The first direct enzymatic hydrolysis of alicyclic β-amino esters: A route to enantiopure cis and trans β-amino acids

Article abstract of DOI:10.1002/chem.200700257

The first direct enzymatic method is reported for the synthesis of cis and trans βamino acid enantiomers through the lipase-catalyzed enantioselective hydrolysis of alicyclic β esters in organic media. High enantioselectivities (E usually > 001) were observed when the Candida antarctica lipase B catalyzed reactions were performed with H₂O (0.5 equivalents) in iP iPr₂O at 5°C. The resolved products, obtained in good yields (≥42%), could be easily separated.

Full text of DOI:10.1002/chem.200700257

FULL PAPER
DOI: 10.1002/chem.200700257
The First Direct Enzymatic Hydrolysis of Alicyclic b-Amino Esters:
ARoute to Enantiopure cis and trans b-Amino Acids
[
a]
Enikð Forró and Ferenc Fülçp*
Abstract: The first direct enzymatic method is reported for the synthesis of cis and
trans b-amino acid enantiomers through the lipase-catalyzed enantioselective hy-
drolysis of alicyclic b-amino esters in organic media. High enantioselectivities (E
usually >100) were observed when the Candida antarctica lipase B catalyzed reac-
tions were performed with H O (0.5 equivalents) in iPr O at 658C. The resolved
Keywords:
cispentacin
enzymes · hydrolysis
amino
· enantioselectivity
acids
·
·
2
2
products, obtained in good yields (ꢁ42%), could be easily separated.
[
10]
Introduction
the synthesis of cispentacin). The great advantages of this
method are that the lactam ring need not necessarily be acti-
vated and the product cis b-amino acids are obtained in
good yields.
Alicyclic b-amino acids are important compounds from both
pharmacological (for example, cispentacin) and chemical
aspects. They can serve as building blocks for the synthesis
of modified peptides with increased activity and stability
[1]
[
2]
Few articles have dealt with the enzymatic hydrolysis of
amino esters. For example, Katayama et al. described the
enzyme-catalyzed hydrolysis of methyl 1,2,3,4-tetrahydroqui-
noline-1-acetate through enantioselective hydrolysis (E=94)
of the ester function. The substrate contains one stereo-
genic centre and the ring nitrogen is a secondary one. The
reaction was performed in an organic solvent, with Novo-
[3]
and with well-defined three-dimensional structures (for ex-
ample, b-peptides with possible antibiotic activity) similar to
[4]
[12]
those of natural peptides. They can also be used in hetero-
[
5]
[6]
[7]
cyclic, combinatorial chemistry and in drug research.
In the past few years, several enzymatic syntheses have
been developed for enantiopure alicyclic b-amino acid deriv-
atives. As an example, the indirect enzymatic method
through the lipase-catalyzed asymmetric acylation of the pri-
mary hydroxy group of N-hydroxymethylated b-lactams or
the lipase-catalyzed hydrolysis of the corresponding ester
derivatives, followed by ring opening to the b-amino ester
or acid, respectively, are not too efficient and relatively long
zym 435 as the enzyme and 5% H O as the nucleophile. It
2
is also important to mention that Lloyd et al. prepared alicy-
clic N-Boc-protected g-amino acids through lipase-type VII
(Candida rugosa)-catalyzed methyl ester hydrolysis with
[13]
KH PO (pH 7) at 268C.
2
4
In this paper, we report a new direct enzymatic method
for the enantioselective hydrolysis of both cis b-amino esters
(ꢀ)-1–(ꢀ)-4 (Scheme 1) and trans b-amino esters (ꢀ)-5 and
(ꢀ)-6 (Scheme 2), yielding the b-amino acids 7–12 and un-
reacted b-amino esters 13–18.
[8,9]
procedures.
Kanerva et al. described the enzymatic N-
acylation of cis and trans alicyclic b-amino esters, but hy-
drolysis of the N-acylated products to the corresponding b-
[10]
amino acids was not performed.
We have recently discovered a simple and efficient direct
enzymatic route to alicyclic b-amino acid enantiomers
through lipase-catalyzed enantioselective (E >200) ring
cleavage of b-lactams in an organic medium (for example,
Results and Discussion
Our preliminary experiments were started with enzyme
screening, including lipase PS (Pseudomonas cepacia), lipase
AK (Pseudomonas fluorescens), lipase AY (Candida
rugosa), Chirazyme L-2 (a carrier-fixed lipase B from Candi-
da antarctica), Novozym 435 (lipase B from Candida antarc-
tica immobilized on a macroporous polyacrylic resin), Chira-
zyme L-5 (lipase A from Candida antarctica), Lipolase
(lipase B from Candida antarctica, produced by submerged
[
a] Dr. E. Forró, Prof. Dr. F. Fülçp
Institute of Pharmaceutical Chemistry
University of Szeged, 6701 Szeged
PO Box 427 (Hungary)
Fax : (+36)62-545-705
E-mail: fulop@pharm.u-szeged.hu
Chem. Eur. J. 2007, 13, 6397 – 6401
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6397

in iPr O at 658C (Scheme 1). Lipases PS, AK and AY did
2
not exhibit any activity at 458C (no conversion after 48 h),
while PPL even catalyzed the hydrolysis of 2 at 458C, but
the reaction rate was low (Table 1, entry 17). The Chirazyme
L-5-catalyzed hydrolysis of 2 was slower and much less
enantioselective (entry 3). Chirazyme L-2 (entry 1), Novo-
zym 435 (entry 2) and Lipolase (entry 4) were all promising
catalysts, directing the hydrolysis with slight differences in
enantioselectivities. Lipolase was chosen as the enzyme for
further studies.
