1

Appendix

Auxin transport sites are visualized in planta using fluorescent auxin analogs

Ken-ichiro Hayashi, Shouichi Nakamura, Shiho Fukunaga, Takeshi Nishimura, Mark Jenness, Angus Murphy,

Hiroyasu Motose, Hiroshi Nozaki, Masahiko Furutani, Takashi Aoyama

Supplementary information Supplementary Materials & Methods P. 1-14

Supplementary figures S1-S16 P. 15-30

Plant Materials and Growth Conditions. Arabidopsis thaliana seeds were stratified for 2 days at 4 °C and cultured on germination medium (GM; 0.5× Murashige and Skoog salts [Gibco-BRL], 1% sucrose, 1× B5 vitamins, and 0.5 g/L MES, pH 5.8) in soft gel plates (0.8 g/L gellan gum) or vertical agar plates (12 g/L agar) at 22 °C under continuous light for all assays. Arabidopsis (Arabidopsis thaliana) ecotype Columbia was used for all experiments. The Arabidopsis mutants and the transgenic lines pER8::GH3.6, pIAA3::GUS, BA3::GUS, pIAA12::GUS, DR5::tdTomato-NLS, DII-VENUS, 35S::VHA-a3-mRFP and pin3 pin 7 were gifts from Dr. Hiroyuki Kasahara (Riken PSC, Japan), Dr. Dolf Weijers (ZMBP, University of Tuebingen, Germany), Dr. Teva Vernoux (Université de Lyon, Lyon, France), Prof. Karin Schumacher (University of Heidelberg, Germany), Dr. Tatsuya Sakai (Niigata University, Japan), and Arabidopsis abcb1, abcb19, aux1-7, pin2/eir1-1, CFP-ER (CS16250), DR5::GUS and DR5::GFP lines were obtained from the Arabidopsis Biological Resource Center. Suspension-cultured Arabidopsis MM1 cells and tobacco (Nicotiana tabacum) BY-2 cells were obtained from the Riken Bioresource Center, Japan. Suspension-cultured tobacco cells (cv BY-2) were maintained in modified Murashige and Skoog medium, as described previously (1), on a rotary shaker (100 rpm) at 25°C in the dark. Suspension-cultured Arabidopsis MM1 cells were cultured as described in (2). Imaging of fluorescent auxin analogs. Fluorescence images were recorded under an Olympus BX-50 fluorescent microscope and a confocal laser scanning microscope (FV1200 CLSM, Olympus). Typically, the seedlings were incubated with GM medium containing NBD-analogs for 15－20 min at 24 °C in the dark, and fluorescence images were then immediately recorded. For the co-treatment with NBD-auxin and ER-trackerTM Red (Invitrogen, Japan), the both dyes are simultaneously added to the medium and then incubated for 20 min. For gravitropic stimulation of the hypocotyl, etiolated seedlings grown vertically were rotated to a 90° angle from the vertical direction. After a 24 h incubation (Fig 5F) or 10 h incubation (Fig 5G, 5H), the hypocotyl was treated with NBD-auxins for 15 min or 30 min at 24 °C while maintaining the unidirectional stimulus of gravity, and fluorescence images were then recorded immediately. For counterstaining to visualize root anatomy, co-treatment with NBD-auxins and propidium iodide / FM4-64 would greatly affect the fluorescent image of NBD-auxins. Anionic dye, NBD-auxins might strongly interact with strong cationic dyes, propidium iodide / FM4-64 to form the insoluble precipitate that bind unspecifically to membranes.

DII-VENUS assay 6-d-old DII-VENUS seedlings were incubated in GM liquid medium containing 10 μM auxinole for 6h at 24°C. The DII-VENUS seedlings were washed out well with a fresh medium and incubated in a fresh GM liquid medium for 5 min. Exogenous NAA was added to this medium and then root filled with medium was immediately placed on slide glass. Fluorescent confocal images of roots were recorded by FV1200 CLSM under identical conditions at regular intervals. Yeast two hybrid assay. In this assay, the yeast (EGY48::pJK103) cells expressing a LexA DNA-binding domain (BD)-TIR1 fusion (DBD-TIR1) and

2

activation domain (AD) AtIAA7 fusion was used (3). The interaction between TIR1 and AtIAA7 was evaluated by growing the yeast on SD(Gal)/-Ura/-His/-Trp/-Leu plates with 40 μM NBD-auxins, with/without 50 μM IAA. All plates were incubated for 2–3 days at 30°C. Root Gravitropic Response Assay 6-d-old seedlings grown vertically on GM agar plates under continuous light at 24°C. The seedlings were then transferred to agar plates containing chemicals and cultured vertically for one hour. The plates were rotated to a 90° angle from the vertical direction, followed by incubation for 5 hours in the dark. Photographs of the roots were recorded with a digital camera. NBD-Auxin Transport Assay For assay using etiolated hypocotyl of Col-0 and pin3 pin7 mutant, the seedlings were stratified 2 d at 4 °C on 1% phyta-agar plates containing ¼ MS (pH 5.5) and 1 % sucrose and then vertically grown for 4 days in the dark after the incubation for 18h under light. A 0.2 µl droplette of 0.1% agarose containing 80µM NBD-NAA was placed on the shoot apex, then seedlings were incubated in dark for 4 h. Fluorescent image was taken by Olympus BX-50 fluorescent microscope with FITC filter set. For assay using decapitated etiolated hypocotyl (Fig S13B), the apical ~1mm of 4-days old etiolated hypocotyls (Col-0) were excised with a scalpel. Cut hypocotyls were inverted in 5 µl of GM solution containing 0.1% agar and chemicals (NAA or TIBA) in 0.5 ml microcentrifuge tubes and incubated for 1 h in the dark. The GM solution (5 µl) containing 0.1% agar and 100 µM NBD-NAA was added to the bottom of tube and mixed well by pipetting. The hypocotyls were incubated for additional 3 h in the dark. Hypocotyls were analyzed by or BX-50 fluorescent microscope under settings for FITC fluorescence. For assay using decapitated etiolated hypocotyl (Fig S13C and S13D), the apical ~1mm of 5-6 days-old etiolated hypocotyls (Col-0) were excised with a scalpel. Cut hypocotyls were inverted in 15µl of 1/4 MS solution containing 0.5% sucrose and 1, 10 or 50 µM NBD-IAA or NBD-NAA in 1.5 ml micro-centrifuge tubes and incubated for 2-3 h in the dark. Hypocotyls were analyzed by confocal microscopy on a Zeiss LSM 710 under settings for GFP fluorescence. Quantification of NBD-auxins in suspension-cultured Arabidopsis cell. MM1 cells were incubated at 24°C with/without 50 μM TIBA for 30 min, and 2 μM NBD-analogs were then added to the medium. After incubation for 30 min, the cells (2 mL) were collected via filtration and then subjected to extraction in methanol (2 mL). The fluorescent signals of the NBD-auxins were quantified with a fluorometer (Ex 460 nm, Em 540 nm). HPLC analysis of NBD-NAA in tobacco cultured BY-2 cell. BY-2 cells were incubated at 24°C with 2 μM NBD-NAA, and 0.5 ml aliquots of the cultures were centrifuged at regular intervals. The cells (1 mL) were washed with fresh medium, and methanol was added to extract NBD-NAA metabolites from the cells. The methanol extracts were analyzed via HPLC with fluorescence detector (Ex 460 nm, Em 540 nm, 0.8 mL/min, CH3OH:H2O=3:1+0.05% TFA, 4.5 mm × 150 mm Cosmosil ODS-MS-II column). The retention time of NBD-NAA was 5.6 min under these analytical conditions. Molecular docking calculations Structural data on the ABP1-NAA complex [PDB ID, 1LRH] and the Ask-TIR1-NAA-IAA7 complex [PDB ID, 2P1O] were obtained from a protein data bank and then edited using Discovery Studio visualizer 3.0 (Accelrys). The PDB files were visually inspected. The ligands and all water molecules were removed, and hydrogen atoms were added, as appropriate. The structures of NBD-IAA and NBD-NAA were generated with Chem3D software (PerkinElmer), and the initial structures were optimized via the MM2 method in Chem3D. Molecular docking calculations for NBD-IAA and NBD-NAA at the auxin-binding pockets of Arabidopsis Auxin-Binding Protein 1 (ABP1) and TIR1 were performed with Autodock Vina software (4). The predicted binding conformations of the NBD-auxins were visualized with Discovery Studio visualizer 3.0. The coordinates of the IAA7 degron peptide and the original ligand were superimposed on the docked molecules. Synthesis of chemicals 1. General experimental condition. 1H and 13C NMR spectra were recorded on a JEOL ECS400 and Lambda 500 NMR spectrometer (JEOL, Japan). Chemical shifts are shown as δ values from TMS as the internal reference. Peak multiplicities are quoted in Hz. Mass spectra were measured on a JMS-700 spectrometer (JEOL, Japan). Column chromatography was carried out on columns of silica gel 60 (230–400 mesh, Merck, Japan). All chemicals were purchased from Tokyo Chemical Industry Japan (Tokyo, Japan) and Sigma-Aldrich Japan (Tokyo, Japan) otherwise stated. 2. Synthesis of fluorescent compounds 2.1. Synthetic procedure of 2-(7-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino) ethoxy) naphthalen-1-yl) acetic acid (NBD-NAA: 9)

3

Starting material, ethyl 2-(7-hydroxynaphthalen-1-yl)acetate (5) was synthesized according to the published procedures (5) form 7-hydroxy 1-tetralone.

