Examination of the new α-(2′Z-fluoro)vinyl trigger with lysine decarboxylase: The absolute stereochemistry dictates the reaction course

Article abstract of DOI:10.1021/ja067240k

The first examination of a terminal α-fluorovinyl trigger in an amino acid decarboxylase active site is reported. To investigate the enantiospecificity of inactivation with this new AADC trigger, an enantioselective synthesis of l-α-(2′Z-fluoro)vinyllysine and its d-antipode has been developed. Control of stereochemistry is achieved through introduction of the amino acid side chain via alkylation of a chiral vinylglycine-derived dienolate. Facial selectivity is conferred by a trans-2′(β-naphthyl)-2′-propylcyclohexyl ester auxiliary, available in both antipodal forms (Comins protocol). The alkylation employs a new electrophile, N-p-methoxybenzyl-N-(2′-trimethylsilylethanesulfonyl)-4-iodobutylamine, for convergent installation of the lysine side chain. Vinyl to 2′-fluorovinyl interconversion then provides l- and d-α-(2′Z-fluoro)vinyllysine in 97-99% ee, as demonstrated by chiral HPLC. Both time-dependent enzyme kinetics and ¹⁹F NMR reveal striking differences in the behavior of these two antipodes in the lysine decarboxylase active site. The l-antipode displays time dependent inactivation (t_1/2 = 3 ± 1 min; K_I = 86 ± 22 μM), whereas the d-antipode behaves as a substrate, being completely turned over to α-(2′Z-fluoro)vinylcadaverine. Titration of LDC with varying amounts of l-α-(2′Z-fluoro)vinyllysine provides an estimate of 20 ± 3 for the partition ratio for this antipode. 19F NMR provides a more detailed account of the inactivation with the l-antipode, revealing that 1 in 3.4 turnovers of this mechanism-based inhibitor results in errant protonation (required by design), with 1 in 5 errant protonation events leading to LDC inactivation. This gives an overall partition ratio of 16 ± 2. Fluoride-selective electrode measurements are in agreement with ¹⁹F NMR estimates of [fluoride] released. Copyright

Full text of DOI:10.1021/ja067240k

Published on Web 12/19/2006
Examination of the New r-(2′Z-Fluoro)vinyl Trigger with Lysine
Decarboxylase: The Absolute Stereochemistry Dictates the Reaction Course
Kannan R. Karukurichi, Roberto de la Salud-Bea, Wan Jin Jahng, and David B. Berkowitz*
Department of Chemistry, UniVersity of Nebraska, Lincoln, Nebraska 68588-0304
Received October 9, 2006; E-mail: dbb@unlserve.unl.edu
Mechanism-based enzyme inactivators (MBEIs)1,2 are useful
Scheme 1. Projected Mechanisms for the 2′FV-AADC Trigger
bioorganic tools to study enzyme mechanism and to modulate
metabolic flux. Moreover, of the over 300 enzyme-targeted drugs
in the FDA orange book, most may be considered broadly
“mechanism-based”.3 As such, some combinatorial libraries are now
built around MBEI trigger motifs.4 Recently, “suicide triggers” have
also found application in screens for catalytic antibodies5 and in
activity-based proteomics.6
We describe here the first successful halovinyl trigger for amino
acid decarboxylase (AADC) inactivation, of which we are aware.
Among MBEIs for AADCs, DFMO (R-difluoromethylornithine),
used clinically for parasitic diseases and as a potential chemopre-
ventive agent,7 probably is the benchmark. The mechanism of action
of the R-difluoromethyl trigger in the ornithine decarboxylase
(ODC) active site has now been elucidated by a combination of
MS8 and X-ray methods,9 and the non-enantioselective nature of
its inactivation has been established.10
We set out to develop an R-(2′-fluoro)vinyl (2′FV) trigger for
AADC inactivation with projected mechanism(s) as outlined in
Scheme 1. The design envisions the normal transaldimination (to
I) and R-decarboxylation (gives II) steps, followed by an errant
protonation at either C4′ of the cofactor (to IV) or at Cγ of the
“suicide substrate”. The former pathway finds precedence in the
now crystallographically established11 Michael addition mecha-
nism12 of the antiepileptic drug, Vigabatrin, though for this target,
GABA transaminase, C4′, is the normal locus of protonation. The
latter Cγ pathway can branch into a nucleophilic Metzler enamine
pathway,13 and/or into a second electrophilic pathway, initiated by
γ-fluoride expulsion out of intermediate VIII,14 rather than Mannich
condensation.
Scheme 2. Stereoselective Introduction of the 2′FV-AADC Trigger
Note again that all three putative AADC inactivation pathways
require that the enzyme be directed into aberrant protonation of
quinonoid intermediate II. The literature provides insight as to how
this detour pathway might be promoted. It is known that installation
of an R-methyl group, to generate a quaternary AA, results in
increased errant (C4′) protonation in the cognate AADC (from
typically 0.01-0.1% up to 4% frequency for ODC).15 So, the
placement of the (2′FV) trigger at a quaternary R-center is critical
to our design and is to serve the dual purpose of (i) dictating
specificity for AADCs over enzymes that labilize the R-proton (e.g.,
transaminases, racemases, and â- and γ-replacement enzymes), and
(ii) promoting the requisite errant protonation.
We chose lysine decarboxylase (LDC) as a model enzyme in
which to test this new AADC trigger, as we had access to multi-
milligram quantities of the Hafnia alVei LDC.16 Furthermore, this
class of bacterial LDC was reported to be resistant to covalent
modification with the R-difluoromethyl trigger.17
A stereoselective synthesis of each antipode of the targeted
inactivator was achieved as outlined in Scheme 2. The quaternary
center is introduced by alkylation of a chiral vinylglycine-derived
dianionic dienolate.18 As illustrated, our working model for such
systems involves (i) the use of R-nitrogen-based amidate chelation
to control enolate geometry and (ii) the application of auxiliaries
of the “arylmenthyl” variety to control facial selectivity.
The use of dienolates outfitted with the Comins auxiliary
(available in both antipodal forms)19 here provides a potentially
generalizable method to access both enantiomers of sought after
quaternary AAs. Following side-chain installation, conversion of
the R-vinyl group to a 2′Z-fluorovinyl group proceeds smoothly,
following our recently disclosed protocol.20
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C O M M U N I C A T I O N S
γ-Protonation provides the simplest mechanism for release of four
excess equivalents of fluoride, but this does not exclude any of the
three pathways put forth for the inactivation step itself. This must
await the results of further studies. Given the success in driving
errant protonation with L-FVL and its favorable kinact, it will be of
interest to examine this trigger in other AADC active sites. The
remarkable enantio-discrimination observed stands in stark contrast
to the case of DFMO and underscores the value of interrogating
individual antipodes in MBEI studies.
Figure 1. Titration of LDC with L-2′FVL (two trials, color-coded)
Acknowledgment. We thank the NSF (Grant CHE-0616840)
and the NIH (Grants CA 62034 and RR016544-01) for support
of unnatural AA synthesis and MBEI efforts in our laboratory.
Joseph Dumais, Sara Basiaga and Richard Shoemaker are thanked
for assistance with NMR.
Supporting Information Available: Details of the asymmetric
synthesis, enzyme inactivation, and 19F NMR studies. This material is
available free of charge via the Internet at http://pubs.acs.org.
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