Process for producing polyvalent and physiologically degradable carbohydrate receptor blockers by enzymatic glycosylation reactions, and uses thereof for manufacturing carbohydrate components

Translation of description of equivalent WO 9534673 A1

description

Process for the preparation of polyvalent carbohydrate and physiologically degradable by enzymatic Giycosylierungsreaktionen receptor blockers and the use thereof for the production of carbohydrate building blocks

The present invention relates to a manufacturing method by enzymatic Giycosylierungsreaktionen of polyvalent carbohydrate receptor blocker polymer-based, which still cause in vivo, either in its entirety in the form of degradation products intolerance reactions, as well as their use for the production of carbohydrate building blocks.

The importance of carbohydrates as an information carrier at physiologically relevant recognition processes has been studied and decrypted in more detail in recent years. The arrangement on the cell surface in the form of ligands allows them due to their binding to specific receptors, a vital role in intercellular communication and thus to play in intercellular recognition processes. Carbohydrate ligands on cell surfaces are recognition domains for viruses, bacteria, toxins, and lectins. Therefore, they play a crucial role as bacterial and viral infections and the induction of inflammatory processes such as rheumatoid arthritis, allergies, Nachinfarktsyndrom, shock, stroke, sepsis. Studies have shown that inflammatory processes the expressed endothelial selectins in vivo mediated adhesion of leukocytes via a carbohydrate ligand on inflammation.

Of particular importance for cell adhesion are sialylated and / or fucosylated carbohydrates such as sialyl Lewis X and sialyl-Lewis A. The therapy of inflammatory diseases with free oligosaccharides that are to bind in place of the natural ligand to receptors, fails because of the very high amounts of administered oligosaccharide, since the affinity between the receptor and the oligosaccharide is low (K_D ~ 10^"4M in the interaction between a monovalent galactoside lectin and the corresponding DT Connolly et al. J. Biol. Chem. 257. 939 (1982)).

Divalent structures with some better binding to the respective receptor are described by Wong et al. (J. Am. Chem. Soc. 115. 7549 (1993) and in US 5,254,676 described.

It is also known that an increased interaction between the receptor and ligand by coupling a plurality of ligands is accomplished on a surface. Using the example of the virus hemaglutinin protein, which binds to sialic acid on the cell surface, could be shown, as these polyvalent effect significantly affecting the ligand-receptor interaction by using a polymer (monovalent K_D = 2x20^"4M, polyvalent K_D = 3x10^'7M, A. columns Stone et al. J. Am. Chem. Soc. 113. 686 (1991)).

J. Polym Be .: Part A, polyacrylamides (RC Rathi et al as surfaces of liposomes have so far (WO 91/19502 N. Yamazaki, Int J Biochem 24, 99 (1991);..; WO 91/19501).. :... Polym Chem 29, 1895 (1991), S.-L. Nishimura et al Macromolecules 24, 4236 (1991)), polylysine or sulfated polysaccharides used. These polyvalent structures have the disadvantage either in vivo not very stable or to be incompatible by degradation into toxic metabolites in vivo. In polylysine or sulfated polysaccharides occur nonspecific interactions with cell surface structures. In the European Offenlegungsschriften 0,089,938, 0,089,939 and 0,089,940 carbohydrate compounds of varying chain length are described that respectively identical with the ligands located on cell surfaces located on the microorganisms receptors. The invention idea is that located ends to the microorganisms to block receptors by the carbohydrate compounds in vitro and in vivo to diagnose and treat illnesses can. The carbohydrate compounds may be coupled to a carrier. It is used inter alia for the production of antibodies. In WO 92/02527, a coupled to a solid support Oligosaccharidbaustein is also disclosed, which is used to diagnose inflammatory processes. The solid support is inert to physiological systems, is therefore not degraded physiologically.

In EP 0601417 A2, however, a physiologically degradable polyvalent carbohydrate receptor blocker based on polymers is disclosed, which carries on the polymer surface Oligosaccharidbausteine. An improved effectiveness as drugs is caused by the increased interaction of the polyvalent present on the polymer surface carbohydrate building blocks with receptors and by blocking of specific structures.

The described in the aforementioned EP 0601417 A2

Carbohydrate receptor blocker is physiologically acceptable and preferably has a molecular weight <70 kDa.

Specifically, the method disclosed in the aforementioned EP 0601417 A2, physiologically acceptable and pysiologisch degradable carbohydrate receptor blocker polymer-based structure:

Carbohydrate side chains - Spacer - hydrophilic polymer biodegradables - (optionally) potentiator,

said from 1 to 20 naturally occurring identical or different monosaccharide existing carbohydrate side chains over several bifunctional spacer natural or synthetic origin are linked to a hydrophilic, biodegradables polymer one or, wherein the hydrophilic, biodegradable polymer optionally a potentiator is connected, which consists of one or more groups with hydrophobic, hydrophilic or ionic properties is, a crosslinker, or a solubility enhancing agent is.

The Kohlenhγdratanteil of carbohydrate receptor blocker according to EP 0601417 A2 may for example consist of the following sugar residues:

Galß1-4GlcNAc-; Galß1-3GlcNAc-; SAσ2-6Galß1 -4GlcNAc-; SAσ2-3Galß1 -4GlcNAc-; SAσ2-3Galß1-3GlcNAc-; Galßl -4 (Fucσ1 -3) GlcNAc; Galßl -3 (Fucσ1 -3) GlcNAc; SAσ2-3Galß1 -3 (Fucσ1 -4) GlcNAc; SAσ2-3Galß1 -4 (Fucσ1 -3) GlcNAc;

Further examples of preferred embodiments of the carbohydrate portion are:

Sialyl Lewis X, sialyl Lewis A, VIM-2 and the following

Blood group determinants Lewis A, B, X, Y and A-type¹ , A-type², B-type¹ .

B-type² and H-type¹, H-type² (RU Lemieux, Chem. Soc. Rev., 1978, p 423 and

1989, p 347)

Examples of the particularly preferred embodiment of the carbohydrate portion are sialyl Lewis X, sialyl Lewis A or VIM-2.

The formula of sialyl Lewis X is: NeuNAcσ2-3Galß1-4 (Fucσ1-3) GlcNAc and sialyl Lewis A: NeuNAcσ2-3Galß1-3 (Fucσ1-4) GlcNAc. The formula of VIM-2 is: NeuNAcσ2-3Galß1 -4GlcN Acß1 -3Galß1 -4 (Fucσ1 -3) GlcNAc; Said EP 0601417 A2 discloses a method for producing the carbohydrate receptor blocker.

The synthesis of the carbohydrate receptor blocker is carried out by the process described on a laboratory scale. This means that the carbohydrate receptor blocker invention is synthesized in milligram Close up grams Enge, while necessary for its synthesis intermediates, ie the hydrophilic biodegradable polymer, the bifunctional spacer and the potentiator in gram can be made to the kilogram Enge. However, an exception to the carbohydrate moiety. He can only be synthesized in milligram tightness up to a gram. Here, the known from the literature synthesis approaches to the production of oligosaccharides are applied so that after a reaction which usually does not proceed in quantitative yield, the product mixture obtained is purified on silica gel saulcnchromatographisch.

This purification process is for the production of amounts corresponding to the industrial needs, generally too expensive and complex, and is most used for the purification of final products or valuable intermediates. Moreover, it is very common heavy metal compounds for the synthesis of oligosaccharides. Their use for the synthesis of substances having a pharmaceutical action is very questionable in view of the future approval of the carbohydrate receptor blocker as a medicament.

In the method described, the desired oligosaccharide is linked only after the structure of which numerous chemical and / or chemoenzymatic Syntheseestufen means of the spacer to the biodegradable polymer. This synthesis is due to the specialist admitted problem of the structure of oligosaccharides (protecting groups Anomerenbildung, poor yields at Giycosylierungsreaktionen not stereoselective glycosylation, numerous stages) very tedious and difficult. Given the very high burden to be operated consecutive reaction and purification steps in the chemical and / or chemoenzymatic synthesis of carbohydrate building blocks, some solid phase syntheses have been proposed lately.

In contrast to the established solid phase synthesis of oligonucleotides and peptides, the chemical synthesis of oligosaccharides on polymeric solid phase by the plurality of functionalities and the need for stereoselective knotting the glycosidic bond is very difficult. Danishefsky et al. (Science, 260 1307 (1993)) linked 3,4-protected glycal via Siiyether bonds with a polystyrene copolymer. This is activated as epoxy and can be linked to further glycal acceptors to oligosaccharide. Douglas et al. (J. Am. Chem. Soc. 1 13, 5095 (1991) describe the synthesis of di- and trisaccharides of glucose at a block PEG-bound. Zehavi (J. Am. Chem. Soc. 95, 5673 (1973)) used as polymeric solid phase, a photosensitive styrene-divinylbenzene copolymer. The protected oligosaccharide is cleaved by irradiation of polymer.

