Enzymatic synthesis

Abstract

The invention relates to enzymatic N-acetyllactosaminylation of a terminal galactose in its 6- position.

Description

[0001] ENZYMATIC SYNTHESIS

[0002] FIELD OF THE INVENTION

[0003] This invention relates to enzymatic N-acetyllactosaminylation of a terminal galactose in its 6-position.

[0004] BACKGROUND OF THE INVENTION

[0005] During the past decades, the interest in the preparation and commercialisation of human milk oligosaccharides (HMOs) has been increasing steadily. The importance of HMOs is directly linked to their unique biological activities, therefore HMOs have become important potential products for nutrition and therapeutic uses. As a result, low cost ways of producing industrially HMOs have been sought.

[0006] To date, the structures of more than 140 HMOs have been determined, and considerably more are probably present in human milk. The HMOs comprise a lactose (Gaipi -4Glc) moiety at the reducing end and may be elongated with an N-acetylglucosamine, or one or more N- acetyllactosamine moiety / moieties (Gaipi -4GlcNAc) and / or a lacto-N-biose moiety (Gaipi- 3GlcNAc). Lactose and the N-acetyllactosaminylated or lacto-N-biosylated lactose derivatives may further be substituted with one or more fucose and / or sialic acid residue(s), or lactose may be substituted with an additional galactose, to give HMOs known so far (Urashima et al.: Milk oligosaccharides, Nova Biomedical Books, 2011 ; Chen Adv. Carbohydr. Chem. Biochem. 72, 1 13 (2015)).

[0007] HMOs having branched core structure share the substructure Gaipi-4GlcNAcpi-6Gaipi-4Glc. Branched core HMO structures are listed in Table 1 below. Although the tetrasaccharide Gaipi - 4GlcNAcpi-6Gaipi-4Glc has not been identified as an HMO, it was isolated from goat milk and horse colostrum (Chaturvedi at al. Biochem. Biophys. Acta 967, 115 (1988); Urashima et al. Comp. Biochem. Physiol. 100B, 177 (1991 )). Its chemical total synthesis has been accomplished by Takamura et al. Chem. Pharm. Bull. 29, 587 and 2270 (1981 )). Enzymatically, it has been prepared from GlcNAcpi-6Gaipi-4Glc by galactosylation with bovine milk N-acetyllactosamine synthase and UDP-[U-14C]Gal in radiolabelled form (Renkonen et al. Glycoconj. J. 8, 376 (1991 )).

[0008] Table 1.

[0009] New solutions have been sought for the enzymatic synthesis of the tetrasaccharide Gaipi - 4GlcNAcpi-6Gaipi-4Glc, a useful intermediate towards HMOs having branched core structure.

[0010] SUMMARY OF THE INVENTION

[0011] The invention relates to a method for synthesizing Gaipi-4GlcNAcpi-6Gal1 -A-R comprising the step of reacting an N-acetyllactosaminyl donor and Gal1 -A-R as acceptor in the presence of an enzyme capable of transferring the N-acetyllactosaminyl residue of the donor to the acceptor, wherein the enzyme is selected from the group consisting of:

[0012] - a protein characterized by SEQ ID No. 7 from Planctomycetales bacterium ZRK34 (GenBank nr. QNN21342.1 ) or its functional, optionally truncated functional analogs,

[0013] - a protein characterized by SEQ ID No. 8 from Paludibaculum fermentans P105 (GenBank nr. QOY89255.1 ) or its functional, optionally truncated functional analogs, and

[0014] - a protein characterized by SEQ ID No. 9 from Parabacteroides distasonis CBBP (GenBank nr. QJE27270.1 ) or its functional, optionally truncated functional analogs,

[0015] R means -OH, -N3 or -OR’, wherein R’ is alkyl, aryl, a group removable by hydrogenolysis, allyl optionally substituted by one or more methyl, propargyl optionally substituted by one or methyl, 2- trimethylsilyl-ethyl, -(CH2)n-NH2or -(CH2)n-N3, wherein integer n is 1 to 10, preferably 2 or 3, and

[0016] A is selected from bond and a divalent carbohydrate linker.

[0017] Preferably, the N-acetyllactosaminyl donor is LNnT.

[0018] Also preferably, R is -OH.

[0019] Also preferably, Gal1 -A-R is Gaipi-4Glc1 -R, more preferably lactose.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] Enzymes from the GH20 family have been known to be exo-glycosidases. The present inventors surprisingly found that some enzymes of the GH20 family, including their truncated but still functional versions, can act as N-acetyllactosaminidase and, in addition, can transfer the chopped N-acetyllactosamine in the 6’-position of lactose. The finding has opened a new way of constructing a Gaipi-4GlcNAcpi-6Gaipi-4Glc motif.

