Trimethyl-Substituted Carbamate as a Versatile Self-Immolative Linker for Fluorescence Detection of Enzyme Reactions

Self-immolative linker is a useful building block of molecular probes, with broad applications in the fields of enzyme activity analysis, stimuli-responsive material science, and drug delivery. This manuscript presents N-methyl dimethyl methyl (i.e., trimethyl) carbamate as a new class of self-immolative linker for the fluorescence detection of enzyme reactions. The trimethyl carbamate was shown to spontaneously undergo intramolecular cyclization upon formation of a carboxylate group, to liberate a fluorophore with the second time rapid reaction kinetics. Interestingly, the auto-cleavage reaction of trimethyl carbamate was also induced by the formation of hydroxyl and amino groups. Fluorescent probes with a trimethyl carbamate could be applicable for fluorescence monitoring of the enzyme reactions catalyzed by esterase, ketoreductase, and aminotransferase, and for fluorescence imaging of intracellular esterase activity in living cells, hence demonstrating the utility of this new class of self-immolative linker.


Table of Contents
showed negligible fluorescence after 4 h as compared to that of the parent coumarin 18-2 (10 μM), suggesting that 18 stably existed in the presence of the physiologically relevant concentration GSH.

General materials and methods for organic synthesis
Unless otherwise noted, chemical reagents were purchased from commercial suppliers (FUJIFILM Wako Pure Chemical Corporation, Tokyo Chemical Industry, Sigma-Aldrich, Watanabe Chemical Industries) and used without further purification. Reactions were carried out under a positive atmosphere of nitrogen, unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) carried out on Merck TLC Silica gel 60 F 254 , using shortwave UV light as the visualizing agent. 1 H NMR spectra were recorded using a Varian UNITY-400 (400 MHz) spectrometer or Bruker Avance III HD 500 MHz spectrometer and chemical shifts (δ, ppm) were referenced to residual solvent peak (CDCl 3 : 7.26 ppm; MeOH-d 4 : 3.31 ppm; DMSO-d 6 : 2.50 ppm). ESI mass spectrometry was recorded using a MicroTOF II (Bruker Daltonics) spectrometer. HPLC purification was conducted with a HITACHI L-7000 series (Hitachi).

S5
Preparation of 3

Synthesis of 3-2
To a solution of 3-1 (500 mg, 2.64 mmol) in dry DCM (10 mL) was added TFA (2.0 mL) and the mixture was stirred for 1.5 hr at room temperature. After removal of the solvent by evaporation, the residue was diluted in 2N NaOH aq. (10 mL). To the solution was added benzyl chloroformate (1.13 mL, 7.92 mmol) and the mixture was stirred for 3h at 0℃. The solution was diluted with water and the aqueous layer was washed with ether.
After adjustment of pH of the aqueous layer to 1 by 5 N HCl aq., the product was extracted with ethyl acetate.

Synthesis of 3
To a solution of 2-5 (9.3 mg, 26.8 µmol) in dry DCM (2.0 mL) was added TFA (0.4 mL) and the mixture was stirred for 2hr at room temperature. The solvent was removed by evaporation to give 2 (7.6 mg, 96%) as a white solid. 3 was used for evaluating the reaction kinetics of intramolecular cyclization without further

Synthesis of 4-4
To a solution of 4-3 (370 mg, 1.26 mmol) in MeOH (15.0 mL) was added Pd/C (80.0 mg) and the mixture was stirred for 7.5 hr at room temperature under H 2 atmosphere. After filtration thorough celite, the solvent was removed by evaporation to give 4-4 (122 mg, 61%) as white solid and used next reaction without further

Synthesis of 5
To a solution of 5-3 (5.7 mg, 15.2 µmol) in dry DCM (2.0 mL) was added TFA (0.4 mL) and the mixture was stirred for 2.5 hr at room temperature. The solvent was removed by evaporation to give 5 (6.2 mg, quant) as a white solid. 5 was used for evaluating the reaction kinetics of intramolecular cyclization without further purification. Measurement of NMR spectrum of 5 was not conducted due to rapid intramolecular cyclization of 5. ESI-TOF-MS: m/z for C 16 H 17 NNaO 6 Na [M+Na] + : calcd 342.0954 , observed 342.0887.

S9
Preparation of 6

Synthesis of 6-2
To a solution of 6-1 (500 mg, 2.46 mmol)and KOH (690 mg, 12.30 mmol) in dry DMSO (5 mL) was added CH 3 I (615 µl, 9.84 mmol) and the mixture was stirred for 1 hr at room temperature. The reaction was quenched by addition of water and the mixture was extracted with ether. After removal of the solvent by evaporation, the residue was diluted with dichloromethane (10 mL) and TFA (4 mL) and stirred for 1hr at room temperature.
After removal of the solvent by evaporation, the residue was diluted with water and washed with ethyl acetate.

Synthesis of 7-2
To a solution of 7-1 (300 mg, 2.91 mmol) in 2 N NaOH aq. (10 mL) was added benzyl chloroformate (1.25 mL, 8.73 mmol) and the mixture was stirred for 5.5 hr at 0℃. The mixture was diluted with water and the aqueous layer was washed with ether. After adjustment of pH to 1 by 5 N HCl aq., the product was extracted with ethyl acetate. The solvent was removed by evaporation to give 7-2 (498 mg, 72%) as a white solid. ¹H-

Synthesis of 7-3
To a solution of 7-2 (500 mg, 2.11 mmol) and NaH (253 mg, 6.32 mmol) in dry THF (8.0 mL) was added CH 3 I (2.62 ml, 42.2 mmol) at 0℃ and the mixture was stirred for 12 hr at room temperature. The reaction was quenched by addition of water and the aqueous layer was washed with ethyl acetate. After adjustment of pH to 2 with 5N citric acid aq., the product was extracted with ethyl acetate. The solution was removed by evaporation to give 7-3 (408 mg, 77%) as a yellow oil. ¹H-NMR (400 MHz, DMSO-d 6 ) : δ1.10-1.12 (3H, d, J

Synthesis of 7
To a solution of 7-6 (4.3 mg, 11.4 µmol) in dry DCM (2.0 mL) was added TFA (0.4 mL) and the mixture was stirred for 7 hr at 0℃. The solvent was removed by evaporation to give 7 (4.8 mg, quant) as a white solid. 7 was used for evaluating the reaction kinetics of intramolecular cyclization without further purification. ESI-TOF-MS: m/z for C 16 H 17 NO 6 Na [M+Na] + : calcd 342.0948, observed 342.0953. S12 Preparation of 8

Synthesis of 8-2
To a solution of 8-1 (2.00 g, 13.7 mmol) in MeOH (20 mL) was added H 2 SO 4 (200 µL) and stirred for 18 hr at room temeperature. The solvent was removed by evaporation and the residue was diluted with sat. NaHCO 3 aq., followed by wash with hexane. After adjustment of the pH to 1 with 1N HCl aq., the product was extracted with ethyl acetate. The solvent was removed by evaporation to give 8-2 (1.20 g, 55%) as colorless oil.

Synthesis of 8-3
To a solution of 8-2 (1.20 g, 7.49 mmol) and triethylamine (1.04 ml, 7.49 mmol) in dry Toluene (10 mL) was added diphenylphosphoryl azide (1.78 ml, 8.24 mmol) and the mixture was refluxed for 25 hr. After removal of the solvent by evaporation, the residue was diluted with ethyl acetate and the organic layer was washed with sat. NaHCO 3 aq. and brine. The solvent was removed by evaporation and the mixture was purified by column chromatography on SiO 2 (hexane : ethyl acetate = 10 : 1) to give 8-3 (1.58 g, 80%) as colorless oil. ¹H-NMR

Synthesis of 8-4
To a solution of 8-3 (1.58 g, 5.96 mmol) in MeOH (20 mL) was added 1N NaOH aq. (17.9 mL, 17.9 mmol) and the mixture was stirred for 10 hr at 0℃. After removal of the solvent by evaporation, the residue was diluted with sat. NaHCO 3 aq. and the aqueous layer was washed with ether. After adjustment of the pH to 1 with 1N HCl aq., the product was extracted with ethyl acetate. The solvent was removed by evaporation to To a solution of 8-4 (893 mg, 3.55 mmol), EDC-HCl (1.02 g, 5.33 mmol), DIPEA (1.85 ml, 10.7 mmol) and DMAP (86.9 mg, 0.711 mmol) in dry DCM (15.0 mL) was added tert-BuOH (1.69 ml, 17.8 mmol) and the mixture was stirred for 12 hr at room temperature. The mixture was diluted with chloroform and the organic layer was washed with sat. NaHCO 3 aq., water and brine. After removal of the solvent by evaporation, the residue was purified by column chromatography on SiO 2 (hexane : ethyl acetate = 10 : 1) to give 8-5 (

