Development of amylose- and β-cyclodextrin-based chiral fluorescent sensors bearing terthienyl pendants

Article abstract of DOI:10.3390/molecules21111518

Phenylcarbamate derivatives of amylose and β-cyclodextrin show excellent chiral recognition when used as chiral stationary phases (CSPs) for high-performance liquid chromatography. To open up new possibilities of carbohydrate-based materials, we developed chiral fluorescent sensors based on amylose and β-cyclodextrin (Am-1b and CyD-1b, respectively) by attaching fluorescent π-conjugated units on their side chains. Their recognition abilities toward chiral analytes containing a nitrophenyl unit were evaluated by measuring the enantioselective fluorescence quenching behavior. Both sensors showed the same degree of enantioselective fluorescence response for various aromatic nitro compounds. However, in some cases, their enantioselectivities were different depending on the analytes. The difference in the chiral recognition abilities between Am-1b and CyD-1b seems to be based on the structural difference of their inherent backbones, that is, the one-handed helical structure and cyclic structure, respectively. The study on the resolution ability of the Am-1b-based CSP revealed that the terthienyl-based pendant of Am-1b provides not only a fluorescent functionality but also a different chiral recognition site from that of amylose tris(phenylcarbamate).

Full text of DOI:10.3390/molecules21111518

molecules
Article
Development of Amylose- and β-Cyclodextrin-Based
Chiral Fluorescent Sensors Bearing
Terthienyl Pendants
Tomoyuki Ikai *, Changsik Yun, Yutaka Kojima, Daisuke Suzuki, Katsuhiro Maeda and
Shigeyoshi Kanoh
Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192,
Japan; robiny87@gmail.com (C.Y.); y_kojima_1991@yahoo.co.jp (Y.K.); dsuke3heat@gmail.com (D.S.);
maeda@se.kanazawa-u.ac.jp (K.M.); kanoh@se.kanazawa-u.ac.jp (S.K.)
* Correspondence: ikai@se.kanazawa-u.ac.jp; Tel.: +81-76-234-4781
Academic Editor: Yoshio Okamoto
Received: 13 October 2016; Accepted: 8 November 2016; Published: 11 November 2016
Abstract: Phenylcarbamate derivatives of amylose and β-cyclodextrin show excellent chiral recognition
when used as chiral stationary phases (CSPs) for high-performance liquid chromatography. To open
up new possibilities of carbohydrate-based materials, we developed chiral fluorescent sensors based on
amylose and β-cyclodextrin (Am-1b and CyD-1b, respectively) by attaching fluorescent π-conjugated
units on their side chains. Their recognition abilities toward chiral analytes containing a nitrophenyl
unit were evaluated by measuring the enantioselective fluorescence quenching behavior. Both sensors
showed the same degree of enantioselective fluorescence response for various aromatic nitro compounds.
However, in some cases, their enantioselectivities were different depending on the analytes. The
difference in the chiral recognition abilities between Am-1b and CyD-1b seems to be based on the
structural difference of their inherent backbones, that is, the one-handed helical structure and cyclic
structure, respectively. The study on the resolution ability of the Am-1b-based CSP revealed that the
terthienyl-based pendant of Am-1b provides not only a fluorescent functionality but also a different
chiral recognition site from that of amylose tris(phenylcarbamate).
Keywords: carbohydrates; chirality; fluorescent sensor; helix; higher-order structure; polysaccharides
1. Introduction
Biologically interesting chiral molecules are ubiquitous in the fields of drugs, agrochemicals,
food additives and fragrances. It has been widely recognized that the physiological properties
of two enantiomers are often different owing to the sophisticated chiral discrimination ability of
enzymes and receptors in the body [1–4]. Thus, to eliminate the incorrect use of hazardous isomers
that may have undesired properties, chiral discrimination has been viewed as an important issue,
particularly in the pharmaceutical industries. Polysaccharides, such as amylose and cellulose, are
renewable and abundant resources that are optically active. Although natural polysaccharides
hardly show marked chiral discrimination ability [5,6], their appropriately modified derivatives
(e.g., phenylcarbamate derivatives) are known to show excellent chiral recognition for a wide variety
of racemic compounds when applied to chiral stationary phases (CSPs) for high-performance liquid
chromatography (HPLC) [7–10]. Today, polysaccharide-based CSPs are widely used around the world,
not only for the analysis of enantiomeric compositions but also to obtain optically pure enantiomers in
the bulk. Although great success has been achieved in the application of polysaccharide derivatives in
CSP materials, their application in other fields of chiral materials has been highly restricted. We have
reported functionalized polysaccharide derivatives bearing reactive prochiral units or catalytically
active sites as pendant groups with the aim of developing chiral auxiliaries or asymmetric catalysts,
Molecules 2016, 21, 1518; doi:10.3390/molecules21111518
www.mdpi.com/journal/molecules

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respectively, for asymmetric synthesis [11
,12]. In addition, a cellulose-based chiral fluorescent sensor
(Ce-1b in Figure 1) containing a terthienyl moiety as a fluorescent signaling unit has also been
developed and was found to show apparent enantioselectivity for various types of chiral compounds
containing either central or axial chirality [13]. The significant contribution of the helical chirality
of Ce-1b to the chiral fluorescence sensing was demonstrated by the fact that the corresponding
monosaccharide-based model compound (Gl-1b in Figure 1) showed almost no enantioselectivity.
Figure 1. Structures of previously reported phenylcarbamate derivatives of polysaccharides (Ce-1b
and ATPC) and glucose (Gl-1b).
In the present study, we developed a novel amylose-based chiral fluorescent sensor (Am-1b in
Scheme 1) bearing terthienyl-based
the enantioselective fluorescent responses of Am-1b toward chiral aromatic nitro compounds with that
of the corresponding -cyclodextrin derivative (CyD-1b in Scheme 1), which has the same repeating
unit as Am-1b. Although a large number of chiral fluorescence sensors, including -cyclodextrin-based
sensors, have been developed so far [14 16], to the best of our knowledge, there have been no reports
π-conjugated pendants as fluorescent signaling units. We compared
β
β
–
of the amylose-based chiral fluorescence sensors except for the seminal example of chiral sensing using
amylose-functionalized graphene [17].
Scheme 1. Synthesis of amylose (Am-1b) and
β-cyclodextrin (CyD-1b) derivatives bearing
terthienyl pendants.
2. Results and Discussion
The amylose derivative (Am-1b) bearing fluorescent terthienyl-based π-conjugated pendants at
the 2-, 3- and 6-positions of the glucose unit was synthesized according to our previously reported
procedure (Scheme 1) [13]. Am-1b showed good solubility, not only in common organic solvents,
such as tetrahydrofuran (THF) and chloroform but also in mixtures of these solvents with 90 vol %
hexane. Hexane is typically a poor solvent for the previously reported amylose phenylcarbamates

