The Nitrile Bis-Thiol Bioconjugation Reaction.

Electron-poor aryl nitriles are promising reagents for bioconjugation due to their high electrophilicity and selectivity for reaction with thiols, albeit generally in a reversible manner. A transient species has previously been observed in such reactions, involving the addition of two thiols to the nitrile functional group, forming a tetrahedral amino dithioacetal (ADTA). In this work, the reaction of heteroaryl nitriles with bis-thiols is explored in an attempt to generate stable ADTAs, which could facilitate new bioconjugation protocols. By use of a 1,2-dithiol, or the incorporation of an electrophilic trap into the aryl nitrile design, the formation of stable products is achieved. The resultant "nitrile bis-thiol" (NBT) reaction is then explored in the context of protein modification, specifically to carry out antibody conjugation. By addition of these nitriles to the reduced disulfide bond of an antibody fragment, it is shown that, depending on the reagent design, cysteine-to-lysine transfer or disulfide bridged NBT products can be generated. Both represent site-selective conjugates and are shown to be stable when challenged with glutathione under physiological conditions and upon incubation in serum. Furthermore, the NBT reaction is tested in the more challenging context of a full antibody, and all four disulfide bonds are effectively modified by these new one-carbon bridging reagents. Overall, this reaction of heteroaryl-nitriles with bis-thiols is shown to be highly efficient and versatile, of tunable reversibility, and offers enticing prospects as a new addition to the toolbox of biocompatible "click"-type reactions.

Insights into E.†coli Cyclopropane Fatty Acid Synthase (CFAS) Towards Enantioselective Carbene Free Biocatalytic Cyclopropanation.

Cyclopropane fatty acid synthases (CFAS) are a class of S-adenosylmethionine (SAM) dependent methyltransferase enzymes able to catalyse the cyclopropanation of unsaturated phospholipids. Since CFAS enzymes employ SAM as a methylene source to cyclopropanate alkene substrates, they have the potential to be mild and more sustainable biocatalysts for cyclopropanation transformations than current carbene-based approaches. This work describes the characterisation of E. coli CFAS (ecCFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids. ecCFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 with up to 58 % conversion and 73 % ee and the absolute configuration (9S,10R) was established. Substrate tolerance of ecCFAS was found to be correlated with the electronic properties of phospholipid headgroups and for the first time ecCFAS was found to catalyse cyclopropanation of both phospholipid chains to form dicyclopropanated products. In addition, mutagenesis and in silico experiments were carried out to identify the enzyme residues with key roles in catalysis and to provide structural insights into the lipid substrate preference of ecCFAS. Finally, the biocatalytic synthesis of methyl dihydrosterculate 1 and its deuterated analogue was also accomplished combining recombinant ecCFAS with the SAM regenerating AtHMT enzyme in the presence of CH3I and CD3I respectively.

Simultaneous measurement of free and conjugated estrogens in surface water using capillary liquid chromatography tandem mass spectrometry.

Given detrimental impacts induced by estrogens at trace level, determination of them is significant but challenging due to their low content in environmental samples and inherent weak ionisation. A modified derivatisation-based methodology was applied for the first time to detect estrogen in free and conjugated forms including some isomers simultaneously using liquid chromatography tandem mass spectrometry (LC-MSn). Derivatisation reaction with previously used 1,2-dimethyl-1H-imidazole-5-sulphonyl chloride allowed significant increase of mass spectrometric signal of analytes and also provided distinctive fragmentation for their confirmation even in complicated matrix. Then satisfactory recovery (>75%) for the majority of analytes was achieved following optimisation of solid phase extraction (SPE) factors. The linearity was validated over a wide concentration with the correlation coefficient around 0.995. The repeatability of this methodology was also confirmed via the intra-day and inter-day precision and was less than 11.73%. Validation of method quantification limits (MQLs) for all chosen estrogens was conducted using 1000 mL surface water, ranging from 7.0 to 132.3 pg L-1. The established methodology was applied to profile presence of targeted estrogens in natural surface water samples. Out of the ten compounds of interest, three free estrogens (E1, E2, E3) and two sulphate estrogens (E1-3S and E2-3S) were found over their MQLs, being in the range of 0.05-0.32 ng L-1.

