Chemoenzymatic resolution of epimeric cis 3-carboxycyclopentylglycine derivatives

Article abstract of DOI:10.1016/j.tet.2006.02.006

Epimeric 3-carboxycyclopentylglycines (+)-10/(-)-10 and (+)-11/(-)-11 were efficiently prepared by the way of a sequence of Diels-Alder and retro-Claisen reactions. The synthesis incorporates a concise and inexpensive chemoenzymatic resolution of racemic

Full text of DOI:10.1016/j.tet.2006.02.006

Tetrahedron 62 (2006) 3502–3508
Chemoenzymatic resolution of epimeric cis
3-carboxycyclopentylglycine derivatives
Chiara Cabrele,b Francesca Clerici,a Raffaella Gandolfi,a Maria Luisa Gelmi,a,
Francesco Molinaric and Sara Pellegrinoa
*
a
`
`
Istituto di Chimica Organica ‘A. Marchesini’, Facolta di Farmacia, Universita di Milano, Via Venezian 21, I-20133 Milano, Italy
b
¨ ¨
Fakultat fur Chemie und Pharmazie, Universitat Regensburg, Universitatsstrasse 31, D-93053 Regensburg, Germany
¨
¨
c
`
Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Universita di Milano, Via Celoria 2, I-20133 Milano, Italy
Received 28 November 2005; revised 18 January 2006; accepted 2 February 2006
Abstract—Epimeric 3-carboxycyclopentylglycines (C)-10/(K)-10 and (C)-11/(K)-11 were efficiently prepared by the way of a sequence
of Diels–Alder and retro-Claisen reactions. The synthesis incorporates a concise and inexpensive chemoenzymatic resolution of racemic
compounds 4,5a, the N,O-protected derivatives of amino acids 10,11. Systematic screening with different enzymes and microorganisms was
performed to select a very efficient catalyst for the separation of the racemic mixtures. The reaction conditions allowing deprotection of both
ester and amino functions and to avoiding epimerization processes were studied. Enantiomers (i.e., (C)-10/(K)-10 and (C)-11/(K)-11)
were obtained in high enantiopurity. The absolute configuration of all stereocenters was unequivocally assigned.
q 2006 Elsevier Ltd. All rights reserved.
1. Introduction
(Scheme 3) characterized by the cis relationship between the
two carbon residues.7
Unnatural amino acids have been recognized as major tools
for the preparation of peptidomimetics with enhanced
biological activity and proteolytic resistance. One of the
modern approaches towards the synthesis of modified
peptides is the incorporation of non-proteinogenic amino
acids into the peptide backbone.1 This is a powerful strategy
to overcome the problem that peptides are generally flexible
and do not take the defined conformation necessary for
function.
Chiral synthesis is a powerful tool to produce a chiral
compound, but there are some limitations such as the
expensive chiral reagents, the necessity to repeat the
synthetic protocol twice when both enantiomers are needed,
and most of all and in most cases, the difficulty of separating
the diasteromers. For these reasons, one alternative
approach to overcome all these limitations is the enzymatic
separation of racemic compounds and, in particular, of
amino acids. In fact, isolated enzymes or microbial cells can
catalyse the stereoselective hydrolysis of amino acids with
N-protected amide group or O-protected ester group.8 High
selectivity and mild and safe conditions are typical. In
particular, the use of whole microbial cells often shows
good performance with regard to stability and selectivity
and is advantageous to access intracellular esterase
activity.9
3-Carboxycyclopentylglycines are very promising amino
acids to be used in this field since they are non-proteinogenic
amino acids containing conformational constraints. Further-
more, the skeleton of both 2-aminoadipic acid and 2-amino-
pimelic acid, two natural amino acids of biological
importance, is included in their structure.2–5 Heterosubstituted
3-carboxycyclopentylglycines were prepared and their bio-
logical activity toward neuraminidase was evaluated.6
Recently, we reported on the chiral preparation of epimeric
1S,3R,10R- and 1S,3R,10S-(benzoylamino-ethoxycarbonyl-
methyl)cyclopentanecarboxylic acid derivatives 6 and 7
The importance of amino acid chirality in biological
interactions is well known. The impossibility to forecast a
priori the biological activity of a single stereoisomer
requests the availability of both pure enantiomers. For this
reason, considering the potential biological interest of the
above amino acids and the perspective of using the
3-carboxycyclopentylglycines in peptide syntheses, racemic
epimers (G)-4 and (G)-5a were prepared and the synthetic
Keywords: 3-Carboxycyclopentylglycines; Enzymatic resolution; Asper-
gillus melleus; Absolute configuration.
