Cyclic Dipeptides as Building Units of Nano- and Microdevices

Article pubs.acs.org/crystal

Cyclic Dipeptides as Building Units of Nano- and Microdevices: Synthesis, Properties, and Structural Studies Agata Jeziorna,† Karolina Stopczyk,† Ewa Skorupska,† Katarzyna Luberda-Durnas,‡ Marcin Oszajca,§ Wiesław Lasocha,‡,§ Marcin Górecki,∥ Jadwiga Frelek,∥ and Marek J. Potrzebowski*,† †

Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza112, 90-363 Lodz, Poland Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239 Krakow, Poland § Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland ∥ Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw, Poland ‡

S Supporting Information *

ABSTRACT: In this paper we report the influence of stereochemistry on self-organization in the solid state of cyclic dipeptides (CDP) employing two diastereomeric samples cyclo(L-Tyr-L-Ala), cYA 1, and cyclo(L-Tyr-D-Ala), cY(D)A 2, as models. Both compounds were investigated by means of differential scanning calorimetry (DSC), solid state NMR (SS NMR) spectroscopy, scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), electronic circular dichroism (ECD) spectroscopy, and attenuated total reflectance Fourier transform infrared spectroscopy (ATR−FTIR). It has been found that distinction in chirality of alanine residue causes a significant difference in self-assembling and formation of higher order structures. Sample 1 forms peptide nanotubes (PNT) and nanowires (PNW), while for sample 2 only formation of peptide microtubes (PMT) was observed. Crystal and molecular structures for 1 and 2 were refined using PXRD due to failure in attempts to grow crystals with quality suitable for single crystal studies. Both compounds crystallize in the P21 space group and monoclinic system. The size of the unit cell is highly similar; however small differences in alignment of water molecules in the hydrophilic channels and geometry of diketopiperazine rings were observed. Each technique confirmed high thermal stability of PNT, PNW, and PMT under investigation. The water molecules can be thermally removed from the lattice without destroying the subtle crystal structures of nano- and microdevices. This reversible process observed for sample 2 is a unique feature, rarely occurring for the linear dipeptide devices.

1. INTRODUCTION Cyclic dipeptides (CDPs) or 2,5-diketopiperazines (DKP) have recently received a great deal of attention due to their present and future applications in different, often not related areas, e.g., as active pharmaceutical ingredients (APIs)1 or as building units of nanodevices (peptide nanotubes, peptide nanowires, etc.).2 From a pharmaceutical point of view, CDPs are hormone-like molecules which are handled for cell−cell communication.3 These compounds can be regarded as brain−blood barrier (BBB) permeable drugs with remarkable bioactivity in reducing inflammation and inducing a protective state in neurons.4 Their utility might be expanded beyond the central nervous system (CNS). CDPs can control macrophagelike cells and could be useful in the treatment of peripheral inflammatory diseases.5,6 Another important and perhaps dominating field of CDPs applications is nanotechnology.2 Today, the literature which reports utilization of linear and cyclic dipeptides as a starting material for the formation of nano species is very extensive.7 The model sample, most commonly used for testing the self© XXXX American Chemical Society

organization mechanism and formation of nanostructures, is linear diphenylalanine (FF) dipeptide.8 This hydrophobic compound undergoes self-assembly into tubular crystallites due to an unusual morphology of the assemblies. They can be prepared on both the nano- and microscale, the crystal structure of the assemblies being the same regardless of crystal dimensions. Unfortunately, the nanostructures formed from linear FF are thermally unstable, and their decomposition at the temperature above 120 °C, leading to cyclic dipeptides, is observed.9 Very recently Adler-Abramovich and Gazit have reviewed various applications of nanodevices built from the peptide units.10 In particular, aromatic dipeptide nanostructures can be utilized in many ways in various fields, including energy storage devices, displays and light-emitting devices, piezoelectric components, super hydrophobic surfaces for self-cleaning Received: August 4, 2015 Revised: September 9, 2015

