Recent advances in the chemical synthesis of mucin-like glycoproteins

Lisa A. Marcaurelle and Carolyn R. Bertozzi1

Center for New Directions in Organic Synthesis, Departments of Chemistry and Molecular and Cell Biology, and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA

Accepted on March 11, 2002;

Abstract

This purpose of this mini review is to familiarize readers with the tools currently available for the synthesis of mucin-type glycoproteins. The article will highlight recent approaches to the synthesis of glycopeptide fragments bearing complex O-linked glycans, as well as new strategies for the generation of full-length glycoproteins.

Key words: chemoselective/enzymatic/glycopeptide/mucin/solid-phase synthesis

Introduction

The decoration of serine and threonine residues with {alpha}-linked glycans, initiated by N-acetylgalactosamine (GalNAc), is a ubiquitous posttranslational modification affecting numerous proteins in mammals and other eukaryotes. Because {alpha}-GalNAc-based glycans are abundant in mucins, this form of O-linked glycosylation is often referred to as "mucin-like" glycosylation (Hanisch, 2001Go). Mucins are cell surface or secreted proteins that contain dense clusters of glycosylated serine and threonine residues. Mucins are abundantly produced by epithelial cells that are specialized for mucus production, where they reside at the interface with the extracellular environment. As depicted in Figure 1, branching of the mucin core GalNAc residue (1) can occur at position 3 and/or position 6 to give rise to some common core structures (cores 1–8). In many cases these structures are modified by sulfation and by the addition of NeuAc, Fuc, and/or repeating units of galactose and GlcNAc to give poly-N-acetyllactosamine (LacNAc) chains. It is important to note that GalNAc-based glycans are not restricted to classical mucins, as some proteins contain only discrete O-glycosylated domains in which case they are said to be "mucin-like."



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Fig. 1. Core structures of mucin-type O-linked glycans.

 
Mucin-type oligosaccharides are known to serve as important recognition elements, mediating a multitude of cell–cell interactions. For example, mucin-like glycoproteins carry many of the Lewis and blood group antigens, and serve as ligands for L- and P-selectin during an inflammatory response (McEver and Cummings, 1997Go). In addition to serving as receptor-binding ligands, O-linked glycans can have a profound influence on the structure and stability of the protein to which they are attached. The importance of mucin-type glycosylation has inspired much interest in the synthesis of well-defined glycoproteins for structural and functional studies (Herzner et al., 2000Go). Due to the association of certain mucin-related structures with tumor progression, there has also been a strong interest in the generation of glycopeptide-based tumor vaccines (Danishefsky and Allen, 2000Go).

This article will highlight recent progress in the chemical synthesis of mucin-like glycoproteins with an emphasis on the construction of glycopeptide fragments containing complex O-glycans and the synthesis of full-length O-linked glycoproteins. These synthetic molecules provide homogeneously glycosylated materials for biological and biophysical studies, and some, such as the synthetic vaccines, may find therapeutic applications in the near future.

Chemical synthesis of glycopeptides with complex O-linked glycans

The most common approach to the synthesis of mucin-like glycopeptides involves the use of a suitably protected O-glycosyl amino acid (2, Figure 2) as a building block in solid-phase peptide synthesis (SPPS). In general, the use of fluorenylmethoxy carbonyl (Fmoc)-based chemistry (Fields and Noble, 1990Go) is preferred over tert-butoxycarbonyl (Boc)-based chemistry for the preparation of glycopeptides, because the reaction conditions of the former are more compatible with the presence of acid-sensitive glycosidic bonds; the use of Fmoc-based chemistry avoids repeated exposure to trifluoroacetic acid (TFA) and final deprotection with HF (as used in Boc-based methods). Typically the hydroxyl groups of the pendant glycan are protected as acetyl or benzoyl esters, which can easily be removed by treatment with sodium methoxide or hydrazine following cleavage of the assembled glycopeptide from the resin.



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Fig. 2. General strategy for the synthesis of O-linked glycopeptides by SPPS.