Molecules 2016, 21, 1518
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used as CSPs for HPLC. The ample solubility of Am-1b is probably owing to the branched alkyl chains
at the terminal thiophene ring of the -conjugated pendants. We also prepared the corresponding
-cyclodextrin (CyD-1b) bearing the same terthienyl pendants using a
π
phenylcarbamate derivative of
similar procedure.
β
Figure 2a shows the circular dichroism (CD) and absorption spectra of Am-1b and CyD-1b in
THF. An apparent split-type CD absorption was observed in the absorption region of the terthienyl
pendant of Am-1b (330–460 nm). As the previously reported glucose derivative Gl-1b showed a very
weak CD absorption in this region [13], the CD absorption of Am-1b is considered to not be derived
from the central chirality of the glucose ring. An asymmetric arrangement is most likely induced in
the pendants of Am-1b owing to the helical structure of the amylose phenylcarbamate framework,
which is known to have a left-handed 4/3 helical conformation [18,19]. The chiroptical properties
of Am-1b were also measured in hexane/THF mixtures (hexane: 50 vol %–90 vol %) (Figure 2b),
which is a solvent used in the following section for chiral fluorescent sensing. The CD intensities
of Am-1b increased with increasing hexane content. The hypochromic effect was also observed
in its absorption spectrum that was measured in hexane/THF (90/10, v/v). Figure S1a shows the
dynamic light scattering (DLS) analysis of Am-1b in THF, from which the hydrodynamic diameter
(Dh) of Am-1b in THF was estimated to be approximately 53 nm. This value was almost comparable
to that measured in hexane/THF (90/10, v/v) (approximately 51 nm) (Figure S1b). These results
indicate that the spectral change induced by the addition of high amounts of hexane was mainly
caused by a certain alteration in the arrangement of the
interactions. Importantly, the CD spectral pattern of Am-1b was found to be almost a mirror image of
the corresponding cellulose phenylcarbamate Ce-1b (Figure S2). This suggests that the -conjugated
π-conjugated pendants through intramolecular
π
pendants of Am-1b and Ce-1b are arranged in opposite chiral environments to each other, probably
reflecting their higher-order structures. CyD-1b also showed a similar split-type CD absorption in the
terthienyl chromophore region, although its CD intensity and pattern were different from those of
Am-1b, particularly in the absorption region below 300 nm. In a hexane/THF mixture (90/10, v/v),
the CD intensity of CyD-1b was increased and its pattern showed a close resemblance to that of Am-1b
over the entire absorption region (Figure S3). This indicated that the π-conjugated pendants of CyD-1b
were arranged in a similar manner to those of Am-1b only when the solution contained a high content
of hexane (~90 vol %).
◦
Figure 2.
(
a
) CD and absorption spectra of Am-1b and CyD-1b in THF at 25 C; (
b
) CD and absorption
◦
−5
spectra of Am-1b in hexane/THF (hexane: 0–90 vol %) at 25 C. [Glucose unit] = 1.0 × 10 M.
The fluorescence spectra of Am-1b and CyD-1b measured in THF are shown in Figure 3.
As depicted in the photographs of their solutions that were irradiated at 365 nm (Figure S4), apparent
greenish emissions derived from their π-conjugated pendants were clearly observed in both cases.
Am-1b and CyD-1b exhibited fluorescence quantum yields of 5% and 6%, respectively, in THF and 2%
and 3%, respectively, in hexane/THF (90/10, v/v), which are low but sufficient for use as fluorescent

Molecules 2016, 21, 1518
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sensor materials. As was the case for Ce-1b
[
13], the fluorescence intensities of the present fluorophores
were diminished when nitrobenzene (
a result of photoinduced electron transfer from the fluorescent pendant residues to
quenching behavior prominently appeared in a THF solution containing 90 vol % hexane as a nonpolar
solvent (Figures S4b,d). This result indicated that THF molecules compete with in the interaction
5) was added to the solutions (Figure S4). This was most likely as
5. This fluorescence
5
with Am-1b and that the hydrogen-bonding and/or the dipole–dipole interactions between these
fluorophores and 5 play an important role in the fluorescence quenching.
Figure 3. Fluorescence spectra of Am-−1b5
(
λ
= 364 nm) and CyD-1b
(
λ
ex
= 362 nm) in THF at room
ex
temperature. [Glucose unit] = 1.0 × 10 M.
Next, we investigated the enantioselective fluorescent responses of Am-1b and CyD-1b toward
chiral aromatic nitro compounds ( 11 in Figure 4). When the fluorescence spectra of Am-1b were
measured in the presence of (R)- or (S)- in a hexane/THF (90/10, v/v) mixture (Figure 5a,b),
6–
6
6
the fluorescence intensities gradually decreased with increasing concentration of either enantiomer.
Figure 5c shows the Stern–Volmer plots for the fluorescence quenching of Am-1b by the (R)- and
(S)-isomers of 6. We found that (S)-6 quenched the emission of Am-1b more efficiently than (R)-6 and
that the fluorescence quenching obeys the Stern–Volmer equation (Equation (1)) in the concentration
range from 0 to 0.30 mM of 6, resulting in straight lines for both isomers:
I0/I = KSV[Q] + 1
(1)
In this equation, I and I0 are the fluorescence intensities of Am-1b at 450 nm with and without
a guest quencher, respectively, KSV is the Stern–Volmer constant, and [Q] is the concentration of the
guest. From these plots, the KSV values for (S)-6 and (R)-6 ,
were determined to be 1800 and 1020 M−1
respectively, with a ratio of 1.77.
Figure 4. Structures of chiral aromatic nitro compounds and their analogs (6–11).

