Dual reductive/oxidative electrochemistry/liquid chromatography/mass spectrometry: Towards peptide and protein modification, separation and identification.

A new hyphenated technique based on on-line dual (oxidative and reductive) electrochemistry coupled to liquid chromatography and high resolution electrospray mass spectrometry is presented. Two liquid streams are combined, with one containing a disulfide, which is reduced to the respective thiol in an electrochemical cell based on a titanium working electrode. The other stream contains phenol, which is electrochemically activated to benzoquinone on a boron-doped diamond working electrode. Upon combination of the two streams, a Michael addition takes places, leading to the covalent coupling of thiol to quinone. In continuous flow, the reaction mixture is transferred into an injection valve and the products are separated by reversed phase liquid chromatography and detected by electrospray-high resolution mass spectrometry. Proof of concept is demonstrated for low molecular mass disulfides and peptides, but further optimization will be required in future work to achieve efficient protein labelling.

Comparison of in†Silico, Electrochemical, in†Vitro and in†Vivo Metabolism of a Homologous Series of (Radio)fluorinated σ1 Receptor Ligands Designed for Positron Emission Tomography.

The imaging of σ1 receptors in the brain by fluorinated radiotracers will be used for the validation of σ1 receptors as drug targets as well as for differential diagnosis of diseases in the central nervous system. The biotransformation of four homologous fluorinated PET tracers 1'-benzyl-3-(ω-fluoromethyl to ω-fluorobutyl)-3H-spiro[2]benzofuran-1,4'-piperidine] ([18 F]1-4) was investigated. In silico studies using fast metabolizer (FAME) software, electrochemical oxidations, in vitro studies with rat liver microsomes, and in vivo metabolism studies after application of the PET tracers [18 F]1-4 to mice were performed. Combined liquid chromatography and mass spectrometry (HPLC-MS) analysis allowed structural identification of non-radioactive metabolites. Radio-HPLC and radio-TLC provided information about the presence of unchanged parent radiotracers and their radiometabolites. Radiometabolites were not found in the brain after application of [18 F]2-4, but liver, plasma, and urine samples contained several radiometabolites. Less than 2 % of the injected dose of [18 F]4 reached the brain, rendering [18 F]4 less appropriate as a PET tracer than [18 F]2 and [18 F]3. Compounds [18 F]2 and [18 F]3 possess the most promising properties for imaging of σ1 receptors in the brain. High σ1 affinity (Ki =0.59 nm), low lipophilicity (logD7.4 =2.57), high brain penetration (4.6 % of injected dose after 30 min), and the absence of radiometabolites in the brain favor the fluoroethyl derivative [18 F]2 slightly over the fluoropropyl derivative [18 F]3 for human use.

Differential protein labeling based on electrochemically generated reactive intermediates.

A specific labeling method for cysteine moieties in proteins was developed. Electrochemical oxidation of phenolic compounds such as phenol or acetaminophen leads to the generation of the reactive intermediates benzoquinone and N-acetyl-p-benzoquinone imine, which can subsequently react with nucleophilic thiol functions in peptides or proteins. Differential labeling of cysteine residues was successfully demonstrated with native as well as heavy-isotope labeled forms of the corresponding labeling compounds. The specific mass differences on the peptide level were successfully analyzed by mass spectrometry for the tripeptide glutathione. Free cysteines in various proteins such as ß-lactoglobulin A, human serum albumin, hemoglobin, and human carbonic anhydrase I were successfully labeled. Tryptic digestion of differentially labeled carbonic anhydrase I and hemoglobin allowed the identification of the binding site in the proteins. The obtained mass difference allowed an easy identification of the cysteine containing peptides. With these experiments, it was successfully demonstrated that the developed method can serve as a tool for counting cysteine moieties in proteins and, thus, be used as an additional technique in protein identification experiments.

Mass spectrometric detection of short-lived drug metabolites generated in an electrochemical microfluidic chip.

The costs of drug development have been rising exponentially over the last six decades, making it essential to select drug candidates in the early drug discovery phases before proceeding to expensive clinical trials. Here, we present novel screening methods using an electrochemical chip coupled online to mass spectrometry (MS) or liquid chromatography (LC) and MS, to generate phase I and phase II drug metabolites and to demonstrate protein modification by reactive metabolites. The short transit time (∼4.5 s) between electrochemical oxidation and mass spectrometric detection, enabled by an integrated electrospray emitter, allows us to detect a short-lived radical metabolite of chlorpromazine which is too unstable to be detected using established test routines. In addition, a fast way to screen candidate drugs is established by recording real-time mass voltammograms, which allows one to identify the drug metabolites that are expected to be formed upon oxidation by applying a linear potential sweep and simultaneously detect oxidation products. Furthermore, detoxification of electrochemically generated reactive metabolites of paracetamol was mimicked by their adduct formation with the antioxidant glutathione. Finally, the potential toxicity of reactive metabolites can be investigated by the modification of proteins, which was demonstrated by modification of carbonic anhydrase I with electrochemically generated reactive metabolites of paracetamol. With this series of experiments, we demonstrate the potential of this electrochemical chip as a complementary tool for a variety of drug metabolism studies in the early stages of drug discovery.