Carbazole-Linked Near-Infrared Aza-BODIPY Dyes as Triplet Sensitizers and Photoacoustic Contrast Agents for Deep-Tissue Imaging

Article abstract of DOI:10.1002/chem.201605702

Four new N-ethylcarbazole-linked aza-boron-dipyrromethene (aza-BODIPY) dyes (8 a,b and 9 a,b) were synthesized and characterized. The presence of the N-ethylcarbazole moiety shifts their absorption and fluorescence spectra to the near-infrared region, λ≈650–730 nm, of the electromagnetic spectrum. These dyes possess strong molar absorptivity in the range of 3–4×10⁴ m^?1 cm^?1 with low fluorescence quantum yields. The triplet excited state and singlet oxygen generation of these dyes were enhanced upon iodination at the core position. The core-iodinated dyes 9 a,b showed excellent triplet quantum yields of about 90 and 75 %, with singlet oxygen generation efficiency of about 70 and 60 % relative to that of the parent dyes. Derivatives 8 a,b showed dual absorption profiles, in contrast to dyes 9 a,b, which had the characteristic absorption band of aza-BODIPY dyes. DFT calculations revealed that the electron density was spread over the iodine and dipyrromethene plane of 9 a,b, whereas in 8 a,b the electron density was distributed on the carbazole group and dipyrromethene plane of aza-BODIPY. The uniqueness of these aza-BODIPY systems is that they exhibit efficient triplet-state quantum yields, high singlet oxygen generation yields, and good photostability. Furthermore, the photoacoustic (PA) characteristics of these aza-BODIPY dyes was explored, and efficient PA signals for 8 a were observed relative to blood serum with in vitro deep-tissue imaging, thereby confirming its use as a promising PA contrast agent.

Full text of DOI:10.1002/chem.201605702

A Journal of
Accepted Article
Title: Carbazole Linked NIR Aza-BODIPY Dyes as Triplet Sensitizers
and Photoacoustic Contrast Agents for Deep Tissue Imaging
Authors: Nagaiyan Sekar, Ramaiah D, Yogesh Gawale, Adarsh N.,
Joseph J, M. Pramanik, and S K Kalva
This manuscript has been accepted after peer review and appears as an
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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
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201605702
Link to VoR: http://dx.doi.org/10.1002/chem.201605702
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Carbazole Linked NIR Aza-BODIPY Dyes as Triplet Sensitizers
and Photoacoustic Contrast Agents for Deep Tissue Imaging
[
a]
[b]
[c]
[b]
Yogesh Gawale , Nagappanpillai Adarsh , Sandeep Kumar Kalva , Joshy Joseph , Manojit
[c]*
[d]*
[a]*
Pramanik , Danaboyina Ramaiah and Nagaiyan Sekar
Abstract: Four novel N-ethylcarbazole linked aza-BODIPY dyes
8a-b and 9a-b) were synthesized and characterized. The presence
oxygen through photosensitization is one of the most utilized
method in recent years.[1] Due to the spin-allowed nature of the
reactions, singlet oxygen exhibits high reactivity towards most of
the organic molecules and thus it has the ability to interrupt the
cellular functions in living organisms. Photodynamic therapy is
based on the formation of reactive oxygen species (ROS),
specifically singlet oxygen, upon activation of the photosensitizer
by light.[2] It damages the biological tissues by subsequent
oxidation. Hence, the quantitative generation of singlet oxygen
(
of N-ethylcarbazole moiety shifts their absorption and fluorescence
spectra to the near-infrared region, ca. 650-730 nm, of
electromagnetic spectra. These dyes possess strong molar
4
-1
-1
absorptivity in the range of 3-4 x 10 M cm with low fluorescence
quantum yields. The triplet excited state as well as singlet oxygen
generation of these dyes were enhanced upon iodination at the core
position. The core iodinated dyes 9a-b showed excellent triplet
quantum yield of ca. 90% and 75% with singlet oxygen generation
efficiency of ca. 70% and 60% when compared to the parent dyes.
The derivatives 8a-b showed dual absorption profiles in contrast to
the dyes 9a-b, which has the characteristic absorption band of the
aza-BODIPY dyes. DFT calculations revealed the electron density
spread over the iodine and dipyrromethene plane of 9a-b, whereas
in 8a-b the electron density distributed on carbazole group as well as
dipyrromethene plane of aza-BODIPY. The uniqueness of these
aza-BODIPY systems is that they exhibit efficient triplet state
quantum yields, high singlet oxygen generation yields and good
photostability. Further we explored the photoacoustic (PA)
characteristics of these aza-BODIPY dyes, and observed efficient
PA signals for 8a compared to blood serum with in vitro deep tissue
imaging, thereby confirming its use as a promising photoacoustic
contrast agent.
(Φ

) is one of the important requirement of an efficient
[3]
photosensitizer (PS).
Most of the photosensitizers used for the generation of
singlet oxygen comprises of a common cyclic tetrapyrrolic
structure, derived from porphyrins. Apart from this several non-
porphyrinic systems reported in the literature to serve the
purpose of singlet oxygen generation and these include
methylene blue, Nile blue, Nile red, Rose bengal,
[4]
chalcogenopyrylium salts, squaraine dyes etc. However, the
drawbacks of such classes of PS are low molar absorption
coefficients in the near infra-red (NIR) region and dark toxicity.
An ideal PS should possess good stability and should have high
absorption in the range of 600-800 nm, below which the light will
be absorbed by different tissues and above which it does not
provide energy to excite oxygen to its singlet state.[5]
Recently, the chemistry of dipyrromethene ligands and
their boron complexes (BODIPY) has attracted much attention
due to their favorable photophysical properties such as intense
absorption at long wavelength with high molar absorption
Introduction
coefficients (ε>104 M cm ), moderate to quantitative
fluorescence quantum yields and chemical properties, which
include tunability through substitution at different positions.[
Aza-dipyrromethenes and aza-BODIPY dyes were first
described several years ago as blue pigments, but now are
-1
-1
There are four major reactive oxygen species (ROS) comprising
superoxide (O .-
.
