Photophysical insights and guidelines for blue “turn-on” fluorescent probes for the direct detection of nitric oxide (NO?) in biological systems

Article abstract of DOI:10.1002/poc.3896

Synthesis and photophysical properties of a family of five blue fluorescent “turn-on” probes for the direct detection of nitric oxide (NO^?) is reported. The probes CB1-5 feature a substituted 7-hydroxy coumarin chromophore coupled to 2-methyl-8-aminoquinoline, which act as tridentate ligand for Cu(II) and active site for monitoring NO^? using a replacement strategy. The UV/vis absorption and fluorescence emission characteristics of the probes are significantly influenced by the substitution pattern on the coumarin ring, as well as by solvent polarity and pH. Time-dependent density functional theory (TD-DFT) calculations for CB4 and CB5 showed that the absorptions are due to π-π* transitions localised on coumarin, with a small charge transfer contribution from the quinoline system at higher pH where the 7-hydroxy coumarin moiety is deprotonated, which leads to a bathochromic shift of the absorption. Complexation of the probes with Cu(II) quenches the fluorescence, which is turned back on upon reaction with NO^?, allowing selective detection of this important signalling molecule in μM concentrations. Preliminary experiments revealed that CB4 and CB5 enable to monitor endogenously produced NO^? in living bacterial cells in multi-dye imaging experiments.

Full text of DOI:10.1002/poc.3896

Received: 15 March 2018
DOI: 10.1002/poc.3896
Revised: 31 July 2018
Accepted: 6 August 2018
R E S E A R C H A R T I C L E
Photophysical insights and guidelines for blue “turn‐on”
fluorescent probes for the direct detection of nitric oxide
•
(
NO ) in biological systems
1
1
2
3
Mina Barzegar Amiri Olia | Amber N. Hancock | Carl H. Schiesser | Lars Goerigk
|
1
Uta Wille
1
School of Chemistry, Bio21 Institute, The
Abstract
University of Melbourne, Parkville,
Victoria, Australia
Synthesis and photophysical properties of a family of five blue fluorescent
2
•
Seleno Therapeutics Pty Ltd, Brighton
“turn‐on” probes for the direct detection of nitric oxide (NO ) is reported.
East, Victoria, Australia
The probes CB1‐5 feature a substituted 7‐hydroxy coumarin chromophore
coupled to 2‐methyl‐8‐aminoquinoline, which act as tridentate ligand for
3
School of Chemistry, The University of
Melbourne, Parkville, Victoria, Australia
•
Cu(II) and active site for monitoring NO using a replacement strategy. The
Correspondence
UV/vis absorption and fluorescence emission characteristics of the probes are
significantly influenced by the substitution pattern on the coumarin ring, as
well as by solvent polarity and pH. Time‐dependent density functional theory
Lars Goerigk, School of Chemistry, The
University of Melbourne, Parkville,
Victoria 3010, Australia.
Email: lars.goerigk@unimelb.edu.au
(TD‐DFT) calculations for CB4 and CB5 showed that the absorptions are due
Uta Wille, School of Chemistry, Bio21
Institute, The University of Melbourne,
Parkville, Victoria 3010, Australia.
Email: uwille@unimelb.edu.au
to π‐π* transitions localised on coumarin, with a small charge transfer contri-
bution from the quinoline system at higher pH where the 7‐hydroxy coumarin
moiety is deprotonated, which leads to a bathochromic shift of the absorption.
Complexation of the probes with Cu(II) quenches the fluorescence, which is
Funding information
National Computational Infrastructure;
Victorian Life Sciences Computation Ini-
tiative; High Performance Computing
Facility of the University of Melbourne;
Discovery Scheme, Grant/Award Number:
DP13010007; Centres of Excellence
Scheme, Grant/Award Number:
•
turned back on upon reaction with NO , allowing selective detection of this
important signalling molecule in μM concentrations. Preliminary experiments
•
revealed that CB4 and CB5 enable to monitor endogenously produced NO in
living bacterial cells in multi‐dye imaging experiments.
KEYWORDS
CE0561607; Australian Research Council,
Grant/Award Numbers: CE0561607,
DE140100550 and DP13010007; Univer-
sity of Melbourne
density functional theory calculations, fluorescent probes, multi‐dye imaging, nitric oxide,
photophysical studies
1
| INTRODUCTION
NO• was also identified as a key regulator for the
[
3]
dispersal of bacterial biofilms. Biofilms are compact
colonies of microorganisms that grow on living and
non‐living surfaces and cause serious problems, for exam-
•
The free radical species nitric oxide (NO ) is an important
signalling molecule, which is produced by many cell
types in a variety of tissues. For example, NO is gener-
ated during immune and inflammatory responses and
is essential for the regulation of various physiological pro-
cesses, including cardiovascular function.
•
[4]
[5]
ple in industrial environments and hospitals, as well
[
1]
[6]
as in cultural materials conservation. Biofilms are held
together by a matrix of extracellular polymeric materials
consisting of DNA, polysaccharides, and proteins, which
[2]
Recently,
J Phys Org Chem. 2018;e3896.
wileyonlinelibrary.com/journal/poc
© 2018 John Wiley & Sons, Ltd.
1 of 15
https://doi.org/10.1002/poc.3896

