Efficient access to enantiomerically pure cyclic α-amino esters through a lipase-catalyzed kinetic resolution

Article abstract of DOI:10.1016/j.tetasy.2008.06.010

A series of α-amino acid derivatives containing the 2,3-dihydroindole or octahydroindole core have been chemoenzymatically synthesized in good overall yields and high enantiomeric purity under mild reaction conditions using lipases for the introduction of chirality. Candida antarctica lipase type A has shown excellent activity and high enantiodiscrimination ability toward the two cyclic amino esters used as substrates. The selectivity of the process proved to be greatly dependent on the alkoxycarbonylating agent. Thus, the enzymatic kinetic resolution of methyl indoline-2-carboxylate has been successfully achieved using 3-methoxyphenyl allyl carbonate, whereas (2R,3aR,7aR)-benzyl octahydroindole-2-carboxylate required the less reactive diallyl carbonate.

Full text of DOI:10.1016/j.tetasy.2008.06.010

Tetrahedron: Asymmetry 19 (2008) 1714–1719
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Efficient access to enantiomerically pure cyclic
lipase-catalyzed kinetic resolution
a-amino esters through a
Sergio Alatorre-Santamaría a, María Rodriguez-Mata a, Vicente Gotor-Fernández a,
a,
Marcos Carlos de Mattos b, Francisco J. Sayago c, Ana I. Jiménez c, Carlos Cativiela c, , Vicente Gotor
*
*
a Departamento de Química Orgánica e Inorgánica, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33071 Oviedo (Asturias), Spain
b Departamento de Química Orgânica e Inorgânica, Laboratorio de Biotecnologia e Sintese Orgânica (LABS), Universidade Federal do Ceará, 60451-970, Fortaleza (Ceará), Brazil
c Departamento de Química Orgánica, Instituto de Ciencia de Materiales de Aragón, Universidad de Zaragoza-CSIC, 50009 Zaragoza, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of a-amino acid derivatives containing the 2,3-dihydroindole or octahydroindole core have been
Received 2 June 2008
Accepted 5 June 2008
Available online 28 June 2008
chemoenzymatically synthesized in good overall yields and high enantiomeric purity under mild reaction
conditions using lipases for the introduction of chirality. Candida antarctica lipase type A has shown
excellent activity and high enantiodiscrimination ability toward the two cyclic amino esters used as sub-
strates. The selectivity of the process proved to be greatly dependent on the alkoxycarbonylating agent.
Thus, the enzymatic kinetic resolution of methyl indoline-2-carboxylate has been successfully achieved
using 3-methoxyphenyl allyl carbonate, whereas (2R,3aR,7aR)-benzyl octahydroindole-2-carboxylate
required the less reactive diallyl carbonate.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Kurokawa and Sugai described the enzymatic kinetic resolution
of methyl N-Boc-indoline-2-carboxylate through a Candida antarc-
The preparation of optically active compounds is a great chal-
lenge for organic chemists, and enantiomerically pure amines
and amino acid derivatives represent one of the most important
targets.1 Nowadays, enzyme-mediated processes are becoming
usual techniques for enantioselective transformations and, among
the biocatalysts of synthetic interest, lipases have gained much
attention over the last two decades due to their ease of manipula-
tion, low cost, and wide substrate tolerance.2 Traditionally, most li-
pase-catalyzed enantioselective reactions have corresponded to
the acylation of alcohols or hydrolysis of esters, whereas the prep-
aration of chiral amides or carbamates has been much less investi-
gated in spite of the fact that mild reaction conditions are required
for the formation of these nitrogenated compounds.3 Examples of
enzymatic kinetic or dynamic kinetic resolutions of cyclic second-
ary amines described in the literature are rare,4 even though they
are present in a large number of biologically relevant systems
and are useful building blocks for the preparation of many valuable
products.
tica lipase B (CAL-B) enantioselective hydrolysis, which yielded
both the substrate and the product in enantiopure form.4g In our
ongoing research on the synthesis of optically active secondary
amines, we observed4j a different reactivity of C. antarctica lipase
A (CAL-A) and CAL-B toward the N-protection of indolines. Thus,
whereas CAL-A exhibited a high enantiopreference toward 2-
substituted derivatives, CAL-B showed remarkable stereoselectivi-
ty toward 3-methyl indoline. This fact suggests that CAL-A prefers
to act at sterically hindered positions,7 while the approach of ind-
olines to the active site of CAL-B demands sterically non-hindered
situations.8
Octahydroindole-2-carboxylic acid (Oic), the completely hydro-
genated analogue of indoline-2-carboxylic acid, is the core struc-
ture of numerous compounds with applications in medicinal
chemistry. For example, it is present in marine compounds with
antithrombotic properties;9 in the dipeptide perindropil (a potent
antihypertensive drug used in the prevention of cardiovascular dis-
orders such as heart failure);10 and in the prolyl oligopeptidase
inhibitor S 17092, a compound with antiamnesic and cognitive-
enhancing properties.11 Recently, some of us reported12 the syn-
thesis of enantiomerically pure (2S,3aS,7aS)- and (2R,3aR,7aR)-Oic
derivatives using preparative chiral HPLC,12a and that of the
Among cyclic secondary amines, the indoline (2,3-dihydroin-
dole) subunit is found in a wide range of natural products and bio-
logically active compounds.5 Only a few catalytic methods for the
production of optically active indolines, either through non-enzy-
matic6 or enzymatic4b,g,j methodologies, have been reported.
