Spotlight on lipids: One of the major limitations of tetrazine bioorthogonal cycloadditions is the requirement of bulky dienophile reaction partners. Methylcyclopropene tags were designed capable of reacting rapidly with tetrazines while maintaining stability in aqueous solution. The suitability of these probes for bioconjugation is shown by imaging cyclopropene‐modified phospholipids in live human cancer cells (see picture).

Cell membranes are dynamic structures found in all living organisms. There have been numerous constructs that model phospholipid membranes. However, unlike natural membranes, these biomimetic systems cannot sustain growth owing to an inability to replenish phospholipid-synthesizing catalysts. Here we report on the design and synthesis of artificial membranes embedded with synthetic, self-reproducing catalysts capable of perpetuating phospholipid bilayer formation. Replacing the complex biochemical pathways used in nature with an autocatalyst that also drives lipid synthesis leads to the continual formation of triazole phospholipids and membrane-bound oligotriazole catalysts from simpler starting materials. In addition to continual phospholipid synthesis and vesicle growth, the synthetic membranes are capable of remodeling their physical composition in response to changes in the environment by preferentially incorporating specific precursors. These results demonstrate that complex membranes capable of indefinite self-synthesis can emerge when supplied with simpler chemical building blocks.membranes | autocatalysis | self-assembly | lipids

We demonstrate the site-specific incorporation of nucleobase derivatives bearing fluorophores or affinity labels into a short RNA stem loop recognition motif by exchange of a guanine residue. The RNA-TAG (transglycosylation at guanosine) is carried out by a bacterial (E. coli) tRNA guanine transglycosylase (TGT), whose natural substrate is the nitrogenous base PreQ1. Remarkably, we have successfully incorporated large functional groups including biotin, BODIPY, thiazole orange, and Cy7 through a polyethylene glycol linker attached to the exocyclic amine of PreQ1. Larger RNAs, such as mRNA transcripts, can be site-specifically labeled if they possess the 17-nucleotide hairpin recognition motif. The RNA-TAG methodology could facilitate the detection and manipulation of RNA molecules by enabling the direct incorporation of functional artificial nucleobases using a simple hairpin recognition element.

Tetrazine ligations have proven to be a powerful bioorthogonal technique for the detection of many labeled biomolecules, but the ligating nature of these reactions can limit reaction turnover in templated chemistry. We have developed a transfer reaction between 7-azabenzonorbornadiene derivatives and fluorogenic tetrazines that facilitates turnover amplification of the fluorogenic response in nucleic acid-templated reactions. Fluorogenic tetrazine-mediated transfer (TMT) reaction probes can be used to detect DNA and microRNA (miRNA) templates to 0.5 and 5 pM concentrations, respectively. The endogenous oncogenic miRNA target mir-21 could be detected in crude cell lysates and detected by imaging in live cells. Remarkably, the technique is also able to differentiate between miRNA templates bearing a single mismatch with high signal to background. We imagine that TMT reactions could find wide application for amplified fluorescent detection of clinically relevant nucleic acid templates.

There is tremendous interest in the use of bioorthogonal reactions for imaging of unnatural building blocks that are metabolically incorporated into biosynthetic pathways. Applications include visualizing glycans, imaging proteins tagged with unnatural amino acids, monitoring cellular proliferation, and tracking lipid analogues. [1] For live-cell imaging applications, the azide-cyclooctyne catalyst-free cycloaddition has been extremely useful in providing a rapid and biocompatible method to label small azide tags with fluorophore. [1a] Recently, there has been growing interest in exploring bioorthogonal cycloadditions involving tetrazines for live-cell imaging applications. [2] Tetrazines have been shown to react rapidly through inverse-electron-demand Diels-Alder reactions with a variety of strained alkenes and alkynes including trans-cyclooctenes, norbornenes, and cyclooctynes. These reactions can be used for live-cell imaging, and tetrazines can quench the fluorescence of commonly used imaging probes such as BODIPY dyes and fluoresceins. [3] This leads to a fluorogenic response after reaction, which can improve signal-to-background, which is particularly useful for intracellular live-cell imaging applications. [1b,4] Though tetrazine cycloadditions would be exciting developments for a wide array of metabolic imaging applications, the large size of both the tetrazine and cycloalkene coupling partners has limited their ability to be incorporated into small bio-active molecules. In response to this challenge, we recently developed small and stable methylcyclopropenes as coupling partners for fluorogenic tetrazines. [5] The molecular weight of these tags rivaled those of azides and were used to fluorogenically image lipids in live mammalian cells. However, we were interested in whether methylcyclopropenes could substitute for azides in metabolic imaging applications with stringent steric constraints. Here we demonstrate that unnatural cyclopropenemannosamine derivatives can be used to image glycans on live human cancer cell lines. Tetrazine-based cycloadditions are an emerging class of bioorthogonal reactions that can proceed with rapid rate constants, enable imaging of dienophile tags in live-cells and animals, and be mutually orthogonal to azide-alkyne cycloadditions. [6] Despite these applications, the use of tetrazine cycloadditions has been limited in metabolic imaging. This is due to the size of tetrazines and paired dienophiles such as trans-cyclooctene and norbornene, which are large compared to the azide and alkyne tags commonly used in bioorthogonal chemistry. We recently developed methylcyclopropenes as tetrazine-reactive cellular imaging tags that are comparable to azides in terms of molecular weight (Scheme 1A). [5] Following our work, others have shown that cyclopropene-containing amino acids and cyclopropene-derivatized neuraminic acid analogues can be incorporated into cellular macromolecules and later tagged using photoinitated reactions or tetrazine-biotin-avidin coupling. [7] However, althou...