CA2192956A1 - Process for producing polyvalent and physiologically degradable carbohydrate receptor blockers by enzymatic glycosylation reactions, and uses thereof for manufacturing carbohydrate components - Google Patents

Abstract

A process for preparing polyvalent and physiologically degradable carbohydrate receptor blockers by enzymatic glycosylation reactions and the use thereof for preparing carbohydrate building blocks The invention relates to a process for preparing polyvalent polymer-based carbohydrate receptor blockers which cause no intolerance reactions in vivo either in their entirety or in the form of degradation products. The carbohydrate side chain of the receptor blocker is assembled by enzymatic glycosylation reactions in aqueous buffer systems and homogeneous phase directly on the biodegradable polymer; the yields of the glycosylation reaction being considerably improved by comparison with the yields of known processes, as a rule taking place quantitatively and distinctly increasing the loading densities. Furthermore, a process for preparing the free oligosaccharides by means of the carbohydrate receptor blocker is prepared according to the invention is proposed.

Description

~192~51~
WO 95/34673 - 1 - PCT/BP95/02~85 Description A process for preparing polyvalent and phy3iologically degradable carbohydrate receptor blockers by enzymatic glycosylation reactions and the use thereof for preparing 5 carbohydrate building blocks The present illvention relates to a process for preparing, by enzymatic glyco3ylation reactions, polyvalent polymer-based carbohydrate receptor blockers which cause no intolerance reactions in vivo either in their entlrety or 10 in the ~orm of degrAdation products, and to the use thereof for preparing ca LoLy-l ~.te building blocks.
The importance of carbohydrates aa information carriers in physiologically relevant recognition processes has in rece~t years undergone detailed investigation and 15 ~l~rirh - ~. A~.L-''Sj. t on the cell surface in the _orm of ligands makes it possible for t~em to play, because of their binding to spe~;fir receptors, a crucial part in interc~ r communication and thus in intercellular recognition processes. Carbohydrate ligands on cell 20 surfaces are recognition domains for viruses, bacteria, toxins and lectins. ~ence they play a crucial part ~or example in bacterial and viral infections and in the initiation of ~nfll tory processes, for exaTnple rheuma-toid arthritis, allergies, post-infarct syndrome, shock, 25 stroke and sepsis. Investigations have shown that in ~nfl tory processes the selectins expressed by endothelial cells in vivo mediates the ~h~io~ of leukocytes by a ca Lol.yd ,~te ligand to the ;nfl tory ~ocus .
30 Particularly important ~or ce~ 1 adhesion are sialylated and/or ~ucosylated carbohydr;ltes such as sialyl-Lewis X
and sialyl-~ewis A.
I ~RTr.~N~T. DOC1~63NTS

2192~56 --~- -- 2 -- .-The therapy of ~nfl tory disorders with ~ree oligo-saccharides ;nt~n~l~d to bind to receptors in place of the natural ligands fails becauae o~ the very large amounts of oligo~l rh~ride ~o be administered, because the affinity between receptor and o1;~osa,crh ~ride i5 low 10-4M for the interaction between a monovalent galactoside and the aorr-~p~ ; ns lectin, D . T . Connolly et al. J. Biol. Chem. 257, 939, (1982) ) .
Divalent structures, some of which have better binding to the particular receptor, are desoribed by Wong et al.
(J. Am. Chem. Soc. 115, 7549 (1993) and in US 5,254,676.
It is also known that an increased interaction between receptor and ligand is achieved by co-lrl ;n~ a plurality of ligands to a surface. It has been shown, taking the example of the viral protein hemaglutinin, which binds to n~llr~m;n~ acid on the cell surface, how this poly-valent effeot due to the use of a polymer aisn~f~ ntly affects the ligand-receptor interaction (monovalent R~ = 2x20-~M, polyvalent l~D = 3xlO 7M, A. Spaltenstein et al. J. Am.
Chem. Soc. 113, 686 (1991) ) .
The sur~aces used to date have been liposomes (N.
Yamazaki, Int. ~. Biochem. 24, 99 (1991); W0 91~19501;
W0 91/19502), polyacrylamides (R.C. Rathi et al. J.
Polym. Sci.: Part A: Polym. Chem. 29, 1895 (1991), S.-I.
N; Flh; ra et al . Macromolecules 24, 4236 (1991) ), polylysine or sulfated polysaccharides. These polyvalent structures have the disadvantage of either having only low stability in vivo or not being tolerated in vivo due to degradation to toxic metabolites. In the case of polylysine or sulfated polysaccharides there are non-speci ric interactions with cell surface structures .
European P~hli~he~l Specifications 0 089 938, ~ 089 939 and 0 089 940 describe carbohydrate r~ ds which vary in chain length and which are identical to ligands 3 5 located on cell surf aces or receptors located on micro -organisms. The inventive idea in these cases is to block ~ 2192~

the receptorE located on the~ mi~; vvly-l~isms by the carbohydrate c ~ in vitro and in vlvo in order to be able to diagnose and treat diseases. The carbohydrate C~ may be coupled to l carrier. The latter is used inter alia to produce aDtibodies. WO 92/02527 likewise ~;~rl~8~ an ol;goE~rhs~ride building block which is coupled to a solid carrier a d is used to diagnose ~nfl~ tory proceases. The solid carrier is inert toward physiological systems and is thus not physiologicAlly degraded.
By contrast, EP 0 601 417 A2 discloses a physiologically degradable polymer-based caLLohy-lL~Ite receptor blocker which carries oligo~rrh~ride b~ ;n~ blocks on the polymer surface. 2~ vvesd activity as pharmaceutical is brought about by the e~hanced interaction o~ the carbohydrate b~ ; n~ blocks, which are present poly-valently on the polymer sur~ace, with receptors and by blocki~g speciic structures.
The carbohydrate receptor blocker described in said EP 0 601 417 A2 i~ physiologically tolerated and pre~er-ably has a molecular weight o ~ 70 kD.
In detail, the physiologically tolerated and physiologi-cally degradable, polymer-based carbohydrate receptor blocker disclosed in said EP 0 601 417 A2 has the ollow-ing structure:
carbohydrate side chains - spacer - hydrophilic, bio-degradable polymer - (optional) potentiator, where the carbohydrate side chains consist o 1 to 20 naturally occurring, identical or dierent ~s~rrl~r;de units and are coupled via one or more biurctional 3pacers o natural or synthetic origin to a hydrophilic, biodegradable polymer, where the hydro-philic, biodegradable polymer is optionally also linked to a potentiator which consists o ona or more groups ~ 219295~

with hydrophobic, hydrophilic or ionic properties or i8 a crosslinker or a solubility; ~ VVt:L.
The calLo~ydLc-te portion of the carbohydrate receptor blocker disclosed in EP 0 601 417 A2 can comprise, for example, the following sugar residues:
Gal~ 4GlcNAc-;
Gal,~51-3GlcNAc-;
SAa2 - 6Gal,~51- 4GlcNAc -;
SAa2-3Gal,~51-4GlcNAc-;
1 0 SAa2 - 3 Gal 151 - 3 GlcNAc -;
Gal,~51-4(Fucal-3)GlcNAc-;
Gal~1-3 (Fucal-3)GlcNAc-;
SAa2-3Gal,l~1-3 (Fuca!l-4) GlcNAc-;
SAa2-3Gal,~1-4(Fucal-3)GlcNAc-;
15 Other examples of preferred ~ ts of the carbo-hydrate portion are:
sialyl-Lewis X, sialyl- ~ewis A, VI~-2 and the following blood-group det~n:~nta Lewis A, B, X, Y and A typel, A type2, B type1, B type2 and H type1 and H type2 (R.~.
Lemieux, Chem. Soc. Rev., 1978, page 423 and 1989, page 347 ) -Examples of the particularly preferred ~ t of the ca,~ohydl~te portior, are sialyl-Lewis X, sialyl-Lewis A
or VIM- 2 .
The formula of sialyl-I,ewis X i5: ~ -3Gal,Bl-4-~Fucal-3)GlcNAc and of sialyl-Lewis A: NeuNAca2-3Gal~1-3-(Fucal-4)GlcNAc. The formula of VIM-2 is:
Ne~N~ 2-3Gal~1-4GlcNAcl51-3Gall51-4 (Fucal-3) GlcNAc;
Said EP 0 601 417 A2 also discloses a process for the preparation o~ the carbohydrate receptor blocker.
The synthesis of the carbohydrate receptor blockcr in the described process takes place or. the laboratory scale.

~ 2192~6 This means that the ca bohyd~te receptor blocker according to the invention is syn th~ ocl in an amount in milligrams to an amount iA grams, whereas the int~ tes n~c~sA--y ~or its synthesis~ i.e. t_e 5 hydrophilic biodegradable polymer, the bi~unctional spacer and the potentiator can be prepared in an amount f rom grams to kilograms . ~Iowever, the carbohydrate portion forms an exception. It can be synthesized only in an amount in milligr_ms up to one gram. In this case, the 10 synthetic schemes .1;~ losecl in the literature for the preparation of oligos~ hA-ides are d~ n~cl 80 that the product mixture obtained a~ter a reactio~, which rormally does not take place with a quantitative yield, is purified by column ChL~ ~o~-aphy on silica gel.
15 This puriication process is generally too costly and elaborate for preparing amounta complying with indu3trial needs and is used at the most for puri~ying final pro-ducts or valuable int~ tes. In addition, heavy metal c ~ are very freguently used to synthesize oligo-20 saccharides. Their use for synthe~;7;n~ substances witha phArr~-eutical action is very objec~ nAhle with a view to future approval of the carbohydrate receptor blocker as rhA~ e~t; cal.
In the procesa described, the required oligosA~hAride is 25 linked to the biodegradable polymer by means o~ the 3pacer only after the oligoE~ hA~ide has been A~SI" ' ~
by a large number of chemical and/or chemoenzymatic synthesis 6tages. This synthesia ia very lengthy and rl;ff;r~ t because o the problems, known to the skilled 30 worker, o the aasembly of oligos~Ax ~hA~ides (protective groups, anomer ormation, poor yield~ o~ glycosylation reactions, non-stereoselective glycosylation, numerous 8 tages ) .
In view of the very great efort needed for consecutive 35 reaction and puri~ication steps in the chemical and/or ..h. -A-~7ymatic gyntheE~i8 of carbohydrate building blocks, recently some solid-pha~e ayntheses have also been -~! 2192gS~
proposed .
In contrast to the e3tAhl; ~h~d solid-phase 3ynthese3 of olig n~ leotide3 and peptides, the rh~om;c~l synthesis of 01;~o3~~ h~rides on polymeric solid phases is very 5 ~ff;c"lt owing to the large number of functionalities and the need for stereoselective formation of the glycosidic linkage. D~n;~h~f~ky et al. (Science, 260, 1307 (1993) ) link 3,4-protected glycal via 3ily ether linkages to a polystyrene copolymer. The latter is 10 activated as epoxide and can be linked to further glycal acceptors to give the ~ os~ h~ride. Douqlas et al. (J.
Am. Chem. Soc. 113, 5095 (1991) describe the synthesis of di- and tri~r~ h~rides or, a PEG-bound gluco3e unit.
Zehavi (J. Am. Chem. Soc. 95, 5673 (1973) ) uses as 15 polymeric solid phase a photo3ensitive styrene/
divinylbenzene copolymer . The protected ol; gosa~-h~ride is eliminated from the polymer by irradiation.
The disadvantage of chemical solid-phase 3ynthesis of oligo3accharide3 are:
_ glycosylatio~ reaction3 ;nr , lete in some cases - only a few glycosylation h~ n~ blocks are 3uit-able both o~ the donor side and on the acceptor side .
- Need for protective groups a~yl ~",Liate for the particular reaction.
These di3advantage3 of the rhPm;~1 3ynthe3is of oligo-saccharide3 on polymeric matrice3 are avoided by enzy-matic glycosylation. The reactions take place without protective groups and absolutely stereo3electively and, 30 owing to the large number of available glyco3yltrans-_erase3 and nucleotide-activated sugars as glycosyl donors, can be employed very t idely.
As long ago as 1980, Nunez and sarker (Biochemistry 19, 489 (1980) deacribed the enzymatic galactosylation of 35 N-acetylgl~r 08~mi n~ linked to agaroge via a h~Ys-nr~ m;n~
spacer. }Iowever, very large amounts of galacto3yl-.

