FormylBODIPYs: Privileged Building Blocks for Multicomponent Reactions. the Case of the Passerini Reaction

Article abstract of DOI:10.1021/acs.joc.5b02893

Eleven formyl-containing BODIPY dyes were prepared by means of either the Liebeskind-Srogl cross-coupling reaction or the Vilsmeier reaction. These dyes were used as components in the Passerini reaction to give highly substituted BODIPY dyes. A joined spe

Full text of DOI:10.1021/acs.joc.5b02893

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Article
FormylBODIPYs: Privileged Building Blocks for
Multicomponent Reactions. The case of the Passerini Reaction
Diana E. Ramírez-Ornelas, Enrique Alvarado-Martínez, Jorge Bañuelos, Iñigo Lopez Arbeloa,
Teresa Arbeloa, Héctor M. Mora-Montes, Luis A. Pérez-García, and Eduardo Peña-Cabrera
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b02893 • Publication Date (Web): 11 Mar 2016
Downloaded from http://pubs.acs.org on March 12, 2016
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FormylBODIPYs: Privileged Building Blocks for Multicomponent
Reactions. The case of the Passerini Reaction
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Diana E. RamírezꢀOrnelas,† Enrique AlvaradoꢀMartínez,† Jorge Bañuelos,*,¥ Iñigo López
Arbeloa,¥ Teresa Arbeloa,¥ Héctor M. MoraꢀMontes,§ Luis A. PérezꢀGarcía§ and Eduardo
PeñaꢀCabrera*,†
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† Departamento de Química, Universidad de Guanajuato, Col. Noria Alta S/N,
Guanajuato, Gto. 36050 Mexico.
¥ Departamento de Química Física, Universidad del País VascoꢀEHU, Apartado 644,
48080, Bilbao, Spain.
§ Departamento de Biología, Universidad de Guanajuato, Col. Noria Alta S/N,
Guanajuato, Gto. 36050 Mexico.
Eꢀmail: jorge.banuelos@ehu.es, eduardop@ugto.mx
Abstract
Eleven formylꢀcontaining BODIPY dyes were prepared by means of either the
LiebeskindꢀSrogl crossꢀcoupling reaction or the Vilsmeier reaction. These dyes were used
as components in the Passerini reaction to give highly substituted BODIPY dyes. A
joined spectroscopic and theoretical characterization of the synthesized compounds was
conducted to unravel the impact of the structural rigidity/flexibility on the photophysical
signatures. These dyes were tested as fluorescent trackers for phagocytosis. Additionally,
they proved to be useful to stain different blood cells with intense and stable signal at a
very low exposure time.
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Introduction
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Multicomponent reactions (MCR) are ideal processes to generate chemical complexity in
an efficient and atomꢀeconomical way.1 Several applications of these reactions can be
found in the literature. For example, they have been used to prepare nitrogenꢀenriched
heterocyclic scaffolds,2 imidazo[1,2ꢀa]pyridines,3 dihydropyrroles,4 substituted 1Hꢀ
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chromeno[2,3ꢀd]pyrimidineꢀ5ꢀcarboxamides,5
imidazoles,6
aminoacids,7
1,4ꢀ
benzodiazepineꢀ2,5ꢀdiones,8 and Nꢀsubstituted diketopiperazines,9 just to mention a few.
It is clear that in the majority of the examples reported, aldehydes play a crucial role, and
therefore, it is of paramount importance to be able to prepare the aldehyde component for
the specific application sought after. Many of the complex structures that are constructed
after MCR´s possess important properties, such as cytotoxic activity against the human
lung cancer cell line,10 antimicrobial and antioxidant,11 aggregation-induced
emitters,12 anti-tumor agents,13 just to mention a few examples. An additional
property that would be highly desirable for the MCR adducts to have is fluorescence.
This property would provide plenty of information about processes that take place
on and inside cells.14 Some groups have devoted considerable efforts to prepare
fluorescent molecules using MCR. For instance, Wang et al. have prepared
polysubstituted 2,6-dicyanoanilines and iminocumarins,15 Balakirev and co-workers
developed fluorescent pharmacophores in droplet arrays,16 Müller and
collaborators prepared fluorescent indolizines,17 In a pioneering paper, Vendrell,
Lavilla, and co-workers reported several isonitrile-based MCR to prepare
fluorescent probes for in vivo imaging of phagocytic macrophages.18 They chose to
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use a BODIPY19 (borondipyrromethene)-containing isonitrile 1, to carry out a
Passerini,20 a Bienaymé−Blackburn−Groebcke,21 and variants of the Ugi reaction.22
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Despite the fact that such work was a groundꢀbreaking contribution, no variation of the
functional groups in 1 was presented. In that regard, another good candidate for MCRs
where the final product would be fluorescent (FMCR), is a formylꢀcontaining BODIPY
of the type 2 or 3 (Figure 1).
Figure 1. Formylꢀcontaining BODIPYs suitable for FMCR.
Results and discussion
Our laboratory has devised a versatile method to prepare, in a very straightforward way, a
series of mesoꢀformylhet(aryl)BODIPYs using the LiebeskindꢀSrogl coupling reaction
starting from commercially available Biellmann BODIPYs A-C23 (Scheme 1).24 In those
contributions, we underscore the fact that, with this methodology, the preparation of
formylꢀcontaining BODIPYs becomes trivial and one does not have to face the
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regioselectivity issues posed by the typical Lindsey methodology that entails the
condensations of a benzaldehyde with pyrrole under acidic conditions.
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Scheme 1. Application of the Liebeskind-Srogl coupling in the synthesis of meso-
formylhet(aryl)BODIPYs from the Biellmann BODIPYs A-C
The synthesis of 2ꢀformylBODIPYs is well documented in the literature.25 In this
contribution, we demonstrate the applicability of several highly funtionalized formylꢀ
containing BODIPYs in the Passerini reaction.
FormylBODIPYs of the type 2 were prepared according to Scheme 1, and the results are
shown in Scheme 2.
Scheme 2. FormylBODIPYs of the type 2 synthesizeda
a
Conditions: Biellmann BODIPY (1 equiv), formylboronic acid (3 equiv), Pd2(dba)3 (2.5%), TFP (7.5%),
CuTC (3 equiv), THF, 55 °C.
