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 E. Wright, Andrew C. Runcie, Morgan S. Gadd, William T. A. Harrison, Eun-Jung Ko, Daniel Fletcher, Alessio Ciulli

Research output: Contribution to journalArticle

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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 proper-ties of prolines containing both modifications i.e. fluoro-hydroxyprolines have not been described. Here we present a practical syn-thesis of all four diasteroisomers of 3-fluoro-4-hydroxyprolines (F-Hyps). Conformational preferences and the effect of fluorination on hydrogen bond donating capacity are elucidated by means of X-ray crystallography, NMR spectroscopy and quantum mechani-cal calculations. We exemplify an application of F-Hyps as novel building blocks for biomolecular recognition by incorporating them in HIF-1α peptides and small peptidomimetic ligands targeting the von Hippel-Lindau E3 ligase (VHL) – widely used as PROTAC (PROteolysis TArgeting Chimera) conjugates for targeted protein degradation. We found that VHL exhibits stereoselec-tive recognition of the (3R,4S)-F-Hyp over the corresponding (3S,4S) epimer and rationalize this preference through co-crystal structures and electrostatic potential calculations. Hyp substitution with (3R,4S)-F-Hyp into the BET degrader MZ1 retained bind-ing affinities and cellular degradation activities comparable to those of the parent PROTAC. In spite of a ~20-fold loss in binding affinity to VHL, incorporation of the (3S,4S) epimer into MZ1 led to Brd4-selective degradation at nanomolar concentration. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein deg-radation will be of wide interest to medicinal organic 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 - 2018

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Hydroxyproline
Ubiquitin-Protein Ligases
Proteolysis
Proteins
Degradation
Proline
Fluorination
Halogenation
Ligands
Peptidomimetics
Molecular recognition
Hydroxylation
Stereochemistry
Pharmaceutical Chemistry
X ray crystallography
X Ray Crystallography
Organic chemicals
Biological systems
Static Electricity
Amides

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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 E.; Runcie, Andrew C.; Gadd, Morgan S.; Harrison, William T. A.; Ko, Eun-Jung; Fletcher, Daniel; Ciulli, Alessio.

In: Journal of the American Chemical Society, Vol. 140, No. 29, 2018, p. 9299-9313.

Research output: Contribution to journalArticle

Testa, Andrea ; Lucas, Xavier ; Castro, Guilherme ; Chan, Kwok-Ho ; Wright, Jane E. ; Runcie, Andrew C. ; Gadd, Morgan S. ; Harrison, William T. A. ; Ko, Eun-Jung ; Fletcher, Daniel ; Ciulli, Alessio. / 3-Fluoro-4-hydroxyprolines : Synthesis, Conformational Analysis, and Stereoselective Recognition by the VHL E3 Ubiquitin Ligase for Targeted Protein Degradation. In: Journal of the American Chemical Society. 2018 ; Vol. 140, No. 29. pp. 9299-9313.
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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 proper-ties of prolines containing both modifications i.e. fluoro-hydroxyprolines have not been described. Here we present a practical syn-thesis of all four diasteroisomers of 3-fluoro-4-hydroxyprolines (F-Hyps). Conformational preferences and the effect of fluorination on hydrogen bond donating capacity are elucidated by means of X-ray crystallography, NMR spectroscopy and quantum mechani-cal calculations. We exemplify an application of F-Hyps as novel building blocks for biomolecular recognition by incorporating them in HIF-1α peptides and small peptidomimetic ligands targeting the von Hippel-Lindau E3 ligase (VHL) – widely used as PROTAC (PROteolysis TArgeting Chimera) conjugates for targeted protein degradation. We found that VHL exhibits stereoselec-tive recognition of the (3R,4S)-F-Hyp over the corresponding (3S,4S) epimer and rationalize this preference through co-crystal structures and electrostatic potential calculations. Hyp substitution with (3R,4S)-F-Hyp into the BET degrader MZ1 retained bind-ing affinities and cellular degradation activities comparable to those of the parent PROTAC. In spite of a ~20-fold loss in binding affinity to VHL, incorporation of the (3S,4S) epimer into MZ1 led to Brd4-selective degradation at nanomolar concentration. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein deg-radation will be of wide interest to medicinal organic chemists, chemical biologists and drug discoverers alike",
author = "Andrea Testa and Xavier Lucas and Guilherme Castro and Kwok-Ho Chan and Wright, {Jane E.} and Runcie, {Andrew C.} and Gadd, {Morgan S.} and Harrison, {William T. A.} and Eun-Jung Ko and Daniel Fletcher and Alessio Ciulli",
note = "Correction published online 24th April 2019, in issue 8th May 2019. J. Am. Chem. Soc.2019141187644 Funding Sources 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. C. was supported by a Ph.D. Studentship from the Minist{\'e}rio da Educa{\cc}{\~a}o Coordena{\cc}{\~a}o de Aperfei{\cc}oamento de Pessoal de N{\'i}vel Superior (CAPES, No. 7148-14-3). Biophysics and drug discovery activities are supported by Wellcome Trust strategic awards to Dundee (100476/Z/12/Z and 094090/Z/10/Z, respectively). We thank Professor B. Linclau for helpful discussions, P. Fyfe for support with in-house X-ray facilities, and to the Diamond Light Source for beamtime (BAG proposal MX10071) and beamline support at beamline I04-1.",
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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 E.

AU - Runcie, Andrew C.

AU - Gadd, Morgan S.

AU - Harrison, William T. A.

AU - Ko, Eun-Jung

AU - Fletcher, Daniel

AU - Ciulli, Alessio

N1 - Correction published online 24th April 2019, in issue 8th May 2019. J. Am. Chem. Soc.2019141187644 Funding Sources 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. C. was supported by a Ph.D. Studentship from the Ministério da Educação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, No. 7148-14-3). Biophysics and drug discovery activities are supported by Wellcome Trust strategic awards to Dundee (100476/Z/12/Z and 094090/Z/10/Z, respectively). We thank Professor B. Linclau for helpful discussions, P. Fyfe for support with in-house X-ray facilities, and to the Diamond Light Source for beamtime (BAG proposal MX10071) and beamline support at beamline I04-1.

PY - 2018

Y1 - 2018

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 proper-ties of prolines containing both modifications i.e. fluoro-hydroxyprolines have not been described. Here we present a practical syn-thesis of all four diasteroisomers of 3-fluoro-4-hydroxyprolines (F-Hyps). Conformational preferences and the effect of fluorination on hydrogen bond donating capacity are elucidated by means of X-ray crystallography, NMR spectroscopy and quantum mechani-cal calculations. We exemplify an application of F-Hyps as novel building blocks for biomolecular recognition by incorporating them in HIF-1α peptides and small peptidomimetic ligands targeting the von Hippel-Lindau E3 ligase (VHL) – widely used as PROTAC (PROteolysis TArgeting Chimera) conjugates for targeted protein degradation. We found that VHL exhibits stereoselec-tive recognition of the (3R,4S)-F-Hyp over the corresponding (3S,4S) epimer and rationalize this preference through co-crystal structures and electrostatic potential calculations. Hyp substitution with (3R,4S)-F-Hyp into the BET degrader MZ1 retained bind-ing affinities and cellular degradation activities comparable to those of the parent PROTAC. In spite of a ~20-fold loss in binding affinity to VHL, incorporation of the (3S,4S) epimer into MZ1 led to Brd4-selective degradation at nanomolar concentration. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein deg-radation will be of wide interest to medicinal organic 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 proper-ties of prolines containing both modifications i.e. fluoro-hydroxyprolines have not been described. Here we present a practical syn-thesis of all four diasteroisomers of 3-fluoro-4-hydroxyprolines (F-Hyps). Conformational preferences and the effect of fluorination on hydrogen bond donating capacity are elucidated by means of X-ray crystallography, NMR spectroscopy and quantum mechani-cal calculations. We exemplify an application of F-Hyps as novel building blocks for biomolecular recognition by incorporating them in HIF-1α peptides and small peptidomimetic ligands targeting the von Hippel-Lindau E3 ligase (VHL) – widely used as PROTAC (PROteolysis TArgeting Chimera) conjugates for targeted protein degradation. We found that VHL exhibits stereoselec-tive recognition of the (3R,4S)-F-Hyp over the corresponding (3S,4S) epimer and rationalize this preference through co-crystal structures and electrostatic potential calculations. Hyp substitution with (3R,4S)-F-Hyp into the BET degrader MZ1 retained bind-ing affinities and cellular degradation activities comparable to those of the parent PROTAC. In spite of a ~20-fold loss in binding affinity to VHL, incorporation of the (3S,4S) epimer into MZ1 led to Brd4-selective degradation at nanomolar concentration. We anticipate that the disclosed chemistry of 3-fluoro-4-hydroxyprolines and their application as VHL ligands for targeted protein deg-radation will be of wide interest to medicinal organic chemists, chemical biologists and drug discoverers alike

U2 - 10.1021/jacs.8b05807

DO - 10.1021/jacs.8b05807

M3 - Article

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

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