Synthesis of glucopyranosyl Schiff base zinc(II) complexes capable of interacting with mononucleotides, and their DNA-cleavage activities

Article abstract of DOI:10.1002/cbdv.201100441

New glucopyranosyl Schiff base zinc complexes, [Zn(GlcSal)₂] (1; GlcSalH=N-(2-deoxy-β-D-glucopyranos-2-yl-salicylaldimine) and [Zn(AcOGlcSal)₂] (2; AcOGlcSalH=N-(2-deoxy-β-D-1,3,4,6- tetraacetylglucopyranos-2-yl-salicylaldimine) were synthesized, and characterized by spectral and analytical methods. The interaction between the Zn complexes and mononucleotides was investigated by ¹H-NMR, ³¹P-NMR and UV/VIS spectroscopies. Mononucleotides, cytidine 5'-monophosphate (CMP) and uridyl 5'-monophosphate (UMP), interacted with these complexes to form a 1: 1 complex with 1 and a 1: 2 complex with 2, depending on the presence of the OH group of glucopyranosyl substituents. The DNA-cleavage activities of 1 and 2 were studied using plasmid DNA (pBR322) in a medium of 5 mM Tris·HCl/50 mM NaCl buffer in the presence of H₂O ₂. The DNA-cleavage activity decreased in the order of 2>1>Zn(OAc)₂, indicating the significant promoting effect of the glucopyranosyl Schiff base ligand and the participation of the glucopyranosyl OH groups in the cleavage mechanism. The mechanism of the DNA cleavage by 1 and 2 was investigated by evaluation of the effect of a HO radical scavenger and a singlet-oxygen (¹O ₂) quencher under aerobic conditions. The former exhibited little effect, excluding the HO^. radical as an active species and supporting the hydrolysis mechanism for the main process of the DNA cleavage. The latter quencher somewhat hindered the cleavage, indicating the partial participation of a ¹O₂ as a competitive active species in the present system. Copyright

Full text of DOI:10.1002/cbdv.201100441

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Synthesis of Glucopyranosyl Schiff Base Zinc(II) Complexes Capable of
Interacting with Mononucleotides, and Their DNA-Cleavage Activities
by Misaki Nakai*a), Hironobu Fukudaa), Shigenobu Yanob)c), and Yasuo Nakabayashia)
a) Department of Chemistry and Materials Engineering, Faculty of Chemistry, Materials and
Bioengineering, Kansai University, 3-3-35, Yamatecho, Suita-shi, Osaka, 564-8680, Japan
(phone: þ81-6-6368-1111-5908, e-mail: nakai@kansai-u.ac.jp)
b) Office of Society-Academia Collaboration for Innovation, Kyoto University, Kyoto University
Katsura, Nishikyo-ku, Kyoto-daigaku Katsura, Kyoto 615-8520, Japan
c) Graduate School of Material Science, Nara Institute of Science and Technology, 8916-54,
Takayama-cho, Ikoma, Nara, 630-0192, Japan
New glucopyranosyl Schiff base zinc complexes, [Zn(GlcSal)2] (1; GlcSalH¼N-(2-deoxy-b-d-
glucopyranos-2-yl-salicylaldimine) and [Zn(AcOGlcSal)2] (2; AcOGlcSalH¼N-(2-deoxy-b-d-1,3,4,6-
tetraacetylglucopyranos-2-yl-salicylaldimine) were synthesized, and characterized by spectral and
analytical methods. The interaction between the Zn complexes and mononucleotides was investigated
by 1H-NMR, 31P-NMR and UV/VIS spectroscopies. Mononucleotides, cytidine 5’-monophosphate
(CMP) and uridyl 5’-monophosphate (UMP), interacted with these complexes to form a 1:1 complex
with 1 and a 1:2 complex with 2, depending on the presence of the OH group of glucopyranosyl
substituents. The DNA-cleavage activities of 1 and 2 were studied using plasmid DNA (pBR322) in a
medium of 5 mm Tris·HCl/50 mm NaCl buffer in the presence of H2O2. The DNA-cleavage activity
decreased in the order of 2>1>Zn(OAc)2, indicating the significant promoting effect of the
glucopyranosyl Schiff base ligand and the participation of the glucopyranosyl OH groups in the cleavage
mechanism. The mechanism of the DNA cleavage by 1 and 2 was investigated by evaluation of the effect
.
of a HO radical scavenger and a singlet-oxygen (1O2) quencher under aerobic conditions. The former
.
exhibited little effect, excluding the HO radical as an active species and supporting the hydrolysis
mechanism for the main process of the DNA cleavage. The latter quencher somewhat hindered the
cleavage, indicating the partial participation of a 1O2 as a competitive active species in the present system.
