Evaluation of Anisole-Substituted Boron Difluoride Formazanate Complexes for Fluorescence Cell Imaging

Article abstract of DOI:10.1002/chem.201502821

Evaluation of three subclasses of boron difluoride formazanate complexes bearing o-, m-, and p-anisole N-aryl substituents (Ar) as readily accessible alternatives to boron dipyrromethene (BODIPY) dyes for cell imaging applications is described. While the

Full text of DOI:10.1002/chem.201502821

DOI: 10.1002/chem.201502821
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Evaluation of Anisole-Substituted Boron Difluoride Formazanate
Complexes for Fluorescence Cell Imaging
Ryan R. Maar,[a, b] Stephanie M. Barbon,[a, b] Neha Sharma,[a] Hilary Groom,[c]
Leonard G. Luyt,*[a, c, d] and Joe B. Gilroy*[a, b]
Abstract: Evaluation of three subclasses of boron difluoride
formazanate complexes bearing o-, m-, and p-anisole N-aryl
substituents (Ar) as readily accessible alternatives to boron
dipyrromethene (BODIPY) dyes for cell imaging applications
is described. While the wavelengths of maximum absorption
(lmax) and emission (lem) observed for each subclass of com-
plexes, which differed by their carbon-bound substituents
(R), were similar, the emission quantum yields for 7a–c (R=
cyano) were enhanced relative to 8a–c (R=nitro) and 9a–
c (R=phenyl). Complexes 7a–c and 8a–c were also signifi-
cantly easier to reduce electrochemically to their radical
anion and dianion forms compared to 9a–c. Within each
subclass, the o-substituted derivatives were more difficult to
reduce, had shorter lmax and lem, and lower emission quan-
tum yields than the p-substituted analogues as a result of
sterically driven twisting of the N-aryl substituents and a de-
crease in the degree of p-conjugation. The m-substituted
complexes were the least difficult to reduce and possessed
intermediate lmax, lem, and quantum yields. The complexes
studied also exhibited large Stokes shifts (82–152 nm, 2143–
5483 cmÀ1). Finally, the utility of complex 7c (Ar=p-anisole,
R=cyano), which can be prepared for just a few dollars per
gram, for fluorescence cell imaging was demonstrated. The
use of 7c and 4’,6-diamino-2-phenylindole (DAPI) allowed
for simultaneous imaging of the cytoplasm and nucleus of
mouse fibroblast cells.
Introduction
further development of the chemistry of many BF2 complexes
has often been limited by their challenging syntheses and/or
substantial cost. For example, a few milligrams of commercially
available BODIPYs for cell imaging applications will often cost
several hundred US dollars. Considering these factors, there re-
mains a significant need for readily accessible molecular mate-
rials with similar or improved properties compared to BODIPYs
that may be prepared by relatively simple, low-cost synthetic
pathways.
Boron difluoride (BF2) chelates of N-donor ligands are among
the most widely studied classes of molecular materials due to
their unique, tunable, and potentially useful absorption, emis-
sion, and electrochemical properties.[1,2] These compounds, in-
cluding the ubiquitous boron dipyrromethanes (BODIPYs) 1,[3]
have shown utility as sensors,[4,5] as efficient electrochemilumi-
nescence luminophores,[6,7] as the functional component of or-
ganic electronics,[8] in photodynamic therapy,[9,10] and perhaps,
most commonly, as fluorescence cell imaging agents.[11,12] De-
spite their widespread use in a broad range of applications,
BF2 formazanate complexes meet these criteria and offer the
ability to tune spectroscopic and electrochemical properties
through structural variation.[13] The parent formazans 2,[14]
known primarily for their use in cell viability assays,[15] are typi-
cally prepared using aryl diazonium coupling reactions in
aqueous media. They exist in three different conformations
(i.e., “closed”, “open”, or “linear”) depending on the carbon-
bound substituent (R) present.[14a,16] In recent years, the coordi-
nation chemistry of formazanate ligands 3 has received signifi-
cant attention due to the rich spectroscopic and redox proper-
ties exhibited by the resulting complexes.[17,18] However, their
utility in practical applications such as cell imaging has not
been demonstrated.
[a] R. R. Maar, S. M. Barbon, N. Sharma, Prof. Dr. L. G. Luyt, Prof. Dr. J. B. Gilroy
Department of Chemistry
The University of Western Ontario
1151 Richmond St. N., London, Ontario N6A 5B7 (Canada)
E-mail: lluyt@uwo.ca
joe.gilroy@uwo.ca
[b] R. R. Maar, S. M. Barbon, Prof. Dr. J. B. Gilroy
The Centre for Advanced Materials and Biomaterials Research (CAMBR)
The University of Western Ontario
1151 Richmond St. N., London, Ontario N6A 5B7 (Canada)
[c] H. Groom, Prof. Dr. L. G. Luyt
Herein, taking inspiration from the biocompatibility of for-
mazans, we present a study designed to evaluate a series of
anisole-substituted BF2 formazanate complexes (Ar=o-anisole,
m-anisole, p-anisole; R=cyano, nitro, phenyl) in an effort to
showcase their utility as fluorescence cell imaging agents.
Department of Oncology, London Regional Cancer Program
790 Commissioners Rd. E., London, Ontario N6A 4L6 (Canada)
[d] Prof. Dr. L. G. Luyt
Department of Medical Imaging
The University of Western Ontario
1151 Richmond St. N., London, Ontario N6A 5B7 (Canada)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201502821.
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Table 1. Substituents for formazans 4a–d, 5a–c, and 6a–c and BF2 for-
mazanate complexes 7a–d, 8a–c, and 9a–c.
Compound
Ar
R
4a/7a
4b/7b
4c/7c
4d/7d
5a/8a
5b/8b
5c/8c
6a/9a
6b/9b
6c/9c
o-anisole
m-anisole
p-anisole
o-ethylbenzene
o-anisole
m-anisole
p-anisole
o-anisole
m-anisole
p-anisole
cyano
cyano
cyano
cyano
nitro
nitro
nitro
phenyl
phenyl
phenyl
Results and Discussion
and were highly dependent on the purification methods em-
ployed. All new BF2 formazanate complexes were fully charac-
Synthesis
1
terized by H, 11B, 13C, and 19F NMR spectroscopy, UV/Vis ab-
Anisole-substituted formazans 4a–c, 5a–c, and 6a–c were pre-
pared by adapting established procedures for the coupling of
aryl diazonium salts and activated methylenes such as cyano-
acetic acid, nitromethane, and phenylpyruvic acid in yields
ranging from 18–90%.[16,19] It is worth noting that the variabili-
ty in reaction yields relates mainly to the unpredictable stabili-
ty of the aryl diazonium salts involved.[20] 3-Cyanoformazans
4b–d exist primarily in the “open” conformation in solution
due to the presence of a linear carbon-bound cyano substitu-
ent and were identified by their characteristic orange color
(wavelength of maximum absorption (lmax): 419–453 nm) and
sorption and emission spectroscopy, IR spectroscopy, and
HRMS. The formation of the BF2 complexes was accompanied
by the disappearance of the NH resonance in their 1H NMR
spectra, and the appearance of a 1:2:1 triplet and a 1:1:1:1
quartet in their 11B and 19F NMR spectra, respectively. With the
exception of 7a (Ar=o-anisole, R=cyano), the BF2 complexes
were stable towards hydrolysis. In the presence of 10 equiva-
lents of water, complex 7a slowly hydrolyzed to re-form the
parent formazan 4a (4% degradation after 48 h, 8% degrada-
tion after 96 h). By comparison, complex 7d (Ar=o-ethylben-
zene, R=cyano) was indefinitely stable towards hydrolysis
under the same conditions. Based on this comparison, we con-
clude that the hydrolysis of 7a is not driven by steric interac-
tions alone, but rather that the lone pairs on oxygen likely play
an important role in the decomposition pathway. Furthermore,
the presence of the linear carbon-bound cyano substituent in
7a may facilitate its decomposition as it potentially allows for
“open” and “linear” ligand arrangements to be accessed. Simi-
lar ligand orientations are less likely for reaction intermediates
derived from complexes 8a and 9a, which possess nonlinear
nitro and phenyl carbon-bound substituents.
1
the chemical shift of the H NMR resonance originating from
their NH protons (d=11.75–12.89 ppm). All other formazans
produced during this study exist in the more common “closed”
conformation in solution and exhibit characteristic NH resonan-
1
ces in their H NMR spectra between 13.91 and 15.56 ppm and
lmax values between 448 and 530 nm in their UV/Vis absorption
spectra.
BF2 formazanate complexes 7a–d, 8a–c, and 9a–c were pre-
pared by heating the appropriate formazan in toluene with an
excess of triethylamine and boron trifluoride diethyl etherate
(Scheme 1, Table 1, Figure S1–S36, Supporting Information).
