Dispersity determination of poly(ethylene glycol)s using supercritical fluid chromatography-mass spectrometry and different mass analysers.

RATIONALE: Dispersity values are considered critical quality attributes for the quality control of poly(ethylene glycol) formulations due to the direct impact on drug performance. However, when these polymers are analysed using mass spectrometry, the design of the mass analyser can impact the oligomer response and affect the obtained dispersity values, so further understanding is needed. METHODS: The deconvoluted electrospray ionisation mass spectra of poly(ethylene glycol)s obtained using supercritical fluid chromatography (SFC) hyphenated to different mass analysers were compared, and visualisation diagrams were used to understand the differences in the dispersity value calculations. Five calibration approaches based on a surrogate single oligomer that represents the whole distribution, or the whole distribution itself, for response selection, were used to evaluate ionisation efficiency prior to quantitation. The impact of using an internal standard (ISTD) on the expanded uncertainty was also assessed. RESULTS: Although there were challenges related to the resolution of multiply charged species when low-resolution instruments were used, similar quantitation capabilities were obtained to those when high-resolution mass analysers were used. Evaluation of approaches using a surrogate oligomer or the whole distribution suggested the independence of both approaches and a constant ionisation efficiency across the oligomer chain length. The higher degree of chromatographic resolution of SFC allowed incorporating a monodispersed ISTD to improve the accuracy and precision of the method. CONCLUSIONS: The use of low resolution mass analysers was sufficient to provide accurate and precise dispersity values; however, higher resolution instruments were recommended for characterisation due to the improved mass resolution of ions. The introduction of a monodispersed ISTD improved precision without compromising the calculated dispersity value due to the lack of analyte suppression.

Structure-Reactivity Studies of 2-Sulfonylpyrimidines Allow Selective Protein Arylation.

Protein arylation has attracted much attention for developing new classes of bioconjugates with improved properties. Here, we have evaluated 2-sulfonylpyrimidines as covalent warheads for the mild, chemoselective, and metal free cysteine S-arylation. 2-Sulfonylpyrimidines react rapidly with cysteine, resulting in stable S-heteroarylated adducts at neutral pH. Fine tuning the heterocyclic core and exocyclic leaving group allowed predictable SNAr reactivity in vitro, covering >9 orders of magnitude. Finally, we achieved fast chemo- and regiospecific arylation of a mutant p53 protein and confirmed arylation sites by protein X-ray crystallography. Hence, we report the first example of a protein site specifically S-arylated with iodo-aromatic motifs. Overall, this study provides the most comprehensive structure-reactivity relationship to date on heteroaryl sulfones and highlights 2-sulfonylpyrimidine as a synthetically tractable and protein compatible covalent motif for targeting reactive cysteines, expanding the arsenal of tunable warheads for modern covalent ligand discovery.

Structural Diversity in Divalent Group 14 Triflate Complexes Involving Endocyclic Thia-Macrocyclic Coordination.

A highly unusual series of M(II) (M = Ge, Sn, Pb) complexes with endocyclic thioether macrocyclic coordination and with coordination numbers ranging from three to nine have been prepared by the reaction of [9]aneS3 (1,4,7-trithiacyclononane), [12]aneS4 (1,4,7,10-tetrathiacyclododecane), or [24]aneS8 (1,4,7,10,13,16,19,22-octathiacyclotetracosane) with M(OTf)2 (M = Sn and Pb; OTf = CF3SO3-) or with GeCl2·dioxane and 2 mol equiv of TMSOTf (Me3SiO3SCF3) in a mixture of anhydrous CH2Cl2 and MeCN. The isolated bulk products are characterized by 1H, 13C{1H}, 19F{1H}, and 119Sn{1H} NMR and IR spectroscopy, high-resolution ESI+ MS, and microanalytical data. Crystal structures are also reported for [M(L)][OTf]2 (M = Ge, Sn, Pb; L = [9]aneS3, [12]aneS4) and for [M([24]aneS8)][OTf]2 (M = Sn, Pb). In all cases, the ligand is bound in an endocyclic fashion, but the coordination environment and number are highly dependent on the group 14 ion, the macrocyclic ring size, and the number of S-donor atoms it presents. Solution NMR spectroscopic data suggest that the metal-macrocycle coordination is retained in solution but that the triflate anions are extensively dissociated on the NMR timescale. Density functional theory calculations on the [M([9]aneS3)]2+ and [M([12]aneS4)]2+ (M = Ge, Sn, Pb) dications reveal that the HOMO is centered on the group 14 atom as a directional "lone pair"; it also retains a significant amount of positive charge.

