Enzymatic resolution of hindered cyanohydrins, key precursors of muscarinic receptor antagonists

Article abstract of DOI:10.1016/j.tetasy.2007.04.002

Enantiomerically pure 1-cycloalkyl-1-hydroxy-1-phenylcyanohydrins, key precursors in the synthesis of (S)-oxybutynin and other antimuscarinic agents, have been successfully prepared via lipase-catalyzed kinetic resolutions. Pseudomonas cepacia and Candida antarctica lipase B have shown excellent enantioselectivities in hydrolysis and acylation processes, depending on the substrate structure and the reaction conditions. Furthermore, molecular modeling of phosphonate transition state analogue for the C. antarctica lipase B enzymatic hydrolysis resolution step supports these facts, which were experimentally observed.

Full text of DOI:10.1016/j.tetasy.2007.04.002

Tetrahedron: Asymmetry 18 (2007) 994–1002
Enzymatic resolution of hindered cyanohydrins, key precursors
of muscarinic receptor antagonists
´
´
Veronica Recuero, Miguel Ferrero, Vicente Gotor-Fernandez,
Rosario Brieva and Vicente Gotor*
Departamento de Quı´mica Orga´nica e Inorga´nica and Instituto Universitario de Biotecnologı´a de Asturias,
Universidad de Oviedo, 33006 Oviedo, Asturias, Spain
Received 27 March 2007; accepted 4 April 2007
Abstract—Enantiomerically pure 1-cycloalkyl-1-hydroxy-1-phenylcyanohydrins, key precursors in the synthesis of (S)-oxybutynin and
other antimuscarinic agents, have been successfully prepared via lipase-catalyzed kinetic resolutions. Pseudomonas cepacia and Candida
antarctica lipase B have shown excellent enantioselectivities in hydrolysis and acylation processes, depending on the substrate structure
and the reaction conditions. Furthermore, molecular modeling of phosphonate transition state analogue for the C. antarctica lipase B
enzymatic hydrolysis resolution step supports these facts, which were experimentally observed.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
chiral mandelic acid as a starting material,6 diastereoselec-
tive Michael addition,3a Sharpless asymmetric dihydroxyl-
ation,7 or enantioselective cyanosilylation of cycloalkyl
phenyl ketones catalyzed by a gadolinium complex,8 have
all been reported to synthesize the key a-hydroxy acid pre-
cursor of (S)-oxybutynin and analogues.
Tertiary a-hydroxy acids are constituents of a variety of
pharmaceuticals and important natural products.1 For
example, oxybutynin (Fig. 1),2 such as the majority of mus-
carinic receptor antagonists, is composed of a tertiary
a-hydroxy acid ester.3 Despite the limited therapeutic
applicability of the racemic drug due to side effects, oxy-
butynin has been widely prescribed for the treatment of
urinary tract disorders. However, the (S)-enantiomer is
proposed to display an improved therapeutical profile com-
pared to its racemic counterpart and a number of deriva-
tives have been synthesized mainly by modification of the
ester or the cycloalkyl moieties.4 Several approaches have
been used to construct the quaternary stereocenter of these
compounds. Grignard addition using a chiral auxiliary,5
On the other hand, the resolution of tertiary alcohols by
enzymatic methods is an interesting challenge from a theo-
retical point of view; hydrolytic enzymes are usually unable
to accept sterically hindered substrates bearing fully substi-
tuted quaternary carbons in the vicinity of the function to
be hydrolyzed or acylated.9
Taking into account the importance of enantiomerically
pure tertiary a-hydroxynitriles as precursors of antimuscar-
inic agents and the theoretical interest in their resolution,
we describe a study of their enzymatic resolution. Some
of the results presented here have been rationalized using
a computational approach.
n
n
OH
OH
O
N
N
O
2. Results and discussion
1a−c, n = 1−3
(S)-Oxybutynin (Ditropan), n = 3
First, we studied the transesterification of racemic cyano-
hydrins ( )-1a–c catalyzed by several commercially avail-
able lipases [Candida antarctica lipases A and B (CAL-A
and CAL-B), Pseudomonas cepacia (PSL-C), Candida
rugosa (CRL), Aspergillus Melleus, porcine pancreatic
Figure 1.
*
Corresponding author. Tel./fax: +34 985 103 448; e-mail: vgs@fq.
uniovi.es
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.04.002

V. Recuero et al. / Tetrahedron: Asymmetry 18 (2007) 994–1002
995
O
(PPL)], and also pig liver esterase (PLE). As was expected,
O
none of the enzymes tested catalyzed the process. These re-
sults are probably due to adverse steric interactions caused
by the quaternary stereogenic center in the active site of the
enzyme. Consequently, in order to circumvent this prob-
lem, we proceeded to modify the substrate by the prepara-
tion of several derivatives. First, we prepared the derivative
of the cyclobutyl cyanohydrin ( )-2a (Scheme 1). Thus,
the enzyme catalyzed the hydrolysis of the methyl ester
sited five bonds away from the stereogenic center. The
strategy to turn tertiary alcohols into suitable substrates
for hydrolases was successfully applied by Franssen
et al.,10 nevertheless the processes described by these
authors showed low enantioselectivities.
n
OH
CN
n
O
O
Cl
O
O
O
CN
Py / CH2Cl2
1a-c, n = 1,2,3
( )-4a-c
O
Lipase
n
n
O
OH
Phosphate buffer
pH = 7.0, 1,4-dioxane
O
O
+
O
O
CN
CN
(+) or (−)-4a-c
Scheme 2. Synthesis and enzymatic hydrolysis of ( )-4a–c.
(+) or (−)-5a-c
O
O
O
O
enzymes tested catalyzed the process. The reactions cata-
lyzed by CAL-B and PSL-C were very fast, reaching in
both cases, 100% conversion after 24 h. The reaction rates
of the hydrolysis catalyzed by CCL or CAL-A were slower
but the obtained products were formed in both cases as
racemates. In order to obtain conversions close to 50% in
the hydrolysis catalyzed by CAL-B and PSL-C, we carried
out these processes using less concentration of the buffer
and stopping the reaction after 16 h. The results in both
cases were very successful. As shown in Table 2, good rates
and very high enantioselectivities were reached, especially
for the product that was recovered in enantiopure form.
Cl
OH
CN
O
O
O
CN
Py / CH2Cl2
( )-1a
( )-2a
O
O
O
OH
Lipase
Phosphate buffer
O
O
+
pH = 7.0, 1,4-dioxane
O
O
CN
CN
(+) or (−)-2a
Scheme 1. Synthesis and enzymatic hydrolysis of ( )-2a.
