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Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs
Author(s) -
Janson Nils,
Krüger Tobias,
Karsten Lennard,
Boschanski Mareile,
Dierks Thomas,
Müller Kristian M.,
Sewald Norbert
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000416
Subject(s) - bioconjugation , bioorthogonal chemistry , knoevenagel condensation , chemistry , combinatorial chemistry , sortase , bifunctional , click chemistry , fluorophore , moiety , conjugated system , peptidomimetic , cysteine , azide , fluorescence , peptide , biochemistry , stereochemistry , organic chemistry , enzyme , physics , polymer , quantum mechanics , bacterial protein , gene , catalysis
Formylglycine‐generating enzymes specifically oxidize cysteine within the consensus sequence CxPxR to C α ‐formylglycine (FGly). This noncanonical electrophilic amino acid can subsequently be addressed selectively by bioorthogonal hydrazino‐ iso ‐Pictet‐Spengler (HIPS) or Knoevenagel ligation to attach payloads like fluorophores or drugs to proteins to obtain a defined payload‐to‐protein ratio. However, the disadvantages of these conjugation techniques include the need for a large excess of conjugation building block, comparably low reaction rates and limited stability of FGly‐containing proteins. Therefore, functionalized clickable HIPS and tandem Knoevenagel building blocks were synthesized, conjugated to small proteins (DARPins) and subsequently linked to strained alkyne‐containing payloads for protein labeling. This procedure allowed the selective bioconjugation of one or two DBCO‐carrying payloads with nearly stoichiometric amounts at low concentrations. Furthermore, an azide‐modified tandem Knoevenagel building block enabled the synthesis of branched PEG linkers and the conjugation of two fluorophores, resulting in an improved signal‐to‐noise ratio in live‐cell fluorescence‐imaging experiments targeting the EGF receptor.