Disadvantage of solid phase chemical synthesis of oligosaccharides include., For example T. incomplete Giycosylierungsreaktionen few Glycosγlierungsbausteine ​​both on the donor and the acceptor are suitable. Need of the particular reaction matched protecting groups.

These disadvantages of the chemical synthesis of oligosaccharides on polymeric matrices are avoided by the enzymatic glycosylation. The reactions proceed without protecting groups stereoselectively absolutely and are very widely used by the large number of available glycosyltransferases and nucleotide-activated sugars as glycosyl.

As early as 1980 describe Nunez and Barker (Biochemistry 19, 489 (1980) enzymatic galactosylation of a hexanol spacer to agarose-bound N-acetylglucosamine. However, very large amounts of enzyme used to galactosyltransferase. U. Zehavi describes the enzymatic galactosylation with galactosyltransferase in both light-sensitive water-insoluble and water-soluble polymers. The transfer yields are with <1 to a maximum of 36% very low (U. Zehavi et al., Carbohydrate Res. 124. 23 (1983), U. Zehavi et al. Carbohydrate Res. 128. 160 (1984), U. Zehavi Reactive Polymers f>, 189 (1987), U. Zehavi et al. Glycoconjugate J. 7, 229 (1990), U. Zehavi innovation Perspect. Solid phase Synthesis Collect. Paper, Int. Symp., 1990, 389-396). The disaccharide obtained at the polymer is cleaved by the action of light or through an enzyme (U. Zehavi et al. Carbohydrate Res. 133. 339 (1984)) from the polymer.

Nishimura (Nishimura et al. Biochemical and Biophysical Research Comm. 199, 249-254 (1994))) describes the enzymatic preparation of a water-soluble polyacrylamide with 3'-sialyl-N-acetyllactosamine side chains, wherein a water-soluble, N-acetylglucosamine-carrying polyacrylamide is glγcosγliert gradually enzγmatisch. At low overall yields but low occupancy densities are achieved with this method. A recent work (Wong et al. J. Am. Chem. Soc. 135 116.1 (1994)) describes the enzymatic synthesis of oligosaccharides on a modified silica gel. Due to the insolubility of the silica gel in the space required for the enzymatic carbohydrate synthesis aqueous buffers and low packing density with the linked via a peptide GlcNAc block only small Glycosylierungsausbeuten in all three reaction steps can be achieved. After cleavage of the peptide anchor enzγmatischer you therefore product mixtures obtained with only 20% of the desired product and 45% of the starting material used.

In WO 92/22661, WO 92/22565 and of WO 92/22563 are enzymatic glycosylation with a Sialytransferase of on a "non-natural support" (artificial carrier) bonded disaccharides proposed. A "non-natural carrier" is usually a high or low molecular weight carrier with antigenic properties, such as bovine serum albumin, KLH, HSA, Diphtheria or tetanus toxin, etc., or a solid carrier that is inert to physiological systems.

Starting from the aforementioned prior art, the object of the present invention to provide a process for the preparation of multivalent physiologically tolerated and physiologically degradable carbohydrate receptor blocker described in the introduction, which method is characterized in that the structure of the carbohydrate portion of the receptor blocker by enzymatic Giycosylierungsreaktionen in aqueous buffer systems and homogeneous phase is carried out directly on biodegradable polymer, the yields of glycosylation are compared to the yields of known processes significantly improved and extend typically quantitative and the coverages are increased oligosaccharide significantly on the polymer, and a use of the carbohydrate receptor blocker according to the invention for the preparation of the free propose oligosaccharides.

The object is achieved by a process for preparing a physiologically tolerated and physiologically degradable carbohydrate receptor blocker based on polymers consisting of

a) a hydrophilic, biodegradable polymer unit, b) at least one di- or oligosaccharide unit and c) are linked to at least one bifunctional spacer, via which the di- or oligosaccharide with the polymer unit,

characterized in that by chemical linkage of a mono- or oligosaccharide, of the spacer and of the hydrophilic, biodegradable polymer is prepared first, an acceptor, after which one or more other monosaccharide units are attached by enzymatic glycosylation. The enzymatic glycosylation reaction proceeds stereoselectively and with surprisingly high yields directly on the polymer and, as each Gycosylierungsschritt proceeds quantitatively, be repeated as often with any donors. In this way, the generation of polyvalent carbohydrate compounds by direct construction of the oligosaccharide structures on the polymer, in contrast to prior art, where the yields are ering and therefore product mixtures are formed at other Glycosylierungsschritten, very simple and possible in very high yields. Another advantage is the ease of isolation and the uniformity of polγvalenten carbohydrate compound.

The acceptor for the enzymatic glycosylation is produced by formation of a covalent bond between a mono- or oligosaccharide and the bifunctional spacer, followed by covalent attachment of the mono- or oligosaccharide-spacer complex with the polymer.

The enzymatic glycosylation at acceptor takes place in homogeneous aqueous phase, preferably by means of nucleotide-activated carbohydrates as donors and glycosyltransferases.

The aqueous medium should be a buffer system which is adapted to the respective glycosyltransferase, preferably the buffer system in a concentration of 0.01 M to 1 M and contains advantageously necessary for the activation of the respective glycosyltransferase cations.

The pH is 6.0 to 8.5, preferably 6.0 to 8.5, most preferably between 7.0 and 7.5.

At equimolar or excess addition of the donor to the reaction medium alkaline phosphatase should be added. To the reaction mixture are added 0.01 to 10 units of the glycosyltransferase.

The enzymatic glycosylation is 1 to 5 days at 10 to 40 ° C, preferably at 20 to 37 ° C, more preferably at 25 to 37 ° C is performed.

In the following the invention is explained in detail:

1. Synthesis of the acceptor FOR ENZYMATIC glycosylation

The acceptor for the enzymatic glycosylation reaction consists of a mono- or oligosaccharide which is covalently linked via a spacer to a biodegradables hydrophilic polymer. The polymer may be provided with a potentiator. The synthesis of the acceptor takes place after the specialist admitted methods. The individual building blocks of the acceptor are described.

Biodegradables hydrophilic polymer:

By definition, the polymer consists of at least two identical or different monomer units which are linked to each other linear or branched and may have a molecular weight distribution.

The polymer is preferably a polyamino acid as polyamide or anhydride associated with a molecular weight less than or equal to 70 kD. Preferably, the polymer has a minimum size of 2 kD in order to obtain an increased residence time in blood in comparison to low molecular weight carriers.

For the production of polymer-based carbohydrate receptor blocker on particularly preferably suitable polyamino polyaspartamides, polysuccinimides, polyglutamates and Polylysinfumaramide such as poly-σ, SS (2-hydroxyethyl) -D, L-aspartamide, poly-D, L-succinimide, polyglutamate, poly-L-lysine methyl ester fumaramide, and their copolymers.

The preparation of of biodegradable, hydrophilic polymer is carried out by the person skilled in known processes. These are described for example in: HG Elias, Macromolecules, Vol 1 and 2, Hüthig & Wepf Verlag, Basel, Switzerland, 1991/92 or D. Braun, H. Cherdron, W. Kern, placement of macromolecular organic chemistry, Hüthig. 1979th

For example, poly-D, L-succinimide (PSI) according to the instructions of Neri et al., J. Med. Chem., 16, 893 (1973) by the action of 85% phosphoric acid to aspartic acid at temperatures of 160 ° C is obtained 180 ° C -. By analogous reaction of PSI with hydroxyethylamine at Raumtempertur or slightly elevated temperature to obtain poly-σ, ß (2-hydroxyethyl) -D, L-aspartamide (PHEA) (Neri et al., Ibid). The alcohol groups of PHEA can be esterified by conventional methods (US 5,041,291). In partial implementation of PSI with ethanolamine obtained corresponding copolymers (US 5,229,469). The base hydrolysis of PSI leads to polyaspartic acid (analog Giammona et al., Chem. Pharm. Bull. 37 (8), 2245 (1989).

Analogous to the reaction with hydroxyethylamine may PSI also be reacted with other amines (EP 0 548 794), which allows additional functional groups introduced, which may act as synergists.

Poly-L-lysine methyl ester fumaramide, as another starting polymer is prepared by Grenzphasenpolykondensation of L-lysine methyl ester and fumaryl chloride (US 4834248). The methyl ester groups can be converted directly or after partial hydrolysis and subsequent activation, such as p-nitrophenyl, with the amino mono-, di- and oligosaccharides. Analog, ie by means of p-nitrophenyl, to polymeric carbohydrate receptor blocker based Polyglutarmaten be prepared (polymer synthesis analog: Anderson in "Macromolecules as Drugs and as Carriers for Biologically active material" (Ed:.. DA Tirell), NY Acad Sci, NY, 1985 P. 67 - 75).

