Sonication-induced instant fibrillation and fluorescent labeling of tripeptide fibers

Abstract

The peptide Boc-Phe-Acp-Phe-OMe 1 (Acp = ε-aminocaproic acid), containing a core of conformationally flexible achiral amino acids and two termini of L-phenylalanine residues, behaves differently on ultrasound exposure. The ultrasound provides the energy to reset the normal self-assembly pattern and modify the morphology of the peptides from polydisperse nanospheres to an entangled fiber network. The sonication induces instant fibril formation, organogelation in toluene, xylene and 1,2-dichlorobenzene, and crystallization in cyclohexane. X-ray crystallography reveals that the peptide 1 adopts a kink-like conformation and self-associates to form a parallel sheet-like structure through multiple intermolecular hydrogen bonds, where the phenyl groups are on the surface of the sheet like hot spots. Field emission scanning electron microscopy (FE-SEM) of the peptide xerogel reveals the sonication-induced nanofibrillar morphology. Further, the sonication-induced fibril formation technique has been used for supramolecular fluorescent labeling of the peptide nanofibers with organic dyes, such as 2,3,6,7-tetrabromonaphthalene diimide and coumarin, and the capture and slow-release of the drug carbamazepine.

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Introduction

Ultrasound exposure causes cavitation, where bubbles form and collapse, generating significant local pressures and temperatures for ultrashort time spans.¹ Hence, ultrasonication is a powerful synthetic tool that can be used to break weak noncovalent interactions, such as hydrogen bonding, π–π stacking, electrostatic and van der Waals interactions, and rearrange them to fabricate stimuli-responsive functional materials.^1,2 Although ultrasonication is widely used in medicine for in vivo imaging,³ and in industry and research for rapid dissolution or dispersion,⁴ little is known about its effects on proteins or peptides in solution. Stathopulos and co-workers have reported that ultrasonication resulted in the formation of amyloid-like aggregates from various proteins.⁵ Bardelang et al. have reported that ultrasound exposure leads to unusual sculpting of dipeptide particles and gelation.⁶ Goto and co-workers have reported the ultrasonication-induced amyloid fibril formation of β₂-microglobulin, where the agitation accelerates the formation of aggregates which then operate as nucleation sites for fibril formation.⁷ The sonication-induced fibril formation mechanism has similarity with the crystal growth of compounds, where the nucleation process can be accelerated by the agitation of the solution. Recently, Aizenberg et al. have reported the formation of large crystals by sonication of a mixture of liquid mercury and an excess of neat 1-dodecanethiol.⁸

Diphenylalanine is an interesting motif for biology and materials science. The self-assembled materials from diphenylalanine have remarkable physical and chemical stability⁹ and have unique mechanical,¹⁰ electrical,¹¹ and optical¹² properties. Görbitz has reported the X-ray structure of diphenylalanine nanotubes.¹³ Gazit and co-workers have used the nanotube to cast silver nanowire.¹⁴ Ulijn and coworkers have used an Fmoc-protected diphenylalanine hydrogel for 3D cell culture.¹⁵ Na et al. developed fluorescent microtubes from diphenylalanine.¹⁶ Previously, we have reported the sonication-induced instant amyloid-like fibril formation and organogelation of a tripeptide having sequence similarity with the central hydrophobic fragment Val-Phe-Phe of the amyloid β-peptide Aβ⁴².¹⁷ Inspired by previous studies, we wanted to study the self-assembly propensity of a peptide in which phenylalanine residues are separated by an achiral spacer. We have synthesized Boc-Phe-Acp-Phe-OMe 1. The peptide exhibits a microsphere morphology. However, the sonication of the peptide induces instant fibril formation, organogelation in toluene, xylene and 1,2-dichlorobenzene, and crystallization in cyclohexane. Field emission scanning electron microscopy (FE-SEM) of the peptide xerogel reveals the sonication-induced nanofibrillar morphology. The single crystal X-ray diffraction reveals that the peptide 1 adopts a kink-like conformation and self-associates to form a parallel sheet-like structure through multiple intermolecular hydrogen bonding, π–π stacking and C–H⋯π interactions. The phenyl groups are on the surface of the sheet like hot spots and are available for charge transfer complex formation. We have exploited this possibility to decorate the peptide fibers with organic dyes, like 2,3,6,7-tetrabromonaphthalene diimide and coumarin. We also have used this method to easily capture and slowly release a therapeutically-relevant molecule, carbamazepine.

