1

Electronic Supplementary Information (ESI)

pH-SENSITIVE PERYLENE BISIMIDE PROBES FOR LIVE CELL FLUORESCENCE LIFETIME IMAGING

Daniel Aigner**, Ruslan I Dmitriev**, Sergey M Borisov, Dmitri B Papkovsky and Ingo Klimant*

** Authors contributed equally to this work

Contents:

1. Additional figures (S1-S6)2. HPLC purification of MP3. Structure analysis (S7-S22)

Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B.This journal is © The Royal Society of Chemistry 2014

2

ADDITIONAL FIGURES

Figure S1: Typical fluorescence decay curves in MEF cells stained with NSP, permeabilized with nigericin and exposed to buffer as indicated, obtained by confocal TCSPC-FLIM.

4 5 6 7 8

0.5

0.6

0.7

0.8

0.9

1.0

1.1

(a)

NSP in buffer NSP in 10% FBS NSP in 80% FBS

Nor

mal

ized

Inte

nsity

/ a.u

.

pH

4 5 6 7 84.5

5.0

5.5

6.0

6.5

7.0

NSP in 80% FBS NSP in buffer

Fluo

resc

ence

dec

ay ti

me/

ns

pH

(b)

Figure S2: pH-calibration curves of NSP in aqueous buffer at 37°C, with and without the presence of fetal bovine serum (FBS). A: fluorescence intensity; b: fluorescence decay time.

0 2 4 6 8 10 120.01

0.1

1

pH 8.0

Nor

mal

ized

P

hoto

n C

ount

Time / ns

Measured Decay Curve Fit (Monoexponential)

pH 4.4(NSP in MEF Cells)

3

Figure S3: Confocal TCSPC-FLIM of permeabilized cells stained with NSP or MP, permeabilized with nigericin and exposed to buffer as indicated, performed at 25°C. Left: False-colour images of cells exposed to buffers with different pH; Right: Corresponding distributions of lifetimes within the images.

Figure S4: pH calibration curves in MEF cells stained with NSP or BCECF, permeabilized with nigericin and exposed to buffer as indicated, obtained by confocal TCSPC-FLIM.

NSP (WT)

NSP (CACO2)

MP (WT)

MP (SCO2)

MP (CACO2)

