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Fucose\u2010containing carbohydrates have important biological functions. Alterations in the expression of fucosylated oligosaccharides have been observed in several pathological processes such as cancer and atherosclerosis. Chemical formation of fucosidic bonds is challenging due to its acid lability. Enzymatic construction of fucosidic bonds by fucosyltransferases is highly efficient and selective but requires the expensive sugar nucleotide donor guanosine 5\u2032\u2010diphosphate\u2010<jats:sc>L<\/jats:sc>\u2010fucose (GDP\u2010Fuc). Here, we describe a protocol for applying a one\u2010pot three\u2010enzyme system in synthesizing structurally defined fucose\u2010containing oligosaccharides from free <jats:sc>L<\/jats:sc>\u2010fucose. In this system, GDP\u2010Fuc is generated from <jats:sc>L<\/jats:sc>\u2010fucose, adenosine 5\u2032\u2010triphosphate (ATP), and guanosine 5\u2032\u2010triphosphate (GTP) by a bifunctional <jats:sc>L<\/jats:sc>\u2010fucokinase\/GDP\u2010fucose pyrophosphorylase (FKP). An inorganic pyrophosphatase (PpA) is used to degrade the by\u2010product pyrophosphate (PPi) to drive the reaction towards the formation of GDP\u2010Fuc. In situ generated GDP\u2010Fuc is then used by a suitable fucosyltransferase for the formation of fucosides. The three\u2010enzyme reactions are carried out in one pot without the need for high\u2010cost sugar nucleotide or isolation of intermediates. The time for the synthesis is 4 to 24 hr. Purification and characterization of products can be completed in 2 to 3 days. <jats:italic>Curr. Protoc. Chem. Biol<\/jats:italic>. 4:233\u2010247 \u00a9 2012 by John Wiley &amp; Sons, Inc.<\/jats:p>","DOI":"10.1002\/9780470559277.ch110277","type":"journal-article","created":{"date-parts":[[2012,9,4]],"date-time":"2012-09-04T22:47:04Z","timestamp":1346798824000},"page":"233-247","source":"Crossref","is-referenced-by-count":20,"title":["One\u2010Pot Multienzyme Synthesis of Lewis x and Sialyl Lewis x Antigens"],"prefix":"10.1002","volume":"4","author":[{"given":"Hai","family":"Yu","sequence":"first","affiliation":[{"name":"Department of Chemistry, University of California\u2010Davis  Davis California"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kam","family":"Lau","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of California\u2010Davis  Davis California"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanhong","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of California\u2010Davis  Davis California"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Go","family":"Sugiarto","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of California\u2010Davis  Davis California"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xi","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Chemistry, University of California\u2010Davis  Davis California"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2012,9]]},"reference":[{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.1093\/jn\/137.3.847S"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/5.7.683"},{"key":"e_1_2_6_4_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1106469"},{"key":"e_1_2_6_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1521-3773(20000303)39:5<836::AID-ANIE836>3.0.CO;2-I"},{"key":"e_1_2_6_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrd1751"},{"key":"e_1_2_6_7_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja020887u"},{"key":"e_1_2_6_8_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.272.34.21357"},{"key":"e_1_2_6_9_1","first-page":"447","article-title":"Expression of carbohydrate antigen 19\u20109 and stage\u2010specific embryonic antigen 1 in non\u2010tumorous and tumorous epithelia of the human colon and rectum","volume":"75","author":"Gong E.","year":"1985","journal-title":"J. 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