaction with the model drugs and demonstrate increase the half-life in the bloodstream of a mouse. In Cell. Mol. Life Sci. 57 (2000) 1964-1969 and Biotechnol. Genet. Eng. Rev. 16 (1999) 203-215, Gregoriadis et al. describes the conjugation polisialovoi acids with asparaginase and catalase and it is shown that the rate of clearance from blood flow was decreased, while the enzyme activity was preserved. The authors also described polisialilirovaniya insulin (Biochim. Biophys. Acta 1622 (2003) 42-49) and shows that it is active. The authors also described polisialilirovaniya interferon (AAPS Annual meeting 2002, Toronto, Canada, M1056). The authors also described polisialilirovaniya fragment of the antibody Fab (A. Epenetos Et al., Proceedings of ASCO (Clinical Pharmacy) 21 (2002) 21186).

In all these publications polisialovoi acid gave reactio the ing ability, by obtaining aldehyde groups on nevosstanovlenie late in the oxidation of 7,8-villalbilla periodates sodium. The aldehyde group is then reacted with primary amines of proteins, which are mostly Epsilon-amino lysine residues in proteins or N-terminal amino groups. In the reaction are formed Schiff's base, which restored cyanoborohydride in a secondary amine.

In WO-A-01/87922 the authors also suggested that it is possible to perform the derivatization other molecules in the presence of a denaturing agent in order to increase the degree of derivatization. Examples of other derivatizing agents are compounds of polyethylene glycol. Were given activated connection PEG, such as trisil-PEG and complex succinimidylester ether PEG. Examples of the use of PEG-activated succinimidylester, which is believed to react with the amine groups.

PEG derivative having a functional group for combination with thiol groups, are commercially available. Functional groups can be maleinimide, vinylsulfonate, itacatharina or orthopaedically group. Since these reagents react in a special way with cysteine and since the surface proteins cysteine units less than lysine, who privatizacia is more controlled. In addition, in the absence of free cysteine in the native protein, one or more cysteine can be added by means of genetic engineering. The advantage of this method is that it allows the site-specific derivatization those parts of the protein for which it will be accompanied by minimal loss of biological activity.

Found that PEG-proteins in isolation produce Antipas antibodies, which may also affect the residence time of the conjugate in the bloodstream (T. Cheng, M. Wu, P. Wu, J. Chern, Roffer SR., Accelarated clearece of polyethylene glycol modified proteins by anti-polyethylene glycol lgM. Bioconjugate chemistry, 10 (1999) 520-528). Despite the backstory of the PEG, as parenteral applied polymer associated with therapy, you will need a better understanding of its immunotoxicology, pharmacology, and metabolism. (A.C. Hunter, S.M. Moghimi, Therapeutic synthetic polymers: a game of Russian Roulette. Drug Discovery Today, 7 (2002) 998-1001; Brocchini, S., Polymer in medicine: a game of chess. Drug Discovery Today, 8 (2003) 111-112).

For modification of sialic acid will be useful to focus directly on tirinya (sulfhydryl) groups. It is also desirable to improve the efficiency of the derivatization of sialic acid in the ways described by the authors in the above prior art, requiring high excess activity polisialovoi acid. It is also desirable to avoid the use of cyanoborohydride.

According to the SNO present invention proposes a new connection, comprising a polysaccharide having a portion of the molecule that is associated with one or each end of the link, which contains a functional group selected from N-maleinimide, vinylsulfonic groups, N-iodoacetamide groups and orthopaedically groups.

End link, which connects the molecule may be non-end polisialovoi acid or regenerating end polisialovoi acid. Usually, the end link of sialic acid is subjected to a preliminary chemical reaction for the formation of useful functional groups that can be attached reagent containing maleinimide group. The authors found that it is convenient to use a chemical process described in earlier publications, in which the formation of aldehyde groups is conducted as a preliminary stage in which a functional group through which you can attach a molecule containing maleinimide group.

In earlier publications, cited above, shows that there is non limit level, which results in an aldehyde group in the oxidation of 7,8-villalbilla periodates sodium obtaining compounds with aldehyde group in position 7. A similar reaction used in the present invention.

An alternative, not necessarily to get aldehydo the group to pampering the end link. In this case, preferably (but not necessarily) to conduct a preliminary stage in which non end decontaminated using the preliminary stages of oxidation or recovery. At the first stage of recovery turns catalog group a pampering late in the restored open ring shape having vicinality. Vicinality then oxidized by periodate sodium obtaining aldehyde group at carbon atom, pre-forming after-care group in position 7 of the end link. Where non endnote glycosyl (usually sialic acid) is not deactivated by using the pre-stage oxidation, end link will be simultaneously activated.

In the invention, the portion of the molecule, which includes a functional group can be attached directly to the link polisialovoi acid or, what is more convenient, can be attached via a bifunctional organic group, such as albandeira group, Allenova group or oligo(alkylene)albanova group or, alternatively, Oligopeptide group. Joining polisialovoi acid through a linker or part of a molecule comprising a functional group may be a group of the secondary amine, hydrazone, complex Olkiluoto ether of hydrazine or of peptidyl. The portion of the molecule can be is obtained by the reaction of the substrate polisialovoi acid heterobifunctional reagent. This method represents an additional aspect of the invention.

The reagents used for the introduction of selective functional groups are commercially available. The connection, which will enter maleinimide group in the amine portion of the molecule without the introduction of additional linker fragment is the N-methoxycarbonylmethylene. Typically, the reagents contain a second functional group for reaction with a group polisialovoi acid, which may be aldehyde group described above, or amine group. Suitable second functional group include complex N-hydroxysuccinimide esters and their sulfosuccinimidyl analogues and hydrazides. The preferred compound is a hydrazide N-maleinimide acid or hydrazide N-malenkayayulenka acid, i.e. the compound having the General formula

X-R-Y

in which:

X represents N-maleinimide, N-iodoacetamide, S-vinylsulphonyl or S-orthopaedically group,

R represents landiolol, Allenova or Aracinovo, alkalinous, alkylenediamines or alkylnaphthalenes or alkylresorcinol group; and

Y represents a hydrazide, amine or N-hydroxysuccinimide group.

