Study of the influence of pure ionic liquids on the lipase-catalyzed (trans)esterification of mannose based on their anion and cation nature Short title: Lipase-catalyzed (trans)esterification of mannose in pure ionic liquids Nadine GALONDE *a, Katherine NOTT a,b, Gaëtan RICHARD a,b, Antoine DEBUIGNE c, François NICKS a,b, Christine JERÔME c, Michel PAQUOT b, Jean-Paul WATHELET a (a) Department of General and Organic Chemistry, Gembloux Agro-Bio Tech, University of Liège, Passage des Déportés, 2, B – 5030 Gembloux, Belgium (b) Department of Biological Chemistry, Gembloux Agro-Bio Tech, University of Liège, Passage des Déportés, 2, B – 5030 Gembloux, Belgium (c) Center for Education and Research on Macromolecules,Chemistry Department, University of Liège, SartTilman, B6a, 4000 Liège, Belgium
*
[email protected], telephone: +32 81 622291
1
Abstract A screening of nine ionic liquids (ILs) has been carried out in order to study the influence of the anion's and cation's nature and structure on the synthesis of mannosyl myristate by (trans)esterification catalyzed by Novozym® 435. The best ILs in terms of yield (η) and initial rate (v 0) are those based on the TFO- anion. The 24h yield (24h-η) reached 64.9% in [Bmim][TFO] and 70.9% in [Bmpyrr][TFO] by transesterification while it reached 29.7% and 44.5% respectively in each IL by esterification. [Bmpyrr][TFO] based on the pyrrolidinium cation gave the best results although this cation has been rarely used for biocatalysis. This work has thus highlighted a cation that could be further studied in biocatalysis of glycosylated compounds. The study of the relationship between the structure of the ILs and the v0 and η for the biocatalysis of mannosyl myristate showed that the lipase effectiveness is influenced by the anions while the cations have an indirect influence on the interaction strength between Novozym® 435 and the anions. Keywords Acylation, biocatalysis, carbohydrates, green chemistry, ionic liquids, Novozym® 435, mannosyl myristate, sugar esters
1
Introduction
Fatty acid sugar esters are non-ionic surfactants produced from renewable resources. They are generally considered as odorless, tasteless, non-irritant, non-toxic and biodegradable. Therefore, they are particularly interesting for applications in the pharmaceutical, cosmetic and food industries in which safety for human health and the environment are essential [1, 2]. Most of these compounds are synthesized by chemical methods which present several disadvantages, one of which is the need of fastidious protection/deprotection steps to obtain a high degree of regioselectivity. Furthermore, these energy consuming routes that can lead to the production of undesirable and toxic by-products are often environmentally unfriendly [2, 3]. For the last 20 years, biocatalysis has been considered as an ecofriendly alternative due to many advantages such as the use of mild reaction conditions and high regio-(stereo-) selectivity [2, 4, 5]. However the choice of a suitable solvent for the enzymatic modification of sugars is still a major challenge. The solvent must be able to dissolve polar substrates such as sugars and non-polar ones such as fatty acids or their vinyl esters. Furthermore, the solvents must not deactivate the enzymes which may be the case for some hydrophilic organic solvents such as pyridine, dimethylsulfoxide and dimethylformamide which
2
are able to dissolve saccharides [5-7]. Moreover, by using such solvents, the green character and compatibility with human health of the biocatalytic approach can be discussed. Consequently, it is opportune to search for substitutes to organic solvents for the biocatalyzed modification of sugars. In this context, ionic liquids (ILs) may be good alternatives to organic solvents. This new type of solvents, also called “Room Temperature Ionic Liquids” (RTILs) because of their low melting point, are salt-like materials composed of a large cation combined with a smaller anion [8]. They are able to dissolve a large range of polar and non-polar compounds and present a high thermal stability and a low vapor pressure. Furthermore, they are considered as safe to handle since they are non-flammable and non-explosive [9, 10]. Also, many studies highlighted their capacity, compared to organic solvents, to improve regio-, stereo- and enantioselectivity of biocatalyzed reactions [11, 12]. Therefore, for the last decade, ILs have successfully drawn an increasing interest from researchers mindful to develop “greener” biocatalytic processes in sugar transformation especially in sugar esters synthesis [11-22]. In order to explore the great opportunity offered by ILs to improve the biocatalytic modification of carbohydrates, nine ILs were screened as solvent for the acylation of mannose. This sugar previously studied in biocatalysis [23] was chosen for the first time for biocatalysis in ILs since its derivatives could be of interest as anticancer drug carrier due to potential cell recognition (Prakash et al., 2010). Novozym ® 435 (immobilized lipase B from Candida antarctica) was used to catalyze mannose esterification with myristic acid and its transesterification with vinyl myristate (Figure 1). The influence of the nature and structure of the IL's anion and cation on the initial rate (v0) and 24h-yield (24h-η) of mannose (trans)esterification is discussed.
