Chemoenzymatic synthesis of oligosaccharides and glycoproteins

Chemoenzymatic synthesis ofoligosaccharides and glycoproteinsSarah Hanson, Michael Best, Marian C. Bryan and Chi-Huey Wong

Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA

Oligosaccharides are involved in a wide range of

biological processes including, for example, bacterial

and viral infection, cancer metastasis, the blood-clotting

cascade and many other crucial intercellular recognition

events. The molecular details of these biological recog-

nition events are, however, not well understood. To

express their function, oligosaccharides often occur as

glycoconjugates attached to proteins (called glyco-

proteins) or lipids (called glycolipids) that are often

found on the surface of cells. Such physiological

relevance has stimulated researchers to make signifi-

cant advances in oligosaccharide and glycoprotein

preparation despite the chemically imposing and poly-

disperse nature of these molecules. The chemical and

Chemoenzymatic methods developed recently have

facilitated the synthesis of structurally defined oligo-

saccharides and glycoconjugates such that a more

thorough understanding of their biological function

and potential therapeutic application can be addressed.

The function of a gene is often expressed at the proteinlevel through transcription, translation and, in manycases, posttranslational modification. Among the post-translational events that are known so far, proteinglycosylation is the most complex. More than 50% ofhuman proteins are glycosylated. The effect of glycosyla-tion on the structure and function of proteins is, however,not well understood, mainly because of the often arduoustask of synthesizing and characterizing oligosaccharides.

Unlike nucleic acids and amino acids, oligosaccharideshave no coding template for synthesis [1–3]. It remainsunclear whether oligosaccharide synthesis is involved inthe transfer of biological information, but the complexityand structural diversity of oligosaccharides could surpassthose of proteins and nucleic acids, which have beenselected by nature for the production of informationalmolecules. In oligosaccharide synthesis, different enzymesoften compete for a given acceptor, thereby creating aneven more unpredictable outcome [2].

Characterization is complicated by the one aspect ofthis family that makes them ideal recognition factors:namely, their inherent diversity [2–5]. Unlike nucleosidesor amino acids, monosaccharides can form links at up tofive positions with either a or b stereochemistry. Thismeans that a simple library of tetrasaccharides yieldsmore than 15 million possible combinations from the eight

Corresponding author: Chi-Huey Wong ([email protected]).

www.sciencedirect.com 0968-0004/$ - see front matter Q 2004 Published by Elsevier Ltd. doi:

commonly foundmonosaccharides, although nature mightnot use all of the possible combinations in biologicalsystems. However, a similar library of tetramers of linearpeptides and nucleic acids yields only 160 000 and 256members, respectively.

Oligosaccharide characterization is further compli-cated because several of the eight common monosacchar-ides have identical masses, making identification of thestructural make-up of oligosaccharides exceedingly chal-lenging [3–5]. In addition, oligosaccharides often occur asglycoconjugates attached to proteins (called glycoproteins)or lipids (called glycolipids) on the surface of cells. Theeffect of glycosylation on the structure and function ofthese biomolecules thus remains a subject of intensivestudy.

As we review here, the development of simple andeffective methods for the synthesis of oligosaccharidesand glycoconjugates, especially glycoproteins, is essentialfor a better understanding of glycosylation in biology andfor the development of new diagnostic and therapeuticstrategies.

Programmable ‘one-pot’ approach to oligosaccharide

synthesis

Chemical synthesis

The creation of specific glycosidic bonds presents aformidable synthetic challenge for several reasons. Pro-tecting groups, potential participating groups, promotersor catalysts, reaction conditions, donor leaving groups andacceptors must be carefully designed because they arecrucial for generating the correct regiochemistry andstereochemistry of the created glycosidic bond. Theselection of these factors is further complicated by thefact that most oligosaccharides are branched and notlinear like peptides and nucleic acids [2,6].

Despite these intrinsic difficulties, several strategieshave been developed for oligosaccharide synthesis(Figure 1), including the most recent development ofsolid-phase synthesis [6–10] (Figure 1a). The problem ofspecifically manipulating the protecting group stillremains in solid-phase synthesis, and the complexity ofthis problem increases with the length of the oligo-saccharide chain. Overall, however, the approach offerssimplicity in procedure and flexibility in structure.

