Chemoenzymatic synthesis and biological evaluation of enantiomerically enriched 1-(β-hydroxypropyl)imidazolium- and triazolium-based ionic liquids

Racemic 1-(β-hydroxypropyl)azoles were prepared by solvent-free direct regioselective ring opening of 1,2-propylene oxide with imidazole or 1,2,4-triazole. Lipase-catalyzed transesterification of alcohols with vinyl acetate resulted in kinetic enantiomers resolution. Separated (S)-enantiomers of (+)-1-(1H-imidazol-1-yl)propan-2-ol and (+)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol were quaternized with alkyl bromides or iodides, yielding novel optically active ionic liquids. Racemic salts were tested against a wide range of microorganisms.


Introduction
Aromatic heterocycles play a crucial role in medicinal chemistry [1,2]. More than half of all known drugs contain at least one heterocyclic component. Novel biologically active compounds are often designed as analogues of endogenous ligands that are vital to biochemical processes. Since most of these substances are comprised of heterocycles, such rings by default become core structures of the newly designed therapeutically active compounds.
In turn, many 1,2,4-triazole derivatives exhibit antimicrobial [19,20], antifungal [21], antitumor [22], analgesic [23], antiinflammatory [24], psychoactive [25][26][27], anticonvulsant [28], diuretic [29], and anti-HIV [30] activity. 1,2,4-Triazole derivatives also represent the most important group of herbicides and fungicides [31]. In recent years growing attention has been focused on imidazole-and 1,2,4-triazole-derived ionic liquids (ILs), as well as their chiral derivatives (CILs). CILs have become a subject of intensive study since their potential as catalysts for asymmetric induction [32][33][34], as supplements for influencing reaction stereoselectivity [35], as chiral solvents in stereoselective polymerization [36], as chiral phase for gas chromatography [37], or as chiral differentiating solvents for spectroscopic investigations [38,39] is far from being exhausted. CILs can also act as a tool for organizing the structure of solid polymeric electrolytes (SPE), playing the triple role as plasticizer, solvent for the ions, and medium. Most of the described optically active ILs are prepared from easily accessible natural chiral substrates [40]. The use of ILs in so-called "green chemistry", as for example solvents in inorganic or organic syntheses or as a replacement for many hazardous and volatile organic solvents, raises the question about the toxicity of newly designed compounds. Therefore, it is crucial to select the least toxic ILs with good chemical properties that can be used in industrial processes [41]. Moreover, a number of current studies have demonstrated the potential of certain ionic liquids to exhibit excellent antimicrobial activity, raising the possibility that ionic liquids could find application as biocidal agents in the control of microorganism growth [42][43][44][45].
Herein, we report the results of our investigation on the chemoenzymatic synthesis of new chiral ionic liquids using enzyme-catalyzed kinetic resolution of 1-(β-hydroxypro-pyl)azoles as a key step. We have shown that this attempt is simple and allows the preparation of new types of optically active ionic liquids. Inhibitory activity of the newly synthesized CILs was tested towards gram-negative and gram-positive bacteria and fungi.

