Pd(II)-Mediated C-H Activation for Cysteine Bioconjugation.

Selective bioconjugation remains a significant challenge for the synthetic chemist due to the stringent reaction conditions required by biomolecules coupled with their high degree of functionality. The current trailblazer of transition-metal mediated bioconjugation chemistry involves the use of Pd(II) complexes prepared via an oxidative addition process. Herein, the preparation of Pd(II) complexes for cysteine bioconjugation via a facile C-H activation process is reported. These complexes show bioconjugation efficiency competitive with what is seen in the current literature, with a user-friendly synthesis, common Pd(II) sources, and a more cost-effective ligand. Furthermore, these complexes need not be isolated, and still achieve high conversion efficiency and selectivity of a model peptide. These complexes also demonstrate the ability to selectively arylate a single surface cysteine residue on a model protein substrate, further demonstrating their utility.

Deriving Heterospecific Self-Assembling Protein-Protein Interactions Using a Computational Interactome Screen.

Interactions between naturally occurring proteins are highly specific, with protein-network imbalances associated with numerous diseases. For designed protein-protein interactions (PPIs), required specificity can be notoriously difficult to engineer. To accelerate this process, we have derived peptides that form heterospecific PPIs when combined. This is achieved using software that generates large virtual libraries of peptide sequences and searches within the resulting interactome for preferentially interacting peptides. To demonstrate feasibility, we have (i) generated 1536 peptide sequences based on the parallel dimeric coiled-coil motif and varied residues known to be important for stability and specificity, (ii) screened the 1,180,416 member interactome for predicted Tm values and (iii) used predicted Tm cutoff points to isolate eight peptides that form four heterospecific PPIs when combined. This required that all 32 hypothetical off-target interactions within the eight-peptide interactome be disfavoured and that the four desired interactions pair correctly. Lastly, we have verified the approach by characterising all 36 pairs within the interactome. In analysing the output, we hypothesised that several sequences are capable of adopting antiparallel orientations. We subsequently improved the software by removing sequences where doing so led to fully complementary electrostatic pairings. Our approach can be used to derive increasingly large and therefore complex sets of heterospecific PPIs with a wide range of potential downstream applications from disease modulation to the design of biomaterials and peptides in synthetic biology.

Feedback-amplified electrochemical dual-plate boron-doped diamond microtrench detector for flow injection analysis.

An electrochemical flow cell with a boron-doped diamond dual-plate microtrench electrode has been developed and demonstrated for hydroquinone flow injection electroanalysis in phosphate buffer pH 7. Using the electrochemical generator-collector feedback detector improves the sensitivity by one order of magnitude (when compared to a single working electrode detector). The diffusion process is switched from an analyte consuming "external" process to an analyte regenerating "internal" process with benefits in selectivity and sensitivity.

Signal transduction and amplification through enzyme-triggered ligand release and accelerated catalysis.

Signal transduction and signal amplification are both important mechanisms used within biological signalling pathways. Inspired by this process, we have developed a signal amplification methodology that utilises the selectivity and high activity of enzymes in combination with the robustness and generality of an organometallic catalyst, achieving a hybrid biological and synthetic catalyst cascade. A proligand enzyme substrate was designed to selectively self-immolate in the presence of the enzyme to release a ligand that can bind to a metal pre-catalyst and accelerate the rate of a transfer hydrogenation reaction. Enzyme-triggered catalytic signal amplification was then applied to a range of catalyst substrates demonstrating that signal amplification and signal transduction can both be achieved through this methodology.

Cavity transport effects in generator-collector electrochemical analysis of nitrobenzene.

Two types of generator-collector electrode systems, (i) a gold-gold interdigitated microband array and (ii) a gold-gold dual-plate microtrench, are compared for nitrobenzene electroanalysis in aerated aqueous 0.1 M NaOH. The complexity of the nitrobenzene reduction in conjunction with the presence of ambient levels of oxygen in the analysis solution provide a challenging problem in which feedback-amplified generator-collector steady state currents provide the analytical signal. In contrast to the more openly accessible geometry of the interdigitated array electrode, where the voltammetric response for nitrobenzene is less well-defined and signals drift, the voltammetric response for the cavity-like microtrench electrode is stable and readily detectable at 1 µM level. Both types of electrode show oxygen-enhanced low concentration collector current responses due to additional feedback via reaction intermediates. The observations are rationalised in terms of a "cavity transport coefficient" which is beneficial in the dual-plate microtrench, where oxygen interference effects are suppressed and the analytical signal is amplified and stabilised.

Reproducible product-ion tandem mass spectra on various liquid chromatography/mass spectrometry instruments for the development of spectral libraries.

The results of the comparison of product-ion tandem mass (MS/MS) spectra recorded on three ion trap mass spectrometers, a triple quadrupole mass spectrometer and a Fourier transform ion cyclotron resonance mass spectrometer are reported. The spectra were recorded in accordance with a simple experimental protocol, which involved the collision-induced dissociation (CID) attenuation of the abundance of the [M+H]+ ion to between 10 and 50% of its original abundance. The degree of similarity between the spectra from four of the mass spectrometers was calculated off-line by comparing the five most abundant ions from the spectrum on each instrument. A percentage fit value (20% for each ion that matches) was calculated by comparing each spectrum against the spectra recorded for the same compound on each instrument. The percentage of the inter-library pairwise comparisons (total = 434) that matched to > or = 60% ranged from 64-89%, depending on the instrument pair. A blind trial was also undertaken using five unknown compounds resulting in 1670 pairwise comparisons with the library entries. The blind trial produced no false positives and correct identifications in all cases. The results of the study have established the basis for the construction of a transferable product-ion MS/MS library.