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DOI: 10.1021/acs.cgd.5b01121 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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2. EXPERIMENTAL SECTION

applications, composite reinforcement, scaffolds for inorganic ultrastructures, metal−organic frameworks, ultrasensitive sensors, three-dimensional (3D) hydrogel scaffolds for tissue engineering, and drug delivery agents. For most of the applications discussed supra, the thermal stability of selfassembled samples is a prerequisite. Moreover, when the selforganized nano- and/or micropeptide devices are planned to be used as molecular transporters (e.g., drug delivery systems) the crucial question is whether it is possible to remove water from hydrophilic channels (often formed during self-assembling)11 and make this space available for new medium, without destruction of the subtle structure of the device. The answer to this question is one of the aims of our project. The last few years have witnessed growing interest in the stereochemical aspects of cyclic dipeptides and correlation between chirality and self-organization. This problem was recently discussed by Govindaraju12,13 who investigated (LL)and (DD)-isomers of cyclo (Phe-Phe) and (DL)-and (LL)isomers of cyclo (Phg-Phg) under mild reaction conditions. It has been found that molecular self-assembly leads to production of fiber bundles that resemble natural fibers and further undergo transformation into the gel state under the influence of organic solvents.14 More advanced studies were reported by Manchineella et al.15 who searched [cyclo(L-Lys-D-Tyr), cyclo(L-Lys-D-Phe), cyclo(L-Lys-D-Leu), cyclo(L-Lys-Gly), cyclo(L-Lys-D-Tyr), and cyclo(L-Lys-D-Phe)]. Such CDPs, having different reactive groups, can be considered as building units for synthesis of innovative, functional materials. In our work, we present self-organization of two diastereomeric samples (Scheme 1), cyclo(L-Tyr-L-Ala), cYA 1, and

2.1. Synthesis of cYA 1 and cY(D)A 2 Dipeptides. Cyclo(LTyr-L-Ala) 1 and cyclo(L-Tyr-D-Ala-) 2 were synthesized by cyclization of the appropriate N-tert-butoxycarbonyl dipeptide methyl ester by the two-step procedure of Nitecki.16 Boc-L-Tyr-L-Ala-OMe (10 mmol) or Boc-L-Tyr-L-Ala-OMe (10 mmol) was dissolved in formic acid and stirred for 2 h. The solvent was evaporated, and trace of formic acid was removed by vacuum evaporation with toluene (2 × 100 mL). The residue was dissolved in 4:1 sec-butanol/toluene (200 mL), and the solution was heated at reflux for 3 h. CDPs were precipitated in form of a white solid. The products were collected by filtration and washed with methanol. cYA 1 and cY(D)A 2 were obtained with above 95% yield. cYA 1 was also obtained by the previously described temperature treatment procedure.9 2.2. Differential Scanning Calorimetry (DSC). 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. 2.3. Solid State NMR. The solid-state cross-polarization magic angle spinning (CP/MAS) NMR experiments were performed on a 400 MHz Bruker Avance III spectrometer equipped with a MAS probe head using 4 mm ZrO2 rotors, at a frequency of 100.61 MHz for 13C spectra. 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 product was collected with a SPINAL decoupling sequence.17 The solid-state Ultra-Fast MAS spectra with spin rates up to 50 kHz (with an ultrafast MAS probe head using 1.3- mm ZrO2 rotors) were recorded on Bruker Avance III 600 spectrometer operating at frequencies of 600.1 MHz for 1H. The FIDs were accumulated using a time domain size of 16k data points. 2.4. Scanning Electron Microscopy (SEM). SEM images were made using a Jeol JSM-5500LV apparatus with an acceleration voltage of 10 kV. Samples were applied on copper stubs in solution drop, dried at room temperature, and coated with gold. 2.5. Powder X-ray Diffraction (PXRD). Powdered crystalline samples were packed into 0.5 mm borosilicate glass capillaries and mounted on the goniometer (240 mm radius) of a PANalytical X’Pert PRO MPD powder diffractometer. The instrument was fitted with a sealed copper tube, X-ray focusing mirror (providing a focused monochromatized beam), and a PIXcel solid-state position sensitive detector. 1/2° divergence slit, as well as 0.02 rad. Soller slits in both incident and diffracted beam paths were inserted during the experiment preparation. To limit the registering of unwanted air scattered radiation an extension was fixed in front of the detector mount. Generator settings used in the experiment were 40 kV and 30 mA. Measurements were performed on rotating sample-packed capillaries in the range of 5−85° 2θ with a step of 0.02°. Patterns were collected in a multiscan strategy with ca. 4 h per a whole range scan. After the scans were analyzed for discrepancies, the final pattern was obtained by summation of the individual scans. 2.6. Electronic Circular Dichroism (ECD). The 12.69 mg of cYA 1 and 12.58 mg of cY(D)A 2 were dispersed in 1 mL of water and used for ECD measurements. A single drop containing 100 μL of the above suspension was placed on glass coverslips and dried at room temperature. Next, the dried samples were heated for 1 h at a predetermined temperature, i.e., 80, 120, 140, and 160 °C. Subsequently, each of the resulting structures obtained after heating the samples was cooled down and redissolved in 5 mL of water. Solutions thus obtained were subjected to measurements of the ECD spectra at room temperature in quartz cells of 1 cm (240−400 nm range) or 0.05 cm (180−240 nm range) path length. Spectra of both samples at room temperature (r.t.) were also recorded for comparison. ECD spectra were registered on a Jasco J-715 spectrometer under following conditions: scanning speed of 100 nm min−1, step size of 0.2 nm, bandwidth of 1 nm, response time of 0.5 s, and accumulation of

