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Coupling Reagents

Peptide synthesis is an organic chemistry process in which peptides are produced. A peptide is an organic compound made up of several amino acids that are linked together using amide bonds. If this occurs through a biological process then it is called protein biosynthesis.

Peptides are made via the coupling of one amino acids carboxyl group to the amino group of a second amino acid. To prevent unexpected or unintended reactions, there are protecting groups employed to limit which groups couple with each other.

The Different Kinds of Peptide Synthesis

There are several different kinds of peptide synthesis. Traditionally, liquid phase synthesis was used in laboratories, and this method is still commonly used when peptides are being synthesized for industrial purposes, but for laboratory use solid-phase peptide synthesis is now more popular.

Solid phase synthesis was developed by Robert Bruce Merrifield. His pioneering work made for some massive leaps forward in peptide synthesis technology, and now SPPS is considered to be the standard technique for making proteins and peptides in modern laboratories. The main reason that it is so popular is that it makes it possible to make peptides that would be too difficult to make using bacteria.

It is possible to incorporate amino acids that are unnatural, to modify the backbone of the protein or peptide, and to make D-proteins containing D-amino acids.

Solid phase peptide synthesis (SPPS) is a process that relies on several repeated cycles of peptide coupling, washing, deprotection and then more washing. Two amino acids are coupled and washed, then deprotected to reveal an N-terminal amine so that another amino acid can be coupled to the existing chain.

There are automated solid phase synthesizers available, but a lot of laboratories perform SPPS by hand to ensure maximum possible yields.

Using Peptide Coupling Reagents

To facilitate the coupling of the two amino acids, a peptide coupling reagent is used. There are several different kinds of coupling reagents, including carbodiimides, aminium, phosphonium, uronium and others.

The most successful and widely used peptide synthesis methods use uronium or guandidium salts as their peptide coupling reagents. Carbodiimide mediated peptide coupling is a popular process which relies on benzotriazole derivatives such as HOAt and HOBt.

However, scientists are always looking for new peptide coupling reagents and more efficient processes. One recent study conducted by Prof. Fernando Albericio and Prof. Ayman El-Faham groups has found that incorporating a hydrogen bond acceptor as part of the iminium coupling reagent can produce a significant performance increase.

Bear in mind that if there is a 95 percent yield at each step, then that means a five percent loss – five percent per coupling in a 26 chain peptide would amount to a significant amount of waste. Every percentage increase in efficiency is important.

The groups also found that by replacing one dimethylamino moiety with a morpholino group that is more polar, the enhancement was improved even more.

Another thing that the scientists have been looking at is improving the safety of activating agents. An unusual property of HOBt derivatives is that they could potentially be made into explosives.

Because they are classified as explosives, they are difficult to store and shipping them is expensive. This means that laboratories are eager to find alternative coupling reagents that are equally effective but safer to handle. The El-Faham and Albericio researchers have showed that OxymaPure® is potent and makes a safer alternative to HOAt and HOBt.

This reagent appears to be good at inhibiting racemization when used as an additive for carbodiimide mediated peptide synthesis. Its coupling efficiency, in initial testing, proved superior to that of HOBt and comparable to that of HOAt in both liquid and solid phase peptide coupling. In addition, there appear to be only minimal thermal risks.

Another interesting option is COMU®. This appears to be a safe and efficient reagent. COMU is only found in the reactive uronium structure, but it appears to provide equivalent or better peptide yield than HATU, which is currently thought of as the gold standard in terms of peptide yield.

In pentapeptide yield tests, HBTU produces yields of just 47%, while HATU produces much better yields of 83%. HOTU, the current industry standard, offers 99% yields, and preliminary testing of COMU shows yields of 99.7%.


COMU has remarkable properties, offering fast coupling that is very efficient, and almost no tendency for racemization. Using COMU appears to reduce the risk of Epimerization during coupling as compared to the use of HATU or HOBt. Another benefit of COMU is that it is quite soluble, and it is stable in most of the popular solvents that are used for peptide coupling.

Solvents such as DMF and NMP are used in most laboratories as well as for industrial peptide synthesis. COMU’s relative stability means that it is perfect for use in SPPS and that it can also be used for liquid synthesis. The by-products that are formed by COMU during the synthesis process are water-soluble, so are easy to extract when necessary.

The solution will change color when the reaction takes place, and this side-effect is also a nice benefit because it allows for colorimetric or visual monitoring of the reaction. COMU is considered to be non-explosive so it is much cheaper to ship and much easier to store than many of the traditional regents.

COMU can be used under essentially the same protocols as those that are applied to coupling reagents that are popular in labs and industrial synthesis plants today, including TBTU, HATU, HBTU and PyBOP. Where racemization is an issue, an equivalent of base can be used alongside COMU. The polar morpholino group can contribute as a base.

A Long History of Research

Chemists have been struggling with the issue of synthesizing proteins for more than a century, and Merrifields work from the 1960s was one of the biggest leaps forward in the field. Since then, we have made only incremental improvements in the way that proteins are synthesied, and making long chain proteins is still a very slow and labor intensive process.

While the risk of racemization has been reduced and the discovery of COMU has helped with the issue of solubility of the intermediate synthetics, the fact that most laboratories still prefer to do manual synthesis is a sign of how primitive the synthesis process remains.

Successful synthesis is a multi-step process. It is important to note that in addition to giving a good yield, the carboxylic component’s integrity must be maintained. There are many things that can go wrong with this process. A lot of modern methods for synthesizing proteins involve converting an acid into a derivative that bears a suitable leaving group.

Each leaving group can increase the acidity of the a-proton, and this can favour the formation of an undesirable oxazolone which increases the risk of the loss of configuration of the carboxylic component.

This risk is reduced in SPPS, but there is still some potential for racemization. One feature of most SPPS processes is that they tend to use a large excess of the relevant reagents. The speed with which the reactions occur means that the risk of loss of configuration is minimal, but it is generally accepted that the risk of racemization is near-zero for mose amino acids.

The risk is still present for some particularly sensitive amino acids such as histidine and cysteine, but in general SPPS is considered the safest and most efficient technique. The performance and safety of this technique improves as better reagents are used. Carbodoilimides are among the most widely used today, with phosphonium and aminium also being popular choices.

Both of these sets of coupling reagents have been used in SPPS for decades. The researchers at Albericio and El-Faham proposed better reagents as a part of their research in the mid-late 2000s, but they have not found the final answer to the problem of synthesis.

Another area that is being explored is the option of organophosphorus regents. These reagents are thought to give higher regioselectivity than other methods. One reason that these reagents are considered worthy of research time is that they are crystalline and they are also relatively stable so they could be good for long-term storage. Several research groups have tested organophosphorus reagents and have found their results promising compared to older guanidinium reagents, especially in terms of segment coupling and shelf stability. More research is needed to determine whether such reagents are a viable alternative to COMU however.

Protein synthesis is important for both industrial use and scientific research and it is a field with potential applications in everything from food to medicine. We have a lot to learn about coupling reagents, and hopefully soon there will be a massive leap forward in our understanding of how to efficiently make large and complex protein chains.

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