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Modular synthesis of supramolecular ureidopyrimidinone–peptide conjugates using an oxime ligation strategywz Roxanne E. Kieltyka, Maartje M. C. Bastings, Geert C. van Almen, Pol Besenius, Erwin W. L. Kemps and Patricia Y. W. Dankers* Received 31st July 2011, Accepted 7th October 2011 DOI: 10.1039/c1cc14728e A convenient method to prepare supramolecular bioconjugates in a facile and scalable manner is by a modular approach, whereby self-assembling units and peptides are coupled using oxime chemistry. We here report syntheses of bioactive ureidopyrimidinone-based peptide conjugates, and their resultant self-assembly into fibrous structures. Many different biomaterials have been developed for applications in the field of regenerative medicine, ranging from natural to covalent and hybrid polymeric structures. A relatively new class of materials used in this biomedical field are supramolecular systems in which molecular building blocks are held together via specific non-covalent interactions.1 Self-assembled peptide systems have been studied in great detail using bioactive peptide sequences, or in combinations of bioactive peptides mixed with non-active, filler peptides.2 Previously, we demonstrated a unique approach to prepare bioactive supramolecular materials3 using short oligomeric prepolymers and bioactive peptides, both modified with selfcomplementary four-fold hydrogen bonding ureidopyrimidinone (UPy) units.4 The mixing of these prepolymers with several peptides in a modular way resulted in bioactive biomaterials with a range of properties.3,5 Moreover, the inherent properties of the biomaterial can be manipulated by modifications to the chemical structure of the base prepolymers yielding films,3 membranes,5,6 and hydrogels. Originally, guest molecules were prepared on a solid support by reaction of a UPy-synthon with the peptide.7 However, this method becomes less straightforward with more complex supramolecular synthons. An alternative approach that can aid in larger scale synthesis is a modular methodology, where unprotected peptides are coupled directly in solution using a chemoselective ligation strategy.8

Institute for Complex Molecular Systems, and Laboratory of Chemical Biology, Eindhoven University of Technology (TU/e), PO Box 513, 5600 MB, Eindhoven, The Netherlands. E-mail: [email protected]; Fax: +31 40 247 8367; Tel: +31 40 247 5620 w This article is part of the ChemComm ‘Emerging Investigators 2012’ themed issue. z Electronic supplementary information (ESI) available: Full experimental section, and additional AFM micrographs are shown. See DOI: 10.1039/c1cc14728e

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This method allows the user to flexibly couple a variety of peptides to various self-assembling modules on an as-need basis. Oxime ligation, a reaction between an aminooxyacetyl group and aldehyde or ketone, is a highly efficient conjugation strategy that does not require any pre-activation or stabilization steps. The oxime bond formed shows hydrolytic stabilities over a large pH range from 2 to 9. The conjugation reaction is performed under slightly acidic conditions (pH 4.5–5.7) in order to increase the transimination reaction kinetics of the protonated aniline Schiff base into the oxime.9 Oxime ligations of peptides to proteins,10 peptides,9 polymers,11,12 dendrimers,13,14 oligonucleotides,15 surfaces,16 and discotic molecules17 have been demonstrated. Here, we show the preparation of several UPy-modified peptides using oxime ligation. Sequences applicable in regenerative medicine were used as model peptides, i.e. the integrin binding RGD18 peptide, its synergistic PHSRN19 sequence, a collagen I binding peptide,20 and their scrambled non-active counterparts. The UPy-modified aminooxy units 5a and 5b consist of a UPy-unit that can dimerize,4 an additional urea functionality that takes care of hydrogen bonding in the lateral direction,5 a hydrophobic dodecyl spacer that is proposed to shield the hydrogen bonding in aqueous environments, and hydrophilic oligo(ethylene glycol) (OEG) segments (7 and 15 repeats) that enhance solubility (Scheme 1). Preparation of these UPy-modified aminooxy units commenced with the Boc-protection of the amine on the heterobifunctional OEGs 1a and 1b followed by CDI-activation of the alcohol and reaction with a mono Cbz-protected dodecyldiamine. Subsequent Cbz-deprotection of 2a and 2b was afforded by generation of hydrogen in situ using Et3SiH and Pd/C. The free amine was further reacted with UPy-hexyl-isocyanate to provide the Boc-protected UPy 3a and 3b. The reactive aminooxy moiety was introduced by TFA deprotection of the Boc-protecting group followed by reaction of the free amine with the NHS-ester of a Boc-protected aminooxy moiety to afford the final modular building blocks 4a and 4b. Through these one-pot syntheses good yields ranging from 56–88% were obtained for the various synthetic steps denoting the efficiency and scalability of the synthetic protocols to generate the supramolecular synthons. The peptide sequences 6–11 were prepared by solid phase peptide synthesis using Fmoc-chemistry on a Rink amide This journal is

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Scheme 1 Synthesis of the aminooxy-functionalized UPy-units containing a hydrophobic alkyl spacer and two different oligo(ethylene glycol) (OEG) lengths, n = 6 and n = 14, 5a and 5b, respectively. (i) Boc2(QO), DIPEA, CHCl3, (ii) CDI, (iii) CbzNHC12H24NH2, DIPEA, reflux; 2a = 67%, 2b = 77%, (iv) Et3SiH, Pd/C, MeOH, (v) DIPEA, CHCl3, 3a = 59%, 3b = 89%, (vi) TFA : CHCl3 (3 : 1), 0 1C, (vii) DIPEA, CHCl3, 4a = 61%, 4b = 56%, (viii) TFA, 0 1C.