Preferably, R is a C1-6Alcantara,2-3/sub> alcolocks-C2-3alkylene,2-3albularyo(oxa-C2-3alkylene) or (C2-6alkylammonium.

Preferably, X is an N-maleinimide or orthopaedically.

Preferably, Y is a hydrazide or hydroxysuccinimide.

Compounds that can react with the aldehyde group, and which include linker fragment and include maleinimide group containing hydrazide N-[β-maleinimide acid] hydrazide of 4-(4-maleinimide)butyric acid. Hydrazide group reacts with the aldehyde to the formation of stable gidrazonami group. Suitable heterobifunctional connection, which contains oligo(ethyleneoxy)ethylene group, is a compound containing polietilenglikolya (poly(ethylenoxy)) group on one end of which is N-hydroxysuccinimide (NHS) group and at the other end of the functional group. NHS group reacts with the amine groups with the formation of stable amide bonds. Heterobifunctional the glycols with the NHS at one end or vinylsulfonate or maleinimide at the other end are available. Other examples heterobifunctional reagents include: 3-(2-pyridyldithio)propionylacetate,

N-Succinimidyl-3-[2-pyridyldithio]propionate,

Succinimidyl-H-[N-maleimidomethyl]cyclohexane-1-carbox the lat,

complex m-maleinimides-N-hydroxysuccinimidyl ether,

N-Succinimidyl[4-iodoacetyl]aminobenzoate,

complex N-[gamma-maleinimides]operations ether,

complex N-[Epsilon-maleinimides]operations ether and

N-succinimidylester. Other reagents (polyethyleneglycol) available from Pierce Biotechnology, and Shearwater Corporation.

Sialic acid (known as nonulosonic acid) belong to the family of aminecontaining sugars containing 9 or more carbon atoms. The most important of sialic acid is N-acetylneuraminic acid (also known as 5-(acetylamino)for 3,5-dideoxy-D-glycero-D-Galaktionova, lactamania acid and O-sialic acid), which has the formula:

Policially acid can connect 2→8 and/or 2→9, usually in α-configuration. In some polisialovoi acid compounds are alternative 2→,8 and 2→9. The invention is also useful for heteropolymeric polysaccharides, including glucosamine links other than links sialic acid.

Found that policially acid, in General, are non-toxic and completely non-immunogenic. Moreover, it is unknown to the products of biodegradation sialic acid was toxic, really sialic acid is widely u is ostranenie in animal proteins and cells, including blood cells and circulating proteins.

Polysaccharide compounds containing a lot of links sialic acid, are produced byEscherichia coli, Moraxella nonliquifaciens, Pasteruella aeroginosisandNeisseria meningitidisor their derivatives. For example, colomina acid, obtained by hydrolysis, forming chains of shorter length) of theE. coliK1, includes α-2→8 related links sialic acid. Polysaccharide from strainE. coliK92 includes alternative 2→8 and 2→9 related links sialic acid. Polysaccharides WithN. Meningitidisgroups have 2→9 related links sialic acid.

One group of polysaccharide compounds, which, as mentioned, is partly used in the invention is the group B polysaccharide. These compounds are produced byN. Meningitidis,M. nonliquifaciens, P. AeroginosisA2 andE. coliK1. These compounds include polysaccharide component comprising units of sialic acid, and phospholipid component. Links sialic acid connected α-2→8 polymer of natural origin, including about 200 links. Some of the molecules of glycolipids, especially compounds with a high molecular weight, are covalently linked phospholipids on pampering the end polysaccharide conjugate.

For convenience, it is preferable that during the production of the bacterium from which production is out polysaccharide compound, was not pathogenic. In particular, the polysaccharide is particularly suitable for extraction from non-pathogenic strains ofE. colisuch asE. coliK92 or, preferably, K1, which is non-immunogenic. CultureE. coliK92 and K1 are well known and any such type of any such strains can be used as a suitable source of polysaccharide. Preferably, Polivanova acid had at least 2, preferably at least 5, more preferably at least 10, for example, more than 50 units of sialic acid.

According to the invention also offers protein conjugate comprising a protein and a new activated polysaccharide. New connection comprises a protein containing at least one cysteine group linked through a simple thioester bond of the side chain of cysteine link with polisialovoi acid through groups of one or both end links polisialovoi acid.

Where a derived polisialovoi acid was N-maleinimide, some molecules will include N-Succinimidyl group with kontiainen joining α-carbon atom. Preferably, the portion of the molecule contains a secondary amine, hydrazono or amide bond.

In the invention it is also proposed a new way in which policylevel acid is subjected to inter eastview with heterobifunctional reagent, having the first functional group for reaction with sulfhydryl groups and the second functional group other than the first group where the specified second functional group immediately reacts with the terminal link polisialovoi acid with the formation of the covalent bond and the formation of reactive sulfhydryl functional group polisialovoi acid.

In one embodiment, the second functional group is a nucleophilic group, preferably hydrazine. In particular, it is used where Polivanova acid contains an aldehyde group into the end link, a carbonyl group which is attacked by nucleophilic group.

In another embodiment, the method of the second functional group is an electrophilic group, such as N-amoxilonline, such as a complex N-hydroxysuccinimidyl ether or complex sulfosuccinimidyl ether, or carbodiimide. End group in this case is preferably nucleophilic, such as Amin.

In this way, it is preferable that the reagent contained bifunctional organic group connecting the first and second functional groups. Preferably, the bifunctional group selected from C2-18alkadienes group, Allenova group, oligopeptides and oligo(ALCO is si)alkyl groups.

Examples of suitable reagents listed above.

The most effective way, which includes successive stage, in which functional policylevel acid is subjected to interaction with the polypeptide or protein having at least one free and unprotected Cys link, with which the functional group forms a simple thioester bond with Tilney group of Cys-level education politiaromana.ro polypeptide or protein. How, in particular, is suitable where the protein contains a single Cys link, when this is reached the website is controlled derivatization.

The invention is illustrated in the accompanying examples.

