[9] Chemoenzymatic Synthesis of Glycan Libraries By OLA BLIXT and NAHID RAZI Abstract The expanding interest for carbohydrates and glycoconjugates in cell communication has led to an increased demand of these structures for biological studies. Complicated chemical strategies in glycan synthesis are now more frequently replaced by regio‐ and stereo‐specific enzymes. The exploration of microbial resources and improved production of mammali- an enzymes have established glycosyltransferases as an efficient comple- mentary tool for glycan synthesis. In this chapter, we demonstrate the feasibility of preparative enzymatic synthesis of different categories of glycans, such as blood group and tumor‐associated poly‐N‐acetyllactosa- mines antigens, ganglio‐oligosaccharides, N‐ and O‐glycans. The enzymatic approach has generated over 100 novel oligosaccharides in amounts allow- ing milligram to gram distribution to many researchers in the field. Our diverse library has also formed the foundation for the successful develop- ments of both the noncovalent enzyme‐linked immunosorbent assay glycan array and the covalent printed glycan microarray. Introduction Carbohydrate groups of glycoproteins and glycolipids exhibit a great deal of structural diversity and are major components of the outer surface of animal cells (Sharon and Lis, 1993). The lack of availability and afford- ability of appropriate oligosaccharides has always been a major limitation in the field. To overcome these shortcomings, the Consortium for Functional Glycomics’ (CFG) initiative was launched to generate an efficient and rapid production of essential oligosaccharides in sufficient quantities to build up a compound library for distribution, as well as for building glycan microarray platforms. To date, the library and the developed glycan microarrays (see Chapter 18) have been a tremendous success story and a widely used resource for advancing the research in the field of glycobiology. One of the strategic key points in the formation of a diverse functional glycan library is to build glycan structures that can readily be adapted to various needs—for example, coupling to proteins, solid‐phase or used as is. A short neutral and versatile linker (2‐azidoethyl) or a protected amino acid (F‐moc) was introduced chemically to the penultimate monosaccharide, followed by chemical or enzymatic elongation. The linker and the amino [9] chemoenzymatic synthesis of glycan libraries 137 METHODS IN ENZYMOLOGY, VOL. 415 0076-6879/06 $35.00 Copyright 2006, Elsevier Inc. All rights reserved. DOI: 10.1016/S0076-6879(06)15009-0
Transcript
[9] chemoenzymatic synthesis of glycan libraries 137
[9] Chemoenzymatic Synthesis of Glycan Libraries
By OLA BLIXT and NAHID RAZI
Abstract
The expanding interest for carbohydrates and glycoconjugates in cellcommunication has led to an increased demand of these structures forbiological studies. Complicated chemical strategies in glycan synthesis arenow more frequently replaced by regio‐ and stereo‐specific enzymes. Theexploration of microbial resources and improved production of mammali-an enzymes have established glycosyltransferases as an efficient comple-mentary tool for glycan synthesis. In this chapter, we demonstrate thefeasibility of preparative enzymatic synthesis of different categories ofglycans, such as blood group and tumor‐associated poly‐N‐acetyllactosa-mines antigens, ganglio‐oligosaccharides, N‐ andO‐glycans. The enzymaticapproach has generated over 100 novel oligosaccharides in amounts allow-ing milligram to gram distribution to many researchers in the field. Ourdiverse library has also formed the foundation for the successful develop-ments of both the noncovalent enzyme‐linked immunosorbent assay glycanarray and the covalent printed glycan microarray.
Introduction
Carbohydrate groups of glycoproteins and glycolipids exhibit a greatdeal of structural diversity and are major components of the outer surfaceof animal cells (Sharon and Lis, 1993). The lack of availability and afford-ability of appropriate oligosaccharides has always been amajor limitation inthe field. To overcome these shortcomings, the Consortium for FunctionalGlycomics’ (CFG) initiative was launched to generate an efficient and rapidproduction of essential oligosaccharides in sufficient quantities to build up acompound library for distribution, as well as for building glycan microarrayplatforms. To date, the library and the developed glycan microarrays (seeChapter 18 ) ha ve been a tremendou s succes s story and a widely usedresource for advancing the research in the field of glycobiology.
