NIR absorbing AzaBODIPY dyes for pH sensing

Article abstract of DOI:10.3390/molecules25163689

Two near-infrared (NIR) absorbing di(thien-2-nyl)-di(dimethylanilino)azaBODIPY dyes 2a and 2b were synthesized and characterized that differ depending on whether the dimethylaniline substituents are introduced at the 3,5- or 1,7-positions of the azaBODIPY core. The main spectral bands lie at 824 and 790 nm, respectively, in CH2Cl2. The effect of substituent position on the photophysical and pH sensing properties was analyzed through a comparison of the optical properties with the results of time-dependent density functional theory (TD-DFT) calculations. Protonation of the dimethylamino nitrogen atoms eliminates the intramolecular charge transfer properties of these compounds, and this results in a marked blue-shift of the main absorption bands to 696 and 730 nm, respectively, in CH2Cl2, and a fluorescence "turn-on"effect in the NIR region. The pH dependence studies reveal that the pKa values of the non-protonated 2a and 2b molecules are ca. 6.9 (±0.05) and 7.3 (±0.05), respectively, while that of the monoprotonated species for both dyes is ca. 1.4 (±0.05) making them potentially suitable for use as colorimetric pH indicators under highly acidic conditions.

Full text of DOI:10.3390/molecules25163689

molecules
Article
NIR Absorbing AzaBODIPY Dyes for pH Sensing
Gugu Kubheka 1, John Mack 1,
* *
, Tebello Nyokong 1 and Zhen Shen 2,
1
Institute for Nanotechnology Innovation, Department of Chemistry, Rhodes University,
Grahamstown 6140, South Africa; gugu_kubheka@yahoo.com (G.K.); t.nyokong@ru.ac.za (T.N.)
State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering,
Nanjing University, Nanjing 210046, China
2
*
Correspondence: j.mack@ru.ac.za (J.M.); zshen@nju.edu.cn (Z.S.);
Tel.: +27-46-603-7234 (J.M.); +86-25-8968-6679 (Z.S.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: M. Salomé Rodríguez-Morgade
Received: 23 July 2020; Accepted: 12 August 2020; Published: 13 August 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Two near-infrared (NIR) absorbing di(thien-2-nyl)-di(dimethylanilino)azaBODIPY dyes 2a
and 2b were synthesized and characterized that differ depending on whether the dimethylaniline
substituents are introduced at the 3,5- or 1,7-positions of the azaBODIPY core. The main spectral bands
lie at 824 and 790 nm, respectively, in CH2Cl2. The effect of substituent position on the photophysical
and pH sensing properties was analyzed through a comparison of the optical properties with the
results of time-dependent density functional theory (TD-DFT) calculations. Protonation of the
dimethylamino nitrogen atoms eliminates the intramolecular charge transfer properties of these
compounds, and this results in a marked blue-shift of the main absorption bands to 696 and 730 nm,
respectively, in CH2Cl2, and a fluorescence “turn-on” effect in the NIR region. The pH dependence
studies reveal that the pKa values of the non-protonated 2a and 2b molecules are ca. 6.9 (
±
0.05) and
0.05)
7.3 ( 0.05), respectively, while that of the monoprotonated species for both dyes is ca. 1.4 (
±
±
making them potentially suitable for use as colorimetric pH indicators under highly acidic conditions.
Keywords: aza-BODIPY dye; pH sensing; intramolecular charge transfer; photophysical properties;
pKa values
1. Introduction
Near-infrared (NIR) region absorbing dyes are essential for various applications in fields as diverse
as materials science and medicine [1]. NIR dyes are advantageous in a biomedical context due to
reduced background absorption, fluorescence, and light scattering, resulting in improved sensitivity [
2
].
Boron azadipyrromethene (azaBODIPY) dyes are of particular interest, since the incorporation of an
aza-nitrogen atom into the boron dipyrromethene (BODIPY) chromophore is one of the main strategies
used to red shift the main spectral band into the NIR region [3]. Among the various possible NIR
chromophores, there has been an increasing focus on azaBODIPY dyes in recent years due to their
favorable photophysical properties, such as high photostability, and their structural versatility and
ease of modification at all positions on the azaBODIPY core [4,5]. Different strategies have been
used to develop dyes with highly red-shifted spectral bands, such as attaching electron-donating and
withdrawing group at the 3,5- and 1,7-positions, respectively; replacing the phenyl groups with smaller
five-membered heterocyclic rings [
5–8]; and the addition of fused rings to the pyrrole rings of the
azaBODIDY core [ ]. When electron-donating aryl groups, such as dimethylaniline, are introduced at
9
the 3,5-positions there is a large red-shift of the main spectral band, since there is a marked decrease
in the HOMO–LUMO gap [10]. The red-shifts that are observed when a five-membered ring system
is incorporated into the structure have been ascribed to increased delocalization that arises from
enhanced co-planarity with the azaBODIPY core and reduced torsion angles relative to structures that
Molecules 2020, 25, 3689; doi:10.3390/molecules25163689
www.mdpi.com/journal/molecules

