Preparation and Use of Turn-on Fluorescent Probe for Detection and Live Cell Imaging of Vitamin D Receptor as a Target Protein

SUMMARY Turn-on fluorescent probe mediated by conjugate addition and cyclization (TCC probe) is a small molecule that reacts with a protein of interest in cells. TCC probe is applicable to various types of proteins by exchanging the ligand unit for target proteins. TCC probes are a potent tool for molecular imaging and chemical proteomics. This protocol describes the synthesis of a TCC probe via unstable intermediate and how to use this probe to visualize vitamin D receptor as a target protein. For complete details on the use and execution of this protocol, please refer to Kojima et al. (2020).


KEY RESOURCES
4. Cool the reaction mixture to 0 C by using an ice-water cooling bath. Add 10 mL of distilled water to stop the reaction. Extract the mixture with ethyl acetate three times in a separatory funnel to afford the organic layer. Dry the organic layer with anhydrous magnesium sulfate (3 g) for 15 min and filter to remove the magnesium sulfate. Condense the organic layer using the rotary evaporator to afford the crude product. Characterize the product by 1 H NMR spectroscopy (see the EXPECTED OUT-COMES section). Use the crude compound 1 to next reaction without further purification.
Pause Point: At this point, the product can be stored at -20 C for at least 3-6 months.
5. Slowly add a magnetic stirring bar to a 100 mL two-neck round-bottom flask. Equip the main neck of the two-neck round-bottom flask with an argon-filled balloon. Place a rubber septum on remaining side neck of the flask. This is the reaction flask. 6. Connect the flask to a vacuum pump. Put the flask under reduced pressure for 10 min and then back-fill the flask with argon. 7. Using a syringe, add 5.4 mL of anhydrous THF and 0.95 mL of diisopropylamine (6.77 mmol) through the septum on the side neck. Cool the flask to 0 C by using an ice-water cooling bath. 8. Using a syringe, add 3.1 mL of 2.0 M n-butyllithium in cyclohexane (6.25 mmol) dropwise to above flask. Stir the solution at 0 C for 10 min. 9. Cool the reaction mixture to -78 C by using a dry ice-acetone cooling bath. Using a syringe, add 3.1 mL of 2.0 M (trimethylsilyl)diazomethane in diethyl ether (6.25 mmol) dropwise to the flask. Stir the solution at -78 C for 30 min. 10. Charge a 25 mL two-neck round-bottom flask with a magnetic stirring bar and compound 1 (5.21 mmol). Equip the main neck of the two-neck round-bottom flask with an argon-filled balloon. Place a rubber septum on remaining side neck of the flask. Connect the flask to a vacuum pump. Put the flask under reduced pressure for 10 min and then back-fill the flask with argon. 11. Using a syringe, add 6.1 mL of anhydrous THF to the flask from Step 6. Stir the solution until the compound 1 has dissolved. 12. Using a syringe, add the compound 1 solution dropwise to the reaction flask from Step 5. Allow the reaction mixture to warm to room temperature (15 C-25 C) and stir the solution for 19 h. 13. Add 10 mL of distilled water to stop the reaction. Extract the mixture with chloroform three times in a separatory funnel to afford the organic layer. Dry the organic layer with anhydrous sodium sulfate (5 g) for 15 min and filter to remove the sodium sulfate. Condense the organic layer using the rotary evaporator to afford the crude product. 14. Purify it by NH 2 silica gel column with hexane to obtain the pure compound 2 (1.35 g; yield = 85% from 4-(diethylamino)salicylaldehyde). 15. Characterize the product by NMR spectroscopy ( 1 H NMR and 13 C NMR) and electrospray ionization MS (HRMS, ESI) (see the EXPECTED OUTCOMES section). CRITICAL: n-Butyllithium can burn in the presence of oxygen and moisture. Always use syringes with needles equipped with luer lock fittings when transferring n-butyllithium.
Must avoid any spills with n-butyllithium and keep flammable solvent away to avoid a fire.
CRITICAL: n-Butyllithium should be added slowly in a drop-by-drop manner.
Pause Point: At this point, the product can be stored at -20 C for at least 3-6 months.
16. Weigh 243 mg of compound 2 (0.80 mmol) in a 50 mL two-neck round-bottom flask containing a magnetic stirring bar. Equip the main neck of the two-neck round-bottom flask with an argonfilled balloon. Place a rubber septum on remaining side neck of the flask. This is the reaction flask. 17. Connect the flask to a vacuum pump. Put the flask under reduced pressure for 10 min and then back-fill the flask with argon. 18. Using a syringe, add 3.2 mL of anhydrous THF through the septum on the side neck. Cool the flask to -78 C by using a dry ice-acetone cooling bath. 19. Using a syringe, add 0.57 mL of 2.0 M n-butyllithium in cyclohexane (0.88 mmol) dropwise to above flask. Stir the solution at -78 C for 30 min. Allow the reaction mixture to warm to 0 C by using an ice-water cooling bath. 20. Insert a cannula to the septum on the side neck and flush with CO 2 for 10 min. Stir the solution at 0 C for 1.5 h. 21. Condense the organic layer using the rotary evaporator to afford the crude mixture. Characterize the product by electrospray ionization MS (HRMS, ESI) (see the EXPECTED OUTCOMES section). Use the crude compound 3 to next reaction without further purification. Troubleshooting 1 and 2 CRITICAL: n-Butyllithium should be added slowly in a drop-by-drop manner. The condensation of organic layer should be run as quickly as possible, as the product is prone to degradation when it is left on water bath (35 C) for too long. Pause Point: At this point, the product can be stored at -20 C for at least a week, but it should generally be used in the next step as soon as possible.   48. Culture HeLa cells in a 10-cm cell culture dish containing 10 mL of culture medium in an incubator with 5% CO 2 at 37 C. 49. Aspirate the culture medium from the cell culture dish using a vacuum pump and rinse the cells once with 5 mL of PBS. 50. Add 0.75 mL of trypsin-EDTA solution to the cells and place the cells into the 37 C incubator for 3 min. 51. Resuspend the cells in 10 mL of culture medium and count the number of cells under a microscope using a hemocytometer. 52. Seed 0.5 mL of the cell solution (containing 5 3 10 4 cells) on a poly-L-lysine coated cover slips in a 24-well cell culture plate. Place the plate in an incubator with 5% CO 2 at 37 C for 24 h. 53. HeLa cells should be treated with LCA-TCC probe 6 at a concentration of 20 mM in 0.5 mL of cell culture medium. Place the cells into the 37 C incubator for 8 h. 54. Incubate labeled cells with 0.5 mL of cell culture medium containing 250 nM TMRM at 37 C for 30 min. 55. Rinse each cover slip three times with 0.5 mL of DMEM/Ham's F-12 culture medium. 56. Transfer the cover slips to a glass bottom dish filled with DMEM/Ham's F-12 culture medium.
Take images using confocal microscopy.
CRITICAL: Cell culture medium should be prewarmed to 37 C.

