Received: May 4, 2015 | Accepted: May 26, 2015 | Web Released: June 4, 2015
CL-150428
Synthesis of the Pyridine Hydrazones as Metal-free Artificial Nucleases
Zhifen Li, Jun Qiao, Zhifang Jia, and Shuangming Meng*
School of Chemical and Environmental Engineering, Datong University, Xingyun Street, Shanxi 037009, P. R. China
(E-mail: 200931150019@mail.bnu.edu.cn)
O
O
In this report, four pyridine hydrazones containing anthra-
cene and triphenylamine as a new type of metal-free nucleases
were synthesized. Results indicate that the conjugates can cleave
the plasmid DNA to Form II or Form III under physiological
conditions via hydrolytic pathway.
N
O
O
ii)
i)
EtO
iv)
OEt
O
(NH4)2HPO4
O
CH3CCH2COCH2CH3
N
N
N
N
H
1
O
O
O
O
iii)
CH NHN
R
OEt
EtO
OEt
H2NHN
OEt
N
N
2
N
3
6a-b
v)
O
O
O
O
Intensive research has been focused on the development of
artificial nucleases. These studies are beneficial for shedding
light on cellular and molecular biology and have been applied in
gene therapeutic agents.1-7 With the consideration that many
natural nucleases have been constructed with amino acids and
two or more metal ions in the active sites, many studies have
been devoted to mimic the active sites of natural nucleases.8-10
So most of cleaving agents have been focused on the metal
complexes, including hydrazone complexes,11-13 which need to
be triggered to induce oxidative DNA cleavage by a reducing
agent.14-16 Considering the metal lability and toxicity of metal
complexes in the potential clinical application, the absence of
metal ions in the cleavage reagents were considered safer for
therapy.17-20 Amino acids or various peptide derivatives were
first considered as metal-free artificial nucleases.21-23 Recently,
other metal-free artificial nucleases have also attracted signifi-
cant interest; guanidinium derivatives,24,25 macrocyclic poly-
amines,26-30 cyclodextrin derivatives,31 and hydroxylamine32
have been used as metal-free DNA nucleases, but hydrazones
are rarely reported as metal-free artificial nuclease. Here, we
synthesized the pyridine hydrazones containing anthracene and
triphenylamine and reported the hydrolytic cleavage of super-
coiled plasmid DNA by these compounds.
vi)
H2NHN
NHNH2
R
CH NHN
NHN HC
R
N
N
4
5a-b
5a ,6a R=
5b,6b
R=
N
Scheme 1. i) C2H5OH, H2O, 80 °C; ii) ammonium persulfate,
CH3COCH3, H2O; iii) NH2NH2¢H2O, DMSO; iv) 9-anthracenecar-
boxaldehyde or 4-(diphenylamino)benzaldehyde, H2O and C2H5OH;
v) NH2NH2¢H2O, C2H5OH; vi) 9-anthracenecarboxaldehyde or 4-
(diphenylamino)benzaldehyde, H2O and C2H5OH.
Figure 1. Effect of 5a, 5b, 6a, and 6b (100-400 ¯M) on the
cleavage reactions of pUC 18 DNA (9 ¯g mL¹1) in a HEPES buffer
(20 mM, pH 7.2) at 37 °C for 6 h. (a) Agarose gel electrophoresis
diagram: Lane 1, DNA control; Lane 2-5 5a (100, 200, 300, and
400 ¯M), Lane 6-9 5b (100, 200, 300, and 400 ¯M), Lane 10-13 6a
(100, 200, 300, and 400 ¯M), Lane 14-17 6b (100, 200, 300, and
400 ¯M).35
In this study, we combined 2,6-dimethylpyridine with 9-
anthracenecarboxaldehyde and 4-(diphenylamino)benzaldehyde
together through the -CONHN=CH- linkage and investigated
their feasibility in the DNA cleavage.
(Form II) or the linear DNA (Form III) in the absence of any
external agents or metal ions. Figure 1 shows the agarose gel
of concentration-dependence assays, which indicates that the
supercoiled DNA (Form I) relaxes to form a nicked circular
DNA (Form II) in the presence of 5a, 5b, 6a, and 6b at different
concentrations. The results indicated that the four conjugates
cleaved the plasmid DNA with almost the same activities and the
increase of concentration appeared to be insignificant. Thus,
further investigation of the cleaving ability has been carried out
through the increase of time. Figure 2 shows the agarose gel of
supercoiled plasmid DNA cleavage promoted by 5a, 5b, 6a, and
6b in HEPES buffer (pH 7.2) at 37 °C for 12 h. It was obvious
that all of the plasmid DNA was converted to nicked (Form II)
and linear forms (Form III). Hind III (endonuclease) that was
used to clarify the new band (Figure S3), the result clearly
indicated that the new band produced by 6a was Form II and
Form III.
Firstly, ethyl acetoacetate was reacted with hexamethylene-
tetramine and ammonium phosphate in ethanol and water at
80 °C to afford the desired product 1, which was then oxidized
with ammonium persulfate in acetone and water at room
temperature for 3 h to give 2 (Scheme 1).33 The monocarbohy-
drazide 3 and dicarbohydrazide 4 were obtained by the reaction
between 2 and hydrazine hydrate in different solvents.34 4 and
9-anthracenecarboxaldehyde or 4-(diphenylamino)benzaldehyde
were mixed in ethanol and water to obtain 5a and 5b. 3 and
9-anthracenecarboxaldehyde or 4-(diphenylamino)benzaldehyde
were mixed in ethanol and water to obtain 6a and 6b. The yield
of each step is acceptable. The detailed data were listed in the
Supporting Information.
The DNA catalytic activities of 5a, 5b, 6a, and 6b on the
cleavage of DNA were studied using supercoiled pUC18 DNA
plasmid DNA as a substrate in HEPES (pH 7.2, 37 °C). Agarose
gel electrophoresis was used to assess the conversion of
supercoiled plasmid DNA (Form I) to nicked circular DNA
To verify the catalytic activity of DNA cleavage promoted
by 5a, 5b, 6a, and 6b, several control experiments were carried
© 2015 The Chemical Society of Japan | 1243