Evaluation of snake venom phospholipase A2: hydrolysis of non-natural esters

ARTICLE

Evaluation of snake venom phospholipase A₂: hydrolysis of non-natural esters

Renan A. S. Pirolla; Paulo A. Baldasso; Sérgio Marangoni; Paulo J. S. Moran; José Augusto R. Rodrigues^*

Department of Organic Chemistry, Institute of Chemistry, University of Campinas, PO Box 6154, 13083-970 Campinas-SP, Brazil

ABSTRACT

Phospholipase A₂ from the rattlesnake Crotalus durissus terrificus was employed for the first time to test its enantioseletivity on the hydrolysis of different non-natural esters. It was observed that the structure of this small enzyme is restrictive in the choice of its lipase action with non-natural substrates. Two forms of the enzyme were used; free and as its cross-linked enzyme aggregate (CLEA). With all substrates, the free enzyme showed activity similar to the CLEA preparation. The advantage of the CLEA phospholipase is the possibility to reuse it in several consecutive reactions without a decrease of activity and selectivity with good but higher yields and ee than with the free enzyme.

Keywords: phospholipase A₂, snake venom PLA₂, cross-linking enzyme aggregate, biocatalysis

RESUMO

Fosfolipase A₂ colhida da serpente Crotalus durissus terrificus foi empregada pela primeira vez para testar sua enantiosseletividade na hidrólise de diferentes ésteres não-naturais. Foi observado que a estrutura desta pequena enzima tem sua ação lipase dependente da escolha dos substratos não-naturais. Foram usadas duas formas da enzima; livre e como agregado enzimático formado com ligações cruzadas, CLEA. Com todos os substratos, a enzima livre mostrou atividade similar a da preparada CLEA. A vantagem da fosfolipase CLEA é que permite sua reutilização em várias reações consecutivas sem demonstrar diminuição de atividade e seletividade, propiciando rendimentos bons e superiores, e ee similares quando comparados com os da enzima livre.

Introduction

The phospholipases PLA₂ (EC 3.1.1.4) are calcium-dependent enzymes that catalyze the hydrolysis of 1,2-diacyl-3-syn-glycerol phosphoglycerides, liberating the fatty acid from the 2-position. These enzymes possess interesting properties, such as heat stability, the capacity to act in nonaqueous systems and at the surface of lipid particles and monolayers. There are several recent reviews in the literature on these enzymes reporting on different properties of their action. ¹ These enzymes are common in pancreatic juice in snake and insect venoms. Mammalian PLA₂ are classified into four groups (I, II, III and IV) based on their origin (extracellular or intracellular), primary structure and disulfide bonding. ² Snake venom PLA₂ occurs in groups I (Elapidae and Hydrophidae) and II (Crotalidae and Viperidae). Group II contains the catalytically active D49 PLA₂ as well the K49 isoforms, which has low or no activity. ³ In addition to their enzymatic activity, venom PLA₂ may have neurotoxic, myotoxic, hemolytic, anticoagulant, cardiotoxic, and edematogenic activities as well as platelet aggregatory or antiaggregatory actions. ⁴

Crotoxin, a heterodimeric complex with phospholipase A₂ activity, is the main neurotoxic component in the venom of the South America rattlesnake Crotalus durissus terrificus, accounting for approximately 50% of the venom dry weight. Crotoxin has neurotoxic,⁵ myotoxic,⁶ hemolytic⁷ and platelet aggregating⁸ activities. The crotoxin complex consists of a basic PLA₂ and an acidic, nonenzymatic component known as crotapotin,⁹ which increases the pharmacologic activity of PLA₂ by acting as a chaperone protein for the enzyme. ¹⁰ Crotoxin exists in several isoforms which result from the random association of the PLA₂ with crotapotin¹¹ following the post-translational modification of a single precursor of crotoxin or the expression of different mRNAs. ¹²

The chemical mechanism of the enzymatic catalysis involving PLA₂ is not yet fully explored. In order to obtain more information about the mechanism of the PLA₂-catalyzed reaction, it would be advantageous to use substrates for which structural variations could be correlated with catalytic activity. In the past, Kézdy et al., ¹³ have explored the activity of the enzyme complex in which the glycerol backbone and the phosphate diester were replaced by simpler structures. Since a major role of the phosphate is to complex Ca²⁺ in the active site, they proposed that a carboxylate group could replace the phosphate in this function. In this case, the distance between the reactive ester and the carboxylate group in the new substrate should be similar to that found in the phospholipids. Also, the glycerol would be replaced by a much better leaving group such as a nitrophenol. They choose 4-nitro-3-(octanoyloxy)benzoic acid 1, which presents the essential structural features of a phosphatidylcholine (R, R¹ = fatty acid residue). They argued that while the geometries of the molecules are different, the sums of the bond lengths between the reactive ester and the ligand of Ca²⁺ are approximately the same in both molecules. Ester 1 showed the highest specificity toward the enzyme, suggesting that in this compound the distance between the negative charged carboxylate and the reactive ester approximates that found in the lecithin-enzyme complex. All kinetic characteristics of the enzymatic hydrolysis indicated that the reaction occurs by the same mechanism shown in the hydrolysis of lecithins. Also compound 1 is ideally suited for a rapid and sensitive spectrophotometric assay of phospholipases A₂ carried out at pH 8 containing 10 mmol L^-1 calcium chloride (CaCl₂) and 0.1 mol L^-1 sodium chloride (NaCl) in acetonitrile.

