Meso-ester and carboxylic acid substituted BODIPYs with far-red and near-infrared emission for bioimaging applications

Article abstract of DOI:10.1002/chem.201303868

A series of meso-ester-substituted BODIPY derivatives 1-6 are synthesized and characterized. In particular, dyes functionalized with oligo(ethylene glycol) ether styryl or naphthalene vinylene groups at the α positions of the BODIPY core (3-6) become partially soluble in water, and their absorptions and emissions are located in the far-red or near-infrared region. Three synthetic approaches are attempted to access the meso-carboxylic acid (COOH)-substituted BODIPYs 7 and 8 from the meso-ester-substituted BODIPYs. Two feasible synthetic routes are developed successfully, including one short route with only three steps. The meso-COOH-substituted BODIPY 7 is completely soluble in pure water, and its fluorescence maximum reaches around 650 nm with a fluorescence quantum yield of up to 15 %. Time-dependent density functional theory calculations are conducted to understand the structure-optical properties relationship, and it is revealed that the Stokes shift is dependent mainly on the geometric change from the ground state to the first excited singlet state. Furthermore, cell staining tests demonstrate that the meso-ester-substituted BODIPYs (1 and 3-6) and one of the meso-COOH-substituted BODIPYs (8) are very membrane-permeable. These features make these meso-ester- and meso-COOH-substituted BODIPY dyes attractive for bioimaging and biolabeling applications in living cells. Copyright

Full text of DOI:10.1002/chem.201303868

DOI: 10.1002/chem.201303868
Full Paper
&
Dyes
meso-Ester and Carboxylic Acid Substituted BODIPYs with
Far-Red and Near-Infrared Emission for Bioimaging Applications
Yong Ni,[a, b] Lintao Zeng,[c] Nam-Young Kang,[d] Kuo-Wei Huang,[e] Liang Wang,[c]
Zebing Zeng,[a] Young-Tae Chang,*[a, d] and Jishan Wu*[a, b, f]
Abstract: A series of meso-ester-substituted BODIPY deriva-
tives 1–6 are synthesized and characterized. In particular,
dyes functionalized with oligo(ethylene glycol) ether styryl
or naphthalene vinylene groups at the a positions of the
BODIPY core (3–6) become partially soluble in water, and
their absorptions and emissions are located in the far-red or
near-infrared region. Three synthetic approaches are at-
tempted to access the meso-carboxylic acid (COOH)-substi-
tuted BODIPYs 7 and 8 from the meso-ester-substituted
BODIPYs. Two feasible synthetic routes are developed suc-
cessfully, including one short route with only three steps.
The meso-COOH-substituted BODIPY 7 is completely soluble
in pure water, and its fluorescence maximum reaches
around 650 nm with a fluorescence quantum yield of up to
15%. Time-dependent density functional theory calculations
are conducted to understand the structure–optical proper-
ties relationship, and it is revealed that the Stokes shift is de-
pendent mainly on the geometric change from the ground
state to the first excited singlet state. Furthermore, cell stain-
ing tests demonstrate that the meso-ester-substituted
BODIPYs (1 and 3–6) and one of the meso-COOH-substituted
BODIPYs (8) are very membrane-permeable. These features
make these meso-ester- and meso-COOH-substituted BODIPY
dyes attractive for bioimaging and biolabeling applications
in living cells.
Introduction
avoid the use of light, which may cause phototoxicity and an
autofluorescent background.[1] Although numerous synthetic
far-red and NIR fluorophores with exceptional brightness exist,
some of them tend to be water-insoluble and membrane-im-
permeable owing to their hydrophobic character,[2] others
show poor photostability and undesired aggregation (such as
cyanine, squaraine derivatives),[3] and others exhibit unspecific
binding to cellular components because of a lack of appropri-
ate binding sites.[4]
The remarkable technological advances in the areas of bio-
imaging and sensing have created a demand for biocompat-
ible fluorescent probes with excitation and emission in the far-
red and near-infrared (NIR) region (650–900 nm), because they
[a] Y. Ni, Z. Zeng, Prof. Y.-T. Chang, Prof. J. Wu
Department of Chemistry, National University of Singapore
3 Science Drive 3, Singapore 117543 (Singapore)
Fax: (+65)6779-1691
4,4-Difluoro-4-borata-3a,4a-diaza-s-indacene, abbreviated as
BODIPY, is a well-known fluorophore that is attracting increas-
ing attention in various bioapplications because of its out-
standing photophysical properties such as its excellent envi-
ronmental stability, large molar absorption coefficients, and
sharp fluorescence spectrum with high fluorescence quantum
yield.[1b–d] For example, pH-activated fluorescence probes in-
cluding a BODIPY dye have been developed recently for the
detection of cancers and real-time therapy monitoring.[5] More-
over, new probes generated by combining a BODIPY-based
pH-sensing unit with a bisphosphonate compound have been
used for the selective detection of bone-resorbing osteoclasts
in vivo.[6] However, the undesirable optical property of most
BODIPY dyes, with absorption and emission located below
600 nm, is still one of the severe limitations for their bioimag-
ing applications. In the past few decades, a large number of
far-red and NIR BODIPYs have been synthesized through vari-
ous chemical modification methods (Figure 1): 1) functionaliza-
tion at the a, b, and meso sites of the BODIPY core to extend
p conjugation and generate a push–pull structure;[7] 2) em-
E-mail: chmcyt@nus.edu.sg
chmwuj@nus.edu.sg
[b] Y. Ni, Prof. J. Wu
NUS Environmental Research Institute, National University of Singapore
5A Engineering Drive 1, 02-01, Singapore 117411 (Singapore)
[c] Prof. L. Zeng, Prof. L. Wang
School of Chemical and Chemical Engineering
Tianjin University of Technology
Tianjin 300384 (China)
[d] Dr. N.-Y. Kang, Prof. Y.-T. Chang
Laboratory of Bioimaging Probe Development
Singapore Bioimaging Consortium, A*STAR
[e] Prof. K.-W. Huang
King Abdullah University of Science and Technology (KAUST)
Division of Physical Sciences and Engineering and
KAUST Catalysis Center
Thuwal 23955-6900 (Saudi Arabia)
[f] Prof. J. Wu
Institute of Materials Research and Engineering, A*STAR
3 Research Link, Singapore 117602 (Singapore)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201303868.
Chem. Eur. J. 2014, 20, 2301 – 2310
2301
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
through a condensation reaction at the a positions of the
BODIPY to ensure the compounds are sufficiently hydrophilic
and have good membrane permeability.[12] To the best of our
knowledge, such BODIPY dyes have never been reported, al-
though there are some BODIPYs with an ester or carboxylic
group linked by an alkyl or phenyl spacer.[13,16] In this work,
a series of meso-ester-substituted BODIPY dyes (1–6) and two
meso-carboxylic-acid-functionalized BODIPY dyes (7, 8) were
prepared successfully (Figure 2). These new dyes exhibit tuna-
ble absorption and emission spectra in the far-red or even the
NIR region, with acceptable fluorescence quantum yields and
large Stokes shifts. Most of them have the appropriate hydro-
philicity and are membrane-permeable. The successful conver-
sion of the ester group to a carboxylic group provides many
opportunities for further conjugation and specific binding
studies. Our work started from the synthesis of the simplest
meso-ester BODIPY 1, moving to the more highly p-conjugated
dye 2, then to the push–pull-type dyes 3–6 with oligo(ethylene
glycol) ether groups, and finally to the meso-COOH-substituted
BODIPY dyes 7 and 8, with the aim of tuning their absorption/
emission wavelengths, fluorescence quantum yields, Stokes
shifts, and hydrophilicities, and of introducing specific binding
sites. Time-dependent density functional theory (TD DFT) cal-
culations were conducted to gain more insight into their opti-
cal properties. Finally, cell-staining experiments were per-
formed to probe their potentials for bioimaging and biolabel-
ing applications.
