Geometry relaxation-induced large stokes shift in red-emitting borondipyrromethenes (BODIPY) and applications in fluorescent thiol probes

Article

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Geometry Relaxation-Induced Large Stokes Shift in Red-Emitting

Borondipyrromethenes (BODIPY) and Applications in Fluorescent

Thiol Probes

Yinghui Chen,^†,‡Jianzhang Zhao,^†,* Huimin Guo,^†and Lijuan Xie^‡

^†State Key Laboratory of Fine Chemicals, School of Chemical Engineering, Dalian University of Technology, E-208 West Campus,

2 Ling-Gong Road, Dalian 116024, P. R. China

^‡Institute of Molecular Medicine, Huaqiao University, 269 Chenghua North Road, Quanzhou 362021, P. R. China

S

* Supporting Information

ABSTRACT: 2-Thienyl and 2,6-bisthienyl BODIPY derivatives (BS-SS and BS-

DS) were prepared that show intense absorption (ε = 65000 M⁻¹cm⁻¹at

507 nm) and a large Stokes shift (96 nm) vs the small Stokes shift of typical

BODIPY (<15 nm). Control compounds with a thienyl unit at the 8-position or

phenyl substituents at the 2,6-positions were prepared (BS-1 and 9). BS-1 shows

absorption/emission in the blue-shifted range and a small Stokes shift (12 nm).

Compound 9 shows absorption in the red-shifted range, but the Stokes shift

(<30 nm) is much smaller than that for BS-SS and BS-DS. DFT calculations

propose the large Stokes shifts of BS-SS and BS-DS are due to the remarkable geometry relaxation upon photoexcitation and its

substantial effect on the energy levels of molecular orbitals. For the dyes with small Stokes shifts, much smaller geometry

relaxations were found. The fluorophores were used for fluorescent thiol probes, with 2,4-dinitrobenzenesulfonyl (DNBS) as the

fluorescence switch. Both fluorescence OFF−ON and unprecedented ON−OFF transduction were observed, which are

attributed to the different photoinduced intramolecular electron-transfer (PET) profile. All the photophysics were rationalized by

DFT calculations based on the concept of “electronic states” instead of the very often used approximation of “molecular orbitals”.

this method suffers from the drawback that self-absorption is

still strong and the large Stokes shift is actually a pseudo-Stokes

shift.^{1i,2f,6c,11b,f,12}The inner filter effect cannot be completely

eliminated by FRET if the energy acceptor shows an intrinsic

small Stokes shift.^10,13

1. INTRODUCTION

Borondipyrromethenes (BODIPY) as a versatile fluorophore

has attracted much attention in luminophores,¹molecular probes

or molecular logic gates,²light-harvesting molecular arrays,³

photodynamic therapy (PDT),⁴and more recently, triplet−

triplet annihilation (TTA) upconversions, etc.^5,6BODIPYs are

known for their robust photophysical properties, such as strong

absorption of visible light (for the unsubstituted BODIPY, the

molar extinction coefficient ε is up to 87000 M⁻¹cm⁻¹at

510 nm), high fluorescence quantum yield, good photostability,

etc.^6a,cFurthermore, the chemistry of BODIPY dyes is rich;⁶

thus, the properties of BODIPY, such as the absorption/emission

wavelength, or the hydrophilicity, etc., can be readily tuned by

molecular structural modification.^1o,6,7

The persistent and remarkable disadvantage of BODIPY

dyes, that is, the small Stokes shift, has never been addressed.^6,8

A very small Stokes shift (ca. 10 nm) was usually found for

BODIPY dyes.^{1k,4d,6a,c,7d,9}This small Stokes shift is very often

detrimental to its applications, such as in molecular probes or

intracellular fluorescence imaging. A small Stokes shift reduces

the emission intensity by self-absorption, or the inner filter

effect.¹⁰Unfortunately, the typical chemical modification of the

BODIPY core, i.e., extending the π-conjugation framework,

which leads to red-shifted absorption and emission, is not help-

ful at all in increasing the Stokes shift.^{1b,f,g,i,o,2h,n,4e,5b,7d−f,9,11}

Although the large Stokes shift can be observed with the strategy of

In order to address the small Stokes shift of the BODIPY

dyes, herein we devised new BODIPY derivatives that show

intrinsic large Stokes shift up to 96 nm, with excitation at green

(ca. 530 nm) and emission at red (ca. 620 nm). This large

Stokes shift is achieved by a de novo approach, that is, to

increase the geometry relaxation of the molecular framework

upon photoexcitation. In other words, the geometry of the

energy-minimized first singlet excited state, i.e., the S₁state,

which is responsible for the fluorescence (Kasha’s rule), is

remarkably different from the ground-state geometry (S₀state).

Therefore, the emission energy is much smaller than the excita-

tion energy; i.e., the Stokes shift is large.^10,14For the unsub-

stituted BODIPY fluorophore and the control compounds,

however, we proved that the geometry of the S₁excited state is

similar to that of the S₀state; thus the difference of the

excitation energy (UV−vis absorption) and the emission energy

is small; i.e., the Stokes shift is small. These analyses were

supported by DFT/TDDFT calculations.

Received: November 3, 2011

Published: February 8, 2012

fluorescent-resonance-energy-transfer (FRET, Foster energy transfer),

̈

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a

Scheme 1. Synthesis of the Compounds BS-1-OH, BS-1, BS-2-OH, BS-2, BS-SS, and BS-DS

a

Key: (a) 4-iodophenol, K₃PO₄·3H₂O, Pd(OAc)₂, toluene, ethanol, under Ar, 80 °C, 8 h, yield 40.2%; (b) 2,4-dimethylpyrrole, TFA, THF, under

Ar, overnight; DDQ, 4 h; TEA, BF₃·OEt₂, overnight; yield 30.0%; (c) DNBS, CH₂Cl₂, TEA, 3 h; yield 50.0%; (d) 2,4-dimethylpyrrole, TFA, THF,

under Ar, overnight; DDQ, 4 h; TEA, BF₃·OEt₂, overnight; yield 40.0%; (e) i-Pr₂NEt, MOMCl, CH₂Cl₂, 60 °C; yield 50.0%; (f) NIS, CH₂Cl₂; yield

80.0%; (g) thiophene-2-boronic acid, K₃PO₄·3H₂O, Pd(OAc)₂, toluene, ethanol, under Ar, 80 °C, 8 h; yield 67.0%; (h) 6 N HCl, EtOH, 60 °C, 4 h;

yield 56.7%; (i) DNBS, CH₂Cl₂, TEA, 3 h; yield 34.0%; (j) 2,4-dimethylpyrrole, under Ar, overnight; TEA, BF₃·OEt₂, overnight; yield 55.0%; (k)

NIS, CH₂Cl₂; yield 45.0%; (l) thiophene-2-boronic acid, K₃PO₄·3H₂O, Pd(OAc)₂, toluene, ethanol, under Ar, 80 °C, 8 h; yield 63.4%; (m) NIS,

CH₂Cl₂; yield 77.0%; (n) thiophene-2-boronic acid, K₃PO₄·3H₂O, Pd(OAc)₂, toluene, ethanol, under Ar, 80 °C, 8 h; yield 54.4%; (o) phenylboronic

acid, K₃PO₄·3H₂O, Pd(OAc)₂, toluene, ethanol, under Ar, 80 °C, 8 h; yield 30.5%;

The increased geometry relaxation of the new BODIPY

derivatives is obtained by introducing thienyl substituents at 2-

and 6-position of the BODIPY core (Scheme 1). With DFT

optimizations of the geometry of the S₀and S₁excited states,

we found that the dihedral angle between the thienyl unit and

the BODIPY core decreased from 55° to 39°. The new

BODIPY derivatives that show large Stokes shift and red emis-

sion were used for fluorescent thiol probes, which were based

on the strategy for the 2,4-dinitrobenzenesulfonyl-protected

fluorophore.^2m,11j,15Furthermore, we found that the position of

the thienyl group on the BODIPY core is crucial. Substitution

at the 8-position leads to a fluorophore with a small Stokes

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shift.^6e−gThe thiol probe with this dye shows the unusual

fluorescence ON−OFF switching effect, which is due to the

perturbation of the normal electron transfer of DNBS thiol

probes. The photophysical properties of the dyes and the

probes were fully rationalized by DFT calculations. Our results

are useful for design of new BODIPY derivatives that show

large Stokes shifts and for applications of these dyes in

molecular probes, light-harvesting molecular arrays, etc.

2.2. UV−vis Absorption of the Thienyl-BODIPY

Fluorophores. The UV−vis absorption of the compounds

were studied (Figure 1). Compared to the unsubstituted

2. RESULTS AND DISCUSSION

2.1. Design Rationales and Synthesis of the BODIPY

Derivatives. Our strategy to increase the Stokes shift is based

on the Jablonski diagram; i.e., the Stokes shift is, to a large

extent, dependent on the geometry relaxation of the

fluorophores upon photoexcitation.^10,14Based on the Jablonski

diagram, a large Stokes shift will be produced by large geometry

relaxation; i.e., the geometry difference between the S₀state and

the S₁state. For the unsubstituted BODIPY fluorophore, the

molecular framework is rigid; thus, small geometry relaxation

and small Stokes shifts are expected. With thienyl substitution

at the 2- and 6-positions, the molecular flexibility was increased;

that is, the dihedral angle between the thienyl and the BODIPY

core may be changed upon excitation. This enhanced geometry

relaxation will increase the Stokes shift.^10,14The experimental

results fully proved our postulations.

The synthesis of the compounds is straightforward (Scheme 1).

