November 29
PROGRAM

11Glycoscience : diversity and integration

Chemical andchemoenzymatic synthesis ofGPI anchors and GPI-anchored proteins
Zhongwu Guo, Ph.D.
Professor, Department of Chemistry,
Wayne State University, USA

Zhongwu Guo
ProfileZhongwu Guo received his undergraduate
degree from The Second Military Medical
University in China, his Ph.D. degree inorganic chemistry from the Institute of Organic Chemistry, Polish Academy of Sciences, and his postdoctoral training in glycoscience at Shanghai Institute of Organic
Chemistry (SIOC), Chinese Academy ofSciences. He was appointed Assistant andAssociate Professor at SIOC (China) from1994 to 1996, RIKEN Fellow at RIKEN (Japan) from 1996 to 1997, Assistant Research
Officer at National Research Council ofCanada from 1997 to 1999, and Assistantand Associate Professor at Case WesternReserve University (USA) from 1999 to2005. He is currently Professor of Chemistry at Wayne State University, USA, andthe National Glycoengineering ResearchCenter, Shandong University, China. He is
also serving as the Editor-in-Chief of theJournal of Carbohydrate Chemistry. His
research interests are focused on carbohydrate chemistry, glycobiology, and medicinal chemistry, in particular the development of new methodologies for complexcarbohydrate and glycoconjugate synthesis
and the application of the synthetic glycoconjugates to the investigation of biologicalproblems and to the development of new
therapeutic strategies for various diseases.

Many surface proteins and glycoproteins areanchored onto the cell membrane throughglycosylphosphatidylinositols (GPIs)-a classof complex glycolipids, which share thehighly conserved core structure: H2NCH2CH2-OPO3-6-α-Man-(1→2)-α-Man(1→6)-α-Man-(1→4)-α-GlcNH 2-(1→6)myo-inositol-1-OPO3-glycerolipid (Fig.1A) 1). Proteins and glycoproteins have theirpolypeptide C-termini linked invariably
to the phosphoethanolamine moiety at
the non-reducing end of the GPI core
structure. GPI-anchored proteins and glycoproteins play an important role in various biological and pathological processes.
To study these processes, it is essential tohave access to homogeneous and structurally well-defined GPIs and GPI-anchoredproteins and glycoproteins, as well as theirderivatives, which is currently a significant
challenge. One of the research projects in
my laboratory aims at the developmentof practical methods for the synthesis ofnatural and unnatural and structurally
defined GPI anchors and GPI-anchoredpeptides, glycopeptides, proteins, and glycoproteins, as well as functionalized GPI
derivatives and conjugates, and applications of the synthetic molecules to exploring GPI anchorage to cells and related
biological problems.
First, we developed a highly convergent
strategy for GPI anchor synthesis 2). In
contrast to the traditional strategy where
the phospholipid moiety was introduced at
the final stage of the synthesis, we carriedout the phospholipidation reaction earlywith the pseudodisaccharide to obtain aphospholipidated pseudodisaccharide 1(Figure 1. A), which was used as a common key intermediate in GPI synthesis.
Evidently, an advantage of this synthetic
strategy was that glycosylation of 1 witholigosaccharides could lead to the backbone of desired GPIs in one step. Anotheradvantage of this new synthetic strategy
was that the difficult steps involved inGPI synthesis, such as the installation of40

