Synthesis of chiral non-racemic intermediates and Arg-Gly-Asp mimetics by CaLB-catalyzed resolution

Article abstract of DOI:10.1016/j.tetasy.2009.12.021

The reactivity of both the ester and amine functions present in β-amino esters was tested in order to obtain the synthesis of enantiopure αvβ3 and α5β1 integrin ligands. CaLB successfully catalyzed both the enantioselective transesterification and the N-acylation of racemic β-amino esters, allowing the isolation of intermediates for the preparation of Arg-Gly-Asp (RGD) mimetic compounds. In particular, a CaLB-catalyzed amidation reaction with unprotected p-aminobenzylamine reduced the number of synthetic steps, thus avoiding protection and deprotection of the intermediate compounds. Following this procedure, RGD mimetics were isolated with high yields and enantiomeric purities.

Full text of DOI:10.1016/j.tetasy.2009.12.021

Tetrahedron: Asymmetry 21 (2010) 96–102
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Synthesis of chiral non-racemic intermediates and Arg-Gly-Asp mimetics
by CaLB-catalyzed resolution
*
*
Giuliana Cardillo , Arianna Gennari, Luca Gentilucci, Elisa Mosconi, Alessandra Tolomelli , Stefano Troisi
Department of Chemistry ‘G.Ciamician’, University of Bologna Via Selmi 2, 40126 Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The reactivity of both the ester and amine functions present in b-amino esters was tested in order to
Received 18 November 2009
Accepted 21 December 2009
Available online 3 February 2010
obtain the synthesis of enantiopure
avb3 and a5b1 integrin ligands. CaLB successfully catalyzed both
the enantioselective transesterification and the N-acylation of racemic b-amino esters, allowing the iso-
lation of intermediates for the preparation of Arg-Gly-Asp (RGD) mimetic compounds. In particular, a
CaLB-catalyzed amidation reaction with unprotected p-aminobenzylamine reduced the number of syn-
thetic steps, thus avoiding protection and deprotection of the intermediate compounds. Following this
procedure, RGD mimetics were isolated with high yields and enantiomeric purities.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
different strategies,8 the lipase enzymatic resolution9 is currently
increasing in importance. A recent paper by Escalante et al.10
Peptides display a variety of important biological activities and
most of their physiological functions are regulated by interactions
with specific receptors, through relatively small regions of their
described the preparation of chiral non-racemic N-protected-b-
amino esters by stereoselective transesterification reaction using
lipase from Candida antarctica (CaLB)10 and the lipase-mediated
selective acylation of primary amines has been also described with
the same enzyme.11
On the basis of these data, we herein report on the reactivity of
both the ester and amine functions present in the b-amino ester, to
realize the chemoenzymatic synthesis of RGD ligand intermediates
such as A and B. Moreover, we also report the four-step synthesis
of the mimetic C, performing lipase-catalyzed resolution at differ-
ent levels of the synthetic plan (Fig. 1).
surfaces. For instance,
avb3 and a
5b1 integrins1 are families of
heterodimeric transmembrane glycoproteins that bind to a large
number of extracellular matrix (ECM) proteins through recognition
of Arg-Gly-Asp (RGD) sequence.2 The ligand is connected to the
receptor mainly through electrostatic interactions binding regions
with opposite charges within the protein: Arg interacts with two
Asp groups located in the
a unit of the protein and the Asp with
a metal cation in the b chain.3 Small molecules mimicking the
RGD motif have been designed and synthesized and most of the
structures proposed so far consist of a synthetic sequence contain-
ing a basic appendage corresponding to an arginine side chain and
a carboxylic function mimicking the aspartic acid residue.4 Over
the course of our drug development programme5, in order to
compare the effect of rigid scaffolds to flexible cores, we needed
to synthesize linear molecules containing an enantiomerically pure
b-amino acid as a central fragment. The introduction of b-amino
acids into peptide sequences gives access to molecules with in-
creased resistance towards proteolytic enzymes and enhanced
metabolic stability. Due to the presence of b-amino acids in natural
compounds and to the range of their application in bioactive pep-
tide sequences,6 the resolution of racemic mixtures is an area of
current interest.7 Although the asymmetric synthesis of enantio-
pure amines has been successfully achieved through a number of
According to the requirements reported in the literature for an
effective interaction of ligands to
avb3 and a5b1 integrins, the
malonyl appendage was chosen to mimic aspartic acid on the basis
of its insertion in bioactive sequences, previously tested by our
group.5c,e
2. Results and discussion
2.1. Regio- and stereoselective synthesis of derivative A
At first, we explored the possibility of performing CaLB-cata-
lyzed transesterification with alternative alcohols. For this pur-
pose, we repeated the reaction under the conditions reported9 on
the N-Cbz-derivative 1a. Benzyl alcohol was chosen as an easily
removable protecting group under reducing conditions, and
isopropyl alcohol was selected since it can be removed under basic
conditions. It is known that lipases from microbial sources are
commonly used in organic solvents.12 In particular, CaLB has been
widely used in anhydrous solvents with success.13 Therefore the
* Corresponding author. Tel. +39 051 2099575; fax +39 051 2099456.
E-mail addresses: giuliana.cardillo@unibo.it (G. Cardillo), alessandra.tolomelli@
unibo.it (A. Tolomelli).
0957-4166/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.12.021

G. Cardillo et al. / Tetrahedron: Asymmetry 21 (2010) 96–102
97
O
O
accomplished under basic, acidic or reductive conditions respec-
tively, allows for the linkage of the b-amino acid carbonyl moiety
to different amines or amino acids, thus giving access to a library
of RGD mimetics.
R2
R1
CaLB
NH
O
transesterification
t-BuO
NH
O
R1
OR3
OMe
CaLB
2.2. Stereoselective CaLB-catalyzed formation of malonamide B
A
amidation
O
O
CaLB
amidation
A range of b-amino esters have been reported to react regiose-
lectively at the amino function in the presence of lipase from C.
antarctica with a variety of acyl donors.13
MeO
NH
O
R1
OMe
On the basis of the results reported in the literature on the ster-
eoselective enzymatic resolution of racemic alcohols with acetoac-
etates17 and cyclic diamine hydrochlorides with malonates,18 we
treated the dimethyl malonate with racemic 4a in the presence
of CaLB and TEA, in either dioxane or DME (Scheme 4). Under these
conditions the enzyme successfully catalyzed the acylation of 4a,
affording the malonyl amide (R)-8a and the unreacted b-amino
methyl ester (S)-4a, that was easily separated from the mixture
by acidic extraction. After removal of the solvent, (R)-8a was
isolated and purified by flash chromatography on silica gel. These
results show that the reaction is selective and that the formation
of the acyl-complex with bifunctional dimethyl malonate is faster
than the attack on the methyl function in 4a, since no trace of
products deriving from side reactions was observed.
O
O
B
t-BuO
NH
O
R1
N
H
C
NH2
Figure 1. General structure of b-amino acid containing RGD mimetics.
enzymatic transesterification was performed in dry toluene at
50 °C using 2 equiv of benzyl alcohol in the presence of CaLB
(Scheme 1).