Introdution. – Cisplatin, which is in widespread clinical use, is very efficient and
often used for chemotherapy of cancer treatment. However, cisplatin has serious side
effects such as nephrotoxicity, neurotoxicity, etc. [1]. As cisplatin-resistant cells were
also reported in recent years [2], it is very important to develop new metal complexes
which can act as antitumor drugs. Therefore, several metal complexes with antitumor
activity, such as palladium [3–6], ruthenium [7–11], copper [12–14], zinc, and
vanadium [15–17], were synthesized and investigated, expecting significant interaction
with DNA and high DNA-cleavage activity. In this report, we have focused on Zn
complexes, since Zn is one of the most important metals in the biological system and
plays the role of the active site of more than 200 enzymes [18][19]. Some Zn-containing
enzymes are known to act in the DNA and RNA reactions [20]. Further, as reported by
Arunakaran and co-workers, Zn inhibits proliferation of prostate cancer, and acts as a
potential chemopreventive agent in targeting the prostate cancer [21]. Therefore, Zn
can be expected as an antitumor or a tumor-inhibiting agent.
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Recently, some Schiff base Zn complexes were synthesized and investigated for
their DNA-cleavage and antitumor activities [22–25]. The major mechanism of the
DNA cleavage with these Zn complexes has been explained by hydrolysis of the
phosphate ester [26]. The Zn complex binds to DNA through an O-atom in the
phosphodiester backbone of DNA, resulting in activation of the PꢀO bond to be
cleaved. An adjacent coordinated H2O molecule attacks the P-atom of the phospho-
diester to form a pentacoordinated intermediate, followed by the cleavage of one of the
PꢀO ester bonds via the intramolecular charge transfer to accomplish the DNA
cleavage [26].
On the other hand, carbohydrate is one of the components of cell walls and used as
an energy source in biological system [27]. Because a cancer cell incorporates glucose
much more efficiently than that of a normal tissue, glucose derivatives have been used
as a diagnostic drug for, e.g., positron emission tomography (PET) [28]. In addition,
glucosyl conjugation of metal complexes was found useful for antitumor drugs and
photodynamic-therapy agents [6][29][30]. It has been reported that introduction of
carbohydrate into an antitumor drug is efficient for uptake by cancer cell and antitumor
activities [31]. From this viewpoint of the effective glucosyl conjugation, we here
synthesized new ZnII complexes attached to the glucosamine derivatives. Although
some Schiff base Zn complexes conjugated with glucosamine have been synthesized
[32–34], there are a few reports on their potential for interaction with mononucleo-
tides or for the DNA-cleavage activity. Since it is assumed that a H-bond between a
metal complex and DNA plays an important role for the DNA-binding affinity of the
metal complex [11][35][36], the present study was undertaken to clarify how the
glucopyranosyl conjugation to the Schiff base Zn complex affects the interaction with
DNA and the DNA-cleavage activity. The acetylglucopyranosyl analog was also
studied to understand the effect of the OH groups.
