Chemiluminescent probes for imaging H2S in living animals

Responsive 1,2-dioxetane chemiluminescent probes have been developed that display instantaneous, sensitive, and selective responses to H2S and are capable of imaging H2S in living mice.


Chemiluminescent response
Chemiluminescent responses and time scans were acquired using a Hitachi F-7000 Fluorescence Spectrophotometer (Hitachi, Tokyo, Japan) using the luminescence detection module and setting emission wavelength to 545 nm. 393 µL of a 20 mM HEPES buffered to pH 7.4 ( Figure 1) or 100 mM glycine buffered to pH 10.02, 100 µL Emerald II Enhancer (Life Technologies, Carlsbad, CA), 5 µL of a 20 mM Na 2 S stock solution in DI-H 2 O and 2 µL of a 10 mM stock solution of CHS probes in CH 3 CN were added to a quartz cuvette (Starna, Atascadero, CA). Samples were shaken gently to assure mixing. Then chemiluminescence spectra were acquired immediately after adding the probes. Time scans were acquired using the time scan module. 40 μM CHS-1, CHS-2, and CHS-3 were treated with 0, 5, 10, 20, 40, 80, 100, 150, and 200 μM Na 2 S, 1 and 10 min (Figure 1, Figure S1) or 120 min ( Figure S2) time scans were measured 1 min after adding probes.

Selectivity tests
Selectivity for CHS-1, CHS-2, and CHS-3 was measured by monitoring the time-dependent chemiluminescent emission at 545 nm. All assays were performed in 20 mM HEPES buffered to pH 7.4 with 20% Emerald II Enhancer.  Blank: 2 µL of 10 mM CHS-1, CHS-2, or CHS-3 in CH 3 CN was added to a solution of 398 µL HEPES and 100 µL Emerald II Enhancer.

Computational results
All the geometries were optimized using density functional theory (DFT) with B3LYP 2,3,4,5 functional and Pople basis set 6-311+G(d,p) 6 and integral equation formalism of polarizable continuum model (IEF-PCM) 7 with water as solvent. Atomic charges are calculated using ESP model. 8 The ESP charges were also calculated at using M06 9 and ωB97XD 10 functionals and 6-311+G(d,p) basis set using IEF-PCM with water as solvent at geometries optimized at B3LYP/6-311+G(d,p) level of theory. All the calculations were carried out using Gaussian09. 11

Cellular experiments
Chemiluminescent response using a multi-well plate reader. Chemiluminescent responses were measured using a BioTek plate reader (Winooski, VT) by using the luminescence detection method, endpoint read type, and setting sensitivity to 135. 120 µL, 119 µL, 119 µL, 118 µL, and 116 µL of 20 mM HEPES buffer (pH 7.4) were added into the wells of a black opaque Corning® 96-well plate from A1 to A5 in sequence, 30 µL Emerald II Enhancer was pipetted into each well, then different volumes of a 10 mM Na 2 S solution (0 µL, 0.38 µL, 0.75 µL, 1.5 µL, 3.0 µL) were added into each well. 0.60 µL CHS-3 was injected into each well and the luminescence intensity of the plate was measured every 2 min after addition of probes. The detection limit was estimated as the amount of Na 2 S required to give a chemiluminescent signal above three times the standard deviation of at least 3 independent experiments with 0 µM Na 2 S. The concentration of Na 2 S needed was estimated by fitting a line to the linear region of the curve between the data points corresponding to 0 µM Na 2 S and 25 µM Na 2 S.
Cell culture and detecting cellular H 2 S using a multi-well plate reader. Human lung adenocarcinoma epithelial cell (A549) were purchased from ATCC and cultured in F-12K media supplemented with 10% Fetal Calf Serum (FCS) at 37 °C with 5% CO 2 . Two days before the experiment, cells were passed and plated on Costar® 12-well plates by adding 150K-175K of A549 cells per well, filling each well up to 600 µL of media with FCS, and aspirating the media upon 90-95% confluence. Cells were serum-starved for 18 h prior to the experiment. Stock solutions of 20 mM homocysteine and 20 mM D,L-propargyl glycine (PAG) were prepared in 20 mM HEPES buffer (pH 7.4) and 10 mM CHS-3 was prepared in CH 3 CN. 6 µL PAG (final concentration: 200 µM) was added to C1-C3 of the 12-well plate. After 20 min incubation, 6 µL homocysteine (final concentration: 200 µM) was added into B1-B3 and C1-C3 of the 12-well plate, and 6 µL 20 mM HEPES buffer (pH 7.4) as a vehicle control was added to A1-A3. After another 20 min incubation, cells were washed with 1 x PBS. Then 500 µL PBS media was added into each well after aspirating the media. 125 µL Emerald II Enhancer and 2.7 µL CHS-3 (final concentration: 40 µM) were added to each well and the luminescent intensity was measured every two minutes for 20 minutes. The experiment was repeated with four independent well plates and the peak value for the luminescence emission for each well at 10-12 minutes was normalized to the average luminescence emission at 10-12 minutes for the control replicates of each plate. A single outlier was rejected according to the extreme studentized deviate method (Grubbs' test, p < 0.01).

