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WO2013013244A2 - Chemoenzymatic synthesis of heparin and haparan sulfate analogs - Google Patents

Chemoenzymatic synthesis of heparin and haparan sulfate analogs Download PDF

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Publication number
WO2013013244A2
WO2013013244A2 PCT/US2012/047875 US2012047875W WO2013013244A2 WO 2013013244 A2 WO2013013244 A2 WO 2013013244A2 US 2012047875 W US2012047875 W US 2012047875W WO 2013013244 A2 WO2013013244 A2 WO 2013013244A2
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Prior art keywords
udp
glcnac
substituted
glca
unsubstituted
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PCT/US2012/047875
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French (fr)
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WO2013013244A3 (en
Inventor
Xi Chen
Hai Yu
Yanhong Li
Yi Chen
Jingyao QU
Musleh M. MUTHANA
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The Regents Of The University Of California
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Publication of WO2013013244A2 publication Critical patent/WO2013013244A2/en
Publication of WO2013013244A3 publication Critical patent/WO2013013244A3/en
Priority to US14/160,451 priority Critical patent/US9290530B2/en
Priority to US15/017,365 priority patent/US10160986B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • 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
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods 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
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • 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
    • C12P19/12—Disaccharides
    • 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
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • 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
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/305—Pyrimidine nucleotides
    • 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

Definitions

  • Heparin and heparan sulfate are sulfated linear polysaccharides composed of alternating al-4-linked D-glucosamine (GlcN3 ⁇ 4) residues and l ⁇ linked uronic acid (a- linkage for L-iduronic acid, IdoA, and ⁇ -linkage for D-glucuronic acid, GlcA).
  • Possible modifications are 2-O-sulfation on the uronic acid residues and one or more modifications on the glucosamine residues including N-sulfation, N-acetylation, 6-O-sulfation, and 3-0- sulfation.
  • Heparin is a mixture of polysaccharides that can be considered as special forms of HS with higher levels of sulfation and iduronic acid content per disaccharide repeat unit. Heparin is mostly produced by mast cells and heparan sulfates are produced by different cell types in animals. They are attractive synthetic targets due to their structural complexity which possesses great synthetic challenges and their important roles in regulating cancer growth, blood coagulation, inflammation, assisting viral and bacterial infections, signal transduction, lipid metabolism, and cell differentiation.
  • Heparin pentasaccharide sequence 3 ⁇ 4 also call DEFGH
  • GlcNS6S- GlcA-GlcNS3S6S-IdoA2S-GlcNS6S is essential for antithrombin III binding and thrombin inhibition activities.
  • a new potential antithrombotic, idraparinux was synthesized by replacing TV-sulfate groups in all three glucosamine residues of heparin pentasaccharide DEFGH with O-sulfates and introducing methyl ethers at the available free hydroxyl groups and showed better anticoagulation activity and longer duration of action than DEFGH.
  • Another pentasaccharide sequence HexA-GlcNS-HexA-GlcNS- ldoA2S has high affinity selectively for FGF-2 (fibroblast grow factor 2), while trisaccharide motif IdoA2S-GlcNS6S-IdoA2S is specific for FGF- 1 .
  • the invention provides a method of synthesizing a UDP- sugar.
  • the method includes forming a reaction mixture comprising a first sugar, a nucleotide- sugar pyrophosphorylase, and a first enzyme selected from a kinase and a dehydrogenase under conditions sufficient to form the UDP-sugar.
  • the invention provides a method of preparing an oligosaccharide.
  • the method includes forming a first reaction mixture containing a first sugar, an acceptor sugar, a glycosyltransferase, a nucleotide-sugar pyrophosphorylase, and an enzyme selected from a kinase and a dehydrogenase.
  • the first sugar is selected from a substituted or unsubstituted TV-acetylglucosamine (2-acetamido-2-deoxy glucose, GlcNAc), a substituted or unsubstituted glucosamine (GIcNH 2 ), a substituted or unsubstituted glucuronic acid (GlcA), a substituted or unsubstituted iduronic acid (IdoA), and a substituted or unsubstituted glucose- 1 -phosphate (Glc- l -P), and the acceptor sugar includes at least one member selected from a substituted or unsubstituted jV-acetylglucosamine (GlcNAc), a substituted or unsubstituted glucosamine (GlcNH 2 ), a substituted or unsubstituted glucuronic acid (GlcA), and a substituted or unsubstituted iduronic acid (IdoA).
  • the reaction mixture is formed under conditions sufficent to convert the first sugar to a UDP-sugar, and sufficient to couple the sugar in the UDP-sugar to the acceptor sugar.
  • the first sugar is substituted or unsubstituted GlcNAc or GlcNHh
  • the sugar in the UDP-sugar is coupled to a substituted or unsubstituted GlcA or a substituted or unsubstituted IdoA of the acceptor sugar.
  • the sugar in the UDP-sugar is coupled to a substituted or unsubstituted GlcNH? or a substituted or unsubstituted GlcNAc of the acceptor sugar.
  • Figure 1 shows a sequence alignment of NahK_.JCM 1 217 (GenBank accession no. BAF73925), NahK_ATCC55813, and NahK_ATCC 15697.
  • Figure 2 shows the pH profiles of NahK_ATCC 15697 ( ⁇ , filled diamond) and NahK ATCC55813 (0, open diamond). Buffers used: MES, pH 6.0; Tris-HCI, pH 7.0-9.0; CAPS, pH 10.0-1 1.0.
  • Figure 3 shows the effect of MgCla on the activity of NahKs.
  • Figure 4 shows the one-pot three-enzyme synthesis of UDP-GlcNAc and derivatives.
  • Enzyme used NahK_ATCC55813, an N-acetylhexosamine 1 -kinase cloned from Bifidobacterium longiim ATCC55813 ;
  • PmGlmU Pasteurella multocida N- acetylglucosamine-1 -phosphate uridylyltransferase;
  • PmPpA Pasteurella multocida inorganic pyrophosphatase.
  • Figure 5 shows the chemical diversification at (A) the C-2 of glucosamine and (B) the C-6 of N-acetylglucosamine in UDP-sugar nucleotides.
  • Reagents and conditions a) K 2 C0 3 , CH 3 OH, H 2 0, 20 °C, overnight, 98%; b) PyS0 3 , 2 M NaOH, H 2 0, overnight, 86%; c) RCOCl, NaHC0 3 , CH 3 CN, H 2 0; d) NaOMe, MeOH; e) H 2 , Pd/C, MeOH, H 2 0, 1 h, 96%.
  • Figure 6 shows the pH profile of Bifidobacterium longum UDP-sugar
  • BUSP pyrophosphorylase
  • Figure 7 shows the metal requirements of BLUSP.
  • Figure 8 shows the synthesis of UDP-ManNAc from UDP-ManN 3 in 79% yield via the formation of UDP-ManNH 2 by catalytic hydrogenation followed by acetylation.
  • Figure 9 shows a one-pot, three-enzyme system for the synthesis of UDP- monosaccharides and derivatives.
  • Figure 10 shows the one-pot multienzyme synthesis of UDP-glucuronic acid, UDP- iduronic acid, and UDP-galacturonic acid.
  • Figure 11 shows the results of the substrate specificity assay for the heparosan synthase activity of KfiA (Figure 11A) and PmHS2 ( Figure 11B). Each reaction was performed at 37 °C in MES buffer ( 1 00 mM, pH 6.5) for 30 min, 4 h or 16 h. Enzyme used: KfiA ( 1 .08 ⁇ g/ ⁇ L), PmHS2 (2.5 l O "2 ⁇ / ⁇ ).
  • Figure 12 shows the structures of the substrates tested in the substrate specificity assay for KfiA and PmHS2 in Figure 11.
  • Figure 13 shows the synthetic scheme for preparation of fluorescently labeled GlcA GlcAp2AAMe.
  • Figure 14 shows the synthesis of tetrasaccharides
  • Figure 15 shows the synthesis of GlcA-TEG-PABA-biotin (F15-8).
  • Figure 16 shows the one-pot four-enzyme synthesis of dissacharides with different modification on C2 and C6.
  • Enzymes used NahK_ATCC55813, N-acetylhexosamine 1 - kinase cloned from Bifidobacterium longum ATCC55813; PmGlmU, Pasteurella multocida N-acetylglucosamine-1 -phosphate uri-dylyltransferase; PmPpA, Pasteurella multocida inorganic pyrophosphatase; PmHS2, Pasteurella multocida heparosan synthase 2.
  • FIG 17 shows the structures of UDP-GlcNAc derivatives F17-1-F17-12 including UDP-GlcNAc (F17-1), UDP-GlcNTFA (F17-2), UDP-GlcNGc (F17-3), UDP- GlcNAcN 3 (F17-4), UDP-GlcNH 2 (F17-5), UDP-GlcN 3 (F17-6), UDP-GlcNS (F17-7), UDP- GlcNAc6N 3 (F17-8), UDp-GlcNAc6NGc (F17-9), UDP-GlcNAc6NH 2 (F17-10j), UDP- GlcNAc6NAcN 3 (F17-11), and UDP-GlcNAc6S (F17-12).
  • UDP-GlcNAc UDP-GlcNAc
  • F17-1 UDP-GlcNTFA
  • F17-3 UDP-GlcNGc
  • F17-4 UDP-Gl
  • Figure 18 shows the enzymatic synthesis of the disaccharides.
  • Figure 18A shows the one-pot four-enzyme system of the disaccharides GlcNAcccl -4GlcAp2AAMe (F18-1), GlcNTF Act 1 -4G IcA ⁇ 2 AAMe (F18-2), GlcNAc6N 3 al -4GlcA 2AAMe (F18-3).
  • Figure 18B shows the PmHS2-catalyzed synthesis of the disaccharides GlcNGcal -4GlcA 2AAMe (F18-4), GIcNAcN 3 al -4GlcAp2AAMe (F18-5), GlcNAc6NGcal -4GlcAp2AAMe (F18-6).
  • Figure 19 shows the enzymatic synthesis of trisaccharides from disaccharides via in situ generation of UDP-GIcA from Glc- l -P catalyzed by Echerichia coli glucose- 1 -phosphate uridylyltransferase (EcGalU), Pasteurella multocida UDP-glucose dehydrogenase (PmUgd), and PmHS2.
  • EcGalU Echerichia coli glucose- 1 -phosphate uridylyltransferase
  • PmUgd Pasteurella multocida UDP-glucose dehydrogenase
  • PmHS2 Pasteurella multocida UDP-glucose dehydrogenase
  • Figure 20 shows the one-pot three-enzyme synthesis of trisaccharides GlcA l - 4GlcNAcct l -4GlcAP2AAMe (F20-1), GlcApi -4GlcNTFActl -4GlcAP2AAMe (F20-2), GlcA i -4GlcNAc6N 3 a l -4GlcAp2AAMe (F20-3), GIcA i -4GlcNGca l -4GlcA 2AAMe (F20-4), GlcApi -4GlcNAcN 3 al -4GlcAp2AAMe (F20-5), GlcA i -4GlcNAc6NGca l - 4GlcAp2AAMe (F20-6).
  • Figure 21 shows the one-pot four-enzyme synthesis of tetrasaccharide
  • FIG. 22 shows the synthesis of tetrasaccharides G lcN Ac6N 3 Ct 1 -4G lc ⁇ 1 - 4GlcNH 2 al -4GlcA 2AA (F22-1), GlcNAc6N 3 al -4GlcApi-4GlcNSal -4GlcAp2AA (F22- 2), GlcNAc6NH 2 l -4GlcApl -4GlcNSal -4GlcAp2AA (F22-3), GlcNAc6NSal -4GlcAp i - 4GlcNSal -4GlcAp2AA (F22-4) from GlcNAc6N 3 al -4GlcApl-4GlcNTFAal - 4GlcAp2AAMe (F21-1) by chemical modifications.
  • Reagents and conditions (a) 2 CO3, H 2 0, r.t. overnight, 81%; (b) PyS0 3 , 2 M NaOH, H 2 0, 3d, 70%; (c) H 2 , Pd/C, MeOH, H 2 0, 1 h.
  • Figure 23 shows the inhibitory activities of LMWH or compounds F24-1-F24-16 (see Figure 24 for structures) against the binding of human fibroblast growth factors FGF- 1 ( Figure 23A), FGF-2 ( Figure 23B), or FGF-4 ( Figure 23C) to the heparin-biotin immobilized on NeutrAvidin-coated 384-weIl plates. Samples without LMWH or monosaccharide/tetrasaccharide inhibitors were used as positive controls (P.C.).
  • Figure 24 shows structures of compounds F24-1-F24-16 used in Figure 23 for inhibition studies of the binding of human fibroblast growth factors FGF- 1 , FGF-2, and FGF- 4 to the heparin-biotin immobilized on NeutrAvidin-coated 384-well plates.
  • FIG. 25 shows thin-layer chromatography (TLC) analysis data for AtGlcA reactions.
  • Developing solvent used for running TLC: «-Pr0H:H 2 0:NH 4 0H 7:4:2 (by volume).
  • Figure 26 shows LC-MS assay data for AtGlcA -catalyzed synthesis of siigar- 1 - phosphate from sugar and ATP.
  • Figure 26A AtGlcAK kinase reaction using GlcA as the starting sugar
  • Figure 26B AtGlcAK kinase reaction using GalA as the starting sugar
  • Figure 26C AtGlcAK kinase reaction using IdoA as the starting sugar.
  • Figure 27 shows pH profiles of Kfi A (Figure 27A) and PmHS2 ( Figure 27B).
  • Figure 28 shows metal effects on the heparosan synthase activity of KfiA ( Figure 28A) and PmHS2 ( Figure 28B).
  • Figure 29 shows high-resolution mass spectrometry (Orbitrap HRMS) assay for the synthesis of UDP- GlcNAc3N 3 from GlcNAc3N 3 , ATP, and UTP using one-pot three- enzyme reactions containing NahK, PmGlmU, and PmPpA.
  • Orbitrap HRMS high-resolution mass spectrometry
  • Figure 30 shows LC-MS or high resolution mass spectrometry (Orbitrap HRMS) assay for the synthesis of UDP-sugars from sugar, ATP, and UTP using one-pot three- enzyme reactions containing AtGlcAK, BLUSP, and PmPpA.
  • Figure 30A LC-MS assay and GlcA was used as the starting sugar
  • Figure 30B HRMS assay and GalA was used as the starting sugar
  • Figure 30C HRMS assay and IdoA was used as the starting sugar.
  • Figure 31 shows thin-layer chromatograph analysis of PmHS2-catalyzed reaction for the formation of GlcA-GlcNAc disaccharide derivatives.
  • Figure 32 shows LC-MS analysis of PmHS2-catalyzed reaction for the formation of GlcA-GlcNAc disaccharide derivatives.
  • Figure 32A GlcNAc 2AA was used as the acceptor;
  • Figure 32B GlcNAcpMU was used as the acceptor;
  • Figure 32C GlcNAcaProN 3 was used as the acceptor;
  • Figure 32D Glc AcpProN 3 was used as the acceptor.
  • the present invention provides a convenient and highly efficient one-pot multienzyme system for the synthesis of UDP-sugars and oligosaccharides including heparin and heparosan sulfate (HS) analogs.
  • Kinases or dehydrogenases, nucleotide-sugar pyrophosphorylases, and/or glycosyltransferases are used in one-pot reactions to convert monosaccharide precursors to UDP-sugars and/or oligosaccharides.
  • Chemical diversification of the enzymatically formed UDP-sugars and oligosaccharides can be conducted to produce more structural variations.
  • non-sulfated oligosaccharides can be selectively modified to prepare structurally defined products with desired sulfation patterns.
  • a diverse set of enzymatic substrates can be used in the methods of the invention to prepare a wide range of useful UDP-sugars and oligosaccharides.
  • first sugar refers to a monosaccharide starting material used in the methods of the invention.
  • the monosaccharide can be a hexose or a
  • Pentose.Hexoses include, but are not limited to, glucose (Glc), glucosamine (2-amino-2- deoxy-glucose; GlcNH 2 ), N-acetylglucosamine (2-acetamido-2-deoxy-glucose; GlcNAc), galactose (Gal), galactosamine (2-amino-2-deoxy-galactose; GalNH?), N-acetylgalactosamine (2-acetamido-2-deoxy-galactose; GalNAc), mannose (Man), mannosamine (2-amino-2- deoxy-mannose; ManNH 2 ), .V-acetylmannosamine (2-acetamido-2-deoxy-mannose;
  • ManNAc ManNAc
  • glucuronic acid GlcA
  • iduronic acid IdoA
  • galacturonic acid GalA
  • Pentoses include, but are not limited to, ribose (Rib), xylose (Xyl), and arabinose (Arb).
  • the sugar can be a D sugar or an L sugar.
  • the sugar can be unsubstituted or substituted with moieties including, but not limited to, amino groups, azido groups, amido groups, acylamido groups, 7V-sulfate groups (sulfamate), and O-sulfate groups.
  • a "second sugar” and subsequent sugars are generally defined as for the first sugar, except that they are used after the first sugar in a multi-step synthesis.
  • UDP-sugar refers to a sugar containing a uridine diphosphate moiety.
  • the sugar portion of the UDP-sugar is defined as for the "first sugar” described above.
  • UDP-sugars include, but are not limited to UDP-Glc, UDP-GlcNAc, UDP- GlcNH 2 , UDP-GlcA, UDP-ldoA, UDP-GalA, UDP-Gal, UDP-GalNAc, UDP-GalNH 2 , UDP- Man, UDP-ManNAc, and UDP-ManNH 2 .
  • the UDP-sugar can be unsubstituted or substituted as described above.
  • oligosaccharide refers to a compound containing at least two sugars covalently linked together. Oligosaccharides include disaccharides, trisaccharides, tetrasachharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, and the like. Covalent linkages generally consist of glycosidic linkages (i.e., C-O-C bonds) formed from the hydroxy I groups of adjacent sugars.
  • Linkages can occur between the 1 - carbon and the 4-carbon of adjacent sugars (i.e., a 1 -4 linkage), the 1 -carbon and the 3-carbon of adjacent sugars (i.e. , a 1 -3 linkage), the 1 -carbon and the 6-carbon of adjacent sugars (i. e., a 1 -6 linkage), or the 1 -carbon and the 2-carbon of adjacent sugars (i.e. , a 1 -2 linkage).
  • a sugar can be linked within an oligosaccharide such that the anomeric carbon is in the a- or ⁇ - configuration.
  • the oligosaccharides prepared according to the methods of the invention can also include linkages between carbon atoms other than the 1 -, 2-, 3-, 4-, and 6-carbons.
  • the term "enzyme” refers to a polypeptide that catalyzes the transformation of a starting material, such as a sugar, to an intermediate or product of the one-pot reactions of the invention.
  • enzymes include, but are not limited to, kinases, dehydrogenases, nucleotide-sugar pyrophosphorylases, pyrophosphatases, and glycosyltransferases. Other enzymes may be useful in the methods of the invention.
  • kinase refers to a polypeptide that catalyzes the covalent addition of a phosphate group to a substrate.
  • the substrate for a kinase used in the methods of the invention is generally a sugar as defined above, and a phosphate group is added to the anomeric carbon (i. e. the " 1 " position) of the sugar.
  • the product of the reaction is a sugar- 1 - phosphate.
  • Kinases include, but are not limited to, N-acetylhexosamine 1 -kinases (NahKs), glucuronokinases (GlcAKs), glucokinases (GlcKs), galactokinases (GalKs), monosaccharide- 1 -kinases, and xylulokinases.
  • Certain kinases utilize nucleotide triphosphates, including adenosine-5 '-triphosphate (ATP) as substrates.
  • dehydrogenase refers to a polypeptide that catalyzes the oxidation of a primary alcohol.
  • the dehyrogenases used in the methods of the invention convert the hydroxymethyl group of a hexose (i.e. the C6-OH moiety) to a carboxylic acid.
  • Dehydrogenases useful in the methods of the invention include, but are not limited to, UDP-glucose dehydrogenases (Ugds).
  • nucleotide-sugar pyrophosphorylase refers to a polypeptide that catalyzes the conversion of a sugar- 1 -phosphate to a UDP-sugar. In general, a uridine-5'-monophosphate moiety is transferred from uridine- 5 '-triphosphate to the sugar-1 - phosphate to form the UDP-sugar.
  • nucleotide-sugar pyrophosphorylases include glucosamine uridylyltransferases (GlmUs) and glucose- 1 -phosphate uridylyltransferases (GalUs).
  • Nucleotide-sugar pyrophosphorylases also include promiscuous UDP-sugar pyrophosphorylases, termed "USPs," that can catalyze the conversion of various sugar- 1 - phosphates to UDP-sugars including UDP-Glc, UDP-GlcNAc, UDP-GlcNH 2 , UDP-Gal, UDP-GalNAc, UDP-GalNH 2 , UDP-Man, UDP-ManNAc, UDP-ManNH 2 , UDP-GlcA, UDP- IdoA, UDP-GalA, and their substituted analogs.
  • UDP-Glc promiscuous UDP-sugar pyrophosphorylases
  • pyrophosphatase refers to a polypeptide that catalyzes the conversion of pyrophosphate (i.e. , P 2 0 7 4 , ⁇ 2 0 " , H 2 P 2 0 7 , 3 ⁇ 4 ⁇ 2 ⁇ 7 " ) to two molar equivalents of inorganic phosphate (i.e., PO 4 3" , HPO4 2" , H2PO4 " ).
  • glycosyltransferase refers to a polypeptide that catalyzes the formation of an oligosaccharide from a UDP-sugar and an acceptor sugar.
  • a glycosyltransferase catalyzes the transfer of the monosaccharide moiety of the UDP-sugar to a hydroxyl group of the acceptor sugar.
  • the covalent linkage between the monosaccharide and the acceptor sugar can be a 1-4 linkage, a 1 -4 linkage, a 1 -6-linkage, or a 1 -2 linkage as described above.
  • the linkage may be in the a- or ⁇ -configuration with respect to the anomeric carbon of the monosaccharide.
  • Other types of linkages may be formed by the glycosyltransferases in the methods of the invention.
  • Glycosyltransferases include, but are not limited to, heparosan synthases (HSs) glucosaminyltransferases, N- acetylglucosaminyltransferases, glucosyltransferasess, glucuronyltransferases.
  • HSs heparosan synthases
  • the term "couple” refers to catalyzing the formation of a covalent bond between enzyme substrates.
  • the coupling can take place via the direct reaction of two substrates with each other.
  • the coupling can include the formation of one or more enzyme-substrate intermediates.
  • An enzyme-substrate intermediate can, in turn, react with another substrate (or another enzyme-substrate intermediate) to form the bond between the substrates.
  • UDP-sugars can be synthesized according to the methods of the invention.
  • the UDP-sugars have structures according to Formula I:
  • each of R 1 , R 2 , and R 3 is independently selected from OH, N 3 , NH 2 , NHS0 3 ⁇ OSO3 " , NHC(0)CH 3 , NHC(0)CF 3 , NHC(0)CH 2 OH, and NHC(0)CH 2 N 3 ; and R 4 is selected from CH 2 OH, C0 2 " , C0 2 H, CH 2 N 3 , CH 2 NH 2 , CH 2 NHS0 3 ⁇ CH 2 OS0 3 " , CH 2 NHC(0)CH 3 , CH 2 NHC(0)CF 3 , CH 2 NHC(0)CH 2 OH, and CH 2 NHC(0)CH 2 N 3 .
  • the UDP-sugars have structures according to formula I UDP (la) [0054]
  • a range of oligosaccharides can also be prepared using the methods of the invention.
  • the oligosaccharides contain one or more unit according to Formula II:
  • each of R la , R lb , R 2a , and R 2b is independently selected from OH, N 3 , NH 2 , NHS0 3 " , OS0 3 ⁇ NHC(0)CH 3 , NHC(0)CF 3 ,
  • each of R lc and R 2c is independently selected from CH 2 OH, C0 2 ⁇ C0 2 H, CH 2 N 3 , CH 2 NH 2 , CH 2 NHS0 3 ⁇ , CH 2 0S0 3 ⁇ CH 2 NHC(0)CH 3 , CH 2 NHC(0)CF 3 , CH 2 NHC(0)CH 2 OH, or CH 2 NHC(0)CH 2 N 3 .
  • one of R l c and R 2c can be C0 2 " or C0 2 H, while the other of R lc and R 2c can be CH 2 OH, CH 2 N 3 , CH 2 NH 2 , CH 2 NHS0 3 ⁇ , CH 2 OS0 3 , CH 2 NHC(0)CH 3 , CH 2 NHC(0)CF 3i
  • R includes but not is limited to H, CH 3 , CH 2 CH 3 , CH2CH2N3, CH 2 CH 2 CH 2 N 3 , an aglycon according to Formula B, Formula C, Formula D, or Formula E below, substituted or unsubstituted GlcNAc, substituted or
  • the oligosaccharides have the structure of formula lla:
  • the method provides oligosaccharides with structures according to Formula III: wherein each of R la , R l b , and R 2a is independently selected from OH, N 3 , NH 2 , NHS0 3 ⁇ OS0 3 " , NHC(0)CH 3 , NHC(0)CF 3 , NHC(0)CH 2 OH, or NHC(0)CH 2 N 3 ; and R lc is selected from CH 2 OH, CH 2 N 3 , CH 2 NH 2 , CH 2 NHS0 3 " , CH 2 0S0 3 ⁇ CH 2 NHC(0)CH 3 ,
  • the method provides oligosaccharides with structures according to Formula IV:
  • each of R l , R 2a , and R 2b is independently selected from OH, N 3 , NH 2 , NHS0 3 ⁇ OS0 3 ⁇ NHC(0)CH 3 , NHC(0)CF 3 , NHC(0)CH 2 OH, and NHC(0)CH 2 N 3 ; and R 2c is selected from CH 2 OH, CH 2 N 3 , CH 2 NH 2 , CH 2 NHS0 3 " , CH 2 0S0 3 ⁇ CH 2 NHC(0)CH 3 ,
  • CH 2 NHC(0)CF 3 CH 2 NHC(0)CH 2 0H, or CH 2 NHC(0)CH 2 N 3 .
  • the present invention provides oligosaccharides having the structure of formula IVa:
  • the method provides oligosaccharides with structures according to Formula (V):
  • each of R la , R 2a , R 2b , and R 3a is independently selected from OH, N 3 , NH 2 , NHS0 3 " , OS0 3 " , NHC(0)CH 3 , NHC(0)CF 3 , NHC(0)CH 2 OH, and NHC(0)CH 2 N 3 ; and R 2c is selected from CH 2 OH, CH 2 N 3 , CH 2 NH 2 , CH 2 NHS0 3 " , CH2OSO3 " , CH 2 NIIC(0)CH 3 ,
  • the present invention provides oligosaccharides having a structure of formula Va:
  • the method provides oligosaccharides with structures according to Formula VI:
  • each of R la , R 2a , R 2b , R 3a , R b , and R 4b is independently selected from OH, N 3 , NH 2 , NHS0 3 " , OSO 3 " , NHC(0)CH 3 , NHC(0)CF 3 , NHC(0)CH 2 OH, or NHC(0)CH 2 N 3 ; and each of R 2c , R 4c is independently selected from CH 2 OH, CH 2 N 3 , CH 2 NH 2 , CH 2 NHS0 3 " , CH 2 0S0 3 " , CH 2 NHC(0)CH 3 , CH 2 NHC(0)CF 3 , CH 2 NHC(0)CH 2 0H, or
  • the oligosaccharides has the structure of formula Via:
  • the invention provides a method of synthesizing a UDP- sugar.
  • the method includes forming a reaction mixture comprising a first sugar, a nucleotide- sugar pyrophosphorylase, and a first enzyme selected from a kinase and a dehydrogenase under conditions sufficient to form the UDP-sugar.
  • the first sugar is selected from substituted or unsubstituted glucose (Glc), substituted or unsubstituted glucose- 1 -phosphate (Glc- l -P), substituted or unsubstituted glucuronic acid (GlcA), substituted or unsubstituted glucuronic acid-1 - phosphate (GlcA- l -P), substituted or unsubstituted iduronic acid (IdoA), substituted or unsubstituted iduronic acid- 1 -phosphate (IdoA- l -P), substituted or unsubstituted N- acetylglucosamine (GlcNAc), substituted or unsubstituted vV-acetylglucosain ine- 1 -phosphate (Glc Ac- l -P), substituted or unsubstituted glucosamine (GIcN3 ⁇ 4), substituted or
  • Linsubstituted glucosamine-1 -phosphate (GlcNH 2 -l -P), substituted or unsubstituted galactose (Gal), substituted or unsubstituted galactose- 1 -phosphate (Gal- l -P), substituted or unsubstituted galacturonic acid (GalA), substituted or unsubstituted galacturonic acid-1 - phosphate (GalA- l -P), substituted or unsubstituted N-acetylgalactosamine (GalNAc), substituted or unsubstituted ⁇ -acetylgalactosamine- 1 -phosphate (GalNAc- 1 -P), substituted or unsubstituted galactosamine (GalNH 2 ), substituted or unsubstituted galactosamine- l - phosphate (GalNH l -P), substituted or unsubstituted man
  • the first sugar has the formula VII:
  • each of R 1 , R 2 , and R 3 is selected from OH, N 3 , NH 2 , NHSOy, OS0 3 ⁇ NHC(0)CH 3 ,
  • NHC(0)CF 3 NHC(0)CH 2 OH, and NHC(0)CH 2 N 3 ;
  • R 4 is selected from CH 2 OH, C0 2 " ,
  • the first sugar has the formula VIII or IX: (or OP0 3 H-) (1X) .
  • the reaction mixture formed in the methods of the invention contains a nucleotide-sugar pyrophosphorylase.
  • the nucleotide-sugar pyrophosporylase can be, but is not limited to, a glucosamine uridyltransferase (GlmU), a Glc- l -P uridylyltransferase (GalU), or a promiscuous UDP-sugar pyrophosphorylase (USP).
  • GlmU glucosamine uridyltransferase
  • AlU Glc- l -P uridylyltransferase
  • USP promiscuous UDP-sugar pyrophosphorylase
  • the present inventors have cloned and characterized a GlmU from P. muJtocida (PmGlmU) that is useful for the synthesis of UDP-sugars according to the methods of the invention.
  • Suitable GalUs can be obtained, for example, from yeasts such as Saccharomyces fragilis, pigeon livers, mammalian livers such as bovine liver, Gram-positive bacteria such as Bifidobacterium bifidum, and Gram-negative bacteria such as Echerichia coli (EcGalU) (Chen X, Fang JW, Zhang JB, Liu ZY, Shao J, owal P, Andreana P, and Wang PG. J. Am. chem. Soc. 2001 , 123, 2081 -2082).
  • the nucleotide-sugar pyrophosporylase is a USP.
  • USPs include, but are not limited to, those obtained from Pisum sativum L. (PsUSP) and Arabidopsis thaliana (AtUSP), as well as enzymes obtained from protozoan parasites (such as Leishmania major and Trypanosoma cruzi) and hyperthermophilic archaea (such as Pyrococcus furiosus DSM 3638). USPs also include human UDP-GalNAc pyrophosphorylase AGX l , E. coli EcGlmU, and Bifidobacterium longum BLUSP. BLUSP was cloned and characterized by the inventors.
  • the nucleotide-sugar pyrophosphorylase is selected from AGX l , EcGlmU, EcGalU, PmGlmU, and BLUSP. In some embodiments, the nucleotide-sugar pyrophosphorylase is selected from EcGalU, PmGlmU, and BLUSP. In some embodiments, the nucleotide-sugar pyrophosphorylase is EcGalU. In some embodiments, the nucleotide- sugar pyrophosphorylase is PmGlmU. In some embodiments, the nucleotide-sugar pyrophosphorylase is BLUSP.
  • the reaction mixture formed in the methods of the invention also contains a kinase or a dehydrogenase.
  • the first enzyme in the reaction mixture is a kinase.
  • the kinase can be, but is not limited to, an N-acctylhexosamine 1 -kinase (NahK), a galactokinase (GalK), or a glucuronokinase (GlcA ).
  • the kinase is an NahK.
  • the NahK can be, for example, Bifidobacterium infantis NahK_ATCC 15697 or Bifidobacterium longum NahK_ATCC55813.
  • the kinase is a GalK.
  • the GalK can be, for example, Escherichia coli EcGalK (Chen X, Fang JW, Zhang JB, Liu ZY, Shao J, Kowal P, Andreana P, and Wang PG. J. Am. chem. Soc.
  • Streptococcus pneumoniae TIGR4 SpGalK (Chen M, Chen LL, Zou Y, Xue M, Liang M, Jing L, Guan WY, Shen J, Wang W, Wang L, Liu J, and Wang PG. Carbohydr. Res. 201 1 , 346, 2421 -2425).
  • the UDP-sugar is a substituted or unsubstituted UDP-GlcA.
  • the first sugar employed in the synthesis of UDP-GlcA may vary depending on the enzymes that are used in the one-pot reaction.
  • Glc- l -P can be converted to UDP-Glc using a UDP-sugar pyrophosporylase.
  • UDP-GlcA can be obtained from UDP-Glc using a dehydrogenase.
  • the reaction mixture in some embodiments of the invention includes a dehydrogenase.
  • the dehydrogenase can be, but is not limited to, a UDP-glucose dehydrogenase (Ugd).
  • Ugd UDP-glucose dehydrogenase
  • the dehydrogenase is Pasteur ella multocida PmUgd.
  • the PmUgd was cloned and characterized by the inventors.
  • GlcA can be converted to GlcA- l -P using a GlcAK.
  • the kinase in the reaction mixture is a GlcAK.
  • the GlcAK can be, for example, Arabidopsis thaliana AtGlcAK.
  • the GlcA-l -P is then converted to UDP-GlcA by a UDP-sugar pyrophosphorylase such as Arabidopsis thaliana AtUSP.
  • UDP-sugar pyrophosphorylase such as Arabidopsis thaliana AtUSP.
  • the AtGlcAK was cloned and characterized by the inventors.Other sugars, including iduronic acid (IdoA) and galacturonic acid (GalA), can also be used as substrates for GlcAKs in the methods of the invention.
  • UDP-sugars can be synthesized using the methods of the invention.
  • the UDP-sugar is selected from substituted or unsubstituted UDP-Glc, substituted or unsubstituted UDP-GlcA, substituted or unsubstituted UDP-IdoA, substituted or unsubstituted UDP-GalA, substituted or unsubstituted UDP-GlcNAc, substituted or unsubstituted UDP-GlcNFL, substituted or unsubstituted UDP-Gal, substituted or unsubstituted UDP-GalNAc, substituted or unsubstituted UDP-GalNH 2 , substituted or unsubstituted UDP-Man, substituted or unsubstituted UDP-ManNAc, and substituted or unsubstituted UDP-ManN3 ⁇ 4,.
  • the UPD-sugar is selected from UDP- GlcNAc, UDP-GlcNH 2 , UDP-GlcA, UDP-IdoA, UDP-GalA, UDP-Gal, UDP-Man, and UDP-Glc.
  • the UDP-sugar can also have the structure of formula I described above.
  • the hydroxyl groups, the amino group, and the iV-acetyl amino group in UDP-sugar can be substituted with any suitable substituent.
  • the hydroxyl groups, the amino group, and the V-acetyl amino group in UDP-sugar can be substituted with an azide, an amine, an iV-trifluoroacetyl group, an N-acyl group, an ⁇ -sulfate, or an N-sulfate.
  • the reaction mixture formed in the methods of the invention can further include an inorganic pyrophosphatase (PpA).
  • PpAs can catalyze the degradation of the pyrophosphate (PPi) that is formed during the conversion of a sugar- 1 -phosphate to a UDP-sugar. PPi degradation in this manner can drive the reaction towards the formation of the UDP-sugar products.
  • the pyrophosphatase can be, but is not limited to, Pasteiirella multocida PmPpA (Lau K, Thon V, Yu H, Ding L, Chen Y, Muthana MM, Wong D, Huang R, and Chen X. Chem. Commun. 2010, 46, 6066-6068).
  • the reaction mixture in the present methods can be formed under any conditions sufficient to convert the first sugar to a UDP-sugar or an intermediate such as a sugar- 1 - phosphate.
  • the reaction mixture can include, for example, buffering agents to maintain a desired pH, as well as salts and/or detergents to adjust the solubility of the enzymes or other reaction components.
  • the reaction mixture also includes one or more nucleotide triphosphates (NTPs), such as UTP or ATP, that are consumed during sugar phosphorylation and UDP-sugar formation.
  • NTPs nucleotide triphosphates
  • the reaction mixture can contain a stoichiometric amount of an NTP, with respect to the first sugar, or an excess of the NTP.
  • Divalent metal ions such as magnesium ions, manganese ions, cobalt ions, or calcium ions, may be required to maintain the catalytic activity of certain enzymes.
  • Enzyme cofactors including but not limited to nicotinamide adenine dinucleotide (NAD " ), can also be included in the reaction mixture.
  • the reaction mixture further includes at least one component selected from UTP, ATP, Mn ⁇ , Co" . Ca” , and Mg" .
  • the reaction mixture is held under conditions that allow for the conversion of the first sugar to the U DP sugar. For example, the reaction mixture can be held at 37 °C for 1 min-72 hr to form the UDP-sugar.
  • the reaction mixture can also be held at 25 °C to form the UDP-sugar. Other temperatures and conditions may be suitable for forming the UDP-sugar, depending on the nature of the first sugar and the enzymes used for the synthesis. [0074] In some embodiments, the invention provides a method of synthesizing a UDP- sugar of Formula I:
  • the method includes forming a reaction mixture comprising a first sugar, a nucleotide-sugar pyrophosphorylase, and a first enzyme selected from the group consisting of a kinase and a dehydrogenase under conditions sufficient to form the UDP-sugar.
  • the first sugar has the formula VII:
  • each of R 1 , R 2 , and R 3 is selected from OH, N 3 , NH 2 , NHS0 3 ⁇ OS0 3 ⁇ NHC(0)CH 3 , NHC(0)CF 3 , NHC(0)CH 2 OH, and NHC(0)CH 2 N 3 ;
  • R 4 is selected from CH 2 OH, C0 2 " , C0 2 H, CH 2 N 3 , CH 2 NH 2 , CH 2 NHS0 3 " , CH 2 OS0 3 " , CH 2 NHC(0)CH 3 , CH 2 NHC(0)CF 3 , CH 2 NHC(0)CH 2 OH, and CH 2 NHC(0)CH 2 N 3 ; and
  • R 5 can be H, P0 3 2 or HP0 3 " .
  • Certain enzymes that are useful in the methods of the invention are characterized by a level of substrate promiscuity that allows for the synthesis of various natural and non- natural UDP-sugars.
  • the scope of the products can be widened further by chemically appending a range of functionality to common enzymatically synthesized UDP-sugars.
  • a UDP-sugar containing an azido moiety, for example, can be reduced to form an amino moiety which can be further elaborated via amide bond formation or TV-sulfation to install various functional groups in the UDP-sugar.
  • trifluoracctamido moieties can also be converted to amino moieties for further derivitization.
  • some embodiments of the invention include converting a UDP- azido-sugar or a UDP- trifluoroacetamido-sugar to a UDP-amino-sugar.
  • the UDP amino-sugar is further converted to a UDP-acylamido-sugar or a UDP-TV-sulfated-sugar.
  • some embodiments of the invention provide a method of preparing an oligosaccharide.
  • the method includes forming a first reaction mixture containing a first sugar, an acceptor sugar, a glycosyltransferase, a nucleotide-sugar pyrophosphorylase, and an enzyme selected from a kinase and a dehydrogenase.
  • the first sugar is selected from a substituted or unsubstituted vV-acetylglucosamine (2-acetamido-2- deoxy glucose, GlcNAc), a substituted or unsubstituted glucosamine (GlcNH 2 ), a substituted or unsubstituted glucuronic acid (GlcA), a substituted or unsubstituted iduronic acid (IdoA), and a substituted or unsubstituted glucose- 1 -phosphate (Glc- l -P), and the acceptor sugar includes at least one member selected from a substituted or unsubstituted N- acetylglucosamine (GlcNAc), a substituted or unsubstituted glucosamine (GlcNH 2 ), a substituted or unsubstituted glucuronic acid (GlcA), and a substituted or unsubstituted iduronic acid (Ido
  • the reaction mixture is formed under conditions sufficent to convert the first sugar to a UDP-sugar, and sufficient to couple the sugar in the UDP-sugar to the acceptor sugar.
  • the first sugar is substituted or unsubstituted GlcNAc or GlcNHi
  • the sugar in the UDP-sugar is coupled to a substituted or unsubstituted GlcA or a substituted or unsubstituted IdoA of the acceptor sugar.
  • the sugar in the UDP-sugar is coupled to a substituted or unsubstituted GlcNH 2 or a substituted or unsubstituted GlcNAc of the acceptor sugar.
  • the first sugar has the formula:
  • each of PJ , R 2 , and R 3 is selected from OH, N 3 , NH 2 , NHS0 3 ⁇ OS0 3 ⁇ NHC(0)CH 3 , NHC(0)CF 3 , NHC(0)CH 2 OH, and NHC(0)CH 2 N 3 ;
  • R 4 is selected from CH 2 OH, C0 2 ⁇ C0 2 H, CH 2 N 3 , CH 2 NH 2 , CH 2 NHS0 3 ⁇ CH 2 OS0 3 ⁇ CH 2 NHC(0)CH 3 , CH 2 NHC(0)CF 3 , CH 2 NHC(0)CH 2 OH, and CH 2 NHC(0)CH 2 N 3 ;
  • R 5 can be H, P0 3 2" , or HP0 3 " .
  • the first sugar has the formula VIII or IX: (or OP0 3 H ) (I X)
  • the first sugar is converted to the UDP-sugar by the UDP-sugar pyrophosphorylase and the kinase/dehydrogenase as described above.
  • the first sugar is selected from substituted or unsubstituted glucose (Glc), substituted or unsubstituted glucose- 1-phosphate (Glc-l -P), substituted or unsubstituted glucuronic acid (GlcA), substituted or unsubstituted iduronic acid (IdoA), substituted or unsubstituted glucuronic acid- 1 -phosphate (GlcA- l -P), substituted or unsubstituted iduronic acid (IdoA), substituted or unsubstituted iduronic acid- 1 -phosphate (IdoA- l -P), substituted or unsubstituted jV-acetylglucosamine (GlcNAc), substituted or un
  • the sugar in the UDP-sugar is, in turn, coupled to an acceptor sugar to form an oligosaccharide product.
  • an acceptor sugar can be used as the acceptor sugar.
  • the acceptor sugar can be a monosaccharide, a disaccharide, a tri saccharide, or a tetrasaccharide. Longer oligosaccharides may also be used as the acceptor sugar in the methods of the invention.
  • the oligosaccharide can be a compound of Formula II, III, IV, V, or VI.
  • the sugar in a UDP-sugar is coupled to an acceptor sugar by the glycosyltransferase in the reaction mixture.
  • Any suitable glycosyltransferase can be used in the methods of the invention.
  • Certain glycosyltransfers have exhibited a level of substrate promiscuity that are particularly useful for preparing a variety of oligosaccharide products.
  • Promiscuous glycosyltransferases can utilize a range of UDP-sugars and/or a range of acceptor sugars.
  • the glycosyltransferase can be, for example, P. multocida PmHS l or
  • glycosyltransferase can also be E. coli KfiA or KfiC. Other glycosyltransferases can also be useful in the methods of the invention. In some embodiments, the
  • glycosyltransferase is selected from PmHS l , PmHS2 and KfiA.
  • the UDP-sugar can be formed enzymatically in the one-pot reaction mixture as described above.
  • the nucleotide-sugar pyrophosporylase can be, but is not limited to, a glucosamine uridyltransferase (GlmU), a Glc- l -P uridylyltransferase (GalU), or a promiscuous UDP-sugar pyrophosphorylase (USP).
  • the nucleotide- sugar pyrophosphorylase is selected from AGX 1 , EcGlmU, EcGalU, PmGlmU, and BLUSP. In some embodiments, the nucleotide-sugar pyrophosphorylase is selected from AGX 1 , EcGalU, and BLUSP. In some embodiments, the nucleotide-sugar pyrophosphorylase is selected from EcGalU, PmGlmU, and BLUSP. In some embodiments, the nucleotide-sugar pyrophosphorylase is EcGalU. In some embodiments, the nucleotide-sugar
  • the pyrophosphorylase is PmGlmU.
  • the nucleotide-sugar is PmGlmU.
  • pyrophosphorylase is BLUSP.
  • the kinase in the reaction mixture is selected from an N- acetylhexosamine 1 -kinase (NahK), a galactokinase (GalK), and a glucuronokinase (GlcAK).
  • NahK N- acetylhexosamine 1 -kinase
  • GaK galactokinase
  • GlcAK glucuronokinase
  • the kinase is selected from NahK_ATCC 15697, NahK_ ATCC55813, EcGalK, SpGalK, and AtGlcAK.
  • the kinase is selected from
  • the kinase is selected from NahK ATCC 1 5697, NahK_ATCC5581 3, and AtGlcAK. In some embodiments, the kinase is EcGalK. In some embodiments, the kinase is
  • the kinase is NahK_ATCC 15697. In some embodiments, the kinase is NahK_ATCC55813. In some embodiments, the kinase is AtGlcAK. In some embodiments, the kinase is
  • the dehydrogenase in the reaction mixture is UDP-glucose dehydrogenase (Ugd). In some embodiments, the Ugd is PmUgd.
  • the UDP-sugar formed in the one-pot reaction mixture is selected from substituted or unsubstituted UDP-GlcNAc, substituted or unsubstituted UDP- Glc, substituted or unsubstituted UDP-GlcA, and substituted or unsubstituted UDP-IdoA.
  • the UDP-sugar is substituted with at least one moiety selected from an azide, an amine, an TV-trifluoroacetyl group, an N-acylamido group, an ( -sulfate, and an N- sulfate.
  • the reaction m ixture further contains a pyrophosphatase.
  • the pyrophosphatase is PinPpA.
  • the reaction mixture in the present methods can be formed under any suitable conditions sufficient to prepare an oligosaccharide.
  • the reaction mixture can include, for example, buffering agents to maintain a desired pH, as well as salts and/or detergents to adjust the solubility of the enzymes or other reaction components.
  • the reaction mixture also includes one or more nucleotide triphosphates (NTPs), such as UTP or ATP, that are consumed during sugar phosphorylation and UDP-sugar formation.
  • NTPs nucleotide triphosphates
  • the reaction mixture can contain a stoichiometric amount of an NTP, with respect to the first sugar, or an excess of the NTP.
  • Divalent metal ions such as magnesium ions, manganese ions, cobalt ions, or calcium ions, may be required to maintain the catalytic activity of certain enzymes.
  • Enzyme cofactors including but not limited to nicotinamide adenine dinucleotide (NAD + ), can also be included in the reaction mixture.
  • the reaction mixture further includes at least one component selected from UTP, ATP, Mn 2+ , Co 2+ , Ca 2+ , and Mg 2+ .
  • the reaction mixture After the reaction mixture is formed, it is held under conditions that allow for preparation of the oligosaccharide.
  • the reaction mixture can be held at 37 °C for 1 min-72 hr.
  • the reaction mixture can also be held at 25 °C.
  • Other temperatures and conditions may be suitable for forming the oligosaccharide, depending on the nature of the sugars and the enzymes used for the synthesis.
  • Heparin and heparan sulfate (HS) oligosaccharides have particularly important biological, pathological, and therapeutic properties.
  • Heparin and HS are sulfated linear polysaccharides composed of alternating a 1 -4 linked D-glucosamine (GlcNH ) residues and 1 -4 linked uronic acid resiudes (a-linkage for iduronic acid, IdoA, and ⁇ -linkage for glucuronic acid, GlcA].
  • the methods of the invention can be used to prepare oligosaccharides containing alternating glucosamine and uronic acid residues.
  • the oligosaccharides can contain, for example, alternating GlcNAc residues and GlcA residues.
  • the oligosaccharide is selected from: GlcNAc-GlcA; GlcA-GlcNAc-GlcA; GlcNAc-GlcA- GlcNAc-GlcA; GlcA-GlcNAc-GlcA-GlcNAc-GlcA; GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc- GlcA; GlcA-GlcNAc-GlcA-GlcNAc-GlcA; GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA; GlcNAc-GlcA-GlcNAc-GlcA- GlcNAc-GlcA; GlcNAc-G
  • GlcA-GlcNAc-GlcA-GlcNAc GlcA-GlcNAc-GlcA-GlcNAc; GlcNAc-GlcA- GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc; GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA- GlcNAc; GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc; and GlcA- GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GIcNAc-GlcA-GlcNAc.
  • each GlcA and GlcNAc are optionally independently mono- or multi-substituted with a moiety selected from an azide, an amine, an -trifluoroacetyl group, an N-acyl group, and an N-sulfate.
  • oligosaccharides can also be prepared using the methods of the invention. Oligosaccharides of arbitrary length can be prepared by repeating the one-pot reaction methods as described above. Accordingly, some embodiments of the invention provide a method for preparing an oligosaccharide as described above, wherein the method is repeated with a second sugar in place of the first sugar and the oligosaccharide in place of the acceptor sugar. In this manner, a variety of products can be prepared.
  • the oligosaccharides of the present invention can be a compound of any of Formulas II, III, IV,
  • the present invention provides a method of prepar oligosaccharide of formula II:
  • the method includes forming a first reaction mixture containing a first sugar, an acceptor sugar, a glycosyltransferase, a UDP-sugar pyrophosphorylase, and/or one enzyme selected from a kinase and a dehydrogenase.
  • the first sugar is selected from a substituted or unsubstituted iV-acetylglucosamine (GlcNAc), a substituted or unsubstituted glucosamine (GlcNH 2 ), a substituted or unsubstituted glucoronic acid (GlcA), a substituted or unsubstituted iduronic acid (IdoA), and a substituted or unsubstituted glucose-l -phosphate (Glc- l -P), and the acceptor sugar includes at least one member selected from a substituted or unsubstituted iV-acetylglucosamine (GlcNAc), a substituted or unsubstituted glucosamine (GlcNHb), a substituted or unsubstituted glucuronic acid (GlcA), and substituted or unsubstituted iduronic acid (IdoA).
  • GlcNAc substituted or unsub
  • the reaction mixture is formed under conditions sufficent to convert the first sugar to a UDP-sugar having a structure of formula I : UDP (I), and sufficient to couple the sugar in the UDP-sugar to the acceptor sugar.
