CEM has developed key technology advantages for solid phase peptide synthesis. These technology advantages are utilized in the production process of peptides.


Microwave Enhanced Solid Phase Peptide Synthesis

The use of microwave energy to enhance solid phase peptide synthesis has been established as a valuable technique. It has demonstrated the ability to enhance both the deprotection and coupling steps within each amino acid cycle1. Initially this was shown to push difficult reactions to completion which were a challenge under conventional conditions. At the same time, certain side-reactions had the potential to be increased at elevated temperature and required special conditions. This included primarily epimerization of amino acids during coupling and aspartimide formation of aspartic acid under basic deprotection conditions. In subsequent years the technology was refined to provide highly effective deprotection and coupling processes that minimize these side reactions while still providing acceleration of the steps. In 2014, the High-Efficiency Solid Phase Peptide Synthesis (HE-SPPS) process was developed. The HE-SPPS process was utilized on a next generation microwave peptide synthesizer, Liberty Blue which provided a massive savings in synthesis time and solvent usage. The Liberty Blue was recognized with an R&D 100 award in 2014. In 2018, CEM developed the world’s first production scale automated microwave peptide synthesizer, Liberty PRO. This system allowed for the HE-SPPS process to be scaled up for generation of up to kilogram amounts of peptide.

Further Information
2012
Microwave heating in solid-phase peptide synthesis

2013
Microwave-Enhanced Synthesis of Peptides, Proteins, and Peptidomimetics

2020
New Developments in Microwave-Assisted Solid Phase Peptide Synthesis


CarboMax Coupling Process

CarboMax Coupling Process

Amide bond formation in peptide synthesis has been demonstrated to be dramatically enhanced by microwave irradiation. This results in more complete coupling in a faster time with the potential to use less excess of reagents. Under elevated temperature conditions, carbodiimide based coupling is preferred over onium salt based methods which employ strong bases that can lead to increased epimerization.

In 2017, CEM developed an improvement on the standard carbodiimide based process2. This technology, called CarboMAX improved traditional carbodiimide coupling by increasing the rate of formation of the key intermediate, o-acylisourea. By increasing the formation rate of the o-acylisourea intermediate the rate of formation of the activated amino acid is correspondingly increased. The result is a faster and more complete coupling process. This allows for a reduction in epimerization of amino acids by reducing the average lifetime of activated amino acids before they couple.

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One-Pot Combined Coupling & Deprotection

One-Pot Combined Coupling & Deprotection

Traditional solid phase peptide synthesis involves the use of iterative and separate deprotection and coupling steps with washing in-between. This is based on the assumption that undesirable amino acid insertions can occur without complete draining and washing between each step. With HE-SPPS, it was demonstrated that washing after the coupling step can be eliminated without effect on peptide purity3. This process was taken further by the development of a new one pot coupling and deprotection process4. This technique involves addition of the deprotection reagent (base) directly to the undrained post-coupling mixture. The ability to do this is based on the insight that faster reaction kinetics in the solution phase promote rapid hydrolysis or self-condensation of the active ester, thereby avoiding potential side reactions at the resin bound amino functionality. The Fmoc removal then proceeds uninterrupted at elevated temperature. An optimized use of reagents results in an essentially neutral reaction mixture towards the end of deprotection step.

This new procedure offers major advantages within each iterative cycle from a reduction in solvent requirement for the deprotection step and a faster deprotection step since the microwave ramp time is not needed. At production scale, this can result in significant solvent, time, and energy savings.

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Greener Solvents

Greener Solvents

N-butylpyrrolidinone (NBP; TamiSolve NxG-PS) has demonstrated the ability to work well as a greener alternative apolar diprotic solvent. It’s use in microwave SPPS is easier than traditional methods since the elevated temperature reduces its relatively high viscosity.

Additionally, CEM has developed a unique solvent mixture that is ideal for microwave peptide synthesis5. This is based on the use of anisole and N-butylpyrrolidinone that provides ideal properties for completely replacing traditional solvents. This solvent system has been demonstrated on a range of difficult peptides and provides favorable toxicity, reduced viscosity, and low overall cost.

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View Data

1US7393920; US7582728; US8058393; JP4773695
2US 10,858,390; US 9,969,769; EP17188963.7; JP2016138090
3Collins, J., Porter K., Singh S., Vanier G. Org. Lett. , 940-943 2014
4US20170226152; WO2017070512; WO2018044356A1
5US 11,014,959; WO2019241586A1