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contain phenyl rings [
5]. Substitution with thiophene rings also provides scope for more effectively
conjugated donor-acceptor compounds [11].
The inclusion of dimethylaniline substituents into the structures of azaBODIPY dyes can generate
pronounced photophysical changes as a result of substrate recognition [12,13], due to changes in the
intramolecular charge transfer (ICT) properties of the dyes. Therefore, this ICT switching mechanism
can potentially be used to form pH indicators [12] or in the detection of small metal ions such
as Ca2+, Cu2+, and Mg2+ when a more elaborate amine receptor is used [14]. ICT systems are
advantageous for sensor applications, since the use of highly fluorescent dyes results in inner-filter
effects, due to the bias introduced by the pH-dependent emission [14]. A limited number of pH-sensitive
amino moiety functionalized azaBODIPY dyes have been previously reported as indicator dyes,
and different spectroscopic methods, such as absorption and fluorescence, have been used to measure
the pH of the probes [1,12,14]. For example, McDonnell et al. reported the sensing properties of
1,7-diphenyl-3,5-di-(dimethylanilino)azaBODIPY (1), which is structurally similar to the dyes reported
herein [12].
In this study, we report the synthesis of dimethylaniline and thien-2-yl substituted aza-BODIPY
dyes 2a and 2b as NIR and pH-responsive chromophores by varying the position of the substituents
between 1,7- and 3,5-positions (Figure 1). The aim of the study is to investigate the effect of the
substituent and position on the photophysical properties and pKa values, and to form azaBODIPY
dyes with spectral bands red shifted beyond 750 nm. To the best of our knowledge, there have been
very few reports on non-protonated azaBODIPY dyes that exhibit no fluorescence emission and hence
provide scope for use as “turn-on” fluorescence pH sensing indicators in the NIR region. The goal was
to use the ICT properties associated with the dimethylaniline groups to achieve this.
Figure 1. A comparison of the molecular structures of 2a and 2b with that of 1, which was reported
previously by McDonnell et al. [12].
2. Results
2.1. Synthesis
AzaBODIPYs 2a and 2b were synthesized according to literature procedures (Scheme 1) [5,15,16]
After initially forming chalcone from the appropriate aryl aldehyde and acetophenone and
subsequently converting it to nitro-derivative , azadipyrromethene was obtained by heating
under reflux with ammonium acetate (NH4OAc) in n-butanol (n-BuOH) for 24 h. After separation on
column chromatography, the BF2 moiety was introduced to using boron trifluoroetherate (BF3 OEt2)
.
3
4
5
4
5
·
and diisopropylethylamine (DIPEA) in CH2Cl2 under reflux for 6 h. The products were isolated as
bluish-purple and blue for 2a and 2b, respectively, in moderately high yields. The target compounds
were characterized by 1H NMR and FTIR spectroscopy and mass spectrometry. The 1H NMR spectrum
of 2a (Figures S1 and S2, Supporting Information) contains peaks at 7.24 (double doublet), 7.69 (double
doublet) and 8.06 (double doublet) ppm that can be assigned on the basis of integration to the
thien-2-yl groups, while the corresponding peaks for 2b lie at 7.23 (double doublet), 7.54 (doublet)
and 8.32 (doublet) ppm (1-, 2- and 3-H in Figures S1 and S2, Supporting Information). Singlets at