Spacer:

Covalent attachment of the polymer with the spacer or with a compound consisting of covalently verküpftem Spacer and carbohydrate and a covalent compound of polymer and potentiator with the spacer or with a compound consisting of covalently Linked Spacer and carbohydrate is carried out by reaction between a reactive group and an activated group. Both the reactive group at the end of the spacer or a compound consisting of covalently Linked Spacer and carbohydrate and the activated group on the part of the polymer or a compound consisting of covalently Linked Polymer and. Itself can are potentiator and the activated group at the end of the spacer or a combination of covalently Linked Spacer and carbohydrate and the reactive group on the part of the polymer or a compound consisting of covalently Linked polymer and potentiator. The reaction between the reactive and activated groups carried by the person skilled in known processes for the alkylation, acylation or addition across a double bond. These methods are known to the skilled person from the literature. (Larock, RC Comprehensive Organic Transformations, 1989, VCH Verlagsgesellschaft Weinheim).

The spacer preferably has the formula I

(Mono- or oligosaccharide) -O- [Q¹- (CH₂)_p-Q²]_r- (Polymer unit) I,

which mean O

Q¹ -CH₂- Or -C ¹-, O

Q² -NH- Or -C-NH-, p is an integer from 1 to 6 and r is 1 or 2

Carbohydrate portion of the acceptor

The carbohydrate content of the acceptor for the enzymatic glycosylation reaction may be derived from natural sources or chemically or enzymatically produced chemoenzymatically. Suitable natural sources of carbohydrates are known in the art and can be found in the biochemical literature, there established, known to those skilled methods for purifying oligosaccharides are also described.

A process for the chemical, enzymatic or chemoenzymatic synthesis of carbohydrates, which are recognized by cell surface receptors, are known to the expert from the chemical literature, as well as review articles. For example, chemical synthesis Carbohydrate Research, Elsevier Science Publishers BU Amsterdam; Journal of Carbohydrate Chemistry, Marcel Dekker Inc. New York; H. Paulsen, Angew. Chem. 9__L 184 (1982) and 102 851 (1990); RR Schmidt Angew. Chem 98, 213 (1987). H. Kunz Angew. Chem. 9_8, 247 (1987). For the enzymatic synthesis as Carbohydrate Research, Elsevier Science Publishers BU Amsterdam; Journal of Carbohydrate Chemistry, Marcel Dekker Inc. New York; . Bednarski u Simon Enzymes in Carbohydrate Synthesis, ACS Symposium Series 466 (1991); KGI Nilsson, Applied Biocatalysis 1991. 117; S. David et al. Adv. Carbohydr. Chem. Biochem. 49_, 175 (1991); Y. Ichikawa et al. Anal. Biochem. 202. 215 (1992); DG Drueckhammer et al. Synthesis 1991. 499; EJ Toone et al. Tetrahedron 45, 5365 (1989).

The mono- or oligosaccharides prepared in this way can be obtained both with a free reducing end and in a spacer-linked form. The introduction of the spacer is carried by the person skilled in known processes for the chemical or enzymatic glycosylation.

2. Enzymatic Glycosylation

The method of the invention for producing a polymeric carbohydrate receptor blocker is described by enzymatsiche glycosylation:

The obtained after the first acceptor for the enzymatic glycosylation reaction consists of a mono- or oligosaccharide which is covalently linked via a spacer to a biodegradables hydrophilic polymer. The polymer may be provided with a potentiator. Preferably, the enzymatic glycosylation of the acceptor takes place in homogeneous aqueous phase. Preferably nucleotidaktivierte carbohydrates were used as donors and glycosyltransferases as enzymes.

The acceptor is dissolved in an aqueous buffer system. The buffer is adapted to the particular glycosyltransferase and may consist of 0.01 M to 1 M cacodylate, HEPES, PIPES, MOPS, citrate, bicarbonate, etc.. It contains the necessary to activate the respective glycosyltransferase cations, eg Mn.

The pH-value is also adapted to the particular glycosyltransferase, it is 6.0 to 8.5, preferably 6.5 to 7.8, most preferably between 7.0 and 7.5. After releasing the acceptor in an aqueous buffer system of the donor is added. This is a nucleotide activated sugars or an analog of a nucleotide activated sugar. The nucleotide activated sugars are commercially available, but can also be prepared by the specialist admitted methods by chemical or enzymatic synthesis or isolated from natural sources. This also applies to the analogs. The donor is either added in the 1.1 to 2 fold excess or by known methods regenerated in situ (for example, Y. Ichikawa et al. J. Am. Chem. Soc. 114, 9283 (1992), CH Wong et al. J. Org. Chem . 57, 4343 (1992), Y. Ichikawa et al. J. Am. Chem. Soc. 113, 6300 (1991), CH Wong et al. J. Org. Chem. 47.5416 (1982).

In the enzymatic galactosylation is generally used in the UDP-galactose as a donor. But it can also be assumed by UDP-glucose, which can be Epimerizing by the enzyme UDP-galactose 4-epimerase enzyme in situ to UDP-galactose (J. Thiem et al. Angew. Chem. 102 78 (1990)) ,

When the donor is in the enzymatic glycosylation equimolar or used in excess, J., it is necessary that decompose in the reaction dates are end UDP enzymatically by adding alkaline phosphatase to prevent inhibition of glycosyltransferase (C. Undaunted et al. Am. Chem . Soc. 1_u, 9308 (1990)).

Nucleotide-activated sugars are, for example UDP-glucose, UDP-galactose, UDP-N-acetyl glucosamine, UDP-N-acetylgalactosamine, UDP-glucuronic acid, CMP-neuraminic acid, GDP-fucose, GDP-mannose, dTDP-glucose, UDP d galactose.

Known in the art method for the production of nucleotide-activated sugars are for example: M. Kittel et al. Annals of the New York Academy of Sciences Vol. 6_72 Enzyme Engineering, pp 444-450 (1992), S. Makino et al. Tetrahedron Lett. 34, 2775 (1993), TJ Martin et al. Tetrahedron Lett. 3.4 1765 (1993), European Patent Application 0524143 A1; . K. Ikeda, Carbohydrate Res 224. 123 (1992), EL Kean Glycobiology 1, 441 (1991); Y. Ichikawa et al. J. Org. Chem. 52, 2943 (1992), K. Adel Horst et al. Carbohydrate Research 69 242 (1993), RR Schmidt et al. Lieb. Ann. Chem. 1991. 121, R. Stiller et al. Lieb. Ann. Chem 1992 467th; JE Heidias et al. J. Org. Chem. 52, 146 (1992), JE Heidias Acc. Chem. Res. 25, 307 (1992), ES Simon et al. J. Org. Chem. 55, 1834 (1990), CH Wong et al. J. Org. Chem. 57, 4343 (1992), JE Pallanca et al. J. Chem. Soc. Perkin Trans. 1 1993 3017th

To the dissolved in the aqueous buffer system Acceptor and the nucleotide activated sugars will be 0.01 to 10 units of the glycosyltransferase added, which can transmit the respective nucleotide activated sugars to the acceptor.

The glycosyltransferases are commercially available, can be isolated from natural sources or recombinant available. Glycosyltransferases that can be used for the enzymatic glycosylation within the meaning of the inventive process are, for example beta-1, 4-galactosyl transferase [R. Barker et al. J. Biol. Chem. 247. 7135 (1972), CH Krezhorn et al. EUR. J. Biochem. 212. 1 13 (1993), Gal-ß-1-4-GlcNAc-σ-2-6-sialyltransferase [JC Paulson et al. J. Biol. Chem. 252. 2363 (1977), H. Higa et al. J. Biol. Chem. 260, 8838 (1985), J. Weinstein et al. J. Biol. Chem. 257. 13835 (1982)],

Gal-ß-1-3GalNAc-σ-2-3-sialyltransferase [W. Gillespie et al. J. Biol. Chem. 267. 21004 (1992)],

Gal-ß-1-3 (4) GlcNAc-σ-2-3-sialyltransferase [J. Weinstein et al. J. Biol. Chem. 257. 13835 (1982), M. Nemansky et al. Glycoconjugates J. IQ, 99 (1993)], GalNAc-σ-2-6-Sialytransferase [HJ: Gross et al. Biochemistry 28, 7386 (1989), N-acetylglucosaminyltransferases [R. Oehrlein et al. Carbohydrate Res. 244. 149 (1993), T. Szumilo et al. Biochemistry 26, 5498 (1987), O. Hindsgaul et al. J. Biol. Chem. 266. 17858 (1991), GC Look et al. J. Org. Chem. 58, 4326 (1993)], σ-1-3-Fucosyltransferaεe [BW Weston, J. Biol. Chem. 267 4152 (1992)], σ-1-2 fucosyltransferase [TA Beyer, J. Biol. Chem. 255. 5364 (1980)], σ-3/4-fucosyltransferase [PH Johnson Glycoconjugate J. 9_, 241 (1992)], σ-1-2-mannosyltransferase [P. Wang, J. Org Chem 58, 3985 (1993)]. General:. TA Beyer et al. Advances in Enzymology Vol. 52, 23-175 (1981), WO 93/13198

The enzymatic glycosylation is 1 to 5 days at 10 to 40 ° C, preferably, more preferably performed at 20 to 37 ° C at 25 to 37 ° C.