The reaction was monitored by GC and 1H NMR. After 2 h the
required conversion was achieved, the enzyme was filtered off
and washed with CH2Cl2 for re-use. After removal of the solvent,
compounds (S)-1a and (R)-2a (Scheme 1) were easily separated
by flash chromatography on SiO2, and the enantiomeric excess
was determined by chiral HPLC (Table 1, entry 1).
The absolute configuration of (S)-4a was assigned by making a
comparison of its specific rotation {[
a]D = +25.0 (c 1, H2O)}, with
literature data {[
a
]
D = +34 (c 0.88, H2O)}.19 Good conversion and
enantiomeric excesses were obtained in both dioxane and DME,
confirming that these dipolar solvents are suitable for the kinetic
resolution of the racemic amine.20
Compound 8a has two identical methyl ester functions that
cannot be selectively removed. This molecule represents an inter-
esting building block for the preparation of bioactive heterocyclic
compounds and has been used as an intermediate in the synthesis
of undecaprenyl pyrophosphate synthase (UPPS) selective inhibi-
tors21 or lactam analogues of fentanyl.22
On the other hand, the transformation of enantiomerically en-
riched (S)-b-amino methyl ester 4a (ee 68%) into the corresponding
N-t-butylmalonyl-b-amino methyl ester under usual peptide cou-
pling conditions (Scheme 4) afforded (S)-5a, whose optical rotation
Cbz
Cbz
Cbz
NH
O
NH
O
NH
O
ROH
CALB
+
OMe
OR
OMe
(R)-2a R = Bn
(R)-3a R = iPr
( )-1a
(S)-1a
Scheme 1. CaLB-catalyzed enantioselective transesterification reaction of 1a.
The absolute configuration was assessed for (S)-1a by making a
comparison of the sign of its specific rotation with the data re-
{[
a
]
D = À9.3 (c 0.5, CHCl3)} confirmed the (S)-absolute configura-
ported in the literature, {[
a
]
D = À13.4 (c 1, CHCl3) lit. [ D = À17.3
a
]
tion of 5a already attributed on the basis of Kazlauskas’rule.
The CaLB-catalyzed acylation of 4b with dimethylmalonate un-
der a variety of conditions failed because of the presence of the
bulky isopropyl group. A very low conversion of the starting mate-
rial was observed, but the positive sign of the specific rotation for
(c 1, CHCl3),14 and consequently the (R)-configuration was assigned
to 2a. The (S)- and (R)-configuration of the products were in agree-
ment with the stereochemical preference of CaLB, deduced by
Kazlauskas’rule.15 In a similar way, the treatment of racemic 1a
with CaLB and isopropyl alcohol gave (S)-1a and (R)-3a (Table 1,
entry 2) with excellent enantiomeric excesses.
the recovered 4b ([a]D = +2 (c 0.5, MeOH), Table 2, entry 3), is
indicative of the stereochemical CaLB preference {lit. [a]D = +28.2
(c 0.48, MeOH)}.23
On the basis of these results, we have synthesized 3-(N-t-
butylmalonyl)aminobutyric acid methyl esters ( )-5a–b, introduc-
ing the malonyl chain by either using t-butylmalonyl chloride or
via a coupling reaction between b-amino acid methyl ester hydro-
chlorides 4a–b and mono-t-butyl malonate. The bulky t-butyl ester
group in 5a–b was selected to induce the regioselectivity, driving
the enzymatic resolution exclusively on the smaller methyl ester
(Scheme 2).
CaLB catalyzed the transesterification performed on ( )-5a
(Scheme 3), in the presence of benzyl alcohol, afforded (S)-5a
and (R)-6a in good yields and high enantiomeric excesses (Table 1,
entry 3). Similar results could be observed with isopropyl alcohol
as the nucleophile (Table 1, entry 4). As shown by the data reported
in Table 1, the N-t-butyl-malonyl-b-amino methyl ester 5a proved
to be a good substrate for CaLB.
2.3. Synthesis of peptidomimetic sequence C
The most widely used methods for peptide synthesis involve
chemical steps with selectively protected and activated functions.
Thus, an extensive use of protection and deprotection extra-steps
is required. From the results reported above, showing the useful
resolution of racemic b-amino esters, we hypothesized that malo-
nyl b-amino esters could be adequate substrates for CaLB in the
synthesis of a RGD mimetic sequence. Thus, each easily prepared
N-t-butylmalonyl-b-amino methyl ester ( )-5a or ( )-5b was trea-
ted with p-aminobenzylamine in dioxane in the presence of CaLB
at rt for two days, monitoring the reaction progress by 1H NMR
(Scheme 5).
Following this approach, the regio- and stereoselective synthe-
sis of derivative A was successfully performed. The selective
removal of the methyl, isopropyl, t-butyl or benzyl esters,
The enzyme was then removed and the mixture was separated
by flash chromatography on silica gel, first eluting with ethyl
acetate/cyclohexane 80:20 to obtain the unreacted enantiomer of

98
G. Cardillo et al. / Tetrahedron: Asymmetry 21 (2010) 96–102
Table 1
CaLB-catalyzed enantioselective transesterification reaction of 1a and 5a
Conv.a (%)
50
Yieldb (%)
[a]
D
eed
Ee
c
Entry
1
S.M.
Prod.
( )-1a
(S)-1a
(R)-2a
30
40
À13.4
À7.4
84
>98
531
2
3
4
( )-1a
( )-5a
( )-5a
(S)-1a
(R)-3a
50
50
40
35
42
À14
94
97
235
684
45
À20.2
(S)-5a
(R)-6a
44
30
À13.4
93
>98
+10.4
(S)-5a
(R)-7a
36
20
À9.0
70
91
+16.0
a
b
c
Calculated on the basis of 1H NMR spectra signal integration.
Determined on isolated products, purified by flash chromatography on silica gel.
Values obtained for CHCl3 solutions (see Section 4).
d
Determined by HPLC on a chiral column. For 2a–3a and 6a–7a the ee was calculated after transesterification to methyl esters 1a and 5a, respectively, by treatment with
methanol and pTsOH.
e
Values determined using Sih’s method, from the extent of conversion and enantiomeric excess of the recovered substrate as described and checked via values determined
from the enantiomeric excess of the product.16
single regioisomers. This complete regioselectivity allowed dia-
mine monoprotection to be avoided, and makes this last reaction
particularly interesting. Finally, the t-butyl esters (R)-9a and
(S)-9b were hydrolyzed with H3PO4 in water,25 to afford the RGD
mimetic sequences (R)-10a and (S)-10b (Scheme 6). These com-
pounds were purified on Dowex 50WX2-200 ion exchange resin,
eluting with NH4OH 0.5 M; aqueous solvent was removed by a vac-
uum freeze drying. To evaluate the biological activity of (R)-10a
and (S)-10b in a cellular environment, we tested their ability to
O
O
R
t-Butyl
Cl-H3N+
R
O
malonate
tBuO
NH
O
OMe
HBTU, TEA
DCM/DMF
OMe
( )-4a: R= CH3
( )-4b:
( )-5a-b
R= (CH3)2CH
Scheme 2. Synthesis of ( )-5a–b.
perturb the initial cell attachment mediated by
avb3 integrin and
O
O
a5b1 integrin using cell adhesion assays. These experiments affor-
t-BuO
NH
O
ded IC50 in the micromolar range for both the receptors, thus sug-
gesting that these compounds deserve further studies for their
potential application as dual inhibitors.