2
), hydroxyl radical ( OH), hydrogen peroxide
6]
(Δ
(
H
2
O
2
), and singlet oxygen O
2
g
), but all these species show
different level of activity and kinetics. The hydroxyl radical and
singlet oxygen are more toxic and acutely lethal compared to
superoxide and hydrogen peroxide. The generation of singlet
rapidly emerging as
photophysical properties.
a class with desirable near infrared
[7]
The strong absorption of light in the NIR region has also
finds potential application of these dyes as photoacoustic (PA)
contrast agent.[ PA imaging is gaining attention as an emerging
deep tissue biomedical imaging modality that incorporates rich
optical contrast and high ultrasound resolution. Due to this
added advantage, PA imaging finds many applications in breast
cancer imaging, brain imaging, tumour angiogenesis, molecular
imaging, sentinel lymph node imaging, vascularisation
[
a]
b]
Y. Gawale, Prof. N. Sekar
Dyestuff Technology Department,
Institute of Chemical Technology,
Matunga, Mumbai, 400 019, India,
n.sekar@ictmumbai.edu.in, nethi.sekar@gmail.com
8]
[
Dr. N. Adarsh, Dr. J. Joseph
Chemical Sciences and Technology Division
CSIR-National Institute for Interdisciplinary Science and
Technology, Thiruvananthapuram - 695 019, Kerala, India
[
c]
S. K. Kalva, Dr. M. Pramanik
School of Chemical and Biomedical Engineering
Nanyang Technological University
[9]
monitoring and so on. Moreover, these contrast agents help in
targeted PA imaging and molecular imaging with high contrast
and sensitivity.[
10]
62 Nanyang Drive, Singapore 637459,
manojit@ntu.edu.sg
[d]
Dr. D. Ramaiah
CSIR-North East Institute of Science and Technology,
Jorhat 785 006, Assam, India
d.ramaiah@gmail.com
Supporting information for this article is given via a link at the end of
the document.
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Chemistry - A European Journal
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FULL PAPER
O
Ethyl Iodide/
KOH
Acetone, RT
DMF/POCl3
8
N
N
NH
00C, 4h
3h
1
2
3
O
X
N
N
O N
4
a-b
O
2
O
2.5M KOH,
CH3NO2
4
NH OAc
MeOH
4h
DEA, CH OH
reflux 24h
Butanol, Reflux
24h
N
N
N
X
3
N
X
2
N
HN
5a-b
6a-b
N
7
a-b
X
X
N
N
TEA, BF -OEt2
NIS
3
DCM, 400C
N
B
a) X = H
b) X = Br
N
CHCl -AcOH (3:1),
3
I
I
5
h
25oC, 2h
N
N
N
N
B
F F
F F
X
X
X
8a-b
X
9a-b
Scheme 1: Synthetic route for the aza-BODIPY dyes 8a-b and 9a-b.
Herein, we designed a new series of carbazole linked aza-
BODIPY derivatives with an objective of developing new and
efficient photosensitizers and photoacoustic imaging agents that
exhibit near infrared absorption in the range of ca. 600-800 nm.
We synthesized a series of the N-ethylcarbazole linked aza-
BODIPY dyes, characterised and have investigated their
photophysical properties, triplet excited state and singlet oxygen
generation efficiencies. The photoacoustic characteristics of the
dyes were investigated and the derivative, 8a was found to be
efficient for in vitro deep tissue imaging application.
triethylamine at 40°C in dichloromethane for 5 h. The controlled
iodination of 9a-b was carried out by modifying the reported
procedures using NIS as the iodinating agent and achieved 60-
65% yield.[3a]
Absorption
and
fluorescence
properties:
N-
Ethylcarbazole linked aza-BODIPY dyes, 8a-b showed very
distinct photophysical properties as compared to their iodinated
derivatives, 9a-b. The non-iodinated dyes 8a-b showed dual
absorption in the red region (610-720 nm) and having molar
4
-1
-1
extinction coefficients 3.0 and 3.2 × 10 M cm , (Figure S27,
Supporting Information) respectively. Interestingly, for the core
iodinated aza-BODIPY derivatives 9a-b, we observed a single
absorption profile with a maximum at ca. 645-655 nm (Figure 1A,
S24, Supporting Information), with an enhanced molar
Results and Discussion
4
-1
-1
Synthesis and characterization: The synthetic strategy
adopted for the synthesis of N-ethylcarbazole linked aza-
BODIPY 8a-b and 9a-b using different acetophenone derivatives
is shown in Scheme 1. The aza-BODIPY derivatives 8a-b and
absorptivity of ca. 3.7 and 4.1 × 10 M cm , respectively. All
these aza-BODIPY derivatives exhibited good solubility in
common organic solvents such as CH
DMSO.
3
3
CN, CHCl , THF and
9
a-b were synthesized in a facile six step route starting from
A)
carbazole 1. N-Ethylation of carbazole by ethyl iodide in
acetonitrile yielded 2, which upon Vilsmeier-Haack formylation
gave the corresponding formyl derivative 3, in good yield.
0.8
B)
6
52 nm
705 nm
9
8a
a
610 nm
6
0
0
0
0
.6
.4
.2
8a
Further, aldol condensation reaction between
acetophenone derivatives 4a-b yielded the respective chalcones
a-b. The addition of nitromethane to the chalcone in presence
of diethylamine gave the adducts 6a-b, which upon refluxing
with ammonium acetate in n-butanol for 20 gave the
3
and the
4
2
0
9a
5
.0
4
00
600
, nm
800
750
800
, nm
850
h

corresponding aza-dipyrromethenes 7a-b as condensation
products. The aza-dipyrromethenes 7a-b were subsequently
converted to the targeted aza-BODIPY derivatives 8a-b (35-
Figure 1: A) Absorption and B) fluorescence spectra of the representative
aza-BODIPY derivatives 8a and 9a in acetonitrile (20 µM each).
