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Donor–acceptor–acceptor-type near-infrared
ﬂuorophores that contain dithienophosphole oxideand boryl groups: eﬀect of the boryl group on thenonradiative decay†
Yoshiaki Sugihara,‡a Naoto Inai,‡a Masayasu Taki, b Thomas Baumgartner,
Ryosuke Kawakami,d Takashi Saitou,d Takeshi Imamura,d Takeshi Yanai *aband Shigehiro Yamaguchi *abc

The use of donor–p–acceptor (D–p–A) skeletons is an eﬀective strategy for the design of ﬂuorophores
with red-shifted emission. In particular, the use of amino and boryl moieties as the electron-donatingand -accepting groups, respectively, can produce dyes that exhibit high ﬂuorescence andsolvatochromism. Herein, we introduce a dithienophosphole P-oxide scaﬀold as an acceptor–spacer toproduce a boryl- and amino-substituted donor–acceptor–acceptor (D–A–A) p-system. The thus
obtained ﬂuorophores exhibit emission in the near-infrared (NIR) region, while maintaining highﬂuorescence quantum yields even in polar solvents (e.g. lem ¼ 704 nm and FF ¼ 0.69 in CH3CN). A
comparison of these compounds with their formyl- or cyano-substituted counterparts demonstrated theimportance of the boryl group for generating intense emission. The diﬀerences among these electronaccepting substituents were examined in detail using theoretical calculations, which revealed the crucial
Received 10th February 2021
Accepted 24th March 2021

role of the boryl group in lowering the nonradiative decay rate constant by decreasing the non-adiabatic

DOI: 10.1039/d1sc00827g

coupling in the internal conversion process. The D–A–A framework was further ﬁne-tuned to improve
the photostability. One of these D–A–A dyes was successfully used in bioimaging to visualize the bloodrsc.li/chemical-science

vessels of Japanese medaka larvae and mouse brain.

Introduction
Organic p-conjugated compounds with a donor–p–acceptor (D–p–A) framework have attracted substantial attention due totheir wide range of applications.1–8 The absorption and emission properties of D–p–A compounds can be tuned by structuralmodications of the individual components. By using anappropriate combination of donor, p-spacer, and acceptor
moieties, even near-infrared (NIR) emission can be attained.

aDepartment of Chemistry, Graduate School of Science, Integrated ResearchConsortium on Chemical Sciences (IRCCS), Nagoya University, Furo, Chikusa,
Nagoya, 464-8602, Japan. E-mail: yanait@chem.nagoya-u.ac.jp; yamaguchi@chem.
nagoya-u.ac.jpbInstitute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo,
Chikusa, Nagoya, 464-8602, Japanc
Department of Chemistry, York University, 4700 Keele St., Toronto, ON M3J 1P3,
Canadad

Department of Molecular Medicine for Pathogenesis, Graduate School of Medicine,
Ehime University, Shitsukawa, Toon City, Ehime, 791-0295, Japan† Electronic supplementary information (ESI) available: Experimental details,
photophysical properties, evaluation of the photostability, theoretical
calculations, in vivo imaging, and NMR spectra for all new compounds. See
DOI: 10.1039/d1sc00827g‡ These authors contributed equally to this work.

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

Such NIR-emissive compounds are promising materials for
NIR-emissive OLEDs,9,10 biosensing applications,11 and deeptissue bioimaging given the high biopermeability of the NIR
light.12,13
Even though various D–p–A-type uorophores that exhibit
NIR emission have been developed so far, their uorescencequantum yields (FF) tend to decrease drastically in polar
solvents. However, D–p–A compounds that contain a diarylboryl moiety as the electron-accepting group represent one
exception to this trend.5–8 In 1972, Williams and co-workers
reported a simple p-(dimesitylboryl)-substituted triphenylamine as the rst example of a boron-based D–p–A-type uorophore.14 Since then, boryl-substituted D–p–A dyes have been
extensively studied in order to explore their potential utility as
nonlinear optical materials,15,16 two-photon-emissive materials,17 anion sensors,18 and bioimaging.19 These compoundsoen exhibit high FF values even in polar media, despite thesignicant red-shi of their emission bands.5c Two moieties inthis type of molecular frameworks can be modied to obtain
NIR emission: the aryl group on the boron atom and the pspacer. For example, in 2015, Marder and co-workers reported
D–p–A compounds with an electron-withdrawing peruorophenyl or 3,5-(CF3)2C6H3 group at the para position of theChem. Sci., 2021, 12, 6333–6341 | 6333

