Peptides have a wide range of applications in science today. They are used in drugs and vaccines, to map enzyme binding sites, in the preparation of epitope-specific antibodies and to come up with a variety of novel enzymes among others. Back in the day, the process of peptide synthesis was not only labor-intensive but also resulted in very low peptide yields. Over the years, a lot has changed and the process has become much more efficient, leading to faster synthesis processes and higher yields.
About Peptide Synthesis
Peptide synthesis refers to the processes by which short sequences of polypeptides are added to amino acids one at a time. The process leads to the creation of new and specific sequences that are a representation of the epitopes of a particular protein domain that might or might not have undergone modification by moieties such as phosphate groups. The short sequences after the synthesis are then subjected to a variety of applications, including further research, as well as polyclonal antibody production, receptor ligand assays and cell/tissue culture.
The concept of peptide synthesis has been around for more than 100 years. However, the actual synthesis of real peptides never took place until after another 60 or so years – a subtle demonstration at just how difficult the process of peptide synthesis was. In the last fifty years, however, there have been a lot of scientific and technological advancements that have led to an oversimplification of the synthesis methods that you can now get precisely the kinds of sequences you need with a higher degree of success.
Applications of peptide synthesis
The breakthrough in peptide synthesis led to a myriad of developments in a variety of sectors. Some of the areas with rampant applications of synthetic peptides currently include the identification and characterization of proteins, and studying the functions of proteins. Additionally, synthetic peptides are also being widely used in studying enzyme-substrate relationships – an application that has led to a lot of discoveries and insights about the important role of cell signaling.
In cell biology, it is now possible to study receptor binding and substrate recognition with the help of homologous synthetic peptides. Synthetic peptides may look and act just like natural peptides, and as such, they are being used in a variety of clinical studies and research involving drugs against conditions such as cancer and other major ailments.
Also, synthetic peptides are being used extensively as reagents and standards in processes such as mass spectrometry-based applications. They play a vital role in MS-based discovery, quantitation and characterization of proteins, particularly those that work as early biomarkers of diseases.
The process of peptide synthesis
The process of peptide synthesis usually involves the coupling of the carboxyl group and the N-terminus of an incoming amino acid to extend the peptide chain. This kind of coupling is quite different from protein biosynthesis which usually happens in the reverse i.e. the coupling usually happens between the N-terminus of incoming amino acid and the C-terminus of the protein chain.
Since in vitro protein synthesis is very complex, the addition of amino acids to the already growing peptide chain can be done in a precise, step-by-step and cyclic nature. Though some of the common methods of peptide synthesis differ critically, all of them follow the step by step methods where the amino acid is added to the growing peptide chain step by step and one at a time.
Amino acids have a variety of reactive groups. Due to this, the process of peptide synthesis must be conducted with a lot of caution to avoid any instances of side reactions that may lead to shortening the peptide length as well as making the peptide chain to branch. To ensure that the synthesis happens with minimal side effects, a variety of chemical groups have been developed to bind them to the amino acid reactive groups to protect or block the functional groups from any form of side reactions.
Individual amino acids that have been purified for the peptide synthesis are then reacted with the protective groups before the synthesis process begins. After that, the protective synthesis groups are then removed from the newly added amino acid - a process known as peptide deprotection, immediately after coupling is done so that the next incoming amino acid can easily bind to the growing chain at the right spot.
Following the completion of the peptide synthesis, any remaining protective group is removed. In most cases, there are three major types of protective groups, and the type used will mainly depend on the preferred method of peptide synthesis as briefly explained below.
“Temporary protecting groups” are the ones responsible for protecting the N-terminus of the amino acid. This is because these temporary protecting groups can be removed easily and allow for seamless bond formation. There are two main common temporary protecting groups, and each of the groups come with unique characteristics which heavily determines how they are used.
They include Fmoc and Boc. With Boc, strong acids such as TFA are used to remove it from the newly added amino acid, while for the case of Fmoc, which is a base-labile protecting group, a soft or mild base like piperidine can be used for its removal from the newly formed amino acid.
As noted earlier, the C-terminal protecting group used will be mainly determined by the type of peptide synthesis method used. In the liquid phase of peptide synthesis, for instance, the C-terminus of the first amino acid will have to be protected. This is not the case in solid-phase peptide synthesis where solid support is normally used to protect the C-terminal amino acid only.