40%) by reacting with borontrifluoride diethyletherate and
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Table 1. Summary of photophysical, electrochemical and theoretical parameters for the aza-BODIIPY dyes 8a-b and 9a-b
Triplet and singlet oxygen
parameters
E
HOMO
E
LUMO
E
gap
Stokes
shift
λ
abs
Ɛ
max
λ
em
(nm)
 [
a]
f
Dyes
(
nm)
(M-1cm-1
)
Experi
Experi
Exper
iment
-1
[b]
[c]
[d]
Δ
(
cm )
Theory
Theory
Theory

T

T

ment
-5.322
-5.343
-4.082
-4.16
ment
-3.86
-3.899
-2.69
-2.67
4
4
4
4
8
a
a
705
653
723
649
818
815
820
830
0.22
0.03
0.03
0.32
1959.46
3067.49
1636.14
3360.13
-5.297
-3.583
-5.324
-3.645
-3.381
-1.050
-3.461
-1.102
1.462
1.444
1.39
1.916
2.533
1.863
2.544
e
e
1
70
1
3.0 x 10
3.7 x 10
3.2 x 10
4.1 x 10
9
90
E
1.15
e
8
b
b
9
1.48
75
1.64
61
[
a] Absolute quantum yield in % [b] Quantum yield of triplet state, were calculated by using the triplet–triplet energy transfer in % [c] Triplet state lifetime [d]
1
Quantum yeild of singlet oxygen, were quantified through scavenging of
2
O by DPBF [e] Triplet is not observed in Acetonitrile.
Figure 1B (Figure 26, Supporting Information) shows the
emission spectra of the corresponding N-ethylcarbazole linked
aza-BODIPY derivatives in acetonitrile. The aza-BODIPY
derivatives 8a-b showed emission in the region 815-825 nm with
Stokes shifts of 113 nm and 97 nm, respectively. Interestingly,
the core iodine substituted aza-BODIPY derivatives 9a-b, also
showed emission maxima in the region 815-830 nm marking
large Stokes shifts values of 163 nm and 181 nm, respectively.
Determination of triplet excited state quantum yields:
The transient intermediates such as triplet excited states
involved in these systems were characterized and quantified
through nanosecond laser flash photolysis. The time-resolved
transient absorption spectra of the compounds were studied.
Figure 2 shows the transient absorption spectra of 9a in
acetonitrile obtained after 355 nm laser excitation (10 ns, 50 mJ/
pulse). For the derivative 9a, the transient absorption spectra
has two peaks at 320 nm and 460 nm with a strong bleach in the
region corresponding to its ground state absorption. The
transient was characterized as triplet excited state using oxygen
quenching experiments as reported previously.[11, 6d] The triplet
formed from 9a within laser pulse, decayed by a first-order
process with a lifetime value of 1.15 µs (Inset of Figure 2).
In case of dye 9b, similar transient absorption spectrum
observed having bands at 320 nm and 460 nm formed upon the
excitation with 355 nm laser (Figure S28, Supporting
Information). Triplet quantum yields of 9a and 9b derivatives
were determined using triplet-triplet energy transfer to β-
carotene using tris(bipyridyl)ruthenium(II) complex as the
reference (Table 1).[7c, 12]
Quantification of singlet oxygen generation: Enhanced
triplet quantum yields are favorable for the efficient generation of
singlet oxygen and therefore we have examined the singlet
oxygen generation efficiency of the derivatives 9a and 9b in
acetonitrile. Quantum yields of singlet oxygen generation were
determined by monitoring the photooxidation of 1,3-
diphenylisobenzofuran
spectroscopy.[13] For this,
(DPBF)
through
absorption
a
solution of the aza-BODIPY
derivative and DPBF were irradiated using 630 nm long pass
over a time period of 0-10 s and the decrease in the absorption
band of (~10%) of DPBF at 409 nm was monitored (Figure 3A).
Yields for the generation of singlet oxygen were calculated using
the standard, Methylene Blue (MB), and by plotting the ∆OD of
DPBF against the irradiation time (Figure 3B). The non-iodinated
aza-dipyrromethenes 8a and 8b showed negligible singlet
oxygen generation efficiency.
a
f
460 nm
A)
0
B)
0
0
0
.07
.00
0
s
.3
.2
9
a
.06
.03
0
1
0 s
MB
0

0.1
0.0
0
0
0
.09
.06
.03
0.00

400
600
, nm
800
0
3
6
9
f
t, s
-0.07
0.00

Figure 3: A) Absorption spectra of diphenylisobenzofuran (DPBF) upon
irradiation in presence of 9a from 0 to 10 s (recorded at 2 s interval) in ACN,
B) straight line plot between the changes in absorption of DPBF with the
irradiation time.
0
.0 0.5 1.0 1.5
a
t, s
400
600
800

, nm
From the previous reports, it is clear that the tuning of
singlet oxygen generation efficiencies of the aza-BODIPY
derivatives can be achieved by suitably incorporating the heavier
halogen atoms such as iodine and bromine at the core as well
Figure 2: Transient absorption spectra of A) 9a following 355 nm laser pulse
excitation; time-resolved absorption spectra recorded at (a) 0.5, (b) 0.2 (c) 0.1,
(
d) 0.05, (e) 2 μs. Inset shows the decay of the transient at 460 nm.
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as at the peripheral phenyl rings.[3a] We have observed the
similar effect for the carbazole linked aza-BODIPY dyes 8a-b
and 9a-b. The aza-BODIPY derivative, 8a showed negligible
singlet oxygen generation efficiency while a slight improvement
was observed with peripheral substitution with bromine as in 8b
for dyes 8a and 8b, the electron density in HOMO is localized
only on the pyrrolic position and the aza-BODIPY core.
(
Table 1). On the other hand, a significant enhancement in
singlet oxygen generation was observed upon iodination at core
position (9a). To summarize, the derivatives 9a and 9b, having
two iodine atoms at the core of the pyrrole exhibited singlet
oxygen quantum yield of 70% and 60%, respectively (Figure 3,
S29, Supporting Information) when compared to the standard
MB (Φ
∆
= 52%).[14] The negligible changes in the absorption
spectra after irradiating an oxygen saturated solution of these
dyes for 2 h, indicates their photostability under these conditions.
