Effect of Molecular Structure on the Properties of Naphthalene

Jul 14, 2010 - Lin Chen,† Steven Revel,† Kyle Morris,‡ Louise C. Serpell,‡ and Dave J. Adams*,†. †Department of Chemistry, University of L...
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Effect of Molecular Structure on the Properties of Naphthalene-Dipeptide Hydrogelators Lin Chen,† Steven Revel,† Kyle Morris,‡ Louise C. Serpell,‡ and Dave J. Adams*,† †

‡

Department of Chemistry, University of Liverpool, Crown Street, Liverpool L69 7ZD, U.K., and Department of Chemistry and Biochemistry, School of Life Sciences, University of Sussex, Falmer BN19QG, U.K. Received March 4, 2010. Revised Manuscript Received June 30, 2010

Dipeptide-conjugates can be efficient low molecular weight hydrogelators. However, the effective design of a gelator for a specific application is compromised by the lack of a clear understanding of the design rules that govern assembly and hence gelation. Here, we report a library of naphthalene-dipeptides, their physical chemical properties, and gelation ability. We have varied both the amino acids in the dipeptides and the substitution on the naphthalene ring to allow variation of the structure throughout the molecule. We have examined the effects of these permutations on the critical micelle concentration and air-water partition coefficient at high pH and the apparent pKa. We show that there is a clear link between these properties and the predicted hydrophobicity of the overall conjugates, rather than the properties varying with, for example, the dipeptide sequence. The majority of these dipeptide-conjugates are effective hydrogelators, although there is no apparent link between the solution properties and whether or not a conjugate is a hydrogelator. Nevertheless, where gelation occurs, the link between hydrophobicity and apparent pKa allows the prediction of the pH at which a gel will be formed and hence informed choice of gelator for specific applications.

Introduction Hydrogels are widely used for a number of applications. The majority of these are polymer-based, with the gels being formed via the cross-linking or entanglement of polymer chains.1-3 There has been a recent surge of interest in the preparation of hydrogels via the assembly of low molecular weight hydrogelators (LMWGs).4-7 LMWGs assemble in solution via noncovalent forces such as hydrogen bonding, van der Waals interactions, and π-stacking to give long fibrous structures.8 The entanglement of these long fibers results in immobilization of the solvent and hence gel formation. A number of classes of molecule have been shown to be efficient LMWGs including oligopeptides,9-16 peptide *Corresponding author. E-mail: [email protected]. (1) Jagur-Grodzinski, J. Polym. Adv. Technol. 2010, 21, 27–47. (2) Slaughter, B. V.; Khurshid, S. S.; Fisher, O. Z.; Khademhosseini, A.; Peppas, N. A. Adv. Mater. 2009, 21, 3307–3329. (3) Pal, K.; Banthia, A. K.; Majumdar, D. K. Des. Monomers Polym. 2009, 12, 197–220. (4) Wang, Y. J.; Tang, L. M.; Yu, J. Prog. Chem. 2009, 21, 1312–1324. (5) de Loos, M.; Feringa, B. L.; van Esch, J. H. Eur. J. Org. Chem. 2005, 3615– 3631. (6) Dastidar, P. Chem. Soc. Rev. 2008, 37, 2699–2715. (7) Xu, B. Langmuir 2009, 25, 8375–8377. (8) Ulijn, R. V.; Smith, A. M. Chem. Soc. Rev. 2008, 37, 664–675. (9) Zhang, S. Adv. Cancer Res. 2008, 99, 335–362. (10) Zhang, S. G. Biotechnol. Adv. 2002, 20, 321–339. (11) Krysmann, M. J.; Castelletto, V.; Kelarakis, A.; Hamley, I. W.; Hule, R. A.; Pochan, D. J. Biochemistry 2008, 47, 4597–4605. (12) de Groot, N. S.; Parella, T.; Aviles, F. X.; Vendrell, J.; Ventura, S. Biophys. J. 2007, 92, 1732–1741. (13) Adhikari, B.; Palui, G.; Banerjee, A. Soft Matter 2009, 5, 3452–3460. (14) Naskar, J.; Palui, G.; Banerjee, A. J. Phys. Chem. B 2009, 113, 11787–11792. (15) Palui, G.; Nanda, J.; Ray, S.; Banerjee, A. Chem.—Eur. J. 2009, 15, 6902– 6909. (16) Rodriguez-Llansola, F.; Miravet, J. F.; Escuder, B. Chem. Commun. 2009, 7303–7305. (17) Greenfield, M. A.; Hoffmann, J. R.; de la Cruz, M. O.; Stupp, S. I. Langmuir 2010, 25, 3461–3647. (18) Jayawarna, V.; Ali, M.; Jowitt, T. A.; Miller, A. E.; Saiani, A.; Gough, J. E.; Ulijn, R. V. Adv. Mater. 2006, 18, 611–614. (19) Jayawarna, V.; Richardson, S. M.; Hirst, A. R.; Hodson, N. W.; Saiani, A.; Gough, J. E.; Ulijn, R. V. Acta Biomater. 2009, 5, 934–943. (20) Jayawarna, V.; Smith, A.; Gough, J. E.; Ulijn, R. V. Biochem. Soc. Trans. 2007, 35, 535–537.

