Supramolecular switching of the self-assembly of cyclic peptide–polymer conjugates via host–guest chemistry †

A supramolecular strategy of switching the self-assembly of cyclic peptide–polymer conjugates using host–guest chemistry is proposed. The formation of tubular supramolecular polymers based on cyclic peptide–polymer conjugates can be controlled by reversibly attaching cucurbit[7]uril onto the cyclic peptide via host–guest interactions.

Herein, as a proof of concept, we designed and synthesized a cyclic peptide conjugated with a water-soluble polymer-poly-(ethylene glycol) (PEG), which could form tubular supramolecular polymers in aqueous solution, Scheme 1.It has been reported that CB [7] binds strongly with a phenylalanine moiety, with the binding constant as high as 10 6 M À1 . 45To this end, we introduced two phenylalanine moieties onto the cyclic peptide, enabling us to non-covalently incorporate two bulky CB [7] host molecules onto every cyclic peptide-polymer conjugate.Moreover, a competitive guest molecule (1-adamantanamine, ADA) can be added to disassociate the binding between the cyclic peptidepolymer conjugate and CB [7].In this way, we are able to reversibly tune the self-assembly of the cyclic peptide-polymer conjugate.
A linear peptide with the sequence of H 2 N-L-Lys(Boc)-D-Leu-L-Lys(N 3 )-D-Leu-L-Lys(Boc)-D-Leu-L-Trp(Boc)-D-Leu-COOH was first synthesized by solid-phase peptide synthesis using Fmocdeprotection chemistry.The cyclic peptide (H 2 N) 2 -CP-N 3 was then synthesized by a cyclization reaction of the linear peptide under dilute conditions, followed by deprotection of the protecting groups (see the ESI, † S2).The two 'guest' phenylalanine moieties were introduced onto the cyclic peptide via a two-step synthesis (Phe 2 -CP-N 3 ), which was confirmed by 1 H NMR spectroscopy and ESI-MS (see the ESI, † S2 and Fig. S4).
Conjugating a water-soluble polymer to the cyclic peptide helps improve the solubility of the cyclic peptide and prevent the further lateral aggregation of the formed tubular supramolecular polymers.Here, PEG (5000 g mol À1 ) was chosen to conjugate to Phe 2 -CP-N 3 through the highly efficient strained alkyne/azide group ligation.The cyclic peptide-polymer conjugate Phe 2 -CP-PEG was easily synthesized by reacting Phe 2 -CP-N 3 with mPEG-BCN and purified by precipitation in a mixed solvent.
The conjugate Phe 2 -CP-PEG was thoroughly characterized by HPLC, GPC and 1 H NMR spectroscopy.As shown in Fig. S6 (ESI †), the retention time of Phe 2 -CP-PEG was 16.6 min, and no trace of unreacted Phe 2 -CP-N 3 was observed at 17.2 min, indicating that all the Phe 2 -CP-N 3 was conjugated with mPEG-BCN.GPC analysis gave an increased M n,GPC of 18 800 g mol À1 with a narrow dispersity (Ð) of 1.06 for the Phe 2 -CP-PEG conjugate, while the M n,GPC of mPEG-BCN is 10 800 g mol À1 , as shown in Fig. S7 (ESI †).Finally, the 1 H NMR spectrum of Phe 2 -CP-PEG was measured in TFA-d and proton signals ascribed to the cyclic peptide and PEG could be clearly assigned (Fig. S8, ESI †).
Hydrogen bonding interactions between the amide bonds of cyclic peptides are the driving force to form tubular supramolecular polymers.The self-assembly of Phe 2 -CP-PEG in aqueous solution was first studied by diffusion-ordered NMR spectroscopy (DOSY).DOSY is a widely-used technique in the field of supramolecular chemistry to directly confirm the formation of assemblies in solution. 46As shown in Fig. 1(a), the average diffusion coefficient of Phe 2 -CP-PEG in D 2 O was measured to be 2.2 Â 10 À11 m 2 s À1 , while the diffusion coefficient of mPEG-BCN was 1.0 Â 10 À10 m 2 s À1 (Fig. S10, ESI †).Such a decrease in the diffusion coefficient indicates the formation of large assemblies by Phe 2 -CP-PEG conjugate.Simplistically assuming that all the assemblies are hydrodynamically spherical, the radius R h is estimated to be 11.2 nm based on the Stokes-Einstein equation (Fig. S13 and Table S1, ESI †).
Small angle neutron scattering (SANS) is a powerful tool to characterize supramolecular systems to obtain information about structural parameters in solution.Here, we used SANS to confirm the formation of tubular supramolecular polymers in aqueous solution.Fig. 1(b) shows the reduced, corrected scattering data for Phe 2 -CP-PEG conjugate in D 2 O. Using SASfit software, a cylindrical micelle with attached polymer chains (''CYL + CHAINS(RW)'') (hairy cylinder) model was used, as fitting to a core-shell cylinder.From the fit, the average length of 16.8 AE 0.5 nm for each nanotube could be obtained, with a width of 6.4 AE 0.1 nm.Given that the distance between two cyclic peptides has been previously determined to be 0.47 nm, the average number of aggregation (N agg ) is calculated to be 36.