Results and discussion

For tripeptide 1 the design principle explored the effect of conformationally flexible achiral ε-aminocaproic acid spacers between two L-phenylalanine residues, to study the stimuli-responsive self-assembly behaviour. The central ε-aminocaproic acid will restrict hydrogen bonded turn formation and form an extended structure. The reported tripeptide Boc-Phe-Acp-Phe-OMe 1 was synthesized using conventional solution-phase methodology and purified and characterized using ¹H-NMR, ¹³C-NMR, FT-IR and mass spectrometry (MS) analysis.

Initially, a wide variety of organic solvents were used to make the tripeptide 1 gel, using conventional heating–cooling techniques.¹⁸ However, the tripeptide did not form a gel. For aromatic solvents like toluene, xylene and 1,2-dichlorobenzene, the peptide did not form a gel and in cyclohexane the compound precipitated out of the solution. The field emission scanning electron microscopy (FE-SEM) image shows that the tripeptide forms polydisperse nanospheres in aromatic solvents (Fig. 1a). The average diameter of the peptide 1 nanospheres is ca. 12 nm. However, when solutions of the reported peptide in various aromatic solvents were subjected to sonication at 25 °C for a period of about 40 s, instant homogeneous gelation was observed.¹⁷ The gelation has been confirmed using the inverted vial method.¹⁹ The opaque gels are stable for 2–3 months at room temperature. Moreover, under ultrasound exposure, the peptide 1 did not form an organogel but formed crystals in cyclohexane (ESI Fig. 1†).

Fig. 1 FE-SEM images showing (a) nanospheres of peptide 1 in xylene after a heating–cooling cycle, (b) twisted ribbon-like morphology of tripeptide 1 xerogel from toluene, (c) rod-like morphology of tripeptide 1 xerogel from xylene and (d) long unbranched nanofibers of tripeptide 1 xerogel from 1,2-dichlorobenzene after sonication.

The self-assembly of the tripeptide 1 in the organogels was further studied using FE-SEM. Sonication of peptide 1 in toluene results in a twisted ribbon-like morphology (Fig. 1b). The twisted ribbons have a diameter ca. 500 nm and are several micrometers in length. However, in xylene, the peptide 1 self-assembled to form nanorod-like fibers (Fig. 1c) after 40 s sonication. In 1,2-dichlorobenzene, networks of very long unbranched fibers have been observed (Fig. 1d). The diameter of the fibers is about 125 nm.

The macroscopic properties of the sonication-induced organogel were derived primarily from the tertiary structure using rheology.^10b Both the storage modulus (G′, a measure of the elastic response of the material) and the loss modulus (G′′, a measure of the viscous response) were measured at 25 °C as a function of time. For peptide 1, the storage modulus (G′) of the organogel (15 mg mL⁻¹) was found to be approximately an order of magnitude larger than the loss modulus (G′′), indicative of an elastic rather than viscous material (Fig. 2). Such rheological behavior is a characteristic exhibited by networks that are physically cross-linked through weak cooperative interactions like hydrogen bonding, electrostatic, and/or hydrophobic interactions.^10b

Fig. 2 Mechanical response of tripeptide 1 gel in toluene at 25 °C with small oscillatory shear in the linear viscoelastic regime.