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7.37 and 6.99 ppm, respectively, can be attributed to the 2,6-positions of the aza-BODIPY core (4-H in
Figures S1 and S2, Supporting Information). Two doublets at 6.86 and 8.22 ppm were detected in the
spectrum of 2a with coupling constants of 9.6 Hz indicating the presence of two adjacent protons (5-
and 6-H in Figure S1, Supporting Information) that can be assigned to the dimethylaniline moiety, while
the corresponding peaks for 2b peaks lie at 6.77 and 8.07 ppm (5- and 6-H in Figure S2, Supporting
Information). The methyl protons (7- and 8-H in Figures S1 and S2, Supporting Information) lie at
3.15 ppm for 2a and 3.09 ppm for 2b. The chemical shifts for 2a lie upfield of those observed for 2b
.
This provides an indication of more efficient ICT from the donor to the acceptor group. Molecular ion
peaks are observed at m/z = 594.96 for 2a and 595.00 for 2b (Figures S3 and S4, Supporting Information)
in the MS data in close agreement with the calculated value of m/z = 595.19.
Scheme 1. Synthesis of azaBODIPYs 2a and 2b.
2.2. Photophysical Properties
The photophysical properties of 2a and 2b were investigated by UV-visible absorption and
fluorescence emission spectroscopy in solvents of differing polarity. The absorption spectra of 2a and
2b consist of two prominent bands that lie in the 570–600 and 810–860 nm regions for 2a and 660–690
and 770–850 nm regions for 2b (Figure 2). In comparison to the spectrum of
1 in toluene [12], the
absorption band maximum of the main spectral band for 2a is red shifted by 18 nm (Table 1).
Table 1. Wavelengths of the main spectral bands of 2a and 2b in a range of different solvents.
DMSO
CH2Cl2
THF
Toluene
Benzene
Ref.
1
1
2a
2b
—
824
790
—
819
786
798
816
776
—
814
779
[12]
—
—
—
859
810
1
No value available.
Both 2a and 2b exhibit marked solvatochromism (Figure 2 and Table 1). The solvatochromism is
stronger for 2b where the dimethylaniline groups are substituted at the 1,7-positions, since the ICT
character of the S1 state is expected to be particularly strong due to the effect of the electron-donating
effect of these substituents on the HOMO which is expected to have large MO coefficients at the
3,5-positions [17]. Both dyes exhibit linear Beer-Lambert plots in all solvents studied, which points to
an absence of aggregation. In contrast with
1 [12], when the fluorescence properties of 2a and 2b were
studied in polar, polar aprotic and non-polar solvents, no emission was observed. The fluorescence
quenching is related to ICT character in the S1 state that is associated with the presence of the

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electron-donating thien-2-yl and dimethylanilino groups and an electron-deficient azaBODIPY core [18].
The heavy atom effect of the thiophene sulfur atoms is the most likely explanation for differences in the
fluorescence properties of 2a and 2b relative to those of 1 [12].
Figure 2. Solvatochromism effects on the optical properties of 2a and 2b. Normalized UV-visible
absorption spectra of (A) 2a and (B) 2b in dimethylsulfoxide (DMSO), CH2Cl2 and toluene.
2.3. Effect of Protonation in CH2Cl2
The effect of adding trifluoroacetic acid (TFA) to solutions of 2a and 2b in CH2Cl2 on the absorption
and emission spectra was investigated. Protonation of 2a and 2b is accompanied by visible color

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changes, as shown in (Figure 3). O’Shea et al. have demonstrated that protonation disrupts the
dimethylamino lone pair and this eliminates the ICT character of the S1 state, and this results in a blue
shift of the main spectral band and diminished absorbance [19]. In a similar manner, the addition
of TFA to CH2Cl2 solutions of 2a and 2b results in a large blue shift of the main absorption bands
(Figure 3). Upon addition of TFA to the CH2Cl2 solution of 2a solution, new bands are observed
at 772 and 694 nm representing mono- and diprotonated species, respectively. The non-protonated
species band at 824 nm gradually diminishes in intensity. The reasonably sharp isosbestic points at
790 and 746 nm provide an indication that points of equilibrium are reached during the addition of
TFA (Figure 3). When TFA is added to the CH2Cl2 solution of 2b, a new band appears at 731 nm.
The spectra of the mono- and diprotonated species are not as well defined in this case.
Figure 3. The diprotonated ((i) and (iv)) and non-protonated ((ii) and (iii)) structures of 2a and 2b
and the observed colorimetric changes upon protonation with trifluoroacetic acid TFA (TOP). Effect of
protonation (BOTTOM) on the absorption spectra of 2a (A) and 2b (B) in CH2Cl2.
The fluorescence spectra for H22a2+ and H22b2+ in CH2Cl2 are shown in Figure 4. The fluorescence
quantum yields (ΦF) were determined to be 0.04 and 0.14 in CH2Cl2, respectively. Relatively low ΦF
values are expected for these compounds, since the values for NIR-emitting dyes are generally lower
than for those emitting in the visible region [20]. The absence of fluorescence for 2a and 2b provides