 
Methods for the preparation of simple GalNAc-{alpha}-O-Ser/Thr building blocks for the assembly of mucin-type glycopeptides are well developed and for the most part are based on methodology introduced by Paulsen et al. (1978)Go. GalNAc derivatives 7 and 8 (Scheme S01) are generally prepared using a glycosyl donor (4) that has a nonparticipating azido group at C-2 and a leaving group, such as a halogen, at the anomeric center. The resulting Fmoc-glycosyl amino acids can be incorporated into peptides using standard coupling procedures, such as activation with dicyclohexylcarbodiimide/1-hydroxybenzotriazole or 2-(1H-benzotriazole-1-yl)-1,2,3,3-tetramethyluronium hexafluorophosphate/1-hydroxybenzotriazole. Alternately, "active esters," such as pentafluorophenyl esters can be used. Because building blocks 7 and 8 are now commercially available (from BACHEM and Oxford Glycosciences), simple O-linked glycopeptides can be made even by those with limited expertise in synthetic chemistry.



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Scheme 1. Synthesis of GalNAc derivatives 7 and 8.

 
Though the assembly of glycopeptides bearing simple {alpha}-GalNAc residues is a painless undertaking requiring little experience in carbohydrate chemistry, the construction of glycopeptides containing more elaborate O-linked glycans is still a daunting task. Nonetheless, some impressive mucin-related glycopeptides have been generated through the use of complex glycosyl amino acids. The most common approach that has been adopted for the preparation of these building blocks is outlined in the following paragraph.

One of the main challenges in the synthesis of O-glycosyl amino acids is achieving high stereoselectivity in the formation of the core {alpha}-O-Ser/Thr linkage. Even with simple monosaccharide donors, such as 4, the outcome of the glycosylation reaction can be difficult to predict, often proceeding with only moderate {alpha}-selectivity. This problem is even more pronounced when dealing with large oligosaccharide donors. As a result, the most commonly employed method for the synthesis of complex O-glycosyl amino acids involves the installation of the desired {alpha}-O-Ser/Thr linkage prior to elaboration of additional sugars from the core GalNAc moiety. As outlined in Figure 3, the difficult glycosylation reaction is generally performed with a simple monosaccharide donor (9), and the branching carbohydrate residues are appended to the resulting {alpha}-glycosyl amino acid (10) or {alpha}-O-linked "cassette." Such an approach has been described by several groups for the construction of a variety of mucin-related structures. Meldal and co-workers (Meinjohanns et al., 1996Go; Mathieux et al., 1997Go) first used this cassette methodology for the synthesis of building blocks 14–17 (Figure 4), which correspond to four common O-linked core structures (cores 1–4, Figure 1). The synthesis of all four building blocks required the use of only two glycosyl donors (18 and 20) and one selectively protected {alpha}-O-GalNAc-Thr cassette (19), making this a highly efficient route to the core O-linked glycosyl amino acids. Building blocks 14–17 were used for the construction of a series of decapeptides corresponding to repeating units of the mucins MUC-2 and MUC-3.



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Fig. 3. Synthesis of complex O-glycosyl amino acids by the cassette method.

 


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Fig. 4. Retrosynthesis of building blocks 14–17 (cores 1–4) generated by the cassette method.

 
In a series of recent reports, Danishefsky and colleagues described the use of a related cassette-based approach for the preparation of a variety of complex building blocks bearing tumor-related antigens (Kuduk et al., 1998Go; Schwarz et al., 1999Go; Glunz et al., 2000Go). The target glycosyl amino acids 21–23 (Figure 5), which correspond to the sialyl TN antigen, glycophorin, and Lewis y, respectively, were generated ultimately from Thr and Ser cassettes 24 and 25. Glycosyl amino acids 21 and 23 were incorporated into clustered O-linked glycopeptides as part of a program aimed at the development of anti-tumor vaccines (Danishefsky and Allen, 2000Go).



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Fig. 5. Mucin-related building blocks 21–23, which were generated from {alpha}-O-linked cassettes 24 and 25.

 
As highlighted by these examples, cassette-based strategies have proven to be an efficient method for the assembly of mucin-type glycopeptides carrying defined O-linked glycans. Recent advances in oligosaccharide synthesis, including the use of automated solid-phase methods (Plante et al., 2001Go; Seeberger and Haase, 2000Go) and programmable one-pot methods (Koeller and Wong, 2000Go), should facilitate the production of complex glycosyl amino acids for cassette-based strategies and other related approaches (Winterfeld and Schmidt, 2001Go; Herzner et al., 2000Go).

