Synthesis of the constrained glutamate analogues (2S,1′R,2′R)- and (2S,1′S,2′S)-2-(2′-carboxycyclobutyl)glycines L-CBG-II and L-CBG-I by enzymatic transamination

Article abstract of DOI:10.1016/j.tetlet.2005.10.156

Optically pure trans-cyclobutane analogues of glutamic acid are prepared by highly selective enzymatic transamination of a single racemic trans-cyclobutane α-ketoglutaric acid derivative 5, which is synthesized in five steps from maleic anhydride. (2S,1′R,2′R)- and (2S,1′S,2′S)-2- (2′-carboxycyclobutyl)glycines L-CBG-II and L-CBG-I are obtained using aspartate aminotransferase (AAT) and branched chain aminotransferase (BCAT), respectively.

Full text of DOI:10.1016/j.tetlet.2005.10.156

Tetrahedron
Letters
Tetrahedron Letters 47 (2006) 193–196
Synthesis of the constrained glutamate analogues (2S,10R,20R)-
and (2S,10S,20S)-2-(20-carboxycyclobutyl)glycines L-CBG-II
and L-CBG-I by enzymatic transamination
Xin Gu, Mo Xian, Sophie Roy-Faure, Jean Bolte, David J. Aitken* and Thierry Gefflaut*
Laboratoire SEESIB-CNRS (UMR 6504), De´partement de Chimie, Universite´ Blaise Pascal—Clermont-Ferrand II,
`
24, Avenue des Landais, 63177 Aubiere cedex, France
Received 12 October 2005; revised 26 October 2005; accepted 28 October 2005
Available online 16 November 2005
Abstract—Optically pure trans-cyclobutane analogues of glutamic acid are prepared by highly selective enzymatic transamination of
a single racemic trans-cyclobutane a-ketoglutaric acid derivative 5, which is synthesized in five steps from maleic anhydride.
(2S,10R,20R)- and (2S,10S,20S)-2-(20-carboxycyclobutyl)glycines L-CBG-II and L-CBG-I are obtained using aspartate aminotrans-
ferase (AAT) and branched chain aminotransferase (BCAT), respectively.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
of group II mGluRs while the cis-(2S,10S,20R)-isomer
(L-CCG-III) is an inhibitor of glutamate transport sys-
tems at the excitatory synapses (Fig. 1).4 In contrast,
very limited work has been done on 2-(20-carboxycyclo-
butyl)glycine (CBG) and to our knowledge only two
of the four stereoisomers of the L-series, L-CBG-I and
L-CBG-III, have been synthesized.5
L-Glutamic acid (Glu) is the major excitatory neuro-
transmitter in the central nervous system of vertebrates.
It interacts with two families of receptors, namely iono-
tropic glutamate receptors (iGluRs) and metabotropic
glutamate receptors (mGluRs).1 Several Glu transport-
ers have also been shown to play a crucial role in the
modulation of neurotransmission.2 Glu is involved in
most brain functions such as learning, memory, motor
control, vision and pain sensitivity. The development
of selective ligands for GluRs allows a fine modulation
of the glutamatergic pathways and may provide an
appropriate therapeutic approach for the treatment of
neurodegenerative pathologies or neuropsychiatric dis-
orders.1 Glu is a highly flexible molecule and conforma-
tionally constrained analogues have been designed and
synthesized in order to elucidate the conformational
requirements for binding at individual receptors. For
this purpose, alicyclic Glu analogues are of particular
interest, and intensive studies have been carried out on
three- to seven-membered ring analogues.3–5 Among
them, trans-(2S,10S,20S)-2-(20-carboxycyclopropyl)gly-
cine (L-CCG-I) has been shown to be a potent agonist
Over the last few years, we have been developing a
chemoenzymatic approach for the synthesis of Glu
derivatives, based on the stereoselective aminotransfer-
CO2H
NH2
H
NH2
CO2H
NH2
CO2H
HO2C
H
HO2C
H
CO2H
L-CCG-III
H
L-Glu
L-CCG-1
HO2C
H
NH2
CO2H
NH2
H
HO2C
CO2H
H
H
L-CCG-II
L-CCG-IV
H
CO2H
NH2
CO2H
NH2
HO2C
H
H
*
CO2H
L-CBG-III
H
Corresponding authors. Tel.: +33 4 73 40 74 81; fax: +33 4 73 40 77
17 (D.J.A.); tel.: +33 4 73 40 71 28; fax: +33 4 73 40 77 17 (T.G.);
e-mail addresses: David.AITKEN@univ-bpclermont.fr; Thierry.
