Aminonaphthalimide-based Imidazolium Podands for Turn-On Fluorescence Sensing of Nucleoside

Polyphosphates

Dehui Wang, Xiaolin Zhang, Cheng He, and Chunying Duan*

State Key Laboratory of Fine Chemicals, Dalian University of Technology,

Dalian 116012, China.

E-mail: cyduan@dlut.edu.cn

Contents 1. Experimental Section.

Scheme 1 Synthetic procedure of TIA1 and TIA2. Synthesis of TIA1 and TIA2. Preparation of fluorometric nucleotides titration solutions. NMR titration method. Association constants calculation. Cell incubation and imaging.

2. Figure S1 Family of fluorescence spectra of TIA1 upon the addition of 20 eq ribonucleotide polyphosphates.

3. Figure S2 Family of fluorescence spectra of TIA2 upon the addition of 20 eq ribonucleotide polyphosphates.

4. Figure S3 Absorption spectra of TIA1 upon addition of increasing amounts of ADP. 5. Figure S4 Absorption spectra of TIA2 upon addition of increasing amounts of ADP, GTP and

UTP. 6. Figure S5 Fluorescence spectra of TIA1 upon addition of increasing amounts of ADP. 7. Figure S6 Fluorescence spectra of TIA2 upon addition of increasing amounts of ATP, GTP and

UTP. 8. Figure S7 Partial 1H-NMR spectra for TIA2+ATP, GTP and UTP.

9. Figure S8 HeLa cell imaging of TIA1. 10. Figure S9 HeLa cell imaging of TIA1+ADP. 11. Figure S10 HeLa cell imaging of TIA1+ATP. 12. Figure S11 Competitive experiments of TIA1 toward other nucleosides. 13. Figure S12 1H-NMR and 13C-NMR spectra of the TIA1 in d6-DMSO.

14. Figure S13 Mass spectrum of compound of TIA1. 15. Figure S14 1H-NMR 13C-NMR and Mass spectra of the TIA2 in d6-DMSO. 16. Figure S15 1H-NMR 13C-NMR and Mass spectra of the 1. 17. Figure S16 1H-NMR 13C-NMR and Mass spectra of the 2. 18. Figure S17 1H-NMR 13C-NMR and Mass spectra of the 5. 19. Figure S18 1H-NMR 13C-NMR and Mass spectra of the 6. 20. Figure S19 1H-NMR 13C-NMR and Mass spectra of the 7.

1. Experimental Section.

N

NN

N

N

N

N

NN

N

NN

Br

3Br-+

21

O

O

O

BrO

O

O

NN

O

O

N OHN

O

O

N Br

3 5

Piperidine 2-Aminoethanol PBr3

4

N

O

O

N N=N=N

7

imidazole

N

O

O

N Br

5

K2CO3N

O

O

N N N6

TIA1

NaN3

Br

BrBr

N

N3B(ph)4-N

NO O

H

N

NO

O

N

NNNO

ON

H

H

N

TIA2

+ 6NaB(ph)4

CHCl3

NH

NN

N

N

N

NN

N

NN N

NNN

N

NN

OO

O

O

N

NN

O

O

3Br-

2+7CuI H

HH

Scheme S1 Synthetic procedure of TIA1 and TIA2.

General

Materials unless otherwise stated, were obtained from commercial suppliers and used without

further purification. Adenosine-5’-triphosphate disodium trihydrate (ATP), Adenosine-5’-

diphosphate disodium (ADP), Adenosine-5’-monophosphate acid monodydrate (AMP), Cytidine-

5’-triphosphate disodium dihydrate (CTP), Cytidine-5’-diphosphate disodium hydrate (CDP),

Cytidine-5’-diphosphate acid (CMP), Guanosine-5’-triphosphate disodium hydrate (GTP),

Guanosine-5’-diphosphate disodium (GDP), Guanosine-5’-monophosphate disodium (GMP),

Uridine-5’-triphosphate trisodium (UTP), Uridine-5’-diphosphate disodium (UDP) and

Uridine-5’-monophosphate disodium (UMP) were purchased from Bio Basic Inc.(BBI) company.

