Azole-Linked Coumarin Dyes as Fluoresence Probes of Domain-Forming Polymers
Boston University, Boston, MA 02215
The binding of 7-aminocoumarins, substituted in the 3-position with heterocyclic benzimidazole or benzothiazole groups by domain-forming polymers in water has been studied. The acrylic polyelectrolyte, poly(methacrylic) acid (PMAA) was used as a solubilizing agent for coumarin dyes 6, 7, and 30 in water. The acid-base properties of these bound coumarin dyes were monitored spectroscopically on titration of aqueous solutions. Alterations in the fluorescence wavelength and intensity, quantum yields, lifetimes, and polarization are consistent with the preferential binding of the dyes in compact hydrophobic domains that form at a pH regime in which the polymer is in its protonated (uncharged) state. In this pH range (< 4.0), coumarins 7 and 30 are bound as monocations, whereas coumarin 6 remains in its neutral form. Reduced quantum yields and lifetimes of fluorescence for cationic coumarins can be understood in terms of the imposition of a low-lying electron transfer state, an example of a twisted intramolecular charge transfer (TICT) intermediate. Effects of polymer microenvironment on the rate of TICT state decay (a reverse electron transfer) are observed. Coumarins of the azole type may find use as fluoroprobes of the microenvironments of proteins and other biological macromolecules and as agents for pH sensing.
A number of papers from our laboratory have focused on the fundamental photochemical and photophysical properties of coumarin laser dyes -. Of special interest has been the use of polymeric media in which dyes may be doped (solid acrylics) - or bound in microdomains in water , , . Receiving the most attention in the latter application is the polyelectrolyte, poly(methacrylic) acid (PMAA), which has been shown to increase the solubility and to amplify fluorescence yields of coumarin dyes in water . The ability of this polymer to recognize and complex hydrophobic agents when it assumes a compact hypercoiled conformation is well established -. Studies of PMAA complexation of dyes have also been extended to other structures such as the triarylmethanes , the rhodamines  and the cyanines .
Within the class of 7-amino coumarins that typically display favorable fluorescence and lasing properties, the derivatives that bear an additional heterocyclic (azole) substituent at the 3-position have several differentiating properties. They display smaller Stokes shifts of fluorescence, indicative of a lower degree of charge transfer for low-lying excited species ,. Moreover, they potentially have an additional state of protonation, since the benzimidazole group (for example) is known to have a pKa of 5.48 . The acid-base properties of coumarin 6 have in fact been examined recently, particularly from the point of view of using the dye as a probe of acidic sites in zeolites or clays . Electronic spectral data and NMR results are consistent with protonation of C6 at the benzothiazole ring nitrogen (Scheme 1) with concomitant shifts of absorption and emission bands .
In the present study, we have investigated coumarins 7 (1), 30 (2), and 6 (3) (Scheme 2), in order to determine both the acid-base properties and the potential for intramolecular electron transfer for this important series of dyes. Fluorescence properties, including quantum yield, lifetime, and polarization have been recorded as a function of pH for mixed organic/water media. For water samples in which dyes are solubilized using PMAA, 1 and 2 are shown to form polymer complexes in their protonated forms for moderately acidic pHs, whereas 3 remains neutral when bound to the hypercoiled polymer. A pathway involving charge transfer excitation, followed by non-radiative return to the ground state via electron transfer has been identified for 1 and 2.
The 7-aminocoumarins studied here can be used as fluoroprobes for microenvironments of systems such as thin films, polymers and proteins that are of commercial or biological importance ,. These dyes should serve as sensitive probes of micropolarity and pH for biopolymers and biomembranes, or other self assembled systems -.
2. Experimental Section
Materials. The coumarin dyes used in this study were used as received from Eastman Kodak and Acros Organics. They were determined to be relatively pure by thin layer chromatography, NMR and melting point determination. Trifluoroacetic acid was obtained from Aldrich. Poly(methacrylic acid), PMAA was prepared following a reported procedure  using twice distilled methacrylic acid dissolved in N,N-dimethylformamide (DMF); polymerization proceeded under Ar at 55-65o (20 hr), using the initiator, 2,2'-azobis-isobutyronitrile (AIBN). Fractionation of the polymer  provided a sample that was determined to have a number averaged molecular weight of 25,000 g/mol by viscometry (Ostwald viscometer) . Water used for preparation of aqueous solutions of the samples was generated from Millipore Milli-Q Water system (resistivity, 18 MW/cm2).