GEFFLAUT@univ-bpclermont.fr
L-CBG-I
Figure 1. L-glutamic acid and alicyclic analogues.
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.10.156

194
X. Gu et al. / Tetrahedron Letters 47 (2006) 193–196
ase-catalyzed conversion of a-keto acids to L-amino
acids. We have shown that aspartate aminotransferase
(AAT, EC 2.6.1.1) is a useful tool for the stereospecific
synthesis of a variety of 4-substituted Glu derivatives
from the corresponding 2-ketoglutaric acid (KG) ana-
logues, easily prepared in racemic form.6 The extension
of the method to 3,4-disubstituted derivatives such as
CBG was a stimulating challenge. The present work
describes the original synthesis of the two trans isomers
of 2-(20-carboxycyclobutyl)glycines (CBG) by a highly
selective enzymatic transamination of the appropriate
cyclobutane derivative of a-ketoglutaric acid using one
of two aminotransferases from Escherichia coli: aspar-
tate aminotransferase (AAT) and branched chain amino-
transferase (BCAT).
was carried out in the presence of a stoichiometric
amount of lithium hydroxide in MeOH. In these reac-
tion conditions, regiospecific epimerization a ketone
functionality was observed, furnishing the thermody-
namically more stable trans cyclobutane. The lithium
salt 5 was isolated as a 90:10 trans/cis mixture deter-
mined by 1H NMR. The transamination substrate 5
was thus prepared in five steps with an overall yield of
42% from maleic anhydride.
3. Enzymatic transaminations
E. coli Aspartate aminotransferase (AAT) catalyzes the
equilibrium between Glu + oxalacetate on one side and
KG + aspartic acid (Asp) on the other side. This enzyme
presents a broad substrate spectrum towards 4-substi-
tuted KG analogues and shows marked enantioselectiv-
ity in favour of the 4R configuration of 4-alkyl
derivatives.6c–e,9 Kinetic resolution of racemic 4-alkyl
KGs thus allows the preparation of (2S,4R)-4-alkyl
Glu with very high ee and de. However, the enzyme
specificity towards 3-substituted Glu is restricted to the
(3R)-3-methyl derivative (data not published). E. coli
Branched chain aminotransferase (BCAT) is a broader
spectrum aminotransferase since it is involved in the
metabolism of Glu, Val, Leu, Ile, Phe and Met.10 Sev-
eral examples of BCAT-catalyzed syntheses of unnatu-
ral amino acids, such as L-tert-leucine and L-
phosphinothricine have already been described.11 Enzy-
matic transamination is a useful approach to the synthe-
sis of amino acids, provided that an equilibrium shift is
performed.12 AAT offers the opportunity of decarboxyl-
ation of oxalacetate, formed when Asp is used as the
amino donor substrate. This decarboxylation can be cat-
alyzed chemically or by enzymatic means and generates
pyruvate which is not a substrate for AAT. Alterna-
tively, a close analogue of Asp, cysteine sulfinic acid
(CSA), can be used as the amino donor and furnishes
an even more unstable b-keto sulfinic acid, which is
readily decomposed into pyruvate and sulfur dioxide
without the need for catalysis (Scheme 2).13 Moreover,
because of the high acidity of CSA, the purification of
the amino acid product by ion exchange chromatogra-
phy is highly simplified. Unfortunately, Asp or CSA
are not substrates for BCAT and an alternative strategy
has to be used for the equilibrium shift. This can be
achieved through the enzymatic regeneration of the ami-
no donor Glu used in a catalytic amount. Glutamic
dehydrogenase (EC 1.4.1.3) can thus be used to catalyze
the conversion of KG back to Glu in the presence of
2. Synthesis of the trans-cyclobutane analogue of KG
The four-membered ring derivative of a-ketoglutaric
acid was efficiently synthesized in racemic form as fol-
lows (Scheme 1): a [2+2] photocycloaddition reaction
between maleic anhydride and ethylene was performed
in acetonitrile solution in the presence of acetophenone
as photosensitizer by irradiation for 5 h with a 400 W
medium-pressure mercury lamp fitted with a pyrex filter.
