Turn-on fluorescence sensor for mono- and di-phosphonic acid derivatives using anthracene-based diamidine and its detection of amidinium-phosphonate and amidinium formation

Article abstract of DOI:10.1016/j.tet.2017.12.011

The fluorescence detection of di-phosphonic acid and mono-phosphonic acid derivatives using the anthracene-based diamidine 1 has been investigated. The diamidine 1 forms 1:1 and 1:2 complexes with the di-phosphonic acid and mono-phosphonic acid derivative

Full text of DOI:10.1016/j.tet.2017.12.011

Tetrahedron 74 (2018) 465e476
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Tetrahedron
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Turn-on fluorescence sensor for mono- and di-phosphonic acid
derivatives using anthracene-based diamidine and its detection of
amidinium-phosphonate and amidinium formation
Takahiro Kusukawa*, Hitoshi Nagano, Keita Nakaguchi, Shota Takeshita, Yuya Harumoto
Department of Chemistry and Materials Technology, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku Kyoto
606-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The fluorescence detection of di-phosphonic acid and mono-phosphonic acid derivatives using the
anthracene-based diamidine 1 has been investigated. The diamidine 1 forms 1:1 and 1:2 complexes with
the di-phosphonic acid and mono-phosphonic acid derivatives, respectively, and showed a blue fluo-
rescence (lem ¼ 432e442 nm) in a DMSO solution. The formation of amidinium-phosphonate (complex
formation) and dissociated amidinum (lem ¼ 468 nm as a broad band) were distinguished by the dif-
ference in the fluorescence wavelength, and confirmed by DOSY NMR spectroscopy and TD-DFT calcu-
lations. The formation of a 1:2 complex with diamidine 1 and methylphosphonic acid having additional
intermolecular hydrogen-bonding between the methylphosphonic acids is proposed.
Received 31 October 2017
Received in revised form
4 December 2017
Accepted 6 December 2017
Available online 7 December 2017
Keywords:
Amidinium-phosphonate
Amidinium
© 2017 Elsevier Ltd. All rights reserved.
Fluorescence
Phosphonic acid recognition
1. Introduction
human urine as a “turn-on” type fluorescence sensor.5 In this sys-
tem, the formation of cationic charges after the recognition of the
The development of fluorogenic chemosensors for phosphorus-
related oxoacids (oxoanions) is of particular interest due to their
applications in the chemical, biological and environmental sci-
ences.1 Although there are numerous reports of fluorogenic che-
mosensors for “phosphoric acids (phosphate)”,1a only a few
examples of fluorogenic chemosensors for “phosphonic acids
(phosphonate)” have been reported.2 The phosphonic acid de-
rivatives have a broad range of applications3 where they find utility,
such as in bone cancer treatments and care for diseases related to
osteoporosis.
dicarboxylic acids suppresses the PET (photoinduced electron
transfer6) processes and consequently “turns-on” the fluorescence
after the recognition of carboxylic acids (formation of amidinium-
carboxylate salt bridge, Scheme 1).
We now report the fluorescence detection of “phosphonic acid
derivatives” using the anthracene-based diamidine 1. The
anthracene-based diamidine 1 recognize mono- and di-phosphonic
acid derivatives by the formation of “amidinium-phosphonate” salt
bridges and consequently turn-on the fluorescence. To compare the
recognition ability between “dicarboxylic acids” and “diphosphonic
The amidine is known to bind with oxoacids, such as carboxylic
acids, phosphonic acids, phosphoric acids and sulfonic acids, and
forms salt bridges having twitter ionic charges.4 Due to the for-
mation of the twitter ionic charges, the hydrogen bond of the
amidinium-oxoanion salt bridge shows a stronger binding even in
polar solvents (i.e., charge-assisted hydrogen bond4a). Recently, we
reported the fluorescence detection of a,u-dicarboxylic acids using
an anthracene-based diamidine 1 in a DMSO solution and suc-
ceeded in the quantification of the amount of dicarboxylic acid in
acid derivatives”, we examined the recognition of a,u-diphos-
phonic acid having two hydroxyl groups for one phosphorous atom
(i.e., 1,8-octanediphosphonic acid). However, the precipitation of
the formed complexes was observed and could not characterize the
structure of the binding complex using NMR spectroscopy. To in-
crease the solubility of the formed complexes, we synthesized new
diphosphonic acid diethyl esters 2 having two solubilizing ethyl
groups. The formation of 1:1 complexes of diamidine 1 and
diphosphonic acid diethyl esters 2 was analyzed by 1H, 31P NMR,
DOSY NMR and fluorescence spectroscopies. The formation of the
“stable 1:1 complex 1ꢀ2” or “dissociated amidinium 1ꢀ2Hþ” was
also characterized by DOSY NMR spectroscopy and the differences
* Corresponding author.
E-mail address: kusu@kit.ac.jp (T. Kusukawa).
https://doi.org/10.1016/j.tet.2017.12.011
0040-4020/© 2017 Elsevier Ltd. All rights reserved.

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T. Kusukawa et al. / Tetrahedron 74 (2018) 465e476
DOSY NMR spectra for the complexation of diamidine 1 with the
structurally slightly rigid diphosphonic acid diethyl esters (2a, 2b).
The formation of the 1:1 complex 1ꢀ2a was achieved by mixing the
DMSO-d6 solution (1.89 mM) of 1 and 2a (m-xylylene diphosphonic
acid diethyl ester). The 1H NMR spectrum of the obtained solution
showed the characteristic upfield and downfield shifts with respect
to those of 1 and 2a, respectively (Fig. 1 and Fig. S1). The downfield
shift of the amidine unit (Ha-Hc, He) and the upfield shift of the
phosphonic acid unit (HA-HC, HE, HF) in the 1H NMR indicated the
formation of a cationic charge and an anionic charge in each unit
after complexation. The resonance of the NH protons was observed
at 11.9 and 9.7 ppm (Fig. 1b). Based on the comparison of the for-
mation of “amidinium-carboxylate” binding complexes,5 these NH
proton signals strongly indicated the fomation of “amidinium-
phosphonate” salt bridges (i.e., formation of a stable complex).
Additional proof of the formation of 1ꢀ2a was obtained from the
DOSY NMR spectroscopy. The 1H DOSY spectra showed that the
diffusion coefficient of complex 1ꢀ2a is lower than those of the
corresponding free building blocks (1, 2a), confirming the forma-
tion of the larger-sized complex (Fig. 2). The molecular volume of
1ꢀ2a derived from the observed diffusion coefficient (V ¼ 1062 Å3)
is slightly greater than the sum of the volumes of the free building
blocks (1: V ¼ 280 Å3, 2a: V ¼ 352 Å3), but smaller than the volume
after the formation of the 2:2 complex 12ꢀ(2a)2 (Vcalc ¼ 1264 Å3),
which indicated the formation of the stable 1:1 complex (Fig. 2,
Table 1).7 The complexation of diamidine 1 with the m-phenylene
diphosphonic acid diethyl ester 2b also showed the formation of
the stable 1:1 complex 1ꢀ2b in DMSO, which was confirmed by 1H
NMR and DOSY NMR spectroscopies (Figs. S2 and S26, Table 1).
Scheme 1. Schematic representation of the formation of amidinium-carboxylate salt
bridge and its fluorescence characters.
in the wavelengths of the fluorescence spectra (Scheme 2).
Surprisingly, the formation of 1:2 complexes 1ꢀ(3)2 was
observed for the recognition of the mono-phosphonic acids 3
which have two hydroxyl groups on the phosphorous atom. Similar
1:2 complexes of diamidine 1 with mono-carboxylic acids have
never been observed and form only the dissociated amidinium
1ꢀ2Hþ. In the case of the mono-phosphonic acid binding, the for-
mation of 1:2 complexes 1ꢀ(3)2 is achieved by the formation of
additional intermolecular hydrogen bonding between the phos-
phonic acids after formation of the amidinium-phosphonate salt
bridges.
2. Results and discussion
2.1. Synthesis of diphosphonic acid derivatives
The diphosphonic acid diethyl esters 2 were prepared according
to Scheme 3. The tetraethyl diphosphonate derivatives 5 were
prepared from the corresponding dibromide by the Arbuzov reac-
tion (refluxing in P(OEt)3) or cross-coupling conditions (NiCl2/
P(OEt)3). The obtained tetraethyl diphosphonate derivatives 5 were
refluxed with anhydrous LiBr in 2-hexanone and the precipitates of
2.3. Binding experiments of diamidine 1 with
acid diethyl esters (2c-2h)
a,u-diphosphonic
the dilithium-diphosphonate derivatives
6 were collected by
centrifugation. The diphosphonic acid diethyl esters 2 were pre-
pared from the corresponding dilithium-diphosphonates 6 by the
treatment with an ion exchange resin.
To compare the recognition ability of the diamidine 1 toward
“diphosphonic acid derivatives” and “dicarboxylic acids”, we
examined the recognition of the conformationally flexible
2.2. Binding experiments of anthracene-based diamidine 1 with
diphosphonic acid diethyl esters (2a, 2b)
a
,
u
ꢁdiphosphonic acid derivatives. However, after the complexa-
tion of diamidine 1 with -diphosphonic acid, which has two
a,u
hydroxyl groups for one phosphorous atom (i.e. 1,8-
octanediphosphonic acid), precipitation was observed from the
DMSO solution. To increase the solubility of the formed complexes,
we synthesized
a
,
u
ꢁdiphosphonic acid diethyl esters 2c-2h which
have soluble ethyl groups (protection of the hydroxyl group to
inhibit the formation of a higher aggregate). The formation of the
1:1 complex 1ꢀ2g (n ¼ 7, 0.95 mM) was achieved by mixing the
DMSO-d6 solution of both components. After complexation, the 1H
NMR spectrum of the obtained solution showed the characteristic
upfield and downfield shifts with respect to those of 1 and 2g, and
To investigate the recognition ability of the diamidine 1 toward
the diphosphonic acid derivatives, we observed the 1H NMR and

T. Kusukawa et al. / Tetrahedron 74 (2018) 465e476
467
Scheme 2. Schematic representation of the formation of amidinium-phosphonate and dissociated amidinium, and their fluorescence characters.
resonance of the NH protons was also observed at 12.7 and 9.6 ppm,
respectively, which indicated the formation of the stable complex
(Fig. 3b and Fig. S7). Additional proof of the formation of 1ꢀ2g
(n ¼ 7) in the solution state was also obtained from DOSY NMR
spectroscopy. The DOSY spectra showed that the diffusion coeffi-
cient of complex 1ꢀ2g is lower than those of the corresponding free
building blocks (1, 2g), confirming the formation of the larger-sized
complex (Fig. 4). The molecular volume of 1ꢀ2g derived from the
observed diffusion coefficient (V ¼ 932 Å3) is slightly greater than
the sum of the volumes of the free building blocks (1: V ¼ 297 Å3,
2g: V ¼ 372 Å3), which indicated the formation of the stable 1:1
complex (Table 2).
For the complexation of diamidine 1 with the diphosphonic acid
diethyl esters 2c-2f (n ¼ 3e6) and 2h (n ¼ 8), the 1H NMR spectra of
the formed complexes showed reasonable upfield and downfield
shifts indicating the formation of stable complexes (Figs. S4eS8).
For the 1:1 complexes, 1ꢀ2d, 1ꢀ2f, and 1ꢀ2h, similar diffusion co-
efficients (molecular volume) compared to the formation of 1ꢀ2g
were observed by the DOSY NMR spectroscopy which indicated the
formation of stable 1:1 complexes (Table 2). In the case of the
diphosphonic acid diethyl ester 2c, which has a slightly shorter
methylene chain length compared to the distances between the
Scheme 3. Synthesis of diphosphonic acid diethyl esters 2.
Fig. 1. 1H NMR spectra (1.89 mM in DMSO-d6, 298 K) of a) diamidine 1, b) 1ꢀ2a obtained by mixing equimolar solutions of 1 and 2a, and c) diphosphonic acid diethyl ester 2a.

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T. Kusukawa et al. / Tetrahedron 74 (2018) 465e476
1
Fig. 2. H DOSY spectra (1.89 mM in DMSO-d6, 25 ꢂC, diffusion time
D
¼ 200 ms) of a) complex 1ꢀ2a, b) diphosphonic acid diethyl ester 2a, c) diamidine 1.
Table 1
two amidino groups of 1, different diffusion coefficients were
separately observed indicating the formation of the slightly weaker
1:1 complex 1ꢀ2c. Also, in the case of 1ꢀ2e (n ¼ 5), precipitation
Comparison of diffusion coefficients, molecular radii and volumes.a
Compounds
D (m2 sꢁ1
)
rH (Å)
V (Å3)
was observed within several hours after complexation, and could
1
2a
2.45 0.03 ꢃ 10ꢁ10
2.27 0.03 ꢃ 10ꢁ10
1.57 0.06 ꢃ 10ꢁ10
2.37 0.02 ꢃ 10ꢁ10
1.70 0.06 ꢃ 10ꢁ10
4.06
4.38
6.33
4.20
5.85
280
352
1062
310
839
not observed by DOSY NMR spectroscopy (Figs. S27eS30).
1ꢀ2a
2b
2.4. Fluorescence detection of diphosphonic acid diethyl esters 2:
detection of amidinium-phosphonate and amidinium formation
1ꢀ2b
a
Diffusion coefficients were recorded at 298 K in DMSO-d6 (1.89 mM, diffusion
¼ 200 ms), and the molecular radii were calculated using the Stokes-Einstein
equation.
time
D
To demonstrate the recognition ability of the diamidine 1, we
observed the fluorescence spectra of the diamidine 1 with the
Fig. 3. 1H NMR spectra (0.95 mM in DMSO-d6, 298 K) of a) diamidine 1, b) 1ꢀ2g obtained by mixing equimolar solutions of 1 and 2g, and c) diphosphonic acid diethyl ester 2g.

T. Kusukawa et al. / Tetrahedron 74 (2018) 465e476
469
Fig. 4. 1H DOSY spectra (0.95 mM in DMSO-d6, 298 K, diffusion time
D
¼ 200 ms) of a) complex 1ꢀ2g, b) diphosphonic acid diethyl ester 2g, c) diamidine 1.
Table 2
Comparison of diffusion coefficients, molecular radii and volumes.a
Compounds
D (m2 sꢁ1
)
rH (Å)
V (Å3)
1
2c
1ꢀ2c
2.40 0.02 ꢃ 10ꢁ10
2.56 0.04 ꢃ 10ꢁ10
1.66 0.01 ꢃ 10ꢁ10(A)b
1.85 0.01 ꢃ 10ꢁ10(P)c
2.39 0.03 ꢃ 10ꢁ10
1.67 0.05 ꢃ 10ꢁ10
2.29 0.02 ꢃ 10ꢁ10
1.68 0.05 ꢃ 10ꢁ10
2.23 0.01 ꢃ 10ꢁ10
1.64 0.04 ꢃ 10ꢁ10
2.19 0.05 ꢃ 10ꢁ10
1.54 0.02 ꢃ 10ꢁ10
4.14
3.88
5.99
5.38
4.16
5.95
4.34
5.92
4.46
6.06
4.54
6.46
297
245
900
652
301
882
342
869
372
932
392
1129
2d
1ꢀ2d
2f
1ꢀ2f
2g
1ꢀ2g
2h
1ꢀ2h
a
Diffusion coefficients were recorded at 298 K in DMSO-d6 (0.95 mM, diffusion
¼ 200 ms), and the molecular radii were calculated using the Stokes-Einstein
equation.
time
D
b
Derived from diamidine 1.
