CA2086972A1 - Process for .beta.-galactosidase-catalyzed transglycosidation with unphysiological glycosyl donors - Google Patents

Process for .beta.-galactosidase-catalyzed transglycosidation with unphysiological glycosyl donors

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CA2086972A1
CA2086972A1 CA002086972A CA2086972A CA2086972A1 CA 2086972 A1 CA2086972 A1 CA 2086972A1 CA 002086972 A CA002086972 A CA 002086972A CA 2086972 A CA2086972 A CA 2086972A CA 2086972 A1 CA2086972 A1 CA 2086972A1
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carbon atoms
beta
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ser
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Wolfgang Holla
Gunther Becker
Wilhelm Stahl
Manfred Schudok
Joachim Thiem
Bernd Sauerbrei
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Hoechst AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
    • C07K9/001—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence having less than 12 amino acids and not being part of a ring structure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
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Abstract

Abstract of the disclosure The invention relates to a process for .beta.-galactosidase-catalyzed transglycosidation, wherein unphysiological or unnatural glycosides are employed as glycosyl donors for .beta.-galactosidase.

Description

HOEC~ST AKTIENGESELLSCHAFT HOE 92/F 001 Dr.LP/PP

Description A process for ~-galactosidase-catalyzed transglycosida-tion with unphysiological glycosyl donors The invention relates to a process for ~-galactosidase-catalyzed transglycosidation, wherein unphysiological or unnatural glycosides are employed as glycosyl donors for ~-galactosidase.

Oligosaccharides and glycoconjugates (glycoproteins, glycosphingolipids, glycophospholipids) play a central role in biological recognition processes such as tumorigenesis, bacterial and viral infection, cell-cell recognition, cell growth and cell differentiation. They form the basis for blood group classification and are responsible for the internalization of various macro-molecular substances and pharmaceuticals. An important area of use of glycoproteins is, for example, the selective direction of drugs to the target organ and the protection of pharmaceuticals from proteolytic degradation.

Central problems in the chemical synthesis of oligo-saccharides and glycoconjugates are the stereoselective and regioselective chain formation from monosaccharide units, the stereoselective formation of the glycosidic linkage to the aglycone and not least the synthesis of the aglycone, e.g. of a peptide or ceramide.

The solution of these problems, even in ~he case of relatively small oligosaccharides and glycopeptides, requires sophisticated synthesis designs with customized combinations of protective groups. Classical chemical syntheses are therefore often tedious multistage reactions which frequently yield only small quantities of the desired substance free of all protective groups.

Recently we were able to show that it is possible to synthesize small glycoconjugates and glycoconjugate huilding bloc~s (synthesis precursors) by enzymatic attachment of monosaccharides to derivatives of D- and L-hydroxyamino acids (European Patent Application EP-A-0 455 101).

~hus, N-protected serine esters or N-terminally or C-terminally protected serine peptides were, for example, converted into galactosides using E. coli ~-galacto-sidase. Various ~-D-galactopyranosides may be employed as galactosyl donors, such as, for example, lactose and nitrophenyl galactosides.

Against the background of the biological functions mentioned at the beginning, not only the synthesis of naturally arising oligosaccharides and glycoconjugates, but also, to an increasing extent, the synthesis of analogs, excites considerable interest, since modified sugars in particular, such as, for example, deoxysugars and fluorosugars or the corresponding disaccharides and oligosaccharides and glycoconjugates represent important tools for exploring the abovementioned phenomena and processes. In this connection, general laws of protein-carbohydrate interaction are as much a focus of interest as are specific enzyme-substrate interactions. Thus, information on the active center of enzymes can, for example, be obtained by successive modification of enzyme substrates.

It has now been found, surprisingly, that ~-D-fuco-pyranosides and ~-L-arabinopyranosides as well as 6-O-acetyl-~-D-galactopyranosides and 6-O-formyl-~-D-galactopyranosides can also function as glycosyl donors in preparative transglycosidations catalyzed by ~-galactosidase. In addition, it was possible to transfer ~-D-glucosyl residues and ~-D-mannosyl residues using ~-galactosidases.

As a number of studies on glycosidase-catalysed reverse hydrolyses and transglycosidations demonstrate, various hydroxy compounds (simple, primary and secondary alcohols, monosaccharides, steroids) are accepted by the S enzymes as glycosyl acceptors, that is as nucleophiles.

By contrast, the successful transfer of different glyco-syl residues to alcohols, monosaccharides, hydroxyamino acids, etc. using one and the same glycosidase has not hitherto been described.

Although it is known from hydrolysis experiments that ~-galactosidase from E. coli, for example, will hydrolyse nitrophenyl fucosides and arabinosides - if sometimes only very 510wly - [R.E. Huber, M.T. Gaunt, Archives of Biochemistry and Biophysics 220 (1), 263-271 (1983);
T. Tochikura et al., Agric. Biol. Chem. 50 (9), 2279-85 (1986)], and enzymatic conversion of glycals has been described [J. Lehmann, E. Schroter, Carbohydr. Res. 23, 359-368 (1972) and Carbohydr. Res. 58, 65 (1977)], it could not be predicted from these predominantly bio-chemical findings that transglycosidation, which only takes place in vitro and is in any case very disfavored, with unnatural glycosyl donors produces yields of up to about 40% in preparative batches. Unphysiological and unnatural glycosyl donors are understood to mean glycosides which differ from the natural substrates with regard to the glycosyl residue to be transferred.
Examples of unphysiological glycosyl donors are stereo-isomers of the natural glycosyl donor, i.e. in the case of ~-galactosides derivatives of glucose and mannose or modified/derivatized glycosyl residues, such as, for example, deoxygalactose residues or galactosyl residues supplied with protective groups.

Without doubt, it is a particularly surprising fact that 6-O-acetyl- and 6-O-formyl-~-nitrophenyl-D-galactopyranoside, two monosaccharide derivatives of ; 1 l ` l i ) _ 4 --galactose which do not occur in nature, are accepted as glycosyl donors by the ~-D-galactosidases employed, 50 that partially protected sugars of particular synthetic interest can be transferred to a hydroxy compound, e.g.
another monosaccharide. This could not be predicted, because the 6-O-acetyl- and the 6-O-formyl-galactosyl residue is on the one hand more demanding sterically, and on the other less polar, than the natural glycosyl donor.
Biochemical investigations of 6-O-acylated mono-saccharides as glycosyl donors in glycosidase-catalyzed hydrolyses ha~e likewise not been described.

