Fluorescent detection of amidinium-carboxylate and amidinium formation using a 1,8-diphenylnaphthalene-based diamidine: Dicarboxylic acid recognition with high fluorescence efficiency

Article abstract of DOI:10.1016/j.tet.2014.03.036

A 1,8-diphenylnaphthalene-based diamidine (1) 'turn-on' fluorescent probe for the detection of dicarboxylic acids has been designed and synthesized. The fluorescence spectra of the diamidine 1 with carboxylic acids that showed two different fluorescence bands, which corresponded to the amidinium-carboxylate (λ_em=410-430 nm) and amidinium (λ_em=440-470 nm as a broad band, which consisted from two peaks) formation, were confirmed by DOSY NMR and TD-DFT calculations. The complexation of diamidine 1 with dicarboxylic acids, which have sufficient distances between the two carboxylic groups for binding to the diamidine 1 (dicarboxylic acids 3, 4, and α,ω-dicarboxylic acids 6 (C₆-C₂₀)), showed the formation of 1:1 complexes (i.e., amidinium-carboxylate formation). On the other hand, for the complexation with monocarboxylic acids and dicarboxylic acids having insufficient distances between the two carboxylic groups (benzoic acid 5, acetic acid 7, and α,ω-dicarboxylic acids 6 (C ₃-C₅)), formation of the amidinium (1·2H ⁺) was observed. Relatively similar binding constants (10 ^-5) for the complexation of the diamidine 1 with dicarboxylic acids 6, which depend on their chain length (strain), were observed due to the flexibility of the 1,8-diphenylnaphthalene unit. Additionally, for the complexation of the diamidine 1 with dicarboxylic acids, higher fluorescence quantum yields (Φ_fl: up to 80%) were observed when compared to the binding of the diamidine 2 (Φ_fl: up to 35%).

Full text of DOI:10.1016/j.tet.2014.03.036

Accepted Manuscript
Fluorescent detection of amidinium-carboxylate and amidinium formation using a
1,8-diphenylnaphthalene-based diamidine: dicarboxylic acid recognition with high
fluorescence efficiency
Takahiro Kusukawa, Syugo Tanaka, Kouta Inoue
PII:
S0040-4020(14)00370-6
10.1016/j.tet.2014.03.036
TET 25376
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 28 January 2014
Revised Date: 11 March 2014
Accepted Date: 12 March 2014
Please cite this article as: Kusukawa T, Tanaka S, Inoue K, Fluorescent detection of amidinium-
carboxylate and amidinium formation using a 1,8-diphenylnaphthalene-based diamidine: dicarboxylic
acid recognition with high fluorescence efficiency, Tetrahedron (2014), doi: 10.1016/j.tet.2014.03.036.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Fluorescent detection of amidinium-carboxylate
and amidinium formation using a 1,8-
diphenylnaphthalene-based diamidine:
dicarboxylic acid recognition with high
fluorescence efficiency
Leave this area blank for abstract info.
Takahiro Kusukawa*, Syugo Tanaka and Kouta Inoue
1

ACCEPTED MANUSCRIPT
Fluorescent detection of amidinium-carboxylate and
amidinium
formation
using
a
1,8-
diphenylnaphthalene-based diamidine: dicarboxylic
acid recognition with high fluorescence efficiency
Takahiro Kusukawa*, Syugo Tanaka and Kouta Inoue
Department of Chemistry and Materials Technology,
Graduate School of Science and Technology, Kyoto Institute of Technology,
Matsugasaki, Sakyo-ku Kyoto 606-8585, Japan
TEL/FAX: +81-75-724-7506, e-mail: kusu@kit.ac.jp
-----------------------------------------------------------------------------------------------------------------------------
2

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Abstract: A 1,8-diphenylnaphthalene-based diamidine (1) “turn-on” fluorescent probe for the detection
of dicarboxylic acids has been designed and synthesized. The fluorescence spectra of the diamidine 1
with carboxylic acids that showed two different fluorescence bands, which corresponded to the
amidinium-carboxylate ( em = 410-430 nm) and amidinium (λem = 440-470 nm as a broad band which
λ
consisted from two peaks) formation, were confirmed by DOSY NMR and TD-DFT calculations. The
complexation of diamidine 1 with dicarboxylic acids, which have sufficient distances between the two
carboxylic groups for binding to the diamidine 1 (dicarboxylic acids 3, 4 and
α,ω−dicarboxylic acids 6
(
C -C )), showed the formation of 1:1 complexes (i.e., amidinium-carboxylate formation). On the
6
20
other hand, for the complexation with monocarboxylic acids and dicarboxylic acids having insufficient
distances between the two carboxylic groups (benzoic acid 5, acetic acid 7 and
α
,
ω−dicarboxylic acids 6
+
-5
(
C -C )), formation of the amidinium (1•2H ) was observed. Relatively similar binding constants (10 )
3
5
for the complexation of the diamidine 1 with dicarboxylic acids 6, which depend on their chain length
strain), were observed due to the flexibility of the 1,8-diphenylnaphthalene unit. Additionally, for the
complexation of the diamidine 1 with dicarboxylic acids, higher fluorescence quantum yields (Φ : up to
(
fl
8
0%) were observed when compared to the binding of the diamidine 2 (Φ : up to 35%).
fl
Keywords: Amidinium-carboxylate / Amidinium / Fluorescence / Dicarboxylic acid recognition
----------------------------------------------------------------------------------------------------------------------------
. Introduction
The amidinium-carboxylate salt bridge formation is one of the important noncovalent
-
1
1
interactions, which has been extensively used to construct self-assembled structures, and it also has
2
importance related to the carboxylic acid and carboxylate recognition site in biological systems. The
structures of the amidinium-carboxylate salt have been studied by X-ray crystallographic analysis; the
hydrogen atoms in the amidinium-carboxylate salt are bonded to the nitrogen atoms and generate a
3
,4
cationic charge and an anionic charge as the amidinium and carboxylate pairs.
According to the
formation of twitter ionic charges, the hydrogen bond of the amidinium-carboxylate salt shows a
3

