Solid State NMR Study of Thermal Processes in Nanoassemblies

May 15, 2012 - Solid State NMR Study of Thermal Processes in Nanoassemblies Formed by Dipeptides. Magdalena Jaworska, Agata Jeziorna, Ewelina Drabik, ...
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Solid State NMR Study of Thermal Processes in Nanoassemblies Formed by Dipeptides Magdalena Jaworska, Agata Jeziorna, Ewelina Drabik, and Marek J. Potrzebowski* Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland S Supporting Information *

ABSTRACT: Three linear dipeptides Phe-Phe (FF), Tyr-Ala (YA) and Asp-Phe(OMe) (DF-OMe, also known as aspartame) were investigated via solid state (SS) NMR spectroscopy, differential scanning calorimetry (DSC), mass spectrometry, and scanning electron microscopy (SEM). Both 1D and 2D SS NMR techniques (1H MAS, 13C CP/MAS, and 1 H−13C inverse HETCOR ultrafast MAS) were used to study the thermal stability and chemical processes of the self-assembled structures: peptide nanotubes (PNTs) and peptide nanowires (PNWs). Each of the investigated dipeptides underwent thermal rearrangement to cyclic dipeptides, also known as diketopiperazines (DKP). Employment of variable temperature (VT) 13C NMR measurements revealed that the cyclization of Phe-Phe (FF) PNT began at a temperature of 373 K, which is lower than the temperature reported previously. The process to form FF DKP would be anticipated to occur because of the removal of water from the hydrophilic channel of the PNTs. When FF PNT is thermally treated carefully and the subtle nanostructure is not damaged, the empty channel can be refilled with water during the diffusion process. An analysis of the thermal stability of YA dipeptide revealed that, as in case of FF, a synthesis of YA DKP is a facile process and can be performed in NMR rotor. YA DKP forms PNTs, which are more thermally stable than FF PNTs. Finally, aspartame forms fibrils and peptide nanowires, which is particulary important because it is commonly applied in the food industry.

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Scheme 1. Visualization of FF PNTsa

INTRODUCTION Peptides have recently received great deal of attention as a simple precursors for the manufacture of self-organizing nanostructures.1−4 The attraction of peptide-based nanosystems results from a number of recent practical applications for natural products, such as drug delivery systems5 or models for the study of ion channels,6 membrane pores,7 electronic devices, and others.8−10 Several research groups have investigated the mechanism for the formation of dipeptide supramolecular assemblies and explained the role of noncovalent interactions.11 In the case of peptides consisting of aromatic residues, π-stacking interactions play a major role in self-assembly.12 Probing the role of aromaticity in the design of dipeptide nanostructures was recently discussed by Mishra and Chauhan.13 The most highly characterized nanostructure for dipeptides with aromatic side-chains is Phe-Phe (FF), which is often used as a model to test self-organization procedures.14,15 The supramolecular structure of FF reported by Görbitz had two distinct regions that were clearly observed: an aromatic hydrophobic zone organized by aromatic−aromatic interactions and water channels organized by hydrogen bonding (a hydrophilic zone) (Scheme 1).16 © 2012 American Chemical Society

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The red color represents water molecules in the hydrophilic channels.

One of the most challenging questions in nanotechnology of natural products is thermal stability of self-assembled materials.17 This problem was recently exhaustively discussed Received: March 19, 2012 Revised: May 15, 2012 Published: May 15, 2012 12330