Derived from diphosphonic acid diethyl ester 2c.
c
diphosphonic acid diethyl esters 2 in a DMSO solution. The dia-
midine 1 does not show a fluorescence (turn-off state) in the DMSO
solution under UV light irradiation due to the PET (photoinduced
electron transfer) process6 from the amidino groups (Scheme 1,
Fig. 5). However, after the addition of the diphosphonic acid diethyl
esters 2a (1 equiv.), a blue emission was observed at 440 nm.
Similarly, after the addition of the diphosphonic acid diethyl esters
2b-2h (1 equiv.), a blue emission at 432e442 nm was also observed
Fig. 5. Fluorescence spectra of diamidine 1 (25
addition of diphosphonic acid diethyl ester 2a-2h (1 eq.).
m
M in DMSO, lex ¼ 348 nm) upon the
from TD-DFT calculation and the fluorescence spectrum of dia-
midine dihydrochloride 1ꢀ2HCl in DMSO was observed as a
broadband around at 460 nm (Fig. 5, Table 3). These observations
indicated that complexation of the diamidine 1 with the diphos-
phonic acid diethyl esters 2 produced stable 1:1 complexes in the
even in the flexible a,u-diphosphonic acid diethyl esters (Fig. 5). To
characterize the origin of these fluorescence spectra, we evaluated
the estimated fluorescence wavelengths of 1ꢀ2a, 1ꢀ2b and 1þ2Hþ
(dissociated amidinium) using the time-dependent density func-
tional theory (TD-DFT, B3LYP/6-31G*//CIS/6-31G*) calculations. The
fluorescence wavelengths of the 1:1 complexes 1ꢀ2a
DMSO solution even at a low concentration (25
mM) by the for-
mation of two amidinium-phosphonate salt bridges (Scheme 2).
To obtain additional evidence for the formation of the
amidinium-phosphonate salt bridge, we measured the fluores-
cence spectra of 1ꢀ2d (n ¼ 4) after the addition of H2O (Fig. 6). The
fluorescence spectra of 1ꢀ2d was red-shifted (lmax ¼ 460 nm) by
(
lcalc ¼ 441 nm) and 1ꢀ2b (lcalc ¼ 436 nm) were obtained, and good
agreement was observed with the fluorescence wavelengths of
1ꢀ2a (lexp ¼ 440 nm) and 1ꢀ2b (lexp ¼ 438 nm) in a DMSO solution
(Table 3). The fluorescence wavelength of the dissociate amidinium
the addition of H2O (0e560 mL), which indicated the formation and
dissociation of the amidinium-phosphonate salt bridges in the
DMSO solution.
1þ2Hþ
lcalc ¼ 465 nm, using PCM model in DMSO) was estimated
(

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T. Kusukawa et al. / Tetrahedron 74 (2018) 465e476
Table 3
Results of TD-DFT calculations and experimental wavelengths for fluorescence spectra.
Compounds
Experimental maximal wavelength (nm)a
Calculated maximal wavelength (nm)b
Oscillator strength (f)b
1ꢀ2a
440
438
d
441
436
465c
d
0.08
0.08
0.12
d
1ꢀ2b
1ꢀ2Hþ
1ꢀ2HCl
460
a
Fluorescence spectra were recorded in DMSO (25
TD-PBE0/6-31G*//CIS/6-31G*.
Calculation was performed with PCM model (DMSO).
mM, lex ¼ 348 nm).
b
c
Fig. 7. Fluorescence spectra of diamidine 1 (25
addition of monophosphonic acids 3a-3d (2 equiv.) and ethyl ester 4 (2 equiv.).
m
M in DMSO, lex ¼ 348 nm) upon the
emission band at 441 nm indicating the formation of the stable 1:2
complex (formation of amidinium-phosphonate, Fig. 7). Similar
fluorescence spectra were observed for the recognition of the
monophosphonic acid 3b and 3c, indicating the formation of a
stable 1:2 complex. In the recognition of the phenylphosphonic
acid 3d, the fluorescence spectrum showed slightly broad bands
and it may have caused the dissociation of the amidinium-
phosphonate bonding due to the slightly strong acidity of the
phenylphosphonic acid (pKa(calc) ¼ 1.88)8 compared to the meth-
ylphosphonic acid (pKa(calc) ¼ 2.27)8. The differences in the recog-
nition ability of the diamidine 1 toward the “monophosphonic
acid” and “monocarboxylic acid” may be caused by the presence
and absence of an additional hydroxyl group after amidinium-
phosphonate and amidinium-carboxylate formation. To clarify
this hypothesis, we performed the DFT calculation (B3LYP/6-31G*)
of the 1:2 complex of the diamidine 1 and monophosphonic acid 3.
The optimized structure of 1ꢀ(3a)2 showed the presence of inter-
molecular hydrogen bonds of the hydroxyl groups between the two
methylphosphonic acids 3a (Fig. 8). The calculated fluorescence
wavelengths of 1ꢀ(3a)2 (lobs. ¼ 441 nm, lcalc. ¼ 434 nm) and 1ꢀ(3c)2
Fig. 6. Fluorescence spectra of 1ꢀ2d (25
(0e560 L) with excitation at 348 nm.
mM, DMSO) upon the addition of H2O
m
2.5. Binding experiments of anthracene-based diamidine 1 with
monophosphonic acid derivatives
(lobs. ¼ 439 nm, lcalc. ¼ 433 nm) using the TD-DFT calculation is
very close to the observed fluoresce wavelength, which indicated
the formation of the stable complexes 1ꢀ(3a)2 and 1ꢀ(3c)2 in the
DMSO solution (Table S1). To obtain the experimental proof of the
formation of the 1:2 complex assisted by the additional intermo-
lecular hydrogen bonding of the phosphonic acids, we examined
the recognition of ethyl methylphosphonate 4, which does not have
an additional hydroxyl group on the phosphorous atom after the
Next, we examined the recognition of the monophosphonic
acids with diamidine 1 using fluorescence spectroscopy. According
to our previous results about the recognition of the monocarboxylic
acids,5 the formation of the dissociated amidinium 1ꢀ2Hþ was
observed due to the instability of the formed 1:2 complex. Sur-
prisingly, however, the fluorescence spectra of the diamidine 1 with
methylphosphonic acid 3a (2 equiv.) in DMSO showed a blue

T. Kusukawa et al. / Tetrahedron 74 (2018) 465e476
471
observed in the 1H NMR spectrum due to the fast exchanging with a
water molecule. Additional proof for the formation of 1ꢀ(3a)2 in the
solution state was also obtained from the DOSY NMR spectra. The
molecular volume of 1ꢀ(3a)2 derived from the observed diffusion
coefficient (V ¼ 660 Å3) is slightly greater than the calculated
molecular volume of 1ꢀ(3a)2 (1: V ¼ 280 Å3, 3a: V ¼ 99 Å3, 1ꢀ(3a)2:
Vcalc. ¼ 478 Å3), which indicated the formation of the stable 1:2
complex (Fig. 10, Table 4). The formation of 1ꢀ(3b)2 and 1ꢀ(3c)2 was
also evidenced by the 1H NMR and DOSY NMR spectroscopy
(Figs. S10, S11, S31, S32, Table 4). A small difference in the diffusion
coefficients derived from 1 and 3d was observed for the complex-
ation of 1ꢀ(3d)2, which indicated that the slightly weak stability of
1ꢀ(3d)2, and a similar phenomenon was observed in the fluores-
cence spectra (Fig. 7). To clarify the effect of the intermolecular
hydrogen bonding of the hydroxyl groups on the formation of the
stable 1:2 complex 1ꢀ(3)2, we measured the DOSY spectrum of the
1:2 mixture of diamidine 1 and ethyl methylphosphonate 4 in the
DMSO-d6 solution. The DOSY spectrum of the desired solution
showed two different diffusion signals derived from 1 and 4
(Fig. S34, Table 4). The separately observed DOSY signal for 1ꢀ(4)2
showed that the complex exists in equilibrium with the dissociated
amidinium and phosphonate fragment. These observations agreed
with the difference in the fluorescence spectra of 1ꢀ(3)2 and 1ꢀ(4)2
(Fig. 7).