LIMITATIONS
Structure of ligand unit will be limited to avoid intramolecular reaction of fluorophore unit with highly nucleophilic functional groups. To avoid undesired reaction, we recommend not to use a ligand having extra reactive thiol or amino group.
LCA-TCC probe 6 fluorescently labels not only vitamin D receptor but also mitochondria, which is unfavorable for accurate evaluation of target protein's distribution. Although LCA is a low-affinity ligand for vitamin D receptor (inhibition constant (Ki) = 29 G 6 mM in COS-7 cells) (Makishima et al., 2002), LCA-TCC probe 6 can label vitamin D receptor in living cells. The target selectivity of TCC probe will be improved to use high-affinity and selective ligand unit. Fluorescent labeling selectivity can be ligand unit dependent.

TROUBLESHOOTING
Problem 1 Insufficient yield of compound 2 and 3.
Potential Solution n-Butyllithium is moisture sensitive and is gradually decomposed by reacting with moisture. Estimate the concentration of n-butyllithium by using diphenylacetic acid titration (Kofron and Baclawski, 1976) before it is used.

Potential Solution
Compound 3 is unstable for heat and light. Cover flask with aluminum sheet during the reaction and evaporation. Condense the organic solvent using the rotary evaporator immediately and use the crude product for the next reaction on the day.

OPEN ACCESS
Problem 3 Insufficient yield of LCA-TCC probe 6.
Potential Solution LCA-TCC probe 6 is degraded during the deprotection reaction for tert-butyldimethylsilyl ether protecting group. Reduce reaction time or reaction temperature.

Potential Solution
Reduce LCA-TCC probe 6 concentration and wash out excess probe with PBS. As an alternative approach, optimize ligand unit selectivity and affinity for protein of interest.

Problem 5
To design TCC probe, there are no information about interaction between ligand and target protein.

Potential Solution
We recommend adding fluorophore unit to polar side of the ligand. Because polar side of ligand often faces to surface of protein where many types of nucleophilic amino acid residues exist.