Spontaneous Fiber Formation and Hydrogelation of Nucleotide

Ankita Mitra , Vikramjit Sarkar , Balaram Mukhopadhyay ..... Debapratim Das , Antara Dasgupta , Sangita Roy , Rajendra Narayan Mitra , Sisir Debnath ...
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Chem. Mater. 2002, 14, 3047-3053

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Spontaneous Fiber Formation and Hydrogelation of Nucleotide Bolaamphiphiles Rika Iwaura,†,| Kaname Yoshida,‡,| Mitsutoshi Masuda,‡,| Kiyoshi Yase,§ and Toshimi Shimizu*,‡,| Graduate School of Chemistry, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan, Nanoarchitectonics Research Center (NARC), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan, Photonics Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan, and CREST, Japan Science and Technology Corporation (JST), Tokyo, Japan Received March 12, 2002. Revised Manuscript Received May 7, 2002

Nucleotide-appended bolaamphiphiles 1-7, in which two 3′-phosphorylated thymidine moieties are connected to both ends of a long oligomethylene spacer, have been first synthesized. Their self-assembling behavior in aqueous solutions was investigated in terms of gelling ability of water molecules. The longer homologues 6 and 7 with the C18 and C20 oligomethylene spacers, respectively, proved to be capable of gelling water very effectively (>25 000 water molecules per molecule) through spontaneous formation of a fibrous network. Gelation behavior of both bolaamphiphiles strongly depended on the pH and temperature of the aqueous solutions used. The gel-to-sol transition temperature (TGS) of 7 was determined to be approximately 85 °C. XRD measurement of a freeze-dried hydrogel from 7 suggested the presence of lamellar organization consisting of monolayer sheets. Hydrogen bonds involving the 5′-hydroxyl group of the deoxyribose moiety, hydrophobic interaction between the long oligomethylene chains, and π-π stacking of the thymine residues are responsible for the effective hydrogel formation.

Introduction Gels are now ubiquitous in our daily life, and yet they continue to attract considerable attention in terms of not only familiar foods such as jelly or pudding but also intriguing experimental materials for medical, analytical, and material science.1-5 In particular, hydrogels are of great interest in the near future, due to their capability of entrapping a large number of water molecules per one gelator molecule, which may be the key to solving various agricultural, environmental, and medical problems. A wide variety of polymeric hydrogelators have been described;6-9 to date there have been

some examples of nonpolymeric hydrogels spontaneously formed from small molecules.10-18 Low molecular weight hydrogelators should have a particular advantage in that one can easily control gel characteristics by changing preparation conditions such as the pH, temperature, and composition of the aqueous solutions. In careful view of the structural characteristics of nonpolymeric hydrogelators prepared so far, the rational molecular design should satisfy one more of the following three requirements: (i) the incorporation of a supplementary hydrophilic moiety at the middle position between the hydrophobic spacer and the hydrogen bond functionalities at each end,11,15,16 (ii) the presence

* To whom correspondence should be addressed at Nanoarchitectonics Research Center (NARC), AIST. Telephone: +81-298-61-4544. FAX: +81-298-61-4545. E-mail: [email protected]. † University of Tsukuba. ‡ Nanoarchitectonics Research Center (NARC), AIST. § Photonics Research Institute, AIST. | CREST, JST. (1) Zhang, L.; Yu, K.; Eisenberg, A. Science 1996, 272, 1777. (2) Terech, P.; Weiss, R. G. Chem. Rev. 1997, 97, 3133-3159. (3) Hanabusa, K.; Yamada, M.; Kimura, M.; Shirai, H. Angew. Chem., Int. Ed. Engl. 1996, 35, 1949-1951. (4) Jung, J. H.; Ono, Y.; Sakurai, K.; Sano, M.; Shinkai, S. J. Am. Chem. Soc. 2000, 122, 8648-8653. (5) Wang, R.; Geiger, C.; Chen, L.; Swanson, B.; Whitten, D. G. J. Am. Chem. Soc. 2000, 122, 2399-2400. (6) Chen, G.; Hoffman, A. S. Nature (London) 1995, 373, 49-52. (7) Yoshida, R.; Uchida, K.; Kaneko, Y.; Sakai, K.; Kikuchi, A.; Sakurai, Y.; Okano, T. Nature (London) 1995, 374, 240-242. (8) Wang, C.; Stewart, R. J.; Kopecek, J. Nature (London) 1999, 397, 417-420. (9) Lee, K. Y.; Mooney, D. J. Chem. Rev. 2001, 101, 1869-1880.

(10) Fuhrhop, J.-H.; Boettcher, C. J. Am. Chem. Soc. 1990, 112, 1768. (11) Newkome, G. R.; Baker, G. R.; Arai, S.; Saunders, M. J.; Russo, P. S.; Theriot, K. J.; Moorefield, C. N.; Rogers, L. E.; Miller, J. E.; Lieux, T. R.; Murray, M. E.; Phillips, B.; Pascal, L. J. Am. Chem. Soc. 1990, 112, 8458-8465. (12) Jokic´, M.; Makarevic´, J.; Zˇ inic´, M. J. Chem. Soc., Chem. Commun. 1995, 1723-1725. (13) Oda, R.; Huc, I.; Candau, S. J. Angew. Chem., Int. Ed. Engl. 1998, 37, 2689-2691. (14) Kogiso, M.; Ohnishi, S.; Yase, K.; Masuda, M.; Shimizu, T. Langmuir 1998, 14, 4978-4986. (15) Bhattacharya, S.; Acharya, S. N. G. Chem. Mater. 1999, 11, 3504-3511. (16) Estroff, L. A.; Hamilton, A. D. Angew. Chem., Int. Ed. 2000, 39, 3447-3450. (17) Menger, F. M.; Caran, K. L. J. Am. Chem. Soc. 2000, 122, 11679-11691. (18) Marmillon, C.; Gauffre, F.; Gulik-Krzywicki, T.; Loup, C.; Caminade, A.-M.; Rump, E. Angew. Chem., Int. Ed. 2001, 40, 26262629.

10.1021/cm020263n CCC: $22.00 © 2002 American Chemical Society Published on Web 06/13/2002

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of chiral centers,10,12,13 and (iii) a bola-form amphiphile rather than the mono-head derivative.11,12,14,16,17,19 Nucleotides are some of the most complex among the small biological molecules since they contain the greatest variety of hydrogen-bonding functional groups. Therefore, nucleotides can easily catch water molecules and are generally hydrated in the crystalline state; about 45% of their crystal structures are hydrates, presenting a striking contrast to 20% for carbohydrates.20 Nucleotides can also be very useful as a programmable headgroup when we make use of their multiple complementary hydrogen bonds to obtain nanometer-sized structures through self-assembly.21-25 Considering the promising importance of both hydrogels and nucleotides as new materials, the fusion of these two concepts should afford interesting features applicable to a wide variety of biological and medical uses such as biocompatible materials, gels for electrophoresis, and gene engineering. From this point of view, we first describe herein the synthesis of novel nucleotide bolaamphiphiles with a 3′-phosphorylated thymidine moiety at each end of oligomethylene spacers and their excellent gelling ability as hydrogelators. Most advantageously, the design of this nucleotide bolaamphiphiles is quite compatible with the structural prerequisites mentioned above. In other words, (i) the sugar and phosphodiester moieties play an important role in imparting the additional hydrophilic functionality, (ii) the sugar moiety can provide multiple chiral centers, and (iii) the thymidine moiety is connected to both ends of long oligomethylene chains. Experimental Section Materials and General Methods. 5′-O-dimethoxytrityl2′-deoxythymidine-3′-O-[O-(2-cyanoethyl)-N,N-diisopropyl phosphoramidite] was purchased from Amersham Pharmacia Biotech and was used after drying in a vacuum. The structures of final products were confirmed by NMR, FT-IR, and highresolution ESI-FTMS (electrospray ionization Fourier transform mass spectrometry) spectroscopy. 1H NMR spectra were recorded on a JEOL 600 spectrometer. For the FT-IR measurement, a Jasco FT-IR-620 (resolution 4 cm-1) was used. ESI-FTMS spectra were measured using a Bruker Daltonics Apex II 70e. The UV absorption spectra were recorded at 25 °C on a Hitachi U-3300 spectrometer. For the evaluation of pKa value for the compound 7, aqueous solutions (10 mL) were prepared by addition of degassed distilled water to 7 and subsequent sonication at 25 °C. The obtained aqueous solutions (10 mM) were then titrated with 1 N NaOH. For the transmittance spectra measurement, compound 7 was gelatinized with water in a quartz cell and the transmittance was monitored at 500 nm. The sample was equilibrated at each temperature for 30 min. Molecular mechanics calculations with the dynamics and the molecular force field were conducted with the SONY Tektronix CAChe system (version 4.1.1). An optimized model of the nucleotide (19) Jung, J. H.; Shinkai, S.; Shimizu, T. Chem.sEur. J, in press. (20) Jeffrey, G. A.; Saenger, W. Hydrogen Bonding in Biological Structures; Springer-Verlag: Berlin, 1993. (21) Yanagawa, H.; Ogawa, Y.; Furuta, H.; Tsuno, K. J. Am. Chem. Soc. 1989, 111, 4567-4570. (22) Pincet, F.; Perez, E.; Bryant, G.; Lebeau, L.; Mioskowski, C. Phys. Rev. Lett. 1994, 73, 2780-2783. (23) Bonaccio, S.; Capitani, D.; Segre, A. L.; Walde, P.; Luisi, P. L. Langmuir 1997, 13, 1952-1956. (24) Berti, D.; Barbaro, P.; Bucci, I.; Baglioni, P. J. Phys. Chem. B 1999, 103, 4916-4922. (25) Forman, S. L.; Fettinger, J. C. Pieraccini, S.; Gottarelli, G.; Davis, J. T. J. Am. Chem. Soc. 2000, 122, 4060-4067.

Iwaura et al. bolaamphiphile 7 was constructed using a editor in CAChe system. The starting conformation of the nucleoside was defined so as to involve the C3′- or the C2′-puckering for the deoxyribose moiety and an anti- or a syn-orientation for the thymine moiety. Synthesis of Nucleotide Bolaamphiphiles. The synthetic route to 1-8 is shown in Scheme 1. A 1,ω-alkanediol (0.14 g, 0.7 mmol) and 1H-tetrazol (0.2 mg, 2.9 mmol) were added to a solution of 5′-O-dimethoxytrityl-2′-deoxythymidine3′-O-[O-(2-cyanoethyl)-N,N-diisopropyl phosphoramidite] (1.0 g, 1.4 mmol) in 30 mL of dry tetrahydrofuran, and the reaction mixture was stirred overnight. To the reaction mixture was added 1.6 mL of 70% tert-butylhydroperoxide solution to oxidize phosphite, and then the mixture was allowed to stand for 30 min. The removal of the protecting cyanoethyl group was carried out with 8 mL of 25% ammonia solution. The reaction mixture was stirred for 2 days and evaporated under reduced pressure. The silica gel column chromatography of the residue using a mixture of chloroform and methanol (4/1, v/v) as an eluent gave a compound protected with dimethoxytrityl group. A solution of 5% trifluoroacetic acid in chloroform was then added to remove the protecting group. The residual precipitate obtained by evaporation was washed with chloroform and dried. The compounds 1-7 were obtained as a white powder. The degree of purity was checked by TLC, 1H NMR, and ESI-FTMS, indicating >99.9% and 99% for compounds 1, 2, 3, 4, and 7 and for compounds 5 and 6, respectively. Selected data for 1: yield 33%; mp 232 °C dec; TLC (chloroform-methanol, 1:1, v/v) Rf ) 0.71; 1H NMR (600 MHz in D2O at 25 °C) δ 1.20-1.34 (m, 16H, CH2(CH2)8CH2), 1.59 (dd, 4H, CH2(CH2)8CH2), 1.90 (s, 6H, 5-Me), 2.35-2.54 (m, 4H, 2′-H), 3.75-3.80 (m, 4H, 5′-H), 3.86-3.89 (m, 4H, CH2CH2(CH2)8CH2CH2), 4.19 (br, 2H, 4′-H), 4.77 (br, 2H, 3′-H), 6.28 (t, 2H, 1′-H), 7.66 (s, 2H, 6-H). ESI-FTMS: calcd for [M - H]-, 809.2781; found, 809.2770. FT-IR (KBr): ν 3414, 3069, 2931, 2857, 1685, 1477, 1105 cm-1. All compounds 2-7 also gave satisfactory spectral data (see Supporting Information). Table 1 indicates the total reaction yields and physical properties of a series of nucleotide bolaamphiphiles 1-7. Synthesis of Acetylated Derivative 8. Acetic anhydride (5 g, 50 mmol) was added to a solution of compound 7 (10 mg, 10 µmol) in 7 mL of dry pyridine, and the reaction mixture was stirred overnight and evaporated under reduced pressure. The residue was dissolved in 5 mL of methanol, and the resultant precipitate was collected. The compound 8 was obtained as a white powder: yield 98%; mp 255 °C dec; TLC (chloroform-methanol, 1:1, v/v) Rf ) 0.48; 1H NMR (600 MHz in DMSO at 25 °C) δ 1.20-1.35 (m, 32H, CH2(CH2)16CH2), 1.49 (dd, 4H, CH2(CH2)16CH2), 1.90 (s, 6H, 5-Me), 2.04 (s, 6H, 5′Ac), 2.35-2.54 (m, 4H, 2′-H), 3.86-3.89 (m, 4H, CH2CH2(CH2)16CH2CH2), 4.19 (br, 2H, 4′-H), 4.77 (br, 2H, 3′-H), 6.28 (t, 2H, 1′-H), 7.66 (s, 2H, 6-H), ESI-FTMS: calcd for [M - H]-, 1005.4244; found, 1005.4260. FT-IR (KBr): ν 3060, 2925, 2853, 1746, 1698, 1471, 1102 cm-1. Gelation Test. The aqueous dispersions (0.5 mL) containing 10 mg of compounds 1-7 were heated to boiling and refluxed for 10 min to completely dissolve the compound in water and gradually allowed to cool to room temperature. If the solid aggregate mass was stable to inversion of the container when the test tube was turned upside down, the compound was recognized to form a hydrogel. LM, TEM, SEM, and AFM Observations. Light microscopy (LM) was carried out in a similar way to those described in detail elsewhere.26 The specimens were observed using a LEICA DMRX. The hydrogel formed from 7 was put on a poly(vinyl formal) supporting film for TEM or a sample stage for SEM and AFM, both of which were allowed to stand at room temperature for 24 h and then washed with small amount of water. The specimens were dried at room temperature for further 24 h. TEM, SEM, and AFM were conducted on CarlZeiss LEO912, JEOL JSM-5000, and Digital Instruments Nano Scope III, respectively. (26) Shimizu, T.; Masuda, M. J. Am. Chem. Soc. 1997, 119, 28122818.

Nucleotide Bolaamphiphiles

Chem. Mater., Vol. 14, No. 7, 2002 3049 Scheme 1a

a Key: (i) 1,ω-alkanediol, 1H-tetrazol, THF; (ii) tert-butylhydroperoxide; (iii) 25% aqueous NH ; (iv) 5% TFA; (v) acetic anhydride, 3 pyridine.

Table 1. Reaction Yield and Physical Properties of the Nucleotide Bolaamphiphiles 1-7

Table 2. Gelation Test of 2.0 wt % of the Nucleotide Bolaamphiphiles 1-7a

HRMSa [M - H]compound

n

yield (%)

calcd

found

mp (°C)

1 2 3 4 5 6 7

12 13 14 15 16 18 20

35 40 25 36 30 20 36

809.2781 823.2937 837.3094 851.3250 865.3407 893.3720 921.4033

809.2770 823.2937 837.3074 851.3255 865.3423 893.3719 921.4022

232 dec 225 dec 230 dec 232 dec 227 dec 230 dec 233 dec

a

The high-resolution mode of ESI-FTMS was used.

X-ray Diffraction (XRD). XRD was measured using a Rigaku diffractometer (Type 4037) using graded d-space elliptical side-by-side multilayer optics, monochromated Cu KR radiation (40 kV, 30 mA), and imaging plate (R-Axis IV). Freeze-dried hydrogel from 7 was obtained in a glass tube by vacuum-drying to constant weight and then put in a capillary as it is, without being powdered. Crystalline solid samples 1-7 were used as prepared from synthesis and subjected to the analysis.

Results Gelation Behavior. The gelation behavior of 1-7 was found to strongly depend on the pH of the solution used and the length of oligomethylene spacers (Table 2). The pKa value of 7 was evaluated to be 3.7 by a conventional titration method. Therefore, most of the phosphodiester moieties of 7 should be protonated at pH ) 1.68, about 66% ionized at pH ) 4.01, and fully ionized at pH 7.55 and 9.18. The bolaamphiphiles 1-7 were insoluble in a potassium tetraoxalate buffer at pH 1.68 and quite soluble in more alkaline buffers. In particular, bolaamphiphiles 1-3 remained soluble in buffers (pH 4.01-9.18) and Milli-Q water without

compound

n

1 2 3 4 5 6 7

12 13 14 15 16 18 20

pH 1.68b pH 4.01c pH 7.55d pH 9.18e I I I I I I I

S S S S S V G

S S S S S V G

S S S S S S G

Milli-Q water S S S C V G G

a I: insoluble. S: homogeneous solution. G: opaque gel. C: leaflet crystal. V: giant vesicles. b 0.05 M potassium tetraoxalate buffer. c 0.05 M phthalate buffer. d 0.05 M HEPES buffer. e 0.01 M sodium tetraborate buffer.

producing any self-assembled structures. The compound 4 gave a leaflet crystal as shown in Figure 1a. Interestingly, the formation of giant vesicles with diameters of 1-2 µm was observable for the bolaamphiphile 5 in Milli-Q water and 6 in the buffers (pH 4.01-7.55) (Figure 1b). The vesicles retained their spherical morphologies for several weeks. In contrast, the longer homologue 7 resulted in the formation of robust hydrogels with Milli-Q water and the buffers (>pH 4.01). Gel structure was also stable over a week under neutral or mild alkaline conditions. It should be noted here that the gelation of water can be attained even at concentrations of 0.2 wt % (>25 000 water molecules per one hydrogelator molecule) (Figure 2). Gel-to-Sol Phase Transition. The temperature dependence of the transmittance (Figure 3) for the obtained hydrogel was monitored to clarify the gel-tosol phase transition behavior. The transmittance of the hydrogel from 7 remains constant (∼0%) and then starts to drastically increase at temperatures higher than 80 °C. The hydrogel and a small amount of water were

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Iwaura et al.

Figure 3. Transmittance dependence of the hydrogel from 7 on the temperature upon heating (measured at λ ) 500 nm).

Figure 1. (a) Polarized light micrograph of a leaflet crystal from 4 in Milli-Q water. (b) Dark-field micrograph of a giant vesicle from 5 in Milli-Q water.

Figure 4. (a) EF-TEM, (b) SEM, and (c) AFM images of the hydrogel from 7.

Figure 2. Hydrogel formed from the nucleotide bolaamphiphile 7 (C ) 0.2 wt %).

apparently separated at 85 °C, showing a sudden decrease in viscosity, and eventually turned into a clear solution. Therefore, the gel-to-sol transition temperature (TGS) of 7 was determined to be approximately 85 °C, indicating a considerably higher value among those reported so far.11,15,17

Microscopic Observations of the Hydrogel. Figure 4a displays the energy-filtering transmission electron micrograph (EF-TEM)27 of the hydrogel of 7, which was taken with unstained specimens. Intertwined nanofibers are observable with well-defined size dimensions of ca. 10-30 nm in diameter and several micrometers in length, which eventually form developed fiber networks. Figures 4b and 4c display the scanning electron (27) Reimer, L. Energy-Filtering Transmission electron microscopy; Springer-Verlag: Berlin, 1995.

Nucleotide Bolaamphiphiles

Chem. Mater., Vol. 14, No. 7, 2002 3051 Table 3. Molar Absorptivity (E)a and Hypochromicityb of the Nucleotide Bolaamphiphiles 1-7 compound

n

 (×104)

H (%)

1 2 3 4 5 6 7

12 13 14 15 16 18 20

1.69 1.47 1.54 1.56 1.49 1.33 1.36

3 20 17 18 14 28 27

a Measured at λ b max ) 270 nm. Compared with 5′-thymidinemonophosphate (5′-TMP). The value of hypochromicity H (%) was evaluated according to the following equation: H (%) ) 100[1-(/ 25′-TMP)].

Figure 5. AFM image of the hydrogel from 7 and its crosssection profile. Figure 7. X-ray diffraction pattern of a freeze-dried hydrogel formed from 7.

Figure 6. Dependence of the methylene stretching band frequencies on the olligomethylene spacer length (n) for the self-assemblies from 1-7.

micrograph (SEM) and an atomic force microscopic (AFM) image. No remarkable, helically twisted fibrous morphologies, which often appear in microscopic images of organogels,3,28 were observed here. On the basis of AFM cross-section profile, we found instead ribbon-type fibers with 10 nm thickness and 80 nm width as a typical constituent of the hydrogel (Figure 5). FT-IR Spectroscopy. The CH2 antisymmetric and symmetric FT-IR bands were measured using the dried self-assemblies obtained from nucleotide bolaamphiphiles 1-7 with Milli-Q water (Figure 6). It is well-known that the frequencies of the CH2 stretching bands are very sensitive to the conformation of oligomethylene chain.29,30 The relatively higher frequencies of the CH2 stretching bands for 1 at 2931 and 2857 cm-1 suggest the existence of a gauche conformation on the oligomethylene chain. The CH2 antisymmetric and symmetric stretching bands (28) Snip, E.; Shinkai, S.; Reinhoudt, D. N. Tetrahedron Lett. 2001, 42, 2153-2156. (29) Snyder, R. G.; Strauss, H. L.; Elliger, C. A. J. Phys. Chem. 1982, 86, 5145-5150. (30) Masuda, M.; Vill, V.; Shimizu, T. J. Am. Chem. Soc. 2000, 122, 12327-12333.

tend to decrease with increasing the length of oligomethylene chains. Eventually, the CH2 stretching bands for 7 at 2921 and 2850 cm-1 strongly support the existence of a highly populated all-trans conformation on the oligomethylene chain. UV Spectroscopy. Nucleobase stacking is one of the important interactions in DNA to stabilize a doublehelix structure. To confirm the base stacking in the selfassemblies of the nucleotide bolaamphiphiles, we examined their UV spectra in aqueous solutions (C ) 1 × 10-4 M). On the basis of the concentration dependence of 1H NMR measurement, we confirmed that except for 1 the bolaamphiphiles should self-assemble into aggregates in the solution even at this concentration. The resulting molar absorptivity () and hypochromicity H (%) values are summarized in Table 3. The H (%) values were calculated by a comparison of the absorption intensity with 5′-thymidinemonophosphate (5′-TMP). Compounds 1-5 gave H (%) values varying from 3 to 20%, whereas the bolaamphiphiles 6 and 7 were 28 and 27%, respectively. This finding is well compatible with the fact that the thymine moieties are effectively stacking in the self-assemblies of 6 and 7.31 Considering the excellent hydrogelation ability of the compounds 6 and 7, we can conclude that the base stacking interaction is playing important role in stabilizing the self-assemblies and resultant hydrogelation. XRD Measurement. To get further insight into the molecular packing and orientation within the selfassembled ribbon structures, we measured X-ray diffraction (XRD) of a freeze-dried hydrogel of 7. A sharp reflection peak appears at d ) 3.59 nm in the small angle region together with a single broad reflection band at around d ) 0.43 nm (Figure 7), indicating the structural feature characterized by a long period of 3.59 nm. Molecular mechanics calculations of 7 were carried (31) Itahara, T. Bull. Chem. Soc. Jpn. 1997, 70, 2239-2247.

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Iwaura et al. Table 4. XRD Data of Crystalline Compounds 1-7

compound

n

XRD patterns long spacing d (nm)

1 2 3 4 5 6 7

12 13 14 15 16 18 20

1.63 1.78 1.95 1.97 2.07 2.28 2.24

molecular modelinga fully extended molecular length (nm) 2.62 2.47 2.87 2.72 3.09 3.32 3.51

a Calculated lengths from extended chain CPK molecular models.

molecular packing in the lamellar structure. For comparison, we also looked at the d-spacings of crystalline 1-6, since neither hydrogel nor xerogel was obtainable from 1-6 (Table 4 and Supporting Information). The chain-length (n) dependence of the d-spacings indicated almost regular, linear progression as n increases, suggesting the similar packing feature through the compounds 1-7. As a result, we found no remarkable structural features for 6 and/or 7 in the crystalline state, which may show why the compounds 6 and 7 differ in the gelation ability from others. Discussion Figure 8. (a) Minimized structure of 7 with a hooklike conformation at each end. (b) Detailed conformation of the nucleoside moiety with the C3′-end puckering and an antiorientation using a wire molecular model. Hydrogens are omitted to simplify.

out using the dynamics and the molecular force field. The nucleotide component of the nucleic acids generally occurs as a limited number of preferred conformations.20 Therefore, we used the most plausible C3′- or C2′-end conformation as well as an anti- or a syn-orientation as the puckering of the deoxyribose ring and the base orientation relative to the sugar moiety, respectively. As a result of the careful calculation, we obtained a hooklike conformation for the nucleotide moiety of each end of 7 (Figure 8a). The minimized conformation of the nucleoside was obtained as the C3′-end puckering of the deoxyribose ring and an anti-orientation of the base relative to the sugar moiety (Figure 8b). The evaluated molecular length (3.51 nm) from this model is well consistent with the 3.59 nm long period obtained from XRD measurement. The molecules 7 should, therefore, be arranged parallel with respect to the normal to the monolayer plane, resulting in the formation of the ribbon-type fiber in Figure 5. More interestingly, two hydroxyl groups of the phosphate and the deoxyribose moieties are found to be exposing to external environment from the molecule. This situation is much favorable for the effective hydrogen bonding to water molecules. Furthermore, the thymine group is shielded from the aqueous environment to readily form the base stacking arrangement. On the other hand, the XRD diagram of crystalline 7 obtained just after synthesis was compared with that in Figure 7, giving a broad reflection peak at d ) 2.24 nm in the small angle region. This finding remarkably contrasts to the d value 3.59 nm obtained for the freezedried hydrogel of 7. We thus suppose that the hydrogel formation is accompanied by rearrangement of the

A consideration of the various functionalities including nucleobase, sugar, and phosphodiester moiety found within the nucleotide bolaamphiphile allows us to draw some conclusions concerning their role in the hydrogelation process. When the carbon numbers of the oligomethylene chains are less than C16, no efficient hydrogelation took place at any pH regions of the buffer solutions. In contrast, compounds 6 and 7 with the longer C18 and C20 oligomethylene spacers exhibited excellent gelation ability under neutral and weak alkaline aqueous conditions, suggesting that the fundamental driving force of the hydrogelation is the intermolecular hydrophobic interaction between the long oligomethylene chains. The phosphodiester moiety can drastically change the solubility of the bolaamphiphiles since none of the bolaamphiphiles 1-7 is soluble in aqueous solutions when the phosphodiester moieties are fully protonated under acidic conditions. The increase in the degree of ionization of the phosphodiester moieties allows a gradual increase in solubility, in balance with the hydrophobicity of each molecule. The deoxyribose moiety has triple chiral centers as well as one hydroxyl group easily accessible to water molecules. The acetylated derivative 8 was synthesized to reveal the effect of the 5′-hydroxyl group of the ribose on gel formation. Compound 8 is sparingly soluble in water and found to fail to form hydrogels at any pH regions, indicating that the 5′-hydroxyl group plays an important role in hydration of the nucleotide bolaamphiphiles. A minimized conformation of 7 can rationally support this feature. Simpler, nucleobase-appended bolaamphiphiles with thymine or adenine residues separated by oligomethylene spacers are also known to self-assemble in water to produce supramolecular nanofibers in a crystalline state without forming a hydrogel.32 (32) Shimizu, T.; Iwaura, R.; Masuda, M.; Hanada, T.; Yase, K. J. Am. Chem. Soc. 2001, 123, 5947-5955.

Nucleotide Bolaamphiphiles

For the rational design of efficient hydrogelators, this difference in the self-assembled state between crystalline precipitate and hydrogels supports the necessity of the incorporation of both deoxyribose and phosphodiester groups into the bolaamphiphiles. UV spectra study suggests that π-π stacking interaction of the thymine residues should be also responsible for the hydrogel formation. Actually, no remarkable π-π stacking interaction can be observed in liposomes prepared from phosphatidyl nucleosides, resulting in no hydrogelation.23 In addition, the thymine moiety is involved in hydrogen bonding since the addition of equimolar, complementary adenine molecules to the solution completely inhibits the hydrogelation of the compound 7. Conclusion Novel nucleotide bolaamphiphiles with a 3′-phosphorylated thymidine moiety at each end of long oligom-

Chem. Mater., Vol. 14, No. 7, 2002 3053

ethylene chains have been synthesized. The longer homologues with the C18 and C20 oligomethylene spacers proved to be capable of gelling water through the spontaneous formation of fibrous network, which strongly depend on the pH and temperature of the aqueous solutions. Acknowledgment. We thank Drs. T. Ihara and M. Ohnishi-Kameyama for advice concerned with synthesis and ESI-FTMS measurement, respectively. Supporting Information Available: Text giving additional analytical data for 2-7 and figures showing 1H NMR and ESI-FTMS spectra of all compounds and XRD diagrams of crystalline 1-7. This material is available free of charge via the Internet at http://pubs.acs.org. CM020263N