COMMUNICATION pubs.acs.org/bc
Facile Synthesis of Symmetric, Monofunctional Cyanine Dyes for Imaging Applications Lai-Qiang Ying* and Bruce P. Branchaud Life Technologies, 29851 Willow Creek Road, Eugene, Oregon 97402, United States
bS Supporting Information This paper was withdrawn on September 16, 2011 (DOI: 10.1021/bc200469q).
luorescent dyes are generally well-known for fluorescence labeling and detection of biomolecules, in diverse applications to studies of cells, tissues, proteins, antibodies, and nucleic acids through fluorescence microscopy, fluorescence immunoassays, and flow cytometry.1,2 The labeling of biomolecules with fluorescent dyes is generally achieved via amine-reactive functionalities such as isothiocyanate and N-hydroxysuccinimide esters3 5 or thiol-reactive iodoacetamide and maleimide groups.6 8 Near-infrared (NIR) fluorescent dyes with absorption and emission maxima over 600 nm are particular useful for in vivo imaging due to the minimal autofluorescence of biological samples in the NIR range.9 Among the NIR probes, the cyanine dyes, with large extinction coefficients and high quantum yields, are the most widely used probes for optical imaging in vivo and in single molecule fluorescence microscopy.10 17 The common, commercially available cyanine dyes such as Cy3, Cy5, and Cy7 are monofunctional and asymmetric, with a single activated carboxylic acid group attached to one of the indoleninium or benzindoleninium moieties.3,18 Cyanine dyes are usually prepared by condensing indoleninium or benzindoleninium moieties to a polymethine chain in a stepwise synthetic strategy. That synthetic approach can result in the formation of undesired symmetric difunctionalized or unfunctionalized dyes as byproducts, and the desired monofunctional products often must be purified by HPLC, resulting in lower overall yields (Scheme 1).3 Alternative syntheses of monofunctional symmetric heptamethine cyanine dyes have been developed via the reaction of chloro-substituted cyanine dyes with either (1) nucleophiles or (2) in Suzuki-Miyaura cross-coupling reactions.19 22 Many cyanine dyes exhibit low quantum yields in H2O, and many are also not very stable.23 Recently, a synthetic method has been reported for the preparation of monofunctional
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r 2011 American Chemical Society
(carboxyl, azido, and alkynyl groups) pentamethine carbocyanine dyes, based on modifications of the synthetic method using malonaldehyde dianil dye precursors.24 The compounds prepared by that method have lower quantum yields than Cy5, and they have shown florescence quenching properties when the dye is conjugated to antibodies, similar to the florescence quenching seen with Cy5.25 Development of new NIR probes with increased fluorescence brightness, improved photostability, and lower nonspecific binding is needed for improved imaging applications. Also, better, facile, high-yield syntheses of NIR probes would also be a significant improvement over current methods. In this report, we present a simple two-step procedure for the synthesis of monocarboxylate-functionalized cyanine dyes based on the pyridine-containing malonaldehyde dianil dye precursor. The monocarboxylate-functionalized cyanine dyes can be prepared in high yield (>60%) with excellent water solubility and display similar excitation and emission properties as Cy5 and Alexa Fluor 647. To evaluate the utility of the monocarboxylate functionalized cyanine dyes, these probes were conjugated with goat antimouse IgG, and used for cell imaging. The results of microtubule imaging inside cells demonstrate the potential applications of the monocarboxylate functionalized cyanine dyes for use as NIR probes. The synthesis of asymmetric cyanine dyes such as Cy5 by the stepwise condensation of indoleninium or benzindoleninium moieties to a polymethine chain always produces some of the undesired symmetric dyes as byproducts (Scheme 1). This methodology leads to low yields of the desired products and difficulty in purification. Received: February 24, 2011 Revised: April 14, 2011 Published: April 26, 2011 865
dx.doi.org/10.1021/bc2001006 | Bioconjugate Chem. 2011, 22, 865–869