To the solution of 5 (103 mg, 0.45 mmol) in DMF (2 mL) was added Cs2CO3 (146 mg, 0.45 mmol) and tert-butoxylcarbonylaminoethyl iodide (242 mg, 0.90 mmol), and then stirred for 15h at room temperature. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with saturated NH4Cl solution and brine, and then dried over Na2SO4. The residue was purified by a silica gel column chromatography (n-hexane:acetone=3:1) to give ethyl 2-(7-(2-((tert-butoxycarbonyl) amino)ethoxy) naphthalen-1-yl)acetate (6) as pale yellow oil (136mg, 81 % yield).1H NMR (400 MHz, CDCl3): δ 1.22 (3H, t, J=7.3), 1.45 (9H, s), 3.61 (2H, m), 3.99 (2H, s), 4.14 (4H, m), 5.08 (NH, brs), 7.15 (1H, d, J=9.2), 7.29 (2H, m), 7.39 (1H, d, J=6.9), 7.71 (1H, d, J=8.2), 7.76 (1H, d, J=8.7); 13C NMR (100 MHz, CDCl3): δ 14.18, 26.48, 26.84, 28.38, 39.69, 40.30, 60.89, 67.45, 79.05, 103.26, 118.49, 123.13, 127.64, 128.45, 129.16, 129.34, 130.13, 133.21, 155.97, 157.22, 171.58; HR-EIMS m/z 373.1870 [M]+ (∆mmu 1.9); m/z 373.1889 calcd. for C21H27NO5. The ethyl ester (6) (88 mg, 0.24 mmol) was dissolved in CH2Cl2 (1mL) and then trifluoroacetic acid (0.5mL) was added dorpwise. and the mixture was stirred for 5 min. at room temperature. The reaction mixture was poured into sodium bicarbonate aqueous solution (5 mL) and extracted with EtOAc (20 mL × 3). The organic layer was washed with brine. After dried over Na2SO4, the solvent was removed in vacuo to give ethyl 2-(7-(2-aminoethoxy)naphthalen-1-yl)acetate (7) as pale yellow oil. Amine (7) was used for following reaction without further purification. 1H NMR (500 MHz, CDCl3): δ 1.21 (3H, t, J=7.2), 3.16 (2H, m), 3.99 (2H, s), 4.14 (4H, m), 7.17 (1H, dd, J=8.9, 2.2), 7.29 (2H, m), 7.38 (1H, d, J=6.8), 7.71 (1H, d, J=8.0), 7.75(1H, d, J=8.8); 13C NMR (125 MHz, CDCl3): δ 14.17, 39.66, 41.23, 60.92, 69.54, 103.40, 118.35, 123.26, 127.66, 128.53, 129.26, 129.37, 130.22, 133.15, 157.09, 171.58; FABMS m/z 274 [M+H]+. To the solution of 7 (50 mg, 0.18 mmol) in 4 mL of mixed solvent [THF : 100mM borate buffer pH 9] was gradually added 4-fluoro-7-nitro-2,1,3-benzoxadiazole, NBD-F (44 mg, 0.24 mmol). The reaction mixture was then stirred for 60 min at room temperature. During the reaction, pH of reaction mixture was maintained between pH 8 – 9 by adding 100 mM borate buffer. The reaction mixture was added to saturated NH4Cl solution (20 mL), and extracted with EtOAc (20 mL × 3). The organic layer was washed with and brine, and then dried over Na2SO4. The residue was purified by a silica gel column chromatography (benezne:ethyl actetate=8:1) to give ethyl 2-(7-(2-((7-nitrobenzo[c][1,2,5] oxadiazol-4-yl)amino)ethoxy) naphthalen-1-yl)acetate (8) as amorphous red powder (26 mg, 33 % yield). m.p.= 151−157°C; 1H NMR (400 MHz, DMSO-d6): δ 1.15 (3H, t, J=7.3), 3.37 (2H, s), 4.07 (4H, m), 4.43 (2H, m), 6.59 (1H, d, J=9.2), 7.20 (1H, dd, J=9.2, 2.4), 7.29 (2H, m), 7.40 (1H, d, J=6.7), 7.77 (1H, d, J=7.9), 7.86 (1H, d, J=9.2), 8.55 (1H, d, J=8.6); 13C NMR (100 MHz, DMSO-d6): δ 14.28, 38.56, 43.11, 60.50, 65.67, 99.84, 104.00, 118.24, 121.32, 123.47, 127.49, 128.70, 129.02, 130.07, 130.36, 133.06, 137.98, 144.24, 144.60, 145.48, 156.52, 171.36.; FABMS m/z 437 [M+H]+ . The ethyl ester (8) (34 mg, 0.078 mmol) was dissolved in mixed solution (H2O:MeOH :THF=2:1:1). Lithium hydroxide monohydrate (7 mg, 0.15 mmol) was added to the solution and then stirred at room temperature until the complete hydrolysis of 8. The resulting solution was neutralized and evaporated in vacuo and acidified to pH 3.5 with 6N HCl. The resulting suspension was extracted with EtOAc (20 mL × 3). The organic layer was washed with a brine. After dried over Na2SO4, the solvent was removed in vacuo. The residue was purified by a silica gel column chromatography (benzene:acetone=5:1) to give 2-(7-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy) naphthalen-1-yl) acetic acid (NBD-NAA: 9) as amorphous red powder (7.9 mg, 25 % yield). m.p.= 158−164°C; 1H NMR (400 MHz, DMSO-d6): δ 3.61 (2H, s), 4.10 (2H, m), 4.43 (2H, m), 6.60 (1H, d, J= 9.2), 7.20 (1H, dd, J= 8.8, 2.4), 7.29 (1H, d, J= 2.4), 7.31 (2H, m), 7.78 (1H, d, J= 7.6), 7.86 (1H, d, J= 8.8), 8.56 (1H, d, J= 9.2); 13C NMR (100 MHz, DMSO-d6): δ 38.33, 43.16, 65.74, 99.88, 104.01, 118.25, 121.34, 123.49, 127.54, 128.75, 129.03, 129.97, 130.39, 133.06, 138.06, 144.30, 144.66, 145.54, 156.57, 171.87; FABMS

4

m/z 409 [M+H]+ , HR-FABMS m/z calcd. for C20H17N4O6; 409.1148 [M+H]+, found 409.1162 (∆mmu 1.4). Fluorescent spectrum (Ex 466nm, Em 538 nm in 50mM KPB pH7.0 and 1/2 MS medium pH 5.8) was indicted below. Fluorescent filter sets for GFP or FITC were available for the detection of NBD-NAA (9).

Fluorescent spectra of NBD-NAA (9)

2.2. Synthetic procedure of 2-(7-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino) butoxy) naphthalen-1-yl) acetic acid（NBD-C4-NAA: 13）

To the solution of 5 (45 mg, 0.20 mmol) in DMF (2 mL) was added Cs2CO3 (126 mg, 0.38 mmol) and tert-butoxylcarbonylaminobutyl iodide (140 mg, 0.47 mmol), and then stirred for 15h at room temperature. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with saturated NH4Cl solution and brine, and then dried over Na2SO4. The residue was purified by a silica gel column chromatography (n-hexane:acetone=3:1) to give ethyl 2-(7-(2-((tert-butoxycarbonyl) amino)butoxy) naphthalen-1-yl)acetate (10) as pale yellow oil (74 mg, 95 % yield).1H NMR (400 MHz, CDCl3): δ1.21 (3H, t, J=7.1), 1.48 (9H, s), 1.71 (2H, m), 1.88 (2H, m), 3.22 (2H, m), 3.99 (2H, s), 4.17 (4H, m), 4.71 (NH, brs), 7.14 (1H, dd, J=9.2, 2.8), 7.28 (2H, m), 7.38 (1H, d, J=6.0), 7.70 (1H, d, J=8.3), 7.74 (1H, d, J=9.2); 13C NMR (100 MHz, CDCl3): δ14.18, 26.48, 26.84, 28.39, 39.69, 40.30, 60.89, 67.45, 79.05, 103.26, 118.49, 123.13, 127.64, 128.45, 129.16, 129.34, 130.13, 133.21, 155.97, 157.22, 171.58; HR-EIMS m/z 401.2202 [M]+, m/z 401.2175 calcd. for C23H31NO5; (∆mmu 2.7). The ethyl ester (6) (82 mg, 0.20 mmol) was dissolved in CH2Cl2 (1 mL) and then trifluoroacetic acid (0.5mL) was added dorpwise. The mixture was stirred for 5 min. at room temperature. The reaction mixture was poured into sodium bicarbonate aqueous solution (5 mL) and extracted with EtOAc (20 mL × 3). The organic layer was washed successively with brine. After dried over Na2SO4, the solvent was removed in vacuo to give ethyl 2-(7-(2-aminoethoxy)naphthalen-1-yl)acetate (11) as pale yellow oil (52 mg, 84% yield). Amine (11) was used for following reaction without further purification. 1H NMR (500 MHz, CDCl3): δ 1.21 (3H, t, J=7.0), 1.69 (2H, m), 1.87 (2H, m), 2.61 (2H, m), 3.98 (2H, s), 4.12 (4H, m), 7.14 (1H, dd, J=6.4, 3.0), 7.27 (2H, m), 7.37 (1H, d, J=7.1), 7.69 (1H, d, J=8.0), 7.73 (1H, d, J=8.9); 13C NMR (125 MHz, CDCl3): δ 14.14, 26.47, 29.51, 39.63, 41.52, 60.86, 67.53, 103.21, 118.47, 123.08, 127.61, 128.42, 129.12, 129.29, 130.10, 33.24, 157.24, 171.59; FABMS m/z 302 [M+H]+. To the solution of 11 (39 mg, 0.13 mmol) in 4 mL of mixed solvent [THF : 100mM borate buffer pH 9] was gradually added 4-fluoro-7-nitro-2,1,3-benzoxadiazole, NBD-F (31 mg, 0.17 mmol). The reaction mixture was then stirred for 60 min at room temperature. During the reaction, pH of reaction mixture was maintained between pH 8 – 9 by adding 100 mM borate buffer. The reaction mixture was added to saturated NH4Cl solution (20 mL), and extracted with EtOAc (20 mL × 3). The organic layer was washed with and brine, and then dried over Na2SO4. The residue was purified by silica gel column chromatography (benezne :ethyl actetate=10:1) to give ethyl 2-(7-(2-((7-nitrobenzo[c][1,2,5] oxadiazol-4-yl)amino)butoxy) naphthalen-1-yl) acetate (12) as amorphous red powder (18 mg, 31% yield). m.p.= 131−142°C; 1H NMR (400 MHz, DMSO-d6): δ 1.14 (3H, t,

5

J=4.4), 1.92 (4H, m), 3.35 (2H, s), 4.07 (4H, m), 4.15 (2H, m), 6.49 (1H, d, J=8.8), 7.17 (1H, dd, J=9.0, 2.4), 7.28 (2H, m), 7.39 (1H, d, J=6.4), 7.77 (1H, d, J=8.4), 7.84 (1H, d, J=8.0), 8.50 (1H, d, J=9.2); 13C NMR (100 MHz, DMSO-d6): δ 14.31, 24.57, 26.22, 38.66, 43.24, 60.49, 67.26, 99.30, 103.83, 118.30, 120.80, 123.29, 127.49, 128.63, 128.86, 129.95, 130.27, 133.14, 138.06, 144.32, 144.63, 145.34, 156.85, 171.40; FABMS m/z 465 [M+H]+ .

Ethyl ester (12) (26.5 mg, 0.057 mmol) was dissolved in mixed solution (H2O:MeOH :THF=2:1:1). Lithium hydroxide monohydrate (5 mg, 0.11 mmol) was added to the solution and then stirred at room temperature until the complete hydrolysis of 12. The resulting solution was neutralized and evaporated in vacuo and then acidified to pH 3.5 with 6N HCl. The resulting suspension was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine. After dried over Na2SO4, the solvent was removed in vacuo. The residue was purified by a silica gel column chromatography (benzene:acetone=5:1) to give 2-(7-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)butoxy) naphthalen-1-yl) acetic acid (NBD-C4-NAA: 13) as amorphous red powder (13.5 mg, 42 % yield). m.p.= 138−144°C; 1H NMR (400 MHz, DMSO-d6): δ 1.92 (4H, m), 3.34 (2H, m), 4.08 (2H, s), 4.15 (2H, m), 6.44 (1H, d, J=8.8), 7.19 (1H, dd, J=9.2, 2.4), 7.28 (2H, m), 7.39 (1H, d, J=6.8), 7.77 (1H, d, J=8.4), 7.84 (1H, d, J=9.2), 8.50 (1H, d, J=8.4), 9.58 (NH, brs); 13C NMR (100 MHz, DMSO-d6): δ 24.93, 26.54, 38.77, 43.57, 67.62, 99.68, 104.19, 118.64, 121.15, 123.64, 127.86, 128.84, 129.02, 129.20, 130.19, 133.48, 138.43, 144.65, 144.97, 145.72, 157.24, 172.23; FABMS m/z 437 [M+H]+ , HR-FABMS m/z 437.1487 [M+H]+, (∆mmu 2.6), m/z 437.1461 calcd. for C20H17N4O6;. Fluorescent spectrum (Ex 468 nm, Em 545 nm in 50mM KPB pH7.0 and 1/2 MS medium pH 5.8). 2.3. Synthetic procedure of 2-(5-((1-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)piperidin-4-yl) methoxy)-1H-indol-3-yl)acetic acid (NBD-IAA: 18)

