Retro Diels–Alder Fragmentation of Fulvene–Maleimide Bioconjugates for Mass Spectrometric Detection of Biomolecules

Abstract

Diels−Alder chemistry is a well-explored avenue for the synthesis of bioactive materials; however, its potential applications have recently expanded following the development of reactions that can be performed in buffered aqueous environments at low temperatures, including fulvene−maleimide [4 + 2] cycloadditions. In this study, we synthesized two novel amine-reactive fulvene linkers to demonstrate the application of this chemistry for generating mass spectrometry-cleavable labels ("mass tags"), which can be used for the labeling and detection of proteins. Successful conjugation of these linkers to maleimidelabeled peptides was observed at low temperatures in phosphatebuffered saline, allowing the non-destructive modification of proteins with such mass tags. The labile nature of fulvene−maleimide adducts in the gas phase also makes them suitable for both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometric analysis. Unlike previous examples of MALDI mass tags, we show that fulvene−maleimide cycloaddition adducts fragment predictably upon gas-phase activation without the need for bulky photocleavable groups. Further exploration of this chemistry could therefore lead to new approaches for mass spectrometry-based bioassays.

Ylidenenorbornadiene Carboxylates: Experimental Kinetic Analysis of a Nucleophile-Induced Fragmentation Reaction

“…One commonly employed dynamic covalent system utilizes a furan diene and maleimide dienophile in a [4 + 2] cycloaddition reaction. This chemistry has been studied extensively, and the chemical reaction mechanisms are well understood. , Limitations for this reaction include fragmentation over a broad temperature range. − Fulvenes have previously displayed a wide array of cycloaddition reactivities, − and their use as dienes in place of furan has been shown to narrow this fragmentation temperature window. − …”

Exploring the versatility of pentafulvene–maleimide cycloaddition as a ligation strategy: buffer and pH effects

“…Detailed kinetic studies of the [4 + 2] cycloaddition between 6,6-dimethyl pentafulvenes and maleimide derivatives in PBS (10 mM, pH 7.4) to afford 7-alkylidene norborn-2-ene cycloadducts were previously reported by our group, 24 including cross-reactivity studies of pentafulvenes with a range of functional groups and pentafulvene stability between pH 6-9 over 48 h. In addition, we have also reported the stability of 7-alkylidene norborn-2-ene cycloadducts in aqueous solutions without any evidence for hydrolysis or retro-Diels Alder reactions over at least 24 h. 24 The broadening applications of the pentafulvene-maleimide ligation has been demonstrated for the preparation of hydrogels for cell encapsulation 25,26 and for generating mass spectrometry-cleavable labels (mass tags) for mass spectrometric detection of biomolecules. 27 To further investigate the potential of the pentafulvene-maleimide ligation for different bioconjugation applications, we herein report the stability of the pentafulvene 1 and maleimide 2 reactant partners as a model system in different buffer systems and the rate, efficiency and stereoselectivity of their cycloaddition reaction (Scheme 2).…”