Morphological transition triggered by mannose conjugation to a cyclic hexapeptide

The present manuscript describes a facile synthesis and the self-assembling behavior of a cyclic hexapeptide functionalized with mannose residues. Cyclic hexapeptides synthesized through cyclodimerization of tripeptides was conjugated to carbohydrate using click reaction. When self-assembling property of this novel conjugate was studied it afforded spherical morphology which was drastically different from self-assembled nanotubes formed by pristine cyclic peptide. Thus, functionalization of mannose to cyclic hexapeptide triggers morphological transition from tubular to spherical structure, opening new avenue for controlled manipulation and modification of peptide based self-assembled structures.


Introduction
Cyclic peptides found in variety of bioactive metabolites have been selected as lead compounds for development of various drugs. 1,2They are superior to their linear counterparts for applications in vivo as they are more permeable and more resistant to degradation by both exoand endoproteases due to absence of C-and N-termini. 3Due to conformational rigidity in cyclic peptides various functional groups could be spatially arranged in a predetermined manner and this also makes them useful candidate for studying protein folding. 4part from direct utility as drugs or bioactive agents, self-assembly of cyclic peptides into various structures, notably nanotubes, have gained attention in recent past due to their applicability in various fields.Pioneering work by Ghadiri et al which showed that cyclic peptides containing alternating D-and L-amino acids could self-assemble into nanotubes and can act as trans-membrane ion channels with comparable activity to naturally occurring cyclic peptide gramicidin A; paved way for design of self-assembling cyclic peptides. 5Since then formation of nanotubes and other structures such as nanorods, nanofibers, spherical structures from various cyclic peptides 6 and their potential application had been reported thereof. 7In recent example by Ghadiri et al self-assembled nanotubes from cyclic peptides could block entry of Hepatitis C virus through cell membrane. 8In another report transport of antitumor drug through cyclic peptide nanotube was shown. 9Other important factors those contribute to design of cyclic peptides are their tunable ring size, control over hydrophobicity by altering amino acid side chains. 6Growing interest for transportation through cyclic peptide nanotubes had resulted in modifications of them with unnatural amino acids such as β-amino acids, alternating α-and βamino acids, alternating α-and γ-amino acids, δ-amino acids and carbohydrates, 10 to have specific binding site at the interior of the nanotubes. 7Although having significant utilities, limitation of cyclic peptides arises from difficulty in their synthesis, low yield and requirement of expensive coupling reagents. 1ur group is concerned with the synthesis and design of various peptide based molecules for creation of nanostructured materials for applications such as drug delivery, multivalent receptors etc. 11 In this regard, we are keen to synthesize cyclic peptides for studying self-assembly process as there is a heightened interest of such self-assembled materials in various disciplines due to their immense implication in both technological and medicinal field.Moreover, we also aim to functionalize the cyclic peptides post cyclization to incorporate some bioactive moieties in them which may enable in further enhancing their potential applications.Thus to contrive this aim, firstly, we choose to design a C2 symmetric cyclic hexapeptide cyclo(Gly-L-Ser-Gly)2 since it possess free OH groups which may enable further functionalization after cyclization.But contrary to our belief post modification of cyclo(Gly-L-Ser-Gly)2 was a difficult and challenging task.Henceforth, we decided to modify the OH group prior to cyclization with propargyl group and subsequent cyclization lead to synthesis of cyclo[Gly-L-(O-propargyl)Ser-Gly]2 which could afford conjugation of other molecule through aza-alkyne click reaction for specific applications.Herein, we report functionalization of mannose residues by conjugation to cyclic hexapeptide through click chemistry.The motivation for mannose functionalization originated from our previous studies where we demonstrated self-assembly of glycosylated dipeptides and its application in plasmid encapsulation and for studying its interaction with lectins.11h-i, 12 It is envisaged that glycosylation of peptides could not only induce self-assembling properties, but also alter morphology of soft structures in addition to conferring recognition properties. 13Selfassembly of this novel conjugate afforded very interesting morphological consequence since it afforded spherical morphologies instead of tubular structures formed by self-assembly of pristine cyclic hexapeptide itself.Mannose conjugation to cyclic peptide thus triggers stark change in morphology from tubular to spherical shape.
For the synthesis of symmetric cyclic hexapeptide, two disconnection of the final compound is possible: i) macrocyclization after synthesis of linear hexapeptide, ii) selective cyclodimerization of linear tripeptide (Scheme 1). 14Cyclodimerization had been employed in some cases, but mostly cyclization steps involved disulfide bond formation, 15 depsipeptide formation, 16 click reaction to form triazole, 17 unnatural amino acids, 18 side chains (lysine, glutamic acid) of natural amino acids, 14 metal ions as templates, 19 and peptide containing thiazole ring in side chain. 20heme 1. Disconnection approach for synthesis of symmetric hexapeptides.