Figure 1. Previous and current strategies toward functional BODIPYs with
long-wavelength absorption and emission.
ployment of p-extended pyrrole units instead of simple pyrrole
to extend the conjugation configuration at the a or b bond of
BODIPY;[8] 3) replacement of the meso-carbon with an imide-
type nitrogen atom;[9] and 4) oxidative fusion of aromatic units
at the zigzag edge to extend the p conjugation significantly.[10]
Unfortunately, most of the generated BODIPYs showed low
fluorescence quantum yields with small Stokes shifts, and
tended to be too hydrophobic and membrane-impermeable.
Moreover, the introduction of binding sites (such as carboxylic
acid), which are essential for
connection with some specific
bioprobes to these p-extended
BODIPY systems, was also a big
synthetic challenge.
Herein, we propose a new
design concept involving the in-
troduction of an ester group to
the meso position of the BODIPY
(Figure 1) on the basis of the fol-
lowing considerations: 1) the
ester group is an electron-with-
drawing group, and it is easy to
generate a push–pull-type struc-
ture and extend the p conjuga-
tion through functionalization at
the a position of the BODIPY
core, so it is possible to push the
absorption and emission wave-
lengths to the far-red or NIR re-
gions; 2) the ester group can be
hydrolyzed to form a carboxylic
acid group, which is an impor-
tant binding site for various bio-
molecules
and
biocompo-
versatile
nents,[11] and also
a
functional group for further con-
jugation; 3) hydrophilic groups
such as oligo(ethylene glycol)
ether can be introduced easily Figure 2. Chemical structures of the meso-ester- and COOH-substituted BODIPYs reported in this work.
Chem. Eur. J. 2014, 20, 2301 – 2310 www.chemeurj.org ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2302

Full Paper
tion was performed on the benzyl (Bn)-tert-butyl- and methyl-
protected ester groups, which will be discussed below
(Scheme 1).
Results and Discussion
Synthesis
Palladium-catalyzed reduction was a preferable approach for
achieving a carboxylic acid group from the benzyl ester, be-
cause it is a tidy reaction with a very high yield,[16] so benzyl-
ester-substituted BODIPY was the first choice. Benzyl-2-chloro-
2-oxoacetate 14 was obtained by adding one equivalent of
benzyl alcohol (12) dropwise to the solution of oxalyl chloride
(13) in dry chloroform.[17] The solution was then stirred at RT
for 2 h, and the resulting compound 14 was used directly for
the subsequent condensation reaction with 2,4-dimethylpyr-
role at ꢀ788C, without further purification. After complexation
with BF3·OEt2, compound 15 was separated in a total yield of
45%. A low temperature was necessary for this condensation
reaction, because the high reactivity of benzyl-2-chloro-2-oxo-
acetate generated many byproducts at RT, resulting in a much
lower overall yield of 13%. The sufficient reactivity of the 3,5-
methyl groups in 15 made functionalization at these positions
realistic. Condensation of 15 with 4-(diphenylamino)benzalde-
hyde 16 gave the p-extended BODIPY 17 in 47% yield.[18] Hy-
drogenation of 17 was catalyzed by 10% Pd/C in a H2 atmos-
phere (1 atm),[16] and thin-layer chromatography (TLC) analysis
confirmed that the benzyl group
The meso-ester-substituted BODIPYs 1 and 2 were prepared in
20% and 28% yield, respectively, through the condensation of
ethyl glyoxalate (9) with 2-ethyl pyrrole (10) or 2-(3,5-di-tert-
butylphenyl)pyrrole (11)[10a] in the presence of trifluoroacetic
acid (TFA) in dichloromethane (DCM), followed by oxidative de-
hydrogenation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) and complexation with BF3·Et2O (Scheme 1).[14] Attempt-
ed hydrolysis of the ester groups in 1 and 2 with strong bases
(e.g., KOH, NaOH, and LiOH) in highly polar solvents (THF,
MeOH, and water) at room temperature all resulted in the de-
composition of the starting materials, presumably because of
nucleophilic attack on the BꢀF bonds (see Supporting Informa-
tion).[15] Further modification and functionalization of these
two molecules was difficult. Thus, a slight structural change
was made by replacing the 2-ethyl pyrrole with 2,4-dimethyl-
pyrrole; then, functionalization was possible through conden-
sation with the corresponding aldehyde at the 3,5-methyl sites
of the resulting BODIPYs. On the other hand, in an attempt to
access meso-COOH-substituted BODIPYs, the hydrolysis reac-
was removed. However, the UV/
Vis absorption spectrum and
MALDI-TOF analysis of the prod-
uct demonstrated that the
double bonds linked to the 3,5-
positions of BODIPY were also
hydrogenated at the same time
and gave the major product 18
(Supporting Information). Alter-
natively, the benzyl group in 15
was removed through a similar
hydrogenation to give the meso-
COOH-substituted BODIPY 19 in
nearly quantitative yield. Howev-
er, the subsequent condensation
of compound 19 with 4-(diphe-
nylamino) benzaldehyde did not
occur under similar condensa-
tion conditions.
The tert-butyl ester group was
hydrolyzed quite easily under
acid conditions, so meso-tert-
butyl-ester-substituted BODIPY
could serve as another choice.
The condensation reaction be-
tween tert-butyl-2-chloro-2-oxoa-
cetate and 2,4-dimethylpyrrole
at a low temperature (ꢀ788C)
followed by oxidation and com-
plexation produced the meso-
Scheme 1. Synthesis of the meso-ester and carboxylic-acid-functionalized BODIPYs 1–8: a) i) BF3·Et2O, CH2Cl2, RT,
3 h; ii) DDQ, RT, 1 h; iii) Et3N, BF3·Et2O, RT, 1 h; b) i) 2,4-dimethylpyrrole, ꢀ788C, 4 h; ii) Et3N, BF3·Et2O, BF3·OEt2,
ꢀ788C to RT, 2 h; c) piperidine, AcOH, toluene, reflux, 12 h; d) 10% Pd/C, 1 atm H2, RT, 4 h; e) tert-butanol, DCC,
anhydrous diethyl ether, RT, 5 h; f) HCl, CH3NO2, 08C to RT, 4 h; g) LiI, ethyl acetate, reflux, 8 h.
tert-butyl-ester-substituted
BODIPY 20 in very low yield (<
5%), because the tert-butyl
Chem. Eur. J. 2014, 20, 2301 – 2310
www.chemeurj.org
2303
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
group was removed easily under acidic conditions. On the
other hand, esterification of 19 in an excess of tert-butanol
using N,N’-dicyclohexylcarbodiimide (DCC) as the dehydrating
reagent gave 20 in 85% yield. Subsequent condensation with
the corresponding oligo(ethylene glycol)-ether-substituted
benzaldehyde and naphthalene aldehyde afforded the meso-
tert-butyl ester BODIPYs 3 and 4 in 44% and 39% yields, re-
spectively. Finally, hydrolysis of 3 and 4 with HCl (aq) in nitro-
methane at room temperature[19] gave the meso-COOH
BODIPYs 7 and 8 in 28% and 22% separation yields, respec-
tively. The TLC analysis showed that some of the starting mate-
rial was decomposed; moreover, the high polarity of the car-
boxylic acid led to a significant loss of the compound during
column chromatography on a silica gel column, resulting in
a low separation yield. The generated meso-COOH-substituted
BODIPYs 7 and 8 were fully soluble in pure water thanks to
the carboxylic acid group at the meso site and the oligo(ethy-
lene glycol) ether group localized at the other terminals.
