Bithiophene-Cored, mono-, bis-, and tris-(Trimethylammonium)-Substituted, bis-Triarylborane Chromophores: Effect of the Number and Position of Charges on Cell Imaging and DNA/RNA Sensing

Article abstract of DOI:10.1002/chem.202102308

The synthesis, photophysical, and electrochemical properties of selectively mono-, bis- and tris-dimethylamino- and trimethylammonium-substituted bis-triarylborane bithiophene chromophores are presented along with the water solubility and singlet oxygen s

Full text of DOI:10.1002/chem.202102308

Accepted Article
Accepted Article
Title: Bithiophene-Cored, mono-, bis-, and tris-(Trimethylammonium)-
Substituted, bis-Triarylborane Chromophores: Effect of the
Number and Position of Charges on Cell Imaging and DNA/RNA
Sensing
Authors: Sarina M. Berger, Jessica Rühe, Johannes Schwarzmann,
Alexandra Phillipps, Ann-Katrin Richard, Matthias Ferger,
Ivo Krummenacher, Lidija-Marija Tumir, Željka Ban, Ivo
Crnolatac, Dragomira Majhen, Ivan Barišić, Ivo Piantanida,
Domenik Schleier, Stefanie Griesbeck, Alexandra Friedrich,
Holger Braunschweig, and Todd B. Marder
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202102308
Link to VoR: https://doi.org/10.1002/chem.202102308
01/2020

10.1002/chem.202102308
Chemistry - A European Journal
Bithiophene-Cored, mono-, bis-, and tris-(Trimethylammonium)-Substituted, bis-
Triarylborane Chromophores: Effect of the Number and Position of Charges on Cell
Imaging and DNA/RNA Sensing
Sarina M. Berger,[a] Jessica Rühe,[a] Johannes Schwarzmann,[a] Alexandra Phillipps,[a] Ann-
Katrin Richard,[a] Matthias Ferger,[a] Ivo Krummenacher,[a] Lidija-Marija Tumir,[b] Željka Ban,[b]
Ivo Crnolatac,[b] Dragomira Majhen,[c]* Ivan Barišić,[d]* Ivo Piantanida,[b]* Domenik Schleier,[a]
Stefanie Griesbeck,[a] Alexandra Friedrich,[a] Holger Braunschweig,[a] Todd B. Marder[a]*
[a] S. M. Berger, J. Rühe, J. Schwarzmann, A. Phillipps, A.-K. Richard, Dr. M. Ferger, Dr. I.
Krummenacher, D. Schleier, Dr. S. Griesbeck, Dr. A. Friedrich, Prof. Dr. H. Braunschweig,
Prof. Dr. T. B. Marder
Institut für Anorganische Chemie and Institute for Sustainable Chemistry & Catalysis with
Boron, Julius-Maximilians-Universität Würzburg, Am Hubland, 97074 Würzburg, Germany.
E-mail: todd.marder@uni-wuerzburg.de
[b] Dr. L.-M. Tumir, Dr. Ž. Ban, Dr. I. Crnolatac, Prof. Dr. I. Piantanida
Division of Organic Chemistry and Biochemistry, Ruder Boskovic Institute, Bijenicka c. 54,
10000 Zagreb, Croatia.
E-mail: Ivo.Piantanida@irb.hr
[c] Dr. D. Majhen
Department of Molecular Biology, Laboratory for Cell Biology and Signaling, Ruder Boskovic
Institute, Bijenicka c. 54, 10000 Zagreb, Croatia.
E-Mail: Dragomira.Majhen@irb.hr
[d] Dr. I. Barišić
Molecular Diagnostics, Center for Health and Bioresources, AIT Austrian Institute of
Technology GmbH, Giefinggasse 4, 1210 Wien, Austria.
E-Mail: Ivan.Barisic@ait.ac.at
1
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Abstract
The synthesis, photophysical, and electrochemical properties of selectively mono-, bis- and
tris-dimethylamino- and trimethylammonium-substituted bis-triarylborane bithiophene
chromophores are presented along with the water solubility and singlet oxygen sensitizing
efficiency of the cationic compounds Cat1+, Cat2+, Cat(i)2+, and Cat3+. Comparison with the
mono-triarylboranes reveals the large influence of the bridging unit on the properties of the bis-
triarylboranes, especially those of the cationic compounds. Based on these preliminary
investigations, the interactions of Cat1+, Cat2+, Cat(i)2+, and Cat3+ with DNA, RNA, and
DNApore were investigated in buffered solutions. The same compounds were investigated for
their ability to enter and localize within organelles of human lung carcinoma (A549) and normal
lung (WI38) cells showing that not only the number of charges but also their distribution over
the chromophore influences interactions and staining properties.
Introduction
Three-coordinate boron compounds have an empty p-orbital at the boron center. Hence, such
compounds are good π-acceptors and display interesting photophysical and electrochemical
properties, which lead to many different applications,[1-8] e. g., optoelectronics,[9-11] sensors for
anions or small molecules,[12-17] and cell-imaging agents and biomolecule sensors.[15, 18-33] For
the latter applications, which were summarized very recently by Berger and Marder,[34] focusing
on triarylboranes, water-stable and soluble compounds are required. In 2009, Gabbaï and
coworkers reported water-soluble triarylboranes bearing at least two trimethylammonium
groups at positions para to the boron center.[35] Using this basic concept, we have shown that
tetra-cationic, bis-triarylborane chromophores are water-soluble[26, 29, 36] and water-stable if they
contain at least 5 ortho-methyl groups.[27] Water-stability can also be obtained upon
introduction of other sterically demanding substituents, e. g., anthracene[27] or perylene.[18] It
was also shown that such bis-triarylborane chromophores can be used to stain organelles in
HeLa cells[29, 37] or NIH 3T3, HEK 293T, and HEPG2-16 cells.[26]
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Figure 1: Overview of tetra-cationic, bis-triarylborane-substituted chromophores reported by
Marder and coworkers for biological applications.[26, 29, 37]
Combining these cellular experiments with investigations of the compound in buffered
solutions in the presence of DNA, RNA and/or proteins, the bithiophene-bridged model
compound Cat4+ was shown to stain different cell organelles and to interact with proteins.[38] In
the present work, we demonstrate the influence of the number and position of positively
charged trimethylammonium groups on basic properties such as water-solubility, singlet
oxygen sensitizing efficiency, and photophysical and electrochemical properties of a
bithiophene-bridged, bis-triarylborane chromophore. Additionally, we demonstrate that
biomolecule binding and staining selectivity for these compounds depends on the number and
distribution of the cationic charges. Thus, the neutral compounds Neut1-Neut3 and the
resulting selectively charged compounds Cat1+-Cat3+ (Figure 2), which are analogues of the
previously studied compounds Neut4, and Cat4+,[26] were synthesized, fully characterized, and
their properties were investigated. Neut0 was synthesized as a reference for the investigation
of photophysical and electrochemical properties.