Several solvents were tested to study the solvent effect in
the Lipolase-catalyzed hydrolysis of (ꢀ)-2 at 658C. The re-
action proceeded somewhat more slowly in n-hexane and in
toluene (Table 1, entries 10 and 11) and much more slowly
in dioxane, (CH ) CO and THF (entries 5–7) than those in
3
2
ether-type solvents (entries 4, 8 and 9), which all afforded
high enantioselectivities (E >100).
Next, we analyzed the effects of temperature on the enan-
tioselectivity and reaction rate. The Lipolase-catalyzed hy-
drolysis of (ꢀ)-2 with 0.5 equivalents of H O in iPr O at
Scheme 1. Enantioselective hydrolysis of alicyclic cis-b-amino esters (ꢀ)-
–(ꢀ)-4.
1
2
2
2
58C proved to be a very slow reaction, although the enan-
tioselectivity was high (Table 1, entry 12). When the reaction
was performed at 45 or at 608C (entries 13 and 14), the reac-
tion rate increased and the enantioselectivity remained high.
A further increase in the temperature (708C) did not affect
the enantioselectivity, and the reaction did not become
faster (entry 15). Indeed, the reaction even slowed down
when the reaction temperature was increased to 808C
(
entry 16).
The catalytic activity of the tested Lipolase was not affect-
ed when the reaction was performed without the addition of
H O (Table 2, entry 3): the H O present in the reaction
2
2
medium (<0.1%) or in the enzyme preparation (<5%) was
sufficient for the hydrolysis.
The reaction rate for the hydrolysis in the case of (ꢀ)-2
increased slightly on increase of the amount of enzyme
(
Table 2, entries 1, 2 and 7–9).
Finally, we analyzed the reusability of the enzyme: the hy-
Scheme 2. Enantioselective hydrolysis of alicyclic trans-b-amino esters
ꢀ)-5 and (ꢀ)-6.
drolysis of (ꢀ)-2 was tested with Lipolase that had already
been used in 1, 2 or 3 cycles (Table 2, entries 4–6). The cata-
lytic activities of the tested Lipolase were progressively
slightly lowered, though the enantiomneric excess of the
product was apparently not affected.
Hydrolysis of the other cis ((ꢀ)-1, (ꢀ)-3, and (ꢀ)-4) and
trans b-amino acid esters (Scheme 2, (ꢀ)-5 and (ꢀ)-6) like-
wise exhibited excellent enantioselectivities under these con-
ditions.
(
fermentation of a genetically modified Aspergillus oryzae
microorganism and adsorbed on a macroporous resin) and
PPL (porcine pancreatic lipase). The reactions were per-
formed on model compound 2 with 0.5 equivalents of H O
2
Abstract in Hungarian: HatØkony enzimes módszert dolgoz-
tunk ki elsðkkØnt cisz- Øs transz b-aminosav enantiomerek
elðµllítµsµra aliciklusos b-aminosav Øszterek lipµz-katalizµlta
enantioszelektív hidrolízisØn keresztül. Nagy enantioszelekti-
vitµs (E µltalµban >100) volt elØrhetð, amikor a reakciókat
Candida antarctica B lipµz-katalízissel, víz (0.5 ekv.) hoz-
zµadµsa mellett, diizopropil-Øterben, 658C-on vØgeztük. A jó
termelØssel kapott (ꢁ42%) enantiomerek elvµlasztµsa egy-
szerßen, szerves-vizes extrakcióval tçrtØnt.
On the basis of the preliminary results, the gram-scale res-
olutions of (ꢀ)-1–(ꢀ)-6 were performed with 0.5 equiva-
lents of H O in the presence of Lipolase in iPr O at 658C.
The products were characterized by a relatively good enan-
tiomeric excess next to 50% conversion. The results are re-
ported in Table 3 and the Experimental Section.
It should be mentioned that an attempt was made to im-
prove the enzyme/substrate mass ratio (from 1.8:1 to 1:1) in
the case of (ꢀ)-2 (Experimental Section), and similarly
good results (E >200) were obtained; however, a longer re-
2
2
6398
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 6397 – 6401

A Route to cis and trans b-Amino Acids
FULL PAPER
[
a]
Table 1. Conversion and enantioselectivity of hydrolysis of (ꢀ)-2.
chloride salts 7·HCl–12·HCl.
The absolute configurations
were proved by comparing the
[a] values with the literature
data (Experimental Section),
except in the case of 12, for
which the unsaturated b-amino
acid enantiomer was reduced
catalytically to (1R,2R)-11 by
transfer hydrogenation in the
presence of cyclohexene as a
hydrogen donor. Thus, the abso-
lute configurations indicated
Lipolase-catalyzed S-selective
hydrolysis for the cis com-
pounds, while for the trans com-
pounds the opposite R selectivi-
ty was observed.
