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WO1996006102A1 - Procede de preparation de melanges d'oligosaccharides - Google Patents

Procede de preparation de melanges d'oligosaccharides Download PDF

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Publication number
WO1996006102A1
WO1996006102A1 PCT/EP1995/003254 EP9503254W WO9606102A1 WO 1996006102 A1 WO1996006102 A1 WO 1996006102A1 EP 9503254 W EP9503254 W EP 9503254W WO 9606102 A1 WO9606102 A1 WO 9606102A1
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group
sugar
mixture according
core structure
promoter
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English (en)
Inventor
Ole Hindsgaul
Osamu Kanie
Monica Palcic
Beat Ernst
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Novartis AG
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Ciba Geigy AG
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Priority to AU33464/95A priority Critical patent/AU3346495A/en
Publication of WO1996006102A1 publication Critical patent/WO1996006102A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/12—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by acids having the group -X-C(=X)-X-, or halides thereof, in which each X means nitrogen, oxygen, sulfur, selenium or tellurium, e.g. carbonic acid, carbamic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages

Definitions

  • the present invention relates to an oligosaccharide mixture whose individual constituents have an identical cone structure which is substituted by at least one sugar and in which all positional isomers are represented, and all isomers with respect to the substituent are in the form of ⁇ - or ⁇ -isomers, to a process for the preparation of said oligosaccharide mixture and to a process for the rapid isolation of a biologically active oligosaccharide.
  • carbohydrates have an important part to play as information carriers in intercellular communication and in inter ⁇ cellular recognition processes.
  • their specific interaction with determinants of the cell surface causes signals to be passed on to the cell.
  • neurotrans- mitters, growth factors, protein hormones and cytokins are able - by means of carbo ⁇ hydrate-mediated interaction with the target cell - to transport signals to the cell that result in changes in metabolism.
  • the carbohydrate-mediated adhesion of e.g. viruses, bac ⁇ teria, toxins, tumour cells and immune-competent cells such as lymphocytes and leuco ⁇ cytes are an important factor in pathological processes.
  • oligosaccharide mixtures of this invention enable the number of syntheses and bioassays to be reduced massively. These mixtures make it possible to select core structures very rapidly.
  • This invention makes it possible for the first time to provide mixtures of different oligo ⁇ saccharides in which (a) the oligosaccharides consist of an identical core structure carry ⁇ ing at least one substituent, such that within a mixture the substituents are all identical but are located in different positions and are linked ⁇ - or ⁇ -glycosidically, and (b) all positio ⁇ nal isomers and stereoisomers are present in a mixture.
  • the invention also makes it poss ⁇ ible to provide mixtures whose oligosaccharides have a uniform degree of substitution.
  • the invention relates to a mixture of different oligosaccharides which are derived from at least two sugar monomers, both individual components of said mixture having an identical core structure of identical or different sugar monomers, wherein
  • sugar monomers of the core structure are unprotected or partially protected, but are preferably unprotected,
  • At least one substituent selected from the group consisting of unprotected and protected mono-, di- and trimeric sugars is linked ⁇ - or ⁇ -, -0-, -N-, -S- or -C-glycosidically to the core structure, with the proviso that all substituents are identical if there is more than one substituent,
  • the oligosaccharides consist of 2 to 8, preferably 2 to 6, most preferably 2 to 3, sugar monomers.
  • the sugar monomers are linked ⁇ - or ⁇ -(anomeric centre ⁇ n) glycosi- dically to one another, where n is a number from 1 to 15 and the term (anomeric centre-n) denotes in which positions of the two participating monomers the glycosidic bond is located.
  • the core structure of all oligosaccharides of a mixture is built up from 1 to 5, preferably 1 to 3 and, most preferably, 1 or 2, identical or different unprotected sugar monomers.
  • the core structure may be branched or unbranched.
