Inhibition of Fibril Formation by Tyrosine Modification of Diphenylalanine

Article pubs.acs.org/crystal

Inhibition of Fibril Formation by Tyrosine Modification of Diphenylalanine: Crystallographic Insights Santu Bera, Poulami Jana, Suman K. Maity, and Debasish Haldar* Department of Chemical Sciences, Indian Institute of Science Education and Research−Kolkata, Kolkata, Mohanpur, Nadia, West Bengal−741252, India S Supporting Information *

ABSTRACT: The self-assemblies of diphenylalanine and its tyrosine analogues have been investigated. The peptide Boc-Phe-Phe-OMe (1), having a sequence identity with the central hydrophobic cluster (CHC) of Alzheimer’s β-amyloid diphenylalanine motif, self-assembles to produce twisted fibrils. In contrast, the tyrosine-modified analogues Boc-Phe-Tyr-OMe (2), Boc-Tyr-Phe-OMe (3), and Boc-Tyr-Tyr-OMe (4), self-assemble to form microspheres. The X-ray crystallography reveal that the peptide 1 adopts an inverse γ-turn structure and self-associates as a hydrogen-bonded chain of molecules along a 2-fold screw axis, whereas the tyrosine-modified analogues exhibit parallel β-sheet aggregation and cyclic packing in higher-order assembly. The structural analysis of the peptides as described here can serve as a basis for de novo design and therapeutics.

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INTRODUCTION

Intrigued by the previous report, we wanted to investigate whether analogues of diphenylalanine can be designed with minor chemical modifications and to study their intrinsic folding nature. In this report, we have described the design, synthesis, and structural evaluation of a diphenyalalanine model system and its tyrosine modifications. We show that out of this series, the diphenyalalanine 1 self-assembles to produce twisted fibrils. But, the tyrosine-modified dipeptides 2, 3, and 4 form microspheres under the same condition. The X-ray crystallography shed some light on the atomic level structures and selfassembly of the reported peptides.

Molecular self-assembly is highly important for biological, chemical, and material sciences.1 The building blocks undergo self-association to form hierarchical structures at the nanoscale or macroscale by a combination of noncovalent interactions, including hydrogen bonds, electrostatic interactions, π−π stacking, and hydrophobic interactions.2 The resultant structure may achieve biological function-like biological membranes,3 DNA double helix,4 protein microtubules, and microfilaments. Moreover, the self-assembly processes may be pathogenic like the formation of amyloid fibrils relevant to a variety of neurological diseases.5 In many amyloid proteins like Aβ, amylin (IAPP), calcitonin, and others, π−π interactions are highly important.6 Self-assembly of Aβ peptide fragments and polypeptides can develop nanomaterials.7 In this context, small peptide building blocks of diphenylalanine having sequence identity with the central hydrophobic cluster (CHC) of Alzheimer’s β-amyloid, have been widely studied.8 Various functional nanostructures such as nanofibrils,9 nanowires and nanotubes,10 spherical vesicles,11 and ordered molecular chains have been fabricated by self-assembly of diphenylalanine.12 Even the diphenylalanine-based nanotubes have been used as templates for the fabrication of inorganic hybrid materials.13 The self-assembled diphenylalanine-based materials also have potential applications like drug delivery vehicles,14 3D cell culture,15 bioimaging, biosensing, and encapsulation.16 Previously, we have reported the self-assembly and fibril formation by pentapeptide-containing diphenylalanine moiety17 and its L-Ala modified tetrapeptide analogues.18 We have also reported the sheet to helix modification by incorporation of αaminoisobutyric acid in the moiety.19 © 2014 American Chemical Society