ꢂ1
[b]
[c]
Entry Enzyme (30 mgmL
)
T [8C] Solvent
Reaction t [h] Conv. [%] ee
s
[%] ee
p
[%]
E
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
Chirazyme L-2
Novozym 435
Chirazyme L-5
Lipolase
Lipolase
Lipolase
Lipolase
Lipolase
Lipolase
Lipolase
Lipolase
Lipolase
Lipolase
Lipolase
Lipolase
Lipolase
PPL
65
65
65
65
65
65
65
65
65
65
65
25
45
60
70
80
45
iPr
iPr
iPr
iPr
2
2
2
2
O
O
O
O
26
26
48
25
28
48
47
15
48
19
6
12
45
49
29
38
11 (47)
21
36
36
29
6
89
96
97
58
98
99
99
99
98
96
99
98
147
187
4
87
10
90
23
6
14
80
92
40
61
[
d]
307
249
211
228
245
161
295
185
223 (187)
256
346
172
146
211
dioxane
A
H
R
U
G
3
)
2
CO 28
28
2
O
28
28
28
tBuOMe
toluene
n-hexane 28
0
1
2
3
4
5
6
7
iPr
2
iPr
2
iPr
2
iPr
2
iPr
2
iPr
2
O
O
O
O
O
O
20 (5 d)
20
19
19
19
12 (87)
99 (97)
99
26
55
55
40
6
99
98
98
99
48
[
a] 0.05m substrate, 0.5 equivH
2
O. [b] According to GC. [c] According to GC after double derivatisation.
d] Contains 20% (w/w) of lipase adsorbed on Celite in the presence of sucrose.
[
Conclusion
[
a]
Table 2. Conversion and enantioselectivity for the hydrolysis of (ꢀ)-2.
[
b]
[c]
Entry Lipolase
H
[equiv]
2
O
Reaction Conv. ee
s
ee
[%]
p
E
ꢂ1
The first, simple and efficient direct enzymatic method that
is equally applicable for the synthesis of cis and trans b-
amino acids has been developed. The Lipolase (lipase B
from Candida antarctica) catalyzed highly enantioselective
hydrolysis of both cis (1–4) and trans b-amino esters (5 and
[mgmL
]
t [h]
[%]
[%]
1
2
3
4
5
6
7
8
9
10
20
30
30
30
30
40
50
75
0.5
0.5
–
–
–
25
25
29
24
24
24
25
22
22
42
45
43
43
38
32
48
50
51
70
79
75
74
59
46
91
95
99
98
98
98
98
97
97
97
96
94
208
239
224
244
120
103
209
183
174
[
[
[
d]
e]
f]
6
) (E usually >100), when H O (0.5 equivalents) is used in
2
iPr O at 658C, results in cis (1S,2R)-7–10 and trans b-amino
–
2
0.5
0.5
0.5
acids (1R,2R)-11 and 12, unreacted cis b-amino esters
(1R,2S)-13–16 and trans b-amino esters (1S,2S)-17 and 18 in
good yields (42–48%). The products could be easily separat-
ed. Transformations of esters 13–18 with 18% aqueous HCl
resulted in the enantiomers of b-amino acid hydrochlorides
[
2
a] 0.05m substrate, iPr O, 65 8C. [b] According to GC. [c] According to
GC after double derivatisation. [d] Already used once. [e] Already used
twice. [f] Already used three times.
1
9–24 (ꢁ94% ee, ee=enantiomeric excess). The present
method was successfully used for the synthesis of cispenta-
cin.
action time was necessary (the conversion was 42% after
45 h).
The transformations involving the hydrolysis of b-amino
esters 13–18 with 18% aqueous HCl resulted in the enantio-
mers of the b-amino acid hydrochlorides 19–24 (Schemes 1
and 2, Table 3). Compound 19 is the well-known natural
Experimental Section
Materials and methods: Lipolase (lipase B from Candida antarctica) pro-
duced by submerged fermentation of a genetically modified Aspergillus
oryzae microorganism and adsorbed on a macroporous resin was ob-
tained from Sigma-Aldrich. Lipase PS and lipase AK were obtained from
[1a]
cyclic b-amino acid cispentacin. Treatment of amino acids
7
–12 with 22% HCl/EtOH resulted in enantiopure hydro-
[
a]
Table 3. Lipolase-catalyzed preparative-scale hydrolysis of (ꢀ)-1–(ꢀ)-6
.
t [h]
Conv. [%]
E
b-Amino acid·HCl (19–24)
b-Amino acid (7–12)
[c]
[
b]
25
25
Yield [%]
Isomer
ee [%]
[a]D (H
2
O)
Yield [%]
Isomer
ee [%]
[a]D (H
2
O)
[
c]
[d]
(
(
(
(
(
(
ꢀ)-1
ꢀ)-2
ꢀ)-3
ꢀ)-4
ꢀ)-5
ꢀ)-6
87
31
72
66
68
58
48
49
50
49
49
50
74
>200
110
133
>200
183
42
46
45
45
46
44
1R,2S
1R,2S
1R,2S
1R,2S
1S,2S
1S,2S
94
99
98
98
99
99
ꢂ5
42
47
46
46
48
45
1S,2R
1S,2R
1S,2R
1S,2R
1R,2R
1R,2R
96
98
98
99
99
99
+8
[
e]
[f]
ꢂ8.4
+21
[
[
[
[
g]
[h]
ꢂ28
+121
+51
+34
d]
[g]
ꢂ120
d]
i]
[h]
ꢂ65
[f]
+123
ꢂ152
ꢂ1
[
[
a] 30 mgmL enzyme in iPr
g] c=0.11. [h] c=0.26. [i] c=0.27.