Amino acid chains usually feature a wide range of functional groups and as such, they are targets for considerable side chain reactions during the peptide synthesis process. Due to this, various protecting groups are always used. The respective protective group used during the processes of a given peptide will also vary based on the sequence of the peptide as well as the N-terminal protection used for that particular reaction.
Apart from the use of temporary protecting groups, side chain protecting groups, also known as permanent protecting groups can also be used to prevent chain reactions during the synthesis process. These groups don’t have to be removed after the reaction is over, and they can tolerate multiple cycles of chemical treatments during the synthesis process. Their removal can only be done through a strong acid treatment, but only after the synthesis process is complete.
Since more than one protecting group is used during the process of peptide synthesis, it goes without saying that the groups used must be compatible with one another so that none of the group members are affected in any way. To ensure this, protecting schemes must be established to match the various protecting groups so that the deprotection method will not have any impact on the binding of another group. Since the N-terminus deprotection is usually a continuous process throughout the peptide synthesis process, the protective schemes established must have different types of side chain protecting groups and they must also either match Boc or Fmoc to guarantee a fully optimized deprotection.
Amino Acid Coupling
For synthetic acid coupling, there has to be the activation of the C-terminal carboxylic acid on the incoming amino acid. This is usually done using a coupling reagent such as DCC or DIC which reacts with the carboxyl group leading to the creation of a highly reactive O-acylisourea intermediate. This newly formed intermediate will within a moment displace the nucleophilic attack from the N-terminus of the newly formed amino group, leading to the formation of a nascent peptide bond.
Strategies for peptide synthesis
Liquid phase peptide synthesis was the very first method that was used when the concept of peptide synthesis was first applied. It was a slow and labor-intensive method since the resulting product had to be removed manually from the chain reaction after every step. This process also requires that there has to be another chemical group to protect the C-terminus of the initial amino acid after every reaction. Though hectic, the liquid phase peptide synthesis had one benefit.
Because the resulting product is purified after every step, it is very easy to detect side reactions. Also, it is possible to perform convergent synthesis which involved the synthesis of peptides separately and then coupling them together leading to the creation of larger peptides.
Currently, liquid phase peptide synthesis is still in use, though there is a lot of love and preference for solid-phase synthesis. With solid-phase peptide synthesis, coupling happens between the C-terminus of the first amino acid and active solid support, usually a polyacrylamide or polystyrene. With this type of approach, there are two types of processes taking place: the resin performs the function of the C-terminal to protect the group and also it provides a quick method for separating the incoming and growing peptides from the various reaction mixtures that may occur during the synthesis process. Just like with most of the various biological manufacturing processes, the process of peptide synthesis still offers room for a lot of development, including advanced automation as well as high-throughput production.
The Purification Process of Peptides
Though the methods for peptide synthesis have evolved and improved to allow for the mass production of peptides, they are still not 100% perfect. This simply means that they don’t lead to 100% purity on the resulting peptides. Concerns such as reactions between free protecting groups and incomplete deprotection are known to lead to undesirable effects such as sequence truncation or sequence deletion and other unintended side products.
The occurrence of these events can be at any stage of the synthesis process. Consequently, long peptide sequences have greater chances of encountering these events, which ultimately impacts the overall purity of the resulting peptide. This implies the fact that yields will always be inversely correlated with peptide lengths.
The purification method used will be determined by various separation methods, especially those that can utilize the peptide’s physicochemical properties. Some of the common purification techniques include-: partition chromatography, Ion exchange chromatography, High-performance liquid chromatography, reverse phase chromatography, and size exclusion chromatography. Among all these separation methods, reverse phase chromatography is considered to be the most versatile of them all and is the most widely used.
The peptide purity is usually considered as a ratio of the target peptide to the impurities expressed as a percentage. With commercial peptide purification, there are varying levels of purity, and there are primarily based on the intended application of the peptides. Here is a brief look at the common impurity levels and the likely practical application of the peptides-:
- >95% - these are used for quantitative studies such as those involving NMR, monoclonal antibody production, receptor-ligand binding studies, and in vivo studies among others.
- >80% - peptides with this purity level are likely to be used for western blot analysis, antibody affinity purification, plate coating for cell attachment, and enzyme-substrate studies among others.
- >70% - for these purity levels the peptides may be applied in polyclonal antibody production, used as ELISPOT assays or as ELISA standards.
Choosing the right synthesis service
With many peptide synthesis services available today, it is imperative to choose a service provider that will deliver the desired purity.