What Is The Importance Of Peptide Synthesis?
The twenty-first century has been defined by advancements and developments in biotechnology and bioengineering, regardless of recurring challenges. In the modern age of medicine, researchers utilize peptide applications for therapeutic purposes, such as cancer treatment and diagnosis, epitope mapping, antibiotic drug development, vaccine design, and antibody sequencing services. In addition, the requirements for vaccine development have contributed to the further development of synthetic peptides.
Specifically, peptide synthesis is the production or development of peptides where several amino acids are linked through peptide bonds under the principles of organic chemistry.
Simultaneously, the biological process of developing extended proteins (peptides) typically refers to protein biosynthesis. Even though the concept of amino acids linking into a chain is over a century old, it is essential to know that it took around half a century to find solutions for peptide-specific issues. Robert Bruce Merrifield is one of the researchers who pioneered solid-phase peptide synthesis (SPPS). When using SPPS, a researcher can synthesize peptides with a length of up to 50 amino acids. This allows for the synthesis of natural peptides that are challenging to express in bacterial processes, supporting the integration of unnatural amino acids and distinctive peptides.
The era following the invention of peptide synthesis was a considerable breakthrough. Researchers developed various applications through synthetic peptides, including epitope-specific antibody development against pathogenic proteins, protein function studies, and protein characterization and identification. Additionally, synthetic peptides allow for research on essential cell signaling enzymes such as proteases and kinases, primarily to understand enzyme-substrate interactions.
Introduction
As already discussed, peptide synthesis is the formation of peptide bonds between two specific amino acids. However, when it comes to a definitive definition in the biosciences, peptides are simply flexible, chain-like structures of amino acids. Consequently, scholars first synthesized peptides such as insulin and oxytocin. In addition, advancements in protein chemistry and applications have progressed significantly in recent years. Today, the process has become one of the most common approaches in high-throughput research and antibody production.
The primary advantage of peptide synthesis in the modern age is that, apart from developing peptides found in biological systems, imagination and creativity can be accommodated to produce unique peptides and optimize desired biological responses.
Synthetic Peptide Applications
Investigations in the early sixties introduced various application areas where synthetic peptides are now utilized. In cell biology, the substrate specificity and receptor binding of newly synthesized enzymes can be researched using a combination of homologous peptides. In addition, synthetic peptides may also mirror naturally occurring proteins and serve as products against major diseases like cancer. Finally, one can utilize synthetic peptides as reagents and standards in mass spectrometry applications.
Benefits Of Automating Peptide Synthesis
Reduces error
The solid-phase peptide synthesis (SPPS) process requires intricate attention to detail. When conducted manually, the analyst must closely monitor every step to ensure elements do not become contaminated and that all processes proceed correctly. Furthermore, even the most fastidious analysts are only human and might commit mistakes, compromising the entire throughput and experiment.
Automating the SPPS process can negate the risk of human error entirely. This leads to fewer mistakes and overall losses while increasing the validity and reliability of results.
Reduces experimental costs
The total cost of correcting human errors may sometimes outweigh the potential benefits, leading to setbacks. Not only is the process of fixing errors costly, but it also requires individuals to expend valuable effort and time. By reducing the propensity for error through automated SPPS, labs can reallocate funds and cut expenses previously set aside for such fixes.
Increases research throughput
Another essential advantage of automating custom peptide synthesis is that automated workstations streamline the entire method and enhance research throughput. Consequently, these workstations can maximize the number of specimens analyzed on specific surfaces to increase sample sizes. This process, in turn, increases the overall accuracy of the experiment.
Increases productivity
With a reduced likelihood of error and increased throughput, productivity improves. When there are fewer issues in experiments, analysts can proceed further and allocate more resources, skills, and time to other tasks. Moreover, in an automated SPPS process, analysts can step away from specific protocols with the utmost precision and confidence.
Process of Peptide Synthesis
Synthesizing peptides often occurs by coupling the carboxyl group of an incoming amino acid to the N-terminus of the peptide chain. This C-to-N method is the opposite of protein biochemistry and biosynthesis, during which the N-terminus of the incoming amino acid links to the C-terminus of the peptide chain. Since the in vitro protein process is quite challenging and complex, the inclusion of amino acids in the peptide chain occurs in a stepwise, cyclic, and precise manner.
Moving on, some of the standard methods of custom peptide synthesis address critical issues and differences, though they follow similar steps to include amino acids sequentially into the growing protein chain.
Peptide deprotection
Since amino acids include several reactive groups, researchers must perform peptide synthesis carefully to prevent unnecessary side reactions. This procedure allows one to reduce chain branching. To expedite the production of peptides with negligible side reactions, scholars have developed various chemical groups to interact with the amino acids' reactive groups and protect or block the functional group from nonspecific reactions.
Individual and purified amino acids are reacted with such protecting groups before any synthesis occurs. Afterward, researchers remove these protecting chains from the initially added amino acids, a step referred to as deprotection. This happens after coupling to enable incoming amino acids to interact adequately with the growing peptide chains. Once the peptide synthesis process is complete, all protecting chains or groups are eliminated from the existing peptides.
Once the synthesis of peptides has entered the completion stage, one can remove all the remaining groups from nascent peptides. Earlier, researchers utilized three protecting groups, depending on the peptide synthesis method. If analysts perform the tests, they can preserve the amino acid N-terminus through groups termed "temporary," since they are quickly removed to enable the bond formation of peptides. Two primary N-terminal groups are 9-fluorenyl methoxycarbonyl (Fmoc) and tert-butoxycarbonyl (Boc).
While Boc requires a relatively strong acid like trifluoroacetic acid to be eliminated, Fmoc is a protecting group removed with mild bases like piperidine. Boc chemistry was first described in the early 1950s. Because of the mild deprotection conditions, Fmoc chemistry is gaining more momentum and support from qualified professionals and biosciences departments worldwide. Even though it is more common in commercial settings, this method can also lead to greater yield and higher quality.
Why Are These Methods Essential?
Although scholars and researchers have utilized various unstable protein expression services to find the final output, it is difficult to replicate them without the right tools. This is where the C-terminal makes a significant difference. From synthesizing peptides and proteins to protecting the liquid phase and specific amino acids, robust support can go a long way in strategizing such processes. Meanwhile, amino acid side chains represent an array of available sets referred to as side-chain reactivity. Due to this, investigators require various other protecting groups, such as those based in the tert-butyl or benzyl (Bzl) groups.
Amino acid group protection
Regardless of the method, the side chains and N-termini are preserved with specific chemical bonds to block nonspecific reactions while the synthesis process is underway. Consequently, you can protect the C-terminal amino acid to ensure peptide extensions occur in the correct orientation. Since various groups typically undergo the process of peptide synthesis, it is imperative that such groups are compatible to enable the deprotection of a unique group without affecting others.
Amino acid coupling
Peptide coupling requires C-terminal carboxylic acid activation when utilizing incoming amino acids with reagents like diisopropyl carbodiimide (DIC) or dicyclohexylcarbodiimide (DCC). These coupling reagents react with carboxyl groups to form a reactive O-acylisourea intermediate. During this step, the reagent is displaced by a nucleophilic attack from the deprotected amino groups on the N-terminus.
On the other hand, carbodiimides can form a reactive intermediate that can cause amino acid racemization. As a result, you can add reagents such as 1-hydroxy benzotriazole (HOBt) to form less-reactive intermediates and reduce the possibilities of racemization.
Peptide cleavage
After recurring rounds of amino acid coupling and deprotection, the remaining protecting groups should be eliminated from the nascent peptides. This is usually achieved by acidolysis. The chemicals utilized for peptide cleavage rely on the protection scheme used. Analysts use potent acids like hydrogen bromide (HBr), hydrogen fluoride (HF), or trifluoromethanesulfonic acid to cleave Bzl and Boc groups, while using a comparatively milder acid like TFA to cleave t-Butyl and Fmoc groups.
Peptide synthesis strategies
The liquid-phase method is one of the conventional methods scientists use to produce peptides in vitro. However, it is a labor-intensive and slow process. You have to remove the product manually from the reaction solution after every step, making it a challenging sequence. Additionally, such an approach requires other chemical groups to protect the first amino acid's C-terminus. However, the most significant advantage of liquid-phase synthesis is that the product undergoes purification after every step.
If you're wondering where peptides are synthesized, it would be ideal to check specialized peptide synthesis companies. Additionally, one can perform convergent synthesis, where separate peptides and proteins are coupled to produce multiple peptides.
Peptide purification
Even though you can mass-produce and optimize peptide synthesis strategies, the method is not perfect. Events like incomplete reactions or incomplete deprotection can lead to deletion sequences or truncated isomers. The longer your peptide sequence is, the more probable it is that your target peptide will be negatively affected. Henceforth, the entire product's yield is inversely proportional to the peptide length.
The Final Word
Different peptide synthesis companies have various media and methods to produce peptides. The entire purification strategy is usually based on a blend of separation methods that exploit a peptide's physicochemical characteristics, including charge, size, and hydrophobicity. In the modern age, it has become essential to match the credibility and sophistication of synthetic chemistry. Peptide chemistry is a never-ending research field, and while advances are constant, some still require time to be applied to large-scale peptide manufacturing.