Supporting Information for:

Thiophene-based fluorescent probes with low cytotoxicity and high photostability for lysosomes in living cells

Agostina Lina Capodilupo,1,a Viviana Vergaro,1,a Francesca Baldassarre,b Antonio Cardone,c

Giuseppina Anna Corrente,d Claudia Carlucci,a Stefano Leporatti,a Paride Papadia,e Giuseppe

Gigliafg and Giuseppe Ciccarella.ah *

a Istituto Nanoscienze – CNR, National Nanotechnology Laboratory (NNL), Via Arnesano, 73100

Lecce, Italy. E-mail: giuseppe.ciccarella@unisalento.it

b Università del Salento, via Piazza Tancredi, 7, 73100 Lecce. Italy

c Istituto di Chimica dei Composti Organo Metallici (ICCOM) – Consiglio Nazionale delle Ricerche

CNR, Via Orabona, 4 – 70125 Bari, Italy.

d Università della Calabria, via Pietro Bucci, 87036 Arcavacata di Renze, Cosenza, Italy

f Center for Biomolecular Nanotechnologies (CBN) Fondazione Istituto Italiano di Tecnologia (IIT),

Via Barsanti 1, Arnesano, 73010, Italy

g Dipartimento di Matematica e Fisica “Ennio De Giorgi”, Università del Salento, Via Monteroni,

73100, Lecce, Italy.

h Dipartimento di Ingegneria dell'Innovazione, Università del Salento, Via Monteroni, 73100, Lecce,

Italy.

S1

Contents of Supporting Information

S3 Fig. S3. UV-vis absorption.

S4 Fig. S4 pH sensitivity of TC1 and TC2.

S5 Fig. S5 Qualitative analysis of LTR photobleaching obtained by confocal microscopy

S6 Fig. S6 Qualitative analysis of MTR photobleaching obtained by confocal microscopy

S7 Fig. S7 Qualitative analysis of TC1 photobleaching obtained by confocal microscopy

S8 Fig. S8 Qualitative analysis of TC2 photobleaching obtained by confocal microscopy

S9 1H-NMR, 13C-NMR, spectra of compounds.

S2

Fig.S3 Normalized absorbance (a) and emission (b) spectra in DMSO of TC1 and TC2.

Fig.S3 Normalized absorbance (c) and emission (d) spectra in H2O of TC1 and TC2.

Table 1 Summary of spectroscopic data of TC dyes.dye solvent λabs (nm) Δλ(nm) λem (nm) ε (104 M-1cm-1) ΦPL

TC1 CH2Cl2 442 86 528 1.86 0.051DMSO 444 87 531 2.14 0.079H2Oa 452 150 602 1.60 <0.001

TC2 CH2Cl2 496 40 536 4.27 0.007DMSOH2Oa

498492

31152

529644

3.192.13

0.009<0.001

Standard: [Ru(bpy)3]2+ (Φ =0.028) in H2O. a The measurements were carried out dissolving in DMSO / H2O (1:125 v/v).

S3

pH sensitivity of TC1 and TC2.

Fig.S4 The Absorbance (a) and the fluorescence intensities (b) in H2O of TC1 and TC2 as a function of pH.

S4

Fig S5 Micrographs obtained by confocal microscopy of MCF 7 cells stained with LTR (0.075 μM). To check the photostability of commercial LTR the fluorescence intensity in the consecutive t-scan mode was determined for 30 min (time interval of data determination was indicated above each micrographs). Excitation wavelength was 514 nm. Scale bar, 5 m

S5

Fig. S6 Micrographs obtained by confocal microscopy of MCF 7 cells stained with MTR (0.1 μM). To check the photostability of commercial MTR the fluorescence intensity in the consecutive t-scan mode was determined for 30 min (time interval of data determination was indicated above each micrographs). Excitation wavelength was 514 nm. Scale bar, 5 m

S6

Fig S7 Micrographs obtained by confocal microscopy of MCF 7 cells stained with TC1 (2.0 μM). To check the photostability of commercial TC1 the fluorescence intensity in the consecutive t-scan mode was determined for 30 min (time interval of data determination was indicated above each micrographs). Excitation wavelength was 488 nm. Scale bar, 5 m

S7

Fig. S8 Micrographs obtained by confocal microscopy of MCF 7 cells stained with TC2 (2.0 μM). To check the photostability of commercial TC2 the fluorescence intensity in the consecutive t-scan mode was determined for 30 min (time interval of data determination was indicated above each micrographs). Excitation wavelength was 488 nm

S8

Compound 3

ppm (f1)0.000.501.001.502.002.503.003.504.004.505.005.506.006.507.007.508.008.50

7.601

7.591

7.155

7.145

1.00

1.02

ppm (f1)6.907.007.107.207.307.407.507.607.707.80

1.00

1.02

ppm (f1)0255075100125150175200

175.9

87

143.3

74

133.0

3213

1.024

122.5

97

S9

Compound TC1

ppm (f1)0.000.501.001.502.002.503.003.504.004.505.005.506.006.507.007.508.008.50

7.584

7.573

7.360

7.339

7.321

7.278

7.253

7.186

7.166

7.150

6.439

6.428

1.00

1.944.154.60

1.03

ppm (f1)6.256.506.757.007.257.507.75

1.00

1.94

4.15

4.60

1.03

ppm (f1)255075100125150175

176.6

24

160.5

95

146.4

74

132.7

6013

1.155

129.8

0512

9.558

129.4

3412

5.017

124.6

5811

4.158

S10

Compound TC2

ppm (f1)0.501.001.502.002.503.003.504.004.505.005.506.006.507.007.508.00

7.338

7.248

7.181

6.428

4.179

1.566

1.233

2.00

1.73

3.146.307.49

3.92

ppm (f1)0255075100125150175200

164.4

5716

0.380

152.9

6114

6.154

136.7

6313

5.093

129.4

4612

4.656

120.0

8311

4.591

88.88

7

60.95

3

13.99

8

S11