Electrochemical properties: We have investigated the
oxidation and reduction potentials of 8a-b and 9a-b using cyclic
voltammetry and square wave voltammetry techniques in
acetonitrile using 0.1 M TBAPF
6
as the supporting electrolyte.
and scanned at a rate
Acetonitrile solutions were purged with N
2
-
1
of 100 mV s .The redox potentials were calculated using the
potentials obtained from the square wave voltammograms
(
Figure S30, Supporting Information). Table 1 lists the calculated
HOMO-LUMO energies of the compounds 8a-b and 9a-b. It can
be seen that unlike ferrocene, which showed single oxidation
wave at 0.38 V,[15] all the aza-BODIPY derivatives exhibited two
oxidation and two reduction waves in their cyclic
voltammograms (CV). Incorporation of the iodo atom at core
position of aza-BODIPY 8a and 8b has a negative effect on the
oxidation potentials (EOX). The dyes 8a (EOX= 5.322 V) and 8b
Figure 4: Energy level diagram containing the HOMO-1, HOMO and LUMO
levels of 8a and 9a calculated at B3LYP/6-31g level.
DFT results suggested that iodination at the core position
of aza-BODIPY affects the orientation of carbazole group at the
peripheral position. The dihedral angle of 8a (1c-2c-3c-4c)
(EOX=5.343 V) possess higher oxidation potential as compared
to 9a (EOX= 4.082 V) and 9b (EOX=4.16 V), by 1.24 V and 1.18 V.
This can be attributed to the presence of iodine atom at the core
position, which leads to the rigidification of the core and hence
the reduction processes become more feasible for the aza-
BODIPY 8a and 8b. The HOMO values of the aza-BODIPY
derivatives increases in the order 8b<8a<9b<9a.
0
between the carbazole group and dipyrromethene plane is 21 ,
0
whereas the dihedral angle of 9a increases to 41 (Figure 5).
Similar observations noticed in case of aza-BODIPY 8b and 9b,
0
0 [7i]
in which the dihedral angle increased from 20 to 41 . Hence
we have observed reasonable enhancement in band gap of
iodinated derivatives 9a and 9b as compared to 8a and 8b.
Frontier molecular orbital energies.The energy levels of
the frontier molecular orbitals (FMOs), especially HOMO-1,
HOMO, LUMO and their spatial distributions are useful to predict
the behavior of the molecules in terms of their electronic
structures, photophysical properties, and photostability. Herein
we report the contour plots of the HOMO-1, HOMO and LUMO
of dyes 8a, 9a, 8b and 9b in acetonitrile as shown in (Figure 4,
S31, Supporting Information). These energy values were
calculated by employing B3LYP/6-31G level of theory for aza-
BODIPY 8a, 8b, 9a, and 9b. These states encompasses a
complex orbital contribution, beyond the crude HOMO−LUMO,
so that barely considering these two frontier orbitals would not
provide accurate energy gap [16, 6f] and hence deviated more
compared to the experimental energy gap. HOMO-1 orbital of
dye 8a and 8b clearly indicate the electron density is situated
more on the carbazole ring as compared to the aza-BODIPY
core. Whereas, HOMO-1 orbital of dye 9a and 9b indicate the
whole electron density is situated on pyrrole core towards iodine
atom due to its heaviness. In the case of HOMO of dye 8a and
Figure 5. Optimized geometry of 8a and 9a at B3LYP/6-31g level.
Photoacoustic spectrum (PAS). According to the
Kasha’s rule,[17] the non-radiative decay (S
→S ) is responsible
n 1
for the enhancement in PA signal, followed by the competition
between radiative and non-radiative pathways for the S1→S0
transition. An organic material having strong NIR absorption and
long-lived S1 excited state (life time of excited state) facilitating
excited-state absorption is expected to produce an enhanced PA
8b, the electron density is distributed almost uniformly over the
entire chromophore. Interestingly, for the dyes 9a and 9b, unlike
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Chemistry - A European Journal
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signal.[18] We further attempted to understand the potential of
aza-BODIPY dyes as molecular photoacoustic contrast agents
in solution as well as in the tissue. We have initially performed
the photoacoustic study on the aza-BODIPY dyes (8a-b, 9a-b) in
acetonitrile at a concentration of 30 µM. The PA spectra were
measured in the wavelength range from ca. 670-970 nm with an
interval of 5 nm. The PAS of each sample is compared to that of
animal blood. Figure 6A (Fig. S32, Supporting Information)
shows the PA spectrum of samples 8a-b, 9a-b dissolved in
acetonitrile with respect to the PA spectrum of blood. Peak to
peak normalized PA signal has been calculated at each
wavelength.
(Figure 7B). Figures 7C and 7D show the reconstructed cross-
sectional photoacoustic tomography (PAT) images at 1 cm and
2 cm depths, respectively. The signal-to-noise ratio (SNR) at ca.
1 cm depth for 8a is measured to be 53.15 dB and for blood
29.63 dB. Notably at ca. 2 cm depth the SNR for 8a is found to
be 42.23 dB whereas the blood tube was not visible at all. The
PA signal for the aza-BODIPY derivative, 8a is clearly seen even
at the depth of 2 cm. Hence, the aza-BODIPY dye, 8a can be
used as a promising photoacoustic contrast agent that produces
rich contrast during in vitro PAT imaging
Figure 6: Photoacoustic spectra of aza-BODIPY dyes in acetonitrile (30 uM).
(
(
A) PA spectra of 8a, 8b, 9a, and 9b in solvent ACN compared to that of blood.
B) PA signal comparison of sample 8a in ACN with blood and water (control
data) at 675 nm.
Figure 7. Deep tissue cross-sectional photoacoustic tomography (PAT)
imaging of aza-BODIPY dye, 8a and blood embedded inside chicken breast
tissue using OPO-PAT system. (A) LDPE tubes filled with 8a and blood. (B)
Layers of chicken tissue in stacks inside which blood and 8a were placed. (C)
Cross-sectional PAT image at 1 cm depth. (D) Cross-sectional PAT image at 2
cm depth.
