electronic supplementary information ph fluctuation in subcellular organelles … · 2019-12-29 ·...

Electronic Supplementary Information

Self-calibrating phosphorescent polymeric probe for measuring

pH fluctuation in subcellular organelles and zebrafish digestive

tract†

Zejing Chen,‡a,b Xiangchun Meng,‡a Mingjuan Xie,a Yuxiang Shi,a Liang Zou,a Song

Guo,a Jiayang Jiang,a Shujuan Liu*a and Qiang Zhao*a

a Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors,

Institute of Advanced Materials (IAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road,

Nanjing 210023, P. R. China.

b Jiangxi Key Laboratory for Nano-Biomaterials, Institute of Advanced Materials (IAM), East China Jiaotong

University, 808 Shuanggang East Main Street, Nanchang 330013, P. R. China.

1

Electronic Supplementary Material (ESI) for Journal of Materials Chemistry C.This journal is © The Royal Society of Chemistry 2019

Table of contents

1. Experimental section1.1. Methods

General experimental information

Theoretical calculation

Calculation of pKa value

Cell culture and MTT assay

Cell imaging

Intracellular pH calibration

Establishment of alkalization of lysosomes

Establishment of oxidative stress starvation model

Zebrafish imaging

1.2. Synthesis

Scheme S1. Synthetic procedure of complex Ir1

Scheme S2. Synthetic procedure of complex Ir3 and Ir4

Scheme S3. Synthetic procedure of P-pH

2. Supplementary tables and figures2.1. Supplementary tables

Table S1. Photophysical data for iridium(III) complexes and polymer

Table S2. Calculated phosphorescent emission of protonated/deprotonated forms of complexes

Table S3. Calculated important molecular orbits of protonated/deprotonated forms of complexes

2.2. Supplementary figures

Fig. S1. Absorption spectra of protonated/deprotonated forms of iridium(III) complexes

Fig. S2. Absorption spectra and emission spectra of Ir2 in dichloromethane

Fig. S3. Evaluation of potential of P-pH for biological applications

Fig. S4. CLSM images of Hela cells labeled with P-pH at different pH

Fig. S5. Intracellular pH calibration curve and pKa

2

Fig. S6. CLSM images of Hela cells with internalized Lyso-Tracker Red excited by different lasers

Fig. S7. CLSM images of Hela cells with internalized P-pH and Lyso-Tracker Red

Fig. S8. CLSM images for indicating the photostability of P-pH and Lyso-Tracker Red

Fig. S9. CLSM images of Hela cells labelled with P-pH and MitoTracker-DeepRed

Fig. S10. Ratiometric photoluminescence images of Hela cells and phosphorescence spectra of corresponding ROIs in Hela cells

Fig. S11. CLSM images and ratiometric photoluminescence images of living zebrafishes

3. Characterization3.1. MALDI-TOF mass spectroscopic characterization of related complexes

3.2. NMR spectroscopic characterization of related complexes

3.3. NMR spectroscopic characterization of other chemical intermediates

4. References

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1. Experimental section

1.1. Methods

General experimental information

All reagents and chemicals were procured from commercial sources and used without further

purification unless otherwise noted. All solvents were of analytical grade and purified according to

standard procedures. Cell culture reagents and fetal bovine serum (FBS) were purchased from

Gibcco. The 1H and 13C NMR spectra were recorded on a Bruker Ultra Shield Plus 400 MHz NMR

instrument at 298 K using deuterated solvents. Chemical shifts are given in ppm, and are

referenced against external Me4Si (1H, 13C). Mass spectra were obtained on a Bruker autoflex

matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer. The UV-

visible absorption spectra were obtained with a Shimadzu UV-3600 UV-VIS-NIR spectrophotometer.

Photoluminescence spectra were measured on an Edinburgh FL 920 spectrophotometer. Excited-

state lifetime studies were performed with an Edinburgh LFS-920 spectrometer with a hydrogen-

filled excitation source. The data were analyzed by using a software package provided by Edinburgh

Instruments. The absolute quantum yields of the complexes were determined through an absolute

method by employing an integrating sphere. The methyl thiazolyltetrazolium assay was performed

by a Power Wave XS/XS2 microplate spectrophotometer. The zebrafish microinjection system was

established by Eppendorf. The photoluminescence imaging and time-resolved photoluminescence

imaging of cells and zebrafish larvae were carried out on an Olympus FV1000 confocal laser

scanning microscope. The PLIM setup is integrated with the same Olympus FV1000 confocal laser

scanning microscope. The lifetime values were calculated with professional software provided by

PicoQuant Company.

Theoretical calculation

The ground-state and the lowest-lying triplet excited-state geometries were optimized by density

functional theory (DFT) with Becke’s LYP (B3LYP) exchange-correlation functional and the

unrestricted B3LYP (UB3LYP) approach, respectively. On the basis of ground- and excited-state

optimization, the time-dependent DFT (TDDFT) approach associated with the polarized continuum

model (PCM) in dichloromethane media was carried out to obtain the vertical excitation energies

of triplet (Tn) states. The calculation was performed using the Gaussian 09 suite of programs. The

LANL2DZ basis set was used to treat the iridium atom, whereas the 6-31G* basis set was used to

treat all other atoms. The contours of important molecular orbitals (HOMOs and LUMOs) were

plotted.

