Fluorescent and Circular Dichroic Detection of Monosaccharides by Molecular Sensors: Bis[(Pyrrolyl)ethynyl]naphthyridine and Bis[(Indolyl)ethynyl]naphthyridine

Article abstract of DOI:10.1021/ja039237w

The push-pull conjugated molecules 2,7-bis-(1H-pyrrol-2-yl)ethynyl-1,8-naphthyridine (BPN) and 2,7-bis(1H-indol-2-yl)ethynyl-1,8-naphthyridine (BIN) adopting daad relays of proton donors (d) and acceptors (a) form multiple hydrogen-bonding complexes with various monosaccharides that possess complementary adda sequences. Although the free BPN emits blue light at λ_max = 475 nm in CH₂Cl₂, its complexation with octyl β -D-glucopyranoside gives green fluorescence at λ_max = 535 nm. The excellent photophysical properties make BPN a highly sensitive probe for monitoring glucopyranoside to a detection limit of ～100 pM. On the other hand, the CD-silent BIN molecule binds with monosaccharides to form the CD-active multiple hydrogen-bonding complexes, which exhibit the remarkable chirality dependent helicities consistent with the prediction by the ab initio approaches. On the basis of the similar daad cleft and hence the binding property, the fluorescence and CD absorption methods in BPN and BIN, respectively, are complementary, which, in combination with computational molecular modeling, not only give a detailed insight into the structures of the receptor-saccharide complexes in solution, but also differentiate octyl β-D-glucopyranoside from its enantiomer and other monosaccharides.

Full text of DOI:10.1021/ja039237w

Published on Web 03/02/2004
Fluorescent and Circular Dichroic Detection of
Monosaccharides by Molecular Sensors:
Bis[(Pyrrolyl)ethynyl]naphthyridine and
Bis[(Indolyl)ethynyl]naphthyridine
Jim-Min Fang,*,‡,| Srinivasan Selvi,‡ Jen-Hai Liao,‡ Zdenek Slanina,†,‡
Chao-Tsen Chen,*,‡ and Pi-Tai Chou*,‡
Contribution from the Department of Chemistry, National Taiwan UniVersity,
Taipei, 106, Taiwan and Genomic Research Center, Academia Sinica, Taipei, 115, Taiwan
Received October 26, 2003; E-mail: jmfang@ntu.edu.tw; zdenek@ims.ac.jp
Abstract: The push-pull conjugated molecules 2,7-bis-(1H-pyrrol-2-yl)ethynyl-1,8-naphthyridine (BPN) and
2,7-bis(1H-indol-2-yl)ethynyl-1,8-naphthyridine (BIN) adopting daad relays of proton donors (d) and acceptors
(a) form multiple hydrogen-bonding complexes with various monosaccharides that possess complementary
adda sequences. Although the free BPN emits blue light at λmax ) 475 nm in CH2Cl2, its complexation with
octyl â-D-glucopyranoside gives green fluorescence at λmax ) 535 nm. The excellent photophysical properties
make BPN a highly sensitive probe for monitoring glucopyranoside to a detection limit of ∼100 pM. On the
other hand, the CD-silent BIN molecule binds with monosaccharides to form the CD-active multiple hydrogen-
bonding complexes, which exhibit the remarkable chirality dependent helicities consistent with the prediction
by the ab initio approaches. On the basis of the similar daad cleft and hence the binding property, the
fluorescence and CD absorption methods in BPN and BIN, respectively, are complementary, which, in
combination with computational molecular modeling, not only give a detailed insight into the structures of
the receptor-saccharide complexes in solution, but also differentiate octyl â-D-glucopyranoside from its
enantiomer and other monosaccharides.
Introduction
Sensing of saccharides is essential in a variety of medical,
diagnostic, and therapeutic contexts.1 Expeditious methods that
distinguish D- and L-sugars are particularly crucial because the
present enzymatic protocols2 cannot be exploited to detect
unnatural sugars (e.g., L-glucose). Detection of the nonmetabo-
lizable L-glucose can be applicable to the diagnosis of blood
vessel damage. One approach to make an effective saccharide
binding is to mimic the biotic carbohydrate recognition such as
the protein-saccharide complex that comprises multiple hydrogen-
bonding interactions.3 Sensing of carbohydrates via the covalent
formation of boronic esters relies on a well-defined geometry,4
† Current address of Z. S.: Institute for Molecular Science, Myodaiji,
Okazaki 444-8585, Japan.
Figure 1. (a) Complexation of BPN with alkyl â-D-glucopyranoside via
quadruple hydrogen bondings. (b) Structure of BIN with two indole rings
in lieu of the pyrrole rings in BPN.
‡ National Taiwan University.
| Genomic Research Center.
(1) For reviews, see: (a) Feizi, T.; Mulloy, B. Curr. Opin. Struct. Biol. 2001,
11, 585-586 and succeeding articles. (b) Williams, S. J.; Davies, G. J.
Trends Biotechnol. 2001, 19, 356-362. (c) Roseman, S. J. Biol. Chem.
2001, 276, 41 527-41 542. (d) Dwek, R. A.; Butters, T. D. Chem. ReV.
2002, 102, 283-284 and succeeding articles. (e) Capila, I.; Linhardt, R. J.
Angew. Chem., Int. Ed. 2002, 41, 390-412.
whereas the hydrogen-bonding recognition affords the superior-
ity of the geometrical flexibility in complexation. Herein, we
demonstrate two relatively simple molecular receptors, 2,7-bis-
(lH-pyrrol-2-yl)ethynyl-l,8-naphthyridine (BPN, Figure 1a) and
2,7-bis(1H-indol-2-yl)ethynyl-1,8-naphthyridine (BIN, Figure
1b), both of which possess a well-defined cleft with flexibility
suited to an efficient multiple H-bonds formation with various
guest saccharides.
(2) Bodenner, D. L.; Eastman, R. C. Curr. Opin. Endocrinol. Diabetes 1995,
21, 72-77.
(3) For reviews, see: (a) Aoyama, Y. In ComprehensiVe Supramolecular
Chemistry, Atwood, J. L.; Davies, J. E. D.; MacNicol, D. D.; Vo¨gtle, F.
Eds.; Elsevier: Oxford, 1996, 2, 279-307. (b) Davis, A. P.; Wareham, R.
S. Angew. Chem., Int. Ed. 1999, 38, 2978-2996.
(4) For reviews, see: (a) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S.
Angew. Chem., Int. Ed. 1996, 35, 1911-1922. (b) James, T. D.; Shinkai,
S. In Host-Guest Chemistry: Mimetic Approaches to Study Carbohydrate
Recognition; Penade´s, S., Ed.; Springer-Verlag: Berlin, 2002, 159-200.