In this study we explore the enzymatic reactivity of the phospholipase A₂ from the component venom of the South America rattlesnake Crotalus durissus terrificus, on the hydrolysis of non-natural substrates with structural variations related to 1. We also studied for the first time the potential of that enzyme to steroselectively resolve esters of secondary alcohols. Considering the low availability of the enzyme we immobilized it in order to reuse the biocatalyst in successive reactions. Within the different methods of immobilization we choose precipitation of the enzyme and cross-linking its aggregates to produce the cross-linked enzyme aggregate (CLEA). ¹⁴ Several reports have described the CLEA activity to exceed that of the native enzyme with lipases and other enzymes. ¹⁵ The immobilization of enzymes via cross-linking is also attractive because the final preparation is basically pure protein, with a high concentration of enzyme per unit of volume. ¹⁶

Experimental

General procedures

Proton and carbon nuclear magnetic resonance (¹H NMR and ¹³C NMR) were recorded in CDCl₃ solution on Varian INOVA 500 MHz, Bruker Advance DPX 250 MHz and Bruker Advance II 300 MHz spectrometers. Chemical shifts were expressed in ppm using TMS as internal standard. Infrared spectra were recorded in KBr on a BOMEN MB-100 FTIR spectrophotometer from Hartmann & Braun. The gas chromatography-mass spectrometry (GC MS) analyses were obtained on a Agilent 6890 coupled a to HP 5973 mass detector using a Supelco MDN-5S (30 m × 250 µm × 0.25 µm ) capillary column. Chiral analyses were performed in a Agilent 6850N GC FID, using a MN Hydrodex^® -b-3P (30 m × 250 µm × 0.25 µm ), Chrompack Chirasil-dex CB (30 m × 250 µm × 0.25 µm ) or Lipodex-E 2,6-Pe-3-Bu-g-CD (28 m × 250 µm × 0.25 µm ) capillary columns. Optical rotations were recorded on a PerkinElmer 341 Polarimeter. The spectrophotometric analyses were recorded on an Agilent HP8453 spectrophotometer or FlashScan 530 Analitic Jena. The melting points were determined on a MQAPF-301 Microquimica apparatus.

The absolute configurations of the alcohols were established by comparison of their chromatograms with the products obtained from known reduction reactions of the corresponding ketones with carrot root. E value of the reaction was calculated based on ee of the recovered product and the extent of conversion. ¹⁷

Enzyme and chemicals

The phospholipase A₂ were isolated from Crotalus durissus terrificus venom and purified by a research group from the Biology Institute of Unicamp (Campinas-SP). ¹⁸ All chemicals were purchased from Sigma-Aldrich (São Paulo, Brazil) at least in analytical grade.

CLEA procedure

The CLEA were prepared by dissolving 0.5 mg of phospholipase A₂ in 1 mL of Tris-HCl buffer (10 mmol L^-1) at pH 8.0 and then adding 0.5 mL of precipitant (55% ammonium sulphate solution, polyethylene glycol 600 Da, dimethoxyethane or acetone) and stirred for 40 min to form the aggregate. When an additive was used, the enzyme was dissolved in the presence of 10 mg of Triton-X100 or 0.5 mL of polyethylenediimine (PEI). Then 100 µL of a 25% (v/v) solution of glutaraldehyde was added and the mixture was stirred overnight at room temperature. The solution was centrifuged at 500 rpm and the solid kept in Tris-HCl buffer at 4 ºC.

Synthesis of 4-nitro-3-(octanoyloxy)benzoic acid 1a

3-Hydroxy-4-nitrobenzoic acid (183 mg, 1.0 mmol) and N, N-diisopropylethylamine (259 mg, 2.0 mmol) in 2 mL of dry tetrahydrofuran (THF) was added dropwise to a cooled solution (0 ºC) of octanoyl chloride (163 mg, 1.0 mmol) in 10 mL of dry THF. The reaction was slowly warmed to room temperature and stirred overnight. The reaction mixture was filtered and washed with ether. The crude product was purified by flash chromatography.