The isolated yields for the complexes ranged from 15–91%
X-ray crystallography
Single crystals of complexes 7a and 7b suitable for X-ray dif-
fraction studies were grown by slow evaporation of saturated
dichloromethane solutions in a sealed vessel containing hex-
anes. In the solid-state 7a and 7b exist in “dragonfly” confor-
mations with the nitrogen–nitrogen and carbon–nitrogen
bond lengths of the formazanate ligand backbone falling be-
tween those typically associated with single and double bonds
of the same atoms (Figure 1, Table 2).[21] The N-aryl substituents
are bent out of plane relative to the N4 backbone of the forma-
zanate ligands by an average torsion angle of 61.2 and 47.78
for 7a and 7b, respectively. The boron atoms reside 0.54 and
0.50 Š out of the N4 plane in the structures of 7a and 7b.
These values are substantially larger than the torsion angles
of 18.3 and 37.18 and boron displacements of 0.13 and 0.34 Š
observed for the two crystallographically independent forms of
Scheme 1. General synthetic approach for the synthesis of BF2 formazanate
complexes 7b–d (a) and BF2 formazanate complexes 7a, 8a–c, and 9a–
c (b). See Table 1 for a detailed list of substituents.
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Cyclic voltammetry
The electrochemical properties of BF2 complexes 7a–c were
studied using cyclic voltammetry (Figure 2 and Figure S38,
Supporting Information, Table 3). Each complex underwent
two reversible one-electron reductions to form the correspond-
ing ligand-centered radical anion and dianion (Scheme 2),
Figure 1. Solid-state structures of complexes 7a (a) and 7b (b). Anisotropic
displacement ellipsoids are shown at 50% probability and hydrogen atoms
have been omitted for clarity.
Figure 2. Cyclic voltammograms for BF2 formazanate complexes 7a (Ar=o-
anisole, R=cyano), 7b (Ar=m-anisole, R=cyano), and 7c (Ar=p-anisole,
R=cyano) recorded in dry, degassed acetonitrile containing ~1 mm analyte
and 0.1m nBu4NPF6 at a scan rate of 100 mVsÀ1
Table 2. Selected bond lengths [Š] and angles [8] for BF2 formazanate
complexes 7a–c.
7a
7b
1.2987(6)
7c[a]
1.307(2)
1.304(2)
1.340(3)
1.335(3)
N1ÀN2
1.3040(16)
1.3038(16)
1.3404(19)
1.3438(18)
116.09(11)
101.99(10)
–
1.2987(6)
–
1.3443(5)
–
116.42(4)
–
N3ÀN4
–
C1ÀN2
1.3443(5)
–
116.42(4) 116.75(18)
C1ÀN4
N1-N2-C1
N1-B1-N3
Scheme 2. Electrochemical reduction of BF2 formazanate complexes 7a, 7b,
and 7c (Ar=o-anisole, m-anisole, or p-anisole).
–
106.85(17)
N1-B1-N1’
N2-C1-N4
N2’-C1-N2
boron displacement[b]
dihedral angles[c]
102.63(5)
–
127.22(6)
0.50
–
102.63(5)
–
127.22(6)
0.50
126.85(12)
–
130.0(2)
–
0.13
0.54
while 7c was the only complex in the series that was oxidized
(irreversibly) within the electrochemical window offered by
acetonitrile.
62.0, 60.5
47.7, 47.7
15.3, 21.4 47.7, 47.7
[a] The asymmetric unit determined for BF2 complex 7c contains two
unique molecules. [b] Distance between B1 and the N4 plane. [c] Torsion
angle between N1 and N3 aryl substituents and the N4 plane.
We have previously shown that electron-donating N-aryl
substituents render BF2 formazanate complexes more difficult
to reduce than the phenyl-substituted analogues, and that the
opposite is true for complexes bearing electron-withdrawing
substituents.[13b,c] In the present study, the interpretation is
slightly more complex as both steric and electronic effects
must be considered. Complexes 7a and 7c possess anisole
substituents with o- and p-substitution patterns, which poten-
tially allow for resonance donation of oxygen lone pairs lead-
ing to extended p-conjugation of the formazanate framework.
The m-substituted complex 7b is not resonance delocalized,
and should therefore possess properties purely associated with
the inductive electron-withdrawing character of the methoxy
group. Indeed, 7b is the easiest complex to reduce within the
series (Eꢀred 1 =À0.50 V; Erꢀed 2 =À1.62 V vs. ferrocene/ferroceni-
um). The o-substituted complex 7a was observed to be more
difficult to reduce (Eꢀred 1 =À0.73 V; Erꢀed 2 =À1.88 V) than the p-
7c.[13b] Initially, we were tempted to rationalize these trends
through steric arguments, whereby interactions between the
o-, and to a lesser extent, m-methoxy groups and the fluorine
atoms may lead to twisting of the N-aryl substituents relative
to the formazanate backbone. While this interpretation ap-
pears to hold true for 7a (Ar=o-anisole, R=cyano), the large
torsion angles observed for 7b (Ar=m-anisole, R=cyano)
likely arise due to crystal packing effects including slipped p-
stacking interactions (Figure S37, Supporting Information). This
conclusion is further corroborated by the fact that the me-
thoxy substituents are anti to the BF2 moiety in the solid-state
structure of 7b.
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Table 3. Solution characterization data for BF2 complexes 7a–d, 8a–c, and 9a–c.
[b]
Eꢀred 1
[b]
Solvent
lmax [nm]
e [mÀ1 cmÀ1
]
lem [nm]
Ff[â]
uST [nm]
uST [cmÀ1
]
Eꢀred 2
[V]
[V]
R=cyano
7a (Ar=o-anisole)
THF
456
466
467
510
517
525
556
558
572
433
431
436
10100
15500
16000
20000
18500
21100
33400
35300
42700
12000
14300
14000
590
589
592
639
637
635
662
661
656
562
562
565
0.03
134
123
125
129
120
110
106
103
84
4981
4481
4521
3958
3644
3300
2880
2793
2239
5301
5408
5236
CH2Cl2
toluene
THF
CH2Cl2
toluene
THF
CH2Cl2
toluene
THF
CH2Cl2
toluene
0.04
0.05
0.01
0.03
0.13
0.46
0.65
0.77
0.01
0.02
0.02
À0.73
À0.50
À0.68
À0.66
À1.88
À1.62
À1.82
À1.86
7b (Ar=m-anisole)
7c[13b] (Ar=p-anisole)
7d (Ar=o-ethylbenzene)
129
131
129
R=nitro
8a (Ar=o-anisole)
THF
456
466
468
512
511
533
559
564
572
5900
7100
7500
17700
18300
18400
26700
20100
23600
608
599
603
656
651
644
667
665
661
0.03
0.03
0.05
<0.01
<0.01
0.04
0.17
0.24
0.33
152
133
135
144
140
111
108
101
82
5483
4768
4784
4287
4209
3234
2897
2693
2143
CH2Cl2
toluene
THF
CH2Cl2
toluene
THF
À0.71
À0.43
À0.60
À1.83
À1.54
À1.72
8b (Ar=m-anisole)
8c (Ar=p-anisole)
CH2Cl2
toluene
R=phenyl
9a (Ar=o-anisole)
THF
459
464
467
520
521
530
548
545
551
18900
21600
18800
25000
25000
22900
23400
29300
25500
592
588
596
643
644
644
672
670
672
<0.01
0.01
133
124
129
123
123
114
124
125
121
4895
4545
4635
3679
3666
3340
3367
3423
3268
CH2Cl2
toluene
THF
CH2Cl2
toluene
THF
À1.09
À0.81
À0.94
À1.97
À1.87
À1.93
<0.01
<0.01
0.025
0.012
0.019
0.063
0.028
9b (Ar=m-anisole)
9c (Ar=p-anisole)
CH2Cl2
toluene
[a] Quantum yields were determined according to a published protocol[22a] using ruthenium tris(bipyridine) hexafluorophosphate as a relative standard[22b]
and corrected for wavelength-dependent detector sensitivity (Figure S41, Supporting Information). [b] Cyclic voltammograms were recorded in dry, de-
gassed acetonitrile containing ~1 mm analyte and 0.1m nBu4NPF6 at a scan rate of 100 mVsÀ1 and internally referenced relative to the ferrocene/ferroceni-
um redox couple.
substituted complex 7c (Eꢀred 1 =À0.68 V; Erꢀed 2 =À1.82 V). We
attribute this behavior to twisting of the o-anisole substituents
relative to the formazanate backbone in 7a in solution. This
twisting, which is consistent with the solid-state structure de-
scribed above, limits the degree of pconjugation of the forma-
zanate framework. The reduced degree of p-conjugation re-
sults in destabilization of the LUMO, and, in turn, renders 7a
more difficult to reduce than the p-conjugated complex 7c.
This argument is supported by DFT studies that showed the
LUMO orbital of closely related complexes to include both the
formazanate backbone and N-aryl substituents.[13b,d] To confirm
that the effect of the substituent pattern on the electrochemi-
cal properties of anisole-substituted BF2 formazanate com-
plexes was general, cyclic voltammograms were also collected
for 8a–c (R=nitro) and 9a–c (R=phenyl) (Figure S39 and S40,
Supporting Information, Table 3). Nitro-substituted complexes
8a–c were slightly easier to reduce than the corresponding
cyano-substituted complexes due to the strong electron-with-
drawing nature of the nitro group, while phenyl-substituted
complexes 9a–c were much more difficult to reduce due to
the absence of a strongly electron-withdrawing carbon-bound
substituent on the formazanate backbone. For each series, the
m-substituted complexes were the easiest to reduce and the
o-substituted complexes were more difficult to reduce than
the p-analogues, confirming that the trend discussed above is
general.
