Some reports describe ILs as suitable media to increase the solubility, stability and activity of enzymes. [10,13,40,41,42,43] For example, Constantinescu et al. [41] studied the denaturation of enzymes in ILs including the cations [C₂mim]⁺, [C₄mim]⁺, [C₆mim]⁺, [C₄C₁pyrr]⁺, [C_i,j,k,lN]⁺, [guan]⁺ and the anions [SCN]⁻, [MeOSO₃]⁻, [EtOSO₃]⁻, [OTf]⁻, [NTf₂]⁻, [N(CN)₂]⁻. It was found that the stability at higher temperature (70 °C) was increased compared to aqueous buffer solution, with [C_i,j,k,lN]⁺ and [NTf₂]⁻ showing the best results. These studies were later extended by others, for example to the use of [C₂mim][FSI] (FSI = bis(fluorosulfonyl)imide) for the α-chymotrypsin-catalyzed reaction of Ac-Trp-OEt with Gly-Gly-NH₂ to yield Ac-Trp-Gly-Gly-NH₂ (see also below) [42]. Here, a 16-fold increase in the initial rates for peptide synthesis was observed compared to the reaction carried out in acetonitrile. In addition to these findings, they also found a 17-fold higher activity for α -chymotrypsin in ILs compared to organic solvents.

Besides, other applications of ILs have been reported with respect to proteins, e.g., Summers and Flowers have described the renaturation of proteins in the IL ethylammonium nitrate as a one-step process [44]. This IL prevented aggregation and caused the refolding of proteins, e.g., lysozyme, resulting in its active state with up to 90% of active protein [44]. ILs as suppressors of protein aggregation and useful supports for refolding processes of denatured proteins were also described by Buchfink et al. in 2010 [45]. A series of [C₂mim]⁺ salts was used with a focus on the impact of the anions as refolding enhancers as well as different model proteins, among them recombinant plasminogen activator rPA. It was found that IL co-solvent systems with an intermediate capacity to solubilize proteins are effective as refolding additives, i.e., were found to permit effective renaturation of rPA. The most promising refolding enhancer in this respect was [C₂mim][Cl]. By means of fluorescence correlation spectroscopy Sasmal et al. found that the presence of 6 M guanidine hydrochloride helps a protein, e.g., human serum albumin, to refold after addition of 1.5 M [pmim][Br] [46].

An interesting contribution quantifying the biomolecular interactions of the functional groups in proteins with biocompatible ILs, e.g., diethylammonium and triethylammonium acetate, has been reported recently [47]. Herein, transfer free energies (ΔG’_tr) of compounds such as Gly, Gly-Gly and cyclo(Gly-Gly) from water to aqueous ionic liquid solutions were determined depending on the IL concentration. The solubility in aqueous solutions decreased with increasing IL concentration (salting-out effect). This was attributed to the non-favorable interactions between IL and individual amino acid residue(s). However, overall all ILs could stabilize the structure of the model compounds—with triethylammonium acetate being the strongest stabilizer among the ILs used. On the other hand, a negative contribution (ΔG’tr) of the peptide backbone from water to higher concentration of e.g., TMAA (50–70%) indicated that the interactions between the solvent and backbone unit are favorable.

The first attempts in peptide bond formation by enzymes, however, were already reported by Erbeldinger et al. in 2000 [31]. Here, the synthesis of Z-aspartame in a thermolysin-catalyzed reaction in [C₄mim][PF₆] was demonstrated. Upon addition of 5% of water a biphasic reaction mixture was formed, and water exhibited a stabilizing effect on thermolysin. Also, the activity of the enzyme was dependent on the water content. In addition, an increased solubility was observed for the substrate and product upon addition of water. The reaction was monitored by HPLC after mixing of the reaction mixtures with 60% acetonitrile/water containing 10 mM orthophoshoric acid prior to injection. For the final procedure, however, a step allowing for the recycling of the IL was suggested after the reaction occured. The yield of the product was 95%, i.e., the same as found for organic solvents, e.g., ethyl acetate/water mixtures.

In 2007, Xing et al. reported the use of an α -chymotrypsin-catalyzed synthesis of protected tripeptides, e.g., the fragment Z-Tyr-Gly-Gly-OEt of Leu-enkephalin, in six different 1-alkyl-3-methylimidazolium hexafluorophosphates and tetrafluoroborates [30]. Again, it was found that the water content has a great influence on the two main factors, enzyme activity and coupling yield. At higher water content, the product is hydrolyzed, thus reducing the reaction yield. However, a small amount of water was essential for the reaction to succeed, i.e., for hydrophobic [MOEMIM][PF₆] the minimum content was 3%, while for the more hydrophilic [MOEMIM][BF₄] 8% turned out to be favorable. The authors also reported that [MOEMIM][PF₆] was the most suitable IL among the ILs tested, and peptides were prepared in 68–75% yield. Further enzyme-supported reactions in ILs have been reported by Wehofsky et al. [48] and are in detail in Section 2.2.3, because larger peptide molecules prepared by ligation reactions are in the focus of that study. However, enzymatic acylation of smaller peptides has also been described by other groups, as can be exemplified by the lipase-catalyzed acylation of Lys-Ser x HCl with oleic acid carried out in either organic media (2-methyl-2-butanol) or an ionic liquid ([C₄mim][PF₆]) [49]. Substrate conversion in this case was found to depend on peptide solubility which was improved in the IL. Primarily chemoselective acylation occurred at the N_ε-amino group of lysine, while the side chain acylated serine derivative was not detected at all. Another example came from Malhotra et al., who reported on the immobilized protease-catalyzed synthesis of peptides, e.g., Boc-Leu-Trp-OEt, in the ionic liquids [C₄mim][PF₆], [C₄mim][BF₄], and [C₂pyr][BF₄] [50]. Here, product yields were comparable to those obtained in organic solvents, i.e., a maximum yield of 49% for Boc-Leu-Trp-OEt was obtained.

In summary, 1,3-disubstituted imidazolium-based ionic liquids were successfully applied as additives to enzymatic syntheses. They were described to have a positive effect on enzyme-catalyzed coupling reactions, to increase the enzyme’s activity, the coupling yields, the solubility of reagents as well as the product. Interestingly, the necessity of water for optimal coupling yields has been demonstrated regularly. Obviously a minimum amount of water (3–8%) is favorable for the activation respective enzyme.

Radiolabeling is an established technique with many applications in biological research, such as the investigation of the distribution of labeled markers in vivo (e.g., in humans and in animals) by positron emission tomography [60]. The synthesis of the radiolabeled bioactive compounds, in particular peptides, is usually time consuming and requires complicated procedures and special equipment to purify the product from unwanted non-radioactive and radioactive byproducts. Strictly anhydrous conditions are highly recommended, for example, for fluoride ion displacement reactions in the conventional protocols.

A new approach in this respect has been proposed by Kim et al. in 2003 (Figure 4) [37]. Herein, fluorine-18 labeling of alkyl mesylate or alkyl halides, e.g., 2-(3-methanesulfonyloxypropoxy)-naphthalene, in ILs containing a hydrophobic cation ([C₄mim][X], where X = [BF₄]⁻, [PF₆]⁻, [SbF₆]⁻, [OTf]⁻, [NTf₂]⁻) was described for the first time. This method was successful despite the addition of a small amount of water, which is in contrast to the conventional method used. The ILs enhanced the reactivity of fluorides and also, a reduction of by-product formation (alkenes, alcohols) was observed. This approach led to further developments in this field as reported by Schirrmacher et al. [38]. The IL [C₄mim][OTf] was useful for the labeling of short model peptides, e.g., GSH [38]. Thus, radiolabeling performed in IL (1,3-dialkylimidazolium salts) obviously occurs without the need for complicated procedures and strictly anhydrous conditions, but by direct addition of the IL to the reaction mixture and further simple purification steps.

