A Near-IR Uncaging Strategy Based on Cyanine Photochemistry

The development of photocaging groups activated by near-IR light would enable new approaches for basic research and allow for spatial and temporal control of drug delivery. Here we report a near-IR light-initiated uncaging reaction sequence based on readily synthesized C4′-dialkylamine-substituted heptamethine cyanines. Phenol-containing small molecules are uncaged through sequential release of the C4′-amine and intramolecular cyclization. The release sequence is initiated by a previously unexploited photochemical reaction of the cyanine fluorophore scaffold. The uncaging process is compatible with biological milieu and is initiated with low intensity 690 nm light. We show that cell viability can be inhibited through light-dependent release of the estrogen receptor antagonist, 4-hydroxycyclofen. In addition, through uncaging of the same compound, gene expression is controlled with near-IR light in a ligand-dependent CreERT/LoxP-reporter cell line derived from transgenic mice. These studies provide a chemical foundation that we expect will enable specific delivery of small molecules using cytocompatible, tissue penetrant near-IR light.


S2
General Materials and Methods. Unless stated otherwise, reactions were conducted in ovendried glassware under an atmosphere of nitrogen or argon using anhydrous solvents (passed through activated alumina columns). All commercially obtained reagents were used as received.
Preparative HPLC was performed with a Waters ® 2545 Binary Gradient Module equipped with a Waters ® 2767 Sample Manager fraction collector and a Luna 10 μm C18 110 Å (75 x 30 mm) column obtained from Phenomenex, Inc. High-resolution LC/MS analyses were conducted on a Thermo-Fisher LTQ-Orbitrap-XL hybrid mass spectrometer system with an Ion MAX API electrospray ion source in positive ion mode. Analytical LC/MS was performed using a Shimadzu LCMS-2020 Single Quadrupole utilizing a Kinetex 2.6 μm C18 100 Å (2.1 x 50 mm) column obtained from Phenomenex Inc. Runs employed a gradient of 0→90% MeOH/0.1% aqueous formic acid over 4.5 min at a flow rate of 0.2 mL/min. 1 H NMR and 13 C NMR spectra were recorded on Varian and Bruker spectrometers (at 400 or 500 MHz or at 100 or 125 MHz) and are reported relative to deuterated solvent signals. Data for 1 H NMR spectra are reported as follows: chemical shift (δ ppm), multiplicity, coupling constant (Hz), and integration. Data for 13 C NMR spectra are reported in terms of chemical shift. IR spectra were recorded on a Varian 640-IR spectrometer and are reported in terms of frequency of absorption (cm -1 ). 96-well plate-based absorbance and fluorescence measurements were performed on a BioTek Synergy 2 multi-mode microplate reader operated by Gen5 2.01 software. Coumarin fluorescence was monitored using a 360/40 nm excitation filter and a 460/40 nm emission filter. Absorbance traces for quantum yield measurements were performed on a Shimadzu UV-2550 spectrophotometer operated by UVProbe 2.32 software. Fluorescence traces and quantum yield measurements were recorded on a PTI QuantaMaster steady-state spectrofluorimeter operated by FelixGX 4.0.3 software, with 5 nm excitation and emission slit widths, 0.1 s integration rate, and enabled emission correction. Data analysis and curve fitting were performed using MS Excel 2011 and GraphPad Prism 6. Light intensity measurements were performed with a Thorlabs PM200 optical power and energy meter fitted with an S120VC standard Si photodiode power sensor (200-1100 nm, 50 mW). Flow cytometry was performed at the CCR Flow Cytometry Core (NCI-Frederick) using a BD LSRII SORP analyzer operating laser lines at 488 nm (EGFP) and 561 nm (tdTomato). Cellular imaging was performed using a Zeiss LSM UV510 confocal laser-scanning microscope. Cells S3 were plated on Nunc Lab-Tek ® II chambered #1.5 German borosilicate coverglass (Thermo Fisher Scientific, Inc.). Hoescht 33342 staining (1 µM incubated for 1 h) was imaged using a Diode 405-30 laser (405 nm ex., 420-480 nm bandpass em.), EGFP using an Argon/2 laser (488 nm ex., 505-550 bandpass em.), LysoTracker ® Red DND-99 staining (0.5 µM incubated for 3 h) using a DPSS laser (561 nm ex., 585-600 nm spectral em.), and 11 using a HeNe633 laser (633 nm ex., 650 longpass em.). All images result from 63x magnification using a plan-apochromat oil immersion objective. Image processing was conducted with ImageJ 1.47.