  • At each core structure of a mixture at least one unprotected or protected mono-, di- or trimeric sugar is attached to a hydroxyl group which does not participate in a bond within the core structure.
  • the maximum number of sugars attached to a core structure will depend on the number of these hydroxyl groups.
  • Those mixtures are preferred in which the core structures of their components are substituted by 1 to 6, more particularly by 1 to 3 and, most preferably, by 1 or 2 sugars.
  • the novel mixtures contain only components in which the same sugars are attached to the core structure. The components differ only in the position at which the sugar, or each of the sugars, is attached, and in their stereochemistry.
  • the following different components are present in a mixture whose com ⁇ ponents have a core structure containing three free hydroxyl groups: (1) substitution at hydroxyl group 1, (2) substitution at hydroxyl group 2, (3) substitution at hydroxyl group 3, (4) substitutions at hydroxyl group 1 and hydroxyl group 2, (5) substitutions at hydroxyl group 1 and hydroxyl group 3, (6) substitutions at hydroxyl group 2 and hydroxyl group 3, (7) substitutions at all hydroxyl groups, the variants (1) to (7) being present as ⁇ -, ⁇ -, ⁇ -, ⁇ -, ⁇ -, ⁇ -, ⁇ -, ⁇ -, ⁇ -, ⁇ -, ⁇ - or ⁇ -isomers.
  • Useful mixtures are those in which the variants (1) to (6), preferably (1) to (3), with their respective stereo- isomers, are present. Very particularly preferred mixtures have a uniform degree of substi ⁇ tution. The degree of substitution will be understood as meaning the number of substi ⁇ tuents per core structure.
  • monomeric sugars will be understood as meaning all compounds whose structure conforms to the formula (CH 2 0) m , where m is preferably a natural number from 3 to 15, as well as polyhydroxyaldehydes, polyhydroxyketones, polyhydroxyacids and polyhydroxyamines and derivatives thereof.
  • Illustrative examples are sugar monomers selected from the group consisting of D- and L-aldopyranoses and D- and L-aldofuranoses, including glycerol aldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose and talose, from the group consisting of D- and L-ketopyranoses and D- and L-ketofuranoses, typically including dihydroxyacetone, erythrulose, ribulose, xylulose, psicose, fructose, sorbose and tagatose, as well as from the group consisting of D- and L-diketopyranoses, typically pentodiulose and hexodiulose.
  • D- and L-diketopyranoses typically pentodi
  • sugar monomers comprises also sugar monomers which represent substitutions of the cited examples.
  • these sugar monomers typically include protected, partially protected or unprotected deoxy sugars of the D- and L-configuration, preferably 2-, 3-, 4-, 5- and 6-deoxyaldoses such as fucose, rhamnose and digitoxose, 1,2-dideoxyaldoses such as glucal, galactal and fucal, and 1-, 3-, 4-, 5- and 6-deoxyketo- ses, 1-, 3-, 4-, 5- and 6-deoxyazido, 2-, 3-, 4-, 5- and 6-deoxyamino sugars of the D- and L-configuration, typically glucosamine, mannosamine, galactosamine and fucosamine, deoxyacylamino sugars such as N-acylglucosamine, N-acylmannosamine, N-acylgalactos- amine and N-acylfucosamine, preferably the C r C 4 al
  • Sugar monomers will also be understood as meaning aldonic, aldaric and uronic acids such as gluconic acid or glucuronic acid, as well as ascorbic acid, amino acid-carrying sugar monomers and those that carry lipid, phosphatidyl or polyol substituents.
  • Substituted sugar monomers will also be understood as meaning those having a carbon chain longer than 6 carbon atoms, typically heptoses, octoses, nonoses, heptuloses, octuloses and nonuloses, and also the representatives substituted in accordance with the foregoing criteria, for example ketodeoxyoctanoic acid, ketodeoxynonanoic acid, N-acyl- neuraminic acids and N-acylmuramic acids.
  • di- and trimeric sugars will be understood as meaning those derived from two or three identical or different monomers cited above.
  • the linkage is preferably ⁇ - or ⁇ -O-glycosidic, but S-, N- and C-glycosidic linkages are also possible. All carbon atoms of the one participant of a linkage are suitable. Illustrative examples are in particular (1-2)-, (1-3)-, (1-4)-, (1-5), (1-6), (2-3)- and (2-6)glycosidic linkages.
  • Typical examples of dimeric sugars are those selected from the group consisting of treha- lose, sophorose, kojibiose, laminaribiose, maltose, cellobiose, isomaltose, gentibiose, sac ⁇ charose and lactose.