Yellow solid, mp 142-145 ºC; IR (KBr) ν_max/cm^-1 3453, 3067, 2959, 2855, 1786, 1696, 1595, 1532. ¹H NMR (CDCl₃) δ 0.9 (t, 3H, J 6.7 Hz), 1.2-1.5 (m, 8H), 1.75 (m, 2H, J 7.4), 2.71 (t, 2H, J 7.5 Hz), 7.92 (s, 1H), 8.10 (d, 1H, J 8.2 Hz), 8.15 (d, 1H, J 8.2 Hz), ¹³C NMR(CDCl₃) δ 14.1 (CH₃), 22.6 (CH₂), 24.4 (CH₂), 28.9 (CH₂), 29.0 (CH₂), 31.6 (CH₂), 34.0 (CH₂), 125.9 (CH), 127.3 (CH), 128.1 (CH), 134.6 (C₀), 144.0 (C₀), 145.1 (C₀), 168.9 (C₀), 171.1 (C₀).

Test of phospholipase activity

To a flask was added 100 µL of a solution 2 mg mL^-1 of 4-nitro-3-(octanoyloxy)benzoic acid 1 in acetonitrile, 3 mL of Tris-HCl/CaCl₂/NaCl (10/10/100 mmol L^-1) pH 8.0 buffer. The enzymatic hydrolysis was monitored at 425 nm in a spectrophotometer for 45 min.

General procedure for biocatalysis reactions

The biocatalysis reactions were made by adding the CLEA prepared in 1 mL of Tris-HCl/NaCl/CaCl₂ (10/10/100 mmol L^-1) pH 8.0 buffer and 1 mg of substrate dissolved in 0.5 mL of acetonitrile and were monitored by GC MS. After each reaction, the CLEA biocatalyst was isolated, washed with water, filtered and kept for further use after testing the phospholipase activity (this test was described above). Reactions with free enzymes were made for comparison. Resolution was stopped before 50% conversion.

General procedure for biocatalysis reactions of acidics compounds

The reactions of 3-(2-bromohexanoyloxy)-4-nitrobenzoic acid and 3-(2-methylhexanoyloxy)-4-nitrobenzoic acid were initially made on polypropylene microplates and analyzed in FlashScan at 425 nm. The substrate (0.05 mg dissolved in 12.5 µL of acetonitrile), 0.045 mg of PLA₂ and the Tris-HCl/CaCl₂/NaCl buffer were added to the microplate. The reaction mixtures were stirred at 22 ºC until stabilization.

3-(2-Bromohexanoyloxy)-4-nitrobenzoic acid 2a

3-Hydroxy-4-nitrobenzoic acid (257 mg, 1.4 mmol) and N, N-diisopropylethylamine (363 mg, 2.8 mmol) in 2 mL of dry THF was added dropwise to a cooled solution (0 ºC) of 2-bromohexanoyl bromide (362 mg, 1.4 mmol) in 10 mL of dry THF. The reaction mixture was warmed slowly to room temperature and stirred overnight. The reaction mixture was filtered, and the precipitate was washed with ether (10 mL × 3). The filtrate was concentrated under vacuum, and the residue was taken up by 50 mL of ethyl acetate. The organic layer was washed with 0.01 mol L^-1 (50 mL × 2) HCl, dried over anhydrous sodium sulfate (Na₂SO₄), and evaporated. The residue was recrystallized from ethyl acetate-hexane (1:10 v/v).

Yellow solid; mp 125-125.6 ºC; IR (KBr) ν_max/cm^-1 3282, 3065, 2959, 2875, 2560, 1780, 1697, 1594, 1533, 1306, 1215, 1102, 955, 924, 841, 795, 527. ¹H NMR (CDCl₃) δ 0.97 (t, 3H, J 7.5 Hz), 1.51 (m, 4H), 2.20 (m, 2H), 4.5 (t, 1H, J 7.5 Hz), 8.03 (s, 2H), 8.18 (s, 1H), 10.5 (br, 1H); ¹³C NMR (CDCl₃) δ 13.7 (CH₃), 22.0 (CH₂), 29.2 (CH₂), 34.1 (CH₂), 44.4 (CH), 126.1 (CH), 126.7 (CH), 128.6 (CH), 134.9 (C₀), 143.5 (C₀), 144.8 (C₀), 167.3 (C₀), 168.6 (C₀).

