Preparation of highly enantiopure β-amino esters by Candida antarctica lipase A

Article abstract of DOI:10.1016/S0957-4166(01)00002-7

The enantioselectivities for the reactions of aliphatic β-substituted β-amino esters [RCH(NH₂)CH₂CO₂Et with R = Me, Et, n-Pr, i-Pr, CHEt₂, cyclohexyl and Ph] with butyl butanoate in neat butyl butanoate and with 2,2,2-trifluoroethyl butanoate in diisopropyl ether were studied in the presence of Candida antarctica lipase A. Enantioselectivities ranging from good (E = 70-100) to excellent (E>100) were commonly observed, allowing gram-scale resolution of the substrates.

Full text of DOI:10.1016/S0957-4166(01)00002-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 105–110
Preparation of highly enantiopure b-amino esters by Candida
antarctica lipase A
Szilvia Gedey,a,b Arto Liljeblad,a La´szlo´ La´za´r,b Ferenc Fu¨lo¨pb and Liisa T. Kanervaa,*
aDepartment of Chemistry and Laboratory of Synthetic Drug Chemistry, University of Turku, Lemminka¨isenkatu 2,
FIN-20520 Turku, Finland
bInstitute of Pharmaceutical Chemistry, University of Szeged, PO Box 121, H-6701 Szeged, Hungary
Received 27 November 2000; accepted 21 December 2000
Abstract—The enantioselectivities for the reactions of aliphatic b-substituted b-amino esters [RCH(NH2)CH2CO2Et with R=Me,
Et, n-Pr, i-Pr, CHEt2, cyclohexyl and Ph] with butyl butanoate in neat butyl butanoate and with 2,2,2-trifluoroethyl butanoate
in diisopropyl ether were studied in the presence of Candida antarctica lipase A. Enantioselectivities ranging from good
(E=70–100) to excellent (E>100) were commonly observed, allowing gram-scale resolution of the substrates. © 2001 Elsevier
Science Ltd. All rights reserved.
1. Introduction
the use of highly enantioselective amide formation
between the amino group of racemic cis- and trans-
amino esters and 2,2,2-trifluoroethyl carboxylates in
diisopropyl ether in the presence of Pseudomonas cepa-
cia lipase PS or Candida antarctica lipase A (CAL-
A).11,16 The expectation that this method would
generally allow resolution of aliphatic b-amino esters
proved overly optimistic, as the lipases exhibited either
low chemoselectivity (lipase PS), leading to multiple
products, or low enantioselectivity (CAL-A) when
applied for the reaction of acyclic ethyl 3-aminobu-
tanoate 1a.13 Low chemoselectivity was observed when
the alkyl group of an achiral carboxylic ester differed
Optically active b-amino acids are gaining ever greater
significance in synthetic chemistry in connection with
the design and synthesis of pharmaceutical drugs and
natural products, and with the unique secondary struc-
tures and activation which they offer for peptides.1–8 In
recent years, we have focused on the preparation of
enantiopure b-amino acids and their derivatives by
exploiting the potential of lipases as chiral catalysts.9–13
Special attention has been paid to the kinetic resolution
of alicyclic and acyclic b-amino esters. In work with
such bifunctional substrates, enzymatic chemo- and
regioselectivity are of utmost importance in directing
the reaction to the desired functional group. Another
point is that enzymes, like other catalysts, simply cata-
lyze the approach to equilibrium for a given reaction.
The existence of an equilibrium can easily destroy the
possibility of obtaining enantiopure products. For this
reason, enzymatic resolution based on an acylation
reaction (e.g. at the amino group) with an appropriate
achiral acyl donor (activated esters, acid anhydrides,
etc.) is often more applicable than a deacylation reac-
tion at an ester group.14,15
By means of an enzymatic acylation strategy, ten ali-
cyclic b-amino acids were previously resolved through
Scheme 1. a: R=Me; b: R=Et; c: R=n-Pr; d: R=i-Pr; e:
R=CHEt2; f: R=cyclohexyl; g: R=Ph, R%=CH2CF3 or Bu.
* Corresponding author. E-mail: lkanerva@utu.fi
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00002-7

106
S. Gedey et al. / Tetrahedron: Asymmetry 12 (2001) 105–110
Table 1. Enantioselectivities of CAL-A preparation (40 mg/mL) for the reactions of 1a–g (0.1 M) in butyl butanoate and
with 2,2,2-trifluoroethyl butanoate (0.2 M) in diisopropyl ether at room temperature
R
PrCO2CH2CF3
Time (h)
PrCO2Bu
Time (h)
Conv. (%)
E
Conv. (%)
E
1a
1b
1c
1d
1e
1f
Me
Et
n-Pr
i-Pr
CHEt2
Cyclohexyl
Ph
50
52
55
49
52
53a
52
25
692
168911
72
10698
3893
\100
7594
51
50
50
50
33
23
52
22
24
9
130
16 days
48
3291
256936
\100
11599
2
1.33
1.5
1.5
4
1
0.33
997
2993
1g
70
a Amount of enzyme preparation 30 mg/mL.
from that of the substrate. Enzyme screening revealed
other lipases besides lipase PS, for example C. antarc-
tica lipase B (CAL-B),16 which displayed low chemose-
lectivity for the reactions of 1a. Under optimized
reaction conditions, involving either acylation of the
NH2 group followed by transesterification at the CO2Et
group or transesterification followed by acylation, the
successful sequential resolution of 1a with CAL-B was
achieved.13
the corresponding optically-active amide product (one
of 2a–g). On this basis, solvent effects on enantioselec-
tivity can be postulated as the main cause for the higher
enantioselectivity observed with straight-chain amino
esters such as 1a–c in PrCO2Bu as compared with the
reactions in diisopropyl ether. Similarly, diisopropyl
ether is favorable when the branched-chain (including
cyclic) substrates 1e–g react. The (R)-configuration was
assigned to the reactive enantiomers for compounds 1a
and b, while the (S)-enantiomer was the reactive enan-
tiomer for compounds 1d and g. In this way the steric
arrangement of the substituents around the stereocenter
is the same in the above cases (the priority sequence of
the substituents changes with the structure of b-sub-
stituent R in Scheme 1). The determination of the
above absolute configurations is based on the known
specific rotations of the free acids or esters (see Section
3). Ion-exchange chromatography allowed the prepara-
tion of the free amino acids. In this procedure, some
racemization evidently took place as seen in low values
of specific rotations. On the assumption that the
enzyme has straightforward stereochemical demands, it
is concluded that for the CAL-A-catalyzed acylation of
1c the (R)-enantiomer reacts faster, while for that of 1e
and f the (S)-enantiomer reacts faster.
The effect of substrate structure on the enantioselectiv-
ity of CAL-A was studied in the acylation of 1a–g with
butyl butanoate in neat butyl butanoate and with 2,2,2-
trifluoroethyl butanoate in diisopropyl ether (Scheme
1). Compounds 1b–g were resolved on a gram scale.
2. Results and discussion
In accordance with the previously observed excellent
chemoselectivity of CAL-A,13 the acylation products
2a–g were the only detectable products for the acylation
of racemic 1a–g with 2,2,2-trifluoroethyl butanoate in
diisopropyl ether and with butyl butanoate in neat
butyl butanoate (Table 1; Scheme 1). The reactions of
1a–g with 2,2,2-trifluoroethyl butanoate in diisopropyl
ether approach a conversion of approximately 50%
faster than the reactions with butyl butanoate as acyl
donor and solvent. This can readily be understood
because the 2,2,2-trifluoroethyl ester as an activated
alkyl ester is considerably more prone than the ‘normal’
esters (butyl butanoate) to ester formation with the
Ser-OH of the enzyme, i.e. to the formation of a
butanoyl–enzyme intermediate in the two-step catalytic
mechanism.15
The basic aim for economic enzymatic kinetic resolu-
tion should be conditions where the two enantiomers
are obtained simultaneously in enantiopure form from
one reaction. The enantiomer ratio, E, as a measure of
the enantioselective behavior of the biocatalytic system
can be used in the prediction of such conditions, E
values greater than 100 being excellent. Compounds
1b–g were successfully resolved on a gram scale through
the use of CAL-A, the best method being chosen
according to the E values in Table 1. The results in
Table 2 clearly indicate the applicability of CAL-A for
the resolution of both alicyclic b-amino acid esters11
and acyclic compounds; 1e is an exception in that it
allows only the preparation of the less reactive enan-
tiomer in highly enantiopure form. This was also the
case for the reaction of 1a in butyl butanoate, with
E=32 (Table 1). Compound 1a was earlier successfully
resolved by using CAL-B catalysis in butyl butanoate
(row 1, Table 2).13 This is an in situ sequential resolu-
tion following both transesterification–acylation (the
main route) and acylation–interesterification sequences,
For the CAL-A-catalyzed acylation of 1b–g, good (E=
40–100) to excellent (E>100) enantioselectivities were
observed with at least one of the two acyl donors,
although solvent effects and/or structural effects caused
by an acyl donor can also be seen in the results.
