Noncovalent PEGylation via Sulfonatocalix[4]arene â a

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Noncovalent PEGylation via Sulfonatocalix[4]arene – a Crystallographic Proof Sreenivasu Mummidivarapu, Martin Rennie, Aishling Doolan, and Peter B. Crowley Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.8b00769 • Publication Date (Web): 16 Nov 2018 Downloaded from http://pubs.acs.org on November 19, 2018

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Bioconjugate Chemistry

Noncovalent PEGylation via Sulfonatocalix[4]arene – a Crystallographic Proof V. V. Sreenivasu Mummidivarapu, Martin L. Rennie, Aishling M. Doolan and Peter B. Crowley* School of Chemistry, National University of Ireland Galway, University Road, H91 TK33, Galway, Ireland Correspondence [email protected] +353 91 49 24 80

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Abstract Noncovalent or supramolecular PEGylation, in combination with site of administration, has great potential to increase the half-life of therapeutic proteins. To date, a variety of noncovalent PEGylation strategies have been devised. However, questions remain concerning the nature of the protein-PEG interaction. Here, we report structural analyses of a model system that comprised the lysine-rich cytochrome c and two PEGylated variants of sulfonatocalix[4]arene. Complex formation was characterized in solution by NMR spectroscopy. It was found that mono- or di-PEGylated sulfonatocalix[4]arene bound the protein similar to the parent calixarene. X-ray crystal structures at 2 M) of this ion in the crystallization solution. In a lysozyme - sclx4 crystal grown in 100 mM MgCl2, a Mg2+ ion was observed to complex PEG and calixarene.24 These observations suggest the possibility to control sclx4-PEGn self-encapsulation by the presence of counter-ions. Similar to the sclx4-PEG1 structure only a small portion of the PEG chains was modelled, suggesting that the PEG was mostly disordered. This conclusion is supported by the NMR analysis, in which similar chemical shift perturbations, with respect to the parent sclx4, were observed for both PEGylated forms.

Figure 4. The partial cone sclx4-PEG1 binding site (see Figure 3B) occurs at a three-fold symmetry interface. Symmetry-related calixarenes are shown in blue, and yellow, respectively. Noncovalent PEGylation is a topic of increasing importance.4,6-21 Here, we have demonstrated the potential of (supramolecular) calixarene-mediated noncovalent PEGylation. Solution state NMR and solid state crystallographic studies reveal that sclx4-PEG1 and sclx4-PEG2 recognise surface arginines and lysines similar to the parent compound sclx4 and with comparable affinity. The affinity of this system is modest (mM), however, tighter binding can be achieved by dimethylation of lysine residues25 or by employing larger calixarenes hosts.27,31 Complex formation between the cationic 7 ACS Paragon Plus Environment


protein and the anionic ligand occurred in the solid state despite the high ionic strength of the crystallization condition. The crystal structures provided clear evidence for protein complexation by the PEGylated calixarenes. Interestingly, the calixarenes were self-encapsulated with a fragment of PEG occupying the cavity. Furthermore, there was no evidence of protein-PEG interactions in the crystal structures. The absence of electron density suggests that the PEG chains were disordered. In contrast, PEG fragments are often refined in protein crystal structures where PEG was the precipitant.48 While it may be that the resolution of the sclx4-PEGn structures prevented detection of such interactions we assume that the PEG chains were water-solvated random coils. Previously, it was observed that a protein-PEG conjugate with 5 kDa PEG yielded a porous crystal lattice.36 Here, with shorter PEG chains (550 Da) there was no apparent impact on crystal porosity. Further experiments, including testing the site of administration, are required to develop the biological application of these and related compounds. As the parent molecule (sclx4) has negligible toxicity49 it is likely that the PEGylated compounds will also be biocompatible. The straightforward synthesis of sclx4-PEGn is further conducive to biopharmaceutical applications. Acknowledgements This research was supported by NUI Galway, Irish Research Council (grant GOIPD/2016/542 to VVSM) and Science Foundation Ireland (grants 13/ERC/B2912 and 13/CDA/2168 to PBC). We thank A. R. Khan, SOLEIL synchrotron (Paris) and Advanced Photon Source (Argonne National Laboratory) for beam-time allocation, and the staff at beamlines Proxima2A (Soleil) and 24-ID-C (APS) for their assistance with data collection. The Pilatus 6M detector on 24-ID-C beam line is funded by a NIH-ORIP HEI grant (S10 RR029205). Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: … Complete experimental procedures for ligand synthesis / characterization, NMR binding studies as well as protein-ligand co-crystallization and X-ray structure determination (PDF).


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