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Representative boryl-substituted D–p–A dyes and dithienophosphole P-oxide derivatives and boryl-substituted D–A–A type dyes
and relevant compounds studied in this work.

Fig. 1aryl group on the boron atom (Fig. 1).20 In combination with thestrong donor julolidine, the compounds showed NIR uorescence in CH3CN with maximum emission wavelengths (lem)around 745 nm. On the other hand, in 2014, Zhao and coworkers reported a uorophore that employed 2,1,3-benzothiadiazole as additional electron-accepting p-spacer, emittingin the far-red to NIR region (lem ¼ 669 nm) in CH3CN (Fig. 1).21
Misra and co-workers achieved a further red-shied emission(lem ¼ 692 nm; FF ¼ 0.27 in CH2Cl2) by insertion of acetylene
spacers.22 Such donor–acceptor–acceptor (D–A–A)-type structures should thus be benecial for achieving red-shiedemission.
To develop NIR-uorescent dyes, we have focused ourattention on the modication of the p-spacer by introducing
a phosphine oxide group. We envisioned that the P(]O)Ph
group could bathochromically shi the emission by loweringthe LUMO energy level due to the s*–p* interaction23,24 as wellas the inherent inductive electron-withdrawing eﬀect. The
profound eﬀect of introducing a phosphine oxide group has
already been documented for several uorescent dyes. For
example, uorescein, a xanthene dye, exhibits a uorescencemaximum at 510 nm, while phospha-uorescein, a phosphineoxide-containing uorescein analogue, exhibits an emission
maximum of 656 nm.25 This example aptly illustrates thesubstantial impact of the phosphine oxide group on the electronic structure.
In this context, dithieno[3,2-b:20 ,30 -d]phosphole P-oxide
represents an attractive scaﬀold. Various dithienophospholederivatives have already been developed by expanding its pskeleton or modifying the P-aryl groups.26,27 While6334 | Chem. Sci., 2021, 12, 6333–6341

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dithienophosphole P-oxide itself exhibits an emission
maximum at 453 nm in CH2Cl2, its emission wavelength can be
red-shied by introducing electron-donating triphenylamine
moieties at its termini.28 The incorporation of the stronger
donor moiety aminothiophene further red-shis the emission
to 657 nm with a moderate uorescence quantum yield (FF ¼0.39) in CH2Cl2 (Fig. 1).29
In this article, we report the design and synthesis of borylsubstituted D–A–A-type uorophores, which contain a dithienophosphole P-oxide scaﬀold as an additional acceptor moiety,as highly emissive NIR-uorescent dyes (Fig. 1). The phosphineoxide group was expected to red-shi the emission band,
improve the photostability of the dye, and aﬀord a more rigid
molecular structure, which would suppress the nonradiative
decay process and thus improve the quantum yield. A series of
the D–A–A type dyes 1a–1f that bear various aryl groups on theboron atom, phosphorus-substitution patterns, and electrondonating moieties was synthesized, and their substituent
eﬀects were studied in-depth in order to accomplish intense
NIR emission and high photostability. A comparative study withother D–A–A type analogues 2 and 3 with diﬀerent acceptor
units in place of the boryl group (Fig. 1) revealed the importantrole of a terminal boryl group to suppress the non-radiative
decay process, resulting in the high uorescence quantum
yields, according to excited-state theoretical calculations. To
demonstrate the utility of such dyes, 1c was employed foruorescence imaging in vivo.

