Five-membered ring pucker conformational analysis of novel fluorinated prolines

Fluorination of amino acids has become a relevant tool in protein engineering as a way to induce property changes such as enhanced stability, foldability and hydrophobicity while only introducing a small steric cost.1,2 Furthermore, it also enables the use of 19F NMR which, due to the sensitivity of the 19F nucleus and the low natural abundance of fluorine in biological molecules, can provide well-resolved and site-specific information.3 Fluorinated prolines would thus be suitable probes for structural studies of proline-rich regions in proteins, which are typical sites for protein-protein interactions4. However, due to the strong stereoelectronic effects of fluorine, fluoroprolines are mainly employed as structural modulators, in order to study the relevance of ring conformation and cis/trans interconversion. The strongly conformationally biased (4R) FPro and (4S) FPro residues are generally used for this purpose.4 Yet, fluoroprolines that are only minimally structurally perturbing, such as 4,4 F2Pro, are desired when aiming to use them as 19F NMR reporters.5 Therefore, it is necessary to perform conformational analyses of novel fluorinated prolines relative to proline, prior to their application in peptides and proteins. The standard procedure for conformational analysis of five-membered rings consists of measuring NMR J-couplings and using Karplus relations to translate these couplings to torsion angles. This procedure typically fails for fluoroprolines because of the underlying assumptions of generic 1H-1H and 1H-19F Karplus relations, which are not valid for these compounds, certainly when they are difluorinated. Therefore, we have developed an alternative methodology in which we perform an ab initio analysis of the conformational landscape of the five-membered ring. This allows to define a very precise relation between 1H-1H or 1H-19F J couplings and ring pucker, meaning NMR J-coupling analysis can be used to experimentally evaluate conformational distributions of the five-membered ring of any fluoroproline derivative, also within the context of peptides and proteins. We have used this method to investigate the conformational preferences of Ac-FPro-OMe derivatives in which the proline is either singly or doubly fluorinated at the 3- and/or 4-position.5, 6 The results provide detailed insight into the mechanism of the preorganizing effect of fluorine. References 1. Buer, B. C., Marsh, E. N. G. Protein Sci. 2012, 21, 453-462. 2. Odar, C., Winkler, M., Wiltschi, B. Biotechnol. J. 2015, 10, 427-446. 3. Chen, H., Viel, S., Ziarelli, F., Peng, L. Chem. Soc. Rev. 2013, 42, 7971-7982. 4. Newberry, R. W., Raines, R. T. In: Lubell W. (eds) Peptidomimetics I. Topics in Heterocyclic Chemistry 2016, 48, 1-25, Springer, Cham. 5. Hofman, G.-J., Ottoy, E., Light, M. E., Kieffer, B., Kuprov, I., Martins, J. C., Sinnaeve, D., Linclau, B. Chem. Commun. 2018, 54, 5118-5121. 6. Hofman, G.-J., Ottoy, E., Light, M. E., Kieffer, B., Martins, J. C., Kuprov, I., Sinnaeve, D., Linclau, B. J. Org. Chem. 2019, 84, 3100-3120.

Jaar van publicatie:2019