Clearly, aminopentanoate 1b has the optimal structure
for CAL-A-catalyzed enantioselective acylation. As
concerns the two-step reaction mechanism, enan-
tiodiscrimination takes place in the second step when
the butanoyl–enzyme intermediate selectively reacts
with one of the substrates 1a–g, leading to formation of

S. Gedey et al. / Tetrahedron: Asymmetry 12 (2001) 105–110
107
where the two functional groups (NH2 and ethyl ester)
of 1a have both reacted with the achiral ester. In this
case the preparation of two highly enantiopure frac-
tions [(S)-P2 (Bu in place of Et in 1a)+(S)-(1a) and
(R)-P3 (Bu in place of Et in 2a)] at ca. 80% conversion
becomes possible.
(1+e.e.1/e.e.2)] with c=e.e.1/(e.e.1+e.e.2), as derived from
the original equations of Chen et al.22 Linear regression
was applied to obtain E as the slope of the line.
1H NMR spectra were recorded in CDCl3, DMSO-d6 or
D2O solutions at ambient temperature on Bruker 200/
Aspect 3000 or Bruker AM 400 spectrometers. Chemi-
cal shifts are given in l (ppm) relative to TMS (CDCl3,
DMSO-d6) or to TSP (D2O) as internal standards;
multiplicities were recorded as s (singlet), d (doublet),
dd (double doublet), t (triplet), q (quartet), m (multi-
plet) and om (overlapping multiplet). MS spectra were
3. Materials and methods
3-Amino-4-methylpentanoic acid, 3-amino-4-ethylhex-
anoic acid, 3-amino-3-cyclohexylpropanoic acid, and
3-amino-3-phenylpropanoic acid were prepared by
known procedures,17–20 and transformed to the corre-
sponding ethyl esters with thionyl chloride and dry
ethanol or butanol. Acyl donors were prepared from
the corresponding acid chlorides and alcohols. CAL-A
(C. antarctica lipase A, Chirazyme L5) was purchased
from Boehringer–Mannheim.16 The enzyme was immo-
bilized on Celite in the presence of sucrose before use,
giving a final lipase content of 10% (w/w) in the lipase
preparation.21 The solvents were of the best analytical
grade from Lab Scan Ltd.
recorded on
a VG Analytical 7070E instrument
equipped with a Vaxstation 3100 M 76 computer. Ele-
mental analyses were performed with a Perkin–Elmer
CHNS-2400 Ser II elemental analyzer. Optical rota-
tions were measured with a JASCO DIP-360 polarime-
ter, and [h]D values are given in units of 10−1 deg cm2/g.
3.1. Preparation of 3-aminopentanoic acid and 3-amino-
hexanoic acid
A mixture of (E)-2-pentenoic acid or (E)-2-hexenoic
acid (0.10 mol), benzylamine (10.72 g, 0.1 mol) and dry
pyridine (35 mL) was stirred at 120–130°C for 3 h.
Acetone (50 mL) was then added and the mixture was
cooled to ambient temperature. After standing
overnight, the crystals of 3-(benzylamino)pentanoic or
hexanoic acid that separated out were filtered off,
washed with acetone and recrystallized from methanol–
acetone.
As an example of a typical small-scale experiment, ethyl
3-aminobutanoate (1a, 0.1 M) and hexadecane (0.01 M,
an internal standard) were dissolved in diisopropyl
ether (2.5 mL), and an acyl donor (0.2 M) was added.
The addition of the enzyme preparation (40 mg/mL)
started the reaction. The reaction mixture was shaken
at room temperature. The progress of the reaction and
the e.e. values of the products were followed by taking
samples (0.1 mL) at intervals and analyzing them by
gas chromatography on a Chrompack CP-Chirasil-
DEX CB column (25 m). In the case of ethyl 3-amino-
3-phenylpropanoate 1g, the enantiomers were separated
3-(Benzylamino)pentanoic acid: 14.80 g, 0.071 mol; mp
169–170°C. Analysis calcd for C12H17NO2: C, 69.54; H,
1
8.27; N, 6.76%. Found: C, 69.27; H, 8.04; N, 6.55%. H
NMR (400 MHz, D2O) l (ppm): 0.99 (3H, t, J=7.5,
CH3), 1.69 (1H, m, CH2CH3), 1.88 (1H, m, CH2CH3),
2.50 (1H, dd, J=16.9, 7.4, CHCH2CO), 2.66 (1H, dd,
J=16.9, 4.8, CHCH2CO), 3.40 (1H, m, CHNH), 4.26
(2H, m, CH2C6H5), 7.50 (5H, m, C6H5).
on a Chrompack CP-Chirasil-L-Valine column (25 m).