  • the first sugar can have a structure of the formula VII:
  • R 1 , R 2 , R 3 , R l a , R l b , R 2a , and R 2b is independently selected from OH, N 3 , NH 2 , NHS0 3 " , OSO3 " , NHC(0)CH 3 , NHC(0)CF 3 , NHC(0)CH 2 OH, or NHC(0)CH 2 N 3 ; each of R 4 , R l c , and R 2c is independently selected from CH 2 OH, C0 2 ⁇ C0 2 H, CH 2 N 3 , CH 2 NH 2 , CH 2 NHS0 3 CH2OSO3 " , CH 2 NHC(0)CH 3 , CH 2 i HC(0)CF 3 , CH 2 NHC(0)CH 2 OH, or CH 2 NHC(0)CH 2 N 3 ; R includes but not is limited to H, CH 3 , CH 2 CH 3 , CH 2 CH 2 N 3 ,
  • R 4 is C0 2 " or C0 2 H
  • R 2c is CO, " or C0 2 H
  • R lc is selected from CH 2 OH, CH 2 N 3 , CH 2 NH 2 , CH 2 NHS0 3 ⁇ CH 2 OS0 3 " , CH 2 NHC(0)CH 3 , CH 2 NHC(0)CF 3 , CH 2 NHC(0)CH 2 OH, and
  • R l c is C0 2 " or C0 2 H, and R 2c is R 4 .
  • Heparin and HS generally contain varying levels of sulfated sugar residues.
  • sulfated sugar residues include, but are not limited to, GlcNS, containing an N- sulfate at the 2 position of glucosamine (GICNH2); GlcNS3S, containing an TV-sulfate at the 2 position and an O-sulfate at the 3 position of glucosamine (GlcNH 2 ); GlcNS6S, containing an N-sulfate at the 2 position and an O-sulfate at the 6 position of glucosamine (GICNH2);
  • GlcNS3S6S containing an /-sulfate at the 2 position, an O-sulfate at the 3 position, and an O-sulfate at the 6 position of glucosamine (GlcNH 2 ); GlcNAc3S, containing an O-sulfate at the 3 position of N-acetylglucosamine (GlcNAc); GlcNAc6S, containing an O-sulfate at the 6 position of N-acetylglucosamine (GlcNAc); GlcNAc3S6S, containing an O-sulfate at the 3 position and an -sulfate at the 6 position of ./V-acetylglucosamine (GlcNAc); GlcNH 2 3S, containing an O-sulfate at the 3 position of glucosamine (GICNH2); GlcNH 2 6S, containing an -sulfate at the 6 position of glucos
  • the present inventors have discovered enzymes that exhibit catalytic activity for a number of natural and non-natural UDP-sugar and acceptor sugar substrates.
  • the oligosaccharides that are prepared using these enzymes can contain functional moieties that can be chemically modified to diversify the structure of the products. For example, azido- sugar residues or trifluoroacetamido-sugar residues can be converted to amino-sugar residues.
  • Azido groups and trifluoracetamos groups can be manipulated independently using orthogonal chemical methods to selectively install desired functionality at specific sites on a given oligosaccharide.
  • Amine-containing ol igosaccharides can be further elaborated to form acylamino groups and sulfamate groups.
  • Sulfamate (i.e. ⁇ -sulfate) groups in particular, can be instal led to form heparin and HS analogs.
  • oligosaccharides containing N-sulfate groups demonstrate inhibitory activity aganst the binding of fibroblast growth factors (FGFs) to heparin.
  • FGFs fibroblast growth factors
  • the invention provides convenient and flexible methods for preparation of oligosaccharides with useful biological activity.
  • NahK (EC 2.7. 1.162) catalyzes the direct addition of a phosphate from adenosine 5'- triphophate (ATP) to the anomeric position of ZV-acetylhexosamine for the formation of N- acetylhexosamine- 1 -phosphate and adenosine 5'-diphophate (ADP).
  • ATP adenosine 5'- triphophate
  • ADP adenosine 5'-diphophate
  • Electrocompetent DH5a and chemically competent BL21 (DE3) E. coli cells were from Invitrogen (Carlsbad, CA).
  • Bifidobacterium longum Reuter ATCC#55813 was from American Type Culture Collection (ATCC, Manassas, VA). Genomic DNA of Bifidobacterium longum subsp. infantis
  • Vector plasm id pET22b(+) was from Novagen (EMD Biosciences Inc. Madison, WI).
  • Ni 2+ - NTA agarose nickel-nitrilotriacetic acid agarose
  • QIAprcp spin miniprep kit was from Qiagen (Valencia, CA).
  • Herculase-enhanced DNA polymerase was from Stratagene (La Jolla, CA).
  • T4 DNA ligase and 1 kb DNA ladder were from Promega (Madison, WI).
  • Ndel and Xho ⁇ restriction enzymes were from New England Biolabs Inc. (Beverly, MA).
  • Adenosine-5'-triphosphate disodium salt (ATP), GlcNAc, and GalNAc were from Sigma (St. Louis, MO).
  • GlcNAc, GalNAc, mannose, and ManNAc derivatives were synthesized according to reported procedures.
  • NahK_ATCC 15697 and ahK ATCC55813 were each cloned as a C- His 6 -tagged fusion protein in pET22b(+) vector using genomic DNAs of Bifidobacterium longum subsp. infantis ATCC#15697 and Bifidobacterium longum ATCC#55813, respectively, as the template for polymerase chain reactions (PCR).
  • the primers used for NahK ATCC 15697 were: forward primer 5 '
  • PCR was performed in a 50 ⁇ ⁇ reaction mixture containing genomic DNA ( 1 ⁇ g), forward and reverse primers (1 ⁇ each), 10 x Herculase buffer (5 ⁇ _.), dNTP mixture (1 mM), and 5 U ( 1 ⁇ ) of Herculase-enhanced DNA polymerase.
  • the reaction mixture was subjected to 35 cycles of amplification with an annealing temperature of 52 °C.
  • the resulting PCR product was purified and digested with Ndel and Xhol restriction enzymes.
  • the purified and digested PCR product was ligated with predigested pET22b(+) vector and transformed into electrocompetent E. colt DH5oc cells. Selected clones were grown for minipreps and characterization by restriction mapping and DNA sequencing performed by Davis Sequencing Facility at the University of California-Davis.
  • the cell pellet was re-suspended in lysis buffer (pH 8.0, 100 mM Tris-HCl containing 0.1 % Triton X-100, 20 mL/L cell culture) containing lysozyme (100 ⁇ g/mL) and DNasel (3 ⁇ g/mL). After incubating at 37 °C for 60 min with vigorous shaking (250 rpm), the lysate was collected by centrifugation at 12,000 g for 30 min. His 6 -tagged target proteins were purified from cell lysate using an AKTA FPLC system (GE Healthcare, Piscataway, NJ, USA).
  • lysis buffer pH 8.0, 100 mM Tris-HCl containing 0.1 % Triton X-100, 20 mL/L cell culture
  • lysozyme 100 ⁇ g/mL
  • DNasel 3 ⁇ g/mL
  • the lysate was loaded to a HisTrapTM FF 5 mL column (GE Healthcare) pre-washed and equilibrated with binding buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 7.5). The column was then washed with 8 volumes of binding buffer, 10 volumes of washing buffer (10 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5) and eluted with 8 volumes of e lute- buffer (200 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5). Fractions containing the purified enzyme were combined and dialyzed against dialysis buffer (Tris-HCl containing 10% glycerol, pH 7.5, 20 mM) and stored at 4 °C.
  • binding buffer 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5
  • pH Profile by capillary electrophoresis (CE) assays were performed in a 20 ⁇ L ⁇ reaction mixture containing a buffer (200 mM) with a pH in the range of 6.0-1 1.0, GlcNAc ( 1 mM), ATP ( 1 mM), MgCl 2 (5 mM), and a NahK (0.75 ⁇ ). Buffers used were: MES, pH 6.0; Tris-HCl, pH 7.0-9.0; CAPS, pH 10.0-1 1 .0. Reactions were allowed to proceed for 10 min at 37 °C and were stopped by adding 20 of cold ethanol to each reaction mixture.
  • CE Capillary Electrophoresis
  • PDA Photodiode Array
  • Apparent kinetic parameters were obtained by varying the ATP concentration from 0.1 -5.0 mM (0.1 mM, 0.2 mM, 0.4 mM, 1 mM, 2 mM, and 5 mM) at a fixed concentration of GlcNAc or GalNAc (1 mM), or varying the concentration of GlcNAc or Gal Ac (0.1 mM, 0.2 mM, 0.4 mM, 1 mM, 2 mM, and 5 mM) at a fixed concentration of ATP ( 1 mM) and fitting the data to the Michaelis-Menten equation using Grafit 5.0.
  • NahK_ATCC55813 were each cloned as a C-His 6 -tagged fusion protein in a pET22b(+) vector.
  • Sequence alignment indicates that NahK_ATCC55813 is almost identical to the NahK from Bifidobacterium longum JCM 1217 (NahK_JCM 1217, GenBank accession no. BAF73925) except for a single amino acid difference R348H (R is in NahK_JCM 1217).
  • NahK_ATCC 15697 shares 90% amino acid sequence identity with
  • NahKs were expressed by induction with 0. 1 mM of isopropyl- 1 -thio-P-D- galactopyranoside (IPTG) followed by incubation at 20 °C for 24 h with vigorous shaking (250 rpm). Up to 180 mg and 1 85 mg of Ni 2+ -column purified NahK_ATCC 15697 and NahK_ATCC55813, respectively, could be obtained from one l iter of E. coli culture.
  • IPTG isopropyl- 1 -thio-P-D- galactopyranoside
  • Capillary electrophoresis (CE) assays Based on the detection of ADP and ATP in the reaction mixture by a UV detector, a capillary electrophoresis-based method was developed to directly measure the formation of ADP and N-acetylhexosamine-1 -phosphate from ATP and N-acetylhexosamine for characterizing the activities of NahKs. Both ATP and ADP gave absorbance at 254 nra with equal signal responses.
  • the pH optima of these two enzymes are slight different from that (pH 8.5) of NahK_JCM121 7.
  • the activity of NahK_ATCC55813 is higher than that of NahK_ATCC l 5697 in the pH range of 6.0-10.0 when GlcNAc was used as the substrate and the same molar concentrations of the enzymes were used.
  • NahK_ATCC 15697 and NahK_ATCC55813 require a divalent metal ion for activity.
  • the optimal concentration of Mg 2+ was determined to be 1 mM.
  • the activities of both NahKs in the presence of 0.5 mM of Mg 2+ were about two thirds of those in the presence of 1 .0 mM of Mg 2+ .
  • Increasing the concentration of Mg 2+ from 1 mM to 20 mM caused a slight decrease of the activities of both NahKs.
  • the other substrate is GlcNAc:
  • the other substrate is GalNAc.
  • NahK_ATCC55813 is more reactive towards non-modified GlcNAc (T2-1), GalNAc (T2-11), and some of their C2-modified derivatives with an N-trifluoroacetyl (GlcNTFA T2-2 and GalNTFA T2-12), an N-azidoacetyl group (GlcNAcN 3 T2-3 and GalNAcN 3 T2-13), or an N-butanoyl group (GlcNBu T2-4 and GalNBu T2-14).
  • GlcNAc N-trifluoroacetyl
  • GalNAcN 3 T2-3 and GalNAcN 3 T2-13 an N-azidoacetyl group
  • GlcNBu T2-4 and GalNBu T2-14 N-butanoyl group
  • NahK ATCC 15697 is more reactive than NahK_ATCC55813 for some of C2- modified GlcNAc and GalNAc derivatives such as those with a bulky N-benzoyl group (GlcNBz T2-5 and GalNBz T2-15) and a C2-azido group (GlcN 3 T2-6 and GalN 3 T2-16).
  • C2- modified GlcNAc and GalNAc derivatives such as those with a bulky N-benzoyl group (GlcNBz T2-5 and GalNBz T2-15) and a C2-azido group (GlcN 3 T2-6 and GalN 3 T2-16).
  • NahK_ATCC 1 5697 is also more reactive towards 2-amino-2-deoxy-glucose (GlcNH 2 T2-7), 2-N-sulfo-glucose (GlcNS T2-8), as well as C6-modified GlcNAc derivatives such as 6- deoxy-GlcNAc (GlcNAc6Me T2-9), 6-azido-6-deoxy-GlcNAc (GlcNAc6N 3 T2-10), and 6- 6>-sulfo-GlcNAc (GlcNAc6S T2-17).
  • GlcNAc such as 6-O-sulfo-N-trifluoroacetyl glucosamine (GlcNTFA6S T2-18) and 6-0-sulfo-2-azido-2- deoxy glucose (GlcN 3 T2-19) as well as both C2 and C3-modified GlcNAc derivative 3-0- sulfo-2-azido-2-deoxy glucose (GlcN 3 3 S T2-20) are poor but acceptable substrates for both enzymes.
  • GlcNAc6N 3 NA not assayed; a Reactions were allowed to proceed for 10 min at 37 °C; b Reactions were allowed to proceed for 30 min at 37 °C.
  • mannose (T3-23), its 2-fluoro- (2F-Man T3- 24) and 2-azido- (2N 3 -Man T3-26) derivatives, as well as its 4-deoxy (4-deoxyMan T3-27) derivative are relatively good substrates.
  • 2-methyl modification of mannose (2Me-Man T3-25) decreases its tolerance as the substrate for both NahKs.
  • ManNAc T3-29 and some of its C-2 derivatives (T3-30-T3-32) are poor substrates for the NahKs
  • N-azidoacetylmannosamiiie ManNAcN 3 T3-33, a C2-derivative of ManNAc
  • ManNAc60Me T3-34 C6-derivative N-acetyl-6-O-methylmannosamine
  • NahK_ATCC 15697 shows higher activity than NahK_ATCC55813 for mannose, ManNAc, and their derivatives.
  • AtGIcAK - Arabidopsis thaliana glucuronokinase (EC 2.7.1.43)
  • E. coli BL21 (DE3) chemically competent cells for protein expression.
  • E. coli cells harboring the pETl 5b-AtGlcAK plasmid were cultured in LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) with ampicillin (100 ⁇ g mL) at 37 °C with rigorous shaking at 250 rpm in a C25KC incubator shaker (New Brunswick Scientific, Edison, NJ) until the OD600 nm of the culture reached 0.8-1.0.
  • His ⁇ 5-tagged protein was purified from cell lysate using Ni 2+ -NTA affinity column. To obtain cell lysate, cells were harvested by centrifugation at 4,000 rpm (Sorvall) at 4 °C for 2 h. The cell pellet was resuspended in lysis buffer (pH 8.0, 100 mM Tris-HCl containing 0.1 % Triton X-100). Lysozyme ( 100 ⁇ /ITlL) and DNasel (5 g/mL) were added to the cell suspension. The mixture was incubated at 37 °C for 1 hr with vigorous shaking (200 rpm).
  • Cell lysate was obtained as the supernatant by centrifugation at 1 1 ,000 rpm (Sorvall) at 4 °C for 45 min. Purification was performed by loading the supernatant onto a Ni 2+ -NTA column pre-equilibrated with 10 column volumes of binding buffer (10 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was wash with 10 column volumes of binding buffer and 10 column volumes of washing buffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5).
  • Protein of interest was eluted with Tris-HCl (pH 7.5, 50 mM) containing imidazole (200 mM) and NaCl (0.5 M). The fractions containing the purified enzyme were collected and dialyzed against Tris-HCl buffer (pH 7.5, 20 mM) containing 30% glycerol. Dialyzed proteins were stored at -20 °C. Alternatively, fractions containing purified enzyme were dialyzed against Tris-HCl buffer (pH 7.5, 20 mM) and freeze dried. On average, 57 mg of purified protein was obtained from 1 l iter of cell culture.
  • LC-MS assays for AtGlcAK reactions were also analyzed by LC-MS. 2 ⁇ L ⁇ of sample was diluted 100 fold and 8 ⁇ ⁇ was injected into a Waters spherisorb ODS-2 column (5 ⁇ particles, 250 mm length, 4.6 mm I.D.). The sample was eluted with 30 % acetonitrile in 3 ⁇ 40 with 0.1 % formic acid and detected by ESI- MS in negative mode.
  • PmGimU Pasteurella multocida glucosaminyl uridyltransferase
  • glycosyltransferases are key enzymes for the formation of oligosaccharides and glycoconjugates in nature. Most glycosyltransferases require sugar nucleotides as donor substrates and catalyze the transfer of monosaccharides from sugar nucleotides to acceptors in high regio- and stereoselective manner. Some carbohydrate structures contain post- glycosylational modifications (modifications on carbohydrates and glycoconjugates which take place after the formation of glycosidic bonds).
  • One strategy to obtain naturally existing oligosaccharides and glycoconjugates with modified sugar moieties is to develop novel chemoeiizymatic methods using structurally modified monosaccharides as starting materials and carbohydrate biosynthetic enzymes (the simplest carbohydrate biosynthetic route usually involves a monosaccharide kinase, a nucleotidyltransferase, and a glycosyltransferase) with substrate promiscuities.
  • carbohydrate biosynthetic route usually involves a monosaccharide kinase, a nucleotidyltransferase, and a glycosyltransferase
  • Carbohydrates with non-natural modifications can be synthesized similarly. Some of these compounds are potential drug candidates as they can effectively interfere with carbohydrate-dependent biological processes.
  • Glycosaminoglycans including keratan sulfate, heparan sulfate, and heparin are N- acetylglucosamine (GlcNAc)-containing polysaccharides with post-glycosylational modifications. While GlcNAc and 6-(9-sulfo-GlcNAc are commonly found in kearatan sulfate, additional modified GlcNAc forms such as N-sulfo- and 3-0-sulfo-GlcNAc are common for heparan sulfate and heparin.
  • GlcNAc N- acetylglucosamine
  • UDP- GlcNAc derivatives including UDP-N-suIfo-glucosamine, were also produced by chemical diversification from enzymatica!ly produced UDP-GlcNAc derivatives. These compounds will be tested as potential donor substrates for GlcNAc-glycosyltransferases.
  • Pasteurella multocida subsp. ultocida strain Pm70 was used as a reference for designing primers.
  • the genomic DNA of Pasteurella multocida strain P-1059 was used as a template for polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • uridylyltransferase (PmGlmU) was cloned in pETl 5b and pET22b(+) vectors as N-His 6 - and C-HiS(5 -tagged fusion proteins, respectively.
  • the primers used were: forward primer 5' GATCCATATG
  • E. coli BL21 (DE3) chemically competent cells.
  • E. coli cells harboring the pETl 5b-PmGlmU or pET22b(+)-PmGlmU plasmid were cultured in LB medium (10 g L tryptone, 5 g/L yeast extract, and 10 g/L NaCI) with ampicillin (100 ⁇ g mL) until the ODeoo nm of the culture reached 0.8-1 .0.
  • the cell pellet was resuspended in lysis buffer (pH 8.0, 100 mM Tris-HCl containing 0.1 % Triton X- 100). Lysozyme (100 ⁇ g mL) and DNasel (5 ⁇ g/mL) were then added to the cell suspension. The mixture was incubated at 37 °C for 1 hr with vigorous shaking (200 rpm). Cell lysate was obtained as the supernatant by centrifugation at 1 1 ,000 rpm (Sorvall) at 4 °C for 45 min.
  • lysis buffer pH 8.0, 100 mM Tris-HCl containing 0.1 % Triton X- 100.
  • Lysozyme 100 ⁇ g mL
  • DNasel 5 ⁇ g/mL
  • Purification is performed by loading the supernatant onto a Ni 2+ -NTA column pre-equilibrated with 10 column volumes of binding buffer ( 10 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was wash with 10 column volumes of binding buffer and 10 column volumes of washing buffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). Protein of interest was eluted with Tris-HCl (pH 7.5, 50 mM) containing imidazole (200 mM) and NaCl (0.5 M). The fractions containing the purified enzymes were collected and dialyzed against Tris-HCl (pH 7.5, 25 mM) buffer containing 10% glycerol. Dialyzed proteins were stored at 4 °C. Results and Discussion
  • glycosyltransferases Most glycosyltransferases require monosaccharide nucleotides as the common activated donor substrates. Among monosaccharide nucleotides used by mammalian glycosyltransferases, many are uridine 5'-diphosphate (UDP)-monosaccharides such as UDP- glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-glucuronic acid (UDP-GlcA), UDP-N- acetylglucosamine (UDP-GlcNAc), UDP-jV-acetylgalactosamine (UDP-GalNAc), and UDP- xylose (UDP-Xyl). In addition, UDP-mannose (UDP-Man) has been isolated from UDP-Glc), UDP-galactose (UDP-Gal), UDP-glucuronic acid (UDP-GlcA), UDP
  • UDP-/V-acetylmannosamine UDP-ManNAc
  • UDP-N-acetylmannosaminuronic acid UDP-ManNAcA
  • UDP-ManNAcA UDP-N-acetylmannosaminuronic acid
  • the simplest biosynthetic route for obtaining monosaccharide nucleotides such as UDP-monosaccharides usually involves the formation of a monosaccharide- ] -phosphate catalyzed by a monosaccharide- 1 -phosphate kinase followed by the formation of monosaccharide nucleotides catalyzed by a nucleotidyltransferase (or pyrophosphorylase).
  • UDP-Gal used in galactosyltransferase-catalyzed enzymatic synthesis of galactosides has been more frequently obtained from UDP-Glc by reactions catalyzed by UDP-Gal 4-epimerases or UDP-glucose:galactose- l -phosphate uridylyltransferases (EC 2.7.7.12, GalT or GalPUT) in the Leloir pathway.
  • UDP-galactose pyrophosphorylase activity was identified from yeast Saccharomyces fragilis, pigeon liver, and mammalian livers. The enzyme was purified from bovine liver and Gram-positive bacterium Bifidobacterium bifidum. Recently, promiscuous UDP-sugar pyrophosphoryiases (USPs) (EC 2.7.7.64) that can use various monosaccharide 1 - phosphates in the presence of UTP for direct synthesis of UDP-monosaccharides including UDP-Glc, UDP-Gal, and UDP-GlcA, etc.
  • USPs promiscuous UDP-sugar pyrophosphoryiases
  • Trypanosoma cr zi two trypanosomatid protozoan parasites, and were shown to have good activity towards Gal- l -P and Glc- l -P and weaker activity towards xylose- 1 -phosphate and GlcA- l -P.
  • a USP with broad substrate specificity and optimal activity at 99°C was also cloned from a hyperthermophile archaea Pyrococcus furiosus DSM 3638 for which Glc- l -P, Man- l -P, Gal-l -P, Fuc-l-P, GlcNH 2 -l -P, GalNH 2 -l-P, and GlcNAc- l -P were all shown to be tolerable substrate, and both UTP and dTTP could be used as nucleotide triphosphate substrates by the enzyme. Nevertheless, none of these enzymes has been used in preparative- scale or large-scale synthesis of sugar nucleotides and non-natural derivatives of monosaccharide-1 -P have not been tested as substrates for USPs.
  • BLUSP Full length Bifidobacterium longum UDP-sugar pyrophosphorylase (EC 2.7.7.64) (BLUSP) (encoded by gene ugpA, DNA GenBank accession number: ACHIOI OOO I 19, locus tag: HMPREF0175_1671 ; protein GenBank accession number: EEI80102) was cloned from the genomic DNA of
  • Bifidobacterium longum strain ATCC55813 in pET15b vector as an N-His 6 -tagged fusion protein The primers used were: forward primer 5'
  • coli cells harboring the pET15b-BLUSP plasmid were cultured in LB medium (1 0 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) with ampicillin ( 100 ⁇ g/mL) at 37 °C with rigorous shaking at 250 rpm in a C25 C incubator shaker (New Brunswick Scientific, Edison, NJ) until the ODgoo nm of the culture reached 0.8-1 .0.
  • His6-tagged protein was purified from cell lysate using Ni 2 ' -NTA affinity column. To obtain cell lysate, cells were harvested by centrifugation at 4,000 rpm (Sorvall) at 4 °C for 2 hr. The cell pellet was resuspended in lysis buffer (pH 8.0, 100 mM Tris-HCl containing 0.1 % Triton X- 100). Lysozyme ( 100 ⁇ g/mL) and DNasel (5 ⁇ g/mL) were added to the cell suspension. The mixture was incubated at 37 °C for 1 hr with vigorous shaking (200 rpm).
  • Cell lysate was obtained as the supernatant by centrifugation at 1 1 ,000 rpm (Sorvall) at 4 °C for 45 min. Purification was performed by loading the supernatant onto a Ni 2+ -NTA column pre-equilibrated w ith 1 0 column volumes of binding buffer (10 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was wash with 1 0 column volumes of binding buffer and 1 0 column volumes of washing buffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5).
  • Protein of interest was eluted with Tris-HCl (pH 7.5, 50 mM) containing imidazole (200 mM) and NaCl (0.5 M). The fractions containing the purified enzyme were collected and dialyzed against Tris-HCl buffer (pH 7.5, 25 mM) containing 10% glycerol and 0.25 M NaCl. Dialyzed proteins were stored at 4 °C. Alternatively, fractions containing purified enzyme were dialyzed against Tris-HCl buffer (pH 7.5, 25 mM) and freeze dried. On average, 167 mg of purified protein was obtained from 1 liter of cell culture. Protein concentration was determined in a 96-well plate using bicinchoninic acid with BSA as standard. The absorbance was measured at 562 nm using a plate reader.
  • pH profile study for BLUSP Typical enzymatic assays for pH profile studies were carried out for 10 min at 37 °C in a total volume of 20 ⁇ ⁇ containing Glc-l -P ( 1 mM), UTP ( 1 mM), Mg 2+ (20 mM), and BLUSP (10 ng) in a buffer ( 100 mM) with pH varying from 3.0 to 9.5. The reaction mixture was quenched by boiling for 5 min followed by adding 20 ih of pre-chilled 95% (v/v) ethanol.
  • SDS-PAGE analysis of BLUSP shows that the recombinant BLUSP has a very good expression level in E. coli and has a high solubility. It consists of about 90% of the total protein extracts from E. coli host cells and more than 90% of the soluble protein. The protein size observed is about 60 kDa which is close to 59.7 kDa calculated molecular weight.
  • BLUSP pH profile of BLUSP. As shown in Figure 6, BLUSP is active in a broad pH range of 4.0-8.0 and with optimal activity at pH 6.5 in MES buffer.
  • PmUgd was cloned as a C-Hise-tagged fusion protein in pET22b(+) vector using the genomic DNA of P. multocida P-1059 (ATCC# 15742) as the template for polymerase chain reactions (PCR). Primers used for cloning were: forward primer 5'-GATCCATATGAAGAAAATTACAATTGCTGGGGC-3 ' (Ndel restriction site is underlined) and reverse primer 5'- CCGCTCGAGAGCATCACCGCCAAAAATATCTCTTG-3 ' (Xhol restriction site is underlined).
  • PCR was performed in a reaction mixture of 50 ⁇ containing genomic DNA (1 ⁇ g), forward and reverse primers (1 ⁇ each), l OxHerculase buffer (5 ⁇ ), dNTP mixture ( 1 mM), and 5 U (1 ⁇ ) of Herculase-enhanced DNA polymerase.
  • the reaction mixture was subjected to 30 cycles of amplification with an annealing temperature of 55 °C.
  • the resulting PCR products were purified, digested, and ligated with the corresponding pre-digested vector.
  • the ligation products were transformed into electrocompetent E. coli DH5a cells. Plasmids containing the target genes as confirmed by DNA sequencing (performed by UC-Davis Sequencing Facility) were selected and transformed into E. coli BL21 (DE3) chemically competent cells.
  • the obtained gene of PmUgd has 19 base differences (A357G, C381 A, A390G, A397C, C404A, A406G, T408A, C414T, A420T, A426G, C430T, G438A, C447A, T451 C, C453T, T456C, A464T, C582T, and G807A, the nucleotide before the number is from the DNA sequence of PM0776, the number is based on PM0776 gene) compared to publically available PM0776 gene sequence.
  • PmUgd 127K, N 133H, LI 371, Y 151 H and Y 155F, the amino acid residue before the number is from the protein sequence deduced from PM0776, the number is based on the protein sequence deduced from PM0776) compared to the deduced protein sequence from PM0776 gene.
  • E. coli strains were cultured in LB rich medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g L NaCl) supplemented with ampicillin ( 100 ⁇ g mL).
  • Over-expression of PmUgd was achieved by inducing the E. co/ BL21 (DE3) cell culture with 0.1 mM of isopropyl- 1 -thio-p-D-galactopyranoside (IPTG) when the OD 6 oo nm of the culture reached 0.8-1.0 followed by incubation at 20 °C for 20 h.
  • IPTG isopropyl- 1 -thio-p-D-galactopyranoside
  • Bacterial cells were harvested by centrifugation at 4 °C in a Sorvall Legend RT centrifuge with a hanging bucket rotor at 4000 x rpm for 2 h.
  • Harvested cells were resuspended in lysis buffer (Tris-HCl buffer, 100 mM, pH 8.0 containing 0.1 % Triton X-100) (20 mL for cells collected from one liter cell culture).
  • Lysozyme 100 ⁇ g/mL
  • DNasel 5 ⁇ g mL
  • Cell lysate (supernatant) was obtained by centrifugation at 12000 x rpm for 15 min. Purification was carried out by loading the supernatant onto a Ni 2+ - NTA column pre-equilibrated with 8 column volumes of binding buffer (10 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was washed with 8 column volumes of binding buffer and 8 column volumes of washing buffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The target protein was eluted with Tris-HCl buffer (50 mM, pH 7.5) containing imidazole (200 mM) and NaCl (0.5 M). The fractions containing the purified enzymes were collected and dialyzed against Tris-HCl buffer (20 mM, pH 7.5) containing 10% glycerol. Dialyzed proteins were stored at 4 °C.
  • E. coli electrocompetent DH5 and chemically competent BL21 (DE3) cells were from Invitrogen (Carlsbad, CA).
  • P. miiltocida P-934 (ATCC# 12948) and P. nmltocida P- 1059 (ATCC# 1 5742) were from American Type Culture Collection (ATCC, Manassas, VA, USA).
  • fiA synthetic gene with codons optimized for E. coli expression was synthesized by GeneArt (Grand Island, NY) based on KfiA gene sequence from E. coli N issle 191 7 (GenBank accession number: AJ586888, ORF79).
  • Vector plasmid pFTl 5b was from Novagen (EMD Biosciences Inc. Madison, WI, USA).
  • Vector pMAL-c4X was purchased from New England Biolabs (Ipswich, MA).
  • Nickel-nitrilotriacetic acid agarose Ni 2+ -NTA agarose
  • QIAprep spin miniprep kit Q1AEX II gel extraction kit
  • Herculase-enhanced DNA polymerase was from Stratagene (La Jolla, CA, USA).
  • T4 DNA ligase and 1 kb DNA ladder were from Promega (Madison, WI, USA). Ndel, BamHl, EcoRl, and Hindl
  • restriction enzymes were from New England Biolabs Inc. (Beverly, MA, USA).
  • PmHSl, PmHS2 and KfiA were cloned as N- and C-His 6 - tagged fusion proteins in pET15b and pET22b(+) vector, respectively, using genomic DNAs of P. m ltocida P-l 059 (ATCC# 15742) as the template for polymerase chain reactions (PCR).
  • PmHS l and KfiA were cloned as a fusion protein of an N-terminal with a maltose- binding protein (MBP) and a C-terminal Hise tag in pMAL-c4X vector using the P.
  • MBP maltose- binding protein
  • PCR was performed in a reaction mixture of 50 containing genomic DNA (1 ⁇ g), forward and reverse primers (1 ⁇ each), 10* Herculase buffer (5 ⁇ ), dNTP mixture ( 1 mM), and 5 U ( 1 ⁇ ) of Herculase-enhanced DNA
  • the reaction mixture was subjected to 30 cycles of amplification with an annealing temperature of 55 °C (for PmHSl and PmHS2) or 52 °C (for KfiA).
  • the resulting PCR products were purified, digested, and ligated with the corresponding pre-digested vector.
  • the ligation products were transformed into electrocompetent E. coli DH5cc cells. Plasmids containing the target genes as confirmed by DNA sequencing (performed by UC-Davis Sequencing Facility) were selected and transformed into £. coli BL21 (DE3) chemically competent cells.
  • E. coli strains were cultured in LB rich medium ( 10 g/L tryptone , 5 g/L yeast extract, and 10 g/L NaCl) supplemented with ampicillin ( 1 00 ⁇ g/mL).
  • Over-expression of PmHSl and PmHS2 were achieved by inducing the E. coli BL21 (DE3) cell culture with 0.1 mM of isopropyl-l -thio- -D-galactopyranoside (IPTG) when the ⁇ nm of the culture reached 0.8-1 .0 followed by incubation at 20 °C for 20 h.
  • Overexpression of KfiA was performed by inoculating 10 mL of a fresh overnight bacterial culture grown in LB containing 50 ⁇ g/mL ampicillin and 20 ⁇ g/mL chloramphenicol into 1 L of LB (containing 50 ⁇ g mL of ampicillin, 20 ⁇ g/mL of chloramphenicol and 2 mg/mL of L- arabinose). The culture was incubated at 37 °C with shaking at 250 rpm. When the OD 6 oo of the culture reached 0.4-0.6, expression was induced by adding IPTG to a final concentration of 0.3 mM and then the cell was cultured at 20 °C for 20 h.
  • Bacterial cells were harvested by centrifugation at 4 °C in a Sorvall Legend RT centrifuge with a hanging bucket rotor at 4000 x rpm for 2 h.
  • Harvested cells were resuspended in lysis buffer (Tris-HCl buffer, 100 mM, pH 8.0 containing 0.1 % Triton X- 100) (20 mL for cells collected from one liter cell culture).
  • Lysozyme 100 ⁇ g/mL
  • DNasel 5 ⁇ g/mL
  • Cell lysate (supernatant) was obtained by centrifugation at 12000 x rpm for 15 min. Purification was carried out by loading the supernatant onto a N i 2+ - NTA column pre-equilibrated with 10 column volumes of binding buffer ( 10 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was washed with 10 column volumes of binding buffer and 10 column volumes of washing buffer (20-50 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5).
  • binding buffer 10 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5.
  • the target protein was eluted with Tris-HCl buffer (50 mM, pH 7.5) containing imidazole (200 mM) and NaCl (0.5 M).
  • Tris-HCl buffer 50 mM, pH 7.5
  • imidazole 200 mM
  • NaCl 0.5 M
  • the fractions containing the purified enzymes were collected and dialyzed against Tris-HCl buffer (20 mM, pH 7.5) containing 10% glycerol. Dialyzed proteins were stored at 4 °C.
  • pH profile by HPLC Typical enzymatic assays were performed in a 10 ⁇ reaction mixture containing a buffer ( 100 mM) with a pH in the range of 4.0- 10.0, UDP-GlcNAc ( 1 mM), GlcAp2AA (1 mM), MnCb (10 mM) and KfiA (9.0 ⁇ g) or PmHS2 (0.25 ⁇ £). Buffers used were: Na 2 HP0 4 /citric acid, pH 4.0; MES, pH 5.0-6.5; Tris-HCl, pH 7.0-9.0; and CAPS, pH 10.0.
  • N-His 6 -PmHS2 was routinely obtained from the cell lysate of one liter E. coli cell culture.
  • KfiA was expressed in an N-terminal MBP and a C-terminal six-His fusion protein in BL21 (DE3) cells coexpressed with chaperone protein pGro7.
  • the recombinant KfiA was purified to homogeneity with a Ni 2 ⁇ -affinity column.
  • About 8.0 mg of MBP-KfiA-His6 was routinely obtained from the cell lysate of one liter E. coli cell culture.
  • the size of the protein shown by SDS-PAGE was about 75 kDa and 69 kDa, respectively.
  • the MBP tag was introduced by using pMAL-c4X vector, while the C-His6-tag was introduced by including the His6-tag codons in the 3'-primer used for cloning.
  • Both enzymes can use the UDP-GlcNAc (F12-3), UDP- GlcNTFA (F12-4), UDP-GlcNGc (F12-8), UDP-GlcNAcN, (F12-9), among which the UDP- GlcNAc (F12-3) is the best substrate for both enzymes.
  • UDP- GlcNAc6N 3 (F12-5) is a substrate for PmHS2 but not for KfiA.
  • ATP, UTP, and GlcNAc were purchased from Sigma.
  • GlcNTFA, GlcN 3 , GlcNAc6N 3 , GIcNAc6S, GlcNS were synthesized as described previously.
  • Nan _ATCC55813 and PmPpA were overexpressed as discussed previously.
  • Uridine 5'-diphospho-2-deoxy-2-trifluoroacetamido-ct-D-glucopyranoside (UDP-GlcNTFA, T5b-10). Yield, 97% (699 mg); white foam.
  • Uridine 5'-diphospho-2-azido-2-deoxy-a-D-glucopyranoside UDP-GICN3, T5b-
  • Uridine 5'-diphospho-2-acetamido-6-azido-2,6-dideoxy-a-D-ghicopyranoside (UDP-GIcNAc6N 3 , T5b-12). Yield, 72% (462 mg); white foam.
  • Uridine 5'-diphospho-2-acetamido-2-deoxy-6-0-sulfo-a-D-glucopyranoside (UDP-GlcNAc6S, T5b-13). Yield, 62% (70 mg); white foam.
  • UDP-sugars F5-2-F5-8, and F5-10-F5-15 [0164] Uridine 5'-diphospho-2-amino-2-deoxy-a-D-glucopyranoside (UDP-GICNH2, F5-2). UDP-GlcNTFA F5-1 ( 150 mg, 0.22 mmol) was dissolved in 25 mL of methanol and 5 mL of H 2 0. The pH of the solution was adjusted to 9.5 by adding 2CO3. After being vigorously stirred at r.t. for overnight, the reaction mixture was neutralized with DOWEX HCR-W2 (H + ) resin, filtered and concentrated.
  • DOWEX HCR-W2 (H + ) resin DOWEX HCR-W2 (H + ) resin
  • Uridine 5'-diphospho-2-sulfoamino-2-deoxy-a-D-glucopyranoside (UDP- GlcNS, F5-3).
  • UDP-GlcNH 2 F5-2 (50 mg, 0.082 mmol) was dissolved in 30 mL of water. The pH of the solution was adjusted to 9.5 by adding 2 N NaOH (aq). Sulfur trioxide- pyridine complex (65 mg, 0.41 mmol) was added in three equal portions during 35 minutes intervals at room temperature, and the pH was maintained at 9.5 throughout the whole process using 2 N NaOH (aq). After being stirred at r.t.
  • Uridine 5'-diphospho-2-hydroxyacetamido-2-deoxy-a-D-glucopyranoside (UDP-GlcNGc, F5-5).
  • UDP-GlcNGc Uridine 5'-diphospho-2-hydroxyacetamido-2-deoxy-a-D-glucopyranoside
  • Uridine 5'-diphospho-2-phenylacetamido-2-deoxy-a-D-glucopyranoside (UDP- GlcNAcPh, F5-7).
  • 2-Phenylacetyl acid (33 mg, 0.25 mmol) was dissolved in 10 mL of CH 2 O 2 and two drops of DMF. The mixture was cooled to 0 °C. Oxalyl chloride (28 ⁇ , 0.33 mmol) was slowly added over 15 min using a syringe. The reaction was allowed to warm up to r.t. for overnight. The solvent was then removed under reduced pressure to afford 2-phenylacetyl chloride as a light pink solid.
  • Uridine 5'-diphospho-2-(l,l'-biphenyl-4-yl)acetamido-2-deoxy-a-D- glucopyranoside (UDP-GkNAcPh 2 , F5-8).
  • UDP-GlcNAcPh 2 F5-8 was synthesized from UDP-GlcNH 2 F5-2 using a similar procedure as described above for UDP-GlcNAcPh F5-7g except that the reagent 2-phenylacetyl acid was replaced by 2-([ l , l '-biphenyl]-4-yl)acetic acid.
  • UDP-GlcNAcPh 2 F5-8 was obtained as a white solid in 82% yield (31 mg).
  • Uridine 5'-diphospho-2-acetamido-6-amino-2,6-dideoxy-a-D-gkicopyrarioside (UDP-GlcNAc6NH 2 , F5-10).
  • UDP-GlcNAc6N 3 T5b-12 or F5-9)(100 mg, 0.16 mmol) was dissolved in MeOH-H 2 0 (10 mL, 1:1, v/v) and 20 mg of Pd/C was added. The mixture was shaken under H 2 gas (4 Bar) for 1 hr, filtered, and concentrated.
  • Uridine 5'-diphospho-2-acetamido-6-hydroxyacetamido-2,6-dideoxy-a-D- glucopyranoside (UDP-GlcNAc6NGc, F5-12).
  • UDP-GlcNAc6NGcAc F5-ll was synthesized from UDP-GIcNAc6NH 2 F5-10 using the same process as described above for UDP-GlcNAcNGcAc F5-4.
  • UDP-GlcNAc6NGcAc F5-11 was obtained as a white solid in 91% yield (31 mg).
  • Uridine 5'-diphospho-2-acetamido-6-azidoacetamido-2,6-dideoxy-a-D- glucopyranoside (UDP-GlcNAc6NAcN 3 , F5-13).
  • UDP-GlcNAc6NAcN 3 (F5-13) was synthesized from UDP-GlcNAc6NH 2 (F5-10) using the same process as described above for UDP-GlcNAcN 3 (F5-6).
  • UDP-GlcNAc6NAcN 3 (F5-13) was obtained as a white solid in 61 % yield (21 mg).
  • Uridine 5'-diphospho-2-acetamido-6-phenylacetamido-2,6-dideoxy-a-D- glucopyranoside (UDP-GlcNAc6NAcPh, F5-14).
  • UDP-GlcNAc6NAcPh F5-14 was synthesized from UDP-GlcNAc6NH 2 F5-10 using the same way as described above for UDP-GlcNAcPh (F5-7).
  • UDP-GlcNAc6NAcPh (F5-14) was obtained as a white solid in 86% yield (30 mg).
  • Uridine 5'-diphospho-2-acetamido-6-(l,l'-biphenyl-4-yl)-acetamido-2,6- dideoxy-a-D-glucopyranoside (UDP-GlcNAc6NAcPh 2 , F5-15).
  • UDP-GlcNAc6NAcPh 2 (F5-15) was synthesized from UDP-GlcNAc6NH 2 using the same way as described above for UDP-GlcNAcPh 2 (F5-8).
  • UDP-GlcNAc6NAcPh 2 (F5-15) was obtained as a white solid in
  • the first enzyme was an -acetylhexosamine 1 -kinase cloned from Bifidobacterium longum strain ATCC55813 (NahK_ATCC55813) which showed promiscuous substrate specificity and were able to use N-sulfated, 3-0-sulfated, or 6-0- sulfated GlcNAc and derivatives as substrates for the formation of GlcNAcal -phosphate derivatives.
  • the second enzyme was an N-acetylglucosamine- 1 -phosphate undylyltransferase that we cloned from Pasteurella rnidtocida strain P-l 059 (ATCC 15742) (PmGlmU). It catalyzes the reversible formation of UDP-GlcNAc and pyrophosphate from UTP and GlcNAc l -phosphate with tolerance on some substrate modifications.
  • the third enzyme was an inorganic pyrophosphatase also cloned from Pasteurella multocida strain P- l 059 (PmPpA) for hydrolyzing the pyrophosphate by-product formed to drive the reaction towards the formation of UDP-GlcNAc and derivatives.
  • PmPpA Pasteurella multocida strain P- l 059
  • NahK_JCM 1217 A recombinant Nah cloned from another strain of Bifidobacterium longum (NahK_JCM 1217) was used in the synthesis of GlcNAc- 1 - phosphate, GalNAc- 1 -phosphate, and their derivatives.
  • the purified HexN Ac- 1 -phosphates were then used in a one-pot two-enzyme system containing a commercially available inorganic pyrophosphatase (PpA) and a GlmU cloned from E. coli (EcGlmU) or an AGX 1 cloned from human for the synthesis of UDP-GlcN Ac, dNDP-GlcNAc, dNDP-Glc, UDP- GalNAc, and derivatives. Nevertheless, chemoenzymatic synthesis of UDP-GlcNAc derivatives using all three enzymes in one-pot has not been reported. In addition, UDP- GlcNAc derivatives containing N-sulfated glucosamine or 0-sulfated GlcNAc have not been synthesized using the combination of these three enzymes.
  • the one-pot three-enzyme system ( Figure 4) was quite efficient in synthesizing UDP-GlcNAc (T5b-9, 81 %), its C-2 derivatives such as UDP-N- trifluoroacctylglucosam ine (UDP-GlcNTFA, T5b-10, 97%) and UDP-2-azido-2-deoxy- glucose (UDP-GlcN 3 , T5b-ll, 54%), as well as its C-6 derivatives including UDP-N-acetyl- 6-azido-6-deoxy-glucosamine (UDP-GlcNAc6N 3 , T5b-12, 72%) and UDP-Nacetyl-6-O- sulfo-glucosamine (UDP-GlcNAc6S, T5b-13, 62%) from GlcNAc (T5b-1) and derivatives (T5b-2-T5b-5).
  • C-2 derivatives such as UDP-
  • N-TFA group in UDP-GlcNTFA (T5b-10) as well as the N 3 group in UDP- GlcN 3 (T5b-l l) UDP-GlcNAc6N 3 (T5b-12), and UDP-GlcN 3 6S (T5b-15) can be easily- converted to a free amine, allowing further modifications to generate a diverse array of N- substituted UDP-GlcNAc derivatives.
  • the N-TFA group at C2 of UDP-GlcNTFA T5b-10 (or F5-1) was removed under mild basic condition to produce UDP- glucosamine (UDP-GlcNH , F5-2) in 98% yield.
  • the reaction was carried out by incubating the solution in an isotherm incubator for 24 hr at 37 °C with gentle shaking or without shaking.
  • Glc-l -P commercially available Glc-l -P (55.2 mg), UTP (1.2 eq.), Tris- HCl buffer ( 100 mM, pH 8.0), and MgCl 2 (10 mM) were used along with BLUSP (1 mg) and PmPpA (1.5 mg).
  • BLUSP 1 mg
  • PmPpA 1.5 mg
  • Uridine 5'-diphospho-a-D-galactopyranoside (UDP-Gal, T6-16). 135 mg. Yield, 86%; white foam.
  • Uridine 5'-diphospho-a-D-glucopyranoside (UDP-Glc, T6-21).82 mg. Yield, 99%; white foam.
  • Uridine 5'-diphospho-2-deoxy-a-D-glucopyranoside UDP-2-deoxyGlc, T6-22.
  • Uridine 5'-diphospho-2-aniino-2-deoxy-a-D-glucopyranoside (UDP-GICNH2, T6-23).56 mg. Yield, 43%; white foam.
  • Uridine 5'-diphospho-2-azido-2-deoxy-a-D-glucopyranoside UDP-GICN3, T6-
  • Uridine 5'-diphospho-2-fluoro-2-deoxy-a-D-mannopyranoside (UDP-ManF, T6-27).142 mg. Yield, 92%; white foam.
  • Uridine 5'-diphospho-2-azido-2-deoxy-a-D-mannopyranoside (UDP-ManN3, T6-29).259 mg, Yield, 90%; white foam.
  • UDP- Glc including UDP-2-deoxyGlc (T6-22), UDP-GlcNH 2 (T6-23), and UDP-GlcN 3 (T6-24) were obtained from 2-deoxyGlc (T6-7), glucosamine (GlcNH 2 , T6-8) and GlcN (T6-9) in 56%, 43%, and 61 % yields, respectively.
  • the moderate yields for these three compounds may be attributed by less optimal NahK kinase activity for GlcNH 2 (T6-8) and GlcN ⁇ (T6-9), and the less optimal BLUSP activity for 2-deoxyGlc (T6-7).
  • UDP-Man (T6-26) was synthesized from Man (T6-11) in moderate 60% yield using the one-pot three-enzyme system and the moderate yield was most likely due to the less optimal activity of BLUSP towards Man-l -P.
  • UDP-GlcNAc T6-25
  • UDP- ManNH 2 T6-28
  • UDP-ManNAc T6-30
  • T6-10, T6-13, and T6-15 monosaccharides
  • UDP-ManNH 2 (T6-28) and UDP-ManNAc (T6-30) were not directly available from ManNH 2 (T6-13) and ManNAc (T6-15), respectively, via the one-pot three- enzyme reaction shown in Figure 9, they can be readily prepared via simple chemical modification reactions from UDP-ManN 3 (T6-29) obtained from the one-pot three-enzyme system.
  • UDP-ManN 3 UDP-ManN 3
  • a simple one-step catalytic hydrogenation of UDP-ManN 3 (T6-29) produced UDP-ManNH 2 (T6-28).
  • Acetylation of the amino group in UDP-ManNH 2 (T6-28) provided an easy access of UDP-ManNAc (T6-30).
  • the similar chemical acylation of UDP-ManNH 2 can be used to synthesize other acyl derivatives of UDP-ManNAc.
  • AtGlcAK was shown to be active on GlcA, GalA, and IdoA by TLC and LC-MS analyses.
  • One-pot three-enzyme strategy containing AtGlcAK, BLUSP, and PmPpA ( Figure 10) was shown to be able to produce UDP-GlcA, UDP-GalA, and UDP-IdoA from their corresponding monosaccharides GlcA, GalA, and IdoA respectively in small-scale assays confirmed by LC-MS or HRMS ( Figure 30).
  • glucuronolactone purchased from Sigma.
  • GlcNTFA, GlcNAc6N 3 , UDP-GlcNGc, UDP- GlcNAz, UDP-GlcNAc6NGc were synthesized as described previously.
  • NanK_ATCC55813, PmGlmU and PmPpA were overexpressed as reported.
  • FIG. 18A GlAp2AAMe (F13-8) (5 to 30 mg, 1 eq.), glucosamine derivatives (1.5 eq.), ATP ( 1.8 eq.), and UTP (1 .8 eq.) were dissolved in water in a 1 5 mL centrifuge tube containing Tris-HCl buffer ( 100 mM, pH 7.5) or MES buffer (100 mM, pH 6.5) and MgCl 2 (10 mM).
  • Tris-HCl buffer 100 mM, pH 7.5
  • MES buffer 100 mM, pH 6.5
  • MgCl 2 10 mM
  • Nan ATCC55813 0.5-2.1 mg
  • PmGlmU 1-3 mg
  • PmPpA 0.5-1 .5 mg
  • PmHS2 1-6 mg
  • water was added to bring the concentration of ⁇ 1 ⁇ 2 ⁇ (F13-8) to 5 mM.
  • the reaction was carried out by incubating the solution in an isotherm incubator for 12-36 h at 37 °C with gentle shaking.
  • the reaction was stopped by adding the same volume of ice-cold ethanol and incubating at 4 °C for 30 min.
  • Measured values represent M+Na + , M+2Na + -H + , M+3Na + -2H + .
  • Disaccharide GlcNTFAal -4GIcAp2AAMe F18-2 (30 mg, 1 eq.), Glc- l -P ( 1 .2 eq), UTP ( 1 .5 eq) and NAD + (2.4 eq.) were dissolved in water in a 15 mL centrifuge tube containing Tris- HC1 buffer ( 100 mM, pH 7.0) and MgCl 2 (10 mM). After the addition of appropriate amount of GalU ( 1 mg), PmUgd (3 mg), PmHS2 (4.5 mg), water was added to bring the volume of the reaction mixture to 8 mL.