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stronger binding even in polar solvents. Spectroscopic observation of the formation of the “amidinium-
carboxylate salt bridge” is important in order to understand the formation of the assembled structure and
to also study the carboxylic acid binding in biological research. However, the formation of the
1
3
amidinium-carboxylate salt in the solution state is observed by only H NMR analysis, and the
equilibrium of the formation of amidinium-carboxylate and amidinium species has only been rarely
discussed (Scheme 1).
5
6
On the other hand, dicarboxylic acid and dicarboxylate recognitions have a biological
importance because of their significant roles in metabolic processes. However, most known
5
dicarboxylic acid sensors function only in less polar solvents (such as CHCl ) and show “turn-off type”
3
fluorescent probes for the recognition of the dicarboxylic acids.
Recently, we reported the fluorescence detection of amidinium-carboxylate and amidinium
7
formation using anthracene-based diamidine 2 for the recognition of carboxylic acids. In this system,
the formation of the amidinium-carboxylate and amidinium was characterized by the difference in the
fluorescence wavelength and the formation which was confirmed by DOSY spectroscopy and TD-DFT
calculations. To demonstrate the applicability of this “amidinium-carboxylate and amidinium detection”
toward other fluorophores, we have designed and synthesized a new diamidine 1 having the 1,8-
diphenylnaphthalene unit as a fluorophore. For the recognition of carboxylic acids with diamidine 1, the
formation of the amidinium-carboxylate (λem = 410-430 nm) and amidinium (λem = 440-470 nm as a
broad band which consisted from two peaks) formation was observed in the fluorescence spectra and
confirmed by DOSY NMR and TD-DFT calculations. Moreover, for the recognition of dicarboxylic
acids with the diamidine 1, higher fluorescence quantum yields (Φ : up to 80%) were observed when
fl
compared to the binding of the diamidine 2.
4

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Scheme 1. Schematic representation of the formation of amidinium-carboxylate and amidinium species from
amidine containing a fluorophore and carboxylic acid.
2
2
. Results and Discussion
.1. Synthesis of diamidine 1
The 1,8-diphenylnaphthalene-based diamidine 1 was prepared according to Scheme 2. The 1,8-
bis(4-cyanophenyl)naphthalene was prepared from 1,8-diiodonaphthalene and 4-cyanophenylboronic
acid in 86% yield and the obtained product was treated with Weinreb reagent (NH Cl/Me Al), and
4
3
diamidine dihydrochloride (1•2HCl) was obtained as the crude product. To obtain the pure diamidine 1,
we employed Boc protection of the amidino groups and purification by column chromatography. The
obtained Boc protected diamidine was converted to the diamidine dihydrochloride 1•2HCl in an almost
quantitative yield (98%). The pure diamidine 1 was obtained from diamidine dihydrochloride (1•2HCl)
by treatment with an aqueous NaOH solution.
Scheme 2. Synthesis of diamidine 1.
2.2. Fluorescent detection of amidinium-carboxylate and amidinium formation; binding
experiments of diamidine 1 with carboxylic acids
To investigate the recognition ability of the diamidine 1 toward carboxylic acids, we observed the
fluorescence spectra of the diamidine 1 with several carboxylic acids in a DMSO solution. The
5

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diamidine 1 does not show a fluorescence (turn-off state) in the DMSO solution under UV irradiation
8
due to the PET process from the amidino groups (Figure 1e). However, after the addition of 1,3-
adamantanediacetic acid 3 (1 equiv.), a dark blue emission was observed at 417 nm (Figure 1a).
Similarly, after the addition of m-phenylenediacetic acid 4 (1 equiv.), a dark blue emission at 422 nm
was also observed (Figure 1b). On the other hand, for the complexation with benzoic acid 5
(monocarboxylic acid, 2 equiv.), a broad emission band, which consisted from two peaks, was observed
at 438-470 nm (Figure 1c). The obtained spectrum of the complex of 1 and 5 is similar to the
fluorescence spectrum of diamidine dihydrochloride 1•2HCl which corresponded to the amidinium
+
1
•2H species (Figure 1d).
Figure 1. Fluorescence spectra of diamidine 1 (25
µM in DMSO, λex = 307 nm) upon the addition of carboxylic
acids a) 1,3-adamantanediacetic acid 3 (1 eq.), b) m-phenylenediacetic acid 4 (1 eq.), c) benzoic acid 5 (2 eq.), d)
fluorescence spectrum of 1•2HCl (25
µM in DMSO), e) fluorescence spectrum of diamidine 1 (25 µM in
DMSO).
6

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These observations indicated that the complexation of the diamidine 1 with dicarboxylic acids
(3, 4) produced stable 1:1 complexes in the DMSO solution by the formation of two amidinium-
carboxylate salt bridges. However, for the complexation of 1 with 2 equiv. of benzoic acid 5
(monocarboxylic acid), a fast equilibrium with the amidinium species occurs (Scheme 1). These
7
phenomena are almost consistent with the binding character of the diamidine 2 as previously reported.
Although UV spectra of diamidine 1, complexes 1•3, 1•4, 1•5 and diamidine dihydrochloride 1•2HCl
2
were observed, could not explain the difference of each fluorescence spectra (Figure S39). To confirm
the reason for these two different fluorescence bands, we studied the formation of the 1:1 complexes,
1
•3 and 1•4, and 1:2 complex, 1•5 . The stability of complexes 1•3, 1•4, and 1•5 were qualitatively
2 2
1
characterized by H NMR and DOSY NMR spectroscopies. The complexation of 1•3 was achieved by
1
mixing the DMSO-d solution (2.0 mM) of both components. The H NMR spectrum of the obtained
6
solution showed the characteristic upfield and downfield shifts with respect to those of 1 and 3 (Figures
a
e
2
b, S1b). The downfield shift of the amidine unit (H -H ) and the upfield shift of the carboxylic acid
A
1
unit (H ) in the H 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 as broad singlets at 13.5 and 9 ppm,
1
b,3
indicating the formation of amidinium-carboxylate salt bridges (i.e., formation of a stable complex).
1
Figure 2. H NMR spectra (2.0 mM in DMSO-d , 298 K) of a) diamidine 1, b) 1•3 obtained by mixing equimolar
6
solutions of 1 and 3, and c) dicarboxylic acid 3.
7

