S-1

Supporting Information (SI)

Near-infrared mito-specific fluorescent probe for ratiometric

detection and imaging of alkaline phosphatase activity with high

sensitivity

Qian Zhang, Shasha Li, Caixia Fu, Yuzhe Xiao, Peng Zhang* and Caifeng Ding*

Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education; Shandong Key Laboratory of Biochemical Analysis; Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong; College of Chemistry and Molecular Engineering. Qingdao University of Science and Technology, Qingdao 266042, PR China.

*E-mail: pengzhang@qust.edu.cn; dingcaifeng2003@163.com.

Contents

1. Syntheses of Cy-OP................................................................................................S-2

2. 1H NMR, 13C NMR, and 31P NMR spectra (Figures S1-S3)......................S-4

3. Spectral profiles (Figures S4-S20)………………………………………..............S-5

4. References.............................................................................................................S-14

Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B.This journal is © The Royal Society of Chemistry 2018

S-2

1. Synthesis and characteristic of probe Cy-OP.

N

O

N

P

Cl-

N NI-

Cl

N N

OCH3COONa

DMF, 80 oC, 4h

(a) POCl3(b) H2O

Cy-7 Cy-O

Cy-OP

OHOHO

Scheme S1 Synthesis route of Cy-OP.Synthesis of Cy-7.

Cy-7 was synthesized according to the literature.[1]

Synthesis of Cy-O.

Compound Cy-O was also synthesized according to the literature.[2]

To 0.638 g (1.0 mmol) of Cy-7 and 0.245 g (1.5 mmol) of sodium acetate was

added 20 mL of anhydrous DMF. The contents were heated at 80 oC for 4 h in

nitrogen atmosphere after which the heating was discontinued and allowed to cool to

r.t. Solvents were distilled off in vacuo and the resulting residue was purified by the

silica gel chromatography (CH2Cl2 and MeOH 100:l), compound Cy-O was obtained

as a red solid (0.374 g, 76%).

Synthesis of Cy-OP.

Cy-O (0.246 g, 0.5 mmol) was stirred into dry CH2Cl2 (50 mL) at 0 °C. And

POCl3 (0.2 mL) was added slowly though syringe. After that, the reaction solution

was stirred at room temperature for 1 h in nitrogen atmosphere. Then, ice water (50

mL) was added, and the reaction solution was extracted with CH2Cl2 (3 × 30ml). The

combined organic phase was dried with Na2SO4, and concentrated under reduced

pressure. After purified by the silica gel chromatography (CH2Cl2/MeOH, 3:1, v/v),

the pure compound Cy-OP was obtained as a green solid (0.170 g, 56%). FT-IR: (KBr,

cm-1) v: 3438 (OH), 3050 (ArH), 2964, 2930, 2866 (Alkyl CH), 1626 (C=N), 1577,

1509, 1455 (ArC=C), 1432 (O=P-OH), 1318 (C–O), 1256 (P=O), 1210 (C–N), 1074

S-3

(C–C). 1H NMR (500 MHz, DMSO-d6) δ (ppm) δ 8.36 (d, J = 14.0 Hz, 1H), 8.25 (d, J

= 14.1 Hz, 1H), 7.56 (t, J = 6.6 Hz, 2H), 7.43 – 7.29 (m, 4H), 7.20 (dt, J = 14.2, 7.2

Hz, 2H), 6.09 (dd, J = 24.4, 14.2 Hz, 2H), 4.21 – 4.11 (m, 4H), 2.58 (s, 4H), 1.79 (d, J

= 5.7 Hz, 2H), 1.66 (d, J = 6.2 Hz, 12H), 1.27 (t, J = 7.0 Hz, 6H). 13C NMR (126

MHz, DMSO-d6): δ (ppm) 171.62 (s), 161.26 (s), 145.62 – 143.91 (m), 143.32 (d, J =

112.2 Hz), 142.17 (s), 141.44 (d, J = 93.7 Hz), 128.84 (s), 124.94 (s), 122.85 (s),

122.11 (s), 111.13 (s), 100.01 (s), 49.20 (s), 27.24 (s), 24.62(s), 12.39 (s). 31P NMR

(202 MHz, DMSO-d6): δ (ppm) -5.49 (s). ESI-MS m/z: [M+CH3OH-H2O]+ Calcd for

C35H44N2O4P+ 587.3033; Found 587.3013.

S-4

2. 1H, 13C, and 31P NMR spectra of probe Cy-OP.

N

O

N

P

Cl-

OHOHO

Figure S1 1H NMR spectrum of Cy-OP in DMSO-d6.

N

O

N

P

Cl-

OHOHO

Figure S2 13C NMR spectrum of Cy-OP in DMSO-d6.

S-5

N

O

N

P

Cl-

OHOHO

Figure S3 31P NMR spectrum of Cy-OP in DMSO-d6.

3. Spectral titration profiles

400 480 560 640 720 800 8800.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0No

rmal

ized

inte

nsity

Norm

alize

d ab

sorb

ance

Wavelength / nm

Figure S4 Normalized absorbance (red line) and fluorescence (blue line) spectra of

Cy-O (solid line) and Cy-OP (dotted line) in TBS buffer. λex: 516 nm for Cy-O and

700 nm for Cy-OP.

