NHC-silver(I) complexes as chemical nucleases; Synthesis, crystal structures, and antibacterial studies

Article abstract of DOI:10.1002/hc.21160

A series of N-heterocyclic carbene (NHC) precursors, 1-methoxylethyl-3- allylimidazolium hexafluorophosphate (1), 1-ethyl-3-allylimidazolium hexafluorophosphate (2), and 1-pentyl-3-allylimidazolium hexafluorophosphate (3) were synthesized. These salts were treated with Ag₂O to afford their corresponding mononuclear Ag(I)-NHC complexes, namely 1-methoxylethyl-3- allylimidazolium silver(I) hexafluorophosphate (4), 1-ethyl-3-allylimidazolium silver(I) hexafluorophosphate (5), and 1-pentyl-3-allylimidazolium silver(I) hexafluorophosphate (6), respectively. These compounds were characterized by physicochemical and spectroscopy techniques. Compounds 4 and 5 were structurally characterized by single crystal X-ray diffraction, and their stability in solution was investigated and found to be acceptable for the antibacterial studies. These new NHC precursors and their respective Ag-NHC complexes were screened for their antibacterial activities against Staphylococcus aureus (ATCC 12600) and Escherichia coli (ATCC 25922). Compounds 1-3 showed no inhibition, whereas 4-6 inhibited the growth of these bacteria. The nuclease activities of the reported compounds against plasmid DNA and RNA were assessed by gel electrophoresis, and the results indicate that complexes 5 and 6 can degrade both DNA and RNA in the absence of an oxidant.

Full text of DOI:10.1002/hc.21160

Heteroatom Chemistry
Volume 25, Number 3, 2014
N
HC–Silver(I) Complexes as Chemical
Nucleases; Synthesis, Crystal Structures,
and Antibacterial Studies
1
1
1
Rosenani A. Haque, Patrick O. Asekunowo, Mohd. R. Razali,
2
and Faisal Mohamad
1
The School of Chemical Sciences, Universiti Sains Malaysia, 11800, USM, Penang, Malaysia
2The School of Biological Sciences, Universiti Sains Malaysia, 11800, USM, Penang,
Malaysia
Received 27 January 2014; revised 27 March 2014
DNA and RNA were assessed by gel electrophoresis,
ABSTRACT:
bene (NHC)
allylimidazolium hexafluorophosphate (1), 1-ethyl-
A
series of N-heterocyclic car-
precursors, 1-methoxylethyl-3-
and the results indicate that complexes 5 and 6 can
degrade both DNA and RNA in the absence of an
oxidant. ꢀC 2014 Wiley Periodicals, Inc. Heteroatom
Chem. 25:194–204, 2014; View this article online at
wileyonlinelibrary.com. DOI 10.1002/hc.21160
3
1
-allylimidazolium hexafluorophosphate (2), and
-pentyl-3-allylimidazolium hexafluorophosphate
(
3) were synthesized. These salts were treated with
Ag2O to afford their corresponding mononuclear
Ag(I)-NHC complexes, namely 1-methoxylethyl-
3
-allylimidazolium silver(I) hexafluorophosphate
INTRODUCTION
(
4), 1-ethyl-3-allylimidazolium silver(I) hexafluo-
rophosphate (5), and 1-pentyl-3-allylimidazolium
silver(I) hexafluorophosphate (6), respectively. These
compounds were characterized by physicochemical
and spectroscopy techniques. Compounds 4 and 5
were structurally characterized by single crystal X-ray
diffraction, and their stability in solution was investi-
gated and found to be acceptable for the antibacterial
studies. These new NHC precursors and their re-
spective Ag–NHC complexes were screened for their
antibacterial activities against Staphylococcus aureus
The generation of artificial chemical nucleases is im-
portant in drug design and biotechnology [1–3], and
they offer important new tools for DNA manipula-
tion to molecular biologists [ [4], [5]]. In addition,
the propensity to achieve site-specific nucleic acids
cleavage will certainly break ground for the devel-
opment of new chemotherapeutic agents and an-
timicrobial drugs [6–8]. With inspiration from the
observation that in living beings nucleophilic dis-
placement reactions on phosphate esters are mostly
promoted by metalloenzymes [9]. Attention is cur-
rently addressed to metal ion-based hydrolytic cata-
lysts, and transition metal complexes are very appro-
priate to be used as artificial nucleases, due to their
various structural features and the prospect to tune
their redox potential through the choice of suitable
ligands [10–14]. Among these metals, silver is more
suitable because of its compatibility with biological
systems.
(ATCC 12600) and Escherichia coli (ATCC 25922).
Compounds 1–3 showed no inhibition, whereas 4–6
inhibited the growth of these bacteria. The nuclease
activities of the reported compounds against plasmid
Correspondence to: Rosenani A. Haque; e-mail: rosenani@
usm.my.
Supporting Information is available in the online issue at
www.wileyonlinelibrary.com.
ꢀC
2014 Wiley Periodicals, Inc.
194

NHC–Silver(I) Complexes as Chemical Nucleases 195
Biological studies of Ag(I)–NHC complexes are
established on the knowledge that elemental silver
and its salts have long been recognized for their po-
tential in the protection of the eyes of newborns from
infection, as well as their antimicrobial properties,
essentially against chronic ulcers, extensive burns,
and wounds [15, 16]. This astounding activity of sil-
ver or Ag(I)–NHC complexes against bacteria, fungi,
and yeast is due to slow delivery of silver cations
across the cell membrane, which mostly interferes
with the electron transport system of the cell and
also interacts with the thiol groups of the vital en-
zymes of bacteria [17]. Ag(I)–NHC complexes offers
encouraging results to overcome the problems of
drug resistance of some pathogenic bacteria, which
has so far eluded the effectiveness of sulfonamides.
Recently, Ag(I)–NHC/coordination complexes have
been found to display potential antimicrobial activ-
ity, particularly for the potential treatment of cystic
fibrosis and chronic lung infections [18, 19], which
makes them appropriate entities for drug develop-
ment [20].