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Figure 3. Crystal structure of 1•3. Solvent molecule is omitted for clarity. Selected
distances (Å): N1-O1: 2.768(3); N2-O2: 2.920(3); N3-O3: 2.771(3); N4-O4: 2.710(4).
The single crystal of the complex 1•3 was obtained from a DMSO solution of the diamidine 1
and 1,3-adamantanediacetic acid 3. The crystal structure of 1•3 showed the presence of two pairs of
amidinium-carboxylate salt bridges (Figure 3). The hydrogen atoms in the amidinium-carboxylate
hydrogen bonds were found in the structure (see experimental part), and both are bonded to the nitrogen
atoms. The slightly long N•••O bond distances of 2.710(4)-2.920(3) Å compare to the distances of the
3
a,3c,9
monoamidine and monocarboxylic acid pairs (N•••O distances: 2.723-2.835 Å)
which may be
observed due to the ring strain from the formation of the 1:1 complex 1•3.
1
The H NMR spectrum of 1•4 also showed the formation of a stable complex (NH signals: 13
1
and 9 ppm, Figure S2b). However, in the H NMR spectrum of 1•5 , no NH signals were observed that
2
correspond to the amidinium-carboxylate, which indicated the presence of complex 1•5 in fast
2
+
equilibrium with the amidinium species 1•2H (Figure S3b).
A Job’s plot analysis of 1•3 and 1•4 definitely proved the 1:1 (or 2:2) stoichiometry in the
1
DMSO-d solution by H NMR titration or in the DMSO solution by fluorescence titrations (Figures S8-
6
S10). Additionally, a Job’s plot analysis of 1•5 definitely proved the 1:2 stoichiometry (Figure S11).
2
8

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1
Figure 4. H DOSY spectra (2.0 mM in DMSO-d , 25 °C, diffusion time
∆ = 200 ms) of a) complex 1•3, b) 1,3-
6
adamantanediacetic acid 3, c) diamidine 1.
Additional proof of the stoichiometry of 1•3 was obtained from DOSY NMR spectroscopy. The
DOSY spectra showed that the diffusion coefficient of complex 1•3 is lower than those of the
corresponding free building blocks (1, 3), confirming the formation of the larger-sized complex (Figure
3
4
). The molecular volume of 1•3 derived from the observed diffusion coefficient (V = 780 Å ) is
3
3
slightly greater than the sum of the volumes of the free building blocks (1: V = 317 Å , 3: V = 226 Å ),
3
but smaller than the volume after the formation of the 2:2 complex 1 •3 (V = 1086 Å ), which indicated
2
2
the formation of the 1:1 complex (Figure 4, Table 1). Unfortunately, the DOSY spectra of 1•4 could not
be observed due to the precipitation of the formed complex 1•4. On the other hand, the DOSY spectrum
of 1•5 showed two diffusion signals derived from 1 and 5 that were separately observed during the
2
diffusion time (
∆) of 100-300 ms (Figure S22, Table 1). The separately observed DOSY signal for 1•52
showed that the complex exists in equilibrium with the free building blocks or dissociates into the
+
+ -
amidinium (1•2H ) and carboxylate ([5-H ] ) fragments. These observations agreed with the difference
in the fluorescence bands of 1•3, 1•4 and 1•5 (Figure 1).
2
9

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Table 1. Comparison of diffusion coefficients, molecular radii and volumesa
2
-1
V (Å3)
compound conc. (mM)
D (m s )
rH (Å)
2.35 ± 0.06×10-
10
10
10
10
10
1
3
2.0
2.0
2.0
4.0
4.23
3.78
5.71
2.44
5.59
4.11
317
226
780
61
732
291
-
2.63 ± 0.03×10
1.74 ± 0.07×10-
1
•3
4.07 ± 0.01×10-
5
-
b
c
1
.78 ± 0.03×10 (A)
1•
52
2.0
-10
2
.42 ± 0.02×10 (C)
a
Diffusion coefficients were recorded at 25 °C in DMSO-d (diffusion time
∆ = 200 ms),
6
and the molecular radii were calculated using the Stokes-Einstein equation.
Derived from diamidine 1.
b
c
Derived from benzoic acid 5.
To further confirm the reason for the difference in the fluorescence wavelength, we examined the
+
estimation of the fluorescence wavelengths of 1•3, 1•4, 1•5 and 1•2H using the time-dependent density
2
functional theory (TD-DFT, PBE0/6-31G*) calculations (Figure S27, Table 2). For the 1:1 complexes
1
•3 and 1•4, a slightly shorter fluorescence wavelength (λcalc = 394 nm) compared to the observed
fluorescence wavelengths (1•3: λexp = 417 nm, 1•4: λexp = 422 nm) in a DMSO solution was observed.
These differences may caused by the dissociation of the formed 1:1 complexes in the solution due to the
flexibility of the diamidine 1. On the other hand, the estimated fluorescence wavelength of 1•52 (λcalc
=
4
09 nm) was not consistent with the observed fluorescence wavelength ( exp = 442, 470 nm) in the
λ
DMSO solution. However, the fluorescence wavelength of 1•5 was close to the estimated fluorescent
2
+
wavelength of the amidinium 1•2H (λcalc = 454, 475 nm) and observed fluorescence wavelength of
1
•2HCl (λexp = 443, 468 nm) in the DMSO solution.
10

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Table 2. Results of TD-DFT calculations and experimental
wavelengths for fluorescence spectra
experimental maximal calculated maximal oscillator
compound
wavelength (nm)a
wavelength (nm)b strength ( f )b
1
1•
•
3
4
417
422
394
394
0.43
0.38
4
42
70
1
•
52
409
0.42
4
4
4
54
75
0.15
0.37
1•
2H+
4
43
68
1•
2HCl
4
a
Fluorescence spectra were recorded in DMSO (25
TD-PBE0/6-31G*//CIS/6-31G*.
µM, λex = 307 nm).
b
Based on these results, we concluded that the fluorescence band around 410-430 nm comes from
the formation of the 1:1 complex (i.e., formation of amidinium-carboxylate salt bridges), and the
fluorescence band around 440-470 nm (as a broad band which consisted from two peaks) is due to the
+
formation of the amidinium 1•2H (Scheme 1). Consequently, it is possible to determine the
dicarboxylic acids or monocarboxylic acids based on the fluorescence spectra. The differences between
the calculated and observed fluorescence wavelengths were greater than those of diamidine 2 which has
7
a rigid structure. This may make the flexibility of the structure of the diamidine 2 difficult to get the
ideal minimized structure in the DFT calculations.
2
.3. Binding experiments of diamidine 1 with
To explore the applicability of this “turn-on” fluorescence sensor 1, we examined the recognition
of the conformationally flexible −dicarboxylic acids 6. The fluorescence spectra of the diamidine 1
α,ω−dicarboxylic acids 6
α,ω
and dicarboxylic acid 6d-6l (n = 4-11 and 18, 1 equiv.) in DMSO showed the formation of the 1:1
complex (λem = 410-430 nm, Figure 5). However, the fluorescence spectra of 1 with the dicarboxylic
acid 6a-6c (n = 1-3), which has a shorter distance between the two carboxylic groups than the length
between the two amidino groups of 1, showed the formation of amidinium species (λem = 440-470 nm as
a broad band, Figure 5). Additionally, the fluorescence spectra of 1 with 2 equivalents of acetic acid 7
11