Synthesis of 8-6
To a solution of 8-5 (389 mg, 1.27 mmol) and NaH (152 mg, 3.80 mmol) in dry THF (10 mL) was added CH 3 I (791 µL, 12.7 mmol) at 0℃ and the mixture was stirred for 22 hr at room temperature. The mixture was diluted with ethyl acetate and the organic layer was washed with sat. NaHCO 3 aq., water and brine. After removal of the solvent by evaporation, the residue was purified by column chromatography on SiO 2 (hexane : ethyl acetate in dry DCM (1.5 mL) by dropwise and the mixture was stirred for 1 hr at room temperature. The mixture was diluted with chloroform and the organic layer was washed with sat. NaHCO 3 aq., water and brine. After

Synthesis of 8
To a solution of 8-8 (3.30 mg, 8.47 µmol) in dry DCM (0.8 mL) was added TFA (0.2 mL) and the mixture was stirred for 7 hr at 0℃. The solvent was removed by evaporation to give 8 (3.5 mg, quant) as a white solid. 8 S14 was used for evaluating the reaction kinetics of intramolecular cyclization without further purification.
Measurement of NMR spectrum of 8 was not conducted due to rapid intramolecular cyclization of 8. Preparation of 9

Synthesis of 9-2
To a solution of 9-1 (100 mg, 0.381 mmol) in MeOH (2.0 mL) was added NaIO 4 (97.7 mg, 0.457 mmol) in H2O (1.0 mL) and the mixture was stirred for 70 min at 0 °C. After dilution with H 2 O, the product was extracted with ether and the solvent was removed by evaporation. The residue was diluted wit H 2 O (0.1 mL) and to the solution were added NH 4 Cl (32.2 mg, 0.602 mmol) and cyclopentadiene (127 µL, 1.51 mmol). The mixture was stirred for 18 hr at room temperature, diluted with water, and washed with ether. After adjustment of pH to 12 with 1N NaOH aq., the product was extracted with ethyl acetate to give 9-2 (32.4 mg, 28%) as

Synthesis of 9-3
To a solution of 1 (20.0 mg, 0.114 mmol) and DIPEA (19.8 µL, 0.114 mmol) in dry THF (1.0 mL) was added triphosgene (11.2 mg, 37.8 µmol) in dry DCM (1.0 mL) by dropwise and the mixture was stirred for 4 hr at room temperature. To the solution were added 9-2 (33.2 mg, 0.170 mmol) and DIPEA (29.7 µL, 0.170 mmol) in dry DCM (1.0 mL) by dropwise and the mixture was stirred for 3 hr at room temperature. The mixture was diluted with chloroform and the organic layer was washed with sat. NaHCO 3 aq., water and brine. After

Synthesis of 10
To a solution of 10-6 (5.0 mg, 13.4 µmol) in dry DCM (1.5 mL) was added TFA (0.3 mL) and the mixture was stirred for 7 hr at room temperature. The solvent was removed by evaporation to give 10 (5.2 mg, quant) as a white solid. 10 was used for evaluating the reaction kinetics of intramolecular cyclization without further purification. ESI-TOF-MS: m/z for C 16 H 15 NO 6 Na [M+Na] + : calcd 340.0792, observed 340.0790.

Preparation of 11
Synthesis of 11-1 To a solution of 1 (50.0 mg, 0.284 mmol) and DIPEA (49.5 µL, 0.284 mmol) in dry THF (2.0 mL) was added triphosgene (28.1 mg, 94.7 µmol) in dry DCM (2.5 mL) by dropwise and the mixture was stirred for 3 hr at room temperature. To the solution were added L-Proline tert-butyl ester (72.9 mg, 0.426 mmol) and DIPEA (74.2 µL, 0.426 mmol) in dry DCM (2.5 mL) by dropwise and the mixture was stirred for 1.5 hr at room temperature. The mixture was diluted with chloroform and the organic layer was washed with sat. NaHCO 3 aq., water and brine. After removal of the solvent by evaporation, the residue was purified by column chromatography on SiO 2 (hexane : ethyl acetate = 2 : 1) to give 11-1 (45.6 mg, 43%) as colorless oil. ¹H-NMR

Synthesis of 12-2
To a solution of 12-1 (800 mg, 9.51 mmol) and DIPEA (2.13 mL, 12.4 mmol) in dry THF (20 mL) was added benzyl chloroformate (1.61 mL, 11.4 mmol) and the mixture was stirred for 2.5 hr at 0 °C. After removal of the solvent, the residue was diluted with ethyl acetate and the organic layer was washed with water and brine.