  • Illustrative examples of trimeric sugars are raffinose and melezitose. It has been found that it is advantageous if a substituent is linked ⁇ - or ⁇ -O-, ⁇ - or ⁇ -N-, ⁇ - or ⁇ -S- or ⁇ - or ⁇ -C-glycosidically to the anomeric carbon atom of the core structure direct or through a linking group. This substituent influences a property of the oligosaccharide selected from the group consisting of detectability, separability, bonding and distribution behaviour.
  • substituents that influence the separability of the oligosaccharide are ligands for biopolymers, for example biotin as ligand for streptavidin and hapten as ligand for antibodies.
  • These groups are particularly suitable as lipophilic anchor groups for chromatographic purification, coating lipophilic surfaces, incorporation in liposomes and micelles and transportation through membranes.
  • substituents When these substituents are attached to the oligosaccharide through a linking group, they are protected by (a) groups that can be activated chemically, for example amines, carb- oxylic acids, phenols, aldehydes, mercaptans that are protected by conventional methods, reactive double bonds that are suitable for conversion into functional groups, or (b) groups that can be activated photochemically, for example arylazide and azirinyl groups.
  • groups that can be activated chemically for example amines, carb- oxylic acids, phenols, aldehydes, mercaptans that are protected by conventional methods, reactive double bonds that are suitable for conversion into functional groups
  • groups that can be activated photochemically for example arylazide and azirinyl groups.
  • a linking group will be understood as meaning a bivalent group of formula -(X-A) p -X'- wherein X and X' are each independently of the other C r C 12 alkenylene, C 1 -C 12 alkynylene -(C x H 2 ⁇ O) y -, C 5 -C 8 cyclo- alkylene, C 6 -C 12 arylene or C 7 -C ⁇ 2 aralkylene, A is -0-, -S-, -S-S-, -NR 10 -CO-NR 10 -, -NR 10 -CS-NR 10 -, -NR 10 -, -NR 10 -C(O)-O-, -C(0)0-, -C(0)S-, -C(O)NR 10 -, -C(S)S-, -C(S)0-, -C(S)NR 10 -, -SO 2 NR 10 -, -, -
  • Protective groups and processes for derivatizing the hydroxyl groups with such protective groups belong to the stock of common knowledge of sugar and nucleotide chemistry and are described, inter alia, by Greene, B.T., Protective Groups in Organic Synthesis, Wiley Interscience, New York (1991), by Sonveaux, E., Bioorganic Chemistry 14:274-325 (1986), or by Beaucage, S.L., Iyer, R., Tetrahedron 48:2223-2311 (1992).
  • Such protective groups are: benzyl, methylbenzyl, dimethylbenzyl, methoxy- benzyl, dimethoxybenzyl, bromobenzyl, 2,4-dichlorobenzyl; diphenylmethyl, di(methyl- phenyl)methyl, bis(dimethylphenyl)methyl, di(methoxyphenyl)methyl, bis(dimethoxy- phenyl)methyl, triphenylmethyl, tris-4,4',4"-tert-butylphenylmethyl, di-p-anisylphenyl- methyl, tri(methylphenyl)methyl, tris(dimethylphenyl)methyl, methoxyphenyl(diphenyl)- methyl, di(methoxyphenyl)phenylmethyl, tri(methoxyphenyl)methyl, tris(dimethoxy- phenyl)methyl; triphenylsilyl, alkyldiphenylsilyl, dialky
  • the protective groups may be identical or different.
  • Preferred protective groups are selected from the group consisting of linear and branched C r C 8 alkyl, preferably C 1 -C 4 alkyl, typically methyl, ethyl, n- and isopropyl, n-, iso- and tert-butyl; C 7 -C 12 aralkyl, typically benzyl; trialkylsilyl containing 3 to 20, preferably 3 tol2, carbon atoms, typically trimethylsilyl, triethylsilyl, tri-n-propylsilyl, tri-i-propylsilyl, isopropyldimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, n-octyldimethylsilyl, (1,1,2 ⁇ 2-tetramethyl- ethyl)dimethylsilyl; substituted methylidene groups which are
  • R is C j -C ⁇ alkyl, preferably C r C 6 alkyl, C 5 -C 6 cycloalkyl, phenyl, benzyl, C j -C 12 alkylphenyl, preferably preferably C ⁇ -C 4 alkylbenzyl, for example methylsulfonyl, ethylsulfonyl, propylsulfonyl, butylsulfonyl, phenylsulfonyl, benzylsulfonyl and p-methylphenylsulfonyl.
  • a partially protected mono- or oligosaccharide will be understood as meaning one containing at least 2, preferably at least 3, free hydroxyl groups.
  • the invention relates to a mixture of different oligosaccharides which are derived from at least two sugar monomers, both individual components of said mixture having an identical core structure of identical or different sugar monomers, wherein
  • sugar monomers of the core structure are unprotected or partially protected, but are preferably unprotected,
