A new fluorescent probe for zinc(II): An 8-hydroxy-5-N,N- dimethylaminosulfonylquinoline-pendant 1,4,7,10-tetraazacyclododecane

Article abstract of DOI:10.1002/chem.200600379

A new fluorescent probe for Zn²⁺, namely, 8-hydroxy-5-N,N- dimethylaminosulfonylquinolin-2-ylmethylpendant cyclen (L⁸), was designed and synthesized (cyclen = 1,4,7,10-tetraazacyclododecane). By potentiometric pH, ¹H NMR,

Full text of DOI:10.1002/chem.200600379

DOI: 10.1002/chem.200600379
A New Fluorescent Probe for Zinc(II): An 8-Hydroxy-5-N,N-
dimethylaminosulfonylquinoline-Pendant 1,4,7,10-Tetraazacyclododecane
Shin Aoki,*[a, b] Kazusa Sakurama,[c] Nanako Matsuo,[a] Yasuyuki Yamada,[a, b]
Ryoko Takasawa,[d] Sei-ichi Tanuma,[a, d] Motoo Shiro,[e] Kei Takeda,[c] and
Eiichi Kimura[f]
Abstract: A new fluorescent probe for
Zn2+, namely, 8-hydroxy-5-N,N-dime-
thylaminosulfonylquinolin-2-ylmethyl-
pendant cyclen (L8), was designed and
synthesized (cyclen=1,4,7,10-tetraaza-
cyclododecane). By potentiometric pH,
1H NMR, and UV spectroscopic titra-
tions, the deprotonation constants
pKa1–pKa6 of L8·4HCl were determined
to be <2, <2, <2 (for amino groups of
the cyclen and quinoline moieties),
7.19ꢀ0.05 (for 8-OH of the quinoline
moiety), 10.10ꢀ0.05, and 11.49ꢀ0.05,
respectively, at 258C with I=0.1
analysis, showed that L8 and Zn2+ form
a 1:1 complex [Zn
(Hꢁ1L8)], in which
the unique property of quenching the
fluorescence emission of the quinolinol
moiety when not complexed with metal
cations, but enhancing emission when
complexed with Zn2+ or Cd2+. In addi-
AHCTREUNG
the 8-OH group of the quinoline ring
of L8 is deprotonated and coordinates
to Zn2+, in aqueous solution at neutral
pH. On addition of one equivalent of
Zn2+ and Cd2+, the fluorescence emis-
sion of L8 (5 mm) at 512 nm in aqueous
solution at pH 7.4 [10 mm HEPES with
I=0.1 (NaNO3)] and 258C increased
by factors of 17 and 43, respectively.
We found that the cyclen moiety has
tion, the Zn2+–L8 complex [Zn
is much more thermodynamically and
kinetically stable (Kd{Zn
(Hꢁ1L8)}=
N
AHCTREUNG
[Zn2+
]
free[L8]free/[Zn(Hꢁ1L8)]=8 fm at
U
(Hꢁ1L2)] and [Zn(L3)]). Furthermore,
formation of [Zn
ACHTREUNG
faster than those of [Zn
AHCTREUNG
Keywords: bioinorganic chemistry ·
1
(NaNO3). The results of H NMR, po-
tentiometric pH, and UV titrations, as
well as single-crystal X-ray diffraction
[Zn(L3)]. The staining of early-stage
apoptotic cells with L8 is also descri-
bed.
fluorescent probes
·
host–guest
systems · macrocyclic ligands · zinc
Introduction
hydrase, carboxypeptidase, class II aldolase, alkaline phos-
phatase, and collagenase,[2] and as a structural factor in
many enzymes or proteins, such as zinc finger peptides.[3]
Zn2+ apparently plays important physiological roles in living
systems as a neural signal transmitter[4] and an allosteric reg-
ulator of G protein-coupled receptors (GPCR) such as b2
Zinc(II) is the second most abundant transition metal after
iron and an essential element in natural biological systems.[1]
Most Zn2+ is found as a catalytic or cocatalytic factor in the
active sites of more than 300 enzymes including carbonic an-
[d] Dr. R. Takasawa, Prof. S.-i. Tanuma
[a] Prof. S. Aoki, N. Matsuo, Dr. Y. Yamada, Prof. S.-i. Tanuma
Faculty of Pharmaceutical Sciences, Tokyo University of Science
2641 Yamazaki, Noda 278-8510 (Japan)
Genome and Drug Research Center, Tokyo University of Science
2641 Yamazaki, Noda 278–8510 (Japan)
Fax : (+81)4-7121-3670
[e] Dr. M. Shiro
Rigaku Corporation X-ray Research Laboratory
3-9-12 Matsubaracho, Akishima, Tokyo, 196-8666 (Japan)
E-mail: shinaoki@rs.noda.tus.ac.jp
[b] Prof. S. Aoki, Dr. Y. Yamada
Center for Drug Delivery Research
[f] Prof. E. Kimura
Faculty of Pharmaceutical Sciences, Tokyo University of Science
2641 Yamazaki, Noda 278-8510 (Japan)
Faculty of Integrated Arts and Sciences, Hiroshima University
1-7-1 Kagamiyama, Higashi-Hiroshima, 739-8521 (Japan)
[c] K. Sakurama, Prof. K. Takeda
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Division of Medicinal Chemistry
Graduate School of Biomedical Sciences, Hiroshima University
1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551 (Japan)
9066
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Chem. Eur. J. 2006, 12, 9066 – 9080

FULL PAPER
adrenergic receptors or dopamine D2 receptors.[5] In addi-
tion, Zn2+ is reported to be a key intracellular regulator of
apoptosis, that is, programmed cell death occurring during
embryogenesis, lymphocyte selection in the thymus, immu-
nological responses, and many other physiological and
pathological situations.[6] It is now assumed that an intracel-
lular pool contains Zn2+ (free or loosely bound) in the mi-
cromolar to picomolar range.[7]
To elucidate the biological roles of Zn2+, which is spectro-
scopically silent due to its d10 electron configuration, the
demand for determining the concentration of Zn2+ in
sample solutions and in living cells is growing rapidly.[8] Up
to now, Zn2+-selective fluorescent sensors including 6-me-
thoxy-8-p-toluenesulfonamidoquinoline (TSQ), 6-methoxy-
8-p-toluenesulfonamidoquinoline (2-Me-TSQ), and Zinquin
Scheme 2.
(for 4aQ4b+H+) than the pKa (10.8) of the sulfonamide
proton of 2 (Scheme 1).[17,20]
Recently, we reported a new Zn2+ fluorophore based on
the chelation-controlled twisted intramolecular charge trans-
fer (TICT) utilizing the simple ligand 2-pyridylcyclen (6, L4,
Scheme 3).[21] In the absence of Zn2+, the emission maxi-
have been developed, the fluorescence of which is enhanced
[9,10]
considerably on complexation with Zn2+
.
Zinquin is an
effective apoptosis sensor by detecting free Zn2+ in apoptot-
ic cells at early stages.[10] Since the discovery of TSQ and
Zinquin, the design and synthesis of many Zn2+ fluoro-
phores have been reported, and some of them have been
used for the detection of Zn2+ in cultured cells.[11–14]
Macrocyclic polyamines such as 1,4,7,10-tetraazacyclodo-
decane (cyclen) form very stable Zn2+ complexes such as
[Zn2+(cyclen)] (1, [Zn(L1)]) in aqueous solution at neutral
A
pH (Scheme 1).[15,16] Therefore, cyclen is a promising and
Scheme 3.
mum of 6 was observed at 356 nm at neutral pH. On com-
plexation with Zn2+, the pyridyl nitrogen atom of L4 che-
lates to Zn2+ in 1:1 complex 7 ([Zn(L4)]) to fix a perpendic-
ular conformation of the pyridine ring with respect to the di-
alkylamino group in the 2-position, and the emission shifts
to 430 nm from the TICT state. Furthermore, it was found
that anion (Xꢁ) coordination to Zn2+ of 7 yields [Zn(L4)X]
complex 8, the emission of which shifts to about 350 nm and
allows anion sensing.
Scheme 1.