2
2
O, 0.5 equivH O, 65 8C. [b] According to GC after double derivatisation. [c] c=0.21. [d] c=0.23. [e] c=0.5. [f] c=0.28.
Chem. Eur. J. 2007, 13, 6397 – 6401
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6399

F. Fülçp and E. Forró
Amano Pharmaceuticals, PPL (type II) was obtained from Sigma and
Chirazyme L-5 (lipase A from Candida antarctica) and Novozym 435 as
an immobilized lipase (lipase B from Candida antarctica) on a macropo-
rous acrylic resin from Novo Nordisk. Chirazyme L-2 (a carrier-fixed
lipase B from Candida antarctica) was purchased from Roche Diagnostics
Corporation. Before use, lipase PS, lipase AK and CAL-A (5 g) were dis-
solved in Tris-HCl buffer (0.02m; pH 7.8) in the presence of sucrose
and (1S,2R)-8 in 31 h. Compound (1R,2S)-20: Yield: 482 mg, 46%;
2
5
[11a]
25
[a] =ꢂ8.4 (c=0.5 in H
2
O) (lit.
[a]D =ꢂ8.4 (c=0.4 in H
2
O); m.p.
D
2
229–2318C (recrystallised from EtOH and Et O)); 99% ee. Compound
2
D
5
(1S,2R)-8: Yield: 393 mg, 47%; [a] =+22 (c=0.28 in H
2
O); m.p. 264–
2668C (recrystallised from H O and (CH ) CO); 98% ee.
2
3 2
The hydrolysis of (ꢀ)-2 with
a reduced amount of Lipolase (1 g,
ꢂ1
1
6.6 mgmL
)
under the same conditions afforded (1R,2S)-20 and
(
3 g), followed by adsorption on Celite (17 g) (Sigma). The lipase prepa-
25
(
1S,2R)-8 in 66 h. Compound (1R,2S)-20: Yield: 492 mg, 47%; [a]D
ꢂ8.1 (c=0.35 in H O); m.p. 229–2338C; 99% ee. Compound (1S,2R)-8:
Yield: 390 mg, 47%; [a] =+21.1 (c=0.3 in H
=
rations thus obtained contained 20% (w/w) of lipase. The alicyclic cis
2
[
1a,14]
and trans b-amino esters were prepared according to the literature.
25
O); m.p. 260–2618C;
D
2
The solvents were of the highest analytical grade.
9
9% ee.
In a typical small-scale experiment, racemic b-amino ester (0.05m solu-
When 8 (100 mg) was treated with 22% HCl/EtOH (5 mL), (1S,2R)-
tion) in an organic solvent (2 mL) was added to the lipase tested (10, 20,
25
8
·HCl was obtained. Yield: 110 mg, 88%; [a] =+8.3 (c=0.33 in H
2
O)
D
ꢂ1
3
0, 40, 50 or 75 mgmL ). H
2
O (0 or 0.5 equiv) was added. The mixture
[11a]
25
(
lit.
[a]D =+8.8 (c=0.3 in H O); m.p. 230–2338C (recrystallised from
2
was shaken at 258C, 458C, 60 8C, 65 8C, 70 8C or 808C. The progress of
the reaction was followed by taking samples from the reaction mixture at
intervals and analyzing them by gas chromatography. The ee values for
the unreacted b-amino ester and the b-amino acid enantiomers produced
were determined by gas chromatography on a Chromopak Chiralsil-Dex
CB column (25 m) or Chrompack L-Val (25 m) column after double deri-
vatisation with 1) diazomethane (Caution! The derivatisation with di-
chloromethane should be carried out under a well-working fume hood)
and 2) acetic anhydride in the presence of 4-dimethylaminopyridine and
pyridine (Chirasil-l-Val column, 1208C for 5 min!1908C, rate of tem-
2
EtOH and Et O)); 99% ee.
1
Data for 8: H NMR (400 MHz, D
2
O, 25 8C, TMS): d=1.46–2.02 (m, 8H;
4
2
”CH ), 2.63–2.67 (m, 1H; H-1), 3.45–3.49 ppm (m, 1H; H-2); elemental
7 2
analysis calcd (%) for C H13NO (143.18): C 58.72, H 9.15, N 9.78; found:
C 58.66, H 9.21, N 9.72.