The six-step synthetic approach toward the meso-COOH-sub-
stituted BODIPYs 7 and 8 was tedious and the overall yield
was low. To simplify the synthesis, we finally developed a more
convenient approach with fewer steps and a much higher
overall yield. The meso-methyl-ester-substituted BODIPY 22
was synthesized from methyl chloro-oxalate (21) in 56% yield
by using a similar procedure to that for 15 (Scheme 1). A simi-
lar condensation reaction of 22 with the corresponding alde-
hyde gave the meso-methyl-ester-substituted BODIPYs 5 and 6
in 51% and 48% yield, respectively. The hydrolysis of the
meso-COOMe group again turned out to be the major chal-
lenge. We tried various conditions, but the use of a strong
base resulted in decomposition, and with a strong acid such as
HCl or an organic base such as triethylamine or pyridine, the
methyl group could not be removed.[20] Fortunately, after
many optimizations (see Supporting Information for details),
we found that the use of the Lewis acid lithium iodide as re-
agent and ethyl acetate as solvent under reflux were the best
conditions, providing the desired meso-COOH-substituted
BODIPYs 7 and 8 in 57% and 48% yields, respectively.[21] The
TLC analysis indicated that the conversion yield was nearly
quantitative but the separation yield was much lower owing
to the loss during purification. This approach has made the
water-soluble meso-COOH-substituted BODIPYs 7 and 8 acces-
sible in only three steps, and the separation yield of each step
is around 50%. The structures of all these compounds were in-
dentified unambiguously through NMR spectroscopy and mass
spectrometry (Supporting Information).
Figure 3. Normalized absorption (solid line), and emission (dashed line)
spectra of a) dye 1 (lex =510 nm) and b) dye 2 (lex =560 nm) in CH2Cl2.
Table 1. Summary of optical data of dyes 1–8.
max
abs
max
em
Dye
Solvent
l
log e
[Mꢀ1 cmꢀ1
l
Dn
[cmꢀ1
]
F
[nm]
]
[nm]
1
2
3
4
5
6
7
CH2Cl2
CH2Cl2
MeOH
MeOH
MeOH
MeOH
MeOH
H2O
536
592
657
680
658
682
627
628
645
641
4.70
4.40
4.80
4.89
5.08
4.83
4.48
4.36
4.78
4.38
592
653
684
729
663
727
642
646
676
680
1765
1578
601
998
115
908
373
544
711
0.78
0.17
0.19
0.11
0.06
0.06
0.36
0.15
0.15
0.03
8
MeOH
H2O
895
(labsmax =548 nm).[22] The maximum emission wavelength
(lemmax) is at 592 nm, which is much longer than that of meso-
CF3-substituted BODIPY. The absorption and emission spectra
of 1 are almost completely separate from each other with
a large Stokes shift (Dn=1765 cmꢀ1). The unusually redshifted
emission and large Stokes shift are probably associated with
the remarkable geometrical difference between the first singlet
excited state and the ground state (discussed in the DFT calcu-
lation section, vide infra).[23] In addition, 1 shows high fluores-
cence quantum yields (F=0.63 in methanol and 0.78 in DCM)
with good storage and photostability. The absorption spec-
trum of 2 is centered at 592 nm, whereas the emission maxi-
mum is located at 653 nm, also with a large Stokes shift (Dn=
1578 cmꢀ1). In comparison with 1, the aryl group at the 3,5-po-
sitions of BODIPY cause a notable bathochromic shift (60 nm)
caused by the extended p conjugation. The fluorescence quan-
Optical properties
The absorption and normalized emission spectra of 1 and 2 in
DCM are shown in Figure 3 and the data are collected in
Table 1. For dye 1, the maximum absorption wavelength
(labsmax) is located at 536 nm, which is a bathochromic shift of
40 nm compared to those of the congeners with alkyl substitu-
ents.[4] The moderate bathochromic shift can be attributed to
the inductive effect from the COOEt group, which is consistent
with the results on the meso-CF3-substituted BODIPY derivative
Chem. Eur. J. 2014, 20, 2301 – 2310
www.chemeurj.org
2304
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
rophores. These four dyes show
good solubility in highly polar
solvents such as MeOH, DMSO,
and even in PBS buffer solution
containing 2% DMSO. Mean-
while, the storage and photosta-
bility of these BODIPY dyes are
excellent. Notably, the fluores-
cence goes to the far-red and
NIR region (above 700 nm) with
an acceptable quantum yield
and large Stokes shift. All these
excellent features make these
dyes very attractive for bioimag-
ing applications.
After hydrolysis, the meso-
COOH-substituted BODIPYs
7
and were soluble in both
8
methanol and water, and a blue-
shift of their absorption and
emission spectra was observed
compared with their correspond-
ing meso-ester analogs 3–6
max
(Figure 4 and Table 1). The labs
value of 7 centered at 627 nm in
MeOH and 628 nm in pure
max
water, whereas lem
was locat-
ed at 642 nm in methanol and
646 nm in pure water, with a re-
markable fluorescence quantum
yield (F=36% in methanol and
F=15% in pure water). This can
be regarded as a significant im-
provement for BODIPY dyes, es-
pecially for fluorophores with
Figure 4. a–d) absorption (solid line) and normalized emission (dashed line) spectra of dyes 3–6 in methanol;
e,f) absorption (solid line) and normalized emission (dashed line) spectra of dye 7 and 8 in methanol and pure
water.
emission around or above
max
650 nm. For 8, labs
goes to
645 nm in methanol and 641 nm
max
in pure water, with lem
locat-
tum yield of 2 in DCM (F=0.17) is much lower than that of 1,
which can be attributed to nonirradiative energy loss arising
from the free rotation of the aryl substituents.
ed at 676 nm (F=15%) and 680 nm (F=3%), respectively.
The prominent features of these two meso-COOH-substituted
BODIPY dyes, namely, good water solubility, long wavelength
absorption and emission, appreciable fluorescence quantum
yields in pure water, the presence of carboxylic acid as a bond-
ing site, and excellent storage and photostability, make them
highly attractive for bioapplications.
After functionalization at the 3,5-positions by oligo(ethylene
glycol)-ether-substituted styryl or naphthalene vinylene
groups, the absorption and emission wavelength of dyes 3–6
in methanol displayed remarkable bathochromic shifts
(Figure 4 and Table 1). The maximum absorption wavelength
reaches approximately 658 nm for 3 and 5, with fluorescence
at 684 nm (F=19%) and 663 nm (F=6%), respectively. Dye 3
displays a larger Stokes shift (Dn=601 cmꢀ1) than 5 (Dn=
115 cmꢀ1), but both are significantly smaller than those of
1 and 2. Attachment of naphthalene groups at the 3,5-methyl
position results in an even more significant bathochromic shift,
with the absorption maxima of 4 and 6 at approximately
680 nm and fluorescence at around 728 nm (F=11% for 4
and F=6% for 6). Large Stokes shifts of 998 cmꢀ1 for 4 and
908 cmꢀ1 for 6 were observed, which are desirable for NIR fluo-
DFT calculations
To gain more insight into the optical properties of these new
meso-ester- and carboxylic-acid-substituted BODIPY dyes, we
performed time-dependent (TD) DFT calculations at the B3LYP/
6-31G*level of theory for the model compounds, as imple-
mented in the Gaussian 09 program package.[24] The geometry
optimizations for the first excited states of compounds 1 and 5
(root=1) were conducted using the default SCRF method with
the integral equation formalism variant (IEFPCM) in dichloro-
Chem. Eur. J. 2014, 20, 2301 – 2310
www.chemeurj.org
2305
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
methane and methanol, respec-
tively.[25] UV/Vis/NIR absorption
spectra were generated assum-
ing an average UV/Vis width of
3000 cmꢀ1 at half-height by
using the SWizard program.[26]
The calculated frontier molec-
ular orbital profiles and energy
levels are shown in Figure 5. For
dyes 2–8, the HOMOs are delo-
calized through the BODIPY core
to the aryl substituents, whereas
the LUMOs are located mainly
on the BODIPY core with a partial
disjoint feature, indicating an in-
tramolecular push–pull character,
which also explains the large
redshift in the absorption spec-
tra. TD DFT calculations also pre-
dicted that dyes 1–8 would
show absorption maxima at 423,
505, 596, 634, 592, 631, 594, and
634 nm (under vacuum), respec-
tively, and this trend is consis-
tent with the experimental data.
