Tetracationic Bis-Triarylborane 1,3-Butadiyne as a Combined Fluorimetric and Raman Probe for Simultaneous and Selective Sensing of Various DNA, RNA, and Proteins

Article abstract of DOI:10.1002/chem.201905328

A bis-triarylborane tetracation (4-Ar₂B-3,5-Me₂C₆H₂)-C≡C?C≡C-(3,5-Me₂C₆H₂-4-BAr₂ [Ar=(2,6-Me₂-4-NMe₃-C₆H₂)⁺] (2⁴⁺) shows distinctly different behaviour in its fluorimetric response than that of our recently published bis-triarylborane 5-(4-Ar₂B-3,5-Me₂C₆H₂)-2,2′-(C₄H₂S)₂–5′-(3,5-Me₂C₆H₂-4-BAr₂) (3⁴⁺). Single-crystal X-ray diffraction data on the neutral bis-triarylborane precursor 2 N confirm its rod-like dumbbell structure, which is shown to be important for DNA/RNA targeting and also for BSA protein binding. Fluorimetric titrations with DNA/RNA/BSA revealed the very strong affinity of 2⁴⁺ and indicated the importance of the properties of the linker connecting the two triarylboranes. Using the butadiyne rather than a bithiophene linker resulted in an opposite emission effect (quenching vs. enhancement), and 2⁴⁺ bound to BSA 100 times stronger than 3⁴⁺. Moreover, 2⁴⁺ interacted strongly with ss-RNA, and circular dichroism (CD) results suggest ss-RNA chain-wrapping around the rod-like bis-triarylborane dumbbell structure like a thread around a spindle, a very unusual mode of binding of ss-RNA with small molecules. Furthermore, 2⁴⁺ yielded strong Raman/SERS signals, allowing DNA or protein detection at ca. 10 nm concentrations. The above observations, combined with low cytotoxicity, efficient human cell uptake and organelle-selective accumulation make such compounds intriguing novel lead structures for bio-oriented, dual fluorescence/Raman-based applications.

Full text of DOI:10.1002/chem.201905328

A Journal of
Accepted Article
Title: Tetracationic bis-triarylborane 1,3-butadiyne as a combined
fluorimetric and Raman probe for simultaneous and selective
sensing of various DNA, RNA and proteins
Authors: Hashem Amini, Željka Ban, Matthias Ferger, Sabine
Lorenzen, Florian Rauch, Alexandra Friedrich, Ivo Crnolatac,
Adriana Kenđel, Snežana Miljanić, Ivo Piantanida, 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.201905328
Link to VoR: http://dx.doi.org/10.1002/chem.201905328
Supported by

10.1002/chem.201905328
Chemistry - A European Journal
Tetracationic bis-triarylborane 1,3-butadiyne as a combined fluorimetric and
Raman probe for simultaneous and selective sensing of various DNA, RNA and
proteins
Hashem Amini,[a] Željka Ban,[b] Matthias Ferger,[a] Sabine Lorenzen,[a] Florian Rauch,[a]
Alexandra Friedrich,[a] Ivo Crnolatac,[b] Adriana Kenđel,[c] Snežana Miljanić,[c] Ivo Piantanida,[b]*
and Todd B. Marder[a]*
[a] Institut für Anorganische Chemie, and Institute for Sustainable Chemistry & Catalysis with
Boron, Julius-Maximilians-Universität Würzburg, Würzburg, Germany
[b] Laboratory for Study of Interactions of Biomacromolecules, Division of Organic Chemistry
& Biochemistry, Ruđer Bošković Institute, Zagreb, Croatia
[c] Division of Analytical Chemistry, Department of Chemistry, Faculty of Science, University
of Zagreb, Zagreb, Croatia
Abstract
A new bis-triarylborane tetracation (4-Ar2B-3,5-Me2C6H2)-C≡C-C≡C-(3,5-Me2C6H2-4-
BAr2 [Ar = (2,6-Me2-4-NMe3-C6H2)+] (24+) shows distinctly different behaviour in its
fluorimetric response than that of our recently published bis-triarylborane 5-
(4-Ar2B-3,5-Me2C6H2)-2,2’-(C4H2S)2-5’-(3,5-Me2C6H2-4-BAr2) (34+). Single-crystal X-
ray diffraction data on the neutral bis-triarylborane precursor 2N confirm its rod-like
dumbbell structure, which is shown to be important for DNA/RNA targeting and also
for BSA protein binding. Fluorimetric titrations with DNA/RNA/BSA revealed the very
strong affinity of 24+ and indicated the importance of the properties of the linker
connecting the two triarylboranes. Using the butadiyne- rather than a bithiophene linker
resulted in an opposite emission effect (quenching vs enhancement), and 24+ bound to
BSA 100 times stronger than 34+. Moreover, 24+ interacted strongly with ss-RNA, and
circular dichroism (CD) results suggest ss-RNA chain-wrapping around the rod-like bis-
triarylborane dumbbell structure like a thread around a spindle, a very unusual mode
of binding of ss-RNA with small molecules. Furthermore, 24+ yielded strong
Raman/SERS signals, allowing DNA or protein detection at ca. 10 nM concentrations.
The above observations, combined with low cytotoxicity, efficient human cell uptake
and organelle-selective accumulation make such compounds intriguing novel lead
structures for bio-oriented, dual fluorescence/Raman-based applications.
1
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
Introduction
The use of fluorescent labels to image specific small molecules in live cells is an
essential tool in biology, medicinal chemistry, and many other related fields of
research. However, common fluorescent dyes also have many drawbacks. Due to the
considerable size and aromatic nature, and often also being H-bond donors/acceptors,
they may alter the biological activity, cellular localization, and dynamics of the targeted
bio-molecules.[1] Moreover, even the most advanced fluorescence microscopy
techniques are limited to a maximum simultaneous resolution of six different colors.[2]
This is additionally complicated[3] by unavoidable cross-talk in organic dyes, energy
transfer between quantum dots[4] and the limited number of suitable features for
straightforward decoding in rare-earth nanocrystals and metal nanoparticles.[5]
Therefore, further development in the increasingly complex bioimaging field essentially
requires new fluorophore structures for better bio-target diversification. Even more
useful would be new optical probes based on intrinsically different methods at least
approaching similar sensitivities of common fluorophores. Such an aim recently led to
the development of vibrational spectroscopy methods, particularly Raman bioimaging
microscopy, due to its compatibility with aqueous biological samples.[2d]
Phenyl end-capped polyynes have been employed as alkyne tags for Raman
visualization of mobile small molecules in cells[6] as well as for surface-enhanced
Raman spectroscopy (SERS) multiplex cellular imaging,[7] showing very promising
applications in supermultiplexed optical imaging and barcoding.[2d] However, these
polyyne-probes relied exclusively on their Raman response, and thus were not
combined with other sensing techniques.
Although fluorescence, under certain experimental conditions, interferes with Raman
measurements, by causing significant background signal,[8] for some chromophores it
is possible to obtain a satisfying compromise. In recent years, the combination of
Raman and fluorescence spectroscopy has emerged as a way to circumvent some of
the intrinsic problems of Raman spectroscopy (i.e. low signal strength, long acquisition
times) or to gain additional information on a given system.[9] Thus in the first multimodal
approaches, fluorescence was used as a fast macroscopic scanning method, prior to
a detailed Raman analysis, or to confirm conclusions made via Raman-based
imaging.[10] Raman spectroscopy, in combination with the use of fluorescently labelled
molecules or quantum dots, also proved to be an interesting approach in some cell
2
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
imaging applications.[11] In disease diagnostics, dual Raman and fluorescence
spectroscopy was used by several groups in a complimentary fashion to improve the
accuracy and sensitivity of the diagnosis.[12] Dual fluorescence and Raman
spectroscopy was successfully employed to investigate and visualize intracellular drug
delivery.[13] The design of small molecules with inherently strong Raman and
fluorescence responses is a rather novel approach,[14] which has already produced
much information on the location, environment[14b] or concentration[14a] of the small
molecule inside a cell.