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Figure 5.
Fluorescence spectra of Am-1b
(
a
,
b
) and CyD-1b
) in hexane/THF (90/10, v/v) at room temperature.
ex = 450 nm; ( ) Stern–Volmer plots for the fluorescence
(d,e) upon the addition of
various amounts of (R)-
6
(
a
,
d
) and (S)-6 (b,e
[Glucose unit] = 1.0 × 10−5 M, [
6] = 0–0.30 mM,
λ
c,f
quenching of Am-1b (c) and CyD-1b (f) by (R)-6 (red) and (S)-6 (blue).
Am-1b showed distinct enantioselective fluorescence responses toward all of the tested aromatic
nitro compounds except for 11b (Figure 6). The upward curvatures observed in the Stern–Volmer plots
using 11 as quenchers indicate that the fluorescence quenching of Am-1b by 11 occurs by both dynamic
and static quenching processes (Figure 6e,f) [20]. We also confirmed that an intermediate quenching
between the results obtained using (R)-11a and (S)-11a was observed when rac-11a was used as a
quencher (Figure 6e). In all cases except for 11b, the fluorescence emission from Am-1b was more
strongly quenched by each (S)-isomer. This means that the intermolecular interactions between Am-1b
and the (S)-isomers are more effective than those with the corresponding antipodes. Interestingly,
the enantioselectivities observed for Am-1b were opposite to those observed for the corresponding
cellulose-based sensor (Ce-1b), in which the (R)-isomers induced more noticeable quenching than their
antipodes (Figure S5) [13]. As described in the CD spectral analysis, the π-conjugated pendants in
Am-1b seem to be arranged in opposite chiral environments to those of Ce-1b, which may result in
different enantioselectivities in the fluorescent sensing.
CyD-1b also exhibited an enantioselective fluorescence response to aromatic nitro compounds
(Figures 5d–f and 7). The Stern–Volmer plots obtained using 7a as quenchers show non-linear
relationships, suggesting that some of the π-conjugated pendants were inaccessible or only slightly
accessible to the guest quenchers (Figure 7a) [20]. The chiral recognition ability of CyD-1b was found
to be almost comparable to that of Am-1b, although the enantioselectivities against 7a and 11a were
opposite to each other. As both fluorophores were composed of the same α-D-glucose-based repeating
unit, the characteristic chiral sensing abilities observed for Am-1b and CyD-1b are most likely present
as a result of the formation of a one-handed helical structure and a cyclic structure, respectively, rather
than the contribution from the monomeric glucose unit itself. This reasoning is also supported by
the following results that have been reported previously. (1) A distinct chiral recognition was hardly
observed using Gl-1b as a sensor [13]; (2) Amylose phenylcarbamates showed different resolution
abilities to the corresponding β-cyclodextrin derivatives when used as CSPs for HPLC [21]. It was
also found that rac-7c and rac-11c without a nitro group did not quench the emission of Am-1b and
CyD-1b at all, probably due to a mismatch in the lowest unoccupied molecular orbital levels of the
fluorophores and the guests (Figure S6).

Molecules 2016, 21, 1518
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Figure 6. Stern–Volmer plots for the fluorescence quenching of Am-1b
(
λ
ex
= 450 nm) by the
(R)- (red) and (S)- (blue) isomers of 7a ); ); ); 10 ); 11a ) and 11b (f) in hexane/THF
(
a
8
(b
9
(
c
(d
(e
(90/10, v/v) at room temperature. The result using rac-11a as a quencher is also shown in (
[Glucose unit] = 1.0 × 10 M.
e
).
−5
Figure 7. Stern–Volmer plots for the fluorescence quenching of CyD-1b
(
λ
ex
= 450 nm) by the
(R)- (red) and (S)- (blue) isomers of 7a ); ); ); 10 ); 11a ) and 11b (f) in hexane/THF
(a
8
(b
9
(
c
(
d
(
e
(90/10, v/v) at room temperature. The result using rac-11a as a quencher is also shown in (
[Glucose unit] = 1.0 × 10 M.
e
).
−5
The influence of the fluorescent terthienyl-based pendant on the chiral recognition was
investigated by comparison of the chiral recognition abilities of Am-1b and the previously reported
amylose tris(phenylcarbamate) (ATPC in Figure 1), which contains non-substituted phenylcarbamate
pendants [22]. Because the enantioselectivity of ATPC cannot be evaluated as a chiral fluorescence