The mononitrophenyl and dinitrophenyl ~-L-arabino-pyranosides and mononitrophenyl and dinitrophenyl ~-D-fuco-, gluco- and mannopyranosides which have been lS used are obtainable con~ercially or can be synthesized according to known processes.

The 6-O-acetyl- and 6-O-formyl-mono and -dinitrophenyl ~-D-galactopyranosides were prepared by regioselective, lipase-catalysed acylation of the unprotected ~-nitrophenyl galactosides using lipase OF from Candida cylindracea or vinyl acetate and vinyl formate ~A.M. Rlibanov, J. Am. Chem. Soc. 110 (1988), 584-589;
E.W. Holla, Angew. Chem. 101 (1989), 222-223; H. Schick et al., Synthesis 1991, 533].

The glycosyl acceptors which are employed are short-chain, acyclic or cyclic, saturated or unsaturated mono-, di-, tri- to polyhydroxy com-pounds (mono- to polyhydric alcohols), with Cl to C10, which are optionally substituted by halogen (F, Cl, Br, I) or sulfonyl, cyano, alkyl, alkyloxy, aryl, aryloxy, heteroaryl, heteroalkyloxy, thioalkyl, trialkylsilyl, trialkylsilyloxy, azido, amino, N-acyl, N-tert.- butyloxycarbonyl~N-Boc), N-benzyloxycarbonyl(N-Z) or N-allyloxycarbonyl (N-Aloc) groups or ~ ~S~J ~ 2 simple monosaccharides (e.g. Glc, Gal), 2-deoxy-2-aminosugars. 2-deoxy-2-N-acylsugars (such as, for example, GlcNAc), glycosyl fluorides (such as, for example, ~-F-Glc and ~-F-GlcNAc) as well as alkyl glycosides and aryl glycosides, with alkyl or aryl residues with one to ten carbon atoms, which may be optionally substituted by halogen, trialkylsilyl, cyano, nitro and azido groups (such as, for example, ~-Gal-OMe, ~-Gal-OCH2Ph, ~-Gal-OPh) 10 or compounds of the formula I

NHRl HO~ 1 R2 ( CH2 ) n¦l/
o with n = 1-10 in which R~ is an amino protective group and R2 is a hydroxyl group, an alkoxy or thioalkyl group or an alkenyloxy group, in each case with 1 to 18 carbon atoms, which may be substituted by halogen or cyano, an aryloxy group with 6 to 10 carbon atoms, which may be substituted by alkyl, alkoxy, thioalkyl, in each case with 1 to 5 carbon atoms, and nitro groups, or is the -NHR3 group, in which R3 is an alkyl group with 1 to 5 carbon atoms or a residue of the formula II

~ II

or a dipeptide or tripeptide residue of the formula III
or IV

yCO~NH~COR4 ~CO-NH~CO-NH~COR~ IV, where R4 is a hydroxyl group, an alkoxy, a thioalkyl or a alkenyloxy group, with in each case 1 to 5 carbon atoms, which may be substituted by halogen or cyano, an aryloxy group with 6 to 10 carbon atoms, which may be substituted by alkyl, alkoxy, thioalkyl, with in each case 1 to S
carbon atoms, and nitro groups and R5, R6 and R7 are the same or different and are hydrogen or straight-chain, branched or cyclic alkyl or alkenyl groups with 1 to 10 carbon atoms, which may be substitu-ted by halogen, hydroxyl, alkoxy, thiol, thioalkyl, aryl or heteroaryl.

lS R2 and R4 in formulae II-IV are preferably alkoxy groups with 1-10 carbon atoms or methoxymethyloxy, methylthio-methyloxy, chloroethoxy, bromoethoxy or cyanoethoxy, benzyloxy, p-nitrobenzyloxy, p-methoxybenzyloxy, piperonyloxy, allyloxy or vinyloxy as well as tertiary butyloxy or tertiary butyldimethylsilyloxy groups.

Amino protective groups which are essentially those currently used in peptide and glycopeptide chemistry, such as, for example, acyl groups, and also acyl residues of long-chain fatty acids and alkyl- and aryloxycarbonyl groups, may be employed as the protective group in the R1 position of formula I. Applicable protec~ive groups are described, for example, in the article by H. Hubbuch in Xontakte 3/79, p. 14, in T.W. Greene, Protective Groups .

~)Vv ~1 in Organic Synthesis, John Wiley & Sons 1981, p. 223 ff.
or in Houben-Weyl Vol. 15/1 p. 46. Preferably employed are benzyloxycarbonyl (Z), allyloxycarbonyl (Aloc) and tertiary butyloxycarbonyl (Boc~ as well as formyl, acetyl, chloroacetyl, trifluoroacetyl, phenacetyl, benzoyl or acyl residues of long-chain fatty acids with 6 to 24 carbon atoms.

Particularly preferred as glycosyl acceptors are compounds of the formula I, such as Aloc-Ser-OAll, Boc-Ser-OAll, Z-Ser-OAll, Aloc-Ser-OCH2CH2Br, Aloc Ser-Ala-OMe.

The glycosyl acceptors and glycosyl donors which are used can be employed in the ratio 10:1 to 1:4, preferably 2:3 to 4:1. 10-150 units of the enzyme are expediently used per 0.02 mmol of glycosyl donor.

The reaction can take place within a pH range of 4.5 to 8.0, but advantageously between pH 5.0 and 7.5. The temperature should be kept between about 15C and 50C, preferably between 20C and 35C. Below 15C the activi~y of the enzyme is too low for economic operation, while above 50C the enzyme is inactivated irreversibly to an increasing extent as the temperature rises. The incubation period may be 15 minutes to 200 hours.

The ~-galactosidases which are employed are enzymes from microorganisms, plants or ma~mals, preferably from E. coli, Aspergillus oryzae, Saccharomyces fragilis, jack beans, bovine liver and bovine testes.