(S)-5a
ROH
+
( )5a
OMe
CALB
O
O
(R)-6a R = Bn
(R)-7a R = ipr
t-BuO
NH
O
3. Conclusion
OR
Scheme 3. CaLB-catalyzed enantioselective transesterification reaction of ( )-5a.
We have demonstrated the utility of CaLB-catalyzed enantiose-
lective transesterification or acylation of racemic b-amino esters,
which allows the isolation of interesting pharmacological interme-
diates A, B and C. We have observed that both the malonyl esters
and the b-amino esters can be good substrates for CaLB. Carrying
out an investigation on the reactivity of different alkyl esters, we
decided to introduce the bulky t-butyl ester on the malonyl residue
and the small methyl ester on the b-amino acid, as reported in ( )-
5a, trusting on the ability of the enzyme to exclusively recognize,
under kinetic resolution conditions, the less hindered position. As
a result, an effective selectivity was obtained and the transesterifi-
cation on ( )-5a occurred with good yields and enantiomeric
excesses. In addition we have synthesized in two steps RGD mi-
metic compounds via CaLB-catalyzed amidation reaction, avoiding
protection and deprotection of the intermediate derivatives.
Preliminary studies to establish the ability of these new ligands
to inhibit integrin-mediated cell adhesion gave promising results
and further studies in this area will be reported in due course.
Cl-H3N+
O
O
O
+
OMe
MeO
OMe
O
4a
CALB
TEA
O
Cl-H3N+
O
MeO
+
NH
O
OMe
O
OMe
(S)-4a
(R)-8a
O
t-Butyl
malonate
t-BuO
NH
O
HBTU,
TEA
DCM/DMF
OMe
(S)-5a
4. Experimental
Scheme 4. CaLB-catalyzed enantioselective amidation reaction of 4a and 4b and
conversion of enantiomerically enriched (S)-4a into (S)-5a.
All chemicals were purchased from commercial suppliers and
used without further purification. Lipase B from C. Antarctica, pro-
duced by submerged fermentation of a genetically modified Asper-
gillus oryzae microorganism and adsorbed on a macroporous
acrylic resin (P10,000 U/g, recombinant) was used for enzymatic
reactions. Anhydrous solvents were purchased in sure seal bottles
over molecular sieves and used without further drying. Flash
chromatography was performed on silica gel (230–400 mesh).
malonyl esters (S)-5a or (R)-5b and then with ethyl acetate/meth-
anol/0.5 M ammonium hydroxide 94:5:1 to isolate the amides (R)-
9a or (S)-9b.24 The yields and enantiomeric excesses are reported
in Table 3. The isolated products show that only the benzylamine
function reacted as the nucleophile to afford (R)-9a and (S)-9b as

G. Cardillo et al. / Tetrahedron: Asymmetry 21 (2010) 96–102
99
Table 2
CaLB catalyzed enantioselective amidation reaction of 4a and 4b
Conv.a (%)
44
Yieldb (%)
[a]
D
eed
Ee
c
Entry
1
S.M.
Prod.
( )-4af
(S)-4a
(R)-8a
47
25
+24.2
+21.6
68
>98
410
2
3
( )-4ag
( )-4bh
(S)-4a
(R)-8a
45
5
42
30
+25.0
+21.0
70
>98
418
n.d.
(R)-4b
(S)-8b
60
4
+2
n.d.
7
n.d.
a
b
c
Calculated on the basis of 1H NMR spectra signal integration.
Determined on isolated products, purified by acidic extraction and purification on Dowex 50WX2-200 ion exchange resin.
Values obtained for CHCl3 solutions, except for 4b that was dissolved in methanol.
Determined by HPLC on a chiral column.
d
e
Values determined using Sih’s method, from the extent of conversion and enantiomeric excess of the recovered substrate as described and checked via values determined
from the enantiomeric excess of the product.16
f
Reaction in dioxane for 3 h.
Reaction in DME for 3 h.
Reaction in dioxane for 120 h.
g
h
DOWEXÒ 50WX2-200(H) ion exchange resin was used for purifica-
tion of free amino acids or free amines. NMR Spectra were recorded
with Varian Gemini 200, Mercury Plus 400 or Unity Inova 600 MHz
spectrometers. Chemical shifts were reported as d values (ppm)
relative to the solvent peak of CDCl3 set at d = 7.27 (1H NMR) or
d = 77.0 (13C NMR), CD3OD set at d = 3.31 (1H NMR) or d = 49.0
O
O
R
NH2
t-BuO
NH
O
CALB
dioxane
+
OMe
( )-5a-b
H2N
O
(
13C NMR), D2O set at d = 4.79 (1H NMR). Coupling constants are gi-
O
O
O
ven in hertz. GC–MS analyses were performed on Agilent technol-
ogies GC-6850 MS-5975 (inert XL mass selective detector)
equipped with column HP5 (5% PhMeSi), 30 M Â 0.32 mm,
t-BuO
+
NH
O
t-BuO
NH
O
R
NH
0.25 l, injecting sample with a temperature programme starting
R
OMe
from 50 °C to 280 °C (10 °C/min gradient).
R = Me (R)-9a
R = ipr (S)-9b
R = Me (S)-5a
R = ipr (R)-5b
The enantiomeric excesses of the products were determined by
HPLC analyses performed an HP1100 instrument with UV–vis
detector and equipped with Chiralcel OJ column (25 Â 0.46 cm),
eluted with n-hexane/2-propanol. Optical rotations were mea-
sured in a Perkin–Elmer 343 polarimeter using a 1 dm cuvette
and are referenced to the Na-D line value. Melting points were
NH2
Scheme 5. CaLB-catalyzed enantioselective amidation reaction of 5a and 5b.
determined on
uncorrected.
a Stuart Scientific SMP3 apparatus and are
Table 3
CaLB catalyzed enantioselective amidation reaction of 5a and 5b
Conv.a (%)
35
Yieldb (%)
[a]
D
eed
c
Entry
1
S.M.
Prod.
4.1. Preparation of methyl 3(benzyloxycarbonyl)amino
butanoate 1a
( )-5a
(S)-5a
(R)-9a
45
30
À10.5
80
93
+9.2
To a stirred solution of commercial b-amino acid (10 mmol) and
NaOH (2.2 equiv, 22 mmol, 0.88 g) in water/acetone 1:1 (50 mL),
benzyl chloroformate (1.1 equiv, 11 mmol, 1.57 mL) was added
dropwise at 0 °C. The solution was stirred at room temperature
for 1 h and then acetone was removed under reduced pressure.
The aqueous residue was adjusted to acid pH with 0.5 M HCl and
then extracted twice with CH2Cl2 (2 Â 20 mL), the organic layers
were dried with Na2SO4 and the solvent was removed by rotava-
por. The N-benzyloxycarbonyl derivative was used without further
purification in the following step. Thionyl chloride (15 mmol,
1.1 mL) was added dropwise to stirred methanol (15 mL) at
À15 °C. After 15 min, a solution of the N-benzyloxycarbonyl deriv-
ative in methanol (5 mL) was added in one portion and the mixture
was stirred at room temperature overnight. Removal of the excess
of reagent and of the solvent under reduced pressure afforded
methyl 3(N-benzyloxycarbonyl)amino butanoate 1a in 85% yield.
