Ionic Liquids: Efficient Media for the Lipase-Catalyzed Michael Addition

molecules

Article Ionic Liquids: Efﬁcient Media for the Lipase-Catalyzed Michael Addition

Yunchang Fan *, Dongxu Cai, Xin Wang and Lei Yang College of Chemistry and Chemical Engineering, Henan Polytechnic University, Jiaozuo 454003, China; [email protected] (D.C.); [email protected] (X.W.); [email protected] (L.Y.) * Correspondence: [email protected]; Tel.: +86-391-398-6813

Received: 5 August 2018; Accepted: 25 August 2018; Published: 27 August 2018

Abstract: Recently, ionic liquids (ILs) have been regarded as ideal media for non-aqueous bio-catalysis. In this work, the synthesis of warfarin by the lipase-catalyzed Michael addition in IL media and the parameters that affected the warfarin yield were investigated. Experimental results demonstrated that the chemical structures of the ILs were a major factor for influencing the warfarin yield. The ILs – containing the NTf2 anion were suitable reaction media due to the high chemical stability of this anion. The incorporation of the hydroxyl group on the IL cation significantly improved the lipase activity due to the H2O-mimicking property of this group. The lipase activity decreased by increasing the alkyl chain length on the IL cation due to the non-polar domain formation of the IL cation at the active site entrance of lipase. The ILs and lipase could be reused no less than five times without reduction in the warfarin yield.

Keywords: ionic liquids (ILs); lipase; Michael addition; warfarin; bio-catalysis

1. Introduction In recent decades, enzymes have been regarded as green catalysts because they are recyclable non-toxic materials and the enzymatic reactions are usually performed under mild conditions [1,2]. Lipases are the most used enzymes because of their high stability, activity, wide range of substrates, and low cost [3–6]. They can catalyze many reactions such as the Michael addition [3–5], esterification [6], ester-hydrolysis [7], and transesterification [8]. Among these reactions, Michael addition is one of the most effective ways to form mild C–C bonds and generate interesting, drug-like scaffolds. The synthesis of warfarin by Michael addition of 4-hydroxycoumarin (4-HC) to benzylideneacetone (BA) is a classic case. Warfarin and sodium warfarin are widely used as vitamin K antagonists, which prevent the vitamin K-dependent synthesis of blood-clotting proteins and, thus, decrease blood coagulation [9,10]. Xie et al. [11] reported the synthesis of warfarin and derivatives via the lipase-catalyzed Michael addition in dimethyl sulfoxide (DMSO) media, which presented a yield of 87%. Sano et al. [5] also reported the lipase-catalyzed Michael reaction of 4-HC to BA in anhydrous DMSO and a higher yield (85%) was achieved. Although some interesting results have been reported by these excellent papers, there is still one problem [3,5,11]. Although lipases exhibit the highest activity in DMSO medium, DMSO is flammable and toxic even at low concentrations (<10%, v/v)[12]. Therefore, it is of great importance to find environmentally-benign solvents. In this context, ionic liquids (ILs) are considered as environmentally-benign solvents compared to the conventional solvents because of their negligible vapor pressure, high dissolving power, and high thermal stability and recyclability [13–15]. At present, ILs have found applications in diverse areas such as the conversion of biomass [15] and biocatalysis [16–19]. Schutt et al. studied the impact of water-dilution on the solvation properties of an ester-functionalized IL for model biomass molecules through the use of biased and unbiased molecular dynamics (MD) simulations. It is found that hydrogen-bonding interactions

Molecules 2018, 23, 2154; doi:10.3390/molecules23092154 www.mdpi.com/journal/molecules Molecules 2018, 23, 2154 2 of 11 between the IL anion and water are a major driving force that significantly impacts the solvent properties of the ester-functionalized IL as well as conformational preferences of the cellulosic model compound [15]. Burney et al. studied the impact of imidazolium-based ILs on enzyme functionality by using molecular simulations. It is found that the enzymes lipase and α-chymotrypsin are altered by randomly mutating lysine surface residues to glutamate in aqueous ILs 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium ethyl sulfate. These mutations resemble succinylation of the enzyme by chemical modification, which enhances the stability of both enzymes in ILs [19]. Very recently, the lipase-catalyzed esterification [20], ester-hydrolysis [21], and transesterification [22] reactions in the IL media have been widely reported. Furthermore, many reports have demonstrated – – that the ILs with the anions PF6 and BF4 were suitable reaction media for the lipase-catalyzed – – transesterification [22,23] and esterification [24,25] reactions. However, the anions PF6 and BF4 are hydrolytically unstable, which results in a propensity to decompose and release corrosive HF [26,27]. – It has been proven that bis(trifluoromethanesulphonyl)imide (NTf2 ) has the higher chemical stability – – than PF6 and BF4 [27,28]. Some work suggests that the hydroxyl-functionalized ILs have a good compatibility with biomolecules such as catalase [29] and cytochrome c [30]. Therefore, it will be – expected that the hydroxyl-functionalized ILs containing NTf2 can be used as ideal media for the lipase-catalyzed Michael addition reaction. In this paper, the feasibility of the use of the hydroxyl-functionalized ILs as the reaction media of lipase-catalyzed Michael addition and the parameters affecting the reaction yield have been investigated systematically. Furthermore, the recyclability of ILs and lipase has also been studied. For clarity, the names and abbreviations of the ILs are listed in Table1.

Table 1. Names and abbreviations of the ILs used in this work.

Name Abbreviation

1-Butyl-3-methylimidazolium tetrafluoroborate [C1C4im]BF4 1-Butyl-3-methylimidazolium hexafluorophosphate [C1C4im]PF6 1-Butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [C1C4im]NTf2 1-Methyl-3-(6-hydroxyhexyl) imidazolium bis(trifluoromethylsulfonyl)imide [C1C6OHim]NTf2 1-Butyl-3-(3-hydroxypropyl) imidazolium bis(trifluoromethylsulfonyl)imide [C4C3OHim]NTf2 1-Methyl-3-(3-hydroxypropyl) imidazolium bis(trifluoromethylsulfonyl)imide [C1C3OHim]NTf2 1-Butyl-3-(11-hydroxyundecyl) imidazolium bis(trifluoromethylsulfonyl)imide [C4C11OHim]NTf2 1-Methyl-3-(11-hydroxyundecyl) imidazolium bis(trifluoromethylsulfonyl)imide [C1C11OHim]NTf2