MBHA resin (Scheme 2). Peptides 6–9 were prepared with an N-terminal serine for oxidation to the glyoxylic aldehyde. For 10 and 11 rapid oxidation of methionine to methionine sulfoxide in the presence of sodium m-periodate under several conditions occurred. Consequently, the serine residue was substituted with the ketone containing levulinic acid to prevent this transformation and to expedite purification. Moreover, an additional lysine was incorporated in order to provide a convenient handle for the attachment of for example fluorophores afterwards. In the final step, UPy-functionalization of the peptides was achieved by oxime ligation with the UPy-modified aminooxy

units. Two equivalents of the peptides were reacted with the UPy-units in a DMF containing anilinium acetate buffer, pH 4.5 at 37 1C. DMF was used to help the solubilization of the UPy-units, and to accelerate the ligation reaction. The final products 12–19 were purified by C18 flash chromatography resulting in yields ranging from 33–73%, illustrating the potential of this modular approach for scalable preparation and purification of the UPy–peptide conjugates. Finally, the capacity of the bioconjugates 12, 13, 16–19 to self-assemble on a surface was studied by atomic force microscopy (AFM; Fig. 1). A sonication approach to solubilize the UPy–peptides in pure water was attempted. Unfortunately, scission of the oxime bond occurred after a brief period of sonication (see ESIz). Consequently, a solution-based mixing approach was used in which the compounds were dissolved in a mixture of THF/water (60 : 40 v : v) and drop cast on mica several hours after dissolution. For the RGD conjugates 12 and 17 sheets of fibres were observed that were rigid and continuous in appearance. No differences were found for the two OEG lengths. However fibres formed by the scrambled peptide 13 showed more curvature. On average, the fibres of 12, 17 and 13 were found to be 11–13 nm in width and microns in length. Conversely, using a totally different peptide sequence and ligation via a ketone, as for the collagen I binding conjugates 16 and 18, resulted in much shorter and stiff fibres. Also in this case the length of the OEG did not show a pronounced effect. Also the scrambled sequence 19 shows similar behaviour. The fibres of this set of bioconjugates have a width of 10–40 nm and a length of 60–200 nm. The mechanism of aggregation is currently being investigated in more detail in solution in order to correlate differences in chemical structures of both the UPy-modules and peptides to fibre formation. Moreover, these AFM micrographs show for the first time that UPy-modified bioconjugates can form fibrous structures on a surface. Investigation of the bioactive properties of these bioconjugates was performed on coatings of RGD peptides 12 and 17, and scrambled non-active GDR peptide 13, using a fibroblast adhesion assay to study actin filament organization (Fig. 2).

Scheme 2 (A) Oxime ligation of the aminooxy-functionalized UPy-units 5a or 5b with the N-terminal aldehyde (6 = GGG-GRGDS, 7 = GGGSGDRG, 8 = GGG-PHSRN, 9 = GGG-PNRHS) or ketone containing (10 = KGGG-HVWMQAP, 11 = KGGG-AMPVQWH) peptides. (i) Ligations were carried out in 0.1 M anilinium acetate buffer at pH 4.5 and 50% DMF. (B) Schematic representations of UPy-GGG-GRGDS 12, and UPy-KGGG-AMPVQWH 19.

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ligation with any biomolecule desired. By this approach, we propose to achieve unique supramolecular control over the position and availability of bioactive molecules in biomaterials. This work is supported by the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (CW-NWO), and the European Union by its Marie Curie program. We thank E. W. Meijer for essential discussions, M. Kemerink, J. van Dongen, X. Lou, R. Bovee, and P. Englebienne (all from TU/e) for their experimental assistance and F. Hoeben (SyMO-Chem) for donation of N 0 -Boc-aminooxyacetyl N-hydroxysuccinimide ester.

Notes and references

Fig. 1 Atomic force micrographs of UPy-modified peptides after drop casting on mica from THF/H2O (60 : 40 v : v). Tapping mode height images are shown (scale bars = 200 nm).

Fig. 2 Immunofluorescent images of actin filament organisation in NIH/3T3 fibroblasts on surfaces coated with RGD-peptides 12 and 17, scrambled GDR peptide 13, and fibronectin (Fn), 4 hours after seeding (actin = red, nucleus = blue; scale bars = 100 mm).

The cells adhered and spread on 12 and 17 displaying comparable actin organization as on the fibronectin reference. On 13 the actin organization is clearly absent and no cell spreading occurred. This shows that fibroblasts specifically adhere and spread on RGD peptide coatings 12 and 17 without showing distinct differences between the two OEG lengths of 12 and 17. The synthesis of bioactive supramolecular UPy–peptide conjugates via a modular approach using oxime chemistry and their ability to self-assemble into fibrous structures are reported. This chemo-selective approach enables the scalable synthesis and in situ coupling of biomolecules to UPy-units. Making use of chemical modifications to both the UPy-units and the peptides we propose to be able to control their aggregation behaviour as well as the applicability of these structures in regenerative medicine applications in a modular approach (using mixtures of these UPy–peptides and UPy-modified prepolymers). Furthermore, this convergent synthesis method paves the way to post-modification of UPy-biomaterials after mixing of UPy-prepolymers with these UPy-modified aminooxy modules, and subsequent oxime

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