UV/Vis absorption and emission spectroscopy
The UV/Vis absorption and emission spectra for BF2 complexes
7a–c (R=cyano) are shown in Figure 3 and the data summar-
ized in Table 3. Each of the complexes is strongly absorbing
within the visible region of the electromagnetic spectrum with
wavelengths of maximum absorption (lmax) ranging from 467
to 572 nm and molar extinction coefficients (e) ranging from
16000 to 42700mÀ1 cmÀ1 in toluene. The low-energy absorp-
tion maxima for a similar set of complexes was shown by time-
dependent DFT to involve primarily the HOMO–LUMO transi-
tion.[13d] The same series of compounds exhibited wavelengths
of maximum emission (lem) ranging from 592 to 656 nm and
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ways associated with the carbon-bound phenyl substituent.
Each of the anisole-substituted BF2 complexes reported in this
study exhibited large Stokes shifts (uST) ranging from 82 to
152 nm (2143–5483 cmÀ1). This key characteristic of BF2 forma-
zanate complexes is of significant importance to their potential
use as fluorescence cell imaging agents, in which photon re-
absorption greatly limits emission intensity.
Cell imaging
Based on our evaluation of the anisole-substituted BF2 forma-
zanate complexes described above, we concluded that com-
plex 7c, which can be prepared in two straightforward syn-
thetic steps involving minimal bench time for just a few dollars
per gram, was an excellent candidate for fluorescence cell-
imaging studies. To this end, the uptake of complex 7c into fi-
broblast cells was investigated (Figure 4).
Figure 3. UV/Vis absorption spectra (a) and emission spectra (b) for BF2 for-
mazanate complexes 7a (Ar=o-anisole, R=cyano), 7b (Ar=m-anisole,
R=cyano), and 7c (Ar=p-anisole, R=cyano) recorded for degassed 10 mm
toluene solutions.
quantum yields (Ff) ranging from 0.05 to 0.77. As a result of
the twisted conformation adopted by the N-aryl substituents,
and the reduced degree of p-conjugation that results, the o-
substituted complex 7a had the lowest values of lmax
(467 nm), e (16000mÀ1 cmÀ1), lem (592 nm), and Ff (0.05). Con-
versely, p-substituted complex 7c possessed properties consis-
tent with extended p-conjugation arising from the relatively
coplanar orientation of the N-aryl substituents and the forma-
zanate backbone (lmax =572 nm; e=42700mÀ1 cmÀ1; lem
=
Figure 4. Confocal fluorescence micrographs of mouse fibroblast cells
stained with BF2 formazanate complex 7c and DAPI. Images (a) and (b) were
visualized with excitation at 559 nm and emission collected between 625–
725 nm. Images (c) and (d) are an overlay of images (a) and (b) with those
obtained from excitation at 405 nm and emission collected between 425–
475 nm.
656 nm; and Ff =0.77). The spectroscopic characteristics of
the m-substituted analogue 7b fall between those observed
for 7a and 7c and are dictated by the presence of the induc-
tively electron-withdrawing methoxy group (lmax =525 nm; e=
21100mÀ1 cmÀ1; lem =635 nm; and Ff =0.13). Complex 7d,
which has o-ethylbenzene N-aryl substituents, has lmax
=
436 nm and e=14000mÀ1 cmÀ1, confirming that the twisted
structure postulated for 7a is not unique to anisole-substituted
compounds, and is driven by steric interactions (Figure S42,
Supporting Information). The spectroscopic properties ob-
served for complexes 8a–c (R=nitro) and 9a–c (R=phenyl)
were closely related to those of 7a–c, further demonstrating
the generality of the trend observed (Figures S43 and S44,
Supporting Information). However, it must be noted that the
emission quantum yields calculated for 8a–c and 9a–c, were
lower than those calculated for 7a–c. In particular, 9a–c are
weakly emissive, likely as a result of nonradiative decay path-
Mouse fibroblast cells were incubated with 7c for one hour,
and after washing and fixing, were imaged by confocal fluores-
cence microscopy. The fluorescence images demonstrated that
7c was internalized by the fibroblast cells (Figure 4a,b). As
a result of the low-energy (lem ~660 nm) emission of 7c, this
dye proved to be well suited for orthogonal imaging applica-
tions. We explored this potential by co-staining cells with the
nuclear dye 4’,6-diamidino-2-phenylindole (DAPI), and demon-
strated that 7c and DAPI could be used to image the cell cyto-
plasm and nucleus simultaneously (Figure 4c,d). During the
course of these studies, we frequently observed punctate
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2.50 ppm) and 13C NMR spectra were referenced to CDCl3 (d=
77.0 ppm) or [D6]DMSO (d=39.5 ppm). 11B spectra were referenced
to BF3·OEt2 at d=0.00 ppm, and 19F spectra were referenced to
CFCl3 at d0.00 ppm. MS data were recorded in positive-ion mode
using a high-resolution Finnigan MAT 8200 spectrometer using
electron impact ionization. UV/Vis spectra were recorded using
a Cary 300 Scan instrument. Molar extinction coefficients were de-
termined from the slope of a plot of absorbance against concen-
tration using four solutions with known concentrations ranging be-
tween 10 and 100 mm. IR spectra were recorded on a KBr disk
using a Bruker Vector 33 FTIR spectrometer. Emission spectra were
obtained using a Photon Technology International QM-4 SE spec-
trofluorometer. Excitation wavelengths were chosen based on ab-
sorption maxima from the respective UV/Vis spectrum in the same
solvent. Quantum yields were calculated relative to ruthenium tris(-
bipyridine) hexafluorophosphate by methods described by Fery-
Forgues and co-workers.[22]
staining, which could be due to the dye being localized in
either the endoplasmic reticulum and/or vesicles. Aggregation
of the dye could be another possible explanation for the punc-
tate appearance. However, we were able to rule out aggrega-
tion induced emission enhancement by adding water to THF
solutions of 7c, and demonstrating that emission intensity de-
creases as the volume fraction of water is increased and aggre-
gates begin to form (Figure S45, Supporting Information). We
therefore conclude that the punctate staining is due to high
local concentration of 7c rather than aggregation.
Conclusion
We have systematically evaluated a library of o-, m-, and p-ani-
sole-substituted BF2 formazanate complexes, and, for the first
time, demonstrated their potential as readily accessible fluores-
cence imaging agents. The substitution pattern for each
subset of complexes (R=cyano, nitro, and phenyl) was shown
to have a dramatic effect on their electrochemical and spectro-
scopic properties. In each case, the o-substituted complexes
were the most difficult to reduce electrochemically, had the
shortest lmax and lem, and possessed low emission quantum
yields due to limited p-conjugation of the formazanate frame-
work. By comparison, the p-substituted analogues were slightly
easier to reduce electrochemically, had dramatically redshifted
lmax and lem, and significantly enhanced emission quantum
yields (up to 0.77). Each of these traits were consistent with ex-
tended p-conjugation between the formazanate backbone and
the N-aryl substituents. The spectroscopic properties of the m-
substituted complexes, which were the easiest to reduce, were
intermediate between those of the o- and p- complexes, due
to the absence of resonance delocalization of the oxygen lone
pairs and the inductive electron-withdrawing nature of the me-
thoxy groups. Based on our evaluation, we were able to identi-
fy complex 7c (Ar=p-anisole, R=cyano) as a strong candidate
for use in fluorescence cell imaging applications. We subse-
quently demonstrated its efficacy as a fluorescent dye for or-
thogonal imaging of the cytoplasm (complex 7c) and nucleus
(DAPI) of mouse fibroblast cells. Our future work in this area
will expand on these results and focus on cell imaging applica-
tions involving disease-targeting BF2 formazanate dyes.
Electrochemical methods
Cyclic voltammetry experiments were performed with a Bioanalyti-
cal Systems Inc. (BASi) Epsilon potentiostat and analyzed using
BASi Epsilon software. Typical electrochemical cells consisted of
a three-electrode setup including a silver pseudo reference elec-
trode, glassy carbon working electrode, and platinum counter elec-
trode. Experiments were run at 100 mVsÀ1 scan rate in degassed
acetonitrile solutions of the analyte (~1 mm) and electrolyte (0.1m
tetrabutylammonium hexafluorophosphate). Cyclic voltammo-
grams were referenced relative to the ferrocene/ferrocenium redox
couple (~1 mm internal standard) and corrected for internal cell re-
sistance using the BASi Epsilon software.
