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

Abstract

Many reagents have been developed for cysteine-specific protein modification. However, few of them allow for multi-functionalization of a single Cys residue and disulfide bridging bioconjugation. Herein, we report 3-bromo-5-methylene pyrrolones (3Br-5MPs) as a simple, robust, and versatile class of reagents for cysteine-specific protein modification. These compounds can be facilely synthesized via a one-pot mild reaction and they show comparable tagging efficiency but higher cysteine specificity than the maleimide counterparts. The addition of cysteine to 3Br-5MPs generates conjugates that are amenable to secondary addition by another thiol or cysteine, making 3Br-5MPs valuable for multi-functionalization of a single cysteine and disulfide bridging bioconjugation. The labeling reaction and subsequent treatments are mild enough to produce stable and active protein conjugates for biological applications.

5-Hydroxy-pyrrolone based building blocks as maleimide alternatives for protein bioconjugation and single-site multi-functionalization

“…We found that trace amounts of the exchanged product were observed only after 4 days (see the ESI † ). This observation contrasts with the behavior of the thiol conjugates coming from pyrrolones of type I ( Scheme 1B ), 32 where thiol exchange was observed, most probably due to the presence of the sulfur group at the exocyclic position. As a consequence, 5HP2O building blocks fulfill all the criteria in terms of hydrolytic stability and Michael-addition irreversibility to be able to answer the previously unmet call for a selective method for dual-functionalization of cysteine residues.…”

“…We have also established that the new 5HP2O modifiers have characteristics which offer significant performance enhancement when compared to the alternative maleimide-based bioconjugation methods. We have shown that the resulting conjugates are hydrolytically stable in buffered media and that the protein conjugate is stable towards thiol-exchange over prolonged periods of time which represent considerable advantages when compared to maleimide derivatives or the thiol conjugates of the 3-bromo-5-methylene pyrrolones 32 which degrade through exchange. Furthermore, protein labeling with 5HP2Os resulted in conjugates with high stability.…”

Contemporary Approaches for Site‐Selective Dual Functionalization of Proteins

“…First, a Michael reaction of cysteine and 3Br‐5MPs generated the bioconjugate that is amenable to a second Michael addition with another thiol, allowing protein dual functionalization at a cysteine site. Due to the slow release of the second functionality, a reducing reagent, for example NaBH 4 , was required to retard the elimination reaction to generate a stable and bioactive conjugate for subsequent applications [91] …”

Diethinylphosphinate für die Cystein‐selektive Proteinmarkierung und Disulfid‐Verbrückung

“…[12] Neben der Modifikation einzelner Cys-Reste mit einer spezifischen Nutzlast sind in der Literatur auch Peptid-oder Proteinmodifikationen mit doppelt reaktiven Cys-spezifischen Reagenzien beschrieben worden. [13][14][15][16][17][18] ¾hnlich wie bei normalen Maleimiden sind die mit solchen Reagenzien erzeugten Bindungen jedoch oft instabil gegenüber reduzierenden Bedingungen oder einem Überschuss an kleinen Thiolen. [13,15] Diese bisfunktionellen Reagenzien kçnnen für die intermolekulare Vernetzung von zwei Cys-haltigen Proteinen verwendet werden.…”