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Figure 2: Molecular structures of Neut0-Neut4 and the selectively charged compounds Cat1+-
Cat4+.
The synthesis of Neut1, Neut(i)2, and Neut3 was made possible via a synthetic route to
unsymmetrically-substituted triarylboranes which was previously not well developed,[39] but has
been reported very recently by our group.[40]
Results and Discussion
Synthesis
The synthetic strategy is based on Suzuki-Miyaura cross-coupling reactions as previously
reported for Neut4 and Cat4+.[26] Thus, mono- and di-halogenated bithiophenes (2, 3) as well
as three different, borylated triarylboranes 4b, 5b, and 6b were required (Figure 3).
Figure 3: Starting materials for the synthesis of Neut0-Neut3.
Triarylboranes 6b[26] and 5b[40] and their para-selective C–H borylated analogues were
synthesized according to literature procedures. Mono-[41-42] and di-brominated[42] bithiophenes
2 and 3 were synthesized by routes similar to those in the literature.
4
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Scheme 1: A) Synthetic route to 4a and 4b. B) Synthetic route to Neut0 and Neut(i)2.
For the synthesis of Neut0, 4a was synthesized from mesityl magnesium bromide (MesMgBr),
BF3·OEt2, and 2,6-dimethylphenyl lithium using the general approach developed by Grisdale
and co-workers.[43] Isolation of Mes2BF and subsequent reaction with 2,6-dimethylphenyl
lithium gave the triarylborane 4a in 76% yield over two steps. C–H borylation with [Ir(COD)(µ-
OMe)]2 as the precatalyst, 4,4′-di-tert-butyl-2,2′-dipyridyl (dtbpy) as the ligand, and bis-
pinacolato diboron (B2pin2) gave 4b in 90% yield. The 1H NMR spectrum of 4b matches that
reported in the literature.[44] Subsequently, Suzuki-Miyaura cross-coupling with 5,5’-dibromo-
2,2’-bithiophene 3 using Pd2(dba)3·CHCl3 as the precatalyst, 2-dicyclohexylphosphino-2′,6′-
dimethoxybiphenyl (SPhos) as the ligand, and potassium hydroxide as the base gave Neut0
in 31% yield (Scheme 1B). Similarly, Neut(i)2 was synthesized from 5b and 5,5’-dibromo-2,2’-
bithiophene 3 in 53% yield.
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Scheme 2: Synthetic routes to Neut1, Neut2, and Neut3. a) Pd2(dba)3·CHCl3, SPhos, KOH,
toluene/water 2:1, 85 °C, 2 d; b) NBS, DMF, 0 °C, 15 min then r.t., 2 h; c) nBuLi, THF, -78 °C,
1.5 h, then I2, -78 °C -> r.t., 18 h.
To obtain Neut1, Neut2, and Neut3, 5-bromo-2,2’-bithiophene 2 was cross-coupled with 4b,
5b, and 6b, respectively, in Suzuki-Miyaura reactions using the conditions described above to
give 7a, 8a, and 9a in 71%, 93%, and 80% yields, respectively (Scheme 2). Prior to the second
Suzuki-Miyaura cross-coupling, these intermediates were halogenated at the 5’-position of the
bithiophene. Compound 7a was brominated at this position in 93% yield using NBS in DMF.
For the aminated compounds 8a and 9a, it was not possible to affect bromination with NBS at
the desired position due to the more activated positions at the dimethylamino-substituted
arene. Therefore, the bithiophene moiety was lithiated with nBuLi and then treated with I2. This
gave inseparable mixtures containing 80% of the iodinated compounds 8b and 9b and their
respective starting materials as determined by 1H NMR spectroscopy. As the starting material
cannot undergo Suzuki-Miyaura cross-coupling, these mixtures were used directly, after minor
purification, for the synthesis of Neut3. It was possible to isolate Neut3 from both reaction
sequences (8b + 6b and 9b + 5b). However, the first reaction sequence gave higher yields in
all steps, especially the last one (60% vs. 6% yield).
With all uncharged compounds Neut1-3 in hand, their cationic counterparts were synthesized
using methyl triflate in CH2Cl2 similar to previous reports[26-27, 29, 35, 40] giving Cat1+, Cat2+,
Cat(i)2+, and Cat3+ in 88%, 69%, 49%, and 57% yields, respectively. The neutral triarylboranes
5a and 6a were methylated similarly giving 5c and 6c in 89% and 93% yields, respectively.
With the neutral and cationic triarylboranes and bis-triarylboranes in hand, their photophysical,
and electrochemical properties were investigated as well as the singlet oxygen sensitizing
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efficiency and water-solubility of Cat1+-Cat3+ and their DNA/RNA binding affinities in buffered
solutions. Additionally, the behavior of the most promising compounds Cat(i)2+ and Cat3+ was
investigated in two different cell lines.
Solubility in Water
While Cat4+ is soluble in pure water with a maximum solubility of 1.0 mM,[26] none of the new,
mono-, di- or tri-cationic bis-triarylboranes are soluble in pure water at concentrations of 2.6 –
3.4 µM (Figure S64, Table S2). However, concentrated solutions of Cat1+-Cat3+ in acetonitrile
can be diluted with water to less than 1% without precipitation or aggregation. The
concentrations of the cationic compounds in these diluted solutions were as high as 15 to
34 µM and are suitable for photophysical studies. Thus, aqueous solutions of the cationic bis-
triarylboranes were prepared from concentrated acetonitrile solutions which were diluted with
water until the solution contained ≤1% acetonitrile. For studies of interactions with DNA/RNA
in buffered solutions and cells, stock solutions were prepared in DMSO (c = 1 x 10-2 mol L-1)
and diluted prior to use with the buffered solutions indicated.
