Amino acid recognition of pyridine bis(oxazoline)-copper(II) complex in aqueous solvent

Article abstract of DOI:10.1016/S0040-4039(03)00937-7

Enantioselective recognition of amino acids has been studied with C₂-symmetric chiral pyridine bis(oxazoline)-copper(II) complexes at physiological pH condition. UV-visible titration revealed strong binding of submillimolar dissociation constan

Full text of DOI:10.1016/S0040-4039(03)00937-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 4335–4338
Amino acid recognition of pyridine bis(oxazoline)–copper(II)
complex in aqueous solvent
a
a
a
a,b,
Hae-Jo Kim, Riaz Asif, Doo Soo Chung and Jong-In Hong *
a
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea
b
Center for Molecular Design and Synthesis, KAIST, Taejon 305-701, Republic of Korea
Received 14 March 2003; revised 11 April 2003; accepted 11 April 2003
Abstract—Enantioselective recognition of amino acids has been studied with C -symmetric chiral pyridine bis(oxazoline)–cop-
2
per(II) complexes at physiological pH condition. UV–visible titration revealed strong binding of submillimolar dissociation
constant between pyridine bis(oxazoline)–copper(II) complex and amino acids in aqueous solution. Moderate selectivity of up to
2
:1 between
D- and L-amino acids was achieved. The enantiomers were baseline resolved by capillary electrophoresis, using the
bis( -lysine)–copper(II) complex as a chiral selector. © 2003 Elsevier Science Ltd. All rights reserved.
L
4
Molecular recognition of amino acids and peptides is a
challenging goal because they are vital components of
proteins which play major roles in living organisms.
While many receptors with fairly good residue-selectiv-
ity and stereoselectivity for amino acid derivatives have
to a similar literature procedure. The light green
aqueous solution turned to deep blue-green (m281 nm=
−
1
−1
−1
−1
4525 M cm , m310 nm=3062 M cm ) in aqueous
HEPES buffer (pH 7.4 with 10 mM HEPES,
MeOH:H O=1:1, v/v) during the metal–ligand com-
plexation. Additional evidence for the formation of
2
1
been intensively studied in nonpolar solvents, receptors
pybox-Ph(R)
working in polar and aqueous solvents are less devel-
[CuL
](NO ) came from electrospray ioniza-
3 2
2
oped. As a receptor operating in water, we chose a
tion (ESI) mass analysis (positive, HCO H/MeOH:
2
pybox-Ph(R) +
Cu(II) complex to utilize the water-soluble properties of
observed 477.5 ([CuL
·HCO ] , 15%), observed
2
+
3
pybox-Ph(R)
metalloreceptors. Herein we report a high affinity of
401.5 ([L
·CH OH·H] , 100%), observed 370.5
3
pybox-Ph(R)
+
simple chiral hosts for underivatized amino acids and
their moderate enantioselectivity in aqueous solvents.
The chiral hosts are known for their enantioselective
([L
·H] , 52%)).
pybox-Ph(R)
The metal–ligand complex, [CuL
](NO3)2 in
4
catalytic activities in Diels–Alder and Friedel–Craft
aqueous solvent was assumed to be a square pyramidal
5
pybox-iPr(R)
8
reactions.
structure similar to [CuL
(H O) ]. Upon
2 2
adding amino acid (AA) to aqueous solution of
pybox
pybox-Ph(R)
The ligand, pyridine bis(oxazoline) (L
) was synthe-
[CuL
](NO ) , bidentate ligand AA plausibly
pybox-iPr(R)
3
2
sized according to the modified procedure from the
literature. Conversion of 2,6-pyridine dicarboxylic acid
replaces the two water ligands to afford [CuL
-
6
+
3c
(AA)] as shown in Figure 2.
