Accepted Manuscript

L-Proline induced self-assembly of indolicidin derived palindromic tripeptide

Khashti Ballabh Joshi, Prabhpreet Singh

PII: S0040-4039(14)00727-8DOI: http://dx.doi.org/10.1016/j.tetlet.2014.04.090Reference: TETL 44552

To appear in: Tetrahedron Letters

Received Date: 19 March 2014Revised Date: 26 April 2014Accepted Date: 27 April 2014

Please cite this article as: Joshi, K.B., Singh, P., L-Proline induced self-assembly of indolicidin derived palindromictripeptide, Tetrahedron Letters (2014), doi: http://dx.doi.org/10.1016/j.tetlet.2014.04.090

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L-Proline induced self-assembly of

indolicidin derived palindromic tripeptide

Khashti Ballabh Joshia* and Prabhpreet Singhb ∗ The crystal structure and microscopic studies of the palindromic tripeptide WPW gives interesting insight into contribution

of L-proline and flanking tryptophan residues in the self-assembly process.

Leave this area blank for abstract info.

1

Tetrahedron Letters

jo u r n a l h om e p ag e : w w w .e ls e v ie r .c om

L-Proline induced self-assembly of indolicidin derived palindromic tripeptide

Khashti Ballabh Joshia* and Prabhpreet Singhb∗ a

Department of Chemistry Dr. H.S.G. Central University Sagar-470003, (M.P.)-India b Department of Chemistry, UGC Centre for Advance Studies-I, Guru Nanak Dev University Amritsar-143005 (P.B.)-India

——— ∗ Corresponding author. Tel.: +91-8427101534; e-mail: prabhpreet.chem@gndu.ac.in

Indolicidin (ILPWKWPWWPWRR-NH2), originally isolated from bovine neutrophils, is a 13-mer cationic antimicrobial peptide (AMPs) with a +3 overall charge, an amidated C-terminus and belongs to cathelicidin family.1-3 This short peptide is effective against a broad spectrum of organisms including bacteria, fungi, protozoa and acts as an inhibitor/modulator for various enzymes. The peptide kills the HIV-1 virus but also exhibit cytotoxicity against human T-lymphocytes and erythrocytes.4-5 Due to the presence of high content of tryptophan residues (39%) interspersed with proline residues (23%) throughout the sequence, it probably assumed a structure distinct from the well-known α-helical and β-structured peptides.6 Moreover, the presence of high content of tryptophan is regarded as an essential feature for exhibiting high antimicrobial activity, because these Trp- residues have a natural liking for the interfacial region of lipid bilayers.7

The self-assembly and folding of protein, peptide building blocks to form discrete nanostructured materials (via hydrogen bonding and other non-covalent interactions), their chemical diversity, facile synthesis and biocompatibility make these building blocks very attractive for various bio-nanotechnological applications. These protein and peptide based nanostructures can be further used for patterning through fabrication, useful for micro and nanofluidic sensing or to make responsive materials sensitive to external stimuli for applications in drug delivery systems and (bio)chemical sensors.8-10 Our group11-14 have earlier elucidated that peptide building blocks could form diversity of aggregate morphologies, such as spherical aggregates11, ditryptophan transforms biotin fibers into compact spherical ball-like structures12, nanofibrils13 and hybrid structure14 (templated

growth of fibrils on vesicular platform) on the peptide-peptide interface.

Tryptophan is an interesting aromatic amino acid for determining the 3D structure of protein and play pivotal role in self-assembly of peptide. Like Phe-Phe dipeptide, Trp-Trp dipeptide is not able to form any self-assembled structure in the solution state8a,12, however instantaneous vesicular morphology was observed for tetrapeptide PWWP. The orientation of tryptophan moiety for π-π interaction is highly dependent on two proline residues present at N- and C-terminus of the tetra peptide. This prompted us to investigate the self-assembly of palindromic tripeptide Trp-Pro-Trp (WPW), derived from the antimicrobial peptide indolicidin sequence, in solution and solid state. In this sequence the L-Pro residue situated in between the two L-Trp residues.

O NH

O

O

N

HN

HN

O

O

O

HN

H2N

O

N

HN

HN

O

O

OH

HN

[1] [2] Figure 1: Chemical structure of protected tripeptide Boc-WPW-OEt (1) and unprotected tripeptide NH2-WPW-COOH (2).