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scope for a clear “turn-on” response for protonation in the NIR region. The response was found to be
reversible when triethylamine (TEA) was added to reverse the pH change.
Figure 4. Absorption and emission spectra of H22a2+ (A) and H22b2+ (B) in CH Cl at λ = 600 nm.
ex
2
2
In addition to the UV-visible absorption and fluorescence titration data, 1H NMR spectra were
measured in CDCl3 to identify the site of protonation on 2b by using TFA as a titrant. 2b was
sequentially titrated seven times with 50 µL of concentrated TFA to increase the acidity of the solution,
and a 1H NMR spectrum was obtained at each step. Figure 5 provides a comparison of the 1H NMR
spectra of 2b in CDCl3 after the initial and final titrations. The initial titration results in a 0.14 M TFA
solution and drastically upfield shifted proton signals. It is evident that further titration ultimately
resulted in no significant change in the aromatic and the dimethylamino proton signals, except for the
deshielding effect induced by protonation. This demonstrates that 2b only undergoes protonation at
the amino nitrogen atoms. The shift of the peak for the dimethylamino moieties from 3.08 to 3.37 ppm
demonstrates that protonation occurs at the dimethylamino nitrogens. Initially upon forming the
monocation species, rapid exchange of the proton between the two dimethylamino groups results
in averaged NMR peaks. Subsequently, both groups are protonated when the dication is formed.
The 1H NMR spectra for the TFA titration of the dye (Figure S5, Supporting Information) reveal that an
unusually broad signal due to TFA shifts across the aromatic region to ca. 8.5 ppm with a simultaneous
increase in intensity.
Figure 5. 1H NMR spectroscopic titration of 2b with TFA in CDCl3.

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3. Discussion
3.1. Effect of Protonation in DMSO/Aqueous Solutions
Detailed protonation experiments were carried out in 2:1 (v/v) dimethyl sulfoxide (DMSO)/aqueous
solutions, since both dyes are sufficiently soluble in this medium. In principle, the dyes should be
suitable for use as acidic range pH indicators in the red/NIR spectral region due to the presence of
the dimethylaminostyryl groups at the 1,7- and 3,5-positions. Aliquots of TFA in water were added,
and spectral changes were monitored by UV-visible absorption and emission spectroscopy. The main
azaBODIPY absorption band was found to undergo a significant red shift to 859 nm in 2:1 (v/v)
DMSO/aqueous solution mixtures relative to what is observed in CH2Cl2 (Figure 3). The changes in
absorption spectra in polar solvents upon protonation were similar to those reported above under
TFA titration in CH2Cl2. There was a marked blue shift of the main absorption band to 778 nm for the
monocation species and then to 682 nm for the dication species (Figure 6). Plots of absorbance against
pH were used to identify isosbestic points, which are an indication of complete conversion between
two distinct forms (Figure 6 insets). Reasonably sharp isosbestic points were observed for 2a at pH
1.71 (ca. 800 nm) and at pH 1.14 and 0.23 (ca. 700 nm), and for 2b at pH 1.34 (ca. 745 nm) and pH 1.10
(ca. 700 nm).
Figure 6. The pH dependence of absorbance for 2a in 2:1 (v/v) DMSO/aqueous solution (A) and
the corresponding calibration curve (inset). The pH dependence of absorbance for 2b in 2:1 (v/v)
DMSO/aqueous solution (B) and the corresponding calibration curve (inset).