The xerogel obtained from the 40 s sonicated toluene solution of the peptide was examined using solid-state FT-IR spectroscopy to investigate the structure of the peptide. The region of 3500–3200 cm⁻¹ is important for the N–H stretching vibrations.²⁰ The range 1800–1500 cm⁻¹ is important for the stretching band of amide I and the bending peak of amide II.²¹ For peptide 1, an intense band at 3352 cm⁻¹ indicates the presence of hydrogen-bonded NH groups (ESI Fig. 2†). The amide I and amide II bands appeared at 1631 and 1553 cm⁻¹ (ESI Fig. 2†). This suggests that the peptide 1 adopts an extended backbone conformation and forms an extensively hydrogen-bonded network.²²

To investigate if there are any conformational changes in the peptides under sonication, solution state ¹H NMR experiments were performed. Since in toluene the peptide forms a stable gel after sonication, C₇D₈ has been selected as the solvent system to investigate the process by NMR.¹⁷ 15 mg of peptide 1 was dissolved in 0.8 mL C₇D₈ and ¹H NMR was performed before and after 40 s exposure to ultrasound. On sonication, the C_αH of Phe(1) and Phe(3) shifted from 4.38 to 4.42 (Fig. 3). The peaks responsible for the Phe(1) and Phe(3) NHs of peptide 1 exhibit the strongest modifications and are likely engaged in H bonds (Fig. 7).¹⁷ The peaks responsible for the ε-aminocaproic acid NH shifted with peak deformation. Hence, these results clearly indicate that on sonication the peptide 1 molecules arrange in a more orderly way to enhance intermolecular H-bonding.

Fig. 3 Part of the ¹H NMR spectra of peptide 1 in C₇D₈ (a) after sonication and (b) before sonication, showing down-field shifting of peaks.

The solid state conformation and packing of the reported peptide 1 at the atomic level was studied using single crystal X-ray diffraction analysis. From the X-ray crystallography, it is evident that the asymmetric unit contains one molecule of peptide 1.²³ The ORTEP diagram (Fig. 4) of peptide 1 shows that the peptide backbone adopts a kink-like conformation, though the ε-aminocaproic acid adopts the anti conformation (Fig. 4).²⁴ There is an intramolecular N–H⋯N hydrogen bond.²⁵ For the peptide 1 molecule, the five-membered hydrogen bonded ring between the Phe N and ε-aminocaproic acid NH resulted in a rigid kink-like conformation in the solid state (Fig. 4). The important backbone torsion angles are listed in Table 1.

Fig. 4 The ORTEP diagram of peptide 1 showing the atomic numbering scheme. Ellipsoids are drawn at the 50% probability level. The ε-aminocaproic acid is in the anti conformation. The intramolecular hydrogen bond is shown as a dotted line.

Table 1 Selected backbone torsion angles (deg) for peptide 1

O2–C5–N6–C6	168.2	ω1	C31–C18–C19–C20	179.8	θ4
C5–N6–C6–C15	−67.6	ϕ1	C15–N5–C16–C17	−145.3	ϕ2
N6–C6–C15–N5	−26.5	ψ1	C18–C19–C20–N3	179.8	ψ2
C6–C16–N5–C16	177.3	ω2	C19–C20–N3–C21	−172.9	ω3
N5–C16–C17–C31	176.3	θ1	C20–N3–C21–C29	−110.5	ϕ3
C16–C17–C31–C18	−178.8	θ2	N3–C21–C29-012	18.2	ψ3
C17–C31–C18–C19	−179.0	θ3

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The individual subunits of peptide 1 are themselves regularly interlinked through multiple intermolecular hydrogen-bonding interactions, N6–H10⋯O11, N5–H19⋯O11 and N3–H30⋯O10, and thereby form a supramolecular sheet-like structure along the crystallographic a direction (Fig. 5b). Hydrogen bonding parameters for peptide 1 are listed in Table 2. The higher order packing of the peptide 1 molecules through cooperative multiple intermolecular hydrogen bonding interactions and π-π interactions shows a supramolecular parallel sheet-like structure (Fig. 5a and b). There are two C–H⋯π interactions²⁶ between the Phe(1) ring and ester methyl (C30–H40⋯π, 2.707 Å, 3.562 Å, 148.56°) and the Phe(3) ring and ε-methylene of caproic acid (C16–H21⋯π, 2.833 Å, 3.656 Å, 143.12°) (ESI Fig. 3†). The phenyl groups of the L-phenylalanine residues are on the surface of the sheet-like structure (Fig. 5b). Fig. 5c shows the surface diagram of the sheet-like structure with electropositive and electronegative area in blue and red.