Another strategy for the rapid synthesis of oligo-saccharides is the application of thioglycoside buildingblocks to form glycosidic bonds through an anomericreactivity-based and programmable approach [2,9,10]

Review TRENDS in Biochemical Sciences Vol.29 No.12 December 2004

10.1016/j.tibs.2004.10.004

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O O

OP1

OP2

P3O

OP4O O

OP1

OP2

O

OP4

O

OP5

OP6

P7O

OP8 O O

OP1

OP2

HO

OP4

O O

OP1

OP2

O

OP4

O

OP4

OP5

HO

OP8

O O

OP1

OP2

O

OP4

O

OP5

OP6

O

OP8

O

OP9

OP10

P11O

OP12

OO

OP1P2O

O

OP4

O

OP5P6O

O

OP8

O

OP9P10O

O

OP12

O

OPPO

PO

OP

O

OPOP

PO

OP

STol O

OP9

OP10

HO

OP12

STol O

OP5

OP6

HO

OP8

STol O

OP1

OP2

HO

OP4

O

11

21

1st condition

Differential deprotection1 in 4

2nd condition

Coupling

1

2

1

2

3

3rd condition

4th condition

Coupling

5th condition Differential deprotection1 in 10

6th conditionCoupling

+ + +

Most reactive 2nd most reactive 3rd... 4th...

4 3 2 1

123

One-pot

4

(a) Stepwise solid-phase synthesis

(b) OptiMer programmed one-pot synthesis

Differential deprotection1 in 7

Figure 1. Advances in chemical oligosaccharide synthesis. (a) Oligosaccharide synthesis has been adapted to the solid phase for the production of complex sugars. This

approach eliminates the need for intermediate isolation and offers a high degree of flexibility. It is an effective approach for target synthesis or for the synthesis of a few

oligosaccharides. The requirement of differential deprotection during synthesis is a problem, however, that increases with increasing chain length. (b) Programmable one-

pot synthesis uses a range of differentially protected saccharide building blocks with known reactivities to synthesize complex sugars in one pot. The building blocks

(currently about 400) and their relative reactivities are stored in the OptiMer computer program. For example, when a tetrasaccharide of interest is to be synthesized, the

sequence of the tetrasaccharide is entered as a query to the OptiMer program. Four building blocks will appear on the screen that can be combined sequentially according to

their reactivity order in one vessel to produce the desired protected oligosaccharide in good yield. In the process shown here, the donor–acceptor 3 (sugar 3) is incubatedwith

the most reactive donor (sugar 4). On consumption of the most reactive donor, the third donor–acceptor (sugar 2) is added without intermediary purification of the

trisaccharide, and then finally the least reactive acceptor (sugar 1) is added. After a global deprotection, the oligosaccharide is purified. The whole process can take from

minutes to days, depending on the target. The oligosaccharide fragment used in the one-pot assembly can be also made from monosaccharide building blocks using this

approach. All building blocks contain the same thiophenyl leaving group and all donor–acceptors contain one hydroxyl group. Abbreviations: P and P1-12, protecting group;

STol, s-tolyl.

Review TRENDS in Biochemical Sciences Vol.29 No.12 December 2004 657

(Figure 1b). Activation of the anomeric leaving group can beattenuated through modification of the protecting groupstrategy and through neighboring group participation [9].This reactivity-based strategy has been applied to one-potglycosylation reactions in which a series of building blockswith identical leaving groups react sequentially in onevessel without laborious intermediate purification steps.Traditional carbohydrate synthesis requires intermediatemodifications and protecting group manipulations, and apurification step between each coupling step. This meansthat the synthesis of even small libraries of oligosaccharidesis extremely time-consuming and tedious. A programmableone-pot synthesis using the sequential addition of thioglyco-side building blocks with lessening reactivities eliminatesthe intermediate steps andprotecting groupmanipulations,and provides rapid access to oligosaccharides with a widerange of molecular diversity.

