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Accurate determination of relapses in multiple sclerosis is important for diagnosis, classification of clinical course and therapeutic decision making. The identification of biofluid markers for multiple sclerosis relapses would add to our current diagnostic armamentarium and increase our understanding of the biology underlying the clinical expression of inflammation in multiple sclerosis. However, there is presently no biofluid marker capable of objectively determining multiple sclerosis relapses although some, in particular neurofilament-light chain, have shown promise. In this study, we sought to determine if metabolic perturbations are present during multiple sclerosis relapses, and, if so, identify candidate metabolite biomarkers and evaluate their discriminatory abilities at both group and individual levels, in comparison with neurofilament-light chain. High-resolution global and targeted 1H nuclear magnetic resonance metabolomics as well as neurofilament-light chain measurements were performed on the serum in four groups of relapsing-remitting multiple sclerosis patients, stratified by time since relapse onset: (i) in relapse (R); (ii) last relapse (LR) ≥ 1 month (M) to < 6 M ago; (iii) LR ≥ 6 M to < 24 M ago; and (iv) LR ≥ 24 M ago. Two hundred and one relapsing-remitting multiple sclerosis patients were recruited: R (n = 38), LR 1–6 M (n = 28), LR 6–24 M (n = 34), LR ≥ 24 M (n = 101). Using supervised multivariate analysis, we found that the global metabolomics profile of R patients was significantly perturbed compared to LR ≥ 24 M patients. Identified discriminatory metabolites were then quantified using targeted metabolomics. Lysine and asparagine (higher in R), as well as, isoleucine and leucine (lower in R), were shortlisted as potential metabolite biomarkers. ANOVA of these metabolites revealed significant differences across the four patient groups, with a clear trend with time since relapse onset. Multivariable receiver operating characteristics analysis of these four metabolites in discriminating R versus LR ≥ 24 M showed an area under the curve of 0.758, while the area under the curve for serum neurofilament-light chain was 0.575. Within individual patients with paired relapse–remission samples, all four metabolites were significantly different in relapse versus remission, with the direction of change consistent with that observed at group level, while neurofilament-light chain was not discriminatory. The perturbations in the identified metabolites point towards energy deficiency and immune activation in multiple sclerosis relapses, and the measurement of these metabolites, either singly or in combination, are useful as biomarkers to differentiate relapse from remission at both group and individual levels., Yeo et al. used a metabolomics approach to identify serum metabolite biomarkers for relapses in multiple sclerosis, at both group and individual levels. Four identified metabolites, used individually or in combination, significantly outperformed serum neurofilament-light chain for differentiating relapse from non-relapse., Graphical Abstract Graphical Abstract

The transition from relapsing–remitting multiple sclerosis (RRMS) to secondary progressive MS (SPMS) represents a huge clinical challenge. We previously demonstrated that serum metabolomics could distinguish RRMS from SPMS with high diagnostic accuracy. As differing sample-handling protocols can affect the blood metabolite profile, it is vital to understand which factors may influence the accuracy of this metabolomics-based test in a clinical setting. Herein, we aim to further validate the high accuracy of this metabolomics test and to determine if this is maintained in a ‘real-life’ clinical environment. Blood from 31 RRMS and 28 SPMS patients was subjected to different sample-handling protocols representing variations encountered in clinics. The effect of freeze–thaw cycles (0 or 1) and time to erythrocyte removal (30, 120, or 240 min) on the accuracy of the test was investigated. For test development, samples from the optimised protocol (30 min standing time, 0 freeze–thaw) were used, resulting in high diagnostic accuracy (mean ± SD, 91.0 ± 3.0%). This test remained able to discriminate RRMS and SPMS samples that had experienced additional freeze–thaw, and increased standing times of 120 and 240 min with accuracies ranging from 85.5 to 88.0%, because the top discriminatory metabolite biomarkers from the optimised protocol remained discriminatory between RRMS and SPMS despite these sample-handling variations. In conclusion, while strict sample-handling is essential for the development of metabolomics-based blood tests, the results confirmed that the RRMS vs. SPMS test is resistant to sample-handling variations and can distinguish these two MS stages in the clinics.