Herein we describe the solution phase synthesis, crystal structure and various microscopic techniques for investigating the vesicular morphology obtained from protected tripeptide Boc-WPW-OEt (1) and unprotected tripeptide NH2-WPW-COOH (2)

ARTIC LE INFO ABSTRACT

Article history:

Received Received in revised form Accepted Available online

We describe the synthesis, crystal structure and various microscopic studies of the palindromic tripeptide WPW derived from antimicrobial peptide indolicidin. The present study reveals that tripeptide 1 and 2 undergo self-assembly to form vesicular structures after prolonged incubation, thus gives interesting insight into contribution of L-proline and flanking tryptophan residues in the self-assembly process. These vesicles were also amenable to simple focused ion beam (FIB)-aided bisection and thus possible to mill these vesicles to create different shapes. The circular dichroism (CD) analysis indicates that incubation promotes and stabilizes the more favourable secondary structures for 1 and 2. Preliminary result shows that tripeptide 1 exhibit appreciable interaction with Tb3+ as determined by quenching in tryptophan fluorescence.

2014 Elsevier Ltd. All rights reserved.

Keywords:

Self-Assembly Peptide L-Proline Tryptophan Biofabrication

2sequence. The microscopic studies reveal that presence of proline, in between tryptophan (WPW) delayed the process of self-assembly in solution. The CD spectra of tripeptides after 5 days indicate that incubation promotes and stabilize the more favorable secondary structures for 1 and 2. The vesicles obtained via self-assembly of peptide 1 and 2 were also acquiescent to focused ion beam (FIB)-aided milling to create different shapes. Furthermore, the supramolecular recognition properties towards heavy transition metal ions using tripeptide 1 were studied.

Peptide 1 and 2 (figure 1) were synthesized by solution phase peptide synthesis techniques involving protection/deprotection and condensation methodologies using DCC/HOBT as coupling reagent (see supplementary data for synthesis).

Tripeptide 1 crystallizes in monoclinic space group P1 21 1 (No. 19) (Figure 2). Crystallographic signature of 1 displayed the solid state conformation, which perhaps is the basis of significant ordered self-assembly in solution state. The values of most of the torsional angles (φ1, ψ1, φ2 and ψ2) of the constituent amino acid residues fall within the polyproline II (PP-II) structural region (supplymentary data, CCDC No. 670518). The solid state structure of 1 involved two strong intermolecular interactions involving indolic -NH…O (carbonyl of Boc group) (2.23 Å) and Pro-α-CH…π (indole ring) (2.63 Å) (Figure 2b). Owing to these two interactions the molecule of 1 appeared in ring shape architecture stabilized by pyrrolidine-aromatic interactions as well as hydrogen bonding. We were unable to grow crystals for 2, even after repeated attempt.

Figure 2: (a) ORTEP view of crystal structure of tripeptide 1 and (b) Diamond generated architecture of 1 showing strong Pro-α-CH...π interaction and indolic N-H…O=C hydrogen bonding. tert-Boc group has been removed for sake of clarity

In preliminary experiments we evaluated the time dependent

aggregative propensity of peptide 1 and 2 (1 mM in 60% CH3OH:H2O) under fluorescence optical microscope. The fresh and incubated samples (at different time intervals) of 1 labeled with rhodamine B revealed that the fresh solution of 1 failed to give any significant and noticeable self-assembly. However, in case of incubated samples, we observed ordered vesicular structure after 2 days of incubation at ambient temperature with a diameter of 0.8-2 µM (Figure 3). A continued follow up confirmed that 5 days incubation time period refine the dimension of vesicular structures, however gross morphology remained same and intact. In comparison, peptide 2 did not reveal any vesicular morphology in fresh solution as well as after 2 days incubation of samples at ambient temperature. However, we observed vesicular structures of peptide 2 after 5 days incubation period.

The presence of vesicular structure for 1 (1 mM in 60% CH3OH:H2O) was further confirmed by scanning electron microscope (SEM), where uniform vesicles (more or less like a tennis ball), with a diameter of 1-2 µM, were evident when the

sample was aged for 2 days for peptide 1 and 5 days for peptide 2. Time-dependent follow up confirmed that longer incubation periods simply altered the dimensions of spherical structures without affecting the gross morphology hence SEM investigated data are well corresponded with optical microscopic data.