[0022] Accordingly, the invention relates to a method for synthesizing Gaipi-4GlcNAcpi-6Gal1 -A-R comprising the step of reacting an N-acetyllactosaminyl donor and Gal1-A-R as acceptor in the presence of an enzyme capable of transferring the N-acetyllactosaminyl residue of the donor to the acceptor, wherein the enzyme is a protein comprising an amino acid sequence disclosed as SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3,

[0023] A is selected from bond and a divalent carbohydrate linker, and

[0024] R means -OH, -N3 or -OR’, wherein R’ is alkyl, aryl, a group removable by hydrogenolysis, allyl optionally substituted by one or more methyl, propargyl optionally substituted by one or methyl, 2-trimethylsilyl-ethyl, -(CH2)n-NH2or -(CH2)n-N3, wherein integer n is 1 to 10, preferably 2 or 3.

[0025] The amino acid sequence characterized by SEQ ID No. 1 is the catalytic domain of the protein from Planctomycetales bacterium ZRK34 (GenBank nr. QNN21342.1) described by SEQ ID No. 7, which is believed to be in charge of the desired activity.

[0026] The amino acid sequence characterized by SEQ ID No. 2 is the catalytic domain of the protein from Paludibaculum fermentans P105 (GenBank nr. QOY89255.1) described by SEQ ID No. 8, which is believed to be in charge of the desired activity.

[0027] The amino acid sequence characterized by SEQ ID No. 3 is the catalytic domain of the protein from Parabacteroides distasonis CBBP (GenBank nr. QJE27270.1 ) described by SEQ ID No. 9, which is believed to be in charge of the desired activity.

[0028] Preferably, the enzyme capable of transferring the N-acetyllactosaminyl residue of the donor to the acceptor in the method of the invention is a protein comprising SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

[0029] The amino acid sequence characterized by SEQ ID No. 4 is the protein from Planctomycetales bacterium ZRK34 (GenBank nr. QNN21342.1 ) described by SEQ ID No. 7 without the native signal peptide.

[0030] The amino acid sequence characterized by SEQ ID No. 5 is the protein from Paludibaculum fermentans P105 (GenBank nr. QOY89255.1) described by SEQ ID No. 8 without the native signal peptide.

[0031] The amino acid sequence characterized by SEQ ID No. 6 is the protein from Parabacteroides distasonis CBBP (GenBank nr. QJE27270.1 ) described by SEQ ID No. 9 without the native signal peptide. For avoidance of any doubt, “Gaipi-4GlcNAcpi-6Gal1” in the Gaipi-4GlcNAcpi-6Gal1 -A-R product means 0-p-D-galactopyranosyl-(1 ->4)-0-2-acetamido-2-deoxy-p-D-glucopyranosyl-(1 ->6)- O-p-D-galactopyranosyl moiety which is attached via its 1 -OH to the A-R group; and likewise, Gaipi-4GlcNAcpi-6Gaipi-4Glc is 0-p-D-galactopyranosyl-(1 ->4)-0-2-acetamido-2-deoxy-p-D- glucopyranosyl-(1 ->6)- 0-p-D-galactopyranosyl-(1 ->4)-D-glucopyranose.

[0032] When R -is OH in the Gall -A-R acceptor, the substance is galactose (A is bond) or a di- or oligosaccharide (A is a carbohydrate linker) with a terminal galactose at the non-reducing end. The anomeric OH in the reducing end is free.

[0033] Likewise, when R -is OH in the Gaipi-4GlcNAcpi-6Gal1 -A-R product, the substance is the trisaccharide Gaipi-4GlcNAcpi-6Gal (A is bond) or a tetra- or higher oligosaccharide (A is a carbohydrate linker) with a terminal N-acetyllactosamine moiety at the non-reducing end. The anomeric OH in the reducing end is free.

[0034] When R is -N3, the compound is a glycosyl azide.

[0035] When R means -OR’, the molecule is an O-glycoside. The R’-group is either a common protecting group of the anomeric centre or have further synthetic usefulness. The unsaturated bond of glycosides having -N3,-O-(CH2)n-N3, -O-allyl or -O-propargyl aglycon or the -O-(CH2)n-NH2group can be functionalized by a wide variety of selective and mild water-compatible chemical reactions. Thus e.g. the azido group can be brought into “click chemistry” with an alkyne to form bioconjugates (and so can the propargyl derivatives with an azido reagents). The allyl functionality can be converted to other functional groups by addition reactions to the double bond or by ozonolysis. The amino function can be used to make amide or urea linkage to bind the compound to (bio)macromolecules / polymers.