The much larger Stokes shifts
observed for 1 and 2 in compari-
son with the other dyes (3–8) is
also an interesting feature. For
a further understanding of the
origin of this difference, the
ground state (S0) and the first
excited singlet state (S1) of mol-
ecules 1 and 5 were calculated
in CH2Cl2 and MeOH, respective-
Figure 5. Calculated frontier molecular orbital profiles and the HOMO/LUMO energy levels of 1–8. For simplifica-
tion of the calculations, the ethylene glycol ether groups were replaced with methoxy groups.
ly. The optimized geometry of 1 in the S0 state shows a large
twist of the ester group from the BODIPY plane with a torsion
angle of 64.58, which becomes much smaller (1.78) in the S1
state (Figure 6). Such a significant change in geometry from
the S0 to S1 state should be ascribed to the large Stokes shift
observed in 1. On the other hand, the torsion angle of 5
changes from 87.78 in the S0 state to 61.98 in the S1 state, and
such a small change in geometry can explain the relatively
smaller Stokes shift observed for 5. The two methyl groups at
the b positions in molecules 3–8 are supposed to limit the free
rotation of the ester or carboxylic acid group, thus leading to
a small Stokes shift. The TD DFT calculations in solvents re-
vealed that 1 in CH2Cl2 would show S0!S1 absorption and
S1!S0 emission at 441 and 509 nm, respectively (Stokes
shift=3027 cmꢀ1), and 5 in MeOH would exhibit S0!S1 ab-
sorption and S1!S0 emission at 637 and 685 nm, respectively
(Stokes shift=1100 cmꢀ1). Such a trend is in reasonable agree-
ment with the experimental data. Therefore, for future design,
it would be essential to keep the two b sites of the BODIPY
core unsubstituted if a larger Stokes shift were required.
Figure 6. Calculated geometries of the ground states (S0) and first excited
states (S1) of molecules 1 and 5. For simplification of the calculations, the
ethylene glycol ether groups in molecule 5 were replaced with methoxy
groups.
Chem. Eur. J. 2014, 20, 2301 – 2310
www.chemeurj.org
2306
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Cell-staining tests
HeLa cells loaded with 1 showed a bright red cell profile.
Strong fluorescence from the cellular cytoplasm was observed
for dye-loaded HeLa cells, implying that dye 1 can diffuse effi-
ciently into HeLa cells and be retained within them, thus con-
firming its membrane permeability. However, dye 2 was not
appropriate for cell-imaging experiments owing to its high hy-
drophobicity, and thus, low solubility in cell growth media.
For the BODIPY dyes functionalized at the a positions (3–8),
the extended p conjugation induced a significant redshift in
absorption and emission, whereas the coupled oligo(ethylene
glycol) ether groups would improve the hydrophilicity by form-
ing a hydrophilic terminal within the structure. It was found
that all the meso-ester-substituted BODIPYs 3–6 could stain
HeLa cells successfully. For 3, the Texas Red filter was used be-
cause the emission wavelength is within the far-red region. A
deep red cell profile was observed, and the merged picture in-
dicated that this dye was localized mainly in the cytoplasm,
similarly to dye 1. The NIR filter was employed for the image
of the HeLa cell stained with 4 because of its emission at
727 nm (NIR region); a deep red cell profile was also observed,
with the dyes staining mainly in the cytoplasm. The cell-stain-
ing tests of dyes 5 and 6 showed similar results (the Cy5 filter
was used for 5). The cell-imaging results indicate that all four
functionalized meso-ester dyes are very cell-membrane-perme-
able, which allowed them to diffuse into the HeLa cells and be
retained within them. The cell profiles of deep red color were
observed through epifluorescent microscopy, implying that far-
red or even NIR imaging can be realized by using these meso-
ester-substituted BODIPYs. Cell-staining tests were also con-
ducted for the two water-soluble dyes 7 and 8. The experi-
mental results indicated that 7 cannot penetrate and stain
HeLa cells even if the concentration is raised to 9 mm. The
membrane impermeability of compound 7 may be attributed
to its high polarity and the hindrance from the membrane and
the negatively charged cell components, which would block its
penetration into the cell. However, when live HeLa cells were
treated with 8 (1 mm) for 1 h and an image was taken under
the Cy5 filter, a deep red stained cell profile was displayed, im-
plying that 8 can diffuse efficiently into HeLa cells and remain
within them.
Encouraged by the outstanding features of the meso-ester-
and meso-COOH-substituted BODIPYs, we next employed an
epifluorescent microscope to examine the imaging ability of
these dyes in living HeLa cells (Figure 7). Each dye (1 mm) was
treated in live HeLa cell for 1 h and an image was then taken
under a Cy5, Texas Red, or NIR filter according to the excitation
and emission wavelength of the dye. The nucleus of the cell
was stained with Hoechst, and an image was taken under
a DAPI filter. The images were both taken with a 40ꢁ objective
lens and merged.[27]
Overall, the balance between the hydrophobicity and hydro-
philicity of the new dyes can be adjusted for biological stain-
ing purposes through modification at the meso and a positions.
Furthermore, it is reasonable to expect that after binding with
some specific probes or drug molecules at the meso-COOH
site, the resulting conjugated compounds would tend to be
cell-membrane-permeable, opening the path for targetted bio-
imaging.
Conclusion
In summary, the meso-COOEt-substituted BODIPY derivatives
1 and 2 were first synthesized and characterized. Motivated by
its bright fluorescence with large Stokes shift, we modified the
structure further and prepared four meso-ester-substituted
BODIPYs with extended p conjugation (3–6). After the intro-
duction of oligo(ethylene glycol)-ether-substituted phenylene
Figure 7. Living HeLa cell staining with dyes 1–8. 1 mm of the dye was treat-
ed in live HeLa cells for 1 h and images were taken under a Texas Red filter
(for 1, 3), NIR filter (for 4, 6), or Cy5 filter (for 5, 7, 8). Nuclei were stained
using Hoechst, and images were taken under a DAPI filter. Scale bar: 10 mm.