*
Corresponding author. Tel.: C390250314481; fax: C390250314476;
e-mail: marialuisa.gelmi@unimi.it
0040–4020/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.02.006

C. Cabrele et al. / Tetrahedron 62 (2006) 3502–3508
3503
protocol for their preparation was considerably improved.10
The chemoenzymatic resolution of each epimer (G)-4 and
(G)-5a was planned. Systematic screening with different
enzymes and microorganisms was performed to select a
very efficient catalyst for the separation of the racemic
mixtures.
b-Ketoester (G)-exo-3 was then transformed into a mixture
of epimeric cyclopentylglycines (G)-4 and (G)-5a (1:1;
82%) by a base catalysed retro-Claisen reaction (pyridine/
water at reflux; 1:1) which ensured the cis relationship
between the two carbon residues on the ring (Scheme 2).
2.1. Enzymatic resolution
Furthermore, the absolute configuration of each stereocenter
in the above amino acids was assigned taking advantage of
the availability of chiral derivatives 6 and 7.
Aiming to obtain the enantiopure amino acids, an enzymatic
study was performed starting from racemic compound
(G)-5a. Twelve enzymes (lipases, acylases, proteases) and
fifty microorganisms (yeasts, bacteria) belonging to differ-
ent genera were tested. The screening was carried out using
miniaturized systems and TLC as analytical technique
allowing an evaluation of the activity of a large number of
biocatalysts in a short time. Different enzymes and
microbial cells were able to selectively hydrolyze the ester
function of the cyclopentylglycine 5a to obtain compound
S-9. The enantiomeric excess was evaluated by chiral HPLC
and the results are summarized in Table 1. The hydrolysis of
the amide function was never observed.
2. Results
The key starting material for the preparation of the epimeric
3-carboxy-cyclopentylglycines 4,5a is the racemic ethyl
2-benzoylamino-3-oxo-bicylo[2.2.1]heptane-2-carboxylate
(G)-exo-3 (Scheme 1).
NHCOPh
OCO2Et
EtO2C
+
As shown in Table 1, the best results were achieved by using
PPL and acylase from Aspergillus melleus. In both cases it
was possible to obtain the complete resolution of racemic
(G)-5a. After the extraction of the reaction mixture, the
bicarboxylic acid (K)-9 was separated from the ester
(K)-5a (50%, [a]2D5 K22) by simple crystallization from
dichloromethane. (Scheme 2) Enantiopure acid (K)-9
([a]2D5 K14.5) was obtained in 40% yield. The same results
were obtained starting from (G)-4. Enantiopure acid (K)-8
([a]2D5 K20) was separated from the ester (K)-4 (50%, [a]2D5
K11) by crystallization. (Scheme 2) Interestingly, a
different reaction rate was observed starting from two
epimers, with the 1S*,03R*,10R* epimer being more reactive
(24 h) than 1S*,3R*,1 S* one (48 h).
CO2Et
PhCONH
(±)-exo-1
OCO2Et
(±)-endo-1
Na2CO3
EtOH
1. Na2CO3/EtOH
2. PCC/CH2Cl2
42%
62%
EtO2C
EtO2C
PCC
PhCONH
CH2Cl2
95%
PhCONH
O
OH
(±)-exo-2
(±)-exo-3
Scheme 1.
The acylase from Aspergillus melleus was also most active
also at low concentration (5 mg/mL) for both racemic
compounds. For this reason this enzyme was selected for the
semi-preparative resolution (250 mg) of both (G)-4 and
(G)-5a (Scheme 2).
The synthesis of the corresponding methyl ester, was
already reported by us11 but a more efficient protocol is
described here which allowed to minimize us the reaction
steps, to avoid chromatographic purifications and to
improve the reaction yield (53% instead of 35%).
One problem of biotransformations carried out with
enzymes is the formation of emulsions during the work-up
of the crude reaction mixture. For this reason we screened
whole cells and the best results were found using Pichia
etchellsii MIM and Saccharomyces cerevisiae ZEUS.
(Table 1) This result appears very interesting in the
perspective of scaling up the process to the gram scale.
A mixture of the norbornane derivatives (G)-exo-1 and
(G)-endo-1 (70:30) was the starting material for the
preparation of 3. (Scheme 1) The selective deprotection of
the C-3 carbonate of the exo compound, using sodium
carbonate in ethanol at room temperature, gave the
3-hydroxy derivative (G)-exo-2 which was easily separated
from the endo carbonate (G)-1. Compound (G)-exo-2 was
isolated in 42% yield and than oxidized to ketone (G)-exo-3
(95%) using pyridinium chlorocromate (PCC). Alterna-
tively, the oxidation reaction was performed directly on the
mixture of alcohol (G)-exo-2 and carbonate (G)-endo-1.