2
( )-5b
(R)-5b
(S)-9b
38
35
28
À20.5
80
n.d.
+0.5
Calculated on the basis of 1H NMR spectra signal integration.
Determined on isolated products, purified by flash chromatography on silica
a
b
gel.
c
Values obtained for CHCl3 solutions, except for 9b which was dissolved in
methanol.
Determined by HPLC on chiral column. The ee for 9a and 9b was determined on
the acetamido derivatives (see Section 4)
d
O
O
R
HO
NH
O
(R)-9a
or
H3PO4
H2O
NH
(S)-9b
4.1.1. Methyl 3(benzyloxycarbonyl)amino butanoate 1a
White solid: mp 55–57 °C; IR
m
3324, 2949, 2924, 1731, 1684,
H2N
1540, 1439, 1315, 1261, 1203, 1070, 697 cmÀ1
;
1H NMR
(R)-10a [α]D = + 13.4 (c 1, H2O)
(S)-10b [α]D = + 6.5 (c 1, H2O)
(200 MHz, CDCl3) 1.25 (3H, d, J = 7.0 Hz), 2.54 (2H, d, J = 6.0), 3.68
(3H, s), 4.13 (1H, m), 5.09 (2H, s), 5.27 (1H, br s), 7.27–7.39 (5H,
m). 13C NMR (CDCl3, 75 MHz) dC 20.4, 40.5, 44.2, 51.6, 66.6,
Scheme 6. Hydrolysis of t-butyl esters to give RGD mimetics 10a and 10b.

100
G. Cardillo et al. / Tetrahedron: Asymmetry 21 (2010) 96–102
127.0, 128.1, 128.6, 136.7, 155.7, 171.9. LC–ESI-MS: rt 7.08 min m/z
4.4.1. Compound 5a
252 (M+1). Anal. Calcd for C13H17NO4: C, 62.14; H, 6.82; N, 5.57.
White solid: mp 66–69 °C; IR
m
3307, 2924, 2853, 1736, 1648,
;
Found: C, 62.05; H, 6.80; N, 5.58. (S) Enantiomer: [
a
]D = À14.0 (c
1561, 1459, 1375, 1144, 998 cmÀ1 1H NMR (200 MHz, CDCl3)
1, CHCl3), HPLC on Chiralcel OJ (from 99:1 to 90:10 n-hexane/2-
propanol in 15 min, flow 1.0 mL/min, temp. 40 °C): rt 30.0 min.
(R)-Enantiomer: HPLC on Chiralcel OJ (from 99:1 to 90:10 n-hex-
ane/2-propanol in 15 min, flow 1.0 mL/min, temp. 40 °C): rt
27.3 min.
1.24 (3H, d, J = 7.0 Hz), 1.46 (9H, s), 2.52 (2H, d, J = 6.2), 3.18 (2H,
s), 3.68 (3H, s), 4.37 (1H, m), 7.40 (1H, br s). 13C NMR (CDCl3,
75 MHz) dC 19.8, 27.9 (3C), 39.8, 42.0, 42.6, 51.6, 82.3, 164.7,
168.3, 171.7. LC–ESI-MS: rt 4.75 min m/z 282 (M+Na). Anal. Calcd
for C12H21NO5: C, 55.58; H, 8.16; N, 5.40. Found: C, 55.37; H,
8.17; N, 5.39. (S)-Enantiomer: [
a
]
D = À13.4 (c 0.5, CHCl3), HPLC
on Chiralcel OJ (from 99:1 to 95:5 n-hexane/2-propanol in
20 min, flow 0.6 mL/min, temp. 40 °C): rt 21.3 min. (R)-Enantio-
mer: HPLC on Chiralcel OJ (from 99:1 to 95:5 n-hexane/2-propanol
in 20 min, flow 0.6 mL/min, temp. 40 °C): rt 20.9 min.
4.2. General procedure for the CaLB-catalyzed transesterification
of 1a
To a stirred solution of N-protected-b-amino methyl ester
(2 mmol) in anhydrous toluene (70 mL), benzyl or isopropyl alco-
hol (2 equiv, 4 mmol) and CaLB (1 g) were added at room temper-
ature. The solution was stirred at 50 °C and monitored by analysis
of 0.1 mL samples by GC–MS. To finish the reaction, the enzyme
was filtered and the solvent was removed in vacuo. The ester 2a
or 3a was isolated by purification with flash chromatography on
silica gel (cyclohexane/EtOAc, 8:2 as mobile phase).
4.4.2. Compound 5b
Pale yellow oil; IR
m 2966, 2873, 1736, 1655, 1548, 1437, 1369,
1281, 1149 cmÀ1 1H NMR (200 MHz, CDCl3) d 0.94 (6H, d, J = 6.6),
;
1.48 (9H, s), 1.90 (1H, m), 2.54 (2H, d, J = 5.8), 3.22 (2H, s), 3.68 (3H,
s), 4.14 (1H, m), 7.35 (1H, br d). 13C NMR (CDCl3, 75 MHz) dC 18.4,
19.2, 27.9 (3C), 31.3, 36.6, 42.7, 51.5, 51.7, 82.4, 165.0, 168.5, 172.0.
LC–ESI-MS: rt 6.80 min m/z 310 (M+Na). Anal. Calcd for
C14H25NO5: C, 58.52; H, 8.77; N, 4.87. Found: C, 58.38; H, 8.79;
N, 4.89. (S) Enantiomer: HPLC on Chiralcel OJ (from 99:1 to 90:10
n-hexane/2-propanol in 30 min, flow 0.8 mL/min, temp. 40 °C): rt
12.4 min. (R) Enantiomer: [
cel OJ (from 99:1 to 90:10 n-hexane/2-propanol in 30 min, flow
0.8 mL/min, temp. 40 °C): rt 12.8 min.
4.2.1. Benzyl 3-(benzyloxycarbonyl)amino butanoate 2a
Pale yellow oil; IR
m 3338, 2952, 1716, 1530, 1454, 1299, 1060,
739 cmÀ1 1H NMR (200 MHz, CDCl3) 1.25 (3H, d, J = 7.0 Hz), 2.58
;
a]
D = À20.5 (c 1, CHCl3), HPLC on Chiral-
(2H, d, J = 6.0), 4.20 (1H, m), 5.10 (2H, s), 5.12 (2H, s), 5.25 (1H,
br s), 7.30–7.39 (10H, m). 13C NMR (CDCl3, 75 MHz) dC 20.4, 40.3,
44.0, 51.6, 66.6, 128.0 (4C), 128.1 (2C), 128.5 (4C), 136.5 (2C),
155.5, 171.8. LC–ESI-MS: rt 9.39 min m/z 328 (M+1), 350 (M+Na).
Anal. Calcd for C19H21NO4: C, 69.71; H, 6.47; N, 4.28. Found: C,
4.5. General procedure for the CaLB-catalyzed transesterification
of 5a
69.81; H, 6.47; N, 4.29. (R) Enantiomer: [
a]
D = À7.4 (c 2.7, CHCl3).
The reaction was performed following the procedure already re-
ported for 1a. The ester 6a or 7a was isolated by purification with
flash chromatography on silica gel (cyclohexane/EtOAc, 2:8 as mo-
bile phase).