Photophysical Data
Due to large number of compounds which are discussed in the following sections of this paper,
an overview of their molecular structures and numbering is given in Scheme S1 in the
Supporting Information. The absorption spectra, extinction coefficients, emission spectra,
fluorescence lifetimes, and quantum yields of triarylboranes 4a-6a, 5c, and 6c and of the
cationic and neutral bis-triarylboranes Neut0-Neut3 and Cat1+-Cat3+ were measured in
solvents of different polarity (Table 1 and Figures S65-S78 in the Supporting Information). The
photophysical properties of the triarylboranes and of the neutral bis-triarylboranes are
discussed briefly here and a more detailed discussion is given in the Supporting Information.
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Table 1: Selected photophysical properties of the new triarylboranes and bis-triarylboranes.
apparent
Stokes shift
[cm-1]
푓푙
푚푎푥
푎푏푠
ε
τ
Φf
휆
휆
푚푎푥
Solvent
[L mol-1 cm-1]
[ns]
[nm]
[nm]
4a
5a
6a
Hexane
Hexane
Hexane
326
383
390
15 000
22 000
30 000
367
440
424
3400
3400
2100
1.48
5.17
1.55
0.10
0.34
0.13
MeCN
1%
MeCN in 313
water
MeCN
1%
MeCN in 308
water
314
10 000
420
437
416
425
8 000
9 100
8 300
8 900
3.37
7.49
4.40
6.65
0.11
0.19
0.10
0.11
5c
6c
7 000
309
11 000
10 000
Neut0
Neut1
Neut2
Hexane
Hexane
Hexane
Hexane
Hexane
410
412
405
408
404
66 000
75 000
76 000
103 000
129 000
464
464
464
464
463
2 800
2 700
3 100
2 900
3 100
0.52
0.54
0.54
0.54
0.52
0.31
0.38
0.32
0.39
0.39
Neut(i)2
Neut3
MeCN
1%
MeCN in 415
water
MeCN
1%
MeCN in 424
water
414
52 000
29 500
51 000
31 100
51 000
34 000
51 000
36 000
519
527
584
550
515
510
568
558
4 900
5 100
6 700
5 400
4 500
4 000
6 000
5 700
0.82
0.54 [a]
2.98
0.34
0.07
0.48
0.15
0.35
0.15
0.46
0.08
Cat1+
Cat2+
420
0.76 [a]
0.75
MeCN
1%
MeCN in 424
water
418
Cat(i)2+
Cat3+
0.63 [a]
2.41
MeCN
1%
MeCN in 423
water
424
0.24 [a]
[a] More than one lifetime was observed and only the dominant lifetime (>50%) is listed here.
More information is given in the Supporting Information.
The absorption spectra of the triarylboranes display no solvatochromism. The introduction of
a strong electron-donating functionality, in this case dimethylamino group(s), leads to
bathochromically shifted absorption and emission maxima. Upon methylation, the positive
solvatochromism of the emission is almost lost due to the loss of the charge-transfer process
which was supported by DFT and TD-DFT calculations using the orbital overlap parameter Λ.
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2
∑
푐
⟨∣ 휑 ∣|∣ 휑 ∣⟩ with 휑푎 and 휑푖 representing the occupied
푖,푎
푖,푎
푎
푖
This parameter is defined as  =
2
∑
푐
푖,푎
푖,푎
and unoccupied one-electron wavefunctions, respectively, |⟨휑푎|||휑푎⟩| representing the norm of
the one-electron wavefunction centroid and 푐푖,푎 representing the weight of the one-electron
excitation. Λ takes values between 0 and 1, with 0 corresponding to no overlap and 1 to
complete overlap.[45] For all mono-triarylboranes, these values are smaller than 0.6, thus
suggesting localized orbitals and CT character of the transition.[45] Hence, the photophysical
properties of 4a, 5c, and 6c are very similar, as these compounds are electronically similar
due to the conversion of the electron-donating amine to trimethylammonium groups upon
methylation. The molar extinction coefficient increases almost linearly with the number of
dimethylamino groups in case of the neutral triarylboranes. For 5c and 6c, these values are
lower and independent of the number of trimethylammonium groups.
The properties of the neutral bis-triarylboranes are very similar when dissolved in the same
solvent, except for the molar extinction coefficients, which increase non-linearly with the
number of amino groups. The emission spectra display charge-transfer character for Neut1-
Neut3, but not for Neut0 as the latter bears no amino groups. DFT calculations show the
HOMOs and LUMOs of the neutral bis-triarylboranes to be mainly localized at the bithiophene
bridge. Thus, transitions are of π-π* nature. This is supported by TD-DFT calculations and Λ
values resulting therefrom, which are between 0.66 and 0.72 thus reflecting delocalized
orbitals over the bridge and locally excited transitions.[45] In contrast, for the cationic bis-
triarylboranes, Λ values are between 0.30 and 0.53 reflecting charge-transfer character of the
transitions, and rather localized orbitals. Thus, charge transfer character of the transition of
lowest energy is lost upon methylation, which has been shown previously for very similar
compounds.[26] The orbitals obtained from DFT calculations suggest that the HOMOs of the
cationic bis-triarylboranes are mainly localized at the bithiophene bridge as it is for the neutral
compounds. However, the LUMOs of the cationic compounds are mainly localized at the most
electron deficient boron center (5c for Cat1+; 6c for Cat2+, Cat3+). Therefore, the transition for
most of the cationic bis-triarylboranes is not of π-π* nature but of π-B(p) nature. In the case of
Cat(i)2+ and Cat4+,[26] the LUMO is delocalized from one boron center to the other over the
bithiophene bridge. As a result, the transition is of π-π* nature. Due to the different origins of
the main transitions of the cationic bis-triarylboranes, the photophysical properties are not the
same for these compounds as they depend to some extent on the number and distribution of
the trimethylammonium groups. For example, in acetonitrile, the absorption maxima shift
bathochromically with increasing number of trimethylammonium groups. The emission maxima
shift bathochromically with increasing dipole moment of the cationic bis-triarylboranes in the
order Cat1+≈Cat(i)2+<<Cat3+<Cat2+. Note that the dipole moment of charged compounds is
defined relative to its origin. Thus, it is not an observable quantity. However, the term dipole
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moment will be used herein to describe the distribution of the electron density over the
molecules for convenience. The molar extinction coefficient does not change significantly with
increasing number of trimethylammonium groups. Similar, but less pronounced trends, are
found in 1% MeCN in water and dilutions of DMSO stock solutions with sodium cacodylate
buffer solutions (see Supporting Information).
