Deep eutectic solvents are considered the green solvents of the 21st century with tremendous applicability in all areas of the chemical industry.
Deep eutectic solvents (DES) can be defined as a mixture of two or more primary metabolites (normally solid at room temperature), such as sugars, amino-acids or organic acids, that at certain molar ratios exhibit a high depression of the melting temperature becoming liquid at or near room temperature. At these conditions, the compounds that form the deep eutectic solvents interact between themselves, mainly through hydrogen bonding. This strong interaction is responsible for the components behaving as one single entity and for the depression observed on the melting point of this new entity in comparison with the pure components.
Because the production of these deep eutectic solvents relies solely of the physical mixture of two or more natural components, their production has virtually no impact on the environment. Moreover, because deep eutectic solvents do not need any complex processing and equipment, they are also cheap alternatives to most common green solvents such as ionic liquids.
Deep eutectic solvents
For the above, deep eutectic solvents are considered the green solvents of the 21st century. Research dealing with deep eutectic solvents is relatively recent, with about 85% of the papers in the field being published in the last five years. The world of DES is growing exponentially, with new findings happening every day, either in understanding the fundamentals on how DES are formed, on their properties and behaviour in different conditions, or their applicability in new and exciting fields. From extraction solvents to cell cryopreservants, from therapeutic agents to protein stabilisers, DES have proven to be versatile solvents that transverse different fields of research.
As part of the associated laboratory LAQV in the Faculty of Sciences and Technology – NOVA, the Biocatalysis and Bioenergy (B&B) research group, led by Professor Susana Barreiros and where Dr Alexandre Paiva leads the research in DES, works in collaboration with the DES.SOLVE research group led by Professor Ana Rita Duarte. A Nature-inspired approach has been adopted here in order to develop new DES for different industries such as biomedical, pharmaceutical, cosmetics, and others.
Since the beginning of time, mankind has mimicked Nature in order to develop new technologies, products and solutions for everyday problems. This is especially true for the pharmaceutical and food industries in the search for new, safer and better drugs and food additives. One of the solutions that Nature has to offer comes from the adaptation of animals and plants to extreme environments. To survive in environments with large temperature variations, animals and plants have developed defense mechanism through the production of specific metabolites as surrounding conditions vary.
In 2011, Choi and co-workers related the presence of these naturally occurring molecules and proposed that they formed a third type of liquid, one separate from water and lipids. By mimicking the role of these DES in nature, new nature-inspired applications can be devised. This is the main mission of the B&B and DES.SOLVE research groups. Through two national funded research projects co-ordinated by Paiva (DES-ZYME – PTDC/BBB-EBB/1676/2014 and CryoDES – PTDC/EQU-EQU/29851/2017) and a European Research Council Consolidator grant awarded to Duarte (ERC-2016-COG-725034), both research groups have been fully dedicated in working together towards the advancement of deep eutectic solvents.
Biocatalysis in deep eutectic solvents for the pharmaceutical industry
Many active pharmaceutical ingredients (API) are available as racemic mixtures in which usually only one of the enantiomers has bioactive properties. When applied to medical formulations only 50% is active, which causes the dosage to be twice the amount that is necessary. The unreactive enantiomer can be harmless and eliminated by the organism without any consequences. However, there are historical examples of drugs, such as thalidomide, in which the non-bioactive enantiomer accumulation lead to severe health problems. In this specific case the (+) enantiomer had sedative properties and was used by pregnant women to prevent nausea while the (-) enantiomer accumulated in the organism and caused teratogenic effects to the developing babies.
Over the years, industries have developed several strategies for the resolution of racemic compounds including crystallisation, chromatography and kinetic resolution. Enzymatic kinetic resolution (EKR) is based on the ability of some enzymes to distinguish between enantiomers. Resolution of racemates is one of the most common transformations catalysed by lipases. Lipases are widely used in the industry because they are readily available, inexpensive and are capable to differentiate between enantiomers, hence reacting faster with one of them. Numerous compounds have been separated using enzymes such as Candida antarctica lipase B (CALB), Candida rugosa lipase (CRL) or Thermomyces lanuginosus lipase (TLC).
Most of the reactions, however, are carried out in organic solvents that require purification and sometimes cannot be completely removed.