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A significant increase was observed in the fluorescence intensity at 742 and 754 nm for 2a and 2b
when the acidity of the solutions was increased to very low pH values (Figure 7). Visible color changes
are observed in this context (Figure 8), making the dyes potentially suitable for use as colorimetric
indicators. These changes are consistent with ICT-restricted emission in the presence of dimethylamino
groups, which is more pronounced for 2a.
Figure 7. The pH dependence of emission for (
A
) H22a2+ and (
B
) H22b2+ in 2:1 (v/v) DMSO/aqueous
solution (1.41
(insets).
×
10−6 M), and the corresponding pH calibration curves at the emission band maxima
Figure 8. The colorimetric changes for 2a and 2b under different pH conditions in 2:1 (v/v)
DMSO/aqueous solutions.
3.2. pKa Determination
The dissociation constant (pKa) values were determined to gain insight on their ionization states
with respect to pH. Both dyes were prepared in 2:1 (v/v) DMSO/aqueous buffered solutions (Table S1,

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Supporting Information), and the absorption measurements were made for the dye:buffer mixtures.
Equation (1) was used to obtain approximate pKa values for 2a and 2b [21]:
dM − d
pKa = pH + log
(1)
d − dI
where pH is the value recorded on the pH meter and d is the absorbance of the molecule in the
respective buffers tested. dM and dI are the absorbance of the non-protonated and protonated species,
respectively. To verify the pKa results obtained, the inflection method reported was also used [22].
The inflection point is determined from a plot of absorbance against pH, and a best-fit line is obtained by
fitting the experimental data with a fourth-order polynomial (Figure 9). The results from both methods
were found to be in close agreement. The spectroscopic behavior of both 2a and 2b reveals the presence
of two pKa values. The pKa values of the non-protonated molecules are 6.9 (
for 2a and 2b, respectively. The monoprotonated species for both dyes has a pKa value of 1.4 (
which is an increase of 0.1 compared to the apparent pKa value reported by McDonnell et al. [12] for
the monoprotonated form of 12]. The relative positions of the thien-2-yl and dimethylaniline groups
±
0.05) and 7.3 (
±
0.05)
0.05),
±
1
[
was therefore found to have a negligible effect on the pKa value in this context.
Figure 9. A plot of the observed absorbance values for 2a and 2b at 787 and 708 nm, respectively,
against pH trend for selected buffer solutions (Table S1, Supporting Information). The best-fit line is to
a fourth-order polynomial, the equations are shown, along with the coefficient of determination (R2).
3.3. Molecular Modelling
Theoretical calculations were carried out for the unsubstituted core dye (AzaBDY),
1,3,5,7-tetraphenyl (4Ph), 1,3,5,7-tetradimethylaniline (4An), and 1,3,5,7-tetrathien-2-yl (4Th) substituted
azaBODIPY model complexes, 2a and 2b to obtain additional insight about the electronic structures
and optical spectroscopy of 2a and 2b (Figure 10). The trends predicted in TD-DFT spectra of 2a and
2b match those observed experimentally (Table S2, Supporting Information). The incorporation of
electron-rich dimethylaniline and thien-2-yl rings makes the azaBODIPY ligand results in a large
destabilization of the HOMO energy [23,24]. As can be observed in Figure 10, the substitution of the
thien-2-yl rings at the 1,3,5,7-positions of the 4Th model complex results in a slight stabilization of both
the HOMO and LUMO relative to the 4Ph model complex, while the presence of electron-donating
dimethylaniline rings in the 4An model complex results in a destabilization. In comparison to
AzaBDY, the 4Ph, 4An and 4Th model complexes, and 2a and 2b are predicted to have significantly
smaller HOMO–LUMO energy gaps (Figure 10). The differing arrangements of the substituents at the