4

Calculation of pKa value

The pKa value for P-pH was determined in Britton-Robison buffer and estimated from the changes

in the phosphorescence intensity ratio with various pH values by using the relationship, log[(Rmax –

R)/(R–Rmin)] = pH–pKa. R is the ratio of the emission intensity at 470 nm to the emission intensity at

590 nm. Rmax and Rmin are maximum and minimum limiting values of R, respectively. The pKa value

(y-intercept) was derived from the plot of pH v.s. log[(Rmax–R) / (R–Rmin)].

Cell culture and MTT assay

The Hela cell lines were purchased from the Institute of Biochemistry and Cell Biology, SIBS, CAS

(China). Hela cells were incubated in Dulbecco's modified Eagle’s medium (DMEM) supplemented

with 10% fetal bovine serum (FBS), 100 mg mL-1 streptomycin and 100 U mL-1 penicillin in a

humidified incubator at 37 °C with 5% CO2. The in vitro cytotoxicity toward cells was measured

using the methyl thiazolyl tetrazolium assay. Briefly, cells growing in log phase were seeded into

96-well cell culture plate at 1 × 104/well. P-pH was added to the wells of the treatment groups at

concentrations of 10, 60, 100, 200 and 400 μg mL-1. The cells were incubated for 24 h or 48 h at 25

°C under 5% CO2. The MTT (5 mg mL-1) in PBS solution was added to each well, and incubated for

another 4 h. After removal of the culture solution, 200 μL DMSO was added to each well and then

shaken for 10 min at shaking table. An enzyme-linked immunosorbent assay (ELISA) reader (BioTek

Instruments, Rower Wave XS2) was used to measure the OD570 (Absorbance value) of each well

referenced at 570 nm. The following formula was used to calculate the viability of cell growth:

Viability (%) = (mean of absorbance value of treatment group / mean of absorbance value of

control) × 100.

Cell imaging

Hela cells used for imaging were first incubated in culture dishes until their adherence. The cells

were washed with PBS three times and then incubated with P-pH (0.1 mg mL-1) in serum-free

culture medium for 2 hours at 37 °C and 5% CO2. Cell imaging experiments were then performed

after the cells were washed and covered with 1 mL PBS in the culture dishes for imaging.

Photoluminescence imaging was performed with an Olympus FV1000 confocal laser scanning

microscope, a 40× objective lens for cells. Hela cells containing the P-pH were excited at 405 nm

with a semiconductor laser, and the emission was measured according to the spectral data. The

images were accomplished using the software package provided by Olympus instruments.

Photoluminescence lifetime imaging microscopy and time-gated photoluminescence imaging

techniques for cells were adopted on the platform afforded by Olympus FV1000 confocal laser

5

scanning microscope and PicoQuant Company. The objective lens was 40× and the frequency was

0.5 MHz. Phosphorescence was excited with 405 nm laser, the emission was collected through

bandpass filter (482 ± 32 nm) or longpass filter (≥ 550 nm). The related calculations of the data

were carried out with the software provided by PicoQuant.

Intracellular pH calibration

After incubation with P-pH, the obtained cells were washed with Krebs−Ringer−Britton−Robinson

(KRBR) buffer containing high K+ (120 mM KCl, 30 mM NaCl, 0.5 mM MgSO4, 1.0 mM CaCl2, 5.0 mM

glucose, 20 mM NaOAc) at various pH values (pH was adjusted by adding small amounts of 0.2 N

solution of NaOH or 0.1 N solution of HCl) for three times. Then, nigericin (1 μg mL-1) was added to

the medium to allow a rapid exchange of K+ for H+ which resulted in a rapid equilibration of

external and internal pH. After 5 min, the cell images were obtained using excitation wavelengths

of 405 nm, and collection windows were set at 460−510 nm (green) and 560−610 nm (red),

respectively. The pH calibration curve was established by the calculated Igreen/Ired. The pH values

were estimated from the average intensity ratios with the above established calibration curve.

Establishment of alkalization of lysosomes

The cells were incubated with P-pH (0.1 mg mL-1) in serum-free culture medium for 2 hours and

Lyso-Tracker Red (10 μM) for 20 min at 37 °C and 5% CO2. Hela cells were washed with serum-free

culture medium. Then, NH4Cl (10 mM) was added to culture medium to regulate the intracellular

H+ level.

Establishment of oxidative stress starvation model

Nutrient deprived (ND) Hela cells were established to cause intracellular starvation state. The

nutrient-containing medium was washed and replaced with serum-free KRBR (115 mM NaCl, 5 mM

KCl, 1 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2 and 25 mL of Britton−Robinson buffer) containing

glucagon (1.0 μM) and pepstatin A (7.5 μM). The obtained nutrient deprived cells were incubated

in phorbol myristate acetate (PMA, 6 μg mL-1) for 2 h. The cells were incubated with P-pH (0.1 mg

mL-1) in serum-free KRBR culture medium for 2 hours and MitoTracker-DeepRed (10 μM) for 20 min

at 37 °C and 5% CO2. Hela cells were washed with PBS three times before imaging.