to 2,6-pyridine dicarbonyl dichloride with 2 equiv. of
oxalyl chloride under catalytic DMF, coupling with
amino alcohol in the presence of TEA and activation of
the resulting alcohol with methanesulfonyl chloride,
Complexation between the pybox–copper complex and
amino acids was investigated at physiological condi-
tions (pH 7.4) with HEPES buffer. When adding under-
followed by cyclization under basic conditions afforded
pybox
the desired product, L
in high yield over three steps
7
(
Fig. 1).
pybox
Ligand, L
in dichloromethane was mixed with 1
equiv. of cupric nitrate in aqueous methanol according
*
Corresponding author. Tel.: +82-2-880-6682; fax: +82-2-889-1568;
e-mail: jihong@plaza.snu.ac.kr
pybox
Figure 1. Synthetic scheme of L
.
0
040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)00937-7

4
336
H.-J. Kim et al. / Tetrahedron Letters 44 (2003) 4335–4338
Table 1. Dissociationconstants(K )between[CuL
pybox-Ph(R)
]-
d
a
(
NO ) and amino acids
3 2
Entry
Guest
Kd (M)
5.98(±0.69)×10−
3.12(±0.42)×10−
4.02(±0.89)×10−
4
1
2
3
4
5
6
7
8
9
L
-Ala
-Ala
-Val
-Val
-Phe
-Phe
-Asn
-Asn
-Gln
-Gln
4
4
4
4
4
4
5
D
L
−
Figure 2. Schematic drawing of stepwise metal–ligand and its
amino acid complexation.
D
2.02(±0.43)×10
L
1.95(±0.30)×10−
1.10(±0.08)×10−
D
−
L
2.27(±0.59)×10
ivatized amino acid such as
L
-phenylalanine (
L-Phe) to
9.98(±1.81)×10−
pybox-Ph(R) 2+
] , absorbance at u281
D
[CuL
increases,
nm
−4
−4
−4
−4
L
6.21(±2.04)×10
5.00(±3.16)×10
1.54(±0.26)×10
3.41(±0.51)×10
whereas absorbance at u310 nm decreases. Therefore, an
isosbestic point is observed at u298 nm (Fig. 3). This
isosbestic point is also observed in the case of interac-
tions with other amino acids (Ala, Val, Asn, etc.). The
1
0
D
11
L
L
-Asp
-Glu
1
2
a
pybox-Ph(R)
evident isosbestic point is regarded as the 1:1 host–
UV–vis titration at 298 K, [CuL
amino acid] =10–15 mM. Absorbance changes at u were
281 nm
](NO ) =0.10 mM,
3 2
pybox-Ph(R) 2+
[
guest equilibrium between the host ([CuL
]
o
monitored after each addition of guests to determine dissociation
constants.
and the amino acid guests. The apparent dissociation
constant (K ) was determined by fitting the UV–vis
d
data to the nonlinear regression method. The binding
pybox-Ph(R) 2+
versus 9, 10 and 11 versus 12). It is surprising that this
simple host without a hydrophobic cavity shows sub-
millimolar dissociation constants toward polar amino
acids even in aqueous solvent. In order to understand
why the polar amino acids are strongly recognized by
the pybox–copper complex, thermodynamic driving
forces were examined by isothermal microcalorimetric
constants of the host, [CuL
] , with several
amino acids are summarized in Table 1. The disso-
ciation constants between the host and amino acids
are in the range of submillimolar concentrations:
3c
−
4
−5
K =10 ꢀ10 M. The host with (R,R) configuration,
d
pybox-Ph(R) 2+
[CuL
] , shows a favorable affinity toward D-
amino acids. This enantioselectivity seems to come
from the steric hindrance between one phenyl group of
the host and the a substituents of amino acids (Fig. 4).
