Synthesis, computational modeling, and properties of benzo-appended BODIPYs

Article abstract of DOI:10.1002/chem.201103002

A series of new functionalized mono- and dibenzo-appended BODIPY dyes were synthesized from a common tetrahydroisoindole precursor following two different synthetic routes. Route A involved the assembly of the BODIPY core prior to aromatization, while in Route B the aromatization step was performed first. In general, Route A gave higher yields of the target dibenzo-BODIPYs, due to the ease of aromatization of the BODIPYs compared with the corresponding dipyrromethenes, probably due to their higher stability under the oxidative conditions (2,3-dichloro-5,6-dicyano-1,4-benzoquinone in refluxing toluene). However, due to the slow oxidation of highly electron-deficient BODIPY 3 c bearing a meso-C₆F₅ group, dibenzo-BODIPY 4 c was obtained, in 35 % overall from dipyrromethane, only by Route B. Computational calculations performed at the 6-31G(d,p) level are in agreement with the experimental results, showing similar relative energies for all reaction intermediates in both routes. In addition, BODIPY 3 c had the highest molecular electrostatic potential (MEPN), confirming its high electron deficiency and consequent resistance toward oxidation. X-ray analyses of eight BODIPYs and several intermediates show that benzannulation further enhances the planarity of these systems. The π-extended BODIPYs show strong red-shifted absorptions and emissions, about 50-60 nm per benzoannulated ring, at 589-658 and 596-680 nm, respectively. In particular, db-BODIPY 4 c bearing a meso-C ₆F₅ group showed the longest λ_max of absorption and emission, along with the lowest fluorescence quantum yield (0.31 in CH₂Cl₂); on the other hand monobenzo-BODIPY 8 showed the highest quantum yield (0.99) of this series. Cellular investigations using human carcinoma HEp2 cells revealed high plasma membrane permeability for all dibenzo-BODIPYs, low dark- and photo-cytotoxicities and intracellular localization in the cell endoplasmic reticulum, in addition to other organelles. Our studies indicate that benzo-appended BODIPYs, in particular the highly stable meso-substituted BODIPYs, are promising fluorophores for bioimaging applications. Two synthetic routes to benzo- BODIPYs from a pyrrolic precursor were investigated. Computational calculations showed similar relative energies for all reaction intermediates and products in both routes. X-ray analyses indicated that benzannulation enhances the planarity of the BODIPY core (see figure). The benzo-BODIPYs show red-shifted absorptions and emissions, ca. 50-60 nm per benzoannulated ring, and high fluorescence quantum yields. Copyright

Full text of DOI:10.1002/chem.201103002

FULL PAPER
DOI: 10.1002/chem.201103002
Synthesis, Computational Modeling, and Properties of Benzo-Appended
BODIPYs**
Timsy Uppal,[a] Xiaoke Hu,[a] Frank R. Fronczek,[a] Stephanie Maschek,[b]
Petia Bobadova-Parvanova,[b] and M. GraÅa H. Vicente*[a]
Abstract: A series of new functional-
ized mono- and dibenzo-appended
BODIPY dyes were synthesized from
a common tetrahydroisoindole precur-
sor following two different synthetic
routes. Route A involved the assembly
of the BODIPY core prior to aromati-
zation, while in Route B the aromatiza-
tion step was performed first. In gener-
al, Route A gave higher yields of the
target dibenzo-BODIPYs, due to the
ease of aromatization of the BODIPYs
compared with the corresponding di-
pyrromethenes, probably due to their
higher stability under the oxidative
Route B. Computational calculations
performed at the 6-31G(d,p) level are
596–680 nm, respectively. In particular,
db-BODIPY 4c bearing a meso-C6F5
group showed the longest lmax of ab-
sorption and emission, along with the
lowest fluorescence quantum yield
(0.31 in CH2Cl2); on the other hand
AHCTUNGTRENNUNG
in agreement with the experimental re-
sults, showing similar relative energies
for all reaction intermediates in both
routes. In addition, BODIPY 3c had
the highest molecular electrostatic po-
tential (MEPN), confirming its high
electron deficiency and consequent re-
sistance toward oxidation. X-ray ana-
monobenzo-BODIPY
8 showed the
highest quantum yield (0.99) of this
series. Cellular investigations using
human carcinoma HEp2 cells revealed
high plasma membrane permeability
for all dibenzo-BODIPYs, low dark-
and photo-cytotoxicities and intracellu-
lar localization in the cell endoplasmic
reticulum, in addition to other organ-
elles. Our studies indicate that benzo-
appended BODIPYs, in particular the
AHCTUNGTRENNlGUN yses of eight BODIPYs and several in-
termediates show that benzannulation
further enhances the planarity of these
systems. The p-extended BODIPYs
show strong red-shifted absorptions
and emissions, about 50–60 nm per
benzoannulated ring, at 589–658 and
conditions
(2,3-dichloro-5,6-dicyano-
1,4-benzoquinone in refluxing toluene).
However, due to the slow oxidation of
highly electron-deficient BODIPY 3c
bearing a meso-C6F5 group, dibenzo-
BODIPY 4c was obtained, in 35%
overall from dipyrromethane, only by
highly
stable
meso-substituted
BODIPYs, are promising fluorophores
for bioimaging applications.
Keywords: aromatization · cellular
uptake
· cytotoxicity · dyes/pig-
ments · fluorescence · planarity
Introduction
extinction coefficients, and high fluorescence quantum
yields, BODIPYs have attracted special interest in drug dis-
covery,[4] biomedical imaging,[5] and optical sensing.[6,7] The
success of these applications rely on the syntheses of long-
wavelength absorbing and emitting BODIPYs (ca. 600–
800 nm), for reduced rotational motion and fluorescence
quenching,[8] as well as the incorporation of functionalities
for increased solubility in aqueous media and/or for conju-
gation to target-specific molecules. Strategies toward these
goals include the preparation of: 1) aza-substituted
BODIPYs at the bridging meso-position, designated Aza-
BODIPYs,[9] 2) core-modified BODIPYs bearing substituted
aryl, ethynylphenyl or styryl groups,[10] and 3) functionalized
BODIPYs with p-extended systems through the fusion of
external aromatic rings to the b-pyrrolic positions.[11] The so-
called p-extended BODIPYs form a class of highly con-
strained BODIPYs with rigid aromatic rings fused to the
b,b’-pyrrole carbon atoms (Figure 1).[1a] In general, aromatic
ring fusion at the b-pyrrolic positions provide constrained
and highly planar BODIPY platforms with fluorescence in
the near-IR region of the spectrum.[12,13] Current synthetic
approaches to dibenzo-fused BODIPYs (db-BODIPYs)
Borondipyrromethene (BODIPY) dyes, often referred to as
“semi-porphyrins”,[1] are among the most recent investigated
fluorescence dyes for a variety of analytical and imaging ap-
plications.[2,3] Due to their favorable photophysical and opto-
electronic properties that include high photostability, high
[a] T. Uppal, Dr. X. Hu, Dr. F. R. Fronczek, Prof. M. G. H. Vicente
Department of Chemistry
Louisiana State University
Baton Rouge, LA, 70803 (USA)
Fax : (+1)225-578-3458
E-mail: vicente@lsu.edu
[b] S. Maschek, Prof. P. Bobadova-Parvanova
Department of Chemistry
Rockhurst University
Kansas City, MO, 64110 (USA)
[**] BODIPY=Borondipyrromethene
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103002. It contains additional
X-ray structures, UV/Vis and fluorescence data, cytotoxicity data,
and NMR spectra, Cartesian coordinates of all optimized structures
and schemes of the computed potential energy surfaces.
Chem. Eur. J. 2012, 18, 3893 – 3905
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3893

Figure 1. Synthetic strategies toward symmetric dibenzo-appended
BODIPYs.
from pyrrolic precursors are shown in Figure 1. The “Ono
et al. method”[2] uses
a bicyclo-octadiene-fused pyrrole
(Figure 1, Route A) and its major drawback is the harsh re-
action conditions required for the aromatization step, upon
a thermal retro Diels–Alder reaction. On the other hand the
isoindole precursor required for Route B (Figure 1) is unsta-
ble due to the high reactivity of the pyrrolic a-positions. Al-
ternatively, di- and/or tetra-hydroisoindole precursors can
be used (Routes C and D), as already demonstrated for the
synthesis of tetrabenzoporphyrins (TBP),[14] but these strat-
egies have not yet been developed into a general approach
toward the synthesis of benzo-BODIPY systems. While this
article was in preparation, reports describing the synthesis
of p-extended dipyrrins from a 4,7-dihydroisoindole precur-
sor,[15] and an alternative synthesis of db-BODIPYs from 2-
acetylphenols were published.[16] In the search of practical
and straightforward approaches to benzo-appended
BODIPYs, we investigated two synthetic routes from
a common dipyrromethane precursor, readily available from
a thermodynamically stable tetrahydroisoindole synthon
(Scheme 1). Our goals were to develop a general synthetic
methodology to db-BODIPYs, and to study the effect of
number of fused benzene subunits and the nature of meso-
substituent (A) on the photophysical and biological proper-
ties of BODIPYs.