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by Ryu and Park.18 In cited paper authors have investigated the thermal, chemical, proteolytic stability, and conformational change of Peptide NanoWires (PNWs) and NanoTubes (PNTs). It has been shown that PNWs are highly stable up to 200 °C and remained almost unchanged when incubated in aqueous solutions (from pH 1 to 14) or in various chemical solvents (from polar to nonpolar). In contrast, PNTs started to disintegrate even at 100 °C and underwent a conformational change at an elevated temperature. The problem of thermal stability of self-assembled biological nanosystems was further discussed by Rosenman and coworkers19 who searching the structural transitions in PNTs have proved that FF below the melting point undergoes cyclization forming the appropriate 2,5-diketopiperazine (DKP). During this process the change of nanocrystalline structure from noncentro-symmetric hexagonal space group to a centrosymmetric orthorhombic space group was observed. The linear system possessed a high piezoelectric coefficient and strong optical nonlinearity. The cyclic assembly was characterized by high hydrophobicity, which allowed the possibility for application in hydrophobic biosurfaces. The stability of peptide nanostructures on solid substrates was studied using multiple analytical devices, including electron microscopy, thermal analysis techniques, circular dichroism (CD), X-ray diffraction, and Fourier-transform infrared (FTIR) spectroscopy.20,21 To the best of our knowledge, solid state (SS) NMR spectroscopy was first time used in this study as a basic tool to determine the thermal behavior of peptide nanosystems. SS NMR provided information on the molecular level, gave a “fingerprint” of the local structure and represented the local electronic environment for each nucleus under investigation. In this work we report application of 1D and 2D NMR techniques to study of three different dipeptides: Phe-Phe (FF), Asp-Phe(OMe) (DF-OMe, also known as aspartame), and Tyr-Ala (YA) forming nanotubes and nanowires.

YA DKP. The YA DKP PNTs were obtained by heating a linear sample inside the muffle furnace at 403 K for 0.5 h or in the NMR rotor during a SS NMR experiment. The prepared sample was dissolved in boiling anhydrous methanol (20 mL; T = 338 K; pH 7). The mixture was left under a nonhermetic cover on the air. The precipitate was filtered after slow evaporation occurred after three days. FAB-HRMS: m/z [M +H+] calcd for C12H15N2O3, 253.1082; found: 235.1083. DF-OMe. The DF-OMe PNWs were prepared by dissolving crude commercial sample (100 mg) in boiling HFIP (1 mL; T = 332 K; pH 7) and adding deionized water (50 mL; T = 293 K; pH 7). The mixture was left under a nonhermetic cover on the air. The precipitate was filtered after very slow evaporation that occurred over seven days. Mp = 513−515 K. FAB-HRMS: m/z [M+H+] calcd for C14H19N2O5, 295.1294; found: 295.1289. DF-OMe DKP. The DF-OMe DKP was obtained by heating a linear sample inside the muffle furnace at 463 K for 0.5 h. The prepared sample was dissolved in boiling anhydrous methanol (20 mL; T = 338 K; pH 7). The mixture was left under a nonhermetic cover on the air. The precipitate was filtered after slow evaporation occurred over two days. FAB-HRMS: m/z [M +H+] calcd for C13H15N2O4, 263.1032; found: 263.1041. (iii). Methods. NMR Spectroscopy. The solid-state crosspolarization magic angle spinning (CP/MAS) NMR and onepulse 1H MAS experiments were performed on a 400 MHz Bruker Avance III spectrometer, at a frequency of 100.61 MHz for 13C spectra and was equipped with a MAS probe head using 4-mm ZrO2 rotors. A sample of α-glycine was used to set the Hartmann−Hahn condition for 13C. The conventional 13C CP/ MAS spectra were performed with a proton 90° pulse length of 4 μs, contact time of 2 ms, repetition delay of 3 s, spectral width of 40 kHz and time domain size of 3.5 k data points. The acquisition was collected with a SPINAL decoupling sequence.24 The solid-state Ultra-Fast MAS spectra with spin rates up to 60 kHz (with an ultrafast MAS probe head using 1.3-mm ZrO2 rotors) were recorded on a 500 MHz spectrometer operating at 500.13 MHz for 1H and 125.76 MHz for 13C. The one-pulse 1H MAS were performed at a 55 kHz spin rate with a proton 90° pulse length of 2.5 μs, repetition delay of 2s, spectral width of 12 kHz and time domain size of 4 k data points. The 1H−13C HETCOR (for indirect detection of 13C) experiments were performed using the pulse sequence described by Mao et al.25 The following parameters were used: a 55 kHz spin rate, a proton 90° pulse length of 2.5 μs, first contact time of 1 ms, second contact time of 1 ms or 50 μs and a proton π pulse (5 μs) was used in the middle of the evolution period (instead of CW 1H decoupling as mentioned by Ishii and Tycko).26 Mass Spectrometry. Low-resolution Fast Atom Bombardment mass spectra were recorded on a Finnigan MAT 95 double focusing (BE geometry) mass spectrometer (Finnigan MAT, Bremen, Germany). The methanol solution of samples was mixed with glycerol (the matrix). The sample was bombarded by a beam of Cs+ ions with an energy of 13 keV. Accurate mass measurements were performed by a peak matching technique in FAB positive ion detection mode using glycerol as an internal standard at a resolving power of 8 000 (8% valley definition). Other Methods (DSC, SEM). DSC measurements were recorded using a DSC 2920, TA Instruments. The heating processes investigated for the dipeptides were performed in a muffle furnace (SM-2002) manufactured by Czylok. SEM

