Recognition of Hg2+ using diametrically disubstituted cyclam unit

Article abstract of DOI:10.1021/ic1014797

A newly synthesized 1,4,8,11-tetraazacyclotetradecane derivative (L), functionalized with a diazo moiety as the reporter functionality, is found to bind specifically to Hg²⁺ with an associated change in color that could be visually detected. Wi

Full text of DOI:10.1021/ic1014797

Inorg. Chem. 2010, 49, 11485–11492 11485
DOI: 10.1021/ic1014797
Recognition of Hg2þ Using Diametrically Disubstituted Cyclam Unit
Prasenjit Mahato, Amrita Ghosh, Sukdeb Saha, Sandhya Mishra, Sanjiv K. Mishra, and Amitava Das*
Central Salt and Marine Chemical Research Institute, (CSIR), Bhavnagar -364002, Gujarat, India
Received July 23, 2010
A newly synthesized 1,4,8,11-tetraazacyclotetradecane derivative (L), functionalized with a diazo moiety as the reporter
functionality, is found to bind specifically to Hg2þ with an associated change in color that could be visually detected. With
biologically benign β-CD, it forms an inclusion complex (L 2β-CD), which shows a much higher solubility in water,
3
and this helps in developing a more intense color on binding to Hg2þ in a CH3CN-HEPES buffer medium. The nontoxic
nature of L was checked with the living cells of a Gram negative bacterium, Pseudomonas putida. Further, experiments
revealed that these two reagents could be used as staining agents for the detection of Hg2þ present in this microorganism.
Introduction
methods for the selective detection and visualization of Hg2þ
ions in chemical and biological systems is critically important,
and significant research efforts have been devoted to improve
mercury detection and recognition.7,8 Current industrial ap-
proaches rely on costly, time-consuming methods like atomic
absorption/emission or inductively coupled plasma mass
spectroscopy, which are not very convenient or handy for
“in-the-field” detection. A number of selective Hg2þ sensors
have been devised using either redox,9 chromogenic,10 or
fluorogenic11 changes. Further, chromogenic sensors that
allow visual detection have an edge over others owing to
obvious simplicity in the recognition process; its naked eye
detection offers qualitative and quantitative information
without using expensive equipment. However, such examples
Mercury is a dangerous toxin that has become widespread
throughout our environment and can pose a serious threat to
the food chain and thus human health. Coal-fired power
plants, waste incinerators, chloro-alkali plants, gold mining,1
and sources like oceanic and volcanic emission2 add a sub-
stantial quantity of mercury in various forms to the environ-
ment. Atmospheric oxidation of mercury vapor leads to the
generation of water-soluble Hg2þ ions that deposit onto land
or into water and is assimilated by microorganisms, which
convert some of it to methylmercury, a potent neurotoxin
linked to many cognitive and motion disorders.3 Bioaccumu-
lation of this occurs as bigger animals eat smaller ones.4 While
attempts are being made to curb the mercury emissions
substantially from medical and municipal waste incinerators,5
very little effort has been made to curb the mercury pollution
from coal-fired power plants.6 Thus, the development of new
(8) (a) Mandal, A. K.; Suresh, M.; Suresh, E.; Mishra, S. K.; Mishra, S.;
Das, A. Sens. Actuators, B 2010, 145, 32. (b) Suresh, M.; Mishra, S.; Mishra,
S. K.; Suresh, E.; Mandal, A. K.; Shrivastav, A.; Das, A. Org. Lett. 2009, 11,
2740. (c) Suresh, M.; Shrivastav, A.; Mishra, S.; Suresh, E.; Das, A. Org. Lett.
2008, 10, 3013. (d) Suresh, M.; Mishra, S. K.; Mishra, S.; Das, A. Chem.
Commun. 2009, 2496. (e) Li, J.; Yao, J.; Zhong, W. Chem. Commun. 2009, 4962.
(f) Wu, D.; Descalzo, A. B.; Weik, F.; Emmerling, F.; Shen, Z.; You, X.-Z.;
Rurack, K. Angew. Chem., Int. Ed. 2008, 47, 193. (g) Lim, C. S.; Kang, D. W.;
Tian, Y. S.; Han, J. H.; Hwang, H. L.; Cho, B. R. Chem. Commun. 2010, 2388.
(h) Huang, C.-C.; Yang, Z.; Lee, K.-H.; Chang, H.-T. Angew. Chem., Int. Ed.
2007, 46, 6824. (i) Santra, M.; Ryu, D.; Chatterjee, A.; Ko, S. K.; Shin, I.; Ahn,
K. H. Chem. Commun. 2009, 2115. (j) Huang, W.; Song, C.; He, C.; Lv, G.; Hu,
X.; Zhu, X.; Duan, C. Inorg. Chem. 2009, 48, 5061. (k) Jana, A.; Kim, J. S.; Jung,
H. S.; Bharadwaj, P. K. Chem. Commun. 2009, 4417.
*To whom correspondence should be addressed. Telephone: þ91 278
2567760 [672]. Fax: þ91 278 2656762. E-mail: amitava@csmcri.org.
(1) (a) Malm, O. Environ. Res., Sect. A 1998, 77, 73. (b) Mercury Update:
Impact of Fish Advisories. EPA Fact Sheet EPA-823-F-01-011; EPA, Office of
Water: Washington, DC, 2001. (c) Chu, P.; Porcella, D. B. Water, Air, Soil
Pollut. 1995, 80, 135.
(2) Renzoni, A.; Zino, F.; Fraanchi, E. Environ. Res. 1998, 77, 68.
(3) Clarkson, T. W.; Magoos, L.; Myers, G. J. N. Engl. J. Med. 2003, 349,
1731.
(4) Nendza, M.; Herbst, T.; Kussatz, C.; Gies, A. Chemosphere 1997, 35,
1875.
(9) (a) Wang, J. Acc. Chem. Res. 2002, 35, 811. (b) Caballero, A.; Martinez,
R.; Lioveras, V.; Ratera, I.; Vidal-Gancedo, J.; Wurst, A.; Tarraga, A.; Molina, P.;
Veciana, J. J. Am. Chem. Soc. 2005, 127, 15666. (c) Liu, S.-J.; Nie, H.-G.; Jiang,
J.-H.; Shen, G.-L.; Yu, R.-Q. Anal. Chem. 2009, 81, 5724. (d) Gong, J.; Zhou, T.;
Song, D.; Zhang, L.; Hu, X. Anal. Chem. 2010, 82, 567. (e) Alfonso, M.; T'arraga,
A.; Molina, P. Dalton Trans. 2010, 8637. (f) Zhao, Q.; Cao, T.; Li, F.; Li, X.; Jing,
H.; Yi, T.; Huang, C. Organometallics 2007, 26, 2077. (g) Yang, H.; Zhou, Z.;
Huang, K.; Yu, M.; Li, F.; Yi, T.; Huang, C. Org. Lett. 2007, 9, 4729. (h) Zejli, H.;
Sharrock, P.; Hidalgo-Hidalgo de Cisneros, J. L.; Naranjo-Rodriguez, I.;
Temsamani, K. R. Talanta 2005, 68, 79. (i) Wu, D.; Zhang, Q.; Chu, X.; Wang,
H.; Shen, G.; Yu, R. Biosens. Bioelectron. 2010, 25, 1025. (j) Singh, L. P.;
Bhatnagar, J. M. J. App. Electrochem. 2004, 34, 391.