S-6

400 480 560 640 720 8000.0

0.5

1.0

1.5

2.0

2.5

3.0

0 2 4 6 8 10 12 14 16 180.0

0.5

1.0

1.5

2.0

2.5

3.0

Abs.

736 n

m

[Cy-OP] / M

Abso

rban

ce

Wavelength / nm

18 M

1 M

Figure S5 Absorption spectra of Cy-OP in different concentrations from 1 to 18 μM

in 10 mM TBS buffer of pH 8.0. Inset: Plots of absorbance at 736 nm versus [Cy-OP].

720 760 800 840 8800

1000

2000

3000

4000

5000

0 2 4 6 8 10 12 14 16 18 20

2000

3000

4000

5000

[Cy-OP] / M

Int.

766 n

m

Fluo

resc

ence

inte

nsity

Wavelength / nm

20 M

1 M

Figure S6 Fluorescence spectra of Cy-OP in different concentrations from 1 to 20 μM

in 10 mM TBS buffer of pH 8.0. Inset: Plots of intensity at 766 nm versus [Cy-OP],

λex = 700 nm.[3]

S-7

5 6 7 8 9 100.0

0.2

0.4

0.6

0.8

1.0

pH

I 616n

m\ I

766n

m

Cy-OP Cy-OP+ALP

Figure S7 Plots of I 616 nm/I 766 nm of Cy-OP without (black squares) or with (red dots)

200 mU/mL ALP versus solution pH between 5.0 and 9.5. Solution pH was tuned by

TBS buffer. [Cy-OP] = 5 μM, λex = 516 nm.

(a)

(b)

0 20 40 60 80 100 1200

2

4

6

8

10

A 51

6 nm

/ A

736

nm

Time / min

45 oC 37 oC 20 oC 10 oC 0 oC

0 20 40 60 80 100 1200

1

2

3

4

5

6

7

0 oC

10 oC

20 oC

37 oC

Time / min

I 616n

m / I

766n

m

45 oC

S-8

Figure S8 Plots of A 516 nm/A 736 nm (a) and I 616 nm/I 766 nm (b) of Cy-OP in TBS buffer

solution pH 8.0 upon addition of ALP from 0 to 120 min at different temperatures (0

°C: black line; 10 °C: red line; 20 °C: blue line; 37 °C: pink line; 45 °C: green line).

[Cy-OP] = 5 μM, [ALP] = 400 mU/mL, λex = 516 nm.

500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

Abso

rban

ce

Wavelength (nm)

600 640 680 720 760 800 8400

100

200

300

400

Fluo

resc

ence

Inte

nsity

Wavelength (nm)

0 20 40 60 80 100 1200.0

0.3

0.6

0.9

1.2

A 73

6 nm

Time / min

0 20 40 60 80 100 1200.0

0.1

0.2

0.3

0.4

0.5

0.6

I 61

6 nm

/ I 76

6 nm

Time / min

(a)

(c) (d)

(b)

Figure S9 Time-dependent absorption (a) and fluorescence (c) spectra of Cy-OP in

TBS buffer solution of pH 8.0 at 37 °C from 0 to 120 min. Plots of A 736 nm (b) and I

616 nm/I 766 nm (d) versus time. [Cy-OP] = 5 μM, λex = 516 nm.

0 50 100 150 200 250 3000

1

2

3

4

[ALP] / mU.mL-1

A 51

6 nm

/ A 73

6 nm

Figure S10 Plots of A 736 nm/A 516 nm versus ALP concentration from 0 to 300 mU/mL

in TBS buffer solution pH 8.0 . [Cy-OP] = 5 μM.

S-9

560 600 640 680 7200

500

1000

1500

2000

Flu

ores

cenc

e in

tens

ity

Wavelength / nm

+ALP

720 760 800 8400

1000

2000

3000

4000

5000

Fluo

resc

ence

inte

nsity

Wavelength / nm

+ALP

(a) (b)

Figure S11 Fluorescence spectra of Cy-OP incubated with ALP of increasing

concentration from 0 to 300 mU/mL for 60 min in 10 mM TBS buffer of pH 8.0, λex=

516 nm (a) and 700 nm (b). [Cy-OP] = 5 μM.

0 50 100 150 200 250 3000

2

4

6

8

10

[ALP] / mU.mL-1

F 61

6nm

/ F 76

6nm

Figure S12 Plots of I 766 nm/I 616 nm versus ALP concentration from 0 to 300 mU/mL in

TBS buffer solution pH 8.0 . [Cy-OP] = 5 μM, λex= 516 nm.

S-10

0.0 0.2 0.4 0.6 0.8 1.00

1

2

3

4

5

6

7

(1 /

V) /

min

mM

-1

(1 / [Cy-OP]) / M-1

Equation y = a + b*xAdj. R-Square 0.98842

Value Standard ErrorD Intercept 0.15488 0.15201D Slope 6.56055 0.31719

Figure S13 Lineweaver-Burk plot for the enzyme-catalyzed reaction of Cy-OP. The

Michaelis-Menten equation was described as: V = Vmax [probe]/ (Km + [probe]), where

V is the initial reaction rate, [probe] is the probe concentration (substrate), and the Km

is the Michaelis constant. Conditions: 400 mU/mL ALP, 1, 2, 4, 5, 8, 10 μM of Cy-

OP. The measurements were performed at 37 °C.

