Site-specific Bioorthogonal Protein Labelling by Tetrazine Ligation Using Endogenous β-amino Acid Dienophiles

Overview

Journal Nat Chem

Specialty Chemistry

Date 2023 Jul 3

PMID 37400596

Authors

Daniel Richter

Edgars Lakis

Jorn Piel

Affiliations

Soon will be listed here.

Abstract

The tetrazine ligation is an inverse electron-demand Diels-Alder reaction widely used for bioorthogonal modifications due to its versatility, site specificity and fast reaction kinetics. A major limitation has been the incorporation of dienophiles in biomolecules and organisms, which relies on externally added reagents. Available methods require the incorporation of tetrazine-reactive groups by enzyme-mediated ligations or unnatural amino acid incorporation. Here we report a tetrazine ligation strategy, termed TyrEx (tyramine excision) cycloaddition, permitting autonomous dienophile generation in bacteria. It utilizes a unique aminopyruvate unit introduced by post-translational protein splicing at a short tag. Tetrazine conjugation occurs rapidly with a rate constant of 0.625 (15) M s and was applied to produce a radiolabel chelator-modified Her2-binding Affibody and intracellular, fluorescently labelled cell division protein FtsZ. We anticipate the labelling strategy to be useful for intracellular studies of proteins, as a stable conjugation method for protein therapeutics, as well as other applications.

Citing Articles

Peptide Backbone Editing via Post-Translational O to C Acyl Shift.

Schissel C, Roberts-Mataric H, Garcia I, Kang H, Mowzoon-Mogharrabi R, Francis M J Am Chem Soc. 2025; 147(8):6503-6513.

PMID: 39933167 PMC: 11869294. DOI: 10.1021/jacs.4c14103.

Bioorthogonal Janus microparticles for photothermal and chemo-therapy.

Zhang Q, Kuang G, Chen K, Zhao M, Shang L Smart Med. 2025; 3(4):e20240038.

PMID: 39776589 PMC: 11669771. DOI: 10.1002/SMMD.20240038.

Mechanistic Insights Into Post-Translational α-Keto-β-Amino Acid Formation by a Radical S-Adenosyl Methionine Peptide Splicease.

Vagstad A, Vagstad A, Lakis E, Csizi K, Walls W, Richter D Angew Chem Int Ed Engl. 2024; 64(6):e202418054.

PMID: 39688170 PMC: 11879070. DOI: 10.1002/anie.202418054.

Metagenomic study of lake microbial mats reveals protease-inhibiting antiviral peptides from a core microbiome member.

Padhi C, Field C, Forneris C, Olszewski D, Fraley A, Sandu I Proc Natl Acad Sci U S A. 2024; 121(49):e2409026121.

PMID: 39585984 PMC: 11626197. DOI: 10.1073/pnas.2409026121.

A structurally conserved helix enables leader-independent tyramine splicing of proteins.

Richter D, Courvoisier-Clement A, Vagstad A, Magyari S, Piel J Chem Sci. 2024; .

PMID: 39309086 PMC: 11414181. DOI: 10.1039/d4sc03867c.

References

Ballantyne A, Dhillon S . Trastuzumab emtansine: first global approval. Drugs. 2013; 73(7):755-65. DOI: 10.1007/s40265-013-0050-2. View

Kumar A, Planchais C, Fronzes R, Mouquet H, Reyes N . Binding mechanisms of therapeutic antibodies to human CD20. Science. 2020; 369(6505):793-799. DOI: 10.1126/science.abb8008. View

Alley S, Benjamin D, Jeffrey S, Okeley N, Meyer D, Sanderson R . Contribution of linker stability to the activities of anticancer immunoconjugates. Bioconjug Chem. 2008; 19(3):759-65. DOI: 10.1021/bc7004329. View

Montalban-Lopez M, Scott T, Ramesh S, Rahman I, van Heel A, Viel J . New developments in RiPP discovery, enzymology and engineering. Nat Prod Rep. 2020; 38(1):130-239. PMC: 7864896. DOI: 10.1039/d0np00027b. View