Correction: Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis.

Correction for 'Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis' by Maurício Morais et al., Org. Biomol. Chem., 2017, 15, 2947-2952, DOI: .

Probing cytochrome P450 (CYP) bioactivation with chloromethylindoline bioprecursors derived from the duocarmycin family of compounds.

The duocarmycins belong to a class of agent which has great potential for use in cancer therapy. Their exquisite potency means they are too toxic for systemic use, and targeted approaches are required to unlock their clinical potential. In this study, we have explored seco-OH-chloromethylindoline (CI) duocarmycin-based bioprecursors for their potential for cytochrome P450 (CYP)-mediated cancer cell kill. We report on synthetic and biological explorations of racemic seco-CI-MI, where MI is a 5-methoxy indole motif, and dehydroxylated analogues. We show up to a 10-fold bioactivation of de-OH CI-MI and a fluoro bioprecursor analogue in CYP1A1-transfected cells. Using CYP bactosomes, we also demonstrate that CYP1A2 but not CYP1B1 or CYP3A4 has propensity for potentiating these compounds, indicating preference for CYP1A bioactivation.

Quantitative Proteomic Analysis of the Central Amygdala in Neuropathic Pain Model Rats.

Pain and emotional distress have a reciprocal relation. The amygdala has been implicated in emotional processing. The central nucleus of the amygdala (CeA) receives nociceptive information from the dorsal horn of spinal cord and is responsible for the central plasticity in chronic pain. Neuropathic pain is a type of severe chronic pain and can be strongly influenced by emotional components. Plastic changes in the CeA may play a key role in the development or maintenance or both of neuropathic pain. We studied the expression levels of proteins in the CeA of spinal nerve transection (SNT) model rats. Total tissue lysate proteins were separated by two-dimensional-gel electrophoresis (2D-PAGE). Gels from different time points were compared using Progenesis SameSpot software, and the spots with Fold Change greater than 2 were excised for protein identification by mass spectrometry. We identified more than 50 cytosolic proteins as significantly altered in their expression levels in the CeA of SNT rats, and most of these changes have been validated at mRNA levels by qRT-PCR. We also identified more than 40 membrane proteins as notably up- or down-regulated in the CeA of SNT model rats relative to a control using stable isotope dimethyl labeling nano-LC-MS/MS based proteomics and found that one such protein, doublecortin (DCX), a microtubule-associated protein expressed by neuronal precursor cells during development, is specifically localized in the membrane fraction without changes in total amount of the protein. Immunohistochemistry showed that doublecortin is expressed in processes in the CeA of rats 7 and 21 days after SNT surgery, suggesting that doublecortin is one of the proteins that may contribute to the plastic changes, namely, redevelopment or rewiring of neural networks, in the CeA in the neuropathic pain model. These dysregulated proteins may play roles in reciprocal relationships between pain and psychological distress in the amygdala and contribute to central sensitization. Data are available via ProteomeXchange with identifier PXD017473.

Optimised approach to albumin-drug conjugates using monobromomaleimide-C-2 linkers.

Conjugation of therapeutics to human serum albumin (HSA) using bromomaleimides represents a promising platform for half-life extension. We show here that the Cys-34 crevice substantially reduces the rate of serum stabilising maleimide hydrolysis in these conjugates, necessitating reagent optimisation. This improved reagent design is applied to the construction of an HSA-paclitaxel conjugate, preventing drug loss during maleimide hydrolysis.

Effect of Flavin-Containing Monooxygenase Genotype, Mouse Strain, and Gender on Trimethylamine N-oxide Production, Plasma Cholesterol Concentration, and an Index of Atherosclerosis.