Since our target peptides were rich in glycine, a β-turn inducer, 2 we chose the cyclodimerization route (Scheme1, route a), instead of cyclization of a linear hexapeptide (Scheme1, route b) for imparting enhanced synthetic feasibility and avoiding unnecessary synthetic and purification steps.We were also motivated by our previous work, in which a proline-containing symmetric cyclic hexapeptide was synthesized by cyclodimerisation.11g,f In that case the presence of proline further increased the possibility of cyclodimerization.
Linear tripeptides (4 and 10) were synthesized using standard solution phase peptide synthetic methods with dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) as coupling reagents (Scheme 2, 3).The tripeptides were then activated by synthesizing pnitrophenol esters using DCC as coupling reagent.Cleavage of amine protecting group was accomplished by treatment with trifluoroacetic acid (TFA).At this stage the amine groups are not reactive due to their presence in salt form.Boc-Gly-ONHS and 8 was synthesized following earlier reports. 21propargyl group was incorporated in 10 with the aim to derivatize the cyclic peptide post cyclization using click chemistry.Cyclizations were carried out by dropwise addition of deprotected active ester solutions in DMF at room temperature into a solution of triethylamine in DMF (Scheme 4).4-5 drops were allowed to fall per minute and the whole solution was added over 3-4 h.Dropwise addition maintained very dilute concentrations of the reactants (3-4 µmol), favoring cyclodimerization over oligomerization.Conjugation of mannose with cyclic hexapeptide 13 using aza-alkyne click reaction also became feasible by incorporation of propargyl group prior to cyclization.Thus, tetracetylmannose azide was conjugated to cyclic hexapeptide resulting in formation of conjugate 14 which was purified by silica gel column chromatography purification (Scheme 5).Characterization by 1 H, 13 C NMR spectroscopy, high resolution mass spectroscopy and X-ray crystallography confirmed synthesis of 6 and 15 in accordance with molecular structures.Yield of cyclization were moderate but we were able to achieve it in solution phase without using any expensive coupling reagent or sophisticated experimental set up.Since synthesis of 6 and 13 were accomplished with similar yields it could also be inferred that a substituent at the Ser residue did not affect the reaction process.Scheme 4. Synthetic methodology for cyclic hexapeptides cyclo(Gly-L-Ser-Gly)2 (6) and cyclo[Gly-L-(O-propargyl)Ser-Gly]2 (13).microscopy image also confirmed formation of fibers (Figure 3c).Stacking of cyclic peptide molecules one above another by intermolecular complementary H-bonding of amide functionality facilitates formation of nanotubes in case of cyclic peptides 5 .From the crystal structure we could propose that formation of nanotubes from 6 11e also formed by stacking of cyclic peptide molecules with each other, but stabilization in this case is not by backbone Hbonding between amide bonds; rather it was assisted by the hydroxyl groups of Ser side chains and water molecules (Figures 1a, 2).We studied the self-assembly process of glycopeptide conjugate 15 in aqueous solvents with the help of microscopy techniques. 1 mM solutions of 15 were incubated in methanol: water (1:1) and water for different time intervals at room temperature.Atomic force microscopy (AFM) study on silicon surface revealed formation of spherical morphology with diameter of about 600 nm in both cases (Figure 4).Morphology of the self-assembled structures did not changed with time as could be seen from images of 1 day and 7 days aged samples.Further, clustered aggregates of spherical morphology were also observed in scanning electron microscopy (SEM) for 15 (1 mM in methanol: water, 1:1) at different times of ageing (Figure 5) confirming the solution phase self-assembling behavior of 15 to spherical shape.Moreover, from the structures of 6 and 15, it could be assessed that the change in substituent at the serine residues of 15 is directing the change in self-assembled morphology from tubular structure of 6 to spherical structure of 15.Such substituent dependent morphology of cyclic peptides were recently shown by Zhang et al.where they observed that by changing substituent present in a synthetic cyclic peptide could alter its self-assembly from nano-fibrillar to nanospherical structures.6b A recent report by Gianneschi and coworkers demonstrated the crucial role of hydrophobic to hydrophilic balance in inducing morphological changes from micellar to cylindrical shape. 23In our recent report, we had also shown that conjugation of mannose to diphenylalanine dipeptide exerted morphological changes depending upon number of mannose residues due to increased hydrophilic character.11h Also we observed in the crystal structure that the presence of the hydroxyl group of Ser side-chain is critical in stabilization of stacking of cyclic peptide 6.Thus, we surmise that possibly substitution at Ser OH and presence of hydrophilic mannose groups in 15, as compared to 6, might lead to a different H-bonding pattern resulting in transition of self-assembled morphologies from tubular to spherical shape.

