Rhodamines structure

Four forms of amine-reactive rhodamine probes are commonly available. Two of them are based on the tetramethyl derivatives of the fundamental rhodamine structure, one is based on the sulforhodamine B or Lissamine derivative, and the last is the sulforhodamine 101 or Texas Red-type of derivative. All of them react under alkaline conditions with primary amines in proteins and other molecules to form stable, highly fluorescent complexes. 

The effect of forming a more rigid structure in fluorescent dyes of the rhodamine series has been clearly demonstrated (18) with the remarkable dye designated Rhodamine 101 [41175A3-3] (19). This dye has its terminal nitrogen atoms each held in two rings and has a fluorescence quantum yield of virtually 100% independent of the temperature. 

Eisenthal and coworkers have also measured interfacial friction via the rotational dynamics of probe molecules at the interface. In their first study, Eisenthal and coworkers probed the rotational dynamics of rhodamine 6G (R6G, structure shown in Fig. 2) at the... 

In most cases, the linear absorption is measured with standard spectrometers, and the fluorescence properties are obtained with commercially available spectrofluo-rometers using reference samples with well-known <1>F for calibration of the fluorescence quantum yield. In the ultraviolet and visible range, there are many well-known fluorescence quantum yield standards. Anthracene in ethanol (Cresyl Violet in methanol (commonly used reference samples for wavelengths of 350-650 nm. For wavelengths longer than 650 nm, there is a lack of fluorescence references. Recently, a photochemically stable, D-ji-D polymethine molecule has been proposed as a fluorescence standard near 800 nm [57]. This molecule, PD 2631 (chemical structure shown in Fig. 5) in ethanol, has linear absorption and fluorescence spectra of the reference PD 2631 in ethanol to... 

Fig. 6 General structure of xanthene dyes containing fluorescein, eosin, and rhodamine...

Due to their longer wavelength fluorescence and photostability, rhodamine and its derivatives have often been employed for the labeling of probes tested in living cells. Like fluorescein, the chemical structure of rhodamine consists of an upper xanthene ring and a lower benzene ring. In this case, the xanthene ring is substituted... 

Fig. 6.3. Chemical structures of rhodamine and some derivatives. TAMRA = N,N,TV,A-tetramethylrhodamine. Lissamine rhodamine = 3,5-disulfonyl-N,N, A,A-tetramethylrhodamine.

A different strategy for measuring protease activity is based on the property of xanthene dyes to form H-type dimers (see Sect. 6.2.3) when they are in close proximity. These dimers are accompanied with a characteristic quenching of their fluorescence and, particularly for rhodamines, with a blue shift in the absorption spectrum [121, 122]. The probe D-NorFES-D designed to measure activity of elastase in HL-60 cells consists of an undecapeptide derivatized with one tetramethylrhodamine dye on each side. The sequence contains proline residues to create a bent structure and bring the two fluoro-phores in close proximity. Intact D-NorFES-D shows 90% of its fluorescence quenched plus a blue shift of the absorption spectrum. After addition of the serine protease elastase, an increase in the fluorescence and a bathochromic shift of the absorption spectrum is observed, resulting in an increase in the emission ratio [80],... 

NHS-rhodamine is an amine-reactive fluorescent probe that contains a carboxy-succinimidyl ester group off the No. 5 or 6 carbons on rhodamine s lower-ring structure (Kellogg et al., 1988). The 5- and 6-isomers are virtually identical in their reactivity and fluorescent characteristics. Similar to TRITC (described previously), NHS-rhodamine can be used to label proteins and other macromolecules that contain primary amine groups. The isomeric forms of the fluorescent probe are available in mixed and purified forms (Invitrogen, Thermo Fisher). The pure forms are... 

Texas Red hydrazide is a derivative of Texas Red sulfonyl chloride made by reaction with hydrazine (Invitrogen). The result is a sulfonyl hydrazine group on the No. 5 carbon position of the lower-ring structure of sulforhodamine 101. The intense Texas Red fluorophore has a QY that is inherently higher than either the tetramethylrhodamine or Lissamine rhodamine B derivatives of the basic rhodamine molecule. 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 derivatives of this fluorescent probe among the best choices of labels for use in double-staining techniques. 

