A METHOD FOR SYNTHESIS OF OLIGOSACCHARIDES

Abstract: A method for synthesis of oligosaccharide compounds which either consist of or are fragments or analogs of the carbohydrate part in glycoconjugates is described. The synthesis from donor and acceptor substrates is carried out in that at least one glycosidase and at least one glycosyltransferase are used as catalysts and the oligosaccharide compound is isolated from the reaction mixture.

Description: A METHOD FOR SYNTHESIS OF OLIGOSACCHARIDES Descri tion The present invention relates to a method for enzymatic synthesis of an oligosaccharide compound, which either consists of or is a fragment or an analog of the carbohydrate part in a glycoconjugate Furthermore, the invention relates to the use of the product prepared by this method. It has been found that the oligosaccharide part of various glyco¬ conjugates (especially glycolipids and glycoproteins ) have a number of important functions in vivo (Biology of Carbohydrates, Vol. 2., Ginsburg et al., Wiley, New York (1984); The Glyco ¬ conjugates, Vol. 1-V, Academic Press, New York; S. Hakomori, Ann. Rev. Biochem. Vol.50, pp. 733-64); Feizi. Nature, pp. 314 (1985); S. Hakomori, Chemistry and Physics of Lipids, Vol.42, pp. 209-33). Among other things it was found that - the carbohydrate structures are important for the stability, activity, localisation, immunogenicity and degradation of glycoproteins ; - carbohydrates are antigenic determinants (for example blood group antigens); - carbohydrates function as receptors when bound to cell surfaces for pathogens, proteins, hormones, toxins and during cell-cell interactions ; - carbohydrates are important to oncogenesis, since specific ol igosaccharides have been found to be cancer-assocιated antigenic determinants; - frequently only a smaller sequence (di- or tπsaccharide ) of the carbohydrate part of the glycoconjugate is required for full biological activity (e.g. receptor activity). Universities and industry are at present working intensely on developing the use of biologically active ol igosaccharides within a number of different fields, such as - novel diagnostics and blood typing reagents - highly specific materials for affinity chromatography - cell specific agglutination reagents - targetting of drugs - monoclonal antibodies, specific against e.g. cancer-associated structures - therapy Besides the above-mentioned fields, a considerable future market is envisaged for fine chemicals based on biologically active carbohydrates. The organic chemical techniques used today for synthesis of these carbohydrate structures require an extensive protective group chemistry with many steps of synthesis and expensive catalysts. Low total yields are obtained in these complicated reaction schemes and the technique is not favorable, especially for larger scale work. Enzymes are nature's own catalysts with many attractive characteristics, such as high stereo-, regio- and substrate selectivity as well as high catalytic efficiency under mild conditions. Today, great hopes are therefore placed in being able to utilise enzymes for large-scale selective synthesis of ol igosaccharides with fewer reaction steps and consequently higher total yields than by organic chemical methodology. Both hydrolases (glycosidases, EC 3.2) and glycosyltransferases ( EC 2.4) can be used for synthesis, (glycosidases * - see Nisi∑awa et al. in The Carbohydrates, Chemistry and Biochemistry. 