Allosteric binding of alkali metal ions to a pseudocryptand formed by a C-pivot tripodal ligand containing 3-hydroxy-2(1H)-pyridinone and Ga(III)

Article abstract of DOI:10.3987/COM-05-S(K)20

A novel C-pivot tripodal hexadentate ligand (3,2-HOPOHL) composed of 3-hydroxy-2(1H)-pyridinone as a bidentate ligand, the ethyleneoxy chain as a spacer, and tris(carboxyric acid) as an anchor was synthesized. 3,2-HOPOHL recognized only Na⁺ ion, suggesting that it pre-organized a cavity due to the electrostatic interaction among the 2(1H)-pyridinone rings. UV-VIS spectroscopic analysis indicated that 3,2-HOPOHL formed a stable intramolecular 1:1 Fe(III) complex in aqueous solution. The stability constant (log K) of 3,2-HOPOHL-Fe(III) complex was estimated to be 27.6 from the competitive reaction with EDTA. ¹H-NMR titration of 3,2-HOPOHL-Ga(III) complex with Na⁺ and K⁺ ions in CDCl₃-CD₃CN indicated the formation of 1:1 complexes. The binding constants of Na⁺- and K⁺-3,2-HOPOHL-Ga(III) complexes were estimated to be 3.3×10³ and 7.8×10³ M^-1, respectively, the ion selectivity of K⁺ toward Na⁺ being more than two-fold.

Full text of DOI:10.3987/COM-05-S(K)20

HETEROCYCLES, Vol. 66, 2005
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HETEROCYCLES, Vol. 66, 2005, pp. 285 – 297. © The Japan Institute of Heterocyclic Chemistry
Received, 23rd August, 2055, Accepted, 12th October, 2005, Published online, 14th October, 2005. COM-05-S(K)20
ALLOSTERIC BINDING OF ALKALI METAL IONS TO A PSEUDO-
CRYPTAND FORMED BY A C-PIVOT TRIPODAL LIGAND CONTAIN-
ING 3-HYDROXY-2(1H)-PYRIDINONE AND Ga(III) †
Akira Katoh,* Hidenori Kudo, and Ryota Saito
Department of Materials and Life Science, Faculty of Science and Technology,
Seikei University, Kita-machi 3-3-1, Kichijoji, Musashino-shi, Tokyo 180-8633,
Japan; e-mail: katoh@st.seikei.ac.jp
Abstract – A novel C-pivot tripodal hexadentate ligand (3,2-HOPOHL)
composed of 3-hydroxy-2(1H)-pyridinone as a bidentate ligand, the ethyleneoxy
chain as a spacer, and tris(carboxyric acid) as an anchor was synthesized.
3,2-HOPOHL recognized only Na+ ion, suggesting that it pre-organized a cavity
due to the electrostatic interaction among the 2(1H)-pyridinone rings. UV-VIS
spectroscopic analysis indicated that 3,2-HOPOHL formed
a
stable
intramolecular 1:1 Fe(III) complex in aqueous solution. The stability constant
(log K) of 3,2-HOPOHL-Fe(III) complex was estimated to be 27.6 from the
competitive reaction with EDTA. 1H-NMR titration of 3,2-HOPOHL-Ga(III)
complex with Na+ and K+ ions in CDCl3-CD3CN indicated the formation of 1:1
complexes. The binding constants of Na+- and K+-3,2-HOPOHL-Ga(III)
complexes were estimated to be 3.3×103 and 7.8×103 M-1, respectively, the ion
selectivity of K+ toward Na+ being more than two-fold.
INTRODUCTION
Allostery plays an important role in enzyme regulation. Allosteric regulation is the control of enzyme
activity by effectors (ions or molecules) which bind to the enzyme at a site other than the active site, but
change the conformation of the active site.1 An allosteric enzyme is unable to act directly toward a
substrate. However, three-dimensional change in enzyme’s active site induced by binding of an effector
to the allosteric site results in binding of a substrate to its active site. Such phenomenon is called alloste-
† Dedicated to Dr. Kenji Koga, Emeritus Professor of Tokyo University as the memory.