Example 1

1.1 Synthesis

Three separate preparations were made as follows:

Aldehyde kolominova acid (CAO)obtained according to WO-A-9222331 (100 mg, 4,4×10-6mol) was dissolved in 500 μl of 0.1 m solution of sodium acetate was added to 5 mol equivalents of hydrazide N-[β-maleinimide acid] (6.5 mg), 2,2×10-5mol). The mixture was then mixed by shaking, covered with foil and kept at 37°C for 2 h on a rotary mixer. The polymer is then precipitated with addition of 2 volumes (1.0 ml) of ethanol. The precipitate was separated by centrifugation (13000 rpm for 2 min) in the table microcentrifuge. Supernat the NT was decanted and the precipitate was dissolved in 500 μl of 0.1m acetate. This procedure was repeated an additional 2 times, last precipitate was dissolved in deionized water and placed in the device for freeze-drying overnight.

1.2 analysis of the content of maleinimide

In this analysis cysteine were subjected to interaction with maleinimide on the polymer, preventing further reaction with the reagent Almana (5,5-dithiobis(2-nitrobenzoic acid)), which contains a disulfide, forming an intense yellow color, when it is replaced by a thiol, not adjacent to the aromatic ring. Thus, the content of maleinimide can be calculated by measuring the inhibition of the reaction between cysteine and reagent Almana.

The first initial reaction mass cysteine 12×10-3M (0,145 mg/ml) were prepared in phosphate buffered saline (PBS). In pure titration the microplate volume of 100 µl double-bred from 12×10-3M to 0,375×10-3M from row to row N. In a row And 100 ál of PBS was used as a zero standard. Prepared samples SA and SA-maleinimide (HIMSELF) 5 or 10 mg/ml, and 100 µl of each sample was added in a dual column to determine cysteine dilutions In one column was added 100 μl of PBS without CA as a tablet, covered and left to incubate at 37°C for 1 h and Then 20 µl of reagent Almana (4 mg/ml) was added into each of UNCW and the plate incubated in the dark at room temperature for 15 minutes Measured absorption in the wells at 405 nm. Then for samples constructed standard curves and calculated the number of maleinimide reduction signal.

1.4 Milirovanie Fab and attach to ITSELF.

In the first stage tirinya group was introduced into the model protein tonirovaniem amine of lysine.

Sheep Fab (antideprin/noetically, 4 mg, 7,2×10-3mol) was dissolved in 0.25 ml PBS+10 mm EDTA and added 0,498 mg 2-aminosilane (2-IT or reagent trout 50 mol-equivalents of 3.6×10-6mol) in 0.25 ml of the same buffer. The test tube was covered with foil and left for incubation under stirring flexible stirrer for 1 h at 37°C.

Etiolirovannye Fab was purified from free 2-IT (reagent trout) gel filtration (PD-10) and 0.5 ml fractions were analyzed for the presence of protein (BCA assay) or thiol (analysis of Almana). First suirvey peak containing protein and thiol, summarized and protein and thiol was determined quantitatively.

The conjugation of Fab-thiol with CAM.

To etiolirovannuyu Fab (3.6 mg, 6,6×10-8mol) in 2 ml of PBS/EDTA was added to 22.5 mg CAM (9×10-7mol, 15 mol-equivalents). The tube was sealed and left for incubation at 37°C for 1 h under mild stirring. The resulting conjugate is then purified in accordance with the adopted methodology for the removal of free CAM. The content of CA and Bel is in the conjugate was analyzed to calculate the degree of conjugation.

Control reactions were performed with CA as a negative control.

Prepared several series of CAM-Fab with varying degrees of etiolirovaniya, but maintaining the ratio of 15:1 CAM:Fab. The results are presented in table 1 below:

Table 1
The ratio of thiol:FabThe ratio of conjugate< / br>
(CA:Fab)
1of 0.53:1
20,9:1
5 (three times a duplicate response)1,925:1+/-0,19:1 Fab
103,51:1

Figure 1 shows the SDS-PAGE gel three duplicate samples and relevant controls.

1.6 Conclusion

The results show that all control wells (samples tarirovannogo Fab) movement of the sample is identical with the movement of the sample, freshly cooked FAb (below 50 kDa marker), showing the absence of binding of the FAb molecules during the process of conjugation. In lines replicate there is a marked lane, close to the border with maximum intensity between markers 98 and 250 kDa, which usually show the mass increases, which indicates politicalirony.com product.

1.7 Algae CAM with beta-galactosidase

To E. coli β-galactosidase (β-gal: 5.0 mg, 4,3×10- mol) in 1 ml of PBS was added 15 mg CAM (6,59×10-7mol, 15 mol-equivalents). The test tube was covered with foil and left to incubate at room temperature for 1 h under mild stirring. The resulting conjugate was analyzed by SDS-PAGE and then purified in accordance with the accepted method of removing free HIMSELF. The samples were analyzed for polymer content and protein according to the relevant procedure.

Control reactions were performed with CA as a negative control.

1.8 Analysis of the enzymatic activity

Standards from 60 µg/ml up to 3.75 µg/ml freshly prepared β-galactosidase was prepared in PBS. Sample CAMβ-gal was diluted to 60 µg/ml in the same buffer. Enzymatic activity of the conjugates was measured in the following way: in titrations the microplate, 100 μl of sample or standard was also added 100 ál β-gal substrate (perforation). The microplate was incubated at 37°C for 30 min and the absorption was read at 405 nm. A calibration curve was built using standards and activity of the samples was calculated from the equation for the curve linear regression analysis.

1.9 Results

Analysis for protein and polymer was determined that the ratio of conjugation are 1.23 CAM:1 β-Gal. According to SDS-PAGE samples were marked with a corresponding increase in apparent molecular weight Fig. 2). The calculated enzyme activity of the purified sample was 100.4% compared with the free enzyme.