Zebrafish imaging

The zebrafish larvae (120 hpf) were purchased from Model Animal Research Center of Nanjing

University. All the zebrafish experiments were carried out in accordance with the relevant laws and

the guidelines of Institutional Animal Care and Use Committee. Femtojet (Eppendorf) controlled 6

by a micromanipulator (Eppendorf) was used for microinjection. P-pH (5 mg mL-1) was mixed with

the supersaturated suspension of three acids, respectively, and the mixture of P-pH and fumaric

acid, or P-pH and succinic acid, or P-pH and adipic acid was microinjected into the zebrafish with a

glass capillary needle Femtotips II (Eppendorf). The volume of the injected solution was estimated

to be 1 pL. The zebrafish were used for imaging immediately. After first imaging, the zebrafish were

cultured for another 2 h and used for the second imaging. The zebrafish were anesthetized before

each imaging.

Photoluminescence imaging was performed with an Olympus FV1000 confocal laser scanning

microscope, a 4× objective lens for zebrafish. Zebrafish containing the P-pH were excited at 405 nm

with a semiconductor laser, and the emission was measured according to the spectral data. The

images were accomplished using the software package provided by Olympus instruments.

Photoluminescence lifetime imaging microscopy and time-gated photoluminescence imaging

techniques for cells were adopted on the platform afforded by Olympus FV1000 confocal laser

scanning microscope and PicoQuant Company. The objective lens was 4× and the frequency was

0.5 MHz. Phosphorescence was excited with 405 nm light, the emission was collected through

bandpass filter (482 ± 32 nm) or longpass filter (≥ 550 nm). The related calculations of the data

were carried out with the software provided by PicoQuant.

7

1.2. Synthesis

All reagents and chemicals were procured from commercial sources and used without further

purification unless otherwise noted. All solvents were of analytical grade and purified according to

standard procedures.1

HN

NH2

CHO

NaOH, EtOH, 80 °C, 12 h

Br

O

N

Br

IrCl3·H2O, glycol ether/H2O

110 °C, 24 h

N

Br

IrCl

Cl

N

Br

Ir

2 2

N

N 165 °C, 4 h

HNO3, H2SO4 N

N

NO2 N2H4·H2O, Pd/C, EtOH

70 °C, 6 h

N

N

NH2 Boc2O, TEA, CH3OH

40 °C,12 h

N

N

HN

O

O

NaH, allyl bromide

0 °C, 4 h

N

N

N

O

O CF3COOH, CH2Cl2rt, 4 h

N

N

HN

[Ir(bpq)2Cl]2

50 °C, 6 h rt, 4 h

KPF6[Ir(bpq)2Cl]2, CH2Cl2/CH3OH

N

Br

Ir

2

N

N

HN

80 °C, 12 h

Pd(PPh3)4, Tolune/EtOH/K2CO3(aq.)

N B(OH)2 NIr

2

N

N

N

PF6-PF6-

1 2 3

4 5 6 7

8 9

10 Ir1[Ir(ppq)2(npa)]+PF6-[Ir(bpq)2(npa)]+PF6-

Scheme S1 Synthetic procedure of complex Ir1

2-(4-bromophenyl)quinoline (2). This compound was prepared according to the literature

procedure.2 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.46 (d, J = 8.8 Hz, 1H), 8.23 (m, 2H), 8.15 (d, J =

8.4 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 7.6 Hz, 1 H), 7.78 (ddd, J = 8.4 Hz, J = 7.2 Hz, J = 1.6

Hz, 1H), 7.76-7.72 (m, 2H), 7.60 (ddd, J = 8.0 Hz, J = 7.6 Hz, J = 0.8 Hz, 1H).

[Ir(bpq)2Cl]2 (3). IrCl3• H2O (1140 mg, 3.24 mmol) and compound 2 (2025 mg, 7.14 mmol) were

added into a flask. Then the mixture of 2-ethoxyethanol and water (v/v=3/1) was injected into the

8

reactor and stirred under N2 at 110 °C. After refluxing for 24 h, the reaction was cooled and

filtrated to obtain dark red solid, and then was dried in a vacuum oven to get crude cyclometalated

Ir(III) chloro-bridged dimer.

1,10-phenanthrolin-5-amine (6). This compound was prepared by 1,10-phenanthroline (compound

4) according to the literature procedure.3 1H NMR (400 MHz, CDCl3) δ (ppm): 9.19 (dd, J = 1.6 Hz, J =

4.4 Hz, 1H), 8.94 (dd, J = 1.6 Hz, J = 4.4 Hz, 1H), 8.27 (dd, J = 1.6 Hz, J = 8.0 Hz, 1H), 7.98 (dd, J = 1.6

Hz, J = 8.0 Hz, 1H), 7.64 (dd, J = 4.0 Hz, J = 8.0 Hz, 1 H), 7.50 (dd, J = 4.0 Hz, J = 8.0 Hz, 1H), 6.94 (s,

1H), 4.28 (s, 2H).

tert-butyl 1,10-phenanthrolin-5-ylcarbamate (7). Compound 6 (500 mg, 2.56 mmol), anhydrous

triethylamine (0.53 mL, 3.84 mmol) and anhydrous methanol (5 mL) were added into the round

bottomed flask. Then, the mixture was stirred under N2 at room temperature. After 5 minutes, di-

tert-butyl dicarbonate (2.94 mL, 12.8 mmol) was injected into the reaction, and then the mixture

was heated at 40 °C for 12 h. Next, the reaction solution was concentrated and purified by column

chromatography with dichloromethane/methanol (v/v = 100/1) to give a pale yellow solid. Yield:

75%. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.22 (d, J = 4.4 Hz, 1H), 9.10 (d, J = 4.4 Hz, 1H), 8.33 (d, J =

8.8 Hz, 1H), 8.25 (s, 1H), 8.21 (d, J = 8.0 Hz, 1H), 7.69 (dd, J = 8.8 Hz, J = 4.4 Hz, 1H), 7.60 (dd, J = 8.8

Hz, J = 4.4 Hz, 1H), 6.95 (s, 1H), 1.59 (s, 9H). 13C NMR (100 MHz, CDCl3) δ (ppm): 153.3, 150.2, 149.5,

146.6, 144.0, 135.7, 130.9, 129.1, 128.7, 123.5, 122.7, 89.0, 77.2, 29.6, 28.3.

tert-butyl allyl(1,10-phenanthrolin-5-yl)carbamate (8). Compound 7 (265 mg, 0.90 mmol) and

sodium hydride (0.53 mg, 1.34 mmol) were added to a round bottomed flask. Then anhydrous DMF

(3 mL) was added into the flask and stirred at 0 °C for 0.5 h. Subsequently, 3-bromopropene (0.15

mL, 1.35 mmol) was injected to the reaction solution, and stirred at room temperature. After 4

hours, a little water was added into the reaction to quench the reaction. Then, we extracted the

mixture with dichloromethane and collected organic phase. It was further concentrated and

purified by column chromatography using dichloromethane/methanol (v/v = 200/1) as eluent to

obtain yellow oil product. Yield: 78%. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.17 (d, J = 2.4 Hz, 1H),

9.15 (d, J = 4.8 Hz, 1H), 8.20 (s, 1H), 8.18 (s, 1H), 7.65-7.58 (m, 3H), 6.00-5.90 (m, 1H), 5.08 (s, 1H),

5.05 (s, 1H), 4.50 (dd, J = 16.0 Hz, J = 8.0 Hz, 1H), 4.13 (s, 1H), 1.21 (s, 9H). 13C NMR (100 MHz,

CDCl3) δ (ppm): 154.8, 150.6, 150.3, 146.9, 145.6, 137.2, 136.0, 133.4, 131.8, 128.0, 123.3, 123.0,

118.5, 80.9, 77.3, 55.4, 29.4, 28.3.

N-allyl-1,10-phenanthrolin-5-amine (9). Compound 8 (280 mg, 0.84 mmol), dichloromethane (5

mL) and trifluoroacetic acid (1 mL) was added into the bottomed flask and heated at the room

temperature under N2. After 4 hours, saturated Na2CO3 solution was added into the reaction,

9

and the mixture was extracted with dichloromethane. After collecting and concentrating the

organic phase, it was purified via column chromatography using dichloroMethane/methanol (v/v =

150/1) as an eluent to get pale yellow solid. Yield: 85%. 1H NMR (400 MHz, CD3OD) δ (ppm): 9.06

(dd, J = 1.2 Hz, J = 4.4 Hz, 1H), 8.74 (dd, J = 1.2 Hz, J = 8.4 Hz, 1H), 8.64 (dd, J = 0.8 Hz, 1H), 8.52 (d, J

= 8 Hz, 1H), 7.87-7.83 (m, 2H), 6.63 (s, 1H). 6.07-5.98 (m, 1H), 5.35 (dd, J = 1.6 Hz, J = 17.2 Hz, 1H),

5.22 (dd, J = 1.2 Hz, J = 10.0 Hz, 1H), 4.00 (d, J = 1.2 Hz, 2H).

[Ir(bpq)2(npa)]+PF6- (10). Compound 3 (635 mg, 0.4 mmol) and compound 9 (200 mg, 0.85 mmol)

were added into a round bottomed flask. The mixture of dichloromethane/methanol (v/v = 2/1)

was injected into the flask and stirred at 50 °C under N2 for 4 h. After the mixture was cooled to the

room temperature, KPF6 (3.2 g, 17 mmol) was added to the reaction and further stirred for 2 h.

Then, we removed KPF6 by filtration and collected the organic phase. The organic phase was

concentrated and purified through column chromatography (dichloromethane/methanol = 200/1)

to give orange-red solid. Yield: 70%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.96 (d, J = 8.8 Hz, 1H),

8.61-8.52 (m, 4H), 8.46 (d, J = 4.8 Hz, 1H), 8.31 (dd, J = 10.0 Hz, J = 8.8 Hz, 2H), 8.24 (d, J = 8.0 Hz,

1H), 8.02-7.96 (m, 2H), 7.84 (d, J = 7.6 Hz, 2H), 7.71 (dd, J = 8.0 Hz, J = 4.8 Hz, 1H), 7.45-7.39 (m,

3H), 7.31 (ddd, J = 7.2 Hz, J = 7.2 Hz, J = 2.8 Hz, 2H), 7.14 (d, J = 9.2 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H),

6.94-6.87 (m, 2H), 6.67 (s, 1H), 6.55 (d, J = 1.6 Hz, 1H), 6.51 (d, J = 1.6 Hz, 1H), 5.93-5.83 (m, 1H),

5.19 (dd, J = 17.2 Hz, 1.6 Hz, 1H), 5.10 (dd, J = 10.0 Hz, J = 1.2 Hz, 1H), 3.87 (s, 2H). 13C NMR (100

MHz, DMSO-d6) δ (ppm): 169.4, 169.3, 153.6, 153.1, 148.7, 147.2, 147.1, 147.0, 145.9, 145.8, 143.5,

143.4, 141.4, 141.3, 140.2, 136.3, 136.2, 136.1, 134.5, 134.1, 133.1, 131.6, 131.5, 130.3, 130.2,

129.9, 129.8, 127.9, 127.8, 127.6, 127.5, 127.3, 126.5, 126.4, 126.3, 125.5, 125.4, 124.1, 123.8,

123.6, 119.1, 119.0, 117.0, 98.9, 45.9. MALDI-TOF-MS m/z: 993.32 [M-PF6-]+.