This is a problem, especially for the pharmaceutical industry on which the purity requirements are very high and that are limitations on the use of some solvents. Enzymes are able to retain their activity when dissolved in eutectic mixtures, providing a better reaction media than the conventional organic solvents.
Recently, our group has been focusing on the resolution of racemic menthol using CRL has catalyst. Menthol is one of the most sold aromas in the world. It is used in food industry, in tobacco, is cosmetics and even in the pharmaceutical industry. The compound is available in nature as a racemic mixture, however the (-) enantiomer is the one with the freshness and cooling effect much more accentuated.
The esterification of (-)-menthol with a variety of fatty acids using CRL as catalyst has been reported on organic solvents such as hexane or isooctane. Using a DES composed of menthol and lauric acid, in a 2:1 molar ratio, after adding the CRL, a conversion of nearly 50%, meaning 100 % of the pure enantiomer. The (-)-menthyl laurate is chemically significantly different from the unreacted (+)-menthol so that their separation can be achieve by simple crystallization and filtration. Water is the by-product of this reaction but because the DES is hydrophobic water is naturally removed from the reaction media to a second liquid phase.
Using the same enzyme and using the formed water is used for the hydrolysis of the (-)-menthyl laurate yielding the pure (-)-menthol and lauric acid, thus completing the circle.
Bioactive deep eutectic solvents (THEDES)
Eutectic solvents can find various applications in the pharmaceutical industry, which is known to use a large amount of solvents to produce specialty chemicals. The production yields high added value compounds, but in low amounts, and this often challenges the sustainability of pharma-related processes. The use of DES as solvents for synthetic processes of some active pharmaceutical ingredients (APIs) has been reported to improve their production, benefiting from apparent non-toxicity and biocompatibility of some DES.
However, the most interesting strategy of using DES for pharmaceutical applications is related to the fact that some APIs are able to form a DES with an excipient or another therapeutic compound. This type of DES can even be termed as ‘therapeutic deep eutectic solvents’ (THEDES). In this case, compounds which are normally solid and immiscible are mixed at specific conditions, yielding a liquid homogeneous solution. Obtaining an API or a drug in the liquid form can bring about several advantages, one of which is circumventing the existence of different crystalline polymorphs of the API, which can diminish API efficiency.
By obtaining the drug or API as therapeutic deep eutectic solvents (THEDES), its melting temperature is lowered, and its processing can be performed at milder temperatures, avoiding degradation problems. The obtained THEDES are normally amorphous species, at least in the working temperatures, and so the formation of THEDES is also advantageous for the processing of the API. In the liquid form, THEDES can be impregnated in polymeric matrices or can be encapsulated in particles, making the delivery mechanism of the API much more versatile. This also makes it possible to envisage various routes of the administration of the API.
Examples of THEDES are eutectic solvents composed of APIs such as acetylsalicylic acid, benzoic acid, phenylacetic acid, or even ibuprofen, and other known anti-inflammatory, anti-fungal and antiseptic APIs.
These APIs can be combined with quaternary ammonium salts (such as choline chloride) or terpenes (such as thymol or menthol) among other species, showing that it is possible to obtain a wide variety and tunability of THEDES.
The impregnation of THEDES composed by menthol and ibuprofen in starch-based polymers has been successfully achieved using supercritical carbon dioxide. Using these techniques, it was possible to obtain a system of controlled drug release that is more efficient when compared with ibuprofen in powdered form. Also, in the case of this THEDES, both components have a therapeutic effect, since ibuprofen is a non-steroidal anti-inflammatory drug, and menthol acts as permeation enhancer, making this THEDES a good candidate for transdermal drug delivery applications.
Another advantage of obtaining APIs in the form of THEDES is related to the solubility and permeability enhancement of the API. This has a huge impact in pharmaceutical applications, since by increasing its solubility the bioavailability of the API can be enhanced, and lower dosages of the therapeutic compounds are needed, leading to better patient-compliance, fewer side effects, and a more economic process.
THEDES can open doors to new processing methodologies in the pharmaceutical industry, and at the same time bring about the advantages of being more sustainable solvents with high versatility.
