Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs
Formylglycine‐generating enzymes specifically oxidize cysteine within the consensus sequence CxPxR to Cα‐formylglycine (FGly). This noncanonical electrophilic amino acid can subsequently be addressed selectively by bioorthogonal hydrazino‐iso‐Pictet‐Spengler (HIPS) or Knoevenagel ligation to attach...
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Published in | Chembiochem : a European journal of chemical biology Vol. 21; no. 24; pp. 3580 - 3593 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
WEINHEIM
Wiley
11.12.2020
Wiley Subscription Services, Inc John Wiley and Sons Inc |
Subjects | |
Online Access | Get full text |
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Summary: | Formylglycine‐generating enzymes specifically oxidize cysteine within the consensus sequence CxPxR to Cα‐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.
Increased efficiency: The combination of azide‐functionalized HIPS or tandem Knoevenagel ligation reagents and SPAAC enables the fast and efficient mono‐ and double‐derivatization of proteins with stoichiometric amounts of DBCO‐modified payloads at low concentration. |
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Bibliography: | Thomas Dierks passed away on July 07, 2020 Dedicated to the memory of Thomas Dierks ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1439-4227 1439-7633 |
DOI: | 10.1002/cbic.202000416 |