the 20th Anniversary of the Mizutani Foundation for Glycoscience

the phospholipid and the α-glucosamine
moieties, were realized at the initial stage,
which helped circumvent some issues metin GPI synthesis and improve the overall
synthetic efficiency. This highly convergent synthetic strategy was proved widely
applicable and used to prepare a numberof GPI anchors and GPI analogs. The synthetic GPI anchors and GPI analogs havebeen used to study biological problems,such as how some pore-forming bacterial
toxins interact with GPI anchors 3) .
We also explored the use of para-methoxybenzyl group as the permanent protectionof hydroxyl groups in GPI synthesis 4),
which enabled global deprotection of thetarget molecules under mild conditions
compatible with various functional groups
sensitive to hydrogenation. Combinedwith the convergent synthetic strategy
described above, the new protecting tactic
was successfully applied to the synthesis ofGPI anchors carrying unsaturated lipids
and other functionalities such as the azido
and alkynyl groups 5)6). The latter facilitatedfurther specific modifications of GPIs by“click” chemistry. For example, fluoresceinand biotin as imaging and affinity probes
were effectively coupled to GPIs carrying
the azido and alkynyl groups by “click”
reactions to generate GPI-fluoro and GPIbiotin conjugates 6). These functionalized
GPI anchors and GPI conjugates are usefultools for biological studies of GPI anchorage.
Since all naturally occurring GPI-anchoredproteins and proteins have their polypeptide C-termini linked to the conserved
phosphoethanolamine moiety at the GPI
non-reducing end, for GPI-anchored peptide, glycopeptide and protein synthesis, apotentially universally useful strategy is toprepare structurally defined GPI anchors,
peptides, glycopeptides, and proteinsseparately and then couple them together
site-specifically. In this regard, bothchemical and enzymatic coupling reac-tions were exploited. For the chemical
coupling, extensively protected GPIs
with a free non-reducing end phosphoethanolamine moiety and peptides/
glycopeptides with a free C-terminus
were stitched together in a site-specificmanner under the conditions for conventional amine and carboxylic acidcondensation. Finally, the couplingproducts were globally deprotected toafford the target molecules7). Despite its
success, this synthetic method would
be difficult to apply to full size proteins because of the difficulty to accessextensively protected proteins or toachieve global deprotection of GPIs in
the presence of proteins. To deal withthe problem, an enzyme-mediatedmethod for coupling GPI anchors withpeptides, glycopeptides and proteins
was developed (Figure 1 . B).
The enzyme employed to ligate GPI
anchors with peptides, glycopeptides
and proteins was sortase, a bacterial
Figure 1. Schematic depiction of GPI anchorage of a glycoprotein to the cell membrane, as well as thestrategies developed for the synthesis of GPI anchors and GPI-anchored peptides, glycopeptides, andtranspeptidase used by bacteria to anchorproteins.
surface proteins to the cell wall 8). Sortase(A) Only the conserved core structure of GPI anchors without additional modifications is shown here.
recognizes a unique peptide sequence,
GPI-anchored proteins/glycoproteins always have the polypeptide C-terminus linked to the phosknown as the “sorting signal”, near thephoethanolamine moiety of the GPI core structure. GPIs anchor proteins and glycoproteins onto thetarget protein C-terminus, cleaves acell surface by inserting the lipid chains into the cell membrane lipid bilayer. A potentially widely
applicable method for the synthesis of GPI-linked peptides, glycopeptides, proteins, and glycoprospecific peptide bond of the signal, andteins is to site-specifically couple the C-terminus of a synthetic peptide/glycopeptide or recombinantfinally links the target protein to theprotein/glycoprotein to the phosphoethanolamine moiety of a synthetic GPI anchor. The coupling
terminal glycine residue of peptidoglyreaction was achieved both chemically and enzymatically. Moreover, a highly convergent strategy
cans on the bacterial cell wall. The spewas developed for the synthesis of GPI anchors with phospholipidated psudodisaccharide 1 as thecific enzyme utilized in our researchcommon key building block. Glycosylation of this building block with oligosaccharides can lead towas sortase A (SrtA) originated fromthe skeleton structures of various GPI anchors in one step.
(B) SrtA-mediated ligation of GPI anchors and proteins for the synthesis of GPI-anchored proteins.
Staphylococcus aureus. It recognizes apentapeptide LPXTG, where X can be
D, E, A, N, Q, or K, cleaves the peptide
bond between T and G, and then links theally useful for the synthesis of structurally
peptides, glycopeptides, and proteins 10)11).
carboxyl group of T to an amino group.
defined GPI-anchored peptides and glycoSrtA was also utilized to cyclize peptides
We have proved that SrtA would accept
peptides, as well as GPI-anchored proteinsand glycopeptides for the preparation ofGPI anchors with one or multiple glycine
and glycoproteins that contain complex
macrocyclic peptides and glycopeptides12)
residues linked to their non-reducing end
and to attach proteins to the liposome
GPIs and full size proteins.
phosphoethanolamine moiety as subsurface decorated with glycine residues13).
strates 9) and efficiently couple GPIs toIt is anticipated that SrtA may be generReferences1) 	M.A.J. Ferguson and A.F. Williams, Cell-surface anchoring of proteins via
glycosylphosphatidylinositol structure. Ann. Rev. Biochem. 1988, 57, 285320.
2) 	J. Xue and Z. Guo, Convergent synthesis of a GPI containing an acylated inositol. J. Am. Chem. Soc. 2003, 125, 16334-16339.
3) 	Q. Wu and Z. Guo, Glycosylphosphatidylinositols (GPIs) are potential targets for the development of novel Inhibitors for aerolysin-type of pore-forming bacterial toxins. Med. Res. Rev. 2010, 258-269.
4) 	B.M. Swarts and Z. Guo, Synthesis of a glycosylphosphatidylinositol (GPI)
anchor carrying unsturated lipid chains. J. Am. Chem. Soc. 2010, 6648–6650.
5) 	S. Burgula, B.M. Swarts, and Z. Guo, Total synthesis of a glycosylphosphatidylinositol anchor of the human lymphoctye CD52 antigen. Chem. Eur. J.
2012, 18, 1194-1201.
6) 	B.M. Swarts and Z. Guo, Chemical synthesis and functionalization of clickable glycosylphosphatidylinositol anchors. Chem. Sci. 2011, 2, 2342-2352.
7) 	N. Shao, J. Xue, and Z. Guo, Chemical synthesis of a skeleton structure ofsperm CD52-A GPI-anchored glycopeptide. Angew. Chem. Int. Ed. 2004, 43,1569-1573.

8) 	L.A. Marraffini, A.C. DeDent, and O. Schneewind, Sortases and the art of anchoring proteins to the envelopes of Gram-positive bacteria. Microbiol. Mol.
Biol. Rev. 2006, 70, 192-221.
9) 	X. Guo, Q. Wang, B.M. Swarts, and Z. Guo, Sortase-catalyzed peptide-glycosylphosphatidylinositol analog ligation. J. Am. Chem. Soc. 2009, 131, 98789879.
10) 	Z. Wu, X. Guo, Q. Wang, B.M. Swarts, and Z. Guo, Sortase A-catalyzed transpeptidation of glycosylphosphatidylinositol derivatives for chemoenzymaticsynthesis of GPI-anchored proteins. J. Am. Chem. Soc. 2010, 132, 1567-1571.
11) 	Z. Wu, X. Guo, and Z. Guo, Chemoenzymatic synthesis of glycosylphosphatidylinositol-anchored glycopeptides. Chem. Commun. 2010, 46, 5773-5774.
12) 	Z. Wu, X. Guo, and Z. Guo, Sortase A-catalyzed peptide cyclization for thesynthesis of macrocyclic peptides and glycopeptides. Chem. Commun. 2011,47, 9218-9220.
13) 	X. Guo, Z. Wu, and Z. Guo, New method for site-specific modification ofliposomes with proteins using sortase A-mediated transpeptidation. Bioconjugate Chem. 2012, doi: 10.1021/bc200694t.

Commemorative Symposium

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