[Ir(ppq)2(npa)]+PF6- (Ir1). Compound 10 (200 mg, 0.18 mmol), 4-pyridine boronic acid (55 mg, 0.45

mmol), and Pd(PPh3)4 (50 mg, 0.043 mmol) were placed in a flask. Then, the mixture of toluene (2

mL), K2CO3 solution (2 mL) and ethanol (6 mL) was injected into the reactor, and was heated under

N2 at 80 °C away from light. After 2 h, the reaction liquid was extracted with dichloromethane and

purified via column chromatography (dichloromethane/methanol = 50/1) to obtain orange-red

solid Ir1. Yield: 40%. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.01 (d, J = 12.0 Hz, 1H), 8.47 (d, J = 8.0 Hz,

1H), 8.37-8.35 (m, 4H), 8.27-8.25 (m, 4H), 8.19-8.14 (m, 2H), 8.08 (dd, J = 5.2 Hz, J = 0.8 Hz, 1H),

7.99 (dd, J = 7.6 Hz, J = 0.8 Hz, 1H), 7.85 (dd, J = 12.0 Hz, J = 4.0 Hz, 1H), 7.70-7.65 (m, 2H), 7.52 (dd,

J = 8.0 Hz, J = 3.6 Hz, 1H), 7.47-7.43 (m, 2H), 7.34-7.27 (m, 3H), 7.24 (d, J = 8.0 Hz, 1H), 6.96-6.94 (m,

4H), 6.89-6.82 (m, 4H), 6.49-6.42 (m, 2H), 5.91-5.82 (m, 1H), 5.21 (dd, J = 16.0 Hz, J = 4.0 Hz, 1H),

5.07 (dd, J = 10.4 Hz, J = 0.8Hz, 1H), 3.94 (s, 2H). 13C NMR (100 MHz, CDCl3) δ (ppm): 169.4, 169.3,

151.6, 151.4, 149.7, 149.6, 148.1, 148.0, 147.8, 147.7, 147.6, 147.4, 147.0, 146.9, 143.9, 142.4, 10

141.1, 140.5, 140.2, 139.8, 139.7, 135.3, 134.0, 133.6, 133.1, 132.9, 131.4, 131.0, 129.4, 129.1,

127.7, 127.6, 127.5, 127.4, 127.2, 127.1, 125.8, 125.5, 124.7, 124.2, 124.1, 122.4, 122.2, 121.7,

121.6, 117.9, 117.8, 117.0, 98.7, 46.4. MALDI-TOF-MS m/z: 989.35 [M-PF6-]+.

11

N

N 50 °C, 6 h rt, 4 h

KPF6[Ir(bpq)2Cl]2, CH2Cl2/CH3OHN

Br

Ir

2

N

N 80 °C, 12 h


N B(OH)2 NIr

2

N

N

N

PF6-PF6-

80 °C, 12 h


B(OH)2N

Br

Ir

2

N

N

HN

HNN

Ir

2

N

NPF6-PF6-

10 Ir3

4 11 Ir4

[Ir(mpq)2(npa)]+PF6-

[Ir(ppq)2(prl)]+PF6-

[Ir(bpq)2(npa)]+PF6-

[Ir(bpq)2(prl)]+PF6-

Scheme S2 Synthetic procedure of complex Ir3 and Ir4.

[Ir(mpq)2(npa)]+PF6- (Ir3). Compound 10 (200 mg, 0.18 mmol), phenylboronic acid (55 mg, 0.45





solid Ir3. Yield: 40%. 1H NMR (400 MHz, CD3Cl) δ (ppm):8.79 (d, J = 8.4 Hz, 1H), 8.51 (d, J = 5.2Hz,

1H), 8.23-8.05 (m, 7H), 7.97 (d, J = 8.0Hz, 1H), 7.88-7.81 (m, 1H), 7.70-7.59 (m, 2H), 7.52-7.47 (m,

1H), 7.41 (t, J = 6.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 1H), 7.29-7.24 (m, 3H), 7.20-7.11 (m, 6H), 7.11-7.03

(m, 4H), 6.93-6.78 (m, 4H), 6.49 (s, 1H), 5.99-5.81 (m, 2H), 5.25 (d, J = 16.8, 1H), 5.14 (d, J = 10.0,

1H), 3.95 (s, 2H). 13C NMR (100 MHz, CD3Cl) δ (ppm): 169.8, 169.7, 151.6, 151.5, 147.9, 147.8,

147.7, 147.4, 145.0, 143.6, 142.9, 142.6, 140.6, 140.5, 139.9, 139.6, 133.6, 133.3, 133.2, 129.2,

128.8, 128.5, 128.4, 127.5, 127.4, 127.3, 127.2, 127.1, 125.6, 125.5, 125.4, 124.3, 124.2, 117.79,

117.5, 117.2, 117.1, 98.8, 46.5. MALDI-TOF-MS m/z: 987.01 [M-PF6-]+.