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3,5- and 1,7-positions of the aza-BODIPY mainly influences the energy of the HOMO. The stronger
electron-donating character of the dimethylaniline rings and the presence of large MO coefficients at
the 3,5-positions (Figure 10) result in a larger destabilization of the HOMO of 2a than is the case with
2b. Protonation of the dimethylamine nitrogen shifts the electron density to the thien-2-yl groups in
the HOMO (Figure 10), and this increases the HOMO–LUMO gap in a manner consistent with the
observed blue shifts of the main spectral bands (Figures 3, 4 and 6).
Figure 10. The HOMO and LUMO energies obtained from time-dependent density functional theory
(TD-DFT) calculations at the CAM-B3LYP/5-31G(d) level of theory before (A) and after (B) protonation
are highlighted with red lines. When a comparison is made with diprotonated species, the LUMO
energy is set to zero. Small black squares are used to denote occupied MOs. Red diamonds are used to
highlight the HOMO–LUMO band gap values and are plotted against a secondary axis. The angular
nodal patterns of the HOMO and LUMO of the neutral (
are shown at an isosurface of 0.02 a.u.
A) and diprotonated (B) species of 2a and 2b
4. Materials and Methods
4.1. Materials
4-Dimethylaminoacetophenone, 4-dimethylaminobenzaldehyde, 2-acetylthiophene, thiophene-2-
carboxaldehyde, diethylamine (DEA), potassium hydroxide, nitromethane, potassium carbonate,
NH4OAc, n-BuOH were purchased from commercial suppliers in China. BF3 OEt2, TFA, TEA, NH4OAc,
·
sodium sulfate anhydrous, sodium chloride, potassium hydrogen phthalate, potassium hydrogen
phosphate, ethyl actetate, DMSO and DIPEA were purchased from Sigma Aldrich (St. Louis, MO,
USA) and used as received. CH2Cl2, ethanol (EtOH), methanol, potassium chloride and hydrochloric
acid were purchased from Minema (Johannesburg, South Africa). Sodium acetate was purchased
from Saarchem (Johannesburg, South Africa), and acetic acid was purchased from B&M Scientific

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(Cape Town, South Africa). All chemicals were analytically pure and were used as received. Acetone-d6
and CDCl3 for 1H NMR spectroscopy, and spectroscopic grade solvents for optical spectroscopy were
purchased from Sigma-Aldrich and Merck (Darmstadt, Germany), respectively.
4.2. Equipment
1H NMR spectra were recorded on a Bruker AMX 600 spectrometer (Billerica, MA, USA) at
600 MHz. Mass spectra were determined on a Bruker AutoFLEX III Smartbeam TOF MALDI-TOF
mass spectrometer. The instrument was operated in positive ion mode with a 337 nm nitrogen laser
and the data were obtained by using α-cyano-4-hydroxycinnamic acid as the matrix. UV-visible
absorption spectra were measured on a PerkinElmer Lambda 950 spectrophotometer (Waltham, MA,
USA) operated over a wavelength range of 300–1200 nm. Fluorescence spectra were measured on a
Varian Eclipse spectrofluorometer (Palo Alto, CA, USA). Fourier transform infrafred (FT-IR) spectra
were recorded on an Alpha II (100 FT-IR) spectrometer (Bruker, Billerica, MA, USA) with a universal
attenuated total reflectance (ATR) sampling accessory. All the data were obtained from neat particles.
The pH values for the buffer solutions were measured using an 86555 Laboratory Benchtop digital pH
meter (AZ Instrument Corp., Taichung City, China).
4.3. Synthesis
The first step in the preparation of 2a and 2b was the preparation of the appropriate chalcones 3
and nitro-derivatives 4 (Scheme 1). Potassium hydroxide (0.16 mol) dissolved in 5 mL water was added
at room temperature to a 50 mL solution of the appropriate aryl ketone (11.41 mmol) in ethanol. After
stirring for 5 min, an ethanol solution of the appropriate aryl aldehyde (11.41 mmol), and the mixture
was stirred for 24 h. The precipitated products were filtered off, washed with aqueous ethanol, and
dried. Recrystallization from methanol gave chalcones
3 as light-yellow solids in moderate yields [15].
3
(100 mmol), nitromethane (500 mmol), and K2CO3 (0.2 mmol) were dissolved in ethanol (50 mL)
and heated to reflux for 12 h. After cooling to rt, the solvent was removed in vacuum and the oily
residue obtained was dissolved in ethyl acetate and washed with water three times. The combined
organic layers were washed with brine, dried over sodium sulfate, and concentrated to give
4 as
yellowish oily residues in nearly quantitative yields, which were used in the next step without further
purification [16].
Azadipyrromethenes
5 were used to prepare 2a and 2b (Scheme 1). A mixture of 4 (1 mmol)
and NH4OAc (20 mmol) was refluxed in n-BuOH (50 mL) for 24 h. After cooling to rt, the reaction
mixture was diluted with water and extracted three times with CH2Cl2. The combined organic layers
were washed with water and brine, dried with sodium sulfate, and concentrated to give the crude
intermediate product
prior to use to form the target compounds. DIPEA (0.8 mL, 4.8 mmol) was added to a solution of
(1 mmol) in CH2Cl2 (50 mL), and the mixture was stirred for 1 h. BF3 OEt2 (0.6 mL, 5.0 mmol)
5 as a dark blue-black solid, which was then purified by column chromatography
5
·
was then added at rt, and the resulting mixture refluxed until the starting material was completely
converted (ca. 2 h) to the target product. The progress of the reaction was monitored by thin-layer
chromatography (TLC). The cold reaction mixture was diluted with water and extracted twice with
CH2Cl2 (100 mL). The combined organic layers were dried with sodium sulfate and concentrated under
vacuum. The crude products were purified by flash column chromatography using CH2Cl2/hexane
(2:1) as eluent. Target compounds 2a and 2b were obtained as shiny coppery crystals:
2a was obtained in 62% yield; UV-vis (DMSO)
(=C-H stretch, 858 (-C-H stretch), 1666 (C=N stretch), 1598–1543 (Ar C-C), 1368–1030 (C-N stretch);
1H NMR (600 MHz, Acetone-d6):
/ppm 3.15 (12H, s, NCH3), 6.86 (4H, d, J = 9.6 Hz, Ar-H), 7.24 (2H,
λ/nm (log ε) 859 (4.8); IR (FT-IR) ν
max/cm−1: 2952
δ
dd, J = 0.9 and 5.1 Hz, Ar-H), 7.37 (2H, s, Ar-H), 7.69 (2H, dd, J = 0.9 and 5.1 Hz, Ar-H), 8.06 (2H, dd,
J = 1.2 and 3.6 Hz, Ar-H), 8.22 (4H, d, J = 9.6 Hz, Ar-H); MS (MALDI-TOF): Anal. calc. m/z 595.19;
Found: [M]+ 594.96; Anal. calc. for [C32H28BF2N5S2]: C, 64.54; H, 4.74; N, 11.76; S, 10.77; Found: C,
64.30; H, 4.68; N, 11.80, S, 10.80.

