Developing specific chemical functionalities to deploy in biological environments for targeted enzyme inactivation lies at the heart of mechanism-based inhibitor (MBI) development, but also is central to other protein-tagging methods in modern chemical biology including activity-based protein profiling (ABPP) and proteolysis-targeting chimeras (PROTACS). 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′-fluorovinyl 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 yields 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 (Ki = 630 ± 20 μM; t1/2 = 2.8 min) and D-α-(1′-fluoro)vinyllysine (Ki = 470 ± 30 μM; t1/2 = 3.6 min). The stage is now set for exploration of the efficacy of this trigger in other PLP-enzyme active sites.
The asymmetric synthesis of a Rho kinase/norepinephrine transport inhibitor, netarsudil, the active component in the recently FDA-approved product Rhopressa™, is described herein. This concise six-step synthetic route utilizes the 2,4-dimethylbenzoate ester of a phenylacetic acid as the backbone of the β-amino acid’s framework. A chiral enolate of the Evans auxiliary, (R)-4-benzyloxazolidin-2-one, is used to direct the formation of the (S)-stereocenter by incorporating the N-Boc-protected β-amino methyl arm with high diastereoselectivity (96:4 dr) using N-Boc-1-aminomethylbenzotriazole as the electrophile. Uniquely, 2,2,2-trichloro-1,1-dimethylethyl chloroformate is used as a non-racemizing activating agent for the coupling reaction between the chiral (S)-N-Boc-protected 2,4-dimethylbenzoyloxymethyl phenyl propanoic acid and 6-aminoisoquinoline to provide N-Boc-protected netarsudil in good yield and excellent enantiomeric purity (63%, 98% ee). Final acidic deprotection and recrystallization provides netarsudil (>99% ee), an ophthalmic agent used for the treatment of patients with open-angle glaucoma.
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