To implement this one-pot programmable synthesis,the relative reactivity of each thioglycoside donor must be

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characterized. Relative reactivity values (RRVs) aremeasured by performing a competition reaction betweena given donor and a reference donor with methanol as theacceptor [9]. Changes in the ratio of the donor and thereference through the course of the reaction are thenused to calculate the RRV by high-performance liquidchromatography. The RRV database has been used todevelop the ‘OptiMer’ one-pot synthesis program. Thisprogram contains information on each thioglycosidebuilding block, including its RRV, the position of anyunprotected hydroxyls and the a/b directing nature of theC 02 functionality. OptiMer uses this information toanalyze a given oligosaccharide, determines the bestcombinations of donor–acceptors, and predicts the yield.At present, more than 400 thioglycoside building blockswith defined RRVs are available.

Several reagents are acceptable activators for thioglyco-sides including dimethylthiosulfonium triflate, trimethyl-silyl triflate, methyl triflate,N-iodosuccinimide/ triflic acid,



benzene sulfinylpiperidine/triflic anhydride [2,6,9] and thenewly developed N-(phenylthio)caprolactam [11]. The twolast thioglycoside activators are faster, cause fewer sidereactions, and have been applied to various oligosaccharideand glyconjugate syntheses, including Globo-H, Lewis Y,poly-N-acetyl lactosaminesand fucosylGM1, all ofwhicharecommonly found on the surface of cancer cells [2,12–13].Globo-H was originally identified as an antigen on prostateand breast cancer cells and is being clinically developed as atherapeutic vaccine for the treatment of breast cancer [13].Theone-potsynthesis canalsobeused for themodificationofde novoglycosylation of glycosylated and non-glycosylatednatural products [14].

Enzymatic synthesis

Of growing interest as an alternative to chemicalsynthesis is the use of enzymes in synthesis. Enzymaticcoupling has several advantages over its chemicalcounterpart. Enzymatic glycosylation occurs stereo- andregioselectively under mild conditions without protectinggroup manipulation [2,15]. In addition, even very steri-cally demanding couplings, such as those involving sialicacid glycosylation, can be performed selectively. Enzymescatalyzing such reactions fall into one of two categories:glycosyltransferases or glycosidases.Glycosyltransferases. These enzymes, which catalyze thetransfer of a saccharide from a sugar nucleotide donor to

O

OH

OH

HO

H3C

O

HO2C

HO

OH

O

OH

HO

AcHN

HO

OH

OPO32–

CO2–

CO2–

O

UDP

UTP

-UDP

PPi

Sugar 1 Sugar 2–Sugar 1

P

Sugar 2–P

PEP

Pyr

PiE2E3

E4

Sugar 2

E1

1: Glycosyltransferase

E2: Pyruvate kinase

E3: Sugar nucleotide pyrophosphorylase

E4: Pyrophosphatase

E5: Myokinase

E6: Sugar nucleotide synthase

PEP: phosphoenol pyruvate

Pyr: pyrateSLe

E

Key:

Figure 2. Regeneration of sugar nucleotide in the glycosyltransferase-catalyzed synthes

general scheme shows enzymatic regeneration of a nucleoside diphosphate suga

galactosamine, GDP-fucose and GDP-mannose; left) or a nucleosidemonophosphate sug

The nucleoside phosphate causes severe inhibition of the glycosyltransferases; therefo

phosphate in situ eliminates such product inhibition and also minimizes the cost. Mo

regeneration of the nucleoside phosphate sugar for each glycosyltransferase, to form m

been synthesized in this manner by a multiple enzyme system.


an acceptor, are responsible for the biosynthesis ofoligosaccharides and N- and O-linked glycoconjugates innature [15–18]. Although glycosyltransferase-catalyzedreactions show high stereo- and regiocontrol, both theenzyme and the sugar nucleotide are expensive and theglycosylation pathway can be plagued with feedback inhi-bition by the generated nucleoside phosphate. Regener-ation of the sugar nucleotide substrate from its by-productnucleoside phosphate eliminates the problem of productinhibition and lowers the cost, facilitating the synthesis ofoligosaccharides and polysaccharides on a large scale.

