Synthesis and DNA cleavage activity of piperazine containing guanidinoethyl and hydroxyethyl side arms

Article abstract of DOI:10.1002/jccs.201300321

According to active groups synergetic catalysis principle, a novel phosphodiester receptor 1-(N-guanidinoethyl)-4-(N-hydroxyethyl)-piperazidine hydrochloride was firstly synthesized and the preliminary studies of its DNA cleavage activity. It is characterized and confirmed by methods as ¹H NMR and ^13C NMR. A couple hardness with softness piperazidine is designed to connect guanidinium group and hydroxyl group; The reserch was showed that the best cleaving conditions was 7.2 in pH; Cleaving DNA by the compound reaction was oxidation process that were proved through the free radicals quenches experiments; The compound can cleaved the pUC 19 DNA by phospholipid transferance was proved by BDNPP. A novel DNA cleavage activity of artificial small organic molecule receptor 1-(N-guanidinoethyl)-4-(N- hydroxyethyl)piperazine hydrochloride containing the electrophilic activation guanidinoethyl and the nucleophilic hydroxyl group side arms was synthesized. The results of pH-dependence indicate pH 7.2 is the optimal pH for DNA cleavage in the presence of 3. Transphosphorylation pathway is the mechanism for the DNA cleavage was proved by BDNPP which is used as the DNA mimics successfully. Copyright

Full text of DOI:10.1002/jccs.201300321

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Synthesis and DNA Cleavage Activity of Piperazine Containing Guanidinoethyl and
Hydroxyethyl Side Arms
Dong An and Zhiwen Ye*
Departmrnt of Chemistry Engineering, Nanjing University of Science and Technology, Nanjing, 210094, China
(Received: Jun. 29, 2013; Accepted: Sept. 24, 2013; Published Online: Dec. 2, 2013; DOI: 10.1002/jccs.201300321)
According to active groups synergetic catalysis principle, a novel phosphodiester receptor 1-(N-guani-
dinoethyl)-4-(N-hydroxyethyl)-piperazidine hydrochloride was firstly synthesized and the preliminary
1
studies of its DNA cleavage activity. It is characterized and confirmed by methods as H NMR and
13
C
NMR. A “couple hardness with softness” piperazidine is designed to connect guanidinium group and
hydroxyl group; The reserch was showed that the best cleaving conditions was 7.2 in pH; Cleaving DNA
by the compound reaction was oxidation process that were proved through the free radicals quenches ex-
periments; The compound can cleaved the pUC 19 DNA by phospholipid transferance was proved by
BDNPP.
Keywords: Piperazidine; Phosphodiester bond; Guanidinium group; Spacer.
2
0-22
INTRODUCTION
effect.
Artificial nucleases including some small organic
molecules and metal complexes have been widely investi-
gated for their potential applications in the fields of molec-
RESULTS AND DISCUSSION
Structural information
1
-2
1 13
H NMR and C NMR Spectra
ular biological technology and drug development. How-
ever, the pharmic use of metal complexes is hampered by
concerns over the lability and toxicity due to free-radical
generation during the redox processes of some transition
Although the guanidine group is not a stronger elec-
tron with drawing group compared to the phthalimido group
1
3
according to the C NMR spectra and the alpha methyl
3
-5
1
protons in H NMR. It gives a significant difference of
metal, such as Zn. Metal-free cleaving reagents which
are identified safer for their hydrolytic pathway of cleaving
the P-O bond of phosphodiester in nucleic acids and have
shown clinical potential have been first put forward by
Gobel and coworkers. Such small organic molecules as
0.8-1.1 ppm between two compounds. The capacity of
guanidine groups in the compound 3 to absorb electrons
from methyl is more intensive than phthalimide groups in
the compound 1, which facilitates the reduction of the den-
sity of electron clouds surrounding the piperazine, abating
the shielding that results the movement of electron clouds
to the low-field.
