& Gelation

Tetrapeptidic Molecular Hydrogels: Self-assembly andCo-aggregation with Amyloid Fragment Ab1-40

Marta Tena-Solsona, Juan F. Miravet,* and Beatriu Escuder*[a]

Abstract: A new family of isomeric tetrapeptides containingaromatic and polar amino acid residues that are able toform molecular hydrogels following a smooth change in pHis described. The hydrogels have been studied by spectro-scopic and microscopic techniques showing that the peptideprimary sequence has an enormous influence on the self-as-sembly process. In particular, the formation of extended hy-drophobic regions and the appearance of p-stacking interac-

tions have been revealed as the driving forces for aggrega-tion. Moreover, the interaction of these compounds withamyloid peptidic fragment Ab1-40 has been studied andsome of them have been shown to act as templates for theaggregation of this peptide into non-b-sheet fibrillar struc-tures. These compounds could potentially be used for thecapture of toxic, soluble amyloid oligomers.

Introduction

The self-assembly of peptides into fibrillar aggregates is a cur-rent hot topic in materials science as well as in biomedicine.[1]

In particular, the aggregation and fibrillization of peptides is atthe core of fatal amyloidogenic diseases such as Alzheimer’sdisease.[2] Important studies on the self-assembly of modelpeptides have been carried out to gain an understanding ofthe structures of their aggregates as well as to design inhibi-tors for such an unwanted process. For example, the toxic pep-tides Ab1-40 and Ab1-42 have been extensively studied andthe relationship between the mechanisms of aggregation anddisease has been a matter of intense discussion.[3] Furthermore,shorter peptidic fragments found within these sequences havebeen studied to assess the role of each residue in the self-as-sembly and fibrillization.[4] This information has been used inthe design of inhibitors either peptidic[5] or non-peptidic.[6]

In addition, it is well known that the hierarchical organiza-tion of peptidic fibrils into long fibers and physically cross-linked fibrillar networks leads to the formation of hydrogels.Hydrogel formation has been widely studied for proteins andpeptides,[1a, 7] and more recently for low-molecular-weightamino acid based analogues (e.g. , di- to pentapeptides, pep-tide amphiphiles, peptidomimetics).[1b, 8] Some of these systemshave shown parallelisms with amyloid b-peptide aggregationand may serve as simple models for the development of ag-gregation inhibition strategies.[9]

Herein we report on a family of six tetrapeptides (1–6 ;Scheme 1) that combine nonpolar aromatic phenylalanine (F)and polar aspartic acid (D) residues and are able to form hy-drogels that we have designed foreseeing the parallelism be-tween the hydrogelation of small molecules and the fibrilliza-tion of amyloidogenic peptides. Through this sequence weaimed on one hand to study the role of the hydrophobic/hy-drophilic balance and disposition of the residues in the fibrilli-zation and on the other hand to evaluate the potential ofthese compounds for the interaction with amyloid Ab1-40 byscreening positively charged Lys residues that seem to play animportant role in amyloid misfolding.

Results and Discussion

Compounds 1–6 were prepared by solution peptide synthesisand contain two phenylalanine and two aspartic acid residuesin different positions (see the Supporting Information for de-tails). The chain termini in these compounds were blocked toavoid additional ionization. The benzyloxycarbonyl (Z) protect-ing group was used at the N terminus as an additional aromat-

Scheme 1.

[a] M. Tena-Solsona, Dr. J. F. Miravet, Dr. B. EscuderDepartament de Qu�mica Inorg�nica i Org�nicaUniversitat Jaume I, 12071 Castell� (Spain)Fax: (+ 34) 964728214E-mail : miravet@uji.es

escuder@uji.es

Supporting information for this article is available on the WWW underhttp ://dx.doi.org/10.1002/chem.201302651.