Molecules 2016, 21, 1518
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sensor due to the lack of fluorescence, their chiral recognition abilities were compared on the basis
of their resolution abilities when used as CSPs for HPLC. The Am-1b- and ATPC-based CSPs were
prepared by coating the corresponding polymers on macroporous silica gel. Their resolution abilities
were evaluated using the aromatic nitro compounds and their analogs (rac-6–11). Because Am-1b
had a high affinity for hexane and was soluble in a hexane/alcohol mixture, which is a typical eluent
for chiral HPLC, ethanol was used as the eluent. The CyD-1b-based CSP was slightly soluble in
ethanol; therefore, the evaluation of its resolution ability was impossible. The results of the resolution
of rac-11a
is shown in Figure S7 [23]. The enantiomers were eluted at retention times of t1 and t2, and the
hold-up time (t0) was estimated to be 18.7 min. Therefore, the retention factors, k1 [=(t1 t0)/t0]
–c are summarized in Table 1, and the chromatogram of rac-11a partially resolved on Am-1b
−
and k2 [=(t2 − t0)/t0], were calculated to be 1.15 and 1.35, respectively, resulting in a separation factor
α (=k2/k1) of 1.17.
Table 1. Resolutions of racemates on Am-1- and ATPC-based CSPs.
Am-1
ATPC
Racemates
k1
α
k1
α
11a
11b
11c
1.15 (R)
0.29
0.14
1.17
1.0
1.0
1.40 (R)
0.95 (R)
0.39
ca. 1
1.22
1.0
Column: 25 cm
the absolute configurations of the first-eluted enantiomers.
×
0.20 cm (i.d.). Eluent: ethanol. Flow rate: 0.05 mL
·
min−1. The signs in parentheses represent
The Am-1b-based CSP displayed a clear resolving ability only toward 11a among three racemates
–11c, whereas ATPC resolved only 11b. The different resolution abilities of these CSPs imply
11a
that the terthienyl-based pendant of Am-1b provides not only a fluorescent functionality but also a
totally different recognition site from that of the original ATPC. In the resolution of rac-11a on the
Am-1b-based CSP, the (R)-isomer was eluted first followed by the (S)-isomer, meaning that Am-1b
interacts more favorably with the (S)-isomer. Combined with the finding that the Am-1b-based
CSP could not resolve 11b, the enantioselectivity of the Am-1b-based CSP is in agreement with the
fluorescence sensing results. The fact that Am-1b did not show clear recognition ability toward 11c
suggests that the nitro group of 11a probably acts as a valuable interaction site with Am-1b.
3. Materials and Methods
3.1. General Information
Anhydrous solvents, such as THF, dichloromethane, pyridine and N,N-dimethylacetamide
(DMA), as well as lithium chloride were purchased from Kanto Kagaku (Tokyo, Japan).
Amylose (DP~300) was kindly supplied by Daicel Chemical Industries (Tokyo, Japan). 4-Iodophenyl
isocyanate (
-Cyclodextrin (
(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) and N,N-dimethyl-4-aminopyridine
(DMAP), diisopropylamine (DIPA) and were purchased from Wako Pure Chemical Industries
(Osaka, Japan). Triethylamine was obtained from Kishida (Osaka, Japan). All starting materials for
the synthesis of aromatic nitro compounds and their analogues ( 11) were purchased from Wako
Pure Chemical Industries, Tokyo Kasei Kogyo (TCI) (Tokyo, Japan) and Nacalai (Kyoto, Japan).
Aromatic nitro compounds and their analogues ( 11a and 11b) were prepared according to
2) and copper (I) iodide (CuI) were from Sigma-Aldrich (St. Louis, MO, USA).
β
4), trans-dichlorobis(triphenylphosphine)palladium(II) (Pd(PPh3)2Cl2), 1-ethyl-3-
5
6
–
7–9,
literature procedures [13]. The porous spherical silica gel with a mean particle size of 5 µm and
a mean pore diameter of 100 nm (Daiso gel SP-1000-5) was kindly supplied from OSAKA SODA
(Osaka, Japan). Chiralpak IA (25 cm
×
0.46 cm (i.d.)) was purchased from Daicel (Tokyo, Japan).

