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Improvements in the enzymatic synthesis of phosphatidylserine employing ionic liquids
Journal of Molecular Catalysis B: Enzymatic 84 (2012) 132–135
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb
Improvements in the enzymatic synthesis of phosphatidylserine employing ionic liquids Paola D’Arrigo a,b,∗, Lorenzo Cerioli a, Cinzia Chiappe c, Walter Panzeri d, Davide Tessaro a,b, Andrea Mele a,b a
Dipartimento di Chimica, Materiali ed Ingegneria Chimica “G. Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy The Protein Factory, Politecnico di Milano, Università dell’Insubria, ICRM Istituto di Chimica del Riconoscimento Molecolare Consiglio Nazionale delle Ricerche, via Mancinelli 7, 20131 Milano, Italy c Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via Risorgimento 25, 56126, Pisa, Italy d CNR ICRM, Istituto di Chimica del Riconoscimento Molecolare, via Mancinelli 7, 20131 Milano, Italy b
a r t i c l e
i n f o
Article history: Available online 15 April 2012 Keywords: Phospholipase D Transphosphatidylation Ionic liquid Phosphatidylserine
a b s t r a c t The aim of this work was to investigate innovative reaction media such as ionic liquids for enzymatic phospholipids transformations in order to get a higher selectivity in the enzymatic transphosphatidylation reactions of natural phosphatidylcholine catalyzed by phospholipase D (PLD) from Streptomyces PMF. The stability and the activity of the PLD in the ionic liquid [BMIM][PF6 ] were tested. This ionic liquid used as a cosolvent in the synthesis of phosphatidylserine lead surprisingly to the almost quantitative suppression of unwanted hydrolytic side reaction, thus providing a great improvement in the final product purification. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Phospholipids (PLs), essential constituents of cellular membranes, have been thoroughly investigated and are the topic of many areas of biomedical research [1,2]. Because of their amphiphilic properties, natural and synthetic PLs have attracted considerable interest for the multiple scientific and practical applications [3,4] in pharmaceutical, cosmetic and food industry [5,6]. Their involvement in the formation of natural membranes and ability for self-organisation in water has stimulated a large body of studies on their physical properties [7]. Recent advances in the construction of artificial cell membranes with specific biological functions require tailor-made glycerol-derived lipids [8]. Therefore development of new synthetic methods for the preparation of biologically active phospholipid derivatives is a challenging problem of membrane-chemistry and biochemistry today [9,10]. Natural PLs can be obtained as mixtures of products with different acyl chains and polar heads from manly soy beans, many vegetable oils, egg yolk, biomass. Partial purification of PLs is possible by solvent partitioning and, eventually, chromatography. Total synthesis of PLs is complex and expensive. Nevertheless, all sites
∗ Corresponding author at: Dipartimento di Chimica, Materiali ed Ingegneria Chimica “G. Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy. Tel.: +39 02 23993075; fax: +39 02 23993180. E-mail address: paola.d’[email protected] (P. D’Arrigo). 1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2012.04.011
of PLs can be selectively modified with biocatalytic methods using phospholipases [11–14]. Our attention has mainly been focused on phospholipase D (PLD, EC 3.1.4.4), an enzyme used to modify the polar head of PLs molecules [15,16]. PLD catalyses in vivo the hydrolysis of PLs to phosphatidic acid (PA) but, in presence of an appropriate acceptor (an alcohol), PLD also catalyses the in vitro transphosphatidylation of the molecule polar head in which the exchange of alcohol head groups takes place (see Fig. 1). Polar head modified phospholipids find applications as nutraceuticals, in liposome technology and cosmetics. They can be prepared starting from phosphatidylcholine (PC) from natural sources via PLD catalyzed transphosphatidylation reaction. The reactions were usually carried out in water–organic solvent mixtures and the extent of the competing hydrolytic reaction depends on the source of the enzyme and the nature of the nucleophile [17–20]. In particular, PLD from Streptomyces PMF was directly applied to the transformation of the most abundant natural PL, PC from soy beans into other minor natural products (PX), namely phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) and also in a great number of unnatural compounds [21–23]. The presence of water in the reaction medium always causes the formation of the PA as a by-product of the transesterification, lowering reaction yields and forcing to further purification steps. The purification of this class of compounds is not straightforward and necessitates different chromatographic and selective
P. D’Arrigo et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 132–135
RCOO R'COO
H
O
O P O
O
+
N
PLD/X-OH
133
O
O RCOO R'COO
H
O
P O
OX
+
RCOO R'COO
H
O
P O
O
PA
PX PC XOH= CH3CH2OH, PX= P-Et O O , PX= PS
XOH= HO NH3+
Fig. 1. PLD catalyzed transphosphatidylation of phosphatidylcholine (PC).