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ic fragment that may assist the aggregation through p-stack-ing interactions.[10]

Aggregation studies

Compounds 1–6 are highly insoluble in water and either it isnot possible to dissolve them by heating at concentrations inthe millimolar range or they precipitate after cooling the hotsolutions. However, hydrogels could be prepared after dissolu-tion in basic media by deprotonation of the D residues andsubsequent acidification with HCl (aqueous solution or vapors).In this way, white opaque hydrogels could be prepared. How-ever, it was found that the high local concentration of HClupon acidification of the samples in many cases led to hetero-geneous aggregation and the formation of large particles lead-ing to poor reproducibility.

To obtain homogeneous transparent hydrogels, solutions ofd-gluconolactone (GL) were used as pH-tuning media. As re-ported by Adams et al. , the hydrolysis of GL into gluconic acidin basic aqueous solution may be used to smoothly lower thepH and produce a homogeneous change in pH in the entiresample.[11] In a typical experiment, the hydrogelator was dis-solved in basic media (Na2CO3) and a weighed amount of GLwas added to this solution. The hydrolysis of GL starts immedi-ately, lowering the pH with the concomitant protonation ofthe aspartate residues of the tetrapeptides and the formationof hydrogels with different macroscopic aspects, as shown inFigures 1–3 (see the Supporting Information for details). Thefinal pH of the samples was in the range of 5.5–6.5 in all cases,which indicates an aggregation-induced pKa shift of the car-boxylic acids, which are expected to have pKa values of about4.5.[12]

The minimum gel concentrations (mgc) observed by usingthis methodology are collected in Table 1. The best hydrogela-

tor is compound 1 (1.5 mm) followed by 3 (2.5 mm), bothforming transparent gels. TEM revealed that these hydrogelsare formed by networks of thin fibrils with widths of about 5–8 nm and lengths in the range of micrometers (Figure 1). Thenetworks are highly uniform and the fibers are physicallycross-linked with few defects and branching. A regular meshsize is clearly observed in both cases by SEM. Compound 6also forms transparent hydrogels with an mgc of 3 mm, but in

this case the microscopic network appears less crowded thanin previous compounds. As can be seen in Figure 3, TEM re-vealed the presence of fibers of several micrometers in lengthand around 10 nm in width. These fibers co-exist in two differ-ent shapes, some as loosely curled right-handed helices rang-ing between 200 and 600 nm in pitch and others with a left-handed helical shape with a much shorter pitch of 50 nm. Webelieve that the latter are formed by the assembly of two fi-brils of the former type.

Compounds 2 and 4 present slightly higher mgc values (4.5and 4 mm, respectively) and form translucent hydrogels (seeFigures 1 and 2). TEM revealed that the hydrogel of 2 is com-posed of a mixture of thin tapes of around 5–10 nm in widthand wider ones of around 40–50 nm. Larger tapes of morethan 100 nm in width were also observed by SEM. In this case,the heterogeneity in sizes provokes the scattering of light anda translucent macroscopic aspect. On the other hand, the hy-drogels formed by compound 4 consist of a network of thin fi-brils of about 8 nm in width that co-exist with larger left-handed helical fibers. These nanohelices are formed after theassembly of several fibrils (see Figure 2, inset) and, althoughthey show different pitches, the pitch-to-width ratio is constantand equal to 5. In this case, self-assembly follows a hierarchicalpathway in which the chiral molecular information is not clear-ly transferred to the small fibrils but emerges after the associa-tion of several of them into higher-order helical nanofibers. Fi-nally, compound 5 forms opaque hydrogels with an mgc of7.5 mm. At the microscopic level, these hydrogels are formedby a mixture of fibrils of around 10 nm in width and large

Table 1. Hydrogelation behavior and minimum gel concentrations (mgc)for compounds 1–6 at 25 8C.

Compound Hydrogel[a] mgc [mm]

1 TpG 1.52 TlG 4.53 TpG 2.54 TlG 4.05 OG 7.56 TpG 3.0

[a] Key: TpG, transparent gel ; TlG, translucent gel; OG, opaque gel.

