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Cite this: Chem. Commun., 2014, 50, 6733 Received 27th January 2014, Accepted 9th May 2014

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Introducing charge transfer functionality into prebiotically relevant b-sheet peptide fibrils† Denis Ivnitski,a Moran Amit,b Boris Rubinov,a Rivka Cohen-Luria,a Nurit Ashkenasy*bc and Gonen Ashkenasy*ac

DOI: 10.1039/c4cc00717d www.rsc.org/chemcomm

Incorporation of naphthalene diimide moieties as side chains of short amphiphilic peptide results in the formation of fibrils that exhibit substantial intermolecular p-stacking interactions. These interactions can be manipulated without affecting the structure. The new system is suggested as a first step towards functional self-synthesizing materials.

The self-assembly of amphiphilic peptides and proteins in water is the subject of intense research. Such molecules form b-sheet derived supramolecular structures, including micelles, b-plates, fibrils and nanotubes.1 Since amphiphilic sequences may be formed rather easily by spontaneous coupling of hydrophobic and hydrophilic amino acid segments, several essays have postulated the potential roles of peptide b-sheets in the Origin of Life. Researchers have first highlighted the fibrils protection against decomposition or racemization of polyribonucleotides and polypeptides, and their oligo-ribonucleotides hydrolysis.2 More recently, it was shown that b-sheet plates and fibers can induce chiral amplification via polymerization of amino acids,3 template enzyme-assisted amino acid polymerization,4 and serve as catalysts for various reactions in water.5 We have recently shown that short amphiphilic peptides possessing alternating Glu-Phe dyads can transiently form fibrils that serve as catalysts for native chemical ligation, and thus for self-replication and fibril reproduction.6 Using a related approach, the mechano-sensitive replication of cyclic peptides, also driven by fibril formation, was described.7 Such replication systems were named ‘self-synthesizing materials’ and suggested as models for understanding how molecular assemblies and a

Department of Chemistry, Ben Gurion University of the Negev, Beer Sheva 84105, Israel b Department of Materials Engineering, Ben Gurion University of the Negev, Beer Sheva 84105, Israel c Ilse Katz Institute for Nanoscale Science and Technology, Ben Gurion University of the Negev, Beer Sheva 84105, Israel. E-mail: [email protected], [email protected]; Tel: +972-8-6461637 † Electronic supplementary information (ESI) available: Experimental section and additional results figures. See DOI: 10.1039/c4cc00717d

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cellular machineries emerged in the early evolution, and in particular the crucial transformations from simple singlemolecule replication to reproduction of large structures. It is argued here that the next step towards establishing the utility of self-synthesizing materials will be realized via the introduction of functionality into such replicating and reproducing assemblies. In order to initiate research in this direction, we describe the incorporation of non-natural aromatic groups into a peptide very similar in sequence to the b-sheet replicator. This new peptide is designed to form elongated architectures with longrange charge delocalization. We describe in detail its propensity to form b-sheet fibrils with p interacting moieties. p stacking in these systems is reversibly controlled by reduction–oxidation cycles without altering the overall architecture. The study of b-sheet peptide fibrils offers different implications for bio- and nano-technology. Several groups have investigated the electronic properties of elongated peptide structures, aiming at increasing their conductivity and highlighting the conduction mechanisms. Two different sequence patterns were utilized so far by several research groups, including the authors, for constructing peptide–aromatics conjugates for these studies: (i) cyclic peptides exposing aromatics in their periphery,8 and (ii) amphiphilic9 or bola-amphiphilic10 conjugates with the aromatics at one end of a hydrophilic sequence, or between short hydrophilic sequences, respectively. The sequence pattern of peptide 1NDI studied here (Fig. 1) is substantially different, and is based on the Glu-Phe repeats design of the replicator peptide mentioned above. More specifically, it is derived from the 13 aa peptide 1 (Fig. 1) that has been recently exploited to form stable regular fibril structures.11 Using solution experiments (Fig. S1, ESI†),12 and molecular dynamic simulations,11 we have found that peptide 1 forms super-helical bilayer fibril structures, in which the monomer molecules are perfectly aligned and registered within the monolayer and opposite to monomers in the other layer. Peptide 1NDI contains two naphthalene diimide (NDI) groups attached as side chains to diaminopropionic acids (Dpr), in place of two Phe residues in 1. We note that within anti-parallel