Chemoenzymatic synthesis of glycopeptides with complex O-linked glycans

Glycosyltransferases are powerful synthetic tools for the construction of defined carbohydrate structures, especially in the context of richly functionalized glycopeptides and proteins (Koeller and Wong, 2000Go). The enzymatic transfer of individual monosaccharides to preformed glycopeptides containing simple O-linked glycans is an attractive alternative to the total chemical synthesis of large glycosyl amino acids. Recently, enzymes have been employed for the synthesis of some impressive O-linked structures, most notably several glycopeptides corresponding to the P-selectin glycoprotein ligand-1 (PSGL-1). PSGL-1 is a dimeric, membrane-bound mucin expressed on leukocytes, where it serves to bind to the selectins and initiate the inflammation-adhesion cascade (McEver and Cummings, 1997Go). Interest in the synthesis of this glycoprotein has come from the need for homogeneous material for elucidating the structural requirements for high-affinity binding to P-selectin (Somers et al., 2000Go). In addition, soluble versions of PSGL-1 are of interest as P-selectin inhibitors (Leppänen et al., 1999Go; Koeller et al., 2000Go).

In two recent papers from Cummings and co-workers, the chemoenzymatic synthesis of a panel of PSGL-1 fragments was described (Leppänen et al., 1999Go, 2000). One of the target glycopeptides (29), shown in Scheme S02, contained three sulfated tyrosine (TyrSO3) residues and a core 2–based glycan capped with a sialyl Lewis X (sLex) motif. This glycopeptide represents a partial structure of the N-terminus of PSGL-1 that is known to be important for binding to P-selectin. The synthesis of fragment 29 began with the incorporation of a single GalNAc residue into the peptide using commercially available Fmoc-Thr-({alpha}-Ac3GalNAc)OH (7), as previously discussed. Following cleavage and deprotection of the glycopeptide (26), the appropriate glycosyltransferases were used to elaborate the target hexasaccharide bearing the sLex motif. Sulfation of the three tyrosine residues was also carried out enzymatically using a recently cloned tyrosylprotein sulfotransferase (Ouyang et al., 1998Go). In their most recent report, the authors chose to install the TyrSO3 residues prior to enzymatic glycosylation, using the building block Fmoc-Tyr(SO3)-OH during SPPS (Leppänen et al., 2000Go). Chemical incorporation of the TyrSO3 residues has some advantages over enzymatic sulfation in that the required Tyr derivative is commercially available, whereas the sulfotransferase is not. Even so, care must be taken when using the synthetic approach to sulfotyrosyl peptides due to the acid-sensitive nature of the phenolic sulfate esters; generally, the cleavage and deprotection of the peptide with TFA must be performed at low temperatures (0–4°C) to avoid loss of the sulfate moiety (Kitagawa et al., 2001Go).



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Scheme 2. Synthesis of PSGL-1 fragment 29.

 
The synthesis of a related PSGL-1 fragment (32) has recently been described by Wong and colleagues (Koeller et al., 2000Go) (Scheme S03). Their approach differs from the method reported by Leppänen et al. in that their synthesis began with a synthetic glycopeptide (31) carrying an {alpha}-O-linked disaccharide rather than a simple monosaccharide. Thus the synthesis of the target glycopeptide (32) required the preparation of building block 30. One advantage of this strategy is that it does not require the use of either the core 1 ß1,3-GalT or the core 2 ß1,6-GlcNAcT to elaborate the sLex moiety. This is significant because neither of these enzymes is currently available commercially. Thus, amino acid 30 is a useful building block for generating C-6 branched O-linked glycans when used in combination with enzymatic techniques.



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Scheme 3. Synthesis of PSGL-1 fragment 32.

 
Synthesis of O-linked glycopeptides by chemoselective ligation

The attachment of preformed oligosaccharides to simple glycopeptides by the technique of chemoselective ligation is an attractive method for the rapid assembly of peptides carrying complex O-glycans. Chemoselective ligation reactions are mild and selective, allowing for the coupling of unprotected biomolecules, such as peptides and carbohydrates, in an aqueous environment (Lemieux and Bertozzi, 1998Go; Marcaurelle and Bertozzi, 1999Go; Hang and Bertozzi, 2001Go). Chemoselective ligation reactions offer advantages similar to those of enzymatic reactions—they tolerate a diverse array of functional groups, thereby minimizing the need for protecting groups, but have the potential of a much broader range of substrates for use as coupling partners. Such an approach has recently been employed for the synthesis of O-linked glycopeptide mimetics that possess unnatural bonds at the branch points (C-6 and C-3) of the core GalNAc but retain the native sugar–peptide linkage. As depicted in Figure 6A, a simple O-linked glycopeptide (33) bearing a single GalNAc residue was selectively oxidized using the commercially available enzyme galactose oxidase to generate the C-6 aldehyde (34). Chemoselective ligation with aminooxy-functionalized sugars (35) yielded higher-order glycans (36) bearing an unnatural oxime-linkage (Rodriguez et al., 1997Go). To generate glycopeptides with oligosaccharides extended at C-3 of the core GalNAc residue, glycosyl amino acid 37 (Figure 6B) was synthesized (Marcaurelle and Bertozzi, 2001Go). Building block 37 has a protected thiol group in place of the C-3 hydroxyl group of GalNAc. Following incorporation of 37 into a glycopeptide (38) by Fmoc-based SPPS, the deprotected thiol group was selectively alkylated with N-bromoacetamido sugars (39). These two orthogonal ligation reactions (A and B) could potentially be used in parallel for the one-pot assembly of glycopeptides carrying biantennary O-linked glycans.



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Fig. 6. Synthesis of oxime (A) and thiother-linked (B) glycopeptide mimetics by chemoselective ligation.

 
The process of constructing oxime and thioether-linked glycopeptide mimetics is considerably easier than generating the corresponding glycosyl amino acid building blocks for use in peptide synthesis. The synthesis of aminooxy- and N-bromoacetamido sugars is straightforward; even complex structures, such as sulfated and sialylated oligosaccharides (41–43; Figure 7), can be accessed via chemical and enzymatic methods (Rodriguez et al., 1998Go; Marcaurelle et al., unpublished data). How these unnatural glycans compare with their native counterparts at the structural and functional level is a topic of current investigation. Although a structural modification of the native glycosidic bond has been engendered in these analogs, their considerable synthetic facility allows one to generate such analogs rapidly and in large scale. In cases where the precise structure of the core region of the O-linked glycan of interest is not a determinant of bioactivity, these practical analogs might be preferred to their more synthetically difficult native counterparts.



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Fig. 7. Examples of aminooxy- and N-bromoacetamido sugars for use in chemoselective ligation.

 
Synthesis of full-length glycoproteins by native chemical ligation

The methods described have been used primarily for the construction of relatively short glycopeptide fragments (~20 amino acids). In nature, of course, mucin-type oligosaccharides are found on proteins that far exceed this size. The technique of native chemical ligation (NCL) has found widespread use in the field of protein chemistry for the synthesis of large unglycosylated proteins (Dawson and Kent, 2000Go). The method involves the condensation of two unprotected peptide segments, one bearing a C-terminal thioester and the other an N-terminal cysteine residue, to afford a protein with a native amide bond at the ligation site. The NCL reaction is mild, selective, and compatible with the presence of O-linked glycans, suggesting that extension to glycoprotein synthesis should be feasible. Indeed, this method has recently been applied to the synthesis of two O-linked glycoproteins, lymphotactin (Lptn) (Marcaurelle et al., 2001Go) and diptericin (Shin et al., 1999Go).

Lptn is a 93-amino-acid chemokine that is a potent chemoattractant for both T cells and natural killer cells (Dorner et al., 1997Go; Hedrick et al., 1997Go). An unusual feature of Lptn is a small, mucin-like domain located at its C-terminus; relatively few chemokines are extensively O-glycosylated. To investigate the structural and functional significance of this domain, the synthesis of glycosylated Lptn (46, Figure 8) was undertaken. The strategy required the construction of a 47-residue peptide-{alpha}-thioester (44) and a 46-residue glycopeptide (45) with 8 {alpha}-GalNAc residues. The thioester fragment (44) was synthesized using traditional Boc-based methods, and Fmoc-based chemistry was employed for the synthesis of glycopeptide 45. Ligation of the two fragments proceeded smoothly to give the glycosylated chemokine (46), which was biologically active as assessed by a standard calcium mobilization assay. This NCL strategy provided milligram quantities of homogeneous glycoprotein for both structural and functional studies.



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Fig. 8. Synthesis of glycosylated Lptn by NCL.