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HETEROCYCLES, Vol. 66, 2005
ric effect”. Allosteric enzymes exist in the turning point of the metabolic pathway and involve in the
metabolic regulation in vivo. Metal ions are often seen in allosteric regulation of enzymes.2 Recently,
studies on the reproduction of the allosteric effect by artificial systems have been actively performed in
the field of supramolecular chemistry.3 Nabeshima and co-workers have demonstrated the first example
of pseudocrown ethers for allosteric switching of ion regulation.4 The oligoethyleneglycol-ligating two
bipyridine units formed a complex with Cu(I) and as a result the conformation change occurred to form
the pseudocrown ether which recognizes alkali metal ions. This concept has been also applied to a
pseudocryptands. The recognition of alkali metal ions by pseudocryptands formed by bipyridine-5 or
catechol-armed azacrown ether6 and metal ions has been reported. The recognition of alkali metal ions
by pseudocryptand-like complexes with M2L3 coordination mode (M=Fe(III), Ti(IV), and Ga(III);
L=β-diketone,7 catechol,8 and 8-hydroxyquinoline9) has been also reported. However, only a paper10 on
Cs+ recognition by a pseudocryptand formed by complexation of a N-pivot tripodal hexadentate ligand
having bipyridine with Fe(II) has been reported.
In our laboratory, synthesis of tripodal hexadentate ligands bearing hydroxyazine-type heterocycles and
functional evaluation of their transition metal complexes have been intensively investigated.11 In the
previous paper,12 we reported for the first time the allosteric binding of alkali metal ions to a
pseudocryptand formed by a C-pivot tripodal hexadentate ligand (3,4-HOPOHL) and Ga(III) as shown in
Figure 1.
O
OH
OH
O
N
O
O
HO
OH
N
CH3
HO
OH
H3C
O
OH
N O
OH
Bidentate ligand
Bidentate ligand
N
O
N
N
N
CH3
N
O
3-hydroxy-4(1H)-
3-hydroxy-2(1H)-
CH3
pyridinone
pyridinone
O
O
O
O
O
O
NH2
OH
O
O
O
O
O
O
O
O
NH
HN
NH
O
NH
HN
NH
C
C O C
C
C O C
O
O
O
O
H2N
Spacer
O
O
H3C
H2N
O
Spacer
O
O
O
H3C
COOH
COOH
COOH
COOH
3,2-HOPOHL
3,4-HOPOHL
O
O
O
O
COOH
COOH
Anchor
CH3
C
C
O
H3C
H3C
O
O
O
M
N
M
Anchor
N
O
O
(b) ortho position
(a) para position
Figure 1 Molecular design of C-pivot tripodal hexadentate ligands
The ion selectivity and binding constant, however, are poor and far below compared to a [2.2.2]cryptand.

HETEROCYCLES, Vol. 66, 2005
287
It may be attributable that 3,4-HOPOHL-Ga(III) complex can not construct an ideal three-dimensional
cavity like a cryptand, because the 4(1H)-pyridinone derivative is linked to the spacer group at the para
position toward the carbonyl group with a divergent mode as shown in Figure 1-(a).
In search for new compound showing higher ion selectivity and binding constant compared to
3,4-HOPOHL, we wish to report here synthesis of a novel C-pivot tripodal hexadentate ligand
(3,2-HOPOHL) and the allosteric binding of alkali metal ions to a pseudocryptand formed by
complexation of it with Ga(III) as shown in Figure 1. The 2(1H)-pyridinone derivative has the spacer
group at the ortho position toward the carbonyl group as shown in Figure 1-(b), and thus it would be
expected to construct an ideal three-dimensional cavity like a cryptand.
The difference of
3,4-HOPOHL and 3,2-HOPOHL is also discussed.
RESULTS AND DISCUSSION
Synthesis of a C-pivot tripodal hexadentate ligand: The synthetic procedure for a C-pivot tripodal
hexadentate ligand (3,2-HOPOHL) was depicted in Scheme 1. A commercially available
3-methoxy-2(1H)-pyridinone was allowed to react with benzyl 2-bromoethyl ether in dry DMF to give
compound (1) in 65% yield. The debenzylation of compound (1) by the catalytic hydrogenation
afforded compound (2) in 77% yield. Compound (2) was subjected to the demethylation with 1M
BBr3 in dry CH2Cl2 to give compound (3) in 89% yield. Treatment of compound (3) with benzyl
chloride in the presence of NaOH under reflux gave compound (4) in 57% yield. The condensation of
2-(2-aminoethoxy)ethanol and phthalic anhydride gave compound (5) in 46% yield. Compound (5) was
converted into the corresponding O-tosyl derivative (6) in 55% yield. The reaction of compound (4)
with (6) afforded compound (7) in 62% yield. Subsequently the deprotection of the phthaloyl group with
methylamine gave compound (8) in 73% yield. The coupling of three equimolar amount of compound
(8) with 1,1,1-tris(succinimideoxycarbonylethoxymethyl)- ethane13 in dry DMF at 38 °C for 69 h14 gave
the O-benzyl-protecting tripodal compound (9) in 67% yield, which was purified by gel chromatography
on Toyopearl HW-40 with MeOH as an eluent. Finally, the debenzylation of compound (9) by the
catalytic hydrogenation smoothly proceeded to give the desired C-pivot tripodal hexadentate ligand
(3,2-HOPOHL) in 82% yield as a colorless amorphous solid. The structural assignment of
1
3,2-HOPOHL was carried out by means of H-NMR and IR spectral analyses. Three characteristic
olefinic protons at C-4, C-5 and C-6 positions of the pyridinone ring were observed at δ 6.82, 6.14, and
6.93 ppm, respectively. The ethyleneoxy and the methyl protons of the anchor moiety were observed at
δ 2.43-4.17 and 0.87 ppm, respectively. The absorption bands due to -OH, C=O, and C-O-C stretching
vibrations were observed at 3415, 1646, and 1102 cm-1, respectively. Further, 3,2-HOPOHL showed
the hydroxamic acid test (+).