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Fig. 1 (a) Structure of peptide 1 that served as a design model for 1NDI. (b) Synthesis of 1NDI, modified with the aromatic moieties, suitable for long-range electron transfer along fibrils. Inset: HPLC of the crude mixture and ESI-MS of the pure compound; calculated mass = 2255 g mol 1.

b-sheet assemblies, the NDI moieties from adjacent molecules can optimally overlap. 1NDI was synthesized by first preparing on solid phase the respective sequence with orthogonally protected Dpr side chains (Dpr-Mtt). The Mtt groups were removed under mild acidic conditions, and the NDI moieties attached by coupling premade N-butylimide naphthalene 1,9-dicarboxylic acid to the Dpr amine (Fig. 1). After cleavage and deprotection, the peptide was purified and characterized by HPLC and ESI-MS (Fig. 1 – inset). The selfassembly of peptide 1NDI to form fibrils in aqueous solution was probed by cryo-transmission electron microscopy (Cryo-TEM) (Fig. 2a and Fig. S2, ESI†). The formation of long (4500 nm) and entangled fibril assemblies, including bundles of different widths, was observed. The narrowest mono-fibril assemblies (e.g., Fig. 2a) were found to be 4.9  1.2 nm, correlating very well with the length of 1NDI along the b-strand axis. The formation of b-sheet structures in these solutions was clearly evidenced from circular dichroism (CD) measurements that indicated a Cotton effect with a typical 218 nm minimum (Fig. 2b). The biophysical measurements additionally aimed at detecting and analysing the NDI inter-moieties p-interactions as evidence for long range charge or energy transport. A positive Cotton effect at 330–400 nm of the CD spectra (Fig. 2b) provides an indication for excitation energy sharing between NDI groups, and was associated with their P helicity arrangement.13 This Cotton effect was found to increase with 1NDI concentration, in correlation with more b-sheets formation (Fig. 2b), hence confirming that inter-strand NDI interactions take place. The UV spectrum of 1NDI fibrils showed the typical absorption

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Fig. 2 Self-assembly of 1NDI in acetonitrile–water solutions to form b-sheet fibrils, inducing intermolecular p-stacking interactions. (a) Cryo-TEM image of vitrified 1NDI solution after 30 minutes of equilibration. The black arrows mark a 5 nm wide mono-fibril image. (b) CD spectra at different concentrations. (c) UV/Vis absorption of 1NDI (25 mM; black) in comparison to the spectrum of (50 mM) dibutyl-NDI (green). (d) Fluorescence emission spectra at different concentrations, following excitation at 350 nm.

peaks at the 340–400 nm region (Fig. 2c). These peaks were red-shifted by about 5 nm versus the peaks of a control compound dibutyl-NDI that did not form aggregates (Fig. 2c). Such shifts, indicative of J aggregates,14 were also observed when we characterized 1NDI assembly in different acetonitrile– water mixtures (Table S1, ESI†), reflecting more p-interactions in the aqueous mixtures. The fluorescence measurements of 1NDI (Fig. 2d) showed two emission peaks; one at 397 nm, and a second at 500 nm indicative of excimer band formation due to p–p interactions. The excimer-to-monomer peak ratio increased monotonically with peptide concentration, pointing again towards inter-strand, rather than intramolecular, NDI p-interactions. While these ‘passive’ attempts to form fibrils of 1NDI were successful, we also probed two additional methods to actively affect the self-assembly. Thus, we have studied the co-assembly of peptides 1NDI and 1 in different molar ratios. We predicted that this may further facilitate fibril formation, since structures with ‘relaxed’ NDI steric hindrance are obtained. Indeed, as shown by AFM measurements (Fig. S3, ESI†) fibrils were formed from different 1@1NDI mixtures. Fluorescence measurements of the 1@1NDI 1 : 20 mixture revealed lower excimer-tomonomer peak ratio than for 1NDI alone, namely less efficient NDI p-interactions (Fig. S4, ESI†). This suggests incorporation of 1 strands in between 1NDI monomers. After further 1NDI dilution (1@1NDI 1 : 1), the excimer-to-monomer peak ratio was

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Fig. 4 Cryo-TEM images of (a) 25 and (b) 50 mM vitrified 1NDI solutions, after 30 minutes of equilibration and reduction by sodium dithionite.