The commercially available glycosidases used according to the invention, e.g. E. coli ~-galactosidases, 30 EC 3.2.1.23, supplied by Sigma Chemie GmbH ~grade VI, hOT
2lH6806, activity 475 u/mg; LOT 7lH6831, activity 500 u/mg) and Asp. oryzae ~-galactosidase supplied by Sigma Chemie GmbH (grade XI, LOT 40H0798, activity h o 3~ 7 2 5.6 u/mg) may be employed in an aqueous solution as water-soluble enzymes or in water-insoluble form bound to a carrier by conventional methods (cf. German Patent Application No. 27 32 301). If the enzyme is used in immobilized form, this can be both in the batch process and in the continuous process.

Solvents which have no, or only slight, negative effect on the activity of the enzyme may be used to improve the dissolution of the substrates. These are, for example, acetone, dimethoxyethane and diglyme, but in particular toluene, xylene and acetonitrile. Furthermore, salts which are physiologically compatible with the enzymes employed may be added in order to increase the reaction velocity. Such salts are, for example, MnSO4, CaCl~, ~Cl, NaBr, LiCl, LiBr and KMnO4, but NiSO4 and MgCl2 are preferred.

The course of the reaction can be followed using HPLC, or by monitoring with TLC. Subsequent workup takes place, for example, by extraction with ethyl acetate, freeze-drying of the aqueous phase and purification bypreparative thin-layer chromatography or flash column chromatography on silica gel or polydextrans such as, for example, Sephadex. It is also possible to freeze-dry the reaction solution first and subsequently extract the solid residue with methanol, it being possible to employ ~he abovementioned chromatographic methods for further purification after the methanolic solution has been filtered and concentrated.

Example 1 74.2 mg (0.26 mmol) of ortho-nitro-phenyl ~-D-fuco-pyranoside (~-Fucp-oNP) and 191.2 mg (0.78 mmol) of Boc-Ser-OAll are stirxed with 400 units of E. coli ~-galactosidase (EC 3.2.1.23) in 8 ml of Tris buffer (0.1 M, pH = 7.0) at room temperature for 40 h. Freeze-drying and flash chromatography on silica gel with ,~ fi ..~ .J ~J I ~
g CHCl3/MeOH/hexane 3:1:1 yield 42 mg (41~) of the desired allyl ester of N-tert.butyloxycarbonyl 3-O-~-D-fuco-pyranosyl-L-serine. The compound is homogeneous according to TLC, lH-NMR and l3C-NMR.
FAB-MS: MH~ = 392 13C-NMR (d6-DMSO, 75 MHz):
Chemical shift (ppm) ___ 170.0 'C1-Ser 156.0 CO-Boc 132.8 CH-allyl 117.5 CH~-allyl, terminal 103.9_ C1-fuco 78.8 ~ 3l3-Boc 73.2 C3-fuco 71.0 C4-fuco _ 70.3 C2-fuco 70.2 C5-fuco 68.8 C3-Ser 64.8 CH7-allyl 54.3 C2-Ser 28.4 _ (CH3l~-Boc 16.6 C6-fuco Example 2 25 In an analogous manner to Example 1, 74.2 mg (0.26 mmol) of ~-Fucp-oNP, 213.8 mg (0.78 mmol) of Aloc-Ser-Ala-OMe and 800 units of E. coli ~-galactosidase (EC 3.2.1.23) in 8 ml of imidazole buffer (0.1 M, pH = 7.5) are stirred at room temperature for 76 h. Following workup, as 30 described in Example 1, 14 mg (13%) of the desired glycopeptide with the ~-configuration, which is pure according to TLC, lH-NMR and l3C-NMR, is obtained.

f~ ? ~
~ 3~j S~3 ~ 3 ~

lH-NMR (d6-DMSO, 300 MHz) Shifts and coupling constants tppm) (Hz) 8.15 d, NH Ala (J: 6.91 7.27 d, NH Ser (J: 8.16 5.91 m, CH Aloc 5 30 dm, CH Aloc (J: 16.80 5.18 dm, CH Aloc ~J: 10.34 4.80 OH
4.60 OH
4.49 2 x dt, 2H CH~ Aloc _ 4.31 5L, C~-H Ala 4.30 OH
4.23 dt,_C~-H Ser 4.11 C1-H fuco 3.91_ dd. CB-H Ser (J: 10.25. 5.45 3 ! 62 s, 3H OCH3 3.59 dd, CB-H Ser (J: 10.25. 5.01) 3.53 dq, C5~H fuco (J: 6-36, 0-99!
3.41 s, broad, C4-H fuco 3.29 C3~H fuco 3.29 C2-H fuco 1.29 d, 3H, CH3 Ala (J: 7.25) 1.14 d, 3H, C6 fuco (J: 6.35 !-Example 3 In an analogous manner to Example 2, 74.2 mg (0.26 mmol) of ~-Fucp~oNP, 231 mg (0.78 mmol) of Aloc~Ser-OCH2CH2Br and 1000 units of E. coli ~~galactosidase (EC 3.2.1.23) in 100 ml of Tris buffer (0.1 M, pH = 7.0) are stirred at room temperature for about 72 h and subsequently worked up as described in Example 1. 31.4 mg (27%) are obtained of the expected bromoethyl ester of N~allyl~
oxycarbonyl~3-O-~~D~fucopyranosyl~L-serine, which is homogeneous according to lH~NMR, 13C~NNR and TLC.

ti ~ ~

HRMS: theoretical for C15H24NOgBr 441.0634 found: 441.0659 Example 4 74.2 mg (0.26 mmol) of ~-Fucp-oNP and 217.7 mg (0.78 mmol) of Aloc-Ser-OCH2Ph are stirred in 8.0 ml of Tris buffer (0.1 M, pH = 7.0) with 1000 units of ~-galactosidase (EC 3.2.1.23) at room temperature for 127 h. After the purification described in the preceding examples, 12 mg (11%) of the pure serylglycoside with the B-configuration are obtained.

