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3‑Fluoro-4-hydroxyprolines: Synthesis, Conformational Analysis, and Stereoselective Recognition by the VHL E3 Ubiquitin Ligase for Targeted Protein Degradation
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posted on 2018-06-27, 00:00 authored by Andrea Testa, Xavier Lucas, Guilherme V. Castro, Kwok-Ho Chan, Jane E. Wright, Andrew C. Runcie, Morgan S. Gadd, William T. A. Harrison, Eun-Jung Ko, Daniel Fletcher, Alessio CiulliHydroxylation 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 and peptidomimetic ligands
that form part of PROTAC (proteolysis targeting chimera) conjugates
for targeted protein degradation. 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 organic chemists, chemical biologists,
and drug discoverers alike.
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Keywords
fluorinationligandF-Hyp4- oxo-l-proline derivativesrecognitionPROTAC MZ 1Small-molecule X-ray crystallographyhypoxia-inducible factor 1Targeted Protein Degradation Hydroxylation3- fluoro -4-hydroxyprolinesbioisosteric Hyp substitutionsynthesisE 3 ligaseVHL E 3 Ubiquitin LigaseNMRhydrogen bond donor capacityfashioncalculationC 4protein degradationHIFC 4 hydroxylpyrrolidine ring puckerexo
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