The single crystal of the complex 1ꢀ(3a)2 was obtained from a
CH3OH solution of the diamidine 1 and methylphosphonic acid 3a
(2 equiv.). The crystal structure of 1ꢀ(3a)2 showed the presence of
amidinium-phosphonate salt bridges (Fig. S51). The hydrogen
atoms in the amidinium-phosphonate bonds were found in the
structure (see experimental part), and both are bonded to the ni-
trogen atoms similar to the amidinium-carboxylate salt bridges.
Unfortunately, intermolecular additional hydrogen bonding be-
tween two hydroxyl groups of methylphosphonic acid 3a in the
crystal structure of 1ꢀ(3a)2 could not be observed. Instead, the
intermolecular hydrogen bonding of methylphosphonic acid 3a and
the formation of the one-dimensional network structure of 1ꢀ(3a)2
Fig. 8. Optimized structure of 1ꢀ(3a)2 at B3LYP/6-31G(d) level.
amidinium-phosphonate formation. After mixing the solution of
diamidine 1 and ethyl methylphosphonate 4 (2 equiv.), the broad
emission band (lmax ¼ 468 nm) corresponding to the formation of
the amidinium 1ꢀ2Hþ was observed (Fig. 7). These observations
showed that the remaining hydroxyl group of the monophosphonic
acid 3 stabilizes the formed 1:2 complex 1ꢀ(3)2 (Fig. 8).
To further demonstrate the formation of the 1:2 complex of the
diamidine 1 and monophosphonic acid 3, we measured the 1H NMR
and DOSY NMR spectra. The formation of the 1:2 complex 1ꢀ(3a)2
(1.89 mM) was achieved by mixing the DMSO-d6 solution of both
components. After the complexation, the 1H NMR spectrum of the
obtained solution showed the characteristic upfield and downfield
shifts with respect to those of 1 and 3a, and resonance of the NH
protons was also observed at 12.3 and 9.7 ppm which also indicated
the formation of the stable complex (Fig. 9 and Fig. S9). However,
the two hydrogen atoms for the formation of the intermolecular
hydrogen bonding between two phosphonic acids 3 was not
Fig. 9. 1H NMR spectra (1.89 mM in DMSO-d6, 298 K) of a) diamidine 1, b) 1ꢀ(3a)2 obtained by mixing equimolar solutions of 1 and 3a, and c) methylphosphonic acid 3a.

472
T. Kusukawa et al. / Tetrahedron 74 (2018) 465e476
Fig. 10. 1H DOSY spectra (1.89 mM in DMSO-d6, 298 K, diffusion time
D
¼ 200 ms) of a) complex 1ꢀ(3a)2, b) methylphosphonic acid 3a, c) diamidine 1.
Table 4
acid diethyl esters 2 and monophosphonic acids 3 by fluorescence
titrations, and the pKa values of the monophosphonic acid 3 are
summarized in Table 5. The binding constants of the diamidine 1
and diphosphonic acid diethyl esters 2 with a 1:1 stoichiometry are
on the order of 106 (Mꢁ1) in the DMSO solution and slightly higher
binding constants were observed compared to the dicarboxylic acid
bindings (see ref.5). Almost no influence of the chain lengths of the
Comparison of diffusion coefficients, molecular radii and volumes.a
Compounds
D (m2 sꢁ1
)
rH (Å)
V (Å3)
1
3a
2.45 0.03 ꢃ 10ꢁ10
3.46 0.01 ꢃ 10ꢁ10
1.84 0.01 ꢃ 10ꢁ10
3.08 0.02 ꢃ 10ꢁ10
1.71 0.05 ꢃ 10ꢁ10
3.10 0.01 ꢃ 10ꢁ10
1.75 0.03 ꢃ 10ꢁ10
3.04 0.06 ꢃ 10ꢁ10
1.65 0.02 ꢃ 10ꢁ10(A)b
1.78 0.01 ꢃ 10ꢁ10(P)c
4.62 0.05 ꢃ 10ꢁ10
2.34 0.03 ꢃ 10ꢁ10(A)b
2.94 0.03 ꢃ 10ꢁ10(P)d
4.06
2.87
5.40
3.23
5.82
3.21
5.68
3.27
6.03
5.59
2.15
4.25
3.38
280
99
1ꢀ(3a)2
660
141
826
139
768
146
918
732
42
3b
1ꢀ(3b)2
3c
a,b-diphosphonic acid diethyl esters (2c-2h) on the formation of
1:1 complexes was observed. For the binding of the mono-
phosphonic acids 3, the binding constants were determined by 1:2
(1:3) stoichiometry (K1ꢀK2/Mꢁ2) and almost no influence of the pKa
values of the monophosphonic acid 3 was observed.
1ꢀ(3c)2
3d
1ꢀ(3d)2
4
To demonstrate the selectivity of the diamidine 1 for the
diphosphonic acid 2, we observed the fluorescence spectra of 1ꢀ2d
after the addition of acetic acid. The fluorescence spectra of 1ꢀ2d
were slightly changed by the addition of acetic acid (0e10 equiv.).
However, the fluorescence spectra of 1ꢀ2d showed a profile suffi-
cient for recognizing the presence of the diphosphonic acid
(Fig. S49). These results show that the 1:1 complex of the diamidine
1 and diphosphonic acid 2d is stabler than the 1:2 complex with
acetic acid even in the presence of excess acetic acid. A similar
observation was observed for the 1:2 complex 1ꢀ(3a)2 (mono-
phosphonic acid) after the addition of acetic acid (Fig. S50).
1ꢀ(4)2
322
162
a
Diffusion coefficients were recorded at 298 K in DMSO-d6 (1.89 mM, diffusion
¼ 200 ms), and the molecular radii were calculated using the Stokes-Einstein
equation.
time
D
b
Derived from diamidine 1.
Derived from phosphonic acid 3d.
Derived from ethyl methylphosphonate 4.
c
d
was observed (Fig. S53). These results support that the formation of
the 1ꢀ(3a)2 complex having additional intermolecular hydrogen
bonding between the two phosphonic acids in the solution state.
The single crystal of 1ꢀ(3c)2 was also obtained from a CH3OH so-
lution. The crystal structure of 1ꢀ(3c)2 showed a similar one-
dimensional network structure, and the pseudoesix-membered
ring of the formation of hydrogen bonding between two phos-
phonic acid 3c was observed (Figs. S52 and S54). These results
support the formation of the 1ꢀ(3)2 complex having intermolecular
additional hydrogen bonding between the two phosphonic acids 3
in the solution state.
3. Conclusions
We have investigated the fluorescence detection of the di-
phosphonic acid and mono-phosphonic acid derivatives using the
anthracene-based diamidine 1. The diamidine 1 forms 1:1 and 1:2
complexes with the di-phosphonic acid and mono-phosphonic acid
derivatives, respectively, and showed
a
blue fluorescence
(
lem ¼ 432e442 nm) in the DMSO solution. The formation of
2.6. Fluorescence titration experiments of diamidine 1 with
phosphonic acid derivatives
amidinium-phosphonate (complex formation) and dissociated
amidinum (lem ¼ 468 nm as a broad band) were distinguished by
the difference in the fluorescence wavelength, and confirmed by
DOSY NMR spectroscopy and TD-DFT calculations.