2. Results and Discussion

2.1. Selection of ILs In this work, eight kinds of ILs and DMSO have been investigated systematically in order to ﬁnd the most suitable reaction medium. As shown in Figure1, the lipase activity in dialkylimidazolium-based IL media follows the order: [C1C4im]PF6 > [C1C4im]BF4 > [C1C4im]NTf2. – – It has been reported that the nucleophilicity of anions increases in the following trend: PF6 < BF4 – < NTf2 where the more nucleophilic anions have a tendency to interact with the positively charged sites in the enzyme structure and to change the enzyme’s conformation, which could eventually result – – in a denaturation of the enzyme [31–33]. However, PF6 and BF4 are moisture-sensitive anions and tend to hydrolyze and release toxic HF. From the point of view of safety and environmental protection, – the use of NTf2 is more preferable [26,27]. Molecules 2018, 23, 2154 3 of 11 Molecules 2018, 23, x FOR PEER REVIEW 3 of 11

Figure 1. The correlation of the hydrophobicity of of solven solventsts used used in in this this work work and and the the warfarin warfarin yield. yield. Reaction conditions: BA,BA, 1.01.0 mmol,mmol, water, water, 0.1 0.1 mL, mL, reaction reaction time, time, 96 96 h, temperature,h, temperature, 45 ◦45C, °C, lipase, lipase, 30 mg, 30 mg,4-HC, 4-HC, 0.5 mmol, 0.5 mmol, [C1C [C3OHim]NTf1C3OHim]NTf2, 2.02, g.2.0 g.

An interesting interesting phenomenon, phenomenon, seen seen in in Figure Figure 1,1 ,is is that that the the incorporation incorporation of ofthe the hydroxyl hydroxyl group group on the IL cation significantly improves the enzyme activity ([C1C3OHim]NTf2 versus [C1C4im]NTf2) and on the IL cation signiﬁcantly improves the enzyme activity ([C1C3OHim]NTf2 versus [C1C4im]NTf2) the lipase activity in [C1C3OHim]NTf2 is also much higher than in DMSO. This accounts for the fact and the lipase activity in [C1C3OHim]NTf2 is also much higher than in DMSO. This accounts for thatthe factthe thatessential the essential water layer water around layer the around lipase the mo lipaselecule molecule maintains maintains the enzyme the enzymeactivity in activity organic in media. The IL [C1C3OHim]NTf2 contains a hydroxyl group and holds the H2O-mimicking property organic media. The IL [C1C3OHim]NTf2 contains a hydroxyl group and holds the H2O-mimicking andproperty hydrogen-bonding and hydrogen-bonding functionalit functionality,y, which helps which the helps enzyme the enzymeto maintain to maintain its flexible its ﬂexibleand active and conformationactive conformation [30,34,35]. [30, 34,35]. Lastly, asas shownshown in in Figure Figure1, the1, the lipase lipase activity activi inty the in hydroxyl-functionalizedthe hydroxyl-functionalized ILs follows ILs follows the order: the order: [C1C3OHim]NTf2 > [C1C6OHim]NTf2 > [C4C3OHim]NTf2 > [C1C11OHim]NTf2 > [C1C3OHim]NTf2 > [C1C6OHim]NTf2 > [C4C3OHim]NTf2 > [C1C11OHim]NTf2 > [C4C11OHim]NTf2, [Cwhich4C11OHim]NTf is opposite2, towhich the order is opposite of their to hydrophobicity. the order of thei It isr knownhydrophobicity. that the entrance It is known of active that sitesthe entranceof lipase isof formedactive sites by the of non-polarlipase is formed residues. by th Thee non-polar IL cation canresidues. diffuse The into IL the cation active can sites diffuse of lipase into thedriven active by sites van of der lipase Waals driven interactions. by van der This Waals means interactions. that the substrates This means have that to the compete substrates with have the to IL competecations to with reach the the IL active cations sites to reach [33,36 the]. The active increase sites [33,36]. in the hydrophobicity The increase in results the hydrophobicity in the increase results in the invan the der increase Waals interactionsin the van der between Waals the interactions alkyl chains between of the ILsthe and alkyl the chains active of sites the of ILs lipase. and the Moreover, active sitesstronger of lipase. hydrophobic Moreover, ILs havestronger longer hydrophobic hydrophobic ILs tailshave (alkyl longer chains). hydrophobic Recent studiestails (alkyl suggest chains). that Recentthe hydrophobic studies suggest tails ofthat the the IL hydrophobic cations tend totails cluster of the together IL cations and tend form to non-polarcluster together domains and in form the non-polarIL-water mixtures domains with in the longer IL-water tails mixtures able to do with so morelonger effectively tails able to [33 do,36 so,37 more]. These effectively domains [33,36,37]. are close Theseto the activedomains site are entrance close to of the lipase, active which site mayentrance generate of lipase, a negative which impact may generate on the ability a negative of substrates impact onentering the ability or leaving of substrates the active enteri sitesng of or lipase leaving and reducethe active the sites activity of lipase of lipase and accordingly reduce the [ 33activity,36]. of lipase accordingly [33,36]. Above all, [C1C3OHim]NTf2 can be used in the subsequent experiments as the optimal reactionAbove medium. all, [C1C3OHim]NTf2 can be used in the subsequent experiments as the optimal reaction medium. 2.2. Optimization of the Warfarin Yield 2.2. Optimization of the Warfarin Yield Reaction time and temperature are important factors for the warfarin yield. As can be seen from FigureReaction2a, the warfarintime and yield temperature keeps increasing are important in the factor ranges of for reaction the warfarin time from yield. 0 toAs 168 can h be and seen remains from Figurealmost 2a, constant the warfarin above 168 yield h. Therefore,keeps increasing 168 h is regardedin the range as the of optimalreactionreaction time from time 0 andto 168 is used h and in remainsthe subsequent almost experiments.constant above 168 h. Therefore, 168 h is regarded as the optimal reaction time and is used Thein the effect subsequent of the reaction experiments. temperature on the warfarin yield is shown in Figure2b. The warfarin yieldThe increases effect withof the a reaction rise in temperature temperature from on the 25 ◦ warfarinC to 50 ◦C. yield It is is understood shown in Figure that higher 2b. The temperature warfarin yieldlowers increases the viscosity with ofa rise reaction in temperature media, which from is 25 favorable °C to 50 for°C. reducing It is understood the mass that transfer higher resistance temperature and, lowers the viscosity of reaction media, which is favorable for reducing the mass transfer resistance

Molecules 2018, 23, 2154 4 of 11 Molecules 2018, 23, x FOR PEER REVIEW 4 of 11 thus,and, thus, improves improves the reaction the reaction yield [38yield,39]. [38,39]. When theWh reactionen the reaction temperature temperature is above is 50 above◦C, the 50 warfarin °C, the yieldwarfarin keeps yield constant keeps byconstant further by increasing further increasing the reaction the reaction temperature. temperature. Thus, 50 Thus,◦C is 50 a choice°C is a forchoice the optimalfor the optimal reaction reaction temperature. temperature.