Scheme 1. Chemical Structure of Cyclo(L-Tyr-L-Ala), cYA 1 and Cyclo(L-Tyr-D-Ala), cY(D)A 2 Dipeptides

cyclo(L-Tyr-D-Ala), cY(D)A 2, dipeptides. As we show, the distinct chirality of CDPs under investigation has a significant influence on the formation of higher ordered structures. The explanation of the origin of these differences on the basis of molecular structure and stereochemistry is in our opinion a second important question worth answering. In order to clarify these problems the synthesis, scanning electron microscopy (SEM), solid state NMR, differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and electronic circular dichroism (ECD) studies will be presented. The samples under investigation are additionally attractive due to the presence of the reactive hydroxyl group, which can be involved in the formation of hydrogen bonds and easily chemically modified to open a new field of applications. B



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five scans. The spectra were background-corrected using water as a solvent. UV spectra of the same solutions as for ECD were recorded on a Varian spectrophotometer, model Carry 100E, in the range of 190− 400 nm using 100 nm/min scanning speed, a step size of 0.2 nm, a bandwidth of 2 nm, an accumulation of 1 scans in cuvettes of the 1 or 0.05 cm optical path. 2.7. Infrared Spectroscopy (FT-IR). Solid samples of cYA and cY(D)A 2 were placed on glass coverslips and heated for 1 h at a predetermined temperature, i.e., 80, 120, 140, and 160 °C. Subsequently, each of the resulting structures obtained after heating treatment was cooled down to room temperature and examined by means of Fourier transform infrared spectroscopy (FT-IR) in attenuated total reflectance (ATR) mode using JASCO FTIR-6200 spectrometer equipped with DLATGS detector. All spectra were obtained with 4 cm−1 resolution using a ZnSe single-crystal in the range of 4000−550 cm−1. Spectrum at r.t. was also recorded for comparison. To support the assignment of bands within the range of 1800−1400 cm−1 the theoretical IR spectra for cYA 1 and cY(D)A 2 were generated. The calculations in a vacuum at the DFT/B3LYP/aug-ccpVDZ level of theory were performed using optimized geometries of the relevant compounds taken directly from the X-ray structures.

3. RESULTS AND DISCUSSION 3.1. DSC versus Solid State NMR. As we highlighted in the introduction, nanodevices constructed from CDP are more thermally stable compared to their linear analogues. Thus, in the first step of this project we have tested the thermal behavior of cYA 1 and cY(D)A 2. Figure 1a shows the DSC plot of sample 1 with two clearly observable endotherms occurring at 144 and 282 °C. The second endotherm corresponds to the reported melting point of cYA. An analysis of the first part of the profile in the temperature range between 100 and 200 °C revealed an endothermic change, which is probably related to the removal of water from the hydrophilic zone. Water (Figure 1b) is removed from crystal lattice when the sample is kept for about 1 h at a temperature of 160 °C. In order to check whether the structure of dehydrated sample is renewable in the sense of interactions with water, crystals were kept in a closed vessel in contact with water vapors. It is clear from DSC profile (Figure 1c) that the sample does not contain water molecules. Figure 2 displays high resolution 13C NMR spectra of sample 1 after crystallization (Figure 2a) and after thermal treatment (Figure 2b). It is apparent from these spectra that morphology of samples changes with the migration of water. The most striking differences are seen in aromatic part of spectra. When the thermal phase transition was finished we have tried to introduce water molecules into the lattice by diffusion. Inspection of Figure 2c shows that high crystallinity of sample is preserved, but the subtle structure is not renewed due to absence of water in the crystal lattice. This conclusion is consistent with the DSC studies. The picture of thermal transformations for sample 2 is different. Figure 3a presents the DSC profile for the sample after self-organization. Compared to 1, the peak representing water release is very broad. It means that water molecules are bonded to CDP 2 in a different manner as in the case of 1. It is worth expressing that the release of water is a reversible process. Water molecules can be removed from crystals in a thermal process at 160 °C (Figure 3b) and introduced into the lattice by diffusion of vapors (Figure 3c). These observations are further confirmed by inspection of 13 C CP/MAS spectra (Figure 4). The most significant

