3-Fluoro-4-hydroxyprolines: Synthesis, conformational analysis and stereoselective recognition by the VHL E3 ubiquitin ligase for targeted protein degradation

Andrea Testa, Xavier Lucas, Guilherme Castro, Kwok Ho Chan, Jane Wright, Andrew Runcie, Morgan Gadd, William T.A. Harrison, Eun Jung Ko, Daniel Fletcher, Alessio Ciulli (Lead / Corresponding author)

Research output: Contribution to journalArticle

17 Citations (Scopus)
79 Downloads (Pure)

Abstract

Hydroxylation and fluorination of proline alters the pyrrolidine ring pucker and the trans:cis amide bond ratio in a stereochemistry-dependent fashion, affecting molecular recognition of proline-containing molecules by biological systems. While hydroxyprolines and fluoroprolines are common motifs in medicinal and biological chemistry, the synthesis and molecular properties of prolines containing both modifications, i.e. fluoro-hydroxyprolines, have not been described. Here we present a practical and facile synthesis of all four diastereoisomers of 3-fluoro-4-hydroxyprolines (F-Hyps), starting from readily available 4-oxo-L-proline derivatives. Small-molecule X-ray crystallography, NMR spectroscopy and quantum mechanical calculations are consistent with fluorination at C3 having negligible effects on the hydrogen bond donor capacity of the C4 hydroxyl, but inverting the natural preference of Hyp from C4-exo to C4-endo pucker. In spite of this, F-Hyps still bind to the von Hippel-Lindau (VHL) E3 ligase, which naturally recognizes C4-exo Hyp in a stereoselective fashion. Co-crystal structures and electrostatic potential calculations support and rationalize the observed preferential recognition for (3R,4S)-F-Hyp over the corresponding (3S,4S) epimer by VHL. We show that (3R,4S)-F-Hyp provides bioisosteric Hyp substitution in both hypoxia inducible factor 1 alpha (HIF-1α) substrate peptides as well as peptidomimetic ligands that form part of PROTAC (PROteolysis TArgeting Chimera) conjugates for targeted protein deg-radation. Despite a weakened affinity, Hyp substitution with (3S,4S)-F-Hyp within the PROTAC MZ1 led to Brd4-selective cellular degradation at concentrations >100-fold lower than the binary Kd for VHL. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein degradation will be of wide interest to medicinal organ-ic chemists, chemical biologists and drug discoverers alike.

Original languageEnglish
Pages (from-to)9299-9313
Number of pages15
JournalJournal of the American Chemical Society
Volume140
Issue number29
Early online date27 Jun 2018
DOIs
Publication statusPublished - 25 Jul 2018

Fingerprint

Hydroxyproline
Ubiquitin-Protein Ligases
Proteolysis
Proteins
Degradation
Proline
Fluorination
Halogenation
Substitution reactions
Ligands
Peptidomimetics
Hypoxia-Inducible Factor 1
Molecular recognition
Hydroxylation
Stereochemistry
Molecules
Pharmaceutical Chemistry
X ray crystallography
X Ray Crystallography
Biological systems