8-Hydroxyquinoline (9a, 8-HQ, also called oxine) and its
derivatives such as 9b (L6) and 9c (L7) are classified in the
second most important category of chelating agents after
EDTA and are used as fluorescence sensors of various
metal cations.[8b,22] While 8-HQ generally does not exhibit
good selectivity for specific metal ions, a hexapeptide 10
containing a Val-Pro-d-Ser-Phe-Cys-Ser sequence (for re-
verse-turn formation) with an artificial amino acid having an
8-HQ unit (2-Oxn), designed by Imperiali et al., was shown
suitable chelator for Zn2+ fluorophores. We previously re-
ported that dansylamide-pendant cyclen 2 (L2) quantitative-
ly responds to Zn2+ at submicromolar concentrations in
aqueous solution as a result of dansylamide deprotonation
in 1:1 complex 3 ([Zn
(Hꢁ1L2)]; Scheme 1), the dissociation
A
constant of which Kd{3}, was about 8 pm at pH 7.4.[17,18] We
also reported that 2 is a more stable indicator of apoptosis
than Zinquin[9,10] and acts alone to detect early-stage apop-
totic cells.[19]
to exhibit Zn2+ selectivity.[23] Later, they examined the fluo-
To improve Zn2+ selectivity and sensitivity, (anthrylme-
thylamino)ethyl cyclen 4 (L3) was synthesized (Scheme 2).[20]
The emission enhancement in the 1:1 Zn2+–L3 complex 5
([Zn(L3)]) is due to the retardation of photoinduced elec-
tron transfer (PET) by chelation of a nitrogen atom of the
side chain to Zn2+. Interestingly, 5 is more stable than 3 at a
slightly acidic pH (see below), possibly because of the lower
pKa value (7.2) of the ammonium proton of the side chain
rescence response of 8-HQ derivatives 9a–c to Zn2+
.
[24]
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S. Aoki et al.
At 500 nm, the fluorescence emissions of 9a and 9c ([9a
or 9c]=5 mm) increase by a factor of 8–15 on addition of ten
equivalents of Zn2+ in 10:90 CH3CN/10 mm HEPES [pH 7.4
with I=0.1 (NaNO3)] at 258C (see below). We have thus de-
signed and synthesized new cyclen-based Zn2+ fluorophore
11 (L8) having an 8-hydroxy-5-N,N-dimethylaminosulfonyl-
Results and Discussion
Deprotonation and fluorescence behavior of 8-hydroxy-2-
methylquinoline derivatives: Prior to the synthesis of the
new ligands, we conducted potentiometric pH titrations of
8-hydroxy-2-methylquinoline (9a) and 8-hydroxy-2-methyl-
5-N,N-dimethylaminosulfonylquinoline (9c)[24] with I=0.1
(NaNO3) at 258C and calculated their pKa values, defined
by Equation (1), by using the program BEST.[25] As summar-
ized in Scheme 5, pKa values for L7 of 3.57 (pKa1) and 7.79
(pKa2) were lower than those for L5 of 5.70 (pKa1) and 10.7
(pKa2), due to the electron-withdrawing effects of the N,N-
dimethylaminosulfonyl group at the 5-position. The results
of UV titrations of 9a and 9c ([9a or 9c]=50 mm) with Zn2+
in 99:1 EtOH/10 mm HEPES [pH 7.4 with I=0.1 (NaNO3)]
at 258C (Supporting Information) strongly suggest 2:1 com-
plexation of 9a or 9c with Zn2+. Fluorescence titration of
Scheme 4.
quinoline unit on the side
chain (Scheme 4). We postulat-
ed that 11 would form a 1:1
complex [Zn
(Hꢁ1L8)] (12), in
G
which deprotonation of the hy-
droxyl group of 8-HQ and che-
lation to Zn2+ at neutral pH
allow more sensitive detection
of Zn2+ than 2 and 4. Herein
we describe potent Zn2+ com-
Scheme 5.
plexation by 11 and quantitative Zn2+ sensing in aqueous
solution.
9a and 9c (5 mm) with Zn2+ in 10/90 CH3CN/10 mm HEPES
[pH 7.4 with I=0.1 (NaNO3)] at 258C showed that the emis-
sions of 9a and 9c increase by a factor of 8–15 on addition
of ten equivalents of Zn2+ (excitation at 334–335 nm), as
shown in the Supporting Information (the quantum yield F
of 9c increased from 4.0”10ꢁ3 to 1.6”10ꢁ1). These data indi-
cated that Zn2+ complexes of 9a or 9c are not so stable.
In addition, we synthesized reference compound 13 (L9)
having a di-2-picolylamine (DPA) moiety in order to com-
pare its fluorescence properties with those of 11. The DPA
unit has been widely used for potent Zn2+ chelators such as
N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN,
14),[7c] Zn2+ fluorophores including the Zinpyr series[11] [e.g.,
Zinpyr-1 (15)],[11a–c] the ZnAF series[12] [e.g., ZnAF-R2
(16)],[12d] and 17.[13b]
L ꢂ Hþ Ð L þ Hþ Ka ¼ ½LŠaHþ =½L ꢂ HþŠ
ð1Þ
Synthesis of 11 (L8) and 13 (L9): The new ligand (8-hydroxy-
5-N,N-dimethylaminosulfonylquinoline-pendant cyclen 11
(L8) was synthesized as shown in Scheme 6. Reaction of 2-
bromomethyl-8-benzensulfonyloxy-5-N,N-dimethylaminosul-
fonylquinoline (19) with (Boc)3cyclen 18[26] gave 20, the
PhSO2 group of which was deprotected with aqueous NH3
to afford 21. The Boc groups of 21 were removed with aque-
Scheme 6.
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A New Fluorescent Probe for Zinc(II)
FULL PAPER
Table 1. Deprotonation constants Kai and complexation constants of cyclen (L1), 2 (L2), 4 (L3), and 11 (L8)
with I=0.1 (NaNO3) at 258C.[a]
ous HCl to yield 11 (L8) as
L8·4HCl. Reaction of 19 with
di-2-picolylamine and succes-
Cyclen (L1)[b]
2 (L2)[c]
4 (L3)[d]
11 (L8)
sive removal of PhSO2 and
Boc groups gave reference
compound 13 (L9), which was
isolated as 13·4HCl.[27]
pKa1
<2
<2
9.9
<2
2.2
4.0
9.4
<2
<2
7.2
9.1
<2
<2
<2
7.19ꢀ0.05
7.0ꢀ0.2[e]
10.10ꢀ0.05
11.49ꢀ0.05
22.4ꢀ0.1
14.1
pKa2
pKa3
pKa4
11.0
pKa5
pKa6
10.8
11.8
20.8
11.1
4.0
10.6
Deprotonation constants of 11
(L8) determined by potentio-
logKs([Zn(L)])
16.2
10.6
5.5
17.6
10.7
6.0
logKapp([Zn(L)])[f] at pH 7.4
logKapp([Zn(L)])[g] at pH 5.0
1
metric pH and H NMR titra-
10.8ꢀ0.1
tions: Typical potentiometric
pH titration curves (Figure 1)
[a] For the definition of Ks([Zn(L)]), Kapp([Zn(L)]), and pKa([Zn(L)]), see text. [b] From reference [28].
[c] From reference [17a]. [d] From reference [20]. [e] The pKa4 value was determined based on pH–UV absorp-
tion profiles (see Figure 3a and c). [f] Apparent complexation constants at pH 7.4 with I=0.1 (NaNO3)).
[g] Apparent complexation constants at pH 5.0 with I=0.1 (NaNO3)).
of
a
mixture of 1 mm
L8·4HCl+1 mm HNO3 with
I=0.1 (NaNO3) by addition of
0.1m NaOH at 258C were ana-
lyzed for acid–base equilibri-
um [Eq. (2)]. The deprotona-
tion constants pKai (i=1–6) of
L8·4HCl were determined to
be <2 (pKa1), <2 (pKa2), <2
(pKa2),
7.19ꢀ0.05
(pKa4),
10.10ꢀ0.05 (pKa5), and 11.49ꢀ
0.05 (pKa6) by using the pro-
gram BEST (Table 1).[25] The
pKa1, pKa2, pKa5, and pKa6
values were assigned to depro-
tonation constants of three
secondary amines in a cyclen
ring by comparison with the
Scheme 7.
pKa values for cyclen (L1),[28]
(L2),[17] and 4 (L3)[20] (Table 1).