First total synthesis of type II abyssomicins: (±)-abyssomicin 2 and (±)-neoabyssomicin B.

The intramolecular Diels-Alder reaction (IMDA) of a butenolide derivative, as an entry to the type II abyssomicin scaffold, and the total synthesis of (±)-abyssomicin 2 and (±)-neoabyssomicin B are reported for the first time. A facile route to the IMDA precursor, the formation of a type I intermediate and two paths to (±)-neoabyssomicin B are also discussed.

Controlling the positive ion electrospray ionization of poly(ethylene glycols) when using ultra-high-performance supercritical fluid chromatography-mass spectrometry.

Poly(ethylene glycols) are complex polymers often added to pharmaceutical formulations to improve drug solubility and delivery. One of the main challenges when using chromatographic techniques coupled to mass spectrometry is the unselective ionization of poly(ethylene glycols) oligomers. Additionally, when the chain length is large enough, multiple charged species are formed, further complicating the mass spectra and processing. This study uses the advanced oligomer separation provided by supercritical fluid chromatography with a mass spectrometry approach that selectively ionizes poly(ethylene glycols) as ammoniated molecules to simplify data analysis and facilitate batch-to-batch comparisons. Several visual representations of the response of the ionization events based on the polymer molecular weight and the repeating unit were used to elucidate trends in ionization. Evaluation of the influence of the oligomer length and end-group on the electrospray ionization of the polymer allowed the development of a process to enable selective ionization for these complex polymers.

Development of ultra-high-performance supercritical fluid chromatography-mass spectrometry assays to analyze potential biomarkers in sweat.

Liquid chromatography-mass spectrometry methods were required to afford the rapid separation and detection of purines and small organic acids. These compounds are found in sweat and sebum and are potential biomarkers for the early detection of pressures sores. Two ultra-high-performance supercritical fluid chromatography-mass spectrometry assays have been successfully developed for both classes of compounds. Separation for purines was achieved using a gradient of supercritical carbon dioxide and methanol with a 1-aminoanthracene sub 2 µm particle size column followed by positive ion electrospray ionization. Separation for organic acids was achieved using a gradient of supercritical carbon dioxide and methanol (50 mM ammonium acetate 2% water) with a Diol sub 2 µm particle size column followed by negative ion electrospray ionization. Calibration curves were created in the absence of internal standards and R2 values > 0.96 were achieved using single ion monitoring methods for the protonated purines and the deprotonated acids. The two new assays afford rapid analytical methods for the separation and detection of potential biomarkers in human sweat leading to the early detection and prevention of pressure sores.

Development of ultrahigh-performance liquid chromatography/mass spectrometry and ultrahigh-performance supercritical fluid chromatography/mass spectrometry assays to determine the concentration of Bitrex™ and sodium saccharin in homemade facemask fit testing solutions.

Fast and easily transferable chromatography/mass spectrometry assays were required to detect and quantify the amount of Bitrex™ and sodium saccharin in homemade facemask fit testing solutions. METHODS: Bitrex™ solutions were analysed using reversed-phase ultrahigh-performance liquid chromatography coupled with positive ion electrospray ionisation mass spectrometry (UHPLC/ESI-MS). Separation was achieved using a mobile phase gradient with an Acquity BEH C18-packed column. Sodium saccharin solutions were analysed using ultrahigh-performance supercritical fluid chromatography coupled with negative ion electrospray ionisation (UHPSFC/ESI-MS). Separation was achieved using isocratic elution with an Acquity UPC2 Torus Diol packed column and a methanol (25 mM ammonium acetate) co-solvent. RESULTS: The calibration curves obtained using the ratio of the active compound to an internal standard generated linear regression values (R2 ) >0.99. Samples analysed prior to and after an autoclave sterilisation process and bottling gave repeatable measurements within 10% of the expected concentration. CONCLUSIONS: The two assays afford a fast robust and quantitative analytical method for the detection of the active components used to test the efficacy of the homemade facemask testing solutions.