Cite this

@article{ff59e3b4c4284a669e67263ce5f9031a,
title = "3-Fluoro-4-hydroxyprolines: Synthesis, conformational analysis and stereoselective recognition by the VHL E3 ubiquitin ligase for targeted protein degradation",
abstract = "Hydroxylation and fluorination of proline alters the pyrrolidine ring pucker and the trans:cis amide bond ratio in a stereochemistry-dependent fashion, affecting molecular recognition of proline-containing molecules by biological systems. While hydroxyprolines and fluoroprolines are common motifs in medicinal and biological chemistry, the synthesis and molecular properties of prolines containing both modifications, i.e. fluoro-hydroxyprolines, have not been described. Here we present a practical and facile synthesis of all four diastereoisomers of 3-fluoro-4-hydroxyprolines (F-Hyps), starting from readily available 4-oxo-L-proline derivatives. Small-molecule X-ray crystallography, NMR spectroscopy and quantum mechanical calculations are consistent with fluorination at C3 having negligible effects on the hydrogen bond donor capacity of the C4 hydroxyl, but inverting the natural preference of Hyp from C4-exo to C4-endo pucker. In spite of this, F-Hyps still bind to the von Hippel-Lindau (VHL) E3 ligase, which naturally recognizes C4-exo Hyp in a stereoselective fashion. Co-crystal structures and electrostatic potential calculations support and rationalize the observed preferential recognition for (3R,4S)-F-Hyp over the corresponding (3S,4S) epimer by VHL. We show that (3R,4S)-F-Hyp provides bioisosteric Hyp substitution in both hypoxia inducible factor 1 alpha (HIF-1α) substrate peptides as well as peptidomimetic ligands that form part of PROTAC (PROteolysis TArgeting Chimera) conjugates for targeted protein deg-radation. Despite a weakened affinity, Hyp substitution with (3S,4S)-F-Hyp within the PROTAC MZ1 led to Brd4-selective cellular degradation at concentrations >100-fold lower than the binary Kd for VHL. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein degradation will be of wide interest to medicinal organ-ic chemists, chemical biologists and drug discoverers alike.",
author = "Andrea Testa and Xavier Lucas and Guilherme Castro and Chan, {Kwok Ho} and Jane Wright and Andrew Runcie and Morgan Gadd and Harrison, {William T.A.} and Ko, {Eun Jung} and Daniel Fletcher and Alessio Ciulli",
note = "This project has received funding from the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007−2013) as a Starting Grant to A.C. (grant agreement no. ERC-2012-StG-311460 DrugE3CRLs). X.L. and K.-H.C. were supported by Marie Skłodowska-Curie Actions Individual Fellowships from the European Commission (H2020-MSCA-IF-2015-806323 and H2020-MSCA-IF-2014-655516, respectively). G.V.C. was supported by a Ph.D. Studentship from the Coordenaca̧ o de Aperfeicoamento de Pessoal de Nivel Superior (CAPES, No. 7148-14-3). A.C.R. was supported by a BBSRC EASTBIO Doctoral Training Partnership award (BB/J01446X/1). Biophysics and drug discovery activities were supported by Wellcome Trust strategic awards to Dundee (100476/Z/12/Z and 094090/Z/10/Z, respectively). We thank Kevin Read and the DMPK team of the Dundee Drug Discovery Unit for cellular PK data, Professor B. Linclau for helpful discussions, P. Fyfe for support with in-house X-ray facilities, and the Diamond Light Source for beamtime (BAG proposals MX10071 and MX14980) and beamline support at beamlines I03, I04, and I04-1.",
year = "2018",
month = "7",
day = "25",
doi = "10.1021/jacs.8b05807",
language = "English",
volume = "140",
pages = "9299--9313",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "29",

}

3-Fluoro-4-hydroxyprolines : Synthesis, conformational analysis and stereoselective recognition by the VHL E3 ubiquitin ligase for targeted protein degradation. / Testa, Andrea; Lucas, Xavier; Castro, Guilherme; Chan, Kwok Ho; Wright, Jane; Runcie, Andrew; Gadd, Morgan; Harrison, William T.A.; Ko, Eun Jung; Fletcher, Daniel; Ciulli, Alessio (Lead / Corresponding author).

In: Journal of the American Chemical Society, Vol. 140, No. 29, 25.07.2018, p. 9299-9313.

Research output: Contribution to journalArticle

TY - JOUR

T1 - 3-Fluoro-4-hydroxyprolines

T2 - Synthesis, conformational analysis and stereoselective recognition by the VHL E3 ubiquitin ligase for targeted protein degradation

AU - Testa, Andrea

AU - Lucas, Xavier

AU - Castro, Guilherme

AU - Chan, Kwok Ho

AU - Wright, Jane

AU - Runcie, Andrew

AU - Gadd, Morgan

AU - Harrison, William T.A.

AU - Ko, Eun Jung

AU - Fletcher, Daniel

AU - Ciulli, Alessio

N1 - This project has received funding from the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007−2013) as a Starting Grant to A.C. (grant agreement no. ERC-2012-StG-311460 DrugE3CRLs). X.L. and K.-H.C. were supported by Marie Skłodowska-Curie Actions Individual Fellowships from the European Commission (H2020-MSCA-IF-2015-806323 and H2020-MSCA-IF-2014-655516, respectively). G.V.C. was supported by a Ph.D. Studentship from the Coordenaca̧ o de Aperfeicoamento de Pessoal de Nivel Superior (CAPES, No. 7148-14-3). A.C.R. was supported by a BBSRC EASTBIO Doctoral Training Partnership award (BB/J01446X/1). Biophysics and drug discovery activities were supported by Wellcome Trust strategic awards to Dundee (100476/Z/12/Z and 094090/Z/10/Z, respectively). We thank Kevin Read and the DMPK team of the Dundee Drug Discovery Unit for cellular PK data, Professor B. Linclau for helpful discussions, P. Fyfe for support with in-house X-ray facilities, and the Diamond Light Source for beamtime (BAG proposals MX10071 and MX14980) and beamline support at beamlines I03, I04, and I04-1.

