Two-photon polarity probes built from octupolar fluorophores: Synthesis, structure-properties relationships, and use in cellular imaging

FULL PAPER

DOI: 10.1002/asia.201300735

Two-Photon Polarity Probes Built from Octupolar Fluorophores: Synthesis,

Structure–Properties Relationships, and Use in Cellular Imaging

Cꢀline Le Droumaguet,^[c]Aude Sourdon,^[b]Emilie Genin,^[a]Olivier Mongin,*^{[b, c]}and

Mireille Blanchard-Desce*^{[a, c]}

Abstract: A series of octupolar fluoro-

phores built from triphenylamine

an efficient coupling of the branches

through the TPA core. Increasing the

strength of the EW end groups or the

length of the conjugated spacers and

replacing triple-bond linkers with

double bonds induces both enhance-

ment and broadening of the 2PA re-

sponses, thereby leading to cross-sec-

tions up to 2100 GM at peak and

higher than 1000 GM over the whole

700–900 nm range. All derivatives ex-

hibit intense photoluminescence (PL)

in low- to medium-polarity environ-

ments (with quantum yields in the 0.5–

0.9 range) and display a strong positive

solvatochromic behavior (with Lip-

pert–Mataga specific shifts ranging

from 15000 to 27500 cm^ꢀ1), triple

bonds, and phenyl moieties in the con-

jugated spacers, thereby leading to

larger sensitivities than those of double

bonds and thienyl moieties. More hy-

drophilic derivatives were also shown

to be biocompatible, to retain their

2PA and PL properties in biological

conditions, and finally to be suitable as

polarity sensors for multiphoton cell

imaging.

a

(TPA) core connected to electron-with-

drawing (EW) peripheral groups

through conjugated spacers has been

synthesized. Their photoluminescence,

solvatochromism, and two-photon ab-

sorption (2PA) properties were system-

atically investigated to derive struc-

ture–property relationships. All deriva-

tives exhibit two 2PA bands in the 700–

1000 nm region: a first band at low

energy correlated with a core-to-pe-

riphery intramolecular charge transfer

that leads to an intense 1PA in the

blue-visible range, and a second more

intense band at higher energy due to

Keywords: absorption · bioimaging ·

chromophores

·

fluorescence

·

solvatochromism

Introduction

medicine. These include two-photon excited fluorescence

(2PEF) microscopy for bioimaging,^[2]localized photodynam-

ic therapy (PDT),^[3]localized release of bioactive species (of

major interest in neurosciences),^[4]3D optical data storage,^[5]

optical power limiting,^[6]and 3D microfabrication.^[7]In this

general framework, we have been interested more specifi-

cally in the design of innovative environmental two-photon

fluorescent probes and sensors for biological applications

and/or heavy-metal detection.^[8]The ability of the probe to

generate sufficient fluorescent signal is of major importance

for detecting species of ultra-low concentration (such as

heavy-metal traces or biomolecules in minute concentra-

tions). This requires very large brightness to improve detec-

tion sensitivity (or alternatively the scanning speed in dy-

namic imaging of fast biological events). In this general

framework, we have been interested in the design of sensi-

tive two-photon fluorescent probes that combine very large

2PA responses in the NIR region as well as high sensitivity

of their fluorescence to their microenvironment. With this

aim in mind, we report the synthesis of a library of modular

two-photon octupolar fluorophores with structural diversity

(Figure 1), and the comprehensive study of structure–prop-

erty (fluorescence–2PA–polarity sensitivity) relationships.

By taking advantage of this investigation, we further de-

signed more hydrophilic probes that were successfully ap-

plied to two-photon imaging in biological environments and

micropolarity sensing in living cells.

Over the past decade, significant research efforts have been

devoted to the molecular engineering of novel compounds

that show very large two-photon absorption (2PA) respons-

es.^[1]These efforts were mostly driven by the advantages

that 2PA provides (i.e., intrinsic 3D localization of excita-

tion, excitation in the red-near-infrared (NIR) range to

allow reduction of scattering losses) for applications in vari-

ous domains that range from material science to biology and

[a] Dr. E. Genin, Dr. M. Blanchard-Desce

Universitꢀ Bordeaux, Institut des Sciences Molꢀculaires

CNRS UMR 5255, 351 Cours de la Libꢀration

33405 Talence Cedex (France)

Fax : (+33)5-40-00-69-94

E-mail: m.blanchard-desce@ism.u-bordeaux1.fr

[b] A. Sourdon, Dr. O. Mongin

Institut des Sciences Chimiques de Rennes

CNRS UMR 6226, Universitꢀ de Rennes 1

Campus de Beaulieu, 35042 Rennes Cedex (France)

Fax : (+33)2-23-23-69-55

E-mail: olivier.mongin@univ-rennes1.fr

[c] Dr. C. Le Droumaguet, Dr. O. Mongin, Dr. M. Blanchard-Desce

Chimie et Photonique Molꢀculaires

CNRS UMR 6510, Universitꢀ de Rennes 1

Campus de Beaulieu, F-35042 Rennes Cedex (France)

Supporting information for this article is available on the WWW

under http://dx.doi.org/10.1002/asia.201300735.

1

&

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

These are not the final page numbers! ÞÞ

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

between the dipolar branches

in such branched systems

(Figure 1) and consequently

the photophysical and 2PA

properties is strongly depen-

dent on the nature of the

common (D or A) core.

Strongly nonadditive responses

are observed in the case of the

triphenylamine (TPA) donor

moiety (which leads to the rise

of a more intense 2PA band at

higher energy, thereby corre-

sponding to higher one-

photon-forbidden, two-photon-

allowed excited state, as ex-

pected from octupolar deriva-

tives due to symmetry rea-

sons)^[12]or of the tricyanoben-

zene (TCB) acceptor moie-

ty.^[11a]In contrast, triphenyl-

phosphane oxide (TPPO)- and

triphenylphosphane

sulfide

(TPPS)-accepting cores^[13]as

well as the ambivalent triphe-

nylbenzene (TPB)^[14]core do

not promote major 2PA en-

hancement linked to the rise of

a new 2PA band at higher

energy. Instead an almost addi-

tive behavior is observed and

only broadening of the low-

energy 2PA band (which corre-

sponds to the low-energy, one-

photon-allowed transition) is

obtained, which can be possi-

bly ascribed to through-space

electrostatic interactions be-

tween branches. Hence the

nature of the common D or A

core is of major importance as

Figure 1. Selection of two-photon fluorescent three-branched octupolar compounds reported in the literature

(derived from TCB,^[11a]TPB,^[14]triazine,^[11b]triazinetrione (isocyanurate),^[11c]TPPO,^[13]TPPS,^[13]cyclohexane-

1,3,5-trione (phloroglucinol)^[11d]cores), and the library reported in the present work.

Results and Discussion

long as giant 2PA responses are sought. Interestingly, in sys-

tems in which dipolar branches are assembled through

a common D or A core, a localization of the excitation on

one of the branches is observed prior to emission. As

a result, their fluorescence is closely related to that of the

dipolar chromophoric subunits, thus leading to a polar emis-

sive excited state (and subsequent fluorescence solvato-

chromism).^[12]On the basis of this analysis, we have shown

earlier that elongated octupolar derivatives built from

a TPA core and that bear potent electron-withdrawing

(EW) peripheral moieties could lead to fluorescent probes

that combine a large 2PA response and sensitivity to the po-

larity of their environment.^[8d,10b]Herein, we present our

continuing efforts in the comprehensive studies of structure–

property relationships in a modular series of such deriva-

tives (Figure 1) in which both the conjugated linkers and the

Design

It has been now widely demonstrated that quadrupolar sys-

tems (centrosymmetrical conjugated molecules that bear

two electron-releasing (D) or electron-withdrawing (A) end

groups exhibit larger 2PA cross-section (s₂) values than

push–pull dipolar molecules (D group linked to A group

with a conjugated moiety) in relation to a quadrupolar intra-

molecular charge transfer that takes place between the

edges and the center of the molecules.^[8a,9]Similarly, experi-

mental and theoretical studies revealed that non-centrosym-

metric octupolar systems typically built from the coupling of

dipolar branches through specific common donor (D)^[8d,10]or

acceptor (A) cores^[11]can lead to large 2PA responses. How-

ever, it has to be noted that the nature of the interactions

&

2

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ÝÝ These are not the final page numbers!

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

end groups are varied, focusing in particular on the com-

bined effect of structural variation on 2PA and sensitivity to

environment polarity. Rigid or semirigid conjugated spacers

built from arylene–ethynylene and/or arylene–vinylene olig-

omers have been selected to allow efficient electronic com-

munication between the TPA core and peripheral EW

groups while preserving fluorescence.^[9j]Selected arylenes

are either aromatic (phenylene (P) and fluorenylene (Fl)) or

heteroaromatic with reduced aromaticity (3,4-ethylenedioxy-

thienylene (EDOT)) moieties. This latter heterocyclic

moiety was chosen in light of shifting the emission to the

red region, which is of interest for biological applications. In

pursuit of our earlier efforts, we focused on peripheral moi-

eties from the sulfone family, which show strong EW ability

(with Hammett constants s_plarger than 0.8) and confer suit-

able and tunable solubility to the target octupolar deriva-

tives relative to more classical cyano and nitro moieties.

Electron-withdrawing (EW) groups that show increasing

EW strength were used: from alkylsulfones (SO₂Oct, s_p

ꢁ0.8) to triflyl moiety (SO₂CF₃, s_pꢁ1) and perfluorobutyl-

sulfone (SO₂C₄F₉, s_pꢁ1.1).^[10c,15]In addition, we prepared

derivatives that bear SO₂CH₂CH₂OH peripheral groups to

induce a better solubility of the octupolar molecule in polar

solvents or biological environments and provide the possibil-

ity for further functionalization and grafting onto biomole-

cules.

Scheme 1. Reagents and conditions: a) 1a, 4-iodobenzaldehyde, NaH,

THF, 208C, 15 h (61% of 2a); b) 1b, 4-iodobenzaldehyde, tBuOK,

CH₂Cl₂, 208C, 15 h (52% of 2b); c) 3b, p-vinylbenzaldehyde, K₂CO₃,

Bu₄NCl, PdACHTNURTGNEUNG(OAc)₂, PPh₃, DMF, 1108C, 10 h (26%); and d) methyltriphe-

nylphosphonium iodide (2.4 equiv), tBuOK, CH₂Cl₂, 208C, 4 h (51%).

that bear very powerful electron-withdrawing groups (such

as SO₂CF₃) are tricky substrates and represent a challenge

in this kind of methodology in term of reactivity.

The synthesis of iodinated arm 7 that contained an EDOT

moiety was achieved by Horner–Wadsworth–Emmons con-

densation between aldehyde 6^[17]and phosphonate 1b in the

presence of potassium tert-butoxide. Halogen–metal ex-

change on diiodofluorene 8a,^[9j]followed by quenching of

the monolithio intermediate with DMF, afforded the non-

symmetrical fluorene 8b, which was treated with phospho-

nate 1b to lead to the iodine-bearing extended arm 9 as

a mixture of E/Z stereoisomers (Scheme 2).

Perfluorobutylsulfone 11 building block was prepared ac-

cording to a two-step alkylation/oxidation sequence in very

good yields. Alkylation of p-bromothiophenol 10a with 1-io-

dononafluorobutane led to 10b, which was then oxidized

with hydrogen peroxide in acetic acid to give sulfone 11.

Sulfone 13b, which bears an alkyne group, was obtained

from the corresponding bromo derivative 12^[18]with an ex-

cellent overall yield (91%) by means of a palladium-cata-

lyzed Sonogashira coupling with ethynyltrimethylsilane fol-

Synthesis

We used a modular synthetic strategy to prepare the octupo-

lar fluorophores that consist mainly of threefold coupling

between a TPA core and functionalized building blocks. We

focused on three different electron-releasing precursors,

namely, triiodo-, trivinyl-, and triethynyl triphenylamine.

Considering the need for carrying out three sequential re-

petitive reactions (due to the octupolar symmetry), we

chose to focus on highly efficient methodologies. The elabo-

ration of octupolar compounds with double-bond links was

performed by means of Horner–Wadsworth–Emmons con-

densations or Heck couplings between vinyl and halogenat-

ed aromatic precursors. On the other hand, octupolar com-

pounds that bear triple bonds were prepared by means of

Sonogashira couplings between either the trihalogenated

core and ethynyl arms or triethynyl core and halogenated

derivatives. All new molecules have been fully characterized

by NMR spectroscopy, HRMS, and elemental analysis data.

The extended iodinated building blocks 2a and 2b were

obtained stereoselectively by condensations between p-iodo-

benzaldehyde and the corresponding phosphonates (1a and

1b, respectively) in the presence of a base (sodium hydride

and potassium tert-butoxide, respectively). The elongated

arms 4a and 4b that contain a triflyl electron-withdrawing

group were synthesized from bromo derivative 3b: there-

fore, Heck coupling between 3b^[9j]and p-vinylbenzalde-

hyde^[16]gave aldehyde 4a, which was condensed with meth-

yltriphenylphosphonium iodide to afford vinyl compound

4b (Scheme 1). It should to be noted that these molecules

Scheme 2. Reagents and conditions: a) 1b (1.1 equiv), tBuOK, CH₂Cl₂,

208C, 15 h (43%); b) nBuLi, Et₂O, ꢀ788C to 208C, 30 min, then DMF,

ꢀ788C to 208C, 15 h (27%); and c) 1b (1.1 equiv), tBuOK, CH₂Cl₂,

208C, 15 h (18%). Non=n-nonyl.

3

&

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

These are not the final page numbers! ÞÞ

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

Scheme 3. Reagents and conditions: a) NaH, DMF, 208C, 1 h, then 1-

iodo-nonafluorobutane, DMF, 208C, 2 h (92%); b) H₂O₂, AcOH, reflux,

(PPh₃)₂Cl₂], CuI, Et₃N, toluene, 408C,

3 h (71%); c) HCꢂCSiMe₃, [Pd

ACHTUNGTRENNUNG

16 h (96%); and d) NaOH, THF, 208C, 15 min (95%).

Scheme 4. Reagents and conditions: a) HCꢂCSiMe₃, [Pd

NEt₃, toluene, 408C, 3 h (96%); b) NaOH, THF, 208C, 35 h (87%);

c) 14a (1 equiv), 13b (4 equiv), [Pd(PPh₃)₂Cl₂], CuI, NEt₃, toluene, 408C,

15 h (78% of 15a); d) 14b (1 equiv), 3a or 3b (3.5 equiv), [Pd₂A(dba)₃]

(dba=dibenzylideneacetone), CuI, PPh₃, tetra-n-butylammonium fluo-

ride (TBAF), NEt₃, toluene, 358C, 22 h (65% of 15b, 84% of 15c); and

ACHTNUGTNERN(UGN Ph₃)₂Cl₂], CuI,

lowed by a base-promoted deprotection of the trimethylsilyl

group in the presence of sodium hydroxide (Scheme 3).

