Nitrile-functionalized Hg(II)- and Ag(I)-N-heterocyclic carbene complexes: Synthesis, crystal structures, nuclease and DNA binding activities

Article abstract of DOI:10.1002/aoc.2912

Various nitrile-functionalized benzimidazol-2-ylidene carbene complexes of Hg(II) and Ag(I) were synthesized by the interaction of 1-benzyl/1-butyl-3- (cyano-benzyl)-3 H-benzimidazol-1-ium mono/dihexafluorophosphate with Hg(OAc)₂/Ag₂

Full text of DOI:10.1002/aoc.2912

Full Paper
Received: 3 July 2012
Accepted: 15 July 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/aoc.2912
Nitrile-functionalized Hg(II)- and Ag(I)-N-
heterocyclic carbene complexes: synthesis,
crystal structures, nuclease and
DNA binding activities
Rosenani A. Haquea*, Srinivasa Budagumpia, Sze Yii Chooa,
Mei Kee Choonga, Bhadravathi Eswara Lokeshb and Kumar Sudeshb
Various nitrile-functionalized benzimidazol-2-ylidene carbene complexes of Hg(II) and Ag(I) were synthesized by the interaction of
1-benzyl/1-butyl-3-(cyano-benzyl)-3H-benzimidazol-1-ium mono/dihexafluorophosphate with Hg(OAc)2/Ag2O in acetonitrile.
Two of the benzimidazolium salts were structurally characterized by single crystal X-ray diffraction technique. Structures of
reported compounds were characterized by 1 H, 13C NMR, FT-IR, UV–visible spectroscopic techniques, and molar conductivity
and elemental analyses. For bis-benzimidazolium salt, dinuclear Hg(II)– and Ag(I)–carbene complexes were obtained. Nuclease
activity and binding interactions of the synthesized benzimidazolium salts and their Ag(I)–carbene complexes with DNA were
studied using agarose gel electrophoresis and, absorption spectroscopy and viscosity measurements, respectively. Ag(I)–carbene
complexes showed higher DNA binding activity compared to their respective benzimidazolium salts. However, a benzimidazolium
salt and two of the Ag(I) complexes showed remarkably higher nuclease activity both, in the presence and absence of an oxidizing
agent. Copyright © 2012 John Wiley & Sons, Ltd.
Keywords: Ag(I)–carbene complex; DNA binding; Hg(II)–carbene complex; N-heterocyclic carbene; nuclease activity; X-ray diffraction
In the past few years, functionalized NHCs have found application
in Ag(I)–carbene complexes and their potential in transmetallation
Introduction
The chemistry of N-heterocyclic carbenes (NHCs) is centred on the
availability of a lone pair of electrons on a carbene carbon atom,
and its reactivity towards electropositive metal ions in forming
new bonds.[1,2] NHC derivatives have a major structural difference
from their phosphine analogues, and thus their physical and
chemical behaviours. Since NHC ligands act as excellent s
donors and generally weak p acceptors, they can produce
stable metal–NHC complexes with strong metal–carbon bonds
(Chart 1). Nitrile-functionalized NHCs are potential multidentate
ligands, which can give rise to carbene complexes with increased
stability through ligand chelation. Their reactivity towards Ag(I) or
Hg(II) is interesting and also important, especially on the mode of
chelation. This field of functionalized NHC chemistry is currently
of great interest and activity because of the versatile potential
applications of derived complexes.[3,4]
NHCs with functionalized imidazole core are relatively common;
however, those with nitrile-functionalized benzimidazole derived
NHCs still remain relatively unexplored.[5,6] The choice of functional
group is also crucial; this modification can be easily achieved by
introducing functionalized aliphatic or aromatic groups at the
nitrogen atoms of the benzimidazole ring. Although many functio-
nalized carbene complexes were reported, nitrile-functionalized Hg
(II)–NHC complexes are still very few. Recently, pyridine-derived
pincer complexes of Hg(II) based on NHCs have attracted great
attention as carbene transfer agents.[7] Moreover, the fluorescent
emission property of an anthracene-derived Hg(II)–NHC complex
is found to be stronger than that of its corresponding Ag(I)–NHC
complex.[8]
reactions has greatly increased. Functionalized Ag(I)–NHC organo-
metallic chemistry is a well-established and flourishing field of
research, especially in biomedical and nanomaterial sciences. In
particular, for the possible treatment of chronic lung infections
and cystic fibrosis, and perhaps even in the treatment of different
types of cancer.[9–11] This biological and/or pharmaceutical rele-
vance of Ag(I)–NHC complexes has promoted the synthesis of
model silver compounds containing functionalized NHC ligands.
The stability of the central bioactive benzimidazole core has
inspired medicinal chemists to synthesize new potential Ag(I)–NHC
complexes to explore their anticancer potential.[12–14] Furthermore,
their high stability and ease of derivatization make them suitable
compounds for drug discovery and development.
On the other hand, the interaction of Ag(I)–NHC complexes with
DNA is a major area of research to be explored to a greater extent
due to the utility of these carbene complexes in the development
of spectroscopic probes, diagnostic agents and site-specific nucleic
acid cleavers, among others.[15] Ag(I)–NHC complexes would be
valuable in the rational design of sequence-specific DNA binding
*
Correspondence to: Rosenani A. Haque, The School of Chemical Sciences, Univer-
siti Sains Malaysia, 11800 USM, Penang, Malaysia. E-mail: rosenani@usm.my
a The School of Chemical Sciences, Universiti Sains Malaysia, 11800 USM,
Penang, Malaysia
b Ecobiomaterial Research Laboratory, The School of Biological Sciences, Universiti
Sains Malaysia, 11800 USM, Penang, Malaysia
Appl. Organometal. Chem. 2012, 26, 689–700
Copyright © 2012 John Wiley & Sons, Ltd.

R. A. Haque et al.
Chart 1. (Benz)imidazole derived Hg(II)– and Ag(I)–NHC complexes.
molecules as they proved to be less toxic and more effective
chemotherapeutic agents.[16] Moreover, these complexes can be
applied to the development of tools for biotechnology and
biochemistry. Interactions of Ag(I)–NHC complexes with bacterial
cell walls and the related biochemistry seem to be of more
relevance. In order to find efficient DNA binding agents and to
understand the mode of coordination, we prepared and character-
ized a series of new nitrile-functionalized benzimidazol-2-ylidines
and their Ag(I) and Hg(II) complexes. NHC precursors and Ag(I)
complexes were screened for their in vitro DNA binding activity
using different spectral and analytical methods.
Series CHN/S analyser. Electronic spectra were recorded on a
PerkinElmer Lambda 35 spectrometer in the range 1000–200 nm.
Molar conductivity measurements were made on a Eutech Instru-
ments CON 510 conductivity meter.
Synthesis of Benzimidazolium Salts
Synthesis of 1-(2-cyanobenzyl)-3-benzyl-3 H-benzimidazol-1-ium hexafluor-
ophosphate (3)
A mixture of 2-bromomethyl benzonitrile (0.196 g, 1 mmol) and
1-benzylbenzimidazole (0.208 g, 1 mmol) were stirred in acetonitrile
(30 ml) at 100 ꢁC for 48 h. Solvent was removed under reduced
pressure, and the product was then washed with diethyl ether
(3ꢂ 3 ml). The bromide salt 2 thus obtained was recrystallized from
acetonitrile–diethyl ether (1:10v/v). Salt 2 was directly converted
into its dihexafluorophosphate counterpart by metathesis reaction
using KPF6 (0.276 g, 1.5mmol) in methanol (20 ml). The resultant
mixture was stirred for 3 h and was to stand overnight. The pale-
green precipitate was filtered under reduced pressure, washed with
distilled water (3ꢂ 5 ml) to remove unreacted KPF6, and air dried.