2nd Ed., Vol. IIA, pp. 242-290. Academic Press, New York 1970)). With glycosidases reversed hydrolysis (equilibrium reaction) or transglycosylation (kinetic reaction) are often used to obtain synthesis (see e.g. K.G.I. Nilsson, Carbohydrate Res. , Vol. 167, pp. 95-103 (1987)). With transferases a nucleotide sugar (UDP-Gal, CHP-Sia, UDP-GalNAc, GDP-Fuc, etc), which is relatively expensive, is used as donor. Both types of enzymes have advantages : Glycosidases are abundant and can often be used directly without purification, glycosyltransferases show high regio- and acceptor- selectivity. However, both types of enzymes have disadvantages when used for synthesis. Glycosidases have a low or often wrong regioselectivity which may result in complicated product mixtures and thus purification problems. As a result glycosidases are often not suitable for synthesis of higher ol igosaccharides. Glycosyltransferases are often present in small amounts in living cells and are thus often of low availability. Furthermore, as mentioned above, the transferases are cofactor dependent. One of the objects of the present invention is to use the properties of glycosidases and glycosyltransferases in a- favorable way for efficient synthesis of o1 igosaccharides . This is achieved according to the invention by combining glycosidase-catalysed synthesis of an oligosaccharide compound with glycosyltransferase-catalysed synthesis of the final, higher oligosaccharide. An easily available glycosidase is thus used for synthesis of the shorter oligosaccharide compound and a regiospecific enzyme (i.e. glycosyltransferase) is used when a higher regioselec ivity is required, i.e. for synthesis of the final oligosaccharide. This is illustrated in the following scheme (which is not intended to restrict the scope of the invention) : glycos idase cosyltransferase (DiRi symbolises donor saccharide (oligosaccharide, glycoside) with - or (--bound aglycon CRi), D1AR2 is an 0-, C-, N-, S- or F-glycoside of a di- or higher oligosaccharide, ND 2 is a suitable sugar nucleotide (CHP-Neu5Ac, UDP-Gal, UDP-GalNAc, GDP-Fuc, etc) and D Z 1 R2 is the final oligosaccharide product. The glycosidase reaction can also be an equilibrium reaction. More than one glycosidase and/or transferase can be used for synthesis of higher oligosaccharides. The substrates are selected with regard to the oligosaccharide which is to be synthesised, and are often commercially available or can be synthesised by organic or enzymatic synthesis and therefore do not restrict the use of the invention. The enzymes are selected with regard to the final oligo¬ saccharide uhich is to be synthesised. The enzyme can be used in situ (especially several glycosidases) or after partial or complete purification (especially glycosyltransferases) from their natural environment. The enzyme may be used in soluble form or immobilised to a solid phase by e.g. adsorption, encapsulation, chelation, precipitation or covalent binding. Simultaneous use of glycosidase and glycosyltransferase in soluble form or immobilised to a solid phase (eventually co-immobilised) may be advantageous according to the invention facilitating the conversion of the intermediate oligosaccharide product (e.g. DiARz in the scheme above) to the final product oligosaccharide (e.g. Dz iARz in the scheme above). In this way the method according to the invention gives important advantages compared to previous methods: purification of intermediary product is not necessary, secondary hydrolysis is minimised (i.e. higher yield) and trisaccharides or higher ol igosaccharides can be synthesised in a minimum of "pots" (in some cases one-pot reactions). This is facilitated by the high acceptor specificity of most glycosyltransferases : the transferase in the scheme above does not react with with the wrong isomer of DiARz or with iRi. Thus, for example, CMP-N-acetylneura inate-β-D-galactoside ( 2-3)sialyltransferase (EC 2.4.99.4) prefers Galβ l-3GalNAcR over GalfU-3GlcNAcR as acceptor and Galfϊ l-4GlcNacR and GalR are poor acceptors (Sadler et al. , J. Biol. Chem. , Vol. 254, pp. 4434-43). If glycosides (AR Z ) are used as acceptors in the glycosidase- catalysed reaction, a product glycoside is obtained which is easy to purify