1

Electronic supplementary information

Stimuli-responsive colorimetric and NIR fluorescence combination probe for

selective reporting of cellular hydrogen peroxide

Nagarjun Narayanaswamy,a Sivakrishna Narra,

b Raji R. Nair,

c Deepak Kumar Saini,

c Paturu

Kondaiahb and T. Govindaraju*

a

aBioorganic Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced

Scientific Research, Jakkur P.O., Bengaluru 560064, India.

b,cDepartment of Molecular Reproduction, Development and Genetics, Indian Institute of

Science, Bengaluru 560 012, India.

Electronic Supplementary Material (ESI) for Chemical Science.This journal is © The Royal Society of Chemistry 2016

2

Scheme 1. Synthesis of QCy-BA

Preparation of (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol (1).

BHO OH

HO

MgSO4 / Pinacol

Acetonitrile, Reflux

BO O

HO

1

B

HO

HO OH

MgSO4/Pinacol

Acetonitrile, Reflux

B

HO

OO

NaI/TMSCl

Acetonitrile, 0 oC

B

I

OO

4-hydroxyisophthalaldehydeK2CO3

DMF, RT

O O

O

BO

O

N

S

I

1 2

34

Piperidine

DCM/MeOH Reflux

O

BOH

OH

S

N N

S

I I

QCy-BA

3

To a stirred solution of 4-(hydroxymethyl)phenylboronic acid (0.4 g, 2.63 mmol) in acetonitrile

(15 mL), MgSO4 (3 g) and pinacol (0.37 g, 3.15 mmol) were added. The reaction mixture was

heated up to 80 °C and allowed to reflux for 24 h. After completion of the reaction, solvent was

evaporated under vacuum. The crude mixture was dissolved in dichloromethane and filtered. The

obtained product (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol (1) was used

for further reaction without purification.

Preparation of 2-(4-(iodomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2).

To a stirred solution of (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanol (1)

(0.55 g, 2.35 mmol) in acetonitrile (20 mL), sodium iodide (1.1 g, 7.05 mmol) and Trimethylsilyl

chloride (0.65 mL, 7.05 mmol) were added at 0 °C. The reaction mixture was allowed to stir at

room temperature for 1 h. After completion of the reaction solvent was evaporated under

vacuum. The crude product was dissolved in saturated solution of Na2S2O3 to quench the

unreacted iodide and the product was extracted with dichloromethane. The crude product was

purified by column chromatography on silica gel using ethyl acetate\hexane (5:95) as an eluent

to give product 2-(4-(iodomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2) in

excellent yield (90%). 1H NMR (400 MHz, CDCl3) δppm 7.73 (d, J = 8 Hz, 2H), 7.37 (d, J = 8

BO O

HO

NaI/TMsCl

Acetonitrile, 0 °C

BO O

I

2

4

Hz, 2H), 4.45 (s, 2H), 1.34 (s, 12H). 13

C NMR (100 MHz, CDCl3) δppm 142.3, 135.3, 128.0, 24.9,

5.4

Preparation of 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)benzyloxy)isophthalaldehyde (3).

To a stirred solution of 4-hydroxyisipthaladehyde (0.11 g, 0.73 mmol) in DMF (5 mL), K2CO3

(0.3 g, 2.17 mmol) was added and allowed to stir for 20 min. After 20 min, 2-(4-

(iodomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2) (0.3 g, 0.87 mmol) was added

and stirred overnight at room temperature (RT). The completion of reaction was monitored by

TLC. After completion of the reaction, solvent was evaporated and product was extracted with

diethylether (3×100 mL). The crude product was purified by column chromatography on silica

gel using ethyl acetate\hexane (20:80) as an eluent to obtained compound 3 in good yield (60%).

1H NMR (400 MHz, CDCl3) δppm 10.56 (s, 1H), 9.95 (s, 1H), 8.35 (d, J = 2.4 Hz, 1H), 8.08 (dd, J

= 2 Hz, 8.8 Hz, 1H), 7.86 (d, J = 8 Hz, 2H), 7.44 (d, J = 8 Hz, 2H), 7.17 (d, J = 8 Hz, 1H), 5.32

(s, 2H), 1.35 (s, 12H). 13

C NMR (100 MHz, CDCl3) δppm 190.1, 188.5, 164.9, 137.9, 135.5,

135.3, 131.9, 129.9, 126.5, 125.2, 113.7, 84.0, 71.0, 24.9

BO O

I

DMF, RT

K2CO3O O

O O

O

OH

BO

O

32

5

Preparation of QCy-BA.

