137005-46-0Relevant academic research and scientific papers
Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs
Janson, Nils,Krüger, Tobias,Karsten, Lennard,Boschanski, Mareile,Dierks, Thomas,Müller, Kristian M.,Sewald, Norbert
, p. 3580 - 3593 (2020)
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.
Efficient Site-Selective Immobilization of Aldehyde-Tagged Peptides and Proteins by Knoevenagel Ligation
Janson, Nils,Heinks, Tobias,Beuel, Tobias,Alam, Sarfaraz,H?hne, Matthias,Bornscheuer, Uwe T.,Fischer von Mollard, Gabriele,Sewald, Norbert
, (2021/12/14)
The aldehyde tag is appropriate to selectively label proteins, prepare antibody-drug conjugates or to immobilize enzymes or antibodies for biotechnological and medical applications. The cysteine within the consensus sequence CxPxR of the aldehyde tag is specifically oxidized by the formylglycine-generating enzyme (FGE) to the non-canonical and electrophilic amino acid Cα-formylglycine (FGly). Subsequent reductive amination is a common method for site-directed immobilization, which usually results in poor immobilization efficiency due to the reaction conditions. Here, we introduce a new solid support like agarose modified with an aryl substituted pyrazolone (Knoevenagel reagent) that was obtained in a facile and efficient 2-step synthesis. The modified agarose allowed the site-selective and efficient immobilization of aldehyde-containing small molecules, peptides and proteins – in particular enzymes – at physiological pH (6.2–8.2) without any additive or catalyst needed. In comparison to reductive amination, higher loadings and activities were achieved in various buffers at different concentrations and temperatures.