Results and discussion
Compounds 1a–1f were synthesized using 2,6-dibromodithienophosphole P-oxide 428a as the key precursor (Scheme 1). The
crucial step in the synthesis of 1a–1e is the monofunctionalization of 4. Its reduction with trichlorosilane, followed by treatment with 1.1 equiv. of n-BuLi produced mainly
the mono-lithiated product, which was successively treated with
Mes2BF. Further oxidation with pyridinium chlorochromate(PCC) or sulfur aﬀorded 5 or 6 in 24% and 18% overall yield,
respectively. Suzuki–Miyaura cross-coupling of 5 or 6 with thecorresponding amino-substituted arylboronic acid or boronic
ester aﬀorded 1a–1d in moderate yields. The phosphonium
derivative 1e was obtained by the reduction of 1a, followed bymethylation of the P centre with MeOTf. Subsequent recrystallization from a hexane/CHCl3 mixed solvent aﬀorded 1e.
For the synthesis of 1f, bearing a bulky tri(t-butyl)phenylgroup on the boron atom, mono-arylation of the key precursor 4
was employed. Specically, the Suzuki–Miyaura cross-couplingof 4 with 1.0 equiv. of diphenylaminophenylboronic acid furnished 7, which was subsequently coupled with borylthienylboronic ester 8, obtained in situ from the direct borylationof the corresponding thienylborane precursor using an Ir catalyst, to generate 1f (for details, see the ESI†). Compounds 1a–1f
are suﬃciently stable to be handled in air without any special
precautions.
The formyl- and cyano-substituted analogues 2 and 3 weresynthesized as reference compounds (vide infra) via the monolithiation of 4; i.e., following the reduction of 4 and its© 2021 The Author(s). Published by the Royal Society of ChemistryView Article Online

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Chemical Sciencelithiation with 1.1 equiv. of n-BuLi, DMF was added as theformyl source. Further oxidation of the phosphine moiety with
H2O2 aﬀorded 9. Aer protection of the formyl group, Suzuki–
Miyaura cross-coupling and subsequent deprotection of theacetal moiety aﬀorded 2. Transformation of the formyl group in
2 into a cyano group using hydroxylamine aﬀorded 3.
Photophysical properties of D–A–A dye 1a

Scheme 1 Synthesis of compounds 1a–1f, 2, and 3. Reagents andconditions: (a) (1) HSiCl3, toluene, rt; (2) n-BuLi, THF, 78  C; (3)Mes2BF or DMF, THF, 78  C to rt; (4) PCC, S8, or H2O2, CH2Cl2, rt; (b)4-(Ph2N)C6H4B(OH)2, 4-(Et2N)C6H4B(OH)2, or 2-(Ph2N)-5-(pin)Bthiophene, Pd(PPh3)4, K2CO3, toluene, 110  C; (c) (1) HSiCl3, toluene, rt;(2) MeOTf, CH2Cl2, rt; (d) (1) ethylene glycol, TsOH$H2O, benzene,
100  C, (2) 4-(Ph2N)C6H4B(OH)2, Pd(PPh3)4, Na2CO3, toluene/H2O, (3)HClaq; (e) H2NOH$HCl, TsOH$H2O, MgSO4, toluene, 120  C; (f)Pd(PPh3)4, Na2CO3, toluene/H2O, 110  C.