precipitation steps. Thus, from a synthetic point of view, the possibility to enhance the selectivity of the reaction could allow to simplify the preparation of the compounds on a relatively large scale leading to lower production costs. Ionic liquids (organic salts consisting only of ions, liquid at or near room temperature) are a new class of solvents for enzymatic catalysis [24–28]. Using enzymes in ILs many new applications in the biocatalysis field have came out. In fact these unconventional media have been used mainly with well-investigated hydrolytic enzymes, such as lipases, proteases and glycosidases [29–32] where increased stability, catalytic activity as well as regio- and enantioselectivity have been observed. However, so far, the opportunity of using ionic liquids in biocatalytic modification of phospholipids has not been investigated. The most important advantage of the use of ILs in PLD-catalyzed transesterifications is expected to be the modification of selectivity of the enzyme with suppression of unwanted hydrolytic side reactions, leading to an easier product recovery by a simple solvent extraction. The reduction or absence of water implies the absence of the competitive hydrolysis reaction making the product purification much easier and cheaper, due to the limited use of organic solvents. In particular, we have then tested the possibility of improving the enzymatic preparation of a particular PL, PS from natural PC, by the use of commercial ILs. PS has a great commercial interest because it is a membrane phospholipid ubiquitously present in cellular membranes especially in brain and is involved in many neurological processes [30]. Low levels of PS have been linked to memory problems, Alzheimer’s disease and others mental diseases. Several studies have shown how assuming PS leads to positive benefits in terms of mind and memory enhancement, reduction of age-related decline in mental function, improving thinking skills in young people, attention deficit-hyperactivity disorder (ADHD), depression, preventing exercise-induced stress, and improving athletic performance [31]. The goal of this work has been to explore innovative reaction media for PS preparation in order to get a higher selectivity and a simplification of product isolation and purification with a limited use of organic solvents because the purification steps could be performed without very long chromatographic separations. 2. Materials and methods The ionic liquids 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF4 ]), 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6 ]), 1-ethyl-3-methylimidazolium acetate [EMIm][Ac] and 1-ethyl-2-methylimidazolium diethylphosphate [EMIm][DEP] were purchased from Sigma-Aldrich and used
without further purification. Phosphatidylcholine was supplied from Lucas Meyer (Epikuron 200, soy lecithin, minimum 95 wt% phosphatidylcholine). Others chemicals were purchased from Sigma-Aldrich. All solvents were of analytical grade. 1 H NMR spectra were recorded on a Bruker ARX 400 instrument operating at the 1 H resonance frequency of 400 MHz. Chemical shifts (ı, ppm) are reported relative to tetramethylsilane (TMS) as internal standard. All spectra were recorded in CDCl3 /CD3 OD at 305 K. Mass spectra were recorded on a ESI/MS Bruker Esquire 3000 by direct infusion of methanol solution of compounds. The progress of the hydrolysis of transphosphatidylation reaction in the organic phase was monitored by TLC and HPLC. Silica gel 60 F254 plates (Merck) were used for analytical TLC; mixtures of CHCl3 /CH3 OH/NH3 in different ratios were used as eluant. Detection was achieved with UV light followed by I2 staining. HPLC analysis were performed on a Agilent 1100 series apparatus fitted with a LiChrospher 100 diol 5 m column, length/internal diameter = 125/4, and evaporative light scattering detector (ELSD) (Sedex model 75 Sedere, 41 ◦ C). The operating temperature and pressure were 55 ◦ C and 3.5 bar. A binary solvent system of solvent A: hexane/2-propanol/acetic acid/triethylamine (815/170/15/0.8, v/v/v/v) and solvent B: 2-propanol/water/acetic acid/triethylamine (837/140/15/0.8, v/v/v/v) was used in a gradient mode starting from 60% A, 40% B ramping to 100% B in 16 min. The 20 L samples of a CH2 Cl2 /CH3 OH solution of 1 mg/ml were injected into the column at a mobile phase flow rate of 1.5 mL/min. PLD from Streptomyces PMF (2500 U/g) was a kind gift from Chemi s.p.a. [Patrica (Fr) – Italy]. PLD activity was determined spectrophotometrically in 0.05 M Tris Buffer, pH 8, by monitoring the hydrolysis of phosphatidylp-nitrophenol to phosphatidic acid and p-nitrophenol at 405 nm [33]. 3. Enzymatic reactions 3.1. PLD pretreatment in [BMIm][PF6 ] 0.6 mg of PLD were incubated in 200 L of [BMIm][PF6 ] for 1 h at 40 ◦ C. Then the enzyme was extracted two times with 200 L of sodium acetate (NaAc) buffer 0.1 M. The 400 L of aqueous solution so obtained were used directly as aqueous phase in the transphosphatidylation reaction with ethanol. 3.2. PC transphosphatidylation to phosphatidylethanol (P-Et) The experiment was performed in a 2 mL vial containing PC (20 mg, 27 mol) in 200 L of toluene, ethanol 1 M (24 L,
P. D’Arrigo et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 132–135
400 mol), 400 L NaAc buffer 0.1 M pH 5.6 (3.3 mg, 40 mol), CaCl2 0.1 M (4.4 mg, 40 mol) and 0.6 mg of normal or pretreated PLD. The reaction was magnetically stirred at 40 ◦ C for 24 h. Samples were taken at regular time and the transesterification activity of PLD was evaluated as concentration of P-Et by HPLC). 3.3. PC hydrolysis PC (20 mg, 27 mol) was dissolved with magnetic stirring in 200 L of toluene at 40 ◦ C in vials sealed by septa. Then 400 L of a mixture of IL/buffer 0.1 M sodium acetate buffer pH 5.6 containing 0.1 M of CaCl2 were added and thermostated at 40 ◦ C. Phospholipase D from Streptomyces PMF (0.6 mg, 2500 U/g) was then added and the reaction mixture mixed at 40 ◦ C by magnetic stirring for 24 h. During the reaction, aliquots (10 L) of the organic phase were withdrawn at scheduled times, centrifugated, evaporated, dissolved in CH2 Cl2 /CH3 OH and analyzed by HPLC. 3.4. Transphosphatidylation of PC to phosphatidylserine (PS) PC (20 mg, 27 mol) was dissolved with magnetic stirring in 200 L of toluene at 40 ◦ C in vials sealed by septa. Then 400 L of a mixture of IL/buffer 0.1 M sodium acetate buffer pH 5.6 containing 0.1 M of CaCl2 (4.4 mg, 40 mol), 3 M of l-serine (0.13 g, 1.2 mmol) were added and thermostated at 40 ◦ C. Phospholipase D from Streptomyces PMF (0.6 mg, 2500 U/g) was then added and the reaction mixtures mixed vigorously at 40 ◦ C by magnetic stirring for 24 h. During the reaction, aliquots (10 L) of the organic phase were withdrawn at scheduled times, centrifugated, evaporated, dissolved in CH2 Cl2 /CH3 OH and analyzed by HPLC.
