Thiol Specific and Tracelessly Removable Bioconjugation via Michael Addition to 5-Methylene Pyrrolones

Abstract

5-Methylene pyrrolones (5MPs) are highly thiol-specific and tracelessly removable bioconjugation tools. 5MPs are readily prepared from primary amines in one step. 5MPs exhibit significantly improved stability under physiologically relevant conditions and cysteine specificity compared to commonly used analogues, maleimides. Michael addition of thiol to 5MPs occurs rapidly, cleanly and does not generate a stereocenter. The conjugates efficiently release thiols via retro-Michael reaction in alkaline buffer (pH 9.5) or via thiol exchange at pH 7.5. This unique property makes 5MPs valuable for the controlled release of conjugated cargo and temporary thiol protection. The utilization of 5MPs for protein immobilization and pull-down of active complexes is illustrated using E. coli. acetohydroxyacid synthase isozyme I.

Bioconjugation with Maleimides: A Useful Tool for Chemical Biology

“…Biochemical studies focused on identifying the mechanistic pathways leading to the cleavage of the C4‐oxidized abasic site (C4‐AP) in nucleosomes showed that a 5‐methylene pyrrolone (5MP) modification is generated along with a sugar unit transference into a lysine side‐chain . This observation inspired the use of 5MPs as potential highly specific class of thiol‐tagging reagents, as they present similar structural features when compared to maleimides, are synthetically accessible and do not generate any stereocenter upon bioconjugation (Scheme A) . Experimentally, 5MP reagents evidenced remarkable stability (including hydrolysis) when incubated in a relevant range of pHs (6–9.5, up to 72 h).…”

“…[80] This observation inspired the use of 5MPs as potential highly specific class of thiol-tagging reagents,a st hey presents imilar structural features when compared to maleimides,a re synthetically accessible and do not generate any stereocenter upon bioconjugation (Scheme22A). [81] Experimentally,5 MP reagents evidenced remarkable stability (including hydrolysis)w hen incubated in ar elevant range of pHs (6-9.5, up to 72 h). This feature contrasts with the behavior observed with structurally related N-hydroxyethyl maleimide, which underwent complete ring-opening hydrolysis after 2hours.…”

Cysteine-specific protein multi-functionalization and disulfide bridging using 3-bromo-5-methylene pyrrolones

“…Recently, the need to prepare homogeneous antibody-drug conjugates has inspired a wave of interest in exploring cysteine-specific bioconjugation reagents with improved reactivity, specificity, stability, and biocompatibility 6,7 . To this end, maleimide derivatives [8][9][10] , dehydroalanine 11,12 , perfluoroaromatic reagents [13][14][15][16] , organometallic palladium reagents 17,18 , and many others [19][20][21][22][23] have been developed, and each of these reagents shows unique merits. However, only a few of them permit multifunctionalization of a single Cys residue 22,24,25 , which would be advantageous in certain situations.…”

“…Recently, we reported that 5-methylene pyrrolones (5MPs) are promising protein-modification reagents 10 . These compounds are easy to prepare, stable, and highly thiol-specific.…”

Highly Reactive and Tracelessly Cleavable Cysteine‐Specific Modification of Proteins via 4‐Substituted Cyclopentenone

“…[24,25] Only af ew methods are available for cleavable cysteinespecific modification, including Ellmansr eagent (10), [26,27] bromo-maleimides (11 a), [28] bromo-pyridazinediones (11 b), [29] electron deficient acetylenes (12), [30] and recently reported 5-methylene pyrrolones (13). [31] Herein, the cysteine-specific protein modification with 4substituted cyclopentenone was reported. Compared to many previous reports, [32] this ligation could achieve high reactivity (with arate constant over 100 m À1 s À1 at the peptide level) and stable product at physiological conditions with little impact on the function and structure of the protein.…”