For good baseline separation, the unreacted amino
group in the sample was derivatized with acetic anhy-
dride in the presence of pyridine containing 1% 4-N,N-
dimethylaminopyridine (DMAP). The separation of the
enantiomers of 2c was unsuccessful by the GLC
method so 2c was first separated by column chromatog-
raphy and then converted to the acetamide 3c for e.e.2
determination as described later. Conversions were
determined using e.e.1 and e.e.2 values except for the
reaction of 1c, where the conversion was determined by
internal standard. The determination of E was based on
the equation E=ln[(1−e.e.1)/(1−e.e.1/e.e.2)]/ln[(1−e.e.1)/
3-(Benzylamino)hexanoic acid: 14.65 g, 0.066 mol; mp
160–162°C. Analysis calcd for C13H19NO2: C, 70.56; H,
1
8.65; N, 6.33%. Found: C, 70.39; H, 8.42; N, 6.20%. H
NMR (400 MHz, D2O) l (ppm): 0.93 (3H, t, J=7.3,
CH3), 1.39 (2H, m, CH2CH3), 1.65 (1H, m,
CH2CH2CH3), 1.79 (1H, m, CH2CH2CH3), 2.50 (1H,
dd, J=17.0, 7.5, CHCH2CO), 2.66 (1H, dd, J=17.0,
4.8, CHCH2CO), 3.46 (1H, m, CHNH), 4.27 (2H, m,
CH2C6H5), 7.50 (5H, m, C6H5).
Table 2. Gram-scale resolutions of ethyl 3-aminocarboxylates 1a–g
R
Acyl donor
Enzyme
Time (h)
Conversion (%)
E.e.1 (%)
E.e.2 (%)
1a
1b
1c
1d
1e
1f
Me
Et
n-Pr
i-Pr
CHEt2
Cyclohexyl
Ph
PrCO2Bu
PrCO2Bu
PrCO2Bu
PrCO2CH2CF3
PrCO2CH2CF3
PrCO2CH2CF3
PrCO2CH2CF3
CAL-B
CAL-A
CAL-A
CAL-A
CAL-A
CAL-A
CAL-A
2
65
50
48
53
55
51
52
96 (S)
97 (S)
96 (S)
99 (R)
98 (R)
99 (R)
98 (R)
99 (R)a
97 (R)
98 (R)b
87 (S)
79 (S)
99 (S)
90 (S)
15
9.5
2.5
13
1.2
1
1g
a Product butyl 3-amino-N-butanoylbutanoate after sequential resolution.13
b E.e.2 determined after conversion to the acetamide.

108
S. Gedey et al. / Tetrahedron: Asymmetry 12 (2001) 105–110
3-(Benzylamino)pentanoic or hexanoic acid (0.050 mol)
was dissolved in a 1:1 mixture of methanol and water
(100 mL), and 20% palladium hydroxide on carbon (1.0
g) was added to the solution. The mixture was hydro-
genated in an autoclave at 70°C and 90 bar for 24 h.
The catalyst was removed by filtration, the filtrate was
evaporated to dryness and the residue was crystallized
on treatment with acetone. The crystals were filtered
off, washed with acetone and recrystallized from
methanol–acetone.
m, CHCH2CO), 4.14 (2H, q, J=7.2, OCH2CH3),
ꢀ4.18 (1H, om, CHNH), 6.22 (1H, br s, NH).
For determination of the absolute configuration, the
acetamide of unreacted 1b (30 mg) was boiled in 18%
HCl (5 mL) for 1 h and purified by ion-exchange
chromatography (Dowex 50, basic). (S)-3-Aminopen-
tanoic acid {[h]D25 +40.1 (c=0.99, H2O); literature23
value for the (S)-acid: [h]2D2 +39.5 (c=0.97, H2O), e.e.=
91%}.
3-Aminopentanoic acid: 4.92 g, 0.042 mol; mp 189–
191°C (mp. literature20 179–180°C). 1H NMR (400
MHz, D2O) l (ppm): 0.99 (3H, t, J=7.5, CH2CH3),
1.69 (2H, m, CH2CH3), 2.44 (1H, dd, J=16.6, 8.3,
CHCH2CO), 2.57 (1H, dd, J=16.6, 4.9, CHCH2CO),
3.43 (1H, m, CHNH2).
3.3. Gram-scale resolution of ethyl 3-aminohexanoate
(1c; R=n-Pr)
Racemic 1c (2.0 g, 12.6 mmol) was dissolved in butyl
butanoate (126 mL), and Chirazyme L5 (3.78 g, 30
mg/mL) and heptadecane (0.6 g; internal standard)
were added. The mixture was stirred at room tempera-
ture. The reaction was stopped at 48% conversion after
9.5 h. The unreacted substrate was derivatized with
acetic anhydride, followed by purification of the prod-
ucts by column chromatography as above.