2
of 15
BARZEGAR AMIRI OLIA ET AL.
preserve nutrients and obstruct the diffusion of antibacte-
and its derivatives, which are known blue‐fluorescent
[7,8]
[19]
rial agents.
Thus, biofilm‐based bacteria display
dyes,
would be used as chromophore, some of us
an increased resistance to treatment and are less vulnera-
recently suggested that Lippard's replacement approach
could provide access to blue “turn‐on” fluorescent probes
and hereupon synthesised the probe CB5 to test its poten-
ble to antibacterial agents compared with planktonic sus-
[7,9]
pensions.
Complete eradication of bacterial biofilms is
•
[20]
extremely difficult and often not feasible, in particular
when damage of the underlying surface must be
tial to detect NO (Scheme 1).
In CB5, a substituted coumarin is linked to 2‐methyl‐
8‐aminoquinoline to provide a tridentate ligand for
[10]
avoided.
Inhibition of biofilm formation and biofilm
[
18]
cell dispersal are the most common non‐invasive
Cu(II).
Treatment of CB5 with Cu(II) produces the
[
11]
approaches for biofilm removal to date.
Since the
complex Cu(II)‐CB5 in which fluorescence is quenched,
•
[18,21]
discovery that NO triggers a regulated and coordinated
dispersal of bacterial biofilms, NO donor compounds
presumably by the paramagnetic Cu(II) ion.
It was
•
•
pleasing to see that reaction of Cu(II)‐CB5 with NO
leads to replacement of the metal ion through a redox
reaction, which results in formation of the N‐nitrosamine
have been successfully employed to initiate biofilm break-
down. It is during the transition of the sessile biofilm
colony into free‐swimming planktonic cells that biofilms
[
20]
CB5‐NO and blue fluorescence turn‐on.
[12,13]
become vulnerable to remediation.
In our previous work, we did not explore the
photophysical properties of the probe. However, funda-
mental knowledge whether and how the substitution pat-
tern at the coumarin chromophore affects the UV/vis
absorption and fluorescence emission behaviour is essen-
tial to guide the design and development of selective and
•
Remarkably, the mechanism by which NO mediates
formation and dispersal of biofilms is still not fully under-
stood. The development of new approaches for the effi-
cient eradication of biofilms requires the availability of
•
methods to detect NO in biological systems with high
•
sensitivity and selectivity, and a number of sensors based
sensitive blue “turn‐on” fluorescence sensors for NO in
[
14]
[15]
on electrochemical
and fluorescence
methods have
the future. In this work, we will close this gap by provid-
ing a detailed assessment of the performance of a series of
five coumarin‐based fluorescent probes (CB1‐5), which
all contain a 2‐methyl‐8‐aminoquinoline moiety as coor-
dination site for Cu(II) but differ in the substitution pat-
tern on the coumarin ring. The photophysical studies
are augmented with TD‐DFT calculations to obtain
understanding of the nature of the electronic transitions
in these compounds. Furthermore, we also explored the
been invented. Fluorescence techniques in combination
with laser‐scanning confocal microscopy are particularly
powerful approaches due to the high spatiotemporal res-
[16]
•
olution.
The ideal sensor for detecting NO in biologi-
cal systems should have a very high fluorescence
emission at physiological pH for maximum sensitivity.
The majority of the currently available fluorescent probes
•
for NO emit in the green or red region of the electromag-
[17]
•
netic spectrum,
which is, unfortunately, also the
selectivity of the probes for NO and performed prelimi-
region where most of the DNA, protein, or other cell
function fluorescence stains are emitting. Thus, in order
to enable visualisation of the site of NO production in
nary multi‐dye fluorescence imaging studies to visualise
endogenously produced NO in living bacterial cells.
•
•
cells simultaneously with other cell constituents, the
development of sensitive NO selective probes that fluo-
•
2 | RESULTS AND DISCUSSION
resce in the blue region (λem ~ 470 ± 25 nm) and which
could be used in multi‐dye imaging experiments is highly
desirable.
2
.1 | Synthesis of the probes CB1‐5
Scheme 2 outlines the synthetic strategy to access the
functionalized coumarin dyes CB1‐5. Briefly, the couma-
rin chromophores 1a,c‐d were synthesised through a
Knoevenagel reaction involving 2,4‐dihydroxy benzalde-
hydes and substituted malonates or phenylacetic acid,
Lippard et al developed a strategy to transform
•
fluorophores into sensors for NO , which utilises a non‐
fluorescent copper complex that turns on fluorescence
•
[17,18]
upon replacement of the metal by NO .
If coumarin
SCHEME 1 Mechanism of “turn‐on”
fluorescence through replacement of
•
Cu(II) by NO in the recently developed
[20]
coumarin based probe CB5

BARZEGAR AMIRI OLIA ET AL.
3 of 15
The phenyl substituted probe CB1 has a fluorescence
maximum at λ = 489 nm, which lies in the green‐blue
region of the UV/vis spectrum and is not suitable for
•
monitoring NO in the intended multi‐dye imaging exper-
iments. Replacing the phenyl group at C‐3 by a methyl
group at C‐4 blue shifts the emission band to
λ = 470 nm (CB2). Unfortunately, the absorption band
also shifts to a shorter wavelength to appear at
λ = 339 nm, which is not compatible with experiments
in living cells. On the other hand, having no substituent
at C‐4 in coumarin but electron‐withdrawing ester groups
at C‐3 (CB3, CB4, and CB5) maintains blue fluorescence
emission at λ ~ 450 nm, while red‐shifting the absorption
maximum to λ ~ 410 nm. Thus, the probes C3‐C5 could
be excited with a laser at λ = 405 nm in the confocal
[
20,24]
SCHEME 2 Reagents and conditions: (i) Hexamine, TFA, reflux,
microscope without adverse impact on cells.
2
0 h. (ii) Hexamine, HOAc, 70°C, 6 h. (iii) MeOH, MS (4 Å), rt, 8 h,
then NaBH , 24 h, rt. (iv) MeOH, CH Cl , MS (4 Å) rt, 8 h, then
Na (OAc) BH, 24 h, rt.
Interestingly, at pH 7, both CB3 and CB4 display
two absorption bands, but excitation at either of these
bands resulted in similar fluorescence emission spectra.
4
2
2
3
7
‐Hydroxy coumarin dyes are known to exhibit
pH‐responsive and solvent‐responsive, ratio‐metric
absorption/excitation profiles as result of the
equilibrium between the neutral phenol moiety and the
[
22]
respectively.
Formylation at C‐8 through a Duff
reaction in the presence of trifluoroacetic acid and
hexamine gave aldehydes 2a‐e,
to 2‐methyl‐8‐aminoquinoline via an intermediate Schiff
base, followed by reduction to yield CB1‐5.
a
[23]
which were coupled
[
25]
phenolate anion.
of pH and solvent on λabs,max of our probes in more detail.
We therefore investigated the effect
[18,20]
Details
are given in the Experimental Section (see also
The pK values for CB1‐5 were determined from the UV/
a
Figures S1‐12 in the Supporting Information, SI).
vis absorbance as a function of pH (see Figure S14) and
are included in Table 1. Table 2 presents the λabs,max data
for all probes at selected pH as well as in several non‐
aqueous solvents. The UV/vis absorption spectra are
shown in Figures S15 and S16.
2
.2 | Photophysical properties of CB1‐5 in
•
the absence of Cu(II) and NO
While the pK of phenyl‐substituted CB1 (9.5) is sim-
a
The photophysical properties of the probes were explored
using 100 μM of CB1‐5 in aqueous buffer solution at pH 7
ilar to that of phenol, the strong electron‐withdrawing
ester substituents at C‐3 on the coumarin moiety increase
the acidity of the hydroxyl group by about two orders of
magnitude. Additional electron‐withdrawing substituents
at C‐6 (ortho to OH), for example chlorine in CB5, lead to
a further increase of the acidity by three orders of
magnitude.
(tris‐buffered saline (TBS) with 1% DMSO; details of the
sample preparation are given in the Experimental
Section). The absorption and emission characteristics of
all compounds are compiled in Table 1. The spectra are
shown in Figure S13.
The different acidity of the various probes is reflected
in the absorption spectra. No noticeable change between
pH 1 and 8 was found for CB1, which exhibits one absor-
bance band with a maximum at λ ~ 355 nm. Increasing
the pH to 11 leads to a bathochromic shift of the absorp-
tion maximum to λ = 396 nm, which is due to deproton-
ation of the phenolic hydroxyl group. For CB2, a similar
gradual red shift of the absorption maximum from
λ = 326 nm at pH 3 to λ = 367 nm at pH 11 was found.
The more acidic CB3 and CB4 display only one absorp-
tion band with λmax ~ 355 nm at pH 1‐5. Increasing the
pH to 7‐8 produces a second absorption maximum at
λ = 412 nm and λ = 409 nm respectively, which becomes
the sole absorption peak at pH 11, where the phenolic
TABLE 1 Absorption and emission maxima (in nm) and pK
values of CB1‐5 (100 μM in TBS at pH 7, with 1% DMSO)
a
a
b
c
Compound
λ
abs,max
λ
ex
λ
em,max
pK
a
CB1
CB2
CB3
CB4
CB5
355
339
350
325
489
470
450
450
448
9.5
8
363, 412
366, 409
410
350, 410
360, 410
420
7.4
7
4
a
Maximum absorbance.
b
Excitation wavelength.
c
Maximum fluorescence emission.