1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene was synthesized according to the literature

method.1 1H NMR and 13C NMR spectra were measured on a VARIAN INOVA-400 spectrometer

with chemical shifts reported as ppm (in d6-DMSO, d6-CDCl3 or d6-D2O, TMS as internal

standard). Mass spectrometric data were obtained on a API/MS mass spectrometry, GCT CA156

MS spectrometry and a LCQ-Tof MS spectrometry. The solution fluorescent spectra were

measured on EDINBURGH FS920. Optical absorption spectra were measured on a TU-1900

Uv/Vis spectrophotometer at room temperature. Cell imaging were measured on Nikon eclipase

TE2000-5 inverted fluorescence microscopy or Leica TCS-SP2 confocal microscope.

Synthesis of 1

1 was synthesized according the literature methord.2

Synthesis of 2

A mixture of 1,1',1''-(2,4,6-trimethylbenzene-1,3,5-triyl)tris(methylene)tris(1H-imidazole) (0.36 g,

1 mmol) and Propargyl bromide (0.476 g, 4 mmol) was heated to reflux in acetonitrile (20 mL)

under nitrogen atmosphere for 12 h. The white precipitated formed was filtered, and dried in

vacuo. Yield 0.66 g (76%). m.p. 255–256.6 oC; Anal calc. for C30H33Br3N6: C 50.23, H 4.64, Br

33.42, N 11.72%. Found: C 50.20, H 4.67, Br 33.32, N 11.81%; 1H NMR (d6-DMSO, ppm) δ:

9.29 (s, 1Htriazole), 7.87 (s, 1H, Htriazole), 7.81 (s, 1Htriazole), 5.60 (s, 2HCH2), 5.25 (d, 2HCH2, J =

2.8Hz ), 3.82 (s, 1Hallylene), 2.30 (s, 3HCH3); 13C NMR (d6-DMSO, ppm) δ: 141.63, 135.64, 129.23,

122.85, 122.27, 78.68, 78.65, 76.19, 47.91, 16.27; LCQ-Tof MS: 256.10 [M+2H2O]2+, 279.10

[M+HBr]2+, 302.11 [M+4CH3OH]2+, 311.14 [M+2DMF]2+.

Synthesis of 3

A mixture of 6-bromobenzoisochromene-1,3-dione (5.54 g, 20 mmol) and piperidine (2.23 g, 26.2

mmol) was heated to reflux in methoxylethanol (100 mL) under nitrogen atmosphere for 24 h.

After removal of methoxylethanol, the residues was purified by silica gel column chromatography

using dichloromethane as eluent to afford 3. Compound 3 was used in the next reaction without

any further characterization.

Synthesis of 4

A mixture of 3 (1.50 g, 5.33 mmol) and 2-Aminoethanol (0.4 mL, 6.66 mmol) was heated to reflux

in EtOH (20 mL) under nitrogen atmosphere for 12 h. After cool to room temperature, yellow

solid appeared, filter cake was washed by EtOH to afford compound 4. The yield was 1.28 g

(80%), yellow powder. Compound 4 was used in the next reaction without any further

characterization.

Synthesis of 5

Phosphorus tribromide (2 mL) was added dropwise to 4 (1.46 g, 4.51 mmol) in dry chloroform at

0–5 oC and the mixture was allowed to reflux for 3 h. The reaction solution was washed by water

and then extracted several times with dichloromethane. The extract was dried over sodium sulfate

and was concentrated to give a yellow brown mixture, which was purified by silica gel column

chromatography using petroleum ether/chloroform (1:1) as eluent to afford compound 5. The yield

was 0.8 g (56 %), yellow powder. m.p. 184.3–185.5 oC; Anal calc. for C19H19BrN2O2: C 58.93, H

4.95, Br 20.63, N 7.23, O 8.26%. Found: C 58.92, H 4.97, Br 20.62, N 7.20, O 8.29%; 1H NMR

(d6-CDCl3, ppm) δ: 8.6 (d, 1HAr, J = 6Hz ), 8.52 (d, 1 HAr, J = 8.4Hz), 8.49 (d, 1HAr, J = 7.6Hz),

7.72 (t, 1HAr, J = 7.6Hz), 7.24 (d, 1HAr, J = 8.4Hz), 4.60 (t, 2HCH2, J = 7.2Hz), 3.67 (t, 2HCH2, J =

7Hz), 3.29 (m, 4Hpiperdine), 1.95 (m, 4Hpiperdine), 1.75 (m, 2Hpiperdine); 13C NMR (d6-CDCl3, ppm) δ:

164.38, 163.82, 133.01, 131.39, 131.00, 130.08, 126.25, 125.45, 122.78, 115.00, 114.83, 54.61,

41.07, 27.98, 26.16, 24.29; GCT CA156 MS 386.0637.