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A more functionalized formylBODIPY 2f was prepared according to Scheme 3 using our
recently reported orthogonal reactivity of BODIPY D.26
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Scheme 3. Synthesis of BODIPY 2f
BODIPYs of the type 3 were prepared according to the reported method (Scheme 4).
Scheme 4. Synthesis of 8-(het)aryl-2-formylBODIPYsa,b
a Isolated yields. b Conditions: 1) BODIPY (1 equiv), POCl3/DMF (160 equiv), DCE (10
mL), 80 ºC. 2) sat. aq. K2CO3, 1 h.
FormylBODIPYs 2a-f and 3aꢀe were utilized as components in the Passerini reaction
along with benzoic acid and tꢀbutyl isonitrile. The results are illustrated in Scheme 5.
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Scheme 5. Synthesis of the Passerini BODIPY adductsa
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CHO
Het(Ar)
8
9
O
N
N
O
B
N
R
R
H
F
F
O
OH
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O
Het(Ar)
Het(Ar)
CH2Cl2 / H2O
r.t, 24 h
2
or
NC
O
O
or
N
N
N
N
NH
Het(Ar)
B
B
R
R
O
R
R
F
F
F
F
4
5
CHO
N
N
B
F
F
3
O
N
O
O
O
N
O
O
NH
N
NH
N
O
N
O
N
O
O
N
N
N
N
O
O
H
H
H
H
O
O
O
O
S
O
S
O
Br
Br
N
N
N
N
N
N
B
F
B
B
F
B
B
B
F F
F
F
F F
F F
F F
4f, 84%
4a, 75%
4b, 87%
4c, 90%
4d, 90%
4e,76%
Br
CF3
S
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
N
N
NH
NH
NH
NH
NH
B
B
B
B
B
O
O
O
O
O
F
F
F
F
F
F
F
F
F F
5a, 68%
5bb, 91%
5c, 40%
5d, 38%
5e, 60%
a
Isolated yields. Conditions: formylBODIPY (1 equiv), benzoic acid (3 equiv), tertꢀbutyl isocyanide (3
equiv), CH2Cl2/ H2O (1:1/ 1mL), r.t.
b The reaction took 48 h for this substrate.
In general, the reaction is operationally simple. In all cases, except for 5b, the reaction
went to completion in 24 h. A general trend was observed; the yields were higher when
the formyl group was located on the 8ꢀhet(aryl) ring (compounds 4a-f). When the formyl
group was connected directly to the BODIPY core, the yields were lower (except for 5b)
presumably because of a diminished electrophilicity of the aldehyde group due to the
electronꢀreleasing properties of the BODIPY unit at position 2 (Figure 2).
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Figure 2. Decreased electrophilicity of the formyl group at position 2.
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Additionally, substituents on the mesoꢀaryl group of 2ꢀformylBODIPYs also affected the
chemical yield. When the mesoꢀaryl ring contained electronꢀwithdrawing groups, the
yields dropped (5c and 5d).
Spectroscopical properties
The photophysical signatures of those BODIPYs resulting from the MCR (4aꢀf and 5aꢀe,
in Scheme 5) are summarized in Table 1. Overall, the absorption properties of the
BODIPY are hardly affected by the 8ꢀarylation, owing to the low resonance interaction
between their electronic clouds, at least in the ground state. Indeed, the computing
molecular orbitals (see HOMO of 5aꢀb in Figure 3) predict almost no extension of the
delocalized πꢀsystem from the indacene core to the mesoꢀaryl fragment. However, the
fluorescence response of the BODIPYs bearing an unhindered 8ꢀphenyl (4a and 5a) is
rather low (lower than 4% with lifetime of hundred of picoseconds) owing to a
remarkable high internal conversion probability caused by the free rotational motion of
such ring (energy barrier around 12 kcal/mol in Figure 4).24b,1 Moreover, the LUMO of
5a places electronic density at the 8ꢀaryl upon excitation (Figure 3), suggesting more
degree of electronic coupling with the BODIPY, which eventually could lead to a strong
interaction and cause a severe distortion of the chromophore planarity (Figure 4).27
Indeed, such nonꢀradiative pathways are fully suppressed when the aryl is locked in an
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orthogonal arrangement by methylation at its ortho positions (5b). The high rotational
barrier of the mesityl group (around 45 kcal/mol) disables the free motion of the aryl and
the bright fluorescence efficiency characteristic of BODIPYs is recovered (82% with
long lifetimes, 6.5 ns in Table 1).28 Accordingly, the contribution of the aryl to the
LUMO orbital is almost negligible since the electronic coupling is hampered.
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Figure 3. Computed frontier molecular orbitals for the representative compounds 5aꢀb
and 5e. The corresponding dihedral angle between the indacene and the aryl planes from
the optimized ground and excited state geometries are also included.
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Figure 4. Computed energy surfaces for the rotation of the 8ꢀphenyl (5a) and 8ꢀthenyl
(5e) derivatives with regard to the indacene plane in AcOEt. The corresponding
geometries at the energy maximum and minimum are also shown.
The simple alkylation at the α positions (3 and 5) of pyrroles (see 4b and 4c vs 4a in
Scheme 5) leads to a bathochromic shift of the spectral bands and a slight increase of
both, the absorption probability (with values up to 82400 Mꢀ1 cmꢀ1) and the fluorescence
efficiency (Figure 5), which is ascribed to a more planar structure and thereby a more
aromatic chromophore.29 Whereas the paraꢀfunctionalization of the 8ꢀphenyl (bromine 5c
and trifluoromethyl 5d) does not alter the photophysics of the BODIPYs (being identical
to the 5a counterpart, Scheme 5), it is noteworthy the βꢀbromination (position 2 and 6)
effect (4f, Figure 5). As a common rule, this heavy atom shifts bathochromically the
spectral bands and increases the intersystem crossing.30 As a consequence, the direct
linkage of the bromine heteroatom usually quenches the fluorescence deactivation of the
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BODIPY. However, in the herein reported BODIPYs with unlocked 8ꢀrings the
bromination provides improved fluorescence efficiencies (from 4% in 4b to 25% with
lifetime of 2.45 ns, Table 1). Thus, the deleterious effect of the aryl free motion can be
counteracted not only by steric hindrance, but also with electronegative atoms which
remove electronic density from the meso position.31
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Table 1. Photophysical properties of the resulting BODIPY derivatives after MCR (4aꢀf
and 5aꢀe, Scheme 5) from the corresponding formylated dyes in ethyl acetate; absorption
(λab) and fluorescence wavelength (λfl), molar absorption (εmax), fluorescence quantum
yield (φ), lifetime (τ), radiative (kfl), and nonꢀradiative (knr) deactivation rate constants.