2
pD range of 5.5–8.5 in D2O (Supporting Information) im-
plies that the pKa value for the 8-OH group of 11 is 7.2ꢀ
0.2. This value agrees well with the pKa value (7.0) obtained
H
ð6ꢁiÞL8 Ð Hð5ꢁiÞL8 þ Hþ
ð2Þ
from its pH–UV profile (see below).
Kai ¼ ½Hð5ꢁiÞL8ŠaHþ =½Hð6ꢁiÞL8Š ði ¼ 1 ꢁ 6Þ
The structure of the H
2(Hꢁ1L8) (=HL8) form was con-
G
firmed by single-crystal X-ray diffraction analysis of a fine
colorless prism of 11 obtained from an aqueous solution at
pH 10. Figure 2 shows the hydrogen-bonding network in-
cluding the deprotonated 8-OH group, an ammonium
proton of the cyclen ring, four water molecules, and one Clꢁ
ion. Based on these results, the deprotonation behavior of
L8 is summarized in Scheme 7 and Table 1.
1
Considerable change of the H NMR signal for H7 of the
quinoline ring of 11 (for numbering, see Scheme 7) in the
Because the solubility of reference ligand 13·4HCl (H4L9)
in aqueous solution was low, the pKa value of 8-OH of 13 at
1
258C was determined by the pH-dependent H NMR spec-
tral change (at [13]=1 mm in 20/80 CD3CN/D2O, data not
shown) and pH–UV profile (at [13]=0.1 mm, see Figure 4)
to be 7.2ꢀ0.2 and 7.4ꢀ0.2, respectively.
The pH-dependent change of UV and fluorescence spectra
of 11 (L8) and 13 (L9): The UV spectra of 11 (L8) in the pH
range of 5–11 are shown in Figure 3a ([11]=50 mm). From
the sigmoidal curve of e258 in Figure 3c, the pKa value for 8-
OH in the ground state of 11 was determined to be 7.0ꢀ0.2,
which agrees well with the pKa values obtained from the po-
Figure 1. Typical potentiometric pH titration curves for a mixture of
1 mm H4L8 +1 mm HNO3 (a) and
a
mixture of 1 mm H4L8 +1 mm
HNO3 +1 mm Zn2+ (b) with I=0.1 (NaNO3) at 258C. Equiv(HOꢁ) is the
A
number of equivalents of base (NaOH) added.
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S. Aoki et al.
Figure 2. ORTEP drawing (50% probability ellipsoids) of 11 in the H2-
8
ꢁ ꢁ
A
ꢁ
ꢁ
O(3) 1.4328(14), S(1) N(6) 1.6339(18), C(16) O(1) 1.302(2); selected di-
hedral angles [8]: N(1)-C(1)-C(2)-N(2) 52.7, N(2)-C(3)-C(4)-N(3) 55.0,
N(3)-C(5)-C(6)-N(4) 64.9, N(4)-C(7)-C(8)-N(1) 58.6, C(14)-C(13)-S(1)-
N(6) 86.0.
1
tentiometric pH (7.19) and H NMR titrations (7.0) descri-
bed above.
Figure 3b shows fluorescence emission spectra of 5 mm 11
(L8) in the pH range of 5–12 [10 mm Goodꢁs buffer with I=
0.1 (NaNO3)] at 258C (excitation at 338 nm, which is an iso-
sbestic point obtained from UV titrations; Figure 3a). As
the pH is raised, emission at 478 nm increases with a sigmoi-
dal curve giving a pKa value of 10.0ꢀ0.2 (Figure 3c), which
is different from the pKa value of 7.0–7.2 in the ground state
described above. The quantum yields F of L8 at pH 7.4 and
12 are 2.0”10ꢁ3 and 1.7”10ꢁ1, respectively. This discrepancy
in pKa values is discussed below.
Figure 3. a) Change in UV spectra of 50 mm 11 (L8) in the range pH 4.0–
12.0 (Goodꢁs buffer) with I=
ACHTREUNG
spectra of 5 mm 11 (L8) in the range pH 8.0–12.0 with I=
AHCTREUNG
258C (excitation at 338 nm). c) Comparison of pH-dependent change in
e258 (open circles) and emission intensity at 478 nm (filled circles) of 5 mm
11 (L8) with I=0.1 (NaNO3) at 258C.
The pH-dependent changes in UV absorption and fluores-
cence emission spectra of 13 (L9) in the pH range of 4–11
are summarized in Figure 4 ([13]=50 mm for UV spectra
and 5 mm for emission spectra). From the sigmoidal curve of
e261 (open circles), the pKa value for 8-OH was determined
to be 7.4ꢀ0.2, which is in fair agreement with the pKa value
of 7.2ꢀ0.2 obtained from 1H NMR titrations (data not
shown). The change in emission intensity of 13 at 496 nm
(excitation at 338 nm) plotted in Figure 4 (filled circles) at
pH 4–10 [10 mm Goodꢁs buffer with I=0.1 (NaNO3)] and
258C was somewhat complex, and this hampered the estima-
tion of the pKa value of 13 in the excited state.[29] For com-
parison, emission intensities of 5 mm 9c (L7) at 478 nm
(filled squares in Figure 4) were very low at pH 4–11 (F at
pH 7.4 was 1.0”10ꢁ3).
Figure 4. Comparison of pH-dependent change in e261 (open circles) and
emission intensity at 496 nm (filled circles) of 13 (L9) with I=0.1
(NaNO3) at 258C (excitation at 338 nm; [13]=50 mm for UV spectra and
5 mm for emission spectra). Filled squares indicate emission intensity of
5 mm 9c (L7) at 496 nm (excitation at 338 nm).
9070
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A New Fluorescent Probe for Zinc(II)
FULL PAPER
Discrepancy of the pKa values for the 8-OH group of 12
(L8) determined by pH–UV and pH–emission profiles: It
was unexpected that the pKa value of 10.0 for 8-OH-quino-
line of 11 (L8) in the excited state as determined by pH–
emission profile (Figure 3c) would be different from the
pKa3 value for the 8-OH group in the ground state of 7.2 de-
termined by potentiometric pH titrations and pH-dependent
UV spectra.
With regard to this kind of discrepancy of the pKa values
between ground and excited states, we previously reported a
selective fluorescent probe for La3+ and Y3+, namely, 22
(L10), which has a dansyl group and three carbamoyl groups
as side chains (Scheme 8).[30] The pKa value of the dansyl-
Scheme 9.
Scheme 8.
amide proton of metal-free 22 in the ground state (deter-
mined by potentiometric pH and UV titrations) was 10.6,
which differed from the pKa value of 8.8 for the same
proton in the excited state (obtained by pH–emission pro-
file). We concluded that the dansylamide-deprotonated form
of 22 is stabilized by hydrogen bonds between the dansyl-
amide proton and the carbamoyl groups (hydrogen-bond ac-
ceptors) in the excited state 22*, which result in increased
fluorescence emission above pH 8–9.
The structure of H
2(Hꢁ1L8) (=HL8) was confirmed by
N
single-crystal X-ray diffraction analysis (Figure 2 and
Scheme 9). By analogy with the photochemical properties of
22 (L10), we postulated that the neutral forms of the 8-OH-
quinoline moiety in H
2(Hꢁ1L8) in the excited state (23a and/
A
or its tautomer 23b[31] in Scheme 9) are stabilized by a hy-
drogen-bonding network formed by the 8-OH group and
two ammonium cations of cyclen, which results in repression
of the fluorescent emission of 11 below pH 8. We assume
that further deprotonation of the cyclen ring in H
2(Hꢁ1L8),
A
which gives H
(Hꢁ1L8) (=L8), weakens the hydrogen-bonded
A
network and enhances emission from the excited state of L8
(24 in Scheme 9).
Figure 5. a) Change in UV absorption spectra of 0.1 mm 11 (L8) on addi-
tion of Zn2+ at pH 7.4 [10 mm HEPES with I=0.1 (NaNO3)] and 258C.
