Biocatalytic preparation of chiral 3,4-dihydroxypyrrolidines

Article abstract of DOI:10.1016/j.tet.2010.06.060

Enzymatic acylations and alcoxycarbonylations of cis- and trans-3,4-dihydroxypyrrolidines and hydrolysis of their diacylated or dialcoxycarbonylated derivatives have been studied. High enantioselectivity is obtained using Candida antarctica lipase B as catalyst in the hydrolysis of the trans-diacetyl derivative, while for the desymmetrization of the cis-3,4-dihydroxypyrrolidines the best results are obtained in the acylation process catalyzed by C. antarctica lipase A.

Full text of DOI:10.1016/j.tet.2010.06.060

Tetrahedron 66 (2010) 6789e6796
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Biocatalytic preparation of chiral 3,4-dihydroxypyrrolidines
*
*
Jesús A. Rodríguez-Rodríguez, Rosario Brieva , Vicente Gotor
Departamento de Química Orgánica e Inorgánica and Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006 Oviedo, Asturias, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 April 2010
Received in revised form 16 June 2010
Accepted 22 June 2010
Available online 25 June 2010
Enzymatic acylations and alcoxycarbonylations of cis- and trans-3,4-dihydroxypyrrolidines and hydro-
lysis of their diacylated or dialcoxycarbonylated derivatives have been studied. High enantioselectivity is
obtained using Candida antarctica lipase B as catalyst in the hydrolysis of the trans-diacetyl derivative,
while for the desymmetrization of the cis-3,4-dihydroxypyrrolidines the best results are obtained in the
acylation process catalyzed by C. antarctica lipase A.
Ó 2010 Elsevier Ltd. All rights reserved.
Dedicated to Professor Carmen Nájera in the
occasion of her 60th birthday
1. Introduction
2. Results and discussion
Optically pure 3,4-dihydroxypyrrolidines form an integral feature
of many natural compounds and of synthetic derivatives that exhibit
interesting biological activities.1 They are immediate precursors to
a promising new class of 3,4-disubstituted pyrrolidine HIV-1 pro-
tease inhibitors,2 as well as to diverse analogues of natural nucleo-
sides3 and aza-sugars;4 the often potent inhibitory activity of these
compounds toward glycosidases, suggests an enormous therapeutic
potential in the treatment of many diseases. Moreover, trans-vicinal
diols, diamines, and their derivatives occupy a central role in cata-
lytic asymmetry synthesis: 3,4-dihydroxypyrrolidines have been
used in the preparation of chiral ligands for asymmetric catalysis5 or
Our initial experiments were designed to find the most suitable
lipase for catalyzing the hydrolysis of the diacetylated derivative
(ꢀ)-trans-4. This substrate has been prepared in high yield by
treatment of 1-benzyloxy-carbonyl-3-pyrroline (2) with m-chlor-
operbenzoic acid in CH2Cl2 and subsequent opening of the
resulting epoxide 3 with acetic anhydride and BF3$Et2O (Scheme 1).
10
AcO
OAc
O
Ac2O
CbzCl
m-CPBA
NaHCO3
CH2Cl2
(90 %)
CH2Cl2
(70%)
BF3.Et2O
N
H
N
N
N
chiral ionic liquids;6 a novel single-isomer of
b-cyclodextrin has
AcOH
(80%)
Cbz
( )-trans-4
Cbz
2
Cbz
3
been employed as chiral selector in capillary electrophoresis.7
Typically, enantiomerically enriched 3,4-dihydroxypyrrolidine
derivatives have been synthesized by cyclization of tartaric acid,
followed by a low yield hydride reduction step.1d,5c,8 Biocatalytic
methods, such as the use of microbial epoxide hydrolases, have
also been successfully employed for the asymmetric ring opening
of the meso-epoxide, to obtain optically pure trans-3,4-
dihydroxypyrrolidines.9
1
Scheme 1. Synthesis of (ꢀ)-trans-4.
Taking into account our previous results in the biocatalytic
resolution of trans-3,4-dihydroxytetrahydrofuran11 and trans-1-
benzyloxycarbonyl-3,4-dihydroxypiperidines,12 we studied the
hydrolysis of (ꢀ)-trans-4 in 1,4-dioxane using a small amount of
water (5 equiv) as nucleophile (Scheme 2). We tested several
commercially available lipases [Pseudomonas cepacia (PSL-C and
PSL-D) and Candida antarctica lipase A and B (CAL-A and CAL-B)].
All the processes were carried out at 30 ꢁC.
As a part of our research on the enzymatic preparation of opti-
cally active polyhydroxyheterocycles, we are very interested in the
enzymatic resolution of trans-3,4-dihydroxypyrrolidines, and in the
desymmetrization of the cis-isomer. Lipase-catalyzed processes are
a very advantageous alternative to chemical or microbial methods
because they allow the selective functionalization of one of the two
hydroxyl groups and constitute an easy access to optically pure
functionalized pyrrolidines.
HO
OAc
∗
HO
OH
∗
AcO
OAc
∗
AcO
OAc
∗
∗
∗
Lipase
+
+
Organic solvent / H2O
N
N
N
N
Cbz
Cbz
Cbz
Cbz
(+) or (−)-trans-5
( )-trans-4
(+) or (−)-trans-4
(+) or (−)-trans-6
* Corresponding authors. Tel.: þ34 985 102 994; fax: þ34 985 103 448; e-mail
Scheme 2. Enzymatic hydrolysis of (ꢀ)-trans-4.
addresses: rbrieva@uniovi.es (R. Brieva), vgs@uniovi.es (V. Gotor).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.06.060

6790
J.A. Rodríguez-Rodríguez et al. / Tetrahedron 66 (2010) 6789e6796
Under these conditions only lipases CAL-A and CAL-B catalyzed
entry 5). As in the case of CAL-A, when this reaction was allowed to
continue, the second hydrolytic step takes place affording the cor-
responding diol (3R,4R)-6 (Table 1, entries 6 and 7). The two se-
quential hydrolytic steps showed very high enantioselectivity.
Therefore, it seems that CAL-B shows identical stereochemical
preference in the two sequential hydrolytic steps.
Taking into account that an increment of the water concentra-
tion does not improve the reaction rate (enzymatic processes car-
ried out in a mixture 1,4-dioxane/H2O 2:1 were slower that using
10 equiv of water), we decided to study the influence of different
reaction parameters in the reaction.
the sequential hydrolysis of substrate (ꢀ)-trans-4 (Table 1, entries 1
and 2), whereas, in processes catalyzed by lipases PSL-C and PSL-D
no product was observed after 7 days of reaction. The enantiose-
lectivity of the processes catalyzed by lipases CAL-A and CAL-B was
very high, even though very low conversion was achieved. In-
terestingly, a different behavior and an opposite stereochemical
preference of the two lipases was observed: in the process cata-
lyzed by CAL-A only the monoacetate (3S,4S)-5 was detected (entry
1), whereas a mixture of the monoacetate (3R,4R)-5 and the diol
(3R,4R)-6 was achieved in the hydrolysis catalyzed by CAL-B
(entry 2).