3-(2-Methylhexanoyloxy)-4-nitrobenzoic acid 2b

2-Methylhexanoic acid (221 mg, 1.7 mmol) was dissolved in 17 mL of dry THF. The solution was stirred and cooled to 0 ºC. Then, N-methylmorpholine (172 mg, 1.7 mmol) and isobutyl chloroformate (233 mg, 1.7 mmol) were added successively. The reaction mixture was stirred for 30 min at 0 ºC during which a white precipitate was formed. Next, the reaction mixture was allowed to reach room temperature, and a solution of 3-hydroxy-4-nitrobenzoic acid (330 mg, 1.8 mmol) and N-methylmorpholine (364 mg, 3.6 mmol) in 10 mL of dry THF was added. The resulting mixture was stirred overnight at room temperature. The reaction mixture was filtered, and the precipitate was washed with ether (10 mL × 3). The filtrate was concentrated in vacuum, and the residue was taken up by 50 mL of ethyl acetate. The organic layer was washed with 0.01 mol L^-1 (50 mL × 2) HCl, dried over anhydrous Na₂SO₄, and evaporated. The residue was recrystallized from ethyl acetate-hexane (1:10 v/v).

Pale yellow solid; mp 135-137 ºC; IR (KBr) ν_max/cm^-1 3444, 3096, 2959, 2929, 2863, 1773, 1627, 1594, 1536, 1327, 1242, 1223, 746. ¹H NMR (CDCl₃) δ 0.948 (t, 3H, J 6.9 Hz), 1.35 (d, 3H, J 7Hz), 1.41 (m, 4H), 1.58 (m, 2H), 2.78 (m, 1H, J 7Hz), 7.95 (s, 1H), 8.23 (m, 2H), 10.53 (br, 1H); ¹³C NMR (CDCl₃) δ 13.9 (CH₃), 16.6 (CH₃), 22.6 (CH₂), 29.3 (CH₂), 32.9 (CH), 39.4 (CH), 125.8 (CH), 127.2 (CH), 128.0 (CH), 134.5 (C₀), 144.0 (C₀), 145.3 (C₀), 168.5 (C₀), 174.0 (C₀).

Synthesis of substrates

General procedure for reduction of ketones

The ketone substrate (5.5 mmol) was dissolved in dry methanol with sodium borohydride (NaBH₄) (4.2 mmol) and stirred overnight. Then 20 mL of dilute HCl (pH 4.0) was added and the solution was stirred for 20 min. The solution was extracted with ethyl acetate and the organic layer was dried over anhydrous Na₂SO₄ and evaporated. The alcohol residue was purified by flash chromatography eluting with 20% ethyl acetate in hexane.

1-Phenylethanol 7

From acetophenone: colorless oil; IR (KBr) ν_max/cm^-1 3359, 3063, 3029, 2973, 2927, 2876, 1493, 1451, 1369, 1302, 1204, 1077, 1010, 996, 899. ¹H NMR (CDCl₃) δ 1.48 (d, 3H, J 6 Hz), 2.05 (br, 1H), 4.86 (q, 1H, J 6 Hz), 7.35 (m, 5H); ¹³C NMR (CDCl₃) δ 25.1 (CH₃), 70.3 (CH), 125.3 (CH), 127.4 (CH), 128.4 (CH), 145.7 (C₀); MS m/z (%): 122 (M⁺, 33); 107 (100); 79 (92); 77 (55). ¹⁹

(S)-p-Nitrophenyl-1-ethanol 8

From p-nitroacetophenone:yellow oil; IR (KBr) ν_max/cm^-1 3414, 2978, 2931, 2866, 1735, 1605, 1520, 1347, 1254, 1090, 1046, 855. ¹H NMR (CDCl₃) δ 1.5 (d, 3H, J 6,8 Hz), 2.37 (br, 1H), 5.0 (q, 1H, J 6,5 Hz), 7.52 (d, 2H, J 8.6 Hz), 8.16 (d, 2H, J 8.6 Hz); ¹³C NMR CDCl₃) δ 25.4 (CH₃), 69.4 (CH), 123.7 (CH), 126.1 (CH), 147.1 (C₀), 153.1 (C₀); MS m/z (%): 167 (M⁺, 0,7), 166 (1), 152 (100), 122 (24), 107 (45), 77 (39). ²⁰

1,2,3,4-Tetrahydronaphthalen-1-ol 12

From 1-tetralone: colorless oil; IR (KBr) ν_max/cm^-1 3285, 3041, 2946, 2928, 2855, 2835, 1491, 1453, 1431, 1286, 1267, 1069, 42. ¹H NMR (CDCl₃) δ 1.60-1.70 (m, 2H), 1.70-2.10 (m, 3H), 2.71 (m, 2H), 4.78 (t, 1H), 7.13 (m, 1H), 7.18 (t, 1H, J 3.5 Hz), 7.23 (t, 1H, J 3.5 Hz), 7.44 (t, 1H, J 4.5 Hz); ¹³C NMR (CDCl₃) δ 18.8 (CH₂), 29.2 (CH₂), 32.3 (CH₂), 68.1 (CH), 126.2 (CH), 127.6 (CH), 128.7 (CH), 129.0 (CH), 137.1 (C₀), 138.8 (C₀); MS m/z (%):148 (M⁺, 31), 147 (35), 130 (100), 120 (76), 115 (29), 105 (29), 91 (54). ²¹