λab
ε
max·10ꢀ4
λfl
kfl·10ꢀ7 knr·10ꢀ9
φ
τ
(sꢀ1)
(sꢀ1)
(nm)
(Mꢀ1cmꢀ1)
(nm)
(ns)
499.5
509.5
510.5
509.5
5.8
8.2
7.3
4.6
518.0
525.0
526.0
530.0
657.0
549.0
645.0
561.0
0.01
0.20
9.62
3.81
4.30
ꢀ
7.6
0.9
1.0
ꢀ
4a
4b
4c
0.04
1.05
0.04
0.93
4d*
ꢀ
0.09(68%)ꢀ0.56(32%)
ꢀ
0.30
4e*
4f
520.5
540.0
5.9
7.5
ꢀ
ꢀ
0.15(75%)ꢀ0.37(25%)
ꢀ
ꢀ
0.35
2.45
0.25
10.2
0.3
505.0
505.5
507.0
509.5
513.5
4.7
6.6
5.1
5.1
4.1
525.5
522.0
529.5
530.5
549.0
646.0
0.03
0.82
0.02
0.02
ꢀ
0.34
10.0
12.6
8.33
9.52
ꢀ
2.8
0.03
4.1
4.7
ꢀ
5a
5b
5c
6.51
0.24
0.21
0.18(70%)ꢀ0.47(30%)
0.50
5d
5e*
ꢀ
*In the BODIPYs bearing and 8ꢀthienyl fragment (4dꢀe and 5e) the fluorescence quantum
yield is not given because two overlapped emission bands are detected simultaneously.
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Figure 5. Absorption and fluorescence spectra of the compounds bearing unhindered 8ꢀ
tolyl (4a), with alkyls at positions α (methyl 4b and ethyl 4c) and bromines at positions β
(4f) in diluted solutions of ethyl acetate.
The presence of the thienyl group at the meso position (4dꢀe and 5e) does not affect the
position of the absorption band (Table 1), confirming again the absence of electronic
coupling of aromatic rings at such positions in the ground state (Figure 3). It should be
noticed that thiophene leads to effective resonance interactions to develop redꢀemitting
BODIPYs but at position α or β of the chromophoric core or even fused to it.32 However,
its high electron releasing ability in the highly sensitive meso position induces significant
changes in the emission spectrum (Figure 6). The fluorescence signal from the locally
excited (LE) state of the BODIPY (530ꢀ550 nm) is drastically quenched and a new broad
band appears at 645ꢀ655 nm, which is ascribed to the population of an intramolecular
charge transfer state (ICT) from the donor thienyl group to the acceptor BODIPY.33 The
intensity of this new emission markedly changes with the solvent polarity. Thus, it
decreases progressively as the solvent polarity is increased. In these media the charge
separation is further stabilized (low probability of charge recombination) and the nonꢀ
radiative deactivation from the ICT prevails over the radiative one.34 As a consequence,
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in the more polar solvent the fluorescence signal is hardly detected. As a matter of fact,
note that the additional presence of the inductive donor methyl groups at α positions (4e)
leads to a less intense emission from the ICT than that from the LE, owing to the above
claimed higher stabilization of the ICT state, whereas in 4d (nonꢀalkylated chromophore)
the ICT band is most intense than the emission from the LE state (Figure 6). It is
remarkable that this solvent switchable new emission depends on the geometrical
arrangement of the thienyl fragment, since when it is disposed fully orthogonal (by the
steric hindrance at the adjacent positions 1 and 7) with regard to the BODIPY core, the
ICT is also operative but no new emission bands are detected.35 Moreover, it seems that
the thienyl twisting is involved in the ICT population probability. This ring at the meso
position contributes to the LUMO (see compound 5e in Figure 3), as expected due to its
rotational freedom (rotational barrier of 5.5 kcal/mol, lesser than 5a owing to the smaller
thiophene ring, Figure 4), but what is more striking is the increase of the dihedral angle
upon excitation (from 45º to 65º, Figure 3). Such more perpendicular orientation of the
thienyl group in the excited state is in agreement with a twisted ICT (TICT).36
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Figure 6. Fluorescence spectra of the compounds bearing 8ꢀthienyl (4dꢀe and 5e) and
their corresponding analogues with 8ꢀphenyl (4a and 5a, respectively) in diluted solutions
of ethyl acetate.
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Finally, it should be stressed that the photophysics of the BODIPY are not affected by
the bulky fragment generated after MCR either at the para position of the 8ꢀphenyl
group, or directly at the BODIPY 2ꢀposition since it is separated from the chromophoric
core by a methine unit. Thus, the herein reported synthetic route is a costꢀeffective and
straightforward method to design smart multichromophoric assemblies, from BODIPYs
or other fluorophores with the required functionality, in which each fragment retains its
molecular identity.
Biological Essays
We have tested the ability of the eleven BODIPYs herein described as possible probes for
fluorescence microscopy using human phagocytic cells. First, we aimed to assess whether
BODIPYs were suitable for tracking the fungal cell phagocytic processes in humanꢀ
derived macrophages or not. After 3h of Candida parapsilosisꢀmacrophage interaction
BODIPYs were added and compounds 4b-e, and , 5a-d showed fluorescent signal in the
green channel, nevertheless, it was quenched soon afterwards independent of the
concentration used. In all cases, BODIPYs showed affinity to smallꢀunidentified vesicles
in the periphery of the macrophage but not to the phagolysosome, making unfeasible the
tracking of the phagocytic process (Figure 7). Acridine orange staining was used as a
control to assess phagocytosis.37
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Then, we further investigated the selectivity of the BODIPYs for different blood cells by
performing a staining of monocytes, granulocytes and erythrocytes with them.