Synthesis of 6

Imidazole (2 g, 29.4 mmol) was added to anhydrous potassium carbonate (K2CO3) (1.02 g, 7.34

mmol) and anhydrous tetrahydrofuran (THF) (50 mL). The mixture was stirred at room

temperature for 10 min prior to the addition of 5 (5.69 g, 14.7 mmol). The mixture was then stirred

under reflux for 24 h. After filtration, the THF was removed under vacuum to leave a yellow solid

which was dissolved in dichloromethane (DCM) (40 mL) and washed with water (3×50 mL). The

organic layer was then extracted using hydrochloric acid (HCl) (2 M, 3×30 mL) followed by water

(2×50 mL). The combined acid layer was neutralised with solid saturated sodium bicarbonate

(NaHCO3) and then extracted into DCM (2×40 mL). The combined DCM layer was washed with

water (3×50 mL), dried over anhydrous magnesium sulfate (MgSO4) and filtered. Removal of

DCM under vacuum gave a yellow solid, which was purified by silica gel column chromatography

using CH2Cl2/CH3OH (20:1) as eluent to afford compound 6. The yield was 0.66 g (12 %), yellow

powder. m.p. 147–149 oC; Anal calc. for C22H22N4O2: C 70.57, H 5.92, N 14.96, O 8.55%. Found:

C 70.62, H 5.97, N 14.91, O 8.50%; 1H NMR (d6-CDCl3, ppm) δ: 8.55 (d, 1HAr, J = 8Hz), 8.46 (d,

1HAr, J = 8Hz), 8.40 (d, 1HAr, J = 8Hz), 7.68 (t, 1HAr, J = 8Hz), 7.54 (s, 1Himidazole), 7.17 (d, 1HAr,

J = 8Hz), 7.06 (s, 2Himidazole), 4.55 (t, 2HCH2, J = 6Hz), 4.34 (t, 2HCH2, J = 6Hz), 3.25 (m,

4Hpiperdine), 1.89 (m, 4Hpiperdine), 1.73 (m, 2Hpiperdine); 13C NMR (d6-CDCl3, ppm) δ: 164.45, 163.87,

157.83, 137.37, 133.14, 131.41, 131.23, 130.10, 129.39, 126.27, 125.40, 122.44, 119.38, 115.00,

114.87, 54.54, 44.41, 40.10, 26.20, 24.32; API-MS m/z: 375.5 [M+H]+;

Synthesis of 7

N-bromobutyl-4-piperidine-1.8-naphthalimide (1.1 g, 2.6 mmol), NaN3 (0.8 g, 12 mmol) were

added into 20 mL ethanol. Followed by warmed at 110 oC for 6 hr, the solvent was removed by

rotate evaporator to afford product. m.p. 138–139.6 oC; Anal calc. for C19H19N5O2: C 65.32, H

5.48, N 20.04, O 9.16%. Found: C 65.20, H 5.60, N 20.10, O 9.22%; 1H NMR (CDCl3, ppm) δ:

8.58 (d, 1HAr, J = 8Hz), 8.50 (d, 1HAr, J = 8Hz), 8.40 (d, 1HAr, J = 8Hz), 7.68 (t, 1HAr, J = 16Hz),

7.18 (d, 1HAr, J = 8Hz), 4.43 (t, 2HCH2, J = 12Hz), 3.65 (t, 2HCH2, J = 16Hz), 3.24 (m, 4Hpiperdine),

1.89 (m, 4Hpiperdine), 1.73 (m, 2Hpiperdine); 13C NMR (CDCl3, ppm) δ: 164.62, 164.05, 157.62,

133.03, 131.33, 130.98, 130.08, 126.26, 125.38, 122.73, 115.39, 114.75, 54.54, 48.95, 38.64,

26.21, 24.34; API-MS m/z: 350.3 [M+H]+, 372.3 [M+Na]+, 721.5 [2M+Na]+;