Table 1
Lipase catalyzed hydrolysis of (ꢀ)-trans-4 at 30 ꢁC in 1,4-dioxane
Lipasea
H2O
t (h)
Yieldb (%)
ees (%)
Yieldb (%)
eepc (%)
Yield (%)b
eepc (%)
c
d (%)
Ed
c
Entry
(equiv)
trans-4
trans-4
trans-5
trans-5
trans-6
trans-6
1
2
3
4
5
6
7
CAL-A
CAL-B
CAL-A
CAL-A
CAL-B
CAL-B
CAL-B
5
5
65
26
12
20
4.6
6.6
20
d
90
d
69
d
83
63
7
12
23
38
20
29
73
d
7
d
28
d
8
>99
>99
>99
79
>99
>99
>99
d
4
d
7
>200
d
>99
d
d
19
d
17
d
d
10
10
10
10
10
d
>200
d
3
d
8
>99
d
>200
d
>99
>99
4
33
d
a
Weight ratio enzyme/substrate 1:1.
Determined by GC.
Determined by chiral HPLC.
b
c
d
Conversion, c¼ees/(eesþeep), enantiomeric ratio, E¼ln [(1ꢂc)(1ꢂees)]/ln [(1ꢂc)(1þees)].13 These equations are used when only one product is achieved in the kinetic
resolution.
The absolute configurations of the products and the remaining
First, we increased the weight ratio enzyme/substrate to 2:1
(200 mg of enzyme/100 mg substrate in a typical experiment).
Table 2 summarizes the results of these experiments. For both
enzymatic processes, the reaction rate increased considerably in
these conditions.
substrate were assigned as follows. The hydrolysis of the remaining
substrate-trans-4 or the monoacetate trans-5 using NaOMe in
MeOH, afforded the diol trans-6, whose specific rotation sign was
compared with that reported for (3R,4R)-(þ)-6.9b
Table 2
Lipase catalyzed hydrolysis of (ꢀ)-trans-4 in 1,4-dioxane, using 5 equiv of H2O and a weight ratio enzyme/substrate 2:1
Yielda (%)
ees (%)
Yielda (%)
eepb(%)
Yielda (%)
eepb (%)
cc(%)
Ec
b
Entry
Lipase
T (ꢁC)
t (days)
trans-4
trans-4
trans-5
trans-5
trans-6
trans-6
1
2
3
4
5
6
7
CAL-A
CAL-A
CAL-B
CAL-B
CAL-B
CAL-B
CAL-B
30
30
30
30
50
50
50
5
6
5
11
1
2
d
28
33
79
99
87
d
27
5
2
5
81
76
>99
>99
>99
39
d
2
d
25
d
d
d
d
d
d
12
d
d
d
d
d
d
71
56
45
55
49
39
>99
>99
92
92
87
40
55
40
45
53
98
>99
6
8
5
58
62
a
Determined by GC in the processes where two products are achieved.
Determined by chiral HPLC.
b
c
Conversion, c¼ees/(eesþeep), enantiomeric ratio, E¼ln [(1ꢂc)(1ꢂees)]/ln [(1ꢂc)(1þees)].13 These equations are used when only one product is achieved in the kinetic
resolution.
In order to improve the conversion of this enzymatic hydrolysis
In the reaction catalyzed by CAL-A a conversion of 25% had been
achieved after 5 days of reaction (Table 2, entry 1); the reaction
product showed a moderate enantioselectivity [(3S,4S)-5, ee¼81%].
The selectivity of the process was also moderate and after 6 days of
reaction the diol (3S,4S)-6 was detected, and the enantiomeric
excess of monoacetate (3S,4S)-5 decreased considerably, (Table 2,
entry 2).
Better results were obtained in the process catalyzed by CAL-B
(Table 2, entries 3 and 4). A 40% yield of the optically pure diol
(3R,4R)-6 was achieved after 5 days of reaction (entry 3). When the
reaction was incubated for 11 days, the remaining substrate (3S,4S)-
4 was obtained in an isolated yield higher than 40% and ee of 99%
(entry 4).
we increased the amount of water to 10 equiv. Entries 3e7 of
Table 1 show the progress of the reactions catalyzed by CAL-A and
CAL-B in these conditions. Even though the rates of these processes
were slightly lower using a double amount of water, one can es-
tablish several conclusions from the obtained results. In the hy-
drolysis catalyzed by CAL-A high enantioselectivity was attained.
After 12 days of reaction the optically pure monoacetate (3S,4S)-5
was achieved with 19% conversion, (Table 1, entry 3). When this
reaction was allowed to continue, the second hydrolytic step took
place affording the corresponding diol (3S,4S)-6, (Table 1, entry 4).
The process catalyzed by CAL-B was also slow, after 4.6 days 17%
of optically pure monoacetate (3R,4R)-5 was achieved (Table 1,

J.A. Rodríguez-Rodríguez et al. / Tetrahedron 66 (2010) 6789e6796
6791
For a better appreciation of the reaction progress with time,
Figures 1 and 2 represent percent of substrate and products and the
enantiomeric excess versus time for the reaction catalyzed by
CAL-B at 30 ꢁC.
study of the influence of the organic solvent was carried out for
both lipases, in the same conditions. Table 3 shows the results
obtained for both enzymes after 5.5 and 13 days in different sol-
vents. In general, processes catalyzed by CAL-A (entries 1e8) were
slower and less selective than those catalyzed by CAL-B (entries
9e15). Nevertheless, the result obtained with CAL-A in acetonitrile
was noteworthy, because these conditions allowed us to obtain the
monoacetate (3S,4S)-5 in an enantiopure form with a moderate
yield of 21% (entry 3).
For the processes catalyzed by CAL-B, the best results were
obtained in THF: 30% of the optically pure diol (3R,4R)-6 was ach-
ieved after 5.5 days (entry 14) and a 53% of the remaining substrate
(3S,4S)-4 can be obtained in a 92% of enantiomeric excess after 13
days (entry 15). In general, the results obtained in the processes
catalyzed by CAL-B in different solvents did not improve those
obtained using 1,4-dioxane (Table 2).
In order to better understand the enantiomeric preference and
the enantioselectivity of CAL-A and B in the two sequential
hydrolytic steps, we studied the second step by carrying out the
enzymatic hydrolysis of the racemic monoacetate (ꢀ)-trans-5 in the
optimum conditions for each enzyme. As one can see from
Scheme 3, the reaction rate was moderate in the process catalyzed
by CAL-A and fast in the case of CAL-B. In both processes the
enantioselectivity was low.
These data are in accordance with the results obtained in the
sequential hydrolysis of substrate (ꢀ)-trans-4. For both enzymes,
the first hydrolysis to give the monoacetate 5 must be the slow and
enantioselective step of the process. In the process catalyzed by
CAL-B, the second step was so fast that only a small amount (less
than 10%) of the monoacetate intermediate is detected during the
reaction. In the case of CAL-A, the second step was slower and,
depending on the reaction conditions, a moderate yield of the op-
tically pure monoacetate intermediate can be obtained, as we in-
dicated previously (Table 3, entry 3).
Figure 1. Percent of substrate and products versus time for the CAL-B catalyzed hy-
drolysis 30 ꢁC in 1,4-dioxane, using 5 equiv of H2O and a weight ratio enzyme/sub-
strate 2:1.