pH4.4

pH6.5

pH8

pH4.4

pH6.5

pH8

pH4.4

pH6

pH8

pH4.4

pH6.5

pH8

pH4.4

pH6.5

pH8

3.5 4.0 4.5 5.00.0

0.5

1.0 pH 6.5 pH 4.4

Rel

ativ

e A

bund

ance

Fluorescence Decay Time / ns

pH 8

3.0 3.5 4.0 4.5 5.00.0

0.5

1.0 pH 6.5 pH 4.4

Rel

ativ

e A

bund

ance

Fluorescence Decay Time / ns

pH 8

3.0 3.5 4.0 4.50.0

0.5

1.0pH 8

pH 6

Rel

ativ

e A

bund

ance

Fluorescence Decay Time / ns

pH 4.4

2.5 3.0 3.5 4.00.0

0.5

1.0 pH 8 pH 6

Rel

ativ

e Ab

unda

nce

Fluorescence Decay Time / ns

pH 4.4

3.0 3.5 4.0 4.5 5.00.0

0.5

1.0 pH 8 pH 6

Rela

tive

Abun

danc

e

Fluorescence Decay Time / ns

pH 4.4

3.0 3.5 4.0 4.5 5.00.0

0.5

1.0 pH 8pH 6

Rel

ativ

e A

bund

ance

Fluorescence Lifetime / ns

pH 4.4

3.5 4.0 4.5 5.00.0

0.5

1.0 pH 6.5 pH 4.4

Rel

ativ

e A

bund

ance

Fluorescence Lifetime / ns

pH 8

3.0 3.5 4.0 4.5 5.00.0

0.5

1.0 pH 6.5 pH 4.4

Rel

ativ

e A

bund

ance

Fluorescence Lifetime / ns

pH 8

2.5 3.0 3.5 4.00.0

0.5

1.0 pH 8 pH 6

Rel

ativ

e A

bund

ance

Fluorescence Lifetime / ns

pH 4.4

2.5 3.0 3.5 4.0 4.50.0

0.5

1.0pH 8

pH 6

Rel

ativ

e A

bund

ance

Fluorescence Lifetime / ns

pH 4.4

4 5 6 7 82.5

3.0

3.5

Fluo

resc

ence

Li

fetim

e / n

s

pH

MP (MEF cells)

5 6 7 8

3.53.63.73.83.94.0

Lifetime

Fluo

resc

ence

pH

Life

time

/ ns

0.00.20.40.60.81.0

Intensity

Fluo

resc

ence

In

tens

ity /

a.u.

BCECF (MEF cells)

4

4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

3.8

4.0

4.2

4.4

4.6

4.8

5.0

37°C, replicate 1 37°C, replicate 2 37°C, replicate 3 25°C 25°C, sigmoidal fit 25°C, linear fit 37°C, linear fit

Fluo

resc

ence

Life

time

/ ns

pH

MEF Cells

4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

4.5

5.0

5.5

6.0

6.5

7.0

25°C 37°C

Fluo

resc

ence

Life

time

/ ns

pH

NSP in buffer solution

Figure S5: pH calibration curves of NSP based on fluorescence lifetime in MEF cells (left) and aqueous buffer (right), obtained by confocal TCSPC-FLIM.

Figure S6: Monitoring of intracellular acidification with NSP, obtained by confocal FLIM microscopy. A: false-color images of MEF cells before (resting) and after stimulation with bafilomycin A1 (0.25 µM); B: Corresponding lifetime distributions over the images.

HPLC Purification Details for MP

Table S1: HPLC gradient used for the purification of MP (Temperature 30°C).Time/min Ratio MeOH/% Ratio 0.01% aqueous

formic acid/%Flow rate/mlmin-1

0 35 65 2035 70 70 20

35.1 100 0 2039 100 0 20

RestingMEF Cells

Treated withBafilomycin A1

4.5 5.0 5.5 6.00.0

0.5

1.0Treated withBafilomycin A1

Rel

ativ

e ab

unda

nce

Fluorescence Decay Time / ns

Resting cells

5

STRUCTURE ANALYSIS

NMR spectra:

N-(2,6-Diisopropylphenyl)-N’-(2-dimethylaminoethyl)-1,6,7,12-tetrachloroperylene-3,4:9,10-tertracarboxylic bisimide:

Figure S7: 1H NMR-spectrum (300MHz, CDCl3, TMS) of N-(2,6-Diisopropylphenyl)-N’-(2-dimethylaminoethyl)-1,6,7,12-tetrachloroperylene-3,4:9,10-tertracarboxylic bisimide; δ = 8.71 (4H, 2s, H(1)), 7.52 (1H, t, H(3a)), 7.38 (2H, d, J = 7.7Hz, H(3b)), 4.39 (2H, t, J = 6.3Hz, H(2a)), 2.6 – 2.8 (4H, m, H(2b) and H(3d), 2.37 (6H, s, H(2c)), 1.18 (12H, dd, J1 = 3.7 Hz, J2 = 3.2 Hz, H(3c)).

NN

ClCl

ClClO

O

O

O

NCH3

CH3

CH3CH3

CH3CH3

H

HH

HH

H

HH

H

HH

HH

(1) (1)

(1) (1)

(2a)

(2b)(2c)

(2c)(3a)

(3b)

(3b)

(3d)

(3d)

(3c)

(3c)

CH2Cl2CHCl3

6

Figure S8: 13C-APT NMR-spectrum (300MHz, CDCl3, TMS) of N-(2,6-Diisopropylphenyl)-N’-(2-dimethylaminoethyl)-1,6,7,12-tetrachloroperylene-3,4:9,10-tertracarboxylic bisimide; δc = 162.4 (C=O), 145.6, 133.5, 133.4 (pelylene C(Ar)-H), 133.1 (perylene C(Ar)-H), 130.0 (C(Ar)-H), 129.0, 128.6, 124.3 (C(Ar)-H), 123.4, 123.1; 57.0 (NCH2CH2N(CH3)2), 45.8 (N(CH3)2), 38.5 ((NCH2CH2N(CH3)2), 29.3 (ArCH(CH3)2), 24.0 (ArCH(CH3)2).

7

N-(2,6-diisopropylphenyl)-N’-(2-dimethylaminoethyl)-1,6,7,12-tetraphenoxyperylene-3,4:9,10-tertracarboxylic bisimide (2):

Figure S9: 1H NMR-spectrum (300MHz, CDCl3, TMS) of 2; δ = 8.21 (4H, 2s, H(1)); 7.42 (1H, t, J = 7.8 Hz, H(3a)); 7.20 – 7.33 (10H, m, H(3b,4b)); 7.12 (4H, q, J = 7.8 Hz, H(4c)); 6.96 (8H, q, J = 3.9 Hz, H(4a)); 4.28 (2H, t, J = 6.8 Hz, H(2a)); 2.58 – 2.75 (4H, m, (2b,3d)); 2.34 (6H, s, H(2c)); 1.12 (12H, d, J = 6.8 Hz, H(3c)).

(1)

(1)(1)

(1)

(2a)(2b)

(2c) (2c)

(3a)(3b) (3b)

(3c) (3c)

(3d) (3d)

NCH3CH3

HH

HH

N

N

R

R

O O

O O

R

R

CH3CH3CH3CH3

H H

HH

H

H

H

HH

O

H

H

H

H

H

(4a)

(4a) (4b)

(4b)

(4c)R =

8

Figure S10: 13C-APT NMR-spectrum (300MHz, CDCl3, TMS) of 2; δc = 163.2 (C=O), 155.9, 155.2, 145.6, 133.4, 133.0, 130.7, 130.0 (C(Ar)-H), 129.5 (C(Ar)-H), 124.6 (C(Ar)-H), 123.9 (pelylene C(Ar)-H), 122.8, 122.7, 120.8, 120.5, 120.4 (C(Ar)-H), 120.0 (C(Ar)-H), 119.8; 56.9 (NCH2CH2N(CH3)2), 45.6 (N(CH3)2), 38.2 ((NCH2CH2N(CH3)2), 29.1 (ArCH(CH3)2), 24.0 (ArCH(CH3)2).

9

N-(2,6-Diisopropyl-4-sulfophenyl)-N’-(2-dimethylaminoethyl)-1,6,7,12-tetra(4-sulfophenoxy)perylene-3,4:9,10-tertracarboxylic bisimide (3):

Figure S11: 1H NMR-spectrum (300MHz, (CD3OD:D2O 3:1 (V/V), TMS) of 3; δ = 8.19 (2H, s, H(1)), 8.07 (2H, s, H(1)), 7.81 (4H, d, J = 8.7Hz, H(4b)), 7.68 (4H, d, J = 8.7Hz, H(4b)), 7.44 (1H, t, J = 7.6Hz, H(3a)), 7.31 (2H, d, J = 7.8Hz, H(3b)), 7.05 (8H, 2d, H(4a)), 4.49 (2H, broad s, H(2a)), 3.51 (2H, broad s, H(2b)), 3.00 (6H, s, H(2c)), 2.67 (2H, m, H(3d)), 1.09 (12H, d, J = 6.7Hz, H(3c)).

10

Figure S12: HH-COSY NMR-spectrum (300MHz, (CD3OD:D2O 3:1 (V/V), TMS) of 3.