Synthesis of TIA1

1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene (0.16 g, 0.41 mmol) and 6 (0.46 g, 1.23 mmol)

were dissolved in CHCl3 (20 mL) and stirred at reflux for 15 h. During this time, a white

precipitate formed. The product was filtered off and washed with CHCl3 to give the desired

tribromo anions product as a yellow powder. A solution of the mixture of 3Br- product (0.6 g, 0.39

mmol) and NaB(C6H5)4 (0.88 g, 2.6 mmol) was stirred at room temperature in CH3OH (30 mL)

for 1 hr. The yellow precipitated formed was filtered, washed with methanol and diethyl ether, and

dried in vacuo. Yield 0.66 g (76%). m.p. 190–192 oC; Anal calc. for C150H141B3N12O6: C 80.42, H

6.34, B 1.45, N 7.5, O 4.29%. Found: C 80.40, H 6.36, B 1.42, N 7.52, O 4.30%; 1H NMR

(d6-DMSO, ppm) δ: 9.02 (s, 1Himidazole), 8.27 (d, 1HAr, J = 4Hz), 8.25 (d, 1HAr, J = 4Hz), 8.17 (d,

1HAr, J = 8Hz), 7.77 (s, 1Himidazole), 7.70 (t, 1HAr, J = 8Hz), 7.57 (s, 1Himidazole), 7.17 (m, 1HAr),

5.49 (s, 2HCH2), 4.46 (s, 2HCH2), 4.38 (s, 2HCH2), 3.13 (M, 4Hpiperdine), 2.24 (s, 3HCH3), 3.13(m,

4Hpiperdine), 1.79 (m, 4Hpiperdine), 1.64 (m, 2Hpiperdine); 13C NMR (d6-DMSO, ppm) δ: 164.03, 163.53,

163.04, 162.55, 156.78, 141.10, 136.00, 135.44, 132.22, 130.68, 130.48, 129.16, 125.58, 125.21,

125.19, 125.15, 123.50, 121.97, 121.92, 121.39, 114.64, 114.23, 53.82, 47.69, 25.59, 23.70, 15.86;

LCQ-Tof MS: 427.29 [M]3+.

Synthesis of TIA2

A mixture of 2 (0.33 g 0.416 mmol), 7 (0.53 g 1.5 mmol), CuI (20 mg 0.1 mmol) was heated to

reflux in THF (30 mL) for 48 hr. The solvent was removed by rotate evaporator to afford crude

product. The crude product was recrystallizated in CH3OH and ether. m.p. 203–205 oC; Anal calc.

for C87H90Br3N21O6: C 59.19, H 5.14, Br 13.58, N 16.66, O 5.44%. Found: C 59.17, H 5.16, Br

13.55, N 16.68, O 5.45%; 1H NMR (d6-DMSO, ppm) δ: 9.30 (s, 1Himidazole), 8.39 (s, 1Htriazole), 8.34

(d, 1HAr, J = 8Hz), 8.31 (d, 1HAr, J = 8Hz), 8.24 (d, 1HAr, J = 8Hz), 7.79 (s, 1Himidazole), 7.74 (t,

1HAr, J = 8Hz), 7.62 (s, 1Himidazole), 7.23 (d, 1HAr, J = 8Hz), 5.59 (s, 2HCH2), 5.54 (s, 2HCH2), 4.71

(m, 2HCH2), 4.40 (m, 2HCH2), 3.15 (m, 4Hpiperdine), 2.29 (m, 3HCH3), 1.79 (m, 4Hpiperdine), 1.65 (m,

2Hpiperdine); 13C NMR (d6-DMSO, ppm) δ: 163.50, 162.88, 156.86, 141.26, 140.14, 135.67, 132.37,

130.79, 130.66, 129.36, 129.22, 125.78, 125.52, 125.34, 122.77, 122.31, 122.06, 114.86, 114.43,

53.93, 47.95, 47.57, 43.55, 25.69, 23.82, 16.32; LCQ-Tof MS: 508.42 [M] 3+, 802.57 [M+HBr]2+.

Preparation of fluorometric nucleotides titration solutions.