(+) or (−)-3a
Depending on the biocatalyst employed, an opposite
stereochemical preference in the resolution processes was
observed. CAL-B catalyzed the hydrolysis of the (R)-(+)
enantiomer, while for the PSL-C, product 5a was obtained
with the (S)-(ꢀ)-configuration, leaving the (R)-(+) enantio-
mer of the unreacted substrate 4a.
The hydrolysis of ( )-2a was carried out at 30 ꢁC in a 1:1
mixture of phosphate buffer 1 M and 1,4-dioxane. Under
these conditions, two of the enzymes tested catalyzed the
reaction, even though the enantioselectivities were low
and only moderate conversions were achieved (Table 1).
In view of these promising results, we decided to apply the
same reaction conditions employed in the resolution of
derivative ( )-4a with 4b and 4c. CAL-B and PSL-C
showed high or moderate conversions and the same oppo-
site stereochemical preference as shown in the case of sub-
strate ( )-4a; however, low enantioselectivities were found
(Table 3, entries 1–4). In order to improve the enantioselec-
tivity of these processes, we analyzed the influence of the
organic cosolvent in the hydrolysis of these substrates
(entries 5–11), but none of the solvents tested (t-BuOMe,
toluene, and acetonitrile) improved the results obtained
using PSL-C in 1,4-dioxane.
Table 1. Lipase-catalyzed hydrolysis of ( )-2a at 30 ꢁC in phosphate
buffer 1 M pH = 7.0/1,4-dioxane 1:1, after 65 h
b
Entry
Lipase
t (h)
c (%)a
eesb (%)
eep (%)
Ec
1
2
CAL-B
PSL-C
65
65
36
37
35
16
63
27
6
2
a Conversion, c = ees/(ees + eep).
b Determined by chiral HPLC.
c Enantiomeric ratio, E = ln[(1 ꢀ c)(1 ꢀ ees)]/ln[(1 ꢀ c)(1 + ees)].11
Next, we examined the behavior of acetoxyacetylated
derivatives ( )-4a–c as possible candidates for the enzy-
matic hydrolysis resolution (Scheme 2). First, we carried
out the hydrolysis of the cyclobutyl derivative ( )-4a under
the same conditions as for substrate ( )-2a, and four of the
Taking into account that the spacer inserted between the
quaternary stereogenic center and the reaction center must
be long enough to convert cyanohydrins ( )-1a–c into suit-
Table 2. Lipase-catalyzed hydrolysis of ( )-4a at 30 ꢁC in a mixture 1:2 of 1 M phosphate buffer pH = 7.0/1,4-dioxane, after 16 h
ca (%)
eesb (%)
Conf.
eep (%)
Conf.
Ec
b
Entry
Lipase
Buffer/solvent
1
2
CAL-B
PSL-C
1:2
1:2
42
31
71
45
(S)
(R)
99
99
(R)
(S)
>200
>200
a Conversion, c = ees/(ees + eep).
b Determined by chiral HPLC.
c Enantiomeric ratio, E = ln[(1 ꢀ c)(1 ꢀ ees)]/ln[(1 ꢀ c)(1 + ees)].11

996
V. Recuero et al. / Tetrahedron: Asymmetry 18 (2007) 994–1002
Table 3. Lipase-catalyzed hydrolysis of ( )-4b and ( )-4c at 30 ꢁC in a mixture 1:2 of 1 M phosphate buffer pH = 7.0/1,4-dioxane
ca (%)
eesb (%)
Conf.
eep (%)
Conf.
Ec
b
Entry
Substrate
Lipase
Cosolvent
t (h)
1
2
3
4
5
6
7
8
9
(
(
(
(
(
(
(
(
(
(
(
)-4b
)-4b
)-4c
)-4c
)-4b
)-4b
)-4b
)-4b
)-4c
)-4b
)-4b
CAL-B
PSL-C
CAL-B
PSL-C
CAL-B
PSL-C
CAL-B
PSL-C
PSL-C
CAL-B
PSL-C
1,4-Dioxane
1,4-Dioxane
1,4-Dioxane
1,4-Dioxane
t-BuOMe
t-BuOMe
Toluene
Toluene
Toluene
5
24
7
17
7
6
9
19
11
5
89
35
83
56
56
79
48
39
11
40
41
40
36
40
40
10
48
10
13
5
(S)
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(R)
(S)
(R)
5
66
8
31
8
13
11
20
70
9
(R)
(S)
(R)
(S)
(R)
(S)
(R)
(S)
(S)
(R)
(S)
1
7
2
3
1
2
1
2
2
1
7
10
11
Acetonitrile
Acetonitrile
6
43
24
63
a Conversion, c = ees/(ees + eep).
b Determined by chiral HPLC.
c Enantiomeric ratio, E = ln[(1 ꢀ c)(1 ꢀ ees)]/ln[(1 ꢀ c)(1 + ees)].11
O
able substrates to be accepted for the lipases, while on the
other hand, that the chain length interferes with the chiral
recognition process, we prepared two new derivatives with
different chain sizes (Scheme 3).
n
n
O
OH
CAL-B
O
O
O
O
CN
Phosphate buffer
CN
pH = 7.0, 1,4-dioxane
100% conversion
The enzymatic hydrolysis processes catalyzed by CAL-B or
PSL-C of substrates ( )-6 and ( )-7 were carried out under
the same reaction conditions as described above. Unfortu-
nately, none of the examined enzymes catalyzed the hydro-
lysis of these substrates.
( )-4b,c n= 2, 3
( )-5b,c
O
R
Lipase
n
O
n
O
OH
RCOOR'
O
O
CN
O
+
CN
Another common approach for the resolution of racemic
alcohols is the enzymatic transesterification reaction. In
order to examine these enzymatic processes we used as
substrates the racemic derivatives ( )-5b and ( )-5c,
obtained through the enzymatic hydrolysis of ( )-4b and
( )-4c catalyzed by CAL-B, when the processes were
allowed to react until 100% conversion (Scheme 4).
Organic
solvent
(+) or (−)-4b,c R = CH3
( )-5b,c
(+) or (−)-8b,c R = (CH2)2CH3
(+) or (−)-9b,c R = (CH2)8CH3
(+) or (−)-10b,c R = (CH2)10CH3
(+) or (−)-11b,c R = Ph
Scheme 4. Synthesis and enzymatic acylation of ( )-5b,c.
First, we studied the resolution catalyzed by CAL-B or
PSL-C of substrate ( )-5b using common reaction condi-
tions for an enzymatic transesterification, so vinyl acetate
was chosen as acyl donor and the reactions were carried
out in toluene at 30 ꢁC (Table 4, entries 1 and 2). The reac-
tion catalyzed by CAL-B was very fast and after 30 min a
100% of conversion was achieved. Using PSL-C the process
was slower but also showed a low enantioselectivity.