4
of 15
BARZEGAR AMIRI OLIA ET AL.
TABLE 2 Effect of pH and solvent on the UV/Vis absorption maxima λabs,max (in nm) of CB1‐5 (100‐μM solutions)
Solvent
CB1
CB2
CB3
CB4
CB5
a
b
pH 1
350
352
354
355
358
396
n.d.
326
354
355
352
354
356
362
410
410
n.d.
410
a
pH 3
a
pH 5
n.d.
339
345
367
360
360
a
c
c
pH 7
363, 412
366, 409
a
c
pH 8
413
370 , 411
a
pH 11
413
411
356, 441
360, 417
362, 420
355
DMSO
MeOH
EtOH
348, 445
343
325, 390
n.d.
358, 436
359, 419
353, 414
351
357, 440
357, 417
n.d.
343
327
Et
2
O
347
n.d.
354, 438
353, 435
MeCN
342
321
349, 427
356, 429
a
In TBS with 1% DMSO.
b
n.d. = not determined.
c
Peak appeared as a shoulder.
OH group is fully deprotonated in both probes. CB5, the
CB1‐5. In the case of the least acidic coumarin derivative
CB1, this effect is only observed with the most polar
solvent DMSO, while the strongly acidic CB5 interacts
most acidic compound in this series (pK = 4), exhibits
a
one absorption band with λmax ~ 360 nm under very
acidic conditions (pH 1‐3), which red shifts to
λmax = 410 nm at pH 5 with no further changes at higher
pH. This clearly shows that CB5 is fully deprotonated at
physiological pH.
[
28]
with DMSO, MeOH, MeCN, and even Et O.
2
Since it is known that the fluorescence emission
intensity of unsubstituted 7‐hydroxycoumarin is also pH
[
29]
dependent,
we next explored the effect of pH on the
Table 2 also reveals significant solvent effects on
λabs,max. Thus, the absorption maximum of CB1 in
fluorescence emission spectra of CB1‐5. As expected, in
aqueous solution, the emission intensities of all probes
increased with increasing pH due to progressive depro-
tonation (see Figure S17 for CB1‐4). Figure 1 shows the
fluorescence emission of CB1‐5 at pH 7, which clearly
reveals a significantly higher emission intensity for CB5
compared with CB1‐4.
MeOH, EtOH, Et O, and MeCN is the same as in aqueous
2
buffer at pH 1‐8. However, CB1 exhibits in DMSO not
only a band with λmax = 348 nm (corresponding to “neu-
tral” CB1), but also a second, red‐shifted band centred at
λ = 445 nm. A similar solvent effect was also observed for
CB2, where a second, red‐shifted absorption in DMSO
appears with λmax = 390 nm. Interestingly, CB3 and
CB4 exhibit a second bathochromic absorption band not
only in DMSO, but also in MeCN, EtOH, and MeOH,
which red‐shifts in the order MeOH/EtOH < MeCN <
DMSO. No such solvent effect was observed for the
absorption band that can be assigned to the “neutral”
CB3 and CB4. In contrast to this, CB5 displays two
absorption maxima in all organic solvents. Apart from
the absorption band of the neutral species centred at λ ~
3
55 nm and which does not significantly vary with
solvent, a second red‐shifted absorption appears at
λmax = 417 nm in MeOH and at λmax = 435‐440 nm in
Et O, DMSO, and MeCN. This second, bathochromically
2
shifted band in CB1‐5 could result from a solvent‐induced
ground state deprotonation. A similar solvent depen-
[
26]
dence of λmax has been described for 3‐hydroxyflavone
FIGURE 1 Fluorescence emission intensities of CB1‐5 at pH 7
(100 μM in TBS, with 1% DMSO; λex = 410 nm (CB1,3‐5) or
360 nm (CB2))
[27]
and coumarin‐substituted nicotinamides. The extent of
the solvochromatic shift correlates with the pKa for

BARZEGAR AMIRI OLIA ET AL.
5 of 15
This finding can be rationalised by the degree of
deprotonation of the probes at pH 7. CB1 and CB2 are
fully protonated and show only weak fluorescence. CB3
and CB4 are partially deprotonated, leading to a more
intense fluorescence emission. CB5 exists exclusively as
the phenolate at pH 7, which clearly shows that the
observed strong fluorescence emission is due to the
O‐deprotonated coumarin. Therefore, with regards to
the intended detection of NO• in biological systems,
CB5 appears particularly promising: (1) because of the
complete deprotonation of the coumarin moiety the fluo-
rescence intensity is the highest at physiological pH,
which makes CB5 the most sensitive probe of those stud-
ied here; and (2) the absorption maximum of
λmax = 410 nm remains constant above pH 5 (in contrast
to CB1‐4), which enables examination by confocal laser‐
scanning microscopy through excitation with a 405‐nm
in biological systems, we did not further investigate
this effect.
In comparison with CB1, the generally higher fluores-
cence emission intensities of CB4 in organic solvents
might be due to the solvent‐induced ground state depro-
tonation proposed above (see Table 2) or could suggest a
rapid deprotonation within the lifetime of its singlet
excited state. In fact, it is known that the pK value of
a
phenols decreases by several orders of magnitude upon
electronic excitation, likely due to charge redistribution,
[
29,30]
which is stabilised by polar solvents.
Overall, these
findings confirm the observations made above that the
observed fluorescence arises from probes with an
O‐deprotonated coumarin moiety.
3 | COMPUTATIONAL STUDIES
laser. Overall, these data show that blue coumarin based
•
“
turn‐on” fluorescent probes for NO require electron‐
In order to obtain a fundamental understanding of the
observed absorption and emission properties, DFT calcu-
lations were exemplary performed for CB4 and CB5 in
their neutral and anionic (ie, phenolate) states, as well
as for three different N‐protonated forms (Figure 2).
N‐Protonated derivatives were included in the computa-
tional studies to explore whether such compounds could
contribute to the observed UV/vis absorption spectra
under very acidic conditions.
withdrawing substituents at C‐3 and C‐6 on the coumarin
ring system to ensure full deprotonation of the phenolic
hydroxyl group at physiological pH for high sensitivity.
The influence of the organic solvent on the fluores-
cence emission λem,max was more subtle and is shown in
Table 3 exemplary for CB1 and CB4. Overall, the fluores-
cence emission intensity of CB1 is considerably stronger
in DMSO than in any other organic solvent investigated
in this work (see Figure S18). Compared with the band
at pH 7 in aqueous solution (see Table 1), λem,max
becomes slightly blue‐shifted by up to 20 nm in organic
solvents without any obvious dependence on solvent
polarity. Compared with CB1, the fluorescence emission
intensity of the more acidic CB4 is higher in the most
polar solvents explored here (ie, EtOH, MeOH, DMSO;
see Figure S18), but only a very small solvent variability
We first performed a thorough conformational search
with reliable dispersion‐corrected DFT techniques (see
Computational Methods for details), followed by linear‐
response TD‐DFT treatments. The latter were carried
out with the range‐separated hybrid DFT approximation
[
31]
[32]
CAM‐B3LYP
and the 6‐311G**
atomic‐orbital
(AO) basis set. This method has been shown to be reliable
for the calculation of vertical singlet‐singlet excitation
[
33]
of λem,max was found, except for Et O, where CB4 exhibits
energies in medium‐sized organic dyes
and is suffi-
2
a second, red‐shifted emission band with λmax = 547 nm.
ciently accurate to provide a qualitative analysis of the
systems explored in this study. The first seven excitation
However, since Et O is not a suitable solvent for studies
2
TABLE 3 Solvent effect on the fluorescence emission maxima
λ
em,max (in nm) of CB1 and CB4 (100 μM solutions; λex = 410 nm)
Solvent
CB1
CB4
DMSO
MeOH
EtOH
490
478
478
485
487
477
473
486
462
450
452
450
449
449
MeCN
EtOAC
CHCl
Et
THF
3
2
O
451, 547
450
FIGURE 2 O‐deprotonated and N‐protonated isomers of CB4
and CB5