The same two-step alkylation/deprotection procedure was

applied to convert the triiodo core 14a^[19]into the triethynyl-

phenylamine derivatives 14b and 14c with excellent yields.

The synthesis of the hydrophilic fluorophore 15a was ach-

ieved with a good yield by means of a triple Sonogashira

coupling of triiodo compound 14a with alkyne 13b. Core

14b was used to prepare fluo-

AHCTUNGTRENNUNG

CHTUNGTRENNUNG

e) 14c (1 equiv), 11 (4 equiv), [Pd

(47% of 15d).

₂ACTHUGNTREN(UNGN dba)₃], CuI, PPh₃, NEt₃, toluene

rophores 15b and 15c by treat-

ment with bromo derivatives

3a and 3b, respectively, in

a two-step, one-pot manner in

the presence of fluoride ions,

palladium(0),

and

copper

iodide. This nice in situ depro-

tection/coupling procedure is

a good way to avoid possible

storage problems of the

not-so-stable 14c core. It is

also a very efficient synthetic

method as demonstrated by oc-

tupole 15c, which was obtained

by threefold Sonogashira reac-

tion with 84% yield, thereby

resulting in an average yield of

94% for each arm. Triple So-

nogashira coupling of core

14c with 11 also led efficiently

to fluorophore 15d with

a yield of 78% per branch

(Scheme 4).

Extended octupoles 16a,

16b, and 17 were analogously

synthesized with excellent

yields by treating trialkynes

14b or 14c with extended iodo

derivatives 2a, 2b, and 7, re-

spectively (Scheme 5). Vinylic

fluorophores with alkylsulfonyl

end groups 19a and 19b were

obtained as pure all-E stereo-

Scheme 5. Reagents and conditions: a) 14b (1 equiv), 2a (3.5 equiv), [Pd

ene, 208C, 65 h (89% of 16a); b) 14c (1 equiv), 2b (4 equiv), [Pd₂A(dba)₃], CuI, PPh₃, NEt₃, toluene, 358C, 15 h

(94% of 16b); and c) 14c (1 equiv), 7 (3.5 equiv), conditions as in (b), 408C, 13 h (84% of 17).

₂ACTHUGNTREN(NNUG dba)₃], CuI, PPh₃, TBAF, NEt₃, tolu-

CTHUNGTRENNUNG

&

4

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ÝÝ These are not the final page numbers!

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

with quite good to very good

yields and with all-E stereo-

chemistry. In the same way, oc-

tupolar fluorophore 20, which

bears an extended fluoreny-

lene–vinylene spacer, was syn-

thesized by coupling between

core 18b and iodinated deriva-

tive 9. Remarkably, it was also

obtained with all-E stereo-

chemistry, whereas starting ma-

terial 9 was a mixture of E/Z

stereoisomers. Two possible

Heck couplings were tested for

the preparation of fluorophore

19d that bears two phenylene–

vinylene moieties. The reaction

between 18b and 2b with the

catalytic system previously

used was disappointing in this

case. Alternatively, the assem-

bly of triodo core 14a and

vinyl arm 4b under Jefferyꢂs

conditions^[21]afforded octupole

19d with a 65% yield and with

all-E stereochemistry as well

(Scheme 6).

Finally, a dissymmetrical de-

rivative 19g that bears two

triflyl

end

groups

and

a

SO₂CH₂CH₂OH extremity

linked to phenylene–vinylene

spacer was prepared by using

the same type of Wittig con-

densation/Heck coupling two-

step sequence (Scheme 7). The

key step of this synthesis is the

dissymmetrization of the tri-

phenylamine core. This was

achieved starting from trialde-

hyde 18a by using only

1.5 equivalents of methyltri-

Scheme 6. Reagents and conditions: a) methyltriphenylphosphonium iodide, NaH, THF, RT, 15 h (99%);

b) 18a (1 equiv), 1a or 5 (3.3 equiv), tBuOK, CH₂Cl₂, 208C, 16 h, then reflux (36% of 19a, 27% of 19b);

c) 18b (1 equiv), 3b or 11 or 12 (3.5–4 equiv), Pd

of 19e, 42% of 19 f); d) 14a (1 equiv), 4b (4.5 equiv), Pd

(65% of 19d); and e) 18b (1 equiv), 9 (4 equiv), conditions as in (c), 1108C, 15 h (18% of 20). Non=n-nonyl.

ACHTUNGTRENNUNG

AHCTUNGTRENNUNG

phenylphosphonium

iodide,

and derivative 18c was ob-

isomers, as testified by ¹H NMR spectroscopy, by a triple

Horner–Wadsworth–Emmons condensation of trialdehyde

core 18a^[20]with phosphonates 1a and 5, respectively, in the

presence of potassium tert-butoxide but with quite low

yields (Scheme 6). For the synthesis of the fluorophores

with triflyl and perfluoroalkylsulfonyl end groups 19c–f and

20, an alternative pathway was thus considered, and they

were prepared by means of triple Heck couplings starting

from either triiodotriphenylamine 14a or the trivinyl core

18b, which resulted from the quantitative condensation be-

tween 18a and methyltriphenylphosphonium iodide. Treat-

ment of compound 18b with brominated derivatives 3b, 11,

and 12 afforded octupoles 19c, 19e, and 19 f, respectively,

tained with an isolated yield of 31%. The desired octupole

19g was then synthesized from the intermediate 18c in

three steps that involved double Heck coupling with bromo

derivative 3b, conversion of the remaining aldehyde into

alkene, and final Heck coupling with bromo derivative 12.

Photophysical Properties

The photophysical data (absorption and fluorescence) of the

synthesized octupolar derivatives built from a core TPA

moiety are collected in Table 1 and include fluorescence

quantum yields, lifetimes, and radiative and nonradiative

decay rates values. All compounds display a broad absorp-

5

&

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

These are not the final page numbers! ÞÞ

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

All octupolar derivatives ex-

hibit intense fluorescence emis-

sion in the visible region when

dissolved in low-polarity sol-

vents (such as toluene) or in

ethanol (for compounds that

bear

groups)

SO₂CH₂CH₂OH

with fluorescence

end

quantum yields that range

from 0.52 to 0.93. Both the ab-

sorption and emission of these

molecules can be tuned by

modifying the strength of the

peripheral electron-withdraw-

ing end groups (Figure 2), the

Scheme 7. Reagents and conditions: a) methyltriphenylphosphonium iodide (1.5 equiv), NaH, THF, RT

nature of the connector (or

linker) between the TPA core

and the end groups (Figures 3

and 4), or the length of the

(31%); b) 18c (1 equiv), 3b (3.5 equiv), Pd

ACHTUNGTRENNUNG

phosphonium iodide, NaH, THF, RT (54%); and d) 21b (1 equiv), 12 (1.3 equiv), Pd

AHCTUNGTRENNUNG

DMF, 1008C (53%).

branches (Figure 3). Their fluo-

tion in the near-UV blue-visible region associated with high

molar extinction coefficients (up to 135000m^ꢀ1cm^ꢀ1). A

higher-energy, less-intense absorption band is observed in

the UV region (300–350 nm).

rescence lifetimes (which range from 1 to 2 ns) and quantum

yields (which range from 0.5 to 0.95) are also found to vary

Figure 2. Absorption and emission spectra of 15b, 15c, and 15d in tolu-

ene: EW effect of end groups.

Figure 3. Absorption and emission spectra of 16b, 19c, and 19d in tolu-

ene: Effects of conjugated linker and length.

Table 1. Photophysical properties of the series of octupolar chromophores and hydrophilic three-branched derivatives (in toluene).

k_r^[c]

k_nr

[10⁹s^ꢀ1

]

abs

max

em

max

[d]

No.

Compound

End group

(X=)

l

loge_max

l

Stokes shift

[cm^ꢀ1

F^[a]

t^[b]

[nm]

N

]

[ns]

G

]

U

15b

15c

15d

16a

16b

17

19a

19c

19e

19b

19d

20

N

SO₂Oct

SO₂CF₃

SO₂C₄F₉

SO₂Oct

SO₂CF₃

SO₂Oct

SO₂CF₃

SO₂C₄F₉

388

405

408

397

408

442

410

430

435

426

441

435

389

415

425

4.91

4.78

4.90

5.13

4.97

5.07

4.90

4.91

4.90

5.10

5.07

–

424

450

455

446

474

523

463

494

499

487

518

498

430

469

498

2100

2470

2510

2770

3390

3500

2790

2990

2950

2940

3380

2910

2450

2750

3500

0.77

0.78

0.90

0.79

0.77

0.52

0.72

0.71

0.81

0.77

0.84

0.93

0.70

0.77

1.27

1.49

1.51

1.35

1.29

1.00

1.66

1.85

1.84

1.23

1.51

1.29

1.25

1.94

1.75

0.62

0.52

0.60

0.59

0.60

0.52

0.43

0.38

0.44

0.63

0.56

0.72

0.56

0.36

0.44

0.18

0.15

0.07

0.20

0.18

0.48

0.17

0.16

0.10

0.19

0.11

0.05

0.24

0.15

0.13

A

SO₂Oct

SO₂CF₃

E

15a

19 f

19g

SO₂CH₂CH₂OH

SO₂CF₃/SO₂CH₂CH₂OH

–

4.83

N

[a] Fluorescence quantum yield determined relative to fluorescein in 0.1m NaOH.^[22][b] Fluorescence lifetime determined using time-correlated single-

photon counting (TCSPC). [c] Radiative decay rate, k_r=F/t. [d] Nonradiative decay rate, k_nr=(1ꢀF)/t.

&

6

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ÝÝ These are not the final page numbers!

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

than their alkylsulfone (SO₂Oct) analogues owing to com-

bined reduction of radiative and nonradiative decay rates

(Table 1).

Effect of the Nature of the Conjugated Linker

As expected and already observed in the case of quadrupo-

lar derivatives,^[9j]replacing a triple bond by a double bond

in the conjugated connector induces a bathochromic shift in

both absorption and emission (Figure 3 and Table 1). Once

again the emission band redshifts are usually larger, thus

leading to larger Stokes shift values, which is indicative of

larger nuclear reorganization in the excited state after exci-

tation prior to emission. In contrast, the replacement of

a triple bond with a double bond in the conjugated linkers

does not significantly affect the one-photon absorptivity

(thus leading to almost unchanged extinction coefficients),

nor does it affect the fluorescence quantum yields, which

are maintained. This results from the combined reduction of

radiative (as expected from redshifted emission)^[23]and non-

radiative decay rates. It is important to note that the triple

bond—although it confers improved rigidity (reducing con-

formational flexibility)—does not result in lower nonradia-

tive rates (as commonly expected from the rigidity/fluores-

cence paradigm). Such an effect is not uncommon^[9j]and

provides evidence of active vibrational modes. The fluores-

cence lifetimes are somewhat longer for derivatives with

double bonds instead of triple bonds in the conjugated

linker, thanks to a slower radiative decay in relation to the

redshifted emission. Interestingly, elongated octupolar fluo-

rophores that bear a triflyl moiety, 16b and 19d, display

a singular behavior; the replacement of the triple bond with

a double bond in the conjugated linker leads to an increase

in the quantum yield value in relation to a marked decrease

in nonradiative decay and lack of decrease in the radiative

decay. In that particular case, the effect of redshifted emis-

sion is compensated by a hyperchromic effect, thereby re-

sulting in the conservation of the radiative rate.^[23]

Figure 4. Absorption and emission spectra of 16b and 17 (top); 19d and

20 (bottom) in toluene: effect of arylene connector.

to some extent depending on the octupolar structures

(Table 1).

Effect of End Groups

Comparisons of the series of octupolar derivatives with

a phenylene–ethylene spacer (15b, 15c, 15d) or with a phen-

ylene–vinylene one (19a, 19c, and 19e) show that increasing

the electron-withdrawing strength of the end groups induces

a bathochromic shift of both the absorption and emission

bands (Figure 2 and Table 1). The magnitude of the batho-

chromic effect is correlated to the EW strength of the end

groups, as expected from a periphery-to-core intramolecular

charge-transfer (ptc-ICT) phenomenon.^[10c]The Stokes shifts

values are also found to increase along with the EW

strength, which is indicative of a larger nuclear reorganiza-

tion in the excited state and is in agreement with more pro-

nounced ptc-ICT (or more precisely larger excited-state

dipole moments; see below). We note, however, that the in-

crease in EW strength does not induce an increase in one-

photon absorptivity (Table 1), which is in contrast to what is

obtained when tuning the length and nature of the connect-

ing branches (see below).

In contrast, changing the nature of the arylene connector

in conjugated linkers can significantly affect not only the po-

sition of the absorption and emission bands but also the

fluorescence quantum yield and lifetime values. As illustrat-

ed in Figure 4, the replacement of a phenyl unit with an

EDOT moiety induces a marked bathochromic shift in both

absorption and emission bands as well as a hyperchromic

effect and reduction in both fluorescence quantum yield and

lifetime values (Table 1). The EDOT unit leads to a larger

nonradiative decay rate (probably in connection with fa-

vored intersystem crossing), whereas the radiative decay

rate is maintained.

Interestingly, fluorescence quantum yield values remain

high even though their emission is redshifted. This phenom-

enon is a consequence of the singular decrease in the nonra-

diative decay rates (Table 1), most probably in relation to

the fluorinated nature of the perfluoroakyl sulfone moieties

(SO₂CF₃and SO₂C₄F₉), which are responsible for a reduction

in the vibrational deactivation processes. As a result, these

derivatives also show slightly longer fluorescence lifetimes

On the contrary, both the absorption and emission bands

are slightly blueshifted when the phenyl unit is replaced

with a fluorenylene moiety (Figure 4), which also induces

a marked hypochromic effect. Interestingly, the nonradiative

decay is significantly slowed (most probably in relation with

decreased vibrational deactivation), whereas the radiative

decay rate significantly increases (Table 1). As a result, the

octupolar fluorophore built from the fluorenylene–vinylene

7

&

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

These are not the final page numbers! ÞÞ

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

conjugated linkers 20 displays the largest fluorescence quan-

tum yield in the series. These observations confirm that fine

tuning of the fluorescence properties can be achieved

through systematic structural variations, which are made

possible on account of the modular approach implemented.

optimize the potential of the investigated octupolar deriva-

tives as (micro)polarity probes. As illustrated in Figure 5 for

molecules 16b and 17, all octupolar derivatives behave

nicely as fluorescent polarity probes: increasing the solvent

Effect of the Length of the Conjugated Linker

Increasing the length of the conjugated linker between the

TPA core and the EW peripheral groups by adding a phenyl-

ene–vinylene (PV) unit induces a bathochromic (and hyper-

chromic effect) shift in both the absorption and emission

bands, as well as a marked spectral broadening of the ab-

sorption and increase in the Stokes shift values (Figure 3).