Figure 3: Fluorescent optical micrograph (a-c) and SEM micrograph (d-f) of 1 mM (60% CH3OH:H2O solution) of tripeptide (1) and (2); (a) 2 days aged sample of 1, (b) 5 days aged sample of 1 (arrow indicates almost same size of

vesicles), and (c) 5 days aged sample of 2 (d) 2 days aged sample of 1, [inset] magnified image of vesicular morphology (e) 2 days aged sample of 1 in 100% methanol (f) 5 days aged sample of 2.

Further we studied the effect of solvent (60% to 100%

CH3OH in H2O) and concentration (0.5 - 3 mM) on the morphology of the peptide 1 through SEM. We observed that as the concentration of methanol increased (for one specified concentration of peptide 1) the number of vesicles increased and in 100% methanol, 1 resulted in extensive aggregation of vesicular structure. However, fresh sample of 1 again failed to show any resolvable vesicular structure in 100% methanol. Also, there is no effect of concentration on the morphology of the vesicular structure is observed, when we change the concentration of compound 1 from 3 mM to 0.5 mM. The sizes of the vesicles are about ~1 µm. These vesicular structures were exclusively formed without any indication of other self-assembled structure such as fibrils, filaments etc. Such vesicular morphologies for 1 and 2 were preserved when we observed through TEM and AFM.

Transmission electron micrograph (TEM) also revealed the presence of intersperse, circular structures with an average diameter of 1-2 µm for 1 and 2. Aged solutions of 1 were subjected to AFM analysis at various incubation time points, including a freshly prepared solution. The self-aggregation was observed in the case of 2 and 5 days incubated sample when probed by AFM on a freshly cleaved mica surface whereas, the fresh sample of 1 does not reveal any resolvable structures. As the self-assembly of 2 was poorly resolved under SEM we did not further studied the self-assembly process of 2 under atomic force microscopic events. The observed self-assembly was independent of the surface used, as similar structure was also observed on highly oriented pyrolytic graphite surfaces (HOPG), thus suggesting that the self-organizing property as an inherent property. Further, on incubation or changing the concentration of the peptide does not lead to any deformity in the structure (Figure 4).

3

Figure 4: (a) TEM micrograph of protected tripeptide 1; (b) TEM micrograph of deprotected tripeptide 2 vesicles in 5 days incubated solution; (c) AFM image of tripeptide 1 vesicles in 5 days incubated solution and (d) 3D view of tripeptide 1 vesicles on mica surface

The experiment with focused-ion-beam/high resolution scanning electron microscope (FIB-HRSEM) also revealed vesicular structures with an average diameter of 1-2 µm (Figure 5), which suggests that these spherical structures do not result as drying artifacts.15 We attempted to see the interior core of the spherical ball-like structures with the help of FIB milling experiments.16-18 The microsphere from 1 was bisected by the help of ion beam followed by electron beam imaging at 52º angle, thus revealing interior part of the spherical structure. The reasonable structural integrity of these structures enables them to withstand ion currents for a short time period (Figure 5) compare to WPWWPW sequence.11b These microspheres were also amenable to simple FIB-aided bisection and thus possible to mill these microspheres to create different shapes.

Figure 5. FIB-HRSEM micrograph of protected and deprotected tripeptide (a) 1 and (b) 2 under wet conditions, FIB milling of tripeptide (c) 1 and (d) tripetide 2 shows instability under high beam current and little possibility for

biofabrications.

Circular dichroism (CD) study becomes a powerful analytical

tool in determining the conformational change of protein and peptide with other molecules in the solution. Therefore compound 1 and 2 showed a characteristic CD signal with

negative maxima at 225 nm and positive maxima centered at 210 nm in the far UV-region. Changes in the ellipticity at 225 nm and 210 nm are useful probes for visualizing variation in conformational change and hence self-assembly (Figure 6).

Figure 6. CD spectra of compound 1, 2 conjugated in fresh and aged condition in 60% methanol-water.

