Deep Eutectic Solvents Cut Amide Bond Waste by 91%

Researchers from the University of Calabria and the University of Alicante have demonstrated that combining next-generation carbodiimide coupling reagents with deep eutectic solvents creates a sustainable platform for amide bond formation. Published in Frontiers in Chemistry, the study presents a methodology that significantly reduces the environmental footprint of a reaction central to pharmaceutical and peptide synthesis. The team identified a specific combination of the reagent TBDCTsOH and the solvent reline as the optimal system for efficient carboxylic acid activation and rapid amidation under mild conditions.

Amide bond formation is one of the most frequently performed transformations in organic synthesis, serving as a cornerstone for the production of peptides, proteins, and a large proportion of marketed drugs. Industrial surveys from major pharmaceutical companies indicate that amidation accounts for approximately 16% of all synthetic transformations during medicinal chemistry campaigns. Despite its importance, conventional carbodiimide-mediated coupling protocols often rely on hazardous reagents and large volumes of volatile organic solvents, resulting in significant waste generation and environmental concerns.

The primary challenge in amide synthesis is the direct condensation of carboxylic acids with amines, which is thermodynamically and kinetically unfavorable under mild conditions. This difficulty arises from competitive ammonium carboxylate salt formation and the poor leaving ability of the hydroxyl group. To overcome this, activation strategies using stoichiometric coupling reagents have become the standard approach, allowing the in situ generation of reactive intermediates such as O-acylisoureas or activated esters. Carbodiimide-mediated coupling remains widely adopted due to its operational simplicity and broad substrate scope.

However, classical carbodiimide reagents like dicyclohexylcarbodiimide and EDCHCl have well-recognized limitations. These reagents can undergo hydrolysis or promote the competitive rearrangement of intermediates into unreactive N-acylureas, which reduces coupling efficiency and increases the risk of epimerization in stereochemically sensitive substrates. Additionally, the byproducts of these reactions, such as poorly soluble dicyclohexylurea, complicate product isolation. The traditional use of benzotriazole-based additives to suppress racemization has also become problematic due to concerns regarding thermal instability and potential explosiveness.

To address these issues, the research team investigated a new generation of non-symmetrical carbodiimide coupling reagents, specifically QCD, TBDCHCl, and TBDCTsOH. These reagents were designed to overcome the shortcomings of classical carbodiimides by incorporating quaternary ammonium functionalities and carefully selected counterions. This structural redesign suppresses undesired intramolecular cyclization, enhances hydrolytic stability, and significantly improves solubility in polar media. The study represents the first systematic evaluation of these advanced reagents in selected deep eutectic solvents for sustainable amide and peptide bond formation.

Deep eutectic solvents, or DES, are environmentally benign reaction media formed by hydrogen-bond acceptors and donors. They combine negligible vapor pressure, low volatility, and tunable polarity with excellent synthetic versatility. The team selected three DES based on choline chloride: reline (choline chloride and urea), ethaline (choline chloride and ethylene glycol), and glyceline (choline chloride and glycerol). Solubility screening revealed that while all three next-generation carbodiimides dissolved readily in ethaline at room temperature, complete dissolution in reline and glyceline required mild heating between 30 and 60 degrees Celsius.

Optimization experiments using a model amidation reaction between phenylacetic acid and 2-phenylethylamine identified the TBDC·TsOH-reline combination as the most effective system. This combination afforded the desired amide in 90% isolated yield after just 30 minutes at 60 degrees Celsius. In contrast, reactions in ethaline and glyceline yielded significantly lower results, with ethaline producing side products due to competitive nucleophilic attack by its ethylene glycol component. The superior performance in reline is attributed to its highly organized hydrogen-bonding network, which stabilizes reactive intermediates and facilitates efficient nucleophilic attack by the amine.

A key advantage of the optimized protocol is the elimination of the need for an external base. The team found that comparable reaction times and isolated yields were obtained without the addition of DIPEA, suggesting that the hydrogen-bonding environment of reline actively facilitates proton-transfer events. The high solubility of the urea-derived byproduct in the DES also simplified product isolation, eliminating the need for chromatographic purification. This streamlined work-up represents a significant practical advantage over conventional carbodiimide-mediated protocols, which often require complex purification steps.

The methodology was successfully extended to a broad substrate scope, including aliphatic and aromatic carboxylic acids, primary amines, and protected amino acids. The protocol demonstrated excellent compatibility with Fmoc-, Boc-, and Cbz-protected amino acids, affording dipeptides in 79% to 96% isolated yield. Stereochemical analysis using 1H NMR spectroscopy showed no detectable epimerization under the optimized conditions. The team noted that while a quantitative detection limit was not established, the spectroscopic method was sufficient to distinguish diastereomers, confirming the preservation of stereochemical integrity in the investigated reaction mixtures.

Green metrics analysis revealed that the DES-based methodology maintains high synthetic efficiency while substantially reducing the environmental footprint. Compared with a conventional carbodiimide-mediated protocol, the new method achieved nearly 91% reductions in both process mass intensity (PMI) and E-factor. The conventional protocol exhibited a PMI of 494.6 g/g, reflecting the extensive use of volatile organic solvents, whereas the DES-based methodology displayed a total PMI of only 44.7 g/g. The E-factor decreased from 493 g/g to 44 g/g, demonstrating a considerable reduction in waste generation while maintaining comparable reaction efficiency.

The study acknowledges that DES recycling was not investigated and represents a current limitation of the methodology. The addition of water during the work-up disrupts the hydrogen-bonding network of reline, and the high water solubility of urea may lead to preferential loss of the hydrogen-bond donor. These compositional changes could affect the physicochemical properties of the recovered DES. Future studies will be required to develop a recovery strategy that minimizes by-product accumulation and composition drift while maintaining the original performance of the solvent system.

