Coumarin fluorophores

Based on the preliminary understanding of ICT mechanism of coumarin core skeleton, the first systematic application of combinatorial approach toward the field of fluorescence chemistry was reported by Bauerle and co-workers in 2001 [44]. In their study, the structure-photophysical property relationships (SPR) of coumarin fluorophore were revealed by means of a combinatorial approach. 

Figure 9.21 The basic structural characteristics of coumarin fluorophores.

Sippel, T.O. (1981) New fluorochromes for thiols Maleimide and iodoacetamide derivatives of 3-phenyl-coumarin fluorophore./. Histochem. Cytochem. 29, 314. 

Zhao, Y Zheng, Q Dakin, K Xu, K., Martinez, M.L. and Li, W.-H.D (2004) New caged coumarin fluorophores with extraordinary uncaging cross sections suitable for biological imaging applications. Journal of the American Chemical Society, 126, 4653—4663. 

Lupei D, Nanting N, Minyong L, Wang B (2010) A fluorescent hydrogen peroxide probe based on a click modifled coumarin fluorophore. Tetrahedron Lett 51 1152-1154... 

Danko, M. Szabo, E. Hrdlovic, P. Synthesis and spectral characteristics of fluorescent dyes based on coumarin fluorophore and hindered amine stabilizer in solution and polymer matrices. Dyes Pigm. 2011, 90, 129-138. 

Overall, the significant Stokes shift of 100 nm and the good quantum yields make the coumarin dye a powerful fluorescent probe for nucleic acids assays or cell biology. The postsynthetic click chemistry makes this fluorophore readily accessible for fluorescent labeling of nucleic acids. 

The wide variety of absorption and emission wavelengths has allowed the use of different coumarin derivatives as FRET pairs [37], Contrary to most fluorophores, coumarin is uncharged which makes it intrinsically membrane-permeant. To induce water solubility polar groups are frequently introduced to the basic structure [35],... 

Another important group of hydrolytic enzymes are phospho- and cyclophosphodiesterases. They catalyze the hydrolysis of phospho-diester bonds and many of the most relevant biological substrates are nucleic acids. Phospholipase C and D are also important examples. Initial attempts to measure phosphodiesterase activity placed a phosphodiester between a fluorophore and a quencher and the probe was tested in vitro [146], This system was slightly modified by Caturla and used for the identification of catalysts with phosphodiesterase activity [147], More recently, Nagano and co-workers used a coumarin donor and fluorescein as a FRET... 

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],... 

Aminomethylcoumarin derivatives possess intense fluorescent properties within the blue region of the visible spectrum. Their emission range is sufficiently removed from other common fluorophores that they are excellent choices for double-labeling techniques. In fact, coumarin fluorescent probes are very good donors for excited-state energy transfer to fluoresceins. 

The spectral properties of this fluorophore are similar to those of other coumarin derivatives. The excitation maximum occurs at about 382nm and its emission peak at 472nm, producing light in the blue region of the spectrum. The extinction coefficient of DCIA at its wavelength of maximum absorbance, 382nm, is 33,000 M em-1 (in methanol). 

Blackburn C, Bai M, LeCompte KA et al (1994) Lithium responsive fluorophores derived from monoaza-12-crown-4 and coumarin. The influence of a methoxy side-arm on photophysical properties. Tetrahedron Lett 43 7915-7918... 

Many fluorophores of practical interest are heterocyclic coumarins, rhodamines, pyronines, fluoresceins, oxazines, etc. A few examples will now be presented. 

In contrast to the above-described systems, there are only few systems in which the bound cation can interact with the acceptor part of charge-transfer probes. The case of coumarins linked to crowns (Figure 10.24) is of special interest because the cation interacts directly with the electron-withdrawing group, i.e. the carbonyl group, in spite of the spacer between the fluorophore and the crown. An important consequence is the increase in stability constant of the complexes with respect to the same crown without external complexing atoms. 

Relatively few probes have been widely applied to FlAs. Fluorescein and rho-damine, their derivatives, and to some extent phycoerythrin have dominated immunoassays that are based on steady-state measurements. Dansyl, umbelliferone, coumarins, and pyrenes are among others that have been used. Several more commonly used dyes are shown in Figure 14.2. Shorter-wavelength dyes tend to be less useful since they excite at wavelengths where endogenous chromophores, fluorophores, and... 

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