[Ir(bpq)2(prl)]+PF6- (11). Compound 3 (396 mg, 0.25 mmol) and compound 9 (100 mg, 0.55 mmol)

were added into a round bottomed flask. The mixture of dichloromethane/methanol (v/v = 2/1)

was injected into the flask and stirred at 50 °C under N2 for 4 h. After the mixture was cooled to the

room temperature, KPF6 (1.0 g, 5.3 mmol) was added to the reaction and further stirred for 2 h.

Then, we removed KPF6 by filtration and collected the organic phase. The organic phase was

12

concentrated to give orange-red solid. This compound was not purified and used directly for next

steps. MALDI-TOF-MS m/z: 938.60 [M-PF6-]+.

[Ir(ppq)2(prl)]+PF6- (Ir4). Compound 11 (250 mg, 0.23 mmol), 4-pyridine boronic acid (70 mg, 0.57





solid Ir4. Yield: 33%. 1H NMR (400 MHz, CD3OD) δ (ppm): 8.70 (dd, J = 5.2 Hz, 1.2 Hz, 2H), 8.63 (dd, J

= 8.0 Hz, 1.2 Hz, 2H), 8.53 (d, J = 8.4 Hz, 2H), 8.42 (dd, J = 8.4 Hz, J = 8.4 Hz, 4H), 8.31-8.30 (m, 4H),

8.01 (s, 2H), 7.96 (dd, J = 8.0 Hz, 5.2 Hz, 2H), 7.78 (dd, J = 8.0 Hz, 0.8 Hz, 2H), 7.61 (dd, J = 8.4 Hz, 2.0

Hz, 2H), 7.35 (d, J = 9.2 Hz, 2H), 7.29 (dd, J = 8.4 Hz, J = 8.4 Hz, 2H), 7.15-7.13 (m, 4H) 6.92 (d, J = 1.6

Hz, 2H), 6.87 (ddd, J = 8.4 Hz, 6.8 Hz, 1.2 Hz, 2H). 13C NMR (100 MHz, CD3OD) δ (ppm): 169.6, 150.6,

148.9, 148.6, 147.9, 147.5, 147.5, 146.5, 143.1, 140.4, 139.4, 138.7, 132.4, 131.0, 130.7, 129.1,

127.9, 127.8, 126.8, 126.5, 123.8, 122.2, 121.6, 118.1. MALDI-TOF-MS m/z: 934.11 [M-PF6-]+.

13

P-pH

N O+ +

AIBN, THF

70 °C, 6 h

CH2 CH CH2aCH CH2bN O

CHc

HN

N

Ir

N N

N

PF6-

F

NF

Ir

N

O

O

O

2 2

Ir1 Ir2

HNN

IrN

N

N

PF6-F

N

F

IrN

OO

O

2

2

Scheme S3 Synthetic procedure of P-pH

[Ir(fpp)2(apa)] (Ir2). This compound was prepared according to the literature procedure.4 1H NMR

(400 MHz, DMSO-d6) δ (ppm): 8.60 (d, J = 5.6 Hz, 1 H), 8.26 (dd, J = 8.4 Hz, 18.8 Hz, 2H), 8.09-8.00

(m, 2H), 7.81 (d, J = 8.8Hz, 1H), 7.68 (d, J = 6.0 Hz, 1H), 7.56-7.49 (m, 2H), 7.36-7.30 (m, 2H), 6.86-

6.75 (m, 2H), 6.09-6.00 (m, 1H), 5.70-5.60 (m, 2H), 5.46 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 5.29 (dd, J = 2.4

Hz, 10.8 Hz, 1H), 4.71 (dd, J = 1.6 Hz, 4.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 170.46,

164.12 (d, J =7.0 Hz), 163.50 (d, J = 6.8 Hz), 163.18 ( dd, J = 12.8 Hz, 253 Hz), 162.67 (dd, J = 12.3 Hz,

256 Hz), 161.15 (dd, J = 13.4 Hz, 257.5 Hz), 160.83 (dd, J = 12.9 Hz, 257.4 Hz), 158.50, 155.15 (d, J =

60 Hz), 154.15 (d, J = 6.2 Hz), 149.43, 148.46, 140.72, 139.76 (d, J = 7.2 Hz), 138.31, 132.97, 130.58,

128.57 (t, J = 3.4 Hz), 128.34 (t, J = 3.4 Hz), 125.41, 124.44, 124.01, 123.20 (dd, J = 8.5 Hz, 19.3

Hz),118.12, 114.12 (t, J = 9 Hz), 98.09 (t, J = 27.0 Hz), 97.93 (t, J = 27.0 Hz), 70.73, 22.36, 10. 85. 19F

NMR (376 MHz, DMSO-d6) δ (ppm): -107.65 (d, J = 10.2 Hz), -108.47 (d, J = 9.8 Hz), -109.69 (d, J =

10.2 Hz), -110.26 (d, J = 10.2 Hz). MALDI-TOF-MS m/z: 751.62.

Preparation of P-pH. A mixture of N-vinypyrrolidone (0.55 mL, 5.15 mmol), 2,2-

azobisisobutyronitrile (5.5 mg, 33.5 μmol), complex Ir1 (18 mg, 15.8 μmol) and complex Ir2 (35 mg,

46.6 μmol) was dissolved in anhydrous tetrahydrofuran (3 mL). The solution was bubbled with dry

nitrogen for 2 hours to remove dissolved oxygen and then stirred under nitrogen at 70 °C for 24 h.

