An accurate method for J-coupling based conformational analysis of five-membered rings : study of β- and γ-fluorinated prolines

Fluorinated prolines (FPros) can be exploited in two ways in proteins or peptides [1]. First, they can modulate proline’s cis-trans isomerization and its five-membered ring conformation (pucker) due to the stereoelectronic effect of fluorine. Second, they can serve as 19F NMR reporters to probe intermolecular interactions. For this, the conformational bias to proline should be minimal, which can be achieved by judiciously incorporating two fluorines with opposing effects, such as the (3S,4R) F2Pro variant recently reported by us [2]. However, for both applications, it is necessary to gain insight into the preorganizing effect of fluorine and the (residual) conformational bias to proline, both for model compounds and within the actual peptide sequence of interest. For experimental five-membered ring analysis, current procedures use Karplus relations [3] that connect vicinal scalar couplings to dihedral angles. Unfortunately, the approximate and general nature of state-of-the-art Karplus relations severely limits accuracy, especially in the case of 3JHF couplings, which are particularly important for doubly fluorinated FPro’s. Here, we present an alternative means to translate experimental J-couplings into a probability distribution of five-membered ring conformation (pucker) based on an ab initio full energy landscape analysis of FPro model compounds. In a first step, 901 ring conformations were generated, evenly spaced in the pseudorotation coordinate space [4]. Next, a geometry optimization was performed for each coordinate while constraining the ring pucker. This was done using DFT in Gaussian16, on Ac-FPro-OMe and Ac-FPro-NMe2 model compounds of all β- and/or γ- fluorination patterns. From the resulting energies, a very detailed probability distribution of conformations was obtained. Experimentally measured 1H-1H and 1H-19F scalar couplings from the same model compounds (using PSYCHEDELIC [5]) satisfyingly matched the Boltzmann weighted average of the computed scalar couplings, validating the calculations. The calculated couplings as a function of ring pucker can then be directly compared with experimental coupling data from FPro residues incorporated in peptides, providing a means to accurately evaluate ring pucker in different contexts. Alternatively, we also developed parameterized relations that directly translate the scalar couplings into pseudorotation coordinates rather than individual torsion angles, allowing for through-space effects across the ring to be taken into account. This approach can in principle be applied to any five-membered ring compound. References 1. Newberry R. W., Raines R. T. In: Lubell W. (eds) Peptidomimetics I. Topics in Heterocyclic Chemistry 2016, 48, 1-25, Springer, Cham. 2. Hofman G.-J., Ottoy E., et al., Chem. Commun. 2018, DOI: 10.1039/C8CC01493K 3. Karplus M. J. Chem. Phys. 1959, 30, 11-15. 4. Altona C., Sundaralingam, M., J. Am. Chem. Soc. 1972, 94, 8205-8212. 5. Sinnaeve D., et al., Angew. Chem. Int. Ed. 2016, 55, 1090-1093