For working up, the product solution after the reaction, recognizable by chromatographic methods (TLC, HPLC), against redistilled. Water dialyzed. The carbohydrate receptor blocker can be purified by chromatographic methods such as gel chromatography further subsequently.

The inventive method is particularly suitable for the production of carbohydrate receptor blockers having the following oligosaccharide or disaccharide units:

Galßl -4GlcNAc-; Galßl -3GlcNAc-, SAσ2-6Galß1 -4GlcNAc-; SAσ2-3Galß1 -4GlcNAc-; SAσ2-3Galß1 -3GlcNAc-; Galßl -4 (Fucσ1 -3) GlcNAc; Galßl -3 (Fucσ1 -3) GlcNAc; SAσ2-3Galß1 -3 (Fucσ1 -4) GlcNAc; SAσ2-3Galß1 -4 (Fucσ1 -3) GlcNAc;

Sialyl Lewis X, sialyl Lewis A, VIM-2 and the following 18

Blood group determinants Lewis A, B, X, Y and A-type¹, A-type², B-type¹, B-type² and H-type¹ , H-type² (RU Lemieux, Chem. Soc. Rev., 1978, pp 423 and 1989, p 347) and fernder sialyl Lewix X, sialyl Lewis A or VIM-2.

The formula of sialyl Lewis X is: NeuNAcσ2-3Galß1-4 (Fucσ1-3) GlcNAc and sialyl Lewis A: NeuNAc_r2-3Galß1-3 (Fucσ1-4) GlcNAc. The formula of VIM 2 is: NeuNAcσ2-3Galß1-4GlcNAcß1-3Galß1-4 (Fucσ1-3) GlcNAc;

The following are the rules for the synthesis are described by way of example.

Reaction of the polymer with the carbohydrate portion with bifunctional spacer on the one hand and the potentiator other hand, formation of covalent bonds:

Example 1: 1- (6-aminohexyl) -2-deoxy-2-acetamido-.beta.-D-glucopyranoside

2-amino-2-deoxyglucose hydrochloride is in accordance with the provision of RU Lemieux et al. (ACS Symp. Ser. 39, 90 (1976) converted by reaction with phthalic anhydride and subsequent reaction with acetic anhydride / pyridine in 1, 3,4,6-tetraacetyl-2-N-acetyl-2-deoxyglucose. By treatment with tin tetrachloride / thiophenol by the Nicolaou et al. (J. Am. Chem. Soc. 112, 3695 (1990)) the corresponding 1-Thiophenylderivat. This is according to the rule of BA Silwanis et al. (J. Carbohydr. Chem. 10, 1067 (1991)) (converted N-benzyloxycarbonyl) -aminohexanol. Analogous to the procedure of Nicolaou et al. (J. Am. Chem. Soc. 1 14, 3127 (1992) with 6), the cleavage of the acetyl and the Phthaloylschutzgruppen with hydrazine hydrate. Before cleavage of the benzyl protecting groups (H2 / Pd (OH) 2, MeOH), the free amino group acetylated selectively in the presence of the free hydroxyl groups with excess acetic anhydride. This gives 1- (6-aminohexyl) -2-deoxy-2 acetamido-ß-D-glucopyranoside Example 2: poly-D, L-succinimide-co-σ, ß-D, L-aspartamido-6-hexyl-2-deoxy-2-acet amido-ß-D-Glucopγranose (poly-D, L-succinimide co-σ, ß-D, L-aspartamido-C₆GlcNAc)

PSI (500 mg, MW 24,000) is dissolved in 2 mL DMF and treated with 225 mg (0.71 mmol) of 1- (6-aminohexyl) -2-deoxy-2-acetamido-.beta.-D-glucopyranoside (GlcNAc-C₆NH₂) Was added in 2.5 ml DMF. The batch is 5.5 hours at room temperature under N₂ touched. Thereafter, 40 ml of 1-butanol and washed like the polymer obtained with methanol. After a second precipitation from DMF in 1-butanol is again washed with methanol and then dried in an oil pump vacuum. Yield: 490 mg degree of substitution according to NMR: 12.5%

Example 3: poly-D, L-succinimide-co-σ, ß-D, L-aspartamido-6-hexyl-2-deoxy-2-acet amido-ß-D-glucopyranose (poly-D, L-succinimide co-σ, ß-D, L-aspartamido-C₆GlcNAc)

Analogously to Example 2, 320 mg PSI (MW 24,000) with 300 mg of 1- (6-aminohexyl) -2-deoxy-2-acetamido-.beta.-D-glucopyranoside (GlcNAc-C₆NH₂) And worked up by double precipitation from DMF with 1-butanol. Yield: 383 mg degree of substitution according to NMR: 18.5%

Example 4: poly-D, L-succinimide-co-σ, ß-D, L-aspartamido-6-hexyl-2-deoxy-2-acet amido-ß-D-glucopyranose (poly-D, L-succinimide co-σ, ß-D, L-aspartamido-C₆GlcNAc)

Analogously to Example 2, 480 mg PSI (MW 9600) with 210 mg of 1- (6-aminohexyl) -2-deoxy-2-acetamido-.beta.-D-glucopyranoside (GlcNAc-C₆NH₂) And worked up by double precipitation from DMF with 1-butanol. Yield: 485 mg degree of substitution according to NMR: 12.5%

Example 5: poly-D, L-succinimide-co-σ, ß-D, L-aspartamido-6-hexyl-2-deoxy-2-acet amido-ß-D-glucopyranose (poly-D, L-succinimide co-σ, ß-D, L-aspartamido-C₆GlcNAc)

Analogously to Example 2, 300 mg PSI (MW 9600) with 280 mg of 1- (6-aminohexyl) -2-deoxy-2-acetamido-.beta.-D-glucopyranoside (GlcNAc-C₆NH₂) And worked up by double precipitation from DMF with 1-butanol. Yield: 331 mg degree of substitution according to NMR: 18%

Example 6: Poly-σ, ß (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6 hexyl-2-deoxy-2-acetamido-ß-D-glucopyranose (PHEA-co-aspartamido-C₆GlcNAc)

PSI-co-aspartamido-C₆GlcNAc of Example 2 (200 mg) is dissolved in 2 ml of DMF, and freshly distilled hydroxyethylamine (81 mg). After

Stirring for 16 hours under N₂ at room temperature, precipitated with 1-butanol.

The polymer is washed with methanol in H₂O and freeze-dried.

Yield: 180 mg

Degree of substitution according to NMR: 87.5% HEA, 12.5% ​​GlcNAc-C₆NH₂

Example 7: poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido 6-hexyl-2-deoxy-2-acetamido-.beta.-D-glucopyranose (PHEA-co-aspartamido-C₆GlcNAc)

PSI-co-aspartamido-C₆GlcNAc from Example 3 (100 mg) is dissolved in 2 ml of DMF, and freshly distilled hydroxyethylamine (41 mg). After 16 hours, stirring under N₂ at room temperature, precipitated with 1-butanol. The Polymer is washed with methanol in H₂O and freeze-dried.

Yield: 112 mg

Degree of substitution according to NMR: 81, 5% HEA, 18.5% GlcNAc-C₆NH₂

Example 8: Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido 6-hexyl-2-deoxy-2-acetamido-.beta.-D-glucopyranose (PHEA-co-aspartamido-C₆GlcNAc)

PSI-co-aspartamido-C₆GlcNAc of Example 4 (150 mg) is dissolved in 2 ml of DMF, and freshly distilled hydroxyethylamine (61 mg). After

Stirring for 16 hours under N₂ at room temperature, precipitated with 1-butanol.

The polymer is washed with methanol in H₂O and freeze-dried.

Yield: 147 mg

Degree of substitution according to NMR: 87.5% HEA, 12.5% ​​GlcNAc-C₆NH₂

Example 9: Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido 6-hexyl-2-deoxy-2-acetamido-.beta.-D-glucopyranose (PHEA-co-aspartamido-C₆GlcNAc)

PSI-co-aspartamido-C₆GlcNAc of Example 5 (150 mg) is dissolved in 2 ml of DMF, and freshly distilled hydroxyethylamine (61 mg). After

Stirring for 16 hours under N₂ at room temperature, precipitated with 1-butanol.