1H-NMR (d6-DMSO, 300 MHz):
Shifts and coupling constants !
(ppm) (Hz) 7.63 d, NH (J: 8.85 7.37 5H phenyl 5.91 m, CH Aloc 5.30 ~dm, CH Aloc (J: 16.98) 5.20 d, lH CH~ Bzl (J: 12.97) 5.18 dm, CH Aloc (J: 10.81) 5.15 d, lH CH2 szl (J: 12.97) 4.93 OH
4.60 O~
4.51 m, 2H, CH7 Aloc 4.43 dt, C~-H Ser (J: 8.84, 3.70) 4.37 OH ~J: 4.69) 4.22 dd, CB-H Ser (J: 9.67, 3.95) 4.08 d, Cl fuco (J: 7.40) 3.64 dd, CB-H Ser tJ: 9.64, 3.821 3.53 qd, C5-H fuco (J: 6.43, 0-77 3.41 s, broad, C4-H fuco 3.29 s, C3~H fuco 3 26 C2-H fuco ~overla~ with H70) ~ .

1.14 d, 3H, C6-H fuco (J: 6.36 ~ ~t ~ i' iV

Example 5 In an analogous manner to Example 4, 74.2 mg t0.26 mmol) of ~-Fucp-oNP, 178.8 mg (0.78 mmol~ of Aloc-Ser-OAll and 1600 units of E. coli ~-galactosidase (EC 3.2.1.23) in 8.0 ml of Tris buffer (0.1 M, pH = 7.0) are stirred at room temperature for 51 h. After workup as described in Example 1, 21 mg (22~) are obtained of the desired allyl ester of N-allyloxycarbonyl-3-O-~-D-fucopyranosyl-L-serine, which is pure according to TLC, lH-NMR and l3C-NMR.
HRMS:
theoretical for C16H25NOg: 375.1529 found: 375.1532 lH-NMR (d6-DMSO, 300 MHz):
Shifts and coupling constants (ppm) (Hz) 7.58 d, lH urethane (J: 8.85) 5~89 ml lH vinyl qrouP allYl ester 5.88 m, lH vinyl ~roup Aloc 5 33 dm, lH vinvl group all~l ester (J: 17.38) _ 5.31 dm, lH vinyl qroup Aloc (J: 17.05) 5.19 dm, lH vinyl qroup allyl ester (J: 10.58?_ 5.18 dm, lH vinyl qroup Aloc 4.91 d, OH C2 fuco (J: 3.04?
4.62 m, lH CH7 qroup allyl ester 4.61 m, lH CH2 qrouP allyl ester 4.57 d, OH C3 fuco (J: 5.44) 4.51 m, 2H CH2 qroup Aloc _ _ 4.38 dt, lH Ser-C~-H N (J: 8.85, 4.03 (t)~

4.33 d, OH C4 fuco ~J; 4.66) 4.15 Ser-C~-H NK
4.15 dd, Ser-C~-H (J: 10.1, 4.04 4.06 C1-~ fuco (J: 7.30!

2~'.`~.,ii i~

Shifts and coupling constants _ (ppm) ~Hz) 3.68 dd, Ser-C~-H
3.64 dd, Ser-C~-H (J: 10.05, 4.02) 3.52 q, C5-H fuco ~J: 6.2g, (q) !
_ 3.39 m, C4-H fuco 3.27 m, C3-H fuco 3.24 _ m, C2-H fuco 1.12 d, 3H, C6-H fuco (J: 6.29 Example 6 742 mg (2.6 mmol) of ~-Fucp-oNP, 1.91 g (7.8 mmol) of Boc-Ser-OAll and 4000 units of E. coli ~-galactosidase (EC 3.2.1.23) are stirred in 80 ml of Tris buffer (0.1 M~
pH = 7.0) at room temperature for 40 h and worked up as described in Example 1. 386 mg (38~) are obtained of the expected glycoconjugate, which is homogeneous according to lH-NMR, l3C-NMR and TLC.

13C-NMR td6-DMSO, 75 MHz):
Chemical shift (ppm) 170.0 Cl-Ser 156.0 CO-Boc 25132.8 CH-allyl 117.5 CH2-allyl, terminal 103.9 Cl-fuco 78.8 C~CH3l3-Boc 73.2 C3-fuco 3071.0 C4-fuco 70.3 ~C2-fuco 70.2 C5-fuco 68.8 C3-Ser 64.8 CH7-allyl 3554.3 C2-Ser ' c~ ,i 2 Chemical shift (ppm) -28.4 ¦ (CH3)3-Boc 16.6 I C6-fuco Example 7 70.5 mg (0.26 mmol) of ~-para-nitro-phenyl ~-arabino-pyranoside (~-L-Arap-pNP) and 191 mg (0.78 mmol) of Boc-Ser-OAll are stirred with 400 units o~ E. coli ~-galactosidase (EC 3.2.1.23) in 8 ml of Tris buffer (0.1 M, pH = 7.0) at room temperature for 11.5 h. Freeze-drying and flash chromatography on silica gel and CHCl3tMeOH/hexane 3:1:1 yield 36.4 mg (37%) of the desired allyl ester of N-butyloxycarbonyl-3-O-~-L-arabino-pyranosyl-L-serine. The compound is homogeneous according to TLC, lH-NMR and l3C-NNR

H-NMR (d6-DMSO, 300 MHz):
Shifts and coupling constants (ppm) (Hz) _ 7.17 d! NH (J: 8.68) 5.89 ddt, CH allyl (J: 17.20, 10.55, 5.20 (t) !
5.33 ddt, CH allyl (J: 17.20, 1.65~ 1.72 lt) 5.20 dm, CH allyl (J: 10.71, m 255.00 s OH
4.60 m, 2H CH7 allyl 4.55 d, OH
4.45 d, OH _ _ 4.30 m, C~-H Ser 304.10 dd, C~-H Ser (J: 9.95, 4.12 4.06 d, C1-H Ara lJ: 6.52!
3.67 dd, C5-H Ara (J:_11.95, 3.18) 3.60 m, 2H, C4-H Ara + C~-H Ser 3.34 dd, C5-H Ara (J: 11.96, 1.27?
353.33 m, 2H. C2-H Ara + C3-H Ara 1.39 s, 9H Boc-H

L ~

l3C-NMR ( d6-DNSO, 7 5 MHz ):
Chemical shift (ppm) 1 = 170.0 Cl-Ser _ _ 2 = 155.8 CO-Boc 3 = 132.5 CH-allyl 4 - 117.7 CH7-allyl, terminal 5 = 103.8 Cl-Ara 6 = 78.8 C(CH3~3-Boc 10 7 = 72.5 C3~Ara (aj _ 8 - 70.7 C4-Ara ~_) 9 = 68.8 C3-Ser 10 = 67.8 C4-Ara 11 = 65.7 C5-Ara 1512 = 65.2 CH7-allvl 13 = 54.1 C2-Ser 14 = 28.4 CH3, C ( CH3 ! 3 - Boc (a) = not possible to assign unambiguously Example 8 74.2 mg (0.26 mmol) of ~-Fucp-oNP and 217.8 mg (0.78 mmol) of Z-Ser-OAll are stirred in 8.0 ml of Tris buffer (0.1 M, pH = 7.9) with 800 units of E. coli ~-galactosidase (EC 3.2.1.23) at room temperature for 156 h. After the usual purification, 9 mg (8%) of the desired serylglycoside are obtained.