Fig. 5 (a) Side view showing π–π interaction-mediated packing of peptide 1. (b) Top view packing presentation of the supramolecular sheet-like structure of peptide 1, where the phenyl groups are on the surface of the sheet. The intermolecular hydrogen bonds are presented as black dotted lines. (c) The surface diagram of the peptide 1 sheet-like structure.

Table 2 Hydrogen bonding parameters of tripeptide 1^a

D–H⋯A	D⋯H (Å)	H⋯A (Å)	D⋯A (Å)	D–H⋯A (°)
a Symmetry equivalent a: 1 − x, 1/2 + y, 3/2 − z, b = 1 − x, 1/2 + y, 3/2 − z, c = −x, −1/2 + y, 3/2 − z.
N6–H10⋯O11^a	0.86	2.29	2.773(6)	115
N5–H19⋯N6	0.86	2.43	2.779(5)	105
N5–H19⋯O11^b	0.86	2.18	2.951(6)	150
N3–H30⋯O10^c	0.86	1.93	2.744(6)	159

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Moreover, to gain better insight into the morphological evolution by sonication of the material, X-ray diffraction experiments have been performed on the xerogel from toluene.²⁷ The powder X-ray diffraction (PXRD) pattern (Fig. 6b) of the xerogel clearly shows that the material is crystalline in the xerogel and sharp reflections appeared in the 5–40° 2θ range. A comparison of the spectrum obtained from the powder X-ray diffraction data of the xerogel (Fig. 6b) and the powder pattern from X-ray crystallography of a single crystal of peptide 1 (Fig. 6a) clearly shows the existence of the same structure in both nanofibers and crystal.

Fig. 6 (a) Powder pattern from X-ray crystallography of peptide 1 and (b) powder X-ray diffraction data of the xerogel of peptide 1.

From the intriguing results above we propose a model for this sonication-responsive fiber formation and gelation (Fig. 7). The ultrasound provides the energy to reset the self-assembly pattern of peptide 1 and modify the morphology of the peptides from nanospheres to fibers. The phenyl groups of the L-phenylalanine residues are on the surface of the fibers, like hot spots. The phenyl groups are electron rich and can form charge transfer complexes with electron deficient substrates. We have utilized this possibility to decorate the peptide fibers with organic dyes (Fig. 7).

Fig. 7 Schematic presentation of sonication-induced fiber formation. Phenyl groups are on the surface of the fiber like hot spots and can be decorated with dyes.

The ultrasound-responsive nanofiber formation and organogelation have been used to fabricate a decorated hybrid material with enhance photophysical properties.¹⁶ For this purpose, coumarin and 2,3,6,7-tetrabromonaphthalene diimide have been used. Coumarin exhibits emission at 445 nm upon excitation at 360 nm.²⁸ 2,3,6,7-Tetrabromonaphthalene diimide has a microsphere morphology and exhibits green emission upon excitation at 400 nm.²⁹ The solutions of the reported peptide 1 in toluene (15 mg mL⁻¹) with coumarin or 2,3,6,7-tetrabromonaphthalene diimide (1 mg) were subjected to sonication at 25 °C for a period of about 40 s. Instant homogeneous gelation in toluene was observed. The confocal microscopy images show that the coumarin-appended peptide 1 nanofibers exhibit blue fluorescence upon excitation at 360 nm (Fig. 8a and b). The confocal microscopy images show that with 2,3,6,7-tetrabromonaphthalene diimide present during the self-assembly of peptide 1, the obtained nanofibers show green fluorescence (Fig. 8c and d) upon excitation at 400 nm. Thus, coumarin or 2,3,6,7-tetrabromonaphthalene diimide aggregated with peptide 1 by sonication-induced organogelation and act as a fluorescent label for imaging the peptide nanofibers.

Fig. 8 Confocal microscope image of (a) peptide 1 and coumarin xerogel in bright field and (b) blue fluorescent fibers upon excitation at 360 nm. (c) Image of peptide 1 and 2,3,6,7-tetrabromonaphthalene diimide xerogel in bright field and (d) green fluorescent fibers upon excitation at 400 nm.