The polymer is washed with methanol in H₂O and freeze-dried.

Yield: 127 mg

Degree of substitution according to NMR: 82% HEA, 18% GlcNAc-C₆NH₂ Example 10: Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido 6-hexyl-2-deoxy-2-acetamido-.beta.-D-glucopyranose (PHEA-co-aspartamido-C₆GlcNAc)

PSI-co-aspartamido-C₆GlcNAc of Example 2 (170 mg) is dissolved in 2 ml of DMF, and freshly distilled hydroxyethylamine (43 mg). After 4 hours, stirring under N₂ at room temperature, precipitated with 1-butanol. The polymer is washed with methanol in H₂O and freeze-dried. Yield: 160 mg degree of substitution according to NMR: 61, 5% HEA, 12.5% ​​GlcNAc-C₆NH₂

Example 11: Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido 6-hexyl-2-deoxy-2-acetamido-.beta.-D-glucopyranose (PHEA-co-aspartamido-C₆GlcNAc)

PSI-co-aspartamido-C₆GlcNAc from Example 3 (120 mg) is dissolved in 2 ml of DMF, and freshly distilled hydroxyethylamine (30 mg). After 4 hours, stirring under N₂ at room temperature, precipitated with 1-butanol. The polymer is washed with methanol in H₂O and freeze-dried. Yield: 126 mg degree of substitution according to NMR: 45.5% HEA, 18.5% GlcNAc-C₆NH₂

Example 12: Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido 6-hexyl-2-deoxy-2-acetamido-.beta.-D-glucopyranose (PHEA-co-aspartamido-C₆GlcNAc)

PSI-co-aspartamido-C₆GlcNAc of Example 4 (150 mg) is dissolved in 2 ml of DMF, and freshly distilled hydroxyethylamine (38 mg). After 4 hours, stirring under N₂ at room temperature, precipitated with 1-butanol. The polymer is washed with methanol in H₂O and freeze-dried. Yield: 146 mg degree of substitution according to NMR: 56% HEA, 12.5% ​​GlcNAc-C₆NH₂ Example 13: Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido 6-hexyl-2-deoxy-2-acetamido-.beta.-D-glucopyranose (PHEA-co-aspartamido-C₆GlcNAc)

PSI-co-aspartamido-C₆GlcNAc of Example 5 (150 mg) is dissolved in 2 ml of DMF, and freshly distilled hydroxyethylamine (38 mg). After 4 hours, stirring under N₂ at room temperature, precipitated with 1-butanol. The polymer is washed with methanol in H₂O and freeze-dried. Yield: 143 mg degree of substitution according to NMR: 50% HEA, 12.5% ​​GlcNAc-C₆NH₂

Example 14: Poly-D, L-succinimide σ, SS (5-carboxypentyl) -D, L-aspartamide

500 mg PSI (MG 9600) are dissolved in 2 ml DMF and treated with 1, 38 g

6-aminohexanoic acid, dissolved in 8 ml of formamide and 1 ml of triethylamine added.

It is stirred for 13 hours at 45 ° C and then precipitated with 1-butanol.

After washing the polymer with methanol is taken up in H2O and freeze-dried.

Yield: 350 mg

Degree of substitution according to NMR: 8% aminohexanoic

Example 15: Poly-D, L-succinimide-co-σ, SS (5-carboxypentyl) -D, L-aspartamide

500 mg PSI (MW 24,000) are dissolved in 2 ml DMF and treated with 690 mg

6-aminohexanoic acid, dissolved in 4 ml of formamide and 1 ml of triethylamine added.

It is stirred for 3 d at room temperature and 5 hours at 45 ° C and then precipitated with 1-butanol. After washing the polymer with methanol in H₂O and freeze-dried.

Yield: 470 mg

Degree of substitution according to NMR: 12.5% ​​aminohexanoic Example 16: Poly-D, L-succinimide-co-σ, SS (5-carboxypentyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6-hexyl-2-deoxy-2 acetamido-ß-D-glucopyranose (PCPA-co-aspartamido-C₆GlcNAc)

100 mg PCPA from Example 14 in 2 ml of H₂O and treated with 20 mg

1- {6-aminohexyl) -2-deoxy-2-acetamido-.beta.-D-glucopyranoside (GlcNAc-C₆NH₂) Was added. 36 hours 4 times per 20 mg

1-ethyl-3- (3-dimethylaminopropyl) - carbodiimide hydrochloride (EDC) was added.

After dialysis and lyophilization the product remains.

Yield: 1 12 mg

Degree of substitution according to NMR: 8% GlcNAc-O (CH₂)₆NH₂

Example 17: Poly-D, L-succinimide-co-σ, SS (5-carboxypentyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6-hexyl-2-deoxy-2 acetamido-ß-D-glucopyranose (PCPA-co-aspartamido-C₆GlcNAc)

100 mg PCPA from Example 15 in 2 ml of H₂O and treated with 20 mg

1- (6-aminohexyl) -2-deoxy-2-acetamido-.beta.-D-glucopyranoside (GlcNAc-C₆NH₂) Was added. 36 hours 4 times per 20 mg

1-ethyl-3- (3-dimethylaminopropyl) - carbodiimide hydrochloride (EDC) was added.

After dialysis and lyophilization the product remains.

Yield: 137 mg

Degree of substitution according to NMR: 12.5% ​​GlcNAc-O (CH₂)₆NH₂

Enzymatic galactosylation

Example 18: Enzymatic galactosylation of

Poly-D, L-succinimide-co-σ, ß-aspartamido-C₆GlcNAc

50 mg of polymer from Example 2 are HEPES buffer pH 7.5 0.05 M dissolved in 10 ml with 2 mg MnCl₂ and 55 mg UDP-glucose and 1 mg albumin added. After addition of 2 U of UDP-galactose 4-epimerase, 2 U Galactosyltransferase and 40 U of alkaline phosphatase (from calf intestine) is

8 days incubated at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Sephadex G 10th After further

Freeze-drying remains the product.

Yield: 74 mg

Degree of substitution LacNAc according to NMR: 12.5%

Example 19: Enzymatic galactosylation of

Poly-D, L-succinimide-co-σ, ß-aspartamido-C₆GlcNAc

50 mg of the polymer from Example 3, 0.05 M HEPES buffer pH 7.5 and dissolved in 10 ml with 2 mg MnCl and 55 mg of UDP-glucose and 1 mg offset lactalbumin. After addition of 2 U of UDP-galactose 4-epimerase, 2 U of galactosyltransferase and 40 U of alkaline phosphatase (calf intestine) is incubated for 8 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Sephadex G 10th After repeated freeze-drying the product remains. Yield: 76 mg degree of substitution LacNAc according to NMR: 18.5%

Example 20: Enzymatic galactosylation of

Poly-D, L-succinimide-co-σ, ß-aspartamido-C₆GlcNAc

50 mg of polymer from Example 4 are HEPES buffer pH 7.5 0.05 M dissolved in 10 ml with 2 mg MnCl₂ and 55 mg UDP-glucose and 1 mg albumin added. After addition of 2 U of UDP-galactose 4-epimerase, 2 U of galactosyltransferase and 40 U of alkaline phosphatase (calf intestine) is incubated for 8 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Sephadex G 10th After repeated freeze-drying the product remains. Yield: 77 mg degree of substitution LacNAc according to NMR: 12.5% Example 21: Enzymatic galactosylation of

Poly-D, L-succinimide-co-σ, ß-aspartamido-C₆GlcNAc

50 mg of polymer from Example 5 are dissolved in HEPES buffer pH 7.5 and with 10 ml 0.05M MnCl 2 mg₂ and 55 mg UDP-glucose and 1 mg albumin added. After addition of 2 U of UDP-galactose 4-epimerase, 2 U of galactosyltransferase and 40 U of alkaline phosphatase (calf intestine) is incubated for 8 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Sephadex G 10th After repeated freeze-drying the product remains. Yield: 78 mg degree of substitution LacNAc according to NMR: 18%

Example 22: Enzymatic galactosylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-a, bd, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-.beta.-D-glucopyranose

(PHEA-co-aspartamido-C₆GlcNAc)

50 mg of polymer from Example 6 are dissolved in HEPES buffer pH 7.5 and with 10 ml 0.05M MnCl 2 mg₂ and 40 mg UDP-glucose and 1 mg albumin added. After addition of 2 U of UDP-galactose 4-epimerase, 2 U of galactosyltransferase and 40 U of alkaline phosphatase (calf intestine) is incubated for 7 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Sephadex G 10th After repeated freeze-drying the product remains. Yield: 70.6 mg degree of substitution LacNAc according to NMR: 12.5% Example 23: Enzymatic galactosylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

hexyl-2-deoxy-2-acetamido-ß-D-glucopyranose

(PHEA-co-aspartamido-C₆GlcNAc)