Both BPN and BIN possess a naphthyridine core moiety, in
which two pyridinic nitrogen atoms act as the proton acceptors
9
10.1021/ja039237w CCC: $27.50 © 2004 American Chemical Society
J. AM. CHEM. SOC. 2004, 126, 3559-3566
3559

A R T I C L E S
Fang et al.
Scheme 1. Synthesis of BPN (1) and BIN (2)
(aa). Attached to the C2 and C7 positions of naphthyridine are
two identical arms, each of which consists of an ethynyl bridge
terminated by pyrrole (in BPN) or indole (in BIN) moieties that
function as the proton donors (dd). The molecular framework
for both BPN and BIN thus exhibits a unique property among
the hydrogen-bonding type of artificial receptors, in which the
conjugated daad arrays act intrinsically as the saccharide
receptors as well as the sensing chromophores.5 The relay of
hydroxyl groups in monosaccharides offers an adda motif
complementary to the daad array of BPN (or BIN) to form a
quadruple H-bonds complex.
Complexation of saccharides with a receptor is often detected
1
by the H NMR chemical-shift changes,6 though this method
wavelengths may induce a more distinct CD exciton effect upon
complexation with saccharide. We also anticipate that various
saccharides can be differentiated by the corresponding CD
spectra of their BIN complexes. Thus, the integration of UV-
vis, fluorescence and ICD titration methods, along with the
assistance of molecular modeling, may gain detailed insights
into the structures of the receptor-saccharide complexes in
solution.
shows a limited sensitivity. Alternatively, one can use fluores-
cence spectroscopic methods to improve the detection sensitiv-
ity,7 as that shown in sensing octyl D-glucopyranoside by BPN.8
In this case, BPN provides an integral system for noncovalent
recognition and direct visual detection of carbohydrate. From
another approach, the induced circular dichroic (ICD) method
has recently been explored for the saccharide detection by using
electrostatic interactions as the major recognition motif, for
example, in the cyclophane-phosphate,9 polypyridine,10 quino-
line,11 and porphyrin-type receptors12 bearing boronic acid units.
Though BPN and BIN are CD-silent molecules, their bindings
with chiral monosaccharide may come up with the CD-active
complexes. Theoretically, the two pyrrole rings in BPN may
show a CD exciton coupling effect13 to dictate the chirality of
the BPN-saccharide complex. In lieu of the pyrrole rings, BIN
containing the indole chromophores with absorption at longer
Results and Discussion
Synthesis and Characterization of Molecular Sensors BPN
and BIN. The synthesis of BPN (Scheme 1) incorporated
Sonogashira coupling reaction14 of 2,7-dichloro-1,8-naphthyri-
dine (3) with 2 equiv of (1-tert-butoxycarbonyl-2-ethynyl)-
pyrrole (4). The Boc protective group was readily removed by
stirring with MeONa at room temperature. The preparation of
dichloronaphthyridine 3 was initiated by the condensation
reaction of 2,6-diaminopyridine with malic acid according to
the reported procedures.15 Sonogashira coupling reaction of
2-bromopyrrole-1-carboxylic acid tert-butyl ester with (trimeth-
ylsilyl)acetylene, followed by removal of the trimethylsilyl group
with KF, afforded another starting material, ethynylpyrrole 4.
By the similar procedures, (1-tert-butoxycarbonyl-2-ethynyl)-
indole (5), prepared from 1-tert-butoxycarbonyl-2-iodoindole
and (trimethylsilyl)acetylene, was reacted with dichloronaph-
thyridine 3 to give BIN in 61% overall yield after removal of
the Boc group.
The X-ray diffraction analysis of BPN indicated that the BPN
molecule had an ideal V-shaped cleft to provide as many as
four hydrogen-bonding sites, in which the pyrrole and naph-
thyridine moieties function as the proton donors (d) and
acceptors (a), respectively (Figure 2).16 The two pyrrole rings
disposed their NHs as the preorganized inward conformation
with a slight tilt toward opposite directions. The distance
between two pyrrolyl nitrogen atoms was 12.2 Å, and the
dihedral angles between the pyrrole and naphthyridine rings
were 39.4°.
(5) (a) Bell, T. W.; Beckles, D. L.; Cragg, P. J.; Liu, J.; Maioriello, J.; Papoulis,
A. T.; Santora, V. J. In Fluorescent Chemosensors for Ion and Molecular
Recognition; Czarnik, A. W. Ed., Am. Chem. Soc. Washington DC, Chapter
7, 1993, 85-103. (b) Bell, Z. T.; Hou, W.; Luo, Y.; Drew, M. G. B.;
Chapoteau, E.; Czech, B. P.; Kumar, A. Science 1995, 269, 671-674. (c)
Lu¨ning, U.; Ku¨hl, C. Tetrahedron Lett. 1998, 39, 5735-5738. (d) Corbin,
P. S.; Zimmerman, S. C. J. Am. Chem. Soc. 2000, 122, 3779-3780.
(6) (a) Kobayashi, K.; Asakawa, Y.; Kato, Y.; Aoyama, Y. J. Am. Chem. Soc.
1992, 114, 10 307-10 313. (b) Bhattarai, K. M.; Bonar-Law, R. P.; Davis,
A. P.; Murray, B. A. Chem. Commun. 1992, 752-754. (c) Liu, R.; Still,
W. C. Tetrahedron Lett. 1993, 34, 2573-2576. (d) Huang, C.-Y.; Cabell,
L. A.; Anslyn, E. V. J. Am. Chem. Soc. 1994, 116, 2778-2792. (e) Das,
G.; Hamilton, A. D. J. Am. Chem. Soc. 1994, 116, 11 139-11 140. (f)
Jime´nez-Barbero, J.; Junquera, E.; Mart´ın-Pastor, M.; Sharma, S.; Vicent,
C.; Penade´s, S. J. Am. Chem. Soc. 1995, 117, 11 198-11 204. (g) Mizutani,
T.; Kurahashi, K.; Murakami, T.; Matsumi, N.; Ogoshi, H. J. Am. Chem.
Soc. 1997, 119, 8991-9001. (h) Davis, A. P.; Wareham, R. S. Angew.
Chem., Int. Ed. Engl. 1998, 37, 2270-2273. (i) Inouye, M.; Takahashi,
K.; Nakazumi, H. J. Am. Chem. Soc. 1999, 121, 341-345. (j) Ba¨hr, A.;
Felber, B.; Schneider, K.; Diederich, F. HelV. Chim. Acta 2000, 83, 1346-
1376. (k) Bitta, J.; Kubik, S. Org. Lett. 2001, 3, 2637-2640. (l) Benito, J.
M.; Go´mez-Garc´ıa, M.; Blanco, J. L. J.; Mellet, C. O.; Ferna´ndez, J. M.