Fluorescence remained constant during the whole observation period for all the
compounds. Unlike the rest of the BODIPYs, compounds 4b and 5b preferentially
stained the erythrocyte membrane, but the former caused cell crenation (Figure 8 AꢀD).
The rest of the compounds mostly stained the cytoplasm but not the nuclei of monocytes
and granulocytes cells (Figure 8 EꢀH). BODIPYs 4b and 5b exhibit an excellent
performance at 75 ms of exposition; while the rest of the compounds worked better at
150 ms of exposition. The only exception was the BODIPY 4d that needed 700 ms to
achieve a defined signal.
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Figure 7. Phagocytosis essays in bright field (A) and fluorescence microscopy (B) using
the BODYPI 5b. The compound showed affinity to smallꢀunidentified vesicles in the
macrophage but not to the phagolysosome, making unfeasible the tracking of the
phagocytic process. All the compounds exhibited similar results.
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Figure 8. Compound 4b stained the erythrocyte membrane causing crenation of the cells
(A, B) unlike compound 5b that also stained the membrane of the red blood cells without
crenation (C,D). Similarly, both compounds stained mononuclear cells in a similar way
(G,H). The rest of them, as compound 4e, selectively stained the granulocytes but not the
erithrocytes (E, F), and all of them stained the mononuclear cells.
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Conclusions
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8
We have described the synthesis of formylꢀcontaining BODIPY derivatives using either
the LiebeskindꢀSrogl crossꢀcoupling reaction (6 examples) or the Vilsmeier reaction (5
examples). These dyes were used along with benzoic acid and tꢀbutyl isocyanide in the
Passerini reaction to produce 11 new dyes with rich functionality in a straightforward
manner. The photophysical signatures of the formylated BODIPYs are ruled by the free
motion of the 8ꢀaryl and are kept after MCR reaction. Thus, this synthetic route is a
straightforward method to design smart multichromophoric assemblies. These
compounds are not suitable for phagocytosis tracking, nevertheless some of them can be
used as fluorescent agents to identify blood cells in a very efficient manner since
fluorescence remain constant and intense at a very low exposure time. Other examples of
MCR with formylBODIPYs as well as other formylꢀcontaining fluorophores are currently
under investigation in our laboratories and the results will be reported shortly.
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Experimental Section
Synthesis and characterization: Starting materials and reagents used in the preparation
of BODIPYs are commercially available unless otherwise noted. The solvents were
distilled and dried according to standard procedures before use. Unless stated otherwise,
all reactions were performed without inert atmosphere in ovenꢀ and or flameꢀdried
glassware. Reaction progress was monitored by TLC (silica gel plates with Fꢀ254
indicator) and the spots were visualized under UV light (254 or 365 nm). Flash column
chromatography was performed using silica gel Kieselgel 60 (70ꢀ230 mesh) and mixture
of ethyl acetate and hexanes as the eluent. Spectral data of the known compounds were in
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accordance with the literature data. H and 13C NMR spectra were acquired on
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spectrometers (500 or 400 MHz for 1H and 125 or 101 MHz for 13C) in
deuteriochloroform (CDCl3) with either tetramethylsilane (TMS) (0.00 ppm 1H, 0.00 ppm
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1
13C) or chloroform (7.26 ppm H, 77.16 ppm 13C) or as internal reference unless
otherwise stated.38 Data are reported in the following order: chemical shift in ppm,
multiplicities (br (broadened), s (singlet), d (doublet), t (triplet), q (quartet), sex (sextet),
hep (heptet), m (multiplet), exch (exchangeable), app (apparent)), coupling constants J
(Hz), and integration. Infrared spectra were recorded on a FTIR spectrophotometer.
Peaks are reported (cmꢀ1) with the following relative intensities: s (strong, 67ꢀ100%), m
(medium 40ꢀ66%), and w (weak 20ꢀ39%). Melting points were determined and were
uncorrected. HRMS samples were ionized by ESI+ and recorded via the TOF method.
8ꢀMethylthiobodipys A-C,23 compounds 2a-c, 3aꢀ3c, 3e,39 and CuTC40 were prepared
according to the literature procedures.
Spectroscopic measurements.
The photophysical properties were registered in diluted solutions (around 2×10ꢀ6 M) of
ethyl acetate, using quartz cuvettes with optical pathway of 1 cm. UVꢀVis absorption and
fluorescence spectra were recorded on a spectrophotometer and a spectrofluorimeter,
respectively. Fluorescence quantum yield (φ) was obtained by using the commercial
PM567 dye as reference (φr = 0.84 in ethanol). Radiative decay curves were registered
with the time correlated singleꢀphoton counting technique , equipped with a microchannel
plate detector of picosecond timeꢀresolution (20 ps). Fluorescence emission was
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monitored at the maximum emission wavelength after excitation by means of a laser.
The fluorescence lifetime (τ) was obtained after the deconvolution of the instrumental
response signal from the recorded decay curves by means of an iterative method. The
goodness of the exponential fit was controlled by statistical parameters (chiꢀsquare,
DurbinꢀWatson and the analysis of the residuals). Radiative (kfl) and nonꢀradiative (knr)
rate constants were calculated as follows; kfl =φ/τ and knr = (1ꢀφ)/τ.
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Computational modeling for representative compound 5a, 5b and 5e.
Ground state energy minimizations were performed using the Becke´s Three Paramaters
(B3LYP) Density Functional (DFT) method, whereas excited state geometry optimization
was carried out using the ab initio Configuration Interaction Singles (CIS) method. The
basis set was the double valence 6ꢀ31g* (with a polarization function). The optimized
geometry was taken as a true energy minimum using frequency calculations (no negative
frequencies). Rotational energy barriers in the ground state were calculated from the
potential energy surface, which was simulated by relaxed scans (steps of 10º) of the 8ꢀ
phenyl, 8ꢀthienyl and 8ꢀmesityl twist with regard to the plane of the BODIPY core. The
solvent effect (ethyl acetate) was considered in all the calculations by the Polarizable
Continuum Model (PCM). All the calculations were performed in Gaussian 09, using the
“arina” computational resources provided by the UPVꢀEHU.