To date, a large number of studies have been
carried out on Ag(I) complexes as potential antimi-
crobials, anticancer, and their carbene transfer abil-
ity [21–24]. Also, Ag(I)-complexes have shown inter-
esting DNA-binding ability [25–27]; however, there
is paucity of data on their ability to cleave the nu-
cleic acids (DNA/RNA) especially in the case of Ag–
NHC complexes. On the other hand, the interaction
of Ag(I)–NHC complexes with nucleic acids is a sig-
nificant field of research to be explored to a greater
extent due to the utility of these carbene complexes
in the development of spectroscopic probes, diag-
nostic agents, and site-specific nucleic acid cleavers,
among others [28]. It is worthy of mentioning that
apart from DNA there are some other biological
targets including RNA, enzyme, or protein. There-
fore, it is considered worthwhile to investigate the
interaction of these synthesized compounds with
DNA and/or RNA as a preliminary study for pre-
dicting likely mode of action(s) of the reported
compounds.
EXPERIMENTAL
Materials and Measurements
All chemicals were of reagent grade and were used
as received. All solvents were redistilled except for
acetonitrile and dimethyl sulfoxide, which were of
AR grade. NMR spectra were recorded on a Bruker
500 MHz spectrometer at room temperature (RT)
in DMSO-d , using TMS as an internal standard.
6
FTIR spectra were recorded on a Perkin–Elmer-
2000 system spectrometer in the range of 4000–
−1
400 cm . Elemental analysis was carried out on
a Perkin–Elmer series II 2400 microanalyzer. Melt-
ing points were measured using a Stuart Scientific
SMP-1 (UK) instrument. The instruments are avail-
able at The School of Chemical Sciences, Universiti
Sains Malaysia, Penang, Malaysia. The X-ray sin-
gle crystal structure analysis was obtained using a
Bruker smart ApexII-2009 CCD area detector diffrac-
tometer. For compound 4, in one of the methoxy
group, the oxygen atom is disordered over three po-
sitions and was refined as 33%, 33%, and 34% occu-
pancies, respectively. Hydrogen atoms at this group
are not located, and disordered atoms were refined
isotropically due to a disordered problem.
Synthesis of 1-Methoxylethyl-3-allylimidazolium
Hexafluorophosphate (1)
A mixture of imidazole (0.50 g, 7.34 mmol) and KOH
(0.62 g, 11.00 mmol) in DMSO (30 mL) was stirred
for 1 h at RT. 1-Bromo-2-methoxyethane (1.00 g,
7.34 mmol) was then added dropwise to the solu-
tion mixture. After 2 h, the mixture was poured into
200–300 mL of water and extracted with chloroform
(3 × 30 mL). The extract was filtered thrice through
four plies of Whatman filter paper to dry the ex-
tract and the desired compound, and this was col-
lected as a clear solution, which was subsequently
evaporated under reduced pressure. The com-
pound formed, N-methoxyethyl imidazole (0.8 g,
6.30 mmol), was added dropwise in a stirring so-
lution of allyl bromide (0.81 g, 6.30 mmol) in ace-
tonitrile (30 mL) and refluxed for 20 h. The sol-
vent was removed under reduced pressure to give
1-methoxylethyl-3-allylimidazolium bromide, which
On the basis of these findings, we herein present
information on the synthesis and characterizations
of six new compounds (1–6). The newly investigated
series of compounds were evaluated for their nu-
clease activity in the absence of an oxidizing agent
to evaluate their self-activating properties, which
do not depend on the presence of cofactors. Their
potential as antibacterial agents was also investi-
gated against Staphylococcus aureus (S. aureus) as a
Gram-positive and Eschericia coli (E. coli) as a Gram-
negative bacterial strains.
was then reacted with a solution of KPF (0.65 g,
3.50 mmol) in methanol (20 mL). The mixture was
6
stirred at RT for 3 h and allowed to stand overnight.
Then the solvent was removed under reduced pres-
sure, and the resultant white powder was washed
with distilled water (3 × 5 mL) to remove unreacted
KPF , and air dried. The powder was recrystallized
6
in a solution of acetonitrile/methanol to obtain a pale
Heteroatom Chemistry DOI 10.1002/hc

196 Haque et al.
ꢁ
ꢁ
yellow crystalline compound. Yield: 0.80 g (72%).
Melting point: 120–122°C; H NMR (500 MHz,
zolium H5 ), 7.80 (t, J = 1.5 Hz, H, imidazolium H4 ),
1
ꢁ
13 1
9.25 (s, H, imidazolium H2 ); C { H} NMR (125
DMSO-d6, 298 K, δ ppm): 3.30 (s, 3H, OCH3), 3.70
MHz, DMSO-d6, 298 K, δ ppm): 14.9 (CH3), 40.0 (N–
ꢁ
(
2
t, J = 5.0 Hz, 2H, CH2–OCH3); 4.45 (t, J = 5.0 Hz,
CH2–R), 48.7 (N–CH2–CH), 122.9 (imidazolium C5 ),
ꢁ
ꢁ
H, N–CH2–CH2–OCH3); 4.90 (d, J = 7.0 Hz, 2H, N–
123.2 (imidazolium C4 ), 136.7 (imidazolium C2 ).
2
3
−1
CH2–CH); 5.35 (dd, 1H, JHH = 1.5 Hz, JHH = 10.0
FTIR (KBr disk) cm , ꢀ 3422 (Caliph–Nimid); 2966
2
3
Hz, CH=HHcis); 5.39 (dd, 1H, JHH = 1.5 Hz, JHH
(C–Haliph); 2230 (C=N); 1426, 1400 (Cimid–Nimid);
1679 (C=C, allyl); Anal. Calcd for C8H13N2F6P: C,
34.1; H, 4.6; N, 9.9. Found: C, 34.3; H, 4.7; N, 10.0.
