A novel optically based chemosensor for the detection of blood Na+

Article abstract of DOI:10.1016/S0040-4039(01)00823-1

The azo dye based chemosensor 2 displays extremely good sensitivity and selectivity for Na⁺ over other physiologically important alkali and alkali earth metal ions in the 0.3-1.0×10^-4 M concentration range as well as being pH independent above pH 3.9. This sensitivity coincides with the Na⁺ concentration range found in blood. The sensor exhibits almost 110 nm hypsochromic shift in the absorption spectrum upon Na⁺ detection, with a strong colour change from red to yellow in aqueous solution which is clearly visible to the naked eye even at low concentrations.

Full text of DOI:10.1016/S0040-4039(01)00823-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 4725–4728
A novel optically based chemosensor for the detection
+
of blood Na
a,
b
a
a
Thorfinnur Gunnlaugsson, * Mark Nieuwenhuyzen, Ludovic Richard and Vera Thoss
a
Department of Chemistry, Trinity College Dublin, Dublin 2, Ireland
b
School of Chemistry, Queen’s University of Belfast, BT9 5AG, Northern Ireland, UK
Received 27 February 2001; revised 10 May 2001; accepted 15 May 2001
+
Abstract—The azo dye based chemosensor 2 displays extremely good sensitivity and selectivity for Na over other physiologically
−
4
important alkali and alkali earth metal ions in the 0.3–1.0×10 M concentration range as well as being pH independent above
+
pH 3.9. This sensitivity coincides with the Na concentration range found in blood. The sensor exhibits almost 110 nm
+
hypsochromic shift in the absorption spectrum upon Na detection, with a strong colour change from red to yellow in aqueous
solution which is clearly visible to the naked eye even at low concentrations. © 2001 Elsevier Science Ltd. All rights reserved.
The recognition and sensing of physiologically impor-
tant ions and molecules has been one of the most active
areas of research within the field of supramolecular
where the line-like time delayed emission of the lan-
thanide ion, emitting between 550–730 and 500–650
nm, respectively, was modulated upon ion recognition
1
3
chemistry. Systems responding to concentration varia-
in 100% water. We have also employed this detection
tions by displaying changes in their optical properties,
such as absorption, fluorescence, phosphorescence and
metal based luminescence upon analyte recognition, are
mode to demonstrate the function of molecular logic
4
gates. Concurrently we have been interested in the
+
detection of intra- and extracellular Na concentration
2
very attractive for in vivo applications. Such sensors
by employing such lanthanide based time-delayed
5
+
need to fulfil the criteria of high selectivity and high
sensitivity including good solubility in water or alterna-
tively being easily incorporated into hydrophobic mem-
branes, and they should absorb and/or emit at long
wavelength to overcome undesirable side effects such as
autofluorescence and light scattering in vivo.
detection. Our initial goal was to design a Na selec-
+
tive macrocyclic receptor for blood Na that would
+
+
exhibit good selectivity for Na over K as well as being
easily incorporated into the lanthanide emitting moie-
ties. With this in mind, we synthesised the pivot crown
ether 1, previously designed by Gokel et al. for Na
detection in MeOH. By simple modification, the recep-
+
6
We have recently synthesised several kinetically robust
tor can be easily incorporated into lanthanide-based
macrocyclic systems, or it could be functionalised to
+
2+
Eu(III) and Tb(III) chemosensors for H , O and Zn
2
Scheme 1. (i) Na HPO , KI, ClCH CO CH and CH CN; (ii) LiAlH , THF; (iii) NaH, THF, (TsOCH CH OCH ) ; (iv) a.
2
4
2
2
3
3
4
2
2
2 2
NO C H NH , NaNO , HCl, THF/H O (1:1), b. 1.
2
6
6
2
2
2
*
0
Corresponding author.
040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00823-1

4
726
T. Gunnlaugsson et al. / Tetrahedron Letters 42 (2001) 4725–4728
yield a chromogenic receptor/ionophore that would
7
absorb and emit at long wavelengths. In both cases
interference from the physiological system would be
overcome. In this letter we describe the synthesis and
incorporation of this receptor into an azo dye, yielding
2
, that displays both large changes in the absorption
+
spectra upon Na recognition, while discriminating
+
2+
2+
against K , Ca and Mg .
