Ten carboxymethylindocyanine dyes which form the basis of a new series of fluorescent probes have been synthesized and converted into succinimidyl active esters for fluorescent labeling of proteins or other amino-containing substances. Fluorescence emission maxima for members of the series range from 575 to 780 nm. Hydrophilic, water-soluble reagents have been obtained which yield labeled antibodies with little tendency to form precipitates. The fluorescence intensities achieved are higher than those produced by labeling with the cyanine isothiocyanates described previously (Mujumdar et al.: Cytornetry 1011-19, 1989). The utility of these reagents has been demonstrated in antibody labeling for two-color immunofluorescent imaging of internal structures in a mammalian cell and for two-color flow-cytometry experiments. The use of values of chromophore-equivalent weight (W/Ceq), calculated from quantitative absorption data on dye samples, is proposed as an aid in formulating labeling procedures.Key terms: Fluorescent biolabeling probes, fluorescent cyanine dyes, succinimidyl ester biolabeling probes, twocolor immunofluorescent imaging, longwavelength fluorescent probes, flow cytometry, chromophore equivalent weight Earlier papers from this laboratory (4,111 have described the preparation of two series of cyanine and merocyanine dyes which are useful as fluorescent labeling reagents for biological investigations. Functional groups utilized in these reagents for covalent linking to biomolecules have been the iodoacetamido group, for sulfhydryl tagging, and the isothiocyanato moiety, for attachment to primary or secondary amino groups. The incorporation of carboxylic acid groups into the basic cyanine structure was expected to permit fluorescent labeling through the use of derived active esters. The synthesis of (2,3,3-trimethyl-3-H-indol-5-yl)-acetic acid and derived intermediates suitable for the construction of highly fluorescent carboxyl-containing indocyanine dyes has been described recently (12).Here we describe the preparation of the dyes themselves, the procedures used for converting the carboxylic acid groups into active ester functions (esters of N-hydroxysuccinimide) which are reactive toward proteins, and procedures for attaching these dyes to various proteins. Advantages which characterize reagents in this group include ease of preparation, higher activity toward protein substrates in aqueous media under mild conditions, and the high extinction coefficients and quantum yields required for intense fluorescence. As with the other dyes treated in this series of papers, the fluorescence emission maxima are to be found from the long-wavelength portion of the visible range into the near infrared, well separated from the shorterwavelength autofluorescence which is characteristic of many biological specimens and which presents a difficulty in working with such fluorophores as fluorescein.
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