Esterefication procedure for the alcohols

The esterification of the alcohols with acetyl chloride and propionyl chloride was carried out as described by Vogel's Textbook. ²²

1-Phenylethyl acetate 5

From 1-phenylethanol 7, colorless oil; IR (KBr) ν_max/cm^-1 3034, 2982, 2934, 1744, 1495, 1453, 1371, 1241, 1209, 1065, 1029, 944, 699. ¹H NMR (CDCl₃) δ 1.53 (d, 3H, J 6 Hz), 2.07 (s, 3H), 5.88 (q, 1H, J 6 Hz), 7.35 (m, 5H); ¹³C NMR (CDCl₃) δ 21.3 (CH₃), 22.2 (CH₃), 72.3 (CH), 126.1 (CH), 127.8 (CH), 128.5 (CH), 141.7 (C₀), 170.3 (C₀); MS m/z (%): 164 (M⁺, 21), 149 (1), 122 (95), 104 (100), 91 (2), 77 (36). ²¹

1-Phenylethyl propionate 5b

From 1-phenylethanol 7, colorless oil; IR (KBr) ν_max/cm^-1 3063, 3034, 2982, 2941, 2880, 1738, 1495, 1454, 1368, 1273, 1189, 1064, 1029; ¹H NMR (CDCl₃) δ 1.14 (t, 3H, J 7 Hz), 1.53 (d, 3H, J 6,5 Hz), 2.35 (m, 2H J 7.5 Hz and 3.5 Hz), 5.89 (t, 1H, J 6.5 Hz), 7.32 (m, 5H); ¹³C NMR (CDCl₃) δ 9.0 (CH₃), 22.2 (CH₃), 27.8 (CH₂), 72.0 (CH), 126.0 (CH), 127.8 (CH), 128.4 (CH), 141.8 (C₀), 173.7 (C₀); MS m/z (%): 178 (M⁺, 25), 122 (92), 105 (100), 77 (28). ²³

p-Nitrophenyl-1-ethyl acetate 6a

From p-nitrophenyl-1-ethanol 8, white solid; mp 49-51 ºC; IR (KBr) ν_max/cm^-1 2965, 2923, 2856, 1731, 1602, 1518, 1347; 1248, 1069, 856. ¹H NMR (CDCl₃) δ 1.55 (d, 3H, J 6.8 Hz), 2.11 (s, 3H), 5.92 (q, 1H, J 6.7 Hz), 7.51 (d, 2H, J 8.5 Hz), 8.21 (d, 2H, J 8.5 Hz); ¹³C NMR (CDCl₃) δ 20.8 (CH₃), 22.4 (CH₃), 71.0 (CH), 123.8 (CH), 126.9 (CH), 148.9 (C₀), 170.0 (C₀); MS m/z (%): 209 (M⁺,6); 167 (100); 150 (23); 119 (16); 103 (25); 91 (18); 77 (19). ²⁴

p-Nitrophenyl-1-ethyl propionate 6b

From p-nitrophenyl-1-ethanol 8, white solid; mp 36-37 ºC; IR (KBr) ν_max/cm^-1 3066, 2989, 2946, 1736, 1605, 1524, 1333, 1184, 1086, 1065, 855. ¹H NMR (CDCl₃) δ 1.15 (t, 3H, J 7.6 Hz), 1.55 (d, 2H, J 6.7 Hz), 2.39 (qd, 2H, J 7.58 Hz and J 1.3 Hz), 5.93 (q, 1H, J 6.7 Hz), 7.50 (d, 2H, J 8.8 Hz), 8.19 (d, 2H, J 8.8Hz); ¹³C NMR (CDCl₃) δ 9.0 (CH₃), 22.4 (CH₃), 27.7 (CH₂), 71.8 (CH), 123.9 (CH), 126.7 (CH), 149.2 (C₀), 173.5 (C₀); MS m/z (%): 223 (M⁺, 11); 167 (100); 150 (68); 119 (24); 103 (36); 91 (25).