Figure 2. InfluenceInﬂuence of reaction reaction time ( a) and and temperature temperature ( b) on on the the warfarin warfarin yield. yield. Reaction Reaction conditions: conditions: ◦ ((a)) BA,BA, 1.01.0 mmol,mmol, water,water, 0.10.1 mL,mL, temperature,temperature,45 45 °C,C, lipase,lipase, 3030 mg,mg, 4-HC,4-HC, 0.50.5 mmol,mmol, [C[C11C33OHim]NTf2,, 2.0 g. ( (bb)) BA, BA, 1.0 1.0 mmol, mmol, water, water, 0.1 0.1 mL, mL, reaction time, 168 168 h, lip lipase,ase, 30 30 mg, 4-HC, 0.5 mmol,

[C1C33OHim]NTfOHim]NTf22, ,2.0 2.0 g. g.

Generally, thethe dosagedosage ofof solventsolvent hashas aa signiﬁcantsignificant effecteffect onon thethe reactionreaction yieldyield [[22,40].22,40]. As shown in Figure 3a, the warfarin yield increases with the [C1C3OHim]NTf2 dosage from 0 g to 0.5 g while in Figure3a, the warfarin yield increases with the [C 1C3OHim]NTf2 dosage from 0 g to 0.5 g while warfarin yield will decrease with a further increase in the [C1C3OHim]NTf2 dosage. This can be warfarin yield will decrease with a further increase in the [C1C3OHim]NTf2 dosage. This can be explained by the facts that the addition of the solvent ([C1C3OHim]NTf2) can improve the enzyme explained by the facts that the addition of the solvent ([C1C3OHim]NTf2) can improve the enzyme stability andand activityactivity by by maintaining maintaining an an appropriate appropriate microenvironment microenvironment with with mild mild polarity polarity around around the lipasethe lipase active active sites. sites. However, However, excess excess solvent solvent can can dilute dilute the the concentrations concentrations of of substrates, substrates, whichwhich willwill result in a lower reaction yield [22,40]. Since 0.5 g of [C1C3OHim]NTf2 provides the highest warfarin result in a lower reaction yield [22,40]. Since 0.5 g of [C1C3OHim]NTf2 provides the highest warfarin yieldMolecules (99.1%), 2018, 23 it,it x is,is,FOR thus,thus, PEER selectedselected REVIEW asas thethe optimaloptimal ILIL dosagedosage inin thethe reactions.reactions. 5 of 11 The amount of water plays an important role for the enzymatic catalysis in organic media [22,41]: an essential amount of water can activate the enzyme by increasing the polarity and structural flexibility of the enzyme active sites. Nevertheless, excess water is harmful to the enzyme by facilitating enzyme aggregation, which diminishes the substrate diffusion and eventually leads to the enzyme inactivation. Furthermore, recent studies reported the impact of water dilution on the overall liquid structure and properties of ILs. It is found that the presence of water can reduce the viscosity of ILs and the liquid structure of ILs depends on the IL concentration: there is strong ordering in the local structure of IL at high IL concentrations and a breakdown of the structure at low IL concentrations [15,37]. Therefore, it is necessary to study the impact of the water dosage. The results are shown in Figure 3b. We observed that the addition of an appropriate amount of water can improve the enzyme activity, which achieves the highest warfarin yield (0.1 mL of water), but the warfarin yield decreases with a further increase of the water dosage. Thus, 0.1 mL is regarded as the Figure 3. Influence of the dosages of [C1C3OHim]NTf2 (a) and water (b). Reaction conditions: (a) BA, optimalFigure water 3. Inﬂuence dosage. of the dosages of [C1C3OHim]NTf2 (a) and water (b). Reaction conditions: (a) BA, 1.0 mmol, water, 0.1 mL, reaction time, 168 h, temperature, 50◦ °C, lipase, 30 mg, 4-HC, 0.5 mmol. (b) 1.0 mmol, water, 0.1 mL, reaction time, 168 h, temperature, 50 C, lipase, 30 mg, 4-HC, 0.5 mmol. (b) BA, BA, 1.0 mmol, reaction time, 168 ho, temperature,◦ 50 °C, lipase, 30 mg; 4-HC, 0.5 mmol, 1.0 mmol, reaction time, 168 ho, temperature, 50 C, lipase, 30 mg; 4-HC, 0.5 mmol, [C1C3OHim]NTf2, [C0.51C g.3OHim]NTf2, 0.5 g.

The effect of enzyme dosage on the warfarin yield has also been researched in this work and the The amount of water plays an important role for the enzymatic catalysis in organic media [22,41]: results are shown in Figure 4a. We observed that the warfarin yield increases continuously with the an essential amount of water can activate the enzyme by increasing the polarity and structural ﬂexibility enzyme dosage increasing from 5 mg to 30 mg and decreasing slightly with a further increase of the of the enzyme active sites. Nevertheless, excess water is harmful to the enzyme by facilitating enzyme amount of enzyme. It has been reported that the addition of lipase is essential and higher amounts of aggregation, which diminishes the substrate diffusion and eventually leads to the enzyme inactivation. lipase have higher activity within a certain range. Beyond that range, the addition of excess lipase Furthermore, recent studies reported the impact of water dilution on the overall liquid structure and would no longer enhance the warfarin yield since an optimal amount of water is required to activate properties of ILs. It is found that the presence of water can reduce the viscosity of ILs and the liquid the added enzyme. In this way, at a non-optimal water dosage, some portion of the enzyme may be structure of ILs depends on the IL concentration: there is strong ordering in the local structure of IL at inactive [22,42]. In light of this, 30 mg of lipase is selected for the following experiments. Figure 4b illustrates the effect of the molar ratio of BA to 4-HC on the warfarin yield. The highest yield is obtained at a 2:1 molar ratio of BA to 4-HC. Further increasing the number of equivalents of BA does not improve the warfarin yield. Therefore, 2:1 is taken as the optimal molar ratio of BA to 4- HC.

Figure 4. Influence of enzyme dosage (a) and molar ratio of BA to 4-HC (b). Reaction conditions: (a) BA, 1.0 mmol, water, 0.1 mL, temperature, 50 °C, 4-HC, 0.5 mmol, reaction time, 168 h, [C1C3OHim]NTf2, 0.5 g. (b) Water, 0.1 mL, temperature, 50 °C, lipase, 30 mg, 4-HC, 0.5 mmol, reaction

time, 168 ho, [C1C3OHim]NTf2, 0.5 g.