The synthesis of 1 was achieved in good yield by
6
modification of the procedure by Gokel et. al., Scheme
1
. The sensor 2 was obtained in 52% yield in a one-pot
+
The Na sensing of 1 and 2 was measured at pH 7.5 in
reaction by first carrying out diazoniation on p-nitroani-
Tris buffered (0.01 M) 50:50 MeOH:H O solution, and
in the presence of 0.1 M tetramethyl ammonium chloride
2
line in aqueous THF solution, followed by the addition
8
of 1. The resulting red residue was purified by flash-
to maintain constant ionic strength. The pK for 1 and
a
column chromatography on neutral alumina (30:70
EtOAc:hexane) giving 2 as a red powder. Interestingly,
when other diazonium salts such as p-chloro, p-sulfonic
or p-carboxylic acids were used, no coupling was
observed. All the products and intermediates were
2
was also determined by absorption titration. The
absorption spectra of 1 showed two bands at 245 and 285
nm in alkaline solution, which reduced in intensity upon
addition of acid. A similar effect was observed using
sodium acetate, with ca. 15% reduction in the absorption
1
13
analysed by H and C NMR, ESMS, accurate mass
intensities. From these changes a pK of 5.8 (±0.05) and
a
1
determination and IR. The H NMR of 2 showed five
a log iNa of 2.4 (±0.05) was determined, which is in good
aromatic signals resonating between 8.3 and 7.0 ppm, a
singlet for the methoxy group at 3.91, a multiplet and a
triplet at 3.68 and 3.79 ppm, respectively, corresponding
to the crown ether protons. In the ESMS only two major
peaks were observed at 475.5 and 497.5 corresponding
6
agreement with that observed by Gokel et al. in MeOH.
The changes in the absorption spectra of 2 were more
dramatic as shown in Fig. 1a. The compound displayed
a broad absorption band at 487 and 285 nm in the
absence of Na , whereas upon addition of Na , the 487
nm band hypsochromically shifted to 380 nm (Duꢀ110
nm) with the formation of an isosbestic point at 410 nm.
These changes were also clear to the naked eye where the
deep-red colour solution of 2 changed to a yellow after
+
+
+
to the molecular ion and the Na complex of 2. We were
able to obtain suitable crystals of 2 for crystallographic
evaluation by slow evaporation from ethanol. The X-ray
crystal structure of 2 is shown below, showing the
compound in its trans conformation, with a dihedral
angle of 23.3° for the N11ꢀN10ꢀC7ꢀC8 and −163.3° for
the C6ꢀC7ꢀN10ꢀN11 atoms. The dihedral angle for
C18ꢀC16ꢀC15ꢀN20 was measured to be −4.5°, whereas
the C21ꢀN20ꢀC15ꢀC16 and C34ꢀN20ꢀC15ꢀC14 angles
+
+
−4
addition of Na ([Na ]=1.0×10 to 0.3 M). These
changes are shown in Fig. 1b, where the Na titration
profile is sigmoidal over 2 pM units, from pK =0.5–2.5,
+
a
consistent with a simple ion-binding equilibrium. A
similar effect was seen for the pH titration of 2, with the
exception that the 487 nm band completely disappeared
upon addition of acid to an alkaline solution of 2. From
9–11
were measured to be −44.0 and −12.7°, respectively.
These values indicated that the nitrogen lone pair is not
fully conjugated with the aromatic p-system due to the
steric affect of the o-methoxy group, but the group
should provide an extra chelation site upon complexation
these changes a log iNa of 1.25 (±0.05) and pK of 3.9
a
+
(±0.05) was determined. This Na sensitivity conve-
+
+
to Na . We are presently undertaking further studies of
niently overlaps with the Na concentration range found
the solid-state properties of 2 in the presence of group
I and II ions.
in blood, which has an upper limit of 145 mM (extracel-
lular concentration ranges from 120–450 mM in animal
Figure 1. (a) The absorption spectra of 2 upon addition of sodium acetate [Na]=0“0.5 M; (b) absorption versus pM (−log[M],
+
+
+
2+
2+
M=Li (ꢀ), Na (ꢁ), K (ꢂ) Ca (ꢃ) Mg (×)) profile.

T. Gunnlaugsson et al. / Tetrahedron Letters 42 (2001) 4725–4728
4727
cells), to a background of ca. 4.8–3.5 mM and 2.6–2.2
VCH: Weinheim; Germany, 1998; (b) Chemosensors of
Ion and Molecular Recognition; Desvergne, J. P.; Czarnik,
A. W., Eds.; Kluwer Academic Publishers: Dordrecht,
1997; (c) Fluorescent Chemosensors for Ion and Molecular
Recognition; Czarnik, A. W., Ed.; ACS Books: Washing-
ton, 1993; (d) Hartley, J. H; James, T. D.; Ward, C. J. J.