[0036] Herein, the term “group removable by hydrogenolysis” preferably means a protecting group whose C-0 bond can be cleaved by hydrogen in the presence of a catalytic amount of palladium, Raney nickel or any other conventional hydrogenolysis catalyst to regenerate the protected -OH group. Such protecting groups are described in Wuts and Greene: Protective Groups in Organic Synthesis, John Wiley & Sons, 2007, and include benzyl, diphenylmethyl (benzhydryl), 1 - naphthylmethyl, 2-naphthylmethyl and triphenylmethyl (trityl) groups, each of which can be optionally substituted by one or more of the following groups: alkyl, alkoxy, phenyl, amino, acylamino, alkylamino, dialkylamino, nitro, carboxyl, alkoxycarbonyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, azido, halogenalkyl or halogen. Preferably, such substitution, if present, is on the aromatic ring(s). A preferred protecting group is benzyl optionally substituted with one or more of the following groups: phenyl, alkyl and halogen, particularly unsubstituted benzyl, 4- chlorobenzyl, 3-phenylbenzyl and 4-methylbenzyl groups. These preferred and particularly preferred protecting groups have the advantage that the by-products of their hydrogenolysis are exclusively toluene or substituted toluene. Such by-products can easily be removed, even in multi- ton quantities, from water-soluble oligosaccharide products via evaporation and / or extraction processes.

[0037] The term “alkyl” preferably means a linear or branched hydrocarbon group with 1-6 carbon atoms, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, etc. An alkyl group may be substituted with one or more halogen atoms and / or one or more OH-groups.

[0038] The term “aryl” preferably means an aromatic hydrocarbon group, more preferably phenyl or naphthyl. An aryl group may be substituted with one or more halogen atoms and / or one or more OH-groups.

[0039] The term “divalent carbohydrate linker” above means any mono-, di- or oligosaccharide glycosyl residue which is attached to the R group by the C-1 (in case of aldoses) or C-2 (in case of ketoses) anomeric carbon, and at the same time it is also attached, via one of its non-glycosidic OH-groups, to the galactosyl residue. Preferably, the divalent glycosyl linker or moiety A is a glucosyl moiety that forms a lactosyl moiety with the adjacent galactosyl moiety.

[0040] “Truncated functional analog" means that the remaining truncated protein substantially retains the desired activity. For example, the polypeptide SEQ ID No. 1 is a truncated functional analog of the protein of SEQ ID No. 4 or of SEQ ID No. 7 and the protein of SEQ ID No. 4 is a truncated functional analog of the protein of SEQ ID No. 7. Likewise, the polypeptide SEQ ID No. 2 is a truncated functional analog of the protein of SEQ ID No. 5 or of SEQ ID No. 8 and the protein of SEQ ID No. 5 is a truncated functional analog of the protein of SEQ ID No. 8; and the polypeptide SEQ ID No. 3 is a truncated functional analog of the protein of SEQ ID No. 6 or of SEQ ID No. 9 and the protein of SEQ ID No. 6 is a truncated functional analog of the protein of SEQ ID No. 9.

[0041] The enzymes described above transfer an N-acetyllactosamine residue from a donor substrate to an acceptor. If the acceptor is a carbohydrate (mono-, di- or oligosaccharide) or a carbohydrate derivative, then the enzyme acts as a trans-N-acetyllactosaminidase (able to make an N- acetyllactosamine containing carbohydrate product). On the other hand, the same enzyme can transfer the N-acetyllactosaminyl residue to a water molecule, acting thus as a hydrolase. The two processes take place concurrently. The overall synthetic performance is the ratio of the trans-N- acetyllactosaminidase and hydrolysis activities.

[0042] The enzymes in the method of the invention, including the preferred and more preferred embodiments, may be His-tagged at the N- or C-terminus. A His-tag is a short sequence of DNA that codes for a specific polypeptide, which is frequently inserted into a target gene at the point of coding for expression at either the N- or C-terminus of the protein required. The method of His- tagging is especially useful as it allows for easy purification and detection of the recombinant protein. Preferably, the His-tag is attached to the N-terminus. The presence of a His-tag does not affect markedly the basic characteristics of the enzymes. The His-tagged enzymes are therefore functional analogs. The enzymes in the method of the invention can be made for example in a method comprising the steps of:

[0043] (a) providing a DNA sequence encoding the enzyme, then

[0044] (b) expressing the enzyme in a host cell transformed with the DNA sequence obtained in step (a).

