Oligopyridine ligands derived from amino acid precursors: Their Zn 2+ complexation and effects on hepatic stellate cell functions

Article abstract of DOI:10.1039/b806548a

A series of oligopyridine ligands were derived from amino acid amides in which amide oxygen and ternary nitrogen atoms were combined with pyridine moieties. ¹H NMR and circular dichroism spectroscopic characterizations revealed that they formed stable Zn²⁺ complexes in neutral aqueous solutions and caused Zn²⁺ deficiency in the hepatic stellate cell systems. Since collagen synthesis was effectively promoted in the cells, the present oligopyridine derivatives worked as biocompatible ligands for Zn²⁺ complexation and cell activation.

Full text of DOI:10.1039/b806548a

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PAPER
www.rsc.org/dalton | Dalton Transactions
Oligopyridine ligands derived from amino acid precursors: Their Zn2+
complexation and effects on hepatic stellate cell functions†
a
a
a
a
a
Hiroshi Tsukube,* Yuki Noda, Yumiko Kataoka, Hiroyuki Miyake, Satoshi Shinoda,
b
b
b
Akiko Kojima-Yuasa,* Yoshitake Nishida and Isao Matsui-Yuasa
Received 17th April 2008, Accepted 12th May 2008
First published as an Advance Article on the web 24th June 2008
DOI: 10.1039/b806548a
A series of oligopyridine ligands were derived from amino acid amides in which amide oxygen and
1
ternary nitrogen atoms were combined with pyridine moieties. H NMR and circular dichroism
2
+
spectroscopic characterizations revealed that they formed stable Zn complexes in neutral aqueous
2
+
solutions and caused Zn deficiency in the hepatic stellate cell systems. Since collagen synthesis was
effectively promoted in the cells, the present oligopyridine derivatives worked as biocompatible ligands
2
+
for Zn complexation and cell activation.
Inroduction
significantly depended on the nature of the ligand substituents.
Since they exhibited no cytotoxicity and promoted collagen
synthesis in the hepatic stellate cells, the present oligopyridines
worked as biocompatible ligands for Zn complexation and cell
activation.
2
+
Zn ion is the second most abundant d-block metal cation in
humans, and often plays important roles in DNA synthesis,
apoptosis, gene expression, neurotransmission, signal transduc-
2+
1
tion and other cell events. Although several types of ligand
2
+
have been designed to monitor the Zn cation and to affect cell
2
functions, oligopyridine derivatives are promising candidates as
Results and discussion
2
+
3
biocompatible Zn ligands. Lippard et al. prepared fluorescent
derivatives from tetrakis(2-pyridylmethyl)ethylenediamine 1 (see
Fig. 1) as cell-permeable ligands for in vivo Zn determination.
Nagano et al., Smith et al., Hamachi et al. and others linked
1. Ligand synthesis and characterization
2
+
Amino acids are useful building blocks for the synthesis of
ꢀ
multidentate ligands. Alsfasser et al. prepared a series of 2,2 -
2
+
dipicolyamine 2–Zn complexes with dyes, cholesterols and
5
bispicolylamino acid esters. Ligand 5 was typically demonstrated
3
proteins. We have reported that ligand 1 and diethylenetriamine
2+
to form a trigonal-bipyramidal Zn complex, in which two
2
+
pentaacetic acid caused Zn deficiency in hepatic stellate cell
pyridine, one tertiary nitrogen and one amide oxygen atom were
4
2+
systems. Since the Zn cation is significantly involved in collagen
synthesis and other hepatotoxic metabolic events, its abnormal
metabolism and depletion caused liver fibrosis.
6
significantly involved. Although its solution structure and bio-
activity were not detailed, hard amide and soft pyridine donor
2+
moieties were nicely arranged to offer stable Zn complexes. We
We describe here that a new series of mixed-donor type oligopy-
attached four pyridine rings to the amino acid amide skeletons of
2
+
ridines 3a–3c form stable Zn complexes in neutral aqueous
solutions and promote collagen synthesis in hepatic stellate cells.
