In Fmoc solid-phase peptide synthesis (SPPS), N,N-dimethylformamide (DMF) is used as standard solvent, in academia as well as industry, from research scale to bulk production. Within the EU, the overall market for DMF is currently 20-30 thousand tons per year. However, according to Article 57(c) of REACH (Registration, Evaluation Authorization and Restriction of Chemicals), this solvent is classified i. a. as toxic for reproduction. The REACH regulation handles the use of harmful and environmentally damaging substances in Europe and was established to protect both humans and the environment from hazardous chemicals.

As from December 12, 2023, the EU placed a new restriction on DMF under Annex XVII to REACH regulation. Its use is not prohibited, but the exposure limits were lowered to 6 mg/m³ (2 ppm) for exposure by inhalation and 1.1 mg/kg/day for dermal exposure. An extension of this deadline by one year is given for polyurethane coating processes and membrane fabrication, and by two years for the dry and wet spinning of synthetic fibers. Still, this is just an action within Europe, which is not scheduled for UK or USA.

In view of this stricter regulation, alternative solvents are heavily discussed in literature. However, it is important to not only circumvent the regulatory control, but to understand the potential hazards of an alternative solvent and choose wisely. Besides, especially for SPPS, solvent polarity and – even more for automated synthesizers – viscosity are crucial parameters that need to be evaluated. The choice of the solvent has a drastic impact on resin swelling, diffusion of the solvent into the solid support, and peptide solvation.

In terms of safety and environmental impact, water would be the ideal alternative solvent and has already been reported for the synthesis of short research peptides. Nevertheless, it is – so far – not compatible with standard Fmoc SPPS and protecting groups, resins, and set-ups need to be fine-tuned. As further alternative, organic “green” solvents such as 2-methyltetrahydrofuran (2-MeTHF), gamma-valerolactone (GVL), N-butylpyrrolidone (NBP), methyl-5-(dimethylamino)-2-methyl-5-oxopentanoate (Polarclean), and N-octylpyrrolidone, are under evaluation as DMF substitute. “Green” solvents are specified by, e.g., low toxicity, easy availability, re-usability, non-flammability, and non-volatility thus reducing dangers to health and environment. Already in 2018, there was a detailed solvent comparison by Lopez et al. revealing that NBP could be used as replacement for DMF in the large-scale SPPS of Octreotide. However, reaction rates in NBP are slower compared to DMF due to its increased viscosity limiting its attractiveness.  

Another consideration is the use of solvent mixtures instead of a single solvent to replace DMF. One promising combination might be DMSO/EtOAc. (Interested? Get in contact!) Advantageously, for solvent mixtures, polarity and viscosity can be adjusted by changing the ratios of the components providing additional flexibility depending on the requirements of the performed reaction step.

In conclusion, there is no “one-fits-all” solution. For each peptide, risks given by DMF should be considered carefully and possibilities of alternative solvents should be investigated as much as possible.

References:

N,N-Dimethyl Formamide European Restriction Demands Solvent Substitution in Research and Development; J. Sherwood, F. Albericio, B. G. de la Torre; ChemSusChem 2024; e202301639. https://doi.org/10.1002/cssc.202301639

What is a green solvent? A comprehensive framework for the environmental assessment of solvents; C. Capello, U. Fischer, K. Hungerbühler; Green Chem. 2007; 9:  927-934. https://doi.org/10.1039/B617536H

Green and Sustainable Solvents in Chemical Processes; C. J. Clarke, W.-C. Tu, O. Levers, A. Bröhl, J. P. Hallett; Chem. Rev. 2018; 118(2): 747-800. https://doi.org/10.1021/acs.chemrev.7b00571

Green Solvents. Solvents – Dilute, Dissolve, and Disperse; P. Tomar, D. Jain; IntechOpen 2023. https://doi.org/10.5772/intechopen.1002491

The green solvent: a critical perspective; N. Winterton; Clean Technol Environ Policy 2021; 23(9): 2499-2522. https://doi.org/10.1007/s10098-021-02188-8

Evaluating Green Solvents for Bio-Oil Extraction: Advancements, Challenges, and Future Perspectives; M. Usman, S. Cheng, S. Boonyubol, J. S. Cross; Energies 2023; 16(15): 5852. https://doi.org/10.3390/en16155852

Molecular Solvents - Replacements for DMF, DMAC, NMP; https://reagents.acsgcipr.org/reagent-guides/greener-peptide-synthesis/list-of-reagents/molecular-solvents-replacements-for-dmf-dmac-nmp; 08.01.2024

Replacing DMF in solid-phase peptide synthesis: varying the composition of green binary solvent mixtures as a tool to mitigate common side-reactions; S. Jadhav, V. Martin, P. H. G. Egelund, H. J. Castro, T. Krüger, F. Richner, S. T. Le Quement, F. Albericio, F. Dettner, C. Lechner, R. Schönleber, D. S. Pedersen; Green Chem. 2021; 23: 3312-3321. https://doi.org/10.1039/D1GC00604E

All’s swell: Greener replacements for hazardous solvents in peptide synthesis; K. Wegner, D. Barnes, K. Manzor, A. Jardine, D. Moran; Green Chemistry Letters and Reviews 2021; 14(1): 153-164. https://doi.org/10.1080/17518253.2021.1877363

Evaluation of greener solvents for solid-phase peptide synthesis; K. Wegner, D. Barnes, K. Manzor, A. Jardine, D. Moran; Green Chemistry Letters and Reviews 2021; 14(1): 153-164. https://doi.org/10.1080/17518253.2021.1877363

Peptide synthesis beyond DMF: THF and ACN as excellent and friendlier alternatives; Y. E. Jad, G. A. Acosta, S. N. Khattab, B. G. de la Torre, T. Govender, H. G. Kruger, A. El-Faham, F. Albericio; Org. Biomol. Chem. 2015; 13: 2393-2398. https://doi.org/10.1039/C4OB02046D

Green Solvent Mixtures for Solid-Phase Peptide Synthesis: A Dimethylformamide-Free Highly Efficient Synthesis of Pharmaceutical-Grade Peptides; L. Ferrazzano, D. Corbisiero, G. Martelli, A. Tolomelli, A. Viola, A. Ricci, W. Cabri; ACS Sustainable Chem. Eng. 2019; 7(15): 12867-12877. https://doi.org/10.1021/acssuschemeng.9b01766

N-Butylpyrrolidinone as Alternative Solvent for Solid-Phase Peptide Synthesis; J. Lopez, S. Pletscher, A. Aemissegger, C. Bucher, F. Gallou; Org. Process Res. Dev. 2018; 22: 494-503. https://doi.org/10.1021/acs.oprd.7b00389

Harnessing polarity and viscosity to identify green binary solvent mixtures as viable alternatives to DMF in solid-phase peptide synthesis; V. Martin, S. Jadhav, P. H. G. Egelund, R. Liffert, H. J. Castro, T. Krüger, K. F. Haselmann, S. Thordal Le Quement, F. Albericio, F. Dettner, C. Lechner, R. Schönleber, D. S. Pedersen; Green Chem. 2021; 23: 3295-3311. https://doi.org/10.1039/D1GC00603G