Table 1

Initially, we evaluated the photophysical properties of 1a,summarized in Table 1, and the UV-vis absorption and uorescence spectra of 1a in various solvents are shown in Fig. 2a.
Regardless of the solvent used, 1a showed absorption maxima(labs) at 458–466 nm with molar absorption coeﬃcients>30 000 M1 cm1. In contrast, the uorescence spectra of 1a
exhibited signicant solvatochromism. While 1a showed anemission maximum (lem) at 532 nm with a Stokes shi of3037 cm1 in nonpolar cyclohexane, a signicant bathochromicshi (lem ¼ 665 nm) with a large Stokes shi (6607 cm1) was
observed in polar CH3CN. This result demonstrates that 1a
exhibits a strong intramolecular charge-transfer (ICT) characterin the excited state, which is a typical feature of D–p–A uorophores. The ICT character of 1a is also reected in a large
dipole moment in the excited state (mE ¼ 20.6 D), which wasestimated using the Lippert–Mataga equation (Fig. S8 and Table
S2†). Notably, 1a maintained a high uorescence quantum yieldin polar solvents (e.g., in CH3CN: FF ¼ 0.59) despite its large mEvalue. This behaviour diﬀers signicantly from that commonly
seen in D–p–A-type uorescent dyes.
Eﬀects of the boryl group on the photophysical properties
To investigate the origin of the high uorescence quantum yieldof 1a, especially in polar solvents, its photophysical properties

Photophysical properties of D–A–A-type dyes 1a–1f and reference compounds 2 and 3 in various solvents

CompoundSolvent

labsa (nm)3(104 M1 cm1)lem (nm)

Stokes shi(cm1)FFb

kr (108 s1)knr (108 s1)1aCyclohexane
Toluene
CHCl3
CH2Cl2
CH3CN
Cyclohexane
CH3CN
Cyclohexane
CH3CN
Cyclohexane
CH3CN
Cyclohexane
CH3CN
Cyclohexane
CH3CN
Cyclohexane
CH3CN
Cyclohexane
CH3CN

458
466
466
466
462
470
487
486
477
476
475
488
480
470
474
463
464
453
452

3.56
3.43
3.18
3.32
3.45
3.26
3.28
3.14
2.77
3.07
2.77
—c
2.67
4.47
4.70
2.44
2.70
—c
2.46

532
566
597
626
665
540
695
570
704
567
699
617
748
550
651
537
709
548
681

3037
3791
4709
5485
6607
2758
6145
3032
6760
3372
6848
4284
7464
3095
5736
2976
7447
3827
7440

0.81
0.90
0.90
0.88
0.59
0.81
0.72
0.65
0.67
0.86
0.69
0.62
0.07
0.44
0.71
0.60
0.08
0.59
0.26

2.6
2.8
2.3
2.1
1.5
2.3
1.5
1.9
1.5
2.4
1.5
n.d.
n.d.
2.0
1.8
1.5
0.94
1.4
0.95

0.62
0.31
0.25
0.28
1.0
0.54
0.59
0.99
0.74
0.39
0.67
n.d.
n.d.
2.6
0.72
1.0
11
0.98
2.7

1b
1c
1d
1e
1f
2
3

a
Only the longest absorption maximum wavelengths are shown. b Absolute uorescence quantum yields were determined by a calibrated
integrating sphere system within 3% error. c Not determined due to poor solubility.

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value of 1a in CH3CN is 1.0  108 s1, while those of 2 and 3 are
beyond 108 s1. These results indicate that not only the higher kr
value, but also the suppressed knr value are responsible for thehigh uorescence quantum yield of 1a in polar solvents.
Theoretical examination of the eﬀects of the boryl group

