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

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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
LanguageEnglish
Pages9299-9313
Number of pages15
JournalJournal of the American Chemical Society
Volume140
Issue number29
Early online date27 Jun 2018
DOIs
StatePublished - 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, A, Lucas, X, Castro, G, Chan, K-H, Wright, JE, Runcie, AC, Gadd, MS, Harrison, WTA, Ko, E-J, Fletcher, D & Ciulli, A 2018, '3-Fluoro-4-hydroxyprolines: Synthesis, Conformational Analysis, and Stereoselective Recognition by the VHL E3 Ubiquitin Ligase for Targeted Protein Degradation' Journal of the American Chemical Society, vol. 140, no. 29, pp. 9299-9313. DOI: 10.1021/jacs.8b05807
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 = "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.",
year = "2018",
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T1 - 3-Fluoro-4-hydroxyprolines

T2 - Journal of the American Chemical Society

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 - 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

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JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

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