  • At least one substituent selected from the group consisting of unprotected and protected mono-, di- and trimeric sugars is linked ⁇ - or ⁇ -, -0-, -N-, -S- or -C-glycosidically to the core structure, with the proviso that all substituents are identical if there is more than one substituent,
  • a pharmacologically active substituent will be under- stood as meaning a negatively charged substituent which is introduced by reacting the free hydroxyl groups of the monosaccharides or oligosaccharides with carboxylic acids which in ⁇ -position carry a leaving group, for example a halide, triflate, tosylate, mesylate, brosylate or a diazo group, by reaction with a sulfuric acid derivative (q.v.
  • Example 3 or by reacting the monosaccharides or oligosaccharides with derivatives of phosphoric acid, for example di-0-benzylphosphochloridate, or with derivatives of phosphorous acid such as di-0-benzyl-N,N-dialky-phosphoroamidite, followed by oxidation (I 2 , tBuOOH) and, in both cases, followed by the hydrogenolytic removal of the benzyl protective group.
  • derivatives of phosphoric acid for example di-0-benzylphosphochloridate
  • derivatives of phosphorous acid such as di-0-benzyl-N,N-dialky-phosphoroamidite
  • the maximum number of pharmacologically active substituents attached to an oligo ⁇ saccharide will depend on the number of free hydroxyl groups in the oligosaccharide mix ⁇ ture.
  • Preferred mixtures are those in which the oligosaccharides are substituted by 1 to 6, preferably 1 to 3 and, most preferably, 1 to 2, pharmacologically active substituents.
  • the novel mixtures contain only oligosaccharides in which the pharmacologically active substituents are identical.
  • the oligosaccharides differ only in the position at which the pharmacologically active substituent or each of the pharmacologically active substituents is attached.
  • all stereoisomers are represented.
  • a further object of the invention is a process for the preparation of the novel mixtures, which comprises reacting an unprotected or partially protected mono- or oligosaccharide which has a core structure of identical or different sugar monomers, in the presence of an aprotic polar solvent and optionally a promoter, with an activated, protected mono-, di- or trimeric sugar, and removing any protective groups present.
  • the reaction can be carried out in solution or in immobilized form. Either core structure or sugar substituent can be immobilized. Useful are all solid phase materials normally used in solid phase peptide synthesis.
  • aprotic polar solvents are suitable for use in the inventive process.
  • Particularly useful solvents are those selected from the group consisting of nitriles, sulfoxides, sulfones, N-hydrocarbons, N-dialkylcarboxamides, N-alkyllactams, polyethylene glycol dialkyl ethers, cyclic ethers, N-alkylated cyclic amines and mixtures of said solvents with one an- other.
  • Representative examples of nitriles are acetonitrile, propionitrile, benzonitrile and benzyl nitrile.
  • a suitable sulfoxide is typically dimethyl sulfoxide.
  • sulfones are tetramethylene sulfone and dimethyl sulfone.
  • N-Hydrocarbons are typically nitromethane and nitrobenzene.
  • N-Dialkylcarboxamides are typically dimethyl form ⁇ amide and dimethyl acetamide.
  • a suitable N-alkyllactam is is typically N-methylpy ⁇ oli- done.
  • a suitable polyethylene glycol dialkyl ether is typically diethylene glycol dimethyl ether.
  • Cyclic ethers are typically tetrahydrofuran, dioxane and dioxolane.
  • Typical examples of N-alkylated cyclic amines are N-methylmorpholine and N-methylpyrro- lidone.
  • Preferred solvents are selected from the group consisting of acetonitrile, propio ⁇ nitrile, d methyl formamide and dimethyl formamide in admixture with acetonitrile.
  • a very particularly preferred solvent is dimethyl formamide or a mixture of dimethyl form ⁇ amide and acetonitrile, the ratio of dimethyl formamide to acetonitrile advantageously being 1:10 to 10:1, preferably from 1:1 to 1:5, most preferably from 1:4.
  • the ratio of oligosaccharide to protected mono-, di- or trimeric sugar is from 10:0.1, preferably from 3:1 and, most preferably, from 2:1.
  • the ratio of protected mono-, -di- or trimeric sugar to promoter is from 10:0.1 to 0.1: 10, the preferred ratio being 1:1.
  • the reaction is carried out in the temperature range from -40°C to 100°C, preferably from 0°C to 50°C, most preferably at room temperature.
  • activation means the introduction of an anomeric leaving or acceptor group into the mono-, di- or trimeric sugar.
  • C r C 10 haloalkyl, aryl, aralkyl, heterocycloalkyl or heteroaryl, preferably CF 3 , CC1 3 , methyl, ethyl or benzyl.
