Chemoenzymatic synthesis of GDP-L-fucose and the Lewis X glycan 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 glycans play important roles in numerous Ile-712, providing specificity for Lex over other galactose- 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 antigen- 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 oligosaccharides, Le -bearing glycans are fucose), the universal fucosyl donor, the Lex trisaccharide, and their products of template-independent biosynthetic pathways. The dy- C-5 substituted derivatives can be synthesized on preparative namic process of glycosylation 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 Helicobacter pylori 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-mannose into x x sialyl Le (sLe ), a tetrasaccharide 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 –lectin 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 mass spectrometry (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