Evaluation of Snake Venom Phospholipase A 2 : Hydrolysis of Non-Natural Esters

Fosfolipase A 2 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 PLA 2 (EC 3.1.1.4)are calciumdependent 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. 1These enzymes are common in pancreatic juice in snake and insect venoms.Mammalian PLA 2 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 PLA 2 occurs in groups I (Elapidae and Hydrophidae) and II (Crotalidae and Viperidae).Group II contains the catalytically active D49 PLA 2 as well the K49 isoforms, which has low or no activity. 3In addition to their enzymatic activity, venom PLA 2 may have neurotoxic, myotoxic, hemolytic, anticoagulant, cardiotoxic, and edematogenic activities as well as platelet aggregatory or antiaggregatory actions. 4rotoxin, a heterodimeric complex with phospholipase A 2 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 hemolytic 7 and platelet aggregating 8 activities.The crotoxin complex consists of a basic PLA 2 and an acidic, nonenzymatic component known as crotapotin, 9 which increases the pharmacologic activity of PLA 2 by acting as a chaperone protein for the enzyme. 10Crotoxin exists in several isoforms which result from the random association of the PLA 2 with crotapotin 11 following the post-translational modification of a single precursor of crotoxin or the expression of different mRNAs. 12he chemical mechanism of the enzymatic catalysis involving PLA 2 is not yet fully explored.In order to obtain more information about the mechanism of the Vol.22, No. 2, 2011   PLA 2 -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 Ca 2+ 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, R 1 = 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 Ca 2+ 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 A 2 carried out at pH 8 containing 10 mmol L -1 calcium chloride (CaCl 2 ) and 0.1 mol L -1 sodium chloride (NaCl) in acetonitrile.
In this study we explore the enzymatic reactivity of the phospholipase A 2 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). 14Several reports have described the CLEA activity to exceed that of the native enzyme with lipases and other enzymes. 15The 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

General procedures
Proton and carbon nuclear magnetic resonance ( 1 H NMR and 13  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 A 2 were isolated from Crotalus durissus terrificus venom and purified by a research group from the Biology Institute of Unicamp (Campinas-SP). 18ll 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 A 2 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 °C.

General procedure for biocatalysis reactions
The biocatalysis reactions were made by adding the CLEA prepared in 1 mL of Tris-HCl/NaCl/CaCl 2 (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)-4nitrobenzoic 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 PLA 2 and the Tris-HCl/CaCl 2 /NaCl buffer were added to the microplate.The reaction mixtures were stirred at 22 ºC until stabilization.

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 Na 2 SO 4 , and evaporated.The Vol. 22

General procedure for reduction of ketones
The ketone substrate (5.5 mmol) was dissolved in dry methanol with sodium borohydride (NaBH 4 ) (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 Na 2 SO 4 and evaporated.The alcohol residue was purified by flash chromatography eluting with 20% ethyl acetate in hexane.

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

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 °C 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 Na 2 SO 4 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] D 20 +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] D 20 -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 A 2 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 PLA 2 activity since it shows the highest specificity toward the Ca 2+ dependent phospholipase. 29The 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 A 2 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)-4nitrobenzoic acid 2b.Initially, we tested the hydrolysis of both substrates in a blank reaction (acetonitrile 3%, water in Tris-HCl/CaCl 2 /NaCl at pH 8) to be sure that 2a and The enzymatic hydrolysis of 2b in 3% acetonitirle (v/v) in a buffer of Tris-HCl/CaCl 2 /NaCl at pH 8 with the free phospholipase at 25 o C gave a 25% yield and 7% ee after 4 days (Scheme 1).
When the reaction was carried out at 45 o C 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. 31nally, 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 A 2 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.

Table 1 .
CLEA enzyme activities with several different precipitant and surfactants in the hydrolysis of 4-nitro-3(octanoyloxy)benzoic acid 1 Compared to activity of the same amount of free PLA 2 enzyme; b polyethylenediimine; c 1,2-dimethoxyethane. Vol.22,No.2, 20112b 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. a

Table 3 .
Hydrolysis of esters 5 and 6 with free PLA 2 and with CLEAphospholipase

Table 4 .
Hydrolysis of esters 11a and b with free PLA 2 and with CLEAphospholipase entry substrate enzyme Product, yield (%) Product, ee (%)