Everything You Need to Know About Peptide Synthesis

by | Jun 24, 2022 | Knowledge Center

What is it?

Peptide synthesis, in organic chemistry, is characterized as the formation of a peptide bond between two amino acids. By definition, it is the production of peptides. Chemistry and technology advancements have improved synthesis methods causing peptide synthesis to play a vital role in scientific and medical progress in this modern age. The same could not be said in the beginning due to inefficient production practices at peptide synthesis’ inception. However, the field of peptides has grown much since then.

What is its Value?

Just as peptides have proven to be a critical element in biomedical research, peptide synthesis has continued to prove its value in scientific progress worldwide as well. They have gained the attention of numerous pharmaceutical companies, and several drugs made from peptides have received FDA approval and reached the market due to their therapeutic potential. Peptides will continuously be perused and developed for pharmaceutical and diagnostic purposes due to its efficacy, specificity, and low toxicity. This assures it will be a growing area of biochemical research for years to come.

The Process of Peptide Synthesis

Solution Phase Synthesis (SPS) was the original approach to peptide synthesis. Though this process still has merit in this modern age, for large scale peptide production, Solid-Phase Peptide Synthesis (SPPS) has proven to be the method of choice. SPPS’s advantages are the reason for this. SPPS creates peptides with higher purity, high yield, and faster production time.

There are five steps performed in a cyclical manner that SPPS involves. The first step involves the attachment of an amino acid to a polymer. The second step is the protection of this attachment to prevent unwanted reactions. The next step is coupling the protected amino acids. Once that is done, they deprotect them to allow attachment acids to react to the following amino acid that will be added. And finally, they engage in polymer removal, allowing there to be a free peptide.

SPPS synthesis can also be enhanced by microwave-assisted SPPS. This could be helpful when synthesizing long peptide sequences due to improved yield and speed. Microwave-assisted SPPS can, however, be the more expensive option compared to the traditional SPPS synthesis process.

Though SPSS can offer excellent purity and yield standards, it can still become a victim to impurities and imperfections during the process. The chances increase with a lengthier peptide sequence as more steps are needed to complete the synthesis process. Thus in order to secure optimal quality, specific purification techniques would need to be utilized. Examples would be:

  1. Reverse-Phase Chromatography (RPC)
  2. High-Performance Liquid Chromatography (HPLC)

Reverse-Phase Chromatography (RPC) is today’s most widely used peptide purification method. These purification methods can separate the impure peptides from the desired peptide. Thus benefiting peptides’ physicochemical properties.

Linking two amino acids together is how Peptides are synthesized. Majority of the time this is accomplished by attaching the C-terminus, or carboxyl group, of one amino acid to the N-terminus, or amino group, of another. Unlike protein biosynthesis, which involves N-terminus to C-terminus linkage, peptide synthesis occurs in this C-to-N fashion.

While in the natural world there are 20 amino acids commonly occurring such as arginine, lysine, and glutamine,  many other amino acids are also being synthesized. This creates abundant possibilities for the creation of new peptides. However, amino acids have multiple reactive groups that can negatively interact during the synthesis process. This could lead to unwanted truncating or branching of the peptide chain or causing suboptimal purity or yield. Due to these negative effects, peptide synthesis must be expertly carried out as it is a complex process.

Protecting Groups

Scientists have engineered special and specific chemical groups designed to secure the preferred outcome from the synthesis process and avoid extraneous, unwelcome reactions. Certain amino acid reactive groups must be deactivated or protected from reacting to accomplish this goal. These special groups are known as protecting groups and can be separated into three categories: N-terminal, C-terminal, and Side chain. The N-terminal protecting group protects the N-termini of amino acids. Known as the temporary protecting groups, they are removed easily to aid the formation of peptide bonds. Two frequently used N-terminal protecting groups are:

    1. Tert-butoxycarbonyl (Boc)
    2. 9-fluorenylmethoxycarbonyl (Fmoc).

The C-terminal protecting group protects the C-terminus of amino acids. C-terminal protecting groups are needed in liquid-phase peptide synthesis but not for solid-phase peptide synthesis. Lastly side chain a various and unique protecting groups are needed to protect against unwanted reactions because amino acid side chains are conducive to reactivity during peptide synthesis. It can stay intact during the numerous cycles of chemical treatment during synthesis. These side chain protecting groups are known as permanent protecting groups. Once peptide synthesis is done, side chains are only removed with strong acids.