Chem. Eur. J. 2014, 20, 2301 – 2310
www.chemeurj.org
2307
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
2H), 4.47 (q, J=6.9 Hz, 2H), 3.04 (q, J=7.6 Hz, 4H), 1.44 (m, 3H),
1.34 ppm (m, 6H); 13C NMR (125 MHz, CDCl3): d=166.4, 163.9,
133.1, 130.7, 128.9, 118.5, 62.5, 29.7, 22.3, 14.2, 12.6 ppm; HRMS
(EI): m/z calcd for C16H19BO2F2N2: 320.1510; found: 320.1508 ([M]+).
vinylene or naphthalene vinylene groups, the absorption and
emission maxima shifted to the far-red and NIR region. The re-
sulting meso-ester-substituted BODIPY dyes also became par-
tially water-soluble with appreciable fluorescence quantum
yields. Moreover, cell-staining tests indicated that they suffi-
ciently membrane-permeable to penetrate into living HeLa
cells. All these features make these dyes attractive for bioimag-
ing applications. Three different synthetic approaches to
access the meso-COOH-substituted BODIPYs 7 and 8 were at-
tempted, two of which were feasible, and one of which was
a three-step, highly efficient, convenient synthetic route. The
meso-COOH-substituted BODIPYs 7 and 8 were soluble in pure
water and displayed remarkable fluorescence quantum yields
at wavelengths around or above 650 nm. The carboxylic group
can act as a versatile binding site to connect some specific
probes for biotargeting and sensing applications. In addition,
our synthetic strategies can be adapted easily for the prepara-
tion of a series of meso-COOH-substituted push–pull-type
BODIPY dyes, which have good potential for applications in
dye-sensitized solar cells.[18] The related studies are underway
in our laboratory.
Compound 2: Dry DCM (50 mL) in a round-bottomed flask was
purged with nitrogen for 15 min. To this solution, ethyl glyoxalate
(50% in toluene, 204 mg, 1 mmol), 2-(3,5-di-tert-butylphenyl) pyrro-
le[10a] (561 mg, 2.2 mmol), and TFA (two drops) were added. The re-
action mixture was stirred for 3 h at room temperature. After com-
plete consumption of the aldehyde (monitored by TLC), DDQ
(248 mg, 1.1 mmol) was added to the reaction mixture. After
30 min, triethylamine (4 mL) and BF3·OEt2 (5 mL) were added to the
mixture. The resulting mixture was stirred at room temperature for
1 h. When the reaction was complete, the solvent was removed
under reduced pressure. The residue was purified by column chro-
matography (silica gel, EtOAc/hexane, 1:5, v/v) to give 2 as a blue
solid (179 mg, 28%). 1H NMR (500 MHz, CDCl3): d=7.69 (d, J=
1.9 Hz, 4H), 7.47 (d, J=1.9 Hz, 2H), 7.37 (d, J=3.8 Hz, 2H), 6.64 (d,
J=3.8 Hz, 2H), 4.53 (q, J=7.6 Hz, 2H), 1.49 (t, J=7.6 Hz, 3H),
1.33 ppm (s, 36H); 13C NMR (125 MHz, CDCl3): d=164.1, 162.0,
150.4, 134.9, 131.8, 130.3, 129.5, 124.3, 123.7, 122.1, 62.6, 34.9,
31.4 ppm; HRMS (EI): m/z calcd for C40H51BO2F2N2: 640.4012; found:
640.3999 ([M]+).
Compound 15: Benzyl alcohol (10.8 g, 100 mmol) in dry DCM
(20 mL) was added through a syringe pump to a solution of oxalyl
chloride (12.7 g, 100 mmol) in anhydrous DCM (50 mL) over 0.5 h.
The solvent was removed in vacuo, affording benzyl chlorooxalate
14 as a colorless oil. Compound 14 was then used directly for the
next step of the reaction without further purification. The solution
of previously generated colorless oil (2 g, 10.1 mmol) in anhydrous
DCM (20 mL) was added dropwise to the solution of 2,4-dimethyl-
pyrrole (2.4 g, 25.4 mmol) in dry DCM (50 mL) through a syringe
pump over 1 h at ꢀ788C under an Ar atmosphere. The resulting
mixture was stirred at this temperature for 4 h, after which Et3N
(5.6 mL, 40.4 mmol) was added slowly, and the color of the solu-
tion turned to red–brown. Subsequently, BF3·OEt2 (8.0 mL, excess)
was added, and the solution was warmed slowly to room tempera-
ture and stirred for a further 2 h. After removal of the solvent
under reduced pressure, the residue was purified by column chro-
matography (silica gel, DCM/hexane=1:6 to 1:2) to afford com-
pound 15 as a dark red solid (1.76 g, 45%). 1H NMR (CDCl3
500 MHz): d=7.38–7.43 (m, 5H), 6.03 (s, 2H), 5.38 (s, 2H), 2.52 (s,
6H), 2.02 ppm (s, 6H); 13C NMR (CDCl3 125 MHz): d=165.0, 157.6,
141.2, 133.6, 129.2, 129.1, 128.8, 121.2, 68.6, 14.7, 12.7 ppm; HRMS
(EI): m/z calcd for C21H21BF2N2O2: 382.1664; found: 382.1656 ([M]+).
Experimental Section
General procedures
All reagents were purchased from commercial suppliers, and were
used as received without further purification. Anhydrous dichloro-
methane and DMF were distilled from CaH2. Toluene and THF were
distilled from sodium benzophenone immediately prior to use. The
1H NMR and 13C NMR spectra were recorded on a Bruker DPX 300
or DRX 500 NMR spectrometer with tetramethylsilane as the inter-
nal standard. The chemical shift was recorded in ppm, and the fol-
lowing abbreviations are used for the multiplicities: s=singlet, d=
doublet, t=triplet, q=quartet, m=multiplet. EI mass spectra were
recorded on an Agilent 5975C DIP/MS mass spectrometer. MALDI-
TOF-mass measurements were taken on a Bruker Autoflex instru-
ment with anthracence-1,8,9-triol as the matrix. UV/Vis spectra and
fluorescence spectra were recorded on a Shimadzu UV-1700 spec-
trometer and RF-5301 fluorometer, respectively. The solvents used
for UV/Vis and PL measurements were of HPLC grade. Cell-staining
images were taken under confocal microscopy upon living HeLa
cells; the nuclei were stained with Hoechst and images were re-
corded under a DAPI filter. Images were taken with a 40ꢁ objective
lens and merged.
Compound 22: Compound 22 was synthesized in 56% yield ac-
cording to the same procedure as for compound 15 but with
methyl chorooxalate. 1H NMR (CDCl3, 500 MHz): d=6.06 (s, 2H0,
3.97 (s, 3H), 2.53 (s, 6H), 2.11 ppm (s, 6H); 13C NMR (CDCl3,
125 MHz): d=166.4, 158.3, 141.7, 129.5, 129.4, 121.9, 53.8, 15.4,
13.2 ppm; HRMS (EI): m/z calcd for C15H17BF2N2O2: 306.1351; found:
306.1337 ([M]+).