=
17.5 Hz, CH=CHHtrans); 6.00–6.13 (m, H, CH);
ꢁ
7
.75 (t, J = 2.0 Hz, H, imidazolium H5 ); 7.85 (t,
ꢁ
J = 2.0 Hz, H, imidazolium H4 ); 9.27 (s, H, imi-
ꢁ
13
1
dazolium H2 ); C { H} NMR (125 MHz, DMSO-d6,
98 K, δ ppm): 51.0 (OCH3); 53.62 (CH2–OCH3); 58.0
N–CH2–CH2–OCH3); 60.3 (N–CH2–CH); 70.8 (CO),
Synthesis of 1-Pentyl-3-allylimidazolium
Hexafluorophosphate (3)
2
(
1
ꢁ
ꢁ
18.17 (imidazolium C5 ), 121.6 (imidazolium C4 ),
Salt 3 was prepared in an analogous fashion to 2, but
instead of bromoethane 1- bromopentane (3.32 g,
22.00 mmol) was added. Salt 3 was isolated as a
white solid. Yield: 1.2 g (79%). Melting point: 121–
1
22.7 (=CH2); 130.4 (–CH= ); 135.74 (imidazolium
ꢁ
−1
C2 ). FTIR (KBr disk) cm , ꢀ3432 (C
–N
);
aliph
imid
2
976 (C–Haliph); 2230 (C=N); 1446, 1410 (Cimid–
1
Nimid); 1691 (C=C, allyl); 1307, 1170 (C–O–C, C–O).
Anal. Calcd for C9H17N2OF6P: C, 34.4; H, 5.4; N, 8.9.
Found: C, 34.6; H, 5.7; N, 9.1.
123°C. H NMR (500 MHz, DMSO-d , 298 K, δ ppm):
6
0.80 (t, J = 7.0 Hz, 3H, CH ); 1.15 (m, 2H, CH ); 1.25
3
2
(m, 2H, CH ); 1.85 (m, 2H, CH ); 4.25 (t, J = 7.0
2
2
Hz, 2H, N–CH2–R); 4.90 (d, J = 7.5 Hz, 2H, N–CH2–
2
3
CH); 5.27 (dd, 1H, JHH = 1.2 Hz, JHH = 10.2 Hz,
Synthesis of 1-Ethyl-3-allylimidazolium
Hexafluorophosphate (2)
2
3
CH=HHcis); 5.32 (dd, 1H, JHH = 1.2 Hz, JHH = 17.0
Hz, CH=CHHtrans); 6.00–6.13 (H, m, CH); 7.77 (t, J =
ꢁ
A mixture of imidazole (1.50 g, 22.00 mmol) and
KOH (1.85 g, 33.00 mmol) in DMSO (30 mL) was
stirred for 1 h at RT, and 1-bromoethane (2.40 g,
2.0 Hz, 1H, imidazolium H5 ); 7.87 (t, J = 2.0 Hz, 1H,
ꢁ
ꢁ
13
imidazolium H4 ); 9.37 (s, H, imidazolium H2 );
C
1
{ H} NMR (125 MHz, DMSO-d6, 298 K, δ ppm): 13.9
2
2.00 mmol) was added dropwise. After 2 h, the mix-
(CH3); 21.7 (CH2); 28.3 (CH2); 40.0 (N–CH2); 48.7
(N–CH2–Ar); 122.3 (imidazolium C5 ); 123.2 (imi-
ꢁ
ture was poured into 200–300 mL of water and ex-
tracted with chloroform (3 × 30 mL). The extract
was filtered through four plies of Whatman filter pa-
per to dry the extract. This process of filtration was
repeated thrice to collect a crystal clear solution of
the desired compound, which was evaporated under
reduced pressure. The compound formed, N-ethyl
imidazole (1.0 g, 10.00 mmol), was added dropwise
in a stirring solution of allyl bromide (1.21 g, 10.00
mmol) in acetonitrile (30 mL) and refluxed for 20 h.
The solvent was removed under reduced pressure
to give 1-ethyl-3-allylimidazolium bromide, which
was then reacted with a solution of KPF6 (1.33 g,
ꢁ
ꢁ
dazolium C4 ); 136.8 (imidazolium C2 ). FTIR (KBr
−1
disk) cm , ꢀ 3430 (Caliph–Nimid); 2968 (C–Haliph);
2230 (C=N); 1426, 1410 (Cimid–Nimid); 1690 (C=C,
allyl); Anal. Calcd for C11H19N2F6P: C, 40.7; H, 5.9;
N, 8.6. Found: C, 40.9; H, 6.1; N, 8.9.
Synthesis of 1-Methoxylethyl-3-allylimidazolium
Silver(I) Hexafluorophosphate (4)
An acetonitrile solution (35 mL) of 3 (0.80 g,
3.24 mmol) and Ag2O (0.75 g, 3.24 mmol) was stirred
at 50–60°C for 12 h in the absence of light. The
mixture was then filtered through a pad of celite,
and the resulting colorless solution was evaporated
to give a white solid and was redissolved in ace-
tonitrile, followed by the reduction of solvent to
about 3 mL. Addition of diethyl ether reprecipitated
a solid, which was isolated by filtration and vacuum
dried to yield 4. Single crystals suitable for X-ray
were obtained by slow diffusion of diethyl ether into
acetonitrile solution containing the complex. Yield:
7.24 mmol) in methanol (20 mL). The mixture was
stirred at RT for 3 h and allowed to stand overnight.