Recently, triarylboranes have emerged as a structurally novel class of compounds
suitable for biological imaging applications.[15] Over the last decades, triarylboranes
have also found applications in many other fields, such as anion sensors, OLEDs and
non-linear optical materials.[16] Due to its vacant pz-orbital, the three-coordinate boron
in a triarylborane is a strong -acceptor. However, it is also Lewis-acidic and sensitive
to hydrolysis. Bulky substituents can stabilize three-coordinate boron against
decomposition by air and moisture, while maintaining its -acceptor strength.[17] Using
Gabbaï´s approach[18] we developed the water-soluble, water-stable and non-cytotoxic
tetracationic chromophore 34+ (Scheme 1) which was successfully utilized in live cell
imaging.[19] Further studies revealed 34+ to be a structurally novel DNA/RNA/protein
probe of high affinity and selective fluorimetric and chirooptic response.[20]
The question arose to which extent the chromophoric linker (bithiophene) is
responsible for the observed spectroscopic responses and affinity. In a search for
linkers which could also be non-conventional response probes, 1,3-butadiyne attracted
our attention for several reasons. It is a rigid, rod-like symmetrical linker, excellent for
precise orientation of terminal triarylborane cations (Scheme 1, 24+), and it is
completely inert, in terms of covalent and non-covalent interactions, to
DNA/RNA/protein. Even more importantly, it exhibits an intense Raman band with a
narrow linewidth in the Raman-silent spectral region (2250–2000 cm-1) convenient for
Raman-based probing in aqueous solutions. Additionally, the intensity of response can
be strongly increased by surface-enhanced Raman scattering (SERS) spectroscopy
and related techniques.
To address the above mentioned issues, we designed the new molecule 24+ by
replacing the phenyl end-capping of the shortest polyyne[2d] (1,3-butadiyne) with two
bis-triarylborane cations, acting as a fluorescence probe (Scheme 1). For comparison,
3
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
the dicationic monomer 12+ was also prepared (Scheme 1) as well as their non-charged
precursors 1N and 2N (Scheme 2).
Thus, novel molecule 24+, and studies of its interactions with DNA/RNA/proteins, will
address the potential for the development of innovative combined fluorophore-Raman
probes for the general purpose of non-covalent probing of DNA/RNA/proteins, and the
impact of the nature of the linker connecting two bis-triarylborane cations on the
fluorescent and chirooptic response upon binding to biomacromolecules.
Given the generally weak spontaneous Raman response of molecules in solution,
SERS spectroscopy was used to study the binding to biomolecules in aqueous media
mimicking the biological environment. The enhancement of the Raman scattering in
the vicinity of nanostructured metal surfaces arises from amplification of the
electromagnetic field upon excitation of the localized surface plasmon resonances
(LSPR) of the molecules when physisorbed on a surface, and from transfer of electrons
from the metal to the molecule and vice versa for chemisorbed molecules.[21] Even
though the charge transfer mechanism is considered to contribute to the overall
enhancement to a lesser extent than the electromagnetic one, the total SERS
enhancement factor with respect to the normal Raman signal, in most cases, is the
product of both mechanisms, reaching up to 10.[10] Owing to its high sensitivity and
ability to produce molecularly specific fingerprint spectra, SERS has been successfully
applied for the detection, quantification and biophysical characterization of a variety of
biomolecules.[22]
4
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
Scheme 1. The ethynyl-triarylborane monomer (12+), bis-triarylborane 1,3-butadiyne
(24+) and previously studied bithiophene-linked bis-triarylborane tetracation (34+).
Results and Discussion
Synthesis
The key precursor A, which was previously only accessible via Sandmeyer type
reactions in moderate yields,[23] was reproducibly prepared in 91% yield on a 73 mmol
scale using a new approach based on literature precedents.[24] This was achieved by
direct, regioselective, Ir-catalyzed borylation[25] of the C-H bond of 2-bromo-1,3-
dimethylbenzene at the 5-position. Without any purification, the borylated intermediate
was converted into A, using CuBr2 as a brominating agent. Compound A was then
cross-coupled with trimethylsilylacetylene, at the sterically less hindered bromine,
giving B. For the synthesis of triarylborane C, compound B was lithiated and bis[4-
(N,N-dimethylamino)-2,6-dimethylphenyl]fluoroborane[18] was added. Triarylborane C
was deprotected using KOH in MeOH/THF, to give the neutral terminal alkyne
compound 1N. The neutral 1,3-butadiyne compound 2N was formed by oxidative
homo-coupling of 1N in a Glaser type reaction, using I2 as an oxidant. The two neutral
compounds 1N and 2N were methylated at the amine groups using methyltriflate in
CH2Cl2. The two cationic species 12+ and 24+ were precipitated by addition of Et2O; 12+
was washed with CH2Cl2/Et2O, and 24+ with acetone/Et2O.
5
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
Scheme 2. Synthesis of the compounds 1N, 2N, 12+ and 24+. a) 1. B2pin2,
[Ir(COD)(OMe)]2, dtbpy, hexane, 80 °C; 2. CuBr2, MeOH/H2O, 90 °C, yield: 91% over
two steps; b) trimethylsilylacetylene, Pd(PPh3)2Cl2, CuI, NEt3, 80 °C, yield: 62%; c) t-
BuLi, bis[4-(N,N-dimethylamino)-2,6-dimethylphenyl]fluoroborane, THF, -78 °C to r.t.,
yield: 34%; d) KOH, MeOH/THF, r.t., yield: 97%; e) Pd(PPh3)2Cl2, CuI, I2, NEt3/THF,
r.t., yield: 76%; f) MeOTf, CH2Cl2, r.t., yield: 64% (12+), 23% (24+).
Single crystals suitable for X-ray diffraction analysis were obtained for compounds 1N
(Figure S17) and 2N. The solid state molecular structure of 2N, along with selected
bond lengths and angles, are depicted in Figure 1, to illustrate the shape and size of
the diyne compound. The distance between the two boron atoms B1 and B1’ is
15.457(7) Å. The molecule has an inversion center located between C1 and C1’ and
is almost linear, as the relevant C-C-C bond angles of the diyne bridge are all close to
180°.
6
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
Figure 1. The solid state molecular structure of 2N (50% probability ellipsoids).
Hydrogen atoms and solvent molecules are omitted for clarity. Angles between the
plane of the BC3-core (defined by B1, C6, C11 and C21) and the planes of the adjacent
aryl rings: 57.9(1)° for C6; 49.6(1)° for C11; 48.9(1)° for C21. Selected distances and
angles for 2N: B1C6 1.596(2) Å; B1C11 1.561(2) Å; B1C21 1.570(2) Å; C1C1’
1.373(3) Å; C1C2 1.202(2) Å; C2C3 1.431(2) Å N1C14 1.381(2) Å; N2C24
1.390(2) Å; C6B1C11 116.577(30)°; C6B1C21 117.639(28)°; C11B1C21
125.764(42)°; C1C2C3 177.35(17)°, C1’C1C2 179.6(2)°. Sum of the CBC
angles around B: 359.98(14)°.
Physico-chemical properties
Both positively charged compounds (12+, 24+) are moderately soluble in water (c =
1×10-3 M), their solutions being stable for longer periods when stored in the dark. The
neutral analogue of 1N was dissolved in DMSO to prepare a stock solution (c = 5×10-
3 M) which was further diluted with aqueous buffer prior to every experiment, yielding
stable solutions up to the 0.1 M range. The poor solubility of neutral analogue 2N under
biorelevant conditions hampered further experiments.
The absorbancies of the studied compounds (Figure 2) were proportional to their
concentrations up to c = 210-5 M (Figures S21-S23), and changes in their UV/Vis
absorption spectra upon temperature increases up to 90 °C were minor (Figure S24).
Reproducibility of the UV/Vis spectra upon cooling to 25 °C was excellent. These
findings indicate that the compounds do not aggregate by intermolecular stacking
7
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
under the experimental conditions used. Absorption maxima and corresponding molar
extinction coefficients () are given in Table 1.
6x104
377 nm
5x104
1N
12+
4x104
3x104
2x104
24+

408 nm
335 nm
1x104
0
250
300
350
400
450
500
/ nm
Figure 2. UV/vis spectra of neutral 1N, 12+, 24+ in water.
Comparison of their UV/Vis absorption spectra reveals distinct differences between the
neutral compound 1N and its charged analogue 12+. The neutral molecule 1N shows
an absorption band at 408 nm while the charged analogue 12+ shows a
hypsochromically shifted absorption with a maximum at 315 nm and a shoulder at
335 nm. This absorption behavior is well documented in previous publications from our
group for a series of analogous neutral and charged compounds[17p, 19, 26] and can be
attributed to a loss of the charge transfer transition from the amine to the boron moiety
upon methylation of the amine. The approximate doubling of the extinction coefficient
and the bathochromic shift of > 40 nm (3 300 cm-1) in absorption, when comparing the
monomer 12+ and the dimer 24+, is also consistent with our previous studies[17p, 19] and
can be attributed to a larger π-system in the case of 24+. All photophysical data is
summarized in Table 1.