Molecules 2016, 21, 1518
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ATPC [22] and a terthiophene compound bearing an ethynyl group (
3
) [13] were prepared according
to literature procedures.
3.2. Synthesis
Aromatic nitro compounds
6, 10 and 11c were synthesized thorough common condensation
reactions using the corresponding amine or alcohol compounds as starting materials.
(
−
)-N-(4-Nitrobenzoyl)-D-phenylglycine methyl ester ((R)- ). D-Phenylglycine methyl ester hydrochloride
6
(0.40 g, 2.0 mmol) and triethylamine (0.84 mL, 6.0 mmol) were dissolved in anhydrous THF (5 mL) and
the solution was cooled to 0 ◦C. To this solution was added 4-nitrobenzoyl chloride (0.37 g, 2.0 mmol
dissolved in THF (5 mL) and the mixture was stirred at r.t. for 2 h. The mixture was diluted with THF,
washed with brine and then dried over Na2SO4. The solvent was removed under reduced pressure,
)
followed by recrystallization from a hexane–ethyl acetate mixture to give the desired product as a
25
white solid (0.48 g, 76% yield). m.p.: 189.1–189.4 ◦C. [
DMSO-d6, r.t.):
α
]
−
6.8 (c 1.0, DMSO). 1H-NMR (500 MHz,
D
δ 9.58 (d, J = 6.9 Hz, 1H, NH), 8.32 (d, J = 8.6 Hz, 2H, ArH), 8.14 (d, J = 9.0 Hz, 2H,
ArH), 7.49 (d, J = 7.0 Hz, 2H, ArH), 7.44–7.35 (m, 3H, ArH), 5.69 (d, J = 6.9 Hz, 1H, CH), 3.67 (s, 3H,
OCH3). 13C-NMR (125 MHz, DMSO-d6, r.t.):
δ 170.83, 165.07, 149.25, 139.13, 135.77, 129.35, 128.66,
128.41, 128.33, 123.47, 57.14, 52.42. IR (KBr, cm−1): 3322 (N-H), 1746 (C=O). Calcd for C16H14N2O5: C,
61.14; H, 4.49; N, 8.91. Found: C, 61.03; H, 4.38; N, 8.93.
(+)-N-(4-Nitrobenzoyl)-L-phenylglycine methyl ester ((S)-
6
). The title compound was prepared from
and obtained in 56% yield as
+7.0 (c 1.0, DMSO). H-NMR (500 MHz, DMSO-d6, r.t.):
L-phenylglycine methyl ester hydrochloride in the same way as (R)-
6
◦
25
1
a white solid. m.p.: 189.0–189.3 C. [α]
D
δ
9.58 (d, J = 6.9 Hz, 1H, NH), 8.32 (d, J = 9.2 Hz, 2H, ArH), 8.14 (d, J = 9.2 Hz, 2H, ArH), 7.49 (d,
J = 6.5 Hz, 2H, ArH), 7.44–7.35 (m, 3H, ArH), 5.69 (d, J = 6.9 Hz, 1H, CH), 3.67 (s, 3H, OCH3). 13C-NMR
(125 MHz, DMSO-d6, r.t.):
δ 170.81, 165.05, 149.23, 139.11, 135.75, 129.33, 128.65, 128.40, 128.32, 123.46,
57.13, 52.41. IR (KBr, cm−1): 3321 (N-H), 1746 (C=O). Calcd for C16H14N2O5: C, 61.14; H, 4.49; N, 8.91.
Found: C, 61.06; H, 4.47; N, 8.89.
(+)-N-(4-Nitrobenzoyl)-D-lysine methyl ester ((R)-10). The title compound was prepared from D-lysine
methyl ester hydrochloride in the same way as (R)-
6 and obtained in 20% yield as a white solid. Mp:
201.6–201.9 ◦C. [ 25 + 0.9 (c 1.0, DMSO). 1H-NMR (500 MHz, DMSO-d6, 80 ◦C):
α
]
δ
8.83 (d, J = 6.9 Hz
,
,
,
,
D
1H, NH), 8.55 (s, 1H, NH), 8.27 (d, J = 8.5 Hz, 2H, ArH), 8.25 (d, J = 8.5 Hz, 2H, ArH), 8.08 (d, J = 9 Hz
2H, ArH), 8.03 (d, J = 8.5 Hz, 2H, ArH), 4.54–4.48 (m, 1H, CH), 3.67 (s, 3H, OCH3), 3.32 (q, J = 6.5 Hz
2H, CH2), 1.96–1.81 (m, 2H, CH2), 1.68–1.56 (m, 2H, CH2), 1.54–1.42 (m, 2H, CH2). 13C-NMR (125 MHz
DMSO-d6, r.t.):
δ 172.46, 165.06, 164.46, 149.13, 148.86, 140.23, 139.24, 128.98, 128.62, 123.50, 123.45, 52.77,
51.99, 39.10, 30.03, 28.39, 23.10. IR (KBr, cm−1): 3305 (N-H), 1731 (C=O). Calcd for C21H22N4O8
C, 54.38; H, 4.91; N, 12.08. Found: C, 54.22; H, 4.76; N, 11.97.
·
0.3H2O:
(–)-N-(4-Nitrobenzoyl)-L-lysine methyl ester ((S)-10). The title compound was prepared from L-lysine
methyl ester hydrochloride in the same way as (R)-
6
and obtained in 21% yield as a white solid. m.p.:
8.83 (d, J = 7.4 Hz
25
201.5–201.8 ◦C. [
α
]
−
1.2 (c 1.0, DMSO). 1H-NMR (500 MHz, DMSO-d6, 80 ◦C):
δ
,
D
1H, NH), 8.55 (s, 1H, NH), 8.27 (d, J = 9.5 Hz, 2H, ArH), 8.25 (d, J = 9 Hz, 2H, ArH), 8.08 (d, J = 9 Hz, 2H,
ArH), 8.03 (d, J = 8.5 Hz, 2H, ArH), 4.54–4.48 (m, 1H, CH), 3.67 (s, 3H, OCH3), 3.32 (q, J = 6.5 Hz, 2H,
CH2), 1.96–1.81 (m, 2H, CH2), 1.68–1.56 (m, 2H, CH2), 1.53–1.41 (m, 2H, CH2). 13C-NMR (125 MHz
,
DMSO-d6, r.t.):
δ 172.48, 165.09, 164.49, 149.15, 148.88, 140.24, 139.26, 129.00, 128.64, 123.52, 123.47, 52.79,
52.01, 39.17, 30.04, 28.41, 23.11. IR (KBr, cm−1): 3305 (N-H), 1730 (C=O). Calcd for C21H22N4O8
C, 54.59; H, 4.89; N, 12.13. Found: C, 54.53; H, 4.75; N, 12.05.
·
0.2H2O:
(R)-(+)-1,10-Binaphthyl-2,20-diyl bis(2-nitrobenzoate) ((R)-11c). (R)-1,10-Bi-2-naphthol (0.50 g, 1.8 mmol),
2-nitrobenzoic acid (0.87 g, 5.2 mmol), DMAP (0.64 g, 5.2 mmol) were dissolved in anhydrous
dichloromethane (9 mL) and the solution was cooled to 0 ◦C. To this solution was added EDC-HCl
(1.0 g, 5.2 mmol) and the mixture was stirred at r.t. for 12 h. The mixture was diluted with