When the a substituents of the amino acids are nonpo-
lar, more favorable binding affinities between the host
and guests are observed as in the case of guests with
hydrophobic side chains (entries 1–6). It is also interest-
ing that guests with hydrophilic side chains show
stronger binding affinity to the host when the guests
have shorter side chains: Asp, Asn>Glu, Gln (entry 7, 8
(
ITC) analysis during the host–guest binding. The host–
guest complexation is enthalpically driven due to favor-
9
able metal–ligand interaction: DH=−10.56(±0.18)
kcal/mol, TDS=−5.14(±1.27) kcal/mol and DH=
−
12.99(±0.71) kcal/mol, TDS=−7.48 kcal/mol at 30°C
for L-Ala and D-Ala, respectively (Fig. 5).
Even though chiral discrimination of amino acids by
pybox–copper complex was not large, we were able to
resolve the enantiomers by capillary electrophoresis
(
CE). In order to directly detect the analytes and sup-
press the background UV–vis absorbance, we employed
the selector as a nonchromophoric [Cu(
L
-Lys) ] and the
2
pybox-Ph(R) pybox-Ph(S) 10
analyte as a chromophoric L
Ligand -lysine of the chiral selector, [Cu(
assumed to be exchanged with L
trophoresis. The exchange product, [Cu(
is thought to be less cationic than the selector, [Cu(
/L
.
L
L
-Lys) ], is
2
pybox
during the elec-
pybox
L
-Lys)(L
)]
L
-
Lys) ] at pH 7.4, which implies tighter binding as the
2
copper complex moves faster in normal applied voltage
Figure 4. Modeling structures between host and alanine
Figure 3. UV–vis spectral change upon addition of
L
-Phe to a
guests: (left) calculated structure for [CuL(R)]/(
kcal/mol), (right) calculated structure for [CuL(R)]/(
(28.2 kcal/mol).
D
)-Ala (27.9
pybox-Ph(R)
solution of [CuL
pH 7.4, 10 mM HEPES in MeOH/H O, 1:1, v/v).
](NO ) in aqueous HEPES buffer
L
)-Ala
3
2
(
2

H.-J. Kim et al. / Tetrahedron Letters 44 (2003) 4335–4338
pybox-Ph(R)
4337
In conclusion, [CuL
] shows strong affinities to
amino acids in the range of sub-millimolar concentra-
−
4
−5
tion (K =10 ꢀ10 M) even in aqueous solution.
d
Enantioselectivity was moderate up to Kd(
L)/Kd(D)=2
with favorable affinities of the R-host to -amino acids.
D
pybox-Ph(R) pybox-Ph(S)
CE enables enantiomers of L
/L
to be
baseline separated using [Cu(
L
-Lys) ] as a chiral selec-
2
pybox(R)
tor. Binding of [CuL
] to amino acids was driven
by enthalpy. This leads to higher binding constants
even in aqueous solution.
Acknowledgements
Support of this work by a grant from the KRF (Grant
No. KRF-99-042-D00073) is gratefully acknowledged.
H.-J.K. thanks the Ministry of Education for the BK
pybox-Ph(R)
Figure 5. ITC titrations between [CuL
](NO3)2 and
amino acids under 10 mM HEPES buffer (pH 7.4) in aqueous
2
1 fellowship.
methanol (1:1, v/v) at 30°C. (left)
L
-Ala, (right)
D
-Ala. [H] =
0
0
.10 mM, [G] =4.0 mM (3 mL×40).