21929~6 tr~l~sferase enzyme were uaed. U. Zehavi describes enzy-matic galactoaylation with galactosyltransferase on photosensitive polymers, both insoluble in water and soluble in water. The transfer yields are very low at 5 < 1 to a maximum o~ 3696 (IJ. Zehavi et al., CaLLohy~cste Res. 124, 23 (1983), U. Zehavi et al. Carbohydrate Res.
128, 160~ (1984), ~. Zehavi, Reactive Polymers 6, 189 (1987), IJ. Zehavi et al. Glycoconjugate J. 7, 229 (1990) and U. Zehavi Innovation Perspect. Solid Phase Synthesis Collect. Paper, Int. Symp. 1990, 389 to 396). The result-ing .1~ ~rrh~ide on the polymer is eliminated from the polymer by the action of light or by an enzyme (U. Zehavi et al. Carbohydrate Res. 133, 339 (1984) ) .
N;~h~ c~ (N~h~mllra et al. p~or-h~m;cal and Biophyaical Research Comm. 199, 249-254 (1994) ) describes the enzymatic preparation of a water-soluble polyacrylamide with 3'-sialyl-N-acetyllacto~m~ne side chains, with ~tepwise enzymatic glycosylation of a water-~oluble, N-acetylgl~l-os~minQ-carry-ing polyacrylamide. Eowever, low 20 loading densities are achieved, with low overall yields, in this process. A more recent paper (Wong et al. J. Am.
Chem. Soc. 116,1135 (1994) ) describes the enzymatic synthesis of oligosaccharides on a 'i f ~ ilica gel .
Owing to the insolubility of the silica gel in the 25 aqueous buffers needed for the enzymatic ca LoLyd c.te synthesis, and the low loading density with the GlcNAc building block linked via a peptide, only low glycosylation yields are achieved in all three reaction steps. Thus, after enzymatic cleavage of the peptide 30 anchor, product mixtures with only 2096 of the reguired product and 45% of the precursor used are obtained.
WO 92/22661, WO 92/22565 and WO 92/22563 propose enzymatic glycosylations with a 3ialytransferase of h~ides linked to an ~unnatural carrieT (artifi-35 cial carrier) . An "unnatural carrier" is, as a rule, ahigh or low molecular weight carrier with antigenic properties, for example bovine serum albumin, RL~I, HSA, diphteria or tetanus toxin etc. or a solid carrier which .. . ..

` 2192~
_ -- 8 is inert toward physiologiaal systems.
Based on said state of the art, it is an object of the present invention to provide a proceas for preparing the polyvalent, physiologically tolerated and physiologically degradable carbohydrate receptor blocker described at the outset, which process i~ distinguished i~ that the asse_bly of the ca~L,ohydLclte portion of the receptor blocker takes place by enzymatic glycosylation reactions in aqlleous buf fer systems and homogeneous phase directly on the biodegradable polymer, the yields of the glycosylation reaction ~re r~n~ Prably; _ ~,v~d by comparison with the yields of known processes and, as a rule, take place quantitatively, and the loading den-l3ities of ol; ~sa~h~ride on the polymer are distinctly increased, and to propose a use of the caLLo~ydL~te receptor blocker prepared according to the invention f or preparlng the ~ree oligos~c ~ h~rides.
The object is achieved according to the invention by a process for preparlng a physiologically tolerated and phy,3iolo~ y degradable polymer-based caLl~ohydL~te receptor blocker consisting of a) a hydrophilic, biodegradable polymer u~it, b) at least one di- or oligr s~ ride unit and c) at least one bifunctional spacer by which the di- or oligo~a~h~ride units are linked to the polymer unit, which comprises initially preparing an acceptor by ~-~hPm; c;~l linkage o~ a mono- or oligos~ h~ride, of the spacer and of the hydrophilic, biodegradable polymer, 3 0 af ter which o~e or more other mor~osaccharide units are attached by enzymatic glycosylation.
The enzymatic glycosylation reaction takes place stereo-Relectively and with surprisingly high yields directly on the polymer and can, because each gycosylation step takes 21929~6 `
g place quantitatively, be repeated as of ten as desired with any desired donors. It is po3s;hle in this way to produce polyvalent carbohydrate - __u~ds by direct assembly of the oligos~rrhsride structures on the polymer 5 very simply and in very high yields, by contrast with the state of the art, where the yields are low and therefore product mixtures result with further glycosylation steps.
Another advantage is the simple isolatlon and the homo-geneity of the polyvale~t carbohydrate r ~
10 The aCCQptor for the enzymatic glycosylation is prepared by forming a covalent bond betwee~ a mono- or Ql; ~08arrh~ride and the bifunctional spacer, and subsequently linkirg the mono- or oligosaccharide- spacer complex covalently to the polymer.
15 The enzymatic glycosylation or. the acceptor takes place in homogeneous aqueous phase, preferably using nucleotide-activated carbohydrates as donors and glyco-syltransf erases .
The aqueous medium should consist of a buffer system 20 which is suited to the particular glycosyltransferase, and the buffer system preferably has a conce~tration of 0 . 01 M to 1 ~ and advantageously contains the cations necessary for activating the particular glycosyltrans-f erase .
The p~ is between 6.0 and 8.5, preferably between 6.0 and 8 . 5, very particularly pref erably between 7 . 0 and 7 . 5 .
In the case of equimolar or excess addition of the donor, All~l;n~ pho8phata8e ghould be added to the reaction medium .
0.01 to 10 units of the glycoayltransferase are added to the reaction mixture.
The enzymatic glycosylation is carried out at 10 to 40~C, ~ - 10 - , preferably at 20 to 37C, even more preferably at 25 to 37C, for 1 to 5 days.
The i~ventio~ is ~ n~d in detail hereinafter:
1. SYNTHESIS OF THE ACCEPTOR FOR THE EN~YMATIC
G~YCOSYLATION RE ACTION
The acceptor f or the enzymatic glycosylation reaction con~ists of a mono- or oligosaccharide which is covale~ltly linked via a spacer to a biodegradable hydro-philic polymer. The polymer can be provided with a 10 potentiator. The acceptor is syn~h~ by methods known to the skilled worker. The individual building blocks of the acceptor are described hereinaf ter .
Biodegradable hydrophilic polymer:
By defirition, the polymer conaists of at least two 15 i~nt; C ~l or different monomer units which are linked together in linear or br~n~h~d fashion and may display a molecular weight distribution.
The polymer is preferably a polyamino-acid linked as polyamide or -anhydride wlth a molecular weight less than 20 or equal to 70 kD. The preferred minimum 3ize of the polymer is 2 kD in order to achieve a r~ onre time in the blood which is i~creased by c~mpariaon with low molecular weight carriers.
Polyamino-aclds which are suitable and particularly

2 5 pref erred f or preparing polymer-based carbohydrate receptor blockers are polyaspart~mides, polysuc~-;n~m;d~a, polyglutamates and polylysine-fumaramides, auch as, for ample, poly-~, ~- (2 -l~y.l- o, yethyl) -D, L-aspartamide, poly-D,L-sllc~ ;n;~ , polyglutamate, poly~ lysine methyl 30 eE~ter furamamide, and copolymers thereof.
The biodegradable, hydrophilic polymer is prepared by ~` 21929~

processes known to the skilled worker. These are described, for example, in: H.G. Elias, Makromolekule [Ma, lecules], Volumes 1 and 2 , Huthig ~ Wepf Verlag, Baale, Switzerland, l991/92 c r D. Braun, H. Cherdron and 5 W. Rern, Praktikum der Ma~L, l-~k~ ren Or~n; Rrh~n Chemie [Practical Molecular Organic Chemistry], Huthig Verlag 1979.
Thus, for example, poly-D,L-s~rc~n;~ (PSI) is obtained by the method of Neri et al., J. Med. Chem., 16, 893 (1973) by the action of 8596 strength rl~srhnric acid on aspartic acid at temperatures of 160C-180C. Reaction of PgI polymer with hydroxyethylamine at room temperature or slightly elevated temperature results in poly-al,p- (2-Lydl~y~thyl)-D~L-aspartamide (PHEA) (Neri et al., ibid).
15 The alcohol groups Or the PHEA can be esterified by customary proces6es (US 5,041,291). Partial reaction of PSI with e~hAnnl~ ne results in coLL~ ;n~ copolymers (~S 5,229,469). Basic hydrolysis of PSI leads to poly-aspartic acid (in analogy to G~ - et al., Chem.
20 Pharm. Bull. 37 (8), 2245 (1989).
In analogy to the reaction with hyd ~,~y,athylamine, PSI
can also be reacted with other amines (EP 0 5g8 794), which makes it possible to introduce additional func-tional groups which may act as potentiators.
25 Poly-L-lysine methyl ester fumaramide, as another initial polymer, iB prepared by boundary phase polycnn~nRRtion of ~-lysine methyl ester and fumaryl chloride (US
4,834,248). The methyl ester groups can be reacted directly or af ter partial hydrolysis and s~hseSrl-~nt 30 activation, for exa~ple as p-nitrophenyl ester, with the mono-, di- and oligosaccharides contai~ing amino groups.
n an analogous way, i.e. using p-nitrophenyl esters, it is possible to prepare polymeric carbohydrate receptor blockers based on polyglutarmates (polymer synthesiR in 35 analogy to: Anderson in "Macromolecules as Drugs and as ~192~6 Carriers for Biologically active Naterials" (Ed: D.A.
Tirell), NY Acad. Sci., NY, 1985, pages 67-75).
Spacer:
The covalent linkage of tb.e polymer to the spacer or to a c~ __u.-d consisting of covalently linked spacer and carbohydrate and o~ a covalent ~ ~d of polymer and potentiator to the spacer or to a compound consisting of covalently linked spacer and carbohydrate takes place by reaction between a reactive group and an activated group.
In this connection it is possible both for the reactive group to be located at the end of the spacer or of ~
~_ ~ consisting of covalently linked spacer and carbohydrate and the activated group to be located on the polymer or a c~ a~ consisting of covalently linked polymer and pote~tiator, and _or the activated group to be located at the end of the spacer or of a c~ ,_ a of covalently linked spacer and carbohydrate and the reac-tive group to be located on the polymer or on a compound consisting of covalently linked polymer and potentiator.
The reaction Between reactive and activated groups take place by processes, known to the skilled worker, for alkylation, ~cylation or addition onto a double bond.
These processes are known to the skilled person ~rom the literature. (Larock, R.C. Comprehensive Organic Trans-_ormations, 1989, VC~3 Verlagsg~ - h~ft h'F;nh~;~) .
The spacer preferably has the formula I
(110- o:r nl ~ 5~~ r~ de~ -o- [Ql- (C~2) p-Q~ r- (polyme~ t) I, in which o Q1 i B - CE2 - or ¦¦
-C-, -21~2g~