After the study of the enzymatic hydrolysis of the diacetylated
derivative (ꢀ)-trans-4 we examined the diol (ꢀ)-trans-6 as sub-
strate in enzymatic acylations. We carried out the processes at
30 ꢁC, using different acylating agents (5 equiv) and 1,4-dioxane as
solvent, except when ethyl acetate was used itself as solvent
(Scheme 4).
The results obtained in the processes catalyzed by CAL-A, CAL-B
and PSL-C are summarized in Table 4. In all cases, the stereo-
chemical preferences of the tested enzymes were the same as for
the hydrolytic processes, but lower conversions and enantiose-
lectivities were achieved. When ethyl acetate was used as acyl
donor (Table 4, entries 1e3), only the monoacetylated product was
obtained but the three tested lipases showed low enantiose-
lectivity. The conversion was low in processes catalyzed by CAL-A
and PSL-C but a 47% of conversion can be obtained after 15 days of
reaction using CAL-B (entry 2). When the ethyl acetate was used
itself as solvent (Table 4, entries 4e6), the enzymatic processes
were faster but, in the case of CAL-A and CAL-B, a mixture of the
mono and diacetylated products were obtained, with moderate
enantioselectivity.
Figure 2. Enantiomeric excess of substrate and products versus time for the CAL-B
catalyzed hydrolysis 30 ꢁC in 1,4-dioxane, using 5 equiv of H2O and a weight ratio
enzyme/substrate 2:1.
The effect of the temperature was also analyzed in the process
catalyzed by CAL-B (Table 2, entries 5e7). An increase of the tem-
perature to 50 ꢁC enhanced the reaction rate, even though the
enantiomeric excess of the diol (3R,4R)-6 was slightly lower (entry
5) than at 30 ꢁC. Nevertheless, these are the best conditions to
obtain the remaining substrate (3S,4S)-4 in an enantiopure form
and an excellent yield; after 5 days of reaction 39% of the optically
pure substrate is achieved (entry 7). A considerable improvement
of the yield is possible (49%) with only a slight loss of enantiomeric
excess if the reaction is stopped after 2 days of reaction (entry 6).
Taking into account the good results obtained using 5 equiv of
H2O and a weight ratio enzyme/substrate of 2:1 at 30 ꢁC, a further
Finally, when the vinyl acetate was used as acyl donor (Table 4,
entries 7e9), faster processes were achieved and the optically pure
remaining substrate was obtained, even thought only in a moderate
yield of 19% in the reaction catalyzed by CAL-A (Table 4, entry 7).
It is remarkable that when the acylation was catalyzed by CAL-B,
a 92% yield of monoacetylated (ꢀ)-trans-5 was obtained after 12 h
of reaction (Table 4, entry 8). This lipase-catalyzed process is a very
advantageous alternative to the chemical selective functionaliza-
tion of one of the two hydroxyl groups.
In order to establish the scope of this enantiomer separation
methodology, the best conditions used in the biocatalytic resolu-
tion of trans-3,4-dihydroxypyrrolidines, were applied in the

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J.A. Rodríguez-Rodríguez et al. / Tetrahedron 66 (2010) 6789e6796
Table 3
Lipase catalyzed hydrolysis of (ꢀ)-trans-4 at 30 ꢁC in organic solvents, using 5 equiv of H2O and a weight ratio enzyme/substrate 2:1
Entry Lipase Solvent
t (days) Yielda (%) trans-4 eesb (%) trans-4 Yielda (%) trans-5 eepb (%) trans-5 Yielda (%) trans-6 eepb (%) trans-6
c
c (%) Ec
1
2
3
4
5
6
7
8
CAL-A Toluene
CAL-A Toluene 13
5.5
64
55
d
66
7
d
d
71
87
90
26
42
72
d
29
37
d
18
33
>99
62
36
23
>99
71
7
8
>99
61
d
d
21
d
d
d
9
d
d
CAL-A CH3CN
CAL-A CH3CN
CAL-A tBuOMe
5.5
13
5.5
d
11
55
83
d
4
d
>200
d
23
37
17
d
>99
41
5
d
d
CAL-A tBuOMe 13
d
CAL-A THF
CAL-A THF
5.5
13
10
30
>200
d
25
>99
d
9
CAL-B Toluene 13
>99
72
54
7
12
69
53
d
d
7
6
37
10
2
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
10
11
12
13
14
15
CAL-B CH3CN
CAL-B CH3CN
CAL-B tBuOMe
5.5
13
5.5
53
84
72
85
57
92
>99
>99
36
6
>99
>99
19
40
55
78
30
46
>99
85
41
7
>99
91
CAL-B tBuOMe 13
CAL-B THF
CAL-B THF
5.5
13
2
a
Determined by GC in the processes where two products are achieved.
Determined by chiral HPLC.
b
c
Conversion, c¼ees/(eesþeep), enantiomeric ratio, E¼ln [(1ꢂc)(1ꢂees)]/ln [(1ꢂc)(1þees)].13 These equations are used when only one product is achieved in the kinetic
resolution.
HO
OAc
hydrolysis or acylation of their cis-analogues. Substrates cis-4aec
and cis-6 were synthesized according to Scheme 5. Treatment of 1-
HO
OAc
HO
OH
CAL-A/CH3CN
+
benzyloxycarbonyl-3-pyrroline
2 with OsO4 and N-methyl-
c = 29 %
E = 2
N
N
N
morpholine N-oxide (NMMO) yielded the cis-6, which was acylated
or alcoxycarbonylated to the corresponding derivative cis-4aec.
The enzymatic hydrolysis was carried out at 30 ꢁC in 1,4-dioxane
using 5 equiv of H2O (Scheme 6). The results obtained are sum-
marized in Table 5. For the substrate cis-4a both enzymes, CAL-A
and CAL-B showed low selectivity (Table 5, entries 1 and 2); the
process catalyzed by CAL-B was faster but a high percent of the
dihydroxylated product cis-6 was achieved.
Next we tested substrates cis-4b and cis-4c in the same condi-
tions (Table 5, entries 3e6). No reaction was observed in the en-
zymatic hydrolysis of cis-4b. In the hydrolysis of the dicarbonate
cis-4c the best result was obtained in the reaction catalyzed by
CAL-A, even though only a 35% yield of the monohydrolyzed
product was achieved, with a low enantiomeric excess of 44%
(Table 5, entry 5).
Cbz
Cbz
Cbz
(
)-trans-5
(3R,4R)-5
(3S,4S)-6
ee= 13 %
ee= 32 %
HO
OAc
HO
OAc
HO
OH
CAL-B /1,4-dioxane
+
c = 73 %
E = 2
N
N
N
Cbz
Cbz
Cbz
(
)-trans-5
(3R,4R)-6
(3S,4S)-5
ee= 41 %
ee= 16 %
Scheme 3. Enzymatic hydrolysis of (ꢀ)-trans-5 at 30 ꢁC in organic solvent, using
5 equiv of H2O and a weight ratio enzyme/substrate 2:1; reaction time 18 h.
Enzymatic transesterification or alcoxycarbonylation of sub-
strate cis-6 were also tested. As for the trans-analogues, the pro-
cesses were carried out in 1,4-dioxane at 30 ꢁC using 5 equiv of the
corresponding ester or carbonate (Scheme 7, Table 6).