With iodination of the BODIPY core by N-iodosuccinimide

(NIS), monoiodo- or bisiodo-BODIPY (BS-SS or BS-DS) were

obtained. Then the monothienyl- or bis-thienyl-substituted

derivatives BS-SS and BS-DS were obtained by using Pd(0)-

catalyzed Suzuki cross-coupling reactions with 2-thienyl

boronic acid (Scheme 1). In order to use this BODIPY deriva-

tives to construct a fluorescent thiol probe, we prepared 8-(4′-

hydroxyl)phenyl-substituted 2,6-bisthienyl derivatives BS-2-

OH. Since we found that direct iodination of compound 2

leads to a complicated mixture, the phenolic group was pro-

tected with methyl chloromethyl ether (MOMCl) prior to iodi-

nation with NIS (Scheme 1). After the bis-iodination of

BODIPY, the protection MOM was readily removed by treat-

ment with half-concentrated HCl acid under mild conditions

(Scheme 1). Then the thiol probe BS-2 was obtained by

reaction of BS-2-OH with 2,4-dinitrobenzenesulfonyl chloride

(DNBS-Cl). All of the compounds were obtained in moderate

to satisfying yields (see the Experimental Section for details).

Compound 9 was prepared as a control compound to study the

different effects of the phenyl and thienyl substituents (BS-DS)

on Stokes shifts.

We also prepared a control compound with the thienyl

substituent at the 8-position (BS-1-OH, Scheme 1) to study

the effect of substitution position of the thienyl moiety on the

photophysical properties. The 4′-hydroxylphenyl group at the

5-position of the thienyl moiety is for preparation of the

thiol probe BS-1 (Scheme 1). Direct preparation of BODIPY

derivatives with 2-formyl thienyl was unsuccessful. BS-2 is a

fluorescence OFF−ON thiol probe, but interestingly, BS-1 is a

fluorescence ON−OFF probe. That is, the compound BS-2-

OH is fluorescent and probe BS-2 is nonfluorescent. For dye

BS-1-OH and the probe BS-1, however, the emissive profile is

reversed; i.e., BS-1-OH is nonfluorescent but probe BS-1

is fluorescent. These fluorescence relays can be rationalized

by different photoinduced electron transfer processes of the

compounds, which is supported by the DFT and TDDFT

calculations.

Figure 1. UV−vis absorption spectra of the compounds (in toluene);

7.5 × 10⁻⁶M, 25 °C.

BODIPY,^6a−dthe new derivatives show red-shifted absorption.

For example, BS-SS shows absorption at 514 nm, and the

bisthienyl-substituted derivative BS-DS gives absorption at

529 nm. These red-shifted absorptions are due to the slightly

extended π-conjugation framework, which was proved by the

DFT calculations (discussed later). For the derivatives with a

thienyl substituent at 8-position (BS-1 and BS-1-OH), how-

ever, the absorption/emission wavelengths do not show sig-

nificant red-shifts. DFT calculations indicate that in this case

the thienyl is not involved in the frontier molecular orbitals; i.e.

the π-conjugation framework is not extended to the thienyl

moiety (Supporting Information). The control compound with

phenyl moieties at the 2,6-position was prepared (9 in Scheme 1).

Compound 9 shows similar red-shifted absorption at 530 nm

compared to the unsubstituted BODIPY (compound 6) at

504 nm (Supporting Information).

We noticed that the derivatives with thienyl substitution at

the 8-position of BODIPY core gave a similar absorption wave-

length. For example, BS-1 (with thienyl moiety at the meso-

position) gives absorption maxima at 514 nm.

2.3. Emission of the New Derivatives with Large

Stokes Shifts. The fluorescence of the new derivatives was

studied (Figure 2). For the unsubstituted BODIPY (6), the

typical small Stokes shift (10 nm) was observed (Figure 2c),

with the excitation and emission maxima at 506 and 516 nm,

respectively. Interestingly, much larger Stokes shift were

observed for the new derivatives with the thienyl moiety at 2-

or 2,6-position of the BODIPY core. For example, excitation

and emission maxima of compound BS-SS at 509 and 601 nm

were observed, respectively (Figure 2a). Thu, the Stokes shift is

up to 92 nm. To the best of our knowledge, this is among the

largest Stokes shifts ever reported for the BODIPY dyes,

without resorting to the FRET effect, etc.^1i,6a,c

Interestingly, with thienyl substitution at the 8-position, no

large Stokes shift was observed. For example, the excitation and

emission maxima of BS-1 are at 515 and 527 nm, respectively.

Thus, the Stokes shift of BS-1 is 12 nm (see Figure 12). This

small Stokes shift is typical for BODIPY dyes.^{2a,b,6,11b,g,15f,16}

A

similar emission profile was found for BS-1-OH (Supporting

Information).

We noticed that BODIPY derivatives with thienyl units at

the 3- or 3,5-positions were reported.^11e,17However, those

compounds show a typical small Stokes shift of BODIPY (17−

29 nm), although the emission wavelengths are red-shifted

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Figure 2. Emission and excitation spectra of (a) BS-SS (λ_ex= 520 nm, λ_em= 600 nm); (b) BS-DS (λ_ex= 530 nm, λ_em= 636 nm); and (c) compound

6 (unsubstituted BODIPY) (λ_ex= 480 nm, λ_em= 536 nm). The wavelengths were used for measuring the excitation/emission spectra, they are not

the excitation/emission maxima. c = 7.5 × 10⁻⁶M in toluene, 25 °C.

(587−643 nm) compared to the unsubstituted BODIPY dyes.

It is clear that red-shifted emission does not necessarily induce

a large Stokes shift because the two photophysical parameters

are of entirely different origin.^10,14It should be pointed out that

the red-shifted absorption and emission of the new BODIPY

derivatives are not due to the electron-donating capability of

the moiety at the 8-position of the BODIPY core because

previously blue-shifted absorption and emission were observed

with amino or 4-hydroxylphenyl substituents at the 8-position.^18,2l

Also, the Stokes shift of the BODIPY cannot be increased by

extension of the π-conjugation at the 2,6-postions, such as with

CC triple bonds.^{2m,7a,11h,k,19}

probes or intracellular fluorescent bioimagings.^2k,3cPreviously

the thienyl substituted BODIPY derivatives (at the 2−6 position)

were reported, but the photophysical properties were not studied

in detail.²⁰

2.4. Rationalization of the Large Stokes Shift of the

Thienyl-Substituted BODIPYs with DFT Calculations:

Geometry Relaxation upon Photoexcitation. In order to

rationalize the photophysical properties of the new derivatives,

such as the UV−vis absorption and fluorescence, especially the

large Stokes shift, the geometry of the molecules at the ground

state (S₀state) and the S₁excited state was optimized by

density functional theory (DFT) and time-dependent DFT

(TDDFT) methods. The energy gap between the S₀state and

the S₁excited state (excitation energy) was calculated with the

TDDFT method based on the optimized S₀state geometry (for

absorption) and the S₁state geometry (for fluorescence),

respectively. Revealing the photophysical properties from a

theoretical perspective will be useful for the design of new

fluorophores.

First the ground state (S₀) geometry of BS-DS was opti-

mized (for the molecular structure please refer to Figure 5).

The dihedral angle between the thienyl and BODIPY moiety is

55°. However, HOMO is spread over both the BODIPY and

the thienyl moiety, indicating that the π-conjugation between

the thienyl moiety and the BODIPY core is remarkable even

when the two moieties are not fully coplanar. It is known that

thienyl is an efficient π-conjugation linker and has been widely

used in organic chromophores such as those for dye-sensitized

solar cells.²¹On the basis of the optimized S₀state geometry,

the vertical excitation (UV−vis absorption) was calculated with

the TDDFT method. The calculated absorption bands are

located at 488 and 395 nm (Figure 5), which are in agreement

with the experimental results of 521 and 394 nm (BS-DS)

(Figure 1). These two absorption bands can be assigned to the

S₀→S₁and S₀→S₃transitions, respectively (Table 2). Similar

calculations were carried out for other dyes (Table 2).

In order to study the emission, the S₁state geometry was

optimized.^10,14The energy gap between the S₀state and S₁

state, calculated with the optimized S₁state geometry, is the

fluorescence emission wavelength.^10,14The most prominent

difference between the S₀state geometry and the energy-

minimized geometry of S₁state is the dihedral angle between

the BODIPY core and the thienyl moiety. Compared to the

dihedral angle at S₀state geometry (55°), the two moieties

become more coplanar at the optimized S₁excited state, for

which the dihedral angle is 39°. This geometry relaxation upon

photoexcitation imparts remarkable effect on the energy level of

the molecular orbitals. For example, the LUMO is stabilized by

0.17 eV at the S₁state geometry compared to that at the S₀

state geometry, but the HOMO is destabilized by 0.24 eV for S₁

In principle, the emission wavelength of fluorophore is

related to the size of π-conjugation framework, or the intra-

molecular charge transfer (ICT) feature of the fluorophore.^1g

For the Stokes shift, however, the geometry relaxation at the S₁

state after vertical excitation from S₀state is crucial (from the

Franck−Condon excited state to the energy-minimized

geometry at the S₁state).^10,14It should be pointed out that

no geometry changes occur during the excitation process

(vertical excitation, Franck−Condon principle).^10,14The

geometry relaxation only occurs after the vertical excitation,

i.e., at the S₁state before decay S₁→S₀. In a later section, we

will show that with increased geometry relaxation at the S₁state

the Stokes shift of the fluorophore will increase. This is true for

BS-SS, BS-DS, etc. For the dyes with thienyl moieties at the 3-

or 8-position, however, the geometry changes are smaller; thus

the Stokes shifts of these dyes are small. This is obvious from

the Jablonski diagram (Figure 6 and Supporting Information).^10,14

The control compound 9 shows emission maxima at 555 nm,

which is red-shifted from the thienyl-substituted compounds

BS-DS and BS-SS. However, the Stokes shift of compound 9 is

only 26 nm (Table 1 and Supporting Information).

