Chirality sensing with synthetic pores

Abstract

A concept to determine enantiomeric excess with synthetic multifunctional pores is introduced. To do so, the poor stereoselectivity of molecular recognition by stimuli-responsive pores is coupled with the stereospecificity of enzymes. With substrates as good and products as poor pore blockers, enzymatic conversion of one enantiomer is shown to readily reveal the concentration of the other one. Calculations suggest that high substrate/product discrimination by the synthetic pores may provide access to the accurate detection of the extreme enantiomeric excess that is of interest in chemistry, pharmacology, and medicine, but otherwise possibly problematic to detect. Validity of the introduced concept is experimentally confirmed with poly-L-glutamate and poly-D-glutamate as enantiomeric substrates with high blockage efficiency, L-glutamate and D-glutamate as enantiomeric products with poor blockage efficiency, subtilisin A as enzyme, and a classical rigid-rod beta-barrel as synthetic pore.

Supramolecular Tandem Enzyme Assays for Multiparameter Sensor Arrays and Enantiomeric Excess Determination of Amino Acids

“…A related method for ee determination based on the absolute concentration of only one enantiomer has recently been described by Matile and co-workers in the enantiospecific enzymatic degradation of polymeric analytes, such as poly-l-versus poly-d-glutamate, in which the selective formation of monomeric l-glutamate results in blockage of a synthetic pore in dependence on the ee of the polymer. [55] The method allowed ee determinations between 90-98 %, which have proven difficult to assess by conventional analytical techniques, such as CD, GC, or HPLC. Another example of a related determination of ee involves the enantiospecific enzymatic oxidation of (S)-1-phenylpropanol, [56] in which the formation of the reduced cofactor NADPH, followed spectrophotometrically, provided indirect information about the optical purity of the alcohol with an accuracy of AE 10 % ee.…”

Chirality sensing with pores: Reactive signal amplifiers for otherwise undetectable small molecules

“…However, a comprehensive assessment of the potential of chirality sensing with pores in a more general context and in comparison with established methods was clearly beyond the scope of this proof-of-principle study. Very limited on first view, it is clear that the practical usefulness of this method will depend on the availability of appropriate enzymes 1,2,35,[37][38][39] or alternative stereospecific signal generators as well as on the existence of specific problems of interest that are otherwise difficult to address [40][41][42][43][44][45] (e.g., the detection of extreme ee). It is difficult to generalize at this stage, the question concerning practical usefulness of chirality sensing with pores will thus have to be evaluated from case to case as the number of successful examples grows with time.…”

“…1,2,34 With L-and D-lactate as enantiomers, L-lactate oxidase as signal generator, and CB hydrazide as signal amplifier, synthetic pores are shown to detect the enantiopurity of D-lactate up to at least 99% ee (98.6% ee expected, 98.7 6 0.2% ee found) and to sense trace impurities in commercial samples with reasonable accuracy (99.6% ee expected, 99.3 6 0.1% ee found, Fig. 1).…”

“…The possibility to detect the enantiomeric purity of chiral macromolecules with synthetic multifunctional pores has been reported previously. 2 In this approach, the poor stereoselectivity of molecular recognition by synthetic pores is coupled with the stereospecificity of enzymes. This approach worked well with poly-L-glutamate and poly-D-glutamate as enantiomeric substrates that both inactivate synthetic pores with IC 50 % 10 nM (inactivator concentration needed to reduce pore activity to 50%).…”

“…In practice, 98 6 1% ee was detectable without special optimization. 2 This approach to chirality sensing is limited to molecules that are recognized by synthetic multifunctional pores. Usually, there is no problem with macromolecules such as DNA, RNA, polysaccharides, or polypeptides, whereas smaller and less charged molecules often pass undetected.…”