To the solution of 14 (105 mg, 0.51 mmol) in DMF (2 mL) was added Cs2CO3 (250 mg, 0.77 mmol) and tert-butyl 4-(iodomethyl)piperidine-1-carboxylate (250 mg, 0.77 mmol), and then stirred for 3h at room temperature. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with saturated NH4Cl solution and brine, and then dried over Na2SO4. The residue was purified by a silica gel column chromatography (n-hexane:EtOAc=2:1) to give tert-butyl 4-(2-((3-(2-methoxy-2-oxoethyl) -1H-indol-5-yl)oxy)methyl)piperidine-1-carboxylate (15) as pale yellow oil (173 mg, 78.5 % yield).1H NMR (400 MHz, CDCl3): δ 1.27−1.34 (m, 2H), 1.47 (s, 9H), 1.85 (d, J=12.8, 2H), 2.16 (d, J=2.0, 1H), 2.75 (t, J=12.2, 2H), 3.69 (s, 3H), 3.74 (s, 2H), 3.84 (d, J=6.4, 2H), 6.83 (dd, J=2.4, 2.0, 1H), 7.03 (d, J=2.4, 1H), 7.08 (d, J=2.4, 1H), 7.20 (d, J=8.8, 1H), 8.37 (s, 1H); 13C NMR (100 MHz, CDCl3): δ 28.40, 28.93, 31.15, 36.28, 51.88, 53.73, 73.15, 79.33, 101.64, 107.76, 111.91, 112.71, 123.92, 127.51, 131.29, 153.39, 154.88, 172.52 ; EI-MS m/z 402 [M]+. tert-butyl 4-(2-((3-(2-methoxy-2-oxoethyl)-1H-indol-5-yl)oxy)methyl)piperidine-1-carboxylate (15) (76 mg, 0.18 mmol) was dissolved in 4 mL of mixed solution (2N aqueous KOH:MeOH:THF=1:2:1) and then stirred at 30 °C until the complete hydrolysis of 15. The resulting solution was neutralized and evaporated in vacuo and then acidified to pH 3.5 with 6N HCl. The resulting suspension was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine. After dried over Na2SO4, the solvent was removed in vacuo. The residue was purified by a silica gel column chromatography (chloroform:MeOH=9:1) to give 2-(5-((1-(tert-butoxycarbonyl)piperidin-4-yl)methoxy)-1H-indol-3-yl)acetic acid as colorless powder (63 mg, 86 % yield); m.p.=62-64ºC; 1H NMR (400 MHz, CDCl3): δ1.16−1.26 (m, 2H), 1.47 (s, 9H), 1.78 (d, J=12.4, 2H), 1.90 (s, 1H), 2.71 (s, 2H), 3.72 (s, 2H), 3.78 (s, 2H), 6.80 (dd, J=2.4, 2.4, 1H), 7.00 (d, J=2.4, 1H), 7.03 (d, J=2.0, 1H), 7.16 (d, J=8.8, 1H), 8.41 (s, 1H); 13C NMR (100 MHz, CDCl3): δ 28.42, 28.83, 31.17, 36.12, 43.77, 73.08, 79.59, 101.58, 107.21, 112.00, 112.77, 124.17, 127.42, 131.28, 153.32, 155.05, 177.42; FABMS m/z 411 [M+Na]+ . The carboxylic acid (16) (25 mg, 0.06 mmol) was dissolved in CH2Cl2 (0.5mL) and trifluoroacetic acid (0.5mL) was added dropwise. The solution was stand for 15 min. at room temperature. The reaction mixture was evaporated in vacuo to give 2-(5-(piperidin-4-ylmethoxy)-1H-indol-3-yl)acetic acid (17) as pale yellow oil. Crude compound (18 mg, 0.06 mmol) was dissolved in 4 mL of mixed solvent [CH3CN : 100mM borate buffer pH 9=4:1] and 4-fluoro-7-nitro-2,1,3-benzoxadiazole, NBD-F (15 mg, 0.08 mmol) was gradually added. The reaction mixture was then stirred for 60 min at room temperature.

6

During reaction, pH of reaction mixture was maintained between pH 8 – 9 by adding borate buffer. The reaction mixture was added to saturated NH4Cl solution (20 mL), and extracted with EtOAc (20 mL × 3). The organic layer was washed with and brine, and then dried over Na2SO4. The residue was purified by a silica gel column chromatography (CHCl3:EtOAc=9:1) to give 2-(5-((1-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)piperidin-4-yl)methoxy)-1H-indol-3-yl)acetic acid (18) as amorphous red powder (9 mg, yield 32%). m.p.= 163−164°C; 1H NMR (400 MHz, DMSO-d6): δ 1.51−1.61 (m, 2H), 2.06 (s, 1H), 2.09 (s, 2H), 3.54 (t, J=10.8, 2H), 3.70 (s, 2H), 3.87 (d, J=5.6, 2H), 4.86 (d, J=10.4, 2H), 6.66 (dd, J=2.8, 2.4, 1H), 6.74 (dd, J=2.4, 2.0, 1H), 6.99 (d, J=2.4, 1H), 7.20 (d, J=2.0, 1H), 7.24 (d, J=8.8, 1H), 8.45 (dd, J=2.0, 2.4, 1H), 10.79 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ 28.90, 30.56, 35.15, 49.93, 72.11, 101.79, 103.52, 106.85, 111.80, 112.24, 120.52, 124.98, 127.65, 131.52, 136.56, 144.97, 145.16, 145.44, 152.60, 172.26, HR-FABMS m/z 474.1368 [M+Na]+ (∆mmu 2.2), m/z 474.1390 calcd. for C22H21N5O6Na. Fluorescent spectrum (Ex 517 nm, Em 552 nm in 50mM KPB pH 7.0 and 1/2 MS medium pH 5.8) was indicted below. Fluorescent filter sets for GFP or FITC were available for the detection of NBD-IAA (18).

Fluorescent spectra of NBD-IAA (18)

2.4. Synthetic procedure of 4-(4-(((1H-indol-5-yl)oxy)methyl)piperidin-1-yl) -7-nitrobenzo[c] [1,2,5]oxadiazole (NBD-indole: 13）

NON

O2N

F

NON

O2N

O

N

NH

O

HN

NH

HO

Cs2CO3DMF

O

O

N

IO

O

O

N

NH N

H

TFA

CH2Cl219

2021

To the solution of 5-hydroxyindole (201 mg, 1.51 mmol) in DMF (2 mL) was added Cs2CO3 (975 mg, 3.0 mmol) and tert-butyl 4-(iodomethyl)piperidine-1-carboxylate (982 mg, 3.0 mmol), and then stirred for 3h at room temperature. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with saturated NH4Cl solution and brine, and then dried over Na2SO4. The residue was purified by a silica gel column chromatography (n-hexane:EtOAc=1:1) to give tert-butyl 4-(((1H-indol-5-yl)oxy)methyl) piperidine-1-carboxylate (19) as colorless powder (79 mg, 16 % yield).1H NMR (400 MHz, CDCl3): δ 1.20−1.30 (m, 2H), 1.47 (s, 9H), 1.82 (d, J=12.8, 2H), 1.90−1.97 (m, 1H), 2.73 (t, J=11.2, 2H), 3.80 (d, J=6.4, 2H), 4.15 (s, 2H), 6.43 (d, J=2.4, 1H), 6.82 (dd, J=2.0, 2.4, 1H), 7.07, (d, J=2.4, 1H), 7.10 (t, J=2.8, 1H), 7.21 (d, J=9.2, 1H), 8.55 (s, 1H); 13C NMR (100 MHz, CDCl3): δ 28.36, 28.83, 36.09, 43.81, 73.10, 79.36, 101.81, 103.30, 111.67, 112.47, 124.97, 128.11, 131.00, 153.22, 154.89; EI-MS m/z 330 [M]+.

tert-Butyl 4-(((1H-indol-5-yl)oxy)methyl)piperidine-1-carboxylate (19) (69 mg, 0.21 mmol) was dissolved in CH2Cl2 and trifluoroacetic acid (0.5mL) was added dropwise. The solution was stand for 15 min. at room temperature. The reaction mixture was evaporated in vacuo to give 5-(piperidin-4-ylmethoxy)-1H-indole (20) as pale yellow oil. This crude compound (24 mg) was dissolved in 2 mL of mixed solvent [THF : 100mM borate buffer pH 9=4:1] and 4-fluoro-7-nitro-2,1,3-benzoxadiazole, NBD-F (19 mg, 0.10 mmol) was gradually added. The reaction mixture was then stirred for 30 min at room temperature. During reaction, pH of reaction mixture was maintained between pH 8–9 by adding borate buffer. The reaction mixture was added to saturated NH4Cl solution (20 mL), and extracted with EtOAc (20 mL × 3). The organic layer was washed with and brine, and then dried over Na2SO4. The residue was purified by a silica gel column chromatography (benzene:MeOH=15:1) to give 4-(4-(((1H-indol-5-yl)oxy)methyl) piperidin-1-yl)-7-nitrobenzo[c][1,2,5]oxadiazole (21) as amorphous red powder (18 mg, 43% yield).1H NMR (400 MHz, acetone-d6): δ 1.56 (m, 2H), 2.21(m, 3H), 3.60 (m, 2H), 4.03 (d, J=6.4, 2H), 5.015 (brd, 2H), 6.70 (d, J=9.0, 1H), 7.15 (dd, J=8.8, 2.4, 1H), 7.54, (d, J=8.8, 1H), 7.62 (m, 1H), 7.69 (m, 1H), 7.82 (d, J=2.4), 8.34 (brs, 1H),

7

8.49 (d=9.0, 1H); 13C NMR (400 MHz, acetone-d6): δ 35.75, 49.94, 72.16, 102.98, 104.40, 113.58, 114.69, 127.33, 128.56, 128.68, 131.90, 135.79, 136.40, 145.30, 145.46, 156.61. FABMS m/z 394 [M+H]+, HR-FABMS m/z 394.1482 [M+H]+ (∆mmu 1.3), m/z 394.1515 calcd. for C20H20N5O4. Fluorescent spectrum (Ex 487 nm, Em 545 nm in 50mM KPB pH7.0 and 1/2 MS medium pH 5.8) 2.5. Synthetic procedure of 2-(5-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl) amino)ethoxy)- 1H-indol-3-yl) acetate (NBD-C2-IAA: 25)