Figure 1. TEM (left) and SEM (right) micrographs of xerogels of compounds1–3 (top to bottom) prepared from hydrogels at their mgc. Insets right:Macroscopic aspects of the gels.

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tapes of a few hundreds of nm in width. Closer inspection re-vealed that these tapes are formed by the merging of tens offibrils in the direction of the tape.

Structural studies

The supramolecular structures ofthe aggregates were studied byFTIR and CD spectroscopy. Thehydrogels were filtered andwashed several times with waterto remove nonaggregated com-pounds (GL and salts), and IRspectra were recorded afterdrying (see the Supporting Infor-mation). All the compoundspresent an intense amide Istretching band in the range1634–1641 cm�1 assignable toa b-sheet structure as the majorfeature of these aggregates.[13]

However, this band is at theupper limit for this kind of sec-ondary structure, which suggeststhat the sheets may be twistedor distorted from the idealplanar arrangement. In addition,less intense broad shoulders areobserved at higher wavelengths.These bands, which should cor-respond to vibrations related tocarboxylic acids (1700–1730 cm�1) and to minor secon-dary structure components ofamide groups (random coil orturns), were difficult to assign di-rectly and were analyzed byusing a curve-fitting procedure(JASCO FTIR Curve Fitting soft-ware, see the Supporting Infor-mation for details). The positionof the carboxylic acid vibrationband is quite sensitive to thestrength of the hydrogen bondsinvolving this functional group.For example, non-hydrogen-bonded carboxylic acid mono-mers typically present a band ataround 1730 cm�1, whereas thisband is shifted to lower wave-numbers in hydrogen-bondeddimers (ca. 1720 cm�1 for acyclicdimers, ca. 1700 cm�1 for cyclicdimers).[14] In the current case,two carboxylic groups are pres-ent in each molecule and maybe located in environments withdifferent hydrogen-bonding fea-

tures depending on the sequence. Moreover, the formation ofhydrophobic pockets may also have an influence on thestrength of the hydrogen bonds.[15] Compound 1 with the al-

Figure 2. TEM (top) and SEM (bottom) micrographs of hydrogels of compound 4 prepared at its mgc. Inset rightbottom: Macroscopic aspect of the gel.

Figure 3. TEM (left) and SEM (right) micrographs of hydrogels of compounds 5 (top) and 6 (down) prepared attheir mgc. Insets right: Macroscopic aspects of the gels.

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ternating sequence Z-FDFD shows a single broad band cen-tered at 1701 cm�1, which suggests that both D residues are ina strongly hydrogen-bonding environment. In contrast, com-pound 2 with the reverse alternating sequence Z-DFDF showstwo bands of similar strength centered at 1733 and 1709 cm�1,which suggests two different carboxylic acid components, onein a non-hydrogen-bonded environment leading to the bandwith the higher wavenumber and the other in a medium-strength hydrogen-bonding region. Compound 3 with theblock sequence Z-FFDD shows a single broad band centeredat 1724 cm�1, which indicates that both D residues are in-volved in weak hydrogen bonds. The strength of the hydrogenbonding is increased for compound 4 with the reverse blocksequence Z-DDFF; two different bands are observed, a strongone at 1722 cm�1 and a weaker one at 1704 cm�1. For com-pound 5 with the sequence Z-FDDF, two bands are observedat 1712 and 1703 cm�1, which again suggests a strongly hydro-gen-bonded environment for both D residues. Finally, for com-pound 6 with the sequence Z-DFFD, the major band appearsat 1722 cm�1, which suggests a poor hydrogen-bonding envi-ronment. All the compounds present a weak band between1684 and 1693 cm�1, which is usually indicative of an antiparal-lel b-sheet secondary structure although there is some contro-versy about this assignment.[13]

CD spectra of compounds 1–6 were recorded in solution(0.75 mm) and as hydrogels at their respective mgc.[16] As canbe seen in Figure 4A, in solution these compounds do notshow a defined secondary structure by circular dichroism. Inprinciple, random coil structures are suggested, however, thepresence of several aromatic chromophores in the moleculecould lead to a mixing of excitons and alteration of the spec-tral shape. Remarkably, aggregation into hydrogels producednoticeable changes in the CD spectra for some of the com-pounds (Figure 4B), most significantly for compounds 1 and 4,which show an intense negative band centered at 228 nm re-vealing an important conformational change on going fromsolution to the gel phase and a reorganization of the chromo-phores completely different to the rest of the compounds.These two compounds, with no clear similarities in their pri-mary sequence (1: FDFD; 4 : DDFF), self-assemble into highlyCD-active supramolecular structures. The rest of the hydrogelsare less CD active. Compound 5 presents a broad negativeband of lower intensity between 230 and 250 nm related tothe aromatic side-groups, which would be involved in the con-formational change on going from solution to the hydrogel. Inthe case of compound 3, the spectrum shows a negative bandat 222 nm and a positive lobe at 211 nm, which can be as-signed to a typical b-sheet structure. Compounds 2 and 6 aremuch less active, probably due to the cancellation of excitons,and present small negative bands at 235 and 230 nm, respec-tively.

Fluorescence spectroscopy was used to investigate the envi-ronment of the phenylalanine residues. It has been reportedthat the involvement of the aromatic fragment in p-stackinginteractions can be monitored by the shift of the emissionpeak at 284 nm characteristic of this amino acid residue.[17] Inthe current case we studied the fluorescence of compounds

1–6 in solution (0.75 mm) as well as in the gel state (see theSupporting Information). In the former case, we aimed tostudy the effect of the sequence on the fluorescence of thephenylalanine residues. As can be seen in Table 2, all the com-pounds except 5 show an emission peak at 284–285 nm,which indicates isolated aromatic fragments. However, com-pound 5 presents a peak at 288 nm and this redshift of 4 nmcan be ascribed to an intramolecular interaction between thearomatic fragments. This effect requires the folding of the mol-

Figure 4. Mean-residue molar ellipticity of compounds 1–6 in solution(0.75 mm, A) and as hydrogels at their respective mgc (B).

Table 2. Fluorescence studies of solutions (0.75 mm) and hydrogels (mgc)of compounds 1–6.[a]

Compound lem at0.75 mm [nm]

lem at mgc[nm]

Aggregationinducedredshift[nm]

Additional peak at460 nm

1 285 293 8 no2 284 296 12 yes3 284 295 11 weak4 285 297 12 yes5 288 302 14 yes6 284 295 11 no

[a] lex = 260 nm.

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ecule and should be related to the presence of the DD centralblock.

Fluorescence studies were performed also on the hydrogelsat their respective mgc. As can be seen, a redshift of 8 to14 nm from the solution phase emission band is observed inall cases, which suggests the presence of strong p-stacking in-teractions upon aggregation, as reported for related FF con-taining compounds.[17, 18] In addition, for hydrogels 2, 4, and 5,an additional broad weak band appears at around 460 nm.Some authors have suggested that this peak corresponds tothe disposition of the aromatic residues in J aggregates.[8, 18]

This band is not present in the spectra of compounds 1, 3, and6 (the more efficient gelators : lowest mgc values and transpar-ent). On the other hand, the largest redshift (14 nm) corre-sponds to the least effective gelator 5, which suggestsa strong involvement of aromatic residues in the structural re-arrangement from solution to the gel state.

