Influence of the acyl moiety on the hydrolysis of quinuclidinium esters catalyzed by butyrylcholinesterase

Article abstract of DOI:10.5562/cca1829

Eight chiral esters of quinuclidin-3-ol and butyric, acetic, pivalic and benzoic acid were synthesized as well as their racemic and chiral, quaternary N-benzyl derivatives. All racemic and chiral quaternary compounds were studied as substrates and/or inhi

Full text of DOI:10.5562/cca1829

CROATICA CHEMICA ACTA
CCACAA, ISSN 0011-1643, e-ISSN 1334-417X
Croat. Chem. Acta 84 (2) (2011) 245–249.
CCA-3471
Original Scientific Article
Influence of the Acyl Moiety on the Hydrolysis of Quinuclidinium Esters
Catalyzed by Butyrylcholinesterase†
*
Ines Primožič and Srđanka Tomić
Department of Chemistry, Faculty of Science, University of Zagreb Horvatovac 102a, HR-10000 Zagreb, Croatia
RECEIVED APRIL 15, 2011; REVISED JULY 1, 2011; ACCEPTED JULY 6, 2011
Abstract. Eight chiral esters of quinuclidin-3-ol and butyric, acetic, pivalic and benzoic acid were synthe-
sized as well as their racemic and chiral, quaternary N-benzyl derivatives. All racemic and chiral quater-
nary compounds were studied as substrates and/or inhibitors of horse serum butyrylcholinesterase
(BChE). The best substrate for the enzyme was (R)-N-benzyl butyrate. The rates of hydrolysis decreased
in order (R)-butyrate >> (R)-acetate (7-fold slower) > (R)-pivalate (8-fold slower) > (R)-benzoate (9-fold
slower reaction), while (S)-N-benzyl esters were much poorer substrates (320 (butyrate) - 4360-fold slow-
er (pivalate) than the appropriate (R)-enantiomer). For all (S)-N-benzyl esters excluding (S)-N-benzyl ace-
tate inhibition constants were determined (Ka = 3.3−60 μmol dm−3). The hydrolysis of racemic mixtures of
N-benzyl esters proceeded 1.4 (for acetate) − 5.1 (for benzoate) times slower than that of pure (R)-
enantiomers of the corresponding concentrations due to the inhibition with (S)-enantiomers. Change of the
acyl moiety of the substrate effected both activity and stereoselectivity of the BChE.(doi:
10.5562/cca1829)
Keywords: esters of quinuclidin-3-ol, enzymic resolution, butyrylcholinesterase, hydrolysis kinetics, inhi-
bition constants
INTRODUCTION
lution of racemic nonquaternized quinuclidin-3-yl bu-
tyrate8 and for the hydrolysis of (R)- and (S)-quinuclidin-
3-yl benzoates,14−16 stereoselectivity of hydrolyses being
in favour of the (R)-enantiomer.
Azabicyclo[2.2.2]octan-3-ol, its derivatives and other
quinuclidine compounds display a broad range of bio-
logical activities and pharmaceutical industries have
large interest to explore them.1 Among other activities,
they were shown to be potential antidotes against or-
ganophosphorus poisoning caused by warfare agents.2−4
Quinuclidin-3-ol is a chiral compound which contains an
asymmetric carbon atom at the position 3 of the bicyclic
ring. Since the racemates are regarded with suspicion as
pharmaceuticals (enantiomers can have different activity
or even toxic effects), the issue of quinuclidin-3-ol reso-
lution has been addressed by using a number of chemi-
cal5−7 and biocatalytic8,9 methods. One of the enzymes
tested as a biocatalyst was butyrylcholinesterase from
horse serum (BChE, EC 3.1.1.8.). This enzyme has not
been used as much as some other esterases for biotrans-
formations in organic chemistry10 because of its prefer-
ence toward positively charged substrates which resem-
ble its best substrate: butyryl choline.11−13 Structural
similarity among choline and quinuclidin-3-ol implied
that quinuclidine esters might be good substrates of the
enzyme. Thus, it was possible to use BChE for the reso-
In this paper the synthesis of quaternary, N-benzyl
derivatives of racemic, (R)- and (S)-quinuclidin-3-yl
butyrate, acetate, pivalate and benzoate are reported,
Figure 1. Quaternization with N-benzyl group was used
because it can be considered as a protecting group
which can be successfully removed afterwards.17
All synthesized quaternary compounds were stud-
ied as substrates and/or inhibitors of horse serum BChE
to determine the potential of BChE as a biocatalyst in
kinetic resolution of quinuclidine esters.
Figure 1. Structures of synthesized quinuclidinium esters.
†
This article belongs to the Special Issue Chemistry of Living Systems devoted to the intersection of chemistry with life.
* Author to whom correspondence should be addressed. (E-mail: ines.primozic@chem.pmf.hr)

