Journal
CHEMBIOCHEM
Volume 21, Issue 24, Pages 3580-3593Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.202000416
Keywords
bioconjugation; branched PEG linker; click chemistry; formylglycine; HIPS ligation; Knoevenagel ligation
Funding
- Deutsche Forschungsgemeinschaft [SPP 1623, DI 575/9-1, MU 2286/6-1, SE 609/15-1]
- Projekt DEAL
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Formylglycine-generating enzymes specifically oxidize cysteine within the consensus sequence CxPxR to C-alpha-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 andtandemKnoevenagel 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-modifiedtandemKnoevenagel 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.
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