Multicomponent deep eutectic solvents, their preparation and use

Abstract

The present invention belongs to the fields of chemistry and biotechnology, more precisely to the field of deep eutectic solvents for various applications. The present invention relates to a multi-component deep eutectic solvent (DES), a method for preparing the same, and uses of the multi-component DES. A multi-component DES according to a first aspect of the present invention comprises at least two components, at least one of which is selected from the group consisting of ectoine, trimethylamine N-oxide (TMAO), sarcosine, glycerophosphate choline (GPC), dimethylsulfonopropionate (DMSP), guanidine, arginine, and taurine. A second aspect of the present invention relates to a multi-component DES comprising at least four components. In a preferred embodiment, at least one of the components of the DES is selected from the group consisting of naturally occurring osmolytes.

Description

[Technical Field]

[0001] The present invention belongs to the field of chemistry and biotechnology, more precisely to the field of deep eutectic solvents for various applications. The present invention relates to a multicomponent deep eutectic solvent (DES), a method for its preparation and the use of said multicomponent DES. [Background technology]

[0002] (Background and Technical Issues) Modern systems that effectively mimic the natural environment of various biological systems and the biomolecules (i.e., any organic molecules, including carbohydrates, proteins, lipids, and / or nucleic acids) that compose these systems—so-called deep eutectic solvents (DESs)—have been intensively investigated in the past few years as nontoxic and highly tunable solvents in the production of foods, pesticides, cosmetics, and pharmaceuticals (Vanda et al., 2019: Natural deep eutectic solvents: From their discovery to their applications. in Deep Eutectic Solvents: Synthesis, Properties, and Applications 61–81). Unlike traditional molecular solvents, DESs are mixtures of two or more compounds (usually solids) that are combined in the appropriate molar ratio to produce a liquid at ambient temperature (Hansen et al., 2021: Chem. Rev. 121, 1232–1285). A particular subgroup of these systems, the natural DES (NADES), consists exclusively of compounds that occur in nature (Dai et al., 2013: Anal. Chim. Acta 766, 61-68).

[0003] DESs typically have extremely low volatility, are nonflammable, water-resistant, and are easily prepared with high purity and low cost. Furthermore, DESs are typically considered environmentally friendly solvents due to their typical biodegradability and biocompatibility. Furthermore, DESs are highly tunable by varying their constituent components or the relative ratios of the components, thus promising a wide variety of applications. To meet emerging needs for environmentally friendly solvents, such as those in the pharmaceutical industry, biochemistry, materials science, and biotechnology, novel DES systems with new and / or more diverse properties are required. The present invention aims to address this need. In particular, currently known DESs are primarily used for extraction, after which they must be discarded or recycled. Choline chloride (ChCl) is often used due to its low cost. ChCl is produced from ethylene oxide obtained from ethylene derived from fossil fuels (oil or natural gas). For sustainability reasons, removal of ChCl is desirable. The objective of the present invention is to provide novel ChCl-free DESs that can be used in final products and exhibit novel properties and functions.

[0004] (prior art) Several DES are known and have been reported in scientific papers and patent applications. For example, EP 3870227 relates to a deep eutectic solvent (DES) composition comprising a combination of a glycol and a polymer solubilizer component, the polymer solubilizer component being selected from the group consisting of esters and lactones of organic acids, dicarboxylic acids, esters of dicarboxylic acids, esters, ethers, and carbonates of diols and triols, and mixtures thereof, wherein the molar ratio of glycol to polymer solubilizer component is in the range of 12:1 to 1:10, preferably in the range of 8:1 to 1:2, and more preferably in the range of 4:1 to 1:1, and the composition further comprises at least one DES constituent.

[0005] In European Patent No. 3971230, a DES is · at least one carboxylic acid containing at least two carboxylic acid functional groups, the number of carbon atoms being in the range of 4 to 10; at least one alcohol containing at least two alcohol functional groups, Alcohols with several carbon atoms in the range of 2 to 12 carbon atoms, Polyethylene glycol, and Polypropylene glycol an alcohol selected from the group consisting of: Water in an amount of 10~50wt% of the total weight of the DES DESs including:

[0006] EP 3485874 describes a method for preparing or optimizing a system comprising a NADES and one or more active ingredients, which comprises the following steps: a) analyzing the active ingredient or target compound; b) matching the active ingredient with at least one potential NADES starting composition; c) synthesizing a stable liquid NADES-active ingredient system; and d) optimizing the system prepared in step (c). A method is described which includes:

[0007] International Publication No. 2017134190 discloses a DES comprising (2-hydroxyethyl)trimethylammonium chloride and dithiothreitol in a molar ratio of 1:2 to 1:3, and 0% to 10% of a cosolvent, as well as a method for enzymatically producing a polypeptide using this DES.