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RESULTS AND DISCUSSION Background. The diphenylalanine motif 1 has been adopted from the central hydrophobic cluster (CHC) Aβ19,20 of the amyloid β-peptide, which plays a key role in the process of molecular recognition and fibril formation in Alzheimer’s disease.20 We have replaced the phenylalanine residues by tyrosine and developed peptides Boc-Phe-Tyr-OMe 2, BocTyr-Phe-OMe 3, and Boc-Tyr-Tyr-OMe 4 (Figure 1). The tyrosine-modified peptides have additional hydrogen bonding sites. The aggregation behaviors of the reported peptides were observed by CD spectroscopy, atomic force microscopy (AFM), and field emission scanning electron microscopy (FE-SEM). Solution Analysis. CD is an excellent method to determine the aggregation propensity of the reported peptides. The CD spectra of dipeptides also exhibit general information of the Received: October 10, 2013 Revised: January 30, 2014 Published: February 4, 2014 1032

dx.doi.org/10.1021/cg4015042 | Cryst. Growth Des. 2014, 14, 1032−1038

Crystal Growth & Design

Article

Figure 1. Schematic presentation of peptides 1−4.

secondary structure of the peptides. CD spectra of peptide 1 (Figure 2, black line) in methanol−water (2:1) have positive

Figure 3. (a) AFM image of twisted fibriller aggregates of peptide 1. Inset is a part of the twisted fibril and its schematic diagram. (b) Height profile plot of peptide 1 fibers (green line marked in a). (c−e) AFM images showing polydisperse microspheres morphology of peptides 2, 3, and 4, respectively. (f) Height profile plot of peptide 4 microspheres (green line marked in e).

exhibits the calculated heights of the microspheres were about 150 to 200 nm. The morphology of the reported peptides was also studied by field emission scanning electron microscopy (FE-SEM). A solution containing corresponding peptide in methanol−water (2:1) was incubated at 30 °C over 7 days. A small amount of that solution was drop-casted on a clean silicon wafer and allowed to dry under vacuum at 30 °C for 2 days. FE-SEM images of peptide 1 elucidate the formation of fibrillar morphology (Figure 4a). The length of the twisted fibers is in several micrometer ranges. However, tyrosine-modified peptides 2, 3, and 4 exhibit polydisperse microsphere morphology from methanol−water solution (Figure 4). Solid State Analysis. The aggregated mass obtained from the methanol−water solution of the respective peptides was examined by solid-state Fourier-transform infrared (FT-IR) spectroscopy to study the secondary structure of the peptides.22 The region of 3500−3200 cm−1 is important for the N−H stretching vibrations.23 The range 1800−1500 cm−1 is important for the stretching band of amide I and the bending peak of amide II.24 For peptide 1, an intense band at 3276 cm−1 indicates the presence of strongly hydrogen-bonded NH groups (Figure 5). Another band at around 3393 cm−1 indicates that all NH groups are not involved in intermolecular hydrogen bonding.24 The amide I bands at 1752, 1696, and 1657 cm−1 and the amide II band at 1525 cm−1 suggest that the peptide 1 adopts extensively hydrogen-bonded antiparallel network in fibrils (Figure 5a).25 For peptide 2, an intense band at 3352 cm−1 indicates the presence of strongly hydrogen-bonded NH groups. The amide I bands at 1662 cm−1 and the amide II

Figure 2. CD spectra of peptides 1 (black), 2 (pink), 3 (purple), and 4 (green) from methanol−water (2:1) solution.

bands at 202 and 220 nm, depicting the β-strand structure. However, C terminal tyrosine-modified peptide 2 has a positive band at 204 and 225 nm (Figure 2, pink line). The N terminal tyrosine-modified peptide 3 has a positive band at 200, 205, and 225 nm (Figure 2, purple line). However, the peptide 4 shows positive bands at 196, 203, 207, and 228 nm. For peptides 2, 3, and 4, increased ellipticity around 190 nm indicates coexistence of unordered structure. Hence, the aggregation behaviors of diphenylalanine 1 and its tyrosine analogues are significantly different. Morphology. Furthermore, the morphology of the aggregates was investigated by AFM. A solution containing corresponding peptide in methanol−water (2:1) was incubated at 30 °C over 7 days. A small amount of that solution was dropcasted on a clean microscopic coverslip and allowed to dry under vacuum at 30 °C for 2 days. The atomic force microscopy revealed that the diphenylalanine 1 exhibits twisted fibrillar morphology. The length of the twisted fibers is in several micrometer ranges (Figure 3a).21 The height profile plot (Figure 3b) of peptide 1 fibers (green line marked in Figure 3a) exhibits that the calculated height was about 200 nm. But, the AFM images revealed that the tyrosine modified peptides 2, 3, and 4 exhibit polydisperse microsphere morphology from the methanol−water solution (Figure 3, panels c−e, respectively). The height profile plot (Figure 3f) of peptide 4 microspheres (green line marked in Figure 3e) 1033



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Figure 4. FE-SEM images of (a) peptide 1, (b) peptide 2, (c) peptide 3, and (d) peptide 4.

Crystal Structure Analysis. The conformation and selfassembly of the reported peptides 1, 2, and 4 at the atomic level were further studied by X-ray crystallography.26 Colorless crystals of peptides 1, 2, and 4 suitable for X-ray diffraction studies were obtained from their methanol−water solutions by slow evaporation. Crystals were not obtained for peptide 3 under the same condition. Peptide 1 crystallizes with one peptide molecule in the asymmetric unit (Figure 6a). The solid state conformation of peptide 1 shows that the peptide adopts an inverse γ-turn structure stabilized by seven-membered hydrogen-bonded rings, N1−H30···O2.20 The important backbone torsion angles of peptide 1 are listed in Table 1. The individual subunits of peptide 1 are themselves regularly interlinked through intermolecular hydrogen-bonding interaction, N2−H2···O3, and thereby form a supramolecular antiparallel β-sheet structure along the crystallographic b direction, which supports the FT-IR study.27 The higherorder packing through aromatic−aromatic interactions (C−C distances of 4.001, 4.149, and 4.166 Å) of peptide 1 shows a supramolecular array of columnar structure (Figure 6b).19 The self-assembly pattern reported here is different from the Görbitz NH2-Phe-Phe-COOH hydrogen-bonded cage formation28,29 or the other self-assembled diphenylalanine.30 Peptide 2 crystallizes with one peptide molecule in the asymmetric unit (Figure 7a) and adopts a kink-like conformation. There is no intramolecular hydrogen bond. The important backbone torsion angles of peptide 2 are listed in Table 1. The individual subunits of peptide 2 are themselves regularly interlinked through intermolecular hydrogen-bonding interactions, N1−H1···O4 and N2−H2···O3, and thereby forms a supramolecular parallel β-sheet structure along the crystallographic a direction (Figure 7b). The β-sheet columns are further assembled by O1−H1B···O5 intermolecular hydrogen bonds and aromatic−aromatic interactions (shortest C−C distances: 3.958 and 5.081 Å) to form four-membered cyclic packing (Figure 7b). However, one molecule of peptide 4 crystallizes with one molecule of water in the asymmetric unit. In the solid state,

Figure 5. Solid state FT-IR spectra of aggregated mass obtained from the methanol−water solution of (a) peptide 1, (b) peptide 2, (c) peptide 3, and (d) peptide 4.

band at 1523 cm−1 (Figure 5b) suggest that the peptide 2 has a kink-like structure. Peptide 3 exhibits bands at 3340, 1662, and 1515 cm−1 (Figure 5c), which is structurally very close to peptide 2. Peptide 4 has intense bands at 3390, 1678, and 1515 cm−1, suggesting a sheet-like structure (Figure 5d). 1034



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Figure 7. (a) Solid state conformation of peptide 2. (b) Intermolecular N−H···O hydrogen bonded parallel β-sheet structure of peptide 2, which further self-assemble by O−H···O hydrogen bonds to form four-membered ringlike structures. Hydrogen bonds are shown as black dotted lines. Green lines show the shortest C−C distances for aromatic−aromatic interactions.

Figure 6. (a) Inverse γ-turn conformation of peptide 1 in solid state. (b) Supramolecular array of peptide 1 through intermolecular hydrogen bond and aromatic−aromatic interactions. Hydrogen bonds are shown as black dotted lines. Green lines are showing the shortest C−C distances for aromatic−aromatic interactions.

Table 1. Selected Backbone Torsion Angles (deg) for Peptides 1, 2, and 4 O1−C16−N2−C6 C16−N2−C6−C7 N2−C6−C7−N1

O2−C18−N2−C6 C18−N2−C6−C7 N2−C6−C7−N1

O5−C18−N2− C10 C18−N2−C10− C9 N2−C10−C9−N1

−176.5 −80.4 95.9

−178.6 −120.2 99.5

−173.7 −110.5 96.0

peptide 1 ω1 C6−C7−N1−C8 ϕ1 C7−N1−C8− C17 ψ1 N1−C8−C17− O5 peptide 2 ω1 C6−C7−N1−C8 ϕ1 C7−N1−C8− C21 ψ1 N1−C8−C21− O6 peptide 4 ω1 C10−C9−N1− C8 ϕ1 C9−N1−C8− C23 ψ1 N1−C8−C23− O7

−174.2 −89.2

ω2 ϕ2

170.2

ψ2

−173.1 −120.9

ω2 ϕ2

−28.4

ψ2

−174.6

ω2

−123.7

ϕ2

52.4

ψ2

Figure 8. (a) Kinklike conformation of peptide 4 in the solid state. (b) Intermolecular hydrogen-bonded parallel β-sheet structure of peptide 4 in the crystal. Hydrogen bonds are shown as dotted lines. Tyrosine side chains here appear as violet spheres.

peptide 4 adopts a kinklike conformation (Figure 8a). The important backbone torsion angles of peptide 4 are listed in Table 1. There is no intramolecular hydrogen bond in peptide 4. The peptide 4 molecules organize into a parallel β-sheet assembly by two intermolecular hydrogen-bonding interactions, N1−H9···O2 and N2−H18···O4 (Figure 8b).31 In higher-order aggregation, the peptide 4 forms a water-mediated cyclic packing by intermolecular hydrogen bonds O1−H1···O1S and O3−H3A···O1 and aromatic−aromatic interactions (shortest C−C distances: 4.225 and 4.682 Å) (Figure 9). Hydrogenbonding data for peptides 1, 2, and 4 are also listed in Table 2. Crystal data for these three reported peptides are detailed in Table 3.

Figure 9. Supramolecular water-mediated cyclic packing of peptide 4.

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Table 2. Hydrogen Bonds in Crystal Structures of Peptides 1, 2, and 4 interactions peptide 1 N1− H30....O2 N2−H2....O3 peptide 2 N1−H1···O4 N2−H2···O3 O1−H1B··· O5 peptide 4 N1−H9···O2 N2−H18··· O4 O1−H1··· O1S O3−H3A··· O1

H···A (Å)

D···A (Å)

D−H···A (deg)

2.61

3.04

112

intramolecular

2.12

2.95

164

−1/2 + x, 1/2 − y, −z

2.11 2.11 1.99

2.96 2.90 2.81

172 153 167

1 + x, y, z 1 + x, y, z 2 − x, −1/2 + y, 1 − z

2.11 2.09

2.89 2.91

151 159

x, y, 1 + z x, y, −1 + z

2.01

2.80

165

x, −1 + y, 1 + z

1.97

2.76

162

1/2 + x, 1/2 - y, 2 − z

Table 3. Crystallographic Parameters of Peptides 1, 2, and 4 empirical formula formula weight crystal system space group T (K) a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) V (Å3) Z dc (Mgm−3) observed reflns R1 [I > 2σ(I)] wR2

peptide 1

peptide 2

peptide 4

C24H30N2O5,

C24H30N2O6

2(C24H30N2O7).H2O,

426.50 orthorhombic P212121 100 6.329(5) 17.445(15) 21.917(18) 90 90 90 2420 4 1.171 4490 0.0814 0.2291

442.50 monoclinic P21 100 5.071(1) 13.93(1) 16.36(1) 90 90.01(2) 90 1155.7 2 1.271 3381 0.0680 0.1939

935.02 orthorhombic P21212 100 21.195(4) 23.059(5) 5.105(11) 90 90 90 2495 2 1.244 3631 0.0640 0.1482

Figure 10. (a) PXRD pattern of peptide 1 in fibrils and (b) powder pattern from X-ray crystallography of a peptide 1 single crystal, showing structural similarity.

and 4 have kinklike conformation and self-associate to form a supramolecular parallel β-sheet structure. The results demonstrate how remarkably distinct morphologies originate from analogus building blocks assembled in a subtly different manner.