Recently, we have synthesised the chiral epimeric cyclo-
pentylglycines (C)-6 and (C)-7. Since their separation had
been achieved by semi-preparative HPLC, which required
the use of considerable amounts of solvent and long times,
we planned their separation using enzymes. This target was
successfully achieved using a lipase from Candida
cylindracea, which gave the best selectivity. In fact, this
enzyme is both regioselective and stereoselective. It
hydrolysed only the methyl ester function of stereoisomer
(C)-7 in 72 h with a diastereomeric excess of 97% and
molar conversion of 49%. After column chromatography it
was easily possible to obtain the pure compound (K)-5b
Ethyl 2-phenyl-5-oxo-oxazol-4-methylene-carbonate and
cyclopentadiene were the starting reagents used for the
synthesis of norbornane ring via a Diels–Alder reaction.
Here, we prepared the ketone (G)-exo-3 in four steps and
53% overall yield. This protocol allowed us to avoid one
chromatographic step and, importantly, to perform an easier
separation of carbonate endo-1 from the ketone exo-3.

3504
C. Cabrele et al. / Tetrahedron 62 (2006) 3502–3508
NH3+ Cl-
H
NHCOPh
NH3 + Cl-
R
H
S
R
H
CO2H
CO2H
HO2C
H
H
H
R
S
HCl 6M
95%
R
S
R
S
H CO2H
H CO2H
HO2C
H
(-)-10
(+)-8
(+)-10
BBTO
toluene
HCl 6M
95%
95%
NHCOPh
R
H
NHCOPh
R*
NHCOPh
H
S
H
CO2Et
CO2Et
HO2C
H
H
H
Acylase
R*
S*
R
S
S
R
+
H CO2H
H CO2H
HO2C
H
(±)-4
(-)-4
(-)-8
40%
50%
EtO2C
Py/H2O
82%
PhCONH
O
EtO2C
H
CO2Et
H
HO2C
H
S*
NHCOPh
R
S
(±)-exo-3a
PhCONH
NHCOPh
H
H
H
Acylase
R
S
S
R
R*
S*
+
H CO2H
HO2C H
H CO2H
(±)-5a
(-)-5a
(-)-9
40%
50%
95%
BBTO
toluene
HCl 6M
95%
CO2H
CO2H
HO2 C
H
R
R
Cl- + H3N
H
S
H
NH3+ Cl-
PhCONH
H
H
H
HCl 6M
S
R
S
R
R
S
HO2C H
HO2C
H
95%
H CO2H
(+)-11
(-)-11
(+)-9
Scheme 2.
(30%, [a]2D5 K8) and the methyl ester derivative (C)-6
(40%) (Scheme 3). Considering that both epimer 6 and 7
have the same 1-S stereocenter, we conclude that the remote
amino acid function controls the selectivity of the enzyme.
The epimerization was also observed by hydrolysis of the
ester function of (G)-5a with BBr3 (entry 3).12 Attempts to
first hydrolyze the amide function using Na2O213 gave only
the hydrolysis of the ester function with epimerization
(entry 4). Finally, the best result was achieved by using
a catalytic amount of bis-tributyltin oxide (BBTO)14 in
refluxing toluene (entry 5). Compound (G)-9 was obtained
from 5a with a small amount of 8 (7%). The direct
hydrolysis of both ester and amide function of (G)-5a with
6 M HCl at reflux was possible and a mixture of amino acids
11 and 10 was obtained due to the epimerization process
(entry 6). Instead, the hydrolysis (6 M HCl) of the amide
function of the free bicarboxylic compounds did not
undergo epimerization. In fact, starting from the mixture
of acids 9/8 (93:7) the mixture of amino acids 11 and 10 was
obtained in the same ratio (entry 7).
2.2. Deprotection of the amino acid function
The deprotection of the ester and amide functions in
compounds 4,5a was critical because a partial epimerization
of the amino acid C-a occurred when performing the above
hydrolysis in standard conditions (see below). To avoid this
problem, different reaction conditions were tested starting
from the racemic (G)-4 and (G)-5a. Results are summar-
ized in Table 2 (Scheme 2).
The selective deprotection of the ester function of both
(G)-4 and (G)-5a with LiOH in MeOH at room temperature
gave a mixture of epimeric amino acids (G)-8 and (G)-9,
respectively, in the ratio indicated in the Table (entries 1, 2).
