2,7-Bis(1H-pyrrol-2-yl)ethynyl-1,8-naphthyridine: An Ultrasensitive Fluorescent Probe for Glucopyranoside

Article abstract of DOI:10.1021/ol0264096

(Matrix Presented) A push-pull conjugated molecule, 2,7-bis(1H-pyrrol-2-yl)ethynyl-1,8-naphthyridine (BPN), has been designed to bind selectively with octyl glucopyranoside (OGU). The BPN/OGU quadruple hydrogen-bonding complex adopts a rigid BPN conformation in which the proton donor (d) and acceptor (a) relays (daad) are resonantly conjugated through the ethynyl bridge, inducing π-electron delocalization, i.e., a charge transfer effect. The excellent photophysical properties make BPN a highly sensitive probe for monitoring glucopyranoside to a detection limit of ～100 pM.

Full text of DOI:10.1021/ol0264096

ORGANIC
LETTERS
2002
Vol. 4, No. 18
3107-3110
2,7-Bis(1H-pyrrol-2-yl)ethynyl-1,8-
naphthyridine: An Ultrasensitive
Fluorescent Probe for Glucopyranoside
,†
Jen-Hai Liao,† Chao-Tsen Chen,* He-Chun Chou,† Chung-Chih Cheng,†,§
,†
,†
Pi-Tai Chou,* Jim-Min Fang,* Zdenek Slanina,‡ and Tashin J. Chow‡
Department of Chemistry, National Taiwan UniVersity, Taipei, 106, Taiwan,
and Institute of Chemistry, Academia Sinica, Nankang, 115, Taiwan, Republic of China
jmfang@ccms.ntu.edu.tw
Received June 21, 2002
ABSTRACT
A push−pull conjugated molecule, 2,7-bis(1H-pyrrol-2-yl)ethynyl-1,8-naphthyridine (BPN), has been designed to bind selectively with octyl
glucopyranoside (OGU). The BPN/OGU quadruple hydrogen-bonding complex adopts a rigid BPN conformation in which the proton donor (d)
and acceptor (a) relays (daad) are resonantly conjugated through the ethynyl bridge, inducing π-electron delocalization, i.e., a charge transfer
effect. The excellent photophysical properties make BPN a highly sensitive probe for monitoring glucopyranoside to a detection limit of ∼100
pM.
The hydrogen-bonding recognition for carbohydrates1,2 af-
fords an advantage of geometrical flexibility over that of the
covalent type.3 Multiple hydrogen bonding interactions
provide a general method for the protein-carbohydrate
recognition, in which most water molecules are often
excluded from the cleft of the protein in order to avoid their
possible hydrogen-bonding competition. Thus, many artificial
receptors have been designed to mimic biotic carbohydrate
recognition by using hydrogen bondings in aprotic solvents.1b
troscopic and circular dichroic methods have been utilized
as the detection tools in several examples.4 We report here
(2) (a) Kobayashi, K.; Asakawa, Y.; Kato, Y.; Aoyama, Y. J. Am. Chem.
Soc. 1992, 114, 1030. (b) Bhattarai, K. M.; Bonar-Law, R. P.; Davis, A.
P.; Murray, B. A. Chem. Commun. 1992, 752. (c) Liu, R.; Still, W. C.
Tetrahedron Lett. 1993, 34, 2573. (d) Huang, C.-Y.; Cabell, L. A.; Anslyn,
E. V. J. Am. Chem. Soc. 1994, 116, 2778. (e) Das, G.; Hamilton, A. D. J.
Am. Chem. Soc. 1994, 116, 11139. (f) Anderson, S.; Neidlein, U.; Gramlich,
V.; Diederich, F. Angew. Chem., Int. Ed. Engl. 1995, 34, 1596. (g) Jime´nez-
Barbero, J.; Junquera, E.; Mart´ın-Pastor, M.; Sharma, S.; Vicent, C.;
Penade´s, S. J. Am. Chem. Soc. 1995, 117, 11198. (h) Mizutani, T.;
Kurahashi, K.; Murakami, T.; Matsumi, N.; Ogoshi, H. J. Am. Chem. Soc.
1997, 119, 8991. (i) Davis, A. P.; Wareham, R. S. Angew. Chem., Int. Ed.
1998, 37, 2270. (j) Inouye, M.; Takahashi, K.; Nakazumi, H. J. Am. Chem.
Soc. 1999, 121, 341. (k) Mazik, M.; Sicking, W. Chem. Eur. J. 2001, 7,
664. (l) Kra´l, V.; Rusin, O.; Schmidtchen, F. P. Org. Lett. 2001, 3, 873.
(m) Bitta, J.; Kubik, S. Org. Lett. 2001, 3, 2637. (n) 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.
1
Unfortunately, the detecting method generally relies on H
NMR chemical shift changes and hence is limited in its
sensitivity. To circumvent this problem, fluorescence spec-
* Fax for Prof. Jim-Min Fang: (8862)-2363-6359.
† National Taiwan University.
‡ Academia Sinica.
§ Current address: Department of Chemistry, Fu-Jen Catholic University,
No. 510, Chung Cheng Road, Shih Chuang, Taiwan.
(1) For general 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; Vol. 2, Chapter 9, pp 279-
307. (b) Davis, A. P.; Wareham, R. S. Angew. Chem., Int. Ed. 1999, 38,
2978.
(3) (a) James, T. D.; Sandanayake, K. R. A. S.; Shinkai, S. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 1910. (b) Eggert, H.; Frederiksen, J.; Morin, C.;
Norrild, J. C. J. Org. Chem. 1999, 64, 3846. (c) Kukrer, B.; Akkaya, E. U.
Tetrahedron Lett. 1999, 40, 9125. (d) DiCesare, N.; Lakowicz, J. R. Chem.
Commun. 2001, 2022. (e) DiCesare, N.; Lakowicz, J. R. J. Phys. Chem.
2001, 105, 6834. (f) Yang, W.; He, H.; Drueckhammer, D. G. Angew.
Chem., Int. Ed. 2001, 40, 1714.
10.1021/ol0264096 CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/13/2002