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2b was obtained in 74% yield; UV-vis (THF)
λ/nm (log
ε
) 786 (4.7); IR (FT-IR)
ν
max/cm−1: 2952
(=C-H stretch), 2857 (-C-H stretch), 1667 (C=N stretch), 1598–1543 (Ar C-C), 1368–1030 (C-N stretch);
1H NMR (600 MHz, CDCl3):
δ
/ppm 3.09 (12H, s, NCH3), 6.77 (4H, d, J = 9.0 Hz, Ar-H), 6.99 (2H, s,
Ar-H), 7.23 (2H, dd, J = 3.6 and 4.8 Hz, Ar-H), 7.54 (2H, d, J = 4.2 Hz, Ar-H), 8.07 (4H, d, J = 9.0 Hz,
Ar-H), 8.32 (2H, d, J = 3.6 Hz, Ar-H); MS (MALDI-TOF): Anal. calc. m/z 595.19; Found: [M]+ 595.00;
Anal. calc. for [C32H28BF2N5S2]: C, 64.54; H, 4.74; N, 11.76; S, 10.77; Found: C, 64.50; H, 4.60; N, 11.68,
S, 10.82.
4.4. Photophysical Parameters
A comparative method was used to determine fluorescence quantum yield (ΦF) values:
F Astd η2
ΦF = ΦF(std)
×
(2)
Fstd A η2
std
where F and Fstd are the areas under the fluorescence curves of 2a and 2b and the reference, respectively.
A and Astd are the absorbances of sample and reference at the excitation wavelength, and η2 and η2
std
are the refractive indices of solvents used for the sample and the reference measurements, respectively.
IR-820 was used as the standard; ΦF = 0.044 in methanol [25].
4.5. pH Studies
The effect of protonation on 2a and 2b was investigated through the addition of aliquots of a
dilute TFA solution in CH2Cl2. 3000
water was prepared and used for the pH studies. Concentrations of 3.2
of 2a and 2b, respectively, were used in 4 mL of solution. A total of 50 L of the TFA solution was
µL of TFA in 7000
µL (6000
µ
L in the case of 2b) of Millipore
×
10−6 and 3.6 10−5
×
M
µ
added gradually, and changes in pH were monitored. The buffer solutions were prepared according to
literature procedures [26] with slight modifications (Table S1, Supporting Information). A UV-visible
absorption spectroscopy method was used to determine the pKa value. Stock solutions of 1.5
×
10−6
and 1.9
×
10−5 M of 2a and 2b, respectively, were prepared in DMSO. The vials were filled with different
buffers of constant ionic strength (I = 1 M), ranging from pH 0.4 to 9.0. A fixed amount of stock solution
for both compounds was added to each vial and mixed, except in vials that were used as blank buffers
to provide correction factors.
4.6. Theoretical Calculations
The Gaussian 09 software package [27] was used to carry out density functional theory (DFT)
geometry optimizations at the B3LYP/6-31G(d) level of theory for 2a, 2b and a series of model complexes.
Time dependent-DFT (TD-DFT) calculations were carried out by using the CAM-B3LYP functional
with 6-31G(d) basis sets. The CAM-B3LYP functional was used since it contains a long-range correction
that makes it more suitable for use with dyes that exhibit ICT character.
5. Conclusions
Two pH-sensitive NIR absorbing azaBODIPY dyes have been successfully synthesized and
characterized. The introduction of electron-donating dimethylaniline and thien-2-yl groups results in
unusually large red shifts of the main azaBODIPY absorption band well into the NIR region at 824
and 790 nm, respectively, in CH2Cl2. A larger red shift is observed for 2a, since the more strongly
electron-donating dimethylaniline rings lie at the 3,5-positions, which have large MO coefficients in
the HOMO. A relative destabilization of the HOMO results in a narrowing of the HOMO–LUMO
gap. In bulk solution, the dyes are only fluorescent under acidic conditions, due to suppression of
the ICT process upon protonation. This provides scope for a pH-dependent fluorescence “turn-on”
effect. 2a with strongly electron-donating dimethylaniline rings at the 3,5-positions has a pKa value of
6.9 (±
0.05) and is more strongly acidic than 2b, which has a pKa value of 7.3 ( 0.05). The monoprotonated
±