We can conclude that it is impossible to completely avoid
the epimerization process in compounds in which both

C. Cabrele et al. / Tetrahedron 62 (2006) 3502–3508
3505
Table 1. Hydrolysis of ester of racemic compound (G)-5a with different microbial cells or isolated enzymes
Microorganism
ee S (%)a
ee P (%)a
Molar conversion (%)a
Ea
Time (h)
Streptomyces sp. 27
Streptomyces sp. 52
Streptomyces sp. 103
Streptomyces sp. 34
Pichia etchellsii MIM
Pichia etchellsii MIM
Saccaromyces cerevisiae Zeus
Saccaromyces cerevisiae Zeus
Geobacillus thermoleovorans ATCC
43513
74
22
24
4
36
O99
66
90
51
O99
68
O99
80
O99
/
42
45
30
29
6
26
56
40
/
42
3,8
O200
5,5
O200
65
O200
/
3,6
24
48
24
144
4
24
4
24
24
O99
41
49
Papaine
Papaine
93
93
84
95
84
92
83
O99
99
50
54
51
54
49
55
44
50
94
84
O200
84
69
78
O200
O200
24
96
24
96
4
24
4
24
O99
O99
O99
88
O99
77
Acylase from Aspergillus melleus
Acylase from Aspergillus melleus
Acylase from Asp. sp.
Acylase from Asp. sp.
Porcine pancreatic lipase
Porcine pancreatic lipase
O99
a Conversion and enantioselectivity factor (E) calculated from the ee of the substrate (ee S) and the product (ee P).
compounds (C)-8 (de 86%) and (C)-9 (de 86%) were
obtained, respectively.
NHCOPh
RO2C
H
R CO2R
H
SNHCOPh
H
H
R
S
R
S
+
The hydrolysis of the amide function of the single
stereomers (C)-8, (K)-8, (C)-9 and (K)-9 to the
corresponding amino acids (K)-10 (de 86%, [a]2D5 K4),
(C)-10 ([a]2D5 C5), (K)-11 (de 86%, [a]2D5 K10) and
(C)-11 ([a]2D5 C11) was performed using 6 M HCl.
H CO2Me
H CO2Me
(+)-7
(+)-6
lipase
30%
50%
The absolute configuration of all stereocenters of bicar-
boxylic acids 8 and 9 was unequivocally assigned by
correlation of the [a] values with authentic samples. We
found that starting both from 7,7 by hydrolysis, and from
(G)-5a, by enzymatic resolution, the same epimer (K)-S-9
was obtained. The same S selectivity of the enzymes was
observed in the case of compound (G)-4. Epimer (K)-S-8
was obtained, having the opposite optical rotation with
respect to that found for compound obtained through the
chiral synthesis.
RO2C
H
SNHCOPh
H
Me
R
S
R =
H CO2H
(-)-5b
Me
Ph
Me
Scheme 3.
the amino and the carboxy groups are protected, probably
because of the high acidity of the amino acid a-hydrogen.
Instead, starting from the N-protected acids this problem is
overcome.
3. Conclusion
In conclusion, a highly efficient protocol for the preparation
of four enantiopure stereoisomers of the 3-carboxycyclo-
pentylglycine derivatives 10,11 was achieved starting from
the racemic 3-oxo-norbornaneamino acid derivative 3. The
enzymatic separation of protected cyclopentylglycine
With this information in mind, the chiral esters (K)-4 and
(K)-5a were hydrolyzed with BBTO. Bicarboxylic
Table 2. Chemical deprotection of amino acid function
Entry
Reagent
Reaction conditions
Yield (%)
Productsa (ratio)
1
2
3
4
5
6
7
(G)-4
(G)-5a
(G)-5a
(G)-5a
(G)-5a
(G)-5a
(G)-9/8 (93:7)
LiOH/MeOH, 25 8C
LiOH/MeOH, 25 8C
BBr3/CH2Cl2, 25 8C
Na2O2/H2O, 50 8C
BBTO/toluene/reflux
6 M HCl, reflux
80
80
75
80
95
95
95
8/9 (86:14) b
9/8 (86:14)b
9/8 (85:15) b
9/8 (80:20) b
9/8 (93:7) b
11/10 (87:13) c
11/10 (93:7) c
6 M HCl, reflux
a Isolated compounds.
b HPLC determination.
13C NMR determination.
c

3506
C. Cabrele et al. / Tetrahedron 62 (2006) 3502–3508
derivatives, obtained in high enantiomeric purity, was
assured using the acylase from Aspergillus melleus, which
also operates on semipreparative scale.
a mixture of THF/AcOEt (7.5 mL, 1:1). The organic layer
was separated and dried over Na2SO4 giving a mixture of
the two diastereomeric amino acid derivatives 4 and 5a (1:1,
260 mg, 82%). Compounds (G)-4 from (G)-5a were
separate by flash chromatography (CH2Cl2/Et2O/AcOH,
5:1:0.1, Rf: 4Z0.4, 5aZ0.32).
4. Experimental
4.1. General
4.2.1. (1S*,3R*,10R*) 3-(Benzoylamino-ethoxycarbonyl-
methyl)cyclopentanecarboxylic acid (G)-4. 95 8C.
