Practical, highly enantioselective chemoenzymatic one-pot synthesis of short-chain aliphatic β-amino acid esters

Article abstract of DOI:10.1055/s-0029-1216721

A practical, highly enantioselective method for the synthesis of short-chain aliphatic β-amino acid esters was developed starting from prochiral and easily accessible substrates. This chemoenzymatic approach is based on a nonenzymatic aza-Michael addition

Full text of DOI:10.1055/s-0029-1216721

LETTER
1251
Practical, Highly Enantioselective Chemoenzymatic One-Pot Synthesis of
Short-Chain Aliphatic b-Amino Acid Esters
One-Pot Synthesis of
S
a
hort-Chain
r
A
liphati
k
c
b
-AminoA
u
c
idEsters s Weiß, Harald Gröger*
Department of Chemistry and Pharmacy, University of Erlangen-Nuremberg, Henkestr. 42, 91054 Erlangen, Germany
Fax +49(9131)8521165; E-mail: harald.groeger@chemie.uni-erlangen.de
Received 6 January 2009
enoate and benzylamine as raw materials, a lipase as a
(bio-)catalyst and no solvent (Scheme 1, route A). The
reaction concept is based on an initial Michael addition of
benzylamine (2) to ethyl crotonate (1a) under formation
Abstract: A practical, highly enantioselective method for the syn-
thesis of short-chain aliphatic b-amino acid esters was developed
starting from prochiral and easily accessible substrates. This
chemoenzymatic approach is based on a nonenzymatic aza-Michael
addition of benzylamine to enoates and subsequent lipase-catalyzed of rac-3a, followed by subsequent enantioselective enzy-
matic aminolysis7 with benzylamine as amine component
resolution via enantioselective aminolysis. The two reactions are
carried out as a one-pot synthesis under solvent free-conditions af-
fording the b-amino esters in satisfying to good yields and with ex-
3a. The Michael addition might occur noncatalyzed or
cellent enantioselectivities of up to 99% ee.
under formation of (R)-4a and the (remaining) ester (S)-
lipase-catalyzed (which has recently been reported to be
non-enantioselective for similar Michael additions).8 The
Key words: aminolysis, amino acids, asymmetric catalysis, en-
zyme catalysis, stereoselective synthesis
choice of benzylamine as donor has the advantage that the
resulting N-benzyl moiety can be easily cleaved from the
b-amino acid derivative at a later stage. As an (undesired)
side reaction, however, one could expect the direct lipase-
catalyzed aminolysis of substrate 1a under formation of
enamide 5 (Scheme 1, route B).
Enantiomerically pure b-amino acids are attractive key
building blocks for the synthesis of pharmaceuticals.1 The
tendency to use b-amino acids in drug synthesis has in-
creased over recent years as well as the search for new
synthetic methods for their enantioselective preparation.2
For the synthesis of chiral aryl-substituted b-amino acids
numerous highly enantioselective approaches have been
developed, which are based on the use of chemocatalysts3
or enzymes.4 In part, these methods are applied on techni-
cal scale. In contrast there is a limited number of scalable
and efficient approaches to aliphatic, particularly short-
chain b-amino acids and their esters.5 In general, overall
process efficiency and practicability of syntheses for these
target molecules is limited by numerous workup steps in
multistep syntheses. Furthermore, often enantiomeric ex-
cess of the products is below 99% or even 95%, which
make additional recrystallization step(s) necessary to ob-
tain the (short-chain) aliphatic b-amino acids in enantio-
merically pure form (>99% ee). Thus, among key
challenges for a practical route for these types of b-amino
acids are (i) minimization of solvent use and isolation
steps in multistep approaches and (ii) development of syn-
theses from readily available substrates leading to prod-
ucts with 95% ee, in particular 99% ee, based on the use
of environmental friendly and highly enantioselective cat-
alysts.