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EXPERIMENTAL SECTION (i). Materials. FF (lyophilized), DF-OMe (lyophilized) and HFIP were purchased from Sigma-Aldrich. YA was synthesized employing standard procedure used in our laboratory.22,23 Deionized water (18.2 MΩ/cm) was used in all experiments, which was produced by a Millipore nanopure water system. (ii). Preparation of Samples. FF. The FF PNTs were prepared according to the procedure described by Reches and Gazit:10 the lyophilized form of FF (100 mg) was dissolved in boiling HFIP (1 mL; T = 332 K; pH 7), and then, deionized water (50 mL; T = 293 K; pH 7) was added. The mixture was left under a nonhermetic cover on the air. The precipitate was filtered after 0.5 h. Mp = 575−577 K. FF DKP. The FF DKP PNTs were obtained by heating a linear FF sample inside the muffle furnace at 473 K for 0.5 h. The sample was dissolved in boiling HFIP (1 mL; T = 332 K; pH 7), and then deionized water (50 mL; T = 293 K; pH 7) was added. The mixture was left under a nonhermetic cover on the air. The precipitate was filtered after 0.5 h. YA. The lyophilized form of YA (30 mg) was prepared by dissolving the compound in boiling water (T = 373 K; pH 7) and after slow cooling. The mixture was left under a nonhermetic cover on the air. The precipitate (small white crystals) was filtered. Mp = 543 − 545 K. FAB-HRMS: m/z [M +H+] calcd for C12H17N2O4, 253.1188; found: 253.1196. 12331

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images were made using a Jeol JSM-5500LV apparatus with an acceleration voltage of 10 kV. Prior to the acquisition of the SEM measurements, samples were dried at room temperature on copper stubs and coated with gold.

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RESULT AND DISCUSSION 1. Study of FF Peptides Nanotubes. Scanning electron microscopy (SEM) analysis was used to characterize the structure of the FF PNTs. SEM images are displayed in Figure 1. The FF nanotubes were stable below 373 K (Figure 1A).

Figure 3. 13C CP/MAS NMR spectra recorded at an 8 kHz spin rate for A) FF PNTs and B) DKP obtained after a thermal, solid-state, cyclization process of FF.

temperature limit of 403 K. The difference between the spectra for Figure 3, panels A and B, is apparent. The most striking distinction is the lack of the carboxyl peak at 180 ppm, which disappeared as a result of the cyclization process and formation of appropriate diketopiperazine. Differences in chemical shifts are also seen in the aliphatic and aromatic regions. The molecular structure of the new compound (DKP) is shown in Figure 3B. Figure 4 presents variable temperature (VT) 13C CP/MAS spectra recorded at 298, 333, 353, 373, and 393 K. The top spectrum (Figure 4A) shows the FF nanotube starting material. An analysis of the spectra demonstrated that increased temperatures caused the phenyl rings to become mobile. The

Figure 1. SEM images at different magnifications are shown for (A) FF and (B) FF after heating at 423 K.

After heating at 423 K, the disorganization of PNTs was observed (Figure 1B), which is consistent with previously published results.18 Figure 2 shows the DSC plot of FF with four clearly observable endotherms occurring at 380, 406, 453, and 575 K.

Figure 2. DSC trace of the FF dipeptide (heating rate = 5 K/min).