Then the solvent was removed under reduced pres-
sure, and the resultant water powder was washed
with distilled water (3 × 5 mL) to remove unreacted
KPF6, and air dried. The powder was recrystallized
in a solution of acetonitrile/methanol to obtain a
beige solid. Yield: 1.8 g (70%). Melting point: 105–
1
1
1
07°C. H NMR (500 MHz, DMSO-d6, 298 K, δ ppm):
.40 (t, J = 7.5 Hz, 3H, CH3), 4.20 (q, 2H, N–CH2–R);
1
0.9 g (60%). Melting point: 181–183°C; H NMR (500
4
.90 (d, J = 7.0 Hz, 2H, N–CH2–CH), 5.30 (dd, 1H,
MHz, DMSO-d6, 298 K, δ ppm): 3.30 (s, 6H, 2 ×
OCH3); 3.70 (t, J = 5.0 Hz, 4H, 2 × CH2–OCH3); 4.50
(t, J = 7.0 Hz, 4H, 2 × N–CH2–CH2–OCH3); 4.90
(d, J = 6.0 Hz, 4H, 2 × N–CH2–CH); 5.35 (dd, 2H,
2
3
JHH = 1.5 Hz, JHH = 10.2 Hz, CH=HHcis); 5.34 (dd,
2
3
1
6
H, JHH = 1.5 Hz, JHH = 17.5 Hz, CH=CHHtrans);
.00–6.13 (m, H, CH); 7.70 (t, J = 1.5 Hz, H, imida-
Heteroatom Chemistry DOI 10.1002/hc

NHC–Silver(I) Complexes as Chemical Nucleases 197
2
3
1
JHH = 1.5 Hz, JHH = 10.0 Hz, CH=HHcis); 5.39 (dd,
0.88 g (67%). Melting point: 160–162°C. H NMR
2
3
2
6
2
H, JHH = 1.5 Hz, JHH = 17.5 Hz, CH=CHHtrans);
.00–6.13 (m, 2H, 2 × CH); 7.45 (t, J = 2.0 Hz,
(500 MHz, DMSO-d6, 298 K, δ ppm): 0.85 (t, J =
7.0 Hz, 6H, 2 × CH3); 1.20 (m, 4H, 2 × CH2CH3);
1.30 (m, 4H, 2 × CH2–CH2–CH3); 1.75 (m, 4H,
2 × N–CH2–CH2); 4.15 (t, J = 6.0 Hz, 4H, 2 ×
N–CH2–R); 4.90 (d, J = 7.0 Hz, 4H, 2 × N–CH2–
ꢁ
H, imidazolium H5 ); 7.55 (t, J = 2.0 Hz, 2H, im-
ꢁ
13
1
idazolium H4 ). C { H} NMR (125 MHz, DMSO-
d6, 298 K, δ ppm): 51.0 (OCH3); 53.6 (CH2–OCH3);
5
2
3
8.0 (N–CH2–CH2–OCH3); 70.8 (CO); 118.2 (imida-
CH); 5.25 (dd, 2H, JHH = 1.2 Hz, JHH = 10.2 Hz,
2 3
ꢁ
ꢁ
zolium C5 ); 121.6 (imidazolium C4 ); 180.67 and
CH=HHcis); 5.29 (dd, 2H, JHH = 1.2 Hz, JHH =
17.0 Hz, CH=CHHtrans); 6.00–6.13 (m, 2H, 2 × CH);
1
109
1
1
81.70 [(d, J(C- Ag) = 208.5 Hz and d, J(C-
1
07
ꢁ
ꢁ
Ag) = 180.0 Hz] (imidazolium Ag-C2 ). FTIR (KBr
7.45 (t, 2H, imidazolium H4 ); 7.55 (t, J = 2.0 Hz,
−1
ꢁ
13
1
disk) cm , ꢀ3412 (C
2
allyl); 1307, 1170 (C–O–C, C–O). Anal. Calcd for:
C18H30N4AgO2F6P: C, 36.8; H, 5.1; N, 9.5. Found:
C, 37.1; H, 5.4; N, 9.7.
–N
); 2970 (C–H );
aliph
2H, imidazolium H5 ). C { H} NMR (125 MHz,
DMSO-d6, 298 K, δ ppm): 13.9 (CH3); 21.7 (CH2);
28.3 (CH2); 40.0 (N–CH2); 48.7 (N–CH2–Ar); 122.28
aliph
imid
100 (C–N); 1428, 1400 (Cimid–Nimid); 1691 ν(C=C,
ꢁ
1
ꢁ
(imidazolium C5 ); 123.16 (imidazolium C4 ); 179.90
109
1
and 180.10 [(d, J(C- Ag) = 205.0 Hz and d, J(C-
1
07
ꢁ
Ag) = 185.5 Hz] (imidazolium Ag-C2 ). FTIR (KBr
−
1
disk) cm , ꢀ 3400 (C
–N
); 2965 (C–H
);
aliph
aliph
imid
Synthesis of 1-Ethyl-3-allylimidazolium Silver(I)
Hexafluorophosphate (5)
2215 (C–N); 1400, 1389 (Cimid–Nimid); 1690 (C=C, al-
lyl); Anal. Calcd for C H N AgF P: C, 43.5; H, 5.6;
2
2
34
4
6
N, 9.2. Found: C, 43.8; H, 5.8; N, 9.5.
A mixture of 2 (0.80 g, 3.16 mmol) with Ag2O (0.73 g,
3
5
.16 mmol) in acetonitrile (40 mL) was stirred at
0–60°C for 12 h. The solution was filtered through
a pad celite giving a colorless solution. Compound
was obtained as a white solid after the removal
Antibacterial Studies
5
Stock solutions of all compounds were prepared
using DMSO. Antimicrobial tests were performed
using the Kirby Beur disk diffusion method [29],
Single colonies of E. coli (ATCC 11303) and S. aureus
of volatiles under reduced pressure. The compound
was further purified by acetonitrile/dichloromethane
to give a crystalline solid. Single crystals suitable
for X-ray were obtained by the slow diffusion of
diethyl ether into the acetonitrile solution contain-
(
ATCC 12600) from fresh culture agar plates were,
respectively, cultured in two bottles containing
mL of Luria-Bertani (LB) broth solution (tryp-
ing the complex. Yield: 0.96 g (64%). Melting point:
5
1
1
51–153°C. H NMR (500 MHz, DMSO-d6, 298 K,
tone10 g, yeast extract 5 g, NaCl 10 g/L) and
incubated overnight at 37°C. The turbidity of each
culture was adjusted by comparing it to 0.5 McFar-
land standard, which is equal to 1.58 × 10 CFU/mL
δ ppm): 1.40 (t, J = 7.0 Hz, 6H, 2 × CH3); 4.20
(
q, 4H, 2 × N–CH2–CH3); 4.95 (d, J = 7.0 Hz, 4H, 2 ×
2 3
N–CH2–CH); 5.30 (dd, 2H, JHH = 1.5 Hz, JHH =
8
2
1
0.2 Hz, CH=HHcis); 5.34 (dd, 2H, JHH = 1.5 Hz,
or 0.5 (O.D600 reading). Using sterile cotton buds,
each bacterial lawn culture was spread uniformly on
different agar plates before placing the antimicrobial
assay disks on the plate. Four disks were placed on
the agar plate, and 5 μL volumes of the compounds,
AgNO3 and ciprofloxacin, were loaded on the disks
with concentrations at 100 and 50 μg/mL. The plates
were incubated at 37°C for 24 h, and the diameters of
the inhibition zones were measured in millimeters.
