Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/02—Acyclic radicals, not substituted by cyclic structures
  • C07H15/12—Acyclic radicals, not substituted by cyclic structures attached to a nitrogen atom of the saccharide radical
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/20—Carbocyclic rings
  • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
• • 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
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
• • 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

Definitions

• the present invention relates to novel fucosylated oligosaccharides or oligosaccharide containing compounds which are analogues to natural oligosaccharides and which contain at least one fucosylated monosaccharide unit.
• the invention also relates to a process for the preparation of such oligosaccharides or oligosaccharide containing compounds.
• Fucosylated mammalian glycans have functions in fertilization (1), early differen- tiation of embryo (2), brain development (3,4), and leukocyte extravasation (5,6), the ⁇ l-3/4fucosylated oligo- and polysacharides being conjugated to lipids and proteins or as free oligosaccharides such as the oligosaccharides of human milk.
• the fucosylated N-acetyllactosamines (Lewis x, Gal/31-4(Fuc ⁇ l-3)GlcNAc and Lewis a, Gal/ 1-3(Fuc ⁇ l-4)GlcNAc) and fucosylated N-acetyllactosdiamine (Lex- NAc, GalNAc/31-4(Fuc ⁇ l-3)GlcNAc) occur often as terminal sequences such as Gal 31 -4(Fuc ⁇ 1 -3)GlcNAc/31 -2Man ⁇ - , Gal ⁇ 1 -3(Fuc ⁇ 1 -4)GlcNAc/31 -2Man ⁇ - , Gal- NAc/31 -4(Fuccd-3)GlcNAc/31 -2Man ⁇ - , GaljS 1 -4(Fuc ⁇ 1 -3)GlcNAc/ 1 -3GaI/S- - /GalNAc ⁇ -, Gal/31-3(Fuccd-4)GlcNAc/31-3G
• the ⁇ l-3fuco- sylated epitope at the reducing end of the saccharide is commonly lactose (Gal/31— 4Glc) or its elongated/substituted form such as Gal 31-4(Fuc ⁇ l-3)Glc, Fuc ⁇ l-2Gal- ⁇ 1 -4(Fuc ⁇ 1 -3)Glc, Gal/ 1 -4GlcNAc/31 -3Gal/31 -4(Fuc ⁇ 1-3)- Glc,Gal/31-3GlcNAc/31-3Gal/31-4(Fuc ⁇ l-3)Glc, and Gal/3 l-4(Fuc ⁇ l-3)GlcNACiSl- 3Gal/31-4(Fuc ⁇ l-3)Glc. Analogs of these could be useful for studies of specificities of biological activities of the natural mammalian ⁇ l-3/4fucosylated sequences.
• Fuc-Ts The fucosylation step in the biosynthesis of these glycans is accomplished by the family of ⁇ l-3/4fucosyltransferases (Fuc-Ts). In man, at least Fuc-Ts III- VII and IX are expressed (7-9), and enzymatically active homologs are known in other animals and bacteria.
• ⁇ l-3Fucosyltransferases transfer fucose to position 3 of GlcNAc or Glc residues in Gal/31-4GlcNAc (LacNAc), GalNAc/31-4GlcNAc (LacdiNAc) and Gal/31-4Glc (lactose) to synthesize the bioactive epitopes Gal/31-4(Fuc ⁇ l-3)GlcNAc (Lewis x, Lex), GalNAc/31-4(Fuc ⁇ l-3)GlcNAc (LexNAc), and Gal/31-4(Fuc ⁇ l-3)Glc, respectively. All human Fuc-Ts are known to use Gal/3 l-4GlcN Ac-type acceptors (7,9).
• GalNAc/31-4GlcNAc serves also as an acceptor for the Fuc-Ts of human milk (10).
• Human Fuc-Ts III and V have also ⁇ l-4fucosyltransferase activity using acceptors such as Gal/31-3 GlcNAc (type I N-acetyl-lactosamine) to synthesize Gal/31-3(Fuc ⁇ l-4)GlcNAc (Lewis a).
• acceptors such as Gal/31-3 GlcNAc (type I N-acetyl-lactosamine) to synthesize Gal/31-3(Fuc ⁇ l-4)GlcNAc (Lewis a).
• At least human Fuc-Ts III and V and (weakly VI) are able to fucosylate lactose and related oligosaccharides to structures containing Gal/3 l-4(Fuc ⁇ l-3)Glc sequences.
• the present invention describes saccharide epitope analogues of the mammalian fucosylated saccharide chains, as well as their synthesis.
• An effective method to synthesize such epitopes is to use ⁇ l-3 fucosyltransferases or ⁇ l-3/4fucosyltransfe- rases for fucosylation of novel acceptor sequences.