G. J. Org. Chem. 2001, 66, 1366-1372. (m) Mazik, M.; Radunz, W.;
Sicking, W. Org. Lett. 2002, 4, 4579-4582.
(7) For general reviews, see: (a) Czarnik, A. W. Acc. Chem. Res. 1994, 27,
302-308. (b) James, T. D.; Linnane, P.; Shinkai, S. Chem. Commun. 1996,
281-288. (c) Fluorescence Spectroscopy, Imaging and Probes: New Tools
in Chemical, Physical and Life Sciences; Kraayenhof, R.; Visser, A. J. W.
G.; Gerritscen, H. C.; Eds.; Springer-Verlag: Berlin, 2002.
NMR Study of the Binding Modes of BPN with Octyl â-D-
Glucopyranoside. When a stock solution of BPN in CDCl3
(2 × 10-4 M) was treated with 1 equiv of octyl â-D-
glucopyranoside (as 0.02 M CDCl3 solution), the NHs on the
pyrrole rings showed a large chemical-shift change (∆δ ) 1.4
ppm). The glucoside counterpart also showed significant chemi-
(8) Liao, J.-H.; Chen, C.-T.; Chou, H.-C.; Cheng, C.-C.; Chou, P.-T.; Fang,
J.-M.; Slanina, Z.; Chow, T. J. Org. Lett. 2002, 4, 3107-3110.
(9) (a) Kikuchi, Y.; Kobayashi, K.; Aoyama, Y. J. Am. Chem. Soc. 1992, 114,
1351-1358. (b) Droz, A. S.; Neidlein, U.; Anderson, S.; Seiler, P.;
Diederich, F. HelV. Chim. Acta 2001, 84, 2243-2289.
(10) (a) Tamaru, S.-i.; Yamamoto, M.; Shinkai, S.; Khasanov, A. B.; Bell, T.
W. Chem. Eur. J. 2001, 7, 5270-5276. (b) Tamaru, S.-i.; Shinkai, S.;
Khasanov, A. B.; Bell, T. W. Proc. Natl. Acad. Sci. 2002, 99, 4972-4976.
(11) (a) Mitzutani, T.; Kurahashi, T.; Murakami, T.; Matsumi, N.; Ogoshi, H.
J. Am. Chem. Soc. 1997, 119, 8991-900. (b) Kim, Y.-H.; Hong, J.-I. Angew.
Chem., Int. Ed. 2002, 41, 2947-2950.
(12) (a) Imada, T.; Kijima, H.; Takeuchi, M.; Shinkai, S. Tetrahedron Lett. 1995,
36, 2093-2096. (b) Takeuchi, M.; Imada, T.; Shinkai, S. J. Am. Chem.
Soc. 1996, 118, 10 658-10 659. (c) Sugasaki, A.; Sugiyasu, K.; Ikeda, M.;
Takeuchi, M.; Shinkai, S. J. Am. Chem. Soc. 2001, 123, 10 239-10 244.
(13) Nakanishi, K.; Berova, N.; Woody, R. W. Circular Dichroism: Principles
and Applications; VCH: New York, 1994.
(14) For a recent review, see: Tykwinski, R. R. Angew. Chem., Int. Ed. 2003,
42, 1566-1568.
(15) (a) Newkome, G. R.; Garbis, S. J.; Majestic, V. K.; Fronczek, F. R.; Chiari,
G. J. Org. Chem. 1981, 46, 833-839. (b) Ziessel, R.; Suffert, J.; Youinou,
M.-T. J. Org. Chem. 1996, 61, 6535-6546.
(16) The crystallographic data of BPN have been deposited with the Cambridge
Crystallographic Data Centre as no. CCDC 176324.
9
3560 J. AM. CHEM. SOC. VOL. 126, NO. 11, 2004

Detection of Monosaccharides by Molecular Sensors
A R T I C L E S
Fluorescence Titration of the BPN and BIN Complexes
with Octyl â-D-Glucopyranoside. Dual spectral features were
observed in the fluorescence titration study of BPN with octyl
â-D-glucopyranoside. The characteristic uncomplexed BPN
emission band at 475 nm (τf ≈ 1.25 ns) decreased, accompanied
by the growth of a 535-nm (τf ≈ 0.95 ns) emission band. The
results in combination with different excitation spectra between
monitoring at e.g., 450 and 550 nm led us to conclude that dual
fluorescence originates from different ground-state precursors,
namely the uncomplexed BPN and BPN-saccharide complex,
respectively (Figure 6). The free BPN emits a sky blue light,
whereas its complexation with octyl â-D-glucopyranoside has
a large Stokes' shift (60 nm) to emit a grass green light. The
plot of [F0/(F - F0)] at 535 nm versus the inverse of saccharide
concentrations reconfirmed a 1:1 BPN-saccharide complex.18
The fluorescence yield (i.e., the 535-nm band) for the complex
of BPN and octyl â-D-glucopyranoside was measured to be as
high as 0.25 in CH2Cl2. By selecting the excitation wavelength
at > 460 nm where the absorbance was solely attributed to the
BPN-saccharide complex, a detection limit as low as ∼100
pM of octyl â-D-glucopyranoside could be achieved based on
the nearly background (i.e., the uncomplexed BPN) free 535-
nm emission. The ratiometric fluorescence proved to be a
reliable and ultrasensitive method for the real time detection of
glucopyranoside.
Figure 2. ORTEP drawing of BPN shows a V-shaped cleft.
cal-shift changes of hydroxyl protons (Figure 3). The 1:1
complexation of BPN with octyl â-D-glucopyranoside was
supported by a continuous variation method (Job plot),17 in
which the chemical-shift changes of pyrrole-NH as a function
of saccharide concentrations were monitored. The binding
constant of the BPN-saccharide complex, Ka ) 19 800 ( 810
in CDCl3 solution at 300 K, was determined by nonlinear
regression analyses.17 The temperature dependent (285-315 K)
1H NMR study indicated that the complex formation of BPN
with octyl â-D-glucopyranoside was thermodynamically favored
(∆G300 ) -24.7 kJ/mol), in agreement with the proposed
quadruple hydrogen-bonds formation (Figure 1a). A linear
relationship was obtained by plotting lnKa as a function of 1/T
according to the equation: lnKa ) -∆H/RT + ∆S/R. From the
slope and intercept of the straight line, ∆H and ∆S were
determined to be -51.4 kJ/mol and -91.1 J/mol, respectively.
