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.

中文翻译：

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3-Fluoro-4-hydroxyprolines：VHL E3 泛素连接酶用于靶向蛋白质降解的合成、构象分析和立体选择性识别

脯氨酸的羟基化和氟化以立体化学依赖性方式改变吡咯烷环褶皱和反式：顺式酰胺键比率，影响生物系统对含脯氨酸分子的分子识别。虽然羟脯氨酸和氟脯氨酸是药物和生物化学中的常见基序，但尚未描述含有这两种修饰的脯氨酸（即氟羟脯氨酸）的合成和分子特性。在这里，我们从容易获得的 4-氧代-l-脯氨酸衍生物开始，提出了一种实用且简便的 3-氟-4-羟基脯氨酸 (F-Hyps) 的所有四种非对映异构体的合成方法。小分子 X 射线晶体学、核磁共振光谱学、和量子力学计算与 C3 的氟化一致，对 C4 羟基的氢键供体能力的影响可以忽略不计，但将 Hyp 从 C4-exo 到 C4-endo 褶皱的自然偏好反转。尽管如此，F-Hyps 仍然与 von Hippel-Lindau (VHL) E3 连接酶结合，后者以立体选择性的方式自然识别 C4-exo Hyp。共晶结构和静电势计算支持并合理化了观察到的 (3R,4S)-F-Hyp 优先于 VHL 对相应 (3S,4S) 差向异构体的识别。我们表明 (3R,4S)-F-Hyp 在缺氧诱导因子 1 α (HIF-1α) 底物肽和拟肽配体中提供生物等排 Hyp 取代，这些配体构成 PROTAC（蛋白水解靶向嵌合体）缀合物的一部分，用于靶向蛋白质降解。尽管亲和力减弱，在 PROTAC MZ1 中用 (3S,4S)-F-Hyp 取代 Hyp 导致 Brd4 选择性细胞降解，其浓度比 VHL 的二元 Kd 低 100 倍以上。我们预计，已公开的 3-fluoro-4-hydroxyprolines 化学及其作为 VHL 配体用于靶向蛋白质降解的应用将引起药用有机化学家、化学生物学家和药物发现者等广泛兴趣。