  • Disaccharide GlcNH 2 cd-4GlcAp2AA (F24-9) (15 mg, 1 eq.), Glc-l-P (1.2 eq), UTP (1.5 eq), and NAD + (2.4 eq.) were dissolved in water in a 15 mL centrifuge tube containing Tris-HCl buffer (100 mM, pi I 7.0) and MgC (10 mM). After the addition of appropriate amount of GalU (0.5 mg), PmUgd (1.5 mg), PmHS2 (2.5 mg), water was added to bring the volume of the reaction mixture to 4 mL. The reaction was carried out by incubating the solution in an isotherm incubator at 37 °C for 12 hr with gentle shaking.
  • Trisaccharide GlcAp i ⁇ GlcNTFAal- 4GIcAp2AAMe ⁇ 20-2 (30 mg, 1 eq.), GlcNAc6N 3 (1.5 eq.), ATP ( 1.8 eq.), and UTP ( 1 .8 eq.) were dissolved in water in a 15 mL centrifuge tube containing MES buffer (100 mM, pH 6.5) and MgCl 2 ( 10 mM).
  • Nan _ATCC55813 2.5 mg
  • PmGlmU 3 mg
  • PmPpA 1.5 mg
  • PmHS2 4 mg
  • water was added to bring the volume of the reaction mixture to 6.5 mL.
  • the reaction was carried out by incubating the solution in an isotherm incubator for 18 h at 37 °C with gentle shaking.
  • the reaction was stopped by adding the same volume of ice-cold ethanol and incubating at 4 °C for 30 min.
  • the mixture was concentrated and passed through a BioGel P- 2 gel filtration column to obtain the desired product.
  • Trisaccharide GlcAp i-4GlcNTFAal -4GlcAp2AAMe (Compound F14-1 or F20-2, Figure 14) ( 1 1 mg, 1 eq.), GlcNTFA ( 1.5 eq.), ATP (1.8 eq.), and UTP ( 1 .8 eq.) were dissolved in water in a 15 mL centrifuge tube containing tris buffer (100 raM, pH 7.0) and MgCI 2 ( 10 inM).
  • NanK_ATCC55813 2.5 mg
  • PmGlmU 3 mg
  • PmPpA 1 .5 mg
  • PmHS2 2 mg
  • water was added to bring the volume of the reaction mixture to 10 mL.
  • the reaction was carried out by incubating the solution in an isotherm incubator at 37 °C for 20 hr with gentle shaking.
  • the reaction was stopped by adding the same volume of ice-cold ethanol and incubating at 4 °C for 30 min.
  • the first enzyme was an /V-acetylhexosamine 1 - kinase cloned from Bifidobacterium infantis strain ATCC 15697 (Nah _ATCC l 5697).
  • the second enzyme was an /V-acetylglucosamine- 1 -phosphate uridylyltransferase that we cloned from Pasteurella multocida strain P- 1059 (ATCC 15742) (PmGlmU).
  • the third enzyme was an inorganic pyrophosphatase that we cloned from Pasteurella multocida strain P- 1059
  • the fourth enzyme is a heparosan synthase 2 cloned from Pasteurella multocida strain P- 1059 (PmHS2) for the formation of al-4 linkage.
  • PmHS2 is a bifunctional enzyme which demonstrates al ⁇ GlcNAc and p l ⁇ lGlcA transferase activity. It not only uses UDP- Glc Ac as donor, transferring GlcNAc to GlcA to form al -4 linkage, but also transfers GIcA from donor UDP-GlcA to acceptor GlcNAc to form ⁇ 1— 4 linkage.
  • PmHS2 has been shown to be able to synthesize heparosan polysaccharides, its donor and acceptor specificity has not been investigated in detail.
  • UDP-GlcNAc F17-1 and some of its C2- (UDP-GlcNTFA F17-2, UDP-GlcNGc F17-3, and UDP-GlcNAcN 3 F17-4), and C6- (UDP-GlcNAc6N 3 F17-8 and UDP-GlcNAc6NGc F17-9) derivatives are tolerable donor substrates for PmHS2.
  • UDP-GlcNH 2 F17-5, UDP-GlcN 3 F17-6, UDP-GlcNS F17-7, UDP-GlcNAc6NH 2 F17-10, UDP-GlcNAc6NAcN 3 F17-11 and UDP-GlcNAc6S F17-12 did not serve as donor substrates for PmHS2.
  • GlcNAc6NGccd-4GlcAp2AAMe F18-6 are prepared in 92%, 91 %, and 74% yields, respectively. See Figure 18.
  • the second enzyme was a UDP-glucose dehydrogenase (Ugd) for oxidation of 6-OH in glucose residue of UDP-Glc to form the UDP-glucuronic acid (UDP-GlcA) in the presence of its coenzyme NAD + .
  • the third enzyme is PmHS2 transferring GlcA from UDP-GlcA for the formation of ⁇ — 4 linkage.
  • trisaccharides GlcApi- GlcNAcal- 4GlcAp2AAMe F20-1, GlcApl-4GlcNAc6N 3 al ⁇ GlcAp2AAMe F20-3, GlcApi - 4GlcNAcN 3 al ⁇ lGlcAp2AAMe F20-5 were synthesis by small-scale reaction and analyzed by HPLC method in 100%, 100% and 95% yields, respectively.
  • the relative low yield (72%) for the formation F20-2 was due to the formation of byproduct in which the TFA group was removed.
  • Disaccharide F18-4 with N-glycolyl group in C2 position of glucosamine residue acts as a good acceptor for PmHS2, leading to the formation of GlcAp i ⁇ GlcNGcal -4GlcAp2AAMe F20-4 in 75% yield, but the disaccharide F18-6 with N-glycolyl group in C6 position of GlcNAc was converted to trisaccharide GlcA i - GlcNAc6NGcal -4GlcAp2AAMe F20-6 only in 14% yield.
  • these results indicate that the donor and acceptor substrate activity of PmHS2 can tolerate a limited number of modifications on C-2 and C-6 position of glucosamine residue.
  • Trisaccharide F20-2 was used as the starting material for the synthesis of the tetrasaccharide F21-1 ( Figure 21).
  • the yV-TFA group at C2 of internal GlcNTFA residue of tetrasaccharide F21-1 was removed under mild basic conditions to produce GlcNAc6N 3 al-4GlcAp l-4GlcNH 2 al ⁇ lGlcA 2AA F22-1 in 81 % yield.
  • TFA group was accompanied by demethylation in methyl carboxylic ester
  • tetrasaccharide F22-1 contain a free carboxyl acid in 2AA motif instead of carboxylic ester in tetrasaccharide F21-1.
  • DCM:MeOH:NH 4 OH 9: 1 :0.1-1 : 1 : 0.1
  • both GlcA and GlcNAc can be used as the first sugar for oligosaccharide synthesis by the methods described in this invention.
  • Example 11 Inhibition assays of monosaccharides, disaccharides, trisaccharides, and tetrasaecharides.
  • FGF- 1 , FGF-2, FGF-4, and anti-human FGF- 1 , FGF-2, FGF-4 were purchased from PeproTech Inc (Rocky Hill, NJ). Heparin-biotin was from Sigma (St. Louis, MO). Low molecular weight heparin (LMWH) was bought from AMS Biotechnology (Lake Forest, CA). Alexa Fluor® 488 goat anti-rabbit IgG (H+L) was from Invitrogen (Carlsbad, CA). 384- Well NeutrAvid in-coated plates for the sialidase assays were from Fisher Biotech.
  • each set of duplicate wells were added 20 ⁇ , of human FGF- 1 , FGF-2, or FGF-4 (1 ⁇ ) with or without premixing with LMWH ( ⁇ 22 iiM or 0.1 ⁇ ), monosaccharide, or oligosaccharides (100 ⁇ or 1 niM) and the plate was incubate at r.t. for 1 hr.
  • LMWH ⁇ 22 iiM or 0.1 ⁇
  • monosaccharide, or oligosaccharides 100 ⁇ or 1 niM
  • LMWH was used as a control sample for testing the inhibitory activities of sixteen compounds including seven monosaccharides, two disaccharides, two trisaccharides, and five tetrasaecharides (see Figure 24 for compound structures) against the binding of fibroblast growth factors FGF-1 , FGF-2, and FGF-4 to heparin-biotin immobilized on NeutrAvidin-coated plates. See Table 10 and Figure 23. Table 10. Percentage inhibition of compounds F24-1-F24-16 (1 mM) against the binding of human FGF-1, FGF-2, and FGF-4 to heparin-biotin immobilized on NeutrAvidin- coated plates.
  • Protein sequence of His6-PmGlmU (Note: Italic sections of the sequences are from pET15b vector and primer. N-terminal His6-tag is underlined in the protein sequence)
  • Protein sequence of MBP-KfiA-His 6 (Note: Italic sections of the sequences are from pMAL-c4X vector and primer. The sequences for His6-tag are underlined)

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Abstract

The present invention provides a one-pot, multi-enzyme method for preparing UDP-sugars from simple sugar starting materials. The invention also provides a one-pot, multi-enzyme method for preparing oligosaccharides from simple sugar starting materials.

Description

CHEMOENZYMATIC SYNTHESIS OF HEPARIN AND HEPARAN
SULFATE ANALOGS
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application claims priority to United Station Provisional Patent
Application No. 61/510, 125, filed July 21 , 201 1 , the entirety of which is incorporated herein by reference.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[0002] This work was support by NIH grant R01 HD065122, and NSF grants CHE- 101251 1 and CHE-0548235. The United States government may have certain rights to the invention disclosed herein. BACKGROUND OF THE INVENTION
[0003] Heparin and heparan sulfate (HS) are sulfated linear polysaccharides composed of alternating al-4-linked D-glucosamine (GlcN¾) residues and l^ linked uronic acid (a- linkage for L-iduronic acid, IdoA, and β-linkage for D-glucuronic acid, GlcA). Possible modifications are 2-O-sulfation on the uronic acid residues and one or more modifications on the glucosamine residues including N-sulfation, N-acetylation, 6-O-sulfation, and 3-0- sulfation. Heparin is a mixture of polysaccharides that can be considered as special forms of HS with higher levels of sulfation and iduronic acid content per disaccharide repeat unit.Heparin is mostly produced by mast cells and heparan sulfates are produced by different cell types in animals. They are attractive synthetic targets due to their structural complexity which possesses great synthetic challenges and their important roles in regulating cancer growth, blood coagulation, inflammation, assisting viral and bacterial infections, signal transduction, lipid metabolism, and cell differentiation.
[0004] Currently, more than a hundred heparan sulfate binding proteins have been identified, and the structure-activity relationship studies (SAS) have revealed the interaction pattern between heparan sulfate and protein, and further directed toward discovering and designing HS mimics. Heparin pentasaccharide sequence ¾ (also call DEFGH) GlcNS6S- GlcA-GlcNS3S6S-IdoA2S-GlcNS6S is essential for antithrombin III binding and thrombin inhibition activities. Based on the DEFGH structure, a new potential antithrombotic, idraparinux, was synthesized by replacing TV-sulfate groups in all three glucosamine residues of heparin pentasaccharide DEFGH with O-sulfates and introducing methyl ethers at the available free hydroxyl groups and showed better anticoagulation activity and longer duration of action than DEFGH. Another pentasaccharide sequence HexA-GlcNS-HexA-GlcNS- ldoA2S has high affinity selectively for FGF-2 (fibroblast grow factor 2), while trisaccharide motif IdoA2S-GlcNS6S-IdoA2S is specific for FGF- 1 . N-, 2-0- and 6-0-sulfations of the glucosamine residues in HS have been shown to be required for FGF4 binding. Additionally, it has been suggested that the V-acetylated glucosamine region rich in GlcA residues displays structural plasticity and hence could mediate protein interactions. However, the detailed information about sequence requirement of HS that interact with many other proteins is currently unclear due to the lack of the technology of preparing a w ide range of structurally defined HS.
[0005] Current chemical and enzymatic synthetic methods do not provide convenient access to all possible heparin and HS oligosaccharide sequences. Chemical synthetic approaches are time-consuming and tedious. The production yields decrease dramatically with the increase of the length of the target molecules. Obtaining defined structures longer than octasaccharides remains as a major challenge for chemical synthesis. HS-modifying enzymes have been used with other enzymes to prepare heparin polysaccharides and oligosaccharides with a limited range of sulfation patterns. Due to the complex nature of HS- modifying enzymes, these types of methods do not allow the synthesis of a wide range of HS structures.
[0006] What is needed is a convenient route to form oligosaccharide products containing post-glycosylational modifications from monosaccharide derivatives as starting
materials. Importantly, the intermediates and products should be formed in a highly regio- and stereo-selective manner. The one-pot enzymatic methods of the present invention meet this and other needs. BRIEF SUMMARY OF THE INVENTION
[0007] In a first embodiment, the invention provides a method of synthesizing a UDP- sugar. The method includes forming a reaction mixture comprising a first sugar, a nucleotide- sugar pyrophosphorylase, and a first enzyme selected from a kinase and a dehydrogenase under conditions sufficient to form the UDP-sugar.
[0008] In a second embodiment, the invention provides a method of preparing an oligosaccharide. The method includes forming a first reaction mixture containing a first sugar, an acceptor sugar, a glycosyltransferase, a nucleotide-sugar pyrophosphorylase, and an enzyme selected from a kinase and a dehydrogenase. The first sugar is selected from a substituted or unsubstituted TV-acetylglucosamine (2-acetamido-2-deoxy glucose, GlcNAc), a substituted or unsubstituted glucosamine (GIcNH2), a substituted or unsubstituted glucuronic acid (GlcA), a substituted or unsubstituted iduronic acid (IdoA), and a substituted or unsubstituted glucose- 1 -phosphate (Glc- l -P), and the acceptor sugar includes at least one member selected from a substituted or unsubstituted jV-acetylglucosamine (GlcNAc), a substituted or unsubstituted glucosamine (GlcNH2), a substituted or unsubstituted glucuronic acid (GlcA), and a substituted or unsubstituted iduronic acid (IdoA). The reaction mixture is formed under conditions sufficent to convert the first sugar to a UDP-sugar, and sufficient to couple the sugar in the UDP-sugar to the acceptor sugar. When the first sugar is substituted or unsubstituted GlcNAc or GlcNHh, the sugar in the UDP-sugar is coupled to a substituted or unsubstituted GlcA or a substituted or unsubstituted IdoA of the acceptor sugar. When the first sugar is substituted or unsubstituted Glc- l -P, substituted or unsubstituted GlcA, or substituted or unsubstituted IdoA, the sugar in the UDP-sugar is coupled to a substituted or unsubstituted GlcNH? or a substituted or unsubstituted GlcNAc of the acceptor sugar. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1 shows a sequence alignment of NahK_.JCM 1 217 (GenBank accession no. BAF73925), NahK_ATCC55813, and NahK_ATCC 15697.
[0010] Figure 2 shows the pH profiles of NahK_ATCC 15697 (♦, filled diamond) and NahK ATCC55813 (0, open diamond). Buffers used: MES, pH 6.0; Tris-HCI, pH 7.0-9.0; CAPS, pH 10.0-1 1.0.
[0011] Figure 3 shows the effect of MgCla on the activity of NahKs. [0012] Figure 4 shows the one-pot three-enzyme synthesis of UDP-GlcNAc and derivatives. Enzyme used: NahK_ATCC55813, an N-acetylhexosamine 1 -kinase cloned from Bifidobacterium longiim ATCC55813 ; PmGlmU, Pasteurella multocida N- acetylglucosamine-1 -phosphate uridylyltransferase; PmPpA, Pasteurella multocida inorganic pyrophosphatase.
[0013] Figure 5 shows the chemical diversification at (A) the C-2 of glucosamine and (B) the C-6 of N-acetylglucosamine in UDP-sugar nucleotides. Reagents and conditions: a) K2C03, CH3OH, H20, 20 °C, overnight, 98%; b) PyS03, 2 M NaOH, H20, overnight, 86%; c) RCOCl, NaHC03, CH3CN, H20; d) NaOMe, MeOH; e) H2, Pd/C, MeOH, H20, 1 h, 96%.
[0014] Figure 6 shows the pH profile of Bifidobacterium longum UDP-sugar
pyrophosphorylase (BLUSP).
[0015] Figure 7 shows the metal requirements of BLUSP.
[0016] Figure 8 shows the synthesis of UDP-ManNAc from UDP-ManN3 in 79% yield via the formation of UDP-ManNH2 by catalytic hydrogenation followed by acetylation.
[0017] Figure 9 shows a one-pot, three-enzyme system for the synthesis of UDP- monosaccharides and derivatives. Enzymes used: NahK._ATCC l 5697, Bifidobacterium infantis strain ATCC 15697 /V-acetyl hexosamine 1 -kinase; SpGalK, Streptococcus pneumoniae TIGR4 galactokinase; EcGalK, Echerichia coli galactokinase; BLUSP, Bifidobacterium longum UDP-sugar pyrophosphorylase; PmPpA, Pasteurella multocida inorganic pyrophosphatase.
[0018] Figure 10 shows the one-pot multienzyme synthesis of UDP-glucuronic acid, UDP- iduronic acid, and UDP-galacturonic acid.
[0019] Figure 11 shows the results of the substrate specificity assay for the heparosan synthase activity of KfiA (Figure 11A) and PmHS2 (Figure 11B). Each reaction was performed at 37 °C in MES buffer ( 1 00 mM, pH 6.5) for 30 min, 4 h or 16 h. Enzyme used: KfiA ( 1 .08 μg/μL), PmHS2 (2.5 l O"2 μ /μί).
[0020] Figure 12 shows the structures of the substrates tested in the substrate specificity assay for KfiA and PmHS2 in Figure 11.
[0021] Figure 13 shows the synthetic scheme for preparation of fluorescently labeled GlcA GlcAp2AAMe. [0022] Figure 14 shows the synthesis of tetrasaccharides
Figure imgf000006_0001
4GlcNTFAal—4GlcA 2AAMe (F14-2) and GlcNSal^GlcApl-4GlcNSal^GlcAp2AA (F14-3) from trisaccharide GlcA i^tGlcNTFAal-4GlcAp2AAMe (F14-1).
[0023] Figure 15 shows the synthesis of GlcA-TEG-PABA-biotin (F15-8).
[0024] Figure 16 shows the one-pot four-enzyme synthesis of dissacharides with different modification on C2 and C6. Enzymes used: NahK_ATCC55813, N-acetylhexosamine 1 - kinase cloned from Bifidobacterium longum ATCC55813; PmGlmU, Pasteurella multocida N-acetylglucosamine-1 -phosphate uri-dylyltransferase; PmPpA, Pasteurella multocida inorganic pyrophosphatase; PmHS2, Pasteurella multocida heparosan synthase 2.
[0025] Figure 17 shows the structures of UDP-GlcNAc derivatives F17-1-F17-12 including UDP-GlcNAc (F17-1), UDP-GlcNTFA (F17-2), UDP-GlcNGc (F17-3), UDP- GlcNAcN3 (F17-4), UDP-GlcNH2 (F17-5), UDP-GlcN3 (F17-6), UDP-GlcNS (F17-7), UDP- GlcNAc6N3 (F17-8), UDp-GlcNAc6NGc (F17-9), UDP-GlcNAc6NH2 (F17-10j), UDP- GlcNAc6NAcN3 (F17-11), and UDP-GlcNAc6S (F17-12).
[0026] Figure 18 shows the enzymatic synthesis of the disaccharides. Figure 18A shows the one-pot four-enzyme system of the disaccharides GlcNAcccl -4GlcAp2AAMe (F18-1), GlcNTF Act 1 -4G IcA β2 AAMe (F18-2), GlcNAc6N3al -4GlcA 2AAMe (F18-3). Figure 18B shows the PmHS2-catalyzed synthesis of the disaccharides GlcNGcal -4GlcA 2AAMe (F18-4), GIcNAcN3al -4GlcAp2AAMe (F18-5), GlcNAc6NGcal -4GlcAp2AAMe (F18-6). [0027] Figure 19 shows the enzymatic synthesis of trisaccharides from disaccharides via in situ generation of UDP-GIcA from Glc- l -P catalyzed by Echerichia coli glucose- 1 -phosphate uridylyltransferase (EcGalU), Pasteurella multocida UDP-glucose dehydrogenase (PmUgd), and PmHS2.
[0028] Figure 20 shows the one-pot three-enzyme synthesis of trisaccharides GlcA l - 4GlcNAcct l -4GlcAP2AAMe (F20-1), GlcApi -4GlcNTFActl -4GlcAP2AAMe (F20-2), GlcA i -4GlcNAc6N3a l -4GlcAp2AAMe (F20-3), GIcA i -4GlcNGca l -4GlcA 2AAMe (F20-4), GlcApi -4GlcNAcN3al -4GlcAp2AAMe (F20-5), GlcA i -4GlcNAc6NGca l - 4GlcAp2AAMe (F20-6). [0029] Figure 21 shows the one-pot four-enzyme synthesis of tetrasaccharide
GlcNAc6N3al-4GlcA i-4GlcNTFAal -4GlcA 2AAMe (F21-1) from trisaccharide GlcApi - 4GlcNTFAal -4GlcAp2AAMe (F20-1).
[0030 J Figure 22 shows the synthesis of tetrasaccharides G lcN Ac6N3Ct 1 -4G lc Αβ 1 - 4GlcNH2al -4GlcA 2AA (F22-1), GlcNAc6N3al -4GlcApi-4GlcNSal -4GlcAp2AA (F22- 2), GlcNAc6NH2 l -4GlcApl -4GlcNSal -4GlcAp2AA (F22-3), GlcNAc6NSal -4GlcAp i - 4GlcNSal -4GlcAp2AA (F22-4) from GlcNAc6N3al -4GlcApl-4GlcNTFAal - 4GlcAp2AAMe (F21-1) by chemical modifications. Reagents and conditions: (a) 2CO3, H20, r.t. overnight, 81%; (b) PyS03, 2 M NaOH, H20, 3d, 70%; (c) H2, Pd/C, MeOH, H20, 1 h.
[0031] Figure 23 shows the inhibitory activities of LMWH or compounds F24-1-F24-16 (see Figure 24 for structures) against the binding of human fibroblast growth factors FGF- 1 (Figure 23A), FGF-2 (Figure 23B), or FGF-4 (Figure 23C) to the heparin-biotin immobilized on NeutrAvidin-coated 384-weIl plates. Samples without LMWH or monosaccharide/tetrasaccharide inhibitors were used as positive controls (P.C.).
[0032] Figure 24 shows structures of compounds F24-1-F24-16 used in Figure 23 for inhibition studies of the binding of human fibroblast growth factors FGF- 1 , FGF-2, and FGF- 4 to the heparin-biotin immobilized on NeutrAvidin-coated 384-well plates.
[0033] Figure 25 shows thin-layer chromatography (TLC) analysis data for AtGlcA reactions. Lanes: 1 , ATP; 2, GlcA; 3, reaction with GlcA and ATP; 4, GalA; 5, reaction with GalA and ATP; 6, IdoA; 7, reaction with IdoA and ATP; 8, xylose; 9; reaction with xylose and ATP. Developing solvent used for running TLC: «-Pr0H:H20:NH40H = 7:4:2 (by volume).
[0034] Figure 26 shows LC-MS assay data for AtGlcA -catalyzed synthesis of siigar- 1 - phosphate from sugar and ATP. Figure 26A, AtGlcAK kinase reaction using GlcA as the starting sugar; Figure 26B, AtGlcAK kinase reaction using GalA as the starting sugar; Figure 26C, AtGlcAK kinase reaction using IdoA as the starting sugar.
[0035] Figure 27 shows pH profiles of Kfi A (Figure 27A) and PmHS2 (Figure 27B).
Buffers used were: Na2HP04/cirtic acid, pH 4.0; MES, pH 5.0-6.5; Tris-HCl, pH 7.0- 9.0; and CAPS, pH 10.0. [0036] Figure 28 shows metal effects on the heparosan synthase activity of KfiA (Figure 28A) and PmHS2 (Figure 28B).
[0037] Figure 29 shows high-resolution mass spectrometry (Orbitrap HRMS) assay for the synthesis of UDP- GlcNAc3N3 from GlcNAc3N3, ATP, and UTP using one-pot three- enzyme reactions containing NahK, PmGlmU, and PmPpA.
[0038] Figure 30 shows LC-MS or high resolution mass spectrometry (Orbitrap HRMS) assay for the synthesis of UDP-sugars from sugar, ATP, and UTP using one-pot three- enzyme reactions containing AtGlcAK, BLUSP, and PmPpA. Figure 30A, LC-MS assay and GlcA was used as the starting sugar; Figure 30B, HRMS assay and GalA was used as the starting sugar; Figure 30C, HRMS assay and IdoA was used as the starting sugar.
[0039] Figure 31 shows thin-layer chromatograph analysis of PmHS2-catalyzed reaction for the formation of GlcA-GlcNAc disaccharide derivatives.
[0040] Figure 32 shows LC-MS analysis of PmHS2-catalyzed reaction for the formation of GlcA-GlcNAc disaccharide derivatives. Figure 32A, GlcNAc 2AA was used as the acceptor; Figure 32B, GlcNAcpMU was used as the acceptor; Figure 32C, GlcNAcaProN3 was used as the acceptor; Figure 32D, Glc AcpProN3 was used as the acceptor.
DETAILED DESCRIPTION OF THE INVENTION I. GENERAL
[0041] The present invention provides a convenient and highly efficient one-pot multienzyme system for the synthesis of UDP-sugars and oligosaccharides including heparin and heparosan sulfate (HS) analogs. Kinases or dehydrogenases, nucleotide-sugar pyrophosphorylases, and/or glycosyltransferases are used in one-pot reactions to convert monosaccharide precursors to UDP-sugars and/or oligosaccharides. Chemical diversification of the enzymatically formed UDP-sugars and oligosaccharides can be conducted to produce more structural variations. In particular, non-sulfated oligosaccharides can be selectively modified to prepare structurally defined products with desired sulfation patterns. A diverse set of enzymatic substrates can be used in the methods of the invention to prepare a wide range of useful UDP-sugars and oligosaccharides. II. DEFINITIONS
[0042] As used herein, the term "first sugar" refers to a monosaccharide starting material used in the methods of the invention. The monosaccharide can be a hexose or a
pentose.Hexoses include, but are not limited to, glucose (Glc), glucosamine (2-amino-2- deoxy-glucose; GlcNH2), N-acetylglucosamine (2-acetamido-2-deoxy-glucose; GlcNAc), galactose (Gal), galactosamine (2-amino-2-deoxy-galactose; GalNH?), N-acetylgalactosamine (2-acetamido-2-deoxy-galactose; GalNAc), mannose (Man), mannosamine (2-amino-2- deoxy-mannose; ManNH2), .V-acetylmannosamine (2-acetamido-2-deoxy-mannose;
ManNAc), glucuronic acid (GlcA), iduronic acid (IdoA), and galacturonic acid (GalA). Pentoses include, but are not limited to, ribose (Rib), xylose (Xyl), and arabinose (Arb). The sugar can be a D sugar or an L sugar. The sugar can be unsubstituted or substituted with moieties including, but not limited to, amino groups, azido groups, amido groups, acylamido groups, 7V-sulfate groups (sulfamate), and O-sulfate groups. A "second sugar" and subsequent sugars are generally defined as for the first sugar, except that they are used after the first sugar in a multi-step synthesis.
[0043] As used herein, the term "UDP-sugar" refers to a sugar containing a uridine diphosphate moiety. The sugar portion of the UDP-sugar is defined as for the "first sugar" described above. UDP-sugars include, but are not limited to UDP-Glc, UDP-GlcNAc, UDP- GlcNH2, UDP-GlcA, UDP-ldoA, UDP-GalA, UDP-Gal, UDP-GalNAc, UDP-GalNH2, UDP- Man, UDP-ManNAc, and UDP-ManNH2. The UDP-sugar can be unsubstituted or substituted as described above.
[0044] As used herein, the term "oligosaccharide" refers to a compound containing at least two sugars covalently linked together. Oligosaccharides include disaccharides, trisaccharides, tetrasachharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, and the like. Covalent linkages generally consist of glycosidic linkages (i.e., C-O-C bonds) formed from the hydroxy I groups of adjacent sugars. Linkages can occur between the 1 - carbon and the 4-carbon of adjacent sugars (i.e., a 1 -4 linkage), the 1 -carbon and the 3-carbon of adjacent sugars (i.e. , a 1 -3 linkage), the 1 -carbon and the 6-carbon of adjacent sugars (i. e., a 1 -6 linkage), or the 1 -carbon and the 2-carbon of adjacent sugars (i.e. , a 1 -2 linkage). A sugar can be linked within an oligosaccharide such that the anomeric carbon is in the a- or β- configuration. The oligosaccharides prepared according to the methods of the invention can also include linkages between carbon atoms other than the 1 -, 2-, 3-, 4-, and 6-carbons. [0045] As used herein, the term "enzyme" refers to a polypeptide that catalyzes the transformation of a starting material, such as a sugar, to an intermediate or product of the one-pot reactions of the invention. Examples of enzymes include, but are not limited to, kinases, dehydrogenases, nucleotide-sugar pyrophosphorylases, pyrophosphatases, and glycosyltransferases. Other enzymes may be useful in the methods of the invention.
[0046] As used herein, the term "kinase" refers to a polypeptide that catalyzes the covalent addition of a phosphate group to a substrate. The substrate for a kinase used in the methods of the invention is generally a sugar as defined above, and a phosphate group is added to the anomeric carbon (i. e. the " 1 " position) of the sugar. The product of the reaction is a sugar- 1 - phosphate. Kinases include, but are not limited to, N-acetylhexosamine 1 -kinases (NahKs), glucuronokinases (GlcAKs), glucokinases (GlcKs), galactokinases (GalKs), monosaccharide- 1 -kinases, and xylulokinases. Certain kinases utilize nucleotide triphosphates, including adenosine-5 '-triphosphate (ATP) as substrates.
[0047] As used herein, the term "dehydrogenase" refers to a polypeptide that catalyzes the oxidation of a primary alcohol. In general, the dehyrogenases used in the methods of the invention convert the hydroxymethyl group of a hexose (i.e. the C6-OH moiety) to a carboxylic acid. Dehydrogenases useful in the methods of the invention include, but are not limited to, UDP-glucose dehydrogenases (Ugds).
[0048] As used herein, the term "nucleotide-sugar pyrophosphorylase" refers to a polypeptide that catalyzes the conversion of a sugar- 1 -phosphate to a UDP-sugar. In general, a uridine-5'-monophosphate moiety is transferred from uridine- 5 '-triphosphate to the sugar-1 - phosphate to form the UDP-sugar. Examples of nucleotide-sugar pyrophosphorylases include glucosamine uridylyltransferases (GlmUs) and glucose- 1 -phosphate uridylyltransferases (GalUs). Nucleotide-sugar pyrophosphorylases also include promiscuous UDP-sugar pyrophosphorylases, termed "USPs," that can catalyze the conversion of various sugar- 1 - phosphates to UDP-sugars including UDP-Glc, UDP-GlcNAc, UDP-GlcNH2, UDP-Gal, UDP-GalNAc, UDP-GalNH2, UDP-Man, UDP-ManNAc, UDP-ManNH2, UDP-GlcA, UDP- IdoA, UDP-GalA, and their substituted analogs.
[0049] As used herein, the term "pyrophosphatase" (abbreviated as PpA) refers to a polypeptide that catalyzes the conversion of pyrophosphate (i.e. , P207 4, ΗΡ20 " , H2P207 , ¾Ρ2θ7") to two molar equivalents of inorganic phosphate (i.e., PO4 3", HPO42", H2PO4"). [0050] As used herein, the term "glycosyltransferase" refers to a polypeptide that catalyzes the formation of an oligosaccharide from a UDP-sugar and an acceptor sugar. In general, a glycosyltransferase catalyzes the transfer of the monosaccharide moiety of the UDP-sugar to a hydroxyl group of the acceptor sugar. The covalent linkage between the monosaccharide and the acceptor sugar can be a 1-4 linkage, a 1 -4 linkage, a 1 -6-linkage, or a 1 -2 linkage as described above. The linkage may be in the a- or β-configuration with respect to the anomeric carbon of the monosaccharide. Other types of linkages may be formed by the glycosyltransferases in the methods of the invention. Glycosyltransferases include, but are not limited to, heparosan synthases (HSs) glucosaminyltransferases, N- acetylglucosaminyltransferases, glucosyltransferasess, glucuronyltransferases.
[0051] As used herein, the term "couple" refers to catalyzing the formation of a covalent bond between enzyme substrates. The coupling can take place via the direct reaction of two substrates with each other. Alternatively, the coupling can include the formation of one or more enzyme-substrate intermediates. An enzyme-substrate intermediate can, in turn, react with another substrate (or another enzyme-substrate intermediate) to form the bond between the substrates.
III. MONO- AND OLIGOSACCHARIDES
[0052] A number of UDP-sugars can be synthesized according to the methods of the invention. In general, the UDP-sugars have structures according to Formula I:
Figure imgf000011_0001
wherein each of R1, R2, and R3 is independently selected from OH, N3, NH2, NHS03\ OSO3", NHC(0)CH3, NHC(0)CF3, NHC(0)CH2OH, and NHC(0)CH2N3; and R4 is selected from CH2OH, C02 ", C02H, CH2N3, CH2NH2, CH2NHS03\ CH2OS03 ", CH2NHC(0)CH3, CH2NHC(0)CF3, CH2NHC(0)CH2OH, and CH2NHC(0)CH2N3.
[0053] In some embodiments, the UDP-sugars have structures according to formula I
Figure imgf000011_0002
UDP (la) [0054] A range of oligosaccharides can also be prepared using the methods of the invention. In general, the oligosaccharides contain one or more unit according to Formula II:
Figure imgf000012_0001
[0055] In oligosaccharide units according to Formula II, each of Rla, Rlb, R2a, and R2b is independently selected from OH, N3, NH2, NHS03 ", OS03\ NHC(0)CH3, NHC(0)CF3,
NHC(0)CH2OH, or HC(0)CH2N3; and each of Rlc and R2c is independently selected from CH2OH, C02\ C02H, CH2N3, CH2NH2, CH2NHS03 ~, CH20S03\ CH2NHC(0)CH3, CH2NHC(0)CF3, CH2NHC(0)CH2OH, or CH2NHC(0)CH2N3. In some embodiments, one of Rl c and R2c can be C02 " or C02H, while the other of Rlc and R2c can be CH2OH, CH2N3, CH2NH2, CH2NHS03 ~, CH2OS03 , CH2NHC(0)CH3, CH2NHC(0)CF3i
CH2NHC(0)CH2OH, or CH2NHC(0)CH2N3. R includes but not is limited to H, CH3, CH2CH3, CH2CH2N3, CH2CH2CH2N3, an aglycon according to Formula B, Formula C, Formula D, or Formula E below, substituted or unsubstituted GlcNAc, substituted or
I unsubstituted GlcNH2, substituted or unsubstituted GlcA, or substituted or unsubstituted Ido:
Figure imgf000012_0002
[0056] In some embodiments, the oligosaccharides have the structure of formula lla:
Figure imgf000013_0001
_[0057] In some embodiments, the method provides oligosaccharides with structures according to Formula III:
Figure imgf000013_0002
wherein each of Rla, Rl b, and R2a is independently selected from OH, N3, NH2, NHS03\ OS03 ", NHC(0)CH3, NHC(0)CF3, NHC(0)CH2OH, or NHC(0)CH2N3; and Rlc is selected from CH2OH, CH2N3, CH2NH2, CH2NHS03 ", CH20S03\ CH2NHC(0)CH3,
CH2NHC(0)CF3, CH2NHC(0)CH2OH, or CH2NHC(0)CH2N3.
[0058] In some embodiments, the method provides oligosaccharides with structures according to Formula IV:
Figure imgf000013_0003
wherein each of Rl , R2a, and R2b is independently selected from OH, N3, NH2, NHS03\ OS03\ NHC(0)CH3, NHC(0)CF3, NHC(0)CH2OH, and NHC(0)CH2N3; and R2c is selected from CH2OH, CH2N3, CH2NH2, CH2NHS03 ", CH20S03\ CH2NHC(0)CH3,
CH2NHC(0)CF3, CH2NHC(0)CH20H, or CH2NHC(0)CH2N3.
[0059] In some embodiments, the present invention provides oligosaccharides having the structure of formula IVa:
Figure imgf000013_0004
(IVa) [0060] In some embodiments, the method provides oligosaccharides with structures according to Formula (V):
Figure imgf000014_0001
wherein each of Rla, R2a, R2b, and R3a is independently selected from OH, N3, NH2, NHS03 ", OS03 ", NHC(0)CH3, NHC(0)CF3, NHC(0)CH2OH, and NHC(0)CH2N3; and R2c is selected from CH2OH, CH2N3, CH2NH2, CH2NHS03 ", CH2OSO3", CH2NIIC(0)CH3,
CH2NHC(0)CF3, CH2NHC(0)CH2OH, or CH2NHC(0)CH2 3.
[0061] In some embodiments, the present invention provides oligosaccharides having a structure of formula Va:
Figure imgf000014_0002
[0062] In some embodiments, the method provides oligosaccharides with structures according to Formula VI:
Figure imgf000014_0003
wherein each of R la, R2a, R2b, R3a, R b, and R4b is independently selected from OH, N3, NH2, NHS03 ", OSO3 ", NHC(0)CH3, NHC(0)CF3, NHC(0)CH2OH, or NHC(0)CH2N3; and each of R2c, R4c is independently selected from CH2OH, CH2N3, CH2NH2, CH2NHS03 ", CH20S03 ", CH2NHC(0)CH3, CH2NHC(0)CF3, CH2NHC(0)CH20H, or
CH2NHC(0)CH2N3. [0063] In some embodiments, the oligosaccharides has the structure of formula Via:
Figure imgf000015_0001
IV. ONE-POT METHOD OF MAKING UDP-SUGARS
[0064] In a first embodiment, the invention provides a method of synthesizing a UDP- sugar. The method includes forming a reaction mixture comprising a first sugar, a nucleotide- sugar pyrophosphorylase, and a first enzyme selected from a kinase and a dehydrogenase under conditions sufficient to form the UDP-sugar.
[0065] In some embodiments, the first sugar is selected from substituted or unsubstituted glucose (Glc), substituted or unsubstituted glucose- 1 -phosphate (Glc- l -P), substituted or unsubstituted glucuronic acid (GlcA), substituted or unsubstituted glucuronic acid-1 - phosphate (GlcA- l -P), substituted or unsubstituted iduronic acid (IdoA), substituted or unsubstituted iduronic acid- 1 -phosphate (IdoA- l -P), substituted or unsubstituted N- acetylglucosamine (GlcNAc), substituted or unsubstituted vV-acetylglucosain ine- 1 -phosphate (Glc Ac- l -P), substituted or unsubstituted glucosamine (GIcN¾), substituted or
Linsubstituted glucosamine-1 -phosphate (GlcNH2-l -P), substituted or unsubstituted galactose (Gal), substituted or unsubstituted galactose- 1 -phosphate (Gal- l -P), substituted or unsubstituted galacturonic acid (GalA), substituted or unsubstituted galacturonic acid-1 - phosphate (GalA- l -P), substituted or unsubstituted N-acetylgalactosamine (GalNAc), substituted or unsubstituted ^-acetylgalactosamine- 1 -phosphate (GalNAc- 1 -P), substituted or unsubstituted galactosamine (GalNH2), substituted or unsubstituted galactosamine- l - phosphate (GalNH l -P), substituted or unsubstituted mannose (Man), substituted or unsubstituted mannose- 1 -phosphate (Man- 1 -P), substituted or unsubstituted N- acetylmannosam ine (ManNAc), substituted or unsubstituted N-acetylmannosamine- 1 - phosphate (ManNAc- 1 -P), substituted or unsubstituted mamiosamine (ManNH2), substituted or unsubstituted mannosamine-1 -phosphate (ManNP -l -P). In some embodiments, the first sugar is selected from GlcNAc, Glc- l -P, GlcA, and IdoA.
[0066] In some embodiments, the first sugar has the formula VII:
Figure imgf000016_0001
Wherein each of R1, R2, and R3 is selected from OH, N3, NH2, NHSOy, OS03\ NHC(0)CH3,
NHC(0)CF3, NHC(0)CH2OH, and NHC(0)CH2N3; R4 is selected from CH2OH, C02 ",
C02H, CH2N3, CH2NH2, CH2NHS03 ', CH2OS03 ", CH2NHC(0)CH3, CH2NHC(0)CF3, CH2NHC(0)CH2OH, and CH2NI IC(0)CH2N3; and R5 can be H, P03 2\ or HP03\ In some embodiments, the first sugar has the formula VIII or IX: (or OP03H-) (1X).
Figure imgf000016_0002
[0067] In general, the reaction mixture formed in the methods of the invention contains a nucleotide-sugar pyrophosphorylase. The nucleotide-sugar pyrophosporylase can be, but is not limited to, a glucosamine uridyltransferase (GlmU), a Glc- l -P uridylyltransferase (GalU), or a promiscuous UDP-sugar pyrophosphorylase (USP). The present inventors have cloned and characterized a GlmU from P. muJtocida (PmGlmU) that is useful for the synthesis of UDP-sugars according to the methods of the invention. Suitable GalUs can be obtained, for example, from yeasts such as Saccharomyces fragilis, pigeon livers, mammalian livers such as bovine liver, Gram-positive bacteria such as Bifidobacterium bifidum, and Gram-negative bacteria such as Echerichia coli (EcGalU) (Chen X, Fang JW, Zhang JB, Liu ZY, Shao J, owal P, Andreana P, and Wang PG. J. Am. chem. Soc. 2001 , 123, 2081 -2082). In some embodiments, the nucleotide-sugar pyrophosporylase is a USP. USPs include, but are not limited to, those obtained from Pisum sativum L. (PsUSP) and Arabidopsis thaliana (AtUSP), as well as enzymes obtained from protozoan parasites (such as Leishmania major and Trypanosoma cruzi) and hyperthermophilic archaea (such as Pyrococcus furiosus DSM 3638). USPs also include human UDP-GalNAc pyrophosphorylase AGX l , E. coli EcGlmU, and Bifidobacterium longum BLUSP. BLUSP was cloned and characterized by the inventors. In some embodiments, the nucleotide-sugar pyrophosphorylase is selected from AGX l , EcGlmU, EcGalU, PmGlmU, and BLUSP. In some embodiments, the nucleotide-sugar pyrophosphorylase is selected from EcGalU, PmGlmU, and BLUSP. In some embodiments, the nucleotide-sugar pyrophosphorylase is EcGalU. In some embodiments, the nucleotide- sugar pyrophosphorylase is PmGlmU. In some embodiments, the nucleotide-sugar pyrophosphorylase is BLUSP. [0068] The reaction mixture formed in the methods of the invention also contains a kinase or a dehydrogenase. In some embodiments, the first enzyme in the reaction mixture is a kinase. The kinase can be, but is not limited to, an N-acctylhexosamine 1 -kinase (NahK), a galactokinase (GalK), or a glucuronokinase (GlcA ). In some embodiments, the kinase is an NahK. The NahK can be, for example, Bifidobacterium infantis NahK_ATCC 15697 or Bifidobacterium longum NahK_ATCC55813. NahK ATCCl 5697 and NahK_ATCC55813 were cloned and characterized by the inventors. In some embodiments, the kinase is a GalK. The GalK can be, for example, Escherichia coli EcGalK (Chen X, Fang JW, Zhang JB, Liu ZY, Shao J, Kowal P, Andreana P, and Wang PG. J. Am. chem. Soc. 2001 , 123, 2081 -2082) and Streptococcus pneumoniae TIGR4 SpGalK (Chen M, Chen LL, Zou Y, Xue M, Liang M, Jing L, Guan WY, Shen J, Wang W, Wang L, Liu J, and Wang PG. Carbohydr. Res. 201 1 , 346, 2421 -2425).
[0069] In some embodiments, the UDP-sugar is a substituted or unsubstituted UDP-GlcA. The first sugar employed in the synthesis of UDP-GlcA may vary depending on the enzymes that are used in the one-pot reaction. For example, Glc- l -P can be converted to UDP-Glc using a UDP-sugar pyrophosporylase. UDP-GlcA can be obtained from UDP-Glc using a dehydrogenase. Accordingly, the reaction mixture in some embodiments of the invention includes a dehydrogenase. The dehydrogenase can be, but is not limited to, a UDP-glucose dehydrogenase (Ugd). In some embodiments, the dehydrogenase is Pasteur ella multocida PmUgd. The PmUgd was cloned and characterized by the inventors. Alternatively, GlcA can be converted to GlcA- l -P using a GlcAK. In some embodiments, therefore, the kinase in the reaction mixture is a GlcAK. The GlcAK can be, for example, Arabidopsis thaliana AtGlcAK. The GlcA-l -P is then converted to UDP-GlcA by a UDP-sugar pyrophosphorylase such as Arabidopsis thaliana AtUSP. The AtGlcAK was cloned and characterized by the inventors.Other sugars, including iduronic acid (IdoA) and galacturonic acid (GalA), can also be used as substrates for GlcAKs in the methods of the invention.
[0070] Various UDP-sugars can be synthesized using the methods of the invention. In some embodiments, the UDP-sugar is selected from substituted or unsubstituted UDP-Glc, substituted or unsubstituted UDP-GlcA, substituted or unsubstituted UDP-IdoA, substituted or unsubstituted UDP-GalA, substituted or unsubstituted UDP-GlcNAc, substituted or unsubstituted UDP-GlcNFL, substituted or unsubstituted UDP-Gal, substituted or unsubstituted UDP-GalNAc, substituted or unsubstituted UDP-GalNH2, substituted or unsubstituted UDP-Man, substituted or unsubstituted UDP-ManNAc, and substituted or unsubstituted UDP-ManN¾,. In some embodiments, the UPD-sugar is selected from UDP- GlcNAc, UDP-GlcNH2, UDP-GlcA, UDP-IdoA, UDP-GalA, UDP-Gal, UDP-Man, and UDP-Glc. The UDP-sugar can also have the structure of formula I described above.
[0071] The hydroxyl groups, the amino group, and the iV-acetyl amino group in UDP-sugar can be substituted with any suitable substituent. In some embodiments, the hydroxyl groups, the amino group, and the V-acetyl amino group in UDP-sugar can be substituted with an azide, an amine, an iV-trifluoroacetyl group, an N-acyl group, an < -sulfate, or an N-sulfate.
[0072] The reaction mixture formed in the methods of the invention can further include an inorganic pyrophosphatase (PpA). PpAs can catalyze the degradation of the pyrophosphate (PPi) that is formed during the conversion of a sugar- 1 -phosphate to a UDP-sugar. PPi degradation in this manner can drive the reaction towards the formation of the UDP-sugar products. The pyrophosphatase can be, but is not limited to, Pasteiirella multocida PmPpA (Lau K, Thon V, Yu H, Ding L, Chen Y, Muthana MM, Wong D, Huang R, and Chen X. Chem. Commun. 2010, 46, 6066-6068).
[0073] The reaction mixture in the present methods can be formed under any conditions sufficient to convert the first sugar to a UDP-sugar or an intermediate such as a sugar- 1 - phosphate. The reaction mixture can include, for example, buffering agents to maintain a desired pH, as well as salts and/or detergents to adjust the solubility of the enzymes or other reaction components. In general, the reaction mixture also includes one or more nucleotide triphosphates (NTPs), such as UTP or ATP, that are consumed during sugar phosphorylation and UDP-sugar formation. The reaction mixture can contain a stoichiometric amount of an NTP, with respect to the first sugar, or an excess of the NTP. Divalent metal ions, such as magnesium ions, manganese ions, cobalt ions, or calcium ions, may be required to maintain the catalytic activity of certain enzymes. Enzyme cofactors, including but not limited to nicotinamide adenine dinucleotide (NAD"), can also be included in the reaction mixture. In some embodiments, the reaction mixture further includes at least one component selected from UTP, ATP, Mn~ , Co" . Ca" , and Mg" . After the reaction mixture is formed, it is held under conditions that allow for the conversion of the first sugar to the U DP sugar. For example, the reaction mixture can be held at 37 °C for 1 min-72 hr to form the UDP-sugar. The reaction mixture can also be held at 25 °C to form the UDP-sugar. Other temperatures and conditions may be suitable for forming the UDP-sugar, depending on the nature of the first sugar and the enzymes used for the synthesis. [0074] In some embodiments, the invention provides a method of synthesizing a UDP- sugar of Formula I:
Figure imgf000019_0001
The method includes forming a reaction mixture comprising a first sugar, a nucleotide-sugar pyrophosphorylase, and a first enzyme selected from the group consisting of a kinase and a dehydrogenase under conditions sufficient to form the UDP-sugar. In some embodiments, the first sugar has the formula VII:
Figure imgf000019_0002
Wherein each of R1, R2, and R3 is selected from OH, N3, NH2, NHS03\ OS03\ NHC(0)CH3, NHC(0)CF3, NHC(0)CH2OH, and NHC(0)CH2N3; R4 is selected from CH2OH, C02 ", C02H, CH2N3, CH2NH2, CH2NHS03 ", CH2OS03 ", CH2NHC(0)CH3, CH2NHC(0)CF3, CH2NHC(0)CH2OH, and CH2NHC(0)CH2N3; and R5 can be H, P03 2 or HP03 ".
[0075] Certain enzymes that are useful in the methods of the invention are characterized by a level of substrate promiscuity that allows for the synthesis of various natural and non- natural UDP-sugars. The scope of the products can be widened further by chemically appending a range of functionality to common enzymatically synthesized UDP-sugars. A UDP-sugar containing an azido moiety, for example, can be reduced to form an amino moiety which can be further elaborated via amide bond formation or TV-sulfation to install various functional groups in the UDP-sugar. Similarly, trifluoracctamido moieties can also be converted to amino moieties for further derivitization. Accordingly, some embodiments of the invention include converting a UDP- azido-sugar or a UDP- trifluoroacetamido-sugar to a UDP-amino-sugar. In some embodiments, the UDP amino-sugar is further converted to a UDP-acylamido-sugar or a UDP-TV-sulfated-sugar.
V. ONE-POT METHOD OF MAKING OLIGOSACCHARIDES
[0076] The method described above for preparing UDP-sugars can be extended by incorporating additional enzymes that incorporate the sugar in UDP-sugars into
oligosaccharide products. Accordingly, some embodiments of the invention provide a method of preparing an oligosaccharide. The method includes forming a first reaction mixture containing a first sugar, an acceptor sugar, a glycosyltransferase, a nucleotide-sugar pyrophosphorylase, and an enzyme selected from a kinase and a dehydrogenase. The first sugar is selected from a substituted or unsubstituted vV-acetylglucosamine (2-acetamido-2- deoxy glucose, GlcNAc), a substituted or unsubstituted glucosamine (GlcNH2), a substituted or unsubstituted glucuronic acid (GlcA), a substituted or unsubstituted iduronic acid (IdoA), and a substituted or unsubstituted glucose- 1 -phosphate (Glc- l -P), and the acceptor sugar includes at least one member selected from a substituted or unsubstituted N- acetylglucosamine (GlcNAc), a substituted or unsubstituted glucosamine (GlcNH2), a substituted or unsubstituted glucuronic acid (GlcA), and a substituted or unsubstituted iduronic acid (IdoA). The reaction mixture is formed under conditions sufficent to convert the first sugar to a UDP-sugar, and sufficient to couple the sugar in the UDP-sugar to the acceptor sugar. When the first sugar is substituted or unsubstituted GlcNAc or GlcNHi, the sugar in the UDP-sugar is coupled to a substituted or unsubstituted GlcA or a substituted or unsubstituted IdoA of the acceptor sugar. When the first sugar is substituted or unsubstituted Glc- l -P, substituted or unsubstituted GlcA, or substituted or unsubstituted IdoA, the sugar in the UDP-sugar is coupled to a substituted or unsubstituted GlcNH2 or a substituted or unsubstituted GlcNAc of the acceptor sugar.
