Fluorophore Appended Saccharide Cyclophane: Self-Association

Self-association behavior of the dansyl-appended saccharide cyclophane was ... Such a supramolecular assembling of dansyl-modified cyclophane 1 and CD...
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Fluorophore Appended Saccharide Cyclophane: Self-Association, Fluorescent Properties, Heterodimers with Cyclodextrins, and Cross-Linking Behavior with Peanut Agglutinin of Dansyl-Modified Saccharide Cyclophane Osamu Hayashida* and Itaru Hamachi Institute for Materials Chemistry and Engineering, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan [email protected] Received February 25, 2004

A saccharide cyclophane bearing an environment-sensitive fluorophore (1) was prepared by introducing not only three branches with a terminal galactose residue but also one with a dansyl moiety into a tetraaza[6.1.6.1]paracyclophane skeleton. Self-association behavior of the dansylappended saccharide cyclophane was characterized in aqueous media by fluorescence spectroscopy and dynamic light scattering measurements. At least in the concentrations below 1.0 × 10-5 M, saccharide cyclophane 1 existed in a monomeric state, whereas it tended to form self-aggregated complexes in the higher concentration. Solvent polarity dependency on the emission spectra of 1 was examined by fluorescence spectroscopy. With increasing dioxane contents in dioxane/water solvents, the fluorescence intensity originating from the dansyl moiety of 1 increased along with a concomitant blue shift of the fluorescence maximum (λem). In the monomeric state of 1 in water, the dansyl moiety of 1 was not fully included into its cyclophane cavity but partially exposed to the bulk aqueous phase. In the higher concentration ranges in an aggregate state, however, the dansyl group of 1 was located in the apolar cyclophane cavity whose microenvironment was equivalent to the polarity of 1-butanol evaluated on the basis of a correlation between λem and solvent polarity. This indicates an intermolecular inclusion of the dansyl moiety within the cyclophane. When cyclodextrin (CD) was mixed with 1, the dansyl group of 1 was bound to an internal cavity of CD such as γ-CD, β-CD, 6-O-R-glucosyl-β-CD, and 6-O-R-maltosyl-β-CD with binding constants of 7.5 × 102, 7.8 × 102, 7.7 × 102, and 6.0 × 102 M-1, respectively. Such a supramolecular assembling of dansyl-modified cyclophane 1 and CDs caused changes of the fluorescence spectra as well as appearance of induced CD bands in aqueous media. Furthermore, saccharide cyclophane 1 was selectively bound to peanut agglutinin (PNA), galactoside-binding lectin, which was readily monitored by a visible turbidity of the solution due to a cross-linking agglutination of these components, as well as by fluorescence spectroscopy. Introduction Naturally occurring cell-surface oligosaccharides are claimed to be key elements in a variety of important biological phenomena such as differentiation, proliferation, immunological responses, cell adhesion, cancer transfer, and so on.1,2 In those events on the cell surfaces, the saccharides are recognized by specific proteins. Although isolated saccharide-protein interactions are very weak, so-called glycoside cluster effects3 by multiplying of natural saccharide ligands compensate for the weakness of the interactions to allocate stronger coopera(1) (a) Hakomori, S. Pure Appl. Chem. 1991, 63, 473-482. (b) Hakomori, S. Glycoconjugate J. 2000, 17, 627-647. (2) Kobata, A. Acc. Chem. Res. 1993, 26, 319-324. (3) (a) Lee, Y. C.; Lee, R. T.; Rice, K.; Ichikawa, Y.; Wong, T. C. Pure. Appl. Chem. 1991, 63, 499-506. (b) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321-327. (c) Sigal, G. B.; Mammen, M.; Dahmann, G.; Whitesides, G. M. J. Am. Chem. Soc. 1996, 118, 37893800. (d) Lundquist, J. J.; Toone, E. J. Chem. Rev. 2002, 102, 555578.

tive binding. In recent years, much attention has been focused on the development of artificial compounds having multiple saccharide moieties, to understand molecular details of the glycoside cluster effects and to provide biomimetic structures and functions. On these grounds, many molecular scaffolds such as peptides,4 polymers,5-8 dendrimers,9,10 resorcarenes,11 calix[n]arens,12 and so on13 have been used to attach and display saccharide ligands in a multiple fashion to obtain high affinity between saccharide ligands and receptors. Re(4) (a) Sprengard, U.; Schdok, M.; Kretzschmar, G.; Kunz, H. Angew. Chem., Int. Ed. Engl. 1996, 35, 321-324. (b) Thoma, G.; Ernst, B.; Schwarzenbach, F.; Duthaler, R. O. Bioorg. Med. Chem. Lett. 1997, 7, 1705-1708. (c) Palcic, M. M.; Li, H.; Zanini, D.; Bhella, R. S.; Roy, R. Carbohydr. Res. 1998, 305, 433-442. (d) Wittmann, V.; Seeberger, S. Angew. Chem., Int. Ed. 2000, 39, 4348-4352. (5) (a) Spaltenstein, A.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 686-687. (b) Sigal, G. B.; Mammen, M.; Whitesides, G. M. J. Am. Chem. Soc. 1996, 118, 3789-3800. (c) Choi, S. K.; Mammen, M.; Whitesides, G. M. J. Am. Chem. Soc. 1997, 119, 4103-4111. (d) Li, J.; Zacharek, S.; Chen, X.; Wang, J.; Zhang, W.; Janczuk, A.; Wang, P. G. Bioorg. Med. Chem. 1999, 7, 1549-1558.

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sorcarene- or calix[n]arene-based saccharide clusters, in particular, have advantages from the viewpoint of their well-defined molecular structures and an intrinsic potential to act as a host for inclusion of guest molecules.14 For example, Aoyama and co-workers have demonstrated that resorcarene-based saccharide clusters can deliver guest molecules from an aqueous phase to polar surfaces and also to lectin surfaces,11 with the aim of molecular delivery systems.15 Water-soluble cyclophanes16 having a hydrophobic internal cavity are also favorable candidates as the framework to graft various saccharide residues, because shape and size of the cavity can be easily designed for the capture of target guest molecules.16 Recently, we have developed a saccharide cyclophane having four terminal (6) (a) Kanai, M.; Mortell, K. H.; Kiessling, L. L. J. Am. Chem. Soc. 1997, 119, 9931-9932. (b) Mann, D. A.; Kanai, M.; Maly, D. J.; Kiessling, L. L. J. Am. Chem. Soc. 1988, 120, 10575-10582. (c) Sanders, W. J.; Gordon, E. J.; Dwir, O.; Beck, P. J.; Alon, R.; Kiessling, L. L. J. Biol. Chem. 1999, 274, 5271-5278. (d) Cairo, C. W.; Gestwicki, J. E.; Kanai, M.; Kiessling, L. L. J. Am. Chem. Soc. 2002, 124, 16151619. (7) (a) Wataoka, I.; Urakawa, H.; Kobayashi, K.; Akaike, T.; Schmidt, M.; Kajiwara, K. Macromolecules 1999, 32, 1816-1821. (b) Wataoka, I.; Urakawa, H.; Kobayashi, K.; Ohno, K.; Fukuda, T.; Akaike, T.; Kajiwara, K. Polymer J. 1999, 31, 590-594. (c) Hasegawa, T.; Matsuura, K.; Ariga, K.; Kobayashi, K. Macromolecules 2000, 33, 2772-2775. (d) Sashiwa, H.; Shigemasa, Y.; Roy, R. Macromolecules 2000, 33, 6913-6915. (e) Sashiwa, H.; Shigemasa, Y.; Roy, R. Macromolecules 2001, 34, 3211-3214. (f) Sashiwa, H.; Shigemasa, Y.; Roy, R. Macromolecules 2001, 34, 3905-3909. (8) (a) Nishimura, S.-I.; Nomura, S.; Yamada. K. Chem. Commun. 1998, 617-618. (b) Kurita, K.; Shimada, K.; Nishiyama, Y.; Shimojoh, M.; Nishimura, S.-I. Macromolecules 1998, 31, 4764-4769. (9) (a) Roy, R.; Kim, J. M. Angew. Chem., Int. Ed. 1999, 38, 369372. (b) Andre, S.; Ortega, P. J. C.; Perez, M. A.; Roy, R.; Gabius, H.J. Glycobiology 1999, 9, 1253-1261. (c) Baek, M. G.; Rittenhouse-Olson, K.; Roy, R. Chem. Commun. 2001, 257-258. (10) (a) Miller, T. M.; Kwock, E. W.; Neenan, T. X. Macromolecules 1992, 25, 3143-3148. (b) Peerlings, H. W. I.; Nepogodiev, S. A.; Stoddart, J. F.; Meijer, E. W. Eur. J. Org. Chem. 1994, 9, 1879-1886. (c) Ashton, P. R.; Boyd, S. E.; Brown, C. L.; Nepogodiev, S. A.; Meijer, E. W.; Peerlings, H. W. I.; Stoddart, J. F. Chem. Eur. J. 1997, 3, 974984. (d) Aston, P. R.; Boyd, S. E.; Brown, C. L.; Jayaraman, N.; Stoddart, J. F. Angew. Chem., Int. Ed. Engl. 1997, 36, 732-735. (e) Colonna, B.; Harding, V. D.; Nepogodiev, S. A.; Raymo, F. M.; Spencer, N.; Stoddart, J. F. Chem. Eur. J. 1998, 4, 1244-1254. (f) Aston, P. R.; Hounsell, E. F.; Jayaraman, N.; Nilsen, T. M.; Spencer, N.; Stoddart, J. F.; Young, M. J. Org. Chem. 1998, 63, 3429-3437. (g) Matsuoka, K.; Terabatake, M.; Esumi, Y.; Terunuma, D.; Kuzuhara, H. Tetrahedron Lett. 1999, 40, 7839-7842. (11) (a) Fujimoto, T.; Shimizu, C.; Hayashida, O.; Y. Aoyama, Y. J. Am. Chem. Soc. 1997, 119, 6676-6677. (b) Fujimoto, T.; Shimizu, C.; Hayashida, O.; Aoyama, Y. J. Am. Chem. Soc. 1998, 120, 601-602. (c) Hayashida, O.; Shimizu, C.; Fujimoto, T.; Aoyama, Y. Chem. Lett. 1998, 13-14. (d) Hayashida, O.; Nishiyama, K.; Matsuda, Y.; Aoyama, Y. Tetrahedron Lett. 1999, 40, 3407-3410. (e) Hayashida, O.; Kato, M.; Akagi, K.; Aoyama, Y. J. Am. Chem. Soc. 1999, 121, 11597-11599. (f) Hayashida, O.; Matsuo, A.; Aoyama, Y. Chem. Lett. 2001, 272-273. (g) Hayashida, O.; Mizuki, K.; Akagi, K.; Matsuo, A.; Kanamori, T.; Nakai, T.; Sando, S.; Aoyama, Y. J. Am. Chem. Soc. 2003, 125, 594601. (h) Aoyama, Y.; Kanamori, T.; Nakai, T.; Sasaki, T.; Horiuchi, S.; Sando, S.; Niidome, T. J. Am. Chem. Soc. 2003, 125, 3455-3457. (12) (a) Marra, A.; Scherrmann, M. C.; Dondoni, A.; Casnai, A.; Minari, P.; Ungaro, R. Angew. Chem., Int. Ed. 1994, 33, 2479-2481. (b) Marra, A.; Dondoni, A.; Sansone, F. J. Org. Chem. 1996, 61, 51555158. (c) Dondoni, A.; Marra, A.; Scherrmann, M. C.; Casnai, A.; Sansone, F. Ungaro, R. Chem. Eur. J. 1997, 3, 1774-1782. (d) Dondoni, A.; Kleban, M.; Marra, A. Tetrahedron Lett. 1997, 38, 7801-7804. (e) Consoli, G. M. L.; Cunsolo, F.; Geraci, C.; Mecca, T.; Neri, P. Tetrahedron Lett. 2003, 44, 7467-7470. (13) Glycosylated cyclodextrins, see: (a) Fernandez, J. M. G.; OrtizMellet, C.; Maciejewski, S.; Defaye, J. Chem. Commun. 1996, 27412742. (b) Kassab, R.; Felix, C.; Ptrot-Lopez, H.; Bonaly, R. Tetrahedron Lett. 1997, 38, 7555-7558. (c) Furuike, T.; Aiba, S.; Nishimura, S.-I. Tetrahedron 2000, 56, 9909-9915. (d) Fulton, D. A.; Stoddart, J. F. Org. Lett. 2000, 2, 1113-1116. (14) Pochini, A.; Ungaro, R. In Comprehensive Supramolecular Chemistry; Vo¨gtle, F., Ed.; Pergamon: Oxford, 1996; Vol. 2; Chapter 4, pp 103-142.