PY - 2018/7/25

Y1 - 2018/7/25

N2 - Hydroxylation and fluorination of proline alters the pyrrolidine ring pucker and the trans:cis amide bond ratio in a stereochemistry-dependent fashion, affecting molecular recognition of proline-containing molecules by biological systems. While hydroxyprolines and fluoroprolines are common motifs in medicinal and biological chemistry, the synthesis and molecular properties of prolines containing both modifications, i.e. fluoro-hydroxyprolines, have not been described. Here we present a practical and facile synthesis of all four diastereoisomers of 3-fluoro-4-hydroxyprolines (F-Hyps), starting from readily available 4-oxo-L-proline derivatives. Small-molecule X-ray crystallography, NMR spectroscopy and quantum mechanical calculations are consistent with fluorination at C3 having negligible effects on the hydrogen bond donor capacity of the C4 hydroxyl, but inverting the natural preference of Hyp from C4-exo to C4-endo pucker. In spite of this, F-Hyps still bind to the von Hippel-Lindau (VHL) E3 ligase, which naturally recognizes C4-exo Hyp in a stereoselective fashion. Co-crystal structures and electrostatic potential calculations support and rationalize the observed preferential recognition for (3R,4S)-F-Hyp over the corresponding (3S,4S) epimer by VHL. We show that (3R,4S)-F-Hyp provides bioisosteric Hyp substitution in both hypoxia inducible factor 1 alpha (HIF-1α) substrate peptides as well as peptidomimetic ligands that form part of PROTAC (PROteolysis TArgeting Chimera) conjugates for targeted protein deg-radation. Despite a weakened affinity, Hyp substitution with (3S,4S)-F-Hyp within the PROTAC MZ1 led to Brd4-selective cellular degradation at concentrations >100-fold lower than the binary Kd for VHL. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein degradation will be of wide interest to medicinal organ-ic chemists, chemical biologists and drug discoverers alike.

AB - Hydroxylation and fluorination of proline alters the pyrrolidine ring pucker and the trans:cis amide bond ratio in a stereochemistry-dependent fashion, affecting molecular recognition of proline-containing molecules by biological systems. While hydroxyprolines and fluoroprolines are common motifs in medicinal and biological chemistry, the synthesis and molecular properties of prolines containing both modifications, i.e. fluoro-hydroxyprolines, have not been described. Here we present a practical and facile synthesis of all four diastereoisomers of 3-fluoro-4-hydroxyprolines (F-Hyps), starting from readily available 4-oxo-L-proline derivatives. Small-molecule X-ray crystallography, NMR spectroscopy and quantum mechanical calculations are consistent with fluorination at C3 having negligible effects on the hydrogen bond donor capacity of the C4 hydroxyl, but inverting the natural preference of Hyp from C4-exo to C4-endo pucker. In spite of this, F-Hyps still bind to the von Hippel-Lindau (VHL) E3 ligase, which naturally recognizes C4-exo Hyp in a stereoselective fashion. Co-crystal structures and electrostatic potential calculations support and rationalize the observed preferential recognition for (3R,4S)-F-Hyp over the corresponding (3S,4S) epimer by VHL. We show that (3R,4S)-F-Hyp provides bioisosteric Hyp substitution in both hypoxia inducible factor 1 alpha (HIF-1α) substrate peptides as well as peptidomimetic ligands that form part of PROTAC (PROteolysis TArgeting Chimera) conjugates for targeted protein deg-radation. Despite a weakened affinity, Hyp substitution with (3S,4S)-F-Hyp within the PROTAC MZ1 led to Brd4-selective cellular degradation at concentrations >100-fold lower than the binary Kd for VHL. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein degradation will be of wide interest to medicinal organ-ic chemists, chemical biologists and drug discoverers alike.

UR - http://www.scopus.com/inward/record.url?scp=85049368469&partnerID=8YFLogxK

U2 - 10.1021/jacs.8b05807

DO - 10.1021/jacs.8b05807

M3 - Article

C2 - 29949369

VL - 140

SP - 9299

EP - 9313

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 29

ER -