Results and Discussion. – Syntheses and Characterization. The ligands were
synthesized from d-glucosamine, and characterized by using 1H-NMR. The Zn
complex [Zn(GlcSal)2] (1; GlcSalH¼N-(2-deoxy-b-d-glucopyranos-2-yl)salicylaldi-
mine) was prepared by the reaction of N-(2-deoxy-b-d-glucopyranos-2-yl)salicylaldi-
mine with Zn(OAc)2 according to the procedure reported in [33]. The Zn complex 2
was synthesized in the analogous way: the reaction of N-(2-deoxy-b-d-1,3,4,6-
tetraacetylglucopyranos-2-yl)salicylaldimine (AcOGlcSalH) with Zn(OAc)2 was per-
formed in EtOH to produce [Zn(AcOGlcSal)2] in the 30% yield after reprecipitation
from Et2O (Scheme). HR-ESI-MS of 1 and 2 recorded in the positive-ion mode
exhibited peaks at m/z 651.1071 ([MþNa]þ ) for 1 and 965.1933 (Mþ ) for 2,
respectively, indicating formation of the expected complexes. In the IR spectrum of 1, a
broad band at ca. 3400 cmꢀ1 was assigned to the stretching vibration of the H-bonded
OH groups of the glucosamine moiety. On the other hand, 2 exhibited a strong band at
1750 cmꢀ1, which was ascribed to the C¼O stretching vibration of the Ac group. The
bands attributed to the C¼N stretching vibration of the imino group were observed at
1630 cmꢀ1 [23].
1
In the H-NMR spectra, two sets of signals were observed for the H-atoms of
azomethine and glucose groups of GlcSalH and 1 (in DMSO/D2O), and for the phenol
OH group of GlcSalH (in DMSO), indicating the presence of two isomers. In case of

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Scheme. Syntheses of Zn Complexes 1 (a- and b-anomers) and 2 (b-anomer)
GlcSalH, the coupling constants of the clear two doublets of anomeric H-atoms were
3.4 and 7.9 Hz, which correspond to the typical gauche and trans vicinal H,H coupling
values, respectively [27]. Therefore, these two sets of peaks were attributed to the a-
and b-anomers of glucosamine moieties. The ratio a/b anomers of GlcSalH and 1 was
both ca, 4 :6. On the other hand, in case of AcOGlcSalH and 2, only one set of signals
was observed for the corresponding H-atoms, indicating that the compounds are not
mixtures of isomers. This is compatible with the steric effect of the Ac group in the
glucosamine moiety, and the plausible isomer is the b-anomer. Fig. 1 shows the UV/VIS
spectra of the Zn complexes in DMSO. Two peaks were observed, at ca. 260 and at 320–
380 nm, which were attributed to the pꢀp* transition of the salicyl group and the nꢀp*
transition of the imino group, respectively [37].
Interaction between Zn Complexes and Mononucleotides. Interactions between the
Zn complexes 1 and 2, and mononucleotides, 5’-cytidylic acid (CMP) and 5’-uridylic
acid (UMP), as monomers of DNA and RNA, respectively, were investigated by using
1H-NMR and 31P-NMR in DMSO/D2O 4 :1 (v/v). Since it was reported that Zn
1
complexes with an imino group were often hydrolyzed in H2O [38], the H-NMR
spectral changes of 1 and 2 in DMSO/D2O 4 :1 (v/v) were determined after 0 h and 2 h.
As depicted in Fig. 2, no spectral change was observed, indicating that decomposition
of 1 and 2 by hydrolysis is negligible.
Fig. 3 shows 1H-NMR spectra of 1 and 2 in the presence of CMP. After addition of
mononucleotides, peaks ascribed to free ligands, GlcSalH and AcOGlcSalH, were
observed, suggesting that the CMP coordination to Zn was accompanied by the partial

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1945
Fig. 1. UV/VIS Spectra of complexes 1 and 2 in DMSO solutions. —: 1, ---: 2.
dissociation of the Schiff base ligands. The signals of CMP were shifted and broadened
after coordination to 1 and 2, as observed previously with other Zn complexes [39]. In
case of 2, the signals of CMP coordinated to 2 were not clear in a [CMP]/[2] ratio of 1:1,
but broad peaks of CMP bonded to 2 were clearly observed at [CMP]/[2] 2 :1. This
indicates the coordination of two molecules of CMP to Zn, forming [Zn(AcOGlc-
Sal)2(CMP)2] or [Zn(AcOGlcSal)(CMP)2]. As shown in Fig. 3, the chemical shifts
*
(Dd) of CMP H-atoms (HꢀC(1), HꢀC(5), CH2(6); marked with ) after coordination
to 1 and 2 were less than 0.2 ppm. Since the chemical-shift values of CMP are reported
to be within only 0.2 ppm after coordination to Ru via phosphate anions [40], the
chemical-shift values of CMP and UMP with 1 and 2 suggested the coordination of
mononucleotides to 1 and 2 via phosphate anions.
