Activatable Raman Probes Utilizing Enzyme-Induced Aggregate Formation for Selective Ex Vivo Imaging

Detecting multiple enzyme activities simultaneously with high spatial specificity is a promising strategy to investigate complex biological phenomena, and Raman imaging would be an excellent tool for this purpose due to its high multiplexing capabilities. We previously developed activatable Raman probes based on 9CN-pyronins, but specific visualization of cells with target enzyme activities proved difficult due to leakage of the hydrolysis products from the target cells after activation. Here, focusing on rhodol bearing a nitrile group at the position of 9 (9CN-rhodol), we established a novel mechanism for Raman signal activation based on a combination of aggregate formation (to increase local dye concentration) and the resonant Raman effect along with the bathochromic shift of the absorption, and utilized it to develop Raman probes. We selected the 9CN-rhodol derivative 9CN-JCR as offering a suitable combination of increased stimulated Raman scattering (SRS) signal intensity and high aggregate-forming ability, resulting in good retention in target cells after probe activation. By using isotope-edited 9CN-JCR-based probes, we could simultaneously detect β-galactosidase, γ-glutamyl transpeptidase, and dipeptidyl peptidase-4 activities in live cultured cells and distinguish cell regions expressing target enzyme activity in Drosophila wing disc and fat body ex vivo.


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of the dye at that wavelength was normalized and plotted against the concentration. Three points from the lowest concentration were fitted to Eq. 1 for linear approximation and 10 points from the lowest concentration were fitted to Eq. 2 for nonlinear approximation:

SRS spectral measurements in vitro.
SRS spectra were obtained with the SRS microscope as described above. All samples were held in imaging chambers consisting of two glass coverslips (Matsunami, C218181, C024361) and an imaging spacer (Merck, GBL654002, diam. × thickness 9 mm × 0.12 mm). The chamber was filled with dye solution containing PMMA beads (Sekisui, Techpolymer SSX-110, average particle size: 10 µm) to facilitate finding the position of the solution and sealed with nail polish. Each point of the SRS spectrum was acquired by averaging the pixel values of 5 frames at a particular wavenumber.

RIE (relative Raman intensity vs EdU) 2 measurements.
The SRS peak intensities of DMSO solutions of 100 mM EdU and PBS solutions (containing 30% DMSO) of 100 µM 9CN-rhodols were compared to calculate RIE values. Each measurement was performed under the same condition.

SRS measurements of in vitro enzyme reaction.
200 µM probe was mixed with the corresponding target enzyme in PBS (pH 7.4). The reaction solutions were incubated for a sufficient time for the enzyme reaction to occur at room temperature, then DMSO was added to a final concentration of 30% (v/v) to dissolve the produced dyes. The solutions were sealed in the imaging chambers and SRS measurements were acquired as described above.

LC analysis.
In order to confirm that the water solubilities of 9CN-DEP and 9CN-DER are different, and that

SRS images and spectra of live-cells and ex vivo tissues.
SRS images were constructed by subtracting averaged images at detection wavenumbers with averaged images at background wavenumbers. For the detection wavenumbers, two wavenumbers S6 were selected around the respective probe's SRS intensity maximum. For the background wavenumbers, wavenumbers 20 cm -1 away from the detection wavenumbers were selected. When the background wavenumbers and detection wavenumbers were too close because of cross-talk, the highest background wavenumber and the lowest background wavenumbers were selected as common background wavenumbers for all peaks. The numbers of frames used for averaging at each wavenumber were 300 for live-cultured cells, 1000 for ex vivo tissues and 100 for ex vivo threedimensional imaging. For measuring spectra, the wavenumber was tuned continuously with 3.3 cm -1 intervals and the number of frames used for averaging at each wavenumber was 5.
Live-cell SRS and confocal fluorescence imaging with dual β-Gal probes.  Evaluation of cytotoxicity of 9CN-JCR-based probes.
After incubation for 200 minutes, the absorbance at 450 nm was measured using a plate reader, EnVision (PerkinElmer Co., Ltd.).
For fluorescence imaging of GFP, the excitation wavelength was 488 nm and emission over the range of 500-520 nm was detected.

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Drosophila tissues were stained with 100 µM 9C 15 N-JCR-Bn-βGal as described above. Z-stack images were acquired from z = 0 µm to z = 69 µm with 3 µm steps. Acquisition time for each slice was 13 sec and total acquisition time for all stacks was 320 sec. Detection wavenumbers were 2187 cm -1 and 2190 cm -1 . Background wavenumbers were 2167 cm -1 and 2210 cm -1 . Confocal fluorescence imaging was performed after the acquisition of SRS images. For fluorescence imaging of GFP, the excitation wavelength was 488 nm and emission over the range of 500-520 nm was detected.

Computational details.
We performed calculations using the Gaussian09 program 6 . General geometry optimization and vibrational analysis of local minimum were performed at the B3LYP/6-31G(d) level including implicit water in the PCM model, or correction of dispersive interactions, and we used tight convergence criteria.