Ligands 4a and 4b are compared, because they have two pyridine
rings in addition to one amide oxygen and one tertiary nitrogen
atom. Both types of ligand are derived from amino acid precursors,
and have multidentate coordination modes and tunable lipophilic
properties. NMR and circular dichroism (CD) characterizations
2+
glycine, alanine and methionine (see 3a–3c in Fig. 1). Their Zn
complexation properties were characterized in neutral aqueous
solutions, and biological activities toward hepatic stellate cells were
further investigated. Ligands 4a and 4b were synthesized for com-
parison. Since they have different arrangements of pyridine rings,
one amide oxygen and one tertiary nitrogen atom, they provide
2+
markedly different coordination modes for the Zn cation.
2
+
revealed that ligands 3a–3c and 4a formed stable Zn complexes
in neutral aqueous solutions, though their complexation profiles
The synthetic routes of the employed ligands were straight-
7
forward as reported for related ligands (Fig. 2). Ligands 3a–
3
c were derived from the corresponding glycine, L-alanine and
a
L-methionine amides. Boc-amino acids were first reacted with
dipicolylamine to give amide precursors. After deprotection,
the treatment with chloromethylpyridine yielded ligands 3a–3c.
Reference ligands 4a and 4b were prepared from N,N-diethyl
glycine amide and N,N-dimethylglycine. The ligands obtained
were fully characterized by H and C NMR, IR and HRMS
methods. The log D (distribution coefficient) value of each ligand
was calculated in an n-octanol–water system at pH = 6.3 as
an indication of ligand hydrophobicity. The PALLAS program
(Windows version 3.0, CompuDrug International Inc., Sedona,
Department of Chemistry, Graduate School of Science, Osaka City Univer-
sity, Sugimoto, Sumiyoshi-ku, Osaka, 558-8585, Japan. E-mail: tsukube@
sci.osaka-cu.ac.jp; Fax: +81-6-6605-2560; Tel: +81-6-6605-2560
b
Department of Food and Human Health Sciences, Graduate School of
Human Life Science, Osaka City University, Sugimoto, Sumiyoshi-ku,
Osaka, 558-8585, Japan. E-mail: kojima@life.osaka-cu.ac.jp; Fax: +81-
6
1
13
-6605-2810; Tel: +81-6-6605-2810
Electronic supplementary information (ESI) available: H NMR spectral
complexation in aqueous solution, ESI-MS
1
†
4 2
changes of 3a upon Zn(ClO )
spectrum of an equimolar mixture of 3b and Zn(ClO
4
)
2
in H
2
O, CD spectral
ꢀ
changes and titration curves for log K determination of 2, 3a, 3c and 4a,
1
13
H and C NMR spectra of 3a–3c and 4b. See DOI: 10.1039/b806548a
038 | Dalton Trans., 2008, 4038–4043
4
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Fig. 1 Structures of ligands 1, 2, 3a–3c, 4a, 4b and 5.
Fig. 2 Synthesis of ligands 3a–3c, 4a and 4b. Boc = N-tert-butoxycarbonyl, HOBt = 1-hydroxybenzotriazole, EDCI = 1-[3-(dimethylamino)-
propyl]-3-ethyl carbodiimide.
USA) was employed. Ligands 3a–3c exhibited comparable log D
values to that of ligand 1, while ligand 4b had a lower value: log D =
ligand 4a was confirmed to provide stronger coordination with the
Zn cation than those of ligands 3a and 4b.