100 90 80 [P-Et]/total PLs
134
70 60 50 40 30 20 10 0 0
30
60
90
PLD preincubated in IL
120
150
180
210
240
me (min) PLD without preincubation in IL
Fig. 2. Comparison of PC conversion to P-Et with normal PLD and PLD preincubated for 1 h in [BMIm][PF6 ] (the experiment was performed in a 2 mL vial containing 20 mg of PC in 200 L of toluene, ethanol 1 M, 400 L NaAc buffer 0.1 M, pH 5.6, CaCl2 0.1 M and 0.6 mg of normal or pretreated PLD. The reaction was stirred on a magnetic stirrer at 40 ◦ C for 24 h. Samples were taken at regular time and the transesterification activity of PLD was evaluated as [P-Et] by HPLC).
of its active site which is not able to efficiently bind the water molecules any longer. The conversion of PC to PS has been performed in presence of toluene and different ratios of IL/buffer. For this purpose, a PC solution in toluene was stirred with a mixture of ionic liquid/sodium acetate buffer at pH 5.6 and the amount of PS and PA formed was monitored at different [BMIm][PF6 ]/buffer ratios. The temperature was fixed at 40 ◦ C. In order to measure the transesterification ability two parameters were considered:
4. Results and discussion (a) the ratio between the amount of transesterification product (PS) and the sum of (remaining PC + PA + PS) (b) The PX/PA ratio. Both were determined at 24 h. The results are reported in Fig. 4 and Table 1. If PLD was suspended in pure IL, the total absence of water completely inactivated the enzyme. As already shown, a minimal concentration of at least 2% of water is mandatory for the enzyme activity. If the ratio IL/buffer was 95:5 the quantity of PA formed was less than 1% with a 93% of conversion of PC in PS. The ratio 100 90 80 [PA] / ([PA]+ [PC]) (%)
In the present work we have tested the possibility of using a commercial IL, in particular 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6 ]), as a cosolvent in the enzymatic preparation of phosphatidylserine from natural phosphatidylcholine from soya. This hydrophobic IL has been used in the PS synthesis taking advantage of a three-phase system consisting of toluene, IL and water. No information were available in literature about the effect of ILs on PLD. Thus firstly, the evaluation of enzyme stability and the influence of operation conditions (quantity of water, ionic liquid/buffer ratio, time) has been established using the transphosphatidylation of PC with ethanol (see Fig. 1). This reaction has been selected because ethanol is the best nucleophile for PLD in toluene/buffer mixtures [25]. The stability of PLD in [BMIm][PF6 ] has been studied. The enzyme was incubated for 1 h in pure [BMIm][PF6 ] and then extracted with a pH 5.6 buffer. The enzymatic solution was then directly used for a classical biphasic reaction of transphosphatidylation of PC with ethanol [34]. As indicated in Fig. 2, PLD from Streptomyces PMF was stable in [BMIm][PF6 ] and did not lose its activity when pre-treated in ionic liquid as it happens with others enzymes [35,36]. The reaction rate seemed only to be lower in the first 2 h but the 100% conversion of substrate was reached after 4 h in presence of IL whereas it took 60 min without IL. This first result was very encouraging for the possibility of using this IL for PS preparation. We have then evaluated the influence of the presence of [BMIm][PF6 ] on the hydrolysis of PC catalyzed by PLD (see Fig. 3). The extent of formation of PA was measured after 24 h. The catalytic activity of PLD was greatly inhibited by the presence of increasing quantities of IL. The enzyme in presence of high IL/buffer ratios loses most of its activity probably because of a conformational change
70 60 50 40 30 20 10 0 0
20
50
80
90
100
[BMIm][PF6] / buffer (v/v) (%)
Fig. 3. Influence of the presence of [BMIm][PF6 ] on the hydrolytic activity of phospholipase D from Streptomyces PMF expressed as % of PA formed (the experiment was performed in a 2 mL vial containing 20 mg of PC in 200 L of toluene PLD, IL/water (% NaAc buffer 0.1 M, pH 5.6, CaCl2 0.1 M) in the opportune ratio. After the addition of PLD (0.6 mg) to the mixture, the reaction was stirred on a magnetic stirrer at 40 ◦ C for 24 h. Samples were taken at regular time. The hydrolytic activity of PLD was evaluated by HPLC).
P. D’Arrigo et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 132–135
135
Table 1 Results of conversion and selectivity in the reaction of transphosphatidylation of PC with l-serine in presence of different mixtures IL/buffer. Conversion of PC at 24 h (%)
Chemical yield PS (%)
Selectivity (PS/PA)
90:10 95:5 90:10 90:10 90:10
98.8 92.9 3 49 0.2
91.4 92.1 3 48.8 0
17 115 >200 195 0
100 90 80 70 60 50 40 30 20 10 0
9 8 7 6 5 4 3 2 1 0 0
20 PS
PA
40
60
80
100
[PA] / [total phospholipids] (%)
IL:buffer (v/v)
[BMIm][PF6 ] [BMIm]PF6 ] [BMIm][BF4 ] [EMIm][DEP] [EMIm][Ac]
[PS] / [total phospholipids] (%)
IL
[BMIm][PF6]/([BMIm][PF6] + buffer) (v/v) (%)
Fig. 4. Influence of IL/buffer ratio (reported as volume IL/(volume buffer + IL)) on transphosphatidylation selectivity (expressed as % of PS formed) and on PA formation (expressed as % of PA obtained) (the experiment was performed in a 2 mL vial containing 20 mg of PC in 200 L of toluene, 400 L IL/water (% NaAc buffer 0.1 M, pH 5.6, CaCl2 0.1 M) in different ratios and l-serine 3 M. After adding phospholipase D (0.6 mg) to the mixture, the reaction was stirred on a magnetic stirrer at 40 ◦ C for 24 h. Samples were taken at regular intervals. The transesterification activity of PLD was evaluated by HPLC).