After cooling, the reaction mixture was poured into diethyl ether (250 mL), and the obtained P-pH

14

was purified by reprecipitation using dichloromethane/diethyl ether (5 ml/250 ml) and dialysis.

Yield: 29%. GPC (THF, polystyrene standard): Mn = 11200, PDI =1.93.

The proportions of the iridium(III) complexes units in the copolymers were determined from their

absorbance in dichloromethane with the model iridium(III) complexes. The contents of the N-

vinypyrrolidone units in the copolymers were determined from related calculations.

.

15

2. Supplementary tables and figures2.1. Supplementary tables

Table S1 Photophysical data for iridium(III) complexes and polymer at 298 K

Medium λem (nm) ΦPL τ(s)

Ir1CH2Cl2

CH2Cl2[a]

565

592

0.25

0.10

1.23

0.84

Ir2CH2Cl2

CH2Cl2[a]

470, 492

471, 493

0.35

0.35

0.56

0.55

Ir3CH2Cl2

CH2Cl2[a]

565

565

0.27

0.27

1.38

1.37

Ir4CH2Cl2

CH2Cl2[a]

565

593

0.25

0.11

1.00

0.78

P-pH H2O 470, 493 / 575 0.13 0.46 / 1.06

[a] Containing 0.1 % CF3COOH (V/V)

16

Table S2. Calculated phosphorescent emission of deprotonated/protonated forms of complexes

(Ir1, Ir1-H+, Ir4, Ir4-H+, Ir3 and Ir3-H+) with the TDDFT method.

Complexes State λ/nm Configuration Character

Ir1 T1 683 HOMO→LUMO (62%)

HOMO-1→LUMO (27%)

HOMO-3→LUMO (2%)

MLCT / LLCT

MLCT / LLCT / ILCT

MLCT / lLCT

Ir1-H+ T1 742 HOMO→LUMO (80%)

HOMO-1→LUMO (10%)

HOMO-3→LUMO (2%)

MLCT / LLCT

MLCT / ILCT

MLCT / ILCT


HOMO-1→LUMO (26%)

HOMO-2→LUMO (2%)

MLCT / LLCT

MLCT / LLCT / ILCT

MLCT / LLCT


HOMO-1→LUMO (7%)

HOMO-2→LUMO (3%)

MLCT / LLCT / ILCT

MLCT / ILCT

MLCT / ILCT


HOMO-1→LUMO (19%)

HOMO-4→LUMO (4%)

MLCT / LLCT

MLCT / LLCT

MLCT / LLCT / ILCT


HOMO→LUMO+1 (2%)

MLCT / LLCT

MLCT / LLCT

17

Table S3 Calculated important molecular orbits of Ir1, Ir1-H+, Ir4, Ir4-H+, Ir3 and Ir3-H+ with the

TDDFT method.

Complex Ir1 LUMO (-2.85 eV) HOMO (-5.86 eV)

HOMO-1 (-6.25 eV) HOMO-3 (-6.57 eV)

Complex Ir1-H+ LUMO (-4.12 eV) HOMO (-6.78 eV)

HOMO-1 (-7.28 eV) HOMO-3 (-7.57 eV)

18

Complex Ir4 LUMO (-2.87 eV) HOMO (- 5.87 eV)

HOMO-1 (-6.26 eV) HOMO-2 (-6.59 eV)

Complex Ir4-H+ LUMO (-3.93 eV) HOMO (-6.51 eV)

HOMO-1 (-7.05 eV) HOMO-2 (-7.25 eV)

19

Complex Ir3 LUMO (-2.71 eV) HOMO (-5.71 eV)

HOMO-1 (-6.03 eV) HOMO-4 (-6.40 eV)

Complex Ir3-H+ LUMO+1 (-3.22 eV) LUMO (-3.61 eV)

HOMO (-5.93 eV)

20

2.2. Supplementary figures

Fig. S1 (a) Absorption spectra of iridium(III) complexes Ir1 in dichloromethane (black) and in dichloromethane containing 0.1% CF3COOH (red). (b) Absorption spectra of iridium(III) complexes Ir3 in dichloromethane (black) and in dichloromethane containing 0.1% CF3COOH (red). (c) Absorption spectra of iridium(III) complexes Ir4 in dichloromethane (black) and in dichloromethane containing 0.1% CF3COOH (red).

21

Fig. S2 (a) Absorption spectra of Ir2 in dichloromethane (black) and in dichloromethane containing 0.1% CF3COOH (red). (b) Emission spectra of Ir2 in dichloromethane (black) and in dichloromethane containing 0.1% CF3COOH (cyan).

22

Fig. S3 (a) Phosphorescence spectra of P-pH (0.1 mg mL-1) in the presence of redox molecules and ions (100 µM). (b) Reversibility of the phosphorescence ratio response of P-pH to pH variation. (c) Stability of P-pH (0.1 mg mL-1) in aqueous solutions with different pH value at 25 °C for over 26 hours. Excitation wavelength was 405 nm (d) Cytotoxicity of P-pH on Hela cells determined by MTT assay.