Syntheses
Compound 1: Dry DCM (30 mL) in a round-bottomed flask was
purged with nitrogen for 10 min. To this solution, ethyl glyoxalate
(50% in toluene, 204 mg, 1 mmol), 2-ethyl pyrrole (234 mg,
2.2 mmol), and two drops of TFA were added. The reaction mixture
was stirred for 3 h at RT. After complete consumption of the alde-
hyde (monitored by TLC), DDQ (248 mg, 1.1 mmol) was added to
the reaction mixture. After 60 min, triethylamine (4 mL) and
BF3·OEt2 (5 mL) were added to the mixture. The resulting mixture
was stirred at room temperature for 1 h. When the reaction was
complete, the solvent was removed under reduced pressure. The
residue was purified by column chromatography (silica gel, EtOAc/
hexane, 1:3, v/v) to give 1 as a red solid (64 mg, 20%). 1H NMR
(500 MHz, CDCl3): d=7.26 (d, J=3.2 Hz, 2H), 6.39 (d, J=3.2 Hz,
Compound 20: Compound 20 synthesized by using the same pro-
cedure as for 15 and 22 resulted in a yield of only 4.7%. The pro-
cedure using compound 15 to generate compound 20 is described
below. Compound 15 (500 mg, 1.31 mmol) was dissolved in DCM
(10 mL), and a small amount of 10% Pd/C powder (5 mol%) was
added under an Ar atmosphere. This was followed by the addition
of MeOH (20 mL). The mixture was degassed and then filled with
H2 gas by using a balloon. The resulting mixture was then stirred
at RT under a H2 atmosphere (1 atm) for 5 h until no starting mate-
rial remained. Pd/C was removed through filtration, and the sol-
vent was evaporated under vacuum to afford the crude com-
Chem. Eur. J. 2014, 20, 2301 – 2310
www.chemeurj.org
2308
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
pound 19 as a brown–red solid in almost quantitative yield. With-
out further purification, compound 19 was redissolved in a mixed
solvent of tert-butanol (5 mL) and anhydrous diethyl ether (15 mL),
and then, an excess amount of DCC was added fractionally in 5 h.
The insoluble white solid was removed by filtration, the solvent
was removed under rotary evaporation, and the residue was puri-
fied by column chromatography (silica gel, DCM) to afford com-
pound 20 as a bright red solid (1.76 g, 45%). 1H NMR (CDCl3,
500 MHz): d=6.05 (s, 2H), 2.53 (s, 6H), 2.26 (s, 6H), 1.64 ppm (s,
9H); 13C NMR (CDCl3, 125 MHz): d=166.1, 158.1, 141.5, 129.3,
129.1, 121.7, 53.5, 28.2, 15.2, 13.0 ppm; MS (MALDI-TOF): m/z calcd
for C18H23BF2N2O2: 348.1821; found: 348.1803 ([M]+).
119.2, 117.8, 105.3, 71.9, 71.0, 70.7, 70.6, 69.7, 68.0, 59.0, 53.0,
12.8 ppm; HRMS (ESI): m/z calcd for C51H57BF2N2NaO10: 929.3975;
found: 929.3973 ([M+Na]+).
Synthesis of meso-COOH-substituted BODIPYs
Method 1: Concentrated HCl (1.0 mL) was added to a solution of
meso-COO-tBu-substituted BODIPY 3 or 4 (0.05 mmol) in CH3NO2.
The mixture was then stirred in an ice-water bath for 4 h, and dilut-
ed with DCM (50 mL). The solution was dried over Na2SO4, and the
solvent was removed through rotary evaporation. The residue was
purified by column chromatography (silica gel, DCM/MeOH=10:1)
to give the expected compound. Compound 7, yield 28%; com-
pound 8, yield 22%.
General procedure for the Knoevenagel condensation
Method 2: A mixture of meso-COOMe-substituted BODIPY 5 or 6
(0.10 mmol) and LiI (2.0 mmol) in dry ethyl acetate was heated to
reflux for 16 h under an Ar atmosphere. The mixture was cooled to
room temperature, and a small amount of HCl (0.2 mL) was added
to quench the reaction. The organic layer was dried over Na2SO4
and the solvent was removed under vacuum. The residue was puri-
fied by column chromatography (silica gel, DCM/MeOH=10:1) to
give the target compounds. Compound 7, yield 57%; compound 8,
yield 48%.
Compound 7: 1H NMR (CD3OD, 500 MHz): d=7.77 (d, J=8.25 Hz,
2H), 7.61 (d, J=4.5 Hz, 4H), 7.27 (d, J=8.25 Hz, 2H), 6.95 (d, J=
4.5 Hz, 4H), 6.82 (s, 2H), 4.15 (t, 4H), 3.84 (t, J=5.0 Hz, 4H), 3.69
(m, 8H), 3.53 (m, 4H), 3.33 (s, 6H), 2.41 ppm (s, 6H); 13C NMR
(CD3OD, 75 MHz): d=160.8, 153.9, 140.5, 135.7, 132.6; 131.5, 129.9,
118.9, 117.7, 117.5, 116.0, 115.9, 72.8, 71.6, 71.4, 71.2, 70.7, 68.7,
In a round-bottomed flask equipped with a Dean–Stark apparatus,
the corresponding aldehyde (2.2 equiv), piperidine (2 mL), and
AcOH (2 mL) were added to a solution of BODIPY (1 mmol) in tolu-
ene (20 mL). The solution was heated at reflux for 12 h. The mix-
ture was cooled to room temperature and washed three times
with water. The organic phase was dried over MgSO4 and the sol-
vent was evaporated under reduced pressure. The residue was pu-
rified by silica gel column chromatography to afford the desired
compounds 17, 3, 4, 5, and 6.
1
Compound 17: Yield: 47%; H NMR (CDCl3, 500 MHz): d=6.96–7.55
(m, 37H), 6.66 (s, 2H), 5.40 (s, 2H), 2.08 ppm (s, 6H); 13C NMR
(CDCl3, 125 MHz): d=166.3, 154.7, 149.6, 147.8, 139.9, 137.3, 134.5,
130.8, 130.1, 129.8, 129.7, 129.5, 129.4, 125.8, 124.4, 122.9, 118.2,
117.8, 69.1, 13.6 ppm; MS (MALDI-TOF): m/z calcd for
C59H47BF2N4O2: 892.3760; found: 892.3717 ([M]+).
59.1,
13.3 ppm;
HRMS
(ESI):
m/z
calcd
for
Compound 3: Yield: 43%; 1H NMR (CDCl3, 500 MHz): d=7.57 (m,
6H), 7.23 (d, J=4.8 Hz, 2H), 6.94 (d, J=2.6 Hz, 2H), 6.68 (s, 2H),
4.18 (t, 4H), 3.88 (t, 4H), 3.76 (m, 12H), 3.56 (t, 4H), 3.39 (s, 6H),
2.32 (s, 6H), 1.66 ppm (s, 9H); 13C NMR (CDCl3, 125 MHz): d=164.6,
159.8, 153.9, 139.9, 136.6, 131.0, 129.6, 129.2, 127.7, 117.4, 117.2,
115.0, 72.0, 70.9, 70.7, 70.6, 69.7, 67.6, 59.0, 29.7, 28.5, 14.4 ppm;
MS (MALDI-TOF): m/z calcd for C46H59BF2N2O10: 848.4231; found:
848.4210 ([M]+).
Compound 4: Yield: 39%; 1H NMR (CDCl3, 500 MHz): d=8.35 (d,
J=4.25 Hz, 2H), 8.17 (d, J=4.25 Hz, 2H), 8.06 (d, J=8.0 Hz, 2H),
7.95 (d, J=4.0 Hz, 2H), 7.73 (d, J=8 Hz, 2H), 7.49–7.60 (m, 4H),
6.90 (d, J=8.0 Hz, 2H), 6.84 (s, 2H), 4.36 (t, 4H), 4.02 (t, 4H), 3.82 (t,
4H), 3.66–3.72 (m, 8H), 3.55 (t, 4H), 3.37 (s, 6H), 2.38 (s, 6H),
1.69 ppm (s, 9H); 13C NMR (CDCl3,125 MHz): d=164.6, 155.9, 154.0,
140.0, 133.1, 132.3, 131.2, 127.7, 127.1, 126.2, 125.7, 125.6, 125.3,
122.9, 119.4, 117.7, 105.3, 85.5, 71.9, 71.0, 70.7, 70.6, 69.7, 68.0, 59.0,
28.5, 14.4 ppm; MS (MALDI-TOF): m/z calcd for C54H63BF2N2O10:
948.4544; found: 948.4521 ([M]+).