(5) Shore, M. Out of Control and Close to Home Mercury pollution From
Power Plants; Environmental Defense: New York, 2003.
(6) (a) Spiegel, S. J. Ecol. Econ. 2009, 68, 3072. (b) Baughman, T. A.
Environ. Health Perspect. 2006, 114, 147. (c) Fitzgerald, W. F.; Lamborg, C. H.;
Hammerschmidt, C. R. Chem. Rev. 2007, 107, 641.
(7) (a) Nolan, E. M.; Lippard, S. J. Chem. Rev. 2008, 108, 3443. (b) de Silva,
A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.;
Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515. (c) Callen, J. F.; de Silva,
A. P.; Magri, D. C. Tetrahedron 2005, 61, 8551. (d) Bell, T. W.; Hext, N. M. Chem.
Soc. Rev. 2004, 33, 589. (e) Rurack, K.; Resch-Genger, U. Chem. Soc. Rev. 2002,
31, 116. (f) Haugland, R. P. Handbook of Fluorescent Probes and Research
Porducts, 9th ed.; Molecular Probes: Eugene, OR, 2002; Chapter 20.
r
2010 American Chemical Society
Published on Web 11/15/2010
pubs.acs.org/IC

11486 Inorganic Chemistry, Vol. 49, No. 24, 2010
Mahato et al.
of the reversible recognition of Hg2þ are relatively scarce
compared to those which can detect Hg2þ ions in aqueous
solution by chemodosimetry.8a-e,12 Thus, despite widespread
interest and recent advances, there are only a few examples
of chromogenic probes that bind specifically and reversibly
to Hg2þ in the presence of all other metal ions, like alkali,
alkaline earth, common transition metal (more specifically
metal ions like Pb2þ and Cu2þ), and lanthanide ions, and
are capable of detection of Hg2þ in biological samples.13
Thus, the design and synthesis of such new synthetic mole-
cules remains an outstanding challenge. Recent reports sug-
gest that nitrogen binding sites are a good choice for the selec-
tive recognition of heavy metal ions such as Cd2þ, Pb2þ, and
without sacrificing much of its binding ability to the target
Hg2þ ion. With this aim, we could synthesize a diametrically
disubstituted 1,4,8,11-tetraazacyclotetradecane (cyclam) de-
rivative, functionalized with 4-(4-dimethylamino)phenyl azo-
benzene as the reporter moiety, which has shown remarkable
specificity towards Hg2þ in the presence of a wide range of
interfering metal ions in aqueous solutions (including Cd2þ
,
Pd2þ, Cu2þ, and all lanthanide ions). We are reporting herein
its synthesis, binding studies, and [3]pseudorotaxane forma-
tion in the presence of β-cyclodextrin (β-CD). Further, results
of the experimental studies reveal that [3]pseudorotaxane
formation improves the solubility of the receptor in aqueous
solutions and thereby the colorimetric detection phenomena.
Hg2þ 14
More recently, Ho and his co-workers have shown
.
that 8,80-(1,4,10,13-tetrathia-7,16-diazacyclooctadecane-7,16-
diyl)-bis(methylene)diquinolin-7-ol could be used for the
recognition of Hg(II), present in micromolar concentrations
Experimental Section
1,4,8,11-Tetraazacyclotetradecane, 4-(4-dimethylamino-
phenylazo)-benzenesulphonyl chloride, β-cyclodextrin, Hg-
(ClO4)2, Cu(ClO4)2, Zn(ClO4)2, Ni(ClO4)2, Fe(ClO4)2, Pb-
(ClO4)2, Cd(ClO4)2,Cr(ClO4)3,Ca(ClO4)2, Co(ClO4)2,NaClO4,
in the presence of various cations like Liþ, Naþ, Kþ, Mg2þ
,
Ca2þ, Fe2þ, Cu2þ, Zn2þ, Pb2þ, and Al3þ, while some of the
metal ions are reported to interfere with detection if they are
present in relatively much higher concentrations.14 This
leaves us with the opportunity to design and synthesize a
suitable receptor with a softer base as a coordinating unit,
which may allow us to achieve a better specificity over a wider
range of metal ions including Cd2þ, Cu2þ, and lanthanide ions
KClO4,Mg(ClO4)2, CsClO4, Ba(ClO4)2, Ce(NO3)3 6H2O, Eu-
3
(NO3)3 5H2O,Er(NO3)3 5H2O,Nd(NO3)3 6H2O,Tb(NO3)3
3
3
3
3
5H2O, Yb(NO3)3 5H2O, Pr(NO3)3 6H2O, Ho(NO3)3 6H2O,
3
3
3
Lu(NO3)3 xH2O, Dy(NO3)3 xH2O, Gd(NO3)3 6H2O, Sm-
3
3
3
3
(NO3)3 6H2O, La(NO3)3 6H2O, andTm(NO3)3 5H2O were
3
3
obtained from Sigma-Aldrich and were used as received. All
of the other reagents used were procured from S. D. Fine
Chemicals, India. Acetonitrile, chloroform, methanol (AR;
Merck, India), and ethanol (Spectrosol; Spectrochem, India)
were used as solvents. HPLC-grade water (Merck, India) was
used for experiments and spectral studies. ESI-MS measure-
ments were carried out on Waters QTof-Micro instrument.
Microanalysis (C, H, N) was performedusing a Perkin-Elmer
4100 elemental analyzer. FTIR spectra were recorded as KBr
pellets using a Perkin-Elmer Spectra GX 2000 spectrometer.
1H and31PNMRspectrawererecordedon a Bruker500 MHz
FT NMR machine (model: Avance-DPX 500). Electronic
spectra were recorded with a Shimadzu UV-3101 PC/Varian
Cary 500 Scan UV-vis-NIR Spectrophotometer. The images
of the Pseudomonas putida cells were viewed under a normal
light microscope (AXIO IMAGER-Carl Zeiss).
(10) (a) Li, T.; Li, B.; Wang, E.; Dong, S. Chem. Commun. 2009, 3551.
(b) Sancenon, F.; Martinez-Manez, R.; Soto, J. Tetrahedron Lett. 2001, 42, 4321.
(c) Choi, M. J.; Kim, M. Y.; Chang, S.-K. Chem. Commun. 2001, 1664.
(d) Sancenon, F.; Martinez-Manez, R.; Soto, J. Chem. Commun. 2001, 2262.
(e) Brummer, O.; La Clair, J. J.; Janda, K. D. Org. Lett. 1999, 1, 415. (f) Coronado,
ꢀ
ꢀ
E.; Galan-Mascaros, J. R.; Martí-Gastaldo, C.; Palomares, E.; Durrant, J. R.; Vilar,
R.; Gratzel, M.; Nazeeruddin, M. K. J. Am. Chem. Soc. 2005, 127, 12351. (g) Lee,
S.-H.; Kumar, J.; Tripathy, S. K. Langmuir 2000, 16, 10482. (h) Ren, X.; Xu, Q.-H.
