A water soluble probe with near infra-red two-photon absorption … · A water soluble probe with near infra-red two-photon absorption and polarity-induced fluorescence for cerebral

1

SUPPORTING INFORMATION FOR :

A water soluble probe with near infra-red two-photon absorption

and polarity-induced fluorescence for cerebral vascular imaging

Julien Massin,a Azzam Charaf-Eddin,

b Florence Appaix,

c Denis Jacquemin,

b,d Boudewijn

VanDerSanden,c Yann Bretonnière,

a Chantal Andraud

a and Cyrille Monnereau

a

Figure S1. Plot of emission Stokes shift (top) absorption shift (center), and emission shift(bottom)t to Lippert-

Mataga polarity scale .......................................................................................................................................... 2

Figure S2. Plot of the fluorescence lifetime vs emission energy, and fit of the evolution to an exponential

model ................................................................................................................................................................... 3

Figure S3 Excited state decay’s kinetic constants vs emission wavelength ........................................................ 3

Scheme S1 bond numbering for the values in Table S-1 .................................................................................... 4

Table S1 .............................................................................................................................................................. 4

Figures S4-S5 ...................................................................................................................................................... 4

Figure S4 Tested dihedral angle for excited-state twists. ................................................................................ 5

Figure S5 Evolution of the relative energies of the GS and ES in toluene and DMSO, for twist around 1

and 2. ............................................................................................................................................................. 5

Figure S6 stick and convoluted vibrationally resolved calculated absorption (left) and emission (right) spectra

for Lem in DMSO ............................................................................................................................................... 5

Figures S7. 1H and

13C NMR spectra of all new compounds in this paper ......................................................... 6

Figures S8. GPC traces of Lem-PHEA (detection : RI(main), UV (inset))......................................................... 9

Figure S9: In vivo measurements of the red and near infrared emission of Lem-PHEA in the cerebral cortex

vasculature of a CD1 mouse................................................................................................................................ 9

Figure S10: Local biodistribution of Lem-PHEA in the kidneys and liver. ...................................................... 10

Electronic Supplementary Material (ESI) for Chemical ScienceThis journal is © The Royal Society of Chemistry 2013

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Figure S1. Plot of emission Stokes shift (top) absorption shift (center), and emission shift(bottom)t to Lippert-Mataga polarity scale

Linearization is performed on Lem-In data only. An Onsager’s radius of 8,5 A is considered for the molecule,

on the basis of comparison with literature values for a closely related compound (compound 1f in reference 1,

calculated from crystal structure packing parameters) 1

Df

nab

s-n

em

y = 3088,5x + 4444,7R² = 0,8235

4400

4600

4800

5000

5200

5400

5600

5800

0 0,05 0,1 0,15 0,2 0,25 0,3 0,35

Lippert-Mataga's Stokes shift plot

y = -2399,5x + 21090R² = 0,4087

18500

19000

19500

20000

20500

21000

21500

22000

0 0,05 0,1 0,15 0,2 0,25 0,3 0,35

Lippert-Mataga's absorption shift plotLippert-Mataga's absorption shift plot

y = -5488x + 16645R² = 0,8973

14000

14500

15000

15500

16000

16500

17000

17500

0 0,05 0,1 0,15 0,2 0,25 0,3 0,35

Lippert-Mataga's emission shift plot

nab

sn

em

DmCT = 13.5 D

mGS = 10.5 D

mES = 24.5 D

Lem-PHEA

Lem-In


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Figure S2. Plot of the fluorescence lifetime vs emission energy, and fit of the evolution to an exponential model

Figure S3 Excited state decay’s kinetic constants vs emission wavelength

Plot of the evolution of the radiative (kr : □) and non radiative (knr : ○) kinetic constants for the excited state

decay vs emission wavelength. The y axis is in a logarithmic scale, where kr = f/f, and knr= 1/tf - kr= (1-f)/f

R² = 0,8623

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

14500 15000 15500 16000 16500 17000

Lem-In

Lem-PHEA

Fluo

resc

ence

life

tim

e (n

s)

emission energy (cm-1)

6,00E+07

6,00E+08

6,00E+09

580 600 620 640 660 680 700

Wavelength (nm)

k(s-1

)


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Scheme S1 bond numbering for the values in Table S-1

Table S1 Distance S0 (Theo) S1 (Theo) S0 (Exp)

a

b

c

d

e

f

g

h

i

j

1.372

1.417

1.380

1.405

1.452

1.353

1.446

1.365

1.430

1.377

1.358

1.426

1.370

1.427

1.417

1.397

1.404

1.409

1.397

1.416

1.379

1.395

1.380

1.404

1.444

1.334

1.448

1.364

1.418

1.364

MAD 0.009

BLA 0.061 0.028 0.057

Comparison between theoretical (PCM-CAM-B3LYP) and experimental (XRD) structures for the ground-state

(and excited-state for theory) distance along the conjugated path. MAD is the mean absolute deviation, whereas

BLA is the computed bond length alternation. All values are in Å.

