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Cite this: RSC Adv., 2021, 11, 26403

Received 30th June 2021
Accepted 22nd July 2021

Two-photon excitable boron complex based ontridentate imidazo[1,5-a]pyridine ligand for heavyatom-free mitochondria-targeted photodynamic
therapy†
Keita Hoshi,a Masami Itaya,a Koki Tahara,a Airi Matsumoto, b Atsuhi Tabata,bHideaki Nagamune,b Yasushi Yoshida, c Eiji Hase,d Takeo Minamikawa,dTakeshi Yasui,d Tetsuro Katayama,*d Akihiro Furube, d Keiji Minagawa,aYasushi Imada a and Fumitoshi Yagishita *ad
We have synthesized a cyan ﬂuorescent boron complex based on a tridentate imidazo[1,5-a]pyridine ligand.

DOI: 10.1039/d1ra05059a

The boron complex was found to have potential applications as not only a chiroptical material but also

rsc.li/rsc-advancesa heavy-atom-free mitochondria-targeted photosensitizer for cancer treatment.

Small organic molecules showing photoluminescence havedrawn considerable attention due to their application inmaterial science and bioscience. Among them, introduction ofboron atoms into the p-conjugated system is recognized to
improve the photophysical properties dramatically. Thus,
numerous boron complexes including BODIPY dyes, which are
one of the well-known uorescent boron complexes based on
the dipyrromethene ligand, have been designed and evaluatedfor their potential applications in organic light-emitting diodes(OLEDs),1 solar cells,2 medicinal diagnosis3 and photodynamic
therapy (PDT).4 Various BODIPY analogues based on the
dipyrromethene ligand have been developed to achieve theapplications mentioned above, whereas the research on thedevelopment of novel luminescent boron complexes based onaromatic ligands has emerged to explore and improve thephotofunctionalities. For example, some successful examples ofrigid four-coordinated boron complexes based on tridentate
ligands, which show unique and useful luminescent properties
such as self-assembly,5 aggregation-induced emission6 andchiroptical properties,7 have been reported.

PDT has a huge importance in the healthcare eld due to its
noninvasive treatment for various diseases including cancer.8 In
the PDT process the photosensitizers play a key role to generate
the reactive oxygen species (ROS) via energy transfer from thetriplet photosensitizer to molecular oxygen. Although theintroduction of heavy halogen atoms into the dye is one of thepromising strategies to enhance intersystem crossing for
improving the singlet oxygen generation capability, this methodoen causes dark toxicity. Thus, the research on the development of the heavy-atom-free photosensitizers has become
increasingly important. Recently, some heavy-atom-free triplet
BODIPY dyes have been designed and synthesized based on themolecular concept such as the photo-induced electron transfer(PET) process, reducing the energy gap between the lowest
singlet excited state and the lowest triplet excited state, andreplacement of the oxygen atom of the carbonyl group witha sulfur atom to enhance the intersystem crossing.9 However,the designing of heavy-atom-free photosensitizers is stilla challenging research area.
In this manuscript, we describe the synthesis, crystal structure and photophysical properties of the luminescent boronaDepartment of Applied Chemistry, Tokushima University, 2-1 Minamijosanjima,
Tokushima 770-8506, Japan. E-mail: yagishitaf@tokushima-u.ac.jpbDepartment of Bioscience and Bioindustry,
Minamijosanjima, Tokushima 770-8513, Japan

Tokushima

University,

2-1c
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering,
Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japand

Department of Post-LED Photonics Research, Institute of Post-LED Photonics,
Tokushima University, 2-1 Minamijosanjima, Tokushima 770-8506, Japan† Electronic supplementary information (ESI) available: Additional gures,
crystallographic data in CIF, and NMR spectra. CCDC 2085924. For ESI andcrystallographic data in CIF or other electronic format see DOI:
10.1039/d1ra05059a© 2021 The Author(s). Published by the Royal Society of Chemistry

Scheme 1

Synthesis of the boron complex 1.