X-ray crystallography methods
Single crystals of 7a and 7b were mounted on a Mitegen poly-
imide micromount with a small amount of Paratone N oil. X-ray dif-
fraction measurements for 7a were made on a Nonius KappaCCD
Apex2 and measurements for 7b were made using Bruker a Kappa
Axis Apex2 diffractometer. Both compounds were measured at
a temperature of 110 K. The data collection strategy was a number
of w and f scans which collected data over a range of angles, 2q.
The frame integration was performed using SAINT.[23] The resulting
raw data was scaled and absorption corrected using a multiscan
averaging of symmetry equivalent data using SADABS.[24]
The structures were solved by using a dual space methodology
using the SHELXT program.[25] All nonhydrogen atoms were ob-
tained from the initial solution. The hydrogen atoms were intro-
duced at idealized positions and were allowed to refine isotropical-
ly. The structural model was fit to the data using full matrix least-
squares based on F2. The calculated structure factors included cor-
rections for anomalous dispersion from the usual tabulation. The
structures were refined using the SHELXL-2014 program from the
SHELXTL program package.[26] See Table 4 for crystallographic data
collection and refinement details.
Experimental Section
General considerations
All reactions were carried out under a nitrogen atmosphere using
standard Schlenk techniques unless otherwise stated. Reagents
were purchased from Sigma–Aldrich or Alfa Aesar and used as re-
ceived. Solvents were purchased from Caledon Laboratories, dried
using an Innovative Technologies Inc. solvent purification system,
collected under vacuum, and stored under an inert (nitrogen) at-
mosphere containing 4 Š molecular sieves. The synthesis and char-
acterization of compounds 4c and 7c have been reported pre-
viously.[13b] NMR spectra were recorded on 400 MHz (1H: 399.8 MHz,
11B: 128.3 MHz, 19F: 376.1 MHz) or 600 MHz (1H: 599.5 MHz, 13C:
CCDC-1413166 and -1413167 contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Cell imaging protocols
A stock solution of BF2 formazanate complex 7c in DMSO (10 mm)
was prepared and subsequently diluted with Dulbecco’s modified
Eagle’s medium (DMEM) to obtain final concentrations of 0.5 mm of
1
150.8 MHz) Varian INOVA spectrometers. H NMR spectra were ref-
erenced to residual CHCl3 (d=7.27 ppm) or (CD3)(CD2H)SO (d=
Chem. Eur. J. 2015, 21, 15589 – 15599
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Full Paper
mixture was left to warm slowly with stirring for 18 h before the
solid was isolated by vacuum filtration. The filtrate was neutralized
with 1m HCl (ca. 150 mL), extracted into dichloromethane (3”
150 mL), washed with deionized water (3”150 mL), dried over
MgSO4, gravity filtered, and concentrated in vacuo to afford a red
solid. The crude solids were combined and purified by flash
column chromatography (dichloromethane, neutral alumina) and
recrystallized from a saturated methanolic solution to afford forma-
Table 4. X-ray diffraction data collection and refinement details for 7a
and 7b.
7a
7b
chemical formula
FW [gmolÀ1
C16H14BF2N5O2
357.13
C16H14BF2N5O2
357.13
]
crystal dimensions
0.411”0.149”0.104
0.384”0.324”0.246
[mm]
1
zan 4a as red needles. Yield: 6.10 g, 70%; m.p. 136–1388C; H NMR
crystal habit
crystal system
space group
T [K]
red needle
monoclinic
P21/c
purple prism
monoclinic
C2/m
(599.5 MHz, CDCl3): d=13.91 (s, 1H, NH), 7.78 (d, 3JHH =8 Hz, 2H,
aryl CH), 7.34–7.31 (m, 2H, aryl CH), 7.06–7.02 (m, 4H, aryl CH),
4.00 ppm (s, 6H, OCH3); 13C{1H} NMR (150.8 MHz, CDCl3): d=152.5,
136.1, 130.0, 121.4, 116.5, 116.5, 116.1, 112.0, 56.1 ppm; FTIR (KBr):
n˜ =3074 (m), 3027 (m), 2970 (m), 2944 (m), 2840 (m), 2224 (s),
1584 (s), 1485 (s), 1482 (s), 1258 (s), 1024 cmÀ1 (s); UV/Vis (toluene):
lmax =448 nm (e=19700mÀ1 cmÀ1); MS (EI= +ve mode): exact
mass calculated for [C16H15N5O2]+: 309.1226; exact mass found:
309.1238; difference: +3.9 ppm.
110
110
a [Š]
b [Š]
c [Š]
a [8]
9.784(2)
18.259(5)
10.150(2)
90
13.349(3)
16.206(3)
8.1753(19)
90
b [8]
111.382(6)
90
1688.4(7)
4
1.405
1.54178 (CuKa
0.938
112.872(10)
90
1629.6(6)
4
1.456
0.71073 (MoKa
0.114
g [8]
V [Š3]
Z
Formazan 4b (Ar1 =Ar5 =m-anisole, R3 =CN): From 3.41 g
(27.7 mmol) of m-anisidine. Yield: 0.751 g, 18% of a dark-red micro-
1 [gcmÀ3
l [Š]
]
)
)
1
crystalline solid; m.p. 132–1348C; H NMR (599.5 MHz, CDCl3): open
m [cmÀ1
]
3
isomer (major, 75%): d=12.89 (s, 1H, NH), 7.37 (t, JHH =8 Hz, 2H,
diffractometer type
Nonius KappaCCD
Apex2
Bruker Kappa Axis
Apex2
aryl CH), 7.22–7.21 (m, 2H, aryl CH), 7.19–7.17 (m, 2H, aryl CH), 6.92
3
4
(dd, JHH =8, JHH =3 Hz, 2H, aryl CH), 3.89 ppm (s, 6H, OCH3); linear
Rmerge
0.0380
0.0272
3
isomer (minor, 25%): d=9.14 (s, 1H, NH), 7.53 (d, JHH =8 Hz, 1H,
R1 [I>2s(I)]
wR2 [I>2s(I)]
R1 (all data)
wR2 (all data)
GOF
0.0305
0.0731
0.0376
0.0768
0.0387
0.1127
0.0509
0.1210
aryl CH), 7.43–7.40 (m, 3H, aryl CH), 7.33–7.30 (m, 1H, aryl CH),
7.07–7.06 (m, 1H, aryl CH), 7.02 (m, 1H, aryl CH), 6.74–6.73 (m, 1H,
aryl CH), 3.89 (s, 3H, OCH3), 3.88 ppm (s, 3H, OCH3); 13C{1H} NMR
(150.8 MHz, CDCl3): d=161.0, 160.8, 160.4, 153.2, 147.5, 142.0,
130.5, 130.3, 130.3, 129.9, 125.1, 119.2, 117.7, 115.7, 114.4, 113.0,
111.2, 108.0, 107.9, 105.6, 103.5, 100.9, 55.6 ppm (br); FTIR (KBr):
n˜ =3276 (m), 3086 (m), 3008 (m), 2960 (m), 2839 (m), 2227 (s),
1609 (s), 1535 (s), 1496 (s), 1412 (m), 1240 (s), 1150 (m), 1044 cmÀ1
(s); UV/Vis (toluene): lmax 432 nm (e=20150mÀ1 cmÀ1); MS (EI=
+ve mode): exact mass calculated for [C16H15N5O2]+: 309.1226;
exact mass found: 309.1221; difference: À1.6 ppm.
Formazan 4d (Ar1 =Ar5 =o-ethylbenzene, R3 =CN): From 2.96 g
(24.4 mmol) of 2-ethylaniline. Yield: 0.322 g, 9% of a dark-red
powder; m.p. 89–918C; 1H NMR (599.5 MHz, CDCl3): open isomer
(major, 81%): d=11.75 (s, 1H, NH), 7.67–7.65 (m, 2H, aryl CH),
7.34–7.31 (m, 6H, aryl CH), 2.97 (q, 3JHH =8 Hz, 4H, CH2CH3),
1.35 ppm (m, 6H, CH2CH3); linear isomer (minor, 29%): d=9.17 (s,
1H, NH), 7.77 (d, 3JHH =8 Hz, 1H, aryl CH), 7.72 (m, 1H, aryl CH),
7.44 (m, 2H, aryl CH), 7.40 (m, 2H, aryl CH), 7.30–7.28 (m, 1H, aryl
CH), 7.15 (m, 1H, CH), 3.18 (q, 3JHH =8 Hz, 4H, CH2CH3), 2.80 (q,
3JHH =8 Hz, 2H, CH2CH3), 1.39 ppm (m, 6H, CH2CH3); 13C{1H} NMR
(150.8 MHz, CDCl3): d=149.3, 145.3, 144.2, 138.1, 137.8, 132.3,
131.9, 130.0, 129.6, 129.5, 129.1, 129.0, 127.6, 127.4, 127.2, 126.5,
124.8, 115.6, 115.5, 115.4, 113.3, 108.0, 24.7, 24.2, 23.5, 16.3, 15.0,
13.8 ppm; FTIR (KBr): n˜ =3338 (m), 3077 (m), 2973 (m), 2936 (m),
2878 (m), 2227 (m), 1589 (m), 1529 (s), 1457 (m), 1276 (m), 1201
(m), 1157 (m), 1060 cmÀ1 (w); UV/Vis (toluene): lmax 419 nm (e=
25,500mÀ1 cmÀ1); MS (EI= +ve mode): exact mass calculated for
[C18H19N5]+: 305.1640; exact mass found: 305.1639; difference:
À0.3 ppm.