To the solution of 5-hydroxy indole 3-acetic acid methyl ester (198 mg, 0.97 mmol) in DMF (3 mL) was added Cs2CO3 (627 mg, 1.93 mmol) and tert-butoxylcarbonylaminoethyl iodide (523 mg, 1.93 mmol), and then stirred for 2 h at 70 °C. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with saturated NH4Cl solution and brine, and then dried over Na2SO4. The residue was purified by a silica gel column chromatography (CHCl3:ethyl acetate=6:1) to give ethyl 2-(7-(2-((tert-butoxycarbonyl) amino)butoxy) naphthalen-1-yl)acetate (22) as pale brown oil (290 mg, 86 % yield).1H NMR (400 MHz, CDCl3): δ 1.46 (9H, s), 3.55 (2H, m), 3.70 3H, s), 3.73 (2H, s), 4.05 (2H, m), 5.11 (NH, brs), 6.88 (1H, dd, J=8.7, 2.3), 7.04 (1H, d, J=2.3), 7.11 (1H, d, J=2.3), 7.22 (1H, d, J=8.7), 8.32 (NH, brs), 7.14 (1H, dd, J=9.2, 2.8), 7.28 (2H, m), 7.38 (1H, d, J=6.0), 7.70 (1H, d, J=8.3), 7.74 (1H, d, J=9.2); 13C NMR (100 MHz, CDCl3): δ 28.35, 31.11, 40.23, 51.95, 67.83, 79.37, 101.78, 107.90, 111.96, 112.51, 124.03, 127.52, 131.43, 152.90, 155.95, 172.49; FAB-MS m/z 349 [M+H]+. Methyl ester 22 (100 mg, 0.28 mmol) was dissolved in CH2Cl2 (1 mL) and then trifluoroacetic acid (0.5mL) was added dorpwise and the stirred for 5 min. at room temperature. The reaction mixture was poured into sodium bicarbonate aqueous solution (5 mL) and extracted with EtOAc (20 mL × 3). The organic layer was washed successively with brine. After dried over Na2SO4, the solvent was removed in vacuo to give methyl 2-(5-(2-aminoethoxy)-1H-indol-3-yl)acetate (23) as pale brown oil (41 mg, 57% yield). Amine (23) was used for following reaction without further purification. 1H NMR (500 MHz, CD3OD): δ 3.15 (2H, m), 3.67 (3H, s), 3.73 (2H, s), 4.11 (2H, m), 6.84 (1H, dd, J=8.7, 2.6), 7.06 (1H, d, J=2.6), 7.14 (1H, s), 7.25 (1H, d, J=8.7); 13C NMR (125 MHz, CD3OD): δ 31.89, 41.36, 52.36, 68.79, 102.90, 108.33, 113.05, 113.16, 125.65, 128.89, 133.56, 153.87, 174.78; FABMS m/z 249 [M+H]+. To the solution of 23 (163 mg, 0.65 mmol) and triethylamine (100 mg, 0.65mmol) in CH3CN (4 mL), gradually added 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole, NBD-Cl (131 mg, 0.65 mmol). The reaction mixture was then stirred for 60 min at room temperature. The reaction mixture was added to saturated NH4Cl solution (20 mL), and extracted with EtOAc (20 mL × 3). The organic layer was washed with and brine, and then dried over Na2SO4. The residue was purified by silica gel column chromatography (CHCl3:ethyl acetate=5:1) to give methyl 2-(5-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)-1H-indol-3-yl)acetate (24) as amorphous red powder (43.2 mg, 16 % yield). m.p. >200°C; 1H NMR (400 MHz, DMF-d7): δ 3.55 (2H, m), 3.65 (3H, s), 3.77 (2H, s), 4.43 (2H, t, J=4.3), 6.70 (1H, d, J=8.7), 6.81 (1H, dd, J=8.7, 2.3), 7.16 (1H, d, J=2.3), 7.33 (2H, m), 8.61(1H, m), 10.91 (NH, brs); 13C NMR (100 MHz, DMF-d7): δ 30.01, 43.05, 50.58, 65.68, 98.92, 101.15, 106.71, 111.15, 111.55, 121.23, 124.42, 127.28, 131.50, 137.03, 143.81, 144.21, 145.02, 151.96, 171.63; FAB-MS m/z 412 [M+H]+. Methyl ester 24 (42 mg, 0.10 mmol) was dissolved in mixed solution (2 mL, H2O:MeOH :THF=2:1:1). Lithium hydroxide monohydrate (9 mg, 0.20 mmol) was added to the solution and then stirred at 40 °C until the complete hydrolysis of 24. The resulting solution was neutralized and evaporated in vacuo and then acidified to pH 3.5 with 6N HCl. The resulting suspension was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine. After dried over Na2SO4, the solvent was removed in vacuo. The residue was purified by a silica gel column chromatography (chloroform:acetone:MeOH=8:1:0.2, 1% acetic acid) to give 2-(5-(2-((7-nitrobenzo [c][1,2,5] oxadiazol-4-yl) amino)ethoxy)-1H-indol-3-yl) acetic acid (NBD-C2-IAA: 25) as amorphous red powder (17 mg, 41% yield) . m.p.>200°C; 1H NMR (400 MHz, DMF-d7): δ 3.71 (2H, s), 4.07 (2H, m), 4.42 (2H, m), 6.70 (1H, d, J=8.7), 6.79 (1H, dd, J=8.7, 2.3), 7.18 (1H, d, J=2.3), 7.32 (2H, m), 8.61 (1H, d, J=8.4), 10.86 (NH, brs); 13C NMR (100 MHz, DMF-d7): δ 30.53, 43.20, 65.82, 99.02, 101.44, 107.57, 111.13, 111.59, 121.35, 124.44, 127.58, 131.66, 137.11, 143.72, 144.35, 145.20, 152.01, 172.70; HR-FABMS m/z 420.089 [M+Na]+ , m/z 420.0920, calcd. for C18H15N5NaO6.

8

Fluorescent spectrum (Ex 467 nm, Em 547 nm in 50mM KPB pH7.0 and 1/2 MS medium pH 5.8). 2.6. Synthetic procedure of 2-(5-(4-((7-nitrobenzo[c][1,2,5] oxadiazol-4-yl)amino) butoxy)-1H-indol- 3-yl) acetic acid (NBD-C4-IAA: 29)

To the solution of 5-hydroxyindole 3-acetic acid methyl ester (139 mg, 0.68 mmol) in DMF (2 mL) was added Cs2CO3 (440 mg, 1.35 mmol) and tert-butoxylcarbonylaminobutyl iodide (405 mg, 1.35 mmol), and then stirred for 5h at room temperature. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with saturated NH4Cl solution and brine, and then dried over Na2SO4. The residue was purified by silica gel column chromatography (chloroform:ethyl acetate=6:1) to give methyl 2-(5-(4-((tert-butoxycarbonyl) amino)butoxy) -1H-indol-3-yl) acetate (26) (234 mg, 92% yield).1H NMR (400 MHz, CDCl3): δ1.45 (9H, s), 1.67 (2H, m), 1.81 (2H, m), 3.19 (2H, m), 3.69 (3H, s), 3.73 (2H, s), 3.99 (2H, t, J=6.4), 4.77 (NH, brs), 6.82 (1H, dd, J=8.7, 2.3), 7.03 (1H, d, J=2.3), 7.06 (1H, s), 7.18 (1H, d, J=8.8), 8.41 (NH, brs); 13C NMR (100 MHz, CDCl3): δ 26.65, 26.77, 28.35, 31.14, 40.31, 51.90, 68.17, 79.01, 101.59, 107.69, 111.91, 112.71, 123.90, 127.48, 131.28, 153.19, 156.03, 172.59; FAB-MS m/z 377 [M+H]+. Synthesis of methyl 2-(5-(4-aminobutoxy)-1H-indol-3-yl)acetate (27) The ethyl ester (26) (126 mg, 0.34 mmol) was dissolved in CH2Cl2 (1 mL) and then trifluoroacetic acid (0.5mL) was added dorpwise and the stirred for 5 min. at room temperature. The reaction mixture was poured into sodium bicarbonate aqueous solution (5 mL) and extracted with EtOAc (20 mL × 3). The organic layer was washed successively with brine. After dried over Na2SO4, the solvent was removed in vacuo to give e methyl 2-(5-(4-aminobutoxy)- 1H-indol-3-yl)acetate (27) as pale yellow oil (51 mg, 61% yield). Amine (27) was used for following reaction without further purification. 1H NMR (400 MHz, CD3OD): δ 1.83 (4H, m), 2.97 (2H, m), 3.99 (2H, m), 3.66 (3H, s), 3.71 (2H, s), 6.78 (1H, dd, J=8.7, 2.3), 7.02 (1H, d, J=2.3), 7.11 (1H, s),7.25 (1H, d, J=8.7); 13C NMR (125 MHz, CDCl3): δ 27.44, 30.06, 31.89, 40.65, 52.37, 68.97, 102.63, 108.24, 113.02, 113.16, 125.54, 128.91 ,133.35, 154.14, 174.83; FABMS m/z 277 [M+H]+. To the solution of 27 (44 mg, 0.16 mmol) in 4 mL of mixed solvent [THF : 100mM borate buffer pH 9=4:1] was gradually added 4-fluoro-7-nitro-2,1,3-benzoxadiazole, NBD-F (38 mg, 0.20 mmol). The reaction mixture was then stirred for 60 min at room temperature. During reaction, pH of reaction mixture was maintained between pH 8–9 by adding 100 mM borate buffer. The reaction mixture was added to saturated NH4Cl solution (20 mL), and extracted with EtOAc (20 mL × 3). The organic layer was washed with and brine, and then dried over Na2SO4. The residue was purified by a silica gel column chromatography (benzene :acetone=8:1) to give methyl 2-(5-(4-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)butoxy)-1H-indol-3-yl)acetate (28) as amorphous red powder (9.5 mg, 14% yield). Crude methyl ester (28) was dissolved in mixed solution (2mL, H2O:MeOH :THF=2:1:1) and then lithium hydroxide monohydrate (2 mg) was added to the solution and then stirred at room temperature until the complete hydrolysis of 28. The resulting solution was neutralized and evaporated in vacuo and the acidified to pH 3.5 with 6N HCl. The resulting suspension was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine. After dried over Na2SO4, the solvent was removed in vacuo. The residue was purified by a silica gel column chromatography (chloroform: MeOH=6:1) to give 2-(5-(4-((7-nitrobenzo[c][1,2,5] oxadiazol-4-yl)amino) butoxy)-1H- indol-3-yl) acetic acid (NBD-C4-IAA: 29) as amorphous red powder (8.3 mg, 90 % yield) . m.p.>200°C; 1H NMR (400 MHz, DMF-d7): δ 1.85 (4H, m), 3.48 (2H, s), 3.54 (2H, m), 4.00 (2H, m), 6.30 (1H, d, J=9.5), 6.69 (1H, dd, J=8.8, 2.4), 7.02 (1H, d, J=1.4), 7.13 (1H, d, J=1.6), 7.19 (1H, d, J=8.8), 8.26 (1H, d, J=9.2), 10.70 (NH, brs); 13C NMR (100 MHz, DMF-d7): δ 26.84, 32.79, 35.07, 45.26, 49.44, 67.86, 100.00, 102.16, 109.18, 111.72, 111.92, 120.59, 124.45, 128.04, 131.48, 136.23, 145.08, 145.95, 147.02, 152.27, 172.98; FABMS m/z 426 [M+H]+ , Fluorescent spectrum (Ex 473 nm, Em 538 nm in 50mM KPB pH7.0 and 1/2 MS medium pH 5.8) 2.7. Synthetic procedure of 2-(5-(4-((7-nitrobenzo[c][1,2,5] oxadiazol-4-yl)amino) butoxy) -1H-indol- 3-yl) acetic acid (6-NBD-2-NAA: 33)

9

To the solution of 6-hydroxynaphthalene-1-acetic acid ethyl ester (109 mg, 0.47 mmol) in DMF (2 mL) was added Cs2CO3 (308 mg, 0.95 mmol) and tert-butoxylcarbonylaminobutyl iodide (256 mg, 0.95 mmol), and then stirred for 10h at room temperature. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with saturated NH4Cl solution and brine, and then dried over Na2SO4. The residue was purified by silica gel column chromatography (n-hexane:acetone=3:1) to give [6-(2-tert-butoxycarbonylamino-ethoxy)-naphthalen-1-yl]-acetic acid ethyl ester (30) as colorless oil (114 mg, 64% yield).1H NMR (400 MHz, CDCl3): δ 1.21 (3H, t, J=6.8),1.46 (9H, s), 3.58 (2H, m), 4.00 (2H, s), 4.08 (4H, m), 7.12 (1H, d, J=2.4), 7.18 (1H, dd, J=9.2, 2.4), 7.26 (1H, d, J=5.2), 7.37 (1H, t, J=8.0), 7.64 (1H, d, J=8.4), 7.91 (1H, d, J=8.8); 13C NMR (100 MHz, CDCl3): δ 14.00, 28.24, 39.16, 39.96, 60.77, 67.02, 79.30, 107.40, 118.70, 125.44, 125.79, 126.07, 126.79, 127.54, 130.54, 134.91, 155.8, 156.21, 171.39; EIMS m/z 373 [M]+.