Figure 1. DSC plots of cyclo(L-Tyr-L-Ala) 1 dipeptide: (a) after crystallization (water/methanol,1:1 v/v); (b) after heating (1 h, 160 °C); (c) after heating and diffusion of water vapors (17 h).

differences in the transition state are found in the aromatic region. The splitting of signals in aliphatic and carbonyl regions is observed. We assume that this effect is related to the presence of two independent forms rather than to the specific crystal unit organization (Figure 4b). Figure 4c displays crystals of sample 2 after thermal treatment and diffusion of water. The resemblance between starting material (Figure 4a) and treated sample is apparent. In our previous paper we have proved that proton NMR spectroscopy employing advanced ultra fast magic angle spinning (UF MAS) technique with sample spinning in the range 50−70 kHz can be an invaluable tool in structural studies of peptide nano and micro devices.9 For true solids, the broadening of proton lines is not eliminated by slow or medium magic angle spinning without the application of complex pulse sequences.18 In simple standard experiments assignment of the resonances and quantitative analysis of spectra is challenging C



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Figure 2. 13C CP/MAS NMR spectra of cYA 1; (a) after crystallization (water/methanol,1:1 v/v); (b) after heating (1 h, 160 °C); (c) after heating and diffusion of water vapors (17 h).

due to extremely strong homonuclear dipolar couplings. Under a spinning regime greater than 50 kHz, obtained using commercially available 1.3 mm rotors, the spinning frequency exceeds the strength of homonuclear proton dipolar coupling, significantly improving resolution. Figure 5 shows the 1H UF MAS spectra for sample 1 after crystallization (5a), after thermal treatment (5b), and after contact with vapors of water in the closed vessel (5c). The resolution of spectra is not perfect even though special hardware approach was employed. On the other hand, the position of hydroxyl proton bonded to the phenyl ring of tyrosine is diagnostic and can be unambiguously assigned. This signal, found at δ = 13.2 ppm, was used for analysis of crystal composition and evaluation of water contents. From deconvolution and integration of OH tyrosine peak, it is concluded that one water molecule is bonded to one molecule of cYA 1. With removal of water from the crystal lattice, the OH signal is shifted in the direction of lower 1H chemical shift values. It means that the hydroxyl group in dehydrated crystal contributes to weaker hydrogen bonding compared to hydrated form. It is worth remarking that the release of water is not a reversible process, and a water molecule cannot be introduced into the crystal lattice by diffusion of vapors (Figure 5c). It is consistent with DSC and 13C CP/MAS results. Figure 6 shows 1H spectra of cY(D)A 2 recorded with spinning rate 50 kHz. As in the previous case, the 1H signal of the hydroxyl group (δ = 13.4 ppm) for the freshly crystallized sample is diagnostic. Following the procedure for assignment of crystal contents we integrated the OH proton with respect to other residues. The obtained results suggest that in this case we also have one water molecule in the crystal lattice. At a temperature of 160 °C water is removed from the lattice (Figure 6b). Water molecules can be introduced to the crystal network by diffusion process (Figure 6c) which means that during thermal treatment the lattice of 2 is preserved and dehydration/hydration is reversible. 3.2. SEM for cYA 1 and cY(D)A 2. Scanning electron microscopy (SEM) was used to characterize the morphology of the CDPs structures of 1 and 2 obtained by spontaneous selforganization in methanol/water (1:1) solution (see Supporting

Figure 3. (a) DSC plots of cyclo(Tyr-D-Ala) 2 dipeptide: (b) after heating (1 h, 160 °C); (c) after heating and water diffusion (17 h).

Information, Figure S1). The preliminary study of selfassembling of cYA 1 was reported in our previous paper,9 where we describe the formation of a mixture of nanotubes and nanowires. Although the proportion of both forms is not easy to quantify, it is apparent that the dominating forms are nanotubes. Repeating this procedure, employing 1 synthesized by the wet method we were surprised that nanowires are formed as the major product (Figures 7a, top panels). Because of obtaining different results under similar conditions sample 1 was organized by thermal solid state synthesis. In this case, the nanotubes are the dominating form (Figure 7b), which is consistent with our previous data.9 This observation and apparent distinction are not easy to rationalize, since both samples are analytically pure. Even if they contain traces of impurities, which have an influence on self-assembly, their content is below the detectable limit of spectroscopic techniques. The structure of 2 differs not only in diameter dimension but also in the shape of tubes formed during the self-assembly process. Size of the tubes is defined at the micrometer scale (Figure 8a). Testing the thermal stability of microtubes, we D



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Figure 6. 1H UFMAS NMR spectra of cyclo Y(D)A 2; (a) after crystallization (water/methanol, 1:1 v/v); (b) after heating (1 h, 160 °C), (c) after heating and water diffusion (17 h). Spectra were recorded with spinning rate 50 kHz.

