Method To Diversify Cyanine Chromophore Functionality Enables Improved Biomolecule Tracking and Intracellular Imaging

Abstract

Heptamethine indocyanines are invaluable probes for near-infrared (NIR) imaging. Despite broad use, there are only a few synthetic methods to assemble these molecules, and each has significant limitations. Here, we report the use of pyridinium benzoxazole (PyBox) salts as heptamethine indocyanine precursors. This method is high yielding, simple to implement, and provides access to previously unknown chromophore functionality. We applied this method to create molecules to address two outstanding objectives in NIR fluorescence imaging. First, we used an iterative approach to develop molecules for protein-targeted tumor imaging. When compared to common NIR fluorophores, the optimized probe increases the tumor specificity of monoclonal antibody (mAb) and nanobody conjugates. Second, we developed cyclizing heptamethine indocyanines with the goal of improving cellular uptake and fluorogenic properties. By modifying both the electrophilic and nucleophilic components, we demonstrate that the solvent sensitivity of the ring-open/ring-closed equilibrium can be modified over a wide range. We then show that a chloroalkane derivative of a compound with tuned cyclization properties undergoes particularly efficient no-wash live cell imaging using organelle-targeted HaloTag self-labeling proteins. Overall, the chemistry reported here broadens the scope of accessible chromophore functionality, and, in turn, enables the discovery of NIR probes with promising properties for advanced imaging applications.

Expedient Synthesis and Characterization of π-Extended Luciferins

“…In conclusion, the chemistry of π-extended systems is challenging due to the potential for undesired reactivity on the exposed polymethine system. − Expanding the repertoire of strategies for modifying π-systems has proven to be invaluable to the development of other polymethine-containing fluorescent probes. , To expand the portfolio of BL probes, we developed a two-step synthesis for accessing structurally diverse vinyl-containing luciferins. As a starting point, we utilized this two-step method to improve the synthesis of the extensively characterized luciferin, AkaLumine.…”

“…The title compound was produced by following the general procedure using 3-[7-(diethylamino)-2-oxo-2 H -chromen-3-yl]acrylaldehyde (270 mg, 1.0 mmol) and diethyl cyanomethylphosphonate (250 mg, 1.25 mmol). Purification by flash chromatography on silica gel (hexanes/EtOAc, 0% to 50%) afforded 6g as a red solid (190 mg, 63% yield): 1 H NMR (CDCl 3 , 500 MHz, compound exists as a mixture of isomers, Z isomer denoted by an asterisk, E isomer denoted by §) δ 7.72 (s, 1H*), 7.54 (dd, J = 12.1, 6.9 Hz, 1H § ), 7.48 (s, 1H § ), 7.44–7.41 (m, 1H*), 7.38–7.33 (m, 1H § ), 7.26–7.23 (m, 1H § ), 7.21–7.20 (m, 1H*), 7.17–7.15 (m, 1H*), 6.97 (d, J = 11.1 Hz, 1H*), 6.93 (dd, J = 13.0, 13.0 Hz, 1H § ), 6.83 (dd, J = 11.0, 11.0 Hz, 1H*), 6.59 (d, J = 15.3 Hz, 1H § ), 6.50–6.47 (m, 1H*, 1H § ), 6.36 (s, 1H § ), 6.36 (s, 1H*), 5.29 (d, J = 15.7 Hz, 1H § ), 5.08 (d, J = 10.6 Hz, 1H*), 3.31 (q, J = 7.2 Hz, 2H*, 4H § ), 1.10 (t, J = 7.2 Hz, 3H*, 6H § ); 13 C{ 1 H} NMR (CDCl 3 , 125 MHz, compound exists as a mixture of isomers, Z isomer denoted by an asterisk, E isomer denoted by §) δ 161.32 § , 160.57*, 156.62 § , 156.32*, 151.59 § , 151.55*, 151.30 § , 149.73*, 142.53 § , 139.69*, 136.44 § , 135.69*, 132.27 § , 132.19 § , 132.14*, 132.12*, 129.91*, 129.70 § , 128.71*, 128.61*, 127.39 § , 124.32 § , 118.90 § , 117.12*, 115.60*, 115.52 § , 109.68*, 109.64 § , 108.94*, 108.92 § , 97.73 § , 97.28*, 97.13 § , 95.76*, 45.15 § , 45.14*, 29.83 § , 29.49*, 12.61 § , 12.60*; HRMS (ESI) calcd for C 18 H 18 N 2 O 2 (M + H) + 295.1446, observed 295.1445.…”

Engineered fluorogenic HaloTag ligands for turn-on labelling in live cells

“…5 has excellent properties that will make it the fluorogenic dye of choice for well-established HaloTag systems used in cell biology, pharmacology, and drug design. 19,20,27,28 For example, 5 is smaller than other fluorogenic HaloTag substrates 8,29–31 and the HaloTag· 5 complex is spectrally orthogonal to many commonly used rhodamine dyes. The fluorogenic HaloTag· 5 system will enable many no-wash, real-time assays for subcellular detection and localization.…”

Development of Iodinated Indocyanine Green Analogs as a Strategy for Targeted Therapy of Liver Cancer

“…42 Therefore, we synthesized iodine-containing ICG (I-ICG) analogs (Figure 1) using a synthetic methodology recently reported from our group (Figure 1A). 43 We focused on exploring whether installing iodine within the cyanine structure could improve the in vivo pharmacokinetic and liver tumor targeting properties of ICG.…”

“…Iodine-131 is routinely used alone or conjugated to tumor-selective molecules as a radiopharmaceutical therapy agent, − and iodine-124 can be used for positron emission tomography (PET) imaging . Therefore, we synthesized iodine-containing ICG (I-ICG) analogs (Figure ) using a synthetic methodology recently reported from our group (Figure A) . We focused on exploring whether installing iodine within the cyanine structure could improve the in vivo pharmacokinetic and liver tumor targeting properties of ICG.…”