PS/PA was 115 in these conditions whereas in a normal biphasic system (without the presence of IL) the value of this ratio was much lower, usually around 7–9%. The enhanced selectivity of the reaction is a real improvement in the PS preparation because the step of purification of the product is much more straightforward. Besides to [BMIm][PF6 ] we have checked the possibility of using three hydrophilic commercial ILs, in particular: 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF4 ]), 1-ethyl-3-methylimidazolium acetate [EMIm][Ac] and 1-ethyl-2methylimidazolium diethylphosphate [EMIm][DEP]. The results of the transphosphatidylation in terms of conversion of PC, yield of PS and selectivity (PS/PA ratio) are reported in Table 1. As regards enzyme-IL interactions, the cation and the anion composition of the IL affected in a significant way the PLD catalysis. It appears clearly that the three tested ILs, used in a mixture 90/10 (V/V) with buffer, gave PC conversions at 24 h much lower than [BMIm][PF6 ]. These results seemed to indicate that PLD from Streptomyces PMF is highly inactivated by the presence of hydrophilic ILs. Probably PLD is stabilized by a hydrophobic IL which tends to preserve the essential water layer around the protein molecule as it has been already established for different lipases [37]. 5. Conclusion This work explores the possibility of using ionic liquids in enzymatic phospholipids transformations. The PLD activity in triphasic water/IL/toluene system for the hydrolysis and transphosphatidylation reactions of natural PC is investigated for the first time. The results have demonstrated the stability of the enzyme in presence of [BMIm][PF6 ] and the preference of PLD for hydrophobic ionic liquid [BMIm][PF6 ] versus hydrophilic ILs. A microemulsion of toluene and IL/buffer 90/10 (V/V) seems to be a very promising system for carrying out PC transphosphatidylation, especially with l-serine, with high selectivity. The presence of the IL inhibits quite completely the hydrolysis side-reaction driving the reaction toward transphosphatidylation. Moreover the reaction media can be
easily recycled and reused. We are currently performing a screening of different tailor-made ILs as reaction media for PLs and their polar head modifications. Detailed studies about the interaction of ILs with PLD catalysis are under investigation. In fact, the effect of these novel solvents on the phospholipase’s active site as well as on PLs aggregates is very interesting and hardly predictable. We believe that the results discussed above can stimulate further study on PLs biotransformations in ILs. References [1] D.E. Vance, J.E. Vance, Biochemistry of Lipids, Lipoproteins and membranes, 4th ed., Elsevier Science, Amsterdam, The Netherlands, 2002, pp. 315–340. [2] W. Dowhan, Adv. Lipobiol. 2 (1997) 79–107. [3] Y. Barenholz, Cevc FF G., in: A. Baskin, W. Norde (Eds.), Structure and Properties of Membranes, in Physical Chemistry of Biological Interfaces, Marcel Dekker, New York, NY, USA, 2000, pp. 171–242. [4] W. Dowhan, Annu. Rev. Biochem. 66 (1997) 199–232. [5] T. Stora, Z. Dienes, H. Vogel, C. Duschl, Langmuir 16 (2000) 5471–5478. [6] E.J. Goetzl, S. An, FASEB J. 12 (1998) 1589–1598. [7] L. Vares, A. Koulov, V. Atanas, B.D. Smith, J. Org. Chem. 68 (2003) 10073–10078. [8] M.U. Uhumwangho, R.S. Okor, J. Med. Biomed. Res. 4 (2005) 9–21. [9] A. Gabizon, D. Goren, T. Horowitz, A. Tzemach, A. Losos, T. Siegal, Adv. Drug Deliv. Rev. 24 (1997) 337–344. [10] Z. Reblova, J. Pokorny, in: G. Cevc, F. Paltauf (Eds.), Phospholipids: Characterization, Metabolism, and Novel Biological Applications, AOCS Press, Champaign, IL, USA, 1995, pp. 378–383. [11] W. Van Nieuwenhuyzen, M.C. Tomás, Eur. J. Lipid Sci. Technol. 110 (2008) 472–486. [12] P. D’Arrigo, S. Servi, Trends Biotechnol. 15 (1997) 90–96. [13] P. Mustranta, A.M. Forsell, T. Aura, K. Poutanen, Biocatalysis 9 (1994) 181–194. [14] K. Shimbo, H. Yano, Y. Miyamoto, Agric. Biol. Chem. 54 (1990) 1189–1193. [15] M. Heller, Adv. Lipid Res. 16 (1978) 267–284. [16] R. Ulbrich-Hofmann, A. Lerchner, M. Oblozinsky, L. Bezakova, Biotechnol. Lett. 27 (2005) 535–543. [17] G. Carrea, P. D’Arrigo, V. Piergianni, S. Roncaglio, F. Secundo, S. Servi, Biochim. Biophys. Acta 1255 (1995) 273–279. [18] G. Carrea, P. D’Arrigo, F. Secundo, S. Servi, Biotechnol. Lett. 19 (11) (1997) 1083–1085. [19] P. D’Arrigo, L. de Ferra, V. Piergianni, A. Selva, S. Servi, J. Chem. Soc. Perkin Trans. 1 (1996) 2651–2656. [20] R. Ulbrich-Hofman, Eur. J. Lipid Sci. Technol. 105 (2003) 305–308. [21] P. D’Arrigo, G. Pedrocchi-Fantoni, S. Servi, A. Strini, J. Chem. Soc. Perkin Trans. 