This is consistent with efficient ptc-ICT, thus indicating that

the linkers are indeed acting as efficient conjugating con-

necting modules. Although a lengthening of the conjugated

linker does not influence the nonradiative decay, the radia-

tive decay undergoes a definite enhancement, most probably

in relation to the marked hyperchromic effect that parallels

an increase in the transition dipole between the emitting ex-

cited state and the ground state. This leads to a slight in-

crease in the fluorescence quantum yields and to significant-

ly shorter fluorescence lifetimes (Table 1).

When increasing the length of the conjugated linker be-

tween the TPA core and the EW peripheral groups by

adding a phenylene–ethynylene (PE) unit instead, a marked

hyperchromic shift and spectral broadening is also observed.

However, a hypsochromic shift in both the absorption and

emission bands is observed (Figure 3) in contrast to the

effect of phenylene–vinylene unit insertion. As a result, the

radiative decay rates significantly increase, thereby lead-

ing—also in that case—to the enhancement of fluorescence

quantum yield (but shorter lifetimes than the elongated

compound with a PV2 linker; Table 1). Hence, increasing

the linker length by inserting PE or PV units indeed allows

one to increase the brightness (e_maxF) of the octupolar fluo-

rescent probes reported in this work. This validates the mo-

lecular engineering strategy implemented here for octupolar

derivatives derived from a TPA core, as also observed for

rodlike quadrupolar derivatives built from ambivalent (i.e.,

weak donor/acceptor) biphenyl, fluorenyl,^[9e,j]and dihydro-

phenanthrene^[8a]cores but with similar connecting units be-

tween the core and terminal electroactive end groups. Inter-

estingly, inserting a fluorenylene–vinylene (Fl–V) unit into

the conjugated linker allows one to bathochromically (and

hyperchromically) shift both the absorption and emission

while increasing the fluorescence quantum yield (Table 1,

comparison of compounds 19c and 20). This confirms that

changing the nature of the arylene connecting unit in the

conjugated linker can be used to both tune the position and

the intensity of the emission band.

Figure 5. Solvatochromic behavior of octupolar fluorophores 17 (top) and

16b (bottom).

polarity induces only a slight bathochromic shift in the ab-

sorption band but produces a marked redshift (and broaden-

ing) in the fluorescence emission band. Accordingly, the

Stokes shift values significantly increase along with an in-

crease in solvent polarity. Such positive solvatochromic be-

havior is consistent with a ptc-ICT phenomenon, thereby

leading after excitation, and prior to emission, to the locali-

zation of the excitation on the dipolar chromophoric branch-

es.^[12]As a result, highly polar emissive excited states are ob-

tained, which are stabilized by polar solvents to result in

redshifted emission. Indeed, the Stokes shift values were

found to depend linearly on the polarity–polarizability pa-

rameter Df (Figure 6) in agreement with the Lippert–

Mataga relationship [Eq. (1)].^[24]

Polarity Sensing

n_absꢀn_em¼ 2Dm²Df=ðhca³Þ þ const

ð1Þ

Solvent effects on the photophysical properties of the library

of octupoles were then investigated to gain better insight

into the nature of the emissive excited state and derive

structure–sensitivity relationships, thereby allowing one to

for which n_abs(n_em) is the wavenumber of the absorption

(fluorescence) maximum, Dm is the change in the dipole

moment between the relaxed emissive excited state and cor-

&

8

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ÝÝ These are not the final page numbers!

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

the sensitivity of the fluorescent probes (Table 2). More pre-

cisely, we observe that increasing the electron-withdrawing

strength of the end groups induces a pronounced amplifica-

tion of the fluorescence sensitivity on the solvent polarity as

illustrated by the comparison of compounds 15b–d or 16a–

b. We also observe that in most cases, compounds that bear

a more rigid spacer (i.e., with a triple bond instead of

a double bond) display larger specific shift values when

comparing related octupoles that contain either short (15c,

15d and 19c, 19e, respectively) or extended conjugated

paths (16b and 19d in Figure 6). Interestingly, a further in-

crease in the specific shift values can be achieved by adding

a phenylene–vinylene unit and consequently lengthening the

conjugated spacer as shown by a comparison of compounds

19a and 19b that bear SO₂Oct end groups. This phenomen-

on is even more pronounced with octupoles 19c and 19d,

which contain stronger electron-withdrawing terminal moi-

eties (Table 2).

Finally, the nature of the p connector in the dipolar

branches is found to significantly influence the polarity sen-

sitivity of the octupolar probes. Indeed, replacing a phenyl

unit with a less aromatic EDOT moiety leads to a pro-

nounced decrease in solvatochromic behavior, as indicated

by comparison of 16b and 17 (Figure 5), whereas the re-

placement with a fluorenyl connector induces a slight in-

crease of the solvatochromic behavior, as indicated by com-

parison of 19d and 20 (Figure 6).

Figure 6. Lippert–Mataga correlations for octupolar compounds 16b,

19d, 17, and 20.

responding Frank–Condon ground state, h is the Planck con-

stant, c is the light velocity, a is the radius of the Onsager

spherical cavity, and Df=(eꢀ1)/

(2e+1)ꢀ

E

G

The slope values derived from the Lippert–Mataga linear

correlations for all octupolar chromophores are collected in

Table 2. These specific shift values allow one to quantify the

sensitivity to solvent polarity and rank the different synthet-

ic derivatives as fluorescent (micro)polarity probes.^[25]We

observed that the specific shift values determined for all oc-

tupolar derivatives are quite high (they vary from 15 to 27ꢃ

10³cm^ꢀ1); the largest ones approach that of the best fluores-

cent polarity probes despite their significant size.^[26]This

confirms the interest in such derivatives for polarity sensing.

In that respect, molecular optimization can be achieved as

the sensitivity to solvent polarity is found to be strongly de-

pendent on structural/electronic features. Indeed, both the

strength of the peripheral EW end groups and the length

and nature of the conjugated linkers are found to influence

From the specific shift values derived from the Lippert–

Mataga relationship, which are directly related to Dm/a³

[Eq. (1)], we could derive Dm values by using estimated

a values calculated by considering the dipolar branches as

emissive dipoles (Table 2).^[27]We observe that the octupolar

derivatives that combine the stronger EW peripheral groups

(SO₂CF₃) as well as the longer branches and more rigid con-

nectors led to the largest Dm values (up to 42 D), thereby

providing evidence of highly polarized emissive excited

Table 2. Solvatochromic data of the series of octupolar chromophores and hydrophilic three-branched derivatives.

No.

Compound

End group

(X=)

Specific solvatochromic shift^[a]

[10³cm^ꢀ1

l^[b]

[ꢄ]

a^[c]

[ꢄ]

Dm^[d]

G

]

[D]

15b

15c

15d

16a

16b

17

19a

19c

19e

19b

19d

20

N

SO₂Oct

SO₂CF₃

SO₂C₄F₉

SO₂Oct

SO₂CF₃

SO₂Oct

SO₂CF₃

SO₂C₄F₉

14.6

19.7

23.4

24.4

27.2

15.6

15.5

15.2

16.8

17.9

21.8

23.5

17.0

18.9

20.0

12.9

19.4

18.9

12.5

17.6

23.0

12.9

12.5

5.2

7.7

7.5

5.0

7.0

9.2

5.2

5.0

14.2

16.5

17.9

33.5

35.4

25.8

13.9

13.8

14.5

24.8

27.4

42.6

15.3

15.4

15.8

G

SO₂Oct

SO₂CF₃

E

15a

19 f

19g

SO₂CH₂CH₂OH

SO₂CF₃/SO₂CH₂CH₂OH

N

CHTUNGTRENNUNG

[a] Absolute value of the slope derived from the linear dependence of the Stokes shift on the polarity/polarizability function of the solvent (Df=

(2n²+1), in which e is the dielectric constant and n the refractive index). [b] Distance from the core N atom to the peripheral S

(eꢀ1)/

(2e+1)ꢀ

E

ACHTUNGTRENNUNG

Condon ground state.

9

&

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

These are not the final page numbers! ÞÞ

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

states as the origin of the large sensitivity toward environ-

ment polarity.

Two-Photon Absorption

Thanks to their strong fluorescence, the 2PA response of all

derivatives in the NIR region could be experimentally deter-

mined by using the well-known two-photon-induced fluores-

cence (2PEF) technique following the methodology devel-

oped and fully documented by Webb and collaborators.^[9b,28]

As illustrated in Figures 7 and 8, all molecules display broad

Figure 8. Two-photon absorption spectra of compounds 16b, 17, 20, and

19d: effect of conjugated spacer.

which is promoted by the efficient electronic coupling be-

tween the branches provided by the TPA core.^[12,27]

Effect of End Groups

A comparison of octupoles that bear different EW groups

shows that increasing the acceptor strength leads to both an

enlargement of the 2PA band in the near-IR region and

a significant enhancement of the s₂values, typically by

a factor of 1.5 to 2.5 for the peak 2PA cross-sections, as il-

lustrated in Figure 7 and observed from Table 3. The intro-

duction of the OH functions as appending moieties provides

solubility in hydrophilic and alcoholic environments, which

in turn leads to a sizeable increase in the 2PA responses

(15b and 19a compared to 15a and 19 f, respectively).

Figure 7. Two-photon absorption spectra of compounds 19a, 19b, and

19e: effect of the (EW) strength of the peripheral groups.

and intense 2PA bands in the 700–1000 nm region. We ob-

serve that the first 2PA maxima occurs at about twice the

wavelength of the low-energy intense one-photon absorption

band, which indicates that the lowest excited state is both

one- and two-photon allowed (as reported earlier for octu-

polar derivatives built from TPA core^[12,27]as well for differ-

ent three-branched derivatives^[13,14]). In addition, much more

intense and broad 2PA bands are observed at higher energy,

Effect of the Conjugated Linker

The comparison of compounds 16b and 19d shows that re-

placing a phenylene–ethynylene unit with a phenylene–vi-

nylene unit results classically in a large redshift of the 2PA

spectrum and a pronounced enhancement of the 2PA peak

cross-sections (Figure 8). Nevertheless, octupolar fluoro-

Table 3. Two-photon absorption properties of the series of octupolar chromophores and three-branched hydrophilic derivatives (in toluene).

abs

max

2PA

max1

2PA

max2

No.

Compound

End group

(X=)

2l

l

s₂at l²_m^P_a^A_x1

l

s₂at l²_m^P_a^A_x2

[nm]

[GM]^[a]

[nm]

[GM]^[a]

15b

15c

15d

16a

16b

17

U

SO₂Oct

SO₂CF₃

SO₂C₄F₉

SO₂Oct

SO₂CF₃

SO₂Oct

SO₂CF₃

SO₂C₄F₉

776

810

816

794

816

884

820

860

870

852

882

870

768

822

850

750

800

825

815

800

890

815

880

885

880

885

750

820

180

370

400

590

790

290

280

440

475

1250

1145

270

360

490

–

ꢄ700

ꢄ995

1085

1090

ꢄ1670

ꢄ995

1340

1425

1510

2070

2080

ꢄ300

800

ꢃ705

715

740

ꢃ710

ꢃ705

740

755

770

19a

19c

19e

19b

19d

20

N

R

E

G

SO₂Oct

SO₂CF₃

E

800

740

15a^[b]

19 f^[b]

19g^[b]

SO₂CH₂CH₂OH

SO₂CF₃/SO₂CH₂CH₂OH

ꢃ700

ACHTUNGTRENNUNG

715

N

G

740

950

[a] 1 GM=10^ꢀ50cm⁴s^ꢀ1photon^ꢀ1. [b] In ethanol.

&

10

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ÝÝ These are not the final page numbers!

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

phores that contain triple bonds have the advantage to be

more photostable and rigid than their analogues with

double bonds. We observe from Figure 8 that increasing the

length of the arms induces a further marked enhancement

of the 2PA properties, which follows the trend observed in

the case of 1PA and the extension of the electronic conjuga-

tion. A pronounced bathochromic shift in the 2PA spectrum

associated with a definite broadening of the 2PA band is ob-

tained, whereas the 2PA cross-section values increase signifi-

cantly in the spectral range of interest for biological applica-

tion (700–1000 nm) as illustrated by octupolar compound

19d; its 2PA cross-section value peaks at 2070 GM (800 nm)

and remains superior to 1000 GM from 750 to 950 nm.

Figure 8 shows that the nature of the connector also plays

an important role. Indeed, when comparing compounds 16b

and 17, we observe that the replacement of a phenylene

moiety by a less aromatic EDOT moiety in the conjugated

arms significantly increases the 2PA cross-sections over the

whole spectral range of interest. Hence, fluorophore 17,

which is also the more redshifted emitter of the series, main-

tains sizeable 2PA cross-sections in the whole 700–950 nm

spectral range.

cal microscope and combined transmission and 2PEF

images were acquired. l-Scan experiments were also per-

formed to determine the emission spectrum of the fluoro-

phores in the cellular environment. We checked after each

l-scan experiment that the laser irradiation did not damage

cells by combined transmission and 2PEF imaging, then

compared the images to the previously acquired one. The

three dyes were found to show good to excellent biocompat-

ibility considering the shape and the number of observable

cells in the transmission images. We observed that cells are

grown to 80% confluency in the presence of compounds

15a and 19 f. These two octupolar derivatives thus allow

high cell viability. The dissymmetrical derivative 19g ap-

pears to have slightly lower biocompatibility, because the

cell adhesion on the cover slip after similar incubation time

lessens. This slightly lower biocompatibility can possibly be

ascribed to the presence of the fluorinated moiety.

2PEF images obtained when exciting at 750 or 780 nm (at

which all fluorophores show significant 2PA responses)

clearly reveal that these novel two-photon fluorescent

probes penetrate into the cell, presumably through internali-

zation or endocytosis mechanisms. When using compound

15a as the two-photon probe, the dyes were mainly found in

the cell cytoplasm and do not enter the nucleus as illustrated

in Figure 9. In all imaging experiments, the probes were ob-

The introduction of a fluorenylene–vinylene in the conju-

gated arms also allows one to attain very high 2PA respons-

es over a wide spectral range, with 2PA cross-sections supe-

rior to 1000 GM from 700 to 900 nm for fluorophore 20

(Figure 8). Octupolar compound 20, which is the biggest de-

rivative in the investigated series, is found to exhibit to the

highest two-photon brightness (s₂F^maxꢁ2000 GM).