Two such examples are the enzymatic synthesis ofsialyl Lewis x (Lex), which is responsible for the binding ofneutrophils and leukocytes to the selectins of injuredtissues during the inflammation cascade, and hyaluronicacid, which has essential roles in angiogenesis, hemo-poiesis and adhesion [2] (Figure 2). Both compounds havebeen prepared in one pot by using multiple glycosyltrans-ferases and the regeneration of each sugar nucleotide.Further improvement of the system through the use ofwhole cells or multienzymes on beads has been alsoreported [19].

In addition, glycosyltransferases have been used inenzymatic one-pot strategies for the synthesis of anti-biotics. By using a combination of both natural andunnatural UDP-sugars along with the naturally occur-ring transferases in the vancomycin and teicoplanin

OO

HO

O

COO–

HO HO

HO

AcHN

O

n

O

O

OH

O

NHAcOHO

OH

ATP

CMP

PPi

CTP CDP

ADP

Sugar 2

Sugar 2–Sugar 1Sugar 1

Sugar 2–CMP

Pyr

PEP

PEP

Pyr

i

E1

E5 E2

E6

E2

E4

x Hyaluronic acid, n ~ 1500~

is of the complex oligosaccharides sialyl Lewis x (SLeX) and hyaluronic acid. This

r (e.g. UDP-glucose, UDP-galactose, UDP-N-acetyl glucosamine, UDP-N-acetyl

ar (e.g. CMP-sialic acid; right) used as a substrate for a glycosyltransferase reaction.

re, regeneration of the nucleoside phosphate sugar from its by-product nucleoside

re than one glycosyltransferase can be mixed together in a reaction vessel, with

ultiple glycosidic bonds in one pot. Indeed, the two saccharides shown here have



biosynthetic pathways, analogs of these natural productswith modified oligosaccharide motifs have been prepared[20]. Further in vitro glyco-randomization of the vanco-mycin core using sugar nucleotide libraries coupled withflexible glycosyltransferases associated with secondarymetabolites has produced vancomycin derivatives withincreased potency. [21] Similar approaches have beenimplemented to produce macrolide antibiotics and mightprove useful in the future production of libraries ofantibiotics bearing oligosaccharides, such as the enediyneand bleomycin classes of compound [20], and angucyclicanti-tumor drugs [22].Glycosidases. These enzymes are responsible for thecleavage of glycosidic bonds in vivo but can be used totransfer monosaccharides or oligosaccharides to sacchar-ide acceptors in vitro in a kinetic or thermodynamic mode.Although they lack the regiocontrol of glycosyltrans-ferases, glycosidases are relatively inexpensive, stableand readily available, as are their saccharide donors [16].Some of the regioselectivity issues can be overcome,however, with the selection of proper combinations ofenzyme and substrate [23]. Glycosidases also acceptvarious saccharide donors with different leaving groupsin a kinetic mode [24].

An efficient approach based on the protein engineeringof retaining b-glycosidases has been developed in whichthe a-face nucleophile is mutated from aspartic acid toalanine. The mutant enzyme obtained in this way, called‘glycosynthase’, has been shown to catalyze the formationof b-glycosidic bond using a-glycosyl fluoride as a sub-strate and has no detectable hydrolytic activity [25]. Thedevelopment of glycosynthetases has proved to be useful,and several examples of this utility have been providedincluding the synthesis of polysaccharides with cellulase[26] and the creation of a new class of thioglycoligases [27].

O OR

HO

HO

HO

HO

HO

Transferases

Enzyme recovery

Transferase 1

(a) Immobilized substrate

(b) Immobilized enzymes

Figure 3. Enzymatic saccharide synthesis is amenable to automation by approaches inv

sugars can be passed sequentially through polymer-bound saccharides, facilitating the pr

the growing saccharide chain can be used directly or attached to a water-soluble suppo


Sulfotransferases. A common modification of saccharidesand other biomolecules in nature is sulfonation [28].Modification of saccharides by sulfotransferases, whichinstall sulfate esters, is used to mediate inhibition andbinding in various biological pathways [29]. More recently,sulfatases have been shown to impart physiologicalsignals that are important for proper growth and develop-ment by cleaving sulfate esters from oligosaccharidechains [30]. Much remains unknown, however, about theeffect of this modification in nature [29,30].