1
The H NMR (400 MHz, D
2
O, 25 8C, TMS) data are similar for 8·HCl
), 2.98–3.02 (m, 1H; H-1), 3.55–
.59 ppm (m, 1H; H-2); elemental analysis calcd (%) for C ·HCl
179.64): C 46.80, H 7.86, N 7.80; found for 8·HCl: C 46.57, H 7.69, N
and 20: d=1.43–2.02 (m, 8H; 4”CH
3
(
2
7 2
H13NO
ꢂ1
7.75; found for 20: C 46.59, H 7.88, N 7.91.
perature rise: 208C min , 140 kPa, retention times [min]: 13: 9.20 (anti-
pode 9.40), 7: 8.64 (antipode 8.62), 14: 10.28 (antipode 10.42), 8: 9.74 (an-
tipode 9.65), 9: 9.29 (antipode 9.21), 10: 8.95 (antipode 8.79); CP-Chira-
Preparative-scale resolution of ethyl cis-(2-aminocyclohex-4-ene)-1-car-
boxylate (ꢀ)-3: Following the procedure described above, the reaction of
sil-Dex CB column, 1208C for 2 min!1908C, rate of temperature rise
racemic 3 (1 g, 5.90 mmol) and H
2
O (53.2 mL, 2.95 mmol) in iPr
2
O
ꢂ1
ꢂ1
2
1
8
08C min , 140 kPa, retention times [min]: 15: 10.68 (antipode 10.77),
6: 9.89 (antipode 10.51), 17: 10.81 (antipode 10.93), 11: 8.86 (antipode
.78)). The ee values for 12 and 18 were determined as in the case of 11
(60 mL) in the presence of Lipolase (1.8 g, 30 mgmL ) at 658C afforded
(1R,2S)-21 and (1S,2R)-9 in 72 h. Compound (1R,2S)-21: Yield: 472 mg,
2
D
5
[11c]
25
D
45%; [a]
=ꢂ28 (c=0.11 in H
2
O), lit.
[a]
=ꢂ26 (c=0.25 in H
O); 98% ee. Com-
=+34 (c=0.26 in H O); m.p.
O and (CH CO); 98% ee.
When 9 (100 mg) was treated with 22% HCl/EtOH (5 mL), (1S,2R)-
2
O);
and 17, after catalytic reduction to the corresponding saturated com-
m.p. 205–2108C (recrystallised from EtOH and Et
2
[
8c]
25
D
pounds.
pound (1S,2R)-9: Yield: 383 mg, 46%; [a]
34–2398C (recrystallised from H
2
2
2
3 2
)
Optical rotations were measured with a Perkin–Elmer 341 polarimeter.
1
H NMR spectra were recorded on a Bruker Avance DRX 400 spectrom-
2
D
5
eter. Melting points were determined on a Kofler apparatus.
9·HCl was obtained. Yield: 110 mg, 88%; [a] +28 (c=0.24 in H
2
O);
[
11c]
25
lit.
[a]D =+25.8 (c=0.4 in H O); m.p. 205–2098C (recrystallised from
2
Preparative-scale resolution of ethyl cis-2-aminocyclopentane-1-carboxyl-
ꢂ1
EtOH and Et O), 99% ee.
ate (ꢀ)-1: Lipolase (450 mg, 30 mgmL ) and H
2
O (9.0 mL, 0.50 mmol)
O (15 mL), and
2
1
were added to the racemic 1 (157 mg, 1.00 mmol) in iPr
2
Data for 9: H NMR (400 MHz, D
2
O, 25 8C, TMS): d=2.25–2.51 (m, 4H;
the mixture was shaken in an incubator shaker at 658C for 87 h. The re-
action was stopped by filtering off the enzyme at 48% conversion (13:
2
2”CH ), 2.75–2.77 (m, 1H; H-1), 3.78–3.79 (m, 1H; H-2), 5.64–5.84 ppm
(m, 2H; CHCH); elemental analysis calcd (%) for C H11NO (141.17): C
7 2
9
3% ee). The solvent was evaporated off and the residue (1R,2S)-13 was
59.56, H 7.85, N 9.92; found: C 59.76, H 7.68, N 9.89.
immediately hydrolyzed by refluxing (4 h) with 18% aqueous HCl solu-
tion (7 mL) into b-amino acid hydrochloride (1R,2S)-19. Yield: 69.4 mg,
1
The H NMR (400 MHz, D
2
O, 25 8C, TMS) spectroscopic data are similar
), 3.07–3.12 (m, 1H; H-1),
3.90–3.91 (m, 1H; H-2), 5.66–5.82 ppm (m, 2H; CHCH); elemental anal-
ysis calcd (%) for C ·HCl (177.63): C 47.33, H 6.81, N 7.89; found
for 9·HCl and 21: d=2.32–2.55 (m, 4H, 2”CH
2
2
5
[11a]
25
4
2%; [a] =ꢂ5 (c=0.21 in H
2
O), lit.
[a]D =ꢂ5.1 (c=0.5 in H
2
O);
D
2
m.p. 164–1668C (recrystallised from EtOH and Et O); 94% ee.
7
H11NO
2
The filtered enzyme was washed with distilled H
2
O (3”15 mL), and the
for 8·HCl: C 47.33, H 6.81, N 7.89; found for 21: C 47.45, H 6.77, N 7.99.
2
H O was evaporated off, yielding the crystalline b-amino acid (1S,2R)-7.