8
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
Table 1. Photophysical data for compounds 1N, 2N, 12+ and 24+.
Stoke’s
λabs
ɛ
λem
τ
τ0
[ns]
kr
knr
Solvent
shift
[cm-1]
2 800
Φf
[nm][a] [M-1 cm-1] [nm]
[ns]
[108 s-1] [108 s-1]
Hexane
Toluene
Et2O
397
405
400
408
386
386
386
344
335
384
377
̶
447[b]
496
0.10[b]
0.22
0.27
-
1.5
4.4
7.6
-
15.0
20.0
28.3
-
0.67
0.50
0.35
-
6.0
1.8
0.96
-
25 000
̶
4 500
5 500
4 600
4 400
6 900
7 600
5 100
6 700
1 900
3 100
1N
2N
514
H2O[c]
Hexane
Toluene
Et2O
11 000
̶
502
464
0.10
0.16
0.15
0.15
0.18
0.26
0.25
2.0
4.3
6.2
3.9
6.4
0.94
1.3
20.0
26.9
41.3
26.0
35.5
3.6
0.50
0.37
0.24
0.38
0.28
2.8
4.5
1.9
1.4
2.2
1.3
7.8
5.8
60 000
-
525[b]
546[b]
418
EtOH
H2O
26 000
23 000
45 000
49 000
12+
428
EtOH
H2O
414[b]
431[b]
24+
5.2
1.9
[a] Lowest energy absorption band. [b] For compound 1N in hexane, λex = 385 and
375 nm were used for measuring emission and quantum yield respectively. For
compound 24+, λex = 360 nm (in H2O) and λex = 365 nm (in EtOH) were used for
emission and quantum yield measurements. For compound 2N in toluene and Et2O, a
395 nm cut-off filter was used for the emission measurement. [c] DMSO stock solution
(c = 5×10-3 M) further diluted with aqueous buffer.
Both the neutral and charged compounds are strongly fluorescent, with significant
apparent Stoke’s shifts in all tested solvents (see Table 1 and Figure 3). However,
neutral compound 1N showed pronounced emission quenching upon heating to 90 °C,
whereas the temperature-induced emission changes of 12+ and 24+ were negligible
(Figures S25-S27).
9
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
700
600
500
400
300
200
100
0
1N
12+
24+
3
400
450
500
550
600
650
700
/ nm
Figure 3. Comparison of emission spectra of neutral 1N (λex = 408 nm ), 12+(λex = 335
nm), 24+(λex = 377 nm) and 34+[19] (λex = 425 nm) in sodium cacodylate buffer (pH = 7.0,
I = 0.05 M). c (1N and 34+) = 510-7 M; c (12+ and 24+) = 510-8 M
Study of interactions with DNA, RNA and BSA
To study the interactions of 1 with DNA/RNA, several typical types of DNA and RNA
were chosen (Table S2). Naturally occurring calf thymus (ct)-DNA represents a typical
β-helix structure with a balanced ratio of GC-(48%) and AT-(52%) base pairs. Synthetic
alternating polynucleotides poly (dGdC)2 and poly (dAdT)2 represent two extreme
situations (only AT- or GC-basepairs, respectively), differing significantly in their
secondary structures as well as in the availability of the minor groove for small
molecule binding (the guanine amino group sterically hinders deep molecule
penetration). For comparison between double stranded (ds) DNA and ds-RNA, poly
rA- poly rU RNA was chosen as an α-helical structure characterized by a major groove
available for the binding of bulky small molecules.
Furthermore, to explore the DNA/RNA binding of the novel chromophore to a greater
extent, we also studied the single stranded synthetic ss-RNA polynucleotides poly G,
poly A, poly U and poly C, each of them characterized by different properties. Adenine
ss-RNA mimics 50 to 250 adenine nucleotides at the 3' end of mRNA, poly G is related
to guanine-rich sequences in both DNA and RNA, whereas poly C and poly U are
10
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
significantly more flexible than purine-RNAs, and with less organized secondary
structures.
Due to the possibility that the compounds studied could interact with proteins, we
examined the most naturally abundant protein, bovine serum albumin (BSA), taking
into account its versatility of binding sites.
Thermal denaturation experiments
It is well known that double stranded (ds)-helices of polynucleotides dissociate into two
single stranded polynucleotides upon heating at well-defined temperatures (Tm value).
Non-covalent binding of small molecules to ds-polynucleotides usually increases the
thermal stability of the ds-helices thus resulting in increased Tm values, and the
increase (∆Tm) can (corroborated by other methods) be related to the various binding
modes.[27]
Tetra-charged 24+ stabilized ds-DNA moderately (r
= 0.1; ∆Tm =
[compound] / [polynucleotide]
+4.0 °C), whereas monomer 12+ induced only minor stabilization (r [compound] / [polynucleotide]
= 0.1; ∆Tm = +1.1 °C) and the neutral analogue 1N did not affect the thermal stability
of ds-DNA (Figures S29-S31). The results show a direct proportionality of ds-DNA
stabilization to the number of positive charges. However, tetra-cation 34+ stabilized ds-
DNA much more strongly (r [compound] / [polynucleotide] = 0.1; ∆Tm = +7.3 °C), pointing to the
significant impact of the bithiophene linker.[20]
Fluorimetric titrations with DNA, RNA, BSA
The intrinsic strong fluorescence of the compounds studied allowed fluorimetric
titrations with various ds-DNA/RNA, single-stranded (ss)-RNA and (BSA). Addition of
ds-DNA or ds-RNA caused strong, nonselective quenching of the emission of 12+
(Figures S34 & S35) or 24+ (Figure 4), but no fluorescence change was observed for
neutral 1N (Figure S32). Intriguingly, addition of ss-RNA or BSA caused negligible
changes in the emission from monomer 12+ (Figures S36 & S37), in contrast to the
behavior of dimer 24+, which revealed a strong fluorimetric response (Figures S38-
S42).
Furthermore, 34+ showed an emission increase upon DNA/RNA/BSA complexation,
characterized by distinct differences in fluorescence maxima between DNA/RNA and
protein (BSA), whereas 24+ showed only non-selective emission quenching. Such a
difference in fluorimetric response between 24+ and 34+ might be attributed to the fact
11
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
that bithiophene is a weak donor, whereas the diyne unit does not have an electron-
donating effect.
ct-DNA
poly (dAdT)2
500
400
300
200
100
0
500
400
300
200
100
0
BSA
poly (dGdC)2
poly A - poly U
1,0x10-6
2,0x10-6
3,0x10-6
4,0x10-6
4,0x10-8 8,0x10-8 1,2x10-7 1,6x10-7 2,0x10-7
0,0
0,0
c (polynucleotide) / mol dm-3
c (BSA) / mol dm-3
600
431 nm
ct-DNA
poly (dAdT)2
500
400
300
200
100
poly (dGdC)2
poly A - poly U
BSA
free 24+
0
400
450
500
550
600
/ nm
Figure 4. Top: Comparison of fluorimetric titrations of 24+ (c = 510-8 M, λex = 377 nm)
at λem = 431 nm for all ds-DNA, ds-RNA and BSA (ss-RNA not shown, see Supporting
Information Figures S38-S42). Bottom: Comparison of the fluorescence spectrum of
24+ with spectra of 24+/biomacromolecule complexes at the end of titrations. All
measurements performed at pH = 7.0, in sodium cacodylate buffer, I = 0.05 M.
The non-linear fitting of the DNA or RNA titration data by means of the Scatchard
equation (McGhee, vonHippel formalism)[28, 29] allowed the calculation of binding
constants (Table 2). The BSA titration data fitted excellently to a 1:1 (24+:BSA)
stoichiometry model, pointing to only one dominant binding site of 24+ at BSA.
Table 2. Binding constants (log Ks)[a] of 12+ and 24+ with polynucleotides or BSA
calculated by processing fluorimetric titrations[a]; at pH = 7.0, sodium cacodylate buffer,
I = 0.05 M.