[0077] In some embodiments, the first sugar has the formula:
Figure imgf000020_0001
Wherein each of PJ , R2, and R3 is selected from OH, N3, NH2, NHS03\ OS03\ NHC(0)CH3, NHC(0)CF3, NHC(0)CH2OH, and NHC(0)CH2N3; R4 is selected from CH2OH, C02\ C02H, CH2N3, CH2NH2, CH2NHS03\ CH2OS03\ CH2NHC(0)CH3, CH2NHC(0)CF3, CH2NHC(0)CH2OH, and CH2NHC(0)CH2N3; and R5 can be H, P03 2", or HP03 ". In some embodiments, the first sugar has the formula VIII or IX: (or OP03H ) (I X)
Figure imgf000020_0002
[0078] The first sugar is converted to the UDP-sugar by the UDP-sugar pyrophosphorylase and the kinase/dehydrogenase as described above. In some embodiments, the first sugar is selected from substituted or unsubstituted glucose (Glc), substituted or unsubstituted glucose- 1-phosphate (Glc-l -P), substituted or unsubstituted glucuronic acid (GlcA), substituted or unsubstituted iduronic acid (IdoA), substituted or unsubstituted glucuronic acid- 1 -phosphate (GlcA- l -P), substituted or unsubstituted iduronic acid (IdoA), substituted or unsubstituted iduronic acid- 1 -phosphate (IdoA- l -P), substituted or unsubstituted jV-acetylglucosamine (GlcNAc), substituted or unsubstituted A -acetylglucosamine- l -phosphate (GlcNAc- l -P), substituted or unsubstituted glucosamine (GICNH2), substituted or unsubstituted glucosamine- 1 -phosphate (GlcNH2-l -P), substituted or unsubstituted galactose (Gal), substituted or unsubstituted galactose- 1 -phosphate (Gal- l -P), substituted or unsubstituted galacturonic acid (GalA), substituted or unsubstituted galacturonic acid- 1 -phosphate (GalA- l -P), substituted or unsubstituted N-acetylgalactosamine (GalNAc), substituted or unsubstituted N- acetylgalactosamine- 1 -phosphate (GaFN Ac- 1 -P), substituted or unsubstituted galactosamine (GalNH ), substituted or unsubstituted galactosamine- 1 -phosphate (GalNHo- l -P), substituted or unsubstituted mannose (Man), substituted or unsubstituted mannose-1 -phosphate (Man- 1 - P), substituted or unsubstituted iV-acetylmannosamine (ManNAc), substituted or unsubstituted N-acetylmannosamine- l -phosphate (ManNAc- 1 -P), substituted or unsubstituted mannosamine (ManN¾), substituted or unsubstituted mannosamine- 1 -phosphate (ManNl L- 1-P). In some embodiments, the UDP-sugar is a compound of Formula I .
[0079] The sugar in the UDP-sugar is, in turn, coupled to an acceptor sugar to form an oligosaccharide product. A variety of sugars can be used as the acceptor sugar. For example, the acceptor sugar can be a monosaccharide, a disaccharide, a tri saccharide, or a tetrasaccharide. Longer oligosaccharides may also be used as the acceptor sugar in the methods of the invention. In some embodiments, the oligosaccharide can be a compound of Formula II, III, IV, V, or VI.
[0080] In general, the sugar in a UDP-sugar is coupled to an acceptor sugar by the glycosyltransferase in the reaction mixture. Any suitable glycosyltransferase can be used in the methods of the invention. Certain glycosyltransfers have exhibited a level of substrate promiscuity that are particularly useful for preparing a variety of oligosaccharide products. Promiscuous glycosyltransferases can utilize a range of UDP-sugars and/or a range of acceptor sugars. The glycosyltransferase can be, for example, P. multocida PmHS l or
PmHS2. The glycosyltransferase can also be E. coli KfiA or KfiC. Other glycosyltransferases can also be useful in the methods of the invention. In some embodiments, the
glycosyltransferase is selected from PmHS l , PmHS2 and KfiA. [0081] In general, the UDP-sugar can be formed enzymatically in the one-pot reaction mixture as described above. The nucleotide-sugar pyrophosporylase can be, but is not limited to, a glucosamine uridyltransferase (GlmU), a Glc- l -P uridylyltransferase (GalU), or a promiscuous UDP-sugar pyrophosphorylase (USP). In some embodiments, the nucleotide- sugar pyrophosphorylase is selected from AGX 1 , EcGlmU, EcGalU, PmGlmU, and BLUSP. In some embodiments, the nucleotide-sugar pyrophosphorylase is selected from AGX 1 , EcGalU, and BLUSP. In some embodiments, the nucleotide-sugar pyrophosphorylase is selected from EcGalU, PmGlmU, and BLUSP. In some embodiments, the nucleotide-sugar pyrophosphorylase is EcGalU. In some embodiments, the nucleotide-sugar
pyrophosphorylase is PmGlmU. In some embodiments, the nucleotide-sugar
pyrophosphorylase is BLUSP.
[0082] In some embodiments, the kinase in the reaction mixture is selected from an N- acetylhexosamine 1 -kinase (NahK), a galactokinase (GalK), and a glucuronokinase (GlcAK). In some embodiments, the kinase is selected from NahK_ATCC 15697, NahK_ ATCC55813, EcGalK, SpGalK, and AtGlcAK. In some embodiments, the kinase is selected from
NahK ATCC 1 5697, NahK_ATCC55813, EcGalK, and AtGlcAK. In some embodiments, the kinase is selected from NahK ATCC 1 5697, NahK_ATCC5581 3, and AtGlcAK. In some embodiments, the kinase is EcGalK. In some embodiments, the kinase is
NahK_ATCC 15697. In some embodiments, the kinase is NahK_ATCC55813. In some embodiments, the kinase is AtGlcAK. In some embodiments, the kinase is
NahK_ATCC55813.
[0083] In some embodiments, the dehydrogenase in the reaction mixture is UDP-glucose dehydrogenase (Ugd). In some embodiments, the Ugd is PmUgd.
[0084] In some embodiments, the UDP-sugar formed in the one-pot reaction mixture is selected from substituted or unsubstituted UDP-GlcNAc, substituted or unsubstituted UDP- Glc, substituted or unsubstituted UDP-GlcA, and substituted or unsubstituted UDP-IdoA. In some embodiments, the UDP-sugar is substituted with at least one moiety selected from an azide, an amine, an TV-trifluoroacetyl group, an N-acylamido group, an ( -sulfate, and an N- sulfate.
[0085] In some embodiments, the reaction m ixture further contains a pyrophosphatase. In some embodiments, the pyrophosphatase is PinPpA. [0086] The reaction mixture in the present methods can be formed under any suitable conditions sufficient to prepare an oligosaccharide. The reaction mixture can include, for example, buffering agents to maintain a desired pH, as well as salts and/or detergents to adjust the solubility of the enzymes or other reaction components. In general, the reaction mixture also includes one or more nucleotide triphosphates (NTPs), such as UTP or ATP, that are consumed during sugar phosphorylation and UDP-sugar formation. The reaction mixture can contain a stoichiometric amount of an NTP, with respect to the first sugar, or an excess of the NTP. Divalent metal ions, such as magnesium ions, manganese ions, cobalt ions, or calcium ions, may be required to maintain the catalytic activity of certain enzymes. Enzyme cofactors, including but not limited to nicotinamide adenine dinucleotide (NAD+), can also be included in the reaction mixture. In some embodiments, the reaction mixture further includes at least one component selected from UTP, ATP, Mn2+, Co2+, Ca2+, and Mg2+. After the reaction mixture is formed, it is held under conditions that allow for preparation of the oligosaccharide. For example, the reaction mixture can be held at 37 °C for 1 min-72 hr. The reaction mixture can also be held at 25 °C. Other temperatures and conditions may be suitable for forming the oligosaccharide, depending on the nature of the sugars and the enzymes used for the synthesis.
[0087] Heparin and heparan sulfate (HS) oligosaccharides have particularly important biological, pathological, and therapeutic properties. Heparin and HS are sulfated linear polysaccharides composed of alternating a 1 -4 linked D-glucosamine (GlcNH ) residues and 1 -4 linked uronic acid resiudes (a-linkage for iduronic acid, IdoA, and β-linkage for glucuronic acid, GlcA]. In order to prepare heparin and HS oligosaccharides and their analogs, the methods of the invention can be used to prepare oligosaccharides containing alternating glucosamine and uronic acid residues. The oligosaccharides can contain, for example, alternating GlcNAc residues and GlcA residues. In some embodiments, the oligosaccharide is selected from: GlcNAc-GlcA; GlcA-GlcNAc-GlcA; GlcNAc-GlcA- GlcNAc-GlcA; GlcA-GlcNAc-GlcA-GlcNAc-GlcA; GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc- GlcA; GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA; GlcNAc-GlcA-GlcNAc-GlcA- GlcNAc-GlcA-GlcNAc-GIcA; GlcA-G!cNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc- GlcA; GlcA-GlcNAc; GlcNAc-GlcA-GlcNAc; GlcA-GlcNAc-GlcA-GIcNAc; GlcNAc-
GlcA-GlcNAc-GlcA-GlcNAc; GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc; GlcNAc-GlcA- GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc; GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA- GlcNAc; GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc; and GlcA- GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GIcNAc-GlcA-GlcNAc. In some embodiments, each GlcA and GlcNAc are optionally independently mono- or multi-substituted with a moiety selected from an azide, an amine, an -trifluoroacetyl group, an N-acyl group, and an N-sulfate.
[0088] Other oligosaccharides can also be prepared using the methods of the invention. Oligosaccharides of arbitrary length can be prepared by repeating the one-pot reaction methods as described above. Accordingly, some embodiments of the invention provide a method for preparing an oligosaccharide as described above, wherein the method is repeated with a second sugar in place of the first sugar and the oligosaccharide in place of the acceptor sugar. In this manner, a variety of products can be prepared. In some embodiments, the oligosaccharides of the present invention can be a compound of any of Formulas II, III, IV,
V, or VI.
[0089] In some embodiments, the present invention provides a method of prepar oligosaccharide of formula II:
Figure imgf000024_0001
wherein the method includes forming a first reaction mixture containing a first sugar, an acceptor sugar, a glycosyltransferase, a UDP-sugar pyrophosphorylase, and/or one enzyme selected from a kinase and a dehydrogenase. The first sugar is selected from a substituted or unsubstituted iV-acetylglucosamine (GlcNAc), a substituted or unsubstituted glucosamine (GlcNH2), a substituted or unsubstituted glucoronic acid (GlcA), a substituted or unsubstituted iduronic acid (IdoA), and a substituted or unsubstituted glucose-l -phosphate (Glc- l -P), and the acceptor sugar includes at least one member selected from a substituted or unsubstituted iV-acetylglucosamine (GlcNAc), a substituted or unsubstituted glucosamine (GlcNHb), a substituted or unsubstituted glucuronic acid (GlcA), and substituted or unsubstituted iduronic acid (IdoA). The reaction mixture is formed under conditions sufficent to convert the first sugar to a UDP-sugar having a structure of formula I :
Figure imgf000024_0002
UDP (I), and sufficient to couple the sugar in the UDP-sugar to the acceptor sugar. The first sugar can have a structure of the formula VII:
Figure imgf000025_0001
Each of R1, R2, R3, Rl a, Rl b, R2a, and R2b is independently selected from OH, N3, NH2, NHS03 ", OSO3", NHC(0)CH3, NHC(0)CF3, NHC(0)CH2OH, or NHC(0)CH2N3; each of R4, Rl c, and R2c is independently selected from CH2OH, C02\ C02H, CH2N3, CH2NH2, CH2NHS03 CH2OSO3", CH2NHC(0)CH3, CH2i HC(0)CF3, CH2NHC(0)CH2OH, or CH2NHC(0)CH2N3; R includes but not is limited to H, CH3, CH2CH3, CH2CH2N3,
CH2CH2CH2N3, an aglycon according to Formula B, Formula C, Formula D, or Formula E below, substituted or unsubstituted GlcNAc, substituted or unsubstituted GlcNHo, substituted or unsubstituted GlcA, or substituted or unsubstituted IdoA. When R4 is C02 " or C02H, then R2c is CO," or C02H, and Rlc is is selected from CH2OH, CH2N3, CH2NH2, CH2NHS03\ CH2OS03 ", CH2NHC(0)CH3, CH2NHC(0)CF3, CH2NHC(0)CH2OH, and
CH2NHC(0)CH2N3. Altematively, when R4 is CH2OH, CH2N3, CH2NH2, CH2NHSO3", CH2OSO3", CH2NHC(0)CH3, CH2NHC(0)CF3, CH2NHC(0)CH2OH, or
CH2NHC(0)CH2N3, then Rl c is C02 " or C02H, and R2c is R4.
Figure imgf000025_0002
[0090] Heparin and HS generally contain varying levels of sulfated sugar residues.
Examples of sulfated sugar residues include, but are not limited to, GlcNS, containing an N- sulfate at the 2 position of glucosamine (GICNH2); GlcNS3S, containing an TV-sulfate at the 2 position and an O-sulfate at the 3 position of glucosamine (GlcNH2); GlcNS6S, containing an N-sulfate at the 2 position and an O-sulfate at the 6 position of glucosamine (GICNH2);
GlcNS3S6S, containing an /-sulfate at the 2 position, an O-sulfate at the 3 position, and an O-sulfate at the 6 position of glucosamine (GlcNH2); GlcNAc3S, containing an O-sulfate at the 3 position of N-acetylglucosamine (GlcNAc); GlcNAc6S, containing an O-sulfate at the 6 position of N-acetylglucosamine (GlcNAc); GlcNAc3S6S, containing an O-sulfate at the 3 position and an -sulfate at the 6 position of ./V-acetylglucosamine (GlcNAc); GlcNH23S, containing an O-sulfate at the 3 position of glucosamine (GICNH2); GlcNH26S, containing an -sulfate at the 6 position of glucosamine (GlcNH2); GlcNH23S6S, containing an -sulfate at the 3 position and an O-sulfate at the 6 position of glucosamine (GlcNH2); GlcA2S, containing an O-sulfate at the 2 position of glucuronic acid (GlcA); and IdoA2S, containing an O-sulfate at the 2 position of iduronic acid (IdoA). Substrate preferences for the methods of the invention will vary depending on the specific enzymes employed in the one-pot reactions. As described above, various substituted and unsubstituted sugars can be used in the methods of the invention.
[0091] The present inventors have discovered enzymes that exhibit catalytic activity for a number of natural and non-natural UDP-sugar and acceptor sugar substrates. The oligosaccharides that are prepared using these enzymes can contain functional moieties that can be chemically modified to diversify the structure of the products. For example, azido- sugar residues or trifluoroacetamido-sugar residues can be converted to amino-sugar residues. Azido groups and trifluoracetamos groups can be manipulated independently using orthogonal chemical methods to selectively install desired functionality at specific sites on a given oligosaccharide. Amine-containing ol igosaccharides can be further elaborated to form acylamino groups and sulfamate groups. Sulfamate (i.e. Λ-sulfate) groups, in particular, can be instal led to form heparin and HS analogs.
[0092] The inventors have discovered that certain oligosaccharides containing N-sulfate groups (where O-sulfate groups would normally be present in heparin and HS) demonstrate inhibitory activity aganst the binding of fibroblast growth factors (FGFs) to heparin. FGFs, in turn, have a role in regulating a number of processes including angiogenesis, cell proliferation, differentiation, morphogenesis, and wound healing. As such, the invention provides convenient and flexible methods for preparation of oligosaccharides with useful biological activity.
VI. EXAMPLES
Example 1. Enzymes
NahK ATCC15697 and NahK ATCC55813 - N-acetylhexosamine 1-kinases
[0093] NahK (EC 2.7. 1.162) catalyzes the direct addition of a phosphate from adenosine 5'- triphophate (ATP) to the anomeric position of ZV-acetylhexosamine for the formation of N- acetylhexosamine- 1 -phosphate and adenosine 5'-diphophate (ADP). The only characterized NahK to date is encoded by the InpB gene in the InpABCD operon of Bifidobacterium longum JCM 1217. Herein we report the cloning and characterization of two new NahKs from Bifidobacterium infantis (ATCC 1 5697) and Bifidobacterium longum (ATCC55813), respectively. A new capillary electrophoresis-based assay method has been developed for biochemical characterization of NahKs. We found that in addition to previously reported NahK substrates, various GlcNAc derivatives including those with C2-azido, C6-azido, and 6-0-sulfate groups are tolerable substrates for the newly cloned NahKs. In addition, despite of their low activities toward glucose and galactose, the activities of both NahKs are much higher for mannose and some of its C-2, C-4, and C-6 derivatives including 2-deoxy- mannose or 2-deoxy-glucose.
Experimental
[0094] Bacterial strains, plasmids, and materials. Electrocompetent DH5a and chemically competent BL21 (DE3) E. coli cells were from Invitrogen (Carlsbad, CA).
Bifidobacterium longum Reuter ATCC#55813 was from American Type Culture Collection (ATCC, Manassas, VA). Genomic DNA of Bifidobacterium longum subsp. infantis
(ATCC# 15697) was a kind gift from Professor David Mills (University of California, Davis). Vector plasm id pET22b(+) was from Novagen (EMD Biosciences Inc. Madison, WI). Ni2+- NTA agarose (nickel-nitrilotriacetic acid agarose), QIAprcp spin miniprep kit, and QIAEX II gel extraction kit were from Qiagen (Valencia, CA). Herculase-enhanced DNA polymerase was from Stratagene (La Jolla, CA). T4 DNA ligase and 1 kb DNA ladder were from Promega (Madison, WI). Ndel and Xho\ restriction enzymes were from New England Biolabs Inc. (Beverly, MA). Adenosine-5'-triphosphate disodium salt (ATP), GlcNAc, and GalNAc were from Sigma (St. Louis, MO). GlcNAc, GalNAc, mannose, and ManNAc derivatives were synthesized according to reported procedures.
[00951 Cloning. NahK_ATCC 15697 and ahK ATCC55813 were each cloned as a C- His6-tagged fusion protein in pET22b(+) vector using genomic DNAs of Bifidobacterium longum subsp. infantis ATCC#15697 and Bifidobacterium longum ATCC#55813, respectively, as the template for polymerase chain reactions (PCR). The primers used for NahK ATCC 15697 were: forward primer 5 '
ACCCCATATGAACAACACCAATGAAGCCCTG 3 ' (Ndel restriction site is underlined) and reverse primer 5' TGACCTCGAGCTTGGTCGTCTCCATGACGTCG T(Xho\ restriction site is underlined). The primers used for Nah _ATCC55813 were: forward primer 5' ACCCCATATGACCGAAAGCAATGAAGTTTTATTC 3 ' (Ndel restriction site is underlined) and reverse primer 5' TGACCTCGAGCCTGGCAGCCTCCATGATG V(Xh.ol restriction site is underlined). PCR was performed in a 50 μΐ^ reaction mixture containing genomic DNA ( 1 μg), forward and reverse primers (1 μΜ each), 10 x Herculase buffer (5 μΙ_.), dNTP mixture (1 mM), and 5 U ( 1 μί) of Herculase-enhanced DNA polymerase. The reaction mixture was subjected to 35 cycles of amplification with an annealing temperature of 52 °C. The resulting PCR product was purified and digested with Ndel and Xhol restriction enzymes. The purified and digested PCR product was ligated with predigested pET22b(+) vector and transformed into electrocompetent E. colt DH5oc cells. Selected clones were grown for minipreps and characterization by restriction mapping and DNA sequencing performed by Davis Sequencing Facility at the University of California-Davis.
[0096] Expression and purification. Positive plasmids were selected and transformed into BL21 (DE3) chemically competent cells. The plasmid-bearing E. coli cells were cultured in LB rich medium (10 g L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) supplied with ampicillin ( 100 μg mL). Overexpression of the target protein was achieved by inducing the E. coli culture with 0.1 mM of isopropyl- l -thio- -D-galactopyranoside (IPTG) when the OD6oo nm of the culture reaches 0.8-1 .0 followed by incubation at 20 °C for 24 h with vigorous shaking at 250 rpm in a C25KC incubator shaker (New Brunswick Scientific, Edison, NJ). To obtain the cell lysate, cells were harvested by centrifuge cell culture at 4000 x g for 2 h. The cell pellet was re-suspended in lysis buffer (pH 8.0, 100 mM Tris-HCl containing 0.1 % Triton X-100, 20 mL/L cell culture) containing lysozyme (100 μg/mL) and DNasel (3 μg/mL). After incubating at 37 °C for 60 min with vigorous shaking (250 rpm), the lysate was collected by centrifugation at 12,000 g for 30 min. His6-tagged target proteins were purified from cell lysate using an AKTA FPLC system (GE Healthcare, Piscataway, NJ, USA). To do this, the lysate was loaded to a HisTrap™ FF 5 mL column (GE Healthcare) pre-washed and equilibrated with binding buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 7.5). The column was then washed with 8 volumes of binding buffer, 10 volumes of washing buffer (10 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5) and eluted with 8 volumes of e lute- buffer (200 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5). Fractions containing the purified enzyme were combined and dialyzed against dialysis buffer (Tris-HCl containing 10% glycerol, pH 7.5, 20 mM) and stored at 4 °C.
[0097] Quantification of purified protein. Protein concentration was determined in a 96- well plate using a Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce Biotechnology, Rockford, IL) with bovine serum albumin as a protein standard. The absorbance of each sample was measured at 562 nm by a BioTek Synergy™ HT Multi-Mode Microplate Reader.
[0098] pH Profile by capillary electrophoresis (CE) assays. Typical enzymatic assays were performed in a 20 μL· reaction mixture containing a buffer (200 mM) with a pH in the range of 6.0-1 1.0, GlcNAc ( 1 mM), ATP ( 1 mM), MgCl2 (5 mM), and a NahK (0.75 μΜ). Buffers used were: MES, pH 6.0; Tris-HCl, pH 7.0-9.0; CAPS, pH 10.0-1 1 .0. Reactions were allowed to proceed for 10 min at 37 °C and were stopped by adding 20 of cold ethanol to each reaction mixture. Samples were centrifuged and the supernatants were analyzed by a P/ACE™ Capillary Electrophoresis (CE) system equipped with a Photodiode Array (PDA) detector (Beckman Coulter, Inc., Fullerton, CA). CE conditions were as follows: 75 μιη i.d. capillary, 25 KV/80 μΑ, 5 s vacuum injections, monitored at 254 nm, the running buffer used was sodium tetraborate (25 mM, pH 10.0).
[0099] Effect of MgC on the enzymatic activity. Different concentrations of MgCl? were used in a Tris-HCl buffer (pH 8.0, 200 mM) containing GlcNAc ( 1 mM), ATP ( 1 mM), and a NahK (0.75 μΜ). Reactions were allowed to proceed for 1 0 m in at 37 °C. Reaction without MgCh was used as a control .
[0100] Substrates specificity assays. GlcNAc, GalNAc, and their derivatives (1 mM) were used as substrates in the presence of ATP ( 1 mM) and MgC (5 mM) in a Tris-HCl buffer (pH 8.0, 200 mM) to analyze the substrate specificity of NahKs. Two concentrations (0.75 μΜ or 15 μΜ) of each NahK were used and the reactions were allowed to proceed for 10 min (for 0.75 μΜ NahK) or 30 min (for 15 μΜ NahK) at 37 °C. For substrate specificity studies of Glc, Gal, mannose, ManNAc, and their derivatives, 15 μΜ of NahK was used for each reaction and the reactions were carried out at 37 °C for 30 min. All other conditions were the same as for GlcNAc, GalNAc, and their derivatives.
[0101] Kinetics by CE assays. Reactions were carried out in duplicate at 37 °C for 10 min in a total volume of 20 μΐ in Tris-HCl buffer (200 in , pH 7.5) containing gCl2 (1 mM), ATP, GlcNAc or GalNAc, and NahK (0.25 μΜ when GlcNAc and ATP were used as substrates, 0.5 μΜ when GalNAc and ATP were used as substrates). Apparent kinetic parameters were obtained by varying the ATP concentration from 0.1 -5.0 mM (0.1 mM, 0.2 mM, 0.4 mM, 1 mM, 2 mM, and 5 mM) at a fixed concentration of GlcNAc or GalNAc (1 mM), or varying the concentration of GlcNAc or Gal Ac (0.1 mM, 0.2 mM, 0.4 mM, 1 mM, 2 mM, and 5 mM) at a fixed concentration of ATP ( 1 mM) and fitting the data to the Michaelis-Menten equation using Grafit 5.0.
Results and Discussion
[0102] Cloning, expression, and purification. NahKs from Bifidobacterium, infantis ATCC# 15697 (NahK_ATCC 15697) and Bifidobacterium longum ATCC#55813
(NahK_ATCC55813) were each cloned as a C-His6-tagged fusion protein in a pET22b(+) vector. Sequence alignment (Figure 1) indicates that NahK_ATCC55813 is almost identical to the NahK from Bifidobacterium longum JCM 1217 (NahK_JCM 1217, GenBank accession no. BAF73925) except for a single amino acid difference R348H (R is in NahK_JCM 1217). In comparison, NahK_ATCC 15697 shares 90% amino acid sequence identity with
NahK_JCM 1217.
[0103] Both NahKs were expressed by induction with 0. 1 mM of isopropyl- 1 -thio-P-D- galactopyranoside (IPTG) followed by incubation at 20 °C for 24 h with vigorous shaking (250 rpm). Up to 180 mg and 1 85 mg of Ni2+-column purified NahK_ATCC 15697 and NahK_ATCC55813, respectively, could be obtained from one l iter of E. coli culture. SDS- PAGF analysis shows that both purified proteins migrated to around 41 kDa, matching well to the calculated molecular weights of the translated Hise-tagged fusion proteins of 41.4 and 40.9 kDa for NahK ATCC l 5697 and NahK_ATCC5581 3, respectively.
[0104] Capillary electrophoresis (CE) assays. Based on the detection of ADP and ATP in the reaction mixture by a UV detector, a capillary electrophoresis-based method was developed to directly measure the formation of ADP and N-acetylhexosamine-1 -phosphate from ATP and N-acetylhexosamine for characterizing the activities of NahKs. Both ATP and ADP gave absorbance at 254 nra with equal signal responses.
[0105] pH profile. As shown in Figure 2, both NahKs are highly active in a pH range of 7.0-8.0 with slight variations. The activities of both NahKs drop quickly with either decrease of the pH to lower than 7.0 or increase of the pH to higher than 8.0. About 50% of the optimal activity was observed at pH 6.0 and pH 8.5 for NahK_ATCC 15697. In comparison, about 70% of the optimal activity was observed at pH 6.0 and pH 8.5 for
NahK_ATCC55813. The pH optima of these two enzymes are slight different from that (pH 8.5) of NahK_JCM121 7. Overall, the activity of NahK_ATCC55813 is higher than that of NahK_ATCC l 5697 in the pH range of 6.0-10.0 when GlcNAc was used as the substrate and the same molar concentrations of the enzymes were used.
[0106] Effect of MgCl2. Similar to NahK JCM l 21 7 and other kinases, both
NahK_ATCC 15697 and NahK_ATCC55813 require a divalent metal ion for activity. As shown in Figure 3, the optimal concentration of Mg2+ was determined to be 1 mM. The activities of both NahKs in the presence of 0.5 mM of Mg2+ were about two thirds of those in the presence of 1 .0 mM of Mg2+. Increasing the concentration of Mg2+ from 1 mM to 20 mM caused a slight decrease of the activities of both NahKs.
[0107] Kinetics. The apparent kinetic parameters shown in Table 1 indicate that the activities of two NahKs are close, with NahK_ATCC55813 having 16% or 39% higher activity than NahK_ATCC 15697 when GlcNAc or GalN Ac was used as the substrate in the presence of ATP. Overall, GlcNAc is a more efficient (3.1 -fold for NahK ATCC 15697 and 2.6-fold for NahK_ATCC55813) substrate than GalNAc for both NahKs due to relatively lower Km values and higher (~2-fold) kcu, values obtained when GlcNAc was used. Using ATP and GlcNAc as the substrates, the Km values of ATP (0. 1 0±0.03 mM and 0.1 1 ±0.03 mM) and GlcNAc (0.06±0.01 mM) for both NahKs are lower than those for NahK_JCM1217 (0.172 mM for ATP and 0.1 1 8 mM for GlcNAc) determined by high performance ion chromatography (HPIC) with a pulsed amperometric detector (DX500, Dionex Corporation, Sunnyvale, CA) using a Dionex CarboPac PA 1 column (4 mm * 250 mm). The discrepancies of the parameters may be due to the differences in the assay conditions used.
Table 1. Apparent kinetic parameters of NahKs.
Enzymes Nah l ATCC15697 NahK ATCC55813
Substrate K„ (mM) kc„, ( ]) kcJK,„(s ' mU' ) K„, (mM) (S ) kcJK,„(s ] mM" 1)
ATP" 0.10±0.03 1 .1±0.1 1 1.0 0.1 1±0.03 1.3±0.1 1 1.8 GIoNAc 0.06±0.01 0.95±0.01 15.8 0.06±0.01 1.1±0.1 18.3
ATP" 0.08±0.03 0.38±0.02 4.8 0.06±0.02 0.48±0.03 8.0
GalNAc 0.09±0.05 0.46±0.07 5.1 0.08±0.03 0.57±0.04 7.1
The other substrate is GlcNAc: The other substrate is GalNAc.
[0108] Substrate specificity. The substrate specificity studies using GlcNAc, GalNAc, and their derivatives (Table 2) indicate that both NahKs exhibit promiscuous substrate specificity and have comparable levels of activity toward GlcNAc and GalNAc derivatives. Compared to NahK_ATCC 15697, NahK_ATCC55813 is more reactive towards non-modified GlcNAc (T2-1), GalNAc (T2-11), and some of their C2-modified derivatives with an N-trifluoroacetyl (GlcNTFA T2-2 and GalNTFA T2-12), an N-azidoacetyl group (GlcNAcN3 T2-3 and GalNAcN3 T2-13), or an N-butanoyl group (GlcNBu T2-4 and GalNBu T2-14).
Nevertheless, NahK ATCC 15697 is more reactive than NahK_ATCC55813 for some of C2- modified GlcNAc and GalNAc derivatives such as those with a bulky N-benzoyl group (GlcNBz T2-5 and GalNBz T2-15) and a C2-azido group (GlcN3 T2-6 and GalN3 T2-16). NahK_ATCC 1 5697 is also more reactive towards 2-amino-2-deoxy-glucose (GlcNH2 T2-7), 2-N-sulfo-glucose (GlcNS T2-8), as well as C6-modified GlcNAc derivatives such as 6- deoxy-GlcNAc (GlcNAc6Me T2-9), 6-azido-6-deoxy-GlcNAc (GlcNAc6N3 T2-10), and 6- 6>-sulfo-GlcNAc (GlcNAc6S T2-17). Both C2 and C6-modified derivatives GlcNAc such as 6-O-sulfo-N-trifluoroacetyl glucosamine (GlcNTFA6S T2-18) and 6-0-sulfo-2-azido-2- deoxy glucose (GlcN3 T2-19) as well as both C2 and C3-modified GlcNAc derivative 3-0- sulfo-2-azido-2-deoxy glucose (GlcN33 S T2-20) are poor but acceptable substrates for both enzymes. Overall, some of the C2-modified GlcNAc and GalNAc (T2-1- T2-5 and T2-11- T2-14) are relatively good substrates for both NahKs with yields varied from 5.2-42.3% in a 10 min reaction containing 0.75 μΜ of enzyme. In comparison, other C2-modified GlcNAc and GalNAc (T2-6- T2-8, 15, T2-16), C6- (T2-9, T2-10, T2-17), both C2- and C6- (T2-18, T2-19), as well as both C2- and C3-modified GlcNAc (T2-20) derivatives are poor but tolerable substrates for both NahKs and the assays have to be carried out for a longer reaction time (30 min) with a 20-fold higher concentration ( 1 5 μΜ) of enzyme.
Table 2. Substrate specificity of NahKs using GlcNAc, GalNAc, and their derivatives.
Percentage Conversion (%) Percentage Conversion (%)
Figure imgf000033_0001
Figure imgf000033_0002
Figure imgf000033_0003
GlcNAc6Me
-0
-i-- ^'-,
NH 0 37.2+0.5 0 23.4+0.1 0 4.9±0.1 3.9±0.1
T2-20 GlcN33S
T2-10
GlcNAc6N3 NA: not assayed; aReactions were allowed to proceed for 10 min at 37 °C; bReactions were allowed to proceed for 30 min at 37 °C.
[0109] Among twenty compounds of GlcNAc, GalNAc and their derivatives tested, compounds T2-1, T2-3-T2-5, T2-9-T2-11, T2-13-T2-15 have been reported before as suitable substrates for Nah _JCM1217 [1 1 -12], while other compounds including T2-2, T2- 6-T2-8, T2-12, and T2-16-T2-20 are newly identified substrates for NahKs. It is worth to note that some of these compounds have negatively charged ( -sulfate group at different positions of GlcNAc or its derivatives.
[0110] Quite interestingly, the substrate specificity studies using glucose (Glc T3-21), galactose (Gal T3-28), mannose (Man T3-23), N-acetylmannosamine (ManNAc T3-29), and derivatives of mannose and ManNAc (Table 3) indicate that while both Glc (T3-21) and Gal (T3-28) are poor substrates for both NahKs, 2-deoxy glucose (2-deoxyGlc T3-22) or 2- deoxymannose is a better substrate. In addition, mannose (T3-23), its 2-fluoro- (2F-Man T3- 24) and 2-azido- (2N3-Man T3-26) derivatives, as well as its 4-deoxy (4-deoxyMan T3-27) derivative are relatively good substrates. In comparison, 2-methyl modification of mannose (2Me-Man T3-25) decreases its tolerance as the substrate for both NahKs. Quite surprisingly, while ManNAc (T3-29) and some of its C-2 derivatives (T3-30-T3-32) are poor substrates for the NahKs, N-azidoacetylmannosamiiie (ManNAcN3 T3-33, a C2-derivative of ManNAc) and its C6-derivative N-acetyl-6-O-methylmannosamine (ManNAc60Me T3-34) are better substrates for both NahKs. Overall, except for 2-fluoro-mannose (2F-Man T3-24),
NahK_ATCC 15697 shows higher activity than NahK_ATCC55813 for mannose, ManNAc, and their derivatives.
Table 3. Substrate specificity of NahKs using Glc, Gal, Man, ManNAc, and their derivatives.
Figure imgf000034_0001
Figure imgf000035_0001
°C for 30 min.
AtGIcAK - Arabidopsis thaliana glucuronokinase (EC 2.7.1.43)
Experimental
[0111] Cloning, expression, and purification. Full length Arabidopsis thaliana glucuronokinase (EC 2.7.1.43) (AtGIcAK) (encoded by gene GlcAKl, DNA GenBank accession number: GU599900; protein GenBank accession number: NP 566144) was cloned in pET15b vector from a cDNA library of Arabidopsis thaliana and expressed as an N-Hise- tagged fusion protein. The primers used were: forward primer 5 '
GGAATTCCATATGGATCCGAATTCCACGG 3' {Nde\ restriction site is bold and underlined) and reverse primer 5'
CCGCTCGAGTCATAAGGTCTGAATGTCAGAATCATTC 3 ' [Xho\ restriction site is bold and underlined). The resulting PCR products were digested with restriction enzymes, purified, and ligated with pET1 5b vector predigested with Nde\ and Xho\ restriction enzymes. The ligated product was transformed into electrocompetent E. coli DH5a cells. Selected clones were grown for minipreps and positive clones were verified by restriction mapping and DNA sequencing performed by Davis Sequencing Facility. The DNA sequence of the insert matched to GlcAKl .
[0112] The plasmid was transformed into E. coli BL21 (DE3) chemically competent cells for protein expression. E. coli cells harboring the pETl 5b-AtGlcAK plasmid were cultured in LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) with ampicillin (100 μg mL) at 37 °C with rigorous shaking at 250 rpm in a C25KC incubator shaker (New Brunswick Scientific, Edison, NJ) until the OD600 nm of the culture reached 0.8-1.0.
Overexpression of the targeted proteins was achieved by adding 0.15 mM of isopropyl- 1 - thio-P-D-galactopyranoside (IPTG) followed by incubation at 18 °C for 20 h with rigorous shaking at 250 rpm.
[0113] His<5-tagged protein was purified from cell lysate using Ni2+-NTA affinity column. To obtain cell lysate, cells were harvested by centrifugation at 4,000 rpm (Sorvall) at 4 °C for 2 h. The cell pellet was resuspended in lysis buffer (pH 8.0, 100 mM Tris-HCl containing 0.1 % Triton X-100). Lysozyme ( 100 μ /ITlL) and DNasel (5 g/mL) were added to the cell suspension. The mixture was incubated at 37 °C for 1 hr with vigorous shaking (200 rpm). Cell lysate was obtained as the supernatant by centrifugation at 1 1 ,000 rpm (Sorvall) at 4 °C for 45 min. Purification was performed by loading the supernatant onto a Ni2+-NTA column pre-equilibrated with 10 column volumes of binding buffer (10 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was wash with 10 column volumes of binding buffer and 10 column volumes of washing buffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). Protein of interest was eluted with Tris-HCl (pH 7.5, 50 mM) containing imidazole (200 mM) and NaCl (0.5 M). The fractions containing the purified enzyme were collected and dialyzed against Tris-HCl buffer (pH 7.5, 20 mM) containing 30% glycerol. Dialyzed proteins were stored at -20 °C. Alternatively, fractions containing purified enzyme were dialyzed against Tris-HCl buffer (pH 7.5, 20 mM) and freeze dried. On average, 57 mg of purified protein was obtained from 1 l iter of cell culture.
[0114] Substrates specificity assays by thin-layer chromatography (TLC).
Enzymatic assays were carried out in a total volume of 10 μί in Tris-HCl buffer (100 mM, pH 7.5) containing GlcA (or Gal A, IdoA, xylose) ( 10 mM), ATP (20 mM), MgCl2 (20 mM), and AtGlcA (22 μg). Reactions were allowed to proceed at 37 °C for 1 5 hr and monitored using thin-layer chromatographic (TLC) analysis using «-PrOH:H20:NH4OH = 7:4:2 (by volume) as a developing solvent. For visualizing compounds on TLC plate, />-anisaldehyde sugar stain was used.
[0115] LC-MS assays for AtGlcAK reactions. The AtGlcAK reaction mixtures above were also analyzed by LC-MS. 2 μL· of sample was diluted 100 fold and 8 μΐ^ was injected into a Waters spherisorb ODS-2 column (5 μπι particles, 250 mm length, 4.6 mm I.D.). The sample was eluted with 30 % acetonitrile in ¾0 with 0.1 % formic acid and detected by ESI- MS in negative mode.
Results and Discussion
[0116] Substrates specificity assays. As shown in Figure 25 and Figure 26, thin-layer chromatography results and mass spectrometry (MS) results showed that GlcA, GalA, and IdoA are acceptable substrates for AtGlcAK for the formation of GlcA- l -P, GalA- l -P, and IdoA- l P, respectively.
PmGimU— Pasteurella multocida glucosaminyl uridyltransferase
[0117] Glycosyltranferases are key enzymes for the formation of oligosaccharides and glycoconjugates in nature. Most glycosyltransferases require sugar nucleotides as donor substrates and catalyze the transfer of monosaccharides from sugar nucleotides to acceptors in high regio- and stereoselective manner. Some carbohydrate structures contain post- glycosylational modifications (modifications on carbohydrates and glycoconjugates which take place after the formation of glycosidic bonds). One strategy to obtain naturally existing oligosaccharides and glycoconjugates with modified sugar moieties is to develop novel chemoeiizymatic methods using structurally modified monosaccharides as starting materials and carbohydrate biosynthetic enzymes (the simplest carbohydrate biosynthetic route usually involves a monosaccharide kinase, a nucleotidyltransferase, and a glycosyltransferase) with substrate promiscuities. Carbohydrates with non-natural modifications can be synthesized similarly. Some of these compounds are potential drug candidates as they can effectively interfere with carbohydrate-dependent biological processes.
[0118] Glycosaminoglycans including keratan sulfate, heparan sulfate, and heparin are N- acetylglucosamine (GlcNAc)-containing polysaccharides with post-glycosylational modifications. While GlcNAc and 6-(9-sulfo-GlcNAc are commonly found in kearatan sulfate, additional modified GlcNAc forms such as N-sulfo- and 3-0-sulfo-GlcNAc are common for heparan sulfate and heparin. In addition, 6-O-sulfation on GlcNAc is also common in Lewis x and sialyl Lewis x structures and has been shown to affect the binding affinity of the related carbohydrate-binding proteins such as Selectins and Siglecs. In attempts to synthesizing glycans containing naturally modified GlcNAc and their non-natural derivatives using glycosyltransferase-catalyzed reactions, we applied an efficient one-pot three-enzyme approach to synthesize UDP-GlcNAc derivatives including UDP-6-O-sulfo- GlcNAc, UDP-GlcNTFA, and azido-containing UDP-GlcNAc derivatives. Additional UDP- GlcNAc derivatives, including UDP-N-suIfo-glucosamine, were also produced by chemical diversification from enzymatica!ly produced UDP-GlcNAc derivatives. These compounds will be tested as potential donor substrates for GlcNAc-glycosyltransferases.
Experimental
[0119] Cloning, expression, and purification of PmGlrnU. The gene sequence of
Pml806 from Pasteurella multocida subsp. ultocida strain Pm70 (GenBank accession no. AAK03890) was used as a reference for designing primers. The genomic DNA of Pasteurella multocida strain P-1059 (ATCC 15742) was used as a template for polymerase chain reaction (PCR). Full length Pasteurella ww/toc/da N-acetylglucosamine- 1 -phosphate
uridylyltransferase (PmGlmU) was cloned in pETl 5b and pET22b(+) vectors as N-His6- and C-HiS(5 -tagged fusion proteins, respectively. For cloning into pETl 5b vector as an N-H is6- tagged protein, the primers used were: forward primer 5' GATCCATATG
AAAGAGAAAGCATTAAGTATCGTG 3' (Nde restriction site is bold and underlined) and reverse primer 5' CCGCTCGAGTTACTTTTTCGTTTGTTTAGTAGGGCG 3 ' (Xho\ restriction site is bold and underlined). For cloning into pET22b(+) vector as a C-H is6-tagged protein, the primers used were: forward primer 5 '
GATCC TATG AAAGAGAAAGCATTAAGTATCGTG 3 ' {Nde\ restriction site is bold and underlined) and reverse primer 5' CCGCTCGAG
CTTTTTCGTTTGTTTAGTAGGGCGTTGC 3' (Xho\ restriction site is bold and underlined). The resulting PCR products were digested with restriction enzymes, purified, and ligated with pETl 5b or pET22b(+) vector predigested with Ndel and Xho\ restriction enzymes. The ligated product was transformed into electrocompetent E. coli DH5ot cells. Selected clones were grown for minipreps and positive clones were verified by restriction mapping and DNA sequencing performed by Davis Sequencing Facility.
[0120] Positive plasmids were transformed into E. coli BL21 (DE3) chemically competent cells. E. coli cells harboring the pETl 5b-PmGlmU or pET22b(+)-PmGlmU plasmid were cultured in LB medium (10 g L tryptone, 5 g/L yeast extract, and 10 g/L NaCI) with ampicillin (100 μg mL) until the ODeoo nm of the culture reached 0.8-1 .0. Overexpression of the targeted proteins was achieved by adding 0.1 mM of isoprop l- l -thio-P-D- galactopyranoside (IPTG) followed by incubation at 25°C for 1 8 h with rigorous shaking at 250 rpm in a C25KC incubator shaker (New Brunswick Scientific, Edison, NJ). [0121] His6-tagged proteins were purified from cell lysate using Ni" -NTA affinity column. To obtain cell lysate, cells were harvested by centrifugation at 4,000 rpm (Sorvall) at 4 °C for 2 hr. The cell pellet was resuspended in lysis buffer (pH 8.0, 100 mM Tris-HCl containing 0.1 % Triton X- 100). Lysozyme (100 μg mL) and DNasel (5 μg/mL) were then added to the cell suspension. The mixture was incubated at 37 °C for 1 hr with vigorous shaking (200 rpm). Cell lysate was obtained as the supernatant by centrifugation at 1 1 ,000 rpm (Sorvall) at 4 °C for 45 min. Purification is performed by loading the supernatant onto a Ni2+-NTA column pre-equilibrated with 10 column volumes of binding buffer ( 10 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was wash with 10 column volumes of binding buffer and 10 column volumes of washing buffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). Protein of interest was eluted with Tris-HCl (pH 7.5, 50 mM) containing imidazole (200 mM) and NaCl (0.5 M). The fractions containing the purified enzymes were collected and dialyzed against Tris-HCl (pH 7.5, 25 mM) buffer containing 10% glycerol. Dialyzed proteins were stored at 4 °C. Results and Discussion
[0122] DNA and protein sequences of PmGlmU cloned from Pasteurella multocida strain P-1059 (ATCC 15742). Compared to the sequences of GlmU (gene Pml806) from Pasteurella multocida genomic strain Pm70 (GenBank accession numbers: AE004439 for gene and AAK03890 for protein), there are 13 base differences (C39A, T195C, A333G, G334A, T339C, G636A, G655A, G817C, T882A, A 1006G, A1008T, G 1071 A, and G 1266T) and four amino acid differences (E l 12 , D219N, E273Q, and T336A) (italicized and underlined) in Pasteurella multocida strain P- 1059 (ATCC 15742).
[0123] Expression level and SDS-PAGE of PmGlmU. The N-His6-tagged PmGlmU has a higher expression level than the C-His6-tagged PmGlmU. On average, 170 mg of purified N- His6-tagged PmGlmU was obtained from 1 liter of cell culture. SDS-PAGE analysis shows that both the purified protein migrated to around 55 kDa.
BLUSP Bifidobacterium longum UDP-sugar pyrophosphorylase
[0124] Carbohydrates are widespread in nature and play pivotal roles in biological systems. The key enzymes for the formation of glycosidic bonds in carbohydrates are
glycosyltransferases. Most glycosyltransferases require monosaccharide nucleotides as the common activated donor substrates. Among monosaccharide nucleotides used by mammalian glycosyltransferases, many are uridine 5'-diphosphate (UDP)-monosaccharides such as UDP- glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-glucuronic acid (UDP-GlcA), UDP-N- acetylglucosamine (UDP-GlcNAc), UDP-jV-acetylgalactosamine (UDP-GalNAc), and UDP- xylose (UDP-Xyl). In addition, UDP-mannose (UDP-Man) has been isolated from
Mycobacterium smegmatis and proposed to be an intermediate in the biosynthesis of mycobacterial polysaccharides. Furthermore, UDP-/V-acetylmannosamine (UDP-ManNAc) and UDP-N-acetylmannosaminuronic acid (UDP-ManNAcA) have been used by some bacteria for producing capsular polysaccharides containing anNAc or ManNAcA residues or forming ManNAcpl -4GlcNAc-PP-undecaprenol (lipid II) for the biosynthesis of cell wall teichoic acids of Gram-positive bacteria.
[0125] The simplest biosynthetic route for obtaining monosaccharide nucleotides such as UDP-monosaccharides usually involves the formation of a monosaccharide- ] -phosphate catalyzed by a monosaccharide- 1 -phosphate kinase followed by the formation of monosaccharide nucleotides catalyzed by a nucleotidyltransferase (or pyrophosphorylase). However, the simplest route has not been applied routinely for the formation of UDP-Gal due to the less common access to UTP:galactose- l -phosphate uridylyltransferases or UDP-Gal pyrophosphorylase (EC 2.7.7.1 0) for direct formation of UDP-Gal from Gal- 1 -phosphate and UTP. For example, UDP-Gal used in galactosyltransferase-catalyzed enzymatic synthesis of galactosides has been more frequently obtained from UDP-Glc by reactions catalyzed by UDP-Gal 4-epimerases or UDP-glucose:galactose- l -phosphate uridylyltransferases (EC 2.7.7.12, GalT or GalPUT) in the Leloir pathway.
[0126] Nevertheless, UDP-galactose pyrophosphorylase activity was identified from yeast Saccharomyces fragilis, pigeon liver, and mammalian livers. The enzyme was purified from bovine liver and Gram-positive bacterium Bifidobacterium bifidum. Recently, promiscuous UDP-sugar pyrophosphoryiases (USPs) (EC 2.7.7.64) that can use various monosaccharide 1 - phosphates in the presence of UTP for direct synthesis of UDP-monosaccharides including UDP-Glc, UDP-Gal, and UDP-GlcA, etc. were cloned from plants such as pea (Pisuni sativum L.) sprouts (PsUSP) and Arabidopsis thaliana (AtUSP). Enzymes which share sequence homology to plant USPs were also cloned from Leis nania major and
Trypanosoma cr zi, two trypanosomatid protozoan parasites, and were shown to have good activity towards Gal- l -P and Glc- l -P and weaker activity towards xylose- 1 -phosphate and GlcA- l -P. A USP with broad substrate specificity and optimal activity at 99°C was also cloned from a hyperthermophile archaea Pyrococcus furiosus DSM 3638 for which Glc- l -P, Man- l -P, Gal-l -P, Fuc-l-P, GlcNH2-l -P, GalNH2-l-P, and GlcNAc- l -P were all shown to be tolerable substrate, and both UTP and dTTP could be used as nucleotide triphosphate substrates by the enzyme. Nevertheless, none of these enzymes has been used in preparative- scale or large-scale synthesis of sugar nucleotides and non-natural derivatives of monosaccharide-1 -P have not been tested as substrates for USPs.
[0127] Here we report the cloning of a promiscuous USP from a probiotic Bifidobacterium longum strain ATCC55813 and its application in an efficient one-pot three-enzyme system for preparative-scale synthesis of UDP-monosaccharides and their derivatives from simple monosaccharides or derivatives (except for UDP-Glc which was synthesized from Glc- l -P in a one-pot two-enzyme system as discussed below). These compounds will be tested as potential donor substrates for various glycosyltransferases.