One of the strategic key points in the formation of a diverse functionalglycan library is to build glycan structures that can readily be adapted tovarious needs—for example, coupling to proteins, solid‐phase or used as is.A short neutral and versatile linker (2‐azidoethyl) or a protected amino acid(F‐moc) was introduced chemically to the penultimate monosaccharide,followed by chemical or enzymatic elongation. The linker and the amino
METHODS IN ENZYMOLOGY, VOL. 415 0076-6879/06 $35.00Copyright 2006, Elsevier Inc. All rights reserved. DOI: 10.1016/S0076-6879(06)15009-0
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acid moiety can readily be converted to an amine functionality for furtherderivatization (Blixt et al., 2003, 2004). Chemical synthesis of oligosacchar-ides is now very sophisticated, and it is possible to synthesize essentially anyglycosidic linkage, although with differing degrees of difficulty. Alternativesto traditional chemical solution‐phase synthesis (Garegg, 2004; Lee et al.,2004; Nicolaou and Mitchell, 2001), such as solid‐phase synthesis and one‐pot reactivity‐based glycosylations (Bartolozzi and Seeberger, 2001; Tanakaet al., 2002; Ye and Wong, 2000), have the advantage of avoiding intermedi-ate isolation and several purification steps. Consequently, it dramaticallyshortens and simplifies chemical synthesis. Despite such improvements,chemical synthesis is still hampered by time‐consuming multiple protec-tion/de‐protection building‐block strategies and complex isomeric reactionmixtures (Boons and Demchenko, 2000).
A second powerful and complementary approach to chemical synthesis isto use regio‐ and stereo‐specific glycosylating enzymes, glycosyltransferases(Auge and Crout, 1997; Koeller and Wong, 2001). Glycosyltransferasesgenerate glycosidic linkages in one‐step reactions between an unprotectedacceptor and a sugar donor nucleotide; thus, multistep protection groupstrategies are not required (Hanson et al., 2004). With new advances inmolecular biology, recombinant enzymes and accessory enzymes for sugar‐nucleotide regeneration are nowbeing readily expressed in largequantities forsynthesis of complex oligosaccharides (Blixt and Razi, 2004; Endo et al., 1999;Johnson, 1999).Although enzymes have relatively strict substrate specificities,manyof themoffer substantial acceptor substrateflexibility for synthesis. Theycan be applied directly onto a simple monosaccharide acceptor, followed bysubsequent elongation to amore complex structure, or they can be introducedafter de‐protection of chemically synthesized intermediates. For example,sialic acid, a commonmonosaccharide that frequently terminates oligosaccha-ride sequences on various glycoproteins and glycolipids, is notoriously difficultto apply in chemical glycosylation but can be easily introduced enzymaticallyas a final step by a sialyltransferase (Blixt et al., 2002, 2005).
In this chapter, we illustrate our enzyme production and chemoenzy-matic approach for large‐scale synthesis of terminal glycans commonlyfound on glycoproteins and glycolipids. Many are blood group related,tumor associated, and specific for the C‐type lectins, galectins, and Siglecssubgroup glycan‐binding protein families.
Materials
Enzyme Production Materials
Escherichia coli strain AD202 (CGSG 7297), Aspergillus niger DVK1,Sf‐9 and HiFive insect cells (Invitrogen, CA), and CHO cells were used
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in bacterial, fungal, baculoviral, and mammalian expression systems,respectively. Yeast Extract Trypton, 2xYT (Difco, NJ), Isopropyl‐1‐thio‐�‐D‐galactopyranoside (Calbiochem‐Merck, Germany), HyQ SFX(Hyclone, Utah), DMEM (Invitrogen, CA), FuGene (Roche, IN), Effec-tene (Qiagen, CA), uridine 50‐diphospho‐N‐acetylglucosamine (UDP‐GlcNAc), and guanidine‐5‐diphospho‐fucose (GDP‐Fuc) were a gift fromTokyo Research Laboratories, KyowaHakko Kogyo Co. Ltd. The radioac-tive nucleotide sugars were diluted with unlabeled nucleotide sugars toobtain the desired specific radioactivity. GDP‐[14C]Fuc and CMP‐[14C]NeuAc came from GEHealthcare—formerly Amersham Biosciences (LittleChalfont, UK)—and theUDP‐[3H] sugar nucleotides from PerkinElmer Lifeand Analytical Sciences (Shelton, CT). All other chemicals, supplements,and resins were of highest purity and purchased from Sigma‐Aldrich(St. Louis, MO). Large‐scale enzyme production (100 l, Braun Fermentor),Microfluidizer (Microfluidics,ModelM‐110S), andTangential FlowFiltration(Pall Corporation) were used in the extraction and concentration of theenzyme‐containing media.