Yield 82.67%; m.p. 110–111 ꢁC. 1 H NMR (500 MHz, d6-DMSO):
d 5.85 (2H, s, CH2-benzyl), 6.08 (2H, s, CH2-benzonitrile), 7.40 (3 H,
m, 2-CHBenzyl), 7.55 (3H, m, 3-CHBenzyl), 7.70 (3H, m, CHBenzimidazole),
7.79 (1H, t, CHBenzimidazole, J = 7.8Hz), 7.90 (1H, m, 4-CHBenzonitrile),
8.0 (2H, m, 5-CHBenzonitrile), 10.04 (1H, s, benzimidazolium 2-CH).
13C{1 H}NMR (125 MHz, d6-DMSO): d 48.60 (CH2-benzyl), 50.09
(CH2-benzonitrile), 110.89 (C ꢃ N-nitrile), 113.81, 126.94, 128.75,
133.73, 134.02, (Ar-CBenzyl), 114.18, 127.07, 128.96, 130.96, 133.84,
(Ar-CBenzonitrile), 116.95, 128.31, 129.14, 129.59, 131.35, 136.91
(Ar-CBenzimidazole), 143.49 (benzimidazolium C20). FT-IR (KBr disc)
cmꢀ1: 1602 n(Cꢃ N, benzimidazole), 2224 n(Cꢃ N, benzonitrile),
2964, 3033 n(C-H, aliphatic and aromatic). UV–visible (DMF, nm):
270.1 (p–p*) and 313.3 (n–p*). Molar conductance: 16.76 S cm2
molꢀ1. Further, molar conductance and 1 H, 13C NMR, FT-IR,
UV-visible spectral data of salt 2 has no or negligible changes
compared to salt 3.
Experimental
Reagents and Instruments
All the chemicals used were of reagent grade; solvents were
dried and distilled before use according to standard procedures.
Benzimidazole, 2-bromomethylbenzonitrile, 3-bromomethylben-
zonitrile, ortho-dibromomethylxylene, benzyl bromide, n-butyl
bromide, potassium hexafluorophosphate, silver oxide and
mercury acetate were purchased from Sigma-Aldrich and used
as received. 1-Benzyl benzimidazole and 1-methylbenzonitrile
benzimidazole were prepared according to the literature[17]
method with slight modifications. For X-ray single-crystal struc-
ture analysis, a Bruker SMART APEX2-2009 CCD area-detector
diffractometer was used for data collection. a SAINT Bruker-2009
for cell refinement and SAINT for data reduction, and SHELXTL
(Sheldrick, 2008) was used to solve structures.[18] Calculations, struc-
ture refinement, molecular graphics and material for publication
were performed using the SHELXTL and PLATON (Spek, 2009)
software packages.[18] Structures were solved by direct methods
and refined by full-matrix least-squares against F2. FT-IR
spectra of compounds were recorded in potassium bromide disks
using a PerkinElmer 2000 system spectrometer in the range
4000–400 cmꢀ1 1 H and 13C NMR spectra were obtained at room
.
temperature on a Bruker 500 MHz Ascend spectrometer in DMSO-d6
or CD3CN-d3 using tetramethylsilane as an internal reference. Melting
points were assessed using a Stuart Scientific SMP-1 (UK) instrument.
All reported compounds were analyzed for carbon, hydrogen and
nitrogen by CHN microanalyses using a PerkinElmer 2400 LS
Synthesis of 1-(2-cyanobenzyl)-3-butyl-3 H-benzimidazol-1-ium hexafluoro-
phosphate (5)
A
mixture of 1-methylbenzonitrile benzimidazole (0.233 g,
1 mmol) and n-butyl bromide (0.137 g, 1 mmol) was stirred in
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Copyright © 2012 John Wiley & Sons, Ltd. Appl. Organometal. Chem. 2012, 26, 689–700

Nitrile-functionalized carbene complexes
acetonitrile (30 ml) at 100 ꢁC for 48 h. Solvent was removed under
reduced pressure and the product was washed with diethyl ether
(3 ꢂ 3 ml). The bromide salt thus obtained was recrystallized from
acetonitrile/diethyl ether (1:10 v/v). The bromide salt was directly
converted into its dihexafluorophosphate counterpart by metath-
esis reaction using KPF6 (0.276 g, 1.5 mmol) in methanol (20 ml).
The resultant mixture was stirred for 4 h and was left to stand
overnight. The pale-green solid was filtered under reduced
pressure, washed with distilled water (3 ꢂ 5 ml) to remove
unreacted KPF6, and air dried. Yield 89.02%; m.p. 122 ꢁC. 1 H
NMR (500 MHz, d6-DMSO): d 0.95 (3 H, t, CH3-butyl, J = 7.5 Hz),
1.40 (2 H, m, CH2-butyl), 1.90 (2 H, m, CH2-butyl), 4.60 (2H, t, CH2-
butyl, J =7.3 Hz), 6.03 (2 H, s, CH2-benzonitrile), 7.50 (1H, d, 2-CH
126.92, 130.17, 131.79 (Ar-C, benzonitrile), 127.14, 129.45,
131.92, 135.36 (Ar-C, benzimidazole), 128.86, 132.49, 133.22,
(Ar-C, xylene), 143.29 (benzimidazolium–C2). FT-IR (KBr disc)
cmꢀ1: 1612 n(C N, benzimidazole), 2224 n(C ꢃ N, benzonitrile),
2964, 3099 n(C-H, aliphatic and aromatic). UV–visible (DMF, nm):
292.7 (p–p*) and 315.8 (n–p*). Molar conductance: 37.8 S cm2
molꢀ1. Further, molar conductance and 1 H, 13C NMR, FT-IR, UV–
visible spectral data of bromide salt had no or negligible changes
compared to salt 9.
2.3. Synthesis of NHC Complexes
Synthesis of 1-(2-cyanobenzyl)-3-benzyl-3 H-benzimidazol-1-ium silver(I)
hexafluorophosphate (10)
Benzyl, J = 8 Hz), 7.70 (4 H, m, 3-CHBenzyl), 7.90 (1H, d, 4-CHBenzyl
,
J =8 Hz), 8.00 (1H, d, CHBenzimidazole, J = 7.5 Hz), 8.20 (1H, d, CH
Benzimidazole, J= 8 Hz), 9.88 (1H, s, benzimidazolium 2-CH). 13C{1 H}
NMR (125 MHz, d6-DMSO): d 13.35 (CH3-butyl), 19.02 (CH2-butyl),
30.53 (CH2-butyl), 46.67 (N-CH2-butyl), 48.48 (CH2-benzonitrile),
110.84 (C ꢃ N, nitrile), 113.62, 126.82, 129.11, 131.14, 133.99, (Ar-C,
benzonitrile) 116.89, 126.98, 129.58, 131.25, 133.84, 136.9 (Ar-C,
benzimidazole), 143.16 (benzimidazolium–C2). FT-IR (KBr disc)
cmꢀ1: 1602 n(C N, benzimidazole), 2225 n(Cꢃ N, benzonitrile),
2962, 3043 n(C-H, aliphatic and aromatic). UV–visible (DMF, nm):
275.5 (p–p*) and 317.1 (n–p*). Molar conductance: 12.52 S cm2
molꢀ1. Further, molar conductance and 1 H, 13C NMR, FT-IR, UV–
visible spectral data of bromide salt has no or negligible
changes compared to salt 5.