No reaction or very low yields were achieved when ethyl acetate
or ethyl benzoate were used as acylating agents (results not show in
table).
HO
OAc
∗
HO
OH
∗
AcO
OAc
∗
HO
OH
∗
∗
∗
CH3CO2R
+
+
Lipase
1,4-dioxane, 30 °C
N
N
N
N
Cbz
Cbz
Cbz
Cbz
( )-trans-6
(+) or (−)-trans-5
(+) or (−)-trans-4
(+) or (−)-trans-6
Scheme 4. Enzymatic acetylation of (ꢀ)-trans-6.
The results obtained with activated acylating agents are sum-
marized in Table 6. When CAL-A was used as catalyst and vinyl
Table 4
Lipase catalyzed transesterification of (ꢀ)-trans-6 at 30 ꢁC in organic solvents
Entry
Lipasea
R
Solvent
t (days)
Yieldb (%)
eesc (%)
Yieldb (%)
eepc (%)
Yieldb (%)
eepc (%)
c
d (%)
Ed
trans-4
trans-4
trans-5
trans-5
trans-6
trans-6
1
2
3
4
5
6
7
8
9
CAL-A
CAL-B
PSL-C
CAL-A
CAL-B
PSL-C
CAL-A
CAL-B
PSL-C
Ethyl
Ethyl
Ethyl
Ethyl acetate
Ethyl acetate
Ethyl acetate
Vinyl
Vinyl
Vinyl
1,4-Dioxane
1,4-Dioxane
1,4-Dioxane
15
15
15
10
10
10
4.5
0.5
4.5
d
d
d
72
80
d
29
6
d
d
d
14
13
d
91
79
47
d
d
d
2
78
4
d
d
d
38
7
22
19
2
3
3
4
17
16
d
4
47
5
d
d
29
d
d
d
8
1
85
69
71
d
1
13
d
d
4
d
d
d
9
56
52
92
80
1,4-Dioxane
1,4-Dioxane
1,4-Dioxane
>99
>99
>99
3
13
8
12
a
Weight ratio enzyme/substrate 2:1.
b
c
Determined by GC in the processes where two products are achieved.
Determined by chiral HPLC.
d
Conversion, c¼ees/(eesþeep), enantiomeric ratio, E¼ln [(1ꢂc)(1ꢂees)]/ln [(1ꢂc)(1þees)].13 These equations are used when only one product is achieved in the kinetic
resolution.

J.A. Rodríguez-Rodríguez et al. / Tetrahedron 66 (2010) 6789e6796
6793
R
R
acetate as acyl donor, a fast reaction was observed; in 24 h a 93%
yield of diacetylated product cis-4a was achieved (Table 6, entry 1).
When the process was allowed to react for only 2 h, a 71% yield of
(ꢂ)-cis-5a was obtained, but the enantioselectivity of the process
was low (ee¼33%, entry 2).
O
HO
OH
OsO4
O
O
R
Cl
NMMO
N
Ethyl acetate/H2O
80%
N
N
CH2Cl2/Et3N
70 %
Cbz
2
Cbz
Cbz
cis-4a-c
cis-6
When CAL-B was used as catalyst, in the same conditions, the
formation of the diacetylated product cis-4a was slower and the
monoacetylated derivative (ꢂ)-cis-5a was obtained in 89% yield;
however, the enantioselectivity of the process was only moderate
(ee¼41%, Table 6, entry 3).
a
b
c
R = methyl
R = phenyl
R = allyloxy
Scheme 5. Synthesis of cis-4 and cis-6.
For both catalysts, slower processes were achieved when vinyl
benzoate was used as acyl donor (Table 6, entries 4 and 5). How-
ever, it is noteworthy the enantomeric excess of 75% of the mon-
obezoylated derivative (ꢂ)-cis-5b obtained in the process catalyzed
by CAL-A. Unfortunately, attempts to improve the yield of (ꢂ)-cis-
5b by increasing the reaction time failed because of the formation
of diol cis-6.
Finally, an alcoxycarbonylation was carried out using dia-
llylcarbonate as acylating agent. Product (ꢂ)-cis-5c was obtained in
low yield in both enzymatic processes (Table 6, entries 6 and 7). The
process catalyzed by CAL-A showed a moderate enantioselectivity.
However, as in the case of substrate (ꢂ)-cis-5b, attempts to improve
the yield of the monoalcoxycarbonylated product by increasing the
reaction time, were not possible because of the formation of the
diol cis-6.
R
R
R
O
HO
O
O
OH
HO
Lipase
O
O +
O
Organic solvent / H2O
N
N
N
Cbz
cis-6
Cbz
Cbz
(+) or (−)-cis-5a-c
cis-4a-c
a
b
c
R = methyl
R = phenyl
R = allyloxy
Scheme 6. Enzymatic hydrolysis of cis-4aec.
Table 5
Lipase catalyzed hydrolysis of cis-4aec at 30 ꢁC in 1,4-dioxane. using 5 equiv of H2O
3. Conclusion
Entry Lipasea
R
t (days) (%)b cis-5 eepc (%) cis-5 (%)b cis-6
Thispaperdescribes aneasy methodology for the enantioselective
preparation of both enantiomers of trans-1-benzyloxycarbonyl-3,4-
dihydroxypyrrolidines via a lipase-catalyzed process. The optically
pure diol (3R,4R)-6 can be prepared in an excellent yield by the CAL-B
catalyzed hydrolysis of the diacetyl derivative (ꢀ)-trans-4, in 1,4-di-
oxane at 30 ꢁC. The optically pure (3S,4S)-derivative is obtained as the
remaining substrate in the same process; in order to obtain this iso-
mer in good yield, the reaction must be carried out at 50 ꢁC.
Monoacetate (3S,4S)-5 can be obtained in an enantiopure form
using CAL-A in acetonitrile, although only with a moderate yield of
21%.
1
2
3
4
5
6
CAL-A
CAL-B
CAL-A
CAL-B
CAL-A
CAL-B
Me
Me
Ph
Ph
Allyloxy
Allyloxy
3
3
5
5
8
8
13
5
d
d
35
6
8
6
d
d
44
4
73
15
d
d
13
7
a
b
c
Weight ratio enzyme/substrate 2:1.
Determined by HPLC.
Determined by chiral HPLC.
On the other hand, the CAL-B catalyzed transesterification of the
dihydroxyderivative (ꢀ)-trans-6 allows the preparation the mon-
oacetylated derivative (ꢀ)-trans-5 in an almost quantitative yield.
Finally, the methodology was applied to the preparation of cis
derivatives. Moderate yields and enantiomeric excesses were
obtained in the conditions tested. A more exhaustive study of the
lipase catalyzed desymmetrization of these substrates needs to be
carried out in the future.
R
R
R
O
O
HO
O
OH
O
HO
R'
O
R
O
O
O
+
Lipase
1,4-dioxane
N
N
N
Cbz
cis-4a-c
Cbz
Cbz
(+) or (−)-cis-5a-c
cis-6
a R = methyl
b R = phenyl
c R = allyloxy
R' = vinyl
R' = vinyl
R' = allyl
4. Experimental
Scheme 7. Enzymatic acylation or alcoxycarbonylation of cis-6.