The binding constants of the diamidine 1 with diphosphonic

T. Kusukawa et al. / Tetrahedron 74 (2018) 465e476
473
f
Table 5
Binding constantsa of diamidine 1 with phosphonic acid derivatives and pKa values of phosphonic acids.
Phosphonic acid derivatives
lem/nmb
K1/Mꢁ1
K1ꢀK2c/Mꢁ2
pKa (obs)
pKa (calc)
2a
2b
441
439
432
438
438
441
440
442
443
442
441
451
8.4 ꢃ 105
1.6 ꢃ 106
1.4 ꢃ 106
9.6 ꢃ 105
1.0 ꢃ 106
1.0 ꢃ 106
2.0 ꢃ 106
1.6 ꢃ 106
2c (n ¼ 3)
2d (n ¼ 4)
2e (n ¼ 5)
2f (n ¼ 6)
2g (n ¼ 7)
2h (n ¼ 8)
3a (R ¼ Me)
3b (R ¼ Bu)
3c (R ¼ tBu)
3d (R ¼ Ph)
1.0 ꢃ 1010
1.8 ꢃ 109
2.7 ꢃ 109
3.5 ꢃ 109
2.41,
1.83,
7.54d
7.07e
2.27
2.66
3.50
1.88
a
Binding constants were determined by fluorescence titration in DMSO (1: 3e25
Maximum fluorescence wavelength of titration spectra.
m
M) using the 1:1 and 1:2 models.
b
c
d
e
f
The binding constants of the K1 and K2 values could not be individually determined.
pKa values of phosphonic acid in H2O, taken from ref.9
pKa values of phosphonic acid in H2O, taken from ref.10
Calculated pKa values of phosphonic acids using Advanced Chemistry Development (ACD/Laboratories) Software V11.02 (1994e2017 ACD/Labs) taken from SciFinder
Scholar.
4. Experimental section
1.67e1.58 (m, 4H), 1.52e1.47 (m, 2H), 1.32 (t, J ¼ 7.1 Hz, 12H). 31P
NMR (202 MHz, CDCl3);
d
(ppm) ¼ 32.0.
4.1. General methods
4.1.1.5. 1,6-Bis(diethoxyphosphoryl)hexane (5f). 91% yield; 1H NMR
1H NMR, 13C NMR and 31P NMR spectra were recorded using
Avance III 500 (500, 125 and 202 MHz) spectrometers. The FAB-
mass spectra were recorded by a JEOL JMS-700 mass spectrom-
eter. The absorption spectra were recorded by a SHIMADZU UV-
2550 UVeVisible spectrometer. The fluorescence spectra were
recorded by a JASCO FP-6200 spectrometer, and the fluorescence
spectra were corrected using rhodamine B as the reference. Infrared
spectra were recorded using a Jasco FTIR 4100 spectrometer. All
solvents and reagents were purified according to standard pro-
cedures. For the NMR measurement using CDCl3, the acidic impu-
rity in the CDCl3 solvent was removed by passage through basic
Al2O3 (Merck, 101076). Anthracene-based diamidine 1 was pre-
pared by the published procedure.5
(500 MHz, CDCl3)
d
(ppm) ¼ 4.09 (m, 8H), 1.76e1.67 (m, 4H),
1.65e1.55 (m, 4H), 1.42e1.38 (m, 4H), 1.32 (t, J ¼ 7.1 Hz, 12H). 31P
NMR (202 MHz, CDCl3);
d
(ppm) ¼ 32.2.
4.1.1.6. 1,7-Bis(diethoxyphosphoryl)heptane (5g). 45% yield; 1H
NMR (500 MHz, CDCl3)
(ppm) ¼ 4.1e4.0 (m, 8H), 1.75e1.68 (m,
4H), 1.62e1.57 (m, 4H), 1.40e1.36 (m, 6H), 1.32 (t, J ¼ 7.1 Hz, 12H).
31P NMR (202 MHz, CDCl3);
(ppm) ¼ 32.2.
d
d
4.1.1.7. 1,8-Bis(diethoxyphosphoryl)octane (5h). 71% yield; 1H NMR
(500 MHz, CDCl3)
d
(ppm) ¼ 4.1e4.0 (m, 8H), 1.75e1.68 (m, 4H),
1.62e1.55 (m, 4H), 1.4e1.2 (m, 8H), 1.32 (t, J ¼ 7.1 Hz, 12H). 31P NMR
(202 MHz, CDCl3);
d
(ppm) ¼ 32.5.
4.1.1. Synthesis of diphosphonic acid tetraethyl esters (5)
General Procedure: The dibromides (6e7 mmol) and triethyl
phosphite (10 mL) were stirred and heated to 120 ꢂC for 24 h, then
additionally heated to 156 ꢂC (reflux) for 3 h under an argon at-
mosphere. The consumption of the dibromides was monitored by
gas chromatography, and excess triethyl phosphite was removed by
short pass vacuum distillation. The residue was purified by column
chromatography (AcOEt to AcOEt/MeOH ¼ 10/1) and the corre-
sponding diphosphonic acid tetraethyl esters 5 were obtained.
4.1.1.8. 1,3-Bis(diethoxyphosphoryl)benzene (5b). To a solution of
1,3-dibromobenzene (784 mg, 3.32 mmol) and NiCl2 (864 mg,
6.67 mmol) in benzonitrile (30 mL), P(OEt)3 (2.0 mL, 11.5 mmol)
was added dropwise over 5 min under argon at 160 ꢂC and stirring
was continued for 2 h. The reaction mixture was poured into
toluene (50 mL), the organic layer was washed four times with 10%
aqueous NH3, dried over Na2SO4 and the solvent evaporated. The
remaining benzonitrile was removed under reduced pressure at
60e80 ꢂC. The product 5b (446 mg, 38%) was isolated after column
chromatography (SiO2, AcOEt to AcOEt/MeOH ¼ 10:1) as a colorless
4.1.1.1. 1,3-Bis(diethoxyphosphonomethyl)benzene (5a). 92% yield;
1H NMR (500 MHz, CDCl3)
d
(ppm) ¼ 7.3e7.2 (m, 4H), 4.1e4.0 (m,
oil. 1H NMR (500 MHz, CDCl3)
d
¼ 8.26e8.20 (m, 1H), 8.02e7.97 (m,
2H), 7.61e7.55 (m, 1H), 4.18e4.07 (m, 8H), 1.34 (t, J ¼ 7.1 Hz, 12H).
31P NMR (202 MHz, CDCl3);
¼ 16.8.
2
8H), 3.13 (d, JP-H ¼ 21.9 Hz, 4H), 1.25 (t, J ¼ 7.1 Hz, 12H). 31P NMR
(202 MHz, CDCl3);
d
(ppm) ¼ 26.2.
d
4.1.1.2. 1,3-Bis(diethoxyphosphoryl)propane (5c). 93% yield; 1H
NMR (500 MHz, CDCl3)
d
(ppm) ¼ 4.1e4.0 (m, 8H), 2.0e1.8 (m, 6H),
4.1.2. Synthesis of bis(hydroxyethoxyphosphoryl) dilithium salts (6)
General Procedure: The tetraethyl diphosphonates 5 (1e8 mmol)
and LiBr (2.6 eq.) were refluxed in 2-hexanone (10 mL, distilled
from K2CO3) under an argon atmosphere. After 3 h, the white
precipitate was collected by centrifugation and washed five times
with Et2O. The collected precipitate was dried under vacuum to
give the dilithium salt 6 as a white powder. Inclusion of excess LiBr
in the obtained products was observed by elemental analysis and
the products were only characterized by NMR spectroscopy.