Ethyl ester (30) (84 mg,0.22 mmol) was dissolved in CH2Cl2 (1 mL) and then trifluoroacetic acid (0.5mL) was added dorpwise and then stirred for 15 min. at room temperature. The reaction mixture was poured into sodium bicarbonate aqueous solution (5 mL) and extracted with EtOAc (20 mL × 3). The organic layer was washed successively with brine. After dried over Na2SO4, the solvent was removed in vacuo to give [6-(2-amino-ethoxy)-naphthalen-1-yl]-acetic acid ethyl ester (31) as pale yellow oil (57 mg, 93% yield). Amine (31) was used for following reaction without further purification. 1H NMR (500 MHz, CDCl3): δ 1.17 (3H, t, J=6.8), 3.04 (2H, m), 4.01 (2H, s), 4.08 (4H, m), 7.18 (1H, d, J=2.8), 7.21 (2H, m), 7.35 (1H, t, J=8.0), 7.67 (1H, d, J=8.8), 7.88 (1H, d, J=9.2); 13C NMR (125 MHz, CDCl3) : δ 14.76, 40.10, 41.95, 62.30, 70.10, 108.81, 120.15, 126.86, 127.17, 127.45, 128.19, 129.28, 132.38, 136.92, 158.22, 173.81; FABMS m/z 274 [M+H]+.

To the solution of 31 (37 mg, 0.14 mmol) in 4 mL of mixed solvent [THF : 100mM borate buffer pH 9] was gradually added 4-fluoro-7-nitro-2,1,3-benzoxadiazole, NBD-F (32 mg, 0.17 mmol). The reaction mixture was then stirred for 60 min at room temperature. During reaction, pH of reaction mixture was maintained between pH 8 – 9 by adding borate buffer. The reaction mixture was added to saturated NH4Cl solution (20 mL), and extracted with EtOAc (20 mL × 3). The organic layer was washed with and brine, and then dried over Na2SO4. The residue was purified by silica gel column chromatography (benezne :ethyl actetate=8:1) to give 6-[3-(7-Nitro-benzo[1,2,5]oxadiazol-4-ylamino)-propoxy]-naphthalen-1-yl}-acetic acid ethyl ester (32) as amorphous orange powder (51 mg, 86% yield). m.p.= 197−198°C; 1H NMR (400 MHz, DMSO-d6): δ 1.17 (3H, t, J=6.4), 3.39 (2H, s), 4.05 (4H, m), 4.45 (2H, m), 6.58 (1H, d, J=9.2), 7.22 (1H, dd, J=6.8, 2.8), 7.28 (1H, d, J=6.8), 7.40 (2H, m), 7.76 (1H, d, J=8.4), 7.88 (1H, d, J=9.2), 8.55 (1H, d, J=8.89); 13C NMR (100 MHz, DMSO-d6): δ 13.67, 38.12, 42.85, 60.19, 65.42, 99.51, 107.32, 118.50, 121.09, 125.55, 125.75, 126.02, 126.50, 127.22, 130.90, 134.60, 137.66, 144.17, 144.31, 145.16, 155.81, 171.01; FABMS m/z 437 [M+H]+ .

Ethyl ester (32) (29 mg, 0.067 mmol) was dissolved in mixed solution (H2O:MeOH :THF=2:1:1). Lithium hydroxide monohydrate (5 mg, 0.01mmol) was added to the solution and then stirred at room temperature until the complete hydrolysis of 32. The resulting solution was neutralized and evaporated in vacuo and then acidified to pH 3.5 with 6N HCl. The resulting suspension was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine. After dried over Na2SO4, the solvent was removed in vacuo. The residue was purified by a silica gel column chromatography (benzene:EtOAc=4:1) to give 6-[2-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-ethoxy]-naphthalen-1-yl}-acetic acid（6-NBD-C2-NAA: 33） as amorphous red powder (8.0 mg, 30% yield). m.p.>200°C; 1H NMR (400 MHz, DMSO-d6): δ 3.97 (2H, m), 4.10 (2H, s), 4.43 (2H, t, J=5.6), 6.59 (1H, d, J=9.2), 7.20 (1H, dd, J=9.2, 2.8), 7.27 (1H, d, J=6.4), 7.40 (2H, m), 7.75 (1H. d. J=8.4), 7.85 (1H, d, J=9.2), 8.56 (1H, d, J=8.8), 9.69 (NH, brs); 13C NMR (100 MHz, DMSO-d6): δ 38.17, 43.16, 68.71, 99.92, 107.86, 118.90, 121.35, 125.86, 126.12, 126.39, 126.86, 127.49, 131.10, 134.88, 138.14, 144.31, 144.69, 145.52, 156.10, 171.85; FABMS m/z 431 [M+Na]+ . Fluorescent spectrum (Ex 468 nm, Em 545 nm in 50mM KPB pH7.0 and 1/2 MS medium pH 5.8).

2.6. Synthetic procedure of [7-(2-tert-Butoxycarbonylamino-ethoxy)-naphthalen-1-yl]-acetic acid（BOC-C2-NAA: 34）

10

Ethyl ester (10) (89 mg, 0.262 mmol) was dissolved in methanol (3mL) and 3N NaOH aqueous solution (1mL) was added. The solution was stirred at room temperature for an 1hour. The resulting solution was neutralized and evaporated in vacuo and the acidified to pH 3.5 with 6N HCl. The resulting suspension was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine. After dried over Na2SO4, the solvent was removed in vacuo. The residue was purified by a silica gel column chromatography (benzene:acetone=4:1) to give [7-(2-tert-Butoxycarbonylamino-ethoxy)-naphthalen-1-yl]-acetic acid（BOC-C2-NAA: 34） as amorphous colorless powder (56 mg, 67% yield) . m.p.= 139−142°C; 1H NMR (500 MHz, CDCl3): δ 1.45 (9H, s), 3.56 (2H, m), 4.01 (2H, s), 4.11 (2H, m), 5.13 (NH, brs), 7.13 (1H, d, J=8.0), 7.25 (2H, m), 7.36 (1H, t, J=7.5), 7.64 (1H, d, J=8.2), 7.86 (1H, d, J=8.8); 13C NMR (125 MHz, CDCl3): δ 28.30, 38.86, 40.02, 60.38, 67.10, 79.35, 107.58, 118.84, 125.43, 126.05, 126.123, 127.06, 127.56, 130.06, 134.95, 155.01, 156.27, 171.25, 176.46; FABMS m/z 374 [M+H]+ . 2.8. Synthetic procedure of 3-[2-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-ethoxy]-benzoic acid (39)

To the solution of 35 (109 mg, 0.66 mmol) in DMF (2 mL) was added Cs2CO3 (427 mg, 1.31 mmol) and (2-iodo-ethyl)-carbamic acid tert-butyl ester (355 mg, 1.31 mmol), and then stirred for 4h at room temperature. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with saturated NH4Cl solution and brine, and then dried over Na2SO4. The residue was purified by silica gel column chromatography (n-hexane:acetone=5:1) to give 3-(2-tert-butoxycarbonylamino-ethoxy)- benzoic acid ethyl ester (36) as pale yellow oil (168mg, yield 83% yield).1H NMR (400 MHz, CDCl3): δ 1.38 (3H, t, J=6.8), 1.45 (9H, s), 3.55 (2H, m), 4.06 (2H, t, J=5.6), 4.37 (2H, m), 5.13 (NH, brs), 7.08 (1H, dd, J=8.0, 2.0), 7.33 (1H, t, J=7.8), 7.55 (1H, m), 7.65 (1H, d, J=8.0); 13C NMR (100 MHz, CDCl3): δ14.20, 28.27, 39.93, 60.95, 67.25, 79.57, 114.76, 119.32, 122.16, 129.31, 131.75, 155.78 ,158.41, 166.21; EIMS m/z 309 [M]+. Ethyl ester (36) (76 mg, 0.25 mmol) was dissolved in methanol (3mL) and 3N NaOH aqueous solution (1mL) was added. The solution was stirred at 60°C for an 2hour. The resulting solution was neutralized and evaporated in vacuo and the acidified to pH 3.5 with 6N HCl. The resulting suspension was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine. After dried over Na2SO4, the solvent was removed in vacuo. The residue was purified by a silica gel column chromatography (benzene:acetone=4:1) to give 3-(2-tert-Butoxycarbonylamino-ethoxy)-benzoic acid (37) as amorphous colorless powder (65 mg, 94 % yield) . m.p.= 119−120°C; 1H NMR (400 MHz, CDCl3): δ 3.57 (2H, m), 4.08 (2H, m), 5.16 (NH, brs), 7.13 (1H, dd, J=8.4, 2.0), 7.36 (1H, m), 7.61 (1H, m), 7.71 (1H, d, J=8.0); 13C NMR (100 MHz, CDCl3): δ 28.30, 39.98, 67.31, 115.13, 120.21, 122.79, 128.24, 129.44, 131.00, 158.48, 170.68, 171.24; FABMS m/z 304 [M+Na]+ .

The carboxylic acid (37) (49 mg, 0.17 mmol) was dissolved in trifluoroacetic acid (0.5mL) and stand for 15 min. at room temperature. The reaction mixture was evaporated in vacuo to give 3-(2-amino-ethoxy)-benzoic acid (38) as pale yellow oil. Crude compound 37 (31 mg, 0.17 mmol) was dissolved in 4 mL of mixed solvent [THF : 100mM borate buffer pH 9=4:1] and 4-fluoro-7-nitro-2,1,3-benzoxadiazole, NBD-F (40 mg, 0.22 mmol) was gradually added. The reaction mixture was then stirred for 60 min at room temperature. During reaction, pH of reaction mixture was maintained between pH 8–9 by adding borate buffer. The reaction mixture was added to saturated NH4Cl solution (20 mL), and extracted with EtOAc (20 mL × 3). The organic layer was washed with and brine, and then dried over Na2SO4. The residue was purified by silica gel column chromatography (Chloroform :Methanol=5:1) to give 3-[2-(7-nitro-benzo[1,2,5] oxadiazol-4-ylamino)-ethoxy]-benzoic acid (39) as amorphous oragne powder (21 mg, 36% yield). m.p.= 98−103°C; 1H NMR (400 MHz, DMSO-d6): δ 4.14 (2H, m), 4.34 (2H, m), 6.57 (1H, d, J=8.8), 7.15 (1H, dd, J=8.0, 1.6), 7.37 (1H, d, J=8.0), 7.53 (1H, dd, J=8.0), 7.70 (1H, m), 8.54 (1H, d, J=8.0); 13C NMR (100 MHz, DMSO-d6): δ 43.30, 65.80, 99.97, 114.74, 118.94, 121.13, 122.06, 128.87, 129.69, 131.80, 137.99, 144.34, 144.73, 145.63, 158.31, 167.83; HR-FABMS m/z 345.0843 [M+H]+ (∆mmu 0.8), m/z 345.08351 calcd for C15H13N4O6, Fluorescent spectrum (Ex 485 nm, Em 540 nm in 50mM KPB pH7.0 and 1/2 MS medium pH 5.8).