RSC Adv., 2021, 11, 26403–26407 | 26403

RSC Advancescomplex 1 based on 1,3-bis(o-hydroxyphenyl)imidazo[1,5-a]pyridine ligand 2, which works as a tridentate O,N,O-ligand(Scheme 1). In addition, it was found that the boron complex 1
not only shows a cyan emission from the singlet excited state
but also forms a triplet excited state via intersystem crossing,which is applicable in biological application as a heavy-atomfree photosensitizer for a potent cancer treatment.
The tridentate ligand 2 was synthesized from commerciallyavailable imidazo[1,5-a]pyridine via three steps including theiodination reaction, Suzuki–Miyaura cross coupling reaction
using the o-methoxyphenylboronic acid and demethylation
reaction using boron tribromide (Scheme S1†). As shown in
Scheme 1, the desired boron complex 1 was prepared by thereaction of tridentate ligand 2 with phenylboronic acid in 90%
yield. The structure of 1 was determined by NMR spectroscopy
and HRMS. Fortunately, the single crystals of 1 suitable for the
X-ray crystallographic analysis were prepared from dichloromethane and hexane. The X-ray analysis of 1 revealed that theboron atom is bound to the sp2 nitrogen atom of the imidazo[1,5-a]pyridine ring and two oxygen atoms of the o-hydroxyphenyl groups to form a tetracoordinate boron complex(Fig. 1). The N–B bond length is 1.547 
A and slightly longer thanthat of the O–B bond (1.475 
A for O1–B bond and 1.463 
A for O2–
B bond, respectively). The dihedral angles between the imidazo[1,5-a]pyridine core and each substituted phenyl ring at 1- and3-positions are 10.27 for N–C1–C2–C3 and 14.59 for N–C4–C5–
C6. It should be noted that the rigid perpendicular orientationof the axial phenyl ring at boron atom to the unsymmetricalimidazo[1,5-a]pyridine ligand leads to chiral scaﬀold. Indeed,
the pair of the positive and negative CD peaks at 254 nm, whichindicate the opposite of optical rotation derived from bothenantiomers of 1, were observed by HPLC using a chiralstationary phase (Daicel Corp. CHIRALPAK IB) (Fig. S1†). We
also conducted the optical resolution of 1 and the ECD spectra
were shown in Fig S2.† The experimental ECD spectra weresimilar to the calculated ECD spectra shown in Fig. S8.† The
calculated ECD spectra indicated that the (R)-1 and (S)-1 show
the positive and negative ECD signals at around 370 nm,
respectively. Thus, we speculate that the rst and second fractions observed in HPLC analysis correspond to (R)-1 and (S)-1,
respectively. Although we did not conduct the CPL

ORTEP drawing of 1. Disordered dichloromethane molecule isomitted for clarity.

Fig. 1

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Paper

Fig. 2 (a) Absorption (blue line) and PL (red line) spectra of 1 in DMSO“sh” denotes a shoulder band. (b) Photoluminescence of 1 in DMSO
under 365 nm irradiation.

measurement of the boron complex 1, these ndings indicate
the potential application of 1 as a new class of luminescentboron complex showing a chiroptical property.
Next, we investigated the photophysical property of 1. The
photophysical data of 1 in dimethyl sulfoxide (DMSO) aresummarized in Table S1† accompanied by its absorption andphotoluminescence (PL) spectra in Fig. 2. The absorption
maximum of 1 was observed at 373 nm with moderate molar
extinction coeﬃcient (3max ¼ 15 800 L mol1 cm1) concomitantly with some shoulder bands. The boron complex 1 exhibits
cyan emission, and PL peak was observed at 479 nm. Although
we also examined the solvent eﬀect using some organic
solvents, obvious solvent polarity-dependent absorption and PL
emission of 1 were not observed (Fig. S3†). The absolute PL
quantum yield is found to be 0.34 in DMSO. The uorescent
lifetime of 1 (sf ¼ 5.01 ns) is shorter than those of boron
complexes of 1-(o-hydroxyphenyl)imidazo[1,5-a]pyridine (sf ¼
10.1–12.5 ns),10 the value is within a range of the uorescence
lifetimes of known BODIPY dyes (around 1–10 ns).11 The boron
complex of 1-(o-hydroxyphenyl)imidazo[1,5-a]pyridine showshigh uorescence quantum yields (Fem ¼ up to 0.71),10 whereas
the boron complex 1 shows medium uorescence quantum
yield in solution (Fem ¼ 0.34). The decrease of PL quantum yieldsuggests the increase in non-radiative rate constant. Although
the absolute PL value is decreased in solid state (Fem ¼ 0.05),
the boron complex 1 shows cyan emission even in the solidstate. Decreasing of the PL quantum yield in the solid statesuggests that the structural relaxation process of imidazo[1,5-a]pyridine unit is important for the radiative process. Such a nonradiative process is considered as intramolecular vibrational
redistribution (IVR) or intersystem crossing process. Thus, wemeasured femtosecond transient absorption spectra of 1 in
DMSO to elucidate the non-radiative process on the excitedstate. The setup for the measurement of transient absorption
spectra is shown in Fig. S4,† and the time-resolved transient
spectra of 1 in DMSO are shown in Fig. 3a. The absorption band
appeared immediately around 690 nm aer excitation. Decay
components with a time constant of 480 ps was obtained withexponential tting as shown in Fig. 3b. Sub nanoseconds
relaxation process is slower than general IVR and suggests thechange of intramolecular charge transfer (CT) character.12© 2021 The Author(s). Published by the Royal Society of ChemistryPaper(a) and (c) Femtosecond transient absorption spectra of 1 in
DMSO after excitation at 400 nm. (b) Transient absorption decay
proﬁle (scattered points) at 693 nm and ﬁt (solid line) with an exponential function with a time constant 480 ps.
Fig. 3