• Some of the acceptor sequences can be synthesized from cheap natural polysaccharides such as cellulose, chitin, chondroitin/chondroitin sulphates, or hyaluronic acid, also natural polysaccharides with the sequence Glc/31-(3Glc/31-4Glc/31-) n 3Glc could be used to synthesize acceptors.
• ⁇ l-4GlcNAc transferase and UDP-GlcNAc can be used to generate GlcN- Ac/31-4GlcN Ac/31- linked to Gal, GalNac or Man (11) and these can be used to make other analogues.
• Certain parasites have also been reported to contain N- acetyl-chitooligosaccharides which could be used as acceptors for the fucosylation reaction (12).
• N-acetyl-chitooligosaccharides N-acetyl- chitotriose and larger
• novel fucosylations of N-acetyl-chitooligosaccharides occured to the non-reducing subterminal residue (forming a terminal Lewis x-like structure with a linkage structure similar to human glycans) and not to the reducing-end GlcNAc as in plant N-glycans.
• the present invention is directed to novel fucosylated oligosacch- arides and oligosaccharide type compounds, especially N-acetyl-chitooligosaccharides which are ⁇ l-3 fucosylated in the monosaccharide at the position subterminal to the non-reducing end of the oligosaccharide.
• the present invention is also directed to a process for the preparation of such compounds, which results in a site-specific fucosylation by the use of ⁇ l-3fucosyltransferase or ⁇ l-3/4fu- cosyltransferase enzyme to glycosylate the oligosaccharide acceptor substrate with L-fucose.
• Fig. 1 (A) is a HPAE-chromatography of the neutral Fuc-TV fucosylation products of N-acetyl-chitotetraose.
• Fucosylated product (Glycan 4, FGN 4 ; for structures of Glycans 3-7, see Table 1) eluted at 7.62 min and the putative acceptor at 9.74 min. in the isocratic run with 40 mM NaOH.
• FIG. B shows HPAE-chromatographic purification of fucosylated N-acetyl-chitohexaose (FGN 6 ) eluting at 7.09 min., putative N-acetyl-chitohexaose peak at 9.04 min in the isocratic run with 40 mM NaOH.
• the saccharides were from Fuc-TV reaction.
• Fig. 2 shows MALDI-TOF mass spectra of the key reaction mixtures and purified product saccharides.
• F is L-Fucose
• GN is N-acetyl-D-glucoseamine.
• A shows a mixture of FGN 4 and GN 4 produced by human Fuc-TV
• B shows a mixture of FGN 6 and GN 6 produced by Fuc-TV.
• C shows FGN 6 purified by HPAE-chroma- tography.
• D shows exo-/3-N-acetylhexosaminidase and novel endo-chitinase (the jack bean enzyme, mild conditions) reaction to the mixture of FGN 6 and GN 6 produced by Fuc-TV.
• the major product is surprisingly FGN 5 .
• E shows mild cleavage of the purified FGN 6 to mostly FGN 5 by the jack bean preparation.
• F shows mild treatment of the purified FGN 4 by the jack bean preparation, 91 % of the substrate remains intact.
• Fig. 3. shows MS/MS spectra of reduced and permethylated fucosylated N-acetyl- chitotriose 3 (A), and N-acetyl-chitotetraose 4 (B).
• the fragment ion nomenclature of Domon and Costello is used to denote the generated fragments.
• the present invention concerns fucosylated oligosaccharide compounds having the formula
• A is H or a glycosidically /31-3 linked D-glucopyranosyl residue (Glc/31-3)
• Ri is OH
• R 2 is H and R 3 is OH or acylamido
• -NH-acyl i.e. mono- saccharide 1 is Glc, or GlcNAcyl
• R j is H
• R 2 is OH and R 3 is acetamido - NHCOCH 3 (i.e. monosaccharide 1 is GalNAc)
• B is H, or an ⁇ -L-fucosyl or an ⁇ -L-fucosyl analogue
• R 4 is OH or acetamido -NHCOCH 3 (i.e.
• monosaccharide 2 is optionally fucosylated Glc or GlcNAc), the curved line between the saccharide units indicating that the monosaccharide 1 is ⁇ 1-4 linked to monosaccharide 2 when B is linked to the position 3 of the monosaccharide 2, and the monosaccharide 1 is 1-3 linked to monosaccharide 2 when B is linked to the position 4 of the monosaccharide 2, monosaccharide 1 is GalNAc only when monosaccharide 2 is Glc, n is 1 to 100, with the proviso that there is always at least one ⁇ - fucosyl or ⁇ -fucosyl analogous group present in the molecule, and
• p and k are 0 and m is 1, in which case X is H, an aglycon residue or a mo- nosaccharide selected from the group consisting of Glc, GlcNAc, Gal or GalNAc, optionally in reduced form, or oligosaccharide containing one or more of said monosaccharide units, the monosaccharide 2 being 31-2, /31-3, /31-4 or ⁇ 1-6 linked to saccharide X, with the proviso that X is not H when both monosaccharides 1 and 2 are GlcNAc, B is L-fucosyl and n is 1 , or ii) p is 1 , k is 0 or 1 and lj ⁇ m j 1000, in which case X is a straight bond, or a mono- or oligosaccharide as defined under i),
• Y is a spacer or linking group capable of linking the saccharide 2 or X to Z
• Z is a mono- or polyvalent carrier molecule.