O
Q2 i8 -NEI- or -C-NH-, p is an integer from 1 to 6 a~d r i5 1 or 2 Carbohydrate portion of th- acceptor 5 The carbohydrate portion of the acceptor for the eazymatic glycosylation reactio~ car. be derived fr natural aource3 or be prepared rhAn~ 1y~ r~ ~y-matically or enzymatically. Suitable natural source3 for carbohydrates are known to the 3killed worker z~d can be looked up ln the biorhATn; rAl literature. 'rhe latter likewise describes estAh~; ~hAd processes known to the skilled worke~ for purifyi~g oligo~AcrhArides.
Processes for the rhA"~; c~l, enzymatic or rhl -n ~ym_tic synthesis of carbohydrates which are recogni ~9:1 by cell surface receptors are know to the skilled worker from the rh~ rAl literature and from review articles. For the chemical synthesis for example, CaLLo}.yd~te Research, Elsevier ScieQce P~hl; ~hArs B.U. Amsterdam; Journal of CaLL,o~lyd te Chemistry, Marcel Dekker Irc. New York; }~.
Paulsen, Angew. Chem. 94, 184 (1982) and 102, 851 (1990);
R.R. Schmidt Angew. Chem. 98, 213 (1987); H. ~unz. Angew.
Chem. 98, 247 (1987). For the enzymatic 3ynthesis for example, Carbohydrate Research, Elsevier Scie~ce Pub-lishers B.U. Amsterdam; Journal of Carbohydrate Chemis-try, Marcel Dekker Inc. New York; Bednarski and Simon Enzymes in Carbohydrate Synthesis, ACS Sympo3iu~L Series 466 (1991); R.G.I. Nils~o~, Applied Biocatalysis 1991, 117; S. David et al. Adv. Carbohydr. Chem. Biochem. 49, 175 (1991); Y. IrhikA~a et al. A~al. Biochem. 202, 215 (1992); D.G. Dr~l~or~h~ -- et al. Synthesis 1991, 499;
E.J. Toone et al. Tetrahedron 45, 5365 (1989) .
The mono- or oligos~rhA~ides prepared ir, thi3 way can be 21~29~

obtained both with free reducing end and in a spacer-linked form. The spacer is introduced by processes known to the skilled worker for ~h~ l or enzymatic glyco~ylation .
2. ENZY~L~TIC GLYCOSYLATION
The process according to the invention for preparing a polymeric carbohydrate receptor blocker by enzymatic glycosylation is described hereinaf ter .
The acceptor, obtained as in 1., for the enzymatic glycosylation reaction consists of a mono- or oligos~ h~ride which is covalently linked via a spacer to a biodegradable hydrophilic polymer. The polymer can be provided with a potentiator. The enzymatic glycosylation of the acceptor preferably takes place in homogeneous aqueous phase. Nucleotide-activated carbo-hydrates ar~ preferably used as donors, and glycosyl-transferases are pre~erably used as enzymes.
The acceptor is dissolved in an aqueous buffer system.
The bu~fer is appropriate for the particular glycosyl-tr~nsferase and can consist, for example, of 0.01 M to 1 M cacodylate, ~EPES, PIPES, MOPS, citrate, bi~-~rh~ te etc. It contains the cations n~ s~ry for activating the particular glycosyltransferase, for example Mn.
The p~ is likewise appropriate ~or the particular glyco-syltransferase and i8 between 6.0 and 8.5, preferably between 6.5 and 7.8, very particularly preferably between 7.0 and 7.5-Af ter the acceptor has been dissolved in the aqueous buf ~er system, the donor is added. The latter is a nucleotide-activated sugar or an analog of a nucleotide-~ctivated sugar. The nucleotide-activated sugars can in nome cases be bought, but can also be prepared by methods known to the skilled worker by chemical or enzymatic 21929~6 syntheses or be ~ol7~ted ~rom natural sources. This also applies to the analogs. The donor is either added in an excess o~ 1.1 to 2 fold or regenerated in situ by known processes (~or example Y. Ichikawa et al. J. Am. Chem.
Soc. 114, 9283 (1992), C.EI. Wong et al. J. Org. Chem. 57, 4343 (1992), ~. Ichikawa et al. J. Am. Chem. Soc. 113, 6300 (1991) and C.E. Nong et al. J. Org. Chem. 47,5416 (1982) .
The donor used in the enzymatic galactosylation is, as a rule, UDP-galactose. ~owever, it is also possible to start from UDP-glucose which can be enzymatically epimer-ized 1~ situ by the enzyme UDP-galactose 4-epimerase to ~orm IlDP-galactose (~. Thiem et al. Angew. Chem. 102, 78 (1990) ) .
I~ the donor is used in e~luimolar amount or in excess in the enzymatic glycosylation, it is necessary to .1.", _-se elzzymatically the UDP liberated in the reaction by adding All~ n~ pho8phatase in order to prevent inhibition o~
the glycosyltran3~erase (C. Unverzagt et al. J. Am. Chem.
Soc. 112, 9308 (1990) ) .
Examples of nucleotide-activated sugars are UDP-glucose, UDP-galactose, IJDP-N-acetylglll~os~m~n~, UDP-N-acetyl-galactosAm; n~ UDP-glucuronic acid, CMP-neuraminic acid, GDP-~ucose, GDP-mannose, dTDP-glucose and dUDP-galactose.
25 Processes known to the skilled worker ~or the preparation of nucleotide-activated sugars are, for example:
M. iCittelmann et al. Annals o~ the New York Academy o~
Sciences Vol. 672 Enzy31e ~n~ln~ing~ pages 444 to 450 (1992), S. Makino et al. Tetrahedron Lett. 34, 2775 (1993), T.J. Martin et al. Tetrahedron ~ett. 34, 1765 (1993), Buropean Patent Application 0 524 143 A1;
R. Ikeda, Car~ ohydrate Res. 224, 123 (1992), B.L. ~ean Glycobiology 1, 441 (1991); Y. Ichikawa et al. J. Org.
Chem. 57, 2943 (1992), ~. Adelhorst et al. Carbohydrate Research 242, 69 (1993), R.R. Schmidt et al. Lieb. A~n.
Chem. 1991, 121, R. Stiller et al. Lieb. Ann. Chem. 1992, - 2192~

467; J.E. Heidlas et al. J. Org. Chem. 57, 146 (1992), J.E. Heldlas Acc. Chem. Res. 25, 307 ~1992), E.S. Simon et al. J. Org. Chem. 55, 1834 (1990), C.H. Wong et al.
J. Org. Chem. 57, 4343 (1992) and J.E. Pallanca et al. J.
Chem. Soc. Perkin Trans. 1 1993, 3017.
0 . 01 to 10 u~ita of the glycosyltransferase able to transfer the particular nucleotide-activated sugar to the receptor are added to the acceptor di3aolved in the aqueous buffer system and to the nucleotide-activated 10 sugar.
The glycosyltransfera6es can in some cases be bought, be i~olated from natural sources or be obtained by re~l ' ;nSlnt te~hn;~el~. Examples of glycosyltransferases which can be used for the enzymatic glycosylation for the 15 purpose of the process according to the invention are ~-1,4-galactoayltransferase [R. Barker et al. J. Biol.
Chem. 247, 7135 (1972) and C.H. Rre~horn et al. Eur. J.
Biochem. 212, 113 (1993), Gal-,~5-1-4-GlcNAc ~Y-2-6-sialyltransferase [J.C. Paulson et al. J. Biol. Chem. 252, 2363 (1977), Higa et al. J. Biol.
Chem. 260, 8838 (1985) and J. Weinstein et al. J. Biol.
Chem. 257, 13835 (1982) ], Gal-,~3-1-3-GalNAc -2-3-sialyltrAn~ferase [W. ~ pie et al. J. Biol. Chem. 267, 21004 (1992) ], Gal-,5-1-3 (4) -GlcNAc a-2-3-sialyltransferase [J.Weinstein et al. J. Biol. Chem. 257, 13835 (1982) and N. ~emansky et al. Glycocon~ugate J. 10, 99 (1993) ], GalNAc c~-2-6-sialytransferase [H.J: Gross et al. Bio-chemistry 28, 7386 (1989), 30 N-acetylgl~ os~m;nyltransfera~e~ [R. Oehrlein et al.
Carbohydrate Res. 244, 149 (1993), T. Szumilo et al.
Biochemistry 26, 5498 (1987), o. ~;n~l~g~lll et al. J.
Biol. Chem. 266, 17858 (1991) a~d G.C. Look et al. J.
Org. Chem. 58, 4326 (1993) ], 35 a-1-3-fucosyltransferase [B.W. Weston, J. Biol. Chem.
267, 4152 (1992) ], -1-2-fucosyltransferase [T.A. Beyer, J. Biol. Chem. 255, 5364 (1980) ], 21929~

cY-3/4-fucosyltrans~erase [P.H. ~Johnson Glycoconjugate J~
9, 241 (1992) ], -1-2-mannosyltrans~erase [P. Wang, J. Org. Chem. 58, 3985 (1993) ], 5 General: T.A. Beyer et al. Advances in Enzymology Vol.
52, 23 to 175 (1981), WO 93/13198.
The enzymatic glycosylation is carricd out at 10 to 40C, preferably at 20 to 37C, particularly preferably at 25 to 37C, for 1 to 5 daya.
10 For working up, the product aolution a~ter ~ let;~n o:E
the reaction, ~ n~ f ~ ~hle by chromatography methods (TI,C, EIPLC), is dialyzed againat double-diatilled water.
The carbohydrate receptor blocker can subaequently be further purified by chromatography methods 3uch as, for 15 example, gel chromatography.
The procesa according to the invention ia particularly suitable for preparing ca l,oLydlc.te receptor blockers with the following oligo- or ~ cr~h~ride units:
Gal,151 - 4GlcNAc -;
2 0 Gal~1 - 3 GlcNAc -, SA~2 - 6Gal,~1 -4GlcNAc -;
SA~2 - 3 Gal,~11 - 4GlcNAc -;
SAcY2 - 3Gal,~l - 3GlcNAc -;
Gal,~1-4 (Fuco~1-3) GlcNAc-;
2 5 Gall51- 3 (Fucoll - 3 ) GlcNAc -;
SA~Y2-3Gal,B1-3 (FuccY1-4)GlcNAc-;
SAY2 -3Gal,B1-4 (FUCCY1-3) GlcNAc-;
sialyl-Lewis X, sialyl-Lewis A, VIM-2 and the ~ollowing blood-group det~r~n~nt~ Lewis A, B, X, Y and A typel, A
30 type2, B typel, B type2 and ~I typel, ~I t_pe2 (R.U.
~emieux, Chem. Soc. ~ev., 1978, page 423 and 1989, page 347) and, fur~h~ , Gialyl-~ewix X, sialyl-L~wia A or VIM- 2 .