Since the fairly broad 1H-NMR peaks of CMP and UMP after coordination to 1 and
2 were not suitable to further investigate the coordination mode, the coordination of
CMP or UMP to Zn was examined by 31P-NMR. In the 31P-NMR spectra, the 31P
resonances of CMP and UMP were also shifted to the lower fields and broadened after
addition to the solutions of 1 and 2 (Fig. 4). As seen in the Table, these shifts are more
1
remarkable than those observed with H-NMR and ascribed to the coordination of
CMP and UMP with phosphate anions to 1 and 2, as observed previously with other
Schiff base Zn complexes [25]. In addition, the chemical-shift values of CMP and UMP
are greater after ligation to 2 than with 1, suggesting the stronger binding of 2 than 1
[24].
The interactions of 1 and 2 with mononucleotides were also investigated by UV/VIS
spectroscopy. Fig. 5 shows UV/VIS spectral changes of 1 and 2 after addition of CMP in
DMSO/H2O 4 :1 (v/v). In the 300–500-nm regions, where the nucleotide absorption is

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 2. 1H-NMR Spectra of complex 1 after 0 h (a) and 2 h (b); of complex 2 after 0 h (c) and 2 h in
DMSO/D2O 4 :1 (v/v) (d)
negligible, the intensity of the low-energy band at ca. 380 nm of 1 and 2 decreased,
accompanied with the increase in the new bands at ca. 323 nm, indicating that
nucleotides bind to 1 and 2 as observed with another Schiff base Zn complex [25]. The
spectral change ceased when the concentration of CMP became equivalent to that of 1.
On the other hand, in case of 2, the spectral change continued until the [CMP]/[2] ratio
became 2. The intrinsic-binding-constant (Kb) values were estimated according to
Eqn. 1 [41].
½CMPꢁ
½CMPꢁ
½CMPꢁ
¼
þ
(1)
jea ꢀ ebj jeb ꢀ efj Kbjeb ꢀ efj
where [CMP] represents the concentration of CMP, and ea, ef, and eb are the apparent
extinction coefficient (Aobs/[M]), the extinction coefficient for free metal complex
(M), and the extinction coefficient for the free metal complex (M) in the fully bound
form, respectively. As shown in Table, the Kb value of 2 is greater than that of 1,
supporting the stronger binding affinity of 2 to CMP than 1 as indicated by 31P-NMR.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Fig. 3. 1H-NMR Spectra of a) CMP, b) CMP/1 1:1, c) CMP/2 1:1, d) CMP/2 2 :1 in DMSO/D2O 4 :1
*
(v/v). *: Peaks of the free ligands; : peaks of mononucleotide.
Orvig and co-workers have reported the formation of ReI tricarbonyl complexes of
N-(2’-hydroxybenzyl)-2-amino-2-deoxy-d-glucose [42]. Tridentate coordination of the
glucosamine ligand with C(3)ꢀOH(glucose), NH(amino), and O(phenolato) suggests
the similar tridentate coordination of GlcSal to Zn with C(1) or C(3)ꢀOH(glucose),
N(imino), and O(phenolato) in 1. In case of 2, the absence of the glucose OH groups
after acetylation suggests the bidentate coordination of AcOGlcSal to Zn, leaving open
or solvent coordination sites at the Zn center. Thus, in case of 1, the mono-coordination
of nucleotide to Zn may form a tetracoordinate complex after dissociation of one of
two GlcSal ligands such as [Zn(GlcSal)(CMP)]. Similar dissociation of one of two
tridentate ligands was also reported by Mitra et al., in the reaction of phosphate anions
with the hexacoordinate Zn complex of 1-[2’-deoxy-2’-(iminomethyl)-d-glucopyrano-
syl]-2-hydroxynaphthalene forming a tetrahedral Zn complex, in which case ligations
of three dihydrogen phosphate and one Schiff base ligand in the monodentate form
have been proposed [34]. Since coordination of one molecule of CMP was indicated in
case of 1, the GlcSal ligand seems to keep the tridentate coordination mode. In case of
2, two nucleotide molecules may coordinate to Zn after dissociation of one of two
AcOGlcSal ligands to form a tetracoordinate complex such as [Zn(AcOGlc-
Sal)(CMP)2], but the probability of coordinatiton without dissociation of AcOGlcSal
to form a hexacoordinate complex, e.g., [Zn(AcOGlcSal)2(CMP)2], may not be
excluded.