The inset shows the titration curve (increasing e258) of 11 with Zn2+ at
pH 7.4 [10 mm HEPES with I=0.1 (NaNO3)] and 258C. b) Change in
emission spectra of 5 mm 11 (L8) on addition of Zn2+ at pH 7.0 [10 mm
HEPES with I=0.1 (NaNO3)] and 258C (excitation at 338 nm). The inset
shows the increase in the relative emission intensity (I/I0) of 11 at 512 nm
on addition of Zn2+ at pH 7.4 [10 mm HEPES with I=0.1 (NaNO3)] and
258C, in which I0 =emission intensity of L5 at 512 nm in the absence of
UV and fluorescence titrations of 11 (L8) and 13 (L9) with
Zn2+: Figure 5a shows the results of UV absorption titra-
tions of 0.1 mm 11 (L8) with Zn2+ at pH 7.0 [10 mm HEPES
with I=0.1 (NaNO3)] and 258C with isosbestic points at 268
and 338 nm. The inset shows the linear increase in e258
(e258 =2.5”10ꢁ4 for metal-free 11) with increasing concentra-
tions of Zn2+
.
Zn2+
.
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S. Aoki et al.
The emission of metal-free 11 (5 mm) was very weak at
pH 7.4 (excitation at 338 nm) as described above (Fig-
ure 3b), and it quantitatively increased on addition of Zn2+
and reached a plateau at [11]=[Zn2+]=5 mm (Figure 5b),
which is a strong indication for 1:1 complexation of 11 with
Zn2+. As shown in the inset, the emission of 11 at 512 nm
increased by about 17 times with increasing [Zn2+] (F of 11
increased from 2.0”10ꢁ3 to 4.4”10ꢁ2). The 1:1 complexation
its inset (F decreased from 6.1”10ꢁ2 to 2.1”10ꢁ2), presuma-
bly because metal-free 13 had its own moderate emission.[32]
X-ray crystal structure of [Zn
(Hꢁ1L8)] complex 12: Fine col-
G
orless crystals were obtained from a 1:1 mixture of 11 (L8)
and Zn2+ in aqueous solution at pH 7.0. Single-crystal X-ray
structure analysis disclosed that Zn2+ in the [Zn(Hꢁ1L8)]
U
complex 12 is sixfold-coordinated by deprotonated O(1) at
the 8-position of the quinoline ring, N(5) of the quinoline
1
of 11 with Zn2+ was also confirmed by H NMR titrations
ꢁ
(Supporting Information).
ring, and N(1) N(4)of the cyclen ring (Figure 7). The of
2+
The results of UV titrations of the reference compound
13 (L9) (0.1 mm) with Zn2+ at 7.4 [10 mm HEPES with I=
0.1 (NaNO3)] were similar to those of 11 (L8), as shown in
Figure 6A. The increase in absorption at 261 nm and the
inset of Figure 6a imply quantitative formation of the 1:1
Zn2+ O(1) and Zn
N(5) bond lengths of 2.10 and 2.09 Š,
ꢁ
ꢁ
respectively, are shorter than the average Zn2+
distance of 2.21 Š. Four N atoms of the cyclen ring and the
Zn2+ ion form a tetragonal-pyramidal structure.
N(cyclen)
A
ꢁ
complex [Zn
(Hꢁ1L9)] with deprotonation of the 8-OH group
A
of the quinoline moiety. In contrast, addition of Zn2+ to 13
(5 mm) induced a linear decrease in its fluorescence emission
at 496 nm (excitation at 338 nm), as shown in Figure 6b and
Figure 7. ORTEP drawing (50% probability ellipsoids) of [Zn
(Hꢁ1L8)]
G
ꢁ
ꢁ
(12). Selected bond lengths [Š]: Zn(1) O(1) 2.095(8), Zn(1) N(1)
ꢁ
ꢁ
ꢁ
2.30(1), Zn(1) N(2) 2.162(1), Zn(1) N(3) 2.18(1), Zn(1) N(4) 2.180(8),
ꢁ
Zn(1) N(5) 2.094(9).
Comparison of fluorescence responses of 2 (L2), 4 (L3), 11
2+
(L8), and 13 (L9) to Z n , Cd2+, and Cu2+: Fluorescence re-
sponses of 5 mm 11 (L8) to Zn2+, Cd2+, and Cu2+ at pH 7.4
[10 mm HEPES with I=0.1 (NaNO3)] and 258C are com-
pared with those of 2 (L2), 4 (L3), and 13 (L9) in Figure 8.
As previously reported,[17] 2 and 4 (at 5 mm) responded to
Zn2+, which resulted in a linear increase in emission (Fig-
Figure 6. a) Change in UV absorption spectra of 0.1 mm 13 (L9) on addi-
tion of Zn2+ at pH 7.4 [10 mm HEPES with I=0.1 (NaNO3)] and 258C.
The inset shows the titration curve (increasing e258) of 13 with Zn2+ at
pH 7.4 [10 mm HEPES with I=0.1 (NaNO3)] and 258C. b) Change in flu-
orescent emission of 5 mm 13 upon addition of Zn2+ at pH 7.4 [10 mm
HEPES with I=0.1 (NaNO3)] and 258C (excitation at 338 nm). The inset
shows the change in emission intensity (I/I0) of 13 at 496 nm on addition
of Zn2+ at pH 7.4 [10 mm HEPES with I=0.1 (NaNO3)] and 258C, in
which I0 =emission intensity of the metal-free L9 at 496 nm.
ure 8a and b). Addition of Cd2+ to 2 gave almost the same
[17]
titration curve as that of Zn2+
.
Cu2+ quantitatively
quenched the emission of 2 and 4.
9072
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ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 9066 – 9080

A New Fluorescent Probe for Zinc(II)
FULL PAPER
metric pH titrations, the pKa of the Cd2+
-bound H2O in 25 at 258C [with I=0.1
(NaNO3)] was determined to be 10.7ꢀ
0.1 (see Supporting Information for a
typical potentiometric pH titration
curve for 25), which is higher than that
(7.9)[15,28] of the Zn2+-bound H2O in 1
(see Scheme 1). Therefore, we attributed
the lower emission intensity of Zn2+
complex [Zn
C
G
.
Complexation behavior of 11 (L8) with Zn2+ based on po-
tentiometric pH titrations: Analysis of a potentiometric pH
titration curve for a mixture of 1 mm H5L8 and 1 mm ZnSO4
(curve b in Figure 1) with the program BEST[25] gave a com-
plexation constant logKs([Zn(L)]), defined by Equation (3),
for L8 of 22.4, from which the apparent complexation con-
stants logKapp([Zn(L)]), defined by Equations (4) and (5),
were calculated to be 14.1 and 10.8 at pH 7.4 and 5.0, re-
spectively
(dissociation
constant
Kd([Zn(L)])=1/
Kapp([Zn(L)]) at pH 7.4 is 7.9 fm). These values are much
Figure 8. Comparison of fluorescent response of a) 2 (L2) (from
larger than those for the previous Zn2+ complexes [Zn-
ref. [17a]), b) 4 (L3) (from ref. [20]), c) 11 (L8), and d) 13 (L9) to Zn2+
,
(Hꢁ1L2)] (3) and [Zn(L3)] (5).
Cd2+, and Cu2+ at pH 7.4 [20 mm HEPES with I=0.1 (NaNO3)] and
258C. I0 is the emission intensity of each ligand at the indicated wave-
length (528 nm for L2, 416 nm for L3, 512 nm for L8 and L9). The excita-
tion wavelengths were 330 nm for 2, 368 nm for 4, and 338 nm for 11 and
13.
