Genetic Encoding of Bicyclononynes and trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels–Alder Reactions

Abstract

Rapid, site-specific labeling of proteins with diverse probes remains an outstanding challenge for chemical biologists. Enzyme-mediated labeling approaches may be rapid but use protein or peptide fusions that introduce perturbations into the protein under study and may limit the sites that can be labeled, while many “bioorthogonal” reactions for which a component can be genetically encoded are too slow to effect quantitative site-specific labeling of proteins on a time scale that is useful for studying many biological processes. We report a fluorogenic reaction between bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and tetrazines that is 3–7 orders of magnitude faster than many bioorthogonal reactions. Unlike the reactions of strained alkenes, including trans-cyclooctenes and norbornenes, with tetrazines, the BCN–tetrazine reaction gives a single product of defined stereochemistry. We have discovered aminoacyl-tRNA synthetase/tRNA pairs for the efficient site-specific incorporation of a BCN-containing amino acid, 1, and a trans-cyclooctene-containing amino acid 2 (which also reacts extremely rapidly with tetrazines) into proteins expressed in Escherichia coli and mammalian cells. We demonstrate the rapid fluorogenic labeling of proteins containing 1 and 2 in vitro, in E. coli, and in live mammalian cells. These approaches may be extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on labeling and imaging studies.

Selective chemical protein modification

“…4a). The site-specific incorporation of cyclooctynes 97 and biscyclononynes 98 into proteins by amberstop codon suppression has also recently been reported. However, limitations remain, such as occasional difficulty in the synthesis and handling of strained and unstable compounds, while crucially a degree of incompatibility towards cysteine has been reported 99,100 .…”

“…To generally enable IEDDA protein reactions, key reactive UAAs (tetrazine 105 , norbornene [106][107][108] , cyclooctene 98,107 and biscyclononene 98 ) have been incorporated into proteins by amber-stop codon suppression (see Box 2). Such strain-promoted cycloadditions offer intriguing and exciting possibilities for future protein labelling where the speed of labelling is vital, and recent developments suggest that the cyclooctyne-azide and cyclooctenetetrazine reactions may have a degree of mutual compatibility, thereby allowing multi-site labelling 109,110 .…”

A Minimal, Unstrained S‐Allyl Handle for Pre‐Targeting Diels–Alder Bioorthogonal Labeling in Live Cells

“…Most protein‐labeling approaches through IEDDA ligation rely on strained cyclic alkenes and alkynes, such as norbornene,2 trans ‐cyclooctene (TCO),3 or bicyclo[6.1.0]nonyne,3b that are introduced on proteins by genetic encoding methods for subsequent reaction with tetrazines. Alternatively, tetrazines may also be genetically encoded into proteins and subsequently labeled with TCO 4.…”

18F-Labeling of Mannan for Inflammation Research with Positron Emission Tomography

“…In the original studies, mannan (10 mg per mouse) was intraperitoneally injected into mice to induce psoriasis and psoriatic arthritis. 7 To perform the biological evaluations in similar experimental settings, [ 19 F]fluoromannan (10 mg per mouse) was injected intraperitoneally into mice with a neutrophil cytosolic factor 1 gene mutation (Ncf1 m1J/m1J ), 7 and mannan (10 mg per mouse) was administrated into control mice for comparison. The disease phenotypes were followed by visual observation, and the disease severity was quantified by blinded scoring.…”