Near-infrared dichromic fluorescent carbocyanine molecules.

Central to major advances in biochemical assays, molecular sensor technologies, and molecular optical imaging are fluorescent materials that provide high detection sensitivity for molecular processes. In biological optical imaging, the low tissue autofluorescence and the deep penetration of light into the tissues observed at wavelengths between 650 and 900 nm allow the use of near-infrared (NIR) fluorescent dyes as contrast agents in heterogeneous systems. The ease of synthesis, biocompatibility, tunable spectral properties, and exceptionally high molar absorptivity of NIR fluorescent carbocyanines provide added incentive for their use in cellular and animal imaging applications. For these reasons, a variety of cyanine-based molecular probes are currently used for molecular imaging—including cypate (1), which possesses reactive carboxylic acid groups and the nonspecific indocyanine green (ICG, 2) that incorporates two hydrophilic sulfonate groups (see Scheme 1). To improve the hydrophilicity of cypate and minimize potential side reactions caused by the presence of the two carboxylic acid functions, we synthesized a hybrid of cypate and ICG. The new compound, ICG–CO2H (3), has only one reactive carboxylic acid and a water-solubilizing sulfonate group. Similarly, we prepared an analogous amine derivative, namely, ICG-NH2 (4) for labeling peptides or proteins via their activated carboxylic acid groups. In the course of these studies, we observed unusual spectral properties of the cyanine dyes that are amplified by the nonsymmetrical structural features of compounds 3 and 4. In previous studies, we and others have consistently reported the characteristic broad absorption and fluorescence emission bands of NIR carbocyanine dyes. The spectral profiles of the absorption band typically possess a blue-shifted shoulder, which is generally attributed to the presence of H aggregates or other forms of electronic transitions. Previous literature reports have shown that nonsymmetrical cyanine dyes can generate multiple fluorescence from different excited states. These emissions are generally dependent on the solvents, the pH or the electrolytes used. Here, we present the first examples of NIR dichromic fluorescent molecules based on a carbocyanine dye molecular template. This phenomenon is favored by nonsymmetrical cyanine dye structures and manifested by an increase in the blue-shifted shoulder peak with minimal solvatochromic effect. The absorption and emission spectra of representative compounds studied are shown in Figure 1. In addition to possessing the expected long-wavelength fluorescence, the nonsymmetrical molecular probes 3 and 4 also exhibited a second emission band (at 700 nm), which is blue-shifted relative to the peak at about 800 nm. Initially, we attributed the second peak to the presence of impurities or the half-dye. However, after careful purifications and sample analysis, we confirmed that both emissions originated from the same compound. To further explore the prevalence of this phenomenon in NIR carbocyanine dyes, we prepared and evaluated the spectral properties of the widely used and symmetrical NIR dyes 1 and 2 as well as those of the nonsymmetrical cypate–peptide conjugate 5. As shown in Figure 1, an excellent overlap of the absorbance spectra of the symmetrical (1 and 2) and nonsymmetrical (3 and 4) compounds was observed, with 3 and 4 exhibiting a higher absorbance than other compounds at about 700 nm. This trend persisted in different solvents, thus Scheme 1. Chemical structures of NIR fluorescent carbocyanines and derivatives.