3
Figure 1: Lipase-catalyzed acylation of mannose with myristic acid (esterification) or vinyl myristate (transesterification) in pure ILs.
2
Materials and methods
2.1
Materials
The
ILs
1-butyl-3-methylimidazolium
hexafluorophosphate ([Bmim][TF2N]),
([Bmim][PF6]),
tetrafluoroborate
([Bmim][BF4]),
1-butyl-3-methylimidazolium
1-butyl-3-methylimidazolium
1-butyl-3-methylimidazolium
bis(trifluoromethanesulfonyl)imide
trifluoromethanesulfonate
([Bmim][TFO]),
1-ethyl-3-
methylimidazolium hexafluorophosphate ([Emim][PF6]), 1-hexyl-3-methylimidazolium hexafluorophosphate ([Hmim][PF6]),
1-butyl-3-methylpyrrolidinium
trifluoromethanesulfonate
([Bmpyrr][TFO]),
1-butyl-3-
methylpyrrolidinium bis(trifluoromethanesulfonyl)imide ([Bmpyrr][TF 2N]) and N-trimethyl-N-hexylammonium bis(trifluoromethanesulfonyl)imide ([TMHA][TF2N]) (99.5%) were purchased from Solvionic (Toulouse, France). D-Mannose (> 99%) and myristic acid (> 99%) were purchased from Sigma-Aldrich NV/SA (Bornem, Belgium). Vinyl myristate (> 99%, stabilized with MEHQ) was purchased from TCI Europe NV (Zwijndrecht, Belgium). The lipase B from Candida antartica immobilized on acrylic resin (Novozym® 435) was a generous gift from Novozymes A/S (Bagsvaerd, Denmark). The HPLC grade acetonitrile (ACN) and methanol (MeOH) were purchased from Scharlab SL (Spain).
4
2.2
General procedure for enzymatic acylation
0.05 mmoles of mannose and 0.30 mmoles of myristic acid (esterification) or vinyl myristate (transesterification) were mixed in 0.5 ml of IL in a screw cape tube, vigorously stirred at 600 rpm and thermostated at 60°C. The reaction started by adding 5% (w/v) of Novozym® 435. At a chosen time interval, the reaction was stopped by addition of 4 volumes of an ACN/MeOH mixture (50/50, v/v) followed by centrifugation (3000 rpm, 5min). The supernatant was recovered and filtered on a 0.22 µm pore size nylon membrane before RP-HPLC analysis.
2.3
HPLC analysis method for the determination of the product yield
The quantitative analyses were performed on an Agilent Technologies 1200 series HPLC equipped with an evaporative light scattering detector (ELSD). A Halo® fused-core RP-C18 column (75x4.6 mm, 2.6 µm) from Advanced Materials Technology was thermostated at 30°C. The elution was carried out thanks to a linear gradient of ACN/water (both containing 0.1% of formic acid): the ACN % increased from 60% to 100 % in 2 min at a flow rate of 1.5 ml/min. The ester was quantified by external calibration. Calibration curve was obtained with a series of mannosyl myristate solutions in a concentration range from 0.1 g/l to 10 g/l. The 24 hours yield (24h-η, expressed in percent) was determined as (mmol of sugar ester /mmol of default substrate) x 100. The initial rate (v0) expressed in g.l-1.h-1.mgenz-1 was also determined by HPLC-ELSD: v0 corresponds to the slope of the graph representing the ester concentration as a function of reaction time within the first five hours (or less if non linear).