4.1. General remarks
Enzymatic reactions were carried out in a Gallenkamp in-
cubatory orbital shaker. Immobilized C. antarctica lipase B, CAL-B
(Novozym 435, 7300 PLU/g), was a gift from Novo Nordisk co.,
immobilized CAL-A (lipase NZL-101, 6,2 U/g) is commercialized
by Codexis, immobilized P. cepacia lipase (PSL-C, 783 U/g) is
commercialized by Amano Pharmaceuticals. Chemical reagents
were commercialized by Aldrich, Fluka, Lancaster or Prolabo.
Solvents were distilled over an appropriate desiccant under ni-
trogen. Flash chromatography was performed using Merck silica
gel 60 (230e400 mesh). Optical rotations were measured using
a Perkin/Elmer 343 polarimeter and are quoted in units of
10ꢂ1 deg cm2 gꢂ1.1H 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
Table 6
Lipase catalyzed acylation or alcoxycarbonylation of cis-6, using 5 equiv of acylating
agent and a weight ratio enzyme/substrate 2:1
Entry Lipase
R
R0
t (h) (%)a cis-5 eesb (%) cis-5 (%)a trans-4
1
2
3
4
5
6
7
CAL-A Me
CAL-A Me
CAL-B Me
CAL-A Ph
CAL-B Ph
Vinyl
Vinyl
Vinyl
Vinyl 168 31
Vinyl 216
24
2
24 89
7
71
10
33
41
75
0
93
2
9
d
d
d
d
9
8
CAL-A Allyloxy Allyl
CAL-B Allyloxy Allyl
96
95 12
73
13
a
Determined by HPLC.
Determined by chiral HPLC.
b

6794
J.A. Rodríguez-Rodríguez et al. / Tetrahedron 66 (2010) 6789e6796
values (
d
) are given in parts per million. APCIþ and ESIþ using
0.14 mmol) was added under nitrogen atmosphere. The mixture
was stirred at room temperature for 4 h. Then, the mixture was
washed with 1 N HCl (3ꢃ1 mL), the organic phase was separated
and dried over Na2SO4. The solvent was removed under reduced
pressure and the crude residue was purified by flash chromatog-
raphy on silica gel (hexane/EtOAc 2:3) to afford the product
(ꢀ)-trans-5 as a colorless oil (13 mg, 36%). This compound can be
obtained almost quantitatively by the enzymatic procedure as
a Hewlett/Packard 1100 chromatograph mass detector or EIþ
with a Hewlett/Packard 5973 mass spectrometer were used to
record mass spectra (MS). IR spectra were recorded in a UNICAM
Mattson 3000 FT. The enantiomeric excesses were determined by
chiral HPLC analysis on
a Hewlett/Packard 1100, LC liquid
chromatograph, using a CHIRALPCK IA column (4.5ꢃ250 mm)
and concentrations by GC analyses on a Hewlett Packard 6890
Series II chromatograph equipped with
a
column HP-1
stated above. 1H NMR (CDCl3, 300.13 MHz):
d 7.34e7.31 (m, 5H),
(30 mꢃ0.32 mmꢃ0.25
m
m, 1.0 bar N2). For all the analyses, the
5.14 (s, 2H), 5.04e5.02 (m, 1H), 4.27e4.26 (m, 1H), 3.85e3.80 (m,
injector temperature was 70 ꢁC and 5 ꢁC minꢂ1 until the FID
temperature (230 ꢁC).
1H), 3.60e3.48 (m, 2H), 2.72 (br s, 1H, OH), 2.06 (s, 3H); 13C NMR
(CDCl3, 75.5 MHz):
d 170.4 (CO), 154.9 (CO), 136.5 (C), 128.4 (CH),
127.9 (CH), 127.8 (CH), 77.7 (CH), 77.1 (CH), 67.0 (CH2), 52.1 (CH2),
49.3 (CH2), 20.9 (CH3); IR (neat, NaCl):
n ;
3421, 1743, 1687 cmꢂ1
4.2. Synthesis of 1-benzyloxycarbonyl-3-pyrroline, (2)
HRMS-ESIþ calcd for [C14H17NNaO5]þ (MþNa)þ m/z 302.0999,
found 302.1002.
Benzyl chloroformate (3.0 mL, 36.3 mmol) was added drop wise
to a stirred mixture of 3-pyrroline 1 (1.0 g, 14.5 mmol) and pyridine
(5 mL, 36.3 mmol) in dry CH2Cl2 (50 ml) under N2 atmosphere, the
resulting mixture was stirred 4 h at room temperature. After this
time, 50 mL of 1 N HCl was added, the organic phase was separated
and dried over Na2SO4; the solvent was removed under reduced
pressure and the crude residue was purified by flash chromatog-
raphy on silica gel (hexane/EtOAc 4:1) to afford the Cbz-protected
product 2 as a colorless oil (2.8 g, 95%).
4.6. Preparation of ( )-trans-1-benzyloxycarbonyl-3,4-
dihydroxypyrrolidine, [( )-trans-6]
To a solution of (ꢀ)-trans-4 (1.6 g, 5.07 mmol) in methanol
(50 mL), a 0.5 M solution of sodium methoxide in methanol was
added. The mixture was stirred at room temperature for 5 h. Then,
the solvent was removed under reduced pressure and the crude
residue was purified by flash chromatography on silica gel (EtOAc)
to afford the product (ꢀ)-trans-6 as a colorless oil (0.84 g, 70%).
Analytical and spectroscopical data agree with those reported in
the literature.9a
4.3. Synthesis of 1-benzyloxycarbonyl-3,4-epoxy pyrrolidine, (3)
A solution of m-CPBA (1.7 g, 14.8 mmol) in dry CH2Cl2 (9 mL)
was added drop wise to a stirred solution of 2 (1.5 g, 7.4 mmol) in
dry CH2Cl2 (8 mL) and the mixture was stirred at room temperature
for 12 h. The formed solid was removed by filtration and the so-
lution was washed with a 10% aqueous NaOH solution (2ꢃ20 mL).
The organic phase was dried over Na2SO4, the solvent was removed
under reduced pressure to afford (3) as a crude oil residue in a 70%
yield. The residue was used for the preparation of (ꢀ)-trans-4
without a further purification. Analytical and spectroscopical data
agree with those reported in the literature.10
4.7. General procedure for the enzymatic hydrolysis of
( )-trans-4
The reaction mixture, containing (ꢀ)-trans-4 (50 mg,
0.15 mmol), the appropriate amount of H2O and the lipase
(100 mg) in the corresponding organic solvent (2 mL), was
shaken (generally at 30 ꢁC and 250 rpm) in an orbital shaker. The
progress of the reaction was monitored by HPLC analysis of
samples that were prepared by taking aliquots (0.1e0.2 mL) until
achievement of the required conversion. The enzyme was then
removed by filtration and washed with the corresponding or-
ganic solvent. The solvent was evaporated under reduced pres-
sure and the crude residue was purified by flash chromatography
on silica gel (hexane/EtOAc 2:3). The proportion of products and
the remaining substrate was determined by GC: tR 31.98 min
product-trans-6; tR 32.86 min product-trans-5; tR 33.96 min
substrate-trans-4.