1.31 (t, J ¼ 7.1 Hz, 12H). 31P NMR (202 MHz, CDCl3);
(ppm) ¼ 30.5.
d
4.1.1.3. 1,4-Bis(diethoxyphosphoryl)butane (5d). 83% yield; 1H NMR
(500 MHz, CDCl3)
d
(ppm) ¼ 4.1e4.0 (m, 8H), 1.8e1.6 (m, 8H), 1.32
(t, J ¼ 7.1 Hz, 12H). 31P NMR (202 MHz, CDCl3);
d
(ppm) ¼ 31.5.
4.1.1.4. 1,5-Bis(diethoxyphosphoryl)pentane (5e). 87% yield; 1H NMR
(500 MHz, CDCl3)
(ppm) ¼ 4.1e4.0 (m, 8H), 1.76e1.69 (m, 4H),
d

474
T. Kusukawa et al. / Tetrahedron 74 (2018) 465e476
3
4.1.2.1. 1,3-Bis(hydroxyethoxyphosphorylmethyl)benzene dilithium
2JC-P ¼ 3.8 Hz, CH2), 16.0 (d, JC-O-P ¼ 5.0 Hz, CH3). 31P NMR
salt (6a). 82% yield (including LiBr); colorless solid; 1H NMR
(202 MHz, D2O, H3PO4 in D2O as external standard);
d
(ppm) ¼ 29.5.
(500 MHz, D2O, TMS in CDCl3 as external standard)
d
(ppm) ¼ 7.28
(t, J ¼ 7.9 Hz,1H), 7.18 (brs, 3H), 3.85 (m, 4H), 3.02 (d, 2JP-H ¼ 20.5 Hz,
4H), 1.20 (t, J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz, D2O, TMS in CDCl3 as
4.1.2.8. 1,8-Bis(hydroxyethoxyphosphoryl)octane dilithium salt (6h).
104% yield (including LiBr); colorless solid; 1H NMR (500 MHz, D2O,
external standard)
d
(ppm) ¼ 134.9 (m, Cq), 130.8 (t, 3JC-P ¼ 6.3 Hz,
TMS in CDCl3 as external standard)
d
(ppm) ¼ 3.89 (m, 4H),
CH), 128.5 (CH), 127.3 (t, 3JC-P ¼ 4.4 Hz, CH), 61.3 (d, 2JC-O-P ¼ 5.0 Hz,
OCH2), 34.2 (d, 1JC-P ¼ 130.0 Hz, CH2), 16.0 (t, 3JC-O-P ¼ 3.1 Hz, CH3).
31P NMR (202 MHz, D2O, H3PO4 in D2O as external standard);
1.61e1.53 (m, 4H), 1.53e1.46 (m, 4H), 1.37e1.29 (m, 8H), 1.25 (t,
J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz, D2O, TMS in CDCl3 as external
2
3
standard)
d
(ppm) ¼ 60.4 (d, JC-O-P ¼ 5.0 Hz, OCH2), 30.3 (d, JC-
d
(ppm) ¼ 22.9.
¼ 17.5 Hz, CH2), 28.3 (s, CH2), 26.2 (d, 1JC-P ¼ 132.5, CH2), 22.86 (d,
P
3
2JC-P ¼ 5.0 Hz, CH2), 16.0 (d, JC-O-P ¼ 6.3 Hz, CH3). 31P NMR
4.1.2.2. 1,3-Bis(hydroxyethoxyphosphoryl)benzene dilithium salt
(202 MHz, D2O, H3PO4 in D2O as external standard);
d
(ppm) ¼ 29.6.
(6b). 95% yield (including LiBr); colorless solid; 1H NMR (500 MHz,
D2O, TMS in CDCl3 as external standard)
d
(ppm) ¼ 8.00 (t,
4.1.3. Synthesis of diphosphonic acid P,P0-diethyl esters (2)
J ¼ 12.5 Hz, 1H), 7.87e7.82 (m, 2H), 7.59 (m, 1H), 3.84 (m, 4H), 1.19
General Procedure: An ion exchange resin of sulfonic acid (6 g,
Dowex 50Wx8 200e400 mesh) was activated with 6 M HCl
(25 mL), then washed with distilled water (30 mL) in a disposable
column (16 ꢃ 80 mm). An aqueous solution (5 mL) of the bis(hy-
(t, J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz, D2O, TMS in CDCl3 as external
1
3
standard)
d
(ppm) ¼ 133.5 (m, CH),133.4 (dd, JC-P ¼ 176.3, JC-
¼ 12.5 Hz, Cq),133.0 (t, 2JC-P ¼ 10.0 Hz, CH),128.5 (t, 3JC-P ¼ 13.2 Hz,
P
CH), 61.4 (s, OCH2), 15.8 (t, 3JC-O-P ¼ 3.1 Hz, CH3). 31P NMR (202 MHz,
droxyethoxyphosphoryl) dilithium salts
6
(ca 50 mg,
D2O, H3PO4 in D2O as external standard);
d
(ppm) ¼ 14.8.
0.15e0.19 mmol) was deposited on the resin and eluted with
distilled water (ca 50 mL) until the eluent become neutral (pH ¼ 7).
The eluent was concentrated in vacuo and the residue was
continuously evacuated under vacuum for one week. The purity of
the obtained diphosphonic acid diethyl esters 2 was analyzed by
the 1H NMR integral ratio in comparison with the internal
standards.
4.1.2.3. 1,3-Bis(hydroxyethoxyphosphoryl)propane dilithium salt (6c).
102% yield (including LiBr); colorless solid; 1H NMR (500 MHz, D2O,
TMS in CDCl3 as external standard)
d
(ppm) ¼ 3.91 (m, 4H), 1.8e1.6
(m, 6H), 1.25 (t, J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz, D2O, TMS in
2
CDCl3 as external standard)
d
(ppm) ¼ 60.6 (t, JC-O-P ¼ 3.1 Hz,
1
3
2
OCH2), 27.5 (dd, JC-P ¼ 133.8, JC-P ¼ 15.0 Hz, CH2), 17.6 (t, JC-
¼ 3.8 Hz, CH2), 16.0 (t, 3JC-O-P ¼ 2.5 Hz, CH3). 31P NMR (202 MHz,
4.1.3.1. 1,3-Bis(hydroxyethoxyphosphorylmethyl)benzene
(2a).
P
D2O, H3PO4 in D2O as external standard);
d
(ppm) ¼ 27.6.
Colorless oil; 94% yield; 1H NMR (500 MHz, DMSO-d6)
d
(ppm) ¼ 7.21 (t, J ¼ 7.5 Hz, 1H), 7.13e7.11 (m, 3H), 3.88 (m, 4H),
1
4.1.2.4. 1,4-Bis(hydroxyethoxyphosphoryl)butane dilithium salt (6d).
3.02 (d, JP-H ¼ 21.6 Hz, 4H), 1.16 (t, J ¼ 7.0 Hz, 6H). 13C NMR
102% yield (including LiBr); colorless solid; 1H NMR (500 MHz, D2O,
(125 MHz, DMSO-d6)
d
(ppm) ¼ 133.1 (m, Cq), 131.1 (t, 3JC-P ¼ 6.3 Hz,
TMS in CDCl3 as external standard)
d
(ppm) ¼ 3.90 (m, 4H), 1.7e1.5
CH), 127.8 (CH), 127.3 (m, CH), 60.5 (d, 2JC-O-P ¼ 5.0 Hz, OCH2), 33.6
(m, 8H), 1.25 (t, J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz, D2O, TMS in
(d, JC-P ¼ 132.5 Hz, CH2), 16.3 (t, JC-O-P ¼ 6.3 Hz, CH3). 31P NMR
1
3
2
CDCl3 as external standard)
d
(ppm) ¼ 60.5 (d, JC-O-P ¼ 6.3 Hz,
(202 MHz, DMSO-d6);
d
(ppm) ¼ 23.7; HRMS (FAB, NBA) m/
1
2
3
OCH2), 25.9 (d, JC-P ¼ 133.8, CH2), 24.4 (dd, JC-P ¼ 18.8, JC-
z ¼ 345.0631 (calculated for [MþNa]þ: 345.0633); IR (KBr, cmꢁ1):
n
¼ 4.4 Hz, CH2), 16.0 (d, 3JC-O-P ¼ 5.0 Hz, CH3). 31P NMR (202 MHz,
3389, 2985, 2911, 2360, 2336, 1700, 1167, 1038.