The principles employed in parallel NMR and MRI are applied to NMR supersequences yielding as many as ten 2D NMR spectra in one measurement. We present a number of examples where two NOAH (NMR by Ordered Acquisition using 1H-detection) supersequences are recorded in parallel, thus dramatically increasing the information content obtained in a single NMR experiment. The two parallel supersequences entangled by time-sharing schemes (IPAP-seHSQC, HSQC-COSY, and HSQC-TOCSY) incorporate also modified (sequential and/or interleaved) conventional pulse schemes (modules), including HMBC, TOCSY, COSY, CLIP-COSY, NOESY, and ROESY. Such parallel supersequences can be tailored for specific applications, for instance, the analysis and characterization of molecular structure of complex organic molecules from a single measurement. In particular, the CASPER software was used to establish the structure of a tetrasaccharide, β-LNnTOMe, with a high degree of confidence from a single measurement involving a parallel NOAH-5 supersequence.

Objectives: The toxicity of chemotherapeutic anticancer drugs is a serious issue in clinics. Drug discovery from edible and medicinal plants represents a promising approach towards finding safer anticancer therapeutics. Justicia insularis T. Anderson (Acanthaceae) is an edible and medicinal plant in Nigeria. This study aims to discover cytotoxic compounds from this rarely explored J. insularis and investigate their underlying mechanism of action. Methods: The cytotoxicity of the plant extract was evaluated in human ovarian cancer cell lines and normal human ovarian surface epithelia (HOE) cells using a sulforhodamine B assay. Bioassay-guided isolation was carried out using column chromatography including HPLC, and the isolated natural products were characterized using GC-MS, LC-HRMS, and 1D/2D NMR techniques. Induction of apoptosis was evaluated using Caspase 3/7, 8, and 9, and Annexin V and PI based flow cytometry assays. SwissADME and SwissTargetPrediction web tools were used to predict the molecular properties and possible protein targets of identified active compounds. Key finding: The two cytotoxic compounds were identified as clerodane diterpenoids: 16(α/β)-hydroxy-cleroda-3,13(14)Z-dien-15,16-olide (1) and 16-oxo-cleroda-3,13(14)E-dien-15-oic acid (2) from the Acanthaceous plant for the first time. Compound 1 was a very abundant compound (0.7% per dry weight of plant material) and was shown to be more potent than compound 2 with IC50 values in the micromolar range against OVCAR-4 and OVCAR-8 cancer cells. Compounds 1 and 2 were less cytotoxic to HOE cell line. Both compounds induced apoptosis by increasing caspase 3/7 activities in a concentration dependent manner. Compound 1 further increased caspase 8 and 9 activities and apoptosis cell populations. Compounds 1 and 2 are both drug like, and compound 1 may target various proteins including a kinase. Conclusions: Clerodane diterpenoids (1 and 2) in J. insularis were identified as cytotoxic to ovarian cancer cells via the induction of apoptosis, providing an abundant and valuable source of hit compounds for the treatment of ovarian cancer.

Complementary XFEL methods reveal O2 reaction intermediates and global protein dynamics during Isopenicillin N synthase catalysis., Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.

Methylated flavones, commonly found in many plants of the Brassicaceae family, have potent antioxidant and anticancer activity with diverse therapeutic potential. However, the specific regioisomers of methylated flavones can have significantly different biochemical and potentially therapeutic properties as shown by various bioassays but analytically differentiating these compounds has been technically challenging and rarely reported. In this study, we demonstrate differentiation and identification of selected bioactive methylated flavone regioisomers, namely 5,7,3′-trihydroxy-4′-methoxyflavone, and 5,7,4′-trihydroxy-3′-methoxyflavone extracted from Coronopus didymus, a member of the Brassicaceae family, using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MS/MS). Characteristic MS/MS product ions produced from neutral loss of carbon monoxide, and a methyl radical from the [M-H]– ion, exhibited differential relative abundances attributed to different structural stabilities under the same activation and collision-induced dissociation conditions. MS/MS also provided structural information which was sufficient to differentiate the methylated regioisomers and determine the position of the methyl group based on interpretation of their respective fragmentation patterns. Quantification showed 5,7,4′-trihydroxy-3′-methoxyflavone was at least 1.60 mg per 10 g plant material in C. didymus extracts. This study demonstrates a straightforward and novel approach to rapidly differentiate, identify and quantify regio-isomeric methylated flavone natural products using reversed-phase UPLC-MS/MS.