Figure 4. 13C CP/MAS NMR spectra of cY(D)A 2; (a) after crystallization (water/methanol, 1:1 v/v); (b) after heating (1 h, 160 °C); (c) after heating and water diffusion (17 h).

Figure 7. SEM images of cYA 1 after crystallization in methanol/water (1:1) solution; (a) cYA 1 obtained by general procedure in solution; (b) cYA 1 obtained by thermal treatment of linear dipeptide H2Tyr(L)AlaOH in solid state. (c) Sample (a) after heating at 160 °C.

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Figure 5. H UF MAS NMR spectra of cYA 1; (a) after crystallization (water/methanol,1:1 v/v); (b) after heating (1 h, 160 °C); (c) after heating and water diffusion (17 h). Spectra were recorded with spinning rate 50 kHz.

techniques allowing the X-ray analysis of powdered samples seemed to be justified. Experimentally obtained diffraction patterns for 1 and 2 were independently indexed using N-TREOR0920 and DICVOL21 programs. The resulting crystal cells for both samples (the same for N-treor and DICVOL) were monoclinic and of similar volume (ca. 600 Å3) with high figures of merit. On the basis of the systematic absences the P21 space group was selected in both cases for structure determination. Structures of both measured samples were determined using direct space-based approach implemented in Fox.22 Z-matrices of the CDP molecules were prepared based on the data available from the deposited structure of a similar compound [COYRIR − CSD refcode23]. These molecule models were introduced into the global optimization calculations along with additional oxygen atoms responsible for modeling the water molecules. Water was included in the calculations after determining that the lack of it led to an insufficient agreement between the experimental data

have found that these structures are resistant to heating up to 160 °C. After thermal treatment, the layer construction of the tube is clearly visible (Figure 8c, right corner). 3.3. X-ray Diffraction Studies for Powdered Samples 1 and 2. 3.3.1. PXRD-Based Structure Solution and Refinements. Searching the CSD database we have found four similar compounds created by connection of tyrosine and other amino acids.19 In cited paper the crystal and molecular structure of cyclic dipeptides was refined using single crystal diffraction. Unfortunately, despite a number of attempts and long-time efforts we were not successful in growing crystals of 1 and 2 with a quality suitable for single crystal X-ray diffraction studies. On the other hand, the quality of NMR spectra clearly proved that the powdered material under investigation is highly crystalline for both samples. Hence, the application of E



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Table 1. Crystallographic Data for cYA and cY(D)A compound

cYA

cY(D)A

empirical formula formula weight/g·mol−1 crystal system space group a/Å b/Å c/Å V /Å3 cell angles Rp [%] Rwp [%] RF [%] number of restraints

C12 H13 N2 O4 249.2 monoclinic P21 11.1238(5) 6.1919(3) 8.9149(5) 603.62(5) 90, 100.563(4), 90 3.88 5.51 9.73 39

C12 H13 N2 O4 249.2 monoclinic P21 11.6890(9) 6.1603(5) 8.8736(9) 612.90(9) 90, 106.422(4), 90 4.98 7.49 10.98 40

Figure 8. SEM images of cY(D)A 2 (a) after crystallization in methanol/water (1:1) solution; (b) after heating at 130 °C; (c) after heating at 160 °C.

and the structure models. During the calculations, the flexibility model allowed for limited variations in bonding distances and angles as well as unrestrained optimization of the torsional degrees of freedom (those describing the aromatic ring were rigidified). Resulting structure models were refined using the Rietveld technique as implemented in JANA2006.24 Discrepancy factors obtained in the refinements were sufficiently low, and an inspection of the difference curves showed good agreement between data calculated from the model and the experiments (see Figure 9A,B). The detailed structural information is presented in Table 1. Both structures were tested for structural voids using PLATON;25 however, no sufficiently large volumes to house additional solvent molecules (e.g., water) were found, though in the case of 2 a small void space can be detected. Both models were optimized using GIPAW calculation methods (Figure 10).26 Next, the GIPAW-structures were refined against the experimental data (only atomic displacement parameters were refined along with preferred orientation corrections). In comparison to experimental data, the obtained discrepancy factors are slightly worse (about 2%, though the ones calculated from structure factors actually improve by a similar value), which can be a result of different approaches to defining 3D structures (i.e., atomic positions) in both methods

Figure 10. Molecular and crystal structures for cYA 1 (panels A and C) and for cY(D)A 2 (panels B and D).

