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Introduction
Peptides are an important kind of biomolecule with an increasing importance in drug discovery, (1) cosmetics, (2) agriculture, (3) and material science, (4) among others. This privileged position of peptides in the scientific world, with a strong impact on the markets, is undoubtedly thanks to the development of solid-phase synthesis (SPS) led by Nobel Prize winner R. Bruce Merrifield in the 1960s (5). Without the strategy of solid-phase peptide synthesis (SPPS), peptides would be solely the subject of academic study with some applications as biochemical tools.
In SPPS, the peptide elongation takes place with the growing peptide attached to a solid support/resin. This allows a very easy work-up because the excess reagents and soluble side-products can be removed by simple washing and filtrations. Using solid-phase chemistry, the SPPS process is highly streamlined; the method is an iterative cycle of adding reagents dissolved in the appropriate solvents, followed by filtration and adding solvents for washing until a final cleavage and elution step. This allows automation, which has sped up much of all the peptide research.
For many years, SPPS has been carried out at room temperature (6). However, it is important to take into account that in SPPS, the reagents (amino acids, coupling and deprotecting agents) need to diffuse into the polymeric resin beads to reach the reactive sites (7). Higher temperatures reduce solvent viscosity and increase the rate of diffusion, ensuring that the reagents can effectively reach all available reaction sites within the resin matrix. In addition, SPPS performed at higher temperatures enables the reduction of deprotection and coupling times by speeding up reaction kinetics (8), (9). Heat reduces peptide aggregation on the resin, which causes most of the side reactions; reducing aggregation optimizes the accessibility of free amino acids of the growing peptide chain for subsequent addition steps (10).
Meeting the rising demand for peptide therapeutics and for other applications requires the development of methodologies that increase the productivity of commercial-scale peptide manufacturing facilities. At small-medium scale, several approaches have demonstrated success at improving synthesis speed, including microwave heating, (11) rapid stirring, (12) and variable bed flow reactors; (13) these methods have not been demonstrated to meet the demands of large commercial scale production.
Conduction heating is a low-cost, simple to integrate method of providing heat to peptide synthesis processes. It provides precise, uniform temperature control, in contrast to other heating methods that may potentially generate hotspots (11). As synthesis quality is sensitive to temperature, maintaining tight control over the temperature of the reagents matters for optimizing the full process, reducing the need for double coupling by improving the reaction kinetics and minimizing undesirable reactions such as racemization. Conduction heating can be implemented at all scales, streamlining process adaptation as demands on throughput increase.
Here, we integrated a heat exchanger on the CSBio 136X, a research scale automated peptide synthesizer, to allow preheating of reagents prior to transfer to the heated reaction vessel. Preheating was expected to reduce the time required for reagents to reach the target temperature, speeding up synthesis time, while enabling deprotection and coupling reactions to occur at higher temperatures to improve product yield and purity.
Materials and Methods
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