The fourth endotherm at 575 K corresponds to the reported melting point of FF. An analysis of the first part of profile in the temperature range between 303 and 403 K revealed a smooth endothermic change, which is probably related to the slow removal of water from the hydrophilic zone. 1.1. 13C CP/MAS NMR Study of FF. Figure 3a shows the 13C CP/MAS spectrum of FF nanotubes prepared by previously reported procedures.27 The assignment of signals is straightforward. 13C resonances at a chemical shift of 180 and 168 ppm correspond to carboxylic and amide carbons, respectively. Figure 3B displays the 13C NMR spectrum for the FF sample after heating 30 min at 423 K. The sample in the NMR rotor was stored in an oven outside of the spectrometer because standard solid-state NMR probe heads have an upper

Figure 4. VT 13C CP/MAS NMR spectra of FF recorded at an 8 kHz spin rate at the following temperatures: (A) 298, (B) 333, (C) 353, (D) 373, (E) 393, and (F) 298 K (after 17 h at 393 K). 12332

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Figure 5. VT 1H MAS NMR spectra of FF were recorded at an 8 kHz spin rate at the following temperatures: (A) 303, (B) 323, (C) 333, (D) 353, and (E) 373 K (left column). The right column shows the 1H MAS NMR spectra for a water diffusion process performed in a diffusion chamber for FF nanotubes that underwent heat treatment at 358 K for 0.5 h (F). Water was allowed to diffuse into the system for the following durations at room temperature: (G) 0.5, (H) 1, (I) 3, and (J) 18 h.

Figure 6. 13C CP/MAS NMR spectra (8 kHz spin rate and 298 K) of FF PNT recorded under the following conditions: (A) after heating at 358 K during 0.5 h and (B) after the water diffusion process. (C) The DSC trace of FF PNT after heating at 358 K for 0.5 h, followed by the water diffusion process.

Figure 5A−E displays the VT 1H MAS NMR spectra of selforganized FF NTs recorded at the indicated temperatures. The spin rate was 8 kHz. The 1H MAS spectrum of the FF sample was dominated by a very strong, sharp water signal observed at 4.5 ppm. The water line shape is typical for a very mobile system. Solid-state effects such as chemical shift anisotropy and dipole−dipole interactions, which are responsible for line broadening, are eliminated by the fast molecular motion on the NMR time scale. Increased temperatures caused the water signal to gradually diminish. At 373 K, the mobile water was almost completely removed from the hydrophilic channel. Note that DKP formation starts at this temperature. Further analysis of the DSC profile possibly indicates that the sharp endotherm at 406 K reflects the process of water removal, which was strongly hydrogen bonded with the FF peptide on the border of the hydrophilic wall. “Crystalline” water is more difficult to detect by solid state NMR spectroscopy because of extremely strong homonuclear dipolar coupling, which in many cases, exceeds the range of the chemical shifts for protons.30 The broadening of the proton lines was not removed by slow MAS. Therefore, the spectra recorded under slow conditions were difficult to

phase of the sample changed slightly, but the major motifs were preserved. The spectrum recorded at 373 K was poor quality with a low signal-to-noise ratio, which was likely due to a chemical process starting at this temperature. In particular, the aliphatic part of the spectrum indicated that two compounds (linear and cyclic FF) were present, which supports this assumption. The sample was kept overnight in an NMR spectrometer at a temperature of 393 K exclusively containing DKP. The thermal process at 393 K was not reversible. The spectrum of the sample after it was cooled to room temperature (Figure 4E) confirmed the formation of DKP. 1.2. Water Molecules in Hydrophilic Channel of FF PNT−1H MAS and 13C CP/MAS NMR Study. The role of water in the self-organization of FF PNTs was recently discussed by Kim et al.28 as well Chu and co-workers.29 Water molecules were required to form the hexagonal channels; therefore, the availability of water molecules directly affects the crystal structure. At a low RH/FF ratio (RH = relative humidity), the supply of water to the FF monomers is limited, which resulted in a larger portion of the amorphous phase. In our work, the presence of water molecules in the crystal lattice of FF was monitored by SS 1H NMR spectroscopy. 12333

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reported to form a variety of hydrates, which can easily be resolved by high resolution SS NMR.33 The thermal stability of DF-OMe was reported by Leung and Grant, in which the DSC and TGA analyses were described in detail.34 On the course of our study, we unexpectedly found that aspartame forms nanowires during very slow evaporation of solvents, H2O/ HFIP (v/v, 50:1). Figure 8 shows SEM images for DF-OMe PNWs at various magnifications. The formation of fibers is apparent.