We found that the UV−vis absorption of BODIPY deri-

vatives are not sensitive to solvent polarity (Figure 3a). Inte-

restingly, the emission wavelength of BS-DS is independent of

the solvent polarity (Figure 3b), although the emission

intensity decreased in more polar solvents, such as methanol,

than that in nonpolar solvents, such as toluene. This result indi-

cates that the large Stokes shift of the new BODIPY derivatives

is intrinsic property, not a solvent-polarity induced phenomena,

or any intramolecular charge transfer (ICT) related result.¹⁰

The absorption and emission changes of BODIPY dyes with

thienyl substitution are clearly visible (Figure 4). For example,

the solution of the unsubstituted BODIPY gives light green

color. For the thienyl substituted BODIPY derivatives, how-

ever, purple color was observed. The emission changed from

green to red. The red-shifted absorption and emission of the

BODIPY derivatives, especially the large Stokes shift, will be

useful for the application of the dyes in fluorescent molecular

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Table 1. Photophysical Parameters of the Compounds

a

b

c

d

e

f

compds

solvents

PhCH₃

λ_abs(nm)

λ_em(nm)

Δν (nm)

ε

(M⁻¹cm⁻¹

)

Φ_F(%)

τ (ns)

BS-2

535

533

526

527

530

528

521

522

530

527

521

523

513

511

507

504

501

498

501

518

515

512

514

517

514

511

514

530

528

524

608

607

609

619

604

605

602

609

610

612

591

594

600

603

516

513

509

530

528

523

528

525

521

g

73

74

83

92

74

77

81

87

79

82

89

78

83

93

96

12

11

8

40000

42000

41000

63000

62000

59000

16000

17000

69000

66000

65000

94000

87000

88000

86000

75000

79000

71000

69000

88000

90000

82000

73000

26000

40000

9000

5.9

g

3.6

g

DCM

CH₃OH

g

CH₃OH/H₂O =4/1

PhCH₃

0.7

57.3

g

BS-2-OH

BS-DS

BS-SS

6

3.8

g

DCM

CH₃OH

g

CH₃OH/H₂O =4/1

PhCH₃

8.8

47.2

g

3.4

g

DCM

CH₃OH

g

CH₃OH/H₂O =4/1

PhCH₃

5.1

20.6

g

3.2

g

DCM

CH₃OH

g

CH₃OH/H₂O =4/1

PhCH₃

3.2

85.2

g

3.4

g

DCM

CH₃OH

g

CH₃OH/H₂O =4/1

PhCH₃

g

3.7

g

BS-1

12

13

11

14

11

10

g

6.6

g

DCM

g

CH₃OH

g

DMF/H₂O =4/1

PHCH₃

5.4

6.9

g

BS-1-OH

3.5

g

DCM

CH₃OH

g

DMF/H₂O =4/1

PhCH₃

0.4

32.5

g

9

556

555

550

551

26

27

26

27

4.1

g

DCM

CH₃OH

g

c

g

CH₃OH/H₂O =4/1

10000

g

a

b

λ_abs(nm): absorption wavelength of first absorption maximum. λ_em(nm): emission wavelength (at the maximum intensity). Δν: Stokes shifts.

d

e

ε: extinction coefficient. Φ: fluorescence quantum yields, with unsubstituted BODIPY (compound 6) as the standard (Φ = 72.0% in tetrahy-

f

g

drofuran). τ (ns): fluorescence lifetimes. Not determined.

Figure 4. Fluorescence photographs of the compounds, taken under

ambient light and UV light (hand-held UV lamp, 365 nm). c = 7.5 ×

10⁻⁶M. In toluene. 25 °C.

Figure 3. (a) UV−vis absorption spectra of BS-DS in different

solvents. (b) Fluorescence spectra of BS-DS in different solvents. c =

7.5 × 10⁻⁶M. λ_ex= 530 nm. 25 °C.

We noticed that BODIPY derivatives with thienyl sub-

stituents at the 2,6-position have been reported, but the origin

of the large Stokes shift was not discussed.²⁰Our rationalization

of the Stokes shift by the concept of geometry relaxation at the

S₁state, supported by the DFT calculations, will be useful for

design of fluorophores that show large Stokes shift. To the best

of our knowledge, this rationale has never been used for design

of fluorophores to access large Stokes shifts.

With the thienyl substituent at the 8-position, the derivative

BS-1 gives the typical small Stokes shift of 12 nm (Table 1).

Our later study shows that for BS-1 the thienyl moiety is not

involved in the frontier molecular orbitals of the electronic

state geometry compared to that at the S₀state geometry. As a

result, the energy gap between the HOMO and LUMO is

greatly decreased with geometry relaxation at the S₁state

compared to that at S₀state geometry (UV−vis absorption, i.e.,

excitation). We propose this geometry relaxation is the main

origin of large Stokes shift for BS-DS, which is in agreement

with the Jablonski diagram of the fluorescence.¹⁰The fluo-

rescence wavelength was calculated as 625 nm (in methanol),

which is in very good agreement with the experimental result of

610 nm (Figure 2).

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transitions. Furthermore, the geometric relaxation of BS-1 at

the singlet excited state (S₁state) is very small. For example,

the dihedral angle between the thienyl substituent and the

BODIPY core shows a very small change of 2°, with dihedral

angles of 89° and 87° for the optimized S₀state geometry and

the optimized S₁state geometry, respectively. By comparison,

the compound BS-2, which gives a large Stokes shift of 92 nm,

shows a dihedral angle change (geometry relaxation) of 16°.

Furthermore, the substantial changes of the energy levels of the

molecular orbitals and the excited state contributed to the

Stokes shifts. A rigid fluorophore, without geometry relaxation

upon photoexcitation, will give a small Stokes shift.^10,14It

should be pointed out that the Stokes shift of BODIPYs were

rarely studied by the DFT calculations.²²

We noticed that BODIPY derivatives with thienyl sub-

stitutions at the 3- or 3,5-positions have been reported to give

red-shifted emission in the region 587−643 nm. However, the

Stokes shifts of those BODIPY derivatives are 17−29 nm,^11e

much smaller than the Stokes shift of the current BODIPY deri-

vatives with thienyl substituents at the 2- or 2,6-positions, such

as BS-SS, BS-DS and BS-SS (Stokes shift is up to 96 nm). We

also investigated the geometrical relaxation of BODIPY deri-

vatives with 3,5-thienyl substituents that give smaller Stokes

shift (see the Supporting Information); the results indicate that

those compounds give smaller dihedral angle changes (less than

10°).^11ePreviously, BODIPY derivatives with aryl substituents

at the 3,5-position were prepared to show relatively large Stokes

shifts (up to 65 nm),²³but the origin of the large Stokes shift

was not rationalized.

The geometry changes of the control compound 9, with

phenyl groups at the 2- and 6-positions, which show a small

Stokes shift of ca. 20 nm, was also studied with the DFT calcu-

lations (Supporting Information). Although the dihedral angles

varied significantly upon excitation, the energy levels of the

molecular orbitals gave very small changes, which resulted in a

small Stokes shift for 9 (Supporting Information). This theoret-

ical approach to rationalize the Stokes shift is applicable to the

8-(2-furyl) BODIPY or the 8-(2-thienyl) BODIPY,^6fwhich are

known with relative large Stokes shift (see the Supporting

Information for the detailed calculation results).

Figure 5. Rationalization of the large Stokes shift of BS-DS: the

geometry relaxation upon photoexcitation and the frontier molecular

orbitals (MOs) involved in the vertical excitation (i.e., UV−vis

absorption, the left two columns) and emission (right column) of BS-

DS. The vertical excitations were calculated based on the optimized

ground state geometry (S₀), the emission was calculated based on the

optimized geometry of the excited state (S₁). Methanol was used as

the solvent (PCM model). Calculated at B3LYP/6-31G(d)/level using

Gaussian 09W. Note the energy levels of HOMO and LUMO at S₁

state are different from that at S₀state. IC stands for internal

conversion and CT stands for conformation transformation. Excitation

and radiative processes are marked as solid lines and the nonradiative

processes are marked by dotted lines.

Table 2. Selected Parameters for the Vertical Excitation (UV−vis Absorptions) and Emission of BS-DS and BS-SS; Electronic

Excitation Energies (eV) and Oscillator Strengths (f), Configurations of the Low-Lying Excited States of the Compounds

(Methanol Used as Solvent in All of the Calculations, Calculated at the DFT/TDDFT//B3LYP/6-31G(d) Level with

Gaussian 09W)

TDDFT//B3LYP/6-31G(d)

a

b

c

d

compd

electronic transitions

excitation energy

2.54 eV (488 nm)

f

composition

CI

e

BS-DS

absorption

S₀→S₁

0.5809

0.4395

H-2→L

H→L

0.2219

0.6714

0.6673

−0.2255

0.7031

0.2659

0.6538

0.6517

0.2709

S₀→S₃

3.14 eV (395 nm)

H-2→L

H→L

f

emission

S₁→S₀

S₀→S₁

1.98 eV (625 nm)

2.68 eV (462 nm)

0.6180

0.4755

H→L

e

BS-SS

absorption

H-1→L

H→ L

H-1→L

H→L

S₀→S₂

3.13 eV (396 nm)

2.01 eV (616 nm)

0.3460

0.4292

f

emission

S₁→S₀

H→L

0.7027

a

b

c

Only selected excited states were considered. The numbers in parentheses are the excitation energy in wavelength. Oscillator strength. H stands

d

for HOMO and L stands for LUMO. Only the main configurations are presented. Coefficient of the wave function for each excitations. The CI

coefficients are in absolute values. The calculations on UV−vis absorption were based on the optimized ground-state geometry. The calculations on

fluorescence are based on the optimized S₁state geometry.

e

f

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The origin of the Stokes shifts of fluorophores can be illus-

trated by the simplified Jablonski diagram in Figure 6.^10,14Large

Figure 7. UV−vis absorption of BS-2 (1.0 × 10⁻⁵M) before and after

treatment with Cys (200 equiv) for 20 min. 25 °C. (b) Fluorescence

spectral changes of probe BS-2 (1.0 × 10⁻⁵M) before and after

treating with 200 equiv of Cys. λ_ex= 540 nm. In mixed solvent

MeOH/H₂O (4:1, v/v). 37 °C.