Wide-angle X-ray diffraction (WAXD) analysis was performedon the xerogels of compounds 1–6 (see the Supporting Infor-mation). In general, transparent hydrogels formed from thin fi-brils are poorly crystalline, showing only a broad backgroundsignal in the cases of 1 and 6, and two broad and weak peaksat low angles for compound 3. On the other hand, the xerogelof compound 4 presents a low-angle diffraction peak correlat-ing to a distance of 32 �, which may correspond to the size ofan extended molecule, and a second peak correlating to22.5 �. Finally, compounds 2 and 5, which are formed fromtapes of different widths, exhibit lamellar packing. In particular,compound 5, which is the least effective hydrogelator (highmgc, opaqueness), forms the most crystalline xerogel. It can beenvisaged that crystalline rigid tapes having a low aspect ratioare less prone to physically cross-linking and a larger amountof material is required to form the sample-spanning network.

Taking together all the structural information, tentativemodels for the assemblies consistent with the experimentaldata were constructed by using Macromodel 9.9 (AMBER*force field and a generalized Born surface area (GB/SA) solventsimulation for water ; see Supporting Information for refer-ence). After a conformational search, low-energy mo-lecular structures were used with slight modifications(i.e. , extension of the folded molecule) to constructthe assemblies by using an antiparallel arrangementof the molecules and then these assemblies wereminimized. The resulting distribution of aromatic resi-dues in the assemblies was analyzed in detail as it isknown that p–p and hydrophobic interactions arethe driving forces for self-assembly into higher-orderaggregates. For example, compounds 1 and 2, whichpossess alternating FD sequences, show alternationof polar and nonpolar strands on each face of themodeled sheet (Figure 5). However, the models forcompound 1 present the aromatic Z group close toan F residue, whereas the aggregates of compound 2show a more regular distribution of aromatic groups.These structural differences would have an effect ontheir aggregation into fibrils. For example, in the caseof 2, the models point to a more compact packing

into lamellar aggregates, as suggested by WAXD and the pres-ence of the fluorescence band at 460 nm. Compounds 3 and4, constructed from blocks of FF and DD, show a scrambling ofaromatic residues on both sides of the sheet and the D resi-dues appear to be buried among the aromatic residues, there-by being poorly accessible for hydrogen bonding, in accord-ance with the high wavenumber of their carboxylic IR bands(Figure 6). In the case of 3, the presence of a ZFF block mayalso influence the intersheet assembly, whereas compound 4shows slightly higher crystallinity of the aggregates as well asthe additional fluorescence band at 460 nm.

The models for 5, which bears a central DD block, indicatethat this compound folds in solution into an amphipathicstructure that clusters the aromatic groups on one side of the

molecule minimizing their exposure to water, in agreementwith the results found in fluorescence studies (band at 288 nm

Figure 5. Minimized assembly models for compounds 1 (A) and 2 (B). Topside aromatic (Z, F) and D residues are highlighted in blue and red, respec-tively. Nonpolar hydrogen atoms have been omitted for clarity.

Figure 6. Minimized assembly models for compounds 3 (A) and 4 (B). Top side aromatic(Z, F) and D residues are highlighted in blue and red, respectively. Nonpolar hydrogenatoms have been omitted for clarity.

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and additional broad shoulder at around 400 nm thatis not present for the other compounds, see the Sup-porting Information). A similar arrangement has beenused to construct a packing model that is consistentwith the lamellar structure of the tapes revealed byWAXD. As can be seen in Figure 7A, two sheets ofthis compound can be assembled into a bilayer withthe aromatic groups closely packed in a hydrophobicregion and the aspartic groups on the outer faceavailable for hydrogen bonding. This model is inagreement with the largest shift in the main fluores-cence band of the aromatic groups (14 nm), the pres-ence of an additional band at 460 nm, and the lowwavenumbers of the carboxylic IR vibrations ob-served for this compound. Finally, the models for theaggregation of compound 6 are similar to those ofcompounds 1 and 3, showing segregated blocks ofaromatic and acidic groups on both sides of thesheet leading to low crystallinity (Figure 7B).