11

2.9. Synthetic procedure of 2-(5-(prop-2-yn-1-yloxy)-1H-indol-3-yl)acetic acid and 2-(7-(prop-2-yn-1-yloxy) naphthalen-1-yl)acetic acid

To the solution of 5-hydroxyindole acetic acid methyl ester (220 mg, 1.07 mmol) in DMF (2 mL) was added Cs2CO3 (697 mg, 2.15 mmol) and propargyl bromide (127mg 1.07 mmol), and then stirred for 1h at 50ºC. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with saturated NH4Cl solution and brine. After dried over Na2SO4, the solvent was removed in vacuo, the residue was purified by silica gel column chromatography (n-hexane:EtOAc=1:1) to give (5-prop-2-ynyloxy-1H-indol-3-yl)-acetic acid methyl ester (40) as crystal (196 mg, 75 % yield). mp 79-81 ºC; 1H NMR (400 MHz, CDCl3): δ 8.36 (s, 1H), 7.10 (d, J=2.0, 1H), 7.05 (d, J=8.8, 1H), 6.88 (d, J=2.0, 1H), 6.83 (dd, J=2.4, 1H), 4.66 (d, J=2.0, 2H), 3.68 (s, 2H), 3.63 (s, 3H), 2.49 (dd, J=2.4, 1H); 13C NMR (100 MHz, CDCl3): δ 172.64, 151.62, 131.63, 127.08, 124.25, 112.51, 111.97, 107.31, 102.45, 79.06, 75.11, 56.62, 51.75, 31.43. EIMS m/z 243 [M]+ .

Methyl ester (40) (118 mg, 0.49 mmol) was dissolved in CH3OH (2mL) and 3N NaOH aqueous solution (1mL) was added. The solution was stirred for 30 min. at room temperature. The resulting solution was neutralized and evaporated in vacuo and then acidified to pH 3.5 with 6N HCl. The resulting suspension was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine. After dried over Na2SO4, the solvent was removed in vacuo. The residue was purified by a silica gel column chromatography (CHCl3:CH3OH=7:1) to give (5-prop-2-ynyloxy-1H-indol-3-yl)-acetic acid (41) as crystal (107 mg 96% yield). m.p.= 98−99°C; 1H NMR (400 MHz, acetone-d6): δ 9.93 (s, 1H), 7.28 (d, J=8.8, 1H), 7.24 (dd, J=2.4, 2H), 6.84 (dd, J=2.6, 1H), 4.74 (d, J=2.4, 2H), 3.74 (s, 2H), 2.96 (t, J=2.6, 1H); 13C NMR (100 MHz, acetone-d6): δ173.48, 152.12, 132.46, 128.14, 124.97, 112.33, 112.21, 108.1, 103.09, 79.91, 75.80, 56.61, 30.84; FABMS m/z 230 [M+H]+ .

To the solution of 7-hydroxynapthalene 1-acetic acid ethyl ester (203 mg, 0.88 mmol) in DMF (2 mL) was added Cs2CO3 (573 mg, 1.76 mmol) and propargyl bromide (105 mg 0.88 mmol), and then stirred for 1h at 50ºC. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with saturated NH4Cl solution and brine, and then dried over Na2SO4. The residue was purified by silica gel column chromatography (n-hexane:EtOAc=7:1) to give (7-prop-2-ynyloxy-naphthalen-1-yl)-acetic acid ethyl este (42) as colorless oil (194 mg, 82 % yield); 1H NMR (400 MHz, CDCl3): δ 1.22 (t, J=7.2, 3H), 2.55 (t, J=2.4, 1H), 3.99 (s, 2H), 4.14 (dd, J=6.8, 6.8, 2H), 4.81 (d, J=2.8, 2H), 7.20 (dd, J=2.8, 2.0, 1H), 7.30 (dd, J=6.8, 7.6, 1H), 7.40 (t, J=5.2, 2H), 7.71 (d, J=8.4, 1H), 7.77 (d, J=9.2, 1H); 13C NMR (100 MHz, CDCl3): δ 14.15, 39.20, 55.68, 60.89, 75.63, 78.27, 104.32, 118.20, 123.58, 127.64, 128.55, 129.56, 130.31, 132.97, 155.79, 171.44. EIMS m/z 268 [M]+ .

Ethyl ester (42) (147 mg, 0.55 mmol) was dissolved in CH3OH (2mL) and 3N NaOH aqueous solution (1mL) was added. The solution was stirred for 30 min. at room temperature. The resulting solution was neutralized and evaporated in vacuo and then acidified to pH 3.5 with 6N HCl. The resulting suspension was extracted with EtOAc (20 mL × 3). The organic layer was washed with brine. After dried over Na2SO4, the solvent was removed in vacuo to give (7-prop-2-ynyloxy-naphthalen-1-yl)-acetic acid (43) as crystal (129 mg 98% yield). m.p.= 153−155°C; 1H NMR (400 MHz,

12

acetone-d6): δ 3.07 (t, J=2.6, 1H), 4.06 (s, 2H), 4.90 (d, J=2.4, 2H), 7.22 (dd, J=2.4, 2.8, 1H), 7.31 (dd, J=6.8, 6.8, 1H), 7.44 (d, J=6.0, 1H), 7.55 (d, J=2.8, 1H), 7.76 (d, J=8.0, 1H), 7.84 (d, J=8.4, 1H); 13C NMR (100 MHz, acetone-d6): δ 38.73, 55.89, 76.67, 78.86, 105.13, 118.19, 123.78, 127.64, 128.83, 129.89, 130.49, 130.76, 133.61, 156.15, 172.44; FABMS m/z 241 [M+H]+ . 2.10. Synthetic procedure of [7-(1-{2-[1-(7-Nitro-benzo[1,2,5]oxadiazol-4-yl)-piperidin-4-yl] -ethyl}-1H-[1,2,3] triazol- 4-ylmethoxy)-naphthalen-1-yl]-acetic acid (45)

Starting material, 4-(4-(2-azidoethyl)piperidin-1-yl)-7-nitrobenzo[c][1,2,5]oxadiazole (44) was synthesized according to published procedure (6), To the solution of 2-(7-(prop-2-yn-1-yloxy)naphthalen-1-yl)acetic acid (10 mg, 0.04 mmol) and NBD-azido (44, 15 mg, 0.045 mmol) in THF (2 mL), aqueous solution of Cu2SO4 5H2O (2.7 mg 0.01mmol) and L- sodium ascorbate (4 mg, 0.02mmol) was added dropwise. The solution was stirred for 1h at room temperature. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with brine, and then dried over Na2SO4. The residue was purified by a silica gel column chromatography (Benzene:MeOH=8:1) to give [7-(1-{2-[1-(7-Nitro-benzo[1,2,5] oxadiazol-4-yl)-piperidin-4-yl] -ethyl}-1H-[1,2,3] triazol-4- ylmethoxy)-naphthalen-1-yl]-acetic acid (45) as orange powder (19.4 mg, 84 % yield). m.p. 182-184 ºC ; 1H NMR (400 MHz, DMSO-D6): δ 8.32 (s, 1H), 7.81~7.87 (m, 1H), 7.71 (dd, J=7.6, 10.0, 2H), 7.47~7.56 (m, 1H), 7.17~7.36 (m, 3H), 6.17 (s, 2H), 5.24 (s, 2H), 4.44(t, J=6.8, 2H), 3.94 (d, J=10.8, 2H), 2.90 (t, J=8.0, 2H), 2.39 (s, 6H), 2.29 (s, 6H), 2.10 (d, J=15.2, 2H), 2.03 (t, J=7.6, 2H); 13C NMR (100 MHz, DMSO-D6): δ 173.66, 167.18, 156.01, 153.35, 146.20, 142.64, 141.04, 133.37, 130.83, 130.17, 128.39, 126.79, 124.9, 123.42, 121.88, 118.02, 104.97, 78.58, 56.02, 38.27, 32.31, 29.79, 27.35, 23.44, 15.89, 14.26. FABMS m/z 580 [M+Na]+ .Fluorescent spectrum (Ex 497 nm, Em 538 nm in 50mM KPB pH7.0 and 1/2 MS medium pH 5.8). 2.11. Synthetic procedure of [5-(1-{2-[1-(7-Nitro-benzo[1,2,5] oxadiazol-4-yl)-piperidin -4-yl]-ethyl}-1H-[1,2,3]triazol-4-ylmethoxy)-1H-indol-3-yl]-acetic acid (46)

Starting material, 4-(4-(2-azidoethyl)piperidin-1-yl)-7-nitrobenzo[c][1,2,5]oxadiazole (44) was synthesized according to published procedure (6), To the solution of (5-prop-2-ynyloxy-1H-indol-3-yl)-acetic acid (41, 37 mg, 0.16 mmol) and NBD-azido (44, 40 mg, 0.16 mmol) in THF (2 mL), aqueous solution of Cu2SO4 5H2O (10mg 0.04mmol) and L- sodium ascorbate (16 mg 0.08mmol) was added dropwise. The solution was stirred for 1h at room temperature. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with brine, and then dried over Na2SO4. The residue was purified by silica gel column chromatography (Chloroform:MeOH=9:1) to give [5-(1-{2-[1-(7-Nitro-benzo[1,2,5]oxadiazol-4-yl)-piperidin-4-yl]-ethyl}-1H-[1,2,3]triazol-4-ylmeth oxy)-1H-indol-3-yl]-acetic acid (46) as orange powder (52 mg, 68 % yield). m.p. 178-180 ºC ; 1H NMR (400 MHz, acetone-d6): δ 8.36 (d, J=8.6, 1H), 8.13 (s, 1H), 7.21-7.25 (m, 3H), 6.74 (dd, J=2.0, 2.0, 1H), 6.31 (d, J=8.4, 1H), 5.13 (s, 2H), 4.84 (t, J=6.0, 2H), 4.19 (m, 2H), 3.72 (s, 2H). FABMS m/z 501 [M+Na]+. Fluorescent spectrum (Ex 510 nm, Em 546 nm in 50mM KPB pH7.0 and 1/2 MS medium pH 5.8). 2.12. Synthetic procedure of 10-(4-(4-(((3-(carboxymethyl)-1H-indol-5-yl)oxy)methyl) -1H-1,2,3-triazol-1-yl)butyl) -5,5-difluoro-1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide (50)

