Peptide synthesis is a complex and crucial process most commonly utilized in organic chemistry. During peptide synthesis, multiple amino acids are links by peptide bonds. Linking amino acids has been a common practice in organic chemistry for many years, but the process is evolving. Recent technological advancements and more specialized synthesis processes have enabled researchers to now synthesize natural peptides and generate unique peptides with increased accuracy and reliability. However, in order to answer the question, “what is peptide synthesis?”, one must dive a little deeper into the process’s intricacies and applications.
What are peptides?
One must first gain a base understanding of peptides to properly understand what peptide synthesis is. Peptides are comprised of short chains of amino acids, all of which are linked together by peptide bonds. Structurally speaking, peptides are quite similar to proteins, as they are both made up of amino acid chains which are held together via peptide bonds. The main distinguishing factor between peptides and proteins, however, is their size. Whereas proteins are generally made up of 50 or more amino acids, peptides are often on the smaller side, consisting of only two to 50 amino acids. Additionally, protein structures are slightly more complex and can adopt secondary, tertiary, and quaternary structures. Peptides offer less variation and are typically only divided into two subsets. Oligopeptides are those which possess fewer amino acids, typically between two and 20. Polypeptides are those which contain a higher number of amino acids and, when linked together, produce proteins. In essence, a protein is one large peptide comprised of many polypeptides.
Peptide synthesis processes and procedures
While peptides do occur naturally and can be found in all living organisms, synthetic peptide production is often used by researchers to produce specific peptides. In particular, researchers synthesize those that may be difficult to express in bacteria or to experiment with the incorporation of amino acids that are not typically found in peptides. The most common method for the synthetic peptide production is called solid-phase peptide synthesis.
Solid-phase peptide synthesis
First pioneered by American biochemist Robert Bruce Merrifield, solid-phase peptide synthesis has since become the leading method for the synthetic production of peptides. Solid-phase peptide synthesis, often abbreviated as SPPS, streamlines the process of synthetic peptide production by creating multiple successive amino acid reactions on a singular porous apparatus.
Peptide synthesis occurs when the carboxyl group of an incoming amino acid is coupled to the N-terminus of an existing and growing peptide chain. Amino acids are added one at a time to the growing peptide chain in a precise, cyclic manner, taking special care to follow the necessary steps exactly. Analysts must be extremely cautious when creating this peptide chain, as amino acids possess multiple reactive groups and may cause side reactions which reduce the length of the chain or cause it to branch. In order to reduce the likelihood of these side reactions, the observing analyst must utilize protecting groups such as Fmoc or Boc. These base-labile and acid-labile protecting groups, respectively, help achieve chemoselectivity throughout the synthesis process, effectively reducing the likelihood of any unintended side reactions.
Once the system has been treated with these temporary protecting groups—which can be added and removed with relative ease throughout the entire synthesis process—amino acids can be added to the peptide chain. Carbodiimides are used to activate the C-terminal carboxylic acid of the incoming amino acid, enabling it to link to the N-terminus of the growing peptide chain.
As we’ve stated, the SPPS process is cyclical and these steps are repeated numerous times in order to achieve the desired peptide bond length. Once the peptide bond has reached the desired length it must be cleaved of any remaining protecting groups. Through a process called acidolysis, the peptide bond is stripped of its remaining protecting groups. That is to say, the N-terminal protecting group of the last-added amino acid, the C-terminal protecting group of the first amino acid, and any remaining side-chain protecting groups will be removed. The chemical chosen to complete this process will be dependent upon the protecting groups used in the system. Acid-based protecting groups, such as Boc, will require the use of strong acids, such as hydrogen bromide or hydrogen fluoride. Conversely, base-labile protecting groups, such as Fmoc, can be cleaved using a milder acid, such as trifluoroacetic acid (TFA).
Because SPPS is such a complex procedure which requires extreme care and precision, it can also be quite time consuming. For this reason—among others—many laboratories turn to automated solid-phase extraction systems to streamline the process. These workstations enable the observing analyst to leave the system unattended while maintaining full confidence that the procedures will be completed with accuracy and efficiency. The workstations are able to accommodate larger samples and can be micro-controlled via computer to yield even higher precision and accuracy. For more information on how automated workstations can be utilized for solid-phase peptide synthesis, click here.
Solution-phase peptide synthesis
Another technique in the synthetic production of peptides is solution-phase peptide synthesis. Though less widely used, this technique utilizes many of the same procedures as SPPS. However, this process can be slightly more time consuming, as it requires the product peptide to be isolated from the solution after each reaction step. It is for this reason that solution-phase peptide synthesis has been replaced by SPPS in many laboratories. Solution-phase peptide synthesis is still often used in larger-scale production of peptides, however, which are typically used for industrial applications.
Applications of peptide synthesis
Peptide synthesis is often used in conjunction with epitope mapping and typically sees applications in medical sciences and biotechnology. Synthetic peptides are used to research potential cancer diagnoses and treatments and in the development of antibiotic drugs. When used in conjunction with epitope mapping—which is the process of identifying the bonding sites between an antibody and its antigen—peptide synthesis can be used to produce antibodies and create more intelligent vaccine designs. Synthetically produced peptides are also utilized in mass spectrometry-based applications, in which they can function as both standards and reagents.