Nuclear magnetic resonance (NMR) spectroscopy is a principal analytical technique used for the structure elucidation of molecules. This Primer covers different approaches to accelerate data acquisition and increase sensitivity of NMR measurements through parallelization, enabled by hardware design and/or pulse sequence development. Starting with hardware-based methods, we discuss coupling multiple detectors to multiple samples so each detector/sample combination provides unique information. We then cover spatio-temporal encoding, which uses magnetic field gradients and frequency-selective manipulations to parallelize multidimensional acquisition and compress it into a single shot. We also consider the parallel manipulation of different magnetization reservoirs within a sample to yield new, information-rich pulse schemes using either homonuclear or multinuclear detection. The Experimentation section describes the set-up of parallel NMR techniques. Practical examples revealing improvements in speed and sensitivity offered by the parallel methods are demonstrated in Results. Examples of use of parallelization in small-molecule analysis are discussed in Applications, with experimental constraints addressed under the Limitations and optimizations and Reproducibility and data deposition sections. The most promising future developments are considered in the Outlook, where the largest gains are expected to emerge once the discussed techniques are combined. This Primer on parallel nuclear magnetic resonance describes various parallelization techniques ranging from hardware design to pulse sequence development that allow concurrent acquisition of different signals and lead to an increase in both data acquisition speeds and sensitivity when elucidating molecular structures.

Understanding how catalytic asymmetric reactions with racemic starting materials can operate would enable new enantioselective cross-coupling reactions that give chiral products. Here we propose a catalytic cycle for the highly enantioselective Rh(i)-catalysed Suzuki–Miyaura coupling of boronic acids and racemic allyl halides. Natural abundance 13C kinetic isotope effects provide quantitative information about the transition-state structures of two key elementary steps in the catalytic cycle, transmetallation and oxidative addition. Experiments with configurationally stable, deuterium-labelled substrates revealed that oxidative addition can happen via syn- or anti-pathways, which control diastereoselectivity. Density functional theory calculations attribute the extremely high enantioselectivity to reductive elimination from a common Rh complex formed from both allyl halide enantiomers. Our conclusions are supported by analysis of the reaction kinetics. These insights into the sequence of bond-forming steps and their transition-state structures will contribute to our understanding of asymmetric Rh–allyl chemistry and enable the discovery and application of asymmetric reactions with racemic substrates. A major drive in current chemistry research is to develop asymmetric versions of widely used carbon–carbon bond-forming reactions, such as Suzuki-Miyaura cross-couplings. Now, the origins of diastereo- and enantioselectivity in a Rh-catalysed cross-coupling of boronic acid and racemic allyl halides have been established.

Tropical forests constitute a prolific sanctuary of unique floral diversity and potential medicinal sources, however, many of them remain unexplored. The scarcity of rigorous scientific data on the surviving Mascarene endemic taxa renders bioprospecting of this untapped resource of utmost importance. Thus, in view of valorizing the native resource, this study has as its objective to investigate the bioactivities of endemic leaf extracts. Herein, seven Mascarene endemic plants leaves were extracted and evaluated for their in vitro antioxidant properties and antiproliferative effects on a panel of cancer cell lines, using methyl thiazolyl diphenyl-tetrazolium bromide (MTT) and clonogenic cell survival assays. Flow cytometry and comet assay were used to investigate the cell cycle and DNA damaging effects, respectively. Bioassay guided-fractionation coupled with liquid chromatography mass spectrometry (MS), gas chromatography-MS, and nuclear magnetic resonance spectroscopic analysis were used to identify the bioactive compounds. Among the seven plants tested, Terminaliabentzo&euml, was comparatively the most potent antioxidant extract, with significantly (p &lt, 0.05) higher cytotoxic activities. T. bentzo&euml, extract further selectively suppressed the growth of human hepatocellular carcinoma cells and significantly halted the cell cycle progression in the G0/G1 phase, decreased the cells&rsquo, replicative potential and induced significant DNA damage. In total, 10 phenolic compounds, including punicalagin and ellagic acid, were identified and likely contributed to the extract&rsquo, s potent antioxidant and cytotoxic activities. These results established a promising basis for further in-depth investigations into the potential use of T. bentzo&euml, as a supportive therapy in cancer management.