Methods
Enzyme Expression Methods
Recombinant enzymes were produced in different expression systemsto be used in carbohydrate synthesis. All the bacterial enzymes and a ratUDP Gal/GalNAc epimerase were expressed in E. coli strain AD202.Other enzymes that require posttranslational modifications were producedin A. niger (rat ST3Gal III, human FUT V and FUT VII), insect cells(human ST6GalI, pig ST3GalI, chicken ST6GalNAcI, human FUTs II, III,IV, and VI), and the CHO cells (Core 2 �1,6GlcNAcT). The procedurefor each cloning and expression has been fully described previously(Blixt et al., 2001, 2002, 2005; Fukuta et al., 1997; Schwientek et al., 2000;Uchimura et al., 1998; Vasiliu et al., 2006), as well as being incorporatedinto the CFG database (www.functionalglycomics.org).
General Procedure for the Bacterial Expression
E. coli strain AD202 was used in bacterial expression either in shakerflasks or in a 100‐l Fermentor. Bacteria was cultured in 2�YT/ampicillin(150 �g/ml) and induced with isopropyl‐thiogalactopyranoside (1 �M) atA600 ¼ 0.3–0.6, depending on each enzyme. Cells were harvested byspinning at 5000 g for 30 min after completing the incubation. Pellet wasweighed and resuspended in an appropriate buffer (see assay buffers inTable I) in a 1 g/ml ratio of cell weight to the buffer. The cell paste was
w �2‐6SiaT (hST6GalI) Human Hi Five 14 U/L Lactose Caco/6.5 CMP‐14CSia NaCl Dowex/PO4 SP ion exch.
x �2‐6SiaT(chST6GalNAcI) Chicken Hi Five 20 U/L Asialofetuin Caco/7.5 CMP‐14CSia NaCl Seph. G‐50 CDP affinity
y UDP‐GlcNAc/GalNac‐4‐E Rat AD202 1100 U/L UDPGNAc/LN Hepes/7.5 UDP‐3HGlc MnCl2/NAD Dowex/Cl� Lysate
z UDP Glc/GalE (GalE) P. aeruginosa AD202 205 U/L UDPGlc/lactose Hepes/7.5 UDP‐3HGlc MnCl2/NAD Dowex/Cl� Lysate
a sp; 2‐azidocthyl.bBn; benzyl.cme; methyl.
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aliquoted and stored at �20�. The enzymes remained stable in cell pastefor at least 1 year.
General Procedure for the Baculoviral Expression System
The baculoviral expression was performed in Spodoptera frugip, Sf‐9,and HiFive cells at 27� in serum‐free media, HYQ SFX‐INSECT supple-mented with 10% antibiotic/antimycotic. Insect cell growth and mainte-nances require special care (see Notes). Viral amplification was generallyperformed in Sf‐9 cells with a multiplicity of infection, MOI ¼ 1 and up to48 hours’ incubation. Enzyme expression was performed in either Sf‐9 orHiFive cells with the MOI about 3 to 10, empirically determined for eachenzyme. The production was regularly monitored by the enzyme assayfor up to 7 to 8 days. When the activity started to decrease, the mediawas collected by centrifugation at 5000g for 30 minutes. The media wasconcentrated and was either directly used in synthesis or was furtherpurified by different methods.