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EXPERIMENTAL SECTION

General. All L-amino acids were purchased from Sigma Chemicals. 1-Hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC) were purchased from SRL. Peptide Synthesis. The peptides were synthesized by conventional solution-phase methods using racemization free fragment condensation strategy. The Boc group was used for N-terminal protection, and the C-terminus was protected as a methyl ester. Coupling was mediated by dicyclohexylcarbodiimide/1-hydroxyl benzotriazole (DCC/HOBt). The intermediates and final compounds were fully characterized by 500 MHz 1H NMR spectroscopy, 125 MHz 13C NMR spectroscopy, mass spectrometry, and FT-IR spectroscopy. The products were purified by column chromatography using silica (100−200 mesh size) gel as a stationary phase and an nhexane−ethyl acetate mixture as an eluent. NMR Experiments. All NMR studies were carried out on a Brüker AVANCE 500 MHz spectrometer at 278 K. Compound concentrations were in the range of 1−10 mmol in CDCl3 and (CD3)2SO. FT-IR Spectroscopy. All reported solid-state FT-IR spectra were obtained with a Perkin-Elmer Spectrum RX1 spectrophotometer with the KBr disk technique. Mass Spectrometry. Mass spectra were recorded on a Q-Tof Micro YA263 high-resolution (Waters Corporation) mass spectrometer by positive-mode electrospray ionization. X-ray Crystallography. Intensity data were collected with Mo Kα radiation at room temperature using a Bruker APEX-2 CCD diffractometer. Data were processed using Bruker SAINT, and the structure solution and refinement procedures were performed using SHELX97.32 The nonhydrogen atoms were refined with anisotropic thermal parameters. The hydrogen atoms were included in geometric

Moreover, the X-ray diffraction experiment of the fibril of peptide 1 obtained from methanol solution has been performed. A comparison of the spectra obtained by X-ray diffraction data of the fibers (Figure 10a) and the powder pattern from X-ray crystallography of a single crystal of peptide 1 (Figure 10b) clearly exhibits the existence of the same structure in both fibril and crystal.

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CONCLUSIONS In conclusion, by preparing a series of tyrosine modified analogues of diphenylalanine, we found inhibition of fibril formation. The diphenylalanine 1 self-assembles to produce twisted fibrils. The atomic force microscope (AFM) and scanning electron microscope (SEM) images showed the approximate size and shape of these fibers. However, the tyrosine-modified analogues 2, 3, and 4 form polydisperse microspheres under the same condition. In addition, from X-ray crystallography, the diphenylalanine 1 adopts an inverse γ-turn conformation and self-associates to form a supramolecular antiparallel β-sheet structure. But tyrosine-modified peptides 2 1036



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positions and given thermal parameters equivalent to 1.5 times those of the atom to which they were attached. The data have been deposited at the Cambridge Crystallographic Data Center with reference number CCDC 948376, 948377, and 926837 for peptides 1, 2, and 4, respectively. Atomic Force Microscopy. The morphology of the reported compound was investigated by atomic force microscopy (AFM). A small amount of solution (1 mg/mL MeOH:H2O 2:1 v/v) of the corresponding peptides was incubated at 30 °C over 7 days and placed on a clean microscope cover glass and then dried by slow evaporation. The material was then allowed to dry under vacuum at 30 °C for two days. For AFM, images were taken with an NTMDT instrument, model no. AP-0100 in the semicontact mode. Field Emission Scanning Electron Microscopy. Morphologies of all reported peptides were investigated using field emission-scanning electron microscopy (FE-SEM). A small amount of solution (1 mg/ mL MeOH:H2O 2:1 v/v) of the corresponding peptides was incubated at 30 °C over 7 days and placed on a clean silicon wafer and then dried by slow evaporation. The material was then allowed to dry under vacuum at 30 °C for two days. The materials were platinum-coated, and the micrographs were taken in an FE-SEM apparatus (Jeol Scanning Microscope-JSM-6700F).

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures, spectral characterization, and crystallographic data in CIF format of peptides 1, 2, and 4. 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]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the CSIR, New Delhi, India, for financial assistance [project no. 01(2507)/11/EMR-II]. S.B. thanks UGC, India, for a fellowship. S.K.M. and P.J. acknowledge C.S.I.R., New Delhi, India.

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REFERENCES

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Inhibition of Fibril Formation by Tyrosine Modification of Diphenylalanine

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