1 (1996) 2657–2660. [22] Y. Yamamoto, H. Kurihara, K. Miyashita, M. Hosokawa, New Biotechnol. 28 (2011) 1–6. [23] P. Wang, M. Schuster, Y.F. Wang, C.H. Wong, J. Am. Chem. Soc. 115 (1993) 10487–10491. [24] Z. Yang, W. Pan, Enzyme Microb. Technol. 37 (2005) 19–35. [25] M. Monizzuraman, N. Kamiya, M. Goto, Organ. Biomol. Chem. 8 (2010) 2887–2899. [26] U. Kragl, M. Eckstein, N. Kaftzik, Curr. Opin. Biotechnol. 13 (2002) 565–571. [27] C. Chiappe, D. Pieraccini, J. Phys. Org. Chem. 18 (4) (2005) 275–297. [28] C. Chiappe, E. Leandri, S. Lucchesi, D. Pieraccini, B.D. Hammock, C. Morisseau, J. Mol. Catal. B: Enzym. 27 (4-6) (2004) 243–248. [29] C. Roosen, P. Muller, L. Greiner, Appl. Microbiol. Biotechnol. 81 (2008) 607–614. [30] I.V. Pavlidis, D. Gournis, G.K. Papadopoulos, H. Stamatis, J. Mol. Catal. B: Enzym. 60 (2009) 50–56. [31] M.L. Bader Lange, D. Clair, W.R. Markesbery, C.M. Studzinski, M.P. Murphy, A.D. Butterfield, Neurobiol. Dis. 38 (1) (2010) 104–115. [32] P. Hara, E. Hanefeld, L.T. Kanerva, Green Chem. 11 (2009) 250–256. [33] P. D’Arrigo, V. Piergianni, A. Ricci, D. Scarcelli, Anal. Chim. Acta 304 (1995) 249–254. [34] P. D’Arrigo, L. de Ferra, V. Piergianni, A. Ricci, D. Scarcelli, S. Servi, J. Chem. Soc. Chem. Commun. (1994) 1709–1710. [35] Y. Abe, K. Kude, S. Hayase, M. Kawatsura, K. Tsunashima, T. Itoh, J. Mol. Catal. B: Enzym. 51 (2008) 81–85. [36] J.E. Vance, R. Steenbergen, Prog. Lipid Res. 44 (2005) 207–234. [37] M. Singh, R.S. Singh, U.C. Banerjee, J. Mol. Catal. B (2009) 294–299.
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb
Improvements in the enzymatic synthesis of phosphatidylserine employing ionic liquids Paola D’Arrigo a,b,∗, Lorenzo Cerioli a, Cinzia Chiappe c, Walter Panzeri d, Davide Tessaro a,b, Andrea Mele a,b a
Dipartimento di Chimica, Materiali ed Ingegneria Chimica “G. Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy The Protein Factory, Politecnico di Milano, Università dell’Insubria, ICRM Istituto di Chimica del Riconoscimento Molecolare Consiglio Nazionale delle Ricerche, via Mancinelli 7, 20131 Milano, Italy c Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via Risorgimento 25, 56126, Pisa, Italy d CNR ICRM, Istituto di Chimica del Riconoscimento Molecolare, via Mancinelli 7, 20131 Milano, Italy b
a r t i c l e
i n f o
Article history: Available online 15 April 2012 Keywords: Phospholipase D Transphosphatidylation Ionic liquid Phosphatidylserine
a b s t r a c t The aim of this work was to investigate innovative reaction media such as ionic liquids for enzymatic phospholipids transformations in order to get a higher selectivity in the enzymatic transphosphatidylation reactions of natural phosphatidylcholine catalyzed by phospholipase D (PLD) from Streptomyces PMF. The stability and the activity of the PLD in the ionic liquid [BMIM][PF6 ] were tested. This ionic liquid used as a cosolvent in the synthesis of phosphatidylserine lead surprisingly to the almost quantitative suppression of unwanted hydrolytic side reaction, thus providing a great improvement in the final product purification. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Phospholipids (PLs), essential constituents of cellular membranes, have been thoroughly investigated and are the topic of many areas of biomedical research [1,2]. Because of their amphiphilic properties, natural and synthetic PLs have attracted considerable interest for the multiple scientific and practical applications [3,4] in pharmaceutical, cosmetic and food industry [5,6]. Their involvement in the formation of natural membranes and ability for self-organisation in water has stimulated a large body of studies on their physical properties [7]. Recent advances in the construction of artificial cell membranes with specific biological functions require tailor-made glycerol-derived lipids [8]. Therefore development of new synthetic methods for the preparation of biologically active phospholipid derivatives is a challenging problem of membrane-chemistry and biochemistry today [9,10]. Natural PLs can be obtained as mixtures of products with different acyl chains and polar heads from manly soy beans, many vegetable oils, egg yolk, biomass. Partial purification of PLs is possible by solvent partitioning and, eventually, chromatography. Total synthesis of PLs is complex and expensive. Nevertheless, all sites
∗ Corresponding author at: Dipartimento di Chimica, Materiali ed Ingegneria Chimica “G. Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano, Italy. Tel.: +39 02 23993075; fax: +39 02 23993180. E-mail address: paola.d’[email protected] (P. D’Arrigo). 1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2012.04.011