• Glc means a D-glucose residue and Gal means a D-galactose residue.
• Fuc or F means a L-fucose residue.
• GlcNAc or GN means a N-acetyl-D-glucose amine residue.
• the monosaccharides are in pyranose form when glycosidically linked.
• B as an analogue to the L-fucosyl residue is preferably a compound that contains a hydroxy-methyl group in place of the methyl group in 6 position of fucosyl, that is L-galactosyl, or a deoxy derivative of L-fucosyl, or an analogue where a di- to tetrasaccharide is linked to C6.
• B is H or L-fucosyl.
• R 3 as an acylamido group is preferably an alkanoylamido group with 2 to 24 carbon atoms and 0 to 3 double bonds between carbon atoms in a straight chain.
• R 3 is acetamido -NH-COCH 3 , or an alkanoylamido group with 8 to 24 carbon atoms, and 1 to 3 double bonds
• m is preferably 1 to 100 and most preferably 1 to 10
• n is preferably 1 to 10.
• An oligosaccharide in the meaning of X contains preferably from 2 to 10 mo- nosaccharide units, the monosaccharide units preferably being glycosidically /31-4 or ⁇ 1-3 linked Glc or GlcNAc residues.
• An aglycon group is preferably a hydrocarbon group, such as a C- ⁇ o alkyl or C 2 _ 20 alkenyl group, a C 3 . 10 cycloalkyl or cycloalkenyl group, an aryl or aralkyl group containing up to 10 carbon atoms in the aromatic ring, alkyl having the meaning given above, for example a phenyl group or benzyl group, or a heterocy-rod group, that is a cycloalkyl or an aryl group as defined containing one or more heteroatoms O, S or N in the ring(s).
• a hydrocarbon group such as a C- ⁇ o alkyl or C 2 _ 20 alkenyl group, a C 3 . 10 cycloalkyl or cycloalkenyl group, an aryl or aralkyl group containing up to 10 carbon atoms in the aromatic ring, alkyl having the meaning given above, for example a
• a prefened aglycon group is a lower alkyl or alkenyl group of 1 to 7 , or 2 to 7 carbon atoms, respectively, or a phenyl or benzyl group.
• a prefened heterocyclic aglycon group is 4-methylumbelliferyl.
• the spacer group Y can be any group that is capable of linking the group X or saccharide 2 to the carrier molecule Z, and such groups and methods of linking are known in the art, and also commercially available.
• X is a saccharide
• a bond between X and Y can be formed by reacting an aldehyde or a carboxylic acid with X at its C j or by introducing any aldehyde or carboxylic acid group in X through oxidation, to form a suitable bond such as - NH-, -N(R)- where R is an alkyl group, or a hydroxyalkylamine, an amide, an ester, a thioester or thioamide.
• a suitable bond is also -O- or -S-, see e.g. Stowell et al Advances in Carbohydrate Chemistry and Biochemistry, 37 (1980), 225-.
• the carrier molecule can be mono- or polyvalent, and is preferably selected from polymers, such as polyacrylami- des, polyols, polysaccharides, such as agarose, biomolecules, including peptides and proteins, bovine or human serum albumin being commonly used carriers for example in immunoassays.
• a prefened group of compounds with the formula I comprises the following
• the monosaccharides 1 and 2 are preferably indepen- dently Glc and GlcNAc, B is L-fucosyl, and X is Glc or GlcNAc or a 01-3 or 01- 4 linked oligomer comprising up to 10 units of Glc and/or GlcNAc.
• the symbols have the same meanings as given above in the formula IA, or X can also be H provided the monosaccharides 1 and 2 are both Glc.
• the compounds of the formula IA and IB are thus oligosaccharides that are fucosylated in the subterminal or penultimate non-reducing end monosaccharide. According to the invention it has been discovered that this specific fucosylation results in highly stable oligosaccharides, especially N-acetyl-chitooligosaccharides.
• the saccharides have the formula
• n' is the integer 1 to 8, preferably 1 to 6.
• the saccharides have the formula
• n' has the meaning given above and acyl is an alkanoyl group which preferably contains 8 to 24 carbon atoms and 1 to 3 double bonds.
• acyl is an alkanoyl group which preferably contains 8 to 24 carbon atoms and 1 to 3 double bonds.
• the compounds of the formula I are prepared by fucosylating a compound of the formula I wherein B is always H, with a donor nucleotide sugar containing L-fucose, or an analogue thereof, in the presence of a fucosyltransferase enzyme, and optionally recovering the fucosylated saccharide so prepared.