5-(Ethylsulfonyl)-2-(naphthalen-2-yl)benzo[d]oxazole (ezutromid, 1) is a first-in-class utrophin modulator that has been evaluated in a phase 2 clinical study for the treatment of Duchenne muscular dystrophy (DMD). Ezutromid was found to undergo hepatic oxidation of its 2-naphthyl substituent to produce two regioisomeric 1,2-dihydronaphthalene-1,2-diols, DHD1 and DHD3, as the major metabolites after oral administration in humans and rodents. In many patients, plasma levels of the DHD metabolites were found to exceed those of ezutromid. Herein, we describe the structural elucidation of the main metabolites of ezutromid, the regio- and relative stereochemical assignments of DHD1 and DHD3, their de novo chemical synthesis, and their production in systems in vitro. We further elucidate the likely metabolic pathway and CYP isoforms responsible for DHD1 and DHD3 production and characterize their physicochemical, ADME, and pharmacological properties and their preliminary toxicological profiles.

A series of NMR supersequences are presented for the time-efficient structure characterisation of small molecules in the solution state. These triplet sequences provide HMBC, HSQC, and one homonuclear correlation experiment of choice according to the NMR by Ordered Acquisition using 1 H detection principle. The experiments are demonstrated to be compatible with non-uniform sampling schemes and may be acquired and processed under full automation.

In animals, the response to chronic hypoxia is mediated by upregulation of the α,β-heterodimeric hypoxia-inducible factors (HIFs). Levels of HIFα isoforms, but not HIFβ, are regulated by their post-translational modification as catalysed by prolyl hydroxylase domain enzymes (PHDs). Different roles for the human HIF-1/2α isoforms and their two oxygen-dependent degradation domains (ODDs) are proposed. We report kinetic and NMR analyses of the ODD selectivity of the catalytic domain of wild-type PHD2 (which is conserved in nearly all animals) and clinically observed variants. Studies using Ala scanning and "hybrid" ODD peptides imply that the relatively rigid conformation of the (hydroxylated) proline plays an important role in ODD binding. They also reveal differential roles in binding for the residues on the N- and C-terminal sides of the substrate proline. The overall results indicate how the PHDs achieve selectivity for HIFα ODDs and might be of use in identifying substrate-selective PHD inhibitors.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. Open access article Even though NMR has found countless applications in the field of small molecule characterization, there is no standard file for the NMR data relevant to structure characterization of small molecules. A file format is introduced to associate the NMR parameters extracted from 1D and 2D spectra of organic compounds to the assigned chemical structure. These NMR parameters, which we shall call NMReDATA, include chemical shift values, signal integrals, intensities, multiplicities, scalar coupling constants, lists of 2D correlations, relaxation times and diffusion rates. The file format is an extension of the existing SDF (Structure Data Format), which is compatible with the commonly used MOL format. The association of an NMReDATA file with the raw and spectral data from which it originates constitutes an NMR record. This format is easily readable by humans and computers and provides a simple and efficient way for disseminating results of structural chemistry investigations, automating the verification of published result, and for assisting the constitution of highly needed open-source structural databases.

Niemann-Pick type C1 (NPC1) disease is a rare autosomal recessive, neurodegenerative lysosomal storage disorder, which presents with a range of clinical phenotypes and hence diagnosis remains a challenge. In view of these difficulties, the search for a novel, NPC1-specific biomarker (or set of biomarkers) is a topic of much interest. Here we employed high-resolution 1H nuclear magnetic resonance spectroscopy coupled with advanced multivariate analysis techniques in order to explore and seek differences between blood plasma samples acquired from NPC1 (untreated and miglustat treated), heterozygote, and healthy control subjects. Using this approach, we were able to identify NPC1 disease with 91% accuracy confirming that there are significant differences in the NMR plasma metabolic profiles of NPC1 patients when compared to healthy controls. The discrimination between NPC1 (both miglustat treated and untreated) and healthy controls was dominated by lipoprotein triacylglycerol 1H NMR resonances and isoleucine. Heterozygote plasma samples displayed also increases in the intensities of selected lipoprotein triacylglycerol 1H NMR signals over those of healthy controls. The metabolites identified could represent useful biomarkers in the future and provide valuable insight in to the underlying pathology of NPC1 disease.

WaterLOGSY is a popular ligand-observed NMR technique to screen for protein-ligand interactions, yet when applied to measure dissociation constants (KD) through ligand titration, the results were found to be strongly dependent on sample conditions. Herein, we show that accurate KDs can be obtained by waterLOGSY with optimised experimental setup.