2.3. Reusability of Lipase and [C1C3OHim]NTf2

The reuse of lipase and [C1C3OHim]NTf2 is clearly preferable from both environmental and economic points of view. To reuse lipase, the reaction mixture is dissolved with acetone after the end of the reaction. After centrifugation, the upper phase (acetone phase) is collected to recover [C1C3OHim]NTf2 and warfarin. The bottom phase is lipase, which is washed with acetone several times until no [C1C3OHim]NTf2, BA, 4-HC, and warfarin are detected in the washings. After being dried at 25 °C for 24 h, the recovered lipase is loaded into the reaction mixture for a new reaction cycle under the optimal reaction conditions. The catalytic performance of lipase after recycling is

Molecules 2018, 23, x FOR PEER REVIEW 5 of 11

Molecules 2018, 23, 2154 5 of 11

Figure 3. Influence of the dosages of [C1C3OHim]NTf2 (a) and water (b). Reaction conditions: (a) BA, high1.0 IL concentrationsmmol, water, 0.1 andmL, areaction breakdown time, 168 of theh, temperature, structure at 50 low °C, ILlipase, concentrations 30 mg, 4-HC, [ 150.5,37 mmol.]. Therefore, (b) it is necessaryBA, 1.0 mmol, to study reaction the impact time, of 168 the waterho, temperature, dosage. The 50 results °C, lipase, are shown 30 mg; in Figure 4-HC,3 b.0.5 We mmol, observed that the[C1C addition3OHim]NTf of an2, 0.5 appropriate g. amount of water can improve the enzyme activity, which achieves the highest warfarin yield (0.1 mL of water), but the warfarin yield decreases with a further increase of the waterThe effect dosage. of enzyme Thus, 0.1 dosage mL is on regarded the warfarin as the yield optimal has water also been dosage. researched in this work and the resultsThe are effect shown of enzyme in Figure dosage 4a. We on observed the warfarin that yieldthe warfarin has also yield been increases researched continuously in this work with and thethe resultsenzyme are dosage shown increasing in Figure from4a. We 5 mg observed to 30 mg that and the de warfarincreasing yield slightly increases with a continuously further increase with of the the enzymeamount dosageof enzyme. increasing It has been from reported 5 mg to that 30 mg the and addition decreasing of lipase slightly is essential with a and further higher increase amounts of the of amountlipase have of enzyme. higher activity It has been within reported a certain that range. the addition Beyond of that lipase range, is essential the addition and higherof excess amounts lipase ofwould lipase no have longer higher enhance activity the withinwarfarin a certain yield since range. an Beyondoptimal that amount range, of thewater addition is required of excess to activate lipase wouldthe added no longer enzyme. enhance In this the way, warfarin at a non-optimal yield since water an optimal dosage, amount some ofportio watern of is requiredthe enzyme to activatemay be theinactive added [22,42]. enzyme. In light In this of this, way, 30 at mg a non-optimal of lipase is waterselected dosage, for the some following portion experiments. of the enzyme may be inactiveFigure [22, 424b]. illustrates In light of the this, effect 30 mgof the of lipasemolar isratio selected of BA for to 4-HC the following on the warfarin experiments. yield. The highest yieldFigure is obtained4b illustrates at a 2:1 the molar effect ratio of theof BA molar to 4- ratioHC. of Further BA to 4-HCincreasing on the the warfarin number yield. of equivalents The highest of yieldBA does is obtained not improve at a 2:1 the molar warfarin ratio yield. of BA Therefore, to 4-HC. Further 2:1 is taken increasing as the the optimal number molar of equivalents ratio of BA of to BA 4- doesHC. not improve the warfarin yield. Therefore, 2:1 is taken as the optimal molar ratio of BA to 4-HC.

FigureFigure 4.4. InﬂuenceInfluence of of enzyme enzyme dosage dosage (a ()a and) and molar molar ratio ratio of BAof BA to 4-HCto 4-HC (b). ( Reactionb). Reaction conditions: conditions: (a) BA, (a) ◦ 1.0BA, mmol, 1.0 mmol, water, 0.1water, mL, temperature,0.1 mL, temperature, 50 C, 4-HC, 50 0.5 °C, mmol, 4-HC, reaction 0.5 mmol, time, 168 reaction h, [C1C 3time,OHim]NTf 168 h,2, ◦ 0.5[C1C g.3OHim]NTf (b) Water,2 0.1, 0.5 mL, g. (b temperature,) Water, 0.1 mL, 50 temperature,C, lipase, 30 50 mg, °C, 4-HC, lipase, 0.5 30 mmol,mg, 4-HC, reaction 0.5 mmol, time, reaction 168 ho,

[Ctime,1C3 OHim]NTf168 ho, [C1C2,3OHim]NTf 0.5 g. 2, 0.5 g.

1 3 2 2.3.2.3. Reusability ofof LipaseLipase andand [C[C1C3OHim]NTfOHim]NTf2

1 3 2 TheThe reusereuse ofof lipaselipase andand [C[C1CC3OHim]NTf2 is clearly preferable fromfrom bothboth environmentalenvironmental andand economiceconomic points of of view. view. To To reuse reuse lipase, lipase, the the reacti reactionon mixture mixture is dissolved is dissolved with with acetone acetone after after the end the endof the of thereaction. reaction. After After centrifugation, centrifugation, the theupper upper phase phase (acetone (acetone phase) phase) is is collected collected to recoverrecover [C1C3OHim]NTf2 and warfarin. The bottom phase is lipase, which is washed with acetone several [C1C3OHim]NTf2 and warfarin. The bottom phase is lipase, which is washed with acetone several times until no [C1C3OHim]NTf2, BA, 4-HC, and warfarin are detected in the washings. After being times until no [C1C3OHim]NTf2, BA, 4-HC, and warfarin are detected in the washings. After being drieddried atat 2525◦ C°C for for 24 24 h, h, the the recovered recovered lipase lipase is loadedis loaded into into the reactionthe reaction mixture mixture for a for new a reactionnew reaction cycle undercycle under the optimal the optimal reaction reaction conditions. conditions. The catalytic The ca performancetalytic performance of lipase of after lipase recycling after isrecycling shown inis Figure 5a. As can be seen, no obvious changes in warfarin yield are observed after ﬁve consecutive cycles, which exhibits excellent reusability of lipase. The FT-IR spectra of lipase before and after use were measured and the results shown in Figure6 indicate that the FT-IR spectrum of the recovered lipase is identical to that of the fresh one, which suggests that lipase remains its native conformation after reaction. This explains why the lipase can be reused without the loss of its catalytic performance. MoleculesMolecules 20182018,, 2323,, xx FORFOR PEERPEER REVIEWREVIEW 66 ofof 1111 shownshown inin FigureFigure 5a.5a. AsAs cancan bebe seen,seen, nono obviousobvious chchangesanges inin warfarinwarfarin yieldyield areare observedobserved afterafter fivefive consecutiveconsecutive cycles,cycles, whichwhich exhibitsexhibits excellentexcellent reusabireusabilitylity ofof lipase.lipase. TheThe FT-IRFT-IR spectraspectra ofof lipaselipase beforebefore andand afterafter useuse werewere measuredmeasured andand thethe resultsresults shownshown inin FigureFigure 66 indicateindicate thatthat thethe FT-IRFT-IR spectrumspectrum ofof thethe recoveredrecovered lipaselipase isis identicalidentical toto thatthat ofof thethe freshfresh one,one, whichwhich suggestssuggests thatthat lipaselipase remainsremains itsits nativenative conformationconformation afterafter reaction.reaction. ThisThis explainsexplains whywhy thethe lipalipasese cancan bebe reusedreused withoutwithout thethe lossloss ofof itsits catalyticcatalytic Molecules 2018, 23, 2154 6 of 11 performance.performance.