246
I. Primožič et al., Stereoselective BChE-Catalyzed Ester Hydrolysis
EXPERIMENTAL SECTION
(m, 1H, H3); 13C NMR (CDCl3)  / ppm: 13.41 (−CH3),
18.27 (C8), 19.20 (C5), 24.15 (−CH2CH2CH3), 24.91
(C4), 36.20 (−CH2CH2CH3), 46.16 (C6), 47.07 (C7),
55.20 (C2), 70.62 (C3), 173.47 (C=O); ESMS: m/z (m/z
Melting points were determined in open capillaries
using a Büchi B-540 melting point apparatus and are
uncorrected. Specific optical rotation values were de-
termined with an Optical Activity LTD automatic pola-
rimeter AA-10 on 589 nm at ambient temperature (≈24
°C) in methanol. Elemental analyses were performed
with a Perkin-Elmer PE 2400 Series II CHNS/O Ana-
lyser. IR spectra were recorded with a Perkin-Elmer
+
calcd. for C11H20NO2 , 198.14) found 198.2.
3-Butyryloxy-1-benzylquinuclidinium bromide
(BuBzl): recrystallization from methanol/ether gave
white crystals, yield 94 %; m.p. 169.1−170.0 °C (de-
[α] 2D3
[α] 2D3
comp.); (R)-enantiomer (RBuBzl):
EtOH); (S)-enantiomer (SBuBzl):
+19.0° (c = 1,
−19.0° (c = 1,
1
FTIR 1725 X spectrometer. H and 13C 1D and 2D
EtOH); UV (EtOH) max/nm: 257, 262 and 269 (log  /
−1
−1
dm3 mol−1 cm : 2.50; 2.55 and 2.42); IR (KBr) ν /cm :
3022, 2955, 2877, 1726, 1461, 1453, 1378, 1283, 1156,
1002, 980, 706; 1H NMR (CDCl3)  / ppm: 0.86 (t, 3H,
−CH3, 3J = 7,4 Hz), 1.50−1.65 (m, 2H, −CH2CH3),
1.87−2.49 (m, 5H, H5, H8 and H4), 2,34 (t, 2H,
NMR spectra were recorded with Varian XL-GEM 300
spectrometer at room temperature. Chemical shifts are
given in ppm downfield from TMS as internal standard.
HPLC analyses (Thermo Separation Products, Spectra-
SYSTEM 2000) were performed on a RP18 (Waters,
SymmetryShield, 5 μm, 3.9×150 mm) column (40 °C).
The mobile phase used was water/ methanol/ acetoni-
trile/ acetic acid/ triethylamine (60/25/15/0.33/0.2), flow
rate 1 ml/min. The reactions were carried out in Hei-
dolph UNIMAX 1100 Shaker. BChE (EC 3.1.1.8), type
IV-S lyophilized powder from horse serum (Sigma
Chemical Co.) was used without further purification.
The hydrolysis of esters catalyzed by BChE was moni-
tored by following the production of N-benzyl-
quinuclidin-3-ol by HPLC analysis as described pre-
viously.15 All experiments for (R)-enantiomers were
performed with 1.5 × 10−9 mol dm−3 and for (S)-en-
antiomers with 1.5 × 10−8 mol dm−3 concentration of
BChE. The dissociation constants of enzyme-inhibitor
complex for (S)-enantiomers were determined from
Hunter-Downs plot, using benzoyl choline (BzCh) as a
substrate.