Biological essays
Peripheral blood mononuclear cells (PBMCs) were isolated from human blood as
previously described.41 The differentiation to macrophages was induced in serumꢀfree
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medium supplemented with 1 % (v/v) penicillinꢀstreptomycin solution and 10 ng/mL
recombinant human granulocyteꢀmacrophage colonyꢀstimulating factor, under incubation
at 37 °C, 5 % (v/v) CO2 for 6ꢀ7 days, replacing the medium every 2ꢀ3 days.42
Macrophage infection with Candida parapsilosis yeast cells was performed at a MOI of
1:5, respectively. After 3 hours of incubation at 37 °C, 5 % (v/v) CO2, cells were treated
with the BODIPY at a final concentration of either 1 ꢀg/mL or 5 ꢀg/mL in phosphate
saline buffer (PBS) pH 7.4, letting the compound interact for 5 minutes at room
temperature. Cells were washed and crystal violet solution (0.03% in PBS) was added for
contrast. After 3 minutes, cells were washed again and placed onto slides to be inspected
under fluorescence microscopy. For interaction of BODIPYs with monocytes,
granulocytes and erythrocytes, cells were suspended in PBS containing the BODIPY at a
final concentration of 1 ꢁg/mL. After 15 minutes of incubation at room temperature, cells
were washed twice with PBS and placed onto slides for inspection under the fluorescent
microscopy.
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All preparations were examined using a microscope. Acquisition of images was achieved
by means of AxioVs40 V 4.6.1.0 software and a camera.
Synthesis and characterization of compounds
General Procedure for the LiebeskindꢀSrogl crossꢀcoupling reaction (GP1). An ovenꢀ
dry Schlenk tube, equipped with a stir bar, was charged with either 8ꢀmethylthiobodipy A
or B (1.0 equiv.), the corresponding boronic acid (3.0 equiv.), and dry THF (0.03 M)
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under N2. The mixture was sparged with N2 for 5 min, whereupon Pd2(dba)3 (2.5 mol%),
trifurylphosphine (7.5 mol%), and CuTC (3.0 equiv.) were added followed by 5 min N2
purge. The reaction mixture was immersed into a pre heated oil bath at 55 ºC. After TLC
showed that the reaction went to completion, the reaction mixture was allowed to reach
room temperature and was adsorbed on SiO2ꢀgel. After flashꢀchromatography (SiO2ꢀgel,
EtOAc/hexanes gradient) purification, meso substituted BODIPYs were obtained as
highlyꢀcolored solids.
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General Procedure for the synthesis of β-FormylBODIPYs (GP2). In a roundꢀ
bottomed flask equipped with a stir bar, DMF (3.0 mL, 39 mmol) and POCl3 (3.0 mL, 32
mmol) were combined at 0 ºC (iceꢀbath) under N2 for 5 min. After warming to room
temperature, the reaction mixture was further stirred for 30 min. Then, the corresponding
8ꢀ(het)arylꢀBODIPY (0.2 mmol) in ClCH2CH2Cl (10 mL) was added to the reaction
mixture. After raising the temperature at 80 °C, the reaction mixture was further stirred
for 16 h, cooled to room temperature and slowly poured into a saturated aqueous K2CO3
solution (100 mL) cooled in an ice bath. After warming to room temperature, the reaction
mixture was further stirred for 1 h and extracted with CH2Cl2 (3x50 mL). The organic
layers were combined, washed with water (2x50 mL), dried with anhydrous MgSO4,
filtered, and the solvent was removed under reduced pressure. The crude product was
further purified by column chromatography on silica gel using a mixture of ethyl acetate
and hexanes as eluent to give the βꢀformylated BODIPYs as dark red crystals.
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General Procedure for the Passerini reaction (GP3). To a solution of corresponding
formylꢀBODIPY 2 or 3 (1.0 equiv.) in dichloromethane (0.5 mL), were sequentially
added benzoic acid (3.0 equiv.) and tertꢀbutyl isocyanide (3.0 equiv.) at room
temperature, followed by addition of H2O (0.5 mL). The reaction mixture was stirred at
room temperature for 24 h until completion (TLC monitoring). When the reaction was
completed, the product was extracted with ethyl acetate, washed with brine (3x10 mL),
and dried with anhydrous MgSO4 and filtered. The solvent was removed under reduced
pressure, and the crude product was purified by silicaꢀgel column chromatography
(EtOAc/hexane gradient) to obtain the desired product.
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The Liebeskind-Srogl cross-coupling reaction (LSCC) (Scheme 1).
According to GP1. A (70 mg, 0.2940 mmol, 1.0 equiv.), 5ꢀformylꢀ2ꢀ
thienylboronic acid (138 mg, 0.8821 mmol, 3.0 equiv.), CuTC (168
mg, 0.8821 mmol, 3.0 equiv.), Pd2(dba)3 (6.7 mg, 0.0074 mmol, 2.5
mol%), and triꢀ2ꢀfurylphosphine (5.1 mg, 0.0220 mmol, 7.5 mol%).
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The desired product was obtained as a dark red crystals (69 mg, 78 % yield); mp > 150
°C (subl.); TLC (30% EtOAc/hexanes, Rf = 0.2); IR (KBr, cmꢀ1): 3111 (w), 3094 (w),
1672 (s), 1657 (s), 1548 (s), 1414 (s), 1386 (s), 1264 (s), 1212 (s), 1113 (s), 1076 (s), 969
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(m), 776 (m), 666 (w), 630 (w), 583 (w); H NMR (400 MHz, CDCl3): δ 10.02 (s, 1H),
7.97 (s, 2H), 7.88 (d, J = 4.0 Hz, 1H), 7.58 (d, J = 4.0 Hz, 1H), 7.21 (d, J = 4.4 Hz, 2H),
13
6.60 (d, J = 4.0 Hz, 2H); C NMR (101 MHz, CDCl3): δ 182.9, 147.1, 145.6, 142.7,
137.5, 135.7, 134.4, 132.8, 131.6, 119.4; HRMS (ESI+): m/z calcd. for C14H10BF2N2OS
[M+H]+, 303.0572; found 303.0574.
According to GP1. B (80 mg, 0.3006 mmol, 1.0 equiv.), 5ꢀformylꢀ2ꢀ
thienylboronic acid (141 mg, 0.9018 mmol, 3.0 equiv), CuTC (172
mg, 0.9018 mmol, 3.0 equiv.), Pd2(dba)3 (6.9 mg, 0.0075 mmol, 2.5
mol%), and triꢀ2ꢀfurylphosphine (5.2 mg, 0.0225 mmol, 7.5 mol%).
The desired product was obtained as a dark red crystals (86 mg, 87 %
yield); mp > 180 °C (subl.); TLC (30% EtOAc/hexanes, Rf = 0.3); IR (KBr, cmꢀ1): 3111
(w), 2924 (w), 2852 (w), 1668 (s), 1552 (s), 1493 (s), 1457 (s), 1280 (s), 1211 (s), 1152
1
(s), 1012 (m), 817 (m), 729 (m), 675 (w); H NMR (500 MHz, CDCl3): δ 9.99 (s, 1H),
7.82 (d, J = 4.0 Hz, 1H), 7.46 (d, J = 3.5 Hz, 1H), 6.98 (d, J = 4.0 Hz, 2H), 6.32 (d, J =
13
4.0 Hz, 2H), 2.65 (s, 6H); C NMR (126 MHz, CDCl3): δ 182.8, 159.4, 146.1, 143.6,
135.6, 134.1, 132.7, 131.8, 130.3, 120.4, 15.2; HRMS (ESI+): m/z calcd. for
C16H14BF2N2OS [M+H]+, 331.0885; found 331.0886.
Synthesis of BODIPY 2f (Scheme 3).
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BODIPY D. To a solution of B (50 mg, 0.1879 mmol, 1.0
equiv.) in THF (3.0 mL) was added dropwise a solution of
NBS (70 mg, 2.20 mmol, 2.1 equiv.) in THF (1.5 mL) at
room temperature. The mixture was stirred at room
SMe
Br
Br
N
N
B
F F
D
temperature for 1 h. The product was diluted with ethyl acetate (5.0 mL), washed with
brine (2x10mL) and dried over MgSO4, and filtered. The solvent was removed in vacuo
to provide the desired product as a dark pink solid (74 mg, 93% yield); mp 109 °C; TLC
(15% EtOAc/hexanes, Rf = 0.6); IR (KBr, cmꢀ1): 3540 (w), 2929 (w), 1739 (m), 1532
(m), 1450 (m), 1372 (m), 1314 (w), 1243 (s), 1174 (m), 1141 (s), 1090 (m), 1011 (m),
1
895 (m), 678 (w); H NMR (200 MHz, CDCl3) δ: 7.43 (s, 2H), 2.82 (s, 3H), 2.6 (s, 6H);
13C NMR (50 MHz, CD3CN), δ: 154.8, 145.6, 133.8, 128.3, 108.9, 21.7, 13.6; HRMS
(ESI+): m/z calcd. for C12H12BBr2F2N2S [M+H]+, 424.9125; found 424.9121.
According to GP1. D (100 mg, 0.2359 mmol, 1.0 equiv.), 4ꢀ
CHO
formylphenylboronic acid (88 mg, 0.5897 mmol, 2.5 equiv.),
CuTC (135 mg, 0.7077 mmol, 3.0 equiv.), Pd2(dba)3 (5.4 mg,
Br
Br
0.0059 mmol, 2.5 mol%), and triꢀ2ꢀfurylphosphine (4.1 mg,
0.0018 mmol, 7.5 mol%). The desired product was obtained
N
N
B
F F
2f
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as dark purple crystals (102 mg, 90% yield); mp > 210 °C (subl.); TLC (30%
EtOAc/hexanes, Rf = 0.6); IR (KBr, cmꢀ1): 3122 (w), 2836 (w), 2737 (w), 1703 (s), 1575
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(s), 1559 (s), 1452 (m), 1246 (s), 1130 (s), 1004 (m), 988 (m), 823 (m), 485 (m); H
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NMR (500 MHz, CDCl3): δ 10.13 (s, 1H), 8.03 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 8.0 Hz,
13
2H), 6.74 (s, 2H), 2.64 (s, 6H); C NMR (126 MHz, CDCl3): δ 191.2, 157.0, 140.2,
139.0, 137.8, 133.0, 131.0, 130.3, 129.8, 109.7, 13.7; HRMS (ESI+): m/z calcd. for
C18H14BBr2F2N2O [M+H]+, 482.9512; found 482.9506.
Synthesis of 2-formylBODIPYs (Scheme 4)
According to GP2. DMF (3.0 mL, 39.0 mmol, 218.5 equiv.),
POCl3 (3.0 mL, 32.0 mmol, 179.3 equiv.), pꢀ
trifluoromethylphenylBODIPY (60 mg, 0.1785 mmol, 1.0
equiv.), DCE (20.0 mL). The desired product was obtained as a
dark red crystals (26 mg, 40% yield); mp > 210 °C (dec.); TLC
(30% EtOAc/hexanes, Rf = 0.4); IR (KBr, cmꢀ1): 3109 (w), 2922 (m), 2851 (m), 1677 (s),
1544 (s), 1398 (s), 1329 (s), 1283 (m), 1171 (m), 1123 (m), 1098 (m), 976 (m), 915 (m),
855 (m), 740 (m), 718 (m), 649 (w); 1H NMR (400 MHz, CDCl3): δ 9.86 (s, 1H), 8.31 (s,
1H), 8.23 (s, 1H), 7.85 (d, J = 8.0 Hz, 2H), 7.72 (d, J = 8.0 Hz, 2H), 7.24 (s, 1H), 7.10 (d,
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J = 4.4 Hz, 1H), 6.76 (d, J = 4.4 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ 184.8, 150.7,
5
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7
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3
147.4, 143.7, 137.1, 136.4, 134.7, 134.5, 133.6, 133.3, 132.2, 130.8, 128.7, 126.0 (q, JCF
= 3.7 Hz), 125.0, 122.3; HRMS (ESI+): m/z calcd. for C17H11BF5N2O [M+H]+, 365.0882;
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found 365.0897.
The Passerini reaction (Scheme 5).