FigureFigure 5. 5. ReusabilityReusabilityReusability studystudy study ofof of lipaselipase (( aa)) and andand [C [C11CC33OHim]NTfOHim]NTfOHim]NTf22 2 ((b(bb)) ) underunder under thethe the optimaloptimal reaction reaction conditions.conditions.

FigureFigure 6.6. TheTheThe FT-IRFT-IR FT-IR spectraspectra ofof thethe originaloriginal lipaselipase andand thethe recoveredrecovered one. one.

ToTo reusereuse [C[C11CC33OHim]NTfOHim]NTf22,, thethe acetoneacetone phase,phase, whichwhich isis obtainedobtained fromfrom thethe recyclingrecycling ofof lipase,lipase, isis To reuse [C1C3OHim]NTf2, the acetone phase, which is obtained from the recycling of lipase, distilleddistilled toto removeremove acetone.acetone. TheThe residueresidue isis mixedmixed withwith NaOHNaOH solutionsolution (the(the molarmolar amountamount ofof NaOHNaOH is distilled to remove acetone. The residue is mixed with NaOH solution (the molar amount of NaOH is isis equalequal toto thethe theoreticaltheoretical amountamount ofof warfarin),warfarin), thethe upperupper phasephase (NaOH(NaOH solution)solution) ofof thethe resultantresultant equal to the theoretical amount of warfarin), the upper phase (NaOH solution) of the resultant mixture mixturemixture isis collectedcollected toto isolateisolate warfarin,warfarin, andand thethe bottombottom phasephase isis [C[C11CC33OHim]NTfOHim]NTf22,, whichwhich containscontains is collected to isolate warfarin, and the bottom phase is [C1C3OHim]NTf2, which contains 5.5% of BA, 5.5%5.5% ofof BA,BA, 2.0%2.0% ofof 4-HC,4-HC, andand 1.2%1.2% ofof warfarin.warfarin. AfterAfter beingbeing◦ drieddried atat 6060 °C°C forfor 2424 h,h, thethe recoveredrecovered 2.0% of 4-HC, and 1.2% of warfarin. After being dried at 60 C for 24 h, the recovered [C1C3OHim]NTf2 [C[C11CC33OHim]NTfOHim]NTf22 cancan bebe usedused forfor thethe nextnext reactionreaction cycle.cycle. FiFiguregure 5b5b showsshows thatthat thethe warfarinwarfarin yieldyield staysstays can be used for the next reaction cycle. Figure5b shows that the warfarin yield stays almost constant almostalmost constantconstant afterafter fivefive runs,runs, whichwhich suggestssuggests thatthat [C[C11CC33OHim]NTfOHim]NTf22 alsoalso possessespossesses excellentexcellent after five runs, which suggests that [C1C3OHim]NTf2 also possesses excellent reusability. reusability.reusability. 2.4. Separation of Warfarin from the Reaction Mixture 2.4.2.4. SeparationSeparation ofof WarfarinWarfarin fromfrom thethe ReactionReaction MixtureMixture As mentioned above, after the end of the reaction, acetone is used to dissolve the product mixture As mentioned above, after the end of the reaction, acetone is used to dissolve the product and theAs resultantmentioned acetone above, phase after isthe distilled end of to the remove reaction, acetone. acetone The is resultant used to residuedissolve is the mixed product with mixture and the resultant acetone phase is distilled to remove acetone. The resultant residue is mixed mixtureNaOH solution and the toresultant generate acetone the aqueous phase is phase distilled containing to remove warfarin aceton sodium.e. The resultant In doing residue so, the is product mixed with NaOH solution to generate the aqueous phase containing warfarin sodium. In doing so, the withcan be NaOH separated solution from to the generate reaction the mixture. aqueous Then, phas thee containing pH of the aqueouswarfarin phasesodium. is adjusted In doing to so, about the product can be separated from the reaction mixture. Then, the pH of the aqueous phase is adjusted product3.0 with can acetic be acidseparated and a from light yellowthe reaction precipitate mixture. is obtainedThen, the (crude pH of warfarin).the aqueous The phase resultant is adjusted crude to about 3.0 with acetic acid and a light yellow precipitate is obtained (crude warfarin). The resultant warfarinto about 3.0 is washedwith acetic with acid the and mixture a light of yellow ethanol prec andipitate acetic is acidobtained solution (crude (pH warfarin). 2.5) (3:5, Thev/v resultant) several crude warfarin is washed with the mixture of ethanol and acetic acid solution (pH 2.5) (3:5, v/v) crudetimes untilwarfarin white is powder washed is with obtained. the mixture Part of warfarinof ethanol is and lost duringacetic acid washings solution and (pH the final2.5) (3:5, warfarin v/v) several times until white powder is obtained. Part of warfarin is lost during washings and the final yieldseveral is times 83.7% until and thewhite purity powder of warfarin is obtained. (determined Part of warfarin via the HPLCis lost method)during washings is 99.3%. and The the specific final warfarin yield is 83.7% and the purity of warfarin (determined via the HPLC method) is 99.3%. The warfarinrotation ofyield the synthesizedis 83.7% and warfarin the purity is −of1.8 warfarin◦. It is known (determined that the via specific the HPLC rotations method) of S-warfarin is 99.3%. andThe

R -warfarin are −25.5 ± 1◦ and +24.8 ± 1◦, respectively [43]. Therefore, the synthesized warfarin is a racemic mixture, which is in agreement with the clinically available warfarin [44]. Molecules 2018, 23, 2154 7 of 11

3. Materials and Methods

3.1. Reagents Lipase from Candida ruosa (type VII, activity, ≥700 unit mg–1) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 4-Hydroxycoumarin (4-HC, 98%), warfarin sodium (98%), and benzylideneacetone (BA, ≥99%) were obtained from Aladdin Reagent Co. (Shanghai, China). n-Octanol (98%), sodium 1-heptanesulfonate (99%), and acetonitrile (99.9%) were obtained from Energy Chemical Co. (Shanghai, China). The ILs, [C1C4im]BF4 (99%), [C1C4im]PF6 (99%), and [C1C4im]NTf2 (99%) were supplied by the Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (Lanzhou, China). The ILs, [C1C6OHim]NTf2, [C1C3OHim]NTf2, [C4C3OHim]NTf2, ([C4C11OHim]NTf2, and [C1C11OHim]NTf2 were synthesized on the basis of our early work [45]. All the other reagents were of an analytical grade unless stated otherwise. Ultrapure water (18.2 MΩ cm) used throughout the experiments was produced by an Aquapro puriﬁcation system (Aquapro International Co., Ltd., Dover, DE, USA).