Ksð½ZnðL8ÞŠÞ ¼ ½ZnðHꢁ1L8ފ=½Hꢁ1L8Š½Zn2þ
Š
ð3Þ
ð4Þ
Kappð½ZnðL8ÞŠÞ ¼ ½ZnðHꢁ1L8ފ=½L8Š ½Zn2þ
Š
free
free
ðat designated pHÞ
Figure 8c shows that 11 (L8) linearly responds not only to
Zn2+ (17-fold increase in emission) but also to Cd2+ (43-
fold increase). We assume that the stronger response to
Cd2+ is not a problem, because this metal is not present in
significant amounts in biological cells. The UV titrations of
11 (L8) with Cd2+ and Cu2+ gave almost the same results as
those in Figure 5a, and this strongly suggests that the 8-OH
group of 11 is deprotonated on complexation with Cd2+ and
Cu2+ (data not shown). The X-ray crystal structure analysis
½L8Š
¼
½H5L8Š þ ½H4L8Š þ ½H3L8Š þ ½H2L8Šþ
½H2ðHꢁ1L8ފ þ ½HðHꢁ1L8ފ þ ½Hꢁ1L8Š
free
ðat designated pHÞ
ð5Þ
A distribution diagram for a mixture of 11 (L8, 5 mm) and
Zn2+ (5 mm) is presented in Figure 9a with a comparison
with those for a mixture of 2 (L2, 5 mm) and Zn2+ (5 mm; Fig-
ure 9b) and for a mixture of 4 (L3, 5 mm) and Zn2+ (5 mm,
of the [Cu2+(Hꢁ1L8)] complex (30) obtained from an aque-
G
ous solution at pH 7 revealed that Cu2+ is sixfold-coordinat-
ed by deprotonated O(1), the N atom of the quinolinol
moiety, and the four N atoms of the cyclen ring (Supporting
Information).[33,34] In contrast, emission of reference com-
pound 13 decreased on addition of Zn2+, Cd2+, and Cu2+
(Figure 8d). These results enabled us to conclude that 11
(L8) is a better Zn2+ fluorophore than DPA-based fluoro-
phore 13.
Figure 9c). It is apparent that [Zn
tively formed in the pH range of 5–10 at micromolar con-
centrations and is much more stable than [Zn
(Hꢁ1L2)] (3)
and [Zn(L3)] (5) at pH 5–9. Indeed, the fluorescent emission
A
AHCTREUNG
of [Zn
(Hꢁ1L8)] (12) at pH 4.5–10 was almost constant.[35]
G
Therefore, 11 would be useful for determining [Zn2+] over a
much wider pH range (pH 5–8) than 2 (L2) and 4 (L3) (the
emission of 11 is silent below pH 8, as shown in Figure 3c).
Comparison of Lewis acidity of Zn2+ and Cd2+ based on pH
titrations of Zn2+–cyclen and Cd2+–cyclen: Figure 8c
showed that emission intensity at 512 nm of the Cd2+ com-
plex of 11 was much larger than that of the Zn2+ complex of
11 (12). We assumed that the emission intensity of the metal
complexes of 11 is dependent on the Lewis acidity of the
central metal cation. Thus, we compared the pKa value for
the Cd2+-bound water of Cd2+–cyclen 25 (CdL1) with that
of the Zn2+-bound water in Zn2+–cyclen 1a.[28] By potentio-
Fluorescent response of 11 (L8) and 13 (L9) to various metal
ions in aqueous solution: Next, we examined the fluorescent
response of L8 and L9 (5 mm) to other metal ions including
Co2+, Ni2+, Fe3+, Al3+, Y3+, Ca2+, and Mg2+ at pH 7.4
[10 mm HEPES with I=0.1 (NaNO3)] and 258C. The I/I0
values (I0 and I are the emission intensities of each ligand in
the absence and presence of one equivalent of metal ions:
at 528 nm for L2, at 416 nm for L3, and at 512 nm for L8 and
L9) are presented in Figure 10. Quantitative emission
Chem. Eur. J. 2006, 12, 9066 – 9080
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9073

S. Aoki et al.
and 100 mm Zn2+ (Figure 11). The k2 value for the formation
of 12 ([Zn
C
The k2 for the Zn2+-13 complex ([Zn(Hꢁ1L9)]) was much
faster than that for 12 (k2 for Zn2+-13 >1”106 mꢁ1 sꢁ1).
Because the pKa values for 4 and 11 are almost identical
(7.2), we assumed that the much larger value of k2 for 11 is
because the 8-OH group of the quinolinol moiety of 11 is
fixed close to Zn2+-bound water in [Zn(L8)
Zn2+-bound hydroxide in [Zn(L8)
nation of the quinolinol nitrogen atom to Zn2+ (Scheme 10).
In contrast, the flexible conformation of the anthrylmethyl
group of 25a ([Zn(L3)
sults in rather slow deprotonation of the anthrylmethylam-
monium moiety.
Kinetic stability of [Zn
A
kinetic stability of [Zn
AHCTREUNG
stant logKs of TPEN (14) with Zn2+ was reported to be
15.2,[7c] which is smaller than logKs of 22.4 for 12 (Table 1).
As shown in the Supporting Information, addition of TPEN
(5 mm) to a solution of 12 (5 mm) at pH 7.4 [10 mm HEPES
with I=0.1 (NaNO3)] caused negligible change in the emis-
sion spectra of 12 even after 3 h. Alternatively, addition of
12 (5 mm) to an aqueous solution of Zn2+–TPEN complex
(5 mm) under the same conditions did not enhance emission.
These results strongly indicate that both [Zn
(Hꢁ1L8)] (12)
A
and the Zn2+–TPEN complex are kinetically inert.
Fluorescent staining of Zn2+-loaded and apoptotic HeLaS3
cells with 11: Incubation of HeLaS3 cells with 11 (L8, 50 mm)
and 2 (L2, 50 mm) gave fluorescent images indicating that
Zn2+ concentrations of 11 in intact HeLaS3 cells are very
low (Figure 12a and b). Figure 12c and d show HeLaS3 cells
incubated with Zn2+ (20 mm) and Zn2+ ionophore pyrithione
(2-mercaptopyridine N-oxide, 200 mm) for 30 min to intro-
duce Zn2+ into HeLaS3 cells and then treated with 2 and
11, respectively. They indicate that 2 and 11 respond to Zn2+
transported into cells, while emissions of cells treated with
11 are less bright than those of treated with 2,[19] possibly be-
cause of the smaller fluorescent quantum yield (F=4.4”
Figure 9. Distribution diagrams for a mixture of 5 mm 11 (L8)+5 mm Zn2+
(a), 5 mm 2 (L2)+5 mm Zn2+ (b), and 5 mm 4 (L3)+5 mm Zn2+ (c) with I=
0.1 (NaNO3) at 258C. Species of less than 10% relative concentration are
omitted for clarity.
quenching was observed on addition of Cu2+; other metal
ions induced negligible changes in the emission spectra of
L8.
Kinetics of Zn2+ complexation of 11 (L8) in comparison
with 2 (L2) and 4 (L3): The kinetics of Zn2+ complexation of
11 (L8) were compared with those of our previous Zn2+
10ꢁ2) of [Zn
G
of [Zn
AHCTREUNG
A
the cell-permeation of 11 is less efficient than that of 2, due
to the somewhat hydrophilic properties of the 8-quinoline
group of 11. Although incubation of HeLaS3 cells with 2
and 11 for 36 h tend to induce apoptosis, a short exposure
(e.g., 1 h) did not cause significant damage to HeLaS3 cells.
Apoptosis is a mechanism of cell suicide that eliminates
excess cells during development and is different from ne-
crosis. Apoptosis includes a unique series of morphologic
changes such as cell shrinkage and budding of the cell con-
with I=0.1 (NaNO3)]. We previously reported that the
second-order rate constants k2 for formation of 3 ([Zn-
A
respectively, which implies that 5 is formed three times
faster than 3.[20] These phenomena are explained by the
lower pKa value of the anthrylmethylamino group (pKa =
7.2) of 4 (Scheme 2) compared to that (10.8) of the dansyl-
amino group of 2 (Scheme 1).
Surprisingly, we found that complexation of 11 (5 mm)
with Zn2+ and Cd2+ was complete within 1 min. For accu-
rate determination of k2 for 12, we performed stopped-flow
measurements of emission change in 10 mm 11 at pH 7.4
[10 mm HEPES with I=0.1 (NaNO3)] on addition of 10, 50,
tents into membrane-enclosed vesicles (blebbing).[36]
A
common methods for the detection of apoptosis is visualiz-
ing DNA fragmentation in apoptotic cells by agarose gel
electrophoresis, which is not suitable for monitoring individ-
ual cells undergoing apoptosis.[10d,37] A second method is
9074
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Chem. Eur. J. 2006, 12, 9066 – 9080

A New Fluorescent Probe for Zinc(II)
FULL PAPER
cent cells in seen in Figure 13b
but not in Figure 13c are early-
stage apoptotic cells (indicated
by full arrows) and that the
fluorescent cells seen in both
Figure 13b and c are late-stage
apoptotic
cells
(dashed
arrows). We consider that 11 is
barely able to discriminate
early and late stages of apopto-
sis. It should also be noted that
incubation of HeLaS3 cells
with 2 and 11 for 1–3 h did not
cause significant damage to
cells, although incubation with
those ligands for 36 h tends to
induce apoptosis.[19]
Conclusion
We have designed and synthe-
sized a new Zn2+ fluorophore,
namely
dimethylaminosulfonylquinoli-
n)
ylmethyl cyclen 11 (L8). UV
2-(8-hydroxy-5-N,N-
AHCTREUNG
absorption, fluorescence emis-
1
sion, and H NMR titrations of
11 with Zn2+ indicated that 11
forms the 1:1 complex [Zn-
A
which N(1) and deprotonated
Figure 10. Fluorescent response of 2 (L2) at 528 nm (a), 4 (L3) at 416 nm (b), 11 (L8) at 512 nm (c), and 13 (L9)
at 512 nm (d) to 1 equiv of various metal cations at pH 7.4 [10 mm HEPES with I=0.1 (NaNO3)] at 258C
([ligand]=5 mm). I0 and I are the emission intensities of each ligand at the indicated wavelengths in the ab-
sence and presence of metal ions, respectively. The excitation wavelengths were 330 nm for 2, 368 nm for 4,
and 338 nm for 11 and 13.
O(8) of the quinolinol moiety
coordinate to Zn2+, as proven
by single-crystal X-ray diffrac-
tion analysis. Interestingly, 11
is almost silent in fluorescent
emission in the pH range of 4–
8 due to the cyclen moiety. By
A
rescent compounds,[39] which detect phospholipid scrambling
on the membranes of apoptotic cells. However, these meth-
ods do not reflect intracellular events. Hence, Zn2+ fluoro-
phores such as Zinquin[10] and dansylamidocyclen 2[17] have
been developed as effective sensors for early-stage apoptosis
by detecting free Zn2+ in cells.
Figure 13 shows phase-contrast and fluorescence micro-
graphs (”400) of HeLaS3 cells that were treated with tumor
necrosis factor a (TNFa, 1 ngmLꢁ1) and actinomycin D
(0.3 mgmLꢁ1) for 6 h and then dually stained with propidium
iodide (PI),[19] which stains late-stage apoptotic cells, and 11
(L8). The typical morphological features of apoptotic cells
were observed in Figure 13a (cell shrinkage, decrease in cell
volume, and blebbing of membrane). Figure 13b shows fluo-
rescent HeLaS3 cells (actually green) exposed to UV at
330–350 nm to irradiate 11 (L8), and Figure 13c a fluorescent
image (actually red) showing emissions only from PI (excita-
tion at 460–490 nm). We presume that the partially fluores-
comparison with the photochemical behaviors of 9c (L7)
and DPA-based fluorophore 13 (L9), we conclude that the
cyclen moiety has unique properties that quench fluores-
cence emission of a quinolinol moiety when it is not com-
plexed with metal cations, but enhances emission when com-
plexed with Zn2+ or Cd2+. The fluorescent emission of 11 at
512 nm (excitation at 338 nm) increased 17- and 43-fold on
complexation with Zn2+ and Cd2+, respectively, in aqueous
solution at neutral pH. The smaller enhancement in emis-
sion by complexation with Zn2+ was attributed to the higher
Lewis acidity of Zn2+ compared to Cd2+. The results of po-
tentiometric pH titrations of 11 with Zn2+ strongly suggest
that 12 is much more stable than our previous Zn2+ fluoro-
phores. The emission of [Zn
(Hꢁ1L8)] (12) at pH 5–10 was
A
almost constant, and this implies that 11 would be useful to
determine [Zn2+] over a wide pH range from 5 to 8. We ob-
served a more sensitive response of 11 to Cd2+, whose con-
centration in cells is very low. Therefore, 11 might offer a
Chem. Eur. J. 2006, 12, 9066 – 9080
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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9075

S. Aoki et al.
Figure 12. Fluorescent images (”400) of HeLaS3 cells stained with 50 mm
2 before (a) and after (b) incubation with 20 mm Zn2+ and 200 mm pyri-
thione for 30 min and those stained with 50 mm 11 before (c) and after
(d) incubation with 20 mm Zn2+ and 200 mm pyrithione for 30 min.
Figure 11. Typical results of time-dependent formation of [Zn
(Hꢁ1L2)] (3)
G
(dashed curve) and 12 (Zn
(Hꢁ1L8)) monitored by change in their fluores-
U
cent emissions at 510 nm on addition of Zn2+ at pH 7.4 [10 mm HEPES
with I=0.1 (NaNO3)] and 258C (initial [2 (L2)] and [11 (L8)] were 5 mm;
excitation at 330 nm). The inset shows the results of stopped-flow mea-
A
Figure 13. Fluorescent images (”400) of HeLaS3 cells treated with TNFa
(1 ngmLꢁ1) and actinomycin D (0.3 mgmLꢁ1) for 6 h to induce apoptosis
and then dually stained with 20 mm propidium iodide (PI) and 50 mm 11
(L8). Phase-contrast image (a), a fluorescent image (by 11) irradiated
with UV light at 330–350 nm) showing early- and late-stage apoptotic
cells (b), and a fluorescent image (by PI) irradiated with UV light at
460–490 nm showing late-stage apoptotic cells (plain arrows indicate
early-stage apoptotic cells and dashed arrows indicate late-stage apoptot-
ic cells) (c).
fortunately, 11 was not as selective for early-stage apoptotic
cells over late-stage apoptotic cells as the previous Zn2+
Scheme 10.
A
bility and/or the low quantum yield of 11. Modification of
11 to improve its quantum yield, membrane permeation,
and photochemical properties is underway.
new method of detecting Cd2+ in environment samples, in-
dustrial waste effluent, and tissue samples.[40]
We also observed that [Zn
(Hꢁ1L8)] (12) is both thermody-
A
namically and kinetically stable and that the response of 11
to Zn2+ is very quick. Therefore, fast intracellular events in-
volving changes in free [Zn2+] could be detected by utilizing
11.
Experimental Section
General information: All reagents and solvents were purchased at the
highest commercial quality and used without further purification.
