In recent years, it has become increasingly important to produce cutting-edge medications using sophisticated synthetic chemistry while developing creative ways to lessen their environmental impact.
Among the many methodologies in bioorganic chemistry, Peptide synthesis stands out as essential. It probes biological structure and function and is a vital intermediate for developing enzyme inhibitors and peptidomimetics as therapeutic agents.
Moreover, automated peptide synthesis has permitted many researchers to utilize synthetic peptides. The Association of Biomolecular Resource Facilities or ABRF, for instance, has several laboratories that synthesize peptides and structural analysis of peptides and proteins as a service in academic, government, and research institutions and private industry.
The purpose of this peptide synthesis guide is to provide information on all you need to know about peptide synthesis.Let's first take a closer look at peptide bonds.
What Are Peptide Bonds?
Peptides or peptide bonds are chains of amino acids comprising two or more amino acids connected by an amide bond. Peptide coupling is the process used to join two amino acid residues to create a peptide. The main distinctive feature between peptides and proteins is that they do not require folding for biological activity.
It occurs endogenously as a peptide hormone, such as LHRH, enkephalin, and angiotensin, and as a toxin in animals and plants. Peptides are highly sought-after as lead chemicals in drug research and standalone medications. They are widely employed as antigens to produce antibodies and have uses in vaccinations, biomaterials, and histology probes.
The creation of peptides is through using two techniques: Fmoc and Boc. However, most peptides are made using the Fmoc method rather than the Boc approach, which calls for employing highly toxic, corrosive liquid anhydrous HF in specialized apparatus for the final cleavage and deprotection.
Peptides are chemically synthesized either in a solid phase or in a solution. With the exclusion of existing procedures of large-scale custom peptide synthesis for industrial reasons, solid-phase peptide synthesis has replaced liquid-phase peptide synthesis, which typically uses Boc or Z-amino protection.
The process involves a directed and selective formation of an amide bond. This occurs between an amino acid bearing a free amino group, an N-protected amino acid, and a protected carboxylic acid. The carboxyl protecting group links to the polymer support in solid-phase synthesis. The next N-protected amino acid also connects to the group after removing the amino-protecting group of the dipeptide.
Consequently, scientists use liquid-phase peptide synthesis when creating longer proteins. They do this through native chemical ligating completely deprotected peptides in solution. This technique makes it feasible to develop cyclic, tagged, branching, and post-translationally modified and natural peptides that are challenging to express in bacteria.
Overview of Peptide Synthesis
The synthesis of peptides refers to forming peptide bonds between two amino acids. This art of creating bonds of peptides to connect amino acids has existed for over a century. However, some aspects of manual peptide synthesis, like synthesizing insulin and oxytocin, only occurred in the last six decades after significant medical advancements.
They indicate the complexity of chemically synthesizing various amino acid chains.
Peptide synthesis is now a popular tool in advanced biology research and the developing of new products and medications. This development has resulted from advancements in the chemistry and procedures of custom protein synthesis over the past 50 years.
The advantage of modern peptide synthesis is that you can use them to create original peptides optimized for a particular response or other outcomes.
The following sections outline the history of synthesizing peptides, their critical elements, the strategies for purifying and synthesizing, and their pros and cons.
A Brief Historical Perspective on Peptide Synthesis
Emil Fischer invented it in the first decade of the 20th century. At the time, scientists assumed peptide synthesis supported the polypeptide theory of protein structure. The scientific community had irrational expectations of it to the point where they believed it would ultimately result in the forming of synthetic biological beings.
Peptide chemistry didn't have a solid basis or well-defined objectives until the 1950s, when the first precise amino acid sequences were created. This likely gave rise to modern organic synthesis. It became possible to completely synthesize peptide hormones and antibiotics, which provided helpful information for illuminating structure-functional linkages and the biological action mechanisms.
Peptides gained notoriety as the most prevalent, all-pervasive class of low molecular bioregulators as the number of peptides extracted from various biological sources increased rapidly in the following years.