A suspension of silver oxide (0.126 g , 0.055 mmol) and 3 (0.469 g,
1 mmol) in methanol (30 ml) was stirred in the absence of light
at 50–60 ꢁC for 24 h, after which the mixture was filtered through
a pad of celite to remove the unreacted silver oxide. Filtrate
was evaporated to dryness under reduced pressure to afford
the product as an off-white light-sensitive solid. The complex
thus obtained was recrystallized by repeated precipitation in
methanol and diethyl ether solution (1:10 v/v). Yield 70.86%;
m.p. 278–279 ꢁC. 1 H NMR (500 MHz, d3-CD3CN): d 5.59 (4 H, s,
CH2-benzyl), 5.77 (4 H, s, CH2-benzonitrile), 7.15 (1 H, d, CHBenzyl
J = 8 Hz), 7.40 (2 H, m, CHBenzimidazole), 7.55 (2 H, m, CHBenzonitrile).
13C{1 H}NMR (125 MHz, d3-CD3CN):
49.49 (CH2-benzyl),
50.98 (CH2-benzonitrile), 111.76, 115.02 (Cꢃ N, nitrile), 127.98,
130.51, 131.82 (Ar-C,benzonitrile), 129.67, 134.91, 137.71 (Ar-C,
,
d
Synthesis of 1-(3-cyanobenzyl)-3-benzyl-3 H-benzimidazol-1-ium hexafluor-
ophosphate (7)
benzimidazole), 179.41 (Ag– benzimidazolium C2). FT-IR (KBr disc)
cmꢀ1: 1638 n(C N, benzimidazole), 2224 n(Cꢃ N, benzonitrile),
2923, 3022 n(C-H, aliphatic and aromatic). UV–visible (DMF, nm):
273.3 (p–p*) and 261.0 (n–p*). Molar conductance: 15.2S cm2
molꢀ1. Anal. Calcd for C44H34N6Ag1F6P: C 58.7, H 3.8, N 9.3 %.
Found: C 58.2, H 3.1, N 9.6%.
This compound was prepared in a manner analogous to that
for 3, only with 3-bromomethyl benzonitrile (0.196 g, 1 mmol)
instead of 2-bromomethyl benzonitrile. Yield 78.8%: m.p.
115 ꢁC. 1 H NMR (500 MHz, d6-DMSO): d 5.81 (2 H, s, CH2-benzyl),
5.87 (2 H, s, CH2-benzonitrile), 7.45 (3 H, m, 2-CHBenzyl), 7.55
(2 H, t, 3-CHBenzyl, J = 7.3 Hz), 7.65 (3 H, m, CHBenzimidazole), 7.90
Synthesis of 1-(2-cyanobenzyl)-3-butyl-3 H-benzimidazol-1-ium silver(I) hex-
afluorophosphate (11)
(2 H, t, CHBenzimidazole, J = 8.0 Hz), 7.90 (2 H, d, CHBenzonitrile
,
J = 7.5 Hz), 10.05 (1 H, s, benzimidazolium 2-CH). 13C{1 H}NMR
(125 MHz, d6-DMSO): d 49.62 (CH2-benzyl), 50.63 (CH2-benzonitrile),
112.41 (C ꢃ N, nitrile) 127.35, 129.26, 131.62, 133.00 (Ar-C, benzoni-
trile), 114.36, 127.40, 129.48, 131.52, 133.75 (Ar-C, benzyl), 118.87,
128.84, 130.70, 132.50, 134.27, 135.27 (Ar-C, benzimidazole),
143.55 (benzimidazolium–C2). FT-IR (KBr disc) cmꢀ1: 1605 n(C N,
benzimidazole), 2229 n(Cꢃ N, benzonitrile), 2967, 3032 n(C-H,
aliphatic and aromatic). UV–visible (DMF, nm): 249.6 (p–p*) and
285.2 (n–p*). Molar conductance: 12.9S cm2 molꢀ1. Further, molar
conductance and 1 H, 13C NMR, FT-IR, UV–visible spectral data of
bromide salt 6 had no or negligible changes compared to salt 7.
This compound was prepared in a manner analogous to that for
10, only with 5 (0.435 g, 1 mmol) instead of 3. Yield 68.56 ; m.p.
165–166 ꢁC. 1 H NMR (500 MHz, d3-CD3CN): d 0.85 (3 H, t, CH3-
butyl, J = 7.3 Hz), 1.30 (2 H, m, CH2-butyl), 1.80 (2 H, m, CH2-butyl),
4.50 (2 H, t, CH2-butyl, J = 7.3 Hz), 5.98 (2 H, s, CH2-benzonitrile),
7.05 (1 H, d, CHBenzimidazole, J = 7.8 Hz), 7.45 (3 H, m, CHBenzimidazole),
7.62 (2 H, m, CHBenzonitrile), 7.90 (2 H, t, CHBenzonitrile, J = 7.5 Hz). 13
C
{1 H}NMR (125 MHz, d3-CD3CN): d 14.37 (CH3-butyl), 20.29 (CH2-
butyl), 32.91 (CH2-butyl), 49.57 (CH2-butyl), 51.118 (CH2-benzoni-
trile), 111.34 (C ꢃ N, nitrile), 112.97, 113.18, 125.16, 128.65,
134.25 (Ar-C, benzonitrile), 117.99, 129.71, 134.46, 134.57 (Ar-C
benzimidazole), 180.37 (benzimidazole–C2–Ag). FT-IR (KBr disc)
cmꢀ1: 1638 n(C N, benzimidazole), 2224 n(C ꢃ N, benzonitrile),
2965, 3118 n(C-H, aliphatic and aromatic). UV–visible (DMF, nm):
287.7 (p–p*) and 267.0 (n–p*). Molar conductance: 17.90 S cm2
molꢀ1. Anal. Calcd for C38H38N6Ag1F6P: C 54.9, H 4.6, N 10.1%.
Found: C 55.4, H 4.9, N 9.7%.
Synthesis of 1,2-bis-(3-cyanobenzyl-3 H-benzimidazolium-1-ylmethyl)ben-
zene bis-(hexafluorophosphate) (9)
This compound was prepared in a manner analogous to that for
3, only with 1,2-bis-(3 H-benzimidazole-1-ylmethyl)benzene
(0.338 g, 1 mmol) and 3-bromomethyl benzonitrile (0.392 g,
2 mmol) instead of 1-benzyl benzimidazole and 2-bromomethyl
benzonitrile, respectively. Yield 80.31%; m.p. 145–146 ꢁC. 1 H
NMR (500 MHz, d6-DMSO): d 5.86 (4 H, s, CH2-benzyl), 6.02 (4 H,
s, CH2-benzonitrile), 7.30 (4 H, m, 3-CHXylyl), 7.45 (2 H, m, 4-CHXylyl),
7.70 (6 H, m, CHBenzimidazole), 7.90 (6 H, m, CHBenzimidazole), 8.00
(2 H, t, CHBenzonitrile, J = 8.50 Hz), 9.89 (2 H, s, benzimidazolium
2-CH). 13C{1 H}NMR (125 MHz, d6-DMSO): d 47.72 (CH2-benzyl),
49.20 (CH2-benzonitrile), 111.87, 113.93 (C ꢃ N, nitrile), 118.38,
Synthesis of 1-(3-cyanobenzyl)-3-benzyl-3 H-benzimidazol-1-ium silver(I)
hexafluorophosphate (12)
This compound was prepared in a manner analogous to that for
10, only with 7 (0.469 g, 1 mmol) instead of 3. Yield 71.56%, m.p.