The antibacterial test was calculated as a mean of
three replicates. The effectiveness of these com-
pounds in relation to an inhibition zone was
compared to silver nitrate based on its established
antimicrobial properties [30] and ciprofloxacin,
which were used as positive controls. The minimum
inhibitory concentration (MIC) of each compound
was determined based on the lowest concentration
of the compound that inhibited the growth of bac-
teria using the broth dilution method [31]. Single
colonies of S. aureus, and E. coli were isolated
3
JHH = 17.5 Hz, CH = CHHtrans); 6.00–6.13 (m, 2H,
ꢁ
2
× CH); 7.45 (t, J = 1.5 Hz, 2H, imidazolium H4 );
ꢁ
13
1
7
.55 (t, J = 1.5 Hz, 2H, imidazolium H5 ). C { H}
NMR (125 MHz, DMSO-d6, 298 K, δ ppm): 14.9
CH3), 40.0 (N–CH2); 48.7 (N–CH2–CH); 122.3 (im-
(
ꢁ
ꢁ
idazolium C5 ); 123.2 (imidazolium C4 ); 180.3 (im-
ꢁ
−1
idazolium Ag-C2 ). FTIR (KBr disk) cm , ꢀ 3387
Caliph–Nimid); 2960 (C–Haliph); 2105 (C–N); 1416,
380 (Cimid–Nimid); 1679 (C=C, allyl); Anal. Calcd for
C16H24N4Ag F6P: C, 36.7; H, 4.2; N, 10.7. Found: C,
0.0; H, 4.3; N, 10.9.
(
1
4
Synthesis of 1-Pentyl-3-allylimidazolium
Silver(I) Hexafluorophosphate (6)
Complex 6 was prepared according to the same
procedure for 5 except that 2 was replaced with 3
(
0.70 g, 2.70 mmol) and Ag2O (0.63g, 2.70 mmol).
Complex 6 was obtained as a gray solid. Yield:
Heteroatom Chemistry DOI 10.1002/hc

198 Haque et al.
from agar plates and were grown in a 5.0-mL LB
broth (tryptone 10 g, yeast extract 5 g, NaCl 10
g/L). The solutions were incubated at 37°C and
shaken at 180 rpm overnight to yield bacteria
solutions. Stock solutions of the compounds were
prepared by dissolving each in DMSO to prepare
stock concentrations at 50 mg/mL. Respectively
from each stock solution, 2 μL of the compounds,
AgNO3 and ciprofloxacin, were dissolved in 2 mL
of the broth culture and used to prepare six serial
dilutions of 100, 50, 25, 12.5, 6.25, and 3.125 μg/
ml. Serial dilutions were done for each compound
by transferring 1 mL (100 μg/mL) of the compound
solution from the first tube to the second tube
already containing 1 mL of nutrient broth. Next,
R
NH
N
a: KOH, DMSO
b: Br-R
N
N
a: Reflux, 20 h
N
+
N
R
.
PF
6
Br
b: KPF6
1
, 2, 3
Stir,
o
1
2 h, 50 C
Ag O
2
+
R
N
N
Ag
1
mL from tube 2 was again transferred into tube 3
N
R
N
and same is repeated for tubes 4, 5, and 6 to give the
concentration of 50, 25, 12.5, 6.25, and 3.125 μg/mL,
respectively. Prepared bacteria solution (5 μL) was
added to each tube, and the tubes were incubated
at 37°C for 16 h in a shaking incubator at 180 rpm.
Bacteria growth was indicated by turbidity of the
solution in the tubes, and MIC was determined by
the lowest concentration lacking turbidity.
-
PF
6
4
, 5, 6
1
2
3
, 4: R = CH COCH
2
3
, 5: R = CH CH
2
3
, 6: R = CH CH CH CH CH
2
2
2
2
3
SCHEME 1 Synthetic pathway to imidazolium salts, 1–3 and
their respective Ag(I)–NHC complexes, 4–6, respectively.
Gel Electrophoresis
plexes (4–6) are outlined in Scheme 1. A mixture of
imidazole and KOH was stirred in DMSO for 1 h,
followed by the addition of bromoethyl methylether,
bromoethane, and 1-bromopentane to give the ap-
propriate N-methoxylethyl, ethyl, and pentyl im-
idazole, respectively. The N-substituted imidazole
derivatives are converted into their respective qua-
ternary salts by the reaction of allyl bromide in ace-
tonitrile to afford corresponding imidazolium bro-
mide salts, followed by treatment with KPF6 in
methanol to obtain the respective hexafluorophos-
phate salts (1–3) in good yield. These salts are readily
soluble in common organic solvents such as acetoni-
trile, DMF, and DMSO, but are insoluble in toluene,
methanol, and 1,4-dioxane. The mononuclear Ag(I)–
NHC complexes were obtained by treating com-
pounds 1, 2, and 3 with Ag2O in refluxing acetonitrile
for 12 h with a ligand to metal ratio of 1:1 to give light
sensitive complexes 4, 5, and 6, respectively, in mod-
erately good yields (76–79%). The apparatus were
wrapped with aluminum foil to exclude the light.
Perhaps due to the simple imidazolium salts, the re-
action conditions were simple and convenient. In all
reactions, a black suspension resulted, which was
filtrated through a pad of celite followed by evapora-
tion of the solvents under vacuum. The Ag(I)–NHC
complexes are soluble in polar organic solvents such
as acetonitrile, DMF, and DMSO, but are insoluble
in toluene, diethyl ether, benzene, and hexane.
The electrophoresis method was employed to study
the efficiency of cleavage by the test compounds
[32]. The extracted plasmid DNA and RNA, pTS414
(10 μg/mL), and 1 μL of the test sample (50 μg/mL),
were mixed in 50 mM Tris–HCl buffer (pH 8.00).
The contents were incubated for 8 h at 37°C. Plas-
mid extraction was done using a plasmid purifica-
tion kit (Intron Biotechnology, Kyungki-Do, Korea)
without the addition of RNase to extract both RNA
and DNA. The electrophoresis was performed us-
ing 0.8% agarose gel. For each sample, 5 μL of the
mixture was loaded into the well. The voltage used
was 90 V running on Tris-acetate ethylenediaminete-
traacetic acid buffer (4.84 g Tris base, PH 8.0, 0.5
M EDTA/1L), and the gel was stained with ethidium
bromide solution (10 μg/mL) for 15 min. The gel was
subsequently exposed to UV light and captured by
a gel documentation system (FluorChem HD2; Pro-
teinSimple, Santa Clara, CA). The nucleating ability
of the synthesized imidazolium salts/Ag-NHC com-
plexes (1–6) was determined by its efficiency in cleav-
ing/degrading the plasmid DNA and RNA.