(R)-1,2,3,4-Tetrahydronaphthalen-1-yl acetate 11a

Yellow oil; IR (KBr) ν_max/cm^-1 3064, 3023, 2940, 2859, 2838, 1731, 1492, 1455, 1371, 1238, 1210, 1154, 1030, 1016, 882. ¹H NMR (CDCl₃) δ 1.70-1.95 (m, 4H), 2.10 (s, 3H), 2.85 (m, 2H), 6.02 (t, 1H, J 4.2 Hz), 7.25 (m, 4H); ¹³C NMR (CDCl₃) δ 18.8 (CH₂), 21.5 (CH₃), 29.0 (CH₂), 29.1 (CH₂), 70.0 (CH), 126.1 (CH), 128.1 (CH), 129.1 (CH), 129.4 (CH), 134.6 (C₀), 137.9 (C₀), 170.8 (C₀); MS m/z (%): 190 (M⁺, 1), 148 (19), 130 (100), 115 (32); 91 (20). ²⁵

1,2,3,4-Tetrahydronaphthalen-1-yl propionate 11b

Colorless oil; IR (KBr) ν_max/cm^-1 3023, 2979, 2941, 2870, 2840, 1731, 1492, 1455, 1271, 1185, 1152, 1080, 766. ¹H NMR (CDCl₃) δ 1.17 (t, 3H, J 6.8 Hz), 1.80-2.05 (m, 4H), 2.36 (q, 2H, J 6.8 Hz), 2.82 (m, 2H), 6.01 (t, 1H; J 4.3 Hz), 7.12-7.29 (m, 4H); ¹³C NMR(CDCl₃) δ : 9.2 (CH₃), 18.8 (CH₂), 28.2 (CH₂); 29.0 (CH₂), 29.1 (CH₂), 69.8 (CH), 126.0 (CH), 128.0 (CH), 129.0 (CH), 129.4 (CH), 134.8 (C₀), 138.0 (C₀), 174.3 (C₀); MS m/z (%): 204 (M⁺, 0.8), 148 (27), 130 (100), 115 (29), 91 (24). ²⁶

Determination of absolute configurations

Bioreduction of α -tetralone with Daucus carota

α -Tetralone (20 mg, 0.14 mmol) dissolved in 1 mL of ethanol was added to a suspension of freshly cut carrot root (30 g) in 80 mL of distilled water, and the reaction mixture was incubated on an orbital shaker (180 rpm) at 30 ºC for 6 days. Finally, the suspension was filtered, and the carrot root was washed three times with water. Filtrates were extracted with ethyl acetate (3 × 125 mL), the organic phase was dried over anhydrous Na₂SO₄ and then evaporated. The residue was analyzed by GC MS and GC FID with a chiral column. The product (S)-1,2,3,4-tetrahydronaphtalen-1-ol 12 was obtained in 64% yield and ee 65% with [α]_D²⁰ +15 (c 0.59 in THF).

Bioreduction of p-nitroacetophenone with daucus carota

Using a procedure similar to the describe above, 100 mg (0.60 mmol) of p-nitroacetophenone was reacted with 30 g of carrot root. Following separations and GC analysis the product (S)-p-nitrophenyl-1-ethanol 8 was obtained in 81% yield and ee 96% with [α]_D²⁰ -24 (c 1.48 in THF).

Results and Discussion

Considering that snakes in general produce only small amounts of venom, we attempted to immobilize the purified phospholipase A₂ in order to reuse it in successive batch reactions. CLEAs were prepared varying several parameters like precipitant and additives, such as surfactants and then, their activities were investigated (Table 1). The activities of the resultant CLEA were compared with those of the free enzyme, which was considered as 100%. The kinetics of hydrolysis of 4-nitro-3-(octanoyloxy)benzoic acid 1 were monitored on a UV-spectrophotometer at 425 nm at pH 9. This compound is currently used to assay PLA₂ activity since it shows the highest specificity toward the Ca²⁺ dependent phospholipase. ²⁹ The preparation of CLEA was taken from the simplified procedure of Sheldon et al., ³⁰ by combining the aggregation and cross-linking steps. The precipitants ammonium sulfate, PEG 600, 1,2-dimetoxyethane or acetone were added simultaneously with the surfactant and then the glutaraldeyde. The results (Table 1) show the highest activity was obtained without addition of any surfactant and the most effective precipitant was ammonium sulfate (entry 1). In further experiments we adopted this procedure (Table 1, entry 1) since this CLEA has hydrolysis activity comparable with that of the free enzyme.

One of our objectives was to investigate the capability of the phospholipase A₂ of the rattlesnake Crotalus durissus terrificus to produce chiral compounds through stereoselective hydrolysis. With this in mind, we prepared two substrates closed related to acid 1, with a substituent at α -position of the ester moiety. We choose the commercially available 2-bromohexanoic acid 4a and 2-methylhexanoic acid 4b to prepare the substrates 3-(2-bromohexanoyl)-4-nitrobenzoic acid 2a and 3-(2-methylhexanoyl)-4-nitrobenzoic acid 2b. Initially, we tested the hydrolysis of both substrates in a blank reaction (acetonitrile 3%, water in Tris-HCl/CaCl₂/NaCl at pH 8) to be sure that 2a and 2b are stable in that reaction medium. The spontaneous hydrolysis of 2a was very fast in the reaction medium, avoiding carrying out the enzymatic hydrolysis with this substrate, but slow with 2b.