H-NMR ( d6-DMSO , 300 MElz ):
Shifts and coupling constants .
(ppm) (Hz ) 7.63 d, NH (J: 8.95) _ 7.36 _ m, 5H ~Z) 5.88 ddm, lH allyl 5.33 dm, lH allyl, (J: 17-05!
355.19 dm, lH allvl, (J: 10.69) 2 ~ ~ J

Shifts and coupling constants .
(ppm) (Hz) 5.08 A part 2xd CH7 (Z) (J: 12.2) 5.06 B part 2xd CH~ (Z) tJ: 12.2) 4~90 d, OH bound to v 4.61 m, CH~ allyl 4.57 d, OH bound to u 4.40 dt, Ser-C~-H (J 8.95 ca. 4.0 (t~) 4.32 _ d, OH bound to t (J: 4.51) 4.18 m lH NK. Ser C~-H
4.15 dd Ser C~-H1 (J: 10.25, ca. 4.0) 4.06 d C1-H fuco (J: 7.11) 3.68 2H NK: Ser-C~-H
3.64 dd, Ser CB-H2 (J: 9.79, ca. 3.6) 3.51 q, C5-H fuco (J: 6.44 (q)) 3.39 m, C4-H fuco _ 3.26 m C3-H fuco 3.23 m, C2-H fuco 1.12 d 3H C6-H fuco (J: 6.45 Example 9 In an analogous manner to Example 7, 70.5 mg ~0.26 mmol) of ~-L-Arap-pNP, 213.8 mg (0.78 mmol) of Aloc-Ser-Ala-OMe and 400 units of E. coli ~-galactosidase (EC 3.2.1.23:
Sigma, grade VI) are stirred in 8 ml of imidazole buffer (0.2 M, pH = 7.5) at room temperature for 1.5 h. After workup as described in Example 7, 156 mg (14.8~) are obtained of the desired glycopeptide with the ~-configuration, which is pure according to TLC, lH-NMR
and l3C-NMR

~u~ iJJ ~2 lH-NMR (d6-DMSO, 300 MHz):
Shifts and coupling constants (ppm) ~Hz) 8.19 d, NH amide (J:_7.21) 7.31 d, NH urethane (J: 9.32) 5.91 m, lH allyl-CH
5.31 dm, lH allyl-CH
5.18 dm, lH allyl-CH
4.49 dt, 2H, allyl-CH~
4.32 d~, lH C~-H Ala (J: 7.38, 7.21) 4.25 m, lH C~H Ser 4.14 dm, lH C1-H Ara 3.90 dd, lH, CB-H Ser (J: 10.39. 5.97) 3.69 dd, lH, C5-H Ara (J: 11.43, 3.38) 3.64 m, lH, C4-H Ara 3.62 s. 3H, OCH3 3.59 dd. lH, C~-H Ser (J: 10.39, 4.93) 3.40 dd, lH, (J: 11.43, 2.66~
3.37 m, 2H C2-H Ara + C3-H Ara 1.29 d, 3H CH~ Ala 13C-NMR (d6-DMSO, 75 MHz):
Chemical shift (ppm) 1 = 172.9 Cl-Ser, (a) 2 = 169.6 Cl-Ala, (a) 3 = 135.2 CO-Aloc 4 = 133.5 CH-allyl 305 = 121.7 CH~-allyl 6 = 117.2 Cl-Ara 7 = 103.3 C3-Ara 8 = 72.4 C2-Ara 9 = 70.5 C3-Ser 3510 = 68.6 C4-Ara 11 = 67.4 C5-Ara 12 = 65.2 CH~-Aloc Chemical shift (ppm) 13 = 64.7 C2-Ser 14 = 54.5 CH~-Ala 15 = 51.9 C2-Ala 16 = 47.8 C3-Ala (a) = not possible to assign unambiguously Example 10 352.5 mg (1.3 mmol) of ~-L-Arap-pNP, 1.089 g (3.9 mmol) 10 of Aloc-Ser-OCH2-Ph and 2000 units of E. coli ~-galactosidase (EC 3.2.1.23: Sigma, grade VI) are stirred in 40 ml of Tris buffer (0.1 M, pH = 7.0) at room temperature for 7 h. After workup as described in Example 1, 145 mg (27%) are obtained of the desired glycopeptide with the ~-configuration, which is pure according to T~C, H-NMR and l3C-NMR

1H-NMR (d6-DMSO, 300 MHz):
Shifts and coupling constants (ppm) (Hz) 7.64 d, lH NH
7.4-7.2 m, 5H ~henyl (Z) 5.91 m, lH CH allyl 5.30 dm, lH CH allyl 5.18_ dm, lH CH allyl 5.17 2xd, 2H: CH2 (Z!
4.99 OH
4.55 OH
4.51 m, 2H CH~ allyl 4.46 OH
4.43 m, lH, C~-H Ser 4.18 dd, C5-H Ara 4.07 m, C1-H Ara 3.70 dd, C5-H Ara 3.65 dd, C~-H Ser û ~

