Abstract
Convenient labeling of proteins is important for observing its function under physiological conditions. In tissues particularly, heptamethine cyanine dyes (Cy-7) are valuable because they absorb in the near-infrared (NIR) region (750–900 nm) where light penetration is maximal. In this work, we found Cy-7 dyes with a meso-Cl functionality covalently binding to proteins with free Cys residues under physiological conditions (aqueous environments, at near neutral pH, and 37 °C). It transpired that the meso-Cl of the dye was displaced by free thiols in protein, while nucleophilic side-chains from amino acids like Tyr, Lys, and Ser did not react. This finding shows a new possibility for convenient and selective labeling of proteins with NIR fluorescent probes.
Highlights
Hydrophilic near-infrared (NIR) fluorescent dyes are valued for in-depth imaging in tissues, and heptamethine cyanines, or Cy-7 dyes, which absorb in the NIR region (700–900 nm), are amongst the most widely used [1]
Many applications of Cy-7 dyes require that they be covalently conjugated to, for example, antibodies, cell surface targeting peptides/biomarkers, and small molecule substrates. This is often achieved by modifying Cy-7 derivatives with coupling functionalities such as maleimide, succinimide esters, isocyanates, or sulfonyl halides
The challenge with strategies like this is balancing the demands of experimental convenience with selectivity towards targeted amino acid types
Summary
Hydrophilic near-infrared (NIR) fluorescent dyes are valued for in-depth imaging in tissues, and heptamethine cyanines, or Cy-7 dyes, which absorb in the NIR region (700–900 nm), are amongst the most widely used [1]. Indocyanine green (ICG, Figure 1), the only FDA-approved Cy-7 dye, has been widely used in medical and clinical diagnostics [2,3,4]. Many applications of Cy-7 dyes require that they be covalently conjugated to, for example, antibodies, cell surface targeting peptides/biomarkers, and small molecule substrates. This is often achieved by modifying Cy-7 derivatives with coupling functionalities such as maleimide, succinimide esters, isocyanates, or sulfonyl halides. The challenge with strategies like this is balancing the demands of experimental convenience with selectivity towards targeted amino acid types.
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