Langmuir 2009, 25, 29. (i) Hirayama, T.; Taki, M.; Kashiwagi, Y.; Nakamoto, M.;
Kunishita, A.; Itoh, S.; Yamamoto, Y. Dalton Trans. 2008, 4705. (j) Zhang, D.;
Deng, M.; Xu, L.; Zhou, Y.; Yuwen, J.; Zhou, X. Chem.;Eur. J. 2009, 15, 8117.
(k) Liu, X.; Tang, Y.; Wang, L.; Zhang, J.; Song, S.; Fan, C.; Wang, S. Adv. Mater.
2007, 19, 1471. (l) Lee, H.; Lee, S. S. Org. Lett. 2009, 11, 1393. (m) Zhou, Y.;
Zhu, C.-Y.; Gao, X.-S.; You, X.-Y.; Yao, C. Org. Lett. 2010, 12, 2566.
(11) (a) Huang, J.; Xu, Y.; Qian, X. J. Org. Chem. 2009, 74, 2167. (b) Chan,
D. S.-H.; Lee, H.-M.; Che, C.-M.; Leung, C.-H.; Ma, D.-L. Chem. Commun.
2009, 7479. (c) Rurack, K.; Resch-Genger, U.; Bricks, J. L.; Spieles, M. Chem.
Commun. 2000, 2103. (d) Chae, M.-Y.; Czarnik, A. W. J. Am. Chem. Soc. 1992,
114, 9704. (e) Dave, N.; Chan, M. Y.; Huang, P.-J. J.; Smith, B. D.; Liu., J. J. Am.
Chem. Soc. 2010, 132, 2668. (f) Zhang, X.; Xiao, Y.; Qian, X. Angew. Chem.,
Int. Ed. 2008, 47, 8025. (g) Joshi, B. P.; Lohani, C. R.; Lee, K.-H. Org. Biomol.
Chem. 2010, 3220. (h) Yoon, S.; Miller, E. W.; He, Q.; Do, P. H.; Chang, C. J.
Angew. Chem., Int. Ed. 2007, 46, 6658. (i) Ho, M.-L.; Chen, K.-Y.; Wu, L.-C.;
Shen, J.-Y.; Lee, G.-H.; Ko, M.-J.; Wang, C.-C.; Lee, J.-F.; Chou, P.-T. Chem.
Commun. 2008, 2438. (j) Che, Y.; Yang, X.; Zang, L. Chem. Commun. 2008,
1413. (k) Wang, Z.; Lee, J. H.; Lu, Y. Chem. Commun. 2008, 6005. (l) Lee, M. H.;
Lee, S. W.; Kim, S. H.; Kang, C.; Kim, J. S. Org. Lett. 2009, 11, 2101. (m) Jiang,
W.; Wang, W. Chem. Commun. 2009, 3913. (n) Guo, X.; Qian, X.; Jia, L. J. Am.
Chem. Soc. 2004, 126, 2272. (o) Ma, L.-J.; Li, L.; Sun, J.; Tian, C.; Wu, Y. Chem.
Commun. 2008, 6345. (p) Song, K. C.; Kim, J. S.; Park, S. M.; Chung, K.-C.;
Ahn, S.; Chang, S.-K. Org. Lett. 2006, 8, 3413. (q) Rurack, K.; Kollmannsberger,
M.; Resch-Genger, U.; Daub, J. J. Am. Chem. Soc. 2000, 122, 968. (r) Vaswani,
K. G.; Keranen, M. D. Inorg. Chem. 2009, 48, 5797. (s) Suresh, M.; Mandal,
A. K.; Saha, S.; Suresh, E.; Mandoli, A.; Liddo, R. D.; Parnigotto, P. P.; Das, A.
Org. Letts. 2010, DIO: 10.1021/ol102204r.
Synthesis of L. 1,4,8,11-Tetraazacyclotetradecane (cyclam;
247 mg, 1.23 mM) was dissolved in dry chloroform (40 mL) in a
250 mL round-bottomed flask. A solution of 4-(4-dimethylamino-
phenylazo)-benzenesulphonyl chloride (800 mg, 2.46 mM) and
triethyl amine (Et3N) (1 mL) in 60 mL of dry chloroform was
added to the above solution under ice-cold conditions with stirring.
Then, the resulting mixture was allowed to stir at room tempara-
ture for 10 h, and then it was further refluxed for 1 h. The mixture
was cooled to room temperature and evaporated to dryness using
a rotary evaporator. Then, the crude product was purified by
column chromatography using silica gel as a stationary phase and
a chloroform/methanol mixed solvent (98:2, v/v) as the eluent.
Isolated yield of the compound L (yield was calculated on the basis
1
of the starting compounds): 48%. H NMR (500 MHz, CDCl3,
SiMe4, J (Hz), δ (ppm)): 7.96 (4H, d, J = 8.5, Ar-H16), 7.92-7.88
(8H, m, Ar-H17,20), 6.76 (4H, d, J = 9.0, Ar-H21), 3.38 (4H, br,
H
2,9), 3.21 (12H, br, H3,5,7,10,12,14), 3.13 (12H, s, H23 -N(CH3)2),
2.26 (4H, br, H6,13). 13C NMR (DMSO-d6, 500 MHz, SiMe4, δ
(ppm)): 155.05, 153.19, 142.56, 135.29, 128.90, 125.48, 122.32,
111.54, 48.11, 47.84, 47.63, 45.80, 39.8, 26.43. ESI-MS (þve
mode): m/z 775.93 (100%) (Mþ). Calcd for C38H50N10O4S2:
776.36. Elemental Anal. Calcd: C, 58.74; H, 6.75; N, 18.03.
Found: C, 58.9; H, 6.8; N, 18.1.
(12) (a) Choi, M. G.; Kim, Y. H.; Namgoong, J. E.; Chang, S. K. Chem.
Commun. 2009, 3560. (b) Zhao, Y.; Lin, Z.; He, C.; Wu, H.; Duan, C. Inorg.
Chem. 2007, 46, 1538. (c) Suresh, M.; Ghosh, A.; Das, A. Chem. Commun. 2008,
3906.
(13) Xue, X.; Wang, F.; Liu, X. J. Am. Chem. Soc. 2008, 130, 3244.
(14) (a) Kim, S. H.; Kim, J. S.; Park, S. M.; Chang, S.-K. Org. Lett. 2006,
8, 371. (b) McCleverty, J. A.; Meyer, T. J. Comprehensive Coordination
Chemistry, II; Elsevier: New York, 2003; Vol. 6. (c) Ho, M.-L.; Chen, K.-Y.;
Lee, G.-H.; Chen, Y.-C.; Wang, C.-C.; Lee, J.-F.; Chung, W.-J.; Chou, P.-T. Inorg.