In summary, the absorption and emission maxima are solvent dependent as expected for
donor-acceptor (D–A) compounds in which the boron center is the electron-acceptor. The
electron-donor for the neutral compounds is the dimethylamine moiety. In the case of 4a and
5c, the mesityl moiety acts as an electron-donor, whereas for 6c, only the 2,6-dimethylphenyl
motif serves as the donor, as supported by DFT and TD-DFT calculations. Upon methylation
of the neutral bis-triarylboranes, the bithiophene becomes the electron-donating motif. The
absorption and emission maxima of the cationic bis-triarylboranes Cat1+, Cat2+, Cat(i)2+, and
Cat3+ are bathochromically shifted compared to their respective mono-triarylboranes.
However, no additive effect from combining the triarylboranes with the bithiophene bridge was
observed for the molar extinction coefficient. Thus, it can be concluded that the photophysical
properties mainly result from the interaction of the most electron-poor boron center with the
most electron-rich donating moiety for all compounds investigated.
Figure 4: Absorption (solid lines) and emission (dotted lines; excitation at 흀풎풂풙
풂풃풔 ) spectra of A)
the neutral bis-triarylboranes Neut0-Neut3 and B) the neutral triarylboranes 4a-6a in hexane.
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Figure 5: Absorption (solid lines) and emission (dotted lines; excitation at 흀풎풂풙
풂풃풔 ) spectra of A)
the cationic bis-triarylboranes Cat1+-Cat3+ and B) the cationic triarylboranes 5c and 6c in
acetonitrile.
Figure 6: Absorption (solid lines) and emission (dotted lines; excitation at 흀풎풂풙
풂풃풔 ) spectra of A)
the cationic bis-triarylboranes Cat1+-Cat3+ and B) the cationic triarylboranes 5c and 6c in 1%
MeCN in water.
Electrochemistry
Electrochemical measurements were performed in the respective solvent (THF or acetonitrile)
with [nBu4N][PF6] as the electrolyte and a scan rate of 250 mV s-1. Each measurement is
referenced to the ferrocene/ferrocenium (Fc/Fc+) couple. The redox potentials of uncharged
and cationic triarylboranes 4a-6a, 5c, and 6c were examined as well as of the neutral and
cationic bithiophene bis-triarylboranes Neut0-4 and Cat1+-Cat2+. Potentials of the neutral
compounds were measured in THF, and those of the cationic compounds in MeCN. All cyclic
voltammograms are shown in the Supporting Information (Figures S82-S99) together with an
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extended discussion thereof. The electrochemical data are summarized in Table 2. No
oxidation potentials are expected for 4a, 5c and 6c, within the electrochemical window of the
solvent used. In the case of 5c and 6c, the presence of ca. 0.5% starting material was
confirmed by 1H NMR spectra. Thus, the minor oxidation waves observed for both compounds
can be attributed to the presence of non- or partially methylated starting materials.
Table 2: Reduction and oxidation potentials for neutral compounds 4a-6a, Neut0-Neut4
determined in THF, and cationic compounds 5c, 6c, Cat1+-Cat4+ determined in MeCN.
E1/2 (red1)
[V]
E1/2 (red2)
[V]
E1/2 (red3)
[V]
E1/2 (ox1)
[V]
E1/2 (ox2)
[V]
4a
5a
−2.67
−2.78
0.34
0.28
0.51
6a
−2.86
(irrev.)
5c
6c
−2.24
−2.02
Neut0
Neut1
Neut2
Neut(i)2
−2.31
−2.34
−2.33
−2.39
−2.95 (irrev.)
−3.03 (irrev.)
−2.96 (irrev.)
−3.09 (irrev.)
0.39 (irrev.)
0.32 (irrev.)
0.34 (irrev.)
0.49
Neut3
Neut4
−2.40
−2.40
−3.11 (irrev.)
−3.03 (irrev.)
0.40 (irrev.)
0.32 (irrev.)
(irrev.)
Cat1+
−2.13
−1.94
−2.07
0.64
0.66
0.66
0.98
0.99
0.98
Cat2+
−2.18
Cat(i)2+
−2.76 (irrev.)
−2.80
Cat3+
Cat4+
−1.94
−1.93
−2.07
0.67
0.68
1.01
1.10
(irrev.)
−2.78 (irrev.)
For the triarylboranes 4a, 5a, 6a, 5c, and 6c one partially reversible 1e- reduction potential for
the boron center and one 1e- oxidation potential per amino group was found. The reduction
potentials decrease with increasing electron density at the boron center which increases in the
order 6c<5c<4a<5a<6a. The electrochemical data are in good agreement with values reported
by Gabbaï and coworkers for very similar trimethylammonium-substituted triarylboranes.
However, the trend that each trimethylammonium group leads to a decrease of 0.26 V in
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MeCN[35] was only observed for the addition of the second cationic group (5c vs. 6c) but not
for the addition of the first one (4a vs. 5c).
Figure 7: Cyclic voltammograms of Neut0-Neut4 in THF.
For all neutral bis-triarylboranes in THF (Figure 7), one partially reversible reduction at
ca. -2.35 V was observed, which consists of two simultaneous 1e- reductions (Table S20)
suggesting negligible delocalization between the two boron atoms. Furthermore, a second
(irreversible) 1e- reduction at ca. -3.05 V was observed. For all dimethylamino-substituted bis-
triarylboranes, an irreversible oxidation occurred at ca. 0.35 V; only Neut3 has an additional
irreversible oxidation at 0.49 V.
Figure 8: Cyclic voltammograms of Cat1+-Cat4+ in MeCN.
For all cationic bis-triarylboranes (Figure 8), two fully reversible 1e- oxidation processes were
observed at ca. 0.65 V and 1.0 V, respectively, resulting from the bithiophene bridge. However,
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for Cat1+, Cat2+, and Cat3+, two partially reversible 1e- reductions were observed for each
compound whereas Cat(i)2+ and Cat4+ display only one reduction potential. These result from
two simultaneous 1e- reductions (Table S20), as the two boron centers in the latter are the
same on both sides of the molecule. It was not possible to improve the resolution of the quasi-
reversible reduction waves of Cat1+ by pulse voltammetry experiments (Figure S94D).
HOMO-LUMO Gaps
HOMO-LUMO gaps (ΔEcalc, ΔEopt, ΔECV) were obtained from DFT calculations and
experimental values. The absorption spectra were used to determine the optical bandgap ΔEopt
and ΔECV was calculated from the HOMO and LUMO energies according to a literature
procedure (see Supporting Information).[46-49] The results are summarized in Table 3. ΔEopt
does not change significantly within a series of neutral or cationic bis-triarylboranes but
decreases by ca. 0.5 eV upon introduction of the strong dimethylamino electron donor in the
mono-triarylboranes, due to destabilization of the HOMO more than the LUMO, as supported
by DFT calculations. The values of ΔEopt and ΔECV are of the same magnitude and display the
same trends. For the cationic compounds, only the cationic moiety was used in the
calculations, so ion-ion-interactions were not considered.