6
-8
9-11
guanidinium derivatives, cyclodextrin derivatives,
1
macrocyclic polyamines,
2-14
15-17
and dipeptides
have been
used to cleave phosphodiester. Guanidinium receptors as
nuclease mimics for cleavage of active phosphodiester,
such as bis(p-nitrophenyl) phosphate (BNPP) and bis(2,4-
dinitrophenyl) phosphate (BDNPP), have been reported by
many research workers. DNA clevage activity of artificial
receptors 1,4,7-triazacyclononane and tetrahydropyrimi-
din containing guanidinoethyl and hydroxyethyl side arms
have been reported as phosphodiester receptor success-
Scheme I Synthetic routes of target compound 3
1
8-19
fully.
In the cmpound, the guanidinium group serves to
recognize, bind, and electrophilically activate the anionic
phosphodiester through hydrogen bonding and electro-
static interaction. The hydroxyl group works as a nucleo-
philic groupin the transphosphorylation reaction, which is
expected to be highly efficient because of the proximity
*
Corresponding author. E-mail: yezw@njust.edu.cn
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Article
An and Ye
The chemical shift of the hydrogen in the same sites is
Fig. 3a is the agarose gel of pH-dependence assays
which indicates that the supercoiled DNA (form I) relaxes
to form a nicked circular DNA(form II) in the presence of 3
and thelinear DNA (form III) forms when the pH is in the
range of 6.5 to 9.0. The pH dependence data of 3 (Fig. 3b)
presents a bell shaped profile which indicates that pH 7.2 is
the optimal pH for DNA cleavage in the presence of 3. A
relatively low NaCl concentration (10 mM) was selected to
control the ionic strength in all experiments.
relatively large under the existent of deuterium replaced
water solvent and chlorine anions. So, the NMR chemical
shift of methyl group of piperazinein compound 3 and com-
pound 1 are downshift to around 3.2-3.5 ppm and 2.4-2.6
ppm due to spatial effect, respectively.
The hydrogen on hydroxyl and N-H of guanidine ex-
hibit no chemical shift due to proton exchange effect when
1
heavy water is used as a solvent for H NMR nuclear mag-
netic resonance. (For NMR and ESI-MS data, see the Sup-
porting Information.)
Free radicals quenches experiments
(a) Histogram representing of pUC19 plasmid DNA
(
0.05 mM bp) cleaved by 3 (0.042 mM) in the presence of
standard radical scavengers incubated for 24 h at 37 °C in
pH 7.2 buffer (50 mM Tris–HCl). (b) Agarose gel of radical
scavengers inhibition reactions. Lanes 1–6, DNA control,
DNA + 3, DNA + 3 + NaN
3
(10 mM), DNA + 3 + KI (10
mM), DNA + 3 + t-BuOH (1 mM), and DNA + 3 + DMSO
(
1 mM), respectively.
Fig. 1. Structure of the title compound 1.
Fig. 4 shows the agarose gel of radical scavengers in-
hibition reactions which indicate that the oxidative species
Fig. 2. Packing diagram of the title compound 1 of a
unit cell.
In the molecule of the title compound 1, the piper-
azine ring adopts a chair conformation, with its N-C bonds
in pseudoequatorial orientations the phthalimide fragment
is planar, structure shown in Fig. 1. In the crystal, the crys-
tal packing is stabilized by O-H···N hydrogen bonding in-
teractions and p–p interactions involving the benzene ring,
see Fig. 2.
Fig. 3. The pH-dependence profile for DNA cleavage
promoted by 4.
Agarose gel of pH dependence
(
a) Agarose gel (1%) of pUC 19 DNA (0.05 mmol/L
bp) incubated for 15 h at 37 °C with 0.042 mmol/L 4 in dif-
ferent pH buffer: Lanes 1-10, DNA control, pH 6.5, 6.75,
7
.0, 7.25, 7.75, 8.0, 8.25, 8.5 and 9.0, respectively; (b) pH
dependent profile for DNA cleavage promoted incubated
for 24 h by 0.042 mmol/L 4 (n) in different pH buffer (50
mmol/L) at 37 °C.
Fig. 4. The radical scavengers inhibition reactions for
DNA cleaved by 4.
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Artificial DNA Cleavage
effect was eliminated in the system. The reaction should be
through a non redox process for DNA cleavage in the pres-
ence of 3.
Systhetic procedure and analysis. 1-(N-(2-Ethyl)phth-
alimido)-4-(N-hydroxyethyl)-piperazine (1): A stirred solution of
compounds N-(2-hydroxyethyl)piperazine (0.36 g, 2.75 mmol),
N-(2-bromoethyl)phthalimide (0.63 g, 2.50 mmol) and anhydrous
Reaction mechanism
To study the cleavage mechanism, BDNPP was used
as the DNA mimics. BDNPP and 3 were dissolved in DMF/
3
potassium carbonate (0.90 g) in dry CH CN (15 mL) was heated
at 80-85 °C for 10 h. The mixture was then allowed to cool to
room temperature and filtered. Filter cake was washed with
2
H O, and after 0.5 h equilibration time at room tempera-
ture, ESI-MS analysis was carried out. In ESI-MS spec-
3
CH CN (3 × 5 mL) .The filtrate was concentrated under reduced
trum, the peaks at m/z 428.9(-) and 182.95(-) show the sig-
-
nals of BDNPP (calcd [BDNPP-H] , 429.0) and 2,4-dini-
-
trophenol (DNP) (calcd [DNP-H] , 183.0), respectively.
pressure to give a flaxen solid. This solid was dissolved in de-
ionized water (5 mL) and then a small amount of unreacted N-(2-
bromoethyl)phthalimide was precipitated. The mixture was fil-
tered and the filtrate was collected. After the water was removed
under reduced pressure, a pale oil was obtained and purified by
silica gel chromatographic column (petroleum ether/methanol,
2/1 then 1/1) to obtain compound 1 as a white needle solid (0.55 g,
The phosphodiester bond of BDNPP would be cleaved by 3
via a transphosphorylation pathway (Scheme IIa). Thus,
similar to BDNPP, transphosphorylation is one of the pos-
sible mechanisms for the DNA cleavage promoted by 3,
which is schematically depicted in Scheme IIb.
1
1.80 mmol). Yield: 72.4%. m.p.: 133.2-133.7 °C. H NMR (500
MHz/CDCl
3
): d 2.50-2.52 (m, 10H, 5 × NCH
CH N), 3.59 (t, J = 5.3 Hz, 2H, CH
t, J = 6.5 Hz, 2H, CONCH
2
), 2.64 (t, J = 6.5
OH), 3.82
), 7.71-7.73 (m, 2H, 2 × Ar-H),
Scheme II The mechanism for DNA cleavage pro-
moted by 3
Hz, 2H, CONCH
2
2
2
(
2
3
1
7
.84-7.86 (m, 2H, 2 × Ar-H). C NMR (125 MHz/CDCl
3
): d 35.3
(
CONCH
2
CH
2
), 52.8 (NCH
2
), 53.1 (NCH
2
), 55.7 (NCH
2
CH OH),
2
5
1
9.1 (HOCH ), 123.2 (Ar-C), 132.2 (Ar-C), 133.86 (Ar-C),
+
68.35 (CO). ESI-MS: m/z [M+H] , calcd. 304.17, found 304.05.
2
1
-(N-guanidinoethyl)-4-(N-hydroxyethyl)piperazine hy-
drochloride (3): Compound 2 (0.80 g, 4.62 mmol) was dissolved
in water (5 mL) and then O-Methylisourea sulfate (0.54 g, 2.20
mmol) dissolved in sodium bicarbonate solution (10 mL, 5 M)
was added. The mixture was stirred for 20 h at room temperature
EXPERIMENTAL
under an atmosphere of dry N and then evaporated to remove the
2
Materials and Apparatus. All reagents and chemicals
were obtained from commercial sources and used without further
purifications. N-(2-bromoethyl)phthalimide and N-(2-Hydroxy-
ethyl)piperazine were purchased from Aladdin. pUC 19 plasmid
DNA was purchased from TaKaRa Biotechnology (Dalian) Co.