12
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
polynucleotide
ct-DNA
24+
6.9
7.7
12+
6.0
-
poly dAdT- poly dAdT
poly dGdC –poly dGdC 7.8
-
poly A – poly U
poly G
7.6
7.5
7.2
7.4
6.7
8.2[b]
6.0
-
[c]
poly A
poly C
-
poly U
-
BSA[b]
[c]
[a] Analyses of titration data by means of the Scatchard equation[28, 29] gave values of
the ratio n [bound 24+] / [polynucleotide] = 0.2 - 0.5; for easier comparison, all log Ks
values were re-calculated for fixed n = 0.25 (ds-polynucleotides) and n = 0.5 (ss-RNA).
Correlation coefficients were > 0.99 for all calculated Ks values. [b] Fitted for Langmuir
isotherm for 24+:BSA 1:1 stoichiometry. [c] Negligible emission change did not allow
analysis of titration data.
The strong, submicromolar affinities of 24+ to ds-DNA and ds-RNA were within the
same order of magnitude, while its affinity to ss-RNA was approximately an order of
magnitude lower. The affinity of 24+ to BSA was an order of magnitude higher than its
affinity towards ds-DNA/RNA (Table 2). The excellent fit of the titration data (Figure
S42) strongly supported a single dominant binding site on BSA for 24+, although other
binding sites with several orders of magnitude lower affinities cannot be excluded.
Comparison of the affinities between 24+ and bithiophene analogue 34+ revealed similar
binding constants for ds-DNA/RNA and ss-RNA, but a significant difference in binding
to BSA, with 24+ showing an affinity two orders of magnitude higher than 34+.
Monomer 12+ revealed somewhat lower affinity to ds-DNA/RNA, but still in the
micromolar range, albeit having only half of the positive charge, suggesting that
electrostatic interactions with the negatively charged DNA backbone are not the
dominant binding interactions. Intriguingly, the emission of 12+ did not change with BSA
or ss-RNA addition, indicating that the long rod-like structure of dimer 24+ is essential
for efficient binding to both targets.
CD experiments
This far, we had studied the non-covalent interactions at 25 °C by monitoring the
spectroscopic properties of the compounds upon addition of the polynucleotides. In
13
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
order to obtain insight into the changes of polynucleotide properties induced by small
molecule binding, we chose CD spectroscopy as a highly sensitive method for the
examination of conformational changes in the secondary structure of
polynucleotides.[30] In addition, 12+ or 24+ as achiral small molecules, could display
induced circular dichroism (ICD) within their absorption spectra upon binding to
polynucleotides, which could provide useful information about modes of
interaction.[31, 32]
Addition of 12+ did not significantly change the CD spectra of ds-DNA or ds-RNA
(Figures S44-S46), and no induced (I)CD bands > 300 nm were observed. In contrast,
tetracation 24+ induced a significant decrease in intensity in the CD spectra of all ds-
DNA/RNA (230-300 nm range; Figure 5), attributed to a pronounced decrease in ds-
polynucleotide chirality.[31, 32]
Only poly (dAdT)2 (Figure 5) revealed significant induced (I)CD bands at λ > 300 nm,
which could be attributed to the uniformly oriented binding of 24+ within a well-defined
DNA binding site.[32] Taking into account the structure of the molecule 24+, the DNA
minor groove is the most plausible binding site. Closer inspection of the ICD bands and
comparison with the UV/vis titration data (Figure 5, Inset: black line) revealed a mixed
binding mode of 24+, dependent on a ratio r [compd.] / [poly (dAdT)2]; whereby for r < 0.2 ICD
bands were negative and for r ≥ 0.3 ICD bands were positive. Such a change of the
ICD sign is commonly attributed to single molecule binding at an excess of DNA
(r < 0.2) and molecular aggregation within the DNA grooves at an excess of small
molecule (r > 0.3).[31, 32]
14
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
1.0
0.8
0.6
0.4
2
1
0.2
3
2
0.0
-0.2
-0.4
-0.6
-0.8
UV/vis
CD; r=0.1
CD; r=0.3
1
300
325
350
375
400
/ nm
0
0
-1
-2
-3
ct-DNA
r=0.05
r=0.1
r=0.2
r=0.3.
r=0.4
-1
-2
-3
-4
poly (dAdT)2
r=0.05
r=0.1
r=0.2
r=0.3
r=0.4
r=0.5
r=0.5
-4
250
300
350
400
250
300
350
400
/ nm
/ nm
6
5
5
4
poly (dGdC)2
poly A - poly U
r=0.05
r=0.1
r=0.2
r=0.3
r=0.05
r=0.1
r=0.2
r=0.3
r=0.4
r=0.5
4
3
3
2
r=0.4
r=0.5
2
1
1
0
0
-1
-2
-3
-1
-2
-4
250
300
350
400
250
300
350
400
/ nm
/ nm
Figure 5. CD titration of ctDNA, poly (dAdT)2, poly (dGdC)2, poly A - poly U (all
DNA/RNA c = 210-5 M) with 24+ at molar ratios r = [24+] / [polynucleotide] (pH = 7.0,
buffer sodium cacodylate, I = 0.05 M).
Further, dominant ICD bands at 300-330 nm could be partially attributed to electronic
transition vectors along the boron-nitrogen axes (see Table S5), which seem to be well
oriented with respect to the DNA chiral axis. The maxima at λmax = 360-380 nm), giving
negligible ICD band, were attributed to the electronic transition along the long axis of
24+, parallel to the diyne-linker (see Table S5).
The almost negligible intensity of ICD bands for the analogue 24+/GC-DNA complex
could be attributed to the sterically crowded minor groove with the amino groups of
guanine, not allowing deep insertion of 24+ and, thus, diminishing the induced CD
effect.[32] The broad and shallow minor groove of AU-RNA is a poor binding site for
small molecules, in contrast to the major groove of RNA, which has a width similar to
15
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
that of the minor groove of DNA (Table S2) and could be an efficient binding site for
24+. However, the large depth of the major groove allows heterogeneous orientation of
24+ molecules with respect to the ds-RNA chiral axis, thus resulting in negligible ICD
bands.
poly U
r=0.05
r=0.1
5
3
4
5
3
r=0.2
r=0.3
r=0.4
r=0.5
4
3
2
1
2
1
0
2
-1
0.0
0.1
0.2
0.3
0.4
0.5
1
r
0
0
poly A
-1
-2
-3
-4
r=0.05
r=0.1
r=0.2
r=0.3
r=0.4
r=0.5
-1
-2
253 nm
250
300
350
400
250
300
350
400
/ nm
/ nm
7
6
poly G
r=0.05
r=0.1
r=0.2
r=0.3
r=0.4
r=0.5
11
poly C
r=0.05
r=0.1
r=0.2
r=0.3
r=0.4
r=0.5
10
9
5
4
8
7
3
6
2
5
1
4
3
0
2
-1
-2
-3
-4
1
0
-1
-2
250
300
350
400
250
300
350
400
/ nm
/ nm
Figure 6. CD titration of poly U, poly A, poly C and poly G (all RNA c = 210-5 M) with
24+ at molar ratios r = [24+] / [polynucleotide] (pH = 7.0, buffer sodium cacodylate, I =
0.05 M).
16
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
Particularly intriguing results were obtained for the 24+/ss-RNA complexes (Figure 6).
Addition of 24+ completely disordered the CD spectrum of poly A and also that of poly
U, while the CD spectra of poly G and poly C were less affected. For the poly A titration,
the isoelliptic point at λ = 253 nm strongly supported only one type of 24+/poly A
complex. The poly U titration showed a systematic shift of the spectral crossing points
during titration, which is typical for mixed binding modes.
Complete loss of the helical chirality of A or U ss-RNA upon binding to 24+,
accompanied with a rather high affinity of 24+ (Table 2) suggested wrapping of the ss-
polynucleotide chain around the cylindrically shaped compound 24+. Such a binding
mode would maximize the efficiency of electrostatic interactions between four positive
charges of 24+ and the negative polynucleotide backbone. This binding mode would be
additionally supported by an energetically favorable exclusion of the hydrophobic diyne
linker from hydrophilic solvent molecules. Such a complex of achiral 24+ serving as a
spindle for ss-RNA would not give any chiral response, which is in accordance with the
CD titration experiments (Figure 6).
Another proof of the proposed binding mode is that monomer 12+ did not change the
CD spectrum of ss-RNA, which could be attributed to the globular shape of 12+ with its
centered positive charge, not supporting ss-polynucleotide wrap-around.
Raman spectroscopy
Raman spectra of 24+ were measured in water (110-4 M) and Na-cacodylate buffer,
(pH = 7.0, 110-4 M and 510-5 M) (Figure 7 and Table S4). In the Raman spectra of
all solutions, a broad band around 3220 cm-1 and a medium band around 1640 cm-1
were observed and assigned to the water stretching and bending modes, respectively.