Scheme 1. Reaction conditions: i) CH2ACTHNUTRGNEUNG(OMe)2, TsOH, HOAc, RT, 24 h
(92%); ii) Ar-CHO, TsOH, CH2Cl2, Bu4Cl, RT, 12 h, (70-95%); iii) DDQ
(1.2 equiv), dry CH2Cl2, 08C, 20 min; iv) Et3N, BF3·OEt2, 08C, 30 min to
overnight, RT (55-90%); v) DDQ (9 equiv), dry toluene, reflux, 45 min
to 4 h (56-92%).
viated here as bc-BODIPYs) 3a–e were obtained as green
solids from the corresponding dipyrromethanes 2a–e in 60–
80% yields, after purification by column chromatography on
silica gel and recrystallization from dichloromethane/hex-
anes mixtures. All bc-BODIPYs with the exception of 3c,
produced the corresponding fluorescent db-BODIPYs
4a,b,d,e as deep blue solids in 67–92% yields, upon DDQ
oxidation in refluxing toluene. The highest aromatization
yield was obtained for BODIPY 4d (92%), bearing the
electron-donating 4-methoxyphenyl group, and the lowest
for BODIPY 4c (0%, even upon prolonged heating in the
presence of a large excess of DDQ) bearing the strong elec-
tron-withdrawing pentafluorophenyl group. It is possible
that the highly electron-deficient BODIPY 3c has a much
slower rate of oxidation, due to its lower tendency to form
a benzylic cation intermediate, either by single-electron
transfer, or through hydride transfer, to DDQ.[19]
Results and Discussion
Synthesis of benzo-BODIPYs: The key synthetic precursors
to BODIPYs 4a–e, dipyrromethanes 2a–e, were prepared in
high yields by acid-catalyzed condensation of aldehydes
with ethyl-4,5,6,7-tetrahydroisoindole ester 1[17] (generated
using the Barton–Zard reaction) according to a modified lit-
erature procedure.[18] Two synthetic routes were investigated
for the generation of db-BODIPYs 4a–e, as shown in
Scheme 1. In Route A, the BODIPY core was assembled
first after mild oxidation of 2a–e to the corresponding dipyr-
romethenes 2’a–e and reaction with BF3·OEt2 in a one-flask
two-step procedure, followed by aromatization with 2,3-di-
chloro-5,6-dicyano-1,4-benzoquinone (DDQ), similar to the
TBP synthetic methodology. The bicyclo-BODIPYs (abbre-
These results are in agreement with previous observations
on metallo-TBP synthesis, showing that metalloporphyrins
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Dyes/Pigments
FULL PAPER
containing electron-withdrawing meso-substituents have de-
creased aromatization reaction rates, leading to lower yields
of the target products.[20] Additional oxidizing agents and re-
action conditions were investigated for the aromatization
step, including KMnO4/K10 clay, AgNO3/acetone, and
FeCl3/methanol, but lower yields for the target db-
BODIPYs were obtained.
presence of POCl3 to produce the corresponding dipyrrome-
thene, which reacted with BF3·OEt2 in a one-flask two-step
procedure producing BODIPY 7 in 40% overall yield.[21]
Aromatization using DDQ (5 equiv) in refluxing toluene for
20 min generated the target BODIPY 8 as a green solid in
60% yield.
The structures of all compounds synthesized were con-
firmed by 1H and 13C NMR spectroscopy, MS, and X-ray
analysis (see Supporting Information). For all the
BODIPYs, single crystals suitable for X-ray structure analy-
sis (Figures 2 and 3) were obtained by slow diffusion of hex-
In Route B (Scheme 1) dipyrromethanes 2a–e were first
subjected to oxidative dehydrogenation with DDQ in reflux-
ing toluene affording the p-extended benzodipyrrins 5a–e as
deep green crystals. The yields obtained for the oxidation
reactions of 2a–e (56-69%) or 2’a–e (53-65%) to the corre-
sponding benzodipyrrins 5a–e were lower than those ob-
tained from BODIPYs 3a,b,d,e to 4a,b,d,e (67–92%), with
the exception of 3c probably due to its high electron-defi-
ciency.[19] Reaction of dipyrrins 5a–e with BF3·OEt2 and tri-
ACHTUNGTRENNUNG
ethylamine in refluxing toluene[21] afforded all the target db-
BODIPYs 4a–e in 55–90% yields. These are similar to the
yields obtained (60–80%) from complexation of boron to 2
or 2 by means of Route A. Although db-BODIPY 4c could
only be prepared by Route B, in general Route A gave
higher overall yields for the target db-BODIPYs (41–67%
from 2) compared with Route B (31–62% from 2) due to
the easier aromatization of BODIPYs 3 (67–92%) com-
pared with the corresponding dipyrromethenes 2’ (53–
65%); this result is probably due to the higher stability of
BODIPYs 3 compared with the corresponding intermediates
2 and 2 under the oxidative reaction conditions, and conse-
quently lower tendency for formation of side products. The
highest overall yield was obtained for BODIPY 4d bearing
the electron-donating 4-methoxyphenyl group, and the
lowest for BODIPY 4c bearing the strongest electron-with-
drawing pentafluorophenyl group, suggesting that electron-
rich bc-BODIPYs are easier to aromatize (vide infra).
Figure 2. Molecular structures of BODIPYs 3b, 3c, 3d and 3e.
In order to investigate the effect of number of fused ben-
zene rings on the photophysical properties of BODIPYs, we
also prepared monobenzo-fused BODIPY 8 (Scheme 2), fol-
lowing a route similar to Route A in Scheme 1. Tetrahydro-
ACHTUNGTRENNUNG
isoindole ester 1[17] and 5-formyl-pyrrole 6[22] reacted in the
Scheme 2. Reaction conditions: i) POCl3, CH2Cl2, 24 h, RT; ii) Et3N,
BF3·OEt2, CH2Cl2, 24 h, RT (40% for two steps); iii) DDQ, toluene,
reflux, 10 min (60%).
Figure 3. Molecular structures of BODIPYs 4b, 4d, 4e and 8.
Chem. Eur. J. 2012, 18, 3893 – 3905
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3895

M. G. H. Vicente et al.
anes into a solution of compound in dichloromethane. Sev-
eral molecular structures were also obtained for BODIPY
precursors, and these are shown in the Supporting Informa-
tion (Figures S1 and S2). Due to the central sp3 C atom and
the presence of two NH groups, the two pyrrole rings of the
dipyrromethanes 2a–d are markedly nonparallel (Figure S1).
In these compounds, dihedral angles formed by the two pyr-
role rings fall in the range 53.2(1)–86.01(5)8, with average
value 68.58. Both the coordination to BF2 in the BODIPYs
(Figures 2 and 3) and the intramolecular hydrogen bond for-
mation in the dipyrromethenes (Figure S2 in the Supporting
Information) cause the pyrrole rings to be more coplanar.
The dihedral angles in the 3b–e BODIPYs have an average
value of 7.78, in the benzo-BODIPYs 4b,d,e and 8 an aver-
age value of 4.58, and in the benzo-dipyrromethenes 5b–d
an average value of 7.08. In the corresponding dipyrrome-
thenes with saturated 6-rings (see Figure S2), dihedral
angles have an average value of 8.68. Thus, from the average
values, pyrrole rings in benzo-dipyrromethenes tend to be
slightly more coplanar than in their saturated derivatives,
the same is true of the corresponding BODIPYs, and the
BODIPYs tend to exhibit slightly higher coplanarity than
their free-pyrrole analogs. These trends generally also hold,
with a few exceptions, when comparing individual pairs of
compounds for which we have determined both crystal
structures. These include three pairs of benzo- versus satu-
rated BODIPYs and five pairs of BODIPYs versus their un-
coordinated analogs.
indicates that the significant differences in the yields ob-
tained experimentally for the target BODIPYs 4b–e are
probably a result of different potential energy barriers.
To examine the solvent effect, structures 2b, 2’b, 3b, 4b,
and 5b were re-optimized in dichloromethane and toluene,
respectively, since reactions 2!2 and 2!3 were carried out
in dichloromethane, while reactions 3!4, 2!5, and 5!4,
were carried out in toluene. The results are shown in
Table 2. The presence of the solvent stabilizes all structures.
Table 2. Relative energies [kcalmolꢀ1] of BODIPY reaction species fol-
lowing the two synthetic routes with and without the accounting of the
solvent effect. The solvent is dichloromethane for reaction 2!2’, trietha-
nolamine for 2’b!3b and 5b!4b, and toluene for 3b!4b and 2b!5b.
BODIPY
In gas-phase
In solvent
Route A
reactant
2b
2’b
3b
4b
0
ꢀ14
ꢀ8
0
ꢀ16
ꢀ8
intermediate
intermediate
product
ꢀ127
ꢀ131
Route B
reactant
intermediate
product
2b
5b
4b
0
ꢀ138
ꢀ145
0
ꢀ145
ꢀ136
However, in Route A the effect on the relative energies is
rather small—between 2–4 kcalmolꢀ1 and close to the calcu-
lation error. In Route B the effect is more pronounced, es-
pecially for step 2. The presence of toluene affects the ener-
getics of step 2 with only 2 kcalmolꢀ1, but step 1 by 7 kcal
molꢀ1. Thus, in overall Route B is 9 kcalmolꢀ1 more exother-
mic in toluene solvent than in gas phase mainly due to the
increased exothermicity of step 1. The performed in-solvent
calculations suggest that in general, modeling of the reaction
in gas phase will give a realistic description. However, care
must be taken when Route B, step 1 is studied. For this step
solvent effects are more pronounced and result in higher
exothermicity than in gas phase.
In order to obtain further insight on the different reactivi-
ty observed between 2b–e (or 2’b–e) and 3b–e, we investi-
gated the properties of the cyclohexenyl hydrogen atoms in
these molecules by calculating the average molecular elec-
trostatic potentials at their nuclei (MEPN). This data are
summarized in Table 3. The calculated MEPN showed sig-
nificant differences when different meso-aryl substituents
were used. The third place after the decimal point is reliable
and can be used for tendency examination. The comparison
Computational studies: The computational comparison be-
tween the two reaction routes, Routes A and B, were aimed
at examining the effect of different meso-substituents on re-
action energetics. Four different aryl substituents were used:
C6H5, C6F5, 4-OMe-C6H4, and 4-CO2Me-C6H4, correspond-
ing to structures 2b–e, 2’b–e, 3b–e, 4b–e, and 5b–e, respec-
tively. Calculated relative energies are listed in Table 1 and
in Scheme S1 of the Supporting Information. As can be
seen, there is no significant difference when different sub-
stituents are used. The energy differences are between 1 and
4 kcalmolꢀ1, whereas the accuracy of thermochemical calcu-
lations is generally considered to be around 1 kcalmolꢀ1.
Therefore, thermodynamically there is no observable differ-
ence when different aryl substituents are used. This result
Table 1. Relative energies [kcalmolꢀ1] of BODIPY reaction species fol-
lowing the two synthetic routes when different substituents are used.
Substituent
BODIPY C6H5 C6F5 4-OMe-C6H4 4-CO2Me-C6H4
Route A
reactant
intermediate 2’
intermediate
product
Route B
reactant
intermediate
product
Table 3. Average molecular electrostatic potential at nuclei (MEPN) at
the cyclohexenyl hydrogen atoms of 2’b–e, 3b–e, and 2b–e.