Extraction of bioactive compounds
Bioactive compounds are usually secondary metabolites that have a vital importance for living organisms. They are essential for stress-response or natural defense mechanisms and can be found in diverse organisms (fungi, plants, fruits and others). According to research, 60% of these natural bioactive compounds are already approved as anticancer drugs.
The application of these bioactive compounds is so vast that besides pharmaceuticals, these compounds are also applicable in industries such as nutraceutical, agrochemical, or cosmetic.
Phenolics and its derivatives are usually extracted through two different methods, the conventional ones (using solvent extraction, maceration and others) or through more modern extraction techniques (using enzyme-assisted or ultrasound-assisted extractions, amongst others). However, solvent toxicity, thermal stability, poor selectivity, structural modifications, and compound recovery are problems that need to be addressed for the implementation of these techniques at the industrial scale.
The necessity to be innovative and overcome the typical problems are urgent. Reducing waste and starting to use more sustainable and greener techniques/solvents is crucial. DES have been gathering interest in the research community as extraction agents. They are versatile, and most have low toxicity and pose no environmental problem. Moreover, there
are millions of combinations possible to form DES. Consequently, this allows the production of mixtures with many different properties. DES can be designed to have a wide range of hydrophobicity, meaning that they are able to extract different chemical families of bioactive compounds.
In most extraction techniques, one common drawback is the need to remove the solvent in order to use the extracted bioactive compound. Because DES are based on biodegradable and biocompatible compounds, they are normally also biocompatible and sometimes even have bioactivity themselves, so there is no need to remove the extraction solvent. One can imagine the potential of using a DES-phenolic mixture for the formulation of a cream or even as a nutraceutical.
It is known that phenolics has a short shelf life, since when in contact with oxygen, light or any other external factor, the antioxidant property is lost. Currently, the possibility of using DES not only as solvent extraction but also as a stabilising agent which is able to extend or even improve phenolics antioxidant activity, is being study.
It is very important to lay emphasis on the raw material that it is used to extract bioactive compounds. Any by-product that results from the agricultural industry is suitable to be used as raw material to extract these types of interest compounds. Taking in consideration the amount of waste that is daily produced in the agricultural industry, using it will not only help to reduce the waste produced, but the final product/application will also fit easier into the FDA’s regulations and requirements.
What about taking a shot of antioxidants that results from agro-industrial by-products, without any solvent contamination?
CO2 Capture and utiliSation using deep eutectic solvents
One of the areas where DES have also been used as absorption/adsorption solvents or in the preparation of materials is with regard to applications in carbon dioxide (CO2) capture and its further utilisation. It is now known that CO2 emissions are mainly caused by fossil fuel combustion, which has a profound effect in our society and economy, having already been responsible for different climate related catastrophe. Carbon capture and storage (CCS) and utilisation (CCU) approaches are realistic and economic and can be seen as an alternative process to deal with CO2 emissions.
The chemical post-combustion absorption of CO2 in aqueous amine is the benchmark process for CO2 removal. However, it presents major drawbacks, being poorly sustainable, unstable and requiring a high energy input for CO2 desorption. Alternative solvents for CO2 capture are needed, but they also need to have high CO2 affinity. DES, being present as designer solvents and more sustainable solvents, can play an important role in this field.
Various DES have already been tested as CO2 absorbents, most of which are composed of choline chloride and urea, glycerol and glycol species, showing good values of CO2 solubility.
Hydrophilic DES, based on choline chloride (ChCl) show satisfactory CO2 solubility values, combined with urea or ethylenegylcol, for example. DES with other ammonium salts acting as hydrogen bond acceptor, are also reported to have promising performance for CO2 solubilisation. Hydrophobic DES, and NADES (natural deep eutectic solvents) composed of glycerol and aminoacids, also show promising results for CO2 capture. Since DES show very low volatilities, its reuse in CO2 solubilisation is possible, and the exhaustion of the solvent is not a problem, as in the case of aqueous amine solutions. This also allows for the system to be reusable, leading to more efficiency and lower costs.