Compound 5: Yield: 51%; 1H NMR (CDCl3, 500 MHz): d=7.60 (m,
6H), 7.29 (m, 2H), 7.00 (d, J=4.35 Hz, 4H), 6.74 (s, 2H), 4.23 (m,
4H), 4.0 (s, 3H), 3.94 (m, 4H), 3.70–3.80 (m, 12H), 3.62 (m, 4H),
3.45 (s, 6H), 2.22 ppm (s, 6H); 13C NMR (CDCl3, 125 MHz): d=166.1,
159.9, 154.1, 139.3, 136.9, 130.8, 129.4, 129.2, 129.0, 117.5, 117.0,
115.0, 71.9, 70.8, 70.6, 70.5, 69.6, 67.5, 59.0, 52.9, 12.7 ppm; HRMS
(ESI): m/z calcd for C43H53BF2N2NaO10: 829.3661; found: 829.3685
([M+Na]+).
C42H51BF2N2NaO10:815.3503; found: 815.3534 ([M+Na]+).
1
Compound 8: H NMR (CD3OD, 500 MHz): d=8.34 (d, 4H), 8.14 (d,
J=8.0 Hz, 2H), 8.03 (d, J=4.25 Hz, 2H), 7.98 (d, J=8.0 Hz, 2H),
7.64 (t, J=4.13 Hz, 2H), 7.53 (t, J=4.13 Hz, 2H), 7.10 (s, 2H), 7.05
(d, J=4.25 Hz, 2H), 4.41 (m, 4H), 4.05 (m, 4H), 3.81 (m, 4H), 3.71
(m, 4H), 3.65 (m, 4H), 3.53 (m, 4H), 3.36 (s, 6H), 2.47 ppm (s, 6H);
13C NMR (CD3OD, 125 MHz): 162.8, 155.5, 139.2, 132.3, 131.5, 126.9,
126.5, 125.6, 125.0, 124.9, 122.9, 122.3, 119.8, 116.94, 116.86, 105.1,
71.5, 70.5, 70.2, 70.0, 69.5, 68.0, 57.8, 11.9 ppm; HRMS (ESI): m/z
calcd for C50H55BF2N2NaO10: 891.3816; found: 891.3788 ([M+Na]+) .
Acknowledgements
The work was supported financially by the A*STAR BMRC grant
(10/1/21/19/642), the Singapore-Peking-Oxford Research Enter-
prise (SPORE) (COY-15-EWI-RCFSA/N197–1), MOE Tier 2 grant
(MOE2011-T2–2–130), and IMRE core funding (IMRE/13–
1C0205).
Keywords: bioimaging · BODIPY · dyes · fluorescent probes ·
membrane permeability · near-infrared
Compound 6: Yield: 48%; 1H NMR (CDCl3, 300 MHz): d=8.30 (d,
J=3.75 Hz, 2H), 8.12 (d, J=4.05 Hz, 2H), 8.02 (d, J=7.95 Hz, 2H),
7.90 (d, J=4.2 Hz, 2H), 7.66 (d, J=8.1 Hz, 2H), 7.43–7.56 (m, 4H),
6.85 (d, J=4.05 Hz, 2H), 6.79 (s, 2H), 4.32 (m, 4H), 3.95 (m, 7H),
3.77 (m, 4H), 3.63 (m, 8H), 3.50 (m, 4H), 3.33 (s, 6H), 2.17 ppm (s,
6H); 13C NMR (CDCl3, 75 MHz): d=166.2, 156.0, 154.3, 139.5, 139.4,
133.4, 132.3, 131.0, 127.1, 126.1, 125.73, 125.70, 125.3, 125.0, 122.8,
[1] a) J. Fabian, H. Nakanzumi, M. Matsuoka, Chem. Rev. 1992, 92, 1197;
b) A. Loudet, K. Burgess, Chem. Rev. 2007, 107, 4891; c) R. Ziessel, G.
Ulrich, A. Harriman, New J. Chem. 2007, 31, 496; d) G. Ulrich, R. Ziessel,
A. Harriman, Angew. Chem. 2008, 120, 1202; Angew. Chem. Int. Ed. 2008,
47, 1184; e) G. Qian, Z. Wang, Chem. Asian J. 2010, 5, 1006; f) N. Boens,
V. Leen, W. Dehaen, Chem. Soc. Rev. 2012, 41, 1130; g) K. D. Piatkevich,
F. V. Subach, V. V. Verkhusha, Chem. Soc. Rev. 2013, 42, 3441; h) L. Yuan,
W. Lin, K. Zheng, L. He, W. Huang, Chem. Soc. Rev. 2013, 42, 622.
Chem. Eur. J. 2014, 20, 2301 – 2310
www.chemeurj.org
2309
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
[2] a) L. Lavis, R. T. Raines, ACS Chem. Biol. 2008, 3, 142; b) V. P. Boyarskiy,
V. N. Belov, R. Medda, B. Hein, M. Bossi, S. W. Hell, Chem. Eur. J. 2008, 14,
1784; c) J. Jose, K. Burgess, J. Org. Chem. 2006, 71, 7835.
[3] a) R. P. Haughland, Handbook of Fluorescent Probes and Research Chemi-
cals, 9th ed., Molecular Probes, Eugene, OR, 2002; b) A. Mishra, R. K.
Behera, P. K. Behera, B. K. Mishra, G. B. Behera, Chem. Rev. 2000, 100,
1973; c) A. Ajayaghosh, Acc. Chem. Res. 2005, 38, 449; d) J. J. McEwen,
K. J. Wallace, Chem. Commun. 2009, 6339.
[4] a) G. T. Hermanson, Bioconjugate Techniques, 2nd ed., Thermo Fisher Sci-
entific: Rockford, IL, 2008; b) S. A. Farber, M. Pack, S. Y. Ho, I. D. Johnson,
D. S. Wagner, R. Dosch, M. C. Mullins, H. S. Hendrickson, E. K. Hendrick-
son, M. E. Halpern, Science 2001, 292, 1385; c) J. M. Palomo, M. Lum-
bierres, H. Waldmann, Angew. Chem. 2006, 118, 491; Angew. Chem. Int.
Ed. 2006, 45, 477; d) V. Ntziachristos, J. Ripoll, L. V. Wang, R. Weissleder,
Nat. Biotechnol. 2005, 23, 313; e) J. Murtagh, D. O. Frimannsson, D. F.
O’Shea, Org. Lett. 2009, 11, 5386; f) M. Tasior, D. F. O’Shea, Bioconjugate
Chem. 2010, 21, 1130.
Kurth, Bioconjugate Chem. 2011, 22, 2593; c) D. Das, Z. Tnimov, U. T. T.
Nguyen, G. Thimmaiah, H. Lo, D. Abankwa, Y. Wu, R. S. Goody, H. Wald-
mann, K. Alexandrov, ChemBioChem 2012, 13, 674; d) Z. Zhang, P. Gao,
X. Guo, H. Wang, H. Zhang, Anal. Bioanal. Chem. 2011, 401, 1905; e) S.
Hoogendoorn, K. L. Habets, S. Passemard, J. Kuiper, G. A. van der Marel,
B. I. Floreaa, H. S. Overkleeft, Chem. Commun. 2011, 47, 9363.
[12] a) S. Atilgan, T. Ozdemir, E. U. Akkaya, Org. Lett. 2008, 10, 4065; b) S.
