Bioconjugate Chem. 2007, 18, 1841–1846
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Synthesis and Biosensor Performance of a Near-IR Thiol-Reactive Fluorophore Based on Benzothiazolium Squaraine Joseph Thomas,* Douglas B. Sherman, Terry J. Amiss, Sandra A. Andaluz, and J. Bruce Pitner* BD Technologies, 21 Davis Drive, Research Triangle Park, North Carolina 27709. Received April 24, 2007; Revised Manuscript Received June 29, 2007
Environmentally sensitive near-IR (NIR) dyes are useful fluorophores for various biosensor applications when tissue absorption, scattering, and autofluorescence are a leading concern. Biosensors operating in the NIR region (generally wavelengths >650 nm) would avoid interference from biological media and thereby facilitate relatively interference free sensing. Squaraine dyes are potential candidates to serve as reporter molecules due to their spectral properties in the NIR region, but none is commercially available for site-specific coupling to proteins through native or engineered thiols on cysteine. In this context, we have synthesized a thiol-reactive squaraine that displays fluorescence emission above 650 nm and have coupled the dye site-specifically to various mutants of glucose/galactose binding protein that contained an engineered cysteine for attachment. Mutant E149C/A213R/ L238S ISQ GGBP gave a fluorescence change of +50% and a binding constant of 12 mM, which is in the human physiological range for glucose.
INTRODUCTION Fluorescent dyes that emit in the near-infrared region have been long sought-after for biosensor development due to their specific advantages when operating in biological media. A unique feature that makes the near-IR (NIR) dyes attractive is that their spectral properties are generally independent from biological interferences, such as tissue autofluorescence and scattering. Biological interference is minimized in the 600–900 nm wavelength region (1–4). In order for the dye to qualify as a reporter molecule in a fluorescence-based sensor assay, at least one of the fluorescence properties, such as intensity, lifetime, or wavelength, should undergo a significant change in a variety of environments. However, the availability of such dyes is limited for a number of reasons, including poor photostability, solubility, and toxicity. Moreover, the design of these dyes is complicated, since the increased number of conjugated double bonds required to achieve the NIR spectral properties leads to destabilization of the structure. Squaraines are a class of dyes that could potentially serve as reporter molecules for biosensors in the NIR region. Squaraines are 1,3-disubstituted squaric acid derivatives in which the substituents can be introduced through nucleophilic substitution reactions (5). The intense absorption of squaraine dyes arises from charge transfer transitions between the central cyclobutane ring and the oxygen moieties with a minor contribution from the substituents on either side (6). Dyes that undergo charge transfer transitions during electronic excitation are generally solvatochromic and environmentally sensitive (7). For example, bis(2,4,6-trihydroxy)phenylsquaraine has a fluorescence quantum yield of 4 Å from the rim of the binding pocket of the C-terminal domain. For N211C-ISQ, the benothiazolium ring containing the linker was located inside the binding pocket, while the remainder of the dye structure was solvent-exposed (Figure 4b). Finally, the dye in the conformers of E149C/A213R/
The squaraine dye was chosen due to its sharp absorption and fluorescence properties in the >600 nm region, which is highly sensitive to the environment. In general, the synthesis of ISQ (1) proceeded smoothly. Deprotection of the phthalimide in 1h to form 1i was first attempted using methylamine. This led to a half-protected species as identified by mass spectral and NMR analysis. Using hydrazine in place of methylamine resulted in a cleaner deprotection. In the final step to purify 1, the high reactivity of the iodoacetamide group prevented purification using column chromatography; thus, the final product was obtained by repeated precipitation from hexane and methylene chloride. The spectrophotometric properties of ISQ in chloroform and aqueous medium demonstrated the environmental sensitivity of the dye as well as the potential of ISQ for biosensor applications in the NIR region. The dye had Stokes shifts of g10 nm, and the absorption maxima in chloroform and aqueous medium were above 650 nm. The fluorescence maxima were also above 665 nm. In addition, ISQ showed remarkable fluorescence properties when conjugated to GGBP, demonstrating that the fluorescence response was significantly affected by the glucose binding event. Of the mutants studied, N211C-ISQ had the largest negative change (-45%). E149C-ISQ had a lower fluorescence change of 37% and a binding constant of 5 uM, which is slightly weaker than the binding constant of wild-type GGBP (0.2 uM) (33), but it is consistent with binding constants reported for other dyes (26) and bioconjugates (28) at this site. As shown in Figure 2, the triple mutant E149C/A213R/L238S ISQ had a larger fluorescence change of +50%, and the binding constant was 12 mM (Figure 3), which is near the midpoint of the human physiological range. We also wanted to examine molecular models of several dye–protein conjugates to relate the experimental sensor performance to the structures. The chromophoric unit of benzothiazolium squaraine dyes is highly hydrophobic. It tends to be encapsulated within hydrophobic matrices and polymers such as β-cyclodextrin (8) and polymers such as polyvinyl pyrrolidone (34). Due to this nature, to a first approximation it would be expected that movement of the dye from a more hydrophilic environment to a more hydrophobic environment would result in a positive fluorescence change, whereas movement from a more hydrophobic to a more hydrophilic environment would induce a negative change. Other types of dye–protein interactions, such as hydrogen bonding, can also play an important role in the nature of the fluorescence change (35). Conformational analysis of the dye attached to a number of sites provided qualitative support to this hypothesis. For example, ISQ was solvent-exposed and had very little interaction with the protein in both the open and closed forms of K92CISQ, which would be expected to produce little to no change in fluorescence as was observed. D14C-ISQ and N211C-ISQ had significant negative fluorescence changes. For these bioconjugates, the dye was positioned either within the binding pocket near the surface of the C-terminal domain (D14C) or partially buried inside the binding pocket (N211C) of the open form. As with K92C, the dye was solvent-exposed in the closed form for both mutants with only 1 to 2 residues
