Part of what distinguishes us from bacteria is that the proteins in our bodies are decorated with elaborate arrays of sugars. Protein glycosylation — the attachment of sugars to the amino-acid building-blocks of proteins — plays a crucial role in such diverse processes as protein folding, cell–cell communication and viral invasion of cells. Yet it is conspicuously absent in many simple, unicellular organisms. Understanding the roles of these sugars and how their complex, disparate structures modulate the activities of proteins has been a long-standing challenge. Reporting in the Journal of the American Chemical Society1, Brik and colleagues bring us a step closer to this goal by devising a clever strategy for generating glycopeptides — short sequences of amino acids with sugars attached — that may one day permit the tailored synthesis of glycoproteins.

Glycopeptides and glycoproteins are notoriously difficult to obtain as pure compounds, because they are naturally expressed as inseparable mixtures of different structures (glycoforms) that bear various sugars. This complexity makes it difficult to study how any specific glycoform affects a protein's function, which in turn complicates efforts to generate protein-based medicines. Indeed, most therapeutic glycoproteins are sold as mixtures of glycoforms, the active components of which are often unknown. One approach to solving this problem is to use chemical synthesis to create single structures.

Brik and colleagues1 have now developed a strategy for assembling glycopeptides using a process known as peptide ligation. In their method, one peptide is attached to another that incorporates a modified sugar. A unique feature of this approach is that the sugar assists the process by positioning the two peptides in close proximity to each other. Traditional glycopeptide synthesis is cumbersome, requiring excesses of reagents to drive reactions to completion, and often producing low yields of the desired products. Furthermore, strategies involving 'protecting groups' have been necessary to mask reactive chemical groups that do not participate directly in the reaction sequence. These requirements increase the complexity and the cost of glycopeptide synthesis. But by actively engaging a sugar in the ligation process, Brik et al. demonstrate that a variety of glycopeptides can be made in just a few steps and in high yield, without the need for protecting groups.

The authors' strategy is a clever twist on a well-established method for peptide synthesis known as native chemical ligation2. In this process, two peptide fragments are joined together to form a larger fragment via a two-step mechanism. The first step involves the transient formation of a thioester bond between the two fragments (Fig. 1a), mediated by a reactive sulphur atom on one of the fragments. The resulting intermediate then undergoes a rapid, spontaneous rearrangement to form a peptide bond. The net result is the direct connection of two peptide fragments to form a larger polypeptide. Moderately sized proteins have been produced in this way by sequential ligation of several peptide fragments, or through the coupling of a peptide to a larger protein fragment. Crucially, native chemical ligation provides exquisite control over the protein structure being formed, and allows the incorporation of various useful groups — such as synthetic amino acids, biophysical probes or stable isotopes of atoms used for structural studies — into selected sites within proteins3,4.

Figure 1: Glycopeptide synthesis.
figure 1

a, Native chemical ligation is a well-established method for preparing peptides. A reactive sulphur atom (red) on the side-chain of a cysteine amino acid attacks another peptide (where R is typically a phenyl ring), producing a thioester intermediate that spontaneously rearranges to yield a peptide bond. b, Brik et al.1 have modified this method to prepare glycopeptides, in which sugars are attached to peptide chains. A reactive sulphur atom (red) attached to an appended sugar (green) acts as a surrogate for the cysteine side-chain. Peptide bonds can thus be formed between a greater variety of amino acids. R1 represents an amino-acid side-chain.

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Building on this approach, Brik and colleagues1 attached a reactive sulphur group to a sugar within a peptide (Fig. 1b). In a process similar to the two-step mechanism for native chemical ligation, the authors reacted this sulphur group with a second peptide to form a thioester intermediate. This intermediate subsequently rearranges to give the desired product, in which the two starting materials are linked by a peptide bond. This strategy1 has several remarkable features. Native chemical ligation requires cysteine — a sulphur-containing amino acid — to be at the reacting end of one of the peptides being joined together. By placing a reactive sulphur group on the sugar of a glycopeptide, rather than in an amino acid, the authors circumvent this requirement, thus allowing bonds to be formed between a broader range of amino acids.

Moreover, a surprisingly wide array of amino acids is tolerated at the reaction site, thus permitting access to glycopeptides that are difficult to synthesize using other methods. Amino acids with small side-chains and those (such as histidine or aspartate) with side-chains that can serve as a base in the ligation pathway are favoured substrates in the reaction. Finally, the sulphur atom on the sugar provides a convenient handle for subsequent chemical manipulation — for example, it can be removed to give a naturally occurring sugar, reacted to append fluorescent dyes or other groups to the glycopeptide, or elaborated to form more complex sugars by using glycosyltransferase enzymes1.

Further investigations are needed to assess the full scope of Brik and colleagues' reaction1 and its potential application to glycoprotein synthesis. Nonetheless, the emergence of this and other methods5,6,7,8 for constructing pure peptides and proteins with sugars installed at preselected sites has many implications. For example, such techniques could transform the way therapeutic glycoproteins are discovered, developed and manufactured. Many of these proteins are obtained only as a mixture of glycoforms, just a fraction of which may be biologically active9. But if drug-regulation authorities start to impose stringent regulations on glycoproteins (as they currently do for traditional 'small molecule' drugs, where the purity of the active form is paramount), then single glycoforms will be required. Furthermore, the ability to fine-tune the biological properties of therapeutic proteins by modifying their attached sugars could lead to exciting advances in drug discovery.

More fundamentally, having access to pure glycoproteins would help to elucidate the role of specific sugars in regulating protein structure and function. This could help to reveal how bacteria manage without these sweet appendages. Brik and colleagues' method1 for making pure glycopeptides (and possibly glycoproteins) is truly a milestone achievement in this rapidly developing field.