General Procedure for the Fungus Expression System
Enzyme expression in A. niger DVK1 was performed by conventionalfungal protein expression in baffled Fernbach Flask (2.8 L) in 1 l growthbroth of Sheftone media supplemented by a series of nutritional additives(see ‘‘protocols’’ in www.functionalglycomics.org). An initial pre‐culture(inoculum) was prepared by adding 300 �lA. niger spore stock into a 100‐mlmedia in a 500‐ml shaker flask and incubated for 48 h, shaking at 180 rpmat 37�. Nine hundred milliliters of Sheftone broth was inoculated by 100 mlof the prepared inoculum and the incubation continued at 32�, shaking at200 rpm, and the pH was monitored and adjusted to 6.00 twice daily. After48 h of incubation, the expression was monitored by the enzyme assay untilthe activity remained constant for 12 h. At this stage, the product wascollected by spinning at 5000g for 30min, and the supernatant was aliquotedand stored at�20�. The enzyme remained stable in this condition for at least1 year.
General Procedure for the Mammalian Expression System
Transient transfection of CHO cells was performed by Effectene accord-ing to the manufacturer’s instruction. Stable transfection of CHO‐K1 cellswas performed by FuGene 6, according to the instruction, using the con-ventional mammalian single‐colony selection by filter paper. Transfectedcolonies were selected using Geneticin (G418).
Enzyme Extraction, Concentration, and Purification
Different methods of extraction, concentration, and purification wereused based on the expression systems and the enzymes specificities andstability. Except for the �‐1,4 GalNAcT mutant that was extracted from theinclusion bodies (Ramakrishnan and Qasba, 2002), all the bacterial enzymeswere extracted by sonication or the microfluidizer method (see later).
Bacterial Enzyme Extraction by Microfluidizer
The bacterial enzymes were extracted from the cell paste by breakingthe cells using microfluidizer with 1/1 volume of the buffer to the resus-pended cell paste. The released enzyme was used either as a cell lysate orwas partially purified on different columns. The cell lysate remained stablefor at least 1 year in 20�.
Precipitation and Purification of Fungal Expressing Enzymes
The A. niger–expressed recombinant enzymes were separated from theharvest supernatant by filtration and precipitation with ammonium sulfate,followed by sulfo‐propyl (SP)‐sepharose ion‐exchange chromatography.The precipitation is performed in two steps. First, 20% (114 g/L) NH4SO2
wasmixed for 30min at room temperature or over night at 4�. The pellet wascollected and the supernatant was further precipitated with 60% NH4SO2
(276 g/l). Pellets obtained from both precipitants were mixed and resus-pended in 20 mM Mes (pH, 6.0). The suspension was diafiltered with thesame buffer to adapt the conductivity of the buffer. The suspension neededto be passed a few times through a 5‐�m filter before the final filtration on a1.0‐�m filter. The filtered product was further purified on a SP‐sepharoseion‐exchange column, equilibrated with 20mMMes, 50mMNaCl (pH, 6.0),and eluted with 1 M NaCl.
Enzyme Concentration
The media from the baculovirus expression was concentrated byTangential Flow Filtration. The concentrated fucosyltransferases weredirectly used in glycan synthesis and sialyltransferases (chicken ST6Gal-NAc‐I and pig ST3Gal‐I) were further purified on a cytodine‐50‐diphosphate‐Sepharose after concentrating the cultured media.
Enzyme Purification
Different methods of chromatography were applied for purification ofthe enzymes. The process was selected based on the enzyme specificity and
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stability in crude mixture. The required method for each enzyme is shownin Table I.
Enzyme Assays
The enzyme assays were generally performed in 100 �l total volume ofthe appropriate buffer, 3H or 14C sugar nucleotide donor substrate with theappropriate specific activities, metal ions, acceptor substrate, and the desiredenzyme (see Table I). The incubation continued at 37� for 60 min, and thereactionwas stopped by adding 700�l of coldwater. The reactionmixturewasloaded on a 2‐ml Dowex ion exchange, a 10 � 0.5‐cm Sepharose G‐50size exclusion, or C18 reverse phase, depending on the acceptor substrateand the enzyme reaction (see Table I). The radioactive incorporation to thesubstrate was detected by scintillation counter.