(2R,3aS,7aS) isomer by selective formation of
a trichloro-
methyloxazolidinone.12b The only enzymatic process for producing
optically active Oic derivatives was described by Hirata13 and is
based on the enzymatic hydrolysis of N-protected octahydroin-
dole-2-carboxylates.
* Corresponding authors.
E-mail address: vgs@fq.uniovi.es (V. Gotor).
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.06.010

S. Alatorre-Santamaría et al. / Tetrahedron: Asymmetry 19 (2008) 1714–1719
1715
Our interest in indoline-2-carboxylic acid and octahydroindole-
2-carboxylic acid to be used as proline analogues together with our
experience in the enzymatic resolution of secondary amines,
prompted us to apply procedures to the isolation of these com-
pounds in enantiomerically pure form. Herein, we report the appli-
cation of a straightforward and highly selective lipase-catalyzed
methodology for the alkoxycarbonylation of the secondary amino
group in these compounds.
diallyl carbonate 4b (entries 5 and 6). The use of this reagent
avoids the need for the purification step to separate the 3-
methoxyphenol released during the enzymatic process from the
reaction crude. However, this change resulted in lower kinetics
(entry 5), while higher temperatures led to a decrease in the
enantioselectivity value (entry 6) probably due to the inactivation
of the enzyme.
Thus, under the optimal conditions, we were able to isolate one
enantiomer of both the substrate and the product in enantiomeri-
cally pure form and good yield (Table 1, entry 4). The absolute con-
figuration of the amino ester 2 remaining from the enzymatic
kinetic resolution was determined by comparing its HPLC retention
time15 with that of a sample of enantiopure (S)-2 obtained by
esterification of commercially available (S)-indoline-2-carboxylic
acid, (S)-1. This allowed us to assign an (R)-configuration to the
unreacted amine 2 and an (S)-stereochemistry to the isolated car-
bamate 3 (Scheme 1).
We next undertook the development of adequate chemoenzy-
matic procedures to obtain Oic derivatives in enantiopure form
(Scheme 2). Hydrogenation of racemic indoline-2-carboxylic acid
rac-1 following our previously described methodology,12a afforded
rac-5, which was transformed into the corresponding methyl rac-
6a and benzyl rac-6b esters using standard procedures. Reaction
with allyl chloroformate led to carbamates rac-7a and rac-7b,
respectively, which were analyzed by HPLC to find adequate condi-
tions for the separation of their enantiomers.
For the enzymatic kinetic resolution of rac-6a, CAL-A from Cod-
exis was initially chosen as the biocatalyst, since it gave the best
results in the resolution of 2, and different carbonates were tested.
No reaction was observed when using dibenzyl carbonate, even
when the temperature was raised to 45 °C. In contrast, the more
reactive methoxyphenyl allyl carbonate 4a led to the formation
of racemic carbamate 7a (Table 2, entry 1). The use of diallyl car-
bonate 4b, which is known to exhibit intermediate reactivity, al-
lowed the formation of 7a with excellent selectivity, although
2. Results and discussion
Initially, we focused on the enzymatic resolution of methyl ind-
oline-2-carboxylate rac-2. This compound was obtained in excel-
lent yield by treatment of commercially available indoline-2-
carboxylic acid rac-1 with thionyl chloride in refluxing methanol
(Scheme 1). Prior to the enzymatic resolution assays, compound
rac-2 was transformed into carbamate rac-3 by reaction with allyl
chloroformate, in order to establish the appropriate analytical con-
ditions for HPLC separation of the two enantiomers.
The enzymatic kinetic resolution of rac-2 was then attempted
using C. antarctica lipase type A (CAL-A) from different sources
and C. antarctica lipase type B (CAL-B) as biocatalysts. The data
are summarized in Table 1. Based on previous results with 2-
substituted indoline derivatives,4j 3-methoxyphenyl allyl carbon-
ate14 4a was initially selected as the alkoxycarbonylating reagent
for enzyme activity screening, which was carried out at 30 °C using
dry tert-butyl methyl ether (TBME) as the solvent and a 1:2.5 ami-
no ester to carbonate ratio (entries 1–4). For all the tested CAL-A
preparations, a complete enantioselectivity was observed in the
alkoxycarbonylation process. Lipase from Biocatalytics showed a
lower reaction rate than the ones from Roche or Codexis, although
all of them reached 50% conversion (entries 1–3). An excellent
selectivity was also observed for CAL-B, but low conversions were
reached in longer reaction times (entry 4). Finally, the reaction was
carried out using CAL-A from Codexis and commercially available
O
CO2Me
SOCl2
N
O
Cl
O
CO2H
CO2Me
O
N
N
H
MeOH
reflux (97%)
Pyridine
CH2Cl2
r.t. (90%)
H
rac-1
rac-2
rac-3
CO2Me
O
Lipase
TBME
O
N
O
CO2Me
CO2Me
+
+
N
H
N
RO
O
H
30 ˚C, 250 rpm
rac-2
4a: R= 3-MeO-C6H4-
4b: R= CH2=CHCH2-
(S)-3
(R)-2
Scheme 1. Chemical synthesis and enzymatic kinetic resolution of methyl indoline-2-carboxylate.