analyze and usually did not contain subtle structural information. The empty hydrophilic channel can be refilled by water during the diffusion process. The column to the right of Figure 5 shows the 1H NMR spectra of the sample heated to 358 K. The sample was stored in a closed vessel at room temperature with water outside of NMR rotor. As can be observed from the spectra, water gradually penetrated the empty space of the FF, and the water content was significantly higher after 18 h. Thermal treatment of the FF sample can easily destroy the fragile nanostructure. Control of the process by 13C CP/MAS measurements is recommended. Figure 6A displays a 13C CP/ MAS NMR spectrum with clearly observable structural changes, particularly for the carboxyl/carbonyl carbons. The splitting of signals was due to water deficiency in the channel. Not all carboxyl/carbonyl residues contributed to hydrogen bonding, but the major motif of the structure was preserved. This observation was supported by the 13C CP/MAS NMR measurement of the sample after 18 h in the diffusion chamber. The obtained spectrum was the same as for the starting material (Figure 6B). However, the DSC profile (Figure 6C) is slightly different compared with the trace shown in Figure 2. SEM image of thermally treated FF nanotube’s channel filled with water after the diffusion process is shown in the Supporting Information. It is concluded that after refilling the morphology of FF nanotubes is not changed. It is worthy to note that water can be partially replaced during heating and diffusion processes by other solvents, e.g., methanol. Figure 7 shows proton spectrum of FF nanotube,

Figure 8. SEM images at different magnifications for DF-OMe.

Figure 9A shows the 13C CP/MAS spectrum of a commercial sample of the dipeptide with a spectral pattern that is typical for a semihydrate.33 As the temperature was increased, the water molecules were removed from the crystal lattice. At 403 K, aspartame existed in the anhydrous form (Figure 9B). At higher temperatures (above 463 K), the formation of DKP was observed. Figure 9C shows the spectrum of cyclic dipeptide. FAB MS of dipeptide DF-OMe and DF DKP are shown in the column to the right. The SEM images of thermally treated DFOMe nanowires at temperatures 403 and 463 K are attached as the Supporting Information. As mentioned in the previous section for condensed matter, 1 H MAS NMR spectra recorded under slow spinning conditions do not contain subtle structural information. Therefore, 1H NMR investigations of thermal processes for DF-OMe which contain crystalline water in the lattice, required the “ultra-fast” (UF) regime with sample spinning rates greater than 50 kHz performed with commercially available 1.3-mm rotors. 35 This frequency exceeds the strength of the homonuclear proton dipolar coupling and would be expected to enter a new regime for spin dynamics. Figure 10 shows 500 MHz 1H MAS NMR spectra of DFOMe. The 1H resonances of water molecules are labeled. An inspection of the spectra in Figure 10A−C, clearly show that an increase in temperature gradually removed water from the crystal lattice. The spectrum in Figure 10A shows the sample with no observable proton signals in the region above 11 ppm, which suggests that DF-OMe in the basic form exists as a zwitter-ion with an acidic proton located on the NH3+ residue. The strong acidic signal at δ1H = 13.5 ppm occurred after

Figure 7. 1H MAS NMR spectra of FF nanotube recorded at 8 kHz spin rate (A) sample A after heating at 358 K during 0.5 h (B) sample stored in a closed vessel at room temperature with methanol outside of NMR rotor. Signals of methanol and water are labeled. SEM image of thermally treated FF nanotube’s channel filled with methanol after diffusion process is shown in the Supporting Information.

first gently heated to 358 K (Figure 7A) and next stored in closed vessel at room temperature with methanol outside of rotor (Figure 7B). The presence of methanol and water (which in trace amount is present in vapor) is apparent. Sharp proton signals proves that both methanol and water inside the FF nanotube are under fast regime exchange. Such modification of PNT can greatly influence its physicochemical properties. 2. Study of Aspartame Nanowires. Aspartame (DFOMe) is commonly used in the food industry31 as a sweetener and was intensively investigated by a number of techniques, including solid-state NMR spectroscopy.32 Aspartame has been 12334

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Figure 9. 13C CP/MAS NMR spectra (8 kHz spin rate and 298 K) of DF-OMe recorded under the following conditions: (A) prior to the thermal processes (commercial sample), (B) after heating at 403 K for 0.5 h, and (C) after heating at 463 K for 0.5 h. FAB MS spectra of DF-OMe and DF DKP are presented in the column to the right.

Figure 10. 500 MHz 1H MAS NMR spectra recorded at a 55 kHz spin rate for DF-OMe (A−C). The signals caused by the protons of water molecules are labeled by asterisks. The temperatures of the heating processes are indicated in the figure.

Figure 11. SEM images at different magnifications for cyclic YA.

temperature to allow the slow evaporation of the solvent to occur.36 The standard procedure involving the application of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was not useful in this case. At the right side of Figure 11 (the highest magnification), the nanotube can be observed to be constructed of smaller elements that resemble nanostraws. At higher temperatures (ca. 400 K), the nanotubes were destroyed. The preliminary results prompted us to more detailed study of thermal processes of YA peptide. Figure 12 shows the DSC plot of YA with two clearly observable endotherms occurring at 503 and 543 K. The second endotherm at 543 K corresponds to melting point of YA. Figure 13A shows the 13C CP/MAS spectrum of the linear dipeptide with sharp signals, which are typical for a wellorganized crystalline phase. The thermal product formed from a solid state synthesis at 403 K is shown in Figure 13B. This process was performed in the NMR rotor. An inspection of the

thermal cyclization (Figure 10C). Hence, we can conclude that during heating of aspartame we have two chemical processes; first formation of DKP and second demethylation of ester group related with proton transfer from amine to carboxyl. From this result it is apparent that UF MAS 1H NMR spectroscopy can be a useful tool for monitoring the thermal synthetic progress of diketopiperazines. 3. Study of Tyr-Ala. The case of third dipeptide under investigation, Tyr-Ala is different compared to those reported supra. The linear YA dipeptide does not undergo selforganization processes. The formation of well organized PNTs we observed only for sample after thermal treatment. Figure 11 shows SEM images of a unique structure for the product of thermal transformation. The sample was dissolved in pure methanol and stored for three days at ambient 12335

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Figure 12. DSC profile of the YA dipeptide (heating rate = 5 K/min).

spectrum in Figure 13B indicates that the crystalline structure was preserved, which was similar to the Phe-Phe cyclic dipeptide. The X-ray structure of YA was not previously reported. In the previous section searching aspartame we have proved that ultra fast MAS proton NMR spectroscopy can a be a source of important structural information regarding the phase transitions of dipeptides. Unfortunately, analysis of the 1H MAS spectra of YA (Figure 14) is more complex. The assignment of the signal at 11.5 ppm is ambiguous because this resonance can represent the OH signal of tyrosine and/or the C(O)OH proton of the YA if this dipeptide existed in the non zwitterionic form. Moreover, the signal disappeared after thermal transformation from a linear to a cyclic dipeptide. The problem of assignment was solved by employing a more advanced approach: 2D NMR inverse detected 1H−13C HETCOR correlations.37,38 Figure 15B−E shows 2D HETCOR spectra recorded with a sample spinning rate of 55 kHz and the pulse sequence in Figure 15A. Figure 15, panels B and C, display spectra of the linear peptide with the second contact time (τ2) equal to 50 and 1000 μs, respectively. Only directly bonded C−H protons were observed for the short contact time, whereas distant

Figure 14. 500 MHz 1H MAS NMR spectra recorded at a 55 kHz spin rate for YA. The signals caused by the protons of water molecules are labeled by asterisks. The temperatures of the heating processes are indicated in the figure.

connections (including correlations between quaternary carbons and protons) could be observed for long contact times In Figure 15B, only C−H cross peaks were observed, which were used for the assignment of resonance positions in the proton NMR spectra. In Figure 15C additional correlations, including a cross peak between the proton at 11.5 ppm and the C−O of tyrosine, were observed. The C(O) and C(O)O groups only showed correlations with NH protons and aliphatic signals. These correlations indicate that linear YA in the crystal lattice exists as a zwitter-ion. A similar approach was used for the study of cyclic YA. Only direct C−H correlation peaks were observed for short contact times (Figure 15D). With a contact time equal to 1000 μs (Figure 15E), remote interactions were observable. The position of the correlation peak that reflected the tyrosine

Figure 13. 13C CP/MAS NMR spectra (8 kHz spin rate and 298 K) of YA recorded under the following conditions: (A) before thermal processes (crystalline sample) and (B) after heating at 403 K for 0.5 h. The FAB MS spectra of YA and the corresponding DKP are presented to the right. 12336

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Figure 15. (A) Pulse sequence of 1H−13C HETCOR NMR.25 1H−13C HETCOR NMR spectra were acquired at a 55 kHz spin rate and at 298 K for YA (B and C) and the corresponding DKP (D and E) with the following contact times (τ2): panels B and D were 50 μs and panels C and E were 1000 μs.