Establishing efficient strategies for the synthesis of cysteine-rich peptides has always been a challenging task in the past. Generally, the incorporation of cysteine residues in peptide sequences is difficult as it requires the protection of the highly reactive thiol group [18,61,62,63]. Cysteine, however, is very important since many naturally occurring peptides contain intramolecular disulfide bonds that stabilize the biologically active conformation [18,64,65]. Thus, one application of cysteine-residues in peptide chemistry represents disulfide bridge formation that may be achieved at various stages of the synthesis, on-resin as well as in solution [62,66,67,68]. Protecting group strategies differ according to the number of cysteine residues and disulfide bonds to be formed. While peptides containing one disulfide bridge are mainly prepared by oxidation (e.g., air, H₂O₂, iodine) in solution at low concentrations (10⁻³ to 10⁻⁴ M), for peptides with two or more disulfide bridges the situation is more complicated since intramolecular mispairing (scrambling) and/or intermolecular dimerization or oligomerization may occur [61,62,63].

The major disadvantages of these conventional methods are: (i) the requirement for a high dilution of the reaction mixture, (ii) very long reaction times, (iii) inadequate solubility of hydrophobic peptides in buffer solutions (including the need for organic solvents: isopropanol, methanol, acetonitrile), and (iv) the difficulty to control oxidation that requires the reaction to be carried out at low temperatures (usually 4 °C). Consequently, there are huge problems for producing such peptides in large amounts and sufficient purity. Over the years, many scientists were searching for a method that can overcome these problems. In our own work [14], we recently described for the first time the use of 1-ethyl-3-methylimidazolium-based ILs with different anions including small hydrogen-bond acceptors (acetate [OAc]⁻, diethyl phosphate [DEP]⁻) as well as larger, less-coordinating anions (tosylate [OTs]⁻, dicyanamide [N(CN)₂]⁻) for the oxidative folding of neuropeptides (Figure 5).

A very good solubility of hydrophobic peptides, e.g., δ-EVIA and δ-SVIE, containing six cysteine residues was observed in these ILs. The reaction was performed in ILs of low water content (between 0.5–3%), in a very small volume and a rather high concentration of the peptides (10–15 mM) [14]. Thus, this seems to be a promising method for up-scaling the production of cysteine-rich peptides, also because the formation of by-products and misfolded species was minimized in comparison to conventional methods (oxidation in buffer solution [69,70]). In contrast to the latter, there is no need for additional redox reagents, thus the reaction can be performed in the pure IL. A stabilization of pre-formed secondary structures of the peptides investigated by the ILs was suggested to be the cause of these observations.

Later, different naturally occurring cysteine-rich peptides were studied to understand the nature of interaction between ILs and peptides. Various model peptides—short (10 amino acids) and medium-sized (20–40 amino acids) native peptides with varying numbers of cysteine residues in their structure (from two to six) were subjected to these investigations. Depending on the net charge, amino acid content and hydrophobicity of the peptide, the efficiency in the ILs used differed strongly. In contrast to conotoxins µ-SIIIA, µ-PIIIA, δ-EVIA, and δ-SVIE that showed best results when using [C₂mim][OAc] as reaction medium [14], optimum conversion of linear CCAP-vil [39] was observed in [C₂mim][OTs] (yield >90% according to HPLC analysis) and to a lesser extent in [C₂mim][DEP] (yield >80% according to HPLC analysis). Interestingly, the target peptide did not form the oxidized version in [C₂mim][OAc] in acceptable yields (57%), and the yield was even lower in case of [C₂mim][N(CN)₂] (approximately 44%). When increasing the lipophilicity of the ionic liquids (by increasing the alkyl substituent from [C₂mim][OAc] to [C₄mim][OAc]), the yield of the oxidized product decreased to approximately 47% [39].

It is clear from these studies that oxidative folding depends on the proper choice of the ionic liquid. Also, peptides with a high net charge are apparently more efficiently oxidized in ILs containing more basic anions, such as [C₂mim][OAc], compared to small uncharged peptides such as CCAP-vil, where the most suitable ionic liquid contains a less basic anion, i.e., [C₂mim][OTs]. Unpublished data related to our former work showed interesting dependencies of the reaction outcome and product purities on water content and reaction temperature (Figure 6). This can be exemplified for the conopeptide µ-SIIIA, where already an increase of the water content from 3-10% was disadvantageous for product formation. In contrast, an increase in temperature up to 80 °C was favorable for the oxidation reaction.