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HETEROCYCLES, Vol. 66, 2005
OCH3
O
OCH3
OCH3
O
OBn
NaH
H2/10% Pd-C
in MeOH
N
N
O
N
H
in dry DMF
Br
OH (2)
OBn
(1)
OBn
OH
O
BBr3
BnCl, NaOH
in MeOH
N
O
N
(2)
in dry CH2Cl2
(4)
OH
(3)
OH
O
OH
O
O
p-TsCl
O
O
O
O
N
N
O
in dry pyridine
O
O
H2N
(5)
(6)
OH
OTs
OBn
O
OBn
O
N
N
O
40% MeNH2
in THF
NaH
O
(6)
(4)
in dry THF
O
O
N
O
(8)
O
NH2
(7)
RO
OR
OR
N
O
O
O
N
O
N
O
COOSu
COOSu
O
O
O
3 x (8)
O
in dry DMF
38oC
COOSu
C
H3C
O
O
O
69 h
NH
C
HN
C
NH
C O
O
O
O
O
O
H3C
(9) R=Bn
H2/10%Pd-C in MeOH
3,2-HOPOHL R=H
Scheme 1 Synthetic procedure for C-pivot tripodal hexadentate ligand (3,2-HOPOHL)

HETEROCYCLES, Vol. 66, 2005
289
Properties of 3,2-HOPOHL
Conformation in solution: 1H-NMR spectrum of 3,2-HOPOHL in DMSO-d6 solution at 20 °C
exhibited one set of signals, indicating that it possesses the pseudo-C3-symmetrical structure. The
temperature dependence of the amide proton chemical shift was measured at various temperatures from
20 to 80 °C, and plots of chemical shifts vs. temperatures gave a straight line. (not shown) The
temperature dependence coefficient for a strong intramolecular hydrogen bond has been reported to be
usually less than -3×10-3 ppm deg-1.15 A large temperature dependence coefficient (-6.0×10-3 ppm deg-1)
indicated that no strong intramolecular hydrogen bond exists in DMSO-d6 solution.
Recognition of Na+ ion by the pre-organization
In the case of 3,4-HOPOHL, any change in the chemical shift was not observed upon adding an
equimolar amount of alkali metal ions in CDCl3-CD3CN solution. On the other hand, in the case of
3,2-HOPOHL, the apparent lower magnetic field shift was observed when an equimolar amount of
NaClO4 was added to the solution. Plots of Δδ vs. [Na+]/[3,2-HOPOHL] showed a curvature as shown
in Figure 2, and the intersection point of the extrapolation of two lines was 1.06, indicating the formation
1
of a 1:1 Na+ complex. The binding constant K was determined by the H-NMR titration of host
(3,2-HOPOHL) with guest (Na+ ion) in CDCl3-CD3CN solution. Under the Benesi-Hildebrand
conditions,16 the following equation is used;
1/ΔδobsH=1/ΔδcompH + 1/ΔδcompH×1/K×1/[guest]
Where ΔδobsH is the observed chemical shift change, and [guest] is the total concentration of alkali metal
ion. A straight line was obtained when 1/[guest] was plotted against 1/ΔδobsH as shown in Figure 3.
The binding constant K was calculated to be 160 M-1 from the slope and intercept.
10-2
10
10
20
20
19
18
18
17
9
8
8
7
6
6
5
16
16
s
b
o
15
4
4
14
14
13
3
2
1
2
12
12
11
0
0
10
10
100
100
150
150
200
250
250
300
300
0
1
2
3
4
4
0
1
2
3
200
[Na+] / [3,2-HOPOHL]
1 / [Na+]
Figure 2 Plots of ΔδobsH vs. the molar ratio
of Na+ ion to 3,2-HOPOHL
Figure 3 The Benesi-Hildebrand plot of
Na+-3,2-HOPOHL