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amphiphiles,17 and dipeptides conjugated to a large aromatic group.18-29 For the dipeptide-conjugate hydrogelators, assembly has been carried out using a thermal,30 pH,18,26 or enzymatic31 trigger. The majority of examples are for pH-based assembly, and it has been reported that many dipeptides assemble below a pH of 4 with only a few examples assembling at physiological pH.18,25 This clearly restricts the range of materials available for many applications such as cell culturing. For all LMWGs, the effective design of a gelator for a specific application is compromised by the lack of a clear understanding of the design rules that govern assembly and hence gelation.32 As an additional complication, it is clear that not all dipeptide-conjugates are effective hydrogelators. There are examples where a change in the order of the amino acids can change an efficient gelator (for example, Fmoc-phenylalanineglycine (FmocFG))18 into a nongelator (Fmoc-GF).18,33 Recently, a number of studies have closely followed the assembly process for (21) Smith, A. M.; Williams, R. J.; Tang, C.; Coppo, P.; Collins, R. F.; Turner, M. L.; Saiani, A.; Ulijn, R. V. Adv. Mater. 2008, 20, 37–41. (22) Tang, C.; Smith, A. M.; Collins, R. F.; Ulijn, R. V.; Saiani, A. Langmuir 2009, 25, 9447–9453. (23) Zhou, M.; Smith, A. M.; Das, A. K.; Hodson, N. W.; Collins, R. F.; Ulijn, R. V.; Gough, J. E. Biomaterials 2009, 30, 2523–2530. (24) Liang, G. L.; Yang, Z. M.; Zhang, R. J.; Li, L. H.; Fan, Y. J.; Kuang, Y.; Gao, Y.; Wang, T.; Lu, W. W.; Xu, B. Langmuir 2009, 25, 8419–8422. (25) Yang, Z. M.; Liang, G. L.; Ma, M. L.; Gao, Y.; Xu, B. J. Mater. Chem. 2007, 17, 850–854. (26) Banwell, E. F.; Abelardo, E. S.; Adams, D. J.; Birchall, M. A.; Corrigan, A.; Donald, A. M.; Kirkland, M.; Serpell, L. C.; Butler, M. F.; Woolfson, D. N. Nature Mater. 2009, 8(7), 596–600. (27) Chen, L.; Morris, K.; Laybourn, A.; Elias, D.; Hicks, M. R.; Rodger, A.; Serpell, L. C.; Adams, D. J. Langmuir 2010, 26, 5232–5242. (28) Mahler, A.; Reches, M.; Rechter, M.; Cohen, S.; Gazit, E. Adv. Mater. 2006, 18, 1365–1370. (29) Bhuniya, S.; Seo, Y. J.; Kim, B. H. Tetrahedron Lett. 2006, 47, 7153–7156. (30) Vegners, R.; Sheshtakova, I.; Kalvinsh, I.; Ezzell, R. M.; Janmey, P. A. J. Peptide Sci. 1995, 1, 371–378. (31) Williams, R. J.; Smith, A. M.; Collins, R.; Hodson, N.; Das, A. K.; Ulijn, R. V. Nature Nanotechnol. 2009, 4, 19–24. (32) van Esch, J. H. Langmuir 2009, 25, 8392–8394. (33) Orbach, R.; Adler-Abramovich, L.; Zigerson, S.; Mironi-Harpaz, I.; Seliktar, D.; Gazit, E. Biomacromolecules 2009, 10, 2646–2651.

Published on Web 07/14/2010

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Article Table 1. Substituents and Predicted logP for Each NaphthaleneDipeptide dipeptide

Figure 1. (a) General structure of naphthalene-dipeptides 1-18 (see Table 1 for R1, R2, and R3 for each dipeptide). (b) Photograph of example self-supporting hydrogels (from left to right) formed from dipeptides 7, 8, and 9 at a pH of 4 and a concentration of 0.5 wt %. (c) TEM of fibers formed on decrease in pH of a solution of 7. (d) TEM of fibers formed on decrease in pH of a solution of 8. (e) TEM of fibers formed on decrease in pH of a solution of 9.