Experimental
[0128] Cloning, expression, and purification of BLUSP. Full length Bifidobacterium longum UDP-sugar pyrophosphorylase (EC 2.7.7.64) (BLUSP) (encoded by gene ugpA, DNA GenBank accession number: ACHIOI OOO I 19, locus tag: HMPREF0175_1671 ; protein GenBank accession number: EEI80102) was cloned from the genomic DNA of
Bifidobacterium longum strain ATCC55813 in pET15b vector as an N-His6-tagged fusion protein. The primers used were: forward primer 5'
GGAATTCCATATGACAGAAATAAACGATAAGGCC 3 ' (Ndel restriction site is bold and underlined) and reverse primer 5' CGCGGATCCTCACACCCAATCGTCCG 3 '
(BamHl restriction site is bold and underlined). The resulting PCR products were digested with restriction enzymes, purified, and ligated with pETl 5b vector predigested with Ndel and BamHl restriction enzymes. The ligated product was transformed into electrocompetent E. coll DH5a cells. Selected clones were grown for minipreps and positive clones were verified by restriction mapping and DNA sequencing performed by Davis Sequencing Facility. The DNA sequence of the insert matched to BL0739 (ugpA) gene in the genomic sequence of Bifidobacterium /cwgw/n NCC2705. Compared to the BL0739 (ugpA) gene sequence of Bifidobacterium longum NCC2705 (GenBank accession number: AE014295) which was annotated to encoding a hypothetic UTP:glucose- l -phosphate uridylyltransferase (GenBank accession number: AAN24556), there are 4 base differences (T35C, A47G, C228T, A465C) resulting in one amino acid difference (D16G) in the protein sequence of BLUSP. [0129] The plasmid was transformed into E. coli BL21 (DE3) chemically competent cells for protein expression. E. coli cells harboring the pET15b-BLUSP plasmid were cultured in LB medium (1 0 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) with ampicillin ( 100 μg/mL) at 37 °C with rigorous shaking at 250 rpm in a C25 C incubator shaker (New Brunswick Scientific, Edison, NJ) until the ODgoo nm of the culture reached 0.8-1 .0.
Overexpression of the targeted proteins was achieved by adding 0.15 mM of isopropyl- 1 - thio-p-D-galactopyranoside (IPTG) followed by incubation at 18 °C for 20 hr with rigorous shaking at 250 rpm.
[0130] His6-tagged protein was purified from cell lysate using Ni2 ' -NTA affinity column. To obtain cell lysate, cells were harvested by centrifugation at 4,000 rpm (Sorvall) at 4 °C for 2 hr. The cell pellet was resuspended in lysis buffer (pH 8.0, 100 mM Tris-HCl containing 0.1 % Triton X- 100). Lysozyme ( 100 μg/mL) and DNasel (5 μg/mL) were added to the cell suspension. The mixture was incubated at 37 °C for 1 hr with vigorous shaking (200 rpm). Cell lysate was obtained as the supernatant by centrifugation at 1 1 ,000 rpm (Sorvall) at 4 °C for 45 min. Purification was performed by loading the supernatant onto a Ni2+-NTA column pre-equilibrated w ith 1 0 column volumes of binding buffer (10 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was wash with 1 0 column volumes of binding buffer and 1 0 column volumes of washing buffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). Protein of interest was eluted with Tris-HCl (pH 7.5, 50 mM) containing imidazole (200 mM) and NaCl (0.5 M). The fractions containing the purified enzyme were collected and dialyzed against Tris-HCl buffer (pH 7.5, 25 mM) containing 10% glycerol and 0.25 M NaCl. Dialyzed proteins were stored at 4 °C. Alternatively, fractions containing purified enzyme were dialyzed against Tris-HCl buffer (pH 7.5, 25 mM) and freeze dried. On average, 167 mg of purified protein was obtained from 1 liter of cell culture. Protein concentration was determined in a 96-well plate using bicinchoninic acid with BSA as standard. The absorbance was measured at 562 nm using a plate reader.
[0131 ] pH profile study for BLUSP. Typical enzymatic assays for pH profile studies were carried out for 10 min at 37 °C in a total volume of 20 μΐ^ containing Glc-l -P ( 1 mM), UTP ( 1 mM), Mg2+ (20 mM), and BLUSP (10 ng) in a buffer ( 100 mM) with pH varying from 3.0 to 9.5. The reaction mixture was quenched by boiling for 5 min followed by adding 20 ih of pre-chilled 95% (v/v) ethanol. The samples were then kept on ice until analyzed by a Beckman Coulter P/ACE MDQ Capillary Electrophoresis system equipped with a UV detector and a 50 cm capillary tubing (75 μηι I.D., Beckman Coulter). Assays were run at 25 kV with 25 mM sodium borate buffer (pH 9.8) for 22 min. Percent conversions were calculated from peak areas of UDP-sugar and UTP monitored by UV absorbance at 254 nm. All assays were carried out in duplicate.
[0132] Effects of metal ions and EDTA. EDTA (5 mM), different concentrations (0.5, 1 , 5, 10, 20, 50 mM) of MgCl2, and various divalent metal cations (CaCl2, CoCl2, CuS04, MnCl2, ZnCl2) were used in a MES buffer (pH 6.5, 100 mM) to analyze their effects on the uridylyltransferase activity of BLUSP (10 ng in 20 total volume) using Glc- l -P (1 mM) as the acceptor. Other components are the same as those described for the pH profile studies. Reaction without EDTA or metal ions was used as a control.
[0133] Capillary electrophoresis (CE) and thin-layer chromatograph (TLC) assays for kinase reactions. Kinase reactions were carried out at 37 °C in a total volume of 30 μΐ^ in Tris-HCl buffer (100 mM, pH 8.0) containing monosaccharide (15 mM), ATP (18 mM, 1.2 eq.), MgCl? (10 mM), and a kinase (6 μg). These conditions were similar to those used for preparative-scale synthesis. After 1 hr, 4 hr, and 24 hr, an aliquot of 8 μΐ^ was withdrawn from each reaction mixture, boiled in a water bath for 5 min and stored at -20 °C until being analyzed by capillary electrophoresis (CE) and TLC. For TLC analysis, 0.5 μΐ^ of each sample was directly spotted on TLC plates, developed using suitable developing solvents, and stained with anisaldehyde sugar stain. For CE analysis, 1 .5 μL· of each sample was diluted into 30 μΐ^ and subjected to CE analysis as described above for pH profile studies.
Results and Discussion
[0134] SDS-PAGE analysis of BLUSP. SDS-PAGE analysis shows that the recombinant BLUSP has a very good expression level in E. coli and has a high solubility. It consists of about 90% of the total protein extracts from E. coli host cells and more than 90% of the soluble protein. The protein size observed is about 60 kDa which is close to 59.7 kDa calculated molecular weight.
[0135] pH profile of BLUSP. As shown in Figure 6, BLUSP is active in a broad pH range of 4.0-8.0 and with optimal activity at pH 6.5 in MES buffer.
[0136] Effects of metal ions and EDTA. As shown in Figure 7, a divalent metal cation such as Ca2+, Co2+, Mg2+, or Mn2+ is required for the activity of BLUSP. BLU SP is inactive in the absence of a divalent metal cataion or in the presence of EDTA. At 20 mM concentration, Mg" was the best among all divalent metal cations tested including Ca , Co2+, Mg2+, or Mn2+, Cu2+, and Zn2+. The optimal Mg2+ concentration for BLUSP activity was found to be 20 mM.
PmUgd - Pasteurella multocida UDP-glucose dehydrogenase
[0137] Cloning of PmUgd. PmUgd was cloned as a C-Hise-tagged fusion protein in pET22b(+) vector using the genomic DNA of P. multocida P-1059 (ATCC# 15742) as the template for polymerase chain reactions (PCR). Primers used for cloning were: forward primer 5'-GATCCATATGAAGAAAATTACAATTGCTGGGGC-3 ' (Ndel restriction site is underlined) and reverse primer 5'- CCGCTCGAGAGCATCACCGCCAAAAATATCTCTTG-3 ' (Xhol restriction site is underlined). PCR was performed in a reaction mixture of 50 μΐ containing genomic DNA (1 μg), forward and reverse primers (1 μΜ each), l OxHerculase buffer (5 μΐ), dNTP mixture ( 1 mM), and 5 U (1 μΐ) of Herculase-enhanced DNA polymerase. The reaction mixture was subjected to 30 cycles of amplification with an annealing temperature of 55 °C. The resulting PCR products were purified, digested, and ligated with the corresponding pre-digested vector. The ligation products were transformed into electrocompetent E. coli DH5a cells. Plasmids containing the target genes as confirmed by DNA sequencing (performed by UC-Davis Sequencing Facility) were selected and transformed into E. coli BL21 (DE3) chemically competent cells.
[0138] Compared to the DNA sequence of PM0776 gene from P. multocida strain Pm70 (its genomic DNA sequence is available on NCBI), the obtained gene of PmUgd has 19 base differences (A357G, C381 A, A390G, A397C, C404A, A406G, T408A, C414T, A420T, A426G, C430T, G438A, C447A, T451 C, C453T, T456C, A464T, C582T, and G807A, the nucleotide before the number is from the DNA sequence of PM0776, the number is based on PM0776 gene) compared to publically available PM0776 gene sequence. Furthermore, the C at position 401 in PM0776 is missing in PmUgd and PmUgd has an extra A between 408 and 409 of PM0776. Overall, there are five amino acid differences in PmUgd ( 127K, N 133H, LI 371, Y 151 H and Y 155F, the amino acid residue before the number is from the protein sequence deduced from PM0776, the number is based on the protein sequence deduced from PM0776) compared to the deduced protein sequence from PM0776 gene.
[0139] Expression and purification. E. coli strains were cultured in LB rich medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g L NaCl) supplemented with ampicillin ( 100 μg mL). Over-expression of PmUgd was achieved by inducing the E. co/ BL21 (DE3) cell culture with 0.1 mM of isopropyl- 1 -thio-p-D-galactopyranoside (IPTG) when the OD6oo nm of the culture reached 0.8-1.0 followed by incubation at 20 °C for 20 h.
[0140] Bacterial cells were harvested by centrifugation at 4 °C in a Sorvall Legend RT centrifuge with a hanging bucket rotor at 4000 x rpm for 2 h. Harvested cells were resuspended in lysis buffer (Tris-HCl buffer, 100 mM, pH 8.0 containing 0.1 % Triton X-100) (20 mL for cells collected from one liter cell culture). Lysozyme ( 100 μg/mL) and DNasel (5 μg mL) were added to the cell resuspension. The resulting mixture was incubated at 37 °C for 1 h with shaking at 200 rpm. Cell lysate (supernatant) was obtained by centrifugation at 12000 x rpm for 15 min. Purification was carried out by loading the supernatant onto a Ni2+- NTA column pre-equilibrated with 8 column volumes of binding buffer (10 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was washed with 8 column volumes of binding buffer and 8 column volumes of washing buffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The target protein was eluted with Tris-HCl buffer (50 mM, pH 7.5) containing imidazole (200 mM) and NaCl (0.5 M). The fractions containing the purified enzymes were collected and dialyzed against Tris-HCl buffer (20 mM, pH 7.5) containing 10% glycerol. Dialyzed proteins were stored at 4 °C.
[0141] About 23 mg PmUgd can be routinely expressed and purified from 1 L of £. coli culture under expression conditions described above.
PmHSl, PmHS2, KfiA Materials and Methods
[0142] Bacterial strains, plasmids, and materials. E. coli electrocompetent DH5 and chemically competent BL21 (DE3) cells were from Invitrogen (Carlsbad, CA). P. miiltocida P-934 (ATCC# 12948) and P. nmltocida P- 1059 (ATCC# 1 5742) were from American Type Culture Collection (ATCC, Manassas, VA, USA). fiA synthetic gene with codons optimized for E. coli expression was synthesized by GeneArt (Grand Island, NY) based on KfiA gene sequence from E. coli N issle 191 7 (GenBank accession number: AJ586888, ORF79). Vector plasmid pFTl 5b was from Novagen (EMD Biosciences Inc. Madison, WI, USA). Vector pMAL-c4X was purchased from New England Biolabs (Ipswich, MA).
Nickel-nitrilotriacetic acid agarose (Ni2+-NTA agarose), QIAprep spin miniprep kit, and Q1AEX II gel extraction kit were from Qiagen (Valencia, CA, USA). Herculase-enhanced DNA polymerase was from Stratagene (La Jolla, CA, USA). T4 DNA ligase and 1 kb DNA ladder were from Promega (Madison, WI, USA). Ndel, BamHl, EcoRl, and Hindl
restriction enzymes were from New England Biolabs Inc. (Beverly, MA, USA).
[0143] Cloning of PmHSl, PmHS2 and KfiA. PmHS2 were cloned as N- and C-His6- tagged fusion proteins in pET15b and pET22b(+) vector, respectively, using genomic DNAs of P. m ltocida P-l 059 (ATCC# 15742) as the template for polymerase chain reactions (PCR). PmHS l and KfiA were cloned as a fusion protein of an N-terminal with a maltose- binding protein (MBP) and a C-terminal Hise tag in pMAL-c4X vector using the P. multocida P-934 (ATCC# 12948) and KfiA synthetic gene as template, respectively. Primers used for cloning are summarized in Table 8. PCR was performed in a reaction mixture of 50 containing genomic DNA (1 μg), forward and reverse primers (1 μΜ each), 10* Herculase buffer (5 μί), dNTP mixture ( 1 mM), and 5 U ( 1 μί) of Herculase-enhanced DNA
polymerase. The reaction mixture was subjected to 30 cycles of amplification with an annealing temperature of 55 °C (for PmHSl and PmHS2) or 52 °C (for KfiA). The resulting PCR products were purified, digested, and ligated with the corresponding pre-digested vector. The ligation products were transformed into electrocompetent E. coli DH5cc cells. Plasmids containing the target genes as confirmed by DNA sequencing (performed by UC-Davis Sequencing Facility) were selected and transformed into £. coli BL21 (DE3) chemically competent cells.
Table 4: Primers used for cloning PmHSl, PmHS2 and KfiA.
Primers Sequences (5 -3 ')
KfiA_pMAL-c4X_F_EcoRI GACCG^ZTCATGATTGTTGCAAATATGAGC KfiA_pMAL-c4X_R_HindIlI GTCGA4GC77TTAGTGGTGGTGGTGGTGGTGACCTT
CCACATTATAC
PmHS 1 _ PMAL-c4X F BamHI CGCGG-47UCATGAGCTTATTTAAACGTGCTAC PmHS 1 _pMAL-c4X_R_HindIII GATC^ iGCrTTTAGTGATGATGATGATGATGCTCGT
TATAAAAAGATAAACACGG
PmHS2_pET15b/22b+_F_NdeI GATCC47L47GAAGGGAAAAAAAGAGATGAC PmHS2_pETl 5b_R_BamHI AAGGG^ TCCTTATAAAAAATAAAAAGGTAAACAGG PmHS2_pET22b+_R_BamHI AAGGG 17UCTTAGTGGTGGTGGTGGTGGTGTAAAA
AATAAAAAGGTAAACAGG
[0144] Expression and purification. E. coli strains were cultured in LB rich medium ( 10 g/L tryptone , 5 g/L yeast extract, and 10 g/L NaCl) supplemented with ampicillin ( 1 00 μg/mL). Over-expression of PmHSl and PmHS2 were achieved by inducing the E. coli BL21 (DE3) cell culture with 0.1 mM of isopropyl-l -thio- -D-galactopyranoside (IPTG) when the Οϋήοο nm of the culture reached 0.8-1 .0 followed by incubation at 20 °C for 20 h. Overexpression of KfiA was performed by inoculating 10 mL of a fresh overnight bacterial culture grown in LB containing 50 μg/mL ampicillin and 20 μg/mL chloramphenicol into 1 L of LB (containing 50 μg mL of ampicillin, 20 μg/mL of chloramphenicol and 2 mg/mL of L- arabinose). The culture was incubated at 37 °C with shaking at 250 rpm. When the OD6oo of the culture reached 0.4-0.6, expression was induced by adding IPTG to a final concentration of 0.3 mM and then the cell was cultured at 20 °C for 20 h.
[0145] Bacterial cells were harvested by centrifugation at 4 °C in a Sorvall Legend RT centrifuge with a hanging bucket rotor at 4000 x rpm for 2 h. Harvested cells were resuspended in lysis buffer (Tris-HCl buffer, 100 mM, pH 8.0 containing 0.1 % Triton X- 100) (20 mL for cells collected from one liter cell culture). Lysozyme ( 100 μg/mL) and DNasel (5 μg/mL) were added to the cell resuspension. The resulting mixture was incubated at 37 °C for 1 h with shaking at 200 rpm. Cell lysate (supernatant) was obtained by centrifugation at 12000 x rpm for 15 min. Purification was carried out by loading the supernatant onto a N i2+- NTA column pre-equilibrated with 10 column volumes of binding buffer ( 10 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was washed with 10 column volumes of binding buffer and 10 column volumes of washing buffer (20-50 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The target protein was eluted with Tris-HCl buffer (50 mM, pH 7.5) containing imidazole (200 mM) and NaCl (0.5 M). The fractions containing the purified enzymes were collected and dialyzed against Tris-HCl buffer (20 mM, pH 7.5) containing 10% glycerol. Dialyzed proteins were stored at 4 °C.
[0146] pH profile by HPLC. Typical enzymatic assays were performed in a 10 μΐ reaction mixture containing a buffer ( 100 mM) with a pH in the range of 4.0- 10.0, UDP-GlcNAc ( 1 mM), GlcAp2AA (1 mM), MnCb (10 mM) and KfiA (9.0 μg) or PmHS2 (0.25 μ£). Buffers used were: Na2HP04/citric acid, pH 4.0; MES, pH 5.0-6.5; Tris-HCl, pH 7.0-9.0; and CAPS, pH 10.0. Reactions were allowed to proceed for 30 min at 37 °C and were quenched by adding ice-cold 10% (v/v) acetonitrile to make 100-fold dilutions. The samples were then kept on ice until an aliquot of 8 μΐ was injected and analyzed by a Shimadzu LC-201 OA system equipped with a membrane on-line degasser. a temperature control unit and a fluorescence detector. A reverse phase Premier C I 8 column (250 9 4.6 mm I.D., 5 μπι particle size, Shimadzu) protected with a C I 8 guard column cartridge was used. The mobile phase was 25% (v/v) acetonitrile. The fluorescent compounds GlcA 2AA and GlcNAcal- 4GlcA 2AA were detected by excitation at 315 nm and emission at 400 nm.
[0147] Effects of metal ions. Different concentrations (1 , 5, 10, and 20 mM) of MgCl2 , MnCl2, CaCl2, or CuCl2 were used in a MES buffer (pH 6.5, 100 mM) to analyze their effects on the activity of KfiA (0.9 μg μΓ') or PmHS2 (2.5* 10"2 μg μΓ]). Reaction without metal ions was used as a control. The assay was performed as above pH profile.
[0148] Substrate specificity of KfiA and PmHS2. All reactions were carried out in duplicate at 37 °C in MES (100 mM, pH 6.5) containing an UDP-GlcNAc or its derivatives (1 mM), GlcAcc2AA ( 1 mM), MnCl2 ( 10 mM) and KfiA (1.08 μg μΓ1) or PmHS2 (2.5x 10"2 μg μΓ1). At 30 min, 4 h or 16 h, aliquots of reaction mixture were withdrawn and were quenched by adding ice-cold 10% (v/v) acetonitrile to make 100-fold dilutions. The assays were analyzed by HPLC.
Results
[0149] Cloning, expression and purification of recombinant proteins. PmHS2 was cloned as an N- or a C-Hise-tagged protein using pETl 5b and pET22b (+) vectors, respectively. Both N- and C-Hisg-tagged proteins were able to be expressed as soluble forms in E. coli BL21 (DE3) cells by induction 0.1 mM IPTG. Both could be easily purified using Ni2+-affinity chromatography. The expression level of the soluble and active N-His6-tagged form was relatively higher than its C-His6-tagged counterpart and N-His6-PmHS2 was studied in detail. About 1 1 mg of N-His6-PmHS2 was routinely obtained from the cell lysate of one liter E. coli cell culture. KfiA was expressed in an N-terminal MBP and a C-terminal six-His fusion protein in BL21 (DE3) cells coexpressed with chaperone protein pGro7. The recombinant KfiA was purified to homogeneity with a Ni2~-affinity column. About 8.0 mg of MBP-KfiA-His6 was routinely obtained from the cell lysate of one liter E. coli cell culture. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that one-step Ni2+-column purification was efficient to provide pure PmHS2 and KfiA. As expected from the calculated molecular weight of PmHS2 and KfiA, the size of the protein shown by SDS-PAGE was about 75 kDa and 69 kDa, respectively. To obtain a soluble and active recombinant PmHS 1 in E. coli expression system, the MBP tag was introduced by using pMAL-c4X vector, while the C-His6-tag was introduced by including the His6-tag codons in the 3'-primer used for cloning. Expression was achieved by incubating E. coli BL21 (DE3) cells at 20 °C for 20 h with vigorous shaking (250 rpm) after the addition of IPTG (0.1 m ) for induction. Although it has activity, SDS-PAGE analysis indicated that only a small portion of the recombinant protein was seen in the cell lysate, the soluble portion of the cell extraction.
[0150] pH profile of KfiA and the -acetylglucosaminyltransferase activity of PmHS2. As shown in Figure 27, when GlcA 2AA was used as an acceptor, both KfiA and PmSH2 were found to be active in a pH range of 5.0-9.0 with an optimal activity at pH 5.0. The activities of both enzymes decreased dramatically when pH was below 5.0.
[0151] Effects of metal ions on the heparosan synthase activity of KfiA and PmHS2.
The effects of different metal ions, Mg~ , Mir , Ca~ and Cu" on the heparosan synthase activity of KfiA and PmHS2 were investigated. Reaction without metal ions was used as a control. As shown in Figure 28, no activity was detected without metals. Both enzymes showed best activities in the presence of Mn2+. Increasing Mn2+ from 1 mM to 20 mM increased the activity of both KfiA and PmHS2 first and then decreased the activity of both enzymes. Compared with Mn2+, Mg2+ and Ca2+ showed much less efficiency for the activity of KfiA and PmHS2. No activity was shown in the presence of Cu2+.
[0152] Substrate specificity of KfiA and PmHS2. Using the I IPLC method described above, the substrate specificities of KfiA and Pml IS2 were examined using GlcAa2AA and twenty two compounds of UPD-GlcNAc or UDP-Mannose or their derivatives. Experimental data is shown in Figure 11. Among the tested compounds (see Figure 12), both enzymes exhibited quite narrow substrate specificities. However, the catalytic efficiency of PmHS2 was much high than that of KfiA . Both enzymes can use the UDP-GlcNAc (F12-3), UDP- GlcNTFA (F12-4), UDP-GlcNGc (F12-8), UDP-GlcNAcN, (F12-9), among which the UDP- GlcNAc (F12-3) is the best substrate for both enzymes. Besides these four compounds, UDP- GlcNAc6N3 (F12-5) is a substrate for PmHS2 but not for KfiA.
Example 2. Preparation of UDP-GlcNAc and Derivatives
[0153] General methods for compound purification and characterization. Chemicals were purchased and used without further purification. Ή NMR and l 3C NMR spectra were recorded on a 600 MHz NMR spectrometer. High resolution electrospray ionization (ESI) mass spectra were obtained at the Mass Spectrometry Facility in the University of California, Davis. Silica gel 60 A (Sorbent Technologies) was used for flash column chromatography. Analytical thin-layer chromatography (Sorbent Technologies) was performed on silica gel plates using anisaldehyde sugar stain for detection. Gel filtration chromatography was performed with a column (100 cm * 2.5 cm) packed with BioGel P-2 Fine resins. ATP, UTP, and GlcNAc were purchased from Sigma. GlcNTFA, GlcN3, GlcNAc6N3 , GIcNAc6S, GlcNS were synthesized as described previously. Nan _ATCC55813 and PmPpA were overexpressed as discussed previously.
[0154] Synthesis of GlcNTFA6S T5b-6. GlcNTFA T5b-2 (300 mg, 1 .09 mmol) was dissolved in 15 mL of anhydrous DMF. Anhydrous Et3N (5 mL) and sulfur trioxide pyridine complex (1 .2 eq.) were added at 0 °C. After being stirred at room temperature for overnight, the reaction was stopped by adding MeOH and concentrated. The residue was purified by flash column chromatography (EtOAc:MeOH:H20 = 8:2: 1 , by volume) to afford 6-O-sulfo- GlcNTFA T5b-6 (243 mg, 63%). Ή NMR (600 MHz, D20) δ 5.25 (d, J= 2.4 Hz, 0.6H),
4.84 (d, .7 = 8.4 Hz, 0.4H), 4.26-3.51 (m, 6H). ,3C NMR ( 1 50 MHz, D20) δ 159.75 (J = 37.5 Hz), 159.69 (7 = 37.5 Hz), 1 17.01 (J = 284.7 Hz), 1 16.93 (J= 284.7 Hz), 94.46, 80.59, 74.03, 73.24, 70.21 , 70.00, 69.84, 96.75, 67.22, 67.18, 57.34, 54.87.
Figure imgf000050_0001
T5b-2, GlcNTFA T5b-6, GlcNTFA6S
[0155] Synthesis of GlcN36S T5b-7. 6-0-Sulfo-GlcN3 T5b-7 was synthesized from GlcN3 T5b-3 (300 mg, 1 .46 mmol) in 54% yield (224 mg) and the procedures were similarly as described above for GlcNTFA6S T5b-6. Ή NMR (600 MHz, D20) δ 5.35 (d, J= 2.9 Hz, 0.4H), 4.71 (d, J = 8.4 Hz, 0.6H), 4.21-3.82 (m, 3H), 3.65-3.28 (m, 3H). I 3C NMR ( 1 50 MHz, D20) δ 95.3 1 , 91.39, 74.32, 73.94, 71 .55, 69.80, 69.61 , 69.27, 67. 15, 67.12, 66.85, 63.55.
Figure imgf000050_0002
T5b-3, GlcN3 T5b-7, GlcN36S
[0156J One-pot three-enzyme synthesis of UDP-sugars T5b-9 T5b-13. This was carried out as shown in Figure 4. Glucosamine derivatives T5b-l-T5b-5 (50 to 300 mg, 1 .0 eq.), ATP ( 1 .2 eq.), and UTP ( 1 .2 eq.) were dissolved in water in a 50 mL centrifuge tube containing Tris-HCl buffer ( 1 00 mM, pH 8.0) and MgCI2 (10 mM). After the addition of appropriate amount of NanK_ATCC55813 (3.2^1.8 mg), PmGlmU (5-7.5 mg), and PmPpA (2.5-5 mg), water was added to bring the volume of the reaction mixture to 20 mL. The reaction was carried out by incubating the solution in an isotherm incubator for 24 hr to 48 hr at 37 °C with gentle shaking. Product formation was monitored by TLC (EtOAc:MeOH:H 0 = 3 :2: 1 by volume) with /?-anisaldehyde sugar staining. The reaction was stopped by adding the same volume of ice-cold ethanol and incubating at 4 °C for 30 min. The mixture was concentrated and passed through a BioGel P-2 gel filtration column to obtain the desired product. Silica gel column purification (EtOAc:MeOH:H20 = 4:2: 1 ) was applied when necessary to achieve further purification.
[0157] One-pot three-enzyme synthesis of UDP-GlcNAc3N3 was also conducted using GlcNAc3N3 as the staring sugar (See Table 5a) for reaction conditions). As shown in Figure 29, the formation of UDP-GlcNAc3N3 was confirmed by high-resolution mass spectrometry [HR S (ESI) m/z calcd for C17H25N6016P2 (M-H) 63 1.0802, found 631 .0817].
Table 5a. Reaction conditions for the synthesis of UDP-GlcNAc3 3.
Figure imgf000051_0001
[0158] Uridine 5'-diphospho-2-acetamido-2-deoxy-a-D-glucopyranoside (UDP-
GIcNAc, T5b-9). Yield, 81 % (445 mg); white foam. Ή NMR (600 MHz, D20) 5 7.97 (d, J = 8.4 Hz, 1 H), 5.97-6.00 (m, 2H), 5.53 (dd, J = 6.6, 3.0 Hz, 1 H), 4.37-4.40 (m, 2H), 4.21 -4.31 (m, 3H), 3.81-3.75 (m, 5H), 3.58 (t, J = 9.0 Hz, 1 H), 2.09 (s, 3H). 13C NMR ( 150 MHz, D20) δ 1 74.94, 166.39, 1 51.99, 141.82, 1 02.85, 94.68, 88.72, 83.40 (d, J = 8.7 Hz), 73.96, 73.20, 71 .13, 69.83, 65.18, 65.15, 60.53, 53.88 (d, J = 8.4 Hz), 22.29. HRMS (ESI) m/z calcd for C, 7H27N30,7P2 (M+H) 608.0894, found 608.0906.
[0159] Uridine 5'-diphospho-2-deoxy-2-trifluoroacetamido-ct-D-glucopyranoside (UDP-GlcNTFA, T5b-10). Yield, 97% (699 mg); white foam. Ή NMR (600 MHz, D20) δ 7.95 (d, J= 7.8 Hz, 1H), 5.97-5.98 (m, 2H), 5.64 (dd, J= 6.6, 3.0 Hz, 1H), 4.35^.39 (m, 2H), 4.18-4.29 (m, 3H), 4.12 (d, J= 10.8 Hz, 1H), 3.93-3.98 (m,2H),3.91 (dd,J= 12.6, 1.8 Hz, 1H), 3.85 (dd,y= 12.0, 4.2 Hz, 1 H), 3.61 (t, J=9.0Hz, 1H).13CNMR(150 MHz, D20) 5166.39, 159.73 (d, .7=37.5 Hz), 151.94, 141.83, 116.88 (d, J = 284.6 Hz), 102.79, 93.91, 88.79, 83.22(d, J=9.0 Hz), 73.92, 73.23, 70.35, 69.76, 69.68, 65.12, 60.42, 54.53 (d, J=8.9 Hz). HRMS (ESI) iw/z calcd for C17H24F3N3O17P2 (M+H) 662.0611 , found 662.0615.
[0160] Uridine 5'-diphospho-2-azido-2-deoxy-a-D-glucopyranoside (UDP-GICN3, T5b-
11). Yield, 54% (124 mg); white foam. Ή NMR (600 MHz, D20) 57.96 (d, J= 8.4 Hz, 1 H), 5.97-5.96 (m, 2H), 5.68 (dd, J= 7.2, 3 Hz, 1H), 4.34-4.37 (m, 2H), 4.18-4.27 (m, 3H), 3.89- 3.93 (m, 2H), 3.85 (dd, J= 12.6, 2.4 Hz, 1H), 3.79 (dd, J= 12.0, 4.2 Hz, 1H), 3.54 (t,J=9.6 Hz, lH),3.38(d, J= 10.2 Hz, 1H).13CNMR(150 MHz, D20) δ 166.39, 151.96, 141.84, 102.79, 94.60, 88.64, 83.34 (d, J = 9 Hz), 73.91, 73.07, 70.85, 69.77, 69.49, 65.07, 62.93 (d, J= 8.6 Hz), 60.29. HRMS (ESI) m/z calcd for
Figure imgf000052_0001
633.0959, found 633.0960.
[0161] Uridine 5'-diphospho-2-acetamido-6-azido-2,6-dideoxy-a-D-ghicopyranoside (UDP-GIcNAc6N3, T5b-12). Yield, 72% (462 mg); white foam. Ή NMR (600 MHz, D20) δ 7.93 (d,J=7.8 Hz, 1H), 5.96-5.94 (m, 2H), 5.15 (s, 1H), 4.32-4.36 (m, 2H), 4.17-4.24 (m, 3H), 4.00-4.04 (m, 2H), 3.79 (t, J= 9.6 Hz, 1H), 3.72 (dd, J= 13.2, 2.4 Hz, 1H), 3.55-3.62 (m, 2H), 2.06 (s, 3H).13C NMR (150 MHz, D20) δ 174.86, 166.33, 151.86, 141.82, 102.76, 94.53, 88.92, 83.15 (d,J = 8.9 Hz), 73.93, 71.85, 70.28, 70.28, 69.68, 65.16, 53.77 (d, J= 7.4 Hz), 50.71 , 22.22. HRMS (ESI) m/z calcd for C17H26N60,6P2 (M+H) 592.0693, found 592.0698.
[0162] Uridine 5'-diphospho-2-acetamido-2-deoxy-6-0-sulfo-a-D-glucopyranoside (UDP-GlcNAc6S, T5b-13). Yield, 62% (70 mg); white foam. Ή NMR (600 MHz, D20) δ 7.96 (d, J= 7.8 Hz, 1 H), 5.97-5.99 (m, 2H), 5.55 (dd, J= 7.2, 3.0 Hz, 1 H), 4.35^1.38 (m, 3H), 4.26-4.30 (m, 3H), 4.18^1.22 (m, 1H), 4.12 (d, J = 9.6 Hz, 1H), 4.04 (d, J= 10.8 Hz, 1H), 3.84 (t, J= 9.6 Hz, 1H), 3.68 (t, J= 9.6 Hz, 1H), 2.09 (s, 3H). I3C NMR (150 MHz, ϋ20)δ 174.84, 166.40, 151.93, 141.73, 102.76,94.57, 88.72, 83.15 (d, J= 9.3 Hz), 73.89, 70.83, 69.70, 69.04, 66.56, 65.16, 65.13, 53.67 (d, J= 8.1 Hz), 22.17. HRMS (ESI) m/z calcd for Ci7H27N302oP2S (M+H) 688.0462, found 688.0471.
[0163] Chemical derivatization of UDP-sugars F5-2-F5-8, and F5-10-F5-15. [0164] Uridine 5'-diphospho-2-amino-2-deoxy-a-D-glucopyranoside (UDP-GICNH2, F5-2). UDP-GlcNTFA F5-1 ( 150 mg, 0.22 mmol) was dissolved in 25 mL of methanol and 5 mL of H20. The pH of the solution was adjusted to 9.5 by adding 2CO3. After being vigorously stirred at r.t. for overnight, the reaction mixture was neutralized with DOWEX HCR-W2 (H+) resin, filtered and concentrated. The residue was purified by flash column chromatography (EtOAc:MeOH:H20 = 1 : 1 : 1 , by volume) to afford UDP-GlcNH2 F5-2 as white solid in 98% yield (122 mg). Ή NMR (600 MHz, D20) δ 7.90 (d, J = 7.8 Hz, 1H), 5.89-5.92 (m, 2H), 5.79 (dd, J= 6.0, 3.0 Hz, 1 H), 4.30-4.32 (m, 2H), 4.16-4.24 (m, 3H), 3.86-3.90 (m 2H), 3.81 (dd, J= 12.6, 1.8 Hz, 1 H), 3.77 (dd, J = 12.6, 4.2 Hz, lH), 3.52 (t, J = 9.6 Hz, 1H), 3.33 (d, J = 10.8 Hz, 1 H). , 3C NMR (150 MHz, D20) δ 166.40, 151.93,
141 .75, 102.71 , 92.87, 88.74, 83.21 (d, J= 9 Hz), 73.91 , 73.39, 69.85, 69.69, 69.16, 65.23, 60.09, 54.27 (d, J = 8.4 Hz). HRMS (ESI) m/z calcd for C I 5H25N3016P2 (M+H) 566.0788, found 566.0791.
[0165] Uridine 5'-diphospho-2-sulfoamino-2-deoxy-a-D-glucopyranoside (UDP- GlcNS, F5-3). UDP-GlcNH2 F5-2 (50 mg, 0.082 mmol) was dissolved in 30 mL of water. The pH of the solution was adjusted to 9.5 by adding 2 N NaOH (aq). Sulfur trioxide- pyridine complex (65 mg, 0.41 mmol) was added in three equal portions during 35 minutes intervals at room temperature, and the pH was maintained at 9.5 throughout the whole process using 2 N NaOH (aq). After being stirred at r.t. for overnight, the reaction mixture was neutralized with DOWEX HCR-W2 (H ) resin, filtered, concentrated, and purified using silica gel column (EtOAc:MeOH:H20 = 3 :2: 1 , by volume) to obtain the UDP-GlcNS F5-3 in 86% yield (46 mg). Ή NMR (600 MHz, D20) δ 7.90 (d, J = 7.8 Hz, 1 H), 5.92-5.93 (m, 2H), 5.71 (s, 1 H), 4.31-4.33 (m, 2H), 4.16-4.23 (m, 3H), 3.73-3.86 (m, 3H), 3.66 (t, J= 9.6 Hz, 1 H), 3.51 (t, J= 9.6 Hz, 1 H), 3.24 (d, .7 = 9.6 Hz, 1 H). 13C NMR (150 MHz, D20) δ 166.49, 152.13, 141.99, 103.04, 95.50, 88.86, 83.53 (J = 8.9 Hz), 74.02, 73.06, 71 .73, 69.98, 69.96, 65.38, 60.73, 58.1 1 (J = 9.2 Hz). HRMS (ESI) m/z calcd for C 15H25N3O 19P2S (M+H) 646.0356, found 646.0373.
[0166] Uridine 5'-diphospho-2-hydroxyacetamido-2-deoxy-a-D-glucopyranoside (UDP-GlcNGc, F5-5). To a solution of UDP-GlcNH2 F5-2 (30 mg, 0.049 mmol) in CH3CN- H20 (30 mL, 1 : 1 , v/v) in the presence of NaHC03 (40 mg, 0.49 mmol), the Acetoxyacetyl chloride (6.9 μί, 0.098 mmol) in CH3CN (5 mL) was added. The reaction mixture was stirred for 4 hours at 0 °C and was neutralized with DOWEX HCR-W2 (H+) resin, filtered, and concentrated. The residue was purified by flash column chromatography (EtOAc:MeOH:H20 = 5:2: 1 , by volume) to afford UDP-GlcNGcAc Γ5-4 in 95% yield (31 mg). ]H NMR (600 MHz, D20) δ 7.99 (d, J = 7.8 Hz, 1 H), 6.03-6.04 (m, 2H), 5.62 (dd, J= 6.6, 3.6 Hz, 1 H), 4.41^.45 (m, 2H), 4.24^1.35 (m, 3H), 4.13 (d, J= 10.2 Hz, 1 H), 4.01 (d, J = 7.8 Hz, 1 H), 3.86-3.96 (m, 3H), 3.63 (t, J = 9.6 Hz, 1 H), 2.25 (s, 3H). UDP-GlcNGcAc F5-4 was dissolved in dry methanol (50 mL) containing analytic amount of sodium methoxide. The resulted mixture was stirred at r.t. for overnight. The reaction mixture was then neutralized with DOWEX HCR-W2 (H+) resin, filtered, and concentration to give product UDP-GlcNGc F5-5 in 98% yield (28 mg). Ή NMR (600 MHz, D20) δ 7.92 (d, J = 7.8 Hz, 1H), 5.93-5.95 (m, 2H), 5.52 (dd, J = 7.2, 3.0 Hz, 1 H), 4.3 1-4.35 (m, 2H), 4.09^.25 (m, 5H), 4.02 (d, J = 10.2 Hz, 1 H), 3.83-3.91 (m, 3H), 3.79 (dd, ./= 12.6, 4.2 Hz, 1 H), 3.55 (t, J= 9.6 Hz, 1 H). 13C NMR ( 1 50 MHz, D20) δ 175.47, 166.37, 151 .92, 141.74, 101 .73, 94.36, 88.53, 83.28 (d, J = 8.4 Hz), 73.86, 73.09, 70.69, 69.71 , 69.54, 65.05, 61.1 1 , 60.36, 53.46 (d, J = 7.7 Hz). HRMS (ESI) m/z calcd for C7H27N3O18P2 (M+H) 624.0843, found 624.0847.
[0167] Uridine 5'-diphospho-2-azidoxyacetamido-2-deoxy-a-D-glucopyranoside
(UDP-GlcNAz, F5-6). Sodium azide (62 mg, 0.98 mmol) was dissolved in 5 mL of distilled ¾0 and the mixture was cooled to 0 °C. Bromoacetic acid (68 mg, 0.49 mmol) was then added over 10 min and the reaction was allowed to slowly warm up to r.t. for overnight. The reaction was acidified to pH 1.0 and extracted three times with 5 mL of diethyl ether. The organic portions were combined, dried over MgSC>4 and concentrated. The crude mixture was dissolved in 10 mL of CH2CI2 and two drops of DMF and cooled to 0 °C. Oxalyl chloride (54\L, 0.64mmol) was slowly added over 15 min using a syringe. The reaction was allowed to warm up to r.t. for overnight. The solvent was removed under reduced pressure to afford the crude oil azidoacetyl chloride. To a solution of UDP-GICNH2 F5-2 (30 mg, 0.049 mmol) in CH3CN-H2O (30 mL, 1 : 1 , v/v) in the presence of NaHCOs (40 mg, 0.49 mmol), the azidoacetyl chloride in CH3CN (5 mL) was added. The reaction mixture was stirred for 4 hours at 0 °C and was neutralized with DOWEX HCR-W2 (H+) resin, filtered, and concentrated. The residue was purified by flash column chromatography (EtOAc:MeOH:H20 = 5:2: 1 , by volume) to afford UDP-GlcNAz F5-6 in 68% yield (22 mg). Ή NMR (600 MHz, D20) δ 7.92 (d, J = 8.4 Hz, 1 H), 5.91-5.94 (m, 2H), 5.49 (dd, J = 7.2, 3.6 Hz, 1 H), 4.30-4.36 (m, 2H), 4.00-4.24 (m, 6H), 3.75-3.89 (m, 4H), 3.53 (t, ./ = 9.6 Hz, 1 H). 13C NMR (1 50 MHz, D20) δ 171.13, 166.41 , 151.98, 141.86, 102.84, 94.59, 88.80, 83.34 (d, J= 9.0 Hz), 73.94, 73.25, 71.02, 69.81 , 69.66, 65.24, 60.51 , 53.94 (d, , J = 9.0 Hz), 52.69, 51.80. HRMS (ESI) m/z calcd for Ci7H26N6017P2 (M+H) 649.0908, found 649.0917.
[0168] Uridine 5'-diphospho-2-phenylacetamido-2-deoxy-a-D-glucopyranoside (UDP- GlcNAcPh, F5-7). 2-Phenylacetyl acid (33 mg, 0.25 mmol) was dissolved in 10 mL of CH2O2 and two drops of DMF. The mixture was cooled to 0 °C. Oxalyl chloride (28 μιΤ, 0.33 mmol) was slowly added over 15 min using a syringe. The reaction was allowed to warm up to r.t. for overnight. The solvent was then removed under reduced pressure to afford 2-phenylacetyl chloride as a light pink solid. To a solution of UDP-GlcNH2 F5-2 (30 mg, 0.049 mmol) in CH3CN-H20 (30 mL, 1 : 1 , v/v) in the presence of NaHC03 (40 mg, 0.49 mmol), the 2-phenylacetyl chloride in CH3CN (5 mL) was added. The reaction mixture was stirred for 4 hours at 0 °C and was neutralized with DOWEX HCR-W2 (H+) resin, filtered, and concentrated. The residue was purified by flash column chromatography
(EtOAc:MeOH:H20 = 6:2: 1 , by volume) to afford white solid UDP-GlcNAcPh F5-7 in 79% yield (26 mg). Ή NMR (600 MHz, D20) δ 7.83 (d, J= 8.4 Hz, 1 H), 7.32-7.35 (m, 2H), 7.26-7.29 (m, 3H), 5.90 (d, J = 3.6 Hz, 1 H), 5.83 (d, J= 7.8 Hz, 1 H), 5.54 (dd, ./= 6.6, 3.0 Hz, 1 H), 4.16-4.30 (m, 5H), 3.77-4.00 (m, 5H), 3.67 (s, 2H), 3.54 (t, J = 9.6 Hz, 1 H). 13C NMR ( 150 MHz, D20) δ 175.33, 166.16, 151 .75, 141 .64, 135.13, 129.36, 128.90, 127.26, 102.74, 94.77, 88.80, 83.08 (d, J= 9 Hz), 73.80, 73.25, 70.97, 69.74, 69.65, 65.07, 60.50, 53.89 (d, J= 8.7 Hz), 42.21. HRMS (ESI) m/z calcd for C23H3iN3Oi7P2 ( +H) 684. 1207, found 684.1215.
[0169] Uridine 5'-diphospho-2-(l,l'-biphenyl-4-yl)acetamido-2-deoxy-a-D- glucopyranoside (UDP-GkNAcPh2, F5-8). UDP-GlcNAcPh2F5-8 was synthesized from UDP-GlcNH2 F5-2 using a similar procedure as described above for UDP-GlcNAcPh F5-7g except that the reagent 2-phenylacetyl acid was replaced by 2-([ l , l '-biphenyl]-4-yl)acetic acid. UDP-GlcNAcPh2 F5-8 was obtained as a white solid in 82% yield (31 mg). Ή NMR (600 MHz, D20) δ 7.69 (d, J= 8.4 Hz, 1 H), 7.64 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 7.2 Hz, 2H), 7.45-7.47 (m, 2H), 7.34-7.41 (m, 3H), 5.79 (d, J= 4.2 Hz, 1 H), 5.64 (d, ./ = 7.2 Hz, 1 H), 5.53 (dd, J = 6.6, 3.0 Hz, 1 H) 4.14-4.19 (m, 5H), 4.01 (d, J = 10.2 Hz, 1 H), 3.92 (d, J = 9.6 Hz, 1 H), 3.65-3.85 (m, 5H), 3.53 (t, J = 9.0 Hz, 1 H). 13C NMR ( 1 50 MHz, D20) δ 175.19, 165.92, 15 1.44, 141 .31 , 140.08, 139.12, 134.51 , 129.90, 129.16, 127.75, 127.14,
126.80, 102.49, 94.76, 88.77, 82.87 (d, J = 8.6 Hz), 73.82, 73.22, 71.01 , 69.70, 69.44, 64.89, 60.46, 53.88 (d, J= 8.4 Hz), 41.80. HRMS (ESI) m/z calcd for C29H35N30,7P2 (M+H) 760.1520, found 760.1534.
[0170] Uridine 5'-diphospho-2-acetamido-6-amino-2,6-dideoxy-a-D-gkicopyrarioside (UDP-GlcNAc6NH2, F5-10). UDP-GlcNAc6N3 (T5b-12 or F5-9)(100 mg, 0.16 mmol) was dissolved in MeOH-H20 (10 mL, 1:1, v/v) and 20 mg of Pd/C was added. The mixture was shaken under H2 gas (4 Bar) for 1 hr, filtered, and concentrated. The residue was purified by flash column chromatography (EtOAc:MeOH:H20 = 3:2:1, by volume) to afford UDP- GlcNAc6NH2 F5-10 in 96% yield (93 mg). Ή NMR (600 MHz, D20) δ 7.90 (d, J= 8.4 Hz, 1H), 5.89-5.93 (m, 2H), 5.48 (dd, J= 6.6, 3.0 Hz, 1H), 4.30-4.32 (m, 2H), 4.20-4.23 (m, 2H), 4.08-4.15 (m, 2H), 3.99 (d, J= 10.8 Hz, 1H), 3.77 (t, J= 9.6 Hz, 1H), 3.45 (d, J= 13.2 Hz, 1H),3.40 (t, J=9.6Hz, 1H), 3.11 (t, J= 12.6 Hz, 1H), 2.02 (s, 3H). °CNMR(150 MHz, D20) 5174.94, 166.39, 151.96, 141.84, 102.79,94.33,88.82, 83.29 (J= 9.0 Hz), 73.92, 71.82, 69.80, 69.28, 65.30, 53.64 (J= 8.9 Hz), 40.70, 22.21. HRMS (ESI) m/z calcd for Ci7H28N40,6P2(M+H)" 607.1054, found 607.1068. [0171] Uridine 5'-diphospho-2-acetamido-6-hydroxyacetamido-2,6-dideoxy-a-D- glucopyranoside (UDP-GlcNAc6NGc, F5-12). UDP-GlcNAc6NGcAc F5-llwas synthesized from UDP-GIcNAc6NH2 F5-10 using the same process as described above for UDP-GlcNAcNGcAc F5-4. UDP-GlcNAc6NGcAc F5-11 was obtained as a white solid in 91% yield (31 mg). 'HNMR (600 MHz, D20) δ 7.91 (d,J=7.8Hz, 1H), 5.91-5.93 (m, 2H), 5.46 (dd, J= 6.6, 3.0 Hz, 1H), 4.62 (s, 2H), 4.30-4.34 (m, 2H), 4.14-4.24 (m, 3H), 3.95 (m, 2H), 3.76 (t,J=9.0 Hz, 1H), 3.61 (dd,J= 14.4, 6.0 Hz, 1H),3.54 (dd,J= 14.4,2.4 Hz, 1H), 3.35 (dd, J= 14.4, 4.2 Hz, 1H), 2.15 (s, 3H), 2.03 (s, 3H). I C NMR (150 MHz, D20) δ 174.85, 173.43, 170.60, 166.44, 151.95, 141.74, 102.70, 94.40, 88.70, 83.16, 73.87,71.33, 70.89, 70.68, 69.66, 65.05, 62.88, 53.67, 39.59, 22.14, 20.13. UDP-GlcNAc6NGc F5-12 was synthesized from UDP-GlcNAc6NGcAc F5-11 using the same process as described above for UDP-GlcNGc F5-5 and obtained as a white solid in 98% yield (29 mg). Ή NMR (600 MHz, D20) δ 8.09 (d, J= 7.8 Hz, 1H), 6.11-6.13 (m, 2H), 5.66 (dd, J= 6.6, 3.0 Hz, 1 H), 4.50-4.53 (m, 2H), 4.33-4.43 (m, 3H), 4.26 (s, 2H), 4.14-4.16 (m, 2H), 3.95 (t, J= 9.9 Hz, 1H), 3.82 (d,./= 14.4 Hz, 1H), 3.73 (dd, J= 13.8,6 Hz, 1H), 3.56 (t, J= 10.2 Hz, 1H), 2.22 (s, 3H). ,3C NMR (150 MHz, D20) δ 175.55, 175.05, 166.60, 152.20, 141.97, 102.99,94.62, 88.99, 83.50 (d, J= 8.9 Hz), 74.06, 71.47, 71.06, 69.97, 65.33, 61.35, 53.98 (d, J= 8.4 Hz), 39.76, 22.42. HRMS (ESI) m/z calcd for
Figure imgf000056_0001
665.1109, found 665.1113. [0172] Uridine 5'-diphospho-2-acetamido-6-azidoacetamido-2,6-dideoxy-a-D- glucopyranoside (UDP-GlcNAc6NAcN3, F5-13). UDP-GlcNAc6NAcN3(F5-13) was synthesized from UDP-GlcNAc6NH2 (F5-10) using the same process as described above for UDP-GlcNAcN3 (F5-6). UDP-GlcNAc6NAcN3 (F5-13) was obtained as a white solid in 61 % yield (21 mg). Ή NMR (600 MHz, D20) δ 7.89 (d, J = 7.8 Hz, 1 H), 5.91 (m, 2H), 5.43 (dd, J = 6.6, 3.0 Hz, 1 H), 4.32-4.29 (m, 2H), 4.19-4.22 (m, 3H), 4.00 (s, 2H), 3.92-3.95 (m, 2H), 3.74 (t, J = 10.2 Hz, 1 H), 3.54 (s, 1H), 3.34 (t, ./= 9.6 Hz, 1 H), 2.01 (s, 3H). 13C NMR (150 MHz, D20) 5 174.85, 170.93, 166.40, 151 .92, 141.75, 104.99, 102.71 , 94.42, 88.71 , 83.16 (J = 8.9 Hz), 73.87, 71.27, 71 .01 , 70.71 , 69.67, 65.10, 53.72 (J = 8.1 Hz), 51.80, 39.92, 22.14. HRMS (ESI) m/z calcd for C,9H29N7017P2 (M+H) 690.1 173, found 690.1 180.