  • the anomeric acceptor group is conveniently selected from a subgroup of the group
  • the promoter is preferably selected from the group consisting of
  • ( ⁇ ) mineral and organic acids such as HC1, H 2 S0 4 , H 3 P0 4 , H 2 S0 4 , trifluoromethane- sulfonic acid, p-toluenesulfonic acid and methanesulfonic acid;
  • ( ⁇ ) BF 3 and the complexes thereof preferably BF 3 -diethylether, trialkylsilyl- or triarylsilyltrifluoromethanesulfonate, preferably trimethyl-, triethyl-, triisopropyltriflu- oromethanesulfonate; alkyl- and arylsulfonic acids, preferably CF 3 S0 3 H and ArS0 3 H;
  • N-alkylating reagents of the structure RX where R is alkyl or aryl and X is a leaving group, preferably methyl iodide, methyl trifluoromethane sulfonate, trityl perchlorate, RCOX and RS0 2 X;
  • thiophilic and phosphophilic reagents such as BF 3 -diethylether, NOBF 4 , trialkylsilyl trifluoromethane sulfonate, wherein R is alkyl or aryl, dimethylmethylthiosulfonium tetrafluoroborate, methylsulfenyl bromide, methylsulfenyl trifluoro methane sulfonate, dimethylmethylthiosulfonium trifluoromethane sulfonate, S0 2 Cl 2 /trifluoromethane sulfonic acid, phenyltrifluoromethane sulfonate, Cl 2 , Br 2 , 1 2 , Cl + , Br + , I + , preferably iodonium collidinium perchlorate and iodonium collidinium trifluoromethane sulfonate, N-chloro-, N-bromo- or N
  • trifluoromethane sulfonic acid sulfur and phosphor- alkylating reagents of the general structure RX, where X is a leaving group (e.g. methyl iodide, methyl trifluoromethane sulfonate, trityl perchlorate), alkyl and aryl selenium and sulfenium cations and their precursors (e.g. phenylselenyl trifluoromethane sulfonate);
  • X is a leaving group (e.g. methyl iodide, methyl trifluoromethane sulfonate, trityl perchlorate), alkyl and aryl selenium and sulfenium cations and their precursors (e.g. phenylselenyl trifluoromethane sulfonate);
  • RX alkyl and X is a leaving group (e.g methyl trifluoromethane sulfonate).
  • the inventive process can be carried out under the action of heat, light or electrical fields.
  • the procedure in the case of photochemical activation is described by Hashimoto, S., Kurimoto, I., Fujii, Y., Noyori, R., in J. Amer. Chem. Soc. 107:1427 (1985) and, in the case of electrochemical activation, in J. Org. Chem. 4320 (1993).
  • the invention relates to a process for the rapid isolation of a biologically active oligosaccharide by
  • this process is one by means of which it is possible to identify and isolate very rapidly particularly active oligosaccharides by comparing the activities of very many oligosaccharide mixtures with one another and carrying out further processing only with the most active mixtures and isolating therefrom the most active oligosaccharide.
  • the pharmacological activities of the mixtures of this invention are determined typically by assessing the inhibition of a molecular or cellular response induced by them. Within the scope of this invention, a mixture is always held to be active when it leads to a significant reduction of the molecular or cellular response.
  • Chromatographic methods of isolating oligosaccharides are known. In these methods the oligosaccharides are separated in accordance with their size, their electrical charge and their binding properties. The methods employed are typically affinity, molecular sieve and ion exchange chromatography. HPLC (high pressure liquid chromatography) is especially useful.
  • Example 1 Preparation of l-(4-methoxyphenoxyl)-oct-8-yl 2-acetamido-2-de- oxy-X-0-(Y-D-galactopyranosyl)- ⁇ -D-glucopyranoside (X: 3,4,6; Y: ⁇ , ⁇ )
  • Example 3 Preparation of l-(methoxyphenoxyl)-oct-8-yl 2-acetamido-2-de- oxy-X-0-( ⁇ -L-fucopyranosyI)-4-O-( ⁇ -D-galactopyranosyl)- ⁇ -D-glucopyranoside Z-O-sulfate ester (X: 3, 6, 2', 3', 4', 6'; Y: ⁇ , ⁇ ; Z ⁇ X, 3, 6, 2 ⁇ 3 4 ⁇ 6')
  • the trisaccharide mixture 7 (98 mg, 0.095 mmol) is charged to dry pyridine (5 ml) at room temperature and sulfur trioxide-pyridine complex (15 mg, one equivalent) is added. The reaction mixture is then stirred for 14 hours at 50°C.
  • reaction mixture is afterwards filtered over Celite and the filter product is cautiously washed with 250 ml of methanol and the combined solutions are concentrated to dryness.
  • the crude product is charged to an ion exchange column (0.5 g DEAEA-50), the nonsulfated products are eluted with deionised water and the sulfated products with 1 M NaCl.