[0045] Step (a) can be carried out in a conventional manner by making the DNA sequence encoding the enzyme. In step (b) the DNA sequence is then introduced at the gene level by usual molecular- biological methods. The DNA sequence of the enzyme can be cloned in an expression vector which can be introduced in an appropriate host expression strain such as E. coli, containing DNA plasmids with the required information for regulation of expression of the enzyme variant. The sequence encoding the enzyme can be placed under the control of an inducible promoter. As a result, by adding an inducer, the expression of the enzyme variant can be controlled (generally, isopropyl-p-D-thiogalactopyranoside (IPTG) is used). The so-transformed host cells are then cultured in conventional nutrient media (e.g. Lennox broth, minimal medium M9) and induced with IPTG. After expression, the biomass can be harvested by centrifugation. The mutated enzyme can be isolated from the biomass after appropriate cell lysis and purification. In this process, conventional centrifugation, precipitation, ultrafiltration and / or chromatographic methods can be used.

[0046] The carbohydrate acceptor Gal1 -A-R used in the method of the invention can be any mono-, di- or oligosaccharide that has a terminal galactosyl unit at the non-reducing terminal where an N- acetyllactosaminyl moiety can be transferred by the enzymes disclosed above. In one preferred embodiment, the galactose-containing acceptor is lactose or a lactosyl glycoside, preferably lactose.

[0047] The enzymes in the method of the invention described above are able to create, surprisingly, a pi- 6 interglycosidic linkage when carrying out the method.

[0048] The N-acetyllactosaminyl donor used in the method of the invention can be any N- acetyllactosaminyl compound from which the enzymes are able to transfer the N- acetyllactosaminyl residue to the carbohydrate acceptor as described above. Suitably, the N- acetyllactosaminyl donor can be a compound of formula 1 or 2 wherein X is selected from the group consisting of a mono-, di- or oligosaccharide, azide, fluoro, optionally substituted phenoxy, optionally substituted pyridinyloxy, group A, group B, group C and group D

[0049] A BCD wherein Rais independently H or alkyl, or two vicinal Ragroups represent a =C(Rt>)2 group, wherein Rbis independently H or alkyl, Rcis independently selected from the group consisting of alkoxy, amino, alkylamino and dialkylamino, Rd is selected from the group consisting of H, alkyl and -C(=O)Re, wherein Reis OH, alkoxy, amino, alkylamino, dialkylamino, hydrazino, alkylhydrazino, dialkylhydrazino or trialkylhydrazino, preferably a compound of formula 1 wherein X is selected from the group consisting of a mono-, di- or oligosaccharide, phenoxy-, p-nitrophenoxy-, 2,4-dinitrophenoxy- and 2-chloro-4-nitrophenoxy- group. Advantageously, X in formula 1 is galactose or lactose meaning that the N- acetyllactosaminyl donor is Gaipi-4GlcNAcpi-4Gal or LNnT, respectively.

[0050] EXAMPLES

[0051] Example 1

[0052] The trans-N-acetyllactosaminidase activity of selected enzymes was investigated in the LNnT + lactose reaction. Starting conditions: LNnT 100 mM, lactose 500 mM, medium: UB4 buffer 50 mM, pH 6, temperature: 40 °C, enzyme concentration: 0.5, 2.5 and 5.0 pM. Product formation was examined with HLPC. The structure of the product Gaipi-4GlcNAcpi-6Gaipi-4Glc was confirmed with HPLC-MS and using the chemically prepared standard.

[0053] The following enzymes were used:

[0054] PlbaGH20: an enzyme characterized by SEQ ID No. 4 which is a protein from Planctomycetales bacterium ZRK34 (GenBank nr. QNN21342.1 ) without its native signal peptide

[0055] PafeGH20: an enzyme characterized by SEQ ID No. 5 which is a protein from Paludibaculum fermentans P105 (GenBank nr. QOY89255.1 ) without its native signal peptide

[0056] PadiGH20: an enzyme characterized by SEQ ID No. 6 which is a protein from Parabacteroides distasonis CBBP (GenBank nr. QJE27270.1 ) without its native signal peptide

