A Glutathione-Based Hydrogel and Its Site-Selective Interactions with

pyrenyl moiety in order to test the ability of GSH to direct noncovalent self-assembly in H2O, when combined with a hydrophobic driving force for ...
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Bioconjugate Chem. 2005, 16, 1019−1026

1019

A Glutathione-Based Hydrogel and Its Site-Selective Interactions with Water Sumit S. Mahajan,†,§ Rajan Paranji,‡,§ Ranjana Mehta,† Robert P. Lyon,† and William M. Atkins*,† Department of Medicinal Chemistry, Box 357610, and Department of Chemistry, Box 351700, University of Washington, Seattle, Washington 98195-7610. Received April 27, 2005; Revised Manuscript Received June 9, 2005

Glutathione (γ-glutamyl-cysteinyl-glycine; GSH) is ubiquitous biological tripeptide with multiple functions and possible therapeutic uses. The oxidized disulfide form (GSSG) self-assembles into fibrillar aggregates and gels in organic solvents, but not in solvent mixtures with high water content. Here, the disulfide bond has been replaced with a pyrenyl moiety in order to test the ability of GSH to direct noncovalent self-assembly in H2O, when combined with a hydrophobic driving force for aggregation. The resulting GSH-pyrene forms gels in 95% H2O:5% DMSO. The γ-glutamyl group is critical for gelation, as it is with GSSG organo-gels, inasmuch as neither S-(pyrenyl)-cysteinyl-glycine nor the iodo-acetamido-pyrene precursor gels under any conditions studied. Circular dichroism and fluorescence spectroscopy indicate that the pyrene moieties cluster within the gels. Scanning and transmission electron microscopy reveal fibrous networks with individual strands of ∼50-100 nm diameter. Saturation transfer difference (STD) NMR studies demonstrate that water interacts strongly with GSH-borne protons in both solution and gel states, but only the gels include water-pyrenyl interactions with significant residence times.

INTRODUCTION

Self-assembling gels have potential use in numerous bio-nanotechnologies including tissue engineering, biosensors, bioadhesives, and drug delivery (1-13). For several applications, bioactive peptide hydrogels with tunable sol-gel behavior are the desired endpoint. Recent examples of ‘responsive’ gels include vancomycin-sensitive Fmoc-D-ala-D-ala gels (14), and peptide gels triggered by enzymatic activity (15) or IR radiation (16). A significant challenge has been the design of low molecular weight (< ∼1000 kDa) peptide-based systems that selfassemble in water, although a few examples based on amino acids or short peptides have been described (1620). A design strategy that has yielded elegant selfassembled mesoscopic structures involves grafting orthogonal self-recognition elements into single molecules (6, 8, 14). Here, we report the utility of this strategy with the highly polar peptide of biological interest, glutathione (γ-glutamyl-cysteinyl-glycine, GSH), grafted onto pyrene. This expands the pool of low molecular weight hydrogelators to include a ubiquitous biomolecule with extremely diverse functions. We previously characterized organo-gels derived from GSSG (1, Figure 1, 19). Upon gelation in DMF, DMSO, or MeOH, a specific local conformation of the -S-S- dihedral angle is favored, as suggested also for aroyl-cysteine derivatives (21). Furthermore, inter- and intramolecular hydrogen bonds at the γ-glutamyl group drive self-assembly of 75 nm wide nanofibers, which coincides with gelation. Therefore, we hypothesized that the highly polar γ-glutamyl group could be exploited as a self-recognition element, and, when combined with a sufficient hydrophobic driving * To whom correspondence should be addressed. Tel: (206) 685-0379. Fax: (206) 685-3252. E-mail: [email protected]. † Department of Medicinal Chemistry. ‡ Department of Chemistry. § These authors contributed equally to this work.