(electron density modeled with spherical atoms vs pseudopotentials locations). X-ray measurements were also carried out on samples of 1 and 2 that underwent temperature treatment (Figure 11). Direct comparison of obtained diffraction patterns reveals clear structural changes, though due to weak diffraction of the materials and present broad peaks, despite many trials, we were unable to determine unequivocally the periodicity (cell parameters) of the resulting structures.

Figure 9. Rietveld plots for cY(D)A 2 (A) and cYA 1 (B). F



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Figure 11. XRD patterns of start samples and after temperature treatment cY(D)A (left) 2; cYA 1(right).

3.3.2. Self-Assembly of cYA 1 and cYA 2. Molecular selfassembly of peptides is driven by different kinds of noncovalent interactions. It is the main driving force leading to production of various forms of nano- and micropeptide devices. In the case of peptides containing aromatic amino acids, the π−π interactions between side chains are known to induce peptide fibrillation. Another important interaction is hydrogen bonding, in particular, in those cases when the water molecules are present in the crystal lattice. The self-organization effect is strengthened if amino acid contains hydroxyl group which can behave like a donor and/or acceptor in hydrogen bridging (e.g., serine, threonine, tyrosine). Other functional residues (SH or NH) can act in a similar way. Formation of various selfassembled structures such as nanotubes, nanofibers, and nanospheres by cyclic and acyclic peptides has already been reported before.2,7,8 The cYA 1 and cY(D)A 2 cyclic dipeptides belong to the bifunctional residues, which possess aromatic and hydroxyl groups in their structure. Both contribute to intermolecular contacts. Figure 12 shows a supramolecular array of cYA sample constructed by hydrogen bonding with contribution of water. As one can see, the water molecules are located in the hydrophilic channel (see Supporting Information, Figure S2). They interact with neighboring water and the adjacent hydroxyl group of tyrosines. The hydrogen bonding is additionally strengthened by π−π interactions of phenyl residues. Aromatic rings are aligned in distorted planes with a small shift between them. As shown by Hunter and Sanders, in some cases offset or slipped geometry is a stabilizing factor.27 The final structure has a layered geometry. The supramolecular array for sample 2 created employing the same structural motif (crystallization water included in the asymmetric unit), expanded as in the case of 1 via hydrogen bonding, reveals a slightly different molecular arrangement (Figure 13). The aromatic rings are in parallel planes but in gauche orientations. As in the previous structure, water molecules are aligned in the hydrophilic channel. Its architecture is different even though water molecules for 2 are also involved in hydrogen bonding with the hydroxyl group of tyrosine as we concluded from 1H SS NMR measurements. The distinct localization of water in the self-assembled structures of 1 and 2 is consistent with our DSC and NMR data. Another factor that can influence the formation of different motifs for diastereo-isomeric samples 1 and 2 during self-

Figure 12. Supramolecular array of cYA 1 sample; (top) projection along [100] direction; (bottom) projection along [001] direction.

organization is the different geometry of diketopiperazine rings. For 1 the six-membered ring is almost ideally flat with C−N− C(O)−C torsional angles equal to −0.07° and −0.02°. For 2 the corresponding torsional angles are found to be −14.02° and −5.70°. We conclude that these small differences in the synergic mechanism can cause significant consequences in the formation of higher order peptide structures. 3.4. Investigation of Thermal Stability of cYA 1 and cY(D)A 2 Employing ECD and FT-IR Methods. Electronic circular dichroism (ECD) spectroscopy has repeatedly demonstrated its extreme usefulness in the study of conformaG