23

Fig. S4 Confocal laser scanning microscopy images (CLSM) images of Hela cells labeled with P-pH at different pH. The green channels were acquired by collecting the luminescence from 460 to 510 nm and the red channels were from 560 to 610 nm. Excitation wavelength was 405 nm.

24

Fig. S5 Intracellular pH calibration curve was established by average phosphorescence intensity ratios of green and red channels. The green channels were acquired by collecting the phosphorescence from 460 to 510 nm and the red channels were from 560 to 610 nm. Excitation wavelength was 405 nm. pKa values are given in parentheses inside the graph.

25

Fig. S6 Cells imaging experiments for verifying that Lyso-Tracker Red can excited by 559 nm but cannot excited by 405 nm. (I) Bright field images of Hela cells; (II) CLSM images of Hela with internalized Lyso-Tracker Red and excitation wavelength was 405 nm; (III) Merge images of (I) and (II). (IV) Bright field images of Hela cells; (V) CLSM images of Hela with internalized Lyso-Tracker Red and excitation wavelength was 559 nm; (VI) Merge images of (V) and (VI).

26

Fig. S7 CLSM images of Hela cells (up) and Hela cells treated with NH4Cl (10 mM) (down) stained with the P-pH (0.1 mg mL-1) and Lyso-Tracker Red (0.1 μM). The green channels were acquired by collecting the luminescence from 460 to 510 nm and the red channels were from 560 to 610 nm. Excitation wavelength was 405 nm. The purple channels were acquired by collecting the luminescence from 570 to 620 nm and excitation wavelength was 559 nm.

27

Fig. S8 The CLSM images (I, II, IV) and bright field images (III) of Hela cells with internalized P-pH and Lyso-Tracker Red. The cells indicated by the white dotted lines had been used for high-magnification imaging in lysosomal pH determination experiments. In the process, 559 nm was adopted for exciting the lysosomal trackers to locate lysosomes and 405 nm was used for exciting P-pH to detect intracellular pH. Then, low-magnification images were obtained as above, in which the phosphorescence from P-pH can still be recorded while the fluorescence from Lyso-Tracker Red was hardly observed. (I) The green channels were acquired by collecting the luminescence from 460 to 510 nm and excitation wavelength was 405 nm. (II) The red channels were acquired by collecting the luminescence from 560 to 610 nm and excitation wavelength was 405 nm. (III) Bright field images of Hela cells. (IV) The purple channels were acquired by collecting the luminescence from 570 to 620 nm and excitation wavelength was 559 nm.

28

Fig. S9 CLSM images of Hela cells labelled with P-pH and MitoTracker-DeepRed (up) and CLSM images of Hela cells pretreated with glucagon (1 μM), pepstatin A (7.5 μM), PMA (6 μg mL-1) and labelled with P-pH and MitoTracker-DeepRed (down). The green channels were acquired by collecting the luminescence from 460 to 510 nm and the red channels were acquired by collecting the luminescence from 560 to 610 nm. Excitation wavelength was 405 nm. The purple channels were acquired by collecting the luminescence from 650 to 700 nm and excitation wavelength was 635 nm.

29

Fig. S10 (a) Ratiometric photoluminescence images of Hela cells (up) and Hela cells treated with glucagon (1 μM), pepstatin A (7.5 μM), PMA (6 μg mL-1) (down) stained with the P-pH (0.1 mg mL-1) and MitoTracker-DeepRed. The ratiometric photoluminescence images were the green channel (460 to 510 nm) to red channel (560 to 610 nm). The scale bar is 10 μm (b) The phosphorescence spectra of corresponding ROIs in Hela cells (up) and Hela cells with pretreatment (down).

30

Fig. S11 (a) CLSM images and ratiometric photoluminescence images of living zebrafishes injected with P-pH for 0 h and 2 h. (b) images of living zebrafishes injected with the mixture of P-pH and fumaric acid for 0 h and 2 h. (c) images of living zebrafishes injected with the mixture of P-pH and succinic acid for 0 h and 2 h. (d) images of living zebrafishes injected with the mixture of P-pH and adipic acid for 0 h and 2 h. The green channels were acquired by collecting the photoluminescence from 460 to 510 nm and the red channels were from 560 to 610 nm. The ratiometric photoluminescence images were the green channel to red channel. Excitation wavelength was 405 nm.

31

3. Characterization3.1. MALDI-TOF mass spectroscopic characterization of related complexes

32

3.2. NMR spectroscopic characterization of related complexes

35

3.3. NMR spectroscopic characterization of other chemical intermediates

39

4. References1 W. L. F. Armarego and C. L. L. Chai, Purification of Laboratory Chemicals, 6th ed., Elsevier, Oxford,

2009.

2 W. Xu, S. Liu, Q. Zhao, T. Ma, S. Sun, X. Zhao and W. Huang, Sci. China Chem., 2011, 54, 1750.

3 D. García-Fresnadillo and G. Orellana, Helv. Chim. Acta, 2001, 84, 2708.4 Z. Chen, K. Y. Zhang, X. Tong, Y. Liu, C. Hu, S. Liu, Q. Yu, Q. Zhao and W. Huang, Adv. Funct.

Mater., 2016, 26, 4386.

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