1.054
1.058
1
2
2
R1 =S(jFo jÀjFc j)/SFo;
wR2 =[S(w(Fo ÀFc2)2)/S(wFo4)] =
;
GOF=
1
2
[S(w(Fo ÀFc2)2)/(no. of reflns.Àno. of params.)] =
2
dye. Mouse fibroblast C3H/10T1/2 cells (ATCC, Manassas, VA) were
released from the tissue culture flask by trypsinization and seeded
onto cover slips in a 12-well tissue culture plate at an approximate
cell density of 50000 cells per well. The cells were incubated over-
night in DMEM containing 10% fetal bovine serum (FBS) at 378C
in a 5% CO2 atmosphere. The serum containing DMEM in each
well was removed and replaced with serum free DMEM containing
a concentration of 0.5 mm of dye and incubated at 378C for 1 h.
The cells were then washed three times with phosphate buffer
saline (PBS), fixed with 4% paraformaldehyde and mounted onto
slides containing Pro-Long Antifade mounting medium with DAPI.
Images were then obtained using an Olympus FluoView FV 1000
confocal microscope.
Representative procedure for the preparation of formazans
4a, 4b, and 4d
Formazan 4a (Ar1 =Ar5 =o-anisole, R3 =CN): In air, cyanoacetic
acid (2.40 g, 28.2 mmol) and sodium hydroxide (11.4 g, 285 mmol)
were dissolved in deionized water (140 mL). The resulting colorless
solution was cooled to 08C and stirred for 2 h. In a separate flask,
o-anisidine (6.92 g, 56.2 mmol) was combined with concentrated
hydrochloric acid (14.0 mL, 168 mmol) in deionized water (55 mL)
and stirred for 1.5 h at 08C. A solution of sodium nitrite (5.83 g,
84.5 mmol) in deionized water (17 mL) was cooled to 08C in an ice
bath for 1.5 h before it was added dropwise to the o-anisidine so-
lution over a 25 min period. The resulting yellow/brown diazonium
salt solution was stirred for 1 h at 08C before it was added to the
alkaline cyanoacetic acid solution. Upon addition, the solution
turned blood red and a precipitate of the same color formed. The
Representative procedure for the preparation of formazans
5a and 5b
Formazan 5a (Ar1 =Ar5 =o-anisole, R3 =NO2): The following pro-
cedure was adapted from a protocol developed by von Eschwege
and co-workers.[19b] In air, o-anisidine (3.63 g, 29.5 mmol) was
added to deionized water (28 mL) containing concentrated hydro-
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chloric acid (16.0 mL, 192 mmol). The mixture was cooled to
À108C in an acetone/ice bath and stirred for 1 h. In a separate
flask, sodium nitrite (3.22 g, 46.7 mmol) was dissolved in deionized
water (9.20 mL) and cooled to À108C in an acetone/ice bath for
30 min. The sodium nitrite solution was added to the o-anisidine
mixture dropwise over the course of 30 min. The resulting yellow/
brown diazonium salt solution was then added to a flask contain-
ing sodium acetate (28.5 g, 347 mmol), glacial acetic acid (29.0 mL),
and deionized water (14 mL) and stirred at room temperature for
5 min. Nitromethane (8.98 g, 7.97 mL, 147 mmol) was then added
and the solution was stirred for 1 h before deionized water (1.60 L)
was added. The solution was stirred for an additional 30 min and
vacuum filtered to afford a red solid. The filtrate was extracted into
dichloromethane (3”250 mL), washed with deionized water (4”
200 mL), dried over MgSO4, gravity filtered and concentrated in
vacuo to afford a dark-red solid. The crude solids were combined
and purified by flash column chromatography (dichloromethane,
neutral alumina) to afford formazan 5a as a dark-red microcrystal-
18400mÀ1 cmÀ1); MS (EI, +ve mode): exact mass calculated for
[C15H15N5O4]+: 329.1124; exact mass found: 329.1123; difference:
À0.3 ppm.
Representative procedure for preparation of formazans 6a–c
Formazan 6a (Ar1 =Ar5 =o-anisole, R3 =Ph): In air, phenylpyruvic
acid (1.0 g, 6.1 mmol) and sodium hydroxide (1.83 g, 45.7 mmol)
were combined with deionized water (150 mL) and cooled to 08C
in an ice bath for 30 min. In a separate flask, o-anisidine (1.50 g,
12.2 mmol) and concentrated hydrochloric acid (3.5 mL, 42 mmol)
were mixed in deionized water (10 mL), and cooled to 08C in an
ice bath for 10 min. A cooled solution (08C) of sodium nitrite
(0.97 g, 14.0 mmol) in deionized water (5 mL) was added slowly to
the amine solution over a 10 min period. This yellow mixture was
left to stir at 08C for 30 min, at which time it was added dropwise
to the phenylpyruvic acid reaction mixture described above over
a 10 min period. The resulting solution was left to warm slowly
with stirring for 18 h, at which time a purple precipitate had
formed. The purple solid was isolated by filtration and purified by
flash column chromatography (dichloromethane, neutral alumina)
to afford formazan 6a as a purple microcrystalline solid. Yield:
1
line solid. Yield: 4.01 g, 82%; m.p. 176–1788C; H NMR (599.5 MHz,
CDCl3): d=14.87 (s, 1H, NH), 7.97–7.95 (m, 2H, aryl CH), 7.38–7.35
(m, 2H, aryl CH), 7.08–7.05 (m, 2H, aryl CH), 7.04–7.03 (m, 2H, aryl
CH), 4.02 ppm (s, 6H, OCH3); 13C{1H} NMR (150.8 MHz, CDCl3): d=
153.0, 136.2, 130.8, 121.6, 117.0, 112.0, 56.1 ppm; FTIR (KBr): n˜ =
3057 (m), 2990 (m), 1660 (m), 1578 (s), 1423 (m), 1267 (m),
897 cmÀ1 (m); UV/Vis (toluene): lmax 472 nm (e=8000mÀ1 cmÀ1);
MS (EI= +ve mode): exact mass calculated for [C15H15N5O4]+:
329.1124; exact mass found: 329.1121; difference: À0.9 ppm.
1
1.98 g, 90%; m.p. 120–1228C; H NMR (599.5 MHz, CDCl3): d=15.01
(s, 1H, NH), 8.24–8.23 (m, 2H, aryl CH), 7.98–7.97 (m, 2H, aryl CH),
7.49–7.46 (m, 2H, aryl CH), 7.38–7.36 (m, 1H, aryl CH), 7.28–7.25
(m, 2H, aryl CH), 7.10–7.07 (m, 2H, aryl CH), 7.04–7.02 (m, 2H, aryl
CH), 4.02 ppm (s, 6H, OCH3); 13C{1H} NMR (100.6 MHz, CDCl3): d=
152.0, 142.2, 138.0, 137.9, 128.3, 128.0, 127.3, 125.8, 121.3, 115.7,
111.7, 56.0 ppm; FTIR (KBr): n˜ =3076 (m), 3008 (m), 2915 (m), 1659
Formazan 5b (Ar1 =Ar5 =m-anisole, R3 =NO2): From 2.19 g
(17.8 mmol) of m-anisidine. Yield: 1.77 g, 60% of dark-red micro-
(m), 1267 (m), 1106 (s), 752 cmÀ1 (m); UV/Vis (toluene): lmax
=
1
crystalline solid; m.p. 134–1368C; H NMR (599.5 MHz, CDCl3): d=
507 nm (e=22900mÀ1 cmÀ1); MS (EI, +ve mode): exact mass calcu-
lated for [C21H20N4O2]+: 360.1586; exact mass found: 360.1580; dif-
ference: À1.7 ppm.
15.08 (s, 1H, NH), 7.35 (t, 3JHH =8 Hz, 2H, aryl CH), 7.24–7.23 (m,
4
2H, aryl CH), 7.21–7.19 (m, 2H, aryl CH), 6.92 (dd, 3JHH =8, JHH
=
2 Hz, 2H, aryl CH), 3.85 ppm (s, 6H, OCH3); 13C{1H} NMR (150.8 MHz,
CDCl3): d=160.8, 147.2, 130.3, 116.4, 113.8, 103.3, 55.5 ppm; FTIR
(KBr): n˜ =3058 (m), 3018 (m), 2973 (m), 2858 (m), 1661 (m), 1610 (s)
1552 (m), 1493 (w), 1434 (w), 1360 (w), 1267 (s), 1143 (w), 1045 (w),
896 (w), 826 cmÀ1 (s); UV/Vis (toluene): lmax =466 nm (e=
21200mÀ1 cmÀ1); MS (EI= +ve mode): exact mass calculated for
[C15H15N5O4]+: 329.1124; exact mass found: 329.1118; difference:
À1.8 ppm.