[0008] Esquembre et al. (2013; Phys. Chem. Chem. Phys., 15, 11248–11256) studied the stability of hen egg white lysozyme in various choline chloride-based pseudo-concentrated and solvent-free DESs using intrinsic fluorescence and CD spectroscopy. Thermal unfolding experiments performed in undiluted urea:choline chloride and glycerol:choline chloride eutectic solvents (UChCl-DES and GChCl-DES, respectively) showed that at certain temperatures, a separate, partially folded intermediate with a high content of secondary structure and disrupted tertiary structure accumulated. Under these conditions, the reversibility of the unfolding process was incomplete, and urea-based DESs showed a higher destabilization of protein structure upon heat treatment. On the other hand, dilution of the eutectic mixture with water allowed recovery of the reversible two-state denaturation process. Lysozyme activity was also affected in the undiluted and pseudo-concentrated GChCl-DESs, with activity recovery increasing upon dilution with water and fully recovering after extensive DES removal by dialysis. The results of Esquembre et al. suggest that protein interactions at room temperature are reversible and depend on the DES components and the water content of the original DES dilution.

[0009] Similarly, Banerjee et al. (Adv. Healthcare Mater., 2017, 6, 1601411) observed that a DES consisting of a 1:2 choline:geranate solution preserved the physical integrity of insulin while maintaining its activity. Similarly, Lee et al. (J. Ind. Eng. Chem., 2018, 65, 343-348) used a sugar-based DES to improve the stability of human interferon. Improved stability of several other proteins (i.e., chemotrypsin and Trp-cage proteins) at high temperatures was ensured by choline-based DESs (Yadav et al. (ACS Sustainable Chem. Eng., 2020, 8, 10151-10160); Pal et al. (J. Phys. Chem. B, 2020, 124, 7598-7610)).

[0010] Sanchez-Fernandez et al. (2022; Green Chem., 24, 4437-4442) used lysozyme as a useful model protein to demonstrate the potential of a choline chloride:glycerol DES (molar ratio 1:2) as a synthetically viable medium for the storage and preservation of proteins in the concentrated range without the need for other excipients. The behavior of lysozyme in choline chloride:glycerol was investigated under dilute conditions (1.4 mg ml). -1 , 0.100 mM) to concentrated conditions (142 mg ml -1 Lysozyme was studied over a wide range of concentrations, ranging from 0.1 mM to 9.9 mM, and compared with its behavior in aqueous buffer. It was shown that lysozyme maintained a globular shape in DES, with an overall structure similar to that in aqueous phosphate buffer, potentially allowing the protein to remain physically stable even after prolonged storage in DES (40 days at room temperature). Importantly, the enzymatic activity of the reconstituted, hydrated lysozyme solution was fully retained after storage in DES.

[0011] Nardecchia et al. (2012; Biomacromolecules 13, 7, 2029–2036) reported that UChCl-DES, obtained by heating urea and choline chloride in a 2:1 molar ratio at 80°C and stirring until a homogeneous liquid was formed, promoted the stabilization of the collapsed state of elastin-like recombinamers and the subsequent formation of aggregates due to the loss of structural water molecules responsible for hydrophobic hydration. Cryo-etch scanning electron microscopy enabled the observation of these aggregates in pure DES. The presence of the elastin-like recombinamers in a collapsed state was confirmed by the suppression of the lower critical solution temperature transition observed by differential scanning calorimetry and dynamic light scattering. The transition from the collapsed state to the swollen state was suppressed even after moderate water dilution (water contents ranging from 0 to approximately 45 wt%) and was only restored by the addition of further water (above 50 wt%). These features reveal that the collapsed state is preferentially stabilized not only in pure deep eutectic solvents but also in partially hydrated DESs.