Molecules 2016, 21, 1518
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dichloromethane, washed with 1 N HCl aqueous solution and water and then dried over Na2SO4.
The solvent was removed under reduced pressure and the crude product was purified by silica gel
chromatography using hexane–dichloromethane (1/4, v/v) as the eluent to give the desired product
25
1
as a pale yellow solid (0.81 g, 79% yield). m.p.: 169.4–169.7 ◦C. [
400 MHz, CDCl3, r.t.):
1.5 Hz, 2H, ArH), 7.77 (d, J = 10.5 Hz, 2H, ArH), 7.54–7.29 (m, 10H, ArH), 6.76 (dd, J = 9.0, 2.5 Hz, 2H,
α
]
+ 9.3 (c 1.0, CHCl3). H-NMR
D
(
δ 8.11 (d, J = 11.0 Hz, 2H, ArH), 7.98 (d, J = 10.5 Hz, 2H, ArH), 7.91 (dd, J = 10.0,
ArH). 13C-NMR (125 MHz, CDCl3, r.t.):
δ 164.36, 146.75, 146.54, 133.65, 133.35, 131.92, 131.42, 130.23,
129.24, 128.23, 127.84, 127.29, 126.31, 126.21, 124.02, 123.50, 121.36. IR (KBr, cm−1): 1752 (C=O). Calcd
for C34H20N2O8: C, 69.86; H, 3.45; N, 4.79. Found: C, 69.96; H, 3.51; N, 4.75.
(S)-(
−
)-1,10-Binaphthyl-2,20-diyl bis(2-nitrobenzoate) ((S)-11c). The title compound was prepared from
0
(S)-1,1 -bi-2-naphthol in the same way as (R)-11c and obtained in 84% yield as a pale yellow solid. m.p.:
169.2–169.5 ◦C. [
α
]
−
9.4 (c 1.0, CHCl3). H-NMR (500 MHz, CDCl3, r.t.): δ 8.11 (d, J = 9.5 Hz, 2H,
25
1
D
ArH), 7.98 (d, J = 8.0 Hz, 2H, ArH), 7.91 (d, J = 8.0 Hz, 2H, ArH), 7.77 (d, J = 8.5 Hz, 2H, ArH), 7.53–7.30
(m, 10H, ArH), 6.76 (d, J = 7.5 Hz, 2H, ArH). 13C-NMR (125 MHz, CDCl3, r.t.):
δ
164.34, 146.75, 146.53,
133.63, 133.34, 131.91, 131.41, 130.23, 129.23, 128.23, 127.82, 127.28, 126.31, 126.20, 124.00, 123.49, 121.35.
IR (KBr, cm−1): 1747 (C=O). Calcd for C34H20N2O8: C, 69.86; H, 3.45; N, 4.79. Found: C, 70.00; H, 3.48;
N, 4.75.
Synthesis of Am-1a: Amylose (0.40 g, 2.5 mmol) was first dissolved in a mixture of DMA (12 mL),
lithium chloride (0.80 g) and pyridine (6.0 mL). Then, 2 (1.80 g, 7.35 mmol) was added, and the mixture
was stirred for 24 h at 80 ◦C. After the reaction, the target amylose derivative Am-1a (1.10 g, 50%)
1
was isolated as the methanol-insoluble fraction. H-NMR (400 MHz, pyridine-d5, 80 ◦C):
δ 10.70–9.80
(br, 3H, NH), 7.90–7.00 (br, 12H, ArH), 6.00–4.10 (br, 7H, glucose protons). IR (KBr, cm−1): 3386 (N-H),
1718 (C=O). Calcd for C27H22I3N3O8
N, 4.63.
·
0.2H2O: C, 36.00; H, 2.51; N, 4.66. Found: C, 35.72; H, 2.60;
Synthesis of Am-1b: To a solution of Am-1a (0.10 g, 0.11 mmol) and
degassed THF/DIPA (5/1, v/v) (3 mL) were added Pd(PPh3)2Cl2 (23 mg, 33
3
(0.25 g, 0.50 mmol) in
mol) and CuI (19 mg
µ
,
0.10 mmol), and then the mixture was heated to 60 ◦C. After 12 h, the reaction mixture was diluted
with dichloromethane and passed through Celite to remove the metal catalyst. The solution was
concentrated and poured into a large amount of ethanol, and the resulting polymer was collected by
centrifugation and washed with ethanol for 12 h and then with hexane for 48 h in a Soxhlet apparatus
to remove any by-products. After the polymer was collected in THF, the solution was concentrated
and poured into methanol. The precipitate was collected by centrifugation and dried in vacuo to give
1
Am-1b as an brown solid (0.16 g, 72%). H-NMR (500 MHz, CDCl3, 55 ◦C):
δ
10.0–9.0 (br, 3H, NH),
8.15–6.00 (br, 30H, ArH), 6.20–3.25 (br, 7H, glucose protons), 2.83 (br, 6H, CH2), 1.60 (br, 3H, CH),
1.65–1.05 (br, 72H, CH2), 0.95–0.70 (br, 18H, CH3). IR (KBr, cm−1): 3398 (N-H), 2199 (C
C), 1719 (C=O).
Calcd for C117H139N3O8S9·0.5H2O: C, 69.81; H, 7.01; N, 2.09. Found: C, 69.53; H, 6.86; N, 2.05.
≡
Synthesis of CyD-1a: The title compound was prepared from
4
in the same way as Am-1a and obtained
9.46 (br, 7H, NH), 9.33
1
in 52% yield as a pale white solid. H-NMR (500 MHz, DMSO-d6, 80 ◦C):
δ
(br, 7H, NH), 7.96 (br, 7H, NH), 7.53 (d, J = 8.5 Hz, 14H, ArH), 7.31 (d, J = 9.0 Hz, 14H, ArH), 7.27 (d,
J = 8.5 Hz, 14H, ArH), 7.20 (d, J = 8.5 Hz, 14H, ArH), 7.01 (d, J = 8.5 Hz, 14H, ArH), 6.75 (d, J = 8.5 Hz
,
14H, ArH), 5.48 (t, J = 9.5 Hz, 7H, CH), 5.33 (br, 7H, CH), 4.83 (d, J = 13.0 Hz, 7H, CH), 4.70–4.55
(m, 14H, CH), 4.30 (d, J = 9.0 Hz, 7H, CH), 4.05 (t, J = 9.5 Hz, 7H, CH). IR (KBr, cm−1): 3394 (N-H),
1738 (C=O).
Synthesis of CyD-1b: The title compound was prepared from CyD-1a in the same way as Am-1b and
1
◦
obtained in 72% yield as a yellow solid. H-NMR (500 MHz, CDCl3, 55 C):
δ 8.00–6.35 (m, 231H,
ArH, NH), 5.60 (br, 7H, CH), 5.31 (br, 7H, CH), 5.10 (br, 7H, CH), 4.85 (br, 7H, CH), 4.64 (br, 7H, CH),
4.49 (br, 7H, CH), 3.93 (br, 7H, CH), 2.75–2.55 (m, 42H, CH2), 1.61 (br, 21H, CH), 1.40–1.10 (br, 504H,

