Want to Get Started with Peptide Synthesis for Your Research? Have A Look at Here Before

Peptide synthesis joins two particular amino acids together to form a peptide bond. However, when it comes to a precise definition of peptides in bioscience, it simply refers to the flexible little chain-like structures of amino acids. As a result, researchers first created peptides such as insulin and oxytocin. Furthermore, protein chemistry and applications have significantly progressed in recent years. Today, the method is used frequently in high-throughput research and antibody production.

The sole benefit of peptide synthesis in the modern era is that, in addition to developing peptides found in biological illustration, you can accommodate imagination and creativity to produce unique peptides and optimize desired biological responses.

  1. Process of Synthesizing Peptides

Normally, during peptide synthesis, the carboxyl group of the incoming amino acid is coupled to the N-terminus of the developing peptide chain. This process is the inverse of protein biosynthesis, in which the N-terminus of the incoming amino acid is linked to the C-terminus of the protein chain (N-to-C). Because of the complexities of in vitro protein synthesis, amino acid added to the growing peptide chain occurs precisely, step-by-step, and cyclic. While the common peptide synthesis methods differ in important ways, they all follow the same step-by-step method of adding amino acids one at a time to the growing peptide chain. 

  1. Peptide Deprotection
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Because amino acids have multiple reactive groups, you must do peptide synthesis carefully to avoid side reactions that can shorten and branch the peptide chain. Chemical groups that bind to the amino acid reactive groups and block or protect the functional group from nonspecific reactions have been developed to facilitate peptide formation with minimal side reactions.

Before synthesis, these protecting groups are reacted with individually purified amino acids, and after coupling, specific protecting groups are removed from the newly added amino acid (a process known as deprotection). This allows the subsequent amino acid to bind to the developing peptide chain in the correct orientation. Following the completion of peptide synthesis, all remaining protecting groups are removed from the nascent peptides. Depending on the peptide synthesis method, three types of protecting groups are commonly used and are described below.

“Temporary” protecting groups protect the N-termini of amino acids because you can easily remove them to allow peptide bond formation. Tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc) are two common N-terminal protecting groups, each with unique properties that determine their use. Boc is removed from the newly added amino acid with a moderately strong acid such as trifluoracetic acid (TFA), whereas Fmoc is a base-labile protecting group removed with a mild base such as piperidine.

In contrast to Fmoc, which cleaves under mild, basic conditions and was not reported for another 20 years, boc chemistry was first described in the 1950s and required acidic conditions for deprotection. Due to its higher quality and yield, fmoc chemistry is more frequently used in commercial settings. Boc, on the other hand, is preferred for complex peptide synthesis or when base-sensitive non-natural peptides or analogs are needed.

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Depending on the peptide synthesis method employed, a C-terminal protecting group may or may not be needed. Solid-phase peptide synthesis does not require the protection of the C-terminus of the first amino acid (C-terminal amino acid), as the only C-terminal amino acid that needs protection is protected by solid support (resin).

Because amino acid side chains represent a diverse range of functional groups, they are sites of significant side chain reactivity during peptide synthesis. As a result, various protecting groups are required, though they are typically based on the benzyl (Bzl) or tert-butyl (tBu) groups. Different protecting groups are used during peptide synthesis depending on the peptide sequence and N-terminal protection strategy. Because they can withstand numerous chemical cycles during the synthesis phase and are only eliminated after synthesis is complete by treatment with strong acids, side chain protecting groups are also referred to as permanent protecting groups.

Because multiple protecting groups are typically used in peptide synthesis, it is obvious that these groups must be compatible for distinct protecting groups to be deprotected without affecting other protecting groups. So that the deprotection of one group does not affect the binding of the other groups, protecting schemes are established to match protecting groups. Due to the continuous nature of N-terminal deprotection during peptide synthesis, protecting schemes have been developed in which different kinds of side chain protecting groups (tBu or Bzl) are matched to either Boc or Fmoc to optimize deprotection.

  1. Amino Acid Coupling

Synthetic peptide coupling necessitates the activation of the incoming amino acid’s C-terminal carboxylic acid with carbodiimides such as dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide. These coupling agents react with the carboxyl group to produce the highly reactive O-acylisourea intermediate, swiftly displaced by the nucleophilic attack of the primary amino group left unprotected on the N-terminus of the developing peptide chain to create the nascent peptide bond.

Carbodiimides form such a reactive intermediate that amino acid racemization can occur. As a result, reagents that react with the O-acylisourea intermediate, such as 1-hydroxybenzotriazole (HOBt), are frequently added, forming a less reactive intermediate that reduces the risk of racemization. Other coupling agents, such as benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), which both require activating bases to mediate amino acid coupling, have also been investigated as a result of side effects.

  1. Peptide Cleavage

It would be best to remove all remaining protecting groups from the nascent peptide after several cycles of coupling and deprotection of the amino acids. Strong acids like hydrogen fluoride (HF), hydrogen bromide (HBr), or trifluoromethane sulfonic acid (TFMSA) are used to cleave Boc and Bzl groups. When performed correctly, cleavage removes the N-terminal protecting group of the most recently added amino acid, the C-terminal protecting group (either chemical or resin) of the first amino acid, and any side-chain protecting groups. Scavengers, like deprotection, are included in this step to react with free-protecting groups. Because cleavage is important in peptide synthesis, you should optimize this step to avoid acid-catalyzed side reactions.

  1. Benefits of Automating Peptide Synthesis
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  1. Reduces Error

The solid-phase peptide synthesis process necessitates meticulous attention to detail. When done manually, the observing analyst should monitor each step to ensure that no elements become contaminated and that all other processes run smoothly. Furthermore, even the most meticulous analysts are only human and may make mistakes that jeopardize the entire throughput and experiment.

When the entire SPPS process is automated, the risk of error is eliminated. This results in fewer errors and overall losses, as well as increased validity and reliability of results.

  1. Increases Productivity

Productivity can be increased by lowering the likelihood of error and increasing throughput. It means that when there are fewer issues in experiments, analysis can proceed further, and more resources, including skills and time, can be allocated. Furthermore, in an automated SPPS process, analysts can deviate from specific protocols with pinpoint accuracy and confidence.


Understanding that peptide synthesis companies use different mediums and methods to produce peptides is critical. Furthermore, the entire purification strategy is typically based on a combination of separation methods that can exploit a peptide’s physicochemical properties, which include charge, size, and hydrophobicity. However, in the modern era, it has become critical to match the credibility and sophistication of synthetic chemistry, where several researchers and labs are constantly looking for novel ways to produce cutting-edge medicine. Nonetheless, peptide chemistry is a never-ending research field, and most advances take time to apply to good peptide manufacturing.