DCM / MeOH

Piperidine, Reflux QCy-BAO O

O

BO

O

N

S

I

3 4

S

N N

S

O

BOH

OH

I I

To a stirred solution of compound (4)1 (80 mg, 0.27 mmol) in methanol (10 mL) and

dichloromethane (5 mL), piperidine (8 µL) was added and allowed to stir for 10 minutes. After

10 min, 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyloxy)isophthalaldehyde (3) (45

mg, 0.12 mmol) in DCM (2 mL) was added and heated up to 50 °C for 3 h. After the completion

of reaction solvent was evaporated. The crude brown color solid was washed with diethylether

(50 mL) to remove the unreacted starting materials. The brown solid was dissolved in

acetonitrile/water mixture and purified by reverse phase HPLC using 0.1% trifluoroacetic acid

(TFA) in water/acetonitrile (50-100%) as a mobile phase to obtained boronic acid conjugate

(QCy-BA) in moderate yield 30%. 1H-NMR (400 MHz, DMSO-d6) δppm 8.69 (d, J = 2Hz, 1H),

8.44 (ddd, J = 2.8 Hz, J = 4.0 Hz, J = 7.6 Hz, 2H), 8.34-8.25 (m, 4H), 8.21 (d, J = 4.2 Hz, 1H),

8.16 (d, J = 12.4 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.92-7.88 (m, 4H), 7.81 (td, J = 0.8 Hz, J =

7.6 Hz, 2H ), 7.56 (dd, J = 4 Hz, J = 8.4 Hz, 3H), 5.48 (s, 2H), 4.39 (s, 3H), 4.25 (s, 3H). 13

C-

NMR (100 MHz, DMSO-d6) δppm 171.9, 171.8, 160.6, 158.3, 158.0, 147.0, 142.1, 142.0, 141.6,

137.5, 135.0, 134.5, 132.3, 129.5, 129.4, 128.6, 128.4, 127.9, 127.8, 127.3, 127.0, 124.3, 124.2,

123.1, 118.1, 117.0, 116.9, 115.6, 115.1, 114.4, 113.0, 71.0, 36.4, 36.2. HRMS (ESI-MS): found

288.0875, calcd m/z = 288.0851 for C33H29BN2O3S2 [M-2I]2+

.

6

Fig. S1 (a) Absorption spectra of QCy-BA (5 M) in presence of H2O2 (1 mM). (b) Emission

spectra of QCy-BA (5 M) in presence of H2O2 (1 mM) in PBS-buffer solution as a function of

time.

300 400 500 600 700

0.10

0.15

0.20

Ab

so

rba

nc

e (

a.u

)

Wavelength (nm)

QCy-BA+H2O

2

a b

500 550 600 650 700 750 800 8500

3

6

9

12

Flu

ore

sc

en

ce

in

ten

sit

y (

a.u

)

Wavelength (nm)

QCy-BA+H2O

2

7

Fig. S2 Mechanism of selective reaction of hydrogen peroxide-assisted oxidation of boronic acid

in QCy-BA followed by hydrolysis and 1,6-elimination of p-quinone methide moiety to release

DNA minor groove binder QCy-DT.2

S

N N

S

QCy-BA

O

BHO

OH

O O

H

S

N N

S

O

BHO

OHO

OH

S

N N

S

O

OB

HO

OH

S

N N

S

O

O

S

N N

S

O

OQCy-DT

p-Quinone-methide

1, 6-elimination

Nucleophilic migration

Nucleophilic attack

Tetrahedral boronate complex

Hydrolysis

8

Detection limit of H2O2 in presence of QCy-BA. Concentration dependent studies were

performed using microplate reader. In the well-plates, first we have taken QCy-BA (5 M) in

buffer solution, then we added increasing concentration of H2O2 from 0 to 100 M. Upon

excitation at 400 nm, we have collected the emission at 565 nm as function time after addition of

H2O2.The fluorescence intensity at 565 nm was plotted as a function of concentration of

hydrogen peroxide and each experiment was done in triplicates.

Fig. S3 Plot of the fluorescence intensity at 565 nm against the concentration of [H2O2] PBS-

buffer solution. Each data point was acquired after addition H2O2 at 25 °C. The detection limit

(5.3 μM) was calculated with 3σ/k; where σ is the standard deviation of blank measurement, k is

the slop (-0.30).

Detection Limit

0 5 10 15 2018

20

22

24

26

28

H2O2 (M)

Flu

ore

scen

ce i

nte

nsit

y(a

.u)

9

Fig. S4 (a) Absorption spectra of QCy-BA (2 M) in presence of Drew-AT (2 M). (b)

Emission spectra of QCy-BA (2 M) in presence of Drew-AT (2 M) in PBS-buffer solution.

Fig. S5 (a) Fluorescence response of the combination probe QCy-BA⊂Drew-AT (2 M) in

presence of H2O2 upon excitation with wavelength corresponding to isosbestic point at 456 nm.