The PA signal of dyes 8a and 8b are higher than that of
blood compared to the rest of the samples at 675 nm. The PA
signal of the core iodinated derivatives 9a-b are much lower
than that of blood which may be due to the enhanced
intersystem crossing of 9a-b, that results in the shortening of S1
lifetime. Also the iodo atom at the core position of the aza-
BODIPYs 9a and 9b push the carbazole group out of plane to a
major extent and does not allow taking part in direct conjugation
with dipyrromethene ring which may reduce vibrations and thus
the thermoelastic expansion in the molecule. The dyes 8a and
Conclusion
In conclusion, we have designed and synthesized four
novel N-ethylcarbazole linked aza-BODIPY dyes and
investigated their efficiency to produce triplet excited state and
singlet oxygen generation. All these aza-BODIPY derivatives
showed strong absorption in the NIR region with high molar
8
b, which are free from the core substitution showed strong PAS
signal when compared to blood. These can be attributed to their
low intersystem crossing efficiency, which leads to an increase
in the singlet excited state life time. The peak to peak PA signal
amplitude for the sample 8a in acetonitrile is found to be 12
times higher than that of blood (Figure 6B), and the signal
intensity gets increased upon increasing the concentration of the
extinction coefficients (3-4x104 M cm ). The substitution of
-1
-1
these derivatives with heavy atoms such as iodine at the core
positions resulted in
a
significant enhancement in the
intersystem crossing efficiency of 9a-b. The triplet excited state
quantum yields of ca. 90% and singlet oxygen generation
efficiency of ca. 70% were observed for the core iodinated
derivative 9a. The orientation of carbazole group and the
localisation of electron density in the molecule were confirmed
from the optimised geometries through DFT calculations. Strong
photoacoustic signal observed in the NIR region makes these
molecules ideal for deep tissue contrast agent for photoacoustic
sample. Therefore, the derivative, 8a can be used as
a
promising photoacoustic contrast agent in deep tissue imaging.
Deep tissue photoacoustic imaging. To demonstrate the
potential of the aza-BOIDPY derivative, 8a as a promising PA
contrast agent, and to determine its imaging depth, a study was
conducted to acquire the PA images of 8a (in acetonitrile) and
blood embedded inside chicken breast tissue (CBT). Two low
density polyethylene (LDPE) tubes of 0.38 mm inner diameter
were filled with blood and 8a and placed on layer of CBT as
shown in Figure 7A. PA data was acquired using a 2.25 MHz
ultrasound transducer (UST) at various depths of ca. 1cm and
ca. 2 cm by stacking layers of CBT one on top of the other
imaging. Up to
2 cm deep photoacoustic imaging was
successfully demonstrated by using 8a as contrast agent. Our
results demonstrate that these derivatives can have potential for
theranostic application of simultaneous imaging and therapy by
combining both photoacoustic imaging and photodynamic
therapy.
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1
of singlet oxygen generation (Φ[ O2]) were calculated by a relative
method using optically matched solutions and comparing the quantum
yield of photooxidation of DPBF sensitized by the dye of interest to the
Experimental Section
General Techniques: The melting points were determined on
a
1
[14]
quantum yield of MB (Φ[ O2] = 52%) as the reference. The following eq.
was used,
µThermoCal10 apparatus. The electronic absorption spectra were
recorded on a Shimadzu UV-3101 or 2401 PC UV-VIS-NIR scanning
spectrophotometer. The fluorescence spectra were recorded on a SPEX-
Fluorolog F112X spectrofluorimeter. 1H, 13C, 11B and 19F NMR (Fig. S1-
S22, Supporting Information) were recorded on a 500 MHz Agilent NMR
VNMRS spectrometer. Mass spectra were recorded on Bruker (maxis
impact), sr. no. 282001.00081. All the solvents used were purified and
distilled before use. Fluorescence quantum yields were measured by the
integration of the available area of fluorescence spectra by absolute
method using an integration sphere. Tris-(8-hydroxyquinoline)aluminium
2
mꢀꢁꢂ Fꢆꢇ
Φ∆ꢀꢁꢂ = Φ∆ꢆꢇ
...... (eq. 2)
mꢆꢇ Fꢀꢁꢂ
where superscripts ‘bod’ and ‘MB’ designate aza-BODIPY derivatives
and MB, respectively, Φ∆ is the quantum yield of singlet oxygen, ‘m’ is
the slope of a plot of difference in change in absorbance of DPBF (at 418
nm) with the irradiation time and ‘F’ is the absorption correction factor,
which is given by F = 1 – 10–OD (OD at the irradiation wavelength).
(
Alq3) was used for the calibration of integration sphere before starting
the experiments.[21]
Photoacoustic imaging system: Each sample was injected into a
transparent LDPE tube of 0.38 mm inner diameter and was irradiated
with Optical Parametric Oscillator (OPO) laser (Continuum, Surelite)
pumped with 10 Hz repetition rate Q-switched Nd:YAG laser maintaining
5 ns pulse width and pulse energy density of ~5-10 mJ/cm2. The laser
Quantification of triplet state properties: The nanosecond laser flash
photolysis experiments were carried out by employing an Applied
Photophysics model LKS-20 laser kinetic spectrometer using OCR-12
Series Quanta Ray Nd:YAG laser. Laser beams were fixed at right
angles to each other. The laser energy was 64 mJ at 355 nm. The triplet
yields (ΦT) of the synthesized aza-BODIPYs were measured employing
energy transfer to β-carotene using Ru(bpy)32+as the reference.[12] For
these experiments, an optically matched (355 nm) solutions of
Ru(bpy)32+/aza-BODIPY derivatives were mixed with a known volume of
β-carotene solution (end concentration of β-carotene was 2.0 × 10-4 M).