UV-vis absorption and emission spectra of (a) 1a in various
solvents, (b) 1a, 2, and 3 in CH3CN, and (c) emission spectra of 1a–1e in
CH3CN.
Fig. 2were compared to those of reference compounds 2 and 3, whichcontain a formyl and cyano group instead of the boryl group,
respectively. While cyano analogue 3 showed a maximum
emission wavelength slightly longer than that of 1a in CH3CN,
formyl analogue 2 exhibited a more red-shied emission (Dl ¼
44 nm; Fig. 2b). Moreover, 2 and 3 also showed signicantsolvent eﬀects, i.e., large dipole moments (2: mE ¼ 23.8 D; 3: mE¼ 21.0 D) were estimated using the Lippert–Mataga equation(Fig. S8†), which suggests strong ICT character in the excitedstate, akin to that of 1a. However, their uorescence quantum
yields decreased drastically in polar solvents such as CH3CN (2:FF ¼ 0.08; 3: FF ¼ 0.26), which is typical for D–p–A-type dyes.
To gain further insight into the features of boryl-substituted
uorescent dye 1a, we examined its excited-state dynamics interms of its radiative (kr) and nonradiative (knr) decay rate
constants from the excited singlet state (S1). These values are
determined by the FF values and the uorescence lifetimes s.
For 1a, 2, and 3, the kr values decrease with increasing solvent
polarity (Table 1). Boryl derivative 1a exhibits a higher kr value
than 2 or 3 in CH3CN, which is at least partially responsible forits higher FF value. Moreover, 1a shows the lowest knr value inCHCl3, which increases slightly in CH3CN. Importantly, the knr

6336 | Chem. Sci., 2021, 12, 6333–6341

A theoretical analysis of the photophysical properties of thediarylboryl and dithienophosphole-based D–A–A type molecules
was carried out using time-dependent density functional theory(TD-DFT). As the characteristic eﬀects of the boryl group in 1a
on FF were experimentally observed in comparison with reference compounds 2 and 3, this computational study was focusedon the elucidation of the role of the boryl group in increasing
FF. The solvent eﬀects were considered using the polarizable
continuum model (PCM) to verify their impact on FF. The
calculations were designed to identify the major factors that
determine kr and knr (for the details of the computational
calculations, see the ESI†).
First, the radiative transition relevant to the kr value was
examined based on the TD-DFT results in S0 and S1 (Fig. 3).
Boryl derivative 1a exhibits a slightly higher oscillator strength (f¼ 1.19) for the Franck–Condon transition from S0 to S1 relative
to 2 (f ¼ 1.02) and 3 (f ¼ 0.97). This result is consistent with thefact that 1a exhibits a 50% higher molar absorption coeﬃcientthan 2 and 3. Notably, the f value for the vertical transition from
S1 to S0 for 1a with the optimized S1 geometry is also higher thanthose for 2 and 3 (1a: f ¼ 1.42; 2: 1.34; 3: 1.26, Fig. S13†). As kr is
proportional to n2f, where n is the wavenumber of the emission,
the higher f value in 1a should be partially responsible for thehigher kr value relative to those of 2 and 3.
Next, we examined the non-radiative transition relevant to
the knr value, which may occur through various decay
processes. The possible pathways include the internal

Fig. 3 Energy diagrams for the frontier orbitals of 1a, 2, and 3 in
CH3CN using the optimized ground state structures. Calculations werecarried out at the CAM-B3LYP/6-31G(d) level of theory including the
PCM model.

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conversion (IC) from S1 to S0 and/or the intersystem crossing(ISC) to the triplet states.30 We performed rate-constantcalculations on the S1 / S0 IC process and the S1 / T2 ISC
process; hereaer, their rates are denoted as kIC and kISC,
respectively. With the relaxed S1 geometry, S1 and T2 lie
closely in energy and largely away from other states, whichwere thus neglected (Fig. S14†). The kIC and kISC of 1a in
CH3CN were estimated to be 1.4  108 and 1.0  107 [s1],respectively, using MOMAP-2020A program31 (for details, see
the ESI†). This implies that the main nonradiative decay
pathway of these dyes in CH3CN is the S1 / S0 IC.
To discuss the relative trend of the experimentally obtained
knr values among 1a, 2, and 3, which are assumed to be mainly
kIC, the normal-mode contribution of the nonadiabatic
coupling (NAC) calculated with the S1-optimized geometry was
analysed as shown in Fig. 4a. For all compounds, the normal
mode with a frequency of ca. 1570 cm1 was found to have oneof the largest NAC values (Fig. S15†). These modes were attributed mainly to the C5–C6 and C7–C8 stretching vibration in thebithiophene moiety of the dithienophosphole scaﬀold (for theatom labelling, see Fig. 4b). Because the largest geometry
changes occur in the C5–C6 bond length between the optimized

