Abstract

The first part of this dissertation describes the design, synthesis and evaluation of α-(2'Z-fluoro)vinyllysine as an inactivator for L-lysine decarboxylase (LDC) from Hafnia alvei. An asymmetric synthesis of both enantiomers of α-(2'Z-fluoro)vinyllysine has been accomplished. The L-absolute stereochemistry was unequivocally established via anomalous dispersion studies on the crystalline bis (hydrochloride) salt of the final product. The L-antipode is a suicide inactivator for LDC. A functional requirement for the actuation the (2'Z-fluoro)vinyl trigger is thought to be "errant protonation" either at Cγ or at C _4' of the pyridoxal cofactor, following decarboxylation, leading to the release of fluoride. ¹⁹F NMR studies indicate high-levels (∼1 in 3 protonations) of "errant protonation" induced by this trigger, in the L-antipode. Moreover, parallel examination of LDC activity reveals that ∼1 in 5 decarboxylations lead to enzyme inactivation, providing a partition ratio (total turnovers per inactivation event) of 16 ± 2. The D-antipode does not inactivate LDC and interestingly, ₁₉F NMR shows that it is a substrate rather than a suicide substrate.

The second part of this dissertation describes the development of an "in situ" enzymatic screen (ISES) to estimate the sense and magnitude of enantioselection in the hydrolytic kinetic resolution (HKR) of (±)-propylene oxide. Equine liver alcohol dehydrogenase [NAD ⁺ dependent, (S)-selective] and alcohol dehydrogenase from T. brockii [NADP⁺ dependent, (R)-selective] are used as reporters with opposite enantiopreferences for the 1,2-propanediol product. A focused 7x7 salen array from seven chiral, non-C₂-symmetric 1,2-diamines and seven electronically and sterically different salicylaldehydes was examined. Co(III) complexes obtained from these salens were screened for HKR by ISES in a "double-cuvette" mode. A good correlation was observed between the predicted and measured values for both relative rate and enantioselectivity. Among other interesting discoveries from the screen, an unusual salicylaldehyde (3,5-di- tert-butyl vs. 3,5-diiodo) dependent "enantio-switch" was seen in catalysts bearing identical chiral elements (β-fructopyranose-derived diamine). Synthesis and evaluation of the carbacyclic analogues of these catalysts reinforced this combinatorial find.