ACCEPTED MANUSCRIPT
+
(
monocarboxylic acid) also showed the formation of the amidinium 1•2H (λem = 439 and 469 nm,
Figure 5). To obtain additional evidence for the formation of the amidinium-carboxylate salt bridge, we
measured the fluorescence spectra of 1•6e after the addition of H O (Figure S26). The fluorescence
2
spectra of 1•6e was red-shifted ( max
λ
= 441 and 473 nm) by the addition of H O, which indicated the
2
formation and dissociation of the amidinium-carboxylate salt bridges in the DMSO solution.
HN NH
2HN
NH
2
H
N
H
O
O
a: n = 1, b: n = 2
O
O
N
H
c: n = 3, d: n = 4
e: n = 5, f: n = 6
g: n = 7, h: n = 8
H
CH
2
+ HO
n
H
DMSO
N
H
H
O
O
OH
n
N
i: n = 9, j: n = 10
k: n = 11 l: n = 18
H
6
1

6
1
Figure 5. Fluorescence spectra of diamidine 1 (25
−dicarboxylic acid 6 (1 eq.) and acetic acid 7 (2 eq.).
µM in DMSO, λex = 307 nm) upon the addition of
α,ω
To demonstrate the formation of the 1:1 complex 1•6 using NMR spectroscopy, we prepared
DMSO-d solutions (2.0 mM) of the complexes 1•6. However, in almost all cases, precipitation of the
6
1
formed complexes was observed. Only in the case of the dicarboxylic acid 6h (n = 8), the H NMR
1
spectrum of 1•6h was observed without precipitation in a DMSO-d (2.0 mM) solution. The H NMR
6
spectrum of the obtained solution showed the characteristic upfield and downfield shifts with respect to
12

ACCEPTED MANUSCRIPT
a
e
those of 1 and 6h (Figure S4). The downfield shift of the amidine unit (H -H ) and the upfield shift of
1
the carboxylic acid unit in the H NMR indicated the formation of a stable complex. The resonance of
the NH protons was observed as broad singlets at 13.5 and 9 ppm, indicating the formation of
amidinium-carboxylate salt bridges (i.e., formation of a stable complex). For the 1:1 complexes 1•6e,
1
•6g and 1•6i, the NMR spectra were observed in a DMSO-d -D O (9:1) mixed solvent without
6 2
precipitation (Figures S5-S7). To obtain further evidence for the formation of the 1:1 complex, we
evaluated the Job’s plot analysis (Figures S12-S21) and also the DOSY NMR measurements (Figures
S23-S25, Table 3). The DOSY spectra of 1•6e (n = 5) showed the formation of the stable 1:1 complex
3
even in water (D O) containing mixed solvents and the molecular volume of 1•6e (n = 5, V = 530 Å )
2
3
that is slightly greater than the sum of the volumes of the free building blocks 1 (V = 291 Å ) and 6e (V
3
=
110 Å ), but much smaller than the volume of the 2:2 complex 1 •6e (V = 802, Table 3). For the
2
2
complexation of 1•6g (n = 7) and 1•6i (n = 9), the DOSY signals are separately observed due to the
weaker binding (Figures S25-S26, Table 3). However, the diffusion coefficients indicated the formation
of the 1:1 complexes 1•6g and 1•6i.
Table 3. Comparison of diffusion coefficients, molecular radii and volumesa
2
-1
V (Å3)
compound
D (m s )
rH (Å)
-
-
-
-
-
10
10
10
10
10
10
1
1.64 ± 0.06×10
2.27 ± 0.05×10
1.34 ± 0.03×10
2.09 ± 0.05×10
4.11
2.97
5.02
3.22
5.30
5.07
3.53
291
110
530
140
624
546
184
438
364
6e
1 6e
•
6g
b
c
1
.27 ± 0.02×10 (A)
1•6g
-
1
.33 ± 0.01×10 (C)
-
10
6
i
1.91 ± 0.03×10
-
10
10
b
1
1
.43 ± 0.02×10 (A)
4.71
4.43
1
•6i
-
c
.52 ± 0.04×10 (C)
a
Diffusion coefficients were recorded at 25 °C in DMSO-d -D O mixed solvent (9:1, 2.0 mM),
6
2
and the molecular radii were calculated using the Stokes-Einstein equation (the viscosity of
mixed solvents is taken from reference 10).
b
c
Derived from diamidine 1.
Derived from dicarboxylic acid 6.
13

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The estimation of the fluorescence wavelengths of 1•6 using the time dependent density
functional theory (TD-DFT) calculations was unsuccessful due to the difficult calculation of a fully
minimized structure due to the higher flexibility of the diamidine 1 and dicarboxylic acids 6.
The binding constants (K ) of the diamidine 1 and dicarboxylic acids 3, 6d-6l (n = 4-11, 18)
1
:1
with a 1:1 stoichiometry determined by fluorescence titrations and the pK values of the carboxylic acids
a
are summarized in Table 4 (also see fluorescence titration curves and UV titration experiments in
5
-1
Figures S28-S38). In almost all cases, the binding constants are on the order of 10 (M ) in the DMSO
solution. These binding constants are almost not affected by the chain lengths of the dicarboxylic acids
6
(n = 4-11) compared to the binding of the anthracene-based diamidine 2 (ref. 7, Table 4). This
phenomenon may caused by the flexibility of the diphenylnaphthalene unit compared to the rigid
anthracene unit. The binding constants of the −dicarboxylic acid 6 were not affected by the pKa
values of the dicarboxylic acids 6 (Table 4). However, in the case of the m-phenylenediacetic acid 4 that
shows a low pK value (pK = 3.39), dissociation of the formed complex 1•4 was observed in the
α,ω
a
a (calc)
presence of an excess amount of the dicarboxylic acid 4 (Figure S29).
a
b
Table 4. Binding constants (K ) of diamidine 1 with carboxylic acids and pK values of carboxylic acids
1:1
a
K1:1 / M-1
carboxylic acids
λem / nmc
pKa (obs)
pKa (calc)
1
,3-Adamantanediacetic acid (3)
421
422
430
4.98 x 105
4.42
3.39
m-Phenylenediacetic acid (4)
Adipic acid (6d, n = 4)
4.12 x 105
4.41 5.41
4.39
5
Pimelic acid (6e, n = 5)
Suberic acid (6f, n = 6)
Azelaic acid (6g, n = 7)
Sebacic acid (6h, n = 8)
Undecanedioic acid (6i, n = 9)
Dodecanedioic acid (6j, n = 10)
Tridecanedioic acid (6k, n = 11)
Eicosanedioic acid (6l, n = 18)
421
428
426
424
428
425
3.81 x 10
4.71 5.58
4.52
4.53 5.33
4.59 5.59
4.43
4.46
4.47
4.48
4.48
4.48
4.48
4.48
5
3.88 x 10
5
4.52 x 10
5
2.69 x 10
5
3.56 x 10
4.07 x 105
5
428
430
3.72 x 10
1.25 x 10
5
a
Binding constants were determined by fluorescence titration in DMSO (1: 20-25
µ
M,) using the 1:1 model.
b
th
pKa (obs): pK values of carboxylic acids in H O, Lide D. R. Ed., CRC Handbook of Chemistry and Physics, 88
a
2
ed., CRC Press, Boca Raton, FL, 2007-2008; pKa (calc): Calculated pK values of carboxylic acids using Advanced
a
Chemistry Development (ACD / Laboratories) Software V11.01 (1994-2012 ACD / Labs) taken from SciFinder
Scholar.
14