Example 2

The way of synthesis 2

Stage 1: amination of CA-aldehyde (CAO)

Stage 2: Introduction maleinimide rings

2. Synthesis

2.1. Stage 1: amination of oxidized CA

Oxidized clomidbuy acid (CAO) 10-100 mg/ml was dissolved in 2 ml deionized water with a 300-fold molar excess of NH4Cl in 50 ml test tube and then added NaCNBH3(5M mother solution in 1N NaOH (aq.)) to a final concentration of 5 mg/ml of the Mixture incubated at room temperature for 5 days. A control reaction was also performed with kolominova acid (A) instead of CAO. The product was besieged in the form of amine derivative of kolominova acid by adding 5 ml ice ethanol. The precipitate was separated by centrifugation at 4000 rpm for 30 minutes at room temperature in a tabletop centrifuge. The precipitate in the test tube was kept and resuspendable in 2 ml of deionized water, and then laid siege to 5 ml ice ethanol in 10 ml ultracentrifuge vitro. The precipitate was collected by centrifugation at 30,000 rpm for 30 minutes at room temperature. The precipitate in the test tube again resuspendable in 2 ml Denisova the Noi water and dried by freezing.

2.2. Analysis on the amine content

TNBS (perilalbany acid or 2,4,6-trinitrobenzenesulfonic acid) analysis was used to determine the number of amino groups present in the product.

In well tiralongo microplate to 90 μl of 0,1M borate buffer with a pH of 9.5 was added TNBS (0,5 ál of 15 mm TNBS). To this was added 10 μl of 50 mg/ml CA-amide, the tablet was left to stand for 20 minutes at room temperature before reading the absorbance at 405 nm. Glycine was used as standard in the concentration range from 0.1 to 1 mm. TNBS trinitroaniline primary amine groups. Determined adduct TNP Amin.

Tested product, purified by double precipitation of ice ethanol, using TNBS analysis shows close to 90% conversion.

2.3. Maleinimide CA-amine

CA-amine (17 mg) was dissolved in 1 ml deionized water, to this was added 6 mg of methoxycarbonylmethylene (MCM). The mixture was left to react at room temperature for 30 minutes To the sample was added 100 μl of water and 200 μl of acetonitrile and then incubated at room temperature for 4 h, then was added 300 μl of CHCl3the test tube was shaken and the aqueous fraction was collected. Then the fraction was purified on a PD-10 column to remove small molecules. The eluate was dried by freezing and analyzed for the content is maleinimide. Molar concentration of maleinimide was 44 mol.%.

Example 3: Preparation iodoacetate derivatives of kolominova acid (CAI).

3.1 Synthesis

To 40 mg of the amine kolominova acid (85 mol.% amine)as described in Example 2.1, dissolved in 1 ml of PBS pH of 7.4, was added 5 mg of N-succinimidylester (SIA). The mixture was left to react for 1 h at 37°C, then the excess SIA was removed by gel-filtration on a 5 ml HightrapTmDesalting column (AP Bioscience) by elution PBS. 0.5 ml fractions collected from the column, samples of each fraction were tested on the content of kolominova acid (razorenova test) and reactivity with cysteine, revealing iodide (analysis of Almana). Fractions positive for both iodide and CA, United.

3.2 Conjugation CAI with β-galactosidase

To E. coli β-galactosidase (5.0 mg, 4,3×10-8mol) in 1 ml of PBS 15 mg CAI added (6,59×10-7mol, 15 mol-equivalent). The tube was closed by wrapping in foil and left to incubate at room temperature for 1 h under mild stirring. The resulting conjugate was analyzed using SDS PAGE and then purified in accordance with the accepted method of removing free CAI. Samples were analyzed on the content of the polymer and protein, as described in the methods.

Control reactions n is bodily with CA as a negative control. All samples were analyzed on a β-gal activity, as in the above example 1.8.

3.3 Conclusion

Fractions 3-6 were positive for both polymer and iodoacetate, and were United. SDS PAGE (4-12% Bis/Tris gel; figure 2) shows the increase in apparent molecular weight for the samples incubated with iodoacetamide derived, but not with the control polymer. From analyses of the protein and polymer, it was found that the ratio of conjugation is 1,63 CAI:1 β-gal. It is calculated that β-gal activity is to 100.9% for the conjugate of the sample compared with the free enzyme.

Example 4

4.1 Synthesis: Activation of non end with a hydrazide of 4-(4-N-maleinimide)butyric acid (CA-MBPH) and 3-(2-pyridyldithio)propionitrile (CA-PDPH)

The synthesis was performed as follows:

Aldehyde kolominova acid (CAO; an increase of 22.7 kDa, Camida, Ireland), obtained according to WO-A-9222331 (73 mg for MBPH tube A; 99,3 mg for PDPH; tube B), was dissolved separately in 800 μl of 0,1M solution of sodium acetate (pH 5.5). In test tube A was added 15 mg MBPH (linker:CA is 15:1), dissolved in 200 μl DMSO. In test tube B was added 15 mg (linker:CA is 15:1) PDPH, dissolved in 200 μl DMSO. In each case, for each reaction vessel, the pH was brought to 5.5. These mixtures were then mixed by shaking, covered with foil and astable and for incubation at 37° C for 2 h on epicycle stirrer. The solution of each polymer was purified by gel-filtration (column PD-10), was suirable PBS at pH 7.4 and were collected 1 ml fractions containing CA (razorenova test). The samples were dried by freezing during the night and analyzed for the content of maleinimide as described in example 1.2.

4.2 Synthesis: Activation of the reducing end with a hydrazide of 4-(4-N-maleinimide)butyric acid (MBPH-CA), (2 pyridyldithio)propionitrile (PDPH-CA) and hydrazide N-β-maleinimide acid (BMPH-CA).

All syntheses were performed at a molar ratio of 25:1 linker to the CA as follows:

Clomidbuy acid (CA; 22,7 kDa; 73 mg for MBPH tube A; 99,3 mg for PDPH; tube B, and 76.6 mg for BMPH; tube C) was dissolved separately in 800 μl of 0,1M sodium acetate (pH 5.5). Added to test tube A 25 mg MBPH (dissolved in 200 μl DMSO), in test tube B 25 mg PDPH (dissolved in 200 μl DMSO) and in the vessel C 25 mg BMPH (dissolved in 200 μl of acetate buffer). the pH of the reaction mixture is brought to 5.5. Each mixture was then mixed by shaking, wrapped in foil and left for incubation at 37°C for 72 h on epicycle stirrer. Each polymer solution was purified by gel-filtration (column PD-10), elwira PBS at pH 7.4 and were collected 1 ml fractions containing CA (razorenova test). These samples were dried by freezing during the night and analyzed for the contents of maleinimide, as described in example 1.2.

