α‐Methylene‐β‐Lactone Scaffold for Developing Chemical Probes at the Two Ends of the Selectivity Spectrum

Abstract

The utilities of an α‐methylene‐β‐lactone (MeLac) moiety as a warhead composed of multiple electrophilic sites are reported. We demonstrate that a MeLac‐alkyne not only reacts with diverse proteins as a broadly reactive measurement probe, but also recruits reduced endogenous glutathione (GSH) to assemble a selective chemical probe of GSH‐β‐lactone (GSH‐Lac)‐alkyne in live cells. Tandem mass spectrometry reveals that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael or acyl addition. A peptide‐centric proteomics platform demonstrates that the proteomic selectivity profiles of orlistat and parthenolide, which have distinct reactivities, are measurable by MeLac‐alkyne as a high‐coverage probe. The GSH‐Lac‐alkyne selectively probes the glutathione S‐transferase P responsible for multidrug resistance. The assembly of the GSH‐Lac probe exemplifies a modular and scalable route to develop selective probes with different recognizing moieties.

Single-Sequence Identification of Modified Peptides: A One-Pot Method Using Homologous Biotinyl Azides

“…We have recently demonstrated that the multi-electrophilic MeLac warhead can probe protein nucleophiles of different types in the live cell proteome. 14 MeLac labels reactive cysteine, serine, threonine, tyrosine, lysine residues, and the N-terminal amino group through the Michael acceptor or the acyl carbon atom on the warhead (Scheme 1A). When protein nucleophiles react at the Michael addition site of MeLac, a characteristic hydrolyzed product of the β-lactone ring can be observed to verify the Michael addition mechanism, resulting in a mass increase of 568.32206 Da.…”

“…When protein nucleophiles react at the Michael addition site of MeLac, a characteristic hydrolyzed product of the β-lactone ring can be observed to verify the Michael addition mechanism, resulting in a mass increase of 568.32206 Da. 14 Although nucleophilic thiols, amines, and hydroxyls can all react via the Michael addition, protein adducts of the addition reaction are dominantly attributed to MeLac reacting to nucleophilic cysteines. 14 We used CuAAC to attach desthiobiotin-PEG3-azide to MeLacA-reacted proteins.…”

“…14 Although nucleophilic thiols, amines, and hydroxyls can all react via the Michael addition, protein adducts of the addition reaction are dominantly attributed to MeLac reacting to nucleophilic cysteines. 14 We used CuAAC to attach desthiobiotin-PEG3-azide to MeLacA-reacted proteins.…”

“…MSFragger open search [22][23][24] observed global MeLacA modifications on proteins, as previously reported. 14 MSFragger 25,26 offset search was set for cysteine reacting with the Michael acceptor of MeLacA, which gave an incremental mass of 568.32206 Da (the hydrolyzed adduct of MeLacA with the attached desthiobiotin tag) for the probe-modified peptides. Labile modification search mode was also enabled, requiring the modified peptides to produce one of the two diagnostic ions at m/z 197.12845 and 240.17065; these are diagnostic fragment ions from the desthiobiotin moiety 14 (Scheme 2).…”

“…13 In this technical note, we demonstrate a strategy for applying the SSI principle using a model clickable proteome: live HT-29 cells reacted with a racemic probe of 4-(3-butynyl)-3methyleneoxetan-2-one (α-methylene-β-lactone (MeLac) alkyne (MeLacA); Scheme 1A). 14 The MeLac warhead has multiple reactive sites and is broadly reactive to various residues. We present a one-pot, multiplexing click conjugation method using homologous biotinyl azides.…”