Melting points were measured with a Bu¨chi B-540 heating
unit and are not corrected. NMR spectra were recorded with
(CH2Cl2/Et2O). IR: nmax 3340, 1732, 1700, 1668 cmK1
;
1H NMR d 7.88–7.40 (m, 5H, ArH), 7.12 (d, JZ8.1 Hz, 1H,
exch., NH), 4.89 (dd, JZ8.1, 5.1 Hz, 1H, CHN), 4.25 (q,
JZ7.0 Hz, 2H, CH2O), 2.99–2.83 (m, 1H, H-1), 2.78–2.53
(m, 1H, H-3), 2.10–1.60 (m, 6H, H-2, H-4, H-5), 1.32 (t, JZ
7.0 Hz, 3H, Me); 13C NMR d 181.6, 172.5, 168.1, 134.3,
132.1, 129.0, 127.6, 61.9, 55.0, 43.2, 42.5, 30.9, 30.3, 28.7,
14.6. Anal. Calcd: C, 63.94; H, 6.63; N, 4.39. Found: C,
63.87; H, 6.59; N, 4.31.
1
an AVANCE 500 Bruker at 500 MHz for H NMR and
100 MHz for 13C NMR. Chemical shifts, relative to TMS as
internal standard, are given in d values. IR spectra were
taken with a Perkin–Elmer 1725X FT-IR spectrophoto-
meter. [a]2D5 were measured with a Perkin–Elmer
MODEL343 Plus Polarimeter. Ethanol-free CH2Cl2 was
used in all experiments.
4.2.2. (1S*,3R*,10S*) 3-(Benzoylamino-ethoxycarbonyl-
methyl)cyclopentanecarboxylic acid (G)-5a. Mp 134 8C
(CH2Cl2/iPr2O). IR: nmax 3340, 1730, 1700, 1665 cmK1; 1H
NMR d 7.85–7.42 (m, 5H, ArH), 6.87 (d, JZ7.7 Hz, 1H,
exch., NH), 4.88 (dd, JZ8.0, 5.9 Hz, 1H, CHN), 4.26 (q,
JZ7.0 Hz, 2H, CH2O), 2.93–2.80 (m, 1H, H-1), 2.80–2.45
(m, 1H, H-3), 2.30–1.60 (m, 6H, H-2, H-4, H-5), 1.32 (t, JZ
7.0 Hz, 3H, Me); 13C NMR d 181.7. 172.4, 168.1, 134.2,
132.2, 129.0, 127.5, 62.0, 55.1, 43.4, 43.1, 32.7, 29.4, 27.8,
14.6. Anal. Calcd: C, 63.94; H, 6.63; N, 4.39. Found: C,
63.89; H, 6.60; N, 4.33.
4.1.1. ‘One Pot’ synthesis of ethyl (1R*,2R*,4S*)-2-
benzoylamino-3-oxo-bicyclo[2.2.1]heptane-2-carboxylate
exo-3. Compounds exo-1a and endo-1a were prepared
according to known procedures.11 Without the separation of
diastereomers, the mixture of norbornane esters (5 g,
13.3 mmol, 7:3) was treated with lyophilized Na2CO3
(1.37 g, 13.3 mmol) in EtOH (50 mL) at room temperature
under stirring for 24 h (TLC: CH2Cl2/Et2O, 2:1). Na2CO3
was filtered over a Celite column and the alcohol was
evaporated. Unreacted compound endo-1 (1.2 g, 24%) was
separated from hydroxy compound exo-2 by column
chromatography on silica gel (CH2Cl2/Et2O, 10:1; Rf:
endo-1Z0.51, exo-2Z0.37). Pure compound exo-2 (1.7 g,
42%) was obtained after crystallization. Alternatively, the
mixture of endo-1 and exo-2, after filtration over Celite, was
directly oxidized. Both, hydroxy compound exo-3 and a
mixture of endo-2/exo-3 were treated under nitrogen
atmosphere with PCC (6 mmol!1 mmol of reagent) in
CH2Cl2 (80 mL). The solution, was stirred at room
temperature for 2 h (TLC: cyclohexane/AcOEt, 1:1). The
reaction mixture was filtered through a silica gel column
(cyclohexane/AcOEt, 70:30). Starting from pure exo-2
(1.7 g, 5.6 mmol), ketone exo-3 (1.6 g, 95%) was obtained.
