TY  - JOUR
AB  - Formylglycine-generating enzymes specifically oxidize cysteine within the consensus sequence CxPxR to C alpha -formylglycine (FGly). This non-canonical electrophilic amino acid can subsequently be addressed selectively via bioorthogonal Hydrazino- iso -Pictet-Spengler (HIPS) or Knoevenagel ligation to attach payloads like fluorophores or drugs to proteins, while obtaining a defined payload-to-protein ratio. However, disadvantages of these conjugation techniques include the need of 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. © 2020 Wiley-VCH GmbH.
DA  - 2020
DO  - 10.1002/cbic.202000416
LA  - eng
IS  - 24
M2  - 3580
PY  - 2020
SP  - 3580-3593
T2  - ChemBioChem
TI  - Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs
UR  - https://nbn-resolving.org/urn:nbn:de:0070-pub-29455318
Y2  - 2024-11-22T04:18:32
ER  -