A change in the concentration of free Zn2+ is reported to
be an important factor in the early process of apoptosis. The
question whether the changes in free [Zn2+] are a cause or a
consequence of apoptosis has yet to be answered.[10] Un-
Zn
G
ZnSO4·7H2O,
Fe
A
3CdSO4·8H2O,
CuSO4·5H2O, and AgNO3 were purchased from Kanto Chemical Co.;
NiSO4·6H2O and HgCl were purchased from Yoneyama Yakuhin Kogyo
Co.; Al
chased from Sigma-Aldrich Chemical Co.;
Y
ACHTREUNG
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Chem. Eur. J. 2006, 12, 9066 – 9080

A New Fluorescent Probe for Zinc(II)
FULL PAPER
A
G
A mixture of 18 (0.59 g, 1.2 mmol),[26] 19 (0.66 g, 1.4 mmol), and Na2CO3
(0.2 g, 2.1 mmol) in CH3CN (50 mL) was stirred at 608C under an argon
atmosphere overnight. After insoluble inorganic salts were filtered off,
the filtrate was concentrated under reduced pressure. The residue was
purified by silica-gel column chromatography (hexane/AcOEt) to afford
ACHTREUNG
tries, Co. Anhydrous acetonitrile (CH3CN) was obtained by distillation
from calcium hydride or LiAlH4. All aqueous solutions were prepared
using deionized and distilled water. The Goodꢁs buffer reagents (Do-
jindo) were commercially available: MES (2-morpholinoethanesulfonic
acid, pKa =4.8), HEPES (N-(2-hydroxyethyl)piperazine-N’’-2-ethanesul-
fonic acid, pKa =7.5), EPPS (3-(4-(2-hydroxyethyl)-1-piperazinyl)pro-
panesulfonic acid, pKa =8.0), TAPS (N-(tris(hydroxymethyl)methyl-
amino)-3-propanesulfonic acid, pKa =8.4), and CHES (2-(cyclohexyl-
20 as
a
pale-yellow amorphous solid (1.0 g, 92% yield). 1H NMR
(400 MHz, CDCl3/TMS): d=1.42–1.54 (br, 27H), 2.83 (s, 6H), 2.78–3.56
(br, 16H), 3.96 (s, 2H), 7.53 (dd, J=8.0, 8.0 Hz, 2H; ArH), 7.61 (d, J=
8.3 Hz, 2H; ArH), 7.65 (dd, J=4.4 Hz, 1H; ArH), 8.00 (dd, J=7.1,
1.2 Hz, 2H; ArH), 8.11 (d, J=8.0 Hz, 1H; ArH), 8.96 ppm (d, J=8.9 Hz,
1H; ArH); 13C NMR (100 MHz, CDCl3/TMS): d= 28.46, 28.69, 36.62,
37.38, 47.78, 49.89, 54.21, 55.14, 57.05, 79.50, 81.30, 120.4, 124.4, 128.3,
128.5, 129.1, 129.5, 131.7, 133.5, 134.3, 136.3, 141.3, 149.1 ppm; IR (KBr):
n˜ =2975, 2931, 1685, 1415, 1365, 1250, 1154, 1059, 976, 956, 808, 727,
ACHTREUNG
JASCO UV/VIS spectrophotometer V-550, and fluorescence (excitation
and emission) spectra were recorded on a Hitachi F-4500 fluorescence
spectrophotometer and JASCO FP-6500 spectrofluorometer at 25ꢀ
0.18C. The data from UV titrations (increases or decreases in e values at
a given wavelength) and fluorescence titrations (increases or decreases in
fluorescence emission intensity at a given wavelength) were analyzed for
apparent complexation constants Kapp by using the program Bind Works
(Calorimetry Sciences Corp). The fluorescence quantum yield F was de-
termined by comparison with the integrated corrected emission spectrum
of a quinine sulfate standard, whose quantum yield in 0.1m H2SO4 was
assumed to be 0.55 (excitation at 366 nm). IR spectra were recorded on a
Horiba FTIR-710 spectrophotometer at room temperature. 1H
(400 MHz) and 13C (100 MHz) NMR spectra at 35ꢀ0.18C were recorded
on a JEOL Lambda 400 spectrometer. 1H (300 MHz) and 13C (75 MHz)
NMR spectra were recorded on a JEOL Always 300 spectrometer. Tetra-
methylsilane was used as an internal reference for 1H and 13C NMR
measurements in CDCl3 and CD3CN. [2,2,3,3-D4]-3-(Trimethylsilyl)pro-
pionic acid sodium salt (TSP) was used as external reference for 1H and
13C NMR measurements in D2O. The pD values in D2O were corrected
for deuterium isotope effect by using pD=(pH-meter reading)+0.40. El-
emental analyses were performed on a Perkin-Elmer CHN 2400 analyzer.
TLC and silica-gel column chromatographies were performed using a
Merck 5554 (silica gel) TLC plates and Fuji Silysia Chemical FL-100D,
respectively.
599 cmꢁ1
.
1-(8-Hydroxy-5-N,N-dimethylaminosulfonylquinolin-2-ylmethyl)-1,4,7,10-
tetraazacyclododecane (21): A 28% aqueous NH3 solution (35 mL) was
added to a solution of 20 (1.0 g, 1.1 mmol) in MeOH (40 mL), and the
mixture was stirred at reflux for 5 d. The reaction mixture was concen-
trated under reduced pressure, and the residue was purified by silica-gel
column chromatography (CH2Cl2/MeOH) to afford 21 as a pale yellow
1
amorphous solid (0.82 g, 97% yield): H NMR (400 MHz, CD3OD/TMS):
d=0.91–1.49 (m, 27H), 2.68–2.77 (br, 10H), 3.30 (s, 6H), 3.31–3.64 (br,
10H), 4.12 (s, 2H), 7.20 (d, J=8.3 Hz, 1H; ArH), 7.75 (d, J=8.5 Hz, 1H;
ArH), 8.10 (d, J=8.5 Hz, 1H; ArH), 9.01 ppm (d, J=9.0 Hz, 1H; ArH);
13C NMR (100 MHz, CD3OD/TMS): d=28.9, 29.1, 37.8, 56.0, 56.1, 57.4,
57.5, 57.7, 59.1, 81.2, 81.3, 95.5, 95.6, 97.3, 110.2, 115.4, 120.7, 123.0, 125.8,
126.2, 134.0, 135.4, 139.2, 140.9, 157.4, 157.5, 157.9, 159.2, 159.6 ppm; IR
(KBr): n˜ =2974, 1689, 1564, 1502, 1462, 1415, 1365, 1320, 1251, 1154, 959,
858, 773, 716, 548 cmꢁ1
1-(8-Hydroxy-5-N,N-dimethylaminosulfonylquinolin-2-ylmethyl)-1,4,7,10-
tetraazacyclododecane tetrahydrochloride salt (11·4HCl·2H2O·
.
0.2EtOH): Aqueous HCl (1m, 10 mL) was added dropwise to a solution
of 21 (0.5 g, 0.68 mmol) in MeOH (30 mL) at 08C, and the mixture was
stirred overnight at room temperature. The reaction mixture was concen-
trated under reduced pressure, and the remaining solids were recrystal-
lized from EtOH/H2O to give 11·4HCl·2H2O·0.2EtOH as pale yellow
needles (0.39 g, 89% yield). M.p. 196–1978C; 1H NMR (400 MHz, D2O/
TSP) : d=2.81 (s, 6H), 3.08–3.37 (m, 16H), 4.24 (s, 2H), 7.34 (d, J=
3.3 Hz, 1H; ArH), 7.66 (d, J=9.0 Hz, 1H; ArH), 8.17 (d, J=8.6 Hz, 1H;
ArH), 9.00 ppm (d, J=9.0 Hz, 1H; ArH); 13C NMR (100 MHz, D2O):
d=39.6, 44.9, 45.3, 52.3, 60.4, 98.7, 113.7, 123.7, 126.3, 128.3, 135.7, 137.8,
141.0, 159.8, 161.2 ppm; IR (KBr): n˜ =3433, 2963, 1643, 1560, 1456, 1383,
8-Benzenesulfonyloxy-2-bromomethyl-5-N,N-dimethylaminosulfonylqui-
noline (19): Benzenesulfonyl chloride (1.3 g, 7.1 mmol) was added drop-
wise to a solution of 8-hydroxy-2-methyl-5-N,N-dimethylaminosulfonyl-
quinoline[24] (780 mg, 6.4 mmol) and Et3N (780 mg, 7.7 mmol) in CHCl3
(13 mL) at 08C over 1 h. After the reaction mixture had been stirred for
1.5 h at room temperature, H2O (5 mL) was added and stirring was con-
tinued for 0.5 h. The reaction mixture was extracted from saturated
K2CO3 with CH2Cl2, and the organic layer was dried with anhydrous
K2CO3, filtered, and concentrated under reduced pressure. The residue
was purified by silica-gel column chromatography (hexane/AcOEt) to
give 8-benzenesulfonyloxy-2-methyl-5-N,N-dimethylaminosulfonylquino-
line (2.5 g, 96% yield) as a colorless amorphous solid. A mixture of 8-
benzenesulfonyloxy-2-methyl-5-N,N-dimethylaminosulfonylquinoline
(1.5 g, 3.7 mmol), N-bromosuccinimide (380 mg, 2.6 mmol), and AIBN
(40 mg, 0.24 mmol) in distilled CCl4 (190 mL) was stirred at reflux under
an argon atmosphere for 8 h (N-bromosuccinimide was added portion-
wise), and the reaction mixture was concentrated under reduced pressure.
The residue was dissolved in CH2Cl2 and washed with a mixture of
Na2CO3 and Na2S2O4 in aqueous solution. The residue was purified by
silica-gel column chromatography (hexane/AcOEt) to afford 19 as a col-
orless amorphous solid (250 mg, 14% yield). The starting material (8-
benzenesulfonyloxy-2-methyl-5-N,N-dimethylaminosulfonylquinoline)
1330, 1156, 951, 721, 474 cmꢁ1
20.4H43.2Cl4N6O6.2S (645.67): C 37.95, H 6.74, N 13.02, S 4.97; found:
C 37.50, H 7.23, N 13.21, S 5.39.
