TY - JOUR
T1 - Fluorescent rhodol derivatives
T2 - Versatile, photostable labels and tracers
AU - Whitaker, James E.
AU - Haugland, Rosaria P.
AU - Ryan, Diane
AU - Hewitt, Peter C.
AU - Haugland, Richard P.
AU - Prendergast, Franklyn G.
N1 - Funding Information:
We gratefully acknowledge the technical support of Salah Sedar-ous, Phyllis Fischer, John Hedstrom, and Peter Callahan of the Mayo Foundation as well as Leslie Daniel, Fred Warner, Christina Fox, Wojciech Szalecki, Bruce Bondurant, John Metsner, Ian MacCou-brey, Eugene Pustay, Nan Minchow, and Lisa Wright of Molecular Probes, Inc. This work was supported by NIH Grant GM37347 and DOE Grant FGO6-SSER-60684.
PY - 1992/12
Y1 - 1992/12
N2 - A series of chemically reactive, fluorescent rhodol derivatives was prepared and evaluated. Reactive functional groups included activated esters, amines, haloacetamides, fixable hydrazide derivatives, acrylamides, and photoaffinity reagents. Depending on the choice of substituents, absorption maxima of the dyes varied from 490 to 550 nm with extinction coefficients that were generally greater than 50,000 m-1 cm-1 in aqueous solution and emission maxima from 520 to 580 nm. Most of the compounds investigated exhibited fluorescence lifetimes between 3 and 4 ns. Individual derivatives were suitable for excitation with the 488 and 514-nm lines of the argon ion laser and the 546-nm line of the mercury arc lamp and were compatible for use with standard fluorescein and rhodamine filter sets. The rhodol dyes were more photostable and less sensitive to pH changes in the physiological range than fluorescein derivatives. Some examples show absorption maxima at or near 514 nm, an excitation wavelength that is useful for multicolor fluorescence microscopy, flow cytometry, and DNA sequencing. Derivatives were also prepared that exhibit absorption and emission maxima similar to those of tetramethylrhodamine (TMR) analogs but with higher quantum yields in aqueous solution. A number of the dyes had higher solubilities in aqueous systems and were less quenched on conjugation to proteins than TMR derivatives. Appropriate substitution results in a wider range of solubilities in hydrophilic or lipophilic solvents than is easily accomplished with fluorescein or TMR derivatives. Conjugates of a number of the rhodol fluorophores were generally more photostable and less pH sensitive than fluorescein conjugates and more fluorescent than TMR conjugates.
AB - A series of chemically reactive, fluorescent rhodol derivatives was prepared and evaluated. Reactive functional groups included activated esters, amines, haloacetamides, fixable hydrazide derivatives, acrylamides, and photoaffinity reagents. Depending on the choice of substituents, absorption maxima of the dyes varied from 490 to 550 nm with extinction coefficients that were generally greater than 50,000 m-1 cm-1 in aqueous solution and emission maxima from 520 to 580 nm. Most of the compounds investigated exhibited fluorescence lifetimes between 3 and 4 ns. Individual derivatives were suitable for excitation with the 488 and 514-nm lines of the argon ion laser and the 546-nm line of the mercury arc lamp and were compatible for use with standard fluorescein and rhodamine filter sets. The rhodol dyes were more photostable and less sensitive to pH changes in the physiological range than fluorescein derivatives. Some examples show absorption maxima at or near 514 nm, an excitation wavelength that is useful for multicolor fluorescence microscopy, flow cytometry, and DNA sequencing. Derivatives were also prepared that exhibit absorption and emission maxima similar to those of tetramethylrhodamine (TMR) analogs but with higher quantum yields in aqueous solution. A number of the dyes had higher solubilities in aqueous systems and were less quenched on conjugation to proteins than TMR derivatives. Appropriate substitution results in a wider range of solubilities in hydrophilic or lipophilic solvents than is easily accomplished with fluorescein or TMR derivatives. Conjugates of a number of the rhodol fluorophores were generally more photostable and less pH sensitive than fluorescein conjugates and more fluorescent than TMR conjugates.
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U2 - 10.1016/0003-2697(92)90011-U
DO - 10.1016/0003-2697(92)90011-U
M3 - Article
C2 - 1481981
AN - SCOPUS:0026987490
SN - 0003-2697
VL - 207
SP - 267
EP - 279
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -