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) adoptingdaadrelays of proton donors (d) and acceptors (a) form multiple hydrogen-bonding complexes with various monosaccharides that possess complementary addasequences. Although the free BPN emits blue light atλmax)475 nm in CH2Cl2, its complexation with octylβ-D-glucopyranoside gives green fluorescence atλmax)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 similardaadcleft 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 octylβ-D-glucopyranoside from its enantiomer and other monosaccharides.
Introduction
Sensing of saccharides is essential in a variety of medical, diagnostic, and therapeutic contexts.1Expeditious methods that
distinguishD- andL-sugars are particularly crucial because the
present enzymatic protocols2 cannot be exploited to detect
unnatural sugars (e.g.,L-glucose). Detection of the nonmetabo-lizableL-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.3Sensing of carbohydrates via the covalent
formation of boronic esters relies on a well-defined geometry,4
whereas the hydrogen-bonding recognition affords the superior-ity of the geometrical flexibilsuperior-ity 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.
Both BPN and BIN possess a naphthyridine core moiety, in which two pyridinic nitrogen atoms act as the proton acceptors
†Current address of Z. S.: Institute for Molecular Science, Myodaiji,
Okazaki 444-8585, Japan.
‡National Taiwan University.
|Genomic Research Center.
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Figure 1. (a) Complexation of BPN with alkylβ-D-glucopyranoside via quadruple hydrogen bondings. (b) Structure of BIN with two indole rings in lieu of the pyrrole rings in BPN.
(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.5The 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 by the1H NMR chemical-shift changes,6though this method
shows a limited sensitivity. Alternatively, one can use fluores-cence spectroscopic methods to improve the detection sensitiv-ity,7as that shown in sensing octylD-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,9polypyridine,10
quino-line,11and porphyrin-type receptors12bearing 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 effect13to dictate the chirality of
the BPN-saccharide complex. In lieu of the pyrrole rings, BIN containing the indole chromophores with absorption at longer
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.
Results and Discussion
Synthesis and Characterization of Molecular Sensors BPN and BIN. The synthesis of BPN (Scheme 1) incorporated Sonogashira coupling reaction14of
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-iodo(1-tert-butoxycarbonyl-2-ethynyl)-indole 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).16The 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°.
NMR Study of the Binding Modes of BPN with Octylβ-D -Glucopyranoside. When a stock solution of BPN in CDCl3
(2 × 10-4 M) was treated with 1 equiv of octyl β-D
-glucopyranoside (as 0.02 M CDCl3solution), the NHs on the
pyrrole rings showed a large chemical-shift change (∆δ ) 1.4
ppm). The glucoside counterpart also showed significant
chemi-(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.; Martı´n-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-Garcı´a, 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.
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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.
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and Applications; VCH: New York, 1994.
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(16) The crystallographic data of BPN have been deposited with the Cambridge Crystallographic Data Centre as no. CCDC 176324.
Scheme 1. Synthesis of BPN (1) and BIN (2)
cal-shift changes of hydroxyl protons (Figure 3). The 1:1 complexation of BPN with octyl β-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.17The temperature dependent (285-315 K) 1H NMR study indicated that the complex formation of BPN
with octylβ-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 lnKaas 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 Octylβ-D-Glucopyranoside. Upon adding octylβ-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 β-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 octylβ-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.
Fluorescence Titration of the BPN and BIN Complexes with Octylβ-D-Glucopyranoside. Dual spectral features were observed in the fluorescence titration study of BPN with octyl β-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 octylβ-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 octylβ-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 octylβ-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.
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.18Upon 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 alternadelocaliza-tion on the fluorescent property (Figure 7a).19Furthermore,
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 β-D-glucopyranoside in CDCl3 solution was relatively
weak (Ka≈ 1000 M-1at 300 K) as derived from the1H 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-pullπ-system prohibited the multiple hydrogen-bonds induced charge-transfer effect.
(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.
(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.
(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. Figure 2. ORTEP drawing of BPN shows a V-shaped cleft.