Quantitative UHPSFC-MS analysis of elemental sulfur in mineral oil via derivatisation with triphenylphosphine: application to corrosive sulfur-related power transformer failure.

An ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS) method has been developed as a rapid and reliable analytical method for the detection and quantification of elemental sulfur in mineral transformer oil. The method described in this paper is based on the selective reaction of elemental sulfur with triphenylphosphine (TPP). The derivatisation of elemental sulfur requires minimal sample preparation and resulted in the formation of a single compound, namely triphenylphosphine sulfide (TPPS). This derivative is quantified from the complex oil composition using electrospray ionisation-mass spectrometry (ESI-MS) in selected ion monitoring (SIM) mode, and the reported UHPSFC-MS method allows detection and quantification of the derivative at ppb levels. As sulfur contamination in mineral transformer oil has been linked to costly failures of oil/paper-based power transformers due to corrosion, the analytical approach is demonstrated through its application to mineral oil samples from in service and decommisioned power transformers. The method is ideal as a routine test or to confirm the presence of elemental sulfur in samples where corrosion has occurred.

First Synthesis and Characterization of CH4 @C60.

The endohedral fullerene CH4 @C60 , in which each C60 fullerene cage encapsulates a single methane molecule, has been synthesized for the first time. Methane is the first organic molecule, as well as the largest, to have been encapsulated in C60 to date. The key orifice contraction step, a photochemical desulfinylation of an open fullerene, was completed, even though it is inhibited by the endohedral molecule. The crystal structure of the nickel(II) octaethylporphyrin/ benzene solvate shows no significant distortion of the carbon cage, relative to the C60 analogue, and shows the methane hydrogens as a shell of electron density around the central carbon, indicative of the quantum nature of the methane. The 1 H spin-lattice relaxation times (T1 ) for endohedral methane are similar to those observed in the gas phase, indicating that methane is freely rotating inside the C60 cage. The synthesis of CH4 @C60 opens a route to endofullerenes incorporating large guest molecules and atoms.

Open Access UHPSFC/MS - an additional analytical resource for an academic mass spectrometry facility.

RATIONALE: Many compounds submitted for analysis in Chemistry at the University of Southampton do not retain, elute or ionize using open access reversed-phase ultra-high-performance liquid chromatography/mass spectrometry (RP-UHPLC/MS) and require analysis via infusion. An ultra-high-performance supercritical fluid chromatography mass spectrometry approach was implemented to afford high-throughput analysis of these compounds with chromatographic separation. METHODS: A UPC(2) -TQD MS system has been incorporated into the open access MS provision within Chemistry at the University of Southampton, using an ESCi source (electrospray and atmospheric pressure chemical ionization) and an atmospheric pressure photoionization (APPI) source. Access to instrumentation is enabled via a web-based interface (RemoteAnalyzer™). RESULTS: Compounds such as fluorosugars, fullerenes, phosphoramidites, porphyrins, and rotaxanes exhibiting properties incompatible with RP-UHPLC/MS have been analyzed using automated chromatography and mass spectrometry methods. The speedy return of data enables research in these areas to progress unhindered by sample type. The provision of an electronic web format enables easy incorporation of chromatograms and mass spectra into electronic files and reports. CONCLUSIONS: The implementation of UHPSFC/MS increases access to a wide range of chemistries incompatible with reversed-phase chromatography and polar solvents, enabling more than 90% of submitted samples to be analyzed using an open access approach. Further, chromatographic separation is provided where previously flow injection or infusion analyses were the only options. Copyright © 2016 John Wiley & Sons, Ltd.