Furthermore, the aggregation of compounds 1–6was tested by the thioflavin T (ThT) binding assay. ThT is a ben-zothiazole dye that experiences an enhancement of fluores-cence as a consequence of binding amyloid-like fibrils. Thisassay has been extensively used to quantify the degree of fi-brillar aggregation in amyloidogenic peptides and is based onthe specific enhancement of the emission band at 482 nmcaused by the restriction of the free rotation of the two ringsof ThT upon binding to the amyloid hydrophobic surfaces.[19]

In our studies, we employed this assay to gain information onthe presence of accessible aromatic surfaces in the fibrils. Ina typical experiment the hydrogels were prepared at theirmgc, 20 mL of a stock solution of 0.5 mm ThT in PBS wereadded, and their fluorescence spectra were acquired. The finalconcentration of ThT was set to 5 mm to avoid uncontrolled ef-fects observed by some authors at higher concentrations.[18, 20]

There is intense debate regarding the aggregationstate of bound ThT when higher concentrations(above 30 mm) are used relating to the formation ofThT dimers and other aggregates that produce a de-crease in fluorescence intensity. In our case, we ob-served that for compound 1 the fluorescence in-creased with ThT concentration up to 24 mm andthen a progressive decrease was observed (see theSupporting Information). As a consequence, we useda lower concentration (5 mm) to ensure that thiseffect does not alter our results. As can be seen inFigure 8, hydrogels 6, 1, and 3 show a higher affinityfor ThT, followed by 5, with hydrogels 4 and 2 beingthe least effective binders of ThT. It can be notedthat compounds 1, 3, and 6, the strongest binders,present extended aromatic blocks in their aggregatesand a ZF terminal block in the cases of 1 and 3. Incontrast, compounds 2 and 4, which are the poorestbinders, contain an alternating sequence with no aro-matic blocks. It is also evidenced that compound 5

behaves differently, as commented before. In this case it seemsthat the aromatic groups are so closely packed within the ag-gregates that the access of ThT to the hydrophobic layer couldbe quite difficult. These data give us valuable information onthe presence of hydrophobic regions in the aggregates andare in agreement with the results discussed before: A goodThT binder should have aromatic blocks and these blocksshould be accessible.

Interaction of compounds 1–6 with peptide Ab1-40

Compounds 1–6 have shown different abilities to aggregateinto hydrogels and bind to ThT, which suggests a clear se-quence–properties relationship. These compounds possesstwo features that have been shown crucial in amyloid misfold-

Figure 7. Minimized assembly models for compounds 5 (A) and 6 (B). Aromatic (Z, F) andD residues are highlighted in blue and red, respectively. Nonpolar hydrogen atoms havebeen omitted for clarity.

Figure 8. Fluorescence emission spectra (lex = 450 nm) of the hydrogels 1–6 after the ad-dition of ThT (5 mm, 20 min)

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ing and aggregation: Aromatic residues involved in the forma-tion of large hydrophobic regions and carboxylate side-chainsthat may participate in salt bridges. It has been reported thatthe neurotoxic forms of the Ab peptide are most probablybased on dimeric structures formed by monomers with an an-tiparallel b-sheet structure that is stabilized by the formationof salt bridges between lysine and glutamic or aspartic resi-dues. Further propagation of these dimers into cross-b-sheetsleads to the formation of oligomers as well as insoluble fi-brils.[3] On the other hand, thereis still some controversy as towhether aromatic interactionsbetween F residues are necessa-ry for amyloid aggregation orthe presence of generic hydro-phobic residues is sufficient topromote aggregation in differentamyloidogenic peptides.[21] Forthe compounds studied here,not only the presence of F resi-dues but also their disposition inthe primary structure has a clearinfluence on their aggregationproperties. These differencesmade us envisage a potential se-quence-dependent effect on theinteractions of these compoundswith amyloid peptides, that is,Ab1-40. To investigate this possi-bility, a qualitative study was un-dertaken by using TEM and CD.

For this study, samples of Ab1-40 (50 mm), compounds 1–6(50 mm), and equimolar mixturesof both in phosphate-bufferedsaline (PBS) were incubated at37 8C for 7 days. The aggregationof Ab1-40 can be very sensitive to small environmentalchanges and polymorphism has been reported.[22] For thisreason all the experiments were performed under strictly thesame conditions (see the Supporting Information). Samples forTEM were prepared at different times (6 h, 30 h, 3 days, and7 days) to determine the kinetics of aggregation. In addition,CD spectra of all samples were recorded after 7 days of stabili-zation because no major changes in the solutions of Ab1-40alone were observed in that time (see below) assuming thatthe aggregates are then in local/global minima. As can beseen in Figure SI8 in the Supporting Information, after 6 h ofincubation of Ab1-40, only a few short fibrils were observed byTEM, the sample being formed mainly of unstructured materi-al. After 30 h, the number of short fibrils having a width ofaround 20 nm had increased. After 3 days, long fibers witha dendritic aspect were observed and single fibrils were nolonger detected. These fibers formed an entangled networkafter 7 days that formed a foamy precipitate.

On the other hand, compounds 1–6 were also studied at50 mm, a concentration far below their minimum gel concen-

tration in water. It was observed that some of them main-tained their tendency to form aggregates in PBS at 37 8C evenat this low concentration. For example, compound 1 hadformed thin fibrils of around 6 nm even after 6 h of incubation(Figure 9A), compound 3 had formed a mesh of fibrils after30 h (Figure SI10A), and compound 5 had formed 2D aggre-gates after 30 h (Figure 10A). On the other hand, compounds2, 4, and 6 showed a lesser tendency to aggregate at this con-centration and only a few objects were observed by TEM (see

the Supporting Information). For example, compound 2showed few isolated fibrils with widths of around 5 nm andsamples of compound 6 were almost empty. In the case ofcompound 4, few aggregates were observed initially and iso-lated rope-like aggregates were observed after 7 days.

Equimolar mixtures of Ab1-40 and compounds 1–6 werethen studied by TEM. It was observed that compounds 2 and6, which are poorly aggregating, did not have any appreciableeffect on the aspect of amyloid aggregates after 7 days (seethe Supporting Information) and the micrographs are similarto those of the blank sample of Ab1-40. For the rest of thecompounds, a general increase in the rate of aggregation wasobserved and abundant aggregates were observed even after6 h. Particularly interesting are the cases of compounds 1 and5. In both cases, with time, the aggregates found in the mix-tures took the shape of the pure tetrapeptidic compounds,thin fibrils in the case of 1 (Figure 9) and 2D sheets in the caseof compound 5 (Figure 10). These results point to the co-as-sembly of both peptides and Ab1-40 ruled by the templatingeffect of the tetrapeptide. For mixtures of compounds 3 or 4

Figure 9. TEM micrographs of compound 1 after 6 h (A) and equimolar mixture of Ab1-40 and compound 1 after6 h (B) and 7 days (C). CD spectra of Ab1-40, compound 1, and their equimolar mixture after 7 days (D).

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with Ab1-40, neither seems to dominate and no informationcould be obtained in this respect (see the Supporting Informa-tion).

CD spectra of blank solutions as well as of mixtures were re-corded after 7 days of incubation. The spectrum of the blanksolution of Ab1-40 shows a negative band centered at around225 nm, zero molar ellipticity at 208 nm, and a positive lobe at194 nm typical of a b-sheet structure. This spectrum clearlychanged after incubation with some of the tetrapeptides. Inparticular, incubation with compound 1 led to the disappear-ance of the characteristic b-sheet amyloid band at 225 nm andthe appearance of the spectral shape of pure compound1 with two negative bands at 231 and 205 nm (Figure 9). Inthe case of compound 5, the pure tetrapeptide is not veryactive towards CD, in contrast to the pure amyloid. However, itcan be seen that the intensity of the CD signal of Ab1-40 de-creases considerably in the presence of this compound, whichsuggests that the secondary structure of Ab1-40 has beenmodified in the co-assembly with 5 (Figure 10). A similar effectwas observed for the mixture with compound 4. For the re-maining compounds (2, 3, and 6), the spectrum of Ab1-40 isalmost unaffected, which suggests a weak interaction betweenthese peptides and the amyloid, in agreement with the obser-vations made by TEM. In summary, there is a clear influence ofthe amino acid sequence on the interaction of compounds 1–6 with Ab1-40 and in particular the presence of the Z-FDmoiety at the N terminus in the most effective compounds1 and 5.

