Chemoenzymatic synthesis of GDP-L-fucose and the Lewis X derivatives

Wei Wanga, Tianshun Hua, Patrick A. Frantoma, Tianqing Zhenga, Brian Gerweb, David Soriano del Amoa, Sarah Garretb, Ronald D. Seidel IIIb, and Peng Wua,1

aDepartment of Biochemistry and bMacromolecular Therapeutics Development Facility, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461

Communicated by K. Barry Sharpless, The Scripps Research Institute, La Jolla, CA, July 24, 2009 (received for review April 21, 2009) Lewis X (Lex)-containing play important roles in numerous Ile-712, providing specificity for Lex over other - cellular processes. However, the absence of robust, facile, and containing glycans (10). cost-effective methods for the synthesis of Lex and its structurally Despite the pathophysiological significance of the Lex - related analogs has severely hampered the elucidation of the containing glycans, progress toward delineating these glycans’ spe- specific functions of these glycan epitopes. Here we demonstrate cific functions has been hampered by their complexity and heter- x that chemically defined guanidine 5؅-diphosphate-␤-L-fucose (GDP- ogeneity. Like all , Le -bearing glycans are fucose), the universal fucosyl donor, the Lex , and their products of template-independent biosynthetic pathways. The dy- C-5 substituted derivatives can be synthesized on preparative namic process of orchestrated by glycosyltransferases scales, using a chemoenzymatic approach. This method exploits and the organ-specific expression of these enzymes are responsible L-fucokinase/GDP-fucose pyrophosphorylase (FKP), a bifunctional for the microheterogeneity of these fucosylated glycoconjugates enzyme isolated from Bacteroides fragilis 9343, which converts obtained from the mammalian sources. There is currently no facile x L-fucose into GDP-fucose via a fucose-1-phosphate (Fuc-1-P) inter- and cost-effective chemistry for synthesizing Le -bearing glycocon- mediate. Combining the activities of FKP and a jugates and their derivatives on preparative scales for functional ␣1,3 fucosyltransferase, we prepared a library of Lex trisaccharide studies. Poor selectivity of fucoside coupling reactions and tedious