Like other transferases, sulfotransferases are sensitiveto feedback inhibition by the product of their sulfonationreaction, 3 0-phophoadenosine-5 0-phosphate (PAP). Thesulfate donor, 3 0-phophoadenosine-5 0-phosphosulfate(PAPS), is also expensive, making these types of reactionideal candidates for regeneration of the donor in situ. Assuch, p-nitrophenyl sulfate has been successfully used toregenerate PAPS [28,29]. Indeed, the continued discoveryand characterization of sulfotransferases and sulfatasesshould provide useful tools for creating further diversity inoligosaccharide libraries, including those containing com-plex glycosaminoglycans, and should facilitate a greaterunderstanding of the roles of sulfonation in nature [29,30].Solid-phase synthesis. Similar to the chemical synthesis ofoligosaccharides, enzymatic synthesis is conducive toautomation through the use of solid phase. Solid-phaseenzymatic synthesis of oligosaccharides gives a distinctadvantage over either solution-phase synthesis or chemi-cal solid-phase synthesis: namely, facile purification ofthe product with stereo- and regiocontrol and without theneed for intermediary protecting groupmanipulation. Oneof two solid-phase methods can be applied: either theacceptor saccharide or the enzyme can be attached to thesolid support (Figure 3), although both enzymes and

Ti BS

O O

O OOO

O OOOO O

O OROOO OOO

Nucleotide sugarTransferase 1

Nucleotide sugarTransferase 2

Transferase 2 Transferase 3

olving immobilized substrate or immobilized enzymes. (a) Enzymes and nucleotide

oduction of complex oligosaccharides and the recovery of catalyst. (b)Alternatively,

rt and passed through columns of immobilized transferases.



acceptors have been used in conjunction on differingsupports [31].

When the acceptor saccharide is bound to the solidphase, the glycosyltransferases or glycosidases with theirsubstrate and any necessary cofactors are either runthrough a column containing the immobilized acceptor ormixed with a polymer-bound acceptor in a homogeneoussolution [32]. By running excess equivalents through acolumn or in solution, the reaction can be forced towardscompletion [33]. The use of resin-bound enzymes is wellaccepted in the synthesis of oligosaccharides and is knownto slow down deactivation by proteases, general denatura-tion and feedback inhibition. Incorporating the enzymeinto the solid phase also makes enzyme recovery muchmore facile [31,34–36]. Notably, not all enzymes that areconducive to solution-phase synthesis are applicable to thesolid phase owing to either denaturation or to stereo- orregiochemical constraints, and further development of thestrategy is necessary [37].

Glycoarrays

A principal challenge in cell biology is to define theinteractions that take place between oligosaccharides andproteins in many biological processes. A technique forattaching oligosaccharides to microplates or glass slides toproduce glycoarrays offers a solution to the high-through-put analysis and profiling of such interactions. Recentstudies have shown that glycoarrays have emerged as anew set of tools to facilitate both studies of carbohydrate–protein interactions and the identification of optimal sugarligands and inhibitors [38–49]. For example, N-acetylactosamine bound to microtiter plates has been treatedwith GDP-fucose and a fucosyltransferase to form Lex,which can be detected by a fucose-binding lectin. Thisformat has been used to identify potent inhibitors offucosyltransferase [46,50].

Glycoarrays have also found a use in the study ofinteractions between RNA and aminoglycosides [51]. Asmore genetic sequences and their functions have becomeavailable, RNA has emerged as a new target for drugdiscovery [52–54]. Glycoarrays based on aminoglycosidesoffer a platform for the high-throughput analysis ofinteractions between RNA and aminoglycosides or othersmall molecules.

In the future, glycoarrays might be used to comparethe glycosylation patterns between tumor cells and theirnoncancerous counterparts and could provide informationregarding signaling regulation, cellular transport, cata-lytic activity, targeting, protein fusion and binding, andother biological reactions [3]. In addition, glycoarrays canbe used to detect the presence of antibodies, T lymphocytesand other immune cells that recognize antigens associatedwith cancer and pathogens. One such antigen that iscommonly overexpressed in breast cancer is Globo-H(see above). Conjugating Globo-H or sections of the hexa-saccharide to carrier proteins induces an immuneresponse to the saccharide in humans, indicating thepotential use of this approach for cancer vaccines [55].Glycoarrays can be also used to monitor the level ofantibody in the blood after vaccination.