Table 1
Lipase-catalyzed kinetic resolution of amino ester rac-2 using carbonates 4a–b (ratio 1:2.5) in TBME
a
a
Entry
Enzyme
R
T (°C)
t (h)
eeP (%)
eeS (%)
cb (%)
Ec
1
2
3
4
5
6
CAL-A Biocatalytics
CAL-A Roche
CAL-A Codexis
CAL-B
CAL-A Codexis
CAL-A Codexis
3-MeO–C6H4
3-MeO–C6H4
3-MeO–C6H4
3-MeO–C6H4
CH2@CHCH2
CH2@CHCH2
30
30
30
30
30
45
20
8
4
60
74
49
>99
>99
>99 (92)d
99
92
94
98
98
50
50
50
46
15
8
>200
>200
>200
>200
29
>99 (86)d
85
16
8
35
a
Calculated by HPLC.
c = eeS/(eeS + eep).
b
c
E = ln[(1 ꢀ c)ꢁ (1 ꢀ eeP)]/ln[(1 ꢀ c)ꢁ(1 + eeP)].
d
Isolated yields in brackets.

1716
S. Alatorre-Santamaría et al. / Tetrahedron: Asymmetry 19 (2008) 1714–1719
O
H
H
H
H
MeOH, SOCl2
r.t.
H2, PtO2
Cl
O
COOR1
COOH
COOH
COOR1
O
or
CH2Cl2, r.t.
N
N
N
AcOH, 60 ˚C
(70%)
N
H
H
H
H
BnOH, TsOH
toluene, reflux
H
O
rac-1
rac-6a R1=Me (84%)
rac-6b R1=Bn (90%)
rac-7a (40%)
rac-7b (49%)
rac-5
H
COOR1
N
H
O
H
O
(2S,3aS,7aS)-7b
O
CAL-A
COOR1
+
+
R2O
O
TBME
30 ˚C, 250 rpm
N
H
H
H
H
H
4a: R2= 3-MeO-C6H4-
4b: R2= CH2=CHCH2-
(Boc)2O, DMAP
COOR1
COOR1
rac-6a-b
THF, r.t.
(81%)
N
H
N
H
Boc
(2R,3aR,7aR)-6b
(2R,3aR,7aR)-8b
Scheme 2. Chemical synthesis and enzymatic kinetic resolution of Oic derivatives.
Table 2
Lipase-catalyzed kinetic resolution of amino esters rac-6a–b using carbonates 4a–b (ratio 1:2.5) in TBME
R1
R2
T (°C)
t (h)
eeP (%)
eeS (%)
cb (%)
Ec
a
a
Entry
Enzyme
1
2
3
4
5
CAL-A Codexis
CAL-A Codexis
CAL-A Codexis
CAL-A Codexis
CAL-A Biocatalytics
Me
Me
Me
Bn
3-MeO–C6H4
CH2@CHCH2
CH2@CHCH2
CH2@CHCH2
CH2@CHCH2
30
30
45
30
30
23.5
23
48
57
72
—
—
100
20
12
53
50
—
>99
>99
88
25
13
>99
>200
>200
121
>200
Bn
98 (91)d
>99 (90)d
a
Calculated by HPLC.
c = eeS/(eeS + eep).
b
c
E = ln[(1 ꢀ c)ꢁ(1 ꢀ eeP)]/ln[(1 ꢀ c)ꢁ(1 + eeP)].
d
Isolated yields in brackets.
only 20% conversion was reached after 23 h at 30 °C (entry 2). An
increase in the temperature (entry 3) did not result in a higher con-
version but rather in a loss of enzyme activity, as observed before
for 2.
fully achieved through enzymatic kinetic resolution using lipases
in organic solvents. The different lipases tested exhibited distinct
behaviors in the alkoxycarbonylation process, depending on the
structure of the starting materials. In all cases, CAL-A has been
found to be the most efficient biocatalyst. The choice of the appro-
priate allyl carbonate also proved crucial to reach a good selectivity
and conversion. Under the best conditions, all substrates and prod-
ucts were isolated in good yields and enantiomerically pure form
(98% ee in one case). This methodology represents an excellent
alternative to those previously described for the preparation of
these biologically significant compounds.