Preparative-scale resolution of ethyl cis-(2-aminocyclohex-3-ene)-1-car-
boxylate (ꢀ)-4: Following the procedure described above, the reaction of
2
5
Yield: 54.1 mg, 42%; [a] =+8 (c=0.23 in H
2
O); m.p. 218–2198C with
D
sublimation (recrystallised from H
2 3 2
O and (CH ) CO); 96% ee.
racemic 4 (1 g, 5.90 mmol) and H
2
O (53.2 mL, 2.95 mmol) in iPr
2
O
ꢂ1
When 7 (30 mg) was treated with 22% HCl/EtOH (3 mL), (1S,2R)-7·HCl
(60 mL) in the presence of Lipolase (1.8 g, 30 mgmL ) at 658C afforded
(1R,2S)-22 and (1S,2R)-10 in 66 h. Compound (1R,2S)-22: Yield: 472 mg,
2
D
5
[11a]
was obtained. Yield: 28 mg, 73%; [a] =+5 (c=0.22 in H
2
O), lit.
2
5
25
[11c]
25
[
a] =+5.2 (c=0.5 in H
2
O); m.p. 162–1668C (recrystallised from EtOH
45%; [a]D =+121 (c=0.21 in H
2
O) (lit.
[a]D =+121.7 (c=0.4 in
O); 98% ee.
D
and Et
2
O); 96% ee.
H
2
O)); m.p. 204–2108C (recrystallised from EtOH and Et
2
2
D
5
1
Compound (1S,2R)-10: Yield: 383 mg, 46%; [a] =ꢂ120 (c=0.25 in
Data for 7: H NMR (400 MHz, D
2
O, 25 8C, TMS): d=1.70–2.12 (m, 6H;
2 2 3 2
H O); m.p. 234–2368C (recrystallised from H O and (CH ) CO); 99% ee.
3
”CH
2
), 2.84–2.89 (m, 1H; H-1), 3.72–3.73 ppm (m, 1H; H-2); elemental
(129.08): C 55.80, H 8.58, N 10.84;
analysis calcd (%) for C
6
H
11NO
2
When 10 (100 mg) was treated with 22% HCl/EtOH (5 mL), (1S,2R)-
2
D
5
found: C 55.69, H 8.50, N 10.96.
10·HCl was obtained. Yield: 98 mg, 78%; [a] =ꢂ120 (c=0.24 in H
2
O)
[
11c]
25
1
(lit.
[a]D =ꢂ121.4 (c=0.4 in H O)); m.p. 205–2118C (recrystallised
The H NMR (400 MHz, D
2
O, 25 8C, TMS) spectroscopic data are similar
), 3.10–3.16 (m, 1H; H-
), 3.84–3.85 ppm (m, 1H; H-2); elemental analysis calcd (%) for
·HCl (165.62): C 43.51, H 7.30, N 8.46; found for 7·HCl: C
3.41, H 6.97, N 8.46; found for 19: C 43.28, H 7.11, N 8.61.
2
2
from EtOH and Et O); 99% ee.
for 7·HCl and 19: d=1.75–2.17 (m, 6H; 3”CH
2
1
1
Data for 10: H NMR (400 MHz, D
2
O, 25 8C, TMS): d=1.85–2.18 (m,
C
6
H
11NO
2
4H; 2”CH
6.15 ppm (m, 2H; CHCH); elemental analysis calcd (%) for C
141.17): C 59.56, H 7.85, N 9.92; found: C 59.65, H 7.70, N 10.07.
2
), 2.74–2.78 (m, 1H; H-1), 3.97–3.99 (m, 1H; H-2), 5.74–
4
7 2
H11NO
(
Preparative-scale resolution of ethyl cis-2-aminocyclohexane-1-carboxyl-
ate (ꢀ)-2: Following the procedure described above, the reaction of race-
1
The H NMR (400 MHz, D
and 22: d=1.92–2.21 (m, 4H; 2”CH
(m, 1H; H-2), 5.73–6.16 ppm (m, 2H; CHCH); elemental analysis calcd
2
O, 25 8C, TMS) data are similar for 10·HCl
mic 2 (1 g, 5.83 mmol) and H
the presence of Lipolase (1.8 g, 30 mgmL ) at 658C afforded (1R,2S)-20
2
O (52.5 mL, 2.91 mmol) in iPr
2
O (60 mL) in
2
), 3.04–3.08 (m, 1H; H-1), 4.08–4.09
ꢂ1
6400
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 6397 – 6401

A Route to cis and trans b-Amino Acids
FULL PAPER
(
8
%) for C
7
H
11NO
2
·HCl (177.63): C 47.33, H 6.81, N 7.89; found for
ier Science Publishers, 2000, pp. 273–306; c) K. Park, M. J. Kurth,
Tetrahedron 2002, 58, 8629–8659; d) J. Mittendorf, J. Benet-Buch-
holz, P. Fey, K. H. Mohrs, Synthesis 2003, 136–140; e) A. Kuhl,
M. G. Hahn, M. Dumic, J. Mittendorf, Amino Acids 2005, 29, 89–
·HCl: C 47.22, H 6.97, N 7.81; found for 21: C 47.33, H 6.82, N 7.69.
