Molecular Fluorescence
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10.4 Fluorescence molecular sensors of anions 315
given cation in a specific range of concentration can be very di erent according to the application. In this respect, the choice of the recognition moiety is of major importance but it is also important to note that the fluorophore itself often participates in the complexation and thus plays a role in the selectivity. Cryptands are known to be very selective to alkali ions but they often contain a tertiary amine that is pH sensitive. Calixarenes with appropriate appended groups including fluorophores have great potential in terms of molecular design.
10.4
Fluorescence molecular sensors of anions
Anions play key roles in chemical and biological processes. Many anions act as nucleophiles, bases, redox agents or phase transfer catalysts. Most enzymes bind anions as either substrates or cofactors. The chloride ion is of special interest because it is crucial in several phases of human biology and in disease regulation. Moreover, it is of great interest to detect anionic pollutants such as nitrates and phosphates in ground water. Design of selective anion molecular sensors with optical or electrochemical detection is thus of major interest, however it has received much less attention than molecular sensors for cations.
The methods of anion detection based on fluorescence involve quenching, complex formation, redox reactions and substitution reactions (Fernandez-Gutierrez and Mun˜oz de la Pen˜a, 1985). This chapter will be restricted to anion molecular sensors based on collisional quenching (in general, they exhibit a poor selectivity) and on recognition by an anion receptor linked to a fluorophore (fluoroionophore).
10.4.1
Anion sensors based on collisional quenching
Many fluorescent molecular sensors for halide ions (except F ) are based on collisional quenching of a dye. In particular, the determination of chloride anions in living cells is done according to this principle. Examples of halide ion sensors are given in Figure 10.29.
The drawback of these molecular sensors is their lack of selectivity, as shown by the Stern–Volmer constants (Table 10.4). For instance A-1, 6-methoxy-N- (3-sulfopropyl)quinolinium (SPQ) is mainly used as a Cl -sensitive fluorescent indicator, but its fluorescence is also quenched by several other anions (I , Br and SCN , but not by NO3 ).
Another feature is that the absence of spectral change precludes ratiometric measurements. However, dual-wavelength Cl sensors have been constructed. For instance, in compound A-6 (Figure 10.30), 6-methoxyquinolinium (MQ) as the Cl - sensitive fluorophore (blue fluorescence) is linked to 6-aminoquinolinium (AQ) as the Cl -insensitive fluorophore (green fluorescence), the spacer being either rigid or flexible.
10.4 Fluorescence molecular sensors of anions 317
10.4.2
Anion sensors containing an anion receptor
There are a limited number of fluorescent sensors for anion recognition. An outstanding example is the diprotonated form of hexadecyltetramethylsapphyrin (A-7) that contains a pentaaza macrocyclic core (Figure 10.31): the selectivity for fluoride ion was indeed found to be very high in methanol (stability constant of the complex @105) with respect to chloride and bromide (stability constants 102). Such selectivity can be explained by the fact that F (ionic radius @1.19 A˚ ) can be accommodated within the sapphyrin cavity to form a 1:1 complex with the anion in the plane of the sapphyrin, whereas Cl and Br are too big (ionic radii 1.67 and 1.82 A˚ , respectively) and form out-of-plane ion-paired complexes. A two-fold enhancement of the fluorescent intensity is observed upon addition of fluoride. Such enhancement can be explained by the fact that the presence of F reduces the quenching due to coupling of the inner protons with the solvent.
Phosphate groups have attracted much attention because of their biological relevance. They can be recognized by anthrylpolyamine conjugate probes A-8 (Figure 10.32). The choice of pH is crucial: at pH 6, a fraction of 70% of A-8 exists as a triprotonated form, the nitrogen atom close to the anthracene moiety being unprotonated. The very low fluorescence of this compound is due to photoinduced electron transfer from the unprotonated amino group to anthracene. This trication can bind a complementary structure like monohydrogenophosphate whose three oxygen atoms interact with the three positive charges; the remaining phosphate OH group is in a favorable position to undergo intracomplex proton transfer to the unprotonated amino group, which eliminates intramolecular quenching. Then, binding is accompanied by a drastic enhancement of fluorescence. A-8 can also bind ATP, citrate and sulfate. This mode of recognition is conceptually very interesting but the stability of the complexes is low.
Fig. 10.31. Selective sensor for fluoride ion (from Shionoya M. et al. (1992) J. Am. Chem. Soc. 111, 8735).
318 10 Fluorescent molecular sensors of ions and molecules
Fig. 10.32. Sensors for phosphate groups. (A-8: Huston M. E. et al. (1989) J. Am. Chem. Soc. 111, 8735. A-9: Vance D. H. and Czarnik A. W. (1994) J. Am. Chem. Soc. 116, 9397. A-10:
Nishizawa S. et al. (1999) J. Am. Chem. Soc. 121, 9463).