Molecules 2016, 21, 1518
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CH2), 0.95–0.83 (br, 126H, CH3). IR (KBr, cm−1): 3394 (N-H), 2200 (C
C819H973N21O56S63: C, 70.13; H, 6.99; N, 2.10. Found: C, 69.84; H, 7.04; N, 1.87.
≡
C), 1744 (C=O). Calcd for
3.3. Sample Preparation for Fluorescence Measurements
All solutions were prepared using volumetric syringes and flasks. The stock solutions of
the fluorophores and quenchers were freshly prepared and used for each measurement. For the
fluorescence quenching study, a fluorophore solution (10−4 M) in THF or hexane/THF was mixed
with a THF solution of the quencher in a 5 mL volumetric flask and diluted with THF and hexane to
the desired concentration. The resulting solutions were allowed to stand at 25 ◦C for 1 h before the
fluorescence measurement to allow the interactions to reach equilibrium.
3.4. Preparation of HPLC Columns
The chiral packing materials were prepared by coating Am-1b on macroporous silica gel according
to a method reported previously [24,25], followed by evaporation of the solvent under reduced pressure.
The weight ratio of amylose derivative/silica gel was approximately 1:4, which was estimated by
thermogravimetric analysis (TGA). After fractionating with sieves, the obtained silica gel was packed
into a stainless steel column (25 cm
×
0.20 cm (i.d.)) by a slurry packing technique using a Chemco
ECONO-PACKER MODEL CPP-085 (Chemco, Osaka, Japan). The number of theoretical plates per
column was estimated to be approximately 3000 for benzene using ethanol as the eluent at a flow
rate of 0.05 mL
min−1. 1,3,5-Tri-t-butylbenzene was used as a non-retained compound to estimate the
·
hold-up time (t0) [26].
3.5. Instruments
NMR spectra were taken on a JNA-LA 400 (JEOL, Tokyo, Japan) (400 MHz for 1H) or a JNM-ECA
500 (JEOL) (500 MHz for 1H, 125 MHz for 13C) spectrometer in CDCl3, pyridine-d5 and DMSO-d6 using
tetramethylsilane or a solvent residual peak as the internal standard. Melting points were measured
on a Yanako melting point apparatus and were uncorrected. TGA was conducted with a TG/DTA6200
(SII NanoTechnology, Chiba, Japan) at a heating rate of 10 ◦C min−1 under a nitrogen flow. IR spectra
·
were obtained using a JASCO (Hachioji, Japan) Fourier Transform IR-460 spectrophotometer with
◦
a KBr pellet. Absorption and CD spectra were measured at 25 C in a 10 mm quartz cell using a
JASCO V-570 and a JASCO J-725 spectrometers, respectively. The temperature was controlled using a
JASCO ETC-505T (absorption spectroscopy) and a JASCO PTC-348WI apparatus (CD spectroscopy).
DLS measurements were performed on a Nano partica SZ-100 (Horiba, Kyoto, Japan) equipped with
◦
a 10 mW a diode pumped solid state laser (532 nm) at 25 C. Fluorescence emission spectra were
measured with a JASCO FP-6300. The fluorescence quantum yield was estimated using quinine sulfate
in ethanol as a standard material. Chromatographic separations of enantiomers were performed using
a JASCO PU-2080 Intelligent HPLC pump equipped with a JASCO MD-2018 and a CD detector (JASCO
CD-2095) at ca. 20 ◦C. A solution of a chiral compound was injected into the chromatographic system
by using a Rheodyne Model 7125 injector (Rheodyne, Rohnert Park, CA, USA). Elemental analyses
were performed by the Research Institute for Instrumental Analysis of Advanced Science Research
Center, Kanazawa University, Kanazawa, Japan.
4. Conclusions
We synthesized fluorescent phenylcarbamate derivatives of amylose (Am-1b) and
β-cyclodextrin
(CyD-1b) containing terthienyl-based -conjugated pendant groups. The chiral recognition abilities
π
of Am-1b and CyD-1b were investigated by measuring their enantioselective fluorescence responses
toward chiral aromatic nitro compounds. Am-1b and CyD-1b exhibited characteristic enantioselectivity
for various types of aromatic nitro compounds containing either central or axial chirality. Because both
fluorophores consist of the same
α-D-glucose-based repeating unit, we can conclude that the chiral
recognition abilities of Am-1b and CyD-1b are mainly derived from their characteristic backbone