2
0
ꢀ14
ꢀ8
0
ꢀ13
ꢀ6
0
ꢀ15
ꢀ9
0
ꢀ14
ꢀ7
Dipyrro-
methene
MEPNH
BODIPY
MEPNH
Dipyrro-
methane
MEPNH
3
4
ꢀ127 ꢀ124 ꢀ124
ꢀ126
2’b
2’c
2’d
2’e
ꢀ1.136
ꢀ1.128
ꢀ1.138
ꢀ1.134
3b
3c
3d
3e
ꢀ1.128
ꢀ1.120
ꢀ1.130
ꢀ1.126
2b
2c
2d
2e
ꢀ1.137
ꢀ1.132
ꢀ1.138
ꢀ1.135
2
5
4
0
0
0
0
ꢀ137
ꢀ126
ꢀ138 ꢀ136 ꢀ135
ꢀ127 ꢀ124 ꢀ124
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Dyes/Pigments
FULL PAPER
of MEPN data clearly indicates that dipyrromethenes 2’, in
general, have more negative MEPNs compared with the cor-
responding BODIPYs 3, making them less electron-deficient
and more likely to undergo oxidation. Therefore, the higher
yields obtained from oxidation of 3b,d,e compared with
2b,d,e and 2’b,d,e are probably due to the higher stability of
BODIPYs and their lower tendency to undergo side re-
ACHTUNGTRENNUNGactions. On the other hand, the calculated MEPN for
BODIPY 3c has the highest value of all MEPNs, suggesting
that this compound is too electron-deficient to undergo oxi-
dation, according to the experimental observations.
Spectroscopic properties: The photophysical properties of
BODIPYs 3a–e, 4a–e, 7 and 8 were investigated in dichloro-
methane, toluene, and in a protic solvent (methanol) and
the results are summarized in Tables 4 and S1 (Supporting
Information). Figures 4, S3 and S4 (Supporting Information)
show the normalized absorption and fluorescence spectra
obtained for the benzo-appended BODIPYs, in dichlorome-
thane, methanol, and toluene, respectively. All BODIPYs
showed absorptions with high molar absorption coefficients
(loge=3.5–4.4) and relatively narrow fluorescence emission
bands in the three solvents, characteristic of this type of
compound.[23] The strong BODIPY absorption bands are
due to the S0–S1 (p–p*) transition, and the shoulders at low
wavelength are due to the 0–1 vibrational transition, as pre-
viously reported.[23,24]
In dichloromethane and toluene, the absorption bands for
the BODIPY core, monobenzo- and dibenzo-appended
BODIPYs were centered at about 540, 590 nm and 640 nm,
respectively, and the fluorescence emission maxima at about
560, 595 nm and 665 nm, respectively. Changing the solvent
polarity from dichloromethane to methanol, caused 4–7 nm
blue-shifts of the absorption and emission bands, in agree-
ment with previous reports.[23] As expected, benzannulation
caused a large bathochromic shift in the absorption and
emission bands, of about 50–60 nm per ring, in both solvents.
For example, the aromatization of 7 to produce monobenzo-
Figure 4. Normalized UV/Vis (a) and fluorescence (b) spectra of BODI-
PYs 8 (black), 4a (red), 4b (blue), 4c (purple), 4d (green) and 4e
(brown) in dichloromethane at 258C.
BODIPY 8 lead to nearly a 60 nm red-shift in the absorp-
tion and emission bands, whereas dibenzannulation from 3a
to 4a lead to an approximate 90 nm red-shift (Table 4).
Even larger bathochromic shifts
were observed for the BODI-
Table 4. Spectral properties of BODIPYs 3a–e, 4a–e, 7 and 8 in dichloromethane and methanol (in parenthe-
sis), at room temperature.
PYs bearing a meso-aryl group,
for example, about 104 nm
from 3b,d,e to 4b,d,e, respec-
tively. Furthermore, BODIPY
3c and 4c bearing a strongly
Absorbance
e [mꢀ1 cmꢀ1
]
Emission
Ff[a]
Stokes shift [cmꢀ1
]
lmax [nm]
lmax [nm]
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
7
553 (545)
540 (535)
563 (557)
539 (535)
542 (538)
642 (635)
643 (635)
658 (652)
643 (635)
644 (636)
530 (523)
589 (582)
10700 (11300)
20300 (21700)
10700 (12500)
17600 (22800)
17300 (20500)
19800 (3300)
24400 (17200)
19200 (nd)
15300 (18600)
26300 (8800)
20700 (28500)
14900 (16700)
567 (562)
561 (555)
586 (579)
559 (556)
562 (560)
664 (651)
665 (654)
680 (nd)
664 (655)
666 (654)
541 (536)
596 (591)
0.35 (0.23)
0.34 (0.29)
0.18 (0.06)
0.34 (0.20)
0.30 (0.22)
0.38 (0.11)
0.38 (0.45)
0.31 (nd)
0.43 (0.38)
0.43 (0.36)
0.53 (0.32)
0.99 (0.77)
446 (555)
693 (673)
697 (682)
664 (706)
656 (730)
516 (387)
515 (457)
492 (nd)
492 (481)
513 (433)
384 (464)
199 (262)
electron-withdrawing
penta-
fluorophenyl group showed ab-
sorption and emission bands
red-shifted by about 20 nm
compared with the other 8-aryl
substituted BODIPYs, indicat-
ing a better stabilization of the
LUMO and decreased HOMO–
LUMO energy gap.[24] Howev-
er, the change in the para-
phenyl substituent (H, OMe or
CO2Me) only caused 1–4 nm
8
[a] Fluorescence quantum yields for BODIPYs 7 (lexc =481 nm) and 3 (lexc =500 nm) were calculated using
rhodamine 6G (0.80 in methanol) as standard, while those for BODIPY 8 (lexc =530 nm) and 4 (lexc =600 nm)
were calculated using cresyl violet perchlorate (0.54 in ethanol) and methylene blue (0.03 in mehanol) as the
reference,[29] respectively.
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M. G. H. Vicente et al.
shifts in the absorption/emission maxima, in agreement with
previous observations.[25]
BODIPYs in three cell lines, with exception of a few iodi-
nated derivatives.[31]
The fluorescence quantum yields determined in dichloro-
methane were higher for the db-BODIPYs 4 (0.31–0.43)
compared with the corresponding bc-BODIPYs 3 (0.18–
0.35). The highest quantum yields, 0.53 and 0.99, were deter-
mined for BODIPYs 7 and 8, respectively, probably due to
the presence of a methyl rather than a carbonyl group at the
pyrrolic a-position,[2,26] and the lowest for BODIPY 3c. The
lower fluorescence quantum yields observed for BODIPYs
3c and 4c could be due to additional aryl group rota-
The time-dependent uptake of BODIPYs was also evalu-
ated at a concentration of 10 mm over a period of 24 h
(Figure 5). BODIPY 4c bearing a pentafluorophenyl group,
was clearly taken-up by HEp2 cells to a higher extent than
ACHTUNGTRENNUNG
tion[25a,27] and/or to the heavy atom effect,[25] although re-
cently high fluorescence quantum yields were reported for
a a,a’-dimethyl-BODIPY bearing a meso-C6F5 group, and
its derivatives.[28] The fluorescence quantum yields deter-
mined in non-polar toluene varied between 0.60 (for 4c)
and 0.96 for BODIPY 8 (Table S1, Supporting Information).
In methanol, the fluorescence quantum yields were general-
ly observed to decrease probably due to an increase in the
rate of non-radiative deactivation in a more polar solvent.[25]
The ester groups at the pyrrolic a-positions of BODIPYs 3,
4, 7, and 8 confer water solubility for the biological investi-
gations (vide infra) but generally decrease fluorescence
quantum yields, compared with for example, a methyl
group, because of the relative high flexibility of the ethoxy
carbonyl substituent.
Figure 5. Time-dependent uptake of BODIPYs 8 (black), 4a (red), 4b
(blue), 4c (purple), 4d (green) and 4e (brown) at 10 mm by HEp2 cells.
all other BODIPYs, at all time points investigated. This ob-
servation is probably due to the polar hydrophobic nature
of the fluorinated BODIPY 4c, which enhances plasma
membrane permeability and increases cellular uptake.[32]
The meso-free monobenzo- and dibenzo-BODIPYs 8 and
4a accumulated the least within cells, probably due to their
lower stability compared with the meso-substituted db-
BODIPYs. It has been reported that the presence of
Cellular studies: The dark and phototoxicity of BODIPYs
4a–e, and 8 toward human carcinoma HEp2 cells was evalu-
ated using the Cell Titer Blue assay, and the results are sum-
marized in Table 5 and Figures S6 and S7 (in the Supporting
a
meso-aryl group increases the photostability of
BODIPYs.[6a] Significant fluorescence quenching of
BODIPYs 8 and 4a was observed in DMSO with time, as
shown in Figure S4 of the Supporting Information. However
no fluorescence quenching was observed under the same
conditions for the meso-substituted BODIPYs 4b–e. After
24 h the amount of BODIPY 4c found within cells was
about 4 times higher than that of 4a and about 40 times
higher than that of 8.
The subcellular localization of BODIPYs was investigated
using fluorescence microscopy and the results obtained are
shown in Figures 6 and 7 and summarized in Table 5. The
organelle specific fluorescent probes ERTracker (ER), Lyso-
Sensor Green (lysosomes), Mitotracker Green (mitochon-
dria) and BODIPY Ceramide (Golgi) were used in the over-
lay experiments. All BODIPYs were found to localize in the
ER. This result is in agreement with previous observations
that BODIPYs bearing alkyl and aryl groups tend to local-
ize in the cell ER.[33] In addition, 4a–d and 8 were also
found in mitochondria, 4a and 4c–e in the lysosomes and
4a and 4d,e in the Golgi. Our results are in agreement with
literature reports showing that BODIPYs readily accumu-
late within cells,[31,33,34] and can be found in multiple organ-
elles, including the ER, Golgi, mitochondria and lysosomes.