The transient absorbance (∆A) of the β-carotene triplet, formed by the
energy transfer from Ru(bpy)32+ or the aza-BODIPY triplet, was monitored
at 510 nm. The plateau absorbance following the completion of triplet
formation were compared and properly corrected for the decay of the
donor triplets in competition with energy transfer to β -carotene, and thus
calculated the ΦT of aza-BODIPY derivatives based on eq. 1.
irradiation results in thermoelastic expansion of the sample which in turn
generates PA waves. These PA waves are detected using a single-
element UST (Olympus NDT, V306-SU) of 2.25 MHz central frequency
with ~70% nominal bandwidth. The UST and the LDPE tubes were
enclosed in a water bath for better acoustic coupling. The acquired PA
signal was first amplified and band pass filtered (1-10 MHz) using a pulse
amplifier (Olympus-NDT, 5072PR) and then recorded using a data
acquisition card (GaGe, compuscope 4227) inside a desktop. All PA
signals were acquired with a sampling rate of 25 MHz. The laser
wavelength was varied from 670-970 nm with step size of 5 nm. Each PA
signal was averaged 10 times and normalised with laser power and
amplifier gain.For deep tissue imaging experiments, we have used the
OPO-PAT system (Fig 1. in [19]). The PA signal was acquired by rotating
the UST continuously in a full 3600 around the sample. The acquired PA
ꢀꢁꢂ
obs
ꢃꢄꢅ
obs
ꢃꢄꢅ
ΔAꢀꢁꢂ
ΔAꢃꢄꢅ
k
k
− k0
ꢃꢄꢅ
ꢀꢁꢂ
ꢃꢄꢅ
= ΦT
Φ
… … … . (eq. 1)
T
ꢀꢁꢂ
obs
ꢀꢁꢂ
data was then amplified and filtered. A simple delay-and-sum
reconstruction algorithm was used to obtain the PAT images.[20] SNR of
k
− k0
k
obs
the PAT images were calculated as the ratio of peak-to-peak amplitude
of the PA signal (V) to the standard deviation of background noise (n) i.e.,
SNR (in dB) = 20 log10 (V/n).
where superscripts ‘bod’ and ‘ref’ designate the different aza-BODIPY
derivatives and Ru(bpy)32+, respectively, kobs, is the pseudo-first-order
rate constant for the growth of the β-carotene triplet and k0 is the rate
constant for the decay of the donor triplets, in the absence of β-carotene,
observed in solutions containing Ru(bpy)32+ or aza-BODIPY dye at the
Materials and Methods: The starting materials. carbazole,
acetophenone, 4-bromoacetophenone, ethyl iodide, nitromethane,
diethylamine, ammonium acetate, borontrifluoride diethyl etherate,
same optical density (OD) as those used for sensitization. The direct
excitation of β-carotene did not result in any significant triplet formation
under these experimental conditions, because of negligible triplet yield.
The ΦTref in methanol for Ru(bpy)32+ was taken to be unity. The ΦT data
obtained in this manner are reliable to the extent to which the assumption
regarding 100% efficiency of energy transfer to β-carotene is valid.
triethylamine,
N-iodosuccinimide,
methylene
blue,
1,3-
diphenylisobenzofuran, β-carotene were purchased from S. D. Fine and
sigma Aldrich Chemicals, India.1,3-diphenylisobenzofuran (DPBF) was
recrystallized from a mixture (1:1) of ethanol and chloroform. The
0
0
compounds 2 (mp: 71-73 C) and 3 (mp: 85-87 C) were synthesized in
good yields by following the literature reported procedures.[22]
Quantification of singlet oxygen generation: Singlet oxygen
generation studies were carried out with a light source 200 W mercury
lamp (model 3767) on an Oriel optical bench (model 11200) with a
grating monochromator (model 77250). The intensity of light was
maintained constant throughout the irradiations by measuring the output
using an Oriel photodiode detection system (model 7072). Quantum
yields for singlet oxygen generation in DMSO were determined by
monitoring the photooxidation of DPBF sensitized by the aza-BODIPY
derivatives. Singlet oxygen quantum yields were measured at low dye
concentrations (optical density 0.2-0.3 at the irradiation wavelengths
General procedure for the synthesis of 5a-b
5a-b were synthesized by modifying the reported procedures [22a, 23], 9-
ethyl-9H-carbazole-3-carbaldehyde, 2 (0.022 mol) and acetophenone
derivatives 3a-b (0.022 mol) were dissolved in methanol (300 mL) and
2.5 molar aq. KOH (150 ml) added dropwise while cooling. After the
complete addition, reaction mixture allowed to stir for 24 h. The
precipitated product was filtered, washed with cold methanol, dried and
recrystallized from ethanol to give 5a-b as yellow solid.
>630 nm) to minimize the possibility of singlet oxygen quenching by the
dyes. The photooxidation of DPBF was monitored between 2 s to 2 min.
depending on the efficiency of the dye sensitizer. No thermal recovery of
DPBF (from a possible decomposition of endoperoxide product) was
observed under the conditions of these experiments. The quantum yields
5a: 63%, mp 67-69 °C; 1H NMR (500 MHz, CDCl ) δ 8.17 (m, 1H), 8.04
(d, J = 7.5 Hz, 1H), 7.92 (d, J = 8.0 Hz, 2H), 7.53 – 7.49 (m, 2H), 7.39-
7.36 (dd, J=8.2 1H), 7.18 – 7.10 (m, 4H), 7.06-7.04 (m, 2H), 7.00 (d, 1H),
3
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1
4
.25 (q, J=7.2 Hz 2H), 1.14 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz,
CDCl3) 189.04, 146.50, 142.34, 140.83, 139.57, 135.92, 130.28, 125.65,
7b: 66%, m.p. 272-274 °C;. H NMR (500 MHz, CDCl3) δ 8.58 (s, 2H),
8.13 (d, J = 7.6 Hz, 2H), 8.10 (d, J = 7.8 Hz, 4H), 7.50 (d, J = 7.3 Hz, 2H),
7.46 (m, 4H), 7.43 (d, 2H), 7.42 – 7.39 (m, 4H), 7.35 (m, 2H), 7.26 (s, 1H),
7.19 (t, J = 7.4 Hz, 2H), 4.31 (q, J = 7.3 Hz, 4H), 1.46 (t, J = 7.3 Hz, 6H).
13C NMR (126 MHz, CDCl3) δ 154.76, 149.49, 141.50, 140.97, 140.47,
1
1
3
24.31, 122.67, 121.04, 119.8, 118.4, 113.08, 111.44, 110.47, 109.0,
08.60, 47.10, 38.80, 15.19 cm ; ESI-MS m/z Cald for C23H19NO
25.147, Found 326.02.