Figure 6. Simplified Jablonski diagram for the origin of the Stokes

shifts of thienyl-substituted BODIPYs. (a) Small geometry relaxation

upon photoexcitation produces small Stokes shift, which is applicable

to the unsubstituted BODIPY fluorophore and BS-1. (b) Large

geometry relaxation upon photoexcitation leads to large Stokes shift,

which is applicable to BS-2, BS-DS, and BS-SS. GR stands for

geometry relaxation.

(ε = 59000 M⁻¹cm⁻¹) vs the probe BS-2 at 527 nm (ε =

41000 M⁻¹cm⁻¹).

The probe BS-2 gives a weak emission at 619 nm. In the

presence of cysteine, however, the emission intensity was

enhanced 11-fold (Figure 7b). The sensing mechanism of

probe BS-2 is the cleavage of the DNBS moiety by the thiols,

thus the release of the free fluorophore BS-2-OH.^{11j,15a−c,f,24e}

The ideal fluorescent probes will be those that show pH-

independent fluorescence transduction.^16bThus, the pH-

dependency of the emission of fluorophore BS-2-OH was

studied (Figure 8a). For BS-2, the background emission is weak

geometry relaxation at the excited state (S₁state) and the

change of the energy levels of the electronic states along with

the geometry relaxation will produce large Stokes shift.

Conversely, small geometry relaxation at the excited state will

produce a small Stokes shift. The geometry relaxation is the

geometry difference between the energy-minimized S₀state and

the energy-minimized S₁state, which can be predicted by the

DFT and the TDDFT calculations. This de novo approach to

access the large Stokes shift of a fluorophore by introduce of

remarkable geometry relaxation at the S₁excited state will be

useful for design of new fluorophores that show large Stokes

shifts. This remains a challenge for chemists.

2.5. Application of the New BODIPY Derivatives for

Fluorescent Thiol Probe. Fluorophores with large Stokes

shift and long emission wavelength are ideal for applications in

fluorescent molecular probes because of the elimination of the

inner filter effect, etc.^10,14Thus, the new BODIPY derivatives

described herein (BS-SS and BS-DS, Scheme 1) are ideal for

construction of molecular probes. Detection of thiols with

fluorescent molecular probes have attracted much attention.²⁴

DNBS-protected fluorophore is a straightforward strategy

for construction of fluorescent OFF−ON switching thiol

probes.^{2l,15a,b,d,e,24e,25}Previously, we prepared fluorescent thiol

probes with pyrene, BODIPY, and styryl-BODIPY as the

fluorophores, and fluorescence OFF−ON switching effect was

achieved.^11j,15c,fWith DFT/TDDFT calculations, we proposed

that the fluorescence OFF feature of this kind of thiol probes is

caused by the dark excited states of the probes, whereas the

fluorescence switching-ON effect in the presence of thiols is

caused by the emissive S₁excited state of the cleavaged product

(the released free fluorophore).^11j,15c,fThus, the photophysical

properties of the probes can be rationalized by the concept of

“electronic state” instead of the very often used approximation

of “molecular orbitals”. Herein we devised the thiol probe BS-2

to explore the new fluorophores that show large Stokes shift for

construction of fluorescent thiol probes.

Figure 8. (a) Fluorescence response of BS-2 and BS-2-OH (1.0 ×

10⁻⁵M) to different pH. (b) Fluorescence response of BS-2 (1.0 ×

10⁻⁵M) before and after incubation with 200 equiv of different

analytes for 180 min. Inset of (b): reaction kinetic of BS-2 (1.0 × 10⁻⁵

M) with 200 equiv of Cys. λ_ex= 540 nm. λ_em= 608 nm. In MeOH/water

(4/1, v/v). 37 °C.

in the range of pH 3.0−10.0. The emission increased slightly at

high pH range, probably because of the hydrolysis of the

sulfonic ester at basic conditions. The emission is stable in a

wide range of pH 3.0−10.0, which is sufficient for in vivo

fluorescence imaging studies.

The selectivity of BS-2 toward different biologically related

analytes was studied (Figure 8b). The probe gives a similar

response to thiols with small molecular size, such as cysteine,

homocysteine, and 3-mercaptopropanoic acid. No response was

found for other analytes, such as glycine or lysine, etc. Thus, the

probe BS-2 is selective toward thiols and has good immunity

toward biologically related neucleophiles.^{2l,11j,15,24e}It should be

pointed out that the probe does not show any selectivity

between different small molecular thiols, such as cysteine,

homocysteine, or 3-mercaptopropanoic acid; however, this is

expected because of the specific sensing mechanism.^{2l,11j,15,24e}

First, the UV−vis absorption of BS-2 was studied (Figure 7).

In the presence of thiols such as cysteine, the absorption of

BS-2 at 527 nm was enhanced. This is in line with the stron-

ger absorption of the free fluorophore BS-2-OH at 522 nm

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Probe BS-2 was used for fluorescent imaging of intracellular

thiols (Figure 9). With excitation at 543 nm, red emission was

of the probes, such as the fluorescence OFF−ON switching

effect, were rarely studied from a point of view of theoretical

chemistry, such as by using DFT calculations.²⁷Recently, we

studied the fluorescence OFF−ON switching effect of the

DNBS-based thiol probes.^11j,15c,fWe and others used the

concept of “electronic state”, instead of the “molecular orbitals”

(such as HOMO and LUMO), to rationalize the photophysical

properties of the molecular porbes.^{11j,15c,f,27c}We proposed that

the fluorescence OFF state of the probes is due to the dark

excited state of the probes, that is, the S₁state is nonemissive,

which is deduced from the oscillator strength of the S₀→S₁

transition and the molecular orbitals involved in the transition

(usually an electron transfer process, leading to a transition

integer to be zero, makes the S₀→S₁transition a forbidden

transition). S₁→S₀will follow the same rule; thus, the probe is

nonfluorescent.^11j,15c,fThe fluorescence switching ON effect of

the thiol probes in the presence of thiols is due to the cleavage

of the DNBS moiety off the fluorophore, thus the dark excited

state was removed, the S₁state of the free fluorophore is

emissive, and the fluorescence of the fluorophore is switched

on. All these rationalizations are based on DFT and TDDFT

calculations.^11j,15c,fClearly, more examples are required to

confirm this concept.

Figure 9. Fluorescence images of Hela cells. (a) Fluorescence image of

cells in the absence of probe BS-2; (d) fluorescence image of cells

incubated with probe BS-2 (20 μM) for 45 min; (g) fluorescence

image of cells pretreated with N-methylmaleimide (0.5 mM, to remove

the intracellular thiols) for 1 h and then incubated with probe BS-2

(20 μM) for 45 min; (b, e, h) corresponding bright field images of

(a, d, g); (c, f, i) overlays of the fluorescent and bright field images.

λ_ex= 543 nm. 37 °C.

The UV−vis absorption and the emission properties of BS-2

were studied with DFT calculations. First, the ground-state

geometry was optimized and the UV−vis absorption was

calculated by the TDDFT method based on the ground-state

geometry (vertical excitation, Franck−Condon principle). The

components of the transitions are listed in Table 3, and the

frontier molecular orbitals involved in the transitions are pres-

ented in Figure 10.

observed. N-Methylmaleimide was used as control to

demonstrate that the emission observed is due to the presence

of intracellular thiols.

2.6. DFT/TDDFT Calculations: Rationalization of the

Fluorescence OFF−ON Effect of the Thiol Probe BS-2. In

recent years, fluorescent molecular probes have been investi-

gated extensively.^26,24eHowever, the fluorescence transduction

We observed two main absorption bands for BS-2 with the

calculations, transitions of S₀→S₄and S₀→S₉), which are

located at 492 and 398 nm, respectively. These calculated

absorption bands are in good agreement with experimental

results (527 and 401 nm, Figure 7). The S₀→S₁transition of

the BS-2 is a forbidden transition. The molecular orbitals that

are involved in the transition are HOMO→LUMO, which

Table 3. Selected Parameters for the Vertical Excitation (UV−vis Absorptions) and the Emission of Thiol Probe BS-2 and BS-2-

OH; Electronic Excitation Energies (eV) and Oscillator Strengths (f), Configurations of the Low-Lying Excited States of the

Compounds; Calculation of the S₀−S₁Energy Gaps Based on Optimized Ground-State Geometries (UV-vis Absorption) and

the Optimized Excited-State Geometries (Fluorescence Emission) (Methanol Used as Solvent, Calculated at TDDFT//B3LYP/

6-31G(d) Level with Gaussian 09W)

TDDFT//B3LYP/6-31G(d)

a

b

c

d

compd

electronic transitions

excitation energy

2.54 eV (489 nm)

f

composition

CI

e

BS-2-OH

absorption

S₀→S₁

0.5846

0.4322

H-2→L

H→L

0.2167

0.6731

0.6690

0.2202

0.7031

0.7061

0.2253

0.6690

0.6660

0.2289

0.7067

0.7033

S₀→S₄

3.14 eV (395 nm)

H-2→L

H→L

f

emission

S₁→S₀

S₀→S₁

S₀→S₄

2.00 eV (620 nm)

1.84 eV (675 nm)

2.52 eV (492 nm)

0.6154

0.0001

0.5611

H→L

e

BS-2

absorption

H→L

H-2→L+2

H→L+2

H-2→L+2

H→L+2

H→L

S₀→S₉

3.12 eV (398 nm)

0.4369

f

emission

S₁→S₀

0.92 eV (1353 nm)

2.18 eV (569 nm)

0.0002

0.8694

g

S₅→S₀

H→L+2

a

b

c

Only selected excited states were considered. The numbers in parentheses are the excitation energy in wavelength. Oscillator strength. H stands

d

for HOMO and L stands for LUMO. Only the main configurations are presented. Coefficient of the wave function for each excitations. The CI

coefficients are in absolute values. The calculations on UV−vis absorption are based on the optimized ground-state geometry. The calculations on

fluorescence emissions are based on the optimized S₁state geometry. The emission was calculated at the optimized S₅state geometry.

e

f

g

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most probably responsible for the emission of probe BS-2 at

619 nm (the calculated emission wavelength is 631 nm with

optimized S₅state geometry). Emission from higher excited

state other than the S₁state usually is weak, especially with the

S₁state as a dark excited state because the nonemissive internal

conversion (IC) is competitive to the radiative decay process of

S₅→S₀.^10b,14

In the presence of thiols (such as cysteine), the emission of

probe is switched on after cleavage of the DNBS moiety (which

is a strong intramolecular electron trap) (Figure 7). In order to

rationalize this fluorescence switching ON effect, the UV−vis

absorption and the fluorescence of the released fluorophore

were also studied with the DFT/TDDFT calculations (Figure 11

and Table 3).