Chemical Synthesis of Functionalized Acceptor Substrates
Each synthesized compound is linked to a short neutral flexible spacer2‐azidoethyl (sp1 ¼ OCH2CH2N3) or with the �‐threonine aglycon (Blixtet al., 2002), which enables broad diversification such as attachment toproteins, solid supports (e.g., affinity matrices, polystyrene), and biotin orother functional groups (Blixt and Razi, 2004; Blixt et al., 2005; Eklind et al.,1996). These spacered precursor glycans were subsequently elongated usingrecombinant glycosyltransferases as follows.
General Isolation and Purification of Synthesized Glycans
Enzymatic synthesis of carbohydrates amounts measured in milligramsis generally a straightforward procedure in terms of isolation and purifica-tion of the final structure. Ion exchange and size exclusion chromatographyare the common procedures for product isolation. However, serious purifi-cation problems can be encountered when scaling up to multi‐gram reac-tions. Additives such as nucleotide sugars, buffer salts, and crude enzymesare among the factors that interfere with efficient purification with theconventional chromatographic techniques used on milligram scale. In gen-eral, the isolation strategy used depends on the scale and the compounds tobe prepared.
Dowex Resin and Size Exclusion Chromatography
Typically, the enzymatic reaction mixture (1 to 20 g solid) was centri-fuged and then loaded (5 to 10 ml) onto a column of Sephadex G15 (5 to1.8 cm � 170 cm) equilibrated and eluted with 5% nBuOH in water.
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Appropriate fractions were collected, passed through a Dowex (formate,1 � 3 cm) column, and lyophilized. The residue was further purified bySephadex G15 as described, and appropriate fractions were collected togive 0.5 to 5 g of oligosaccharide in about 70% to 90% yields.
Peracetylation and Extraction
The enzymatic mixture was lyophilized, and solids were suspendedand coevaporated three times in pyridine (10 g solids/200 ml), followed byaddition of pyridine/acetic anhydride mixture (2:1, 10 g solid/300 ml). Themixture was agitated for 24 hrs, at which point TLC (Tol:EtOAc, 1:1 byvolume) indicated complete acetylation of the oligosaccharide. The reactionwas evaporated and coevaporated two times with toluene. The residueswere dissolved in dichloromethane (500 ml) then washed one time withaqueous sulfuric acid (1 M), two times with aqueous sodium bicarbonate(1 M), and one time with ice‐cold water. The pooled organic layers weredried over anhydrous magnesium sulphate, then filtered, and the filtrate wasevaporated to dryness. The residual syrup was dissolved in methanol(200 ml) followed by the addition of sodium methoxide (20 ml, 0.5 M).The mixture was left overnight at room temperature, neutralized with amethanol‐washed cat‐ion exchange resin Dowex Hþ, filtered, and evapo-rated to dryness. The deacetylated white‐yellowish, residue was taken upinto water and loaded onto a column of Sephadex G15 (5 � 160 cm)equilibrated and eluted with 5% BuOH. Fractions containing product werecollected then lyophilized to give 3 to 6 g of oligosaccharide in typically 60%to 80% yields.
High‐performance liquid chromatography (HPLC) was carried out onan Alltech system equipped with an HPLC pump model 627, an Alltechautomated gradient controller, and a Rheodyne injector (3725i‐038). Theoligosaccharide sampleswere run at 5ml/minona 5‐�mAltimaamino column(250 � 10 mm). Up to 500 mg/5 ml samples were manually injected, with aninjection loop of 10 ml. Isocratic or a gradient of acetonitrile and deionizedwater (with or without 0.1% trifluoroacetic acid [TFA]) varying from 95:5 to70:30 was used as mobile phase at room temperature. Standard compoundswere also run in similar conditions. Chromatographic separations were moni-tored by an evaporative light‐scattering detector (Alltech ELSD, 2000). Thechromatographic systemwas connected to a personal computer for data acqui-sition and analysis using the scientific software EZChrom Elite. The elutionof the separated oligosaccharides was collected using a Bio‐Rad BiologicBioFrac fraction collector. Appropriate fractions were pulled and lyophilized.