Formazan 6b (Ar1 =Ar5 =m-anisole, R3 =Ph): From 1.50 g
(12.2 mmol) of m-anisidine. Yield: 1.38 g, 63% as a dark-red solid;
1
m.p. 99–1018C; H NMR (599.5 MHz, CDCl3): d=15.35 (s, 1H, NH),
8.15–8.14 (m, 2H, aryl CH), 7.48–7.46 (m, 2H, aryl CH), 7.39–7.36
(m, 3H, aryl CH), 7.31 (s, 2H, aryl CH), 7.24–7.23 (m, 2H, aryl CH),
6.87–6.85 (m, 2H, aryl CH), 3.91 ppm (s, 6H, OCH3); 13C{1H} NMR
(100.6 MHz, CDCl3): d=160.7, 149.1, 141.0, 137.3, 130.0, 128.4,
127.6, 125.8, 113.4, 112.6, 102.9, 55.4 ppm. FTIR (KBr): 3057 (m),
2991 (m), 1660 (m), 1643 (m), 1426 (m), 1268 (s), 1103 (s), 898 (m),
745 cmÀ1 (m); UV/Vis (toluene): lmax =496 nm (e=21600mÀ1 cmÀ1);
MS (EI, +ve mode): exact mass calculated for [C21H20N4O2]+:
360.1586; exact mass found: 360.1584; difference: À0.6 ppm.
Preparation of Formazan 5c (Ar1 =Ar5 =p-anisole, R3 =NO2): In
air, nitromethane (1.23 g, 1.08 mL, 20.0 mmol) was combined with
deionized water (150 mL), and sodium hydroxide (1.76 g,
44.0 mmol) before the mixture was cooled to 08C in an ice bath. In
a separate flask, p-anisidine (5.00 g, 41.0 mmol) and concentrated
hydrochloric acid (11.1 mL, 133 mmol) were mixed with deionized
water (15 mL) and cooled to 08C in an ice bath. A cooled solution
(08C) of sodium nitrite (3.35 g, 49.0 mmol) in deionized water
(10 mL) was added slowly to the amine solution. This mixture was
left to stir at 08C for 30 min, at which time the dark-yellow diazoni-
um salt solution was added dropwise to the nitromethane solution
described above over a 10 min period. The resulting mixture was
left to warm slowly with stirring for 18 h, during which time
a dark-red precipitate had formed. The dark-red solid was isolated
by vacuum filtration and purified by flash column chromatography
(dichloromethane, neutral alumina) to afford formazan 5c as
a dark-red microcrystalline solid. Yield: 6.59 g, 55%; m.p. 162–
Formazan 6c (Ar1 =Ar5 =p-anisole, R3 =Ph): From 1.48 g
(12.0 mmol) of p-anisidine. Yield: 1.69 g, 82% as a dark-red solid;
1
m.p. 122–1248C; H NMR (599.5 MHz, CDCl3): d=15.56 (s, 1H, NH),
3
3
8.13 (d, JHH =7 Hz, 2H, aryl CH), 7.65 (d, JHH =9 Hz, 4H, aryl CH),
3
7.45–7.43 (m, 2H, aryl CH), 7.34 (t, JHH =8 Hz, 1H, aryl CH), 6.99 (d,
3JHH =9 Hz, 4H, aryl CH), 3.88 ppm (s, 6H, OCH3); 13C{1H} NMR
(100.6 MHz, CDCl3): d=159.2, 142.0, 140.6, 137.8, 128.3, 127.2,
125.6, 120.0, 114.7, 55.6 ppm; FTIR (KBr): n˜ =3263 (br, s), 2984 (m),
2884 (w), 2794 (s), 1594 (s), 1573 (m), 1507 (s), 1495 (s), 1226 (s),
1176 cmÀ1 (m); UV/Vis (toluene): lmax =530 nm (e=23400mÀ1 cmÀ1);
MS (EI, +ve mode): exact mass calculated for [C21H20N4O2]+:
360.1586; exact mass found: 360.1581; difference: À1.4 ppm.
1
1648C; H NMR (599.5 MHz, [D6]DMSO): d=14.61 (s, 1H, NH), 7.86
(d, 3JHH =8 Hz, 4H, aryl CH), 7.11 (d, 3JHH =8 Hz, 4H, aryl CH),
3.85 ppm (s, 6H, OCH3); 13C{1H} NMR (150.8 MHz, [D6]DMSO): d=
160.5, 140.1, 121.8, 114.9, 55.7 ppm; FTIR (KBr): 3061 (m), 3018 (m),
2995 (m), 1659 (m), 1574 (w), 1428 (m), 1348 (w), 1268 (s), 1159
(w), 899 (m), 745 cmÀ1 (m); UV/Vis (toluene): lmax 504 nm (e=
Representative procedure for the preparation of formaza-
nate BF2 complexes 7a and 7d
Formazanate BF2 complex 7a (Ar1 =Ar5 =o-anisole, R3 =CN): For-
mazan 4a (0.500 g, 1.61 mmol) was dissolved in toluene (250 mL)
Chem. Eur. J. 2015, 21, 15589 – 15599
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Full Paper
before triethylamine (1.45 g, 2.00 mL, 14.2 mmol) was added and
the solution stirred for 30 min. Boron trifluoride diethyl etherate
(3.45 g, 3.00 mL, 24.3 mmol) was then added and the solution
heated to 1058C for 36 h. During this time, the solution gradually
changed color from dark-red to red/orange. The solution was
cooled to 208C and deionized water (25 mL) was added to quench
any remaining reactive species. The mixture was then washed with
deionized water (3”50 mL), dried over MgSO4, gravity filtered, and
concentrated in vacuo to afford 7a as a red/orange solid. The
crude solid was purified by flash column chromatography (di-
chloromethane, neutral alumina) to afford 7a as a bright-orange
powder. This compound hydrolyzes slowly. Analyses were per-
formed using dry solvents. Yield: 0.353 g, 61%; m.p. 134–1368C;
1H NMR (599.5 MHz, dry CDCl3): d=7.46–7.43 (m, 2H, aryl CH),
7.30–7.28 (m, 2H, aryl CH), 7.05–7.01 (m, 4H, aryl CH), 3.91 ppm (s,
6H, OCH3); 13C{1H} NMR (150.8 MHz, CDCl3): d=154.8, 132.5, 132.4,
127.2, 120.8, 113.8, 112.8, 56.4 ppm; 11B NMR (128.3 MHz, CDCl3):
d=À1.9 ppm (t, 1JBF =20 Hz); 19F NMR (376.1 MHz, CDCl3): d=
À154.3 ppm (q, 1JFB =19 Hz); FTIR (KBr): n˜ =3072 (m), 3017 (m),
2960 (m), 2853 (m), 2237 (s), 1605 (s), 1488 (s), 1340 (s), 1260 (s),
1195 (s), 1164 (s), 1030 cmÀ1 (s); UV/Vis (toluene): lmax 467 nm (e=
16000mÀ1 cmÀ1); MS (EI= +ve mode): exact mass calculated for
[C16H14BF2N5O2]+: 357.1209; exact mass found: 357.1212; differ-
ence: +0.8 ppm.
2958 (m), 2850 (m), 2237 (s), 1605 (s), 1582 (s), 1487 (s), 1456 (m),
1339 (s), 1293 (m), 1260 (s), 1195 (m), 1164 (m), 1031 cmÀ1 (s); UV/
Vis (toluene): lmax 525 nm (e=21100mÀ1 cmÀ1); MS (EI= +ve
mode): exact mass calculated for [C16H14BF2N5O2]+: 357.1209; exact
mass found: 357.1198; difference: À3.1 ppm.
Formazanate BF2 complex 8b (Ar1 =Ar5 =m-anisole, R3 =NO2):
From 0.398 g (1.21 mmol) of formazan 5b. Yield: 0.111 g, 24% of
dark-purple microcrystalline solid; m.p. 78–808C; 1H NMR
(599.5 MHz, CDCl3): d=7.64–7.62 (m, 2H, aryl CH), 7.52–7.50 (m,
2H, aryl CH), 7.43 (t, 3JHH =8 Hz, 2H, aryl CH), 7.09 (dd, 3JHH =8,
4JHH =2 Hz, 2H, aryl CH), 3.90 ppm (s, 6H, OCH3); 13C{1H} NMR
(150.8 MHz, CDCl3): d=160.5, 144.2, 130.3, 118.5, 116.3 (4JCF =4 Hz),
1
108.0, 55.7 ppm; 11B NMR (128.3 MHz, CDCl3): d=À0.7 (t, JBF
=
=
1
29 Hz); 19F NMR (376.1 MHz, CDCl3): d=À134.6 ppm (q, JFB
29 Hz); FTIR (KBr): n˜ =3076 (m), 3013 (m), 2863 (m), 1658 (m), 1580
(m), 1424 (m), 1266 (m), 1095 (s), 745 cmÀ1 (m); UV/Vis (toluene):
lmax 533 nm (e=18400mÀ1 cmÀ1); MS (EI= +ve mode): exact mass
calculated for [C15H14BF2N5O4]+: 377.1107; exact mass found:
377.1104; difference: À0.8 ppm.
Formazanate BF2 complex 8c (Ar1 =Ar5 =p-anisole, R3 =NO2):
From 1.19 g (3.60 mmol) of formazan 5c. Yield: 0.291 g, 21% of
dark-purple microcrystalline solid; m.p. 154–1568C; 1H NMR
3
(599.5 MHz, CDCl3): d=7.99 (d, JHH =9 Hz, 4H, aryl CH), 7.01 (dd,
3JHH =9, 4JHH =2 Hz, 4H, aryl CH), 3.91 ppm (s, 6H, OCH3);
13C{1H} NMR (150.8 MHz, CDCl3): d=162.4, 137.0, 125.2, 114.9,
Formazanate BF2 complex 7d (Ar1 =Ar5 =o-ethylbenzene, R3 =
CN): From 0.366 g (1.20 mmol) of formazan 4d. Yield: 0.095 g, 22%
of dark-orange oil; 1H NMR (599.5 MHz, CDCl3): d=7.47–7.45 (m,
2H, aryl CH), 7.42–7.39 (m, 4H, aryl CH), 7.34–7.31 (m, 2H, aryl CH),
2.65 (q, 3JHH =8 Hz, 4H, CH2CH3), 1.24 ppm (t, 3JHH =7 Hz, 6H,
CH2CH3); 13C{1H} NMR (150.8 MHz, CDCl3): d=140.9, 139.7, 131.0,
130.5, 126.6, 126.3, 113.4, 24.3, 14.9 ppm; 11B NMR (128.3 MHz,
CDCl3): d=À1.9 (t, 1JBF =24 Hz); 19F NMR (376.1 MHz, CDCl3): d=
À146.9 ppm (q, 1JFB =24 Hz); FTIR (KBr): n˜ =3077 (m), 2975 (s),
2938 (s), 2879 (m), 2247 (m), 1453 (s), 1336 (s), 1217 (s), 1084 (s),
1025 (s), 970 cmÀ1 (s); UV/Vis (toluene): lmax 436 nm (e=
14000mÀ1 cmÀ1); MS (EI= +ve mode): exact mass calculated for
[C18H18BF2N5]+: 353.1623; exact mass found: 353.1626; difference:
+0.8 ppm.
100.0, 55.8 ppm; 11B NMR (128.3 MHz, CDCl3): d=À0.7 ppm (t,
1
1JBF =30 Hz); 19F NMR (376.1 MHz, CDCl3): d=À137.1 ppm (q, JFB
=
29 Hz); FTIR (KBr): n˜ =3058 (m), 3011 (m), 2993 (m), 1656 (m), 1580
(w), 1420 (m), 1340 (w), 1270 (s), 1151 (w), 900 (m), 745 cmÀ1 (m);
UV/Vis (toluene): lmax 579 nm (e=23600mÀ1 cmÀ1); MS (EI= +ve
mode): exact mass calculated for [C15H14BF2N5O4]+: 377.1107; exact
mass found: 377.1101; difference: À1.6 ppm.
Formazanate BF2 complex 9a (Ar1 =Ar5 =o-anisole, R3 =Ph): This
complex required additional purification by flash column chroma-
tography (toluene, silica gel). From 0.396 g (1.10 mmol) of forma-
zan 6a. Yield: 0.212 g, 47% of an orange solid; m.p. 158–1608C;
1H NMR (399.5 MHz, CDCl3): d=8.08–8.06 (m, 2H, aryl CH), 7.46–
7.38 (m, 5H, aryl CH), 7.23–7.22 (m, 2H, aryl CH), 7.04–6.99 (m, 4H,
aryl CH), 3.92 ppm (s, 6H, OCH3); 13C{1H} NMR (100.6 MHz, CDCl3):
d=155.0, 151.5, 133.3, 133.1, 130.9, 129.2, 128.5, 127.3, 125.7,
120.6, 112.5, 56.3 ppm; 11B NMR (128.3 MHz, CDCl3): d=À1.2 ppm
(t, 1JBF =19 Hz); 19F NMR (376.1 MHz, CDCl3): d=À160.6 ppm (q,
1JFB =19 Hz); FTIR (KBr): n˜ =3057 (m), 2988 (m), 2967 (w), 2843 (m),
1666 (m), 1496 (s), 1441 (m), 1359 (m), 1268 (s), 1124 (s), 1050 (m),
897 (m), 745 cmÀ1 (m); UV/Vis (toluene): lmax =467 nm (e=
18,800mÀ1 cmÀ1); MS (EI, +ve mode): exact mass calculated for
[C21H19BF2N4O2]+: 408.1569; exact mass found: 408.1564; differ-
ence: À1.2 ppm.
Representative procedure for the preparation of formaza-
nate BF2 complexes 7b, 8b,c, and 9a–c
Formazanate BF2 complex 7b (Ar1 =Ar5 =m-anisole, R3 =CN):
Formazan 4b (0.398 g, 1.28 mmol) was dissolved in toluene
(175 mL) before triethylamine (0.391 g, 0.540 mL, 3.86 mmol) was
added and the solution stirred for 30 min. Boron trifluoride diethyl
etherate (0.92 g, 0.80 mL, 6.5 mmol) was then added and the solu-
tion heated to 808C for 18 h. During this time, the solution gradu-
ally changed color from dark-red to purple. The solution was
cooled to 208C and deionized water (15 mL) was added to quench
any remaining reactive species. The mixture was then washed with
deionized water (3”50 mL), dried over MgSO4, gravity filtered, and
concentrated in vacuo to afford 7b as a purple solid. The crude
solid was purified by flash column chromatography (dichlorome-
thane, neutral alumina) and recrystallized from a saturated metha-
nolic solution to afford 7b as a purple microcrystalline solid. Yield:
Formazanate BF2 complex 9b (Ar1 =Ar5 =m-anisole, R3 =Ph):
From 0.613 g (1.70 mmol) of formazan 6b. Yield: 0.621 g, 90% of
1
a purple/red solid; m.p. 76–788C; H NMR (399.8 MHz, CDCl3): d=
8.14–8.13 (m, 2H, aryl CH), 7.57–7.56 (m, 2H, aryl CH), 7.52–7.49
(m, 2H, aryl CH), 7.47–7.46 (m, 3H, aryl CH), 7.41–7.39 (m, 2H, aryl
CH), 7.02–7.01 (m, 2H, aryl CH), 3.89 ppm (s, 6H, OCH3);
13C{1H} NMR (100.6 MHz, CDCl3): d=160.1, 148.9, 145.0, 133.5,
129.8, 129.3, 128.7, 125.5, 116.1 (m), 116.0, 108.5, 55.5 ppm;
1
1
0.221 g, 48%; m.p. 143–1458C; H NMR (599.5 MHz, CDCl3): d=7.55
11B NMR (128.3 MHz, CDCl3): d=À0.6 ppm (t, JBF =29 Hz); 19F NMR
(d, 3JHH =9 Hz, 2H, aryl CH), 7.42–7.40 (m, 4H, aryl CH), 7.08 (dd,
3JHH =8, 4JHH =3 Hz, 2H, aryl CH), 3.89 ppm (s, 6H, OCH3);
13C{1H} NMR (150.8 MHz, CDCl3): d=160.4, 144.1, 130.2, 118.2, 116.0
(376.1 MHz, CDCl3): d=À144.2 ppm (q, JFB =28 Hz); FTIR (KBr): n˜ =
1
3058 (m), 2989 (m), 2900 (m), 2841 (w), 1667 (m), 1605 (m), 1494
(m), 1424 (m), 1357 (w), 1267 (s), 1119 (m), 897 (w), 745 cmÀ1 (m);
UV/Vis (toluene): lmax =530 nm (e=22900mÀ1 cmÀ1); MS (EI, +ve
mode): exact mass calculated for [C21H19BF2N4O2]+: 408.1569; exact
mass found: 408.1570; difference: +0.2 ppm.
4
(t, JCF =3 Hz), 114.0, 107.6, 55.7 ppm; 11B NMR (128.3 MHz, CDCl3):
d=À0.8 ppm (t, 1JBF =31 Hz); 19F NMR (376.1 MHz, CDCl3): d=
À132.6 ppm (q, 1JFB =29 Hz); FTIR (KBr): n˜ =3072 (m), 3016 (m),
Chem. Eur. J. 2015, 21, 15589 – 15599
www.chemeurj.org
15597
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
J. Org. Chem. 2013, 78, 7058–7067; c) G. Nawn, S. R. Oakley, M. B. Ma-
jewski, R. McDonald, B. O. Patrick, R. G. Hicks, Chem. Sci. 2013, 4, 612–
621; d) C. Bonnier, D. D. Machin, O. Abdi, B. D. Koivisto, Org. Biomol.
Chem. 2013, 11, 3756–3760; e) D. Frath, A. Poirel, G. Ulrich, A. De Nico-
la, R. Ziessel, Chem. Commun. 2013, 49, 4908–4910; f) A. Díaz-Moscoso,
E. Emond, D. L. Hughes, G. J. Tizzard, S. J. Coles, A. N. Cammidge, J. Org.
Chem. 2014, 79, 8932–8936; g) I.-S. Tamgho, A. Hasheminasab, J. T.