cis-1,2-Cyclobutane dicarboxylic anhydride
1 was
isolated in 75% yield.7 Methanolysis of this anhydride
moiety gave the monoester cis-2-(methoxycarbonyl)cyc-
lobutanecarboxylic acid 2. Homologation of 2 into an
a-ketoester was performed using WassermanÕs method-
ology:8 the acid function of 2 was coupled with (triphen-
ylphosphoranylidene)acetonitrile to yield the a-keto
cyanophosphorane 3 in 91% yield. Ozonolysis of inter-
mediate 3 in a 3:1 mixture of dichloromethane/methanol
at ꢀ78 °C provided cis-2-oxalylcyclobutanecarboxylic
dimethyl ester 4 in 81% yield. Final ester hydrolysis
O
O
MeO2C
CO2H
(i)
(ii)
+
O
O
O
O
( )-2
(iii)
O
O
MeO2C
OMe
(iv)
CN
MeO2C
O
PPh3
( )-4
(v)
( )-3
R4
O
R4 NH2
*
CO2H
O
AAT
LiO2C
OLi
trans/cis
90:10
HO2C
CO2H
HO2C
*
R3
R3
O
KG analogue
Glu analogue
( )-5
NH2
CO2H
O
HO2S
Scheme 1. Reagents and conditions: (i) acetophenone, CH3CN, hm,
pyrex, 5 h, 75%; (ii) MeOH, reflux, 16 h, 77%; (iii) (triphenylphos-
phoranylidene)-acetonitrile, EDCI, DMAP, CH2Cl2, rt, 4 h, 91%; (iv)
O3, CH2Cl2/MeOH 3:1, ꢀ78 °C, 81%; (v) LiOH, MeOH, rt, 2 h then
Dowex resin H+, 100%.
O
SO2
+
HO2S
CO2H
CO2H
CSA
Scheme 2. Aspartate aminotransferase-catalyzed transamination.

X. Gu et al. / Tetrahedron Letters 47 (2006) 193–196
195
R4
O
H
NH2
CO2H
R4 NH2
*
BCAT
HO2C
HO2C
CO2H
NH2
AAT
HO2C
CO2H
R3
*
R3
H
L-CBG-II
H
O
O
HO2C
CO2H
HO2C
CO2H
HO2C
CO2H
NH2
CO2H
( )-5
HO2C
GluDH
BCAT
+
H
NH4
L-CBG-I
Scheme 4. Enzymatic transamination of trans-2-oxalylcyclobutane
carboxylic acid.
NAD+
NADH
FDH
form) followed by elution with an AcOH gradient
(0 ! 1 M). CBG-II was thus isolated as a white solid
in 32% yield with a very good purity (Scheme 4).15
-
HCO2
CO2
Transamination with BCAT was achieved on substrate 5
(0.3 mmol) with a catalytic amount of Glu (0.06 mmol)
as the amino donor and E. coli BCAT (0.5 mg, 4 UI).
The regeneration system comprised NADH (0.03 mmol),
ammonium formate (0.6 mmol), pig liver glutamic dehy-
drogenase (0.4 mg, 9 UI), yeast formate dehydrogenase
(2 mg, 12 UI). The reaction was followed by TLC during
48 h. The cyclobutane Glu analogue was purified by ion
exchange chromatography as previously described and
isolated as a white solid in 26% yield (Scheme 4).16
Scheme 3. Branched chain aminotransferase-catalyzed transamin-
ation.
ammonium ions and of the reduced coenzyme NADH.