Shifts and coupling constants (ppm) ~Hz) 3.58 m, C4-H Ara 3.40 d, CB-H Ser 3.34 m, C2-H Ara, C3-H Ara l3C-NMR (d6-DMSO, 75 MHz):
Chemical shift (ppm) 1 = 170.2 C1-Ser 2 = 156.1 CO-Aloc 3 = 136.0 C1-aromatic 4 = 133.4 CH-allYl 155 = 128.4 C3-aromatic 6 = 127.9 C4-aromatic 7 = 127.5 C2-aromatic 8 = 117.3 CH7-allyl, terminal 9 = 103.7 C1-Ara 2010 = 72.4 C3-Ara 11 = 70 6 C2-Ara 12 = 68.6 C3-Ser 13 = 67.7 C4-Ara 14 = 66.1 CH~-allyl _ -2515 = 65.7 CS-Ara Example 11 141 mg (0.52 mmol) of ~-L-Arap-pNP and 435.6 mg (1.56 mmol) of Z-Ser-OAll are stirred at room temperature for 33 h with 800 units of E. coli ~-galactosidase (EC 3.2.1.23) in 16 ml of Tris buffer (0.1 M, pH = 7.0).
Following workup as described in Example 1, 26 mg (12%) of the desired glycopeptide are obtained.

.

~ ~ J u ~ l 2 1H-NMR (d6-DMSO, 300 MHz)~
Shifts and coupling constants (ppm) (Hz) ~
7.66 d, NH
7.4-7.2 m, SH tz) 5.89 m, lH (allyl) 5.33 dm, lH (allyl 5.20 _ dm, lH (allvl!
5.07 2xd, CH~ (Z) _ _ 4.98 OH
4.61 m, CH7 allyl 4.50 2 OH
154.41 dt, lH C~-H Ser, (J: 8.73, 4.31, 4.16 4.12 dd~ lH, C~-H Ser, (J: 9.91, 4.31) _4.07 m, lH C1-H Ara 3.67 dd, lH C5-H Ara (J: 11.70. 2.90 3.64 dd. lH C~-H Ser, (J: 9.97, 4.16 203.61 m, lH C4-H Ara 3.37 _ ¦dd. lH,_C5-H Ara (J: 11.70. 1.45) 3.33 Im, 2H, C2-~I Ara + C3-H Ara Example 12 In an analogous manner to Example 7, 70.5 mg (0.26 mmol) of para-nitro-phenyl ~-L-arabinopyranoside (~-L-Arap-pNP) and 230.9 mg (0.78 mmol) of Aloc-Ser-OCH~CH2Br are stirred at room temperature for 31 h with 600 units of E. coli ~-galactosidase (EC 3.2.1.23) in 8 ml of Tris buffer (0.05 M, pH = 7.0). Freeze-drying and flash chromato-graphy lead to 26 mg (23%) of the bromoethyl ester of N-allyloxycarbonyl-3-O-~-L-arabinosyl-L-serine.
FAB-MS: MH~ = 428 Example 13 In an analogous manner to Example 7, 70.5 mg (0.26 mmol) of para-nitro-phenyl ~-L-arabinopyranoside (~-L-Arap-pNP) and 178.8 mg (0.78 mmol) of Aloc-Ser-OAll are stirred at ?

room temperature for 26 h with 600 units of E. coli ~-galactosidase (EC 3.2.1.23) in 8 ml of Tris buffer (0.1 M, pH = 7.0). Freeze-drying and flash chromatography lead to 38.7 mg (41%) of the allyl ester of N-allyloxy-carbonyl-3-O-~-L-arabinosyl-L-serine.
FAB-MS: MH~ = 362 Example 14 70.5 mg (0.26 mmol) of para-nitro-phenyl ~-L-arabino-pyranoside (~-L-Arap-pNP) and 195.2 mg (0.78 mmol) of N-Boc-L-phenylalaninol are stirred at room temperature for 30.5 h with 600 units of E. coli ~-galactosidase (EC 3.2.1.23) in 8 ml of Tris buffer (0.1 M, pH = 7.0) and 1 ml of acetone. Freeze-drying, taking up the residue in MeOH, filtering off insoluble components, concentrating in vacuo and flash chromatography on silica gel (CHCl3/MeOH/i-hexane 9:1:3) lead to 51 mg (51%3 of the desired glycosidated amino alcohol.

HRMS: theoretical for C1gH29NO7: 383.1944 found: 383.1939 Example 15 70.5 mg (0.26 mmol) of para-nitro-phenyl ~-L-arabino-pyranoside (~-L-Arap-pNP) and 45.3 mg (0.78 mmol) of allyl alcohol are stirred at room temperature for 6.5 h with 400 units of E. coli ~-galactosidase (EC 3.2.1.23) 25 in 4 ml of K phosphate buffer (0.15 M, pH = 7.0). After freeze-drying, taking up the residue in MeOH, filtering off insoluble components, concentrating in vacuo and flash chromatography on silica gel (CHCl3/MeOH/i-hexane 9:1:3), 17 mg (35%) of the desired allyl glycoside are obtained.

h ~ 4 2 lH-NMR:
Shifts and coupling constants _ (ppm) (Hz) a 5.90 ddt, CH allyl (J: 17.26, 10.48, 5.33 (t)) b 5.30 ddt, CH allyl (J: 17.26, 2.06, 1.37 (t)?
c 5.13 ddt, CH allyl (J: 10.48, 1.72, 1.55 (t!) d 4.86 OH (J: 3.55) _ e 4.51 OH (J: 4.74~
f 4.42 OH (J: 4.34) g 4.18 dddd, CH CH2 group allyl (J: 13.40, 5.04, 2x 1.45) h 4.12 C1-H (J, 6.37) i 4.00 dddd, CH CH2 group allyl (J: 13.40, 5.57, 2x 1.46) k 3.68 C5-H (J: 11.63. 3.08) l 3.63 C4-H
m 3.37 C5-H (J: 11.63, 1.54) n 3.35 C3-H, C2-H

Example 16 89.2 mg (0.26 mmol) of ortho-nitrophenyl ~-D-6-O-acetyl-galactoside and 357.6 mg (1.56 mmol) of Aloc-Ser-OAll are stirred with 1200 units of ~sp. oryzae ~-galactosidase (EC 3.2.1.23) at about 37 to 40C for 0.5 h in 8 ml of K phosphate buffer (0.15 M, pH = 5.5). After completion and workup of the reaction as described in Example 1, 14.5 mg (13%) of the desired 6-O-acyl-protected serylgalactoside are obtained.