force, GSH could provide a scaffold for low molecular weight peptide hydrogels. GSH is a major determinant of cellular thiol redox status, and it is a well-established antioxidant (22). GSH also has potential utility in cystic fibrosis therapy (23, 24), and it is exploited widely as an affinity ligand for immobilizing fusion proteins on surfaces (25, 26). Thus, GSH could provide a scaffold for hydrogels with novel therapeutic value or material applications. Although there are an increasing number of examples of hydrogels incorporating biomolecules, very few mechanistic details of the self-assembly process have been elucidated, and kinetically relevant intermediates en route to gel formation have not been documented in any cases, to our knowledge. In particular it is unclear how solvent water is affected by the gelation agents and whether specific functional groups within the gelators are responsible. To address this aspect of gelation, we have utilized saturation-transfer NMR methods to monitor the site-specific interactions between water and gelator within the hydrogels. This method may be generally useful for understanding sol-gel behavior. EXPERIMENTAL PROCEDURES

Materials and Chemicals. GSH, S-hexyl-GSH (5) and β-mercaptoethanol were purchased from Sigma (St. Louis, MO) and used without further purification. N-(1-Pyrene)iodoacetamide (6) was purchased from Molecular Probes (Eugene, OR). The synthesis of 2 was achieved as follows: N(1-pyrene)iodoacetamide (10 mgs, 0.025 mmol) was dissolved in DMSO (400 µL). To this solution was added GSH (2-fold molar excess), and the reaction mixture was stirred overnight protected from light. Excess water (10-15 mL) was added followed by centrifugation to isolate the GSH-pyrene conjugate as a solid pellet. The above procedure was repeated at least two times to ensure complete removal of excess GSH. The pellet

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cover slip. Images were obtained at 15 keV on a JEOL 6300F scanning microscope. 1 H NMR. Saturation transfer difference experiments were performed on a Bruker AV500 system equipped with a H, C, F triple resonance probe, at 301 or 320 K at six saturation times (250 ms, 500 ms, 1 s, 2 s, 3 s, 4 s). Selective saturation of the water resonance was obtained by using a Gaussian cascade for the necessary saturation duration, followed by excitation sculpting for water suppression (29-31). Control spectra were obtained by placing the saturation frequency to -1.0 ppm followed by shifting the saturation frequency to the water resonance. Each experiment was conducted in an interleaved manner by collecting the control and saturated spectra in alternate scans and storing them in separate memory blocks, to average the effects of instrument instability over the period of the data collection. The STD amplification factor is defined here to be

% amplification factor ) [I0 - Isat]/I0 × 100 where I0 and Isat are the spectral intensities of any single proton without and with water saturation, respectively. Also, T1 relaxation measurements at 301 K were performed on a Bruker AV300 system. An inversion recovery sequence was employed to collect T1 data with a recycle delay of 10 s to ensure the complete recovery of magnetization at the end of each scan. The magnetization recovery curve given by

Mz ) M0 [1 - 2 × exp(-t/T1)]

Figure 1. Structures of compounds compared in this work. 1 has been previously shown to form organo-gels.19 2 is a hydrogelator, as described within. Compounds 3-6 do not induce gelation under the conditions studied here.

obtained was then washed successively with acetonitrile and diethyl ether to obtain a gray solid. The solid was further dried to remove traces of water and ether (12 mgs, 82% yield). MS: calcd M+ ) 564, obsvd. (M + 1)+ ) 565.2. Compounds 3 and 4 were synthesized by an identical procedure, substituting either L-cysteine or β-mercaptoethanol for GSH. Circular Dichroism. CD was performed with a Jasco J-720 spectropolarimeter with a scan rate of 50 nm/min and a path length of 2 mm, at 25 C. Samples were 1 mL containing 0.75 mM 2 in solvents described in the Figure 2 legend. Fluorescence Spectroscopy. Fluorescence emission spectra were obtained with an SLM-Aminco 8100 fluorimeter, with slit widths of 4 nm, and excitation at 340 nm. Temperature was controlled with a circulating water bath. Electron Microscopy. TEM was performed with a JEOL electron microscope at 80 keV. Gel samples were ‘smeared’ on a Formvar-coated copper grid (200 mesh, Fullam, Inc. Latham, N.Y.). The surface was washed briefly with EtOH and then stained for 1 min with 2% uranyl acetate in 95% EtOH. For SEM, gel samples were dried, sputter-coated, and placed on a polylysine-coated