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It is noteworthy that the three ring protons of 2(1H)-pyridinone of 3,2-HOPOHL largely shifted to the
lower magnetic field upon complexation with Na+ ion. Only difference between two C-pivot tripodal
hexadentate ligands is the structure of heterocyclic bidentate ligands, that is, 3-hydroxy-4(1H)-pyridinone
in 3,4-HOPOHL vs. 3-hydroxy-2(1H)-pyridinone in 3,2-HOPOHL. This striking difference in the
binding property toward Na+ ion may be explained by the pre-organization of 3,2-HOPOHL suitable for
Na+ accommodation, although the supporting spectral data could not be obtained.
.
Fe(III) complex formation: UV-VIS spectra of a 1:1 molar mixture of 3,2-HOPOHL and Fe(III) in
aqueous solution were measured in the range from pH 2.9 to 8.4 as shown in Figure 4. The absorption
maxima due to the ligand-to-metal charge transfer were observed at 500-515 and 410-420 nm. The
absorption maxima did not change even at increasing pH, although the absorbance decreased with an
increase of pH, indicating that 3,2-HOPOHL makes a very stable Fe(III) complex even at the acidic
region. λmax and ε values of the complex at pH 2.9 were 515 (4300 dm3mol-1cm-1) and 420 nm (3300
dm3mol-1cm-1). These values are comparable to the reported values17,18 of a 3,2-HOPO-Fe(III) (3:1)
complex, suggesting that 3,2-HOPOHL forms a stable intramolecular 1:1 Fe(III) complex. The 1:1
stoichiometry was also supported by the result of the molar ratio method as shown in Figure 5, in which
an intersection point was 1.03.
The stability constant of Fe(III) complex: The relative stability constant (KFe(L)) of a hexadentate
ligand with Fe(III) is defined by the following equation.
Fe3+ + L3-
Fe(L)
KFe(L) = [Fe(L)]/[Fe3+][L3-]
The competitive Fe(III) exchange reaction19 between EDTA and 3,2-HOPOHL was carried out in order
to obtain the stability constant of Fe(III) complex. Three pKa values of the ligand are necessary for
calculation. These values, however, were approximated by the pKa value (8.66)18 of the model
bidentate ligand, 3-hydroxy-2(1H)-pyridinone, owing to the limitation of the solubility. Absorbance at
515 nm was monitored in order to determine the equilibrium point. (not shown) The relative stability
constant was calculated from pKa values19 of EDTA, pKa of 3-hydroxy-2(1H)-pyridinone, the stability
constant of Fe(EDTA) (logK=25.120), and the equilibrium constant. The relative stability constant was
calculated to be 27.6 in logK, being smaller than natural ferrioxamine B (logK=30.520).
Ga(III) complex formation: 1H-NMR spectrum of Fe(III) complex could not be measured due to the
paramagnetic character of Fe(III). Ga(III) ion is diamagnetic and its ion radius is very close to Fe(III).
Ga(III) complex, therefore, was prepared by mixing an equimolar amount of 3,2-HOPOHL and
1
Ga(acac)3 in CDCl3 solution.12 On H-NMR spectrum of the Ga(III) complex, two signals due to an
olefinic proton and methylene protons adjacent to N-1 of the 2(1H)-pyridinone ring apparently shifted
to the down field compared to those of free ligand; Δδ 0.25 for 5-H, 0.04 ppm for -CH2-N-. On the
other hand, two signals due to olefinic protons of the 2(1H)-pyridinone ring shifted to the upper field

HETEROCYCLES, Vol. 66, 2005
291
compared to those of free ligand; Δδ 0.11 for 4-H, 0.17 ppm for 6-H. Further, the broadening of all
signals of the Ga(III) complex was observed owing to a decrease of flexibility upon complexation.
Figure 4 Spectral change of 3,2-HOPOHL-Fe(III) Figure 5 Plots of absorbance at 515 nm
complex in aqueous solution; 1(pH 2.9), 2(3.3), 3(3.9)
4(5.9), 5(7.1), 6(8.0), and 7(8.4)
vs. the molar ratio of Fe(III) to
3,2-HOPOHL at pH 2.9
Recognition of alkali metal ions by a pseudocrypyand formed by 3,2-HOPOHL and Ga(III): A
change in the chemical shift of each signal was observed when 3,2-HOPOHL-Ga(III) complex was
mixed with alkali metal ions, suggesting the cooperative recognition of alkali metal ions with the Ga(III)
complex. In the case of Na+, the formation of 1:1 complex was confirmed by the molar ratio plot; plots
of ΔδobsH vs. the molar ratio [Na+]/[3,2-HOPOHL-Ga(III) complex] gave an intersection point at 0.99.
The binding constant K was determined by 1H-NMR titration of 3,2-HOPOHL-Ga(III) complex with Na+
ion in CDCl3-CD3CN solution.
a
O
O
O
O
b
Ga3+
O
Ga3+
O
c
O
O
O
N
N
N
O
N
N
O
N
O
d
M+
O
e
O
O
O
O
O
M+
O
O
O
O
O
O
f
NH
C
NH
C
HN
C
NH
C
NH
C
HN
C
O
O
O
O
O
O
O
O
O
O
O
O
CH3
CH3
Figure 6 A possible structure of the allosteric binding of alkali metal ions by pseudocryptand formed
by 3,2-HOPOHL-Ga(III) complex