UV-Vis Spectra of the BPN and BIN Complexes with
Octyl â-D-Glucopyranoside. Upon adding octyl â-D-glucopy-
ranoside, the 410-nm free BPN band (ꢀ ) 44 700) in CH2Cl2
solution decreased significantly along with the growth of a new
absorption band at 435 nm (Figure 4). An isosbestic point at
415 nm throughout the titration was attributed to the formation
of BPN-saccharide hydrogen-bonding complexes in equilibrium
with free BPN. The measured absorbance [A0/(A - A0)] as a
function of the inverse of the concentrations of octyl â-D-
glucopyranoside fit a linear relationship, indicating the 1:1
stoichiometry of BPN-saccharide complex.18
The UV-vis spectrum of BIN in CH2Cl2 showed the
absorption maxima at 319 nm (ꢀ ) 47 700) and 413 nm (ꢀ )
71 280) attributable to the indole and naphthyridine chro-
mophores, respectively. As the amounts of octyl â-D-glucopy-
ranoside increased, new absorption maxima at 325 and 440 nm
appeared with a gradual increase of the absorbance, whereas
the absorptions at 319 and 413 nm for the free BIN receptor
decreased concomitantly (Figure 5). The occurrence of an
isosbestic point at 426 nm throughout the titration supported
the formation of the BIN-saccharide complex in equilibrium
with free BIN.
The intrinsic conjugated daad type chromophore in BPN
molecule is of key importance to account for the remarkable
spectral differences. The acidity of pyrrole and basicity of
naphthyridine could be enhanced at excited-state through the
conjugated dual hydrogen-bonding effect.18 Upon complexation,
the unusual push-pull daad relay would form multiple hydrogen-
bonds with saccharide and induce the n,π-electrons delocaliza-
tion along the ethynyl bridge to account for a drastic alternation
on the fluorescent property (Figure 7a).19 Furthermore, rigidi-
fication upon formation of the BPN-saccharide complex might
also contribute a certain role in enhancing the fluorescence as
that shown in the previous studies on the bindings of ions20
and carbohydrates.19
For a comparison, the nonconjugated daad molecule of 2,7-
bis(3-hydroxy-3-methylbutynyl)-1,8-naphthyridine (BHN) was
prepared (Figure 7b). The crystal structure of BHN also exhibits
a cleft similar to BPN. The distance between the two oxygen
atoms of BHN is 12.0 Å, close to that between the two pyrrolyl
nitrogen atoms in BPN (12.2 Å). Complexation of BHN with
octyl â-D-glucopyranoside in CDCl3 solution was relatively
weak (Ka ≈ 1000 M-1 at 300 K) as derived from the 1H NMR
titrations, in comparison with that of BPN complex (Ka ) 19 800
M-1). The fluorescence intensity (λmax ) 387 nm in CH2Cl2)
did increase upon complexation, albeit no significant spectral
shift was observed. The enhanced fluorescence could be
rationalized by the conformational rigidification, whereas the
lack of a continuous push-pull π-system prohibited the multiple
hydrogen-bonds induced charge-transfer effect.
(19) (a) Sandanayaka, K. R. A. S.; Nakashima, K.; Shinkai, S. Chem. Commun.
1994, 1621-1622. (b) Takeuchi, M.; Yoda, S.; Imada, T.; Shinkai, S.
Tetrahedron 1997, 53, 8335-8348.
(17) Connors, K. A. Binding Constants; Wiley: New York, 1987.
(18) Chou, P. T.; Wu, G. R.; Wei, C. Y.; Cheng, C. C.; Chang, C. P.; Hung, F.
T. J. Phys. Chem. B 2000, 104, 7818-7829. A0, F0, A, and F denote
respectively the absorbance and fluorescent intensity of free BPN, and
solution after adding monosaccharides at a selective wavelength.
(20) (a) McFarland, S. A.; Finney, N. S. J. Am. Chem. Soc. 2001, 123, 1260-
1261. (b) Mello, J. V.; Finney, N. S. Angew. Chem., Int. Ed. 2001, 40,
1536-1538. (c) Choi, K.; Hamilton, A. D. Angew. Chem., Int. Ed. 2001,
40, 3912-3915. (d) McFarland, S. A.; Finney, N. S. J. Am. Chem. Soc.
2002, 124, 1178-1179.
9
J. AM. CHEM. SOC. VOL. 126, NO. 11, 2004 3561

A R T I C L E S
Fang et al.
1
Figure 3. Induced H NMR changes of BPN (5 × 10-4 M in CDCl3, 300 K) on addition of octyl â-D-glucopyranoside: (a) free BPN, (b) BPN with octyl
â-D-glucopyranoside (1 equiv), and (c) free octyl â-D-glucopyranoside (1 × 10-3 M in CDCl3, 300 K). The pyrrole protons of BPN are denoted as N3, N4;
the hydroxyl protons at the C-2, C-3, C-4, and C-6 positions of saccharide are denoted as Ha, Hb, Hc, and Hd, respectively.
Figure 6. Fluorescence spectra of BPN (1.2 × 10-5 M) in CH2Cl2 by
Figure 4. Absorption spectra of BPN (5.4 × 10-6 M) in CH2Cl2 by adding
adding various concentrations (Cg) of octyl â-D-glucopyranoside. The
various concentrations (Cg) of octyl â-D-glucopyranoside. Insert: The plot
at 435 nm shows a linear relationship of [A0/(A - A0)] vs 1/Cg, indicating
the 1:1 stoichiometry of BPN-saccharide.
fluorescence spectra (λex ) 365 nm) show the growth of 535-nm emission
band on complexation, accompanied by decrease of 475-nm band for
uncomplexed BPN. * denotes Raleigh scattering. Insert: The plot at 535
nm shows a linear relationship of [F0/(F - F0)] vs 1/Cg, indicating the 1:1
stoichiometry of BPN-saccharide complex.
â-D-glucopyranoside in different concentrations without obvious
change of wavelength. The 1:1 stoichiometry and the binding
constant (Ka ) 11 700 ( 1280 M-1) for the complexation of
BIN with octyl â-D-glucopyranoside in CH2Cl2 solution were
determined by the fluorescence titration method. Complexation
of BIN with other glycosides also caused certain degrees of
fluorescence quenching with a small red shift (3-5 nm). It
appeared that the stronger the host-guest intermolecular
hydrogen bondings induced a dominant quenching effect.
Comparison of the Binding Strengths of Different Sac-
charide Complexes. The examined alkyl monosaccharides
6-13 are either commercially available or prepared by known
procedures.21 The preferable 1:1 complexation of respective
monosaccharide with BPN or BIN was similarly determined
by absorption, fluorescence and1H NMR titration methods. The
binding constants and free energy changes for various BPN and
Figure 5. UV-vis titration of BIN (5 × 10-6 M) with octyl â-D-glucoside
(5 × 10-3 M) in CH2Cl2 solution.