Then, the absorption band at 545 nm arose in place of thedecrease in band around 690 nm according to an increase in the
delay time aer excitation. Because the transient absorption
band at 545 nm survives substantially longer than the uorescence lifetime, the transient absorption spectrum observed ata time delay of 20 ns is ascribed to the triplet excited state of 1
(Fig. 3c). Intersystem crossing process might be induced with
the increase of intramolecular charge transfer character.13 The
yield of intersystem crossing is roughly estimated to be up to
0.36 from PL yield and deactivation process in transientabsorbance at 693 nm. This nding encouraged us to elucidate
the potential application of 1 as a heavy-atom-free photosensitizer for PDT treatment through triplet state.
To perform the biological application, the dark toxicity isundesirable nature. Thus, we evaluated the cytotoxicity of 1 to
HeLa cells using CCK-8 assay. The viabilities of HeLa cells
incubated for 1 hour (for acute toxicity) and 24 hours (forsubacute toxicity) with various concentrations of 1 aresummarized in Fig. S5.† As a result, the boron complex 1 did notshow the signicant toxicity to HeLa cells in each concentration
tested in this study under a dark. We next applied the boron
complex 1 to a uorescence bioimaging experiment of HeLa
cells because the cellular localization of photosensitizer isimportant to achieve the eﬀective PDT treatment. Among theorganelle, mitochondria, which plays a key role in activating
apoptosis, has been recognized as a promising target for thedesigning PDT agents.14 When HeLa cells were incubated with 1(5.0  105 mol L1) at 37  C for 1 hour under 5% CO2 conditions, the cells were labelled with cyan uorescence signals
indicating 1. To elucidate the cellular localization of 1, we
conducted the co-staining experiment of HeLa cells using 1 anda mitochondrial marker (MitoTracker Red, ThermoFisher
Scientic). When HeLa cells were incubated with boron
complex 1 and the mitochondrial marker at 37  C for 1 hour
under 5% CO2 conditions, it was found that the cyan uorescence signals indicating 1 co-localized with the red uorescencesignals indicating the mitochondrial marker (Fig. 4a). It is reported that the neutral BODIPY dye tends to localize into themitochondria of cells to its low electron density nature.15
Because the TD-DFT calculation indicates the ICT nature of 1 in

© 2021 The Author(s). Published by the Royal Society of Chemistry

RSC AdvancesFig. 4 (a) Observed images focused on mitochondria in HeLa cellswith 1 and a mitochondrial marker. Cyan ﬂuorescence of 1 wasobserved through a WU ﬁlter and red ﬂorescence of a mitochondrial
marker was observed through a WIY ﬁlter. (b) Cell viabilities of HeLa
cells after UVA-LED irradiation with (+) or without () staining by 1.