- ~` 21~29~
~, -- 18 -- _ The iormula of sialyl-Lewis X i8: Ne~N~ Gall51-4-(Fucal-3)GlcNAc and of sialyl-Lewis A: NeuNAc~2-3Gal,51-

3(Fuc~Yl-4)GlcNAc. The formula of VIM-2 is: NeuNAcal2-3Gal,~1-4GlcNAc,Bl-3Gal,~1-4 (Fuc~1-3)GlcNAc:
5 Examples of the methods for the sy~thesis are described hereir~af ter .
Reaction of the polymer with the ca Lol:.y~ te portio~
with bifunctional spacer on the one hand and with the potentiator on the other hand to form covalent bonds:
Example 1: 1- (6-Am~ noh~Yyl) -2-deoxy-2-acetamido-,~-D-glucopyranoside 2-Amino-2-deoY~yglucose hydrochloride i~ converted by the method o~ R.U. I,emieux et al. (ACS Symp. Ser. 39, 90 (1976) intol,3,4,6-tetr~acetyl-2-N-acetyl-2-deoxyglucose by reaction with phthalic a~hydride and subse~uent reaction with acetic anhydride/pyridine. Treatment with tin tetrachloride/thiophenol by the method of Nicolaou et al. (J. Am. Chem. Soc . 112, 3695 (1990) ) results in the corr~ap~n~ng 1-~h;or7~Pnyl derivative. The latter is reacted with 6- (N-benzyloxycarbonyl)~nohPY~nol by the method of B.A. Silwanis et al. (J. Carbohydr. Chem. 10, 1067 (1991) ) . The acetyl and phthaloyl protective groups are cleaved with hydrazine hydrate in analogy to the method of K.C. Nicolaou et al. (J. Am. Chem. Soc. 114, 3127 (1992) ) . Before elimination of the benzyl protective groups (}I2/Pd(0EI)2, ~eO~I), the free amino group is selec-tively acetylated, in the pre3ence of the ~ree hydroxyl groups, with excess acetic anhydride. 1- (6-~ nrlhPYyl) -2-deoxy-2-acetamido-~-D-glucopyranoside is obtained.

Example 2: Poly-D,L-~ ;nim~d~-co~ -D~L-aspartamid 6-hexyl-2-deoxy-2-acetamido-~-D-glucuuy~ ~01 (Poly-D,L-succinimide-co-tY, ,~-D, IL-aspart-amido - C6 - GlcNAc ) PSI (500 mg, MW 24,000) i8 di3solved in 2 ml o~ DMF, and 225 mg (0.71 ~3mol) of 1- (6-~m;noh~yl) -2-deoxy-2-acet-amido-,~-D-glucopyranoside (GlcNAc-C6-NH2) ill 2.5 ml o~
DMF are added. The mixture is stirred at room temperature ur,der N2 for 5.5 hours. Precipltation is ther~ carried out with 40 ml o$ 1-butanol, a~d the resulting polymer i8 washed with mt thanol. After a secord precipitation ~rom DMF in l-buta~ol, the product is again washed with methanol and subsequently dried under oil pump vacuum.
Yield: 490 mg Degree o~ substitution aacording to NMR: 12.5%
Example 3: Poly-D,I,-sur~in;~;de-co-o~"5-D,L-aspartamido-6 -hexyl-2 -deoxy-2 -acetamido-~-D-gluco-pyranose (Poly-D,L-succinimide-co-a,,~-D,L-aspart-amido-C6-GlcNAc) }n analogy to Example 2, 320 mg o~ PSI (MW 24, 000) are reacted with 300 mg o~ 1- (6-Am;n~h~Yyl) -2-deoxy-2-acet-amido-~-D-glucuuyl-..03ide (GlcNAc-C6-NEI2) and worked up by precipitation twice ~rom DMF with l-butanol.
Yield: 383 mg Degree of substitutio~ accordi~g to NMR: 18.5%
Example 4: Poly-D,~-sl~-r;nim;~l~-co-o~"~-D,L-aapartamido-6 -hexyl-2 -deoxy-2-acetamido-~-D-gluco-pyra~ose (Poly-D, L-succinimide-co-~, j5-D,L-aspart-amido - C6 - GlcNAc ) In ar~alogy to Example 2, 480 mg of PSI (MW 9, 600) are reacted with 210 mg o~ 1- (6-~m~n~h~Yyl) -2-deoxy-2-acet-amido-~-D-glucopyraroside (GlcNAc-C6-NE~2) and worked up 2192~6 ao by precipitation twice from DMF ,with 1-butanol.
Yield: 485mg Degree of substitution according to NMR: 12.5%
Example 5: Poly-D,L-3~ ;n;m;~ -co-~ -D,L-aspartamido-6-hexyl-2-deoxy-2-acetamido-,~-D-gluco-pyranose (Poly-D, L-succinimide-co-cY, ~3-D, L-aspart-amido-C6-GlcNAc) Tn aralogy to Example 2, 300 mg of PSI (MW 9,600) are reacted with 280 mg of 1- (6-~m;nr~h~ -deoxy-2-acet-amido-~-D-glucopyranoside (GlcNAc-C6-NEI2) and worked up by precipitation twice ~rom DMF with l-butanol.
Yield: 331 mg Degree of substitution according to NMR: 18%
Example 6: Poly-a,l5- (2-11ydLv~yethyl) -D,L-aspartamide-co-a,,l3-D,L-aspartamido-6-hexyl-2-deoxy-2-acetamido-,5 -D -glucopyranose (P~EA-co-a8partamido-C6-GlcNAc) PSI-co-aspartamido-C6-GlcNAc from Example 2 (200 mg) is dissolved in 2 ml of DMF, and ~reshly distilled hydroxy-ethylamine ( 81 mg) is added. Stirring uslder N2 at room temperature for 16 hours is ~ollowed by precipitation with l-butanol; ~he polymer is washed with methanol, dissolved in ~20 and freeze-dried.
Yield: 180 mg Degree of substitution accordiIlg to NMR: 87.5% EEA, 12.5%
GlcNAc - C6 - NH2 Example 7: Poly-~,~- (2-hydroxyethyl) -D,L-aspartamide-co-ol"~-D, L-aspartamido-6 -hexyl-2 -deoxy-2-3 0 5 cetamido - ,~ -D - glucopyrano~e (PHEA-co-aspartamido-C6-GlcNAc) PSI-co-aspartamido-C6-GlcNAc from Example 3 (100 mg) is dissolved in 2 ml of DMF, a~d freshly distilled hydroxy-~1929~
ethylamine (41 mg) i8 added. Stirring under N2 at room temperature ~or 16 houra i3 ~ollowed by precipitation .-ith l-butanol. The polymer is washed with methanol, dissolved in ~I20 and ~reeze-dried.
5 Yield: 112 mg Degree of substitution according to NNR: 81.5% ~IEA, 18 . 5% GlcNAc-C6-NE~2 Example 8: Poly-a"B- (2-l-y~o~ye~thyl) -D, ~-aspartamide-co-a,,~g-D, L-aspartamido-6-hexyl-2-deoxy-2-acetamido-13-D-glucopyranose (PHEA-co-~spartamido-C6 -Glc-NAc) PSI-co-aspartamido-C6-GlcNAc from Example 4 (150 mg) is dissolved in 2 ml of DMF, and ~reshly distilled hydroxy-ethylamine (61 mg) is added. Stirring under N2 at room 15 temperature for 16 hours is ~ollowed by precipitation with l-butanol. The polymer is washed with methanol, dissolved in ~I20 and ~reeze-dried.
Yield: 147 mg Degree of substitution according to NMR: 87.5% ~EA, 12.5%
2 0 GlcNAC - C 6 - N~2 Example 9: Poly-a"~- (2-hydroxyethyl) -D,~-aspartamide-co-a,,3-D,L-aspartamido-6-hexyl-2-deoxy-2-acetamido - ,~ -D -glucopyranose (P~IEA-co-aspartamido-C6-GlcNAc) PSI-co-aspartamido-C6-GlcNAc ~rom Example 5 (150 mg) is dissolved in 2 ml of DMF, and ~reshly distilled hydroxy-ethylamine ( 61 mg) is added . Stirring under N2 at room temperature for 16 hours is ~ollowed by precipitation with l-butanol. The polymer is washed with methanol, dissolved in E120 and ~reeze-dried.
Yield: 127 mg Degre~ o~ substitution according to NMR: 82% EIEA, 18%
GlcNAc -C6-NE~2 21~2~
Example 10: Poly~ -(2-l-yd~v~yethyl)-D~L-aspartamide-co-~"l5-D,L-aspartamido-6-hexyl-2-deoxy-2-acetamido-,~-D-glucopyranose (P~IEA-co-a8partamido-C6-GlcNAc) 5 PSI-co-aspartamido-C6-GlcNAc from Example 2 (170 mg) is dissolved ln 2 ml of DMF, and ~reahly diatilled hydroxy-ethylamine (43 mg) is added. Stirring under N2 at room temperature ~or 4 hours is ~ollowed by precipitation with l-butanol. The polymer is washed with methanol, dissolved 10 in ~20 and freeze-dried.
Yield: 16 0 mg Degree of substitution according to NMR: 61. 5% ~EA, 12 . 596 GlcNAC - C6 -N~2 Example 11: Poly-~Y"15- (2-IIYd~J .y~thyl) -D, L-aspartamide-co-~,,~-D,L-aspartamido-6-hexyl-2-deoxy-2-acetamido-,~-D-glucopyranoae (PlIEA-co-aspartamido-C6-GlcNAc) PSI-co-aspartamido-C6-GlcNAc from Example 3 (120 mg) is dissolved in 2 ml o~ DMF, and ~reshly distilled hydroxy-ethylamine (30 mg) i5 added. Stirring u~der N2 at room temperature ~or 4 hours is followed by precipitation with 1-buta~ol. The polymer is wa3hed with methanol, dissolved in ~I20 and iree~e-dried.
Yield: 126 mg Degree o~ substitution accordi~g to NMR: 45.5% lIEA, 18.5%
GlcNAc - C6 -NEI2 Example 12: Poly-a~,F-(2-hydroxyethyl)-D,L-aspartamide-co-~ -D, L-aspartamido-6 -hexyl-2-deoxy-2 -acetamido-~-D-glucopyranose (P~EA-co-aspartamido-C6-GlcNAc) PSI-co-aspartamido-C6-GlcNAc from Example 4 (150 mg) is dissolved in 2 ml o~ DMF, and freshly distilled hydroxy-ethylamine (38 mg) is added. Stirring under N2 at room temperature for 4 houra is ~ollowed by precipitation with _ _ _ 21929~6 1-butar,ol. The polymer is washed with methanol, dissolved i~ ~I20 and freeze-dried.
Yield: lg6 mg Degree of substitutior. ac~or~;n~ to NNR: 56% EEA, 12.5%
5 GlCNAC-c6-N~2 Example 13: Poly-~,,15- (2-hydroxyethyl) -D,L-aspartamide-co-~Y"5-D,L-aspartamido-6-hexyl-2-deoxy-2-acQtamido-,~-D-glucopyranose (P~IEA-co-aspartamido-C6 -GlcNAc) PSI-co-aspartamido-C6-GlcNAc from Example 5 (150 _g) is dis301ved ln 2 ml of DMF, and freshly distilled hydroxy-ethylamine (38 mg) is added. Stirring under N2 at room temperature for 4 hours is followed by precipitation with l-butanol. The polymer i8 washed with methanol, dissolved 15 in ~20 and fr~eze-dried.
Yield: 143 mg Degree of substitution according to N21R: 50% ~EA, 12.5%
GlcNAc - C6 -NE~2 Example 14: Poly-D,L-succin;m;t3~ - (5-caLl,o~y~e~tyl) -D,L-aspartamide 500 _g of PSI (MW 9,600) are dissolved in 2 ml o~ DMF, and 1.38 g of 6-~m;noh~T~nnic acid dissolved ir. 8 ml of formide, and 1 ml o~ triethylamine are added. The mixture is stirred at 45C for 13 hours and sub~equently precipi-25 tated with 1-butanol. The polymer is washed with methanol and then taken up in ~I20 and freeze-dried.
Yield: 350 mg Degree of substitutiorL according to ~IMR: 8% ~m;nnh~Ts~nn;C
acid 30 Example 15: Poly-D,~-succi~imide-co-~Y"~- (5-carboxy-p êntyl ) - D, ~ - a spar tamide 500 mg o~ PSI (DIW 24,000) are dissolved in 2 ml of DMF, and 6gO mg of 6-~m; nnh~T~nnic acid dissolved in 4 ml of 21929~
.