Preparative-scale resolution of ethyl trans-2-aminocyclohexane-1-carbox-
ylate ((ꢀ)-5): Following the procedure described above, the reaction of
1
00; f) F. Fülçp, T. A. Martinek, G. K. Tóth, Chem. Soc. Rev. 2006,
racemic 5 (1 g, 5.83 mmol) and H
2
O (52.5 mL, 2.91 mmol) in iPr
2
O
ꢂ1
35, 323–334.
(
(
4
60 mL) in the presence of Lipolase (1.8 g, 30 mgmL ) at 658C afforded
[
2] a) Z. Szakonyi, S. Gyónfalvi, E. Forró, A. HetØnyi, F. Fülçp, Eur. J.
Org. Chem. 2005, 4017–4023; b) F. Fülçp, M. Palkó, E. Forró, M.
Dervarics, T. Martinek, Eur. J. Org. Chem. 2005, 3214–3220.
1S,2S)-23 and (1R,2R)-11 in 68 h. Compound (1S,2S)-23: Yield: 482 mg,
2
5
2
6%; [a] =+51 (c=0.21 in H O); m.p. 198–2018C (recrystallised from
D
EtOH and Et
2
O); 99% ee. Compound (1R,2R)-11: Yield: 404 mg, 48%;
2
5
[15]
25
[3] a) K. M. Brashear, C. A. Hunt, B. T. Kucer, M. E. Duggan, G. D.
Hartman, G. A. Rodan, S. B. Rodan, C. Leu, T. Prueksaritanont, C.
Fernandez-Metzler, A. Barrish, C. F. Homnick, J. H. Hutchinson,
P. J. Coleman, Bioorg. Med. Chem. Lett. 2002, 12, 3483–3486;
b) D. L. Steer, R. A. Lew, P. Perlmutter, A. I. Smith, M. I. Aguilar,
Current Med. Chem. 2002, 9, 811–822; c) C. Palomo, I. Ganboa, M.
Oiarbide, G. T. Sciano, J. L. Miranda, ARKIVOC 2002, Part V, 8–16;
d) F. Fülçp, E. Forró, G. K. Tóth, Org. Lett. 2004, 6, 4239–4241.
4] a) D. H. Appela, L. A. Christianson, D. A. Klein, D. R. Powell, X. L.
Huang, I. J. Barchi, S. H. Gellman, Nature 1997, 387, 381–384;
b) E. A. Porter, X. F. Wang, H. S. Lee, B. Weisblum, S. H. Gellman,
Nature 2000, 404, 565–565; c) T. Martinek, G. K. Tóth, E. Vass, M.
Hollósi, F. Fülçp, Angew. Chem. 2002, 114, 1794–1797; Angew.
Chem. Int. Ed. 2002, 41, 1718–1721; d) T. A. Martinek, I. M.
Mµndity, L. Fülçp, G. K. Tóth, E. Vass, M. Hollósi, E. Forró, F.
Fülçp, J. Am. Chem. Soc. 2006, 128, 13539–13544.
[5] a) S. G. Davies, G. Bhalay, Tetrahedron: Asymmetry 1996, 7, 1595–
1596; b) Z. Szakonyi, T. Martinek, A. HetØnyi, F. Fülçp, Tetrahe-
dron: Asymmetry 2000, 11, 4571–4579.
[6] a) S. Gedey, J. van der Eycken, F. Fülçp, Org. Lett. 2002, 4, 1967–
1969; b) I. Kanizsai, Z. Szakonyi, R. Sillanpää, F. Fülçp, Tetrahedron
Lett. 2006, 47, 9113–9116; I. Kanizsai, S. Gyónfalvi, Z. Szakonyi, R.
Sillanpää, F. Fülçp, Green Chem. 2007, 9, 357–360.
[
2
a] =ꢂ65 (c=0.26 in H
2
O), lit.
[a]D =ꢂ57.8 (c=0.5 in H
2
O); m.p.
D
2 3 2
63–2658C (recrystallised from H O and (CH ) CO); 99% ee.
When 11 (100 mg) was treated with 22% HCl/EtOH (5 mL), (1R,2R)-
1
m.p. 194–1978C (recrystallised from EtOH and Et O); 99% ee.
2
2
D
5
1·HCl was obtained. Yield: 111 mg, 89%; [a] =ꢂ51 (c=0.21 in H
2
O);
1
Data for 11: H NMR (400 MHz, D
2
O, 25 8C, TMS): d=1.29–2.11 (m,
8
H; 4”CH
mental analysis calcd (%) for C
.78; found: C 58.84, H 9.16, N 9.60.
2
), 2.14–2.23 (m, 1H; H-1), 3.20–3.27 ppm (m, 1H; H-2); ele-
7
H13NO
2
(143.18): C 58.72, H 9.15, N
[
9
1
The H NMR (400 MHz, D
for 11·HCl and 23: d=1.27–2.18 (m, 8H, 4”CH
), 3.35–3.42 ppm (m, 1H; H-2); elemental analysis calcd (%) for
·HCl (179.64): C 46.80, H 7.86, N 7.80; found for 11·HCl: C
6.57, H 7.69, N 7.75; found for 23: C 46.77, H 7.91, N 7.82.