Engle, V. N. Nemykin, C. J. Ziegler, J. Am. Chem. Soc. 2014, 136, 5623–
5626; h) S. T. Manjare, J. Kim, Y. Lee, D. G. Churchill, Org. Lett. 2014, 16,
520–523.
Formazanate BF2 complex 9c (Ar1 =Ar5 =p-anisole, R3 =Ph):
From 1.00 g (2.80 mmol) of formazan 6c. Yield: 0.711 g, 62% of
a dark-purple solid; m.p. 170–1728C; 1H NMR (399.8 MHz, CDCl3):
d=8.12 (d, 3JHH =7 Hz, 2H, aryl CH), 7.88 (d, 3JHH =9 Hz, 4H, aryl
CH), 7.49–7.41 (m, 3H, aryl CH), 6.98 (d, 3JHH =9 Hz, 4H, aryl CH),
3.88 ppm (s, 6H, OCH3); 13C{1H} NMR (100.6 MHz, CDCl3): d=160.7,
137.6, 134.0, 128.9, 128.6, 125.4, 124.9, 114.3, 55.6 ppm; 11B NMR
(128.3 MHz, CDCl3): d=À0.4 ppm (t, 1JBF =29 Hz); 19F NMR
1
(376.1 MHz, CDCl3): d=À145.3 ppm (q, JFB =29 Hz); FTIR (KBr): n˜ =
[3] Reviews: a) R. Ziessel, G. Ulrich, A. Harriman, New J. Chem. 2007, 31,
496–501; b) A. Loudet, K. Burgess, Chem. Rev. 2007, 107, 4891–4932;
c) Y. Ni, J. Wu, Org. Biomol. Chem. 2014, 12, 3774–3791.
[4] Review: N. Boens, V. Leen, W. Dehaen, Chem. Soc. Rev. 2012, 41, 1130–
1172.
[5] Selected examples: a) T. W. Hudnall, F. P. Gabba¨ı, Chem. Commun. 2008,
4596–4597; b) A. Nierth, A. Y. Kobitski, G. U. Nienhaus, A. Jäschke, J. Am.
Chem. Soc. 2010, 132, 2646–2654; c) M. A. H. Alamiry, J. P. Hagon, A.
Harriman, T. Bura, R. Ziessel, Chem. Sci. 2012, 3, 1041–1048; d) S. T.
Manjare, S. Kim, W. D. Heo, D. G. Churchill, Org. Lett. 2014, 16, 410–412;
e) S. Raut, J. Kimball, R. Fudala, H. Doan, B. Maliwal, N. Sabnis, A. Lacko,
I. Gryczynski, S. V. Dzyuba, Z. Gryczynski, Phys. Chem. Chem. Phys. 2014,
16, 27037–27042; f) X.-D. Jiang, Y. Su, S. Yue, C. Li, H. Yu, H. Zhang, C.-L.
Sun, L.-J. Xiao, RSC Adv. 2015, 5, 16735–16739.
2948 (m), 2900 (m), 2834 (m), 1641 (s), 1596 (s), 1508 (s), 1313 (m),
1249 (s), 1164 cmÀ1 (s); UV/Vis (toluene): lmax 551 nm (e=
25500mÀ1 cmÀ1); MS (EI, +ve mode): exact mass calculated for
[C21H19BF2N4O2]+: 408.1569; exact mass found: 408.1576; differ-
ence: +1.7 ppm.
Preparation of formazanate BF2 complex 8a (Ar1 =Ar5 =o-ani-
sole, R3 =NO2): Formazan 5a (1.00 g, 3.04 mmol) was dissolved in
toluene (250 mL) before triethylamine (2.82 g, 3.90 mL, 27.9 mmol)
was added and the solution stirred for 30 min. Boron trifluoride di-
ethyl etherate (6.44 g, 5.60 mL, 45.4 mmol) was added and the so-
lution heated with stirring at 1058C for 144 h at which time the so-
lution was dark-purple with a blue hue on the sides of the flask.
The solution was cooled to 208C and poured directly onto
a column containing neutral alumina. After the toluene solution
was adsorbed onto the alumina, dichloromethane was used as the
eluent. The crude product was isolated as a bright-orange solution,
which was washed with deionized water (3”1.00 L). The organic
layer was collected, dried over MgSO4, gravity filtered, and concen-
trated in vacuo to afford 8a as a red-orange solid. The crude solid
was purified by flash column chromatography (toluene, neutral
alumina, Rf =0.33) to afford 8a as a bright orange powder. Yield:
[6] Review: A. B. Nepomnyashchii, A. J. Bard, Acc. Chem. Res. 2012, 45,
1844–1853.
[7] Selected examples: a) A. B. Nepomnyashchii, S. Cho, P. J. Rossky, A. J.
Bard, J. Am. Chem. Soc. 2010, 132, 17550–17559; b) A. B. Nepomnyash-
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8645; c) M. Hesari, S. M. Barbon, V. N. Staroverov, Z. Ding, J. B. Gilroy,
Chem. Commun. 2015, 51, 3766–3769; d) M. Hesari, J.-s. Lu, S. Wang, Z.
Ding, Chem. Commun. 2015, 51, 1081–1084.
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tailleau, R. Ziessel, Adv. Funct. Mater. 2008, 18, 401–413; b) T. Rousseau,
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1673–1675; c) H. Usta, M. D. Yilmaz, A.-J. Avestro, D. Boudinet, M.
Denti, W. Zhao, J. F. Stoddart, A. Facchetti, Adv. Mater. 2013, 25, 4327–
4334; d) N. J. Findlay, J. Bruckbauer, A. R. Inigo, B. Breig, S. Arumugam,
D. J. Wallis, R. W. Martin, P. J. Skabara, Adv. Mater. 2014, 26, 7290–7294;
e) J. J. Chen, S. M. Conron, P. Erwin, M. Dimitriou, K. McAlahney, M. E.
Thompson, ACS Appl. Mater. Interfaces 2015, 7, 662–669.
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Kamkaew, S. H. Lim, H. B. Lee, L. V. Kiew, L. Y. Chung, K. Burgess, Chem.
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7608–7614; b) Y.-C. Lai, S.-Y. Su, C.-C. Chang, ACS Appl. Mater. Interfaces
2013, 5, 12935–12943; c) R. L. Watley, S. G. Awuah, M. Bio, R. Cantu,
H. B. Gobeze, V. N. Nesterov, S. K. Das, F. D’Souza, Y. You, Chem. Asian J.
2015, 10, 1335–1343.
1
0.163 g, 15%; m.p. 166–1688C; H NMR (599.5 MHz, CDCl3): d=7.47
3
3
(t, JHH =8 Hz, 2H, aryl CH), 7.36 (d, JHH =8 Hz, 2H, aryl CH), 7.06–
7.03 (m, 4H, aryl CH), 3.92 ppm (s, 6H, OCH3); 13C{1H} NMR
(150.8 MHz, CDCl3): d=155.0, 132.9, 132.7, 127.3, 120.9 (2C), 112.9,
1
56.5 ppm; 11B NMR (128.3 MHz, CDCl3): d=À1.6 ppm (t, JBF
=
=
1
19 Hz); 19F NMR (376.1 MHz, CDCl3): d=À156.0 ppm (q, JFB
18 Hz); FTIR (KBr): n˜ =3035 (m), 2974 (m), 2845 (m), 1660 (m), 1587
(m), 1557 (m), 1494 (m), 1332 (m), 1260 (m), 1100 (s), 1021 (m), 814
(m), 751 cmÀ1 (m); UV/Vis (toluene): lmax 468 nm (e=
7500mÀ1 cmÀ1); MS (EI= +ve mode): exact mass calculated for
[C15H14BF2N5O4]+: 377.1107; exact mass found: 377.1101; difference:
À1.6 ppm.
[11] Review: T. Kowada, H. Maeda, K. Kikuchi, Chem. Soc. Rev. 2015, 44,
4953–4972.
Acknowledgements
[12] Selected examples: a) T. Komatsu, Y. Urano, Y. Fujikawa, T. Kobayashi, H.
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This work was supported by the Natural Sciences and Engi-
neering Research Council (NSERC) of Canada Discovery Grants
(L.G.L. and J.B.G.), Undergraduate Student Research Award
(R.R.M.), and Canada Graduate Scholarships program (S.M.B.),
the University of Western Ontario, and a Petro-Canada Young
Innovator Award (J.B.G.). We thank Profs. E.R. Gillies and M.S.
Workentin for access to instrumentation within their labs.
Keywords: boron
·
cell imaging
·
fluorescent dyes
·
formazanate ligands · substituent effects
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2014, 53, 2290–2310; Angew. Chem. 2014, 126, 2322–2342.
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Chem. Eur. J. 2015, 21, 15589 – 15599
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Received: July 17, 2015
Published online on September 23, 2015
Chem. Eur. J. 2015, 21, 15589 – 15599
www.chemeurj.org
15599
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