Plasmid DNA-Cleavage Activity. The chemical nuclease activities of 1 and 2,
together with Zn(OAc)2 and ligands, GlcSalH and AcOGlcSalH, as reference, were

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 4. 31P-NMR Spectra of a) CMP, CMP in the presence of b) 1 or c) 2; 31P-NMR spectra of d) UMP;
UMP in the presence of e) 1 or f) 2 in DMSO/D2O 4 :1 (v/v) (mononucleotide/Zn complex 1:1). All
spectra were recorded with H3PO4 in DMSO/D2O 4 :1 (v/v) as an external reference.
Table. 31P-NMR Chemical Shifts (Dd) Observed for Mononucleotide with Complex 1 and 2, and the
Binding-Constant (Kb) Values of 1 and 2 for CMP in DMSO/D2O 4 : 1 (v/v)
Complex
Dd [ppm] (CMP)
Dd [ppm] (UMP)
Kb [mꢀ1
]
1
2
0.98
4.22
1.06
3.62
7.13ꢂ103
8.46ꢂ103

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Fig. 5. UV/VIS Spectral change of a) 1 and b) 2 with increasing concentration of CMP in DMSO/H2O 4 :1
(v/v). Insets: plots of [DNA]/ea –ef vs. [DNA] for the titration of CT-DNA with complexes at 323 nm.
[Complex]¼1.0 ꢂ10ꢀ4 m, [CMP]¼0 a) ca. 1.0 ꢂ10ꢀ4 m, and b) 0–2.0ꢂ10ꢀ4 m.
studied using plasmid DNA (pBR322) in a medium of 5 mm Tris·HCl/50 mm NaCl
buffer (pH 7.5) at 378 and for 8 h incubation in the presence of H2O2. Substrates were
dissolved in 5 mm Tris·HCl/50 mm NaCl buffer containing 1% DMSO solutions
adjusted to 100 mm/l. Results are shown in Fig. 6.
As shown in Fig. 6, Lane 1, the control experiment only with plasmid in the absence
of H2O2 indicates the mobility of both the supercoiled form (form I) as a major
component and the circular form (form II) as a minor component. The other control
experiments under the conditions using only H2O2 (Fig. 6, Lane 2), AcOGlcSalH with
H2O2 (Fig. 6, Lane 4), GlcSalH with H2O2 (Fig. 6, Lane 5), 1 without H2O2 (Fig. 6,
Lane 8), and 2 without H2O2 (Fig. 6, Lane 9), did not show any DNA-cleavage activity.
However, in the presence of H2O2, Zn(OAc)2 (Fig. 6, Lane 3), 1 (Fig. 6, Lane 6), and 2

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 6. The cleavage patterns of the agarose gel electrophoresis for pBR322 plasmid DNA in 5 mm Tris·
HCl/50 mm NaCl (pH 7.5) solutions. Lane 1: DNA control; Lane 2: DNA with H2O2 (1.0 mm); Lane 3:
DNA with H2O2 and Zn(OAc)2 (100 mm); Lane 4: DNA with H2O2 and AcOGlcSalH (100 mm); Lane 5:
DNA with H2O2 and GlcSalH (100 mm); Lane 6: DNA with H2O2 and 1 (100 mm); Lane 7: DNA with
H2O2 and 2 (100 mm); Lane 8: DNA with 1 (without H2O2); Lane 9: DNA with 2 (without H2O2).
(Fig. 6, Lane 7) showed the component formed by the single-strand breaks. Especially,
the complex 2 exhibited the highest DNA-cleavage activity, in the order of 2>1>
Zn(OAc)2. The different activities, i.e., 2>1, pallalel the different binding constants of
these complexes with nucleotide, CMP, suggesting that the DNA-cleavage activity
depends on the binding affinity of these complexes with DNA. The different activities,
i.e., 1>Zn(OAc)2, indicate that the glucosamine ligand enhances the DNA-cleavage
activity, probably because the glucosamine OH groups assist the proximity of 1 to DNA
by the H-bond formation.