6
of 15
BARZEGAR AMIRI OLIA ET AL.
energies were calculated together with their oscillator
strengths (f). These results were used to simulate the
UV/vis absorption spectra. Details are given in the
Computational Methods section.
Comparison of the simulated spectra for different con-
formers of the various protonation states of CB4 and CB5
revealed similarity in the absorption range of interest.
Therefore, it is unlikely that the observed spectral
features shown in Tables 1 and 2 can be explained with
contributions stemming from different conformers of
CB4 and CB5, respectively (see Figures S19‐S21). The role
of the solvent was studied by performing calculations for
very similar, we will restrict the following discussion to
CB5 using DMSO as the solvent.
Figure 3 shows the computed lowest‐energy confor-
−
mations for the neutral (CB5), deprotonated (CB5‐O )
+
and the different protonated forms (CB5‐N(1′)H , CB5‐
+
2+
N(8′)H , and CB5‐N(1′,8′)H2 ). A significant change
of the relative arrangement between the coumarin and
quinoline moieties in dependence of the protonation state
is apparent. While in neutral CB5 hydrogen bonding
between the coumarin OH and the NH group at C‐8′ in
quinoline leads to a twisted arrangement of the two aro-
matic ring systems that can be characterised by the dihe-
dral angle d(8, 11, 8′, 7′) of 51°, lack of such hydrogen
the gas phase and for DMSO, H O, and MeOH, using a
2
−
continuum solvation model (see Computational Methods
for details), but no considerable differences for the gas
phase and in solution were found (see Figure S22). This
result could indicate that continuum solvation models
may not describe all possible solvent effects adequately
when CB4,5 is the solute. However, it should be noted
that explicit solvation was not explored in this work,
because we were interested in obtaining a qualitative
understanding rather than a detailed characterisation of
the role of solvent on the electronic transitions. Further-
more, since the calculated data for CB4 and CB5 were
bonding in the phenolate CB5‐O puts the planes of the
coumarin and quinoline rings nearly orthogonal with
d(8, 11, 8′, 7′) = 80°. The geometries of the N‐protonated
derivatives are governed by various hydrogen bonds
between NH groups and the lactone moiety in coumarin,
causing rotation of the quinoline framework towards cou-
marin. It should be noted that protonation of the NH
group at C‐8′ in quinoline leads to considerable elongation
+
of the C(11)‐N bond by about 0.06 Å (in CB5‐N(8′)H )
2
+
or by 0.09 Å (in CB5‐N(1′,8′)H2 ) compared with
+
CB5‐N(1′)H . Both singly protonated forms can be
FIGURE 3 Calculated lowest‐energy conformations for the different protonation states in CB5, optimised at the TPSS‐D3/def2‐TZVP level
of theory; distances in A; d = dihedral angle (for atom numbering see also Scheme 2)

BARZEGAR AMIRI OLIA ET AL.
7 of 15
described as a folded conformer with favourable disper-
sion interactions between the two aromatic moieties.
In order to predict the preferred N‐protonation site,
we determined the adiabatic proton affinities (PA) from
the energies of the two singly protonated species relative
(
(
(
A)
B)
C)
to the neutral system (see Computational Methods).
+
For the formation of CB5‐N(1′)H ,
a value of
−
1
PA = −247.1 kcal mol
was obtained, which is
−
1
7
.5 kcal mol lower in energy than the value for the for-
+
−1
mation of CB5‐N(8′)H (PA = −239.6 kcal mol ). The
calculated more favourable protonation of the ring nitro-
gen agrees well with the experimentally determined
[
34]
higher basicity (pKa = 4.94), compared with the exocy-
[
35]
clic amino group in 8‐amino quinoline (pKa ~ 3.05).
According to the computations, the first absorption
band of CB5 has the highest oscillator strength of
f = 0.660 and an excitation energy of 3.80 eV
(λmax = 326 nm). The dominant contribution to λmax is a
transition from a π orbital (HOMO‐1 for our chosen level
of theory) to a π* orbital (LUMO), which are both localised
on the coumarin system (Figure 4A), and where “HOMO”
stands for the highest occupied molecular orbital and
“
LUMO” for the lowest unoccupied molecular orbital.
−
In the case of CB5‐O , the strongest transition has an
oscillator strength of f = 0.758 with an excitation energy
of 3.38 eV, which corresponds to λ = 366 nm. Interest-
max
−
ingly, the HOMO‐1 in CB5‐O is delocalized over both
coumarin and quinoline moieties, whereas the LUMO is
localised on coumarin only (Figure 4B). This indicates a
small CT contribution to the transition, which leads to a
bathochromic shift in the UV/vis spectrum of about
(
D)
0
.41 eV (ca. 40 nm) compared with the neutral CB5. This
finding is in very good qualitative agreement with the
experimentally found red shift of 0.46 eV (ca. 50 nm) for
the absorption maximum of CB5 at pH > 5 (see Table 2),
−
which can therefore be clearly assigned as CB5‐O .
Figure 5A compares the simulated UV/vis spectra for
CB5 and CB5‐O with the experimentally determined
spectra at pH 1 and 7. The computed spectra are 0.30 to
.37 eV blue‐shifted compared with the experimentally
FIGURE
4
Molecular orbital (MO) pairs with dominant
−
−
contributions to the λmax TD‐DFT excitations of A, CB5; B, CB5‐O ;
+
+
C, CB5‐N(1′)H ; and D, CB5‐N(8′)H displayed with an isovalue of
−
[36]
0
ca. 0.02 e /Å (TD‐CAM‐B3LYP/6‐311G** level of theory)
determined data. The expected average error for TD‐
[
29]
CAMB3LYP is about 0.2 eV,
while errors for the
delocalized over the entire molecule. Figure 4C shows this
exemplary for CB5‐N(1′)H , which exhibits a strong
+
PCM continuum solvation model can vary between 0.05
[37]
and 0.1 eV.
Our computational results are therefore
in excellent agreement with the reported accuracy for this
method and enable to obtain a reliable qualitative under-
absorption with an oscillator strength of f = 0.384 and an
excitation energy of 3.90 eV, corresponding to λmax = 318 nm.
The calculated spectra shown in Figure 5B reveal that CB5
[38]
+
standing of these systems.
and CB5‐N(1′)H are virtually indistinguishable in this
The major contributions to λmax differ partially among
the three N‐protonated forms. While all three forms are
characterised by π‐π* transitions, we observe only for
wavelength region, clearly showing that protonation of the
aminoquinoline moiety has no influence on the absorption
in the visible region of the electromagnetic spectrum.
Additional geometry optimizations of the relevant
excited states of the five protonated/deprotonated CB5
species revealed little change in the overall relative
+
CB5‐N(8′)H that the involved MO pair is localised entirely
on the coumarin moiety (Figure 4D). The excitations for
+
+
2
CB5‐N(1′)H and CB5‐N(1′,8′)H involve MOs that are

8
of 15
BARZEGAR AMIRI OLIA ET AL.
(A)
(B)
−
FIGURE 5 A, Simulated UV/vis spectrum (left y‐axis) of CB5 (‐ ‐ ‐) and CB5‐O (‐ • ‐) vs the experimentally measured absorbance (right
_
____
+
y‐axis) at pH 1 (
) and pH 7 (‐ ‐ ‐). B, Simulated UV/vis spectrum (left y‐axis) of CB5 (‐ ‐ ‐) and CB5‐N(1′)H (‐ • ‐) vs the experimentally
_____
measured absorbance (right y‐axis) at pH 1 (
)
arrangement of the two conjugated ring systems compared
quenching. In contrast to this, considerable background
fluorescence remained when this reaction was performed
in aqueous TBS buffer at pH 7 (with 1% DMSO). Here, the
with the ground‐state geometries. The resulting Stokes
−
shifts range from 0.28 eV (34 nm) for CB5‐O to 0.49 eV
+
(
44 nm) for CB5‐N(8′)H (see Table S4 for all data). Since
largest drop in fluorescence upon reaction with CuCl was
2
the experimental emission spectrum was measured at pH 7,
only the computed data for CB5‐O are of relevance. The
found with the most acidic probes CB4 and CB5 (44% and
43% respectively), whereas in the case of the considerably
less acidic CB1 and CB3 systems fluorescence quenching
was less efficient (25% and 18%, respectively).
−
bright emission for this system is reflected by a large calcu-
lated oscillator strength (f = 0.990). The computed emission
energy of 400 nm is by 48 nm blue shifted compared with
the experiment (see Table 1), however, when converted to
eV, this deviation is within the expected error for the cho-
sen method (see Computational Methods). Despite this
deviation in the emission energies, we observe nearly per-
fect agreement between the calculated Stokes shift
•
The response of Cu(II)‐CB1‐5 to NO was explored
using
6‐(2‐hydroxy‐1‐methyl‐2‐nitrosohydrazino)‐N‐
methyl‐1‐hexanamine (MAHMA NONOate) as a source of
•
[39]
NO .
Freshly prepared Cu(II)‐CB1‐5 (100 μM in TBS
at pH 7 with 1% DMSO) was treated with 400 equivalents
of MAHMA NONOate (using stock solutions in NaCl,
followed by dilution with TBS at pH 7.6), and fluorescence
emission was measured after 30 minutes. As shown in
Figure 6B, an increase in fluorescence intensity was
observed, ranging from 1.4‐fold in the case of CB1 to 2.7‐
fold in the case of CB5, indicating successful detection of
(0.28 eV or 34 nm) and the experimental value (0.26 eV or
3
8 nm). This finding again underlines the robustness of
our chosen computational approach as well as the validity
of the calculated data and their interpretation.
•
NO . For CB2, the reaction was performed in neat DMSO,
4
| ASSESSMENT OF CB1‐5 AS
which showed a more pronounced increase in fluorescence
upon reaction with NO . The lower background fluores-
•
•
PROBES FOR DETECTING NO
cence of Cu(II)‐CB2 in this solvent and the generally
higher fluorescence emission intensity of these probes in
DMSO are likely responsible for this effect. Measurements
of the fluorescence emission intensity of Cu(II)‐CB5 at
After having obtained detailed knowledge of the
photophysical properties of CB1‐5 in the absence of both
•
Cu(II) and NO , we next assessed their performance as
•
•
probes for sensing NO through replacement of complex
different concentrations of the NO donor revealed that this
bound Cu(II) via an irreversible redox process that is
associated with fluorescence turn‐on (see Scheme 1).
Cu(II)‐complexes of CB1‐5, eg, Cu(II)‐CB1‐5, were pre-
probe, which is the most emissive in this series at physiolog-
•
ical pH, enables detection of [NO ] in μM concentrations
[
40]
(Figure S23).
Time‐dependent fluorescence emission
•
pared by mixing equimolar amounts of CuCl with the
studies revealed that the reaction of Cu(II)‐CB5 with NO
2
respective probe. As Figure 6A clearly shows, the fluores-
cence intensity was quenched upon addition of CuCl2,
but the extent to which quenching occurred depended
on the conditions. Thus, when DMSO was used as sole
solvent (CB2 was explored as example), reaction of Cu(II)
with the probe resulted in almost complete fluorescence
was complete after about 9 minutes (Figure S24).
•
The selectivity of the CB probes towards NO was
examined by measuring the fluorescence emission of
Cu(II)‐CB1‐5 upon addition of 400 equivalents of a selec-
tion of reactive nitrogen and oxygen species (RNS and
−
−
−
ROS), ie, ClO , H O , K O , NO , and NO , which
2
2
2
2
3
2

