How a Peptide Synthesizer Works & How We Save You Time & Money

The different resins used during SPPS provide different functions. One of the most popular is polystyrene, which dissolves in hydrophobic solvents and precipitates in protic solvents (hydrogen atom bound to an oxygen atom). This resin typically contains 1% or 2% divinylbenzene (DVB) as a cross-linking agent. Cross-linked polystyrenes are insoluble in all common solvents.

However, even cross-linked polystyrene resins that are insoluble in organic solvents can be solvated and swollen by aprotic solvents such as toluene and dimethylformamide (DMF), and dichloromethane (DCM). Swelling the resin is essential in solid-phase synthesis since reaction kinetics is diffusion controlled.

Consequently, a resin that swells more will have a higher diffusion rate of reagents into the core of the matrix, resulting in shorter reaction times and more complete chemical conversions.

In general, to swell the resin, you must first accurately weigh the resin into the reaction vessel. Next, you must add an approximate amount of DMF. Afterward, you let the mixture stand at room temperature for 10 minutes or so; that way, the resin beads can swell. Lastly, you drain the DMF.

Peptide deprotection is a secondary process in synthesizing peptides where a target nucleophile and a target electrophile are isolated to promote reaction specificity. Amino acids contain a variety of reactive groups. When creating the proper peptide chain without adverse side reactions, chemical groups that block specific reactive groups are necessary. This is called blocking or protecting.

Deprotecting is the process of removing the protecting groups just after coupling to allow the other amino acids to bind, properly growing the peptide chain.

Because synthesis typically occurs by coupling the carboxyl group of the incoming amino acid to the N-terminus of the growing peptide chain (C-to-N), there are specific protecting groups for both C-termini and N-termini.

Two of the most commonly used protecting groups for the N-terminus include tert-butoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Each group has distinct characteristics that determine its use. For example, Boc requires a strong acid such as trifluoracetic acid (TFA) to be removed from the newly added amino acid. At the same time, Fmoc is a base-labile protecting group that can be removed with a mild base like piperidine.

For C-termini, the protecting group used depends on the type of peptide synthesis used. Solid-phase peptide synthesis does not require a protecting group because the solid support acts as the protecting group.

It is important to note that virtually all processes of synthesis require the use of multiple protecting groups. Amino acid side chains represent a broad range of functional groups. They are, therefore, an area where considerable side chain reactivity occurs during peptide synthesis. For this reason, many different protecting groups are required. However, the various groups used are usually based on the benzyl (Bzl) or tert-butyl (tBu) group.

Furthermore, having groups that are entirely compatible and do not interfere with one another’s functions is essential to building the peptide chain correctly.

Amino acid coupling requires the activation of carboxylic acid on the amino acid added to the reaction by using peptide coupling reagents, such as HBTU. In other words, these peptide coupling reagents are used to form the amide bonds that link separate amino acids into peptides and proteins.

This allows the N-terminus of the growing peptide chain to connect with the C-terminal of the amino acid so that the peptide bond can form and the chain can continually grow. Next, the N-terminus of the newly created peptide chain is deprived, and peptide deprotection is repeated until the entire length of the peptide is completed.

After coupling and deprotection cycles are completed, the remaining protecting groups have to be removed from the peptide that is being formed.

Cleaving out these groups is accomplished by acidolysis. The specific chemical used to remove the groups is entirely dependent on the protection groups used. For example, strong acids such as hydrogen fluoride (HF), hydrogen bromide (HBr), or trifluoromethane sulfonic acid (TFMSA) are used to cleave Boc groups. In contrast, a relatively milder acid such as TFA is used to cleave Fmoc groups.

When peptide cleavage is correctly performed, the N-terminal and C-terminal protecting groups of the last amino acid are both removed, as is any side-chain protecting groups. As is the case with deprotection, there are scavengers included during this step to react with free protecting groups.

While peptide synthesis can be relatively chemically complex, a synthesizer makes the process much simpler. Two main methods are used to generate peptides and proteins, so it is vital to know the difference if you are looking to lease:

• Liquid Phase: This is the classic methodology that scientists implemented when they first discovered that they could generate peptides in vitro. While this solution-phase method is not as standard today, it is still used quite often when performing large-scale synthesis. Unfortunately, the difficulty and length of time needed to use this method have made it less popular since the advent of SPPS.
• Solid Phase: Solid-phase, or SPPS, is the more common method used to create peptides presently. The main reason for the success of this method is due to peptide synthesizers, the easy automation, and the high throughput that they offer. This is in line with many other biological manufacturing processes and is a massive boon to scientific and medical professionals working in molecular biology and biotechnology.

Crude peptides can be purified using various chromatography techniques. This includes high-performance liquid chromatography (HPLC), size-exclusion chromatography, ion-exchange chromatography (IEC), partition chromatography, flash chromatography, and gel permeation chromatography.

The most widely used and most powerful method for peptide purification is a mode of HPLC known as reversed-phase liquid chromatography (RPC, or RP-HPLC). It is highly versatile and employs a non-polar stationary phase alongside a polar, aqueous mobile phase made up of water and, in some cases, a water-miscible organic solvent that acts as a modifier.

For purification, RPC separates the target peptides from impurities left over from the synthesis steps, such as isomers and peptide products from side reactions with free coupling and protecting groups, among other impurities, through the use of columns packed with spherical particles during the stationary phase.