Commercially available BODIPYs are known for the specif-
ic labeling of Golgi and/or the ER, for example BODIPY
FL Ceramide.[3b] The low cytotoxicity, high photostability
Table 5. Cytotoxicity (Cell Titer Blue assay, light dose ca. 1.5 Jcmꢀ2) and
subcellular localization (HEp2 cells) for benzo-appended BODIPYs.
BODIPY
Dark toxicity
Phototoxicity
Major sites
IC50 [mm]
IC50 [mm]
of localization
4a
4b
4c
4d
4e
8
>400
236
>400
>400
373.5
400
>100
>100
>100
>100
>100
>100
ER, Lyso, Golgi, Mito
ER, Mito
ER, Lyso, Mito
ER, Lyso, Golgi, Mito
ER, Lyso, Golgi
ER, Mito
Information). Although BODIPYs 4a–e contain two a-
ethoxy carbonyl substituents, they were poorly soluble in
water and required dimethyl sulfoxide (DMSO) and Cremo-
phor EL as delivery vehicles (never exceeding 0.9%
DMSO/0.1% Cremophor).[30] All BODIPYs were found to
have very low cytotoxicities, with half maximal inhibitory
concentrations (IC50) (dark)> 200 mm and IC50 (ca.
1.5 Jcmꢀ2 light dose)>100 mm. BODIPY 4b was found to
have the highest dark toxicity of this series, probably due to
its lower solubility compared with the other BODIPYs, par-
ticularly at the higher concentrations investigated during the
dark toxicity experiments. The low cytotoxicity of the
BODIPY dyes is in agreement with previous investigations,
which report dark IC50 values >100 mm for a series of 15
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Dyes/Pigments
FULL PAPER
and plasma membrane permeability of benzo-BODIPYs
suggest that these compounds could be used for drug deliv-
ery into cells, or as cation sensors within living cells, in par-
ticular the meso-aryl-substituted benzo-BODIPYs.
Conclusion
Two alternative synthetic routes
were investigated for the prepa-
ration of p-extended BODIPYs
bearing
b,b’-fused
benzene
rings, with fluorecence emissons
in the 596–680 nm region, from
a
common
intermediate.
Route A involved boron com-
plexation to the dipyrrome-
thene (BF3·OEt2/NEt3), fol-
lowed by aromatization with
DDQ in refluxing toluene. In
Route B the aromatization step
to the corresponding dibenzo-
dipyrromethenes
was
per-
formed first, followed by com-
plexation with boron. In gener-
al, Route A gave higher yields
of the dibenzo-BODIPYs (41–
67% from dipyrromethane 2)
because of the higher yields ob-
tained from aromatization of
the BODIPYs compared with
the corresponding dipyrrome-
thenes; this might be due to the
higher
BODIPYs under the oxidative
reaction conditions. There was
however one exception
stability
of
the
AHCTUNGTRENNUNG
(BODIPY 4c), due to the
slow oxidation rate of the
highly electron-deficient 3c,
bearing
a
meso-pentafluoro-
phenyl group; in this case, di-
benzo-BODIPY 4c was rather
obtained by Route B, in 35%
overall yield from dipyrrome-
thane 2. Computational model-
Figure 6. Subcellular localization of
A) BODIPY 4a B) BODIPY 4b,
C) BODIPY 4c, and D) BODIPY 4d
in HEp2 cells at 10 mm for 6 h.
a) Phase contrast, b) overlay of the
BODIPY fluorescence and phase con-
trast, c) ER tracker blue/white fluores-
cence, e) MitoTracker green fluores-
cence, g) BODIPY Ceramide, i) Lyso-
Sensor green fluorescence, and d), f),
h), and j) overlays of organelle tracers
with the BODIPY fluorescence. Scale
bar: 10 mm.
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M. G. H. Vicente et al.
with estimated IC50 values
above 100 mm. BODIPY 4c
bearing
a
pentafluorophenyl
group accumulated within the
HEp2 cells the most of all
BODIPYs investigated, while 8
accumulated the least followed
by 4a, probably due to their
lower stability compared with
the meso-aryl dibenzo-BODI-
PYs. All BODIPYs localized in
the cell ER, and in addition
were also found in other organ-
elles, such as mitochondria, ly-
sosomes and Golgi. Our results
suggest that benzo-appended
BODIPYs, in particular the
meso-substituted BODIPYs, are
promising fluorophores for bio-
imaging applications.
Experimental Section
Syntheses. All air and moisture sensi-
tive reactions were performed under
argon atmosphere in oven-dried glass-
ware. Common reagents were ob-
tained from commercial source and
used without further purification,
unless otherwise stated. Dry solvents
were collected from PS-400 Solvent
Purification System from Innovative
Technology, Inc. Melting points were
Figure 7. Subcellular localization of A) BODIPY 4e and B) BODIPY 8 in HEp2 cells at 10 mm for 6 h.
a) Phase contrast, b) overlay of the BODIPY fluorescence and phase contrast, c) ER tracker Blue/White fluo-
rescence, e) MitoTracker Green fluorescence, g) BODIPY Ceramide, i) LysoSensor Green fluorescence, and
d), f), h), and j) overlays of organelle tracers with the BODIPY fluorescence. Scale bar: 10 mm.
ing of both synthetic approaches demonstrated that chang-
determined in open capillary and are uncorrected. Column chromatogra-
phy was performed on silica gel (Sorbent Technologies, 60 ꢁ, 40–63 mm)
slurry packed into glass columns. Analytical thin-layer-chromatography
(TLC) was carried out using polyester backed TLC plates 254 (precoat-
ed, 200 mm, Sorbent Technologies). 1H NMR and 13C NMR spectra were
recorded on Bruker DPX-400 (operating at 400 MHz for 1H NMR and
100 MHz for 13C NMR) in CDCl3 (7.26 ppm, 1H and 77.0 ppm,13C) with
tetramethylsilane as internal standard. All spectra were recorded at 258C
and coupling constants (J values) are given in Hz. Chemical shifts are
given in parts per million (ppm). High resolution mass spectra were ob-
tained at the LSU Department of Chemistry Mass Spectrometry Facility
using an ESI or MALDI-TOF method, and a peak matching protocol to
determine the mass error range of the molecular ion. Ethyl 4,5,6,7-tetra-
hydroisoindole ester (1)[17] 5-formyl-pyrrole 6[22] and bis(3-ethoxycarbon-
yl-4,5,6,7-tetrahydro-2H-isoindolyl)methane (2a)[17a] were synthesized as
previously described.
ing the meso-substituents did not influence significantly the
energetics of the reaction regardless of the route; therefore
thermodynamically there was no significant difference when
different substituents are used. Furthermore, for Route B
the solvent effects were more pronounced and resulted in
higher exothermicity for the overall reaction. The calculated
MEPN for the butano hydrogen atoms of BODIPY 3c con-
firmed the high electron-deficiency of this compound (high-
est MEPN), which might explain its stability under the oxi-
dation reaction conditions. On the other hand, X-ray analy-
ses of several BODIPYs and intermediates showed that ben-
zannulation further enhances the coplanarity of BODIPY
systems.
The p-extended BODIPYs all showed strong red-shifted
absorptions and fluorescence emissions, about 50–60 nm per
benzoannulated ring. The dibenzo-BODIPY 4c bearing
a meso-pentafluorophenyl group showed the longest lmax ab-
sorption and emission, together with the lowest fluorescence
quantum yield (0.31 in CH2Cl2); on the other hand mono-
benzo-BODIPY 8 showed the highest quantum yield (0.99),
probably due to the presence of a methyl rather than the
more flexible ethoxycarbonyl group at the pyrrolic a-posi-
tion. The p-extended BODIPYs 4a–e, and 8 showed very
low dark- and phototoxicities toward human HEp2 cells
General procedure for the synthesis of 8-aryl-bis(3-ethoxycarbonyl-
4,5,6,7-tetrahydro-2H-isoindolyl)methanes (2b–e): Pyrrole
1 (0.1007 g,
0.5 mmol) was treated with p-toluenesulfonic acid (0.0047 g, 0.025 mmol),
n-tetrabutylammonium chloride (0.0036 g, 0.01 mmol) and THE corre-
sponding aldehyde (0.3 mmol) in CH2Cl2 (50 mL). The mixture was
stirred overnight at room temperature under inert atmosphere, washed
with aqueous saturated NaHCO3 (2ꢂ30 mL), brine (1ꢂ30 mL) and dried
over anhydrous Na2SO4. The solvent was evaporated under vacuum and
the resulting residue was chromatographed on a silica gel column (elu-
tion: hexanes/ethyl acetate 5:1 for 2b, 2d, 2e and hexanes/ethyl acetate
7:3 for 2c). The product was recrystallized from CH2Cl2/hexanes.
Data for 2b: Yield 117 mg (95%); m.p. 110–1128C; 1H NMR (CDCl3):
d=8.74 (brs, 2H), 7.32–7.08 (m, 5H), 5.42 (s, 1H), 4.22–4.15 (m, 4H),
3900
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Dyes/Pigments
FULL PAPER
2.78 (m, 4H), 2.21 (m, 4H), 1.68 (m, 8H), 1.28 ppm (t, J=7.12 Hz, 6H);
13C NMR (CDCl3): d=161.8, 139.1, 130.8, 129.2, 128.9, 128.2, 127.3,
119.7, 116.7, 59.7, 40.6, 23.3, 23.1, 21.2, 14.5 ppm; ESI-HRMS: m/z calcd
for C29H34N2O4 [M+H+]: 475.2575; found: 474.25.
Data for 2c: Yield 123 mg (87%); m.p. 106–1088C; 1H NMR (CDCl3):
d=8.71 (brs, 2H), 5.79 (s, 1H), 4.27 (q, J=7.12 Hz, 4H), 2.27 (m, 4H),
2.26 (m, 2H), 2.12 (m, 2H), 1.70 (m, 8H), 1.33 ppm (t, J=7.12 Hz, 6H);
13C NMR (CDCl3): d=161.4, 146.0, 143.6, 141.9, 139.2, 139.1, 136.5,
128.9, 126.3, 120.2, 117.6, 113.4, 113.3, 113.1, 60.0, 29.8, 23.1,23.0, 22.8,
21.0, 20.1, 14.4 ppm; ESI-HRMS: m/z calcd for C29H29F5N2O4 [M+Na+]:
587.1946; found: 564.20.