2
+
1
.91 for 1; 1.26 for 3a; 1.80 for 3b; 2.14 for 3c; 1.18 for 4a; and 0.03
for 4b. Table 1 summarizes some spectroscopic characteristics of
the ligands. Ligand 4a exhibited an NMR signal for C=O carbon
2+
2
. Zn Complexation profiles in aqueous solution
1
at higher field (d = 169.3 ppm) than ligands 3a (d = 171.5 ppm)
and 4b (d = 171.0 ppm). A similar trend was observed in their
When ligand 3a formed a Zn(ClO
4
)
2
complex in D O, several H
2
2
+
NMR signals appeared separately at [Zn ]/[3a] = 0.5, which were
8
IR characteristic C=O bands. Thus, the amide oxygen atom of
assigned to the free and 1 : 1 complex forms (Fig. S1 in ESI†). In
This journal is © The Royal Society of Chemistry 2008
Dalton Trans., 2008, 4038–4043 | 4039

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9
Table 1 Characteristics of ligands and conditional formation constants
of their Zn complexes in neutral aqueous solutions (pH = 6.3)
exist in their monoprotonated forms at pH = 6.3, the following
2+
competitive displacement took place:
2
+
+
+
2+
[
3b–Zn ] (CD active) + 2–H ꢀ 3b–H + [2–Zn ] (CD inactive)
d
C
(C=O) in
m
max(C=O)
Conditional formation
constant (log K )
2+
−
1
ꢀ
The concentration of the 2–Zn complex was evaluated by
Ligand
CDCl
3
(ppm)
in KBr/cm
ꢀ
monitoring the CD signal intensity at 262 nm, and the log K value
for the 2–Zn complex was estimated as 6.6. Other combinations
of chiral and achiral ligands were examined and the determined
log K values are listed in Table 1. When ligands 3a–3c were
compared, their conditional formation constants with Zn(ClO4)2
clearly depended on the bulkiness of the ligand substituents: log
a
1
2
14.6 ± 0.1
2+
10
6.6 ± 0.2
7.2 ± 0.2
6.1 ± 0.2
5.4 ± 0.1
3
3
3
4
4
a
b
c
171.5
173.6
172.3
169.3
171.0
1651
1648
1648
1643
1651
ꢀ
a
b
8.3 ± 0.2
b
ꢀ
K = 7.2 for 3a (R = –H), > 6.1 for 3b (R = –CH
3
), > 5.4 for 3c (R =
a
ꢀ
Calculated from protonation and formation constants reported in ref. 9
–CH CH SCH ). Ligand 4a exhibited a much larger log K value,
2
2
3
b
and 10. Formation constants of 1 : 1 and 1 : 2 complexes were too small
to be determined.
indicating that its two pyridine moieties effectively coordinated
the Zn ion. In contrast, the log K value of 4b was too small to be
2
+
ꢀ
2
+
spectroscopically determined. Since ligand 1 formed a stable Zn
complex, the two pyridine moieties on the amide sides of ligands
a–3c and 4b were thought to inhibit coordination. Interestingly,
the CD spectrum of an equimolar mixture of ligand 3b and
Zn(ClO was only slightly changed (< 10%) by the addition of a
2
+
contrast, only one set of NMR peaks remained at [Zn ]/[3a] =
3
2
+
1
.0 and 2.0, indicating that ligand 3a formed a 1 : 1 Zn complex.
1
H NMR and ESI-MS characterizations revealed that ligands 3b,
c and 4a also formed 1 : 1 complexes with Zn(ClO . Ligand 3b
typically exhibited ESI-MS signals due to 1 : 1 complexation: [3b +
] and [3b + Zn ] were detected at m/z 615.3 and
58.2 (Fig. S2 in ESI†). Ligand 4b rarely formed Zn complexes.
gave somewhat different H
NMR spectral changes. Further addition of equimolar amounts
of ligand 4a to the mixture gave only signals due to the 4a–Zn
complex. Since no ESI-MS signal for 4b–Zn complex species was
)
4 2
3
)
4 2
large excess (2000 equivalents) of NaCl. This is a common salt in
biological cell systems, but has hardly perturbed the structure of
2
+
−
+
2+ 2+
Zn + ClO
4
2
+
the 3b–Zn complex.
2
+
2
+
Alsfasser et al. isolated the trigonal-bipyramidal [5–ZnCl]
1
A mixture of ligand 4b and Zn(ClO
4
)
2
6
ꢀ
complex. Each ligand exhibited a similar log K value with ZnCl
2
ꢀ
to that with Zn(ClO
Zn(ClO
.3 ± 0.2 for Zn(ClO
4
)
2
. For ligand 3b, log K = 6.1 ± 0.2 for
2
+
ꢀ
)
2
and 6.0 ± 0.4 for ZnCl
2
; for ligand 4a, log K =
2
+
4
8
4
)
2
and 8.4 ± 0.4 for ZnCl
2
. Since these
observed, it is assumed that the two pyridine units attached to the
1
ligands exhibited similar H NMR spectra upon complexation
2
+
amide nitrogen atom rarely coordinate the Zn cation.