Chem. 2009, 48, 10304. (d) Boiocchi, M.; Fabbrizzi, L.; Licchelli, M.; Sacchi, D.;
Vazquez, M; Zampa, C. Chem. Commun. 2003, 1812.
Synthesis of L1. A total of 100 mg (0.31 mM) of 4-(4-dimethy-
lamino-phenylazo)-benzenesulphonyl chloride was added to 15 mL
of pyrrolidine (excess) under an inert atmosphere and heated to
reflux for 24 h. Then, the reaction mixture was evaporated to

Article
Inorganic Chemistry, Vol. 49, No. 24, 2010 11487
Scheme 1. Methodology Adopted for the Synthesis of (A) L and (B) L1
dryness. The crude product was recrystallized from methanol
to obtain L1 in pure form with 85% yield (yield was calculated on
the basis of the starting compounds). 1H NMR (500 MHz,
CD3OD, SiMe4, J (Hz), δ (ppm)): 7.93 (2H, d, J = 8.4, Ar-Hh),
7.87-7.8 (4H, m, Ar-Hd,g), 6.83 (2H, d, J = 9, Ar-Hc), 3.27-
3.32 (4H, t, Hj(-CH2)), 3.1 (6H, s, Ha -N(CH3)2), 2.03-1.96
(4H, m, Hk(-CH2)). ESI-MS (þve mode): m/z 359.18 (5%)
(Mþ þ Hþ). Calcd for C18H22N4O2S: 358.46. Elemental Anal.
Calcd: C, 60.3; H, 6.2; N, 15.6. Found: C, 60.4; H, 6.1; N, 15.5.
Spectrophotometric Titration. Evaluation of the Associa-
limiting value. CM is the metal ion concentration, and n is the
stoichiometry of the complex formed between the ligand and
metal ion.
The following equations were used for the nonlinear least-
squares analysis to determine the association constants of the
complexes formed between L and β-CD:16
1
1
1
1
¼
ꢀ
þ
ð2Þ
A - A0
K1ðA1 - A0Þ ½β-CDꢁ0 A1 - A0
tion Constant for L Hg2þ Formation. A 11.5 μM solution of L
3
in an acetonitrile-HEPES buffer (pH 7.2, 2:3 (v/v)) was prepared
and stored under dark conditions. These solutions were used for
all spectroscopic studies after appropriate dilution. A 16.62 mM
solution of Hg(ClO4)2 was prepared using the same solvent com-
position. A solution of L was used for spectroscopic titrations
after appropriate dilution, and the effective final concentration
was adjusted to 2.3 μM, while the final analyte (Hg2þ) concen-
tration for titration was varied from 0 to 13.3 mM.
1
1
1
1
¼
ꢀ
þ
ð3Þ
2
A - A0
ðA2 - A0ÞK1K2
ðA2 - A0Þ
½β-CDꢁ0
A0 and A1 denote the absorbance of free L and L in the complex,
and K1 is the association constant for 1:1 complexation. A2 is the
absorbance in the complex, and K2 is the association constant
for 1:2 complexation.
Biological Study. Pseudomonas putida (P. putida) was cul-
tured in King’s B (KB) medium (peptone, 20 g; glycerol, 15 g;
K2HPO4, 1.5 g; MgSO4 7H2O, 1.5 g; distilled water, 1000 mL;
pH 7.2). The cells were harvested and vortexed for making the
homogeneous suspension in sterile distilled water.
Evaluation of the Association Constant for [L 2β-CD] Forma-
3
tion. A 21.15 μM solution of L in an acetonitrile-HEPES buffer
(pH 7.2, 2:3 (v/v)) was prepared and stored under dark condi-
tions. These solutions were used for all spectroscopic studies
after appropriate dilution. A 3.64 mM solution of β-CD was
prepared using the same solvent composition. A solution of L
was further diluted for spectroscopic titrations, and the effective
final concentration was adjusted to 4.23 μM, while the effective
[β-CD] was varied from 0 to 2.91 mM.
3
The cultured cells were first exposed to different concentra-
tions of Hg2þ (6 ppb to 3.75 ppm) in a ethanol/HEPES buffer (2:3,
v/v; pH 7.2) for 30 min at 25 °C. Then, free Hg2þ that was present
in the solution was removed through centrifugation. This process
was repeated after adding ∼5 mL of a ethanol-HEPES buffer
(pH 7.2, 2:3 (v/v)) to remove traces of Hg2þ ions that may still be
present on the microorganism surfaces in order to avoid the back-
ground color while recording optical microscope images. The cen-
trifuged bacterial cells were finally exposed to L (12 μM) in a 2:3
ethanol/HEPES buffer (v/v; pH 7.2). The treated bacterial cells
were monitored through an optical microscope (Carl Zeiss;
40 X), and microscopic images of bacterial cells in the absence
(control) and presence of Hg2þ were observed for comparison.
Evaluation of the Association Constant for Hg2þ [L 2β-CD].
3
3
A 34.5 μM solution of L in an acetonitrile-HEPES buffer (pH
7.2, 1:9 (v/v)) was prepared in the presence of 83 μM β-CD and
stored under dark conditions. This solution was used for all spectro-
scopic studies after appropriate dilution. A 6.03 mM solution of
Hg2þ was prepared using the same solvent acetonitrile-HEPES
buffer (pH 7.2, 1:9 (v/v)) composition. A solution of L 2β-CD
3
was further diluted for spectroscopic titrations. The effective final
concentration was adjusted to 6.9 μM, and the [Hg2þ] concentra-
tion was varied from 0 to 2.41 mM.
For checking the suitability of L 2β-CD as the staining agent,
similar experiments were repeated; L 2β-CD was used instead
of L. The centrifuged bacterial cells were finally exposed to
3
3
Calculations for the Binding Constants Using Spectrophoto-
metric Titration Data. The following equation was used for the
nonlinear least-squares analysis to determine the association
constants as well as the stoichiometries for the formation of the
complexes L Hg2þ and Hg2þ L 2β-CD:15
L 2β-CD (11.5 μM L in the presence of 10 molar equivalent of β-
3
CD) in an ethanol-HEPES buffer (pH 7.2, 1:9 (v/v)) and were
monitored through an optical microscope (Carl Zeiss; 40 X),
and microscopic images of bacterial cells in the absence and
presence of Hg2þ were observed for comparison (Figures S5 and
S6 in the Supporting Information).
Live cultures of Pseudomonas fluorescens and Pseudomonas
putida were used for our studies. Pseudomonas putida showed
better tolerance to high concentrations than Pseudomonas fluor-
escens. Earlier report revealed that the Pseudomonas putida
3
3
3
n
n
A ¼ ðA0 þ AlimKnCM Þ=ð1 þ KnCM
Þ
ð1Þ
where A0, A, and Alim are the absorbances of free L, L present
in the form of L Hg2þ in the complex, and L in presence of
3
excess amounts of Hg2þ ions where the absorbance reaches a
(15) Valeur, B.; Pouget, J.; Bouson, J. J. Phys. Chem. 1992, 96, 6545.
(16) Nigam, S.; Durocher, G. J. Phys. Chem. 1996, 100, 7135.