The coenzyme is also used in catalytic amount and is
in turn regenerated using a stoichiometric amount of
formic acid and formate dehydrogenase (EC 1.2.1.2).
This last irreversible step affords the overall equilibrium
shift (Scheme 3).
Since the cyclobutane KG analogue 5 was isolated as a
mixture of cis and trans stereomers, we could not deter-
mine reliable kinetic parameters for the trans derivative.
However, analytical measurements allowed us to evi-
dence that sample (+/ꢀ)-5 was a substrate for E. coli
AAT and BCAT with, for each enzyme, a relative activ-
ity of around 2% of that for the natural substrate KG.
As these aminotransferases are characterized by high
specific activities, this activity proved to be sufficient
for synthetic purposes.
Absolute configurations were attributed as follows.
Physicochemical and spectroscopic data for the isomer
obtained with BCAT were comparable with those for
the trans isomer already described in the literature.5 It
was shown to be (2S,10S,20S)-2-(20-carboxycyclobu-
20
tyl)glycine L-CBG-I with optical rotation ½aꢁD +96.4
20
(c 0.7, H2O) [lit. ½aꢁD +97.1 (c 0.49, H2O)] and mp over
245 °C (dec.) [lit. mp 258–263 (dec.)] (Scheme 4). The
isomer obtained with AAT displayed different spectral
25
data and had ½aꢁD ꢀ53.0 (c 0.7, H2O) and mp 164–
166 °C. Owing to AAT selectivity for L-amino acids,
the (2S,10R,20R) configuration was assigned to this sec-
ond trans isomer L-CBG-II (Scheme 4). To our knowl-
edge, this compound has not been described before. In
order to confirm this stereochemistry, enantiomerically
pure (1R,2R)-2-oxalylcyclobutane carboxylic acid 5
was prepared as described in Scheme 5 and evaluated
as a substrate for AAT. The synthesis involved the se-
quence already described for racemic compound 5 from
enantiomerically pure cis-(1R,2S)-2. This compound
was prepared by desymmetrization of meso-diester 6
by pig liver esterase-catalyzed hydrolysis as described
in the literature.14 Analytical rate measurement of
AAT-catalyzed transamination of (ꢀ)-(1R,2R)-5 gave
relative activity very close from that measured with race-
mic compound 5. (ꢀ)-(1R,2R)-5 is thus the enantiomer
recognized by the enzyme confirming our configuration
assignment for L-CBG-II.
With E. coli AAT, the synthesis was carried out in water
(140 mL) at pH adjusted to 7.6 with a-keto carboxylate
5 (cis/trans 10:90) (2.8 mmol) in the presence of a stoi-
chiometric amount of CSA as the amino donor and
aminotransferase (5 mg, 250 UI). Acetaldehyde (2.8
mmol) was added to the reaction mixture to scavenge
sulfite ions produced from CSA and thus increase the
reaction rate by preventing partial inhibition of the
enzyme by this product. The conversion rate was deter-
mined by titration of pyruvate formed from CSA using
the couple lactic dehydrogenase (LDH, EC 1.1.1.27) and
NADH: NADH consumed for pyruvate reduction was
titrated in small aliquots by absorbance measurements
at 340 nm. After 4 h, near 45% conversion, the reac-
tion slowed markedly and was stopped by passing the
reaction mixture directly through a short column of
Dowex 50 resin (H+ form). Unreacted a-ketoacid, pyru-
vate and excess CSA were eluted simply with water and
the adsorbed glutamic acid analogue was further eluted
with a 1 M ammonium hydroxide solution. The Glu
analogue was further purified by adsorption onto a
short column of strongly basic Dowex 2 resin (AcOꢀ
In conclusion, aminotransferases AAT and BCAT pres-
ent complementary enantioselectivities towards trans-2-
oxalylcyclobutanecarboxylic acid 5, allowing selective
access to isomeric (2S,10R,20R)- and (2S,10S,20S)-CBGs.