Instrumentation and Methodology. Absorption spectra were recorded on DU-7 and DU-640B Beckman spectrophotometers. Steady state emission, fluorescence polarization, quantum yield and lifetime measurements were measured on SLM Aminco 48000 and Felix/Timemaster (Photon Technology International, Inc.) fluorometers. The PTI LaserStrobe fluorescence lifetime and steady state fluorimetry system is described in a forthcoming paper . Aqueous solutions were prepared by combining stock solutions of the coumarins (2 mM in DMF) and solutions of the polyelectrolytes (50 mM) appropriate for producing proper ratios, R/D, the ratio of molar concentrations of polymer (monomer residues) and dye. The solutions were sonicated (typically for 5 min) to achieve complete dissolution. Dilute aqueous solutions of HCl and KOH were utilized for pH adjustment. Quartz cells (1 cm) were used for spectral measurements at room temperature. Fluorescence quantum yield measurements (+ 10%) were carried out using dilute solutions (OD < 0.2) and referenced against coumarin 153 in ethanol, Ff = 0.38 . Fluorescence polarization values were measured in the L-format on the SLM 48000 fluorometer, or using the StrobeMaster polarization option of the PTI fluorometer. Fluorescence lifetimes were recorded using nitrogen laser (337 nm) and dye laser (503 nm) sources. A colloidal solution of silica gel was used as a scatterer to obtain the lamp profile. Fitting of the data was done for functions having single and double exponentials using Timemaster software (c2 values, 0.9 - 1.2).
3. Results and Discussion
Spectral properties of 1-3 in aqueous poly(methacrylic) acid, PMAA. Azole-linked coumarins 1-3 are nearly insoluble in water (< 10 mM) and produce slightly turbid aqueous solutions. Solubilization in aqueous PMAA  leads to the sequestering of dye in hydrophobic domains of the polymer (the hypercoil conformation at pH < 4) (Scheme 3) , , ; complexation of dye and polymer in its acid form also led to a significant increase in emission intensity. Since the conditions required for formation of hydrophobic domains for PMAA are moderately acidic, it was important to determine the state of protonation for 1-3 when they are polymer bound. For comparison purposes, spectra were obtained for 30% EtOH/H2O solutions (in which dyes are more readily soluble) and for 30% EtOH/H2O with added trifluoroacetic acid (TFA). The series of absorption and emission spectra for the three solvent media are shown in Figures 1, 2 and 3, including aqueous PMAA, where the concentration ratio of polymer residues to dye, R/D = 1000. The important trends in the photophysical data are also summarized in Table 1.
There are noticeable effects upon addition of acid to aqueous ethanol/dye solutions, including a red shift of both the visible absorption bands and the emission maxima (Figs 1,2,3), complemented by increases in absorptivity and a reduction in emission. For PMAA samples at pH 3, the pattern of spectral changes diverged. For the benzimidazoles 1 and 2, solutions containing the polymer developed the signature of the acid form noted for EtOH/H20/TFA, whereas dye 3 underwent a more subtle change, with absorption and emission bands shifting modestly to the blue. The generous pattern of spectral shifts and effects on emission intensity can be understood in terms of the acid-base properties associated with the azole dyes and the photophysical properties expected for different protonated forms. We assign the bands observed for dyes in neutral aqueous ethanol solutions to transitions involving low lying intramolecular charge transfer states (ICT) associated primarily with the coumarin moiety (eqn 1).