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HETEROCYCLES, Vol. 66, 2005
Under the Benesi-Hildebrand conditions,16 the binding constant K of the host with Na+ ion was calculated
to be 3.3×103 M-1 from the slope and intercept. In the case of K+ ion, the formation of 1:1 complex was
also confirmed by the molar ratio plot; an intersection point at 1.02. The binding constant K was
calculated to be 7.8×103 M-1. The binding constants toward Na+ and K+ ions of 3,2-HOPOHL-Ga(III)
complex are far larger than those12 (K=5.9x102 for Na+ and 5.8x102 M-1 for K+) of 3,4-HOPOHL-Ga(III)
complex. It may be attributable that 3,2-HOPOHL-Ga(III) complex can construct a suitable cavity like
a cryptand compared to 3,4-HOPOHL-Ga(III) complex. The spacer group at the ortho position toward
the C=O group would be favorable to form a cryptand-like cavity. Further, large downfield shifts are
observed toward protons of the 3-hydroxy-2(1H)-pyridinone ring and of the ethyleneoxy moiety (a-f in
Figure 6), indicating that Na+ and K+ ions locate at nearly central position upon complexation.
EXPERIMENTAL
1-(2-Benzyloxyethyl)-3-methoxypyridin-2(1H)-one (1)
To a suspension of NaH (60% in an oil, 1.05 g, 23.9 mmol), which was washed with hexane, was slowly
added a solution of 3-methoxy-2(1H)-pyridinone (3.0 g, 23.9 mmol) in dry DMF (7 mL), and then the
mixture was stirred for 1 h on an ice bath. To the mixture was added a solution of benzyl 2-bromoethyl
ether (5.15 g, 23.9 mmol) in dry DMF (5 mL), and then the reaction mixture was stirred for 24 h at rt.
After evaporation of the solvent, the residue was dissolved in H2O (10 mL), and then extracted with
AcOEt (50 mLx3). The combined organic layers were dried over anhydrous Na2SO4. After
evaporation of the solvent, the residual oil (6.75 g) was purified by column chromatography on silica gel
60 (60-200 μm) with CHCl3:acetone:EtOH (100:5:1) mixture to give the product (1) (4.04 g, 65%) as a
1
yellow oil; IR(neat): 3062, 2940, 1654, 1101, 1052, and 740 cm-1 ; H-NMR(δ, CDCl3, 400 MHz): 3.79
(2H, t, J=5.0 Hz, N- CH2), 3.82 (3H, s, O-Me), 4.19 (2H, t, J=5.0 Hz, BnO-CH2), 4.47 (2H, s, Ph-CH2),
6.09 (1H, t, J= 7.3 Hz, 5-H), 6.63 (1H, dd, J=1.7 and 7.3 Hz, 4-H), 7.02 (1H, dd, J=1.7 and 7.3 Hz, 6-H),
and 7.23-7.32 ppm (5H, m, Ph). Anal. Calcd for C15H17NO3・0.5H2O: C, 67.15; H, 6.76; N, 5.22.
Found: C, 67.01; H, 6.58; N, 5.38.
1-(2-Hydroxyethyl)-3-methoxypyridin-2(1H)-one (2)
A suspension of 10% Pd-C (80 mg) in MeOH (15 mL) was prehydrogenated with H2 for 30 min. To the
suspension was added a solution of 1 (800 mg, 3.08 mmol) in MeOH (35 mL). After hydrogenation
with H2 under atmospheric pressure for 3 h at rt, the catalyst was removed by filtration. The filtrate was
evaporated to give the residue, which was purified by column chromatography on silica gel 60 with
CHCl3:MeOH (9:1) mixture to give the product (2) (405 mg, 77%) as a colorless solid; mp: 109-112 °C;
IR(KBr): 3322, 2954, 1652, 1191, 1072, and 740 cm-1; 1H-NMR(δ, CDCl3, 400 MHz): 3.83 (3H, s, O-Me),
3.96 (2H, t, J=5.0 Hz, N-CH2), 4.18 (2H, t, J=5.0 Hz, CH2-OH), 6.17 (1H, t, J=7.3 Hz, 5-H), 6.67 (1H, dd,