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glucose residues (2) and reported its following unique functions:17 (i) Amine-coupling fluorescent molecules such as 6-(5-dimethyl-aminonaphthalene-1-sulfonylamino)hexanoic acid succinimidyl ester (dansyl-SE) were effectively incorporated by 2 through hydrophobic interactions. (ii) The fluorescent dye molecules were carried by 2 from the aqueous phase to the glucoside-binding site of Concanavalin A (Con A), a glucoside- and mannosidebinding lectin18 and reacted at the proximal binding site. On the other hand, fluorescence techniques including molecular probes have been widely used to investigate interactions of biomolecular complexes and assemblies.19 For instance, environment-sensitive probes such as 8-anilino-naphthalene-1-sufonate (ANS) or dansyl derivatives, have the following characteristic properties in their fluorescence spectra.20 With decreasing microenvironment polarity exercised by the ANS molecule, its emission intensity increases with a concomitant blue shift in its wavelength, while its fluorescence intensity is hardly detectable in water as the most polar solvent. Therefore, hydrophobic domains/surfaces of biomolecules such as cell membranes and lipophilic proteins were detected and visualized by means of fluorescence imaging spectroscopy and microscopy, owing to the hydrophobicity of the probes.20 In the course of our ongoing research on saccharide cyclophanes, we became interested in developing fluorescent-type saccharide cyclophanes capable of capturing and delivering guest molecules to specific proteins. To develop fluorophore appended saccharide cyclophanes, we have adopted a simple strategy by conjugating the saccharide cyclophane with an environment-sensitive fluorescence probe. We report here the design and preparation of a saccharide cyclophane bearing a dansyl group and its self-association behavior and fluorescence properties in aqueous media. Furthermore, formation of heterodimers with cyclodextrins and cross-linking behavior with carbohydrate-binding proteins18 were demonstrated as unique features of the fluorophore appended saccharide cyclophane. Results and Discussion Design and Syntheses of Saccharide Cyclophanes. We have focused on tetraaza[6.1.6.1]paracyclophane (4)21 as a macrocylic framework on which to assemble saccharide residues and a fluorescence moiety. Compound 4 was first synthesized by Koga and Odashima and found to (15) (a) Parrot-Lopez, H.; Galons, H.; Coleman, A. W.; Mahuteau, J.; Miocque, M. Tetrahedron Lett. 1992, 33, 209-212. (b) Leray, E.; Parrot-Lopez, H.; Auge, C.; Coleman, A. W.; Finance, C.; Bonaly, R. J. Chem. Soc., Chem. Commun. 1995, 1019-1020. (c) Garcia F. J. M.; Mellet, C. O.; Maciejewski, S.; Defaye, J. J. Chem. Soc., Chem. Commun. 1996, 2741-2742. (16) Odashima, K.; Koga, K. In Comprehensive Supramolecular Chemistry; Vo¨gtle, F., Ed.; Pergamon: Oxford, 1996; Vol. 2, Chapter 5, pp 143-194. (17) Hayashida, O.; Hamachi, I. Chem. Lett. 2003, 32, 632-633. (18) (a) Lis, H.; Sharon, N. Chem. Rev. 1998, 98, 637-674. (b) Dam, T. K.; Brewer, C. F. Chem. Rev. 2002, 102, 387-429. (19) Garcia-Parajo, M. F.; Veerman, J. A.; Bouwhuis, R.; Vallee, R.; van Hulst, N. F. ChemPhysChem 2001, 2, 347-360. (20) Slavik, J. Biochim. Biophys. Acta 1982, 694, 1-25. (21) (a) Odashima, K.; Itai, A.; Iitaka, Y.; Koga, K. J. Am. Chem. Soc. 1980, 102, 2504-2505. (b) Odashima, K.; Itai, A.; Iitaka, Y.; Arata, Y.; Koga, K. Tetrahedron Lett. 1980, 21, 4347-4350. (c) Koga, K.; Odashima, K. J. Inclusopm Phenom. 1989, 7, 53-60. (d) Odashima, K.; Itai, A.; Iitaka, Y.; Koga, K. J. Org. Chem. 1991, 50, 4478-4484.

Dansyl-Modified Saccharide Cyclophane

form 1:1 adducts with durene as confirmed by X-ray crystallography.21 Inclusion of various guest molecules within the hydrophobic cavity of 4‚nH+ in an acidic aqueous solution were confirmed by fluorescence and NMR spectroscopy. The tetraazacyclophanes can be modified by N-derivatization with side chains having various functional groups, via four nitrogens.22 A dansyl group and terminal galactose groups were adopted as fluorophore and saccharide residues, respectively, to introduce into the macrocycle. Dansyl fluorophores are utilized as an environmentally sensitive probe, whose emission is sensitive to change in microenvironmental polarity.17,23 On the other hand, terminal galactose residues were found to play biologically important rolls; e.g., naturally occurring receptors such as asialoglycoproteins24 on hepatocyte cell surfaces show terminal galactose specificity. On these grounds, we have now designed a new saccharide cyclophane bearing a dansyl group (1), which is composed of a tetraaza[6.1.6.1]paracyclophane skeleton and three branches with a terminal galactose residue and one with a dansyl derivative. The saccharide cyclophane bearing a dansyl group (1) was prepared from the tetraammonium derivative of tetraaza[6.1.6.1]paracyclophane (6)22c by a reaction with an equimolar amount of 5-(dimethylamino)-naphthalene1-sulfonyl chloride (dansyl-Cl) and subsequent aminolysis with lactonolactone, as shown in Scheme 1. We also prepared a saccharide cyclophane having four galactose residues 3 in a manner similar to that applied to the synthesis of 2.17 Even though compound 1 contains a hydrophobic cavity as well as a dansyl group, the nonionic cyclophane 1 is soluble in aqueous neutral media at biological pH owing to three saccharide moieties. Guest-Binding, Self-Association, and Fluorescent Properties of Dansyl-Modified Saccharide Cyclophane. We have previously clarified that water-soluble saccharide cyclophane 2 binds hydrophobic guests such as Dansyl-SE, 6-p-toluidinonaphthalene-2-sulfonate (TNS), and ANS in aqueous HEPES (N-(2-hydroxyethyl)piperazine-N′-2-ethane sulfonate) buffer (0.01 M, pH 7.0, µ 0.1 with NaCl) with binding constants (K) of 1.2 × 104, 3.1 × 103, and 1.0 × 103 M-1, respectively, at 298 K as evaluated by fluorescence spectroscopy.17 Guest-binding behavior by saccharide cyclophane having a terminal galactose group 3 was also examined by the identical methods. Upon addition of 3 to aqueous solutions containing each of the guests, a fluorescence intensity originating from the guest molecules was subjected to increase along with a concomitant blue shift of the fluorescence maximum, showing that the guest molecules are incorporated into the hydrophobic cavity provided by 3. The K values were evaluated on the basis of the Benesi-Hildebrand relationship25 and found to be 1.0 × 104, 3.0 × 103, and 1.1 × 103 M-1 for dansyl-SE, TNS, and ANS, respectively, which were almost comparable to those exercised by 2. Saccharide cyclophane 1 in a (22) (a) Murakami, Y.; Hayashida, O.; Nagai, Y. J. Am. Chem. Soc. 1994, 116, 2611-2612. (b) Ariga, K.; Terasaka, Y.; Sakai, D.; Tsuji, H.; Kikuchi, J. J. Am. Chem. Soc. 2000, 122, 7835-7836. (c) Hayashida, O.; Hamachi, I. Chem. Lett. 2003, 32, 288-289. (23) Mayer, R. T.; Himel, C. M. Biochemistry 1972, 11, 2082-2090. (24) Spiess, M. Biochemistry 1990, 29, 10009-10018. (25) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703-2707.

SCHEME 1. Preparation of Saccharide Cyclophanes

monomeric state (vide infra) also binds TNS and ANS with binding affinities of 9.7 × 103 and 3.6 × 103, respectively.26 Combination of these results and investigation of CPK molecular modeling indicated that compound 1, having a water-soluble cavity and a hydrophobic dansyl moiety that act as host and guest, respectively, was expected to form intermolecular complexes (self-aggregates) in a higher concentration of 1. First, concentration depen(26) For the binding experiments of 1 with TNS and ANS, upon addition of TNS (ANS) to aqueous solutions of 1 (5.0 × 10-6 M), a fluorescence intensity at 537 nm was increased along with a blue shift of the λem. The apparent binding constants were evaluated on the basis of the Benesi-Hildebrand relationship.

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FIGURE 2. Size distribution (in reference to number of particles) for aggregates of 1 in HEPES (0.01 M, pH 7.0, µ 0.1 with NaCl) buffer at 5.0 × 10-4 M as evaluated by DLS with 50 mW Ar laser at 298 K.

SCHEME 2. Schematic Representation for Self-Association of 1 and Heterodimers Formed with 1 and Cyclodextrins

FIGURE 1. (a) Fluorescence spectra for an aqueous HEPES (0.01 M, pH 7.0, µ 0.1 with NaCl) buffer of 1 at various concentrations at 298 K: (A) [1] ) 0, (B) 4.0 × 10-6, (C) 6.3 × 10-6, (D) 7.7 × 10-6, (E) 1.0 × 10-5, (F) 1.3 × 10-5, (G) 1.7 × 10-5, (H) 2.0 × 10-5, (I) 2.5 × 10-5, (J) 3.3 × 10-5, (K) 4.0 × 10-5, (L) 5.0 × 10-5, and (M) 6.7 × 10-5 M. (b) Correlation of the fluorescence maximum (λem) with logarithm of [1]. (c) Correlation of its intensity at 506 nm with [1].

dency of fluorescence spectra was investigated for an aqueous HEPES (0.01 M, pH 7.0, µ 0.1 with NaCl) buffer of 1 at various concentrations at 298 K (Figure 1). At least in the concentration below 1.0 × 10-5 M 1, the fluorescence intensity originating from the dansyl group of 1 increased in a linear fashion as its concentration increased without any change in its maximum (λem, 537 nm), as shown in Figure 1. In the higher concentrations in the range of 2.0 × 10-5 to 6.7 × 10-5 M, however, a large blue shift of the fluorescence maxima (∆λem, up to ∼30 nm at 6.7 × 10-5 M) and deviation from the linear concentration dependency of the fluorescence intensity were observed for 1 (Figure 1). These results indicate that 1 is in a monomeric state under these conditions at least in a concentration below 1.0 × 10-5 M and tends to form 3512 J. Org. Chem., Vol. 69, No. 10, 2004

intermolecular complexes in the higher concentrations.27 Dynamic light-scattering measurements (DLS) showed the presence of molecular aggregates with an average diameter of 7.7 ( 2 nm at a higher (5.0 × 10-4 M) concentration of 1 (Figure 2).28 In view of investigation of its CPK molecular models, the molecular size of 1 in the extended conformation is 2.6-3.3 nm (vide infra, see Figure 4). Accordingly, the molecular aggregates are considered to be a dimer (12) or oligomer (1n) of 1, as shown in Scheme 2. The emission originating from the dansyl group is sensitive to change in microenvironmental polarity, as mentioned above. We examined the solvent polarity dependency of fluorescence spectra of 1 (Figure 3). Figure 3b shows a correlation of maximum wavelength (λem) of fluorescence spectra of 1 and its intensity at 506 nm with a dioxane content [% (v/v)] in aqueous media. For all of the dioxane/water solutions, the concentration of 1 was maintained at 5.0 × 10-6 M in a monomeric species. With increasing dioxane contents, a fluorescence intensity originating from 1 increased along with a concomitant blue shift of the λem. The λem (537 nm) of 1 in aqueous (27) Similar concentration dependency was also confirmed by electronic absorption spectroscopy. In the higher (>1.2 × 10-5 M) concentrations of 1, their optical density was deviated from Beer’s law. (28) Because of detection limits of DLS equipment, meaningful DLS signals were not obtained for 1 under the diluted condition (1.0 × 10-5 M).

Dansyl-Modified Saccharide Cyclophane

FIGURE 3. Solvent polarity dependency of fluorescence

spectra of 1. (a) Fluorescence spectra of 1 (5.0 × 10-6 M) in dioxane/water solvents at 298 K by the dioxane content (%, v/v) in dioxane/water mixture: (A) 0, (B) 10, (C) 20, (D) 30, (E) 40, (F) 50, (G) 60, (H) 70, (I) 80, and (J) 90. (b) Correlations of the maximum wavelength (λem) of fluorescence spectra of 1 and its intensity at 506 nm with dioxane content (%, v/v).

HEPES buffer at the concentration of 5.0 × 10-6 M was also extrapolated on the above correlation plot. This result indicates that the dansyl moiety of 1 in a monomeric species was not included in its cyclophane cavity but partially exposed to the bulk aqueous phase, as shown in Scheme 2. In light of the CPK molecular model, the branched dansyl moiety of 1 does not have enough conformational flexibility for self-inclusion, because of the rigid amide and sulfonamide linkages. However, the λem (506 nm, see Figure 1) at the higher concentration of 6.7 × 10-5 M in aqueous HEPES buffer suggests the following properties: (i) In the concentration range for selfaggregates, the dansyl group of 1 was located in the apolar cyclophane cavity, as shown in Scheme 2 (dimer 12 and oligomer 1n). (ii) On the basis of a correlation between λem and solvent polarity, the microenvironmental polarity parameter,29 ETN, experienced by the dansyl moiety of 1 in the self-aggregate state was estimated to be 0.6, which was equivalent to values of 90% (v/v) dioxane-water and 1-butanol (ETN ) 0.58 and 0.60, respectively). Clearly, the dansyl moiety was well shielded from the bulk aqueous phase. Formation of Saccharide Cyclophane-Cyclodextrin Heterodimers. Cyclodextrins (CDs) are cyclic glucose oligomers with cylindrical shapes having the hydroxyl groups at the rims of the cylinder. The three most (29) Reichardt, C. Solvents and Solvent Effects in Organic Chemistry; VCH Verlagsgesellschaft: Weinheim, 1988; Chapter 7.

FIGURE 4. (a) Fluorescence spectral changes for aqueous

solution of 1 (6.0 × 10-6 M) upon addition of γ-CD in HEPES (0.01 M, pH 7.0, µ 0.1 with NaCl) buffer at 298 K; [γ-CD] ) (A) 0, (B) 2.4 × 10-4, (C) 4.8 × 10-4, (D) 7.2 × 10-4, (E) 9.6 × 10-4, (F) 1.2 × 10-3, (G) 1.4 × 10-3, (H) 1.7 × 10-3, and (I) 1.9 × 10-3 M. (b) A Benesi-Hildebrand plot for the fluorescence spectral changes (r ) 0.999).

common CDs are R-, β-, and γ-species, which are composed of six, seven, and eight glucopyranose units, respectively. R-, β-, and γ-CDs provide internal cavities with inner diameters of ∼5.7, ∼7.8, and ∼9.5 Å, respectively.30 The internal cavities are apolar relative to the bulk aqueous phase, so that CDs are capable of forming inclusion complexes with various hydrophobic guest molecules in aqueous media. For instance, β-CD moderately binds naphthalene with a binding constant (K) of 6.3 × 102 M-1, whereas γ-CD binds 1,5-dimethylnaphthalene with a K of 1.0 × 103 M-1 in water.31 Bioorganic chemists have long used natural and modified CDs in order to mimic a binding pocket of enzymes or proteins. On these grounds, we investigated complexation behavior of cyclophane 1 with CDs having a hydrophobic pocket by fluorescence spectroscopy. Upon addition of γ-CD to an aqueous HEPES (0.01 M, pH 7.0, µ 0.1 with NaCl) buffer containing 1 (6.0 × 10-6 M) in a monomeric species, a fluorescence intensity originating from the dansyl group of 1 increased, along with a concomitant blue shift of the λem (up to 525 nm), reflecting formation of the corresponding 1‚γ-CD complex, as shown in Figure 4 as a typical (30) Szejtli, J. In Comprehensive Supramolecular Chemistry; Szejtli, J., Ed.; Pergamon: Oxford, 1996; Vol. 3, Chapter 2, pp 5-56. (31) Fujiki, M.; Deguchi, T.; Sanenaga, I. Bull. Chem. Soc. Jpn. 1988, 61, 1163-1167.

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example. Similar complexation behavior of 1 was also confirmed with β-CD, 6-O-R-glucosyl-β-CD (glc-β-CD), and 6-O-R-maltosyl-β-CD (mal-β-CD). Binding constants (K) for the formation of 1:1 inclusion complexes of 1 with γ-CD, β-CD, glc-β-CD, and mal-β-CD were evaluated on the basis of the Benesi-Hildebrand25 relationship and found to be 7.5 × 102, 7.8 × 102, 7.7 × 102 and 6.0 × 102 M-1, respectively (r > 0.998). These values were comparable to those for naphthalene and 1,5-dimethyl-naphthalene with β- or γ-CD, despite the steric hindrance between 1 and CDs. These results suggested again that the dansyl moiety of 1 in a monomeric species was exposed to the bulk aqueous phase to bind readily with CDs. The dansyl group of 1 was bound to a cavity provided by CDs. The ETN values experienced by the dansyl moiety of 1 in the complexes was estimated to be 0.83-0.86 (λem ) 525-528 nm) by using a correlation between λem and solvent polarity (Figure 3), in a manner as described previously.32 These values were comparable to that provided by 1 in 50% (v/v) dioxane-water (ETN ) 0.83). Schematic representation for the heterodimers formed with cyclophane and cyclodextrin are shown in Scheme 2. Such complexation of 1 with β- and γ-CDs was confirmed by induced circular dichroism spectra in an aqueous HEPES (0.01 M, pH 7.0, µ 0.1 with NaCl) buffer at 298 K. Induced circular dichroism bands in the absorption range of the dansyl chromophore of 1 was observed for solutions of 1 in the presence of a large excess of β- or γ-CDs, which were attributable to the inclusion phenomena performed by CDs: [θ], 6.3 × 102 and 6.7 × 102 deg cm2 dmol-1 at 342 nm for β-CD and γ-CD, respectively. The supramolecular assembling of dansyl-modified cyclophane 1 and CDs occurred by inclusion of the dansyl moiety into the CD cavity, accompanying changes of fluorescence spectra as well as appearance of induced CD bands in aqueous media. On the other hand, R-CD having a smaller cavity shows less binding affinity for 1 in aqueous media; K value was not determined because of the low affinity. A possible computer-generated CPK model for the heterodimer composed of 1 and mal-β-CD is also shown in Figure 5.33 Such heterodimers are expected to be utilized as supramolecular saccharide clusters. By using this method, supramolecular saccharide clusters having various kinds of saccharide residues can be readily obtained in aqueous media, i.e., three terminal galactose moieties with a glucose residue and three galactose moieties with a maltose residue for 1‚GLC-β-CD and 1‚mal-β-CD, respectively. Cross-Linking Behavior of Saccharide Cyclophanes with Lectins. Carbohydrate-binding protein, socalled lectin, is normally composed of several subunits, each having a saccharide-binding site.18 Recently, various types of saccharide molecules based on polymers,6 dendrimers,9,10 and calixarenes12 have been developed in order to investigate saccharide-lectin interactions. On the other hand, compound 1 provides three terminal galactose residues with reasonably separated distances, which can be expected to act as multivalent ligands or (32) Hayashida, O.; Ono, K.; Murakami, Y. Tetrahedron 1995, 51, 8423-8436. (33) Optimized using MM2 force fields: Aped, P.; Allinger, N. L. J. Am. Chem. Soc. 1992, 114, 1-16.

3514 J. Org. Chem., Vol. 69, No. 10, 2004

FIGURE 5. A possible computer-generated CPK model for the heterodimer composed of 1 having three terminal galactose (gal) residues and mal-β-CD having a maltose (mal) branch. Carbon, hydrogen, oxygen, nitrogen, and sulfur atoms are shown in green, white, red, blue, and yellow, respectively.

tags for the specific lectins. In general, interactions between lectins and multivalent ligands are conveniently monitored by solution turbidity due to a cross-linked agglutination of these components.11b Agglutination behavior of 1 toward peanut agglutinin (PNA),34 galactoside-binding lectin, was examined by turbidity monitoring and fluorescence spectroscopy in aqueous HEPES buffer (0.01 M, pH 7.4, with 1 mM MnCl2, 1 mM CaCl2, µ 0.1 NaCl) at 298 K. Upon addition of PNA (0.13 mM, subunit basis) to the HEPES buffer containing 1 (0.33 mM) in an aggregated states, the solution becomes turbid very readily to give an insoluble material, whereas Con A, mannose-binding lectin, is never turbid with 1 as shown in Figure 6. Fluorescence spectroscopy is also a convenient method for evaluating the interactions between 1 and PNA. After centrifugation to remove the above insoluble materials (3‚PNA aggregates), the resulting solution shows lessened emission intensity as compared to those of the control experiments in the absence of PNA or in the presence of Con A. The decrease in fluorescence intensity indicates that 60% of 1 was complexed with PNA and removed as insoluble materials under the conditions. Moreover, the turbid solution obtained from 1 and PNA becomes again clear, showing deagglutination, upon addition of a large excess amount of lactose (0.07 M) as a competitive inhibitor (Figure 6). These results indicate that 1 specifically binds with PNA through saccharidelectin interaction. This agglutination/deagglutination is independent of the order of addition of the components, indicating equilibrium control. Similar agglutination behavior of 3 with PNA was confirmed by the turbiditymonitoring method. (34) Lotan, R.; Skutelsky, E.; Danon, D.; Sharon, N. J. Biol. Chem. 1975, 250, 8518-8523. (b) Neurohr, K. L.; Young, N. M.; Mantsch, H. H. J. Biol. Chem. 1980, 255, 9205-9209. (c) Banerjee, R.; Mande, S. C.; Ganesh, V.; Das, K.; Dhanaraj, V.; Mahanta, S. K.; Suguna, K.; Surolia, A.; Vijayan, M. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 227231. (d) Ravishankar, R.; Surolia, A.; Vijayan, M.; Lim, S.; Kishi, Y. J. Am. Chem. Soc. 1998, 120, 11297-11303.

Dansyl-Modified Saccharide Cyclophane

FIGURE 6. Photographs for aqueous HEPES buffers (0.01 M, pH 7.4, with 1 mM MnCl2, 1 mM CaCl2, µ 0.1 with NaCl) containing 1. Samples for entries 1-4 were prepared with the components indicated as crosses under the photos: final concentrations [1] ) 0.33 mM, [PNA] ) [Con A] ) 0.13 mM (subunit basis), and [lactose] ) 0.07 M. Photo B was taken upon irradiation with UV lights for the respective supernatants obtained after centrifugation.