Upon excitation at 413 nm, BIN showed a fluorescence
emission at λmax ) 485 nm in CH2Cl2 solution. Unlike BPN or
BHN, the fluorescence intensity decreased on binding with octyl
9
3562 J. AM. CHEM. SOC. VOL. 126, NO. 11, 2004

Detection of Monosaccharides by Molecular Sensors
A R T I C L E S
Table 1. Binding Constants and Free Energy Changes for BPN-Saccharide and BIN-Saccharide Complexes
alkyl saccharide
K /M-1 (BPN complex)a
−∆G/kJ mol-1 (BPN complex)b
K /M-1 (BIN complex)c
−∆G/kJ mol-1 (BIN complex)b
a
a
â-D-glucoside 6
R-D-glucoside 8
R-D-mannoside 9
â-D-galactoside 10
â-L-fucoside 11
â-D-fructoside 12
â-D-riboside 13
19 800 ( 810d
12 600 ( 450
25 100 ( 220
6190 ( 330e
1950 ( 60
24.7
23.6
25.3
21.8
18.9
15.5
16.0
11 700 ( 1280
5690 ( 560
13500 ( 680
7940 ( 290
2160 ( 270
1540 ( 100
23.1
21.3
23.4
22.1
18.9
18.1
490 ( 7f
605 ( 30
a Derived by 1H NMR titrations in CDCl3 solutions at 300 K. b -∆G ) RT lnKa. c Derived by fluorescence titrations in CH2Cl2 solutions at 296 K. The
data represent the average values of two independent titrations. d The binding constant in CH2Cl2 solution at 300 K was estimated to be 5500 M-1 by
fluorescence titrations, and 4800 M-1 by UV-vis titrations. e The binding constant in CH2Cl2 solution at 300 K was estimated to be 1600 M-1 by absorption
and fluorescence titrations. f The binding constant in CH2Cl2 solution at 300 K was estimated to be 190 M-1 by absorption and fluorescence titrations.
Figure 7. (a) A large Stokes' shift (60 nm) in the fluorescence spectrum
of BPN-saccharide complex is attributed to the multiple hydrogen-bonds
induced charge-transfer. (b) Complexation of BHN with saccharide does
not show significant spectral shift because BHN lacks a continuous push-
pull π-system to account for charge transfer.
BIN-saccharide complexes are collected in Table 1. The BIN
and BPN showed the similar trend of binding strengths toward
the examined monosaccharides, i.e., R-D-mannopyranoside >
â-D-glucopyranoside > (R-D-glucopyranoside, â-D-galactopy-
ranoside) . (â-L-fucopyranoside, â-D-fructopyranoside, â-D-
ribofuranoside). It is thus reasonable to assume that BIN and
BPN molecules behave similarly to bind with various monosac-
charides.
While the only structural difference between octyl â-D-
galactopyranoside and octyl â-D-glucopyranoside is the spatial
orientation of 4-OH, the binding constant for â-galactoside is
much smaller than that of â-glucoside on complexation with
showed a smaller chemical-shift change than the â-anomer on
complexation with BPN. Among the examined saccharides, octyl
R-D-mannopyranoside exhibited the highest affinity toward BPN
and BIN. The 2-OH group in mannoside was free from the
intramolecular hydrogen bonding with octoxy group, and thus
exerted a strong hydrogen bonding with the pyrrolyl (or indolyl)
NH of BPN (or BIN).10b,22
Computational Molecular Modeling of BPN-Saccharide
Complexes. Computations were carried out at ab initio levels
using selected approximations implemented in the Gaussian
program package.23 The complete geometry optimizations were
performed with four increasingly more advanced methods. Three
of them are ab initio Hatree-Fock (HF) methods, namely with
the minimal STO-3G basis set (HF/STO-3G), with the 3-21G
either BPN or BIN. This binding selectivity is comparable to
those reported in the literature.5,10b For octyl â-L-fucopyranoside
in which one hydrogen bond is eliminated by modifying the
C(6)CH2OH to CH3 group, the binding constants with either
BPN or BIN decrease dramatically. The binding of methyl â-D-
ribofuranoside with BPN is weak presumably because the
complexation can at most accommodate three hydrogen bond-
ings. The binding constant of octyl R-D-glucopyranoside is less
than that of â-anomer. It is assumed that a cis-gauche type
hydrogen bonding occurs between the C-1 octoxy group and
the C-2 hydroxyl group in the R-D-glucopyranoside, and thus
renders a less efficient hydrogen bonding between the 2-OH
group and the pyrrolyl (or indolyl) NH of BPN (or BIN).5m,10b,22
As supportive evidence, the 1H NMR titration experiments
indicated that the 2-OH group of octyl R-D-glucopyranoside
(22) (a) Lo´pez de la Paz, M.; Ellis, G.; Pe´rez, M.; Perkins, J.; Jime´nez-Barbero,
J.; Vicent C. Eur. J. Org. Chem. 2002, 840-855. (b) Baggett, N.; Bukhari,
M. A.; Foster, A. B.; Lehmann, J.; Webber, J. M. J. Chem. Soc. 1963,
4157-4160. (c) Alonso, J. L.; Wilson, E. B. J. Am. Chem. Soc. 1980, 102,
1248-1251.
(23) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M.
A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann,
R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin,
K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi,
R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.;
Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.;
Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz,
J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng,
C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.;
Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.; Head-
Gordon, M.; Replogle, E. S.; Pople, J. A. GAUSSIAN 98, Revision A.7,
Gaussian, Inc., Pittsburgh, PA, 1998.
(21) (a) Nicolaou, K. C.; Hummel, C. W.; Nakada, M.; Shibayama, K.; Pitsinos,
E. N.; Saimoti, H.; Mizuno, Y.; Baldenius, K.-U.; Smith, A. L. J. Am.
Chem. Soc. 1993, 115, 7625-7635. (b) Zhang, X.; Kamiya, T.; Otsubo,
N.; Ishida, H.; Kiso, M. J. Carbohydrate Chem. 1999, 18, 225-239. (c)
Ferrie`res, V.; Bertho, J.; Plusquellec, D. Tetrahedron Lett. 1995, 36, 2749-
2752. (d) Ferrie`res, V.; Benvegnu, T.; Lefeuvre, M.; Plusquellec, D.;
Mackenzie, G.; Watson, M. J.; Haley, J. A.; Goodby, J. W.; Pindak, R.;
Durbin, M. K. J. Chem. Soc., Perkin Trans. 2 1999, 951-959.
9
J. AM. CHEM. SOC. VOL. 126, NO. 11, 2004 3563

A R T I C L E S
Fang et al.
Figure 8. (a) Scheme for the geometrically optimized BPN/methyl â-D-glucopyranoside complex derived by the B3LYP/6-31G** method. The distances
(Å) of the hydrogen bonds are shown. (b) The geometrically optimized BPN/â-D-glucopyranoside complex that shows a P-helicity with a clockwise twist
of the BPN backbone. (c) The geometrically optimized BPN/â-D-galactoside complex with the M-helicity. (d) The geometrically optimized BPN/R-D-
mannoside complex with the P-helicity. Black balls: carbon atoms in BIN; green balls: carbon atoms in saccharide; yellow balls: hydrogen atoms; blue
balls: nitrogen atoms, and red balls: oxygen atoms.