11488 Inorganic Chemistry, Vol. 49, No. 24, 2010
Mahato et al.
strain could tolerate heavy metals up to a concentration of
3 mM/L.17
To check the nontoxic nature of the reagents L towards
Pseudomonas putida, cultures (500 μL) of P. putida were incu-
bated in the absence (control) and presence of 5.0 mg of the
respective dye for 2 h in an incubator at 34 °C. The control and
the other culture treated with dye (L) were separately poured on
three different King’s B agar plates, and then these two plates
were incubated at 34 °C for 24 h. The growth of Pseudomonas
putida was observed on both agar plates to examine whether L
had any toxic effect towards the viability of this microbe (Figure
S7 in the Supporting Information).
Results and Discussion
Synthesis of the new sensor molecule (L, Scheme 1A) was
achieved by reacting 1,4,8,11-tetraazacyclotetradecane (cyclam)
and 4-(4-dimethylamino-phenylazo)-benzenesulphonyl chlor-
ide in an appropriate molar ratio. After necessary purification
by column chromatography, L was achieved in pure form,
which was confirmed by various analytical data. L1 was isolated
in pure form after recrystallization of the crude product that was
obtained through the reaction of the sulphonyl chloride frag-
ment with excess pyrrolidine (Scheme 1B).
Figure 1. (A) Absorbance spectra of L (2.3 μM) with varying [Hg2þ
(0-13.3 mM) in CH3CN-aq. HEPES buffer (0.01 M, pH 7.2; 2:3, v/v).
Inset: Least square plot of Δ[A - A0] vs [Hg2þ]. (B) Change in color of L
(5 μM) in a CH3CN-aq. HEPES buffer (0.01 M, pH 7.2; 2:3, v/v) in the
presence of various metal ions (2.5 mM).
]
center as the most preferred conformation in solution.18,19 Thus,
it would not be unreasonable to propose an R,S,R,S config-
uration at the N-stereocenter for L, when bound to Hg2þ in
solution (Scheme 2). This conformation further favors the
enhancement of the dipolar vector for L on coordination to
Hg2þ (Scheme 2) and is also expected to contribute to the
observed red shift in the absorption maxima on coordina-
tion to the Hg2þ ion. A more significant red shift in this ICT
UV-vis spectra for L (CH3CN-aq. HEPES buffer, 2:3
(v/v), pH 7.2) show an intraligand π-π* charge transfer band
with λmax at 469 nm (Figure 1A), which predominantly
involves πNMe [HOMO]- and π[NdN]*[LUMO]-based frontier
2
orbitals. Analogous absorption band maxima for L1 appeared
at 452 nm in a CH3CN-aq. HEPES buffer medium (2:3 (v/v),
pH 7.2). A greater electron withdrawing ability of the sub-
stituted azamacrocyclic moiety in L than that of the pyrrolidine
moiety in L1 is expected to inflict a narrower HOMO-LUMO
gap for L, which can be accounted for by its observed red-
shifted absorption maxima for L (Figure S8 in the Supporting
Information).
band maximum for Hg2þ L (474 to 491 nm), as compared to
3
L (441 to 450 nm), on increasing the solvent polarity from
THF to CH3CN further supports this configuration (Figure
S10 in the Supporting Information). Spectrophotometric
and ESI-MS studies revealed that the binding of Hg2þ to
L followed a 1:1 stoichiometry (Figure S11 in the Supporting
Information).
Electronic spectra for L1 (2.45 ꢀ 10-5 M) were also checked
in the absence and presence of a large excess of Hg2þ (0.02 M)
in a CH3CN-aq. HEPES (2:3, v/v) buffer solution (pH 7.2;
Figure S12 in the Supporting Information). The absorption
maximum for L1 was found to be 452 nm, and an insignificant
shift of 4 nm (λmax = 456 nm) was observed in the presence of
a large excess of Hg2þ (Figure S12 in Supporting Informa-
tion). This tends to nullify the possibility of the binding of
Hg2þ either to the -NMe2 or to the azo- functionality of L1.
Thus, this observation confirms that the observed red shift
of absorption maxima for L in the presence of Hg2þ is due
to the coordination of Hg2þ to the N atoms that belong to
the azamacrocycle moiety. The noncoordinating or at best
weakly coordinating nature of L1 to the Hg2þ ion was also
evident in the 1H and 13C NMR spectra recorded in the ab-
sence and presence of excess Hg2þ ions (vide infra).
This binding ability of L towards various metal ions (all
alkali, alkaline earth, all common transition metal, and lantha-
nide ions) was checked by recording the electronic spectra of L
in the absence and presence of respective metal ions. No change
in spectra for L could be observed in the presence of any one
of these cations, except Hg2þ (Figure S9 in the Supporting
Information). Electronic spectra of L in a CH3CN-aq.
HEPES (2:3, v/v) buffer solution (pH 7.2) with varying
[Hg2þ] are shown in Figure 1A. A new spectral band with
maxima at 494 nm developed (redshift of∼25 nm) along with
the appearance of an isosbestic point at 466 nm. This suggests
that the new complex Hg2þ L was formed and existed in
3
equilibrium with L. Simultaneously, a color change of the
solution from dark yellow to pink-red could be visually
detected, while no such color change could be observed for
all of the other metal ions added (Figure 1B). The observed
red shift could be attributed to the more favored ICT process
The possibility of any involvement of the sulphonyl (SdO)
functionality in L for a probable Hg2þ-OdS sulphonyl-type
coordination could be nullified on the basis of the FTIR
studies. The FTIR spectrum for L shows bands at 1364 and
1134 cm-1, which are characteristic for asymmetric and sym-
metric stretching frequencies, respectively, for the sulphonyl
group. These band positions remained practically unchanged
and appeared at 1365 and 1133 cm-1 even in the presence of
33 molar equivalents of Hg2þ (Figure S13 in the Supporting
Information). Thus, one would rule out any possibility of the
for Hg2þ L as compared to that for L.
3
Previous experimental and theoretical reports on Hg2þ
-
cyclam0 (cyclam0 is 1,8-dimethyl-4,11-tetraazacyclotetradecane)
complexes suggest an R,S,R,S configuration at the N-stereo-
(17) Husseina, H.; Faraga, S; Kandilb, K.; Moawadc, H. Proc. Biochem.
2005, 40, 955.
(18) (a) Enoki, O.; Imaoka, T.; Yamamoto, K. Org. Lett. 2003, 5, 2547.
(b) Liang, X.; Sadler, P. J. Chem. Soc. Rev. 2004, 33, 246. (c) Hunter, T. M.;
McNae, I. W.; Simpson, D. P.; Smith, A. M.; Moggach, S.; White, F.; Walkinshaw,
M. D.; Parsons, S.; Sadler, P. J. Chem.;Eur. J. 2007, 13, 40.
(19) Hrobarik, P.; Kaupp, M.; Riedel, S. Angew. Chem., Int. Ed. 2008, 47,
8631.
involvement of a sulphonyl moiety in coordination to Hg2þ
,
as for such a situation an appreciable shift in IR frequency for
the sulphonyl functionality would be expected.

