Double-Cuvette ISES:  In Situ Estimation of Enantioselectivity and Relative Rate for Catalyst Screening

Abstract

Described is a new method for the screening of an array of catalysts, in situ, to estimate enantioselectivity and relative rates. We term this approach "double-cuvette ISES (in situ enzymatic screening)". The Co(III)-salen mediated hydrolytic kinetic resolution (HKR) of (+/-)-propylene oxide is used as a model reaction to demonstrate proof of principle. In two parallel cuvettes, a lower CHCl3-based organic layer is loaded with the epoxide and the chiral salen catalyst. Aqueous reporting layers, containing distinct "reporting enzymes" and their nicotinamide cofactors, are layered above the organic layers. The 1,2-propanediol enantiomers formed by the chiral catalyst diffuse into the aqueous layer and are oxidized there by the reporting enzymes at rates dependent upon the diol concentration, the R:S ratio of the diol, and the enantioselectivity of the reporting enzymes. A focused chiral salen library was constructed from seven chiral 1,2-diamines, derived from amino acid, terpenoid, and carbohydrates skeletons, and seven salicylaldehyde derivatives. Double-cuvette ISES identified a couple of interesting combinatorial hits in this salen array, wherein either the sense or magnitude of enantioselection for a given chiral diamine depends significantly upon the choice of "salicylaldehyde" partner. A comparison of predicted ee's and relative rates using this new screening tool with those independently measured is provided.

“Cassette” In Situ Enzymatic Screening Identifies Complementary Chiral Scaffolds for Hydrolytic Kinetic Resolution Across a Range of Epoxides

“…In our ISES approach, typically the reaction product [4] or by-product [5] diffuses from an organic layer into an aqueous layer containing the "reporting enzyme". [6][7][8][9] There, an enzyme-catalyzed reaction leads to a spectroscopic signal that is monitored in real time.…”

“…This allows one to obtain simultaneous enantioselectivity readouts on two distinct substrates for the Co IIIsalen-mediated (salen = (salicylidene)ethylenediamine) hydrolytic kinetic resolution (HKR) of epoxides, [17] presenting both "short" (propylene oxide; R = Me) [4] and long (hexene oxide: R = Bu) R groups. In this way, one can begin to address the question of substrate generality, which is so important in asymmetric catalysis today.…”

The In Situ Enzymatic Screening (ISES) Approach to Reaction Discovery and Catalyst Identification

“…This protocol requires two cuvettes per reaction condition, one containing the ( R )-selective enzyme (TBADH) and the other requires the ( S )-selective enzyme (HLADH) for the 1,2-propanediol product (Dey et al, 2005). Equal numbers of ( R )-1,2-propanediol units of individual reporting enzyme are added to the respective reporting cuvettes.…”

“…The Sel parameter in this case was set as the enantioselectivity at 40 mM substrate concentration as obtained from enantioselectivity characterization (HLADH = 0.34, TBADH = 8.4) while v is the initial velocity measured (ΔAbs 340 /time) by either enzyme-1 or enzyme-2 in the ISES screen. For complete details of the reporting enzyme characterization, and of the derivation of the expression for enantioselectivity, see the SI for the initial report on double cuvette-ISES (Dey et al, 2005)…”

Two Methods for the Determination of Enantiomeric Excess and Concentration of a Chiral Sample with a Single Spectroscopic Measurement

“…[11][12][13] Few other methods have been shown to have this capacity. [14][15][16][17] In an eIDA, two spectroscopic measurements are taken. First, the concentration of a chiral sample ([G] t ) is determined with an indicator-displacement assay (IDA) [18] using an achiral host (H, Scheme 1).…”