Table 2. Computed Lengths of Hydrogen Bonds (in Å) and the
in gas phase. Moreover, the dimerization energies should be
Dimerization Energies (Edim, in kJ/mol) for the Complex of BPN
somewhat reduced by addition of the vibrational zero-point
with Methyl â-D-Glucopyranoside, Methyl â-D-Galactopyranoside
and Methyl R-D-Mannopyranosidea
energies.
Though BPN is an achiral molecule, it is expected to form
methyl saccharide N(1)−HO(3) N(2)−HO(4) N(3)H−O(2) N(4)H−O(6)
Edim
chiral complexes with chiral saccharides. On the basis of the
B3LYP/6-31G** computation, the complex of BPN with â-D-
glucopyranoside exhibited a P-helicity24 with clockwise twist
of the BPN backbone, i.e., the upward pyrrole on the left-hand
side moving clockwise along the ethynyl-naphyridine-ethynyl
backbone to the downward pyrrole on the right-hand side (Figure
8b). Although â-D-galactopyranoside differed from â-D-glu-
copyranoside simply by the orientation of the 4-OH group, the
molecular model of BPN/â-D-galactoside complex indicated a
counterclockwise twist of backbone (M-helicity, Figure 8c) with
the upward pyrrole on the right-hand side and the downward
pyrrole on the left-hand side. The molecular modeling also
predicted that the complex of BPN with R-D-mannopyranoside
would have a P-helicity (Figure 8d) as that of BPN/â-D-
glucoside complex.
Application of CD Spectroscopy to Distinguish Different
BIN-Saccharide Complexes. We conducted the CD measure-
ments for the complexation of BIN with several octyl monosac-
charides (Figure 9) in order to substantiate the above structural
modeling. The results turned out to be in good agreement with
the prediction by ab initio computations. In lieu of the pyrrole
rings in BPN, the BIN molecule containing two indole rings
would induce a larger CD exciton coupling effect on binding
with chiral saccharides.
Addition of octyl â-D-glucopyranoside to BIN produced a
positive ICD signal at about 442 nm, as well as an exciton
couplet at about 325 nm (intersection point) with the first
negative and second positive Cotton effects at 344 nm ([θ] )
-16 900) and 317 nm ([θ] ) 500), respectively (Table 3). As
the two indole rings could be brought into an inward conforma-
tion upon binding with a chairlike pyranoside, the exciton
coupling of the two indole chromophores (λabs ≈ 325 nm) would
reflect the helicity of the BIN backbone, i.e., a negative Cotton
effect for the P-helicity. On the other hand, the complex of BIN
and octyl â-L-glucopyranoside (the enantiomer of octyl â-D-
â-D-glucoside
â-D-galactoside
R-D-mannoside
1.970
2.098
2.165
1.827
1.840
1.842
2.140
2.141
1.951
1.813
1.826
1.809
-149.9
-146.9
-135.2
a Molecular calculations were conducted with B3LYP/6-31G** method.
basis set (HF/3-21G), and with the 6-31G** basis set (HF/
6-31G**). Moreover, density functional theory was also applied,
namely Becke’s three parameter functional with the nonlocal
Lee-Yang-Parr correlation functional in the standard 6-31G**
basis set (B3LYP/6-31G**), as the approach, in contrast to the
HF treatments, can include correlation energy (with reasonable
computational demands). In evaluations of the dimerization
energies (Edim), the basis set superposition error (BSSE) was
estimated using the approximative counterpoise method.
The molecular modeling of BPN-saccharide complexes
implied the importance of multiple hydrogen-bonds formation
as well as the geometrical fitness to accommodate each hydrogen
bond (Figure 8a). All calculated OH‚‚‚N and NH‚‚‚O distances
are significantly shorter than the sum of van der Waals radii of
H and N (2.7 Å) and H and O (2.6 Å). At the highest level of
computations, B3LYP/6-31G**, the computed lengths of hy-
drogen bonds and the dimerization energies for the complexes
of BPN with methyl â-D-glucopyranoside, methyl â-D-galac-
topyranoside and methyl R-D-mannopyranoside are listed in
Table 2. The most prominent hydrogen bonding occurs between
C-6 oxygen atom and the pyrrolyl NH. This computation result
supports that the binding of octyl â-L-fucopyranoside with BPN
is weak due to a lack of this hydrogen bond. In comparison
with the galactose complex, all the four computed hydrogen-
bond distances are bit shorter and the computed depth of the
energy minimum is slightly deeper in the glucose complex. The
mannoside complex shows somewhat a shallower minimum at
the B3LYP/6-31G** level, even the hydrogen bond between
N(3)H and O(2) is short. The relative stabilities of the three
species could be still influenced by two factors, entropy and
environmental effects (e.g., solvent and temperature), not
considered in our calculations of the potential energy changes
(24) Eliel, E. L.; Wilen, S. H.; Mander, L. N. Stereochemistry of Organic
Compounds; Wiley: New York, 1994; pp 1012 and 1200.
9
3564 J. AM. CHEM. SOC. VOL. 126, NO. 11, 2004

Detection of Monosaccharides by Molecular Sensors
A R T I C L E S
logues of BPN/BIN receptors for direct sensing of sugars
without prior derivatization to alkyl saccharides. We are
currently engaged in this research work.
Experimental Section
General. All reactions requiring anhydrous conditions were con-
ducted in flame-dried apparatus under an inert atmosphere using
standard techniques. Syringes and needles for the transfer of reagents
were dried at 100 °C and allowed to cool in a desiccator over P2O5
before use. All the reagents and solvents were reagent grade and were
used without further purification unless otherwise specified. Ethers were
distilled from sodium benzophenone ketyl; (chlorinated) hydrocarbons,
and amines from CaH2. Reactions were monitored by TLC using
aluminum plates precoated with a 0.25 mm layer of silica gel containing
a fluorescent indicator (Merck Art. 5544). Kieselgel 60 (40-63 µm)
and neutral aluminum oxide (Acros, 50-200 µm) were used for column
chromatography. Melting points are uncorrected. Chemical shifts (δ)
are given in parts per million (ppm) relative to δH 7.24/δC 77.0 (central
line of t) for CHCl3/CDCl3 and δH 2.49 (m)/δC 39.5 (m) for DMSO-
d6. The splitting patterns are reported as s (singlet), d (doublet),
t (triplet), and multiplet (m). Coupling constants (J) are given in Hz.
Distortionless enhancement polarization transfer (DEPT) spectra were
taken to determine the types of carbon signals.