Article
Inorganic Chemistry, Vol. 49, No. 24, 2010 11489
Scheme 2. Schematic Presentation of L, Hg2þ L, L 2β-CD, Hg2þ [L 2β-CD]
3
3
3
3
The association constant for the complex (Hg2þ L) for-
mation was evaluated from systematic titration using the
nonlinear least-squares method and was found to be (4.91 (
H17, H20, H21) on the successive addition of Hg2þ, and
eventually upfield shifts of about 18 to 20 Hz were observed
on complete binding to Hg2þ (97 molar equivalent). How-
ever, these upfield shifts were more significant for aliphatic
H atoms (Figures S18 and S19 in the Supporting In-
formation). Such upfield shifts upon metal ion binding
are not very uncommon in the literature.8a,20 A shift of ∼28
Hz was observed for H7 and H14 hydrogen atoms on the
binding of L to Hg2þ. Signals for H5 and H12 became very
broad on coordination to Hg2þ; however, the shift was>36
Hz. The shift for H6 and H13 atoms was also ∼36 Hz, while
that for H23 was 25.8 Hz, which was comparable to the shift
observed for other aromatic H atoms in L. The relative shift
data in 1H NMR spectra tend to nullify the possibility of
coordination through the -NMe2 of L and, thus, it was not
considered. All of these tend to indicate that L was bound
to Hg2þ through four nitrogens of the tetrazamacrocyclic
component, and this accounted for the higher upfield shift
for the aliphatic H atoms of the macrocyclic ring. To ascer-
3
0.08) ꢀ 103 M-1 15
.
A relatively lower association constant
value (Kassoc) obtained for Hg2þ L, compared to the classical
3
cyclam-Hg2þ complex, may be ascribed to the attachment
of two electron-withdrawing sulphonyl moieties to the two N
atoms of the cyclam unit.14c The presumably soft nature of
two out of four N atoms of L accounts for the unusual
specificity towards Hg2þ. The lowest detection limit for Hg2þ
was found to be 28 ppb under these experimental conditions
(Figures S14 and S15 in the Supporting Information). On
adding an CH3CN-HEPES (2:3, v/v; pH = 7.2) buffer
solution of Na2EDTA (15.5 mM) to Hg2þ L (3.8 mM,
3
CH3CN-aq. HEPES buffer, (2:3, v/v; pH 7.2), the absorp-
tion band with λmax at 490 nm (characteristic for Hg2þ L)
3
disappeared, and spectra for L with a λmax of 469 nm were
restored (Figure S16 in the Supporting Information) with a
change in color from pink-red to pale yellow. This confirmed
the reversible binding nature of L towards Hg2þ. Further,
the specificity towards the Hg2þ ion was established, as no
1
tain this, we recorded the H NMR spectra for L1 in the
absence and presence of Hg2þ (Figure S20 in the Support-
ing Information). The shift of signals for Hh, Hd, and Hg of
L1 on the addition of excess (100 molar equivalents) Hg2þ
was ∼0.2 Hz, a value within the limit of experimental
error, while that for Hc was 1.6 Hz. This shift in the Δδ
value for Hj is 0.4 Hz. Considering these minor shifts for
L1, as compared to the shifts observed for L on binding
to Hg2þ, led us to confirm that neither the aromatic part
change in absorbance at 494 nm for Hg2þ L was observed
3
(Figure S17 in the Supporting Information) in the presence of
a 10 molar excess of each of the metal ions studied (Naþ, Kþ,
Csþ, Ca2þ, Mg2þ, Ba2þ, Sr2þ, Pb2þ, Cr3þ, Fe3þ, Cu2þ, Cd2þ
Co2þ, Zn2þ, Ni2þ, Pr3þ, Eu3þ, Er3þ, Nd3þ, Dy3þ, Yb3þ
,
,
Ce3þ, Ho3þ, Lu3þ, Tm3þ, Tb3þ, La3þ, Sm3þ, and Gd3þ) or in
the presence of a mixture of all of these metal ions, while each
of these metal ions were present in a 1.2 molar excess relative
tothat of [Hg2þ]. Thus, experimental studies revealed that the
reagent L could be used for specific recognition of Hg2þ inthe
presence of a wide range of metal ions that belong to groups I
and II, common transition metal ions, and all lanthanide ions
in a mixed aqueous-acetonitrile medium.
nor the NNMe part of L or L1 was involved in coordination
2
to Hg2þ
.
13C NMR Spectra. 13C NMR spectra for L and L1 were
recorded in absence and presence of Hg2þ and are shown
in Supporting Information (Figures S21, S22 and S23 in
Supporting Information). Upfiled shifts in the range of
225-625 Hz were observed for aliphatic C-atoms of the
tetraazamacrocyclic ring in L; while those for different
aromatic C-atoms lies within the range of 25 - 85 Hz.
Similar studies with L1 revealed shifts of 0.5-8.5 Hz.
1H NMR Studies. 1H NMR spectra for L and L1 were
recorded in CDCl3 and CD3OD, respectively; assignments
of the individual H atoms of these compounds are provided
in the Experimental Section. 1H NMR spectra for L and L1
were also recorded in the presence of varying [Hg2þ] in
DMSO-d6 and DMF-d7 solvents, respectively. Gradual
upfield shifts were observed for all aromatic H atoms (H16,
(20) Shiraishi, Y.; Maehara, H.; Ishizumi, K.; Hirai, T. Org. Lett. 2007, 9,
3125.

11490 Inorganic Chemistry, Vol. 49, No. 24, 2010
Mahato et al.
Figure 2. (A) Partial 1H NMR spectra of L (8 mM) with varying [β-CD] (0-15.2 mM; in CD3CN-D2O, 1:1, v/v). (B) Partial 1H NMR spectra of
L 2β-CD for δ = 3.45-5 ppm, a region where L does not have any resonance signal.
3
This comparison clearly shows that even if L1 had any
interaction with Hg2þ, the extent of interaction was un-
important as compared to that between L and Hg2þ
.
Thus, any possibility of the observed binding of Hg2þ to L
through the aromatic moiety can be ignored on the basis
of the results of the 1H and 13C NMR spectral studies.