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Table 3: Comparison of HOMO-LUMO gap energies obtained from DFT calculations (ΔEcalc),
absorption (ΔEopt) and CV (ΔECV) measurements.
ΔEcalc
ΔEopt
ΔECV
[eV]
[eV]
[eV]
4a
5a
6a
4.34
3.66
3.63
3.49
2.92
2.92
3.12
3.14
5c
6c
3.87
3.72
3.42
3.47
Neut0
Neut1
Neut2
Neut(i)2
Neut3
Neut4
3.01
2.99
2.99
3.07
3.07
3.10
2.65
2.65
2.67
2.66
2.66
2.73
2.65
2.73
2.80
2.72
2.77
2.60
2.73
2.61
2.60
[a]
-
Cat1+
Cat2+
Cat(i)2+
Cat3+
Cat4+
2.09
1.32
2.58
1.83
2.62
2.56
2.58
2.56
[b]
-
2.55 [c]
[a] No data were reported for this compound.[26] [b] A different basis set was used.[26] [c]
Calculated from reported absorption spectra.[26]
Singlet Oxygen Sensitization
The ability to sensitize singlet oxygen in O2-saturated acetonitrile solutions was investigated
for the triarylboranes 4a, 6c, and 5c as well as for the charged bis-triarylboranes Cat1+-Cat4+
1
by monitoring the weak O2 phosphorescence emission at 1275 nm vs. that obtained from a
solution of the known sensitizer perinaphthenone[50] with an estimated error of ±0.1.
Triarylboranes 4a, 6c, and 5c sensitize 1O2 with efficiencies of 0.3, 0.5, and 0.6, respectively,
(Figures S79) which increases with increasing number of trimethylammonium substituents
(Table S17). The corresponding fluorescence quantum yields in acetonitrile decrease in the
same order from 0.34 to 0.11 and 0.10. Thus, the sum of singlet oxygen efficiency ΦΔ and
fluorescence quantum Φf yield for these compounds is ca. 0.6 (Table S17).
The charged bis-triarylboranes sensitize 1O2 with efficiencies of 0.6, 0.8, 0.7, 0.6, and 0.6 for
Cat1+, Cat2+, Cat(i)2+, Cat3+, and Cat4+, respectively (Figure 9, Table 4). The values for Cat2+
and Cat3+ may be overestimated due to tailing of the compound’s emission into the NIR region
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of the singlet oxygen emission (Figure S80), which is the reason for the raised baseline
between 1230 and 1260 nm in Figures 9B and 9D. Thus, ΦΔ of ca. 0.6 is estimated for all
charged bis-triarylboranes.
Figure 9: Emission spectra from singlet oxygen sensitization of A) Cat1+, B) Cat2+, C) Cat(i)2+,
D) Cat3+, and E) Cat4+ relative to perinaphthenone in acetonitrile.
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Table 4: Summary of singlet oxygen sensitization of Cat1+-Cat4+ relative to perinaphthenone in
acetonitrile.
Compound
Cat1+
ΦΔ
Φf
0.6
0.34
0.48
0.35
0.46
0.41[26]
Cat2+
0.8 [a]
Cat(i)2+
Cat3+
0.7
0.6 [a]
0.6
Cat4+
[a] Values obtained from singlet oxygen sensitization measurements overestimate the actual
value due to tailing of the emission spectrum of the compound between 1230 nm and 1330 nm.
The S1 excited states of Cat1+-Cat4+ thus decay ca. 40% by fluorescence and ca. 60% by
1
intersystem crossing to the triplet state, which very efficiently sensitizes O2. Attaching two
triarylboranes to a bithiophene motif leads to a more efficient energy transfer to triplet oxygen
than for the mono-triarylboranes. Thus, interactions with DNA, RNA and DNApore and cell
imaging studies were only examined for the cationic bis-triarylboranes.
Studies in Buffered Solutions
Interactions of the cationic bis-triarylboranes Cat1+-Cat3+ and naturally occurring calf thymus
DNA (ctDNA; typical B-helical structure with a balanced ratio of GC-(48%) and AT-(52%) base
pairs), double stranded (ds) poly rA – poly rU (pApU; A-helical structure characterized by its
major groove being available for binding of bulky small molecules) and DNApore (Figure 10A)
were investigated in aqueous buffered solutions. Experimental details and all spectra are given
in the Supporting Information. DNApore was chosen as Cat1+-Cat4+ should fit in the cavity of
the structure (Figure 10B).
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Figure 10: A) Schematic presentation of DNApore. The composition of oligonucleotides P1-P6
is given in Table S21. B) Schematic depiction showing that Cat4+ and the analogues Cat1+-
Cat3+ (width ca. 1.2 nm) fit into the cavity of the DNApore (width ca. 2 nm).
The stability of Cat1+-Cat3+ in buffered solutions was confirmed by temperature dependent
absorption and emission spectra (Figures S101-S108).
Changes in the fluorescence of Cat2+-Cat3+ upon addition of increasing amounts of DNA, or
RNA were studied at pH 7 and/or pH 8. In combination with the results of thermal denaturation
(Tm) and circular dichroism (CD) experiments of the polynucleotides, which are known to be
unique for the respective molecule, conclusions about the mode of interaction (intercalation,
major or minor groove binding, or external binding) can be drawn.[51-54] For example, increasing
denaturation temperatures of polynucleotides in the presence of small molecules, as observed
for our bis-triarylboranes Cat2+-Cat3+, suggest non-covalent binding of the latter, characterized
by the type of interactions which can increase the stability of DNA/RNA (e. g. intercalation, H-
bonding, and/or cation-anion interactions). However, the emission of Cat1+ showed negligible
changes in the presence of any polynucleotide at any pH (Figure 11, Figure 13, Table 6). In
some cases, precipitation of Cat1+ was observed. In addition, the denaturation temperatures
(Figure 12, Figure 109, Figure S125, Table 5) or the CD spectra (Figures S121, Figure S141,
Figure 14) of the polynucleotides were not influenced by the presence of the mono-cation. This
behavior is similar to that of neutral analogues investigated previously.[37] Thus, one positive
charge is not sufficient for the bis-triarylboranes to provide efficient interaction with
polynucleotides.