3
−CH2CH2CH3, J = 7,4 Hz), 3.31−4.02 (m, 5H, H6, H7
and 1H2 cis), 4.20−4.40 (m, 1H, H2 trans), 5.05−5.21
(m, 3H, −CH2− bzl and H3), 7.25−7.71 (m, 5H, bzl); 13
C
NMR (CDCl3)  / ppm: 13.38 (−CH3), 17.99 (C8), 18.21
(−CH2CH2CH3), 21.20 (C5), 24.53 (C4), 35.68
(−CH2CH2CH3), 53.23 (C6), 53.64 (C7), 59.74 (C2),
66.31 (−CH2− bzl), 66.69 (C3), 126.59 (C1 bzl), 129.00
(C3, C5 bzl), 130.34 (C4 bzl), 133.18 (C2, C6 bzl), 172.56
+
(C=O); ESMS: m/z (calcd. for C12H22NO2 288.20)
found 288.3.
Anal. Calcd. mass fractions of elements, w/%, for
C18H26BrNO2 (Mr = 368.31) are C 58.70, H 7.12, N
3.80; found: C 57.21; H 7.03; N 3.89.
Quinuclidin-3-yl pivalate, colourless oil, yield 97 %,
(R)-enantiomer:
27 +18.4° (c=2.0; EtOH); (S)-
[] D
[] 2D7

enantiomer
−17.9° (c=2.0, EtOH); IR (NaCl) ν
/cm−1: 2954, 2871, 1725, 1668, 1480, 1457, 1396, 1285,
1
1163, 1033, 981, 773; H NMR (DMSO-d6)  / ppm:
Preparation of substrates
1.19 (s, 9H, (CH3)3C), 1.31−1.77 (m, 4H, H5 and H8),
1.85−1.92 (m, 1H, H4), 2.38−2.76 (m, 5H, H6, H7 and
(R)- and (S)- quinuclidin-3-ol were prepared by a reso-
lution procedure as described previously using L- and
D-tartaric acid.5 All esters were prepared according to
the procedure described for quinuclidin-3-yl benzoate.14
N-quaternary derivatives were prepared by the addition
of benzyl bromide (2 equivalents) to the solution of
appropriate chiral quinuclidin-3yl ester in dry ether.
Synthesis and physical properties of benzoates
(BzBzl)15 and acetates (AcBzl)18 were described pre-
viously.
2
3
H2 cis), 3.09 (dd, 1H, H2, J=14.5 i J = 8.2 Hz, trans),
4.60−4.67 (m, 1H, H3); 13C NMR (DMSO-d6)  / ppm:
19.41 (C5), 24.05 (C8), 24.98 (C4), 26.91 ((CH3)3C),
38.28 (C(CH3)3), 45.94 (C6), 46.97 (C7), 55.30 (C2),
70.71 (C3), 177.46 (C=O); ESMS: m/z (calcd. for
C12H22NO2+ 212.16) found 212.2.
3-Pivaloyloxy-1-benzylquinuclidinium bromide
(PivBzl): recrystallization from methanol/ether gave
white crystals, yield 86 %; m.p. 166.4−167.4 °; (R)-
enantiomer (RPivBzl):
Quinuclidin-3-yl butyrate, colourless oil, yield 95 %,
[α] 2D8
–15.0° (c = 1.0, MeOH);
(S)-enantiomer (SPivBzl):
28 +15.2° (c = 1.0, MeOH);
[α] D
[α] 2D3
(R)-enantiomer:
–22.0° (c = 3, EtOH), (S)-enan-
UV (EtOH) max/nm: 257, 262 and 269 (log /dm3 mol−1
−1
[α] 2D3