Although there is still much information needed on this subject, it has already been reported that some parameters have influence in the DES performance as CO2 sorbents, such as the molar ratio between DES’ components, temperature, pressure, viscosity of the DES and the amount of water present in the DES. One of the main difficulties of CO2 capture from flue gases, for instance, is the low partial pressure of CO2 at normal conditions. If the DES can concentrate the gas, the driving force for its capture will increase, thereby improving the process.
The addition of a catalytic process which is able to transform the captured CO2 in situ will therefore be an improvement. Metallic catalysts as well as biocatalysts have been reported to fulfil this, although their studies in DES are still very scarce. The transformation of CO2 into high added value chemicals will not only influence CO2 emissions, but also transform CO2 in a valuable resource.
Although there is still information lacking on the specific interactions established between DES and the solubilised CO2, as well as the mechanisms by which this phenomenon occurs, the scientific community shows that they are viable solvents for CO2 solubilisation and its transformation, paving the way for a CO2-based circular technology.
Perspectives on the application of deep eutectic solvents
It is the right of any human being to fulfil her/his potential in dignity, equality and health. This is one of the main goals of the UN2030 agenda. And it should also be the goal of the scientific community to direct its research towards creating a high impact in the environment, society, and economy. There are three main topics that are foreseen as new research areas where DES can offer solutions to serious challenges.
One of these challenges is food preservation. In the EU, about 90 million tonnes of food, or 180 kg/capita, is wasted annually. A major part of this wastage could be avoided. Only about 30% of food waste is generated in its production and processing; more than half of the it occurs in the household. In the EU, about a third of the food wasted in the household comes from discarded food over its expiration date. A higher shelf-life of perishable food products would thus significantly decrease food wastage.
The main reaction responsible for the degradation of the quality of food is oxidation. Antioxidants prevent and inhibit this reaction and therefore preserve the quality of food for longer periods. However, antioxidants avoid the oxidation of food by being oxidised themselves when exposed to light and heat. By developing new DES based in antioxidants such as ascorbic acid, the stability of the antioxidant is increased, maintaining its activity for longer periods of time. Therefore, these new DES can be safely applied as food preservatives.
Another challenge is vaccine stabilisation. Millions of deaths are prevented every year due to vaccination. Although in developed countries immunisation is a routine procedure, in developing countries vaccination remains a challenge.
According to UNICEF, more than 30 million children in underdeveloped/developing countries are not immunised due to a lack of access to vaccines, poorly provided healthcare services, or even due to misinformation.
One of the major drawbacks of vaccines is their conservation, especially when the cold chain refrigeration system might fail or might even be bon-existent, which is often the case in underdeveloped countries. When this is not observed, vaccines loose their efficacy. New effective vaccine stabilisation systems will allow for the easier transportation and storage of vaccines in developing countries where proper storage conditions are not available.
Most vaccines are composed of dead or live viruses. A virus is a simple infectious agent mainly formed by proteins. If DES can stabilise proteins, it can therefore be foreseen that they play an important role in the stabilisation of vaccines by allowing for a much longer storage period without the need for refrigeration.
Finally, DES can also contribute to the search for a solution to the cryopreservation of cells and organs. Organ transplant is an effective treatment for patients with organ failure. However, due to the lack of organs available, combined with the short storage time, only about 10% of these patients will ever receive a new organ. As such, the extension of organ storage time will give a new hope to these patients.
However, existing preservation techniques are ineffective in the long term. Cryopreservation is one of the most promising techniques, but it is necessary to avoid the water crystallisation responsible for cell disruption, the use of chemicals and solvents with toxicity, and environment issues.
The solution to this may be found in Nature, based on the strategy that living organisms have found a way to survive extreme temperature amplitudes. It has been observed that animals and plants rely on the orchestrated production of different biological metabolites such as sugars and polyols. These metabolites have also been reported to have the ability to form DES. In fact, these DES are related to the ability to transport and dissolve poorly water-soluble molecules in cells and it has been observed that they may also play a role in the protection of cell integrity during the freezing and thawing processes.
As such, if we can replace the solvents and surfactants used in the preservation of cells and tissues with Nature-based deep eutectic solvents, then it may be possible to discover new strategies to enhance the shelf life of organs ready to be transplanted.
Alexandre Babo de Almeida Paiva
LAQV@REQUIMTE: Faculty of Sciences and Technology – NOVA