4.3 Results

Molar concentration of maleinimide on non (reactive) the end was 49,0 and to 35.0 mol.% for MBPH and PDPH, respectively. Molar concentration of maleinimide a pampering (laborative) end amounted to 41.5, 32,5 and 48.3 mol.% for MBPH, PDPH and BMPH, respectively. Value on pampering the end are the average of the two values in each case.

4.4 Algae β-galactosidase (β-gal) with maleinimide, activated kolominova acid (regenerating end and non end)

It β-gal (1.0 mg; 1 ml in PBS) in a separate test tube was added a 15 molar excess of each maleinimide activated CA (MBPH, PDPH and BMPH on non or pampering the end, in the examples above). Each tube was sealed and left for incubation at 37°C for 1 h under mild stirring. The resulting conjugate is then purified in accordance with the adopted methodology for the removal of free activated polymer. All samples were analyzed using SDS PAGE β-gal activity, as in example 1.8.

4.5 Results

The results (figure 3) show that all the test holes (with β-gal) movement of the sample are similar to freshly prepared β-gal. In lines replicate there is a notable bands who, bordering the maximum intensity between markers 98 and 250 kDa, which usually indicate an increase in mass, which is politicalarena β-gal. The calculated activity β-gal was 91,0-106% for conjugated samples.

1. Polysaccharide, representing policylevel acid having at least two link sialic acid attached one to another in the position of 2.8 and/or 2.9, and having a lateral portion of the associated at least with one end of the link obtained from the level of sialic acid, which includes a functional group selected from N-maleinimide groups, vinylsulfonic groups, N-iodated groups and orthopaedically groups.

2. The polysaccharide according to claim 1, in which the side connected with restoring an end link of the polysaccharide.

3. The polysaccharide according to claim 1 or 2, wherein a portion of the molecule is connected with a non terminal link of the polysaccharide.

4. The polysaccharide according to claim 1, in which the portion of the molecule contains landiolol and/or Allenova group and communication, which is a secondary amine linkage, hydrazono, acylhydrazone bond or a peptide bond, optionally in combination with oxyalkylene or oligoastrocytomas group.

5. The polysaccharide according to claim 1, in which the functional group is N-maleinimide.

6. The policy shall harid according to claim 1, in which the polysaccharide is Polivanova acid.

7. The polysaccharide according to claim 6, in which the polysaccharide preferably consists essentially only of units of sialic acid.

8. The polysaccharide according to claim 1, which has the formula

where (i) R1represents H or-SNONCE2HE, R2is a HE, and R3is or-CH2CHR4R5or-CH(CH2OH)CHR4R5where R4and R5together represent =N-NR6or R4is H and R5is-NR6R7where R6is an organic group containing the specified functional group selected from N-maleinimide, vinylsulfonic, N-iodoacetamide and autodependency group or H, and R7is H, or R6and R7together represent 1,3-buta-2-andyellow group;

ii) R1and R2together represent =N-NR6or R1is N, and R2is-NR6R7where R6is an organic group containing the specified functional group selected from N-maleinimide, vinylsulfonic, N-iodoacetamide and autodependency group or H, and R7is H, or R6and R7together represent 1,3-buta-2-andyellow group;

Gly-O is part of sialic acids;

n equal 1-74; and

AC is acetyl.

9. The polysaccharide of claim 8, in which each Gly is part of sialic acid.

10. Polysaccharide containing protein with at least one free cysteine link and is connected through sluinotify bond with the side chain of cysteine link polisialovoi acid attached to one or each end of the link polisialovoi acid, where Polivanova acid has at least two links of sialic acid attached one to another in the position of 2.8 and/or 2.9 and having a lateral portion of the associated at least with one end of the link obtained from the level of sialic acid, which includes a functional group selected from N-maleinimide groups, vinylsulfonic groups, N-iodated groups and orthopaedically groups.

11. The polysaccharide according to claim 1 or 10, in which the polysaccharide has at least 10 units of sialic acid.

12. The polysaccharide according to claim 1 or 10, which has at least 50 units of sialic acid.

13. A method of obtaining a functional polysaccharide, in which the polysaccharide having at least two interconnected link sialic acid attached in position 2.8 and/or 2.9, and containing at least one terminal element, which is a derivative of sialic acid, is subjected to the interaction with heterobifunctional R the agent, having the first functional group selected from N-maleinimide groups, vinylsulfonic groups, N-iodated groups and orthopaedically groups and the second functional group and second functional group other than the first group where the specified second functional group interacts with the specified terminal link, a derivative of sialic acid, with the formation of the covalent bond and the formation of functional polysaccharide suitable for selective conjugation with Tilney group.

14. The method according to item 13, in which the specified second functional group is a nucleophilic group.

15. The method according to 14, in which the nucleophilic group is hydrazine.

16. The method according to item 13, in which the end link of the polysaccharide has a carbonyl group, which interacts with the specified nucleophilic group.

17. The method according to item 13, in which the specified second functional group is an electrophilic group.

18. The method according to 17, in which the electrophilic group is an N-alkoxycarbonylmethyl or carbodiimide group.

19. The method according to 17, in which the end link of the polysaccharide has an amine group, which interacts with the specified electrophilic group.

20. The method according to claim 19, in which is formed a peptide or urethane link.

21. The method according to item 13, in which the reagent sod is RIT bifunctional organic group, connecting the first and second functional groups.

22. The method according to item 21, in which a bifunctional organic group selected from C2-18alkadienes group, Allenova group, oligopeptides groups and oligo(alkoxy)alkyl groups.

