Department of Chemistry, University of Nebraska , Lincoln, Nebraska 68588-0304, United States.
J Am Chem Soc. 2017 Oct 11;139(40):14077-14089. doi: 10.1021/jacs.7b04690. Epub 2017 Sep 28.
Developing specific chemical functionalities to deploy in biological environments for targeted enzyme inactivation lies at the heart of mechanism-based inhibitor development but also is central to other protein-tagging methods in modern chemical biology including activity-based protein profiling and proteolysis-targeting chimeras. We describe here a previously unknown class of potential PLP enzyme inactivators; namely, a family of quaternary, α-(1'-fluoro)vinyl amino acids, bearing the side chains of the cognate amino acids. These are obtained by the capture of suitably protected amino acid enolates with β,β-difluorovinyl phenyl sulfone, a new (1'-fluoro)vinyl cation equivalent, and an electrophile that previously eluded synthesis, capture and characterization. A significant variety of biologically relevant AA side chains are tolerated including those for alanine, valine, leucine, methionine, lysine, phenylalanine, tyrosine, and tryptophan. Following addition/elimination, the resulting transoid α-(1'-fluoro)-β-(phenylsulfonyl)vinyl AA-esters undergo smooth sulfone-stannane interchange to stereoselectively give the corresponding transoid α-(1'-fluoro)-β-(tributylstannyl)vinyl AA-esters. Protodestannylation and global deprotection then yield these sterically encumbered and densely functionalized quaternary amino acids. The α-(1'-fluoro)vinyl trigger, a potential allene-generating functionality originally proposed by Abeles, is now available in a quaternary AA context for the first time. In an initial test of this new inhibitor class, α-(1'-fluoro)vinyllysine is seen to act as a time-dependent, irreversible inactivator of lysine decarboxylase from Hafnia alvei. The enantiomers of the inhibitor could be resolved, and each is seen to give time-dependent inactivation with this enzyme. Kitz-Wilson analysis reveals similar inactivation parameters for the two antipodes, L-α-(1'-fluoro)vinyllysine (K = 630 ± 20 μM; t = 2.8 min) and D-α-(1'-fluoro)vinyllysine (K = 470 ± 30 μM; t = 3.6 min). The stage is now set for exploration of the efficacy of this trigger in other PLP-enzyme active sites.
开发针对生物环境的特定化学功能，以实现靶向酶失活，这是基于机制的抑制剂开发的核心，但也是现代化学生物学中其他蛋白质标记方法的核心，包括基于活性的蛋白质谱分析和蛋白水解靶向嵌合体。我们在这里描述了一类以前未知的潜在 PLP 酶失活剂; 即，一类带正电荷的α-(1'-氟)乙烯基氨基酸，带有对应氨基酸的侧链。这些是通过适当保护的氨基酸烯醇化物与β,β-二氟乙烯基苯砜（一种新的（1'-氟）乙烯基阳离子等价物）和以前难以合成、捕获和表征的亲电试剂捕获而得到的。大量具有生物相关性的 AA 侧链被容忍，包括丙氨酸、缬氨酸、亮氨酸、蛋氨酸、赖氨酸、苯丙氨酸、酪氨酸和色氨酸。加成/消除后，所得反式α-(1'-氟)-β-(苯磺酰基)乙烯基 AA-酯通过磺酰基-锡烷交换立体选择性地得到相应的反式α-(1'-氟)-β-(三丁基锡基)乙烯基 AA-酯。然后进行原锡烷脱保护和全局脱保护，得到这些空间位阻大和密集官能化的季铵氨基酸。α-(1'-氟)乙烯基引发剂是 Abeles 最初提出的潜在丙二烯生成官能团，现在首次在季铵 AA 环境中可用。在对这种新抑制剂类别的初步测试中，α-(1'-氟)乙烯基赖氨酸被证明是海氏杆菌赖氨酸脱羧酶的时间依赖性、不可逆失活剂。抑制剂的对映异构体可以被拆分，并且每种对映异构体都被证明可以使该酶发生时间依赖性失活。Kitz-Wilson 分析为两种对映异构体（L-α-(1'-氟)乙烯基赖氨酸（K = 630 ± 20 μM;t = 2.8 min）和 D-α-(1'-氟)乙烯基赖氨酸（K = 470 ± 30 μM;t = 3.6 min））提供了相似的失活参数。现在已经为探索这种引发剂在其他 PLP 酶活性位点中的功效奠定了基础。