Our attention was then focused on the resolution of amino ester
rac-6b that, with it being more sterically hindered than rac-6a,
could be a better substrate for the active site of CAL-A.7 Indeed,
the enzymatic kinetic resolution of rac-6b using CAL-A from Cod-
exis was found to proceed with very high enantiopreference, thus
allowing recovery of the remaining starting material in enantio-
pure form (entry 4). Alternatively, when CAL-A from Biocatalytics
was used, a 50% conversion was reached, with both the product
and the starting material being obtained in almost enantiomeri-
cally pure form and excellent isolated yields (entry 5). The absolute
configuration of both compounds was assigned after transforma-
tion of the remaining substrate into a derivative of known stereo-
chemistry. Thus, the unreacted enantiomerically pure amino ester
6b was subjected to N-tert-butoxycarbonyl protection, as previ-
ously described for the racemic compound,12a to afford 8b (Scheme
2); the HPLC retention time of the latter compound was found to be
identical16 to that of a sample of (2R,3aR,7aR)-8b prepared in our
previous work.12a Thereafter, we concluded that the unreacted
amino ester 6b has a (2R,3aR,7aR)-configuration and the carbamate
formed in the enzymatic process 7b is (2S,3aS,7aS).
4. Experimental
4.1. General
C. antarctica lipase type B (CAL-B, Novozyme 435, 7300 PLU/g)
was a gift from Novo Nordisk Co. C. antarctica lipase type A was ac-
quired from different commercial sources:17 CAL-A Chirazyme L-5,
c-f, lyophilized, 1000 U/g using tributyrin from Roche, CAL-A
immobilized NZL-101, 5.0 U/g using 1-butanol and ethyl laurate
from Codexis; CAL-A IMB-104, 2.6 U/mg using tributyrin from Bio-
catalytics. All other reagents were purchased from Aldrich and
used without further purification. Solvents were distilled over an
adequate desiccant under nitrogen. Flash chromatography was
performed using Silica Gel 60 (230–240 mesh). High performance
liquid chromatography (HPLC) analyses were carried out at 20 °C
in a Hewlett Packard 1100 chromatograph under the conditions
specified for each substrate and with UV monitoring at 210 nm.
IR spectra were recorded by using NaCl plates or KBr pellets in a
3. Conclusions
In conclusion, the development of a practical synthetic route for
the preparation of optically active indoline-2-carboxylic acid and
octahydroindole-2-carboxylic acid derivatives has been success-

S. Alatorre-Santamaría et al. / Tetrahedron: Asymmetry 19 (2008) 1714–1719
1717
Perkin–Elmer 1720-X FT. 1H, 13C NMR, DEPT, and 1H–13C heteronu-
clear experiments were obtained using AC-200 (1H, 200.13 MHz
and 13C, 50.3 MHz), AC-300 (1H, 300.13 MHz and 13C, 75.5 MHz),
DPX-300 (1H, 300.13 MHz and 13C, 75.5 MHz) or AV-400 (1H,
400.13 MHz and 13C, 100.6 MHz) Bruker spectrometers. The chem-
ical shifts are given in delta (d) values and the coupling constants
(J) in Hertz (Hz).18 HP1100 chromatograph mass detector was used
to record mass spectra experiments (MS) through APCI+ or ESI+
experiments. Measurement of the optical rotation was done in a
Perkin–Elmer 241 polarimeter.
system was shaken at 30 °C and 250 rpm. The course of the reac-
tion was followed by HPLC till conversions were around 50% (see
Table 1). The mixture was then filtered and the filtrate was concen-
trated under reduced pressure. The crude product was purified by
flash chromatography (eluent gradient 5–15% EtOAc/hexane) to
give (R)-2 {½a 2D0
¼ ꢀ47:3 (c 0.28, CH2Cl2) for >99% ee} and (S)-3
ꢂ
{½a 2D0
¼ ꢀ106:5 (c 1.2, CH2Cl2) for >99% ee}.
ꢂ
4.5. Synthesis of (2S*,3aS*,7aS*)-methyl octahydroindole-2-
carboxylate rac-6a
4.2. Synthesis of methyl indoline-2-carboxylate rac-2
Thionyl chloride (1.18 mL, 16.18 mmol) was added dropwise to
a solution of rac-5 (1.32 g, 7.81 mmol) in dry methanol (20 mL) at
0 °C. The resulting solution was stirred at room temperature for
24 h. The solvent was evaporated to dryness and the resulting res-
idue was dissolved in water and lyophilized. The white solid ob-
tained was partitioned between saturated aqueous NaHCO3
(25 mL) and CH2Cl2 (50 mL). The organic layer was separated and
the aqueous phase was further extracted with CH2Cl2 (2 ꢁ 50 mL).