Figure 9. CD spectra of BIN (1 × 10-5 M) and its complexation with
saccharides (2 × 10-2 M) in CH2Cl2 solutions.
Table 3. CD Spectral Data for BIN/Octyl Saccharide Complexes
in CH2Cl2 Solutionsa
b
BIN/saccharide
λ
max/nm ([θ]/deg cm2 dmol-1
)
∆[θ]
â-D-glucoside 6
â-L-glucoside 7
442 (10 100), 344 (-16 900), 317 (500)
442 (-10 100), 344 (17 700), 317 (300)
-17 400
+17 400
R-D-mannoside 9 442 (-10 900), 344 (-17 400), 317 (1500) -18 900
â-D-galactoside 10 442 (-41 300), 344 (30 600), 317 (-9500) +40 100
2,7-Bis-(1H-pyrrol-2-yl)ethynyl-1,8-naphthyridine (BPN, 1). To
a suspension of 2,7-dichloro-1,8-naphthyridine (3, 137 mg, 0.72 mmol)
in anhydrous THF (2 mL) was added triethylamine (1.38 mL),
Pd(PPh3)2Cl2 (20.1 mg) and CuI (11 mg) under argon. After stirring at
room temperature for 10 min, 2-ethynylpyrrole-1-carboxylic acid tert-
butyl ester (4, 276 mg, 1.45 mmol) in anhydrous THF (10 mL) was
added slowly. The resulting red solution was stirred at room temperature
for 40 h. The reaction mixture was passed through a short plug of
diatomaceous earth, and washed with EtOAc. The combined organic
phase was evaporated to dryness under reduced pressure to give pale
brown solid. After washing with hot hexane the crude product was
recrystallized from EtOAc/hexane to give 2,7-bis-(1-tert-butoxycar-
bonylpyrrol-2-yl)ethynyl-1,8-naphthyridine (BPN-Boc, 189 mg). The
filtrate was concentrated to dryness, and followed the previous
procedure to give the second crop (97 mg). Total yield: 78%.
To a solution of BPN-Boc (100 mg, 0.197 mmol) in anhydrous THF
(5 mL) was added MeONa (63.8 mg, 6 equiv) in anhydrous MeOH (5
mL). The resulting mixture was allowed to stir at room temperature
for 1 h. After evaporation to dryness the residue was treated with water
(30 mL) and CH2Cl2 (50 mL). The mixture was neutralized with 1 N
HCl aqueous solution. The lower layer was separated, and the aqueous
layer was extracted with another portion of CH2Cl2 (30 mL). The
combined organic phase was dried (Na2SO4), filtered, and evaporated
to dryness under reduced pressure. The crude product was purified by
column chromatography with elution of 2.5-10% MeOH/CH2Cl2 to
give BPN (45 mg, 74%). Orange crystal, mp > 300 °C (darken above
160 °C); TLC (MeOH/CH2Cl2 (9:1)) Rf ) 0.57; IR (KBr) 3215, 2196,
a The CH2Cl2 solution of BIN (1 × 10-5 M) and saccharide (2 × 10-2
M) was used. b The difference of absorptions at two extremes of the exciton
couplet. Plus value refers to positive exciton coupling, whereas negative
value refers to negative exciton coupling.
glucopyranoside) furnished a reversed ICD band and a positive
exciton couplet with the same amplitudes. These two CD curves
looked like mirror images of each other. Thus, D- and L-
saccharides can be easily distinguished from their BIN com-
plexes by using a CD method. The positive Cotton effect at
λmax ≈ 344 nm accounts for the M-helical structure of BIN/
â-D-galactoside complex, whereas the negative Cotton effect
for the BIN/R-D-mannoside complex indicated its P-helicity that
is also supported by computation.
Conclusion. Our results demonstrate the selective affinity and
ultrasensitivity for probing the BPN/â-glucoside complex. Visual
change of free BPN (cyanic color) in CH2Cl2 to green color
upon addition of octyl glucopyranoside was obvious. Our study
evokes a novel concept of complementary proton donor (d) and
acceptor (a) relays in designing molecular receptors of carbo-
hydrates. The glycopyranoside is considered as an adda substrate
to bind efficiently with the daad receptors BPN and BIN via
multiple hydrogen bondings. This binding mode is supported
by NMR, absorption, and fluorescence spectral analyses. The
CD spectra and molecular computations give a consistent picture
of the three-dimensional structures of complexes in solutions,
which is a problem not easily tackled by X-ray diffraction and
other methods. In combination of the high fluorescence sensitiv-
ity of BPN and the CD exciton effect with BIN complexation,
sensing of â-D-glucopyranoside at low concentration (∼100 pM)
is achieved, and the differentiation of â-D-glucopyranoside from
its enantiomer and other monosaccharides is realized. This study
can be advanced by using the fluorescence detected circular
dichroism (FDCD),25 or by designing the water-soluble ana-
1
1593 cm-1; H NMR (DMSO-d6, 400 MHz) δ 11.86 (2 H, s), 8.43 (2
H, d, J ) 8.2 Hz), 7.66 (2 H, d, J ) 8.2 Hz), 7.00 (2 H, m), 6.67 (2
H, m), 6.19 (2 H, m); 13C NMR (CD3OD, 100 MHz) δ 156.5 (C),
149.0 (2 × C), 138.8 (2 × CH), 125.9 (2 × CH), 123.1 (2 × CH),
122.1 (C), 118.5 (2 × CH), 112.2 (2 × C), 110.4 (2 × CH), 91.6 (2 ×
C), 88.5 (2 × C); FAB-MS m/z (rel intensity) 309 (M+ + 1, 43%);
HRMS calcd for C20H13N4 (M+ + 1) 309.1140, found 309.1141. Anal.
Calcd for C20H12N4: C, 77.91; H, 3.92; N, 18.17. Found: C, 77.60; H,
4.08; N, 18.07.
2,7-Bis-(1H-indole-2-yl)ethynyl-1,8-naphthyridine (BIN, 2). By
a procedure similar to that for BPN-Boc, Sonogashira coupling reaction
of 2,7-dichloronaphthyridine (300 mg, 1.5 mmol) with 2-ethynylindole-
1-carboxylic acid tert-butyl ester (5, 725 mg, 3 mmol) using Pd(PPh3)2-
Cl2, CuI and i-Pr2NH in THF gave 2,7-bis-[1-(tert-butoxycarbonyl)-
indole-2-yl]ethynyl-1,8-naphthyridine (BIN-Boc, 630 mg, 69%). The
Boc group was removed by MeONa in MeOH, by a procedure similar
(25) (a) Dong, J.-G.; Wada, A.; Takakuwa, T.; Berova, N.; Nakanishi, K. J.
Am. Chem. Soc. 1997, 119, 12 024-12 025. (b) Nehira, T.; Parish, C. A.;
Jockusch, S.; Turro, N. J.; Nakanishi, K.; Berova, N. J. Am. Chem. Soc.