Figure 10. Rationalization of the UV−vis absorption and the weak

emission of BS-2: the frontier molecular orbitals (MOs) involved in

the vertical excitation (i.e., UV−vis absorption, the left two columns)

and emission (the right column). Methanol was used as the solvent for

the calculations (PCM model). The vertical excitation related

calculations are based on the optimized ground state geometry (S₀),

the emission related calculations were based on the optimized excited

state (S₁), at the B3LYP/6-31G(d)/level using Gaussian 09W. Note

the energy levels of the HOMO and LUMO at S₁state are different

from that at the S₀state. IC stands for internal conversion and CT

stand for conformation transformation. Excitation and radiative decay

processes are marked as solid lines and the nonradiative processes are

marked by dotted lines.

indicated a complete electron transfer (Figure 10). The

calculated absorption band is at 675 nm. Experimentally, we

did not observe this absorption band in the UV−vis absorption

spectra (Figure 7).

In order to study the emission of BS-2, the geometry of S₁

excited state was optimized and the emission was calculated

with the TDDFT method (usually S₁state is responsible for the

fluorescence, Kasha’s role) (Table 3). We found that S₁state is

a dark state (f = 0.0001), that is, the S₁→S₀is most probably a

forbidden transition. The calculated energy gap between the S₀

state and the S₁state is 1353 nm. This dark excited state is

responsible for the experimentally observed weak emission of

probe BS-2.

Figure 11. Rationalization of the UV−vis absorption, strong emission

and the large Stokes shift of BS-2-OH: the geometry relaxation and

the frontier molecular orbitals (MOs) involved in the vertical

excitation (i.e., UV−vis absorption, the left two columns) and

emission (the right column) of BS-2-OH. The vertical excitation

related calculations are based on the optimized geometry of the

ground state (S₀), the emission related calculations were based on the

optimized geometry of the excited state (S₁). Methanol was used as

solvent for the DFT calculations. At the B3LYP/6-31G(d)/level using

Gaussian 09W. Note the energy levels of HOMO and LUMO at S₁

state are different from that at the S₀state. CT stands for conformation

transformation. Excitation and radiative processes are marked as solid

lines and the nonradiative processes are marked by dotted lines.

However, emission at 619 nm was observed for BS-2. In

order to rationalize this emission band, we examined the higher

excited state of probe BS-2. We found that S₅→S₀transition is

Similar to the result of probe BS-2, two absorption bands at

392 and 489 nm were found for BS-2-OH, which are close to

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the experimental results (395 and 522 nm, Figure 7). The

transitions are localized on the thienyl-BODIPY framework, the

p-hydroxylphenyl moiety at the 8-position of the BODIPY core

does not contribute to the transitions. This result is in agree-

ment with the reported results of BODIPY dyes.^6a,c,28

Furthermore, the emission of fluorophore was also studied,

based on the optimized S₁excited-state geometry (Table 3 and

Figure 11). In stark contrast to the probe BS-2, the S₁excited

state of fluorophore BS-2-OH is probably an emissive excited

state (S₁→S₀, f = 0.6154). The calculated emission is at 620 nm,

which is close to the experimental results of 602 nm (Table 1,

Figure 7). Thus, the fluorescence OFF-ON switching effect can

be fully rationalized by the transformation from a dark S₁ex-

cited state of the probe to the emissive S₁excited state of the

released free fluorophore, which is the result of cleavage of

DNBS moiety by thiols, such as cysteine. To the best of our

knowledge, the studies of fluorescence emission of a fluoro-

phore with DFT calculations, based on the “electronic excited

state”, rather than “molecular orbitals”, are rare. Herein the

UV−vis absorption and the fluorescence emission are ration-

alized by a perspective of excited states, not molecular orbitals.

Our result will inspire more investigations on the molecular

probes by theoretical calculations. The in-depth understandings

of the fluorescent probe from a perspective of theoretical

chemistry will be useful for rational design of new fluorescence

OFF−ON molecular probes with predetermined photophysical

properties.

Figure 13. Fluorescence response of probe BS-1 (1.0 × 10⁻⁵M)

before and after treatment with Cys (200 equiv) for 20 min. The

emission of BS-1-OH is presented for comparison. In mixed solvent

DMF/H₂O (4:1, v/v). λ_ex= 517 nm. 25 °C.

The 8-thienyl moiety is isolated from the π-conjugation core

of the BODIPY core; thus, the thienyl moiety acts as an

electron donor. As a result, the normal photoinduced electron

transfer (PET) in the DNBS-protected fluorophore type of

thiol probe, i.e., with the fluorophore as the electron donor, is

interrupted by the thienyl moiety. The frustrated PET may be

responsible for the abnormal fluorescence transductions of

probe BS-1 and fluorophore BS-1-OH, that is, the fluorescence

ON feature of the probe and the fluorescence OFF character of

the free fluorophore. In toluene solution, the free fluorophore

BS-1-OH gives smaller fluorescence quantum yield than the

BODIPY derivatives with thienyl moieties at the 2- and 6-

positions, such as BS-SS and BS-DS, etc. (Table 1). These

features are opposite to the normal properties of the DNBS

thiol probe; i.e., the probe is usually nonfluorescent but the free

fluorophore is fluorescent.^15a,b,d,29

2.7. Thiol Probe with Thienyl Substitution at the 8-

Position: Small Stokes Shift, Opposite Electron-Transfer

Direction, and ON−OFF Fluorescence Transduction. In

order to study the effect of substitution position of thienyl

moiety on the photophysical properties of the thiol probe,

fluorophore BS-1-OH was used to prepare a thiol probe BS-1.

The UV−vis absorption of BS-1 show maxima at 512 nm

(Figure 12a). The probe BS-1 shows an emission band at

Probe BS-1 shows faster recognition kinetics than probe BS-

2 (Figure 14). The selectivity of the probe toward different

thiols and other biologically related analytes, such as glycine,

etc., was also studied (Figure 14b). Good selectivity was found

for the probe toward thiols over other biologically related

molecules.

In order to rationalize the emission property of probe BS-1

and the fluorophore BS-1-OH, the UV−vis absorption and the

fluorescence emission were studied with DFT calculations

(Figure 15 and Table 1). The S₁state of BS-1-OH is a dark

state, which means that the direct photoexcitation into S₁state

(S₀→S₁transition) and radiative S₁→S₀transition are for-

bidden, thus this fluorophore is most probably nonfluorescent.

The calculated UV−vis absorption band of BS-1-OH is located

at 419 nm.

By examination of the molecular orbitals involved in the S₁

excited state, it is clear that the thienyl moiety is an electron

donor for PET (Figure 15). Thus the lack of fluorescence of

fluorophore BS-1-OH is due to the photoinduced electron

transfer from the thienyl moiety to the BODIPY chromophore.

The UV−vis absorption and emission of probe BS-1 were

also studied with the TDDFT methods (Figure 16 and Table 4).

The calculated UV−vis absorption are close to the experimen-

tal results. For the emission, we observed a dark excited state

(S₁state) (f = 0.0000). However, the radiative decay from S₆

state is possible, and it is probably responsible for the emission of

the probe at 528 nm (Figure 16).

2.8. Conclusion. In conclusion, new BODIPY fluorophores

with thienyl substituents at the 2- or 2,6-position of BODIPY

core (via C−C single bond connection) that show large Stokes

shifts (96 nm) were prepared by a de novo approach, that is,

by increasing the geometry relaxation of the fluorophore upon

Figure 12. (a) UV−vis absorption spectra of probe BS-1 (1.0 × 10⁻⁵M)

before and after treatment with Cys (200 equiv) for 20 min. The

absorption of BS-1-OH is presented for comparison, 25 °C. (b) Emission

and excitation spectra of probe BS-1 (1.0 × 10⁻⁵M). In mixed solution

(DMF/H₂O = 4/1, v/v). λ_ex= 517 nm, 25 °C.

528 nm; thus, a small Stokes shift of 16 nm was observed,

which is in stark contrast to the large Stokes shift of probe BS-

2. These results imply that the 8-thienyl substitution is not

involved in the frontier molecular orbitals of BS-1.^6aThis con-

clusion is supported by a similar thienyl-substituted BODIPY

fluorophore, which shows similar emission wavelength and

small Stokes shifts (<30 nm).^6g

Interestingly, the fluorescence of BS-1 was decreased in the

presence of thiols, such as cysteine (Figure 13). This is in stark

contrast to the fluorescence switch ON effect with probe BS-2

in the presence of thiols (Figure 7).