2.4 1
Structural analysis of 6-O-tetradecanoyl-α/β-mannopyranose by NMR
H and 13C NMR spectra were recorded at 600 and 150 MHz respectively, at 25°C on a Varian spectrometer. The
chemical shifts are in parts per million downfield from TMS and are referenced by the residual solvent peak. Data are reported as follows: chemical shift, integration, multiplicity (s, singlet; d, doublet; m, multiplet; and bs, broad singlet), coupling constant (Hz) and attribution. 6-O-tetradecanoyl-D-mannopyranose (α and β anomers mixture) 1
H NMR (600 MHz, CD3OD): δ 0.92 (3Hα and 3Hβ, t, J = 7.0 Hz, H-14'), 1.27-1.39 (20Hα and 20Hβ, m, H-4' to
H-13'), 1.60-1.67 (2Hα and 2Hβ, m, H-3'), 2.28 (2Hβ, t, J = 7.4 Hz, H-2'), 2.36 (2Hα, t, J = 7.5 Hz, H-2'), 3.393.44 (1Hβ, m, H-5), 3.48 (1Hβ, dd, J3,4 = 9.4 Hz and J3,2 = 2.9 Hz, H-3), 3.57 (1Hβ, "t", J = 9.6 Hz, H-4), 3.64 (1Hα, "t", J = 9.5 Hz, H-4), 3.78 (1Hα, dd, J3,4 = 9.5 Hz and J3,2 = 3.3 Hz, H-3), 3.81 (1Hα, dd, J2,3 = 3.3 Hz and J2,1 = 1.3 Hz, H-2), 3.83 (1Hβ, d, J2,3 = 2.9 Hz, H-2), 3.91-3.96 (1Hα, m, H-5), 4.21 (1Hβ, dd, J6b,6a = 11.8 Hz and
5
J6b,5 = 6.7 Hz, H-6b), 4.23 (1Hα, dd, J6b,6a = 11.7 Hz and J6b,5 = 6.1 Hz, H-6b), 4.40 (1Hα, dd, J6a,6b = 11.7 Hz and J6a,5 = 1.9 Hz, H-6a), 4.44 (1Hβ, dd, J6a,6b = 11.8 Hz and J6a,5 = 1.9 Hz, H-6a), 4.76 (1Hβ, s, H-1), 5.07 (1Hα, d, J1,2 = 1.3 Hz, H-1) ppm. 13
C NMR (150 MHz, CD3OD): δ 14.4 (C-14'), 23.7 (C-3'), 26.0, 26.3, 30.3, 30.4, 30.5, 30.6, 30.7, 30.75, 30.76,
30.8, 33.1, 35.0, 65.1 (C-6β), 65.2 (C-6α), 68.5 (C-4β), 68.9 (C-4α), 71.7 (C-5α), 72.2 (C-3α), 72.8 (C-2α), 73.0 (C-2β), 75.3 (C-3β), 75.6 (C-5β), 95.7 (C-1β), 95.9 (C-1α), 175.65 (C=O) ppm. The 1H and 13C NMR spectra analysis of the 6-O-tetradecanoyl-D-mannopyranose showed an α/β anomer ratio of 75/25.
3
Results
Nine ILs were chosen (see structures in Table 1) and their efficiency compared to tert-BuOH for mannosyl myristate synthesis by (trans)esterification.
Table 1: Structures and abbreviations of the ILs chosen for the enzymatic acylation of mannose catalyzed by Novozym® 435.
The time courses of the production of mannosyl myristate by (trans)esterification in the pure ILs and tert-BuOH are shown in Figure 2.
6
Esterification
A 20
1
Concentration (g/l)
15
2 3
10
5
4-9 0 0
5
10
15
1
[Bmpyrr][TFO]
2
[Bmim][TFO]
3
[Bmim][BF4]
4
[Bmim][TF2N]
5
[Bmim][PF6]
6
[Bmpyrr][TF2N]
7
[TMHA][TF2N]
8
[Hmim][PF6]
9
[Emim][PF6]
1
[Bmpyrr][TFO]
2
[Bmim][TFO]
3
[Bmim][BF4]
4
[Bmim][TF2N]
5
[Bmpyrr][TF2N]
6
[Bmim][PF6]
7
[Hmim][PF6]
8
[TMHA][TF2N]
9
[Emim][PF6]
20
Reaction time (h)
B
Transesterification
35
Concentration (g/l)
30
1 2
25
20
15
3 10
4 5
5-9 0 0
5
10
15
20
Reaction time (h)
Figure 2: Time courses for the lipase-catalyzed (trans)esterification of mannose in pure ILs. A: Esterification, B: Transesterification.