3.2. Determination of Warfarin To monitor the reaction, the ultraviolet-visible (UV-Vis) spectra of the raw materials and the products were measured by a TU-1810 spectrophotometer (Purkinje General Instrument Co., Beijing, China) (Figure S1). As can be seen from the spectra of the reaction mixture (Figure S1), after reaction, the absorbance of BA decreases remarkably and the absorption peaks of warfarin appear, which suggests that the reaction occurred. To more accurately measure the concentrations of warfarin in the reaction media, an Agilent 1200 high performance liquid chromatograph (HPLC, Santa Clara, CA, USA) equipped with a variable wavelength detector (VWD) and an auto-sampler was used. An Amethyst C18-H column (4.6 mm × 150 mm, 5 µm, Sepax Technologies Inc., Newark, NJ, USA) was used to separate warfarin. The mobile phase was the mixture of acetonitrile and 0.10% (v/v) acetic acid aqueous solution (55:45, v/v), the ﬂow rate was 0.70 mL min−1, the injection volume was 5.0 µL, the column temperature was 30 ◦C, and the detection wavelength was set at 308 nm.

3.3. Determination of the Hydrophobic Parameters of the ILs

The n-octanol–water partition coefﬁcients (Pow), which are usually expressed as logPow values, are regarded as measures of hydrophobicity of organic substances. The logPow values of the ILs used in this work were measured according to the reported methods [46,47]: the IL solutions (5.0 × 10–4 mol L–1 for each) and the n-octanol were presaturated with n-octanol and water, respectively. As an example, the mixture composed of IL solution (10.0 mL) and n-octanol (10.0 mL) was stirred for 30 min at 25 ◦C. Then the n-octanol and the water phases were separated by centrifugation. The concentrations of the ILs both in the n-octanol and water phases were determined by the HPLC method. The chromatographic conditions were listed as follows: injection volume, 2.0 µL, ﬂow rate, 0.80 mL min–1, detection wavelength, 220 nm, mobile phase, a mixture of acetonitrile and 2.0 × 10–3 mol L–1 of aqueous sodium 1-heptanesulfonate solution (50% (v/v) of acetonitrile for determining [C4C11OHim]NTf2 and [C1C11OHim]NTf2; 40% (v/v) of acetonitrile for measuring the remaining ILs). The Pow value is calculated according to the equation below [46,47].

Cn−octanol Pow = (1) Cwater

In the equation, Cn-octanol and Cwater are the concentrations of a given IL in the n-octanol and water phases, respectively. The logPow value of DMSO is from the reported literature [48]. Molecules 2018, 23, 2154 8 of 11

3.4. Synthesis of Warfarin Typically, a mixture composed of 4-HC (0.5 mmol), BA (1.0 mmol), IL (0.5 g), lipase (30 mg), and water (0.1 mL) was kept stirring for 168 h at 50 ◦C. After the reaction, the concentration of warfarin in the prepared product was measured by the HPLC method. The product pre-processing procedure was as follows: at the end of the reaction, the product mixture was diluted to 20 mL with ethanol, ﬁltrated to remove lipase, and then the resulting ethanol solution was further diluted 10 times with ethanol before HPLC measurements. The percent yield is deﬁned as the ratio of the actual amount of warfarin to the theoretical amount.

3.5. Measurements of Fourier Transform Infrared (FT-IR) Spectra The FT-IR spectra were measured by the KBr pressed disc method on a Bruker V70 FT-IR spectrophotometer (Bruker Optic GmbH, Ettlingen, Germany).

3.6. Measurements of the Speciﬁc Rotation of the Synthesized Warfarin The measurements of the speciﬁc rotation of the synthesized warfarin were performed on a SGW-1 automatic polarimeter (Shanghai instrument physical optics instrument Co., Ltd., Shanghai, China). The temperature was 25 ◦C and the concentration of warfarin was 25 g L–1 (solvent, ethanol). All the above experiments were conducted in triplicate and the data presented in this work are averages of the obtained values.

4. Conclusions In this work, a series of ILs were used as the reaction media of the lipase-catalyzed Michael addition of 4-HC to BA. Compared with the conventional solvent DMSO and the dialkylimidazolium-based ILs, the hydroxyl-functionalized IL [C1C3OHim]NTf2 is a more ideal reaction medium because the hydroxyl group in the IL greatly promotes the lipase activity due to its H2O-mimicking property and hydrogen-bonding functionality. The lipase activity decreases with an increasing alkyl chain length of the IL cation, which can be ascribed to the direct interactions of the IL cations with the active sites of lipase and the non-polar domain formation of the IL cations at the active site entrance of lipase. After the end of the reaction, lipase and [C1C3OHim]NTf2 could be recovered and reused no less than ﬁve times without a decrease of the catalytic performance of lipase.

Supplementary Materials: The following are available online, Figure S1: UV-Vis absorption spectra of warfarin, 4-HC, BA, and the reaction mixture before and after the reaction. Author Contributions: Y.F. conceptualization and writing-original draft. D.C. investigation. X.W. data curation. L.Y. writing-review and editing. Funding: This research was funded by the National Natural Science Foundation of China (21307028), the Foundation of Henan Province (182102310728), and the Program for Innovative Research Team of Henan Polytechnic University (T2018-3). Conﬂicts of Interest: The authors declare no conﬂict of interest.