Ph
NH
O
Δ
route A
and/or
lipase-
catalyzed
Me
OEt
lipase
PhCH2NH2
Ph
NH
rac
O
(S)-3a
+
Me
OEt
rac-3a
Ph
NH
O
Ph
NH2
2
Me
N
Ph
H
+
(R)-4a
O
Me
OEt
O
1a
lipase
Me
N
H
Ph
route B
5
Scheme 1 Proposed reaction course
In our initial enzyme screening we could identify lipase
from Candida antarctica B (CAL-B) as a biocatalyst and
benzylamine as a suitable amine donor, which led to the
formation of the desired aza-Michael adduct (S)-3a in
enantiomerically enriched form (Scheme 2).
In the following we report our preliminary results on a sol-
vent-free, two-step, one-pot synthesis of enantiomerically
pure aliphatic b-amino esters through combination of aza-
Michael addition and enzymatic aminolysis.6 As target
process we envisioned a one-pot approach for enantio-
merically pure b-amino acid esters 3 using only prochiral
When carrying out the reaction under neat conditions at
60 °C, the desired b-amino ester (S)-3a is obtained in 38%
yield and with 88% ee.9 As expected, formation of amide
(R)-4a (48% yield) as well as crotonamide 5 (10% yield)
was also observed. The mechanistic picture of this process
can be proposed to proceed according to the concept
shown in Scheme 1. In accordance with route A, initial
aza-Michael addition occurs both noncatalyzed and li-
pase-catalyzed. Comparison experiments with and with-
out enzyme supported the significant contribution of
SYNLETT 2009, No. 8, pp 1251–1254
x
x.
x
x.
2
0
0
9
Advanced online publication: 08.04.2009
DOI: 10.1055/s-0029-1216721; Art ID: G03309ST
© Georg Thieme Verlag Stuttgart · New York

1252
M. Weiß, H. Gröger
LETTER
selectivity for rac-3 (93.5%) over rac-4a (1.5%). The res-
olution (second step) was stopped at a conversion of
59.8%.
Ph
NH
O
Me
OEt
(S)-3a
The substrate spectrum turned out to be interesting in par-
ticular with respect to the synthesis of aliphatic short-
chain b-amino acid esters. When using the homologue
enoate 1b as a Michael acceptor, the two-step, one-pot
process under neat conditions furnished the b-amino acid
ester (S)-3b in 30% yield and also with an excellent enan-
tiomeric excess of 98% (Table 1, entry 3). The conversion
was 97.5% for the initial aza-Michael addition and 63.1%
for the subsequent aminolysis resolution. Extending the
chain length of the enoate led to a decreased enantioselec-
tivity, for example, a conversion of 63.7% for the resolu-
tion step in the one-pot synthesis was found for the
synthesis of 3-aminohexanoate derivative (S)-3c, which
was formed with an enantiomeric excess of 93% (entry 4).
38% yield, 88% ee
lipase
Ph
NH2
+
(CAL-B)
2
no solvent
60 °C, 23 h
Ph
NH
O
(2.2 equiv)
Me
N
Ph
Ph
+
96% overall
conversion
H
O
(R)-4a
48% yield
Me
OEt
+
1a
O
Me
N
H
5
10% yield
Scheme 2 Initial screening result9
lipase-catalyzed Michael addition (e.g., conversion of
31% with enzyme compared to 17% without enzyme after
6 h at r.t.). Taken into consideration yields and enantio-
meric excesses of both (S)-3a (59% yield, 25% ee) and
(R)-4a (18% yield, 77% ee) in a further experiment, it be-
comes evident that enzymatic Michael addition (and, thus,
initial Michael addition in general) proceed without or
with negligible enantioselectivity being in the range of the
deviation of the analytical methods chosen.10 Importantly,
however, subsequent enzymatic aminolysis of Michael
adduct rac-3a with 2 as amine component proceeds with
high enantioselectivity forming the product (S)-3a with
38% yield and 88% ee (Scheme 2). Notably, highest effi-
ciency of the one-pot synthesis was found under solvent-
free conditions. This was underlined by a study of the im-
pact of solvents. The presence of an organic solvent led to
less satisfactory results independent of the choice of sol-
vent, in particular with respect to activity (data not
shown). As solvents n-hexane, toluene, methyl tert-butyl
ether, and (deuterated) chloroform were used. Besides the
desired enzymatic aminolysis resolution, however, the li-
pase also catalyzes the formation of amide 5 as the expect-
ed undesired side product according to Scheme 1, route B.