with Zn(ClO
4
)
2
and ZnCl , they were proposed to form complexes
2
The complexation between chiral ligand 3b and Zn(ClO
)
4 2
2
+
of the type [ligand–Zn–H
2
O] . Based on this assumption, their
Zn complex structures were optimized using the DFT B3LYP/6-
was quantitatively characterized in neutral aqueous solution by
monitoring CD spectral changes (Fig. 3). When the CD intensity
at 262 nm was plotted against a function of the added amount
, the obtained titration curve gave a good fit for 1 : 1
. To characterize
Zn complexation properties in neutral aqueous solution, the
2
+
1
1
+
6
3
1G* method. The crystal structure of the [5–ZnCl] complex
was used in the construction of the initial geometry with CAChe
PM3 calculations (Version 3.2, Oxford Molecular Ltd.), in which
of Zn(ClO
4
)
2
complexation between the ligand and Zn(ClO
4 2
)
2
+
the Zn cation was coordinated by a tetradentate ligand and
2
+
12,13
one H
structure of the [3b–Zn–H
c gave similar complex structures. In the penta-coordinated
2
O molecule.
Fig. 4a typically illustrates the optimized
ꢀ
conditional formation constant K was calculated using the curve-
2
+
O] complex, and ligands 3a and
ꢀ
ꢀ
2
fitting method: log K = 6.1 at pH = 6.3. log K of ligand 3c was
3
similarly estimated as 5.4 (Fig. S3 in ESI†). Since achiral ligand
complex, three nitrogen atoms of the dipicolylamine unit and
the amide oxygen atom are coordinated, while the two pyridine
units connected to the amide nitrogen atom were not involved in
2
was also confirmed to form 1 : 1 complexes with Zn(ClO
4
2
) by
ꢀ
NMR experiments, its K value was determined by competitive
binding experiments with chiral ligand 3b (Fig. S4 in ESI†).
When achiral ligand 2 was added to an equimolar solution of
, the CD signal intensity of the 3b–Zn
complex around 262 nm decreased. Since both ligands practically
2
+
coordinating the Zn center. As shown in Fig. 4b, five-membered
2
+
ring chelation was suggested to stabilize the Zn complex and
lock the rotation around the C–N bond indicated. Since ligand
2
+
chiral 3b and Zn(ClO
4
)
2
3
c derived from a methionine precursor exhibited a smaller log
ꢀ
K value than ligands 3a and 3b from glycine and alanine ones,
its bulky subustituent –(CH
2
)
2
–S–CH was proposed to cause
3
the large steric repulsion with one of the –CH
dipicolylamine part.
2
Py groups of the
2
+
3
. Zn Deficiency in hepatic stellate cells
2
+
When Zn cation homeostasis is not maintained, a broad range
of defects occur in living cells. We have previously reported
2
+
that ligand 1 caused Zn deficiency in the hepatic stellate cells
4
and induced several cell events followed by collagen synthesis.
ꢀ
Although ligands 3a–3c have different complex structures from
ligand 1, their cytotoxic and cell activation abilities toward the
hepatic stellate cells were characterized.
Fig. 3 CD spectral changes and titration curve for the log K determi-
nation of the 3b–Zn(ClO
4
)
2
complex in aqueous solutions. [3b] = 3.0 ×
−
5
−1
1
0
mol L
.
4
040 | Dalton Trans., 2008, 4038–4043
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−
6
−1
−6
−1
1
3
0
mol L for ligand 3b; and 9.1 × 10 mol L for ligand
c. Although the employed ligands had various log D values,
2
+
they had high enough activities to cause Zn deficiency in the
hepatic stellate cells. Further biological analysis is required, but
previous biochemical studies supported the fact that the depletion
of intracellular glutathione levels triggered the progression of
2
+
4
collagen synthesis in Zn deficient cells.
Experimental section
General
1
13
H and C NMR spectra were recorded on JEOL LA-300 and
00 spectrometers. CD spectra were obtained on a JASCO J-820
4
spectrometer and UV-visible spectra were recorded with a Hitachi
U-3500 apparatus. ESI-MS spectra were recorded with a JEOL
JMS-700 instrument. TLC plates were purchased from Merck
KGaA (neutral alumina, No. 5550).
Ligand synthesis
2
+
Ligands 1 and 2 were commercially available, while ligands 3a–
3c and 4b were synthesized in a similar fashion to ligand 4a as
reported earlier. Their synthetic routes are summarized in Fig. 2
2
Fig. 4 (a) Optimized structure of the [3b–Zn–H O] complex and (b)
schematic illustration of its geometry.
7
The hepatic stellate cells were isolated from the liver of male
Wistar rats, incubated and their purity confirmed to be higher than
5% (see Experimetnal section). They were incubated in a culture
and selected specroscopic data are described below.