where Mz and M0 are the magnetization vectors along the ‘z’ axis at time t and M0 is in the initial net magnetization, respectively, was fitted to the data and T1 values were extracted. Care was taken to fit this single exponential recovery curve only in the initial rate regime to avoid the contamination of exchange rates on the measured T1 values (27-29). The NMR samples were prepared by using 30 mM solution of GSH pyrene in DMSO-d6 followed by addition of water to get a 50:50 mixture. The samples were mixed well to get a homogeneous solution and then transferred immediately to a NMR tube. The pulse sequence for the STD NMR experiment was adopted from 1D NOE difference pulse sequence developed by Meyer and co-workers (27). The on-resonance irradiation of water was performed at a chemical shift of 4.45 ppm. To minimize the error due to magnet instability, a reference spectrum was also collected simultaneously with off-resonance irradiation applied at -1 ppm, as no signals of GSH pyrene were observed in that region. The total numbers of scans was 256 with 16 ‘dummy’ scans. RESULTS

On the basis of the apparent selectivity of the γ-glutamyl group of GSSG in directing supramolecular self-assembly of organogels (19), we hypothesized that a hydrophobic moiety attached to GSH could provide a thermodynamic driving force for gelation in water. Analogues containing the γ-glutamyl group could provide a versatile range of gelators, in a manner analogous to the organo-gels derived from GSSG. Therefore, 2 was synthesized and its hydrogelation properties were compared to the desγ-glutamyl analogue 3, and to 4, 5, and 6 (Figure 1). Addition of H2O to stock solutions of 2 in DMSO immediately yielded transparent, fluorescent gels. In solvents ranging from 95:5-40:60 (vol/vol) H2O:DMSO, at

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Figure 2. Left Panel: Fluorescence emission of 2 (0.75 mM) in different sol-gel states. The emission from excimers (485 nm) correlates with formation of gels. Black, 100% DMSO; red, 90:10 H2O:DMSO; blue, 50:50 H2O:DMSO. Inset: The maximal E/M ratio, 50:50 H2O:DMSO yields the stiffest gels, but gels are formed even at 95:5 H2O:DMSO. E/M ) intensity at 485 nm/intensity at 390 nm. Excitation ) 340 nm. Right Panel: Photograph of a fluorescent hydrogel (95:5 H2O:DMSO) formed from 1 mM 2 in a cuvette lying on its side.

final concentrations of 1 mM to 10 mM, only 2 yielded transparent, fluorescent gels; in contrast, 1, 3, 4, 5, and 6 yielded clear solutions or precipitates, and no gels under any conditions studied. The uniqueness of 2 as a hydrogelator among the other analogues is interesting and reminiscent of the specificity observed for GSSG as an organogelator among structural congeners. The γ-glutamyl linkage may be useful in driving gelation in a wide variety of systems. Fluorescence Characterization. Other pyrenecontaining gelators have yielded excimer emission upon gelation, and this provides a convenient low resolution probe of pyrene-pyrene interactions (7, 30). Gelation of 2 coincided with formation of a pyrene excimer fluorescence spectrum (485 nm) in contrast to monomer emission at 370-390 nm for the solution state (Figure 2). Although the absolute fluorescence intensity changed with solvent composition, the excimer:monomer ratio (E/ M) clearly increased at higher ratios of H2O:DMSO, with a maximum at 50:50 (Figure 2, inset). The E/M ratio corresponded well with the apparent stiffness of the gels, which was also maximal in this range. At 1 mM 2, the gels were sufficiently stiff to remain in an inverted vessel even at 95:5 H2O:DMSO. Circular Dichroism. Ground-state chromophore interactions were monitored by circular dichroism (CD). Pyrene CD excitation bands at 290-400 nm were observed in the gel state, but not the solution state. The CD bands are characteristic of chiral pyrene complexes and indicate direct ground-state chromophore interaction (Figure 3). Although the rheological properties of these gels remain to be studied in detail, the temperaturedependent CD spectrum (Figure 3, inset) yields a Tm for gelation of 46 °C for 1 mM 2 in 90:10 H2O:DMSO. Interestingly, there was a modest increase in ellipticity at 366 nm with increasing temperature, until the gel melted at ∼46 °C. Perhaps, the hydrophobic effect resulted in stronger, or more complete, pyrene-pyrene interactions with increasing temperature, until the gels melt at 46 °C. In the previous work with GSSG gels a negative CD band at 240-250 nm was observed only in the gel state. In the sol state, a positive band was observed at this wavelength. On the basis of this, and comparison to CD spectra of cysteine-containing peptides (31), we assigned the negative CD band to the γ-Glu-Cys bond, which apparently is conformationally restricted in the gels. Due to the intense CD band of the pyrene groups, we were unable to observe this spectroscopic signature in the hydrogels from 2.

Figure 3. Sol-gel transition of 2 (0.75 mM) monitored by circular dichroism. The spectra are color coded as follows: Black, 20 °C, 90:10 H2O:DMSO; red, 20 °C, 50:50 H2O:DMSO; blue, 20 °C 100% DMSO. Inset: The CD bands at 260-400 nm are observed only in the gel state, and are lost at a Tm of 46 °C (90:10 H2O:DMSO). The molar ellipticity at 366 nm represents the y-axis.

The temperature-dependent sol-gel conversion was reversible, although with a pronounced hysteresis requiring up to a few hours for gelation. At identical concentrations, neither 3, 4, 5, or 6 in any H2O/DMSO combination yielded fluorescent excimers or CD bands, further indicating the specificity of the γ-glutamyl-cys linkage in directing the self-assembly of pyrene complexes. As with the previously observed organo-gels (19), the γ-glutamylcys linkage is critical for the aqueous sol-gel transition of 2 among the pyrene derivatives studied. Electron Microscopy. The mesoscale morphology of the gel state was monitored by transmission electron microscopy (TEM) and scanning electron microscopy of gel slices, which revealed highly branched fibrous networks, as observed for the GSSG organo-gels (Figure 4). Based on TEM and SEM, the width of individual strands is 50-100 nm, as also observed with the GSSG organogels. Possibly, the organo-gels and hydrogels share a similar supramolecular structure. Also evident in EM images were spherical masses of ∼50 nm diameter decorating the filamentous structures. Under all conditions studied, both morphologies were observed, suggesting multiple possible high order structures. We found no clear correlation between the relative fiber vs sphere morphology upon changing solvent composition or temperature. Both morphologies were observed also with the GSSG organo-gels, but with a significantly greater preference for fibers. The fiber morphology is clearly preferred in the hydrogels as well. The relative homogeneity of the fiber diameter suggests a highly specific self-assembly process, rather than a nonspecific aggrega-

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Figure 4. Electron microscopy of gels formed from 2. Left three panels: TEM at increasing magnification. The branched fibrous network consists of strands approximately 50-100 nm in diameter. The bar in the center panel of the TEM micrographs is 1 µm. Right Panel: Scanning EM of a gel formed from 10 mM 2.

Figure 5. 1H NMR spectrum of 2 in 50:50 H2O:DMSO after gelation has started. The chemical shifts of the assignable protons are annotated on the chemical structure.

tion. Also, a high degree of branching is evident, which suggests the possibility of obtaining thermostable gels, by manipulation of the gelation kinetics. Although the EM images do not directly address the structure of gels in the hydrated state, the fact that no such fibers are observed in samples generated from 3, 4, 5, or 6 suggests that fibril formation correlates with gelation. 1 H NMR. The properties of water within hydrogels remain uncharacterized. Because it is ultimately desirable to ‘tune’ these properties, additional methods are required to monitor solvent dynamics and solventgelator interactions (32). To identify specific watergelator interactions with 2, we performed saturation transfer difference (STD) 1H NMR experiments, wherein the solvent water was irradiated with a saturating pulse, and the subsequent transfer of magnetization to GSHpyrene protons was monitored. An analogous technique has been used to monitor the dynamics of protein-ligand interactions, with saturation transfer from the protein to the bound ligand (27-29). Because H2O is absolutely required for the hydrogel formation from 2, we have adapted the strategy to saturate water in the gels, and to look for differential saturation transfer or magnetization to the protons on 2. In this strategy, water is the

