Quencher dissociation constants

The transient absorption spectra similar to that of the ion-pair state of indole cation radical and flavin anion radical were also observed in D-amino acid oxidase (5), although the spectra were not so clear as those of flavodoxin. In D-amino acid oxidase, the coenzyme, flavin adenine dinucleotide (FAD), is wealtly fluorescent. The fluorescence lifetime was reported to be 40 ps (16), which became drastically shorter (less than 5 ps) when benzoate, a competitive inhibitor, was combined with the enzyme at FAD binding site (17). The dissociation constant of FAD was also marlcedly decreased by the binding of benzoate (17). These results suggest that interaction between isoalloxazine and the quencher became stronger as the inhibitor combined with the enzyme. Absorbance of the transient spectra of D-amino acid oxidase-benzoate complex was remarkably decreased. In this case both rate constants of formation and decay of the CT state could become much faster than those in the case of D-amino acid oxidase free from benzoate. 

When the quencher contains heavy atoms nonradialive relaxation of the exciple occurs via the triplet state (heavy atom perturbation). A second mode of exciplex dissociation is through electron transfer between the excited molecule and the quencher. Ionization potential of the donor, electron affinity of the acceptor and solvent dielectric constant are important parameters in such cases. 

The value of is related to the reactivity within the supramolecular system. This rate constant will depend on the mobility of the quencher with respect to the probe inside the supramolecular structure, as well as on the chemical reactivity for the quenching process. Several models have been described for the mobility of quenchers with respect to probes in micelles [58-65]. Thus, the value of has a dynamic component to it, but it is not related to the association or dissociation processes of the quencher with the supramolecular system, which is the focus of this review. The values of will only be discussed when relevant to the association/dissociation studies. 

Models with increasing sophistication for the analysis of dynamic processes in supramolecular systems, notably micelles, as well as for the determination of other parameters have been developed over the past two decades. The basic conceptual framework has been described early on [59,60,95,96] and has been classifred into different cases which take into account the extent of quencher mobility and the mechanism of quenching [95]. Two of those cases lead to information about mobility and will be discussed. It is important to emphasize that this analysis is only applicable to self-assembled system such as micelles and vesicles it cannot be applied to host-guest complexes. This model assumes that the probe is exclusively bound to the supramolecular system and that no probe migration occurs during its excited state lifetime. The distribution of probe and quencher has been modeled by different statistical distributions, but in most cases, data are consistent with a Poisson distribution. The Poisson distribution implies that the quencher association/dissociation rate constants to/from the supramolecular system does not depend on how many... 

The second case firom which dynamic information can be recovered is also the more general description of the model. The quencher is assumed to be mobile and the dissociation rate constant of the quencher from the supramolecu-lar system ( g ) competes with the quenching process The mechanistic scheme of Fig. 1 is valid taking into account the general assumptions mentioned above. The fluorescence decay can be described by a function with four parameters [60,97] ... 

In conclusion, the limited examples described show that quenching studies can provide some useful information on accessibility of quencher molecules to probes in micelles. However, the rate constants for association and dissociation are not recovered, and interpretation of the quenching results is not always straightforward. 

The values for the association (kg+) and dissociation (kg ) rate constants of the quenchers can be determined when the conditions discussed in Section II are met. Most of the studies in micelles have been based on the model that leads to Eq. (8), where rate constants are recovered from a four-parameter fit of the fluorescence decay in the presence of quencher. The assumptions of this basic model have been discussed in Section II. This model and inclusion of additional processes, such as probe and quencher migration, have been employed for over... 

The exchange mechanism has been occasionally disputed [168], and recently a competing model which claims that the change in parameter D is due to changes in the micellar surface potential has been presented [169,170]. The dynamics of iV-ethylpyridinium ion and Cu used as quenchers for pyrene in SDS micelles was studied for various salt and micelle concentrations [169]. Data were analyzed using Eq. (8). The dissociation rate constant for the quenchers were shown to be strongly dependent on the salt concentration and moderately... 

Iodide was used as a quencher that has an opposite charge to the alkyltri-methylanunonium chloride surfactants. The dissociation rate constants for sur-... 

In addition to comparing overall quenching rate constants, it is also possible to recover the values of the quencher association and dissociation rate constants from quenching experiments. The same model that was employed for fluorescent probes can be employed. This model considered that the probe was immobile. The general solution to this model is given by Eq. (8), which has four parameters defined by the rate constants for the processes described in Fig. 1. However, the experimental results showed that the triplet state decayed by pseudo-first-order kinetics, suggesting that once the quenchers enter the supramolecular system, quenching occurred with an efficiency of unity. Under these conditions, Eq. (5) can be applied. In addition, if the condition that [H] holds, Eq. (5) can be reduced to... 

