Materials:

1. D-cysteine hydrochloride (36 mg，0.20 mmol)
2. potassium carbonate (28 mg，0.20 mmol)
3. the solution of cycloaminobenzothiazol-2-nitrile (0.10 mmol) in 2 mL DCM/MeOH (1:1)
4. 2 mL H₂O/MeOH (1:1)
5. 0.1 M aqueous HCl solution

Procedures:

(s)-2-(6-(pyrrolidin-1-yl)benzo[d]thiazol-2-yl)-4, 5-dihydrothiazole-4-carboxylic acid (5-cyL)

1. Add the mixture of D-cysteine hydrochloride (36 mg，0.20 mmol) and potassium carbonate (28 mg，0.20 mmol) to the solution of cycloaminobenzothiazol-2-nitrile (0.10 mmol) in 2 mL DCM/MeOH (1:1).
2. Dissolve it in 2 mL H₂O/MeOH (1:1).
3. Stir vigorously for 20 minutes at room temperature.
4. DCM and MeOH were then removed from the flask under low pressure
5. Add 0.1 M aqueous HCl solution until pH reaches 5-6.
6. Precipitate formed immediately.
7. Filter the precipitate off and wash it with ice water.
8. Dry the precipitate under reduced pressure, 28 mg of pure product as a yellow solid is provided. (Yield=84%.)

(S)-2-(6-(piperidin-1-yl)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid(6-cyL)

The synthesis was analogous to 5-cyL. The product was obtained as a yellow-white solid. Yield = 68%.

(S)-2-(6-(azepan-1-yl)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid(7-cyL)

The synthesis was analogous to 5-cyL. The product was obtained as an orange-red solid. Yield = 68%.

(S)-2-(6-(azocan-1-yl)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (8-cyL)

The synthesis was analogous to 5-cyL. The product was obtained as a brown solid. Yield = 86%

Reagent:

1. 5-cyL, 6-cyL, 7-cyL, 8-cyL, D-luciferin and aminoluciferin
2. 50 mM Tris-HCl (pH7.4) buffer solution
3. Firefly luciferase solution(20 μg/mL, containing 2 mM ATP)

Device:

1. Quartz cuvettes
2. 2F-2500 FL Spectrophotometer
3. Xenogen IVIS Spectrum imaging system

Procedures:

1. Prepare 5-cyL, 6-cyL, 7-cyL, 8-cyL, D-luciferin and aminoluciferin
2. Prepare the solution of them by with 50 mM Tris-HCl (pH7.4) buffer solution
3. Add the firefly luciferase solution(20 μg/mL, containing 2 mM ATP) to these substrates (100 μM, 0.5 mL) in quartz cuvettes.
4. Measure the bioluminescence emission spectra immediately by a F-2500 FL Spectrophotometer.

Cellular level

Reagent:

1. Luciferase-transfected U-78 cells
2. RPMI 1640
3. 10% fetal bovine serum (FBS)

Device:

1. CO₂ incubator
2. Centrifuge

Procedures:

1. Cultured the expressing firefly luciferase cells in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (37°C, humidified atmosphere, 5% CO₂ incubator. )
2. These cells in logarithmic phase after trypsinization and centrifugation
3. Diluted the cells into RPMI 1640 supplemented with 10% FBS for a cells-suspension (2x 10⁶ /mL)
4. 200 μL cells suspension were added into black 96-well plates (4x 10⁴cells per well)
5. Incubating for 24 h in the incubator
6. Add the different concentrations of 5-cyL, 6-cyL, 7-cyL, 8-cyL, D-luciferin and aminoluciferin (50 μL, 0.1−20 μM) to each well
7. Acquire the bioluminescence images using a Xenogen IVIS Spectrum imaging system. Images were acquired every 1 min for 30 min (3s- exposure per image).
8. 20 minutes later, measure the luciferase activity.
9. Luminescent signal (photons per second) for each well was measured and plotted as average values (experiments conducted in triplicate).

Nude Xenograft Tumor Model Bioluminescence Imaging

Animal level

Reagent:

1. 6-week-old female nude mice
2. luciferase-expressing U-87 cells (1×10⁷)
3. 100 µL of 5-cyL, 6-cyL, 7-cyL, D-luciferin and Aminoluciferin (1 µM to 10 mM)

Devices:

1. puncture needle
2. Xenogen IVIS Spectrum imaging system
3. Anesthesia machine

Procedures:

1. The 6-week-old female nude mice were inoculated subcutaneously with luciferase-expressing U-87 cells (1×10⁷) in normal saline (0.1 mL)
2. When the size of the tumors is 1 cm³, harvest them
3. Cut the tumors into pieces
4. Disinfected the nude mice locally
5. Use the puncture needle to implant 10 mg tumour pieces subcutaneously under the right armpit region
6. Two weeks later, injected i.p. with 100 µL of 5-cyL, 6-cyL, 7-cyL, D-luciferin and Aminoluciferin (1 µM to 10 mM) to the nude mice with tumor xenografts
7. Anesthetized the mice with isoflurane (2% in 1 l/min oxygen)
8. Acquire the bioluminescence images using the Xenogen IVIS Spectrum imaging system. (Images were acquired every 5 min for 500 min)

FVB-Tg mice Bioluminescence Imaging

Reagent:

1. FVB-Tg transgenic nude mice
2. 100 µL of 1 mM 7-cyL, D-luciferin and aminoluciferin dissolved in saline and DMSO (10:1).

Devices:

1. Anesthesia machine
2. Xenogen IVIS Spectrum imaging system.

Procedures:

1. FVB-Tg transgenic nude mice is injected i.v. with 100 µL of 1 mM 7-cyL, D-luciferin and aminoluciferin dissolved in saline and DMSO (10:1), respectively.
2. The animals were anesthetized with isoflurane (2% in 1 L/min oxygen)
3. Bioluminescence images were acquired using the Xenogen IVIS Spectrum imaging system.

Brain Tumor Bioluminescence Imaging

Reagent:

1. female nude mice (6 weeks old, 18-20 g)
2. luciferase-expressing U87-Luc cells (8 × 10³ cells in 2 µL saline)
3. 100 µL of 1 mM 7-cyL, D-luciferin and aminoluciferin
4. saline solution
5. DMSO

Devices:

1. Xenogen IVIS Spectrum imaging system.
2. Anesthesia machine

Procedures:

1. The female nude mice (6 weeks old, 18-20 g) were inoculated with luciferase-expressing U87-Luc cells (8 × 10³ cells in 2 µL saline) in brain hippocampus.
2. Establish the brain tumors for 2 weeks
3. The nude mice with tumors in brain hippocampus were injected i.p. with 100 µL of 1 mM 7-cyL, D-luciferin and aminoluciferin dissolved in saline and DMSO (10:1), respectively.
4. The animals were anesthetized with isoflurane (2% in 1 L/min oxygen)
5. Bioluminescence images were acquired using the Xenogen IVIS Spectrum imaging system. (Images were acquired every 5 min for 120 min)