large reservoir of magnetization, which can be transferred to GSH-pyrene. With this strategy we were able to compare STD spectra in the gel state, in a sample that was just beginning to gel, and of the solution state, using the same sample after raising the temperature to 46 °C, just above the transition temperature. The former case represents a sample in the process of gelling, rather than a fully gelled sample. Therefore, the comparison between the spectra is only a qualitative indication of which protons are effected upon gelation and not a quantitative measure of changes in solvent interaction. To our knowledge, the dynamics of water in hydrogels have not been studied previously by this method. The 1H NMR spectrum of 2 in viscous fluid ‘gels’ is shown in Figure 5. It was not possible to record wellresolved NMR spectra in stiff gels in 95:5 H2O:DMSO, so the NMR experiments were conducted at higher DMSO content. Under these conditions, gels begin to form but they are not sufficiently rigid to afford complete line broadening. To generate the STD spectrum, first the water signal was preirradiated for a sufficiently long time to achieve saturation, and a 1D spectrum was recorded. Next, a control spectrum was recorded without any presaturation on the water signal. The difference in

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Figure 6. Aromatic region of the 1H NMR spectrum of 2 in 50:50 H2O:DMSO with off-resonance saturation (control) and onresonance saturation of water (saturated) in ‘fluid’ gels. The inset is the STD spectrum for this spectral region.

Figure 7. STD amplification factors vs tsat in solution (left panel) and in gels (right panel). The red curves correspond to aliphatic protons on the GSH group, the blue curves are the aromatic protons on the pyrenyl group, and the green curves are the exchangeable amide protons. The specific protons are 8.24 ppm, open circles; 8.16 ppm, open triangles; 8.08 ppm, X’s; 8.05 ppm, open diamonds; 8.01 ppm, crosses; 7.98 ppm, dashes; 3.17 ppm, closed squares; 2.93 ppm, closed triangles; 8.47 ppm, open squares; 8.37 ppm, closed circles.

intensity of resonances for the native spectrum of 2, with off-resonance saturation minus the intensity of the spectrum with on-resonance water saturation yields an STD spectrum as also shown in Figure 6. The largest differences were observed for the protons on the GSH moiety, but they were readily apparent also for the aromatic pyrenyl protons in the gels, as shown in Figure 6. The intensity of any individual proton resonance in the difference STD spectrum depends on the off-rate of the water from that site, the saturation time, and the concentration of the proton in the gel. Water molecules tightly bound to specific protons, on the appropriate NMR time scale, can transfer magnetization. However, if the gelator protons interact too strongly with the water, then the magnetization will be transferred only from the small subset of water that is bound to it. More efficient saturation transfer is obtained for the entire population if the off-rate of water from the proton of interest is fast relative to the saturation time (tsat), so that many water protons can interact with gelator, but also slow enough to allow transfer of magnetization. The STD amplification factor was calculated, as described in Experimental Procedures, for most of the protons in the 1D spectrum. We recorded the results for a range of presaturation times i.e., 250 ms to 4 s, and the STD amplification factors as a function of saturation times are plotted in Figure 7 for all the resolvable peaks from the GSH group, pyrenyl protons, and the observed

amide protons of the GSH-pyrene gel. A few of the aliphatic peaks were not selected because they were too close to the water peak, thus hampering accurate determination of the peak intensity after saturation. Also, a few of the protons were not unambiguously assigned. For all protons, we observed that the maximum saturation transfer is already reached for a saturation time of 2 s. In the solution state, all of the pyrenyl aromatic protons have very low STD amplification factors; some are 4-8% and others are essentially zero. For the GSHborne aliphatic protons (δ ) 2-3 ppm), the amplification factors range from 30% to 70%, with the greatest magnetization transfer occurring with the protons on the β-carbon of the cysteinyl group. It is clear from these results that water only very transiently interacts with the aromatic protons (blue), if at all, but water does have significant residence times on the aliphatic protons in the GSH-moiety (red). The residence times of solvent water on aliphatic groups in peptides has been shown to increase when nearby groups are polar, as with the GSH peptide. In solution, only one amide proton is observed (8.37 ppm, cysteinyl amide). The other amides presumably are severely broadened due to chemical exchange. Upon gelation, there is a dramatic redistribution of STD amplification factors. The maximum STD amplification factor ranges from 45% to 65% for the aliphatic peaks of interest in the GSH group (Figure 7, red). For the pyrenyl protons (Figure 7, blue) this range increases even more, to 15% - 30%. In the case of the amide protons,

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Figure 8. Schematic of changes in STD amplification factors upon gelation. The protons are color-coded as in Figure 7. The relative size reflects the relative amplification factors. The aromatic protons are strongly affected by gelation. Table 1. Representative T1 Relaxation Times and STD Amplification Factors peak position (ppm)

T1 (s)

STD amplification factor

2.37 (GSH) 8.16 (pyrene) 8.47 (NH)

0.26 0.97 0.44

48% 18% 21%

the maximum STD amplification factors are 18% for the peak at 8.37 ppm and 20% for the peak at 8.47 ppm, which is now observed. Presumably, the amide proton at 8.47 ppm in the gel, which is not observed in solution, reflects a decrease in the chemical exchange rate of this proton. Regardless of the complexity for amide protons, as discussed further below, there is a clear and significant difference in saturation transfer from water to different protons of GSH-pyrene upon gelation with a marked increase in the amplification factors for the aromatic pyrenyl protons (Figure 7). It is striking that the data cluster into groups defined by the ‘type’ of proton. The aliphatic GSH protons (red) have the highest amplification factors in both solution and gel states. The aromatic pyrenyl protons (blue) have near zero amplification factors in solution but become spread over a wide range including some with factors as high as 30%, in the gels. For the single amide proton observed in both states, the amplification factor actually decreases upon gelation. These data are presented schematically in Figure 8, where the structure of 2 includes protons with sizes scaled according to their STD amplification factors, in the solution vs gel state. The recovered STD amplification factors must be considered in the context of T1 relaxation processes. The slower on/off rate of H2O from the atoms it interacts with can lead to larger transfer of saturation from H2O, whereas efficient T1 relaxation process counteracts the saturation transfer process. Therefore, we measured T1 relaxation times. Table 1 summarizes the results, with exemplary values, and demonstrates that GSH aliphatic protons relax most efficiently and in contrast the pyrenyl protons relax slowly. The NH proton’s relaxation time is intermediate between them. Despite the efficient relaxation of GSH-associated protons, their maximum achievable STD amplification factor for the gels is about 2- to 2.5-fold larger than for pyrenyl protons or the NH protons. On the basis of the relative T1 and STD amplification factors of GSH and pyrene, it must be true that H2O has higher affinity for GSH backbone than for pyrene group, and the residence time of H2O interacting with the GSH group is longer. This leads to increased saturation transfer efficiency for the GSH group protons (48%) when compared with the pyrenyl group (18%),

despite the faster T1 relaxation processes. A similar conclusion is true for all other aliphatic, nonexchangeable, GSH-associated protons as seen from Figure 7. Figure 7 clearly shows that the STD amplification factors are distinctly different for GSH-borne protons, when compared with pyrenyl protons. Apparently, solvent water does not interact with the pyrenyl protons, or it does only extremely transiently in solution. Most importantly, gelation correlates specifically with stronger water interactions with the pyrenyl group, without much effect on the GSH-water interactions. Considering the NH protons, their chemical exchange with a large reservoir of ‘saturated’ protons should lead to larger saturation transfer and large amplification factors. This situation is in stark contrast to the scenario, where the faster on/off rate of water leads to less efficient saturation transfer efficiency in the case of protons of GSH or pyrene. Given the fact that distinct NH resonances are observed in the case of the GSH-pyrene gel system, the chemical exchange of the NH protons falls in the ‘infrequent’ or the so-called ‘slow’ exchange regime (33-35). In fact, with identical solvent conditions, but in the sol state, the amide resonances are not observed and they appear only when the sample begins to gel. Clearly, the amide-solvent chemical exchange reactions are much slower in the gel state than in the sol state, as expected. This may indicate the participation of the amides in intermolecular hydrogen bonds that are formed uniquely in the gel state. Thus, the gelation process itself would be expected to decrease the amplification factors from a theoretical maximum at which chemical exchange was sufficiently slow to allow for transfer, but sufficiently fast to allow transfer to a large population of GSH amide protons. In short, the magnetization transfer from solvent to amide protons is more complex than the other protons on the gelator, and the interpretation is ambiguous. Table 1 suggests that the efficiency of the exchange process of NH with water seems to lie between the saturation transfer efficiency of GSH and pyrene. For a gel system like the GSH-pyrene, the average viscosity is clearly higher than that of a solution. This also means that the time scale of random fluctuations in such gels is larger than that of solutions, and therefore spin lattice relaxation times for gel systems around roomtemperature lie close to the T1 minimum on the T1 vs correlation time graph (33). From this perspective, when the T1 values for two groups such as GSH and pyrene within the same molecule differ substantially, it generally translates into a significant difference in their time scales of motion. Apparently, the GSH portion of the molecule is more mobile than the pyrenyl group in the gels. DISCUSSION

A common design strategy exploited for the preparation of self-assembling hydrogels includes the combination within a single amphiphilic molecule of hydrophobic groups and water-soluble components. Here, we have applied the term ‘hydrogel’ to our system which includes 5% organic solvent as a lower limit, but which has an absolute requirement for the addition of water to initiate gelation. Numerous examples of hydrogelators include short peptides or sugars as the hydrophilic component (1). We have extended this strategy by incorporation of the polycyclic aromatic hydrocarbon pyrene to the ‘unusual’ peptide GSH. The gels described here are interesting for several reasons. Of primary interest is the use of GSH derivatives. GSH has numerous biological functions including antioxidant

Glutathione-Based Hydrogels

roles, detoxification, and enzyme regulation. Of course, many of these functions require the free thiol group contained in GSH, and the hydrogelator described here (2) has lost this group. However, these results provide proof-of-principle that GSH conjugates can be hydrogelators, and is it reasonable to expect that the thiol group of other hydrogelating GSH-conjugates could be recovered by simple changes in pH or temperature, or enzymatically. Several types of chemical linkage to the GSH thiol are known to be reversible (36). Thus, although we have initiated the exploration of GSH-based hydrogels with 2, other GSH-conjugates are easily imagined that could be ‘cycled’ through the hydrogel state and the biologically functional state with a free thiol. Speculatively, in fact, GSH-conjugates with nontoxic, or even therapeutically useful, hydrophobic removable groups could be used as slow release formulations for delivery of GSH itself, which has been widely used as an antioxidant and potential therapeutic for cystic fibrosis (23, 24). Furthermore, GSH-conjugates may be directly useful as therapeutic anticancer adjuvants, without the need for a free thiol group (37, 38), and it is conceivable that hydrogel formulations could have utility. In such applications, the GSH released from the ‘conjugate gels’ would not reform gels, but would simply mix with the endogenous pools of GSH. Furthermore, immobilized GSH analogues are widely used in protein purification protocols that exploit glutathione S-transferase (GST)-fusion technology (25, 26). For these purposes, a free thiol group on GSH is not a strict requirement, and it is possible that hydrogels bearing pendant S-linked GSH peptides could immobilize GST-fusion proteins, thus providing a strategy for delivery of engineered proteins to hydrogel surfaces. Although the current work does not achieve such putative ‘applications’, it does suggest their feasibility. A second important aspect of this work is the extension of the previously reported specificity of the γ-glutamyl group as a self-assembly unit to hydrogels. As we observed previously with GSSG in organic solvent, the γ-glutamyl group is uniquely able to drive hydrogelation within the series of analogues studied here that contain the same hydrophobic moiety. Although the molecular basis for this apparent specificity remains unknown, it could, in principle, be exploited in non-GSH γ-glutamylcontaining hydrogelators, once it is adequately understood. We are careful to note that, although the previously described organo-gels and the gelation of 2 in water both exhibit an apparent specificity for the γ-glutamyl group and both exhibit similar gross structures, the specific molecular interactions in the two type of gel may be different. A critical, and general, aspect of hydrogels that has not been experimentally addressed in sufficient detail is the structure and dynamics of water sequestered in them (32). It is likely that a complete understanding of solvent dynamics in hydrogels would facilitate control over their sol-gel properties. Although it is generally accepted that hydrogels ‘entrap’ large amounts of water on a molar basis, there are few experimental data that provide details of the behavior of solvent at the molecular level. The STD NMR spectra represent initial steps toward such an understanding, and a potential general method to probe local solvent dynamics in other hydrogel systems. In the case of covalently bonded 1H atoms, which are part of either the GSH or the pyrene group, the two important factors that affect the efficiency of magnetization transfer from the H2O are the on/off rate of water to/from these atoms and the spin-lattice relaxation time

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(T1) experienced by these atoms. While the on/off rates determine the affinity with which water is binding to the group of atoms under consideration and the residence time of their complex, the T1 value is dependent on the dynamic state of the same. As a matter of fact, these two processes counteract each other so that, while the resonances from the atoms that have strong affinity to the saturated water get saturated themselves, the spinlattice relaxation process restores the magnetization of these atoms to their equilibrium values. While similar processes exist also for the exchangeable NH protons, it is necessary to recognize a subtle difference between the mechanism of saturation transfer from H2O to the amide protons and the covalent-bonded protons in GSH or pyrene. In the case of NH protons the ‘chemical exchange’ process includes detachment of the proton from the NH group and replacement by a solvent proton. If the spin of this exchanged 1H were to be in a ‘saturated‘ state, the resulting NH group inherits this state of saturation. Thus, for the amide protons the STD amplification factor is also a function of the chemical exchange rates. Although the spectroscopic methods used here do not provide a high-resolution structural model for the arrangement of 2 within gels, taken together they do suggest a general picture in which pyrenyl groups are aggregated within a central core of fibers that are coated on the periphery with the GSH moieties. Obviously, the dimensions of fibers observed by SEM are too large to correspond to individual molecule of 2 arranged in a linear array, and many molecules of 2 must self-assemble laterally as well as along the major axis of the fibers. The ‘close-packed’ pyrenes, apparently, do not interact with solvent as effectively, and they are relatively rigid compared to the more mobile GSH moieties, which lie on the outside of the fibers and have access to solvent. However, although the interaction of water with the pyrenyl groups is ‘less strong’ the formation of gels coincides with a significant increase in the pyrenylwater interactions, which are essentially absent in the solution state. It is likely that solvent networks are stabilized in the gels, and these networks span the hydrogen bonding groups on GSH and the aromatic pyrenyl groups. Further structural characterization is required to understand how this ‘low resolution’ model accommodates the apparent essentiality of the γ-glutamyl groups and their presumed participation in intermolecular hydrogen bonds. A critical demonstration of the STD results is that the hydrophobic moieties are not excluded from interactions with solvent water in the gels. Rather, the pyrene groups specifically participate in ‘new’ solvent interactions in the gel state, presumably through cooperative interactions with the GSH groups. Furthermore, the STD experiments here suggest parallels between hydrogelation and protein solvation (39), wherein hydrophobic aromatic groups can interact strongly with water when nearby polar residues can also participate, and this is likely to be the critical feature of gelators. LITERATURE CITED (1) Supramolecular Self-assembly for Biological Applications (Yui, N., Ed.) CRC Press, Boca Raton, FL. (2) Kobayashi, H., Friggeri, A., Kuomoto, K., Amaike, M., Shinkai, S. and Reinhoudt, D. N. (2002) Molecular design of “super” hydrogelators: understanding the gelation process of azobenzene-based sugar derivatives in water. Org. Lett. 4, 1423-1426. (3) Schneider, J. P., Pochan, D. J., Ozbas, B., Rajagopal, K., Pakstis, L., and, Kretsinger, J. (2002) Responsive hydrogels from the intramolecular folding and self-assembly of a designed peptide. J. Am. Chem. Soc. 124, 15030-15037.

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