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dipeptide molecular modification.32 An additional measurement after 1 h heating at 150 °C was carried out for peptide cYA 2 to verify the result obtained for 160 °C. As can be seen in Figure 14, the ECD spectra of both samples cYA 1 and cY(D)A 2 are very similar in the whole spectral range. The spectra both before and after the thermal treatment exhibit four Cotton effects (CEs) at ∼275, 215, 200, and 195 nm with positive, negative, positive, and negative sign sequence, respectively. The long-wavelength positive CE arising at ∼275 nm is related to 1Lb aromatic excitation of tyrosine. A minimum ellipticity occurring at ∼215 nm is indicative of an n−π* transition, whereas a second maximum at ∼200 nm is attributed to a π−π* transition of the amide group. The shortwavelength negative CE can be ascribed to 1La aromatic excitation of tyrosine. However, due to the small energy difference between the amide and aromatic excitations resulting in overlapping of ECD bands in the range of 250−190 nm, their precise assignment is thus significantly impeded. The remarkably strong positive band at ca. 200 nm, amounting at r.t. to 56 and 40 mdeg for cYA 1 and cY(D)A 2, respectively, suggests the presence of a defined secondary structure. The shape of the spectra with its negative CE at ∼219 nm, a positive CE at ∼198 nm, and another negative CE at ∼185 nm gives an indication of a dominant β-sheet structure.33,34 Measurements of samples preheated to the desired temperature show almost no changes in the ECD band position and a slight increase in the intensity of the bands only at temperatures ranging from r.t. to +140 °C. However, at higher temperatures, both of 150 °C and of 160 °C, a marked reduction in the intensity of the ECD bands is observed (Figure 14, see inset). In the temperature range r.t. to 140 °C, the relative change of magnitude of 274 nm band in temperature going from low to high, amounts to 9% for cYA 1 and 32% for cY(D)A 2. This result points to the lack of optical activity dependence on the effect of temperature and indicates distinct conformational stability of both samples.30−36 However, the data obtained show up to three times greater stability of cYA 1 versus cY(D)A 2 (Figure 14). Nonetheless, increase in bands intensity with temperature, within the range of 0−40%, is considered to be conformational stability indicator.28−30 It follows from this that the ECD results corroborate the thermal stability of both the test samples. At temperatures above 140 °C, a clear decrease in the intensity of the ECD bands for both samples was observed. As

Figure 13. Supramolecular array of cY(D)A 2 sample; (top) projection along [100] direction; (bottom) projection along [001] direction.

tional homogeneity as well as the thermal stability of a great variety of investigated samples, including short peptides.28−32 Therefore, in the course of this work ECD measurements were performed to test the thermal stability of cyclic peptides cYA 1 and cY(D)A 2, prepared for chiroptical measurements as described in the Experimental Section. In our study, solid samples obtained by evaporating water from the suspension deposited on the glass coverslips were heated successively at five temperatures, namely, r.t., 80, 120, 140, and 160 °C. Next, the samples were cooled down, then dissolved in water, and measured at room temperature. Recently such a procedure demonstrated its usefulness in the study of linear-to-cyclic

Figure 14. ECD (top) and UV (bottom) spectra of preheated samples of cYA 1 (A, left) and cY(D)A 2 (B, right) recorded at r.t. in 180−250 (a) and 240−330 (b) nm spectral regions. Insets in ECD parts show the dependence of ECD band intensities at ∼200 nm (parts Aa and Ba) and ∼275 nm (parts Ab and Bb) on temperature. H



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Figure 15. FT-IR spectra of cYA 1 (A) and cY(D)A 2 (B) at r.t. and after thermal treatment recorded in ATR mode in the 1800−1400 cm−1 range. All curves are normalized.

1505 cm−1 contains partly the vibration of the amide II in the form of a shoulder; however, its relative participation is comparatively low. In both of the dipeptides, the tyrosine vibrations appear to be unaffected throughout the used temperature range. On the basis of calculation results we can conclude that in the case under examination the amide II absorption position is shifted to the 1525−1425 cm−1 range. In this area at r.t. both of the samples exhibit three vibrational modes peaking at 1485, 1468, 1438 cm−1 in the case of cYA 1 and at 1472, 1456, and 1436 cm−1 for cY(D)A 2. These bands can be assigned to N−H bending and C−N stretching vibrations as well as can arise predominantly from the complex peptide backbone. At 120 °C the vibration observed in cYA 1 at 1485 cm−1 disappears and the one centered at 1468 cm−1 shifts to 1475 cm−1, while the last band peaking at 1438 cm−1 remains almost unchanged. In the case of cY(D)A 2, changes occur in the region of 1525−1425 cm−1 already at 80 °C. The vibrations at 1456 and 1436 undergo blue-shift by 10 and 6 cm−1, respectively, whereas the vibration at 1472 cm−1 shall be subject to red-shift by 6 cm−1. Further thermal treatment of both samples investigated does not cause subsequent changes in their IR spectra. In summary, it can be concluded that for both the amide I and II regions, IR spectra do not prove the occurrence of conformational changes as a consequence of heat treatment. Thus, conclusions derived from the ATR-FTIR confirm the results obtained from ECD of the significant thermal stability of the two dipeptides. Moreover, careful observation of the FTIR spectra in the area of amide I band, which is mainly used for secondary structure determinations, allowed the validation of dominated β-structure previously assigned on the basis of the ECD studies.