-1
134.18, 128.98, 128.11, 127.51, 126.16, 124.62, 123.59, 123.35, 123.01,
1
120.64, 119.47, 118.86, 114.09, 108.95, 108.86, 37.70, 13.89; ESI-MS
m/z Cald for C48H35Br2N5 841.124, Found 842.0.
5
(
b: 58%, mp 125-127 °C; H NMR (500 MHz, CDCl3) δ 8.38 (s, 1H), 8.15
d, J = 7.6 Hz, 1H), 8.06 (d, J = 15.5 Hz, 1H), 7.94 (d, J = 8.4 Hz, 2H),
7.79 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.53 (dd, J = 15.3, 6.0
Hz, 2H), 7.46 – 7.41 (m, 2H), 7.30 (m, 1H), 4.40 (q, J = 7.3 Hz, 2H), 1.47
General procedure for the synthesis of 8a-b
1
(t, J = 7.2 Hz, 3H). H NMR (126 MHz, CDCl3) δ; 189.46, 147.13, 141.59,
1
40.51, 137.52, 131.84, 129.98, 127.40, 125.70, 123.51, 122.78, 121.78,
a-b were synthesized by modifying the reported procedures.[25, 15c]
8
-1
120.67, 119.82, 118.38, 109.13, 108.93, 108.17, 37.81, 13.85 cm ; ESI-
Compounds 7a-b (0.62 mmol) dissolved in dry dichloromethane (100 mL)
was treated with triethylamine (1.14 mL, 8.1 mmol) and stirred for 10 min
at 30°C. To this reaction mixture, boron trifluoride diethyl etherate (4.65
mL, 13.1 mmol) was added and heated at 40°C for 5 h. The solvent was
MS m/z Cald for C23H18BrNO 403.057, Found 404.06.
General procedure for the synthesis of 6a-b
evaporated, washed with water (2
× 50 mL) and extracted with
a-b were synthesized by modifying the reported procedures [24, 3a] To a
chloroform. Removal of the solvent gave a residue, which was separated
by column chromatography over silica gel. Elution of the column with a
mixture (1:9) of ethyl acetate and hexane gave the products 8a-b as
metallic blue solid.
6
solution of 5a-b (0.02 mol) dissolved in 100 mL of methanol was added
diethyl amine (9 mL) and nitromethane (4.5 mL) and refluxed for 24 h.
The mixture was cooled and neutralized using 1N HCl and extracted with
chloroform. The removal of the solvent gave a residue which was
separated by column chromatography over silica gel. Elution of the
column with a mixture (1:9) of ethyl acetate and hexane gave 6a-b in
good yields.
8a: 34%, m.p. 234-236°C; 1H NMR (500 MHz, DMSO) δ 9.06 (s, 2H),
8.47 (d, J = 8.7 Hz, 2H), 8.12 (m, 4H), 8.07 (d, J = 7.7 Hz, 2H), 7.66 (m,
6H), 7.57 (m, 6H), 7.47 (m, 2H), 7.12 (m, 2H), 4.44 (m, 4H), 1.31 (t, J =
7
1
1
3
.1 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 158.59, 145.78, 145.01,
40.76, 140.42, 132.18, 130.38, 129.49, 128.45, 128.00, 127.44, 126.03,
24.03, 123.64, 123.32, 122.21, 120.88, 119.25, 117.18, 108.97, 108.73,
7.74, 13.95. 11B NMR (160 MHz, DMSO) δ 1.22, 1.02, 0.83. 19F NMR
6
a: 69%, Viscous liquid; 1H NMR (500 MHz, CDCl3) δ 8.46 (m, J = 8.4
Hz, 2H), 7.97 (m, 1H), 7.82 (m, 1H), 7.69 (m, 2H), 7.62 (m, 1H), 7.46 (m,
2
2
1
1
3
3
H), 7.29 (m, 2H), 7.08 (m, 1H) 4.87 (m, 2H), 4.75 (m, 2H), 4.19 (m, 1H),
.97 (d, 2H), 1.44 (t, J = 7.2 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ
(470 MHz, DMSO) δ -128.40, -128.47, -128.54, -128.61; ESI-MS m/z
Cald for C48 731.303, Found 732.310.
H
2 5
36BF N
96.56, 163,41 139.21, 137.72, 133.50, 131.67, 129.51, 129.09, 127.89,
26.93, 125.15, 124.78, 122.78, 121.04, 118.19, 118.08, 80.46, 42.21,
9.46, 37.56, 36.51, 31.3, 30.48, 13.76; ESI-MS m/z Cald for C24H22N2O3
86.163, Found 387.1
8b: 41%, m.p. 289-291 °C; 1H NMR (500 MHz, DMSO) δ 9.06 (d, 2H),
8.47 (d, J = 8.5 Hz, 2H), 8.30 (s, 2H), 8.04 (m, 6H), 7.79 (m, 3H), 7.68
(
m, 4H), 7.48 (m, 3H), 7.12 (m, 2H), 4.45 (m, 4H), 1.31 (d, J = 7.2 Hz,
6
1
1
H); 13C NMR (126 MHz, DMSO) δ 158.40, 145.46, 145.03, 140.94,
40.55, 131.89, 131.29, 129.80, 129.07, 127.80, 126.81, 123.66, 123.39,
22.88, 122.56, 121.02, 119.85, 118.57, 110.13, 37.69, 14.33. 19F NMR
6b: 80%, m.p. 69-71 °C; 1H NMR (500 MHz, CDCl3) δ 8.03 (dd, J = 7.4
Hz, 2H), 7.69 (dd, J = 8.6 Hz, 2H), 7.47 (m, 1H), 7.39 (m, 1H), 7.29 (m,
2
1
1
1
3
H), 7.08 (m, 1H), 4.80 (m, 1H), 4.41 (m, 2H), 3.56 (m, 2H), 2.92 (d, 2H),
.42 (t, J = 7.2 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 196.34, 163.0,
(470 MHz, DMSO) δ -128.28, -128.34, -128.41, -128.48; ESI-MS m/z
Cald for C48 889.122, Found 890.130.