Inset: Fluorescence intensity ratio of I650/I500 as function of time (0 to 80 min). (b) Fluorescence

response of the combination probe QCy-BA⊂Drew-AT (2 M) with increasing concentration of

H2O2 from 0 to 200 M upon excitation at 456 nm. Inset: Fluorescence intensity ratio of I650/I500

as function of concentration of H2O2.

300 400 500 600 7000.0

0.1

0.2

0.3

0.4A

bs

orb

an

ce

(a

.u)

Wavelength (nm)

QCy-BA

QCy-BA+Drew-ATa

500 550 600 650 700 750 8000

5

10

15

20

25

30

Flu

ore

sc

en

ce

in

ten

sit

y (

a.u

)

Wavelength (nm)

QCy-BA

QCy-BA + Drew-ATb

500 550 600 650 700 750 800 850 9000

20

40

60

80

100

120

Flu

ore

sc

en

ce

in

ten

sit

y (

a.u

)

Wavelength (nm)

H2O

2

500 550 600 650 700 750 800 850 9000

20

40

60

80

100

Flu

ore

sc

en

ce

in

ten

sit

y (

a.u

)

Wavelength (nm)

Time

0 20 40 60 800

5

10

15

20

25

30

I 65

0/I

50

0

Time (min)

a b

0 50 100 150 2000

10

20

30

40

I 65

0/I

50

0Conc. of H

2O

2 (M)

10

Fig. S6 Fluorescence response of combination probe QCy-BA⊂Drew-AT (2 M) to various

ROS at individual concentrations of 100 µM upon excitation at 564 nm.

Fig. S7 Time-dependent fluorescence spectra of QCy-BA (2 M) in presence of Drew-AT (2

M) after the addition of H2O2 (100 M) upon excitation at 564 nm.

0

50

100

150

200

250

300

Flu

ore

scen

ce in

ten

sit

y

at

650 n

m (

a.u

)

0 40 80 120 160 200 240

10

20

30

40

50

60

70

80 QCy-BA + Drew-AT + H

2O

2 (100 M)

Flu

ore

sc

en

ce

in

ten

sit

y (

a.u

)

Time (min)

11

Fig. S8 Plot of ln(F∞–F) of combination probe QCy-BA⊂Drew-AT (2 M) as a functions of

time at 650 nm upon addition of H2O2 (1 mM), where F∞ and F are the fluorescence intensities at

650 nm at time t∞ (= 60 min) and t, respectively. The kobs calculated from the slope of this plot is

1.04 × 10-3

s-1

.

Fig. S9 (a) Fluorescence spectra of combination probe QCy-BA⊂Drew-AT in presence of GOx

(4 U/mL) and glucose (1 mM) with time, upon excitation at 400 nm. (b) Plot of fluorescence

intensity of combination probe QCy-BA⊂Drew-AT at 650 nm as function of time, in presence

of GOx (4 U/mL) with increasing concentration of glucose from 0 to 1 mM upon excitation at

564 nm.

0 800 1600 2400 32000.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

QCy-BA+Drew-AT

Time (sec)

ln(F

∞-F

)

500 550 600 650 700 750 800 8500

6

12

18

24

30

36

Flu

ore

sc

en

ce

in

ten

sit

y (

a.u

)

Wavelength (nm)

0 20 40 60 80 100

10

20

30

40

50

60

Flu

ore

sc

en

ce

in

ten

sit

y (

a.u

)

Time (min)

Glucose

a b

12

Fig. S10 The plot of the fluorescence intensity of combination probe QCy-BA⊂Drew-AT in the

presence of GOx (4 U/mL) against the concentration of glucose at 650 nm. Each data point was

acquired 1 h after addition of glucose and GOx at 25 °C. The detection limit (6.11 μM) was

calculated by 3σ/k, where σ is the standard deviation of blank measurement and k is the slop

(0.12).

Fig. S11 Plot of ln(F∞–F) of combination probe QCy-BA⊂Drew-AT in presence of GOx (4

U/mL) as a functions of time at 650 nm upon addition of glucose (1 mM), where F∞ and F are the

fluorescence intensities at 650 nm at time t∞ (= 100 min) and t, respectively. The kobs calculated

from the slope of this plot is 6.87 × 10-4

s-1

.

Detection Limit

0 50 100 150 2000

10

20

30

40

Glucose (M)

Flu

ore

sc

en

ce

in

ten

sit

y(a

.u)

0 1000 2000 3000 4000 5000 60000.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

QCy-BA + Drew-AT + GOx + Glucose

Time (sec)

ln(F

∞-F

)

13

Fig. S12 (a) Schematic diagram represents the catalase activity in presence of H2O2 and

combination probe QCy-BA⊂Drew-AT. (b) Fluorescence intensity of QCy-BA at 565 nm.