3-Aminohexanoic acid: 5.68 g, 0.043 mol; mp 210–
211°C. Analysis calcd for C6H13NO2: C, 54.94; H, 9.99;
1
N, 10.68%. Found: C, 55.14; H, 9.80; N, 10.45%. H
NMR (400 MHz, D2O) l (ppm): 0.94 (3H, t, J=7.3,
CH3), 1.41 (2H, m, CH2CH3), 1.63 (2H, m,
CH2CH2CH3), 2.43 (1H, dd, J=16.6, 8.3, CHCH2CO),
2.57 (1H, dd, J=16.6, 4.9, CHCH2CO), 3.51 (1H, m,
CHNH2).
Compound (R)-2c: 0.46 g, 2.01 mmol; [h]2D0 +19.5 (c=
1.02, MeOH); e.e. 98% (compound 2c (0.75 g) was
converted to the acetamide through acidic hydrolysis
followed by esterification and derivatization with acetic
anhydride); M=229 according to MS. Analysis calcd
for C12H23NO3: C, 62.85; H, 10.11; N, 6.11%. Found:
C, 62.47; H, 9.85; N, 6.35%. 1H NMR (400 MHz
3.2. Gram-scale resolution of ethyl 3-aminopentanoate
(1b; R=Et)
Racemic 1b (1.32 g, 9.1 mmol) was dissolved in butyl
butanoate (90 mL) and CAL-A (3.6 g, 40 mg/mL) was
added. The mixture was stirred at room temperature.
The reaction was stopped at 50% conversion after 15 h
by filtering off the enzyme. After evaporation, the
residue was dissolved in diisopropyl ether (50 mL) and
the unreacted substrate was derivatized with acetic
anhydride (0.5 mL; 5.3 mmol) in the presence of pyri-
dine containing 1% DMAP (0.1 mL) by stirring
overnight. The products were separated on silica gel by
elution with petroleum ether–propan-2-ol (100:6), the
elution sequence being product 2b before the acetamide
of unreacted 1b.
(CDCl3)
CHCH2CH2CH3),
l
(ppm):
0.94
0.91
(3H,
(3H,
t,
t,
J=7.3,
J=7.4,
COCH2CH2CH3), 1.26 (3H, t, J=7.1, OCH2CH3), 1.35
(2H, m, CHCH2CH2CH3), 1.51 (2H, m,
CHCH2CH2CH3), 1.65 (2H, m, COCH2CH2CH3), 2.14
(2H, t, J=7.5, COCH2CH2CH3), 2.49 (1H, dd, J=15.8,
5.2, CHCH2CO), 2.55 (1H, dd, J=15.8, 5.2,
CHCH2CO), 4.14 (2H, m, OCH2CH3), 4.26 (1H, m,
CHNH), 6.06 (1H, br d, J=8.3, NH).
Compound (S)-3c: 0.45 g, 2.24 mmol; [h]2D0 −19.8 (c=
1.00, MeOH); e.e. 96%; M=201 according to MS.
Analysis calcd for C10H19NO3: C, 59.68; H, 9.52; N,
1
6.96%. Found: C, 59.49; H, 9.93; N, 6.58%. H NMR
Compound (R)-2b: 0.73 g, 3.39 mmol; [h]2D0 +21.4 (c=
1.00, MeOH); e.e. 97%; M=215 according to MS.
Analysis calcd for C11H21NO3: C, 61.37; H, 9.83; N,
(400 MHz) (CDCl3) l (ppm): 0.91 (3H, t, J=7.3,
CH2CH2CH3), 1.27 (3H, t, J=7.1, OCH2CH3), 1.35
(2H, m, CH2CH2CH3), 1.51 (2H, m, CH2CH2CH3),
1.97 (3H, s, COCH3), 2.48 (1H, dd, J=15.9, 5.1,
CHCH2CO), 2.55 (1H, dd, J=15.9, 5.1, CHCH2CO),
4.14 (2H, m, OCH2CH3), 4.25 (1H, m, CHNH), 6.06
(1H, br d, J=7.5, NH).
1
6.51%. Found: C, 61.11; H, 9.35; N, 6.13%. H NMR
(200 MHz, CDCl3) l (ppm): 0.92 (3H, t, J=7.4,
CHCH2CH3), 0.94 (3H, t, J=7.3, CH2CH2CH3), 1.27
(3H, t, J=7.1, OCH2CH3), 1.62 (4H, m, CHCH2CH3,
CH2CH2CH3), 2.15 (2H, t, J=7.4, CH2CH2CH3), 2.52
(2H, dd, J=5.2, 1.0, CHCH2CO), 4.14 (2H, q, J=7.1,
OCH2CH3), ꢀ4.2 (1H, om, CHNH), 6.14 (1H, br d,
J=7.8, NH).