HETEROCYCLES, Vol. 66, 2005
293
J=1.4 and 7.3 Hz, 4-H), and 6.95 ppm (1H, dd, J=1.4 and 7.3 Hz, 6-H). Anal. Calcd for C8H11NO3: C,
56.80; H, 6.55; N, 8.28. Found: C, 56.49; H, 6.38; N, 8.21.
3-Benzyloxy-1-(2-hydroxyethyl)pyridin-2(1H)-one (4)
To a solution of 2 (2 g, 11.8 mmol) in dry CH2Cl2 (20 mL) was added a solution of 1M BBr3 in dry
CH2Cl2 (11.8 mL) at -30 °C. The reaction mixture was stirred for 24 h at rt, and then MeOH was added
to the mixture at -30 °C. After evaporation of the solvent, the residue was dissolved in H2O and
neutralized with 1M NaOH. After removal of the solvent, the residue was recrystallized from EtOH to
give 3-hydroxy-1-(2-hydroxyethyl)pyridine-2(1H)-one (3) (1.60 g, 89%) as a colorless solid; hydroxamic
acid test: positive; mp: 140-142 °C; IR(KBr): 3394, 2979, 2888, 1647, 1553, and 1471 cm-1; 1H-NMR(δ,
CD3OD, 400 MHz): 3.82 (2H, t, J=5.1 Hz, -CH2-OH), 4.10 (2H, t, J=5.1 Hz, N-CH2), 6.21 (1H, t, J=7.0
Hz, 5-H), 6.82 (1H, dd, J=1.6 and 5.6 Hz, 4-H), and 7.08 ppm (1H, dd, J= 1.6 and 5.6 Hz, 6-H ).
To a solution of 3 (1.6 g, 10.3 mmol) in MeOH (20 mL) was added 4M NaOH (3 mL) and benzyl
chloride (5 mL), and then the reaction mixture was refluxed for 24 h. After evaporation of the solvent,
the residue was dissolved in H2O (50 mL), and extracted with CH2Cl2 (50 mLx3). The combined organic
layers were washed with saturated NaCl solution (30 mL), and then dried over anhydrous Na2SO4. After
evaporation of the solvent, the residual oil was purified by column chromatography on silica gel 60 with
CHCl3:MeOH (9:1) mixture to give the product (4) (1.41 g, 57%) as a colorless solid; mp: 89-90 °C;
IR(neat): 3370, 2948, 2875, 1648, 1593, 1554, 1497, 1454, 1231, 744, and 698 cm-1; 1H-NMR(δ, CDCl3,
400 MHz): 3.96 (2H, t, J=4.4 Hz, N-CH2), 4.17 (2H, t, J=4.4 Hz, -CH2-OH), 5.11 (2H, s, -CH2-), 6.08
(1H, t, J=7.0 Hz, 5-H), 6.69 (1H, dd, J=1.4 and 7.0 Hz, 4-H), 6.94 (1H, dd, J=1.4 and 7.0 Hz, 6-H), and
7.30-7.44 ppm (5H, m, Ph). Anal. Calcd for C14H15NO3・0.1H2O: C, 68.06; H, 6.20; N, 5.67. Found: C,
68.14; H, 6.15; N, 5.82.
2-(2-(2-Hydroxyethoxy)ethyl)isoindoline-1,3-dione (5)
A mixture of 2-(2-aminoethoxy)ethanol (5.00 g, 47.5 mmol) and phthalic anhydride (7.04 g, 47.5 mmol)
was heated at 180 °C on an oil bath. The residue was recrystallized from H2O to give the product (5)
(5.21 g, 46%) as a colorless solid; mp: 61.5-63.0 °C; IR(KBr): 3417, 2940, 2873, 1641, 1120, and 794
cm-1; 1H-NMR(δ, CDCl3, 400 MHz): 3.60 (2H, t, J=5.3 Hz, CH2-O), 3.68 (2H, t, J=5.3 Hz, O-CH2), 3.75
(2H, t, J=5.3 Hz, N-CH2), 3.91 (2H, t, J=5.3 Hz, CH2-OH), 7.72 (2H, q, J=3.1 Hz, 5-H and 6-H
isoindoline-1,3-dione), and 7.85 ppm (2H, q, J=3.1 Hz, 4-H and 7-H isoindoline-1,3-dione). Anal.
Calcd for C12H13NO4: C, 61.27; H, 5.57; N, 5.95. Found: C, 60.97; H, 5.32; N, 5.87.
2-(2-(2-O-p-Toluenesulfonylethoxy)ethyl)isoindoline-1,3-dione (6)
To a solution of p-toluenesulfonyl chloride (4.05 g, 21.2 mmol) in dry pyridine (40 mL) was slowly
added 5 (5 g, 21.2 mmol) on an ice bath, and then the reaction mixture was stirred for 6 h. To the
mixture was added ice water, and then the aqueous solution was extracted with CH2Cl2 (25 mLx4). The