The enzymatic hydrolysis of 2b in 3% acetonitirle (v/v) in a buffer of Tris-HCl/CaCl₂/NaCl at pH 8 with the free phospholipase at 25 ºC gave a 25% yield and 7% ee after 4 days (Scheme 1).

When the reaction was carried out at 45 ºC the yield increased to 52% but the stereoselectivity decreased to 3% ee. With CLEA, the yield increased as is shown in Table 2. When compared with substrate 1 for which the reaction is fast, about 2 h, the hydrolysis of 2b was much slower (3-4 days) and gave good yields and low ee. We assume that the decrease of reactivity is a consequence of the presence of the methyl group near the reaction center of the hydrolysis. The absolute configuration of the 2-methylhexanoic acid 4b was not determined considering that it was isolated with low enantioselectivity (closed to a racemate). The yield of 4b with CLEA increased when compared with the free enzyme (entries 3 an 4 of Table 2), but the ee was very low.

Considering that the substituents of the alkyl moiety of substrates 2a and 2b were not suitable for our objective to produce enantioselective products by phospholipase hydrolysis, we choose esters with different structures to test enzyme reactivity. The compounds 5 and 6 (Scheme 2) were prepared by esterification of the parent alcohols which were prepared from the corresponding ketones by reduction with sodium borohydride. We employed the same methodology used for 2. The results of the hydrolysis are summarized in Table 3. For most cases, the reaction using CLEA gave good yields (> 60%) but superior than with the free enzyme. As before the enantioselectivity of the hydrolyzed products was poor nearly racemic. Only in two examples did the enantioselectivity gave better ee, compound 5a with 16% ee for product 7 (entry 2) and 6a with 19% ee for product 8 (entry 5). A low E value (E = 3) for 5a and 6a after 43% conversion were calculated for both reactions. The E-value and the ee of 5a, 5b, 6a and 6b were the same after each cycle, that is, < 3 and 20%, respectively. The absolute configuration of the alcohols (S)-7 and (S)-8 was determined by comparison of GC of the alcohols from authentic samples obtained by carrying out the known bioreduction of acetophenone and p-nitroacetophenone with Daucus carota.³¹

Finally, we carried out the hydrolysis of α -tetralol esters 11a and 11b with free phospholipase and CLEA. The results are summarized in Table 4. For both substrates, (S)-α -tetralol 12 was isolated in good yields (> 50%) but with CLEA phospholipase the yields were superior to those with the free enzyme. Also, the acetyl derivative 11a gave higher yields than the propyl ester 11b. Only with acetyl derivative 11a a modest enantioselectivity was obtained, with 16% ee and a low E value (E = 3) (entry 2) when CLEA was employed (Scheme 3). The enantiomeric ratio E was very low reflecting the low especificity of the enzyme, both free and immobilized (CLEA).

As in the case of 1-phenylethanol 7, the absolute configuration of the (S)-α -tetralol 12 was determined by comparison the GC of the alcohol obtained with an authentic sample prepared by carrying out the known bioreduction of α -tetralone with carrot root (Scheme 3). ²⁶

Conclusions

For the first time, the phospholipase A₂ from rattlesnake Crotalus durissus terrificus has shown some enantioseletivity in the hydrolysis of different esters. It was observed that this small protein is restrictive in the choice of its lipase action with non-natural substrates. With all substrates the free enzyme showed reactivity similar to the CLEA preparation. The advantage of the CLEA phospholipase is to reuse it in several consecutive reactions without a decrease of activity, having a higher yield and similar ee when compared with the free enzyme.

Acknowledgments

This work was financially supported by FAPESP and CNPq. R. A. S. Pirolla thanks CAPES for fellowship.

References

1. Nevalainen, T. J.; Graham, G. G.; Scott, K. F.; Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2008, 1781, 1.

2. Dennis, E. A.; J. Biol. Chem. 1994, 269, 13037.

3. De Araujo, H. S. S.; White, S. P.; Ownby, C. L.; Toxicon 1996, 34, 1237.

4. Kini, R. M.; Evans, H. J.; Int. Peptide Protein Res. 1989, 34,277; Arni, R. K.; Ward, R. J.; Toxicon 1996, 34, 827.

5. Bom, C.; Changeux, J. P.; Jeng, T. W.; Fraenkel-Conrat, H.; Eur. J. Biochem. 1979, 99, 471.

6. Azevedo-Marques, M. M.; Cupo, P.; Coimbra, T. M.; Hering, S. E.; Rossi, M. A.; Laure, C. J.; Toxicon 1982, 23, 631.

7. Rosenfeld, G. I, Venomous Animals and their Venoms; Buücheri, W.; Buckley, E. E., eds.; Academic Press: New York, Vol. II, p. 345.

