Electrochemical detection methods for biologically-active molecules

Pulsed Amperometric Detection (PAD) has proven to be applicable to the determination of a number of organic compounds. One difficulty has been calibration of PAD for quantitative analysis over a wide concentration range. By pairing PAD in series with Conductivity Detection (CD), a much wider linear calibration range was found for carbohydrates and amino acids. Both compound groups were separated via anion-exchange chromatography and detected at Au electrodes;In an attempt to decrease amino acid detection limits, phenylthiohydantoin and methylthiohydantoin derivatives were examined. PAD was found to be more sensitive to these derivatives than the free amino acids. DC amperometry was also applicable to the thiohydantoin derivatives, with no instability or sensitivity loss with time observed. Detection limits as low as two picomoles were determined. Employment of a commercially available C-18 column allowed separation of nearly all amino acids using gradient elution liquid chromatography;Improvement in detection of underivatized amino acids was made by applying Pulsed Coulometric Detection (PCD) and Indirect Coulometric Adsorption Detection (ICAD). Both could be used following separation on anion-exchange columns. PCD was coupled with a glass reference electrode to allow anion-exchange separation of amino acids using gradient elution, and baseline perturbation during the gradient was minimal. Up to 20 amino acids were separated in less than one hour

Investigating the trafficking of Golgi-resident glycosylation enzymes

The Golgi is a major site of glycan processing mediated by a large number of Golgi-resident glycosylation enzymes. These enzymes are largely type II transmembrane domain (TMD) proteins consisting of a short N-terminal cytosolic tail, a short TMD and a lumenal ‘stem’ region which separates the catalytic domain and the lipid bilayer. The cytosolic tail, TMD, and stem together make the CTS domain which is responsible for the Golgi-targeting of these enzymes via multiple COPI-dependent mechanisms. What is not clear is the relative contribution of each mechanism to Golgi retention or how they work to fine tune the sub-Golgi localisation of these enzymes. To explore these nuances of intra-Golgi traffic, a MitoID proteomic screen was applied to the intra-Golgi golgin tethers GMAP-210 and golgin-84 to identify novel intra-Golgi vesicle-resident factors. The screen identified the Rab2 effector of unknown function FAM114A2. FAM114A2 and paralogous protein FAM114A1 were shown to specifically associate with early-Golgi membranes. A predicted ALPS motif was necessary for membrane association, presumably through non-specifically absorbing onto highly-curved membranes. Other regions of the proteins were required to mediate a specific association with membranes of the early-Golgi. GST affinity chromatography and mass spectrometry revealed that FAM114A1 pulled down Rab2A and Rab2B while FAM114A2 pulled down several COPI cargo proteins including many glycosylation enzymes. FAM114A2 interacted directly with the cytoplasmic tails of cargo which had a membrane-proximal polybasic motif. Similar binding studies revealed that the COPI adaptors GOLPH3+3L pulled down the COPI coat and over a hundred novel clients. GOLPH3+3L were shown to interact promiscuously with the cytoplasmic tails of clients with a total predicted charge of ≥4+. An in vivo Golgi retention assay revealed that tails which satisfy the GOLPH3+3L charge threshold are sufficient to impart Golgi-retention in a GOLPH3+3L-dependent manner. The deletion of FAM114A genes had no impact on retention in this assay. The assay also showed that the TMD of ST6GAL1 was sufficient to impart Golgi retention in a GOLPH3+3L-independent manner. The deletion of GOLPH3+3L, but not FAM114A genes, triggered the lysosomal degradation of COPI cargo and caused a global defect in glycosylation in U2OS cells. In contrast, deletion of the D. melanogaster FAM114A ortholog CG9590 caused a defect in mucin-type glycosylation in adult flies. CG9590 interacted directly with the COPI coat and pulled-down a host of COPI cargo proteins including mucin-type glycosylation enzymes. It is likely that FAM114A is an evolutionary-conserved COPI adaptor which acts to recycle a specific subset of COPI cargo from the early-Golgi. In contrast, GOLPH3 proteins are promiscuous COPI adaptors which function as master gatekeepers at the late-Golgi to recycle and retain a broad array of Golgi-residents

Metal-Free Dihydrogen Oxidation by a Borenium Cation:A Combined Electrochemical/Frustrated Lewis Pair Approach

In order to use H2 as a clean source of electricity, prohibitively rare and expensive precious metal electrocatalysts, such as Pt, are often used to overcome the large oxidative voltage required to convert H2 into 2 H+ and 2 e−. Herein, we report a metal-free approach to catalyze the oxidation of H2 by combining the ability of frustrated Lewis pairs (FLPs) to heterolytically cleave H2 with the in situ electrochemical oxidation of the resulting borohydride. The use of the NHC-stabilized borenium cation [(IiPr2)(BC8H14)]+ (IiPr2=C3H2(NiPr)2, NHC=N-heterocyclic carbene) as the Lewis acidic component of the FLP is shown to decrease the voltage required for H2 oxidation by 910 mV at inexpensive carbon electrodes, a significant energy saving equivalent to 175.6 kJ mol−1. The NHC–borenium Lewis acid also offers improved catalyst recyclability and chemical stability compared to B(C6F5)3, the paradigm Lewis acid originally used to pioneer our combined electrochemical/frustrated Lewis pair approach

H2 activation using the first 1:1:1 hetero-tri(aryl)borane

The novel 1:1:1 hetero-tri(aryl)borane (pentafluorophenyl){3,5-bis(trifluoromethyl)phenyl}(pentachlorophenyl)borane has been synthesised and structurally characterised. This has been show to act as the Lewis acidic component in FLPs for the heterolytic cleavage of H2 with three Lewis bases

A combined "electrochemical-frustrated Lewis pair" approach to hydrogen activation: surface catalytic effects at platinum electrodes

Herein, we extend our “combined electrochemical–frustrated Lewis pair” approach to include Pt electrode surfaces for the first time. We found that the voltammetric response of an electrochemical–frustrated Lewis pair (FLP) system involving the B(C6F5)3/[HB(C6F5)3]− redox couple exhibits a strong surface electrocatalytic effect at Pt electrodes. Using a combination of kinetic competition studies in the presence of a H atom scavenger, 6-bromohexene, and by changing the steric bulk of the Lewis acid borane catalyst from B(C6F5)3 to B(C6Cl5)3, the mechanism of electrochemical–FLP reactions on Pt surfaces was shown to be dominated by hydrogen-atom transfer (HAT) between Pt, [Pt[BOND]H] adatoms and transient [HB(C6F5)3]⋅ electrooxidation intermediates. These findings provide further insight into this new area of combining electrochemical and FLP reactions, and proffers additional avenues for exploration beyond energy generation, such as in electrosynthesis

Mode Combinations and International Operations: Theoretical Issues and an Empirical Investigation

An enduring characteristic of extant literature on foreign operation modes is its discrete choice approach, where companies are assumed to choose one among a small number of distinctive alternatives. • In this paper, detailed information about the operations of six Norwegian companies in three key markets (China, UK and USA) is used as the basis for an exploration of the extent to which, and how and why, companies combine clearly different foreign operation modes. We examine their use of foreign operation mode combinations within given value activities as well as within given countries. • The study reveals that companies tend to combine modes of operation; thereby producing unique foreign operation mode “packages” for given activities and/or countries, and that the packages are liable to be modified over time – providing a potentially important optional path for international expansion. • The data show considerable variation across cases; ranging from extensive use of mode combinations to a singular focus on a specific mode of operation. The study contributes to a refinement of our understanding of the path of internationalisation, and throws up a number of awkward theoretical questions about the process