The charge transfer absorption and its accompanying emission are sensitive to solvent polarity. This solvatochromism is typical of the 7-aminocoumarins in that more polar media bring about red shifts due to the higher polarity associated with the ICT state vs the ground state ,,. The effect of incorporation of 3 in PMAA (pH 3) is to provide a microdomain of lower polarity for sequestering neutral dye molecules. For comparison, the peak emission for 3 in a non-polar solvent, hexane, occurs at 490 nm (468 nm, sh). However, for the level of acidity that is associated with the acid form of PMAA, the protonated form of dyes 1 and 2 is dominant and a different type of excited state becomes important. As noted by others  and by us in work reported elsewhere , added acid leads to protonation of azole ring nitrogens, a step confirmed in part by observation of proton NMR chemical shift changes. Imposition of the charged (cationic) heterocycle at position-3 results in relative stabilization of the ICT excited state with an accompanying increase in absorptivity (e.g. for 1 in 30% EtOH/H2O, an increase of 9.1 x 104 to 1.2 x 105 M-1cm-1 is observed on addition of 1.7 M TFA). The ICT absorption and emission bands associated with the cationic form of 1 and 2 shift modestly to the blue, when dye is bound in the folded (less polar) PMAA microdomain.
Titration curves tend to support the model regarding the role of different states of protonation in controlling polymer binding and spectral trends (Fig 4). For aqueous ethanol solutions, shifts occur for 1 and 2 at points expected for protonation of a benzimidazole ring nitrogen (pKa= 5.48) . The similar shift of absorption and emission bands to the red signals the appearance of 5 and the electronic transition to the intramolecular charge transfer (ICT) state 6. Nominal pKa values, unadjusted for H+ activity in the EtOH/H2O medium, fall in the range of 4.5-5.0 for 1 and 2. Similar protonation of the ring nitrogen for 3 occurs at a lower pH (< 2.0), consistent with the lower basicity of the benzothiazole ring (nearly complete conversion to the acid form shown in Fig 3a) ,.
When the PMAA is in its hypercoil conformation at pH < 4, the dye experiences a local medium polarity where most water molecules are excluded . As the polymer starts to uncoil in the transitional region at pH 5 - 6, shifts of the absorption and emission maxima occur until the conformation of PMAA is altered to an elongated rod, when the polyelectrolyte is fully charged (anionic) at pH > 7 (Scheme 3) ,. The absorption and emission shifts observed for the coumarins 1 and 2 as the polymer uncoils is from red to blue because the primary effect of higher basicity is to produce the conjugate base forms of the imidazole dyes. The opposite is seen for the benzothiazole coumarin. At neutral and slightly basic pH, dye molecules released to the bulk aqueous environment are exposed to a more polar medium and typical solvatochromism is observed (red shifts). The spectral trends observed for 3 are similar to data obtained for other coumarin derivatives for which a change in state of dye protonation does not occur .
Fluorescence polarization measurements for 1-3 in aqueous PMAA were obtained to determine the effectiveness of the incorporation of coumarin species into the polymer domain and the orientational distribution of the fluorophore in the polymer microenvironment . An increase in polarization values was observed, proportional to the R/D ratio for the coumarins, as illustrated in Fig 5. The pH of the medium varies in these experiments from about 7.0 at R/D 10 to 3.0 at R/D 3000. When coumarins are bound to PMAA in its compact conformation (pH < 4), rotational depolarization of fluorescence emission is not very effective because the mobility of the dye moiety is hindered due to an increase in local viscosity; thus, high polarization (anisotropy) values are obtained . A limiting concentration of the polymer is achieved at about R/D 500, at which point virtually all dye species (cations or neutral molecules) are complexed in the polymer microdomain.
Fluorescence quantum yield and lifetime. Emission lifetimes were recorded using a stroboscopic technique  that is employed with the PTI LaserStrobe fluorimeter. Good fits to single exponential decay functions were obtained for nearly all solvent media. Since dye species could, in principle, take up different types of binding sites when incorporated as PMAA complexes , lifetime heterogeneity was expected and, indeed in some cases, was observed. For example, a double exponential fitting of 1 in PMAA gave lifetime decays of 3.9 ns (0.97) and 1.8 ns (0.03) (c2 = 1.2) compared to a single exponential fit that yielded 3.9 ns (c2 = 1.2). Nonetheless, all data for both solvents and aqueous PMAA could be reasonably fit to single exponentials (c2 = 1.1) with inclusion of a second component providing only modest improvement at best. Lifetimes that are based on single component decay are thus used for further comparison.