4.4. Preparation of ( )-trans-3,4-diacetoxy-1-
benzyloxycarbonylpyrrolidine, [( )-trans-4]
To a solution of 3 (1.4 g, 6.5 mmol) in acetic acid (55 mL), acetic
anhydride was added (2.5 mL, 26.1 mmol). Boron trifluoride
etherate (600 mL, 6.5 mmol) was slowly added, and the mixture was
stirred at room temperature for 12 h. An aqueous saturated solution
of NaHCO3 (10 mL) was added, and the mixture extracted with
EtOAc (20 mL). The organic phase was washed with saturated
aqueous Na2CO3 solution (20 mL) followed by saturated aqueous
NaCl solution (20 mL), and then was dried over Na2SO4. The solvent
was removed under reduced pressure and the crude residue was
purified by flash chromatography on silica gel (hexane/EtOAc 1:1)
to afford the product (ꢀ)-trans-4 as a white solid (1.7 g, 80%). Mp
(hexane/EtOAc) 61e62 ꢁC. 1H NMR (CDCl3, 300.13 MHz):
4.7.1. Determination of the ee by HPLC analysis. Chiralpack IA, 20 ꢁC,
hexane/2-propanol (95:5), UV 210 nm, 0.8 mL minꢂ1, tR 23.85 min
[(3R,4R)-4]; tR 25.93 min [(3S,4S)-4] tR 40.18 min [(3S,4S)-5]; tR
43.29 min [(3R,4R)-5]; tR 51.75 min [(3R,4R)-6]; tR 58.63 min
[(3S,4S)-4].
(þ)-(3S,4S)-3,4-Diacetoxy-1-benzyloxycarbonyl-pyrrolidine,
[(þ)-(3S,4S)-4]. [
a
]
25 þ17.0 (c 1, EtOH), ee >99%.
D
d
7.39e7.35 (m, 5H), 5.16 (br s, 4H), 3.79e3.74 (m, 2H), 3.64e3.54
(m, 2H), 2.08 (s, 6H); 13C NMR (CDCl3, 75.5 MHz):
169.6 (CO),154.6
(CO), 136.4 (C), 128.4 (CH), 128.0 (CH), 127.9 (CH), 74.9 (CH), 74.0
(þ)-(3R,4R)-1-Benzyloxycarbonyl-3,4-dihydroxy-pyrrolidine
d
25
[(þ)-(3R,4R)-6]. [
a
]
þ6.7 (c 1, CHCl3), ee >99%.
D
(CH), 67.0 (CH2), 50.2 (CH2), 50.0 (CH2), 20.8 (CH3); IR (KBr): n 1744,
4.8. General procedure for the enzymatic acylation of
( )-trans-6
1708 cmꢂ1; HRMS-ESIþ calcd for [C16H19NNaO6]þ (MþNa)þ m/z
344.1105, found 344.1110.
The reaction mixture containing (ꢀ)-trans-6 (25 mg,
0.10 mmol), the lipase (50 mg) and the acylating agent in the
corresponding organic solvent (1 mL), was shaken (generally at
30 ꢁC and 250 rpm) in an orbital shaker. The progress of the re-
action was monitored by HPLC analysis of samples that were
prepared by taking aliquots (0.1e0.2 mL) until achievement of
4.5. Preparation of ( )-trans-3-acetoxy-1-benzyloxycarbonyl-
4-hydroxypyrrolidine, [( )-trans-5]
To a solution of (ꢀ)-trans-6 (30 mg, 0.13 mmol) in CH2Cl2
(1 mL) and pyridine (11 mL, 0.14 mmol), acetyl chloride (10 mL,

J.A. Rodríguez-Rodríguez et al. / Tetrahedron 66 (2010) 6789e6796
6795
the required conversion. The enzyme was then removed by fil-
4.12. Synthesis of cis-3-acetoxy-1-benzyloxycarbonyl-4-
hydroxypyrrolidine, ( )-cis-5a
tration and washed with the corresponding organic solvent. The
solvent was evaporated under reduced pressure and the crude
residue was purified by flash chromatography on silica gel
(hexane/EtOAc 2:3).
Analogous procedure to the synthesis of (ꢀ)-trans-5. Colorless
oil, yield 35%. 1H NMR (CDCl3, 300.13 MHz):
7.34e7.33 (m, 5H),
5.19e5.07 (m, 3H), 4.44e4.39 (m, 1H), 3.79e3.69 (m, 2H),
3.57e3.38 (m, 2H), 2.14 (s, 3H); 13C NMR (CDCl3, 75.5 MHz:)
169.4
d
d
4.9. Synthesis of cis-3,4-diacetoxy-1-
benzyloxycarbonylpyrrolidine, (cis-4a)
(CO), 153.9 (CO), 135.3 (C), 127.5 (CH), 127.0 (CH), 126.8 (CH), 76.9
(CH), 76.1 (CH), 66.1 (CH2), 51.1 (CH2), 48.4 (CH2), 19.8 (CH3); IR
(neat, NaCl):
n
3421, 1743, 1687 cmꢂ1; MS (APCIþ, m/z): 280
[(MþH)þ, 84%], 236 (100%). HRMS-ESIþ calcd for [C14H17NNaO5]þ
To a solution of cis-6 (0.2 g, 0.8 mmol) in CH2Cl2 (10 mL) and
pyridine (0.34 mL, 4.2 mmol), acetyl chloride (0.3 mL, 4.2 mmol)
was added. The resulting mixture was stirred at room temper-
ature for 12 h. The solvent was then removed under reduced
pressure and the crude residue was purified by flash chroma-
tography on silica gel (hexane/EtOAc 3:2) to afford the product
cis-4 as a colorless oil (1.5 g, 70%); 1H NMR (CDCl3, 300.13 MHz):
(MþNa)þ m/z 302.0999, found 302.0992.
4.13. Synthesis of cis-3-benzoyloxy-1-benzyloxycarbonyl-4-
hydroxypyrrolidine, ( )-cis-5b
To a solution of cis-6 (0.1 g, 0.42 mmol) in CH2Cl2 (4 mL),
pyridine (67 mL, 0.84 mmol), benzoylchloride (98 mL, 0.84 mmol)
d
7.4e7.3 (m, 5H), 5.4e5.3 (m, 2H), 5.15 (br s, 2H), 3.81e3.74 (m,
2H), 3.56e3.45 (m, 2H), 2.08 (s, 6H); 13C NMR (CDCl3,
75.5 MHz): 168.9 (CO), 153.6 (CO), 135.4 (C), 127.5 (CH), 127.1
(CH), 127.0 (CH), 69.7 (CH), 69.1 (CH), 66.1 (CH2), 47.2 (CH2), 47.0
was added. The resulting mixture was stirred under nitrogen
atmosphere at room temperature for 12 h. Then, 5 mL of CH2Cl2
was added and the resulting mixture was washed with 1 N HCl
(3ꢃ10 mL). The organic phase was separated and dried over
Na2SO4. The solvent was removed under reduced pressure and
the crude residue was purified by flash chromatography on silica
gel (hexane/EtOAc 2:3) to afford the product (ꢀ)-cis-5b as
d
(CH2), 19.6 (CH3); IR (neat, NaCl):
n
1744, 1708 cmꢂ1; HRMS-ESIþ
calcd for [C16H19NNaO6]þ (MþNa)þ m/z 344.1105, found
344.1112.