Results and Discussion
Synthesis and enzymatic resolution of the N-2-hydroxypropylazoles In this paper we report the synthesis of racemic 1-(1H-imidazol-1-yl)propan-2-ol and 1-(1H-1,2,4-triazol-1-yl)propan-2-ol and the procedure for lipase-catalyzed kinetic separation of their enantiomers. These enantiomers can be used as chiral synthons for new drugs, pesticides or task-specific ionic liquids. The 1-(β-hydroxypropyl)-imidazole (±)-3a and -triazole (±)-3b used in this study were prepared as racemic mixtures according to the method described by Yus and co-workers [46] (Scheme 1). Next, these compounds were used as a new type of substrate in lipase-catalyzed transesterification, yielding both enantiomers with good optical purity.
The syntheses of the secondary alcohols (±)-3a and (±)-3b were accomplished by the addition of imidazole (2a) or 1,2,4-triazole (2b) to 1,2-propylene oxide (1) under solvent-free conditions. The epoxide ring-opening reactions were carried for 24 h at elevated temperature (32 °C) and resulted in the formation of the appropriate alcohols (±)-3a and (±)-3b in high yields (Scheme 1). In the next step, the influence of crucial parameters in enzymecatalyzed reactions such as the type and quantity of the biocatalyst and solvent on the enantiomers resolution was investigated.
The aim was to achieve a conversion close to 50% and 99% ee for the slow-reacting substrates and product enantiomers. Four different lipases, i.e., two native (Amano PS, Amano AK) and two immobilized enzymes (Novozym SP 435, Amano PS-IM), were chosen for the transesterification studies. The screenings were performed with a 5-fold molar excess of vinyl acetate as an irreversible acyl donor at ambient temperature. The selection of a proper solvent is a significant factor in biocatalysis [47,48], since solvents are able to change the activity [49][50][51] and enantioselectivity [52][53][54] of an enzyme. Conformational changes in the enzyme structure under the influence of solvent can even invert the substrate specificity and the enzyme-substrate affinity [55]. The ability to control the direction of an enzymatic reaction and its specificity in a nonaqueous environment has resulted in the development of so-called "solvent engi-neering". After testing several solvents for lipase-catalyzed acetylation of (±)-3a and (±)-3b with vinyl acetate, we determined the following order of their utility as the reaction media: MTBE ~ 2-methyl-2-butanol > acetone > 1,4-dioxane >> acetonitrile. Further optimization revealed that MTBE and 2-methyl-2-butanol are unsurpassed, not only from the viewpoint of substrate solubility and reactivity, but also due to the highest enantiomeric excesses of the separated enantiomers. Results of the lipase-catalyzed transesterification of the racemic (±)-3a are summarized in Table 1.
The experiments demonstrated that the reaction proceeded particularly efficiently when Amano AK or Amano PS-IM were used. These enzymatic preparations also gave excellent reaction enantioselectivities (  the reaction was arrested close to 45% conversion. In turn, conversion exceeding 57% was beneficial for high optical purity of the remaining alcohol (+)-5a (Table 1, entries 6 and 7, >97% ee).
As usual, the optical purities of the acetate, as well as of the remaining alcohol, were dependent on the enzyme used and the conversion rate. For example, with Amano AK the reaction reached 37% conversion after 30 h giving product (+)-6b in 90% ee ( Table 2, entry 4), and after 49 h the stereochemical course reached 54% conversion yielding the slower reacting enantiomer (+)-5b with high enantiomeric excess (Table 2, entry 5, 98% ee). The highest optical purity for (+)-6b (90% ee) and the shortest reaction time (10 h) were obtained by the reac-tion catalyzed by Amano PS-IM, which was much faster than that catalyzed by native Amano PS. The reaction enantioselectivities were fairly good (E = 20-56) for all of the tested lipases except Novozyme SP 435, which showed very high activity but low stereoselectivity (E = 6).
(MPA, Mosher reagent) (Scheme 2). The utility of this method applied to secondary alcohols possessing heterocyclic azole rings has been demonstrated by us previously [58].
The absolute configuration of the substrate is deduced by interpretation of the signs of the Δδ RS values, by using an empirical model that assumes that in MPA esters of secondary alcohols, the most representative conformer has the methoxy group of MPA, the carbonyl group, and a proton bonded to the stereogenic center of the alcohol in the same plane.
The differences in the chemical shifts (Δδ RS ) observed in the spectra of the esters prepared from the chiral auxiliaries [(R)and (S)-MPA acids, respectively], were calculated separately for the protons attached to the carbon atoms situated on both sides of the chirality center, as shown by the following equations: The signs of these parameters bear the information necessary for the configurational assignment, since they indicate the relative position of L1/L2, with respect to the anisotropic group (phenyl group of MPA). The positive value of Δδ RS , which corresponds to the signal of the protons of the substituent L1, and the negative sign for the protons L2 indicate an (S)-configuration for the enantiomers of both types of investigated alcohols (+)-5a and (+)-5b according to Figure 1. The 1 H NMR spectra recorded for the resulting diastereomeric derivatives (Figure 2) confirmed the results of the above equations.
The chiral environment provided by the derivatizing agent (MPA) covalently associated with the respective alcohol leads

Antimicrobial activity of racemic ionic liquids
Antibacterial and antifungal activities of the synthesized racemic CILs were evaluated against a wide range of microorganisms. The studies were conducted on three strains of gramnegative bacteria, two strains of gram-positive bacteria, and eight strains of fungi. For the preliminary screening of bacteria and yeasts we used the agar diffusion test (Table 4) in order to select the most promising compounds for further determination of minimal inhibitory concentrations (MICs) by the broth dilution method (Table 5). In turn, the antifungal activity was tested by the agar dilution method. The results are listed in Table 6.
The antimicrobial and antifungal activities of tested CILs are significantly dependent on the alkyl chain length, as was demonstrated in numerous previous studies [59]. The high toxicity of CILs was noted for CILs with alkyl-chain substituents of 10-16 carbon atoms (Table 4 and 5). Moreover, the chemical nature of the cationic head group influenced the overall toxicity of the CILs, which is in good agreement with several previous studies [60]. The imidazolium CILs exhibited visibly stronger   antibacterial and antifungal activity than triazolium CILs (Tables 4-6). However, the filamentous fungi displayed high tolerance towards the tested CILs. Nevertheless, the relation between the alkyl chain substituent and the antifungal activity of the tested compounds could still be observed, with the strongest toxicity demonstrated by CILs with an alkyl chain substituent of 12 carbon atoms (Table 6). This high tolerance of filamentous fungi against CILs could be explained by the ability
Resolution of (±)-3b proceeded with good yield, but the enantiomeric excess of the slower reacting enantiomer (alcohol (+)-5b) was less than 98%. The faster reacting ester (+)-6b barely reached 90% ee. Nevertheless, the procedure presented here is simple and efficient, and, after some additional optimization, can be readily extended to other substrates of this type. According to 1 H NMR investigations, the slower reacting enantiomers of the alcohols (+)-5a and (+)-5b possess (S)-configuration. This assignment is in good agreement with Kazlauskas's rule [62,63], where in lipase-catalyzed esterification of secondary alcohols the (R)-ester and (S)-alcohol enantiomers are obtained. The imidazolic and triazolic chiral salts derived from alcohols (S)-(+)-5a and (S)-(+)-5b were obtained in high to excellent isolated yields. The core structures of these salts were modified by using, for quaternization reaction, haloalkanes or haloalkenes with various chain lengths. Their antibacterial and antifungal properties were evaluated by three different methods. Some of these compounds exhibited biological activity that was significantly dependent on the alkyl chain length, with considerably high toxicity of the substituents with 10-16 carbon atoms. The imidazolium salts revealed stronger antibacterial activity than their triazolium analogues.

Supporting Information
Supporting Information File 1 Complete experimental procedures and characterization data.