The objectives of the study were to determine the contribution, in mice, of members of the flavin-containing monooxygenase (FMO) family to the production of trimethylamine (TMA) N-oxide (TMAO), a potential proatherogenic molecule, and whether under normal dietary conditions differences in TMAO production were associated with changes in plasma cholesterol concentration or with an index of atherosclerosis (Als). Concentrations of urinary TMA and TMAO and plasma cholesterol were measured in 10-week-old male and female C57BL/6J and CD-1 mice and in mouse lines deficient in various Fmo genes (Fmo1-/- , 2-/- , 4-/- , and Fmo5-/- ). In female mice most TMA N-oxygenation was catalyzed by FMO3, but in both genders 11%-12% of TMA was converted to TMAO by FMO1. Gender-, Fmo genotype-, and strain-related differences in TMAO production were accompanied by opposite effects on plasma cholesterol concentration. Plasma cholesterol was negatively, but weakly, correlated with TMAO production and urinary TMAO concentration. Fmo genotype had no effect on Als. There was no correlation between Als and either TMAO production or urinary TMAO concentration. Our results indicate that under normal dietary conditions TMAO does not increase plasma cholesterol or act as a proatherogenic molecule.

Optimisation of the dibromomaleimide (DBM) platform for native antibody conjugation by accelerated post-conjugation hydrolysis.

Disulfide bridging offers a convenient approach to generate site-selective antibody conjugates from native antibodies. To optimise the reagents available to achieve this strategy, we describe here the use of dibromomaleimides designed to undergo accelerated post-conjugation hydrolysis. Conjugation and hydrolysis, which serve to 'lock' the conjugates as robustly stable maleamic acids, is achieved in just over 1 h. This dramatic acceleration is also shown to infer significant improvements in homogeneity, as demonstrated by mass spectrometry analysis.

The structure of tagetitoxin.

Based on detailed analysis of newly acquired NMR data, we show that the previously revised structure of tagetitoxin is incorrect. A new structure of tagetitoxin is proposed which is consistent with the NMR and MS data.

Analysis of transferred fragrance and its forensic implications.

Perfumes are widely used by many people in developed countries, and a large number of both men and women wear perfumes on a daily basis. Analysis of perfume trace materials from clothing is not commonly employed within forensic casework, yet as a form of trace evidence it has the potential to provide valuable intelligence. In order to appreciate the value of trace evidence there is a fundamental need for an evidence base that can both offer insight into how a trace material behaves under different scenarios and activities, and from which inferences can be made. With this purpose a gas chromatography-mass spectrometry method for trace analysis of perfumes was developed. This paper presents two different series of experiments that investigate the dynamics of perfume transfer as a factor of perfume ageing time, and as a factor of perfume contact time. Empirical data showed that both perfume ageing time, and perfume contact time play a key role in the number of perfume components transferred. These studies have implication for forensic protocols, specifically for perfume trace evidence collection, analysis, interpretation, and presentation, and there is potentially great value in analysing perfumes from clothing exhibits in forensic enquiries that involve close contact between individuals, such as sexual assaults.

Brain endogenous liver X receptor ligands selectively promote midbrain neurogenesis.

Liver X receptors (Lxrα and Lxrß) are ligand-dependent nuclear receptors critical for ventral midbrain neurogenesis in vivo. However, no endogenous midbrain Lxr ligand has so far been identified. Here we used LC/MS and functional assays to identify cholic acid as a new Lxr ligand. Moreover, 24(S),25-epoxycholesterol (24,25-EC) was found to be the most potent and abundant Lxr ligand in the developing mouse midbrain. Both Lxr ligands promoted neural development in an Lxr-dependent manner in zebrafish in vivo. Notably, each ligand selectively regulated the development of distinct midbrain neuronal populations. Whereas cholic acid increased survival and neurogenesis of Brn3a-positive red nucleus neurons, 24,25-EC promoted dopaminergic neurogenesis. These results identify an entirely new class of highly selective and cell type-specific regulators of neurogenesis and neuronal survival. Moreover, 24,25-EC promoted dopaminergic differentiation of embryonic stem cells, suggesting that Lxr ligands may thus contribute to the development of cell replacement and regenerative therapies for Parkinson's disease.

Identification of a receptor for neuropeptide VGF and its role in neuropathic pain.

VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca(2+) levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca(2+) levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.

24S,25-Epoxycholesterol in mouse and rat brain.