of PLs can be selectively modified with biocatalytic methods using phospholipases [11–14]. Our attention has mainly been focused on phospholipase D (PLD, EC 3.1.4.4), an enzyme used to modify the polar head of PLs molecules [15,16]. PLD catalyses in vivo the hydrolysis of PLs to phosphatidic acid (PA) but, in presence of an appropriate acceptor (an alcohol), PLD also catalyses the in vitro transphosphatidylation of the molecule polar head in which the exchange of alcohol head groups takes place (see Fig. 1). Polar head modified phospholipids find applications as nutraceuticals, in liposome technology and cosmetics. They can be prepared starting from phosphatidylcholine (PC) from natural sources via PLD catalyzed transphosphatidylation reaction. The reactions were usually carried out in water–organic solvent mixtures and the extent of the competing hydrolytic reaction depends on the source of the enzyme and the nature of the nucleophile [17–20]. In particular, PLD from Streptomyces PMF was directly applied to the transformation of the most abundant natural PL, PC from soy beans into other minor natural products (PX), namely phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG) and also in a great number of unnatural compounds [21–23]. The presence of water in the reaction medium always causes the formation of the PA as a by-product of the transesterification, lowering reaction yields and forcing to further purification steps. The purification of this class of compounds is not straightforward and necessitates different chromatographic and selective
P. D’Arrigo et al. / Journal of Molecular Catalysis B: Enzymatic 84 (2012) 132–135
RCOO R'COO
H
O
O P O
O
+
N
PLD/X-OH
133
O
O RCOO R'COO
H
O
P O
OX
+
RCOO R'COO
H
O
P O
O
PA
PX PC XOH= CH3CH2OH, PX= P-Et O O , PX= PS
XOH= HO NH3+
Fig. 1. PLD catalyzed transphosphatidylation of phosphatidylcholine (PC).
precipitation steps. Thus, from a synthetic point of view, the possibility to enhance the selectivity of the reaction could allow to simplify the preparation of the compounds on a relatively large scale leading to lower production costs. Ionic liquids (organic salts consisting only of ions, liquid at or near room temperature) are a new class of solvents for enzymatic catalysis [24–28]. Using enzymes in ILs many new applications in the biocatalysis field have came out. In fact these unconventional media have been used mainly with well-investigated hydrolytic enzymes, such as lipases, proteases and glycosidases [29–32] where increased stability, catalytic activity as well as regio- and enantioselectivity have been observed. However, so far, the opportunity of using ionic liquids in biocatalytic modification of phospholipids has not been investigated. The most important advantage of the use of ILs in PLD-catalyzed transesterifications is expected to be the modification of selectivity of the enzyme with suppression of unwanted hydrolytic side reactions, leading to an easier product recovery by a simple solvent extraction. The reduction or absence of water implies the absence of the competitive hydrolysis reaction making the product purification much easier and cheaper, due to the limited use of organic solvents. In particular, we have then tested the possibility of improving the enzymatic preparation of a particular PL, PS from natural PC, by the use of commercial ILs. PS has a great commercial interest because it is a membrane phospholipid ubiquitously present in cellular membranes especially in brain and is involved in many neurological processes [30]. Low levels of PS have been linked to memory problems, Alzheimer’s disease and others mental diseases. Several studies have shown how assuming PS leads to positive benefits in terms of mind and memory enhancement, reduction of age-related decline in mental function, improving thinking skills in young people, attention deficit-hyperactivity disorder (ADHD), depression, preventing exercise-induced stress, and improving athletic performance [31]. The goal of this work has been to explore innovative reaction media for PS preparation in order to get a higher selectivity and a simplification of product isolation and purification with a limited use of organic solvents because the purification steps could be performed without very long chromatographic separations. 2. Materials and methods The ionic liquids 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF4 ]), 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6 ]), 1-ethyl-3-methylimidazolium acetate [EMIm][Ac] and 1-ethyl-2-methylimidazolium diethylphosphate [EMIm][DEP] were purchased from Sigma-Aldrich and used