Data for 2d: Yield 112 mg (89%); m.p. 113–1168C; 1H NMR (CDCl3):
d=9.39 (brs, 2H), 7.91 (m, 2H), 7.15 (m, 2H), 5.50 (s, 1H), 4.18–4.06
(m, 4H), 3.91 (s, 3H), 2.74 (m, 4H), 2.26 (m, 4H), 1.69 (m, 8H),
1.24 ppm (t, J=7.12 Hz, 6H); 13C NMR (CDCl3): d=166.6, 161.9, 144.9,
130.2, 129.9, 129.2, 128.9, 128.1, 120.0, 117.2, 59.8, 52.1, 40.1, 23.3, 23.1,
23.1, 21.4, 14.4 ppm; ESI-HRMS: m/z calcd for C30H36N2O5 [M+H+]:
504.2576 ; found: 504.26.
duced pressure and the residue was dissolved in ethyl acetate and ex-
tracted with 0.1m HCl (2ꢂ30 mL) to remove excess DDQ, brine (1ꢂ
30 mL) and dried over anhydrous Na2SO4. The solvent was evaporated
under vacuum and the resulting residue was purified by column chroma-
tography (elution: hexanes/ethyl acetate 3:2). The product was recrystal-
lized from CH2Cl2/hexanes.
Data for 3a: Yield 149 mg (67%); m.p. 169–1718C; 1H NMR (CDCl3):
d=5.30 (s, 1H), 4.42 (q, J=7.11 Hz, 4H), 2.65 (m, 8H), 1.76 (m, 8H),
1.42 ppm (t, J=7.11 Hz, 6H); 13C NMR (CDCl3): d=161.7, 144.1, 142.6,
134.4, 132.8, 129.6, 128.7, 128.6, 126.1, 121.2, 61.6, 60.7, 29.7, 23.1, 22.9,
22.8, 22.7, 22.4, 22.0, 21.5, 14.3, 14.1, 14.0 ppm; ESI-HRMS: m/z calcd for
C23H27BF2N2O4 [M+Na+]: 467.1917; found: 444.20.
Data for 3b: Yield 208 mg (80%); m.p. 209–2118C; 1H NMR (CDCl3):
d=7.52 (m, 3H), 7.23 (m, 2H), 4.44 (q, J=7.12 Hz, 4H), 2.57 (m, 4H),
1.57 (m, 8H), 1.42 (t, J=7.12 Hz, 6H), 1.39 ppm (m, 4H); 13C NMR
(CDCl3): d=161.6, 147.6, 143.8, 143.6, 134.6, 132.9, 132.5, 129.6, 129.5,
127.2, 61.7, 24.4, 22.6, 22.3, 22.0, 14.0 ppm; MALDI-TOF: m/z calcd for
C29H31BF2N2O4 [M+Na+]: 543.237; found: 520.23.
Data for 3c: Yield 229 mg (75%); m.p. 202–2048C; 1H NMR (CDCl3):
d=4.45 (q, J=7.12 Hz, 4H), 2.60 (m, 4H), 1.81 (m, 4H), 1.65 (m, 4H),
1.59 (m, 4H), 1.45 ppm (t, J=7.12 Hz, 6H); 13C NMR (CDCl3): d=161.0,
145.6, 142.3, 137.8, 133.9, 133.5, 132.6, 128.3, 62.0, 29.6, 23.3, 23.2, 23.0,
22.8, 22.4, 22.1, 22.0, 14.0 ppm; MALDI-TOF: m/z calcd for
C29H26BF7N2O4 [M+]: 610.1876; found: 610.19.
Data for 3d: Yield 200 mg (73%); m.p. 181–1838C; 1H NMR (CDCl3):
d=7.12 (m, 2H), 7.03 (m, 2H), 4.43 (q, J=7.1 Hz, 4H), 3.89 (s, 3H), 2.76
(m, 4H), 2.57 (m, 4H), 1.64 (m, 4H), 1.58 (m, 4H), 1.41 (t, J=7.1 Hz,
6H), 1.40 ppm (m, 4H); 13C NMR (CDCl3): d=161.5, 160.6, 147.9, 143.7,
143.4, 133.3, 132.4, 128.6, 126.5, 114.8, 61.6, 55.3, 24.7, 22.6, 22.3, 22.0,
14.0 ppm; ESI-HRMS: m/z calcd for C30H33BF2N2O5 [M+Na+]: 573.2361;
found: 550.25.
Data for 3e: Yield 174 mg (60%); m.p. 173–1758C; 1H NMR (CDCl3):
d=8.21 (m, 2H), 7.36 (m, 2H), 4.43 (q, J=7.12 Hz, 4H), 3.99 (s, 3H),
2.57 (m, 4H), 1.56 (m, 8H), 1.44 (t, J=7.12 Hz, 6H), 1.39 ppm (m, 4H);
13C NMR (CDCl3): d=166.6, 161.4, 146.0, 144.1, 143.5, 139.2, 132.8,
132.4, 131.4, 130.7, 127.6, 61.8, 52.5, 24.6, 22.5, 22.3, 21.9, 14.0 ppm; ESI-
HRMS: m/z calcd for C31H33BF2N2O6 [M+H+]: 577.2299; found: 578.24.
1
Data for 2e: Yield 93 mg (70%); m.p. 104–1068C; H NMR (CDCl3): d=
8.92 (brs, 2H), 7.94 (m, 2H), 7.16 (m, 2H), 5.47 (s, 1H), 4.17 (m, 4H),
3.91 (s, 3H), 2.76 (m, 4H), 2.21 (m, 4H), 1.68 (m, 8H), 1.27 ppm (t, J=
7.12 Hz, 6H); 13C NMR (CDCl3): d, 166.3, 161.1, 146.2, 143.4, 142.0,
138.6, 132.3, 131.1, 129.8, 129.5, 127.6, 61.2, 52.5, 24.5, 23.2, 22.5, 21.7,
14.0 ppm; ESI-HRMS: m/z calcd for C31H36N2O6 [M+H+]: 533.2649;
found: 532.26.
General procedure for the synthesis of 8-aryl-bis(3-ethoxycarbonyl-
4,5,6,7-tetrahydro-2H-isoindolyl)methenes (2’b–e): A solution of DDQ
(0.36 mmol, 1.2 equiv) in dry dichloromethane (10 mL) was added to a so-
lution of 2’b–e (0.3 mmol, 1.0 equiv) in dry dichloromethane (20 mL) at
08C. The solution was stirred under argon for 15–20 min until TLC analy-
sis indicated reaction completion. The reaction mixture was washed with
aqueous saturated NaHCO3 (2ꢂ30 mL), brine (1ꢂ30 mL) and dried over
anhydrous Na2SO4. The solvent was evaporated under vacuum and the
residue was chromatographed on a silica gel column using CH2Cl2/petro-
leum ether 5:1 for elution. The product was recrystallized from CH2Cl2/
hexanes.
Data for 2’b: Yield 106 mg (75%); m.p. 164–1668C; 1H NMR (CDCl3):
d=7.45 (m, 3H), 7.27 (m, 2H), 4.37 (q, J=7.12 Hz, 4H), 2.69 (m, 4H),
1.57 (m, 4H), 1.51 (m, 4H), 1.42 (t, J=7.12 Hz, 6H), 1.36 ppm (m, 4H);
13C NMR (CDCl3): d=162.3, 143.8, 141.7, 139.7, 139.0, 137.0, 133.0,
129.1, 128.8, 128.7, 60.6, 24.2, 23.2, 23.1, 22.3, 14.4 ppm; ESI-HRMS: m/z
calcd for C29H32N2O4 [M+H+]: 473.2447; found: 472.2362.
Data for 2’c: Yield 118 mg (70%); m.p. 158–1608C; 1H NMR (CDCl3):
d=4.37 (q, J=7.12 Hz, 4H), 2.67 (m, 4H), 1.67 (m, 4H), 1.60 (m, 4H),
1.52 (m, 8H), 1.41 ppm (t, J=7.12 Hz, 6H); 13C NMR (CDCl3): d=161.8,
145.8, 143.2, 138.6, 137.8, 134.0, 132.4, 127.4, 60.9, 29.4, 23.3, 23.2, 23.0,
22.8, 22.4, 22.2, 22.0, 14.1 ppm; ESI-HRMS: m/z calcd for C29H27F5N2O4
[M+H+]: 563.1978; found: 562.1891.
Data for 2’d: Yield 123 mg (82%); m.p. 172–1748C; 1H NMR (CDCl3):
d=7.28 (m, 2H), 7.11 (m, 2H), 4.49 (q, J=7.12 Hz, 4H), 4.07 (s, 3H),
3.05 (m, 4H), 1.96 (m, 4H), 1.53 (m, 8H), 1.22 ppm (t, J=7.12 Hz, 6H);
13C NMR (CDCl3): d=161.6, 160.3, 147.2, 142.7, 139.6, 133.0, 132.1,
129.2, 126.2, 114.2, 60.6, 55.3, 24.5, 23.2, 23.1, 22.4, 14.4 ppm; ESI-HRMS:
m/z calcd for C30H34N2O5 [M+H+]: 503.2560; found: 502.2467.
General procedure for the synthesis of BODIPYs
4 by Route A:
BODIPYs 3a–e (112 mg, 0.2665 mmol) were dissolved in toluene
(30 mL) and heated to 1108C. A solution of DDQ (9 equiv) in toluene
(20 mL) was added and the final mixture was refluxed under argon. The
reaction was monitored by UV/Vis spectroscopy. Upon reaction comple-
tion, the mixture was cooled to room temperature and the solvent was re-
moved under reduced pressure. The residue was dissolved in CH2Cl2
(30 mL) and extracted with 10% aqueous NaHCO3 (3ꢂ30 mL), brine
(1ꢂ30 mL) and dried over anhydrous Na2SO4. The solvent was removed
under reduced pressure and the residue was purified by column chroma-
tography (elution: CH2Cl2 to 0.1% MeOH in CH2Cl2). The product was
recrystallized from CH2Cl2/hexanes.