Then, we tested non-activated esters, which are less reactive
acylating agents, observing that with ethyl acetate, CAL-B
showed a moderate rate and low enantioselectivity (entry
3). No reaction was observed with PSL-C (entry 4). When
ethyl acetate was used also as the solvent, PSL-C catalyzed
the reaction with a moderate rate but a low enantioselectiv-
ity (entry 5). Slightly better was the enantioselectivity ob-
tained with ethyl butyrate and PSL-C (entry 7). Using
this acylating agent, 23% conversion was obtained after
24 h, with a low but promising enantioselectivity of
E = 10. The use of longer chain esters seems to lead to bet-
ter results in the enzymatic process catalyzed by PSL-C.
We then tested this reaction using ethyl and vinyl esters
of decanoic acid, and also vinyl laurate and vinyl benzoate.
The results are summarized in entries 8–11 of Table 4. The
best results were obtained using vinyl decanoate since 37%
conversion was achieved after only 1.5 h of reaction with
good enantioselectivity (E = 41, entry 9).
O
O
O
O
Cl
OH
CN
O
Lipase
O
O
CN
Phosphate buffer
pH = 7, 1,4-dioxane
(1:2)
Py / CH2Cl2
( )-1b
( )-6
O
O
O
O
O
OH
CN
O
Lipase
Cl
O
CN
Phosphate buffer
pH = 7, 1,4-dioxane
(1:2)
Py / CH2Cl2
( )-7
( )-1b
A similar study was carried out for the enzymatic resolu-
tion of substrate ( )-5c catalyzed by PSL-C. As in the case
Scheme 3. Synthesis and enzymatic hydrolysis of derivatives with different
chain lengths, ( )-6 and ( )-7.

V. Recuero et al. / Tetrahedron: Asymmetry 18 (2007) 994–1002
997
Table 4. Lipase catalyzed acylation of ( )-5a–c at 30 ꢁC in toluene, using 5 equiv of the corresponding acylating agent
b
Entry
Substrate
Lipase
Acylating agent
t (h)
0.5
0.5
96
120
17
24
24
24
6
6
48
3
24
24
24
1
15
72
ca (%)
eesb (%)
Conf
eep (%)
Conf
Ec
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
)-5b
)-5b
)-5b
)-5b
)-5b
)-5b
)-5b
)-5b
)-5b
)-5b
)-5b
)-5c
)-5c
)-5c
)-5c
)-5c
)-5c
)-5c
CAL-B
PSL-C
CAL-B
PSL-C
PSL-C
CAL-B
PSL-C
PSL-C
PSL-C
PSL-C
PSL-C
PSL-C
PSL-C
PSL-C
PSL-C
PSL-C
PSL-C
PSL-C
Vinyl acetate
Vinyl acetate
Ethyl acetate
Ethyl acetate
Ethyl acetated
Ethyl butyrate
Ethyl butyrate
Ethyl decanoate
Vinyl decanoate
Vinyl laurate
Vinyl benzoate
Vinyl acetate
Ethyl acetate
Ethyl butyrate
Ethyl decanoate
Vinyl butyrate
Vinyl decanoate
Vinyl benzoate
100
14
48
—
26
9
—
8
51
—
20
6
—
—
50
56
—
57
64
77
86
92
9
70
49
70
90
72
43
15
96
—
—
3
6
—
4
5
10
14
41
4
8
4
7
21
9
4
3
(S)
(R)
—
(R)
(S)
—
(S)
(R)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(R)
(S)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
23
6
23
5
37
92
32
48
20
10
37
54
85
30
54
99
33
45
17
10
42
51
81
40
73
a Conversion, c = ees/(ees + eep).
b Determined by chiral HPLC.
c Enantiomeric ratio, E = ln[(1 ꢀ c)(1 ꢀ ees)]/ln[(1 ꢀ c)(1 + ees)].11
d Ethyl acetate was used as solvent and acylating agent.
of ( )-5b we observed a strong influence of the acylating
agent in the rate and enantioselectivity of the reaction.
The most significant results are summarized in entries 12–
18 of Table 4. The best results were obtained for this sub-
strate using vinyl benzoate as the acylating agent. In this
case, a moderate rate and a high enantioselectivity
(E = 73, entry 18) were achieved.
The starting point for modeling was a simple phosphonate,
which mimics the transition state for the hydrolysis of
methyl acetate. Geometry optimization of this simple phos-
phonate formed all six essential hydrogen bonds for catal-
ysis (d1–d6 in Scheme 5).15 To model the cycloalkyl
acetoxyacetylated substrates, we added the quaternary car-
bon and its substituents (cyano, cycloalkyl, and phenyl)
sequentially until we obtained the phosphonate shown in
Scheme 5 and systematically searched for catalytically pro-
ductive conformations. We were forced to use the chloride
instead of the cyanide because the force field (AMBER16)
could not apply a potential to the latter.17 These intermedi-
ates mimic the hydrolysis of substrates ( )-4a–c.14 We de-
fine the catalytically productive conformations as those
A further optimization of other reaction parameters such
as solvent, acylating agent concentration or temperature
could be carried out in order to improve the results
obtained for both substrates.
The high stereoselectivity observed with the CAL-B-cata-
lyzed hydrolysis of the cyclobutyl acetoxyacetylated 4a
and not with their homologous cyclopentyl 4b and cyclo-
hexyl 4c derivatives motivated us to undertake a modeling
study of this reaction to understand better the results.
Thus, we obtained the X-ray structure of CAL-B12 from
the Protein Data Bank.13 This structure presents a phos-
phonate linked to the active site of the lipase. The catalytic
triad is formed by residues Asp187-His224-Ser105 and the
oxyanion hole is formed by the main chain NH groups of
residues Gln106, Thr40, and the hydroxyl group of the
later.
Large pocket
Ser105
Gln106
HN
d4
Me
O
O
d2
d3
O
P
d5
d1
O
C
N
NH
H
δ
OH
NH
Asp187
O
ε
d6
O
n
His224
O
Ph Cl
Thr40
To qualitatively explain the stereoselectivity of CAL-B, we
modeled phosphonate analogues of the key intermediates
in the hydrolysis of both stereoisomers of each of the three
cycloalkyl acetoxyacetylated derivatives (i.e., cyclobutyl 4a,
n = 1; cyclopentyl 4b, n = 2; cyclohexyl 4c, n = 3). These
phosphonates were able to mimic the key features of the
intermediates in hydrolysis reactions and allowed the com-
putationally simpler molecular mechanics approach to be
used.14 This approach focuses on how the substrate fits in
the active site of the enzyme, but might omit subtle details
about the transition state.
Medium
pocket
Scheme 5. Phosphonate analogue intermediates of the transition state for
the hydrolysis of cycloalkyl acetoxyacetylated derivatives 4a–c (n = 1,2,3).