 
The second O-linked glycoprotein that has been synthesized by NCL, diptericin, is an 82-residue antimicrobial protein that contains two sites of O-linked glycosylation at Thr11 and Thr54 (Scheme S04) (Bulet et al., 1995Go). To synthesize diptericin by NCL, a glycopeptide-{alpha}-thioester (48) was needed. Because glycopeptides must be synthesized using Fmoc-based chemistry, established Boc-based methods for generating peptide-{alpha}-thioesters could not be used. Thus, a new synthetic route to peptide-{alpha}-thioesters by Fmoc-based SPPS was developed (Shin et al., 1999Go). The strategy capitalized on Ellman’s modification of Kenner’s sulfonamide "safety-catch" linker (Scheme S04). The C-terminal residue of the glycopeptide was attached to the resin via an acid- and base-stable N-acyl sulfonamide linkage. Following standard Fmoc-based SPPS, the sulfonamide (47) was activated by cyanomethylation and then cleaved with a thiol nucleophile, benzyl mercaptan, to give the protected glycopeptide-{alpha}-thioester. Removal of the amino acid side chain protecting groups with TFA yielded the desired glycopeptide-{alpha}-thioester (48) for use in NCL with glycopeptide 49. Using this straightforward approach, the total chemical synthesis of diptericin (50) was achieved.



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Scheme 4. Synthesis of diptericin.

 
Semi-synthesis of large glycoproteins by expressed protein ligation

Even in conjunction with native chemical ligation, the limitations of SPPS make proteins larger than 20 kDa in size difficult to access synthetically. Expressed protein ligation (EPL), a method for generating recombinant thioesters that is based on the phenomenon of protein splicing (Noren et al., 2000Go) has been used to a great extent for the semi-synthesis of large biologically active proteins (Muir et al., 1998Go; Muir, 2001Go). The application of EPL to the synthesis of glycoproteins is advantageous because it enables the fusion of synthetic glycopeptides with recombinant protein fragments. Recently this method has been used for the construction of GlyCAM-1, a 132-residue endothelial-derived ligand for L-selectin (Lasky et al., 1992Go). To elucidate the importance of its two mucin domains, a panel of GlyCAM-1 glycoforms was constructed by EPL (Macmillan and Bertozzi, 2000Go; Macmillan et al., unpublished data). The strategy used for the semi-synthesis of GlyCAM-1 involved the tandem ligation of three fragments as depicted in Figure 9. The required synthetic glycopeptides, 52 and 54, were generated by Fmoc-based SPPS through the use of building blocks 7 and 8 (see Scheme S01). The safety-catch method of Shin et al. (1999)Go was used for the construction of the glycopeptide thioester (54). The recombinant thioester fragment 51 was generated using a commercially available intein-mediated expression system. Ligation of the three fragments yielded the target GlyCAM-1 (55) containing 12 O-linked GalNAc residues.



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Fig. 9. Tandem ligation approach for the construction of GlyCAM-1. The recombinant thioester fragment (51) was generated using the commercially available pTYB-1 expression vector. After affinity purification of the fusion protein 51 using chitin resin, the chemical ligation step was performed by incubating the resin-bound protein with glycopeptide 52 in the presence of 2-mercaptoethanesulfonic acid. Factor Xa was used to expose the N-terminal cysteine residue of the resulting protein, which was then ligated to glycopeptide-thioester 54 to give the fully glycosylated GlyCAM-1.

 
Conclusions

As illustrated by the examples presented in this mini review, there currently exist powerful chemical and enzymatic methods for the construction of mucin-type glycopeptides bearing complex O-glycans. The recent application of techniques developed by protein chemists, such as native and expressed protein ligation, has also facilitated the generation of full-length glycoproteins bearing simple, yet defined O-linked glycans. Work by Wong and co-workers indicates that this strategy should also be suitable for the synthesis of N-linked glycoproteins (Tolbert and Wong, 2000Go). Through a combination of the approaches described here, the synthesis of large mucin-type glycoproteins bearing complex oligosaccharides should be possible.

Acknowledgments

The Center for New Directions in Organic Synthesis is supported by Bristol-Myers Squibb as Sponsoring Member. The authors’ work presented in this review was supported by a grant from the National Science Foundation (CAREER Award CHE-9734439). L.A.M. was supported by a predoctoral fellowship from the American Chemical Society Division of Organic Chemistry.

Abbreviations

Boc, tert-butoxycarbonyl; EPL, expressed protein ligation; Fmoc, fluorenylmethoxy carbonyl; LacNAc, N-acetyllactosamine; Lptn, lymphotactin; NCL, native chemical ligation; PSGL-1, P-selectin glycoprotein ligand-1; SPPS, solid-phase peptide synthesis; TFA, trifluoroacetic acid; TPST-1, tyrosylprotein sulfotransferase-1.

Footnotes

1 To whom correspondence should be addressed; E-mail: bertozzi{at}cchem.berkeley.edu Back

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