specific dipeptide-conjugates via a pH switch.21,22,27 These have shown that the apparent pKa of the terminal carboxylic acid of the dipeptide-conjugates is higher than expected for a dipeptide. We recently showed that the apparent pKa was higher than expected for a range of Fmoc-dipeptides and that this apparent pKa correlated crudely with the hydrophobicity of the dipeptides.34 We also found for these Fmoc-dipeptide hydrogels that there was a crude link between the hydrophobicity of the dipeptides and the gel strength at a pH of 4. Nonetheless, we are still some way from a generic understanding that allows the successful design of a LMWG from first principles that will assemble under predictable conditions (pH, concentration, temperature, etc.) to give a hydrogel with well-defined material properties. The ability to predict the conditions under which a gel would be formed is critical for the successful application of these LMWGs. Here, in an effort to address this issue, we have examined a library of naphthalene-dipeptides, varying both the amino acid sequence and the substituents on the naphthalene ring. This has allowed variation in structure at all positions throughout the conjugate. We have examined the effects of these permutations on the critical micelle concentration (cmc) and air-water partition coefficient at high pH and the apparent pKa. We show that there is a clear link between these properties and the predicted hydrophobicity of the overall conjugates, rather than the properties varying with for example the dipeptide sequence. The link between apparent pKa and hydrophobicity allows the prediction of the pH at which a gel will be formed, which is a major step forward in the design of these LMWGs.

Results and Discussion A series of dipeptides conjugated to a naphthalene ring were prepared using established protocols. A OCH2 linker between the dipeptide and the naphthalene ring was used as this has been shown elsewhere to allow effective hydrogelation.25 Such naphthalenedipeptides can form self-supporting hydrogels when the pH of a solution of the conjugate at high pH is lowered below a critical value.25,27 Hydrophobic amino acids were used to prepare the dipeptides with three different naphthalene derivatives: 2-naphthol, 6-bromo-2-naphthol, and 6-cyano-2-naphthol. Hence, 18 different (34) Adams, D. J.; Mullen, L. M.; Berta, M.; Chen, L.; Frith, W. F. Soft Matter 2010, 6, 1971–1980.

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R1

R2

R3

clogPa

1 H CH3 H 0.05 CH3 -0.16 2 H CH3 CH(CH3)2 0.62 3 H CH3 H 1.51 4 H CH2Ph CH(CH3)2 2.08 5 H CH2Ph CH2Ph 2.76 6 H CH2Ph H 0.84 7 Br CH3 CH3 0.63 8 Br CH3 9 Br CH3 CH(CH3)2 1.40 H 2.30 10 Br CH2Ph CH(CH3)2 2.87 11 Br CH2Ph CH2Ph 3.55 12 Br CH2Ph H -0.22 13 CN CH3 CH3 -0.43 14 CN CH3 CH(CH3)2 0.35 15 CN CH3 H 1.24 16 CN CH2Ph CH(CH3)2 1.81 17 CN CH2Ph CH2Ph 2.50 18 CN CH2Ph a Values for c log P were calculated using an online prediction program.35

Table 2. Calculated Parameters from the Gibbs Adsorption Equation for Dipeptides 1-18 with Calculated logP and Measured Apparent pKa Valuesa dipeptide

clogP

As (A˚2)

Kaw (1/au)

cmc (wt %)

pKa

1 0.05 25.5 ( 1.3 0.8 ( 0.02 n.d. 4 2 -0.16 43 ( 0.8 5.45 ( 0.5 2.85 ( 0.1 5.1 3 0.62 65.5 ( 0.5 25.5 ( 2.3 2.65 ( 0.1 4.2 4 1.51 32.9 ( 0.7 26.8 ( 3.7 0.4 ( 0.1 5 5 2.08 70.4 ( 0.7 71.9 ( 6.9 0.75 ( 0.1 6.5 6 2.76 83.5 ( 3.5 237 ( 20 0.5 ( 0.1 6 7 0.84 39 ( 3 6.8 ( 0.8 0.67 ( 0.01 5 8 0.63 45 ( 2 5.55 ( 0.3 1.1 ( 0.2 4.9 9 1.40 41.5 ( 2 13.2 ( 0.8 0.51 ( 0.02 5.8 10 2.30 101 ( 10 108 ( 6.5 0.53 ( 0.06 5.5 11 2.87 62.55 ( 0.3 175 ( 30 0.3 ( 0.04 6.6 12 3.55 85.7 ( 2.0 602 ( 140 0.04 ( 0.01 6.8 b 5.9 ( 1.7 n.d. 5 13 -0.22 n.d. 14 -0.43 n.d. 5.0 ( 0.5 n.d. 5 15 0.35 43 ( 2 120 ( 2 0.9 ( 0.04 5.8 16 1.24 55.5 ( 0.6 56 ( 0.1 0.9 ( 0.02 5 17 1.81 57.5 ( 1.2 66.5 ( 3.2 0.81 ( 0.1 6.1 18 2.50 103 ( 1.4 117.5 ( 40 1.4 ( 0.2 6.6 a Errors were calculated from at least three repeats for each measureb ment. n.d. = not determined due to the high solubility of the dipeptide.