Key hydrogen bonds between CAL-B and the tetrahedral intermediate
are: one from Nd of His224 to the carboxylate of Asp187, two from Ne of
His224 to the oxygen of Ser105 and the O-alkyl group of the tetrahedral
intermediate, and three from the oxyanion to Gln106 (one) and to Thr40
(two). For computer modeling the different groups are introduced in a
stepwise fashion as described in the text forming both enantiomers.

998
V. Recuero et al. / Tetrahedron: Asymmetry 18 (2007) 994–1002
that (a) contained all six key catalytic hydrogen bonds, (b)
avoided steric clashes between the phosphonate and the li-
pase, and (c) avoided steric clashes within the phosphonate.
tures some of the key H-bond interactions were lost. In the
best of them (Fig. 2B), the enzyme situates both cyclobutyl
and phenyl rings inside the large hydrophobic pocket but
in its edge with the chloride pointing to the right
inside the hydrophobic pocket. The binding alkane rings
appears to be a key factor in explaining the enzyme
enantioselectivity.
When geometry optimization was carried out with the cyc-
lobutyl acetoxyacetylated (R)-enantiomer (Fig. 2A), the
tetrahedral intermediate for the hydrolysis reaction situates
both cyclobutyl and phenyl rings on the right side of the
large hydrophobic pocket with the chloride pointing out
to the pocket, making it the most productive conformation
due to better enzyme substrate interaction, since this struc-
ture contains all six catalytically essential hydrogen bonds
in the catalytic site. However, when the tetrahedral inter-
mediate for the hydrolysis reaction of cyclobutyl acetoxy-
acetylated (S)-enantiomer was built and minimized, no
single productive conformation was obtained. In all struc-
Conversely, the geometry optimization of both enantio-
mers of cyclopentyl or cyclohexyl acetoxyacetylated deriv-
atives yielded structures containing all six catalytically
essential hydrogen bonds in the catalytic site, showing no
enzyme selectivity for either of the enantiomers in both
cases. Thus, modeling explains the experimentally observed
facts.
3. Conclusions
Herein we have reported an easy methodology for the
resolution
of
three
1-phenyl-1-hydroxy-1-cyclo-
alkylcyanohydrins via a lipase-catalyzed hydrolysis or
acylation of a suitable derivative. Thus, among all the lip-
ases tested, CAL-B or PSL-C presented the best results and
an opposite stereochemical preference. Enzymatic hydroly-
sis shows the highest enantioselectivity for the cyclobutyl
derivative, whereas the acylation catalyzed by PSL-C is
apparently more adequate for the resolution of cyclopentyl
and cyclohexyl derivatives. The high enantioselectivity ob-
tained in the acylation process of the cyclohexyl derivative
should be noted. Taking into account the collected data, we
can conclude that the resolution of tertiary alcohols by
enzymatic methods is possible, even though hydrolytic
enzymes are usually unable to accept hindered substrates
when the quaternary carbons are in the vicinity of the func-
tion to be hydrolyzed or acylated. As is usual for enzymatic
processes, all reaction parameters must be optimized for
each substrate.
The applicability of this method for the preparation of
antimuscarinic agents that include a tertiary a-hydroxy
acid ester in their structure due to the simplicity and easy
scale-up of lipase-catalyzed reactions should be noted.
Results obtained from the molecular modeling study on
the CAL-B-catalyzed hydrolysis of cycloalkyl acetoxyacet-
ylated derivatives are in agreement with the observed
experimental data. Thus, CAL-B showed high stereoselec-
tivity in the case of the cyclobutyl derivative but failed
with cyclopentyl or cyclohexyl acetoxyacetylated
derivatives.
Figure 2. The best conformations of cyclobutyl acetoxyacetylated (R)-
enantiomer (A) and (S)-enantiomer (B) intermediates in CAL-B are shown
above. The tetrahedral intermediate for the favored (R)-enantiomer
hydrolysis reaction (A) situates both cyclobutyl and phenyl rings on the
right side of the large hydrophobic pocket and the chloride pointing out to
the pocket, making it the most productive conformation due to a better
enzyme substrate interaction. The tetrahedral intermediate for the (S)-
enantiomer hydrolysis reaction (B) situates both cyclobutyl and phenyl
rings inside the large hydrophobic pocket, but in its edge with the chloride
pointing to the right inside the hydrophobic pocket. The binding alkane
rings appears to be the key factor in explaining the enzyme enantioselec-
tivity. Darker spheres indicate the amino acids that are the limits of the
medium and large hydrophobic size pockets around the CAL-B active site.
The above image displays Glu188 and Ile189 in a line representation to
allow a better view of the large pocket of the lipase.
This study constitutes another good example of how
remote interactions can affect the stereo- and/or regioselec-
tivity of an enzyme.18 The key difference between the
intermediates obtained for both enantiomers is the superior
binding of the (R)-enantiomer caused at a site remote from
the reaction center. Such remote binding can lower the free
energy of the corresponding transition state by favoring a
catalytically productive orientation, and at the same time
disfavoring the intermediate for the observed (S)-
enantiomer.

V. Recuero et al. / Tetrahedron: Asymmetry 18 (2007) 994–1002
999
4. Experimental
The crude was purified by flash chromatography on silica
gel (hexane/EtOAc 7:3) to afford the corresponding
product ( )-1a–c.
4.1. General remarks
Enzymatic reactions were carried out in a Gallenkamp
incubatory orbital shaker. Immobilized C. antarctica lipase
B, CAL-B Novozym 435 (7300 PLU/g), was a gift from
Novo Nordisk co. and immobilized P. cepacia lipase
(PSL-C, 783 U/g) is commercialized by Amano Pharm-
aceuticals.
4.2.1.1. ( )-1-Cyclobutyl-1-hydroxy-1-phenylacetonitrile
( )-1a. White solid. Mp: 73–75 ꢁC. IR (KBr): 2275,
1
3480 cmꢀ1. H NMR (CDCl3, 300 MHz): d 1.71–2.29 (m,
6H), 2.74–2.86 (m, 1H), 2.99 (br s, 1H), 7.31–7.44 (m,
3H), 7.47–7.59 (m, 2H). 13C NMR (CDCl3, 75.5 MHz): d
16.5 (CH2), 23.1 (CH2), 23.4 (CH2), 45.5 (CH), 76.8 (C),
119.9 (CN), 124.8 (CH), 128.7 (CH), 128.9 (CH), 138.5
(C); MS (ESI+, m/z): 210 [(M+Na)+, 40], 188 [(M+H)+,
10].