Bioapplication: Cellular Imaging

By taking advantage of the demonstrated brightness and po-

larity sensitivity of the series of octupolar fluorescent dyes,

we decided to investigate their potential use as in cellulo flu-

orescent two-photon markers. Toward this aim, we selected

Figure 9. Transmission (left), 2PEF (middle, excited at 750 nm), and

merged images (right) of HEK 293 cells labeled with hydrophilic fluores-

cent probe 15a.

the

two

hydrophilic

chromophores

that

bear

SO₂CH₂CH₂OH peripheral groups, that is, octupolar com-

pounds 15a, 19 f, and one amphiphilic dissymmetrical deriv-

ative (19g) that bear two strong EW peripheral moieties

(SO₂CF₃) and one hydrophilic one (SO₂-CH₂-CH₂-OH).

These derivatives, which combine good fluorescence, signifi-

cant 2PA response in the 700–900 nm region, and marked

polarity sensitivity (with specific shift values higher than

17000 cm^ꢀ1) were thus suitable as two-photon markers and

appeared to be promising probes for cellular imaging. We

stress that, upon going from 15a to 19 f and 19g, the emis-

sion is nicely redshifted (Table 1) and the 2PA responses are

further increased.

These dedicated dyes were then tested in 2PEF micro-

scopic imaging of live cells and sensing of the polarity of cel-

lular microenvironments. In each case, human embryonic

kidney 293 cells (HEK293) were incubated with a 3 mm solu-

tion of the fluorescent dye for 1.5 h, then washed with phos-

phate-buffered saline (PBS) solution, and finally incubated

in a culture-cell medium for one night to allow further cell

growth. The obtained cover slips were analyzed with a confo-

served to display an intense 2PEF response, which demon-

strates that they retain their excellent 2PA and photolumi-

nescence properties in biological conditions. Moreover, the

2PEF images obtained after incubation with the three differ-

ent hydrophilic fluorescent two-photon probes (15a, 19 f,

and 19g), when using the same probe concentration and

laser power for 2P excitation at the same wavelength, show

relative intensities that match the 2P brightness (s₂F) of the

different fluorescent dyes (i.e., 250, 268, and 120 GM at

750 nm in ethanol), which suggests that uptake ratios are

similar for the three fluorescent dyes. The fluorescence in-

tensities were further normalized to allow comparison of the

emission spectra collected by means of l-scan experiments

(Figure 10). The three fluorescent dyes allow monitoring of

cytoplasmic micropolarity in living cells: all three com-

pounds display a broad emission spectrum with a maximum

at 460, 510, and 520 nm for derivatives 15a, 19 f, and 19g,

respectively. A comparison of these spectra and those ob-

11

&

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

These are not the final page numbers! ÞÞ

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

tained in solvents of varying polarity (Figure 10 bottom, see

Conclusion

also the Supporting Information) reveals that the HEK293

cytoplasm behaves like a low-polarity environment. In the

case of amphiphilic derivative 19g, 2PEF images also clearly

reveal brighter spots that suggest that the Y-shaped two-

photon dye tends to accumulate in specific intracellular

compartments, most likely lysosomes (Figure 11).

A series of octupolar fluorophores built from a TPA core

and that bear different EW peripheral groups and conjugat-

ed spacers has been synthesized. Structure–property rela-

tionships were derived from the comprehensive study of the

absorption, photoluminescence, and two-photon absorption

properties of these compounds. The low-energy transition is

correlated to a core-to-periphery intramolecular charge-

transfer phenomenon that leads to intense absorption in the

visible near-UV blue-visible range. In addition, thanks to an

efficient coupling of the branches through the TPA core, all

derivatives show an additional intense 2PA band located at

higher energy than that which corresponds to the lowest ex-

cited state. As a result, all derivatives exhibit significant

2PA responses in the spectral range of interest for biomedi-

cal applications, with two broad bands in the 700–900 nm

region. All derivatives were found to show intense photolu-

minescence in low- to medium-polarity environments (with

fluorescence quantum yields ranging from 0.5 to 0.9)

Absorption, 2PA, and emission can be tuned by playing

on the structural parameters and taking advantage of the

highly modular and flexible synthesis approach. In analogy

with quadrupolar derivatives designed from a comparable

modular approach, increasing the electron-withdrawing

strength of the end groups or the conjugated spacer length

and replacing a triple bond linker with a double bond

induce a redshift of the emission, and both an enhancement

and broadening of the 2PA responses in the 700–1000 nm

spectral range. As a result, a number of octupolar deriva-

tives (i.e., those that combine powerful EW peripheral

groups and with the longest conjugated spacers) exhibit

large 2PA responses (superior to 1000 GM over the whole

700–900 nm range with up to 2100 GM peak 2PA response).

Excitation localization on the dipolar branches occurs

after absorption prior to emission, thereby leading to highly

polar emissive excited states. Photoluminescence of these

octupolar chromophores is thus highly sensitive to the polar-

ity of the environment. All compounds display a strong posi-

tive solvatochromic behavior that fits the Lippert–Mataga

relationship, thereby allowing us to quantify (by means of

the slope or specific shift values) the sensitivity to polarity.

Interestingly, the probe sensitivity was found to depend

strongly on the strength of the peripheral groups and on the

nature of the conjugated spacer, with specific shift values

ranging between 15000 and 27500 cm^ꢀ1(i.e., much higher,

for instance, than that reported for a standard probe such as

Nile red: 1700 cm^ꢀ1). The larger sensitivity is obtained for

elongated derivatives with a triple bond in the conjugated

spacer, whereas replacement of a phenyl moiety with a less

aromatic EDOT (which efficiently redshifts the emission)

results in a marked decrease in sensitivity (thus leading to

a specific shift that is almost twice as small).

Figure 10. Top: emission spectra of fluorophores 15a, 19 f, and 19g in

HEK 293 cytoplasm obtained from l-scan experiments excited at 750 nm.

Bottom: emission spectra of hydrophilic fluorescent probe 15a in various

environments.

Finally, three more hydrophilic derivatives (either octupo-

lar or dissymmetrical) that combine significant lumines-

cence, 2PA in the 700–850 region, and polarity sensitivity

were tested as two-photon markers for cell imaging by

Figure 11. Transmission (left), 2PEF (middle, excited at 750 nm), and

merged images (right) of HEK 293 cells labeled with amphiphilic fluores-

cent probe 19g.

&

12

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ÝÝ These are not the final page numbers!

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

129.2, 128.7, 127.4, 126.7, 119.8 (q, J=325.8 Hz), 94.8 ppm; HRMS (EI):

m/z calcd for C₁₅H₁₀F₃IO₂S: 437.9398 [M⁺·]; found: 437.9374.

means of 2PEF microscopy. These two-photon probes were

shown to be biocompatible and to retain their 2PA and lu-

minescence properties under biological conditions. Indeed,

2PEF images show that these probes enter the cells and

label the cytoplasm. Furthermore, their environment-depen-

dent emission reveals intracellular media of low polarity.

These derivatives open a promising route to the design of

organelle-selective, two-photon probes. Further functionali-

zation with targeting moieties will be studied as well as their

potential use as polarity sensors for (nano)materials.

4-[(1E)-2-{4-[(Trifluoromethyl)sulfonyl]phenyl}ethenyl]benzaldehyde

(4a)

Air was removed from a solution of 3b^[9j](630.4 mg, 2.18 mmol), p-vinyl-

benzaldehyde^[16](432.2 mg, 3.17 mmol), and K₂CO₃(452 mg, 3.27 mmol)

in anhydrous DMF (10 mL) by blowing argon for 30 min. Then nBu₄NCl

(606 mg, 2.18 mmol), PPh₃(57.2 mg, 0.218 mmol), and PdACHTUNGTRENNUNG(OAc)₂

(24.5 mg, 0.109 mmol) were added, and the mixture was stirred at 1108C

for 10 h. The solvent was removed by distillation under vacuum. After

washing with heptane, the crude product was purified by column chroma-

tography (heptane/CH₂Cl₂40:60) to yield 4a (190.5 mg, 26%). M.p. 161–

1628C; ¹H NMR (200.13 MHz, CDCl₃): d=10.04 (s, 1H), 8.04 and 7.80

(AA’XX’, JACTHUNRTGENNUG(A,X)=8.5 Hz, 4H), 7.93 and 7.72 (AA’XX’, JACHTUGNTREN(NUGN A,X)=8.3 Hz,

Experimental Section

4H), 7.40 (d, J=16.4 Hz, 1H), 7.29 ppm (d, J=16.4 Hz, 1H); ¹³C NMR

(75.47 MHz, CDCl₃): d=191.4, 144.8, 141.7, 136.3, 132.9, 131.3, 130.3,

129.8, 129.1, 127.8, 127.6, 119.8 ppm (q, J=325.9 Hz); HRMS (EI): m/z

calcd for C₁₆H₁₁F₃O₃S: 340.0381 [M⁺·]; found: 340.0380; elemental analy-

sis calcd (%) for C₁₆H₁₁F₃O₃S (340.32): C 56.47, H 3.26, S 9.42; found: C

56.68, H 3.48, S 9.42.

Synthetic Procedures

All air- or water-sensitive reactions were carried out under argon. Sol-

vents were generally dried and distilled prior to use. Reactions were

monitored by thin-layer chromatography on Merck silica gel or neutral

aluminum oxide 60 F₂₅₄precoated aluminum sheets. Column chromatog-

raphy: Merck silica gel Si 60 (40–63 mm, 230–400 mesh). Melting points

were determined with an Electrothermal IA9300 digital melting point in-

strument. NMR: Bruker AM 200 (¹H: 200.13 MHz), AM250 (¹H:

250.13 MHz, ¹³C: 62.90 MHz, ³¹P: 101.25 MHz), ARX 200 (¹H:

200.13 MHz, ¹³C: 50.32 MHz), or Avance AV 300 (¹H: 300.13 MHz, ¹³C:

75.48 MHz, ¹⁹F: 282.38 MHz, ³¹P: 121.50 MHz), in CDCl₃solutions; ¹H

chemical shifts (d) are given in ppm relative to TMS as internal standard,

J values in Hz, ¹³C chemical shifts relative to the central peak of CDCl₃

at d=77.0 ppm, ³¹P relative to H₃PO₄as external standard, and ¹⁹F rela-

tive to CFCl₃as internal standard. High- and low-resolution mass spectra

measurements were performed at the Centre Rꢀgional de Mesures Physi-

ques de lꢂOuest (C.R.M.P.O., Rennes) with a Micromass MS/MS ZAB-

Spec TOF instrument with EBE TOF geometry; liquid secondary-ion

mass spectrometry (LSIMS) at 8 kV with Cs⁺in m-nitrobenzyl alcohol

(mNBA); ES⁺(electrospray ionization, positive mode) at 4 kV; electron

ionization (EI) at 70 eV. Elemental analyses were performed at I.C.S.N.–

C.N.R.S. (Gif-sur-Yvette, France) or at the C.R.M.P.O. Phosphonates

1a,b and 5 were prepared analogously to Ref. [29] and Ref. [30], respec-

tively. Compounds 3a,b,^[9j]6,^[17]8a,^[9j]12,^[18]14,^[19]18a,^[20]19 f,^[27]and

19g,^[27]were synthesized according to the respective literature proce-

dures.

1-Ethenyl-4-[(1E)-2-{4-[(trifluoromethyl)sulfonyl]phenyl}ethenyl]benzene

(4b)

tBuOK (70 mg, 624 mmol) was added to a solution of 4a (68.8 mg,

202 mmol) and methyltriphenylphosphonium iodide (162.1 mg, 401 mmol)

in anhydrous CH₂Cl₂(4.5 mL). The mixture was stirred at 208C for 3 h.

After filtration through Celite (CH₂Cl₂), the solvent was evaporated, and

the crude product was purified by column chromatography (heptane/

CH₂Cl₂70:30) to yield of 4b (34.8 mg, 51%). M.p. 138.5–139.58C;

¹H NMR (200.13 MHz, CDCl₃): d=8.00 and 7.74 (AA’XX’, J

8.5 Hz, 4H), 7.53 and 7.44 (AA’XX’, J(A,X)=8.5 Hz, 4H), 7.32 (d, J=

ACHTUNGTRENNUNG(A,X)=

AHCTUNGTRENNUNG

16.3 Hz, 1H), 7.14 (d, J=16.3 Hz, 1H), 6.74 (dd, J=17.6, 10.9 Hz, 1H),

5.81 (dd, J=17.6, 0.7 Hz, 1H), 5.31 ppm (dd, J=10.9, 0.7 Hz, 1H);

¹³C NMR (75.47 MHz, CDCl₃): d=145.8, 138.4, 136.2, 135.3, 134.1, 131.2,

128.9, 127.4, 127.3, 126.8, 125.7, 119.9 (q, J=326.1 Hz), 114.8 ppm;

HRMS (EI): m/z calcd for C₁₇H₁₃F₃O₂S: 338.0588 [M⁺·]; found:

338.0589; elemental analysis calcd (%) for C₁₇H₁₃F₃O₂S (338.35): C 60.35,

H 3.87, S 9.48; found: C 60.53, H 3.82, S 9.49.

2-Iodo-5-[(1E)-2-{4-[(trifluoromethyl)sulfonyl]phenyl}ethenyl]-3,4-

ethylenedioxythiophene (7)

tBuOK (232 mg, 2.072 mmol) was added to a solution of 6^[17](498 mg,

1.382 mmol) and 1b (450 mg, 1.520 mmol) in anhydrous CH₂Cl₂. The re-

action mixture was stirred in the dark at 208C for 15 h. Then the mixture

was filtered through Celite, the solvent was evaporated, and the crude

product was purified by column chromatography (heptane/CH₂Cl₂, gradi-

ent from 80:20 to 60:40) to yield 7 (302 mg, 43%). M.p. 171–1728C;

1-Iodo-4-{(1E)-2-[4-(octylsulfonyl)phenyl]ethenyl}benzene (2a)

NaH (273 mg, 60% dispersion in mineral oil) was added to a solution of

1a (1.702 g, 4.208 mmol) and 4-iodobenzaldehyde (1.057 g, 4.556 mmol)

in anhydrous THF (16 mL). The mixture was stirred at 208C for 15 h.