Where Q: QCy-BA, QH: QCy-BA+H2O2, QHD: QCy-BA+H2O2+Drew-AT, QCH: QCy-

BA+Catalase+H2O2, QCHD: QCy-BA+Catalase+H2O2+Drew-AT. (c) Time dependent

fluorescence of combination probe QCy-BA⊂Drew-AT in presence and absence of catalase

upon addition of H2O2 (1 mM) upon excitation at 564 nm.

0

25

50

75

100

125

Q QH QHD QCH QCHD

Rela

tive i

nte

nsit

y (

a.u

)

0 20 40 60 80 100 1200

10

20

30

40

50

60

70

80

Flu

ore

sc

en

ce

in

ten

sit

y (

a.u

)

Time (min)

QCy-BA+Catalase+H2O

2+Drew-AT

QCy-BA+H2O

2+Drew-AT

b c

a

H2O2

H2O O2+ Catalase

X Drew-AT

QCy-DT⊂Drew-AT

QCy-BA

o

14

Fig. S13 Dose dependent cell viability of HeLa cells by taking 0.0-25 µM of probe QCy-BA for

24 h. Error bars represent ±standard deviation.

Fig. S14 (a) Flow cytometry plots of HeLa cells in the absence and presence of QCy-BA (5

M). (b) FACS-analysis shows the PerCP mean fluorescence intensity in HeLa cells in presence

of QCy-BA (5 M).

00

20

40

60

80

100

120

0.0 3.125 6.25 12.5 25 50

Conc. of QCy-BA (M)

% C

ell V

iab

ilit

y

00

1000

2000

3000

4000

Cells Cells +QCy-BAFlu

ore

sc

en

ce

in

ten

sit

y(a

.u)

ba

15

Fig. S15 (a) Flow cytometry plots of probe QCy-BA (5 M) treated HeLa cells in the presence

of H2O2 (100 M) and NAC (8 mM). (b) Flow cytometry plots of probe QCy-BA (5 M) treated

HeLa cells in the presence of epidermal growth factor (EGF) (50 ng/mL) and NAC (8 mM).

1

2

34 1. Cells

2. QCy-BA

3. QCy-BA+ NAC +EGF

4. QCy-BA +EGF

Co

un

t

PerCP-A

b

1. QCy-BA

2. QCy-BA +H2O2

3. QCy-BA + NAC

4. QCy-BA + NAC + H2O2

a

16

Fig. S16 H2O2 detection in attached live HeLa cells using DCFDA after treatment with BrdU

(100 µM) and doxorubicin (0.1 µM) for 48 h. Fold change of fluorescence per cell is normalized

to 1 for control cells (n=3).

Table S1. Comparison of probe QCy-BA with Naphtho-Peroxyfluor-1 (NPF-1).

a A. E. Albers, B. C. Dickinson, E. W. Miller and C. J. Chang, Bioorg. Med. Chem. Lett., 2008, 18, 5948–5950.

b

Present work.

0

2

4

6

8

Control BrdU Dox

Fo

ld c

han

ge o

f fl

uo

rescen

ce

per

cell

Property Naphtho-Peroxyfluor-1 (NPF1) a QCy-BA b

Synthesis Metal-catalyst required Metal-catalyst not required

Solubility DMSO Water

Stokes shift ∆λmax = ~62 nm ∆λmax = ~280 nm

Detection Fluorescence Fluorescence as well as naked eye

Incubation time ~60-120 min ~15-30 min

Concentration required for cell

imaging

~20 µM ~5 µM

17

Reference

1. N. Narayanaswamy, M. Kumar, S. Das, R. Sharma, P. K. Samanta, S. K. Pati, S. K. Dhar, T.

K. Kundu and T. Govindaraju. Sci. Rep., 2014, 4, 6476.

2. A. R. Lippert, G. C. Van de Bittner and C. J. Chang, Acc. Chem. Res., 2011, 44, 793–804.

18

1H NMR spectrum of compound 2

13C NMR spectrum of Compound 2

B

I

OO

B

I

OO

19

1H NMR spectrum of Compound 3

13C NMR spectrum of Compound 3

O O

O

BO

O

O O

O

BO

O

20

1H NMR spectrum of QCy-BA

13C NMR spectrum of QCy-BA

O

BOH

OH

S

N N

S

I I

O

BOH

OH

S

N N

S

I I

21

HPLC trace of QCy-BA

HRMS mass data of QCy-BA

0.0 2.5 5.0 7.5 10.0 min

0

100

200

300

400mAU

O

BOH

OH

S

N N

S

O

BOH

OH

S

N N

S

[M-2I]2+

Exact Mass: 288.0851