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c
Maximum fluorescence wavelength of titration spectra.
To demonstrate the selectivity of the diamidine 1 toward a dicarboxylic acid 6, we observed
the fluorescence spectra of 1•6e after the addition of acetic acid 7 (monocarboxylic acid). The
fluorescence spectra of 1•6e were slightly changed by the addition of acetic acid 7 (0-5 equiv.).
However, the fluorescence spectra of 1•6e showed a profile sufficient for recognizing the presence of the
dicarboxylic acid (Figure S40). These results show that the 1:1 complex of the diamidine 1 and
dicarboxylic acid 6e (i.e., pimelic acid) is more stable than the 1:2 complex with acetic acid 7
(monocarboxylic acid) even in the presence of excess monocarboxylic acid 7.
2.4. Fluorescence quantum yields of carboxylic acid-binding complexes derived from diamidines 1
and 2
The fluorescence quantum yields (Φ ) of dicarboxylic acid-binding complexes 1•3, 1•4 and 1•6e
fl
derived from diamidine 1 and dicarboxylic acids in DMSO solutions were determined by a relative
1
1
method using anthracene in EtOH (Φ = 0.27) as a reference (Table 5). For the comparison, the
fl
fluorescence quantum yields of the dicarboxylic acid binding complexes (2•3, 2•4, 2•6e) of the
diamidine 2, and 1,8-diphenylnaphthalene were also observed. The dicarboxylic acid-binding
complexes (1•3, 1•4 and 1•6e) derived from the diamidine 1 showed relatively high fluorescence
quantum yields (Φ = 0.77-0.83) compared to the corresponding carboxylic acid-binding complexes of
fl
the diamidine 2 (2•3, 2•4 and 2•6e, Φ = 0.29-0.35). Moreover, 1,8-diphenylnaphthalene itself showed
fl
1
2
a very low quantum yield (Φ = 0.03) in a DMSO solution (Φ = 0.12 in benzene , Φ = 0.03 in
fl
fl
fl
1
3
cyclohexane ). These findings showed that the introduction of amidinium-carboxylate substituents into
the 1,8-diphenylnaphthalene unit dramatically improved the fluorescence quantum yield in the DMSO
solution. However, in the case of the diamidine 2, the fluorescence quantum yields of 2•3, 2•4, and 2•6e
(
Φ = 0.29-0.35) were not increased by the introduction of the amidinium-carboxylate substituents
fl
(
anthracene: Φ = 0.27 in EtOH). The reason for these phenomena is unclear at the present time.
fl
15

ACCEPTED MANUSCRIPT
Table 5. Fluorescence quantum yields (Φ ) of dicarboxylic acid binding
fl
a
complexes of diamidine, and reference compound in DMSO
Φfl
1
,8-diphenylnaphthalene
0.03
1
1
1
2
2
2
•
•
•
•
•
•
3
4
6e
3
4
0.77
0.83
0.80
0.33
0.35
0.29
6e
a
Fluorescence quantum yields were determined using anthracene
Φ = 0.27 in EtOH) as a standard.
(
fl
3. Conclusions
We have designed and synthesized a “turn-on” fluorescence sensor 1 having a flexible 1,8-
diphenylnaphthalene unit and succeeded in the detection of the amidinium-carboxylate (λem = 410-430
nm) and amidinium (λem = 440-470 nm as a broad band which consisted from two peaks) species by
fluorescence spectroscopy. This fluorescent detection of the amidinium-carboxylate (i.e., detection of
a stable 1:1 complex) was applicable to the detection of α,ω−dicarboxylic acids. Additionally, for the
complexation of the diamidine 1 with dicarboxylic acids, higher fluorescence quantum yields (Φ : up
fl
to 80%) were observed compared to the binding of the diamidine 2 (Φ : up to 35%). The synthesis of
fl
diamidine having different fluorophores and the detection of diacrboxylic acid in highly polar solvents
are currently under investigation.
16

ACCEPTED MANUSCRIPT
4
. Experimental Section
.1. General Methods
4
Melting points were determined by a Yanaco micro melting point apparatus and are uncorrected.
1
13
The H NMR and C NMR spectra were recorded using Bruker DRX-500 and Avance 500 (500, 125
MHz) spectrometers. The FAB-mass spectra were recorded by a JEOL JMS-700 mass spectrometer.
Elemental analyses were obtained using a Yanaco MT-5 CHN recorder. The absorption spectra were
recorded by a Shimadzu UV-Visible spectrometer UV-2550. 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 procedures. 1,8-Diiodonaphthalene was prepared by the
1
4
published procedure.
4.1.1 1,8-Bis(4-cyanophenyl)naphthalene. 1,8-Diiodonaphthalene (602 mg, 1.59 mmol), 4-
cyanophenylboronic acid (704 mg, 4.79 mmol) and Pd(PPh ) (87.5 mg, 0.076 mmol) were mixed in 40
3
4
mL DME, and then a 0.5 M Na CO aqueous solution (15 mL) was added. The obtained mixture was
2
3
degassed 3 times by the freeze-pump-through method and the mixture was heated at 85 °C for 18h in an
Ar atmosphere. After the addition of water (50 mL), the mixture was extracted with CH Cl (60 mL),
2
2
1
3
dried over anhydrous Na SO and the solvent evaporated. 1,8-Bis(4-cyanophenyl)naphthalene was
2
4
isolated (452 mg, 86%) by column chromatography (SiO , AcOEt/hexane 1/1).
2
1
Pale yellow needles; H NMR (500 MHz, CDCl ) δ = 8.03 (dd, J = 8.3, 1.3 Hz, 2H), 7.62 (dd, J = 8.3,
3
1
3
7
.1 Hz, 2H), 7.40 (dd, J = 7.1, 1.3 Hz, 2H), 7.29 (d, J = 8.5 Hz, 4H), 7.09 (d, J = 8.5 Hz, 4H). C NMR
(
125MHz, CDCl ); δ = 147.6 (C ), 137.8 (C ), 135.4 (C ), 131.4 (CH), 131.2 (CH), 130.4 (CH), 129.9
3
q
q
q
-1
(
CH), 128.4 (C ), 125.6 (CH), 118.4 (C ), 110.0 (C ); IR (KBr, cm ):
ν
2229, 1606, 1501, 1398, 1177,
q
q
q
1106, 1019, 842, 827, 777.
17

ACCEPTED MANUSCRIPT
4
.1.2 1,8-bis(tert-butoxycarbonylamino-imino-methyl)naphthalene To a suspension of NH Cl (735
4
mg, 13.7 mmol) in dry toluene (8 mL) at 0 °C, Me Al (2 M in toluene, 5.7 mL, 11.4 mmol) was slowly
3
added (15 min), the mixture was stirred for 30 min at 0 °C, then allowed to warm to room temperature.
To this solution, 1,8-bis(4-cyanophenyl)naphthalene (150 mg, 0.46 mmol) in toluene (50 mL) was added
and the resulting solution was heated under reflux for 17 h in an Ar atmosphere. The reaction was
1
monitored by H NMR (DMSO-d , a few drops of D O were added to remove the NH signals of the
6
2
excess reagents) after treatment of a small portion of the reaction mixture with MeOH and the Al O
2
3
filtered off. After consumption of the cyano compounds (mono and di-cyano compounds), the reaction
mixture was cooled to 0 °C, quenched with MeOH (20 mL), and the resulting suspension was filtered
over Celite. The filtrate was vacuum evaporated to afford a colorless solid. The obtained crude product
was suspended in THF (20 mL) at 0 °C, then 1 M NaOH (16 mL) and Boc O (300 µL, 1.31 mmol) were
2
added. After stirring at 0 °C for 2 h, then at room temperature for 2 h, ethyl acetate was added and the
organic phase was separated, washed with a saturated NaCl solution, dried (Na SO ), and evaporated
2
4
under reduced pressure. 1,8-Bis(tert-butoxycarbonylamino-imino-methyl)naphthalene (223 mg, 87%)
was isolated by column chromatography (SiO , toluene/MeOH 9/1).
2
1
Colorless solid; mp > 300 °C; H NMR (500 MHz, DMSO-d ) δ = 8.9-8.6 (br, 4H), 8.11 (dd, J = 7.1,
6
1.2 Hz, 2H), 7.66 (dd, J = 8.2, 7.1 Hz, 2H), 7.45 (m, 6H), 7.46 (d, J = 8.1 Hz, 4H), 7.44 (dd, J = 7.1, 1.3
1
3
Hz, 2H), 7.01 (d, J = 8.1 Hz, 4H), 1.44 (s, 18H). C NMR (125 MHz, DMSO-d ); δ = 166.6 (C ),
6
q
1
63.5 (C ), 145.7 (C ), 138.7 (C ), 135.0 (C ), 132.3 (C ), 130.9 (CH), 129.3 (CH), 129.0 (CH), 128.3
q q q q q
(
C ), 126.4 (CH), 125.5 (CH), 77.7 (C ), 28.0 (CH ). HRMS (FAB, NBA) m/z = 565.2811 (calculated
q q 3
＋
for [M+H] : 565.2815). Anal. Calcd. for C H N O ・0.5 H O: C:71.18, H:6.50, N:9.77. Found:
3
4
36
4
4
2
-1
C:71.42, H:6.47, N:9.32. IR (KBr, cm ):
ν 3379, 3236, 2979, 2929, 1654, 1610, 1287, 1170, 1147,
828, 776.
18

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4.1.3. 1,8-bis(4-amidinophenyl)naphthalene dihydrochloride (1•2HCl). To a solution of 1,8-bis(tert-
butoxycarbonylamino-imino-methyl)naphthalene (220 mg, 0.39 mmol) in THF (30 mL) at 0 °C was
added 18 mL of 4 M HCl. The reaction mixture was stirred at 0 °C for 2 h, then at room temperature for
1
h. After the addition of additional distilled water (30 mL), the water phase was concentrated to
provide the title compound (167 mg, 98%).
１
Colorless solid; mp > 300 °C; H NMR (500 MHz, D O, TMS in CDCl as external standard) δ = 8.20
2
3
(d, J = 8.2 Hz, 2H), 7.75 (t, J = 7.6 Hz, 2H), 7.60 (d, J = 7.1 Hz, 2H), 7.43 (d, J = 7.9 Hz, 4H), 7.25 (d, J
1
3
=
7.9 Hz, 4H). C NMR (125 MHz, D O, TMS in CDCl as external standard) δ = 166.1 (C ), 148.9
2 3 q
(
C ), 137.8 (C ), 135.2 (C ), 131.4 (CH), 131.0 (CH), 129.9 (CH), 128.2 (C ), 126.9 (CH), 125.9 (CH),
q q q q
1
24.9 (C ). Anal. Calcd. for C H Cl N ・0.7H O: C:64.06, H:5.24, N:12.45. Found: C:64.48, H:5.16,
q 24 22 2 4 2
-1
N:12.07. IR (KBr, cm ):
ν 3344, 3061, 1663, 1611, 1537, 1488, 856, 832, 779.
4.1.4. 1,8-bis(4-amidinophenyl)naphthalene (1). To a stirred solution of 1 M NaOH (7 mL, 7 mmol)
was dropwise added an aqueous solution (20 mL) of 1,8-bis(4-amidinophenyl)naphthalene
dihydrochloride 1•2HCl (20.5 mg, 46.9
µmol) at 0 °C over 5 min. The resulting precipitate was filtered
1
and washed with water to give a white solid 1 (14.2 mg, 83%). colorless solid; mp 282.5 °C (dec.); H
NMR (500 MHz, DMSO-d , TMS in CDCl as external standard) δ = 8.09 (d, J = 7.7 Hz, 2H), 7.63 (dd,
6
3
1
3
J = 8.1, 7.1 Hz, 2H), 7.40 (d, J = 7.0 Hz, 2H), 7.24 (d, J = 7.3 Hz, 4H), 6.91 (d, J = 7.3 Hz, 4H);
C
NMR (125 MHz, DMSO-d , TMS in CDCl as external standard) δ = 163.1 (C ), 143.8 (C ), 139.2 (C ),
6
3
q
q
q
1
35.0 (C ), 134.2 (C ), 130.7 (CH), 129.1 (CH), 128.8 (CH), 128.5 (CH), 125.4 (CH), 125.2 (CH).
q q
+
HRMS (FAB, NBA) m/z = 365.1766 (calculated for [M+H] : 365.1766). Anal. Calcd. for C H N ・
2
4
20
4
-1
0
.8 H O: C:76.09, H:5.75, N:14.79. Found: C:75.94, H:5.49, N:14.76. IR (KBr, cm ):
ν
3338, 3195,
2
1650, 1589, 1550, 1439, 1210, 829, 777.
1
4
.2. Jobs plot analysis ( H NMR)
19