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Interaction of Cat2+-Cat3+ with ctDNA, and pApU at pH 7
In the presence of Cat2+, Cat(i)2+, and Cat3+, Tm is increased compared to pure ctDNA or pApU
at pH 7. Thus, these three compounds efficiently bind to ctDNA and pApU and stabilize these
polynucleotides (Table 5; Figure S110-S112). However, the stabilizing effects of Cat2+,
Cat(i)2+, and Cat3+ are lower than that reported for Cat4+.[38]
Table 5: ∆Tm values [a] (°C) of polynucleotides upon addition of Cat1+-Cat4+ at pH indicated.
r [b] ctDNA pH 7 polyAU pH 7 ctDNA pH 8 DNA-pore pH 8
0.1
0.2
0
0
0
0
-
-
-
-
0/0
-
Cat1+
0.25
0.3
-
-
0.7
0
0
-
0.1
0
0
-
-
0.2
5.9
-
2.0
-
-
-
Cat2+
3.2/1.2 [c]
-
0.25
0.3
8.7
11.8
1.7
2.9
-
11.1
1.3
13.0
-
-
0.1
-
-
0.2
-
-
Cat(i)2+
0.25
0.3
5.5
1.2/3.3 [c]
3.8
3.6
3.5
-
13.0
0.6
6.0
-
-
-
0.1
-
-
0.2
-
-
Cat3+
Cat4+
0.25
0.3
3.4
1.4/6.6 [c]
3.2
7.3[38]
-
5.5
9.5[38]
-
-
-
-
-
-
0.1
0.25
8.0
[a] Error of ∆Tm = ±0.5 °C; [b] r = [compound]/[polynucleotide]; [c] Biphasic transitions.
Upon addition of ctDNA or pApU, the emission of Cat2+-Cat3+ increases depending on the
structure of the polynucleotide and the number of charges. Using the Scatchard equation[55]
and the McGhee, von Hippel formalism,[56] binding constants were obtained, which are very
similar for all compounds and polynucleotides investigated (Table 6). Such non-selective
affinity with respect to the secondary structure of the ds-polynucleotide and the composition of
its respective base pairs was reported previously for structurally related compounds,[36-38] and
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suggests that Cat2+, Cat(i)2+, and Cat3+ bind in the minor groove of DNA or the major groove
of RNA.
1.6
1.4
1.2
Cat3+
1.0
1.8
1.5
Cat(i)2+
1.2
1.4
1.2
Cat2+
1.0
ctDNA pH 7
0.9
0.6
0.3
pApU
Cat1+
1.0x10-5
2.0x10-6
4.0x10-6
6.0x10-6
8.0x10-6
0.0
c (polynucleotide) [mol L-1]
Figure 11: Normalized fluorimetric titration of Cat1+ (c = 110-7 mol L-1; λexc = 414 nm), Cat2+
(c = 110-7 mol L-1; λexc= 422 nm), Cat(i)2+ (c = 110-7 mol L-1; λexc = 417 nm), Cat3+ (c =
110-7 mol L-1; λexc = 421 nm) with ctDNA and pApU at pH 7.
Table 6: Binding constants[a] (log Ks) of Cat1+, Cat2+, Cat(i)2+, Cat3+, and Cat4+ with
polynucleotides calculated from fluorimetric titrations according to literature procedures.[55-56]
Cat1+
Cat2+
Cat(i)2+
Cat3+
Cat4+
7.0 [c]
~7 [c]
-
[b]
ctDNA
pApU
6.4
6.9
6.5
pH 7
pH 8
[b]
7.0
7.8
7.5
[b]
ctDNA
-
6.8
5.1
[b]
DNApore
7.5
6.7
7.5
7.8
[a] Analyses of titration data by means of the Scatchard equation[55] with von Hippel
formalism[56] gave values of the ratio r = [bound compound]/[polynucleotide] = 0.2 - 0.3; for
easier comparison, all log Ks values were re-calculated for fixed r = 0.25 (ds-polynucleotides).
Correlation coefficients were > 0.99 for all calculated Ks values.
[b] Negligible emission change. [c] Values reported previously.[38]
Upon addition of Cat2+, Cat(i)2+, and Cat3+ to ctDNA or pApU, a small decrease of the intensity
of the CD spectra between 230 nm and 300 nm (Figures S122-S124) was observed. This can
be attributed to an unwinding of the double helix of the polynucleotides upon insertion of the
bis-triarylboranes. In contrast to the previously reported tetra-cationic compound Cat4+,[38] no
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ICD bands were observed at 400-500 nm. Thus, Cat2+, Cat(i)2+, and Cat3+ might be non-
uniformly oriented with respect to the chiral axis of the polynucleotide, but it was not possible
to derive direct structural information. Nevertheless, in analogy with our previous binding
studies,[36-38] groove binding of Cat2+, Cat(i)2+, and Cat3+ can be assumed.
Interaction of Cat1+-Cat3+ with ctDNA, and DNApore at pH 8
Similarly, as described above for sodium cacodylate solutions, thermal denaturation
temperatures of ctDNA and DNApore in Tris-buffer at pH 8 (15 mmol L-1 Tris-HCl,
300 mmol L-1 KCl) were increased in the presence of Cat2+, Cat(i)2+, Cat3+, and Cat4+ (Table
5, Figure 12). The denaturation curve of DNApore is slightly biphasic, most likely due to the
presence of two different conformers. Both transitions were stabilized by interaction with the
bis-triarylboranes, with the effect on the melting temperature of DNApore being proportional to
the number of cationic charges in the order Cat1+<Cat2+≈Cat(i)2+<Cat3+<Cat4+. The unusual
drop of absorbance observed for Cat(i)2+ upon addition of DNA is likely due to precipitation of
a Cat2+/DNA complex upon complete unwinding.