formide, and 1 ml o~ triethylamine are added. The mixture is stirred at room temperature for 3 d and at 45C for 5 hours, and subsequently preaipitated with 1-butanol.
The polymer is washed with methanol and then taken up in 5 !I2O and freeze-dried.
Yield: 470 mg Degree of substitution according to NMR: 12.5% amino-h~YAnr~ir acid Example 16: Poly-D,~-succinimide-co-a,~- (5-carboxy-pentyl)-D,L-aspartamide-co-c~"~-D,L-aspart-;~mido-6-hexyl-2 -deoxy-2 -acetamido-,13-D-gluco-pyranose (PCPA-co-aspartamido-C6-GlcNAc) 100 mg o~ PCPA from Example 14 are dissolve~ in 2 ml of ~2~ and 20 mg of 1- (6-Am~noh~Yyl) -2-dexoy-2-acetamido-~-D-glucopyranoside (GlcNAc-C6-NH2) are added. 20 mg por-tions of 1-ethyl-3-(3-dimethylAm~nnpropyl)carbo~l;;m;d~
hydrochloride (EDC) are added 4 times over the course o~
36 hours. The product remains after dialysis _nd freeze-drying .
Yield: 112 mg Degree of substitution according to NMR: 8% GlcNAc-o ( c~I2 ) 6NEI2 Example 17: Poly-D, L-succinimide-co-a, ~- (S-carboxy-pentyl) -D,~-aspartamide-co-a,,l~-D,L-aspart-amido-6-hexyl-2-deoxy-2-acetamido-,~5-D-gluco-pyranose (PCPA-co-aspartamido-C6-GlcNAc) 100 mg o~ PCPA ~rom Example 15 are dissolved in 2 ml of ~2' and 20 mg of 1- (6-Am;n~Yyl) -2-dexoy-2-acet_mido-15-D-glucopyrano~ide (GlcNAc-C6-NEI2) are added. 20 mg por-tions of 1-ethyl-3- (3-dimethyl Am~nnpropyl) carbodiimide hydro~ o~j rl~ (EDC) are added 4 times over the course of 36 hours. The product remains after dialysis and freeze-drying .
Yield: 137 mg Degree of substitution accordir,g to NMR: 12 . 5% GlcNAc-~ 21~2~
-- 25 -- ~-o ( C~I2 ) 6NEI2 Enzymatic galactosylation Example 18: Enzymatic galactosylation of poly-D~ 8u~cin; Tn~ de- co -~ -a8partamid Glc~Ac 50 mg of the polymer from Example 2 are di8solved in 10 ml of 0.05 M ~IEPES buffer p~I 7.5, and 2 mg of MnCl2 and 55 mg of UDP-glucose plus 1 mg of lact~lh~-min are added. After addition of 2 IJ of UDP-galactose

4-epimerase, 2 U of galactosyltransferase and 40 IJ of AlkAlln~ phogphatage (from calf integtine) the mixture is incubated at 25C for 8 days. Dialysis against water is ~ollowed by freeze-drying and subsequent chromatography on Sephadex G 10. The product remains after freeze-drying again.
Yield: 74 mg Degree of LacNAc substitution according to NMR: 12.5%
Example 19: Enzymatic galactosylation of poly-D,L-succinimide-co-ol, j5-aspartamido-C6-GlcNAc 50 mg of the polymer from Example 3 are dissolved in 10 ml of 0.05 M ~IEPES buffer p~I 7.5, and 2 mg of MnC12 and ~5 mg of UDP-glucose plU8 1 mg of lactAlhl-m;n are added. After A~ n of 2 U of IJDP-galactose 4-epimer-25 aae, 2 U of galactogyltransferage and 40 ~ of AlkAl;phosphatase (from calf inteatine) the mixture is incu-bated at 25C for 8 days. Dialysi~ against water is followed by freeze-drying and subsequent chromatography on Sephadex G 10. The product remains after freeze-drying 3 0 again .
Yield: 76 mg Degree of I~acNAc substitution according to NMR: 18 . 5%

- 2192~
a~ ~ 26 --Example 20: Enzymatic galactosylation of poly-D, L-s~cr; n;m; l~-Co-a!, ~3-a8partamido-C6-QlcNAc 50 mg of the polymer from Example 4 are dissolved in lO ml of 0.05 M ~IEPES buffer p~I 7.5, and 2 mg of MnC12 and 55 mg of UDP-glucose plus 1 mg of lactAlh~m;n are added. After additio~ of 2 U of I~DP-galactose 4-epimer-ase, 2 U of galactosyltran8fera8e and 40 U of A~ ne phosphatase (from calf intestine) the mixture is incu-bated at 25C for 8 days. Dlalysis against water is followed by freeze-drying and subsequent chromatogr~phy on Sephadex G 10. The product remai~s after freeze-drying again .
Yield: 7 7 mg Degree of LacNAc substitution according to ~R: 12 . 5%
Example 21: Enzymatic galactosylation of poly-D,L-~ cin;m;cle-co-cY,~-aspartamido-C6-Glc~Ac 50 mg o~ the polymer from Example 5 are dissolved i~
lO ml of 0.05 M ~IEPES bu~fer p}I 7.5, and 2 mg of rOnC12 and 55 mg of ~DP-glucose plus 1 mg of lactalbumin are added. After addition of 2 U of ~DP-galactose 4-epimer-ase, 2 ~ of galactosyltrarlsferase and 40 a of ~ ne phosphatase (from calf intestine) the mixture is incu-bated at 25DC for 8 days. Dialysis ~gainst water is followed by freeze-drying and subse5[uent chromatography orL Sephadex G 10. The product remains after freeze-drying again .
Yield: 78 mg Degree of LacNAc substitutio~ ~ccordi~g to NMR: 18%

- ~ 21~2~6 Example 22: Erzy~atie galactosylation of poly-~"i,- (2-hydroxyethyl) -D,L-aspartamide-eo-a,b-D,L-aspartamido-6-hexyl-2-deoxy-2-aeetamido-,~-D-glu~opyranose (P~EA-eo-aspartamido-C6-GleNAc) 50 mg of the polymer from Example 6 are dissolved in 10 ml of 0.05 M ~EPES buffer p~ 7.5, and 2 mg of MnCl2 and 40 mg of UDP-gluco~e plus 1 mg of lactalbumin are added. After addition of i U of UDP-galactose 4-epimer-ase, 2 U of galactosyltrangferage and 40 ~ of AlkAl ;n~
phosphatase ( Erom calf intestine) the mixture is incu-bated at 25C for 7 days. Dlalysis against water is followed by ~reeze-drying and suhses~uent chromatography on Sephadex G 10 . The product remains af ter freeze-drying again.
Yield: 70 . 6 mg Degree of LacNAc suhstitution according to NMR: 12 . 5%
Example 23: Enzymatic galaetosylation of poly-~"5- (2-1-yd ~y~:thyl) -D,L-aspartamide-co-~Y"5-D,L-aspartamido-6-hexyl-2-deoxy-2-acetamido - ~ -D - glucopyranose (P~EA-co-a8partamido-C6-GlcNAc) 50 mg of the polymer from Example 7 are dissolved in 10 ml of 0.05 M ~IEPES buffer plI 7.5, and 2 mg of MnC12 and 40 mg of IJDP-glueose plus 1 mg of laetAlh--min are added. Af ter addition of 2 IJ of IJDP-galactose 4-epimer-ase, 2 U of galaetosyltransferase and 40 IJ of AlkAl ;nA
phosphatase (from ealf intestine) the mixture is ineu-bated at 25C for 7 days. Dialysis against water is followed by freeze-drying and subseguent chromatography on Sephadex G 10. ~he product remains after freeze-drying again .
Yield: 69 mg Degree of LacNAc substitution a~c~r~l; n~ to NMR: 18 . 5%

- ~ 2192~
~ 28 r Example 24: E~zymatl c galactosylatioA of poly-a!, ,lS- (2 -L~dLv~ye thyl ) -D, L-aspartamide-co-a,,~-D,L-aspartamido-6-hexyl-2-deoxy-2-acetamido-,~3-D-gly..,yyL~ose (PHEA)-co-aspartamido-C6-GlcNAc) 50 mg o~ the polymer from Example a are dissolved in 10 ml of 0.05 M HEPES buffer pH 7.5, and 2 mg of MnC12 and 40 mg of l~DP-glucose plus 1 mg o~ lactAlh~n;n are added. After addition of 2 U of IJDP-galactose 4-epiner-;~se, 2 U of galactosyltrangferage and 40 U of ~lkAl;n~
phoaphatase (from calf intestine) th~ mi7cture is incu-b;~ted at 25C for 7 days. Dialysis against water is followed by freeze-drying and sub~equent chronatography on Sephadex G 10. The product remains after free~e-drying again.
Yield: 55 mg Degree of LacNAc substitution according to N~: 12 . 596 Example 25: Enzymatic galactosylation of poly-a, ,~- (2 -hydL~J~y~thyl) -D, L-aspartamide-co-a"~-D,I,-aspartamido-6-hexyl-2-deoxy-2-acetamido -,~1 -D -gluc~,yyL ~ ~ose (PHEA-co-aspartamido-C6-61cNAc) 50 mg of the polymer from Example 9 are dissolved in 10 ml of 0.05 M HEPES buffer pH 7.5, and 2 mg of MnCl2 2~ and 40 mg of UDP-gl lcose plus 1 mg of lactalbumin are added. After addition of 2 U of IJDP-galactose 4-epimer-ase, 2 IJ of galactosyltrangfera~e and 40 IJ of ~ A1 ;ne phosphatase (from calf i~testine) the mixture is incu-bated at 25C for 7 days. Dialysis against water is followed by freeze-drying and s~hse~nt chromatography on Sephadex G 10. The product remains after free2e-drying again .
Yield: 55 mg Degree of LacNAc ~ubstitution according to NMR: 18%

