Dramatic increase of quench efficiency in “spacerless” dimaleimide fluorogens

Abstract

In this post-genomic era, new techniques are needed to cope with the task of assigning functional roles to the huge number of identified putative gene products. We have developed a minimalist labelling strategy based on the use of synthetic fluorogenic probe reagents that fluoresce only after their reaction with a target peptide sequence. The probe reagents have fluorescent cores and bear two maleimide groups, such that their latent fluorescence is quenched by a photoinduced electron transfer (PET) to the pendant maleimide groups, until both of these groups undergo a specific thiol addition reaction. The efficiency of the fluorescence quenching is critical to the practicality of this labelling method, and has been predicted to be related to the intramolecular distance between the fluorophore and the maleimide groups. We have conducted the first direct test of this hypothesis by preparing a series of novel fluorogens that differ only by the spacer moiety separating their coumarin fluorophore and their dimaleimide fragment. A striking correlation was observed between intramolecular distance and the fluorescence enhancement (FE) observed after reaction with two equivalents of thiol. Guided by this observation, we then designed 'spacerless' fluorogens, of which a dansyl derivative shows an FE ratio of >300, the largest recorded for dimaleimide fluorogens. The trends observed herein provide valuable lessons for subsequent fluorogen design, and the novel fluorogens developed in the course of this study are currently being applied to protein labelling applications.

DBD Dyes as Fluorescence Lifetime Probes to Study Conformational Changes in Proteins

“…We first investigated the spectroscopic properties of 17 in detail. It is known that maleimides often quench the luminescence of chromophores,23, 31, 32 and as compound 17 shows almost no fluorescence at all, it is indicated that such quenching is observed here. The fluorescence intensity and lifetime of 17 increase strongly upon reaction of the double bond of the maleimide with a thiol (e.g., cysteine) to form the corresponding thioether 18 .…”

Evaluation of a 7‐Methoxycoumarin‐3‐carboxylic Acid Ester Derivative as a Fluorescent, Cell‐Cleavable, Phosphonate Protecting Group

“…[16] This carboxylic acid was treated with base and chloromethyl chlorosulfate according to the procedure of Graham et al [17] to obtain the chloromethyl ester 6 . To determine if this coumarin derivative would be cleaved within a cell to release a phosphoantigen, we prepared a derivative of the phosphonate 7 , which we have found does not itself stimulate Vγ9Vδ2 T-cells.…”

Tris(triazolo)benzene and Its Derivatives: High‐Density Energetic Materials

“…The structures of the tris(triazolo)benzene and its derivatives are supported by IR, 1 H, 13 C NMR and, in some cases, 15 N NMR spectroscopic data as well as elemental analysis (or high-resolution mass spectrometry, HRMS). The 13 C NMR spectra of 8 showed only a single peak at 132 ppm in [D 6 ]DMSO (dimethylsulfoxide), which suggests that the hydrogen is shifting from the N1 to the N3 nitrogen atom of the triazole rings of tris(triazolo)benzene.…”

“…When the potassium salt of tris(triazolo)benzene was treated with oxone, the trihydroxy derivative (15) was not produced (Scheme 4). When the potassium salt of tris(triazolo)benzene was treated with oxone, the trihydroxy derivative (15) was not produced (Scheme 4).…”