Figures S4-S5 In order to assess the possibility of rotation at the excited-state, we have performed rigid-rotor scans of the

dialkylamino and dicyano terminal groups (see dihedral in in S3) using the CAM-B3LYP functional within a

LR-PCM solvent model approach. As can be seen in S4, a maximal energy value for deformation close to 90°

have been systematically predicted for the 1, irrespective of the solvent or state (GS versus ES). On the

contrary for 2, we found an extra (less stable minima) for the perpendicular structure only in toluene. For the

records, we underline that 1) SS-PCM calculations failed to converge in several cases (hence explaining the use

of the LR-PCM model); 2) calculations performed with a range-separated hybrids presenting a phyisically

correct asymprotic behavior, namely B97X-D, lead to similar trends for 2.


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Figure S4 Tested dihedral angle for excited-state twists.

Figure S5 Evolution of the relative energies of the GS and ES in toluene and DMSO, for twist around 1 and 2.

Figure S6 stick and convoluted vibrationally resolved calculated absorption (left) and emission (right) spectra for Lem in DMSO


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Figures S7. 1H and 13C NMR spectra of all new compounds in this paper

1H NMR in d6-DMSO

13C NMR in d6-DMSO


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1H NMR in CDCl3

13C NMR in CDCl3


8

1H NMR in CD3OD

13C NMR in CD3OD


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Figures S8. GPC traces of Lem-PHEA (detection : RI(main), UV (inset))

Mn = 4737

Mw/Mn (PDI) = 2.09

Figure S9: In vivo measurements of the red and near infrared emission of Lem-PHEA in the cerebral cortex vasculature of a CD1 mouse. S8a) The cutoff wavelength of the dichroic mirror in front of the 20x objective was 760 nm

and the cutoff wavelength of the dichroic mirror in between the near infrared and red channel

was 650 nm. In near infrared PMT (without emission filter), all fluorescence emissions larger

than 650 and smaller than 760 nm were measured, whereas in the red PMT (with emission

filter) photon wavelengths between 576 and 660 nm were detected. The emission spectrum of

Lem-PHEA is superposed. S8b-d) The fluorescence intensity profile of a vein showed

approximately a 2.5x higher intensity profile in the near IR than in the red channel.

Re

fra

ctive

In

de

x R

esp

on

se

(m

V)

Retention Volume (mL)

5,20 10,40 15,60 20,80 26,00 31,20 36,40 41,60 46,80

1049,30

1038,67

1028,04

1017,41

1006,78

996,15

985,53

974,90

964,27

Data File: 2012-02-08_19;18;32_CYR_148_01.vdt Method: Calib conv PMMA_38mL18_0212-0000.vcm

1059,92

953,64

0,00 52,00

Ultra

Vio

let R

esp

on

se

(m

V)

Retention Volume (mL)

5,20 10,40 15,60 20,80 26,00 31,20 36,40 41,60 46,80

108,53

95,06

81,59

68,11

54,64

41,17

27,70

14,23

0,76

Data File: 2012-02-08_19;18;32_CYR_148_01.vdt Method: Calib conv PMMA_38mL18_0212-0000.vcm

122,00

-12,71

0,00 52,00


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Figure S10: Local biodistribution of Lem-PHEA in the kidneys and liver. S9a) Two-photon image of a kidney slice below the cortex showing the blue

endofluorescence of the tubules (excitation wavelength 740 nm) and the red fluorescence of

Lem-PHEA that accumulated in the renal tubules as early as 30 min after i.v. injection. Scale

bar = 50 µm. S9b) Two-photon image of the renal cortex in situ (whole kidney) at 2 hours

after dye injection. The dye was only present in the tubules and absent in the blood vessels.

The excitation wavelength was 950 nm. Scale bar = 100 µm. S9c) Detail of the tubules in the

renal cortex at 2h after dye injection at an excitation wavelength of 950 nm. Scale bar = 50

µm. S9d) Two-photon image of a liver slice at 2h after dye injection. Lem-PHEA stayed in

the vascular compartment and no dye accumulation was observed in hepatic cells.

1. - Chem. Mater.,

2010, 23, 862-873.


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A water soluble probe with near infra-red two-photon absorption … · A water soluble probe with near infra-red two-photon absorption and polarity-induced fluorescence for cerebral