since no anomerisation of the product glycoside occurs. Furthermore, the same glycosidase may be used for predominant synthesis of several isomers. since the regioselectivity may be changed by the use of different aglycons and by changing the configura ion (α- or β-) of the glycosidic linkage between for example A and Ez in the scheme above (K.G.I. Nilsson, Carbohydrate Res., Vol. 167, pp. 95-103). The aglycon R z may be an organic compound of varying type (aliphatic, aromatic, heterocyc1 ic, or variations thereof) which is 0-,N-, C-, S-glycosidical ly bonded to A. z may also be glycosidical ly bound F or an -OH group. As examples of suitable organic aglycons mention may be made of CHs (CHz)n-groups (methyl, ethyl, etc. ), 2-bromoethy1 , allyl. trimethylsi lylethy1 , 2-(2-carbometoxiethy1thio )ethy1 , amino acids (seryl, threonyl, asparaginyl, etc) or derivatives thereof, peptides, phenyl, bensyl, nitrophenyl, lipids and analogs thereof. Examples of α- and β-glycosidases which may be used according to the invention are D-mannosidases, D-galactosidases, L-fucosidases, N-acetyl-D-galactosaminidases. hexosa inidases and other glycosidases in the EC group 3.2 (Enzyme Nomenclature, Academic Press, 1984). Examples of sialyl-, galactosyl-, fucosyl-, N-acetyl- glucosa inyl-, N-acetyl-galactosaminyl- and mannosyltransferaser which can be used according to the invention are found in the EC group 2.4 (Enzyme Nomenclature, Academic Press, 1984). Recombinant enzymes can be used according to the invention. The synthesis method according to invention is generally applicable to the synthesis of oligosaccharide sequences included in glycoconjugates (see examples of structures given in references on page 1 one above). Of special interest are the minutest fragments of these structures, which are sufficient to transfer biological activity and the choice of Di and A in the scheme above is determined by this. Examples of interesting structures are blood group determinants, cancer-associated oligosaccharide structures and structures with biological receptor activity (see references on p. 1). Some examples of how the invention may be used in actual practice are described in the following Examples, which however are in no way intended to restrict the scope of the invention ( abbreviations according to IUPAC-IUB's recommendations, J- Biol. Che ., Vol. 257, pp. 3347-3354 (1932)). EXAMPLE 1 Synthesis of Neu5Ac (α2-3)Gal (β l-3)GalNAc (β )-OEtBr. GalNAc(β)-OEtBr was obtained by mixing GalNAc (β )-OPhNOz-p (1.2 g) in 100 ml sodium phosphate buffer (0.05 H, pH 5.2 ) with 2-bromoethanol (10 ml) and adding N-acety1-β-D- -glucosa inidase (EC 3.2.1.30; 70 U). After 48 h at room temperature 500 mg of GalNAc (β )-OEtBr was isolated by column chromatography (Kieselgel 60, Merck; methylene chloride-methanol-water ) . GalNAc (β )-0EtBr (400 mg) and Gal (β )-0PhN0 2 -o (1 g) were suspended in 13 ml 0.03 M sodium phosphate buffer, pH 6.5, and di ethyIformamide (4 ml) and 7.2 ml β-D-galactosidase from bovine testes (2 U; Sigma) were added. After 4 days at 37 °C the product was isolated by column chromatogrphy as above. The fractions containing product was acetylated and further purified by column chromatography. Deacetylation gave 40 mg pure Gal (β 1-3)GalNAc (β )-OEtBr which was characterised with NMR ( 1 C, 1 H). CMP-Neu5Ac (4 mg, enzymatical ly prepared) and the above disaccharide (4 mg) were dissolved in 2 ml 0.1 M MES-HC1. pH6.7, CMP-N-acetylneura inyl-β-D-galactoside (α2-3)sialyl ransferase (EC 2.4.99.4, porcine submaxillary gland, 20 mU, 0.17 ml, Genzyme) was added together with 10 μ.1 Triton X-100 and 2 mg bovine serum albumin. More CMP-Neu5Ac (4 mg) was added after 20 h. After a total reaction time of 72 h at 37 °C 5 mg of Neu5Ac( 2-3)Gal ( l-3)GalNAc ( )-0EtBr was isolated by column chromatography (Kieselgel 60, acetonitri