Nanosecond Time‐Resolved Fluorescence Protease Assays

Abstract

Arguably, fluorescence-based assays are on the rise and, stepby-step, will replace alternative radioactive or absorptionbased assays on a laboratory microscale as well in highthroughput screening (HTS) for the pharmaceutical industry. [1][2][3][4][5][6] One of the more recent developments is the use of time-resolved fluorescence (TRF) assays, in which a long fluorescence lifetime is utilized to distinguish the probe fluorescence from any other short-lived background emission. [7][8][9][10] Another independent trend is the use of fluorescent peptide substrates, which are based on intramolecular probe/quencher contact, [11][12][13][14] rather than on quenching by conventional fluorescence resonance energy transfer (FRET). [15][16][17][18] Herein, we introduce an assay type that combines the assets of time-resolved fluorescence detection with the advantages of collision-induced quenching and the use of a highly biocompatible fluorophore:We have previously introduced DBO derivatives as an unconventional class of fluorescent probes; they have been termed fluorazophores.[19] The most remarkable feature is their exceedingly long fluorescence lifetime (up to 1 ms, l max = 430 nm, f f = 0.26), [20] which we have previously exploited for mechanistic investigations in the area of supramolecular and biomolecular chemistry, namely for the determination of the association kinetics of water-soluble host molecules, [21][22][23][24][25] the diffusional properties of antioxidants in membrane models, [26][27][28] and the kinetics of oligonucleotide and polypeptide folding. [29][30][31] This study describes an implementation of these basic research efforts for fluorescence-based assays with immediate biochemical relevance for studies on enzymatic activity and drug discovery.TRF assays have become popular for lanthanide emitters. Their fluorescence, which should be more generally referred to as luminescence due to the nature of the electronic transitions involved, has lifetimes in the ms range that can be easily detected, given the presence of suitable enhancement agents or chelation, and differentiated from background emission. [7][8][9][10] This background is ubiquitous and originates from unconverted substrate, scattered excitation light, sample container materials, enzyme cofactors, impurities, and, most relevant for inhibitor screening and HTS, library compounds to be screened for biological activity. [5] When employing genuine fluorescence of organic chromophores, fluorescence lifetimes above 100 ns and time gates in the nanosecond range are required to allow the same measurement mode, which we refer to as Nano-TRF. Resting on the possibility of laser excitation, highly sensitive detection, and electronic time gating, the pertinent lifetime regime is sufficiently long to allow an accurate discrimination from unconverted substrate as well as background. DBO presents the first organic fluorophore to be utilized for Nano-TRF assays, and DBO-labeled asparagine ("Dbo"), which was previously synthesized in Fmoc-protected form, [29] c...

Temperature‐dependent loop formation kinetics in flexible peptides studied by time‐resolved fluorescence spectroscopy

“…Dbo is an attractive chromophore for biological studies due to its hydrophilicity, its small size, and its versatility in peptide synthesis. In fact, as an important practical application of our peptide flexibility studies, we have recently introduced a novel line of biological assays [25], which exploit the intramolecular quenching and concomitant lifetime shortening of Dbo in conformationally flexible Trp-labeled peptides (Scheme 3). Enzymatic cleavage removes this quenching pathway and results in the recovery of the exceedingly long fluorescence lifetime of Dbo (Scheme 3), which can be detected very accurately by employing the technique of nanosecond time-resolved fluorescence (Nano-TRF).…”

Noncovalent Chirality Sensing Ensembles for the Detection and Reaction Monitoring of Amino Acids, Peptides, Proteins, and Aromatic Drugs

“…As expected, its cleavage into phenol and galactose was accompanied by a decrease in the ICD signal, as the emerging receptor⋅phenol complex is achiral (see Figure S7 in the Supporting Information). Although BSA gives no ICD effect (see above), the addition of the protease pepsin destroys its compact tertiary structure such that the aromatic residues become accessible to the receptor. This structural and chemical change can be continuously monitored through emerging ICD effects with the reporter pair (see Figure S8 in the Supporting Information). Since the ICD patterns of peptides are sequence‐selective, a new line of label‐free enzyme assays can be set up, which remain in high demand, for example, for exopeptidases such as carboxypeptidase A 19. This enzyme cleaves C‐terminal Phe residues preferentially, as could be directly monitored when the digestion of the model substrate hippuryl‐Phe was monitored by the ICD assay (Figure 5 b).…”

“…Since the ICD patterns of peptides are sequence‐selective, a new line of label‐free enzyme assays can be set up, which remain in high demand, for example, for exopeptidases such as carboxypeptidase A 19. This enzyme cleaves C‐terminal Phe residues preferentially, as could be directly monitored when the digestion of the model substrate hippuryl‐Phe was monitored by the ICD assay (Figure 5 b).…”

Bridgehead carboxy-substituted 2,3-diazabicyclo[2.2.2]oct-2-enes: synthesis, fluorescent properties, and host-guest complexation

“…12,13 Derivatives of 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO, 1) have recently become valuable structural and dynamic probes 14 as well as sensors for supramolecular [15][16][17][18][19][20][21][22][23] and biomolecular assemblies. [23][24][25][26][27][28][29][30][31][32][33][34][35] Until now, the derivatives with 1-hydroxymethyl (2) [23][24][25][26] and 1-aminomethyl groups (3) 30 have been employed as key precursors for subsequent derivatization, e.g., for the introduction into polypeptides, [30][31][32][33][34][35] polynucleotides, 24 or biomembranes. [26][27][28][29] Herein, we describe the DBO carboxylic acid 4 and the DBO acetic acid 5.…”