[0012] US Patent Application Publication No. 2022305401 discloses various combinations of two-component DESs and suggests the use of three-component DESs, but does not specifically disclose any combinations of three components. According to this solution, the following combinations are disclosed: betaine:urea, choline chloride:urea, glycerol:betaine, glycerol:choline chloride, malic acid:chloride, lactic acid:betaine, levulinic acid:betaine, pyruvic acid:betaine, urea:betaine HCl, betaine:sorbitol, proline:levulinic acid, betaine:proline, proline:glucose, betaine:glucose, lysine:levulinic acid, sorbitol:levulinic acid, xylitol:levulinic acid, glucose:levulinic acid, glucose:levulinic acid, glycerol:sorbitol, glycerol:lactic acid, sorbitol:lactic acid, and glycerol:levulinic acid.

[0013] The table below summarizes some disclosures of NADES in the scientific literature. These citations are: 1. Yancey and Somero; Biochem. J. 183, 317-323 (1979); 2.Liang,Y.et al.Bioresour.Technol.310,123389(2020) 3. Zeng et al. J. Mol. Liq. 219, 74-78 (2016). 4. Benlebna, M. et al. Journal of Agricultural and Food Chemistry vol.66 (2018). 5. Syakfanaya et al. Pharmacogn. J. 11, 267-271 (2019). 6.Jesus et al. Cryobiology 101,95-104(2021). 7. Panic, M. et al. Waste Manag. 120, 340-350 (2021). 8. Simeonov and Afonso, RSC Adv.6, 5485-5490 (2016). 9. Zhong, L. et al. Bioresour. Technol. 343, 126022 (2022). 10. Pedro, S. N. et al. Pharmaceutics 14, 827 (2022). 11. Karadendrou et al. Catalysts 12, (2022). 12. Sut, S. et al. Molecules 22, 1-11(2017). 13. Faggian, M. et al. Molecules 21, 1-11(2016). 14. Ilgen and Koenig, Green Chem. 11, 848-85 (2009). 15. Zdanowicz Int. J. Biol. Macromol. 176, 387-393 (2021). 16. Wang, T. et al. J.Pharm.Biomed.Anal.145,339-345(2017) is.

[0014] [Table 1]

[0015] [Table 2] [Prior art documents] [Patent documents]

[0016] [Patent Document 1] European Patent No. 3870227 [Patent Document 2] European Patent No. 3971230 [Patent Document 3] European Patent No. 3485874 [Patent Document 4] International Patent Application Publication No. 2017134190 Brochure [Patent Document 5] US Patent Application Publication No. 2022305401 [Non-patent literature]

[0017] [Non-Patent Document 1] ベンダ(Vanda) et al;2019:Natural deep eutectic solvents:From their discovery to their applications.in Deep Eutectic Solvents:Synthesis,Properties,and Applications 61-81. [Non-licensed document 2] Hansen et al., 2021: Chem. Rev. 121, 1232-1285. [Non-licensed document 3] ダイ(Dai) et al., 2013: Anal. Chim. Acta 766, 61-68.

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0018] The present invention aims to expand the use of DES systems in various fields and to provide an alternative DES that eliminates choline chloride. This technical problem is solved as defined in the independent claims, and preferred embodiments of the invention are defined in the dependent claims. [Means for solving the problem]

[0019] It is a multi-component deep eutectic solvent (DES) that contains at least four components. DETAILED DESCRIPTION OF THE INVENTION

[0020] A multi-component DES according to a first aspect of the present invention comprises at least two components, at least one of which is selected from the group consisting of ectoine, trimethylamine N-oxide (TMAO), sarcosine, glycerophosphocholine (GPC), dimethylsulfonopropionate (DMSP), guanidine, arginine, and taurine. Preferably, the multi-component DES comprises three components, more preferably four components. Optionally, the DES also comprises water. The multi-component DES has been observed to improve the stability of biomolecules and the stability and viability of cryopreserved cells, and to maintain or even improve the function of biomolecules. Therefore, a possible embodiment of the present invention is also a medium for biomolecule stabilization or cell / tissue cryoprotection, comprising a DES comprising at least two components, at least one of which is selected from the group consisting of ectoine, TMAO, sarcosine, GPC, DMSP, guanidine, arginine, and taurine. A method for stabilizing a biological molecule or a biological system, such as an organism, organ, organelle, cell, or tissue, includes at least exposing the biological molecule, or the biological system, cell, or tissue, or an artificially engineered construct, to the aforementioned medium. The medium may be supplemented with any other components, and preservation of the mixture of the medium and the biological molecule, biological system, cell, or tissue is carried out by any suitable method. The term "preservation" refers to any process for preserving a biological system or a biological molecule, with the aim of maintaining the normal biological, chemical, and / or physical properties of the biological system or the biological molecule, respectively. The term "preservation" may refer to freezing at low temperatures, typically in the range of -80°C to -196°C, or storage at any given temperature, such as storage at temperatures above -80°C.