tiomer
+22.0° (c = 3, EtOH); IR (NaCl) ν /cm :
−1
−1

cm : 2.50; 2.55 and 2.42); IR (KBr) ν /cm : 3032,
2948, 2873, 1731 1457, 1310,1257, 1185, 1081, 979,
779, 753; 1H NMR (CDCl3)  / ppm: 0.93 (t, 3H, −CH3,
3J = 7.4 Hz), 1.35−1.95 (m, 6H, H5, H8 and −CH2CH3),
2955, 2877, 1729, 1465, 1455, 1376, 1284, 1157, 1005,
990, 704; H NMR (DMSO-d6)  / ppm: 1.19 (s, 9H,
1
3
(CH3)3C-), 1.81−2.08 (m, 4H, H5, H8), 2.27−2.28 (m,
1H, H4), 3.42−3.56 (m, 5H, H6, H7, 1H2 cis), 3.87 (dd,
1H, H2, 2J = 12.9 and 3J = 8.7 Hz, trans), 4.61−4.72 (m,
1.95−2.01 (m, 1H, H4), 2.27 (t, 2H, −CH2CH2CH3, J =
7,4 Hz), 2.62−2.90 (m, 5H, H6, H7 and 1H2 cis), 3.21
(dd, 1H, H2, 2J = 14.6 and 3J = 8.5 Hz, trans), 4.66−4.81
Croat. Chem. Acta 84 (2011) 245.

I. Primožič et al., Stereoselective BChE-Catalyzed Ester Hydrolysis
247
Figure 2. The initial rates of BChE catalyzed hydrolysis of A) (R)-esters (A(BChE)/μmol dm−3 min−1 mg−1 equals 2069 for
RBuBzl, 280 for RAcBzl, 263 for RPivBzl and 236 for RBzBzl) and B) (S)-esters (A(BChE)/μmol dm−3 min−1 mg−1 equals
6.480 for SBuBzl, 0.625 for SAcBzl, 0.063 for SPivBzl and 0.203 for SBzBzl). Monitoring was done by the HPLC analysis of the
products. Concentration of all substrates was 4 mmol dm−3. Each data point represents the average value of three measurements.
2H, −CH2− bzl), 4.99−5.00 (m, 1H, H3), 7.50−7.59 (m,
5H, bzl); 13C NMR (DMSO-d6) /ppm: 18.08 (C5),
20.49 (C8), 24.20 (C4), 26.79 ((CH3)3C−), 38.28
((CH3)3C−), 52.10 (C6), 53.80 (C7), 59.83 (C2), 65.64
(−CH2− bzl), 67.07 (C3), 127.70 (C1 bzl), 129.00 (C3, C5
bzl), 130.24 (C4 bzl), 133.21 (C2, C6 bzl), 176.91 (C=O);
represent free enzyme, substrate, Michaelis complex
and acyl-enzyme intermediate, respectively. P1 (alcohol)
and P2 (acid) are products of the hydrolysis. In the case
of
tested
quinuclidinium
esters,
1-benzyl-3-
hydroxyquinuclidinium is P1 and P2 are acetic, pivalic
butyric and benzoic acid respectively.
+
ESMS: m/z (calcd. for C19H28NO2 302.21) found 302.2.
All tested chiral esters were substrates of the
BChE, Figure 2. Analyses of the initial rates revealed
that the hydrolysis of butyric esters was the fastest
among all examined enantiomers, and that in general the
(R)-enantiomers are significantly better substrates for
BChE then (S)-enantiomers. The rates of hydrolysis for
(R)-enantiomers decreased in order (R)-butyrate >> (R)-
acetate (7-fold slower) > (R)-pivalate (8-fold slower) >
(R)-benzoate (9-fold slower reaction). The enzyme was
even more selective in case of (S)-enantiomers: the rates
of hydrolysis decreased in order (S)-butyrate > (S)-
acetate (10-fold slower) > (S)-benzoate (32-fold slower)
> (S)-pivalate (103-fold slower), Figure 2, chart B. On
the other hand, difference in the initial rates for each
pair of enantiomers was the biggest for pivalates
(AR/S=4360), then benzoates (AR/S=1180), acetates
(AR/S=450) and butyrates (AR/S=320).
Anal. Calcd. mass fractions of elements, w/% , for
C19H28BrNO2 (Mr = 382.34) are C 59.69, H 7.38, N
3.66; found: C 60.38, H 7.12, N 3.59.
RESULTS AND DISCUSSION
Synthesis of quaternary quinuclidinium esters
Chiral (R)- and (S)-quinuclidin-3-ols were obtained by
the resolution of racemic quinuclidin-3-yl acetates with
L- and D-tartaric acid.5 N-benzyl derivatives of racemic,
(R)- and (S)-quinuclidin-3-yl acetates, benzoates,
pivalates and butyrates were synthesized by the esterifi-
cation of quinuclidin-3-ol with appropriate anhydride in
good yields. Quaternization of racemic and chiral esters
with benzyl bromide followed. The structure and purity
of all compounds were determined by HPLC, IR, MS,
Inhibition of BChE by (S)-enantiomers
1
elemental analyses, one- and two-dimensional H and
Since the rates of hydrolysis of (S)-enantiomers were
much slower than the rate of hydrolysis of benzoyl
choline (BzCh) which can be used to monitor enzyme
13C NMR.
Hydrolyses of quaternary esters with BchE
The overall catalytic process of BChE proceeds in three
steps: initial formation of an enzyme–substrate com-
plex, an acylation step, and deacylation by hydrolysis,19
Scheme 1. In the reaction sequence, E, S, ES, and EA
Scheme 1.
Croat. Chem. Acta 84 (2011) 245.