196
X. Gu et al. / Tetrahedron Letters 47 (2006) 193–196
Med. Chem. 1997, 40, 3119–3129; (h) Monn, J. A.; Valli,
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MeO2C
CO2Me
M. J.; Massey, S. M.; Wright, R. A.; Salhoff, C. R.;
Johnson, B. G.; Howe, T.; Alt, C. A.; Rhodes, G. A.;
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(i)
O
6
O
(ii)
4. (a) Chavan, S. P.; Sharma, P.; Sivappa, R.; Bhadbhade,
M. M.; Gonnade, R. J.; Kalkote, U. R. J. Org. Chem.
O
MeO2C
CO2H
(iii), (iv), (v)
LiO2C
OLi
´
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˜
Chem. 1999, 64, 8958–8961; (c) Shimamoto, K.; Ohfune,
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O
(-)-(1R,2R)-5
(-)-(1R,2S)-2
trans/cis 90:10
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Scheme 5. Reagents and conditions: (i) MeOH, c. H2SO4, reflux, 4 h,
60% (ii) PLE, pH 7, 20 °C, 78%; (iii) (triphenylphosphoranylidene)ace-
tonitrile, EDCI, DMAP, CH2Cl2, rt, 4 h, 85%; (iv) O3, CH2Cl2/MeOH
3:1, ꢀ78 °C, 1 h 30 (v) LiOH, MeOH then Dowex resin H+, 2 h, 87%
(two steps).
´
6. (a) Helaine, V.; Rossi, J.; Gefflaut, T.; Alaux, S.; Bolte, J.
Adv. Synth. Catal. 2001, 343, 692–697; (b) Bessis, A. S.;
Bolte, J.; Pin, J. P.; Acher, F. Bioorg. Med. Chem. Lett.
Biological activities of these compounds towards gluta-
mate receptors and transporters are under investigation.
This study shows the potential of AAT and BCAT for
chemoenzymatic synthesis of alicyclic analogues of L-
glutamic acid, a goal which we are currently pursuing.
´
2001, 11, 1569–1572; (c) Helaine, V.; Rossi, J.; Bolte, J.
´
Tetrahedron Lett. 1999, 6577–6580; (d) Helaine, V.; Bolte,
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Acknowledgements
9. E. coli AAT was produced and purified following a
described procedure from an overexpressing E. coli strain
TY103 transformed with pUC19-aspC: Kamitori, S.;
Hirotsu, K.; Higuchi, T.; Kondo, K.; Inoue, K.; Kura-
mitsu, S.; Kagamiyama, H.; Higuchi, Y.; Yasuoka, N.;
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We are grateful to the CNRS for financial support. We
would like to thank Pr. KagamiyamaÕs group (Osaka
Medical College) for providing us with E. coli strains
which over express AAT and BCAT.
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20
15. L-CBG-II: white solid; mp 164–166 °C; ½aꢁD ꢀ53.0 (c 0.7,
H2O); 1H NMR (400 MHz, D2O) d 1.79–2.13 (m, 4H),
2.86 (dq, J = 8.0, 9.2 Hz, 1H), 3.07 (q, J = 9.2 Hz, 1H),
3.68 (d, J = 7.6 Hz, 1H). 13C NMR (100 MHz, D2O) d
21.0 (CH2), 21.6 (CH2), 38.0 (CH), 41.3 (CH), 57.4 (CH),
172.8 (C), 178.5 (C).
20
16. L-CBG-I : white solid ; mp > 245 °C (dec.); ½aꢁD +96.4 (c
`
Pellicciari, R.; Marinozzi, M.; Camaioni, E.; Nunez, M.
0.7, H2O); 1H NMR (400 MHz, D2O) d 1.79–1.95 (m, 2H),
1.96–2.11 (m, 2H), 2.78 (dq, J = 8.8, 9.2 Hz, 1H), 3.15 (q,
J = 9.2 Hz, 1H), 3.67 (d, J = 8.8 Hz, 1H). 13C NMR
(100 MHz, D2O) d 20.9 (CH2), 21.4 (CH2), 38.9 (CH), 41.7
(CH), 57.8 (CH), 172.6 (C), 178.5 (C).
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