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Figure 14. (a) Reaction kinetic curve of BS-1 (1.0 × 10⁻⁵M) with 200 equiv of Cys. (b) Fluorescence response of BS-1 (1.0 × 10⁻⁵M) to

different analytes before and after incubation in the presence of 200 equiv of analytes for 180 min in DMF/water (4/1, v/v). λ_ex= 517 nm.

λ_em= 526 nm. 37 °C.

cm⁻¹at 513 nm), and red emission (λ_em= 591 nm, Φ_F= 20.6%

in toluene). Control compounds with thienyl substituents at 8-

position show the typical small Stokes shift (ca. 12 nm). DFT

and TDDFT calculations proved that large geometry relaxation

upon photoexcitation is responsible for the large Stokes shifts.

Conversely, small geometry relaxation upon photoexcitation

leads to small Stokes shifts.

The new BODIPY derivatives were used for design of thiol

probes based on the 2,4-dinitrobenzenesulfonyl (DNBS) pro-

tection group (the fluorescence is modulated by the attachment

or removal of DNBS moiety from the fluorophore). With

different thienyl substitution profiles, fluorescence OFF−ON

and ON−OFF responses were observed for the probes. For

the probe with 2,6-dithienyl, thienyl moiety is involved in

π-conjugation framework and thus the thienyl/BODIPY core

acts as an integrated electron donor. As a result fluorescence

OFF−ON switching was observed upon recognition of thiols

such as cysteine. For the probe with 8-thienyl substitution,

however, no π-conjugation between the thienyl and BODIPY

core exists, thus the isolated thienyl acts as an electron donor.

As a result, BS-1 gives fluorescence ON−OFF response upon

recognition of thiols, which is opposite to probe BS-2. To the

best of our knowledge, this is the first time that a fluorescence

ON-OFF DNBS-protected fluorophore thiol probe was

reported, illustrating the elusive photophysics of the thiol probes

that based on the DNBS protection strategy.

Our de novo approach to access large Stokes shift of BODIPY

derivatives by increasing the geometry relaxation of the fluoro-

phores upon photoexcitation, the design of the thiol probes, the

Figure 15. Rationalization of the UV−vis absorption and non-

fluorescent feature of BS-1-OH: Frontier molecular orbitals (MOs)

fluorescence ON−OFF effect of the DNBS-based thiol probe,

involved in the vertical excitation (i.e., UV−vis absorption, the left two

rationalization of the photophysical properties by DFT calcula-

tions, is useful for design of fluorophores that show large Stokes

shift, as well as the related fluorescent molecular probes.

columns) and emission (the right column) of BS-1-OH. Methanol

was used as the solvent (PCM model). The vertical excitation related

calculations are based on the optimized ground state geometry

(S₀state), the emission related calculations were based on the opti-

mized excited state (S₁state), at the B3LYP/6-31G(d) level using

Gaussian 09W. Note the energy levels of HOMO and LUMO at S₁

state are different from that at S₀state. CT stands for conformation

transformation. Excitation and radiative processes are marked as solid

lines and the nonradiative processes are marked by dotted lines. The

lack of emission of BS-1-OH is due to the dark excited state (S₁).

3. EXPERIMENTAL SECTION

3.1. Synthesis of 1. To the solution of 4-iodophenol (1.1 g,

5 mmol) in toluene (25 mL), ethanol (25 mL), 5-formylthiophene-2-

boronic acid (1.56 g, 10 mmol) and K₃PO₄·3H₂O (2.67 g, 10 mmol)

were added. Pd(OAc)₂(58.4 mg, 0.6 mmol) was added under Ar. The

mixture was refluxed at 80 °C for 8 h. After complete consumption of

the starting material, the solution was evaporated under reduced pres-

sure. The residual was purified by column chromatography (silica gel,

CH₂Cl₂/CH₃OH = 100:1, v/v). A red solid was obtained, 410.0 mg,

photoexcitation. BODIPY with thienyl substitution at 2-position,

BS-SS, shows strong absorption in the green range (ε = 69000 M⁻¹

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with saturated NaHCO₃(100 mL) and water (100 mL). The

combined organic layers were dried over anhydrous Na₂SO₄, and the

solvent was evaporated under reduced pressure. The crude product

was purified by column chromatography (silica gel, CH₂Cl₂) to obtain

a red powder (235.6 mg), yield 30.0%. Mp >250 °C. ¹H NMR (400 M

Hz, CDCl₃): δ 7.52 (d, 2H, J = 8.6 Hz), 7.21 (d, 1H, J = 3.7 Hz); 6.91

(d, 1H, J = 3.7 Hz), 6.86 (d, 2H, J = 8.5 Hz), 6.01 (s, 2H), 4.98

(s, 1H), 2.56 (s, 6H), 1.73 (s, 6H); ¹³C NMR (100 MHz, CDCl₃):

δ 161.1, 159.9, 151.0, 147.6, 138.0, 136.4, 135.8, 132.7, 131.1, 129.2,

125.7, 125.4, 119.7, 18.3, 17.6. TOF HRMS ES⁺(C₂₃H₂₁N₂BF₂OS)

calcd 422.1436, found 422.1440.

3.3. Synthesis of BS-1. To the solution of BS-1-OH (60.0 mg,

0.14 mmol) in a mixture of CH₂Cl₂(5 mL) and TEA (0.1 mL) was

added dropwise 2,4-dinitrobenzenesulfonyl chloride (DNBS, 112.0 mg,

0.42 mmol) in CH₂Cl₂(5 mL). The mixture was stirred at rt for 4 h.

When TLC showed that the reaction was completed, the reaction

mixture was washed with water (3 × 20 mL) and the aqueous layers

were extracted with CH₂Cl₂. The combined organic layers were dried

over anhydrous Na₂SO₄and evaporated under reduced pressure. The

crude product was purified by column chromatography (silica gel, n-

hexane/CH₂Cl₂= 2/3, v/v) to give a red powder (45.6 mg), yield

1

50.0%. H NMR (400 MHz, CDCl₃): δ 8.68 (d, 1H, J = 2.0 Hz),

8.53− 8.51 (m, 1H), 8.26 (d, 1H, J = 8.5 Hz), 7.64 (d, 2H, J = 8.8 Hz),

7.33 (d, 1H, J = 3.7 Hz), 7.27 (d, 2H, J = 8.8 Hz), 6.98 (d, 1H, J = 3.7

Hz), 6.02 (s, 2H), 2.56 (s, 6H), 1.70 (s, 6H). Mp 130.8 °C−132.4 °C.

¹³C NMR (100 MHz, CDCl₃): δ 156.5, 144.3, 143.3, 135.1, 133.9,

133.7, 129.2, 127.4, 126.4, 124.3, 122.7, 121.7, 120.4, 14.7, 13.9. TOF

LD⁺(C₂₉H₂₃BN₄F₂S₂O₇) calcd 652.1069, found 652.1130.

3.4. Synthesis of 2. The synthesis procedure is similar to that of

BS-1-OH. The crude product was purified by column chromatography

(silica gel, CH₂Cl₂) to obtain a red solid powder (1.22 g), yield 40.0%.

¹H NMR (400 M Hz, CDCl₃): δ 7.14 (d, 2H, J = 8.5 Hz), 6.96 (d, 2H,

J = 8.5 Hz), 5.98 (s, 2H), 5.04 (s, 1H), 2.55 (s, 6H), 1.44 (s, 6H). ¹³C

NMR (100 MHz, CDCl₃and CD₃OD): δ 157.6, 155.0, 143.4, 142.5,

131.9, 129.1, 125.8, 121.1, 116.1, 46.7, 14.5. TOF HRMS ES⁺

(C₁₉H₁₉BN₂OF₂): calcd 340.1559, found 340.1570.

3.5. Synthesis of 3. Compound 2 (200.0 mg, 0.58 mmol) was

dissolved CH₂Cl₂(10 mL). N,N-Diisopropylethylamine (i-Pr₂NEt)

(1.28 mmol, 0.21 mL) and chloromethyl ether (MOMCl, 0.87 mmol,

65.2 μL) were added. The reaction mixture was stirred at 60 °C for 6

h. When TLC (n-hexane/CH₂Cl₂= 1/2 as eluent) showed that the

reaction was complete, a saturated NH₄Cl solution (10 mL) was added

in an ice−water bath and the mixture was stirred for 10 min. The

solution was extracted with CH₂Cl₂and dried over anhydrous Na₂SO₄.

The solvent was evaporated under reduced pressure. The crude

product was purified by column chromatography (silica gel, n-hexane/

Figure 16. Rationalization of the UV−vis absorption, emission and the

small Stokes shift of BS-1: frontier molecular orbitals (MOs) involved

in the vertical excitation (i.e., UV−vis absorption, the left two

columns) and emission (the right column) of BS-1. Methanol was

used as solvent for DFT calculations. The vertical excitation related

calculations are based on the optimized ground-state geometry

(S₀state), the emission related calculations were based on the optimi-

zed excited state (S₁state), at the B3LYP/6-31G(d)/level using

Gaussian 09W. CT stands for conformation transformation. Excitation

and radiative processes are marked as solid lines, and the nonradiative

processes are marked by dotted lines.