• a reaction may be carried out on the starting oligosaccharide prior to the optional binding the oligosaccharide to the carrier molecule Y.
• Such a reaction is carried out essentially in the same manner as with non-bound oligosaccharides.
• a N-acetyl-chitooligosaccharide such as a N-acetyl-chitot- riose
• BSA bovine serum albumin
• the fucosylation reaction preferably an excess GDP-Fuc and a lower concentration of acceptor sites (0.1 - 1 mM) are used. If BSA is the carrier, the reaction buffer contains no non-glycosylated BSA.
• the products can be purified by methods of protein chemistry and the level of fucosylation can be checked by MALDI-TOF mass spectrometry, as described in more detail below. The fucosylation can be repeated if the reaction is incomplete.
• Oligosaccharides with an aglycon group as X can be fucosylated essentially as has been described, preferably using lower acceptor concentrations (0.1 - 1 mM).
• the products can be purified using chromatographic methods including gel filtration and with partial reaction cleavage with N-acetylhexosaminidase from jack beans. As an example, one obtains GlcNAc01-4(Fuc ⁇ l-3)GlcNAc01-benzyl from
• GlcNAc ⁇ 1 -4GlcNAc01 -benzyl GlcNAc ⁇ 1 -4(Fuc ⁇ 1 -3)GlcNAc01 -O-methyl from GlcNAc ⁇ l-4GlcNAc01-O-methyl
• GlcNAc ⁇ l-4(Fuc ⁇ l-3)GlcNAc01- 4GlcNAc 1 -O-4-methylumbelliferyl from GlcNAc ⁇ 1 -4GlcN Ac ⁇ 1 -4GlcNAc01 -O- 4-methylumbelliferyl.
• the fucosyltransferase is human ⁇ l-3-fucosyltransferase or human ⁇ l-3/4fucosyltransferase, especially one of human fucosyltransferases III- VII, IX, or ⁇ l-3/4fucosyl transferase of human milk.
• the L-fucose is in the form of GDP-L-fucose.
• the starting oligosaccharide contains 3 to 10, especially 3 to 8 01-4 saccharide units. Such units are preferably N-acetyl-D-glucosamine residues, GlcNAc, the conesponding starting materials thus being N-acetyl-chitooligomers with formula (GlcNAc01-4) 3 _ 1O( - 8 .
• the oligosaccharides contain 2 to 10, especially 2 to 6 01-4 D-glucose units, Glc, the conesponding starting materials thus being cellooligomers with formula (Glc01-4) 2 . 10(8) -
• the invention it is also possible to include a further step of reacting the product obtained with a 0-N-acetyl-hexosaminidase under sufficently strong conditions and in an amount sufficient to release the non-reducing terminal mo- nosaccharide. It is also possible to react the product obtained with a 0-N-acetyl- hexosaminidase under less severe conditions in order to release a monosaccharide from its reducing terminal. In this manner, a product is obtained which contains one saccharide unit less than the primary oligosaccharide product. This latter reac- tion may also be controlled in such a manner that the enzyme primarily degrades any remaining non-fucosylated substrate. This method thus provides a convenient way of purifying the reaction mixture after the fucosylating step.
• the novel oligosaccharides are stable compounds and as such useful in a number of applications. As such they can, for example, be used as substrates for testing, identifying and differentiating enzymes, such as chitinases, and when bound to a support they find use in immunoassays and affinity chromatography. They can also find use in a agrobiology, as stable plant protectants, as activators of the defence mechanisms and growth regulators of the plant cell simi- larly as has been described with N-acetyl-chitooligosaccharides and their acyl derivatives (13). In such use, incorporation of the fucose group in the oligosaccharide will in practice protect the oligosaccharide from enzymatic degradation.
• the ⁇ l-3/4fucosylated analogues of animal oligosaccharides are useful for studies of biological interactions involving their natural counterparts.
• the natural ⁇ l-3- /4fucosylated oligosaccharides are known to be ligands or counteneceptor of lectins mediating cell adhesion and other intercellular interactions. These saccharides are also important antigenic epitopes recognized by anti-cancer or allergy-related antibodies. Some of these interactions are of special medical interest such as the leukocyte adhesion to blood vessels mediated by the binding of the selectin proteins to their ⁇ l-3/4fucosylated counteneceptor oligosaccharides linked to proteins or lipids.
• the free oligosaccharide analogues can be tested in in vitro or in vivo assays to find out their abilities to inhibit the binding between the lectins and their counterreceptors or alternatively the direct binding of the oligosacchari- des to the lectins can be measured for example by affinity chromatography.
• the data obtained with the analogues in comparison to free saccharide epitopes identi ⁇ cal to the natural counteneceptor oligosaccharides reveal part of the specificity of the interaction. It shows which modifications in the molecule are useful and which are not tolerated when better medical lead compounds for antagonists of the interaction are designed.
