Fluorescein derivatives

The following sections describe the most important fluorescein derivatives commonly used to label biomolecules. 

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Utilization of resonance effects can facilitate unenhanced Raman measurement of surfaces and make the technique more versatile. For instance, a fluorescein derivative and another dye were used as resonantly Raman scattering labels for hydroxyl and carbonyl groups on glassy carbon surfaces. The labels were covalently bonded to the surface, their fluorescence was quenched by the carbon surface, and their resonance Raman spectra could be observed at surface coverages of approximately 1%. These labels enabled assess to changes in surface coverage by C-OH and C=0 with acidic or alkaline pretreatment [4.293]. 

Fluorescein absorbance is sensitive to pH. This property is utilized to measure cytosolic pH changes. Fluorescein derivatives that contain esters on the carboxyl groups have been constructed. These compounds partition through the cell membrane and, once inside the cell, the esters are cleaved by nonspecific esterases in the cytosol, leaving free carboxyl groups thus the probe cannot diffuse out of the cell (or at least does so slowly). Commonly used derivatives are 6-carboxyfluorescein (21-23) and the more recently developed probe 2, 7 -bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein (13, 24,25). 

Among the commercially available fluorescein derivatives that have been widely used are fluorescein isothiocyanate (FITC) 49, carboxyfluorescein (FAM) succini-midyl ester 50, and fluorescein dichlorotriazine (DTAF) 51. FITCs are the most commonly used fluorescein derivatives. They have been used to react with sulfhy-dryl [127], targeting reduced cysteine chains and especially amino groups in peptides or proteins [128]. 

Kamoto and collaborators [150] designed and synthesized a new tum-on fluorescent probe 54, which is excitable by visible light (kex 490 nm), based on a fluorescein derivative 55. It is composed of a metal-nitrilotriacetic (NTA) complex as the hexahistidine tag recognition site, fluorescein as the fluorophore, and a linker. 

One of the interesting features in the structure-photophysical property relationship of fluorescein is that the quantum yield of fluorescein increases under the basic condition. Therefore, many of fluorescein derivatives have been used as pH sensors to measure intracellular pH due to their pH-responding photophysical property [53]. Although fluorescein itself is slightly fluorescent in alcoholic solutions, the addition of alkali (pH > 8) to the fluorescein solution produces the very intense fluorescent alkali salt. The salt form of fluorescein... 

Fig. 8 Structure-photophysical properties relationship of fluorescein derivatives. Measured in 0.1 N NaOH(aq). bOxidation potential of corresponding benzene moiety, obtained in acetonitrile containing 0.1 M TBAP. °HOMO energy level of the corresponding benzene moiety, calculated with B3LYP/6-31G(d)//B3LYP/6-31G(d) by Gaussian 98 W...

Fig. 9 Structure-photophysical properties relationship of benzannulated fluorescein derivatives...

Fabian WMF, Schuppler S, Wolfbeis OS (1996) Effects of annulation on absorption and fluorescence characteristics of fluorescein derivatives a computational study. J Chem Soc Perkin Trans 2(5) 853-856... 

Fujita, S., Nakanishi, S. and Torn, T. (1998). Convenient preparation of fluorescein derivatives. Synth. Commun. 28, 387-393. 

Figure 9.4 Fluorescein derivatives are produced through modification at the C-5 or C-6 positions on the lower ring.

Aldehyde/Ketone and Cytosine-Reactive Fluorescein Derivatives... 

Figure 9.11 This carbohydrazide-containing fluorescein derivative can be used to modify aldehyde-containing molecules. Glycoconjugates may be labeled with this reagent after treatment with sodium periodate to produce aldehydes.

The spectral characteristics of protein conjugates made with Lissamine rhodamine B derivatives are of longer wavelength than those of tetramethylrhodamine—more toward the red region of the spectrum. In addition, modified proteins have better chemical stability and are somewhat easier to purify than those made from TRITC (discussed previously). Lissamine derivatives also make more photostable probes than the fluorescein derivatives (Section 1, this chapter). 

Although FITC and other reactive fluorescein derivatives still are widely used to label (strept)avidin and other proteins, better fluorescence yield and stability will be obtained if one of the newer hydrophilic fluorescein dyes is used. See Chapter 9, Section 1, for additional details on labeling proteins with fluorescein. 

Brauer D, Uknalis J, Triana R, Tu S-I. Subcellular compartmentation of different fluorescein derivatives in maize root epidermal cells. Protoplasma 1996 192 70-79. 

It was demonstrated that poly(styrene-b/oc -vinylpyrrolidone) beads (0 220 nm) are suitable for preparation of pH nanosensors [12]. Various fluorescein derivatives were embedded and did not leach out of the beads due to functionalization with highly lipophilic octadecyl anchor. The pK., of the indicators inside the nanobeads varied from 5.8 to 7.7 making them suitable for various biotechnological, biological and marine applications. The beads based on a lipophilic l-hydroxypyrene-3,6, 8-trisulfonate (pKa 6.9) were also manufactured. 

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