Cat4+
Cat3+
Cat(i)2+
Cat2+
Cat1+
DNApore
40
50
60
70
80
T [°C]
Figure 12: Thermal denaturation curves of DNApore (c = 2  10-5 M at r[dye]/[DNApore] = 0.25) at
pH 8.0 (15 mM Tris-HCl, 300 mM KCl) upon addition of Cat1+-Cat4+. Error in Tm values:
0.5 C.
In contrast to the emission increase of Cat2+ and Cat(i)2+ upon addition of ctDNA at pH 7, at
pH 8, the emission of these compounds decreases (Figure 13A), while the emission of Cat3+
increases in both solutions. However, upon addition of DNApore, the emission of Cat2+,
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Cat(i)2+, Cat3+, and Cat4+ decreased (Figure 13B). Binding constants are very similar for the
different bis-triarylboranes at pH 8 (Table 6). However, upon changing the pH from 7 to 8, the
binding constants of ctDNA are lower, especially for Cat3+. In contrast, for all compounds,
binding constants to the DNApore are larger than those for ctDNA at pH 8. Thus, the
chromophores bind more strongly to DNApore than to ctDNA.
Figure 13: A) Normalized fluorimetric titration of Cat2+ (c = 5  10-7 M; λexc = 422 nm), Cat(i)2+
(c = 5  10-7 M; λexc = 417 nm), and Cat3+ (c = 5  10-7 M; λexc = 421 nm) with ctDNA (pH 8 in
15 mM Tris-HCl, 300 mM KCl) and of B) Cat1+ (c = 5  10-7 M; λexc = 414 nm), Cat2+ (c =
5  10-7 M; λexc = 422 nm), Cat(i)2+ (c = 5  10-7 M; λexc = 417 nm), Cat3+ (c = 5  10-7 M; λexc
=
421 nm), and Cat4+ (c = 4  10-7 M; λexc = 425 nm) with DNApore (pH 8 in 15 mM Tris-HCl,
300 mM KCl).
Addition of Cat1+ to ctDNA at pH 8 resulted in changes of the CD spectrum at <300 nm which
were not observed at pH 7. Thus, a change of the interaction between this bis-triarylborane
and ctDNA takes place upon increasing the pH from 7 to 8. For Cat(i)2+ and Cat3+, a negative
band at ca. 425 nm was observed, indicating a perpendicular orientation of the transition
moments to the chiral axis of ctDNA at pH 8.
The chiral axis of the DNApore cannot be directly correlated to the usually well-defined chiral
axes of typical ds-DNAs due to the six intertwined 50-mer oligonucleotides and, consequently,
the tilted angles of the double helices with respect to the central axis of the DNApore. Thus,
the following ICD signal assignments do not necessarily match those described in the
literature.[54, 57-58] However, due to the similar structures of the bis-triarylboranes studied herein,
and the same experimental procedures applied, the results of these measurements can be
used for comparisons within this series of compounds. For Cat2+, weak, negative ICD bands
were observed which is characteristic for a perpendicular orientation of the transition moment
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of the chromophore with respect to the chiral axis of the DNApore. In contrast to the CD
measurements at pH 7, which did not change at wavelengths >300 nm, addition of Cat(i)2+ to
DNApore at pH 8 induced strong ICD bands at 400-450 nm (Figure 14). These changes fit the
absorption spectra of the corresponding bis-triarylboranes perfectly, which shows that the ICD
signals are derived from our chromophores. The positive ICD bands suggest a tilted orientation
by ca. 60° to the chiral axis of the DNApore. For Cat3+, complete disintegration of the CD bands
of DNApore suggests strong changes of the secondary structure of the DNApore upon binding.
DNApore
r=0.1
r=0.3
Cat4+
1
r=0.5
0
-1
DNApore
r = 0.1
r = 0.3
r = 0.5
Cat3+
1
0
-1
DNApore
r=0.1
r=0.3
r=0.5
Cat(i)2+
1
0
-1
DNApore
r=0.3
r=0.5
r=1
Cat2+
Cat1+
1
0
-1
DNApore
r=0.1
r=0.3
1
0
-1
r=0.5
250
300
350
400
450
500
l [nm]
Figure 14: CD titration of DNApore (c = 5  10-5 M) with Cat1+-Cat4+ at molar ratios
r[dye]/[polynucleotide] = 0.1 - 1 in 15 mM Tris-HCl, 300 mM KCl solutions at pH 8.
Thus, Cat2+, Cat(i)2+, Cat3+, and Cat4+ interact with all polynucleotides investigated. Although
there are some similarities, e. g. interaction with DNApore leads to emission decrease of the
compounds, the number and distribution of charges affects the interaction of bis-triarylboranes
with different polynucleotides. In particular, the induced CD bands of various derivatives
(Figure 14) can be used for fine sensing of the DNApore shape. Thus, Cat2+, Cat(i)2+, Cat3+,
and Cat4+ are useful probes for further studies of the DNApore structure and interactions with
novel ligands.
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Cell Toxicity and Imaging Studies
Studies were conducted on the cationic bis-triarylboranes to see whether they penetrate cell
membranes, stain specific organelles therein, or if they have anti-proliferative effects to A549
and WI38 cells. Confocal microscopy studies showed that the mono-cationic compound Cat1+
does not penetrate the cells, in contrast to Cat2+-Cat3+. Thus, this compound was not further
investigated in biological studies. For Cat2+, only preliminary results were obtained, suggesting
localization at the cellular membrane and certain compartments in the cell nucleus. However,
due to the low stability of this compound in Dulbecco Modified Eagle’s Medium (DMEM)
containing 10% fetal bovine serum (FBS), only the staining patterns of Cat(i)2+ and Cat3+ were
further investigated.
The cytotoxicity of Cat(i)2+ and Cat3+ was tested using the MTT assay against human lung
carcinoma (A549) and normal lung (WI38) cell lines (Figure S146). In the dark, Cat(i)2+ is not
cytotoxic in all concentrations tested (0.1 µM, 1 µM, 10µM). However, when irradiating the
incubated cells of both cell lines in the presence of 1 µM Cat(i)2+ at 400-700 nm for 5 min, the
viability of the cells was reduced drastically with increasing concentration of Cat(i)2+.
In the dark, Cat3+ is not cytotoxic to both cell lines in concentrations of 0.1 µM and 1 µM.
However, at 10 µM concentrations, Cat3+ is cytotoxic to A549 cells. This toxicity is even more
pronounced for WI38 cells. For both cell lines, the toxicity increases under irradiation (400-
700 nm) at 1 µM and 10 µM concentrations.
The increased toxicity of these compounds when irradiated with UV light results most likely
from their ability to sensitize singlet oxygen (vide supra) as the latter is a highly reactive
compound leading to cell death.[59] This assumption was confirmed by monitoring cells
incubated with Cat2+, Cat(i)2+, or Cat3+ by confocal microscopy while irradiating at 405 nm at
the full power of the light source (LED). Within 2 min, significant changes in the cell morphology
were observed (cell blebbing, cell contraction; Figures S148, S150, S152) suggesting strong
cellular damage. Simultaneously, the emission of the dyes bleached rapidly within 30 s to 1 min
(Figures S147, S149, S151). The stability of the emission increases with the number of cationic
charges. After the emission is quenched completely, no additional cell damage was observed.