The same strategy has been applied to the recognition of pyrophosphate ions P2O74 (PPi). A-9 (Figure 10.32) binds these ions over 2000 times more tightly than phosphate ions, permitting the real-time monitoring of pyrophosphate hydrolysis.
Detection of pyrophosphate has also been demonstrated by a simple selfassembling system A-10 (Figure 10.32) with a pyrene-functionalized monoguanidinium receptor. This receptor was found to self-assemble to form a 2:1 (host:- guest) complex with high selectivity for biologically important pyrophosphate ions in methanol. A sandwich-like ground-state pyrene dimer is formed. The character-
320 10 Fluorescent molecular sensors of ions and molecules
Fig. 10.34. Sensors based on acyclic, macrocyclic and calixarene ruthenium–bipyridyl (from Beer, P. D. (1996) Chem. Commun. 689).
The fluorescence spectrum of the tris-acridine cryptand A-13 shows the characteristic monomer and excimer bands. Upon complexation with various organic anions (carboxylates, sulfonates, phosphates), the monomer band increases at the expense of the excimer band. The stability of the complexes depends on the contribution of the electrostatic and hydrophobic forces and on the structural complementarity. Stability constants of the complexes ranging from 103 to 107 have been measured. In particular, A-13 binds tightly to monoand oligonucleotides, and it can discriminate by its optical response between a pyridimic and a purinic sequence.
Another interesting class of anion receptors (Beer, 1996) consists of acyclic, macrocyclic and lower-rim calix[4]arene structures in which the Lewis-acidic redox and photoactive ruthenium(II) bipyridyl moiety is introduced. These sensors o er the dual capability of detection of anionic species via either electrochemical or optical methods. Examples are given in Figure 10.34. Determination of the stability constants of the complexes in dimethyl sulfoxide reveals a high selectivity of the calixarene-based receptor for H2PO4 . It is worth noting that hydrogen bonding plays an important role in the stabilization of the complexes. Six hydrogen bonds stabilize the Cl anion (two amide and four CaH groups) in the complex with A-14. Three hydrogen bonds (two amide and one calix[4]arene hydroxy) permit stabilization of H2PO4 in the complex with A-16. The fluorescence spectrum of the ligands undergoes significant blue-shift upon anion binding (16 nm for addition of
10.4 Fluorescence molecular sensors of anions 321
Fig. 10.35. Recognition of carboxylate ions by a complex of an anthrylamine with ZnII (De Santis G. et al. (1996) Angew. Chem. Int. Ed. 35, 202).
H2PO4 to A-16) with a concomitant large increase in fluorescence quantum yield. Such an increase may be due to the rigidification of the receptor by the bound anion, which decreases the e ciency of non-radiative de-excitation.
Carboxylate ions can be recognized and sensed by a complex of an anthrylamine (analogous to A-8) with ZnII (Figure 10.35). The resulting four-coordinate metal center (A-17) has a vacant site for coordination of an anion to give a trigonalbipyramidal arrangement. A nity towards anions bearing a carboxylate group is strong. Recognition is signaled via fluorescence quenching of the appended fluorophore as a result of intramolecular electron transfer, e.g. from a bound 4-N,N- dimethylaminobenzoate to the excited anthracene moiety. Such a transfer is favored by the stacking of benzoate and anthracene. The selectivity is essentially determined by the energy of the metal–anion coordinative interaction; moreover, only the interactions with anions displaying distinctive electron donor or electron acceptor tendencies cause fluorescence quenching of anthracene. For instance, NO3 and SCN do not a ect the anthracene emission and do not compete with dimethylaminobenzoate for the binding to the metal center, whereas Cl causes an intensity decrease of less than 5% and competes for binding. The acetate ion behaves in a similar way to Cl .
According to the same strategy, the cooperative action of boronic acid and zinc chelate was used in the design of the fluorescent sensor A-18 for recognition of uronic and sialic acid salts (Figure 10.36). The zinc-phenanthroline moiety is the fluorophore whose fluorescence is quenched by the tertiary amine in the free ligand state. Upon addition of uronic and sialic acids at pH 8, the carboxylate functions of these acids can ligate the zinc atom while the hydroxyl functions of the saccharide ring form a boronate ester at the saccharide recognition site. Because the interaction between boronic acid and amine is intensified, the PET process is suppressed and the fluorescence intensity increases.
322 10 Fluorescent molecular sensors of ions and molecules
Fig. 10.36. Recognition of uronic and sialic acid salts by the cooperative action of boronic acid and zinc chelate (Yamamoto M. et al. (1996) Tetrahedron 54, 3125).