7
Initial rates (v0) and 24h yields (24h-η) of the reactions are presented in Table 2. Esterification 24h-η Initial rate, v 0 Ionic liquids
Imidazolium
(g.l-1.h-1.g enz-1)
[Bmim][TFO] [Bmim][BF4]
29.7 28.2
± 0.4 ± 0.5
[Bmim][TF2N]
5.5
± 0.6
32.4 13.3
[Bmim][PF6]
5.1
± 0.4
13.0
[Hmim][PF6]
3.2
± 0.1
2.9 44.5 5.1
[Emim][PF6] [Bmpyrr][TFO] Pyrrolidinium [Bmpyrr][TF2N] Ammonium
Standard Error (SE)
(%)
[TMHA][TF2N]
Transesterification 24h-η Initial rate, v 0 Standard Error (SE)
(%)
(g.l-1.h-1.g enz-1)
64.9 34.3
± 1.0 ± 0.8
214.8 33.2
20.5
± 0.5
29.0
5.7
± 0.3
13.6
10.8
5.6
± 0.2
12.3
± 0.2 ± 0.2 ± 0.1
8.0 77.8 18.3
3 70.9 8.4
± 0.2 ± 0.5 ± 0.8
8.0 148.0 17.4
3.6
± 0.1
8.0
3.7
± 0.1
12.5
50.4
± 1.9
406.2
58.0
± 1.7
575.7
99.3
Organic solvent Tert-BuOH
Table 2: 24h yield (24h-η) and initial rate (v0) of the (trans)esterification of mannose catalyzed by Novozym® 435 in pure ILs.
4 4.1.1
Discussion Synthesis of mannosyl myristate by esterification
The v0 vary from 99.3 to 8.0 g.l-1.h-1.mgenz-1 and decrease in the following order: [Bmim][TFO] > [Bmpyrr][TFO] > [Bmim][BF4] > [Bmpyrr][TF2N] > [Bmim][PF6] ≈ [Bmim][TF2N] > [Hmim][PF6] > [TMHA][TF2N] ≈ [Emim][PF6]. The 24h-η vary from 44.5% to 3.2% and diminish in the following order: [Bmpyrr][TFO] > [Bmim][TFO] > [Bmim][BF 4] > [Bmim][TF2N] > [Bmim][PF6] > [Bmpyrr][TF2N] > [TMHA][TF2N] > [Hmim][PF6] > [Emim][PF6]. [Bmpyrr][TFO] gives the best 24h-η, 44.5% while only 29.7% is reached in [Bmim][TFO] despite its higher v0 of 99.3 g.l-1.h-1.mgenz-1 compared to the 77.8 g.l-1.h-1.mgenz-1 obtained in [Bmpyrr][TFO]. The 24h-η in [Bmpyrr][TFO] is close to the one measured in tert-BuOH (50.4%) although the v0 in this IL is 5 times lower than in tert-BuOH due to a higher enzymatic reactivity in this organic solvent. These results are particularly interesting if we consider that pyrrolidinium based ILs have not been explored in biocatalysis of sugar derivatives until now. 4.1.2
Synthesis of mannosyl myristate by transesterification
For the transesterification of mannose by vinyl myristate in the nine pure ILs tested, the v 0 vary from 214.8 to 8.0 g.l-1.h-1.mgenz-1 and decrease according to the following order: [Bmim][TFO] > [Bmpyrr][TFO] > [Bmim][BF4] > [Bmim][TF2N] > [Bmpyrr][TF2N] > [Bmim][PF6] > [TMHA][TF2N] ≈ [Hmim][PF6] > [Emim][PF6]. The 24h-η follow the decreasing order: [Bmpyrr][TFO] > [Bmim][TFO] > [Bmim][BF4] > [Bmim][TF2N] > [Bmpyrr][TF2N] > [Bmim][PF6] > [Hmim][PF6] > [TMHA][TF2N] > [Emim][PF6]. The ILs
8
based on TFO- anion give the best results as already observed for the esterification. Once again, [Bmpyrr][TFO] gives very good results. The 24h-η reached 70.9% in [Bmpyrr][TFO] and 64.9% in [Bmim][TFO] whereas the v 0 attained in these IL was respectively 148.0 g.l-1.h-1.mgenz-1 and 214.8 g.l-1.h-1.mgenz-1. The v0 obtained in tertBuOH is approximately three times higher than with the best IL ([Bmim][TFO]). However, the 24h-η reached in this IL and in [Bmpyrr][TFO] are superior to the one obtained in tert-BuOH. The use of these TFO- based ILs instead of tert-BuOH is consequently more interesting for transesterification than for esterification for which no improvement was observed compared to tert-BuOH. For half of the ILs tested ([Bmpyrr][TFO], [Bmim][TFO], [Bmim][BF 4], [Bmim][TF2N]), the transesterification v0 is greater than that obtained with the same IL for the esterification. For the other half ([Bmpyrr][TF2N], [Bmim][PF6], [Hmim][PF6], [TMHA][TF2N], [Emim][PF6]), the v0 are similar for the two synthesis routes. Furthermore, the 24h-η for 7 ILs are significantly better than those obtained by esterification. For [Bmim][PF6] and [Emim][PF6] only, the 24h-η are similar for the 2 synthesis routes. In tert-BuOH, as previously observed by Nott et al (2012) [23], the v0 and η are also higher for transesterification than for esterification (see results compiled in Table 2). Better results are observed for transesterification because alcohols (vinyl alcohols in this case) are better leaving groups than water released by the esterification route. In addition, the transesterification with vinyl esters produces vinyl alcohol which converts spontaneously into acetaldehyde that can evaporate easily from the medium (due to a boiling point lower than the reaction temperature). This drives the reaction towards the production of the desired ester and limits the reverse reaction. In the case of esterification, the water produced leads to reverse hydrolysis of the ester. Also since the solubility of fatty acids are lower in ILs compared to organic solvents, the v0 will consequently be less important [18]. However, although better results are obtained by transesterification with vinyl myristate, the esterification with myristic acid is not to be overlooked as it produces a harmless by-product. The fatty acid is less expensive than its vinyl ester derivative [5, 24].