Table 1 One-Pot Synthesis of b-Amino Esters (S)-3
Ph
NH
O
1. 60 °C, t1
conv.1
Ph
NH2
2
R
OEt
(2.2 equiv)
step 1
(S)-3a–c, (R)-3d,e
+
+
2. CAL-B,
60 °C, t2
conv.2
O
Ph
NH
O
R
OEt
R
N
Ph
H
1
step 2
(R)-4a–c, (S)-4d,e
Entrya
R
t1 (h) Conv.1 t2 (h) Conv.2 Product 3e,f Yield ee
(%)
(%)
(%) (%)
1
Me 30 95.0c
Me 30 91.0c
18
18
24
48
23
24
59.8 (S)-3a
60.3 (S)-3a
63.1 (S)-3b
63.7 (S)-3c
3.8d (R)-3d
60.4 (R)-3e
36
33
30
19
99
99
98
93
2b
3
Et
30 97.5c
4
n-Pr 48 99.0c
5
Ph 96 30.0
n.d.g n.d.g
6
CF3
3
99.0c
36 95
a The experimental procedure and analytic data of products (S)-3 are
described in the section References and Notes.11
In order to make this two-step, one-pot process more at-
tractive for preparative use, avoidance of the undesired
side product 5 is a prerequisite. Since formation of 5 only
occurs via lipase-catalyzed aminolysis of 1a, the reaction
protocol has been modified as follows: In a first step,
Michael addition of 2.2 equivalents of 2 to 1a is carried
out in the absence of the enzyme and solvent. After (ide-
ally complete) consumption of 1a under formation of rac-
3a, the lipase is added catalyzing the aminolysis reaction
enantioselectively with the remaining 1.2 equivalents of
2. Under solvent-free reaction conditions, this one-pot
process with enzymatic aminolysis resolution as a key
b This reaction was carried out on a 20 mmol scale.
c Selectivities related to conversions: entry 1: 93.5% rac-3a, 1.5% rac-
4a; entry 2: 89.4% rac-3a, 1.6% rac-4a; entry 3: 85.0% rac-3b, 12.5%
rac-4b; entry 4: 76.0% rac-3c, 23.0% rac-4c; entry 6: 99.0% rac-3e,
0.0% rac-4e.
d Entry 4: Due to low conversion in step 1, the second step was studied
using isolated rac-3d as a substrate.
e The corresponding amides (R)-4 were also isolated; yields and ee
were as follows: entry 1: (R)-4a, 55% yield, 60% ee; entry 2: (R)-4a,
52% yield, 61% ee; entry 3: (R)-4b, 46% yield, 57% ee; entry 4: (R)-
4c, 65% yield, 36% ee; entry 5: (R)-4d, yield and ee not determined;
entry 6: (R)-4e, 57% yield, 64% ee.
f The different assignment of the absolute configuration in case of 3d
step proceeds with high conversion and delivers ethyl 3- and 3e [both (R)-configuration] compared with the S-configuration in
the case of 3a–c is caused by different priorization of the substituents
aminobutyrate (S)-3a in 36% yield and with an excellent
enantiomeric excess of 99% (Table 1, entry 1). For the
first step a conversion of 95.0% was observed with a high
according to the rules of the Cahn–Ingold–Prelog nomenclature.
g Not determined.
Synlett 2009, No. 8, 1251–1254 © Thieme Stuttgart · New York

LETTER
One-Pot Synthesis of Short-Chain Aliphatic b-Amino Acid Esters
1253
In contrast to the highly enantioselective preparation of
short-chain aliphatic b-amino acid esters, this synthetic
methodology is not suitable for the preparation of aromat-
ic b-amino acid esters. A very low conversion was ob-
served for the resolution of racemic ethyl 3-benzylamino-
3-phenylpropanoate (rac-3d) with <5% (entry 5). How-
ever, fluorosubstituted aliphatic short-chain b-amino acid
esters can be also prepared very well using the developed
one-pot, two-step synthesis. When starting from 4,4,4-tri-
fluorocrotonate (1e), aza-Michael addition and subse-
quent enzymatic aminolysis gave the fluorosubstituted b-
amino acid ester (R)-3e in 36% yield and with high enan-
tiomeric excess of 95% (entry 6). The conversion was
99.0% for the aza-Michael addition and 60.4% for the
subsequent aminolysis resolution step.