• oligosaccharides In search of better medical derivatives of the oligosaccharides the reducing end of the oligosaccharide chain can be modified by numerous non-carbohydrate structures, aglycons. Free oligosaccharides can also be useful as mixtures of known composition (so called libraries) in the tests of biolological activities. Mixtures of positional fucosyl isomers are easily obtained by incubating oligosaccharide with multiple acceptor sites with less GDP-Fuc than the amount of the acceptor sites or by following the reaction level by MALDI-TOF analysis of the reaction mixture and limiting the reaction time (14).
• Multi- or polyvalent conjugates of the oligosaccharides can also be more active antagonists of biological interactions when they are of natural and non-antigenic type.
• Antigenic polyvalent conjugates such as oligosaccharides coupled to bovine serum albumin or to keyhole limpet hemocyanin can be used to raise antibodies against the saccharide epitopes.
• Polyvalent conjugates conjugated to solid supports, such as agarose affinity chromatography media or plastic micro titer plates, are also useful for assaying and purification of lectins and other proteins binding the saccharides.
• Fucosylated N-acetyl-chitooligosaccharide epitopes -Man01-4Glc- NAc(Fuc ⁇ al-3)GlcNAc01-Asn of plant and insect proteins (15) are potent and cross reactive human allergens and GlcNAc(Fuc ⁇ l-3)GlcNAc01-conjugates can be useful for assaying allergy antibodies recognizing the epitope.
• the selectin proteins mediating vascular leukocyte adhesions are known to bind sialyl-Lewis x [sLex, NeuNAc ⁇ 2-3Gal01-4(Fuc ⁇ l-3)GlcNAc], especially in sLex- 01-3Lex01- (16), and sialyl-Lewis a [NeuNAc ⁇ 2-3Gal01-3(Fuc ⁇ l-4)GlcNAc] oligosaccharides (17) and in some reports also Lewis x [Lex, Gal01-4(Fuc ⁇ l-3)Glc- NAc] (18) or VIM-2 -epitopes [NeuNAc ⁇ 2 ⁇ 3Gal01-4GlcNAc01-3Gal01-4(Fuc- ⁇ l-3)GlcNAc] (19).
• GalNAc-analogue of Lewis x GalNAc01-4(Fuc ⁇ l-3)GlcNAc has been reported to be better selectin ligand than the sialyl-Lewis x (20, 21).
• the following examples are intended to illustrate the invention.
• DQFCOSY double quantum filtered conelation spectroscopy
• ESI-CID electros- pray ionization-collision induced decay
• Fuc & F L-fucose
• Fuc-Ts III- VIII and IX human ⁇ l-3fucosyltransferases/ ⁇ l-3/4fucosyltransferases III-VII and IX
• Fuc- Thm ⁇ l-3/o -3/4fucosyltransferases of human milk
• Gal D-galactose
• GalNAc D-N-acetylgalacosamine
• GlcNAc & GN D-N-acetylglucosamine
• HPAEC-PAD high pH anion exchange chromatography - pulsed ampero metric detection
• Lac- NAc, Gal01-4GlcNAc LacdiNAc, GalNAc01-4GlcNAc
• MALDI-TOF MS matrix-assisted laser desorption/ionization
• N-acetyl-chitotriose, N-acetyl-chitotetraose, and N-acetyl-chitohexaose were from Seikagaku (Tokyo, Japan).
• Cellobiose was from Thomas Kerfoot and Co. Ltd.
• 0-N-acetylhexosaminidase from jack beans was from Sigma (St. Louis, MO, USA).
• GDP-fucose (used in human milk experiments) was a kind gift from Prof. B. Ernst (Universitat Basel, Switzerland).
• Partially purified human milk fucosyltransferases were prepared as described in (22) and assayed as described in (23), 1 mU conesponds to transfer of 1 nmol of fucose to 190 mM lactose / minute at 37 °C, pH 7.5.
• Fucosylation of N-acetyl-chitooligosaccharides with human milk fucosyltransferases (EC 2.4.1.152 and EC 2.4.1.65) was carried out essentially as described in (24), but with 2 * 360 ⁇ U of the enzyme (adding half of the enzyme after 2 days)/ 100 ⁇ l of reaction mixture and the acceptor concentrations were 5 mM and by incubating reactions at 37 °C for four days.
• Fucosyltransferase V (Fuc-TV, EC 2.4.1.152, recombinant, Calbiochem) reactions were carried out under similar conditions but with 12.5 mU of the enzyme/100 ⁇ l, and the reaction mixtures were incubated at room temperature for five days.
• Fucosyltransferase VI (EC 2.4.1.152, recombinant, Calbiochem) reactions were carried out under the same reaction conditions as with Fuc-TV except 2 mM acceptor and 4 mM GDP-Fucose concentration and 10 mU of the enzyme/ 100 ⁇ l, and incubation for 3 days at 37°C. The reactions were terminated by boiling for 3 minutes, the reaction mixtures were stored frozen until analysed.