BARZEGAR AMIRI OLIA ET AL.
9 of 15
(A)
(B)
FIGURE 6 A, Fluorescence quenching of CB1‐5 by addition of equimolar amounts of CuCl
2
(100 μM in TBS at pH 7 with 1% DMSO) CB1‐
•
5
(solid line) and Cu(II)‐CB1‐5 (dotted line). B, Selectivity of Cu(II)‐CB1‐5 towards NO and a series of RNS and ROS (100 μM in TBS at
pH 7 with 1% DMSO; 400 equiv. of oxidant). The reactions with CB2 in (A) and (B) were performed in neat DMSO
have been typically used to assess probe selectivity for
presented in Figure 7 for CB4 (top) and CB5 (bottom). Blue
•
[41]
NO .
The data in Figure 6B clearly show that none of
fluorescence, which is due to reaction of Cu(II)‐CB4 or
•
these ROS and RNS led to a fluorescence increase, which
Cu(II)‐CB5 with endogenously produced NO , is clearly
[
17,18]
is similar to the sensors developed by Lippard et al.
visible in Figure 7A,D. It should be noted that previous
studies with Cu(II)‐CB5 in macrophages confirmed that
other constituents of the cytoplasm, for example glutathi-
In most cases, the fluorescence dropped, except for the
reaction of Cu(II)‐CB4 with K O , where a slight
2
2
[
20,45]
increase (21%) was observed. However, this fluorescence
one, did not lead to a fluorescence turn‐on.
increase was significantly less than the increase observed
for the reaction of Cu(II)‐CB4 with NO (61%), which
The overlaid images in Figure 7C,F demonstrate con-
vincingly that CB4 and CB5 are both cell permeable and
that the blue fluorescing cells can be easily distinguished
from the HCS stained red fluorescing cells (Figure 7B,E,
respectively). Finally, qualitative comparison of the fluo-
rescence intensities in these images reveals that CB5 is
much brighter than CB4. This finding is in excellent
agreement with the results of the fluorescence emission
studies shown in Figure 1, clearly confirming the
importance of a fully deprotonated coumarin moiety at
physiological pH to achieve the highest sensitivity for
•
demonstrates the generally high selectivity of all CB
•
probes studied here for NO .
•
5
| NO DETECTION IN
BIOLOGICAL SYSTEMS
Finally, preliminary fluorescence imaging studies were per-
formed to obtain a qualitative assessment of the capacity of
•
•
the probes to detect NO in living bacterial cells. For this, we
NO detection in bacterial cell systems.
compared the two most emissive probes, eg, the previously
[42]
reported CB5,
and CB4, using 24‐hour‐old biofilms of
Pseudomonas aeruginosa as the model organism, by
6 | CONCLUSIONS
employing a protocol previously devised by one of us (see
[41]
•
Experimental Section).
Briefly, NO production was
We have synthesised the coumarin‐based probes CB1‐5 and
investigated their photophysical properties to provide
guidelines for the design of blue fluorescence “turn‐on”
stimulated by treating the cells with a 10 mM aqueous solu-
•
tion of KNO , which is reduced to produce NO in mM con-
centrations.
3
[
43,44]
•
After 3 hours, these cells were treated
sensors for the detection of endogenously produced NO
with freshly prepared Cu complexes of the probes and incu-
bated for 1 hour at 37°C. To demonstrate the performance
of the probes in multi‐dye imaging experiments, the cells
were also treated with HCS (NuclearMask Deep Red stain),
which stains DNA in both living and dead cells. The cells
were subsequently examined at 37°C using confocal laser‐
scanning microscopy. Representative cell images are
using the replacement strategy. Electron‐withdrawing ester
groups at C‐3 of the coumarin moiety (as in CB3, CB4, and
CB5) lead not only to blue fluorescence emission at λ ~
450 nm but also enable excitation at λ = 405 nm that is suit-
able for confocal laser scanning microscopy studies in
multi‐dye imaging experiments. Experiments and TD‐DFT
calculations revealed that the observed fluorescence is due

10 of 15
BARZEGAR AMIRI OLIA ET AL.
FIGURE 7 NO• detection in 24‐h P. aeruginosa biofilms that were up‐regulated for NO• production using confocal laser‐scanning
•
microscopy. NO detected with CB4 A, and CB5 D, HCS stained live and dead cells in the presence of CB4 B, and CB5 E, overlaid
images of CB4 and HCS C, and CB5 and HCS F
to the O‐deprotonated coumarin moiety, whereas the “neu-
tral” probes exhibit only a weak fluorescence intensity.
These findings clearly show that coumarin‐based probes
must be fully deprotonated at physiological pH to ensure
oven‐dried or flame‐dried glassware under argon. NMR
spectra were recorded on an Agilent NMR400, Agilent
DD2 or Bruker Advance IIIHD instrument. Chemical
shifts (δ) are reported in parts per million (ppm).
High‐resolution mass spectroscopy (HRMS) was con-
ducted on a Finnigan hybrid linear triple‐quadrupole
(LTQ) Fourier transform ion cyclotron resonance
(FTICR) mass spectrometer. Reverse phase preparative
HPLC was performed on an Agilent 1200 series
HPLC system with a Phenomenex Luna C18(2) 100A
packed (50 mm × 21.2 mm × 5 μm) Axia column.
Compound purity was assessed by analytical reverse
phase HPLC on an Agilent 1100 series HPLC system
with a Phenomenex Aeris peptide XB‐C18 packed
(250 mm × 4.6 mm × 3.6 μm) column. 7‐Hydroxy‐4‐
methylcoumarin 1b was purchased from Sigma Aldrich
and directly used. The 7‐hydroxycoumarins 1a, 1c, and
•
a high sensitivity for NO in biological systems. This depro-
tonation can be achieved by electron‐withdrawing substitu-
ents at C‐6 on the coumarin moiety (ortho to the hydroxyl
group), for example chlorine, as in CB5. Generally, all of
•
the probes CB1‐5 enabled selective detection of NO with-
out interference by a variety of ROS and RNS. Preliminary
fluorescence imaging studies demonstrated that both CB4
and CB5, which are the most acidic and therefore most
emissive probes studied in this work, can be successfully
•
used to detect NO in living P. aeruginosa cells. Future work
is aimed at improving the complexation properties of the
probes to reduce the background fluorescence of the Cu(II)
complexes, while maintaining or even further increasing
the acidity of the OH group on the coumarin moiety to
[
22]
1d were prepared according to literature.
Synthesis of
•
enhance their sensitivity for detecting NO beyond the μM
the formylated coumarins 2a and 2b was performed as
[
23]
concentration range.
described in Huang et al.
The NMR spectra and HPLC
traces for CB1‐4 are given in the ESI. CB5 was obtained
[
20]
as described in Barzegar Amiri Olia et al.
EXPERIMENTAL SECTION
a. Ethyl 7‐hydroxy‐8‐formyl‐coumarin‐3‐carboxyl-
ate (2c). A solution of 1c (1 g, 4.27 mmol) and
hexamine (898 mg, 6.4 mmol) in trifluoroacetic acid
(9 mL) was heated under reflux for 20 hours, and
then 18‐mL water was added. The solution was fur-
ther stirred for 30 minutes at 60°C. After cooling,
1
. Synthetic Materials and Methods
Unless otherwise noted, reagents were obtained from
commercial suppliers and were used without further
purification. All anhydrous reactions were performed in