P
D2O, H3PO4 in D2O as external standard);
d
(ppm) ¼ 28.8.
4.1.3.2. 1,3-Bis(hydroxyethoxyphosphoryl)benzene (2b). Colorless oil;
3
4.1.2.5. 1,5-Bis(hydroxyethoxyphosphoryl)pentane dilithium salt (6e).
69% yield; 1H NMR (500 MHz, DMSO-d6)
d
(ppm) ¼ 8.01 (t, JP-
109% yield (including LiBr); colorless solid; 1H NMR (500 MHz, D2O,
¼ 12.9 Hz, 1H), 7.85 (m, 2H), 7.63 (m, 1H), 3.91 (m, 4H), 1.18 (t,
H
TMS in CDCl3 as external standard)
d
(ppm) ¼ 3.90 (m, 4H), 1.7e1.4
J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz, DMSO-d6)
d
(ppm) ¼ 133.9 (m,
(m, 10H), 1.25 (t, J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz, D2O, TMS in
CH), 133.4 (t, JC-P ¼ 10.0 Hz, CH), 131.7 (dd, JC-P ¼ 181.3, JC-
2
1 3
2
CDCl3 as external standard)
d
(ppm) ¼ 60.4 (d, JC-O-P ¼ 6.3 Hz,
¼ 12.5 Hz, Cq), 128.7 (t, 3JC-P ¼ 13.1 Hz, CH), 60.8 (s, OCH2), 16.2 (s,
P
OCH2), 31.7 (t, 3JC-P ¼ 16.3 Hz, CH2), 26.1 (d, 1JC-P ¼ 133.8, CH2), 22.5
CH3). 31P NMR (202 MHz, DMSO-d6);
d
(ppm) ¼ 14.1; HRMS (FAB,
(d, JC-P ¼ 5.0 Hz, CH2), 16.0 (d, JC-O-P ¼ 6.3 Hz, CH3). 31P NMR
NBA) m/z ¼ 238.9866 (calculated for [MþH]þ: 238.9874); IR (KBr,
2
3
(202 MHz, D2O, H3PO4 in D2O as external standard);
d
(ppm) ¼ 29.2.
cmꢁ1):
n 3463, 2987, 2908, 2360, 2335, 1699, 1166, 1112, 1032.
4.1.2.6. 1,6-Bis(hydroxyethoxyphosphoryl)hexane dilithium salt (6f).
4.1.3.3. 1,3-Bis(hydroxyethoxyphosphoryl)propane (2c). 91% yield;
109% yield (including LiBr); colorless solid; 1H NMR (500 MHz, D2O,
colorless oil; 1H NMR (500 MHz, DMSO-d6)
d
(ppm) ¼ 3.90 (m, 4H),
TMS in CDCl3 as external standard)
d
(ppm) ¼ 3.89 (m, 4H),
1.8e1.6 (m, 6H), 1.20 (t, J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz, DMSO-
1.62e1.55 (m, 4H), 1.55e1.49 (m, 4H), 1.38 (m, 4H), 1.26 (t, J ¼ 7.1 Hz,
d6)
d
(ppm) ¼ 59.8 (t, 2JC-O-P ¼ 2.5 Hz, CH2), 26.5 (dd, 1JC-P ¼ 136.3,
6H). 13C NMR (125 MHz, D2O, TMS in CDCl3 as external standard)
3JC-P ¼ 15.0 Hz, CH2), 16.5 (t, JC-P ¼ 4.4 Hz, CH2), 16.4 (t, JC-O-
2
3
2
3
d
(ppm) ¼ 60.4 (d, JC-O-P ¼ 5.0 Hz, OCH2), 29.9 (d, JC-P ¼ 17.5 Hz,
¼ 3.1 Hz, CH3). 31P NMR (202 MHz, DMSO-d6);
d
(ppm) ¼ 28.5;
P
CH2), 26.1 (d, 1JC-P ¼ 133.8, CH2), 22.8 (d, 2JC-P ¼ 5.0 Hz, CH2), 16.0 (d,
HRMS (FAB, NBA) m/z ¼ 283.0476 (calculated for [MþNa]þ:
3JC-O-P ¼ 5.0 Hz, CH3). 31P NMR (202 MHz, D2O, H3PO4 in D2O as
283.0476); IR (KBr, cmꢁ1):
1204, 1165, 1043, 997.
n 3464, 2985, 2911, 2360, 2335, 1699,
external standard);
d
(ppm) ¼ 29.4.
4.1.2.7. 1,7-Bis(hydroxyethoxyphosphoryl)heptane dilithium salt (6g).
4.1.3.4. 1,4-Bis(hydroxyethoxyphosphoryl)butane (2d). 87% yield;
99% yield (including LiBr); colorless solid; 1H NMR (500 MHz, D2O,
colorless waxy solid; 1H NMR (500 MHz, DMSO-d6)
d
¼ 3.89 (m,
TMS in CDCl3 as external standard)
d
(ppm) ¼ 3.89 (m, 4H),
4H), 1.62e1.47 (m, 8H), 1.18 (t, J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz,
2
1
1.62e1.55 (m, 4H), 1.55e1.46 (m, 4H), 1.40e1.32 (m, 6H), 1.25 (t,
DMSO-d6)
d
(ppm) ¼ 59.9 (d, JC-O-P ¼ 6.3 Hz, CH2), 25.5 (d, JC-
J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz, D2O, TMS in CDCl3 as external
¼ 137.5 Hz, CH2), 23.4 (dd, 2JC-P ¼ 5.0 Hz, 3JC-P ¼ 16.3 Hz, CH2), 16.4
P
2
3
standard)
d
(ppm) ¼ 60.4 (d, JC-O-P ¼ 5.0 Hz, OCH2), 30.1 (d, JC-
(d, 3JC-O-P ¼ 6.3 Hz, CH3). 31P NMR (202 MHz, DMSO-d6);
d
¼ 29.2;
¼ 16.3 Hz, CH2), 28.0 (s, CH2), 26.2 (d, 1JC-P ¼ 132.5, CH2), 22.8 (d,
HRMS (FAB, NBA) m/z ¼ 297.0637 (calculated for [MþNa]þ:
P

T. Kusukawa et al. / Tetrahedron 74 (2018) 465e476
475
297.0633); IR (KBr, cmꢁ1):
1682, 1241, 1191, 1165, 1042, 992, 967.
n
3427, 2993, 2946, 2910, 2364, 2340,
ramp. The gradient length was selected between 2.0 and 3.0 ms
with a diffusion time of 200 ms and an eddy current delay of 5 ms.
4.1.3.5. 1,5-Bis(hydroxyethoxyphosphoryl)pentane (2e). 93% yield;
4.4. Fluorescence titrations
colorless waxy solid; 1H NMR (500 MHz, DMSO-d6)
d
(ppm) ¼ 3.90
(m, 4H),1.59e1.52 (m, 4H),1.49e1.39 (m, 6H),1.20 (t, J ¼ 7.1 Hz, 6H).