50 mg of polymer from Example 7 are dissolved in HEPES buffer pH 7.5 and with 10 ml 0.05M MnCl 2 mg₂ and 40 mg UDP-glucose and 1 mg albumin added. After addition of 2 U of UDP-galactose 4-epimerase, 2 U of galactosyltransferase and 40 U of alkaline phosphatase (calf intestine) is incubated for 7 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Sephadex G 10th After repeated freeze-drying the product remains. Yield: 69 mg degree of substitution LacNAc according to NMR: 18.5%

Example 24: Enzymatic galactosylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

hexyl-2-deoxy-2-acetamido-ß-D-glucopyranose

(PHEA-co-aspartamido-Cg-GlcNAc)

50 mg of the polymer from Example 8 were HEPES buffer pH 7.5 0.05 M dissolved in 10 ml with 2 mg MnCl₂ and 40 mg UDP-glucose and 1 mg albumin added. After addition of 2 U of UDP-galactose 4-epimerase, 2 U of galactosyltransferase and 40 U of alkaline phosphatase (calf intestine) is incubated for 7 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Sephadex G 10th After repeated freeze-drying the product remains. Yield: 55 mg degree of substitution LacNAc according to NMR: 12.5% Example 25: Enzymatic galactosylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

hexyl-2-deoxy-2-acetamido-ß-D-glucopyranose

(PHEA-co-aspartamido-Cg-GlcNAc)

50 mg of the polymer from Example 9 are HEPES buffer pH 7.5 0.05 M dissolved in 10 ml with 2 mg MnCl₂ and 40 mg UDP-glucose and 1 mg albumin added. After addition of 2 U of UDP-galactose 4-epimerase, 2 U of galactosyltransferase and 40 U of alkaline phosphatase (calf intestine) is incubated for 7 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Sephadex G 10th After repeated freeze-drying the product remains. Yield: 55 mg degree of substitution LacNAc according to NMR: 18%

Example 26: Enzymatic galactosylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

hexyl-2-deoxy-2-acetamido-ß-D-glucopyranose

(PHEA-co-aspartamido-Cg-GlcNAc)

50 mg of the polymer from Example 10 were HEPES buffer pH 7.5 0.05 M dissolved in 10 ml with 2 mg MnCl₂ and 40 mg UDP-glucose and 1 mg albumin added. After addition of 2 U of UDP-galactose 4-epimerase, 2 U of galactosyltransferase and 40 U of alkaline phosphatase (calf intestine) is incubated for 7 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Sephadex G 10th After repeated freeze-drying the product remains. Yield: 54 mg degree of substitution LacNAc according to NMR: 12.5% Example 27: Enzymatic galactosylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

hexyl-2-deoxy-2-acetamido-ß-D-glucopyranose

(PHEA-co-aspartamido-Cg-GlcNAc)

50 mg of the polymer of Example 1 1, 0.05M HEPES buffer pH 7.5 and dissolved in 10 ml with 2 mg MnCl₂ and 40 mg UDP-glucose and 1 mg albumin added. After addition of 2 U of UDP-galactose 4-epimerase, 2 U of galactosyltransferase and 40 U of alkaline phosphatase (calf intestine) is incubated for 7 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Sephadex G 10th After repeated freeze-drying the product remains. Yield: 71 mg degree of substitution LacNAc according to NMR: 18.5%

Example 28: Enzymatic galactosylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

hexyl-2-deoxy-2-acetamido-ß-D-glucopyranose

(PHEA-co-aspartamido-C₆GlcNAc)

50 mg of the polymer from Example 12 are dissolved in HEPES buffer pH 7.5 and with 10 ml 0.05M MnCl 2 mg₂ and 40 mg UDP-glucose and 1 mg albumin added. After addition of 2 U of UDP-galactose 4-epimerase, 2 U of galactosyltransferase and 40 U of alkaline phosphatase (calf intestine) is incubated for 7 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Sephadex G 10th After repeated freeze-drying the product remains. Yield: 66 mg degree of substitution LacNAc according to NMR: 12.5% Example 29: Enzymatic galactosylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

hexyl-2-deoxy-2-acetamido-ß-D-glucopyranose

(PHEA-co-aspartamido-C₆GlcNAc)

50 mg of the polymer from Example 13 are dissolved in HEPES buffer pH 7.5 and with 10 ml 0.05M MnCl 2 mg₂ and 40 mg UDP-glucose and 1 mg albumin added. After addition of 2 U of UDP-galactose 4-epimerase, 2 U of galactosyltransferase and 40 U of alkaline phosphatase (calf intestine) is incubated for 7 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Sephadex G 10th After repeated freeze-drying the product remains. Yield: 64 mg degree of substitution LacNAc according to NMR: 18%

Enzymatic sialylation

Example 30: Enzymatic sialylation of

Poly-D, L-succinimide-co-σ, ß-aspartamido-C₆-LacNAc

35 mg of the polymer from Example 18 is added sodium cacodylate buffer pH dissolved in 2 ml 0.05M 7,8 and 1, 5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP-neuraminic acid was added. After addition of 20 mU 2-6-sialyltransferase and 20 U of alkaline phosphatase is incubated at 25 ° C 8 days. After dialysis against water is freeze-dried and then chromatographed on Biogel P2. After repeated freeze-drying the product remains. Yield: 36.6 mg degree of substitution of 2,6-sialyl LacNAc: 12.5% ​​(0.65 / ymol / mg polymer) Example 31: Enzymatic sialylation of

Poly-D, L-succinimide-co-σ, ß-aspartamido-C₆-LacNAc

30 mg of the polymer from Example 19 0.05 M sodium cacodylate buffer pH 7.8 and dissolved in 2 ml of 1, 5 mg bovine serum albumin, 2 mg MnCl added and 5 mg of CMP-neuraminic acid. After addition of 20 mU 2-6-sialyltransferase and 20 U of alkaline phosphatase is incubated at 25 ° C 8 days. After dialysis against water is freeze-dried and then chromatographed on Biogel P2. After repeated freeze-drying the product remains. Yield: 32 mg degree of substitution of 2,6-sialyl LacNAc: 18.5% (0.77 mol / mg polymer)

Example 32: Enzymatic sialylation of

Poly-D, L-succinimide-co-σ, ß-aspartamido-C₆-LacNAc

30 mg of the polymer from Example 20 is added sodium cacodylate buffer pH dissolved in 2 ml 0.05M 7,8 and 1, 5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP-neuraminic acid was added. After addition of 20 mU 2-6-sialyltransferase and 20 U of alkaline phosphatase is incubated at 25 ° C 8 days. After dialysis against water is freeze-dried and then chromatographed on Biogel P2. After repeated freeze-drying the product remains. Yield: 21 mg degree of substitution of 2,6-sialyl LacNAc: 12.5% ​​(0.65 mol / mg polymer)

Be ispiel 33 Enzymatic sialylation of

Poly-D, L-succinimide-co-σ, ß-aspartamido-C₆-LacNAc

35 mg of the polymer from Example 21 is added sodium cacodylate buffer pH dissolved in 2 ml 0.05M 7,8 and 1, 5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP-neuraminic acid was added. After addition of 20 mU 2-6-sialyltransferase and 20 U of alkaline phosphatase is incubated for 8 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Biogel P2. After repeated freeze-drying the product remains.

Yield: 38 mg

Degree of substitution of 2,6-Siaiyl LacNAc: 18% (0.76 mol / mg polymer)

Example 34: Enzymatic sialylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

-hexyl-O- (beta-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-.beta.-D-Glu glucopyranose

(PHEA-co-aspartamido-C₆-LacNAc)

35 mg of the polymer from Example 22 is added sodium cacodylate buffer pH dissolved in 2 ml 0.05M 7,8 and 1, 5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP-neuraminic acid was added. After addition of 20 mU 2-6-sialyltransferase and 20 U of alkaline phosphatase is incubated at 25 ° C 8 days. After dialysis against water is freeze-dried and then chromatographed on Biogel P2. After repeated freeze-drying the product remains. Yield: 29 mg degree of substitution of 2,6-sialyl LacNAc: 12.5% ​​(0.51 / mol / mg polymer)

Example 35: Enzymatic sialylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

-hexyl-O- (beta-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-.beta.-D-Glu glucopyranose

(PHEA-co-aspartamido-C₆-LacNAc)

35 mg of the polymer from Example 23 is added sodium cacodylate buffer pH dissolved in 2 ml 0.05M 7,8 and 1, 5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP-neuraminic acid was added. After addition of 20 mU 33

2-6-sialyltransferase and 20 U of alkaline phosphatase is incubated for 8 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Biogel P2. After repeated freeze-drying the product remains.

