4 Tips to Know When Performing SPPS | RespectYourHealth.eu

4 Tips to Know When Performing SPPS

4 Tips to Know When Performing SPPS 

The regulatory mechanisms of most biological processes depend on peptide synthesis. This naturally involves the breakdown of α–amino acids to produce peptides and proteins, making peptides essential building blocks in many biological products. 

Most medicines nowadays are manufactured from peptides or their derivatives. This means custom peptide synthesis is an essential technology in medicinal chemistry, biotechnology, biochemistry, and organic chemistry. Most blood pressure drugs, anti-cancer agents, and even some antibiotics are also made from peptides. 


Solid-phase peptide synthesis (SPPS) technology is now automated for the commercial production of synthetic peptides and used for biological and industrial applications. Synthetic peptides can synthesize whole enzymes, protein- or peptide mimetics, examine enzyme binding sites, map antibody epitopes, and even produce epitope-specific antibodies.

Solid-phase peptide synthesis SPPS is a long process, yet the points below will help maneuver the most challenging stages and save time.


  • Consider Amino Acid Concentration. 


During a peptide synthesis, the typical amino acid solution concentration has a standard range between 0.1 and 0.5 M in dimethylformamide (DMF). Since manufacturers use automated peptide synthesizers that come with standardized amino acid concentrations, the concentrations are of little significance. 

While these stoichiometry equivalents are the main aspect considered in the synthesis process, the liquid volume of the molecules also matters in the SPPS process. In the synthesis of short peptides, the concentration of amino acids has little impact on the crude peptide purity and doesn’t have a significant impact on the overall success of the process. As a result,  high yields are still possible with high concentrations of amino acids. When DIC and Oxyma are used as the coupling reagents, it’s possible to produce high yields and high-crude purity, regardless of the amino acid concentrations. 


In long-chain peptides synthesis, when using a low concentration of amino acids, a high crude peptide is attained, but there will also be many other by-products in the crude sample. With a high concentration of amino acids, a higher crude purity is attained. 

The use of high concentrations of amino acids can result in high-crude peptide purity in line chain peptide synthesis. This can save a lot of time as there are higher yields and less time is spent purifying the samples. However, this comes with other challenges since the resin may not be free-flowing as desired and the total liquid volume might weaken kinetic energy.


2. Use In-Situ Activating Reagents

As in-situ reagents are easy to use, they are utilized for a variety of applications. The salts give a fast reaction without any detrimental side reactions and two of the common types include phosphonium and uranium reagents. When introduced into a tertiary base, these salts can synthesize protected amino acids to various activated types. 

3. The Difference Between Phosphonium Vs. Uronium Reagents. 

If a more efficient reagent is required, consider uronium-based reagents. Most procedures use coupling reagents that come in pre-made solutions, which makes uronium-based reagents ideal due to their high stability. 

The possibility of chain termination when using uronium-based reagents, however, makes them unsuitable for cyclization and fragment reactions as they require the use of excess uronium reagent. They will also be problematic when synthesizing peptides with positive charges (such as long peptides) as the positive charge can disguise the target ion, concealing it in the ESI mass spectrum. 

When using long peptides, it’s advisable to use phosphonium-based reagents. This is because phosphonium salts contain excess PyAOP, which doesn’t undergo side-reaction at the N-terminal amino group and is an unfavorable process that hinders the continuation of chain assembly.

Phosphonium-based salts, contrarily, are less stable and can only be used within two days and must be stored in a concealed bottle. Phosphonium coupling reagents, however, have an advantage over uronium reagents as they provide cleaner reactions.  Consequently, phosphonium-based reagents are better suited for solid-phase phosphate synthesis for several peptides (especially cyclic peptides) that contain difficult short sequences, and hindered amino acids. 


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4. Synthesis For Difficult Couplings. 

If synthesizing difficult sequences is required, it’s important to understand what strategies to use. 

This is a challenge many face but never get the right answer. Some experts have suggested making amino acid chains of shorter fragments, then using native chemical ligation to join them. Well, the right results would be attained, but it requires a lot of work. 

A simple method is to use a coupling reagent that produces esters that have higher reactivity than OBt as opposed to those that produce OBt-esters. These include HCTU, PyAOP, and HATU, and not the common HBTU, PyBOP, and BOP. These reagents produce O-6-ClBt and OAt esters, which have lower pKa of HO-6-ClBt and HOAt and are, therefore, more reactive. 

In addition, HOAt contains the pyridine nitrogen, which gives the coupling reaction anchimeric assistance. These qualities make HATU and PyAOP better coupling reagents when synthesizing difficult coupling. 

You could also use Oxyma Pure leaving group coupling reagents though there are mixed reactions to their viability. This category includes reagents such as PyOxim and COMU. Some studies indicate they have higher reactivity than HOAt-based reagents, while others suggest COMU is superior. However, there is a consensus that Oxyma-based reagents are superior to the HOBt and O-6-ClBt groups of reagents. 

Bonus Tip!

The esterification process that anchors the amino acid to its solid support is normally problematic and can be dangerous when using certain residues. The process can also form dipeptide and carries the risk of epimerization and low substitution. Based on these concerns, it is advisable to buy commercial C-terminal N-protected amino acid preloaded resins. 

The anchoring reaction in an anhydrous medium must be performed to achieve the best results. For this reason, dry any amino acids that contain water before the procedure. Most prefer the symmetrical anhydride method for the esterification process. If this procedure is applied, use the Fmoc release measurement to determine the loading. In the event the anchoring turns out to be difficult, you can repeat the process using fresh solutions.  


Emma Willson
Emma is a passionate writer & guest blogger. Writing helps her to improve her knowledge, skills & understanding about the specific industry. She loves writing & sharing her knowledge mostly in the fashion & lifestyle industry. She is passionate about spreading her knowledge & tips across the world. Apart from writing, she loves traveling, styling & cooking.