Glycoprotein synthesis

Protein glycosylation is perhaps the most complex post-translational event. More than 50% of human proteins areglycosylated, and this modification affects numerousprotein functions including folding, secretion, targeting,stability in circulation, and many other intercellularcommunication processes. Glycoproteins are often pro-duced as a mixture of glycoforms, however, making itdifficult to isolate individual species for studies ofstructure and function [5]. In addition, there is a lack ofeffective methods available for synthesizing glycoproteinswith a well-defined carbohydrate structure.

Recent advances in the field have provided some newstrategies to tackle this formidable problems [2]. As shownin Figure 4, several methods for synthesizing glyco-proteins in vitro are now available [56], includingremodeling of recombinant glycoproteins using glyco-sidases and glycosyltransferase [2]; ligation of syntheticglycopeptides by enzymatic [2] or chemical [57] methods;intein-mediated coupling of glycopeptides to larger pro-teins expressed as fusion proteins of intein [58,59];ligation of glycopeptides to larger proteins containingN-terminal cysteine expressed as tobacco etch virus (TEV)protease-cleavable fusion proteins [60]; in vitro trans-lation [61]; and pathway re-engineering in yeast systemsto produce human-type N-linked glycoforms [62,63].

Recently, in vivo suppressor tRNA technology has beenexploited for the recombinant production of neoglyco-proteins and glycoproteins [64,65]. Successful in vivoincorporation of unnatural amino acids in Escherichia colihas been achieved systematically by, first, evolving anorthogonal tRNA synthetase and tRNA pair fromMethanococcus jannaschii that are capable of acceptingand charging an unnatural amino acid onto the tRNACUA

in response to the stop codon TAG; and second, intro-ducing the stop codon (TAG) into a protein of interest thatfunctions to direct site-specifically incorporation of theunnatural amino acid [61] (Figure 5). By this method,p-acetyl phenylalanine has been incorporated into pro-teins and subsequently derivatized with aminooxy sac-charides to produce homogenous neoglycoproteins [64].

In addition, a naturally occurring homogeneous glyco-protein population has been produced inE. coli for the firsttime via the direct incorporation of the core glycosylaminoacidN-acetyl glucosamine-b-serine [65]. The glycoproteinswere easily isolated and the sugar chains were furtherelongated using glycosyltransferase in vitro. This methodhas been further extended to the synthesis of mucin-typeglycoproteins containing N-acetyl galactosamine [66].Although the current production level is relatively low,about 1–2 mg/l, this new method might lead eventually tothe development of fermentation method for the large-scale production of glycoproteins with well-defined carbo-hydrates at genetically controlled positions.

Concluding remarks and future perspectives

Development of synthetic oligosaccharide strategies hasreceived considerable attention over the past several yearsas the biological impact of oligosaccharides and glycocon-jugates has become increasingly apparent. Scientists nowhave the ability to synthesize complex oligosaccharides


AUC

OSerH2N

OOHO

HOAcHN

OH

5′3′

TGA-3′5′-AUGGUUCUGUAGGAAGGU

Homogeneousglycoprotein

Glyosyltransferase

Glyosyltransferase

Neoglycoprotein

ONH2

O NO

OOHO

HOAcHN

OH

Sugar

OAcOAcO

AcHNO

H2N COOH

OAc

H2N COOH

O

In vivo translationE. coli +

evolved MjTyrRS

Glycoprotein

evolved MjTyrRS

(ii) In vivo translation

Key:

(i) E. coli +

(a)

(b)

Figure 5. Homogeneous neoglycoproteins and glycoproteins can be produced by in vivo suppressor tRNA technology. An orthogonal tRNA synthetase and tRNA pair can be

evolved to accept and to supply the unnatural amino acidsN-acetyl glucosamine-b-serine (a) and p-acetyl phenylalanine (b) in response to the stop codon TAG during protein

biosynthesis in vivo. The ketone handle can be derivatized with aminooxy saccharides, glycosyltransferases are then used to form extended glycans. Abbreviation: MjTyrRS,

Methanococcus jannaschii tyrosine-tRNA synthetase.