[0057] The enzymes with a His-tag on the N-terminus (MGSSHHHHHHSSGLVPRGSH) were produced in E. coli BL21 (DE3). Overnight precultures of transformed cells grown at 37 °C in LB medium containing 50 pg / ml kanamycin were used to inoculate 500 ml LB medium supplemented with 50 pg / ml kanamycin and incubated at 37 °C. When the OD6oo of each culture reached a value of 0.6- 0.8, gene expression was induced using 1 mM IPTG for 20 h at 25 °C. Cells were harvested by centrifugation (5000xg, 15 min), and the pellet was resuspended in binding buffer (20 mM sodium phosphate buffer, 500 mM NaCI, 20 mM imidazole, pH 7.4) before sonication. Cell debris was removed by centrifugation (20,000xg, 30 min at 4 °C). Proteins were purified on Ni2+Sepharose columns, previously equilibrated with binding buffer (20 mM Tris-HCI (pH 7.4), 500 mM NaCI, and 20 mM imidazole). The His-tagged proteins were eluted using 250 mM of imidazole the same buffer. Imidazole was then removed using PD-10 columns; the resulting protein buffer was 20 mM sodium phosphate with 100 mM NaCI at pH 7.4. The proteins were cleaved from the His-tag using 0.5 U / ml thrombin protease and incubated at 4 °C overnight. Cleaved proteins were further purified through gel filtration using a HiLoad Superdex 200 PG 16 / 600 column, equilibrated with a 20 mM Tris-HCI buffer (pH 7.4) containing 100 mM NaCI.

[0058] HPLC conditions:

[0059] An Accucore-150-Amide-HILIC 2.6pm column (150x3 mm) was used at 25 °C with a flow-rate of 0.8 ml / min using acetonitrile-water as mobile phase in gradient elution mode. Eluted substrates and products were detected with CAD. The product was identified based on retention time comparison with the chemically synthesized Gaipi-4GlcNAcpi-6Gaipi-4Glc and LC-MS, and the concentration was calculated using a quadratic calibration curve with its intercept forced to zero. The calibration solutions were prepared using LNnT analytical standard.

[0060] The results are summarized in the table below.

[0061] I can be concluded that PlbaGH20 performed best, followed by PafeGH20 and PadiGH20. At low enzyme dosage, however, the highest yield was obtained with PadiGH20. In addition, PlbaGH20 also released more N-acetyllactosamine (by hydrolysis), especially at high enzyme dosage. This could point to PafeGH20 and PadiGH20 having a more convenient transglycosidase / hydrolysis ratio.

Claims

CLAIMS1. A method for synthesizing Gaipi-4GlcNAcpi-6Gal1-A-R comprising the step of reacting an N-acetyllactosaminyl donor and Gal1 -A-R as acceptor in the presence of an enzyme capable of transferring the N-acetyllactosaminyl residue of the donor to the acceptor, wherein the enzyme is a protein comprising an amino acid sequence disclosed as SEQ ID No. 1 , SEQ ID No. 2 or SEQ ID No. 3,A is selected from bond and a divalent carbohydrate linker, andR means -OH, -N3 or -OR’, wherein R’ is alkyl, aryl, a group removable by hydrogenolysis, allyl optionally substituted by one or more methyl, propargyl optionally substituted by one or methyl, 2-trimethylsilyl-ethyl, -(CH2)n-NH2or -(CH2)n-N3, wherein integer n is 1 to 10, preferably 2 or 3.

2. The method according to claim 2, wherein the enzyme is a protein comprising an amino acid sequence disclosed as SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6.

3. The method according to claim 1 or 2, wherein R is -OH.

4. The method according to any one of the precedent claims, wherein the acceptor is lactose and the product is Gaipi-4GlcNAcpi-6Gaipi-4Glc.

5. The method according to any one of the precedent claims, wherein the N- acetyllactosaminyl donor is a compound of formula 1 or 2wherein X is selected from the group consisting of a mono-, di- or oligosaccharide, azide, fluoro, optionally substituted phenoxy, optionally substituted pyridinyloxy, group A, group B, group C and group Dwherein Rais independently H or alkyl, or two vicinal Ragroups represent a =C(Rt>)2 group, wherein Rbis independently H or alkyl, Rcis independently selected from the group consisting of alkoxy, amino, alkylamino and dialkylamino, Rd is selected from the group consisting of H, alkyl and -C(=O)Re, wherein Reis OH, alkoxy, amino, alkylamino, dialkylamino, hydrazino, alkylhydrazino, dialkylhydrazino or trialkylhydrazino.

6. The method according to claim 5, wherein the donor is a compound of formula 1.

7. The method according to claim 6, wherein X is selected from the group consisting of a mono-, di- or oligosaccharide, phenoxy-, p-nitrophenoxy-, 2,4-dinitrophenoxy- and 2-chloro- 4-nitrophenoxy-group.

8. The method according to claim 7, wherein X is a galactose moiety meaning that the N- acetyllactosaminyl donor is Gaipi-4GlcNAcpi-4Gal.

9. The method according to claim 7, wherein X is lactose moiety meaning that the N- acetyllactosaminyl donor is LNnT.

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