A tool for the selective sequestration of ATP and PP_i to aid in-solution phosphopeptide detection assays

Abstract

The presence of small phospho-anions, such as PPi and ATP in protein samples often complicates the robust detection of phosphoproteins by metal-based chemosensors and receptors. We herein report the development of a bis(Zn(2+)-cyclen)-triethylbenzene scaffold which can selectively sequester PPi and ATP without affecting the detection of a di-phosphorylated peptide by a ProxyPhos chemosensor.

Steric Effects on the Binding of Phosphate and Polyphosphate Anions by Zinc(II) and Copper(II) Dinuclear Complexes of m-Xylyl-bis-cyclen

“…A quantitative [HPPi 3– ] determination can be easily performed using a ratiometric assay with the absorbances at 397 and 557 nm of the [Zn 2 bmce (Ind)] entity in MOPS at pH 7.4 upon calibration (a [HPPi 3– ] range of (1.8–7.0) × 10 –5 M was used; see Figure S11). The limit of detection found (LOD = 2.5 nM) in this experiment is, to the best of our knowledge, the lowest value already obtained in the visible region of the spectrum by IDA in pure water. , Additionally, a dinuclear complex of m -xylyl-bis-cyclen was used for the first time in this work for the detection of pyrophosphate in pure aqueous solution. − , Very recently, dicopper­(II) complexes of p -xylyl-bis-cyclam (Mozobil) and of dizinc­(II) complexes of bis-cyclen with a triethylbenzene scaffold and methyl-indole-carboxamide linkers were used as receptors for pyrophosphate and for both pyrophosphate and ATP, respectively, but in both studies mixed solvents were used (namely, MeOH/water and 50 mM 4-(2-hydroxyethyl)-1-piperazine­ethanesulfonic acid (HEPES) with 5% dimethyl sulfoxide (DMSO)). In spite of the several studies of the interaction and detection of pyrophosphate by colorimetric techniques, only a few were investigated at physiological conditions. ,− …”

“…26,27 Additionally, a dinuclear complex of m-xylyl-bis-cyclen was used for the first time in this work for the detection of pyrophosphate in pure aqueous solution. [9][10][11][12]21 Very recently, dicopper(II) complexes of p-xylylbis-cyclam (Mozobil) 35 and of dizinc(II) complexes of bis-cyclen with a triethylbenzene scaffold and methyl-indole-carboxamide linkers 36 were used as receptors for pyrophosphate and for both pyrophosphate and ATP, respectively, but in both studies mixed solvents were used (namely, MeOH/water and 50 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) with 5% dimethyl sulfoxide (DMSO)). In spite of the several studies of the interaction and detection of pyrophosphate by colorimetric techniques, only a few were investigated at physiological conditions.…”

“…The chemosensing ensemble strategy was used to develop indicator displacement assays (IDA) for the detection of different anions of interest in aqueous media. − The sensing of HPPi 3– is of great interest given the relevance of this anion in several biological processes and diseases . Furthermore, while a large number of studies reporting HPPi 3– sensing was published, only a few of them were actually performed using physiological conditions. − Moreover, the use of dinuclear complexes derived from nonrigid bis-macrocyclic structures as receptors for HPPi 3– has very few precedents in the literature and was performed in mixed solvents media. , …”

“…26−34 Moreover, the use of dinuclear complexes derived from nonrigid bis-macrocyclic structures as receptors for HPPi 3− has very few precedents in the literature and was performed in mixed solvents media. 35,36 Given the promising behavior of dinuclear metal complexes in the recognition of phosphates, we sought to study the impact that small backbone modifications on the spacer of bis-cyclen compounds has on the complexation properties and in the uptake of anions. With this in mind, a new member of the mxylyl-bis-cyclen family with a 1,3,5-triethylbenzene as spacer (tbmce, Chart 1) was synthesized and compared with the already known receptor bmce.…”

Optical chemosensors for the detection of proximally phosphorylated peptides and proteins

“…Chemosensor 8 was employed alongside a sequestering receptor to improve sensitivity for proximal phosphates over common physiological off-targets, including ATP and pyrophosphate (PPi). 70 The sequestering receptor was designed as a triethylbenzene scaffold functionalized with three indoles, where two of the three indoles were appended with Zn( ii )–cyclen receptors. The cavity induced by the indoles would allow for the binding of low molecular weight phosphoanions such as ATP and PPi but prevent the binding of larger phosphopeptides or proteins.…”