In addition, in the Raman spectra of the buffered solutions, bands originating from
cacodylate ions were obtained: a medium methyl asymmetric stretching band at 2935
cm-1, a weak methyl deformation band at 1414 cm–1 and an intense As=O stretching
band at 605 cm-1. Nevertheless, bands distinctive of 24+ were observed in all Raman
spectra, even at a concentration as low as 510-5 M (Table S4). Hence, the band
around 2220 cm-1 was assigned to the stretching of the C≡C triple bonds in bisaryl-
substituted diynes,[6] while the band around 1600 cm-1, overlapped by the water band,
was attributed to a phenyl ring stretching mode (Table S4).[33] Based on the calculated
Raman spectrum of 24+ (Figure S49), the band around 1355 cm-1 was associated with
17
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
mixed stretching vibrations of the triple bonds and phenyl rings, whereas stretching of
the bonds between the boron atom and three aromatic substituents contributed to the
band around 1070 cm-1. The strong Raman scattering of 24+ was attributed to the diyne
moiety conjugated to the aromatic rings,[34] allowing detection of the molecules in
solution at a micromolar concentration range.
*
a
*
b
2500
2000
1500
1000
500
Wavenumber / cm-1
Figure 7. a) Raman spectra of 24+ (c = 110-4 M) in water (blue line) and Na-cacodylate
buffer, pH = 7.0 (red line). b) SERS spectra of 24+ (c = 110-6 M), in the silver colloid
not containing Na-cacodylate buffer (blue line) and containing Na-cacodylate buffer,
pH = 7.0 (red line). λex = 758 nm. The bands labelled with asterisk originate from the
buffer. The spectra are displaced for visual clarity.
SERS experiments
The SERS spectra of 24+ were measured in the 510-8  510-6 M concentration range
(Figure 8). The characteristic SERS bands of 24+ were obtained at a concentration as
low as 510-8 M, whereas the Raman scattering enhancement was the highest for the
110-6 M sample. Referring to the Raman bands assignment, the strong band at 2215
cm-1 was assigned to the stretching of the C≡C triple bonds, while the intense band at
1596 cm-1 was attributed to the stretching of the phenyl moieties. The medium band at
1354 cm-1 was assigned to the stretching modes of the central conjugated part of the
molecule, including phenyl rings and triple bonds, whereas the moderate band at 1070
18
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
cm-1 was associated with the stretching of the bonds between the boron atom and
aromatic substituents. Aside from the few 24+ bands observed in the Raman spectra,
some additional bands were obtained in the SERS spectra, corresponding mainly to
phenyl ring vibrations (Table S4). The bands at 1286 cm-1 and 1155 cm-1 which
appeared at low 24+ concentrations (110-7 M and 510-7 M) were attributed to the
symmetric stretchings of CF3 and SO3 groups, respectively, of
trifluoromethanesulfonate anions.[35] At low concentrations of 24+, the counterions
came close to the enhancing silver surface and, consequently, their vibrational modes
were enhanced.
5x10-8
1x10-7
M
M
5x10-7
1x10-6
5x10-6
M
M
M
2500
2000
1500
1000
500
Wavenumber / cm-1
Figure 8. The concentration dependent SERS spectra of 24+ in the silver colloid not
containing Na-cacodylate buffer, c = 510-8510-6 M. λex = 758 nm. The spectra are
displaced for visual clarity.
Considering the surface selection rules according to which polarizability changes
perpendicular to the metal surface contribute the most to scattered radiation, the
prominent C≡C (2215 cm-1) and phenyl (1596 cm-1) stretching bands implied that at a
concentration of 110-6 M, the molecules adopted an optimal position with the triple
bonds and phenyl rings oriented perpendicular to the silver surface (Scheme 3a).
19
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
Scheme 3. SERS experiments: Depiction of the molecular orientation of compound
24+ with respect to the silver surface at high concentrations of 24+ (a), low
concentrations of 24+ (b), and in the complex with ds-DNA (c). The 24+/BSA complex
(not shown) is detached from the Ag-surface and, thus, gives no SERS signal.
Moreover, the positive charge on each side of the molecule facilitated its positioning in
hot spots between two nanoparticles. It can be assumed that adsorption of the 24+
molecules was electrostatically driven by the positively charged trimethylamino groups
attracted to the citrate anions on the silver nanoparticles, followed by direct interactions
with the silver surface. A band observed in the low wavenumber region (216 cm-1) was
assigned to AgN stretching, indicating interaction between the nitrogen atom and the
silver surface (Figure 7b). By decreasing the concentration, the intensity of the
characteristic vibrational bands diminished, most likely due to first tilted and then in-
plane positioning of the molecules on the enhancing surface (Scheme 3b).
In order to investigate the effect of the buffer on the SERS response, the spectrum of
24+ (110-6 M) in Na-cacodylate buffer (pH = 7.0) was acquired (Figure 7b). In general,
the spectrum was slightly more intense and the bands more defined, when compared
to the spectrum of 24+ in water, though positively charged 24+ molecules most likely
acted as aggregating agents of the silver colloid. Salts in the buffer composition
additionally induced aggregation of the silver nanoparticles resulting in stronger
enhancement of the Raman scattering. A strong band observed at 231 cm–1 was
assigned to the stretching of the AgCl bond formed between the chloride ions from
the buffer and the silver surface (Figure 7b).
20
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
To study the binding of 24+ to ds-DNA, the SERS spectra of the 24+/ct-DNA complexes
in r [24+] / [ct-DNA] molar ratios of 1, 0.2 and 0.1 were measured at two different 24+
concentrations: a) c(24+) = 110-6 M, which produced the strongest SERS response,
and b) at the lowest concentration at which 24+ was detected (510-8 M) (Figures 9a
and 9b). In both cases, the SERS spectra obtained correspond to the SERS spectrum
of the small molecule.
The SERS bands were more intense for the 24+/ct-DNA complex than for 24+ alone.
For example, at c(24+) = 510-8 M, the intensity of the triple C≡C stretching band at
2214 cm–1 for the complexes of r [24+] / [ct-DNA] = 1 and 0.2 was enhanced 1.4 and
1.3 times, respectively, relative to that of the free 24+ molecules. The increase in
intensity obtained indicated interactions of 24+ with the nucleic acid, owing to which the
small molecules adopted a more optimal orientation with respect to the enhancing
silver surface and/or were placed closer to the silver nanoparticles (Scheme 3c). On
the other hand, the excess of highly negatively charged DNA in the 24+/ct-DNA sample
of the r = 0.1 most likely caused less efficient adsorption of the complex on the silver
surface, reducing the SERS intensity.
Furthermore, the SERS response of 24+ (c = 110-6 M) upon binding to BSA was
studied for the 24+/BSA complexes prepared in r [24+] / [BSA] molar ratios of 1, 0.2 and
0.1 (Figure 9.c). Unlike the complexes of 24+ with the nucleic acids, the SERS intensity
significantly diminished regardless of the complex molar composition. The
characteristic band of the triple bond stretching at 2215 cm–1 almost completely
vanished from the spectrum of the 24+/BSA complex of the molar ratio 0.1. Due to
strong interactions of the small molecules with the protein, and likely deep insertion of
the molecule within the BSA binding site, compound 24+ was removed from the silver
nanoparticles responsible for the SERS effect.
21
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
a
24+
24+/ct-DNA
1
0.2
0.1
b
24+
24+/ct-DNA
1
0.2
0.1
c
24+
24+/BSA
1
0.2
0.1
2500
2000
1500
1000
500
Wavenumber / cm-1
Figure 9. a) SERS spectra of 24+ and 24+/ct-DNA complexes of ratio r [24+] / [ct-DNA]
= 1, 0.2 and 0.1, in the silver colloid containing Na-cacodylate buffer, pH = 7.0; c(24+)
= 510-8 M. b) SERS spectra of 24+ and 24+/ct-DNA complexes of ratio r = 1, 0.2 and
0.1, in the silver colloid containing Na-cacodylate buffer, pH = 7.0; c(24+) = 110-6 M.
c) SERS spectra of 24+ and 24+/BSA complexes of ratio r = 1, 0.2 and 0.1, in the silver
colloid containing Na-cacodylate buffer, pH = 7.0; c(24+) = 110-6 M. λex = 758 nm. The
spectra are displaced for visual clarity.