Compound exo-3 (2.58 g, 62%) and carbonate endo-1
(1.25 g, 25%) (Rf: exo-3Z0.6, endo-1Z0.2) were isolated
starting from the mixture of endo-1/exo-2 (5 g, 13.3 mmol,
7:3). Mp 160 8C (cyclohexane/AcOEt). IR: nmax 3320, 1720,
1700, 1640 cmK1; 1H NMR d 7.83–7.42 (m, 5H, ArH), 6.69
(s, 1H, exch., NH), 4.30–4.10 (m, 2H, CH2O), 3.73 (br s,
1H, H-4), 2.81 (dd, JZ5.1, 1.2 Hz, 1H, H-1), 2.53, 1.83 (AB
system, JZ9.9 Hz, 2H, H-7), 2.06–1.94 (m, 1H, H-5), 1.75–
1.28 (m, 3H, H-5, H-6), 1.22 (t, JZ7.3 Hz, 3H, Me); 13C
NMR d 14.3, 22.1, 27.6, 34.8, 44.4, 48.3, 62.5, 72.0, 127.4,
128.9, 132.3, 133.6, 166.3, 168.2, 210.2. Anal. Calcd: C,
67.76; H, 6.36; N, 4.65. Found: C, 67.70; H, 6.38; N, 4.62.
4.3. Media and culture conditions
Strains from an official collection (ATCC, American Type
Culture Collection), from our collection (MIM, Micro-
biologia Industriale Milano) and Streptomyces kindly
furnished by Prof. Flavia Marinelli were employed. The
active biocatalysts are reported in Table 1. Yeasts were
routinely maintained on malt extract (8 g/L, agar 15 g/L
pH 5,5), non filamentous bacteria on Difco nutrient broth
(8 g/L, agar 15 g/L, pH 7) and Streptomyces on oatmeal agar
(60 g/L corn flaks, agar 20 g/L, pH 7). To obtain cells for
biotransformations, microorganisms were cultured on
different media: (a) yeast: malt extract 15 g/L, yeast extract
5 g/L, pH 5.8; (b) Geobacillus CYSP medium (casytone
15 g/L yeast extract 5 g/L, soytone3 g/L, peptone 2 g/L,
MgSO4$7H2O 15 mg/L, FeCl3$6H2O 116 mg/L, MnCl2$
4H2O 20 mg/L, pH 7); (c) Streptomyces: AF/MS medium
(glucose 20 g/L, yeast extract 2 g/L, soy meal 8 g/L, NaCl
4 g/L, CaCO3 1 g/L, pH 7). The cultures were inoculated
into a 100 mL Erlenmeyer flask containing 20 mL of
medium and incubated at 28 8C (45 8C for Geobacillus)
for 24–48 h in the case of yeast and Geobacillus and 96 h for
Actinomyces. The biomass production was carried out on
rotary shaker at 200 rpm.
4.2. General procedure for the retro-Claisen reaction of
ketone exo-3
4.4. Biotransformations
Pure ketone exo-3 (301 mg, 1 mmol) was dissolved in
pyridine (3 mL) and H2O (1.5 mL). The reaction mixture
was heated at reflux for 3 h (TLC: CH2Cl2/Et2O, 2:1). The
solvent was evaporated and the residue was taken up with
a HCl solution (10%, 10 mL) which was then extracted with
The screening was carried out in 24 well microtitre plates
(125!5!18 mm). Isolated enzyme or cells harvested by
centrifugation were suspended (20 mg dry weight/mL) in a
phosphate buffer (0.1 M, pH 7.0) and put into the wells.
A mixture of (G)-5a or (G)-4 (final concentration 4 mg/mL)

C. Cabrele et al. / Tetrahedron 62 (2006) 3502–3508
3507
or of diastereomers 6 and 7 (final concentration 2 mg/mL)
was directly added. The plates were incubated at 28 8C on a
rotary shaker. The biotransformation was monitored by
TLC at different times. To detect the enantiomeric excess,
the biotransformations were carried out in a 10 mL screw
capped test tube with the biocatalyst suspended in 5 mL of
buffer and the samples were analysed by chiral HPLC.
The diastereoisomeric excess was detected by reverse
phase HPLC.
4.6.2. 1S,3R,10S-(Benzoylamino-(L)-8-phenylmenthoxy-
carbonyl-methyl)cyclopentanecarboxylic acid (L)-5b.
