Alkyl Chain Length Dependent Hydrogelation of l-Tryptophan-Based

Langmuir 2007, 23, 11769-11776

11769

Alkyl Chain Length Dependent Hydrogelation of L-Tryptophan-Based Amphiphile Sangita Roy, Antara Dasgupta, and Prasanta Kumar Das* Department of Biological Chemistry, Indian Association for the CultiVation of Science, JadaVpur, Kolkata-700 032, India ReceiVed May 29, 2007. In Final Form: August 27, 2007 The search for low molecular weight hydrogelators (LMWHs) with varying structural motif is getting intense because of its potential application in biomedicines as well as the diversified area of nanobiotechnology. Hydrophobic interaction is one of the most crucial parameters in the design and development of such LMWHs. To this notion, a methodical investigation was carried out to find the influence of varying alkyl chain length of amphiphile on water gelation efficacy, which has been only marginally addressed in the literature to date. We have synthesized a series of low molecular weight L-tryptophan-based gelators, some of which are excellent gelator for plain water, an essential criterion for biological use. The alkyl chain induced hydrophobicity at the molecular level has remarkable influence in modulating water immobilization. Water gelation efficiency was improved more than 100 times on moving from 10 to 18 carbon atoms. The self-aggregation behavior of these thermoreversible hydrogelators investigated through different spectroscopic and microscopic techniques showed that an optimum balance between hydrophilicity and hydrophobicity is indeed essential, which can be largely regulated by varying the alkyl chain length. Thus, the study offers better understanding toward tailoring the properties of gel in plain water and thereby paving the way for potential applications.

Introduction Although “gels” are quite prevalent in our daily life, they continue to evoke intense interest to modern day chemists owing to their intriguing properties along with versatile applicability in wide ranging fields starting from advanced materials to biotechnology.1 Among them gels of aqueous solutions (hydrogels) are of particular interest, as this is an essential criteria for biomedicinal use.1,2 Hydrogels, capable of entrapping a large number of water molecules per gelator molecule, are rapidly emerging as an important class of soft materials in recent years both in the field of material science as well as biomedicinal chemistry. They offer excellent potential in tissue engineering, biosensing, controlled drug delivery, and biomedicinal implants as well as in nanobiotechnology.1-3 Classically, hydrogels are made from high molecular weight natural polymers4 (collagens, polysaccharides) and also from hydrophilic synthetic polymers5 [poly(acrylic acid) and derivatives and polypeptides]. Although gels derived from low molecular weight hydrogelators (LMWHs) have been known over a 100 * Corresponding author. Also at Centre for Advanced Materials, Indian Association for the Cultivation of Science. Fax: +(91)-33-24732805. E-mail: [email protected]. (1) (a) Estroff, L. A.; Hamilton, A. D. Chem. ReV. 2004, 104, 1201. (b) Lee, K. Y.; Mooney, D. J. Chem. ReV. 2001, 101, 1869. (c) Tiller, J. C. Angew. Chem., Int. Ed. 2003, 42, 3072. (d) van Bommel, K. J. C.; van der Pol, C.; Muizebelt, I.; Friggeri, A.; Meetsma, A.; Feringa, B. L.; van Esch, J. Angew. Chem., Int. Ed. 2004, 43, 1663. (e) Sangeetha, N. M.; Maitra, U. Chem. Soc. ReV. 2005, 34, 821 and references therein. (f) Bhattacharya, S.; Srivastava, A.; Pal, A. Angew. Chem., Int. Ed. 2006, 45, 2934. (g) George, S. J.; Ajayaghosh, A.; Jonkheijm, P.; Schenning, A. P. H. J.; Meijer, E. W. Angew. Chem., Int. Ed. 2004, 43, 3422. (h) Vemula, P. K.; John, G. Chem. Commun. 2006, 2218. (i) Vemula, P. K.; Aslam, U.; Ajay Mallia, V.; John, G. Chem. Mater. 2007, 19, 138. (2) (a) Ko¨hler, K.; Fo¨rster, G.; Hauser, A.; Dobner, B.; Heiser, U. F.; Ziethe, F.; Richter, W.; Steiniger, F.; Drechsler, M.; Stettin, H.; Blume, A. J. Am. Chem. Soc. 2004, 126, 16804. (b) Suzuki, M.; Yumoto, M.; Kimura, M.; Shirai, H.; Hanabusa, K. Chem. Eur. J. 2003, 9, 348. (c) Estroff, L. A.; Hamilton, A. D. Angew. Chem., Int. Ed. 2000, 39, 3447. (d) Herres, A.; van der Pol, C.; Stuart. M.; Friggeri, A.; Feringa, B. L.; van Esch, J. J. Am. Chem. Soc. 2003, 125, 14252. (e) Estroff, L. A.; Leiserowitz, L.; Addadi, L.; Weiner, S.; Hamilton, A. D. AdV. Mater. 2003, 15, 38. (f) Nakashima, T.; Kimizuka, N. AdV. Mater. 2002, 14, 1113. (g) Kobayashi, H.; Friggeri, A.; Koumoto, K.; Amaike, M.; Shinkai, S.; Reinhoudt, D. N. Org. Lett. 2002, 4, 1423. (h) Bieser, A. M.; Tiller, J. C. Chem. Commun. 2005, 3942.

years,1a,6 active investigation on them have only started in the last couple of decades,2,3 presumably because they could not match the superior material properties of the macromolecular hydrogelators.7a-c Nevertheless, polymeric hydrogelators often have certain limitations owing to their constrained synthesis imposed by their thermosetting nature.7d,e Nonpolymeric hydrogelators are less abundant compared to small molecule organogelators, which have drawn considerable attention in soft material research from long back.8 However, LMWHs (“supramolecular gels” or “physical gels”) are considered to be a (3) (a) Yang, Z.; Xu, B. Chem. Commun. 2004, 2424. (b) Kohler, K.; Forster, G.; Hauser, A.; Dobner, B.; Heiser, U. F.; Ziethe, F.; Richter, W.; Steiniger, F.; Drechsler, M.; Stettin, H.; Blume, A. Angew. Chem., Int. Ed. 2004, 43, 245. (c) Wang, G.; Hamilton, A. D. Chem. Commun. 2003, 310. (d) Vinogradov, S. V.; Bronich, T. K.; Kabanov, A. V. AdV. Drug. DeliV. ReV. 2002, 54, 135. (e) Friggeri, A.; Feringa, B. L.; van Esch, J. J. Controlled Release 2004, 97, 241. (f) Park, S. M.; Lee, Y. S.; Kim, B. H. Chem. Commun. 2003, 2912. (g) John, G.; Vemula, P. K. Soft Matter 2006, 2, 909. (h) Vemula, P. K.; Li, J.; John, G. J. Am. Chem. Soc. 2006, 128, 8932. (4) Chirila, T. V.; Constable, I. J.; Crawford, G. J.; Vijayasekaran, S.; Thompson, D. E.; Chen, Y.-C.; Fletcher, W. A.; Griffin, B. J. Biomaterials 1993, 14, 26. (b) Tabata, Y.; Ikada, Y. AdV. Drug DeliVery ReV. 1998, 31, 287. (c) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. Macromolecules 1994, 27, 7654. (d) Akiyoshi, K.; Deguchi, S.; Tajima, H.; Nishikawa, T.; Sunamoto, J. Macromolecules 1997, 30, 857. (5) (a) Chu, Y. H.; Chen, J. K.; Whitesides, G. M. Anal. Chem. 1993, 65, 1314. (b) Lee, K.; Asher, S. A. J. Am. Chem. Soc. 2000, 122, 9534. (c) Pan, G.; Kesavamoorthy, R.; Asher, S. A. J. Am. Chem. Soc. 1998, 120, 6525. (d) Sano, M.; Okamura, J.; Shinkai, S. Langmuir 1999, 15, 7890. (e) Philippova, O. E.; Rulkens, R.; Kovtunenko, B. I.; Abramchuk, S. S.; Khokhlov, A. R.; Wegner, G. Macromolecules 1998, 31, 1168. (f) Li, Y.; Tang, Y.; Narain, R.; Lewis, A. L.; Armes, S. P. Langmuir 2005, 21, 9946. (6) von Lipowitz, A. Ann. Chem. Pharm. 1841, 38, 348. (7) (a) Nishikawa, T.; Akiyoshi, K.; Sunamoto, J. J. Am. Chem. Soc. 1996, 118, 6110. (b) Osada, Y.; Gong, J.-P. AdV. Mater. 1998, 10, 827. (c) Novick, S. J.; Dordick, J. S. Chem. Mater. 1998, 10, 955. (d) Shah, K. R. In Polymeric Materials Encyclopedia; Salamone, J. C., Ed.; CRC Press: Boca Raton, FL, 1996; p 3092. (e) Bhattacharya, S.; Acharya, S. N. G. Chem. Mater. 1999, 11, 3504. (8) (a) Terech, P.; Weiss, R. G. Chem. ReV. 1997, 97, 3133. (b) Weiss, R. G.; Ramamurthy, V.; Hammond, G. S. Acc. Chem. Res. 1993, 26, 530. (b) de Loos, M.; van Esch, J.; Stokroos, I.; Kellogg, R. M.; Feringa, B. L. J. Am. Chem. Soc. 1997, 119, 12675. (c) Jung, J. H.; Ono, Y.; Hanabusa, K.; Shinkai, S. J. Am. Chem. Soc. 2000, 122, 5008. (d) Xing, B.; Choi, M.-F.; Xu, B. Chem. Commun. 2002, 362. (e) Placin, F.; Desvergne, J.-P.; Lassegues, J.-C. Chem. Mater. 2001, 13, 117. (f) Xing, B.; Choi, M.-F.; Xu, B. Chem. Eur. J. 2002, 8, 5028.

10.1021/la701558m CCC: $37.00 © 2007 American Chemical Society Published on Web 10/06/2007

11770 Langmuir, Vol. 23, No. 23, 2007 Chart 1

Roy et al.

explore the influence of alkyl chain length with its hydrophobic domain on water gelation efficacy, in the present study, we have investigated the hydrogelation ability of L-tryptophan-based amphiphiles by systematically varying the alkyl chain length from 10 to 18 carbon atoms (1-6, Chart 1). How the minute structural changes at the molecular level tune the supramolecular arrangement in the respective hydrogels was addressed using several spectroscopic and microscopic techniques. The study reveals that an optimal chain length of 12 carbons is essential to immobilize water while maintaining a balance between the hydrophilic and hydrophobic groups. Gelation ability increases with chain length, which led to the development of gelator 5 (0.2% w/v), with an alkyl chain of 18 carbon atoms, the best among all the amphiphilic hydrogelators. Experimental Section

better choice over the polymeric counterpart, primarily because of their rapid response to external stimuli and thermoreversible nature due to noncovalent supramolecular association within self-aggregates as well as possible biodegradibilty.1,2,3,9 Supramolecular gels are basically an entangled threedimensional (3D) network formed by aggregation of small molecules where intermolecular interactions, such as π-π stacking, hydrogen bonding, hydrophobic, electrostatic, and dipole-dipole interactions, play a key role. Attempts toward understanding such interactions help to design an excellent gelator for water rather than getting it serendipitously. Furthermore, a major challenge is to search out efficient gelators from an abundant precursor that also would be ecofriendly in nature. To this end, amino acid based compounds are one of the potent candidates to be gelators, because of their inherent biocompatibility, leading to their application in the biomedicinal arena. Also synthetic methodologies on amino acid based chemistry are relatively simple and well-established. To this end, recently, we have reported the excellent water gelation ability of a L-tryptophan (4, Chart 1) amphiphile in plain water.10a The presence of both hydrophilic and hydrophobic groups within the same molecule allows them to get aggregated in an ordered fashion to induce immobilization of water.1a,11 Our previous work was mainly focused to understand the influence of head groups in regulating the gelation properties of amphiphiles, and notably, the existing major thrust in the literature is also on the functional moieties to understand the mechanism of gelation.10a Only a very few reports are available on systematic alteration in the alkyl chain length part to tune water gelation ability.12 It is quite lucid from the earlier literature that primarily it is not the hydrogen bonding but rather the hydrophobic interactions that are the key parameter to control the water gelation of the LMWH.12a,13 In order to (9) (a) Kawano, S.; Fujita, N.; Shinkai, S. J. Am. Chem. Soc. 2004, 126, 8592. (b) Xing, B.; Yu, C. W.; Chow, K. H.; Ho, P. L.; Fu, D.; Xu, B. J. Am. Chem. Soc. 2002, 124, 14846. (c) Kiyonaka, S.; Sugiyasu, S.; Hamachi, I. J. Am. Chem. Soc. 2002, 124, 10954. (d) Fuhrhop, J.-H.; Schnieder, P.; Rosenberg, J.; Boekema, E. J. Am. Chem. Soc. 1987, 109, 3387. (e) Hanabusa, K.; Hiratsuka, K.; Kimura, M.; Shirai, H. Chem. Mater. 1999, 11, 649. (10) (a) Das, D.; Dasgupta, A.; Roy, S.; Mitra, R. N.; Debnath, S.; Das, P. K. Chem. Eur. J. 2006, 12, 5068. (b) Das, D.; Roy, S.; Das, P. K. Org. Lett. 2004, 6, 4133. (c) Roy, S.; Das, D.; Dasgupta, A.; Mitra, R. N.; Das, P. K. Langmuir 2005, 21, 10398. (d) Dasgupta, A.; Mitra, R. N.; Roy, S.; Das, P. K. Chem. Asian J. 2006, 1, 780. (11) (a) Moniruzzaman, M.; Sundararajan, P. R. Langmuir 2005, 21, 3802. (b) Hafkamp, R. J. H.; Feiters, M. C.; Nolte, R. J. M. J. Org. Chem. 1999, 64, 412. (c) Jung, J. H.; Shinkai, S.; Shimizu, T. Chem. Eur. J. 2002, 8, 2684. (12) (a) Khatua, D.; Maiti, R.; Dey, J. Chem. Commun. 2006, 4903. (b) Mohmeyer, N.; Schmidt, H-W. Chem. Eur. J. 2005, 11, 863. (13) (a) Israelchivili, J. Intermolecular and Surface Forces, 2nd ed.; Academic Press: New York, 1991. (b) Gaspar, L. J. M.; Baskar, G. J. Mater. Chem. 2005, 15, 5144.

Materials. L-Tryptophan, n-dodecylamine, n-tetradecylamine, n-hexadecylamine, n-octadecylamine, dicyclohexylcarbodiimide (DCC), 4-N,N-(dimethylamino)pyridine (DMAP), iodomethane, sodium hydride, solvents and all other reagents were procured from SRL. HPLC grade water was purchased from Qualigens. All the deuteriated solvents for NMR experiments, 8-anilino-1-naphthalenesulfonic acid (ANS), and n-decylamine were obtained from Aldrich Chemical Co. Thin layer chromatography was performed on Merck precoated silica gel 60-F254 plates. Amberlyst A-26 chloride ionexchange resin was obtained from BDH. 1H NMR spectra were recorded on AVANCE 300 MHz (BRUKER) spectrometer. Mass spectrometric (MS) data were acquired by the electron spray ionization (ESI) technique on a Q-tof-Micro Quadruple mass spectrophotometer (Micromass). Synthesis of [2-(1H-Indol-3-yl)-1-alkylcarbamoylethyl]trimethylammonium Chloride (Chart 1, 1-5). Boc-protected Ltryptophan was coupled with the corresponding n-alkylamine using DCC (1 equiv) and a catalytic amount of DMAP in the presence of 1 equiv of N-hydroxybenztriazole in dry DCM. Boc-protected amide was then purified through column chromatography using 60-120 mesh silica gel and acetone/hexane as the eluent. Amide was then subjected to deprotection by trifluoroacetic acid (4 equiv) in dry DCM. After 2 h of stirring, solvents were removed on a rotary evaporator, and the mixture was taken in ethyl acetate. The EtOAc part was thoroughly washed with aqueous 10% sodium carbonate solution followed by brine to neutrality. The organic part was dried over anhydrous sodium sulfate and concentrated to get the corresponding amine. The primary amine (1 equiv) thus obtained was quaternized with excess iodomethane using 2.2 equiv of anhydrous potassium carbonate and a catalytic amount of 18-crown-6-ether in dry DMF for 2 h. The reaction mixture was taken up in ethyl acetate and washed with aqueous thiosulfate solution and water, respectively. The concentrated ethyl acetate part was then subjected to column chromatography using 60-120 mesh silica gel and methanol/ chloroform as the eluent and, finally, was crystallized from methanol/ ether to obtain solid quaternized iodide salt (synthetic scheme for 1-5 is given in the Supporting Information). The iodide salt thus obtained was subjected to ion exchange on Amberlyst A-26 chloride ion-exchange resin column to get the pure chloride (1-5). The overall yield was ∼60-70%. Synthesis of [2-(1H-Indol-3-yl)-1-octadecylcarbamoylethyl]ammonium Chloride (Chart 1, 6). Boc-protected L-tryptophan was coupled with the corresponding n-alkylamine using DCC (1 equiv) and a catalytic amount of DMAP in the presence of 1 equiv of N-hydroxybenztriazole in dry DCM. Boc-protected amide was then purified through column chromatography using 60-120 mesh silica gel and acetone/hexane as the eluent. Amide was then subjected to deprotection by trifluoroacetic acid (4 equiv) in dry DCM. After 2 h of stirring, solvents were removed on a rotary evaporator, and the mixture was taken up in ethyl acetate. The EtOAc part was thoroughly washed with aqueous 10% sodium carbonate solution followed by brine to neutrality. The organic part was dried over anhydrous sodium sulfate and concentrated to get the corresponding amine. The primary

Hydrogelation of L-Tryptophan-Based Amphiphile amine was protonated with concentrated hydrochloric acid in methanol for 12 h. The ammonium salt thus obtained was crystallized from dry ether to get the pure chloride (6). Overall yield was ∼70%. Compound 1: 1H NMR (300 MHz, CDCl3) δ ) 0.84 (t, 3H), 0.97-0.99 (br, 2H), 1.20-1.62 (br, 14H), 2.88-3.10 (m, 4H), 3.29 (s, 9H), 5.32 (br, 1H), 7.06-7.11 (br, 2H), 7.34-7.36 (d, 1H), 7.43 (br, 1H), 7.52-7.57 (d, 1H); MS (ESI) m/z calcd for C24H40N3O (the 4° ammonium ion, 100%): 386.317, found 386.4493 [M+]. Anal. Calcd (%) for C24H40N3OCl: C, 68.30; H, 9.55; N, 9.96. Found: C, 68.45; H, 9.60; N, 9.99. Compound 2: 1H NMR (300 MHz, CDCl3) δ ) 0.87 (t, 3H), 0.97-0.99 (br, 2H), 1.18-1.33 (br, 16H), 1.34-1.45 (br, 2H), 2.86 (br, 2H), 3.06 (br, 2H), 3.39 (s, 9H), 5.35 (br, 1H), 7.05-7.14 (br, 2H), 7.34-7.36 (d, 1H), 7.45-7.60 (br, 2H); MS (ESI) m/z calcd for C26H44N3O (the 4° ammonium ion, 100%) 414.348, found 414.525 [M+]. Anal. Calcd (%) for C26H44N3OCl: C, 69.38; H, 9.85; N, 9.34. Found: C, 69.50; H, 9.96; N, 9.45. Compound 3: 1H NMR (300 MHz, CDCl3) δ ) 0.88 (t, 3H), 0.90-1.05 (br, 2H), 1.25-1.38 (br, 20H), 1.35-1.47 (br, 2H), 2.91 (m, 4H), 3.38 (s, 9H), 5.71 (br, 1H), 7.1-7.17 (br, 2H), 7.37-7.40 (br, 2H), 7.55-7.57 (d, 1H); MS (ESI) m/z calcd for C28H48N3O (the 4° ammonium ion, 100%) 442.379, found 442.5803 [M+]. Anal. Calcd (%) for C28H48N3OCl: C, 70.33; H, 10.12; N, 8.79. Found: C, 70.48; H, 10.23; N, 8.80. Compound 4: 1H NMR (300 MHz, CDCl3) δ ) 0.83 (t, 3H), 0.97-0.99 (br, 2H), 1.11-1.32 (br, 24H), 1.66-1.73 (br, 2H), 2.822.91 (m, 2H), 3.20 - 3.24 (m, 2H), 3.31 (s, 9H), 5.60 (br, 1H), 7.01-7.06 (br, 1H), 7.29-7.31 (d, 2H), 7.41 (d, 1H), 7.48-7.51 (d, 1H); MS (ESI): m/z calcd for C30H52N3O (the 4° ammonium ion, 100%): 470.41; found: 470.5699 [M+]. Anal. Calcd (%) for C30H52N3OCl: C, 71.18; H, 10.35; N, 8.30. Found: C, 71.28; H, 10.43; N, 8.35. Compound 5: 1H NMR (300 MHz, CDCl3) δ ) 0.87 (t, 3H), 0.97-1.02 (br, 2H), 1.11-1.32 (br, 28H), 1.38-1.45 (br, 2H), 2.803.07 (m, 4H), 3.30 (s, 9H), 5.39-5.41 (br, 1H), 7.05-7.15 (br, 2H), 7.36-7.42 (br, 2H), 7.48-7.54 (d, 1H). MS (ESI) m/z calcd for C32H56N3O (the 4° ammonium ion, 100%) 498.442, found 498.2522 [M+]. Anal. Calcd (%) for C32H56N3OCl: C, 71.94; H, 10.57; N, 7.87. Found: C, 71.90; H, 10.66; N, 8.06. Compound 6: 1H NMR (300 MHz, CDCl3): δ ) 0.87 (t, 3H), 1.25-1.38 (br, 30H), 1.38-1.49 (br, 2H), 3.07-3.17 (m, 2H), 3.383.41 (d, 2H), 4.22 (t, 1H), 7.04-7.16 (m, 2H), 7.33-7.39 (br, 2H), 7.66-7.69 (d, 1H); MS (ESI) m/z calcd for C29H50N3O (the 4° ammonium ion, 100%) 456.395, found 456.7159 [M+]. Anal. Calcd (%) for C29H50N3OCl: C, 70.77; H, 10.24; N, 8.54. Found: C, 70.90; H, 10.48; N, 8.75. Preparation of the Hydrogel. The aqueous dispersions of the required amount of compounds 1-5 were slowly heated to dissolve them in plain water and then allowed to cool slowly (undisturbed) to room temperature in a vial with i.d. of 10 mm. After 20-30 min a colorless and transparent gel was obtained, which was verified as stable by inversion of the glass vial: when the glass vial was turned upside down, the stable aggregate did not flow downward. Determination of Gel-Sol Transition Temperature (Tgel). The gel to sol transition temperature (Tgel) was determined by placing a hydrogel-containing inverted screw-capped glass vial with i.d. of 10 mm in a thermostated oil bath, and then the temperature was raised at a rate of 2 °C/min. Here, the Tgel was defined as the temperature ((0.5 °C) at which the hydrogel melts and starts to flow out of the gel. Microscopic Study. Field emission scanning electron microscopy (SEM) measurements were performed on a JEOL-6700F microscope. A piece of gel was mounted on a glass slide for SEM sampling and dried for a few hours under vacuum before imaging. Atomic Force Microscopic Measurements. Atomic force microscopic images (AFM) were acquired using a Nanosurf easyscan AFM system. A piece of gel was mounted on a silicon wafer and dried for a few hours under vacuum before imaging. FTIR Measurements. FTIR measurements of the gelators in CHCl3 solution and dried gel from D2O were carried out in a Shimadzu

Langmuir, Vol. 23, No. 23, 2007 11771 FT-IR 8100 spectrophotometer using a 1 mm KBr cell and silicon wafer, respectively, at the minimum gelation concentration. NMR Measurements. 1H NMR and 2D-NOESY spectra of 3 and 5 were recorded on an AVANCE 300 MHz (Bruker) spectrometer at 1.5% (w/v, in plain water) for 3 and 1% (w/v, in plain water) for 5, respectively. Circular Dichroism (CD). CD spectra of both the aqueous solutions of all four amphiphiles at varying concentration as well as 0.04% (w/v) aqueous solutions of 5 with varying temperatures from 20 to 80 °C were recorded by using a quartz cuvette of 1 mm path length with a JASCO J-815 spectropolarimeter. X-ray Powder Diffraction (XRD). XRD measurements were taken with a Seifert XRD 3000P diffractometer, and the source was Cu KR radiation (R ) 0.15406 nm) with a voltage and current of 40 kV and 30 mA, respectively. Dried gels of 2-5 were mounted on the aluminum holder and scanned from 1 to 10°. Fluorescence Spectroscopy. The emission spectra of ANS were recorded on Varian Cary Eclipse luminescence spectrometer by adding the probe molecules in aqueous solutions of hydrogels 5 at varying concentrations at room temperature. ANS was initially dissolved in MeOH and from this superstock solution the required amount of ANS solution was added to the experimental solutions [5 µL of superstock (0.01 M) was added to a 5 mL aqueous solution of the gelators to reach the probe concentration of 1 × 10-5 M]. The ANS solutions were excited at λex ) 360 nm. The intrinsic fluorescence of 5 due to tryptophan moiety was measured by using varying concentrations of 5 at room temperature. A superstock solution of 5 was prepared, which was further diluted as required. Solutions were excited at λex ) 280 nm.

Results and Discussion Despite low molecular weight hydrogelators being long known, the literature available till to date still lacks a proper systematic study on the structure-function relationship that controls the self-aggregation of LMWGs. It has already been revealed that while hydrogen bonding appears to be the common driving force for gelation of organic liquids, hydrophobic forces become the major regulator in an aqueous environment.12a,13 Since aggregation of LMWHs need a critical balance between hydrogen bonding and hydrophobic effect, we thought to systematically vary the alkyl chain length of our L-tryptophan-based hydrogelator to see the influence of this hydrophobic domain on gelation efficiency and the corresponding molecular arrangement at the aggregated structure. To this notion, five positively charged amphiphiles (1-5) (Chart 1) were synthesized whose hydrogelation ability in plain water was tested by the “stable-to-inversion” of the container method (Table 1).14a Interestingly, the minimum gelation concentration (MGC) was found to vary drastically from 1 to 5 with hydrophobic chain length. Amphiphile 1, with an alkyl chain length of 10 carbon atoms, formed a very weak opaque gel with a MGC of 15% w/v (only ∼160 water molecules per gelator molecule at the MGC), having a poor water retention capacity. However, 2 formed a relatively stable transparent gel with a moderately high water retention capacity (∼560 water molecules per gelator molecule) at a MGC of 5% w/v, suggesting that an optimal chain length of 12 carbon atoms is essential to show water-gelating capacity. MGCs were rather low, 1.1 and 0.3% w/v, respectively for 3 and 4 with an increase in alkyl chain length. The observed trend led to the development of the efficient gelator 5, with an alkyl chain of 18 carbon atoms, that formed a transparent gel with a further low MGC of 0.2% w/v. This gel can hold a large number (∼14 800 water molecules per gelator molecule at MGC) of water molecules in the interstitial spaces (14) (a) Menger, F. M.; Caran, K. L. J. Am. Chem. Soc. 2000, 122, 11679. (b) Mukhopadhyay, S.; Maitra, U.; Ira; Krishnamoory, G.; Schmidt, J.; Talmon, Y. J. Am. Chem. Soc. 2004, 126, 15905.

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Roy et al. Table 1. Gelation Results of 1-5 in Plain Water

gelator

statea

MGC (% w/v)

1 2 3 4 5

OG TG TG TG TG

15 5 1.1 0.3 0.2

H2O per gelatorb

Tgel at zMGC (°C)

representative X-ray diffraction (nm) d-spacing

molecular modelingc fully extended molecular length (nm)

160 560 2400 9300 14800

40 45 48 39 40

3.25 3.46 3.89 3.92

2.12 2.37 2.74 2.85

a OG, opaque hydrogel; TG, transparent hydrogel. b Number of water molecules entrapped by one gelator molecule. c Molecular modeling was carried out using CS Chem Office 3D MOPAC (AM1 method).

Figure 1. Variation of the Tgel with concentration of 1, 2, 3, and 5.

(Table 1). Also, the gelation ability of 5 did not change across a pH range of 2-10 (10 mM). Thus, the variation in the alkyl chain length can improve the gelation efficiency more than 100 times with the alteration in hydrophobic domain in the selfaggregate of LMWHs. All hydrogels are thermoreversible in nature, melting upon slow heating and again turning to gel on cooling. The gel melting temperatures (Tgel) of all five compounds were almost in the comparable range as their MGC, only being slightly higher for 3 (Figure 1). In concurrence with the literature, Tgel was found to increase with gelator concentration.14 The above study reveals the dramatic influence of alkyl chain length induced hydrophobicity on the intermolecular interaction among the individual monomer in the 3D network of the supramolecular gels. All four transparent gels were quite stable at room temperature for several months. The morphology of the supramolecular network was inspected through SEM, where all xerogels except 1 displayed a typical entangled fibrous network (Figure 2) of varying thickness at their MGC (SEM picture of 4 has been reported in our previous study10a). Gelator 1 having a hydrocarbon chain of only 10 carbon atoms formed a very weak gel due to immobilization of a small quantity of water. Accordingly, a very loosely packed thin fibrous 3D network formed within the selfassembly of 1 could not be visualized prominently through SEM. 2, with a relatively better water retention capacity, also formed thin fibrous network of 100-150 nm compared to 3 and 5, where a thick fibrous morphology of twisted ribbon-like network of thickness 300-500 nm was observed (Figure 2). Morphology of the gel surface was further investigated by determining the surface roughness using noncontact mode atomic force microscopy (AFM) for the most efficient gelator, 5. The topography in AFM images of the dried gel 5 revealed that it was quite

rougher at MGC (0.2%, w/v), where the root-mean-square (rms) roughness (Rq)15 was 67.3 nm compared to that observed at the nongelated state (0.02%, w/v), Rq, 13.2 nm (Figure 3). The gelator molecule probably shows the propensity to aggregate into a helical fiber at a much lower concentration, which gets cross-linked noncovalently at the MGC to form a hydrogel. To obtain insight into the induced helicity in the aggregate structure expected to originate from the chiral monomer during the process of gelation, expression of supramolecular chirality was measured by taking the circular dichroism (CD) spectra of 1-5 (Figure 4a for 5; see Supporting Information for other gelators). A positive cotton effect was observed in the amide absorption region, i.e., at 220-225 nm, that could be attributed to the π-π* transition of the amide bond along with a shoulder at longer wavelength from the n-π* transition of the same bond.16a-c These transitions are extremely sensitive to coupling with neighboring amides. An increase in the molar ellipticity at 225 nm with gelator concentration also suggests a highly ordered superhelical arrangement of chiral planes at the supramolecular level induced by the L-tryptophan residues.16a-c The observed supramolecular chirality that emerged through noncovalent organized packing of molecular components was again supported by a variable temperature CD study of 5 at 0.05% w/v (Figure 4b). The intensity of the CD peak at 220-225 nm gradually decreased with an increase in temperature from 20 to 80 °C as a result of the gel to sol state transition, leading to the destruction of the three-dimensional aggregate structure (Figure 4b). This study clearly depicts that it is the supramolecular chirality of the self-assembled aggregates in the gel state responsible for the observed helicity.16d Even at 80 °C, a slight CD signal exists, probably due to the fact that though the molecule 5 was in the solution state, it may contain small self-aggregates. Now, it is essential to have a better understanding of the intermolecular interactions that primarily regulate such supramolecular arrangement of individual gelators in the 3Dframework, as seen in the preceding paragraphs. In this context, a NMR technique is known to impart a quantitative idea on the orientation of gelator molecules in the self-assembled state. In concurrence with our previous observation for amphiphiles 4, here, too, a 1H NMR study using 1.5 and 1.0% w/v of 3 and 5, respectively, in [D6]DMSO with varying water content showed the contribution of both amide N-H (Ha) and indole N-H (Hb) toward controlling the self-assembly of the amphiphiles (Figure 5).10a The amide N-H shifted upfield (from 8.48 to 8.19 ppm) for 5, as the water content increased up to 10% and then it remained almost constant up to 30% water content (from 8.19 to 8.14 ppm). It shifted further upfield to 7.93 ppm with an increase in (15) Zhang, X.-Z.; Yang, Y.-Y.; Chung, T.-S.; Ma, K.-X. Langmuir 2001, 17, 6094. (16) (a) Moffitt, W. J. Chem. Phys. 1956, 25, 467. (b) Gratzer, W. B.; Holzwarth, G. M.; Doty, P. Proc. Natl. Acad. Sci. U.S.A. 1961, 47, 1785. (c) Zhang, Y.; Gu, H.; Yang, Z.; Yu, B. J. Am. Chem. Soc. 2003, 125, 13680. (d) Friggeri, A.; van der Pol, C.; van Bommel, K. J. C.; Heeres, A.; Stuart, M. C. A.; Feringa, B. L.; van Esch, J. Chem. Eur. J. 2005, 11, 5353.

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Figure 2. (A-C) FESEM images of the dried samples of 2, 3, and 5 at the MGC.

Figure 3. AFM images of 5 (a) at 0.02% and (b) at 0.2% (w/v).

water proportion up to 50%. On the other hand, indole N-H showed a continuous upfield shift from 11.01 to 10.25 ppm with an increase in water content up to 50%. Similar phenomenon was observed for 3, where amide N-H shifted from 8.32 to 7.96 ppm, being constant (∼8.15 ppm) over 10-30% water content, and indole N-H also shifted upfield from 10.98 to 10.31 ppm from 0 to 50% water content (Figure 5). Upon initiation of the self-assembling process with increasing water content, the bulky indole group presumably twisted toward the hydrophobic domain of the self-aggregate, thereby exposing the carbonyl group in the aqueous phase.17 Such change in orientation of the gelator molecule possibly localize the amide and indole N-H in the hydrophobic region, resulting in an upfield shift of both protons.17 Also, with an increase in water content, the ammonium segment of the molecule gets hydrated, leading to the upfield shift of the neighboring amide and indole N-H. Within 10-30% water content amide proton possibly starts participating in the intermolecular hydrogen bonding with the carbonyl oxygen, which may lead to local dehydration around this proton. Consequently, that would initiate the formation of a fibrous network and is also expected to result in a downfield shift of amide proton. However, the continuous shielding effect due to the increasing water at the headgroup region and the development of hydrophobic domain as a result of the increased π-π interaction between the parallel indole moieties compete with the deshielding effect, yielding almost no change in the chemical shift of amide protons within 10-30% water content. Indole N-H was continuously upfield shifted, as it probably has not participated in intermolecular hydrogen bonding. Above 30%, the shielding (17) Billiot, F. H.; McCarroll, M.; Billiot, E. J.; Rugutt, J. K.; Morris, K.; Warner, I. M. Langmuir 2002, 18, 2993.

effect plays the predominant role, where both protons showed upfield shift. In continuation of the preceding observation, we did further investigation on the possible intermolecular interactions between gelator and its neighbor molecules using 2D NOESY experiments with 3 (1.5% w/v) and 5 (1.0% w/v) in [D6]DMSO and also in presence of 40% water. As expected, no off-diagonal cross-peak was observed for the nongelated state of the amphiphiles in [D6]DMSO. However, at 40% water content, where self-assembling process has already initiated toward gelation, the off-diagonal cross-peaks were observed between both indole N-H and aromatic protons and also with hydrogens of quaternary ammonium methyl and aromatic protons (Figure 5). These strong interactions, consistent with that observed in the 1D NMR study, indicate the significance of these two sets of protons in playing a crucial role toward gelation through space. No such long-range interactions were observed in only [D6]DMSO experiment which additionally complement the obvious contribution of N-H and C-H‚‚‚π interaction in gelation.18 To find out how important is the participation of the H’s of the methyl substitution of the ammonium segment in the gelation mechanism, we have synthesized compound 6, which is complementary to amphiphile 5 with an unsubstituted ammonium group (Chart 1). However, compound 6 failed to gelate water, indicating that methyl substitutions are probably necessary at the head group region for a strong interaction between its H’s and the aromatic ring, as supported by the 2D NMR study. Presumably, the absence of methyl groups changes the polarity of compound 6, which largely affects its solubility in water. It has a very low solubility in water at room temperature, and on heating, though it dissolves, it again precipitated out on cooling to room temperature. Since hydrophobic interaction as mentioned in the 1H NMR study is playing a central role in the hydrogelation of LMWGs, it was further probed by luminescence spectra using ANS in the gelation process of 5. With an increase in concentration of 5 from 0.001 to 0.05% w/v, the ANS intensity at first rapidly increases to a maximum with the blue shift from 510 nm (in plain water) to ∼480 nm. Thereafter, up to 0.4% w/v only a moderate blue shift was observed to ∼476 nm, with almost no change in the emission intensity (Figure 6a). Such luminescence behavior of ANS is a distinct feature of the existence of the hydrophobic environment, indicating its participation in hydrogelation.2b,14b,19 The observed steady intensity at a concentration ∼10 times lower than the MGC of 5 showed its propensity to aggregate further into fibers, as observed in AFM and CD study. Besides ANS, we have decided to probe the gelation (18) (a) Yang, Z.; Xu, K.; Wang, L.; Gu, H.; Wei, H.; Zhang, M.; Xu, B. Chem. Commun. 2005, 4414. (b) Schoonbeek, F. S.; van Esch, J. H.; Hulst, R.; Kellogg, R. M.; Feringa, B. L. Chem. Eur. J. 2000, 6, 2633. (19) Maitra, U.; Mukhopadhyay, S.; Sarkar, A.; Rao, P.; Indi, S. S. Angew. Chem. Int. Ed. 2001, 40, 2281.

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Figure 4. (a) CD spectra of 5 with varying concentrations in water at room temperature. (b) CD spectra of 5 at 0.05% (w/v) in plain water with varying temperature from 20 to 80 °C.

Figure 5. (a) Change in 1H NMR chemical shift of 3 (1.5%, w/v) in [D6]DMSO with varying H2O content and 2D-NOESY spectra of 3 (1.5%, w/v) in [D6]DMSO with 40% water. (b) Change in 1H NMR chemical shift of 5 (1%, w/v) in [D6] DMSO with varying H2O content and 2D-NOESY spectra of 5 (1%, w/v) in [D6]DMSO with 40% water.

mechanism by exploiting the intrinsic fluorescence of tryptophan residue. Initially, at a low concentration of gelator 5, i.e., when aggregation has not yet started, the emission intensity at 350 nm was found to gradually increase with concentration up to 0.01% w/v, 10-15 times below the MGC. Next, the intensity decreased

with a further increase in gelator concentration till the MGC (0.2% w/v) (Figure 6b). As long as the amphiphile 5 was in a nonaggregated state, the emission intensity increased with the molecular concentration of the gelator. However, at the beginning of the self-assembling process, π rings of tryptophans and also

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Figure 8. Schematic presentation of the possible network in the hydrogel of 5.

ammonium groups are coming close to each other to gradually quench the fluorescence ability of the tryptophan moiety, as reflected by its reduced intensity.20 To ascertain further the involvement of intermolecular hydrogen bonding between amide N-H and carbonyl oxygen

in the gelation process as observed in the 1H NMR study, FT-IR spectroscopy was performed for all the amphiphiles at the gel and solution state. Although hydrogen bonding may have a less significant role in hydrogelation, it is still known to take essential participationintheself-aggregationprocesstoformnanofibers.2a,11c,21 FT-IR spectra of all hydrogels were recorded in D2O and of the non-self-assembled state of the gelators in CHCl3. The CdO stretching band (amide I) for the dried gels appeared at 1670, 1666, 1662, 1660, 1654 cm-1 for 1-5, respectively, which became fixed at a value of 1676 cm-1 for all of them in the non-assembled condition (representative spectra are given in the Supporting Information). The decrease in absorption frequency delineates the gradual increase in hydrogen-bonding strength with alkyl chain length.21a In addition to a broad amide N-H stretching band at ∼3250-3300 cm-1 with increased intensity, absorption frequencies in dried gels are always lower for amide I and higher for the N-H bending band (amide II) compared to that in CHCl3. The observed ranges in gels are characteristics of hydrogenbonded amide groups. Also, an increase in intensity of the methylene scissoring vibration δ (CH2) band at ∼1460 cm-1 in D2O as well as C-H stretching bands at 2925 and 2854 cm-1 indicates the high trans conformational packing of alkyl chain.2a,21 Thus, the FT-IR study illustrates the essential participation of hydrogen bonding as well as van der Waals interactions in the gelation process. In addition to the preceding spectroscopic and microscopic studies, we have measured the X-ray diffraction (XRD) of the dried gels to establish their molecular packing and the possible orientation of the gelator in the supramolecular twisted ribbon structure. XRD of the dried gels at MGC showed a sharp

(20) (a) Lenz, T.; Bonnist, E. Y. M.; Pljevaljcic, G.; Neely, R. K.; Dryden, D. T. F.; Scheidig, A. J.; Jones, A. C.; Weinhold, E. J. Am. Chem. Soc. 2007, 129, 6240. (b) Bera, S.; Thampi, P.; Cho, W. J.; Abraham, E. C. Biochemistry 2002, 41, 12421.

(21) (a) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds, 5th ed.; John Wiley & Sons: New York, 1991; Chapter 3. (b) Kogiso, M.; Hanada, T.; Yase, K.; Shimizu, T. Chem. Commun. 1998, 1791. (c) Shimizu, T.; Masuda, M. J. Am. Chem. Soc. 1997, 119, 2812.

Figure 6. (a) Luminescence spectra of ANS (1 × 10-5 M) with varying concentrations of 1 in water at room temperature. [5] (% w/v): a, 0; b, 0.001; c, 0.005; d, 0.01; e, 0.05; f, 0.075; g, 0.1; h, 0.4. (b) Luminescence spectra of [5] (% w/v): a, 0.001; b, 0.0025; c, 0.005; d, 0.0075; e, 0.01; f, 0.025; g, 0.05; h, 0.075; i, 0.1; j, 0.2.

Figure 7. Powder XRD diagrams of the dried gels 2-5, prepared in water.

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diffraction peak, indicating the ordered arrangement in the gel state (Figure 7). A series of d-spacings was obtained in the smallangle region for all the amphiphiles (except 1) and were much smaller than twice of the fully extended molecular length of each individual amphiphile (by MOPAC AM1 method, CS Chem Office, Table 1) but longer than the length of one molecule. This dictates us to conclude that the aggregates have an interdigitated bilayer structure (Figure 8).11c,22 The weak gelator 1 cannot produce precise diffraction in XRD study, presumably due to its loosely bound interdigitated bilayer structure. However, with an increase in alkyl chain length, more methylene units were merged to induce a continuous enhancement of the hydrophobic interaction between the corresponding -CH2 groups, which led to the development of the excellent water gelator 5. Overall spectroscopic and microscopic studies indicate that an optimum balance between hydrophilicity and hydrophobicity that can be largely regulated by varying the alkyl chain length is necessary for the development of an efficient hydrogelator. In addition to the intermolecular hydrogen bonding, π-π stacking, and N-H‚‚‚π interactions, the alkyl chain induced hydrophobicity at the molecular level has remarkable influence in modulating water immobilization. (22) (a) Gaspar, L. J. M.; Baskar, G. Chem. Commun. 2005, 3603. (b) Kim, C.; Kim, K. T.; Chang, Y.; Song, H. H.; Cho, T.-Y.; Jeon, H.-J. J. Am. Chem. Soc. 2001, 123, 5586. (c) Jung, J. H.; John, G.; Masuda, M.; Yoshida, K.; Shinkai, S.; Shimizu, T. Langmuir 2001, 17, 7229.

Roy et al.

Conclusion In summary, a systematic investigation was carried out to draw a possible correlation between the hydrophobic chain length of an amino acid amphiphile and the water gelating efficiency, which will definitely serve an essential purpose in the structurefunction relationship of LMWHs. It was evident from all the studies that the gelation ability increases more than 100 times with chain length, leading to the development of a gelator having 18 carbon atoms. We successfully presented how a minute structural orchestration in the molecular architecture dramatically influences their self-assembling behavior. Furthermore, our ongoing research findings in application of these cationic amphiphilic hydrogelators as a potent antibacterial agent will definitely add a new horizon to these soft materials in biomedicinal chemistry. Acknowledgment. P.K.D. is thankful to Department of Science and Technology, India, for financial assistance through Ramanna Fellowship (No. SR/S1/RFPC-04/2006). S.R. and A.D.G. acknowledge Council of Scientific and Industrial Research, India, for their Research Fellowships. We thank Anshupriya Shome, for her help in synthesizing compounds. Supporting Information Available: Concentration-dependent CD spectra, representative FT-IR spectra, and synthetic schemes for 1-5. This material is available free of charge via the Internet at http://pubs.acs.org. LA701558M