Influence of the Acyl Moiety on the Hydrolysis of Quinuclidinium Esters Catalyzed by Butyrylcholinesterase

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 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 slower 
(pivalate) than the appropriate (R)-enantiomer). For all (S)-N-benzyl esters excluding (S)-N-benzyl acetate 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.


INTRODUCTION
−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 resolution has been addressed by using a number of chemical 5−7 and biocatalytic 8,9 methods.One of the enzymes tested as a biocatalyst was butyrylcholinesterase from horse serum (BChE, EC 3.1.1.8.).−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-lution of racemic nonquaternized quinuclidin-3-yl butyrate 8 and for the hydrolysis of (R)-and (S)-quinuclidin-3-yl benzoates, 14−16 stereoselectivity of hydrolyses being in favour of the (R)-enantiomer.
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. 17ll synthesized quaternary compounds were studied as substrates and/or inhibitors of horse serum BChE to determine the potential of BChE as a biocatalyst in kinetic resolution of quinuclidine esters.

EXPERIMENTAL SECTION
Melting points were determined in open capillaries using a Büchi B-540 melting point apparatus and are uncorrected.Specific optical rotation values were determined with an Optical Activity LTD automatic polarimeter 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 Analyser.IR spectra were recorded with a Perkin-Elmer FTIR 1725 X spectrometer. 1 H and 13 C 1D and 2D 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/ acetonitrile/ acetic acid/ triethylamine (60/25/15/0.33/0.2),flow rate 1 ml/min.The reactions were carried out in Heidolph 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 monitored by following the production of N-benzylquinuclidin-3-ol by HPLC analysis as described previously. 15All experiments for (R)-enantiomers were performed with 1.5 × 10 −9 mol dm −3 and for (S)-enantiomers 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.

Preparation of substrates
(R)-and (S)-quinuclidin-3-ol were prepared by a resolution procedure as described previously using L-and D-tartaric acid. 5All esters were prepared according to the procedure described for quinuclidin-3-yl benzoate. 14-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 previously.

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. 5N-benzyl derivatives of racemic, (R)-and (S)-quinuclidin-3-yl acetates, benzoates, pivalates and butyrates were synthesized by the esterification 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, elemental analyses, one-and two-dimensional 1 H and 13 C NMR.

Hydrolyses of quaternary esters with BchE
The overall catalytic process of BChE proceeds in three steps: initial formation of an enzyme-substrate complex, an acylation step, and deacylation by hydrolysis, 19 Scheme 1.In the reaction sequence, E, S, ES, and EA represent free enzyme, substrate, Michaelis complex and acyl-enzyme intermediate, respectively.P 1 (alcohol) and P 2 (acid) are products of the hydrolysis.In the case of tested quinuclidinium esters, 1-benzyl-3hydroxyquinuclidinium is P1 and P2 are acetic, pivalic butyric and benzoic acid respectively.

Inhibition of BChE by (S)-enantiomers
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 activity, it was possible to determine enzyme-inhibitor dissociation constant K i .The enzyme-inhibitor dissociation constant K i was calculated from equation K app =K i +(K a /K m )•s, where K app is the apparent enzymeinhibitor dissociation constant at a given substrate concentration (s) and K m 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 concentration of BzCh was higher, Figure 3. Therefore, it was not possible to determine the enzyme-inhibitor dissociation constant for that compound.
All other (S)-esters proved to be μmol dm −3 inhibitors, (S)-benzoate was the best inhibitor, closely followed by (S)-pivalate.(S)-butyrate had the lowest affinity toward BChE, Table 1.The calculated values of K m for the substrate used (BzCh) obtained from kinetic studies with inhibitors can be compared with ones obtained from data without inhibitors (K m (BzCh) = 0.17±0.01). 14It 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.
To further explore the hydrolysis reaction of racemic N-benzyl quinuclidin-3-ol esters catalysed by BChE, kinetic experiments were carried out to deter-mine the differences in rates of hydrolysis of pure (R)enantiomers (2 mmol dm −3 solution) and racemic mixtures 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 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 accordance 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 activation with present SAcBzl because RAcBzl was the substrate 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 Table 1.Inhibition constants of BChE by (S)-N-benzyl esters: the enzyme-inhibitor dissociation constant K i and standard deviation was obtained from four experiments on average.The dissociation constant of enzyme-inhibitor complex was determined from Hunter-Downs plots.The value for SBzBzl was published previously 15 Inhibitor K m /mmol dm −3 K i /μmol dm −3 SBuBzl 0,19 ± 0,01 60,5 ± 0,5 SPivBzl 0,79 ± 0,02 12,3 ± 0,2 SBzBzl 15 1,6 ± 0,6 3,3 ± 0,4  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.

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 affinity toward BChE and all were determined as µmol dm −3 inhibitors of the enzyme with the exception of the acetate 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 selectivity towards enantiomers.Smaller acetate group and bigger pivalate and benzoate could not realize a maximum possible interactions and the activity of the enzyme was lower.Among (S)-enantiomers, the differences in the activity of BChE were even more pronounced and partly related to their inhibitory properties.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. 14,16ue to the satisfactory difference in the rates of hydrolysis of enantiomers, BChE can be used as a biocatalyst 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 N-benzyl quinuclidinium esters can be successfully achieved by the stereoselective hydrolysis catalysed with BChE.

Figure 3 .
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 .

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.A R/racemate corresponds to the ratio of BChE activity for (R)-enantiomer and racemate Croat.Chem.Acta 84 (2011) 245.