OBJECTIVES: Over 20% of cancer patients develop brain metastases. Current MRI diagnostic techniques only detect late stage metastases, since they rely on blood-brain-barrier permeability to allow contrast enhancement. Thus, new methods enabling earlier diagnosis are urgently needed. We have previously shown that it is possible to discriminate between different inflammatory lesions in the CNS in rats (1), as well as between different stages of multiple sclerosis in patients (2), through biofluid (blood/urine) metabolomics. We believe that this ability is due, at least in part, to alterations in CNS metabolism, which provoke a specific metabolic signature in the biofluids studied. It is known that tumour metabolism differs markedly from normal brain and that brain metabolism itself will be altered by tumour presence. On the basis of the above, therefore, we hypothesised that the presence of brain metastases could be detected, in vivo, through NMR analysis of biofluids. METHODS: Metastatic mammary carcinoma cells (murine 4T1-GFP) were injected into BALB/c mice, via intracerebral, intracardial or intravenous routes to induce differing cerebral and systemic tumour burdens. Urine was collected at days 0 and 10 from all animals, and at days 5, 21 and 35 in animals injected intracerebrally. Samples from naïve, day 0 and vehicle-injected mice served as combined control cohorts. Urine metabolite composition was analysed using 1H-NMR spectroscopy, and statistical pattern recognition and modelling was applied to identify spectral differences and to identify commonalities indicative of brain metastasis burden. A robust method using unknown samples was used to validate model predictions. RESULTS: Significant metabolic profile separations were found between control cohorts and animals with tumour burdens at all timepoints for the intracerebral 4T1-GFP metastasis model (q2 = 0.59, 0.70, 0.81 and 0.80 for days 5, 10, 21 and 35, respectively; q2 > 0.4 considered significant). Models became stronger, with higher sensitivity and specificity, as the timecourse progressed indicating a more severe tumour burden. Sensitivity and specificity for predicting a blinded testing set were 0.89 and 0.82, respectively, at day 5, but both rose to 1.00 at day 35. Key metabolites driving the separations were identified and quantified relative to control (Fig. 1). Significant separations were also found between control and day 10 animals for all 4T1-GFP injected mice irrespective of route (q2 = 0.70, 0.63 and 0.78 for intracerebral, intracardiac and intravenous routes, respectively). The metabolites underpinning separations in each case differed indicating differentiation between systemic and CNS metastatic burden, but with common patterns that suggest a “fingerprint” for brain metastasis. CONCLUSIONS: Our data indicate that animals with brain metastases can be identified using urinary metabolic profiles with NMR metabolomics, and differentiated from animals with a predominantly systemic metastasis burden. This approach may identify a set of biomarkers for the diagnosis of brain metastasis earlier than is currently possible, and may provide insight into metabolic disruption in brain metastasis.

The mechanism of Amifostine (WR-2721) mediated radioprotection is poorly understood. The effects of amifostine on human basal metabolism, mouse liver metabolism and on normal and tumor hepatic cells were studied. Indirect calorimetric canopy tests showed significant reductions in oxygen consumption and of carbon dioxide emission in cancer patients receiving amifostine. Glucose levels significantly decreased and lactate levels increased in patient venous blood. Although amifostine in vitro did not inhibit the activity of the prolyl-hydroxylase PHD2, experiments with mouse liver showed that on a short timescale WR-1065 induced expression of the Hypoxia Inducible Factor HIF1α, lactate dehydrogenase LDH5, glucose transporter GLUT2, phosphorylated pyruvate dehydrogenase pPDH and PDH-kinase. This effect was confirmed on normal mouse NCTC hepatocytes, but not on hepatoma cells. A sharp reduction of acetyl-CoA and ATP levels in NCTC cells indicated reduced mitochondrial usage of pyruvate. Transient changes of mitochondrial membrane potential and reactive oxygen species ROS production were evident. Amifostine selectively protects NCTC cells against radiation, whilst HepG2 neoplastic cells are sensitized. The radiation protection was correlates with HIF levels. These findings shed new light on the mechanism of amifostine cytoprotection and encourage clinical research with this agent for the treatment of primary and metastatic liver cancer.