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The stock solutions of 1 and carboxylic acids in DMSO-d were prepared in separate volumetric
6
flasks. Several sample solutions containing both the diamidine 1 and the carboxylic acids in different
1
ratios (1/9 to 9/1) were prepared and maintained at 2 mM. The H NMR spectra of the mixtures were
1
5
recorded, and the chemical shifts of 1 and the carboxylic acids were analyzed by the Job’s method.
4
.3. Jobs plot analysis (fluorescence spectroscopy)
The stock solutions of 1 and carboxylic acids in DMSO were prepared in separate volumetric
flasks. Several sample solutions containing both the diamidine 1 and carboxylic 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 the Job’s method.
(
4.4. DOSY measurements
In the DOSY experiments with the free building blocks as well as the complexes, the
concentrations of 1, 3, 6e, 6g, 6i, 1•3, 1•5 , 1•6e, 1•6g and 1•6i were kept constant at 2.0 mM (the
2
1
concentrations of 5 was maintained at 4.0 mM). The H 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
1
6
diffusion ordered spectra were collected on samples using the LEDbp pulse sequence. Pulse-fields
were incremented in 50 steps from 2% to 95% of the maximum gradient strength in a linear ramp. The
gradient length was selected between 2.0 and 3.0 ms with a diffusion time of 100-300 ms and an eddy
current delay of 5 ms.
4.5. Fluorescence titrations
A solution of the diamidine 1 was prepared (20-25 µM in DMSO), then an aliquot (3 mL) was
transferred to a 1-cm fluorescence tube. To this solution was dropwise added a stock solution of the
20

ACCEPTED MANUSCRIPT
carboxylic acid (2.0-2.5 mM in DMSO) in small portions. The fluorescence spectra were recorded by
1
7
excitation at 307 nm. The association constants were calculated using the program HYPSPEC and are
summarized in Table 4.
4.6. Computational methods
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 using the time-
dependent density functional theory (TD-DFT) with the PBE0 functional at the 6-31G* basis set level.
1
8
All of the calculations were performed with Gaussian 09W.
4
.7. Crystallographic analysis
Crystals suitable for X-ray crystallography was grown from the solutions of 1•3 in DMSO that
were kept in a small vial and left to stand in the sample bottle in the presence of molecular sieves 4A for
several days. For crystallization of 1•3, the solution of diamidine 1 (3.82 mg, 10.49 mol) and
dicarboxylic acid 3 (2.65 mg, 10.49 mol) in DMSO (4.5 mL) was stored for a few days. 1•3: 63%
yield as 1•3•DMSO (4.60 mg), colorless prisms, mp: > 300 °C, Anal. Calcd. for C H N O ・
µ
µ
3
8
40
4
4
DMSO・0.5H O: C:68.25, H:6.73, N:7.96. Found: C:68.20, H:6.59, N:7.91.
2
For the crystallographic data collection, a Rigaku AFC-7R with graphite-monochromated Cu-Kα
radiation was used. Calculations were performed using the Crystal Structure 3.8 crystallographic
1
9
20
software package. The crystal structures were solved by a direct method using SIR2002 , and the
2
1
structure refinements were performed by a full-matrix least squares method using Shelx-97. Details of
the data are summarized in Table S1. All non-hydrogen atoms were anisotropically refined. The
21

ACCEPTED MANUSCRIPT
hydrogen atoms of the amidino groups were located in a difference Fourier map and were isotropically
refined. All other hydrogen atoms were placed in idealized positions and were included in the structure
factor calculations, but not refined. CCDC-982289 contain the supplementary crystallographic 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.
http://beta-
www.ccdc.cam.ac.uk/Community/Requeststructure/Pages/Requeststructure.aspx.
4.8. Fluorescence quantum yield measurement
The fluorescence quantum yields (Φ ) of the carboxylic acid binding complexes 1•3, 1•4, 1•6e,
fl
2
•3, 2•4, 2•6e and 1,8-diphenylnaphthalene were calculated by measuring the integrated emission area of
the corrected spectra and comparing that value to the area measured for anthracene in EtOH (Φ =
fl
1
1
0
.27) when excited at 320 nm (for 1•3, 1•4, 1•6e, and 1,8-diphenylnaphthalene) and 335 nm (for 2•3,
2
•4, 2•6e) at different concentrations in spectral grade DMSO after purging the samples for 15 min with
argon gas. The quantum yields were calculated using eq. 1, where F represents the area under the
emission spectra for the standard and samples, n is the refractive index of the solvent, and Abs is the
absorbance at the excitation wavelength selected for the standard and samples.
2
sample
standard
sample
sample
Φfl
standard
= Φfl
F
F
Abs
Abs
n
n
(1)
standard
sample
standard
Acknowledgments
This study was supported in part by the Kyoto Institute of Technology Research Fund. We thank Prof.
T. Harada (Kyoto Institute of Technology) for his helpful discussions.
Supplementary data
Supplementary data related to this article can be found online at doi:101016/j.tet.000000.
22