Supporting Notes.
The appropriate wavelength region for electronic pre-resonance (620-750 nm) was calculated according to the literature 7 . The EPR-SRS excitation region is defined by Eq. 5, where ω0 and ωpump denote the molecular absorption peak energy and the pump laser energy (843.26 nm for our SRS system), respectively. Γ represents the homogeneous linewidth, typically about 700 cm -1 .
Absorbance was normalized based on the absorbance at the absorption maximum of 5 µM solution.
(e) The relationship between dye concentration and normalized absorbance at the absorption maximum of 5 µM solution. The black dotted line represents a linear relationship between dye concentration and normalized absorbance.     Absorbance was normalized at the wavelength of the absorption maxima shown in Table 1.              Table S2. Kinetic parameters of 9CN-JR-Bn-βGal, 9C 15 N-JCR-Bn-βGal and βGal-9 13 C 15 N-JCP.

Compound 2.
10 mL dry DMF was added dropwise to POCl3 (10 mL, 108 mmol) at 0 ℃ under an argon atmosphere. To the solution, compound 1 (3.0 g, 18 mmol) in 10 mL dry DMF was added at the same temperature. Then, the mixture was heated to 75 ℃ and stirring was continued for 3 h. After cooling to room temperature, the mixture was slowly added to ice water and neutralized with sat. NaHCO3 aq. The precipitate was collected by filtration to give pure compound 2 (3.1 g, 90%) as a brownish solid without further purification. 1

9CN-DER.
To a solution of compound 6 (21 mg, 0.079 mmol) in 5 mL MeCN was added 0.3 M KCN aq. (530 µL, 0.16 mmol), and the mixture was stirred for 10 min at room temperature. Then, 1 N HCl aq. was added, and the mixture was extracted with DCM. The organic layer was washed with brine, dried over Na2SO4 and evaporated to dryness. The crude compound was dissolved in 5 mL DCM and 1 S40 mL MeOH, and chloranil (10 mg, 0.040 mmol) was added to the solution at room temperature. The mixture was stirred for 5 min and then evaporated. The residue was roughly purified by preparative HPLC using eluent A (H2O with 1% MeCN and 0.1% TFA) and eluent B (MeCN with 1% H2O) (A/B = 90/10 to 0/100 for 40 min). The eluate was evaporated and the residue was purified by column chromatography (silica gel, DCM/MeOH = 100/0 to 93/7) to remove remaining compound 6.
To the solution, compound 10 (500 mg, 2.6 mmol) in 10 mL dry DMF was added at the same temperature. The mixture was heated to 60 ℃ and stirring was continued for 1 h. After cooling to room temperature, the mixture was slowly added to ice water. The precipitate was collected by filtration to give pure compound 11 (316 mg, 55%) as a brownish solid without further purification. 1
To the solution, compound 13 (1.4 g, 6.9 mmol) in 5 mL dry DMF was added at the same temperature. Then, the mixture was heated to 100 ℃ and the stirring was continued for 1 h. After cooling to room temperature, the mixture was slowly added to water and extracted with nhexane/AcOEt = 4/1. The organic layer was washed with brine, dried over Na2SO4 and evaporated to dryness. The crude intermediate was dissolved in 5 mL DCM and 5 mL MeOH at 0 ℃, then NaBH4 (51 mg, 1.4 mmol) was added. After warming to room temperature, the reaction mixture was stirred for 2 h. The reaction was quenched with water and the whole was extracted with DCM. The organic layer was washed with brine, dried over Na2SO4 and evaporated to dryness. The residue was purified by column chromatography (silica gel, n-hexane/AcOEt = 74/26 to 53/47) to give compound 14 (886 mg, 56%). 1

Compound 22.
Compound 21 (330 mg, 1.3 mmol) and compound 15 (120 mg, 1.3 mmol) were dissolved in 5 mL dry DCM under an argon atmosphere. The mixture was cooled to 0 ℃ and then BF3·OEt2 (480 µL, 3.8 mmol) was added dropwise. After warming to room temperature, the reaction mixture was stirred for 45 h, then the reaction was quenched with water and the mixture was extracted with DCM.

9CN-DECR.
To a solution of compound 24 (21 mg, 0.071 mmol) in 5 mL MeCN was added 0.3 M KCN aq. (470 µL, 0.14 mmol), and the mixture was stirred for 10 min at room temperature. Then, 1 N HCl aq. was added, and the mixture was extracted with DCM. The organic layer was washed with brine, dried over Na2SO4 and evaporated to dryness. The crude compound was dissolved in 5 mL DCM and 1 mL MeOH, and chloranil (10 mg, 0.040 mmol) was added at room temperature. The mixture was stirred for 5 min and then evaporated. The residue was purified by preparative HPLC using eluent A Compound 25 was synthesized according to the reported protocol 8 .