2
2
N,N,N ,N -Tetrakis(pyridin-2-ylmethyl)glycinamide (3a). Ye-
9
max(neat)/cm−
1
−
5
−1
2+
llow oil (15%); R = 0.22 (EtOAc), m
1651 (C=O)
solution containing 1.6 × 10 mol L of Zn and other essential
f
14
and 1593, 1571, 1476, 1436 and 764 (py); d (300 MHz; CDCl ;
ingredients. After incubation with the ligand, the expression of
type I collagen was detected using immunohistochemical staining
H
3
Me
4
Si) 3.62 (2 H, s, CH
2
), 3.95 (4 H, s, CH
2
N
amine), 4.73 (2 H, s,
−
5
−1
CH N ), 4.78 (2 H, s, CH Namide), 7.00 (1 H, d, J 7.8, py), 7.08–
(
Fig. 5). When 1.0 × 10 mol L of ligand 3a, 3b or 3c was added,
2
amide
2
7
.12 (4 H, m, py), 7.28 (1 H, d, J 7.8, py), 7.50–7.61 (6 H, m, py)
and 8.42–8.46 (4 H, m, py); d (75 MHz; CDCl ; CDCl ) 51.4, 52.6,
6.3, 60.5, 120.8, 122.1, 122.3, 122.3, 122.6, 123.8, 136.5, 136.7,
136.8, 149.1, 149.3, 149.8, 156.9, 157.5, 159.1 and 171.5; MS(EI)
no ligand exhibited cytotoxicity in the hepatic stellate cell system.
Rather strong immunoreactivity was observed in filaments in each
ligand system, though the control cells were almost negative.
C
3
3
5
−
5
−1
When 1.0 × 10 mol L of ZnSO
4
was supplemented after
treatment with the ligand, the staining was comparable to that at
+
m/z 438.2170 (M . C26
H
26ON requires 438.2168).
6
control level. Thus, ligands 3a–3c induced progression of collagen
2
2
N,N,N ,N -Tetrakis(pyridin-2-ylmethyl)-L-alaninamide (3b).
4
ꢀ
synthesis in a similar fashion to ligand 1. Based on the log K
−1
Colorless oil (8%); R
C=O) and 1591, 1570, 1475, 1433 and 764 (py); d
CDCl ; Me Si) 1.40 (3 H, d, J 6.8, CH ), 3.85 (2 H, d, J 14,
CH amine), 4.05 (3 H, d, J 14, CH amine + CH), 4.13 (1 H, d, J
4, CH amide), 4.41 (1 H, d, J 17, CH amide), 5.23 (1 H, d, J 14,
CH amide), 5.34 (1 H, d, J 17, CH amide), 6.95 (1 H, d, J 9.0, py),
.05–7.13 (4 H, m, py), 7.24–7.40 (4 H, m, py), 7.50–7.55 (3 H,
m, py), 8.39–8.46 (4 H, m, py); d (100 MHz; CDCl ; CDCl ) 9.4,
1.4, 52.3, 55.4, 56.9, 120.5, 121.9, 122.0, 122.2, 122.5, 123.5,
36.4, 136.6, 136.7, 149.0, 149.1, 149.6, 157.3, 157.6, 159.4 and
f
= 0.30 (EtOAc), mmax(neat)/cm 1648
2
+
values determined above, the free Zn concentration in the culture
(
H
(300 MHz;
−
5
−1
was roughly estimated to be lower than 1.6 × 10 mol L : 6.0 ×
mol L for ligand 1; 6.1 × 10 mol L for ligand 3a; 7.0 ×
3
4
3
−
6
−1
−6
−1
10
2
N
2
N
1
2
N
2
N
2
N
2
N
7
C
3
3
5
1
1
4
+
73.6; MS(FAB) m/z 453.2417 (M + H . C27
53.2403).
H
29ON requires
6
2
2
N,N,N ,N -Tetrakis(pyridin-2-ylmethyl)-L-methioninamide (3c).
