Rapid, Stoichiometric, Site-Specific Modification of Aldehyde-Containing Proteins Using a Tandem Knoevenagel-Intra Michael Addition Reaction

Abstract

A site-specific modification of aldehyde-containing proteins using a tandem Knoevenagel-intra Michael addition reaction was developed. The reaction featured fast kinetics (50 M s) and favorable stoichiometry. Various functionalities could be introduced into the protein with little impact on its function and conformation. The reaction was successfully applied in the labeling of living cells.

Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs

“…The Fmoc protected hydrazine 1 (Scheme 2A) and the pyrazolone‐type Knoevenagel core segments 8 and 11 (Scheme 3A and B) were synthesized similar to literature protocols [7–8,28,44–45] . Subsequently, bifunctionalized HIPS ligation reagents were obtained containing the N , N′ ‐dimethylhydrazine group for FGly conjugation together with an azide, a bicyclononyne (BCN), a dibenzocyclooctyne (DBCO) or a 6‐methyl‐tetrazine (mTet) moiety for SPAAC or iEDDA, respectively.…”

“…The Fmoc protected hydrazine 1 (Scheme 2A) and the pyrazolone-type Knoevenagel core segments 8 and 11 (Scheme 3A and B) were synthesized similar to literature protocols. [7][8]28,[44][45] Subsequently, bifunctionalized HIPS ligation reagents were obtained containing the N,N'-dimethylhydrazine group for FGly conjugation together with an azide, a bicyclononyne (BCN), a dibenzocyclooctyne (DBCO) or a 6-methyltetrazine (mTet) moiety for SPAAC or iEDDA, respectively. Amide couplings were accomplished using DCC/EDC and pentafluorophenol/HOBt to form the corresponding PFP or HOBt ester, [28] which reacts with amine functionalized azide-, BCN-, DBCO-or 6-methyl-tetrazine derivatives.…”

“…[44] N-aryl substitution of those moieties further increases reactivity. [45] HIPS and Knoevenagel ligation have already been used in combination with the aldehyde-tag to produce site-specific ADCs with defined drugto-antibody ratios (DAR). However, a large excess of the expensive drug-containing building block and long reaction times were necessary.…”

“…In the latter case, a double bond is formed at neutral pH, which can either be attacked by an intramolecular thiol ( trapped Knoevenagel) [8] or a second pyrazolone building block ( tandem Knoevenagel) [44] . N‐ aryl substitution of those moieties further increases reactivity [45] . HIPS and Knoevenagel ligation have already been used in combination with the aldehyde‐tag to produce site‐specific ADCs with defined drug‐to‐antibody ratios (DAR).…”

Detection of Biothiols with a Fast‐Responsive and Water‐Soluble Pyrazolone‐Based Fluorogenic Probe

“…Rabuka and co‐workers have recently reported site‐specific derivatization of aldehyde‐modified proteins with pyrazolones through a tandem reaction involving a Knoevenagel condensation followed by a fast intramolecular thio‐Michael addition (Scheme A) . Inspired by this work, we designed a pyrazolone‐based Michael acceptor 1 as a fluorogenic probe, composed of a coumarin dye whose fluorescence would be quenched by an intramolecular charge transfer (ICT) and/or photoisomerization process (Scheme B).…”