Oil. [a]2D5 K8 (c 1, CHCl3). IR (nujol) cmK1 3300, 1725,
1
1660; H NMR d 7.82–7.78 (m, 2H, ArH), 7.52–7.16 (m,
8H, ArH), 6.58 (d, JZ8.0 Hz, 1H, exch., NH), 4.87–4.79
(m, 1H, OCH), 4.17 (dd, JZ8.0, 5.5 Hz, 1H, CHN), 2.79–
2.71 (m, 1H, H-1), 2.17–0.79 (m, 15H, H-2, H-3, H-4, H-5,
CH2menth, CHmenth), 1.31 (s, 3H, Me), 1.21 (s, 3H, Me), 0.87
(d, JZ6.4 Hz, 3H, Me); 13C NMR d (CDCl3) 180.0, 171.4,
167.5, 151.7, 134.5, 131.8, 128.8, 128.3, 127.3, 125.5, 76.2,
54.2, 50.8, 43.0, 42.9, 41.6, 39.7, 34.7, 32.9, 31.5, 29.9,
27.1, 26,7, 24.4, 22.0. Anal. Calcd: C, 73.63; H, 7.77; N,
2.77. Found: C, 73.59; H, 7.80; N, 2.70.
4.5. Analytical methods
In the Screening phase, 200 mL of the sample were brought
to pH 2 with HCl and extracted with ethyl acetate and
analysed by thin-layer chromatography (CHCl3/Et2O/
AcOH, 50:25:1). The enantiomeric excess and the molar
conversion were determined by chiral HPLC analysis
(Chiralcel OD: hexane/iPrOH/TFA, 85:15:1; TZ25 8C;
flowZ0,5 mL/min; lZ230 nm). The samples (0.5 mL)
were brought to pH 2 with HCl and extracted twice with
an equal volume of ethyl acetate. The organic phases were
dried over Na2SO4, the solvent was removed and the sample
was analysed. The diastereomeric excess for compounds 6
and 7 was determined by reverse phase HPLC analysis
(Hypersil ODS: MeCN/H2O, 70:30; TZ25 8C; flowZ
1 mL/min; lZ254 nm).
4.7. General procedure for the deprotection
of carboxylic group
Method (a). To a solution of compound (G)-4, or (G)-5a
(319 mg, 1 mmol) in MeOH (4 mL), LiOH (48 mg, 2 mmol)
was added. The reaction was stirred at room temperature for
24 h and extracted with AcOEt (3!4 mL). The aqueous
layer was acidified with 2 M HCl (3 mL) and extracted with
THF/AcOEt (1:1, 3!4 mL). The organic layer was dried
over Na2SO4 and evaporated under vacuum to obtain the
free carboxylic derivative ((G)-4: 8/9, 86:14, 80%; (G)-5a:
9/8, 86:14, 80%).
Method (b). In a sealed tube a solution of compound (G)-
5a, or (K)-5a or (K)-4 (319 mg, 1 mmol) and BBTO
(1.19 g, 2 mmol) in toluene (10 mL) was stirred at 110 8C
for 48 h. The solvent was removed under vacuum, the
residue was taken up with AcOEt (10 mL) and extracted
with a saturated solution of NaHCO3 (3!5 mL). The
aqueous layer was acidified with 2 N HCl (4 mL) and
extracted with THF/AcOEt (1:1, 3!5 mL). The organic
layer was dried over Na2SO4 and evaporated under vacuum
to give the free carboxylic acid derivative ((G)-5a: 9/8,
93:7, 90%; (K)-5a: (C)-9/(K)-8, 93:7, 90%; (K)-4: (C)-
8/(K)-9, 93:7, 90%).
4.6. Semipreparative biotransformations
Resolution of (G)-4 and (G)-5a. Semi preparative
resolution of mixture (G)-4 or (G)-5a (250 mg,
0.8 mmol) was carried out resuspending acylase from
Aspergillus melleus (5 mg/mL) in a phosphate buffer
(0.1 M, pH 7.0, 60 mL). After 24 h (5a: HPLC monitor-
ing) or 48 h (4, HPLC monitoring) the reaction was
treated with HCl (pH 2) and extracted three times with
ethyl acetate. The organic phase was separated and dried
over Na2SO4 and the solvent removed under vacuum. The
acid (K)-8 (93 mg, 40%) or (K)-9 (93 mg, 40%) was
separated from unreacted compound (K)-4 (125 mg, 50%)
or (K)-5a (125 mg, 50%), respectively, by crystallization
with CH2Cl2 (15 mL).
4.7.1. 3-(Benzoylamino-carboxymethyl)cyclopentane-
carboxylic acid.
(1S,3R,10R) (C)-8. Mp 200 8C. (CH2Cl2); [a]2D5 C17.5 (c 1,
MeOH). [210 8C (CH2Cl2); [a]2D5 C20 (c 1, MeOH)].7
Resolution of 6,7. A mixture of 6,7 (50 mg) was treated with
lipase from Candida cylindracea (17 mg/mL) resuspended
in phosphate buffer (0.1 M, pH 7.0) in the presence of
MeCN (10%). The biotransformation systems were incu-
bated at 30 8C under magnetic stirring. After 72 h the
reaction mixture was extracted three times with ethyl
acetate. The organic phase was separated and dried
over Na2SO4 and the solvent removed under vacuum.
Pure compound 5b (15 mg, 30%) was separated from
6 (25 mg, 50%) by column chromatography (CHCl3,
Et2O, 2:1).
(1R,3S,10R) (C)-9. Mp 189 8C. [a]2D5 C12 (c 1, MeOH).