1

CH₂Cl₂= 1/2, v/v) to give red powder (111.4 mg), yield 50.0%. H

NMR (400 M Hz, CDCl₃): δ 7.19−7.13 (m, 4H); 5.98 (s, 2H); 5.23

(s, 2H); 3.52 (s, 3H); 2.55 (s, 6H); 1.43 (s, 6H). ¹³C NMR (100

MHz, CDCl₃): δ 157.9, 155.3, 143.1, 141.7, 131.8, 129.2, 128.3, 121.1,

116.8, 94.6, 56.2, 29.7, 14.5. TOF HRMS ES⁺(C₂₁H₂₃BN₂O₂F₂):

calcd 384.1821, found 384.1827.

yield 40.2%. ¹H NMR (400 MHz, CDCl₃): δ 9.86 (s, 1H); 7.72(d, 1H,

J = 3.8 Hz), 7.58 (d, 2H, J = 8.5 Hz), 7.30 (d, 1H, J = 4.0 Hz); 6.91(d,

2H, J = 8.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 183.3, 158.6, 155.7,

140.6, 138.6, 127.9, 127.1, 124.5, 122.7, 121.2, 116.0. TOF MS EI⁺

(C₁₁H₈O₂S): calcd 204.0245, found 204.0252.

3.2. Synthesis of BS-1-OH. Compound 1 (400.0 mg, 1.86 mmol)

was dissolved in THF (100 mL). 2,4-Dimethylpyrrole (0.72 mL,

5.9 mmol) and several drops of trifluoroacetic acid were added under

Ar atmosphere. The reaction mixture was stirred at rt overnight, a

solution of 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) (0.46 g,

1.96 mmol) in THF (100 mL) was added, and the mixture was stirred

for 4 h. Triethylamine (TEA) (5.9 mL) and BF₃·OEt₂(5.9 mL) were

consecutively added dropwise to the mixture cooled by an ice−water

bath. The mixture was stirred overnight until thin-layer chromato-

graphy (TLC) (CH₂Cl₂/n-hexane =10/1, v/v) showed the reaction

was completed. The solvent was removed under reduced pressure. The

residue was dissolved in CH₂Cl₂, and the organic phase was washed

3.6. Synthesis of 4. To the solution of compound 3 (100.0 mg,

0.26 mmol) in CH₂Cl₂(10 mL) was added N-iodosuccinimide (NIS)

(572.4 mg, 2.54 mmol), and the mixture was stirred for 2 h. After

completely consumption of 3, the solvent was evaporated under

reduced pressure. The residual was purified by column chromato-

graphy (silica gel, n-hexane/CH₂Cl₂= 1/1, v/v) to give a red solid

1

(132.3 mg), yield 80.0%. H NMR (400 MHz, CDCl₃): δ 7.19 (m,

4H); 5.25 (s, 2H); 3.53 (s, 2H); 2.64 (s, 6H); 1.45 (s, 6H). ¹³C NMR

(100 MHz, CDCl₃): δ 158.3, 156.7, 145.4, 141.4, 131.6, 129.1, 127.9,

117.1, 94.6, 56.2, 29.7, 17.2, 16.0. TOF HRMS ES⁺(C₂₁H₂₁N₂O₂BF₂I₂):

calcd 635.9754, found 635.9757.

3.7. Synthesis of 5. The synthesis procedure is similar to that of 1.

The crude product was purified by column chromatography (silica gel,

n-hexane/CH₂Cl₂= 2/1, v/v) to give a red solid (58.8 mg), yield

67.0%. ¹H NMR (400 MHz, CDCl₃): δ 7.35 (d, 2H, J = 5.2 Hz), 7.24

(d, 2H, J = 8.6 Hz), 7.17 (d, 2H, J = 5.8 Hz), 7.09 (t, 2H, J = 3.5 Hz),

6.86 (d, 2H, J = 3.5 Hz), 5.23 (s, 2H), 3.50 (s, 2H), 2.60 (s, 6H), 1.44

(s, 6H). ¹³C NMR (100 MHz, CDCl₃): δ 158.1, 155.1, 142.5, 140.8,

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Table 4. Selected Parameters for the Vertical Excitation (UV−vis Absorptions) and the Emission of the BODIPY Derivatives:

Electronic Excitation Energies (eV) and Oscillator Strengths (f ), Configurations of the Low-Lying Excited States of the

Compounds; Calculation of the S₀/S₁Energy Gaps Based on the Optimized Ground-State Geometries (UV−vis Absorption)

and the Optimized Excited-State Geometries (Fluorescence) (Methanol Was Used as Solvent, Calculated at TDDFT//B3LYP/

6-31G(d) Level with Gaussian 09W)

TDDFT//B3LYP/6-31G(d)

a

b

c

d

compound

electronic transitions

excitation energy

f

composition

CI

e

BS-1-OH

absorption

S₀→S₁

S₀→S₂

2.43 eV (511 nm)

2.85 eV (436 nm)

0.0032

0.5686

H-1→L

H-2→L

H→L

0.6930

0.1240

0.6943

0.7021

0.7055

0.7014

0.7020

0.6964

0.3135

0.6317

0.7068

0.1250

0.6997

0.7069

0.7070

f

BS-1-OH

emission

S₁→S₀

S₂→S₀

S₃→S₀

S₄→S₀

S₅→S₀

S₆→S₀

1.81 eV (686 nm)

2.59 eV (478 nm)

3.03 eV (409 nm)

3.29 eV (376 nm)

3.45 eV (359 nm)

3.57 eV (347 nm)

0.0001

0.7776

0.0290

0.0136

1.1380

0.0005

H→L

H-1→L

H-2→L

H-3→L

H→L+1

H-6→L

H-4→L

H→L

e

BS-1

absorption

emission

S₀→S₁

S₀→S₅

1.98 eV (626 nm)

2.84 eV (436 nm)

0.0000

0.5650

H-2→L+2

H→L+2

H-3→L

H→L

S₀→S₆

2.31 eV (535 nm)

1.16 eV (1068 nm)

2.53 eV (491 nm)

0.0000

0.0001

0.7213

f

S₁→S₀

g

S₆→S₀

H→L+2

0.7018

a

b

c

Only selected excited states were considered. The numbers in parentheses are the excitation energy in wavelength. Oscillator strength. H stands

d

for HOMO and L stands for LUMO. Only the main configurations are presented. Coefficient of the wave function for each excitations. The CI

coefficients are in absolute values. The calculations on UV−vis absorption are based on the optimized ground-state geometry. The calculations on

fluorescence are based on the optimized S₁state geometry. The calculations on fluorescence are based on the optimized S₆state geometry.

e

f

g

134.3, 131.6, 129.3, 128.3, 127.7, 127.2, 126.0, 171.0, 94.6, 56.2, 29.7,

13.5, 13.1. TOF HRMS ES⁺(C₂₉H₂₇N₂O₂F₂S₂B): calcd 548.1575,

found 548.1577.

3.10. Synthesis of 6. Benzoyl chloride (1.4 g, 11 mmol) was

dissolved in CH₂Cl₂(150 mL), and 2,4-dimethylpyrrole (2.0 mL,

20 mmol) was added under Ar atmosphere. After the reaction mix-

ture was stirred at rt overnight, triethylamine (TEA) (10 mL) and

BF₃·OEt₂(10 mL) were added dropwise in an ice−water bath, and the

mixture was stirred overnight until thin-layer chromatography (TLC)

(CH₂Cl₂) showed that the reaction was completed. The solvent

was removed under reduced pressure to obtain a residue which was

dissolved in CH₂Cl₂and washed with saturated Na₂CO₃(100 mL)

and water (100 mL). The combined organic layers were dried over

anhydrous Na₂SO₄and evaporated under reduced pressure. The crude

product was purified by column chromatography (silica gel, CH₂Cl₂)

3.8. Synthesis of BS-2-OH. To the solution of 5 (77.1 mg, 0.14

mmol) in ethanol (10 mL) was added 6 mol/L of hydrochloric acid

(42 μL, 0.25 mmol) in an ice−water bath, and the mixture was stirred

at 60 °C for 4 h. The solvent was evaporated under reduced pressure.

The residue was washed with water (3 × 20 mL), followed by

extraction with CH₂Cl₂. The organic layers were dried over anhydrous

Na₂SO₄and evaporated under reduced pressure. The crude product

was purified by column chromatography (silica gel, n-hexane/CH₂Cl₂=

1/1, v/v) to give a red powder (40.0 mg), yield 56.7%. mp >250 °C. ¹H

NMR (400 MHz, CDCl₃): δ 7.36 (d, 2H, J = 5.5 Hz), 7.20 (d, 2H, J =

8.3 Hz), 7.10 (t, 2H, 4.0 Hz), 6.99 (d, 2H, J = 7.5 Hz), 6.86

(s, 2H), 2.60 (s, 6H), 1.45 (s, 6H). ¹³C NMR (100 MHz, CDCl₃):

δ 151.9, 149.1, 146.4, 140.7, 126.9, 124.7, 123.4, 91.9, 29.3, 26.8, 26.6,

20.2, 11.6. TOF HRMS ES⁺(C₂₇H₂₃N₂OS₂BF₂): calcd 504.1313, found

504.1323.

1

to obtain a red powder (1.96 g), yield 55.0%. H NMR (400 MHz,

CDCl₃): δ 7.49−7.47 (m, 3H), 7.29−7.26 (m, 2H), 5.98 (s, 2H), 2.56

(s, 6H), 1.37 (s, 6H).

3.11. Synthesis of 7. To the solution of 6 (200.0 mg, 0.62 mmol)

in CH₂Cl₂(50 mL) was added dropwise N-iodosuccinimide (NIS)

(140.0 mg, 0.62 mmol) in CH₂Cl₂(20 mL), and the mixture was

stirred for 1 h. After complete consumption of 6, the reaction mixture

was evaporated under reduced pressure. The residual was purified by

column chromatography (silica gel, n-hexane/CH₂Cl₂= 2/1, v/v) to

3.9. Synthesis of BS-2. To the solution of BS-2-OH (35.0 mg,

0.10 mmol) in CH₂Cl₂(5 mL) and TEA (0.1 mL) was added

dropwise a solution of 2,4-dinitrobenzenesulfonyl chloride (DNBS.