indicated above, this decrease should be linked with loss of water molecule/s at higher temperatures resulting in a weight reduction of the samples. This weight loss directly relates to the magnitude decrease of the ECD bands at temperatures 150 and 160 °C, as can be seen in Figure 14. Since in our experiments the exact concentration of the sample is not known, the measured ellipticity is given in mdegs without conversion into molar optical constants, i.e., molar ellipticity [Θ /deg cm2 dmol−1] or molar circular-dichroic absorption [Δε /dm3 mol−1 cm−1]. Thus, the results at temperatures of 150 and 160 °C are not linked with conformational changes associated with a decrease in thermal stability of the samples. The insensitivity of the ECD spectra pattern on increase of temperature indicates high thermal stability of cyclic dipeptides cYA 1 and cY(D)A 2 at all tested temperatures. Therefore, it can be assumed that even at high temperatures both peptides maintain their initial structures. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR−FTIR) was additionally used to study the thermal stability of cYA 1 and cY(D)A 2 samples prepared for measurements as described in the Experimental Section. As can be seen in Figure 15, the amide I region (1800−1600 cm−1) of both samples at r.t. is dominated by a broadband with an intensity maximum at 1665 cm−1 and shoulders at the higher wavenumber side. To a significant extent the band and the shoulder consist of the amide I band, which is primarily governed by the stretching vibration of CO and C−N bonds. On the basis of the literature data and taking into account differences in the positions of absorption bands in solvents and solid phase, the bands in the amide I range can be assigned to a β-sheet conformation.10 In the case of cYA 1, at 120 °C the shoulder at 1678 cm−1 and absorption at 1665 cm−1 merge into one broadband, a maximum of which is now found near 1673 cm−1. At higher temperatures, i.e., at 120, 140, and 160 °C, the IR spectra of this dipeptide in the amide I spectral range are not subject to further changes. The amide I region of cY(D)A 2 behaves quite similarly with the exception that the shoulder at 1678 cm−1 disappears already at 80 °C and a single maximum evolves in its place. Typical amide II spectral region (1600−1480 cm−1)37 in the IR spectra of cYA 1 and cY(D)A 2 is dominated by the vibration of the tyrosine ring with bands arising at 1617, 1593, and 1516 cm−1. Besides absorbance from tyrosine, the peak at

4. CONCLUSIONS A multitechnique approach joining differential scanning calorimetry (DSC), solid state NMR (SS NMR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction of powders (PXRD), electronic circular dichroism (ECD) spectroscopy, and attenuated total reflectance Fourier transform infrared spectroscopy (ATR−FTIR) was used to study the influence of stereochemistry on self-organization in the solid state of cyclic dipeptides (CDPs) employing two diastereomeric samples cyclo(L-Tyr-L-Ala), cYA 1, and cyclo(L-Tyr-DI



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Ala-), cY(D)A 2. It has been found that distinction in chirality of alanine residue causes significant difference in self-assembling of CDPs and formation of higher order structures. Sample 1 forms peptide nanotubes (PNT) and nanowires (PNW), while for sample 2 only formation of microtubes (PMT) was observed. High thermal stability of PNT, PNW, and PMT under investigation was proven. The water molecules can be thermally removed from the crystal lattice without destroying the subtle crystal structures of nano- and microdevices. This process is reversible for sample 2 which is a unique feature rarely observed for linear dipeptides forming nano- and microdevices. The crystal and molecular structure for 1 and 2 was refined using PXRD due to failure in attempts to grow crystals with a quality suitable for single crystal studies. Both compounds crystallize in the P21 space group and monoclinic system. The size of the unit cell is highly similar; however, small differences in the alignment of water molecules in the hydrophilic channels and geometry of diketopiperazine rings were observed. It is worth highlighting that in this project we have proven the usefulness of 1H ultra fast MAS NMR spectroscopy to establish the crystal contents and quantify the water molecules in the lattice. Our study clearly shows that subtle stereochemical differences can cause significant structural consequences and that the choice of models with defined chirality can be a key decision in a strategy of synthesis of peptide devices with tailored morphology and defined functions.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b01121. Photo of cYA and cY(D)A crystals obtained from water/ methanol (1:1) solution; Iintermolecular interaction in cYA (A) and cY(D)A (B) molecules (PDF) Crystallographic information files (CIF) CCDC 1412179−1412182 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

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AUTHOR INFORMATION

Corresponding Author

*E-mail; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are indebted to Mr. Tomasz Pawlak for help in GIPAW calculations. REFERENCES

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Cyclic Dipeptides as Building Units of Nano- and Microdevices

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