[0021] A second aspect of the present invention, which exhibits the same technical effect as the above-described DES, is a multicomponent DES containing at least four components. In a preferred embodiment of a multicomponent DES containing at least four components, at least one of the components of the DES is selected from the group including naturally occurring osmolytes. Therefore, another possible embodiment of the present invention is also a medium for stabilizing biomolecules or preserving (e.g., cryoprotecting) biological systems, cells, or tissues, comprising a DES containing at least four components. The method for stabilizing biomolecules or preserving / cryoprotecting biological systems, cells, or tissues includes at least the step of exposing the biomolecules or cells / tissues to the above-described medium. This medium can be supplemented with any other components, and preservation of the mixture of the medium and the biomolecule, biological system, cell, or tissue is carried out by any suitable method.

[0022] In a preferred embodiment of the present invention, the naturally occurring osmolyte is preferably (i) Polyols and sugar polyols, for example, glycerol, sorbitol, xylitol; (ii) sugars and their derivatives, e.g., glucose, sucrose, trehalose; (iii) amino acids and their derivatives, such as glycine, proline, ectoine, taurine; (iv) methylamines, e.g., trimethylamine N-oxide (TMAO), sarcosine, betaine, glycerophosphate choline (GPC); (v) methylsulfonium compounds, such as dimethylsulfoniopropionate (DMSP); (vi) Y-conjugated compounds, such as urea, guanidine, and arginine Preferably, the naturally occurring osmolyte is selected from the group consisting of ectoine, TMAO, sarcosine, GPC, DMSP, guanidine, arginine, and taurine. The DES may optionally contain water.

[0023] The remaining components of the multi-component DES may be selected from common DES / IL compounds, such as methylamines, methylsulfonium compounds, organic acids, sugars, polyols, amino acids, and Y-conjugate compounds. As mentioned above, in the methods or uses defined herein, the DES may be formulated with or without water, e.g., the solution comprising the DES is essentially free of water.

[0024] Methylamines may be selected from trimethylamine N-oxide (TMAO), betaine, glycerophosphate choline (GPC), carnitine, homarine, and derivatives thereof, such as their halide forms, such as betaine halide (betaine HCl).

[0025] The methylsulfonium compound may be selected from dimethylsulfonopropionate (DMSP) and other compounds containing a methylsulfonium moiety.

[0026] The organic acid may be selected from levulinic acid, lactic acid, malic acid, maleic acid, pyruvic acid, fumaric acid, succinic acid, citric acid, citraconic acid, glutaric acid, glycolic acid, acetic acid, aconitic acid, tartaric acid, ascorbic acid, malonic acid, oxalic acid, glucuronic acid, neuraminic acid, sialic acid, shikimic acid, phytic acid, galacturonic acid, iduronic acid, hyaluronic acid, hydroxycitric acid, lactone derivatives, and derivatives thereof.

[0027] The sugar may be selected from trehalose, glucose, sucrose, lactose, ribose, galactose, fructose, etc., and derivatives thereof.

[0028] The polyol may be selected from glycerol, erythritol, mannitol, sorbitol, xylitol, ethylene glycol, propylene glycol, ribitol, aldonitol, propanediol, inositol, pentylene glycol, and derivatives thereof (eg, o-methyl inositol).

[0029] The amino acids may be selected from glycine, proline, taurine, lysine, etc., and derivatives thereof (eg, ectoine, sarcosine, theanine, dimethylglycine, etc.).

[0030] The Y-conjugate compound may be selected from urea, guanidine, arginine, and compounds containing a guanidino moiety (e.g., creatine, glycocyamine, agmatine, 4-guanidinobutanoic acid, etc.), and derivatives thereof (e.g., guanidinium halides (e.g., guanidine HCl)).

[0031] According to the present invention, the molar ratio of each DES component to the total number of moles of DES can be in the range of 0.001 to 0.9. The molar ratio is calculated by dividing the total amount of all components in the mixture (also expressed in moles) n tot A unit of quantity (expressed in moles) of a component divided by n i In a preferred embodiment, the molar ratio of each DES component to the total number of moles of DES may be 0.003 to 0.7. In a more preferred embodiment, the molar ratio of each DES component to the total number of moles of DES may be 0.005 to 0.6. The preferred ratio may also depend on the combination of components in the DES and the intended use of the DES.

[0032] As described above, DES can be formulated with or without water (e.g., a solution containing DES but essentially free of water). The amount of water used in the DES can be 0% to 90%, for example, about 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.5, 1, 5, 10, 15, 20, 30, or about 40% to about 90, 80, 70, 60, or about 50%. In a preferred embodiment, the amount of water used is about 5% to about 30%, for example, about 10%, 20%, 25%, or about 30%. The prepared DES can also be further diluted.

[0033] In the most preferred embodiment of the present invention, the multi-component DES comprises the components in the combinations shown in Tables 2 and 3 below.

[0034] [Table 3]

[0035] [Table 4]

[0036] [Table 5]

[0037] The DES according to the present invention can be prepared by the following steps: a) mixing the components in the required molar ratio; and b) heating the mixture to 60°C with stirring until a colorless, homogeneous liquid is formed. It is prepared by a method comprising:

[0038] The uses of the multi-component DES according to the present invention are many and include at least the following uses: Stabilization of biomolecules, Preservation of biological systems, e.g., cryoprotection, Pharmaceutical formulations, e.g. pharmaceutical excipients and similar components, ·cosmetics Food, as well as Extract preparation is preferred, where DES is optionally a constituent of the final extract.

[0039] The DES system of the present invention can be used to extract, stabilize, analyze, formulate, preserve, and / or culture, monitor, handle, process, and use organisms, organs, tissues, organelles, biological systems, cells, biomolecules, and bioactive compounds in any form, including, but not limited to, plants, animals, algae, bacteria, fungi, and other microorganisms, whole cells, membranes and other cellular components, proteins, enzymes, antibodies, peptides, and other molecules composed of amino acids or amino acid derivatives, polysaccharides and biopolymers of any composition, lipids, and lipid-containing supramolecular structures, nucleotides, nucleosides, and their precursors and derivatives, polynucleotides of any length, sense, molecular weight, and composition, such as, but not limited to, any type of DNA and RNA, any type of gel, emulsion, dispersion, liquid, and / or solid composition containing the DES of the present invention and / or biomolecules and / or other payloads, such as any type of bioactive compound. The present invention, or formulations containing the DES of the present invention, can be used as pharmaceutical excipients, cosmetic compositions, nutritional compositions, feed compositions, biomass extracts, nutritional products, etc.

[0040] Thus, one aspect of the present invention is a stabilizing composition comprising any of the above-described DESs for use in stabilizing biomolecules (particularly proteins, most preferably enzymes). Another aspect of the present invention is a composition or medium for preserving (e.g., cryoprotecting) biological systems, cells, tissues, and molecules, comprising any of the above-described DESs. The medium may be supplemented with any other components, and preservation of the mixture of the medium and the biomolecule, biological system, cell, or tissue is carried out in any suitable manner. An additional aspect of the present invention is also a method for stabilizing a biomolecule or preserving / cryoprotecting a biological system, cell, or tissue, which method comprises at least the step of exposing the biomolecule, biological system, cell, or tissue to the above-described medium.

[0041] The invention will now be explained in more detail on the basis of exemplary embodiments and examples.

[0042] In the most preferred embodiment of the present invention, the multi-component DES comprises the components in the combinations and proportions shown in Table 4 below.

[0043] [Table 6]

[0044] [Table 7]

[0045] In the most preferred embodiment of the present invention, a multi-component DES comprising at least four components comprises the components in the combinations and proportions shown in Table 5 below.

[0046] [Table 8] [Example]

[0047] Example 1: Preparation of an exemplary DES according to the present invention To test the prepared DES according to the present invention and preferred embodiments and demonstrate their ability to stabilize biomolecules, a group of multi-component DES were prepared according to the combinations and ratios shown in Table 6 below.

[0048] [Table 9]

[0049] These DES were prepared by calculating the appropriate amount of each component and adding it to the mixture and heating it at a temperature up to 60°C to obtain a liquid solution, which was used for further experiments.

[0050] Example 2: Stabilization of lysozyme at 25°C by selected DES prepared in Example 1 Lysozyme activity was determined according to the method of Shugar et al. (1952, Biochim Biophys Acta 8:302-9), which is based on the decrease in turbidity of the substrate Micrococcus lysodeikticus in suspension at a concentration of 0.1 mg ml -1 Lysozyme solutions were prepared in various DES and 10 mM sodium phosphate buffer (pH 7). The solutions were incubated at various temperatures (25, 45, 70°C) for 1, 3, 5, and 7 days (Examples 2 and 3) or 15, 60, and 120 minutes (Example 4). Briefly, a Micrococcus lysodeikticus bacterial suspension (7 mg ml) in sterile PBS buffer was added to 525 μl of 10 mM sodium phosphate buffer (pH 7) in a plastic disposable cuvette. -1 ) and 30 μl of lysozyme solution were added. Immediately after mixing, the cuvette was placed in a UV / VIS spectrophotometer and the absorbance was measured at a wavelength of 450 nm over a period of linear turbidity decrease. Relative activity (%) was calculated as the initial reaction velocity (ΔA450 min) obtained by the enzyme after incubation. -1 The response rates were calculated from the variances (calculated from the previous data) compared to those obtained without previous exposure.

[0051] [Table 10]

[0052] As can be seen from this table, DES containing sarcosine and glycerol at any water content allowed for higher residual lysozyme activity compared to both controls, especially after 5 and 7 days. This effect was more pronounced for compositions with higher water content. Similar Sar:Gly 40 values ​​were observed for DES containing DMSP and glycerol, DES containing betaine, sorbitol, GPC, urea, and taurine, and DES containing betaine, sorbitol, and glycerol. However, the presence of ectoine was most favorable for stabilizing lysozyme, as all analyzed DES containing ectoine showed residual activity greater than 100% even after 1 week. These results suggest that the prepared DES, in all its variability, stabilizes lysozyme stored at 25°C significantly better and for longer periods than the best currently known stabilizing medium containing ChCl and glycerol.

[0053] Example 3: Stabilization of lysozyme at 45°C by selected DES prepared in Example 1 This experiment was carried out in the same manner as described in Example 2, except that the incubation temperature was 45°C.

[0054] [Table 11]

[0055] The results, presented in Table 8, show that all DESs enabled significantly higher residual lysozyme activity compared to both controls, even after 1 day (especially after 5 and 7 days). Much of the residual activity was above 100%, consistent with previous observations (Delorme et al., 2020: doi:10.1016 / j.ijbiomac.2020.07.022; Kaar et al., 2022; J.AM.CHEM.SOC. 2003, 125, 4125-4131; Varriale et al., 2022: 10.1021 / acssuschemeng.1c07104). Overall, these results suggest that the prepared DESs, in all their variability, stabilized lysozyme stored at 45°C significantly better and for longer periods than the best currently known stabilizing media, which contain ChCl and glycerol.

[0056] Example 4: Stabilization of lysozyme at 70°C by selected DES prepared in Example 1 This experiment was carried out in the same manner as described in Example 2, except that the incubation temperature was 70° C. and the incubation times were 15, 60, and 120 minutes, respectively.

[0057] [Table 12]

[0058] The results, presented in Table 9, suggest that all DESs enabled significantly higher resistance of lysozyme to heat shock compared to both controls. Many of the residual activities were greater than 100%. The significant stabilizing effect was least significant for DESs containing sarcosine and glycerol, and for DESs consisting of betaine, glycerol, sorbitol, and ectoine. These results suggest that the prepared DESs, in all their variability, stabilized lysozyme treated at 70°C significantly better than currently known media, but that some DESs performed better at lower temperatures and some provided better protection against heat shock.

[0059] Example 5: Stability of yeast alcohol dehydrogenase (YADH) by selected DES prepared in Example 1 YADH activity was determined according to the method of Walker et al. (Doi:10.1016 / 0307-4412(92)90021-D). -1 YADH solutions were prepared in various DES and glycine pyrophosphate buffer (pH 9). The solutions were incubated at various temperatures (25 and 45°C) for 2, 5, 8, 24, and 48 hours (at 25°C) or 1, 2, 4, 8, and 24 hours (at 45°C).

[0060] Briefly, 975 μl of glycine pyrophosphate buffer (pH 9) in a plastic disposable cuvette was added to NAD in PBS buffer. + (50mgml -1 5 μl of ethanol (96%), 10 μl of ethanol (96%), and 10 μl of YADH solution were added. Immediately after mixing, the cuvette was placed in a UV / VIS spectrophotometer and the absorbance was measured at a wavelength of 340 nm over a period of 2 minutes. The relative activity (%) was calculated as the initial reaction velocity (ΔA340min) obtained by the enzyme after incubation. -1 The response rates were calculated from the variances (calculated from the previous data) compared to those obtained without previous exposure.

[0061] [Table 13]

[0062] [Table 14]

[0063] The results shown in Tables 10 and 11 indicate that some DESs according to the present invention are able to stabilize YADH to a greater extent than conventionally used buffers or stabilizing media. Among the DESs tested, the most excellent stabilizing properties of Sar:Gly were observed.

[0064] Based on known results with other DES, stabilization of other proteins as well as DNA and other biomolecules is expected. Due to the presence of osmolytes in the DES of the present invention, cryoprotection of cells and tissues is made possible by the present invention.

Claims

1. A multi-component deep eutectic solvent (DES) characterized in that it comprises at least four components.

2. 2. A multi-component DES according to claim 1, characterized in that at least one of the components of the DES is selected from the group comprising naturally occurring osmolytes.

3. 3. A multi-component DES according to claim 1 or 2, wherein the naturally occurring osmolyte is Polyols and sugar polyols, for example, glycerol, sorbitol, xylitol; Sugars and their derivatives, such as glucose, sucrose, trehalose; Amino acids and their derivatives, such as glycine, proline, ectoine, taurine; Methylamines, such as trimethylamine N-oxide (TMAO), sarcosine, betaine, glycerophosphate choline (GPC); Methylsulfonium compounds, such as dimethylsulfoniopropionate (DMSP); Y-conjugated compounds, such as urea, guanidine, and arginine; A multi-component DES, characterized in that it is selected from the group comprising:

4. 1. A multi-component deep eutectic solvent (DES) comprising at least two components, characterized in that at least one of the components is selected from the group consisting of ectoine, trimethylamine N-oxide (TMAO), sarcosine, glycerophosphate choline (GPC), dimethylsulfonopropionate (DMSP), guanidine, arginine, and taurine.

5. 5. A multi-component DES according to claim 4, characterized in that it has at least three components, preferably at least four components.

6. A multi-component DES according to any one of claims 1 to 5, characterized in that the remaining components of the multi-component DES are selected from common DES / IL compounds, such as methylamines, methylsulfonium compounds, organic acids, sugars, polyols, amino acids, and Y-conjugated compounds.

7. 7. A multi-component DES according to claim 6, characterized in that the methylamine is selected from N-trimethylamine oxide (TMAO), betaine, glycerophosphate choline (GPC), carnitine, homarine, and derivatives thereof, for example their halide forms such as betaine halide (betaine HCl).

8. 7. A multi-component DES according to claim 6, characterized in that the methylsulfonium compound is dimethylsulfonopropionate (DMSP).

9. 7. A multi-component DES according to claim 6, characterized in that the organic acid is selected from the group comprising levulinic acid, lactic acid, malic acid, maleic acid, pyruvic acid, fumaric acid, succinic acid, citric acid, citraconic acid, glutaric acid, glycolic acid, acetic acid, aconitic acid, tartaric acid, ascorbic acid, malonic acid, oxalic acid, glucuronic acid, neuraminic acid, sialic acid, shikimic acid, phytic acid, galacturonic acid, iduronic acid, hyaluronic acid, hydroxycitric acid, lactone derivatives and derivatives thereof.

