A Hammett Study of Clostridium acetobutylicum Alcohol Dehydrogenase (CaADH): An Enzyme with Remarkable Substrate Promiscuity and Utility for Organic Synthesis

Abstract

Described is a physical organic study of the reduction of three sets of carbonyl compounds by the NADPH-dependent enzyme Clostridium acetobutylicum alcohol dehydrogenase (CaADH). Previous studies in our group have shown this enzyme to display broad substrate promiscuity, yet remarkable stereochemical fidelity, in the reduction of carbonyl compounds, including α-, β- and γ-keto esters (d-stereochemistry), as well as α,α-difluorinated-β-keto phosphonate esters (l-stereochemistry). To better mechanistically characterize this promising dehydrogenase enzyme, we report here the results of a Hammett linear free-energy relationship (LFER) study across three distinct classes of carbonyl substrates; namely aryl aldehydes, aryl β-keto esters and aryl trifluoromethyl ketones. Rates are measured by monitoring the decrease in NADPH fluorescence at 460 nm with time across a range of substrate concentrations for each member of each carbonyl compound class. The resulting v 0 versus [S] data are subjected to least-squares hyperbolic fitting to the Michaelis–Menton equation. Hammett plots of log(V max) versus σX yield the following Hammett parameters: (i) for p-substituted aldehydes, ρ = 0.99 ± 0.10, ρ = 0.40 ± 0.09; two domains observed, (ii) for p-substituted β-keto esters ρ = 1.02 ± 0.31, and (iii) for p-substituted aryl trifluoromethyl ketones ρ = –0.97 ± 0.12. The positive sign of ρ indicated for the first two compound classes suggests that the hydride transfer from the nicotinamide cofactor is at least partially rate-limiting, whereas the negative sign of ρ for the aryl trifluoromethyl ketone class suggests that dehydration of the ketone hydrate may be rate-limiting for this compound class. Consistent with this notion, examination of the 13C NMR spectra for the set of p-substituted aryl trifluo­romethyl ketones in 2% aqueous DMSO reveals significant formation of the hydrate (gem-diol) for this compound family, with compounds bearing the more electron-withdrawing groups showing greater degrees of hydration. This work also presents the first examples of the CaADH-mediated reduction of aryl trifluoromethyl ketones, and chiral HPLC analysis indicates that the parent compound α,α,α-trifluoroacetophenone is enzymatically reduced in 99% ee and 95% yield, providing the (S)-stereoisomer, suggesting yet another compound class for which this enzyme displays high enantioselectivity.

Biomacromolecule-Assisted Screening for Reaction Discovery and Catalyst Optimization

“…Namely, once such enantioselective enzymes have been identified, they cannot only be used to screen for a targeted enantioselective organometallic transformation but can also be exploited in and of themselves for asymmetric biocatalysis. This approach has been highlighted for several ISES reporting enzymes in the Berkowitz group, including recombinant KRED enzymes from Codexis, , the hyperthermophilic SSADH-10 from the archeon, Sulfolobus solfataricus , and the remarkable Clostridial enzyme, CaADH that displays unusual substrate promiscuity yet stereochemical fidelity. − …”

Biocatalytic asymmetric reduction of fluoroalkyl ketones to access enantiopure fluoroalkyl secondary alcohols

“…Ketoreductases (KREDs) are now the main biocatalysts applied for ketone bioreduction, as well as for fluoroalkyl ketones. 9 In this context, an arsenal of enzymes from different origins is now available for the synthesis of fluoroalkyl secondary alcohols with high optical purity, including PpKR8 from Paraburkholderia phymatum , 10 CaADH from Clostridium acetobutylicum , 11 Ras-ADH from Ralstonia sp., 12 KR01 from Leifsonia sp. S749, 13 PFADH from the hyperthermophilic archaeon Pyrococcus furiosus , 14 TtADH from Thermus thermophilus , 15 SsCR from S. salmonicolor , 16 KmCR2 from K. marxianus CBS4857, 17 etc .…”

“…9 In this context, an arsenal of enzymes from different origins is now available for the synthesis of fluoroalkyl secondary alcohols with high optical purity, including PpKR8 from Paraburkholderia phymatum, 10 CaADH from Clostridium aceto- Shanghai Engineering Research Center of Green Fluoropharmaceutical Technology, School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China. E-mail: wujj@sit.edu.cn, wfh@sit.edu.cn butylicum, 11 Ras-ADH from Ralstonia sp., 12 KR01 from Leifsonia sp. S749, 13 PFADH from the hyperthermophilic archaeon Pyrococcus furiosus, 14 TtADH from Thermus thermophilus, 15 SsCR from S. salmonicolor, 16 KmCR2 from K. marxianus CBS4857, 17 etc.…”

Whole-Cells of Yarrowia lipolytica Applied in “One Pot” Indolizine Biosynthesis

“…The application of whole microbial-cell biocatalysis methods is steadily increasing as an environment-friendly alternative to dangerous solvents and catalysts [1][2][3][4]. The use of enzymes in synthetic transformations has many advantages, such as mild and eco-friendly conditions, high regioand stereoselectivities [5][6][7][8][9], but it also has drawbacks in the preparation of enzymes that require the cultivation of microorganisms, purification and crystallization of enzymes [2,10]. Therefore, the use of whole-cell systems for conducting chemical reactions offers some great advantages such as decreased reaction time, considerable decrease of costs, fewer steps, elimination of toxic solvents, mild conditions, high purity and yields of the obtained compounds and ecofriendly transformations [10][11][12][13]].…”