11

Figure S13: 13C-APT NMR-spectrum (300MHz, CD3OD:D2O 3:1 (V/V), TMS) of 3; δc = 164.9 (C=O), 158.4, 158.2, 156.6, 156.1, 147.2, 142.7, 142.6, 131.1 (C(Ar)-H), 129.4 (C(Ar)-H), 125.1 (C(Ar)-H), 124.4, 122.9 (pelylene C(Ar)-H), 122.4 (perylene C(Ar)-H), 122.2, 122.0, 120.0 (C(Ar)-H); 58.0 (NCH2CH2N(CH3)2), 44.4 (N(CH3)2), 37.3 ((NCH2CH2N(CH3)2), 30.3 (ArCH(CH3)2), 24.3 (ArCH(CH3)2). Undelined peaks are absent in the 13C-APT spectrum but can be found in the HSQC spectrum (Figure S13).

12

Figure S14: HSQC-NMR (300MHz, CD3OD:D2O 3:1 (V/V), TMS) of 3.

13

N-(2,6-Diisopropyl-4-([N-(1-ethoxycarbonyl-4-guanidinylbutyl)amino]sulfonyl)phenyl)-N’-(2-dimethylaminoethyl)-1,6,7,12-tetra(4-([N-(1-ethoxycarbonyl-4-guanidinylbutyl)amino]sulfonyl)phenoxy)perylene-3,4:9,10-tertracarboxylic bisimide (MP):

Figure S15: 1H NMR-spectrum (300MHz, CD3OD, TMS) of MP; δ = 8.19 (4H, d, H(1)), J = 8.4Hz), 7.81 (8H, dd, J1 = 6.2Hz, J2 = 8.5Hz, H(4b)), 7.45 (1H, t, J = 7.8Hz, H(3a)), 7.30 (2H, d, J = 7.6Hz, H(3b)), 7.12 (8H, dd, J1 = 8.6Hz, J2 = 16.5Hz, H(4a)), 4.50 (2H, broad s, H(2a)), 3.85 – 4.10 (12H, m, H(5a) and H(5e)), 3.51 (2H, broad s, H(2b)), 3.22 (8H, m, H(5d)), 2.97 (6H, s, H(2c)), 2.69 (2H, p, J = 6.8Hz, H(3d)), 1.6 – 1.9 (16H, m, H(5b) and H(5c)), 1.0 – 1.23 (24H, m, H(3c) and H(5f)).

SH

H

H

HO

NH

NHOO

OO

NH2

NH2+

NN

RR

RRO

O

O

O

NCH3

CH3

CH3CH3

CH3CH3

H

HH

HH

H

HH

H

HH

HH

R =

(1) (1)

(1) (1)

(2a)

(2b)(2c)

(2c)(3a)

(3b)

(3b)

(3d)

(3d)

(3c)

(3c)

(4a)

(4a)

(4b)

(4b)

(5a)(5b)

(5c)

(5d)

(5e)(5f)

DMFDMF (N,N-dimethylformamide)

CH2Cl2Acetone

14

Figure S16: HH-COSY-NMR-spectrum (300MHz, CD3OD, TMS) of MP.

15

Figure S17: 13C-APT NMR-spectrum (300MHz, CD3OD) of MP; δc = 172.7 (COOR), 164.6 (C=O), 160.5, 160.2, 156.1, 155.8, 147.2, 138.2, 131.7, 130.9 (C(Ar)-H), 130.8 (C(Ar)-H), 125.2, 125.1 (C(Ar)-H), 124.7, 122.7 (pelylene C(Ar)-H), 122.5, 120.6 (C(Ar)-H); 62.7 (O-CH2-CH3), 57.0 (NCH2CH2N(CH3)2), 56.9 (CH), 44.6 (N(CH3)2), 41.7 (CH2-NH), 37.0 ((NCH2CH2N(CH3)2), 31.1 (CH2), 30.3 (ArCH(CH3)2), 26.1 (CH2), 24.3 (ArCH(CH3)2), 14.5 (O-CH2-CH3). Undelined peaks are absent in the 13C-APT spectrum but can be found in the HSQC spectrum (Figure S18).