without further purification. Phosphatidylcholine was supplied from Lucas Meyer (Epikuron 200, soy lecithin, minimum 95 wt% phosphatidylcholine). Others chemicals were purchased from Sigma-Aldrich. All solvents were of analytical grade. 1 H NMR spectra were recorded on a Bruker ARX 400 instrument operating at the 1 H resonance frequency of 400 MHz. Chemical shifts (ı, ppm) are reported relative to tetramethylsilane (TMS) as internal standard. All spectra were recorded in CDCl3 /CD3 OD at 305 K. Mass spectra were recorded on a ESI/MS Bruker Esquire 3000 by direct infusion of methanol solution of compounds. The progress of the hydrolysis of transphosphatidylation reaction in the organic phase was monitored by TLC and HPLC. Silica gel 60 F254 plates (Merck) were used for analytical TLC; mixtures of CHCl3 /CH3 OH/NH3 in different ratios were used as eluant. Detection was achieved with UV light followed by I2 staining. HPLC analysis were performed on a Agilent 1100 series apparatus fitted with a LiChrospher 100 diol 5 m column, length/internal diameter = 125/4, and evaporative light scattering detector (ELSD) (Sedex model 75 Sedere, 41 ◦ C). The operating temperature and pressure were 55 ◦ C and 3.5 bar. A binary solvent system of solvent A: hexane/2-propanol/acetic acid/triethylamine (815/170/15/0.8, v/v/v/v) and solvent B: 2-propanol/water/acetic acid/triethylamine (837/140/15/0.8, v/v/v/v) was used in a gradient mode starting from 60% A, 40% B ramping to 100% B in 16 min. The 20 L samples of a CH2 Cl2 /CH3 OH solution of 1 mg/ml were injected into the column at a mobile phase flow rate of 1.5 mL/min. PLD from Streptomyces PMF (2500 U/g) was a kind gift from Chemi s.p.a. [Patrica (Fr) – Italy]. PLD activity was determined spectrophotometrically in 0.05 M Tris Buffer, pH 8, by monitoring the hydrolysis of phosphatidylp-nitrophenol to phosphatidic acid and p-nitrophenol at 405 nm [33]. 3. Enzymatic reactions 3.1. PLD pretreatment in [BMIm][PF6 ] 0.6 mg of PLD were incubated in 200 L of [BMIm][PF6 ] for 1 h at 40 ◦ C. Then the enzyme was extracted two times with 200 L of sodium acetate (NaAc) buffer 0.1 M. The 400 L of aqueous solution so obtained were used directly as aqueous phase in the transphosphatidylation reaction with ethanol. 3.2. PC transphosphatidylation to phosphatidylethanol (P-Et) The experiment was performed in a 2 mL vial containing PC (20 mg, 27 mol) in 200 L of toluene, ethanol 1 M (24 L,
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400 mol), 400 L NaAc buffer 0.1 M pH 5.6 (3.3 mg, 40 mol), CaCl2 0.1 M (4.4 mg, 40 mol) and 0.6 mg of normal or pretreated PLD. The reaction was magnetically stirred at 40 ◦ C for 24 h. Samples were taken at regular time and the transesterification activity of PLD was evaluated as concentration of P-Et by HPLC). 3.3. PC hydrolysis PC (20 mg, 27 mol) was dissolved with magnetic stirring in 200 L of toluene at 40 ◦ C in vials sealed by septa. Then 400 L of a mixture of IL/buffer 0.1 M sodium acetate buffer pH 5.6 containing 0.1 M of CaCl2 were added and thermostated at 40 ◦ C. Phospholipase D from Streptomyces PMF (0.6 mg, 2500 U/g) was then added and the reaction mixture mixed at 40 ◦ C by magnetic stirring for 24 h. During the reaction, aliquots (10 L) of the organic phase were withdrawn at scheduled times, centrifugated, evaporated, dissolved in CH2 Cl2 /CH3 OH and analyzed by HPLC. 3.4. Transphosphatidylation of PC to phosphatidylserine (PS) PC (20 mg, 27 mol) was dissolved with magnetic stirring in 200 L of toluene at 40 ◦ C in vials sealed by septa. Then 400 L of a mixture of IL/buffer 0.1 M sodium acetate buffer pH 5.6 containing 0.1 M of CaCl2 (4.4 mg, 40 mol), 3 M of l-serine (0.13 g, 1.2 mmol) were added and thermostated at 40 ◦ C. Phospholipase D from Streptomyces PMF (0.6 mg, 2500 U/g) was then added and the reaction mixtures mixed vigorously at 40 ◦ C by magnetic stirring for 24 h. During the reaction, aliquots (10 L) of the organic phase were withdrawn at scheduled times, centrifugated, evaporated, dissolved in CH2 Cl2 /CH3 OH and analyzed by HPLC.
100 90 80 [P-Et]/total PLs
134
70 60 50 40 30 20 10 0 0
30
60
90
PLD preincubated in IL
120
150
180
210
240
me (min) PLD without preincubation in IL
Fig. 2. Comparison of PC conversion to P-Et with normal PLD and PLD preincubated for 1 h in [BMIm][PF6 ] (the experiment was performed in a 2 mL vial containing 20 mg of PC in 200 L of toluene, ethanol 1 M, 400 L NaAc buffer 0.1 M, pH 5.6, CaCl2 0.1 M and 0.6 mg of normal or pretreated PLD. The reaction was stirred on a magnetic stirrer at 40 ◦ C for 24 h. Samples were taken at regular time and the transesterification activity of PLD was evaluated as [P-Et] by HPLC).