[9] chemoenzymatic synthesis of glycan libraries 145
General Enzymatic Glycosylation
The general approach can be taken for most enzymatic reactions:Reaction mixtures containing acceptor substrates (20 to 50 mM) andcorresponding sugar‐nucleotide donor substrate (1 to 3 mole equivalents)were dissolved in enzyme specific buffer (see Table I for details). Reactionwas initiated by adding enzymes (typically 1 to 5 U/mmole acceptor) andslowly agitating at room temperature for 24 to 48 h. UDP‐Gal(NAc)‐40‐epimerase (Table I, entry y) (30 to 50 U/mmole donor) was added whenUDP‐Glc(NAc) donors were used. Depending on oligosaccharide product,the mixture was purified as described previously. Typical yields are 50% to90% with a purity of 90% to 95%.
Synthesis of Poly‐N‐Acetyllactosamine of Type 1 and Type 2 Series
Elongated type 1 (Gal�1–3GlcNAc‐) and type 2 (Gal�1–4GlcNAc‐)core structures, poly‐N‐acetyllactosamines (poly‐LacNAc), are known torepresent the backbone or branched epitopes on many N‐linked andO‐linked glycans—where they participate in intercellular signaling viabinding to various lectins, such as selectins or galectins (Leppaenen et al.,2002; Niemela et al., 1998), inflammation processes, and cancer (Hakomori,2001; Ujita et al., 1999). Type 1 chains are also present at the periphery oftype 2 chains (Gal�1–4GlcNAc‐), as well as in their sialylated and/orfucosylated forms. Chemical syntheses of poly‐LacNAc have been devel-oped using a variety of advanced synthetic strategies, yet the methodsalways involve tedious multiple protection/de‐protection steps (Aly et al.,2000; Buskas et al., 2005; Misra et al., 2001; Mong et al., 2003). A limitednumber of structures were synthesized in small amounts using glycosyl-transferases as an alternative approach to the chemical synthesis (Barstromet al., 2000; Bintein et al., 2003; Koeller and Wong, 2000; Zeng and Uzawa,2005). In Scheme 1 (Fig. 1) and in support of Table I, we describe severalenzymatic routes to synthesize blood group type 1 and type 2 oligosacchar-ides and derivatives thereof.
Notes
1. Elongation of type 1 and type 2 terminal sequences with �1–3GlcNAc‐T. Previous specificity studies of the �3GlcNAcT (Table I, entry a) reveal thatabout 7% to 8% of enzyme activity is found with type 2N‐acetyllactosamineand 4% to 5%with type 1 lacto‐N‐biose relative to lactose, respectively. Still,with increased reaction times and additional enzymes (10 to 20 U/moleacceptor), we have demonstrated that �3GlcNAcT is very useful forpreparative synthesis of various carbohydrate structures, including type2 poly‐LacNAc and type 1 poly‐LacNAc (see Fig. 1).
FIG. 1. SCHEME 1. Enzymatic synthesis of sialylated and fucosylated poly‐N‐acetyllactosamines. See Table I for enzyme conditions
(entries a–z).
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Ganglio‐Oligosaccharide Synthesis
Gangliosides are glycolipids that comprise a structurally diverse set ofsialylated molecules that are found in most cells but are particularly abun-dant in neuronal tissues. They have been found to act as receptors forgrowth factors, toxins, and viruses and to facilitate the attachment ofhuman melanoma and neuroblastoma cells. Specific gangliosides are alsopresent in early stages of human neural development and affect majorcellular processes, including proliferation, differentiation, survival, andapoptosis. They are also well‐known tumor‐associated antigens, and activeimmunization using gangliosides may suppress melanoma growth (Gagnonand Saragovi, 2002; Hakomori and Zhang, 1997; Kannagi et al., 2004;Ravindranath et al., 2000; Schnaar, 2000; Svennerholm, 2001). One of thecrucial steps to synthesize the gangliosides is the introduction of sialic acid.Our enzymatic approach has significantly reduced the complications andlabor for synthesis of these sialylated glycans from years to weeks ofexperimental work (Blixt et al., 2005). We summarize our chemoenzymaticapproach for synthesis of the ganglio‐oligosaccharide family in Scheme 2(Fig. 2) and Table I (Blixt et al., 2005).