Chemical reagents were commercialized by Aldrich, Fluka,
Lancaster or Prolabo. Solvents were distilled over an
appropriate desiccant under nitrogen. Flash chromatogra-
phy was performed using Merck Silica Gel 60 (230–
400 mesh). Optical rotations were measured using a Per-
kin–Elmer 241 polarimeter and are quoted in units of
4.2.1.2. ( )-1-Cyclopentyl-1-hydroxy-1-phenylacetonitrile
( )-1b. Colorless oil. IR (NaCl) 2225, 3654 cmꢀ1 1H
.
NMR (CDCl3, 300 MHz): d 1.39–1.71 (m, 8H), 2.38–2.43
(m, 1H), 3.87 (br s, 1H), 7.36–7.53 (m, 5H). 13C NMR
(CDCl3, 100.5 MHz): d 26.0 (CH2), 26.2 (CH2), 28.6
(CH2), 29.0 (CH2), 52.1 (CH), 78.7 (C), 121.0 (CN),
125.9 (2CH), 129.3 (2CH), 129.5 (CH), 140.6 (C). MS
(ESI+m/z): 224 [(M+Na)+, 100], 202 [(M+H)+, 20].
1
10ꢀ1 deg cm2 gꢀ1. H NMR, 13C NMR and DEPT spectra
were recorded in a Bruker AC-300, Bruker AC-300 DPX or
Bruker NAV-400 spectrometer using CDCl3 as solvent.
The chemical shift values (d) are given in ppm. Positive
electrospray ionization (ESI+) was used to record mass
spectra on a Hewlett-Packard 110 LC/MSD Series spec-
trometers. The enantiomeric excesses were determined by
chiral HPLC analysis on a Hewlett-Packard 1100, LC
liquid chromatograph, using a CHIRALCEL OD for the
acylated derivatives 4 and 8–11, and CHIRALPAK AS
for hydroxylated derivatives 5a–c. Absolute configurations
were determined after refluxing the derivatives 5a–c in HCl
6 M to obtain the corresponding known acid and compar-
ison of their optical rotation with reported values: cyclo-
butylmandelic acid,6a cyclopentylmandelic acid,19a and
cyclohexylmandelic acid.19b
4.2.1.3. ( )-1-Cyclohexyl-1-hydroxy-1-phenylacetonitrile
( )-1c. Colorless oil. IR (NaCl): 2195, 3492 cmꢀ1 1H
.
NMR (CDCl3, 300 MHz): d 1.11–2.00 (m, 10 H), 2.35
(m, 1H), 3.71 (br s, 1H), 7.35–7.50 (m, 5H). 13C NMR
(CDCl3, 100.5 MHz): d 14.3 (CH2), 21.2 (CH2), 26.0
(CH2), 26.1 (CH2), 27.4 (CH2), 27.7 (CH), 49.1 (C), 61.0
(C), 78.6 (CN), 120.6 (CH), 125.9 (CH), 128.7 (CH),
129.1 (CH), 139.5 (C). MS (ESI+ m/z): 238 [(M+Na)+,
100], 216 [(M+H), 5].
4.2.2. General procedure for the preparation of derivatives
( )-2a, ( )-4a–c, ( )-6, and ( )-7. The corresponding acyl
chloride (14 mmol) was added dropwise to a stirred solu-
tion of the corresponding 1-cycloalkyl-1-hydroxy-1-phenyl-
acetonitrile ( )-1a–c (7 mmol) in pyridine (14 mmol), and
dry CH2Cl2 (13 mL). The resulting mixture was heated
and stirred for 24 h at 60 ꢁC. After this time, the mixture
was extracted with CH2Cl2 (3 · 20 mL). The organic
phases were combined and dried over Na2SO4. The solvent
was removed under reduced pressure and the crude residue
purified by flash chromatography on silica gel (hexane/
EtOAc 7:3) to afford the corresponding product.
4.2. Molecular modeling
The program INSIGHT II, version 2000.1, was used for
viewing the structures. The geometric optimizations and
molecular dynamics were performed using Discover, ver-
sion 2.9.7 (Accelrys, San Diego, CA, USA), using the AM-
BER16 force field. The distance dependent dielectric
constant was set to 4.0 to mimic the electrostatic shielding
of the solvent and the 1–4 van der Waals interactions were
scaled to 50%. The crystal structure for the CAL-B
(1LBS12) was obtained from the Protein Data Bank,13
and includes a phosphonate. The methodology followed
for the localization of energy minima structures, by means
of a systematic search has been described elsewhere.20 Pro-
tein structures in Figure 2 were generated using PyMOL
0.99.21
4.2.2.1. ( )-(1-Cyano-1-cyclobutyl-1-phenylmethoxy)car-
bonylmethyl acetate ( )-4a. White solid. Mp: 89–92 ꢁC.
IR (KBr): 3032, 2255, 1739, 1742 cmꢀ1. 1H NMR (CDCl3,
300 MHz): d 1.78–1.85 (m, 3H), 1.92–2.07 (m, 3H), 2.14 (s,
3H), 2.91–3.02 (m, 1H), 4.69 (s, 2H), 7.39–7.42 (m, 5H).
13C NMR (CDCl3, 100.5 MHz): d 16.5 (CH3), 20.2
(CH2), 23.1 (CH2), 23.9 (CH2), 44.6 (CH), 60.2 (CH2),
80.3 (C), 115.9 (CN), 124.4 (CH), 128.8 (CH), 129.1
(CH), 135.0 (C), 165.4 (CO), 170.0 (CO). MS (ESI+,
m/z): 288 [(M+H)+, 100].
4.2.1. General procedure for the synthesis of cycloalkyl-1-
hydroxy-1-phenylacetonitriles ( )-1a–c. Tetramethyl-silyl-
cyanide (3.8 mmol) was added dropwise to a stirred solu-
tion of the corresponding ketone (3.2 mmol) in dry
CH2Cl2 (12 mL). A catalytic amount of ZnI2 was also
added and the reaction mixture was stirred for 24 h at
room temperature. The solvent was then evaporated under
reduced pressure and the crude residue hydrolyzed with
HCl (3 M) and extracted with EtOAc (3 · 20 mL). The
organic phases were combined and dried over Na2SO4,
and the solvent was removed under reduced pressure.
(R)-(+)-4a: Determination of the ee by HPLC analysis:
Chiralcel OD, 25 ꢁC, hexane/2-propanol (90:10), UV
210 nm, 0.5 mL minꢀ1, tR 14.8 min (major); tR 20.5 min
20
(minor). ½aꢁD ¼ þ39 (c 0.10, CHCl3) for 70% ee.