Stock solutions (10 mM) of the sodium salts of nucleotides of ADP, ATP, AMP, CDP, CTP, CMP,

GDP, GTP, GMP, UDP, UTP, UMP in 1:1 DMF/H2O (v:v) solvents were prepared. Stock solution

of TIA1 and TIA2 (1 mM) were also prepared in distilled CH3CN solution. Test solutions were

prepared by placing 40 uL of host stock solution into a quartz cell of 1 cm optical path length

including 2 mL distilled CH3CN or aqueous solution, and then adding an appropriate aliquot of

each nucleotides stock by using a micro-syringe. In competition experiments, 4 equiv. amounts of

sodium salts of nucleotides stock was added to the solution of TIA1 with 4 equiv. amounts of ADP

in CH3CN.

NMR Titration Method

All NMR spectra were measured on a VARIAN INOVA-400 spectrometer at 298 K. (a) A solution

(1 mM) of host TIA1 in d6-DMSO was titrated with 3 eq ADP in d6-DMSO. (b) A solution (1 mM)

of host TIA2 in d6-DMSO:D2O = 8:2 (v:v) was titrated with appropriate aliquot of ATP, GTP and

UTP stock in D2O by using a micro-syringe. The chemical shift changes of the proton of

imidazolium and 1, 8-naphthalimides units were monitored.

Association Constants Calculation:

The binding constant was calculated from the fluorescent titration curve according to

the equation.

Log ((F-Fmin)/(Fmax-F))=log k + n log [c]

where A is fluorescent of TIA1 (TIA2) at 548 nm (557 nm, 560 nm and 558 nm) upon

addition of different amount of nucleoside polyphosphates. [c] stands for the

concentration of nucleoside polyphosphates.

Cell incubation and imaging

HeLa cells were cultured in 1640 supplemented with 10% FCS (Invitrogen). Cells were seeded on

18 mm glass coverslips for confocal fluorescence imaging and in 24-well flat-bottomed plates for

Nikon eclipase TE2000-5 inverted fluorescence microscopy. After 12 h, HeLa cells were

incubated with 10 μM compound TIA1 or TIA2 (in the culture medium containing 0.5% DMSO)

for 30 min at 37oC under 5% CO2 and then washed with phosphate-buffered saline (PBS) three

times before incubating with 40 eq ATP and ADP for another 30 min, and cells were rinsed with

PBS three times again. The fluorescence imaging of intracellular ADP in HeLa cells was observed

under Nikon eclipase TE2000-5 inverted fluorescence microscopy with a 20×objective lens

(excited with blue light). For all images, the microscope settings, such as brightness, contrast, and

exposure time were held constant to compare the relative intensity of intracellular ADP

fluorescence. Confocal fluorescence imaging of intracellular ATP in HeLa cells was observed

under a Leica TCS-SP2 confocal microscope. Excitation wavelength of laser was 458 nm.

Emissions were centered at 510±15nm and 590±15nm (double channel). MetaFluor (Universal

Imaging Corp.) was used as imaging analysis software.

2. Figure S1 Family of Fluorescence spectra of TIA1 (red line) in

CH3CN solution (20 µM) upon the addition of 20 eq various

ribonucleotide polyphosphates(black line).

500 550 600 650 7000.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

F/F 0

Wavelength(nm)

ADP

500 550 600 650 7000.0

0.2

0.4

0.6

0.8

1.0

F/F 0

Wavelength(nm)

AMP

500 550 600 650 7000.0

0.2

0.4

0.6

0.8

1.0

1.2

F/F 0

Wavelength(nm)

ATP

500 550 600 650 7000.0

0.2

0.4

0.6

0.8

1.0

1.2

F/F 0

Wavelength(nm)

CDP

500 550 600 650 7000.00.10.20.30.40.50.60.70.80.91.01.1

F/F 0

Wavelength(nm)

CMP

500 550 600 650 7000.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

F/F 0

Wavelength(nm)

CTP

500 550 600 650 7000.0

0.2

0.4

0.6

0.8

1.0

1.2

F/F 0

Wavelength(nm)

GDP

500 550 600 650 7000.00.10.20.30.40.50.60.70.80.91.0

F/F 0

Wavelength(nm)

GMP

500 550 600 650 7000.0

0.2

0.4

0.6

0.8

1.0

1.2

F/F 0

Wavelength(nm)

GTP

500 550 600 650 7000.0

0.2

0.4

0.6

0.8

1.0

F/F 0

Wavelength(nm)

UDP

500 550 600 650 7000.00.10.20.30.40.50.60.70.80.91.01.1

F/F 0

Wavelength(nm)

UMP

500 550 600 650 7000.0

0.2

0.4

0.6

0.8

1.0

F/F 0

Wavelength(nm)

UTP

3. Figure S2 Family of Fluorescence spectra of TIA2 (red line) in

aqueous solution (20 µM) upon the addition of 20 eq various

ribonucleotide polyphosphates(black line).