Inclusion Complex Formation with β-CD. The limited
solubility of the ligand L in water did not allow us to
check the recognition process in either a pure aqueous
solution or a pure HEPES buffer medium. Derivatives of
4,40-bipyridine, like (E)-1,2-di(pyridin-4-yl) diazene, ana-
logous to L are known to form stable [3]pseudorotaxane-
type inclusion complexes with β-cyclodextrin (β-CD),
and the formation of such a complex is expected to
enhance the solubility of these bipyridyl derivatives in
water.21 We studied the formation of such an inclusion
Figure 3. Absorbance spectra of L (4.23 μM) with varying [β-CD]
(0-0.247 mM) in a CH3CN-aq. HEPES buffer (0.01 M, pH 7.2; 2:3,
v/v). Inset: plot of change in absorbance (A - A0) of L against varied
concentrations of β-CD.
complex (L 2β-CD, Scheme 2) by 1H NMR and electronic
3
tion). A mass spectral signal (m/z) at 3044.9 was observed,
and this corresponds well with the 1:2 adduct formation, i.e,
a [3]pseudorotaxane formation (Scheme 2). The binding
process of β-CD and the azobenzene guest is expected to be
driven by favorable enthalpy changes, which could attribute
to the significant contribution of hydrophobicity, hydrogen
spectroscopy (Figures 2A, B and 3). A gradual upfield shift
for all aromatic hydrogen atoms (H16-H21) of L (8 mM)
was observed on the addition of increasing amounts of
β-CD, and these shifts were more prominent for H17 and
H20 (∼ 11 Hz, Figure 2A). For the same study, changes in
the positions for aliphatic hydrogen atoms of the tetraaza
macrocyclic component were almost negligible (∼ 4 Hz).
Upfield shifts of ∼15 Hz were also observed for the
hydrogens (H1-H6) of the β-CD moiety (Figure 2B).
Similar upfield shifts were also reported for an analogous
[3]pseudorotaxane formation with 4,40-bipyridine deriva-
tives and β-CD.21,22 The presence of the deshielding effect
due to diamagnetic anisotropy of the benzenoid moiety
of the included azo compound could account for such up-
field shifts. Thus, the 1H NMR study tends to confirm the
inclusion complex formation, while the 1:2 binding stoichi-
ometry was confirmed by ESI-MS spectra recorded for L
(8.7μMinCH3CN/H2O of 1:1, v/v) in the presence of excess
β-CD (1.14 mM; Figure S24 in the Supporting Informa-
bonds, and van der Waals interactions, and C-H
π
3 3 3
interactions between host and guest molecules. Inclusion
complex formation between L and β-CD was also studied
spectrophotometrically. A little red shift of 3 nm was ob-
served in the electronic spectra for L on the addition of
excess β-CD (Figure 3). A very close examination revealed
the appearance of two consecutive isosbestic points at 387
and 544 nm, with an increase in [β-CD]. This tends to
suggest the presence of two different equilibrium processes.
On the basis of the previous reports,21,22 it may be argued
that various noncovalent interactions,23 which account for
the stability of the [3]pseudorotaxane (L 2β-CD), could
3
lower the energy gap between the frontier orbitals and were
reflected in the little red-shifted absorption band. Further,
the apolar interior of β-CD requires less reorganization to
(21) (a) Wylie, R. S.; Macartney, D. H. Inorg. Chem. 1993, 32, 1830.
(b) Baer, A. J.; Macartney, D. H. Inorg. Chem. 2000, 39, 1410. (c) Wenz, G.
Angew. Chem., Int. Ed. 1994, 33, 803. (d) Sanchez, A. M.; de Rossi, R. H.
J. Org. Chem. 1996, 61, 3446. (e) Liu, Y.; Zhao, Y.-L.; Chen, Y.; Guo, D.-S. Org.
Biomol. Chem. 2005, 3, 584. (f) Zhang, X.; Gramlich, G.; Wang, X.; Nau, W. M.
J. Am. Chem. Soc. 2002, 124, 254. (g) Macartney, D. H.; Waddling, C. A. Inorg.
Chem. 1994, 33, 5912. (h) Smith, A. C.; Macartney, D. H. J. Org. Chem. 1998,
63, 9243. (i) Abou-Hamdan, A; Bugnon, P.; Saudan, C.; Lye, P. G.; Merbach,
A. E. J. Am. Chem. Soc. 2000, 122, 592. (j) Yamamoto, Y.; Kanda, Y.; Inoue, Y.;
Chujo, R.; Kobayashi, S. Chem. Lett. 1988, 495. (k) Steefkerk, D. G.; De Bie,
M. J. A.; Vliegenthart, J. F. G. Tetrahedron 1973, 29, 833.
accompany the electronic transition(s) in L 2β-CD, than in
3
the solvent surroundings for L.24 Although small, this may
also contribute to lowering the energy of activation for
optical electron transfer. Interestingly, least-squares analy-
sis16 of the spectrophotometric titration data revealed two
equilibrium processes with binding constants of (1.1 (
(23) (a) Connors, K. A. Chem. Rev. 1997, 97, 1325 and references therein.
(b) Nepogodiev, S. A.; Stoddart, J. F. Chem. Rev. 1998, 98, 1959. (c) Siddarth, P.;
Marcus, R. A. J. Phys. Chem. 1990, 94, 2985.
(22) (a) Shukla, A. D.; Bajaj, H. C.; Das, A. Angew. Chem., Int. Ed. 2001,
40, 446. (b) Shukla, A. D.; Bajaj, H. C.; Das, A. Proc. Ind. Acad. Sci., Chem. Sci.
2002, 114, 431.
(24) Robinson, E. A.; Earley, J. E. Inorg. Chem. 1999, 38, 4128.

Article
Inorganic Chemistry, Vol. 49, No. 24, 2010 11491
Scheme 3. Schematic Representation of (A) Pyranose Structure and (B)
Gauche Conformation of β-CD with θ as the Dihedral Angle Involving
Two Vicinal Hydrogen Atoms, H1 and H2, of the Pyranose Ring
0.02) ꢀ 104 (K1) and 205 (5(K2) M-1. Two different equili-
brium constants could account for the stepwise formation
of L β-CD and L 2β-CD. However, it is not possible to
Figure 4. Spectrophotometric titration of L 2β-CD ([L], 6.9 μM; [β-
3
CD], 16.6 μM) with varying [Hg2þ] (0-2.41 mM) in a CH3CN-aq.
HEPES buffer (0.01 M, pH 7.2 ;1:9, v/v). Inset: plot of Δ(A - A0) vs
[Hg2þ].
3
3
completely rule out the possibility of two-step inclusion
reactions of β-CD and azobenzene that involve a hasty
inclusion in the first step and slower solvational and/or
conformational changes after inclusion in the second
step.21i A similar explanation was offered for the observed
two-step inclusion process involving a related sulphona-
mide derivative of the azobenzene compound and R-CD
conformational change may have some influence on the
observed spectral changes and contribute to the observed
second equilibrium; however, it is difficult to visualize
and predict the extent of the effect of this conformational
change on the overall changes in electronic spectra.
The affinity of L 2β-CD towards Hg2þ was examined. It
1
3
on the basis of detailed H NMR and variable pressure
was observed that in the presence of β-CD, L was found to
be soluble in a mixed solvent medium with an even higher
proportion (1:9, v/v) of water in a CH3CN-water/aq.
HEPES buffer medium. Systematic changes in the spec-
kinetic studies.21i,25 In the present situation, after initial
inclusion of L in the β-CD cavity, a possible polar inter-
action between the -SO2- moiety of the sulphonamide
fragment and -CH2OH functionality of the β-CD larger
rim could induce conformational changes of the host
cavity and thus a new equilibrium process withsome minor
spectral shifts.
tral pattern for L 2β-CD were recorded with increasing
3
amounts of added [Hg2þ] in a CH3CN/aq. HEPES buffer
medium (1:9, v/v; Figure 4), and the absorption max-
imum for L 2β-CD at 380 nm was found to decrease with
3
Further support for the conformational change(s) of
a concomitant increase in absorbance at 420 and 502 nm.