After the addition of water, the product was extracted with CH₂Cl₂and

was then purified by column chromatography (heptane/CH₂Cl₂30:70) to

yield 2a (1.242 g, 61%). M.p. 158–1598C; ¹H NMR (200.13 MHz,

¹H NMR (200.13 MHz, CDCl₃): d=7.94 and 7.65 (AA’XX’, J

ACHTUNGTRENNUNG(A,X)=

8.6 Hz, 4H), 7.30 (d, J=16.1 Hz, 1H), 6.79 (d, J=16.1 Hz, 1H), 4.32 ppm

(s, 4H); ¹³C NMR (75.47 MHz, CDCl₃): d=145.7, 144.7, 140.0, 131.2,

128.3, 126.8, 123.4, 122.9, 121.0, 119.9 (q, J=326 Hz), 65.2, 64.9,

51.7 ppm; ¹⁹F NMR (282.37 MHz, CDCl₃): d=ꢀ78.47 ppm; HRMS (ES⁺,

MeOH): m/z calcd (%) for C₁₅H₁₀O₄F₃NaIS₂: 524.8915 [M+Na]⁺; found:

524.8922; elemental analysis calcd (%) for C₁₅H₁₀O₄F₃IS₂(525.26): C

35.87, H 2.01, S 12.77; found: C 36.09, H 2.01, S 12.47.

CDCl₃): d=7.88 and 7.66 (AA’XX’, J

(AA’XX’, J

ACHTUNGTRENNUNG

2H), 1.24 (m, 10H), 0.86 ppm (t, J=5.7 Hz, 3H); ¹³C NMR (50.32 MHz,

CDCl₃): d=142.1, 137.8, 137.6, 135.7, 131.2, 128.4, 127.2, 126.9, 94.0, 56.3,

31.5, 29.6, 28.84, 28.77, 22.6, 22.4, 14.0 ppm; HRMS (LSIMS⁺, mNBA):

m/z calcd for C₂₂H₂₈IO₂S: 483.0855 [M+H]⁺; found: 483.0864.

1-Iodo-4-[(1E)-2-{4-[(trifluoromethyl)sulfonyl]phenyl}ethenyl]benzene

7-Iodo-9,9-dioctyl-9H-fluorene-2-carboxaldehyde (8b)

(2b)

A solution of n-butyllithium 2.5m in hexanes (2.19 mL, 5.481 mmol) was

added dropwise to a solution of 8a^[9j](3.5 g, 5.22 mmol) in anhydrous di-

ethyl ether (40 mL) at ꢀ788C. The reaction mixture was stirred at ꢀ788C

for 30 min, then allowed to cool to 208C, and stirred for 30 min. There-

after the mixture was cooled at ꢀ788C and DMF was added (0.64 g,

8.73 mmol), then it was allowed to cool to room temperature for 15 h.

HCl (2n, 25 mL) was added and the organic layer extracted. The aque-

ous layer was washed with diethyl ether (50 mL). The collected organic

layers were dried (Na₂SO₄). After evaporation of the solvents, the crude

product was purified by column chromatography (heptane/CH₂Cl₂, gradi-

tBuOK (48 mg, 428 mmol) was added to

a solution of 1b (100 mg,

278 mmol) and 4-iodobenzaldehyde (99 mg, 427 mmol) in anhydrous

CH₂Cl₂(5 mL). The mixture was stirred at 208C for 15 h. After the addi-

tion of water (10 mL) and extraction with CH₂Cl₂, the crude product was

purified by column chromatography (CH₂Cl₂/heptane 30:70) to yield 2b

(63 mg, 52%). M.p. 142–1438C; ¹H NMR (200.13 MHz, CDCl₃): d=8.00

and 7.74 (AA’XX’, JACHTUNGTRENNUNG(A,X)=8.6 Hz, 4H), 7.73 and 7.29 (AA’XX’, J-

ACHTUNGTRENNUNG(A,X)=8.3 Hz, 4H), 7.25 (d, J=16.4 Hz, 1H), 7.13 ppm (d, J=16.4 Hz,

1H); ¹³C NMR (75.47 MHz, CDCl₃): d=145.3, 138.0, 135.4, 133.3, 131.3,

13

&

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

These are not the final page numbers! ÞÞ

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

1

ent from 75:25 to 65:35) to yield 8b (0.8 g, 27%). H NMR (200.13 MHz,

117.8, 104.8, 93.9, 0.0 ppm; HRMS (LSIMS⁺, mNBA): m/z calcd for

C₃₃H₃₉NSi₃: 533.2390 [M⁺·]; found: 533.2390.

CDCl₃): d=10.06 (s, 1H), 7.83 (m, 3H), 7.70 (m, 2H), 7.51 (d, J=8.4 Hz,

1H), 3.95 (m, 4H), 1.26–1.03 (m, 24H), 0.83 (t, J=7.3 Hz, 6H), 0.56 ppm

(m, 4H); ¹³C NMR (50.32 MHz, CDCl₃): d=191.9, 154.3, 150.8, 146.3,

139.1, 136.2, 135.7, 132.3, 130.3, 123.0, 122.4, 120.0, 94.8, 55.4, 39.9, 31.7,

29.7, 29.3, 29.0, 29.0, 23.6, 22.5, 14.0 ppm; HRMS (ES⁺, MeOH): m/z

calcd for C₃₂H₄₅OINa: 595.2413 [M+Na]⁺; found: 595.2410.

Tris[4-(ethynylphenyl)]amine (14c)

NaOH (1m, 30 mL) was added to a solution of 14b (2.33 g, 4.36 mmol) in

THF (30 mL). The reaction mixture was stirred for 35 h at 208C. The sol-

vent was evaporated, the product was extracted with CH₂Cl₂, and the or-

ganic layer washed with water. The residue was purified by column chro-

matography (CH₂Cl₂/heptane, gradient from 10:90 to 15:85) to yield 14c

(1.20 g, 87%). M.p. 113–1148C (Ref. [32] 110–1128C); ¹H NMR

1-Bromo-4-[(nonafluorobutyl)thio]benzene (10b)

NaH (305 mg, 12.69 mmol) was added to

a solution of 10a (2 g,

(200.13 MHz, CDCl₃): d=7.38 and 7.02 (AA’XX’, JACHTNUTRGNEUNG(A,X)=8.9 Hz, 12H),

10.58 mmol) in anhydrous DMF (2 mL) cooled in an ice water bath. The

mixture was stirred for 1 h at 208C. Then 1-iodononafluorobutane

(4.026 g, 11.64 mmol) was added dropwise, and the solution was stirred

for 2 h at 208C. The solvent was removed by distillation under vacuum.

After adding water, the product was extracted with EtOAc, and the resi-

due was purified by column chromatography (heptane) to yield 10b

(3.95 g, 92%).^{[31] 1}H NMR (200.13 MHz, CDCl₃): d=7.55 and 7.51 ppm

3.05 ppm (s, 3H); ¹³C NMR (50.32 MHz, CDCl₃): d=147.0, 133.3, 123.9,

116.8, 83.4, 76.97 ppm; HRMS (LSIMS⁺, mNBA): m/z calcd for

C₂₄H₁₅N: 317.12045 [M⁺·]; found: 317.1212; elemental analysis calcd (%)

for C₂₄H₁₅N (317.39): C 90.82, H 4.76, N 4.41; found: C 90.39, H 4.93, N

4.28.

Tris[4-({4-[(2-hydroxyethyl)sulfonyl]phenyl}ethynyl)phenyl]amine (15a)

(AA’XX’, JACHTUNGTRENNUNG(A,X)=8.8 Hz, 4H).

Air was removed from a solution of 14a (182 mg, 0.288 mmol) and 13b

1-Bromo-4-[(nonafluorobutyl)sulfonyl]benzene (11)

(243 mg, 1.152 mmol) in toluene/Et₃N (5:1; 9 mL) by blowing argon for

20 min. Then CuI (3.3 mg, 17.3 mmol) and [PdACTHNUGTRNEG(UN PPh₃)₂Cl₂] (12.1 mg,

A solution of 10b (2 g, 4.913 mmol) in glacial acetic acid (80 mL) and

35% aqueous H₂O₂(27 mL) was heated to reflux for 3 h. After cooling,

the product was extracted with CH₂Cl₂, and the organic layers were

washed first with a saturated solution of NaHCO₃(three times) then with

water (three times). After evaporation of solvent, the residue was puri-

fied by column chromatography (heptane) to yield 11 (1.531 g, 71%).

M.p. 57–588C (Ref. [31], 568C); ¹H NMR (200.13 MHz, CDCl₃): d=7.90

17.3 mmol) were added. The reaction mixture was stirred at 408C for

15 h. The solvents were removed, and the crude product was purified by

column chromatography (CH₂Cl₂/EtOH 95:5) to yield 15a (251 mg,

78%). M.p. 1468C; ¹H NMR (200.13 MHz, CDCl₃): d=7.91 and 7.70

(AA’XX’,

JACTHNGUTERNNU(G A,X)=8.4 Hz, 12H), 7.48 and 7.11 (AA’XX’, JACHTUNGTRENNUNG(A,X)=

8.6 Hz, 12H), 4.03 (m, 6H), 3.38 (m, 6H), 2.75 ppm (s, 3H); ¹³C NMR

(75.47 MHz, CDCl₃): d=147.1, 137.8, 133.2, 132.2, 129.6, 128.0, 124.1,

117.1, 93.6, 87.7, 58.3, 56.3 ppm; HRMS (ES⁺, MeOH): m/z calcd for

C₄₈H₃₉NO₉NaS₃: 892.1685 [M+Na]⁺; found: 892.1675.

and 7.84 ppm (AA’XX’, JACTHNUGRTNEUNG(A,X)=8.9 Hz, 4H).

2-({4-[(Trimethylsilyl)ethynyl]phenyl}sulfonyl)ethanol (13a)

Tris(4-{[4-(octylsulfonyl)phenyl]ethynyl}phenyl)amine (15b)

Air was removed from a solution of 12^[18](1.0053 g, 3.79 mmol) in tolu-

Air was removed from a solution of 14b (55.5 mg, 104 mmol) and 3a^[9j]

(125 mg, 375 mmol) in toluene/Et₃N (5:1, 6 mL) by blowing argon for

ene/Et₃N (5:1, 10 mL) by blowing argon for 20 min. Then CuI (14.4 mg,

75.8 mmol), [PdACHTUNGTRENNUNG(PPh₃)₂Cl₂] (53.2 mg, 75.8 mmol), and ethynyltrimethylsi-

lane (810 mL, 5.688 mmol) were added. The mixture was heated for 16 h

at 408C. The solvents were evaporated, and the residue was purified by

column chromatography (CH₂Cl₂/EtOAc 90:10) to yield 13a (1.024 g,

96%). M.p. 93–948C; ¹H NMR (200.13 MHz, CDCl₃): d=7.86 and 7.65

20 min. Then PPh₃(6.6 mg, 25.2 mmol), [Pd₂ACTHNUGTRNENUG(dba)₃] (2.9 mg, 3.2 mmol),

CuI (1.7 mg, 8.9 mmol), and tetrabutylammonium fluoride (1m solution in

THF, 312 mL) were added. The reaction mixture was heated at 358C for

22 h. The solvent was removed, and the crude product was purified by

column chromatography (heptane/CH₂Cl₂, gradient from 50:50 to 40:60)

to yield 15b (73 mg, 65%). M.p. 72–748C; ¹H NMR (200.13 MHz,

(AA’XX’, JACHTUNGTRENNUNG(A,X)=8.4 Hz, 4H), 3.99 (m, 2H), 3.34 (t, J=5.3 Hz, 2H),

2.66 (t, J=6.5 Hz, 1H), 0.27 ppm (s, 9H); ¹³C NMR (75.47 MHz, CDCl₃):

d=138.1, 132.6, 129.3, 128.8, 102.7, 99.6, 58.2, 56.2, 0.0 ppm; HRMS (EI):

m/z calcd for C₁₃H₁₈O₃SiS: 282.07459 [M⁺·]; found: 282.0746; elemental

analysis calcd (%) for C₁₃H₁₈O₃SiS (282.44): C 55.28, H 6.42, S 11.35;

found: C 55.34, H 6.35, S 11.25.

CDCl₃): d=7.88 and 7.68 (AA’XX’, J

ACHTUNGTRNE(NUNG A,X)=8.4 Hz, 12H), 7.48 and 7.12

(AA’XX’, J(A,X)=8.6 Hz, 12H), 3.10 (m, 6H), 1.71 (m, 6H), 1.35 (m,

AHCTUNGTRENNUNG

6H), 1.24 (m, 24H), 0.87 ppm (t, J=6.3 Hz, 9H); ¹³C NMR (50.32 MHz,

CDCl₃): d=147.1, 138.1, 133.1, 132.0, 129.1, 128.1, 124.1, 117.2, 93.2, 87.8,

56.3, 31.6, 28.9, 28.8, 28.2, 22.6, 22.5, 14.0 ppm; HRMS (LSIMS⁺,

mNBA): m/z calcd for C₆₆H₇₅NO₆S₃: 1073.4756 [M⁺·]; found: 1073.4765;

elemental analysis calcd (%) for C₆₆H₇₅NO₆S₃(1074.50): C 73.77, H 7.04,

N 1.30, S 8.93; found: C 73.31, H 7.21, N 1.66, S 8.65.

2-[(4-Ethynylphenyl)sulfonyl]ethanol (13b)

NaOH (1m, 9.5 mL, 9.5 mmol) was added to a solution of 13a (1.004 g,

3.55 mmol) in THF (19 mL). The mixture was stirred for 15 min at 208C.

THF was removed, then the product was extracted by CH₂Cl₂, and the

residue was purified by column chromatography (CH₂Cl₂/EtOAc 90:10)

to yield 13b (709 mg, 95%). M.p. 73–748C; ¹H NMR (200.13 MHz,

Tris[4-({4-[(trifluoromethyl)sulfonyl]phenyl}ethynyl)phenyl]amine (15c)

Air was removed from a solution of 14b (90.6 mg, 170 mmol) and 3b^[9j]

(180.8 mg, 625 mmol) in toluene/Et₃N (5:1, 6 mL) by blowing argon for

20 min. Then PPh₃(12.6 mg, 48 mmol), [Pd₂ACTHNUTRGNEUNG(dba)₃] (5.9 mg, 6.4 mmol),

CuI (2.0 mg, 10.5 mmol), and tetrabutylammonium fluoride (1m solution

in THF, 510 mL) were added. The reaction mixture was heated at 358C

for 22 h. The solvent was evaporated, and the crude product was purified

by column chromatography (heptane/CH₂Cl₂50:50) to yield 15c

(134.4 mg, 84%). M.p. 255–2578C; ¹H NMR (200.13 MHz, CDCl₃): d=

CDCl₃): d=7.90 and 7.69 (AA’XX’, JACTHNUGRTNEUNG(A,X)=8.6 Hz, 4H), 4.02 (m, 2H),

3.36 (t, J=5.3 Hz, 2H), 3.31 (s, 1H), 2.65 ppm (t, J=6.4 Hz, 1H);

¹³C NMR (50.32 MHz, CDCl₃): d=138.7, 132.9, 128.2, 127.9, 81.6, 81.6,

58.2, 56.2 ppm; HRMS (EI): m/z calcd for C₁₀H₁₀O₃S: 210.03507 [M⁺·];

found: 210.0360; elemental analysis calcd (%) for C₁₀H₁₀O₃S (210.25): C

57.13, H 4.79, S 15.25; found: C 57.21, H 4.86, S 15.66.