60.0 mg, 0.3 mmol) in CH₂Cl₂(5 mL), and the mixture was stirred at

rt for 3 h. When TLC showed complete consumption of the starting

material, the solution was washed with water (3 × 20 mL), and the

aqueous layers were extracted with CH₂Cl₂. The combined organic

layers were dried over anhydrous Na₂SO₄and evaporated under

reduced pressure. The crude product was purified by column

chromatography (silica gel, n-hexane/CH₂Cl₂= 3/2, v/v) to give

1

give a bright red solid (125.6 mg), yield 45.0%. H NMR (400 MHz,

CDCl₃): δ 7.51−7.48 (m, 3H), 7.27−7.25 (m, 2H), 6.04 (s, 1H), 2.63

(s, 3H), 2.57 (s, 3H), 1.38 (s, 6H). TOF LD⁺([C₁₉H₁₈BF₂IN₂]⁺):

calcd 450.0576, found 450.0535.

3.12. Synthesis of 8. The synthesis procedure is similar to that of

4. The crude product was purified by column chromatography (silica

gel, n-hexane/CH₂Cl₂= 1/1_,v/v). Red solid was obtained (137.5 mg),

1

yield 77.0%. H NMR (400 MHz, CDCl₃): δ 7.54−7.51 (m, 3H),

1

7.26−7.24 (m, 2H), 2.65 (s, 6H), 1.38 (s, 6H). TOF LD⁻

a red powder (25.0 mg), yield 34.0%. Mp: 96.3−99.5 °C. H NMR

([C₁₉H₁₇BF₂I₂N₂]⁻): calcd 575.9542, found 575.9528.

(400 MHz, CDCl₃): δ 8.67 (s, 1H), 8.47 (d, 1H, J = 8.7 Hz), 8.18

(d, 2H, J = 8.5 Hz), 7.41 (s, 4H), 7.38 (d, 2H, J = 4.1 Hz), 7.11 (t, 2H,

J = 3.7 Hz), 6.86 (d, 2H, J = 2.6 Hz), 2.59 (s, 6H), 1.34 (s, 6H).

¹³C NMR (100 MHz, CDCl₃): δ 156.2, 151.1, 149.6, 149.4, 140.1,

135.5, 133.8, 133.8, 133.4, 131.0, 130.4, 128.0, 127.4, 127.0, 126.3,

123.3, 120.5, 41.9, 31.6, 29.7, 22.7, 13.6, 13.2. TOF MS ES⁺

(C₃₃H₂₄BN₄O₇F₂S₃Na⁺) calcd 757.0844, found 757.0531. TOF LD⁻

(C₃₃H₂₃BN₄0₇F₂S₃⁺): calcd 733.0868, found 733.0892.

3.13. Synthesis of BS-SS. The synthesis procedure is similar to

that of 1. The crude product was purified by column chromatography

(silica gel, n-hexane/CH₂Cl₂= 1/1, v/v) to give a red solid (56.6 mg),

yield 63.4%. Mp: 155.2−157.2 °C. ¹H NMR (400 MHz, CDCl₃):

δ 7.50 (d, 3H, J = 5.9 Hz); 7.33−7.30 (m, 3H); 7.08 (t, 1H, J = 3.6

Hz); 6.84 (d, 1H, J = 3.40 Hz); 6.02 (s, 1H), 2.58 (s, 6H), 1.39 (d, 2H,

J = 11.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 156.7, 154.3, 144.1,

2204

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142.3, 140.3, 135.2, 134.7, 132.1, 130.9, 129.4, 129.2, 128.1, 127.8,

127.3, 126.0, 121.9, 30.5, 14.9, 14.7, 13.5, 12.9. TOF HRMS ES⁺

(C₂₃H₂₁BF₂N₂S): calcd 406.1487, found 406.1498.

Hochstrasser, R. M.; Burgess, K. Chem.Eur. J. 2003, 9, 4430−4441.

(d) Azov, V. A.; Schlegel, A.; Diederich, F. Angew. Chem., Int. Ed. 2005,

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F.; Ziessel, R.; Campagna, S. Chem.Eur. J. 2008, 14, 4381−4392.

(f) Zhu, M.; Jiang, L.; Yuan, M.; Liu, X.; Yang, C.; Zheng, H.; Yin, X.;

Zuo, Z.; Liu, H.; Li, Y. J. Polym. Sci.: Part A: Polym. Chem. 2008, 46,

7401−7410. (g) Rachford, A. A.; Ziessel, R.; Bura, T.; Retailleau, P.;

Castellano, F. N. Org. Lett. 2008, 10, 1581−1584. (h) Nastasi, F.;

Puntoriero, F.; Campagna, S.; Diring, S.; Ziessel, R. Phys. Chem. Chem.

Phys. 2008, 10, 3982−3986. (i) Belman, N.; Israelachvili, J. N.; Li, Y.;

Safinya, C. R.; Bernstein, J.; Golan, Y. J. Am. Chem. Soc. 2009, 131,

9007−9013. (j) Descalzo, A. B.; Xu, H; Xue, Z.; Hoffmann, K.; Shen,

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Psycharis, V.; Saridakis, E.; Griebel, J.; Hermann, R.; Pistolis, G. J. Am.

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Ng, D. K. P. Chem.Eur. J. 2011, 17, 1605−1613. (o) Gresser, R.;

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17, 2939−2947.

3.14. Synthesis of BS-DS. The synthesis procedure is similar to

that of 1. The crude product was purified by column chromatography

(silica gel, n-hexane/CH₂Cl₂= 1/1, v/v) to obtain a red solid (45.1

mg), yield 54.4%. Mp: >250 °C. ¹H NMR (400 MHz, CDCl₃): δ 7.51

(d, 3H, J = 6.6 Hz), 7.35−7.33 (m, 4H), 7.09 (t, 2H, J = 3.7 Hz), 6.86

(d, 2H, J = 3.5 Hz), 2.60 (s, 6H), 1.38 (s, 6H). ¹³C NMR (100 MHz,

CDCl₃): δ 155.5, 141.0, 129.5, 128.2, 127.9, 127.4, 126.2, 29.9, 13.7,

13.1. TOF HRMS ES⁺(C₂₇H₂₃BN₂F₂S₂): calcd 488.1364, found

488.1385.

3.15. Fluorescent Imaging of the Intracellular Thiols. The

photographs were obtained using a Nikon ECLIPSE-Ti confocal laser

scanning microscopy. The Hela cells were incubated with probes BS-2

in PBS (phosphate buffered solution, pH 7.4) for 45 min at 37 °C, and

red emission was observed with 543 nm laser excitation. In the control

experiments, cells were incubated in the presence of 0.5 mM

N-methylmaleimide in DMSO−PBS solution (1:200, v/v) for 1 h to

remove the intracellular thiols. Then the cells were washed with PBS

buffer for three times, and the probe (20 μM, 0.4: 200 DMSO/PBS,

v/v, pH 7.4) was added and incubated at 37 °C for 45 min.

Fluorescence imaging was taken after the cells were washed with PBS

buffer for three times.

3.16. DFT Calculations. The ground-state structures of BODIPY

derivatives and the thiol probes were optimized using DFT with

B3LYP functional and 6-31G (d) basis set. The initial geometries of

the compounds were generated by the GaussView software. The

excited-state related calculations (UV−vis absorptions) were carried

out with the time-dependent DFT (TDDFT) with the optimized

structure of the ground state (DFT/6-31G(d)). The emission of the

fluorophores were calculated based on the optimized S₁excited state

geometry, optimized by the TDDFT method. In some cases, higher

excited states were optimized. There are no imaginary frequencies in

frequency analysis of all calculated structures. All of these calculations

were performed with Gaussian 09W.³⁰

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ASSOCIATED CONTENT

* Supporting Information

■

F. A.; García-Moreno, I.; Costela, A.; Martin, V.; Sastre, R.; Banuelos,

̃

S

J.; Arbeloa, F. L.; Arbeloa, I. L.; Pena-Cabrera, E. Chem. Commun.

̃

Synthesis and structural characterization data of the fluo-

rophores and the probes; solvent-dependent fluorescence

spectra and DFT calculation information. This material is

available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: zhaojzh@dlut.edu.cn. Group homepage: http://finechem.

dlut.edu.cn/photochem.

■

A.; Perez-Bolívar, C. Jr.; Burgess., P. A. J. Am. Chem. Soc. 2011, 133,

́

51−55. (d) Lazarides, T.; McCormick, T. M.; Wilson, K. C.; Lee, S.;

McCamant, D. W.; Eisenberg, R. J. Am. Chem. Soc. 2011, 133, 350−

364.

Notes

The authors declare no competing financial interest.

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M.; O’Shea, D. F. J. Am. Chem. Soc. 2004, 126, 10619−10631.

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F. N. J. Am. Chem. Soc. 2008, 130, 16164−16165. (b) Singh-Rachford,

T. N.; Castellano, F. N. Coord. Chem. Rev. 2010, 254, 2560−2573.

(c) Wu, W.; Guo, H.; Wu, W.; Ji, S.; Zhao, J. J. Org. Chem. 2011, 76,

7056−7064. (d) Zhao, J.; Ji, S.; Guo, H. RSC Adv. 2011, 1, 937−950.

(e) Ji, S.; Wu, W.; Wu, W.; Guo, H.; Zhao, J. Angew. Chem., Int. Ed.

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ACKNOWLEDGMENTS

■

We thank the NSFC (20972024 and 21073028), the Royal Society

(UK) and NSFC (China−UK Cost-Share Science Networks,

21011130154), the Fundamental Research Funds for the Central

Universities (DUT10ZD212 and DUT11LK19), the State Key

laboratory of Fine Chemicals (KF0802 and KF0901), Ministry of

Education (SRFDP-200801410004 and NCET-08-0077), and

Dalian University of Technology for financial support.

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Geometry relaxation-induced large stokes shift in red-emitting borondipyrromethenes (BODIPY) and applications in fluorescent thiol probes

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