The combined organic layers were dried over anhydrous MgSO4, fil-
tered, and evaporated to afford rac-6a as an oil (1.20 g; 6.56 mmol;
To a solution of compound rac-1 (1.0 g, 6.13 mmol) in methanol
(100 mL) under a nitrogen atmosphere, thionyl chloride (671 lL,
9.19 mmol) was added dropwise and the reaction mixture was
heated at reflux for 2 h. The solvent was removed, and the resulting
residue was suspended in saturated aqueous NaHCO3 and extracted
with CH2Cl2 (3 ꢁ 30 mL). The organic fractions were combined,
dried, and evaporated to dryness. The crude product was purified
by flash chromatography (30% EtOAc/hexane) to afford 1.01 g of
rac-2 as an oil (97%). Rf (40% EtOAc/hexane): 0.44; IR (NaCl):
3335, 2950, 2356, 1732, 1684, 1652, 1609, 1531, 1487, 1436,
1313, 1257, 1207, 1151, 992, 773, 748 cmꢀ1 1H NMR (CDCl3,
m
84%). Rf (5% MeOH/CH2Cl2): 0.24; IR (NaCl):
2856, 1733, 1454, 1191, 750 cmꢀ1 1H NMR (CDCl3, 400.13 MHz):
d 3.81 (dd, J = 10.0, 6.0 Hz, 1H, H2), 3.74 (s, 3H, 3H10), 3.09 (m, 1H,
7a), 2.38 (br s, 1H, NH), 2.17 (ddd, J = 12.8, 10.0, 6.8 Hz, 1H, H3),
2.03 (m, 1H, H3a), 1.74–1.60 (m, 3H, H3 + 2H7), 1.52-1.30 (m, 3H,
H4 + H5 + H6), 1.30–1.15 (m, 3H, H6 + H4 + H5); 13C NMR (CDCl3,
100.6 MHz): d 176.2 (C8); 58.4 (C2); 58.0 (C7a); 52.0 (C10); 37.9
(C3a); 35.6 (C3); 27.9 (C7); 27.0 (C4); 23.4 (C5); 21.5 (C6); MS (APCI+,
m/z): 184 [(M+H)+, 100%], 124 [(MꢀCO2Me)+, 32%]. HPLC elution
times: first enantiomer 8.7 min, second enantiomer 10.7 min; con-
ditions: 25 ꢁ 0.46 cm ID Chiralcel OD column, eluent n-hexane/2-
propanol (95:5) at 0.8 mL/min flow rate.
m 3319, 3033, 2927,
;
;
300.13 MHz): d 7.02–7.11 (m, 2H, H4 + H6), 6.71–6.79 (m, 2H,
H5 + H7), 4.39 (dd, J = 9.0, 6.0 Hz, 1H, H2), 3.80 (s, 3H, H10), 3.28–
3.45 (m, 2H, 2H3); 13C NMR (CDCl3, 75.5 MHz): d 174.5 (C8), 149.9
(C7a), 127.6 (C5), 126.5 (C3a), 124.4 (C4), 119.4 (C6), 110.0 (C7),
59.7 (C2), 52.4 (C10), 33.6 (C3). MS (APCI+, m/z): 200 [(M+Na)+,
100%], 178 [(M+H)+, 12%]. HPLC elution times: (S)-2 15.7 min, (R)-
2 17.1 min; conditions: 25 ꢁ 0.46 cm ID Chiralcel OD column, elu-
ent n-hexane/ethanol (97:3) at 0.8 mL/min flow rate.
H
4.3. Synthesis of methyl N-(allyloxycarbonyl)indoline-2-
carboxylate rac-3
4.6. Synthesis of (2S*,3aS*,7aS*)-benzyl octahydroindole-2-
carboxylate rac-6b
Pyridine (14.5 lL, 0.18 mmol) was added to a solution of amino
ester rac-2 (30 mg, 0.17 mmol) in dry CH2Cl2 (1.13 mL) under a
nitrogen atmosphere and the mixture was cooled in an ice bath. Al-
lyl chloroformate (15.3 lL, 0.18 mmol) was added dropwise and
the system was allowed to warm up to room temperature. After
stirring for 4 h, no starting material was detected by TLC analysis.
The solvent was evaporated under reduced pressure and the resi-
due obtained was purified by flash chromatography (30% EtOAc/
hexane) to afford 40 mg of carbamate rac-3 as a pale yellow oil
To a solution of rac-5 (1.00 g, 5.92 mmol) in toluene (21 mL), p-
toluenesulfonic acid monohydrate (1.63 g, 8.59 mmol) and benzyl
alcohol (2.30 mL, 22.21 mmol) were added. The resulting system
was heated at reflux for 4 h using a Dean–Stark trap. After evapo-
ration to dryness, the residue was triturated with diisopropyl ether
and the white solid formed was collected by filtration. It was then
partitioned between saturated aqueous NaHCO3 (25 mL) and
CH2Cl2 (50 mL). The organic layer was separated and the aqueous
phase was further extracted with CH2Cl2 (2 ꢁ 50 mL). The com-
bined organic extracts were dried over anhydrous MgSO4, filtered,
and evaporated to afford rac-6b as an oil (1.38 g; 5.33 mmol; 90%).