Chemical Sciencestructures of S0 and S1, while they are nearly identical among 1a,2, and 3 (Fig. S16†), this quinoidal mode should thus make thelargest contribution to kIC through large nonadiabatic interstate
coupling.32
The NAC values along with the quinoidal mode were calculated to be 113.9 cm1, 124.6 cm1 and 120.8 cm1 in CH3CN
for 1a, 2, and 3, respectively. Assuming that this mode is thepromoting mode of the S1 / S0 IC and vibrational terms arecomparable among these dye molecules, the kIC should be
proportional to the square of NAC along with this mode. The
square of these NAC values provides the following relation: 1a <3 < 2 (Fig. 4c). This trend qualitatively matches the relative
magnitude of the experimental knr among the three compounds
in CH3CN. For the CH3CN solutions, we can thus deduce thatthe substituent-dependence of kIC, which almost coincides withknr, is determined by the NACs associated with the quinoidal
vibration.
The spin–orbit coupling (SOC) between S1 and T2 was also
computed. In the procedure here, SOC was treated asa constant. This means that kISC is proportional to the square of
SOC when vibrational terms are comparable among these dyemolecules. Compound 1a has a smaller square of the SOC thanthe other molecules (Fig. 4c), which implies that 1a has smaller
kISC than the others.
Considering that the experimentally obtained knr for 2 wasabout 10 times larger than that for 1a, our prediction mightunderestimate the interstate interaction of 2. This seemingly
arises from the limitation of our model, where the terms thatcan be important in the calculation of kISC with small direct SOC(see the ESI† for details) were neglected.
Thus, the question of why boryl derivative 1a exhibits a larger
FF (¼ kr/(kr + knr)) compared to 2 and 3 can be partiallyaddressed by our theoretical model, which shows that 1a in S1
undergoes decay processes with a larger radiative decay rate
constant kr and a smaller non-radiative decay rate constant knr.
The latter can be rationalized in terms of a suppression of thenonadiabatic IC process with the smaller NACs and the ISC
process with smaller SOC.

Structural modication to achieve NIR emission

Fig. 4 (a) Computed NAC and frequencies along with the normal
modes of 1a, 2 and 3 in CH3CN. (b) A representative vibrational modewith large NAC of 1a in CH3CN, which has a large contribution of theC5–C6 and C7–C8 stretching and a frequency of 1572 cm1; therepresentative vibrational modes of the other molecules are shown in
Fig. S15.† (c) Comparison of the square of the largest NAC in Fig. 4a(left) and the square of the SOC between S1 and T2 (right) among 1a, 2
and 3 in CH3CN. Quantum mechanical calculations were conducted atthe (TD-)CAM-B3LYP/6-31G(d) level of theory using Gaussian 16 Rev.
B.01 program.33 SOC were computed using PySOC program.34© 2021 The Author(s). Published by the Royal Society of Chemistry

To accomplish a more red-shied emission, we modied thedonor moiety, p-spacer, and/or the phosphorus moiety of the
D–A–A framework (Fig. 2c). The photophysical properties of thecorresponding compounds 1b–1e are summarized in Table 1(for the full data in various solvents, see Table S1†). Compound
1b, which contains ethyl groups in place of phenyl groups on
the amino moiety, and 1c, which contains a thiophene p-spacer,
exhibited more red-shied emission bands than 1a in CH3CN
due to their enhanced electron-donating character (1b: lem ¼
695 nm; 1c: lem ¼ 699 nm). Compound 1d, in which the P]Obond of 1c is replaced with a P]S bond, exhibited a comparable
emission to that of 1c. Although 1e, with a quaternized P centre,
showed the longest wavelength emission (lem ¼ 748 nm in
CH3CN), its FF value was low (0.07). Notably, all these derivatives, except for 1e, retained high quantum yields even in
CH3CN, despite their long-wavelength emission.