BARZEGAR AMIRI OLIA ET AL.
11 of 15
3
3
the precipitate was collected by filtration to give 2c as
7.27 (t, J = 8 HZ, 1H), 7.01 (d, J = 7.5 Hz, 1H), 6.95
1
3
3
yellow solid (60%). H NMR (400 MHz, CDCl ): δ
(d, J = 7 Hz, 1H), 6.90 (d, J = 9 Hz, 1H), 6.54 (NH,
1H), 6.15 (s, 1H), 4.58 (s, 2H), 2.57 (s, 3H), 2.34 ppm
(s, 3H); C NMR (125 MHz, DMSO‐d ): δ 160.46,
159.91, 155.78, 154.34, 153.5, 143.91, 137.24, 136.71,
127.10, 126.71, 125.80, 122.72, 114.09, 112.73, 112.69,
112.55, 110.57, 106.41, 35.69, 25.18, 18.69 ppm. HRMS
3
3
1
6
1
0.38 (s, 1H), 8.72 (s, 1H), 8.02 (d, J = 8 Hz, 1H),
.98 (d, J = 8.8 Hz, 1H), 4.25 (q, J = 7.2 Hz, 2H),
3
3
13
6
3
.27 ppm (t, J = 7.2 Hz, 3H).
b. Methyl 7‐hydroxy‐8‐formyl‐coumarin‐3‐carbox-
ylate (2d). A solution of 1d (1 g, 4.54 mmol) and
hexamine (956 mg, 6.82 mmol) in trifluoroacetic acid
+
+
+
(ESI ); calcd. for C H N O 347.13902 [M + H] ,
21 19 2 3
(9 mL) was heated under reflux for 20 hours, and
found 347.13855.
then 60‐mL water was added. The mixture was
stirred for a further 30 minutes at 60°C. After cooling,
e. CB3. To a stirred solution of 2c (100 mg, 0.38 mmol)
in dry dichloromethane (30 mL) at room temperature
was added a solution of 2‐methyl‐8‐aminoquinoline
(67 mg, 0.42 mmol) in dry methanol (20 mL) and
activated MS (4 Å). The mixture was stirred at room
temperature for 8 hours under argon. The solution
was cooled to 0°C, sodium triacetoxy borohydride
(161 mg, 0.76 mmol) was added, and the reaction
mixture was stirred overnight at room temperature.
The solvent was evaporated in vacuo and the crude
material purified by column chromatography (SiO2,
the precipitate was collected by filtration to give 2d as
1
a yellow solid (53%). H NMR (500 MHz, CDCl ): δ
3
1
2.53 (s, 1H), 10.61 (s, 1H), 8.56 (s, 1H), 7.73 (d,
3
3
J = 8.8 Hz, 1H), 6.96 (d, J = 8.8 Hz, 1H),
3.97 ppm (s, 1H).
c. CB1. To a stirred solution of 2a (210 mg, 0.79 mmol) in
anhydrous methanol (32 mL) at room temperature
was added 2‐methyl‐8‐aminoquinoline (125 mg,
0.79 mmol). The mixture was stirred at room tempera-
ture for 8 hours under argon. The solution was cooled
to 0°C, sodium borohydride (230 mg, 6 mmol) was
added, and the reaction mixture was stirred overnight
at room temperature. The solvent was evaporated in
vacuo, and the crude material purified by column
ethyl acetate/petroleum spirits 3:2) to give CB3 as a
1
yellow solid (70%). H NMR (500 MHz, CDCl ): δ
3
3
8.65 (s, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.69 (d,
J = 9 Hz, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.27 (t,
J = 7.5 Hz, 1H), 7.01 (d, J = 7.5 Hz, 1H), 6.93 (d,
J = 7.5 Hz, 1H), 6.55 (NH, 1H), 4.57 (s, 2H), 4.25
(q, J = 7 Hz, 1H), 2.58 (s, 3H), 1.28 ppm (t,
J = 7.5 Hz, 3H). C NMR (125 MHz, CDCl ): δ
3
3
3
3
3
3
chromatography (SiO , ethyl acetate/petroleum spirits
2
1
3
1
:1) to give CB1 as a yellow solid (80%). H NMR
3
13
(
500 MHz, DMSO‐d ): δ 8.15 (s, 1H), 8.06 (d,
6
3
3
3
3
3
3
J = 10.5 Hz, 1H), 7.70‐7.68 (m, 2H), 7.54 (d,
J = 11 Hz, 1H), 7.45‐7.41 (m, 2H), 7.37 (d,
163.31, 162.90, 156.68, 155.81, 155.64, 150.25, 143.89,
137.34, 136.62, 131.38, 127.07, 126.7, 122.74, 114.16,
113.87, 112.57, 112.27, 110.92, 106.29, 61.28, 35.48,
3
J = 9.5 Hz, 1H), 7.33 (d, J = 10.5 Hz, 1H), 7.28 (t,
3
+
J = 9.5 Hz, 1H), 7.01 (d, J = 10.5 Hz, 1H), 6.97 (d,
25.25, 14.59 ppm. HRMS (ESI ); calcd. for
3
+
+
J = 9.5 Hz, 1H), 6.91 (d, J = 10.5 Hz, 1H), 6.54 (t,
C H N O 405.14450 [M + H] , found 405.14398.
23 21 2 5
3
J = 7 Hz, NH), 4.6 (d, J = 6.5 Hz, 2H), 2.56 ppm
f. CB4. To a stirred solution of 2d (100 mg, 0.4 mmol)
in dry dichloromethane (30 mL) at room temperature
was added a solution of 2‐methyl‐8‐aminoquinoline
(60 mg, 0.4 mmol) in dry methanol (20 mL) and acti-
vated MS (4 Å). The mixture was stirred at room tem-
perature for 8 hours under argon. The solution was
cooled to 0°C, sodium triacetoxy borohydride
(109 mg, 0.8 mmol) was added, and the reaction mix-
ture was stirred overnight at room temperature. The
crude material was purified by column chromatogra-
13
(s, 3H). C NMR (125 MHz, DMSO‐d ): δ 160.35,
6
1
1
1
3
4
55.79, 153.62, 143.99, 143.98, 137.35, 136.63, 135.45,
29.27, 128.70, 128.65, 128.48, 127.11, 126.71,
22.74, 114.09, 113.38, 112.49, 112.41, 106.33,
+
+
5.62, 25.25 ppm. HRMS (ESI ); calcd. for C H N O
6 21 2 3
2
+
09.15467 [M + H] , found 409.15422.
d. CB2. To a stirred solution of 2b (80 mg, 0.39 mmol)
in dry methanol (16 mL) at room temperature
was added 2‐methyl‐8‐aminoquinoline (68 mg,
0
.43 mmol). The mixture was stirred at room temper-
phy (SiO , ethyl acetate/petroleum spirits 3:2) to give
CB4 as a yellow solid (64%). H NMR (500 MHz,
2
1
ature for 8 hours under argon. The solution was
cooled to 0°C, sodium borohydride (114.2 mg, 3 mmol)
was added, and the reaction mixture was stirred over-
night at room temperature. The solvent was evaporated
in vacuo and the crude material purified by column
CDCl ): δ 8.66 (s, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.67
3
3
3
(d, J = 8.5 Hz, 1H), 7.32 (d, J = 8.5 Hz, 1H), 7.27
(t, J = 8 Hz, 1H), 7.01 (d, J = 8 Hz, 1H), 6.94‐
3
3
6.92(m, 2H), 6.53 (NH, 1H), 4.56 (s, 2H), 3.78
13
chromatography (SiO , ethyl acetate/petroleum spirits
2
(s, 3H), 2.58 ppm (s, 3H). C NMR (125 MHz,
2
1
:3) to give CB2 as a yellow solid (51%). H NMR
CDCl ): δ 163.87, 163.01, 156.65, 155.81, 155.68,
3
3
(500MHz,DMSO‐d ):δ10.88(s,1H),8.06(d, J=8.5Hz,
150.58, 143.87, 137.33, 136.61, 131.42 127.06, 126.69,
122.74, 114.17, 113.86, 112.56, 111.94, 110.93, 106.29,
6
3
3
1H), 7.54 (d, J = 8.5 Hz, 1H), 7.33 (d, J = 8 Hz, 1H),