of its active site which is not able to efficiently bind the water molecules any longer. The conversion of PC to PS has been performed in presence of toluene and different ratios of IL/buffer. For this purpose, a PC solution in toluene was stirred with a mixture of ionic liquid/sodium acetate buffer at pH 5.6 and the amount of PS and PA formed was monitored at different [BMIm][PF6 ]/buffer ratios. The temperature was fixed at 40 ◦ C. In order to measure the transesterification ability two parameters were considered:
4. Results and discussion (a) the ratio between the amount of transesterification product (PS) and the sum of (remaining PC + PA + PS) (b) The PX/PA ratio. Both were determined at 24 h. The results are reported in Fig. 4 and Table 1. If PLD was suspended in pure IL, the total absence of water completely inactivated the enzyme. As already shown, a minimal concentration of at least 2% of water is mandatory for the enzyme activity. If the ratio IL/buffer was 95:5 the quantity of PA formed was less than 1% with a 93% of conversion of PC in PS. The ratio 100 90 80 [PA] / ([PA]+ [PC]) (%)
In the present work we have tested the possibility of using a commercial IL, in particular 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIm][PF6 ]), as a cosolvent in the enzymatic preparation of phosphatidylserine from natural phosphatidylcholine from soya. This hydrophobic IL has been used in the PS synthesis taking advantage of a three-phase system consisting of toluene, IL and water. No information were available in literature about the effect of ILs on PLD. Thus firstly, the evaluation of enzyme stability and the influence of operation conditions (quantity of water, ionic liquid/buffer ratio, time) has been established using the transphosphatidylation of PC with ethanol (see Fig. 1). This reaction has been selected because ethanol is the best nucleophile for PLD in toluene/buffer mixtures [25]. The stability of PLD in [BMIm][PF6 ] has been studied. The enzyme was incubated for 1 h in pure [BMIm][PF6 ] and then extracted with a pH 5.6 buffer. The enzymatic solution was then directly used for a classical biphasic reaction of transphosphatidylation of PC with ethanol [34]. As indicated in Fig. 2, PLD from Streptomyces PMF was stable in [BMIm][PF6 ] and did not lose its activity when pre-treated in ionic liquid as it happens with others enzymes [35,36]. The reaction rate seemed only to be lower in the first 2 h but the 100% conversion of substrate was reached after 4 h in presence of IL whereas it took 60 min without IL. This first result was very encouraging for the possibility of using this IL for PS preparation. We have then evaluated the influence of the presence of [BMIm][PF6 ] on the hydrolysis of PC catalyzed by PLD (see Fig. 3). The extent of formation of PA was measured after 24 h. The catalytic activity of PLD was greatly inhibited by the presence of increasing quantities of IL. The enzyme in presence of high IL/buffer ratios loses most of its activity probably because of a conformational change
70 60 50 40 30 20 10 0 0
20
50
80
90
100
[BMIm][PF6] / buffer (v/v) (%)
Fig. 3. Influence of the presence of [BMIm][PF6 ] on the hydrolytic activity of phospholipase D from Streptomyces PMF expressed as % of PA formed (the experiment was performed in a 2 mL vial containing 20 mg of PC in 200 L of toluene PLD, IL/water (% NaAc buffer 0.1 M, pH 5.6, CaCl2 0.1 M) in the opportune ratio. After the addition of PLD (0.6 mg) to the mixture, the reaction was stirred on a magnetic stirrer at 40 ◦ C for 24 h. Samples were taken at regular time. The hydrolytic activity of PLD was evaluated by HPLC).
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135
Table 1 Results of conversion and selectivity in the reaction of transphosphatidylation of PC with l-serine in presence of different mixtures IL/buffer. Conversion of PC at 24 h (%)
Chemical yield PS (%)
Selectivity (PS/PA)
90:10 95:5 90:10 90:10 90:10
98.8 92.9 3 49 0.2
91.4 92.1 3 48.8 0
17 115 >200 195 0
100 90 80 70 60 50 40 30 20 10 0
9 8 7 6 5 4 3 2 1 0 0
20 PS
PA
40
60
80
100
[PA] / [total phospholipids] (%)
IL:buffer (v/v)
[BMIm][PF6 ] [BMIm]PF6 ] [BMIm][BF4 ] [EMIm][DEP] [EMIm][Ac]
[PS] / [total phospholipids] (%)
IL
[BMIm][PF6]/([BMIm][PF6] + buffer) (v/v) (%)
Fig. 4. Influence of IL/buffer ratio (reported as volume IL/(volume buffer + IL)) on transphosphatidylation selectivity (expressed as % of PS formed) and on PA formation (expressed as % of PA obtained) (the experiment was performed in a 2 mL vial containing 20 mg of PC in 200 L of toluene, 400 L IL/water (% NaAc buffer 0.1 M, pH 5.6, CaCl2 0.1 M) in different ratios and l-serine 3 M. After adding phospholipase D (0.6 mg) to the mixture, the reaction was stirred on a magnetic stirrer at 40 ◦ C for 24 h. Samples were taken at regular intervals. The transesterification activity of PLD was evaluated by HPLC).
PS/PA was 115 in these conditions whereas in a normal biphasic system (without the presence of IL) the value of this ratio was much lower, usually around 7–9%. The enhanced selectivity of the reaction is a real improvement in the PS preparation because the step of purification of the product is much more straightforward. Besides to [BMIm][PF6 ] we have checked the possibility of using three hydrophilic commercial ILs, in particular: 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF4 ]), 1-ethyl-3-methylimidazolium acetate [EMIm][Ac] and 1-ethyl-2methylimidazolium diethylphosphate [EMIm][DEP]. The results of the transphosphatidylation in terms of conversion of PC, yield of PS and selectivity (PS/PA ratio) are reported in Table 1. As regards enzyme-IL interactions, the cation and the anion composition of the IL affected in a significant way the PLD catalysis. It appears clearly that the three tested ILs, used in a mixture 90/10 (V/V) with buffer, gave PC conversions at 24 h much lower than [BMIm][PF6 ]. These results seemed to indicate that PLD from Streptomyces PMF is highly inactivated by the presence of hydrophilic ILs. Probably PLD is stabilized by a hydrophobic IL which tends to preserve the essential water layer around the protein molecule as it has been already established for different lipases [37]. 5. Conclusion This work explores the possibility of using ionic liquids in enzymatic phospholipids transformations. The PLD activity in triphasic water/IL/toluene system for the hydrolysis and transphosphatidylation reactions of natural PC is investigated for the first time. The results have demonstrated the stability of the enzyme in presence of [BMIm][PF6 ] and the preference of PLD for hydrophobic ionic liquid [BMIm][PF6 ] versus hydrophilic ILs. A microemulsion of toluene and IL/buffer 90/10 (V/V) seems to be a very promising system for carrying out PC transphosphatidylation, especially with l-serine, with high selectivity. The presence of the IL inhibits quite completely the hydrolysis side-reaction driving the reaction toward transphosphatidylation. Moreover the reaction media can be
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