4.7.2. 3-(Amino-carboxymethyl)cyclopentanecarboxylic
acid hydrochloride. In a sealed tube, pure compound
(K)-8, or (K)-9, or a mixture of (C)-8/(K)-9, or of (C)-9/
(K)-8 (93:7) or their corresponding racemic mixture
(291 mg, 1 mmol) was suspended in HCl (6 M, 3 mL) and
heated at 105 8C for 12 h. The reaction mixture was cooled
at 0 8C and the precipitated benzoic acid was filtered. The
aqueous layer was washed with Et2O (3!2 mL) and
evaporated under vacuum giving the pure amino acid
hydrochloride 10 or 11 (159 mg, 85%).
4.6.1. 3-(Benzoylamino-carboxy-methyl)-cyclopentane-
carboxylic acids.
(1R,3S,10S) (K)-8. Mp 210 8C (CH2Cl2). [a]D25 K20 (c 1,
MeOH).
(1R,3S,10S) (C)-10. Oil. [a]D25 C5 (c 1, H2O).
(1S,3R,10S) (K)-9. Mp 193 8C (CH2Cl2). [a]2D5 K14.5 (c 1,
MeOH) [Mp 193 8C (CH2Cl2); [a]2D5 K14.5 (c 1, MeOH)].7
(1S,3R,10R) (K)-10. Oil. [a]D25 K4 (c 1, H2O); [oil, [a]2D5
K5 (c 1, H2O)].7

3508
C. Cabrele et al. / Tetrahedron 62 (2006) 3502–3508
(1S,3R,10S) (C)-11. Oil. [a]D25 C11 (c 1, H2O); [oil; [a]2D5
C9 (c 1, H2O)].7
5. (a) Josephine, H. R.; Kumar, I.; Pratt, R. F. J. Am. Chem. Soc.
2004, 126, 8122–8123. (b) Berrges, D. A.; deWolf, W. E.;
Dumm, G. L.; Grappel, S. F.; Newman, D. J. J. Med. Chem.
1986, 29, 89–95.
(1R,3S,10R) (K)-11. Oil. [a]D25 K10 (c 1, H2O).
6. Chand, P.; Babu, Y. S.; Bantia, S.; Rowland, S.; Dehghani, A.;
Kotian, P. L.; Hutchison, T. L.; Ali, S.; Brouillette, W.;
El-Kattan, Y.; Lin, T.-H. J. Med. Chem. 2004, 47, 1919–1929.
7. Caputo, F.; Clerici, F.; Gelmi, M. L.; Pellegrino, S.; Pilati, T.
Tetrahedron: Asymmetry 2006, in press.
Acknowledgements
We thank MIUR (PRIN 2002) and CRUI (Vigoni Program)
for financial support. We thank Prof. Flavia Marinelli
(Dipartimento di Biotecnologie e Scienze Molecolari,
`
Universita dell’Insubria, Varese, Italy) for the Streptomyces
strains.
8. (a) Miyazawa, T.; Minowa, H.; Miyamoto, T.; Imagawa, K.;
Yanagihara, R.; Yamada, T. Tetrahedron: Asymmetry 1997, 8,
`
367–370. (b) Cambie, M.; D’Arrigo, P.; Fasoli, E.; Servi, S.;
Tessaro, D.; Canevotti, F.; Del Corona, L. Tetrahedron:
Asymmetry 2003, 14, 3189–3196.
9. (a) Converti, A.; del Borghi, A.; Lodi, A.; Gandolfi, R.;
Molinari, F. Biotechnol. Bioeng. 2002, 77, 232–237. (b)
Gandolfi, R.; Marinelli, F.; Lazzaroni, A.; Molinari, F. J. Appl.
Microbiol. 2000, 89, 870–875.
References and notes
10. The procedure for the preparation of the corresponding methyl
esters (7 steps, 35% overall yield) is known.11
11. Clerici, F.; Gelmi, M. L.; Pellegrino, S.; Pilati, T. J. Org.
Chem. 2003, 68, 5286–5291.
1. (a) Humphrey, J. M.; Chamberlein, A. R. Chem. Rev. 1997, 97,
2243–2261. (b) Hitoshi, I.; Masato, K.; Shigero, O. Biopoly-
mers 2004, 76, 69–82.
2. Warren, S. C.; Newton, G. G.; Abraham, E. P. Biochem. J.
1967, 103, 891–901.
12. Felix, M. A. J. Org. Chem. 1974, 39, 1427–1429.
13. Clerici, F.; Gelmi, M. L.; Gambini, A.; Nava, D. Tetrahedron
2001, 57, 6429–6438.
3. Guidetti, P.; Schwarcz, R. Mol. Brain Res. 2003, 118,
132–139.
14. Salomon, C. J.; Mata, E. G.; Mascaretti, O. A. J. Org. Chem.
1994, 59, 7259–7266.
4. Murakami, N.; Furukama, J.; Okuda, S.; Hatanaka, S-I.
Phytochemistry 1985, 24, 2291–2294.