0
References
pybox-Ph(R)
(
Fig. 6). It was confirmed that L
is the first
pybox-Ph(R) pybox-Ph(S)
eluent by feeding on L
/L
=2 as ana-
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lytes. ITC titration between host ([CuL
] ) and
11
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7. Typical synthetic procedure for L
2
pybox-Ph(S)
Figure 6. Electropherograms of the chiral separations of
is as follows:
pybox-Ph(R) pybox-Ph(S)
L
/L
by CZE (bare fused silica 57 cm×50
To a white suspension of 340 mg (2.00 mmol) of 2,6-
pyridinedicarboxylic acid in 10 mL of dichloromethane
were added 2.2 mL of oxalyl chloride (2 M in DCM) and
catalytic DMF, which was stirred at rt for additional 8 h
to afford a clear yellow solution. All volatiles were evap-
orated under reduced pressure and dried in vacuo to
produce a white 2,6-pyridinedicarbonyl dichloride in
mm I.D., applied +23 kV, detection at 280 nm, 6 mM of
2+
[
Cu(L-Lys)2] as a chiral selector under 10 mM HEPES
buffer (pH 7.4) in aqueous methanol (1:1, v/v) at 23°C. (Red)
without a chiral selector, (blue) with a chiral selector in the
blank run buffer, (green) with a chiral selector in the selector
run buffer.

4
338
H.-J. Kim et al. / Tetrahedron Letters 44 (2003) 4335–4338
quantative yield. To a solution of 604 mg (4.4 mmol) of
R)-2-phenyl glycinol and 2 equiv. (0.7 mL, 5 mmol) of
S.; Campos, K. R.; Connell, B. T.; Staples, R. J. J. Am.
Chem. Soc. 1999, 121, 669–685.
(
TEA in 5 mL of dichloromethane was dropwise added a
solution of 2,6-pyridinedicarbonyl dichloride in 5 mL of
dichloromethane at 0°C under nitrogen. Stirring was
continued at rt for additional 3 h. After all volatiles were
removed under reduced pressure, the white solid of the
bisalcohol was redissolved in 10 mL of CH Cl and 4 equiv.
of TEA (1.3 mL, 9.3 mmol) was added. To the clear
solution was dropwise added 2 equiv. of methanesulfonyl
chloride (340 mL, 4.39 mmol) at 0°C under nitrogen.
Resulting solution was stirred for additional 24 h under
nitrogen. All volatiles are removed under reduced pressure.
Column chromatography in silica gel (EtOAc:Hex=2:1,
9. Host–guest complexation was driven by both enthalpy and
entropy with some guests with hydrophilic side chains such
as Gln and Asn, but the major driving force was the
enthalpy: DH=−3.52 kcal/mol, TDS=0.83 kcal/mol for
pybox-Ph(R) 2+
[CuL
] /D-Gln, DH=−5.52 kcal/mol, TDS=0.93
pybox-Ph(R) 2+
kcal/mol for [CuL
mol, TDS=1.35 kcal/mol for [CuL
30°C. For ITC data for Lys, see Ref. 11.
] /D-Asn, DH=−5.01 kcal/
pybox-Ph(R) 2+
2
2
] /L-Asn at
10. Lu, X.; Chen, Y.; Guo, L.; Yang, Y. J. Chromatogr. A
2002, 945, 249–255.
11. To 0.10 mM of [CuL
pybox-Ph(R)
](NO
)
were stepwise added
-Lys in aqueous
3
2
3 mL×40 times of 4.0 mM of
D
-Lys or L
R =0.18) afforded the desired product as a white solid in
f
methanol at 30°C (10 mM HEPES buffer pH 7.4, MeOH/
a 70% yield over three steps. Further purification was
carried out by recrystallization in MeOH/CH Cl . All the
H
2
O=1/1). After each addition of guests, the evolved heat
was monitored for 180 s to reach equilibration. Nonlinear
regression analysis afforded K
(DH=−4.93(±0.79) kcal/mol and TDS=0.298 kcal/mol
for
2
2
4
−1
NMR spectroscopic and optical properties are in accor-
=1.61(±0.39)×10
a
M
6a,b
+
dance with the reported values.
Mass (ESI , MeOH):
3
−1
m/z 370.5 ([M+H], 100%).
. Evans, D. A.; Kozlowski, M. C.; Murry, J. A.; Burgey, C.
L
-Lys) and K =7.37(±0.73)×10
a
M
(DH=−6.06(±
-Lys).
8
0.75) kcal/mol and TDS=−0.166 kcal/mol for
D