3.4. Gram-scale resolution of ethyl 3-amino-4-methyl-
pentanoate (1d; R=i-Pr)
Racemic 1d (2.10 g, 13.2 mmol) was dissolved in diiso-
propyl ether (130 mL), and 2,2,2-trifluoroethyl
butanoate (4.49 g, 26.4 mmol) and CAL-A (5.2 g, 40
mg/mL) were added. The reaction was stopped at 53%
conversion after 2.5 h. The unreacted substrate was
derivatized with acetic anhydride, followed by purifica-
tion of the products by column chromatography as
above.
Compound (S)-3b: 0.21 g, 1.12 mmol; [h]2D0 −27.5 (c=
1.01, MeOH); e.e. 97%; M=187 according to MS.
Analysis calcd for C9H17NO3: C, 57.73; H, 9.15; N,
1
7.48%. Found: C, 57.49; H, 9.43; N, 7.63%. H NMR
(400 MHz) (CDCl3) l (ppm): 0.92 (3H, t, J=7.4,
CHCH2CH3), 1.27 (3H, t, J=7.2, OCH2CH3), 1.56
(2H, m, CHCH2CH3), 1.98 (3H, s, COCH3), 2.52 (2H,

S. Gedey et al. / Tetrahedron: Asymmetry 12 (2001) 105–110
109
Compound (S)-2d: 1.09 g, 4.75 mmol; [h]2D5 +8.0 (c=
0.99, MeOH); e.e. 87%; M=229 according to MS.
Analysis calcd for C12H23NO3: C, 62.85; H, 10.11; N,
CHCH2CO), 4.14 (2H, m, OCH2CH3), 4.30 (1H, m,
CHNH), 6.04 (1H, br d, J=8.9, NH).
1
6.11%. Found: C, 63.14; H, 10.05; N, 6.42%. H NMR
3.6. Gram-scale resolution of ethyl 3-amino-3-cyclo-
hexylpropanoate (1f; R=cyclohexyl)
(200 MHz, CDCl3)
CH(CH3)2, CH2CH2CH3), 1.18 (3H, t, J=7.1,
OCH2CH3), 1.47–1.85 (3H, om, CH(CH3)2,
l
(ppm): 0.75–95 (9H, om,
Racemic 1f (3.00 g, 15 mmol) was dissolved in diiso-
propyl ether (150 mL), and 2,2,2-trifluoroethyl
butanoate (5.13 g, 30 mmol) and CAL-A (3.0 g, 20
mg/mL) were added. The reaction was stopped after 70
min at 51% conversion by filtering off the enzyme. The
unreacted substrate was derivatized with acetic anhy-
dride, followed by purification of the products by
column chromatography as above. The eluted products
were recrystallized.
CH2CH2CH3), 2.10 (2H, t, J=7.4, COCH2CH2CH3),
2.44 (2H, d, J=5.5, CHCH2CO), 4.00 (1H, om,
CHNH), 4.06 (2H, q, J=7.1, OCH2CH3), 6.13 (1H, br
d, J=8.3, NH).
Compound (R)-3d: 1.08 g, 5.37 mmol; [h]2D5 −11.0 (c=
0.99, MeOH); e.e. 99%; M=201 according to MS.
Analysis calcd for C10H19NO3: C, 59.68; H, 9.52; N,
1
6.96%. Found: C, 59.27; H, 9.41; N, 6.81%. H NMR
(200 MHz, CDCl3) l (ppm): 0.83 (6H, d, J=6.7,
CH(CH3)2), 1.17 (3H, t, J=7.2, OCH2CH3), 1.72 (1H,
m, CH(CH3)2), 1.89 (3H, s, COCH3), 2.42 (2H, d,
J=5.6, CHCH2CO), 4.00 (1H, om, CHNH), 4.04 (2H,
q, J=7.2, OCH2CH3), 6.09 (1H, br s, NH).
Compound (S)-2f: 0.75 g, 2.78 mmol; mp 70–72°C
(n-hexane); [h]2D5 −9.8 (c=1.00, MeOH); e.e. 99%; M=
269 according to MS. Analysis calcd for C15H27NO3: C,
66.88; H, 10.10; N, 5.20%. Found: C, 66.79; H, 9.72; N,
4.84%. 1H NMR (400 MHz, CDCl3) l (ppm): 0.90–1.04
(2H, om, CH(CH2)5), 0.94 (3H, t, J=7.4,
CH2CH2CH3), 1.05–1.30 (3H, om, CH(CH2)5), 1.26
(3H, t, J=7.1, OCH2CH3), 1.47 (1H, m, CH(CH2)5),
1.60–1.80 (7H, om, CH(CH2)5, CH2CH2CH3), 2.15
(2H, t, J=7.5, CH2CH2CH3), 2.52 (2H, m,
CHCH2CO), 4.07 (1H, om, CHNH), 4.14 (2H, om,
OCH2CH3), 6.02 (1H, br d, J=9.2, NH).