OH contact could be assigned. The −OH signal was upfield by approximately 2 ppm, which indicates that the hydroxyl group of the DKP in the crystal lattice contributes to a much weaker hydrogen bond compared with the linear form. Moreover, we assume that the less acidic character of tyrosine hydroxyl groups can be one of the factors making easier self-assembly of YA DKP. In general, the self-assembly of peptides can be influenced by various factors such as nature and topography of substrate, pH, temperature, concentration of the peptide and medium. The three dipeptides investigated in our work differ in the number of aromatic amino acids and type of interaction: FF (two phenyl groups), YA (one phe-OH, one apolar amino acid), and DF-OMe (one aromatic, one polar side chain). The selforganization of FF was investigated in detail and reported in a number of papers. Very recently Krishnan and co-workers searching mechanism of self-assembly of FF have shown that with evaporating of solvent, the individual FF molecules tend to form intermo-

lecular hydrogen bonds between the carbonyl oxygen and the amide NH hydrogen.39 The electrostatic interactions between the terminal NH3+ and COO− groups and the strong hydrogen bonds between them further promote aggregation of the peptide molecules in a head-to-tail fashion. The secondary aromatic−aromatic interactions result in the formation of higher order structure which stacks vertically via π−π stacking of the aromatic rings resulting in lengthening of the peptide nanostructures. Lateral stacking of the peptide nanostructures may also occur due to electrostatic and hydrogen bonding interactions. Similar mechanism can be valid for selforganization of DF-OMe, which forms nanowires. The case of cyclic YA seems to be different. For apparent reason, for this sample the electrostatic interactions are not the driving force of self-organization. On the other hand the unique feature is contribution of hydroxyl group of tyrosine in formation of intermolecular hydrogen bonding. According to our NMR data (see Figure 15 E) the carbonyl groups of cyclic YA contribute as donors in the −O−H···OC hydrogen bridge. 12337

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To the best of our knowledge, this work shows the first application of DKPs of YA as a substrate forming PNT. The self-assembly of other cyclic dipeptides was recently reported by Joshi and Verma40 as well Govindaraju.41 General procedure, leading to formation of PNTs employing cyclic peptides was published by Ghadiri et al.42

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CONCLUSIONS Both 1D and 2D solid-state NMR techniques (1H MAS, 13C CP/MAS, and 1H−13C inverse HETCOR ultrafast MAS) were used to study the thermal stability and chemical processes for selected PNTs and PNWs. VT 1H and 13C NMR measurements showed that the cyclization of Phe-Phe (FF) PNTs began at 373 K, which was lower than previously reported.18,19 The process to form FF DKP was anticipated to occur by removing water from the hydrophilic channel of the PNT. The synthesis of FF DKP was performed in an NMR rotor under constant monitoring of progress. When the FF PNT was thermally treated carefully and the subtle nanostructure was not damaged, the empty channel could be refilled by the diffusion of water or methanol. The thermal stability of the Tyr-Ala (YA) dipeptide was investigated. As in the case of Phe-Phe, a high yielding synthesis of YA DKP was easily accomplished and was performed in an NMR rotor. The obtained YA DKP formed PNTs that were thermally more stable than FF PNTs. The formation of DKP was also observed for DF-OMe. The aspartame formed fibrils and peptide nanowires, which is interesting because of the common use of aspartame in the food industry.

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ASSOCIATED CONTENT

* Supporting Information S

SEM image of thermally treated FF nanotube’s channel filled with water after the diffusion process. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +48 42 680-3240. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the Polish National Center of Sciences (NCN) for financial support, Grant No. 2011/01/B/ ST4/02637. We also thank Dr. Monika Kozak and Mr. Lukasz Pietrzak for technical assistance.

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dx.doi.org/10.1021/jp302616n | J. Phys. Chem. C 2012, 116, 12330−12338