RESULTS AND DISCUSSION
Synthesis
Reactions involving the synthesis of the imidazolium
salts, namely 1–3, and their respective Ag–NHC com-
Heteroatom Chemistry DOI 10.1002/hc

NHC–Silver(I) Complexes as Chemical Nucleases 199
1
FIGURE 1 Representative H NMR spectrum of complex 4 after 48 h.
SPECTROSCOPIC STUDIES
Hydrolysis Test
ing vibrations; this observation is in accordance with
the similar reported compounds [33, 34]. However,
in the complex spectra these bands showed a shift
of ꢀ120 and ꢀ25 cm , respectively, indicating the
formation of free C–N module in the imidazole core,
suggesting dequarternization followed by coordina-
tion of the carbene carbon to Ag(I). However, metal-
to-carbene carbon vibrations are normally observed
in the far-IR region and, therefore, could not be pos-
sibly assign in the available IR spectral data.
−1
Solution studies of these complexes were essential
to identify the bioactive species resulting from the
dissolution of the solid complex and so understand
the underlying biochemical mechanism involved in
the antibacterial activity. This study is also relevant
to the antibacterial studies, since the complexes have
to be maintained in culture medium for at least 24 h.
Therefore, we have determined the stabilities of the
Ag–NHC complexes in both 10% and 100% DMSO-d6
aqueous solution. The complexes are stable in aque-
NMR Spectra Studies
1
13
ous solution for 48 h, since their H NMR and
C
1
The H NMR spectra of imidazolium salts 1–3 in
NMR spectra remained unchanged after 48 h. The
DMSO-d6 exhibit a characteristic NCHN proton res-
onance at ca. δ 9.25–9.37, suggesting the successful
formation of the desired salts, which is quite con-
sistent with those of other imidazole-based NHC
precursors [35]. Two distinguished triplets at ca.
δ 7.75 and 7.85 were observed for imidazolium
C4H and C5H resonances, respectively. Further-
more, methoxy protons and ethylene protons of
ether functionality were resonated as a singlet and
two triplets at ca. δ 3.28 and 3.68–4.47, respectively.
Three usual signals owing to allyl protons appeared
at ca. δ 4.90, 6.10, and the allylic protons (=CHcis)
appeared at ca. δ 5.26–5.33 whereas (=CHtrans) ap-
peared at ca. δ 5.32–5.40 respectively. Finally, alkyl
proton resonances were found in the range ca. δ 0.8–
1
H NMR spectrum of complex 4 after 48 h in 10%
aqueous DMSO is shown in Fig. 1 and is identical to
the spectrum obtained after 30 min (see the Supple-
mentary Information).
To further assess the stability of the complexes
in the broth mixture, 10 mg/mL stock solutions of
the complexes in DMSO were prepared and added
in a 1:1 ratio to LB broth prepared in DMSO. This
was done to imitate the conditions of the MIC evalu-
1
13
ation experiments. The H NMR and C NMR spec-
tra were taken after 15 min and 48 h. The complexes
demonstrated stability in the LB/DMSO broth mix-
1
13
ture at 37°C, as shown by the unchanged H and
C
NMR spectra after 48 h. However the circumstances
under physiological conditions may vary, but these
preliminary results are apparently noteworthy.
4
.4, which is in agreement with the similar structures
1
3
found in the literature [36]. The C NMR spectra of
the salts displayed prominent peaks in the downfield
region at ca. δ 135.74–136.80 attributed to the imada-
zole ring carbene carbon [37–39]. The signals for the
alkyl chain (–CH2–) were observed at ca. δ 10.00–
FTIR Spectra Studies
The spectra of all the compounds showed a band
of medium intensity in the range 2960–2976 cm
−
1
1
,
49.87. As expected in the complex H NMR spectra,
which is assigned to aliphatic C–H vibrations. Two
sharp and medium intensity bands appeared in the
range 2100–2230 and 1380–1428 cm , were as-
cribed to the imidazole ring C=N and C–N stretch-
the absence of the resonances for the imidazolium
carbene protons (NCHN) confirms the formation of
the expected Ag(I)–NHC complexes. This is further
confirmed by the downfield shift of carbene carbon
−1
Heteroatom Chemistry DOI 10.1002/hc

200 Haque et al.
TABLE 1 Crystal Data and Structure Refinement Details for Complexes 4 and 5
Characteristic
4
5
Formula
C
18
H
28
N
4
AgO
573.06
Monoclinic
P2 /n
2
F
6
P
C
16
H
24
N
4
AgF
525.23
Monoclinic
P2 /n
6
P
Formula wt
Crystal system
Space group
1
1
˚
a (A)
10.9696(4)
11.1202(3)
9.55286(3)
11.1592(3)
˚
b (A)
˚
c (A)
α (
20.2531(7)
90
98.214(2)
90
2445.21(14)
4
20.3809(5)
90
95.950(2)
90
2155.46(10)
4
o
o
β ( )
o
γ ( )
3
˚
V (A )
Z
cal
−3
ρ
(gcm
)
1.557
1.618
Temperature (K)
100
1.557
135
1.068
−
1
μ (mm
)
Crystal size (mm)
θ range ( )
0.14 × 0.16 × 0.38
0.08 × 0.21 × 0.31
o
2.0–27.49
2.0–30.1
R(int)
F(000)
0.071
1104
0.079
1056
R, WR
2
, S
0.0670, 0.1301, 1.05
0.0550, 0.1412, 1.02
nuclei to ca. δ 189.00 for complex 5 in the 13C NMR
spectra. In the case of complex 4 and 6, the 13C NMR
spectra displayed a most downfield resonance for the
C2-carbon nuclei as two doublets centered at 180.67,
5 were obtained by slow diffusion of the complexes
grown from a concentrated solution of acetonitrile
in diethyl ether at ambient temperature. Complex 4
crystallizes in the monoclinic space group P21/n with
an entire molecule in the asymmetric unit. The pres-
ence of one hexafluorophosphate anion in the lattice
balances the charge on the metal complex (Fig. 2).