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The improved photostability of 1f suggests that the stericcongestion around the boron atom greatly enhances its
photostability.
Application to bioimaging

Fig. 5 (a) Chemical structure of 10 and (b) monitoring the absorption
decay upon exposure to irradiation from LED light (lem ¼ 449 nm).

Evaluation of the photostability
The introduction of additional heteroatoms can be expected toaﬀect the photostability of the present compounds.35 Therefore,
the photostability of 1a, 3, and reference compound 10 (Fig. 5a),which does not contain a P(]O)Ph group, was evaluated indegassed CH3CN under irradiation from a 449 nm high-power
LED lamp equipped with a 450/10 nm hard-coated bandpass
lter. We have previously demonstrated that the introduction ofa bulky substituent on the B atom results in increased photostability.36 Accordingly, we designed 1f, which should exhibit
improved photostability relative to 1a, on account of the bulky
substituent at the boron atom, and evaluated its photophysicalproperties. To quantitatively evaluate the photostability of thesecompounds, we attempted to determine their total quantum
yield of photodecomposition (Fdec), which is dened as the sumof the quantum yields of all photoreactions producing products
under light irradiation.37–39 The Fdec values estimated wereconsidered to be the minimum possible values, as Fdec isunderestimated when the decomposition products absorb lightat the monitored wavelength.
The results of the irradiation experiments are shown in
Fig. 5b. While the change in absorbance over time was almost
linear for 3 and 1f, 1a and 10 showed non-linear behaviour,
probably due to the inuence of the absorption of the photodecomposed product(s). Although the Fdec values for 1a and10 cannot be determined quantitatively, given that a linear
slope value is required for the calculation, a qualitative
comparison demonstrates that 1a is substantially more photostable than 10, conrming that the introduction of the P(]O)Ph
group improves the photostability of the uorophore. Furthermore, bulky aryl-substituted 1f showed improved photostability(Fdec ¼ 5.8  106). This value approaches that of 3 (2.7  106)and is two orders of magnitude smaller than that of Alexa Fluor
488 (2.3  104 in DMSO/buﬀer ¼ 7/3), which is widely used asa representative photostable dye in bioimaging. However, it
should be noted here that these values cannot be compareddirectly as diﬀerent solvents were used in the measurements.

6338 | Chem. Sci., 2021, 12, 6333–6341

The bright far-red to NIR emission of the D–A–A dyes 1, even inpolar solvents, suggest promising potential for uorescenceimaging of biological samples. To examine the utility of thesedyes in such applications, 1c was employed as a representative
example. Prior to conducting the imaging experiments, weconrmed the solubility of 1c in phosphate-buﬀered saline(PBS; pH ¼ 7.4) containing 2% bovine serum albumin (BSA),
which is the most abundant protein in the blood plasma, bydynamic light scattering (DLS) measurements (Fig. S17†). The
solution of 1c in the presence of BSA only showed a peakcomparable to that of a solution of BSA without 1c, indicatingthat 1c was solved under these conditions. Moreover, the solution exhibited red uorescence with a lem of 632 nm (Fig. S18†),
which is slightly shorter than that in CHCl3. This fact implies
that 1c is bound to the hydrophobic pocket of BSA.40
With these results in hand, we tested 1c in two kinds ofimaging. First, we used 1c for the in vivo imaging of bloodvessels in Japanese medaka (Oryzias latipes) larvae one weekaer hatching. For that purpose, a solution of 1c in DMSO (<1mL, 1 mM) was directly injected into the peritoneal cavity of thesh using a microinjection system. The sh was then placed
into water with 0.3% salinity and cultured for 1 h. We conducted
a whole-body imaging analysis of the larvae using a confocal
microscopy system (lex ¼ 488 nm; emission collection: 570–620
nm). Fig. 6a shows a 3D image of the sh that was reconstructedby combining ve 1272.8 mm  1272.8 mm  918 mm images; asshown, the blood vessels are clearly visible in the living sh.
This result suggests that 1c is rapidly absorbed into the bloodstream, where it most likely binds to proteins such as albumin.
Then, we performed deep imaging of the blood vessels inmouse brain using two-photon excitation microscopy. Immediately aer administering 100 mL of 1c (0.9 mM in PBS containing 18% DMSO and 1.6% BSA) to a mouse via intravenous
injection, images were recorded through an open-skull window
at a two-photon excitation wavelength of 880 nm (Fig. 6b).
Notably, in addition to emission of the dye in the bloodstream
in the red region (lem ¼ 601–657 nm), some blood vessels also
exhibited green uorescence in the 500–550 nm region, whichindicates that the microenvironment of the dye is considerably(a) Confocal image of the entire body of a Japanese medaka
larvae and (b) two-photon excitation image of blood vessels in mousebrain.