Data for 4a: Yield 81 mg (69%); m.p. >2508C; 1H NMR (CDCl3): d=
8.45 (m, 2H), 8.15 (m, 2H), 7.55 (m, 2H), 7.41 (m, 2H), 4.60 (q, J=
7.11 Hz, 4H), 1.55 ppm (t, J=7.3 Hz, 6H); 13C NMR (CDCl3): d=160.2,
140.2, 133.8, 130.9, 127.2, 124.5, 122.9, 62.4, 14.1 ppm; MALDI-TOF: m/z
calcd for C23H27BF2N2O4 [M+H+]: 444.20; found: 444.20.
Data for 4b: Yield 91 mg (67%); m.p. >2508C; 1H NMR (CDCl3): d=
8.11 (m, 2H), 7.72 (m, 3H), 7.52 (m, 2H), 7.26 (m, 2H), 7.08 (m, 2H),
6.16 (m, 2H), 4.62 (q, J=7.12 Hz, 4H), 1.55 ppm (t, J=7.12 Hz, 6H);
13C NMR (CDCl3): d=160.6, 141.2, 140.2, 134.7, 134.2, 131.2, 130.1,
129.8, 129.7, 129.1, 128.5, 126.5, 124.1, 121.7, 62.2, 14.1 ppm; ESI-HRMS:
m/z calcd for C29H23BF2N2O4 [M+Na+] 535.1633; found: 512.17.
Data for 4d: Yield 132 mg (92%); m.p. >2508C; 1H NMR (CDCl3): d=
8.11 (m, 2H), 7.26 (m, 8H), 6.30 (m, 2H), 4.61 (q, J=7.12 Hz, 4H), 4.02
(s, 3H), 1.55 ppm (t, J=7.12 Hz, 6H); 13C NMR (CDCl3): d=160.8,
160.6, 141.4, 139.9, 134.7, 131.1, 129.8, 129.6, 129.5, 126.4, 126.2, 124.0,
121.8, 115.1, 62.2, 55.5, 14.1 ppm; MALDI-TOF: m/z calcd for C30H25
BF2N2O5 [M+]: 542.260; found: 542.18.
Data for 2’e: Yield 108 mg (68%); m.p. 155–1578C; 1H NMR (CDCl3):
d=8.18 (m, 2H), 8.01 (m, 2H), 4.49 (m, 4H), 3.95 (s, 3H), 2.67 (m, 4H),
1.65 (m, 4H), 1.53 (m, 4H), 1.42 (t, J=7.12 Hz, 6H), 1.31 ppm (m, 4H);
13C NMR (CDCl3): d=166.3, 162.1, 146.2, 143.6, 142.2, 138.6, 133.0,
130.1, 129.8, 129.5, 128.0, 61.2, 52.5, 24.5, 23.2, 23.1, 22.4, 14.0 ppm; ESI-
HRMS: m/z calcd for C31H34N2O6 [M+H+]: 531.2480; found: 530.2417.
General procedure to BODIPYs 3: A solution of DDQ (0.6 mmol,
1.2 equiv) in dry THF (10 mL) was added to a solution of 2a (0.5 mmol,
1.0 equiv) in dry THF (10 mL) for 2a or CH2Cl2 for 2b–e, at 08C. The
solution was stirred under argon for 20 min. Et3N (3 mmol, 6 equiv) and
BF3·OEt2 (5 mmol, 10 equiv) were added dropwise and the solution was
stirred for 20 min at 08C and then overnight at room temperature. Once
TLC indicated reaction completion, the solvent was removed under re-
Chem. Eur. J. 2012, 18, 3893 – 3905
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3901

M. G. H. Vicente et al.
Data for 4e: Yield 103 mg (68%); m.p. >2508C; 1H NMR (CDCl3): d=
8.39 (m, 2H), 8.12 (m, 2H), 7.65 (m, 2H), 7.27 (m, 2H), 7.09 (m, 2H),
6.12 (m, 2H), 4.62 (q, J=7.12 Hz, 4H), 4.07 (s, 3H), 1.55 ppm (t, J=
7.12 Hz, 6H); 13C NMR (CDCl3): d=166.3, 160.5, 140.6, 139.7, 138.8,
134.4, 131.8, 131.2, 131.0, 130.0, 128.9, 128.5, 126.7, 124.3, 121.4, 62.3,
52.6, 14.1 ppm; MALDI-TOF: m/z calcd for C31H25 BF2N2O6 [M+H+]:
570.213; found: 570.18;.
128.5, 127.3, 125.1, 122.5, 119.5, 62.6, 14.1 ppm; MALDI-TOF: m/z calcd
for C29H18 BF7N2O4 [M+]: 602.179; found: 602.12.
BODIPY 7: Pyrrole
1
(150 mg, 0.7762 mmol) and
6
(162 mg,
0.7762 mmol) were dissolved in dry dichloromethane (20 mL) at 08C and
POCl3 (0.14 mL, 0.9314 mmol) was added dropwise over 5 min. The reac-
tion mixture was stirred at 08C for 10 min and then stirred overnight at
room temperature. After TLC indicated reaction completion, Et3N
(0.5 mL, 3.9 mmol) was added dropwise at 08C, followed by BF3·OEt2
(0.8 mL, 6.2 mmol) at 08C. After the addition was complete, the mixture
was stirred for 16 h at room temperature. The solvent was removed
under reduced pressure and the residue was dissolved in dichlorome-
thane (20 mL) and washed with 10% aqueous NaHCO3 (2ꢂ20 mL),
brine (2ꢂ20 mL) and dried over anhydrous Na2SO4. The solvent was
evaporated to dryness under reduced pressure and the resulting residue
was purified by column chromatography (elution with hexanes/ethyl ace-
tate 3:2). The fraction containing the product was collected, dried under
vacuum and recrystallized from CH2Cl2/hexanes to yield pure BODIPY 7
(134 mg, 40%) as a lustrous green solid. M.p. 135–1378C; 1H NMR
(CDCl3, 400 MHz): d=7.04 (s, 1H), 4.37 (q, J=7.12 Hz, 4H), 3.67 (s,
1H), 2.72 (m, 4H), 2.65 (overlapped m, 2H), 2.61 (s, 3H), 2.46 (m, 2H),
2.21 (s, 3H), 1.75 (m, 1H), 1.38 ppm (t, J=7.12 Hz, 3H); 13C NMR
(CDCl3, 100 MHz): d=172.6, 165.5, 161.1, 141.7, 136.7, 136.5, 132.2,
131.9, 131.7, 120.8, 60.6, 51.8, 33.4, 23.3, 23.1, 22.4, 21.5, 19.4, 14.2, 13.7,
9.6 ppm; MALDI-TOF: m/z calcd for C22H27BF2N2O6 [M+Na+]: 455.251;
found: 432.27.
General procedure to dibenzo-fused dipyrrins (5a–e): Dipyrromethanes
(2a–e) (112 mg, 0.2665 mmol) were dissolved in dry toluene (30 mL) and
heated to 1108C. A solution of DDQ (9 equiv) in dry toluene (20 mL)
was added and the mixture was refluxed under argon. The reaction was
monitored by UV/Vis spectroscopy and upon completion the solvent was
removed under reduced pressure. The purple residue was dissolved in
ethyl acetate and extracted with 0.1m HCl to remove traces of DDQ (3ꢂ
50 mL), brine (1ꢂ50 mL) and dried over anhydrous Na2SO4. The solvent
was removed under reduced pressure and the residue was purified by
column chromatography (elution: CH2Cl2 for 5a, 5b and hexanes/ethyl
acetate, 2:1 for 5c,d,e). The product was recrystallized from CH2Cl2/
methanol.
Data for 5a: Yield 57 mg (56%); m.p. 168–1698C; UV/Vis (CH2Cl2): lmax
(e)=568 nm (13710mꢀ1 cmꢀ1); 1H NMR (CDCl3): d=8.48 (m, 2H), 8.23
(m, 2H), 7.40 (m, 4H), 4.55 (q, J=7.07 Hz, 4H), 1.55 ppm (t, J=7.3 Hz,
6H); 13C NMR (CDCl3): d=161.6, 137.1, 134.5, 133.3, 133.0, 131.3, 130.8,
128.7, 127.7, 127.0, 126.5, 126.4, 123.1, 123.0, 122.8, 118.9, 61.5, 61.2, 61.1,
29.6, 14.3, 14.5 ppm; ESI-HRMS: m/z calcd for C23H20N2O4 [M+H+]:
388.14; found: 388.14.
BODIPY 8: BODIPY 7 (100 mg, 0.2313 mmol) was dissolved in toluene
(15 mL) and heated to 1108C. A solution of DDQ (262 mg, 1.1566 mmol,
5 equiv) in toluene (15 mL) was added and the mixture was refluxed
under argon. The reaction was monitored using UV/Vis. Once the re-
Data for 5b: Yield 81 mg (66%); m.p. 146–1478C; UV/Vis (CH2Cl2): lmax
(e)=569 nm (24136mꢀ1 cmꢀ1); 1H NMR (CDCl3): d=8.19 (m, 2H), 7.66
(m, 5H), 7.24 (m, 2H), 6.97 (m, 2H), 6.12 (m, 2H), 5.30 (s, 1H), 4.58 (q,
J=7.12 Hz, 4H), 1.56 ppm (t, J=7.12 Hz, 6H); 13C NMR (CDCl3): d=
161.8, 137.7, 136.4, 135.5, 135.0, 131.6, 129.5, 129.4, 129.1, 126.9, 126.0,
122.8, 122.0, 61.2, 14.4 ppm; ESI-HRMS: m/z calcd for C29H24N2O4
[M+H+]: 465.1788; found: 464.17.
AHCTUNGERTGaNNUN ction was complete, it was allowed to cool down to room temperature
and the solvent was removed under reduced pressure. The residue was
dissolved in dichloromethane (20 mL) and extracted with aqueous satu-
rated NaHCO3 (20 mL), brine (20 mL) and dried over anhydrous
Na2SO4. The solution was evaporated to dryness and the residue was pu-
rified by column chromatography (elution: hexanes/ethyl acetate 3:2).