le-2-propano1-2.5 M NH4OH and Sephadex G-15). The product was pure according to NMR ( 1 H, 13 C) and the structure was confirmed by ethylation analysis. EXAMPLE 2 Synthesis of Neu5Ac(α2-3)Gal (β l-3)GlcNAc(β)-0Me. This substance was prepared analogously. β-D-galactoside (o_2-3) sialyltransferase (0.34 ml, 40 mU) was added to 0.7 ml 0.1 M MES-HC1, pH 6.7, which contained 30 mg Gal (β1-3)GlcNAc(β )-0Me (synthesised as described above for Gal (β l-3)GalNAc(β )-OEtBr but with GlcNAc(β)-0Me as acceptor), 10 mg CMP-Neu5Ac, 5 ^1 Triton X-100 and 1 mg albumin. More CMP-Neu5Ac (10 mg) was added after 30 h. After five days at 37 °C column chromatography as described above gave 10 mg NeuδAc(α2-3)Gal ( l-3)GlcNAc( )-0Me which was pure according to NMR. The structure was confirmed with NMR and methylation analysis. The acceptor selec ivitity of the glycosyltransferases in several of the examples is such that co-immobilised glycosidase and glycosyltransferase can be used and in some cases one-pot reactions are possible (enzymes, glycosidase substrate and nucleotide sugar which are used are mixed directly or nucleotide sugar and glycosyltransferase are added after the glycosidase). In some cases the glycosidase product is only partially purified (i.e. Sephadex G10 column) before further reaction with the glycosyltransferase. EXAMPLE 3 Synthesis of Neu5Acc2-3Galβ l-4GlcNAcβ-0Me. β -D-Galactosiάase from bovine testes was used as in Example 2 for synthesis of Galβl-4GlcNAc-0Me (the enzyme gives this isomer in addition to the βl-3 isomer; β-galactosidas from another source (lactobaci1 lus or sporobolomyces 5 is used for more specific synthesis of Galβl-4GlcNAc-0Me) . 2-3Sialyltransferase (EC 2.4.99.5) is used for the sialylation. EXAMPLE 4 Synthesis of Neu5Ac 2-6Galβ l-4GlcNAcβ-0Me . Synthesis as in example 3 but with 2-6s ialyl ransferase (EC 2.4.99.1). EXAMPLE 5 Synthesis of Neu5Ac 2-3Galβ l-3(Neu5Acα2-6)GalNAcα-0EtBr. Synthesis as in example 1 using GalNAcα-OEtBr as acceptor and in addition 2-6sialyltransferase (EC 2.4.99.7) as catalyst. EXAMPLE 6 Synthesis of Fuc l-2Galβ l-3GlcNAcβ-0Me. Synthesis as in example 2 but with l-2fucosyltransferase (EC 2.4.1.69) and GDP-Fuc as donor instead of α2-3sialyl- transferase and CMP-Neu5Ac, respectively. EXAMPLE 7 Synthesis of Fuc l-2Gal l-4GlcNAcβ-0Me. Synthesis as in example 6 but with l-4 isomer as acceptor EXAMPLE 8 Synthesis of Galβ l-4(Fucαl-3)GlcNacβ-0Me. Synthesis as in example 7 but with 2-3fucosyltransferase (EC 2.4.1.152 or EC 2.4.1.65 with which also Galβ l-3(Fuc l-4)GlcNAc can be synthesised as in example 6) as catalyst. EXAMPLE 9 Synthesis of Galαl-3Gal l-4GlcNAcβ-0Me. Synthesis as in example 3 but instead of EC 2.4.99.6 and CMP-Neu5Ac, l-3galactosyltransferase (EC 2.4.1.151) and UDP-Gal are used. EXAMPLE 10 and 11 GalNAcαl-3Galβl-3GlcNAcβ-0Me (10) and GalNAcocl-3Galβ l-4GlcNAcβ-0Me ( 2 2 Fucαl Fuc l Synthesis as in example 6 (substance 10; blood group A, type 1) and as in example 7 (substance 11; blood group A, type 2), respectively, and in addition to EC 2.4.1.69, αl-3N-acetyl- galactosaminyltransferase (EC 2.4.1.40, from e.g. human milk) and UDP-GalNAc are used. EXAMPLE 12 and 13 Galαl-3Galβl-3GlcNAcβ-0Me (12) och Galotl-3Galβl-4GlcNAcβ-0Me (13) 2 2 Fucαl Fucαl Substance 12 and 13 (blood group B. type 1 and 2, respectively) are synthesised as 10 and 11, respectively, but with EC 2.4.1.37 and UDP-Gal instead of EC 2.4.1.40 and UDP-GalNAc. In the above examples various methyl and bromoethyl glycosides were prepared. The expert can easily synthesise other interesting glycosides exemplified in the description and choose optimal conditions for the reactions. The sugar nucleotides are obtained with e.g. enzymatic synthesis (nucleotidyltransferase + nucleotide + onosaccharid (or monosaccharide-1-phosphate) for example CTP + Neu5Ac + CMP-Neu5Ac-syntas (EC 2.7.7.43).