~ 21929~

Example 2 6: Enzymatic galactosylatio~ of poly~ - (2-l~yd o,.y~thyl) -D,L-aspartamide-co-~ -D, L-aspartamido-6-hexyl-2 -deoxy-2 -acetamido-~-D-glucopyranose (P~IEA-co-aspartamido-C6-GlcNAc) 50 mg of the polymer from 3xample 10 are di8solved in 10 ml of 0.05 M ~EPES buffer p~ 7.5, and 2 mg of MnCl2 and 40 mg of UDP-glucose plu8 1 mg of lac~lh~m;n are added. After addition of 2 U of UDP-galactose 4-epimer-a3e, 2 U of galactosyltrangferage and 40 U of ~1 ks~l; ne phosphatase (from calf irtesti~e) the mixture is incu-bated at 25C for 7 days. Dialysls against water is followed by freeze-dryi~g and subsQquent chromatography on Sephadex G 10. The product remains after freeze-drying again.
Yield: 54 mg Degree of LacNAc substitution aacording to NMR: 12 . 596 Example 27: Enzymatic galacto~ylation of poly-ol"~- (2-hyd u~y~thyl) -D, L-aspartamide-co-~, ,~-D, L-aspartamido-6-hexyl-2 -deoxy-2 -acetamido-,~-D-glucopyranose (P~IEA-co-aspartamido-C6-Glc~Ac) 50 mg of the polymer from Example 11 are dissolved i~
10 ml o~ 0.05 ~ ~IEPES buffer pII 7.5, and 2 mg of MnCl2 and 40 mg of UDP-glucose plus 1 mg of lact~lhllrn;n are added. After addition of 2 U of ~DP-galactose 4-epimer-asQ~ 2 ~ of galactosyltrangfera8e and 40 U of ~ l ;n~
phosphatase (from calf intestine) the mixture is incu-bated at 25C for 7 days. Dialysis against water is followed by Ereeze-drying and subsequent chromatography on S~ph d~Y G 10. The product remai~s after freeze-drying again .
Yield: 71 mg Degree of ~acNAc substitution accordir~g to NM~: 18.596 - 2192g~6 Example 2 8: Enzymatic galactosylation of poly-a, ,~- (2 -LydLv~y~thyl) -D, L-aspartamide-co-a, ,15-D, L-aspartamido-6-hexyl-2 -deoxy-2 -acetamido-,15-D-glucopyranose (PHEA-co-aspartamido-C6-GlcNAc) 50 mg of the polymer from Example 12 are di6solved in 10 ml of 0.05 M HEPES buffer pH 7.5, and 2 Ing of MnC12 and 40 mg of UDP-glucose plus 1 mg of lactalbumin are added. Af ter addition of 2 U of UDP-galactose 4-epimer-ase, 2 U of galactogyltrangferage and 40 ~ of :~lk:~l ;n~
phosphatase (~rom calf intestine) the mixture i~ incu-bated at 25C for 7 days. Dialysis against water is followed by freeze-drying and subsequent chromatography on Sephadex G 10. The product remains after freeze-drying again.
Yield: 66 mg Degree of LacNAc substitution according to NMR: ~2.5%
3xample 29: Enzymatic galactosylation of poly-a,,B- (2-LydLv~Ly~thyl) -D,~-aspartamide-co-a"~-D,L-aspartamido-6-hexyl-2-d~oxy-2-acetamido-,~l-D-glucopyranose (PHEA-co-a8partamido-C6-GlcNAc) 50 mg of the polymer from Example 13 are dissolved in 10 ml of 0.05 M HEPES buffer pH 7.5, and 2 mg of MnCl2 and 40 mg of UDP-glucose plu3 1 mg of lacl ~lh~ n are added. A~ter addition of 2 U of UDP-g~lactose 4-epimer-ase, 2 U o~ galactosyltransferase and 40 U of "1~1 ;ne phosphatase (from calf intestine) the mixture is incu-bated at 25C for 7 days. Dialysis against water is followed by freeze-drying and subse~luent chromatography on Sephadex G 10. q~he product remains after freeze-drying again .
Yield: 64 mg Degree o~ LacNAc suhstitution accordi~g to NMR: 18%
Enzymatic sialylation 21929~
Eexample 30: Enzymatic sialylation of poly-D~ L-s~ n;m~ -co-c~ ~-aapartamid LacNAc 35 mg o~ the polymer from Example 18 are dissolved in 2 ml o~ 0.05 ~ sodium cacodylate buffer pH 7.8, and 1. 5 mg of bovine serum al}~umin, 2 mg of MnC12 and 5 mg o~
CMP-neuraminic acid ~re added. Addition o~ 20 mU of 2-6-sialyltranaferase and 20 U of ~lk~l;n~. phosphatase is followed by incubation at 25C for 8 days. Dialysis 10 against water is followed by freeze-drying and subseguent chromatography on Biogel P2 . The product remains af ter ~reeze-drying again.
Yield: 36 . 6 mg Degree o~ 2, 6-sialyl-LacNAc substitution: 12.5%
(0.65 ~mol/mg polymer) 3xample 31: Enzymatic sialylation of poly-D~L-a~ in;rn;~ -co-~-aspartamid ~acNAc 30 mg o~ the polymer from Example 19 are diaaolved in 2 ml of 0.05 ~ sodium cacodylate buffer pH 7.8, and l . 5 mg of bovine serum albumin, 2 mg of MnCl2 and 5 mg of CMP-neuraminic acid are added. Addition of 2 0 mU of 2-6-sialyltransferase and 20 U of ~lk~l;ne phosphatase is followed by incubation at 25C for 8 days. Dialysis against water is followed by freeze-drying and subsequent chromatography on siogel P2 . The product remains af ter ~reeze-drying again.
Yield: 32 mg Degree of 2,6-sialyl-~acNAc substitution: 18.5%
30 ~0.77 ~mol/mg polymer) Example 32: Enzymatic sialyl._ion of poly-D,L-EIuccinimide-co-cY-aspartamido-C6 -~acNAc 30 mg of the polymer from Example 20 are dissolved in -. ' 2192g~6 2 ml of 0.05 M aodium cacodylate buffer pH 7.8, and 1.5 mg of bovine serum albumin, 2 mg of MnCl2 and 5 mg of CMP-neuraminic acid are added. Addition of 20 mU of 2-6-sialyltransferase and 20 IJ of ~lkAl;ne phosphatase is

5 followed by incubation at 25C for 8 days. Dialysi3 againat water i8 followed by freeze-drying and s~hseQ~l~nt chromatography on Biogel P2 . The product remaina af ter freeze-drying again.
Yield: 21 mg Degree of 2,6-sialyl-LacNAc substitution: 12.5%
(0 . 65 ~mol/mg polymer) Example 33: Enzymatic sialylation of poly-D,~-sl~r~;n;m;A~-co-a"(5-aspartamido-C6-LacNAc 15 35 mg of the polymer from Ex_mple 21 are dissolved in 2 ml of 0.05 M sodium cacodylate buffer p~ 7.8, and 1. 5 mg of bovine serum albumin, 2 mg of MnCl2 and 5 mg of CNP-n~llrAm;n;c acid are added. Addition of 20 mU of 2-6-sialyltransferase and 20 U of AlkAl ;n-~ phosphatase is 20 followed by incubation at 25C for 8 days. Dialysis against water is followed by freeze-drying and su~sequent chromatography on Biogel P2. The product remains after freeze-drying again.
Yield: 3 8 mg 25 Degree of 2,6-sialyl-~acNAc substituticn: 18%
( O . 7 6 ~Lmol /mg polymer) Example 34: Enzymatic sialylation of poly-~,~g- (2-hydroxyethyl) -D,L-aspartamide-co -~ "~ -D, L -aspartamido- 6 -hexyl -0- (~-D-3 0 galactopyr_nosyl ) - ( 1- 4 ) - 2 - deoxy - 2 - acetamido --D-glucopyranose (PEEA-co-aspartamido-C6-LacNAc) 35 mg of the polymer from Example 22 are dissolved in 2 ml of 0.05 M sodium cacodylate buffer p~ 7.8, and 1.5 mg of bovine serum albumin, 2 mg of MnClz and 5 mg of ~. 21g29~6 -- 3 3 -- ~ -CMP-neuraminic acid are added. Addition of 20 mU of 2-6-sialyltransferase and 20 U of ~1kAl;n~ phosphataae i8 fol 1 _ l_1 by incubation at 25C for 8 days. Dialysis against water is followed by freeze-drying and subsequent 5 chromatography on Biogel P2 . The product remains af ter freeze-drying again.
Yield: 29 mg Degree of 2,6-sialyl-LacNAc substitution: 12.5%
(0.51 ~mol/mg polymer) 0 Example 35: 3nzymatic siaiylation of poly-al, ~1- (2 -hy~LG~Lyethyl) -D, L-aspartamide-co-a,,15-D,I,-aapartamido-6-hexyl-O- (~-D-galactopyranosyl) - (1-4) -2-deoxy-2-acetamido-1.-D-glucopyranoae (PHEA-co-aspartamido-C6-LacNAc) 35 mg of the polymer from Example 23 are dissolved in 2 ml of 0.05 M aodium cacodylate buffer p~I 7.8, and 1.5 mg of bovine serum albumin, 2 mg of MnCl2 and 5 mg of CMP-neuraminic acid are added. Addition of 2D mU of 20 2-6-aialyltranaferaae and 20 U of s~lk:~l;n-. phoaphataae ia followed by incubation at 25C for 8 daya. Dialysia against water is followed by freeze-drying and subsequent chromatography on Biogel P2. The product remaina a~ter freeze-drying again.
Yield: 29 mg Degree of 2,6-aialyl-I,acNAc substitution: 18.596 (0 . 64 ~mol/mg polymer) Example 36: Enzymatic sialylation of poly-~Y"~- (2-1~ydLO~Ly~thyl) -D,L-aDpartamide-co-~, ~-D, L-aapartamido-6-hexyl-O- (~3-D-galact~,~yL~loDyl) - (l-g) -deoxy-2-acetamido-,l5-D -glucopyranoae (PHEA-co-aapartamido-C6-I,acNAc) 35 mg o~ the polymer from Example 24 are dissolved in 35 2 ml of 0.05 M sodium cacodylate buffer p}I 7.8, and ~ . 21929~G
~ - 34 -1. 5 mg of bovine serum albumin, 2 mg of MnCl2 and 5 mg of CMP-neuraminic acid are added. Additlon of 20 mU of 2-6-sialyltran3ferase and 20 U of s~lk:~l;n-. phosphatase i8 followed by incubation at 25C for 8 days. Dialysis 5 again~t water is followed by freeze-drying and aubsequent chromatography on Biogel P2 . The product remains af ter freeze-drying again.
Yield: 34 mg Degree of 2,6-sialyl-LacNAc substitution: 12.5%
10 (0.51 ~cmol/mg polymer) 3xample 37: Enzymatic sialylation of poly-~, ~1- (2-hyd~O~y ~thyl) -D, L-aspartamide-co-~Y,,5-D,L-a~partamido-6-hexyl-O- (,d-D-galactu~y~ o~yl) - (1-4) -2-deoxy-2-~c~tamido-,15-D-gluc~y~ ose (PHEA-co-a~partamido-C6-LacNAc, 25 mg of the polymer from Example 25 are di~solved in 2 ml of 0.05 M sodium cacodylate buffer pH 7.8, and 1. 5 mg of bovine serum albumin, 2 mg of MnCl2 and 5 mg of 20 CMP-neuraminic acid are added. Addition of 20 mU of 2-6-sialyltransferase and 20 U of ~lk~l~ne phosphatase is followed by incubation at 25C for 8 days. Dialysis against water is followed by freeze-drying and subsequent chromatography on Biogel P2. The product remains after 2 5 f reez e - drying again .
Yield: 25 mg Degree of 2,6-~ialyl-BacNAc substitution: 1896 (0 . 63 ~mol/mg polymer) 3xample 38: 2nzymatic sialylation of poly-~"B- (2 -hydroxyethyl) -D, L-aspartamide-co-~"l~-D,r,-aspartamido-6-hexyl-O- (15-D-galact.,~,,yLeulo~yl) - (1-~) -2-deoxy-2-acetamido-. -D - glucopyranos e (PHEA-co-a8partamido-C6-~acNAc) 35 mg of the poly~mer from Example. 26 are dissolved in 2 ml of 0.05 M sodium cacodylate buffer pH 7.8, and 2~ 929~