An interesting practical application is the detection of the citrate anion in soft drinks, as shown in Box 10.3. The strategy is quite di erent from that of the preceding examples because the anion receptor is not linked to a fluorophore. The latter simply acts in competition with the citrate anion in a fashion that resembles fluorescence-based immunoassays.
There a great need for selective anion sensors, but the number of available sensors is rather limited because of di culties in their design. However, new selective sensors are expected because of the considerable progress made in the synthesis of anion receptors.
10.5
Fluorescent molecular sensors of neutral molecules and surfactants
Recognition of neutral organic molecules in solution is a much greater challenge than recognition of ionic species because the involved interactions (Van der Waals interactions, hydrogen bonds) are much weaker than those existing with charged species and the electronic changes induced by complexation are smaller.
A few examples will be presented in this section. Among them, detection of steroid molecules (e.g. cortisone, hydrocortisone, progesterone, etc.) is of particular interest because of their biological relevance. Because saccharides play a significant role in the metabolic pathways of living organisms, it is necessary to detect the presence and to measure the concentration of biologically important sugars (glucose, fructose, galactose, etc.) in aqueous solutions. Determination of enantiomeric purity of synthetic drugs and monitoring of fermentation processes are examples of applications.
Another example of practical interest is the detection of surfactants that are extensively used in domestic and industrial applications; their slow degradation poses a severe problem of environmental pollution.
10.5 Fluorescent molecular sensors of neutral molecules and surfactants 323
Box 10.3 Detection of citrate in beveragesa)
At neutral pH, citrate bears three negative charges and is thus quite distinctive from interfering species like monoand dicarboxylates, phosphates, sugars and simple salts. This observation led Metzger and Anslyn to design the A-19 receptor, consisting of three guanidinium groups that form hydrogen bonds and ion pairs with carboxylate groups (Figure B10.3.1). The positively charged groups are arranged on one face of a benzene ring. This conformation leads to good binding of citrate in water ðlog Ks ¼ 3:83Þ.
None of the involved species are fluorescent. Therefore, for fluorescence signaling of citrate recognition, carboxyfluorescein is first added to the medium because binding to the receptor in the absence of citrate is possible and causes deprotonation of carboxyfluorescein, which results in high fluorescence. Citrate is then added, and because it has a better a nity for the receptor than carboxyfluorescein, it replaces the latter, which emits less fluorescence in the bulk solvent as a result of protonation. Note that this molecular sensor operates in a similar fashion to antibody-based biosensors in immunoassays. It was succesfully tested on a variety of soft drinks.
Fig. B10.3.1. Formation of a complex between the A-19 receptor and citrate iona).
a)Metzger A. and Anslyn E. V. (1998) Angew. Chem. Int. Ed. 37, 649.
10.5.1
Cyclodextrin-based fluorescent sensors
a-, b- and g-cyclodextrins (CDs) are toroidal molecules containing six, seven and eight glucopyranose units, respectively. The internal diameters of the cavities are approximately 5, 6.5 and 8.5 A˚ , respectively, and the depth is about 8 A˚ (Figure 10.37). Their ability to form inclusion complexes with various organic compounds in aqueous solutions is of major interest for molecular recognition. Numerous modified CDs have been designed for improving the selectivity of binding. CDs
324 10 Fluorescent molecular sensors of ions and molecules
Fig. 10.37. Formulae and dimensions of a, b and g-cyclodextrins.
can be transformed into fluorescent sensors by attaching one, two or more fluorophores.
Figure 10.38 shows modified b- and g-cyclodextrins with two identical appended fluorophores that are able to form excimers (Ueno et al., 1997). They have been studied in 10% ethylene glycol aqueous solutions. b-cyclodextrins with two 2- naphthylsulfonyl moieties linked to the smaller rim (compounds b-CD1, b-CD2, b- CD3), have a cavity that is too small to include both fluorophores; one of them is outside the cavity and the other is inside. The latter can be excluded from the cavity upon inclusion of a guest molecule. Therefore, the excimer band in the fluorescence spectrum increases upon guest inclusion.
In contrast, the fluorescence spectra of the parent g-cyclodextrins (compounds g- CD1, g-CD2, g-CD3, g-CD4) exhibit both monomer and excimer bands in the absence of guests because the cavity is large enough to accommodate both fluorophores (Figure 10.38). The ratio of excimer and monomer bands changes upon guest inclusion. The ratio of the intensities of the monomer and excimer bands was used for detecting various cyclic alcohols and steroids (cyclohexanol, cyclododecanol, l-borneol, 1-adamantanecarboxylic acid, cholic acid, deoxycholic acid and parent molecules, etc.).
Various CDs with a single appended fluorophore were also designed (Ueno et al., 1997). For b-CDs, the principle is the following: in the absence of guest, the fluorophore is encased in the cavity and exhibits photophysical properties that are characteristic of such a nonpolar restricted microenvironment. Upon addition of a