Synthesis and Evaluation of Ubiquitin–Dioxetane Conjugate as a Chemiluminescent Probe for Monitoring Deubiquitinase Activity

Abstract

The removal of ubiquitin (Ub) from a modified protein or Ub chain is a process that occurs regularly by the ubiquitin–proteasome system. This process is known to be mediated by various deubiquitinating enzymes (DUBs) in order to control the protein’s half-life and its expression levels among many other signaling processes. Since the function of DUBs is also involved in numerous human diseases, such as cancer, there is an obvious need for an effective diagnostic probe that can monitor the activity of these enzymes. We have developed the first chemiluminescence probe for detection of DUBs activity. The probe was prepared by conjugation of the chemically synthesized C-terminally activated Ub(1-75) with a Gly-enolether precursor. Subsequent oxidation, under aqueous conditions, of the enolether conjuagate with singlet-oxygen furnished the dioxetane probe Ub-CL . This synthesis provides the first example of a dioxetane–luminophore protein conjugate. The probe’s ability to detect deubiquitinating activity was successfully validated with three different DUBs. In order to demonstrate the advantage of our new probe, comparison measurements for detection of DUB UCH-L3 activity were performed between the chemiluminescent probe Ub-CL and the well-known Ub-AMC probe. The obtained data showed significantly higher S/N, for probe Ub-CL (>93-fold) in comparison to that observed for Ub-AMC (1.5-fold). We anticipate that the successful design and synthesis of the turn-ON protein–dioxetane conjugate probe, demonstrated in this work, will provide the insight and motivation for preparation of other relevant protein–dioxetane conjugates.

Recent advances in self-immolative linkers and their applications in polymeric reporting systems

“…For example, Brik and Shabat reported that attaching the C-terminal sequence of ubiquitin protein (Ub(1–75)) to the aniline nitrogen through a glycine residue ( 85a ) allows an assay for deubiquitinase activity to be developed. 127 Since deubiquitinating enzymes are involved in various human diseases, monitoring of their activity is valuable. Probe 85a was found to be effective at detecting activity from three different deubiquitinases by cleavage of the scissile bond: UCH-L3, UCH-L1 and USP-2, with the first two giving the stronger response.…”

Synthesis and Chemiexcitation of a Distinct Chemiluminescent Luminophore based on a Curcumin Scaffold

“… [6] Importantly, this groundbreaking development enabled the use of chemiluminescent probes as a sole component with no required additives. Numerous research groups worldwide, including ours, took advantage of the ortho ‐substituted phenoxy‐adamantyl‐1,2‐dioxetane luminophore to develop new useful chemiluminescent probes for detection and imaging of a variety of enzymes and analytes in living systems [7–21] …”

“…Numerous research groups worldwide, including ours, took advantage of the ortho-substituted phenoxy-adamantyl-1,2-dioxetane luminophore to develop new useful chemiluminescent probes for detection and imaging of a variety of enzymes and analytes in living systems. [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] Despite the encouraging progress in the field, further advancement has been limited by the synthetic inaccessibility of new dioxetane compounds. Typical synthetic methods for the preparation of phenoxy-dioxetane compounds are laborious and cannot be performed on substrates with unprotected functional groups.…”

Dual Chemiexcitation by a Unique Dioxetane Scaffold Gated by an OR Logic Set of Triggers

“… [6] Importantly, this game‐changer development enabled the use of chemiluminescent probes as a sole component with no required additives. Numerous research groups worldwide, including ours, took advantage of the ortho ‐substituted phenoxy‐adamantyl‐1,2‐dioxetane luminophore to develop useful chemiluminescent probes for detection and imaging of a variety of enzymes and analytes in living systems [7–27] …”