glycans bearing a wide variety of functional groups at the fucose protecting group manipulations are major challenges for synthetic CHEMISTRY C-5 position. These neoglycoconjugates will be invaluable tools for chemists preparing these formidable targets. In contrast, enzymatic studying Lex-mediated biological processes. glycosylation by fucosyltransferases overcomes laborious and ex- pensive chemical routes and produces Lewis antigen-containing glycobiology ͉ catalysis ͉ enzyme glycans in a regio- and stereospecific manner (13). This approach requires fucose or its synthetic analogs in the nucleotide-activated Ј ␤ L x form—guanidine 5 -diphosphate- - -fucose (GDP-fucose) ewis X (Le ), a fucosylated trisaccharide glycan epitope distrib- analogs—as the substrate for fucosyltransferases. GDP-fucose, Luted throughout eukaryotes and certain bacteria, is a determi- although commercially available, is prohibitively expensive for nant of many functional glycoconjugates that play central roles in large-scale synthesis. While Wong and co-workers developed numerous physiological and pathological processes. For example, an enzymatic approach for converting GDP- into x x sialyl Le (sLe ), a constitutively expressed on white GDP-fucose (14), the preparation was air sensitive and per- blood cells such as granulocytes and monocytes, governs leukocyte formed on an analytical scale only. Alternatively, GDP-fucose rolling and extravasation (1–3); up-regulation of this glycan is and its synthetic analogs can be produced in milligram quan- strongly correlated with the transformed phenotype of tumors of tities via a coupling reaction between guanosine 5Ј- diverse tissue origin, including pancreas, breast, colon, and lung monophosphomorpholidate and fucose-1-phosphate (Fuc- tumors (4, 5). Lex-bearing glycans are also found in the infectious 1-P) (15). However, the shortest synthetic route for generating bacterium Helicobacter pylori and the parasite Schistosoma mansoni Fuc-1-P requires six consecutive steps (16). The length of this (6, 7). In the former case, these glycans are hypothesized to mask route prevents its practical application. Therefore, there is an the pathogenic bacterium from the host immune surveillance, while urgent need to develop new strategies for facile synthesis of in the latter situation, the Lex-containing glycans are found to GDP-fucose and its derivatives as tools for large-scale prep- down-regulate the host’s protective immune responses against the aration of structurally defined fucosides, including the Lex- parasite, largely via induction of anti-inflammatory cytokine bearing glycan epitopes. interleukin-10. Here, we report a chemoenzymatic method for the preparative- As revealed by x-ray crystallographic and NMR analyses, the Lex scale synthesis of a diverse array of GDP-fucose derivatives (Fig. 1). trisaccharide assumes a well-defined 3-dimensional structure, with This method exploits L-fucokinase/GDP-fucose pyrophosphorylase its fucose ring stacking on top of the galactose residue (8–10). The (FKP), a bifunctional enzyme isolated from Bacteroides fragilis exocyclic C-5 methyl group of the fucose forms key van der Waals 9343, which converts fucose to Fuc-1-P and thence to GDP-fucose contacts with the galactose, stabilizing this highly compact struc- (17). This transformation is found in the salvage pathway of B. ture. Removal of the methyl group leads to a 5-fold decrease in fragilis 9343 fucosylation and is conserved in all Bacteroides species binding affinity of sLex to its target protein E-selectin (11). This (17). As revealed by sequence alignment, the N terminus (1–430) highly conserved structure is equally important for the specific Lex– of FKP shares 20% amino acid identity to the human GDP-fucose dendritic cell-specific ICAM-3-grabbing nonintegrin (Lex–DC- pyrophosphorylase, while its C terminus (584–949) is similar to SIGN) recognition (8), a unique interaction that is responsible for inducing cellular immunity upon pathogen recognition by dendritic Author contributions: P.W. designed research; W.W., T.H., P.A.F., T.Z., B.G., D.S.d.A., S.G., cells (12). A number of studies have demonstrated that the fucose R.D.S., and P.W. performed research; W.W., T.H., P.A.F., T.Z., B.G., R.D.S., and P.W. analyzed x C-5 methyl group may also directly participate in Le – rec- data; and W.W., P.A.F., T.Z., D.S.d.A., R.D.S., and P.W. wrote the paper. x ognition. For example, the crystal structure of a Le -bound scav- The authors declare no conflict of interest. enger receptor C-type lectin (SRCL) revealed that the terminal 1To whom correspondence should be addressed. E-mail: [email protected]. fucose resides in the secondary binding site of the protein, where This article contains supporting information online at www.pnas.org/cgi/content/full/ the methyl group forms tight van der Waals interactions with 0908248106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908248106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 24, 2021 Fig. 1. A chemoenzymatic approach for the synthesis of GDP-fucose and Lex trisac- charide derivatives. A short 2-azidoethyl spacer is introduced to the accepter sub- strate N-acetyllactosamine to allow further modification.

