A.M. Escorcia et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 21–29
23
Blank experiments were carried out in addition, using the same
reaction conditions but without addition of the enzyme. No acety-
lated product of propranolol was detected under these conditions.
CalB was purified for being used in the acetylation of (R,S)-
propranolol. The purification was done by chromatography based
on the adsorption of the lipase on octyl agarose beads at low ionic
strength [61]. A solution of CalB (20 mL, 0.7 mg/mL) in sodium phos-
phate buffer (5 mM, pH 7) having an activity of 167 U mg−1 in pNPB
assay (see below) was incubated with octyl agarose beads (4 g) dur-
ing 2 h. Periodically, the activity of suspensions and supernatants
was assayed using the p-nitrophenylbutyrate (p-NPB) assay as
described below. The protein concentration in the supernatants
was also determined, using Bradford’s method [62]. After immobi-
lization, the adsorbed lipase was filtered and washed with distilled
water. Then the enzyme was desorbed using sodium phosphate
buffer (20 mL, 5 mM, pH 7) containing Triton X-100 (1%, v/v) [61].
This enzyme solution was diluted 5-fold in sodium phosphate
buffer (5 mM, pH 7) to dilute the detergent. A fraction (50 mL) of
this diluted enzyme solution was dialyzed to remove the deter-
gent. The rest was kept as it was. Finally, both enzyme fractions
were lyophilized and used for the acetylation of propranolol. These
enzyme preparations are referred here as CalB-I and CalB-II respec-
tively. As Triton X-100 has been shown to promote an increase
of the activity of CalB [63], these two enzyme preparations were
obtained to evaluate the effect of this detergent on the activity, the
chemo- and the enantioselectivity of CalB.
2.1.6. HPLC analysis
The conversion of the enzyme reaction was determined by
HPLC (Agilent 1100) using a Silica gel C-18 column (Zorbax C-
18, Agilent Technologies). The mobile phase was composed of
acetonitrile and phosphate buffer pH 6 (70:30) and the samples
a chiral column (25 cm, ES-OVM, Agilent-Technologies, USA) with
methanol/phosphate buffer pH 6 (30:70) at 1.0 mL min−1 as mobile
phase [13]. The enantioselectivity (E) was calculated using the
equation reported by Chen et al. [65].
2.1.7. NMR analysis
1H, 13C, DEPT135◦, HMBC and HSQC NMR spectra were recorded
on
a Bruker Avance III, 400 MHz spectrometer, using CDCl3
as the solvent and tetramethylsilane (TMS) as internal refer-
ence. The experimental conditions were: for 1H, spectral width
(SW) = 4400 Hz (−0.5 to 10.5 ppm), Bruker pulse program zg30
and number of scans = 16; the quantitative 13C NMR spectra were
obtained in the inverse gated mode for fully decoupled spec-
tra with no Nuclear Overhauser Effect (NOE) with spectral width
(SW) = 24,038 Hz (−20 to 219 ppm), Bruker pulse program zgig30
and number of scans = 4096; the two-dimensional phase-sensitive
gradient selected edited heteronuclear single quantum coherence
– HSQC- spectra were obtained with the Bruker pulse program
hsqcedetgp; for the two-dimensional heteronuclear multiple bond
correlation – HMBC- spectra the Bruker pulse program hmbcg-
plpndqf was used; for distortionless enhancement by polarization
transfer – DEPT135◦- spectra the Bruker pulse program dept135
and 2048 scans were used. The products were identified by per-
forming a full assignment of 1H and 13C chemical shifts using
DEPT135◦ as well as 1H–13C HMBC and HSQC heteronuclear cor-
relation techniques (see Supplementary Data for details).
2.1.3. Hydrolysis of p-NPB
This assay was performed by measuring the increase in
absorbance at 348 nm produced by the released p-nitrophenol
in the hydrolysis of p-NPB (0.4 mM) in sodium phosphate buffer
(25 mM, pH 7.0) at 25 ◦C. To start the reaction, the lipase solution or
suspension (0.02 mL) was added to the substrate solution (2.5 mL).
One international unit of activity (U) was defined as the amount
of enzyme that hydrolyzes 1 mol of p-NPB per minute under the
conditions described previously.
2.1.4. Chemical acetylation of (R,S)-propranolol
This reaction was carried out using an analogous procedure
to the classical methodology of Gatterman [64] for the synthe-
sis of (R,S)-O-butyryl propranolol (RS-O-BP): (R,S)-propranolol-HCl
(0.2 g; 0.76 mmol) was refluxed with dichloromethane (20 mL) and
acetyl chloride (0.08 mL; 0.76 mmol) was very slowly added. After
two hours, the reaction mixture was washed successively with
equal volumes of saturated aqueous sodium bicarbonate and brine.
The organic layer was dried over anhydrous sodium sulfate and
evaporated to dryness under reduced pressure to afford N-acetyl
propranolol (N-AP) and O-acetyl propranolol (O-AP).
2.2. Computational methods
The computational part of this study involved the following
stages: preparation of the starting structures corresponding to the
protein and the substrate (R or S propranolol), modeling of the
acetylated CalB (the docking target), docking of both enantiomers
of propranolol, optimization and structural analysis of the poses
with highest interaction free energy from the docking procedure,
and finally molecular dynamics simulations to check the reliability
of the final models.
2.1.5. Lipase-catalyzed acetylation of (R,S)-propranolol
Different experiments were carried out in order to study the
effect of the reaction conditions on the velocity, the chemo-
and the enantioselectivity of the reaction. As propranolol-HCl is
poorly soluble in toluene (<0.5 mg/mL), methanol was added as
a cosolvent according to the amount of propranolol to be sol-
vated: (a) (R,S)-propranolol (39 mM) was dissolved in a solution
of toluene/methanol (20 mL, 93/7, v/v) containing vinyl acetate
(117 mM). (b) (R,S)-propranolol (8–28 mM) was dissolved in a solu-
tion of toluene/methanol (5 mL, 96/4, v/v) containing vinyl acetate
(54 mM). To start these reactions the purified enzyme was added
(3 mg). The reaction mixtures were continuously shaken at 200 rpm
and 25 ◦C.
2.2.1. Preparation of the acetylated CalB (AcCalB) structure
The enzymatic mechanism of lipases (including CalB) involves a
catalytic triad consisting of Serine, Histidine and Aspartate (Ser105,
step is the addition of an acyl-group to the catalytic serine of the
enzyme, yielding the acyl-enzyme. In the second step, the acyl-
group can react with several nucleophiles, such as water, alcohols,
amines or peroxides [19]. According to the general mechanism
of lipase catalyzed acylations, acylation as well as deacylation,
proceed via a tetrahedral intermediate (TI), which is stabilized by
NH and OH functions in the so-called oxyanion hole of the enzyme,
constituted by the residues Thr40 and Gln106 in CalB. In the CalB-
catalyzed acetylation of (R,S)-propranolol, the deacylation part of
the reaction mechanism (reaction 2 in Scheme S2) may be chemo-
(N- or O-acetylation) and stereoselective (acetylation of R- or
Methanol was chosen as cosolvent based on the solubility of
propranolol in this solvent (200 mg/mL). THF, hexane and ethanol
were also tested, but the solubility of propranolol in these solvents
is 9 mg/mL, 3 mg/mL and 10 mg/mL, respectively.