Conclusion The dansyl-branched saccharide cyclophane 1 shows unique fluorescence properties in a monomeric species, reflecting microenvironmental polarity, which was demonstrated by changing the solvent systems and assembling with CDs. Moreover, the saccharide-lectin interactions were retained by the present cyclophane in aqueous media. To the best of our knowledge, the dansylbranched saccharide cyclophane 1 can be cited as the first example of performing microenvironment-dependent fluorescence sensing as well as binding with PNA. We believe that our concept of molecular design by the conjugation of an environment-sensitive fluorophore and saccharide cyclophanes provides a useful guidepost for development of fluorescent probes toward specific cell-surface receptors, directed by the choice of saccharide units displayed on the periphery of the cyclophane. The present dansylbranched saccharide cyclophane has also a hydrophobic cavity suitable for sequestering a guest molecule (drug) within. Development of saccharide-directed molecular/ drug delivery systems15 toward a specific cell is quite promising by using this strategy.

Experimental Section Binding Constants for Cyclophane-Cyclodextrin Heterodimers. To a solution of 1 (6.0 × 10-6 M) in HEPES (0.01

M, pH 7.0, µ 0.1 with NaCl) buffer were added increasing amounts of CDs at 298 K, and the dansyl fluorescence intensity (537 nm) was monitored after each addition. The binding constants were calculated on the basis of the Benesi-Hildebrand relationship for the titration data. N,N′,N′′,N′′′-Tetrakis[2-(tert-butoxycarbonylamino)propionyl]-1,6,20,25-tetraaza[6.1.6.1]pacacyclophane (5). Dicyclohexylcarbodiimide (740 mg, 3.6 mmol) was added to a solution of Boc-β-alanine (605 mg, 3.2 mmol) in dry dichloromethane (10 mL) at 0 °C, and the mixture was allowed to stand at the same temperature while being stirred for 20 min. 1,6,20,25-Tetraaza[6.1.6.1]pacacyclophane (4) (0.2 g, 0.40 mmol) in dry dichloromethane (10 mL) was added to the mixture, and the resulting mixture was stirred for 4 h at 0 °C and for an additional 18 h at room temperature. Precipitates that formed (N,N′-dicyclohexylurea) were removed by filtration, the solvent was eliminated under reduced pressure, and the residue was dissolved in ethyl acetate (60 mL). The solution was then washed with 10% aqueous citric acid (30 mL), saturated aqueous sodium chloride (30 mL), and 5% aqueous sodium hydrogen carbonate (60 mL) in this sequence. After being dried (MgSO4), the solution was evaporated to dryness under reduced pressure. The residue was chromatographed on a column of silica gel (SiO2) with ethyl acetate as eluent. The product fraction was dried in vacuo to give a white solid (0.37 g, 78%): Rf (Wako Silica Gel 70FM, ethyl acetate) 0.32. 1H NMR (600 MHz, CDCl , 303 K) δ 1.40 (s, 36H), 1.4 (m, 8H), 3 2.07 (m, 8H), 3.24 (m, 8H), 3.61 (m, 8H), 3.94 (s, 4H), 5.34 (m, 4H, NHCO), 6.92 and 7.19 (16H, ArH). 13C NMR (150 MHz, CDCl3, 298 K) δ 25.2, 28.8, 35.2, 36.7, 41.4, 48.9, 79,4, 128,8, 130.6, 140.6, 140.9, 156.3, 171.8. Found: C, 64.99; H, 7.74; N, 8.96. Calcd for C66H92N8O12‚2H2O: C, 64.68; H, 7.90; N, 9.14. IR (ATR) 2976, 2932, 1704, 1644 cm-1. MS (FAB) m/z 1190 [M + H1]+, 1212 [M + Na]+. HRMS (FAB) calcd for C66H93N8O12 1189.6913, found 1189.6915. N,N′,N′′,N′′′-Tetrakis[4-(amino)-propionyl]-1,6,20,25tetraaza[6.1.6.1]pacacyclophane Tetratrifluoroacetic Acid (6). Trifluoroacetic acid (0.3 mL) was added to a solution of compound 5 (0.1 g, 0.08 mmol) in dry dichloromethane (20 mL), and the mixture was stirred for 2 h at room temperature. After the solvent was evaporated off under reduced pressure, dichloromethane (20 mL) was added to the residue, and this procedure was repeated three times to remove remaining trifluoroacetic acid. Evaporation of the solvent under reduced pressure gave a glassy solid as the trifluoroacetic acid salt (0.1 g, quantitative): 1H NMR (600 MHz, CD3OD, 298 K) δ 1.50 (m, 8H), 2.43 (m, 8H), 3.13 (m, 8H), 3.69 (m, 8H), 4.04 (s, 4H), 5.2 (NH3), 7.13 and 7.33 (16H, ArH). 13C NMR (150 MHz, CD3OD, 298 K) δ 24.8, 31.5, 36.1, 40.9, 48.9, 113.6, 115.5, 117.4, 119.3, 128.4, 130.6, 139.8, 142.2, 160.2, 160.4, 160.7, 160.9, 170.5. IR (ATR) 2926, 1631 cm-1. MS (FAB) m/z 789.5 [M + H1]+. HRMS (FAB) calcd for C46H61N8O4 789.4816, found 789.4795. Saccharide Cyclophane Having Four Branches with a Terminal Glucose Residue (2). Maltose (6.0 g, 16.5 mmol) was oxidized with I2 (8.5 g, 67 mmol) in a methanol solution of KOH (4%) containing a minimal amount of water at room temperature. The carboxylate salt as initial product was recovered by filtration, washed repeatedly with cold methanol and ether, dried, taken in water, and upon treatment with an ion-exchange resin in the H+ form (Dowex 50) converted to the carboxylic acid, which was taken in methanol or ethanol and evaporated. This procedure of dissolution in alcohol followed by evaporation was repeated approximately 10 times until the material became completely methanol-soluble, thereby converting the carboxylic acid to the lactone (4.5 g). A solution of tetraamine 6 (0.1 g, 0.08 mmol) and triethylamine (0.1 mL, 0.8 mmol) in methanol (5 mL) was added to the methanol solution of the lactone (0.17 g, 0.48 mmol), and the mixture was stirred at 60 °C for 2 h and cooled to room temperature. The precipitates that separated were collected by filtration, washed with methanol, and dried in vacuo. Insoluble materials

J. Org. Chem, Vol. 69, No. 10, 2004 3515

Hayashida and Hamachi were removed by filtration, and the filtrate was evaporated to dryness in vacuo to give compound 2 (80 mg, 40%) as a white solid: 1H NMR (600 MHz, DMSO-d6, 298 K) δ 1.29 (m, 8H), 2.0 (m, 8H), 3.10 (m), 3.5 (m), 3.9 (s, 4H), 3.10, 3.18, 3.33, 3.39, 3.61, 3.93, 4.44, 4.66, 4.92, 5.38, 5.53 (carbohydrate), 7.1 (m, 8H), 7.3 (m, 8H), 7.5 (m 4H). 13C NMR (150 MHz, DMSO-d6, 298 K) δ 25.4, 34.8, 35.5, 48.9, 61.5, 63.5, 70.8, 72.3, 72.8, 73.1, 74.2, 83.8, 101.6, 129.1, 130.7, 140.8, 141.5, 170.8, 172.8. IR (ATR) 3320, 2927, 1634 cm-1. Found: C, 51.61; H, 6.69; N, 5.34. Calcd for C94H140N8O48‚2H2O: C, 51.64; H, 6.64; N, 5.13. MS (FAB) m/z 2151 [M + H1]+. Saccharide Cyclophane Having Four Branches with a Terminal Galactose Residue (3). Lactobionic acid (0.14 g, 0.4 mmol) was taken in methanol or ethanol and evaporated. This procedure of dissolution in alcohol followed by evaporation was repeated approximately 20 times until the materials became completely methanol-soluble, thereby converting the carboxylic acid to the lactone. A solution of tetraamine 6 (0.1 g, 0.08 mmol) and triethylamine (0.1 mL, 0.8 mmol) in methanol (5 mL) was added to the methanol solution of lactonolactone, and the mixture was stirred at 60 °C for 2 h and cooled to room temperature. The precipitates were collected by filtration, washed with methanol, and dried in vacuo. Insoluble materials were removed by filtration, and the filtrate was evaporated to dryness in vacuo to give compound 3 (80 mg, 40%) as a white solid: 1H NMR (600 MHz, DMSO-d6, 298 K) δ 1.29 (m, 8H), 2.03 (m, 8H), 3.2 (m), 3.5 (m), 4.0 (s, 4H), 3.2, 3.33, 3.44, 3.6, 3.65, 3.92, 3.96, 4.24, 4.51, 4.81, 5.19 (carbohydrate), 7.06 (m, 8H), 7.34 (m, 8H), 7.6 (m 4H). 13C NMR (150 MHz, DMSO-d6, 298 K) δ 25.4, 34.7, 35.6, 48.8, 61.5, 63.2, 69.1, 71.2, 71.9, 72.1, 72.9, 74.2, 76.5, 83.9, 105.9, 129.2, 130.7, 140.7, 141.5, 170.8, 172.9. IR (ATR) 3320, 2926, 1630 cm-1. Found: C, 51.81; H, 6.61; N, 5.25. Calcd for C94H140N8O48‚ H2O: C, 52.07; H, 6.60; N, 5.17. Dansyl-Appended Saccharide Cyclophane Having Three Branches with a Terminal Galactose Residue (1).

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Dansyl-Cl (25 mg, 0.09 mmol) was added to 6 (0.1 g, 0.08 mmol) dissolved in dry dichloromethane (5 mL) in the presence of triethylamine (0.1 mL), and the mixture was stirred for 2 h at room temperature. After the mixture was evaporated to dryness under reduced pressure, the resulting material was dissolved in methanol (5 mL). Lactonolactone (0.14 g, 0.4 mmol) and triethylamine (0.1 mL) dissolved in methanol (5 mL) were subsequently added to the mixture, and the resulting mixture was stirred for 18 h at room temperature. An insoluble material (saccharide cluster) was removed by filtration, and the solvent was evaporated off under reduced pressure. The crude product was purified by gel filtration chromatography on a column of Sephadex LH-20 with methanol as an eluant. Evaporation of the product fraction under reduced pressure gave a white solid (40 mg, 24%): 1H NMR (600 MHz, CD3OD, 313 K) δ 1.43 (m, 8H), 1.9-2.1 (m, 8H), 2.90 (s, 6H), 3.05 (m), 3.20 (m), 3.5, 3.6, 3.7, 3.6, 3.8, 3.85, 3.94, 4.02, 4.34, 4.50 (carbohydrate), 6.7 (m), 6.85-7.05 (m), 7.2 (m, 8H), 7.3 (m, 8H), 7.6 (m), 8.1 (m), 8.3 (m), 8.6 (m). 13C NMR (150 MHz, CD3OD, 318 K) δ 27.8, 34.2, 35.3, 39.2, 40.8, 44.9, 61.8, 62.9, 69.4, 71.4, 71.9, 72.3, 72.9, 73.9, 76.3, 82.3, 104.8, 115.5, 119.5, 123.4, 128.3, 128.5, 129.1, 129.9, 130.4, 136.2, 140.0, 141.5, 152.3, 172.1, 173.8. IR (ATR) 3330, 2926, 1632 cm-1. Found: C, 53.75; H, 6.68; N, 6.08. Calcd for C94H131N9O39S‚3H2O: C, 53.83; H, 6.58; N, 6.01. MS (FAB) m/z 2043 [M + H1]+, 2065 [M + Na]+. HRMS (FAB) calcd for C94H132N9O39S 2042.8343, found 2042.8325.

Acknowledgment. This work was partially supported by Izumi Science and Technology Foundation. Supporting Information Available: NMR spectra for compounds 1-6. This material is available free of charge via the Internet at http://pubs.acs.org. JO0496852