It is interesting to note that the characteristic SERS response of 24+ was observed upon
NIR excitation (785 nm) in the simply prepared and widely used silver colloid, even at
a nanomolar concentration range. Thereby the distinctive band of the C≡C triple bond
at 2215 cm–1, which does not interfere either with the bands of its own, or with the
bands of the other species, in the measured system, allowed easy detection of the
small molecules. The intense SERS response obtained for 24+ interacting with DNA,
and its loss upon binding with BSA, is characteristic of binding with the nucleic acid
and the protein, respectively. Owing to the sensitivity obtained and minimal spectral
interference, the molecules studied could potentially be used as alkyne-coded SERS
tags for live cell imaging.[2d]
Preliminary biological screening
The aims of the biological experiments performed were to verify the actual capability
of studied DNA/RNA/protein binder to penetrate into the cells, to visualize its
22
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
intracellular location and subcellular targets, and evaluate the anti-proliferative effect,
in order to identify further potential applications, either as a cytotoxic lead compound
toward theranostic[36] applications (combining dual fluorescent/Raman monitoring with
an anti-proliferative action), or as a non-cytotoxic dye suitable for intracellular
applications or in biochemical studies.
To examine toxicity, i.e., the effect of 24+ on the proliferation of human cell lines, we
used the MTT test. The compound was tested on two human cell lines (HeLa and HEK
293). As an indicator of anti-proliferation activity we used the IC50 value, which
corresponds to the concentration of the compound that inhibits proliferation to 50%
compared to the control cells (proliferating without the compound in the medium). The
results we obtained showed negligible antiproliferative activity of 24+ even at the highest
concentration we used in the test (1x10-4 M, results not shown).
We also checked the ability of the compound to cross the cellular membrane and
penetrate the HeLa cells, by using the intrinsic fluorescence of 24+ in fluorescent
confocal microscopy experiments. Confocal microscopy also allows us to propose the
possible intracellular localization of the compound. The compound 24+ entered cells
very efficiently within 2 hours of cell immersion in c(24+) = 1x10-6 M and the observed
fluorescence of the compound coincided with the area of the cell where the
endoplasmic reticulum is located, and the grainy dispersion of the fluorescence signal
indicates possible endosomal accumulation (Figure 10).
Figure 10. Confocal microscopy of live HeLa cells (c = 50000 cells/mL) taken on a
Leica SP8 X confocal microscope, cells stained for 2h in the c(24+) = 1x10-6 M , λexc
360-400 nm; λem = 415-471 nm.
=
23
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
As titration experiments of 24+ with DNA/RNA and BSA revealed strong quenching of
the emission of 24+ upon binding, the strong fluorescence in the cell suggests that
emission from the compound is not significantly affected by its proposed intracellular
localization. This would be consistent with the endosomal hydrophobic environment,
which is devoid of any DNA/RNA or albumin-like binding sites. This could spur
intriguing future studies of 24+ and its analogues in dual fluorescence/Raman-based
SRS microscopy,[2d] whereby intracellular location with quenched fluorescence and
active SRS signal could be easily differentiated from intracellular location with strong
fluorescence.
The cell nucleus is completely void of any emission which, in combination with its
negligible anti-proliferative activity, strongly suggests that 24+ does not bind to or
interfere with genomic DNA or RNA processes, which are essential for cell viability.
However, for a more accurate determination of its cellular localization, further, more
detailed biological experiments are planned.
Conclusion
As a follow-up to our work on novel applications of bis-triarylboranes as
DNA/RNA/protein binders and bright intra-cellular probes,[17p, 19, 20, 26] we designed and
studied new derivatives to investigate the influence of the linker type on
DNA/RNA/protein interactions and to compare (4-Ar2B-3,5-Me2C6H2)-C≡C-C≡C-(3,5-
Me2C6H2-4-BAr2 (24+) with its corresponding monomeric (4-Ar2B-3,5-Me2C6H2)-C≡CH
(14+) analogue.
It can be safely assumed that positively charged analogue 24+ retains structural
features very similar to those of its neutral analogue 2N (see Figure 1); thus, both
compounds could be considered as rod-like dumbbell structures. Compound 24+ is also
characterized by four terminal positive charges. Only 24+ was soluble in water and
therefore further studied for biorelevant applications using, as a reference, the
corresponding monomer 12+.
One of the main aims of the 24+ design was to study the impact of a linker connecting
two triarylborane units upon the binding to DNA/RNA or protein, in comparison to
previously studied bithiophene-linker analogue 34+.[20] Addition of any type of
DNA/RNA/protein induced quenching of both 24+ and 12+ fluorescence, in contrast to
the strong emission enhancement of 34+. Such an opposite effect demonstrates the
24
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
pronounced impact of the linker on the triarylborane fluorescence emission.
Furthermore, globular-shaped monomer 12+ showed only negligible interaction with
protein BSA, stressing that the highly hydrophobic linker in “dimers” (24+ or 34+) is
essential for efficient binding to the hydrophobic pocket of BSA. Moreover, the linker
structure (24+ diyne vs 34+ bithiophene) strongly influenced the BSA affinity (24+
logKs = 8.2 vs. 34+ logKs = 5.9), suggesting that linear, aliphatic low-volume linker (24+
diyne) is highly prefered for insertion into BSA binding site.
Both cationic compounds (24+, 12+) showed remarkably high affinity toward various
types of DNA/RNA (logKs = 6-7.5 range) and, intriguingly, dicationic monomer 12+
showed only an order of magnitude lower affinity than tetracationic 24+. This implies
only a minor contribution of electrostatic interactions with the negatively charged
DNA/RNA backbone and suggested binding within ds-DNA/RNA grooves, within which
the highly hydrophobic linker and extensive interactions of aromatic units surrounding
the boron atoms strongly contribute to overall DNA/RNA affinity.
However, tetracationic 24+ also strongly interacted also with ss-RNA, which does not
possess any groove as a binding site. The CD results strongly supported ss-RNA chain
wrapping around tetracationic 24+ as a thread around the spindle, which is a very
unusual mode of binding of ss-RNA with small molecules. The absence of interaction
of ss-RNA with globular-shaped structure of 12+ additionally stressed the nature of the
rod-like dumbbell structure of 24+ being crucial for strong interaction with ss-RNA.
Another major design feature of 24+ was the ability to use the diyne-linker as a Raman-
probe, complementing the 24+ fluorescence response. Thus, in aqueous solution 24+
gave rise to several remarkably strong Raman bands in the 2220 cm–1 - 1355 cm–1
range, allowing accurate monitoring at as low as 10 micromolar concentration. Even
more interesting was the response of 24+ in the SERS spectra, leading to a detection
limit below 10-nanomolar concentrations. Most intriguingly, addition of DNA actually
increased the SERS signal intensity slightly, whereas BSA completely quenched it.
This specific response is very useful and complementary to that of the 24+ fluorescence
response (in which DNA/RNA and BSA cause similar emission quenching).
Finally, preliminary biological activity screening showed that 24+ entered human cells
very efficiently while not interfering with cell viability up to 10-4 M concentrations. Its
bright fluorescence is proposed to originate from localization along the endoplasmic
reticulum (possibly via intra-ribosomal or endosomal accumulation).
25
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
Our results strongly support further development of bis-triarylborane
Raman/fluorescence chromophores as dual probes for simultaneous confocal
fluorescence microscopy and SRS microscopy of cell lines, whereby careful choice of
a linker can finely tune DNA-protein selectivity (note the difference between 24+ diyne
vs. 34+ bithiophene) and consequently the intracellular accumulation of a probe. The
Raman-response of 24+-analogues in SRS microscopy could, thus, efficiently
complement the fluorescence response when the studied system is turbid/non-suitable
for fluorescence imaging (e.g. cell-organoid agglomerates), some other fluorescence
probe is used simultaneously, or one of the cellular targets quenches the fluorescence
emission. Moreover, the unique binding mode of ss-RNAs with very high affinity makes
bis-triarylborane tetracations very promising ss-RNA delivery systems, particularly as
some of them have already shown very efficient cellular uptake and negligible
toxicity.[17p, 19, 26]
Acknowledgements
The financial support of the Croatian Science Foundation project IP-2018-01-5475, the
DAAD, and the Julius-Maximilians-Universität Würzburg are gratefully acknowledged.