The fractions containing the title BODIPY were collected, dried under
vacuum and recrystallized from CH2Cl2/hexanes, yielding pure BODIPY
8 (59.4 mg, 60%) as a lustrous green solid. M.p. 162–1648C; 1H NMR
(CDCl3, 400 MHz): d=8.22 (m, 1H), 7.79 (m, 1H), 7.46 (s, 1H), 7.41 (m,
1H), 7.34 (m, 1H), 4.55 (q, J=7.12 Hz, 2H), 3.69 (s, 1H), 2.75 (m, 2H),
2.64 (s, 3H), 2.49 (m, 2H), 2.28 (s, 3H), 1.51 ppm (t, J=7.12 Hz, 3H);
13C NMR (CDCl3, 100 MHz): d=172.6, 163.0, 160.4, 140.7, 135.8, 133.3,
131.3, 130.5, 128.5, 127.9, 126.3, 123.9, 118.7, 118.3, 61.4, 51.8, 33.6, 19.4,
14.3, 13.5, 9.6 ppm; MALDI-TOF: m/z calcd for C22H23BF2N2O4 [M+]
428.224; found: 428.24.
Data for 5c: Yield 91 mg (62%); m.p. 224–2258C; UV/Vis (CH2Cl2): lmax
(e)=583 nm (34069mꢀ1 cmꢀ1); 1H NMR (CDCl3): d=8.25 (m, 2H), 7.33
(m, 2H), 7.18 (m, 2H), 6.34 (m, 2H), 4.56 (q, J=7.12 Hz, 4H), 1.56 ppm
(t, J=7.12 Hz, 6H); 13C NMR (CDCl3): d=161.4, 139.3, 134.6, 134.3,
131.8, 128.2, 126.8, 123.7, 119.8, 61.5, 14.4 ppm; ESI-HRMS: m/z calcd
for C29H19F5N2O4 [M+Na+]: 555.1328; found: 554.13.
Data for 5d: Yield 90 mg (69%); m.p. 197–1998C; UV/Vis (CH2Cl2): lmax
( e)=569 nm (31070mꢀ1 cmꢀ1); 1H NMR (CDCl3): d=8.20 (m, 2H), 7.43
(m, 2H), 7.24 (m, 2H), 7.18 (m, 2H), 7.02 (m, 2H), 6.28 (m, 2H), 4.57
(q, J=7.12 Hz, 4H), 4.01 (s, 3H), 1.56 ppm (t, J=7.12 Hz, 6H);
13C NMR (CDCl3): d=161.8, 135.7, 131.7, 130.6, 126.9, 126.0, 122.7,
122.1, 114.8, 61.2, 55.5,14.4 ppm; MALDI-TOF: m/z calcd for
C30H26N2O5 [M+H+] 495.258; found: 494.18.
Crystal data: Diffraction data were collected at low temperature on
either a Nonius KappaCCD diffractometer equipped with MoKa radiation
(l=0.71073 ꢁ) or a Bruker Kappa Apex-II diffractometer equipped with
CuKa radiation (l=1.54178 ꢁ). Refinement was by full-matrix least
squares using SHELXL, with hydrogen atoms in idealized positions,
except for NH hydrogen atoms, which were located by difference maps
and in most cases their positions refined. Disorder is present in several of
the structures.
Data for 5e: Yield 81 mg (58%); m.p. 208–2098C; UV/Vis (CH2Cl2): lmax
(e)=572 (30918mꢀ1 cmꢀ1), 1H NMR (CDCl3): d=8.35 (m, 2H), 8.18 (m,
2H), 7.65 (m, 2H), 7.23 (m, 2H), 6.96 (m, 2H), 6.06 (m, 2H), 4.57 (q, J=
7.12 Hz, 4H), 4.06 (s, 3H), 1.56 ppm (t, J=7.12 Hz, 6H); 13C NMR
(CDCl3): d=166.6, 161.7, 141.1, 138.1, 137.8, 135.3, 134.4, 131.7, 131.2,
130.7, 129.5, 128.8, 127.1, 126.2, 123.0, 121.7, 61.3, 52.5, 14.4 ppm; ESI-
HRMS: m/z calcd for C31H26N2O6 [M+H+] 522.1831; found: 522.18;.
Crystal data for 2a: C23H30N2O4, monoclinic space group P21/c, a=
8.3020(6), b=18.1194(14), c=13.5258(10) ꢁ, b=98.449(5)8, V=
General procedure for synthesis of BODIPYs 4 by Route B: A solution
of db-dipyrrins 5a–e (0.1 mmol) in toluene (20 mL) was stirred at 08C
for 15 min under argon. Et3N (0.089 mL, 0.64 mmol) and BF3·OEt2
(0.13 mL, 1.024 mmol) were added and the mixture was stirred at room
temperature for 30 min. The reaction mixture was then refluxed until
UV/Vis spectroscopy indicated reaction completion. The solution was
washed with 10% aqueous NaHCO3 (3ꢂ20 mL), brine (1ꢂ20 mL) and
dried over anhydrous Na2SO4. The solvent was evaporated under reduced
pressure and the product was obtained after recrystallization from
CH2Cl2/hexanes mixtures. Yields obtained: 4a, 24 mg (55%): 4b, 40 mg
(80%); 4c, 34 mg (56%); 4d, 49 mg (90%); 4e, 43 mg (75%).
2012.6(3) ꢁ3, T=90.0(5) K, Z=4, 1calcd =1.315 gcmꢀ3
,
mACHTUNGTRENNUNG(CuKa)=
0.73 mmꢀ1. A total of 13,289 data was collected at to q=69.08. R=0.062
for 2895 data with I>2s(I) of 3597 unique data and 270 refined parame-
ters.
Crystal data for 2b EtOAc solvate: C29H34N2O4·1/2C4H8O2, triclinic space
¯
group P1, a=8.698(3), b=14.961(5), c=21.472(9) ꢁ, a=86.86(3), b=
88.15(2), g=89.83(2)8, V=2788.5(18) ꢁ3, T=90.0(5) K, Z=4, 1calcd
=
1.235 gcmꢀ3 (CuKa)=0.67 mmꢀ1
, mACHUTNRTEGNNUNG . 24,515 total data, qmax =58.88. R=
0.087 for 3895 data with I>2s(I) of 7545 unique data and 692 refined pa-
rameters.
Data for 4c: M.p. >2508C; 1H NMR (CDCl3): d=8.16 (m, 2H), 7.38–
7.25 (m, 4H), 6.39 (m, 2H), 4.62 (q, J=7.12 Hz, 4H), 1.54 ppm (t, J=
7.12 Hz, 6H); 13C NMR (CDCl3): d=160.1, 142.0, 133.8, 131.3, 131.2,
Crystal data for 2c pentafluorobenzaldehyde co-crystal: C29H29F5N2O4·1/
2C7HF5O, tetragonal space group I41/a, a=24.931(2), c=40.838(4) ꢁ,
V=25,383(4) ꢁ3, T=90.0(5) K, Z=32, 1calcd =1.387 gcmꢀ3
, mACHTUNGTRENNUNG(CuKa)=
3902
www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 3893 – 3905

Dyes/Pigments
FULL PAPER
1.06 mmꢀ1. 95523 total data, qmax =68.98. R=0.068 for 8230 data with I>
2s(I) of 11630 unique data and 838 refined parameters.
1.08 mmꢀ1. 26194 total data, qmax =68.68. R=0.034 for 3823 data with I>
2s(I) of 4508 unique data and 367 refined parameters.
¯
Crystal data for 2d: C30H36N2O5, triclinic space group P1, a=10.7765(10),
Crystal data for 5d: C30H26N2O5, monoclinic space group P21/n, a=
b=11.1382(12), c=14.2929(13) ꢁ, a=100.304(5), b=103.378(4), g=
9.456(2),
b=13.896(3),
c=18.304(4) ꢁ,
b=93.890(15)8,
V=
102.828(4)8, V=1578.7(3) ꢁ3, T=90.0(5) K, Z=2, 1calcd =1.189 gcmꢀ3, m-
93.890(15) ꢁ3, T=90.0(5) K, Z=4, 1calcd =1.369 gcmꢀ3
,
mACHTUNGTRENNUNG(MoKa)=
ACHTUNGTRENNUNG
(CuKa)=0.58 mmꢀ1. 17121 total data, qmax =68.48. R=0.060 for 4767 data
with I>2s(I) of 5533 unique data and 354 refined parameters.
0.09 mmꢀ1. 14344 total data, qmax =26.08. R=0.051 for 3109 data with I>
2s(I) of 4714 unique data and 339 refined parameters.
Crystal data for 3b: C29H31BF2N2O4, monoclinic space group P21/c, a=
11.3252(15), b=24.088(5), c=9.778(2) ꢁ, b=108.409(10)8, V=
Crystal data for 5d precursor: C30H34N2O5, orthorhombic space group
Pbca, a=21.665(2), b=9.1272(10), c=26.015(2) ꢁ, V=5144.2(8) ꢁ3, T=
2530.9(8) ꢁ3, T=150.0(5) K, Z=4, 1calcd =1.366 gcmꢀ3
,
mACHTNGUTRENNU(G MoKa)=
ACTHNUTRGNEUNG
90.0(5) K, Z=8, 1calcd =1.298 gcmꢀ3, m(CuKa)=0.71 mmꢀ1. 49,707 total
0.10 mmꢀ1. 8398 total data, qmax =27.98. R=0.053 for 3731 data with I>
2s(I) of 6018 unique data and 392 refined parameters.
data, qmax =59.08. R=0.034 for 3043 data with I>2s(I) of 3696 unique
data and 345 refined parameters.
¯
Crystal data for 3c: C29H26BF7N2O4, triclinic space group P1, a=
CCDC-839637 (2a), CCDC-839638 (2b), CCDC-839639 (2c), CCDC-
839640 (2d), CCDC-840083 (3c), CCDC-840084 (3b), CCDC-840085
(3d), CCDC-840086 (3e), CCDC-840120 (4b), CCDC-840121 (4d),
CCDC-840122 (4e), CCDC-840123 (8), CCDC-840124 (5b), CCDC-
840125 (5b precursor), CCDC-840126 (5c), CCDC-840127 (5c precur-
sor), CCDC-840128 (5d), and CCDC-840129 (5d precursor) contain the
supplementary crystallographic data for this paper. These data can be ob-
tained free of charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif.
