The 5 Basic Steps of Peptide Synthesis

The 5 Basic Steps of Peptide Synthesis

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A peptide comprises two or more amino acids joined together by an amide bond to form a chain of amino acids typically 2 to 70 long. Peptides differ from proteins because they do not need to be folded for biological activity. Significantly, peptides are found endogenously in plants and animals as peptide hormones such as angiotensin, LHRH, enkephalin, and toxins.  
Notably, peptides are very interesting as lead compounds for drug discovery. They are also used as antigens to generate antibodies and in vaccines, biomaterials, and histological probes. 
This article will discuss the five basic steps of peptide synthesis. 

1. Peptide deprotection 
Peptide synthesis must be done carefully to prevent side reactions that could shorten and branch the peptide chain because amino acids have numerous reactive groups. 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 peptide synthesis, these protecting groups react with the purified, individual amino acids used. After coupling, certain protecting groups are removed from the newly added amino acid (a procedure known as deprotection), enabling the subsequent amino acid to bind to the lengthening peptide chain in the correct orientation. When the Peptide Synthesis Service is finished, the nascent peptides are stripped of any remaining protecting groups. 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 they can be easily removed 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 trifluoroacetic acid (TFA). In contrast, Fmoc is a base-labile protecting group removed with a mild base such as piperidine.  2. 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 (DPC) (DIC). 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. Furthermore, side effects caused by carbodiimides prompted the investigation of 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), both of which require activating bases to mediate amino acid coupling. 
3. Peptide cleavage 
After several cycles of amino acid deprotection and coupling, the nascent peptide must have all protecting groups removed. Strong acids like hydrogen fluoride, bromide (HBr), or trifluoromethane sulfonic acid cleave Boc and Bzl groups. Fmoc and tBut groups are split apart using TFA. When carried out properly, cleavage eliminates any side-chain protecting groups, the C-terminal protecting group (either chemical or resin) of the first amino acid, and the N-terminal protecting group of the most recently added amino acid. 

4. Peptide synthesis strategies 
The first technique scientists used to figure out how to produce peptides in vitro was liquid-phase peptide synthesis, which is still widely used for large-scale synthesis. Because the product needs to be manually removed from the reaction solution after each step, this method is labor- and time-intensive. Furthermore, this approach requires another chemical group to protect the first amino acid's C-terminus.  
However, solid-phase peptide synthesis is by far the most common method today. Instead of a chemical group protecting the C-terminus, the first amino acid's C-terminus is coupled to activated solid support, such as polystyrene or polyacrylamide. This method serves two purposes: the resin is the C-terminal protecting group. It provides a quick way to separate the growing peptide product from the various reaction mixtures during synthesis. Like many other biological manufacturing processes, Peptide synthesizers have been developed for automation and high-throughput peptide production. 

5. Peptide purification 
The peptide production process is far from ideal, even though peptide synthesis methods have been improved and can be mass-produced. Incomplete deprotection or reactions with free-protecting groups can result in truncated or deleted sequences, isomers, or other byproducts. These events can occur at any point during peptide synthesis, so the longer the peptide sequence, the more likely something will interfere with the synthesis of the target peptide. As a result, peptide yield is inversely related to peptide length. 
Purification strategies are typically based on a combination of separation methods that take advantage of peptide physicochemical properties such as size, charge, and hydrophobicity. Purification methods include: 

          *Size-exclusion chromatography 
          *Ion exchange chromatography (IEC) 
          *Partition chromatography 
          *High-performance liquid chromatography (HPLC) 

The most versatile and widely used method of peptide purification is reverse-phase chromatography (RPC). Conventional HPLC techniques capture polar, hydrophilic molecules in the stationary phase by increasing the concentration of polar solvents in the mobile phase. These molecules are then differentially eluted. As the name suggests, the stationary phase binds hydrophobic C4, C8, or C18 n-alkyl hydrocarbon ligands to capture hydrophobic molecules from aqueous solutions. The hydrophobicity of the molecule and the mobile phase determines how long they remain in the solution. 

The ability to synthesize high-purity peptides on a large scale and multiple sequences in parallel has revolutionized many fields of basic research and provided advanced weapons in the fight against the disease. Significantly, peptide synthesis companies provide you with the peptide synthesizers you require to meet your requirements in terms of yield, purity, scale, and parallel synthesis. 

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