H. A. thanks the Alexander von Humboldt Foundation for a postdoctoral fellowship.
Keywords
Borane; Fluorescent probe; Raman probe; DNA/RNA Sensor; Protein Sensor; Circular
Dichroism
References
[1] (a) H. Yamakoshi, H. Ohori, C. Kudo, A. Sato, N. Kanoh, C. Ishioka, H Shibata, Y. Iwabuchi,
Bioorg. Med. Chem. 2010, 18, 1083-1092; (b) F. Thuaud, Y. Bernard, G. Turkeri, R. Dirr, G.
Aubert, T. Cresteil, A. Baguet, C. Tomasetto, Y. Svitlin, N. Sonenberg, C. G. Nebigil, L.
Dꢀsaubry, J. Med. Chem. 2009, 52, 5176−5187.
[2] a) K. M. Dean and A. E. Palmer, Nat. Chem. Biol. 2014 10, 512–523; b) A. M. Valm, S.
Cohen, W. R. Legant, J. Melunis, U. Hershberg, E. Wait, A. R. Cohen, M. W. Davidson, E.
Betzig and J. Lippincott-Schwartz, Nature 2017, 546, 162-167; c) T. Niehorster, A.
Loschberger, I. Gregor, B. Kramer, H. J. Rahn, M. Patting, F. Koberling, J. Enderlein and M.
Sauer, Nature Methods 2016, 13, 257-262; d) F. H. Hu, C. Zeng, R. Long, Y. P. Miao, L. Wei,
Q. Z. Xu and W. Min, Nature Methods 2018, 15, 194-200.
[3] H. Q. Nguyen, B. C. Baxter, K. Brower, C. A. Diaz-Botia, J. L. DeRisi, P. M. Fordyce and K.
S. Thorn, Adv. Opt. Mater. 2017, 5, 1600548.
26
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
[4] a) S. Fournier-Bidoz, T. L. Jennings, J. M. Klostranec, W. Fung, A. Rhee, D. Li and W. C.
W. Chan, Angew. Chem. Int. Ed. 2008, 47, 5577-5581; b) M. Y. Han, X. H. Gao, J. Z. Su and
S. Nie, Nat. Biotechnol. 2001, 19, 631-635.
[5] a) Y. Q. Lu, J. B. Zhao, R. Zhang, Y. J. Liu, D. M. Liu, E. M. Goldys, X. S. Yang, P. Xi, A.
Sunna, J. Lu, Y. Shi, R. C. Leif, Y. J. Huo, J. Shen, J. A. Piper, J. P. Robinson and D. Y. Jin,
Nature Photonics 2014, 8, 33-37; b) Y. W. C. Cao, R. C. Jin and C. A. Mirkin, Science 2002,
297, 1536-1540; c) R. C. Jin, Y. C. Cao, C. S. Thaxton and C. A. Mirkin, Small 2006, 2, 375-
380.
[6] H. Yamakoshi, K. Dodo, A. Palonpon, J. Ando, K. Fujita, S. Kawata and M. Sodeoka, J.
Am. Chem. Soc 2012, 134, 20681-20689 .
[7] Y. Chen, J. Q. Ren, X. G. Zhang, D. Y. Wu, A. G. Shen and J. M. Hu, Anal. Chem. 2016,
88, 6115-6119.
[8] T. Hirschfeld, Appl. Spectrosc. 1977, 31, 328-329.
[9] K. N. Das, Y. Dai, P. Liu, C. Hu, L. Tong, X. Chen, J. Z. Smith, Sensors 2017, 17, 1592.
[10] a) N. Uzunbajakava, C. Otto, Opt. Lett. 2003, 28, 2073-2075; b) H.-J. van Manen, Y. M.
Kraan, D. Roos, C. Otto, Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 10159.
[11] a) H.-J. van Manen, C. Otto, Nano Lett. 2007, 7, 1631-1636; b) R. P. Carney, S. Hazari,
M. Colquhoun, D. Tran, B. Hwang, M. S. Mulligan, J. D. Bryers, E. Girda, G. S. Leiserowitz, Z.
J. Smith, K. S. Lam, Anal. Chem. 2017, 89, 5357-5363.
[12] a) Z. Huang, H. Lui, D. I. McLean, M. Korbelik, H. Zeng, Photochem. Photobiol. 2005, 81,
1219-1226; b) O. R. Šćepanović, Z. Volynskaya, C.-R. Kong, L. H. Galindo, R. R. Dasari, M.
S. Feld, Rev. Sci. Instrum. 2009, 80, 043103; c) O. R. Scepanovic, M. Fitzmaurice, A. Miller,
C. R. Kong, Z. Volynskaya, R. R. Dasari, J. R. Kramer and M. S. Feld, J. Biomedi. Optics 2011,
16; d) S. Jeong, Y.-i. Kim, H. Kang, G. Kim, M. G. Cha, H. Chang, K. O. Jung, Y.-H. Kim, B.-
H. Jun, D. W. Hwang, Y.-S. Lee, H. Youn, Y.-S. Lee, K. W. Kang, D. S. Lee, D. H. Jeong, Sci.
Rep. 2015, 5, 9455.
[13] a) B. Kang, M. M. Afifi, L. A. Austin, M. A. El-Sayed, ACS Nano 2013, 7, 7420-7427; b)
E.-O. Ganbold, J. Yoon, D. Kim, S.-W. Joo, Phys. Chem. Chem. Phys. 2015, 17, 3019-3023.
[14] a) X. Li, M. Jiang, J. W. Y. Lam, B. Z. Tang, J. Y. Qu, J. Am. Chem. Soc. 2017, 139,
17022-17030; b) J. Gala de Pablo, D. R. Chisholm, A. Steffen, A. K. Nelson, C. Mahler, T. B.
Marder, S. A. Peyman, J. M. Girkin, C. A. Ambler, A. Whiting, S. D. Evans, Analyst 2018, 143,
6113-6120.
[15] a) X. Li, X. Guo, L. Cao, Z. Xun, S. Wang, S. Li, Y. Li, G. Yang, Angew. Chem. Int. Ed.
2014, 53, 7809-7813; b) J. Liu, X. Guo, R. Hu, X. Liu, S. Wang, S. Li, Y. Li, G. Yang, Anal.
Chem. 2016, 88, 1052-1057; c) J. Liu, S. Zhang, C. Zhang, J. Dong, C. Shen, J. Zhu, H. Xu,
M. Fu, G. Yang, X. Zhang, Chem. Commun. 2017, 53, 11476-11479; d) J. Liu, S. Li, S. Zhang,
C. Shen, J. Zhu, G. Yang, X. Zhang, Sens. Actuator. B Chem. 2018, 261, 531-536; e) J. Liu,
X. Guo, R. Hu, J. Xu, S. Wang, S. Li, Y. Li, G. Yang, Anal. Chem. 2015, 87, 3694-3698; f) X.
Guo, X. Zhang, S. Wang, S. Li, R. Hu, Y. Li, G. Yang, Anal. Chim. Acta 2015, 869, 81-88; g)
B. Chen, G. Feng, B. He, C. Goh, S. Xu, G. Ramos-Ortiz, L. Aparicio-Ixta, J. Zhou, L. Ng, Z.
Zhao, B. Liu, B. Z. Tang, Small 2016, 12, 782-792; h) J. Liu, C. Zhang, J. Dong, J. Zhu, C.
Shen, G. Yang, X. Zhang, RSC Advances 2017, 7, 14511-14515; i) J. Liu, C. Zhang, J. Dong,
J. Zhu, C. Shen, G. Yang, X. Zhang, New J. Chem. 2017, 41, 4733-4737; j) S. Pagidi, N. K.
Kalluvettukuzhy, P. Thilagar, Langmuir 2018, 34, 8170-8177; k) J. Liu, K. Cheng, C. Yang, J.
Zhu, C. Shen, X. Zhang, X. Liu, G. Yang, Anal. Chem. 2019, 7, 3694-3698.
[16] For reviews see: a) C. D. Entwistle, T. B. Marder, Angew. Chem. Int. Ed. 2002, 41, 2927-
2931; b) C. D. Entwistle, T. B. Marder, Chem. Mater. 2004, 16, 4574-4585; c) L. Ji, S.
Griesbeck, T. B. Marder, Chem. Sci. 2017, 8, 846-863;
27
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
[17] a) J. C. Doty, B. Babb, P. J. Grisdale, M. Glogowski, J. L. R. Williams, J. Organomet.
