Cyclodextrin fluorophore

Kinsland, L. N. Wiechelman, K. J. Use of P-cyclodextrin fluorophore complexes to improve the efficiency of fluorescent label incorporation into proteins. J. Biochem. Biophys. Methods 1984, 9, 81-83. 

The data were collected using fluorescence measurements, which allow both identification and quantitation of the fluorophore in solvent extraction. Important experimental considerations such as solvent choice, temperature, and concentrations of the modifier and the analytes are discussed. The utility of this method as a means of simplifying complex PAH mixtures is also evaluated. In addition, the coupling of cyclodextrin-modified solvent extraction with luminescence measurements for qualitative evaluation of components in mixtures will be discussed briefly. 

In a similar fashion, steroids are molecules that have been investigated by disruption of FRET. The sensor is a double labeled peptide with cyclodextrin bound to one side chain. The latter keeps the fluorophores closely together by accommodating the coumarin into its cavity thereby ensuring efficient FRET. Steroids compete for the cavity of cyclodextrin and displace the coumarin reducing FRET efficiency. This model, although useful for in vitro applications, seems to be poorly selective for its application in biological samples [95],... 

The same authors studied the CL of 4,4,-[oxalylbis(trifluoromethylsulfo-nyl)imino]to[4-methylmorphilinium trifluoromethane sulfonate] (METQ) with hydrogen peroxide and a fluorophor in the presence of a, p, y, and heptakis 2,6-di-O-methyl P-cyclodextrin [66], The fluorophors studied were rhodamine B (RH B), 8-aniline-l-naphthalene sulfonic acid (ANS), potassium 2-p-toluidinylnaph-thalene-6-sulfonate (TNS), and fluorescein. It was found that TNS, ANS, and fluorescein show CL intensity enhancement in all cyclodextrins, while the CL of rhodamine B is enhanced in a- and y-cyclodextrin and reduced in P-cyclodextrin medium. The enhancement factors were found in the range of 1.4 for rhodamine B in a-cyclodextrin and 300 for TNS in heptakis 2,6-di-O-methyl P-cyclodextrin. The authors conclude that this enhancement could be attributed to increases in reaction rate, excitation efficiency, and fluorescence efficiency of the emitting species. Inclusion of a reaction intermediate and fluorophore in the cyclodextrin cavity is proposed as one possible mechanism for the observed enhancement. 

Figure 10.38 shows modified / - and y-cydodextrins with two identical appended fluorophores that are able to form extimers (Ueno et al., 1997). They have been studied in 10% ethylene glycol aqueous solutions. /J-cyclodextrins with two 2-naphthylsulfonyl moieties linked to the smaller rim (compounds / -CDl, / -CD2, / -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 y-cyclodextrins (compounds y-CD1, y-CD2, y-CD3, y-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, cyclo-dodecanol, i-borneol, 1-adamantanecarboxylic acid, cholic acid, deoxycholic acid and parent molecules, etc.). 

Fig. 10.41. Detection of cationic surfactants by a //-cyclodextrin with seven appended naphthoate fluorophores (Choppinet P. et al. (1996) J. Chem. Soc. Perkin Trans. 2, 249).

A prospective sensor made of a couple 159 consisting of p-cyclodextrin 11 and calix[4]arene 18 bearing a fluorophoric substituent was reported by Bugler and coworkers [68]. The compound forms fibers which change into vesicles upon complexation, forcing the fluorophore out of the cyclodextrin cavity. As a consequence, the intensity of fluorescence is reduced. In another approach to... 

Marchelli used the copper(II) complex of histamine-functionalized P-cy-clodextrin for chiral recognition and separation of amino acids [27]. The best results were obtained for aromatic amino acids (Trp). Enantioselective sensing of amino acids by copper(II) complexes of phenylalanine-based fluorescent P-cyclodextrin has been recently published by the same author [28, 29]. The host containing a metal-binding site and a dansyl fluorophore was shown to form copper(II) complexes with fluorescence quenching. Addition of d- or L-amino acids induced a switch on of the fluorescence, which was enantioselective for Pro, Phe, and Trp. This effect was used for the determination of the optical purity of proline. 

Excitation Energy Transfer in an Inclusion Complex of a Multichromophoric Cyclodextrin with a Fluorophore... 

As an example of excitation energy transfer studied by time-resolved fluorescence, let us take again the case of the inclusion complex of the multichromophoric cyclodextrin CD-St with oxazine 725 described in Section 7.2.4.2 [15]. Figure 7.9 shows the fluorescence decay of CD-St the very first part of the decay is due to energy transfer [13] from the steroidic naphthalene fluorophores to oxazine 725. Data analysis led to an average decay time for transfer of about 25 ps, which is quite fast, as expected from the short average distance between donor and acceptor ( 9-10 A). 

To illustrate the use of fluorescence anisotropy in the elucidation of energy transfer between identical fluorophores, multichromophoric cyclodextrins [29] similar to the CD-St described in Section 7.2.4.2 are considered, but now in the absence of a dye. 

In these multichromophoric cyclodextrins the fluorophores are randomly oriented. Excitation of one of the naphthoate fluorophores is followed by efficient dipole-dipole excitation energy transfer between the seven fluorophores, with a Forster radius of 14 A. This process is not detectable by fluorescence intensity measurements, as neither the intensity nor the decay law are affected by energy transfer between identical fluorophores (also called homotransfer. The dynamics of energy hopping are on the other hand reflected in the fluorescence anisotropy. To avoid depolarization by rotational motion of the fluorophores, experiments were conducted in a low temperature and optically clear rigid glass (9 1 ethanol-methanol at 110 K). 

The binding of fluorophores 6-(4-toluidino)naphthalene-2-sulfonate (TNS) and 6-(4-ZerZ-butylanilino)naphthalene-2-sulfonate (BNS) by bis(cyclodextrin)s has been studied by Sikorski and Petter [10]. The spacer length between the... 

In order to obtain independent evidence for the involvement of the cyclodextrin cavity, fluorescence measurements were carried out for copper(II) ternary complexes with L- or D-tryptophan. In fact, the fluorescence spectrum of tryptophan has already been shown to be sensitive to the polarity of the microenvironment in which it is located and has been used in many studies as a probe for the conformation of proteins and peptides [53]. As for many fluorophores, the indole fluorescence of Trp is quenched by the copper(II) ion this effect has been used as a measure of the stability constants of copper(II) complexes [54, 55]. In a recent work, it has been shown that the fluorescence of dansyl derivatives of amino acids undergo enantioselective fluorescence quenching by chiral copper(n) complexes and that fluorescence measurements can be used for the study of enatioselectivity in the formation of ternary complexes in solution [56]. Bearing this in mind, we performed the same type of experiments by adding increasing amounts of the [Cu(CDhm)] + complex to a solution of D- or L-tryptophan [36]. The fluorescence titration curve shows that the artificial receptor inhibits the indole... 

Some remarkable examples are given by inclusion of a variety of fluorescent guests into toroidal-shaped hosts such as cyclodextrins [27, 28]. The inclusion process protects the fluorophore from collisional quenching by the solvent and changes the polarity of the microenvironment, thus inducing both enhancement and... 

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