(90%). Rf (40% EtOAc/hexane): 0.51; IR (NaCl):
2365, 2342, 1756, 1701, 1648, 1604, 1530, 1487, 1400, 1268,
1205, 826, 749 cmꢀ1 1H NMR (CDCl3, 400.13 MHz): d 7.70 (t,
m 3336, 2954,
;
J = 8.0 Hz, 1H, H7), 7.43 (d, J = 8.3 Hz, 1H, H4), 7.33 (t, J = 15.1 Hz,
1H, H5), 7.16 (t, J = 15.1 Hz, 1H, H6), 6.00–6.25 (m, 1 H, H14),
5.25–5.60 (m, 2H, H15), 5.05–5.15 (m, 1H, H2), 4.70–5.00 (m, 2H,
Rf (5% MeOH/CH2Cl2): 0.41; IR (NaCl):
m 3319, 3033, 2927, 2856,
1733, 1454, 1191, 750 cmꢀ1 1H NMR (CDCl3, 400.13 MHz): d
;
7.37–7.28 (m, 5H, Ph); 5.20 (s, 2H, H10); 3.88 (dd, J = 10.1, 5.9 Hz,
1H, H2); 3.12 (m, 1H, H7a); 2.52 (br s, 1H, NH); 2.20 (ddd,
J = 12.8, 10.1, 6.9 Hz, 1H, H3); 2.04 (m, 1H, H3a); 1.77–1.65 (m,
3H, H3 + 2H7); 1.48 (m, 3H, H4 + H5 + H6); 1.39 (m, 1H, H6); 1.26
(m, 2H, H4 + H5); 13C NMR (CDCl3, 100.6 MHz): d 175.7 (C8);
135.7 (CipsoPh), 128.5, 128.2, 128.1 (5C, Ph), 66.7 (C10); 58.6 (C2);
58.1 (C7a); 37.9 (C3a); 35.8 (C3); 27.9 (C7); 27.0 (C4); 23.5 (C5);
21.5 (C6). MS (APCI+, m/z): 260 [(M+H)+, 100]. HPLC elution times:
(2S,3aS,7aS)-6b 8.2 min, (2R,3aR,7aR)-6b 9.6 min; conditions:
25 ꢁ 0.46 cm ID Chiralcel OD column, eluent n-hexane/2-propanol
(90:10) at 0.8 mL/min flow rate.
H13), 3.90 (s, 3H, H10), 3.69 (t, J = 12.0 Hz, 1H, H3), 3.30 (d,
J = 15.0 Hz, 1H, H3); 13C NMR (CDCl3, 100.6 MHz): d 172 (C8), 152
(C11), 142.1 (C7a), 132.1 (2C, C14, and C3a), 127.9 (C5), 124.3 (C4),
122.9 (C6), 117.7 (C15), 114.7 (C7), 66.0 (C13), 59.9 (C2), 52.9 (C10),
32.8 (C3); MS (APCI+, m/z): 300 [(M+K)+, 34%], 284 [(M+Na)+,
100%], 262 [(M+H)+, 51%]. HPLC elution times: (S)-3 12.3 min,
(R)-3 14.9 min; conditions: 25 ꢁ 0.46 cm ID Chiralcel OD column,
eluent n-hexane/ethanol (97:3) at 0.8 mL/min flow rate.
4.4. Typical procedure for the enzymatic kinetic resolution of
methyl indoline-2-carboxylate rac-2
4.7. Synthesis of carbamates rac-7a–b
To a suspension of compound rac-2 (50 mg, 0.28 mmol) and li-
pase (100 mg) in TBME (1.9 mL) kept under a nitrogen atmosphere
was added the corresponding carbonate 4a–b (0.70 mmol) and the
Allyl chloroformate (18.0
to a solution of the amino ester rac-6a–b (0.15 mmol) in dry CH2Cl2
lL, 0.17 mmol) was added dropwise

1718
S. Alatorre-Santamaría et al. / Tetrahedron: Asymmetry 19 (2008) 1714–1719
(1.03 mL) kept under a nitrogen atmosphere at 0 °C and the
system was allowed to warm up to room temperature. After stir-
ring for 4 h, no starting material was detected by TLC analysis.
The solvent was evaporated under reduced pressure and the resi-
due obtained was purified by flash chromatography (20% EtOAc/
hexane) to afford the corresponding carbamate rac-7a–b as an oil
(40–49%).