Conclusions
We synthesized two glycine rich cyclic hexapeptides in solution phase employing cyclodimerization as the macrocyclization step with moderate yields.The cyclization was achieved without using any expensive coupling reagents.Conjugation of mannose to one of the cyclic peptide was done through aza-alkyne click reaction.Conjugation of mannose induces a dramatic shift from tubular morphology of pristine cyclic hexapeptide to spherical shape.These results are very interesting and open new avenues for post synthetic modification methodologies and also controlled manipulation of morphological consequences of self-assembled structures.
Our future endeavors will include a further study of the potential of these morphologies as delivery vehicles.Facile modification of cyclic peptide with mannose residue via aza-alkyne reaction motivates us to further use this methodology for functionalizing cyclic peptide with bioactive moieties to yield novel conjugates of immense potential for applications in biomedicine and tissue engineering for instance.

Microscopic techniques
Optical microscopy.10 μL solution of 6 (1 mM in water) was spread on a glass slide, dried at room temperature, and imaged under fluorescence microscope (Leica DM2500M) using 40× lens.Atomic force microscopy (AFM).Atomic force microscopy was carried out in air using an Agilent Technologies AFM (5500 AFM/SPM) operating under the Acoustic AC mode (AAC).The sample was mounted on the XY stage of the AFM and the integral video camera (NAVITAR, Model N9451A-USO6310233) with the Fibre-light source, MI-150 high intensity illuminator from Dolan-Jenner Industries) was used to locate the regions of interest.Silicon nitride cantilevers with resonant frequency of 150 kHz were used.The average dimension thickness, width and length of cantilever were approximately 2.0, 51 and 446 µm, respectively.The scanner model N9524A-USO7480132.xml/N9520A-USO7480152.xml was calibrated and used for imaging.The images were taken in air at room temperature, with the scan speed of 1.5-2.2lines/sec.Data acquisition and analysis was carried out using Pico View® 1.8 Basic software, respectively.10 µL aliquot of solutions of 6 and 15 were deposited on onto silicon wafer at room temperature.The sample-coated silicon surfaces were dried in air at room temperature for 2 h, followed by AFM imaging.Scanning electron microscopy (SEM).Field emission scanning electron microscopy images acquired on Supra 40 VP SEM (Zeiss, Germany), equipped with a tungsten filament gun, operating at WD 10.6 mm and 20 kV. 10 μL aliquots of the fresh and aged solutions of 15 were placed on silicon wafer.The samples were dried at room temperature for 2 h and then vacuum dried for another 30 minutes.The dried samples were then gold coated and imaged in FE-SEM.

Figure 1 .
Figure 1.(a) Stacking of molecules of 6 through H-bonding assisted by hydroxyl group of Ser side chain.(b) Numbering scheme for 6.(c) Diagrammatic representations for stacking interactions in 6 to form nanotubular structure.

Figure 2 .
Figure 2. Water assisted interconnection of cyclic peptide molecules.

Figure 3 .
Figure 3. AFM images of 1 mM 6 in water on (a) glass & (b) silicon wafer after 7 days ageing; inset shows magnified image of a fiber.c) Optical microscopy image of 7 days aged sample.Scale bar 10 μm.

Figure 4 .
Figure 4. AFM images of 1 mM 15 in 1:1 methanol: water on silicon surface: (a) 1 day (c) 7 day and in water (b) 1 day (d) 7 days.Height profiles are shown below each image.Scale bar 2 µm.