1.5 mg of bovine serum albumin, 2 mg of MnCl2 and 5 mg of C~$P-neuraminic acid are added. Addition of 20 mU of 2-6-sialyltransferase and 20 U of AlkAl;n~ phosphatase ia followed by incubation at 25-C for 8 days. Dialysis 5 against water is followed by freeze-drying and subsequent chromatography on Biogel P2 . The product remains af ter freeze-drying again.
Yield: 28 mg Degree of 2,6-sialyl-LacNAc aubstitution: 12.596 (O . 54 ILmol/mg polymer) 3xample 39: Enzymatic sialylation of poly-a, p- ( 2 -l~ydL u~ye thyl ) -D, L-aspartamide-co-a, p-D, L-aspartamido-6-hexyl-0- (,l5-D-gala~:t~yL~o~,yl) - (1-4) -2-deoxy-2-acetamido-,15-D-glucopyr~nose (P~IEA- co - aspartamido - C6 - I-acNAc ) 35 mg of the polymer from Example 27 are dissolved in 2 ml of 0.05 ~ sodium cacodylate buffer plI 7.8, and 1.5 mg of bovine serum albumin, 2 mg of NnCl2 and 5 mg of 20 C~6P-neuraminic acid are added. Addition of 20 mU of 2-6-sialyltransferase and 20 IJ of AlkAlin~ phosphatase is followed by incubation at 25C ~or 8 days. Dialysis agalnst water is followed by freeze-drying and subsequent chromatography on Biogel P2 . The product remains af ter freeze-drying again.
Yi~ld: 33 mg Degree of 2,6-sialyl-LacNAc substitution: 18.596 (0 . 69 ~mol/mg polymer) Example 40: Enzymatic sialylation of poly-~"~- (2-l~ydLo~yethyl) -D,L-aspartamide-co-a, p-D, I--aspartamido-6-hexyl-0- (,~-D-galact~,~yL~,Llo..yl) - (1-4) -2-deoxy-2-acetamido-,~-D -glucopyranose (P~EA-co-a8partamido-C6-LacNAc) 35 35 mg of the polymer from Example 28 are dissolved in 2 ml o~ 0.05 M sodi~m cacodylate buffer p~ 7.8, and ~. 21g2g5~

1.5 mg of bovine s~ru~ albumin, ,2 mg of MnC12 and 5 mg of C'MP-neuraminic acid are added. Addition of 20 mU of 2-6-sialyltran3ferase and 20 U of ~lk~l;ne phosphatase i3 followed by incubation at 25C for 8 days. Dialysis 5 against water is followed by freeze-drying and subsequent chromatography on Biogel P2. The product remains after ~reeze-drying again.
Yield: 31 mg Degree of 2,6-sialyl-LacNAc substitution: 12.5%
10 (O . 55 fLmol/mg polymer) Example 41: Enzymatic sialylation of poly-a, ,l~- (2 -hydroxyethyl) -D, L - aspartamide -co-a~, ~-D, ~-aspartamido-6-hexyl-0- (~-D-galactv~,yl~.o~,yl) - (1-4) -2-deoxy-2-acetamido-,~-D-glucv~yL~ose (P~EA-co-a8partamido-C6-LacNAc) 35 mg of the polymer from Example 29 ~re dissolved in 2 ml of 0.05 M sodium cacodylate buffer p~ 7.8, and 1.5 mg of bovine serum albumin, 2 mg of MnC12 and 5 mg of 2 0 CMP -neuraminic acid are added . Addition of 2 0 mU of 2-6-sialyltransferase and 20 U of :~lk~lin~ phosphatase is followed by incubation at 25C for 8 days. Dialysis J~gainst water is followed by freeze-drying and subsequent chromatography on Biogel P2. The product remains after 2 5 f reez e - drying again .
Yield: 15 mg Degree of 2, 6-sialyl-LacNAc substitution: 18%
(O . 68 ~mol/mg polymer) Exampl e 4 2 30 A: PRIrSARY ASSAYS FOR INVESTIGATING T~E EFFECT OF
POLYMERIC ~'ARRrlT~)RZ~l'P! ~E~ R BLOCRERS ON CELL ADEESION
TO RECf~ N~NT SOLUBLE SELEC IN FlJSION PROTEINS
This assay is used to detect the effect of polymer-bound carbohydrate unitEI on cell ~ n of promyelocytic 35 cells by mean~ of selectins:

- 219295~

The assay used to test the aGtivity of polymer-bound carbohydrate units on the interaction between E- and P-selecting (old n~ rlAture ELAM-1 and GMP-140 respec-tively) with their ligands is spe~ i~;c for only one of these interactions $n each case . The ligands are of f ered in their naturll form as surface structures on promyelo-cytic HI 60 cells . Since ~IL60 cells have ligands and A~hP~ n le~ul~ which dif~er widely in ~p~;f;city, the required specificity o~ the assay can be provided only via the binding partner. Used as binding partners were genetically P~;nPP~ed soluble fusion protei~s from the cxtracytoplA~n;c domain in each case of E- and P-selectin and the constant region of a human immunoglobulin of subclass IgG1.
Al. PREPARATION OF L-SE1ECTIN-IGG1 The genetic construct "EI,AM-Rg" pl-hl; ~hecl by Walz et al., l9gO, was used to prepare soluble I.-selection-IgG1 fusion protein .
For the expression, the plasmid DNA was transfected into COS-7 cells (ATCC) using DEAE-dextran (Molecular Bio-logical Methods: see Ausubel, F.M., Brent, R., Ringston, R.E., Moore, D.D., Seidman, J.G., Struhl, R.
~nd Smith, J.A., l990. Current Protocols in Molecular Biology, ~ohn Wiley, New York). Seven days after the transfection, the culture supernatant is obtained, centrifuged to remove cells and cell fragments and adjusted to 25 mM Hepes p~I 7.0, 0.3 mM PMSF, 0.02% sodium ~zide and stored at l4C.
Walz, G., Aruffo, A., Rolanus, W., Bevilacqua, M. and Seed, B. 1990. Recognition by ELAM-1 of the sialyl-Lex detPrm;nAnt on myeloid and tumor cells. ~ ience 250, 113 2 - 1135 .

A2. PR3PARATION OF P-S3L3CTIN-IGGl The genetic conatruct "CD62Rg" pllhli~h~d by Aruffo et al., 1991, i8 uaed to prepare the aoluble P-selectin-IgGl fuaion protein. The subsequent ~ uc~ lu e co. ~ ds to the preparation o~ L-selectin-IgG1 described under Al.
Aruffo, A., Rolanus, W., Walz, G., FredT~an, P. and Seed, B. 1991. CD62/P-selectin recognition of myeloid and tumor cell sulfatidesl Cell 67, 35-44.
A3 . PR3PARATION OF CD4 - IGGl The genetic construct "CD4: IgGl hinge" p--hl; ~h~d by Zett~ al et al., 1990, is used to prepare the soluble CD4-IgGl fusion protein. The subsequent procedure corres-ponda to the preparation of L-aelectin-IgGl described under Al.
ZettQl- ;~81, G., Gregeraen, J.-P.-, Duport, J.M., Mehdi, S., Reiner, G. nd Seed, B. 1990. 3xpression and characterization of huT~Ian CD4 T ,3lnhulin Fusion Proteins. DNA and Cell Biology 9, 347-353.
A4. PROC3DURE FOR TH3 HL60 C3LL ADHESION ASSAY FOR
2 0 T~T.~c~M~T~NT SOLUBL3 ADH3SION MOLT~'~TJT T q 1. 96-well microtiter assay plates (Nunc Maxiaorb) are incubated with 100 ~Ll of a goat anti-human IgG
antibody (Sigma) diluted (1+100) in 50 mM Tria pll 9 . 5 at room teT~perature f or 2 h . Removal of the antibody solution ia followed by one wash with PBS.
2. 150 ~1 o~ the blocking buffer are left in the wella at room tenperature ~or 1 h. The composition of the blocking buf f er is:
0.1% gelatin, 1% BSA, 5% calf serum, 0.2 mM PNSF, 0.02% aodiuT~ azide. Removal of the hlo~lr;ns buffer is followed by one waah with PBS.

2~929~
3. 100 ~Ll of cell culture supernatant from ~
ately transfected and expressing COS cells are pipetted into each of the wella. The incubation takes place at room temperature for 2 h. Removal of the cell culture supernatant is followed by one wash with PBS.
4. 20 ~1 of binding buffer are placed in the wells. A
binding buffer has the composition: 50 mM ~Iepes, p~
7 . 5; 10 0 mM NaCl; 1 mg/ml BSA; 2 mM MgCl2; 1 mM
CaC12; 3 mM MnC12; 0.0296 sodium azide; 0.2 mM PMSF.
5 ~1 o~ the test subatance are added by pipette, mlxed by agitating the plate and incubated at room temperature for 10 min.
5 . 50 ml of an ~IL60 cell culture with 200, 000 cells/ml are centrifuged at 350 g for 4 min. The pellet is resuspended in 10 ml of RPMI 1640 and the cells are again centrifuged. For labeling of the cells, 50 ~g of BCECF-AM (Molecular Probes) are dissolved in 5 ~1 of anhydrous DMS0; subseguently 1. 5 ml of RPMI 1640 are added to the BCECF-AM/DMS0 solution. The cells are resuspended in this 601ution and incubated at 37C for 30 min. After centrifugation at 350 g for 2 minutes, the labeled cell pellet is resuspended in 11 ml of binding buffer, and the r~ np~nrled cells are distributed in 100 ~ul aliquots in the well3 of the microtiter plate. The plate i~ left to stand at room temperature for 10 min in order to allow the cells to sediment to the bottom of the test plate.
This provides the cells with the ~1'~~ ity to adhere to the coated plastic.

6. To stop the assay, the microtiter plate is CCIIL-pletely immersed at an angle of 45 in the stop buffer (25 mM Tri3, p~ 7.5; 125 mM NaCl; 0.1% BSA;
2 mM MgC12; 1 mM CaC12; 3 mM MnC12; 0 . 0296 sodium a~ide). The stop buf~er is removed from the wells l~y inversion, and the procedure is repeated twice more.

2~2~56

7. The BCECF-AM-labeled cells which are ~irmly adherent in the wells are measured in a cyto~luorimeter (M; 1 l; rnre) with a 8engitivity setting o~ 4, an excitation wavelength of 485/22 nm and an emission wavelength o~ 530/25 nm.
IC50 of ~L60 cell adhesion to 3-selectin-IgG:
C _ 1 ~rom Ex . 3 0: 6 0 yN
31: 60 ~N
32: 100 IlM
33: 60 ~M
34: 150 f~M
Comparative value:
ICs of ~L60 cell ~h~sinn to E-selectin-IgG:
slalyl-LeX-O (C~2) 6NH2: l mM
(cf, EPA 93 119 098.7) B. S3CONDARY ASSAY TO INVESTIGATE T~IE EFFECT OF POLY-MERIC ~ ~R~RY7 pl~T~ RE~:~TO~ BBOCKERS ON CELL AD~ESION TO
STTMTTT ~T~n HU2aN ENDOTEIELIAL CELLS
The test of the activity o~ polymeric carbohydrate receptor blockers on cell ~rlh~ n to rec~ ' in:~nt soluble fu~ion proteins is a highly specific assay which is based on the interaction of one type of ~h~inn molecules with the corresponding ligands. In order to simulate the in vivo situation of cell-cell interactions, we use an assay in which ~60 cellg adhere to 8timulated human, endothelial cells.
1. Obtaining the human umbilical endothelial cells (EUVEC) .
IJmbilical cords are stored af ter delivery in PBS contain-ing 100, 000 IU/B penicillin, 100 mg/[ streptomycin, 50 mg/~ gentamicin and 50,000 IU/L mycostatin at +4C
until further processed.
The longest undamaged pieces are cut out of the umbilical cord with a scalpel and placed on fresh aluminum foil.

.
~ 2192~

One end o_ the umbilical cord is closed with a clip. At the other end, a suitable tube is inserted into the u~bilical vein and _ixed by ligating the end o_ the umbilical cord.
5 The vein is filled through the piece o_ tube with col-lagenase aolu~ n (50 mg collagenaDe/100 ml 25 mM ~epes, p~ 7.0) and incubated at 37C _or 15 min.
In order to increase the cell yield, the umbilical cord is gently ma3saged af ter the incubation in order to 10 detach still adherent endothelial cells.
The cell 3~pon~inn is subsequently allowed to run out o_ the vein into a culture tube containing cell culture medium and 10% fetal calf serum. The vein is washed with PBS in order to obtain the L-= in;ng cell3.
15 The cell suspension is centrifuged at 500 g for 5 min;
the cell pellet i8 subsequently resl~p~n~ in 4 ml of culture mediu~ and the cells are plated out . Af ter 3-4 days there i3 conf~ nt growth of the cells and they can be passaged.
20 To check the purity of the endothelial cell culture, the culture ia stained with an antibody against factor VIII
_or the immunofluore3cence. A po3i~ive reaction is shown only by endothelial cells but not by contaminating _ibroblas ts .
25 2. Procedure for the assay 20, 000 endotholial cells are plated out per well in a 96-well microtiter plate and incubated at 37~C _or 24 h.
The endothelial cella for thl: purpose must not have been pasaaged more than 5-6 times. Four hours be_ore the assay 30 the endothelial cells are stimulated by addition o_ Il-l (_inal c~nr~-ntration: 15 U/ml~ . A_ter removal of the culture medium, the cells are wa3hed once to me RPMI

~lF
2~2~&

medium without aerum. Removal ,and renewed pipetting of 20 ~1 of RPMI medium are followed by addition of test substances .
3. The ~urther steps in the assay, lAh-~l;n~ of the hB60 5 cells and introduction of the EIL60 CellB, are carried out Aa under A4. Items 5. - 7-C. SECf~ RY ASSAY TO INVESTIGATE ThE EFFECT OF POLY-MERIC ~RR~IWYr~Ri~'rR k~i~8-l~L~R BLOCRERS ON CE~L ADEIESION TO
FROZEN SECTIONS OF LYMP~ATIC q~ISSV13 10 It i8 possible in vitro to investigate the extent to which leukocytes bind to endothelial cells on frozen sections of lymphatic tissue. These c~ll-cell inter-actions are baaed on the interaction between :~h~8ir~n molecules on ~he surface of the endothelial cells in the 15 frozen section and the COLLF'A~ ;n~ ligands on the surface of leukocytes. It is possible to use as substi-tute ~or primary leukocytes ~IL60 cells whose sur~ace ligands are well described in the scientific literature.
It is possible to determine whether A~hF~si~n of }A~B60 20 cells to lymph node ~rozen sections takes place from the number of bou~d ~IL60 cells.
1. Axillary, cervical or -- te~ic lymph nodes are dissected out of freshly sacrificed rats and rapidly frozen in liquid nitrogen.
25 2. 10 ~m-thick cryostat sections are prepared from the frozen lymph nodes, transferred to circular cover glasses (diameter 18 mm) and dried at room temperature for 2 h.
3. 20 ~Ll of binding buffer are pipetted onto the sections. The test substances are added and incubated at 30 room temperature for 10 min. hL60 cells are labeled as under A4. Item 5. 200,000 labeled ~60 CellB in 100 ~L1 o~
binding buffer are added to each cover glass and le~t to stand for 10 min. This allows the sedimenting cells to ~ 21929~
-- 43 -- ~_ reach endothelial cells to which some of them adhere.
4. The cover glasses are; ~ed at an angle of 45~ in stop buf~er in order to rinse off the non-adherent cells.
The cover glasses are subsequently fixed in 4% form-5 aldehyde in PBS at room temperature for 10 min.
5. Cross sections of lymphatic blood vessela are recorded by photography under an immunof luorescence microscope (FITC excitation). The adherent ~IL60 cells are clearly distinct from the unstained background. The 10 result is expressed as bound HL60 cells per unit area of erdothelium.
D. TERTIARY ASSAY TO INVESTIGATE T~IE EFFECT OF POLY-MERIC r~R~ T~ oR BOCRERS ON L~;UKO~:~L~ AD~ESION
IN RATS IN VIVO
15 The method detailed hereinafter is able to establish the in vivo activity of substanceg which illhibit the ol~lh~n;~n of leukocytes to the vessel intima.
It is known that some of the circulating white blood cells have th~ls tendency to adheIe to the intima of the 20 blood vessels. This tendency is considerably ~n1~Anre~ in ;nfl tory processes. Leukocytes normally impinge continually on the blood vessel walls, but this collision is elastic 80 that the cells rebound to a certain extent and return to the circulation. In ;nfl tory processes, 25 ~iorh~Tn; r:~l c~ange~, both in the leukocytes and in the endothelial cells lining the blood vessels, lead to changes in the surfaoe properties of both types o~ cell.
The cells' behavior becomes more adhesive. This adhesive-ness is initially expressed by the tendency of the 30 leukocytes after collision with the endothelium to roll on the endothelial cell~. This rolling of the leukocytes on the endothelium induces further biorh~' rs~l reactions on both binding partner~, as a conse5~uence o~ which cell ~lh~nion is enhanced. This further enhancement of the ~ 21928~

~dhesiveness slows down the ro,lling of th~ leukocytes until they adhere f irmly to the endothelium. The f irm adherence is followed by, a3 furl~her step, the migration o~ the leukocytes out of the blood vessel. The rolling of 5 the leukocytea, the firm ;~h~ n and the migration out of the blood vessels can be induced with leukocyte-stimulating factors such as FMLP (f-Met-Leu-Phe), LPS
(lipopolysaccharides) or TNF (tumor necrosis factor).
Microscopic recording of these processes i8 possible on 10 dissected mesenteric tissue, for example from rats.
Substances incected into the bloodstream can therefore be investigated to find whether they are able to influence induced leukocyte ~lh~Ri~
Rats are anesthetized with [lacuna] for carrying out this 15 investigation in practice. The abdominal height is opened and a section of the small intestine i 8 pulled out . The section of small intestine is continuously kept moist on a heatable mi- Lvscv~e stage. For microscopic inspection, a region of mesenteric tissue is covered with liquid 2 0 paraf f in . For the control, all the a&erent - non-stimulated - leukocytes in this region are counted every 5 min for a period of 30 min. In parallel with this, the blood pressure, body temperature and flow rate of the blood are recorded. The test substance is ad~inistered by 25 continuous venous in~usion tllLvuyllvut the test. After application of leukocyte stimulants, which are added dropwiae to the mesenteric tissue, the adherent leuko-cytes are counted every 5 min for a period of 90 min.
The investigation is carried out on test groups each 30 composed of three animala. The first animal receives only the vehicle in order to determine the spontaneous adhesion. The second animal receives only the leukocyte stimulation in order to determine the pathogenic control.
The third animal receives leukocyte stimulant and test 35 substance.
The number of adherent leukocytes in the pathogenic 21929~6 control ia aet equal to 100%. The perce~tage change in the leukocyte ~hP~ n on adminiatration of test sub-stance compared with the pathogenic control indicates the activity of a test substance.

Claims (14)

claims:

1. A process for preparing a physiologically tolerated and physiologically degradable polymer-based carbo-hydrate receptor blocker consisting of a) a hydrophilic, biodegradable polymer unit, b) at least one di- or oligosaccharide unit and c) at least one bifunctional spacer by which the di-or oligosaccharide units are linked to the poly-mer unit, which comprises initially preparing an acceptor by chemical linkage of a mono- or oligosaccharide, of the spacer and of the hydrophilic, biodegradable polymer, after which one or more other monosaccharide units are attached by enzymatic glycosylation.

2. The process as claimed in claim 1, wherein the enzymatic glycosylation of the acceptor takes place in homogeneous aqueous phase.

3. The process as claimed in claim 2, wherein the enzymatic glycosylation of the acceptor takes place with the aid of nucleotide-activated carbohydrates as donors and glycosyltransferases.

4. The process as claimed in claim 3, wherein the enzymatic glycosylation of the acceptor takes place in a buffer system appropriate for the particular glycosyltransferase.

5. The process as claimed in claim 4, wherein the buffer system has a concentration of 0.01 M to 1 M.

6. The process as claimed in claim 4 or 5, wherein the buffer system contains the cations necessary for activating the particular glycosyltransferase.

7. The process as claimed in any of claims 2 to 6, wherein the pH of the aqueous medium is between 6.0 and 8.5.

8. The process as claimed in any of claims 3 to 7, wherein alkaline phosphatase is added to the reac-tion medium when the donor is added in equimolar amount or excess.

9. The process as claimed in any of claims 3 to 8, wherein 0.01 to 10 units of the glycosyltransferase are added to the acceptor dissolved in the agueous buffer system and to the nucleotide-activated carbo-hydrate.

10. The process as claimed in any of claims 2 to 9, wherein the enzymatic glycosylation is carried out at 10 to 40°C for 1 to 5 days.

11. The process as claimed in any of claims 1 to 10, wherein a polycarbonate, polyester, polyamide, polyanhydride, polyiminocarbonate, polyanhydride, polyorthoester, polydioxanone, polyphosphazene, polyhydroxy-carboxylic acid, polyamino-acid or a polysaccharide is used as hydrophilic, biodegradable polymer.

12. The process as claimed in claim 11, wherein a polyamino-acid with a molecular weight less than or equal to 70 kD, which is in polyamide or poly-anhydride form, is used as hydrophilic, biodegradable polymer.

13. The process as claimed in claim 12, wherein the polyamlno-acid is poly-.alpha.,.beta.-(2-hydroxyethyl)-D, L-aspartamide, poly-D,L-succinimide, polyglutamate, poly-L-lysine methyl ester fumaramide or a copolymer of these polyamino-acids.

14. The process as claimed in any of claims 1 to 13, wherein the spacer of the acceptor has formula I

(mono- or oligosaccharide-O-[Q1-(CH2)p-Q2]r-(polymer unit) I, in which Q1 is -CH2- or Q2 is -NH- or , p is an integer from 1 to 6 and r is 1 or 2.