References

1. Ghaffari-Moghaddam, M.; Eslahi, H.; Aydin, Y.A.; Saloglu, D. Enzymatic processes in alternative reaction media: A mini review. J. Biol. Meth. 2015, 2, e25. [CrossRef] 2. Solano, D.M.; Hoyos, P.; Hernáiz, M.J.; Alcántara, A.R.; Sánchez-Montero, J.M. Industrial biotransformations in the synthesis of building blocks leading to enantiopure drugs. Bioresour. Technol. 2012, 115, 196–207. [CrossRef][PubMed] 3. Yuan, Y.; Yang, L.; Liu, S.; Yang, J.; Zhang, H.; Yan, J.; Hu, X. Enzyme-catalyzed Michael addition for the synthesis of warfarin and its determination via ﬂuorescence quenching of L-tryptophan. Spectrochim. Acta A 2017, 176, 183–188. [CrossRef][PubMed] Molecules 2018, 23, 2154 9 of 11

4. Steunenberg, P.; Sijm, M.; Zuilhof, H.; Sanders, J.P.M.; Scott, E.L.; Franssen, M.C.R. Lipase-catalyzed aza-Michael reaction on acrylate derivatives. J. Org. Chem. 2013, 78, 3802–3813. [CrossRef][PubMed] 5. Sano, K.; Saito, S.; Hirose, Y.; Kohari, Y.; Nakano, H.; Seki, C.; Tokiwa, M.; Takeshita, M.; Uwai, K. Development of a novel method for warfarin synthesis via lipase-catalyzed stereoselective Michael reaction. Heterocycles 2013, 87, 1269–1278. [CrossRef] 6. Stergiou, P.Y.; Foukis, A.; Filippou, M.; Koukouritaki, M.; Parapouli, M.; Theodorou, L.G.; Hatziloukas, E.; Afendra, A.; Pandey, A.; Papamichael, E.M. Advances in lipase-catalyzed esterification reactions. Biotechnol. Adv. 2013, 31, 1846–1859. [CrossRef][PubMed] 7. Sekiya, M.; Osuga, J.I.; Yahagi, N.; Okazaki, H.; Tamura, Y.; Igarashi, M.; Takase, S.; Harada, K.; Okazaki, S.; Iizuka, Y.; et al. Hormone-sensitive lipase is involved in hepatic cholesteryl ester hydrolysis. J. Lipid Res. 2008, 49, 1829–1838. [CrossRef][PubMed] 8. Canet, A.; Bonet-Ragel, K.; Benaiges, M.D.; Valero, F. Lipase-catalysed transesterification: Viewpoint of the mechanism and influence of free fatty acids. Biomass Bioenergy 2016, 85, 94–99. [CrossRef] 9. Danziger, J. Vitamin K-dependent proteins, warfarin, and vascular calcification. Clin. J. Am. Soc. Nephrol. 2008, 3, 1504–1510. [CrossRef][PubMed] 10. Anderes, E.; Nand, S. Commonly used drugs in hematologic disorders, Neurologic Aspects of Systemic Disease Part II. In Handbook of Clinical Neurology; Elsevier: Amsterdam, The Netherlands, 2014; pp. 1125–1139. 11. Xie, B.H.; Guan, Z.; He, Y.H. Promiscuous enzyme-catalyzed Michael addition: Synthesis of warfarin and derivatives. J. Chem. Technol. Biotechnol. 2012, 87, 1709–1714. [CrossRef] 12. Galvao, J.; Davis, B.; Tilley, M.; Normando, E.; Duchen, M.R.; Cordeiro, M.F. Unexpected low-dose toxicity of the universal solvent DMSO. FASEB J. 2014, 28, 1317–1330. [CrossRef][PubMed] 13. Raut, D.G.; Sundman, O.; Su, W.; Virtanen, P.; Sugano, Y.; Kordas, K.; Mikkola, J.P. A morpholinium ionic liquid for cellulose dissolution. Carbohydr. Polym. 2015, 130, 18–25. [CrossRef][PubMed] 14. Vijaykumar, B.V.D.; Premkumar, B.; Jang, K.; Choi, B.I.; Falck, J.R.; Sheldrake, G.N.; Shin, D.S. Environmentally benign perfluorooctanesulfonate alternatives using a Zn/CuI mediated Michael-type addition in imidazolium ionic liquids. Green Chem. 2014, 16, 2406–2410. [CrossRef] 15. Schutt, T.C.; Hegde, G.A.; Bharadwaj, V.S.; Johns, A.J.; Maupin, C.M. Impact of water-dilution on the solvation properties of the ionic liquid 1-methyltriethoxy-3-ethylimidazolium acetate for model biomass molecules. J. Phys. Chem. B 2017, 121, 843–853. [CrossRef][PubMed] 16. Itoh, T. Ionic liquids as tool to improve enzymatic organic synthesis. Chem. Rev. 2017, 117, 10567–10607. [CrossRef][PubMed] 17. Lozano, P.; Bernal, J.M.; Garcia-Verdugo, E.; Sanchez-Gomez, G.; Vaultier, M.; Burguete, M.I.; Luis, S.V. Sponge-like ionic liquids: A new platform for green biocatalytic chemical processes. Green Chem. 2015, 17, 3706–3717. [CrossRef] 18. Xu, P.; Zheng, G.W.; Du, P.X.; Zong, M.H.; Lou, W.Y. Whole-cell biocatalytic processes with ionic liquids. ACS Sustain. Chem. Eng. 2016, 4, 371–386. [CrossRef] 19. Burney, P.R.; Nordwald, E.M.; Hickman, K.; Kaar, J.L.; Pfaendtner, J. Molecular dynamics investigation of the ionic liquid/enzyme interface: Application to engineering enzyme surface charge. Proteins Struct. Funct. Bioinform. 2015, 83, 670–680. [CrossRef][PubMed] 20. Shi, Y.G.; Wu, Y.; Lu, X.Y.; Ren, Y.P.; Wang, Q.; Zhu, C.M.; Yu, D.; Wang, H. Lipase-catalyzed esterification of ferulic acid with lauryl alcohol in ionic liquids and antibacterial properties in vitro against three food-related bacteria. Food Chem. 2017, 220, 249–256. [CrossRef][PubMed] 21. Xin, J.Y.; Zhao, Y.J.; Shi, Y.G.; Xia, C.G.; Li, S.B. Lipase-catalyzed naproxen methyl ester hydrolysis in water-saturated ionic liquid: Significantly enhanced enantioselectivity and stability. World J. Microb. Biotechnol. 2005, 21, 193–199. [CrossRef] 22. Su, F.; Peng, C.; Li, G.L.; Xu, L.; Yan, Y.J. Biodiesel production from woody oil catalyzed by Candida rugosa lipase in ionic liquid. Renew. Energy 2016, 90, 329–335. [CrossRef] 23. Liu, Y.; Chen, D.; Yan, Y.; Peng, C.; Xu, L. Biodiesel synthesis and conformation of lipase from Burkholderia cepacia in room temperature ionic liquids and organic solvents. Bioresour. Technol. 2011, 102, 10414–10418. [CrossRef][PubMed] 0 24. Fischer, F.; Happe, M.; Emery, J.; Fornage, A.; Schütz, R. Enzymatic synthesis of 6- and 6 -O-linoleyl-α-D- maltose: From solvent-free to binary ionic liquid reaction media. J. Mol. Catal. B 2013, 90, 98–106. [CrossRef] Molecules 2018, 23, 2154 10 of 11