[0173] Uridine 5'-diphospho-2-acetamido-6-phenylacetamido-2,6-dideoxy-a-D- glucopyranoside (UDP-GlcNAc6NAcPh, F5-14). UDP-GlcNAc6NAcPh F5-14 was synthesized from UDP-GlcNAc6NH2 F5-10 using the same way as described above for UDP-GlcNAcPh (F5-7). UDP-GlcNAc6NAcPh (F5-14) was obtained as a white solid in 86% yield (30 mg). Ή NMR (600 MHz, D20) 5 7.87 (d, J = 8.4 Hz, 1 H), 7.36-7.38 (m, 2H), 7.29-7.32 (m, 3H), 5.87-5.89 (m, 2H), 5.48 (dd, J = 6.6, 2.4 Hz, 1 H), 4.16-4.29 (m, 5H), 3.92-3.98 (m, 2H), 3.78 (t, J= 9.6 Hz, 1 H), 3.53-3.65 (m, 4H), 3.30 (t, J= 9.6 Hz, 1 H), 2.05 (s, 3H). 13C MR ( 150 MHz, D2O) 5 175.37, 174.89, 166.26, 1 51 .75, 141.64, 135.34, 129.22, 129.05, 127.38, 102.66, 94.50, 88.93, 83. 12 (.7 = 8.6 Hz), 73.94, 71 .48, 71.06, 70.60, 69.51 , 64.99, 53.79 (J = 8.3 Hz), 42.43, 40.01 , 22.19. HRMS (ESI) m/z calcd for
C25H34N40,7P2 (M+H) 725.1473, found 725.1484.
[0174] Uridine 5'-diphospho-2-acetamido-6-(l,l'-biphenyl-4-yl)-acetamido-2,6- dideoxy-a-D-glucopyranoside (UDP-GlcNAc6NAcPh2, F5-15). UDP-GlcNAc6NAcPh2 (F5-15) was synthesized from UDP-GlcNAc6NH2 using the same way as described above for UDP-GlcNAcPh2 (F5-8). UDP-GlcNAc6NAcPh2 (F5-15) was obtained as a white solid in
88% yield (35 mg). Ή NMR (600 MHz, D20) δ 7.75 (d, J= 7.8 Hz, 1 H), 7.63 (d, J = 7.2 Hz, 2H), 7.60 (d, J = 7.2 Hz, 2H), 7.44-7.45 (m, 2H), 7.34-7.45 (m, 3H), 5.77-5.80 (m, 2H), 5.44 (dd, J = 7.2, 3.6 Hz, 1 H), 4.03-4.1 8 (m, 5H), 3.89-3.96 (m, 2H), 3.75 (t, ./ = 9.6 Hz, 1 H), 3.49-3.63 (m, 4H), 3.28 (t, J= 9.0 Hz, 1 H), 2.01 (s, 3H). I3C NMR (1 50 MHz, D20) δ 175.17, 174.79, 166.28, 151.63, 141.34, 140.12, 139.3 1 , 134.70, 129.74, 129.18, 127.75,
127.30, 126.88, 102.44, 94.39, 88.87, 82.62 (d, J = 8.7 Hz), 73.93, 71.18, 70.54, 69.27, 64.80, 53.77 (d, J= 8.4 Hz), 42.03, 40.16, 22.09. HRMS (ESI) m/z calcd for C3i H38N4017P2 (M+H) 801.1785, found 801 .1807.
Results and Discussion
[0175] As shown in Figure 4, three enzymes were used in one-pot to synthesize UDP- GlcNAc and derivatives. The first enzyme was an -acetylhexosamine 1 -kinase cloned from Bifidobacterium longum strain ATCC55813 (NahK_ATCC55813) which showed promiscuous substrate specificity and were able to use N-sulfated, 3-0-sulfated, or 6-0- sulfated GlcNAc and derivatives as substrates for the formation of GlcNAcal -phosphate derivatives. The second enzyme was an N-acetylglucosamine- 1 -phosphate undylyltransferase that we cloned from Pasteurella rnidtocida strain P-l 059 (ATCC 15742) (PmGlmU). It catalyzes the reversible formation of UDP-GlcNAc and pyrophosphate from UTP and GlcNAc l -phosphate with tolerance on some substrate modifications. The third enzyme was an inorganic pyrophosphatase also cloned from Pasteurella multocida strain P- l 059 (PmPpA) for hydrolyzing the pyrophosphate by-product formed to drive the reaction towards the formation of UDP-GlcNAc and derivatives. A recombinant Nah cloned from another strain of Bifidobacterium longum (NahK_JCM 1217) was used in the synthesis of GlcNAc- 1 - phosphate, GalNAc- 1 -phosphate, and their derivatives. The purified HexN Ac- 1 -phosphates were then used in a one-pot two-enzyme system containing a commercially available inorganic pyrophosphatase (PpA) and a GlmU cloned from E. coli (EcGlmU) or an AGX 1 cloned from human for the synthesis of UDP-GlcN Ac, dNDP-GlcNAc, dNDP-Glc, UDP- GalNAc, and derivatives. Nevertheless, chemoenzymatic synthesis of UDP-GlcNAc derivatives using all three enzymes in one-pot has not been reported. In addition, UDP- GlcNAc derivatives containing N-sulfated glucosamine or 0-sulfated GlcNAc have not been synthesized using the combination of these three enzymes.
[0176] As shown in Table 5b, the one-pot three-enzyme system (Figure 4) was quite efficient in synthesizing UDP-GlcNAc (T5b-9, 81 %), its C-2 derivatives such as UDP-N- trifluoroacctylglucosam ine (UDP-GlcNTFA, T5b-10, 97%) and UDP-2-azido-2-deoxy- glucose (UDP-GlcN3, T5b-ll, 54%), as well as its C-6 derivatives including UDP-N-acetyl- 6-azido-6-deoxy-glucosamine (UDP-GlcNAc6N3, T5b-12, 72%) and UDP-Nacetyl-6-O- sulfo-glucosamine (UDP-GlcNAc6S, T5b-13, 62%) from GlcNAc (T5b-1) and derivatives (T5b-2-T5b-5). An interesting observation was that the yield of the one-pot three-enzyme reaction was improved from 81 % to 97% when the TV-acetyl group of GlcNAc was substituted by an N-trifluoroacetyl group in GlcNTFA (T5b-2). However, while 6-C-sulfated GlcNAc (GlcNAc6S, T5b-5) was used as a substrate to produce UDP-GlcNAc6S (T5b-13) in 62% yield, the synthesis of its -trifluoroacetyl analogue UDP-6-O-sulfo-GlcNTFA (UDP-GlcNTFA6S, T5b-14) from 6-O-sulfo-GlcNTFA (GlcNTFA6S, T5b-6) was not successful. In addition, although both 2-azido-2-deoxy-glucose (T5b-3) and 6-<2-sulfo- GlcNAc (T5b-5) could be used for the synthesis of the corresponding UDP-GlcNAc derivatives UDP-GlcN3 T5b-ll and UDP-GlcNAc6S T5b-13 in 54% and 62% yields, respectively, the synthesis of UDP-2-azido-2-deoxy-6-0-sulfo-glucose (UDP-GICN36S, T5bl5) from GICN36S (T5b-7) with the combined modifications at C-2 and C-6 was not successful. Furthermore, the one-pot three-enzyme synthesis of UDP-/V-sulfo-glucosamine (UDP-GlcNS, T5b-16) from N-sulfo-glucosamine (GlcNS, T5b-8) was not achieved. As compounds T5b-3- T5b-8 have all been shown to be weak substrates for
Nah _ATCC55813, the successful synthesis of compounds T5b-ll-T5b-13 and the unsuccessful synthesis of compounds T5b-14-T5b-16 by the one-pot three-enzyme system indicate that the substrate specificity of PmGlmU is most likely the limiting factor. system shown in Figure 4. ND, not detected.
Figure imgf000059_0001
ND
NHSO3- T5b-8 G!cNS T5b-16 UDP-GlcNS
[0177] Taking advantage of the substrate promiscuity of NahK_ATCC55813 and
PmGImU, UDP-GlcNAc and a number of its natural and non-natural derivatives were synthesized efficiently by the one-pot three-enzyme system illustrated in Scheme 1.
However, the success of the approach relied on the substrate promiscuity of all enzymes used. In order to increase the size of the library of UDP-GlcNAc derivatives with various modifications that can be used to test the activity of diverse GlcNAc-transferases, we further carried out chemical diversification of chemoenzymatically-produced UDP-GlcNAc derivatives.
[01781 The N-TFA group in UDP-GlcNTFA (T5b-10) as well as the N3 group in UDP- GlcN3 (T5b-l l) UDP-GlcNAc6N3 (T5b-12), and UDP-GlcN36S (T5b-15) can be easily- converted to a free amine, allowing further modifications to generate a diverse array of N- substituted UDP-GlcNAc derivatives. As shown in Figure 5A, the N-TFA group at C2 of UDP-GlcNTFA T5b-10 (or F5-1) was removed under mild basic condition to produce UDP- glucosamine (UDP-GlcNH , F5-2) in 98% yield. Selective acylation of the free amine group in F5-2 using various acyl chlorides produced C-2 modified UDP-GlcNAc derivatives UDP- N-acetoxyacetylglucosamine (UDP-GlcNGcAc, F5-4), UDP-7V-azidoacetylglucosamine (UDP-GlcNAz, F5-6), UDP-N-phenylacetylglucosamine (UDP-GlcNPh, F5-7), and UDP-7V- (l J '-biphenyl-4-yl)acetylglucosamine (UDP-GlcNPh2, F5-8) in 68-95% yields.
Deacetylation of compound F5-4 using catalytic amount of NaOMe in MeOH provided UDP- N-hydroxyacetylglucosamine (UDP-GlcNGc, F5-5) in 98% yield. In addition, although UDP- GlcNS T5b-16 (or F5-3) was unable to be prepared from GlcNS (T5b-8) (Table 5b) in the one-pot three-enzyme system, it was readily obtained by N-sulfation of compound F5-2 with PyS03 in 2 M of NaOH aqueous solution in a very good yield (86%) (Figure 5A). Similarly as shown in Figure 5B, catalytic hydrogenation of the azido group at the C-6 of UDP- GlcNAc6N3 T5b-12 (or F5-9) generated UDP-6-amino-6-deoxyl-./V-acetylglucosamine (UDP-GlcNAc6NH2, F5-10) with an excellent yield (96%). Selective acylation of the free am ino group of F5-10 using various acyl chlorides produced C-6 modified UDP-GlcNAc derivatives including UDP-6-acetoxyacetamido- -acetylglucosamine (UDP- GlcNAc6NGcAc, F5-11), UDP-e-azidoacetamido-A'-acetylglucosamine (UDP- GlcNAc6NAz, F5-13), UDP-6-phenylacetamido-N-acetylglucosamine (UDP-GlcNAc6NPh, F5-14), and UDP- V-( 1 , 1 '-biphenyl-4-yl)acetamido-iV-acetylglucosamine (UDP- GlcNAc6NPh2, F5-15) in 61-91 % yields. Finally, C-6 modified derivative UDP-N- hydroxyacetamido-N-acetylglucosamine (UDP-GlcNAc6NGc, F5-12) was obtained in 98% yield by treating compound F5-11 in NaOMe and methanol.
Example 3. Preparation of UDP-GalNAc
[0179] One-pot three-enzyme synthesis of uridine 5'-diphospho-2-acetamido-2-deoxy- a-D-glacopyranoside (UDP-GalNAc). GalNAc (100 mg, 1.0 eq), ATP (1.2 eq.), and UTP (1.2 eq.) were dissolved in water in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mM, pH 8.0) and MgCl2 ( 10 mM). After the addition of NanK_ATCC55813 (3.5 mg), PmGlmU (5 mg), and PmPpA (2.5 mg), water was added to bring the volume of the reaction mixture to 20 mL. The reaction was carried out by incubating the solution in an isotherm incubator at 37 °C for 24 h with gentle shaking. Product formation was monitored by TLC (EtOAc eOILP O = 3:2: 1 by volume) with /?-anisaIdehyde sugar staining. The reaction was stopped by adding the same volume of ice-cold ethanol and incubating at 4 °C for 30 min. The mixture was concentrated and passed through a BioGel P-2 gel filtration column to obtain the desired product. Silica gel column purification (EtOAc:MeOH:H20 = 4:2: 1 ) was applied for further purification to give pure target compound. Yield, 83% (228 mg); white foam. Ή NMR (600 MHz, D20) δ 7.93 (d, ./ = 8.4 Hz, 1 H), 5.94-5.96 (m, 2H), 5.55 (dd, J = 6.6, 3.0 Hz, 1H), 4.27-4.36 (m, 2H), 4.22-4.27 (m, 3H), 4.16-4.1 8 (m, 2H), 4.02 (d, J = 3.0 Hz, 1 H), 3.95 (dd, J = 10.8, 3.0 Hz, 1 H), 3.71 -3.78 (m, 2H), 2.06 (s, 3H). 13C NMR ( 150 MHz, D20) 5 175.05, 166.31 , 151 .85, 141 .89, 102.82, 94.75, 88.70, 83.03 (d, J= 8.6 Hz), 73.97, 72.32, 69.79, 68.50, 67.64, 65.14, 61.17, 49.95 (d, J = 7.8 Hz), 22.24. HRMS (ESI) m/z calcd for C17H28 3O17P2 (M+H) 608.0894, found 608.0906.
Example 4. Preparation of UDP-Sugars using Sugar-l-P kinases, BLUSP. and PmPpA
[0180] General methods for compound purification and characterization. Chemicals were purchased and used without further purification. Ή NMR and l jC NMR spectra were recorded on a Varian Mercury 600 NMR spectrometer. High resolution electrospray ionization (ESI) mass spectra were obtained in negative mode using Thermo Electron LTQ- Orbitrap mass spectrometer. Silica gel 60 A (Sorbent Technologies) was used for flash column chromatography. Thin-layer chromatography (TLC) was performed on silica gel plates 60 GF254 (Sorbent Technologies) using anisaldehyde sugar stain for detection. Gel filtration chromatography was performed with a column (100 cm χ 2.5 cm) packed with BioGel P-2 Fine resins (Bio-Rad). GlcN3 (T6-9) (Lau K, Thon, V, Yu H, Ding L, Chen Y, Muthana MM, Wong D, Huang R, and Chen X. Chem. Commun. 2010, 46, 6066-6068), ManF (T6-11) (Burkart MD, Zhang Z, Hung S-C, and Wong C-H. J. Am. Chem. Soc. 1997, 1 19, 1 1743-1 1746; Cao H, Li Y, Lau K, Muthana S, Yu H, Cheng J, Chokhawala HA, Sugiarto G, Zhang L, and Chen X. Org. Biomol. Chem. 2009, 7, 5137-5145), GahN3 (T6-4) and ManN3 (T6-14) (Yu H, Yu H, Karpel R, cand Chen X. Bioorg. Med. Chem. 2004, 12, 6427-6435) were previously synthesized using reported methods. Nah ATCC 15697, EcGalK, SpGalK, and PmPpA were overexpressed as described previously.
[0181] One-pot multienzyme synthesis of UDP-sugars. Monosaccharides and derivatives (30-100 mg, 1.0 eq.), ATP (1 .2 eq.), and UTP ( 1 .3 eq.) were dissolved in water in a 15 mL centrifuge tube containing Tris-HCl buffer ( 100 mM, pH 8.0) and MgCl2 (10 mM). After the addition of appropriate amount of NahK_ATCC 15697, EcGalK, or SpGalK (1.3-4.5 mg), BLUSP (1.0-2.5 mg), and PmPpA (1 .5-2.5 mg), millipore water was added to bring the total volume of the reaction mixture to 10 mL. The reaction was carried out by incubating the solution in an isotherm incubator for 24 hr at 37 °C with gentle shaking or without shaking. In the synthesis of UDP-Glc, commercially available Glc-l -P (55.2 mg), UTP (1.2 eq.), Tris- HCl buffer ( 100 mM, pH 8.0), and MgCl2 (10 mM) were used along with BLUSP (1 mg) and PmPpA (1.5 mg). The reaction was left for 2 hr at 37 °C in isotherm with gentle shaking. Product formation was monitored by TLC (EtOAc:MeOH:H20:AcOH = 5:3:3 :0.3 by volume) with /?-anisaldehyde sugar staining. The reaction was terminated by adding the same volume of ice-cold ethanol and incubating at 4 °C for 30 min followed by centrifugation remove the enzymes. The supernatant was collected and concentrated and passed through a BioGel P-2 gel filtration column to afford the product. Silica gel column purification (EtOAc:MeOH:H20 = 7:3 :2) was applied when necessary to achieve further purification. [0182] Uridine 5'-diphospho-a-D-galactopyranoside (UDP-Gal, T6-16). 135 mg. Yield, 86%; white foam. Ή NMR (600 MHz, D20) δ 7.93 (d, ./ = 8.4 Hz, 1 H), 5.97-5.95 (m, 2H), 5.63 (dd, J = 7.2, 3.6 Hz, 1 H), 4.37-4.35 (m, 2H), 4.28-4.1 8 (m, 3H), 4.16 (t, J = 6 Hz, 1 H), 4.02 (d, J = 3 Hz, 1 H), 3.90 (dd, J = 10.2, 3.6 Hz, 1 H), 3.80 (dt, J = 10.2, 3.3 Hz, 1 H), 3.76- 3.71 (m, 2H). I3C NMR (1 50 MHz, D20) δ 166.39, 1 51 .96, 141 .78, 102.80, 96.01 (d, J = 6.6 Hz), 88.65, 83.32 (d, J = 8.9 Hz), 73.93, 72.1 1 , 69.78, 69.43, 69.24, 68.50 (d, J = 7.8 Hz), 65.15 (d, J = 5.0 Hz), 61.16. HRMS (ESI) m/z calcd for Ci5H24N2Oi7P2 (M-H) 565.0472, found 565.0453. [0183] Uridine 5'-diphospho-a-D-glucopyranoside (UDP-Glc, T6-21).82 mg. Yield, 99%; white foam. Ή NMR (600 MHz, D20) δ 7.94 (d, J= 8.4 Hz, 1H), 5.98-5.96 (m, 2H), 5.59 (dd, J= 7.2, 3.6 Hz, 1H), 4.37-4.35 (m, 2H), 4.28-4.18 (m, 3H), 3.9-3.83 (m, 2H), 3.78-3.74 (m, 2H), 3.53 (dt, J= 9.6, 3.3 Hz, 1H), 3.46 (t, J= 9.6 Hz, 1H).13C NMR (150 MHz, D20) δ 166.20, 151.75, 141.52, 102.57, 95.51(d, =6.8 Hz), 88.35, 83.07 (d,J= 8.9 Hz), 73.67, 72.72 (2C), 71.45 (d, J= 8.4 Hz ), 69.52, 69.05, 64.86 (d,J= 5.6 Hz), 60.20. H MS (ESI) OT/Z calcd for C^H^O^ (M-H) 565.0472, found 565.0458.
[0184] Uridine 5'-diphospho-2-deoxy-a-D-glucopyranoside (UDP-2-deoxyGlc, T6-22).
96 mg. Yield, 56%; white foam. Ή NMR (600 MHz, D20) δ 7.95 (d, ./= 8.4 Hz, HI), 5.96- 5.95 (m, 2H), 5.70 (dd,J = 7.2, 1.8 Hz, 1H), 4.36^1.33 (m, 2H), 4.27-4.16 (m, 3H), 4.0-3.95 (m, 1H), 3.86-3.75 (m, 3H), 3.39 (t, J= 9.6 Hz, 1H), 2.28-2.24(m, 1H), 1.74-1.68 (m, 1H), (150 MHz, D20)5168.99, 154.54, 144.38, 105.38, 97.63(d, J= 5.7 Hz), 91.25, 85.86 (d, J = 9.0 Hz), 76.51, 76.13, 73.33, 72.32, 70.46, 67.63 (d, J= 5.0 Hz), 63.21, 40.18(d,J= 7.2 Hz), HRMS (ESI) m/z calcd for C15H24N2O16P2 (M-H) 549.0523, found 549.0513. [0185] Uridine 5'-diphospho-2-aniino-2-deoxy-a-D-glucopyranoside (UDP-GICNH2, T6-23).56 mg. Yield, 43%; white foam. Ή NMR (600 MHz, D20) δ 7.93 (d, J= 7.8 Hz, 1H), 5.97-5.94 (m, 2H), 5.82 (d, J= 6.0 Hz, 1H), 4.36^1.34 (m, 2H), 4.28-4.17 (m, 3H), 3.92-3.90 (m2H),3.86 (dd, J= 12.0, 2.4 Hz, 1H), 3.81 (dd, J= 12.6,4.2 Hz, 1H), 3.55 (t, J = 9.9 Hz, 1H), 3.37 (d,J= 10.8 Hz, 1H).13C NMR (150 MHz, D20) δ 166.40, 151.93, 141.75, 102.71, 92.87, 88.74, 83.21 (d, J= 9 Hz), 73.91, 73.39, 69.85, 69.69, 69.16, 65.23, 60.09, 54.27 (d, J= 8.4 Hz). HRMS (ESI) m/z calcd for
Figure imgf000063_0001
(M-H) 564.0632, found 564.0619.
[0186] Uridine 5'-diphospho-2-azido-2-deoxy-a-D-glucopyranoside (UDP-GICN3, T6-
24).88 mg, Yield, 61%; white foam. Ή NMR (600 MHz, D20) δ 7.95 (d, J= 8.4 Hz, 1H), 5.96-5.95 (m, 2H), 5.67 (dd,./= 7.2, 3 Hz, 1H), 4.36-4.33 (m, 2H), 4.27-4.18 (m, 3H), 3.93- 3.88 (m, 2H), 3.85-3.76 (m, 2H), 3.53 (t, J= 9.6 Hz, 1 H), 3.38 (d, ./= 10.8 Hz, 1 H). I3C NMR(150MHz, D20) δ 166.14, 151.75, 141.61, 102.79, 94.39 (d, J= 4.5 Hz), 88.47, 83.48 (d, J= 8.4 Hz), 73.68, 72.85, 70.61, 69.53, 69.24, 64.87, 62.71 (d, J= 7.8Hz), 60.05. HRMS (ESI) m/z calcd for
Figure imgf000063_0002
(M-H) 590.0537, found 590.0524. [0187] Uridine 5'-diphospho-a-D-mannopyranoside (UDP-Man, T6-26).60 mg. Yield, 60%; white foam. Ή NMR (600 MHz, D20) δ 7.93 (d, J = 8.4 Hz, 1H), 5.96-5.94 (m, 2H), 5.51 (d, J= 7.2, 1H), 4.35-4.18 (m, 5H), 4.02 (m, 1H), 3.89-3.82 (m, 3H), 3.75 (dd, J= 12, 4.8 Hz, 1H), 3.67 (t, J=9.9 Hz, 1H).13C NMR (150 MHz, D20) δ 166.40, 151.94, 141.77,
102.79, 96.64 (d, J= 5.5), 88.70, 83.24 (d, J= 8.7 Hz), 73.93, 73.91, 70.38 (d, J= 9.3 Hz), 69.98, 69.74, 66.56, 65.15 (d, J= 4.7 Hz), 60.92. HRMS (ESI) m/z calcd for C15H24 2O17P2 (M-H) 565.0472, found 565.0467.
[0188] Uridine 5'-diphospho-2-fluoro-2-deoxy-a-D-mannopyranoside (UDP-ManF, T6-27).142 mg. Yield, 92%; white foam. Ή NMR (600 MHz, D20) δ 7.94 (d, J= 8.4 Hz, 1H), 5.97-5.95 (m, 2H), 5.70 (t, J= 6.3 Hz, 1H), 4.39-4.35 (m, 2H), 4.36-4.33 (m, 2H), 4.28-4.16 (m, 3H), 4.00 (ddd, J= 30.6, 9.6, 2.4 Hz, 1H), 3.88-3.86 (m, 2H), 3.79 (d, J = 12.6, 4.8 Hz, 1H), 3.74 (t, J= 9.9 Hz, 1H),.13C NMR (150 MHz, D20) δ 166.42, 151.98,
141.80, 102.84, 93.75 (dd, J=31.2, 5.7Hz), 89.75 (dd, J= 173.6, 10.5 Hz), 88.75, 83.26 (d, J= 9.0 Hz), 73.95, 73.84, 69.76, 69.32 (d, J= 17.3 Hz), 66.46, 65.15 (d, J= 5.1 Hz) 60.46. HRMS (ESI) m/z calcd for CI5H23FN20,6P2 (M-H) 567.0429, found 567.0426.
[0189] Uridine 5'-diphospho-2-azido-2-deoxy-a-D-mannopyranoside (UDP-ManN3, T6-29).259 mg, Yield, 90%; white foam. Ή NMR (600 MHz, D20) δ 7.96 (d, J = 8.4 Hz, 1H), 6.00-5.98 (m, 2H), 5.62 (d,J- 7.2 Hz, 1H), 4.39-4.35 (m, 2H), 4.31-4.18 (m, 3H), 4.16-4.13 (m, 2H), 3.87-3.83 (m, 2H), 3.77 (dd, J= 12.6, 4.8 Hz, 1H), 3.70 (t,./= 9.6 Hz, 1H),. I C NMR (150 MHz, D20) δ 166.42, 151.97, 141.81, 102.84, 94.86 (d, 7=5.7 Hz), 88.80, 83.24 (d, J= 8.9 Hz), 73.96, 73.95, 70.09, 69.74, 66.49, 65.16 (d,J= 5.0 Hz), 64.18 (d, ./= 9.5 Hz) 60.64. HRMS (ESI) m/z calcd for C15H23 5O16P2 (M-H) 590.0537, found 590.0532.
[0190] Uridine 5'-diphospho-2-acetamido-2-deoxy-a-D-mannopyranoside (UDP- ManNAc, T6-30). Yield for two steps from UDP-ManN3 (T6-29), 79%; white foam.Ή NMR (600 MHz, D20) δ 7.96 (d, J= 7.8 Hz, 1H), 5.98-5.95 (m, 2H), 5.44 (dd, J= 7.8, 1.8 Hz, 1H), 4.43 (dd, J= 4.8, 1.8 Hz, 1H), 4.37-4.34 (m, 2H), 4.28-4.22 (m, 2H), 4.19-4.15 (m, 1H), 4.11 (dd,J= 10.2, 4.8 Hz, 1H),3.90 (dt,J= 10.2, 3.0 Hz, 1H), 3.85 (d, J= 3.6 Hz, 1H), 3.62 (t,J= 10.2 Hz, HI), 2.03 (s, 3H).13CNMR(150 MHz, D20) δ 175.59, 166.16, 151.75, 141.55, 102.57, 95.35, 88.23, 83.17 (d, J= 8.9 Hz), 73.73, 73.20, 69.58, 68.72, 66.29, 64.82, 59.23, 52.94 (d, J= 8.9 Hz), 21.85. HRMS (ESI) m/z calcd for Ci7H27 30,7P2 (M-H) 606.0737, found 606.0723. Table 6. Synthesis of UDP-monosaccharides using the one-pot three-enzyme system shown in Figure 9. ND, not detected.
Figure imgf000065_0001
[0191] We were able to identify at least one of these kinases for each monosaccharide that gave a yield higher than 58% for the formation of the corresponding monosaccharide- 1- phosphate. The observed results from thin-layer chromotography (TLC) and capillary electrophoresis (CE) (Table 7) confirmed the previously reported activities of NahK, SpGalK, and EcGalK toward their respective substrates except for mannosamine (T6-13), a NahK substrate which was not previously tested.
Table 7. Yields of the kinase reactions monitored by the conversion of ATP to ADP in capillary electrophoresis (CE) assays. Abbreviation: NA, not assayed.
ATP Conversion (%)
Substrate Kinase
1 hr 4 hr 24 hr
No No < 2 < 5 1 1.1
T6-1 Gal SpGalK 92.2 NA NA
T6-1 Gal EcGalK 90.3 NA NA
T6-2 2-deoxyGal SpGalK 80.3 89.5 NA
T6-2 2-deoxyGal EcGalK 78.5 87.6 NA
T6-3 GalNH, EcGalK 90.2 NA NA
T6-4 GalN3 SpGalK 45.7 79.0 81.2
T6-5 GalNAc SpGalK 1 1 .8 24.7 69.5
Glc EcGalK 8.2 13.0 66.4
Glc SpGalK 6.9 13.8 75.5
Glc NahK 10.3 18.6 82.2
T6-7 2-deoxyGlc NahK 36.8 69.6 79.4
T6-8 GlcNH2 NahK 1 1 .9 28.0 67.1
T6-9 GlcN3 NahK 12.4 25.9 71.2
T6-10 GlcNAc NahK 72.6 84.6 85.5
T6-11 Man NahK 29.6 69.3 75.1
T6-12 Man2F NahK 57.9 67.9 78.2
T6-13 ManNH? NahK 10.3 22.8 58.0
T6-14 ManN3 NahK 34.9 65.9 76.4
T6-15 ManNAc NahK 1 1 .4 26.1 73.8 [0192] The synthesis of all other UDP-sugars in Table 6 was carried out using the one-pot three-enzyme system shown in Figure 9. As shown in Table 6, the one-pot three-enzyme system provided excellent yields for the formation of UDP-Gal (T6-16, 86%), UDP-ManF (T6-27, 92%), and UDP-ManN3 (T6-29, 90%) from the corresponding monosaccharides Gal (T6-1), ManF (T6-12), and ManN3 (T6-14), respectively. Three of the derivatives of UDP- Glc including UDP-2-deoxyGlc (T6-22), UDP-GlcNH2 (T6-23), and UDP-GlcN3 (T6-24) were obtained from 2-deoxyGlc (T6-7), glucosamine (GlcNH2, T6-8) and GlcN (T6-9) in 56%, 43%, and 61 % yields, respectively. The moderate yields for these three compounds may be attributed by less optimal NahK kinase activity for GlcNH2 (T6-8) and GlcN} (T6-9), and the less optimal BLUSP activity for 2-deoxyGlc (T6-7). UDP-Man (T6-26) was synthesized from Man (T6-11) in moderate 60% yield using the one-pot three-enzyme system and the moderate yield was most likely due to the less optimal activity of BLUSP towards Man-l -P. The synthesis of four UDP-Gal derivatives including its 2-deoxy, 2-deoxy-2- amido-, 2-deoxy-2-azido-, and 2-deoxy-2-acetamido-derivatives (T6-17-T6-20) using the one-pot three-enzyme system was not successful. In addition, UDP-GlcNAc (T6-25), UDP- ManNH2 (T6-28), and UDP-ManNAc (T6-30) could not be produced from the corresponding monosaccharides (T6-10, T6-13, and T6-15) using the one-pot three-enzyme system. These were most likely due to the substrate restriction of BLUSP instead of kinases used.
[0193] Although UDP-ManNH2 (T6-28) and UDP-ManNAc (T6-30) were not directly available from ManNH2 (T6-13) and ManNAc (T6-15), respectively, via the one-pot three- enzyme reaction shown in Figure 9, they can be readily prepared via simple chemical modification reactions from UDP-ManN3 (T6-29) obtained from the one-pot three-enzyme system. As shown in Figure 8, a simple one-step catalytic hydrogenation of UDP-ManN3 (T6-29) produced UDP-ManNH2 (T6-28). Acetylation of the amino group in UDP-ManNH2 (T6-28) provided an easy access of UDP-ManNAc (T6-30). The similar chemical acylation of UDP-ManNH2 can be used to synthesize other acyl derivatives of UDP-ManNAc.
Example 5: Synthesis of UDP-Uronic Acids using AtGlcAK, BLUSP, and PmPpA
[0194] Mass spectrometry analysis of one-pot multienzyme synthesis of UDP-GlcA, UDP-IdoA, and UDP-GalA. Enzymatic assays were carried out at in a total volume of 10 in Tris-HCl buffer (100 mM, pH 7.5) containing GlcA (GalA, or IdoA) (10 mM), ATP (20 mM), MgCl2 (20 mM), and AtGlcAK (23 μg). Reactions were allowed to proceed at 37 °C for 15 hr and monitored using thin-layer chromatographic analysis using n- PrOH:H20:NH4OH = 7:4:2 (by volume) as a developing solvent. /?-Anisaldehyde sugar stain followed by heating the TLC plates on hot plate was used for visualizing compounds on the TLC plates. After 24 hr, BLUSP (5 μg), PmPpA (5 μg) and UTP ( 12 mM) were added to the reaction mixture. The reactions were allowed to proceed at 37 °C for another 24 hr. The reactions were then quenched with the same volume of 100% ethanol, centrifuged at 13000 rpm for 2 min, and the reaction mixtures were stored at -20 °C.
[0195] For LC-MS analysis, 2 μΐ^ of each sample was diluted 100-fold and 8 μΐ^ was injected to a Waters spherisorb ODS-2 column (5 μιτι particles, 250 mm length, 4.6 mm I.D.). Each sample was eluted with 30 % acetonitrile in H?0 and detected by ESI-MS in the negative mode.
[0196] AtGlcAK was shown to be active on GlcA, GalA, and IdoA by TLC and LC-MS analyses. One-pot three-enzyme strategy containing AtGlcAK, BLUSP, and PmPpA (Figure 10) was shown to be able to produce UDP-GlcA, UDP-GalA, and UDP-IdoA from their corresponding monosaccharides GlcA, GalA, and IdoA respectively in small-scale assays confirmed by LC-MS or HRMS (Figure 30).
Example 6: Preparation of GlcNAcal^lGIcA Disaccharide Derivatives
[0197] General methods for compound purification and characterization. Chemicals were purchased and used without further purification. Ή NMR and l 3C NMR spectra were recorded on Varian VNMRS 600 MHz and Bruker Avance 800 MHz spectrometer. High resolution electrospray ionization (ESI) mass spectra were obtained at the Mass Spectrometry Facility in the University of California, Davis. Silica gel 60 A (Sorbent Technologies) was used for flash column chromatography. Analytical thin-layer chromatography (Sorbent Technologies) was performed on silica gel plates using anisaldehyde sugar stain for detection. Gel filtration chromatography was performed with a column (100 cm x 2.5 cm) packed with BioGel P-2 Fine resins. ATP, UTP, GlcNAc, Glc-l -P, NAD+, and
glucuronolactone were purchased from Sigma. GlcNTFA, GlcNAc6N3, UDP-GlcNGc, UDP- GlcNAz, UDP-GlcNAc6NGc were synthesized as described previously. NanK_ATCC55813, PmGlmU and PmPpA were overexpressed as reported.
[0198] Chemical synthesis of GlcAp2AAMe. GlcAp2AAMe was synthesized as outl ined in Figure 13.
[0199] Synthesis of F13-2. Glucuronolactone F13-1 (2.0 g, 1 1.3 mmol) was dissolved in dry MeOH (12 mL) under 2. To the solution, 20 mg of sodium methoxide was added. The reaction was stirred at room temperature for 3 hours, and then MeOH was removed in vacuo. The resulting syrup was further dried under high-vacuum. The above product was dissolved in pyridine ( 10 mL) and acetic anhydride (8 mL) under 0 °C and N2. The reaction was stirred from 0 °C to room temperature overnight. The mixture was concentrated and purified by flash column chromatography (Hexane : EtOAc: = 1 : 1 , by volume) to provide white solid F13-2 in 67% yield, β-isomer: Ή NMR (600 MHz, CDC13) δ 5.76 (d, ./= 7.8 Hz, 1 H), 5.30 (t, .7 = 9.6 Hz, 1 H), 5.25 (t, .7 = 9.6 Hz, 1 H), 5.13 (t, J = 7.8 Hz, 1 H), 4.17 (d, J= 9.6 Hz, 1 H), 3.73 (s, 3H), 2.10 (s, 3H), 2.03 (s, 6H), 2.02 (s, 3H). 13C NM (1 50 MHz, D20) δ 170.13, 169.65, 169.53, 168.61 , 167.37, 88.90, 70.51 , 69.24, 69.07, 69.00, 53.17, 20.95, 20.79, 20.61 , 20.55. a-isomer: Ή NMR (600 MHz, CDC13) δ 6.39 (d, J = 3.6 Hz, 1 H), 5.51 (t, J = 10.2 Hz, 1 H), 5.22 (t, J = 10.2 Hz, 1 H), 5.12 (dd, J = 10.2, 3.6 Hz, 1 H), 4.41 (d, / = 10.2 Hz, 1 H), 3.74 (s, 3H), 2.15 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H). C NMR (1 50 MHz, CDC13) δ 170.04, 169.56, 169.32, 168.98, 166.95, 91.48, 73.1 1 , 71 .94, 70.27, 69.06, 53.18, 20.93, 20.72, 20.70, 20.63.
[0200] Synthesis of methyl 2,3,4-tri-O-acetyl-D-glucopyranuronate F13-3. Methyl 1 ,2,3,4-tetra-O-acetyl-D-glucopyranuronate F13-2 ( 1.2 g, 3.2 mmol) was dissolved in dry DMF ( 10 mL) under N2. To the solution, benzylamine (0.42 mL, 3.8 mmol) was added. The mixture was stirred at r.t. for 16 hours. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (Hexane : EtOAc: = 1 :2, by volume) to afford a white solid F13c in 84% yield. Ή NMR (600 MHz, CDC13) δ 5.50-5.55 (m, 1 H), 5.1 1 -5.27 (m, 1 H), 4.85^1.91 (m, 1 H), 5.39-4.56 (m, 2H), 3.70-3.72 (m, 3H), 1 .99-2.05 (m, 9H). 13C NMR (150 MHz, CDCl3) 5 170.68, 170.45, 170.34, 170.30, 169.94, 169.81 , 168.77, 167.83, 95.59, 90.36, 72.98, 72.66, 71.81 , 70.97, 69.73, 69.61 , 69.34, 68.12, 53.21 , 53.1 1 , 20.85, 20.84, 20.77, 20.70, 20.66, 20.65.
[0201] Synthesis of methyl 2,3,4-tetra-0-acetyl-l-0-(3-chloropropyl)-P-D- glucopyranuronate F13-5. Methyl 2,3,4-tri-O-acetyl-D-glucopyranuronate F13-3 (800 mg, 2.4 mmol) was dissolved in 8 mL dichloromethane. Trichloroacetonitrile (1 .3 mL, 12 mmol) was added under N2. After cooling to 0 °C, l ,8-diazabicyclo[5.4.0] undee-7-ene (1 ,8-DBU) was added in a drop-wise manner until the color of sodium changes to brown. The reaction mixture was allowed to stir for 1 h and the mixture was concentrated to afford a sticky dark brown residue. The flash column chromatography (Hexane : EtOAc = 3 :2, by volume) gives an off-white product F13-4 in 88% yield. To the mixture of S4 (200 mg, 0.42 mmol) and MS 4A, 8 mL dichloromethane was added, followed by 3-chloropropanol (0.25 mL, 2.1 mmol). The mixture was stirred for 30 min at room temperature under N2. After cooling to 0 °C, boron trifluoride ether complex (0.06 mL, 0.42 mmol) was added drop-wisely. The reaction was stirred at 0 °C for 3 hours. After the TLC showed the reaction is completed, the mixture was filtered and the filtrate was washed with saturated NaHC03. The organic layer was evaporated to give a crude residue which was purified by silica gel chromatography (Hexane : EtOAc: = 3:2, by volume) to provide the product F13-5 in 64% yield. Ή NMR (600 MHz, CDCI3) δ 5.17-5.28 (m, 2H), 4.97-5.00 (dd, J= 9.6, 7.8 Hz, I H), 4.53^1.54 (d, J= 7.8 Hz, IH), 4.02-4.04 (d, J= 9.6 Hz, IH), 3.99-4.01 (dd, J= 9.6, 4.8 Hz, 1 H), 3.27 (s, 3H), 3.66- 3.70 (m, IH), 3.57-3.59 (m, 2H), 2.05-2.09 (m, I H), 2.04 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.91-1 .95(m I H). 13C NMR (150 MHz, CDC13) δ 170.30, 169.59, 169.52, 167.37, 101.21 , 72.78, 72.14, 71.33, 69.62, 66.79, 53.13, 41.48, 32.29, 20.83, 20.82, 20.71.
[0202] Synthesis of methyl 2,3,4-tetra-0-acetyl-l-0-(3-azidoopropyl)-p-D- glucopyranuronate F13-6. Methyl 2,3,4-tetra-0-acetyl-l -0-(3-chloropropyl)-P-D- glucopyranuronate F13-5 (412 mg, 1.0 mmol) was dissolved in 10 mL of DMF. To the solution, sodium azide (325 mg, 5.0 mmol) was added. The reaction was stirred at 65 °C overnight. The solvent was removed under reduced pressure and the residue was purified by flash chromatography (Hexane:EtOAc = 3:2, v/v) to afford a white solid in 92% yield. Ή NMR (600 MHz, CDCI3) δ 5.19-5.27 (m, 2H), 4.99-5.02 (t, J= 7.8 Hz, I H), 4.54-4.55 (d, J = 7.8 Hz, I H), 4.02^1.04 (d, J= 9.6 Hz, I H), 3.94-3.95 (m, I H), 3.75 (s, 3H), 3.58-3.62 (m, I H), 3.32-3.39 (m, 2H), 2.04 (s, 3H), 2.01 (s, 3H), 1.78-1.89 (m 2H). 13C NMR (150 MHz, CDC13) 5 170.27, 169.53, 169.40, 167.34, 101.01 , 72.82, 72.23, 71 .39, 69.60, 66.92, 53.09, 48.10, 29.1 1, 20.81 , 20.79, 20.68.
[0203] Synthesis of l-C-(3-azidoopropyl)-p-D-glucopyranuronic acid F13-7. Methyl 2,3,4-tetra-0-acetyl4 -0-(3-azidoopropyl)- -D-glucopyranuronate F13-6 (350 mg, 0.84 mmol) was dissolved in 5 mL MeOH. To the solution, sodium methoxide was added until the pH go to 9.5. The reaction was stirred at room temperature for 1 hr. After the TLC showed the reaction is completed, potassium hydroxide (60mg, 2.52 mmol) and 10 mL water was added. After stirred at r.t. for 3 hours, the mixture was neutralized with DOWEX HCR-W2 (H+) resin, filtered, and concentrated. The residue was purified by flash column
chromatography (EtOAc:MeOH:H20 = 6:2: 1 , by volume) to afford white solid F13-7 in 79% yield. Ή NMR (600 MHz, D20) δ 4.45-4.47 (d, J = 7.8 Hz, I H), 3.95-3.99 (m, I H), 3.81- 3.82 (d, J = 9.0 Hz, I H), 3.71 -3.75 (m, I H), 3.49-3.54 (m, 2H), 3.42-3.45 (t, J= 6.6 Hz, 2H), 3.28-3.31 (t, J = 8.4 Hz, I H), 1 .86-1.91 (m, 2H). I3C NMR ( 1 50 MHz, D20) 5 175.17, 102.36, 75.91 , 75.64, 73.06, 71.86, 67.57, 48.05, 28.39.
[0204] Synthesis of GlcAp2AAMe (F13-8). l -0-(3-Azidoopropyl)-P-D-glucopyranuronic acid F13-7 (100 mg, 0.44 mmol) was dissolved in 1 0 mL MeOH and 20 mg of Pd/C was added. The mixture was shaken under H2 gas (4 Bar) for 1 h, filtered, and concentrated. The residue was further dried in high-vacuum. To a solution of the amine residue in 10 mL of DMF-MeOH (1 : 1 , v/v), dry triethylamine (61 μί) was added under N2. Then 2- (methoxycarbonyl) succinanilic acid NHS ester3 (2AA-OSu, 306 mg, 0.88 mmol) was added at 0 °C. The reaction mixture was stirred at room temperature for overnight. The reaction mixture was concentrated and the residue was purified by silica gel chromatography
(EtOAc:MeOH:H20 = 8:2: 1 , by volume) to afford white solid GlcAp2AAMe (F13-8) in 83% yield. !H NMR (600 MHz, D20) 8 7.88-7.89 (d, J= 8.4 Hz, 1 H), 7.79-7.80 (d, J= 7.8 Hz, 1 H), 7.48-7.51 (t, ./= 7.8 Hz, 1 H), 7.12-7.15 (t, J= 7.8 Hz, 1 H), 4.27-4.29 (d, J= 7.8 Hz, 1H), 3.83-3.86 (m, 1 H), 3.81 (s, 3H), 3.58-3.60 (d, J= 9.6 Hz, 1H), 3.53-3.57 (m, 1 H), 3.40-3.47 (m, 2H), 3.24-3.30 (m, 2H), 3.1 8-3.22 (m, 1 H), 2.61-2.64 (t, J= 7.2 Hz, 2H), 2.52-2.54 (t, J= 7.2 Hz, 2H), 1.72-1.77 (m, 2H). I C NMR (150 MHz, D20) δ 175.17, 174.18, 172.66, 168.75, 137.89, 134.16, 130.81 , 124.41 , 121 .83, 1 18.36, 101.96, 75.66, 75.46, 72.83, 71 .68, 67.35, 52.63, 36.04, 32.61 , 30.83, 28.23.
[0205] One-pot four-enzyme synthesis of disaccharides F18a-F18c. As shown in
Figure 18A, GlAp2AAMe (F13-8) (5 to 30 mg, 1 eq.), glucosamine derivatives (1.5 eq.), ATP ( 1.8 eq.), and UTP (1 .8 eq.) were dissolved in water in a 1 5 mL centrifuge tube containing Tris-HCl buffer ( 100 mM, pH 7.5) or MES buffer (100 mM, pH 6.5) and MgCl2 (10 mM). After the addition of appropriate amount of Nan ATCC55813 (0.5-2.1 mg), PmGlmU (1-3 mg), PmPpA (0.5-1 .5 mg), and PmHS2 ( 1-6 mg), water was added to bring the concentration of β1Αβ2ΑΑΜε (F13-8) to 5 mM. The reaction was carried out by incubating the solution in an isotherm incubator for 12-36 h at 37 °C with gentle shaking. Product formation was monitored by TLC (EtOAc:MeOH:H20 = 4:2: 1 by volume) with p- anisaldehyde sugar staining. The reaction was stopped by adding the same volume of ice-cold ethanol and incubating at 4 °C for 30 min. The mixture was concentrated and passed through a BioGel P-2 gel filtration column to obtain the desired product. Silica gel column purification (EtOAc:MeOH :H20 = 5 :2: 1 ) was applied when necessary to achieve further purification.
[0206] GlcNAcal-4GlcAp2AAMe F18-1. Yield: 95%; white foam. Ή NMR (600 MHz, D20) δ 7.95-7.96 (d, J = 8.4 Hz, 1 H), 7.81-7.83 (d, J= 8.4 Hz, 1 H), 7.61-7.64 (t, J= 7.8 Hz, 1 H), 7.29-7.32 (t, J= 8.4 Hz, 1 H), 5.38-5.39 (d, J= 3.6 Hz, 1 H), 4.32-4.33 (d, J= 7.8 Hz, 1 H), 3.89 (s, 3H), 3.79-3.88 (m, 4H), 3.70-3.73 (m, 4H), 3.60-3.63 (m, 1 H), 3.55-3.58 (m, 1 H), 3.45-3.48 (t, J= 9.6 Hz, I I I), 3.20-3.32 (m, 3H), 2.73-2.75 (t, J= 6.6 Hz, 2H), 2.58- 2.61 (t, J= 7.2 Hz, 2H), 2.04 (s, 3H), 1.73-1.78 (m, 2H). 13C NMR (150 MHz, D20) δ 175.15, 174.61, 174.51, 173.61, 169.24, 137.16, 134.12, 130.99, 125.45, 123.57, 121.10, 102.21 , 96.98, 76.96, 76.69, 75.90, 73.56, 72.01, 70.86, 69.80, 67.58, 60.22, 53.83, 52.93, 36.23, 32.59, 31.20, 28.40, 22.06. HRMS (ESI) m/z calcd for C29H42N3016 (M+H) 688.2560, found 688.2563.
[0207] GlcNTFAal-4GlcAp2AAMe F18-2. Yield: 84%; white foam. Ή NMR (600
MHz, D20) 5 7.95-7.97 (d, J = 7.8 Hz, 1H), 7.88-7.89 (d, J= 7.8 Hz, 1 H), 7.62-7.65 (t, J = 7.8 Hz, 1H), 7.30-7.32 (t, J= 7.8 Hz, 1H), 5.50-5.51 (d, J= 3.6 Hz, 1H), 4.34-4.35 (d, J = 7.8 Hz, 1H), 4.02-4.04 (dd, J= 10.8, 4.2 Hz, 1H), 3.91 (s, 3H), 3.83-3.88 (m, 4H), 3.75-3.77 (m, 3H), 3.63-3.66 (m, 1 H), 3.57-3.61 (m, 1 H), 3.51-3.55 (t, J= 9.6 Hz, 1 H), 3.23-3.34 (m, 3H), 2.74-2.76 (t, J= 6.6 Hz, 2H), 2.61-2.63 (t, J= 7.2 Hz, 2H), 1 .77-1 .81 (m, 2H). 13C NMR ( 150 MHz, D20) δ 175.15, 174.63, 173.50, 169.26, 159.45 (q, J = 37.6 Hz), 137.49, 134.28, 131 .09, 125.33, 123.27, 120.52, 1 17.02 (q, J = 284.7 Hz), 102.29, 96.51 , 76.90, 76.73, 76.13, 73.61 , 72.21 , 70.40, 69.85, 67.73, 60.32, 54.54, 53.00, 36.34, 32.75, 3 1 .26, 28.51 . HRMS (ESI) m/z calcd for C29H39F3N3O16 (M+H) 742.2277, found 742.2284.