Zhu, J. Zhang, G. Vegesna, F. Luo, S. A. Green, H. Liu, Org. Lett. 2011, 13,
438; c) M. Isik, T. Ozdemir, I. S. Turan, S. Kolemen, E. U. Akkaya, Org. Lett.
2013, 15, 216; d) G. K. Vegesna, S. R. Sripathi, J. Zhang, S. Zhu, W. He, F.
Luo, H. Liu, ACS Appl. Mater. Interfaces 2013, 5, 4107; e) T. Bura, P. Retail-
leau, G. Ulrich, R. Ziessel, J. Org. Chem. 2011, 76, 1109.
[13] a) D. Wang, J. Fan, X. Gao, B. Wang, S. Sun, X. Peng, J. Org. Chem. 2009,
74, 7675; b) F. Wang, X. Xiong, X. Guo, H. Wang, H. Zhang, J. Chroma-
togr. A 2013, 1291, 84–91.
[14] a) S. L. Niu, G. Ulrich, R. Ziessel, A. Kiss, P. Y. Renard, A. Romieu, Org. Lett.
2009, 11, 2049;b) L. Li, J. Han, B. Nguyen, K. Burgess, J. Org. Chem.
2008, 73, 1963.
[15] K. Krumova, G. Cosa, J. Am. Chem. Soc. 2010, 132, 17560.
[16] T. Komatsu, D. Oushiki, A. Takeda, M. Miyamura, T. Ueno, T. Terai, K. Ha-
naoka, Y. Urano, T. Mineno, T. Nagano, Chem. Commun. 2011, 47, 10055.
[17] R. M. Denton, J. An, B. Adeniran, A. J. Blake, W. Lewis, A. M. Poulton, J.
Org. Chem. 2011, 76, 6749.
[18] a) T. Bura, N. Leclerc, S. Fall, R. Ziessel, J. Am. Chem. Soc. 2012, 134,
17404; b) S. Kolemen, O. A. Bozdemir, Y. Cakmak, G. Barin, S. Erten-Ela,
M. Marszalek, J. Yum, S. M. Zakeeruddin, M. K. Nazeeruddin, E. U.
Akkaya, Chem. Sci. 2011, 2, 949.
[19] E. C. Taylor, G. S. K. Wong, J. Org. Chem. 1989, 54, 3618.
[20] A. K. Chakraborti, M. K. Nayak, L. Sharma, J. Org. Chem. 2002, 67, 1776.
[21] J. W. Fisher, K. L. Trinkle, Tetrahedron Lett. 1994, 35, 2505.
[22] L. N. Sobenina, A. M. Vasil’tsov, O. V. Petrova, K. B. Petrushenko, I. A. Ush-
akov, G. Clavier, R. Meallet-Renault, A. I. Mikhaleva, B. A. Trofimov, Org.
Lett. 2011, 13, 2524.
[23] Y. Chen, J. Zhao, H. Guo, L. Xie, J. Org. Chem. 2012, 77, 2192.
[24] a) A. D. Becke, J. Chem. Phys. 1993, 98, 5648; b) C. Lee, W. Yang, R. G.
Parr, Phys. Rev. B 1988, 37, 785; c) R. W. Ditchfield, J. Hehre, J. A. Pople,
J. Chem. Phys. 1971, 54, 724; d) W. J. Hehre, R. Ditchfield, J. A. Pople, J.
Chem. Phys. 1972, 56, 2257; e) P. C. Hariharan, J. A. Pople, Theor. Chim.
Acta 1973, 28, 213.
[5] Y. Urano, D. Asanuma, Y. Hama, Y. Koyama, T. Barrett, M. Kamiya, T.
Nagano, T. Watanabe, A. Hasegawa, P. L. Choyke, H. Kobayashi, Nat.
Med. 2009, 15, 104.
[6] T. Kowada, J. Kikuta, A. Kubo, M. Ishii, H. Maeda, S. Mizukami, K. Kikuchi,
J. Am. Chem. Soc. 2011, 133, 17772.
[7] a) A. Burghart, H. Kim, M. B. Welch, L. H. Thoresen, J. Reibenspies, K. Bur-
gess, F. Bergstrorm, L. B. A. Johansson, J. Org. Chem. 1999, 64, 7813;
b) Y.-H. Yu, A. B. Descalzo, Z. Shen, H. Rçhr, Q. Liu, Y.-W. Wang, M.
Spieles, Y.-Z. Li, K. Rurack, X.-Z. You, Chem. Asian J. 2006, 1, 176; c) T.
Rohand, M. Baruah, W. Qin, N. Boens, W. Dehaen, Chem. Commun.
2006, 266; d) M. Baruah, W. Qin, R. A. L. VallQe, D. Beljonne, T. Rohand,
W. Dehaen, N. Boens, Org. Lett. 2005, 7, 4377.
[8] a) J. Chen, J. Reibenspies, A. Derecskei-Kovacs, K. Burgess, Chem.
Commun. 1999, 2501; b) K. Umezawa, Y. Nakamura, H. Makino, D. Citter-
io, K. Suzuki, J. Am. Chem. Soc. 2008, 130, 1550; c) A. Matsui, K. Umeza-
wa, Y. Shindo, T. Fujii, D. Citterio, K. Oka, K. Suzuki, Chem. Commun.
2011, 47, 10407; d) S. G. Awuah, J. Polreis, V. Biradar, Y. You, Org. Lett.
2011, 13, 3884; e) A. Wakamiya, T. Murakami, S. Yamaguchi, Chem. Sci.
2013, 4, 1002; f) Y. Ni, W. Zeng, K. Huang, J. Wu, Chem. Commun. 2013,
49, 1217.
[9] a) W. Zhao, E. M. Carreira, Angew. Chem. 2005, 117, 1705; Angew. Chem.
Int. Ed. 2005, 44, 1677; b) W. Zhao, E. M. Carreira, Chem. Eur. J. 2006, 12,
7254; c) S. O. McDonnell, D. F. O’Shea, Org. Lett. 2006, 8, 3493.
[10] a) C. Jiao, K.-W. Huang, J. Wu, Org. Lett. 2011, 13, 632; b) C. Jiao, L. Zhu,
J. Wu, Chem. Eur. J. 2011, 17, 6610; c) L. Zeng, C. Jiao, X. Huang, K.-W.
Huang, W.-S. Chin, J. Wu, Org. Lett. 2011, 13, 6026; d) Y. Hayashi, N.
Obata, S. Seki, Y. Kureishi, S. Saito, S. Yamaguchi, H. Shinokubo, Org.
Lett. 2012, 14, 866; e) C. Jones, T. Suarez, J. Org. Chem. 1972, 37, 3622.
[11] a) M. Tera, K. Iida, K. Ikebukuro, H. Seimiya, K. Shin-yac, K. Nagasawa,
Org. Biomol. Chem. 2010, 8, 2749; b) H. R. Davison, S. Taylor, C. Drake, P.-
W. Phuan, N. Derichs, C. Yao, E. F. Jones, J. Sutcliffe, A. S. Verkman, M. J.
[25] J. Tomasi, B. Mennucci, R. Cammi, Chem. Rev. 2005, 105, 2999–3093.
[26] Gorelsky, S. I., SWizard program, http://www.sg-chem.net/, University of
Ottawa, Ottawa, Canada, 2010.
[27] J. Lee, N. Kang, Y. K. Kim, A. Samanta, S. Feng, H. K. Kim, M. Vendrell,
J. H. Park, Y. Chang, J. Am. Chem. Soc. 2009, 131, 10077.
Received: October 3, 2013
Published online on January 21, 2014
Chem. Eur. J. 2014, 20, 2301 – 2310
www.chemeurj.org
2310
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim