Genetic code expansion has bestowed researchers with numerous functionalities relevant to bioorthogonal chemistry, including azides, alkynes, tetrazines, cyclooctenes, and ketones. Considering the latter, ketones are considered useful bioorthogonal handles for bioconjugation, but these reactions often require lower pH and/or extended reaction times which may not be amenable to more sensitive proteins. Aldehydes offer a way to circumvent the limitations mentioned above; their electrophilic nature allows similar reactions to occur in shorter time and under potentially milder conditions. Consequently, their utility as bioorthogonal reagents have been explored, but current methodologies to install this functional group have their own limitations. Aldehyde installation can be achieved via mild oxidation with small molecules or enzymatic processes, but these strategies are indirect and, in the latter case, require special recognition sequences in the protein of interest. To this end, an aldehyde-based phenylalanine has been synthesized and directly incorporated into protein using amber suppression technology. This aldehyde is on the meta position of phenylalanine, and can be incorporated into superfolder green fluorescent protein (sfGFP) using our rationally-designed N346A/C348A pyrrolysyl-tRNA synthetase. To the best of our knowledge, this is the first time the direct genetic incorporation of an aldehyde has been reported. This aldehyde has been shown to persist in the conditions necessary for protein expression, as confirmed by ESI-MS. Furthermore, in vitro, analine-catalyzed hydroxylamine labeling of sfGFP-aldehyde was found to be quite rapid, with labeling observed in as little as five minutes, whereas sfGFP-ketone is unreactive under these conditions. An experimentally determined pseudo first-order rate constant of 182 M-1s-1 has been assigned, verifying the rapid nature of this bioconjugation. In addition, whole-cell labeling of the membrane protein OmpX has been achieved with this strategy. We anticipate this aldehyde to be a useful tool for chemical biologists and we are currently exploring further applications.

Genetic incorporation of a benzaldehyde residue for rapid bioorthogonal labeling at neutral pH

247th National Meeting (2014)

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