The DNA-cleavage mechanism by glucosamine Zn complexes was investigated by
.
evaluation of the effect of a HO radical scavenger (PhCOONa) and a singlet-oxygen
(1O2) quencher (NaN3) on the cleavage activity [43][44]. Fig. 7 shows the cleavage
patterns of the agarose gel electrophoresis for pBR322 plasmid by the complex 2 under
different conditions. As evident from the patterns of Fig. 7, Lane 2 (without any
scavenger), and Fig. 7, Lane 3 (with PhCOONa), any significant effect of PhCOONa
.
was not observed. This result rules out the probability of the DNA cleavage by the HO
radical. Another finding that the DNA cleavage proceeds in the solution containing 1%
.
DMSO (Fig. 7, Lane 2) supports the non-HO radical mechanism, because DMSO is
.
known as a HO radical scavenger. The addition of NaN3 led to a pattern shown in
Fig. 7, Lane 4, and clearly decreased the extent of form II relative to form I in
comparison with the pattern in Fig. 7, Lane 2. This indicates that 1O2 oxygen
1
participates in the DNA cleavage as an active species, though the O2 process is not
1
a main process. To ascertain the participation of O2 under the conditions for Fig. 7,
Lane 2, the experiment was performed in the dark (Fig. 7, Lane 5). Similarly to the
effect of the NaN3 addition, the slight inhibition of conversion to form II was observed,
Fig. 7. The cleavage patterns of the agarose gel electrophoresis for pBR322 plasmid by 2. Lane 1: DNA
control; Lane 2: DNA with H2O2 and 2 (100 mm) alone; Lane 3: DNA with 2 and PhCOONa (50 mm);
Lane 4: DNA with 2 and NaN3 (50 mm); Lane 5: DNA with H2O2 and 2 (100 mm) under dark.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1951
indicating the light-dependent generation of 1O2. Probability of the DNA cleavage by
1O2 was also reported by Chang and co-workers using Zn complexes [44]. Salen-type
Zn complexes were reported to exhibit fluorescence which originates from the singlet
ligand-centered excited state [37][45]. Since this energy gap is enough to generate 1O2,
it is very probable that the Zn complexes 1 and 2 generate 1O2 with the light irradiation.
1
The present results using 1 and 2 indicated that the O2 process is minor in
.
comparison with the process which is not sensitive to either scavenger, i.e., the HO
1
radical or O2. Alternatively, a hydrolytic mechanism is the most probable major
process as proposed for cleavage by various Zn complexes [26][44][46]. Formation of
ZnꢀOH has been proposed as an active species in the hydrolytic mechanism [46][47],
but the present results indicated the participation of H2O2 in the formation of the active
species. Since the addition of H2O2 is effective to generate the DNA cleavage activities
of 1 and 2 (Fig. 6, Lanes 6–9), it is probable that the peroxide ion coordinated to ZnII
works as an active species for the DNA cleavage. ZnꢀOOH is the probable peroxide
species, and its enhanced nucleophilicity and depressed electrophilicity seem to be
responsible for the present enhanced DNA cleavage activity [48]. The peroxide species
may be formed via Zn-(H2O2)2 in H2O [49], but it is rather difficult to accumulate
direct evidences for these active species.
Conclusions. – Glucopyranosyl-conjugated Schiff base Zn complexes were synthe-
sized. The 31P-NMR and UV/VIS spectroscopic studies indicated that the complex 2
was bound more tightly to mononucleotides compared with the complex 1. The DNA-
leavage activity was in the order 2>1>Zn(OAc)2, depending on the interaction
affinity with DNA. The cleavage is assumed to proceed with the hydrolytic mechanism,
in which H2O2 plays an important role for formation of an active species. For
application of the present complexes to the antitumor-drug design, further research in
vivo and in vitro is necessary, since the antitumor activity of the metal complex may
depend on factors other than interaction with DNA, e.g., cell uptake [50].
This work was supported by Kyoto-Advanced Nanotechnology Network at Nara Institute of Science
and Technology. The authors thank Prof. Shun Hirota and Prof. Takashi Matsuo of Nara Institute of
Science and Technology for ESI mass spectra. We thank also Prof. Takuzo Funabiki for helpful
discussions.
Experimental Part
General. All chemicals and reagents were purchased from commercial sources and were used
without further purification. N-(2-Deoxy-b-d-glucopyranos-2-yl)salicylaldimine (GlcSalH),
[Zn(GlcSal)2] [33], and 1,3,4,6-tetra-O-acetyl-b-d-glucosamine hydrochloride [51] were prepared
according to the methods reported previously. UV/VIS Spectra: JASCO V-670 spectrophotometer
equipped with a temp. controller. IR Spectra: Varian 660-IR, as KBr disks. 1H-NMR Spectra: in
(D6)DMSO or (D6)DMSO/D2O 4 :1 (v/v); JEOL GX 400 MHz spectrometer; all chemical shifts (d) rel.
to TMS. 31P-NMR Spectra: JEOL JNM-ECA600; recorded with H3PO4 in DMSO/D2O 4 :1 (v/v) as
external reference (NAIST). HR-ESI-MS: JEOL AccuTOFJMS-T100LC (NAIST).
Synthesis of [Zn(GlcSal)2] (1). A MeOH soln. (20 ml) of Zn(OAc)2 (0.19 g, 1.0 mmol) was added to
a MeOH soln. (20 ml) of GlcSalH (0.28 g, 0.4 mmol). After 2 h of vigorous stirring, the solvent was
evaporated in vacuo. The light-yellow precipitate that appeared after the addition of Et2O was filtered
and washed with Et2O. The complex was dried overnight in vacuo. Yield: 0.11 g (38%). UV/VIS: 366
(9.2ꢂ103), 270 (1.15ꢂ104). IR (KBr): 3464 (OH), 1627 (N¼C), 1448 (C¼C), 1084 (CꢀO), 1043 (CꢀO).

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1H-NMR (D2O/(D6)DMSO 4 :1 (v/v)): 8.25 (s, azometine); 8.12 (s, azometine); 7.22–7.18 (m, salicyl);
6.63–6.61 (d, J¼8.7, salicyl); 6.53 (t, J¼7.1, salicyl); 5.31–5.22 (m, HꢀC(1) of Glc); 3.91 (dd, J¼9.3,
HꢀC(3) of Glc); 3.59–3.70 (m, HꢀC(4), HꢀC(5), 1 H of CH2(6) of Glc); 3.50–3.42 (m, HꢀC(2), 1 H of
CH2(6) of Glc). HR-ESI-MS: 651.1071 ([MþNa]þ , C26H32N2NaO12Znþ ; calc. 651.1144).
Synthesis of N-(1,3,4,6-Tetra-O-acetyl-2-deoxy-b-d-glucopyranos-2-ylsalicylaldimine (AcOGlc-
SalH). 1,3,4,6-Tetra-O-acetyl-b-d-glucosamine hydrochloride (0.38 g, 1.0 mmol) and NaHCO3 (84 mg,
1.0 mmol) were dissolved in 30 ml of H2O/MeOH 1:2 (v/v), and salicylaldehyde (126 ml) was added. The
mixture was stirred vigorously at r.t. and, after 2 h, afforded a yellow precipitate. The yellow precipitate
was filtered, washed with cold H2O, and dried in vacuo. Yield: 0.32 g (70%). UV/VIS: 319 (6.5ꢂ103), 260
(1.75ꢂ104). 1H-NMR ((D6)DMSO): 12.24 (br., OH of phenyl); 8.59 (s, 1 H of azometine); 7.51–7.48 (m,
1 H of salicyl); 7.38–7.32 (m, 1 H of salicyl); 6.93–6.85 (m, 2 H of salicyl); 6.14 (d, J(1,2)¼8.3, HꢀC(1) of
Glc); 5.58 (dd, J(2,3)¼9.5, J(3,4)¼9.6, HꢀC(3) of Glc); 4.98 (dd, J(3,4)¼9.6, J(4,5)¼9.8, HꢀC(4) of
Glc); 4.31–4.18 (m, HꢀC(5), 1 H of CH2(6) of Glc); 4.01 (dd, J(6,6)¼1.8, J(5,6)¼12.2, 1 H of CH2(6) of
Glc); 3.56 (dd, J(1,2)¼8.3, J(2,3)¼9.5, HꢀC(2) of Glc); 2.01 (s, AcO); 2.00 (s, AcO); 1.97 (s, AcO); 1.85
(s, AcO). ESI-MS: 474.1346 ([MþNa]þ , C21H25NNaO1þ0 ; calc. 474.1376).
Synthesis of [Zn(AcOGlcSal)2]. A EtOH soln. (20 ml) of Zn(OAc)2 (55 mg, 0.3 mmol) was added to
a EtOH soln. (20 ml) of AcOGlcSalH (113 mg, 0.3 mmol). After 2 h of vigorous stirring, the solvent was
evaporated in vacuo. The light-yellow precipitate that appeared after the addition of Et2O was filtered
and washed with Et2O. The complex was dried overnight in vacuo. Yield: 73 mg (30%). UV/VIS: 375
(9.5ꢂ103), 279 (1.09ꢂ104). IR (KBr): 1750 (C¼O), 1629 (N¼C), 1448 (C¼C), 1151 (CꢀO), 1033
(CꢀO). 1H-NMR ((D6)DMSO): 8.20 (s, 1 H of azometine); 7.10–7.04 (m, 2 H of salicyl); 6.50 (d, J¼9.1,
1 H of salicyl); 6.34 (t, J¼7.1, 1 H of salicyl); 5.43–5.06 (m, HꢀC(3), HꢀC(4) of Glc); 4.87–4.80 (m,
HꢀC(5) of Glc); 4.36–4.19 (m, HꢀC(1), 1 H of CH2(6) of Glc); 3.95–3.89 (m, 1 H of CH2(6) of Glc);
3.43–3.40 (m, HꢀC(2) of Glc); 1.97 (s, AcO); 1.90 (s, AcO); 1.85 (s, AcO); 1.82 (s, AcO). HR-ESI-MS:
965.1933 (Mþ, C42H48N2O20Znþ ; calc. 695.2170).
Absorption-Spectral Studies [41]. The interactions between CMP and Zn complexes were evaluated
in DMSO/D2O 4 :1 (v/v) by using UV/VIS spectroscopy. The intrinsic binding constant Kb of the complex
to CMP was determined from Eqn. 1, through a plot of [CMP]/ea –ef vs. [CMP], where [CMP] represents
the concentration of CMP, and ea, ef, and eb, the apparent extinction coefficient (Aobs/[M]), the extinction
coefficient for free metal complex (M), and the extinction coefficient for the free metal complex (M) in
the fully bound form, resp. In plots of [CMP]/ea –ef vs. [CMP], Kb is given by the ratio slope/intercept.
These absorption-spectral studies were performed on JASCO V-670 spectrophotometer. Absorption-
spectral titration experiments were performed by maintaining a constant concentration of the complex
and varying mononucletides.
DNA-Cleavage Activities. The cleavage experiments of supercoiled pBR322 DNA by Zn complexes
or ligands (100 mm), and H2O2 (1 mm) in 5 mm Tris·HCl 50 mm NaCl buffer at pH 7.5 were performed by
agarose gel electrophoresis. All compounds were dissolved in 5 mm Tris·HCl 50 mm NaCl buffer
containing 1% DMSO solns. The samples were incubated for 8 h at 378. DNA Restriction fragments were
loaded on 1% (w/v) agarose gel containing 0.5 mg mlꢀ1 EtBr in TBE buffer (89 mm Tris·borate/2 mm
EDTA) and after running the gel at 100 V for 1 h. The bands were visualized by photographing the
fluorescence of intercalated EtBr under a UV (312 nm) illuminator. The DNA cleavages with added
inhibitors were monitored as in case of cleavage experiment without added inhibitors using agarose gel
electrophoresis. Reactions using pBR322 DNA in 5 mm Tris·HCl/50 mm NaCl buffer at pH 7.5 was
treated with complex 2 (100 mm), and PhCOONa (50 mm) or NaN3 (50 mm). The samples were
incubated for 8 h at 378, and analyzed for cleaved products using gel electrophoresis as described above.
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Received December 26, 2011