a
colorless oil (53 mg, 37%); 1H NMR (CDCl3, 300.13 MHz):
4.10. Synthesis of cis-3,4-dibenzoyloxy-1-
benzyloxycarbonylpyrrolidine, (cis-4b)
d
8.12e7.96 (m, 2H), 7.58e7.57 (m, 1H), 7.48e7.34 (m, 2H),
7.37e7.35 (m, 5H), 5.45e5.39 (m, 1H), 5.22e5.08 (m, 2H),
4.57e4.52 (m, 1H), 3.83e3.46 (m, 4H), 2.51 (br s, 1H, OH); 13C
To a solution of cis-6 (0.1 g, 0.42 mmol) in CH2Cl2 (5 mL) and
pyridine (169 mL, 2.1 mmol), benzoylchloride (245 mL, 2.1 mmol)
NMR (CDCl3, 75.5 MHz):
d 171.13 (CO), 166.1 (CO), 154.8 (C),
136.5 (C), 136.4 (CH), 130.0 (CH), 129.7 (CH), 129.1 (CH), 128.5
(CH), 128.4 (CH), 128.3 (CH), 128.0 (CH), 73.7 (CH), 70.5 (CH),
was added. The resulting mixture was stirred at room temperature
for 12 h. Then, 5 mL of CH2Cl2 was added and the resulting mixture
was washed with 1 N HCl (3ꢃ10 mL). The organic phase was sep-
arated and dried over Na2SO4. The solvent was removed under
reduced pressure and the crude residue was purified by flash
chromatography on silica gel (hexane/EtOAc 7:3) to afford the
product cis-4b as a white solid (131 mg, 70%). Mp (hexane/EtOAc)
67.0 (CH2), 50.4 (CH2), 48.6 (CH2); IR (KBr):
n ;
3423, 1706 cmꢂ1
HRMS-ESIþ calcd for [C19H19NNaO5]þ (MþNa)þ m/z 364.1155,
found 364.1153.
4.14. Synthesis of cis-3-allyloxy-1-benzyloxycarbonyl-4-
hydroxypyrrolidine, ( )-cis-5c
124e127 ꢁC. 1H NMR (CDCl3, 300.13 MHz)
5.76e5.68 (m, 2H), 5.24e5.15 (m, 2H), 4.06e3.99 (m, 2H),
3.83e3.70 (m, 2H); 13C NMR (CDCl3, 75.5 MHz):
165.5 (CO), 154.7
d 8.14e7.33 (m, 15H),
To a solution of cis-6 (0.1 g, 0.42 mmol) in CH2Cl2 (4 mL),
pyridine (101 mL, 1.26 mmol), allyl chloroformate (135 mL,
d
(CO), 136.3 (C), 133.6 (CH), 133.4 (CH), 130.1 (CH), 129.7 (CH), 129.5
(CH), 128.5 (C), 128.4 (CH), 128.1 (CH), 128.0 (CH), 71.6 (CH), 70.9
1.26 mmol) was added under nitrogen atmosphere. The
resulting mixture was stirred at room temperature for 2 h.
Then, 6 mL of CH2Cl2 was added and the resulting mixture was
washed with 1 N HCl (3ꢃ10 mL). The organic phase was sepa-
rated and dried over Na2SO4 and the solvent was removed
under reduced pressure. The crude residue was purified by
flash chromatography on silica gel (hexane/EtOAc 2:3) to afford
the product (ꢀ)-cis-5c as a colorless oil (55 mg, 41%); 1H NMR
(CH), 67.2 (CH2), 48.5 (CH2), 48.3 (CH2); IR (KBr): n ;
1733, 1700 cmꢂ1
HRMS-ESIþ calcd for [C26H24NO6]þ (MþH)þ m/z 446.1598, found
446.1600.
4.11. Synthesis of cis-3,4-diallyloxy-1-benzyloxy-
carbonylpyrrolidine, (cis-4c)
(CDCl3, 300.13 MHz):
d 7.43e7.30 (m, 5H), 6.02e5.88 (m, 1H),
To a solution of cis-6 (0.1 g, 0.42 mmol) in CH2Cl2 (5 mL), pyr-
idine (169 mL, 2.1 mmol), allyl chloroformate (224 mL, 2.1 mmol)
5.42e5.41 (m, 1H), 5.37e5.27 (m, 1H), 5.19e5.14 (m, 2H),
5.09e5.02 (m, 1H), 4.70e4.61 (m, 1H), 4.47e4.11 (m, 1H),
4.17e4.09 (m, 1H), 3.82e3.36 (m, 4H), 1.84 (br s, 1H, OH); 13C
was added under nitrogen atmosphere. The resulting mixture was
stirred at room temperature for 24 h. Then, 5 mL of CH2Cl2 was
added and the resulting mixture was washed with 1 N HCl
(3ꢃ10 mL). The organic phase was separated and dried over
Na2SO4 and the solvent was removed under reduced pressure to
afford the product cis-4c as colorless oil (119 mg, 70%); 1H NMR
NMR (CDCl3, 75.5 MHz):
d 154.2 (CO), 135.8 (CO), 130.9 (C),
128.6 (CH), 128.4 (CH), 128.3 (CH), 128.1 (CH), 128.0 (CH), 127.9
(CH), 119.6 (CH2), 69.1 (CH), 68.9 (CH), 67.0 (CH2), 48.6 (CH2),
48.2 (CH2); IR (neat, NaCl):
n
1807, 1743, 1703 cmꢂ1; HRMS-ESIþ
calcd for [C16H19NNaO6]þ (MþNa)þ m/z 344.1105, found
(CDCl3, 300.13 MHz):
d
7.39e7.32 (m, 5H), 6.00e5.87 (m, 2H),
344.1109.
5.41e5.10 (m, 6H), 5.20e5.10 (m, 2H), 4.66e4.64 (m, 4H),
3.85e3.81 (m, 2H), 3.68e3.57 (m, 2H); 13C NMR (CDCl3,
4.15. Synthesis of cis-1-benzyloxycarbonyl-3,4-
dihydroxypyrrolidine, (cis-6)
75.5 MHz):
d 154.4 (CO), 154.0 (CO), 136.3 (C), 131.1 (CH), 128.5
(CH), 128.1 (CH), 128.0 (CH), 127.1 (CH), 119.3 (CH2), 73.9 (CH), 73.4
(CH), 69.0 (CH2), 66.2 (CH2), 48.0 (CH2), 47.6 (CH2); IR (neat, NaCl):
n
To a solution of 2 (1 g, 5 mmol) in acetone/H2O (1:1, 10 mL),
N-methylmorpholine N-oxide (0.87 g, 7.4 mmol) was added, fol-
lowed by a solution of OsO4 in tert-butanol (0.87 mL, 2.5% w/v).
1756, 1712 cmꢂ1; HRMS-ESIþ calcd for [C20H23NNaO8]þ (MþNa)þ
m/z 428.1321, found 428.1336.

6796
J.A. Rodríguez-Rodríguez et al. / Tetrahedron 66 (2010) 6789e6796
The mixture was stirred for
2
days at room temperature.