248
I. Primožič et al., Stereoselective BChE-Catalyzed Ester Hydrolysis
Table 1. Inhibition constants of BChE by (S)-N-benzyl esters:
the enzyme-inhibitor dissociation constant Ki and standard
deviation was obtained from four experiments on average. The
dissociation constant of enzyme-inhibitor complex was deter-
mined from Hunter-Downs plots. The value for SBzBzl was
published previously15
Inhibitor
SBuBzl
Km/mmol dm−3
0,19 ± 0,01
0,79 ± 0,02
1,6 ± 0,6
Ki/μmol dm−3
60,5 ± 0,5
12,3 ± 0,2
3,3 ± 0,4
SPivBzl
SBzBzl15
Km corresponds to (k−1/k1 + k2/k1)[k3/(k2 + k3)]
activity, it was possible to determine enzyme-inhibitor
dissociation constant Ki. The enzyme-inhibitor dissocia-
tion constant Ki was calculated from equation
Figure 3. The kinetic study of inhibitory effectiveness of
SAcBzl toward BChE in catalyzed hydrolyses of BzCh. Each
data point represents the average value of three measurements.
Concentrations of SAcBzl are expressed in mmol dm−3.
Kapp=Ki+(Ka/Km)·s, where Kapp is the apparent enzyme-
inhibitor dissociation constant at a given substrate con-
centration (s) and Km is the Michaelis constant for the
substrate. All (S)-esters acted as reversible inhibitors of
the enzyme, Table 1. Only SAcBzl inhibited the enzyme
when concentrations of BzCh were lower than 0.22
mmol dm−3, and activated the enzyme when the concen-
tration of BzCh was higher, Figure 3. Therefore, it was
not possible to determine the enzyme-inhibitor dissocia-
tion constant for that compound.
mine the differences in rates of hydrolysis of pure (R)-
enantiomers (2 mmol dm−3 solution) and racemic mix-
tures which contained the same concentration of (R)-
enantiomer (4 mmol dm−3 solution of the racemate). The
obtained values for enzymic activity are presented in
Table 2.
All other (S)-esters proved to be μmol dm−3 inhibi-
tors, (S)-benzoate was the best inhibitor, closely fol-
lowed by (S)-pivalate. (S)-butyrate had the lowest affin-
ity toward BChE, Table 1. The calculated values of Km
for the substrate used (BzCh) obtained from kinetic
studies with inhibitors can be compared with ones ob-
tained from data without inhibitors (Km (BzCh) =
0.17±0.01).14 It can be concluded that SBuBzl competed
with BzCh only in the active site while SPivBzl and
especially SBzBzl competed with the substrate in the
peripheral site as well.
All reactions of racemate mixtures were slower
than the ones of pure (R)-enantiomers. The hydrolysis
of RBuBzl was 2.0-fold, RPivBzl 2.7-fold faster, while
that of RBzBzl was the fastest (5.1-fold) in comparison
to the appropriate racemate. These data are in accor-
dance with the measured inhibition constants for (S)-
enantiomers, SBzBzl beeing the best inhibitor, followed
by (S)-pivalate and (S)-butyrate. It is interesting that in
the case of racemic AcBzl there was no effect of activa-
tion with present SAcBzl because RAcBzl was the sub-
strate instead of BzCh, Figure 3., in the enzyme activity
measurements. The hydrolysis of RAcBzl was 1.4-fold
faster when (S)-enantiomer was not present, thus the
inhibition of SAcBzl was the lowest in magnitude
To further explore the hydrolysis reaction of ra-
cemic N-benzyl quinuclidin-3-ol esters catalysed by
BChE, kinetic experiments were carried out to deter-
Table 2. Activity of BChE (A) measured for racemic mixtures and (R)-enantiomers of all compounds: 4 mmol dm−3 solution of
racemate and 2 mmol dm−3 solution of (R)-enantiomer. Each data point represents the average value of three measurements.
AR/racemate corresponds to the ratio of BChE activity for (R)-enantiomer and racemate
A(BChE)/mmol dm−3 min−1 mg−1
Substrate
Inhibition by (S)-enantiomer /
(R)-enantiomer
Racemate
AR/racemate
%
BuBzl
AcBzl
PivBzl
BzBzl
0.993
0.184
0.252
0.406
0.502
0.129
0.094
0.080
2.0
1.4
2.7
5.1
50
29
63
80
Croat. Chem. Acta 84 (2011) 245.