Notes
1. �2‐8‐sialylation using cst‐II (Table I, entry u). With excess amountsof CMP‐Neu5Ac, the cst‐II demonstrates extensive �2‐8 multi‐sialylationactivities. Therefore, the synthesis of GD3‐, GT3‐, and tetra‐sialyllacto‐oligosaccharides required carefully controlled conditions using restrictedamounts of CMP‐Neu5Ac. 2‐azidoethyllactoside was elongated with Cst‐IIand 2.5 equivalents of CMP‐Neu5Ac to produce 2.4 g (34 %) of GD3 and
FIG. 2. SCHEME 2. Enzymatic synthesis of ganglio‐oligosaccharides. See Table I for enzyme
conditions (entries a–z).
148 carbohydrate synthesis and antibiotics [9]
0.39 g (6 %) of GT3 (see Fig. 2). GM3 was completely consumed andconverted toGD3 andGT3 using this molar ratio of CMP‐Neu5Ac donor. Byincreasing the molar ratio of CMP‐Neu5Ac to 4 equivalent, the reaction wasdriven to greater than an 80% conversion of the newly formed GD3 togenerate larger amounts of GT3 (1.0 g, 20%). Smaller amounts of the tetra‐sialic acid (Sia�2–8)3Sia�2–3lactose (0.25 g, 4%)werealso isolated, and tracesof higher sialylated fractions were also detected but not purified. Productswere subject to ion‐exchange chromatography and loaded onto a Dowex1 � 8‐400 formate ion exchange resin (30 � 2.5 cm). Compound GM3oligosaccharide and free Neu5Ac were in the void fractions, whereasGD3 oligosaccharide, GT3 oligosaccharide, and nucleotides absorbed tothe resin. The column was washed with water (500 ml) followed by elutionwith a gradient of aqueous sodium formate (20 to 80 mM [pH, 7.5], 2.5 l).Fractions containing separated compounds were lyophilized and furtherpurified by size‐exclusion chromatography to give pure material (>90%)per nuclear magnetic resonance (NMR) and mass spectrometry analysis.
2. �1‐3‐galactosylation using cgtB (Table I, entry f). Branched GalN-Ac�1‐3 oligosaccharides could be further elongated to the Gal�1‐3GalNAc�1‐4 branch by using the �1‐3GalT (cgtB), UDP‐glucose (2 equiv),and GalNAcE in high yields. Initiating the reaction at high concentrationsof acceptor substrates (>40 mM) is critical for enzyme activity. After48 hrs’ reaction at room temperature, the reaction was purified asdescribed in the General Isolation and Purification section.
O‐Glycans
The most common O‐linked carbohydrates are based on core structuresrepresented by the Tn‐(GalNAc�1‐1Thr/Ser) and T‐(Gal�1‐3GalNAc�1‐1Thr/Ser) antigens (Scheme 3; Fig. 3). Sialylated versions of theseO‐antigensare expressed at low levels by many normal tissues, but they become highlyexpressed in many types of human malignancies, including colon, breast,
FIG. 3. SCHEME 3. Synthesis of O‐glycans. See Table I for enzyme conditions (entries a–z).
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pancreas, ovary, stomach, and lung adenocarcinomas, as well asmyelogenousleukemias (Dabelsteen, 1996; Itzkowitz et al., 1989; Mori et al., 1995). On theother hand, O‐glycans based on the Core‐2 epitope (Gal�1‐3[GlcNAc�1‐6]GalNAc�1‐1Thr/Ser) are expressed in normal tissues. We have been synthe-sizing various compounds using the key enzymes chST6GalNAc‐I and Core‐2‐�1‐6GlcNAcTon acceptors containing the threonine aglycon (seeFig. 3 andTable I).