Appearances of two simultaneous isosbestic points at 394
and 576 nm revealed the presence of only two interacting
components and an equilibrium, which are involved in
this binding process. The binding stoichiometry and
association constant for the formation of the complex,
Hg2þ [L 2β-CD], was evaluated using the nonlinear least-
1
the host β-CD cavity was available in the detailed H
1
NMR studies. H NMR spectra of β-CD (3.5 mM) and
β-CD in the presence of L (7.0 mM) were recorded in
CD3CN-D2O (1:1, v/v). A critical examination, a compar-
ison of these two 1H NMR spectra, and an evaluation of the
coupling constants for the interaction between vicinal H1
and H2 hydrogenofthepyranosestructuresuggested adefi-
3
3
squares method and found to be 1:1 and 3.52 ꢀ103 (35 M-1
,
3
nite change in the coupling constant, J12, for the β-CD
respectively.15 A little lower binding affinity of L 2β-CD, as
3
moiety (Scheme 3). This change in coupling constant value
(Δ3J12) can be used to calculate the change in dihedral
angle (θ) involving two vicinal H1 and H2 atoms of the
pyranose ring of the host β-CD moiety on forming an
inclusion complex with L. The modified Karplus equa-
tion was used for the calculation of θ is shown and this can
be expressed by the following equation:21k,25
compared to L, towards Hg2þ under identical experimental
conditions could account for the higher solvation and thereby
the slower diffusion of L 2β-CD compared to L. The lowest
3
detection limit for Hg2þ was found to be 34.4 ppb under these
experimental conditions (Figure S25 in the Supporting
Information). The more intense absorption band tail in
the longer wavelength region was attributed to an even
more prominent color change for Hg2þ [L 2β-CD] than
3J 12
¼ ð6:6 - 1:0 cos θ þ 5:6 cos 2θÞ
3
3
that for Hg2þ L (Figure S26 in the Supporting Information).
3
ꢀ f1 - Σ4i ¼ 1ðfiΔXiÞg
ð4Þ
This prompted us to explore the possibility of using these as
colorimetric reagents for the detection of the uptake of Hg2þ
by microbes. Pseudomonas putida is known to be resistant
towards Hg2þ and to adsorb Hg2þ at the cell surface and cell
membrane.26 Thus, we have used cells of Pseudomonas putida
to check for such a possibility. We have used pre-exposed
(0-45 min at 25 °C) bacterial cell (Pseudomonas putida,
Figure 5; Figures S5 and S6 in the Supporting Information)
suspensions of the Hg2þ ion ([Hg2þ] was 6 ppb) in an
aqueous medium for studies.
where ΔX=X - XH represents the difference in electro-
negativity between a substituent and hydrogen (the electro-
negativities used are XH = 2.1, XOr, XOCH = 3.3, and
3
X-C-O-=2.5, and the electronegativity factor fi=0.1).21k
When these electronegativity values and J12 values are
3
used before and after the inclusion of L in the β-CD cavity,
Δθ = 1 was evaluated (Table S1 in the Supporting Infor-
mation). The Δθ = 1 value for the present study is less than
what was reported for the formation of an inclusion com-
plex between an analogous sulphonamide derivative of a
diphenyl azo fragment and R-CD,21i which is understand-
able considering the larger cavity diameter of β-CD. This
Each culture was exposed separately to a 12 μM solu-
tion of L and L 2β-CD (11.5 μM) and was viewed through
3
(26) (a) Clark, D. L.; Weiss, A. A.; Silverj, S. J. Bacteriol. 1977, 132, 186.
(b) Rezaee, A.; Derayat, J.; Mortazavi, S. B.; Yamini, Y.; Jafarzadeh, M. T. Am. J.
Environ. Sci. 2005, 1, 102. (c) Nascimento, A. M. A.; Chartone-Souza, E. Genet.
Mol. Res. 2003, 2, 92.
(25) Durette, L.; Horton, D. Org. Magn. Res. 1971, 3, 417.

11492 Inorganic Chemistry, Vol. 49, No. 24, 2010
Mahato et al.
Figure 5. Lightmicroscopeimages of(A) controlcellsofPseudomonasputida, (B) cellsexposedtoHg2þ (6ppb), (C) cellsexposedtoanaqueoussolutionof
Hg2þ (6 ppb) for 45 min and then to L (12 μM, ethanol-aq. HEPES buffer (0.01 M, pH 7.2; 2:3 (v/v)), and (D) cells exposed to an aq. solution of Hg2þ
(6 ppb) for 45 min andthentoL 2β-CD (11.5 μM L in the presenceof10molarequivalentsofβ-CD, ethanol-aq. HEPES buffer(0.01M, pH7.2) (1:9, v/v)).
3
An isolated experiment revealed that L was nontoxic
to the living bacterial cells used. Luxuriant growth was
observed for both the King’s B agar plates (incubated at
34 °C for 24 h) having a culture of Pseudomonas putida in
the absence and presence of the added dye (L) (Figure 6).
It establishes that the culture is viable with the ligand, and
the receptor molecule L does not show any harmful effect
on the growth of the bacteria.
Thus, we could demonstrate that L could be used for
the detection of Hg2þ adsorbed on the cell surface of
microorganisms such as Pseudomonas putida. The extent of
staining and visual detection by optical microscopy could be
further improved upon by using its [3]pseudorotaxane form
Figure 6. Culture of Pseudomonas putida on a King’s B agar plate (a) in
the absence and (b) in the presence of L after incubation at 34 °C for 24 h.
an optical microscope (Carl Zeiss; 40 X). Respective
images of the control and cells in the presence of Hg2þ
and other reagents are shown in Figure 5. This clearly
revealed that the staining of the cells (pre-exposed to
(L 2β-CD) in the presence of biologically benign reagents
3
like β-CD. Such an example of in vivo detection of Hg2þ, in a
lower organism, using a nontoxic colorimetric reagent is not
common in the literature.
6 ppb of Hg2þ ion) could be achieved by L or L 2β-CD,
3
while, staining was more prominent for L 2β-CD. It is
3
Acknowledgment. DST and CSIR have supported this
work. P.M., A.G., S.S., and S.K.M. acknowledge CSIR for
their fellowship. A.D. thanks Dr. P. K. Ghosh (Director),
CSMCRI, Bhavnagar for his keen interest in this work.
evident from our study that microorganisms should not
be overlooked when studying metal contaminants in the
soil. It is worth mentioning that a [Hg2þ] of 6 ppb is much
lower than the international norm for STLC regulatory
levels (0.2 ppm).27
Supporting Information Available: Characterization data of
L, L1, and L 2β-CD; spectral details; and biological studies.
(27) Soluble Threshold Limit Concentration (STLC). Chapter 11, Article
3, Identification and Listing of Hazardous Waste; California Code of
Regulations, Title 22, 2005.
3
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