a novel saccharide probe, 2,7-bis(lH-pyrrol-2-yl)ethynyl-l,8-
naphthyridine (BPN, Figure 1).5 BPN exhibits a unique
Figure 1. (a) Free BPN showing the conjugated daad array with
two pyrrole rings slightly tilted toward opposite directions. (b) BPN
and its associated BPN/â-D-glucoside hydrogen-bonding complex.
The geometrically optimized complex (R ) CH3) derived by the
B3LYP/6-31G** method shows the distances (Å) of the hydrogen
bonds.
Figure 2. ORTEP drawing of BPN (side view).
four hydrogen-bonding sites. Through the multiple hydrogen
bonds effect the unusual push-pull daad relay conjugated
through the ethynyl bridge induces the π-electron delocal-
ization.7b Furthermore, rigidification upon formation of the
BPN/pyranoside complex might also contribute a role in
enhanced fluorescence, as shown in previous studies on the
bindings of ions6 and carbohydrates.7 In combination, ultra-
sensitive detection can thus be achieved via the remarkable
change of the electronic spectroscopy.
Upon adding octyl â-D-glucopyranoside (OGU), the 410-
nm free BPN band (log ꢀ ≈ 4.65) in CH2Cl2 solution
decreased significantly along with the growth of a new
absorption band at ∼435 nm (Figure 3). An isosbestic point
at ∼415 nm throughout the titration was attributed to the
formation of BPN/OGU hydrogen-bonding complexes in
equilibrium with free BPN. The measured absorbance [A0/
(A - A0)] as a function of the inverse of OGU concentrations
fit a linear relationship, indicating the 1:1 stoichiometry of
the BPN/OGU complex.8 On the basis of 1:1 stoichiometry
of BPN/OGU, the relationship between the measured ab-
property among the hydrogen-bonding type of artificial
receptors in which the conjugated daad array acts intrinsically
as a saccharide receptor as well as a sensing chromophore.
As an alternative to the extensively studied boronic acid
receptors,3 we brought noncovalent recognition and direct
visual detection of carbohydrate into an integral system. The
new concept evokes monosaccharide as an adda molecule
complementary to the daad array of BPN to form a complex
with quadruple hydrogen bondings.
Synthesis of BPN incorporated the coupling reaction of
2,7-dichloro-1,8-naphthyridine with 2 equiv of (1-tert-
butoxycarbonyl-pyrrol-2-yl)acetylene, followed by removal
of the Boc protective group (Scheme 1). Two pyrrole rings
Scheme 1. Synthetic of
2,7-Bis(1H-pyrrol-2-yl)ethynyl-1,8-naphthyridine (BPN)
(4) (a) Kikuchi, Y.; Kobayashi, K.; Aoyama, Y. J. Am. Chem. Soc. 1992,
114, 1351. (b) Inouye, M.; Miyake, T.; Furusyo, M.; Nakazumi, H. J. Am.
Chem. Soc. 1995, 117, 12416. (c) Mizutani, T.; Kurahashi, T.; Murakami,
T.; Matsumi, N.; Ogoshi, H. J. Am. Chem. Soc. 1997, 119, 8991. (d) Tamaru,
S.-i.; Yamamoto, M.; Shinkai, S.; Khasanov, A. B.; Bell, T. W. Chem. Eur.
J. 2001, 7, 5270. (e) Tamaru, S.-i.; Shinkai, S.; Khasanov, A. B.; Bell, T.
W. Proc. Natl. Acad. Sci. 2002, 99, 4972.
(5) (a) Newkome, G. R.; Garbis, S. J.; Majestic, V. K.; Fronczek, F. R.;
Chiari, G. J. Org. Chem. 1981, 46, 833. (b) Ziessel, R.; Suffert, J.; Youinou,
M.-T. J. Org. Chem. 1996, 61, 6535. The carbohydrate recognition properties
of some naphthyridine derivatives have been demonstrated in refs 2k, 4d,
and 4e. Compounds containing pyrrole moieties have been used to bind
fluoride ion. See a recent example: (c) Mizuno, T.; Wei, W.-H.; Eller, L.
R.; Sessler, J. L. J. Am. Chem. Soc. 2002, 124, 1134.
(6) (a) McFarland, S. A.; Finney, N. S. J. Am. Chem. Soc. 2001, 123,
1260. (b) Mello, J. V.; Finney, N. S. Angew. Chem., Int. Ed. 2001, 40,
1536. (c) Choi, K.; Hamilton, A. D. Angew. Chem., Int. Ed. 2001, 40,
3912. (d) McFarland, S. A.; Finney, N. S. J. Am. Chem. Soc. 2002, 124,
1178.
in BPN are slightly tilted toward opposite directions (X-ray
analysis, Figure 2), but reorganize to a rigid inward
conformation upon binding with a chairlike pyranoside
(Figure 1). The pyrrole and naphthyridine moieties function
as the proton donor (d) and acceptor (a), respectively, and
also develop an ideal V-shaped cleft to provide as many as
(7) (a) Sandanayaka, K. R. A. S.; Nakashima, K.; Shinkai, S. Chem.
Commun. 1994, 1621. (b) Takeuchi, M.; Yoda, S.; Imada, T.; Shinkai, S.
Tetrahedron 1997, 53, 8335.
3108
Org. Lett., Vol. 4, No. 18, 2002