Circular dichroism studies of fresh and incubated samples confirmed that the 1 and 2 undergoes a more ordered conformational state after aging the sample for 1-5 days.19 The increase in ellipticity values at 225 nm (negative maxima) in the CD spectra of 5 days incubated samples indicates that incubation promotes and stabilize the more favorable secondary structures for 1 and 2 whereas the values decreases at 210 nm indicates that there is change in the secondary structures. Further investigation is necessary to understand the mechanism of time dependent conformational changes in various biomolecules and their small derivatives and, detailed work is being carried out in our group.

The chemical structure of a tripeptide 1 and 2 typically consists of key structural units namely the L-Pro and L-Trp residue. The presence of L-Pro in between two Trp residue makes it specific owing to the β-turns inducing nature of L-Proline. Therefore in the solid state structure we have seen intramolecular Pro-αH..π interaction and indolic N-H…O=C hydrogen bonding along with intermolecular π- π stacking interactions. Such kind of structures have propensity to form intermolecular non-covalent interactions in the polar solvents thus leads to the unique feature of 1D self-assembled nanostructures which will further entangle into 3D networks and perhaps converted to vesicular morphology, confirmed by microscopic investigation. Perhaps a delicate balance of these different interactions will finally lead to vesicular assemblies in the solution (Figure 7).

Figure 7. Proposed model for the morphological changes occurred during self-assembly of tripeptide 1 and 2 in 60% methanol-water.

4In the control experiments, we observed that the protected and de-protected dipeptides of Trp-Pro (WP) and Pro-Trp (PW) sequence were unable to show any kind of defined aggregates (data not shown). These results demonstrated that the position of tryptophan and proline in a truncated sequence playing a crucial role in self-assembly process. Truncated PWWP and WPWWPW sequence11, where two tryptophans are present in between proline, results in instantaneous formation of vesicular structures in fresh solution whereas; the presence of proline in between the tryptophan (WPW) delayed the process of self-assembly in solution.

Careful use of proline and aromatic amino acids systems will not only help us to understand its role as a stabilizing feature in aggregation, but also aid in de novo design of self-assembled structures. Such kinds of nanomaterials have wide applications in drug design/delivery for observation of the biological process at nanoscale and further they can exhibit specific biomolecular or supramolecular recognition properties using multifunctional peptides.

As a proof of concept, supramolecular recognition properties of peptide 1 towards various metal ions were studied in CH3OH/H2O (1:1 v/v, 50 mM HEPES buffer, pH 7.0). Tripeptide 1 in the UV-vis spectrum, displayed a typical absorption peak at 277 nm, due to the Trp indole moiety. In a preliminary fluorescence assay, the fluorescence of 1 (10 µM) was screened in the presence of 10 µM of various metal ions (alkali/alkaline earth metal, transition metal and lanthanide metals) and of the various metals studied, Tb3+ exhibited appreciable interaction with the tripeptide. The original fluorescence intensity of 1 decreased to 60% on addition of Tb3+, 20% decrease on addition of La(III) and Au(I) whereas only 5-10% decrease in fluorescence was observed on addition of other metal ions viz. Ca2+, Zn2+, Cd2+, Ag+, Li+, Sr2+, Pb2+, Mg2+, Eu3+, Cu2+, Co2+, Mn2+, Hg2+, K+, Ni2+. So, the tripeptide 1 showed good chelation towards the heavy transition metal ions20f. The fluorescence emission changes can be rationalized by considering the fact that Trpytophan indole engaged in metal ion binding instead of heavy atom effect and thus complexation has significant effect on Trp emission properties (Figure 8).

Figure 8. Fluorescence quenching of tripeptide 1 (10 µM) by various metal

ions (10 µM) in methanol/water (1:1) (1:1 v/v, 50 mM HEPES buffer, pH 7.0); λex =280 nm and λem =320 nm.

In conclusion, this study gives interesting insight into contributions of proline and indole aromatic side chains of amino acid residues in providing driving force and stability to self-

assembled vesicles formation process, involving tryptophan rich peptide model systems. Such spherical morphologies were preserved when experiments were performed on the copper surface (SEM), on highly oriented pyrolytic graphite surface (HOPG) or under wet conditions (AFM), focused-ion-beam/high resolution scanning electron microscope (FIB-HRSEM).

Acknowledgements

We thank Prof. Sandeep Verma, Department of Chemistry, IIT Kanpur, for his constant support and suggestions. Financial support from UGC and IIT-Kanpur is also gratefully acknowledged.

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