For determination of the absolute configuration,
hydrolysis of 2d (30 mg) was carried out in 18% HCl (5
ml) by heating under reflux for 1 h, and the product
was purified by ion-exchange chromatography (Dowex
50, basic). (S)-3-Amino-4-methylpentanoic acid {[h]D25
−18.1 (c=1.05, H2O); literature23 value for the (R)-acid:
([h]2D2 +40.3 (c=1.02, H2O), e.e.=98%}.
Compound (R)-3f: 0.61 g, 2.53 mmol; mp 103–104°C
(n-hexane–EtOAc); [h]2D5 +6.3 (c=1.00, MeOH); e.e.
99%; M=241 according to MS. Analysis calcd for
C13H23NO3: C, 64.70; H, 9.61; N, 5.80%. Found: C,
3.5. Gram-scale resolution of ethyl 3-amino-4-ethylhex-
anoate (1e; R=CHEt2)
1
Racemic 1e (2.54 g, 13.6 mmol) was dissolved in diiso-
propyl ether (136 ml), and 2,2,2-trifluoroethyl
butanoate (4.63 g, 27.2 mmol) and CAL-A (5.44 g, 40
mg/ml) were added. The reaction was stopped at 55%
conversion after 13 h by filtering off the enzyme. The
unreacted substrate was derivatized with acetic anhy-
dride, followed by purification of the products by
column chromatography as above.
64.39; H, 9.48; N, 5.72%. H NMR (400 MHz, CDCl3)
l (ppm): 0.97 (2H, m, CH(CH2)5), 1.05–1.30 (3H, om,
CH(CH2)5), 1.26 (3H, t, J=7.1, CH2CH3), 1.47 (1H, m,
CH(CH2)5), 1.60–1.82 (5H, om, CH(CH2)5), 1.98 (3H,
s, COCH3), 2.52 (2H, m, CHCH2CO), 4.05 (1H, m,
CHNH), 4.14 (2H, m, OCH2CH3), 6.05 (1H, br d,
J=9.2, NH).
3.7. Gram-scale resolution of ethyl 3-amino-3-phenyl-
Compound (S)-2e: 1.40 g, 5.44 mmol; [h]2D5 +4.3 (c=
1.02, MeOH); e.e. 79%; M=257 according to MS.
Analysis calcd for C14H27NO3: C, 65.33; H, 10.57; N,
propanoate (1g; R=Ph)
Racemic 1g (2.51 g, 13 mmol) was dissolved in diiso-
propyl ether (130 mL), and 2,2,2-trifluoroethyl
butanoate (4.42 g, 26 mmol) and CAL-A (3.9 g, 30
mg/mL) were added. The reaction was stopped after 1
h at 52% conversion by filtering off the enzyme. The
solvent was evaporated off and the residue was dis-
solved in diethyl ether. Gaseous hydrogen chloride
bubbled through the mixture precipitated the unreacted
substrate. The solvent fraction was evaporated and the
residue was purified by column chromatography, with
petroleum ether–propan-2-ol (10:1) as eluent.
1
5.44%. Found: C, 64.94; H, 10.13; N, 5.58%. H NMR
(400 MHz, CDCl3) l (ppm): 0.89 (3H, t, J=7.5,
CHCH2CH3), 0.90 (3H, t, J=7.4, CHCH2CH3), 0.94
(3H, t, J=7.5, CH2CH2CH3), 1.26 (3H, t, J=7.1,
OCH2CH3), 1.28–1.48 (5H, om, CH(CH2CH3)2), 1.65
(2H, m, CH2CH2CH3), 2.16 (2H, t, J=7.4,
CH2CH2CH3), 2.51 (2H, d, J=5.5, CHCH2CO), 4.13
(2H, m, OCH2CH3), 4.32 (1H, m, CHNH), 6.06 (1H,
br d, J=8.9, NH).
Compound (R)-3e: 0.77 g, 3.36 mmol; [h]2D5 −8.7 (c=
1.02, MeOH); e.e. 98%; M=229 according to MS.
Analysis calcd for C12H23NO3: C, 62.85; H, 10.11; N,
Compound (S)-2g: 1.50 g, 5.70 mmol; [h]2D5 −60.6 (c=
1.02, MeOH); e.e. 90%; M=263 according to MS.