[0208] GlcNAc6N3al-4GlcAp2AAMe F18-3. Yield: 89%; white foam. Ή NMR (600 MHz, D20) δ 7.97-7.98 (d, J= 7.8 Hz, 1 H), 7.82-7.84 (d, J= 8.4 Hz, 1 H), 7.63-7.66 (t, J = 7.2 Hz, 1 H), 7.32-7.34 (t, J= 7.2 Hz, 1 H), 5.40-5.41 (d, J= 3.6 Hz, 1 H), 4.34-4.35 (d, J = 7.8 Hz, 1 H), 3.91 (s, 3H), 3.83-3.90 (m, 3H), 3.70-3.73 (m, 3H), 3.57-3.63 (m, 4H), 3.47- 3.51 (t, J = 9.6 Hz, 1H), 3.22-3.33 (m, 3H), 2.74-2.77 (t, J = 6.6 Hz, 2H), 2.60-2.62 (t, J = 7.2 Hz, 2H), 2.05 (s, 3H), 1.75-1.79 (m, 2H). 13C NMR ( 150 MHz, D20) δ 175.06, 174.64, 174.53, 173.69, 169.28, 137.07, 134.10, 130.99, 125.54, 123.75, 121.38, 102.21 , 97.07, 76.88, 76.80, 76.04, 73.57, 70.85, 70.65, 70.44, 67.60, 53.76, 52.94, 50.62, 36.26, 32.58, 31 .23, 28.41 , 22.07. HRMS (ESI) m/z calcd for C2 H41 6O15 (M+H) 713.2625, found 713.2630.
[02091 Chemical derivatization of GlcNTFAcd-4GlcAp2AAMe (F18-2) to form disaccharide ΟΙοΝΗ2α1-401οΑβ2ΑΑ (F24-9). Disaccharide GlcNTFAal ^lGlcAp2AAMe (F18-2) (20 mg, 0.027 mmol) was dissolved in 8 mL of H20. The pH of the solution was adjusted to 10 by adding 1 N NaOH. After being vigorously stirred at r.t. for 1 .5 hr, the reaction mixture was neutralized with DOWEX HCR-W2 (H+) resin, filtered and concentrated. The residue was purified by flash column chromatography (EtOAc:MeOH:H20 = 3:2: 1 , by volume) to obtain a white solid GlcNH2al-4GlcAp2AA (F24-9) in 86% yield. Ή NMR (600 MHz, D20) δ 8.14-8.15 (d, J= 8.4 Hz, 1 H), 7.87-7.88 (d, J= 7.8 Hz, 1 H), 7.49- 7.51 (t,J=7.8Hz, 1H), 7.20-7.22 (t, J= 6.6 Hz, 1H), 5.65-5.66 (d, J= 3.0 Hz, 1H),4.26- 4.27 (d,J= 7.8 Hz, 1H), 3.72-3.85 (m, 8H), 3.66-3.67 (t,J= 8.4 Hz, 1H), 3.46-3.52 (m, 2H), 3.20-3.35 (m, 3H), 2.74-2.76 (t, J= 6.6 Hz, 2H), 2.61-2.63 (t, J= 6.6 Hz, 2H), 1.73- 1.74 (m, 2H).13C NMR (150 MHz, D20) δ 175.15, 174.77, 174.68, 173.01, 137.67, 131.91, 130.82, 125.32, 124.25, 120.95, 102.28, 97.77, 76.77, 76.51, 76.32, 73.27, 72.32, 69.60, 67.61, 61.37, 60.24, 54.95, 36.21, 33.50, 31.57, 28.43. HRMS (ESI) w/zcalcd for
C26H38N30,5(M+H) 632.2303, found 632.2321.
[0210] PmHS2-catalyzed synthesis of disaccharides F18-4— Π86. As shown in Figure 18B,GlA 2AAMe (F13-8) (5 to 10 mg, 1 eq.) and UDP-GlcNAc derivatives (1.2 eq.) were dissolved in water in a 15 mL centrifuge tube containing Tris-HCl buffer (100 mM, pH 7.5) and MgCl2 (10 mM). After the addition of appropriate amount PmHS2 (1-2 mg), water was added to bring the volume of the reaction mixture to 10 mL. The reaction was carried out by incubating the solution in an isotherm incubator for 12 to 36 h at 37 °C with gentle shaking. Product formation was monitored by TLC (EtOAc:MeOH:H20 = 4:2: 1 by volume) with p- anisaldehyde sugar staining. The reaction was stopped by adding the same volume of ice-cold ethanol and incubating at 4 °C for 30 min. The mixture was concentrated and passed through a BioGel P-2 gel filtration column to obtain the desired product. Silica gel column purification (EtOAc:MeOH:H20 = 5:2:1) was applied when necessary to achieve further purification.
[0211] GlcNGcal-4GlcAp2AAMe F18-4. Yield: 92%; white foam. Ή NMR (600 MHz, D20) δ 7.97-7.98 (d, J= 7.8 Hz, 1H), 7.81-7.82 (d, J= 8.4 Hz, 1H), 7.62-7.65 (t, J= 7.2 Hz, 1H), 7.31-7.34 (t, J =8.4 Hz, 1H), 5.39-5.40 (d,J=3.6 Hz, 1H), 4.32-4.33 (d, J= 7.8 Hz, 1H), 4.13 (s, 2H), 3.94-3.96 (dd,J= 10.8, 4.2 Hz, 1H), 3.90 (s, 3H), 3.77-3.86 (m, 4H), 3.71-3.75 (m,3H), 3.61-3.64 (m, 1H), 3.55-3.59 (m, 1 H), 3.48-3.51 (t,J=9.61Iz, 111), 3.20-3.30 (m,3H), 2.74-2.76 (t,J= 6.6 Hz, 2H), 2.59-2.61 (t, J= 7.2 Hz, 2H), 1.74-1.78 (m, 2H).13CNMR(150 MHz, D20) δ 175.18, 175.12, 174.62, 173.67, 169.26, 137.05, 134.08, 130.97, 125.52, 123.72, 121.35, 102.19, 97.05, 76.93, 76.66, 76.03,73.48, 72.06, 70.81, 69.72, 67.57, 61.03, 60.17, 53.45, 52.92, 36.22, 32.56, 31.20, 28.38. HRMS (ESI) m/z calcd for C29H42N30,7 (M+H) 704.2509, found 704.2516.
[0212] GlcNAzal-4GlcAp2AAMe F18-5. Yield: 91%; white foam. Ή NMR (600 MHz, D20) δ 7.97-7.99 (d, J= 7.8 Hz, 1H), 7.82-7.83 (d, J= 7.8 Hz, 1H), 7.64-7.66 (t, J= 7.2 Hz, 1H), 7.33-7.35 (t, J= 7.8 Hz, 1H), 5.41-5.42 (d, J= 3.6 Hz, 1H), 4.33-^.34 (d, J= 7.8 Hz, 1H), 4.08 (s, 2H), 3.95-3.97 (dd, J= 7.8, 3.6 Hz, 1H), 3.91 (s, 3H), 3.82-3.86 (m, 1 H), 3.73- 3.80 (m, 6H), 3.62-3.65 (m, 1 H), 3.56-3.60 (m, 1H), 3.48-3.51 (t, J= 9.0 Hz, 1H), 3.22-3.33 (m, 3H), 2.75-2.77 (t, J = 6.6 Hz, 2H), 2.60-2.62 (t, J = 6.6 Hz, 2H), 1.75-1.79 (m, 2H). 13C NMR (150 MHz, D20) δ 175.1 5, 174.64, 173.71 , 1 70.83, 169.28, 137.04, 134.09, 130.98, 125.56, 123.77, 121.44, 102.21 , 96.88, 76.90, 76.70, 75.99, 73.52, 72.06, 70.72, 69.77, 67.60, 60.20, 53.87, 52.93, 51 .93, 36.24, 32.57, 31.23, 28.40. HRMS (ESI) m/z calcd for
C29H4iN60i6 (M+H) 729.2574, found 729.2582.
[0213] GlcNAc6NGcal-4GlcAp2AAMe F18-6. Yield: 74%; white foam. Ή NMR (600 MHz, D20) δ 7.98-7.99 (d, J= 7.8 Hz, 1 H), 7.82-7.83 (d, J = 7.8 Hz, 1H), 7.64-7.66 (t, J = 7.8 Hz, 1H), 7.33-7.35 (t, J= 7.8 Hz, 1 H), 5.3 1 -5.32 (d, J= 3.6 Hz, 1 H), 4.33-4.35 (d, J = 8.4 Hz, 1 H), 4.12 (s, 2H), 3.91 (s, 3H), 3.81-3.90 (m, 4H), 3.68-3.74 (m, 3H), 3.56-3.63 (m, 3H), 3.50-3.53 (dd, J = 13.8, 2.4 Hz, 1 H), 3.22-3.32 (m, 3H), 2.76-2.78 (t, J = 6.6 Hz, 2H), 2.60-2.63 (t, ./ = 7.2 Hz, 2H), 2.04 (s, 3H), 1 .75-1 .79 (m, 2H). 13C NMR (150 MHz, D20) δ 175.48, 175.29, 174.65, 174.55, 173.71 , 169.28, 137.03, 134.09, 130.99, 125.57, 123.81 , 121.48, 102.16, 97.49, 77.08, 76.86, 76.73, 73.57, 71 .51 , 70.72, 70.51 , 67.57, 61 .18, 53.77, 52.94, 39.62, 36.24, 32.57, 31.24, 28.41 , 22.08. HRMS (ESI) m/z calcd for C31H45N40, 7 (M+H) 745.2780, found 745.2787.
Example 7: Preparation of Trisaccharide Derivatives
[0214] Small scale one-pot three-enzyme synthesis of trisaccharides F20-1-F20-6 by HPLC and MALDI-TOF MS analysis. As shown in Figure 19, Typical enzymatic assays were performed in a total volume of 20 μί, in Tris-HCl buffer (100 mM, pH 7.5) containing MgCh (10 mM), UTP (7.5 mM), disaccharides (5 mM), Glc- l -P (6 mM), NAD+ (12 mM), GalU (2.5 μg), PmUgd (8 μg) and PmHS2 (1 1.5 μg). Reactions were allowed to proceed for 12 hr at 37°C and quenched by adding ice-cold ethanol (20 μί) and water (1 .96 mL) to make 100-fold dilution. The samples were then kept on ice until aliquots of 5
Figure imgf000074_0001
were injected and analyzed by a Shimadzu LC-2010A system equipped with a membrane on-line degasser, a temperature control unit (maintained at 30°C throughout the experiment), and a fluorescence detector. A reverse phase Premier C I 8 column (250 * 4.6 mm I.D., 5 ΐΉ particle size,
Shimadzu) protected with a C I 8 guard column cartridge was used. The mobile phase was 10% acetonitrile. The fluorescent compounds 2AA derivatives were detected by excitation at 305 nm and emission at 415 nm. The MS data of the products were acquired using MALDI Mass. See Table 8.
Table 8. HPLC and MALDI-TOF MS analysis data for the synthesis of trisaccharides F20-1-F20-6
Starting Retention Product Retention Cal. Mass Measured Mass *
Material Time (min) (Yield) Time (min) M+Na" M+Na+-H+ M+2Na+-2H+ M+3Na+-3H'
Compound 8.8 Compound 4.7 885.2627 885.4562 907.4189 929.2829 F18-1 F20-1 (100%)
Compound 13.5 Compound 4.8 940.2423 939.3854 961.3454 983.3108 F18-2 F20-2 (72%)
Compound 9.3 Compound 3.9 91 1.277 910.4507 932.4137 954.3776 F18-3 F20-3 (100%)
Compound 8.1 Compound 4.9 902.2655 901.4351 923.3950 945.3587 FI8-4 F20-4 (75%)
Compound 10.5 Compound 4.8 927.2719 926.4009 948.3610 970.3205 F18-5 F20-5 (95%)
Compound 9.2 Compound 5.1 943.2920 942.4578 964.41 17 — - F18-6 F20f ( 14%)
* Measured values represent M+Na+, M+2Na+-H+, M+3Na+-2H+.
[0215] Preparative-scale preparation of trisaccharide GlcApi-4GlcNTFActl- 4GlcAp2AAMe F20-2 in a one-pot three-enzyme system as shown in Figure 19.
Disaccharide GlcNTFAal -4GIcAp2AAMe F18-2 (30 mg, 1 eq.), Glc- l -P ( 1 .2 eq), UTP ( 1 .5 eq) and NAD+ (2.4 eq.) were dissolved in water in a 15 mL centrifuge tube containing Tris- HC1 buffer ( 100 mM, pH 7.0) and MgCl2 (10 mM). After the addition of appropriate amount of GalU ( 1 mg), PmUgd (3 mg), PmHS2 (4.5 mg), water was added to bring the volume of the reaction mixture to 8 mL. The reaction was carried out by incubating the solution in an isotherm incubator at 37 °C for 12 hr with gentle shaking. Product formation was monitored by TLC (EtOAciMeOHiH O = 3:2: 1 by volume) with 7-anisaldehyde sugar staining. The reaction was stopped by adding the same volume of ice-cold ethanol and incubating at 4 °C for 30 min. The mixture was concentrated and passed through a BioGel P-2 gel filtration column to obtain the desired product. The trisaccharide was further purified by silica gel column chromatography (EtOAcMeOHTLO = 4:2: 1 ) to obtain white solid trisaccharide GlcAp l -4GlcNTFAal-4GlcAp2AAMe F20-2 in 87% yield. Ή NMR (600 MHz, D20) δ 7.96-7.97 (d, J= 7.8 Hz, 1 H), 7.80-7.82 (d, J = 8.4 Hz, 1 H), 7.61-7.64 (t, J = 7.8 Hz, 1 H), 7.31-7.33 (t, .7 = 7.2 Hz, 1 H), 5.44-5.45 (d, .7 = 3.6 Hz, 1 H), 4.94-4.51 (d, J = 7.8 Hz, 1 H), 4.30-4.31 (d, J= 7.8 Hz, 1 H), 3.99-4.01 (dd, J = 1 1 .4, 3.6 Hz, 1 H), 3.94-3.97 (m, 1 H), 3.90 (s, 3H), 3.80-3.85 (m, 4H), 3.70-3.75 (m, 4H), 3.57-3.60 (m, 1H), 3.53-3.56 (m, 1H), 3.48- 3.52 (m, 2H), 3.35-3.37 (t, J= 7.8 Hz, 1H), 3.20-3.31 (m, 3H), 2.73-2.76 (t, J= 7.2 Hz, 2H), 2.58-2.61 (t,J=7.2Hz, 2H), 1.73-1.77 (m, 2H).13CNMR(150 MHz, D20) 5174.88 (2C), 174.40, 173.45, 169.04, 159.11 (q, J= 37.7 Hz), 136.81, 133.86, 130.75, 125.31, 123.50, 121.14, 116.69 (q, J =284.6 Hz), 102.27, 101.95,96.01,78.16,76.60,76.50,76.02,75.75, 75.05, 73.31, 72.82, 71.68, 70.58, 68.59, 67.36, 59.22, 53.85, 52.70, 36.00, 32.34, 30.99, 28.16. HRMS (ESI) m/z calcd for C35H47F3N3O22 (M+H) 918.2603, found 918.2613.
[0216] Preparative-scale preparation of trisaccharide 01οΑ 1-401οΝΗ2 1- 4GlcA 2AA (F24-11) in a one-pot three-enzyme system as shown in Figure 19.
Disaccharide GlcNH2cd-4GlcAp2AA (F24-9) (15 mg, 1 eq.), Glc-l-P (1.2 eq), UTP (1.5 eq), and NAD+ (2.4 eq.) were dissolved in water in a 15 mL centrifuge tube containing Tris-HCl buffer (100 mM, pi I 7.0) and MgC (10 mM). After the addition of appropriate amount of GalU (0.5 mg), PmUgd (1.5 mg), PmHS2 (2.5 mg), water was added to bring the volume of the reaction mixture to 4 mL. The reaction was carried out by incubating the solution in an isotherm incubator at 37 °C for 12 hr with gentle shaking. Product formation was monitored by TLC (EtOAc:MeOH:H20 = 3:2: 1 by volume) with 7-anisaldehyde sugar staining. The reaction was stopped by adding the same volume of ice-cold ethanol and incubating at 4 °C for 30 min. The mixture was concentrated and passed through a BioGel P-2 gel filtration column to obtain the desired product. The trisaccharide was further purified by silica gel column chromatography (EtOAc:MeOH:H20 = 3:2:1) to obtain white solid GlcApi-
4GlcNH2al^lGIcA 2AA (F24-11) in 84% yield. Ή NMR (600 MHz, D20) δ 7.96-7.97 (d, J=6. Hz, 1H), 7.81-7.82 (d, .7=6.6 Hz, 1H), 7.62-7.65 (t,J=6.6Hz, 1H), 7.31-7.34 (d,J = 6.6Hz, 1H), 5.63-5.64 (d, J=3.6Hz, 1H), 4.49-4.50 (d, J= 6.6 Hz, 1H), 4.35-4.37 (d, J = 7.8 Hz, 1H), 3.9-3.98 (t, J= 9.0 Hz, 1H), 3.91 (s, 3H), 3,82-3.88 (m, 4H), 3.67-3.78 (m, 6H), 3.49-3.59 (m, 3H), 3.28-3.38 (m, 3H), 3.21-3.25 (m, 1H), 2.74-2.76 (t, J= 7.2 Hz,
2H), 2.60-2.62 (t, J= 6.6 Hz, 2H), 1.75-1.77 (m, 2H).13C NMR (150 MHz, D20) 5175.82, 175.08, 174.65, 173.66, 169.27, 137.14, 134.14, 131.01, 125.51, 123.66, 121.23, 102.52, 102.23, 97.66, 78.32, 76.76, 76.61, 76.51, 75.90, 75.33, 73.20, 73.09, 71.93, 70.99, 67.62, 59.48, 58.73, 54.66, 52.95, 36.24, 32.60, 31.23, 28.41. HRMS (ESI) m/z calcd for
C32II46N3O21 (M+H) 807.2546, found 807.2557. Example 8: Preparation of Tetrasaccharides
[0217] One-pot four-enzyme synthesis of tetrasaccharide
Figure imgf000077_0001
4GlcNTFAal-4GlcAp2AAMe F21-1. Trisaccharide GlcAp i^GlcNTFAal- 4GIcAp2AAMe Γ20-2 (30 mg, 1 eq.), GlcNAc6N3 (1.5 eq.), ATP ( 1.8 eq.), and UTP ( 1 .8 eq.) were dissolved in water in a 15 mL centrifuge tube containing MES buffer (100 mM, pH 6.5) and MgCl2 ( 10 mM). After the addition of appropriate amount of Nan _ATCC55813 (2.5 mg), PmGlmU (3 mg), PmPpA (1.5 mg), and PmHS2 (4 mg), water was added to bring the volume of the reaction mixture to 6.5 mL. The reaction was carried out by incubating the solution in an isotherm incubator for 18 h at 37 °C with gentle shaking. Product formation was monitored by TLC (EtOAc:MeOH:H20 = 4:2: 1 by volume) with -anisaldehyde sugar staining. The reaction was stopped by adding the same volume of ice-cold ethanol and incubating at 4 °C for 30 min. The mixture was concentrated and passed through a BioGel P- 2 gel filtration column to obtain the desired product. The tetrasaccharide was further purified by silica gel column chromatography (EtOAc:MeOH:H20 = 5 :2: 1 ) to obtain white solid tetrasaccharide GlcNAc6N3al-4GlcAp i -4GlcNTFAal^lGlcAP2AAMe F21-1 in 93% yield. Ή NMR (600 MHz, D20) δ 7.94-7.96 (d, J= 7.8 Hz, 1 H), 7.81-7.83 (d, J = 8.4 Hz, 1H), 7.60-7.63 (t, J = 7.8 Hz, 1 H), 7.29-7.3 1 (t, .7 = 7.2 Hz, 1 H), 5.43-5.44 (d, .7 = 3.6 Hz, 1 H), 5.40-5.41 (d, J = 4.2 Hz, 1 H), 4.47-4.49 (d, J = 7.8 Hz, 1 H), 4.29-4.31 (d, J = 8.4 Hz, 1 H), 3.93-4.00 (m, 211), 3.88-3.90 (m, 4H), 3.78-3.86 (m, 6H), 3.66-3.75 (m, 6H), 3.61- 3.62 (d, J = 2.4 Hz, 2H), 3.57-3.60 (m, 1H), 3.53-3.56 (m, 1H), 3.45-3.48 (t, J= 9.0 Hz, 1 H), 3.34-3.36 (t, J= 7.8 Hz, 1 H), 3.19-3.30 (m, 3H), 2.72-2.74 (t, J= 6.6 Hz, 2H), 2.57- 2.60 (t, J = 6.6 Hz, 2H), 2.03 (s, 3H), 1.72-1.76 (m, 2H). 13C NMR (150 MHz, D20) δ 174.85, 174.78, 174.36, 174.29, 173.36, 169.00, 159.08 (q, 7 = 284.4 Hz), 136.91 , 133.88, 130.75, 125.20, 123.30, 120.83, 1 16.67 (q, = 37.7 Hz), 102.29, 101 .93, 96.83, 96.00, 78.08, 76.57, 76.46, 76.30, 76.28, 76.01 , 75.77, 73.36, 73.29, 70.65, 70.55, 70.35, 70.22, 68.47,
67.35, 59.17, 53.76, 53.47, 52.67, 50.38, 35.98, 32.35, 30.95, 28.15, 21 .79. HRMS (ESI) m/z calcd for C43H59F3N7O26 (M+H) 1 146.3462, found 1 146.3478.
[0218] Synthesis of tetrasaccharide GlcNAc6N3al-4GlcApl-4GlcNH2al-4GlcAp2AA F22-1. Compound GlcNAc6N3al-4GlcAp i -4GlcNTFAal-4GlcAp2AAMe F21-1 (30 mg, 0.029 mmol) was dissolved in 8 mL of H20. The pH of the solution was adjusted to 10 by adding 1 N NaOH. After being vigorously stirred at r.t. for 1.5 hr, the reaction mixture was neutralized with DOWEX HCR-W2 (H+) resin, filtered and concentrated. The residue was purified by flash column chromatography (EtOAc:MeOH:H20 = 4:2:1, by volume) to obtain a white solid GlcNAc6N3al^GlcApi^tGlcNH2al-4GlcAp2AA F22-1 in 81% yield. Ή NMR (600 MHz, D20) δ 8.12-8.13 (d, J= 7.8 Hz, 1H), 7.85-7.87 (d, J= 7.8 Hz, 1H), 7.48- 7.50 (t,J = 7.2 Hz, 1H), 7.20-7.22 (t, J= 7.8 Hz, 1H), 5.59-5.60 (d, J= 3.6 Hz, 1H), 5.41- 5.40 (d, J= 4.2 Hz, 1H), 4.45^.47 (d, J= 7.8 Hz, 1H), 4.27-4.28(d, ./= 7.8 Hz, 1H), 3.88- 3.94 (m, 2H), 3.79-3.88 (m, 6H), 3.66-3.78 (m, 8H), 3.62-3.64 (m, 2H), 3.46-3.52 (m, 2H), 3.34-3.37 (t,J = 7.8 Hz, 1H), 3.19-3.33 (m, 3H), 2.73-2.75 (t, J= 6.6 Hz, 2H), 2.60-2.62 (t, J= 6.6 Hz, 2H), 2.03 (s, 3H), 1.71-1.75 (m, 2H).13C NMR (150 MHz, D20) δ 174.89, 174.87, 174.67, 174.47, 174.29, 172.74, 137.25, 131.59, 130.48, 125.10, 124.00, 120.70, 102.27, 101.92, 96.84, 95.32, 77.32, 76.22, 76.15, 76.10, 76.07, 75.79, 73.38, 73.03, 70.90, 70.65, 70.34, 70.20, 68.24, 68.22, 67.30, 58.92, 53.82, 53.45, 50.38, 35.90, 33.19, 31.28,
28.10, 21.77. HRMS (ESI) m/zcalcd for C4oH58N7025 (M+H) 1036.3482, found 1036.3497.
[0219] Synthesis of tetrasaccharide GlcNAc6N3al-4GlcApl-4GlcNSal-4GlcAp2AA F22-2. Compound GlcNAc6N3al^GlcApi-4GlcNH2al-4GlcA 2AA F22-1 (20 mg, 0.018 mmol) was dissolved in 10 mL of H20. The pH of the solution was adjusted to 9.5 by adding 2 N NaOH (aq). Sulfur trioxide-pyridine complex (58 mg, 0.36 mmol) was added in three equal portions during 35 minutes intervals at room temperature, and the pH was maintained at 9.5 throughout the whole process using 2 N NaOH (aq). After being stirred at r.t. for 24 hr, the reaction mixture was neutralized with DOWEX HCR-W2 (H+) resin, filtered, concentrated. The process has been repeated for three times and purified using silica gel column (EtOAc:MeOH:H20 = 5:2:1, by volume) to obtain a light yellow solid
GlcNAc6N3al-4GlcA i-4GlcNSal^lGlcAp2AA F22-2 in 70% yield. Ή NMR (800 MHz, D20) 58.13-8.14 (d, J= 8.0 Hz, 1H), 7.87-7.88 (d, J= 7.2 Hz, 1H), 7.52-7.50 (t, J= 8.0 Hz, 1H), 7.22-7.24 (t, J= 7.2 Hz, 1H), 5.60-5.61 (d,J=4.0 Hz, 1H), 5.42-5.43 (d,J=3.2Hz, 1H), 4.50-4.51 (d,J=8.0Hz, 1H), 4.34-4.35 (d, J = 8.0 Hz, 1H), 3.86-3.92 (m, 3H), 3.80- 3.83 (m, 4H), 3.73-3.79 (m, 4H), 3.68-3.72 (m, 4H), 3.64-3.65 (d, J= 3.2 Hz, 2H), 3.56- 3.53 (m, 111), 3.48-3.50 (t, J= 9.6 Hz, HI), 3.36-3.39 (t, J= 8.0 Hz, 1H), 3.19-3.28 (m, 4H), 2.75-2.77 (t,J= 7.2 Hz, 2H), 2.62-2.63 (t, J= 7.2 Hz, 2H), 2.04 (s, 3H), 1.75-1.77 (m, 1H). ,3CNMR(200 MHz, D20) δ 174.89, 174.51, 174.32, 174.30, 172.82, 172.78, 137.13, 131.55, 130.41, 125.21, 124.02, 120.77, 102.11, 101.88,96.88,96.79,77.70,76.48,76.29,76.15,
76.11, 75.77, 73.28, 72.51, 70.58, 70.30, 70.21, 70.15, 69.38, 67.25, 59.16, 57.45, 53.43, 50.30, 35.89, 33.09, 31.19, 28.09, 21.73. HRMS (ESI) m/z calcd for C^H^O^S (M+H) 1116.3051, found 1116.3076. [0220] Synthesis of tetrasaccharide GlcNAc6NH2al-4GlcAp l-4GlcNSal-4GlcAp2AA F22-3. Compound GlcNAc6N3al-4GlcAp i-4GlcNSal-4GlcAp2AA F22-2 (17 mg, 0.015 mmol) was dissolved in 10 mL ¾0/ MeOH (1 : 1 ) and 20 mg of Pd/C was added. The mixture was shaken under Ha gas (4 Bar) for 1 hr, filtered, and concentrated to produce F22-3 as a white solid in quantitative yield. Ή NMR (800 MHz, D20) δ 8.04-8.05 (d, J = 8.0 Hz, 1 H), 8.02-8.03 (d, J= 8.0 Hz, 1H), 7.64-7.67 (t, J= 8.0 Hz, 1 H), 7.32-7.34 (t, J= 7.2 Hz, 1 H), 5.56-5.57 (d, J= 4.0 Hz, 1H), 5.34-5.35 (d, J = 4.0 Hz, 1H), 4.56-4.57 (d, J = 8.0 Hz, 1 H), 4.34-4.35 (d, J= 8.0 Hz, 1H), 3.92-3.95 (m, 3H), 3.88-3.90 (dd, J= 12.0, Hz, 2.4H), 3.76- 4.84 (m, 4H), 3.68-3.75 (m, 4H), 3.55-3.58 (m, 1 H), 3.42-3.45 (dd, J= 13.6, 3.2 Hz, 1H), 3.35-3.38 (m, 2H), 3.30-3.32 (m, 2H), 3.19-3.27 (m, 4H), 3.12-3.15 (dd, J = 12.8, 8.8 Hz, 1H), 2.77-2.79 (t, = 6.4 Hz, 1 H), 2.61-2.62 (t, .7= 6.4 Hz, 1 H), 2.05 (s, 3H), 1 .75-1.78 (m, 2H). 13C NMR (200 MHz, D20) δ 174.77, 174.47, 174.42, 173.26, 172.64, 170.76, 137.73, 133.92, 131.18, 127.27, 124.85, 122.56, 102.1 8, 102.87, 97.64, 97.48, 77.66, 77.32, 76.56, 75.76, 75.73, 75.70, 74.58, 73.42, 72.45, 71.70, 70.65, 70.08, 69.22, 68.16, 67.53, 59.22, 57.60, 53.33, 46.57, 40.22, 35.88, 31 .12, 28.13, 21 .78. HRMS (ESI) m/z calcd for
C40H60N5O28S (M+H) 1090.3 146, found 1 190.3 171 .
[0221] Synthesis of tetrasaccharide GlcNAc6NSal-4GlcApi-4GIcNSal^tGlcAp2AA F22-4. Compound GlcNAc6NH2al-4GlcAp l-4GlcNSal-4GlcAp2AA F22-3 (14 mg, 0.013 mmol) was dissolved in 5 mL of H2O. The pH of the solution was adjusted to 9.5 by adding 2 N NaOH. Sulfur trioxide-pyridine complex (30 mg, 0.18 mmol) was added in three equal portions during 1 hr intervals at rt. The pH was maintained at 9.5 throughout the whole process by adding 2 N NaOH. After being stirred at r.t. for overnight, the reaction mixture was neutralized with DOWEX HCR-W2 (H+) resin, filtered, concentrated. The process has been repeated for three times and purified by preparative HPLC using C I 8 column to give white solid GlcNAc6NSal^GlcApi-4GlcNSal^GlcAp2AA F22-4 in 65% yield. Ή NMR (800 MHz, D20) δ 8.06-8.07 (d, J = 8.0 Hz, 1 H), 8.03-8.04 (d, J = 8.0 Hz, 1 H), 7.66-7.68 (t, J= 7.2 Hz, 1 H), 7.33-7.34 (d, J = 7.2 Hz, 1 H), 5.54-5.55 (d, J = 3.2 Hz, 1 H), 5.35-5.36 (d, J = 3.2 Hz, 1 H), 4.61—4.62 (d, J = 8.0 Hz, 1 H), 4.34-4.35 (d, J = 8.0 Hz, 1 H), 4.1 1-4.12 (d, J = 9.6 Hz, 1 H), 3.81-3.93 (m, 4H), 3.73-3.81 (m, 5H), 3.64-3.71 (m, 5H), 3.55-3.58 (m, 1 H), 3.50-3.53 (t, J = 9.6 Hz, 1 H), 3.39-3.41 (t, J= 8.0 Hz, 1 H), 3.28-3.33 (in, 3H), 3.21-3.27 (m, 3H), 2.77-2.79 (t, J = 7.2 Hz, 2H), 2.61-2.62 (t, J = 6.4 Hz, 2H), 2.05 (s, 3H), 1 .75-1.78 (m, 2H). 13C NMR (200 MHz, D20) 5 174.48, 174.34, 173.25, 1 71.88, 1 71 .81, 170.41 , 137.88, 134.18, 131.28, 127.18, 124.87, 122.59, 102.27, 102.04, 97.78, 97.46, 77.88, 76.83, 76.46, 75.65, 75.49, 73.91 , 73.88, 73.14, 72.41, 70.83, 70.69, 70.46, 70.13, 69.14, 67.62, 59.21, 59.15, 57.67, 53.42, 43.29, 35.85, 32.71, 31.09, 28.13, 21.81. HRMS (ESI) m/z calcd for C4oH6o 503iS2 (M+H) 1 170.2714, found 1 170.2730.
[0222] Alternative route for synthesizing tetrasaccharide F22-4 from F21-1.
Compound GlcNAc6N3al-4GlcApl^GlcNH2al-4GlcAp2AAMe F21-1 (10 mg, 0.009 mmol) was dissolved in 5 mL of H2G7MeOH (1 : 1 ) and 5 mg of Pd/C was added. The mixture was shaken under H2 gas (4 Bar) for 1 hr, filtered and concentrated to provide
GlcNAc6NH2al-4GlcApi-4GlcNH2al-4GlcAp2AA. The residue was dissolved in 5 mL of H20. The pH of the solution was adjusted to 9.5 by adding 2 N NaOH. Sulfur trioxide- pyridine complex (15 mg, 0.09 mmol) was added in three equal portions during 1 h intervals at rt. The pH was maintained at 9.5 throughout the whole process by adding 2 N NaOH. After being stirred at r.t. for overnight, the reaction mixture was neutralized with DOWEX HCR- W2 ( T) resin, filtered, and concentrated to give a mixture of GlcNAc6NSa l-4GlcAp i- 4GlcNH2al -4GlcA 2AA, GlcNAc6NH2al-4GlcA i-4GlcNSal-4GlcAp2AA (F22-3), and GlcNAc6NSal- GlcApl-4GlcNSal-4GlcAp2AA (F22-4) which can be separated by HPLC using a C I 8 column.
.[0223] Synthesis of GlcNSal-4GlcApi-4GlcNSal^GlcAp2 AA. The synthesis was conducted as outlined in Figure 14.
[0224] Trisaccharide GlcAp i-4GlcNTFAal -4GlcAp2AAMe (Compound F14-1 or F20-2, Figure 14) ( 1 1 mg, 1 eq.), GlcNTFA ( 1.5 eq.), ATP (1.8 eq.), and UTP ( 1 .8 eq.) were dissolved in water in a 15 mL centrifuge tube containing tris buffer (100 raM, pH 7.0) and MgCI2 ( 10 inM). After the addition of NanK_ATCC55813 (2.5 mg), PmGlmU (3 mg), PmPpA ( 1 .5 mg), and PmHS2 (2 mg), water was added to bring the volume of the reaction mixture to 10 mL. The reaction was carried out by incubating the solution in an isotherm incubator at 37 °C for 20 hr with gentle shaking. Product formation was monitored by TLC (EtOAc:MeOH:H20 = 4:2: 1 by volume) with /7-anisaldehyde sugar staining. The reaction was stopped by adding the same volume of ice-cold ethanol and incubating at 4 °C for 30 min. The mixture was concentrated and passed through a BioGel P-2 gel filtration column to obtain the desired product. The tetrasaccharide was further purified by silica gel column chromatography (EtOAc:MeOH:H20 = 5:2: 1) to obtain a white solid of GlcNTFAal - 4GlcApi^GlcNTFAal-4GlcAp2AAMe (Compound F14-2, Figure 14). 1 1.8 mg, 84% yield. Ή NMR (600 MHz, D20) δ 8.04-8.03 (d, J = 8.4 Hz, 1 H), 7.87-7.85 (d, J = 7.8 Hz, 1H), 7.72-7.69 (t, J= 7.2 Hz, 1 H), 7.41-7.38 (t, J= 7.8 Hz, 1H), 5.54-5.53 (d, J= 4.2 Hz, 1H), 5.50-5.49 (d, J= 3.6 Hz, 1H), 4.56-4.55 (d, J = 7.8 Hz, 1H), 4.38-4.36 (d, J= 7.8 Hz, 1 H), 4.00-3.26 (m, 24H), 2.83-2.81 (t, J= 6.9 Hz, 2H), 2.68-2.66 (t, J = 6.9 Hz, 2H), 1.82- 1.80 (m, 2H).
[0225] GlcNTFAal-4GlcAp l-4GlcNTFAal-4GlcA 2AAMe (Compound F14-2, Figure 14) (1 1 mg) was dissolved in 7.5 mL solution of ( MeOHiHaOitriethylamine = 1 : 1 :0.5). The reaction was stirred overnight and monitored until completion as indicated by TLC. The solution was then rotovaped and re-dissolved in water and lyophilized to afford free amines as a white foam. The free amine was then dissolved in 7 mL of water and the pH of the solution was adjusted to 9.5 by adding 2 N NaOH (aq). Sulfur trioxide-pyridine complex (60 mg, 0.37 mmol) was added in three equal portions during 35 minutes intervals at room temperature, and the pH was maintained at 9.5 throughout the whole process using 2 N NaOH (aq). After being stirred at r.t. for 24 hr, the reaction mixture was neutralized with DOWEX HCR-W2 (H+) resin, filtered, concentrated. The crude product was purified using silica gel column (EtOAc:MeOH:H20 = 5 :3 :2, v/v) to obtain a light yellow solid GlcNSal - 4GlcA i-4GlcNSal^lGlcAp2AA (tetrasaccharide F14-3, Figure 14). MS (ESI) m/z calcd for C38Hs6N403i S2 (M-H) 1 127.23, found 1 127.23. C38H56N4O31 S2 (M/2 -H) 563.1 1 , found 563. 1 .
Results and discussion
[0226] As shown in Figure 16, four enzymes were used in one-pot to synthesize
GlcNAcal-4GlcA disaccharide derivatives. The first enzyme was an /V-acetylhexosamine 1 - kinase cloned from Bifidobacterium infantis strain ATCC 15697 (Nah _ATCC l 5697). The second enzyme was an /V-acetylglucosamine- 1 -phosphate uridylyltransferase that we cloned from Pasteurella multocida strain P- 1059 (ATCC 15742) (PmGlmU). The third enzyme was an inorganic pyrophosphatase that we cloned from Pasteurella multocida strain P- 1059
(PmPpA) for hydrolyzing the pyrophosphate by-product formed to drive the reaction towards the formation of UDP-Glc Ac and derivatives.
[0227] The fourth enzyme is a heparosan synthase 2 cloned from Pasteurella multocida strain P- 1059 (PmHS2) for the formation of al-4 linkage. PmHS2 is a bifunctional enzyme which demonstrates al ^GlcNAc and p l^lGlcA transferase activity. It not only uses UDP- Glc Ac as donor, transferring GlcNAc to GlcA to form al -4 linkage, but also transfers GIcA from donor UDP-GlcA to acceptor GlcNAc to form β 1— 4 linkage. Although PmHS2 has been shown to be able to synthesize heparosan polysaccharides, its donor and acceptor specificity has not been investigated in detail.
[0228] Prior to applying the one-pot three-enzyme system shown in Figure 16 to the preparative-scale synthesis of the disaccharides, UDP-GlcNAc and derivatives F17-1- F17- 12 were tested as donor substrates for PmHS2 in small-scale reaction containing Tris-HCl buffer ( 100 mM, pH 7.5), GlcA 2AAMe (10 mM), UDP-GlcNAc or a derivative (15 mM), MgCli ( 10 mM), and PmHS2 (0.5 mg/mL). See Figure 17. The reactions were earned out at 37 °C for 12 hr and analyzed by thin layer chromatography (TLC). UDP-GlcNAc F17-1 and some of its C2- (UDP-GlcNTFA F17-2, UDP-GlcNGc F17-3, and UDP-GlcNAcN3 F17-4), and C6- (UDP-GlcNAc6N3 F17-8 and UDP-GlcNAc6NGc F17-9) derivatives are tolerable donor substrates for PmHS2. UDP-GlcNH2 F17-5, UDP-GlcN3 F17-6, UDP-GlcNS F17-7, UDP-GlcNAc6NH2 F17-10, UDP-GlcNAc6NAcN3 F17-11 and UDP-GlcNAc6S F17-12 did not serve as donor substrates for PmHS2.
[0229] As shown in Figure 18, preparative-scale transfer of monosaccharide GlcNAc, GlcNTFA and GlcNAc6N3 to fluorescent labeled glucuronide GlcAp2AAMe as an acceptor for PmHS2 successfully produced disaccharides GlcNAcal— 4GlcAp2AAMe F18-1,
GlcNTFAal^GlcAp2AAMe F18-2, and GlcNAc6N3al-4GlcA 2AAMe F18-3 in 95%, 84%, and 89% yields, respectively. It was found that the N-TFA group removal was significant at pH 7.5. Nevertheless, the removal of N-TFA was not significant when the pH of the reaction mixture was changed from 7.5 to 6.5 and the reaction time was shortened. Three additional disaccharides (supporting information) were also synthesized by PmHS2-catalyzed reaction using UDP-GlcNAc derivatives UDP-GlcNGc F17-3, UDP-GlcNAcN3 F17-4, and UDP-GlcNAc6NGc F17-9, since the three sugar nucleotides were prepare form LJDP- GlcNTFA F17-2 by the removal of TFA and acylation of amine with proper acyl chloride. GlcNGcal- GlcAp2AAMe F18-4, GlcNAcN3al-4GlcA 2AAMe F18-5, and
GlcNAc6NGccd-4GlcAp2AAMe F18-6 are prepared in 92%, 91 %, and 74% yields, respectively. See Figure 18.
[0230] Acceptor specificit of the p i^4GlcA transferase activity of PmHS2 was also explored in one-pot three-enzyme system, as shown in Figure 19. The first enzyme was a glucose- 1 -phosphate uridylyltransferase (GalU) which catalyzes the reversible conversion of Glc- l -P in the presence of UTP to produce UDP-Glc and inorganic pyrophosphate. The second enzyme was a UDP-glucose dehydrogenase (Ugd) for oxidation of 6-OH in glucose residue of UDP-Glc to form the UDP-glucuronic acid (UDP-GlcA) in the presence of its coenzyme NAD+. The third enzyme is PmHS2 transferring GlcA from UDP-GlcA for the formation of βΐ— 4 linkage. As shown in Figure 20, trisaccharides GlcApi- GlcNAcal- 4GlcAp2AAMe F20-1, GlcApl-4GlcNAc6N3al^GlcAp2AAMe F20-3, GlcApi - 4GlcNAcN3al^lGlcAp2AAMe F20-5 were synthesis by small-scale reaction and analyzed by HPLC method in 100%, 100% and 95% yields, respectively. The relative low yield (72%) for the formation
Figure imgf000083_0001
F20-2 was due to the formation of byproduct
Figure imgf000083_0002
in which the TFA group was removed. Disaccharide F18-4 with N-glycolyl group in C2 position of glucosamine residue acts as a good acceptor for PmHS2, leading to the formation of GlcAp i^GlcNGcal -4GlcAp2AAMe F20-4 in 75% yield, but the disaccharide F18-6 with N-glycolyl group in C6 position of GlcNAc was converted to trisaccharide GlcA i - GlcNAc6NGcal -4GlcAp2AAMe F20-6 only in 14% yield. Taken together, these results indicate that the donor and acceptor substrate activity of PmHS2 can tolerate a limited number of modifications on C-2 and C-6 position of glucosamine residue.
[0231] Preparative-scale synthesis of trisaccharide F20-2 was also achieved. The removal of TFA group was significantly reduced when the pH of the reaction mixture was change for 7.5 to 7.0, and the yield increased to 87% from 72%. Trisaccharide F20-2 was used as the starting material for the synthesis of the tetrasaccharide F21-1 (Figure 21). In the one-pot four-enzyme system, monosaccharide GlcNAc6N3 was converted to GlcNAc6N3- l -P by NanK, followed by the formation of UDP-GlcNAc6N3 by PmGlmU, and transferred to trisaccharide F20-2 to obtain GlcNAc6N3al ^GlcApi-4GlcNTFAal- GlcAp2AAMe F21- 1 in 93% yield. [0232] The N-TFA group as well as the N3 group can be easily converted to a free amine, allowing sequential sulfation to generate a diverse array of HS tetrasaccharides. As shown in Figure 22, the yV-TFA group at C2 of internal GlcNTFA residue of tetrasaccharide F21-1 was removed under mild basic conditions to produce GlcNAc6N3al-4GlcAp l-4GlcNH2al^lGlcA 2AA F22-1 in 81 % yield. As the removal of TFA group was accompanied by demethylation in methyl carboxylic ester, tetrasaccharide F22-1 contain a free carboxyl acid in 2AA motif instead of carboxylic ester in tetrasaccharide F21-1. Conversion of F22-1 to
Figure imgf000083_0003
F22-2 (70%)) needed a larger excess of sulfating reagent (60 equiv.) and prolonged reaction time (3 d). Catalytic hydrogenation of the azido group at the C6 of non-reduced end GlcNAc6N3 generated GlcNAc6NH2al-4GlcA i-4GlcNSal^GlcA 2AA F22-3 and followed by the sulfation to produce GlcNAc6NSal-4GlcApi^tGlcNSal-4GlcAp2AA F22-4.
Example 9: Preparation of GleA-TEG-PABA-biotin.
[0233] The synthesis was conducted as outlined in Figure 15.
[0234] 2-(2-(2-Tosylethoxy)ethoxy)ethanol. Compound F15-1 (51 grams) was dissolved in 10 mL of water containing 2.2 grams of NaOH. The reaction mixture was cooled in an ice bath. To the reaction mixture tosyl chloride (6.5 g, 34.1 mmol) in 80 mL of THF was added drop-wisely in 1 hr period. The reaction was left in ice bath off for 2 hr. The reaction was worked up with 150 mL of DCM and 150 mL of cold water. The organic layer was collected and saved. The aqueous layer was extracted twice with DCM (150 mL) and the organic layers were collected and washed twice with water (200 mL). The organic portions were combined, dried with magnesium sulfate, and rotovoped to provide crude 2-(2-(2- tosylethoxy)ethoxy)ethanol (9.684 grams, 93% yield).
[0235] Compound F15-2. 2-(2-(2-Tosylethoxy)ethoxy)ethanol (9.684 g, 31.8 mmol) was dissolved in 25 mL of DMF. Sodium azide (10.34 g, 159.1 mmol) was added to the reaction solution. The reaction was left for 3 h at 80 °C. The reaction mixture was worked up with EtOAc/water. The organic layer was collected and dried over magnesium sulfate and purified by silica gel column (Hexane:EtOAc= 2: 1 -0: 1 ) to produce compound F15-2 (4.96 g, 89 % yield).
[0236] Azitlotriethylene glycol-Boc. Compound F15-2 (2.334, 13.3 mmol) was dissolved in 40 mL of DMF and cooled in ice bath. Sodium hydride 50% immersed in mineral oil (959 mg, 19.9 mmol) was added slowly. The reaction was allowed to sit for 20 minutes followed by addition of /-butyl bromoacetate (3.93 mL, 39.9 mmol). The reaction was left for four hours and extracted with ethylacetate and water. The organ ics were dried over magnesium sulfate and purified by silica gel column (Hexane:EtOAc = 5 : 1-1 : 1 ) to afford
azidotriethylene glycol-Boc ( 1 .54 g; Yield: 40%; clear oil). Ή NMR (600 MHz, CDCh) δ 4.01 (s, 2H), 3.71 -3.65 (m, 10H), 3.38-3.36 (t, J = 4.5 Hz, 1 H), 1.45 (s, 9H), 13C NMR ( 150 MHz, CDCI3) δ 169.75, 81.61 , 70.83, 70.79, 70.78, 70.76, 70.13, 69.15, 50.79, 28.21.
[0237] Compound F15-3. Azidotriethylene glycol-Boc (1 .19 g, 4.1 mmol) was dissolved in 15 mL of ethyl acetate and of Pd/C catalyst (240 mg) was added under hydrogen gas in a double balloon. Reaction was stirred until reaction was completed as monitored by TLC. The reaction mixture was filtered over celite and the filtrate was rotovaped to afford crude compound F15-3. Yield: quant; clear oil.
[0238] Compound F15-5. D-Biotin F15-4 (538 mg, 2.2 mmol) was dissolved in 10 mL of hot DMF. The solution was allowed to cool to room temperature and HATU (838 mg, 2.2 mmol) was added and allowed to preactivate for 15 minutes. To The reaction mixture diisopropylethylamine (0.968 mL, 2.4 mmol) and para-ammo benzoic acid (335 mg, 2.4 mmol) were added. Reaction was allowed to react for 24 hr in which then 70 mL of dichloromethane was added to precipitate the product. The precipitate was collected via suction filtration and washed three times with ethyl acetate (50 mL) to attain NMR pure compound F15-5 (687 mg, Yield: 86%; white solid). ]H MR (600 MHz, DMSO) δ 10.19 (s, N-H), 7.88-7.86 (d, J= 8.4 Hz, 2H), 7.71-7.69 (d, J= 9.0 Hz, 2H), 6.46 (s, N-H), 6.38 (s, N- H), 4.32-4.29 (m, 1 H), 4.15-4.12 (m, 1 H), 3.13-3.10 (m, 1 H), 2.83-2.80 (dd, J = 12.6 Hz, 5.4 Hz, 1 H), 2.59-2.57 (d, J = 12.6, 1 H), 2.36-2.33 (t, J= 7.5 Hz, 2H), 1.67-1 .34 (m, 6H). 13C MR (150 MHz, DMSO) δ 171.75, 166.97, 162.77, 143.35, 130.38, 124.90, 1 18.26, 61 .07, 59.22, 55.40 39.94, 36.33, 28.23, 28.1 1 , 24.99.
[0239] Compound F15-6. Compound F15-5 ( 1.1 16 g, 3.1 mmol) was dissolved in 1 5 mL of DMF. carbonyldiimidazole (547 mg, 3.4 mmol) was added to reaction mixture and allowed to preactivate for 40 minutes followed by the addition of compound F15-3 (977 mg, 3.7 mmol) in 5 mL of DMF. The reaction was left at room temperature for 40 hours and was passed through silica gel column (DCM:MeOH:NH4OH = 9: 1 :0.1-1 : 1 : 0.1 ) to afford F15-6 (738 mg; Yield: 40%; yellow flakes). Ή NMR (600 MHz, DMSO) δ 10.13 (s, N-H), 8.40 (m, N-H), 7.89-7.77 (d, J = 8.4 Hz, 2H), 7.65-7.64 (d, .7 = 9.0 Hz, 2H), 6.45 (s, N-H), 6.38 (s, N- H), 4.32^.30 (m, 1 H), 4.15-4.13 (m, 1 H), 3.95 (s, 2H) 3.53-3.50 (m, 10H), 3.40-3.38 (m, 2H), 3.13-3.10 (m, 1 H), 2.83-2.80 (dd, J= 12.6 Hz, 4.8 Hz, 1 H), 2.59-2.57 (d, J= 12.6, 1 H), 2.34-2.31 (t, J = 7.2 Hz, 2H), 1.67-1.34 (m, 15H). 13C NMR (150 MHz, DMSO) δ 1 71 .86, 169.57, 166.07, 163.03, 141 .91 , 128.64, 128.06, 1 1 8.18, 80.89 70.0, 69.85, 69.83, 69.75, 69.1 , 68.26, 61 .28, 59.42, 55.56, 40.00, 39.25, 36.43, 28.42, 28.25, 27.90 25.19.
