High‐Throughput Method for Determining the Enantioselectivity of Enzyme‐Catalyzed Hydroxylations Based on Mass Spectrometry

Abstract

Up to speed: An accurate, sensitive, high‐throughput, and simple method for measuring the product ee value of enzyme‐catalyzed hydroxylations (see scheme) is based on the use of enantiopure or enantioenriched deuterated substrates and mass spectrometric detection.

Directed evolution of RebH for catalyst-controlled halogenation of indole C–H bonds

“…Li and coworkers reported a method to determine the enantioselectivity of C–H/D hydroxylation reactions conducted on deuterium-substituted substrates using mass spectrometry (GC/MS or LC/MS). 49 We envisioned that deuterated tryptamines could be used in a similar fashion to identify RebH variants with altered regioselectivity. RebH halogenates tryptamine with the same high 7-selectivity that it exhibits on tryptophan, 33 but deuterated tryptamines are more readily prepared than the corresponding tryptophans.…”

“…S1 and S2 † ), so rate differences of the isotopomers did not have to be taken into account. 49 Halogenation selectivity could be reliably determined by MALDI-MS analysis of crude reaction mixtures arrayed onto a standard 384-spot sample targets, which allowed for rapid evaluation of halogenase libraries.…”

Sulphide as a leaving group: highly stereoselective bromination of alkyl phenyl sulphides

“…Optically active benzylic bromides have been reported to be prone to racemisation 1c,e,10. For instance, a sample of ( R )-1-(bromoethyl)benzene having 88% ee kept at 0 °C was shown to fully racemise within 8 hours 1 c .…”

“…The above methods showcase the recent progress made in the deoxybromination of alkyl alcohols; however, challenges remain such as alkene side product formation from elimination sensitive bromides, limited scope and functional group tolerance, difficulties in removing by-products such as phosphine oxides, requirement of multiple reagents and bromide racemisation from optically active alkyl alcohols 6a,10…”

A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

“…In short, we have introduced the PECANm ass-spectrometric enzyme activity assay,and demonstrated its suitability for high-throughput screening of P450s in complex matrices, such as cell lysates and microsomes.W hile we have demonstrated the applicability of PECANonly to P450s,webelieve that, after the appropriate method development, this technology could be applicable to any enzyme (or set of enzymatic reactions), that changes the mass of its substrate.F urthermore,i tm ay be possible to monitor isomerization reactions through the application of tandem MS,o re nantioselective reactions through the use of isotopically labeled substrates. [47] As expected from any high-throughput assay,o ur PECAN screen yielded some false positives,a nd we re-screened as mall subset of the library using low-throughput analytical technologies to verify the hits,asisroutine in this field. Like spectrometric assays,t he PECANt echnology requires the synthesis of as pecialized probe and enzymatic reactions are performed in microtiter plates.Therefore,weexpect PECAN to have roughly equivalent throughput to-and act as ac omplementary method to-spectrometric assays.W e expect PECANt ob eavaluable technology for drug discovery or directed-evolution campaigns that focus on enzymes for which no high-throughput screen currently exists.…”