1000
V. Recuero et al. / Tetrahedron: Asymmetry 18 (2007) 994–1002
4.2.2.2. ( )-(1-Cyano-1-cyclopentyl-1-phenylmethoxy)-
125.2 (CH), 129.2 (CH), 129.6 (CH), 137.0 (C), 171.3
(CO). MS (ESI+, m/z): 282 [(M+Na)+, 100], 260
[(M+H)+, 20].
carbonylmethyl acetate ( )-4b. White solid. Mp: 78–
80 ꢁC. IR (KBr): 2235, 1740, 1745 cmꢀ1. 1H NMR (CDCl3,
300 MHz): d 1.28–1.69 (m, 5H), 1.79–1.93 (m, 3H), 2.12 (s,
3H), 2.58–2.64 (m, 1H), 4.65 (s, 2H), 7.31–7.43 (m, 5H).
13C NMR (CDCl3, 100.5 MHz): d 21.1 (CH3), 25.9
(CH2), 26.2 (CH2), 28.5 (CH2), 29.4 (CH2), 51.5 (CH),
61.1 (CH2), 82.6 (CN), 116.9 (C), 125.6 (CH), 129.6
(CH), 129.9 (CH), 137.3 (C), 166.1 (CO), 170.8 (CO). MS
(ESI+ m/z): 324 [(M+Na)+, 100], 302 [(M+H)+, 20], 340
[(M+K)+, 5].
(S)-(+)-5b: Determination of the ee by HPLC analysis:
Chiralpak AS, hexane/EtOH 96:4, UV 210 nm, 0.5
mL minꢀ1, tR 29.9 min (minor); tR 34.3 min (major).
20
½aꢁD ¼ þ13 (c 0.10, CHCl3) for 66% ee.
4.2.3.3. ( )-1-Cyano-1-cyclohexyl-1-phenylmethyl hydr-
oxyacetate ( )-5c. Pale yellow oil. IR (NaCl): 3647,
1
1731 cmꢀ1. H NMR (CDCl3, 300 MHz): d 1.09–1.25 (m,
4.2.2.3. ( )-(1-Cyano-1-cyclohexyl-1-phenylmethoxy)car-
bonylmethyl acetate ( )-4c. White solid. Mp: 77–79 ꢁC.
IR (KBr): 3140, 2230, 1730, 1690 cmꢀ1. 1H NMR (CDCl3,
300 MHz): d 1.11–1.18 (m, 3H), 121–1.25 (m, 3H), 1.63–
1.69 (m, 2H), 1.72–1.84 (m, 1H), 1.85–1.88 (m, 2H), 2.01
(s, 3H), 4.66 (s, 2H), 7.37–7.43 (m, 5H). 13C NMR (CDCl3,
100.5 MHz): d 25.5 (CH3), 25.6 (CH2), 26.6 (CH2), 27.6
(CH2), 48.3 (CH), 60.2 (CH2), 81.8 (C), 115.8 (CN),
125.2 (CH), 128.6 (CH), 129.1 (CH), 135.4 (C), 165.2
(CO), 169.9 (CO). MS (ESI+, m/z): 338 [(M+Na)+, 100],
316 [(M+H)+, 20].
4H), 1.65–1.71 (m, 2H), 1.84–1.87 (m, 1H), 1.98–2.14 (m,
4H), 4.07–4.28 (m, 2H), 7.37–7.43 (m, 5H Ar). 13C NMR
(CDCl3, 100.5 MHz): d 21.3 (CH2), 25.9 (CH2), 25.9
(CH2), 26.9 (CH2), 28.0 (CH2), 48.5 (CH), 60.8 (CH2),
82.3 (C), 116.3 (CN), 125.5 (CH), 129.1 (CH), 129.6
(CH), 136.1 (C), 171.4 (CO). MS (ESI+, m/z): 296
[(M+Na)+, 100].
(S)-(+)-5c: Determination of the ee by HPLC analysis:
Chiralpak AS, hexane/EtOH 96:4, UV 210 nm, 0.5
mL minꢀ1, tR 31.8 min (minor); tR 35.1 min (major).
20
½aꢁD ¼ þ7:5 (c 0.10, CHCl3) for 83% ee.
4.2.3. General procedure for enzymatic hydrolysis. The
reaction mixture containing the corresponding substrate
(0.15 mmol), 1 mL of 1 M phosphate buffer (pH 7.0), the
lipase (100 mg), and the corresponding organic solvent
(2 mL) was shaken at 30 ꢁC and 250 rpm in an orbital sha-
ker. The progress of the reaction was monitored by TLC
(hexane/EtOAc 7:3) until the required conversion was ob-
tained. The enzyme was then removed by filtration and
washed with CHCl3 (3 · 5 mL). The product was extracted
with EtOAc (3 · 5 mL), the organic phases were combined
and dried over Na2SO4. The solvent was removed under re-
duced pressure and the crude residue purified by flash chro-
matography on silica gel (hexane/EtOAc 7:3).
4.2.4. General procedure for enzymatic acylation. The
reaction mixture containing the corresponding substrate
(0.15 mmol), the corresponding acylating agent (0.75
mmol), the lipase (100 mg) and the corresponding organic
solvent (4 mL) was shaken at 30 ꢁC and 250 rpm in an orbi-
tal shaker. The progress of the reaction was monitored by
TLC (hexane/EtOAc 7:3) until the achievement of the re-
quired conversion. The enzyme was then removed by filtra-
tion and washed with CHCl3 (3 · 5 mL). The solvent was
removed under reduced pressure and the crude residue
was purified by flash chromatography on silica gel (hex-
ane/EtOAc 7:3).
4.2.3.1. ( )-1-Cyano-1-cyclobutyl-1-phenylmethyl hydr-
oxyacetate ( )-5a. White solid. Mp: 74–76 ꢁC. IR (KBr):
4.2.4.1. ( )-(1-Cyano-1-cyclopentyl-1-phenylmethoxy)-
carbonylmethyl butyrate ( )-8b. Pale yellow oil. IR
1
3640, 3015, 1735 cmꢀ1
.
1H NMR (CDCl3, 300 MHz): d
(NaCl): 2189, 1732 cmꢀ1. H NMR (CDCl3, 300 MHz): d
1.76–1.88 (m, 3H), 2.06 (m, 2H), 2.25–2.32 (m, 1H), 2.54
(br s, 1H), 2.93–3.03 (m, 1H), 4.18–4.26 (m, 2H), 7.61–
7.85 (m, 5H). 13C NMR (CDCl3, 100.5 MHz): d 16.5
(CH2), 23.0 (CH2), 24.02 (CH2), 44.5 (CH), 60.4 (CH2),
80.5 (C), 115.9 (CN), 124.4 (CH), 128.8 (CH), 129.2
(CH), 135.2 (C), 171.1 (CO). MS (ESI+, m/z): 256
[(M+Na)+, 100], 234 [(M+H)+, 5].
0.96 (t, 3H), 1.35–1.60 (m, 8H), 1.68–1.72 (m, 2H), 2.25
(t, 2H), 2.5 (m, 1H), 5.04 (s, 2H), 7.19–7.25 (m, 5H Ar).