For further investigation of the phenyl–dye interaction, UV-vis and fluorescence experiments were performed, as it is a very sensitive technique for studying the changes in the microenvironment. The fluorescence intensity at 287 nm due to the phenyl groups decreases with increasing coumarin concentration. The emission peak responsible for coumarin also shifted from 454 nm to 464 nm, which indicates an interaction between the dye molecules and peptide 1 (Fig. 9). The spectroscopy study with 2,3,6,7-tetrabromonaphthalene diimide and peptide 1 shows similar results.

Fig. 9 Fluorescence spectra of peptide 1 with increasing concentrations of coumarin. Excitation at 255 nm.

Moreover, we have used this method to easily capture and slowly release a therapeutically relevant molecule. We have demonstrated the sonication-induced capture of carbamazepine, a drug for the treatment of seizure disorders, neuropathic pain and extreme cases of schizophrenia. The drug has common side effects, such as nausea, sedation and skin rashes. Therefore, a controlled or slow-release formulation is required for less side effects. The sonication-induced peptide fibers with phenyl group “hot spots” on the surface can easily capture carbamazepine. The drug binding efficacy of the sonication-induced fibers of peptide 1 was studied using absorption and emission spectroscopy. Fig. 10a shows the UV-vis titration spectra of peptide 1 with gradual addition of carbamazepine. The decrease in the emission intensity of the phenyl group (287 nm) of peptide 1 (ESI Fig. 5†) and the drug with gradual addition of carbamazepine indicates the interaction between peptide 1 and the drug (Fig. 10b).

Fig. 10 (a) UV-vis spectra and (b) fluorescence spectra of peptide 1 with increasing concentrations of carbamazepine. The excitation wavelength is 270 nm. (peptide 1 1.25 × 10⁻⁵ M).

The concentration of the drug bound with peptide was quantified using UV-visible spectroscopy and the results were expressed as loading efficiency ((amount of drug added − amount of free drug)/(amount of drug added in %)). The loading efficiency is 65.6%. The drug loading content (i.e. (weight of the bound drug/weight of xerogel used) × 100) for the fibers obtained from the peptide is 6.64%. The experimental procedures described in the experimental section were applied to calculate the loading efficiency and drug loading. We have also studied the release of the encapsulated carbamazepine from the peptide fiber. 2 mg of the carbamazepine-loaded peptide fibers were immersed into 5 mL sodium phosphate buffer (pH 6.0) in a 15 mL centrifuge tube and centrifuged at 4000 rpm for 10 min and monitored by UV-visible spectroscopy at different time intervals. Under slightly acidic conditions (sodium phosphate buffer of pH 6.0), the protonation of the carbamazepine NH₂ increases the hydrophilicity of the drug molecules and governs the slow release from the surface of the fibers. Fig. 11 shows that the surface of the peptide fibers slowly releases the encapsulated drug, with a complete release after 14 h. The pH value of 6 is very close to that of in vivo conditions. At strongly acidic or basic pH, deprotection of the Boc and –OMe groups affected the self-assembly of the peptide and the fibrous structure and the encapsulated drug was released very fast (ESI Fig. 4†). At the more acidic and basic pH values (i.e. pH 2 and 10) the encapsulated drug is completely released in the time period of 5–6 h.

Fig. 11 Drug release profile of carbamazepine-loaded peptide 1 fibers, obtained from UV-vis spectroscopy.

Conclusions

In conclusion, the stimuli-responsive self-assembly propensity of a tripeptide containing L-phenylalanine and ε-aminocaproic acid has been reported. The sonication energy has been used to cleave and homogenize noncovalent interactions in the peptide and then reorganize the molecules to form elongated nanofibers, organogels and crystals. X-ray crystallography reveals that the tripeptide 1 adopts a kink-like conformation and self-associates to form a sheet-like structure through multiple intermolecular hydrogen bonding and π–π interactions. The phenyl groups of the L-phenylalanine residues are on the surface of the sheet like hot spots for charge transfer complex formation. The ultrasound-responsive nanofibers have been decorated with coumarin or 2,3,6,7-tetrabromonaphthalene diimide to develop hybrid materials with enhance photophysical properties. This method has also used to easily capture and slowly release a therapeutically relevant molecule, carbamazepine. The results presented here may foster new uses of ultrasound energy for the fabrication of hybrid materials with advanced applications.