23. The method according to item 13, in which the first functional group is N-maleinimide group.

24. The method according to item 13, in which the reagent has the General formula

X-R-Y,

in which X represents N-maleinimide, N-iodated, S-vinylsulphonyl or S-orthopaedically group,

R represents landiolol, Allenova or Aracinovo, alkalinous, alkylenediamines or alkylnaphthalenes, or alkylresorcinol group; and

Y represents a hydrazide, amine or N-hydroxysuccinimide group.

25. The method according to item 13, in which the polysaccharide has at least 10 units of sialic acid.

26. The method according A.25, in which the polysaccharide has at least 50 units of sialic acid.

27. The method according to p. 25 or 26, in which the links of sialic acid have 2→8 and/or 2→9 linking with each other.

28. The method of obtaining politiaromana.ro polypeptide or protein, in which maleimidopropionamide policylevel acid according to item 23 is subjected to interaction with the polypeptide or protein having at least one free unprotected Cys link where maleinimide group forms a simple thioester bond with Tilney group of Cys-level education politiaromana.ro polypeptide or protein.

29. The method of obtaining the derivative of a polypeptide or protein with a polysaccharide-based, in which the polysaccharide according to claim 1 is subjected to interaction with the polypeptide or protein having at least one free unprotected Cys link where the specified functional group forms a simple thioester bond with Tilney group of Cys-level, with the formation of conjugate polysaccharide with a polypeptide or protein.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to medicine and describes method of glycosaminoglycan release from mineralised connective tissue, containing enzymatic hydrolysis, deproteinisation, deposition, and characterised by the fact that demineralisation is performed within 20 h using 0.5 H HC1, enzymatic hydrolysis is performed within 18 h using pepsin enzyme, deproteinisation is performed by adding of ammonium sulphate to 75% saturation, glycosaminoglycans are deposited with 4% potassium acetate in 96% ethanol, re-deposited with ethanol and lyophilised. Glycosaminoglycans are exposed to chromatographic separation using "ДЕАЕ"-sephadex A-25, and lyophilised. This method is easy-to-use, realised in laboratory environment of patient care institutions, and time-saving.

EFFECT: easy and saving method of glycosaminoglycan release from mineralised connective tissue.

1 ex, 2 dwg

FIELD: organic chemistry.

SUBSTANCE: graft method of functional group to polysaccharide polymer of galactomannan type, preferentially to guar by means of high-energy electron beams radiation with non-saturated monomer occurrence containing specified functional group. Method can contain depolymerization of graft-polymer up to specified molecular weight. Prefential galactomannan processed under this method is guar gum, guar endosperm and hydroxypropylguar. According to preferential variant of method realization guar gum is also depolymerized, preferentially to molecular weight lower than approximately 700000 daltons and the most preferentially to molecular weight from approximately 100000 daltons to approximately 250000 daltons. According to the most preferable variant depolymerized guar has polydispersity lower, approximately 3.0. Graft-polysaccharide is applied in cosmetic formulations such as moisturizing stick.

EFFECT: possibility for functional groups graft to polysaccharide polymer by means of high-energy electron beams radiation.

17 cl, 1 dwg, 6 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: method for depolymerisation of polysaccharides of galactomannan, as well as xanthan, type, preferably galactomannans, to specified molecular weight by high-energy electron beam treatment. Preferential galactomannans for the said type of treatment are guar gum, guar endosperm and hydroxypropylguar. According to preferential version of the method, guar gum is depolymerised, preferably, to molecular weight of ca. 100000 daltons to ca. 200000 daltons.

EFFECT: depolymerised guar has lower polydispersity and can be used for oil well fracturing in order to gain in oil yield.

16 cl, 15 tbl, 6 dwg

FIELD: food industry; oat enzymatic treatment.

SUBSTANCE: using this method it is possible to obtain new improved oat products containing modified starch, the products have increased glucose and β-glucan content. The present invention is also related to the food products and food compositions, including oat with modified starch or oat liquid containing modified starch.

EFFECT: use of enzymes at their optimal temperatures; efficiency and effectiveness.

24 cl, 1 dwg, 1 tbl, 3 ex

FIELD: biotechnology, microbiological industry.

SUBSTANCE: invention relates to a novel culture of microorganism producing high-molecular exopolysaccharide. Invention proposes the strain of microorganism Paracoccus denitrificans VKPM B-8617 that produces exopolysaccharide possessing cross-linking properties in aqueous and water-containing hydrocarbon systems. Exopolysaccharide is formed by residues of glucose, galactose, mannose and rhamnose in the ratio = 52:4:1, respectively, and comprises glucuronic and pyruvic acids, and acyl groups also and has molecular mass (0.5 x 106)-(2 x 107) Da. This exopolysaccharide is able to form pseudoplastic and thixotropic highly viscous solutions showing stable values of dynamic viscosity in the range of temperature from 20°C to 90°C and unstratifying emulsions. Proposed exopolysaccharide can be used in building, paper, textile, perfume-cosmetic, food, chemical, oil- and gas-extracting industry, agriculture, and in pharmaceutics and medicine.

EFFECT: valuable properties of strain and exopolysaccharide.

2 cl, 9 ex

FIELD: technology of natural substances.

SUBSTANCE: invention relates to a method for isolating pectin from vegetable raw used for production of products of prophylactic and curative designation. Invention describes a method for preparing pectin. Method involves treatment of preliminary milled vegetable raw, for example, dried sunflower heads with a mixture of chloroform and ethanol followed by extraction in water bath at 70-80°C for 30-40 min, filtration and separation of the first extract. Filtered off vegetable raw is poured with distilled water at temperature 60-70°C and extracted in water bath at temperature 90-95°C for 1 h followed by filtration and separation of the second extract. Filtered off vegetable raw is poured with 0.3-0.5% ammonium oxalate solution, extracted in water bath at temperature 75-80°C for 30-40 min, filtered and the third extract is separated. Filtered off vegetable raw is poured with a 0.4-0.5% hydrochloric acid solution, heated at temperature 75-80°C for 1 h followed by filtration and separation of the fourth extract that is mixed with prepared first, second and third extracts. Prepared solution is neutralized with ammonium hydroxide to pH 7 and precipitated with 96% ethyl alcohol, precipitate is squeezed our and dried at temperature 4-6°C. Proposed method provides increasing yield of pectin and its solubility.

