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

Article abstract of DOI:10.1590/S0103-50532011000200016

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.

Full text of DOI:10.1590/S0103-50532011000200016

J. Braz. Chem. Soc., Vol. 22, No. 2, 300-307, 2011.
Printed in Brazil - ©2011 Sociedade Brasileira de Química
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Article
A
Evaluation of Snake Venom Phospholipase A2: Hydrolysis of Non-Natural Esters
Renan A. S. Pirolla, Paulo A. Baldasso, Sérgio Marangoni, Paulo J. S. Moran and
José Augusto R. Rodrigues*
Department of Organic Chemistry, Institute of Chemistry, University of Campinas, PO Box 6154,
13083-970 Campinas-SP, Brazil
FosfolipaseA2 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.
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: phospholipaseA2, snake venom PLA2, cross-linking enzyme aggregate, biocatalysis
Introduction
enzymatic activity, venom PLA2 may have neurotoxic,
myotoxic, hemolytic, anticoagulant, cardiotoxic, and
edematogenic activities as well as platelet aggregatory
or antiaggregatory actions.4
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
Crotoxin, a heterodimeric complex with phospholipase
A2 activity, is the main neurotoxic component in the
venom of the SouthAmerica 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
*e-mail: jaugusto@iqm.unicamp.br

Vol. 22, No. 2, 2011
Pirolla et al.
301
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 phospholipasesA2 carried out
at pH 8 containing 10 mmol L-1 calcium chloride (CaCl2)
and 0.1 mol L-1 sodium chloride (NaCl) in acetonitrile.
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
Protonandcarbonnuclearmagneticresonance(1HNMR
and 13C NMR) were recorded in CDCl3 solution on Varian
INOVA 500 MHz, Bruker Advance DPX 250 MHz and
BrukerAdvance 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 mm ×
0.25 mm) capillary column. Chiral analyses were performed
in aAgilent 6850N GC FID, using a MN Hydrodex®-b-3P
(30 m × 250 mm × 0.25 mm), Chrompack Chirasil-dex CB
(30 m × 250 mm × 0.25 mm) or Lipodex-E 2,6-Pe-3-Bu-g-
CD (28 m × 250 mm × 0.25 mm) 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
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
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
phospholipaseA2 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

302
Evaluation of Snake Venom Phospholipase A2
J. Braz. Chem. Soc.
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.
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.
Synthesis of 4-nitro-3-(octanoyloxy)benzoic acid 1a
3-(2-Bromohexanoyloxy)-4-nitrobenzoic acid 2a
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 °C) 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 °C; IR (KBr) nmax/cm-1 3453,
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 °C) 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).
1
3067, 2959, 2855, 1786, 1696, 1595, 1532. H NMR
(CDCl3) d 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) d 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).
Yellow solid; mp 125-125.6 °C; IR (KBr) nmax/cm-1
3282, 3065, 2959, 2875, 2560, 1780, 1697, 1594, 1533,
1
1306, 1215, 1102, 955, 924, 841, 795, 527. H NMR
(CDCl3) d 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) d 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).
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.
3-(2-Methylhexanoyloxy)-4-nitrobenzoic acid 2b
General procedure for biocatalysis reactions
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
The biocatalysis reactions were made by adding the
CLEA prepared in 1 mL ofTris-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-

Vol. 22, No. 2, 2011
Pirolla et al.
303
residue was recrystallized from ethyl acetate-hexane
(1:10 v/v).
(%):148 (M+, 31), 147 (35), 130 (100), 120 (76), 115 (29),
105 (29), 91 (54).21
Pale yellow solid; mp 135-137 °C; IR (KBr) nmax/cm-1
3444, 3096, 2959, 2929, 2863, 1773, 1627, 1594, 1536, 1327,
1242, 1223, 746. 1H NMR (CDCl3) d 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) d 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).
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)
Synthesis of substrates
n
max/cm-1 3034, 2982, 2934, 1744, 1495, 1453, 1371, 1241,
1209, 1065, 1029, 944, 699. 1H NMR (CDCl3) d 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) d 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
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-Phenylethyl propionate 5b
From 1-phenylethanol 7, colorless oil; IR (KBr)
n
max/cm-1 3063, 3034, 2982, 2941, 2880, 1738, 1495, 1454,
1368, 1273, 1189, 1064, 1029; 1H NMR (CDCl3) d 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) d 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
1-Phenylethanol 7
From acetophenone: colorless oil; IR (KBr) nmax/cm-1
3359, 3063, 3029, 2973, 2927, 2876, 1493, 1451, 1369,
1
1302, 1204, 1077, 1010, 996, 899. H NMR (CDCl3) d
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) d 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
p-Nitrophenyl-1-ethyl acetate 6a
From p-nitrophenyl-1-ethanol 8, white solid; mp
(S)-p-Nitrophenyl-1-ethanol 8
49-51 °C; IR (KBr) nmax/cm-1 2965, 2923, 2856, 1731,
1602, 1518, 1347; 1248, 1069, 856. H NMR (CDCl3)
1
From p-nitroacetophenone: yellow oil; IR (KBr)
n
max/cm-1 3414, 2978, 2931, 2866, 1735, 1605, 1520, 1347,
d 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) d 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
1254, 1090, 1046, 855. 1H NMR (CDCl3) d 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) d 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
p-Nitrophenyl-1-ethyl propionate 6b
1,2,3,4-Tetrahydronaphthalen-1-ol 12
From p-nitrophenyl-1-ethanol 8, white solid; mp
From 1-tetralone: colorless oil; IR (KBr) nmax/cm-1
3285, 3041, 2946, 2928, 2855, 2835, 1491, 1453, 1431,
1286, 1267, 1069, 42. 1H NMR (CDCl3) d 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) d 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
36-37 °C; IR (KBr) nmax/cm-1 3066, 2989, 2946, 1736, 1605,
1
1524, 1333, 1184, 1086, 1065, 855. H NMR (CDCl3) d
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) d
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).