4.2.2. Isopropyl 3-(benzyloxycarbonyl)amino butanoate 3a
Pale yellow oil; IR 3338, 3033, 2975, 2936, 1755, 1725, 1529,
1375, 1245, 1107, 1059, 737 cmÀ1 1H NMR (200 MHz, CDCl3) d
1.25 (9H, m), 2.48 (2H, d, J = 6.0), 4.05 (1H, m), 5.06 (1H, m), 5.10
(2H, s), 5.29 (1H, br s), 7.30 (5H, m). 13C NMR (CDCl3, 75 MHz) dC
20.3, 21.6, 21.7, 40.8, 44.0, 66.4, 67.9, 127.9 (2C), 128.3 (2C),
128.4, 136.5, 155.4, 170.8. LC–ESI-MS: rt 8.80 min m/z 280 (M+1),
302 (M+Na). Anal. Calcd for C15H21NO4: C, 64.50; H, 7.58; N, 5.01.
m
.
4.5.1. Compound 6a
Pale yellow oil; IR
m 3311, 2977, 2825, 1735, 1686, 1561, 1368,
1170, 733 cmÀ1 1H NMR (200 MHz, CDCl3) d 1.15 (3H, d, J = 6.6),
.
1.38 (9H, s), 2.50 (2H, d, J = 5.4), 3.08 (2H, s), 4.32 (1H, m), 5.05
(2H, s), 7.24–7.28 (6H, m). 13C NMR (CDCl3, 75 MHz) dC 19.9, 27.9
(3C), 40.2, 42.2, 42.5, 66.4, 82.4, 128.3 (2C), 128.5 (2C), 128.6,
137.8, 164.2, 169.8, 171.4. LC–ESI-MS: rt 8.5 min m/z 358
(M+Na). Anal. Calcd for C18H25NO5: C, 64.46; H, 7.51; N, 4.18.
Found: C, 64.32; H, 7.58; N, 5.03. (R) Enantiomer: [
0.3, CHCl3).
a]
D = À20.2 (c
4.3. Synthesis of methyl ester hydrochlorides 4a and 4b
Found: C, 64.34; H, 7.51; N, 4.19. (R) Enantiomer: [a]D = +10.4 (c
1, CHCl3).
Thionyl chloride (15 mmol, 1.1 mL) was added dropwise to stir-
red pre-cooled methanol (15 mL) at À15 °C. After 15 min, a solu-
tion of commercial b-amino acid (10 mmol) in methanol (5 mL)
was added in one portion and the mixture was stirred at room
temperature overnight. Removal of the excess of reagent and of
the solvent under reduced pressure afforded the methyl ester
hydrochloride 4 in almost quantitative yield. Analytical data found
for 4a and 4b were in agreement with those reported in the
literature.18,22
4.5.2. Compound 7a
Pale yellow oil; IR
m
.
3303, 2980, 2935, 1731, 1650, 1548, 1370,
1294, 1148, 967 cmÀ1
1H NMR (200 MHz, CDCl3) d 1.24 (6H, d,
J = 6.6), 1.48 (9H, s), 2.49 (2H, d, J = 5.8), 3.17 (2H, s), 4.39 (1H,
m), 5.04 (1H, m), 7.54 (1H, br s). 13C NMR (CDCl3, 75 MHz) dC
19.8, 21.7, 21.8, 27.9 (3C), 40.4, 42.1, 42.5, 68.0, 82.3, 164.7,
168.4, 176.5. LC–ESI-MS: rt 7.16 min m/z 310 (M+Na). Anal. Calcd
for C14H25NO5: C, 58.52; H, 8.77; N, 4.87. Found: C, 58.68; H,
4.4. Preparation of 3(N-tbutylmalonyl)amido esters 5a–b
8.74; N, 4.86. (R) Enantiomer: [a]D = +16.0 (c 0.3, CHCl3).
At first, HBTU (7.5 mmol, 2.84 g) was added to a solution of t-
butyl monomalonate (5 mmol, 0.8 g) and triethylamine (4 equiv,
20 mmol, 2.8 mL) in CH2Cl2/DMF (10:1, 11 mL). After stirring at
room temperature for 20 min, methyl ester 4 (5 mmol) was added
in one portion. The reaction mixture was stirred overnight and
then washed with water (15 mL), 0.5 M HCl (15 mL), 0.5 M NaOH
(15 mL), dried over Na2SO4 and concentrated in vacuo. The residue
obtained was purified by flash chromatography on silica gel (cyclo-
hexane/EtOAc, 2:8) affording 5a–b in 60% yield
4.6. General procedure for the CaLB catalyzed amidation
reaction of 4a and 4b
To a stirred solution of methyl ester hydrochloride 4a or 4b
(2 mmol) and triethylamine (1 equiv, 2 mmol, 0.28 mL) in anhy-
drous dioxane or dimethoxyethane (10 mL) under an inert atmo-
sphere, dimethylmalonate (1.5 equiv, 3 mmol, 0.34 mL) and CaLB
(0.15 g) were added at room temperature. The solution was moni-
tored by analysis of 0.1 mL samples by 1H NMR. To finish the

G. Cardillo et al. / Tetrahedron: Asymmetry 21 (2010) 96–102
101
reaction, enzyme was filtered, the residue was diluted with EtOAc
(15 mL) and it was washed twice with 0.5 M HCl (2 Â 15 mL). The
organic layer was dried over Na2SO4 and concentrated in vacuo.
The residue obtained was purified by flash chromatography on sil-
ica gel (cyclohexane/EtOAc, 2:8) affording 8a–b. The amino esters
4a–b were recovered from the acid aqueous layer by treatment
with DOWEXÒ 50WX2–200(H) ion exchange resin, eluting with di-
luted NH4OH,26 and removing the aqueous phase with a vacuum
freeze dryer.
(of the compound derivatized as acetamide) on Chiralcel OJ (from
90:10 to 70:30 n-hexane/2-propanol in 30 min, flow 1.0 mL/min,
temp. 40 °C): rt 13.8
4.7.2. Compound 9b
Yellow oil; IR
m
3352, 2924, 2852, 1732, 1652, 1558, 1519, 1456,
1260, 1020, 799 cmÀ1
.
1H NMR (200 MHz, CDCl3) d 0.93 (6H, d,
J = 6.8), 1.47 (9H, s), 1.89 (1H, m), 2.46 (2H, m), 3.14 (2H, s), 4.02
(1H, m), 4.28 (2H,d, J = 5.4), 6.38 (1H, br t), 6.63 (2H, d, J = 8.4),
7.06 (2H, d, J = 8.4), 7.48 (1H, bd, J = 9.2). 13C NMR (CD3OD,
75 MHz) dC 16.4, 17.8, 26.3 (3C), 31.2, 37.6, 42.0, 51.9, 61.8, 81.0,
114.7 (2C), 127.7, 127.9 (2C), 146.4, 166.6, 168.8, 171.2. Anal. Calcd
for C20H31N3O4: C, 63.64; H, 8.28; N, 11.13. Found: C, 63.81; H,
4.6.1. Compound 8a
Pale yellow oil; IR
m 3302, 2955, 1736, 1655, 1561, 1439, 1200,
1020 cmÀ1 1H NMR (200 MHz, CDCl3) d 1.23 (3H, d, J = 6.6), 2.54
.