Ti BS

OH3N

HS

CO2

H3N SR

O

Thioester exchange H3N

HS

ONH

OCO2

H3N COONH

OHS

Intein H3N CO2NH

OHS

FmocHN O

O

NH2

O

FmocHN

O

NH

NH2

O

CO2H3N

HS

H3N NH2

O

HS

ONH

CO2H3N ENLYFQ

TEV

Protease

Endoglycosidase

Subtilisin, DMF/H2O

Glycosyltransferase

Key:

Protein

Sugar

(ii) Glycosyltransferase

(i)


(i)


(i)

(a) Glycoprotein remodeling

(b) Glycoprotein semi-synthesis (N-terminal)

(c) Intein-mediated glycopeptide synthesis (C-terminal)

(d) Subtilisin-mediated glycopeptide synthesis

Figure 4. Methods developed for the in vitro synthesis of homogeneous glycoproteins. (a) Remodeling of protein glycoforms can be achieved by initially clipping

heterogeneous populations with a series of glycosidases down to a homogenous monoglycan core, followed by controlled re-elaboration with known glycosyltransferases

and glycosynthetases. (b) Chemically synthesized N-terminal glycopeptides can be coupled to larger synthetic or recombinant protein fragments bearing an N-terminal

cysteine, which can bemasked by a tobacco etch virus (TEV) protease site, by using native peptide ligation strategies. (c) Alternatively, C-terminal glycopeptides can be used

to swap out C-terminal intein–protein fusions in expressed chemical ligation. (d) Finally, proteases such as subtilisin can be optimized to catalyze peptide ligation.

Abbreviation: DMF, N,N-dimethyl formamide.





usingnewlydeveloped chemical andChemoenzymatic tools.Oligosaccharides can be created rapidly in a programmablemanner that should lead to larger libraries of oligosacchar-ide arrays for biological studies. Enzymes have facilitatedthe large-scale synthesis of difficult saccharide linkages andcomplex molecules such as glycoproteins, and newmethodshave been developed through directed evolution to produceglycoproteins in E. coli. Together, these approaches providea platform for glycobiology research and for the productionof glycoprotein- and other carbohydrate-containingpharmaceuticals.

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Getting animated

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Interaction ofBy E. Handman and D.V

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glycoproteins: towards the synthesis of GlyCAM-1 by using expressedprotein ligation. Angew. Chem. Int. Ed. Engl. 43, 1355–1359

60 Tolbert, T.J. and Wong, C-H. (2004) Conjugation of glycopeptidethioesters to expressed protein fragments: semisynthesis of glyco-sylated interleukin-2. Methods Mol. Biol. 283, 255–266

61 Hendrickson, T.L. et al. (2004) Incorporation of nonnatural aminoacids into proteins. Annu. Rev. Biochem. 73, 147–176

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63 Choi, B-K. et al. (2003) Use of combinatorial genetic libraries tohumanize N-linked glycosylation in the yeast Pichia pastoris. Proc.Natl. Acad. Sci. U. S. A. 100, 5022–5027

64 Liu, H. et al. (2003) A method for the generation of glycoproteinmimetics. J. Am. Chem. Soc. 125, 1702–1703

65 Zhang, Z. et al. (2004) A new strategy for the synthesis ofglycoproteins. Science 303, 371–373

66 Xu, R. et al. Site-specific incorporation of the mucin-type N-acetyl-galactomsamine-a-O-threonine into protein in E. coli. J. Am. Chem.Soc. (in press)

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ia: how can they invade other cells?Trends Parasitol. 20, 10.1016/j.pt.2004.04.009]e.bmn.com/supp/part/franzen.html

Leishmania with the host macrophage.R. Bullen [(2002) Trends Parasitol. 18, 332–334]ve.bmn.com/supp/part/swf012.html


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Chemoenzymatic synthesis of oligosaccharides and glycoproteins