A strategy has been established for the synthesis of peptidomimetics derived from unsaturated carbohydrates, and exemplified by the use of methyl 2,6-anhydro-7-azido-3,7-deoxy-4,5-O-isopropylidene-D-lyxo-hept-2-enonate 9 as a dipeptide ‘monomer’ which can be elaborated from either end. Selective reduction of 9 gives a protected pseudodipeptide ester suitable for use as an amino component, and saponification gives an azido acid suitable for use as a carboxyl component. The ‘dimer’ product of coupling these two components with TBTU can be similarly elaborated at either end to give a ‘trimer’, and a further cycle of selective reduction and coupling gave a ‘tetramer’, 17, a pseudo-octapeptide. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd.

1 H NMR (500 MHz) analyses reveal that in phosphate buffer, N-arylsulfonyl β-lactams such as ethyl N-(p-nitrophenylsulfonyl)-2-oxoazetidine-4- carboxylate and (4S)-ethyl 3-ethyl-N-(p-nitrophenylsulfonyl)-2- oxoazetidine-4-carboxylate undergo hydrolysis through (minimally) two mechanisms: via direct hydrolysis and via an unstable acyl phosphate intermediate. The acyl phosphate intermediates can be trapped using hydrazine.

the synthesis and 1H n.m.r. analysis of peptidomimetics of Leu-enkephalin containing bicyclic lactams is described.

The New Delhi metallo-β-lactamase (NDM-1) is involved in the emerging antibiotic resistance problem. Development of metallo-β-lactamases (MBLs) inhibitors has proven challenging, due to their conformational flexibility. Here we report site-selective labeling of NDM-1 with 1,1,1-trifluoro-3-bromo acetone (BFA), and its use to study binding events and conformational changes upon ligand-metal binding using (19) F NMR spectroscopy. The results demonstrate different modes of binding of known NDM-1 inhibitors, including L- and D-captopril by monitoring the changing chemical environment of the active-site loop of NDM-1. The method described will be applicable to other MBLs and more generally to monitoring ligand-induced conformational changes.

Asparagine-803 in the C-terminal transactivation domain of human hypoxia-inducible factor (HIF)-1 α-subunit is hydroxylated by factor inhibiting HIF-1 (FIH-1) under normoxic conditions causing abrogation of the HIF-1α/p300 interaction. NMR and other analyses of a hydroxylated HIF fragment produced in vitro demonstrate that hydroxylation occurs at the β-carbon of Asn-803 and imply production of the threo-isomer, in contrast with other known aspartic acid/asparagine hydroxylases that produce the erythro-isomer.

Cyclodextrins (CDs) have been widely used in host-guest molecular recognition because of their chiral and hydrophobic cavities. For example, β-cyclodextrin (βCD) lodged as a molecular adaptor in protein pores such as α-hemolysin (αHL) is used for stochastic sensing. Here, we have tuned the cavity and overall size of βCD by replacing a single oxygen atom in its ring skeleton by a disulfide unit in two different configurations to both expand our ability to detect analytes and understand the interactions of βCD with protein pores. The three-dimensional structures of the two stereoisomeric CDs have been determined by the combined application of NMR spectroscopy and molecular simulation and show distorted conformations as compared to natural βCD. The interactions of these synthetic βCD analogues with mutant αHL protein pores and guest molecules were studied by single-channel electrical recording. The dissociation rate constants for both disulfide CDs from the mutant pores show ∼1000-fold increase as compared to those of unaltered βCD, but are ∼10-fold lower than the dissociation rate constants for βCD from wild-type αHL. Both of the skeleton-modified CDs show altered selectivity toward guest molecules. Our approach expands the breadth in sensitivity and diversity of sensing with protein pores and suggests structural parameters useful for CD design, particularly in the creation of asymmetric cavities.