A solution of the diamidine 1 was prepared (3e25 mM in DMSO),
2
13C NMR (125 MHz, DMSO-d6)
d
(ppm) ¼ 59.8 (d, JC-O-P ¼ 6.3 Hz,
and an aliquot (3 mL) was transferred to a 1-cm fluorescence tube.
To this solution was dropwise added a stock solution of the phos-
phonic acid (0.6e5 mM in DMSO) in small portions. The fluores-
cence spectra were recorded by excitation at 348 nm. The
association constants were calculated using the program HYP-
SPEC12 and are summarized in Table 5.
CH2), 30.7 (t, 3JC-P ¼ 15.6 Hz, CH2), 25.6 (d, 1JC-P ¼ 137.5, CH2), 22.0
(d, JC-P ¼ 5.0 Hz, CH2), 16.4 (d, JC-O-P ¼ 6.3 Hz, CH3). 31P NMR
2
3
(202 MHz, DMSO-d6);
d
(ppm) ¼ 29.3; HRMS (FAB, NBA) m/
z ¼ 311.0792 (calculated for [MþNa]þ: 311.0789); IR (KBr, cmꢁ1):
n
3444, 2985, 2944, 2871, 2363, 2340, 1682, 1247, 1188, 1042, 982.
4.1.3.6. 1,6-Bis(hydroxyethoxyphosphoryl)hexane (2f). 75% yield;
colorless waxy solid; 1H NMR (500 MHz, DMSO-d6)
(ppm) ¼ 3.90
4.5. Computational methods
d
(m, 4H), 1.61e1.52 (m, 4H), 1.49e1.39 (m, 4H), 1.36e1.29 (m, 4H),
The ground-state geometries were fully optimized using the
density functional theory (DFT) with the B3LYP hybrid functional at
the basis set level of 6-31G*. The excited-state geometries were
optimized by the ab-initio configuration interaction singles method
(CIS/6-31G*); the frequency calculations allowed verification that
those structures did not present imaginary frequencies and we had
global minima. With this optimized geometry, we calculated the
emission spectrum in the time-dependent density functional the-
ory (TD-DFT) with the B3LYP functional at the 6-31G* basis set
level.13,14 All of the calculations were performed with Gaussian
09 W.15
1.20 (t, J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz, DMSO-d6)
(ppm) ¼ 59.8
d
(d, 2JC-O-P ¼ 5.0 Hz, CH2), 29.4 (d, 3JC-P ¼ 16.3 Hz, CH2), 25.7 (d, 1JC-
¼ 137.5, CH2), 22.2 (d, 2JC-P ¼ 5.0 Hz, CH2), 16.4 (d, 3JC-O-P ¼ 6.3 Hz,
P
CH3). 31P NMR (202 MHz, DMSO-d6);
d
(ppm) ¼ 29.4; HRMS (FAB,
NBA) m/z ¼ 325.0948 (calculated for [MþNa]þ: 325.0946); IR (KBr,
cmꢁ1):
n 3427, 2991, 2939, 2864, 2362, 2339, 1652, 1234, 1187, 1036,
986, 965.
4.1.3.7. 1,7-Bis(hydroxyethoxyphosphoryl)heptane (2g). 87% yield;
colorless waxy solid; 1H NMR (500 MHz, DMSO-d6)
d
(ppm) ¼ 3.90
(m, 4H), 1.61e1.52 (m, 4H), 1.50e1.39 (m, 4H), 1.35e1.28 (m, 4H),
1.28e1.22 (m, 2H), 1.20 (t, J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz,
4.6. Crystallographic analysis
2
3
DMSO-d6)
d
(ppm) ¼ 59.8 (d, JC-O-P ¼ 5.0 Hz, CH2), 29.7 (d, JC-
¼ 15.0 Hz, CH2), 28.2 (s, CH2), 25.8 (d, 1JC-P ¼ 136.3, CH2), 22.4 (d,
P
Crystals suitable for X-ray crystallography were grown from the
solutions of 1ꢀ(3a)2 and 1ꢀ(3c)2 in MeOH that were stored in a
small vial and left to stand in the sample bottle in the presence of
molecular sieves 4A for several days. For the crystallographic data
2JC-P ¼ 3.8 Hz, CH2), 16.4 (d, JC-O-P ¼ 6.3 Hz, CH3). 31P NMR
3
(202 MHz, DMSO-d6);
d
(ppm) ¼ 29.5; HRMS (FAB, NBA) m/
z ¼ 339.1115 (calculated for [MþNa]þ: 339.1102); IR (KBr, cmꢁ1):
n
3445, 2984 2932, 2864, 2362, 2339, 1683, 1653, 1245, 1186, 1049,
984.
collection, a Rigaku AFC-7R with graphite-monochromated Cu-K
a
radiation was used. Calculations were performed using the Crystal
Structure 3.8 crystallographic software package.16 The crystal
structures were solved by a direct method using Shelxs-9717, and
the structure refinements were performed by a full-matrix least
squares methods using Shelx-97. Details of the data are summa-
rized in Table S2. All non-hydrogen atoms were anisotropically
refined. The hydrogen atoms of the amidino groups were located in
a difference Fourier map and were isotropically refined. All the
other hydrogen atoms were placed in idealized positions and were
included in structure factor calculations, but were not refined.
CCDC-1582835 and -1582836 contain the supplementary crystal-
lographic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via www.
ccdc.cam.ac.uk/data request/cif.
4.1.3.8. 1,8-Bis(hydroxyethoxyphosphoryl)octane (2h). 86% yield;
colorless waxy solid; 1H NMR (500 MHz, DMSO-d6)
d
(ppm) ¼ 3.89
(m, 4H), 1.60e1.51 (m, 4H), 1.50e1.39 (m, 4H), 1.35e1.28 (m, 4H),
1.26e1.22 (m, 4H),1.19 (t, J ¼ 7.1 Hz, 6H). 13C NMR (125 MHz, DMSO-
d6)
d
(ppm) ¼ 59.8 (d, 2JC-O-P ¼ 5.0 Hz, CH2), 29.9 (d, 3JC-P ¼ 16.3 Hz,
CH2), 28.4 (s, CH2), 25.8 (d, 1JC-P ¼ 136.3, CH2), 22.4 (d, 2JC-P ¼ 5.0 Hz,
CH2), 16.4 (d, 3JC-O-P ¼ 5.0 Hz, CH3). 31P NMR (202 MHz, DMSO-d6);
d
(ppm) ¼ 29.4; HRMS (FAB, NBA) m/z ¼ 353.1269 (calculated for
[MþNa]þ: 353.1259); IR (KBr, cmꢁ1):
n 3463, 2986, 2926, 2849,
2361, 2338, 1671, 1119, 1048, 998.
4.2. Jobs plot analysis (fluorescence spectroscopy)
The stock solutions of 1 and phosphonic acids (2, 3) in DMSO
(1.0e15 mM) were prepared in separate volumetric flasks. Several
sample solutions containing both the diamidine 1 and phosphonic
acids in different ratios (1/9 to 9/1) were prepared and maintained
at 0.2 mM. The fluorescence spectra of the mixtures were recorded,
and the intensities were analyzed by Job's method.
Acknowledgments
This study was supported in part by the Kyoto Institute of
Technology Research Fund. We thank Prof. T. Harada (Kyoto Insti-
tute of Technology) for his helpful discussions.
Appendix A. Supplementary data
4.3. DOSY measurements
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.tet.2017.12.011.
The 1H DOSY experiments were carried out at 298 K using a
Bruker Avance III 500 MHz spectrometer equipped with a 5-mm
BBFO probe with a z-axis gradient coil. Data were acquired and
processed using the Bruker TopSpin 3.0 software. A series of
diffusion ordered spectra were collected of the samples using the
LEDbp pulse sequence.11 The pulse-fields were incremented in 50
steps from 2% to 95% of the maximum gradient strength in a linear
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