Notes
1. �2‐6‐sialylation using chST6GalNAc‐I (Table I, entry x). The enzy-matic activity was previously evaluated on a set of small acceptor molecules(Blixt et al., 2002), and it was found that an absolute requirement forenzymatic activity is that the anomeric position on GalNAc be �‐linked tothreonine. Thus,O‐linked sialosides terminating with a protected threoninecould successfully be synthesized ona gram‐scale reactions (seeFig. 3). Tobeable to attach these compounds to other functional groups, the N‐acetylprotecting group on threonine could be substituted with a biotin derivativebefore enzymatic extension with chST6GalNAc‐I.
Other Glycans
We have demonstrated efficient strategies for generatingN‐acetyllactos-amines, ganglio‐oligosaccharides, and O‐glycans. Our enzymatic approachhas also been used to expand the library with other glycans as well, such asthe globoside series, chemically and enzymatically sulfated glycans, and inthe modification of isolated N‐glycans. Due to space limitations, thesecategories are only briefly summarized later.
Globoside Glycans
The P blood group antigens are glycan structures displayed bymembrane‐associated glycosphingolipids present on red cells and on othertissues (Hellberg et al., 2002; Ziegler et al., 2004) . The P1 antigen is formedby the addition of a galactose in an �1,4‐linkage to the paragloboside by theP1 �1,4galactosyltransferase, forming the pentasaccharide Gal�1‐4Gal�1‐4GlcNAc�1‐3Gal�1‐4Glc. The physiological functions of the P bloodgroup antigens are not known, but these molecules have been implicatedin the pathophysiology of urinary tract infections and parvovirus infections(Yang et al., 1994). We have successfully generated various structures ofthe globoside series using the key enzymes �1,4galactosyltransferase‐GalE(Table I, entry g) and �1,3‐N‐acetylgalactosaminyltransferase (Table I,entry d).
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Sulfated Glycans
In addition to the glycan structural linkage diversity, sulfation alsopossesses a large amount of biological information. A number of proteogly-cans, glycoproteins, and glycolipids contain sulfated carbohydrates. Theirsulfate groups provide a negative charge and play a role in a specific molec-ular recognition process (Honke and Taniguchi, 2002).We have synthesizeda number of sulfated disaccharides of Gal�1‐3GlcANc, Gal�1‐4GlcNAc,and Gal�1‐4Glc at various positions and further performed enzymaticelongation with sialyltransferases and fucosyltransferases from Table I(to be published elsewhere). In addition, we have also begun expressingthe sulfotransferases KSGal6ST (Fukuta et al., 1997; Torii et al., 2000) andGlcNAc6ST (Uchimura et al., 1998) for sulfation of poly‐LacNAc.
N‐Glycans
Isolation and purification of natural glycans are complicated andtedious. Nevertheless, it is important to include them in existing and futureglycan libraries. Several recent reports demonstrate that isolated N‐glycanscan be efficiently modified with various glycosyltransferases (Kajiharaet al., 2005). We have also taken on this task to expand our library withmilligram quantities of isolated and enzymatically diversified N‐glycansusing the produced enzymes in Table I.
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[10] Glycoconjugate Vaccines AgainstHaemophilus influenzae Type b
By VIOLETA FERNANDEZ SANTANA, LUIS PENA ICART, MICHEL BEURRET,LOURDES COSTA, and VICENTE VEREZ BENCOMO
Overview
Glycoconjugation technology leads to the development of prophylacticvaccines against infectious diseases. Several vaccines were introducedstarting in the 1980s to fight Haemophilus influenzae type b. Their compo-sitions are very diverse, from those using the capsular polysaccharidealmost intact before the modification, through oligosaccharide fragmentsobtained by fragmentation of the capsular polysaccharide, to the mostrecent, which contain oligosaccharides prepared by chemical synthesis.We describe here some examples that illustrate the methods used in thedevelopment of such vaccines.
Glycoconjugates composed of a capsular polysaccharide or its oligosac-charide fragments covalently linked to a protein carrier have been shown tobe excellent tools as preventive vaccines against infectious diseases (Jenningsand Pon, 1996; Pozsgay, 2000). The first vaccine of this type was introduced for
METHODS IN ENZYMOLOGY, VOL. 415 0076-6879/06 $35.00Copyright 2006, Elsevier Inc. All rights reserved. DOI: 10.1016/S0076-6879(06)15010-7
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