Synthesis of 12-3
To a solution of 12-2 (1.80 g, 8.29 mmol) in dry DMF (10 mL) was added and NaH (199 mg, 8.29 mmol) and the mixture was stirred for 10 min at 0℃. To the solution was added CH 3 I (4.13 mL, 66.3 mmol) and the mixture was stirred for 16 hr at 0℃. After quenching the reaction by addition of water, the mixture was extracted with ethyl acetate and the organic layer was washed with water and brine. After removal of the solvent by evaporation, the residue was purified by column chromatography on SiO 2 (hexane : ethyl acetate = 5 : 1) to give 12-3 (1.35 g, 70%) as brown oil.

Synthesis of 12-4
To a solution of 12-3 (750 mg, 3.22 mmol) in dry THF (20 mL) was added BH 3 -THF (20.0 mL, 6.37 mmol) and the mixture was stirred for 17 hr at room temperature. After quenching the reaction by addition of MeOH, 1N HCl (15 mL) was added and the mixture was refluxed for 1hr. After removal of THF and methanol by evaporation, the aqueous layer was washed with chloroform. After adjustment of the pH to 12 with 1N NaOH aq., the crude product was extracted with chloroform and purified by column chromatography on SiO 2 (chloroform : methanol : NH 3 aq. = 100 : 10 : 1) to give 12-4 (140 mg, 18 %) as brown oil. To a solution of 12-4 (40 mg, 0.169 mmol) and Et 3 N (46.9 µL, 0.339 mmol) in dry DCM (3 mL) was added Boc 2 O (40.6 mg, 0.186 mmol) and the mixture was stirred for 23 hr at room temperature. After dilution with DCM, the mixture was washed with water and brine. After removal of the solvent, the crude product was purified by column chromatography on SiO 2 (hexane : ethyl acetate = 5 : 1 to 1 : 1) to give 12-5 (43.1 mg, 76 %) as colorless oil.

Synthesis of 12
To a solution of 13 (5.0 mg, 12.4 µmol) in dry DCM (1.5 mL) was added TFA (0.3 mL) and the mixture was stirred for 7 hr at room temperature. The solvent was removed by evaporation to give 12 (5.0 mg, quant) as a white solid. 12 was used for evaluating the reaction kinetics of intramolecular cyclization without further purification. Measurement of NMR spectrum of 12 was not conducted due to rapid intramolecular

Synthesis of 14-3
To a solution of 14-2 (600 mg, 2.15 mmol) and NaH (260 mg, 6.45 mmol) in dry THF (5.0 mL) was added CH 3 I (1.3 mL, 21.5 mmol) and the mixture was stirred for 13 hr at room temperature. After quenching the reaction by addition of water, the mixture was diluted with ethyl acetate and the organic layer was washed with water and brine. After removal of the solvent by evaporation, the residue was purified by column chromatography on SiO 2 (hexane : ethyl acetate = 20 : 1) to give 14-3 (506 mg, 80%) as colorless oil. ¹H-NMR

Synthesis of 14
To a solution of 14-6 (4.3 mg, 0.011 mmol) in MeOH (1.0 mL) was added Pd/C (5.0 mg) under N 2 atmosphere and the mixture was stirred for 9 hr at 0 °C under H 2 atmosphere. Pd/C was removed by filtration through celite to give 14 (3.3 mg, quant). 14 was used for evaluating the reaction kinetics of intramolecular cyclization