Chem. 1972, 38, 229-236; b) Z. Yuan, N. J. Taylor, T. B. Marder, I. D. Williams, S. K. Kurtz,
L.-T. Cheng, J. Chem. Soc., Chem. Commun. 1990, 1489-1492; c) M. Lequan, R. M. Lequan,
K. C. Ching, J. Mater. Chem. 1991, 1, 997-999; d) Z. Yuan, N. J. Taylor, R. Ramachandran, T.
B. Marder, Appl. Organomet. Chem. 1996, 10, 305-316; e) T. Noda, Y. Shirota, J. Am. Chem.
Soc. 1998, 120, 9714-9715; f) W.-L. Jia, D.-R. Bai, T. McCormick, Q.-D. Liu, M. Motala, R.-Y.
Wang, C. Seward, Y. Tao, S. Wang, Chem. Eur. J. 2004, 10, 994-1006; g) Z. Yuan, C. D.
Entwistle, J. C. Collings, D. Albesa-Jové, A. S. Batsanov, J. A. K. Howard, N. J. Taylor, H. M.
Kaiser, D. E. Kaufmann, S.-Y. Poon, W.-Y. Wong, C. Jardin, S. Fathallah, A. Boucekkine, J.-
F. Halet, T. B. Marder, Chem. Eur. J. 2006, 12, 2758-2771; h) H. Li, A. Sundararaman, K.
Venkatasubbaiah, F. Jäkle, J. Am. Chem. Soc. 2007, 129, 5792-5793; i) J. C. Collings, S.-Y.
Poon, C. Le Droumaguet, M. Charlot, C. Katan, L.-O. Pålsson, A. Beeby, J. A. Mosely, H. M.
Kaiser, D. Kaufmann, W.-Y. Wong, M. Blanchard-Desce, T. B. Marder, Chem. Eur. J. 2009,
15, 198-208; j) Y. Kim, H. Zhao, F. P. Gabbaï, Angew. Chem. Int. Ed. 2009, 48, 4957-4960; k)
M. Varlan, B. A. Blight, S. Wang, Chem. Commun. 2012, 48, 12059-12061; l) X. Yin, J. Chen,
R. A. Lalancette, T. B. Marder, F. Jäkle, Angew. Chem. Int. Ed. 2014, 53, 9761-9765; m) Z.
Zhang, R. M. Edkins, J. Nitsch, K. Fucke, A. Steffen, L. E. Longobardi, D. W. Stephan, C.
Lambert, T. B. Marder, Chem. Sci. 2015, 6, 308-321; n) Z. Zhang, R. M. Edkins, J. Nitsch, K.
Fucke, A. Eichhorn, A. Steffen, Y. Wang, T. B. Marder, Chem. Eur. J. 2015, 21, 177-190; o) Z.
Zhang, R. M. Edkins, M. Haehnel, M. Wehner, A. Eichhorn, L. Mailänder, M. Meier, J. Brand,
F. Brede, K. Müller-Buschbaum, H. Braunschweig, T. B. Marder, Chem. Sci. 2015, 6, 5922-
5927; p) S. Griesbeck, M. Ferger, C. Czernetzi, C. Wang, R. Bertermann, A. Friedrich, M.
Haehnel, D. Sieh, M. Taki, S. Yamaguchi, T. B. Marder, Chem. Eur. J. 2019, 25, 7679-7688.
[18] C.-W. Chiu, Y. Kim, F. P. Gabbaï, J. Am. Chem. Soc. 2009, 131, 60-61.
[19] S. Griesbeck, Z. Zhang, M. Gutmann, T. Lühmann, R. M. Edkins, G. Clermont, A. N. Lazar,
M. Haehnel, K. Edkins, A. Eichhorn, M. Blanchard-Desce, L. Meinel, T. B. Marder, Chem. Eur.
J. 2016, 22, 14701.
[20] Ž. Ban, S. Griesbeck, S. Tomić, J. Nitsch, T. B. Marder, I. Piantanida, submitted 2019.
[21] a) S. Schlücker, Surface-Enhanced Raman Spectroscopy: Analytical, Biophysical and Life
Science Applications, Wiley-VCH, Weinheim, 2011; b) S. Schlucker, Angew. Chem. Int. Ed.
2014, 53, 4756-4795.
[22] a) M.M. Harper, K.S. McKeating, K. Faulds, Phys. Chem. Chem. Phys., 2013, 15, 5312‒
5328; b) D. Cialla, S. Pollok, C. Steinbrücker, K. Weber, J. Popp Nanophotonics, 2014, 3, 383‒
411; c) I. Bruzas, W. Lum, Z. Gorunmez, L. Sagle, Analyst, 2018, 143, 3990‒4008.
[23] a) T. Itoh, K. Matsuda, H. Iwamura, K. Hori, J. Am. Chem. Soc. 2000, 122, 2567-2576; b)
K. Toyota, S. Kawasaki, M. Yoshifuji, J. Org. Chem. 2004, 69, 5065-5070; c) B. Kang, J. W.
Kurutz, K.-T. Youm, R. K. Totten, J. T. Hupp, S. T. Nguyen, Chem. Sci. 2012, 3, 1938-1944;
d) S. Takahashi, Y. Kagami, K. Hanaoka, T. Terai, T. Komatsu, T. Ueno, M. Uchiyama, I.
Koyama-Honda, N. Mizushima, T. Taguchi, H. Arai, T. Nagano, Y. Urano, J. Am. Chem. Soc.
2018, 140, 5925-5933.
[24] a) A. N. Nesmejanow, W. A. Ssasonowa, V. N. Drosd, Chem. Ber. 1960, 93, 2717-2729;
b) A. L. S. Thompson, G. W. Kabalka, M. R. Akula, J. W. Huffman, Synthesis 2005, 2005, 547-
550; c) J. M. Murphy, X. Liao, J. F. Hartwig, J. Am. Chem. Soc. 2007, 129, 15434-15435; d)
A. G. Crawford, Z. Liu, I. A. I. Mkhalid, M.-H. Thibault, N. Schwarz, G. Alcaraz, A. Steffen, J.
C. Collings, A. S. Batsanov, J. A. K. Howard, T. B. Marder, Chem. Eur. J. 2012, 18, 5022-
5035.
[25] I. A. I. Mkhalid, J. H. Barnard, T. B. Marder, J. M. Murphy, J. F. Hartwig, Chem. Rev. 2010,
110, 890-931.
28
This article is protected by copyright. All rights reserved.

10.1002/chem.201905328
Chemistry - A European Journal
[26] a) S. Griesbeck, E. Michail, C. Wang, H. Ogasawara, S. Lorenzen, L. Gerstner, T. Zang,
J. Nitsch, Y. Sato, R. Bertermann, M. Taki, C. Lambert, S. Yamaguchi, T. B. Marder, Chem.
Sci. 2019, 10, 5405-5422; b) S. Griesbeck, E. Michail, F. Rauch, H. Ogasawara, C. Wang, Y.
Sato, R. Edkins, Z. Zhang, M. Taki, C. Lambert, S. Yamaguchi, T. B. Marder, Chem. Eur. J.
2019, 10.1002/chem.201902461.
[27] J. L. Mergny, L. Lacroix, Oligonucleotides 2003, 13, 515-537.
[28] G. Scatchard, Ann. N.Y. Acad. Sci. 1949, 51, 660–672.
[29] J. D. McGhee, P. H. V. Hippel, J. Mol. Biol. 1974, 86, 469–489.
[30] A. Rodger, B. Norden, In Circular Dichroism and Linear Dichroism; Oxford University
Press: New York, 1997, Chapter 2.
[31] M. Eriksson, B. Nordén, in Methods in Enzymology, Chaires, J. B.; Waring, M. J. Eds.
Academic Press, San Diego 2001, 340, 68–98.
[32] T. Šmidlehner, I. Piantanida, G. Pescitelli, Beil. J. Org. Chem. 2018, 14, 84–105.
[33] F.R. Dollish, W.G. Fateley, F.F. Bentley, Characteristic Raman Frequencies of Organic
Compounds, John Wiley & Sons, New York, 1974.
[34] A. Lucotti, M. Tommasini, D. Fazzi, M. Del Zoppo, W.A. Chalifoux, R.R. Tykwinski, G.
Zerbi, J. Raman Spectrosc., 2012, 43, 1293–1298.
[35] M.E. Tuttolomondo, A. Navarro, E.L. Varetti, A. Ben Altabef, J. Raman Spectrosc. 2005,
36, 427–434.
[36] C. H. Shi, J. B. Wu, D. F. Pan, J. Biomed. Opt. 2016, 21, 050901.
29
This article is protected by copyright. All rights reserved.