H
2 2 5
34BBr F N
40.28, 139.40, 135.30, 132.06, 129.54, 129.04, 128.70, 126.01, 124.88,
23.30, 122.41, 120.46, 119.15, 118.95, 80.42, 42.19, 39.55, 37.60,
6.47, 31.42, 30.48, 13.83; ESI-MS m/z Cald for C24H21BrN2O3 464.074,
General procedure for the synthesis of 9a-b
Found 465.0.
To a solution of 8a-b (0.27 mmol) in a mixture (60 mL, 3:1) of chloroform
and acetic acid, N-iodosuccinimide (154 mg, 0.68 mmol) was added and
stirred at 30°C for 2 h. The reaction mixture was washed with sodium
thiosulphate followed by sodium bicarbonate solution and extracted with
chloroform. Removal of the solvent gave a residue which was separated
by column chromatography over silica gel. Elution of the column with a
mixture (1:9) of methanol and chloroform gave 60-65% of 9a-b.
General Procedure for the synthesis of 7a-b
The nitromethane derivatives 6a-b (0.01 mol) and ammonium acetate
(
27.30 g, 0.314 mol) were dissolved in n-butanol (60 mL) and heated
under reflux for 24 h.[15c] The precipitated product was filtered, washed
with cold ethanol, dried and recrystallized from chloroform to give 7a-b as
brown solid.
9
a: 61%, m.p. >300 °C; 1H NMR (500 MHz, DMSO) δ 8.69 (d, 2H), 8.61
(m, 2H), 8.14 (m, 2H), 8.08 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H),
a: 57%, m.p. 225-227 °C; 1H NMR (500 MHz, CDCl3) δ 8.84 (s, 2H),
7
8
7
8
1
1
1
7
6
7.76 (m, 2H), 7.59 (m, 4H), 7.53 (m, 6H), 7.48 (m, 2H), 7.43 (m, 2H),
4
.30 (m, 4H), 1.06 (t, J = 7.1 Hz, 6H); 13C NMR (126 MHz, DMSO) δ
158.40, 145.46, 145.03, 140.94, 140.55, 131.89, 131.29, 129.80, 129.07,
.33 (d, J = 8.5 Hz, 2H), 8.02 (d, J = 7.3 Hz, 4H), 7.86 (d, J = 7.6 Hz, 2H),
.55 (t, J = 7.6 Hz, 4H), 7.47 (t, J = 7.3 Hz, 2H), 7.39 (m, 4H), 7.31 (d, J =
.5 Hz, 2H), 7.27 (s, 2H), 7.01 (t, J = 7.3 Hz, 2H), 4.27 (q, J = 7.2 Hz, 4H),
.38 (t, J = 7.2 Hz, 6H); 13C NMR (126 MHz, CDCl3) δ 154.79, 149.83,
127.80, 126.81, 123.66, 123.39, 122.88, 122.56, 121.02, 119.85, 118.57,
1
1
9
10.13, 37.69, 14.33; 19F NMR (470 MHz, DMSO) δ -130.01, -130.08, -
30.13, -130.18; ESI-MS m/z Cald for C48
84.033.
43.80, 140.26, 139.83, 132.57, 129.70, 129.04, 127.21, 126.51, 125.47,
25.20, 123.36, 123.20, 121.48, 120.77, 118.71, 113.37, 108.52, 108.37,
7.24, 76.99, 76.74, 37.58, 13.87; ESI-MS m/z Cald for C48H37N5
83.305, Found 684.312.
H34BF
2
I
2
N
5
983.096, Found
9
b: 58%, m.p. >300 °C; 1H NMR (500 MHz, CDCl3) δ 8.59 (s, 1H), 8.53
(s, 1H), 8.48 (s, 1H), 8.17 (d, 2H), 8.10 – 8.08 (m, 2H), 7.85 (m, 1H), 7.77
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
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(
4
m, 1H), 7.66 (m, 1H), 7.59 (m, 6H), 7.30 (m, 4H), 7.05 (m, 2H), 4.15(m,
1109.
1
1
1
9
[7]
a) W. M. Gallagher, L. T. Allen, C. O’Shea, T. Kenna, M. J. Hall, J.
Killoran, D. F. O’Shea, Br. J. Cancer 2005, 92, 1702-1710; b) A. T.
Byrne, A. E. O'Connor, M. Hall, J. Murtagh, K. O'Neill, K. M. Curran,
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c) J. Joseph, N. V. Eldho, D. Ramaiah, Chem. Eur. J. 2003, 9,
H
)
,
0
.
8
5
(
s
,
6
H
)
.
B
N
M
R
(
1
6
0
M
H
z
,
C
D
C
l
3
)
δ
0
.
3
8
,
0
.
2
3
,
0
.
0
3
.
F
NMR (470 MHz, CDCl3) δ -131.69, -131.75, -131.81, -131.87; ESI-MS
m/z Cald for C48 1140.915, Found 1141.925.
H
2 2 2 5
32BBr F I N
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(
relevant financial interests in the manuscript and no other
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Chemistry - A European Journal
10.1002/chem.201605702
FULL PAPER
Table of Contents
FULL PAPER
The carbazole linked aza-BODIPY
dyes showed favorable photophysical
properties and efficient triplet as well
as singlet oxygen generation quantum
yields. Furthermore, the efficient
photoacoustic signals produced by
these dyes makes them as excellent
candidates for both photodynamic
therapy and contrast agents for
photoacoustic imaging.
Y. Gawale, N. Adarsh, S. K. Kalva, J.
Joseph, M. Pramanik , D. Ramaiah and
N. Sekar*
sampleA-8
sampleB-8
sampleC-8
sampleD-8
BLOOD
*
*
3
5
2
5
1
5
xxxxx – xxxxx
PA signals
6
700
750
800
850
900
950
Wavelength (nm)
9
a
Carbazole Linked NIR Aza-BODIPY
Dyes as Triplet Sensitizers and
Photoacoustic Contrast Agents for
Deep Tissue Imaging
(
(Insert TOC Graphic here: max. MB
width: 5.5 cm; max. height: 5.0 cm))
0
2
4
6
8
10
Irrradiation tim e/s
This article is protected by copyright. All rights reserved.