Upon excitation at 413 nm, BIN showed a fluorescence emission atλmax) 485 nm in CH2Cl2solution. Unlike BPN or
BHN, the fluorescence intensity decreased on binding with octyl
β-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 octylβ-D-glucopyranoside in CH2Cl2solution 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 monosacSac-charides 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 3. Induced1H NMR changes of BPN (5× 10-4M in CDCl
3, 300 K) on addition of octylβ-D-glucopyranoside: (a) free BPN, (b) BPN with octyl
β-D-glucopyranoside (1 equiv), and (c) free octylβ-D-glucopyranoside (1× 10-3M 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 4. Absorption spectra of BPN (5.4× 10-6M) in CH2Cl2by adding
various concentrations (Cg) of octylβ-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.
Figure 5. UV-vis titration of BIN (5× 10-6M) with octylβ-D-glucoside (5× 10-3M) in CH2Cl2solution.
Figure 6. Fluorescence spectra of BPN (1.2× 10-5M) in CH2Cl2by
adding various concentrations (Cg) of octyl β-D-glucopyranoside. The 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.
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 > β-D-glucopyranoside > (R-D-glucopyranoside,β-D -galactopy-ranoside) . (β-L-fucopyranoside, β-D-fructopyranoside, β-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 β-D -galactopyranoside and octylβ-D-glucopyranoside is the spatial orientation of 4-OH, the binding constant forβ-galactoside is much smaller than that of β-glucoside on complexation with either BPN or BIN. This binding selectivity is comparable to those reported in the literature.5,10bFor octylβ-L-fucopyranoside
in which one hydrogen bond is eliminated by modifying the C(6)CH2OH to CH3group, the binding constants with either
BPN or BIN decrease dramatically. The binding of methylβ-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 β-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
showed a smaller chemical-shift change than theβ-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.23The 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
(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.
(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.
Table 1. Binding Constants and Free Energy Changes for BPN-Saccharide and BIN-Saccharide Complexes
alkyl saccharide Ka/M-1(BPN complex)a −∆G/kJ mol-1(BPN complex)b Ka/M-1(BIN complex)c −∆G/kJ mol-1(BIN complex)b
β-D-glucoside 6 19 800 ( 810d 24.7 11 700 ( 1280 23.1 R-D-glucoside 8 12 600 ( 450 23.6 5690 ( 560 21.3 R-D-mannoside 9 25 100 ( 220 25.3 13500 ( 680 23.4 β-D-galactoside 10 6190 ( 330e 21.8 7940 ( 290 22.1 β-L-fucoside 11 1950 ( 60 18.9 2160 ( 270 18.9 β-D-fructoside 12 490 ( 7f 15.5 1540 ( 100 18.1 β-D-riboside 13 605 ( 30 16.0 aDerived by1H NMR titrations in CDCl
3solutions at 300 K.b-∆G ) RT lnKa.cDerived by fluorescence titrations in CH2Cl2solutions at 296 K. The
data represent the average values of two independent titrations.dThe binding constant in CH
2Cl2solution at 300 K was estimated to be 5500 M-1by
fluorescence titrations, and 4800 M-1by UV-vis titrations.eThe binding constant in CH
2Cl2solution at 300 K was estimated to be 1600 M-1by absorption
and fluorescence titrations.fThe binding constant in CH
2Cl2solution at 300 K was estimated to be 190 M-1by 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-pullπ-system to account for charge transfer.
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 methylβ-D-glucopyranoside, methyl β-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 octylβ-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
in gas phase. Moreover, the dimerization energies should be somewhat reduced by addition of the vibrational zero-point energies.
Though BPN is an achiral molecule, it is expected to form chiral complexes with chiral saccharides. On the basis of the B3LYP/6-31G** computation, the complex of BPN withβ-D
-glucopyranoside exhibited a P-helicity24with 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 β-D-galactopyranoside differed from β-D
-glu-copyranoside simply by the orientation of the 4-OH group, the molecular model of BPN/β-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/β-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 β-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 β-L-glucopyranoside (the enantiomer of octyl β-D
-(24) Eliel, E. L.; Wilen, S. H.; Mander, L. N. Stereochemistry of Organic Compounds; Wiley: New York, 1994; pp 1012 and 1200.