(2H, d, J = 5.6), 3.29 (2H, s), 3.68 (3H, s), 3.74 (3H, s), 4.37 (1H,
m), 7.45 (1H, br s). 13C NMR (CDCl3, 75 MHz) dC 19.9, 39.7, 41.1,
42.2, 51.7, 52.3, 164.1, 169.6, 171.8. LC–ESI-MS: rt min m/z 218
(M+1), 240 (M+Na). Anal. Calcd for C9H15NO5: C, 49.76; H, 6.96;
N, 6.45. Found: C, 49.82; H, 6.98; N, 6.46. (S) Enantiomer: HPLC
on Chiralcel OJ (from 99:1 to 90:10 n-hexane/2-propanol in
15 min, flow 1.0 mL/min, temp. 40 °C): rt 20.2 min. (R) Enantiomer:
[a]D = +21.6 (c 1, CHCl3), HPLC on Chiralcel OJ (from 99:1 to 90:10
n-hexane/2-propanol in 15 min, flow 1.0 mL/min, temp. 40 °C): rt
19.6
8.27; N, 11.11. (R) Enantiomer: [a]D = +0.5 (c 1, CH3OH).
4.8. Hydrolysis of t-butyl esters 9a–b
t-Butyl esters 9a–b (1 mmol) were dissolved in H2O (3 mL) and
H3PO4 (85% in H2O, 7 equiv, 0.5 mL) was slowly added. The reac-
tion was stirred at room temperature for 1 h and organic solvent
was removed under reduced pressure. The residue was purified
by treatment with DOWEXÒ 50WX2–200(H) ion exchange resin,
eluting with diluted NH4OH.
4.6.2. Compound 8b
4.8.1. Compound 10a
Pale yellow oil; IR
m 3303, 2960, 2875, 1740, 1657, 1549, 1438,
Yellow oil; IR
m 3208, 2923, 2853, 1641, 1648, 1547, 1461, 1377,
1261, 1170, 1018, 796 cmÀ1. 1H NMR (200 MHz, CDCl3) d 0.96 (6H,
d, J = 7.0), 1.87 (1H, m), 2.54 (2H, d, J = 5.8), 3.32 (2H, s), 3.68 (3H,
s), 3.76 (3H, s), 4.14 (1H, m), 7.50 (1H, br s). 13C NMR (CDCl3,
75 MHz) dC 18.4, 19.2, 31.3, 36.4, 41.1, 51.6, 51.7, 52.3, 164.4,
169.7, 172.1. LC–ESI-MS: rt 2.86 min m/z 246 (M+1). Anal. Calcd
for C11H19NO5: C, 53.87; H, 7.81; N, 5.71. Found: C, 58.75; H,
7.84; N, 5.72.
721 cmÀ1.1H NMR (200 MHz, D2O) d 1.17 (3H, d, J = 6.6), 2.43(2H, d,
J = 10.5), 3.03 (2H, s), 4.25 (3H, m), 6.91 (2H, d, J = 8.2), 7.17 (2H, d,
J = 8.2). 13C NMR (CD3OD, 75 MHz) dC 20.9, 43.7, 45.0, 55.9, 61.9,
122.7 (2C), 129.8, 130.3 (2C), 147.5, 167.3, 168.0, 176.7. LC–ESI-
MS: rt 1.30 min m/z 294 (M+1). Anal. Calcd for C14H19N3O4: C,
57.33; H, 6.53; N, 14.33. Found: C, 57.53; H, 6.52; N, 14.29. (R)-
Enantiomer: [a]D = +13.4 (c 1, H2O).
4.7. General procedure for the CaLB-catalyzed amidation
reaction of 5a and 5b
4.8.2. Compound 10b
Yellow oil; IR 3300, 2923, 2853, 1652, 1648, 1546, 1461,
1376 cmÀ1 1H NMR (200 MHz, D2O) d 0.87 (6H, d, J = 6.2), 1.73
(1H, m), 2.36 (1H, dd, J = 14.4, 10.0), 2.47 (1H, dd, J = 14.4, 4.8),
2.99 (2H, s), 4.01 (1H, m), 4.13 (1H, d, J = 14.6),), 4.29 (1H, d,
J = 14.6), 6.80 (2H, d, J = 8.2), 7.09 (2H, d, J = 8.2). 13C NMR (D2O + C-
D3OD, 75 MHz) dC 17.6, 19.0, 32.4, 39.4, 43.2, 53.2, 62.6, 117.2 (2C),
129.3, 129.6 (2C), 145.9, 163.1, 168.7, 174.2. LC–ESI-MS: rt
1.28 min m/z 322 (M+1), 665 (2 M+Na). Anal. Calcd for
C16H21N3O4: C, 59.80; H, 7.21; N, 13.08. Found: C, 59.99; H, 7.23;
m
.
To a stirred solution 5a or 5b (2 mmol) in anhydrous dioxane
(10 mL) under nitrogen atmosphere, p-aminobenzylamine
(1.5 equiv, 3 mmol, 0.34 mL) and CaLB (0.2 g) were added at room
temperature. The solution was monitored by analysis of 0.1 mL
samples by 1H NMR. To finish the reaction, enzyme was filtered,
the solvent was concentrated in vacuo and the residue obtained
was purified by flash chromatography on silica gel eluting with
cyclohexane/EtOAc, 8:2 to recover 5a–b then ethylacetate/metha-
nol/0.5 M ammonium hydroxide 94:5:1 to recover 9a–b. To obtain
N, 13.09. (S)-enantiomer: [a]D = +6.5 (c 1, H2O).
a
good separation on HPLC chiral column, acetic anhydride
Acknowledgements
(1.5 equiv, 3 mmol, 0.28 mL) and pyridine (1.5 equiv, 3 mmol,
0.24 mL) were added to 9a–b and stirred for 1 h. The mixture
was then diluted with water (5 mL), extracted with EtOAc (5 mL),
dried over Na2SO4 and the solvent was removed under reduced
pressure, to give N-acetamide samples for HPLC analysis.
This study has been carried out with the fundamental contribu-
tion of ‘Fondazione del Monte di Bologna e Ravenna’. We also
thank MAE (Italian Minister for Foreign Affair, General Direction
for the Cultural Promotion and Cooperation) for financial support
to a bilateral projects of between Italy and Mexico. MIUR (PRIN
2006 prot.n. 2006030449_003) and University of Bologna (Strate-
gic project ID 450) are also acknowledged for financial support.
Mr. Andrea Garelli is gratefully acknowledged for the LC–ESI-MS
analysis.
4.7.1. Compound 9a
Yellow oil; IR
m 3304, 3075, 2977, 2931, 1727, 1658, 1518, 1452,
1369, 1150, 951, 832 cmÀ1. 1H NMR (200 MHz, CD3OD) d 1.17 (3H,
d, J = 7.0), 1.45 (9H, s), 2.28 (1H, dd, J = 13.8, 6.8), 2.45 (1H, dd,
J = 13.8, 6.2), 3.12 (2H, s), 4.19 (2H, s), 4.23 (1H, m), 6.66 (2H, d,
J = 8.2), 7.02 (2H, d, J = 8.2). 13C NMR (CD3OD, 75 MHz) dC 20.1,
28.2 (3C), 43.2, 43.9, 44.7, 48.6, 82.8, 116.5 (2C), 129.1, 129.8
(2C), 147.9, 167.8, 169.1, 172.6 LC–ESI-MS: rt 3.15 min m/z 350
(M+1). Anal. Calcd for C18H27N3O4: C, 61.87; H, 7.79; N, 12.03.