Chemists use template-directed synthesis to position molecular components so that they can be covalently linked into complex molecules that are not readily accessible by classical synthetic methods. But as larger structures are targeted, the synthesis of the templates themselves becomes challenging. O'Sullivan et al. now show that 'molecular Verniers', based on the principle of moire pattern formation, can solve this problem. Using a Vernier template with six binding sites and molecular building blocks with four porphyrins acting as binding sites, the authors create a 12-porphyrin nano-ring with a diameter of 4.7 nanometres. The ease and efficiency of this synthesis establishes Vernier templating as a powerful new strategy for producing large monodisperse macromolecules. Templates are widely used to arrange molecular components so they can be covalently linked into complex molecules that are not readily accessible by classical synthetic methods. But, as larger structures are targeted, the synthesis of the templates themselves becomes challenging. It is now shown that 'molecular Verniers' can solve this problem: using a template with six binding sites and molecular building blocks with four porphyrins acting as binding sites, a 12-porphyrin nano-ring with a diameter of 4.7 nm is created. The ease and efficiency of this synthesis establishes Vernier templating as a powerful new strategy for producing large monodisperse macromolecules. Templates are widely used to arrange molecular components so they can be covalently linked into complex molecules that are not readily accessible by classical synthetic methods1,2,3,4,5,6,7. Nature uses sophisticated templates such as the ribosome, whereas chemists use simple ions or small molecules. But as we tackle the synthesis of larger targets, we require larger templates—which themselves become synthetically challenging. Here we show that Vernier complexes can solve this problem: if the number of binding sites on the template, nT, is not a multiple of the number of binding sites on the molecular building blocks, nB, then small templates can direct the assembly of relatively large Vernier complexes where the number of binding sites in the product, nP, is the lowest common multiple of nB and nT (refs 8, 9). We illustrate the value of this concept for the covalent synthesis of challenging targets by using a simple six-site template to direct the synthesis of a 12-porphyrin nano-ring with a diameter of 4.7 nm, thus establishing Vernier templating as a powerful new strategy for the synthesis of large monodisperse macromolecules.

Section 1. NMR measurements. Mixtures of the E and Z-isomers of 3 and 3⊂α -CD were generated by irradiating solutions of each E-isomer in N 2 -saturated water (ca 1 mM) in 10 mm silica cuvettes at 340 and 347 nm respectively using monochromated light from a xenon lamp in a Jobin Yvon Ltd. FluoroMax-2 spectrofluorometer (5 nm excitation bandwidth). At this concentration it was necessary to continue irradiation for 2 days to obtain a substantial mole-fraction of the Z-isomer. After irradiation, samples were evaporated and made up in D 2 O for NMR analysis. The 1 H resonances of the E and Z-isomers are well resolved, as illustrated for the rotaxane in Figure S1. The E:Z

Intermolecular ligand exchange, which is slow on the NMR timescale, has been observed for lead(IV) carboxylate species containing acetate and benzoate ligands by the use of 207Pb NMR spectroscopy.

The efficient synthesis of an octameric furanose carbopeptoid, readily purified by chromatography in ethyl acetate:hexane (2:1), is reported. Extensive NMR studies suggest that two tetrameric 5-aminomethyltetrahydrofuran-2-carboxylates are prone to adopt solution conformations reminiscent of a repeating β-turn, a third tetramer and the corresponding octamer may tend towards a left-handed α-helix.

Dynamic duo: The reversible reaction of boronic acids with alcohols to form boronate esters, coupled to protein mass spectrometry analyses, was used to discover potent oxygenase inhibitors. This dynamic combinatorial mass spectrometry technique could potentially be applied to the identification of other protein inhibitors. Copyright © 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim.

The binding of aryl boronic acids to α-chymotrypsin and subtilisin has been studied by 11B NMR spectroscopy; evidence is provided for the direct spectroscopic observation of a tetrahedral enzyme/boromate complex.

The solid state and solution phase conformational preferences of a homologous series of β-peptides derived from (S,S)-2- aminocyclopentanecarboxylic acid (transpentacin) have been investigated using a variety of spectroscopic and crystallographic techniques. These studies indicate that the hexamer and pentamer persist as a 12-helix in both the solid state and solution phase. Although the conformational traits of a 12-helix are exhibited by oligomers with as few as three residues in the solid state, in solution the trimer exists as an equilibrium of many alternative conformers whilst the tetramer has been shown to predominantly exist in either a 12-helix or a turn-type conformation. © 2010 Elsevier Ltd. All rights reserved.

Methylation of 2,8-dimethyl-6H,12H-5,11-ethanodibenzo[b,f][1,5]-diazocine (ethano-Troger's base) with methyl iodide followed by ion metathesis and fluorination with N-fluoro-2,3,4,5,6-pentachloropyridinium triflate affords a new electrophilic N-F reagent, that is more reactive than Selectfluor. 2D (19)F-(15)N HMQC experiments provide (1)JNF coupling constants which are diagnostic for the N-F functional group.