214 ꢁC. 1 H NMR (500 MHz, d6-DMSO): d 5.77 (2 H, s, CH2-benzyl),
5.82 (2 H, s, CH2-benzonitrile), 7.30 (3 H, m, CHBenzyl), 7.36 (2 H, d,
CHBenzyl, J = 7.00 Hz), 7.41 (2 H, m, CHBenzimidazole), 7.55 (1 H, d,
Appl. Organometal. Chem. 2012, 26, 689–700
Copyright © 2012 John Wiley & Sons, Ltd.
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R. A. Haque et al.
CHBenzimidazole, J = 8.00 Hz), 7.64 (1 H, d, CHBenzimidazole, J = 8.00 Hz),
7.72 (2 H, m, CHBenzonitrile), 7.76 (2 H, m, CHBenzonitrile). 13C{1 H}NMR
(125 MHz, d6-DMSO): d 49.51 (CH2-benzyl), 50.41 (CH2-benzoni-
trile), 113.36 (C ꢃ N, benzonitrile), 126.10, 128.01, 130.32, 132.05
(Ar-C, benzonitrile), 126.15, 128.58, 128.94, 134.30 (Ar-C, benz-
imidazole), 164.92 (Ag–benzimidazolium–C2). FTIR (KBr disc)
cmꢀ1: 1605 n(C N, benzimidazole), 2230 n(C ꢃ N, benzonitrile),
2924, 3062 n(C-H, aliphatic and aromatic). UV–visible (DMF, nm):
257.2 (p–p*) and 261.9 (n–p*). Molar conductance: 13.55 S cm2
molꢀ1. Anal. Calcd for C44H34N6Ag1F6P: C 58.7, H 3.8, N 9.3%.
Found: C 59.0, H 3.7, N 9.8%.
7.51 (5 H, m, CHBenzyl), 7.60 (2 H, t, CHBenzimidazole, J = 7.8 Hz), 7.73
(2 H, m, CHBenzimidazole), 7.83 (2 H, m, CHBenzonitrile), 7.93 (1 H, s,
CHBenzonitrile). 13C{1 H}NMR (125 MHz, d3-CD3CN): d 50.78 (CH2-
benzyl), 51.88 (CH2-benzonitrile), 111.74, 113.52 (C ꢃ N, benzoni-
trile), 118.44, 126.25, 128.75, 129.94, 132.15, 132.54, 132.70,
136.28 (Ar-C, benzonitrile), 127.68, 131.25, 132.65, 134.60 (Ar-C,
benzimidazole), 174.01 (Hg–benzimidazolium C2). FTIR (KBr disc)
cmꢀ1: 1574 n(C N, benzimidazole), 2225 n(C ꢃ N, benzonitrile),
2925, 3069 n(C-H, aliphatic and aromatic). UV–visible (DMF, nm):
280.8 (p–p*) and 293.0 (n–p*). Molar conductance: 30.7 S cm2
molꢀ1. Anal. Calcd for C44H34N6Hg1F12P2: C 46.5, H 3.0, N 7.4%.
Found: C 46.6, H 3.3, N 7.7%.
Synthesis of 1-(2-cyanobenzyl)-3-benzyl-3 H-benzimidazol-1-ium mercury(II)
bis-hexafluorophosphate (13)
Synthesis of 1-(2-((1 H-benzimidazol-1-yl)methyl)benzyl)-1 H-benzimid-
azole disilver(I) bis-hexafluorophosphate (16)
Mercury acetate (0.159 g, 0.5 mmol) was added to a solution
of 3 (0.469 g, 1 mmol) in methanol (20 ml). The mixture was
heated at 80–90 ꢁC for 24 h, after which the solvent was removed
under reduced pressure to give a pale-yellow powder. The
complex thus obtained was collected and recrystallized by
repeated precipitation from methanol and diethyl ether mixture
(1:10 v/v) to gave the complex as a white powder. Yield 70.14%,
m.p. 255–256 ꢁC. 1 H NMR (500 MHz, d3-CD3CN): d 5.98 (4 H, s,
This compound was prepared in a manner analogous to that for
10, only with 9 (0.862 g, 1 mmol) instead of 3. Yield 70.6%; m.p.
251–252 ꢁC. 1 H NMR (500 MHz, d6-DMSO): d 5.49 (2 H, s, CH2-ben-
zyl), 5.67 (2 H, s, CH2-benzonitrile), 7.16 (3 H, m, 3-CHXylyl), 7.40
(3 H, m, 4-CHXylyl), 7.66 (3 H, m, CHBenzimidazole), 7.69 (1 H, s, CHBen-
zimidazole), 7.77 (1 H, m, CHBenzonitrile). 13C{1 H}NMR (125 MHz, d6-
DMSO): d 50.96 (CH2-benzyl), 51.66 (CH2-benzonitrile), 111.71,
CH2-benzyl), 6.24 (4 H, s, CH2-benzonitrile), 6.85 (1 H, d, CHBenzyl
J = 8.0 Hz), 7.37 (8 H, m, CHBenzyl), 7.50 (4 H, d, CHBenzimidazole
J = 8.50 Hz), 7.55 (6 H, m, CHBenzimidazole), 7.70 (2 H, d, CHBenzonitrile
,
,
,
112.25 (C ꢃ N, nitrile), 118.40, 124.22, 126.49, 129.02, (Ar-CXylyl
)
124.32, 127.98, 130.81, 133.48, 134.72 (Ar-CBenzimidazole), 126.85,
136.49, 137.85 (Ar-CBenzonitrile), 171.48 (Ag–benzimidazolium 2C).
FTIR (KBr disc) cmꢀ1: 1618 n(C N, benzimidazole), 2229 n(C ꢃ N,
nitrile), 2917, 3033 n(C-H, aliphatic and aromatic). UV–visible
(DMF, nm): 256.61 (p–p*) and 261.28 (n–p*). Molar conductance:
28.61 S cm2 molꢀ1. Anal. Calcd for C76H56N12Ag2F12P2: C 55.6, H
3.4, N 10.2%. Found: C 56.8, H 3.7, N 10.7%.
J = 8.0 Hz), 7.79 (2 H, s, CHBenzonitrile). 13C{1 H}NMR (125 MHz,
CD3CN-d3): d 51. 8 (CH2-benzyl), 52.08 (CH2-benzonitrile), 110.01,
113.51 (C ꢃ N, nitrile), 117.11, 126.30, 127.72, 128.43, 133.62
(Ar-C, benzonitrile), 126.40, 128.67, 128.79, 133.51, (Ar-C, benz-
imidazole), 176.51 (Hg–benzimidazolium C2). FT-IR (KBr disc)
cmꢀ1: 1569 n(C&dtbond;N, benzimidazole), 2225 n(C ꢃ N, benzo-
nitrile), 2925, 3069 n(C-H, aliphatic and aromatic). UV–visible
(DMF, nm): 278.2 (p–p*) and 290.7 (n–p*). Molar conductance:
31.4 S cm2 molꢀ1. Anal. Calcd for C44H34N6Hg1F12P2: C 46.5,
H 3.0, N 7.4%. Found: C 46.0, H 3.7, N 7.8%.
