Synthesis and Deployment of an Elusive Fluorovinyl Cation Equivalent: Access to Quaternary α-(1'-Fluoro)vinyl Amino Acids as Potential PLP Enzyme Inactivators

Abstract

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_i = 630 ± 20 μM; t_1/2 = 2.8 min) and D-α-(1'-fluoro)vinyllysine (K_i = 470 ± 30 μM; t_1/2 = 3.6 min). The stage is now set for exploration of the efficacy of this trigger in other PLP-enzyme active sites.

Figure 1

Potential PLP-enzyme inactivation mechanisms for quaternary, α-(1′-fluoro)vinyl amino acids, following the design of Abeles for α-(1′-fluoro)vinylglycine, illustrated for an AADC mechanism, highlighting both γ-conjugate addition (via an alleneimine intermediate) and β-conjugate addition (both via an alleneimine and via γ-protonation) pathways.

Figure 2

A-Synthesis of β,β-difluorovinyl phenyl sulfone 5 which following Kuglerohr distillation is isolated as a transparent oil. B-¹H NMR spectrum of 5 after purification; reaction run for 20 s. C-¹³C NMR spectrum of 5 showing both geminal and vicinal C–F splitting.

Figure 3

A-Schematic representation of the ¹⁴CO₂-capture assay employed for LDC activity. LDC from Hafnia alvei was incubated with U-C-labeled L-lysine (2.5 mM conc; 7.14 nCi in 200 μL total volume) in an Eppendorf tube. Evolved ¹⁴CO₂ was captured with base (benzethonium hydroxide) soaked filter paper… B-Schematic of the complementary UV-based Lenhoff assay that measures formation of cadaverine product with time (details in the SI). C-Primary and secondary Kitz-Wilson plots for the time dependent inactivation of H. alvei LDC by L- and D-α-(1′-fluoro)vinyllysine. D-Results of dialysis experiments. LDC (1.5 U) was first inactivated for 1 h with 10 mM (±)-α-(1′-fluoro)vinyllysine (98.9% inactivation). Each cycle was run as a 1:250-fold dilution against 100 μM PLP, 100 mM KPO₄, pH 6.0. A total of 9 cycles of dialysis (~10²¹-fold dilution) was run. The insert (lower right corner) shows an expansion of the cpm vs. time data for the inactivated enzyme samples as a function of dialysis cycle.

Scheme 1. Toward Quaternary, Fluorovinyl Amino Acids

A – Quaternary, α-(2′Z-fluoro)vinyl AAs via excision/(2′-fluoro)methylenation of α-vinyl AAs B – Quaternary, α-(1′-fluoro)vinyl AAs via convergent, α-(1′-fluoro)vinylation of AA-enolates

Scheme 2

Double Add’n/Elimination with AA-Derived Dianions

Scheme 3

Single vs. Double Addition/Elimination

Scheme 4

α-(1′-Fluoro)vinylation of AA-Derived Enolates with 5

Scheme 5

α-Fluoro Sulfone-Stannane Interchange: Entry into Quaternary, Fluoro-Trialkylstannylvinyl AAs

Scheme 6

Global Deprotection to Quaterary, α-(1′-Fluoro)vinyl AAs a – In these cases, though the fluorovinyl AA-HCl salt was clean by NMR, we elected to generate the free, quaternary, α-(1′-fluoro)vinyl AA via ion exchange chromatography [Dowex-50; 96% from 11a-HCl; (Ala); 94% from 11e-HCl (Met)]. b – In this case, Dowex 50 cation exchange chromatography also led to an improvement in purity (~15%, by NMR) of the fluorovinyl AA (76% from 11f-HCl). c – In this case, MOM cleavage was performed with TFA in CH₂Cl₂, with accompanying protodestannylation and aldimine cleavage, followed by via base-catalyzed ester saponification. The crude product was acidified and purified via Dowex 50 cation exchange chromatography (66% yield of 11h).