The role of ILs in oxidative folding of peptides is still sparsely investigated, since further candidates are necessary to derive a scientific explanation for these observations, yet the application of ILs for this reaction clearly has the potential to be developed as a key technology in peptide chemistry in the future. However, an interesting aspect with respect to the folding behavior of peptides was recently presented by Huang et al., who reported on the investigation of the folding of model peptides in neat ionic liquids by CD spectroscopy [71]. This study was motivated by the fact that previous IL studies have almost exclusively focused on large functional proteins, such as lysozyme, ribonuclease A and human serum albumin. Indeed, the authors were correct in stating that the interpretation of the results obtained for proteins are complicated by their size and the different degrees of freedom available to either a peptide or a protein. Thus, they focused on small peptides, e.g., α -helical AKA₂, miniprotein Trp-cage (contains e.g., α -helical part and 3₁₀ helix), and Trpzip4 (β-hairpin), in order to understand the effect of ILs, in this case [C₄C₁pyrr][NTf₂], on well-designed secondary structures. The model sequences used represent optimized native folds in an aqueous environment. Huang et al. characterized the thermal transitions of these peptides using circular dichroism (CD) spectroscopy and found that, in contrast to the aqueous data, the far-UV CD spectra of AKA₂ and Trp-cage in [C₄C₁pyrr][NTf₂] indicated only minimal helical structure at low temperatures. However, in both cases, this data and the temperature-dependent far-UV CD spectra suggested apparent heat-induced structure formation with structure persisting to the highest temperatures recorded (92 °C). In contrast to the results of the helical peptides, the CD data for Trpzip4 indicated huge destabilization by the IL medium with increasing temperature [71]. In addition to this report, Debeljuh et al. studied the impact of triethylammoniummesylate (TeaMS) on the structure of the Abeta(1–40) peptide for Alzheimer’s disease [72]. They found that the IL was able to induce a conformational change and also that this structural change influences the self-assembly of the peptide into amyloid fibrils. In our opinion, these studies are an important starting point for further investigations on peptide conformations and peptide folding observed in ionic liquids.

Chemical ligation reactions are useful tools for the assembly of small to medium-sized proteins and protein domains starting from synthetic peptide fragments [73,74,75]. This type of chemical protein synthesis allows the modification of the molecular structure offering new insights into protein functions. Though 50 to 100-mer peptides can be assembled on a solid support, synthesis of longer peptides and proteins by the Merrifield method is often limited (see section 2.1 and section 2.1.2). Methods of segments ligation have been developed primarily in order to circumvent limitations in chain length. These segment condensation reactions, on the other hand, may be hampered by the low solubility of peptide fragments for the condensation reaction carried out in water or aqueous/organic solvent mixtures, and the stability of biomolecules. Both aspects are very important for the ligation reaction itself, and have an impact on later purification steps. One of the most widely used methods in this respect is the so-called “Native Chemical Ligation” (NCL) introduced by Kent and coworkers [73,74,75]. Here, an unprotected peptide with a thioester at the C-terminal and an unprotected peptide with an N-terminal cysteine react to yield a peptide bond. The mechanism of this reaction is shown in Figure 7. Related methods are the Staudinger ligation, which occurs between a peptide azide and a phosphinothioester, and protease-catalyzed ligations, where specific peptide esters (e.g., substrate mimetics) react regioselectively and stereospecifically with nucleophilic peptide species [73,76,77].

In section 2.1.1, we described examples of thermolysin- and α-chymotrypsin-catalyzed couplings of amino acids to generate short peptides. Bordusa and co-workers, however, highlighted the protease-catalyzed ligation using ILs for the first time in 2008 [48]. The reaction was performed in a mixture of buffer and 1,3-dimethylimidazolium dimethylphosphate [C₁C₁IM][Me₂PO₄] (40:60) (Figure 8). Several positive effects were observed for this reaction in the IL mixture in comparison to conventional methods, e.g., (i) the complete suppression of proteolytic side reactions (proteolytic hydrolysis and decreases competing hydrolysis activity of proteases toward the acyl donor esters to some extent), (ii) high turnover rates, (iii) protease stability, and (iv) good solubility of the reactants and also of the reaction products.