13C NMR (CDCl3, 100.5 MHz): d 13.5 (CH3), 18.3
(CH2), 26.6 (2CH2), 27.5 (2CH2), 35.9 (CH2), 40.8 (CH),
61.7 (CH2), 81.6 (C), 118.1 (CN), 126.1 (CH), 128.2
(CH), 128.5 (2CH), 128.9 (CH), 146.3 (C), 172.9 (CO).
MS (ESI+, m/z): 330 [(M+H)+, 25].
(S)-(ꢀ)-5a: Determination of the ee: the compound was
(R)-(ꢀ)-8b: Determination of the ee by HPLC analysis:
Chiralcel OD, 25 ꢁC, hexane/2-propanol (90:10), UV
converted into the acetylated derivative (ꢀ)-4a and ana-
20
lyzed by HPLC analysis. ½aꢁD ¼ ꢀ49 (c 0.12, CHCl3) for
210 nm, 0.5 mL minꢀ1, tR 11.7 min (major); tR 14.7 min
20
99% ee.
(minor). ½aꢁD ¼ ꢀ5:1 (c 0.10, CHCl3) for 64% ee.
4.2.3.2. ( )-1-Cyano-1-cyclopentyl-1-phenylmethyl hydr-
oxyacetate ( )-5b. White solid. Mp: 79–81 ꢁC. IR (KBr)
4.2.4.2. ( )-(1-Cyano-1-cyclohexyl-1-phenylmethoxy)car-
bonylmethyl butyrate ( )-8c. Pale yellow oil. IR (NaCl):
1
3652, 1741 cmꢀ1
.
1H NMR (CDCl3, 300 MHz): d 1.32–
2193, 1740 cmꢀ1. H NMR (CDCl3, 300 MHz): d 0.96 (t,
1.62 (m, 7H), 1.93–2.02 (m, 1H), 2.48 (br s, OH), 2.61–
2.66 (m, 1H), 4.15 (dd, 1H, J = 17.46 Hz), 4.25 (dd, 1H,
J = 17.46 Hz), 7.37–7.49 (m, 5H). 13C NMR (CDCl3,
100.5 MHz): d 25.4 (CH2), 25.7 (CH2), 28.1 (CH2), 29.1
(CH2), 50.9 (CH), 60.9 (CH2), 82.4 (C), 116.5 (CN),
3H), 1.32–1.65 (m, 10H), 1.67–1.71 (m, 2H), 2.24 (t, 2H),
2.51 (m, 1H), 5.05 (s, 2H), 7.19–7.31 (m, 5H). 13C NMR
(CDCl3, 100.5 MHz): d 13.5 (CH3), 18.3 (CH2), 26.6
(2CH2), 27.5 (2CH2), 29.8 (CH2), 35.4 (CH2), 40.7 (CH),
60.7 (CH2), 81.7 (C), 118.0 (CN), 126.1 (CH), 128.2

V. Recuero et al. / Tetrahedron: Asymmetry 18 (2007) 994–1002
1001
(CH), 128.5 (CH), 128.9 (CH), 146.1 (C), 173.2 (CO). MS
300 MHz): d 1.35–1.74 (m, 10H), 2.56 (m, 1H), 4.91 (d,
2H), 7.19–7.47 (m, 8H), 7.97 (m, 2H). 13C NMR (CDCl3,
100.5 MHz): d 25.4 (CH2), 25.7 (CH2), 27.6 (CH2), 28.0
(CH2), 28.9 (CH2), 51.1 (CH), 62.1 (CH2), 81.5 (C), 116.6
(CN), 125.3 (CH), 128.9 (CH), 128.8 (CH), 129.2 (CH),
129.7 (CH), 130.3 (CH), 134.0 (CH), 136.8 (C), 141.8 (C),
165.5 (CO), 166.1 (CO). MS (ESI+, m/z): 400 [(M+Na)+,
60], 416 [(M+K)+, 5].
(ESI+, m/z): 344 [(M+H)+, 60].
(R)-(ꢀ)-8c: Determination of the ee by HPLC analysis:
Chiralcel OD, 25 ꢁC, hexane/2-propanol (90:10), UV
210 nm, 0.5 mL minꢀ1, tR 11.2 min (major); tR 14.6 min
20
(minor). ½aꢁD ¼ ꢀ6:2 (c 0.10, CHCl3) for 90% ee.
4.2.4.3. ( )-(1-Cyano-1-cyclopentyl-1-phenylmethoxy)-
carbonylmethyl decanoate ( )-9b. Pale yellow oil. IR
(ꢀ)-11c: Determination of the ee by HPLC analysis:
Chiralcel OD, 25 ꢁC, hexane/2-propanol (90:10), UV 210
1
(NaCl): 2228, 1736 cmꢀ1. H NMR (CDCl3, 300 MHz): d
0.96 (t, 3H), 1.29–1.68 (m, 22H), 2.25 (m, 2H), 2.49 (m,
1H), 5.04 (s, 2H), 7.18–7.41 (m, 5H). 13C NMR (CDCl3,
100.5 MHz): d 14.5 (CH3), 22.8 (CH2), 25.1 (CH2), 26.7
(2CH2), 27.7 (2CH2), 29.1 (CH2), 29.4 (2CH2), 29.7
(CH2), 31.9 (CH2), 33.7 (CH2), 40.7 (CH), 61.8 (CH2),
81.9 (C), 118.0 (CN), 126 (CH), 128.2 (2CH), 128.6
(2CH), 146.1 (C), 168.9 (CO), 173.1 (CO). MS (ESI+,
m/z): 414 [(M+H)+, 100].
nm, 0.5 mL minꢀ1, tR 13.014 min (minor); tR 14.415 min
20
(major). ½aꢁD ¼ ꢀ12 (c 0.12, CHCl3) for 96% ee.
References
1. See for example: (a) Visser, T. J.; Van Waarde, A.; Doze, P.;
Wegman, T.; Vaalburg, W. Synapse 2000, 35, 62–67; (b)
Subrahmanyam, D.; Sarma, V. M.; Venkateswarlu, A.;
Sastry, T. V. R. S.; Srinivas, A. S. S. V.; Krishna, C. V.;
Deevi, D. S.; Kumar, S. A.; Babu, M. J.; Damodaran, N. K.
Bioorg. Med. Chem. Lett. 2000, 10, 369–371; (c) Hauser, F.
M.; Ganguly, D. J. Org. Chem. 2000, 65, 1842–1849; (d)
Vladu, B.; Woynarowski, J. M.; Manikumar, G.; Wani, M.
C.; Wall, M. E.; Von Hoff, D. D.; Wadkins, R. M.