Synthesis of 1-(2-((1 H-benzimidazol-1-yl)methyl)benzyl)-1 H-benzimid-
azole dimercury(II) tetra-hexafluorophosphate (17)
This compound was prepared in a manner analogous to that for
13, only with 9 (0.862 g, 1 mmol) instead of 3. Yield 71.18%; m.p.
256–257 ꢁC. 1 H NMR (500 MHz, d6-DMSO): d 6.51 (2 H, s, CH2-ben-
zyl), 6.58 (2 H, s, CH2-benzonitrile), 7.92 (4 H, m, CHXyly), 8.24 (7 H,
m, CHXylyl), 8.35 (1 H, m, CHBenzimidazole, J = 8.00 Hz), 8.45 (1 H, m,
CHBenzonitrile, J = 8.00 Hz). 13C{1 H}NMR (125 MHz, d6-DMSO): d
49.40 (CH2-benzyl), 51.50 (CH2-benzonitrile), 112.59, 113.21 (C ꢃ
N, nitrile), 117.89, 126.96, 129.53, 131.22 (Ar-CXylyl), 127.69,
129.69, 130.08, 132.32, 135.27 (Ar-CBenzimidazole), 129.87, 130.69,
131.85, 132.97, 133.78, (Ar-CBenzonitrile), 176.49 (Hg–benzimidazo-
lium 2C). FTIR (KBr disc) cmꢀ1: 1578 n(C N, benzimidazole), 2232
n(C ꢃ N, benzonitrile), 2965, 3030 n(C-H, aliphatic and aromatic).
UV–visible (DMF, nm): 265.11 (p–p*) and 293.8 (n–p*). Molar
Synthesis of 1-(2-cyanobenzyl)-3-butyl-3 H-benzimidazol-1-ium mercury(II)
bis-hexafluorophosphate (14)
This compound was prepared in a manner analogous to that for
13, only with 5 (0.435 g, 1 mmol) instead of 3. Yield 56.07%, m.p.
182 ꢁC. 1 H NMR (500 MHz, d3-CD3CN): d 1.00 (3 H, t, CH3-butyl,
J = 7.3 Hz), 1.14 (2 H, m, CH2-butyl), 1.97 (2 H, m, CH2-butyl), 4.65
(2 H, t, CH2-butyl, J = 7.8 Hz), 6.04 (2 H, s, CH2-benzonitrile), 7.39
(2 H, m, CHBenzyl), 7.52 (2 H, t, CHBenzyl, J = 7.5 Hz), 7.60 (4 H, t,
CHBenzimidazole, J = 8.3 Hz), 7.95 (2 H, d, CHBenzonitrile, J = 7.0 Hz).
13C{1 H}NMR (125 MHz, CD3CN-d3): d 13.27 (CH3-butyl), 19.95
(CH2-butyl), 32.33 (CH2-butyl), 49.79 (N-CH2-butyl), 65.65 (CH2-
benzonitrile), 113.66, 113.87 (C ꢃ N, nitrile), 127.13, 133.68,
134.33 (Ar-C, benzonitrile), 128.38, 129.80, 134.15, 137.54 (Ar-C,
benzimidazole), 176.20 (Hg–benzimidazole C2). FT-IR (KBr disc)
cmꢀ1: 1588 n(C N, benzimidazole), 2224 n(C ꢃ N, benzonitrile),
2963, 3108 n(C-H, aliphatic and aromatic). UV–visible (DMF, nm):
292.2 (p–p*) and 281.4 (n–p*). Molar conductance: 35.19 S cm2
molꢀ1. Anal. Calcd for C38H38N6Hg1F12P2: C 42.7, H 3.6, N 7.9%.
Found: C 42.4, H 3.9, N 7.7%.
conductance: 143.25 S cm2 molꢀ1
. Anal. Calcd for C76H56N12
Hg2F12P2: C 49.9, H 3.1, N 9.2%. Found: C 50.6, H 3.6, N 9.8%.
Methodology for Nuclease Activity
Supercoiled plasmid DNA pUC19 was used for DNA cleavage
experiments using agarose gel electrophoresis. Plasmid DNA
(pDNA) from the overnight-grown E. coli cells were isolated using
WizardW Plus SV Minipreps DNA purification system (Promega,
Madison, WI, USA). pDNA (200 ng) in 5 mM Tris–HCl/50 mM NaCl
buffer (pH 7.2) was treated with ligands and its Ag complexes
(150 mM) with or without H2O2 (100 mM) for 2 h at 37 ꢁC. After
incubation, 20 ml of the reaction mixture was loaded on to 1%
agarose gel prepared with 1ꢂ TAE (Tris–acetic acid–ethylenedia-
minetetraacetic acid (EDTA)) buffer solution and electrophoresed
Synthesis of 1-(3-cyanobenzyl)-3-benzyl-3 H-benzimidazol-1-ium mercury(II)
bis-hexafluorophosphate (15)
This compound was prepared in a manner analogous to that for
13, only with 7 (0.469 g, 1 mmol) instead of 3. Yield 58.02%, m.p.
252–253 ꢁC. 1 H NMR (500 MHz, d3-CD3CN): d 5.97 (2 H, s, CH2-
benzyl), 6.00 (2 H, s, CH2-benzonitrile), 7.37 (4 H, m, CHBenzyl),
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Nitrile-functionalized carbene complexes
for 50 min at 70 mV in a gel electrophoresis apparatus (Bio-Rad,
California, USA) with 0.5ꢂ TAE buffer (pH 8.3). The gel was then
stained with 1 mg mlꢀ1 ethidium bromide and visualized under
UV light using a gel documentation system (FluorChem HD2, Cell
Biosciences) and photographed. The nucleating ability of the
synthesized benzimidazolium salts/Ag complexes was deter-
mined by its efficiency in cleaving the supercoiled (SC) plasmid
DNA into open circular (OC) or nicked circular (NC) forms.
the complex at a fixed concentration contained in a quartz cell.
The UV–visible spectra were recorded after equilibration at
27 ꢁC for 10 min after each addition. The intrinsic binding
constant Kb was determined from the plot of [DNA]/(ea ꢀ ef) vs.
[DNA] according to equation (1):
½DNAꢄ=ðea ꢀ efÞ ¼ ½DNAꢄ=ðeb ꢀ efÞ þ 1=½Kbðeb ꢀ efÞꢄ
(1)
where [DNA] is the concentration of DNA in base pairs, the apparent
absorption coefficients ea, ef and eb correspond to Aobs/[complex], ex-
tinction coefficient for the free complex and the extinction coefficient
of the complex in the totally bound form, respectively. The data were
fitted to equation (1), with a slope equal to 1/(eb ꢀ ef) and y-intercept
equal to 1/[Kb(eb ꢀ ef)] and Kb was obtained from the ratio of the
slope to the intercept.
Methodology for DNA Binding Activity
Isolation of genomic DNA
Escherichia coli genomic DNA (gDNA) was utilized for the benzi-
midazolium salts/Ag complexes DNA binding studies. E. coli
JM109 strain was grown overnight and the gDNA was extracted
using commercially available DNA isolation kit (DNeasyW, QIA-
GEN, Hilden, Germany).