Compound 30
To a solution of compound 12 (15 mg, 0.050 mmol) and K2CO3 (83 mg, 0.75 mmol) in 5 mL DMF was added methyl iodide (62 µL, 1.0 mmol), and the mixture was stirred at 100 ℃ for 41 h. After cooling to room temperature, the mixture was evaporated and the residue was purified by preparative

Compound 33.
Compound 32 (43 mg, 0.13 mmol) was dissolved in 1.5 mL 80% (v/v) H2SO4 aq. at 0 ℃ and the solution was stirred for 15 min, then allowed to warm to room temperature, and stirring was continued for 1 h. The reaction was quenched with water and the mixture was roughly purified by preparative HPLC using eluent A (H2O with 1% MeCN and 0.1% TFA) and eluent B (MeCN with 1% H2O) (A/B = 90/10 to 0/100 for 40 min) to give a mixture of leuco compound 33 and compound 33. After evaporation, the residue was dissolved in 5 mL DCM and 1 mL MeOH, and chloranil (16 mg, 0.065 mmol) was added at room temperature. The mixture was stirred for 5 min, and then evaporated. The residue was purified by preparative HPLC using eluent A (H2O with 1% MeCN and

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was added. The product became colored by spontaneous oxidation, and the mixture was extracted with DCM. The organic layer was washed with brine, dried over Na2SO4 and evaporated to dryness.
The residue was roughly purified by preparative HPLC using eluent A (H2O with 1% MeCN and Scheme S10. Synthesis of 9CN-JR-Bn-βGal.

Compound 39.
Compound 38 (80 mg, 0.27 mmol), compound 37 (210 mg, 0.41 mmol) and NaH (10 mg, 0.41 mmol) were dissolved in 2 mL dry DMF at 0 ℃ under an argon atmosphere in the dark, then allowed to warm to room temperature, and stirring was continued for 18 h. The reaction was quenched with sat. NH4Cl aq. and the mixture was extracted with DCM. The organic layer was washed with brine, dried over Na2SO4 and evaporated to dryness. The residue was dissolved in 5 mL DCM and 5 mL MeOH, and chloranil (66 mg, 0.27 mmol) was added at room temperature. The mixture was stirred for 10 min and then evaporated. The residue was roughly purified by preparative

Compound 40.
To a solution of compound 39 (31 mg, 0.043 mmol) in 5 mL MeCN was added 0.3 M KCN aq. (300 µL, 0.086 mmol), the mixture was stirred for 10 min at room temperature. Then, the reaction was quenched with eluent A (H2O with 1% MeCN and 0.1% TFA) for HPLC, and the mixture was extracted with DCM. The organic layer was washed with brine, dried over Na2SO4 and evaporated to dryness. The crude compound was dissolved in 5 mL DCM and 1 mL MeOH, and chloranil (5 mg, 0.020 mmol) was added at room temperature. The mixture was stirred for 5 min and then evaporated.
The mixture was stirred for 15 min at the same temperature. Then, the reaction was quenched with

Compound 42.
Compound 41 (163 mg, 0.51 mmol), compound 37 (612 mg, 1.2 mmol) and NaH (18 mg, 0.76 mmol) were dissolved in 10 mL dry DMF at 0 ℃ under an argon atmosphere in the dark. The mixture was allowed to warm to room temperature, and stirring was continued for 18 h. The reaction was quenched with sat. NH4Cl aq. and the mixture was extracted with DCM. The organic layer was washed with brine, dried over Na2SO4 and evaporated to dryness. The residue was dissolved in 5 mL DCM and 5 mL MeOH, and chloranil (62 mg, 0.25 mmol) was added at room temperature. The mixture was stirred for 10 min, and then evaporated. The residue was roughly purified by preparative HPLC using eluent A (H2O with 1% MeCN and 0.

Compound 43.
To a solution of compound 42 (52 mg, 0.069 mmol) in 5 mL MeCN was added 0.3 M KC 15 N aq.
(470 µL, 0.14 mmol), and the mixture was stirred for 10 min at room temperature. Then, the reaction was quenched with eluent A (H2O with 1% MeCN and 0.1% TFA) for HPLC, and the mixture was extracted with DCM. The organic layer was washed with brine, dried over Na2SO4 and evaporated to dryness. The crude compound was dissolved in 5 mL DCM and 1 mL MeOH, and chloranil (17 mg, 0.069 mmol) was added at room temperature. The resulting mixture was stirred for 5 min and then evaporated. The residue was purified by preparative HPLC using eluent A (H2O with 1% MeCN and
The mixture was stirred for 15 min at the same temperature. Then, the reaction was quenched with HPLC eluent A (H2O with 1% MeCN and 0.1% TFA) until the solution became colored. After evaporation, the residue was purified by preparative HPLC using eluent A (H2O with 1% MeCN and