Figure 4. Fluorescence spectra of BPN (1.2 × 10-5 M) in CH2Cl2
by adding various concentrations (Cg) of octyl glucopyranoside.
The fluorescence spectra (λex ) 430 nm) show the growth of a
535-nm emission band on complexation, accompanied by decrease
of the 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/
OGU.
Figure 3. Absorption spectra of BPN (5.4 × 10-6 M) in CH2Cl2
by adding various concentrations (Cg) of octyl 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/OGU.
sorbance A as a function of the added OGU concentration,
Cg, can be expressed by A0/(A - A0) ) [ꢀM/(ꢀC
-
ꢀM)](Ka-1Cg + 1) where ꢀM and ꢀC are molar extinction
coefficients of the BPN monomer and hydrogen-bonding
complex, respectively, at a selected wavelength.8 A0 denotes
the absorbance of the free BPN at that specific wavelength.
The ratio for the intercept versus slope deduced the associa-
tion constant Ka of (4.8 ( 0.8) × 103 M-1.
-1
at >460 nm where the absorbance was solely attributed to
the BPN/OGU complex, a detection limit as low as ∼100
pM OGU could be achieved on the basis of the nearly
background (i.e., the uncomplexed BPN)-free 535-nm emis-
sion. The ratiometric fluorescence proved to be a reliable
and ultrasensitive method for the real time detection of BPN/
OGU complexation.
Dual spectral features were observed in the fluorescence
titration study. 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/OGU complex, respectively (Figure 4). The plot of [F0/
(F - F0)] at 535 nm versus the inverse of OGU concentra-
tions reconfirmed a 1:1 BPN/OGU complex.8 The relation-
ship between the measured fluorescence intensity F and Cg
in a selected wavelength can be expressed by F0/(F - F0)
) [ΦMꢀM/(ΦMꢀM - ΦCꢀC)](Ka-1Cg-1 + 1), where F0 denotes
the fluorescence intensity of free BPN. ΦM and ΦC are
fluorescence quantum yields of the free BPN and complex,
respectively, and are assumed to be constant throughout the
titration.8 The ratio for the intercept versus slope gave a Ka
value of (5.5 ( 0.5) × 103 M-1, which within experimental
error is consistent with the value deduced from the absorption
titration.
The 1:1 BPN/OGU complexation was further supported
by the NMR study using a continuous variation method (Job
plot),9 in which the chemical shift changes of pyrrol-NH as
a function of sugar concentrations were monitored. The
1
temperature-dependent (285-315 K) H NMR study indi-
cated that the formation of the BPN/OGU complex was
thermodynamically favored (∆G300 ) -5.79 kcal/mol), in
agreement with the proposed quadruple hydrogen bond
formation. The association constants and thermodynamic
parameters are collected in Table 1. A molecular modeling
of BPN/methyl â-D-glucoside complex (Figure 1b) implied
Table 1. Association Constants and Thermodynamic
Parameters for the Complexes of BPN with Octyl Glucoside
(OGU), Octyl Galactoside (OGA), and Octyl Furanoside (OFU)
Ka
(M-1
)
∆H300
∆S300
∆G300
complex
(kcal/mol) (cal/mol) (kcal/mol)
BPN/OGU 5000a (20000)b
BPN/OGA 1600a (6200)b
BPN/OFU
-12.3c
-21.8c
-5.79c
-5.21d
-3.69d
The fluorescence yields for BPN (i.e., the 475 nm band)
and the BPN/OGU complex (i.e., the 535-nm band) were
measured to be as high as 0.35 ( 0.01 and 0.23 ( 0.02 in
CH2Cl2, respectively. By selecting the excitation wavelength
190a (490)b
a The association constant was derived from the absorption and fluo-
rescence titrations in CH2Cl2 solution. Estimated errors are within (20%.
b The association constant was derived from the 1N MR titrations in CDCl3
solution. c The thermodynamic data were deduced from the temperature-
(8) 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. A0, F0, A, and F denote,
respectively, the absorbance and fluorescent intensity of free BPN and in
solution after adding monosaccharides at a selective wavelength.
1
dependent H NMR studies. ∆G ) ∆H - T∆S. d The value was derived
from ∆G ) -RT lnKa (in CDCl3 solution).
Org. Lett., Vol. 4, No. 18, 2002
3109

