No abstract

Despite the contribution of changes in pancreatic β-cell mass to the development of all forms of diabetes mellitus, few robust approaches currently exist to monitor these changes prospectively in vivo. Although magnetic-resonance imaging (MRI) provides a potentially useful technique, targeting MRI-active probes to the β cell has proved challenging. Zinc ions are highly concentrated in the secretory granule, but they are relatively less abundant in the exocrine pancreas and in other tissues. We have therefore developed functional dual-modal probes based on transition-metal chelates capable of binding zinc. The first of these, Gd⋅1, binds ZnII directly by means of an amidoquinoline moiety (AQA), thus causing a large ratiometric Stokes shift in the fluorescence from λem=410 to 500 nm with an increase in relaxivity from r1=4.2 up to 4.9 mM−1 s−1. The probe is efficiently accumulated into secretory granules in β-cell-derived lines and isolated islets, but more poorly by non-endocrine cells, and leads to a reduction in T1 in human islets. In vivo murine studies of Gd⋅1 have shown accumulation of the probe in the pancreas with increased signal intensity over 140 minutes.

Activity-based protein profiling (ABPP) research is directed towards the development of tools and techniques that report on enzyme activity in complex biological samples.[1-4] With the aid of activity-based probes (ABPs)-small molecules designed to react specifically, covalently, and irreversibly with the active site residues of an enzyme or enzyme family-enzymatic activity levels are detected, rather than the protein expression levels that are measured by means of conventional proteomics techniques. A typical ABP consists of three parts: 1) a "warhead", the reactive group that binds covalently and irreversibly to the enzyme active site, 2) a recognition element targeting the ABP to a certain enzyme (family), and 3) an affinity tag or a fluorophore for visualization and/or enrichment purposes. In most ABPs that report on enzyme activity, the reporter group is directly attached to the probe, with obvious advantages with respect to experimental design. Incorporation of, for instance, a biotin or large fluorophore in an ABP, however, might have a detrimental effect either on bioavailability (cell permeability) or on enzyme reactivity of the probe, or on both. With the aim of alleviating these problems, the two-step labeling approach is an important alternative in ABPP. We and Cravatt and co-workers simultaneously reported that this approach is also versatile in the profiling of enzyme families: namely the proteasome and serine hydrolases, respectively.[5,6] In two-step ABPP approaches a small biocompatible reactive group, normally an azide or an acetylene, is introduced into an ABP. After covalent modification of a target protein (family), a reporter group is introduced in a chemoselective manner, by means either of Staudinger-Bertozzi ligation[6-8] or of Huisgen [2+3] cycloaddition (the "click reaction", of which both copper(I)-catalyzed[5,9-13] and copper-free[14,15] versions exist). Key to the success of such two-step ABPP experiments are the selectivity (in terms of cross-reactivity towards endogenous functional groups in a biological sample) and efficiency (in terms of chemical yields with which the azide-or acetylene-modified proteins are converted) of the chemoselective ligation step by which the reporter group is attached to the modified proteins. There are several reports on the selectivity of both Staudinger-Bertozzi and click ligations.[11,14] Here we describe a compatible set of one-step and two-step proteasome

A series of gadolinium complexes were synthesised in order to test the design of dual-modal probes that display a change in fluorescence or relaxivity response upon binding of zinc. A dansyl-DO3ATA gadolinium complex [GdL1] displayed an increase and a slight blue-shift in fluorescence in the presence of zinc; however, a decrease in relaxation rate was observed. Consequently, the ability of the well-known zinc chelator, BPEN, was assessed for relaxivity response when conjugated to the gadolinium chelate. The success of this probe [GdL2], lead to the inclusion of the same zinc-probing moiety alongside a longer wavelength emitting fluorophore, rhodamine [GdL3], to arrive at the final iteration of these first generation dual-modal zinc-sensing probes. The compounds give insight into the design protocols required for the successful imaging of zinc ions.