Figure 8. (a) Scheme for the geometrically optimized BPN/methylβ-D-glucopyranoside complex derived by the B3LYP/6-31G** method. The distances (Å) of the hydrogen bonds are shown. (b) The geometrically optimized BPN/β-D-glucopyranoside complex that shows a P-helicity with a clockwise twist of the BPN backbone. (c) The geometrically optimized BPN/β-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 Dimerization Energies (Edim,in kJ/mol) for the Complex of BPN with Methylβ-D-Glucopyranoside, Methylβ-D-Galactopyranoside and MethylR-D-Mannopyranosidea
methyl saccharide N(1)−HO(3) N(2)−HO(4) N(3)H−O(2) N(4)H−O(6) Edim
β-D-glucoside 1.970 1.827 2.140 1.813 -149.9
β-D-galactoside 2.098 1.840 2.141 1.826 -146.9
R-D-mannoside 2.165 1.842 1.951 1.809 -135.2
aMolecular calculations were conducted with B3LYP/6-31G** method.
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/ β-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/β-glucoside complex. Visual change of free BPN (cyanic color) in CH2Cl2to 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 ofβ-D-glucopyranoside at low concentration (∼100 pM)
is achieved, and the differentiation ofβ-D-glucopyranoside from
its enantiomer and other monosaccharides is realized. This study can be advanced by using the fluorescence detected circular dichroism (FDCD),25or by designing the water-soluble
ana-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 (40-63µm)
and neutral aluminum oxide (Acros, 50-200µm) were used for column
chromatography. Melting points are uncorrected. Chemical shifts (δ)
are given in parts per million (ppm) relative toδH7.24/δC77.0 (central
line of t) for CHCl3/CDCl3andδH2.49 (m)/δC39.5 (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.
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/CH2Cl2to
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, 1593 cm-1;1H NMR (DMSO-d 6, 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 (CD 3OD, 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.
Figure 9. CD spectra of BIN (1× 10-5M) and its complexation with saccharides (2× 10-2M) in CH2Cl2solutions.
Table 3. CD Spectral Data for BIN/Octyl Saccharide Complexes in CH2Cl2Solutionsa
BIN/saccharide λmax/nm ([θ]/deg cm2dmol-1) ∆[θ]b
β-D-glucoside 6 442 (10 100), 344 (-16 900), 317 (500) -17 400 β-L-glucoside 7 442 (-10 100), 344 (17 700), 317 (300) +17 400 R-D-mannoside 9 442 (-10 900), 344 (-17 400), 317 (1500) -18 900 β-D-galactoside 10 442 (-41 300), 344 (30 600), 317 (-9500) +40 100
aThe CH
2Cl2solution of BIN (1× 10-5M) and saccharide (2× 10-2
M) was used.bThe 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.
to that for BPN, to give BIN in 89% yield. Pale yellow solid, mp 278 °C (decomp.). TLC (EtOAc/hexane (4:6)) Rf) 0.53. IR (KBr) 3435, 2213, 1602 cm-1;1H NMR (CDCl 3, 400 MHz)δ 11.97 (s, 2 NH), 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 (CDCl 3, 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).
1H NMR Titration Studies.1H NMR spectra were measured on
Brucker Avance-400 NMR spectrometer. A typical experiment was performed as follows. A solution of BPN in CDCl3was prepared (2×
10-4M), and a 0.4-mL portion was transferred to a 5-mm NMR tube. A small aliquot of the CDCl3solution (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.
The stock solutions of BIN (1× 10-6M) and octyl saccharides (5× 10-3 M) were prepared by using spectroscopic grade dichlo-romethane. The BIN solutions in the range of 1× 10-6M 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 400µL; 1 µL corresponds to 2.5 equiv) and their
corresponding UV-vis curves were recorded.
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 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
andλex) 413 nm for BIN) were taken as a function of saccharide
concentrations.
The relationship between the measured fluorescence intensity F and Cgin a selected wavelength can be expressed by F0/F - F0) ΦMM/ (ΦMM- Φpp)(1/KaCg+ 1) where F0denotes the fluorescence intensity
of free BPN.ΦMandΦpare 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 β-D -glucopyranoside were recorded.
Circular Dichroic Studies. The stock solutions of BIN (1× 10-5 M) and octyl saccharides (2× 10-2M) 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. 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-9726and 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.
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.
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.