The host-guest interaction between Phe 2 -CP-PEG and CB [7]  was established by 1   conjugate showed similar changes.As indicated in Fig. 2(a), after adding CB [7], characteristic peaks at 7.73, 6.58 and 6.43 ppm were observed, which could be ascribed to the phenyl groups on the cyclic peptide encapsulated into the CB [7] cavity.Moreover, isothermal titration calorimetry (ITC) was used to thoroughly characterize the host-guest interactions between the conjugate and CB [7] (Fig. 2(b)).A two sequential-binding sites model was used to fit the data, yielding thermodynamic parameters for the interaction between the Phe 2 -CP-PEG conjugate and CB [7], K 1 = (1.1 AE 0.2) Â 10 6 M À1 and K 2 = (7.1 AE 0.4) Â 10 3 M À1 .It is worth pointing out that the binding constant of the second step K 2 is significantly lower than that of the first step K 1 , which is believed to be related to the disassembly of the tubular supramolecular polymers upon the addition of CB [7].In other words, the second step K 2 is in fact a combination of the binding between the phenylalanine moiety and CB [7] and the disassociation of the cyclic peptides.
To investigate whether introducing bulky CB [7] onto the cyclic peptide could lead to the disassembly of the tubular supramolecular polymers formed by the Phe 2 -CP-PEG conjugate, DOSY was conducted with Phe 2 -CP-PEG in the presence of excess CB [7] in D 2 O (Fig. 3(a)).The diffusion coefficient was measured to be 6.5 Â 10 À11 m 2 s À1 , significantly higher than that of the Phe 2 -CP-PEG conjugate.The R h is estimated to be 3.8 nm based on the Stokes-Einstein equation.The disassembly of the tubular supramolecular polymers was further proved by SANS.As shown in Fig. 3(b), whilst a hairy cylinder model fit is not valid anymore, a polymer chain model (DozierStar) gives a better fitting, suggesting that the tubular supramolecular polymers underwent disassembly due to the steric hindrance caused by CB [7] between the cyclic peptides.
More importantly, due to the dynamic nature of the hostguest interactions causing the disassembly of the tubular supramolecular polymers, the tubular supramolecular polymers could be reversibly reformed.Herein, ADA, which has a much stronger binding strength than a phenylalanine moiety, was used as a competitive guest to dissociate the binding between Phe 2 -CP-PEG and CB [7].As shown by 1 H NMR spectroscopy (Fig. S12, ESI †), after adding ADA into the solution of Phe 2 -CP-PEG and CB [7], peaks at 7.73, 6.58 and 6.43 ppm vanished and a broad peak at B7.2 ppm was observed instead, suggesting that CB [7] was dissociated from Phe 2 -CP-PEG and therefore the tubular supramolecular polymers were reformed.In conclusion, we have successfully fabricated tubular supramolecular polymers on the basis of cyclic peptide-polymer conjugates.By introducing binding sites of CB [7] onto the cyclic peptide, the self-assembly of a cyclic peptide-polymer conjugate could be easily tuned using CB based host-guest chemistry.Considering that various functional polymers could be conjugated onto cyclic peptides to construct tubular supramolecular polymers with various functionalities, it is anticipated that this type of supramolecular host-guest strategy will provide a new perspective towards fine control of the structure and function of such tubular supramolecular polymers.
The Royal Society Wolfson Merit Award (WM130055; SP), the European Research Council (TUSUPO 647106; QS, JYR and SP) and the Horizon 2020 Marie-Sklodowska-Curie action (TSPBNTM, JY) are acknowledged for funding.The authors also thank Dr Robert Dalgliesh (ISIS, Oxford, UK), as well as Dr Edward Mansfield and Mr Sean Ellacott for assistance with SANS.We also appreciate the help of Dr Ivan Prokes with DOSY.
Scheme 1 (a) Schematic representation of the reversible self-assembly of a cyclic peptide-polymer conjugate via host-guest chemistry; (b) chemical structures of cyclic peptide-polymer conjugate Phe 2 -CP-PEG, CB[7], and ADA.

Fig. 1
Fig. 1 (a) DOSY spectrum of the Phe 2 -CP-PEG conjugate.(b) Reduced SANS scattering data for the Phe 2 -CP-PEG conjugate.The line corresponds to a fit to the hairy cylinder model.