8.02 and 7.76 (AA’XX’,

(AA’XX’, J

(A,X)=8.6 Hz, 12H); ¹³C NMR (50.32 MHz, CDCl₃): d=

147.3, 133.4, 132.4, 132.3, 130.7, 129.7 (d, J=1.6 Hz), 124.2, 119.7 (q, J=

JACTHNGUTER(NNUG A,X)=8.3 Hz, 12H), 7.50 and 7.14 ppm

Tris{4-[(trimethylsilyl)ethynyl]phenyl}amine (14b)

AHCTUNGTRENNUNG

Air was removed from a solution of 14a^[19](1.0 g, 1.605 mmol) in tolu-

ene/Et₃N (5:1, 8 mL) by blowing argon for 20 min. Then CuI (37 mg,

0.194 mmol), [PdACHTUNGTRENNUNG(PPh₃)₂Cl₂] (68 mg, 0.097 mmol), and ethynyltrimethylsi-

lane (1.15 mL, 8.14 mmol) were added. The reaction mixture was heated

at 408C for 3 h. The solvent was evaporated under vacuum, and the

crude product was purified by column chromatography (heptane/CH₂Cl₂

80:20) to yield 14b (820 mg, 96%). M.p. 172–1758C; ¹H NMR

19

325.9 Hz), 116.9, 95.6, 87.5 ppm;

F NMR (282.38 MHz, CDCl₃): d=

ꢀ78.23 ppm; HRMS (LSIMS⁺, mNBA): m/z calcd for C₄₅H₂₄F₉NO₆S₃:

941.0622 [M⁺·]; found: 941.0618; elemental analysis calcd (%) for

C₄₅H₂₄F₉NO₆S₃(941.85): C 57.39, H 2.57, N 1.49, S 10.21; found: C 57.19,

H 2.78, N 1.39, S 10.22.

(200.13 MHz, CDCl₃): d=7.34 and 6.95 (AA’XX’, JACHTNUTRGNEUNG(A,X)=8.5 Hz, 12H),

0.24 ppm (s, 27H); ¹³C NMR (50.32 MHz, CDCl₃): d=146.8, 133.1, 123.8,

14

Chem. Asian J. 2013, 00, 0 – 0

&

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ÝÝ These are not the final page numbers!

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

Tris[4-({4-[(nonafluorobutyl)sulfonyl]phenyl}ethynyl)phenyl]amine (15d)

Tris(4-ethenylphenyl)amine (18b)

Air was removed from a solution of 14c (41.7 mg, 0.131 mmol) and 11

(230.8 mg, 0.525 mmol) in toluene/Et₃N (5:1, 7.5 mL) by blowing argon

for 20 min. Then PPh₃(10.3 mg, 39 mmol), CuI (1.5 mg, 7.88 mmol), and

Sodium hydride (0.80 g, 33.398 mmol) was added to a solution of 18a^[20]

(1.1 g, 3.339 mmol) and methyltriphenylphosphonium iodide (10.79 g,

26.72 mmol) in anhydrous THF (25 mL). The reaction mixture was

stirred for 15 h at 208C. After filtration through Celite, the crude product

was purified by column chromatography (CH₂Cl₂) to yield 18b (1.07 g,

99%). M.p. 81–828C; ¹H NMR (200.13 MHz, CDCl₃): d=7.36 and 7.11

[Pd₂ACHTUNGTRENNUNG(dba)₃] (3.6 mg, 3.94 mmol) were added. The reaction mixture was

heated at 358C for 20 h. After evaporation of solvents, the residue was

purified by column chromatography (heptane/CH₂Cl₂50:50) to yield 15d

(85 mg, 47%). M.p. 174–1758C; ¹H NMR (300.13 MHz, CDCl₃): d=8.00

(AA’XX’, JACTHNUTRGENUG(N A,X)=8.6 Hz, 12H), 6.73 (dd, J=17.6, 10.9 Hz, 3H), 5.72

and 7.75 (AA’XX’, J

A

(d, J=17.6 Hz, 3H), 5.23 ppm (d, J=10.9 Hz, 3H); ¹³C NMR

(75.47 MHz, CDCl₃): d=147.0, 136.2, 132.4, 127.1, 124.1, 112.4 ppm;

HRMS (EI): m/z calcd for C₂₄H₂₁N: 323.1659 [M⁺·]; found: 323.1659.

JACHTUNGTRENNUNG

6F); HRMS

(ES⁺, MeOH/CH₂Cl₂95:5):

m/z calcd

for

Tris(4-{(1E)-2-[4-(octylsulfonyl)phenyl]ethenyl}phenyl)amine (19a)

C₅₄H₂₄NO₆F₂₇NaS₃: 1414.0232 [M+Na]⁺; found: 1414.0199.

tBuOK (151.5 mg, 1.35 mmol) was added to a solution of tris(4-formyl-

phenyl)amine^[20](18a) (98.8 mg, 0.3 mmol) and 1a (400.5 mg, 0.99 mmol)

in anhydrous CH₂Cl₂(10 mL). The mixture was stirred at 208C for 16 h,

then heated to reflux for 2 h, and the solvent was removed under reduced

pressure. After the addition of water, extraction with CH₂Cl₂, and drying

(Na₂SO₄), the solvent was evaporated. The crude product was purified by

column chromatography (heptane/CH₂Cl₂, 20:80) to yield 19a (116 mg,

36%). M.p. 888C; ¹H NMR (200.13 MHz, CDCl₃): d=7.87 and 7.65

Tris{4-[(4-{(1E)-2-[4-

(octylsulfonyl)phenyl]ethenyl}phenyl)ethynyl]phenyl}amine (16a)

Air was removed from a solution of 14b (100.1 mg, 187 mmol) and 2a

(321.6 mg, 656 mmol) in toluene/Et₃N (5:1, 6 mL) by blowing argon for

20 min. Then PPh₃(14.6 mg, 45 mmol), [Pd₂ACTHNUTRGNEUNG(dba)₃] (5.2 mg, 5.6 mmol),

CuI (2.1 mg, 11.2 mmol), and tetrabutylammonium fluoride (1m solution

in THF, 562 mL) were added. The reaction mixture was stirred at 208C

for 65 h. The solvents were removed, and the residue was purified by

column chromatography (heptane/CH₂Cl₂, gradient from 30:70 to 0:100)

to yield 16a (230 mg, 89%). M.p. 118–1208C; ¹H NMR (200.13 MHz,

(AA’XX’,

JACTHNGUTERNNU(G A,X)=8.5 Hz, 12H), 7.47 and 7.14 (AA’XX’, JACHTUNGTRENNUNG(A,X)=

8.7 Hz, 12H), 7.23 (d, J=16.0 Hz, 3H), 7.05 (d, J=16.0 Hz, 3H), 3.09 (m,

6H), 1.73 (m, 6H), 1.24 (m, 30H), 0.86 ppm (t, J=6.5 Hz, 9H); ¹³C NMR

(50.32 MHz, CDCl₃): d=147.1, 142.8, 137.1, 132.1, 131.5, 128.5, 128.0,

126.7, 125.4, 124.3, 56.3, 31.6, 28.9, 28.8, 28.2, 22.6, 22.5, 14.0 ppm; HRMS

(LSIMS⁺, mNBA): m/z calcd for C₆₆H₈₁NO₆S₃: 1079.5226 [M⁺·]; found:

1079.5245; elemental analysis calcd (%) for C₆₆H₈₁NO₆S₃(1080.57): C

73.36, H 7.56, N 1.30, S 8.90; found: C 73.08, H 7.49, N 1.15, S 9.08.

CDCl₃): d=7.89 and 7.68 (AA’XX’, J

7.46 and 7.10 (AA’XX’, J(A,X)=8.7 Hz, 12H), 7.26 (d, J=16.4 Hz, 3H),

ACHTUNGTRNE(NUNG A,X)=8.5 Hz, 12H), 7.54 (s, 12H),

AHCTUNGTRENNUNG

7.15 (d, J=16.4 Hz, 3H), 3.10 (m, 6H), 1.73 (m, 6H), 1.24 (m, 30H),

0.86 ppm (t, J=6.5 Hz, 9H); ¹³C NMR (50.32 MHz, CDCl₃): d=146.7,

142.4, 137.6, 136.0, 132.8, 131.9, 131.7, 128.6, 127.3, 127.0, 126.9, 124.0,

123.5, 117.8, 90.8, 89.3, 56.4, 31.6, 28.94, 28.87, 28.3, 22.7, 22.5, 14.0 ppm;

HRMS (LSIMS⁺, mNBA): m/z calcd for C₉₀H₉₃NO₆S₃: 1379.6165 [M⁺·];

found: 1379.6172; elemental analysis calcd (%) for C₉₀H₉₃NO₆S₃

(1380.91): C 78.28, H 6.79, N 1.01; found: C 78.35, H 6.83, N 0.96.

Tris{4-[(1E)-2-(4-{(1E)-2-[4-

(octylsulfonyl)phenyl]ethenyl}phenyl)ethenyl]phenyl}amine (19b)

tBuOK (70.7 mg, 0.63 mmol) was added to a solution of tris(4-formylphe-

nyl)amine^[20](18a) (46.1 mg, 0.14 mmol) and 5 (234 mg, 0.462 mmol) in

anhydrous CH₂Cl₂(8 mL). The mixture was stirred at 208C for 16 h, then

heated to reflux for 4 h, and the solvent was removed under reduced

pressure. After the addition of water, extraction with CH₂Cl₂, and drying

(Na₂SO₄), the solvent was evaporated. The crude product was purified by

column chromatography (heptane/CH₂Cl₂, 40:60) and washed with pen-

Tris[4-({4-[(1E)-2-{4-

[(trifluoromethyl)sulfonyl]phenyl}ethenyl]phenyl}ethynyl)phenyl]amine

(16b)

Air was removed from a solution of 14c (72.4 mg, 0.228 mmol) and 2b

(328 mg, 0.748 mmol) in toluene/Et₃N (5:1, 13 mL). Then PPh₃(17.9 mg,

68.4 mmol), CuI (2.6 mg, 13.7 mmol), and [Pd₂ACTHNUTRGNEUNG(dba)₃] (6.3 mg, 6.8 mmol)

were added. The reaction mixture was heated at 358C for 15 h. After re-

moving solvents, the residue was purified by column chromatography

(heptane/CH₂Cl₂, gradient from 50:50 to 25:75) to yield 16b (267 mg,

1

tane to yield 19b (52 mg, 27%). M.p. 127–1298C; H NMR (200.13 MHz,

CDCl₃): d=7.87 and 7.66 (AA’XX’, J

ACHTUNGTRNE(NUNG A,X)=8.4 Hz, 12H), 7.52 (s, 12H),

7.44 and 7.12 (AA’XX’, J(A,X)=8.7 Hz, 12H), 7.25 (d, J=16.3 Hz, 3H),

AHCTUNGTRENNUNG

7.20 (d, J=16.0 Hz, 3H), 7.06 (d, J=16.0 Hz, 3H), 7.02 (d, J=16.3 Hz,

3H), 3.09 (m, 6H), 1.72 (m, 6H), 1.23 (m, 30H), 0.86 ppm (t, J=6.3 Hz,

9H); ¹³C NMR (50.32 MHz, CDCl₃): d=146.7, 142.7, 137.9, 137.3, 135.4,

132.1, 128.5, 127.6, 127.3, 126.9, 126.8, 126.2, 124.3, 56.4, 31.6, 28.93,

28.86, 28.2, 22.7, 22.5, 14.0 ppm; HRMS (ES⁺, CH₂Cl₂/MeOH, 20:80): m/

z calcd for C₉₀H₉₉NO₆NaS₃: 1408.6532 [M+Na]⁺; found: 1408.6560.

1

94%). M.p. 139–1418C; H NMR (200.13 MHz, CDCl₃): d=8.01 and 7.76

(AA’XX’, J

ACHTUNGTRENNUNG(A,X)=8.4 Hz, 12H), 7.55 (s, 12H), 7.46 and 7.10 (AA’XX’,

JACHTUNGTRENNUNG(A,X)=8.5 Hz, 12H), 7.33 (d, J=16.3 Hz, 3H), 7.16 ppm (d, J=

16.3 Hz, 3H); ¹³C NMR (75.47 MHz, CDCl₃): d=146.8, 145.5, 135.6,

133.6, 132.9, 132.0, 131.3, 129.2, 127.4, 127.1, 126.6, 124.1, 124.0, 119.8 (q,

J=326.2 Hz), 117.8, 91.1, 89.3 ppm; ¹⁹F NMR (282.35 MHz, CDCl₃): d=

ꢀ78.39 ppm; HRMS (LSIMS⁺, mNBA): m/z calcd for C₆₉H₄₂F₉NO₆S₃:

1247.2031 [M⁺·]; found: 1247.1990; elemental analysis calcd (%) for

C₆₉H₄₂F₉NO₆S₃(1248.29): C 66.39, H 3.39, N 1.12, S 7.71; found: C 66.09,

H 3.46, N 0.75, S 7.77.

Tris{4-[(1E)-2-{4-[(trifluoromethyl)sulfonyl]phenyl}ethenyl]phenyl}amine

(19c)

Air was removed from a solution of 18b (60 mg, 185 mmol) and 3b^[9j]

(187 mg, 649 mmol) in anhydrous DMF (1 mL) and Et₃N (0.1 mL,

742 mmol) by blowing argon for 20 min. Then (o-tol)₃P (17 mg, 55 mmol)

Tris[4-({5-[(1E)-2-{4-[(trifluoromethyl)sulfonyl]phenyl}ethenyl]-3,4-

ethylenedioxythien-2-yl}ethynyl)phenyl]amine (17)

and PdACTHUNTGRNE(UGN OAc)₂(7 mg, 28 mmol) were added. The reaction mixture was

heated for 3 h at 1008C. After evaporation of the solvents, the crude

product was purified by column chromatography (heptane/CH₂Cl₂50:50)

to yield 19c (133 mg, 75%). M.p. 135–1368C; ¹H NMR (300.13 MHz,

The reaction of 14c (22 mg, 69 mmol) with 7 (121 mg, 241 mmol) was as

described for 16b, for 13 h at 408C, with subsequent purification by

column chromatography (heptane/CH₂Cl₂, gradient from 30:70 to 0:100)

to afford 17 (84 mg, 84%). M.p. 120–1218C; ¹H NMR (300.13 MHz,

CDCl₃): d=8.00 and 7.74 (AA’XX’, J

ACHTUNGTRNE(NUNG A,X)=8.5 Hz, 12H), 7.52 and 7.17

(AA’XX’, J(A,X)=8.5 Hz, 12H), 7.33 (d, J=16.5 Hz, 3H), 7.09 ppm (d,

AHCTUNGTRENNUNG

J=16.5 Hz, 3H); ¹³C NMR (75.47 MHz, CDCl₃): d=147.5, 145.8, 133.6,

131.2, 128.7, 128.4, 127.1, 127.1, 124.9, 124.5, 119.9 ppm (q, J=325.9 Hz);

¹⁹F NMR (282.37 MHz, CDCl₃): d=ꢀ78.42 ppm; HRMS (LSIMS⁺,

mNBA): m/z calcd for C₄₅F₉H₃₀NO₆S₃: 947.1092 [M⁺·]; found: 947.1105;

elemental analysis calcd (%) for C₄₅F₉H₃₀NO₆S₃(947.93): C 57.02, H

3.19, N 1.48; found: C 56.63, H 3.47, N 1.31.