• Reactions performed under exhaustive reactions contained 300 mU of the 0-N- acetylhexosaminidase in 4.8 ⁇ l of 2.5 M (NH 4 ) 2 SO 4 , 2 nmol of fucosylated N- acetyl-chito-oligosaccharides, and 40.2 ⁇ l of 50 mM sodium citrate pH 4.0.
• the reactions were incubated at 37°C, 300 mU of fresh enzyme in 2.5 M (NH 4 ) 2 SO 4 was added on days 2,3,5 and 7, and the reactions were stopped at day 8 by adding 1 vol of ice-cold ethanol and 8 volumes of ice-cold water.
• Oligosaccharides were reduced with NaBH 4 essentially as described in (26). The alditols were purified by gel filtration and the completeness of the reactions was verified by MALDI-TOF mass spectrometry.
• Samples from the enzymatic reactions were desalted by passing them in water through 1.5 ml of Dowex AG-50 (H+ , 200-400 mesh) and 1.5 ml of Dowex AG- 1 (AcO-, 200-400 mesh) and then purified by gel filtration HPLC in a column of Superdex Peptide HR 10/30, with ultra pure water or 50 mM NH 4 HCO 3 as eluant, at a flow rate of 1 ml/min.
• Gel filtration in a Biogel P-2 column (1 x 142 cm) was performed with ultrapure water, UV-absorbance was monitored at 214 nm.
• High- pH anion exchange chromatography with pulsed amperometric detection was performed on a (4x250 mm) Dionex CarboPac PA-1 column (Dionex, CA), the samples were run isocratically with 40 or 60 mM NaOH.
• Mass spectrometry Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed in the positive ion delayed extraction mode with a BIFLEXTM mass spectrometer (Bruker-Franzen Analytik, Bremen, Germany) using 2,5-dihydroxybenzoic acid as the matrix. The matrix peaks at the low mass region were "suppressed” by using unusually high sample concentrations (100 pmol/ ⁇ l).
• Electrospray mass spectra were collected using an API365 triple quad- rupole mass spectrometer (Perkin-Elmer instruments, Thornhill, Ontario, Canada). Samples were dissolved in 50 % aqueous methanol containing 0.5 mM sodium hydroxide, and injected into the mass spectrometer with a nanoelectrospray ion source (Protana A/S, Odense, Denmark) at a flow rate of about 30 nl/min. MS/MS spectra were acquired by colliding the selected precursor ions to nitrogen collision gas with acceleration voltages of 35 V (doubly-charged precursors) or 55 V (singly charged precursors).
• NMR spetroscopy Prior to NMR experiments the saccharides were lyophilized twice from D 2 O and then dissolved in 300 ⁇ L of D 2 O (99.996 atom % , Cambridge Isotope Laboratories, Woburn, MA, USA). The NMR experiments were carried out on a Varian Unity 500 spectrometer at 23 °C using Shigemi tubes (Shigemi Co. , Tokyo, Japan). In recording ID proton spectra a modification of the WEFT sequence (27) was used. For the DQFCOSY (28) and TOCSY (29) experiments (32 scans per t ⁇ value) matrices of 2k*256 and 4k*256 points were collected, and zero-filled to 2k*512 and 4k*512 points, respectively.
• a 90° shifted sine-bell weighting function was employed in both dimensions.
• TOCSY a spin-lock time of 100 ms (MLEV-17) was used.
• ID selective TOCSY (30) spectra were recorded with mixing times varying from 10 ms to 140 ms and a gaussian selective pulse.
• the transmitter was placed outside the signal area at 5.750 ppm and a continuous-wave spin-lock with spin-lock time of 300 ms was employed.
• a matrix of 2k*256 was collected and zero-filled to 2k*512 points.
• a 90° shifted sine-bell weighting func- tion was used in both dimensions. Additionally, the t j time domain data was doubled using forward-backward linear prediction.
• the monofucosylated N-acetyl-chitotriose was purified from the rest of the 0-N- acetylhexosaminidase digest by gel filtration HPLC.
• the total yield of the purified Glycan 3 was 191 nmol (19 %).
• the Y j ions at m/z 316.2 and m/z 338.2 indicate that the GlcNAc alditol carried only one monosaccharide substituent. Furthermore, theY 2 ⁇ /B 2 ion at m/z 442.4 can only arise by loss of terminal unsubstituted GlcNAc and the reduced GlcNAc residue. No fragments were observed even in closer inspection, which would represent fucosylated reduced end (i.e. Fuc ⁇ l -GlcNAcol, m/z 490.4) or fucosylated nonredu- cing end GlcNAc (terminal Fuc l -GlcNAc, m/z 456.2).