The most significant parent structure for pigments of this group is obtained by a slightly modified route by simply using phthalic anhydride instead of aldehyde. Reaction with m-diethylaminophenol at 180°C in the presence of sulfuric acid or zinc chloride and subsequent oxidation with iron(III)chloride thus affords a dye known as Rhodamine B (132), the basis of Pigment Violet 1 ... 

Figure 5 shows two typical core-shell structures (a) contains a metal core and a dye doped silica shell [30, 32, 33, 78-85] and (b) has a dye doped silica core and a metal shell [31, 34]. There is a spacer between the core and the shell to maintain the distance between the fluorophores and the metal to avoid fluorescence quenching [30, 32, 33, 78-80, 83]. Usually, the spacer is a silica layer in this type of nanostructures. Various Ag and Au nanomaterials in different shapes have been used for fluorescence enhancement. Occasionally, Pt and Au-Ag alloys are selected as the metal. A few fluorophores have been studied in these two core-shell structures including Cy3 [30], cascade yellow [78], carboxyfluorescein [78], Ru(bpy)32+ [31, 34], R6G [34], fluorescein isothiocyanate [79], Rhodamine 800 [32, 33], Alexa Fluor 647 [32], NIR 797 [82], dansylamide [84], oxazin 725 [85], and Eu3+ complexes [33, 83]. 

TLC coupled with mass spectrometry employing desorption electrospray ionization has been used for the separation of synthetic dyes. The chemical structures of dyes included in the investigation are shown in Fig. 3.7. ODS HPTLC plates (10 X 10 cm) were used as the stationary phase the mobile phase consisted of methanol-tetrahydrofuran (60 40, v/v) containing 50-100 mM ammonium acetate for the positive-ion test and of methanol-water (70 30, v/v) for the negative-ion test. Test mixtures for negative- and positive-ion mode detection consisted of methyleneblue, crystal violet, rhodamine 6G... 

Another study employed CE for the determination of the stoichiometry of the conjugation reaction between immonuglobulin and Lissamine rhodamine-B sulphonyl chloride (LRSC). The chemical structure of the dye is shown in Fig. 3.162. Separation of the unconjugated dye from the conjugated end product was performed by CE using an uncoated fused-silica capillary column (60 cm X 75 //m i.d.). The running buffer consisted of 10 rnM borate and 0.5 mM sodium dodecyl sulphate. The separation voltage was 20 kV and analytes were detected by a fluorescence detector. It was concluded from the results that the CE method combined with... 

Figure 5.16. Various structures of triphenylmethane and Rhodamine dyes.

M. Vogel, W. Rettig, R. Sens, and K. H. Drexhage, Structural relaxation of rhodamine dyes with different N-substitution patterns A study of fluorescence decay times and quantum yields. Chem. Phys. Lett. 147,452-460 (1988). 

Figure 14.2. Chemical structures of some commonly used organic fluorescent probes 1, fluorescein-5-isothiocyanate (FITC) 2, tetramethylrhodamine-5-isothiocyanate (TRITC) 3, 5-carboxyrhodamine B 4, rhodamine X isothiocyanate (XRITC) 5, malachite green isothiocyanate 6, eosin-5-isothiocyanate 7, 1-pyreneisothiocyanate 8, 7-dimethylaminocoumarin-4-acetic acid 9, CY5.180Su.

Liposomes (without peptide) were labelled with rhodamine-PE in the lipid bilayer and in the inner compartment using FITC-dextrane 9000 and incubated with DCs for one hour. Figure 3 shows clearly that only in the case of AVE 3 and AVE 43 could a significant uptake be observed. The fluorescence inside the DCs is in the case of AVE 3 homogenously distributed in the cytosol, whereas in the case of AVE 43 the liposomes seem to be caught in granular structures, presumably endosomes. The PS causes the liposomes... 

Figure 1 shows the chemical structure of representative mitochondriotropic molecules. The most widely used among them is Rhodamine 123... 

Figure 1 Chemical structures of commonly used typical mitochondriotropic molecules (A) rhodamine 123 (B) methyltriphenylphosphonium (Q dequalinium chloride.

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