Figure 8 shows the effect of varying the values for kp and kp,. These parameters are intrinsic to the system being studied and cannot be adjusted by changing experimental conditions as in the case for k (eff), by the choice of an appropriate quencher, or the concentration of the supramolecular system. However, this Fig. 8 shows the limitations of this method. When the association rate is much faster than the dissociation process, information on kp is lost (Fig. 8A). For low kp values, the quencher concentration can be lowered and the parameters related to dynamics can still be recovered. The determination of the association rate constant can to some extent be fme-tuned by changing the concentration of the supramolecular system, but the condition [H] [P] always...

Quenching studies were also employed to determine the dissociation rate constant of triplet xanthone from CD cavities. These apparent rate constants were obtained by measuring the decay of triplet xanthone at high concentrations of cupric ions. The values for kp were respectively (9 2) x 10 s" and (15 2) X 10 s for P- and y-CDs [185]. The higher value obtained in the quenching experiments for y-CD indicated that the quencher had access to the probe within the CD cavity. Unfortunately, no detailed analysis employing Eq. (25) was performed and, for this reason, a comprehensive comparison between the two methods is not possible. 

Most of the reported data involving triplet states were obtained using quenching processes to recover the association/dissociation rate constants for the excited state triplet probes and/or quenchers. As in the case of singlets, overall quenching rate constants can yield limited information on accessibility, and representative examples have been included where relevant. 

This equation, as a double reciprocal plot, is similar to Eq. (5) but is applied to probe mobility, not quencher dynamics. The same values for the dissociation rate constants (Table 17) were obtained when employing these two different methods with quenchers in different phases, suggesting that the underlying assumptions for the derivation of Eqs. (27) and (28) were reasonable. The entry rate constants were diffusion controlled, and the exit rate constants varied by a factor of 3. In analogous fashion to the polycyclic aromatic hydrocarbon probes, the exit rate constants were faster for the more polar ketones p-methoxyacetophenone and acetophenone compared to isobutyro-phenone or propiophenone [193,194],... 

Frequently, co-solvents are added to aqueous cyclodextrin solutions in most cases, these co-solvents are employed to solubilize the probes. In Section IV.C, the decrease of the exit rate constant of triplet xanthone from CDs with the addition of alcohols was described. This effect was also apparent when studying the dynamics of 1-halonaphthalenes with P-CD in the presence of acetonitrile [141]. When the nitrite ion was used as quencher, the association rate constants decreased in the presence of the organic solvent while the dissociation rate constant increased (Table 21). The main rationalization to explain the change in mobility properties was that acetonitrile was small enough to coinclude inside the cavity a small amount of acetonitrile could preferentially solvate the entrances of the CD thereby leading to a different environment for the probe. 

Turro et al. [17] also mention cases of fast exchange of quencher among micelles and of the escape of the fluorophore from the micelles during its lifetime with subsequent interaction with the quencher in the bulk solution. The latter situation was observed for the photoprotolytic dissociation of 1-naphthol in SDS and Triton X-micelles [16]. The escape of an excited molecule of 1-naphthol from the micelle leads to its instant dissociation. It gave the possibihty of measuring the escape rate constant of 1-naphthol from SDS micelles which equals to 2 x 10 s" [16]. 

The exit rate constants of the excited anions after the photoprotolytic dissociation of l,4-dichloro-2-naphthol within decylsulfate, dedecylsulfate, and cetylsulfate micelles were measured with a fluorescence quencher hardly penetrating the micelles, - the nitrate ion [121]. The addition of nitrate into the solution quenched the fluorescence of those anions which escape from the micelles within the lifetime of the excited state only. The exit rate constant of the naphtholate anion increases with increasing length of the hydrocarbon radical in the micelle-forming surfactant. The exit rate is thus controlled by the lowering of the micelle polarity (i.e. by the free energy of the exit process) rather than by the micelle size or the distance that the anion must diffuse. Perhaps one can establish a kind of correlation between the rate constant of this process and its free energy as was done for photochemical electron transfer [126] and proton transfer [156,157]. 

For protolytic photodissociation of hydroxyaromatic compounds we may expect that in anionic micelles the excited anions of hydroxyaromatic compounds having the same sign of charge will leave the micelles, and in the cationic micelles - hydrogen ions will leave micelles. Exit rate constants of excited anions of hydroxyaromatic compound from the micelles were determined using the non-solubilized fluorescence quenchers. Simultaneously, we have proved that it is in the micellar phase that protolytic dissociation does proceed and not as a result of the preliminary exit of excited molecules of hydroxyaromatic compounds from the micellar phase to an aqueous one. 

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