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combined organic layers were washed with cold 6M HCl (30 mLx7), saturated NaCl solution (30 mL),
and then dried over anhydrous Na2SO4. After evaporation of the solvent, the residue was recrystallized
from AcOEt to give the product (6) (4.57 g, 55%) as a colorless solid; mp: 81.0-83.0 °C; IR(KBr): 2913,
2871, 1715, 1351, 1178, 1116, 813, and 777 cm-1; 1H-NMR(δ, CDCl3, 400 MHz): 2.43 (3H, s, CH3), 3.66
(4H, m, CH2-O-CH2), 3.83 (2H, t, J=5.6 Hz, N-CH2), 4.11 (2H, t, J=5.6 Hz, O-CH2), 7.32 (2H, d, J=8.0
Hz, 3-H and 5-H p-tosyl), 7.72 (2H, q, J=3.1 Hz, 5-H and 6-H indoline-1,3-dione), 7.75 (2H, d, J=8.0 Hz,
2-H and 6-H p-tosyl), and 7.84 ppm (2H, q, J=3.1 Hz, 4-H and 7-H indoline-1,3-dione). Anal. Calcd for
C19H19NO6S: C, 58.60; H, 4.92; N, 3.60. Found: C, 58.38; H, 4.72; N, 3.74.
1-(2-(2-(2-Phthalimidoethoxy)ethoxy)ethyl)-3-benzyloxypyridine-2(1H)-one (7)
To a suspension of NaH (60% in an oil, 179 mg, 4.48 mmol), which was washed with hexane, was slowly
added a solution of 4 (1.00 g, 4.07 mmol) in dry THF (10 mL). The mixture was stirred for 5 h at -10 °C,
and then a solution of 6 (1.58 g, 4.07 mmol) in dry THF (10 mL) was added to the mixture at -10 °C.
The reaction mixture was heated to reflux for 18 h. After evaporation of the solvent, the residue was
dissolved in H2O (10 mL), extracted with AcOEt (50 mLx3). The combined organic layers were washed
with saturated NaCl solution (30 mL), and then dried over anhydrous Na2SO4. After evaporation of the
solvent, the residual oil (2.16 g) was purified by column chromatography on silica gel 60 with
AcOEt:hexane (5:1) mixture to give the product (7) (1.18 g, 62%) as a yellow oil; IR(neat): 2953, 2919,
2865, 1711, 1643, 1601, 1493, 1431, 1278, 1189, 1114, 777, 746, and 723 cm-1; 1H-NMR(δ, CDCl3, 400
MHz): 3.51 (2H, t, J=3.4 Hz, CH2O), 3.57 (2H, t, J=3.4 Hz, OCH2), 3.69 (2H, t, J=5.8 Hz, CH2O), 3.72
(2H, t, J=5.8 Hz, N-CH2), 3.87 (2H, t, J=5.8 Hz, CH2-N(C=O)2), 4.10 (2H, t, J=5.8 Hz, OCH2), 5.98 (1H,
t, J=7.0 Hz, 5-H), 6.63 (1H, dd, J=1.4 and 5.8 Hz), 6.99 (1H, dd, J=1.4 and 5.8 Hz), 7.29-7.44 (5H, m,
Ph), 7.71 (2H, q, J=2.4 Hz, 5-H and 6-H isoindoline-1,3-dione), and 7.84 (2H, q, J=2.4 Hz, 4-H and 7-H
isoindoline-1,3-dione). Anal. Calcd for C26H26N2O6・0.5H2O: C, 66.23; H, 5.77; N, 5.94. Found: C,
66.39; H, 5.76; N, 5.69.
1-(2-(2-(2-Aminoethoxy)ethoxy)ethyl)-3-benzyloxypyridine-2(1H)-one (8)
To a solution of 7 (850 mg. 1.83 mmol) in THF (10 mL) was added 40% aqueous MeNH2 solution (200
mL), and then the reaction mixture was heated for 18 h at 60 °C. After evaporation of the solvent, the
residual oil was purified by column chromatography on aluminum oxide (63-200 μm) with CHCl3:MeOH
(10:1) mixture to give the product (8) (450 mg, 73%) as a yellow oil; IR(neat): 3428, 2925, 1647, 1233,
1
1114, and 755 cm-1; H-NMR(δ, CDCl3, 400 MHz): 2.82 (2H, t, J=5.3 Hz, CH2NH2), 3.45 (2H, t, J=5.3
Hz, OCH2), 3.57 (4H, m, O-CH2-CH2-O), 3.80 (2H, t, J=5.1 Hz, CH2O), 4.17 (2H, t, J=5.1 Hz, N-CH2),
5.99 (1H, t, J=7.0 Hz, 5-H), 6.65 (1H, dd, J=1.6 and 5.8 Hz, 4-H), 7.02 (1H, dd, J=1.6 and 5.8 Hz, 6-H),
and 7.30-7.44 ppm (5H, m, Ph). Anal. Calcd for C18H24N2O4・H2O: C, 61.70; H, 7.48; N, 7.99. Found:
C, 61.68; H, 7.19; N, 7.92.