304
Evaluation of Snake Venom Phospholipase A2
J. Braz. Chem. Soc.
(R)-1,2,3,4-Tetrahydronaphthalen-1-yl acetate 11a
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.
ThiscompoundiscurrentlyusedtoassayPLA2 activitysince
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.
Yellow oil; IR (KBr) nmax/cm-1 3064, 3023, 2940, 2859,
2838, 1731, 1492, 1455, 1371, 1238, 1210, 1154, 1030,
1016, 882. 1H NMR (CDCl3) d 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) d 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) nmax/cm-1 3023, 2979, 2941,
2870, 2840, 1731, 1492, 1455, 1271, 1185, 1152, 1080,
766. 1H NMR (CDCl3) d 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) d: 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
Table 1. CLEA enzyme activities with several different precipitant and
surfactants in the hydrolysis of 4-nitro-3(octanoyloxy)benzoic acid 1
Determination of absolute configurations
Activitya of
CLEA, %
entry
Precipitant
(NH4)2SO4
Surfactant
Bioreduction of α-tetralone with Daucus carota
1
Without
Triton X-100
PEIb
95
40
13
60
7
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 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).
2
3
4
PEG 600
DMEc
Without
Triton X-100
PEI
5
6
0
7
Without
Triton X-100
PEI
25
20
6
8
9
10
11
12
Acetone
Without
Triton X-100
PEI
16
0
6
aCompared to activity of the same amount of free PLA2 enzyme;
bpolyethylenediimine; c1,2-dimethoxyethane.
Bioreduction of p-nitroacetophenone with daucus carota
Using a procedure similar to the describe above, 100 mg
(0.60mmol)ofp-nitroacetophenonewasreactedwith30gof
carrotroot.FollowingseparationsandGCanalysistheproduct
(S)-p-nitrophenyl-1-ethanol 8 was obtained in 81% yield
and ee 96% with [α]D20 -24 (c 1.48 in THF).
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
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,

Vol. 22, No. 2, 2011
Pirolla et al.
305
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).
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
Scheme 1.
Table 3. Hydrolysis of esters 5 and 6 with free PLA2 and with CLEA-
phospholipase
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.
entry Substrate Enzyme Product, Yield (%)
Product, ee (%)
1
2
3
4
5
6
7
8
5a
5a
5b
5b
6a
6a
6b
6b
free PLA2 7, 40
CLEA 7, 67
free PLA2 7, 20
CLEA 7, 64
free PLA2 8, 55
CLEA 8, 83
free PLA2 8, 34
5a, 35
5a, 53
5b, 20
5b, 48
6a, 68
6a. 79
6b, 35
6b, 70
7, 3
7, 16
7, 3
5a, 2
5a, 3
5b, 20
5b, 28
6a, 24
6a, 4
7, 6
8, 19
8, 5
8, 10
8, 6
6b, 15
6b, 7
CLEA
8, 72
Table 2. Hydrolysis of 3-(2-methylhexanoyl)-4-nitrobenzoic acid 2b with
free PLA2 and with CLEA-phospholipase
entry
Enzyme
Temperature
(oC)
Yield (%)
ee (%)
1
2
3
4
Free PLA2
Free PLA2
CLEA
25
45
25
45
25
52
55
76
7
3
9
5
Scheme 2.
CLEA
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).
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

306
Evaluation of Snake Venom Phospholipase A2
J. Braz. Chem. Soc.
Table 4. Hydrolysis of esters 11a and b with free PLA2 and with CLEA-
phospholipase
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;
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entry substrate enzyme Product, yield (%)
Product, ee (%)
15, 3 11a, 3
15, 16 11a, 10
1
2
3
4
11a
11a
11b
11b
PLA2 free 12, 53
CLEA 12, 85
11a, 55
11a, 86
PLA2 free 12, 28 11b, 20
15, 1
15, 3
11b, 1
11b, 3
CLEA
12, 60 11b, 70
8. Landucci, E. C. T.; Condino-Neto, A.; Perez, A. C.; Hyslop,
S.; Corrado, A. P.; Novello, J. C.; Marangoni, S.; Oliveira, B.;
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10. Breithaupt, H.; Toxicon 1976, 14, 221; Habermann, E.;
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Scheme 3.
11. Faure, G.; Guillaume, J. L.; Camoin, L.; Saliou, B.; Bom,
C.; Biochemistry 1991, 30, 8074.
As in the case of 1-phenylethanol 7, the absolute
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Guillaume, J. L.; Monegier, B.; Vuilhorgne, M.; Bon, C.; Eur.
J. Biochem. 1994, 223, 161.
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
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.
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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.
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.
Acknowledgments
20. Saito, T.; Nishimoto, Y.; Yasuda, M.; Baba, A.; J. Org.
Chem. 2006, 71, 8516.
This work was financially supported by FAPESP and
CNPq. R. A. S. Pirolla thanks CAPES for fellowship.
21. Ohkuma, T.; Hattori, T.; Ooka, H.; Inoue, T.; Noyori, R.;
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Submitted: June 16, 2010
Published online: September 30, 2010
FAPESP has sponsored the publication of this article.