Experimental Procedures
General Procedure for Synthesis of Carbamates 6-8       The green solution was heated to 90 °C in a sealed vial for 10 min as the reaction color transitioned from green to dark blue. LC/MS analysis showed the consumption of 5. The reaction was cooled to room temperature and precipitated into Et 2 O (40 mL). The slurry was centrifuged, the supernatant discarded, and the blue pellet was resuspended in Et 2 O (20 mL). The procedure was repeated, and the blue pellet was dissolved in DMF (5 mL). Diisopropylethylamine (44 µL, 0.25 mmol) was added, followed by dropwise addition of phenylchloroformate (17 µL, 0.14 mmol) in DMF (0.1 mL). The reaction was stirred for 10 min at room temperature. After this time the solution was precipitated into Et 2 O (80 mL), centrifuged, and the crude pellet was purified by reversed-phase preparative HPLC (10→95% (MeCN/0.1% v/v NH 4 OH/water). After lyophilization, 9 was obtained (60 mg, 52%) as a dark blue solid. 1  2 With N-methyl carbamates 9, 10, and 11, high temperature NMR (65 °C) was required to resolve the carbamate rotomers. However, these sulfonates proved unstable over many hours at these elevated temperatures in CD 3 OD (as well as several other solvents), which precluded obtaining 13 C NMRs.

Determination of Quantum Yields and Molar Absorption Coefficients
Quantum yields (Φ f ) were determined in DMSO or PBS relative to ICG (Φ f = 0.13 in DMSO 6 , 0.012 in PBS 7 ), from plots of integrated fluorescence intensity vs. absorbance, according to the following relationship: where subscripts st and x denote standard and test sample, respectively, Φ is the fluorescence quantum yield, Grad is the gradient of the integrated fluorescence intensity vs. absorbance plot, and η is the refractive index of the solvent. 8,9,10,11 Measurements were performed in 10 mm path length quartz cuvettes (Hellma 111-QS), maintained at 25 °C, with the absorbance of all dye solutions ≤ 0.08 in order to maximize illumination homogeneity and optical transparency and minimize reabsorption effects. 10 (Table S1).

Procedure for Relative Ion Analysis of Photolysis of 10
A 20 mM stock of 10 in DMSO was diluted into water to yield a 10 µM solution.
Phenylalanine (40 µM) was used as an internal standard. The photolysis was run for 30 min at rt with 10 mW/cm 2 690 nm (± 20 nm) light in a 12-well plate. After 30 min, the reaction was  Mouse embryonic fibroblasts (MEFs) were obtained as described below and maintained at 37 °C in an atmosphere of 20% O 2 and 5% CO 2 in DMEM supplemented with 4 mM Lglutamine, 25 mM D-glucose, 44 mM sodium bicarbonate, 1% (v/v) penicillin-streptomycin, and 10% heat-inactivated fetal bovine serum. Stock cultures were maintained as above.

Cell Photolysis and Cytotoxicity
Cells were seeded into 96-well plates and allowed to adhere overnight. Initial seeding densities were kept sufficiently low to ensure cells were in exponential growth for the duration of the assay. Media was replaced with that containing caged 11, caged 13, or 4-hydroxycyclofen.
Media alone (no inhibitor) was included in all assays as a negative control. Plates were exposed to 10 mW/cm 2 from a 690 nm LED for 30 min or kept dark. Following a 72 h incubation period, 20 µL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) from a 5 mg/mL stock in PBS was added to each well and incubated for 4 h at 37 °C. Media was removed, 100 µL of DMSO added to each well to solubilize MTT formazan, and absorbance at 550 nm was read using a microplate reader. Drug effects were expressed as % cell viability relative to the no inhibitor control. Half-maximal inhibitory concentrations (IC 50 ) were obtained from sigmoidal curve fits of % viability vs. concentration data.

MEF Line Isolation
All animal studies were approved by the NCI ACUC. Primary MEFs were isolated from E13.5 Rosa CreER/mTmG embryos generated by crosses between homozygous Rosa26CreER T and Rosa-mT/mG mice. Time-mated females were euthanized and embryos collected in sterile PBS.
The head and viscera were removed and body was minced in 1 mL of 0.25% trypsin without EDTA and incubated at 37 °C for 5 min in a 15 mL conical tube. Tissue was triturated by pipetting vigorously and the trypsin reaction was stopped by addition of 5 mL DMEM with 15% S22 FBS. Cells were plated 1 embryo per 25 cm 2 flask and then passaged the next day and subsequently as described above.

MEF Photoactivation
Cells were seeded into 6-well plates and allowed to reach near confluency overnight. Media was replaced with DMEM containing 11, 13, or 4-hydroxycyclofen at the indicated concentration and incubated for 2 h. The cells were washed with warm PBS and covered with fresh media.