A significant area of peptide chemistry has emerged around developing and synthesizing new peptide-based medicines. However, the peptide synthesis companies or the scientific community at large currently face the challenge of analyzing the structures and bioactivities of complete sets of peptides, or peptidomics, prevalent in specific tissues or cell groups.
Most organic syntheses occur through solution-phase techniques. However, each independent chemical reaction follows a purification step and the characterization of the resulting synthetic intermediate. This leads to the development of a more effective synthetic method due to two peculiarities of peptide chemistry.
Contrary to most attempts at complete synthesis, the synthesis of peptides is an iterative process that involves successive alpha-amino deprotection and amide couplings until the required generation of the full-length target peptide.
In addition, regardless of side-chain protection strategies, most biologically relevant peptides are severely insoluble in most organic solvents. In the late 1950s, R.B. Merrifield (a biochemist) made significant contributions and advancements in the combined effect of these two distinctive peptide properties.
Merrifield put out a brand-new approach to organic synthesis. However, his proposal to carry out all synthetic manipulations using the C-terminus of the target peptide connected to insoluble solid support faced heavy criticism, as is frequently the case when an outsider looks into an exclusive field and pursues a strategy that is damaging to the existing experts in the area of science.
However, it took only a short time for traditional solution-phase techniques to lose out to the speed and adaptability of solid-phase peptide synthesis (SPPS), thanks to its refined chemistry.
The initial "Merrifield" version of SPPS, more appropriately known as Boc/Benzyl chemistry, used a graduated acid lability mechanism to control all protecting groups and was essentially completed in the late 1960s.
This method eliminates alpha-amino t-butoxy carbonyl (Boc) protection using TFA. At the same time, there is a requirement for substantially more severe acidic conditions to break down side-chain defenses and peptide-resin anchoring (the linker). Liquid HF, which has an acidity function of -11 and TFA's acidity function of 0.1, is used to complete this stage.
By enabling purification by filtration, Boc/Benzyl SPPS streamlines all of the processes involved in peptide synthesis such that extra reagents can be used and eliminated by straightforward washing. It is crucial to stress that solution-phase peptide synthesis uses a chemistry fundamentally similar to that of SPPS.
Peptide Synthesis Procedure
The carboxyl group of the amino acid couples the N-terminus of a developing peptide chain throughout the peptide synthesis process. Protein biosynthesis, in which an amino acid's N terminus links to the C terminus of an existing protein chain, is the polar opposite of this C-to-N synthesis.
Since protein synthesis in vitro is intricate, adding amino acids to an expanding peptide chain happens precisely, step-by-step, and repeatedly. It’s vital to keep repeating the peptide synthesis steps until they produce the desired sequence.
Even though the prevalent peptide synthesis techniques have some significant variances, they all use the exact step-by-step process to gradually add amino acids.
Uses for Synthetic Peptides
Synthetic peptides have a variety of application areas, including:
- Creation of epitope-specific antibodies against pathogenic proteins
- Facilitates the study of protein functions or protein expression service
- Help with the identification and characterization of proteins
The applications above came about through the discovery of peptide synthesis in the 1950s and 1960s. Synthetic peptides also investigate enzyme-substrate interactions within significant enzyme classes, including kinases and proteases, which are essential for cell signaling.
The creation of high-quality antibody production services depends on the selection of appropriate peptides. So, researchers often use similar synthetic peptides to study how receptors bind to proteins or how newly found enzymes recognize their substrates to facilitate custom antibody production. Synthetic peptides can mimic naturally occurring peptides and have therapeutic effects against cancer and other severe disorders.
In applications based on mass spectrometry (MS), one can employ synthesized peptides as standards and reagents. Especially for proteins that function as early disease indicators, synthetic peptides are essential for discovering, characterizing, and quantifying proteins using MS. Peptide synthesis is also crucial for Recombinant Antibody Production and Recombinant Protein Production.