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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 as ring conformation and cis/trans interconversion, with the (4R) FPro and (4S) FPro residues the principally used examples in literature [4]. Furthermore, towards their use as 19F NMR reporters, FPros with minimal structural perturbation would be very useful. The only known example of such an FPro is the doubly fluorinated 4,4 F2Pro, where the two fluorines have counteracting stereoelectronic effects. However, the very large 2JFF coupling (ca. 250 Hz) and limited chemical shift difference between both 19F nuclei complicates 19F NMR experimental setup. We recently achieved synthesis of (3S,4R) F2Pro as an alternative [5], where the 2JFF coupling is small and the chemical shift difference is large. However, conformational analyses relative to proline of both compounds are still elusive, which is a prerequisite for their application in peptide and protein chemistry. Here, we present a new approach to perform an ab initio analysis of the conformational landscape of single FPro amino acids – or any other proline derivative – with unprecedented level of detail, focusing in particular on the five-membered pyrrolidine ring pucker. In addition, 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 in great detail the ring pucker of Pro or any of its derivatives within peptides or proteins, where the context further influences the ring pucker. This topples the state-of-the-art 1H-1H and 1H-19F Karplus relations, which typically fail for FPro residues – especially when difluorinated – because these were based on too generalized data. We apply this method to Ac-X-OMe molecules in which X is Pro, (4R) FPro, (4S) FPro, (3R) FPro, 4,4 F2Pro, or (3S,4R) F2Pro, providing detailed insight into the conformational preference 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., Kieffer, B., Kuprov, I., Martins, J. C., Sinnaeve, D., Linclau, B., submitted for publication.

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MLA
Ottoy, Emile, Steven Worswick, Chris Brett, et al. “Full Conformational Characterization of Novel Fluorinated Prolines.” Belgian Peptide Group, 4th Meeting, Abstracts. 2018. Print.
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. Belgian Peptide Group, 4th Meeting, Abstracts. Presented at the 4th Belgian Peptide Group meeting.
Chicago author-date
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 Belgian Peptide Group, 4th Meeting, Abstracts.
Chicago author-date (all authors)
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 Belgian Peptide Group, 4th Meeting, Abstracts.
Vancouver
1.
Ottoy E, Worswick S, Brett C, Hofman G-J, Kieffer B, Martins J, et al. Full conformational characterization of novel fluorinated prolines. Belgian Peptide Group, 4th Meeting, Abstracts. 2018.
IEEE
[1]
E. Ottoy et al., “Full conformational characterization of novel fluorinated prolines,” in Belgian Peptide Group, 4th Meeting, Abstracts, Brussels, Belgium, 2018.
@inproceedings{8553623,
  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 as ring conformation and cis/trans interconversion, with the (4R) FPro and (4S) FPro residues the principally used examples in literature [4]. Furthermore, towards their use as 19F NMR reporters, FPros with minimal structural perturbation would be very useful. The only known example of such an FPro is the doubly fluorinated 4,4 F2Pro, where the two fluorines have counteracting stereoelectronic effects. However, the very large 2JFF coupling (ca. 250 Hz) and limited chemical shift difference between both 19F nuclei complicates 19F NMR experimental setup. We recently achieved synthesis of (3S,4R) F2Pro as an alternative [5], where the 2JFF coupling is small and the chemical shift difference is large. However, conformational analyses relative to proline of both compounds are still elusive, which is a prerequisite for their application in peptide and protein chemistry. 
Here, we present a new approach to perform an ab initio analysis of the conformational landscape of single FPro amino acids – or any other proline derivative – with unprecedented level of detail, focusing in particular on the five-membered pyrrolidine ring pucker. In addition, 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 in great detail the ring pucker of Pro or any of its derivatives within peptides or proteins, where the context further influences the ring pucker. This topples the state-of-the-art 1H-1H and 1H-19F Karplus relations, which typically fail for FPro residues – especially when difluorinated – because these were based on too generalized data. We apply this method to Ac-X-OMe molecules in which X is Pro, (4R) FPro, (4S) FPro, (3R) FPro, 4,4 F2Pro, or (3S,4R) F2Pro, providing detailed insight into the conformational preference 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., Kieffer, B., Kuprov, I., Martins, J. C., Sinnaeve, D., Linclau, B., submitted for publication.},
  author       = {Ottoy, Emile and Worswick, Steven and Brett, Chris and Hofman, Gert-Jan and Kieffer, Bruno and Martins, José and Linclau, Bruno and Kuprov, Ilya and Sinnaeve, Davy},
  booktitle    = {Belgian Peptide Group, 4th Meeting, Abstracts},
  language     = {eng},
  location     = {Brussels, Belgium},
  title        = {Full conformational characterization of novel fluorinated prolines},
  year         = {2018},
}