Mol. Pharmacol. 2000, 57, 243–251; (e) Di Bugno, C.;
Colombani, S. M.; Dapporto, P.; Garzelli, G.; Giorgi, R.;
Paoli, P.; Subissi, A.; Turbanti, L. Chirality 1997, 9, 713–
721; (f) Kanda, Y.; Fukuyama, T. J. Am. Chem. Soc. 1993,
115, 8451–8452; (g) Atkinson, E. R.; McRitchie, D. D.;
Shoer, L. F.; Harris, L. S.; Archer, S.; Aceto, M. D.;
Pearl, J.; Luduena, F. P. J. Med. Chem. 1977, 20, 1612–
1617.
(R)-(ꢀ)-9b: Determination of the ee by HPLC analysis:
Chiralcel OD, 25 ꢁC, hexane/2-propanol (90:10), UV
210 nm, 0.5 mL minꢀ1, tR 9.1 min (major); tR 13.0 min
20
(minor). ½aꢁD ¼ ꢀ26 (c 0.15, CHCl3) for 92% ee.
4.2.4.4. ( )-(1-Cyano-1-cyclohexyl-1-phenylmethoxy)car-
bonylmethyl decanoate ( )-9c. Pale yellow oil. IR (NaCl):
2192, 1745, 1695 cmꢀ1 1H NMR (CDCl3, 300 MHz): d
.
0.95 (t, 3H), 12.8–1.65 (m, 24H), 2.21 (m, 2H), 2.47 (m,
1H), 5.10 (s, 2H), 7.17–7.39 (m, 5H). 13C NMR (CDCl3,
100.5 MHz): d 14.0 (CH3), 22.5 (CH2), 24.6 (CH2), 24.9
(CH2), 25.3 (CH2), 27.7 (2CH2), 28.7 (CH2), 28.9 (CH2),
29.1 (2CH2), 29.3 (2CH2), 31.7 (CH2), 33.8 (CH2), 40.6
(CH), 61.8 (CH2), 82.0 (C), 116.1 (CN), 124.8 (CH),
128.8 (2CH), 129.2 (2CH), 136.6 (C), 170.9 (CO), 173.1
(CO). MS (ESI+, m/z): 428 [(M+H)+, 100].
2. Thompson, I. M.; Lauvetz, R. Urology 1976, 8, 452–
454.
3. (a) Mitsuya, M.; Ogino, Y.; Ohtake, N.; Mase, T. Tetra-
hedron 2000, 56, 9901–9907; (b) Mitsuya, M.; Kawakami, K.;
Ogino, Y.; Miura, K.; Mase, T. Bioorg. Med. Chem. Lett.
1999, 9, 2037–2038; (c) McPherson, D. W.; Knapp, F. F. J.
Org. Chem. 1996, 61, 8335–8337.
4. (a) Kaiser, C.; Audia, V. H.; Carter, J. P.; McPherson, D. W.;
Waid, P. P.; Lowe, V. C.; Noronha-Blob, L. J. Med. Chem.
1993, 36, 610–616; (b) Carter, J. P.; Noronha-Blob, L.;
Audia, V. H.; Dupont, A. C.; McPherson, D. W.; Natalie, K.
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C. J. Med. Chem. 1991, 34, 3065–3074; (c) Mehta, A.;
Silamkoti, A. V.; Kaur, K.; Gupta, J. B. WO 2004014853,
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Bruhaspathy, M.; Gupta J. B. PCT WO 014363, 2004, CAN
140:181321.
5. Senanayake, C. H.; Fang, K.; Grover, P.; Bakale, R. P.;
Vandenbossche, C. P.; Wald, S. A. Tetrahedron Lett. 1999,
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6. (a) Grover, P. T.; Bhongle, N. N.; Wald, S. A.; Senanayake,
C. H.; Chris, H. J. Org. Chem. 2000, 65, 6283–6287; (b) Su,
X.; Bhongle, N. N.; Pflum, D.; Butler, H.; Wald, S. A.;
Bakale, R. P.; Senanayake, C. H. Tetrahedron: Asymmetry
2003, 14, 3593–3600.
(R)-(ꢀ)-9c: Determination of the ee by HPLC analysis:
Chiralcel OD, 25 ꢁC, hexane/2-propanol (90:10), UV
210 nm, 0.5 mL minꢀ1, tR 8.6 min (major); tR 9.9 min (min-
20
or). ½aꢁD ¼ ꢀ6:8 (c 0.12, CHCl3) for 72% ee.
4.2.4.5. ( )-(1-Cyano-1-cyclopentyl-1-phenylmethoxy)-
carbonylmethyl benzoate ( )-11b. Pale yellow solid. Mp:
1
108–110 ꢁC. IR (KBr): 2232, 1741, 1690 cmꢀ1. H NMR
(CDCl3, 300 MHz): d 1.37–1.73 (m, 8H), 2.56 (m, 1H),
4.91 (d, 2H), 7.19–7.47 (m, 8H), 7.97 (m, 2H). 13C NMR
(CDCl3, 100.5 MHz): d 25.4 (CH2), 25.7 (CH2), 28.1
(CH2), 28.9 (CH2), 51.1 (CH), 61.2 (CH2), 81.6 (C), 116.6
(CN), 125.7 (CH), 128.8 (CH), 128.9 (CH), 129.2 (CH),
129.5 (CH), 130.2 (CH), 133.9 (CH), 136.9 (C), 141.7 (C),
165.6 (CO), 166.0 (CO). MS (ESI+, m/z): 386 [(M+Na)+,
100], 402 [(M+K)+, 20].
(R)-(ꢀ)-11b: Determination of the ee by HPLC analysis:
Chiralcel OD, 25 ꢁC, hexane/2-propanol (90:10), UV
210 nm, 0.5 mL minꢀ1, tR 12.7 min (major); tR 15.8 min
7. Gupta, P.; Fernandes, R. A.; Kumar, P. Tetrahedron Lett.
2003, 44, 4231–4232.
8. Masumoto, S.; Suzuki, M.; Kanai, M.; Shibasaki, M.
Tetrahedron Lett. 2002, 43, 8647–8651.
9. Pogorevc, M.; Faber, K. J. Mol. Catal. B: Enzym. 2000, 10,
357–376.
20
(minor). ½aꢁD ¼ ꢀ6:1 (c 0.10, CHCl3) for 70% ee.
4.2.4.6. ( )-(1-Cyano-1-cyclohexyl-1-phenylmethoxy)car-
bonylmethyl benzoate ( )-11c. Pale yellow solid. Mp:
115–117 ꢁC. IR (KBr) 3195, 1724 cmꢀ1. 1H NMR (CDCl3,

1002
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´
´
´
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¨
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´
20. (a) Lavandera, I.; Fernandez, S.; Magdalena, J.; Ferrero, M.;
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˚
´
less than 3.20 A and a donor–hydrogen–acceptor angle of
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