Conclusion

We have prepared six isomericZ-protected tetrapeptides con-taining aromatic (F) and polar(D) residues that are able toform low-molecular-weight hy-drogels following a smoothchange of pH caused by the insitu hydrolysis of d-gluconolac-tone into gluconic acid. We stud-ied these hydrogels by spectro-scopic and microscopic tech-niques and have shown that theprimary sequence of the tetra-peptides has an enormous influ-ence on the supramolecular be-havior of these compounds. Inaddition, spectroscopic andpowder diffraction studies ofthese hydrogels revealed a crucialinfluence of hydrophobicity andp-stacking interactions. For thebest hydrogelators, the presenceof segregated blocks of aromatic

and polar fragments was shown to be a determining factor.The presence of extended aromatic regions in the aggregateswas also confirmed by the binding of ThT. The binding ofthese compounds to amyloid peptide Ab1-40 was also studiedand revealed a remarkable preference for the interaction ofAb1-40 with compound 1, even at an equimolar concentration,with a less pronounced effect for compounds 5 and 4. Remark-ably, compounds 1 and 5 act as seeds for the aggregation ofAb1-40 and, moreover, have a significant influence on the sec-ondary structure domains of this peptide, which changed fromthe typical cross-b-amyloid structures into a non-b-sheet struc-ture.

These results are of great interest because the capture ofamyloids to form large aggregates through their interactionswith other molecules has been reported as a strategy toreduce the quantity of toxic soluble oligomers.[5b, 23] Althoughthere are previous reports suggesting a potential parallelismbetween the hydrogelation of small molecules and the fibrilli-zation of amyloidogenic peptides, to the best of our knowl-edge, this is the first time that the intermolecular interaction ofa simple molecular hydrogelator, as opposed to larger oligo-peptides, and a b-amyloid fragment has been studied. It hasbeen shown that a low-molecular-weight compound can havea crucial effect on the molecular conformation of a larger pep-tide.

Currently we are investigating in detail the interaction ofcompound 1 and amyloid compounds Ab1-40 and pentapep-tide KLVFF at the molecular level. We believe that the fact thatthis compound forms fibrillar aggregates under physiologicalconditions may also be an advantage for its potential use asan amyloid scavenger because aggregation or hydrogelationwould retard the degrading activity of proteases.[24] Moreover,

Figure 10. TEM micrographs of compound 5 after 30 h (A) and equimolar mixture of Ab1-40 and compound 5after 30 h (B), 3 days (C), and 7 days (inset to C). CD spectra of Ab1-40, compound 5, and their equimolar mixtureafter 7 days (D).

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it is known that molecular hydrogels are reversible dynamicsystems in which free molecules are constantly exchangingfrom solution to the aggregate phase and this feature hasbeen recently used in drug delivery.[25] In this sense, hydrogelsof compounds 1 and 5 could allow a smooth release of theactive free tetrapeptide into, for example, the blood stream inwhich it could capture misfolded amyloids.

Acknowledgements

This work was supported by the Ministry of Economy andCompetitiveness of Spain (Grants CTQ2009-13961 andCTQ2012-37735) and Universitat Jaume I (Grant P1-1B2009-42).M.T.-S. thanks the Ministry of Education, Culture and Sport ofSpain for an FPU fellowship.

Keywords: aggregation · fibers · gels · peptides · self-assembly

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Received: July 8, 2013Published online on December 11, 2013

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