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hydrophobic. Although the identity of the biological component
that is stained with 1c to produce this green uorescence has
not yet been claried, its observation suggests that the hydrophobic dye may have detached from the albumin and thenadsorbed on the hydrophobic region of the blood vessel wall.

Conclusions
We have developed a series of diarylboryl and dithienophosphole P-oxide-containing D–A–A-type uorophores, which exhibit
far-red to NIR emissions. Thus, the tuning of the acceptor–
spacer is a complementary useful strategy for gaining such redshied emissions with the conventional modication of theterminal acceptor boryl group in the D–p–A scaﬀolds.20 Unlike
analogues that bear formyl or cyano groups in place of the boryl
group, the boryl-substituted D–A–A-type uorophores retain
a high uorescence quantum yield even in polar solvents suchas CH3CN. An analysis of the excited-state dynamics revealed
that the higher kr and the lower knr values of the developed dyes
are responsible for the high quantum yields. TD-DFT calculations demonstrated that the higher kr values can be attributed
to higher oscillator strengths for the electronic transition from
S1 to S0. In terms of nonradiative decay, the internal conversion
from S1 to S0 is likely a major pathway, in which the boryl groupcontributes to decreasing the non-adiabatic coupling associatedwith the quinoidal stretching mode of the dithienophospholeoxide moiety. This result should provide an important insight
into the eﬀect of the boryl groups in the widely studied boronbased D–p–A uorophores. The introduction of the P(]O)Ph
group also greatly inuences the photostability of the dyes; thepresence of bulky substituents on the boron atom improves thephotostability. Moreover, one of the synthesized dyes, 1c, wassuccessfully applied for whole-body imaging of blood vessels
and two-photon bioimaging.

Author contributions
S. Y. and Y. S. conceived the idea. Y. S. synthesized all thecompounds and evaluated their properties. N. I. and T. Y.
conducted the theoretical calculations. M. T., R. K., T. S., and T.
I. conducted imaging experiments. S. Y. and T. B. discussed thep-electron systems studied. Y. S., N. I., M. T., T. Y., and S. Y.
wrote the manuscript, and all authors discussed and commented on the manuscript. S. Y. directed the project.

Ethical statement
All animal experiments were approved by the Ethics Committee
for Animal Experiments of Ehime University (#05-RE-4-16). The
experimental procedures we employed were conducted inaccordance with the approved guidelines.

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

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

Chemical Science

Acknowledgements
This work was supported by MEXT/JSPS KAKENHI Grant
Numbers JP18H03909 and JP18H05261 (to S. Y.), JP16H06280(ABiS), JP15H05952 (to T. I. and T. S.), JP19K12218 and
JP20H05038 (to T. S.) and by AMED Grant Number
JP20gm1210001 (to T. I.). This work was also funded by Nakatani Foundation for Advancement of Measuring Technologies
in Biomedical Engineering, and Kato Memorial Bioscience
Foundation (to T. S.). ITbM is supported by the World PremierInternational Research Center (WPI) Initiative, Japan. T. B.
thanks the Canada Research Chairs program for support.

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