10. 7. A multi-component DES according to claim 6, characterized in that the sugar is selected from the group comprising trehalose, glucose, sucrose, lactose, ribose, galactose, fructose, etc., and derivatives thereof.

11. 7. A multi-component DES according to claim 6, characterized in that the polyol is selected from the group comprising glycerol, erythritol, mannitol, sorbitol, xylitol, ethylene glycol, propylene glycol, ribitol, aldonitol, propanediol, inositol, pentylene glycol, and derivatives thereof, such as o-methyl-inositol.

12. 7. A multi-component DES according to claim 6, characterized in that the amino acids are selected from the group comprising glycine, proline, taurine, lysine, etc., and derivatives thereof, such as ectoine, sarcosine, theanine, dimethylglycine, and the like.

13. 7. A multi-component DES according to claim 6, characterized in that the Y-conjugated compound is selected from the group comprising urea, guanidine, arginine and compounds containing a guanidino moiety such as creatine, glycocyamine, agmatine, 4-guanidinobutanoic acid, and derivatives thereof, such as guanidinium halides, for example guanidine HCl.

14. A multi-component DES according to any one of claims 1 to 13, characterized in that the molar ratio of each DES component to the total number of moles of the DES can be in the range of 0.001 to 0.

9.

15. A multi-component DES according to any one of claims 1 to 14, characterized in that the DES also contains water.

16. 16. A multi-component DES according to claim 15, characterized in that the amount of water used in the DES is between 0% and 90%, for example between about 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.5, 1, 5, 10, 15, 20, 30, or about 40% to about 90, 80, 70, 60, or about 50%.

17. 17. A multi-component DES according to claim 16, characterized in that the amount of water used is between about 5% and about 30%, for example about 10%, 20%, 25%, or about 30%.

18. A multi-component DES according to any one of claims 1 to 17, characterized in that the prepared DES is diluted.

19. A multi-component DES according to any one of claims 1 to 18, comprising: Table 1 Table 2 A multi-component DES, characterized in that it is selected from the group consisting of:

20. A multi-component DES according to any one of claims 1 to 19, comprising: Table 3 Table 4 A multi-component DES, characterized in that it is selected from the group consisting of:

21. A multi-component DES according to any one of claims 1 to 20, comprising: Table 5 A multi-component DES, characterized in that it is selected from the group consisting of:

22. A process for the preparation of a multi-component DES according to any one of claims 1 to 21, comprising the following steps: a) mixing the components in the required molar ratios; and b) heating the mixture to 60°C with stirring until a colorless, homogeneous liquid is formed. A method comprising:

23. stabilization of biomolecules, Preservation of biological systems, Pharmaceutical formulations, e.g., pharmaceutical excipients and similar components; cosmetics, Food industry, as well as Preparation of extracts, 23. Use of a multi-component DES according to any one of claims 1 to 22 in the preparation of a pharmaceutical composition, characterized in that said DES is optionally a constituent of a final extract.

24. A medium for the stabilization of biomolecules or the preservation of biological systems, characterized in that it comprises a multicomponent DES according to any one of claims 1 to 23.

25. 25. The method of claim 24, wherein the biological system is an organism, an organ, an organelle, a cell, a tissue, or an engineered construct.

26. 25. The method of claim 24, wherein the biomolecule is selected from the group comprising cellular components, proteins, enzymes, antibodies, peptides and other molecules consisting of amino acids or amino acid derivatives, polysaccharides, biopolymers, lipids, lipid-containing supramolecular structures, nucleotides, nucleosides and their precursors and derivatives, polynucleotides of any length, sense, molecular weight and composition, DNA and RNA.

27. A method for the stabilization of a biological molecule or the preservation of a biological system, characterized in that it comprises at least the step of exposing said biological molecule or said biological system to the medium described above.

28. 28. The method of claim 27, wherein the biological system is an organism, an organ, an organelle, a cell, a tissue, or an engineered construct.

29. 28. The method of claim 27, wherein the biomolecule is selected from the group comprising cellular components, proteins, enzymes, antibodies, peptides and other molecules consisting of amino acids or amino acid derivatives, polysaccharides, biopolymers, lipids, lipid-containing supramolecular structures, nucleotides, nucleosides and their precursors and derivatives, polynucleotides of any length, sense, molecular weight and composition, DNA and RNA.

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