CDCl₃): d=7.95 and 7.66 (AA’XX’, J

ACHTUNGTRNE(NUNG A,X)=8.4 Hz, 12H), 7.42 and 7.05

(AA’XX’, J(A,X)=8.5 Hz, 12H), 7.34 (d, J=16.1 Hz, 3H), 6.88 (d, J=

ACHTUNGTRENNUNG

16.1 Hz, 3H), 4.34 ppm (s, 12H); ¹³C NMR (75.47 MHz, CDCl₃): d=

146.7, 145.7, 144.2, 140.4, 132.7, 131.2, 129.0, 128.3, 126.9, 124.0, 123.0,

122.0, 119.9 (q, J=325.9 Hz), 117.5, 116.8, 98.3, 80.1, 65.1, 64.8 ppm;

¹⁹F NMR (282.37 MHz, CDCl₃): d=ꢀ78.34 ppm; HRMS (LSIMS,

mNBA): m/z calcd for C₆₉H₄₂NO₁₂F₉S₆: 1439.08876 [M⁺·]; found:

1439.0885.

15

&

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

These are not the final page numbers! ÞÞ

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

Tris{4-[(1E)-2-(4-{(1E)-2-[4-

(trifluoromethylsulfonyl)phenyl]ethenyl}phenyl)ethenyl]phenyl}amine

(19d)

tive reconvolution methods implemented in the software of the fluorime-

ter. All compounds displayed strictly monoexponential fluorescence

decays (c²<1.1). The reported lifetimes are within ꢅ0.1 ns.

Air was removed from

a

solution of 14a (20.3 mg, 32.6 mmol), 4b

Two-Photon Absorption Experiments

(50.2 mg, 148 mmol), and K₂CO₃(24.5 mg, 177 mmol) in anhydrous DMF

(1.5 mL) by blowing argon for 20 min. Then nBu₄NCl (41.1 mg,

148 mmol), PPh₃(2.6 mg, 9.9 mmol), and PdACTHNUTRGNEUNG(OAc)₂(1.1 mg, 4.9 mmol)

were added. The reaction mixture was heated at 908C for 10 h. After

elimination of solvent, the crude product was purified by column chroma-

tography (heptane/CH₂Cl₂50:50) to yield 19d (26.5 mg, 65%). M.p. 199–

2018C; ¹H NMR (200.13 MHz, CDCl₃): d=8.00 and 7.75 (AA’XX’, J-

To span the 700–980 nm range, a Nd:YLF-pumped Ti:sapphire oscillator

was used to generate 150 fs pulses at a 76 MHz rate. The excitation was

focused into the cuvette through a microscope objective (10ꢃ/NA 0.25).

The applied average laser power that arrived at the sample was between

0.5 and 15 mW, thereby leading to a time-averaged light flux in the focal

volume on the order of 0.1–1 mWmm^ꢀ2. The generated fluorescence was

collected in epifluorescence mode, through the microscope objective, and

reflected by a dichroic mirror (675dcxru, Chroma Technology Corpora-

tion, USA). This makes it possible to avoid the inner filter effects related

to the high dye concentrations used (10^ꢀ4m) by focusing the laser near

the cuvette window. Residual excitation light was removed using a barrier

filter (e650-2p, Chroma), and the fluorescence was coupled into a 600 mm

multimode fiber by an achromatic doublet. The fiber was connected to

a compact CCD-based spectrometer (BTC112-E, B&WTek, USA), which

measured the two-photon excited emission spectrum. Total fluorescence

intensities were obtained by integrating the corrected emission spectra

measured by this spectrometer. 2PA cross-sections (s₂) were determined

from the two-photon excited fluorescence (2PEF) cross-sections (s₂F)

and the fluorescence emission quantum yield (F). 2PEF cross-sections of

10^ꢀ4m toluene solutions were measured relative to fluorescein in 0.01m

aqueous NaOH for 715–980 nm^[9b,28]by using the well-established method

described by Xu and Webb^[28]and the appropriate solvent-related refrac-

tive index corrections.^[34]The experimental uncertainty on the absolute

action cross-sections determined by this method has been estimated to be

ꢅ20%.^[28]The quadratic dependence of the fluorescence intensity on the

excitation power was checked for each sample and all wavelengths (see

examples of quadratic dependences in the Supporting Information), thus

indicating that the measurements were carried out in intensity regimes in

which saturation or photodegradation did not occur.

ACHTUNGTRENNUNG(A,X)=8.5 Hz, 12H), 7.55 (s, 12H), 7.45 (d, J=8.7 Hz, 6H), 7.34 (d, J=

16.3 Hz, 3H), 7.19–7.11 (m, 12H), 7.03 ppm (d, J=16.3 Hz, 3H);

¹⁹F NMR (282.38 MHz, CDCl₃): d=ꢀ78.44 ppm; HRMS (LSIMS⁺,

mNBA): m/z calcd for C₆₉H₄₈F₉NO₆S₃: 1253.2500 [M⁺·]; found:

1253.2522; elemental analysis calcd (%) for C₆₉H₄₈F₉NO₆S₃(1254.33): C

66.07, H 3.86, N 1.12, S 7.67; found: C 65.92, H 3.98, N 1.22, S 7.54.

Tris{4-[(1E)-2-{4-[(nonafluorobutyl)sulfonyl]phenyl}ethenyl]phenyl}amine

(19e)

The reaction of 18b (41.7 mg, 0.129 mmol) with 11 (226.5 mg,

0.516 mmol) was as described for 19c, with subsequent purification by

column chromatography (heptane/CH₂Cl₂60:40), to afford 19e (100 mg,

1

55%). M.p. 137–1388C; H NMR (200.13 MHz, CDCl₃): d=7.99 and 7.74

(AA’XX’,

JACHTUNGTRENNUNG(A,X)=8.2 Hz, 12H), 7.51 and 7.17 (AA’XX’, JACTHUNGTRENUN(NG A,X)=

8.6 Hz, 12H), 7.32 (d, J=16.2 Hz, 3H), 7.08 ppm (d, J=16.2 Hz, 3H);

¹⁹F NMR (282.37 MHz, CDCl₃): d=ꢀ80.63 (m, 9F), ꢀ110.66 (t, J=

13.8 Hz, 6F), ꢀ120.79 (m, 6F), ꢀ125.8985 ppm (m, 6F); HRMS (ES⁺,

MeOH/CH₂Cl₂, 95:5): m/z calcd for C₅₄H₃₀NO₆F₂₇KS₃: 1436.0441

[M+K]⁺; found: 1436.0479.

Tris{4-[(1E)-2-{9,9-dinonyl-7-[(1E)-2-{4-

[(trifluoromethyl)sulfonyl]phenyl}ethenyl]-9H-fluoren-2-

yl}ethenyl]phenyl}amine (20)

Cell Imaging Methods

tBuOK (120 mg, 1.07 mmol) was added to a solution of 8b (450 mg,

0.786 mmol) and 1b (257 mg, 0.714 mmol) in anhydrous CH₂Cl₂(30 mL).

The reaction mixture was stirred for 15 h in the dark. After evaporation

of the solvent, the residue was purified by column chromatography (hep-

tane/CH₂Cl₂60:40) to yield 9 (102 mg, 18%; as a mixture of E/Z stereo-

isomers). The reaction of 18b (10 mg, 31 mmol) with 9 (97 mg, 124 mmol)

was as described for 19c, for 15 h at 1108C, with subsequent purification

by column chromatography (heptane/CH₂Cl₂, gradient from 70:30 to

50:50) to afford 20 (12 mg, 18%). ¹H NMR (500.13 MHz, CDCl₃): d=

HEK293 was routinely cultured in Dulbeccoꢂs modified eagleꢂs medium

(DMEM) supplemented with 10% fetal calf serum (FCS), d-glucose,

sodium pyruvate, penicillin/streptomycin, and nonessential amino acids,

and maintained in a humid incubator at 378C in 5% CO₂, in a 75 cm²

tissue culture flask. For confocal analysis, HEK cells were grown on

phenol-red-free DMEM supplemented with 10% FCS without steroid

(FCS-DCC) in a cover slip in a six-well culture plate. Cells were split at

a ratio of 1/2 or 1/3 every 3 days. One day after the split, a solution

(6 mL) of 1 mm fluorophores was added into phenol-red-free DMEM

(2 mL) for 2 h incubation. The well was washed with PBS, then fresh

phenol-red-free DMEM (2 mL) supplemented with FCS-DCC was added

and incubated for 24 h. The old media was removed and the cover slip

was analyzed with a confocal microscope.

7.99 and 7.77 (AA’XX’, J

7.67 (d, J=8.2 Hz, 3H), 7.54 (d, J=8.2 Hz, 3H), 7.52 (d, J=8.2 Hz, 3H),

7.49 and 7.14 (AA’XX’, J(A,X)=8.4 Hz, 12H), 7.49–7.42 (m, 12H), 7.42

ACHTUNGTRENUN(NG A,X)=8.4 Hz, 12H), 7.69 (d, J=8.2 Hz, 3H),

AHCTUNGTRENNUNG

(d, J=16.3 Hz, 3H), 7.19 (d, J=16.3 Hz, 3H), 2.01 (m, 12H), 1.23–1.05

(m, 72H), 0.79 (t, J=7 Hz, 18H), 0.66 ppm (m, 12H); ¹⁹F NMR

(282.37 MHz, CDCl₃): d=ꢀ78.40 ppm; HRMS (ES⁺, MeOH): m/z calcd

for C₁₄₄H₁₆₈NO₆S₃F₉K: 2313.15272 [M+K]⁺; found: 2313.1865.

Spectroscopic Measurements

Acknowledgements

All photophysical properties were performed with freshly prepared air-

equilibrated solutions at room temperature (298 K). UV/Vis absorption

spectra were recorded with a Jasco V-570 spectrophotometer. Steady-

state and time-resolved fluorescence measurements were performed on

dilute solutions (ca. 10^ꢀ6m, optical density <0.1) contained in standard

1 cm quartz cuvettes with an Edinburgh Instruments (FLS920) spectrom-

eter in photon-counting mode. Fully corrected emission spectra were ob-

tained for each compound under excitation at the wavelength of the ab-

sorption maximum, with A_lex<0.1 to minimize internal absorption. Fluo-

rescence quantum yields were measured according to literature proce-

dures using fluorescein in 0.1m NaOH as a standard (quantum yield F=

0.90).^[33]Fluorescence lifetimes were measured by time-correlated single-

photon counting (TCSPC). Excitation was achieved by a hydrogen-filled

nanosecond flashlamp (repetition rate 40 kHz). The instrument response

(FWHM ca. 1 ns) was determined by measuring the light scattered by

a Ludox suspension. The TCSPC traces were analyzed by standard itera-

Rꢀgion Bretagne is acknowledged for a fellowship to C.L.D and A.S.

(DEMASIDA and BIPHOBIO grants). M.B.D. gratefully thanks the

Conseil Rꢀgional dꢂAquitaine for generous funding (Chaire dꢂAccueil

grant). We gratefully thank Charlotte Brigand and Frꢀdꢀric Percevault

(IRSET INSERM U 1085, University of Rennes 1) for cell culture and

staining, and Emmanuel Schaub from the PIXEL platform (multiphoton

microscopy facilities, University of Rennes 1) for expert assistance in bio-

imaging experiments.

[1] a) G. S. He, L.-S. Tan, Q. Zheng, P. N. Prasad, Chem. Rev. 2008 , 108 ,

124 5 –1 330; b) F. Terenziani, C. Katan, E. Badaeva, S. Tretiak, M.

Blanchard-Desce, Adv. Mater. 2008 , 20 , 464 1 –4 678; c) H. M. Kim,

B. R. Cho, Chem. Commun. 2009, 153–164; d) M. Pawlicki, H. A.

&

16

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ÝÝ These are not the final page numbers!

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

Collins, R. G. Denning, H. L. Anderson, Angew. Chem. 2009 , 121 ,

329 2 –3 316; Angew. Chem. Int. Ed. 2009 , 48 , 324 4 –3 266.

na, R. Bozio, A. Facchetti, C. Ferrante, G. A. Pagani, D. Pedron, R.

Signorini, Org. Lett. 2002 , 4 , 149 5 –1 498; g) W. J. Yang, D. Y. Kim,

M.-Y. Jeong, H. M. Kim, S.-J. Jeon, B. R. Cho, Chem. Commun.

2003 , 261 8 –2 619; h) S. K. Lee, W. J. Yang, J. J. Choi, C. H. Kim, S.-J.

Jeon, B. R. Cho, Org. Lett. 2005 , 7 , 32 3 –3 26; i) C. B. Nielsen, M.

Johnsen, J. Arnbjerg, M. Pittelkow, S. P. McIlroy, P. R. Ogilby, M.

Jorgensen, J. Org. Chem. 2005 , 70 , 706 5 –7 079; j) O. Mongin, L.

Porrꢅs, M. Charlot, C. Katan, M. Blanchard-Desce, Chem. Eur. J.

2007 , 13 , 148 1 –1 498; k) S. Yao, H.-Y. Ahn, X. Wang, J. Fu, E. W.

Van Stryland, D. J. Hagan, K. D. Belfield, J. Org. Chem. 2010 , 75 ,

396 5 –3 974; l) C. D. Andrade, C. O. Yanez, L. Rodriguez, K. D. Bel-

field, J. Org. Chem. 2010 , 75 , 397 5 –3 982; m) H.-Y. Ahn, S. Yao, X.

Wang, K. D. Belfield, ACS Appl. Mater. Interfaces 2012 , 4 , 284 7 –

2854.

[2] a) W. Denk, J. H. Strickler, W. W. Webb, Science 1990 , 248 , 7 3 –7 6;

b) C. Xu, W. Zipfel, J. B. Shear, R. M. Williams, W. W. Webb, Proc.