The Ag(I) forms a linear geometry, coordinated by
two carbene carbon atoms of two ligands 1 with the
1
81.70 and 179.90, 180.10 ppm, respectively. This is
1
3
109
13
107
due to the presence of C– Ag and C– Ag with
1
109
1
the coupling constants of J(C– Ag) 188.5, J(C–
1
07
1
109
1
107
Ag) 180.00, and J(C– Ag) 180.00, J(C– Ag)
75.10 Hz, respectively. This observation is in accor-
1
˚
˚
dance with the similar reported compounds [36,40].
Apart from these major changes, there are no ob-
servable changes observed in both spectra.
distance of 2.088(6) A and 2.085(7) A for Ag1–C1 and
Ag1–C10, respectively, whereas the angle of C1–Ag1–
o
C10 is 176.59(5) . The planes of the imidazole rings
in this complex are perpendicular to each other and
forming π–π interactions with two imidazole rings
of adjacent molecules.
Single-Crystal X-Ray Diffraction Studies
Complex 5 crystallizes in the monoclinic space
group P21/n with one bis-carbene complex cation
and one hexafluorophosphate anion contained in the
lattice (Fig. 3). The distance of Ag(I) to two carbon
The crystal data of complexes 4 and 5 are shown in
Table 1, whereas the selected bond lengths and bond
angles are given in Tables (2 and 3). Crystals suitable
for single crystal X-ray diffraction studies for 4 and
o
o
˚
˚
TABLE 2 Selected Bond Lengths (A) and Angles ( ) for 4
TABLE 3 Selected Bond Lengths (A) and Angles ( ) for 5
Bond Angle (o)
Bond Angle (o)
˚
˚
Bond Length (A)
Bond Length (A)
Ag1–C1
2.082 (5)
2.089 (4)
1.396 (7)
1.471 (7)
1.349 (4)
1.392 (4)
1.459 (8)
1.337 (9)
1.346 (8)
1.506 (9)
C1–Ag1–C4
176.2 (2)
125.9 (2)
129.9 (4)
111.2 (5)
124.1 (5)
124.4 (3)
104.1 (4)
111.5 (5)
111.7 (5)
127.9 (4)
Ag1–C1
2.084 (4)
2.090 (4)
1.352 (5)
1.388 (6)
1.350 (6)
1.478 (5)
1.463 (5)
1.327 (6)
1.533 (10)
1.480 (8)
C1–Ag1–C9
175.64 (18)
126.1 (3)
129.9 (3)
110.9 (3)
124.7 (3)
124.2 (3)
104.0 (3)
124.4 (3)
124.3 (3)
124.2 (3)
Ag1–C4
N1–C2
N1–C7
N2–C1
N2–C7
N2–C10
C2–C3
C5–C6
C7–C8
Ag1–C1–N1
Ag1–C1–N2
C1–N1–C2
C1–N2–C10
C3–N2–C10
N1–C1–N2
N3–C13–C14
N4–C16–C17
N4–C4–Ag1
Ag1–C1b
N1–C1
N1–C2
N2–C1
N2–C6
N3–C12
C2–C3
C4–C5
C6–C7
Ag1–C1–N1
Ag1–C1–N2
C1–N1–C2
C1–N1–C4
C3–N2–C6
N1–C1–N2
C1–N2–C6
C2–N1–C4
C3–N2–C6
Heteroatom Chemistry DOI 10.1002/hc

NHC–Silver(I) Complexes as Chemical Nucleases 201
FIGURE 2 Structure of 4 with ellipsoids shown at 50% prob-
6
ability. Hydrogen atoms and PF in the lattice were omitted
for clarity.
FIGURE 3 Structure of 5 with ellipsoids shown at 50% prob-
6
ability. Hydrogen atoms and PF in the lattice were omitted
for clarity.
˚
˚
carbene atoms is 2.084(4) A and 2.090(4) A. The bond
angle of Ag(I) and two carbene carbon atoms (C1–
o
Ag1–C9) is 175.65(9) , and it facilitates the formation
of a linear coordination geometry. Both imidazole
rings in each complex form a π–π interactions with
next nearest molecules.
Antibacterial Activities
Stock solutions of all the compounds were prepared
in DMSO. The concentrations of the test compounds
−1
are 3.125, 6.25, 12.5, 25, 50, and 100 μg mL
,
using silver nitrate for positive control and
ciprofloxacin as the standard drug. Compounds were
screened for their antibacterial activities against E.
coli and S. aureus using the disk diffusion method,
and the MIC was determined based on the lowest
concentration that inhibited the growth of bacteria.
The antibacterial activities of the complexes
showed good inhibition against both bacteria
(Table 4, Figs. 4, 5, and 6), whereas no inhibition was
Heteroatom Chemistry DOI 10.1002/hc

202 Haque et al.
FIGURE 4 Representative image showing inhibition against
E. coli at 50 μg/mL of the test compounds (ligand precur-
sor 1; complexes 4–6); see the Supporting Information for
the MIC of the complex 6 activity against E. coli at 12.5 μg/
mL.
FIGURE 6 Antibacterial activities of investigated com-
pounds against S. aureus.
TABLE 5 Minimum Inhibitory Concentrations (MIC) against
E. coli and S. aureus
MIC
Test Compound
E. coli (μg/mL)
S. aureus (μg/mL)
4
5
6
25
100
12.5
50
25
50
12.5
50
AgNO
Ciprofloxacin
3
6.25
3.125
Compounds 1–3 showed no activity.
the metal complexes relative to their corresponding
ligands could be justified on the basis of the over-
tone concept [46]. Therefore, the influence of the
substituents on the N-position may have modulated
the bonding between silver–carbene (Ag–C) by fine-
tuning the lipophilicity of the complexes. As it was
observed for the order of antibacterial activities of
the complexes (5 < 4 < 6) in this study, which shows
that an increase in terminal chain lengths and types
of substituent can be used to tune the lipophilicity of
the reported complexes, and their transport across
the cell wall barrier can be managed. In all cases, the
complexes have shown a good-to-moderate bacterio-
static effect against both strains of bacteria when
compared to the standard drug, ciprofloxacin (MIC,
FIGURE 5 Antibacterial activities of investigated com-
pounds against E. coli
observed for the imidazolium salts. The antibacte-
−1
rial activity of complex 6 (MIC, 12.5 μg mL ) was
the highest for both bacteria and quite significant
−1
when compared with AgNO3 (MIC, 50 μg mL ). The
MIC values for the compounds against both bacteria
−1
are in the range of 12.5–100 μg mL . These values
are comparable to other related results in the litera-
ture; in some cases, the complexes have shown better
results [41–44], indicating that these new Ag–NHC
complexes are quite potent. The complexes showed
greater bacteriostatic activities against both bacteria
when compared with their ligand precursors, which
is in general agreement with the literature [45]. Com-
plexes 4 and 6 showed inhibition against E. coli at 25
−1
3.125–6.25 μg mL ).