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1,1,1-Tris{carbonylethoxymethyl-7-amino-10,13-dioxaoctyl-16-(1’,2’-dihydro-3’-hydroxy-2’-oxo-
pyridin-1’-yl)}ethane (3,2-HOPOHL)
To a solution of compound (8) (232 mg, 0.698 mmol) in dry DMF (5 mL) was added a solution of
1,1,1-tris(succinimideoxycarbonylethoxymethyl)ethane13 (146 mg, 0.233 mmol) in dry DMF (5 mL), and
then the reaction mixture was stirred for 69 h at 38 °C. After evaporation of the solvent under reduced
pressure, H2O (50 mL) was added to the residue, and the aqueous solution was extracted with CHCl3 (50
mLx3). The combined organic layers were successively washed with H2O (25 mL), 5% NaHCO3 (50
mLx2), 5% citric acid (50 mLx2), saturated NaCl solution (50 mL), and then dried over anhydrous
Na2SO4. After evaporation of the solvent, the residue was purified by gel chromatography on Toyopearl
HW-40 with MeOH as an eluent to give 1,1,1-tris{carbonylethoxymethyl-7-amino-10,13-dioxaoctyl-16-
(3’-benzyloxy-1’,2’-dihydro-2’-oxo-pyridin-1’-yl)}ethane (9) (200 mg, 67%) as a yellow oil; IR(neat):
3300, 1648, 1109, 751, and 699 cm-1; 1H-NMR (δ, CDCl3, 400 MHZ): 0.87 (3H, s, -C-CH3), 2.41 (6H, t,
J=6.1 Hz, -CH2CO), 3.23 (6H, s, O-CH2-C), 3.39 (6H, t, J=5.4 Hz, CH2-NH), 3.47 (6H, t, J=5.4 HZ,
O-CH2-), 3.53 (12H, m, O-CH2-CH2-O), 3.62 (6H, t, J=6.1 HZ, CH2-O), 3.79 (6H, t, J=5.1 Hz, -N-CH2-),
4.14 (6H, t, J=5.1 HZ, O-CH2-), 5.09 (6H, s, CH2-Ph), 6.01 (1H, t, J=7.0 Hz, 5-H), 6.66 (1H, dd, J=1.4
and 5.8 Hz, 4-H), 6.99 (1H, dd, J=1.4 and 5.8 Hz, 6-H), and 7.27-7.43 ppm (5H, m, Ph).
A suspension of 10% Pd-C (20 mg) in MeOH (20 mL) was prehydrogenated with H2 for 30 min. To the
suspension was added a solution of 9 (200 mg, 0.156 mmol) in MeOH (10 mL). After hydrogenation
with H2 under atmospheric pressure for 2 h at rt, the catalyst was removed by filtration. The filtrate was
evaporated to give the crude product, which was purified by gel chromatography on Toyopearl HW-40
with MeOH as an eluent to afford the desired product (3,2-HOPOHL) (131 mg, 82%) as a colorless
amorphous solid; hydroxamic acid test: positive; IR (KBr): 3415, 1646, 1595, 1264, and 1102 cm-1;
1H-NMR (δ, CDCl3, 400 MHZ): 0.87 (3H, s, -C-CH3), 2.43 (6H, t, J=5.8 Hz, -CH2CO), 3.25 (6H, s,
O-CH2-C), 3.41 (6H, t, J=5.1 Hz, CH2-NH), 3.49 (6H, t, J=5.1 HZ, O-CH2-), 3.55 (12H, m,
O-CH2-CH2-O), 3.64 (6H, t, J=5.8 HZ, CH2-O), 3.81 (6H, t, J=5.3 Hz, -N-CH2-), 4.17 (6H, t, J=5.3 HZ,
O-CH2-), 6.14 (1H, t, J=7.0 Hz, 5-H), 6.82 (1H, dd, J=1.4 and 5.6 Hz, 4-H), and 6.93 ppm (1H, dd, J=1.4
and 5.6 Hz, 6-H). Anal. Calcd for C47H72N6O18・2.5H2O: C, 53.55; H, 7.36; N, 7.97. Found: C, 53.60;
H, 7.04; N, 7.67.
Measurement of UV-VIS spectra of Fe(III) complex: 3,2-HOPOHL (109 mg, 0.108 mmol) was
dissolved in deionized H2O (50 mL). A 1.0 mL volume of the sample was mixed with an equimolar
amount of the standard Fe(NO3)3 solution (2.17 mM) and diluted to 10.0 mL (2.17x10-4 M); λmax(ε)= 515
(4300 dm3mol-1cm-1) and 420 nm (3300 dm3mol-1cm-1).
The molar ratio plot: 3,2-HOPOHL (109 mg, 0.108 mmol) was dissolved in deionized H2O (50 mL).
A 0.5 mL volume of the standard aqueous Fe(NO3)3 solution (2.17 mM) was mixed with an appropriate

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amount of the sample solution. Visible spectra of the mixture were measured. Absorbance of each
solution at 515 nm was plotted against the molar ratio of [3,2-HOPOHL]/[Fe(III)]; intersection
point=1.03.
Fe(III) exchange reaction: A buffer solution (5 mL phosphate buffer (pH=6.5, and [KNO3]=0.23 M)
containing 4.34x10-6 mol of Fe(III) complex was prepared. An EDTA solution was prepared by
dissolving EDTA・2Na (2.91 mg, 8.68x10-6 mol) in phosphate buffer, and diluting to 10.0 mL. The
Fe(III) exchange reaction was initiated by mixing of the complex solution (2.0 mL) with the EDTA
solution (2.0 mL), and followed by monitoring a decrease of absorbance at 515 nm.
Measurement of the binding constant of Na+ ion to 3,2-HOPOHL: 3,2-HOPOHL (2.354 mg, 2.33
1
μmol) was dissolved in CDCl3:CD3CN (1:1) mixture (0.78 mL), and then H-NMR spectrum was
measured. 4μL portions of stock NaClO4 solution (2.859 mg, 2.33x10-5 mol/200 μL) in CDCl3:CD3CN
(1:1) mixture was directly added to the solution, and then 1H-NMR spectrum of the resulting solution was
again measured. ΔδobsH vs. molar ratio of [Na+]/[3,2-HOPOHL] was plotted. The binding constant was
calculated according to the Benesi-Hildebrand equation; K=160.
Measurement of the binding constant of Na+ and K+ ions to 3,2-HOPOHL-Ga(III) complex:
3,2-HOPOHL (2.354 mg, 2.33 μmol) and Ga(acac)3 (0.832 mg, 2.33 μmol) were dissolved in
1
CDCl3:CD3CN (1:1) mixture (0.78 mL), and then H-NMR spectrum was measured. 4 μL portions of
the stock NaClO4 solution (2.859 mg, 2.33×10-5 mol, 200 μL) or 9 μL portions of the stock KClO4
solution (1.436 mg, 1.04×10-5 mol, 700 μL) in CDCl3:CD3CN (1:1) mixture was directly added to the
3,2-HOPOHL-Ga(III) complex solution, and then 1H-NMR spectrum of the resulting solution was again
measured. ΔδobsH vs. molar ratio of [K+ or Na+]/[3,2-HOPOHL-Ga(III) complex] was plotted. The
binding constant was calculated according to the Benesi-Hildebrand equation.
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