Site-Specific Labeling of Proteins with Near-IR Dyes

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 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 near-IR fluorescent probes.

Role of Albumin in Accumulation and Persistence of Tumor-Seeking Cyanine Dyes

“…Instantaneous red-shift from 780 to 805 nm, followed by a slower blue-shift to 791 nm, can be explained in the following way. Albumin rapidly forms a noncovalent adduct with MHI-148, 1-Cl, followed by slower transformation into a Cys 34 -bound covalent adduct, consistent with the data we reported previously, 34,35 and the work of Canovas et al 37 Support for this assertion comes from the corresponding experiment with the meso-blocked dye 1-Ph (Figure 3). For 1-Ph, Figure 3a shows addition of albumin led to instantaneous formation to a new UV spectrum with a red-shifted absorption maximum, which is invariant over 5 h, the time span of the experiment.…”

“…Covalent bond formation of dyes 1-Cl−4-Cl was supported via HPLC analyses, and previous work by our group 34,35 and by Conovas et al 37 The current study (Figure S11) showed that relative rates for the formation of covalent adducts were (at 37 °C with 2 equiv of HSA): 4-Cl (IR780; t 1/2 2 min) ≫ 1-Cl (6 h) > 3-Cl (24 h) > 2-Cl (72 h), and formation of an adduct from 5-Cl only proceeded to ~30% after 72 h under these conditions (data not shown). In vivo we expect the dyes to convert to covalent adducts more rapidly due to the high concentration of albumin in serum.…”

“…As mentioned above, 1-Cl reacts relatively rapidly under physiological conditions with the free Cys 34 thiol of albumin via displacement of the mesochloride. 34,35 Reaction of 5-Cl (QuatCy) 38 with albumin is much slower under physiological conditions, but it will eventually form an analogous covalent adduct. 39 This difference is related to the extra positive charge on the QuatCy core (5) relative to framework 1 which impacts the electrostatic affinity of the two dyes toward {protein-based} nucleophiles, their water solubilities, logP (reflecting partitioning of ions) and logD (partitioning of all neutral species and ions) values.…”

“…Previous work 34,35 had already established that 1-Cl covalently binds albumin under physiological conditions, and that reaction is not instantaneous, so UV and fluorescence spectroscopies were used to probe the short-term effects of albumin and serum on this dye. Combinations of 1-Cl with HSA and of 1-Cl with serum (i.e., 10% FBS in DMEM buffer) both gave an instant red shift in the UV (and fluorescence).…”

“…Recently, we discovered 35 that the meso-chloride of 1-Cl is displaced by S-nucleophiles under physiological conditions, including the free Cys residue in serum albumins (Cys 34 in human serum albumin, HSA). Thus, in serum, where there is an extremely high concentration of albumin (around 35−50 g/L or 0.53−0.73 mM in humans), 36 1-Cl may form a covalent adduct with a half-life of just over 30 min.…”