Can binary solvents be used in automated solid-phase peptide synthesis?

The increasing demand for peptide therapeutics highlights the urgent need for green peptide synthesis to minimize environmental impact. DMF, a widely used solvent in solid-phase peptide synthesis (SPPS), is highly toxic but essential due to its ability to solubilize Fmoc-protected amino acids and coupling reagents. As industries shift toward sustainable alternatives, evaluating binary mixture solvents to replace DMF has proven to be a critical step toward more eco-friendly peptide manufacturing, but also at research scales.

When evaluating a DMF replacement, considerations must include solubility of Fmoc-protected amino acids, coupling reagents, compatibility with deprotection conditions as well as resin swelling characteristics. Fortunately, significant effort has already been devoted to exploring DMF alternatives, but they have been principally focused on large scale manufacturing processes. In this post, we’ll evaluate previously reported binary solvent mixtures 9:1 EtOAc: DMSO, a combination of binary solvents (9:1 EtOAc: DMSO and 6:4 EtOAc: DMSO), and 7:3 BtOAc: DMSO for use in automated peptide synthesis. Synthesis of the ACP 10mer was used to evaluate the compatibility of each binary solvent mixture with automated SPPS.

As a first step, the default protocol was adjusted to reduce or eliminate the wash steps between the deprotection and coupling reactions. Adjusting these protocols on the Biotage^® Initiator+ Alstra^™ cut solvent consumption by over 50% without a significant loss in synthesis efficiency, enhancing peptide synthesis sustainability significantly.

Maintaining the minimal washing strategy, the binary solvent mixtures mentioned previously, were then tested for synthesis efficiency with an automated synthesizer, Table 1.

Table 1. Sustainable synthesis evaluation using ACP as a test sequence.

Key findings:

• Solvent consumption was reduced by more than 50% by reducing resin washes between chemistry steps.
• Synthesis time was shortened by 29.33%.
• Crude purity reduction was minimal with the 7:3 BtOAc: DMSO solvent system (13.94% reduction relative to default) performed with minimal method changes.

Among the binary solvent mixtures tested, 7:3 BtOAc: DMSO proved to be the most suitable alternative, yielding a 4.99% reduction in crude purity (Table 1) compared to DMF-based synthesis with minimal resin washing. Notably, BtOAc exhibits a higher boiling point than EtOAc, resulting in a slower evaporation rate during synthesis. This reduced volatility helps maintain consistent solvent volume and consumption throughout the synthesis cycle—an advantage over EtOAc, which is likely to evaporate too quickly at elevated coupling temperatures. The stability of BtOAc under reaction conditions may contribute to improved peptide purity and consistency. Additionally, BtOAc’s hydrophobic nature influences resin swelling behavior and therefore improves solvent accessibility and coupling efficiency in SPPS.

Figure 1 provides an HPLC overlay comparing peptide ACP synthesized under default DMF conditions (orange) and using 7:3 BtOAc:DMSO with reduced washes (blue).

Figure 1. HPLC overlay of ACP comparing synthesis using default conditions in DMF (orange) and reduced washing in 7:3 BtOAc: DMSO (blue).

The observed reduction in purity is expected due to fewer post-deprotection washes rather than a fundamental limitation of the binary solvent system. While reducing DMF usage is a priority, transitioning to binary mixture solvents presents a more viable and environmentally friendly alternative.

This study highlights the importance of proper solvent selection and optimized wash steps to ensure efficient and sustainable automated SPPS. The 7:3 BtOAc: DMSO binary solvent system has demonstrated here compatibility with automated peptide synthesis, reducing environmental impact while maintaining high crude purity with minimal method optimization.

Have you explored alternative solvents for a greener approach to automated SPPS? Click here to learn more about sustainable peptide synthesis, and how you can reduce DMF usage in your workflows.