Synthesis and Evaluation of Novel Ring‐Strained Noncanonical Amino Acids for Residue‐Specific Bioorthogonal Reactions in Living Cells

Abstract

Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels–Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)‐a method that allows to incorporate noncanonical amino acids (ncAAs) site‐specifically into proteins in vivo. These reactions enable residue‐specific fluorophore attachment to proteins in living mammalian cells. Several SPIEDAC capable ncAAs have been presented and studied under diverse conditions, revealing different instabilities ranging from educt decomposition to product loss due to β‐elimination. To identify which compounds yield the best labeling inside living mammalian cells has frequently been difficult. In this study we present a) the synthesis of four new SPIEDAC reactive ncAAs that cannot undergo β‐elimination and b) a fluorescence flow cytometry based FRET‐assay to measure reaction kinetics inside living cells. Our results, which at first sight can be seen conflicting with some other studies, capture GCE‐specific experimental conditions, such as long‐term exposure of the ring‐strained ncAA to living cells, that are not taken into account in other assays.

Minimal genetically encoded tags for fluorescent protein labeling in living neurons

“…4 ). The former has been previously successfully used for intracellular protein labeling 39 , 40 and the latter has been recently rediscovered as a very stable UAA for click labeling 41 . Experiments in ND7/23 cells show that both can be genetically encoded into NFL K363TAG and specifically labeled with SiR-tetrazine (Supplementary Fig.…”

“…In contrast to the results obtained with ND7/23 cells, TCO4en/eq-Lys was not efficiently incorporated in NFL K363TAG in primary neurons. This is probably related to the fact that the PylRS AF that we used poorly accepts TCO4en/eq-Lys 41 . Consequently, we found only a few neurons with fully translated and click-labeled NFL K363TAG→TCO4en/eq-Lys .…”

“…The literature on UAA stability is somewhat conflicting, mainly because different methods were used to study reactions rates and stability. Recent in cellulo experiments represent a very useful resource in this regard and “reidentify” previously neglected TCO4en/eq-Lys as a particularly stable UAA 41 . Furthermore, to reduce the background originating from free UAA molecules, UAAs that show a faster washout, such as BCN-Lys 39 , 40 and more hydrophilic dioxo-TCO-Lys 42 could be used.…”

“…Furthermore, to reduce the background originating from free UAA molecules, UAAs that show a faster washout, such as BCN-Lys 39 , 40 and more hydrophilic dioxo-TCO-Lys 42 could be used. In any case, to incorporate these UAAs efficiently in neurons would require further optimizations including using recently developed PylRS synthetase for TCO4en/eq-Lys 41 , selecting a more efficient synthetase for hydrophilic dioxo-TCO 42 , as well as titrating the concentration of BCN-Lys to allow prolonged experiments with primary neurons. With regards to the tetrazine dyes, the number of cell-permeable dyes that can be used for intracellular labeling in living cells is still limited.…”

Dual film-like organelles enable spatial separation of orthogonal eukaryotic translation

“…3-IF is accepted by PylRS AA and TCO ∗ A-K by PylRS AF ( Figure S1 D). TCO ∗ A-K rapidly reacts with a tetrazine in a strain-promoted inverse electron-demand Diels-Alder cycloaddition ( Nikić et al., 2014 ; Plass et al., 2012 ; Reinkemeier et al., 2021 ; Wagner et al., 2015 ) ( Figure 6 A), which can be selectively detected by fluorescence if a suitable tetrazine-dye conjugate is used. The nonreactive 3-IF cannot be easily detected by such an unambiguous method, and it is thus not easy to confirm that the other dimeric film-like organelle also performs as selectively as the FFC and imaging experiments described above indicate.…”