DNA binding analysis using viscosity measurements
Viscosity measurements were carried out using an Oswald micro-
viscometer, maintained at constant temperature (29ꢁC) in a
thermostat. DNA concentration was kept constant in all samples,
but the complex concentration was increased each time (from
200 to 1000 mM). Mixing of the solution was achieved by bubbling
nitrogen gas through the viscometer. The mixture was left for
10min at 29 ꢁC after addition of each aliquot of complex. The flow
time was measured with a digital stopwatch. The experiment was
repeated in triplicate to obtain the concurrent values. Data are
presented as (ꢀ/ꢀ0)1/3 vs. the ratio [complex]/[DNA], where ꢀ and
ꢀ0 are the specific viscosity of DNA in the presence and in absence
of the complex, respectively. The values of ꢀ and ꢀ0 were calculated
using equation (2):
DNA binding analysis using electronic spectral method
The concentration of gDNA per nucleotide [C(p)] was measured
ꢀ1
by using its known extinction coefficient at 260 nm (6600 M
cmꢀ1). The absorbance at 260 nm (A260) and at 280 nm (A280
)
for DNA was measured to check its purity, and was found to be
satisfactorily free from protein. Buffer (5 mM tris(hydroxymethyl)
aminomethane, pH 7.2, 50 mM EDTA] was used. In absorption
studies the complex was dissolved in DMF to obtain the desired
concentration. Spectroscopic titrations were carried out by
adding increasing amounts (20–100 ml) of DNA to a solution of
Scheme 1. Synthetic route to the preparation of benzimidazolium salts 2–9.
Appl. Organometal. Chem. 2012, 26, 689–700
Copyright © 2012 John Wiley & Sons, Ltd.
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R. A. Haque et al.
ꢀ ¼ ðt ꢀ tbÞ=tb
(2)
where tb is the observed flow time of DNA containing solution and
t is the flow time of buffer alone. Relative viscosities for DNA were
calculated from the relation ꢀ/ꢀo.
Results and Discussion
Syntheses
1-(2-nitrilebenzyl) benzimidazole was prepared according to the
similar method reported for the preparation of 1-benzyl benzimid-
azole and ortho-xylyl-bis-benzimidazole with slight modifica-
tions.[17] A mixture of benzimidazole and NaOH (1:2) was stirred in
THF at 70ꢁC for 3 h. 2-Bromomethylbenzonitrile (1 equiv.) in THF
was added dropwise to the mixture and further stirred under the
same conditions for 24h. The light-brown solid was obtained after
solvent removal using a rotary evaporator, washed with distilled
water to remove the unreacted NaOH and air dried.
Scheme 2. Synthesis of mononuclear Ag(I)– and Hg(II)–NHC complexes
10–15.
The synthetic route for both the 2-nitrile and 3-nitrile-substituted
benzimidazolium salts is shown in Scheme 1. 2-Nitrile-substituted
benzimidazolium bromide salts 2 and 4 were prepared by the
reaction of 1-benzyl benzimidazole and 1-methylbenzonitrile
benzimidazole with 2-bromomethyl benzonitrile and butyl bromide,
respectively, in acetonitrile at refluxing conditions for 48 h. Similarly,
3-nitrile-substituted bromide salts were prepared by the reaction
of 3-bromomethylbenzonitrile with 1-benzylbenzimidazole or 1,2-
bis-(3 H-benzimidazole-1-ylmethyl)benzene in acetonitrile under
reflux conditions. Hexafluorophosphate salts of the NHC precursors
are found to be considerably more stable than the corresponding
bromide salts in common organic solvents such as acetonitrile
and acetone. Therefore the bromide salts were converted into their
hexafluorophosphate counterparts by the salt metathesis reaction
using KPF6 in methanol to afford salts 3, 5, 7 and 9 in good yields.
These salts are readily soluble in common organic solvents such
as acetonitrile, DMF and DMSO, but are insoluble in toluene, meth-
anol and 1,4-dioxane.
Reactions involved in the preparation of mononuclear and
binuclear carbene complexes are shown in Schemes 2 and 3,
respectively. Ag(I) and Hg(II)–carbene complexes have been
prepared by the in situ deprotonation method using basic
metal sources.[19] Reactions of benzimidazolium salts 3, 5, 7
and 9 in acetonitrile at refluxing conditions for 24–36 h with
Ag2O/Hg(OAc)2 yield off-white, light-sensitive Ag(I)–carbene
complexes 10, 11, 12 and 16, and Hg(II)–carbene complexes
13, 14, 15 and 17, respectively, in moderately good yields
(55.0–70.9%). All the carbene complexes were purified by
repeated precipitation in acetonitrile by the addition of diethyl
ether. In the case of Ag(I) complexes, the unreacted silver oxide
was filtered through a pad of celite. However, in the case of Hg
(II) complexes, the excess or residual Hg(OAc)2 was removed by
repeated washing with distilled water. Both, Ag(I)– and Hg(II)–
carbene complexes are soluble in polar organic solvents such as
acetonitrile, DMF and DMSO, but are insoluble in toluene, diethyl
ether, benzene and hexane.
Scheme 3. Synthesis of binuclear Ag(I)– and Hg(II)–NHC complexes
16 and 17.
presence of benzylic and benzonitrilic protons. The multiplets in
the range d 7.41–8.19 and 1.3–4.2 are due to the resonance of
aromatic and aliphatic protons. Spectra also evidenced a
characteristic downfield resonance in the range d 9.87–10.06
corresponding to the NCHN proton, indicating the successful
formation of benzimidazolium salts.[20–22] This down-field shift
of the NCHN protons is due to their deshielding by two adjacent
electronegative nitrogen atoms that withdraw higher electron
density from around the benzimidazolium proton compared to
other protons. The 13C NMR spectra of the benzimidazolium
salts display characteristic downfield resonances in the range
d 143.47–148.15 for the NCN carbon nuclei.[23,24] In addition,
spectra also evidenced aromatic, nitrile, benzylic and aliphatic
carbon resonances in the range d 118–138, 111–114, 48–53 and
13–43, respectively.
The successful formation of carbene complexes is indicated by
the disappearance of the characteristic acidic 2H-benzimidazolium
protons in the 1 H NMR spectra of complexes 10–17 via deprotona-
tion. In addition, in the 13C NMR spectra of the complexes, the
characteristic carbene carbon (Ag-C and Hg-C) value in the
range d 164–189 is consequently observed. These observations
collectively confirm the formation of desired Ag(I)– and Hg(II)–
NHC complexes.[25] Apart from this major change, both 1 H and
13C NMR spectra of the complexes showed resonances of aromatic,
nitrile, benzylic and aliphatic carbon nuclei in the range d 118–138,
112–114, 49–53 and 12–41, respectively. These values are in agree-
ment with the reported Ag(I)– and Hg(II)–NHC complexes.[26–28]
NMR Spectral Studies
The NMR spectrum of all the compounds has been analysed in
appropriate deuterated solvents over the scan ranges d 0–16
and 0–200 for 1 H and 13C NMR spectral studies, respectively.
The 1 H NMR spectrum of benzimidazolium salts shows two
distinctive singlets in the range d 5.71–6.02, indicating the
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Nitrile-functionalized carbene complexes
FT-IR Spectral Studies
benzimidazolium salt 9 and its Ag(I)–carbene complex 16, and Hg
(II)–carbene complexes 13, 14 and 15, this value is almost double,
indicating that they are 2:2 electrolytes. Finally, a value of 143.25 S
cm2 molꢀ1 was observed in the case of Gg(II)–carbene complex
17, indicating a 4:4 electrolytic nature. Furthermore, we also tried
to deduce the structure of compounds on the basis of their molar
conductivity measurements. It is evident from this study that the
mononuclear Ag(I)– and Hg(II)–carbene complexes have one
and two hexafluorophosphate counterions, respectively, which in
turn reflects the formation of a bis-carbene complex. Similarly,
complexes 16 and 17 were also found to be bis-carbene analogues
containing a bimetallic core.