the importance of multiple hydrogen bond formation as well
as the geometrical fitness to accommodate each hydrogen
bond. 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).
The intrinsic conjugated daad type chromophore2d,j,k,10 in
BPN is of key importance to account for the remarkable
spectral differences. The acidity of pyrrole and basicity of
naphthyridine could be enhanced at the excited state through
the conjugated dual hydrogen-bonding effect.8 The com-
plexation with saccharide thus provides multiple hydrogen
bond induced charge transfer to account for a drastic
alternation on the fluorescent property. To support this
viewpoint, a nonconjugated daad molecule of 2,7-bis(3-
hydroxy-3-methylbutynyl)-1,8-naphthyridine (BHN) was pre-
pared (Figure 5). Upon forming the BHN/OGU complex (Ka
or fluorescence spectra. The enhanced fluorescence could
be rationalized by the conformational rigidification, whereas
the lack of a continuous push-pull π-system prohibits the
multiple hydrogen bond induced charge-transfer effect.
The preferable 1:1 complexation of BPN with octyl â-D-
galactopyranoside (OGA) or octyl â-L-fucopyranoside (OFU)
was similarly determined by absorption, fluorescence, and1H
NMR titrations (see Table 1 and Supporting Information).
While the only structural difference between OGA and OGU
is the spatial orientation of 4-OH, the association constant
for OGA is smaller than that of OGU by ca. 3-fold. This
binding selectivity of BPN/OGU over BPN/OGA is com-
parable or superior to those reported in the literature.2,4e For
OFU in which one hydrogen bond is eliminated by modifying
the C(6)CH2OH to CH3 group, Ka decreases by a factor of
∼35 in comparison with that of the BPN/OGU complex.
In summary, our results demonstrate the selective affinity
and ultrasensitivity for probing the BPN/OGU complex.
Visual change of free BPN (cyanic color) in CH2Cl2 (1.2 ×
10-5 M) to green color upon addition of octyl gluco-
pyranoside (5.2 × 10-4 M) was obvious (see Abstract
graphic). Since the synthesis of BPN was straightforward,
chemical modification including the preparation of water-
soluble derivatives would be feasible. We are currently
engaged in the research of such aspects in order to develop
practical carbohydrate sensors.
Acknowledgment. We thank the National Science Coun-
cil for financial support.
Supporting Information Available: Detailed experi-
mental procedures, absorption, fluorescence, and H NMR
spectra, molecular calculations, and X-ray diffraction data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
1
Figure 5. (a) A large Stoke shift (60 nm) in the fluorescence
spectrum of BPN/OGU complex is attributed to the multiple
hydrogen bond induced charge transfer. (b) Complexation of BHN
with OGU does not show significant spectral shift because BHN
lacks a continuous push-pull π-system to account for charge
transfer.
OL0264096
(10) (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.; American Chemical Socicty:
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Luo, Y.; Drew, M. G. B.; Chapoteau, E.; Czech, B. P.; Kumar, A. Science
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(d) Corbin, P. S.; Zimmerman, S. C. J. Am. Chem. Soc. 2000, 122, 3779.
1
≈ 1000 M-1 measured by H NMR in CDCl3) the fluores-
cence intensity (λmax 387 nm) did increase, albeit no
significant spectral shift was observed in either absorption
(9) Connors, K. A. Binding Constants; Wiley: New York, 1987.
3110
Org. Lett., Vol. 4, No. 18, 2002