EFFECT: improved preparing method.

2 cl, 1 ex

FIELD: chemical technology.

SUBSTANCE: invention relates to the inclusion complex of cyclodextrins with elemental sulfur. Complex can be prepared using different homologues of cyclodextrins, for example, beta- and gamma-cyclodextrins and hydroxypropylated forms of gamma- and beta-cyclodextrins. Proposed complex can be used as a biologically active compound for medicinal, veterinary and agricultural designation. Invention provides the possibility for further preparing true solutions of elemental sulfur in water in the concentration up to 250-300 mg/l.

EFFECT: improved preparing method, valuable properties of complex.

7 cl, 7 ex

FIELD: medicine, polymeric materials, pharmacy.

SUBSTANCE: invention describes biomaterial as a deposit comprising at least one suitable anion-active polymeric water-soluble component and amphiphilic component of ammonium type comprising a cationic surfactant. Deposit is prepared in the following steps: (1) contacting anion-active polymeric component and cyclodextrin component in aqueous medium, and (2) addition of the above said amphiphilic component of ammonium type to mixture prepared in the step (1) wherein components are taken in amounts providing formation of above said deposit. Biomaterial is used for preparing a depot-composition with regulated release and acceptable for prolonged feeding pharmaceutical substances. Also, invention describes a medicinal agent comprising biomaterial and representing a sheet material or film for closing and healing wounds, or surgery thread, and medicinal implant, or insert comprising the preliminary molded biomaterial.

EFFECT: improved preparing method, valuable properties of material.

27 cl, 34 ex

FIELD: chemistry of polymers.

SUBSTANCE: invention describes a composition for using as a conditioner and comprising water and at least one cationic polygalactomannan or derivative of cationic polygalactomannan. A lower limit of average molecular mass of these cationic substances is 5000 Da and the upper limit is 200000 Da, transparency value of 10% aqueous solution is above 80% at wavelength 600 nm. The protein content is less 1.0% as measured for polysaccharide mass and the content of aldehyde groups is at least 0.01 ml-eqiv./g. Residue for modification of cationic derivative of polygalactomannan is chosen from group comprising alkyl, hydroxyalkyl, alkylhydroxyalkyl and carboxymethyl wherein alkyl comprises in chain from 1 to 22 carbon atoms, Hydroxyalkyl is chosen from group comprising hydroxyethyl, hydroxypropyl and hydroxybutyl, and cation residue is chosen from compounds of quaternary ammonium but with exception of hydroxypropyl trimethyl ammonium chloride. Proposed compositions are used for preparing a clear final product, for example, for personal hygiene agent and household chemistry agent.

EFFECT: improved preparing method, improved and valuable properties of composition.

55 cl, 11 tbl, 14 ex

FIELD: chemical-pharmaceutical industry.

SUBSTANCE: invention relates to a method for synthesis of arabinogalactan-base sulfated biopolymers wherein arabinogalactan is the main polysaccharide from Siberian larch. Invention describes a method for preparing sulfated derivatives of arabinogalactan that involves interaction of arabinogalactant in dimethylsulfoxide medium in the ratio 1:3 with a sulfating agent representing complex SO3 - dimethylformamide in the concentration of SO3 ≥18%. The process is carried out at continuous stirring, at temperature 20°C for 30 min followed by isolation of product both in acid and saline forms. The substitution degree of biopolymer macromolecule is 8.1-12.65% as measured for the quantitative content of sulfur. Synthesized sulfated derivatives of arabinogalactan retain the structural organization, water-solubility and membranotropicity of natural polysaccharide, and elicit high physiological (anticoagulating and hypolipidemic) activity also.

EFFECT: improved method of synthesis.

2 cl, 3 dwg, 3 ex

FIELD: technological processes; pharmacology.

SUBSTANCE: antagonist of human interleukine-1 receptor is prepared with the help of recombinant strain E. coli that contains plasmid, which provides production of this protein. For this purpose the strain is cultivated. Then from bacterial cells the target product is separated with application of three-stage chromatography and concentration on hydrophobic sorbent. At that the first and the third stage of mentioned chromatographic purification is performed on cation-exchanging resin, and in the second stage of chromatography the anion-exchanging resin is used.

EFFECT: application of invention allows to prepare antagonist of human receptor of high purity.

7 cl, 1 tbl, 4 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention group refers to compositions containing hapten-carrier conjugate within arranged and repeating matrix, and method of related composition production. Offered hapten-carrier conjugate used for induction of agent-specified immune reaction in case of addiction or abuse, contains cortex particle including at least one first apposition site, where specified cortex particle is virus-like particle of RNA-phage, and at least one nicotine hapten with at least one second apposition site, where specified second apposition site is associated by at least one covalent non-peptide bond with specified first apposition site, thus forming arranged and repeating hapten-carrier conjugate. Offered conjugates and compositions under this invention can include virus-like particles connected to various haptens including hormones, toxins and agent, especially agents causing addiction, as nicotine and can be applied for induction of hapten immune reaction for therapeutic, preventive and diagnostic purposes.

EFFECT: vaccines can induce stable immune reactions for nicotine and fast reduce nicotine availability for brain absorbing.

31 cl, 6 dwg

FIELD: medicine; pharmacology.

SUBSTANCE: invention relates to the composition containing the virus-like particle AP205 (VLP) and antigen. The invention also relates to the method of production of the antigen or antigen determinant, bound to VLP AP205. Antigen- bound VLP AP205 are used for production of compositions for immune response induction. The compositions are used in prevention or treatment of diseases, abnormalities or conditions, including infectious diseases, allergy, cancer, drug abuse, intoxications.

EFFECT: invention is used for efficient induction of auto-specific immune responses.

43 cl, 15 dwg, 1 tbl, 13 ex1

FIELD: medicine; pharmacology.