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References and notes
1.
(a) Yashima E.; Maeda K.; Furusho Y. Acc. Chem. Res. 2008, 41, 1166 and references cited
therein; (b) Katagiri H.; Tanaka Y.; Furusho Y.; Yashima E. Angew. Chem. Int. Ed. 2007, 46,
2
435.
2
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(a) Papoutsakis D.; Kirby J. P.; Jackson J. E.; Nocera D. G. Chem. Eur. J. 1999, 5, 1474; (b)
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4
.
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Ferretti V.; Bertolasi V.; Pretto L. New. J. Chem. 2004, 28, 646.
5
Turn-off fluororeceptor for dicarboxylic acid: (a) Ghosh K.; Masanta G. Tetrahedron. Lett. 2006,
4
7, 2365; (b) Ghosh K.; Adhikari S. Tetrahedron. Lett. 2006, 47, 3577.; turn-on fluororeceptor
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d) Ray J. K.; Gupta S.; Pan D.; Kar G. K. Tetrahedron 2001, 57, 7213.
(
6.
Turn-off fluororeceptor for dicarboxylate in organic solvents: (a) Ghosh K.; Masanta G.;
Chattopadhyay A. P. Eur. J. Org. Chem. 2009, 4515; (b) Kim S. K.; Singh N. J.; Kim S. J.;
Swamy K. M. K.; Kim S. H.; Lee K.-H.; Kim K. S.; Yoon J. Tetrahedron 2005, 61, 4545; (c)
Gunnlaugsson T.; Davis A. P.; O’Brien J. E.; Glynn M. Org. Lett. 2002, 4, 2449; (d) Mei M.;
Wu S. New. J. Chem. 2001, 25, 471; turn-on fluororeceptor for dicarboxylate in organic solvents:
(e) Singh N.; Lee G. W.; Jang D. O. Tetrahedron 2008, 64, 1482; turn-on and turn-off
fluororeceptor for dicarboxylate in aqueous solvents: (f) Liu S.-Y.; Fang L.; He Y.-B.; Chan W.-
H.; Yeung K.-T.; Cheng Y.-K.; Yang R.-H. Org. Lett. 2002, 4, 2449; (g) Raker J.; Glass T. E. J.
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A. Angew. Chem. Int. Ed. 2004, 43, 3847; (i) Hu M.; Feng G. Chem. Commun. 2012, 48, 6951.
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9
1
1
1
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2
000, 2, 2081.
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1
1
1
1
1
3.
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6.
7.
Feng J.; Chen X.; Han Q.; Wang J.; Lu P.; Wang Y. J. Lumin. 2011, 131, 2775.
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A.; Cheeseman J. R.; Scalmani G.; Barone V.; Mennucci B.; Petersson G. A.; Nakatsuji H.;
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24

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2
0.
1.
SIR2002: Burla M. C.; Camalli M.; Carrozzini B.; Cascarano G. L.; Giacovazzo C.; Polidori G.;
Spagna R. J. Appl. Crystallogr. 2003, 36, 1103.
2
SHELXL-97: Sheldrick G. M. Acta Crystallogr., Sect. A 2008, 64, 112.
25

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Fluorescent detection of amidinium-carboxylate and
amidinium
formation
using
a
1,8-
diphenylnaphthalene-based diamidine: dicarboxylic
acid recognition with high fluorescence efficiency
Takahiro Kusukawa*, Syugo Tanaka and Kouta Inoue
Department of Chemistry and Materials Technology,
Graduate School of Science and Technology, Kyoto Institute of Technology,
Matsugasaki, Sakyo-ku Kyoto 606-8585, Japan
TEL/FAX: +81-75-724-7506, e-mail: kusu@kit.ac.jp
-----------------------------------------------------------------------------------------------------------------------------
1

ACCEPTED MANUSCRIPT
a
Table 1. Comparison of diffusion coefficients, molecular radii and volumes
2
-1
3
compound
conc. (mM)
2.0
D (m s )
rH (Å)
V (Å )
1
-10
-10
-10
-10
2
2
1
4
.35 ± 0.06×10
.63 ± 0.03×10
.74 ± 0.07×10
.07 ± 0.01×10
4.23
3.78
5.71
2.44
5.59
317
226
780
61
3
2.0
2.0
4.0
1
•3
5
-
10
b
c
1
.78 ± 0.03×10 (A)
732
291
1•52
2.0
-10
2
.42 ± 0.02×10 (C)
4.11
a
Diffusion coefficients were recorded at 25 °C in DMSO-d (diffusion time
∆
= 200 ms),
6
and the molecular radii were calculated using the Stokes-Einstein equation.
Derived from diamidine 1.
b
c
Derived from benzoic acid 5.
2

ACCEPTED MANUSCRIPT
Table 2. Results of TD-DFT calculations and experimental wavelengths for fluorescence spectra
compound
experimental maximal calculated maximal
oscillator
strength ( f )
0.43
a
b
b
wavelength (nm)
wavelength (nm)
1
1
•3
•4
417
422
394
394
0.38
4
4
42
70
1
•52
409
0.42
+
454
0.15
0.37
1
•2H
475
4
4
43
68
1
•2HCl
a
Fluorescence spectra were recorded in DMSO (25
TD-PBE0/6-31G*//CIS/6-31G*.
µM, λex = 307 nm).
b
3

ACCEPTED MANUSCRIPT
a
Table 3. Comparison of diffusion coefficients, molecular radii and volumes
2
-1
3
compound
D (m s )
rH (Å)
4.11
2.97
5.02
3.22
5.30
5.07
3.53
4.71
4.43
V (Å )
-
-
-
-
-
10
1
1.64 ± 0.06×10
2.27 ± 0.05×10
1.34 ± 0.03×10
2.09 ± 0.05×10
291
110
530
140
624
546
184
438
364
10
10
10
10
6
e
1
•6e
6
g
b
1
.27 ± 0.02×10 (A)
1
•6g
-10
c
1
.33 ± 0.01×10 (C)
-10
-10
-10
6
i
1.91 ± 0.03×10
b
1
.43 ± 0.02×10 (A)
1
•6i
c
1
.52 ± 0.04×10 (C)
a
Diffusion coefficients were recorded at 25 °C in DMSO-d -D O mixed solvent (9:1, 2.0 mM),
6
2
and the molecular radii were calculated using the Stokes-Einstein equation (the viscosity of
mixed solvents is taken from reference 10).
b
c
Derived from diamidine 1.
Derived from dicarboxylic acid 6.
4