Synthesis of 15-2
To a solution of 15-2 (1.0 g, 6.06 mmol) in acetone (50 mL) and water (5 mL) was added NaN 3 (394.0 mg, 6.06 mmol) and the mixture was refluxed for 17 hr. After removal of the solvent by evaporation, the residue S22 was diluted with ethyl acetate and the organic layer was washed with water and brine. The solvent was removed by evaporation at 20 °C and the mixture was diluted with dry THF (30 mL). To the solution was added LiAlH 4 (414.0 mg, 10.91 mmol) and the mixture was stirred for 6.5 hr at room temperature. The reaction was quenched by addition of water and the precipitate was filtrated thorough celite. After removal of THF by evaporation, the product was extracted with chloroform to give 15-2 (422.0 mg, 67%) as yellow oil.¹H-NMR (400 MHz,

Synthesis of 15-4
To a solution of 15-3 (292.5 mg, 1.44 mmol) and KOH (557.1 mg, 7.20 mmol) in dry DMF (4 mL) was added benzyl bromide (513.3 µL, 4.32 mmol) and the mixture was stirred for 3hr at room temperature. After removal of the solvent by evaporation, the mixture was diluted with ethyl acetate and the organic layer was washed with water and brine. The solvent was removed by evaporation and the residue was purified by column

Synthesis of 15-5
To a solution of 15-4 (344.0 mg, 1.17 mmol) and NaH (141.1 mg, 3.51 mmol) in dry THF (4 mL) was added CH 3 I (707.4 µL, 11.7 mmol) and the mixture was stirred for 19 hr at 40℃. After quenching the reaction by addition of water, the mixture was diluted with ethyl acetate and the organic layer was washed with water and brine. After removal of the solvent by evaporation, the residue was purified by column chromatography on SiO 2 (hexane : ethyl acetate = 20 : 1) to give 15-5 (217.3 mg, 60%) as colorless oil. ¹H-NMR (500 MHz,

Synthesis of 15-6
To a solution of 15-5 (217.3 mg, 0.71 mmol) in dry DCM (4 mL) was added TFA (1 mL) and the mixture was stirred for 2.5 hr at room temperature. After removal of the solvent by evaporation, the residue was diluted with ethyl acetate. The organic layer was washed with sat. NaHCO 3 aq. and brine to give 15-6 (150.1 mg,

S24
Preparation of 18

Synthesis of 17-1
To a solution of 5-1 (1.00 g, 4.50 mmol) and K 2 CO 3 (1.87 g, 13.5 mmol) in dry DMF (15.0 mL) was added methylamine (665 μL, 6.75 mmol) and the mixture was stirred for 45 hr at 60 °C. After dilution with 1N citric acid aq., the aqueous layer was washed with chloroform. The pH of the aqueous layer was adjusted to pH 12 with 5 N NaOH aq. and the product was extracted with ether. The solvent was removed by evaporation without heating and the residue was diluted with 2N NaOH aq. (15 mL). To the solvent was added benzyl chloroformate (2.27 mL, 13.5 mmol) and the mixture was stirred for 1.5 hr at 0 °C. After dilution with ethyl acetate, the organic layer was washed with sat. NaHCO 3 aq., water and brine. After removal of the solvent by evaporation, the crude product was purified by column chromatography on SiO 2 (hexane : ethyl acetate = 10 : 1) to give 17-

Synthesis of 17-4
To a solution of 17-3 (46.3 mg, 0.141 mmol) and formic acid (5 drops) in MeOH (2.0 mL) was added Pd/C (20.0 mg) under N 2 atmosphere and the mixture was stirred for 1 hr at 0 °C under H 2 atmosphere. After removal of Pd/C by filtration through celite, the solvent was removed by evaporation without heating to give 17-4 (27.2 mg, quant). 17-4 was used to the next reaction without further purification due to the instability of 17-4,. ESI-TOF-MS: m/z for C 11 H 16 NO 2 : calcd 194.1181 [M+H] + , observed 194.1187.

Synthesis of 17
To a solution of 1 (24.9 mg, 0.141 mmol) and DIPEA (36.9 µl, 0.212 mmol) in dry THF (1.0 mL) was added triphosgene (14.0 mg, 47.1 µmol) in dry DCM (1.0 mL) by dropwise and the mixture was stirred for 5.5 hr at room temperature. To the solution were added 17-4 (62.1 mg, 0.30 mmol) and DIPEA (52.7 µL, 0.30 mmol) in dry DCM (1.5 mL) by dropwise and the mixture was stirred for 1 hr at room temperature. The mixture was diluted with chloroform and the organic layer was washed with sat. NaHCO 3 aq., water and brine. After