[0240] Compound F15-7. Compound F15-6 (701 mg) was dissolved in 7 mL mixture of DCM/TFA( 2: 1 ) and was left for 3 hr. The crude product was passed through silica gel column (DCM:MeOH:NH4OH = 7:3:0.1-0: 1 : 0.1 ) to afford the free acid (562 mg, 88% yield). The free acid (150 mg, 0.27 mmol) and N-hydroxy succamide (32 mg, 0.3 mmol) were dissolved in hot DMF. The reaction mixture was cooled to room temperature and Ν,Ν'- dicyclohexylcarbodiimide (67 mg, 0.35 mmol) was added and left for 18 h. the reaction mixture was filtrate over celite and the filtrate was rotovaped and the triturated with diethyl ether and collected by suction filtration to afford crude compound F15-7 ( 172 mg; Yield: 98%; white solid). [0241] Glucoronic acid-P-propylamine (200 mg, 0.79 mmol) (obtained by reduction from compound F13-7) and compound F15-7 (672 mg, 1.03 mmol) were dissolved in dry methanol (15 mL) and stirred overnight. The reaction mixture was rotpavoped and purified by silica gel column (DCM:MeOH:NH4OH = 7:3 :0.1-0: 1 : 0.1 ) to provide GlcA-TEG- PABA-biotin (F15-8) (192 mg; Yield: 31 %; white foam). Ή NMR (600 MHz, D20) 8 7.74- 7.73 (d, J= 7.8 Hz, 2H), 7.56-7.54 (d, J = 9.0 Hz, 1 H), 4.56-4.54 (m, 1 H), 4.39^.38 (d, J = 7.8, 1 H), 4.36-4.34 (m, 1 H), 3.92 (s, 2H) 3.91-3.88 (m, 1 H), 3.71-3.47 (m, 18H), 3.37-3.35 (t, J = 6.3 Hz, 2H), 2.94-2.9 l (dd, J = 12.6 Hz, 4.8 Hz, 1 H), 2.74-2.72 (d, J= 12.0, 1 H), 2.39-2.36 (t, J = 7.5 Hz, 2H), 1.80-1.38(m, 8H), ,3C NMR ( 150 MHz, D20) δ 175.60, 175.21 , 172.09, 169.54, 165.15, 140.69, 129.21 , 128.16, 120.29, 1 02.43, 1 02.02, 75.95, 75.47, 72.90, 72.87, 71.76, 70.19, 69.36, 68.74, 67.50, 61.98, 60.15, 55.29, 39.63, 39.45 36.29, 35.79, 28.37, 28.13, 27.96, 27.67, 24.93.
Example 10: Preparation of GIcA 1-4GIcNAc Disaccharide Derivatives
J0242] PmHS2 acceptor substrate specificities using UDP-GlcA as a donor substrate and a- and β-linked GlcNAc derivatives as acceptors were studied. Conversion to the disaccharide products was estimated by LCMS and TLC analysis as outlined in Table 9 below.
Table 9. Reaction conditions and yields for the formation of GlcA-GlcNAc dissacharide
Figure imgf000086_0001
[0243] As shown in Figure 31 and Figure 32, TLC and LC-MS data indicated that both a- and β-linked GlcNAc with different aglycons are suitable acceptor substrates for PmHS2. Therefore, both GlcA and GlcNAc can be used as the first sugar for oligosaccharide synthesis by the methods described in this invention.
Example 11: Inhibition assays of monosaccharides, disaccharides, trisaccharides, and tetrasaecharides.
[0244] Materials. Recombinant human fibroblast growth factors FGF- 1 , FGF-2, FGF-4, and anti-human FGF- 1 , FGF-2, FGF-4 were purchased from PeproTech Inc (Rocky Hill, NJ). Heparin-biotin was from Sigma (St. Louis, MO). Low molecular weight heparin (LMWH) was bought from AMS Biotechnology (Lake Forest, CA). Alexa Fluor® 488 goat anti-rabbit IgG (H+L) was from Invitrogen (Carlsbad, CA). 384- Well NeutrAvid in-coated plates for the sialidase assays were from Fisher Biotech.
[0245] Methods. All assays were carried out in duplicate in 384-well NeutrAvidin coated plates. Heparin-biotin (20 μί, 2 μΜ) was added to each well and the plate was incubated at 4 °C for overnight. The plate was washed with 3 rounds of 1 xPBS buffer containing 0.05% Tween-20 and blocked with 1 % BSA (50
Figure imgf000087_0001
for each well) and incubated at r.t. for 30 min. After the plate was washed three times with 1 *PBS buffer containing 0.05%o Tween-20, each set of duplicate wells were added 20 μΐ, of human FGF- 1 , FGF-2, or FGF-4 (1 μΜ) with or without premixing with LMWH (~ 22 iiM or 0.1 μΜ), monosaccharide, or oligosaccharides (100 μΜ or 1 niM) and the plate was incubate at r.t. for 1 hr. After washing three times with I xPBS buffer containing 0.05% Tween-20, anti-human FGF- 1 , FGF-2 or FGF-4 (20 μί) was added and the plate was incubated at r.t. for 1 hr. After th plate was washed three times with I PBS buffer containing 0.05% Tween-20, Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (20 μΐ^) was added and the plate was incubated at r.t. for 1 hr. After washed three times with 1 xPBS buffer containing 0.05% Tween-20 and once with water, the fluorescent signals of wells in the plate were measured using a microtiter plate reader.
[0246] Results. LMWH was used as a control sample for testing the inhibitory activities of sixteen compounds including seven monosaccharides, two disaccharides, two trisaccharides, and five tetrasaecharides (see Figure 24 for compound structures) against the binding of fibroblast growth factors FGF-1 , FGF-2, and FGF-4 to heparin-biotin immobilized on NeutrAvidin-coated plates. See Table 10 and Figure 23. Table 10. Percentage inhibition of compounds F24-1-F24-16 (1 mM) against the binding of human FGF-1, FGF-2, and FGF-4 to heparin-biotin immobilized on NeutrAvidin- coated plates.
Compounds Structures FGF-1 (% FGF-2 (% FGF-4 (% inhibition) inhibition) inhibition)
F24-1 GlcA 65 68 54
F24-11 GlcApi-4GlcNH2a14GlcA 2AA 76 - -
F24-13 G!cNAc6N3 1-4GlcAp1-4GlcNH2a1 GicAp2AA 62 - -
F24-15 GlcNAc6NH2oc1 -4&οΑβ 1 -4GlcNSa1 -4αοΑβ2ΑΑ - 68 43
F24-16 GlcNAc6NSa1-4GlcApi-4GlcNSa1 -4GlcA 2AA 58 55 -
DNA AND PROTEIN SEQUENCES FOR GENES AND ENZYMES
DNA sequence of NahK_ATCC15697 (Note: The sequences for His6-tag are underlined) ATGAACAACACCAATGAAGCCCTGTTCGACGTCGCTTCGCACTTCGCGCTGGAAGGCACCGT CGACAGCATCGAACCATACGGAGACGGCCATATCAACACCACCTATCTGGTGACCACGGACG GCCCCCGCTACATCCTCCAACGGATGAACACCGGCATCTTCCCCGATACGGTGAATCTGATG CGCAATGTCGAGCTGGTCACCTCCACTCTCAAGGCTCAGGGCAAAGAGACGCTGGACATCGT GCGCACCACCTCCGGCGACACCTGGGCCGAGATCGACGGCGGCGCATGGCGCGTCTACAAGT TCATCGAACACACCATGTCATACAACCTCGTGCCGAACCCGGACGTGTTCCGCGAAGCCGGC AGGGCGTTCGGTGATTTCCAGAACTTCCTGTCCGGGTTCGACGCCAACCAGCTGACCGAGAC CATCGCCCACTTCCACGACACCCCGCACCGCTTCGAGGACTTCAAGAAGGCGCTCGCCGCGG ACGAGCTCGGGCGTGCCGCCGGGTGCGGCCCGGAGATCGAGTTCTATCTGAGTCACGCCGAC CAGTACGCCGTCGTGATGGATGGGCTCAGGGATGGTTCGATCCCGCTGCGCGTGACCCACAA CGACACCAAACTCAACAACATCCTCATGGATGCCACCACCGGCAAGGCCCGTGCGATCATCG ATCTAGACACCATCATGCCGGGGTCCATGCTCTTCGACTTCGGCGATTCCATCCGTTTCGGC GCGTCCACGGCCTTGGAGGATGAGCGGGATCTGGACAAGGTGCATTTCAGCACCGAGCTGTT CCGCGCCTACACGGAAGGCTTCGTGGGCGAACTACGCGACAGCATCACCGCGCGCGAGGCCG AACTGCTGCCGTTCAGCGGCAACCTGCTCACCATGGAATGCGGCATGCGCTTTCTCGCCGAC TACCTGGAAGGCGACGTCTACTTCGCCACCAAGTACCCCGAGCATAACCTGGTGCGCTCCCG CACCCAGATCAAGCTCGTGAGGGAGATGGAGCAGCGAGCCGATGAGACCCGCGCCATCGTGG CCGACGTCATGGAGTCGACCAAGCTCGAGCACCACCACCACCACCACTGA Protein sequence of NahK ATCC15697 (Note: The sequences for His6-tag are underlined)
MNNTNEALFDVASHFALEGTVDSIEPYGDGHINTTYLVTTDGPRYILQR NTGI FPDTVNLM RNVELVTSTLKAQGKETLDIVRTTSGDTWAEIDGGAWRVYKFIEHTMSYNLVPNPDVFREAG RAFGDFQNFLSGFDANQLTETIAHFHDTPHRFEDFKKALAADELGRAAGCGPEIEFYLSHAD QYAVVMDGLRDGSIPLRVTHNDTKLNNIL DATTGKARAIIDLDTI PGSMLFDFGDSIRFG ASTALEDERDLDKVHFSTELFRAYTEGFVGELRDSITAREAELLPFSGNLLT ECGMRFLAD YLEGDVYFATKYPEHNLVRSRTQIKLVREMEQRADETRAIVADVMESTKLEHHHHHH DNA sequence of NahK_ATCC55813 (Note: The sequences for His6-tag are underlined)
ATGACCGAAAGCAATGAAGTTTTATTCGGCATCGCCTCGCATTTTGCGCTGGAAGGTGCCGT GACCGGTATCGAACCTTACGGAGACGGCCACATCAACACCACCTATCTGGTGACCACGGACG GCCCCCGCTACATCCTCCAGCAGATGAACACCAGCATCTTCCCCGATACGGTGAATCTGATG CGCAATGTCGAACTGGTCACCTCCACTCTCAAGGCTCAGGGCAAAGAGACGCTGGACATTGT GCCCACCACCTCAGGCGCCACCTGGGCCGAGATCGATGGCGGCGCATGGCGCGTCTACAAGT TCATCGAACACACCGTGTCCTACAACCTCGTGCCGAACCCGGACGTGTTCCGCGAAGCCGGC AGCGCATTCGGCGACTTCCAGAACTTCCTGTCCGAATTCGACGCCAGCCAGCTGACCGAAAC CATCGCCCACTTCCACGACACCCCGCATCGTTTCGAGGACTTCAAGGCCGCCCTCGCCGCGG ACAAGCTCGGCCGCGCCGCCGCATGCCAGCCGGAAATCGACTTCTATCTGAGTCACGCCGAC CAGTATGCCGTCGTGATGGATGGGCTCAGGGACGGTTCGATTCCGCTGCGCGTGACCCACAA TGACACCAAGCTCAACAACATCCTCATGGACGCCACCACCGGCAAGGCGCGTGCGATCATCG ATCTCGACACCATCATGCCCGGCTCCATGCTGTTCGACTTCGGCGATTCCATACGCTTTGGT GCGTCCACTGCTCTGGAAGACGAAAAGGACCTCAGCAAGGTGCATTTCAGCACCGAGCTGTT CCGCGCCTACACGGAAGGCTTCGTGGGCGAACTACGCGGCAGCATCACCGCGCGCGAGGCCG AACTGCTGCCGTTCAGCGGCAACCTGCTCACCATGGAATGCGGCATGCGCTTTCTCGCCGAC TACTTGGAAGGCGATATCTACTTTGCCACCAAGTACCCCGAGCATAATCTGGTGCGCACCCG CACCCAGATCAAACTCGTGCAGGAGATGGAGCAGAAGGCCAGTGAAACCCACGCCATCGTAG CCGACATCATGGAGGCTGCCAGGCTCGAGCACCACCACCACCACCACTGA
Protein sequence of NahK_ATCC55813 (Note: The sequences for Hise-tag are underlined)
MTESNEVLFGIASHFALEGAVTGIEPYGDGHINTTYLVTTDGPRYILQQMNTSIFPDTVNLM RNVELVTSTLKAQGKETLDIVPTTSGAT AEI DGGAWRVYKFIEHTVSYNLVPNPDVFREAG SAFGDFQNFLSEFDASQLTETIAHFHDTPHRFEDFKAALAADKLGRAAACQPEIDFYLSHAD QYAVV DGLRDGSIPLRVTHNDTKLN ILMDATTGKARAII DLDTIMPGSMLFDFGDSIRFG ASTALEDEKDLSKVHFSTELFRAYTEGFVGELRGSITAREAELLPFSGNLLTMECGMRFLAD YLEGDIYFATKYPEHNLVRTRTQIKLVQEMEQKASETHAIVADIMEAARLEHHHHHH
DNA sequence of AtGlcAK (Note: Italic sections of the sequences are from pETl 5b vector and primer).
ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATAT GGATCCGAATTCCACGGTTTCCGGCGATGGTCAGGCGACGGCGGCGATAGAGCATCGGTCCT TCGCTCGGATCGGATTTCTCGGAAACCCGAGCGATGTATACTTCGGGCGAACCATATCATTG ACCATCGGAAACTTCTGGGCATCCGTGAAGCTGGAGCCATCGGAGCATCTCGTAATCAAGCC TCATCCATTCCATGATCTCGTCCAGTTCACCTCTCTCGACCATCTCCTGAATCGTTTGCAAA ATGAAGGATACTACGGTGGGGTAAGGTTGCTAATGGCGATATGTAAAGTATTCCGTAACTAT TGCAAAGAGAATGACATTCAACTTCACCAAGCCAACTTCTCTCTTTCTTATGATACCAATAT CCCTAGGCAGACAGGGCTTTCGGGTTCTAGTGCCATCGTATCCGCTGCCCTTAACTGCCTTC TCGACTTCTACAATGTCAGGCATTTGATCAAAGTACAAGTCCGCCCTAACATTGTTCTCAGT GCTGAGAAAGAACTTGGCATTGTTGCTGGTCTTCAGGACAGGGTTGCTCAGGTCTATGGTGG TCTTGTTCACATGGATTTTAGCAAGGAGCACATGGATAAATTGGGGCATGGGATTTACACTC CTATGGATATCAGTCTCCTCCCTCCTCTGCATCTCATCTATGCTGAGAATCCGAGCGACTCA GGGAAGGTACATAGTATGGTTCGGCAAAGATGGTTAGACGGTGATGAGTTTATAATCTCATC AATGAAAGAAGTCGGAAGTCTAGCAGAAGAAGGTCGAACTGCATTACTCAACAAGGACCATT CCAAACTTGTGGAACTCATGAACCTTAATTTCGACATTCGGAGGCGGATGTTTGGGGATGAA TGCTTAGGAGCAATGAACATGGAGATGGTGGAAGTAGCAAGGAGGGTTGGTGCAGCCTCAAA GTTCACTGGAAGTGGAGGAGCAGTGGTGGTTTTCTGCCCTGAAGGTCCATCTCAGGTGAAAC TTCTGGAAGAAGAATGCAGGAAAGCGGGATTTACGCTTCAGCCGGTAAAAATTGCGCCTTCA TGTTTGAATGATTCTGACATTCAGACCTTATGA Protein sequence of AtGlcAK (Note: Italic sections of the sequences are from pETl 5b vector and primer. N-terminal His6-tag is underlined in the protein sequence)
MGSSHHHHHHSSGLVPRGSflMDPNSTVSGDGQATAAIEHRSFARIGFLGNPSDVYFGRTISL TIGNFWASVKLEPSEHLVIKPHPFHDLVQFTSLDHLLNRLQNEGYYGGVRLLMAICKVFRNY CKENDIQLHQANFSLSYDTNIPRQTGLSGSSAIVSAALNCLLDFYNVRHLIKVQVRPNIVLS AEKELGIVAGLQDRVAQVYGGLVHMDFSKEHMDKLGHGIY PMDISLLPPLHLIYAENPSDS GKVHSMVRQRWLDGDEFI I SSMKEVGSLAEEGRTALLNKDHSKLVELMNLNFDIRRR FGDE CLGAMNMEMVEVARRVGAASKFTGSGGAVVVFCPEGPSQVKLLEEECRKAGFTLQPVKI PS CLNDSDIQTL DNA sequence of His6-PmGImU (Note: Italic sections of the sequences are from pET15b vector and primer)
ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATAT GAAAGAGAAAGCATTAAGTATCGTGATTTTAGCGGCAGGTAAAGGGACGCGGATGTATTCTG ATTTACCAAAAGTGCTACATAAAATTGCCGGAAAACCGATGGTAAAACATGTGATCGATACG GTGAAATCCATTCATGCAAAAAATATCCATTTAGTGTATGGACATGGTGGGGAAGTGATGCA AACTCGCTTGCAAGATGAACCTGTGAATTGGGTCTTACAAGCCGAGCAATTAGGTACGGGGC ATGCTATGCAGCAAGCAGCCCCGTTTTTTGCAGATGATGAAAATATTTTGATGCTTTATGGT GATGGACCATTAATTACTGCGAAAACCTTACAAACATTAATTGCGGCAAAACCTGAACATGG TATTGCATTATTGACCGTCGTATTAGATGACCCAACTGGTTATGGGCGTATTGTGCGTGAAA ATGGCAATGTGGTGGCGATTGTGGAACAAAAAGATGCCAATGCAGAGCAATTAAAAATCCAA GAAATTAACACAGGCTTGTTAGTGGCAGACGGTAAAAGTTTGAAAAAATGGTTATCACAGTT AACCAACAACAATGCACAGGGAGAATATTATATTACGGATGTGATCGCCTTAGCGAATCAAG ACGGTTGCCAAGTAGTGGCGGTACAAGCCAGTAACTTTATGGAAGTAGAGGGCGTGAATAAC CGTCAGCAATTAGCGCGTTTAGAGCGTTATTATCAGCGCAAACAAGCAGACAATTTATTATT GGCTGGGGTGGCATTAGCGGATCCTGAGCGTTTTGATTTACGCGGGGAACTAAGCCATGGGA AAGACGTGCAAATTGATGTGAACGTGATTATCGAGGGCAAAGTCAGCTTAGGTCACCGAGTT AAAATTGGAGCAGGTTGTGTGTTAAAAAATTGCCAGATTGGTGATGATGTAGAAATTAAACC TTATTCTGTGTTGGAAGAGGCGATTGTTGGACAAGCTGCGCAAATTGGACCCTTCTCTCGTT TGCGTCCGGGGGC rGCATTAGCCGACAACACTCAT TTGGTAATTTCGTTGAAATTAAGAAA GCGCATATTGGGACAGGCTCGAAAGTAAACCATTTAAGTTATGTGGGAGATGCCGAAGTCGG GATGCAATGTAATATTGGTGCCGGCGTGATCACTTGTAACTATGATGGCGCAAATAAATTTA AGACCATTATTGGTGATAATGTGTTTGTAGGGTCTGATGTACAACTCGTGGCACCGGTTACC ATCGAAACGGGTGCAACCATTGGTGCGGGGACTACGGTGACCAAAGATGTGGCTTGTGATGA GTTAGTGATTTCACGTGTTCCTCAACGTCATATTCAAGGTTGGCAACGCCCTACTAAACAAA CGAAAAAGTAA
Protein sequence of His6-PmGlmU (Note: Italic sections of the sequences are from pET15b vector and primer. N-terminal His6-tag is underlined in the protein sequence)
MGSSHHHHHHSSGLVPRGSHMKEKR SIVILAAGKGT MYSDLPKVLHKIAGKPMVKHVIDT VKSIHAKNIHLVYGHGGEVMQTRLQDEPVNWVLQAEQLGTGHAMQQAAPFFADDENILMLYG DGPLITA TLQTLIAAKPEHGIALLTVVLDDPTGYGRIVRENGNVVAIVEQKDANAEQLKIQ EINTGLLVADGKSLK WLSQLTNNNAQGEYYITDVIALANQDGCQVVAVQASWFMEVEGVNN RQQLARLERYYQRKQADNLLLAGVALADPERFDLRGELSHGKDVQIDVNVIIEGKVSLGHRV KIGAGCVLKNCQIGDDVEIKPYSVLEEAIVGQAAQIGPFSRLRPGAALADNTHIGNFVEIKK AHIGTGSKVNHLSYVGDAEVGMQCNIGAGVI CNYDGANKFK I IGDNVFVGSDVQLVAPVT IETGATIGAGTTVTKDVACDELVISRVPQRHIQGWQRPTKQTKK
DNA sequence of BLUSP (Note: The sequences for His6-tag are underlined)
ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATAT GACAGAAATAAACGATAAGGCCCAACTGGATATCGCCGCCGCCGGCGACACCGACGCCGTTA CCTCGGACACCCCCGAAGAAACCGTAAACACCCCCGAAGTGGATGAGACTTTCGAGCTTTCG GCCGCCAAGATGCGCGAGCATGGCATGAGCGAAACCGCCATCAACCAGTTCCACCATTTGTA TGACGTATGGCGCCATGAAGAAGCCTCCAGCTGGATTCGTGAGGACGACATCGAGCCGCTTG GCCACGTGCCCAGCTTCCACGACGTCTATGAGACCATCAACCACGACAAGGCCGTGGACGCC TTCGCCAAGACCGCATTCCTCAAGCTCAATGGCGGTCTGGGCACCTCCATGGGATTAGACAA GGCCAAGTCGCTGTTGCCGGTGCGTAGGCACAAGGCCAAGCAGATGCGCTTCATCGACATCA TCATCGGTCAGGTGCTTACCGCTCGCACCCGCCTGAACGTCGAACTGCCGCTGACGTTCATG AACTCCTTCCACACTTCGGCGGACACGATGAAGGTGCTCAAGCATCATCGCAAGTTCAGTCA GCATGACGTGCCGATGGAAATCATCCAGCATCAGGAACCCAAGCTCGTGGCCGCCACCGGCG AACCTGTGAGCTACCCTGCGAACCCGGAGCTGGAATGGTGCCCGCCCGGCCACGGCGACCTG TTCTCCACCATCTGGGAGTCTGGTCTGCTTGACGTATTGGAGGAGCGCGGCTTCAAGTATCT GTTCATCTCCAATTCCGACAATCTCGGTGCGCGCGCCTCGCGTACGTTGGCCCAGCACTTCG AAAACACAGGTGCCCCGTTTATGGCTGAAGTGGCCATCCGCACCAAGGCCGATCGCAAGGGC GGCCATATTGTACGAGACAAGGCCACTGGTCGCCTAATACTGCGTGAAATGAGCCAGGTCCA TCCGGACGATAAGGAAGCGGCCCAAGACATCACCAAGCATCCTTACTTCAACACCAACTCAA TCTGGGTTCGCATCGACGCTTTGAAAGACAAGCTCGCCGAATGCGATGGTGTGTTGCCGTTG CCGGTGATTCGTAACAAAAAGACCGTGAATCCCACGGACCCGGATTCCGAACAGGTGATTCA GCTGGAAACCGCCATGGGCGCCGCAATCGGTCTGTTCAACGGTTCTATCTGCGTCCAAGTGG ATCGTATGCGCTTCCTTCCGGTGAAAACCACCAATGATTTGTTCATTATGCGTTCCGATCGA TTCCACCTGACGGACACGTATGAGATGGAAGACGGCAATTACATCTTCCCGAACGTCGAACT TGATCCGCGATACTACAAGAACATCCACGATTTCGACGAACGGTTCCCCTACGCCGTGCCAT CTTTGGCCGCAGCCAACTCGGTTTCCATTCAGGGCGACTGGACATTCGGACGTGACGTCATG ATGTTCGCCGACGCCAAACTGGAAGATAAAGGCGAGCCAAGCTATGTGCCGAACGGCGAATA CGTTGGTCCGCAAGGCATCGAACCGGACGATTGGGTGTGA
Protein sequence of BLUSP (Note: The sequences for His6-tag are underlined)
MGSSHHHHHHSSGLVPRGSHMTEINDKAQLDIAAAGDTDAVTSDTPEETVNTPEVDETFELS AAK REHGMSETAINQFHHLYDVWRHEEASSWIREDDIEPLGHVPSFHDVYETINHDKAVDA FAKTAFLKLNGGLGTSMGLDKAKSLLPVRRHKAKQMRFI DI I IGQVLTARTRLNVELPLTFM NS FHTSADTMKVLKHHRKFSQHDVPMEI IQHQEPKLVAATGEPVSYPANPELEWCPPGHGDL FSTI ESGLLDVLEERGFKYLFISNSDNLGARASRTLAQHFENTGAPFMAEVAIRTKADRKG GHIVRDKATGRLILREMSQVHPDDKEAAQDITKHPYFNTNSIWVRIDALKDKLAECDGVLPL PVrRNKKTV PTDPDSEQVIQLETAMGAAIGLFNGSICVQVDRMRFLPVKTTNDLFIMRSDR FHLTDTYEMEDGNYIFPNVELDPRYYKNIHDFDERFPYAVPSLAAANSVSIQGDWTFGRDVM MFADAKLEDKGEPSYVPNGEYVGPQGIEPDD V
DNA sequence of PmUgd-His6 (Note: The sequences for Hise-tag are underlined)
ATGAAGAAAATTACAATTGCTGGGGCTGGCTATGTTGG TTATCCAATGCAGTATTATTAGC TCAACACCACAATGTGATCTTATTAGATATTGATCAAAATAAAGTTGATTTAATTAATAATA AAAAATCGCCCATCACAGATAAAGAAATCGAAGATTTCTTACAAAATAAATCACTGACAATG ATGGCAACAACAGATAAAGAAGTGGCATTAAAAAACGCAGACTTTGTCATCATCGCAACGCC AACAGACTATAATACCGAAACAGGTTATTTTAATACATCCACTGTTGAAGCTGTCATTGAAC AAACCCTTTCAATCAATCCACAAGCAACGATTATTATAAAATCAACGATTCCCGTTGGTTTT ACCGAAAAAATGCGTGAGAAATTTCATACCAAGAACATTATTTTTTCTCCTGAGTTTTTAAG AGAAGGAAAAGCACTTCATGACAATTTGTTTCCAAGCAGAATTATTGTTGGCAGTACTTCTT ATCAAGCAAAAGTATTTGCCGATATGTTGACACAGTGTGCCAGAAAAAAAGATGTAACTGTT TTATTTACACACAATACTGAGGCTGAAGCTGTTAAATTATTTGCAAATACGTATCTCGCAAT GCGAGTTGCCTTTTTTAATGAATTAGATACTTATGCGAGTCTTCACCATTTAAATACAAAAG ACATTATCAATGGTATTTCTACTGATCCTCGCATTGGTACACACTACAATAACCCAAGTTTC GGCTATGGCGGTTATTGTTTACCCAAAGACACTAAACAGTTACTGGCTAACTATGCTGACGT ACCTCAAAATCTCATTGAAGCCATTGTCAAATCTAATGAAACCAGAAAACGTTTCATTACTC ATGATGTATTAAATAAGAAACCTAAAACTGTTGGTATTTATCGTTTAATCATGAAGTCAGGT TCTGATAACTTCAGAGCTTCTGCTATTCTCGATATTATGCCGCATCTCAAAGAAAACGGTGT TGAGATTGTGATTTATGAGCCAACCTTAAATCAACAGGCATTTGAGGACTACCCCGTTATTA ATCAACTCTCTGAATTTATTAATCGCTCTGATGTCATTCTCGCTAATCGTTCTGAGCCAGAT TTAAATCAATGTTCCCATAAAATCTATACAAGAGATATTTTTGGCGGTGATGCTCTCGAGCA CCACCACCACCACCACTGA
Protein sequence of PmUgd-HiS6 (Note: The sequences for Hisg-tag are underlined)
MKKITIAGAGYVGLSNAVLLAQHHNVILLDIDQNKVDLINNKKSPITDKEIEDFLQN SLTM MATTDKEVALKNADFVI I TPTDYNTETGYFNTSTVEAVIEQTLSI PQATI IIKSTI PVGF TEKMREKFHTK I IFSPEFLREGKALHDNLFPSRI IVGSTSYQAKVFADMLTQCARKKDVTV LFTHNTEAEAVKLFANTYLAMRVAFFNELDTYASLHHLNTKDIINGI STDPRIGTHYN PSF GYGGYCLPKDTKQLLANYADVPQNLIEAIVKSNETRKRFITHDVLNKKPKTVGIYRLIMKSG SDNFRASAILDIMPHLKENGVEIVIYEPTLNQQAFEDYPVINQLSEFINRSDVILANRSEPD LNQCSHKIYTRDIFGGDALEHHHHHH
DNA sequence of MBP-PmHSl-His6 (Note: Italic sections of the sequences are from pMAL-c4X vector and primer. The sequences for His6-tag are underlined)
CTCGGGATCGAGGGAAGGATTTCAGAATTCGGATCCATGAGCTTAT TAAACGTGCTACTGA GCTATTTAAGTCAGGAAACTATAAAGATGCACTAACTCTATATGAAAATATAGCTAAAATTT ATGGTTCAGAAAGCCTTGTTAAATATAATATTGATATATG AAAAAAAAT TAACACAATCA AAAAG AA AAATAGAAGAAGATAATATTTCTGGAGAAAACAAATTTTC GTATCAATAAA AGATCTATATAACGAAATAAGCAATAGTGAATTAGGGATTACAAAAGAAAGACTAGGAGCCC CCCCTCTAGTCAGTATTATAATGACTTCTCATAATACAGAAAAATTCATTGAAGCCTCAATT AATTCACTATTATTGCAAACAT C AT ACTTAGAAGTT TCGTTGTAGATG TTATAGCAC AGATAAAACAT TCAGATCGCATCCAGAATAGCAAAC C ACAAGTAAAGTAAAAACATTCC GATTAAACTCAAATCTAGGGACATACTTTGCGAAAAATACAGGAATTTTAAAGTCTAAAGGA GATATTATTTTCTTTCAGGATAGCGATGATGTATGTCACCATGAAAGAATCGAAAGATGTGT TAATGCATTATTATCGAATAAAGATAATATAGCTGTTAGATGTGCATATTCTAGAATAAATC TAGAAACACAAAATATAATAAAAGTTAATGA AATAAATACAAATTAGGATTAATAACTTTA GGCGTTTATAGAAAAGTATTTAATGAAATTGGTTTTTTTAACTGCACAACCAAAGCATCGGA TGATGAATTTTATCATAGAATAATT AATACTATGGTAAAAATAGGATAAATAACTTATTTC TACCACTGTATTATAACACAATGCGTGAAGATTCATTATTTTCTGATATGGTTGAGTGGGTA GATGAAAATAATATAAAGCAAAAAACCTCTGATGCTAGACAAAATTATCTCCATGAATTCCA AAAAATACACAATGAAAGGAAATTAAATGAATTAAAAGAGATTTTTAGCTTTCCTAGAATTC ATGACGCCTTACCTATATCAAAAGAAATGAGTAAGCTCAGCAACCCTAAAATTCCTGTTTAT ATAAATATATGCTCAATACCTTCAAGAATAAAACAACTTCAATACACTATTGGAGTACTAAA AAACCAATGCGATCATTTTCATATTTATCTTGATGGATATCCAGAAGTACCTGATTTTATAA AAAAACTAGGGAATAAAGCGACCGTTATTAATTGTCAAAACAAAAATGAGTCTATTAGAGAT AATGGAAAGTTTATTCTATTAGAAAAACTTATAAAGGAAAATAAAGATGGATATTATATAAC TTGTGATGATGATATCCGGTATCCTGCTGACTACATAAACACTATGATAAAAAAAATTAATA AATACAATGATAAAGCAGCAATTGGATTACATGGTGTTATATTCCCAAGTAGAGTCAACAAG TATTTTTCATCAGACAGAATTGTCTATAATTTTCAAAAACCTTTAGAAAATGATACTGCTGT AAAT TATTAGGAACTGGAACTGTTGCCTTTAGAGTATCTATTTTTAATAAATTTTCTCTAT CTGATTTTGAGCATCCTGGCATGGTAGATATCTATTTTTCTATACTATGTAAGAAAAACAAT ATACTCCAAGTTTGTATATCACGACCATCGAATTGGCTAACAGAAGATAACAAAAACACTGA GACCTTATTTCATGAATTCCAAAATAGAGATGAAATACAAAGTAAACTCATTATTTCAAACA ACCCTTGGGGATACTCAAGTATATATCCATTATTAAATAATAATGCTAATTATTCTGAACTT ATTCCGTGTTTATCTTTTTATAACGAGCATCATCATCATCATCACTAA Protein sequence of MBP-PmHSl-Hise (Note: Italic sections of the sequences are from pMAL-c4X vector and primer. The sequences for His6-tag are underlined)
I/GJEGi^ISiJFGSMSLF RATELFKSGNYKDALTLYENIAKIYGSESLVKYNI DICKKNITQS KS KIEED ISGENKFSVSIKDLYNEISNSELGITKERLGAPPLVSI IMTSHNTEKFIEAS I NSLLLQTYNNLEVIVVDDYSTDKTFQIASRIANSTSKVKTFRLNSNLGTYFAKNTGILKSKG DI IFFQDSDDVCHHERIERCV ALLSNKDNIAVRCAYSRINLETQNI IKV DNKYKLGLITL GVYRKVFNEIGFFNCTTKASDDEFYHRIIKYYGKNRINNLFLPLYYNTMREDSLFSDMVEWV DEN IKQKTSDARQNYLHEFQKIHNERKLNELKEI FSFPRIHDALPISKEMSKLS PKI PVY INICSIPSRIKQLQYTIGVLKNQCDHFHIYLDGYPEVPDFIKKLGNKATVINCQNKNESIRD NGKFILLEKLIKENKDGYYITCDDDIRYPADYINTMIKKINKYND AAIGLHGVI FPSRVNK YFSSDRIVY FQKPLENDTAVNILGTGTVAFRVSI FNKFSLSDFEHPG VDIYFSILCKKNN ILQVCISRPSNWLTEDNKNTETLFHEFQ RDEIQSKLI ISNNPWGYSSIYPLLNNNANYSEL I PCLSFYNEHHHHHH
DNA sequence of His6-PmHS2 (Note: Italic sections of the sequences are from pET15b vector and primer. The sequences for His6-tag are underlined)
ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATAT GAAGGGAAAAAAAGAGATGACTCAAAAACAAATGACTAAAAATCCACCCCAACATGAAAAAG AAAATGAACTCAACACCTTTCAAAATAAAATTGATAGTCTAAAAACAACTTTAAACAAAGAC ATTATTTCTCAACAAACTTTATTGGCAAAACAGGACAGTAAACATCCGCTATCCGAATCCCT TGAAAACGAAAATAAACTTTTATTAAAACAACTCCAATTGGTTCTACAAGAATTTGAAAAAA TATATACCTATAATCAAGCATTAGAAGCAAAGCTAGAAAAAGATAAGCAAACAACATCAATA ACAGATTTATATAATGAAGTCGCTAAAAGTGATTTAGGGTTAGTCAAAGAAACCAACAGCGC AAATCCATTAGTCAGTATTATCATGACATCTCACAATACAGCGCAATTTATCGAAGCTTCTA TTAAT CA TATTGTTACAAACATATAAAAACATAGAAATTATTATTGTAGATG TGATAGC TCGGATAATACATTTGAAATTGCCTCGAGAATAGCGAATACAACAAGCAAAGTCAGAGTATT TAGATTAAATTCAAACCTAGGAACTTACTTTGCGAAAAATACAGGCATATTAAAATCTAAAG GTGACATTATTTTCTTTCAAGATAGTGATGATGTATGTCATCATGAAAGAATAGAAAGATGT GTAAATATATTATTAGCTAATAAAGAAACTATTGCTGTTCGTTGTGCATACTCAAGACTAGC ACCAGAAACACAACATATCATTAAAGTCAATAATATGGATTATAGATTAGGTTTTATAACCT TGGGTATGCACAGAAAAGTATTTCAAGAAATTGGTTTCTTCAATTGTACGACTAAAGGCTCA GATG TGAGT TTTTCATAG ATTGCGAAATATTATGGAAAAGAAAAAATAAAAAATTTACT CTTGCCGTTATACTACAACACAATGAGAGAAAACTCTTTATTTACTGATATGGTTGAATGGA TAGACAATCATAACATAATACAGAAAATGTCTGATACCAGACAACATTATGCAACCCTGTTT CAAGCGATGCATAACGAAACAGCCTCACATGATTTCAAAAATCTTTTTCAATTCCCTCGTAT TTACGATGCCTTACCAGTACCACAAGAAATGAGTAAGTTGTCCAATCCTAAGATTCCTGTTT ATATCAATATTTGTTCTATTCCCTCAAGAATAGCGCAATTACAACGTATTATCGGCATACTA AAAAATCAATGTGATCATTTTCATATTTATCTTGATGGCTATGTAGAAATCCCTGACTTCAT AAAAAATTTAGGTAATAAAGCAACCGTTGTTCATTGCAAAGATAAAGATAACTCCATTAGAG ATAATGGCAAATTCATTTTACTGGAAGAGTTGATTGAAAAAAATCAAGATGGATATTATATA ACCTGTGATGATGACATTATCTATCC AGCGATT C TCAATACGATGATCAAAAAGCTGAA TGAATACGATGATAAAGCGGTTATTGGTTTACACGGCATTCTCTTTCCAAGTAGAATGACCA AATATTTTTCGGCGGATAGACTGGTATATAGCTTCTATAAACCTCTGGAAAAAGACAAAGCG GTCAATGTATTAGGTACAGGAACTGTTAGCTTTAGAGTCAGTCTCTTTAATCAATTTTCTCT TTCTGACTTTACCCATTCAGGCATGGCTGATATCTATTTCTCTCTCTTGTGTAAGAAAAATA ATATTCTTCAGATTTGTATTTCAAGACCAGCAAACTGGCTAACGGAAGATAATAGAGACAGC GAAACACTCTATCATCAATATCGAGACAATGATG GCAAC AACTCAGCTGATCATGGAAAA CGGTCCATGGGGATATTCAAGTATTTATCCATTAGTCAAAAATCATCCTAAATTTACTGACC TTATCCCCTGTTTACCTTTTTATTTTTTATAA Protein sequence of His6-PmHS2 (Note: Italic sections of the sequences are from pET15b vector and primer. The sequences for His6-tag are underlined)
MGSSHHHHHHSSGL yPJ GSJiMKGKKEMTQKQMTKNPPQHEKENELNTFQNKI DSLKTTLNKD IISQQTLLAKQDSKHPLSESLENENKLLLKQLQLVLQEFEKIYTYNQALEAKLEKDKQTTSI TDLYNEVAKSDLGLVKETNSANPLVSI IMTSHNTAQFIEASINSLLLQTYKNIEIIIVDDDS SDNTFEIASRIANTTSKVRVFRLNSNLGTYFAKNTGILKSKGDI IFFQDSDDVCHHERIERC V ILLANKETIAVRCAYSRLAPETQHI IKVNN DYRLGFITLGMHRKVFQEI GFFNCTTKGS DDEFFHRIAKYYGKEKIKNLLLPLYYNTMRENSLFTDMVE IDNHNIIQ MSDTRQHYATLF QAMHNETASHDFKNLFQFPRI YDALPVPQEMSKLSNPKI PVYINICS IPS I AQLQRI IGIL KNQCDHFHI YLDGYVEIPDFIKNLGNKATVVHCKDKDNSIRDNGKFILLEELIEKNQDGYYI TCDDDI I YPSDYINTMIKKLNEYDDKAVIGLHGILFPSRMTKYFSADRLVYSFYKPLEKDKA VNVLGTGTVSFRVSLFNQFSLSDFTHSGMADI YFSLLCKKNNILQICISRPANWLTEDNRDS ETLYHQYRDNDEQQTQLIMENGPWGYSSI YPLVKNHPKFTDLIPCLPFYFL
DNA sequence of MBP-KfiA-His6 (Note: Italic sections of the sequences are from pMAL- c4X vector and primer. The sequences for His6-tag are underlined)
AACCTCGGGATCGAGGGAAGGATTTCAGAATTCATGATTGTTGCAAATA GAGCAGCTATCC TCCGCGTAAAAAAGAACTGGTTCATAGCATTCAGAGCCTGCATGCACAGGTGGATAAAATTA ATCTGTGCCT GAAT GAA T TGAAGAAAT T C CG GAAGAAC T GG AT GGCTTTAG CAAACT G AAT CCGGTTATTCCGGATAAAGATTATAAAGATGTGGGCAAATTTATTTTTCCGTGCGCCAAAAA TGATATGATTGTTCTGACCGATGATGATATTATTTATCCGCCAGATTATGTGGAAAAAATGC TGAATTTTTATAATAGCTTTGCCATTTTTAATTGCATTGTGGGTATTCATGGCTGCATTTAT ATTGATGCCTTTGATGGTGATCAGAGCAAACGTAAAGTGTTTAGCTTTACCCAGGGTCTGCT GCGTCCGCGTGTTGTTAATCAGCTGGGCACCGGCACCGTTTTTCTGAAAGCAGATCAGCTGC CGAGCCTGAAATATATGGATGGTAGCCAGCGTTTTGTGGATGTTCGTTTTAGCCGTTATATG CTGGAAAATGAAATTGGCATGATTTGTGTTCCGCGTGAAAAAAATTGGCTGCGTGAAGTTAG CAGCGGTAGCATGGAAGGTCTGTGGAATACCTTTACCAAAAAATGGCCTCTGGATATCATTA AAGAAACCCAGGCAATTGCCGGTTATAGTAAACTGAATCTGGAACTGGTGTATAATGTGGAA GGT C AC C AC C AC C AC C ACC AC T AA
Protein sequence of MBP-KfiA-His6 (Note: Italic sections of the sequences are from pMAL-c4X vector and primer. The sequences for His6-tag are underlined)
WLGI£GKISEPMIVANMSSYPPRKKELVHSIQSLHAQVDKINLCLNEFEEIPEELDGFSKLN PVIPDKDYKDVGKFI FPCAKNDMI VLTDDDI I YPPDYVEKMLNFYNS FAI FNCIVGIHGCI Y I DAFDGDQSKRKVFSFTQGLLRPRVVNQLGTGTVFLKADQLPSLKYMDGSQRFVDVRFSRYM LENEIGMICVPREKN LREVSSGSMEGLWNTFTKKWPLDI IKETQAIAGYSKLNLELVYNVE GHHHHHH
[0247] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Claims

WHAT IS CLAIMED IS :
1. A method of synthesizing a UDP-sugar, the method comprising forming a reaction mixture comprising a first sugar, a nucleotide-sugar pyrophosphorylase, and a first enzyme selected from the group consisting of a kinase and a dehydrogenase, under conditions sufficient to form the UDP-sugar.
2. The method of claim 1 , wherein the first sugar is selected from the group consisting of substituted or unsubstituted glucose (Glc), substituted or unsubstituted glucose- 1 -phosphate (Glc- l -P), substituted or unsubstituted glucuronic acid (GlcA), substituted or unsubstituted glucuronic acid- 1 -phosphate (GlcA-l-P),, substituted or unsubstituted iduronic acid (IdoA), substituted or unsubstituted iduronic acid- 1 -phosphate (IdoA- l -P), substituted or unsubstituted -acetylglucosamine (GlcNAc), substituted or unsubstituted N-acetylglucosamine- l -phosphate (GlcNAc- 1 -P), substituted or unsubstituted glucosamine (GlcNhb), substituted or unsubstituted glucosamine- 1 -phosphate (GlcNPh- l -P), substituted or unsubstituted galactose (Gal), substituted or unsubstituted galactose- 1 - phosphate (Gal- 1 -P), substituted or unsubstituted galacturonic acid (GalA), substituted or unsubstituted galacturonic acid- 1 -phosphate (GalA- 1 -P), substituted or unsubstituted N- acetylgalactosamine (GalNAc), substituted or unsubstituted N-acetylgalactosamine-1 - phosphate (GalNAc- 1 -P), substituted or unsubstituted galactosamine (GalNH2), substituted or unsubstituted galactosamine- 1 -phosphate (GalNH2-l -P), substituted or unsubstituted mannose (Man), substituted or unsubstituted mannose- 1 -phosphate (Man- 1 -P), and substituted or unsubstituted N-acetylmannosamine (ManNAc), substituted or unsubstituted N- acetylmannosamine-1 -phosphate (ManNAc- 1 -P), substituted or unsubstituted mannosamine (ManN¾), substituted or unsubstituted mannosamine- 1 -phosphate (ManN¾- 1 -P),.
3. The method of claim 1 , wherein the kinase is selected from the group consisting of an N-acetylhexosamine 1 -kinase (NahK), a galactokinase (GalK), and a glucuronokinase (GlcAK).
4. The method of claim 1 , wherein the dehydrogenase is UDP-glucose dehydrogenase (Ugd).
5. The method of claim 4, wherein the dehydrogenase is PmUgd.
6. The method of claim 1, wherein the nucleotide-sugar
pyrophosphorylase is selected from the group consisting of a glucosamine uridyltransferase (GlmU), a Glc- l -P uridylyltransferase (GalU), and a UDP-sugar pyrophosphorylase (USP).
7. The method of claim 1 , wherein the nucleotide-sugar
pyrophosphorylase is selected from the group consisting of AGX1 , EcGlmU, EcGalU, PmGlmU, and BLUSP.
8. The method of claim 1 , wherein the UDP-sugar is selected from the group consisting of substituted or unsubstituted UDP-Glc, substituted or unsubstituted UDP- GlcA, substituted or unsubstituted UDP-IdoA, , substituted or unsubstituted UDP-GalA, substituted or unsubstituted UDP-GlcNAc, substituted or unsubstituted UDP-GlcNH2, substituted or unsubstituted UDP-Gal, substituted or unsubstituted UDP-GalNAc, substituted or unsubstituted UDP-GalNH2, substituted or unsubstituted UDP-Man, and substituted or unsubstituted UDP-ManNAc, substituted or unsubstituted UDP-ManNH2,.
9. The method of claim 1, wherein the UDP-sugar is substituted with at least one member selected from the group consisting of an azide, an amine, an N- trifluoroacetyl group, an N-acyl group, an O-sulfate, and an N-sulfate.
10. The method of claim 1 , wherein the reaction mixture further comprises a pyrophosphatase.
1 1 . The method of claim 10, wherein the pyrophosphatase is PmPpA.
12. The method of claim 1 , wherein the the reaction mixture further comprises at least one member selected from the group consisting of UTP, ATP, Mn2+, Co2+, Ca2+, and Mg2+.
13. A method of preparing an oligosaccharide, the method comprising: forming a first reaction mixture comprising a first sugar, an acceptor sugar, a
glycosyltransferase, a nucleotide-sugar pyrophosphorylase, and an enzyme selected from a kinase and a dehydrogenase,
wherein the first sugar is selected from the group consisting of a substituted or
unsubstituted N-acetylglucosamine (GlcNAc), a susbstituted or unsubstituted glucosamine (GlcNI¾), a substituted or unsubstituted glucuronic acid (GlcA), a substituted or unsubstituted iduronic acid (IdoA), and a substituted or unsubstituted glucose- 1 -phosphate (Glc- 1 -P),
wherein the acceptor sugar comprises at least one member selected from the group consisting of a substituted or unsubstituted N-acetylglucosamine (Glc Ac), a substituted or unsubstituted glucosamine (GlcNHb), a substituted or unsubstituted glucuronic acid (GlcA), and a substituted or unsubstituted iduronic acid (IdoA),
under conditions sufficent to convert the first sugar to a UDP-sugar, and sufficient to couple the sugar in the UDP-sugar to the acceptor sugar such that when the first sugar is substituted or unsubstituted GlcNAc or GlcNE , the sugar in the UDP-sugar is coupled to substituted or unsubstituted GlcA or substituted or ' unsubstituted IdoA of the acceptor sugar, and when the first sugar is selected from the group consisting of substituted or unsubstituted Glc- 1 -P, substituted or unsubstituted GlcA, or substituted or unsubstituted IdoA, the sugar in the UDP-sugar is coupled to substituted or unsubstituted GlcNHb or substituted or unsubstituted GlcNAc of the acceptor sugar,
thereby preparing the oligosaccharide.
14. The method of claim 13, wherein the acceptor sugar is selected from the group consisting of a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide, a hexasaccharide, a heptasaccharide, an octasaccharide, and a
nonasaccharide.
15. The method of claim 13, wherein the glycosyltransferase is selected from the group consisting of PmHS 1 , PmHS2, KfiC, and KfiA.
16. The method of claim 13, wherein the nucleotide-sugar pyrophosphorylase is selected from the group consisting of a glucosam ine uridyltransferase (GlmU), a UDP-glucose pyrophosphorylase (GalU), and a UDP-sugar pyrophosphorylase (USP).
17. The method of claim 13, wherein the nucleotide-sugar pyrophosphorylase is selected from the group consisting of AGX l , EcGlmU, EcGalU, PmGlmU, and BLUSP.
1 8. The method of claim 13, wherein the kinase is selected from the group consisting of an N-acetylhexosamine 1 -kinase (NahK), a galactokinase (GalK), and a glucuronokinase (GlcAK).
19. The method of claim 13, wherein the dehydrogenase is UDP-glucose dehydrogenase (Ugd).
20. The method of claim 1 9, wherein the Ugd is PmUgd.
21. The method of claim 1 3, wherein the UDP-sugar is selected from the group consisting of substituted or unsubstituted UDP-GlcNAc, substituted or unsubstituted UDP-GlcNH2, substituted or unsubstituted UDP-GIc, substituted or unsubstituted UDP-GlcA, and substituted or unsubtitued UDP-IdoA.
22. The method of claim 21 , wherein the UDP-sugar is substituted with at least one member selected from the group consisting of an azide, an amine, an N- trifluoroacetyl group, an 7V-acyl group, an O-sulfate group, and an N-sulfate group.
23. The method of claim 13, wherein the reaction mixture further comprises a pyrophosphatase.
24. The method of claim 23, wherein the pyrophosphatase is PmPpA.
25. The method of claim 1 3, wherein the reaction mixture further comprises at least one member selected from the group consisting of UTP, ATP, Mn2+, Co2+, Ca2+, and Mg2+.
26. The method of claim 13, wherein the oligosaccharide is selected from the group consisting of:
GlcNAc-GlcA,
GlcA-GlcNAc-GlcA,
GlcNAc-GlcA-GlcNAc-GlcA,
GlcA-GlcNAc-GlcA-GlcNAc-GlcA,
GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA,
GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA,
GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GIcA, GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA,
GlcA-GlcNAc,
GlcNAc-GlcA-GlcNAc,
GlcA-GicNAc-GlcA-GlcNAc,
GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc,
GlcA-GlcN Ac-GlcA-GlcN Ac-GlcA-GlcNAc,
GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc,
GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc,
GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc, and
GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc.
27. The method of claim 26, wherein each GlcA and GlcNAc are optionally independently substituted with a member selected from the group consisting of an azide, an amine, an TV-trifluoroacetyl group, an N-acyl group, an O-sulfate group, and an N- sulfate.
28. The method of claim 13, wherein the method is repeated with a second sugar in place of the first sugar and the oligosaccharide in place of the acceptor sugar.
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