mammalian L-fucokinases (18). Connecting these two domains is a produced in the enzymatic reaction were further confirmed by 150-aa linker, whose function is currently unknown. Wang and high-resolution (HR MS) analysis [supporting co-workers demonstrated recently that a His6-tagged recombinant information (SI) Text]. Interestingly, we detected little accumula- FKP, expressed in Escherichia coli, is a promiscuous enzyme with tion of Fuc-1-P intermediate in the successive reaction supplied relaxed specificity toward fucose analogs bearing unnatural sub- with both ATP and GTP, implicating formation of Fuc-1-P as the stituents at the C-5 position (19). rate-limiting step. To fully characterize recombinant FKP activity, GDP-fucose serves as the donor for fucosyltranferases, enzymes we determined the kinetic parameters for both reactions under that attach the activated fucose to cell-surface glycoconjugates. On steady-state conditions (Table 1). the basis of the site of fucose transfer, fucosyltansferases are The kinetic parameters for the B. fragilis FKP are quite different classified as ␣1,2, ␣1,3/4, ␣1,6, and protein O-fucosyltransferases from those of the Arabidopsis FKP, the only other bifunctional FKP (20). In eukaryotes, the former three subfamilies of fucosyltrans- with reported kinetic parameters (26). The B. fragilis enzyme ferases are type II transmembrane proteins, with an N-terminal exhibited 19- and 9-fold greater maximal activities compared to the cytosolic tail, a hydrophobic transmembrane domain, a variable Arabidopsis enzyme for the fucokinase- and GDP-fucose pyrophos- length luminal stem region, and a C-terminal catalytic domain (21). phorylase-catalyzed transformations, respectively. Therefore, B. Their prokaryotic counterparts, however, are usually soluble pro- fragilis FKP is a better choice over the Arabidopsis enzyme for our teins without the transmembrane segment (20, 22, 23). Several ␣1,2, intended preparative-scale production of GDP-fucose and Lex ␣ ␣ 1,3/4 fucosyltransferases have been identified in H. pylori, and 1,6 derivatives. In addition, the KM value determined for L-fucose for fucosyltransferase activities have been observed in various soil the B. fragilis FKP was 25-fold lower than that determined for the bacteria including Bradyrhizobium japonicum, Azorhizobium cauli- Arabidopsis FKP. In this regard, the Michaelis constants for L- nodans, Mesorhizobium loti, and Rhizobium loti (22–25). We dem- fucose and ATP in the phosphorylation reaction were more similar onstrate herein that a recombinant ␣1,3 fucosyltransferase from H. pylori 26695 has broad substrate tolerance toward GDP-fucose analogs modified at the C-5 position and that the action of B. fragilis FKP and H. pylori ␣1,3 fucosyltransferase can be combined in one pot for the facile synthesis of the Lex glycan epitope and its derivatives (Fig. 1). This chemoenzymatic approach creates Lex derivatives with unnatural substituents introduced site specifically at the fucose C-5 position. When presented in a microarray format, these neoglycoconjugates will serve as powerful tools for high- throughput analysis of Lex-mediated lectin recognition. Comple- mentary to lectin mutants generated via site-directed mutagenesis, these unnatural Lex derivatives may shed light on key structural features that are unique to Lex–lectin interactions.

Results and Discussion Determination of the Catalytic Efficiency of B. fragilis FKP. As a first step toward the chemoenzymatic synthesis of Lex derivatives, we expressed an N-terminal His6-tagged B. fragilis FKP in E. coli, using the construct generated by the Comstock lab (17). To confirm the activity of the recombinant FKP toward GDP-fucose production, we incubated L-fucose with FKP in the presence of ATP, GTP, and Fig. 2. TLC analysis of B. fragilis FKP-catalyzed synthesis of GDP-fucose. The MnSO4. We analyzed the crude reaction mixture by qualitative thin layer chromatography (TLC), using commercially available Fuc-1-P plate was stained with p-anisaldehyde stain. Lane 1, reaction mixture in the presence of GTP; lane 2, reaction mixture in the absence of GTP; lane 3, and GDP-fucose (Sigma) as authentic standards (Fig. 2). FKP GDP-fucose; lane 4, Fuc-1-P; lane 5, L-fucose. Both reactions were performed converts L-fucose into GDP-fucose in the presence of both ATP in 50 ␮L Tris-HCl buffer (50 mM, pH 7.5) containing L-fucose (5 mM), ATP (5 and GTP, while in the absence of GTP only the Fuc-1-P interme- mM), and MnSO4 (5 mM). For GDP-fucose production, GTP (5 mM) and diate was formed. The identities of Fuc-1-P and GDP-fucose inorganic pyrophosphatase (1 unit) were included in the reaction mixture.

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908248106 Wang et al. Downloaded by guest on September 24, 2021 Table 1. Kinetic parameters of B. fragilis FKP Table 2. Fucokinase activity and specificity of B. fragilis FKP with

Ϫ1 C-5 substituted fucose analogs Substrate KM (mM) Vmax (min )

Fucokinase activity (Vmax/KM)fucose/ Ϯ Ϯ -1 L-fucose 0.045 0.010 365 22 Substrate KM ( M) Vmax (min ) ATP 1.080 Ϯ 0.21 448 Ϯ 27 (Vmax/KM)fucose analogue GDP-fucose pyrophosphorylase activity OH Fuc-1-P 0.030 Ϯ 0.002 878 Ϯ 18 O GTP 0.012 Ϯ 0.005 800 Ϯ 73 OH OH HO 45 ± 10 365 ± 22 1.0 The fucokinase activity of FKP was measured using a coupled enzymatic assay and the GDP-fucose pyrophosphorylase activity was measured using 1 EnzCheck Pyrophosphate Assay (Invitrogen). All kinetic measurements were OH performed in 50 mM Hepes-KOH, pH 7.5, at 37° C. O OH 2450 ± OH 462 ± 16 43 HO 255 to the values reported for the monofunctional fucokinase isolated 2 from pig kidney (27). Consistent with the previously characterized OH mammalian fucokinase, GDP-fucose pyrophosphorylase, and the O bifunctional FKP from Arabidopsis, the activity of B. fragilis FKP OH OH 60 ± 6 143 ± 3 3.4 requires the presence of divalent metal cations at the active site. HO Importantly, we found that the fucokinase activity of B. fragilis FKP 3 was dependent on the identity of the divalent metal cation used in OH 2ϩ 2ϩ the assay, with Mn being preferred over Mg . The specific O Ϫ1 OH activity of FKP was determined to be 4.5 units mg protein (1 unit OH 100 ± 19 244 ± 14 3.3 is defined as the amount of enzyme that is required to produce 1 HO ␮mol of GDP-fucose per minute at 37 °C). 4

Recently, Wang et al. showed that the His6-tagged recombinant F OH

O CHEMISTRY FKP from B. fragilis has relaxed specificity toward fucose analogs OH OH bearing unnatural substituents at the C-5 position (19). To quan- HO 76 ± 8 195 ± 5 3.2 titatively evaluate the specific activity of the recombinant FKP 5 toward C-5 substituted fucose analogs, we determined the fucoki- OH nase activity of the recombinant FKP toward a panel of unnatural N3 O substrates, using a simple coupled enzymatic assay with a specto- OH OH photometric readout. The production of ADP by B. fragilis FKP is HO 266 ± 25 171 ± 5 12.6 coupled to the oxidation of NADH in the presence of pyruvate 6 kinase and lactate dehydrogenase. The consumption of NADH, and accordingly the fucokinase activity, is monitored by the change in absorbance at 340 nm. All compounds tested as substrates were B. fragilis FKP fucokinase activity and specificity were determined in the competent for catalysis with V values within 3-fold of the value presence of 5 mM ATP under standard assay conditions (see Materials and max Methods). determined for L-fucose (Table 2). Substitutions were generally well tolerated as determined from the ratios of the second-order rate constant of FKP-catalyzed Fuc-1-P formation to those of the pyrophosphate generated in the reaction and to drive the reaction corresponding C-5 substituted analogs. However, removal of the to completion. The syntheses were performed on preparative scales C-5 methyl group resulted in the largest decrease in catalytic (30–50 mg) and the reaction progress was followed by TLC analysis. efficiency (Ϸ43-fold), which corresponds to an energetic penalty of Ϫ After quenching the reaction with ethanol, the crude products were 2.3 kcal mol 1 for this catalytic process. The main factor contrib- purified by gel filtration using BioGel P-2 resin (Bio-Rad). The uting to this result was the 54-fold increase in the KM value, purity and identity of the GDP-fucose derivatives were confirmed suggesting that interaction between the enzyme active site and the by NMR and HR MS analyses. The typical isolated yield of the C-5 methyl group of L-fucose is critical for catalytic efficiency. desired GDP-fucose derivatives was Ͼ75% (Table 3). Remarkably, the alkyne-, alkene-, and fluorine-substituted analogs, 3, 4, and 5, were phosphorylated by FKP almost as efficiently as the Enzymatic Synthesis of the Lex Derivatives. ␣1,3 fucosyltransferases natural substrate L-fucose. are responsible for the last steps in type II Lewis antigen biosyn- thesis (20). Preparative scale synthesis of the Lex glycans using a Enzymatic Synthesis of GDP-Fucose Derivatives. GDP-fucose deriv- chemoenzymatic approach relies on the availability of these en- atives are key intermediates for the synthesis of fucosylated oligo- zymes in milligram quantities. Toward this end, we generated a saccharides and glycoconjugates. Having characterized the activity DNA construct encoding a truncated ␣1,3 fucosyltransferase de- and promiscuity of FKP, we embarked on the synthesis of a panel rived from H. pylori 26695 fucosyltransferase (GenBank accession of GDP-fucose derivatives. Briefly, seven with no. AAD07710), using the commercial pET-24b (ϩ) vector (No- unnatural substituents at the C-5 position were chosen as fucose vagen). Given that high-level expression of a homologous ␣1,3 surrogates. The substituents, which vary in stereoelectronics and fucosyltransferase isolated from H. pylori (NCTC 11639) was hydrophobicity, were chosen to test the scope of this method. impeded by exposed residues at the C terminus (33), we removed Importantly, the monosaccharides used in our synthesis were either the potentially problematic C-terminal tail (residues 434–478), commercially available or readily synthesized in gram scales using which is rich in hydrophobic and positively charged amino acids, well-established chemistry (28–32). In a typical enzymatic reaction, including 6 of the 10 heptad repeats. The truncated protein was a fucose analog was incubated with the recombinant FKP in the highly soluble without a significant change in overall structure presence of one equivalent each of ATP and GTP. An inorganic compared to the full-length enzyme and its enzyme-specific activity pyrophosphatase from Saccharomyces cerevisiae (Sigma), contain- was determined to be Ϸ12 units mgϪ1 protein. ing Mg2ϩ to maintain its maximal activity, was included to hydrolyze With a panel of fucose analogs, a promiscuous FKP, and a

Wang et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 24, 2021 Table 3. Synthesis of GDP-fucose and the Lex trisaccharide derivatives

GDP-fucose derivatives Lex derivatives Fucosyl donor substrate Yield (%) Yield (%)

OH OH OH O O O OH O O GDP HO O O N3 O OH OH NHAc OH OH HO H3C O OH OH HO 9 OH 1 HO 92% yield 17 90% yield OH OH OH O O O OH O GDP HO O O O N O OH OH NHAc 3 OH OH O OH HO HO OH 10 OH 2 HO 85% yield 18 92% yield OH OH OH O O O OH HO O O O O GDP OH N3 O OH NHAc OH OH O OH HO HO OH 11 OH 3 HO 90% yield 19 92% yield OH OH OH O O O OH HO O O N O O O GDP OH NHAc 3 OH OH O OH OH HO HO OH OH 4 12 HO 89% yield 20 91% yield OH OH OH O O O F OH F HO O O O GDP OH N3 O O F NHAc OH OH O OH OH OH HO HO OH 5 13 HO 94% yield 21 90% yield

Table 3 (continued).

recombinant ␣1,3 fucosyltransferase in hand, the stage was set for the fucose C-5 position (Table 3). A number of these fucose analogs the one-pot synthesis of the Lex . This task was were functionalized with elongated hydrocarbon groups that should accomplished by combining a fucose analog and the acceptor enforce different conformational constraints on the final Lex tri- N-acetyllactosamine with the recombinant FKP and saccharides. The resulting Lex derivatives bearing these function- ␣1,3 fucosyltransferase. A short 2-azidoethyl spacer was introduced alities are valuable tools for probing the van der Waals contacts to the acceptor substrate N-acetyllactosamine to allow fast click between the fucose and the galactose and the importance of this modification via Cu(I)-catalyzed azide–alkyne cycloaddition interaction in Lex–lectin binding. Fucose analogs 7 and 8 were (CuAAC) for future applications such as fabricating glyco- modified with hydroxyl and methoxyl groups, respectively. These microarrays (34, 35). Using this method, we synthesized a series of groups may form additional hydrogen bonds with the neighboring the Lex trisaccharide derivatives bearing a variety of substituents at galactose, thereby twisting the natural conformation of Lex.As

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908248106 Wang et al. Downloaded by guest on September 24, 2021 Table 3. Continued

OH OH OH O O OH N HO O O N3 3 O O GDP OH N3 O O N NHAc OH OH 3 O OH OH HO HO OH OH 6 14 HO 94% yield 22 84% yield OH OH OH O O OH HO HO O O HO O OGDP OH N3 O O HO NHAc OH OH O OH HO HO HO OH OH 7 15 HO 75% yield 23 72% yield OH OH OH O O O OH O HO O O O OGDP OH N3 O O O NHAc OH OH O OH HO OH

HO HO CHEMISTRY OH 8 16 HO 77% yield 24 70% yield

previously reported, in a number of cases the C-5 methyl group of Lex derivatives combined with the emerging glyco-microarray tech- the fucose residue is directly involved in lectin binding (10). Thus, nology provides a powerful, rapid means to profile Lex–lectin the Lex derivatives decorated with unnatural functionalities at this interactions and to identify key structural features contributing to position may provide a new platform for examining intermolecular binding. Fabrication of glycodendrimer microarrays using the Lex contacts during receptor binding. The unnatural trisaccharide trisaccharide derivatives for high-throughput screening of the Lex- glycans in Table 3 were typically isolated in 70% yield; in a few cases binding is currently underway. nearly quantitative formation of the desired trisaccharide was achieved. Materials and Methods Measurement of the L-Fucokinase Activity of FKP. The production of ADP was Conclusion measured using a coupled assay system, monitoring the reaction progress by The chemoenzymatic method described here offers a practical and absorbance at 340 nm. Standard conditions were 50 mM Hepes-KOH, pH 7.5, 10 ␮ ␮ versatile approach for the synthesis of GDP-fucose and the Lex mM MnCl2, 5 mM ATP, 100 M NADH, 250 M phosphoenol pyruvate, 0.7 unit of trisaccharide glycan and their derivatives bearing neosubstituents at pyruvate kinase, and 1 unit of lactate dehydrogenase (from a stock solution in 50% glycerol) in a 1-mL reaction at 37 °C. All components except fucose were the fucose C-5 position. The procedure requires only simple cloning mixed in the cuvette and allowed to equilibrate for at least 2 min. Reactions were steps to generate the necessary enzymes for fucose installation. initiated by the addition of fucose. The amount of coupling enzymes was suffi- Many unnatural fucose analogs and acceptor glycans are commer- cient to not limit the rate of reaction. cially available or can be easily synthesized using well-established chemistry. Thus, this procedure can be readily extended for large- Measurement of the GDP-L-Fucose Pyrophosphorylase Activity of FKP. The scale synthesis using these building blocks as substrates. Without production of pyrophosphate was measured using the commercially available any optimization, we obtained a panel of C-5 substituted GDP- EnzCheck Pyrophosphate Assay Kit (Invitrogen). Standard conditions were 50 ␮ fucose derivatives in 90% yield using recombinant FKP. We expect mM Hepes-KOH, pH 7.5, 2 mM MgCl2, 200 M 2-amino-6-mercapto-7- that further engineering of B. fragilis FKP will produce a bifunc- methylpurine ribonucleoside, 1 unit nucleoside phosphorylase, and 0.03 unit tional enzyme that is capable of accepting an even broader spec- inorganic pyrophosphatase. All components except FKP were mixed in the cu- trum of unnatural fucose analogs. Toward this end, we have vette and allowed to equilibrate for at least 2 min. Reactions were initiated by the addition of enzyme. embarked on two complementary approaches—structure-based engineering and directed evolution—to generate a promiscuous Analysis of Kinetic Parameters. To determine the basic kinetic parameters for FKP that can serve as a general tool for synthesis of GDP-fucose each substrate, initial velocities were determined at various concentrations of libraries encompassing diverse structures. In nature, fucosylated one substrate while the concentration of the other substrate was held constant ␣ ␣ glycans are produced by the concerted action of 1,2, 1,3/4, and at saturated levels (5–10 times the KM value) and fit to the Michaelis–Menten ␣1,6 fucosyltransferases. We expect that FKP can be readily cou- equation (Eq. 1). A stands for a substrate [data were analyzed using KaleidaGraph pled with these enzymes in vitro to produce a diverse array of mono- (Synergy Software)]: or difucosylated glycans. V ͓A͔ Structurally defined fucosides are invaluable tools for studying ϭ max v ϩ ͓ ͔ [1] fucose-mediated cellular processes. Access to chemically defined KM A

Wang et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 24, 2021 General Procedure for Preparative Scale (30–50 mg) Synthesis of the GDP- C-5 substituted analogs (2.0 eq, 0.05 or 0.1 mmol), 2-azidoethyl N-acetyllac-

Fucose Derivatives. Reactions were typically carried out in a 15-mL centrifuge tosamine (1.0 eq, 0.025 or 0.05 mmol), ATP (2.0 eq), GTP (2.0 eq), MnSO4 (20 or 40 tube with 5.0 mL Tris-HCl buffer (100 mM, pH 7.5) containing L-fucose or its C-5 mM), inorganic pyrophosphatase (75 units, lyophilized form containing MgCl2), substituted analogs (7.5–10.2 mg, 0.05 mmol), ATP (1.0 eq), GTP (1.0 eq), MnSO4 FKP (9 units), and ␣1,3 fucosyltransferase (2 units). The reaction mixture was (10 mM), inorganic pyrophosphatase (90 units, lyophilized form containing incubated at 37 °C for 2–3 h with vigorous shaking (225 rpm). The reaction was MgCl ), and FKP (9 units). The reaction mixture was incubated at 37 °C for 5–6 h 2 monitored by TLC analysis using EtOAc:MeOH:HOAc:H O (6:3:3:1) as the devel- with shaking (225 rpm). The reaction was monitored by TLC analysis, using 10 mM 2 oping solvent (H SO stain). After adding the same volume of ice-cold ethanol to tetrabutylammonium hydroxide in 80% aqueous acetonitrile as the developing 2 4 solvent (p-anisaldehyde sugar stain). After adding the same volume of ice-cold quench the reaction, the alcoholic mixture was incubated on ice for 30 min. ϫ ethanol to quench the reaction, the alcoholic mixture was incubated on ice for 30 Insoluble material was removed by centrifugation (5,000 g, 30 min) and the min. Insoluble material was removed by centrifugation (5,000 ϫ g, 30 min) and supernatant was concentrated in vacuo to remove volatile ethanol. The aqueous the supernatant was concentrated in vacuo to remove volatile ethanol. Crude residues were lyophilized to dryness. Crude product was purified by Bio-Gel P2 gel X reaction products were purified by Bio-Gel P2 gel filtration chromatography filtration chromatography (1.5 ϫ 75 cm) and eluted with H2O. Lyophilized Le (1.5 ϫ 75 cm) and eluted with H2O. Only the fractions containing the product derivatives were characterized by NMR and HR MS. were collected, lyophilized, and further purified using Bio-Gel P2 gel filtration chromatography (1.5 ϫ 120 cm) and eluted with NH4HCO3 (50 mM). Lyophilized GDP-fucose derivatives were characterized by NMR and HR MS. ACKNOWLEDGMENTS. fkp in pET-16b was provided by Prof. Laurie E. Com- stock. We thank Prof. John S. Blanchard for discussions on FKP kinetics. This General Procedure for Preparative Scale (15–30 mg) Synthesis of the Lex work was supported by startup funds from Albert Einstein College of Medi- Trisaccharide Derivatives. One-pot reactions were performed in 15-mL centri- cine. Patrick A. Frantom was supported by a postdoctoral fellowship from The fuge tubes with 5.0 mL Tris-HCl buffer (100 mM, pH 7.5) containing L-fucose or its Charles H. Revson Foundation.

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