The wall at 50 amino acids

Our earlier series on how peptides are made ended with a limitation. Building a peptide one amino acid at a time on a resin bead works beautifully for short chains. But each step is slightly imperfect, and those imperfections add up. Chapter 7 of Peptide Synthesis and Applications, from Annette Beck-Sickinger's group at the University of Leipzig, puts the practical ceiling at about 50 amino acids in reasonable yield and purity, depending on the sequence.

Many biologically important molecules are longer than that. Hormones, signalling proteins, enzymes. So how do chemists make them?

The answer is one of the most elegant ideas in modern chemistry. Instead of building the whole thing in one line, you make it in pieces and stitch the pieces together. This second series, "Beyond The Straight Chain", starts here, because this idea is what makes much of the rest possible.

The old way: protect everything, then join

Joining two peptide segments is not new. The introductory chapter explains the classic approach, called segment condensation: make two fully protected fragments, purify each, then couple them.

The problem is that fully protected peptides are often poorly soluble. And the activated end of a peptide segment is prone to flipping into the wrong mirror-image form (epimerisation) at the joining point. It can work, but it often needs extensive optimisation.

The breakthrough: join them unprotected

The better idea is to find a reaction so specific that it only happens between two particular chemical groups, one on each fragment, and ignores everything else. Then you do not need to protect anything.

Chemists call this chemoselective ligation. The Leipzig chapter tells the history, which the authors describe as a "fabulous history", with genuine enthusiasm.

1992: Schnölzer and colleagues made the first fully synthetic HIV protease by joining two unprotected fragments. The link they created was not quite a natural peptide bond, but it worked.

1994: Dawson and colleagues published the preparation of interleukin-8, an immune signalling protein, by joining two unprotected segments to form a completely normal peptide bond. Because the backbone was fully natural, they named the method native chemical ligation.

How native chemical ligation works

The requirements are simple, which is why the method spread so quickly:

  • The first fragment must end with a thioester, a sulfur-containing reactive group at its C terminus.
  • The second fragment must begin with a cysteine.

Mix them in water at a suitable pH and two things happen. First, the sulfur on the cysteine swaps onto the thioester, temporarily linking the two fragments through sulfur. That step is reversible. Then, the chain rearranges on its own: the nearby nitrogen of the cysteine takes over the link, forming a normal peptide bond. That step is irreversible.

The result is a single, longer chain with a completely natural backbone, and a cysteine at the join.

The chapter notes the idea traces back to an earlier observation by Wieland and colleagues, who saw an amino acid jump from a thioester onto cysteine and then rearrange. Decades later, that curiosity became a way to build proteins.

Making the thioester is the hard part

The catch is making the fragment with the thioester end. Thioesters do not survive the repeated base treatments used in standard Fmoc synthesis. That is one reason, as our earlier series noted, some labs still use the older Boc method with its hazardous hydrogen fluoride.

Chapter 8 of the book is devoted entirely to clever ways of making thioesters using Fmoc chemistry, including "safety-catch" linkers that stay inert during synthesis and are then chemically switched on at the end to release the peptide as a thioester.

Expressed protein ligation: half biology, half chemistry

The Leipzig chapter describes a powerful variation called expressed protein ligation. One fragment, usually the large one, is made by genetically engineered bacteria. The other, usually small, is made by chemical synthesis and can carry anything the chemist wants: a fluorescent dye, an unnatural amino acid, a chemical tag.

The two are then joined by native chemical ligation. The chapter uses this to make a modified version of SDF-1α (also called CXCL12), a chemokine, which is a signalling protein that guides the movement of immune cells and stem cells. By attaching probes at the C terminus, researchers can study how it behaves using imaging and spectroscopy techniques.

Getting around the cysteine requirement

Native chemical ligation needs a cysteine at the join. Many proteins do not have one in a convenient place. The chapter describes how chemists have worked around this:

  • Using selenocysteine, the so-called 21st amino acid, which reacts the same way but faster.
  • Joining at a cysteine and then chemically converting it into alanine afterwards, which effectively removes the fingerprint of the join.

Why this matters to you

Native chemical ligation is one of the reasons chemists can now make modified proteins that nature never produced, with labels, tags and unnatural building blocks at precise positions. It turned protein chemistry from "whatever cells can make" into something closer to "whatever we can design".

It also sets up the rest of this series. The following parts look at the other ways chemists go beyond a simple straight chain: forming bridges, closing rings, adding methyl groups, fats, sugars and phosphates. Each changes what a peptide can do, and each explains something about peptide medicines you may already know.

This series explains laboratory chemistry for general readers. It is not a guide to making or using any compound. Nothing here is medical advice.