Acknowledgments
We thank Novo Nordisk Co. for the generous gift of CAL-B
(Novozyme 435). This work was supported by the Spanish Ministe-
rio de Educación y Ciencia (Projects CTQ2007-61126 and CTQ2007-
62245) and Gobierno de Aragón (project PIP206/2005 and research
group E40). V.G.-F. thanks the Spanish MEC for a personal grant
(Ramón y Cajal Program), S.A.-S. thanks the Mexican CONACYT
for a pre-doctoral fellowship, and M.C.M. thanks the Brazilian
Agency CAPES for a post-doctoral fellowship. This project has been
funded in whole or in part with Federal funds from the National
Cancer Institute, National Institutes of Health, under contract num-
ber N01-CO-12400. The content of this publication does not neces-
sarily reflect the view of the policies of the Department of Health
and Human Services, nor does the mention of trade names, com-
mercial products, or organization imply endorsement by the US
Government. This research was supported (in part) by the Intramu-
ral Research Program of the NIH, National Cancer Institute, Center
for Cancer Research.
4.7.1. (2S*,3aS*,7aS*)-Methyl N-(allyloxycarbonyl)-
octahydroindole-2-carboxylate rac-7a
Rf (20% EtOAc/hexane): 0.25; IR (NaCl):
m
2929, 2857, 1753,
1705, 1407, 1345, 1303, 1262, 1176, 1123, 1010 cmꢀ1
;
1H NMR
(CDCl3, 300.13 MHz): d 5.98–5.77 (m, 1H, H11), 5.31–5.13 (m, 2H,
2H15), 4.58–4.44 (m, 2H, H13), 4.33–4.25 (m, 1H, H2), 3.93–3.78
(m, 1H, H7a), 3.73 (d, J = 9.8 Hz, 3H, H10), 2.34–2.29 (m, 1H, H3a),
2.14–1.92 (m, 3H, 2H3 + H7), 1.73–1.58 (m, 3H, H5 + 2H4), 1.50–
1.36 (m, 2H, H7 + H6), 1.34–1.08 (m, 2H, H5, H6); 13C NMR (CDCl3,
75.5 MHz): d (duplicate signals are observed for all carbon atoms)
173.6, 173,4 (C8), 154.4, 153.7 (C11), 133.0, 132.7 (C14), 117.1, 116.8
(C15), 65.7, 65.5 (C13), 59.0, 58.9 (C2), 57.7, 57.3 (C7a), 52.1, 52.0
(C10), 37.0, 36.4 (C3a), 32.5, 31.5 (C3), 27.8, 27.2 (C7), 25.7, 25.6
(C4), 23.6, 23.5 (C5), 20.4, 20.3 (C6); MS (APCI+, m/z): 268
[(M+H)+, 100%], 210 [(MꢀCO2Me+2H)+, 10%], 182 [(MꢀCO2CH2-
CHCH2)+, 31%], 124 [(MꢀCO2CH2CHCH2ꢀCO2Me+H)+, 5%]. HPLC
elution times: first enantiomer 9.6 min, second enantiomer
15.6 min; conditions: 25 ꢁ 0.46 cm ID Chiralcel OD column, eluent
n-hexane/2-propanol (95:5) at 0.8 mL/min flow rate.
References
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4.7.2. (2S*,3aS*,7aS*)-Benzyl N-(allyloxycarbonyl)-
octahydroindole-2-carboxylate rac-7b
Rf (20% EtOAc/hexane): 0.29; IR (NaCl):
1703, 1407, 1347, 1303, 1262, 1172, 1124, 989 cmꢀ1
(CDCl3, 300.13 MHz): d 7.43–7.28 (m, 5H, Ph), 5.98–5.68 (m, 1H,
14), 5.32–5.08 (m, 4H, 2H10 + 2H13), 4.67–4.50 (m, 1H, H15),
4.47–4.44 (m, 1H, H15), 4.40–4.30 (m, 1H, H2), 3.95–3.80 (m, 1H,
7a), 2.32–2.23 (m, 1H, H3a), 2.21–2.10 (m, 2H, H3), 2.06–1.96 (m,
m
2929, 2857, 1750,
;
1H NMR
H
H
1H, H7), 1.73–1.58 (m, 3H, H5 + 2H4), 1.50–1.38 (m, 2H, H7 + H6),
1.32–1.14 (m, 2H, H5 + H6); 13C NMR (CDCl3, 75.5 MHz): d (dupli-
cate signals are observed for most carbon atoms) 173.0, 172.8
(C8), 154.4, 153.7 (C11), 135.8, 135.6 (Cipso Ph), 133.0, 132.7 (C14),
128.5, 128.4, 128.2, 128.0, 127.9 (5C, Ph), 117.1, 116.9 (C15), 66.6
(C14), 65.7, 65.6 (C13), 59.1, 59.0 (C2), 57.7, 57.3 (C7a), 37.0, 36.4
(C3a), 32.5, 31.4 (C3), 27.8, 27.2 (C7), 25.7, 25.6 (C4), 23.6, 23.5
(C5), 20.4, 20.3 (C6). MS (APCI+, m/z): 366 [(M+Na+H)+, 70%], 344
[(M+2H)+, 100%]. HPLC elution times: (2S,3aS,7aS)-7b 9.1 min,
(2R,3aR,7aR)-7b 12.1 min; conditions: 25 ꢁ 0.46 cm ID Chiralcel
OD column, eluent n-hexane/2-propanol (90:10) at 0.8 mL/min
flow rate.