500 550 600 650 700 7500

2

4

6

8

10

12

14

16

F/F 0

Wavelength(nm)

ATP

500 550 600 650 700 7500

2

4

6

8

10

12

14

F/F 0

Wavelength(nm)

GTP

500 550 600 650 700 7500

2

4

6

8

10

12

F/F 0

Wavelength(nm)

UTP

500 550 600 650 700 7500.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

F/F 0

Wavelength(nm)

AMP

500 550 600 650 700 7500.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

F/F 0

Wavelength(nm)

ADP

500 550 600 650 700 7500.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

F/F 0

Wavelength(nm)

CDP

500 550 600 650 700 7500.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

F/F 0

Wavelength(nm)

CMP

500 550 600 650 700 7500.0

0.5

1.0

1.5

2.0

2.5

F/F 0

Wavelength(nm)

CTP

500 550 600 650 700 7500.0

0.5

1.0

1.5

2.0

2.5

F/F 0

Wavelength(nm)

GDP

500 550 600 650 700 7500.0

0.2

0.4

0.6

0.8

1.0

1.2

F/F 0

Wavelength(nm)

GMP

500 550 600 650 700 7500.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

F/F 0

Wavelength(nm)

UDP

500 550 600 650 700 7500.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

F/F 0

Wavelength(nm)

UMP

4. Figure S3 Absorption spectra of TIA1 (20 mM) upon addition of

increasing amounts of ADP in DMF/H2O (95:5)

solution.

300 350 400 450 500 5500.0

0.1

0.2

0.3

0.4

0.5

0.6

Abs

orba

nce

Wavelength(nm)

5. Figure S4 Absorption spectra of TIA2 (20 mM) upon addition of

increasing amounts of ATP (top picture), GTP (middle picture) and

UTP (bottom picture) in DMF/H2O (95:5) solution.

.

300 350 400 450 500 550

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Abs

orba

nce

Wavelength(nm)

ATP

300 350 400 450 500 550

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Abs

orba

nce

Wavelength(nm)

GTP

300 350 400 450 500 550

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Abs

orba

nce

Wavelength(nm)

UTP

6. Figure S5 Fluorescence spectra of TIA1 (20 µM) in CH3CN

solution upon addition of increasing concentrations of ADP. Scan

slit:2 nm. liner of log ((F-F0)/(Flim-F) vs. log [ADP]. (A present

fluorescence of TIA1 at 548 nm).

500 550 600 650 7000.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

F/F 0

Wavelength(nm)

ADP

-4.4 -4.2 -4.0 -3.8 -3.6 -3.4 -3.2 -3.0-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5Y = A + B * XA=10.4394B=2.98997R=0.99181

log(

(F-F

0)/(F lim

-F))

log[ADP]

7. Figure S6 Fluorescence spectra of TIA2 (20 µM) in aqueous

solution upon addition of increasing concentrations of ATP, GTP

and UTP with an excitation wavelength at 396 nm. Scan slit:2 nm.

liner of log ((F-F0)/(Flim -F) vs. log [XTP]. (A present fluorescence of

TIA2 at 557 nm, 560 nm and 558 nm, respectily).