dipeptide conjugates were prepared with variation in hydrophobicity at all points in the conjugate (the naphthalene ring, the first amino acid, and the second amino acid). The full range of conjugates is given in Figure 1 and Table 1. Full synthetic and characterization details are available in the Supporting Information. For all dipeptide-conjugates, the adsorption isotherms were measured at pH 10 ( 0.4 and the apparent air-water partition coefficient (Kaw), cross-sectional area (As), and critical micelle concentration (cmc) determined based on the Gibbs adsorption equation (see Experimental Section, Table 2). At a pH of 10, the terminal carboxylic acid of the dipeptides is deprotonated (see below) and free-flowing solutions are formed. The hydrophobicity of the different naphthalene-dipeptides was estimated using an online prediction program as the calculated partition coefficient (clogP).35 Plotting Kaw against this predicted clogP showed a linear relationship between these parameters (Figure 2a), validating the predictions.36 We note however that the prediction for clogP are based on the protonated dipeptides whereas the adsorptions isotherms were measured for the deprotonated molecules. (35) http://www.molinspiration.com. (36) Meylan, W. M.; Howard, P. H. Chemosphere 2005, 61, 640–644.

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Figure 2. (a) Plot of clogP against Kaw for dipeptides 1-18. (b) Plot of clogP against cmc for dipeptides 1-18. Error bars were calculated from at least three repeats for each measurement.

Figure 3. (a) Example titration data for dipeptide-conjugates 7 (black data), 9 (red data), and 11 (blue data). (b) Plot of apparent pKa against clogP for all dipeptide-conjugates. The black data is for the 2-naphthalene derivatives (1-6), the red data for the bromonaphthalene derivatives (7-12), and the blue for the cyanonaphthalene derivatives (13-18). Green data are for example Fmoc-dipeptides. The line is a linear regression to the data for the dipeptides with r2 = 0.622. Data for alkyl soaps38 are also shown (black stars).

The cross-sectional areas in general increase as expected with the increase in size of the amino acids used. The cmc for the dipeptides are plotted against clogP in Figure 2b. Here, it can be seen that the cmc for the dipeptides decreased with increasing hydrophobicity as expected. For 1, 13, and 14, the dipeptide-conjugates were sufficiently hydrophilic that cmc and As values could not be determined in the current experiment. However, these experiments do show that the cmc for each of these three dipeptide-conjugates is greater than 5 wt %. All these data demonstrate that the behavior of the dipeptide-conjugates at pH 10 is strongly related to the hydrophobicity of the molecules. To prepare hydrogels from such naphthalene-dipeptides, solutions of a dipeptide-conjugate were prepared at high pH. The pH was then lowered. On decreasing the pH, the terminal carboxylic acid is protonated and assembly occurs.27 We previously reported that the apparent pKa of dipeptide 9 was 5.8, higher than would be expected for the C-terminus of a peptide (typically around 3.537). The apparent pKa for a small number of Fmoc-dipeptides was also found to be higher than expected.34 These observations are in agreement with recent data showing that the apparent pKa for the pendant carboxylic acid in the Fmocdiphenylalanine is extremely high (ca. 9.9).22 To further investigate the hypothesis that increased hydrophobicity leads to an increase in apparent pKa, we titrated solutions of each dipeptide at a concentration of 0.5 wt % from approximately pH 10 using HCl. This concentration was used to correlate with the gelation studies (see below); we note that this concentration is below the cmc for most of the dipeptides used here apart from 4, 11, and 12 (see Table 2). To ensure that gelation did not lead to artifacts during titration, the solutions were vigorously mixed between

each addition of HCl. Hence, gels were not formed during this titration experiment. Typical titration curves are shown in Figure 3a. The reported apparent pKa was defined as the point at which 50% of the dipeptide-conjugates are ionized. In most cases, this value for the pKa coincided with a plateau in the pH similar to that reported previously27,34 (Figure 3a) (for dipeptides 6 and 12 the plateau was not as pronounced (data not shown)). The apparent pKa values extracted via these titrations are plotted against the predicted logP in Figure 3b. It can be seen that all are higher than typical for the C-terminus of a dipeptide. The apparent pKa increases with clogP, with an apparent linear relationship between these parameters. Interestingly, the apparent pKa seems to scale with clogP of the total dipeptide as opposed to being dependent on either the terminal amino acid or dipeptide sequence. Additionally, there is no relationship between apparent pKa and the presence or absence of a particular substituent on the naphthalene ring. For comparison, we have added data for four Fmoc-dipeptides (Fmoc-leucine-glycine, Fmoc-leucine-phenylalanine, Fmocphenylalanine-glycine, and Fmoc-phenylalanine-phenylalanine). As can be seen, these conjugated-dipeptides also broadly follow the trend set up by naphthalene-dipeptide conjugates despite the different aromatic unit conjugated to the dipeptide. For all the data shown in Figure 3b, there is some scatter, but a linear regression to all data from 1-18 and the Fmoc-dipeptides yields an r2 of 0.622, a relatively close fit to the data considering the differences in molecular structures, the fact that clogP is a calculated value, and the inherent errors involved in the apparent pKa measurement. Further, comparing to literature data for the pKa of a range of alkyl soaps38 to the clogP shows that these data also show the same trend between clogP and pKa.

(37) Ulijn, R. V.; Moore, B. D.; Janssen, A. E. M.; Halling, P. J. J. Chem. Soc., Perkin Trans. 2 2002, 1024–1028.

(38) Kanicky, J. R.; Poniatowski, A. F.; Mehta, N. R.; Shah, D. O. Langmuir 2000, 16(1), 172–177.

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Figure 4. (a) Plot of apparent pKa against concentration for solutions of dipeptide 9. The line has been added as a guide to the eye. (b) Plot of apparent pKa against temperature for solutions of dipeptide 9 at a concentration of 0.5 wt %.

It is known that the pKa can shift dramatically in hydrophobic environments for proteins and peptides.39 Hydrogen bonding between molecules has also been shown to lead to an increase in apparent pKa using a molecular model.40 Similarly, the pK of the lysine unit in a number of amino acid-amphiphiles has been shown to vary with aggregation.41 Also, Shah has reported high apparent pKa values for a number of fatty acid soaps with high alkyl chain length.38,42,43 For these fatty acid soaps, the pKa was found to increase with the chain length; this was attributed to aggregation of the more hydrophobic surfactants leading to the formation of strong ion-dipole interactions between RCOO- and RCOOH, stabilizing the charge and disfavoring the deprotonation of more carboxyl groups. For a number of fatty acid solutions, the pKa was shown to vary with concentration, with extrapolation to low concentration implying a “monomer” pKa of ∼5.0, but observed pKa at higher concentration of up to 7.5.42 Importantly, the high concentration dependent pKa was observed at concentrations several orders of magnitude below the cmc.42 Similarly, acid soap dimers and a high pKa were reported for an N-acylphenylalaninate, showing that such structures can form with amino acid headgroup-based surfactants.44 It is likely therefore that the high apparent pKa values observed here are a result of aggregation of the naphthalene-dipeptides. In light of the above discussion, we have examined the effect of concentration on the measured apparent pKa for dipeptide 9. As can be seen from Figure 4a, the apparent pKa found to be concentration dependent, varying by 0.7 units over a concentration range of 0.1-1.0 wt %. Hence, by analogy with the results from the fatty acids, we interpret that the high apparent pKa values observed here for the naphthalene-dipeptides are a result of aggregation of the naphthalene-dipeptides. In this case, we hypothesized that the aggregation would be induced to some degree by hydrogen bonding between the dipeptide section of the naphthalene-dipeptide conjugate. Hydrogen bonding is strongly temperature dependent. Hence, we measured the apparent pKa of dipeptide 9 at a range of different temperatures (Figure 4b). In line with our hypothesis, the apparent pKa decreased as the temperature increased, implying that interactions between the dipeptideconjugates are reduced at higher temperature. Hence, it is therefore important to note that the correlations between apparent pKa (39) Urry, D. W.; Peng, S. Q.; Parker, T. M.; Gowda, D. C.; Harris, R. D. Angew. Chem., Int. Ed. 1993, 32, 1440–1442. (40) Mafe, S.; Garcia-Morales, V.; Ramirez, P. Chem. Phys. 2004, 296, 29–35. (41) Ariga, K.; Nakanishi, T.; Hill, J. P.; Shirai, M.; Okuno, M.; Abe, T.; Kikuchi, J. J. Am. Chem. Soc. 2005, 127, 12074–12080. (42) Kanicky, J. R.; Shah, D. O. Langmuir 2003, 19, 2034–2038. (43) Kanicky, J. R.; Shah, D. O. J. Colloid Interface Sci. 2002, 256, 201–207. (44) Ohta, A.; Danev, R.; Nagayama, K.; Mita, T.; Asakawa, T.; Miyagishi, S. Langmuir 2006, 22, 8472–8477.

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Table 3. Rheological Data for Hydrogels Formed from Dipeptides 1-18 Where Appropriatea dipeptide

structure formedb

G0 /Pa

G00 /Pa

mgcc/wt %

1 crystal n.d. n.d. n.d. 931 0.10 2 G 8.3  104 3 crystal n.d. n.d. n.d. 4 3 2.1  10 0.10 4 G 2.8  10 1.1  104 0.018 5 TG 2.0  106 1.2  103 0.025 6 G 7.2  104 9.2  102 0.16 7 G 1.5  104 8 G 28 10 n.d. 4 2 5.6  10 0.12 9 G 2.5  10 1.1  103 0.04 10 G 1.8  104 1 n.d. 11 TG 1.0  102 5 700 0.025 12 G 1.8  10 5.6  103 0.10 13 TG 3.0  104 7.8  103 0.16 14 TG 7.9  104 15 crystal n.d. n.d. n.d. 1.7  103 0.12 16 G 2.1  104 5.8  102 0.025 17 TG 2.9  104 2.8  102 0.018 18 G 1.5  104 a All gels were prepared at 0.5 wt % dipeptide with a GdL concentration of 8.75 mg/mL. The final pH in each case is 3.4 ( 0.2. b Visual observations of self-assembled structures formed on GdL addition: Crystal = a crystalline precipitate formed; G = a transparent gel formed; TG = a turbid gel formed; n.d. = not determined. c mgc = minimum gelator concentration.

Figure 5. Plot of minimum gelation concentration against clogP for all dipeptide-conjugates 1-18 that form self-supporting hydrogels excluding 8 and 11.

and clogP will only hold provided the data are compared at the same dipeptide-conjugate concentration and temperature. The majority of the naphthalene-dipeptides 1-18 are efficient hydrogelators. For comparison with other systems,25,27 gelation was examined at a naphthalene-dipeptide concentration of 0.5 wt % in each case. As we have described previously for 9,27 and related systems,25 gelation occurs as the pH of the solution is DOI: 10.1021/la102059x

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Figure 6. (a) Plot of apparent pKa against the pH at which gelation first occurs for naphthalene-dipeptide conjugates and Fmoc-dipeptide

conjugates at a concentration of 0.5 wt %. Dipeptides 1, 3, and 15 did not form gels. The line represents a linear regression to the data with r2 = 0.78 (b) Plot of apparent pKa against the pH at which gelation first occurs for 9. Different apparent pKa values were accessed using different concentrations of 9.

decreased. To do this, we utilized the hydrolysis of glucono-δlactone (GdL) as described elsewhere.26,27,45 GdL hydrolyzes in water to form gluconic acid, with the rate of hydrolysis being slower than the rate of dissolution allowing uniform hydrogels to be prepared.26,27 Table 3 summarizes the gelation behavior of the dipeptide-conjugates, and the full rheological characterization of the hydrogels was carried out at a final pH of 3.4 ( 0.2. At this point, the pH is significantly below the apparent pKa of all of the dipeptides in this study to ensure full gelation. We previously demonstrated for hydrogels prepared from 9 that the rheological properties at a pH of 3.4 were very similar to those formed at higher pH but achieved the final modulus at a faster rate at the lower pH.27 Dipeptide-conjugates 1, 3, and 15 did not form hydrogels, instead forming crystalline material as the pH decreased. All others formed hydrogels as also evidenced by stability to vial inversion.46 Most of these hydrogels were transparent while a few were turbid as noted in Table 3. It is clear that this lack of gelation in the cases of 1, 3, and 15 is not simply down to dipeptide sequence as the same dipeptide sequences with other naphthalene rings did give hydrogels. However, 3 and 15 do have the same sequence alanine-valine suggesting that, despite 9 being an efficient hydrogelator, perhaps this sequence is prone to dipeptide-conjugates forming crystalline materials. The G0 for the majority of the hydrogels were higher than 104 Pa, which is consistent with that reported for other hydrogels prepared with GdL.26,27 However, these values are higher than reported for other related naphthalene-dipeptides where the pH was changed using HCl.25 Two gels, prepared from 8 and 11, exhibited a lower value of G0 . These samples are also the only examples where G0 is not at least an order of magnitude greater than G00 , implying that these may not be true hydrogels, despite the apparent success by vial inversion. The minimum gelator concentrations, mgc, (the minimum concentration required for the formation of a selfsupporting hydrogel as shown by vial inversion) were determined for those dipeptide--conjugates that formed hydrogels (not including 8 or 11 as the gels are very weak even at 0.5 wt %) and are shown in Table 3. There is a general trend that the more hydrophobic dipeptide-conjugates exhibit lower minimum gelator concentrations (Figure 5). The mgc were obtained at a final pH of between 3.2 and 3.6. The values are similar to those reported previously for naphthalene-dipeptide gelators,25 although the more hydrophobic examples here exhibit lower mgc. (45) Sutton, S.; Campbell, N. L.; Cooper, A. I.; Kirkland, M.; Frith, W. F.; Adams, D. J. Langmuir 2009, 25, 10285–10291. (46) Raghavan, S. R.; Cipriano, B. H. In Molecular Gels: Materials with SelfAssembled Fibrillar Networks; Weiss, R. G., Terech, P., Eds.; Springer: New York, 2005.

13470 DOI: 10.1021/la102059x

Importantly from the perspective of potentially predicting the conditions under which these dipeptide-conjugates may form gels, the pH at which a gel is first formed is highly dependent on the molecular structure and correlates well with the apparent pKa of the dipeptide. Generally, the transition between liquid and gel was observed at a pH just below the apparent pKa of the dipeptide with both a liquid and a gel phase observed. At a slightly lower pH, a self-supporting hydrogel is formed. A plot of apparent pKa against the highest pH at which a gel was observed (i.e., a selfsupporting gel was formed stable to vial inversion) is shown in Figure 6a for those dipeptides 1-18 that formed gels at 0.5 wt %. Data for the Fmoc-dipeptides discussed above have also been added. A relatively good correlation (r2 = 0.78) was observed between the apparent pKa and the highest pH at which gelation was observed. In the discussion above, we demonstrated that the apparent pKa for dipeptide 9 was concentration dependent. In line with this, we examined the highest pH at which a gel was formed at different concentrations of 9. As can be seen from Figure 6b, the pH at which a gel was first formed was found to vary with concentration of 9 (and hence the apparent pKa as plotted in Figure 6b).

Conclusions Utilizing a library of naphthalene-dipeptides has allowed the links between molecular structure and physical-chemical properties to be measured. These properties (cmc and Kaw) correlate well with the overall hydrophobicity of the dipeptide-conjugate. No correlation was observed with, for example, a specific dipeptide sequence or terminal amino acid. This behavior holds for conjugates with different substituents on the naphthalene ring. The apparent pKa of the dipeptide conjugates are also higher than might be expected for the C-terminus of a dipeptide, in agreement with other work.22,34 By analogy of work on fatty acids, this high apparent pKa arises from the aggregation of the dipeptideconjugates despite the solutions generally being below the cmc of the dipeptide-conjugates. Interestingly, the apparent pKa correlates well with the predicted clogP and the pH at which a hydrogel is first formed occurs just below the pKa for those conjugates that form hydrogels. The apparent pKa is also dependent on the concentration of naphthalene-dipeptide; the higher the conjugate concentration, the higher the apparent pKa. Also, in line with the high apparent pKa arising from intermolecular aggregates, the apparent pKa is temperature dependent, decreasing as the temperature increases. This implies that the aggregation is hydrogen-bond directed. We note that the correlations between clogP and the apparent pKa and hence the highest pH at which a gel is formed also hold for the Fmoc-dipeptides investigated Langmuir 2010, 26(16), 13466–13471

Chen et al.

Article

here, implying that these correlations are not simply specific to naphthalene-dipeptide gelators. Hence, depending on the application, this correlation between clogP and apparent pKa allows a considered choice of appropriate gelator. This correlation also demonstrates that for systems where a gel is required at physiological pH using a dipeptide-conjugate concentration of 0.5 wt %, a higher clogP will be necessary. Since the most hydrophobic dipeptides used here are diphenylalanine based, which are extremely hydrophobic, a more hydrophobic naphthalene ring will be required. An alternative strategy would be to increase the gelator concentration, and we note that a recent demonstration of naphthalene-dipeptide conjugates that form hydrogels at physiological pH do indeed use a conjugate concentration of 1 wt %,24 in agreement with the predictions of this current work.

Experimental Section Materials. All chemicals and solvents were purchased from Sigma-Aldrich and used as received. Millipore water (resistivity = 18.2 mΩ/cm) was used throughout. Full synthesis and characterization details for the all dipeptides and intermediates are available in the Supporting Information. The Fmoc-dipeptides were synthesized as described elsewhere.34 NMR. 1H NMR spectra were recorded at 400.13 MHz using a Bruker Avance 400 NMR spectrometer. 13C NMR spectra were recorded at 100.6 MHz. Transmission Electron Microscopy. Samples for examination by TEM were prepared on Formvar/carbon film coated 400mesh copper grids (Agar scientific). The required amount of GdL was added to a solution of dipeptide derivative (0.5 wt % solution at pH 10.7) and allowed to gelate. The gel was sampled by removing 10 μL, placing on a grid, and allowing adsorption for 1 min followed by a 1 min wash and two 1 min negative stains using 2% w/v uranyl acetate. Where necessary samples for TEM were prepared in situ by immediately placing grids inverted onto 10 μL droplets of the gelation solution. In a humid chamber at room temperature, material was allowed to adsorb onto the grids and removed after 120 min followed by 5 min of drying and two stains as above. Negatively stained grids were allowed to dry and examined on a Hitachi-7100 TEM operated at 100 kV. Images were acquired digitally using an axially mounted (2000  2000 pixels) Gatan Ultrascan 1000 CCD camera (Gatan, Oxford, UK). Surface Tension Measurements. The surface tension measurements were performed on the high throughput Delta-8 system (Kibron Inc.), and the data were analyzed by the Delta-8 Manger software. The 200 μL concentrated dipeptide samples were transferred onto the first column of a standard Nunclon 96 well plate. A series of concentrations were prepared by sequential dilution with pH 9-10 (tested by pH paper) water across the plate, with the concentration decreased by a dilution factor for each column using a transfer-and-mix protocol. Final 50 μL samples were transferred to the detection plate for measurements. The adsorption isotherm curves of different samples were obtained and the apparent air-water partition coefficient (Kaw), cross-sectional areas (As), and critical micelle concentration (cmc) calculated based on the Gibbs adsorption equation Γ ¼

  -1 dγ 2:3RT d log C

ð1Þ

where Γ is the surface concentration, γ is the surface tension, and C is the concentration of dipeptide-conjugate in solution. The area/molecule at the air/water interface is given by 1/Γ. The slope can be obtained from the surface tension measurements plotted

Langmuir 2010, 26(16), 13466–13471

against log C. All the parameters shown here were obtained using the Delta-8 Manger. Determination of the Apparent pKa. A FC200 pH probe (HANNA instruments) with a (6 mm  10 mm) conical tip was employed for all pH measurements. The stated accuracy of the pH measurements is (0.1. The pKa values of dipeptide solutions were determined by titration via the addition of aliquots of a 0.1 M HCl solution. pH values were recorded until reaching a stable value after each addition during the titration process. To prevent the formation of gel, the solutions were gently stirred, thus keeping the solution liquid during the whole “titration” process. For select samples, heating between aliquot addition and recooling was carried out to check whether mixing and equilibration had occurred.22 No deviation between pKa measurements with or without heating was observed, and hence the values reported are for samples without heating. Gelation Studies. The dipeptide derivative (25.0 mg) was suspended in deionized water (5.00 mL). An equimolar quantity of NaOH (0.1 M, aq) was added and the solution gently stirred for 30 min until a clear solution was formed. The pH of this solution was measured to be 10.7. To prepare hydrogels, solutions were added to measured quantities of glucono-δ-lactone (GdL) according to the requirements of final pH and the samples left to stand for 24 h. To obtain the highest pH of gelation, a series of dipeptide samples were prepared with varying concentrations of GdL such that different final pHs of between pH 8 and pH 3 were achieved. After 24 h, the samples were examined by the vial inversion test. The highest pH of gelation was defined by the pH under which the stable gels were formed, and no flow of liquid was found in the samples.

Determination of the Minimum Gelation Concentration. Stock solutions of dipeptide-conjugates at a concentration of 5 mg/mL (0.5 wt %) were prepared. These were then diluted with pH 10 water to a number of concentrations. To adjust the pH, for each dipeptide-conjugate solution, a number of samples were prepared with different quantities of GdL to ensure the preparation of samples with a final pH between 3.2 and 3.6. All the samples were left to stand at least 24 h. The minimum gelation concentrations were determined by the lowest concentration at which a self-supporting gel was formed as determined by the vial inversion test. Rheology. Dynamic rheological experiments were performed on an Anton Paar Physica MCR101 rheometer. For the oscillatory shear measurements, a sandblasted parallel top plate with a 50 mm diameter and 1.0 mm gap distance were used. Gels for rheological experiments were prepared on the bottom plate of the rheometer by loading a 2.0 mL solution of the gelator immediately after GdL addition. At this point, the sample is still a free-flowing liquid. Hence, sample uniformity and reproducibility are high. Evaporation of water from the hydrogel was minimized by covering the sides of the plate with low-viscosity mineral oil. The measurements of the shear modulus (storage modulus G0 and loss modulus G00 ) with gelation were made as a function of time at a frequency of 1.59 Hz (10 rad/s) and at a constant strain of 0.5% for a period of 24 h. To ensure the measurements were made in the linear viscoelastic regime, an amplitude sweep was performed. The results showed no variation in G0 and G00 at a strain of 0.5% as used above (see Figures S1-S3 for example data, Supporting Information).

Acknowledgment. We thank the EPSRC (EP/G012741/1) for funding.

Supporting Information Available: Full synthetic details for all dipeptides and intermediates. This material is available free of charge via the Internet at http://pubs.acs.org.

DOI: 10.1021/la102059x

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