• the NMR-data confirm that the fucosylated N-acetyl- chitotriose, Glycan 3, generated by Fuc-TV is GlcNAc ⁇ l-4(Fuc ⁇ l -3)GlcNAc01- 4GlcNAc.
• N-acetyl-chitotriose and GDP-Fucose catalyzed by human milk Fuc-Ts Incubation of N-acetyl-chitotriose (1000 nmol, 5 mM) with GDP-fucose (600 nmol, 3 mM) and 1.4 mU of the enzyme for four days at 37°C gave an oligosaccharide mixture. After ion exchange desalting, mild 0-N-acetylhexosaminidase treatment, and repeated gel filtration HPLC runs, a sample of 221 nmol of purified fucosylated N-acetyl-chitotriose was obtained.
• MALDI-TOF MS revealed that the product contained 92 % of fucosylated N-acetyl-chitotriose and 7 % of N- acetyl-chitotriose.
• the ID lH-NMR-spectrum of the product was almost identical with that of Fuc-TV-generated Glycan 3.
• the ESI-MS-data of the Glycan 3 in reduced form, too, were identical to those obtained with reduced derivative of the Fuc-TV-generated Glycan 3 (not shown).
• the oligosaccharide mixture was then fractionated by gel filtration HPLC, yielding a purified product (225 nmol) that consisted of 93 mol % of fucosylated N-acetyl-chitotetraose and 7 % N-acetyl-chitotetraose.
• Another fucosylation mixture consisting of 11 % of the fucosylated N-acetyl-chitotetraose and 89 % of N-acetyl-chitotetraose was separated in an isocratic HPAE run in a (4 x 250 mm) Dionex CarboPac PA-1 column (Dionex, Sunnyvale, CA) column by using with 40 mM NaOH as the eluent (Fig. IA). The peak eluting at 7.62 min contained the fucosylated N-acetyl-chitotetraose.
• the ID 1H-NMR spectrum of the purified product, Glycan 4 synthesized by Fuc-TV was almost identical with that of Glycan 4 synthesized by the Fuc-Ts of human milk (see bellow).
• N-Acetyl-chitotetraose (6.0 ⁇ mol, 5 mM), and GDP-Fuc (3.0 ⁇ mol, 2.5 mM) were incubated with 8.7 mU of the enzyme for four days at 37 °C.
• the fucosylated product was separated from the non-fucos- ylated N-acetyl-chitotetraose by HPAEC using isocratic run with 40 mM NaOH (not shown).
• the purified product was desalted by ion exchange and was further purified by gel filtration HPLC yielding 744 nmol monofucosylated N-acetyl-chitotetraose.
• MALDI-TOF mass spectrometry of the product revealed that it was un- contaminated by N-acetyl-chitotetraose acceptor (not shown).
• the low-mass region of the MS/MS spectrum resembles that of the monofucosylated N-acetyl-chitotriose, showing the same B j fragment s for the unsubstituted terminal GlcNAc unit (m/z 282.2, m/z 260.2), and Y j ions for the reducing end GlcNAc alditol (m/z 316.2, m/z 338.2).
• the B 3 ion (m/z 946.8) verify that the fucose unit is linked to either of the midchain GlcNAc units.
• the ID 1H-NMR spectrum of the purified product, Glycan 4 revealed fucose HI, H5 and H6 signals characteristic to ⁇ 3-linked fucose rather than ⁇ 6- linked fucose in unconjugated N-glycans (36, 37).
• the H4 signal of the distal GlcNAc ⁇ was similar to its analogs in Glycan 3, confirming that the fucose is linked to the penultimate GlcNAc unit close to the non-reducing end.
• the proton signals of the fucose and the non-reducing end GlcNAc ⁇ were assigned from DQFCOSY and TOCSY spectra of Glycan 4.
• the 2D NMR-data strengthen the notion that the non-reducing end elements of Glycan 4 resemble those of Glycan 3.
• the NMR-data confirm the MS-results and the degradation data, establishing that Glycan 4 generated by human milk Fuc-Ts represented also GlcNAc ⁇ 1 -4(Fuc ⁇ 1 -3)GlcNAc01 -4GlcN Ac01 -4GlcN Ac .
• the peak eluting at 7.09 min was pooled, was desalted and was further purified by gel filtration HPLC.
• the product heptasaccharide (195 nmol) showed in MALDI-TOF analysis 96 % of monofucosylated N-acetyl-chitohexaose, Glycan 6, and 4 % of N-acetyl-chitohexaose, Fig. 2C.
• the ID 1H NMR spectrum (Table 2) shows the HI signals in the GlcNAc5, the GlcNAc ⁇ and the fucose units in Glycan 6, which are nearly identical with their counteparts in Glycan 4.
• Glycan 6 is GlcNAc ⁇ l-4(Fuc ⁇ l-3)GlcNAc 1- 4GlcNAc01-4GlcNAc01-4GlcNAc01-4GlcNAcNAc.