25. Yang, Z.; Guo, Z.; Xu, X. Ionic liquid-assisted solubilization for improved enzymatic esterification of phenolic acids. J. Am. Oil Chem. Soc. 2012, 89, 1049–1055. [CrossRef] 26. Freire, M.G.; Neves, C.M.; Marrucho, I.M.; Coutinho, J.A.; Fernandes, A.M. Hydrolysis of tetrafluoroborate and hexafluorophosphate counter ions in imidazolium-based ionic liquids. J. Phys. Chem. A 2010, 114, 3744–3749. [CrossRef][PubMed] 27. Sowmiah, S.; Srinivasadesikan, V.; Tseng, M.C.; Chu, Y.H. On the chemical stabilities of ionic liquids. Molecules 2009, 14, 3780–3813. [CrossRef][PubMed] 28. Somers, A.E.; Howlett, P.C.; MacFarlane, D.R.; Forsyth, M. A review of ionic liquid lubricants. Lubricants 2013, 1, 3–21. [CrossRef] 29. Dong, X.; Fan, Y.; Yang, P.; Kong, J.; Li, D.; Miao, J.; Hua, S.; Hu, C. Ultraviolet-visible (UV-Vis) and fluorescence spectroscopic investigation of the interactions of ionic liquids and catalase. Appl. Spectrosc. 2016, 70, 1851–1860. [CrossRef][PubMed] 30. Papadopoulou, A.A.; Tzani, A.; Alivertis, D.; Katsoura, M.H.; Polydera, A.C.; Detsi, A.; Stamatis, H. Hydroxyl ammonium ionic liquids as media for biocatalytic oxidations. Green Chem. 2016, 18, 1147–1158. [CrossRef] 31. Hernández-Fernández, F.J.; de los Ríos, A.P.; Tomás-Alonso, F.; Gómez, D.; Víllora, G. Stability of hydrolase enzymes in ionic liquids. Can. J. Chem. Eng. 2009, 87, 910–914. [CrossRef] 32. Zhao, H. Methods for stabilizing and activating enzymes in ionic liquids—A review. J. Chem. Technol. Biotechnol. 2010, 85, 891–907. [CrossRef] 33. Klähn, M.; Lim, G.S.; Seduraman, A.; Wu, P. On the different roles of anions and cations in the solvation of enzymes in ionic liquids. Phys. Chem. Chem. Phys. 2011, 13, 1649–1662. [CrossRef][PubMed] 34. Lai, J.Q.; Li, Z.; Lü, Y.H.; Yang, Z. Specific ion effects of ionic liquids on enzyme activity and stability. Green Chem. 2011, 13, 1860–1868. [CrossRef] 35. Zhao, H.; Baker, G.A.; Holmes, S. New eutectic ionic liquids for lipase activation and enzymatic preparation of biodiesel. Org. Biomol. Chem. 2011, 9, 1908–1916. [CrossRef][PubMed] 36. Ventura, S.P.M.; Santos, L.D.F.; Saraiva, J.A.; Coutinho, J.A.P. Concentration effect of hydrophilic ionic liquids on the enzymatic activity of Candida antarctica lipase B. World J. Microb. Biotechnol. 2012, 28, 2303–2310. [CrossRef][PubMed] 37. Hegde, G.A.; Bharadwaj, V.S.; Kinsinger, C.L.; Schutt, T.C.; Pisierra, N.R.; Maupin, C.M. Impact of water dilution and cation tail length on ionic liquid characteristics: Interplay between polar and non-polar interactions. J. Chem. Phys. 2016, 145, 064504. [CrossRef] 38. Zhang, H.; Li, H.; Pan, H.; Liu, X.; Yang, K.; Huang, S.; Yang, S. Efficient production of biodiesel with promising fuel properties from Koelreuteria integrifoliola oil using a magnetically recyclable acidic ionic liquid. Energy Convers. Manag. 2017, 138, 45–53. [CrossRef] 39. Yang, J.; Feng, Y.; Zeng, T.; Guo, X.; Li, L.; Hong, R.; Qiu, T. Synthesis of biodiesel via transesterification of tung oil catalyzed by new Brönsted acidic ionic liquid. Chem. Eng. Res. Des. 2017, 117, 584–592. [CrossRef] 40. Zhang, K.P.; Lai, J.Q.; Huang, Z.L.; Yang, Z. Penicillium expansum lipase-catalyzed production of biodiesel in ionic liquids. Bioresour. Technol. 2011, 102, 2767–2772. [CrossRef][PubMed] 41. Ribeiro, B.D.; Santos, A.G.; Marrucho, I.M. Biocatalysis in ionic liquids. In White Biotechnology for Sustainable Chemistry; Royal Society of Chemistry: Cambridge, UK, 2015. 42. Aghababaie, M.; Beheshti, M.; Razmjou, A.; Bordbar, A.K. Enzymatic biodiesel production from crude Eruca sativa oil using Candida rugosa lipase in a solvent-free system using response surface methodology. Biofuels 2017, 14, 1–7. [CrossRef] 43. Bhonoah, Y. Warfarin: Rat Poison or Wonder Drug? Available online: http://www.ch.ic.ac.uk/local/ projects/bhonoah/characterisation.html (accessed on 17 August 2018). 44. Takahashi, H.; Echizen, H. Pharmacogenetics of warfarin elimination and its clinical implications. Clin. Pharmacokinet. 2001, 40, 587–603. [CrossRef][PubMed] 45. Fan, Y.; Dong, X.; Li, Y.; Zhong, Y.; Miao, J.; Hua, S.; Sun, Y. Extraction of L-tryptophan by hydroxyl-functionalized ionic liquids. Ind. Eng. Chem. Res. 2015, 54, 12966–12973. [CrossRef] 46. Lee, S.H.; Lee, S.B. Octanol/water partition coefficients of ionic liquids. J. Chem. Technol. Biotechnol. 2009, 84, 202–207. [CrossRef] 47. Ropel, L.; Belvèze, L.S.; Aki, S.N.V.K.; Stadtherr, M.A.; Brennecke, J.F. Octanol–water partition coefficients of imidazolium-based ionic liquids. Green Chem. 2005, 7, 83–90. [CrossRef] Molecules 2018, 23, 2154 11 of 11

48. Jamalzadeh, L.; Ghafoori, H.; Sariri, R.; Rabuti, H.; Nasirzade, J.; Hasani, H.; Aghamaali, M.R. Cytotoxic effects of some common organic solvents on MCF-7, RAW-264.7 and human umbilical vein endothelial cells. Avicenna J. Med. Biochem. 2016, 4, e33453. [CrossRef]

Sample Availability: Samples of the compounds are not available from the authors.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).