Recently, we reported on the application of native chemical ligation to produce a 66-mer anticoagulant cysteine-rich peptide (Tridegin) that has been shown to act as an inhibitor of factor XIIIa (Figure 9) [78]. Generally, NCL may be carried out with and without additives, i.e., thiols (e.g., thiophenol, benzyl mercaptan) as catalysts [79,80,81,82]. Therefore, the comparison of segment ligation in buffer and ILs in the presence or absence of additives was performed as well. The results showed that NCL indeed yields the ligated product. The linear Tridegin was identified in [C₂mim][OAc] even in the absence of thiophenol and benzyl mercaptan. In addition, by using additives the ligated product was also formed in [C₂mim][DEP] that correlates well with the results published by Bordusa and co-workers mentioned above [48].

In summary, the results of our and other groups demonstrate that medium-sized and large disulfide-bridged peptides are correctly formed in biocompatible ionic liquids. Moreover, the selection of peptide candidates examined thus far suggests that the correct intramolecular hydrogen bond interactions are not significantly perturbed by the presence of ILs, instead ILs seem to have a favorable effect on stabilizing pre-formed secondary structures of peptides.

HPLC is a widely used method for separation and purification of biomolecules. Especially for peptide analysis RP HPLC is the most common method applied. Some reports have been published about the application of ILs in RP HPLC for separation of nucleotides, geometric isomers, ephedrines or carboxylic acids [84,85,86,87]. These reports are based on the use of ILs as additives for the mobile phase [84] or as a stationary phase [87]. The commonly used ILs for these processes contained 1,3-dialkylimidazolium or N-alkylpyridinium cations and chloride, tetrafluoroborate, hexafluorophosphate, or trifluoroacetate as the anion depending on the analyte [86]. Nevertheless, none of the reports represents an example of an IL-supported optimal separation of peptides. Marszall et al. suggested that it is not possible to find a suitable IL for separation and purification processes [88] according to the principle, which can be expressed by the basic chemical aphorism “similis similibus solvuntur” or “similar dissolves in similar”. Therefore, when the sample, i.e., the peptide, is polar, the use of a polar IL with chaotropic anions, such as hexafluorophosphate or perchlorate was proposed, while a less polar IL with a cosmotropic anion (e.g., chloride) was recommended for a nonpolar analyte [86]. That is exactly why an individual protocol for different analytes is required.

Ionic liquids were not only applied to RP HPLC, but also used for TLC and normal phase HPLC [94,95]. However, characterization of basic or amphoteric samples is challenging if using these methods. The problem is that acidic silanol groups of the stationary phase interact strongly with organic bases (Figure 11) resulting in (i) an increase in retention, and (ii) a broadening and/or tailing of sample peaks [96].

It has already been reported that the use of IL additives may help to overcome these problems. Ionic liquids block silanol groups and in this way provide the desired separation of basic drugs as test analyte [97]. Similarly to RP HPLC, alkyl substituted imidazolium ILs were used for TLC and HPLC as well. Baczek et al. investigated the effect of the percentage amount (0–10%) of the ILs as additives to the eluent on the separation of peptides by TLC. For approximately 50 peptides the R_f values increased with an increased concentration of 1,3-dimethylimidazolium methyl sulfate in a water/0%–5% acetonitrile/0.1% TFA mixture [98]. The influence on the retention of the peptides with an increasing IL concentration was weaker with increasing length of the amino acid sequences. In the study of Baczek et al., 1-ethyl-3-methylimidazolium tetrafluoroborate was applied. Upon addition of acetonitrile (1.5%) a retention coefficient with third-degree polynomial function was derived, however, a quadratic function was needed in case of the experiment without acetonitrile. This was explained by the interaction of the IL with the stationary phase [99].

Another method involved the addition of IL and matrix (α-cyano-4-hydroxycinnamic acid) to the separation mixture. This optimized the chromatographic method allowing for a direct coupling of the thin-layer chromatograms with imaging by MALDI mass spectrometry (see also section 3.2) [100].