24S,25-Epoxycholesterol is formed in a shunt of the mevalonate pathway that produces cholesterol. It is one of the most potent known activators of the liver X receptors and can inhibit sterol regulatory element-binding protein processing. Until recently analysis of 24S,25-epoxycholesterol at high sensitivity has been precluded by its thermal lability and lack of a strong chromophore. Here we report on the analysis of 24S,25-epoxycholesterol in rodent brain where its level was determined to be of the order of 0.4-1.4µg/g wet weight in both adult mouse and rat. For comparison the level of 24S-hydroxycholesterol in brain of both rodents was of the order of 20µg/g, while that of cholesterol in mouse was 10-20mg/g. By exploiting knockout mice for the enzyme oxysterol 7α-hydroxylase (Cyp7b1) we show that this enzymes is important for the subsequent metabolism of the 24S,25-epoxide.

Two-substrate enzyme engineering using small libraries that combine the substrate preferences from two different variant lineages.

Improving the range of substrates accepted by enzymes with high catalytic activity remains an important goal for the industrialisation of biocatalysis. Many enzymes catalyse two-substrate reactions which increases the complexity in engineering them for the synthesis of alternative products. Often mutations are found independently that can improve the acceptance of alternatives to each of the two substrates. Ideally, we would be able to combine mutations identified for each of the two alternative substrates, and so reprogramme new enzyme variants that synthesise specific products from their respective two-substrate combinations. However, as we have previously observed for E. coli transketolase, the mutations that improved activity towards aromatic acceptor aldehydes, did not successfully recombine with mutations that switched the donor substrate to pyruvate. This likely results from several active site residues having multiple roles that can affect both of the substrates, as well as structural interactions between the mutations themselves. Here, we have designed small libraries, including both natural and non-natural amino acids, based on the previous mutational sites that impact on acceptance of the two substrates, to achieve up to 630× increases in kcat for the reaction with 3-formylbenzoic acid (3-FBA) and pyruvate. Computational docking was able to determine how the mutations shaped the active site to improve the proximity of the 3-FBA substrate relative to the enamine-TPP intermediate, formed after the initial reaction with pyruvate. This work opens the way for small libraries to rapidly reprogramme enzyme active sites in a plug and play approach to catalyse new combinations of two-substrate reactions.

Antibiotic Residues in UK Foods: Exploring the Exposure Pathways and Associated Health Risks.

While the use of antibiotics has been reported as extensive in the rearing of agricultural animals, insufficient information is available on the antibiotic residues in animal products and the adverse impact that consistent low-level exposure to antibiotics might have on the human body and its microbiome. The aim of this study was to estimate the antibiotic concentrations that humans are exposed to via their diet using the concentration of antibiotics in animal food products and water and an online survey on dietary intake. A total of 131 participants completed the dietary intake survey, with the majority belonging to the omnivorous diet group (76.3%). Distinct dietary trends were observed in the omnivorous and unknown groups eating animal products, with specific food types dominating each meal: pork (e.g., ham) and dairy products (e.g., milk, yoghurt) during breakfast, beef (e.g., burgers) and chicken (e.g., chicken breast) products during lunch, and fish (e.g., salmon fillet) during dinner. In total, 34 different animal-based food and drink products were tested for the presence of ten different antibiotics. Of all the products tested, over 35% exceeded the acceptable daily antibiotic intake for amoxicillin, ampicillin, and enrofloxacin.

Phytosterols in human serum as measured using a liquid chromatography tandem mass spectrometry.

Phytosterols are lipophilic compounds found in plants with structural similarity to mammalian cholesterol. They cannot be endogenously produced by mammals and therefore always originate from diet. There has been increased interest in dietary phytosterols over the last few decades due to their association with a variety of beneficial health effects including low-density lipoprotein cholesterol lowering, anti-inflammatory and anti-cancerous effects. They are proposed as potential moderators for diseases associated with the central nervous system where cholesterol homeostasis is found to be imperative (multiple sclerosis, dementia, etc.) due to their ability to reach the brain. Here we utilised an enzyme-assisted derivatisation for sterol analysis (EADSA) in combination with a liquid chromatography tandem mass spectrometry (LC-MSn) to characterise phytosterol content in human serum. As little as 100 fg of plant sterol was injected on a reversed phase LC column. The method allows semi-quantitative measurements of phytosterols and their derivatives simultaneously with measurement of cholesterol metabolites. The identification of phytosterols in human serum was based on comparison of their LC retention times and MS2, MS3 spectra with a library of authentic standards. Free campesterol serum concentration was in the range from 0.30-4.10 µg/mL, ß-sitosterol 0.16-3.37 µg/mL and fucosterol was at lowest concentration range from 0.05-0.38 µg/mL in ten individuals. This analytical methodology could be applied to the analysis of other biological fluids and tissues.