Chem. Soc., Perkin Trans. 1 2000, 3155; (e) Czarnik, A.
W. Acc. Chem. Res. 1994, 27, 302.
+
2+
12
mM of K and Ca , respectively (in blood). Further-
more, the sensor is pH independent at physiological pH
as required for blood analysis. However, we do recognise
+
that Na determination in 100% water would yield lower
binding values that call upon some modification of 2 for
such practical application. The spectral changes and the
binding values are substantially different from those seen
for 1, because in 2 the nitrogen lone pair of the anisole
moiety is engaged in a charge push–pull mechanism with
the nitro group. This gives rise to internal charge transfer
excited state (ICT), which gives 2 its intense red colour
2. (a) Parker, D. Coord. Chem. Rev. 2000, 205, 109; (b) de
Silva, A. P.; Fox, B. D.; Huxley, A. J. M.; Moody, T. S.
Coord. Chem. Rev. 2000, 205, 41; (c) 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; (d) Fabbrizzi, L.; Licchelli, M.;
Pallavicini, P. Acc. Chem. Res. 1999, 32, 846; (f) Fab-
brizzi, L.; Poggi, A. Chem. Soc. Rev. 1995, 197.
+
+
prior to the Na complexation. Upon Na complexation,
+
the lone pair is engaged in Na binding reducing its
ability to participate in the ICT mechanism and causing
13
shifts in the absorption spectra to shorter wavelengths.
3
. (a) Gunnlaugsson, T.; Parker, D. Chem. Commun. 1998,
Furthermore, the strongly electron withdrawing nitro
substituents reduce the ability of the nitrogen lone pair
to interact with the ion, yielding weaker binding than that
seen for 1 and shifting the binding constant towards the
511; (b) Reany, O.; Gunnlaugsson, T.; Parker, D. Chem.
Commun. 2000, 473; (c) Reany, O.; Gunnlaugsson, T.;
Parker, D. J. Chem. Soc., Perkin Trans. 2 2000, 1819.
+
4. Gunnlaugsson, T.; MacD o´ naill, D. A.; Parker, D. Chem.
desired range for blood Na detection.
Commun. 2000, 93.
5
. (a) Tahri, A.; Cielen, E.; Van Aken, K. J.; Hoornaert, G.
J.; De Schryver, F. C.; Boens, N. J. Chem. Soc., Perkin
Trans. 2 1999, 1739; (b) Yoshida, K.; Mori, T.; Watan-
abe, S.; Kawai, H.; Nagamura, T. J. Chem. Soc., Perkin
Trans. 2 1999, 393.
The sensitivity of 2 was measured by carrying out
2+
+
+
2+
analogous experiments using Li , K , Ca and Mg
acetate salts. The results are shown in Fig. 1b as the
changes in absorption intensity versus pM, indicating the
+
high sensitivity of 2 for Na over the other cations. This
+
6. Schultz, R. A.; White, B. D.; Dishong, K. A.; Arnold,
high Na selectivity is possible to the size of the crown
W.; Gokel, G. J. Am. Chem. Soc. 1985, 107, 6659.
moiety and the steric effect enforced by the o-methoxy
7
. (a) Oguz, U.; Akkaya, E. U. Tetrahedron Lett. 1997, 38,
4509; (b) Sauve, G.; Kamat, P. V.; Thomas, K. G.;
Thomas, K. J.; Das, S.; George, M. V. J. Phys. Chem.
group, which is also directly involved in the binding of
+
14
the Na . The fluorescence emission spectrum of 2 was
+
also monitored upon addition of Na , showing 60%
1996, 100, 2117; (c) Mitewa, M.; Mateeva, N.; Antonov,
reduction in intensity after the addition of a 0.5 M
concentration of sodium acetate.
L.; Deligeorgiev, T. Dyes Pigments 1995, 27, 219; (d) Li,
H.; Yao, Z.; Liu, R.; Tan, G.; Yu, X. Chem. Lett. 1998,
1
205.
In conclusion, the iNa, the large colour change (red to
yellow) and its resistance to protonation at physiological
pH makes 2 an ideal candidate for the evaluation of
8
. (a) Dix, J. P.; V o¨ gtle, F. Chem. Ber. 1980, 113, 457; (b)
Lohr, H. G.; V o¨ gtle, F. Acc. Chem. Res. 1985, 18, 65; (c)
Dix, J. P.; V o¨ gtle, F. Angew. Chem., Int. Ed. Engl. 1978,
17, 857.