(a) Only Cy-OP

(b) Cy-OP+ALP

(c) NaH2PO4

Figure S14 31P NMR spectra of Cy-OP (a), Cy-OP incubated with 400 mU/mL ALP

for 4h at 37°C (b) and NaH2PO4 (c) in the mixture of DMSO-d6 and D2O (v/v: 4/6).

S-11

[M+H]+

Figure S15 HRMS spectrum of Cy-OP with ALP incubated for 1h at 37 °C in TBS

buffer. [Cy-OP] = 5 μM, [ALP] = 400 mU/mL.

(a) (b)

400 480 560 640 720 8000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 50 100 150 200 250 300

0

1

2

3

4

5

A 51

6 nm

/ A

736 n

m

[Na3VO4] / M

Wavelength (nm)

Abso

rban

ce

560 600 640 680 720 760 800 8400

500

1000

1500

2000

0 50 100 150 200 250 3000

1

2

3

4

5

6

7

I 616

nm /

I 766

nm

[Na3VO4] / M

Wavelength (nm)

Fluo

resc

ence

inte

nsity

Figure S16 Absorbance (a) and fluorescence spectra of of Cy-OP with 400 mU mL−1

ALP pretreated with different concentrations of Na3VO4 from 0 to 300 μM in 10 mM

TBS buffer pH 8.0. ALP was incubated with Na3VO4 for 30 min at 37°C. Inset: Plots

of A 516 nm/A 736 nm (a) and I 616 nm/I 766 nm (b) versus the concentration of Na3VO4. [Cy-

OP] = 5 μM, λex = 516 nm.

S-12

(a) (b)

400 480 560 640 720 8000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.0 0.2 0.4 0.6 0.8 1.0

0

1

2

3

4

5

A 51

6 nm

/ A

736

nm

[NaH2PO4] / mM

Abso

rban

ce

Wavelength (nm)

560 600 640 680 720 760 800 8400

400

800

1200

1600

0.0 0.2 0.4 0.6 0.8 1.0

0

1

2

3

4

5

6

7

I 616

nm /

I 766

nm

[NaH2PO4] / mM

Fluo

resc

ence

Inte

nsity

Wavelength (nm)

Figure S17 Absorbance (a) and fluorescence spectra of of Cy-OP with 400 mU mL−1

ALP pretreated with different concentrations of NaH2PO4 from 0 to 1.0 mM in 10

mM TBS buffer pH 8.0. ALP was incubated with NaH2PO4 for 30 min at 37°C. Inset:

Plots of A 516 nm/A 736 nm (a) and I 616 nm/I 766 nm (b) versus the concentration of

NaH2PO4. [Cy-OP] = 5 μM, λex = 516 nm.

2 4 6 8 10 12 14 16 18 200

1

2

3

4

5

6

I 616

nm/I

766

nm

Cy-OP+competition species Cy-OP+ALP+competition species

Figure S18 I 616 nm/I 766 nm of Cy-OP in 10 mM TBS buffer pH 8.0 in the presence of

400 mU / mL ALP upon addition of 100 μM of competition species from 1 to 20:

none, Na+, K+, Mg2+, Fe3+, Al3+, Ca2+, F-, Cl-, Br-, I-, HCO3-, CO3

2-, NO3-, SO4

2-, AcO-,

BSA, telomerase, lysozyme, and inorganic pyrophosphatase. [Cy-OP] = 5 μM, λex =

516 nm.

S-13

0

20

40

60

80

100

Cell

viab

ility

(%)

[Cy-OP] / M3020105

1 h 2 h 3 h 4 h 5 h 6 h

2

Figure S19 Cytotoxicity of Cy-OP against HeLa cells as determined by CCK-8 (Cell

Counting kit-8) assay: HeLa cells were treated with Cy-OP (2-30 μM) for 1 to 6 hours.

Cy-OP

Brightfield NIR Channel Green Channel Merge

Mito TrackerGreen

Figure S20 Fluorescence images of mitochondria in HeLa cells. HeLa cells were

incubated with Cy-OP (3 μM) or Mito Tracker Green (200 nM) for 30 min,

respectively. Emission from the NIR channel (Cy-OP, λex = 633 nm, λem = 740−800

nm), emission from the green channel (Mito Tracker Green, λex = 488 nm, λem =

500−540 nm), Scale bar = 25 μm.

S-14

4. References

[1] N. Narasimhachari, P. Gabor, J. Org. Chem.,1995, 60, 2391–2395.

[2] Z. Q. Guo, S. W. Nam, S. Park and J. Yoon, Chem. Sci., 2012, 3, 2760-2765.

[3] Zhang, P.; Zhu, M. S.; Luo, H.; Zhang, Q.; Guo, L. E.; Li, Z. and Jiang, Y. B. Anal.

Chem. 2017, 89, 6210-6215.