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Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs.


ABSTRACT: Formylglycine-generating enzymes specifically oxidize cysteine within the consensus sequence CxPxR to C? -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.

SUBMITTER: Janson N 

PROVIDER: S-EPMC7756428 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs.

Janson Nils N   Krüger Tobias T   Karsten Lennard L   Boschanski Mareile M   Dierks Thomas T   Müller Kristian M KM   Sewald Norbert N  

Chembiochem : a European journal of chemical biology 20200918 24


Formylglycine-generating enzymes specifically oxidize cysteine within the consensus sequence CxPxR to C<sup>α</sup> -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  ...[more]

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