21.18 min cis-4b; tR 26.21 min [(ꢂ)-5b]; tR 31.84 min [(þ)-5b]; tR
A saturated solution of Na2S2O5 (50 mL) was added and the
mixture was extracted with EtOAc (3ꢃ50 mL). The organic phase
was dried over Na2SO4, the solvent was removed under reduced
pressure and the crude residue was purified by flash chroma-
tography on silica gel (EtOAc) to afford the product cis-4 as
a white solid (0.95 g, 80%). Mp (hexane/EtOAc) 78e80 ꢁC. Ana-
lytical and spectroscopical data agree with those reported in the
literature.14
23.69 min cis-6b.
(ꢂ)-3-Benzoyloxy-1-benzyloxycarbonyl-4-hydroxypyrrolidine,
[(ꢂ)-5b]. [
a
]
25 ꢂ12.7 (c 1, EtOH), ee¼75%.
D
Acknowledgements
This work was supported by the Ministerio de Ciencia e Inno-
vación (project CTQ2007-61126). We express our appreciation to
Novo Nordisk Co. for the generous gift of the lipase CAL-B.
4.16. General procedure for the enzymatic hydrolysis of cis-
4aec
Supplementary data
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.tet.2010.06.060.
The reaction mixture containing cis-4aec (50 mg), the ap-
propriate amount of H2O, and the lipase (100 mg) in 1,4-dioxane
(2 mL), was shaken (generally at 30 ꢁC and 250 rpm) in an orbital
shaker. The progress of the reaction was monitored by HPLC
analysis of samples that were prepared by taking aliquots
(0.1e0.2 mL) until achievement of the required conversion. The
enzyme was then removed by filtration and washed with 1,4-
dioxane. The solvent was evaporated under reduced pressure and
the crude residue was purified by flash chromatography on silica
gel (hexane/EtOAc 2:3). The proportion of products and the
remaining substrate was determined by GC: tR 31.98 min prod-
uct-cis-6; tR 32.86 min product-cis-5a; tR 33.96 min substrates-
cis-4a.
References and notes
1. (a) Suk, D.-H.; Rejman, D.; Dykstra, C. C.; Pohl, R.; Pankiewicz, K. W.; Patterson, S.
E. Bioorg. Med. Chem. Lett. 2007, 17, 2811e2816; (b) Huebsch, W.; Nell, P.; Su-
essmeier, F.; Vakalopoulos, A; Meibom, D; Albrecht-Kuepper, B; Zimmermann, K.
WO 2,009,100,827 A1. CAN 151: 288977; (c) Huang, H.; Wong, C.-H. J. Org. Chem.
1995, 60, 3100e3106; (d) Kang, Y. K.; Shin, K. J.; Yoo, K. H.; Seo, K. J.; Park, S. Y.; Kim,
D. J.; Park, S. W. Bioorg. Med. Chem. Lett.1999, 9, 2385e2390; (e) Lee, Y. S.; Lee, J. Y.;
Jung, S. H.; Woo, E.-R.; Sik, D. H.; Seo, S. H.; Park, H. J. Antibiot. 1994, 47, 609e612.
€
2. (a) Blum, A.; Bottcher, J.; Heine, A.; Klebe, G.; Diederich, W. E. J. Med. Chem.
€
2008, 51, 2078e2087; (b) Bottcher, J.; Blum, A.; Dórr, S.; Heine, A.; Diederich,
W. E.; Klebe, G. ChemMedChem 2008, 3, 1337e1344.
ꢀ
3. (a) Rejman, D.; Pohl, R.; Kocalka, P.; Masojídková, M.; Rosenberg, I. Tetrahedron
ꢀ
2009, 65, 3673e3681; (b) Rejman, D.; Pohl, R.; Kocalka, P.; Masojídková, M.;
Rosenberg, I. Tetrahedron 2007, 63, 1243e1253.
4. (a) Davis, B. G.; Maughan, M. A. T.; Chapman, T. M.; Villard, R.; Courtney, S. Org.
Lett. 2002, 4,103e106; (b) Lysek, R.; Vogel, P. Helv. Chim. Acta 2004, 87, 3167e3181.
5. (a) Markert, C.; Rósel, P.; Pfaltz, A. J. Am. Chem. Soc. 2008, 130, 3234e3235; (b)
Marson, C. M.; Melling, R. C. Synthesis 2006, 247e256; (c) Rocha Gonsalves,
A. M. d’. A.; Serra, M. E. S.; Murtinho, D.; Silva, V. F.; Matos Beja, A.; Paixão, J. A.;
Ramos Silva, M.; Alte da Veiga, L. J. Mol. Catal. A: Chem. 2003, 195, 1e9.
6. Bonnani, M.; Sodaini, G.; Faggi, C.; Goti, A.; Cardona, F. Synlett 2009, 747e750.
7. Xiao, Y.; Ong, T.-T.; Tan, T.; Ng, S.-C. J. Chromatogr., A 2009, 1216, 994e999.
4.17. General procedure for the enzymatic acylation or
alcoxycarbonylation of cis-6
The reaction mixture containing cis-6 (25 mg, 0.10 mmol), the
lipase (50 mg), 1,4-dioxane (1 mL), and the corresponding acyl-
ation or alcoxycarbonylation agent (5 equiv), was shaken (gen-
erally at 30 ꢁC and 250 rpm) in an orbital shaker. The progress of
the reaction was monitored by HPLC analysis of samples that
were prepared by taking aliquots (0.1e0.2 mL) until the
achievement of the required conversion. The enzyme was then
removed by filtration and washed with 1,4-dioxane. The solvent
was evaporated under reduced pressure and the crude residue
was purified by flash chromatography on silica gel (hexane/
EtOAc 2:3).
_
8. Skarzewski, J.; Gupta, A. Tetrahedron: Asymmetry 1997, 8, 1861e1867.
9. (a) Zhao, L.; Han, B.; Huang, Z.; Miller, M.; Huang, H.; Malashock, D. S.; Zhu, Z.;
Milan, A.; Robertson, D. E.; Weiner, D. P.; Burk, M. J. J. Am. Chem. Soc. 2004, 126,
11156e11157; (b) Chang, D.; Heringa, M. F.; Witholt, B.; Li, Z. J. Org. Chem. 2003,
68, 8599e8606; (c) Chang, D.; Wang, Z.; Heringa, M. F.; Wirthner, R.; Witholt,
B.; Li, Z. Chem. Commun. 2003, 960e961.
10. Xue, F.; Seto, C. T. Bioorg. Med. Chem. 2006, 14, 8467e8487.
11. Recuero, V.; Brieva, R.; Gotor, V. Tetrahedron: Asymmetry 2008, 19, 1684e1688.
12. Solares, L. F.; Lavandera, I.; Gotor-Fernández, V.; Brieva, R.; Gotor, V. Tetrahedron
2006, 62, 3284e3291.
13. Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem. Soc. 1982, 104,
7294e7299.
14. Rosenberg, S. H.; Woods, K. W.; Sham, H. D.; Kleinert, H. D.; Martin, D. L.; Stein,
H.; Cohen, J.; Egan, D. A.; Bopp, B.; Merits, I.; Garren, K. W.; Hoffman, D. J.;
Plattner, J. J. J. Med. Chem. 1990, 33, 1962e1969.
4.17.1. Determination of the ee by HPLC analysis. Chiralpack AS,
30 ꢁC, hexane/2-propanol (90:10), UV 210 nm, 0.8 mL minꢂ1, tR