According to GP3: 2a (30 mg, 0.1013 mmol, 1.0 equiv.),
benzoic acid (37 mg, 0.3039 mmol, 3.0 equiv.) and tertꢀbutyl
isocyanide (34.0 ꢀL, 0.3039 mmol, 3.0 equiv.) in CH2Cl2:H2O
(1.0 mL/1:1). The desired product was obtained as a yellow
solid (38 mg, 75 % yield); mp 220ꢀ222 ºC; TLC (30 %
EtOAc/hexanes, Rf = 0.3); IR (KBr, cmꢀ1): 3305 (m), 3079
(w), 2969 (w), 1727 (s), 1664 (s), 1556 (s), 1414 (s), 1387 (s), 1262 (s), 1112 (s), 1077
1
(s), 985 (w) 709 (m). H NMR (500 MHz, CDCl3): δ 8.13 (d, J = 7.5 Hz, 2H), 7.93 (s,
2H), 7.69 (d, J = 8.0 Hz, 2H), 7.65 (t, J = 7.5 Hz, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.53 (t, J
= 7.8 Hz, 2H), 6.94 (d, J = 4.0 Hz, 2H), 6.53 (d, J = 3.0 Hz, 2H), 6.35 (s, 1H), 6.23 (br,
1H), 1.42 (s, 9H); 13C NMR (126 MHz, CDCl3): δ 167.0, 164.8, 146.7, 144.5, 139.0,
135.0, 134.4, 134.1, 131.8, 130.9, 129.9, 129.2, 129.0, 127.6, 118.8, 75.5, 52.0, 28.9;
HRMS (ESI+): m/z calcd. for C28H27BF2N3O3 [M+H]+, 502.2113; found 502.2112.
According to GP3: 2b (30 mg, 0.0926 mmol, 1.0 equiv.),
benzoic acid (34 mg, 0.2778 mmol, 3.0 equiv.) and tertꢀbutyl
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isocyanide (31.0 ꢀL, 0.2778 mmol, 3.0 equiv.) in CH2Cl2:H2O (1.0 mL/1:1). The desired
product was obtained as an orange solid (43 mg, 87 % yield); mp 201ꢀ203 ºC; TLC (30 %
EtOAc/hexanes, Rf = 0.4); IR (KBr, cmꢀ1): 3299 (m), 3102 (w), 2969 (m), 1723 (s), 1666
(s), 1543 (s), 1413 (s), 1391 (s), 1259 (s), 1224 (s), 1080 (s), 973 (s), 839 (w), 767 (s),
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758 (s), 718 (s). H NMR (500 MHz, CDCl3): δ 8.12 (d, J = 7.0 Hz, 2H), 7.66ꢀ7.63 (m,
3H), 7.53ꢀ7.50 (m, 4H), 6.71 (d, J = 4.0 Hz, 2H), 6.32 (s, 1H), 6.25 (d, J = 4.0 Hz, 2H),
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6.19 (br, 1H), 2.64 (s, 6H), 1.41 (s, 9H); C NMR (126 MHz, CDCl3): δ 167.1, 164.9,
158.0, 141.8, 138.0, 134.8, 134.6, 134.0, 130.8, 130.6, 129.9, 129.3, 128.9, 127.4, 119.6,
75.6, 51.9, 28.9, 15.0; HRMS (ESI+): m/z calcd. for C30H31BF2N3O3 [M+H]+, 530.2426;
found 530.2429.
According to GP3: 2c (21 mg, 0.0596 mmol, 1.0 equiv.),
benzoic acid (22 mg, 0.1789 mmol, 3.0 equiv.) and tertꢀbutyl
isocyanide (20.0 ꢀL, 0.1789 mmol, 3.0 equiv.) in
CH2Cl2:H2O (1.0 mL/1:1). The desired product was obtained
as an orange solid (30 mg, 90 % yield); mp 166ꢀ168 ºC; TLC
(30 % EtOAc/hexanes, Rf = 0.5); IR (KBr, cmꢀ1): 3305 (m),
2971(m), 2935 (w), 1725 (s), 1663 (s), 1555 (s), 1439 (s), 1319 (s), 1260 (s), 1141 (s),
1
1033 (s), 983 (m), 885 (m), 710 (s); H NMR (500 MHz, CDCl3): δ 8.13 (d, J = 7.5 Hz,
2H), 7.66ꢀ7.63 (m, 3H), 7.54ꢀ7.51 (m, 4H), 6.74 (d, J = 4.0 Hz, 2H), 6.34ꢀ6.33 (m, 3H),
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6.19 (br, 1H), 3.07 (q, J = 7.5 Hz, 4H), 1.42 (s, 9H), 1.34 (t, J = 7.6 Hz, 6H); C NMR
(126 MHz, CDCl3): δ 167.1, 164.9, 163.9, 142.2, 138.0, 134.9, 134.3, 134.0, 130.8,
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130.6, 129.9, 129.3, 129.0, 127.4, 117.5, 75.6, 51.9, 28.9, 22.2, 12.9; HRMS (ESI+): m/z
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calcd. for C32H35BF2N3O3 [M+H]+, 558.2740; found 558.2738.
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According to GP3: 2d (20 mg, 0.0662 mmol, 1.0 equiv.),
benzoic acid (24 mg, 0.1986 mmol, 3.0 equiv.) and tertꢀbutyl
isocyanide (23 ꢀL, 0.1986 mmol, 3.0 equiv.) in CH2Cl2:H2O
(1.0 mL/1:1). The desired product was obtained as a red solid
(30 mg, 89 % yield); mp 183ꢀ186 ºC; TLC (30 %
EtOAc/hexanes, Rf = 0.3); IR (KBr, cmꢀ1): 3299 (m), 3102
(w), 2969 (m), 1723 (s), 1666 (s), 1543 (s), 1413 (s), 1391 (s), 1259 (s), 1080 (s), 973 (s),
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767 (s), 718 (s). H NMR (500 MHz, CDCl3): δ 8.12 (d, J = 7.0 Hz, 2H), 7.92 (s, 2H),
7.66 (t, J = 7.5 Hz, 1H), 7.53 (t, J = 7.8 Hz, 2H), 7.45 (d, J = 4.0 Hz, 1H), 7.36 (d, J = 4.0
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Hz, 1H), 7.27 (d, J = 4.0 Hz, 2H), 6.56 (m, 3H), 6.18 (br, 1H), 1.43 (s, 9H); C NMR
(126 MHz, CDCl3): δ 165.8, 164.8, 144.3, 144.2, 139.0, 135.7, 134.4, 134.3, 132.7,
131.7, 130.0, 129.0, 128.8, 128.3, 118.8, 71.7, 52.2, 28.8; HRMS (ESI+): m/z calcd. for
C26H25BF2N3O3S [M+H]+, 508.1677; found 508.1671.