Both benzimidazolium salts and their Ag(I)– and Hg(II)–NHC
complexes were characterized by FT-IR spectral technique over
the range 4000–400 cmꢀ1. IR spectra of the benzimidazolium salts
show a strong band at around 1605cmꢀ1 with medium intensity,
assigned to the stretching vibrations of the benzimidazole ring
C N module.[29] A sharp band of medium intensity was observed
at 2225 cmꢀ1, assigned to the stretching vibrations of nitrile
component. In addition, spectra also evidenced at the two bands
around 2960 and 3030 cmꢀ1, which were assigned to the pure
modes of the C-H stretching vibrations. This variation in the range
is due to the presence of aliphatic and aromatic C-H modules.[30]
However, in the complex spectra, the band due to benzimidazole
ring vibrations showed a negative shift, further indicating the
formation of C-N module and in turn the desired carbene
complexes. Furthermore, a non-ligand band with medium intensity
at around 1100 cmꢀ1 further confirms its formation. Interestingly,
nitrile stretching vibrations remain unaltered in the complex
spectra, indicating their presence outside the coordination spear.
Single-Crystal X-Ray Diffraction Studies
X-ray-quality crystals of salts 2 and 5 were grown by slow evapo-
ration of a salt solution in acetonitrile 1,4-dioxane at ambient
temperature. A perspective view of salts 2 and 5 and their crystal
packing are shown in Figs 1–4. The crystal refinement data and
selected bond lengths and angles of salts 2 and 5 are tabulated
in Tables 1, 2 and 3.
UV–visible Spectral Studies
Salt 2 crystallizes in the monoclinic space group P2(1)/c
with one benzimidazolium cation, one bromide anion and one
water molecule occupying an asymmetric unit. Each repeating
unit of the crystal consists of four benzimidazolium units,
four bromide counterions and four water molecules. Planes of
both the N-substituents on the benzimidazole ring, benzyl and
benzonitrile are almost parallel to each other but perpendicular
to the plane of the central benzimidazole core. In the salt, the
central benzimidazolium ring of the monocation makes dihedral
angles of 131.3(2)ꢁ and 112.45(19)ꢁ with the pendant benzyl
and benzonitrile rings, respectively. The internal ring angle,
N1–C7–N2, at the carbene centre is found to be 110.8(2)ꢁ,
which is consistent with earlier reports.[33,34] In the extended
structure, benzimidazolium cations and bromides are connected
via C-H. . .Br and C-H. . .O hydrogen bonds (2.884 and 3.635 Å),
The electronic spectra of salts and complexes were recorded in
DMF solution (10ꢀ3 M concentration) over the range 1000–200 nm.
All the benzimidazolium salts show two characteristic bands
in the range 255–267 and 290–310 nm assigned to intramolecular
p ! p* and n ! p* transitions originating from the C C, C N and
C ꢃ N modules.[31] These bands show a negative shift of about
18–21nm upon complexation with Ag(I) or Hg(II), indicating the
formation of a C-N module in the benzimidazole ring. In addition,
complex spectra showed a band at around 320 nm and this
accounted for the charge transfer transitions. No bands at lesser
energy level for d–d transition were observed, as expected.
Molar Conductivity Measurements
forming
a three-dimensional array. Each bromide ion is
The molar conductance value of benzimidazolium salts and
complexes was obtained at room temperature in DMF solution at
10ꢀ3 mol dmꢀ3 concentration. The molar conductivity value of
benzimidazolium salts 3, 5, 7 and their Ag(I)–carbene complexes
connected with eight different hydrogen atoms to form the
aforementioned hydrogen bonds, and surprisingly, shows
a weak interaction with the oxygen atom of the water
molecule (Br-O), with a bond distance of 3.112 Å, perpendicular
to the ab plane.
10, 11, 12 is found to be similar, in the range 13.5–18.5 S cm2 molꢀ1
,
indicating that they are 1:1 electrolytes.[32] However, in the case of
Figure 1. Molecular structure of benzimidazolium salt 2 with displacement ellipsoids drawn at 50 % probability.
Appl. Organometal. Chem. 2012, 26, 689–700
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R. A. Haque et al.
Figure 2. The crystal packing of salt 2, showing the C-H. . .Br, C-H. . .O and O-H. . .Br hydrogen bonds as dashed lines.
The asymmetric unit of salt 5 contains one benzimidazolium
cation and one hexafluorophosphate anion. This salt crystallizes
in the monoclinic space group P2(1)/c. Each repeating unit of
the crystal consists of four benzimidazolium units and four
hexafluorophosphate counterions. The benzonitrile ring and
n-butyl chain are inclined at an angle of 113.07(12)ꢁ and 113.23
(13)ꢁ, respectively, with respect to the central benzimidazolium
ring. The internal ring angle, N1–C7–N2, at the carbene centre
is 110.33(14)ꢁ, which is in agreement with the reported
compounds.[33,34] In the extended structure, benzimidazolium
cations and hexafluorophosphate anions are connected via
C-H. . .F and C-H. . .N ꢃ C hydrogen bonds (2.947 and 2.647 Å),
forming a three-dimensional network.
Ag(I)–NHC complexes 10, 11, 12 and 16. Agarose gel images
indicating the nuclease activity of the compounds in the presence
and absence of oxidizing agent H2O2 are shown in Figs 5 and 6,
respectively. When supercoiled pUC19 plasmid DNA was electro-
phoresed, the strands migrated along the gel. This supercoiled
confirmation (form I) had a faster migration when compared to its
closed circular confirmation (form II), which was produced as a
result of the cleavage of one of the strands of the double helix.
However, when both strands were cleaved a linear confirmation
(form III) DNA was formed, which migrated between form I and
form II.[35] By analysing the migration of pDNA in the agarose gel,
the extent of DNA damage can be evaluated. A control experiment
using a mixture of DNA and oxidizing agent, and DNA alone, did
not show any significant cleavage.
Among the tested 2-nitrile-substituted compounds, benzimi-
dazolium salt 5 displayed remarkable DNA cleavage activity in
the absence of oxidizing agent H2O2. Surprisingly, though, its
activity was seen to reduce in the presence of H2O2. Other
tested compounds containing 2-nitrile substitution displayed no
Nuclease Activity of the Benzimidazolium Salts and their
Ag Complexes
Gel electrophoresis was used to evaluate the DNA cleavage proper-
ties of benzimidazolium salts 3, 5, 7 and 9 and the respective
Figure 3. Molecular structure of benzimidazolium salt 5 with displacement ellipsoids drawn at 50 % probability.
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Nitrile-functionalized carbene complexes
Figure 4. The crystal packing of salt 5, showing the C-H. . .F and C-H. . .N hydrogen bonds as dashed lines.
obvious visible activity towards the cleavage of DNA into form III,
either in the presence or absence of H2O2. However, the width of
the DNA band became smaller and intense in appearance and
showed smearing to a lesser extent, indicating their binding
properties rather than cleavage. On the other hand, compounds
derived from 3-nitrile-substituted benzimidazolium salts evidenced
distinct nuclease activity. Salt 9 and its Ag complex 16 efficiently
cleaved supercoiled pDNA form into linear form (form III) in the
presence and absence of oxidizing agent. A smear following the
band indicates further cleavage of the strands. This infers that
further incubation above 2 h could possibly have initiated complete
degradation of the pDNA. Perhaps the observed nuclease activity of
the salts and complex was due to the hydrolytic cleavage of DNA
catalysed by the complex. This could be attributed to the binuclear
nature of the complex and the appropriate substitutions on it.