Found: C, 61.86; H, 7.81; N, 12.04. (S) Enantiomer: HPLC (of the
compound derivatized as acetamide) on Chiralcel OJ (from 90:10
to 70:30 n-hexane/2-propanol in 30 min, flow 1.0 mL/min, temp.
References
1. (a) Hynes, R. O. Cell 1992, 69, 11–25; (b) Curley, G. P.; Blum, H.; Humphries, M.
J. Cell Mol. Life Sci. 1999, 56, 427–441; (c) Gottschalk, K.-E.; Kessler, H. Angew.
Chem., Int. Ed. 2002, 41, 3767–3773.
2. (a) Henry, C.; Moitissier, N.; Chapleur, Y. Mini, Rev. Med. Chem. 2002, 2, 531–
542; (b) Zimmermann, D.; Guthohrlein, E. W.; Malsevic, M.; Sewald, K.; Wobbe,
L.; Heggermann, C.; Sewald, N. ChembioChem 2005, 6, 272–276; (c) Phillips, D.
R.; Conley, P. B.; Sinha, U.; Andre, P. J. Thromb. Haemost. 2005, 3, 1577–1589; (d)
40 °C): rt 12.75 min. (R) Enantiomer: [a]D = +9.2 (c 1, CHCl3), HPLC

102
G. Cardillo et al. / Tetrahedron: Asymmetry 21 (2010) 96–102
Marinelli, L.; Meyer, A.; Heckmann, D.; Lavecchia, A.; Novellino, E.; Kessler, H. J.
1830–1838; (d) Gonzalez-Sabin, J.; Gotor, V.; Rebolledo, F. Tetrahedron:
Asymmetry 2002, 13, 1315–1320; (e) Messina, F.; Botta, M.; Corelli, F.;
Schneider, M. P.; Fazio, F. J. Org. Chem. 1999, 64, 3767–3769; (f) Torre, O.;
Busto, E.; Gotor-Fernandez, V.; Gotor, V. Adv. Synth. Catal. 2007, 349, 1481–
1488; (g) González-Sabín, J.; Lavandera, I.; Rebolledo, F.; Gotor, V. Tetrahedron:
Asymmetry 2006, 17, 1264–1274; For CaLB catalyzed acylations leading to b-
amino acid derivatives see: (h) Sánchez, V. M.; Rebolledo, F.; Gotor, V.
Tetrahedron: Asymmetry 1997, 8, 37–40; (i) Gedey, S.; Liljeblad, A.; Fülöp, F.;
Kanerva, L. T. Tetrahedron: Asymmetry 1999, 10, 2573–2581; (l) Gedey, S.;
Liljeblad, A.; Lázár, L.; Fülöp, F.; Kanerva, L. T. Tetrahedron: Asymmetry 2001, 12,
105–110; (m) Gedey, S.; Liljeblad, A.; Lázár, L.; Fülöp, F.; Kanerva, L. T. Can. J.
Chem. 2002, 80, 565–570; (n) Solymár, M.; Liljeblad, A.; Lázár, L.; Fülöp, F.;
Kanerva, L. T. Tetrahedron: Asymmetry 2002, 13, 1923–1928; (o) Weib, M.;
Groger, H. Synlett 2009, 1251–1254; (p) Xiang-Guo, L.; Kanerva, L. T. Org. Lett.
2006, 8, 5593–5596.
Med. Chem. 2005, 48, 4204–4207; (e) Tucker, G. C. Curr. Oncol. Rep. 2006, 2, 96–
103.
3. Xiong, J.-P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Frach, M.; Goodman, S. L.;
Arnaout, M. A. Science 2002, 296, 151–155. PDB file deposited with code 1L5.
4. (a) Cacciari, B.; Spalluto, G. Curr. Med. Chem. 2005, 12, 51–70; (b) Kubota, D.;
Ishikawa, M.; Yamamoto, M.; Murakami, S.; Hachisu, M.; Katano, K.; Ajito, K.
Bioorg. Med. Chem. 2006, 14, 2089–2108; (c) Perkins, J. J.; Duong, L. T.;
Fernandez-Metzler, C.; Hartman, G. D.; Kimmel, D. B.; Leu, C.-T.; Lynch, J. J.;
Prueksaritanont, T.; Rodan, G. A.; Rodan, S. B.; Duggan, M. E.; Meissner, R. S.
Bioorg. Med. Chem. Lett. 2003, 13, 4285–4288; (d) Biltresse, S.; Attolini, M.; Dive,
G.; Cordi, A.; Tucker, G. C.; Marchand-Brynaert, J. Bioorg. Med. Chem. 2004, 12,
5379–5393; (e) Zartman, A. E.; Duong, L. T.; Fernandez-Metzler, C.; Hartman, G.
D.; Leu, C.-T.; Prueksaritanont, T.; Rodan, G. A.; Rodan, S. B.; Duggan, M. E.;
Meissner, R. S. Bioorg. Med. Chem. Lett. 2005, 15, 1647–1650; (f) van Well, R. M.;
Overkleeft, H. S.; van der Marel, G. A.; Bruss, D.; Thibault, G.; de Groot, P. G.; van
Boom, J. H.; Overhand, M. Bioorg. Med. Chem. Lett. 2003, 13, 331–334.
5. (a) Gentilucci, L.; Cardillo, G.; Tolomelli, A.; De Marco, R.; Garelli, A.;
Spampinato, S.; Spartà, A.; Juaristi, E. ChemMedChem 2009, 4, 517–523; (b)
Gentilucci, L.; Cardillo, G.; Tolomelli, A.; Spampinato, S.; Sparta, A.; Squassabia,
F. Eur. J. Org. Chem. 2008, 4, 729–735; (c) Benfatti, F.; Cardillo, G.; Fabbroni, S.;
Galzerano, P.; Gentilucci, L.; Juris, R.; Tolomelli, A.; Baiula, M.; Spartà, A.;
Spampinato, S. Bioorg. Med. Chem. 2007, 15, 7380–7390; (d) Gentilucci, L.;
Cardillo, G.; Squassabia, F.; Tolomelli, A.; Spampinato, S.; Sparta, A.; Baiula, M.
Bioorg. Med. Chem. Lett. 2007, 17, 2329–2333; (e) Benfatti, F.; Cardillo, G.;
Fabbroni, S.; Gentilucci, L.; Perciaccante, R.; Tolomelli, A.; Baiula, M.;
Spampinato, S. Tetrahedron: Asymmetry 2006, 17, 167–170.
6. (a) Cardillo, G.; Tomasini, C. Chem. Soc. Rev. 1996, 117–128; (b)Enantioselective
Synthesis of b-Amino acids; Juaristi, E., Ed., 1st ed.; Wiley VCH: New York, 1997;
(c) Enantioselective Synthesis of b-Amino acids; Juaristi, E., Soloshonok, V. A., Eds.,
2nd ed.; John Wiley & Sons: New York, 2005; (d) Kiss, L.; Forro, E.; Martinek, T.