Control experiments on untreated cells did not show any sensitivity to light exposure. This is
in good agreement with the cell toxicity of Cat(i)2+ and Cat3+ under UV irradiation found during
the MTT assay.
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Figure 15: Intracellular localization of Cat(i)2+ and Cat3+ in A549 cells. Co-localization with
lysosomes (LAMP1) was monitored by confocal microscopy. Cells were treated with the bis-
triarylboranes at concentrations of 10 µM for 90 min at 37 °C. Nuclei were stained with DAPI.
Co-localization was assessed by determining the Pearson correlation coefficient.
However, when incubating A549 cells for 90 min (37 °C, 5% CO2) with Cat(i)2+, or Cat3+ and
exciting with light (405 nm; LED source), the cells show strong green emission due to the
accumulation of the compounds in some organelles in the cytoplasm. The staining patterns of
both compounds were further analyzed by co-localization experiments (Figure 15; Figures
S153-S154). The degree of co-localization was quantified using the Pearson correlation
coefficient (Rr)[60] and corrected by replicate-based noise correction correlation.[61] For Cat(i)2+
(0.54) and Cat3+ (0.46), the best overlap was obtained for co-localization with LAMP1, a
staining antibody selective for lysosomes. However, the staining pattern of the bis-
triarylboranes suggests further interactions with early endosomes and golgi apparati (Figure
S153-S154; Tables S23-S24). Thus, these new compounds are not specific for one organelle
but distributed between the organelles mentioned with some selectivity for lysosomes.
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Conclusions
The syntheses of selectively trimethylammonium-substituted, bithiophene-bridged, bis-
triarylborane chromophores (Cat1+, Cat2+, Cat(i)2+, Cat3+) and their neutral precursors (Neut1,
Neut2, Neut(i)2, Neut3) are described. The compounds were isolated in good yields and the
influence of different numbers and locations of charges on electrochemical and photophysical
properties and cellular imaging was investigated. Photophysical investigations reveal expected
behavior for D–A compounds wherein the boron center is the electron-acceptor in all
compounds with differing electron-donating moieties, as supported by DFT and TD-DFT
calculations. For the cationic bis-triarylboranes, an increasing hypsochromic shift of the
emission spectra with increasing dipole moment was observed in acetonitrile. CV
measurements of the neutral and cationic bis-triarylboranes show that the π-conjugation, and
therefore the communication between the boron centers, is improved upon methylation of the
dimethylamino groups. Efficient singlet oxygen sensitization was demonstrated for the cationic
bis-triarylboranes Cat1+, Cat2+, Cat(i)2+, and Cat3+. For comparison with the properties of the
bis-triarylboranes, selected properties were also investigated for the mono-triarylboranes 4a-
6a, 5c, and 6c.
Due to the moderate solubilities of Cat1+, Cat2+, Cat(i)2+, and Cat3+ in pure water, photophysical
investigations and biological studies were performed in aqueous environments containing less
than 1% acetonitrile and 0.1% DMSO, respectively. Studies with DNA and RNA in buffered
solutions show negligible binding for the mono-cationic compound Cat1+ but strong binding for
Cat2+, Cat(i)2+, and Cat3+ within the minor groove of DNA or the major groove of RNA exhibiting
a strong fluorescence increase under physiological conditions. The compounds also bind
strongly to DNApore, most likely inside the pore. Thus, they could potentially act as a “stopper”
of ubiquitous material flow, which DNApore enables when inserted in, e. g., bacterial
membranes.[62] Due to their induced CD response, Cat2+, Cat(i)2+, and Cat3+ might be useful
to study the fine structure of DNApore and the interaction with other ligands. Studies with A549
and WI38 cells show all compounds to be cell permeable except for the mono-cation Cat1+.
Under intense irradiation (400-700 nm), the emission of Cat2+, Cat(i)2+, and Cat3+ bleaches
rapidly with simultaneous changes of cell morphology, most likely due to in situ formation of
singlet oxygen. Thus, the cytotoxicity of the compounds is strongly increased upon irradiation
with light (400-700 nm) as demonstrated using the MTT assay. Without irradiation, Cat(i)2+ and
Cat3+ are not cytotoxic up to concentrations of 10 and 1 µM, respectively, for both cell lines.
Co-localization experiments of both compounds in A549 cells demonstrate the lysosome as
the main but not only accumulation site, since Cat(i)2+ and Cat3+ also localize in other
organelles. Thus, the cationic bis-triarylboranes are promising theranostic agents[63] as they
combine the potential for photodynamic therapy due to their relatively high singlet-oxygen
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sensitizing efficiency with high fluorescence quantum yields for simultaneous imaging of the
location of the chromophores in cells or tissues. Furthermore, this study demonstrates that the
number of cationic trimethylammonium groups and their distribution influences all properties
investigated, including intracellular localization. Thus, when designing new bis-triarylborane
chromophores for biological applications, the number and distribution of charges should be
considered as well as the bridging unit.
Crystal Stuctures
“Deposition Number(s)
<urlhref="https://www.ccdc.cam.ac.uk/services/structures?id=doi:10.1002/chem.202102308"
> 2072401 (for 4a), 2072402 (for 4b)</url> contain(s) the supplementary crystallographic
data for this paper. These data are provided free of charge by the joint Cambridge
Crystallographic Data Centre and Fachinformationszentrum Karlsruhe <url href="
http://www.ccdc.cam.ac.uk/structures ">Access Structures service</url>.”
Acknowledgements
We thank the State of Bavaria Ministry of Science, Research, and the Arts, the Julius-
Maximilians-Universität Würzburg, and the Deutsche Forschungsgemeinschaft (DFG,
GRK2112) for financial support. Financial support of the Croatian Science Foundation (project
IP-2018-01-5475), and the bilateral project of the Deutscher Akademischer Austauschdienst
(DAAD) are gratefully acknowledged. We thank Prof. Dr. C. Lambert for providing access to
his FLS980 luminescence spectrophotometer for measurements of static fluorescence
lifetimes ≤0.5 ns.
Keywords boron; bioimaging; luminescence; singlet oxygen; nucleic acid.
Conflict of Interest
No conflicts of interest to declare.
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