Analysis calcd for C15H21NO3: C, 68.42; H, 8.04; N,
1
6.11%. Found: C, 63.13; H, 10.27; N, 6.36%. H NMR
1
(400 MHz, CDCl3) l (ppm): 0.89 (3H, t, J=7.3,
CHCH2CH3), 0.90 (3H, t, J=7.3, CHCH2CH3), 1.26
(3H, t, J=7.1, OCH2CH3), 1.25–1.46 (5H, om,
CH(CH2CH3)2), 1.98 (3H, s, COCH3), 2.50 (2H, m,
5.32%. Found: C, 67.94; H, 8.21; N, 5.64%. H NMR
(200 MHz, CDCl3) l (ppm): 0.93 (3H, t, J=7.1,
CH2CH2CH3), 1.16 (3H, t, J=7.1, OCH2CH3), 1.66
(2H, m, CH2CH2CH3), 2.19 (3H, t, J=7.4,

110
S. Gedey et al. / Tetrahedron: Asymmetry 12 (2001) 105–110
CH2CH2CH3), 2.79 (1H, dd, J=15.5, 6.0, CHCH2CO),
2.91 (1H, dd, J=15.5, 6.1, CHCH2CO), 4.06 (2H, q,
J=7.1, OCH2CH3), 5.43 (1H, m, CHNH), 6.78 (1H, br
d, J=8.3, NH), 7.29 (5H, m, C6H5).
6. Ojima, I.; Lin, S.; Wang, T. Curr. Med. Chem. 1999, 6,
927–954.
7. Scarborough, R. M. Curr. Med. Chem. 1999, 6, 971–981.
8. Juaristi, E. Enantioselective Synthesis of i-Amino Acids;
Wiley-VHC: New York, 1997.
9. Csomo´s, P.; Kanerva, L. T.; Berna´th, G.; Fu¨lo¨p, F.
Tetrahedron: Asymmetry 1996, 7, 1789–1796.
10. Ka´ma´n, J.; Forro´, E.; Fu¨lo¨p, F. Tetrahedron: Asymmetry
2000, 11, 1593–1600.
11. Kanerva, L. T.; Csomo´s, P.; Sundholm, O.; Berna´th, G.;
Fu¨lo¨p, F. Tetrahedron: Asymmetry 1996, 7, 1705–1716.
12. Liljeblad, A.; Kanerva, L. T. Tetrahedron: Asymmetry
1999, 10, 4405–4415.
Hydrochloride of (R)-1g: 1.24 g, 5.40 mmol; mp 115–
116°C; [h]2D5 −5.9 (c=1.01, MeOH), literature24 value
for the hydrochloride of (S)-1g: [h]2D2 +5.8 (c=1.0,
MeOH); e.e. 98%; M=193 according to MS. Analysis
calcd for C11H16ClNO2: C, 57.52; H, 7.02; N, 6.10%.
1
Found: C, 58.09; H, 7.14; N, 6.13%. H NMR (200
MHz, DMSO-d6) l (ppm): 1.06 (3H, t, J=7.1,
CH2CH3), 3.01 (1H, dd, J=15.9, 9.5, CHCH2CO), 3.27
(1H, dd, J=15.9, 5.3, CHCH2CO), 3.97 (2H, q, J=7.0,
CH2CH3), 4.55 (1H, m, CHNH2), 7.39 (3H, m, C6H5),
7.60 (2H, m, C6H5), 8.92 (3H, br s, NH2, HCl).
13. Gedey, S.; Liljeblad, A.; Fu¨lo¨p, F.; Kanerva, L. T. Tetra-
hedron: Asymmetry 1999, 10, 2573–2581.
14. Kanerva, L. T. In Enzymatic Reactions in Organic Media;
Koskinen, A. M. P.; Klibanov, A. M., Eds.; Chapman &
Hall: London, 1996; Chapter 7, p. 170.
15. Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in
Organic Synthesis. Regio- and Stereoselective Biotransfor-
mations; Wiley-VHC: Weinheim, 1999.
Acknowledgements
16. Chirazyme L5 is C. antarctica lipase A, which is similar
to the former lipase SP 526 from Novo-Nordisk. CAL-B
refers to immobilized C. antarctica lipase B with the trade
name Chirazyme L2, which, according to Boehringer–
Mannheim, is a catalyst corresponding to Novozym 435
(the trade name of immobilized C. antarctica lipase SP
525) from Novo-Nordisk. Novo-Nordisk no longer sup-
plies C. antarctica lipases.
S.G. is grateful for a grant from the Centre for Interna-
tional Mobility (CIMO) in Finland. Support from the
Technology Development Centre (TEKES) in Finland
and from MKM (Grant No. FKFP 0535/1999) in
Hungary is also gratefully acknowledged.
17. Zilkha, A.; Rivlin, J. J. Org. Chem. 1958, 23, 94–96.
18. Shih, Y. E.; Wang, J. S.; Chen, C.-T. Heterocycles 1978,
9, 1277–1285.
19. La´za´r, L.; Martinek, T.; Berna´th, G.; Fu¨lo¨p, F. Synth.
Comm. 1998, 28, 219–224.
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