Imaging experiments
Chemiluminescent imaging at pH 7.4. Imaging was carried out with a Caliper Xenogen IVIS Spectrum (Perkin-Elmer, Santa Clara, CA) in black 96-well Costar plates and all the images were analyzed using Living Image 3.1 software. 10 mM CHS-3 in CH 3 CN and 10 mM Na 2 S in DI-H 2 O were prepared prior to imaging. 199 µL, 198 µL, 198 µL, 197 µL and 195 µL of 20 mM HEPES buffer (pH 7.4) were added into wells from A1 to A5 in sequence, 50 µL Emerald II Enhancer was pipetted into each well, then different volume of Na 2 S solution (0 µL, 0.61 µL, 1.25 µL, 2.5 µL, 5.0 µL) were added into each well. 1 µL CHS-3 was injected into the mixture and imaging was performed after 30 seconds using an open filter. All images were acquired with f-stop 1, medium binning, auto exposure and the chamber set to 37 °C.
Imaging H 2 S in mouse carcass. A stock solution of 25 mM CHS-3 in DMSO and 50 mM Na 2 S in DI-H 2 O were prepared in advance. The 50 mM stock solution of Na 2 S was diluted to provide a final concentration of 4 mM Na 2 S in 100 µL (0.4 µmol) to be injected. Images were acquired 30 sec after administering i.p. injections to the carcasses of SCID/BALB-C mice with 0.08 µmol CHS-3 and either 0.4 µmol Na 2 S or a vehicle control (H 2 O) in HEPES buffered at pH 7.4 containing 20% Emerald II Enhancer ( Figure S4a-d). Imaging H 2 S in living mice. The UT Southwestern Institutional Animal Care and Use Committee approved these investigations under APN #2009-0150. A stock solution of 25 mM CHS-3 in DMSO and 50 mM Na 2 S in DI-H 2 O were prepared in advance. The 50 mM stock solution of Na 2 S was diluted to provide a final concentration of 4 mM Na 2 S in 100 µL (0.4 µmol) to be injected. Images were acquired 30 sec after administering i.p. injections to C6 brown mice with 0.08 µmol CHS-3 and either 0.4 µmol Na 2 S or a vehicle control (H 2 O) in HEPES buffered at pH 7.4 containing 20% Emerald II Enhancer ( Figure S5a-f). The skin was raised during injections to avoid puncturing internal organs. The final concentration of Na 2 S in the injection was 4 mM. Figure S5. Imaging H 2 S in living C6 brown mice using CHS-3. Images were taken 30 sec after administering an i.p. injection of 0.08 µmol CHS-3 and (a-c) vehicle control or (d-f) 0.4 µmol Na 2 S in 100 µL 20 mM HEPES at pH 7.4 containing 20% Emerald II Enhancer.