  • the sulfated products are charged to a C-18 Sep Pack column and the column is washed with 120 ml of deionised water and the sulfated products 10 (20.2 mg) l-(methoxyphenoxyl)-oct-8-yl 2-acetamido-2-deoxy-X-0-( ⁇ -L-fucopyranosyl)-4-0-( ⁇ -D-galactopyranosyl)- ⁇ -D-gluco- pyranosid-Z-O-sulfate ester (X: 3, 6, 2 ⁇ 3', 4', 6'; Y: ⁇ , ⁇ ; Z ⁇ X, 3, 6, 2', 3', 4', 6') are eluted with methanol (120 ml).
  • the detection for the ion exchange chromatography and the C-18 chromatography is effected by UV detection at 292nm.
  • Example Bl Primary assay to investigate the activity of the oligosaccharide mixtures
  • This assay demonstrates the activity of the oligosaccharide mixtures on the interaction of the ligands sialyl Lewis A or sialyl Lewis X with selectins (E, P, L).
  • the bonding participants are genetically engineered soluble fusion proteins from each of the extracyto- plasmic domains of E-, P- and L-selectin and the constant region of a human immuno- globulin of the subclass IgG 1.
  • selectin/IgG fusion proteins are prepared by linking the soluble domains of the different selectins to the carboxy-terminal end of the first constant region (CHI) of human IgGl.
  • the construction is carried out in a manner similar to that described by Walz et al. [Walz, G., Aruffo, A., Kolanus, W., Bevilaqua, B., Seed, B., Science 250:1132-1135 (1990)].
  • E-selectin/IgG chimeras To prepare the E-selectin/IgG chimeras, the complete cDNA of E-selectin available from British Biotechnology (Product No. BBG 57) as well as genomic DNA coding for human IgGl are used.
  • the DNA fragments for E-selectin and genomic DNA of human IgGl are amplified by polymerase chain reaction (PCR) and subsequently fused by SOE-PCR [Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K., Pease, L.R., Gene 77:61-68 (1989)].
  • PCR polymerase chain reaction
  • G418 G418
  • stable transfected cell clones are isolated and tested with ELISA for the production of E-selectin/IgG fusion protein.
  • the cell clone with the highest production rate is used for the production of larger amounts of E-selectin/lgG.
  • the transferred CHO-K1 cells are either reproduced in mono- layer culture or in a hollow-fibre system in OptiMEM medium and 2% (v/v) foetal calf-serum and the secreted E-selectin/IgG from the supernatant is purified by affinity chromatography on protein A-sepharose.
  • L-selectin/IgG chimera The construction of the L-selectin/IgG chimera is carried out in analogous manner.
  • the starting basis is the complete cDNA of L-selectin isolated by PCR of HL-60 (ATCC CCL 240) cDNA as well as the above described genomic DNA of human IgGl.
  • the expression of the L-selectin/IgG chimera is effected in a manner similar to that described for E-selectin/IgG using the same vectors, cell lines, selection and purification methods.
  • P-selectin is partially amplified by polymerase chain reaction from the cDNA bank of human lung (KatNr. HL3004b; Clon- tech).
  • the resultant DNA fragment codes for a shortened form of P-selectin with six repeat domains (amino acids 1-568 including signal sequence).
  • the P-selectin fragment - as described for the two other selectins - is fused with genomic DNA of human IgGl.
  • the construct is inserted into pcDNA3 (invitrogen) and transfected to CHO Kl cells. The selection of cell clones producing P-selectin/IgG and chimera purification are likewise effected as described above.
  • blocking buffer (0.1% w/v BSA in PBS) are pipetted into the wells and left for 30 minutes at 37°C. Afterwards the blocking buffer is removed.
  • the HL-60 cells are prepared parallel thereto: HL-60 cells in suspension culture are centriguged for 5 minutes at 350 xg. After removing the medium, the cells are resuspended in RPMI 1640. The cell suspension contains lxlO 6 cells/ml. To the HL-60 cell suspension is added 1/5 volume of freshly prepared MTT solution (thiazolyl blue tetrazolium bromide, Fluka, Buchs, CH, Catalogue No. 88415, 5 mg/ml in PBS) and mixed by pipetting. The marking batch is then incubated for 30 minutes at 37°C.
  • MTT solution thiazolyl blue tetrazolium bromide
  • the marked cells are washed with 2x20 ml of binding buffer [0.1% of BSA and lO ⁇ g/ml of human immunoglobulin (isotype: gamma/lambda; sigma; Catalogue No. 1-2511) in HBSS)] and finally resuspended in binding buffer.
  • the concentration of marked HL60 cells is lxlO 6 cells/ml.
  • the determination of the number of bonded HL-60 cells is made by measuring the absorption of MTT at a wavelength of 600 nm.
  • the sulfate esters 10 and 19 exhibit better inhibition than the comparison substance l-carbomethoxy-oct-8-yl sialyl Lewis x:

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Abstract

Mélanges d'oligosaccharides dont les constituants individuels possèdent une structure de noyau identique substituée par au moins un sucre et dans laquelle tous les isomères de position sont représentés, tous les isomères étant, par rapport au substituant, sous la forme d'isomères α ou β; on décrit également un procédé de préparation de ce mélange d'oligosaccharides ainsi qu'un procédé d'isolation rapide d'un oligosaccharide actif sur le plan biologique.
PCT/EP1995/003254 1994-08-24 1995-08-16 Procede de preparation de melanges d'oligosaccharides Ceased WO1996006102A1 (fr)

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AU33464/95A AU3346495A (en) 1994-08-24 1995-08-16 Process for the preparation of oligosaccharide mixtures

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CH2596/94-5 1994-08-24
CH259694 1994-08-24

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WO1996006102A1 true WO1996006102A1 (fr) 1996-02-29

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PCT/EP1995/003254 Ceased WO1996006102A1 (fr) 1994-08-24 1995-08-16 Procede de preparation de melanges d'oligosaccharides

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
US5780603A (en) * 1996-11-15 1998-07-14 Synsorb Biotech, Inc. Combinatorial synthesis of carbohydrate libraries
US5965719A (en) * 1996-11-15 1999-10-12 Sunsorb Biotech, Inc. Combinatorial synthesis of carbohydrate libraries
WO2004031244A1 (fr) * 2002-10-04 2004-04-15 Glykos Finland Oy Nouvelles compositions glucidiques et processus de preparation de ces compositions
CN108084234A (zh) * 2018-01-23 2018-05-29 佛山科学技术学院 双重随机糖基化合成三糖的方法

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EP0531256A1 (fr) * 1991-09-04 1993-03-10 Ciba-Geigy Ag Méthode pour la préparation des glycosides

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EP0531256A1 (fr) * 1991-09-04 1993-03-10 Ciba-Geigy Ag Méthode pour la préparation des glycosides

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H.PAULSEN ET AL.: "Synthese von Pentasaccharid- und Oligosaccharid-Sequenzen der Kohlenhydrat-Kette von N-Glycoproteinen", CARBOHYDRATE RESEARCH, vol. 125, AMSTERDAM NL, pages 21 - 45 *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780603A (en) * 1996-11-15 1998-07-14 Synsorb Biotech, Inc. Combinatorial synthesis of carbohydrate libraries
US5965719A (en) * 1996-11-15 1999-10-12 Sunsorb Biotech, Inc. Combinatorial synthesis of carbohydrate libraries
WO2004031244A1 (fr) * 2002-10-04 2004-04-15 Glykos Finland Oy Nouvelles compositions glucidiques et processus de preparation de ces compositions
CN108084234A (zh) * 2018-01-23 2018-05-29 佛山科学技术学院 双重随机糖基化合成三糖的方法

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