12 of 15
BARZEGAR AMIRI OLIA ET AL.
+
5
2.58, 35.46, 25.23 ppm. HRMS (ESI ); calcd. For
out with the ORCA 3.0.3 software package with ORCA's
numerical quadrature grid “5” and the resolution‐of‐the‐
identity approximation to speed up the evaluation of
Coulomb integrals and the second‐order perturbative
+
+
C H N O 391.12885 [M + H] , found 391.12844.
22
19 2 5
2
. Photophysical studies
[54]
correlation portion of the PWPB95 double hybrid.
UV/vis absorption spectra were recorded using an
The resulting relative energies and the Cartesian coordi-
nates of all optimised conformers are provided in the
Supporting Information. TD‐DFT calculations were
Agilent 8453 UV/vis absorbance spectrophotometer.
Fluorescence spectroscopy was conducted on
a
[
55]
Horiba Jobin Yvon Fluorolog‐3. Tris (hydroxymethyl)
aminomethane (Tris) (99.8%, Sigma Aldrich) and sodium
chloride were utilised to prepare buffered TBS solutions
carried out with Gaussian09 Revision B.01.
Since the
existence of charge‐transfer (CT) excitations could not
be ruled out, the range‐separated hybrid DFA CAM‐
[
31]
(
50 mM Tris and 150 mM NaCl) in Milli‐Q H O and the
B3LYP
was employed, which has been shown to be
2
pH adjusted to 7.6 with 1.0 M HCl. MAHMA NONOate
reliable for the calculation of vertical singlet‐singlet exci-
tation energies in medium‐sized organic dyes with an
average absolute error of about 0.18 eV and an expected
average overestimation of the excitation energies by
•
(NO donor) was purchased from Sigma Aldrich and
stored in the fridge until used. MAHMA NONOate was
prepared as stock solution (1 mM in aqueous NaCl) and
diluted with TBS to the required concentrations. Stock
solutions of the individual RNS and ROS were prepared
in aqueous media (1 mM in Milli‐Q H O). CuCl ·2H O
[
33]
0.11 eV.
As outlined in the SI, preliminary tests with
an alternative functional approximation gave the same
insights, thus, demonstrating the reliability of our chosen
2
2
2
[
32]
was used to prepare 1 mM CuCl2 stock solutions in
approach. The 6‐311G**
Pople‐type triple‐ζ AO basis
Milli‐Q H O. Stock solutions of CB1‐5 were freshly pre-
pared in DMSO (1 mM) prior to the experiments and then
diluted with TBS as required. Cu(II)‐CB1‐5 solutions
set and the standard Gaussian quadrature integration grid
were used for all calculations. The first seven excitation
energies were calculated together with their oscillator
strengths (f). When required, solvent effects were simu-
lated with Gaussian's default polarizable continuum
2
(
CuCl /CB1‐5 1:1) were prepared immediately prior to
2
the experiments. The solutions for the selectivity mea-
surements were prepared by addition of the RNS and
ROS stock solutions into the Cu(II)‐CB1‐5 solution.
[
56]
solvation model (PCM)
and appropriate dielectric con-
stants and refractive indices for the solvents of interest.
The UV/vis absorption spectra were simulated by the
standard technique of overlapping Gaussian functions
for each transition with a value of 0.4 eV for the half‐
width of the absorption band at a height of 1/e. Excited‐
state geometry optimizations of the first bright state in
each system of the CB5‐type were also carried out at the
TD‐CAM‐B3LYP/6‐311G** level of theory.
3
. Computational methods (see also SI)
Initial sampling of the conformational space of five
different forms of the CB4 and CB5 was accomplished
with the MMX force‐field as implemented in PCMODEL
[46]
9
.3.
Subsequent geometry optimizations were per-
formed at the dispersion‐corrected DFT level with the
[47]
TPSS
density functional approximation (DFA),
4. Confocal microscopy imaging studies
Grimme's DFT‐D3 dispersion correction with Becke‐
[
48]
[49]
Johnson damping
and the def2‐TZVP
triple‐ζ
Overnight cultures of P. aeruginosa were diluted by
100 times in buffered peptone water medium and inocu-
lated in a μ‐slide 8‐well microscopy chamber (Ibidi,
100 μL per well). The chamber was incubated for 24 hours
at ambient temperatures (19°C‐22°C). Then the media
was replaced with freshly prepared 10 mM solution of
Ahlrichs AO basis set (TPSS‐D3/def2‐TZVP). This level
of theory was shown to deliver accurate geometries of
larger molecular systems at low computational cost,
while additionally having an advantage over the com-
monly chosen approaches, such as B3LYP/6‐31G* or
BP86/6‐31G*, by taking into account ubiquitous
London‐dispersion effects and avoiding artefacts due to
buffered peptone water‐KNO and incubated for 3 hours
3
at ambient temperatures (19°C‐22°C). The resultant
[48,50]
basis‐set incompleteness errors.
single‐point calculations at the dispersion‐corrected dou-
Subsequently,
biofilms within the wells were treated with the probes
[
42]
and HCS stains.
The probes (CB4 and CB5) were
[51]
[52]
ble‐hybrid
level (PWPB95 ‐D3/def2‐TZVPP) were
freshly prepared prior to the experiments as 1 mM stock
solutions in DMSO and diluted to 10 μM with TBS for cell
staining. HCS NuclearMaskTM Deep Red stain (HCS)
was purchased from Thermo Fisher Scientific as a
X1000 concentrate in DMSO and stored in the freezer
under exclusion of direct light until used. HCS was
carried out to obtain the relative energies between the
various conformers. This DFA has been proven to be
one of the most accurate DFT methods for the determina-
tion of these types of energies and the calculation of pro-
[53]
ton affinities (PA).
All these calculations were carried

BARZEGAR AMIRI OLIA ET AL.
13 of 15
diluted by a factor of 500 in TBS before use. The resultant
biofilms within the wells were first treated with potas-
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Lars Goerigk http://orcid.org/0000-0003-3155-675X
Uta Wille http://orcid.org/0000-0003-1756-5449
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SUPPORTING INFORMATION
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article.
How to cite this article: Barzegar Amiri Olia M,
Hancock AN, Schiesser CH, Goerigk L, Wille U.
Photophysical insights and guidelines for blue
“
turn‐on” fluorescent probes for the direct detection
•
of nitric oxide (NO ) in biological systems. J Phys Org
Chem. 2018;e3896. https://doi.org/10.1002/poc.3896