1999, 121, 8681-8691.
9
J. AM. CHEM. SOC. VOL. 126, NO. 11, 2004 3565

A R T I C L E S
Fang et al.
to that for BPN, to give BIN in 89% yield. Pale yellow solid, mp 278
The relationship between the measured fluorescence intensity F and
in a selected wavelength can be expressed by F0/F - F0 ) ΦMꢀM/
°C (decomp.). TLC (EtOAc/hexane (4:6)) Rf ) 0.53. IR (KBr) 3435,
Cg
1
2213, 1602 cm-1; H NMR (CDCl3, 400 MHz) δ 11.97 (s, 2 NH),
(ΦMꢀM - Φpꢀp)(1/KaCg + 1) where F0 denotes the fluorescence intensity
of free BPN. ΦM and Φp are fluorescence quantum yields of the free
BPN and complex, respectively, and are assumed to be constant
throughout the titration. The binding constant of complex and 1:1
stoichiometry were determined by curve fitting using the nonlinear least-
squares method.
For determination of detection limit, an Ar+ laser (465.8 nm, 30
mW, Coherent Innova 90) was used as the excitation source. The
resulting luminescence was detected by an intensified charge coupled
detector (ICCD, Princeton Instrument, model 576G/1) operated at a
free run mode. The fluorescence spectra (λex ) 465.8 nm) of BPN
(1.2 × 10-5 M) as a function of the concentrations of octyl â-D-
glucopyranoside were recorded.
Circular Dichroic Studies. The stock solutions of BIN (1 × 10-5
M) and octyl saccharides (2 × 10-2 M) were prepared in spectroscopic
grade CH2Cl2. The BIN stock solution (2 mL) was transferred to a
1-cm length quartz cell, and the CD spectrum was recorded. Then 200
µL (200 equiv) of octyl saccharide solution was introduced into the
same cell, and the corresponding CD spectrum was recorded.
8.56 (d, J ) 8.2 Hz, 2 H), 7.84 (d, J ) 8.2 Hz, 2 H), 7.62 (d, J ) 8.0
Hz, 2 H), 7.40 (d, J ) 8.2 Hz, 2 H), 7.24 (t, J ) 7.6 Hz, 2 H), 7.08 (t,
J ) 7.7 Hz, 2 H), 7.06 (s, 2 H); 13C NMR (CDCl3, 100 MHz) δ 155.0
(2 × C), 146.0 (2 × C), 138.1 (2 × CH), 136.8 (C), 126.9 (C), 125.2
(2 × CH), 123.7 (2 × CH), 121.3 (2 × C), 120.7 (2 × CH), 120 (2 ×
CH), 116.5 (2 × C), 111.4 (2 × CH), 110.1 (2 × CH), 92.0 (2 × C),
84.9 (2 × C); HR-FAB-MS calcd for C28H17N4: 409.15; found: m/z
409.15 (M+ + H).
1
1H NMR Titration Studies. H NMR spectra were measured on
Brucker Avance-400 NMR spectrometer. A typical experiment was
performed as follows. A solution of BPN in CDCl3 was prepared (2 ×
10-4 M), and a 0.4-mL portion was transferred to a 5-mm NMR tube.
A small aliquot of the CDCl3 solution (0.02 M) containing the examined
saccharide was introduced to the BPN solution in an incremental
fashion, and their corresponding spectra were recorded. The chemical
shift of N3,N4-H of BPN was monitored as a function of saccharide
concentrations. Nonlinear regression analyses were used to determine
the binding constants.
UV-Vis Titration Studies. UV-vis spectra were obtained using
Hewlett-Packard-8453 spectrophotometer. A typical experiment was
performed as follows.
X-ray Diffraction Analyses. The crystals were mounted on a glass
fiber. Crystal data were collected on Nonius Kappa CCD or Brucker
SMART CCD diffractometers installed with monochromatized Mo-
KR radiation, λ ) 0.710 73 Å at T ) 295 K. All structures were solved
by using the SHELXS-9726 and refined with SHELXL-9727 by full-
matrix least squares on F2 values. Hydrogen atoms were fixed at
calculated positions and refined using a riding mode.
The stock solutions of BIN (1 × 10-6 M) and octyl saccharides
(5 × 10-3 M) were prepared by using spectroscopic grade dichlo-
romethane. The BIN solutions in the range of 1 × 10-6 M to 5 × 10-6
M exhibited a linear dependence of absorbance. The absorption
spectrum of BIN stock solution (2 mL) in a quartz cell (1 cm length)
was recorded, and then the stock solution of alkyl saccharide was
introduced in an incremental fashion (1, 2, 4, 8, 16, 25, 50, 100, 150,
200, 250, 300, and 400 µL; 1 µL corresponds to 2.5 equiv) and their
corresponding UV-vis curves were recorded.
Acknowledgment. We thank Professor Tashin J. Chow
(Institute of Chemistry, Academia Sinica) for his help in the
initial research work, Prof. Ta-Chau Chang and Mr. Jin-Yi Wu
(IAMS, Academia Sinica) for CD measurements, Mr. Yi-Hung
Liu (Instrumentation Center, National Taiwan University) for
X-ray analyses, and National Science Council for financial
support.
On the basis of 1:1 stoichiometry of complex, the relationship
between the measured absorbance A as a function of the added
saccharide concentration, Cg, can be expressed by A0/A - A0
)
(ꢀM/ꢀC - ꢀM)[1/KaCg + 1], where ꢀM and ꢀC are molar extinction
coefficients of the BPN (or BIN) monomer and hydrogen-bonding
complex, respectively at a selected wavelength. A0 denotes the
absorbance of the free BPN (or BIN) at that specific wavelength.
Fluorescent Titration Studies. Fluorescence spectra were recorded
on Hitachi F-4500 and Edinburgh FS 920 spectrophotometers. The
fluorescence spectra were taken using the same samples employed in
the UV-vis study, i.e., transferring the same cuvette from the UV-
vis spectrophotometer to the fluorescence spectrophotometer for each
incremental addition. The fluorescence spectra (λex ) 415 nm for BPN
and λex ) 413 nm for BIN) were taken as a function of saccharide
concentrations.
Supporting Information Available: Synthetic procedures,
NMR, fluorescence, molecular calculations, and X-ray diffrac-
tion (PDF). These materials are available free of charge via the
Internet at http://pubs.acs.org.
JA039237W
(26) Sheldrick, G. M. Acta Crystallogr. 1990, A46, 467-473.
(27) Sheldrick, G. M. SHELXL-97, Program for the Refinement of Crystal
Structures; University of Go¨ttingen: Germany, 1997.
9
3566 J. AM. CHEM. SOC. VOL. 126, NO. 11, 2004