+
blood Na and serum, especially because it displays such
+
a high selectivity for Na , while discriminating against
9
. X-Ray crystallographic data for C H N O (2) were
23
30
4
7
any other physiologically active group I and II cations.
To the best of our knowledge, there are no such
chromogenic chemosensors in the literature. We are in
the process of evaluating these properties by incorporat-
ing 2 into optodes and membranes for online and
real-time monitoring in vivo.
collected using a Bruker SMART diffractometer with
graphite-monochromated Mo-Ka radiation. The crystal
stability was monitored and there was no significant
decay (±1%). Cell parameters were obtained from 215
accurately centred reflections. Data were collected at ca.
1
20 K in a dinitrogen stream. Phi and omega scans were
employed for data collection and Lorentz and polarisa-
tion corrections were applied. The structure was solved
by direct methods and all non-hydrogen atoms were
refined with anisotropic atomic displacement parameters.
Hydrogen atom positions were added at idealised posi-
tions and a riding model was used in the subsequent
refinement (U =1.2U of the parent atom). The atomic
displacement parameters indicated that the oxygen atom
O30 of the crown moiety was disordered. This disorder
has been modelled over two sites and refined to 75(1)%
for the major component. The function minimised for
Acknowledgements
This work was supported by the Department of Chem-
istry, Trinity College, Dublin, Ecole Nationale
Sup e´ rieure de Chimie de Lille, Kinerton Ltd., and the
European Commission through the SOCRATES and
Interreg Programme. We thank Professor John M. Kelly
TCD) for his support, and Dr. Hazel M. Moncrieff for
valuable discussion.
eq
ij
(
2
2
−1
wR was S[w(ꢀF ꢀ −ꢀF ꢀ )] with reflection weights w =
2
o
c
2
2
2
2
2
[
| ꢀF ꢀ +(g P) +g P] where P=[maxꢀF ꢀ +2ꢀF ꢀ ]/3 for all
o
1
2
o
c
2
References
F and the function minimised for R was S[w(ꢀF ꢀ−ꢀF ꢀ)].
1
o
c
10 11
The SAINT and SHELXTL PC packages were used
for data collection, reduction, structure solution and
refinement. Additional material available from the Cam-
1
. (a) Chemical Sensors and Biosensors for Medical and
Biological Applications; U.S. Spichiger-Keller, Wiley-

4
728
T. Gunnlaugsson et al. / Tetrahedron Letters 42 (2001) 4725–4728
bridge Crystallographic Data Centre includes atomic
Data Collection, Processing, Structure Solution and
Refinement, Bruker Analytical X-Ray Systems, Madison,
WI, 1998.
coordinates, thermal parameters, remaining bond lengths
and angles and structure factors (CCDC number 158714).
Crystal data for C H N O7 (2): M=474.51, mono-
12. Blinks, J. R.; Wier, W.; Hess, P.; Prendergast, F. G.
Prog. Biophys. Mol. Biol. 1982, 40, 1.
13. (a) L e´ tard, J.-F.; Delmond, S.; Lapouyade, R.; Braun,
D.; Rettig, W.; Kreissler, M. Recl. Trav. Chim. Pays-Bas.
1995, 114, 517; (b) L e´ tard, J.-F.; Lapouyade, R.; Rettig,
23
306
4
clinic, space group P2 /c, a=14.161 (5), b=7.521(3),
1
−
3
c=22.967(9) A
, Dc 1.338 Mg m , F(000)=1008, v=0.100 mm ,
crystal dimensions=0.28×0.23×0.10 mm. A total of
4543 reflections were measured for 3<2q<45 and 3075
,
, i=105.703(8)°, U=2354.9(15) A
,
, Z=
−
3
−1
4
1
W. Pure Appl. Chem. 1993, 68, 1705.
1
unique reflections were used in the refinement, the final
parameters were wR =0.2130 and R =0.0695 [I>2|(I)],
14. We have observed that the OCH signal in the H NMR
3
is shifted by 0.16 ppm upon addition of 5 equiv. of
NaPF in CD OD. A similar effect has been seen in a
2
1
S=1.210, 318 parameters.
6
3
+
+
1
1
0. SMART Software Reference Manual, version 5.054,
Bruker Analytical X-Ray Systems, Madison, WI, 1998.
1. Sheldrick, G. M. SHELXTL, An Integrated System for
related Na receptor for intracellular Na detection: de
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Nieuwenhuizen, M. Chem. Commun. 1996, 1967.
.