4.8. Typical procedure for the enzymatic kinetic resolution of
(2S*, 3aS*,7aS*)-methyl and benzyl octahydroindole-2-
carboxylate rac-6a–b
10. (a) Alfakih, K.; Hall, A. S. Expert Opin. Pharmacother. 2006, 7, 63–71; (b) Curran,
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Investigators Lancet 2005, 366, 895–906.; (d) PROGRESS Collaborative Group
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11. (a) Gass, J.; Khosla, C. Cell. Mol. Life Sci. 2007, 64, 345–355; (b) Brandt, I.;
Scharpé, S.; Lambeir, A.-M. Clin. Chim. Acta 2007, 377, 50–61; (c) García-
Horsman, J. A.; Männistö, P. T.; Venäläinen, J. I. Neuropeptides 2007, 41, 1–24;
(d) Bellemère, G.; Vaudry, H.; Morain, P.; Jégou, S. J. Neuroendocrinol. 2005, 17,
To a suspension of the amino ester rac-6a–b (0.12 mmol) and li-
pase (1:2 in weight with respect to the amino ester) in TBME
(1.8 mL) kept under a nitrogen atmosphere was added the corre-
sponding carbonate 4a–b (0.30 mmol) and the system was shaken
at the required temperature and 250 rpm. The course of the reac-
tion was followed by HPLC (see Table 2). After filtration, the solu-
tion was concentrated under reduced pressure and the crude was
purified by flash chromatography (eluent gradient 10–100%
306–313;
(e)
Schneider,
J.
S.;
Giardiniere,
M.;
Morain,
P.
Neuropsychopharmacology 2002, 26, 176–182; (f) Morain, P.; Lestage, P.; De
Nanteuil, G.; Jochemsen, R.; Robin, J. L.; Guez, D.; Boyer, P. A. CNS Drug Rev.
2002, 8, 31–52; (g) Morain, P.; Robin, J. L.; De Nanteuil, G.; Jochemsen, R.;
Heidet, V.; Guez, D. Br. J. Clin. Pharmacol. 2000, 50, 350–359; (h) Barelli, H.;
Petit, A.; Hirsch, E.; Wilk, S.; De Nanteuil, G.; Morain, P.; Checler, F. Biochem.
Biophys. Res. Commun. 1999, 257, 657–661; (i) Portevin, B.; Benoist, A.;
Rémond, G.; Hervé, Y.; Vincent, M.; Lepagnol, J.; De Nanteuil, G. J. Med. Chem.
1996, 39, 2379–2391.
EtOAc/hexane) to afford 6a–b {(2R,3aR,7aR)-6b: ½a D20
¼ þ23:1 (c
ꢂ
1.0, CHCl3) for >99% ee} and 7a–b {one enantiomer of 7a:
½
a 2D0
a 2D0
ꢂ
¼ þ30:4 (c 0.76, CHCl3) for >99% ee; (2S,3aS,7aS)-7b:
½
ꢂ
¼ ꢀ42:8 (c 1.0, CHCl3) for 98% ee}.

S. Alatorre-Santamaría et al. / Tetrahedron: Asymmetry 19 (2008) 1714–1719
1719
12. (a) Sayago, F. J.; Jiménez, A. I.; Cativiela, C. Tetrahedron: Asymmetry 2007, 18,
2358–2364; (b) Sayago, F. J.; Calaza, M. I.; Jiménez, A. I.; Cativiela, C.
Tetrahedron 2008, 64, 84–91.
18. Carbamates 3 and 7a are given as examples of the numerical locants used for
NMR assignment.
13. Hirata, N. U.S. Patent Appl. Publ. 2005106690 A1. CAN 142:462365.
14. 3-Methoxyphenyl allyl carbonate was prepared following the protocol
described in Ref. 4j.
O
O
4
7
4
7
3
3
3a
7a
3a
7a
10
10
5
6
9
O
9
O
2
2
5
6
8
8
1
N
11
1
N
11
15. See Section 4 for elution conditions.
O
16. HPLC retention times: (2R,3aR,7aR)-8b, 8.1 min; (2S,3aS,7aS)-8b, 13.3 min.
Conditions: 25 ꢁ 0.46 cm ID Chiralpak IA column; eluent n-hexane/2-propanol
(95:5) at 0.8 mL/min flow rate.
17. Three years ago, some companies commercialized CAL-A in different
immobilized supports as, for example, Biocatalytics or Roche. Nowadays, this
enzyme is not available from Roche any more. On the other hand, after
Biocatalytics was purchased by Codexis on July 2007, this immobilized enzyme
is produced in a different form.
O
12 O
12 O
13
13
14
14
15
15