; elemental analysis calcd (%) for
C
For X-ray crystal structure analysis, 11·4HCl·3H2O·0.2EtOH (H4L8)
(50 mg, 0.077 mmol) was recrystallized from aqueous solution at pH 10
(adjusted with NaOH) to give 11 in the H
2(Hꢁ1)L8 form (26 mg, 62%
N
yield). M.p. >2508C; 1H NMR (400 MHz, D2O/TSP) : d=2.81 (s, 6H),
3.08–3.37 (m, 16H), 4.24 (s, 2H), 6.85 (d, J=8.8 Hz, 1H), 7.45 (d, J=
8.8 Hz, 1H; ArH), 8.02 (d, J=8.8 Hz, 1H; ArH), 8.86 ppm (d, J=9.1 Hz,
1H; ArH); elemental analysis calcd (%) for C20H41ClN6O7S (545.09): C
44.07; H 7.58; N 15.42; S 5.88; found: C 44.35; H 7.90; N 15.26; S 5.60.
1-(8-Hydroxy-5-N,N-dimethylaminosulfonylquinolin-2-ylmethyl)-1,4,7,10-
tetraazacyclododecane Zn
N
G
An aqueous solution of Zn
AHCTREUNG
(3 mL) was added to a solution of 11·4HCl·3H2O·0.2EtOH (40 mg,
0.062 mmol) in H2O (10 mL) at room temperature, and the pH of the re-
action mixture was adjusted to 7.5 with 0.1m NaOH. After the insoluble
compounds were filtered off, the filtrate was slowly concentrated under
was recovered in 56% yield (860 mg). H NMR (400 MHz, CDCl3/TMS):
d=2.83 (s, 6H), 4.42 (s, 2H), 7.52 (t, J=7.8 Hz, 2H; ArH), 7.65 (d, J=
7.4 Hz, 1H; ArH), 7.67 (d, J=8.9 Hz, 1H; ArH), 7.80 (d,; J=8.3 Hz, 1H
ArH), 8.00 (d, J=8.5 Hz, 2H; ArH), 8.18 (d, J=8.3 Hz, 1H; ArH),
9.05 ppm (d, J=9.0 Hz, 2H; ArH); 13C NMR (100 MHz, CDCl3/TMS):
d=33.08, 37.42, 121.9, 123.4, 125.4, 129.0, 130.2, 132.0, 134.4, 135.9, 140.9,
149.0, 158.1 ppm; IR (KBr): n˜ =3092, 2921, 1706, 1596, 1498, 1460, 1375,
reduced pressure to obtain 12-ClO4 ([Zn
(Hꢁ1L8)]ClO4) as pale yellow
A
prisms (15 mg, 40% yield). M.p. >2508C; 1H NMR (400 MHz, D2O/
TSP): d=2.73 (s, 6H), 2.78–3.25 (m, 16H), 4.32 (s, 2H), 6.96 (d, J=
8.8 Hz, 1H), 7.65 (d, J=8.8 Hz, 1H; ArH), 8.09 (d, J=8.8 Hz, 1H;
ArH), 9.00 ppm (d, J=9.2 Hz, 1H; ArH); 13C NMR (D2O): d=36.41,
43.34, 44.29, 44.57, 52.90, 57.77, 110.6, 121.6, 125.0, 134.9, 136.3, 137.9,
154.8, 165.1 ppm; IR (KBr): 3279, 2915, 2870, 1563, 1469, 1678, 1101, 949,
1343, 1189, 1147, 1054, 957, 799, 619 cmꢁ1
1-(8-Benzenesulfonyloxy-5-N,N-dimethylaminosulfonylquinolin-2-ylmeth-
yl)-4,7,10-tris(tert-butyloxycarbonyl)-1,4,7,10-tetraazacyclododecane (20):
Chem. Eur. J. 2006, 12, 9066 – 9080
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9077

S. Aoki et al.
835, 718, 539 cmꢁ1; elemental analysis calcd (%) for C20H31ClN6O7SZn
(600.39): C 40.01, H 5.20, N 14.00, S 5.34; found: C 39.92, H 5.19, N
13.89, S 5.48.
Observing apoptotic morphology in HeLaS3 cells and detecting apoptosis
with 11: HeLaS3 cells were seeded into 35 mm cell culture dishes each
containing a cover slip. The dishes were incubated under a humidified at-
mosphere of 5% CO2 at 378C. The cells were treated with TNFa
(1 ngmLꢁ1) and actinomycin D (0.3 mgmLꢁ1) for 6 h. The culture medium
was replaced with 1 mL of fresh culture medium containing 50 mm 11 and
30 mm propidium iodide (PI). The reaction mixture was incubated at
378C under a humidified atmosphere of 5% CO2 for 30 min and rinsed
once with 1 mL of PBS for fluorescent microscopy.
Crystallographic study of 11 (H
2(Hꢁ1L8)Cl): C20H41ClN6O7S, Mr =545.09,
A
pale yellow prismatic, crystal size 0.35”0.20”0.10 mm, monoclinic, space
group P21/c (No. 14), a=18.029(20), b=10.785(11), c=13.913(16) Š, b=
90.47(5)8, V=2705(5) Š3, Z=4, 1calcd =1.338 gcmꢁ3, 31462 measured re-
flections, 7808 unique reflections (Rint =0.059), 2qmax =59.98, R1=0.0448
[calculated for 3439 reflections with I>2s(I)], Rw =0.0949 (for all reflec-
tions), GOF=0.982.
Crystallographic study of 12-ClO4 ([Zn
(Hꢁ1L8)]ClO4: C20H31ClN6O7SZn,
U
Mr =600.39, pale yellow prisms, crystal size 0.20”0.07”0.03 mm, ortho-
rhombic, space group Aea2 (No. 41), a=30.575(17), b=12.504(7), c=
13.028(8) Š, V=4981(5) Š3, Z=8, 1calcd =1.601 gcmꢁ3, 20932 measured
reflections, 5602 unique reflections (Rint =0.162), 2qmax =55.08, R1=
0.0752 [calculated for 2695 reflections with I>2s(I)], Rw =0.1413 (for all
reflections), GOF=0.929.
Acknowledgements
S.A. is grateful for a Grant-in-Aid from the Ministry of Education, Sci-
ence and Culture in Japan (No. 15590008) and for grants from the Mitsu-
bishi Chemical Corporation Fund (Tokyo), Toray Science Foundation
(Tokyo), Terumo Life Science Foundation (Kanagawa), Mochida Memo-
rial Foundation for Medical and Pharmaceutical Research (Tokyo), and
The Novartis Foundation (Japan). We thank Mr. Shinya Kondo, Mrs.
Chika Tsuji, and Mr. Yoji Uno (Tega Science Inc., Japan) for stopped-
flow experiments. We thank NMR instruments (for the use of a JEOL
Alpha (400 MHz)) in the Research Center for Molecular Medicine
(RCMM) at Hiroshima University.
All measurements were made on a Rigaku RASXIS-RAPID imaging
plate area detector with graphite-monochromated MoKa radiation at
93 K. The structures were solved by direct methods and refined by full-
matrix least-squares techniques. All calculations were performed using
CrystalStructure crystallographic software package, except for solving
phase problems and refinements, which were done with SHELXS97 and
SHELXL97, respectively.
CCDC 601452 (11, HL8), 601453 (12, [Zn
A
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the calibration method of the electrode system (Potentiometric Automat-
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A
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11, and excitation at 460–490 nm, emission at 590 nm for PI).
9078
www.chemeurj.org
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2006, 12, 9066 – 9080

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ꢁ
[33] Note that the Cu N(1) bond length (2.34 Š) is somewhat longer
ꢁ
than other Cu N
G
ꢁ
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logKs([Cu
(Hꢁ1L8)]) was too large to be determined by potentiomet-
AHCTREUNG
AHCTREUNG
ric pH titrations.
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Received: March 17, 2006
Published online: September 5, 2006
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