13

B

N

NF

F

I

NaN3 B

N

NF

F

N3

B

N

NF

F

N3

NH

O

OH

O

B

N

NF

F

N NN

O

NH

OH

O

DMFrt 0.5h

Cu2SO4 0.25eq

THF/H2Or.t. 1.0h

L-sodium ascorbate

47 48

5041

Molecular Weight: 430.1 Molecular Weight: 345.2

Molecular Weight: 229.2 Molecular Weight: 574.4

To the solution of BODIPY-iodide, 10-(4-azidobutyl) -5,5-difluoro -1,3,7,9-tetramethyl-5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborinin-4-ium-5-uide 47 (100 mg, 0.23 mmol) in DMF (2 mL) was added NaN3 (45 mg 0.70 mmol), and then stirred for 1h at room temperature. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with brine, and dried over Na2SO4. After dried over Na2SO4, the solvent was removed in vacuo, the residue was purified by silica gel column chromatography (n-hexane:EtOAc=5:1) to give 10-(3-azidopropyl) -5,5-difluoro -5H-dipyrrolo[1,2-c:2',1'-f] [1,3,2]diazaborinin-4-ium-5-uide (48) as dark orange paste (74 mg, 92% yield); m.p.= 133−134°C; 1H NMR (400 MHz, CDCl3): δ 6.04 (s, 2H), 3.34 (t, J=9.2, 2H), 2.91 (t, J=8.4, 2H), 2.50 (s, 6H), 2.38 (s, 6H), 1.66~1.77 (m, 4H); 13C NMR (100 MHz, CDCl3): δ153.94, 145.35, 140.19, 131.28, 121.66, 112.59, 50.78, 29.14, 28.66, 27.59, 16.20, 14.37. FABMS m/z 346 [M+H]+. To the solution of (5-prop-2-ynyloxy-1H-indol-3-yl)-acetic acid 41 (50 mg, 0.21 mmol) and BODIPY-C4-azido 48 (75 mg, 0.21 mmol) in THF (2 mL), aqueous solution of Cu2SO4 5H2O (13.6 mg 0.05mmol) and L-sodium ascorbate (21.5 mg 0.11mmol) was added dropwise. The solution was stirred for 1h at room temperature. The reaction mixture was added to water (50 mL), and extracted with EtOAc (50 mL × 3). The organic layer was washed with brine, and then dried over Na2SO4. The residue was purified by silica gel column chromatography (Chloroform:MeOH=9:1) to give 10-(4-(4-(((3-(carboxymethyl)-1H-indol-5-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)butyl)-5,5-difluoro-1,3,7,9-tetramethyl- 5H-dipyrrolo [1,2-c:2',1'-f] [1,3,2]diazaborinin-4-ium-5-uide (50) as orange paste (93 mg, 75 % yield); 1H NMR (400 MHz, DMSO-d6): δ 8.21 (s, 1H), 7.27 (dd, J=5.6, 2H), 7.17 (d, J=2.0, 1H), 6.77~6.18 (m, 2H), 6.17 (s, 2H), 5.10 (s, 2H), 4.43 (t, J=6.6, 2H), 3.19 (s, 2H), 2.88 (t, J=7.6, 2H), 2.40 (s, 6H), 2.29 (s, 6H), 2.02 (t, J=7.2, 2H), 1.52 (s, 2H). FABMS m/z 575 [M+H]+.Fluorescent spectrum (Ex 494 nm, Em 506 nm in 50mM KPB pH7.0 and 1/2 MS medium pH 5.8). 2.13. Synthetic procedure of 2-(5-(2-(3-(3',6'-dihydroxy -3-oxo-3H-spiro [isobenzofuran-1,9'-xanthen] -5-yl)thioureido) ethoxy)-1H-indol-3-yl)acetic acid (53)

2-(5-(2-((tert-butoxycarbonyl)amino)ethoxy)-1H-indol-3-yl)acetic acid (34 mg, 0.10 mmol) was dissolved in CH2Cl2 (1 mL) and then trifluoroacetic acid (0.5mL) was added dorpwise and then stirred for 15 min. at room temperature. The reaction mixture was evaporated in vacuo to give 3-(2-amino-ethoxy)-indole 3-acetic acid (51) as pale yellow oil. Crude compound (31 mg) was dissolved in 2 mL of mixed solvent [100mM aqueous NaHCO3:CH3OH=1:1] and then fluoroscein isothiocynate, isomer I (48 mg 0.123mmol) was gradually added. The reaction mixture was then stirred for 60 min at room temperature. After the

14

evaporation of methanol, the reaction mixture was acidified with 6N HCl, and extracted with EtOAc (20 mL × 3). The organic layer was washed with and brine, and then dried over Na2SO4. The residue was purified by silica gel column chromatography (Chloroform:Methanol=3:1) to give 2-(5-(2-(3-(3',6'- dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl) thioureido)ethoxy)-1H-indol-3-yl)acetic acid (53) as amorphous orange powder (21 mg, 36% yield). m.p.= >200°C; 1H NMR (400 MHz, CD3OD): δ 7.69-7.76 (m, 1H), 7.59-7.62 (m, 1H), 7.23 (d, J=8.8, 1H), 7.12-7.13 (m, 2H), 7.10 (d, J=8.0, 1H), 6.84 (dd, J=2.4, 2.4, 1H), 6.51-6.67 (m, 6H), 4.20-4.27 (m, 2H), 4.03-4.05 (m, 2H), 3.35 (s, 2H). FABMS m/z 624 [M+H]+. Fluorescent spectrum (Ex 488 nm, Em 524 nm in 50mM KPB pH7.0 and 1/2 MS medium pH 5.8). Supporting References 1. Winicur ZM, Zhang GF, & Staehelin LA (1998) Auxin deprivation induces synchronous Golgi differentiation in suspension-cultured tobacco BY-2

cells. Plant Physiology 117(2):501-513. 2. Axelos M, Curie C, Mazzolini L, Bardet C, & Lescure B (1992) A protocol for transient gene expression in Arabidopsis thaliana protoplasts isolated

from cell suspension cultures. Plant Physiol Biochem 30:123-128. 3. Arase F, et al. (2012) IAA8 involved in lateral root formation interacts with the TIR1 auxin receptor and ARF transcription factors in Arabidopsis.

PloS ONE 7(8):e43414. 4. Trott O & Olson AJ (2010) AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and

Multithreading. J Comput Chem 31(2):455-461. 5. Tsuda E, et al. (2011) Alkoxy-auxins Are Selective Inhibitors of Auxin Transport Mediated by PIN, ABCB, and AUX1 Transporters. J Biol Chem

286(3):2354-2364. 6. Li C, et al. (2010) Click Chemistry to Fluorescent Amino Esters: Synthesis and Spectroscopic Studies. Eur J Org Chem (12):2395-2405. Supplementary figures S1-S16

Fig. S1. Structures of fluorescently labeled auxins and fluorescence images of Arabidopsis roots. Fig. S2. Distribution of NBD-benzoic acid and NBD-indole in Arabidopsis root and tobacco BY-2 cells. Fig. S3. Visualization of exogenous NAA distribution by using DII-VENUS system. Fig. S4. Effects of NBD-analogs on auxin-responsive gene expression and molecular docking analysis of the auxin receptors

TIR1 and ABP1. Fig. S5. Distribution of NBD-auxins in GH3-overexpressing roots and stability of NBD-NAA in suspension-cultured tobacco

BY2 cells. Fig. S6. Effects of NBD-auxins and auxin analogs on Arabidopsis root gravitropism. Fig. S7. Effects of auxin and transport inhibitors on the NBD-NAA distribution in Arabidopsis roots. Fig. S8. Effects of auxin and transport inhibitors on NBD-IAA distribution in Arabidopsis roots. Fig. S9. Distribution of NBD-auxins in Arabidopsis transport mutants 35S::PIN1, aux 1-7, abcb1, abcb19, pin3 and pin3pin7. Fig. S10. The excess NAA accumulated NBD-auxins in Arabidopsis cultured cell. Fig. S11. Effects of exogenous auxins on the DR5::GFP expression profile in endogenous auxin-depleted roots. Fig. S12. Distribution of NBD-NAA in Arabidopsis seedlings. Fig. S13. Transport of NBD-auxin in Arabidopsis hypocotyl. Fig. S14. Subcellular distribution of NBD-IAA in tobacco suspension BY-2 cell Fig. S15. Subcellular distribution of NBD-IAA in root expressing tonoplast marker, VHA-a3-mRFP. Fig. S16. Subcellular distribution of NBD-IAA in root expressing ER marker, CFP-HEDL.

15

Fig. S1. Structures of fluorescently labeled auxins and fluorescence images of Arabidopsis roots. 6-d-old seedlings were treated with medium containing 2 μM fluorescent probes for 15 min. Fluorescent microscopy images were generated using FITC filter sets. Compounds 13: 2-(7-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)butoxy) naphthalen-1-yl) acetic acid, 33: 6-[2-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-ethoxy]-naphthalen-1-yl}-acetic acid, 45: [7-(1-{2-[1- (7-Nitro-benzo[1,2,5] oxadiazol-4-yl)-piperidin-4-yl] -ethyl}-1H-[1,2,3] triazol-4- ylmethoxy)- naphthalen-1-yl]-acetic acid, 46: [5-(1-{2-[1- (7-Nitro-benzo [1,2,5]oxadiazol- 4-yl)-piperidin-4-yl]- ethyl}-1H-[1,2,3] triazol-4-ylmethoxy)-1H-indol- 3-yl]-acetic acid, 50: BODIPY-labeled IAA, 10-(4-(4-(((3-(carboxymethyl)- 1H-indol-5-yl)oxy)methyl) -1H-1,2,3-triazol-1-yl) butyl)-5,5-difluoro- 1,3,7,9-tetramethyl- 5H-dipyrrolo [1,2-c:2',1'-f] [1,3,2]diazaborinin-4-ium-5-uide, 53: FITC-labeled IAA, 2-(5-(2-(3-(3',6'- dihydroxy-3-oxo-3H-spiro [isobenzofuran- 1,9'-xanthen]-5-yl)thioureido)ethoxy)-1H-indol-3-yl)acetic acid.

16

Fig. S2. Distribution of NBD-benzoic acid and NBD-indole in Arabidopsis root and tobacco BY-2 cells. (A) Structures of NBD-benzoic acid and NBD-indole used for control. (B) Distribution of NBD-benzoic acid and NBD-indole in Arabidopsis roots. 6-d-old wild-type seedlings were treated with medium containing NBD-labeled chemicals for 15 min. The values presented in parentheses indicate the concentration of chemicals (μM). Scale bar represents 100 μm. (C) The tobacco BY-2 cells were treated with 5 μM NBD-benzoic acid and 5 μM NBD-indole for 15 min. The control compounds NBD-benzoic acid and NBD-indole did not show a clear fluorescent signals. Fluorescence images of roots and cells were recorded under identical conditions by fluorescent microscopy (fluorescent image) and confocal laser scanning microscopy (confocal image). The values in parentheses indicate the concentrations of chemicals (μM).

17

Fig. S3. Visualization of exogenous NAA distribution by using DII-VENUS system. 6-d-old DII-VENUS seedlings were incubated for 6 h with/without 10 μM auxinole, specific antagonist for TIR1/AFB auxin receptor to block the degradation of DII-VENUS reporter protein regulated by endogenous auxin. The DII-VENUS roots were washed out with fresh medium and treated with exogenous NAA. Fluorescent confocal images of roots were recorded under the identical conditions at regular intervals. The values presented in parentheses indicate the concentration of chemicals (μM). Scale bar represents 200 μm. Auxinole uniformly accumulated the DII-VENUS reporter protein by repressing the endogenous IAA activity. The distribution profile of NAA was estimated from the NAA-induced degradation pattern of DII-VENUS protein in root.

18

Fig. S4. Effects of NBD-analogs on auxin-responsive gene expression and molecular docking analysis of the auxin receptors TIR1 and ABP1.