Experimental

General

All L-amino acids were purchased from Sigma chemicals. HOBt (1-hydroxybenzotriazole) and DCC (dicyclohexylcarbodiimide) were purchased from SRL.

Peptide synthesis

The peptides were synthesized using conventional solution-phase methods using a 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 products were purified using column chromatography with silica (100–200 mesh size) gel as the stationary phase and an n-hexane–ethyl acetate mixture as the eluent. The intermediates and final compounds were fully characterized using 500 MHz and 400 MHz ¹H NMR spectroscopy, 125 MHz ¹³C NMR spectroscopy, FT-IR spectroscopy and mass spectrometry. The tripeptide 1 was characterized using X-ray crystallography.

NMR experiments

All NMR studies were carried out on Brüker AVANCE 500 MHz and Jeol 400 MHz spectrometers at 298 K. Compound concentrations were in the range of 1–10 mM in CDCl₃ and (CD₃)₂SO.

FT-IR spectroscopy

All reported solid-state FT-IR spectra were obtained with a Perkin Elmer Spectrum RX1 spectrophotometer using the KBr disk technique.

Mass spectrometry

Mass spectra were recorded on a Q-Tof Micro YA263 high-resolution (Waters Corporation) mass spectrometer using positive-mode electrospray ionization.

UV/vis spectroscopy

UV/Vis absorption spectra were recorded on a Perkin Elmer UV/Vis spectrophotometer.

Fluorescent spectroscopy

Fluorescent spectra were recorded on a Horiba Jobin Yvon fluorescence spectrometer.

Gelation

The solutions of the peptide (15 mg mL⁻¹) in various aromatic solvents were subjected to sonication in a Citizen Ultrasonic Cleaner Bath at 25 °C for a period of 40 s. Instant homogeneous gels appeared.

Rheology

The viscoelastic properties of the sonication-induced gels were measured with a commercial rheometer, AR-G2, TA Instruments, New Castle, USA.

Confocal microscopy

The sonication-induced gels of the reported compound and dye or drug were placed on a glass slide, and then the slides were washed repeatedly with fresh buffer solution. Finally, the slides were dried under vacuum, and images were taken using a Zeiss LSM 710 confocal microscope.

Powder X-ray diffraction study

The xerogel of the peptide was dried under vacuum for 4 days and X-ray data were taken using a Regaku powder X-ray diffractometer.

Field emission scanning electron microscopy

The morphologies of the reported peptide were investigated using field emission-scanning electron microscopy (FE-SEM). A small amount of gel/solution of the peptide was 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 gold-coated, and the micrographs were taken using a FE-SEM apparatus (Jeol Scanning Microscope-JSM-6700F).

Single crystal X-ray diffraction study

Intensity data of peptide 1 was collected with MoKα radiation using a Bruker APEX-2 CCD diffractometer. Data were processed using the Bruker SAINT package and the structure solution and refinement procedures were performed using SHELX97.†

Loading procedure

15 mg of peptide 1 was dissolved in 1 mL toluene containing 1.5 mg of carbamazipine. The solution was sonicated and drop-casted on a Petri dish and dried. Finally, the drug-loaded fibers were dried under vacuum and washed with phosphate buffer solution (several times) to remove unbound drug molecules.

Acknowledgements

We acknowledge the CSIR, India, for financial assistance (Project no. 01/2507/11-EMR-II). A. Pramanik thanks the CSIR, India for research fellowship. A. Paikar acknowledges the UGC, India for fellowship.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Synthesis and characterization of trisamides, ¹H NMR, ¹³C NMR, figures S1–S12. CCDC 1009431. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra07864d

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