8. Landucci, E. C. T.; Condino-Neto, A.; Perez, A. C.; Hyslop, S.; Corrado, A. P.; Novello, J. C.; Marangoni, S.; Oliveira, B.; Antunes, E.; De Nucci, G.; Toxicon 1994, 32, 217.

9. Rübsamen, K.; Breithaupt, H.; Habermann, E.; Naunyn-Schmiedeber's Arch. Pharmacol. 1971, 270, 274; Hendon, R. A.; Fraenkel-Conrat, H.; Proc. Natl. Acad. Sci. U. S. A. 1971, 68, 1560.

10. Breithaupt, H.; Toxicon 1976, 14, 221; Habermann, E.; Breithaupt, H.; Toxicon 1978, 16, 19.

11. Faure, G.; Guillaume, J. L.; Camoin, L.; Saliou, B.; Bom, C.; Biochemistry 1991, 30, 8074.

12. Faure, G.; Choumet, V.; Bouchier, C.; Camoin, L.; Guillaume, J. L.; Monegier, B.; Vuilhorgne, M.; Bon, C.; Eur. J. Biochem. 1994, 223, 161.

13. Cho, W.; Markowitz, M. A.; Kézdy, F. J.; J. Am. Chem. Soc. 1988, 110, 5166.

14. Sheldon, R. A.; Adv. Synth. Catal. 2007, 349, 1289.

15. Schoevaart, R.; Wolbers, M. W.; Golubovic, M.; Ottens, M.; Kieboom, A. P. G.; van Rantwijk, F.; van der Wielen, L. A. M.; Sheldon, R. A.; Biotechnol. Bioeng. 2004, 87, 755.

16. Matijosyte, I.; Arendts, I. W. C. E.; De Vries, S.; Sheldon, R. A.; J. Mol. Catal. B: Enzym. 2010, 62, 142.

17. Chen, C. -H.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J.; J. Am. Chem. Soc. 1982, 104, 7294.

18. Hernandez-Oliveira, S.; Toyama, M. H.; Toyama, D. O.; Marangoni, S.; Hyslop, S.; Rodrigues-Simioni, L.; Protein J. 2005, 24,233; Oliveira, D. G.; Toyama, M. H.; Novello, J. C.; Beriam, L. O. S.; Marangoni, S.; J.Protein Chem. 2002, 21, 161.

19. Mino, T.; Hasegawa, T.; Shirae, Y; Sakamoto, M.; Fujita, T.; J. Organomet. Chem. 2007, 692, 4389.

20. Saito, T.; Nishimoto, Y.; Yasuda, M.; Baba, A.; J. Org. Chem. 2006, 71, 8516.

21. Ohkuma, T.; Hattori, T.; Ooka, H.; Inoue, T.; Noyori, R.; Org. Lett. 2004, 6, 2681.

22. Furniss, B. S.; Hannaford, A. J.; Rogers, V.; Smith, P. W. G; Tatchell, A. R.; Vogel's Textbook of Practical Organic Chemistry; 3^th ed., Longman: London, 1978.

23. Naik, S.; Kavala, V.; Gopinath, R.; Patel, B.; Arkivoc 2006, 1, 119.

24. Ford-Moore, A. H.; J. Chem. Soc. 1946, 679.

25. Buksha, S.; J. Labelled Compd. Radiopharm. 2005, 48, 337.

26. Jansson, A. J. M.; Klunder, A. J. H.; Zwanenburg, B.; Tetrahedron 1991, 47,7645.

27. Ferraz, H. M. C.; Bianco, G. G.; Bombonato, F. I.; Andrade, L. H.; Porto, A. L. M.; Quim. Nova 2008, 31, 813.

28. Yadav, J. S.; Nanda, S.; Reddy, P. T.; Rao, A. B.; J. Org. Chem. 2002, 67, 3900.

29. Holzer, M.; Mackessy, S. P.; Toxicon 1996, 34, 1149.

30. Lópes-Serrano, P.; Cao, L.; van Rantwijk, F.; Sheldon, R. A.; Biotechnol. Lett. 2002, 24, 1379.

31. Yadav, J. S.; Nanda, S.; Reddy, P. T.; Rao, A. B.; J. Org. Chem. 2002, 67, 3900.

Submitted: June 16, 2010

Published online: September 30, 2010

FAPESP has sponsored the publication of this article.

Publication Dates

• Publication in this collection
  14 Feb 2011
• Date of issue
  Feb 2011

History

• Received
  16 June 2010
• Accepted
  30 Sept 2010