the excited state, which may be caused by partial electrondeciency of uorophore, we consider that the boron complex 1
also shows high aﬃnity toward mitochondria (Fig. S6†). In
addition, this nding indicates the potential application of 1 asa heavy-atom-free mitochondria-targeted PDT agent against
cancer cells.
We next focused on the application of 1 as a one-photon PDT
agent using UVA-LED. The photocytotoxicity against HeLa cells
was not observed in the absence of 1 aer the UVA-LED irradiation for 60 minutes (Fig. 4b). Conversely, the cell viabilities
of HeLa cells stained by 1 were gradually decreased according to
the continuous UVA-LED irradiation, and the signicant cytotoxicity of 1 was observed aer UVA-LED irradiation for 60
minutes (Fig. 4b). To validate a role of 1 as mitochondriatargeted PDT agent, we also conducted the JC-1 assay for themeasurement of the mitochondrial membrane potential toevaluate the mitochondrial function aer the continuous UVALED irradiation. When the UVA-LED irradiation on HeLa cells
in the absence of 1 was conducted for 0, 10 and 30 minutes, thechanges of red and green uorescence signals of JC-1 dyes were
not observed (Fig. 5a). On the other hand, the continuous UVALED irradiation on HeLa cells stained by 1 (5.0  105 mol L1)
elevated the depolarization of the mitochondrial membranepotential (Fig. 5b). Mitochondrial damages of HeLa cells weresimilarly observed even in the presence of lower concentrationsof 1 (5.0  106 mol L1) aer 30 min irradiation (Fig. S10†).
The use of UVA light source was unfavorable for PDT treatment due to its low light penetration and potential toxicity. On

Fig. 5 Photographs of HeLa cells incubated with the JC-1 dye (a) in
the absence and (b) presence of 1; ﬂuorescence images through WIG
ﬁlter (upper), those through WIB ﬁlter (middle) and bright ﬁelds (lower).

RSC Adv., 2021, 11, 26403–26407 | 26405

RSC Advances
the other hand, it is known that the two-photon excitation
process enables the excitation of dye at lower energy.16 The twophoton technology realizes the PDT treatment with somebenecial outcomes such as high penetration depth, spatial
selectivity, and low phototoxicity to healthy tissues.17 Thus, weinvestigated the two-photon excitable nature to evaluate thepotential of 1 as two-photon PDT agent for cancer treatment.
The two-photon excitation of 1 was studied using a femtosecond
pulse laser source, and the setup is shown in Fig. S11.† When
the DMSO solution of 1 was excited at 740 nm, cyan uorescence was observed at the focal point of the laser. The log–log
plot of the uorescence intensity against the laser power gavea linear line with a slope of 1.92, which indicates the occurrence
to two-photon absorption of 1 (Fig. S12†). When we conducted
the two-photon imaging of mitochondria in HeLa cells, uorescence image was successfully observed under 740 nm irradiation condition (Fig. S13†). These results strongly indicatedthe potential application of 1 as multi-photon excitable photosensitizer, i.e., NIR excitable PDT agent for cancer treatment.
In conclusion, we have synthesized and characterized theboron complex based on the tridentate imidazo[1,5-a]pyridine
ligand. X-ray analysis of 1 revealed that the perpendicularorientation of the axial phenyl ring at boron atom to theunsymmetrical planar ligand constructs a chiral environment.
The boron complex 1 was found to work as the heavy-atom-freephotosensitizer based on the experimental study of its excitedstate dynamics. The boron complex 1 showed not only a photocytotoxicity through triplet state but also a two-photon
absorption nature. In addition, the PDT agents exhibiting
photoluminescence have the advantages because they canvisualize and dene the aﬀected area during the PDT treatment,
and have potential application in theranostics, which combines
the therapeutic and diagnostic imaging medical techniques.18
Thus, we believe that the boron complex 1 becomes the newmolecular design for a theranostics agent.

Author contributions
F. Y. and T. K. conceived the project, performed all experiments,
and wrote the manuscript. K. H., M. I., H. T. and Y. Y. carriedout the synthesis and characterization of the compounds. A. M.,
A. T. and H. N. carried out the bioassay. E. H., T. M. and T. Y.
carried out the two-photon excitation experiments. A. F
measured the transient absorption spectra. K. M. and Y. I.
supervised the experiments. All authors read and approved thenal manuscript.

Conﬂicts of interest
There are no conicts to declare.

Acknowledgements
The authors would like to thank Dr H. Sato (Rigaku Corp.) for
supporting the X-ray analysis, the NICHIA corp. for supplying
LEDs, Prof. M. Ogasawara (Tokushima Univ.) for the opticalresolution of 1, and a research grant from the Research Clusters

26406 | RSC Adv., 2021, 11, 26403–26407

Paper
Program of Tokushima University (No. 1802001, 1802003, and2003006). This work was supported by the project on the
Promotion of Regional Industries and Universities from the
Cabinet Oﬃce, Japan, by the Creation and Application of NextGeneration Photonics by Tokushima Prefecture from theTokushima Prefectural Government, Japan, and by JSPS
KAKENHI, Grant Numbers 19K20691 and 20H02767.

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