According to GP3: 2e (30 mg, 0.0908 mmol, 1.0 equiv.),
benzoic acid (33 mg, 0.2726 mmol, 3.0 equiv.) and tertꢀ
butyl isocyanide (31.0 ꢀL, 0.2726 mmol, 3.0 equiv.) in
CH2Cl2:H2O (1.0 mL/1:1). The desired product was obtained
as a red solid (37 mg, 76 % yield); mp 210ꢀ213 ºC; TLC (30
% EtOAc/hexanes, Rf = 0.4); IR (KBr, cmꢀ1): 3278 (m),
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3086 (w), 2966 (m), 2918 (m), 2850 (w), 1722 (s), 1665 (s), 1556 (s), 1492 (s), 1452 (s),
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1275 (s), 1143 (s), 1090 (m), 1014 (s), 975 (s), 796 (s), 710 (s); H NMR (500 MHz,
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CDCl3): δ 8.11 (d, J = 7.5 Hz, 2H), 7.65 (t, J = 7.4 Hz, 1H), 7.52 (t, J = 7.8 Hz, 2H),
7.32–7.30 (m, 2H), 7.05 (d, J = 4.0 Hz, 2H), 6.53 (s, 1H), 6.29 (d, J = 4.0 Hz, 2H), 6.13
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(br, 1H), 2.64 (s, 6H), 1.42 (s, 9H); C NMR (126 MHz, CDCl3): δ 166.0, 164.8, 158.2,
142.3, 136.0, 134.3, 134.2, 131.4, 130.6, 130.0, 129.0, 128.6, 128.0, 119.7, 71.8, 52.1,
28.8, 15.1; HRMS (ESI+): m/z calcd. for C28H29BF2N3O3S [M+H]+, 536.1990; found
536.1992.
According to GP3: 2f (30 mg, 0.0622 mmol, 1.0 equiv.),
benzoic acid (23 mg, 0.1866 mmol, 3.0 equiv.) and tertꢀ
butyl isocyanide (21.0 ꢀL, 0.1866 mmol, 3.0 equiv.) in
CH2Cl2:H2O (1.0 mL/1:1). The desired product was
obtained as a fuchsia solid (36 mg, 84 % yield); mp > 280
ºC (subl.); TLC (30 % EtOAc/hexanes, Rf = 0.6); IR (KBr,
cmꢀ1): 3433 (w), 3120 (w), 2967 (w), 1733(s), 1684 (s), 1546 (s), 1451 (s), 1246 (s), 1146
(s), 1011 (s), 715 (m), 489 (m); 1H NMR (500 MHz, CDCl3): δ 8.13 (d, J = 7.0 Hz, 2H),
7.68ꢀ7.64 (m, 3H), 7.53 (t, J = 7.8 Hz, 2H), 7.49 (d, J = 8.5 Hz, 2H), 6.81 (s, 2H), 6.33 (s,
1H), 6.23 (br, 1H), 2.62 (s, 6H), 1.42 (s, 9H); 13C NMR (126 MHz, CDCl3) δ 166.9,
164.8, 156.2, 141.6, 138.9, 134.1, 133.7, 133.2, 130.7, 130.6, 129.9, 129.2, 129.0, 127.7,
109.2, 75.5, 52.0, 28.9, 13.6; HRMS (ESI+): m/z calcd. for C30H29BBr2F2N3O3 [M+H]+,
686.0618; found 686.0617.
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According to GP3: 3a (29 mg, 0.0979 mmol, 1.0 equiv.),
benzoic acid (36 mg, 0.2937 mmol, 3.0 equiv.) and tertꢀ
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CH2Cl2:H2O (1.0 mL/1:1). The desired product was
obtained as an orange solid (33 mg, 68 % yield); mp 172ꢀ
174 ºC; TLC (30 % EtOAc/hexanes, Rf = 0.3); IR (KBr, cmꢀ1): 3394 (w), 2969 (w), 1717
(s), 1687 (s), 1552 (s), 1384 (m), 1259 (s), 1096 (s), 979 (w), 725 (w), 716 (w); 1H NMR
(400 MHz, CDCl3): δ 8.06 (d, J = 7.2 Hz, 2H), 7.99 (d, J = 4.4 Hz, 2H), 7.64ꢀ7.45 (m, 8
H), 7.04 (s, 1H), 6.98 (d, J = 4.4 Hz, 1H), 6.58 (d, J = 3.2 Hz, 1H), 6.23 (s, 1H), 6.04 (br,
1H), 1.36 (s, 9H); 13C NMR (101 MHz, CDCl3): δ 166.8, 165.0, 148.2, 145.7, 141.6,
135.6, 134.6, 134.0, 133.8, 133.6, 132.8, 131.2, 130.7, 130.3, 129.9, 129.1, 128.9, 128.7,
128.6, 119.4, 70.2, 51.8, 28.8; HRMS (ESI+): m/z calcd. for C28H26BF2N3O3Na
[M+Na]+, 524.1932; found 524.1934.
According to GP3: 3b (30 mg, 0.0887 mmol, 1.0 equiv.),
benzoic acid (33 mg, 0.2661 mmol, 3.0 equiv.) and tertꢀ
butyl isocyanide (30.0 ꢀL, 0.2661 mmol, 3.0 equiv.) in
CH2Cl2:H2O (1.0 mL/1:1). The desired product was
obtained as a yellow solid (44 mg, 91 % yield); mp 117ꢀ
120 ºC; TLC (30 % EtOAc/hexanes, Rf = 0.5); IR (KBr, cmꢀ1): 3403 (w), 2973 (w), 2921
(w), 1721 (m), 1694 (m), 1563 (s), 1386 (m), 1364 (m), 1258 (s), 1104 (s), 1069 (m), 706
(m); 1H NMR (500 MHz, CDCl3): δ 8.06 (d, J = 7.0 Hz, 2H), 7.95 (s, 2H), 7.62 (t, J = 7.4
Hz, 1H), 7.49 (t, J = 7.7 Hz, 2H), 6.94 (s, 2H), 6.78 (s, 1H), 6.73 (d, J = 4.0 Hz, 1H),
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