One‐Pot Multienzyme Synthesis of Lewis x and Sialyl Lewis x Antigens

Hai Yu1, Kam Lau1, Yanhong Li1, Go Sugiarto1, Xi Chen1

1 Department of Chemistry, University of California‐Davis, Davis, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110277
Online Posting Date:  September, 2012
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L‐Fucose has been found abundantly in human milk oligosaccharides, bacterial lipopolysaccharides, glycolipids, and many N‐ and O‐linked glycans produced by mammalian cells. Fucose‐containing 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′‐diphosphate‐L‐fucose (GDP‐Fuc). Here, we describe a protocol for applying a one‐pot three‐enzyme system in synthesizing structurally defined fucose‐containing oligosaccharides from free L‐fucose. In this system, GDP‐Fuc is generated from L‐fucose, adenosine 5′‐triphosphate (ATP), and guanosine 5′‐triphosphate (GTP) by a bifunctional L‐fucokinase/GDP‐fucose pyrophosphorylase (FKP). An inorganic pyrophosphatase (PpA) is used to degrade the by‐product pyrophosphate (PPi) to drive the reaction towards the formation of GDP‐Fuc. In situ generated GDP‐Fuc is then used by a suitable fucosyltransferase for the formation of fucosides. The three‐enzyme reactions are carried out in one pot without the need for high‐cost 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. Curr. Protoc. Chem. Biol. 4:233‐247 © 2012 by John Wiley & Sons, Inc.

Keywords: enzymatic synthesis; FKP; fucoside; fucose; fucosylation; fucosyltransferase; Lewis x; one‐pot multienzyme; sialyl Lewis x

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: One‐Pot Multienzyme Synthesis of Fucosides, Lewis x, and Sialyl Lewis x
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: One‐Pot Multienzyme Synthesis of Fucosides, Lewis x, and Sialyl Lewis x

  • 1 M Tris⋅Cl, pH 7.5: prepared from Tris base (Fisher Scientific, cat. no. BP154‐1) by adding HCl to adjust the pH
  • MgCl 2⋅6H 2O (Fisher Scientific, cat. no. MX0045‐2)
  • MnCl 2⋅4H 2O (Fisher Scientific, cat. no. M87‐500)
  • Galβ1–4GlcNAcβProN3 (Lau et al., )
  • Neu5Acα2–3Galβ1–4GlcNAcβProN3 (Yu et al., ; Zhang et al., )
  • L‐Fucose (Sigma‐Aldrich, cat. no. F2252)
  • Adenosine 5′‐triphosphate (ATP; Sigma‐Aldrich, cat. no. A2383)
  • Guanosine 5′‐triphosphate (GTP; Sigma‐Aldrich, cat. no. G8877)
  • Enzymes including:
    • Bacteroides fragilisL‐fucokinase/GDP‐fucose pyrophosphorylase (FKP; Yi et al., )
    • α1–3‐fucosyltransferase from Helicobacter pylori (Hp1–3FTΔ66; Sugiarto et al., )
    • Pasteurella multocida inorganic pyrophosphatase (PmPpA; Lau et al., )
  • Ethyl acetate (EtOAc; Fisher Scientific, cat. no. E14520)
  • Methanol (MeOH; Fisher Scientific, cat. no. A412‐20)
  • p‐Anisaldehyde sugar stain (see recipe)
  • Ice
  • 95% ethanol (Fisher Scientific, cat. no. AC61511‐0010)
  • Deionized water
  • Silica gel 60 (Sorbent technologies, cat. no. 40930‐25)
  • Sand (Fisher Scientific, cat. no. S25‐500)
  • 0.5‐ml microcentrifuge tubes (ITI Scientific, cat. no. MT‐901)
  • Fisher Isotemp economy analog‐control water bath Model 105Q (Fisher Scientific, cat. no. 15‐460‐5)
  • Thin‐layer silica gel plates (silica gel 60 F254; Sorbent technologies, cat. no. 4115126)
  • Glass capillary tubes (VWR, cat. no. 53432‐761)
  • Hotplate (Fisher Scientific, cat. no. 11‐520‐49H) or hair dryer
  • Wide‐mouth straight‐sided glass jar with cap (for developing TLC plates; Fisher Scientific, cat. no. S31963F)
  • UV lamp (Fisher Scientific, cat. no. NC9603820)
  • Sharp pencils
  • Paper towels
  • 50‐ml centrifuge tubes, sterile (Biologix Research Company, cat. no. 10‐9501)
  • Vortex mixer (Fisher Scientific, cat. no. 12‐811SQ)
  • C25KC incubator shaker (Fisher Scientific, cat. no. 14‐278‐178)
  • Sorvall legend T/RT benchtop centrifuge (Fisher Scientific, cat. no. 75‐004‐377)
  • 100‐ and 250‐ml round‐bottom flasks (Quark, cat. no. QF‐1‐15, QF‐1‐20)
  • Buchi rotary evaporator (Fisher Scientific, cat. no. 04‐987‐222)
  • Bio‐Gel P‐2 Gel filtration column (2.5‐cm i.d × 80‐cm length)
  • Model 2110 fraction collector (Bio‐Rad, cat. no. 731‐8120)
  • 10‐ml test tubes (Fisher Scientific, cat. no. 14‐961‐27)
  • Chromatography columns (Quark Glass, cat. no. QCH‐13‐28)
  • Long‐stem funnels (Fisher Scientific, cat. no. 10‐500‐14)
  • Freeze‐dry system (Fisher Scientific, cat. no. 10‐271‐16)
NOTE: Enzymes suitable for this protocol can be recombinant proteins or purified proteins obtained in individual laboratories or from commercially available sources. The enzymes used in this protocol including FKP, PmPpA, and Hp1–3FTΔ66 are examples of suitable recombinant enzymes that are expressed and purified in our laboratory. Commercially, FKP from B. fragilis can be obtained from Accendatech (cat. no. B‐03053, Several inorganic pyrophosphatase are available from Fisher Scientific (inorganic pyrophosphatase from yeast, cat. no. FEREF0221) and Sigma‐Aldrich, including inorganic pyrophosphatase from baker's yeast (S. cerevisiae; cat. no. I1643/I1891), inorganic pyrophosphatase from Escherichia coli (cat. no. I5907), and inorganic pyrophosphatase from Bacillus stearothermophilus (cat. no. I2891).
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