HRMS: theoretical for C1~H27NOl1: 433.1584 found: 433~1564 Example 17 89.2 mg (0.26 mmol) of ortho-nitrophenyl ~-D-6-O-acetyl-galactoside and 461.8 mg (1.56 mmol) of Aloc-Ser-OCHzCH2Br are stirred with 1200 units of Asp. oryæae ~ ~) tl r ,B-galactosidase (EC 3.2.1.23) at about 37 to 40C for 0.5 h in 8 ml of K phosphate buffer (0.15 M, pH = 5.5).
After completion and workup of the reaction as described in Example 1, 8 mg (3%) of the desired 6-O-acyl-protected 5 serylgalactoside are obtained.

HRMS: theoretical for Cl7Hz~NOllBr: 499.0689 found: 499.0685 Example 18 89.2 mg (0.26 mmol) of ortho-nitrophenyl ,B-D-6-O-acetyl-galactoside and 435.4 mg (1.56 mmol) of Aloc-Ser-OBn are stirred with 1200 units of Asp. oryzae ~-galactosidase (EC 3.2.1.23) at about 37 to 40C for 0.5 h in 8 ml of K phosphate buffer (0.15 M, pH = 5.5). P.fter completion and workup of the reaction as described in Example 1~
15 10 mg (8%) of the desired 6-O-acyl-protected seryl-galactoside are obtained.

HRMS: theoretiCal for C22H29NOll: 483.1741 found: 483.1759 Example 19 89.2 mg (0.26 mmol) of ortho-nitrophenyl ,B-D-6-O-acetyl-galactoside and 427.4 mg (1.56 mmol) of Aloc-Ser-AlaOMe are stirred with 1200 units of Asp. oryzae ~-galacto-sidase (EC 3.2.1.23) at about 37 to 40C for 0.5 h in 8 ml of X phosphate buffer (0.15 M, pH = 5.5). After completion and workup of the reaction as described in Example 1, 6.4 mg (5%) of the desired 6-O-acyl-protected serylgalactoside are obtained.

HRMS: theoretical for ClgH30N2Ol2 478.1799 found: 478.1805 Example 20 89.2 mg (O.26 ~unol) of ortho-nitrophenyl ~-D-6-O-acetyl-galactoside and 435.6 mg (1.56 mmol) of Z-Ser-OAll are ,~ ,, J v' stirred with 1200 units of Asp. oryzae ~-galactosi~ase (EC 3.2.1.23) at about 37 to 40C for 0.5 h in 8 ml of K phosphate buffer (0.15 M, pH = 5.5). After completion and workup of the reaction as described in Example 1, 8.2 mg (6.6%) of the desired 6-O-acyl-protected seryl-galactoside are obtained.

HRMS: theoretical for C22H29NO11: 483.1741 found: 483.1734 Example 21 301.3 mg (1 mmol) of para-nitrophenyl ~-glucopyranoside are dissolved in a mixture of 2 ml of acetonitrile and 2 ml of 0.1 M potassium phosphate buffer, pH = 5.0, 2200 units of Aspergillus oryzae ~-galactosidase (EC 3.2.1.23;
Sigma, grade XI) are added and the mixture is stirred at 20C for 3 hours in the presence of 0.26 mmol of Z-Ser-OCH2-Ph as cosubstrate. Stopping the reaction by heating at 95 to 100 DC for 5 minutes, and subsequent workup by concentrating in vacuo, and chromatography on Dowex 1 x 2 and Sephadex G-lO yields 2-O-(~-D-gluco-pyranosyl)-~-D-glucopyranoside in l9~ and para-nitrophenyl 4-O-(~-D-glucopyranosyl)-~-D-glucopyranoside in 11% yield.

Example 22 To a solution of 150 mg ( n . 5 mmol) of p-nitrophenyl D-mannoside (~/~ ratio 1:9) in 1.1 ml of acetonitrile and 0.4 ml of buffer I (O.1 M KH2PO4, pH 5.0) are added 100 mg t550 U) of ~-galactosidase (Aspergillus oryzae EC 3.2.1.23) dissolved in 0.7 ml of buffer I. The reac-tion mixture is incubated at 20DC with shaking for 7 days/ during which gradually a further 150 mg of enzyme in solid form is added. At the end of this period the reaction is stopped by heating for 5 minutes at 80C, the denatured enzyme is centrifuged off and the p-nitrophenol is removed on an anion exchange column (e.g. Dowex lx2).
The concentrated solution is fractionated by gel chromatography ( Sephadex G- 10, 2.5 x 70 cm ) and the product fractions are lyophilized.

Compound A (1.5 mg, 796 yield based on p-nitrophenyl D-mannos ide ):

5 p-nitrophenyl B-D-mannosyl- (1-3) -c~-D-mannoside H-NMR (400 ~z, D20): ~ = 5.68 (d, H-l); 4.18 (dd, H-2);
4.13 (dd, H-3); 3.91 (dd z t, H-4), 3.62-3.70 (mc, 4H, H-5, H-6b, H-6a', H-6b' ); 3.87 (dd, H-6a); 4.68 (d z s, H-l'); 3.96 (dd z d, H-2'); 3.57 (dd, H-3'); 3.48 10 (dd z t, H-4'); 3.36 (ddd, H-5'); J1~ = 1-6; Jz3 = 3-2;
J = 9 5; J4 5 = 9 ; J5 6a = 2.0; J6a 6b = 12 2; J1. ,2. < 1;
J2 3~ = 2-9; J3. 4 = 9~7; J4~ 5~ 9 4i J5~ 6a = 2-0;
J5 6b 7 2; J6a~ 6b~ = 12 2 Hz -Compound B (14.5 mg, 13% yield based on a donor/acceptor 15 ratio of the starting material of 1: 1) Compound B was characterized as peracetylated compound:

p-nitrophenyl 2,3,4,6-tetra-acetyl-B-D-mannosyl-(1-4) -2,3,6-tri-acetyl-B-D-mannoside 1H-NMR (400 MHz, CDCl3): ~ = 5.25 (d z s, H-1) ; 5.58 20 (dd z d, H-2); 4.98 (dd, H-3); 3.98 (dd z t, H-4); 3.80 (ddd, H-5); 4.34 (dd, H-6a); 4.23 (dd, H-6b); 4.69 (d z s, EI-l ' ); 5.37 (dd % d, H-2 ' ); 5.20 (dd, EI-3 ' ); 5.16 (dd z t, H-4'); 3.59 (ddd, H-5'); 4.09 (dd, H-6a'); 4.24 (dd, H-6b'); J1z z 1; J23 = 3-5; J34 9-8; J45 9-2;
25 J5 6a = 3 0; J5 6b = 6 1; J6a 6b = 12.2; J1~ ,2' < 1;
J2 3 = 3.2; J3 4 = 9.2; J4 5 = 9 6; J5 6a = 3 1;
J5 bb 5 6; J6~ bb~ = 12 2 Hz .