16

Figure S18: HSQC-NMR (300MHz, CD3OD) of MP.

17

m/z738 740 742 744 746 748 750 752 754 756 758 760 762 764 766 768 770 772 774 776 778 780 782 784 786 788 790

%

0

100

%

0

100

%

0

100Aigner_4B_alpha (0.014) Is (0.10,0.01) C40H33Cl4N3O4 TOF LD+

2.87e12761.1203

759.1225

763.1184

764.1206

765.1171

Aigner_4B_alpha (0.014) Is (0.10,0.01) C40H32Cl4N3O4 TOF LD+ 2.87e12760.1125

758.1147

762.1106

763.1127

764.1093

Aigner_4B_alpha 18 (0.298) Sb (99,30.00 ); Cm ((7:12+14:16+18:21+28:30+33:36)) TOF LD+ 1.19e3760.1169

758.1191

756.1143

761.1242

762.1205

763.1200

764.1207

765.1219

m/z500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

%

0

100Aigner_4B_alpha 18 (0.298) Sb (99,30.00 ); Cm ((7:12+14:16+18:21+28:30+33:36)) TOF LD+

1.19e3760.1

758.1

756.1

761.1

762.1

763.1

764.1

799.1765.1

MALDI-TOF spectra:

N-(2,6-Diisopropylphenyl)-N’-(2-dimethylaminoethyl)-1,6,7,12-tetrachloroperylene-3,4:9,10-tertracarboxylic bisimide:

Figure S19: MALDI-TOF-spectrum of N-(2,6-Diisopropylphenyl)-N’-(2-dimethylaminoethyl)-1,6,7,12-tetrachloroperylene-3,4:9,10-tertracarboxylic bisimide.

Experimental

Theoretical isotope pattern M+

Theoretical isotope pattern [M-H]+

18

m/z500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700

%

0

100Aigner_5B_DithranolNa 38 (0.632) Sb (99,30.00 ); Cm ((11:16+19:24+31:33+38:40)) TOF LD+

1.06e3990.4

988.4

991.4

992.4

1012.4

1013.4

1014.4

m/z982 984 986 988 990 992 994 996 998 1000 1002 1004 1006 1008 1010 1012 1014 1016 1018 1020 1022 1024

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100Aigner_5B_DithranolNa (0.015) Is (0.10,0.01) C64H52N3O8Na TOF LD+

4.74e121013.3652

1014.3685

1015.3716

Aigner_5B_DithranolNa (0.015) Is (0.10,0.01) C64H51N3O8Na TOF LD+ 4.74e121012.3574

1013.3607

1014.3638

Aigner_5B_DithranolNa (0.015) Is (0.10,0.01) C64H53N3O8 TOF LD+ 4.74e12991.3832

992.3865

993.3897

Aigner_5B_DithranolNa (0.015) Is (0.10,0.01) C64H52N3O8 TOF LD+ 4.74e12990.3754

991.3787

992.3819

Aigner_5B_DithranolNa 38 (0.632) Sb (99,30.00 ); Cm ((11:16+19:24+31:33+38:40)) TOF LD+ 1.06e3990.3752

988.3615

991.3785

992.3820 1012.3609993.3894

1013.3644

Compound 2:

Theoretical isotope pattern [MNa]+

Theoretical isotope pattern [M-H]+

Theoretical isotope pattern M+

Theoretical isotope pattern [(M-2H)Na]+

[MNa]+

[MH]+

Experimental

Figure S20: MALDI-TOF-spectrum of 2

19

Compound 3:

m/z1140 1160 1180 1200 1220 1240 1260 1280 1300 1320 1340 1360 1380 1400 1420 1440 1460 1480 1500 1520 1540 1560