Yield: 29 mg

Degree of substitution of 2,6-sialyl LacNAc: 18.5% (0.64 mol / mg polymer)

Example 36: Enzymatic sialylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

-hexyl-O- (beta-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-.beta.-D-Glu glucopyranose

(PHEA-co-aspartamido-C₆-LacNAc)

35 mg of the polymer from Example 24 is added sodium cacodylate buffer pH dissolved in 2 ml 0.05M 7,8 and 1, 5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP-neuraminic acid was added. After addition of 20 mU 2-6-sialyltransferase and 20 U of alkaline phosphatase is incubated at 25 ° C 8 days. After dialysis against water is freeze-dried and then chromatographed on Biogel P2. After repeated freeze-drying the product remains. Yield: 34 mg degree of substitution of 2,6-sialyl LacNAc: 12.5% ​​(0.51 mol / mg polymer)

Example 37: Enzymatic sialylation of

Poly-σ, ß (2-hydroxyethγl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

-hexyl-O- (beta-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-.beta.-D-Glu glucopyranose

(PHEA-co-Cg-aspartamido LacNAc)

25 mg of the polymer from Example 25, sodium cacodylate buffer pH dissolved in 2 ml 0.05M 7,8 and 1, 5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP-neuraminic acid was added. After addition of 20 mU 2-6-sialyltransferase and 20 U of alkaline phosphatase is incubated for 8 days at 25 ^ C. After dialysis against water is freeze-dried and then chromatographed on Biogel P2. After repeated freeze-drying the product remains.

Yield: 25 mg

Degree of substitution of 2,6-sialyl LacNAc: 18% (0.63 // moles / mg polymer)

Example 38: Enzymatic sialylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

-hexyl-O- (beta-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-.beta.-D-Glu glucopyranose

(PHEA-co-aspartamido-C₆-LacNAc)

35 mg of the polymer from Example 26 w earth pH 7.8 and dissolved with 1, 5 mg bovine serum albumin, 2 mg in 2 ml 0.05 M sodium cacodylate buffer MnCl₂ and 5 mg of CMP-neuraminic acid was added. After addition of 20 mU 2-6-sialyltransferase and 20 U of alkaline phosphatase is incubated at 25 ° C 8 days. After dialysis against water is freeze-dried and then chromatographed on Biogel P2. After repeated freeze-drying the product remains. Yield: 28 mg degree of substitution of 2,6-sialyl LacNAc: 12.5% ​​(0.54 // moles / mg polymer)

Example 39: Enzymatic sialylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

-hexyl-O- (beta-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-.beta.-D-Glu glucopyranose

(PHEA-co-aspartamido-C₆-LacNAc)

35 mg of the polymer from Example 27 is added sodium cacodylate buffer pH dissolved in 2 ml 0.05M 7,8 and 1, 5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP-neuraminic acid was added. After addition of 20 mU 2-6-sialyltransferase and 20 U of alkaline phosphatase is incubated for 8 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Biogel P2. After repeated freeze-drying the product remains.

Yield: 33 mg

Degree of substitution of 2,6-sialyl LacNAc: 18.5% (0.69 // moles / mg polymer)

Example 40: Enzymatic sialylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

-hexyl-O- (beta-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-.beta.-D-Glu glucopyranose

(PHEA-co-aspartamido-C₆-LacNAc)

35 mg of the polymer from Example 28 is added sodium cacodylate buffer pH dissolved in 2 ml 0.05M 7,8 and 1, 5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP-neuraminic acid was added. After addition of 20 mU 2-6-sialyltransferase and 20 U of alkaline phosphatase is incubated at 25 ° C 8 days. After dialysis against water is freeze-dried and then chromatographed on Biogel P2. After repeated freeze-drying the product remains. Yield: 31 mg degree of substitution of 2,6-sialyl LacNAc: 12.5% ​​(0.55 / mol / mg polymer)

Example 41: Enzymatic sialylation of

Poly-σ, SS- (2-hydroxyethyl) -D, L-aspartamide-co-σ, ß-D, L-aspartamido-6

-hexyl-O- (beta-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-.beta.-D-Glu glucopyranose

(PHEA-co-Cg-aspartamido LacNAc)

35 mg of the polymer from Example 29 is added sodium cacodylate buffer pH dissolved in 2 ml 0.05M 7,8 and 1, 5 mg bovine serum albumin, 2 mg MnCl₂ and 5 mg of CMP-neuraminic acid was added. After addition of 20 mU 2-6-sialyltransferase and 20 U of alkaline phosphatase is incubated for 8 days at 25 ° C. After dialysis against water is freeze-dried and then chromatographed on Biogel P2. After repeated freeze-drying the product remains.

Yield: 15 mg

Degree of substitution of 2,6-sialyl LacNAc: 18% (0.68 mol / mg polymer)

example 42

A: PRIMARY ASSAYS FOR THE EXAMINATION OF EFFECT OF POLYMER CARBOHYDRATE receptor blockers ON cell adhesion to recombinant soluble selectin FUSION PROTEIN

With this assay, the effect of polymer-bound carbohydrate building blocks on the Zelianheftung of promyelocytic cells is detected by selectins:

To test the efficacy of the polymer-bound carbohydrate building blocks on the interaction between the E- and P-selectins (old nomenclature ELAM-1 or GMP-140) with its ligand, an assay is used which only one of those interactions is specific. The ligands are offered in their natural form as surface structures on promyelocytic HL60 cells. Since HL60 cells ligands and adhesion molecules of different specificity, the desired specificity of Assaγs can only be provided on the binding partner. As a binding partner soluble fusion proteins prepared by genetic engineering were used from each of the extracytoplasmic domain of E- or P-selectin and the constant region of a human immunoglobulin of the subclass IgGl. A1. Preparation of L-selectin IgG1

For the production of soluble L-selection-IgG1 fusion protein by Walz et al., 1990 published genetic construct "ELAM-Rg" was used.

To express the plasmid DNA into COS-7 cells (ATCC) using DEAE-dextran transfected (Molecular biological methods: see Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Seidman, JG, Struhl, K. and Smith, JA 1990. Current Protocols in Molecular Biology, John Wiley, New York.). Seven days after the transfection, the culture supernatant is recovered, free by centrifugation of cells and cell fragments and brought to 0.02% sodium azide 25 mM Hepes pH 7.0, 0.3 mM PMSF and lifted at + 4 ° C.

Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M. and Seed, B. 1990. Recognition by ELAM-1 of the sialyl-Lex determinant on myeloid and tumor cells. Science 250, 1132-1135.

A2. PREPARATION OF P-selectin IgG1

For the manufacture of the soluble P-selectin-IgG1 fusion protein by Aruffo et al., 1991 published genetic construct "CD62Rg" is used. The further procedure corresponds to the preparation of L-selectin-IgG1 presented under A1.

Aruffo, A., Kolanus, W., Walz, G., Fredman, P. and Seed, B. 1991. CD62 / P- selectin recognition of myeloid and tumor cell sulfatides. Cell 67, 35-44.

A3. PREPARATION OF CD4-IgG1

For the preparation of the soluble CD4-IgG1 fusion protein is that of Zettlemeissl et al, 1990 published genetic construct. "CD4: IgG1 hinge" used. The further procedure corresponds to the preparation of L-selectin-IgG1 presented under A1.

Zettelmeissl, G., Gregersen, J.-P.-, Duport, JM, Mehdi, S., Reiner, G. and Seed, B. 1990. Expression and characterization of human CD4: immunoglobulin fusion protein. DNA and Cell Biology 9, 347-353.

A4. IMPLEMENTATION OF HL60 cell adhesion assays ON RECOMBINANT, SOLUBLE adhesion molecules

1. 96 microtiter test plates (Nunc Maxisorb) were incubated with 100 μ \ of 9.5 diluted in 50 mM Tris pH (1 + 100) goat anti-human IgG antibody (Sigma) for 2 h. At room temperature. After removal of the antibody solution is washed once with PBS.

2. 150 μ \ of the blocking buffer are left for 1 hr. At room temperature in the wells. The composition of the blocking buffer is: 0.1% gelatin, 1% BSA, 5% calf serum, 0.2 mM PMSF, 0.02% sodium azide. After removing the blocking buffer, washed once with PBS.

3. In the cells per 100 μ \ cell culture supernatant of correspondingly transferred and expressing COS cells are pipetted. The incubation is done for 2 h. At room temperature. After removing the cell culture supernatant is washed once with PBS.