1. 6 N HCl, Δ
2. Pd(OH)2/C
H
2 (60 psi)
AcOH–H2O (1:1)
65–70 °C, 22 h
Ph
NH
O
NH2
O
Me
OEt
Me
OH
3. Dowex ion
exchanger
(S)-3a
(>99% ee)
(S)-6
69% yield
[α]D25 +32.1° (c 0.6, H2O)
[ref. 12b: [α]D25 +32.0° (c 0.6, H2O)]
Scheme 3 Synthesis of (S)-b-amino butyric acid
selective aminolysis. The two reactions are carried out as
a one-pot synthesis under solvent free-conditions afford-
ing the b-amino esters in satisfying to good yields and
with excellent enantioselectivities of up to 99%.
In spite of the high enantiomeric excess for the esters 3 in
the one-pot process, the corresponding amides 4 were ob-
tained in much less satisfactory enantiomeric excesses in
the range of 36–64% ee (although good yields were ob-
tained; see Table 1). In addition, E values calculated for
the formation of amides 4 are lower than corresponding E
values calculated from the synthesis of the esters 3. The
reason is the formation of racemic amide rac-4 as a
byproduct in the initial step of formation of the racemic
ester rac-3 (for amount of formed rac-4, see Table 1), and
potentially also during the second reaction step (resolu-
tion). The resulting (R)-4a–c and (S)-4d, e from the enzy-
matic resolution is then isolated jointly with the formed
racemic amide rac-4. Thus, for the isolated products 4 a
lower enantiomeric excess is obtained as calculated from
the enantioselectivity (E value) of the enzymatic resolu-
tion alone.13
Acknowledgment
We thank Evonik Degussa GmbH for generous support. A grant for
M.W. by the Deutsche Bundesstiftung Umwelt (DBU) within the
scholarship programme ‘Nachhaltige Bioprozesse’ (‘Sustainable
Bioprocesses’) is gratefully acknowledged.
References and Notes
(1) Enantioselective Synthesis of b-Amino Acids; Juaristi, E.,
Ed.; Wiley-VCH: New York, 1997.
(2) For reviews, see: (a) Liu, M.; Sibi, M. P. Tetrahedron 2002,
58, 7991. (b) Liljeblad, A.; Kanerva, L. T. Tetrahedron
2006, 62, 5831.
(3) For selected chemocatalytic examples, see: (a) Hsiao, Y.;
Rivera, N. R.; Rosner, T.; Krska, S. W.; Njolito, E.; Wang,
F.; Sun, Y.; Armstrong, J. D.; Grabowski, E. J. J.; Tillyer,
R. D.; Spindler, F.; Malan, C. J. Am. Chem. Soc. 2004, 126,
9918. (b) Berkessel, A.; Cleemann, F.; Mukherjee, S.
Angew. Chem. Int. Ed. 2005, 44, 7466; Angew. Chem. 2005,
117, 7632. (c) Clausen, A. M.; Dziadul, B.; Cappuccio, K.
L.; Kaba, M.; Starbuck, C.; Hsiao, Y.; Dowling, T. M. Org.
Process Res. Dev. 2006, 10, 723. (d) Sibi, M. P.; Itoh, K.
J. Am. Chem. Soc. 2007, 129, 8064.
(4) For selected biocatalytic examples, see: (a) Cohen, S. G.;
Weinstein, S. Y. J. Am. Chem. Soc. 1964, 86, 725.
(b) Soloshonok, V. A.; Svedas, V. K.; Kukhar, V. P.;
Kirilenko, A. G.; Rybakova, A. V.; Solodenko, V. A.;
Fokina, N. A.; Kogut, O. V.; Gulaev, I. Y.; Kozlova, E. V.;
Shishkina, I. P.; Galushko, S. V. Synlett 1993, 339.