2
O, 25 8C, TMS) spectroscopic data are similar
2
), 2.49–2.54 (m, 1H; H-
1
7 2
C H13NO
4
Preparative-scale resolution of ethyl trans-(2-aminocyclohex-4-ene)-1-
carboxylate (ꢀ)-6: Following the procedure described above, the reaction
of racemic 6 (1 g, 5.90 mmol) and H
2
O (53.2 mL, 2.95 mmol) in iPr
2
O
ꢂ1
(
(
4
60 mL) in the presence of Lipolase (1.8 g, 30 mgmL ) at 658C afforded
1S,2S)-24 and (1R,2R)-12 in 58 h. Compound (1S,2S)-24: Yield: 461 mg,
2
5
4%; [a] =+123 (c=0.27 in H
2
O); m.p. 182–1848C (recrystallised from
D
EtOH and Et
2
O); 99% ee. Compound (1R,2R)-12: Yield: 375 mg, 45%;
2
5
[
a] =ꢂ152 (c=0.29 in H
2 2
O); m.p. 268–2708C (recrystallised from H O
D
and (CH
3
)
2
CO); 99% ee.
[7] Enantioselective Synthesis of b-Amino Acids (Eds.: E. Juaristi, V. A.
Soloshonok), Wiley-Interscience, New York, 2005.
[8] a) P. Csomós, L. T. Kanerva, G. Bernµth, F. Fülçp, Tetrahedron:
Asymmetry 1996, 7, 1789–1796; b) J. Kµmµn, E. Forró, F. Fülçp, Tet-
rahedron: Asymmetry 2000, 11, 1593–1600; c) E. Forró, J. rva, F.
Fülçp, Tetrahedron: Asymmetry 2001, 12, 643–649.
When 12 (100 mg) was treated with 22% HCl/EtOH (5 mL), (1R,2R)-
1
m.p. 182–1858C (recrystallised from EtOH and Et O); 99% ee.
2
2
D
5
2·HCl was obtained. Yield: 105 mg, 84%; [a] =ꢂ121 (c=0.25 in H
2
O);
1
Data for 12: H NMR (400 MHz, D
5
CHCH); elemental analysis calcd (%) for C
7
2
O, 25 8C, TMS): d=1.17–2.61 (m,
H; 2”CH
2
and H-1), 3.52–3.58 (m, 1H; H-2), 5.64–6.81 ppm (m, 2H;
(141.17): C 59.56, H
[
9] Reviews: a) E. Forró, F. Fülçp, Mini-Rev. Org. Chem. 2004, 1, 93–
7
H11NO
2
1
5
02; b) A. Liljeblad, L. T. Kanerva, Tetrahedron 2006, 62, 5831–
854.
.85, N 9.92; found: C 59.70, H 7.82, N 9.91.
1
The H NMR (400 MHz, D
2
O, 25 8C, TMS) spectroscopic data are similar
), 2.85–2.89 (m, 1H; H-
), 3.70–3.72 (m, 1H; H-2), 5.67–5.77 ppm (m, 2H; CHCH); elemental
analysis calcd (%) for C ·HCl (177.63): C 47.33, H 6.81, N 7.89;
[
[
10] L. T. Kanerva, P. Csomós, O. Sundholm, G. Bernµth, F. Fülçp, Tetra-
hedron: Asymmetry 1996, 7, 1705–1716.
11] a) E. Forró, F. Fülçp, F. Org. Lett, 2003, 5, 1209–1212; b) E. Forró,
F. Fülçp, Tetrahedron: Asymmetry 2004, 15, 573–575; c) E. Forró, F.
Fülçp, Tetrahedron: Asymmetry 2004, 15, 2875–2880; d) E. Forró, F.
Fülçp, Chem. Eur. J. 2006, 12, 2587–2592.
for 12·HCl and 24: d=2.22–2.57 (m, 4H, 2”CH
2
1
7
H11NO
2
found for 8·HCl: C 47.28, H 6.97, N 7.77; found for 21: C 47.15, H 6.79,
N 7.66.
[
12] S. Katayama, N. Ae, R. Nagata, Tetrahedron: Asymmetry 1998, 9,
4
295–4299.
[
13] R. C. Lloyd, M. C. Lloyd, M. E. B. Smith, K. E. Holt, J. P. Swift,
P. A. Keene, S. J. C. Taylor, R. McCague, Tetrahedron 2004, 60, 717–
Acknowledgements
7
28.
14] G. Bernµth, G. Stµjer, A. E. Szabó, F. Fülçp, P. Sohµr, Tetrahedron,
985, 41, 1353–1365.
[
[
The authors acknowledge receipt of OTKA grants T 046440 and T
49407, GVOP-3.1.1.-2004-05-0255/3.0 and a Bolyai Fellowship for E.F.
1
0
15] J. Priego, P. Flores, C. Ortiz-Nava, J. Escalante, Tetrahedron: Asym-
metry 2004, 15, 3545–3549.
[
1] a) F. Fülçp, Chem. Rev. 2001, 101, 2181–2204; b) F. Fülçp in Studies
in Natural Product Chemistry Vol. 22 (Ed.: Atta-ur-Rahman), Elsev-
Received: February 13, 2007
Published online: May 11, 2007
Chem. Eur. J. 2007, 13, 6397 – 6401
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6401