A.; Bernath, G.; De Kimpe, N.; Fulop, F. Tetrahedron 2008, 64, 5036–5043.
7. (a) Liljeblad, A.; Kanerva, L. T. Tetrahedron 2006, 62, 5831–5854; (b)
Faulconbridge, S. J.; Holt, K. E.; Sevillano, L. G.; Lock, C. J.; Tiffin, P. D.;
Tremayne, N.; Winter, S. Tetrahedron Lett. 2000, 41, 2679–2681; (c) Forro, E.;
Fulop, F. Chem. Eur. J. 2007, 13, 6397–6401; (d) Forro, E.; Fulop, F. Mini. Rev. Org.
Chem. 2004, 1, 93–102. and reference therein cited.
12. (a) Klibanov, A. M. Acc. Chem. Res. 1990, 23, 114–120; (b)Enzymatic Reactions in
Organic Media; Koskinen, A. M. P., Klibanov, A. M., Eds.; Blackie Academic
Professional: New York, 1996.
13. (a) Ottoson, J.; Fransson, L.; King, J. W.; Hult, K. Biochim. Biophys. Acta 2002,
1594, 325–334; (b) Tasnadi, G.; Forro, E.; Fülöp, F. Tetrahedron: Asymmetry
2007, 18, 2841–2844; (c) Anderson, E. M.; Larsson, K. M.; Kirk, O. Biocatal.
Biotransform. 1998, 16, 181–204; (d) De Goede, A. T. J. W.; Benckhuijsen, W.;
Van Rantwijk, F.; Maat, L.; Van Bekkum, H. Recl. Trav. Chim. Pays-Bas 1993, 112,
567–572; (e) Jain, N.; Kumar, A.; Chauhan, S.; Chauhan, S. M. S. Tetrahedron
2005, 61, 1015–1060; (f) Madeira Lau, R.; Van Rantwijk, F.; Seddon, K. R.;
Sheldon, R. A. Org. Lett. 2000, 2, 4189–4191.
14. Podlech, J.; Seebach, D. Liebigs Ann. 1995, 7, 1217–1228.
15. Kazlaukas, R. J.; Weissfloch, A.; Rappaport, A. T.; Cuccia, L. A. J. Org. Chem. 1991,
56, 2656–2665.
16. Chen, C. S.; Wu, S. H.; Girdaukas, G.; Sih, C. J. J. Am. Chem. Soc. 1987, 109, 2812–
2817.
17. Cordova, A.; Janda, K. D. J. Org. Chem. 2001, 66, 1906–1909.
18. Luna, A.; Alfonso, I.; Gotor, V. Org. Lett. 2002, 4, 3627–3629.
19. Juaristi, E.; Escalante, J.; Lamatsch, B.; Seebach, D. J. Org. Chem. 1992, 57, 2396–
2398.
20. It is well known that solvents may influence the enantioselectivity of lipases
when applied to kinetic resolutions of organic compounds. For references see:
(a) Secundo, F.; Riva, S.; Carrea, G. Tetrahedron: Asymmetry 1992, 3, 267–280;
(b) Wescott, C. R.; Klibanov, A. M. J. Am. Chem. Soc. 1993, 115, 1629–1631; (c)
Overbeeke, P. L. A.; Jongejan, J. A.; Heijnen, J. J. Biotechnol. Bioeng. 2000, 70,
278–290; (d) Anthonsen, T.; Jongejan, J. A. Methods Enzymol. 1997, 286, 473–
495.
21. Peukert, S.; Sun, Y.; Zhang, R.; Hurley, B.; Sabio, M.; Shen, X.; Gray, C.; Dzink-
Fox, J. A.; Tao, J.; Cebula, R.; Wattanasin, S. Bioorg. Med. Chem. Lett. 2008, 18,
1840–1844.
22. Micovic, I. V.; Roglic, G. M.; Ivanovic, M. D.; Dosen-Micovic, L.; Kiricojevic, V. D.;
Popovic, J. B. J. Chem. Soc., Perkin Trans. 1 1996, 2041–2050.
8. (a) Dalko, P. I.; Moisan, L. Angew. Chem., Int. Ed. 2004, 43, 5138–5175; (b) Xu, L.-
W.; Xia, C.-G. Eur. J. Org. Chem. 2005, 633–639; (c) Davies, S. G.; Smith, A. D.;
Price, P. D. Tetrahedron: Asymmetry 2005, 17, 2833–2891; (d) Ting, A.; Schaus, S.
E. Eur. J. Org. Chem. 2007, 5797–5815.
9. (a) Schmidt, R. D.; Verger, R. Angew. Chem., Int. Ed. Engl. 1998, 37, 1608–1633.
and reference therein cited; (b) von Raitwijk, F.; Sheldon, R. A. Tetrahedron
2004, 60, 501–519. and reference therein cited; (c) Wiktelius, D. Synlett 2005,
2113–2114; (e) Ghanem, A. Tetrahedron 2007, 63, 1721–1754. and references
therein cited; (f) Hidalgo, A.; Bornscheuer, U. T. Biochem. Pharm. Biotechnol. Ind.
2007, 159–179. and references therein cited; (g) Alfonso, I.; Gotor, V. Chem. Soc.
Rev. 2004, 33, 201–209; (h) Gotor, V. Bioorg. Med. Chem. 1999, 7, 2189–2197; (i)
van Rantwijk, F.; Hacking, M. A. P. J.; Sheldon, R. A. Monatsh. Chem. 2000, 131,
549–569.
23. Caputo, R.; Cassano, E.; Longobardo, L.; Palumbo, G. Tetrahedron 1995, 51,
12337–12342.
24. Lepri, L.; Cincinelli, A., 2nd ed.. In Encyclopedia of Chromatography; Cazes, J., Ed.;
Taylor & Francis Group: Boca Raton (USA), 2005; Vol. 1, pp 75–84.
25. Li, B.; Berliner, M.; Buzon, R.; Chiu, C. K.-F.; Colgan, S. T.; Kaneko, T.; Keene, N.;
Kissel, W.; Le, T.; Leeman, K. R.; Marquez, R.; Morris, L.; Newell, S.;
Wunderwald, M.; Witt, J.; Weaver, B.; Zhang, Z.; Zhang, Z. J. Org. Chem. 2006,
71, 9045–9050.
10. Flores-Sanchez, P.; Escalante, J.; Castillo, E. Tetrahedron: Asymmetry 2005, 16,
629–634.
11. For a general review see: (a) Gotor-Fernandez, V.; Busto, E.; Gotor, V. Adv.
Synth. Catal. 2006, 348, 797–812; For primary amine acylation see: (b)
Skupinska, K. A.; McEachern, E. J.; Baird, I. R.; Skerlj, R. T.; Bridger, G. J. J. Org.
Chem. 2003, 68, 3546–3551; (c) Garcia-Urdiales, E.; Rios-Lombardia, N.;
Mangas-Sanchez, J.; Gotor-Fernandez, V.; Gotor, V. ChemBioChem 2009, 10,
26. Purification of amino esters on ion exchange resin, eluting with 0.5 M NH4OH
caused, in some cases, partial hydrolysis of the ester moiety.