Yellow oil (25%); 0.30 (EtOAc–MeOH 50 : 1),
max(neat)/cm 1638 (C=O) and 1591, 1474, 1433 and 764
py); d (400 MHz; CDCl ; Me Si) 2.04–2.14 (1 H, m, CH ), 2.09
3 H, s, SCH ), 2.37–2.43 (2 H, m, CH ), 2.59–2.66 (1 H, m, CH ),
.85 (2 H, d, J 14, CH amine), 3.93 (1 H, m, CH), 4.03 (2 H, d,
J 14, CH amine), 4.10 (1 H, d, J 15, CH amide), 4.39 (1 H, d, J
7, CH amide), 5.26 (1 H, d, J 18, CH amide), 5.27 (1 H, d, J 15,
CH amide), 6.96–7.30 (8 H, m, py), 7.37–7.58 (4 H, m, py) and
8.38–8.46 (4 H, m, py); d (75 MHz; CDCl ; CDCl ) 15.5, 23.8,
R
f
=
=
−
1
m
(
(
H
3
4
2
3
2
2
3
2
N
2
N
2
N
1
2
N
2
N
Fig. 5 Collagen synthesis of hepatic stellate cells upon addition of ligand
a, 3b, or 3c. The produced collagen was stained.
2
N
3
C
3
3
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4
3
1
1
5
1.4, 51.6, 52.6, 57.2, 59.2, 121.0, 122.0, 122.2, 122.3, 122.6, 123.5,
the liver with pronase and collagenase, as previously described.
36.4, 136.5, 136.7, 149.0, 149.1, 149.5, 156.0, 157.6, 159.4 and
The cells were identified by the typical star-like configuration
and vitamin A autofluorescence, and their purity was confirmed
+
72.3; MS(FAB) m/z 513.2436 (M + H . C29
H
33ON S requires
6
5
−1
13.2437).
as higher than 95%. They were plated at 5 × 10 cells mL
on uncoated culture dishes in 1.5 mL of DMEM (Nissui Phar-
maceutical, Tokyo, Japan) containing 10% fetal bovine serum
Equitech-Bio, Inc., TX, USA) and supplemented with antibiotics
2
2
N ,N -Dimethyl-N,N-bis(pyridin-2-ylmethyl)glycinamide (4b).
14
−
1
Colorless oil (49%); R
f
= 0.17 (EtOAc), mmax(neat)/cm 1651
(400 MHz;
), 4.75 (2 H, s,
amine), 7.14–7.20 (3 H, m, py), 7.31
1 H, d, J 7.8, py), 7.61–7.67 (2 H, m, py), 8.49 (1 H, d, J 4.5, py)
and 8.56 (1 H, d, J 4.5, py). d (100 MHz; CDCl ; CDCl ) 45.7,
1.3, 52.7, 62.2, 121.1, 122.4, 122.5, 122.5, 136.8, 136.8, 149.2,
(
(
(
C=O) and 1592, 1571, 1475, 1436 and 757 (py); d
CDCl ; Me Si) 2.32 (6 H, s, CH ), 3.30 (2 H, s, CH
CH
H
5
−1
−1
10 units L of penicillin G and 500 mg L of streptomycin)
3
4
3
2
2+
for 2 d, then cultured in fresh medium without serum and Zn
salt for 18 h. After pre-incubation, the cells were divided into
2
N
amine), 4.87 (2 H, s, CH N
2
(
2+
three groups: (1) control; (2) ligand (Zn deficient group); and
3) ligand + Zn salt (Zn supplement group). Each culture
C
3
3
2+
2+
(
5
1
C
−5
−1
contained 1.6 × 10 mol L ZnSO
4
and the essentials for cell
+
49.9, 157.1, 157.5 and 171.0; MS(FAB) m/z 285.1705 (M + H .
requires 285.1715).
2+
−5
−1
existence. For the Zn deficient group, 1.0 × 10 mol L ligand
16
H
21ON
4
2+
−5
was added. For the Zn supplement group, 1.0 × 10 mol
−
1
L
ZnSO was further added to the culture 1 h after ligand
4
Determination of conditional formation constants
addition. After incubation for 24 h, the cells were fixed with 4%
paraformaldehyde fixative overnight at 4 C. We used antitype
◦
CD spectra were recorded with non-buffered aqueous solutions
containing ligand 3b and Zn(ClO
temperature. Since the observed UV spectral changes were small,
the CD intensity at 262 nm was plotted against the mole ratio of
I collagen polyclonal antibody (1 : 200 dilution) as the primary
antibody. After incubation with 0.3% hydrogen peroxide to block
endogenous peroxidase and subsequently with normal goat serum
to inhibit non-specific reactions, samples were incubated with
primary antibody for 1 h at room temperature and then with
biotinylated anti-rabbit goat immunoglobulin for 30 min followed
by incubation with horseradish peroxidase-labelled streptavidin-
4
2
) after stirring for 2 h at room
ꢀ
Zn(ClO
4
)
2
to ligand 3b, and the log K value for 1 : 1 complexation
was calculated. Similar procedures were done to determine the
log K value for ligand 3c. Two independent experiments were
conducted for this combination and the calculations were carried
out using IGOR Pro (version 4, WaveMetrics Inc.).
