Fluorophores fluorescein derivatives

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. 

The isothiocyanate derivative of the fluorophore, fluorescein or rhodamine, is coupled to the amino groups of IgG antibody in a one-step procedure and excess label is removed by gel filtration. 

Aminofluorescein has a low quantum yield of 0.015, whereas its amide derivatives fluoresce strongly. To our knowledge, little more is known about the relationship between the chemical structures of fluorescein derivatives and their fluorescent properties. Our working hypothesis is that the fluorescence properties of fluorescein derivatives are controlled by PeT process from donor moiety (benzoic acid) to acceptor moiety (fluorophore) (Fig. la). 

Characteristics of proposed CL reaction. In the preliminary study, CL intensities of several types of fluorophores were measured after mixing with only aryloxalate and without addition of hydrogen peroxide. DBP derivatives and pyrimidopyrimidine derivatives showed CL, but the CL was not observed with typical fluorophores that are frequently used in conventional PO-CL reactions such as dansyl, benzoxazole, lophine and fluorescein derivatives. Therefore, it is proposed that this CL reaction can detect DBP derivatives selectively in the presence of other fluorophores. 

Other fluorophores used include thiazole orange (91) used as an artificial nucleobase, to label aptamers, and with two of the dyes attached to the C5 position of dC to act as a sensitive probe for oligonucleotide hybridisation.Various fluorescein derivatives have been used, including a novel fluorescein phosphoramidite that has been described, and a novel dC-modified derivative as well as some modifications that are formed during oligonucleotide synthesis with the fluorescein HEX. Other derivatives have been used to investigate DNA hybridisation under... 

Fluorescein and rhodamine are common fluorophores used for the fluorescent labeling of proteins in biological applications. This is due to a number of advantageous properties such as long absorption maxima, insensitivity to solvent polarity, high molar extinction coefficient, and the availability of a wide variety of reactive derivatives. Fluorescein derivatives are characterized by a multi-ring aromatic structure, due to the planar nature of an upper, fused, three-ring system (Fig. 2). Derivatives of the basic structure... 

Fig. 6.21. Principle of detection of lipopolysaccharide (LPS) with the CD14-derived probe. It relies on the formation of a ground state complex between fluorescein and rhodamine in aqueous solution with quenching of donor and acceptor fluorescence. Spectrum A shows hypothetical fluorescence emission spectra of this complex. After LPS binding, the peptide sequence gets straightened prohibiting the close contact between the two fluorophores and leading to the recovery of red fluorescence (Spectra B).

The intense Texas Red fluorophore has a QY that is inherently higher than the tetrameth-ylrhodamine or Lissamine rhodamine B derivatives. Texas Red s luminescence is shifted maximally into the red region of the spectrum, and its emission peak only minimally overlaps with that of fluorescein. This makes Texas Red derivatives among the best choices of labels for use in double-staining techniques. 

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 characteristics of Lucifer Yellow iodoacetamide produce luminescence at somewhat higher wavelengths than the green luminescence of fluorescein, thus the yellow designation in its name. The excitation maximum for the probe occurs at 426 nm and its emission at 530 nm. The rather large Stoke s shift makes sensitive measurements of emission intensity possible without interference by scattered excitation light. The 2-mercaptoethanol derivative of the fluorophore has an extinction coefficient at pH 7 of about 13,000 M cm-1 at 426nm. 

DNA modified with a diamine compound to contain terminal primary amines may be coupled with amine-reactive fluorescent labels. The most common fluorophores used for oligonucleotide labeling are the cyanine dyes and derivatives of fluorescein and rhodamine (Chapter 9). However, any of the amine-reactive labels discussed throughout Chapter 9 are valid candidates for DNA applications. 

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