(c) Prashad, M.; Har, D.; Repic, O.; Blacklock, T. J.;
Giannousis, P. Tetrahedron: Asymmetry 1998, 9, 2133.
(d) Cardillo, G.; Gentilucci, L.; Tolomelli, A.; Tomasini, C.
J. Org. Chem. 1998, 63, 2351. (e) Katayama, S.; Ae, N.;
Nagata, R. Tetrahedron: Asymmetry 1998, 9, 4295.
(f) Faulconbridge, S. J.; Holt, K. E.; Sevillano, L. G.; Lock,
C. J.; Tiffin, P. D.; Tremayne, N.; Winter, S. Tetrahedron
Lett. 2000, 41, 2679. (g) Gröger, H.; May, O.; Hüsken, H.;
Georgeon, S.; Drauz, K.; Landfester, K. Angew. Chem. Int.
Ed. 2006, 45, 1645; Angew. Chem. 2006, 118, 1676.
(h) Gröger, H.; Trauthwein, H.; Buchholz, S.; Drauz, K.;
Sacherer, C.; Godfrin, S.; Werner, H. Org. Biomol. Chem.
2004, 2, 1977.
The efficiency of the enantioselective one-pot process for
aliphatic b-amino acid esters has been also demonstrated
on a larger lab scale (Table 1, entry 2). On a 20 mmol
scale the two-step, one-pot synthesis of (S)-3a proceeds
with nearly unchanged performance, leading to the de-
sired b-amino acid in 33% yield and with excellent enan-
tiomeric excess of 99%.
The prepared (S)-amino esters (S)-3 can be easily convert-
ed into the corresponding ‘free’ b-amino acids. This has
been successfully demonstrated for the synthesis of enan-
tiomerically pure b-amino butyric acid, (S)-6, starting
from enantiomerically pure (S)-3a (Scheme 3). After hy-
drolysis in acidic media and cleavage of the benzyl moiety
via Pd-catalyzed hydrogenation subsequent purification
with an ion exchanger furnished the desired (S)-b-amino
butyric acid (S)-6 in 69% yield and with excellent enan-
tiomeric excess (according to the comparison of the mea-
sured optical rotation with the literature known value
given in the literature12b,14).
In conclusion, a practical chemoenzymatic method for the
highly enantioselective synthesis of short-chain aliphatic
b-amino esters has been developed.15 Starting from
prochiral and easily accessible enoates this approach is
based on a nonenzymatic aza-Michael addition to enoates
and subsequent lipase-catalyzed resolution via an enantio-
(5) For selected efficient enzymatic routes, see: (a) Lipase-
catalyzed acylation: Stürmer, R.; Ditrich, K.; Siegel, W.
US 6063615, 2000. (b) PenG acylase-catalyzed hydrolysis,
see ref. 4b.
(6) The term ‘solvent-free synthesis’ refers to the composition
of the reaction mixture (excluding workup), thus enabling a
Synlett 2009, No. 8, 1251–1254 © Thieme Stuttgart · New York

1254
M. Weiß, H. Gröger
LETTER
absolute configuration of the other esters 3b,c,e has been
high space-time yield and free choice of solvent at the
downstream-processing stage.
assigned in analogy to the result in case of 3a. The spectral
(7) For enzymatic aminolysis reactions in general, see:
(a) Puertas, S.; Brieva, R.; Rebolledo, F.; Gotor, V.
Tetrahedron 1993, 49, 4007. (b) Sigmund, A. E.; McNulty,
K. C.; Nguyen, D.; Silverman, C. E.; Ma, P.; Pesti, J. A.;
DiCosimo, R. Can. J. Chem. 2002, 80, 608. (c) López-
Garcia, M.; Alfonso, I.; Gotor, V. J. Org. Chem. 2003, 68,
648. (d) Review: Gotor, V. J. Biotechnol. 2002, 96, 35.
(8) Carlqvist, P.; Svedendahl, M.; Branneby, C.; Hult, K.;
Brinck, T.; Berglund, P. ChemBioChem 2005, 6, 331.
(9) The yields given in Scheme 2 are ‘crude yields’ since the
products have not been isolated.