I. Primožič et al., Stereoselective BChE-Catalyzed Ester Hydrolysis
249
among (S)-enantiomers. The BChE catalysed hydrolysis
of RBzBzl was slower in case of 4mmol dm−3 solution
(Fig. 2.) than in case of 2 mmol dm−3 solution (Table 2.)
indicating inhibition by substrate (RBzBzl) when the
higher substrate concentrations are present.
N-benzyl quinuclidinium esters can be successfully
achieved by the stereoselective hydrolysis catalysed
with BChE.
Acknowledgements. This work was supported by the
Ministry of Science, Education and Sports of the Re-
public of Croatia, Research Project No. 119-1191344-
3121.
CONCLUSION
We have prepared racemic and enantiomerically pure
esters of chiral quaternary N-benzyl quinuclidin-3-ol
and butyric, acetic, pivalic and benzoic acid. Kinetic
studies of BChE-catalyzed ester hydrolysis showed that
the reaction proceeds in a stereoselective manner: (R)-
enantiomers were hydrolyzed preferentially. On the
other hand, (S)-enantiomers showed much higher affin-
ity toward BChE and all were determined as µmol dm−3
inhibitors of the enzyme with the exception of the ace-
tate derivative. It was revealed that there is a great
influence of the acyl moiety both on the activity and
stereoselectivity of the hydrolysis. The best substrates
for the enzyme were butyrates which acyl group is the
same as the one in butyrilcholine. Their acyl group
fitted the best in the acyl binding site of the enzyme
compared to other esters, resulting with the fastest
reactions but, at the same time, with the loss of selec-
tivity towards enantiomers. Smaller acetate group and
bigger pivalate and benzoate could not realize a maxi-
mum possible interactions and the activity of the en-
zyme was lower. Among (S)-enantiomers, the differ-
ences in the activity of BChE were even more pro-
nounced and partly related to their inhibitory proper-
ties. Effective inhibition of the enzyme indicates that
there is a strong non-productive binding of (S)-
enantiomers which can realize stabilizing contacts
within the active site.14,16
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Due to the satisfactory difference in the rates of
hydrolysis of enantiomers, BChE can be used as a bio-
catalyst in preparations of optically pure quaternary
quinuclidin-3-ols. N-benzyl group can be efficiently
removed by catalytic transfer hydrogenation,20 thus
regenerating chiral quinuclidin-3-ols, precursors for the
synthesis of a range of pharmacologically interesting
analogues. Consequently, kinetic resolution of racemic
Croat. Chem. Acta 84 (2011) 245.

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