Ltd., and the purity was checked by agarose gel electrophoresis
and their concentration was determined by UV spectroscopy us-
ing the extinction coefficient appropriate for double-stranded
solvent. The crude product was dissolved in deionized water and
chromatographed on a strong base anion-exchange resin column
(eluted with deionized water). Subsequently, the eluent was evap-
orated under reduced pressure to remove water, and the residue
was washed with ether (3 × 10 mL) to eliminate the unreacted re-
actant 2 and other organic impurities. The residue was then dis-
solved in deionized water (15 mL) and neutralized to pH 7.0 with
5% hydrochloric acid. Water was removed in vacuum to give
compound 3 as strong hygroscopic brown solid (0.11 g, 0.37
-
1
DNA(1.0 OD260 = 50 mg/mL ). Agarose was from Oxoid Limited
of Basingstoke (UK). All solvents were purified by standard pro-
cedure.
1
mmol). Yield: 91.4%. H NMR (500 MHz/D O): d 3.27 (t, J = 6
2
Hz, 2H, NCH ), 3.35 (t, 2H, J = 5 Hz, NHCH CH ), 3.47 (br s,
2
2
2
1
13
H NMR and C NMR data were recorded on a Brucker
2 2
4H, piperzaine-CH ), 3.87 (t, J = 5.0 Hz, 2H, NHCH ), 3.56 (t, J =
1
3
AM 500 spectrometer (Germany). Mass spectra were obtained on
an electrospray mass spectrometer (LCQ, Finnigan) in positive
mode. The pH value was confirmed by ORION868 pH meter with
an Ag/AgCl electrode as the reference electrode in saturated KCl
solution at room temperature. The gelelectrophoresis was con-
ducted by DYY-5 electrophoresis apparatus.
6.0 Hz, 2H, CH OH), 3.63 (brs, 4H, 2 × piperzaine-CH ).
2
C
2
NMR (1250 MHz/D O): d 45.8 (CH OH), 49.6 (NHCH CH ),
2
2
2
2
51.5 (piperzaine-CH ), 55.0 (piperzaine-CH ), 57.5 (NCH CH OH),
2
-
60.7 (NHCH ), 157.12 (guanidine-C). ESI-MS: m/z [M-Cl ]
2
2
2
+
2
calcd. 216.18, found 216.00.
Plasmid DNA cleavage. DNA cleavage experiments were
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Article
An and Ye
performed using 500 ng per reaction of pUC 19 derived plasmid
of 2686 bp length. The DNAfragments after cleavage assays were
separated and monitored by agarose gel electrophoresis. The
supercoiled DNA in 50 mM Tris-HCl buffer containing 10 mM
NaCl was treated with different compound concentrations fol-
lowed by dilution with the buffer to a total volume of 15 mL. The
sample was incubated at 37 °C. The loading buffer (30 mM
EDTA, 0.05% (w/V) glycerol, 36% (V/V) bromophenol blue) 3
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3
% agarose gel containing 1.0 mg/dm EB. Electrophoresis was
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CONCLUSIONS
In this study, design and synthesis of a novel phos-
phodiester receptor 3 containing guanidinoethyl and hy-
droxyethyl side arms was achieved successfully. The reserch
was showed that pH 7.2 is the optimal pH for DNA cleav-
age in the presence of 3; Cleaving DNA by the compound
reaction was oxidation process; The compound can cleave
the pUC 19 DNA by phospholipid transferance. This sub-
stantial acceleration of cleavage reaction is due not only to
the spatial proximity of the nucleophilic hydroxyl group
but also the electrophilic activation for the phosphodiester
of DNA by the binding guanidinium group.
11. Wang, Y. P.; Wu, Q. F.; Xia, Y. J.; Hu, X. L.; Pan, Z. Q. Chem.
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ACKNOWLEDGEMENTS
We acknowledge support of this work by the National
Science Foundation of China (Grant no.11076017).
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