Molecules 2020, 25, 3689
13 of 14
species of 2a and 2b were estimated to have pKa values of 1.4 (
under acidic conditions demonstrate that 2a and 2b are good candidates for use as pH indicators under
±
0.05). The visible colorimetric changes
strongly acidic conditions.
Supplementary Materials: The following are available online. Figure S1: 1H NMR spectrum of 2a in acetone-d6,
Figure S2: 1H NMR spectrum of 2b in CDCl3, Figure S3: MS data for 2a, Figure S4: MS data for 2b, Figure S5:
Stacked 1H NMR spectra for 2b titrated with TFA in CDCl3, Table S1: Preparation of buffer solutions of constant
ionic strength (I = 1 M), Table S2: Calculated electronic excitation spectra of 2a and 2b at the CAM-B3LYP/6-31G(d)
level of theory.
Author Contributions: Conceptualization, G.K., J.M. and Z.S.; methodology, G.K., J.M. and Z.S.; validation, G.K.,
J.M. and Z.S.; formal analysis, G.K.; investigation, G.K.; resources, J.M., T.N. and Z.S.; data curation, G.K., J.M. and
Z.S.; writing—original draft preparation, G.K.; writing—review and editing, G.K., J.M., T.N. and Z.S.; supervision,
J.M. and T.N.; project administration, J.M., T.N. and Z.S.; funding acquisition, J.M., T.N. and Z.S. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by a China-South Africa joint research program (CS08-L07 and uid: 95421) to
Z.S. and J.M., Competitive Support for Unrated Researchers and Incentive Support for Rated Researchers grants
from the National Research Foundation of South Africa (uids: 93627 & 119259) to J.M., by the National Natural
Science Foundation of China (No. 21771102) to Z.S., the Department of Science and Technology (DST) and NRF of
South Africa through a DST/NRF South African Research Chairs Initiative for Professor of Medicinal Chemistry
and Nanotechnology (uid: 62620) to T.N., and an NRF-DAAD bursary to G.K.
Acknowledgments: Photophysical measurements were made possible by the Laser Rental Pool Programme of
the Council for Scientific and Industrial Research (CSIR) of South Africa. The theoretical calculations were carried
out at the Centre for High-Performance Computing in Cape Town.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of compounds 2a and 2b are available from the authors.
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