(A) 6-d-old DR5::GUS (left) and pIAA12::GUS (right) lines were incubated with/without 5 μM IAA, together with NBD-auxins, for 5 h or 16 h, respectively. NBD-IAA, -NAA, -indole and -benzoic acid were inactive regarding IAA-induced gene expression. The values in parentheses indicate the concentrations of chemicals (μM). (B−D) Molecular docking calculations for NBD-IAA and NBD-NAA at the auxin-binding pockets of Arabidopsis Auxin-Binding Protein 1 (ABP1) and TIR1. The NAA molecule within the crystal structures of ABP1 and TIR1 is shown in green. The crystal structure of the ABP1-NAA complex was obtained from a protein data bank [PDB ID, 1LRH]. (B) Blue- and orange-colored molecules represent the predicted binding conformation of NBD-IAA and NBD-NAA to ABP1. The NBD-auxins cannot fit into narrow cavity of the auxin-binding site of ABP1. (C) Structure of the TIR1-NAA complex [PDB ID, 2P1O]. (D) Left panel: The green NAA molecule was placed in a small cavity formed with TIR1 and Aux/IAA (auxin-binding pocket). Middle panel: the blue and orange molecules represent the predicted binding conformations of NBD-IAA and NBD-NAA at the TIR1 auxin-binding site. The NBD-auxins crush the Aux/IAA protein at the TIR1-binding site (red arrow); therefore, the NBD-auxins cannot function as active auxins in the TIR1-Aux/IAA receptor complex.

19

Fig. S5. Distribution of NBD-auxins in GH3-overexpressing roots and stability of NBD-NAA in suspension-cultured tobacco BY2 cells. (A) Arabidopsis estradiol-inducible pER8::GH3.6 overexpression line (GH3ox) showed auxin-deficient root phenotype grown

with 5 μM estradiol. Scale bar represents 5 mm. (B) 5-d-old GH3ox seedling was incubated with liquid GM medium containing 5 μM estradiol for 6 h to induce GH3 protein. After induction of the GH3 enzyme, the seedlings were treated with medium containing NBD-auxins for 20 min (left panel) or 40 min (right panel), and fluorescence images of roots were subsequently recorded. The values in parentheses indicate the concentrations of chemicals (μM). The fluorescence images showed no effects of GH3 overexpression until 40 min, suggesting that NBD-auxins are a poor substrate for the GH3 enzyme. Scale bar represents 500 μm. (C) Fluorescent HPLC chromatogram of extracts from tobacco BY2 cells treated with NBD-NAA. BY-2 cells were incubated with 2 μM NBD-NAA at 24°C, and 0.5 ml aliquots of the cultures were collected at regular intervals. A methanol extract from the harvested cells was analyzed via fluorescent HPLC. As a control, NBD-NAA was added to methanol extracts from non-treated cells. The fluorescent HPLC chromatogram was not altered until 50 min of the incubation period had elapsed. (D) The peak levels of NBD-NAA (HPLC chromatogram) extracted from the cells were maintained during incubation.

20

Fig. S6. Effects of NBD-auxins and auxin analogs on Arabidopsis root gravitropism. (A) Structures of the diffusible fluorescent NBD-auxin NBD-C2-IAA, lipophilic analogs of NBD-NAA BOC-C2-NAA, and auxin transport inhibitors Bz-NAA. (B) Distribution of the diffusible fluorescent NBD-auxin NBD-C2-IAA. 6-d-old seedlings were treated with 10 μM NBD-C2-IAA for 20 min. In contrast to the asymmetric fluorescent signal of NBD-NAA, a uniform fluorescent signal of NBD-C2-IAA was observed in the root tips. Scale bar represents 200 μm. (C) Roots (6-d-old) were placed on GM agar plates containing chemicals. The seedlings were grown in the dark for 5 h after rotating the plates at 90° angle against vertical direction. The values in parentheses indicate the concentrations of chemicals (μM). Bz-NAA, alkoxy-NAA showed potent inhibition of root gravitropism. BOC-C2-NAA, hydrophobic analogs of NBD-NAA, and diffusible NBD-C2-IAA exhibited potent inhibition. These alkoxy auxin analogs are expected to be recognized by auxin transporters and then perturb endogenous auxin movements via competitive inhibition of auxin transport. These data suggest that the membrane permeability of analogs strongly influences inhibitory activity against root gravitropism. (D) Effects of NBD-C2-NAA and BOC-C2-NAA on DR5::GUS expression. NBD-C2-NAA and BOC-C2-NAA did not affect DR5::GUS expression, indicating that both analogs did not act as auxin nor anti-auxin in SCFTIR1/AFB pathway.

21

Fig. S7. Effects of auxin and transport inhibitors on the NBD-NAA distribution in Arabidopsis roots. 6-d-old Arabidopsis seedlings were incubated with liquid GM medium containing auxin or aromatic acids for 30 min or with auxin transport inhibitors for 60 min. NBD-NAA was then added to the medium, followed by incubation for another 20 min. Fluorescence images of roots were recorded under identical conditions. The values in parentheses indicate the concentrations of chemicals (μM). IAA and NAA enhanced NBD-NAA accumulation in the meristematic zone, whereas aromatic acids did not. The auxin transport inhibitors 2,4,5-triiodobenzoic acid (TIBA), brefeldin A (BFA), 1-N-naphthylphthalamic acid (NPA), and 7-benzyloxy-NAA (Bz-NAA) increased NBD-NAA accumulation to the same extent as active auxins.

22

Fig. S8. Effects of auxin and transport inhibitors on NBD-IAA distribution in Arabidopsis roots.

6-d-old Arabidopsis seedlings were incubated with liquid GM medium containing auxin or auxin transport inhibitors for 60 min.

NBD-IAA was then added to the medium, followed by incubation for another 20 min. Fluorescence images of roots were

recorded under identical conditions. The values in parentheses indicate the concentrations of chemicals (μM). NAA and the

auxin transport inhibitors TIBA and BFA enhanced NBD-IAA accumulation in the meristematic zone, whereas benzoic acids

did not. IAA and 5-Benzyloxy-IAA (Bz-IAA) reduced the fluorescent signals via competitive inhibition of AUX1/LAX-mediated

NBD-IAA import. Scale bar represents 200 μm.

23

Fig. S9. Distribution of NBD-auxins in Arabidopsis transport mutants 35S::PIN1, aux 1-7, abcb1, abcb19, pin3 and pin3pin7. (A) 6-d-old Arabidopsis seedlings incubated with liquid GM medium containing 5 μM NBD-IAA and 5 μM NBD-NAA for 20 min. Fluorescent confocal images of roots were recorded under identical conditions. The accumulation of NBD-auxins in the elongation zone was disappeared in PIN1 overexpression (35S::PIN1) mutant. The fluorescent signal of NBD-IAA was reduced in aux1-7 roots due to a loss-of-function mutation in aux1 importer. On the contrary, NBD-NAA is expected to bypass the AUX1 importer in the same manner as original NAA molecule. The abcb1, abcb19, pin3 and pin3 pin7 mutants showed similar distribution profile of NBD-auxins to that in wild-type (Col). Scale bar represents 100 μm. The values in parentheses indicate the concentrations of chemicals (μM). (B) Root phenotypes in 35S::PIN1, aux 1-7, pin2/eir1-1, abcb1, abcb19, pin3 and pin3pin7. The 35S::PIN1, aux 1-7 and pin2/eir1-1 mutants displayed highly agravitropic root phenotype. These agravitropic root phenotype would be correlated with abnormal distribution pattern of NBD-auxins. Scale bar represents 5 mm.

24

Fig. S10. The excess NAA accumulated NBD-auxins in Arabidopsis cultured cell. Arabidopsis MM1 cultured cell were incubated with 100µM NAA for 10 min and then treated with 2 μM NBD-analogs with/without 100 μM NAA for 30 min. The values in parentheses indicate the concentrations of chemicals (μM).

25

Fig. S11. Effects of exogenous auxins on the DR5::GFP expression profile in endogenous auxin-depleted roots. DR5::GFP line were grown for 5 days on medium containing auxin biosynthesis inhibitors (10 μM kynurenine, a TAA1 inhibitor, and 20 μM yucasin, a YUC inhibitor) with/without auxins. (A) DR5::GFP maxima in endogenous auxin-deficient (upper) and control (lower) roots. The DR5::GFP expression profile in the root apex was not fully restored by the application of exogenous auxins. (B) Phenotype of endogenous auxin-deficient and control seedlings in the presence of exogenous auxins. The application of exogenous auxins restored the growth defects observed in auxin-deficient seedlings. The values in parentheses indicate the concentrations of chemicals (μM). (C) The phenotype of auxin biosynthesis mutants wei8-1 tar2-2, yuc 3 5 7 8 9 and wild-type plant (Col-0) treated with kynunrenine and yucain. he values in parentheses indicate the concentrations of chemicals (μM).

26

Fig. S12. Distribution of NBD-NAA in Arabidopsis seedlings. The wild-type seedlings (just germinated and 2-days old) were incubated with liquid GM medium containing 1 μM NBD-NAA for 20 min. The fluorescent image of DR5::GFP seedlings was obtained at same growth period. Scale bar represents 500 μm. The values in parentheses indicate the concentrations of chemicals (μM).

27

Fig. S13. Transport of NBD-auxin in Arabidopsis hypocotyl. (A) Distribution of NBD-NAA in 4-d-old etiolated Arabidopsis hypocotyls of wild-type (Col-0) and pin3 pin7 mutant. 0.2 µl droplette of 0.1 % agarose containing 80µM NBD-NAA was placed on apical cotyledon, and then incubated for 4 h in the dark. (B) Effects of NAA and auxin transport inhibitor on NBD-NAA transport in decapitated etiolated hypocotyls. The decapitated hypocotyls were inverted in 5µl of medium containing NAA or TIBA in 0.5 ml microcentrifuge tubes and incubated for 1 h in the dark. 0.1% agar solution (5µl) of NBD-NAA was added into the tube at 50 µM final concentration, and then incubated for additional 3h. (C, D) Distribution of NBD-auxins in decapitated etiolated hypocotyls. The decapitated hypocotyls were inverted in 15µl of medium containing NBD-auxin in 1.5 ml microcentrifuge tubes and incubated for 2-3 h in the dark. Scale bar represents 50µm. Upper panels show decapitation site, lower panels areas 1-2mm below decapitation site.

28

Fig. S14. Subcellular distribution of NBD-IAA in tobacco suspension BY-2 cells.

(A) Subcellular distribution of NBD-IAA in tobacco BY2 cells. The cells were incubated with NBD-IAA and ER-tracker for 20

min. (B) Auxin transport inhibitors, NPA and TIBA accumulated subcellular NBD-IAA in tobacco BY2 cells. The cells were

pre-incubated with auxin transport inhibitors, NPA or TIBA for 4 h and then NBD-IAA was added. The confocal images were

recorded after 20 min incubation. The values in parentheses indicate the concentrations of chemicals (μM). Scale bar

represents 50 μm.

29

Fig. S15. Subcellular distribution of NBD-IAA in root expressing tonoplast marker, VHA-a3-mRFP.

Subcellular distribution of NBD-auxin in root expressing tonoplast marker, VHA-a3-mRFP. The root was treated with 5 μM

NBD-auxin for 30 min. The values in parentheses indicate the concentrations of chemicals (μM). Scale bar represents 50 μm.

30

Fig. S16. Subcellular distribution of NBD-IAA in root expressing ER marker, CFP-HEDL. (A) Subcellular distribution of NBD-IAA in root expressing ER marker, CFP-HEDL (CFP-ER: ABRC CS16250 line). The root was treated with 5μM NBD-IAA for 20 min. (B) Subcellular distribution of NBD-NAA in root cell expressing ER marker, CFP-ER. The CFP-ER root was treated with 5 μM NBD-NAA for 20 min. (C) The CFP-ER seedling was incubated with 50 μM TIBA for 4h and the NBD-NAA was added to final concentration of 5μM. The root was incubated for additional 20 min. The values in parentheses indicate the concentrations of chemicals (μM). Scale bar represents 20 μm.