Ma X, Ehrhardt D, Margolin W . Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein. Proc Natl Acad Sci U S A. 1996; 93(23):12998-3003. PMC: 24035. DOI: 10.1073/pnas.93.23.12998. View

Rostovtsev V, Green L, Fokin V, Sharpless K . A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes. Angew Chem Int Ed Engl. 2002; 41(14):2596-9. DOI: 10.1002/1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.CO;2-4. View

Reichert A, Poxleitner G, Dauner M, Skerra A . Optimisation of a system for the co-translational incorporation of a keto amino acid and its application to a tumour-specific Anticalin. Protein Eng Des Sel. 2015; 28(12):553-65. DOI: 10.1093/protein/gzv048. View

Lang K, Davis L, Wallace S, Mahesh M, Cox D, Blackman M . 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. J Am Chem Soc. 2012; 134(25):10317-20. PMC: 3687367. DOI: 10.1021/ja302832g. View

Xie H, Zu L, Oueis H, Li H, Wang J, Wang W . Proline-catalyzed direct inverse electron demand diels-alder reactions of ketones with 1,2,4,5-tetrazines. Org Lett. 2008; 10(10):1923-6. DOI: 10.1021/ol800417q. View

10.

Yu J, Liu Y, Yin H, Chang Z . Regrowth-delay body as a bacterial subcellular structure marking multidrug-tolerant persisters. Cell Discov. 2019; 5:8. PMC: 6341109. DOI: 10.1038/s41421-019-0080-3. View

11.

Sletten E, Bertozzi C . Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew Chem Int Ed Engl. 2009; 48(38):6974-98. PMC: 2864149. DOI: 10.1002/anie.200900942. View

12.

Theile C, Witte M, Blom A, Kundrat L, Ploegh H, Guimaraes C . Site-specific N-terminal labeling of proteins using sortase-mediated reactions. Nat Protoc. 2013; 8(9):1800-7. PMC: 3941705. DOI: 10.1038/nprot.2013.102. View

13.

Gardner K, Moore D, Erickson H . The C-terminal linker of Escherichia coli FtsZ functions as an intrinsically disordered peptide. Mol Microbiol. 2013; 89(2):264-75. PMC: 3725778. DOI: 10.1111/mmi.12279. View

14.

Bohne C, Macdonald I, Dunford H . Transient state kinetics of the reactions of isobutyraldehyde with compounds I and II of horseradish peroxidase. J Biol Chem. 1987; 262(8):3572-8. View

15.

Agard N, Prescher J, Bertozzi C . A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J Am Chem Soc. 2004; 126(46):15046-7. DOI: 10.1021/ja044996f. View

16.

Arnison P, Bibb M, Bierbaum G, Bowers A, Bugni T, Bulaj G . Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat Prod Rep. 2012; 30(1):108-60. PMC: 3954855. DOI: 10.1039/c2np20085f. View

17.

Conner K, Rock B, Kwon G, Balthasar J, Abuqayyas L, Wienkers L . Evaluation of near infrared fluorescent labeling of monoclonal antibodies as a tool for tissue distribution. Drug Metab Dispos. 2014; 42(11):1906-13. PMC: 11024893. DOI: 10.1124/dmd.114.060319. View

18.

SAXON E, Armstrong J, Bertozzi C . A "traceless" Staudinger ligation for the chemoselective synthesis of amide bonds. Org Lett. 2000; 2(14):2141-3. DOI: 10.1021/ol006054v. View

19.

Sehlin D, Fang X, Cato L, Antoni G, Lannfelt L, Syvanen S . Antibody-based PET imaging of amyloid beta in mouse models of Alzheimer's disease. Nat Commun. 2016; 7:10759. PMC: 4762893. DOI: 10.1038/ncomms10759. View

20.

Frejd F, Kim K . Affibody molecules as engineered protein drugs. Exp Mol Med. 2017; 49(3):e306. PMC: 5382565. DOI: 10.1038/emm.2017.35. View