• This notion is further supported by the identical H4-signals of the distal GlcNAc ⁇ units and the fucose H5 resonances in the two glycans.
• the similarity of this latter pair of signals in the two glycans is particularly significant, because they are known to interact in Glycan 4 (see above), and are likely to be very sensitive to stuctural differences.
• the similarity of fucose H5 signals in Glycan 6, and Glycan 4 speaks for the identity of the nonreducing area in these three saccharides.
• the resulting digest showed in MALDI- TOF MS only 5 mol % of intact substrate; 85 % of the material was converted into FUC J G1CNAC 5 and 11 % into Fuc j GlcNA ⁇ (Fig. 2E).
• the data confirm that the endochitinase cleaves fast Glycan 6, releasing mainly one GlcNAc unit from the reducing end and the enzyme may be able to cleave off also a N-acetyl-chito- biose unit from the reducing end of Glycan 6, generating Glycan 4.
• Glycan 4 may have been formed by the release of a GlcNAc unit from the reducing end of FUC J G1CNAC5.
• Solvent A was used as described in (38) , using the upper phase of n-butanol-acetic acid: water (4: 1:5, v/v, solvent A) and scintillation counted from small strips of paper.
• Laminaribiose (1000 nmol, 5 mM, Seikagaku) and laminaritetraose (1000 nmol, 5 mM, Seikagaku) were reacted with GDP-[ 14 C]Fuc (1000 nmol, 5 mM, 100 000 cpm) and recombinant fucosyltransferase V (25 mU, 50 ml of the commercial enzyme preparation from Calbiochem) as above.
• the products of both reactions were desalted as above.
• the products from laminaribiose were purified by P-2 gel filtration chromatography and radioactive trisaccharide-like product was obtained.
• the desalted products from laminaritetraose were also purified by P-2 gelfiltration chromatography and a radioactive (174 nmol by radioactivity) peak eluting a pen- tasaccharide product as expected, was obtained.
• 131 nmol of product with monoisotopic m/z [M+Na] + close (difference less than 0.1 %) to 835.3 and m/z [M+- K] + close to 851.3 were pooled from front and middle fractions of the peak, the fractions contained less than 4 % of the acceptor, no other saccharide products were observed in analysis by MALDI-TOF mass spectrometry.
• the purified product saccharides were analyzed by NMR spectroscopy.
• chondroitin sulfate Chondroitin sulfate (shark cartilage, Sigma) was cleaved by hyaluronidase (bovine testes, Sigma; chondroitin sulfates can be also cleaved with chondroitinases giving oligosaccharides with delta-uronic acid at non reducing end) to sulfated oligosaccharides such as[GlcA01-3(sulf-6)GalNAc01-4]nGlcA01-3(sulf-6)GalNAc.
• the reduced tetrasaccharide Glc ⁇ l-3GalNAc ⁇ l-4Glc ⁇ l-3GalNAcol was purified and fucosylated using GDP-Fuc and human milk fucosyltransferase(s).
• the MALDI-TOF mass spectrometry revealed peaks at m/z 919.8 and m/z 935.8 corresponding to the product Glc ⁇ l-3GalNAc ⁇ l-4(Fuc ⁇ l-3)Glc ⁇ l-3GalNAcol (calc. m/z [M+Na] + is 919.7 and m/z [M+K] + is 935.7).
• GlcNAcyl ⁇ l-4GlcNAc ⁇ l-4GlcNAc ⁇ l-4GlcNAc ⁇ l-4GlcNAc wherein Acyl is trans-9-octade- cenoyl, synthesis of the acceptor being described in (13), can be incubated with Fuc-TVI and MnCl 2 under conditions described above for Fuc-TVI, but acceptor concentrations between 0.1-0.5 mM are preferred.
• the product GlcNA- cyl ⁇ l-4(Fuc ⁇ l-3)GlcNAc ⁇ l-4GlcNAc ⁇ l-4GlcNAc is purified chromatographically and analyzed by NMR-spectroscopy and mass spectrometry.
• N-Acetyl-chitotetraose can be incubated with human Fuc-TVI and MnCl 2 under conditions described above for Fuc-TVI.
• the product GlcNAc ⁇ l-4(Fuc ⁇ l-3)Glc- NAc ⁇ l-4GlcNAc ⁇ l-4GlcNAc can be obtained with almost quantitative yield and purified by HPAE-chromatrography.
• the NMR-data and mass spectrometry data of the product are practically identical with the fucosylated N-acetyl-chitotetraose obtained by Fuc-TV.
• Table 1 Structures of the fucosylated N-acetyl-chito-ohgosaccha ⁇ des in present study.
• GN is GlcNAc and Fuc is L-fucose
• line - indicates ⁇ l-4-lmkage
• line / is ⁇ l-3-hnkage
• residue numbering is in italics.

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