8.0664(4), b=14.5015(6), c=23.4495(12) ꢁ, a=92.042(4), b=97.547(4),
g=91.775(3)8, V=2715.8(2) ꢁ3, T=90.0(5) K, Z=4, 1calcd =1.493 gcmꢀ3
,
mACHTUNGTRENNUNG
5350.0(8) ꢁ3, T=90.0(5) K, Z=8, 1calcd =1.367 gcmꢀ3
,
mACHTUNGTRENNUNG
0.84 mmꢀ1. 13921 total data, qmax =68.38. R=0.039 for 4024 data with I>
2s(I) of 4767 unique data and 364 refined parameters.
Computation modeling. The two reaction routes A and B were studied
computationally in the framework of density functional theory.[35] The
Crystal data for 3e: C31H33BF2N2O6, monoclinic space group C2/c, a=
hybrid density functional B3LYP[36] was used at the 6–31G
ACTHNUGRTENUNG(d,p) level.
41.881(3),
b=15.0780(15),
c=27.105(2) ꢁ,
b=97.579(5)8,
V=
16,967(2) ꢁ3, T=150.0(5) K, Z=24, 1calcd =1.359 gcmꢀ3
,
mACHTUNGTRENNUNG
(CuKa)=
The geometry of compounds 2b–e, 2’b–e, 3b–e, 4b–e, and 5b–e were op-
timized and the relative energies were evaluated. Potential energy sur-
face minima were confirmed with frequency calculations. The solvent ef-
fects were accounted for using the Polarizable Continuum Model
(PCM).[37] The molecular electrostatic potentials at the atomic nuclei
were calculated according to the literature.[38] All calculations were per-
formed using Gaussian 09 program package.[39]
0.85 mmꢀ1. 64554 total data, qmax =68.48. R=0.043 for 11391 data with
I>2s(I) of 15206 unique data and 1150 refined parameters.
Crystal data for 4b: C29H23BF2N2O4, orthorhombic space group Pbca, a=
13.0867(9), b=23.649(2), c=15.7556(10) ꢁ, V=4876.2(6) ꢁ3, T=
ACTHNUTRGNEUNG
100.0(5) K, Z=8, 1calcd =1.396 gcmꢀ3, m(CuKa)=0.86 mmꢀ1. 43,842 total
data, qmax =68.38. R=0.036 for 3670 data with I>2s(I) of 4434 unique
Steady-state absorption and fluorescence spectroscopy: The absorption
measurements were carried out on a Varian Cary 50 UV/Vis spectropho-
tometer and the steady-state fluorescence spectroscopic studies in di-
data and 345 refined parameters.
Crystal data for 4d: C30H25BF2N2O5, orthorhombic space group Pbca, a=
16.208(3), b=12.384(2), c=24.802(4) ꢁ, V=4978.3(15) ꢁ3, T=90.0(5) K,
Z=8, 1calcd =1.447 gcmꢀ3, m(CuKa)=0.90 mmꢀ1. 30190 total data, qmax
ACHTUNGTERNUNNG =
chloromethane and methanol were performed on
a PTI Quantum
Master4/2006SE spectrofluorimeter, and in toluene on a Fluorologꢃ-3
Modular spectrofluorometer. For the fluorescence quantum yield meas-
urements, dilute solutions with absorbance between 0.04–0.06 at the exci-
tation wavelength were used. The fluorescence quantum yields of BODI-
PYs 7 and 3a–e were obtained by comparing the area under the correct-
ed emission spectrum of the test sample with that of rhodamine 6G (0.80
in methanol). For BODIPYs 4a–e and 8, methylene blue (0.03 in metha-
nol) and cresyl violet perchlorate (0.54 in ethanol) were used as external
standards, respectively.[29] All spectra were recorded at room temperature
using non-degassed samples, spectroscopic grade solvents and a 10 mm
quartz cuvette. In all cases, correction for the refractive index was ap-
plied.
58.98. R=0.063 for 2995 data with I>2s(I) of 3557 unique data and 397
refined parameters.
Crystal data for 4e: C31H25BF2N2O6, orthorhombic space group Pbca, a=
15.792(2), b=12.530(2), c=26.067(4) ꢁ, V=5158.0(13) ꢁ3, T=90.0(5) K,
Z=8, 1calcd =1.469 gcmꢀ3, m(CuKa)=0.93 mmꢀ1. 34135 total data, qmax
ACHTUNGTERNUNNG =
60.38. R=0.078 for 2405 data with I>2s(I) of 3788 unique data and 382
refined parameters.
Crystal data for 8: C22H23BF2N2O4, monoclinic space group P21/n, a=
11.4027(15), b=7.3503(10), c=24.381(3) ꢁ, b=102.545(6)8, V=
1994.7(5) ꢁ3, T=100.0(5) K, Z=4, 1calcd =1.426 gcmꢀ3
,
mACHTNGUTRENNU(G MoKa)=
0.11 mmꢀ1. 22120 total data, qmax =27.88. R=0.039 for 3415 data with I>
2s(I) of 4679 unique data and 285 refined parameters.
Cell culture. All tissue culture media and reagents were obtained from
Invitrogen. Human carcinoma HEp2 cells were obtained from ATCC
and maintained in a 50:50 mixture of DMEM:Advanced MEM contain-
ing 5% FBS. The HEp2 cells were sub-cultured biweekly to maintain
sub-confluent stocks. the 4th to 15th passage cells were used for all the
experiments.
Crystal data for 5b hexanes solvate: C29H24N2O4·0.2C6H14, monoclinic
space group C2/c, a=18.5304(12), b=20.7226(14), c=6.8836(4) ꢁ, b=
108.738(4)8, V=2503.2(3) ꢁ3, T=90.0(5) K, Z=4, 1calcd =1.285 gcmꢀ3, m-
ACHTUNGTRENNUNG
(MoKa)=0.09 mmꢀ1. 10372 total data, qmax =26.08. R=0.047 for 1634
data with I>2s(I) of 2478 unique data and 162 refined parameters.
¯
Crystal data for 5b precursor: C29H32N2O4, triclinic space group P1, a=
8.5674(5), b=11.1537(9), c=13.4356(10) ꢁ, a=69.244(5), b=89.897(4),
Time-dependent cellular uptake: The HEp2 cells were plated at 7500 per
well in a Costar 96 well plate and allowed to grow for 36 h. BODIPY
stocks were prepared in DMSO containing 1% Cremophor EL, at a con-
centration of 32 mm and then diluted into medium to final working con-
centrations. The cells were exposed to 10 mm of each BODIPY for 0, 1, 2,
4, 8, and 24 h. At the end of the incubation time period the loading
medium was removed and the cells were washed with 200 mL of PBS.
The cells were solubilized upon addition of 100 mL of 0.25% Triton X-
100 (Calbiochem) in PBS. To determine the BODIPY concentration,
fluorescence emission was read at 570/670 nm (excitation/emission) using
a BMG FLUOstar plate reader. The cell numbers were quantified by the
CyQuant cell proliferation assay (Invitrogen) as per the manufacturerꢄs
instructions, and the uptake was expressed in terms of nM compound per
cell.
g=81.625(4)8,
1.323 gcmꢀ3
V=1186.03(15) ꢁ3,
(CuKa)=0.70 mmꢀ1
T=90.0(5) K,
. 14723 total data, qmax =69.38. R=
Z=2,
1calcd =
,
mN
0.038 for 3608 data with I>2s(I) of 4248 unique data and 349 refined pa-
rameters.
Crystal data for 5c: C29H19F5N2O4, monoclinic space group P21/c, a=
15.3125(15), b=11.0509(10), c=15.6714(15) ꢁ, b=115.117(5)8, V=
2401.1(4) ꢁ3, T=90.0(5) K, Z=4, 1calcd =1.534 gcmꢀ3
,
mACHTNGUTRENNU(G CuKa)=
1.11 mmꢀ1. 17798 total data, qmax =68.78. R=0.034 for 3953 data with I>
2s(I) of 4323 unique data and 367 refined parameters.
Crystal data for 5c precursor: C29H27F5N2O4, monoclinic space group P21/
n, a=8.9739(5), b=24.788(2), c=11.3754(10) ꢁ, b=100.947(5)8, V=
22484.4(3) ꢁ3, T=90.0(5) K, Z=4, 1calcd =1.504 gcmꢀ3
,
mACHTNGUTRENNU(G CuKa)=
Chem. Eur. J. 2012, 18, 3893 – 3905
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3903

M. G. H. Vicente et al.
Cytotoxicity: For the dark toxicity, the HEp2 cells were plated as de-
scribed above and allowed 36–48 h to attach. The cells were exposed to
increasing concentrations of BODIPY up to 400 mm and incubated over-
night. The loading medium was then removed and medium containing
Cell Titer Blue (Promega) as per the manufacturerꢄs instructions was
added to the cells. Cell viability was then measured by reading the fluo-
rescence at 570/615 nm using a BMG FLUOstar plate reader. The signal
was normalized to 100% viable (untreated) cells and 0% viable (treated
with 0.2% saponin) cells. For the phototoxicity, the HEp2 cells were pre-
pared as described above for the dark cytotoxicity assay and treated with
BODIPY concentrations of 0, 6.25, 12.5, 25, 50 and 100 mm. After com-
pound loading overnight, the medium was removed and replaced with
medium containing 50 mm HEPES pH 7.4. The cells were exposed to
a NewPort light system with 175 W halogen lamp for 20 min, filtered
through a water filter to provide approximated 1.5 Jcmꢀ2 light dose. The
cells were kept cool by placing the culture on a 58C Echotherm chilling/
heating plate (Torrey Pines Scientific, Inc.). The cells were returned to
the incubator overnight and assayed for viability as described above for
the dark cytotoxicity experiment.
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This work was supported by the National Science Foundation (Grant
CHE-0611629). We are thankful to Dr. Azeem Hassam for assistance
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Boothe Luce Foundation for providing a scholarship for SM.
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Received: September 23, 2011
Published online: February 24, 2012
Chem. Eur. J. 2012, 18, 3893 – 3905
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3905