Fig. 3 Active control over 1NDI fibril architecture and p-stacking interactions by reduction (sodium dithionite) and air oxidation. (a) UV/vis absorption, (b) fluorescence emission (ex = 360 nm), and (c) NIR absorption spectra. Black – original, red – reduced, and blue – reoxidized samples. The decrease in fluorescence after reoxidation (in b) followed over 4 h (1 h intervals), and the final spectrum (green) was obtained after vigorous sonication (see text). Measurements were made in ACN:H2O, ACN:D2O or NH4HCO3 buffer solutions. Insert – chemical structure of the NDI anion radical.

found to be similar to that of 1NDI fibrils, reflecting probably their phase separation. As a second approach to affect the self-assembly of peptide 1NDI, we have considered that the NDI moieties can be reduced in mild conditions, resulting in anionic radicals that are stable in the absence of oxygen. These anion radicals can enhance p-stacking and self-associate, forming aggregates in aqueous solution.15 Accordingly, peptide 1NDI was reduced under anaerobic conditions using an excess of sodium dithionite. Formation of NDI radical anions was confirmed by the appearance of a new peak at 453 nm in the visible region of the absorption spectrum (Fig. 3a), and a new emission band at 600 nm of the fluorescence spectrum (Fig. 3b). This process was found to be reversible by quenching the radical anions upon introduction of air/oxygen into the sample (Fig. 3). The NDI radical anions p-stacking behaviour gives rise to characteristic near-infrared (NIR) absorption bands as a result of electron delocalization.8b The NIR bands wavelength correlate with the aggregate size and the extent of charge delocalization. The NIR spectrum obtained for reduced 1NDI shows a broad absorption with a maximum (lmax) centred at 1120 nm and a long tail continues up to 1500 nm (Fig. 3c). This can be attributed to NDI dimers, and probably also to larger aggregates formed through intermolecular interactions. We have further characterized the reduced structures formed by 1NDI in inert atmosphere by electron microscopy and CD measurements. The Cryo-TEM images (Fig. 4) show that regular elongated fibril and nanotube structures are

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retained. Interestingly, the CD measurements (Fig. S5, ESI†) showed that the Cotton effect at around 380 nm almost disappear, pointing to the formation of less helical inter-NDI arrangement, suggesting better intermolecular overlap and more pronounced p-interactions. This finding was supported by following the structural changes after reoxidation, by fluorescence measurements (Fig. 3b). Right after exposing to air, a large emission peak (B500 nm) was observed, revealing more significant p-interactions than in the original oxidized sample (Fig. 2d). This peak slowly degraded over time in air (Fig. 3b, blue traces), yet even after four hours a pronounced absorption still observed. Only after the sample was vigorously sonicated, this peak was quenched to about the same level as for sample before oxidation, indicating that p stack interactions still exist (Fig. 3b, green). In conclusion, we described a facile derivatization of a potentially prebiotic peptide possessing electronically-active moieties. We showed that while introduction of these NDI moieties do not interfere with efficient self-assembly to form fibril structures, substantial p–p stacking interactions between NDI moieties from adjacent molecules take place. Furthermore, we have shown that it is possible to actively and reasonably reversibly affect the p-stacking and long range conjugation due to better overlap between the p systems but without altering the overall elongated fibril architecture. We suggest that in the next research stages, these molecules, or their close derivatives, will be utilized for replicating electronically active aggregates and devices. This research is supported by the European Research Council (ERC 259204).

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Introducing charge transfer functionality into prebiotically relevant β-sheet peptide fibrils.

Incorporation of naphthalene diimide moieties as side chains of short amphiphilic peptide results in the formation of fibrils that exhibit substantial...
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