4. In the wells 20 μ \ Bindunspuffer be given. The binding buffer had the composition: 50 mM Hepes, pH 7.5; 100 mM NaCl; 1 mg / ml BSA; 2 mM MgCl 2; 1 mM CaCI2; 3 mM MnCI2; 0.02% sodium azide; 0.2 mM PMSF. These 5 μ \ of the test substance are pipetted, mixed by swirling the plate and incubated for 10 min. At room Tempe Artur. 5. 50 ml of HL60 cell culture having 200,000 cells / ml are centrifuged 4 min. At 350 g. The pellet is resuspended in 10 ml RPMI 1640 and the cells were centrifuged again. To label the cells, 50 ug BCECF-AM (Molecular Probes) are dissolved in 5 μ \ anhydrous DMSO; then 5 ml of 1, where RPMI 1640 to the BCECF-AM / DMSO solution. With this solution, the cells are resuspended and 30 min. At 37 ° C. After two minutes of centrifugation at 350 g, the labeled cell pellet is resuspended in 1 1 ml of binding buffer and the resuspended cells distributed in 100 I aliquots in the microtiter plate wells. The plate is 10 min. At room temperature allowed to stand to allow the cells to sediment on the bottom of the test plate. The cells have the opportunity to adhere to the coated plastic.

6. To stop the test, the microtiter plate at a 45 ° angle is completely immersed in the stop buffer (25 mM Tris, pH 7.5; 125 mM NaCl, 0.1% BSA, 2 mM MgCl2, 1 mM CaCI2; 3 mM MnCI2; 0.02% sodium azide). By inverting the stop buffer is removed from the wells and the procedure repeated twice more.

7. Measurement of adherent in the wells, BCECF-AM-labeled cells is performed in a cytofluorimeter (Millipore) at a sensitivity setting of 4, an excitation wavelength of 485/22 nm and an emission wavelength of 530/25 nm.

IC⁵⁰ of HL60 cell adhesion to E-selectin-IgG:

Compound from Ex. 30 60 uM

31 60 uM

32 100 / M

33 60 uM

34,150 / yM Comparison value:

IC⁵⁰ HL60 cell adhesion to E-selectin-IgG:

Sialyl Le^x-O (CH₂)₆NH₂: 1 mm

(See FIG. 1 EPA 93 19 098.7)

B. SECONDARY ASSAY TO INVESTIGATE THE EFFECT OF CARBOHYDRATE POLYME¬ REN receptor blockers ON cell adhesion to STIMULATED HUMAN ENDOTHELIAL

The test of the effectiveness of polymeric carbohydrate receptor blockers on cell adhesion to recombinant, soluble fusion proteins is a highly specific assay based on the interaction of a type of adhesion molecules with the ent speaking ligand. In order to simulate the interactions in vivo situation of cell-Zeil, we use an assay stimulated at at the HL60 cells adhere humane Nabeschnurendothelzellen.

1. Collection of human umbilical vein endothelial cells (HUVEC). Umbilical cords after birth in PBS with 100,000 IU / L penicillin, 100 mg / L streptomycin, canceled 50 mg / L gentamicin, 50,000 IU / L Mycostatin at + 4 ° C until further processing.

The longest intact pieces are cut out of the Nabeschnur with a scalpel and placed on fresh aluminum foil. One end of the cord is closed with a clip. At the other end is inserted a matching hose in the umbilical vein and fixed this by tying the cord end.

The vein is filled by the piece of tubing with collagenase solution (50 mg collagenase / 100 ml 25 mM Hepes, pH 7.0) and 15 min. At 37 ° C.

To increase the cell yield, the umbilical cord is gently massaged after the incubation in order to detach the still attached endothelial cells. Then allowed to the cell suspension from the vein in a culture tube with cell culture medium and 10% fetal calf serum to flow out. The vein is washed with PBS to obtain the residual cells.

The cell suspension is centrifuged for 5 min at 500 g. the cell pellet was then resuspended in 4 ml of culture medium and the cells plated. After 3-4 days, the cells are grown to confluence and be passaged.

To check the purity of endothelial cell culture, the culture is stained with an antibody against factor VIII for immunofluorescence. Only endothelial cells, but not contaminating fibroblasts show a positive reaction.

2. Assay Procedure

20,000 endothelial cells per well of a 96 well plate and incubated for 24 h ausplat¬ Animal T. At 37 ° C. The endothelial cells may this not have been more than 5-6 passaged times. Four hours before the assay, the endothelial cells by the addition of 11-1 (final concentration: 15 U / ml). Stimulated. After removal of the culture medium, the cells are washed once I RPMI medium without serum. After removal and re-pipetting of 20 μ \ RPMI medium, the addition of test substances.

3. The further steps of the assay, labeling of the HL60 cells, application of the HL60 cells, as described under A4. Points 5 - 7 performed. C. SECONDARY ASSAY TO INVESTIGATE THE EFFECT OF PO1 YME- REN CARBOHYDRATE receptor blockers ON cell adhesion to FROZEN SECTIONS OF LYMPHATIC TISSUE

In vitro study can be to what extent on frozen sections bind lymphatic tissue leukocytes to endothelial cells. These cell-cell interactions are based on the interaction between adhesion molecules on the surface of the freeze cut endothelial cells and the corresponding ligands on the surface of leukocytes. Representing primary leukocytes to HL60 cells can be used whose surface ligands are well described in the scientific literature. Whether an attachment of HL60 cells takes place in lymph node frozen sections, can be determined on the number of bound HL60 cells.

1. Axillary, cervical or mesenteric lymph nodes are dissected from rat freshly killed and frozen quickly in liquid nitrogen.

2. From the frozen lymph nodes 10 μ thick cryostat sections are produced, transferred to round cover glasses (diameter 18 mm) and 2 hrs. At room temperature dried.

3. In the sections 20 μ \ of binding buffer are pipetted. The test substances are added and incubated for 10 min. At room temperature. HL60 cells as described in A4. 5. point marked. 200,000 labeled HL60 cells in 100 μ \ of binding buffer are added per cover glass and allowed to stand for 10 minutes.. The sedimented cells arrive here on endothelial cells to which they adhere partially.

4. In the 45 ° angle, the cover slips are dipped in stop buffer rinse the nich chattange attached cells. Subsequently, the coverslips in 4% formaldehyde in PBS for 10 min at room temperature are fixed.. 5. In immunofluorescence microscopy (FITC-excitation) are photographically documented sections of lymphatic blood vessels. The attached HL60 cells stand out clearly from the non-colored background. The result is expressed as bound HL60 cells per surface unit endothelium.

D. TERTIARASSAY TO THE STUDY OF EFFECT OF POLYMERS KOHLENHYDRATREZEPTORBOCKERN TO leukocyte adhesion in the Rat In Vivo

The procedure listed below is able to determine the efficacy of compounds in vivo, the ßinnenwände inhibit the adhesion of leukocytes to the Gefä¬.

It is known that some of the circulating white blood cells have a tendency to adhere to the inner walls of blood vessels. This tendency becomes significant in inflammatory processes. Leukocytes normally encounter constantly against the blood vessel walls, this collision is yet elastic, so that the cells to a certain extent rebounding and returning into the circulation. In inflammatory processes biochemical changes result in both the leukocytes, but also in the endothelial cells lining the blood vessels to changes in the surface properties of both types of cells. The cells behave adhesive. This adhesiveness initially expressed in a tendency of leukocytes after collision with the endothelium to roll on the endothelial cells. This rolling de ~ leukocytes to the endothelium induces further biochemical reactions on both binding partners which resulted in cell adhesion is enhanced. This further enhancement of adhesiveness to the rollers of Leuko¬ slows cytes until they adhere firmly to the endothelium. The solid adhering follows as a further step, the migration of leukocytes from the blood vessel. The rolling of leukocytes, the firm attachment and the migration from the blood vessels can be induced with leukocyte-stimulating factors such as FMLP (f-Met-Leu-Phe), LPS (lipopolysaccharide) or TNF (tumor necrosis factor). A microscopic documentation of these processes is the prepared mesenteric tissue, such as the rat, possible. In the bloodstream inkizierte substances therefore be examined to see whether they are able to influence the induced leukocyte adhesion.

For the practical implementation of this study, rats are thesized with anesthesiology. The abdominal cavity is opened and pulled out a section of the small intestine her¬. The small intestine section is kept constantly moist on a heatable microscope stage. For microscopic viewing an area of ​​mesenteric tissue is covered with paraffin oil. For control in this area every 5 min over a period of 30 minutes all adherent -.. Unstimulated - counted leukocytes. In parallel, blood pressure, body temperature and blood flow rate is recorded. The test substance is administered by continuos venous infusion throughout the test. After application of leukocyte stimulants, which are dropped on the mesenteric tissue, the adherent leukocytes per 5 min. Over a period of 90 min. Counted.

The examination is performed in test groups, each consisting of three animals. The first animal should receive only the vehicle to determine the spontaneous adhesion. The second animal receives only the leukocyte stimulation to determine the pathogenic control. The third animal receives leukocyte stimulant and test substance.

The number of leukocytes in the adhäsierenden pathogenic control is set at 100%. The percentage change in leukocyte adhesion during administration of test substance against the pathogenic control indicates the effectiveness of a test substance.