Claims: AMENDED CLAIMS [received by the International Bureau on 16 August 1989 (16.08.89) original claims 1 - 9 replaced by amended claims 1 - 11 ( 2 pages)] 1. A method of production of an oligosaccharide compound which either consists of or is a fragment or an analog of the carbohydrate part in a glycoconjugate from at least one ol gosaccharide or monosaccharide glycoside. at least one nucleotide sugar (donor substances) and at least one acceptor substance, which is a monosaccharide or oligosaccharide. or glycoside or a saccharide analog, characterised in that at least one glycosidase (E.C. 3.2) and at least one glycosyl¬ transferase (E.C. 2.4) are used and that the oligosaccharide compound is separated from the reaction mixture. 2. A method as claimed in claim 1, characterised m that. the carbohydrate portion of the donor and acceptor substance includes one or more of the monosaccharides D-salactose. D-mannose, N-acetylneuraminic acid. N-acetyl-D-galactosamine. N-acetyl-D-glucosamine and L-fucose. or an analog of these. 3. A method as claimed in claim 1 and 2, characterised in that the acceptor substance is a glycoside in which the aslvcon is glycosidically bound flourine or an 0-. N-, C- or S- glycosidically bound aliphatic or aromatic compound. 4. A method as claimed in claim 3, characte ised in that the aliphatic or aromatic compound is suitable for temporary protection of anomeric carbon, for polymerisation, for construction of affinity labels or of spacer glycosides. for coupling to amino acid, peptide, 1ipid(analo ) . or affmitv support, or is an amino acid, peptide. 1 ipidianalog , or is a compound which facilitates use of the oligosaccharide compound as an inhibitor. 5. A method as claimed in claims 3 or 4. characte sed m that the aliphatic or aroiatic compound is an allvi—. methvl-. ethyl-, bro oethyl-. epoxi-. tri ethyIsi lviethy1—. phenyl-, bensvl-. or a n.itrophenvl _>rouP. 6. A method as claimed m one or more of claims 1-5, characterised in that the nucleotide sugar is one or more of CMP-Neu5Ac, GDP-Fuc, UDP-Gal. UDP-GlcNAc, or UDP-GalNAc. 7. A method as claimed in one or more of claims 1-6. characterised in that at least one glycosidase and at least one glycosyltransferase in soluble or immobilised form are used simultaneous1 for the synthesis of the oligosaccharide compound. 8. A method as claimed in one or more of claims 1-7, characterised in that Neu5Acα2-3Galβ l-3GalNAc-R and Neu5Acα2-3Galβ l-3GlcNAc-R are synthesised employing β-D-galactosidase and β-D-galactoside (α2-3)sιalv1- transferase and R is a glycosidical ly bound aglvcon. 9. A method as claimed in one or more of claims 1-8. characterised in that one of Neu5Acα2-6Galβ l-4GlcNAc-E, Neu5Acα2-3Ga1β 1-3(Neu5Acα2-6)Ga1NAc-R, Fucαl-2Gal l-3GlcNAc-R, Fucαl-2Galβ l-4GlcNAc-R, Galβ 1-4(Fucαl-3)GlcNac-R, Galαl-3Gal l-4GlcNAc-R, GalNAcαl-3Galβ l-3GlcNAc-R. GalNAcαl-3Gal l-4GlcNAc-R. 2 2 7 f Fucαl Fucαl Galαl-3Galβl-3GlcNAc-R or Galαl-3Galβ 1-4GIcNAc-R 2 2 1> f Fucαl Fucαl is synthesised and that R is a glycosidical lv bound aglvcon. 10. A method as claimed in one or more of the preceding claims character sed in that an oligosaccharide compound havme bio- spec fic affinity to another substance is synthesised and isolated. 11. Use of a product obtained m accordance with claim 1. directly or after chemical modification, for affmif" chromatography, affinity distribution m two-phase svεtems. diagnostics, therapy, immunisation, immunological characte isation changing of the properties of enzymes/proteins, measurement of enzyme activitv. or modification of recombinant protenii.

Inventors: NILSSON KURT G I, NILSSON, KURT, G., I.
Applicants: NILSSON KURT G I, NILSSON, KURT, G., I.
Application year: 1989
Main classification: C12P19
IPC codes: C12P19