Claims (10)

1. A process for transglycosidation using .beta.-galacto-sidase, wherein unphysiological glycosides are employed as glycosyl donors for .beta.-galactosidase.
2. The process as claimed in claim 1, wherein 6-O-acetyl-.beta.-D-galactopyranosides or 6-O-formyl-.beta.-D-galactopyranosides are employed as glycosyl donors.
3. The process as claimed in claim 1, wherein .beta.-D-fuco-pyranosides and .alpha.-L-arabinopyranosides are employed as glycosyl donors.
4. The process as claimed in claim 1, wherein .beta.-D-glucopyranosides or .beta.-D-mannopyranosides are employed as glycosyl donors.
5. The process as claimed in any of claims 1, 2 or 3 wherein the glycosyl acceptors employed are short-chain, acyclic or cyclic, saturated or unsaturated mono-, di-, tri- to polyhydroxy compounds (mono- to polyhydric alcohols), with C1 to C10, which are optionally substituted by halogen (F, Cl, Br, I) or sulfonyl, cyano, alkyl, alkyloxy, aryl, aryloxy, heteroaryl, heteroalkyloxy, thioalkyl, trialkyl-silyl, trialkylsilyloxy, azido, amino, N-acyl, N-tert.- butyloxycarbonyl(N-Boc), N-benzyloxycar-bonyl(N-Z) or N-allyloxycarbonyl(N-Aloc) groups, or are simple monosaccharides (e.g. Glc, Gal), 2-deoxy-2-aminosugars. 2-deoxy-2-N-acylsugars (such as, for example, GlcNAc), glycosyl fluorides (such as, for example, .alpha.-F-Glc and .alpha.-F-GlcNAc) as well as alkyl glycosides and aryl glycosides, with alkyl or aryl residues with one to ten carbon atoms, which may be optionally substituted by halogen, tri-alkylsilyl, cyano, nitro and azido groups (such as, for example, .alpha.-Gal-OMe, .alpha.-Gal-OCH2Ph, .alpha.-Gal-OPh), or are compounds of the formula I

I

with n = 1-10 in which R1 is an amino protective group and R2 is a hydroxyl group, an alkoxy or thioalkyl group or an alkenyloxy group, in each case with 1 to 18 carbon atoms, which may be substituted by halogen or cyano, an aryloxy group with 6 to 10 carbon atoms, which may be substituted by alkyl, alkoxy, thioalkyl t in each case with 1 to 5 carbon atoms, and nitro groups, or is the -NHR3 group, in which R3 is an alkyl group with 1 to 5 carbon atoms or a residue of the formula II

II

or a dipeptide or tripeptide residue of the formula III or IV

III

IV, where R4 is a hydroxyl group, an alkoxy, a thioalkyl or a alkenyloxy group, with in each case 1 to 5 carbon atoms, which may be substituted by halogen or cyano, an aryloxy group with 6 to 10 carbon atoms, which may be substituted by alkyl, alkoxy, thioalkyl, with in each case 1 to 5 carbon atoms, and nitro groups and R5, R6 and R7 are the same or different and are hydrogen or straight-chain, branched or cyclic alkyl or alkenyl groups with 1 to 10 carbon atoms, which may be substituted by halogen, hydroxyl, alkoxy, thiol, thioalkyl, aryl or heteroaryl, and R2 and R4 in formula II-IV are preferably an alkoxy group with 1-10 carbon atoms or methoxymethyloxy, methylthiomethyloxy, chloroethoxy, bromoethoxy or cyanoethoxy, benzyloxy, p-nitrobenzyloxy, p-methoxy-benzyloxy, piperonyloxy, allyloxy or vinyloxy as well as tertiary butyloxy or tertiary butyldimethyl-silyloxy groups.
6. The process as claimed in claim 5, wherein amino protective groups which are essentially those currently used in peptide and glycopeptide chemistry are employed as the protective group in the position of formula I, such as, for example, acyl groups, and also acyl residues of long-chain fatty acids and alkyl- and aryloxycarbonyl groups, where benzyloxycarbonyl (Z), allyloxycarbonyl (Aloc) and tertiary butyloxycarbonyl (Boc) as well as formyl, acetyl, chloroacetyl, trifluoroacetyl, phenacetyl, benzoyl or acyl residues of long-chain fatty acids with 6 to 24 carbon atoms are preferred.
7. The process as claimed in one or more of claims 1 to 6, wherein the glycosyl donors employed and the glycosyl acceptors used are employed in the ratio 1:10 to 4:1, and/or toluene, ether, xylene or aceto-nitrile is employed as solvent.
8. The process as claimed in one or more of claims 1 to 7, wherein the reaction takes place within the pH
range 4.5 to 8Ø
9. The process as claimed in one or more of claims 1 to 8, wherein the reaction temperature is held within the range 15°C to 50°C.
10. The process as claimed in one or more of claims 1 to 9, wherein the .beta.-galactosidase is derived from E. coli, Aspergillus niger, coffee beans or bovine testes.
CA002086972A 1992-01-09 1993-01-08 Process for .beta.-galactosidase-catalyzed transglycosidation with unphysiological glycosyl donors Abandoned CA2086972A1 (en)

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US5876981A (en) * 1996-10-17 1999-03-02 The Scripps Research Institute Transglycosylation reactions employing β-galactosidase
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US5149640A (en) * 1988-12-22 1992-09-22 Ajinomoto Co., Inc. Method for producing galactose transfer products
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