Molecules 2016, 21, 1518
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structures, that is, the one-handed helical structure and cyclic structure, respectively, rather than the
chirality of the monomeric units. Although the chiral recognition ability of the present sensors is not
adequate, we believe that an appropriate structural design will provide more potent saccharide-based
chiral fluorescent sensors. Systematic structural modifications are currently in progress and will be
reported in due course.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
11/1518/s1.
Acknowledgments: This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI
Grants-in-Aid for Scientific Research (C), Grant No. 26410129.
Author Contributions: T.I. conceived the project, designed the experiments and wrote the manuscript. C.Y.
principally performed the experiments. Y.K., and D.S. performed experiments on enantioseparations. K.M., and
S.K. performed the data analysis. All authors discussed the results and edited the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References and Note
1.
2.
3.
4.
5.
6.
7.
8.
9.
Ariëns, E.J. Stereochemistry: A source of problems in medicinal chemistry. Med. Res. Rev. 1986, 6, 451–466.
[CrossRef] [PubMed]
Kasprzyk-Hordern, B. Pharmacologically active compounds in the environment and their chirality.
Chem. Soc. Rev. 2010, 39, 4466–4503. [CrossRef] [PubMed]
Brooks, W.H.; Guida, W.C.; Daniel, K.G. The Significance of chirality in drug design and development.
Curr. Top. Med. Chem. 2011, 11, 760–770. [CrossRef] [PubMed]
Zhou, Q.; Yu, L.-S.; Zeng, S. Stereoselectivity of chiral drug transport: A focus on enantiomer–transporter
interaction. Drug Metab. Rev. 2014, 46, 283–290. [CrossRef] [PubMed]
Kotake, M.; Sakan, T.; Nakamura, N.; Senoh, S. Resolution into optical isomers of some amino acids by paper
chromatography. J. Am. Chem. Soc. 1951, 73, 2973–2974. [CrossRef]
Hess, H.; Burger, G.; Musso, H. Complete enantiomer separation by chromatography on potato starch.
Angew. Chem. Int. Ed. Engl. 1978, 17, 612–614. [CrossRef]
Okamoto, Y.; Yashima, E. Polysaccharide derivatives for chromatographic separation of enantiomers.
Angew. Chem. Int. Ed. 1998, 37, 1021–1043. [CrossRef]
Yashima, E. Polysaccharide-based chiral stationary phases for high-performance liquid chromatographic
enantioseparation. J. Chromatogr. A 2001, 906, 105–125. [CrossRef]
Ikai, T.; Okamoto, Y. Structure control of polysaccharide derivatives for efficient separation of enantiomers
by chromatography. Chem. Rev. 2009, 109, 6077–6101. [CrossRef] [PubMed]
10. Shen, J.; Okamoto, Y. Efficient separation of enantiomers using stereoregular chiral polymers. Chem. Rev.
2016, 116, 1094–1138. [CrossRef] [PubMed]
11. Ikai, T.; Moro, M.; Maeda, K.; Kanoh, S. Synthesis of polysaccharide derivatives bearing pyridine N-oxide
groups and their use as asymmetric organocatalysts. React. Funct. Polym. 2011, 71, 1055–1058. [CrossRef]
12. Ikai, T.; Kimura, K.; Maeda, K.; Kanoh, S. Synthesis of polysaccharide serivatives bearing bromobenzoate
pendants for use as chiral auxiliaries. React. Funct. Polym. 2014, 82, 52–57. [CrossRef]
13. Ikai, T.; Suzuki, D.; Kojima, Y.; Yun, C.; Maeda, K.; Kanoh, S. Chiral fluorescent sensors based on cellulose
derivatives bearing terthienyl pendants. Polym. Chem. 2016, 7, 4793–4801. [CrossRef]
14. Pu, L. Fluorescence of organic molecules in chiral recognition. Chem. Rev. 2004, 104, 1687–1716. [CrossRef]
[PubMed]
15. Zhang, X.; Yin, J.; Yoon, J. Recent advances in development of chiral fluorescent and colorimetric sensors.
Chem. Rev. 2014, 114, 4918–4959. [CrossRef] [PubMed]
16. Huang, Q.; Jiang, L.; Liang, W.; Gui, J.; Xu, D.; Wu, W.; Nakai, Y.; Nishijima, M.; Fukuhara, G.; Mori, T.;
et al. Inherently chiral azonia[6]helicene-modified β-cyclodextrin: Synthesis, characterization, and chirality
sensing of underivatized amino acids in water. J. Org. Chem. 2016, 81, 3430–3434. [CrossRef] [PubMed]
17. Wei, W.; Qu, K.; Ren, J.; Qu, X. Chiral detection using reusable fluorescent amylose-functionalized graphene.
Chem. Sci. 2011, 2, 2050–2056. [CrossRef]

Molecules 2016, 21, 1518
12 of 12
18. Vogt, U.; Zugenmaier, P. Presented at the European Science Foundation Workshop on Specific Interaction in
Polysaccharide Systems; European Science Foundation: Uppsala, Sweden, 1983.
19. Yamamoto, C.; Yashima, E.; Okamoto, Y. Structural analysis of amylose tris(3,5-dimethylphenylcarbamate)
by NMR relevant to its chiral recognition mechanism in HPLC. J. Am. Chem. Soc. 2002, 124, 12583–12589.
[CrossRef] [PubMed]
20. Lakowicz, J.R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: Berlin/Heidelberg, Germany, 2006;
Chapter 8.
21. Aburatani, R.; Okamoto, Y.; Hatada, K. Optical resolving ability of 3,5-dimethylphenylcarbamates of
oligosaccharides and cyclodextrins. Bull. Chem. Soc. Jpn. 1990, 63, 3606–3610. [CrossRef]
22. Okamoto, Y.; Kawashima, M.; Hatada, K. Useful chiral packing materials for high-performance liquid
chromatographic resolution of enantiomers: Phenylcarbamates of polysaccharides coated on silica gel. J. Am.
Chem. Soc. 1984, 106, 5357–5359. [CrossRef]
23. Under the chromatographic conditions used, the interactions between Am-1b and the amino acid derivatives
(6–10) were very weak and their k1 values were estimated to be nearly zero. Therefore, we could not discuss
the resolution ability of Am-1b for 6–10.
24. Okamoto, Y.; Kawashima, M.; Yamamoto, K.; Hatada, K. Useful chiral packing materials for
high-performance liquid chromatographic resolution. Cellulose triacetate and tribenzoate coated on
macroporous silica gel. Chem. Lett. 1984, 13, 739–742. [CrossRef]
25. Okamoto, Y.; Aburatani, R.; Hatada, K. Chromatographic chiral resolution. XIV. Cellulose tribenzoate
derivatives as chiral stationary phases for high-performance liquid chromatography. J. Chromatogr. A 1987
389, 95–102. [CrossRef]
,
26. Koller, H.; Rimböck, K.-H.; Mannschreck, A. High-pressure liquid chromatography on triacetylcellulose:
Characterization of a sorbent for the separation of enantiomers. J. Chromatogr. A 1983, 282, 89–94. [CrossRef]
Sample Availability: Samples are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).