The fluorescence data that include quantum efficiencies and emission lifetimes for 1-3 in the various media (Tables 1,2) reveal only a subtle mitigating effect for the polar solvent, aqueous ethanol, on the yield and lifetime of the ICT emission, a trend that is generally observed for aminocoumarins ,,. In the present case, quantum efficiencies are reduced to 0.25-0.50, as compared to a value of 0.80-0.82 in pure ethanol. The acid forms of the dyes (aqueous ethanol with TFA) are still more compromised with regard to fluorescence through the imposition of a second type of charge transfer state involving the azole 3-substituent. Protonation as in structure 5 (eqn 2) results in a fusion of electron donor and acceptor groups through a direct covalent link. For the acid form, the low lying excited state is one that invokes the azole ring as electron acceptor and the coumarin moiety as electron donor .
The non-radiative deactivation that is proposed for the cations thus involves the sequence, 6 7 4, in a series of steps that, in the limit, can be viewed as discrete forward and reverse electron transfer steps . This series stands in contrast to the non-radiative decay that has been identified for the coumarins in which the 7-amino substituent of the coumarin ring twists out of plane ,,. In a complementary way, however, the coumarin and azole substituent in species 7 (one resonance structure shown, eqn 2) are held out-of-plane due to non-bonded interactions of carbonyl and N-H (N-CH3) groups . The electron transfer intermediate 7, can be viewed also as belonging to the class of twisted intramolecular charge transfer (TICT) states ,. Species of this type have been found to be quite general for a wide array of groups, many of which are implicated in pathways responsible for fluorescence quenching.
The remaining important trend is the effect of PMAA binding on the rate of non-radiative decay for 1-3. The reduction in emission quantum efficiency and lifetime observed for cationic forms of 1 and 2 in acidifed EtOH/H2O (H+) (Tables 1,2) is partially restored on incorporation of dyes in the polymer microdomain. These effects can be interpreted in terms of a reduction in rates of non-radiative decay for polymer-bound species (5-10 fold effects in knd) (Table 2). A simple model regarding this effect treats the formation of intermediate 7 as a discrete electron transfer for which Marcus theory may be applied . For a range of circumstances in which the driving force for electron transfer (DGo) is roughly the size of twice the value of the reorganization energy for electron transfer (l), the activation free energy is approximately related to the inverse of l, as in equation 3.
If it is further assumed that the important determinant of the value of l for the step 6 7, involves solvent reorganization, then the lower effective polarity of the microenvironment of PMAA  will result in a higher barrier to deactiviation via electron transfer. For 3 bound to PMAA (pH 3.0), the effects on emission yield and lifetime are more constrained, consistent with the absence of the electron transfer shuttle mechanism involving the protonated azole ring.
Azole-substituted 7-aminocoumarins are solubilized in pure water in the presence of a large excess of poly(methacrylic acid) (PMAA) R/D, [residue]/[dye] > 500, a point at which the polyelectrolyte achieves a compact conformation at pH < 4. Based on the spectral properties exhibited by the benzimidazole compared to the benzothiazole coumarin dyes upon titration, the identity of the bound dye species is established. The more basic benzimidazole coumarins (1 and 2) occupy polymer microdomains as cations, whereas the less basic dye, benzothiazole 3, is bound as the neutral free base. These dyes exhibit significant fluorescence enhancement in the hypercoiled "acid form" of PMAA compared to the unbound dyes in aqueous solution. The conjugate acids of 1 and 2 are shown to have reduced emission quantum yield and lifetime in aqueous media, features that are restored to a degree on incorporation in PMAA. Non-radiative decay for 1 and 2 is associated with a mechanism of electron transfer steps via 6 and 7 (eqn 2) for dye species having severely twisted geometries. The azole derivatives of 7-aminocoumarins show promise for monitoring local acidity and structural details of microdomains of (bio)macromolecules through fluorescence measurements (quantum yield, lifetime, and polarization).
We wish to thank the U.S. Department of Energy, Division of Basic Energy and Sciences, for support of this research. We also extend our gratitude to Dr. Joseph Morais for technical assistance.