Nuclease Activity
Gel electrophoresis was used to evaluate the nucle-
ase properties of the compounds 1–6. An agarose
gel image showing the nuclease activity of the com-
pounds is shown in Fig. 7. Compounds 1–6 were
studied for their nuclease activity by the agarose
gel electrophoresis method against plasmid, pTS414
−
1
and 12. 5 μg mL respectively, while for complex 5
−1
the activity was revealed at 100 μg mL (Table 5).
However, the imidazolium salts showed a poor reac-
tion against both bacteria. Such increased activity of
Heteroatom Chemistry DOI 10.1002/hc

NHC–Silver(I) Complexes as Chemical Nucleases 203
by a hydrolytic path, which remains to be clarified
conclusively. In general, there are three direct modes
of activation that a metal ion can provide for accel-
erating the rate of phosphate ester hydrolysis [48].
They are Lewis acid activation involving the coordi-
nation of phosphoryl oxygen to a metal [49,50], nu-
cleophile activation in which a nucleophile coordi-
nates to the metals [51], and leaving group activation
in which a leaving group oxygen atom coordinates
to the metals [48].
CONCLUSIONS
This study presented a new series of Ag(I)–NHC com-
plexes derived from imidazole-2-ylidenes. All NHC
precursors and their Ag(I) complexes were charac-
terized using different spectral and analytical tech-
niques. The molecular structures of complexes 4 and
FIGURE 7 Nuclease activity; M = marker; lane 1, DNA/RNA
alone; lane 2, DNA/RNA + 1; Lane 3, DNA/RNA+ 2; lane 4,
DNA/RNA + 3; lane 5, DNA/RNA + 4; lane 6, DNA/RNA +
5
; lane 7, DNA/RNA + 6.
5
were structurally determined using single-crystal
X-ray diffraction techniques. The antibacterial ac-
tivities of the prepared compounds were determined
against E. coli and S. aureus. We have shown that
imidazolium salts 1–3 did not act as antibacterial
agents at the working concentrations, but showed
good bacteriostatic activity when combined with sil-
ver. All Ag(I)–NHC complexes are stable in the broth
media, which provides for good systemic delivery
DNA/RNA in the absence of an oxidant. Plasmid
extraction was done using a plasmid purification
kit (Intron Biotechnology) without the addition of
RNase to extract both RNA and DNA. This method
is intended to further investigate the interaction of
these synthesized compounds with DNA and/or RNA
as a preliminary study for predicting likely mode
of action(s) of the reported compounds. The elec-
trophoresis analysis showed that the imidazolium
salt 3 displayed no visible activity toward nucleic
acids (lane 4), whereas salts 1 and 2 showed signs
of RNA cleavage (lanes 2 and 3) as evidenced by the
decrease in intensity of the RNA strands and this is
more pronounced for salt 2. However, all the salts
+
of the Ag . Thus, we have presented evidence of
the steric tuning of the compounds by introducing
varying types of substituents on N positions of the
imidazole ring. The antibacterial properties of the
Ag–NHC complexes were found to vary with the na-
ture of the substituents attached to the N position of
the imidazole ring. The most effective antibacterial
Ag(I)–NHC complex in this study is complex 6 with
an effective concentration of 12.5 μg/mL. The re-
sults demonstrate that Ag(I)–NHC complexes 5 and
6 are extremely efficient in promoting the cleavage
of nucleic acids in the absence of coreactants, i.e., in
the absence of H2O2 and added reductants. Hence,
it seems that there is a positive correlation between
the antibacterial and nuclease activities since com-
plex 6 having the highest antibacterial activity has
also displayed the most efficient nuclease activity in
this study.
(
1–3) showed no apparent interaction with the DNA
strands. Their respective complexes 4, 5, and 6 dis-
played distinct nuclease activity, as can be observed
with their interaction with DNA in lanes 5, 6, and 7
showing complete disappearance of the DNA strands
indicating complete DNA cleavage. Complex 4 do not
show any interaction with the RNA (lane 5), but com-
plexes 5 and 6 exhibited considerable RNA cleavage
activity especially for complex 6, which clearly de-
graded the nucleic acids as shown in lane 7. The plas-
mid DNA was totally degraded leading to the disap-
pearance of bands on agarose gel [47]. There seems
to be a positive correlation between the antibac-
terial and nuclease activities in the present study.
The appearance of smearing in lane 6 reveals a pro-
cess of degradation of RNA. According to Fig. 7, it
shows that the control DNA alone (lane 1) does not
show any apparent cleavage. This observation sug-
gests that the complexes have potential to cleave the
nucleic acids by a nonoxidative mechanism, possibly
SUPPLEMENTARY MATERIAL
Crystallographic data for the structure in this work
has been deposited at the Cambridge crystallo-
graphic data Centre, CCDC 982820 and 982821.
Copies of these materials can be obtained from the
director, CCDC, 12 Union Road, Cambridge CB2
1EZ, UK, (fax: +44-1223-336033; e-mail: deposit@
ccdc.cam.ac.uk/deposit
Heteroatom Chemistry DOI 10.1002/hc

204 Haque et al.
ACKNOWLEDGMENT
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The authors thank Universiti Sains Malaysia for the
Research University Grant 1001/PKIMIA/811217.
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[
24] Tan, S. J.; Yan, Y. K.; Lee, P. P.; Lim, K. H. Future
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Heteroatom Chemistry DOI 10.1002/hc