4.2 4.2.1
Influence of the IL’s ion nature on the v0 and 24h-η Influence of the IL's anion nature
As the activity of lipases in ILs is known to be highly dependent on their anion of the ILs [19, 25], the influence of IL's anion nature on mannose acylation catalyzed by Novozym 435 was studied by varying the anions used with [Bmim] or [Bmpyrr] based ILs. For the former (see Table 2), the 24h-η of mannose acylation by myristic acid or vinyl myristate follow this order: [Bmim][TFO] > [Bmim][BF 4] > [Bmim][TF2N] > [Bmim][PF6].
9
For [Bmpyrr] based ILs, better results were also obtained with TFO - compared to TF2N- (Table 2). For both cations, Novozym® 435 gave the best v0 and 24h-η in hydrophilic ILs such as [Bmpyrr][TFO], [Bmim][TFO] and [Bmim][BF4] rather than in hydrophobic ones such as [Bmim][PF6], [Bmim][TF2N] and [Bmpyrr][TF2N]. These results are in accordance with those obtained for the Novozym® 435 catalyzed transesterification of supersaturated glucose solutions with vinyl laurate or for their esterification with lauric acid in [Bmim] based ILs [16, 18]. Lee et al. (2008) [16] obtained the best results for the transesterification with the water miscible ILs (conversion in [Bmim][TFO] higher than in [Bmim][BF 4]) than in the water immiscible ones ([Bmim][TF 2N] and [Bmim][PF6]). Ha et al. (2010) [18] reached higher esterification yields in [Bmim][TFO] rather than in [Bmim][TF2N]. The same trend was observed for the esterification of methyl- -D glucopyranoside for which a better conversion was obtained in [Bmim][BF4] than in [Bmim][PF6] [19]. However, the opposite tendency has also been observed for Novozym 435 catalyzed (trans)esterifications [25, 26]. For example, the initial reaction rates for the transesterification of benzyl alcohol with vinyl acetate catalyzed by Novozym ® 435 in [Bmim] based ILs follow the decreasing order: [TF2N] > [PF6] > [TFO] > [BF4] [25]. These opposed behaviors can probably be attributed to the polarity of the acyl acceptors. Polar substrates such as mannose in this study and glucose or methyl- -D glucopyranoside in those cited above have a better solubility in hydrophilic ILs than in hydrophobic one, unlike benzyl alcohol. It could be expected that even better results may be obtained in more hydrophilics ILs such as those containing Cl- or dicyanamide anions as their hydrogen bonding abilities render them suitable for dissolution of carbohydrates [9, 17]. But it is not the case since enzyme activity in ILs depends on many factors [19]. Anions which can form strong H bonds, favorable for sugar dissolution, may dissociate the intramolecular H bonds of proteins thus modifying the 3D structure which is unfavorable for the enzyme activity [27, 28]. For ILs, it has been shown by solvatochromic comparison methods that the H bond acceptor ability depends mainly on the anion nature, the cation has only a secondary influence [29]. Molecular dynamics simulations of CALB in various ILs have also shown that the strength of the enzyme-IL interaction is principally determined by the anion which interacts with CAL-B via coulomb interactions and H bonds The cation interacts via weak van der Waals forces. The strong interactions between the anion and the lipase are unfavorable for catalysis as they can lead to enzyme denaturation [30]. FT-IR spectra in the amide I region have shown that the interactions with the ILs do indeed induce conformational changes that result in deactivation of CALB [27]. This probably explains why CALB 's stability in various ILs is strongly influenced by the anion [25, 26, 28]. The selection of the appropriate IL for biotransformation of carbohydrates necessitates a compromise between high substrate dissolution and a maintained lipase activity as function of the anion structure. On one hand, the
10
anions are chosen according to their hydrophilicity which influences their ability to dissolve high concentrations of carbohydrates but on the other hand, the anions should not be too hydrophilic in order to avoid the formation of numerous H-bonds with the enzyme, interfering with its internal hydrogen bonds [28]. Therefore, ILs based on the TFO- anion, [Bmpyrr][TFO], [Bmim][TFO] seem to be a good choice in this present study. 4.2.2 4.2.2.1
Influence of the IL's cation Influence of the IL’s cation nature
Despite the decisive influence of the ILs anions on CALB's activity and stability [19, 30], the influence of their cations is not negligible [19, 30]. The van der Waals interactions between the cation and the enzyme promote the diffusion of the cations into the lipase’s active site whilst the diffusion of the anion is not observed, as this site is negatively charged and its entrance is composed of hydrophobic residues [30]. Furthermore, simulation studies showed that cations indirectly influence the interaction strength between Novozym® 435 and anions: smaller cations lead to stronger Novozym® 435 anion interactions due to the increased number of anions at the enzyme vicinity [30]. To study the influence of cations on the v0 and 24h-η of the synthesis of mannosyl myristate, imidazolium, pyrrolidinium or ammonium based ILs associated with TFO- and TF2N- anions were selected. These anions are the most currently used after BF4- and PF6- in the biocatalysis of sugar derivatives and they tend to be less degraded than BF4- and PF6- which can release HF in the medium by hydrolysis which may deactivate the enzyme [31, 32]. For esterification, Bmim+ or Bmpyrr+ combined with TFO- anion lead to very different results for the synthesis of mannosyl myristate. The 24h-η is 1.5 timeshigher in [Bmpyrr][TFO] than in [Bmim][TFO] despite a lower v0. However, no significant variations are observed when Bmim +, Bmpyrr+ or TMHA+ are combined with TF2N-. In every case, the v0 and 24h-η are very low. For transesterification in the TFO - based ILs, the 24h-η are relatively close (70.9% for [Bmpyrr][TFO] and 64.9% for [Bmim][TFO]) but the v 0 is higher for [Bmim][TFO] (Table 2). For the TF2N- based ILs, no significant effect of the cation is observed on the esterification. But important differences in the transesterification's v0 and 24h-η are observed. The v0 is 29.0 g.l-1.h-1.mgenz-1 for [Bmim][TF2N]. This is 1.6 and 2.3 times higher than the v0 found, respectively in [Bmpyrr][TF2N] and [TMHA][TF2N] (Table 2). The 24h-η reached 20.5% in [Bmim][TF2N], 8.4% in [Bmpyrr][TF2N] and 3.7% in [TMHA][TF2N]. The low v0 and 24h-η observed in [THMA][TF2N] for transesterification (and also
11
esterification) are probably due to its high melting point (59°C) which is very close to the temperature chosen for the reaction (60°C). This makes the solubilization of the substrates difficult. This IL is thus not very suitable for biocatalysis of sugar derivatives under mild conditions. For the TFO- based ILs, Bmpyrr+ cation resulted in the highest 24h-η for both esterification and transesterification of mannose, and Bmim+ the highest v0. For the TF2Nbased ILs, no significant influence of the cation is observed for esterification but for transesterification Bmim + gives better results in terms of v0 and 24h-η. Finally, since Bmpyrr+ cation combined with TFO- anion gave the best 24h-η and v0, this IL appears to be the most appropriate for the synthesis of mannosyl esters. 4.2.2.2
Influence of the alkyl chain length on the imidazolium cation
Since imidazolium based ILs with the PF6- anion are commercially available with a large range of alkyl chain length, these ILs were thus selected to study the influence of the alkyl chain length (from 2 to 6 carbon atoms, see table 1) of the imidazolium cation on the mannosyl myristate synthesis. The v0 and 24h-η for both esterification and transesterification follow this decreasing order: [Bmim][PF 6] > [Hmim][PF6] > [Emim][PF6] (Table 2). The best results are thus obtained for the intermediate chain length. For the esterification, catalyzed by the same enzyme, of glucose saturated solutions with lauric acid in [TFO] based ILs, higher rates and conversions were obtained in [Emim] based IL than in [Bmim] based IL. This can be explained by the solubility difference of the sugar in each IL: 27.8 g/l in [Emim] and 18.1 g/l in [Bmim] at 60°C. The most hydrophilic IL (with the shortest chain on the cation) favors the sugar solubilization. However, in supersaturated glucose solutions, at a sugar concentration of 40 g/l, better results were obtained in [Bmim] than in [Emim] based IL. At the same sugar concentration, the most hydrophobic IL (with the cation having the longest chain) gives the best results as this characteristic is favorable for the lipase activity [17]. A short cation alkyl chain is favorable for saccharide solubilization [27] and thus the displacement of the equilibrium towards the ester formation but, on the other hand, a long chain is favorable for the enzyme activity. Furthermore, a longer chain increases the ILs viscosity and thus this may results in mass transfer limitation which limit the access of the substrate to the active site of the enzyme and thus its activity [25, 31]. Recent studies showed that the alkyl chain of the cation enables the adsorption of the IL at the entrance of the active site of CALB which is non polar. This can limit the substrate accessibility to the active site and thus leads to a lower enzymatic activity [30]. The global effect of cation's chain length on the v0 and η for sugar ester synthesis catalyzed results from a
12
compromise between these opposing effects. This can explain that, in this study, the best results were obtained for the intermediate chain length tested. It is also interesting to note that the effect of the chain length on the cation on the conversion depends on the lipase used. For the kinetic resolution of rac-1-phenylethanol with vinyl acetate by in [Bmim][BF4] [Hmim][BF4] and [Omim][BF4] the conversion increases with the alkyl chain length of the imidazolium with Candida antarctica lipase B and Candida antarctica lipase A. But with Candida rugosa lipase and Pseudomonas sp., the best conversions are observed for the shortest alkyl chain length [33].
5
Conclusion
This study of the mannose acylation with myristic acid or vinyl myristate catalyzed by Novozym® 435 in a large range of pure ILs has given evidence of their usefulness as efficient alternatives to organic solvents. As observed generally in organic solvents, better yields of mannosyl myristate were reached by transesterification rather than by esterification in most of the ILs tested. Furthermore, the ILs which gave the best results in this study are the TFO- based ones: [Bmim][TFO] and [Bmpyrr][TFO]. The latter has been very rarely exploited in biocatalysis with the exception of lipase-catalyzed polytransesterification of divinyl adipate and 1,4-butanediol or the transesterification of methyl methacrylate with 2-ethylhexanol [34]. In this study it gives the best mannosyl myristate 24h-η by both esterification and transesterification. [Bmim][TFO]'ssuitability for carbohydrate biotransformation has already been highlighted in previous studies [16-18, 24]. After comparing the v0 and 24hη obtained in the various ILs as function of their anion, their cation and the alkyl chain length of the imidazolium cation, the strong influence of these structural features of the ILs on the mannose (trans)esterification efficiency has been highlighted. This important effect has several origins such as H-bonds, Coulomb strengths or non-polar interactions occurring between the ILs constituents and the enzyme (at its active site or not) or between these constituents and the substrates. However, even if some studies exist on the interactions between lipase and ILs [30, 35, 36], more investigations focused on the understanding of the influence of anions and cations on the enzymatic activity and stability need to be undertaken in future, in order to design ILs most suitable for biotransformation. Finally, this study provides the evidence that pyrrolidium cations combined with the proper anion may be suitable for biocatalysis as it is the case for imidazolium cations. Acknowledgements
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This work was funded through the ARC grant ‘Superzym’, financed by the French Community of Belgium which is gratefully acknowledged for its financial support. Antoine Debuigne thanks the Fonds National de la Recherche Scientifique (FNRS) from Belgium for his Research Associate position. References
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