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Full conformational characterization of novel fluorinated prolines

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Abstract
Fluorinated amino acids are important tools in protein engineering. Fluorine is almost isosteric to hydrogen, while at the same time it induces property changes such as enhanced stability, foldability and hydrophobicity.1,2 Fluorinated amino acids are also powerful 19F NMR reporters of well-resolved site-specific information, since 19F is sensitive and its spectrum is sparse due to the absence of fluorine in biological molecules.3 In this respect, fluorinated prolines (FPros) would be of high interest,4 especially since proline-rich regions in proteins are typical sites for protein-protein interactions. The stereoelectronic effects of fluorine substitutions in Pro have mainly been used as a means to study the relevance of ring conformation and cis/trans interconversion, with the strongly conformationally biased (4R) FPro and (4S) FPro residues the principally used examples in literature.4 However, towards their use as 19F NMR reporters, fluoroprolines with minimal structural perturbation, such as 4,4 F2Pro, would be very useful.5 Therefore, conformational analyses of novel fluorinated prolines relative to proline are necessary, prior to their application in peptides and proteins, to know which purpose they can serve. Here, we present a new approach to perform an ab initio analysis of the conformational landscape of fluorinated prolines – or any other proline derivative – with much improved level of detail, focusing in particular on the five-membered pyrrolidine ring pucker. The ab initio analysis permits defining very precisely the relation between 1H-1H and 1H-19F J couplings and ring pucker. This allows the use of NMR J-coupling analysis to experimentally evaluate conformational distributions of the proline ring or any of its derivatives within peptides or proteins, where the context further influences the ring pucker. This kind of analysis was not possible with generic 1H-1H and 1H-19F Karplus relations, which typically fail for FPro residues – especially when difluorinated – because their underlying assumptions are typically not valid for these compounds. We apply this new method to Ac-FPro-OMe derivatives in which the proline is either singly or doubly fluorinated at the 3- and/or 4-position. The results provide detailed insight into the conformational preferences of each FPro, but also 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, in press, DOI: 10.1039/c8cc01493k.

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Chicago
Ottoy, Emile, Steven Worswick, Chris Brett, Gert-Jan Hofman, Bruno Kieffer, José Martins, Bruno Linclau, Ilya Kuprov, and Davy Sinnaeve. 2018. “Full Conformational Characterization of Novel Fluorinated Prolines.” In Fluorine Chemistry, 22nd International Symposium, Abstracts.
APA
Ottoy, E., Worswick, S., Brett, C., Hofman, G.-J., Kieffer, B., Martins, J., Linclau, B., et al. (2018). Full conformational characterization of novel fluorinated prolines. Fluorine Chemistry, 22nd International symposium, Abstracts. Presented at the 22nd International symposium on Fluorine Chemistry.
Vancouver
1.
Ottoy E, Worswick S, Brett C, Hofman G-J, Kieffer B, Martins J, et al. Full conformational characterization of novel fluorinated prolines. Fluorine Chemistry, 22nd International symposium, Abstracts. 2018.
MLA
Ottoy, Emile et al. “Full Conformational Characterization of Novel Fluorinated Prolines.” Fluorine Chemistry, 22nd International Symposium, Abstracts. 2018. Print.
@inproceedings{8585127,
  abstract     = {Fluorinated amino acids are important tools in protein engineering. Fluorine is almost isosteric to hydrogen, while at the same time it induces property changes such as enhanced stability, foldability and hydrophobicity.1,2 Fluorinated amino acids are also powerful 19F NMR reporters of well-resolved site-specific information, since 19F is sensitive and its spectrum is sparse due to the absence of fluorine in biological molecules.3 In this respect, fluorinated prolines (FPros) would be of high interest,4 especially since proline-rich regions in proteins are typical sites for protein-protein interactions. The stereoelectronic effects of fluorine substitutions in Pro have mainly been used as a means to study the relevance of ring conformation and cis/trans interconversion, with the strongly conformationally biased (4R) FPro and (4S) FPro residues the principally used examples in literature.4 However, towards their use as 19F NMR reporters, fluoroprolines with minimal structural perturbation, such as 4,4 F2Pro, would be very useful.5 Therefore, conformational analyses of novel fluorinated prolines relative to proline are necessary, prior to their application in peptides and proteins, to know which purpose they can serve. 
Here, we present a new approach to perform an ab initio analysis of the conformational landscape of fluorinated prolines -- or any other proline derivative -- with much improved level of detail, focusing in particular on the five-membered pyrrolidine ring pucker. The ab initio analysis permits defining very precisely the relation between 1H-1H and 1H-19F J couplings and ring pucker. This allows the use of NMR J-coupling analysis to experimentally evaluate conformational distributions of the proline ring or any of its derivatives within peptides or proteins, where the context further influences the ring pucker. This kind of analysis was not possible with generic 1H-1H and 1H-19F Karplus relations, which typically fail for FPro residues -- especially when difluorinated -- because their underlying assumptions are typically not valid for these compounds. We apply this new method to Ac-FPro-OMe derivatives in which the proline is either singly or doubly fluorinated at the 3- and/or 4-position. The results provide detailed insight into the conformational preferences of each FPro, but also 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, in press, DOI: 10.1039/c8cc01493k.},
  author       = {Ottoy, Emile and Worswick, Steven and Brett, Chris and Hofman, Gert-Jan and Kieffer, Bruno and Martins, Jos{\'e} and Linclau, Bruno and Kuprov, Ilya and Sinnaeve, Davy},
  booktitle    = {Fluorine Chemistry, 22nd International symposium, Abstracts},
  language     = {eng},
  location     = {Oxford, UK},
  title        = {Full conformational characterization of novel fluorinated prolines},
  year         = {2018},
}