ꢀ
ꢀ
biotin complex for 30 min. For the peroxidase reaction, 3,3 -
−
1
diaminobenzidine tetrahydrochloride (0.2 mg mL ) with NiCl
2
The competitive method was applied to determine the con-
color modification was incubated for 5 min until the desired color
intensity was obtained. The reproducibility was confirmed by two
independent experiments.
ꢀ
2+
ditional formation constants (K values) of some ligand–Zn
complexes. When achiral ligand 3a was added to the 3b–Zn
complex solution, competitive displacement was successfully
followed by monitoring the decreased CD signal around 262 nm.
Since the resulting 3a–Zn complex was a CD inactive species, the
decreased CD signal was employed in the calculation of the log
K value. We usually added three different amounts of competitive
ligand and averaged the three estimated log K values. Once the log
K value was estimated for the achiral 3a–Zn complex, further
2
+
2
+
Conclusions
ꢀ
We present a new series of mixed donor-type oligopyridines as
biocompatible ligands for Zn complexation and cell activation.
Since the ligands were derived from amino acid precursors, they
had versatile coordination modes and tunable lipophilic proper-
ties. NMR and CD characterization revealed that they formed
ꢀ
2+
ꢀ
2+
ꢀ
2+
combinations allowed log K estimations for Zn complexes with
ligands 2, 4a and 4b. The reproducibility was confirmed to be 0.2
or better in log scale for ligands 2, 3a, 3b, 3c and 4a.
2+
stable Zn complexes in neutral aqueous solution, although their
conditional formation constants significantly depended on the
ligand substituents. The present type of oligopyridines exhibited
no cytotoxicity and promoted collagen synthesis in hepatic stellate
cells.
DFT Calculations
A geometry optimization was performed for the [ligand–Zn–
2
+
11
H O] complex using Gaussian 03 (D.01) starting from coor-
2
+
dinates based on the crystal structure of the [5–Zn–Cl] complex
previously reported. After replacement of Cl with H O, the
initial coordinate was obtained for each complex with the CAChe
program (Version 3.2, Oxford Molecular Ltd.). The DFT calcula-
tion was performed on a singlet electronic state. The method used
was B3LYP with 6-31G* as the basis set.
6
−
2
Notes and references
1
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Cell activation experiments
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allowed free access to water and standard rat chow. Our animal
experiments completely followed the guidelines of Osaka City
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density gradient centrifugation in Nycodenz after digestion of
4
5, 752–760.
3
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042 | Dalton Trans., 2008, 4038–4043
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5
6
7
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005, 44, 4796–4805.
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1
731.
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12 Several tripodal ligands were demonstrated to form penta-coordinated
2+
Zn complexes: C. S. Allen, C.-L. Chuang, M. Cornebise and J. W.
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8
9
DFT calculation indicated that these ligands had almost the same
optimized structures. The amide substituents (C
proposed to cause the electronic effects on these spectral changes.
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.48 and 7.11 for ligand 2: G. Anderegg, E. Hubmann, N. G. Podder
and F. Wenk, Helv. Chim. Acta, 1977, 60, 123–140.
2 5 2
H or CH Py) were
a
2
2+
2+
1
1
0 (a) The log K value of the 1–Zn complex was reported in ref. 9,
13 Tetra-coordinated Zn complexes with tripodal ligands were reported:
2+
while that of the 2–Zn complex was reported in ref. 10(b); (b) D. W.
Gruenwedel, Inorg. Chem., 1968, 7, 495–501.
X. Xu, C. S. Allen, C.-L. Chuang and J. W. Canary, Acta Crystallogr.,
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1 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb,
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14 DMEM contains inorganic salts, amino acids, vitamins and glucose.
See: H. J. Morton, In Vitro, 1970, 6, 89–108.
This journal is © The Royal Society of Chemistry 2008
Dalton Trans., 2008, 4038–4043 | 4043