Table 1. Crystal data and structure refinement details for 2 and 5
2
5
Formula
C
22H19BrN3
C19H20F6N3P
435.35
Formula weight
413.31
Crystal system
Monoclinic
P2(1)/c
Monoclinic
P2(1)/c
Space group
Unit cell dimensions
DNA Binding Activity Using Electronic Spectral Method
a (Å)
b (Å)
c (Å)
a (ꢁ)
b (ꢁ)
g (ꢁ)
12.5545(2)
15.7849(2)
9.6485(2)
90.00
9.52740(10)
8.42460(10)
24.8761(3)
90.00
The UV–visible spectral technique is the most frequently used
method to study the interaction of transition metal complexes with
DNA, since these complexes often have abundant spectroscopic
properties.[36,37] Normally, DNA binding ability of the complexes is
assessed by measuring the effects on their UV spectra in the
presence of DNA.[38] UV–visible absorption titration spectra of
complexes 10 and 16 are shown in Figs 7 and 8, respectively.
Addition of increasing amounts of DNA resulted in a decrease of
absorbance for each investigated benzimidazolium salt and
complex. With increasing concentration of DNA, the absorption
bands of both the benzimidazolium salts and Ag(I) complexes were
affected, resulting in a tendency to hypochromism and a remark-
able red shift. This can be attributed to the intercalative mode of
the binding of tested compounds to the double helix of DNA.[40]
This may be due to the presence of benzimidazole or central xylyl
cores, which may provide an aromatic unit by which overlapping
would occur with the DNA base pairs via an intercalative mode of
binding. Change in absorbance at the peak maximum of benzimi-
dazolium salts and their Ag complexes of the most red-shifted band
of each compound with increasing concentration of DNA has been
monitored for evaluation of the intrinsic binding constant (Kb).
The Kb values (Table 4) of the benzimidazolium salts are sufficiently
less than the corresponding Ag complexes. Similar observations
91.6330(10)
90.00
92.4310(10)
90.00
V (Å3)
Z
1911.28(6)
4
1994.87(4)
4
Density
1.436
1.450
(calcd) (g cmꢀ3
)
Absorp. coeff.
2.164
0.202
(mmꢀ1
F(000)
)
844
896
Crystal size (mm)
Temperature (K)
Radiation (Å)
θ min., max. (ꢁ)
Dataset
0.45 ꢂ 0.31 ꢂ 0.25
100(2)
0.34 ꢂ 0.20 ꢂ 0.11
100(2)
Mo Ka 0.71073
1.62, 33.57
ꢀ19:19, ꢀ24:24,
ꢀ13:14
Mo Ka 0.71073
2.14, 30.32
ꢀ13:12, ꢀ11:11,
ꢀ30:35
Tot.; unique data
28753
22074
R (int.)
0.0321
0.0231
Nref, Npar
7484, 263
5944, 263
R, wR2, S
0.0835, 0.1416, 1.048
0.0753, 0.1079, 1.010
Appl. Organometal. Chem. 2012, 26, 689–700
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R. A. Haque et al.
Table 2. Selected bond lengths (Å) and angles (ꢁ) of 2
C8–C9
1.521(3)
1.514(4)
1.392(3)
131.7(2)
111.3(2)
107.9(2)
108.2(2)
N1–C7
N1–C8
N2–C6
1.334(4)
N2–C7
1.329(4)
1.477(4)
1.152(7)
C16–C17
N1–C1
1.470(3)
N2–C16
N3–C15
112.45(19)
110.8(2)
131.3(2)
178.0(4)
1.398(3)
C2–C1–N1
C5–C6–N2
C7–N1–C1
C7–N2–C6
N1–C8–C9
N2–C7–N1
N2–C16–C17
N3–C15–C14
Table 3. Selected bond lengths (Å) and angles (ꢁ) of 5
C8–C9
1.516(2)
N1–C8
N2–C1
N2–C7
1.4610(19)
1.3282(19)
1.393(2)
N2–C16
N3–C15
1.4734(19)
1.148(2)
N1–C1
1.336(2)
N1–C2
1.392(2)
C1–N1–C8
C1–N1–C2
C1–N2–C7
C1–N2–C16
C2–N1–C8
125.05(14)
108.32(13)
108.31(13)
125.33(14)
126.63(13)
C7–N2–C16
N1–C8–C9
N2–C1–N1
N2–C16–C17
N3–C15–C14
126.30(13)
113.07(12)
110.33(14)
113.23(13)
178.56(18)
Figure 5. Ethidium bromide stained agarose gel (1%) picture of benzimi-
dazolium salts and their Ag(I) complexes in the presence of oxidizing
agent H2O2.
Figure 7. Absorption spectra of complex 10 in the presence of increasing
amounts of E. coli gDNA. Arrow shows the absorption changes upon
changing the DNA concentration.
increased with the concentration of complex tested (50–250 ml);
hence an interactive mode of binding of complexes to DNA is
assigned. Intercalative mode of binding of a compound to DNA is
known to cause a significant increase in the viscosity of a DNA
solution due to an increase in the separation of its base pairs at
the intercalation site and, hence, an increase in the overall DNA
molecular length.[43] Conversely, a compound that binds in the
DNA grooves causes either a less pronounced change or no
significant change in the viscosity of a DNA solution.
Figure 6. Ethidium bromide stained agarose gel (1%) image of benzimi-
dazolium salts and their Ag(I) complexes in the absence of oxidizing
agent H2O2.
were found with late transition metal complexes used as DNA
binding agents.[40]
DNA Binding Activity Using Viscosity Measurements
The binding nature of the benzimidazolium salts and their Ag
complexes was further studied using viscosity measurements. This
method is also a crucial tool to find the nature of binding of metal
complexes to DNA.[41] The viscosity of DNA in a buffer solution is
most sensitive to the changes in its effective length, and is one
of the most significant tests for inferring the binding mode of
compounds with DNA.[42] Plots of relative viscosity of complexes
versus the ratio of concentration of complex and DNA are shown
in Fig. 9. The relative viscosities of the complex-bound DNA solution
Conclusion
Four new non-symmetrically substituted nitrile-functionalized
benzimidazolium salts and their Ag(I)– and Hg(II)–NHC complexes
were prepared and characterized using different spectral and
analytical techniques. All reported compounds were stable to
air and moisture, and were readily soluble in polar and insol-
uble in non-polar organic solvents. Molecular structures of
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Copyright © 2012 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2012, 26, 689–700

Nitrile-functionalized carbene complexes
Further studies on the transmetallation of the newly synthesized
complexes based on the literature method are underway.
Acknowledgements
R. A. Haque thanks Universiti Sains Malaysia (USM) for the short-
term grant (203/PKIMIA/6311123) and the Research University
(RU) grant 1001/PKIMIA/811217. S. Budagumpi thanks USM for a
postdoctoral research fellowship.
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Appendix
CCDC 883342 and 883343 contain the supplementary crystallo-
graphic data for 2 and 5, respectively. These data can be obtained
free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html,
or from the Cambridge Crystallographic Data Centre, 12 Union
Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk.
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Copyright © 2012 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2012, 26, 689–700