%

0

10020140324_Aigner_5O7FlPt1_alpha 31 (0.515) Sb (99,30.00 ); Cm ((16:23+29:36+41:49+57:59)) TOF LD+

1.31e31349.2

1348.2

1311.21310.2

1312.2

1333.2

1332.2

1313.2

1314.2

1334.2

1350.2

1351.2

1370.2

1356.2

1372.2

1373.21387.1

1393.1

m/z1308 1310 1312 1314 1316 1318 1320 1322 1324 1326 1328 1330 1332 1334 1336 1338 1340 1342 1344 1346 1348 1350 1352 1354 1356 1358

%

0

100

%

0

100

%

0

100

%

0

10020140324_Aigner_5O7FlPt1_alpha (0.015) Is (0.10,0.01) C64H51N3O20S4K TOF LD+

3.51e121348.16

1349.16

1350.16

1351.16

1352.16

20140324_Aigner_5O7FlPt1_alpha (0.015) Is (0.10,0.01) C64H51N3O20S4Na TOF LD+ 3.76e121332.18

1333.19

1334.19

1335.191336.19

20140324_Aigner_5O7FlPt1_alpha (0.015) Is (0.10,0.01) C64H51N3O20S4H TOF LD+ 3.76e121310.20

1311.21

1312.21

1313.211314.21

20140324_Aigner_5O7FlPt1_alpha 31 (0.515) Sb (99,30.00 ); Cm ((16:23+29:36+41:49+57:59)) TOF LD+ 1.31e31349.161348.16

1311.211310.20

1312.22 1333.201332.191313.21

1314.20

1334.201335.20

1350.17

1351.171354.181352.16

1353.16

1355.18

1357.17

Figure S21: MALDI-TOF-spectrum of 3. Maxima at m/z = 1270-1272 and 1287.1 correspond to [MNa2K+] and to [MNaK2+], respectively.

Experimental

Theoretical isotope pattern [MH]+

Theoretical isotope pattern [MNa]+

Theoretical isotope pattern [MK]+

20

Compound MP:

m/z1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000

%

0

10020140324_Aigner_GUA_F1R_alpha 41 (0.683) Sb (0,80.00 ); Sm (SG, 1x4.00); Sb (99,30.00 ); Cm ((30:31+36:37+41:45+51:54+78:83)) TOF LD+

3312047.7

2046.7

2033.7

2019.7

2018.6

2048.7

2049.7

2050.7

2069.7

m/z2010 2015 2020 2025 2030 2035 2040 2045 2050 2055 2060 2065 2070 2075 2080 2085

%

0

100

%

0

10020140324_Aigner_GUA_F1R_alpha (0.017) Is (0.10,0.01) C96H115N19O24S4H TOF LD+

2.92e122047.74

2046.732048.74

2049.74

2050.74

2051.74

20140324_Aigner_GUA_F1R_alpha 41 (0.683) Sb (99,30.00 ); Cm ((30:31+36:37+41:45+51:54+78:83)) TOF LD+ 3512047.72

2046.72

2033.71

2019.652018.69

2017.65

2032.692020.70

2021.702031.68

2034.70

2035.71

2036.672042.74

2048.72

2049.72

2050.72

2051.702069.682062.64

2052.712061.74 2063.65

2071.70

2072.71

Figure S22: MALDI-TOF-spectrum of MP. Maxima at m/z = 2061 – 2064 and 2069 – 2073. Correspond to [MNa+] and to [MNK+], respectively. m/z = Those around 2033.71 and 2019.65 most likely originate from small amounts of MP monomethylester and MP monocarboxylate (transesterification or hydrolysis of one of the four ethyl ester groups in MP which can occur during HPLC-chromotography or concentration after purification, catalyzed by formic acid.

Experimental

Theoretical isotope pattern [MH]+