500 550 600 650 700 7500

2

4

6

8

10

12

14

16

ATP

F/F 0

Wavelength(nm)-4.8 -4.6 -4.4 -4.2 -4.0 -3.8 -3.6

-1.5

-1.0

-0.5

0.0

0.5

1.0Y = A + B * XA=8.03024B=1.97776R=0.99749

log(

(F-F

0)/(F

lim-F

))

log[ATP]

500 550 600 650 700 7500

2

4

6

8

10

12

14

F/F 0

Wavelength(nm)

GTP

-4.6 -4.5 -4.4 -4.3 -4.2 -4.1 -4.0 -3.9 -3.8-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2 Y = A + B * XA=8.42072B=1.93901R=0.98989

log(

(F-F

0)/(F

lim-F

))

log[GTP]

500 550 600 650 700 7500123456789

1011

F/F 0

Wavelength(nm)

UTP

-4.8 -4.6 -4.4 -4.2 -4.0 -3.8 -3.6 -3.4-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0 Y = A + B * XA=7.88411B=2.0199R=0.99774

log(

(F-F

0)/(F

lim-F

))

log[UTP]

8. Figure S7 Partial 1H-NMR spectra for TIA2+ATP (top picture),

TIA2+GTP (middle picture) and TIA2+UTP (bottom picture).

9. Figure S8 HeLa cell imaging of TIA1.

10. Figure S9 HeLa cell imaging of TIA1+ADP.

11. Figure S10 HeLa cell imaging of TIA1+ATP.

12. Figure S11. Competitive experiments of ADP (80µM) with TIA1

（20 µM）in CH3CN solution in the presence of various nucleosides

(80µM).

13. Figure S12. 1H-NMR (top picture) and 13C-NMR (bottom picture)

spectra of the TIA1 in d6-DMSO

ppm (t1)2.03.04.05.06.07.08.09.0

9.03

8

8.29

38.

272

8.25

5

8.19

08.

170

7.77

17.

696

7.56

9

5.48

5

4.47

14.

465

4.38

64.

382

3.12

7

2.24

0

1.78

71.

643

1.00

2.011.08

1.011.030.92

6.78

5.862.95

1.95

2.112.39

4.37

3.15

4.162.17

ppm (t1)50100150

14 Figure S13. Mass spectrum of compound of TIA1.

WDH-3

m/z200 300 400 500 600 700 800 900 1000 1100

%

0

10009061410 27 (0.502) Cm (1:31) 1: TOF MS ES+

7.91e3427.2943

191.1212

426.2495

427.6280

427.9618

681.4205680.4098

428.2958

196.0987 658.4315546.3808

498.3529

681.9122

682.4180

15. Figure S14. 1H-NMR (top picture), 13C-NMR (middle picture) and

Mass (bottom picture) spectra of the TIA2 in d6-DMSO

16. Figure S15. H1 NMR spectrum of compound of 1.

17. Figure S16. 1H-NMR (top picture), 13C-NMR (middle picture) and

Mass (bottom picture) spectra of the 2.

WDH

m/z230 240 250 260 270 280 290 300 310 320 330 340 350 360 370

%

0

10010041338 5 (0.094) Cm (1:8) 1: TOF MS ES+

1.27e3279.0993

269.6205

256.0995

256.5995

302.1090

279.6032 311.1376

311.6411

18. Figure S17. 1H-NMR (top picture), 13C-NMR (middle picture) and

Mass (bottom picture) spectra of the 5.

14-Apr-201010:23:39w GCT CA156

25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400m/z0

100

%

dcy100413-1 265 (4.416) Cm (265:272-149:164) TOF MS EI+ 3.28e4386.0637

279.1141

153.0582126.046781.925146.9704

224.0607 236.1072

307.1448

385.0557308.1516

389.0654309.1555

390.0674

19. Figure S18. 1H-NMR (top picture), 13C-NMR (middle picture) and

Mass (bottom picture) spectra of the 6.

m/z250 300 350 400 450 5000

20

40

60

80

100

*MSD1 SPC, time=0.160:0.258 of 10041316.D APCI, Pos, Scan, 50

Max: 123261

375

.5 3

76.3

375

.2 3

73.3

325

.2

461

.5

20. Figure S19. 1H-NMR (top picture), 13C-NMR (middle picture) and

Mass (bottom picture) spectra of the 7.

m/z200 300 400 500 600 7000

20

40

60

80

100

*MSD1 SPC, time=0.349:0.481 of 10041318.D API-ES, Pos, Scan, 50

Max: 4329

350

.3 3

51.2

721

.5

325

.2

536

.0

372

.3

References: (1) A. W. van der Made and R. H. van der Made, J. Org. Chem. 1993, 58, 1262-1263. (2) H. K. Liu, W. Y. Sun, H. L. Zhu, K. B. Yu and W. X. Tang, Inorganica Chimica Acta. 1999,

129-135.