(10) This result is in accordance with previous reports by
Berglund et al. showing a nonenenatioselective course of
another type of CAL-B-catalyzed Michael addition, see
ref. 8.
(11) General Procedure for the One-Pot Synthesis of 3
(According to Table 1, Entries 1, 3–6)
data of the ester products (S)-3a–c, (R)-3e were in
accordance with literature data. The amides 4a–c,e have
been fully characterized (data will be published elsewhere).
(12) (a) Nejman, M.; Sliwinska, A.; Zwierzak, A. Tetrahedron
2005, 61, 8536. (b) Davies, S. G. Tetrahedron: Asymmetry
2007, 18, 1554.
(13) For the enzymatic resolution of rac-3a with benzylamine as
amine and lipase CAL-B as biocatalyst under solvent free
conditions at 60 °C, an E value of 27 has been obtained for
this aminolysis reaction (data not shown).
(14) Procedure for the Synthesis of (S)-b-Amino Butyric Acid,
(S)-6 (According to Scheme 3)
In a 10 mL round-bottom flask 4.32 mmol of (S)-ethyl 3-
(benzylamino)butanoate [(S)-3a, 955 mg, >99% ee] was
dissolved in 25 mL of 6 N HCl and heated to reflux overnight
for 18 h. After completion of the reaction remaining solvent
was evaporated under reduced pressure at 60 °C. The
resulting crude product was dissolved in 15 mL AcOH–H2O
(1:1, v/v) and transferred to a ‘Fischer–Porter bottle’. After
addition of Pd(OH)2/C (450 mg), the bottle was evacuated
and flushed with inert gas for three times. After evacuation
of the inert gas, the bottle was filled with hydrogen (60 psi
corresponding to 0.41 MPa) and heated to 65–70 °C for 22
h. The reaction mixture was filtered through a thin plug of
SiO2. The resulting hydrochloride of (S)-6 was obtained as a
white solid after evaporation of the solvent at reduced
pressure at 60 °C. The desired product (S)-amino butanoic
acid, (S)-6, was obtained in isolated form after Dowex® ion-
exchange chromatography. The spectral data of (S)-6 were in
In a 5 mL round-bottom flask a mixture of enoate 1 (1.0
mmol) and benzylamine (2, 241.5 mL, 2.2 mmol) was stirred
for 3–96 h at 60 °C. After adding 50 mg (entries 1, 3, 5, and
6) or 60 mg (entry 4) of a lipase from Candida antarctica B
(lipase CAL-B, Novozym 435), the reaction mixture was
stirred for further 18–48 h. The crude product was dissolved
in MTBE. After filtration from the solid enzyme, the organic
phase was concentrated to dryness under vacuo. The
resulting oily product was purified by means of column
chromatography [entries 1–4: EtOAc–2-PrOH (95:5, v/v),
0.2% Et2NH; Rf = 3a (0.62), 4a (0.22), 3b (0.66), 4b (0.22),
3c (0.50), 4c (0.16); entry 6: cyclohexane–EtOAc (3:1, v/v);
Rf = 3e (0.60), 4e (0.13)]. The ee was determined by chiral
HPLC chromatography (compounds 3a,b,e, and 4c:
Chiracel OJ-H column; compounds 4a,e: Chiracel AD-H
column) with hexane–2-PrOH–diethylamine in a ratio of
95:5:0.1 or 99:1:0.1 as eluent or NMR spectroscopy with
Eu(hfc)3 (compounds 3c, 4b). The absolute configuration of
3a was assigned according to the direction of optical rotation
of the product 6 after derivatization (see Scheme 3) and its
comparison with the literature value given in ref. 12. The
25
accordance with literature data. Optical rotation data: [a]D
25
+32.1 (c 0.6, H2O); for comparison, see ref. 12b: [a]D
+32.0 (c 0.6, H2O).
(15) During completion of our manuscript we became aware of
the following publication, describing processes related to
our synthesis shown in Scheme 2: Priego, J.; Ortiz-Nava, C.;
Carillo-Morales, M.; Lopez-Munguia, A.; Escalante, J.;
Castillo, E. Tetrahedron 2009, 65, 536.
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