Antibiotic resistance genes and the association with bacterial community in biofilms occurring during the drinking water granular activated carbon (GAC) sandwich biofiltration.

The granular activated carbon (GAC) sandwich modification to slow sand filtration could be considered as a promising technology for improved drinking water quality. Biofilms developed on sand and GAC surfaces are expected to show a functional diversity during the biofiltration. Bench-scale GAC sandwich biofilters were set-up and run continuously with and without antibiotic exposure. Surface sand (the schmutzdecke) and GAC biofilms were sampled and subject to high-throughput qPCR for antibiotic resistance gene (ARG) analysis and 16 S rRNA amplicon sequencing. Similar diversity of ARG profile was found in both types of biofilms, suggesting that all ARG categories decreased in richness along the filter bed. In general, surface sand biofilm remained the most active layer with regards to the richness and abundance of ARGs, where GAC biofilms showed slightly lower ARG risks. Network analysis suggested that 10 taxonomic genera were implicated as possible ARG hosts, among which Nitrospira, Methyloversatilis and Methylotenera showed the highest correlation. Overall, this study was the first attempt to consider the whole structure of the GAC sandwich biofilter and results from this study could help to further understand the persistence of ARGs and their association with the microbial community in drinking water biofiltration system.

Antibacterial Properties of Honey Nanocomposite Fibrous Meshes.

Natural substances are increasingly being developed for use in health-related applications. Honey has attracted significant interest, not only for its physical and chemical properties, but also for its antibacterial activity. For the first time, suspensions of Black Forest honeydew honey and manuka honey UMF 20+ were examined for their antibacterial properties against Escherichia coli and Staphylococcus epidermidis using flow cytometry. The inhibitory effect of honey on bacterial growth was evident at concentrations of 10, 20 and 30 v/v%. The minimum inhibitory effects of both honey types against each bacterium were also investigated and reported. Electrospray ionisation (ESI) mass spectrometry was performed on both Black Forest honeydew honey and manuka honey UMF 20+. Manuka honey had a gluconic concentration of 2519 mg/kg, whilst Black Forest honeydew honey had a concentration of 2195 mg/kg. Manuka honey demonstrated the strongest potency when compared to Black Forest honeydew honey; therefore, it was incorporated into nanofiber scaffolds using pressurised gyration and 10, 20 and 30 v/v% manuka honey-polycaprolactone solutions. Composite fibres were analysed for their morphology and topography using scanning electron microscopy. The average fibre diameter of the manuka honey-polycaprolactone scaffolds was found to range from 437 to 815 nm. The antibacterial activity of the 30 v/v% scaffolds was studied using S. epidermidis. Strong antibacterial activity was observed with a bacterial reduction rate of over 90%. The results show that honey composite fibres formed using pressurised gyration can be considered a natural therapeutic agent for various medicinal purposes, including wound-healing applications.

Unearthing the unique stability of thiophosphonium-C-terminal cysteine adducts on peptides and proteins.

Herein we report a fundamental discovery on the use of tris(dialkylamino)phosphine reagents for peptide and protein modification. We discovered that C-terminal thiophosphonium species, which are uniquely stable, could be selectively and rapidly generated from their disulfide counterparts. In sharp and direct contrast, internal thiophosphonium species rapidly degrade to dehydroalanine. We demonstrate this remarkable chemoselectivity on a bis-cysteine model peptide, and the formation of a stable C-terminal-thiophosphonium adduct on an antibody fragment, as well as characterise the species in various small molecule/peptide studies.