SUBSTANCE: novel micellar preparation is invented, which contains the anticancer agent with increased water-solubility, and which provides its high concentration in blood after the intravenous injection. The micellar preparation is produced from block copolymer with the general formula (1): where. R1 identifies the atom-methyl group; R2 identifies the tri-methyl group; R3 identifies the methylen group; R4 identifies the acetyl group; R5 identifies the hydroxyl group, optionally substituted by aryl C2-8alcoxyl group, substituted C1-4alkylamino group or amino group, containing the residue of amino acid or peptide derivative; n has integral values from 5 to 1000, m has integral values from 2 to 300, and x has integral values from 1 to 300; provided, percentage of hydroxyl groups in R5 is from 0 to 99%, and x has value not more than m; and taxane anticancer agent at concentration from 0.1 to 50 weight% per total mass of the agent and block copolymer.

EFFECT: preparation has high medical activity and reduced side effects.

7 cl, 4 tbl, 3 ex

FIELD: biotechnology, biochemistry.

SUBSTANCE: method involves two-step preparing a complex consisting of protein to be isolated and purified with propeptide of this protein immobilized on sorbent and isolation of protein by elution from the prepared complex. At the first step of preparing a complex propeptide of protein to be isolated and purified is prepared wherein this propeptide is immobilized on sorbent. At the second step of preparing a complex the protein to be isolated and purified is contacted with its propeptide immobilized on sorbent by passing solution of this protein through a filled column. Method provides expanding assortment of agents used for isolation and purification of proteins, to simplify the process and to enhance its effectiveness based on increasing yield of the end product.

EFFECT: improved method of isolation and purification.

7 cl, 3 ex

FIELD: medicine, biotechnology, pharmaceutical industry.

SUBSTANCE: method involves increasing part of the most active conformation of a glycosylated recombinant protein secreted by mammalian cell by its contact with a reagent for coupled oxidation-reduction. The proposed promotion method of the most active conformation of protein is used in a method for preparing a glycosylated recombinant protein in its the most active conformation. Configuration isomer of protein prepared by the indicated method for preparing a glycosylated recombinant protein in it's the most active conformation used in a method for preparing the protein composition for its administration to user and/or a patient or for consumption by user and/or patient. Using of the proposed invention provides enhancing activity of glycosylated protein prepared by a method of recombinant DNAs using a mammalian cell.

EFFECT: improved preparing method, valuable properties of protein.

27 cl, 9 dwg, 2 tbl, 3 ex

FIELD: organic chemistry, peptides.

SUBSTANCE: invention proposes a method for labeling phosphorylated peptides, method for adsorption of phosphorylated peptides and compounds possessing high coordination capacity with respect to phosphorylated peptides that are used in such methods, and compounds used as parent substances. Chelating compound is represented by the formula (I): wherein X represents linker group; Y represents fluorescent group or biotin as labeling group. Compound of the formula (I) provides easy detection and identification of phosphorylated peptide in a sample prepared from a living body.

EFFECT: improved methods of preparing and analysis.

13 cl, 9 dwg, 8 ex

FIELD: pharmaceutical chemistry, chemistry of peptides, hormones.

SUBSTANCE: invention relates to a method for preparing analogs of adrenocorticotropic hormone (ACTH) (4-10) possessing neurotropic activity. Method for preparing analogs of adrenocorticotropic hormone (ACTH), a sequence (4-10), of the general formula (I): A-Glu-His-Phe-Pro-Gly-Pro-OH (I) wherein A means hydrogen atom (H), Met, Met(O), Lys, Ser, Trp, Ala, Gly, Thr is carried out by liquid-phase method by step-by-step splicing peptide chain beginning from C-terminal protected tetrapeptide of the formula: H-Phe-Pro-Gly-Pro-OH (II) wherein X means a protective group and using corresponding fully protected amino acids in activated form followed by removal of protective groups at each step and purification of the end product by liquid chromatography. Method provides simplifying the process and to enhance the yield of the end product.

EFFECT: improved preparing method.

5 cl, 1 tbl, 5 ex

FIELD: biochemistry, medicine, allergology.

SUBSTANCE: invention relates to a method for preparing the hypoallergic birch pollen basic allergen r Bet v 1. Method involves one or more steps of chromatography purification using essentially non-buffered aqueous bases as an eluent and the following neutralization. Hypoallergic basic allergens from birch pollen distinct by absence or decreased binding of immunoglobulin E and simultaneous retaining therapeutically relevant stimulation of T cells. Therefore, prepared preparation can be used as a therapeutic agent with reduced adverse effects for carrying out the specific immunotherapy.

EFFECT: improved and valuable properties of allergen, improved preparing method.

27 cl, 6 dwg, 1 tbl, 6 ex

FIELD: biochemistry, medicine, allergology.

SUBSTANCE: invention relates to a method for preparing the hypoallergic birch pollen basic allergen r Bet v 1. Method involves one or more steps of chromatography purification using essentially non-buffered aqueous bases as an eluent and the following neutralization. Hypoallergic basic allergens from birch pollen distinct by absence or decreased binding of immunoglobulin E and simultaneous retaining therapeutically relevant stimulation of T cells. Therefore, prepared preparation can be used as a therapeutic agent with reduced adverse effects for carrying out the specific immunotherapy.

EFFECT: improved and valuable properties of allergen, improved preparing method.

27 cl, 6 dwg, 1 tbl, 6 ex

FIELD: chemistry; medicine.

SUBSTANCE: description is given of a conjugate of hydroxyalkyl starch and a low-molecular substance, in which the binding interaction between the molecular hydroxyalkyl starch and the low-molecular substance is based on a covalent bond, which forms from a bonding reaction between (i) an end aldehyde group or carboxyl group, formed due to selective oxidation of the end aldehyde group, or activated carboxyl group, obtained from conversion of the carboxyl group of hydroxyalkyl starch molecules and (ii) a functional group of a low-molecular substance, chosen from a group, consisting of an amino group, carboxyl group, thiol group and a hydroxyl group, which is reactive, relative the given aldehyde group or carboxyl group or an activated carboxyl group of hydroxyalkyl starch molecules. The bond directly formed from the bonding reaction can be modified for further reaction until formation of the above mentioned covalent bond, if necessary.

EFFECT: use of the conjugate allows for increasing duration of the presence of the low-molecular substance in blood plasma.

35 cl, 29 ex

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