Overall, the results demonstrate that the synergistic combination of the carbodiimide reagent and the deep eutectic solvent, rather than either component alone, is responsible for the superior performance of the developed methodology. This approach provides a practical strategy for more sustainable amide and peptide bond formation, relevant to pharmaceutical synthesis and fine chemical manufacturing. The integration of reagent engineering and solvent engineering offers a pathway to redesign fundamental organic transformations according to the principles of Green Chemistry.

The research team, led by Maria Luisa Di Gioia from the University of Calabria and Gabriela Guillena from the University of Alicante, conducted a systematic evaluation of three next-generation carbodiimide coupling reagents: QCD, TBDCHCl, and TBDCTsOH. These reagents were selected for their non-symmetrical structures, which incorporate quaternary ammonium functionalities and specific counterions to enhance hydrolytic stability and solubility. The study compared these advanced reagents against the conventional coupling agent EDCHCl, which requires heating to approximately 80 degrees Celsius to dissolve completely in reline. In contrast, the next-generation carbodiimides achieved full solubility at substantially lower temperatures, facilitating homogeneous reaction conditions and reducing the thermal energy required for the coupling process. This improved solubility is a critical factor in sustainable process development, as it allows for more efficient molecular diffusion and interaction within the reaction medium.

The choice of solvent proved to be as critical as the reagent selection in determining reaction efficiency. While ethaline exhibited the highest carbodiimide solubility due to its low viscosity of 36 cP at 25 degrees Celsius, it produced the lowest yields in the model amidation. This discrepancy is attributed to the ethylene glycol component of ethaline, which acts as a competitive nucleophile, attacking the activated acyl intermediate to form 2-hydroxyethyl 2-phenylacetate. Similarly, glyceline, with a viscosity of 376 cP, suffered from reduced molecular diffusion and competitive esterification reactions involving its glycerol component. Reline, despite its higher viscosity of 632 cP, provided the optimal balance of solvation and reactivity, allowing for rapid conversion and high selectivity without significant side-product formation.

The optimized protocol was further validated through its application to the synthesis of long-chain fatty acid amides, which are widely used in cosmetic formulations as surfactants and emulsifiers. The methodology successfully produced these highly hydrophobic compounds in yields ranging from 79% to 97%, demonstrating its versatility beyond pharmaceutical applications. The ability to prepare these industrially relevant compounds using a low-volatility deep eutectic solvent system under mild conditions highlights the potential for sustainable manufacturing in the fine chemical industry. The protocol’s compatibility with multifunctional molecules containing stereochemically sensitive centers and protecting groups further expands its utility in complex molecular synthesis.

Mechanistic considerations suggest that the deep eutectic solvent environment plays an active role in the reaction pathway beyond simple solvation. The hydrogen-bonding network in reline may stabilize polar or charged species and modulate proton-transfer processes, which is consistent with the observation that the reaction proceeds efficiently without an external base. Although direct spectroscopic monitoring of intermediate formation was not performed, the experimental results are consistent with a literature-informed interpretation where the DES facilitates the formation of reactive acyl intermediates. The preservation of stereochemical integrity in the presence of these reactive species is likely due to the rapid acyl activation provided by TBDCTsOH, which limits the lifetime of racemization-prone intermediates such as oxazolones.

The green metrics analysis provides a quantitative assessment of the methodology’s environmental impact, calculated using the CHEM21 Toolkit. The Atom Economy remained at 88% for both the conventional and DES-based protocols, reflecting the unchanged reaction stoichiometry. However, the Reaction Mass Efficiency improved from 71% to 84%, and the Optimum Efficiency increased from 80.8% to 95.8%. These improvements indicate a more efficient use of reagents in the DES-based system. The significant reduction in Process Mass Intensity is partly due to the elimination of chromatographic purification, which is a major contributor to waste in conventional protocols. The study notes that a direct deconvolution of the individual contributions of solvent replacement and purification simplification would require a matched control procedure, which is not straightforward given the different solubility profiles of the reagents.

Despite the demonstrated advantages, the methodology has certain limitations that require further investigation. The high water solubility of urea, a component of reline, poses a challenge for solvent recovery, as the addition of water during work-up can lead to the preferential loss of the hydrogen-bond donor. This compositional drift may alter the physicochemical properties of the recovered DES, potentially affecting its performance in subsequent reaction cycles. The accumulation of urea-derived by-products from the carbodiimide reagent also complicates the recycling process. Future research will need to focus on developing robust recovery strategies that minimize both by-product accumulation and composition drift to fully realize the sustainability potential of this approach.

Subject of Research: Sustainable amide bond formation using deep eutectic solvents and next-generation carbodiimides

Article Title: Sustainable amide bond formation through the combination of deep eutectic solvents and next-generation carbodiimides

Article References: Parrotta, L. C., Pisanelli, D. F., Perri, A., Gencarelli, S., Ramòn, D. J., Guillena, G., & Di Gioia, M. L. (2026). Sustainable amide bond formation through the combination of deep eutectic solvents and next-generation carbodiimides. Frontiers in Chemistry, 14, Article 1964972. https://doi.org/10.3389/fchem.2026.1964972

Image Credits: AI Generated

DOI: 10.3389/fchem.2026.1964972

Keywords: amide bond formation, deep eutectic solvents, green chemistry, carbodiimides, sustainable synthesis, pharmaceutical chemistry, reline, TBDCTsOH, waste reduction, peptide synthesis, Sustainable, amide