Natl. Acad. Sci. USA 1996 , 93 , 1076 3 –1 0768; c) H. M. Kim, B. R.

Cho, Acc. Chem. Res. 2009 , 42 , 86 3 –8 72; d) S. Yao, K. D. Belfield,

Eur. J. Org. Chem. 2012 , 319 9 –3 217.

[3] a) J. D. Bhawalkar, N. D. Kumar, C. F. Zhao, P. N. Prasad, J. Clin.

Laser Med. Surg. 1997, 15, 201–204; b) S. Kim, T. Y. Ohulchanskyy,

H. E. Pudavar, R. K. Pandey, P. N. Prasad, J. Am. Chem. Soc. 2007 ,

129 , 266 9 –2 675; c) H. A. Collins, M. Khurana, E. H. Moriyama, A.

Mariampillai, E. Dahlstedt, M. Balaz, M. K. Kuimova, M. Drobiz-

hev, V. X. D. Yang, D. Phillips, A. Rebane, B. C. Wilson, H. L. An-

derson, Nat. Photonics 2008 , 2 , 42 0 –4 24; d) J. R. Starkey, A. K.

Rebane, M. A. Drobizhev, F. Meng, A. Gong, A. Elliott, K. McIn-

nerney, C. W. Spangler, Clin. Cancer Res. 2008 , 14 , 656 4 –6 573;

e) B. W. Pedersen, T. Breitenbach, R. W. Redmond, P. R. Ogilby,

Free Radical Res. 2010 , 44 , 138 3 –1 397; f) M. Gary-Bobo, Y. Mir, C.

Rouxel, D. Brevet, I. Basile, M. Maynadier, O. Vaillant, O. Mongin,

M. Blanchard-Desce, A. Morꢅre, M. Garcia, J.-O. Durand, L.

Raehm, Angew. Chem. 2011 , 123 , 1162 7 –1 1631; Angew. Chem. Int.

Ed. 2011 , 50 , 1142 5 –1 1429; g) X. Yue, C. O. Yanez, S. Yao, K. D.

Belfield, J. Am. Chem. Soc. 2013 , 135 , 211 2 –2 115.

[4] a) T. Furuta, S. S. H. Wang, J. L. Dantzker, T. M. Dore, W. J. Bybee,

E. M. Callaway, W. Denk, R. Y. Tsien, Proc. Natl. Acad. Sci. USA

1999 , 96 , 119 3 –1 200; b) G. C. R. Ellis-Davies, M. Matsuzaki, M.

Paukert, H. Kasai, D. E. Bergles, J. Neurosci. 2007 , 27 , 660 1 –6 604;

c) M. Matsuzaki, T. Hayama, H. Kasai, G. C. R. Ellis-Davies, Nat.

Chem. Biol. 2010 , 6 , 25 5 –2 57; d) G. C. R. Ellis-Davies, ACS Chem.

Neurosci. 2011 , 2 , 18 5 –1 97.

[10] a) S.-J. Chung, K.-S. Kim, T.-C. Lin, G. S. He, J. Swiatkiewicz, P. N.

Prasad, J. Phys. Chem. B 1999 , 103 , 1074 1 –1 0745; b) L. Porrꢅs, O.

Mongin, C. Katan, M. Charlot, T. Pons, J. Mertz, M. Blanchard-

Desce, Org. Lett. 2004 , 6 , 4 7 –5 0; c) C. Rouxel, C. Le Droumaguet,

Y. Macꢀ, S. Clift, O. Mongin, E. Magnier, M. Blanchard-Desce,

Chem. Eur. J. 2012 , 18 , 1248 7 –1 2497; d) P. Hrobꢆrik, V. Hrobꢆri-

kovꢆ, I. Sigmundovꢆ, P. Zahradnꢇk, M. Fakis, I. Polyzos, P. Persepho-

nis, J. Org. Chem. 2011 , 76 , 872 6 –8 736.

[11] a) B. R. Cho, K. H. Son, S. H. Lee, Y.-S. Song, Y.-K. Lee, S.-J. Jeon,

J. H. Choi, H. Lee, M. Cho, J. Am. Chem. Soc. 2001 , 123 , 1003 9 –

10045; b) Y.-Z. Cui, Q. Fang, G. Xue, G.-B. Xu, L. Yin, W.-T. Yu,

Chem. Lett. 2005 , 34 , 64 4 –6 45; c) G. Argouarch, R. Veillard, T.

Roisnel, A. Amar, H. Meghezzi, A. Boucekkine, V. Hugues, O.

Mongin, M. Blanchard-Desce, F. Paul, Chem. Eur. J. 2012 , 18 ,

1181 1 –1 1827; d) Y. M. Poronik, V. Hugues, M. Blanchard-Desce,

D. T. Gryko, Chem. Eur. J. 2012 , 18 , 925 8 –9 266.

[5] a) D. A. Parthenopoulos, P. M. Rentzepis, Science 1989 , 245 , 84 3 –

845; b) J. H. Strickler, W. W. Webb, Opt. Lett. 1991 , 16 , 178 0 –1 782.

[6] a) G. S. He, G. C. Xu, P. N. Prasad, B. A. Reinhardt, J. C. Bhatt, R.

McKellar, A. G. Dillard, Opt. Lett. 1995 , 20 , 43 5 –4 37; b) J. E. Ehr-

lich, X. L. Wu, I. Y. S. Lee, Z. Y. Hu, H. Rçckel, S. R. Marder, J. W.

Perry, Opt. Lett. 1997 , 22 , 184 3 –1 845.

[12] C. Katan, F. Terenziani, O. Mongin, M. H. V. Werts, L. Porrꢅs, T.

Pons, J. Mertz, S. Tretiak, M. Blanchard-Desce, J. Phys. Chem. A

2005 , 109 , 302 4 –3 037.

[13] V. Alain-Rizzo, D. Drouin-Kucma, C. Rouxel, I. Samb, J. Bell, P. Y.

Toullec, V. Michelet, I. Leray, M. Blanchard-Desce, Chem. Asian J.

2011 , 6 , 108 0 –1 091.

[7] a) S. Maruo, O. Nakamura, S. Kawata, Opt. Lett. 1997 , 22 , 13 2 –1 34;

b) B. H. Cumpston, S. P. Ananthavel, S. Barlow, D. L. Dyer, J. E.

Ehrlich, L. L. Erskine, A. A. Heikal, S. M. Kuebler, I.-Y. S. Lee, D.

McCord-Maughon, J. Qin, H. Rçckel, M. Rumi, X. L. Wu, S. R.

Marder, J. W. Perry, Nature 1999, 398, 51–54; c) S. Kawata, H.-B.

Sun, T. Tanaka, K. Takada, Nature 2001 , 412 , 69 7 –6 98; d) W. Zhou,

S. M. Kuebler, K. L. Braun, T. Yu, J. K. Cammack, C. K. Ober, J. W.

Perry, S. R. Marder, Science 2002 , 296 , 110 6 –1 109; e) F. Claeyssens,

E. A. Hasan, A. Gaidukeviciute, D. S. Achilleos, A. Ranella, C.

Reinhardt, A. Ovsianikov, X. Shizhou, C. Fotakis, M. Vamvakaki,

B. N. Chichkov, M. Farsari, Langmuir 2009 , 25 , 321 9 –3 223; f) M.

Farsari, M. Vamvakaki, B. N. Chichkov, J. Opt. 2010 , 12 , 12400 1 –

124016.

[14] F. Terenziani, C. Le Droumaguet, C. Katan, O. Mongin, M. Blan-

chard-Desce, ChemPhysChem 2007 , 8 , 72 3 –7 34.

[15] C. Hansch, A. Leo, R. W. Taft, Chem. Rev. 1991 , 91 , 16 5 –1 95.

[16] Y. Le Bigot, M. Delmas, A. Gaset, Synth. Commun. 1983 , 13 , 17 7 –

182.

[17] M. Jessing, M. Brandt, K. J. Jensen, J. B. Christensen, U. Boas, J.

Org. Chem. 2006 , 71 , 673 4 –6 741.

[18] C. G. J. Verhart, G. I. Tesser, Recl. Trav. Chim. Pays-Bas 1988, 107,

621–626.

[19] Y. Shirota, T. Kobata, N. Noma, Chem. Lett. 1989 , 114 5 –1 148.

[20] T. Mallegol, S. Gmouh, M. A. A. Meziane, M. Blanchard-Desce, O.

Mongin, Synthesis 2005, 1771–1774.

[21] T. Jeffery, Tetrahedron 1996 , 52 , 1011 3 –1 0130.

[8] a) L. Ventelon, S. Charier, L. Moreaux, J. Mertz, M. Blanchard-

Desce, Angew. Chem. 2001 , 113 , 215 6 –2 159; Angew. Chem. Int. Ed.

2001 , 40 , 209 8 –2 101; b) M. Blanchard-Desce, C. R. Phys. 2002 , 3 ,

43 9 –4 48; c) M. H. V. Werts, S. Gmouh, O. Mongin, T. Pons, M.

Blanchard-Desce, J. Am. Chem. Soc. 2004 , 126 , 1629 4 –1 6295; d) C.

Le Droumaguet, O. Mongin, M. H. V. Werts, M. Blanchard-Desce,

Chem. Commun. 2005 , 280 2 –2 804; e) T. R. Krishna, M. Parent,

M. H. V. Werts, L. Moreaux, S. Gmouh, S. Charpak, A.-M. Cami-

nade, J.-P. Majoral, M. Blanchard-Desce, Angew. Chem. 2006 , 118 ,

476 1 –4 764; Angew. Chem. Int. Ed. 2006 , 45 , 464 5 –4 648; f) M.-H.

Ha-Thi, M. Penhoat, D. Drouin, M. Blanchard-Desce, V. Michelet,

I. Leray, Chem. Eur. J. 2008, 14, 5941–5950.

[22] Fluorescence quantum yields determined relative to quinine bisul-

fate in 0.5m H₂SO₄are in good agreement with those determined

relative to fluorescein in 0.1m NaOH.

[23] S. J. Strickler, R. A. Berg, J. Chem. Phys. 1962 , 37 , 81 4 –8 22.

[24] a) E. Lippert, Z. Naturforsch. A 1955, 10, 541–545; b) N. Mataga, Y.

Kaifu, M. Koizumi, Bull. Chem. Soc. Jpn. 1955 , 28 , 69 0 –6 91.

[25] We also checked that viscosity was not influencing the position of

the fluorescence band but rather the fluorescence quantum yield (in-

creased viscosity would lead to larger fluorescence quantum yield).

[26] G. F. Mes, B. de Jong, H. J. van Ramesdonk, J. W. Verhoeven, J. M.

Warman, M. P. de Haas, L. E. W. Horsman-van den Dool, J. Am.

Chem. Soc. 1984 , 106 , 652 4 –6 528.

[9] a) B. A. Reinhardt, L. L. Brott, S. J. Clarson, A. G. Dillard, J. C.

Bhatt, R. Kannan, L. Yuan, G. S. He, P. N. Prasad, Chem. Mater.

1998 , 10 , 186 3 –1 874; b) M. A. Albota, C. Xu, W. W. Webb, Appl.

Opt. 1998 , 37 , 735 2 –7 356; c) L. Ventelon, L. Moreaux, J. Mertz, M.

Blanchard-Desce, Chem. Commun. 1999 , 205 5 –2 056; d) P. K. Fred-

eriksen, M. Jørgensen, P. R. Ogilby, J. Am. Chem. Soc. 2001 , 123 ,

121 5 –1 221; e) O. Mongin, L. Porrꢅs, L. Moreaux, J. Mertz, M. Blan-

chard-Desce, Org. Lett. 2002 , 4 , 71 9 –7 22; f) A. Abbotto, L. Beveri-

[27] C. Katan, M. Charlot, O. Mongin, C. L. Le Droumaguet, V. Jouikov,

F. Terenziani, E. Badaeva, S. Tretiak, M. Blanchard-Desce, J. Phys.

Chem. B 2010 , 114 , 315 2 –3 169.

[28] C. Xu, W. W. Webb, J. Opt. Soc. Am. B 1996 , 13 , 48 1 –4 91.

[29] A. Ulman, C. S. Willand, W. Kçhler, D. R. Robello, D. J. Williams,

L. Handley, J. Am. Chem. Soc. 1990 , 112 , 708 3 –7 090.

[30] M. S. Wong, M. Samoc, A. Samoc, B. Luther-Davies, M. G. Hum-

phrey, J. Mater. Chem. 1998 , 8 , 200 5 –2 009.

17

&

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

These are not the final page numbers! ÞÞ

www.chemasianj.org

Olivier Mongin, Mireille Blanchard-Desce et al.

[31] S. Rozen, Y. Bareket, J. Org. Chem. 1997 , 62 , 145 7 –1 462.

[32] F. Wꢈrthner, H.-W. Schmidt, F. Haubner, Ger. Offen. 1998, DE 96–

19643097.

[34] M. H. V. Werts, N. Nerambourg, D. Pꢀlꢀgry, Y. Le Grand, M. Blan-

chard-Desce, Photochem. Photobiol. Sci. 2005 , 4 , 53 1 –5 38.

Received: May 30, 2013

Revised: July 3, 2013

[33] a) J. N. Demas, G. A. Crosby, J. Phys. Chem. 1971, 75, 991–1024;

b) D. F. Eaton, Pure Appl. Chem. 1988 , 60 , 110 7 –1 114.

Published online: && &&, 0000

&

18

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

ÝÝ These are not the final page numbers!

FULL PAPER

Bioimaging

Cꢀline Le Droumaguet, Aude Sourdon,

Emilie Genin, Olivier Mongin,*

Mireille Blanchard-Desce* &&&& — &&&&

Two-Photon Polarity Probes Built

from Octupolar Fluorophores:

Synthesis, Structure–Properties Rela-

tionships, and Use in Cellular Imaging

Core values: A series of octupolar flu-

orophores built from a triphenylamine

core connected to electron-withdraw-

ing peripheral groups through conju-

gated spacers has been synthesized.

Their photoluminescence, solvato-

chromism, and two-photon absorption

properties were systematically investi-

gated to derive structure–property

relationships.

19

&

Chem. Asian J. 2013, 00, 0 – 0

ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

These are not the final page numbers! ÞÞ

Article Doi

Two-photon polarity probes built from octupolar fluorophores: Synthesis, structure-properties relationships, and use in cellular imaging

DOI: 10.1002/asia.201300735

Source and publish data:

Authors:

Article abstract of DOI:10.1002/asia.201300735

Full text of DOI:10.1002/asia.201300735

Products guided by the article

R&D Labs maybe for 189178-08-3

Relevant to this article

Hot Product