3-Fluoro-4-hydroxyprolines: Synthesis, Conformational Analysis, and Stereoselective Recognition by the VHL E3 Ubiquitin Ligase for Targeted Protein Degradation.

Andrea Testa,Guilherme V Castro,Morgan S Gadd,William T A Harrison,Andrew C Runcie,Alessio Ciulli,Daniel Fletcher,Eun-Jung Ko,Xavier Lucas,Jane E Wright,Kwok-Ho Chan

doi:10.1021/jacs.8b05807

Andrea Testa, Guilherme V Castro + Show 9 more

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https://doi.org/10.1021/jacs.8b05807

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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 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.

Highlights

(2S,4R)-4-Hydroxyproline (Hyp) is a nonessential amino acid, prevalently found in collagen and produced by the most common, irreversible, posttranslational modification in animals: proline hydroxylation.[1−3] Introduction of the hydroxyl group on the five-membered ring of proline at the 4R position affects the pyrrolidine ring puckering, shifting the C4-endo pucker preference of proline to the C4-exo pucker of Hyp.[4−6] The C4-exo conformer in Hyp is stabilized by the well-known gauche effect (Chart 1A).[7]
We hypothesized that the addition of the highly electronegative F atom adjacent to the hydroxyl group on the pyrrolidine ring could significantly alter the puckering preference of the ring and affect the cis:trans amide ratio
We show that (3R,4S)-3-fluoro-4-hydroxyproline incorporation achieved ligands and proteolysistargeting chimeras (PROTACs) with affinities and cellular activities comparable to those of the parent Hyp-containing compounds, while incorporation of the (3S,4S) epimer led to Brd4-selective degradation at nanomolar concentration in spite of a loss of ∼20-fold binding affinity to von Hippel−Lindau (VHL)

Summary

Introduction

(2S,4R)-4-Hydroxyproline (Hyp) is a nonessential amino acid, prevalently found in collagen and produced by the most common, irreversible, posttranslational modification in animals: proline hydroxylation.[1−3] Introduction of the hydroxyl group on the five-membered ring of proline at the 4R position affects the pyrrolidine ring puckering, shifting the C4-endo pucker preference of proline to the C4-exo pucker of Hyp.[4−6] The C4-exo conformer in Hyp is stabilized by the well-known gauche effect (Chart 1A).[7]. Fluorination at C4 or C3 causes stabilization of the C4-endo/C4-exo ring pucker in a similar fashion to hydroxylation, according to the same gauche effect.[13,14] To this end, 4-fluoro and 3-fluoro prolines have been incorporated in proteins such as collagen,[16] ubiquitin,[17] and GFP,[18] used to probe prolyl isomerase enzyme activity by NMR19 and used as PET probes.[20] Beyond proteins, fluorinated prolines have found applications as building blocks for medicinal chemistry,[21] reflecting the wide interest in fluorination as a strategy to finely tune conformational and physicochemical properties of biologically active small molecules and peptides.[22−25] For instance, the incorporation of (4S)- or (4R)-4-fluoroprolines into inhibitors of fibroblast activation protein (FAP)[26] and thrombin[27] Previous reports have studied the influence of fluorine on the −OH group acidity in the context of 2-fluorocyclohexanols and have shown that fluorination can lead to a reduction of alcohol Hbond acidity as a result of intramolecular F···HO interactions.[30]

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