Two‐Step Protein Labeling by Using Lipoic Acid Ligase with Norbornene Substrates and Subsequent Inverse‐Electron Demand Diels–Alder Reaction

Abstract

Inverse-electron-demand Diels-Alder cycloaddition (DAinv ) between strained alkenes and tetrazines is a highly bio-orthogonal reaction that has been applied in the specific labeling of biomolecules. In this work we present a two-step labeling protocol for the site-specific labeling of proteins based on attachment of a highly stable norbornene derivative to a specific peptide sequence by using a mutant of the enzyme lipoic acid ligase A (LplA(W37V) ), followed by the covalent attachment of tetrazine-modified fluorophores to the norbornene moiety through the bio-orthogonal DAinv . We investigated 15 different norbornene derivatives for their selective enzymatic attachment to a 13-residue lipoic acid acceptor peptide (LAP) by using a standardized HPLC protocol. Finally, we used this two-step labeling strategy to label proteins in cell lysates in a site-specific manner and performed cell-surface labeling on living cells.

Inverse electron demand Diels–Alder reactions in chemical biology

“…83 Similarly, by using LplA W37V, Wombacher et al successfully ligated a norbornene derivative with a transmembrane protein with an extracellular LAP-tag in HEK293T cells. 169 4.2.3 Protein farnesyltransferase. Protein farnesyltransferase (PFTase) has been used to covalently attach farnesyl diphosphate site-specifically at the cysteine residue of the recognizing sequence CAAX (Fig.…”

Peptide-tags for site-specific protein labelling in vitro and in vivo

“…25,26 LplA specifically recognizes the LAP-tag, which consists of 22 aa, and accepts different lipoic acid derivatives containing alkyne, azide, trans-cyclooctene, aryl aldehyde or aryl hydrazine groups as well as fluorophores. 25,[27][28][29] Strain-promoted alkyne-azide cycloaddition (SPAAC), hydrazone formation or Diels-Alder cycloaddition 30 enabled the subsequent chemical modification of the lipoic acid appendage on LAP-tagged proteins such as CFP, 25 BFP, 28 low-density lipoprotein receptor (LDLR), 25 neurexin-1b 27 or actin. 28 The LplA mutant LplA W37V has been reported to provide higher ligation yields than the wild-type enzyme.…”

“…32 Wombacher et al reported norbornene-conjugated lipoic acid analogues, which were introduced into LAP-tagged CFP and dihydrofolate reductase from E. coli (eDHFR) and fluorescently labelled via inverse-electron demand Diels-Alder reaction. 30 The enzyme tubulin tyrosine ligase (TTL) allows the C-terminal labelling of proteins with a 14 aa hydrophilic recognition sequence called Tub-tag. TTL is a regulator of microtubule homeostasis and usually promotes the coupling of tyrosine to the a-tubulin C-terminus.…”

Inverse‐Electron‐Demand Diels–Alder Reactions: Principles and Applications

“…Other variants of trans ‐cyclooctene have also been tested, but compound 57 is the most effectively ligated. Other research groups have made use of norbornene instead of trans ‐cyclooctene as the iEDDA partner . LplA is able to recognize a LplA acceptor peptide (LAP) and site‐selectively tag the protein of interest.…”