Whereas trimers and tetramers of β- C - d -arabinofuranosyl carbohydrate amino acids adopt well defined conformations based around a repeating β-turn like structure, stabilised by (i, i-2) inter-residue hydrogen bonds, there is no indication of any secondary structure in the tetramer of the epimeric α- C - d -arabinofuranosyl carbohydrate amino acid, where the C-2 and C-5 substituents of the tetrahydrofuran ring are trans to each other.

The trisubstituted enolate- and C-C bond-forming capacities of engineered carboxymethylproline synthases CMPSs are coupled with the malonyl-CoA synthetase MatB to enable stereoselective preparation of 5- and 6-membered N-heterocycles functionalised with alkyl-substituted carboxymethyl side chains, starting from achiral alkyl-substituted malonic acids and L-amino acid semialdehydes. The results illustrate the biocatalytic utility of crotonases in tandem enzyme-catalysed reactions for stereoselective synthesis.

A synthesis of the title phosphinamine (PHENAP) is described, following previously established methodology, Resolution via the C,N-palladocycle derived from ( R )-N,N-dimethyl-α-methyl-1-naphthylamine led to two crystallographically defined complexes with the ( R , S )-form posessing a chelated ligand and the ( R , R )-form open with only the phosphine coordinated. Palladium allyl complexes of the ligand were strikingly different from those of the corresponding isoquinoline ligand QUINAP, and the differences could be rationalised by recourse to molecular models, with the additional fused arene ring of PHENAP playing a critical part in defining the coordination sphere through its steric pressure. A brief appraisal of Pd-catalysed allylic alkylation reactions was performed with the new ligand.

We present direct investigations of the conformational preferences of sugars with a positively charged substituent at their anomeric centre, C-1, which display in solution, a preference for an equatorial conformation - an apparent reversal of the normal anomeric effect. The investigations focus on the protonated monosaccharide, d-xylopyranosyl imidazolium in its α and β forms, first probed in a range of different solvents through NMR measurements and then in the gas phase, free of solvent or counterion interactions, through infrared multiphoton dissociation spectroscopy. The results, when compared and discussed in the light of density functional theory, ab initio and natural bond orbital calculations, expose the possible origins of the reversed conformational preference and provide a better understanding of the factors controlling conformational choice. This journal is © The Royal Society of Chemistry 2012.

25, 3R, 4S- and 25, 38, 4R-epoxy-L-prolines were synthesised from trans- 4-hydroxy-L-proline. Assignment of the stereochemical configurations of the epoxy prolines was achieved by n.O.e. studies and chemical correlation. The synthetic utility of the protected epoxides was investigated briefly by ring opening with NAN 3, followed by deprotection to give aminohydroxy prolines.

The absolute configuration of the marine sponge alkaloid pyrinodemin A is established by organic synthesis. © 2003 Elsevier Ltd. All rights reserved.

The biomimetic synthesis of the manzamine-related alkaloid keramaphidin B is described. The key intramolecular Diels-Alder reaction proposed in the Baldwin and Whitehead hypothesis is demonstrated. An investigation of the modified biomimetic hypothesis proposed by Marazano et al. is also reported. Finally, an alternative synthesis of keramaphidin B by Grubbs methathesis is presented.

Deacetoxycephalosporin C synthase (DAOCS) catalyzes the oxidative ring expansion of penicillin N (penN) to give deacetoxycephalosporin C (DAOC), which is the committed step in the biosynthesis of the clinically important cephalosporin antibiotics. DAOCS belongs to the family of non-heme iron(II) and 2-oxoglutarate (2OG) dependent oxygenases, which have substantially conserved active sites and are proposed to employ a consensus mechanism proceeding via formation of an enzyme·Fe(II)·2OG·substrate ternary complex. Previously reported kinetic and crystallographic studies led to the proposal of an unusual "ping-pong" mechanism for DAOCS, which was significantly different from other members of the 2OG oxygenase superfamily. Here we report pre-steady-state kinetics and binding studies employing mass spectrometry and NMR on the DAOCS-catalyzed penN ring expansion that demonstrate the viability of ternary complex formation in DAOCS catalysis, arguing for the generality of the proposed consensus mechanism for 2OG oxygenases.

The utility of wild-type and variant carboxymethylproline synthases for biocatalysis was demonstrated by preparing functionalised 5-, 6- and 7-membered N-heterocycles from amino acid aldehydes and (alkylated) malonyl-coenzyme A derivatives; the N-heterocycles produced were converted to the corresponding bicyclic beta-lactams by a carbapenem synthetase.