Self-reporting hybridisation assay for miRNA analysis.

Hybridisation assays, which are commonly used to analyse oligonucleotides such as siRNAs and miRNAs, often employ detection probes with fluorescent tags. The signal emitted by a fluorescent tag covers a broad range of wavelengths and this limits the multiplexing potential due to overlapping signals. A novel method of indirect oligonucleotide analysis has been developed which combines a hybridisation assay with cleavable small molecule mass tags using HPLC-ESI MS detection. A self-reporting detection probe has been designed which incorporates a DNA/RNA chimeric oligonucleotide sequence in the reporter region, which generates small nucleotide products upon RNase cleavage of the ribose-phosphate backbone. These small nucleotides can then serve as mass tags for the indirect detection of oligonucleotide analytes. The narrow mass range covered by a small molecule mass tag combined with the wide range of possible mass tags provides a high degree of multiplexing potential. This approach has been demonstrated for the analysis of a synthetic miRNA.

Acylthioureas as anion transporters: the effect of intramolecular hydrogen bonding.

Small molecule synthetic anion transporters may have potential application as therapeutic agents for the treatment of diseases including cystic fibrosis and cancer. Understanding the factors that can dictate the anion transport activity of such transporters is a crucial step towards their application in biological systems. In this study a series of acylthiourea anion transporters were synthesised and their anion binding and transport properties in POPC bilayers have been investigated. The transport activity of these receptors is dominated by their lipophilicity, which is in turn dependent on both substituent effects and the formation and strength of an intramolecular hydrogen bond as inferred from DFT calculations. This is in contrast to simpler thiourea systems, in which the lipophilicity depends predominantly on substituent effects alone.

Synthesis and properties of metal trifluoride complexes with amide-functionalised tacn macrocycles and radiofluorination of [GaF3(L1)] by 18F/19F isotopic exchange.

Three amide-functionalised tacn macrocyclic derivatives (tacn = 1,4,7-triazacyclononane) are reported, two tris-amide derivatives, L1 containing three -CH2C(O)NHPh pendant arms, L2 containing three -CH2CH2C(O)NHiPr pendant arms, and one mono-amide, L3, containing iPr groups on two of the tacn amine functions and a single -CH2C(O)NHPh function on the third. The reactions of these new ligands towards [MF3(dmso)(OH2)2] (M = Al, Ga) and towards FeF3·3H2O in alcoholic solution afford the complexes [MF3(L)] (L = L1-L3) in good yields as powdered solids. These complexes are characterised by IR and multinuclear NMR spectroscopy (diamagnetic species only) and mass spectrometry. [GaF3(L1)], [GaF3(L3)] and [FeF3(L3)] are also characterised by single crystal X-ray analysis. The corresponding reactions involving [InF3(dmso)(OH2)2] yield mixtures of products (along with F-), consistent with the M-F bond strengths decreasing as group 13 is descended. A few crystals of the target complex, [InF3(L2)], were also obtained from one such reaction. All of the complexes adopt fac-octahedral coordination via the amine N-donor atoms and retain the three fluoride ligands both in solution and in the solid state. Extensive intramolecular hydrogen-bonding involving the amide NH pendant groups and the MF3 moieties is evident in the crystal structures. In the isostructural [MF3(L3)] (M = Ga, Fe) complexes the head-to-tail C(O)NH⋯F H-bonded dimers observed in the solid state resemble those found frequently in organic compounds and that form the cornerstone of many supramolecular assemblies. This is consistent with the MF3 fragments being strong H-bond acceptors. Radiofluorination of [GaF3(L1)] by 18F/19F isotopic exchange in MeOH at 3 µmol mL-1 precursor concentration and using aqueous [18F]F- in target water (75% : 25%) with gentle heating (80 °C, 10 min) gave ca. 20% radiochemical yield of [Ga18FF2(L1)]. In contrast, no 18F incorporation occurs with [GaF3(L3)] under any of the conditions explored.

A predictive science approach to aid understanding of electrospray ionisation tandem mass spectrometric fragmentation pathways of small molecules using density functional calculations.

RATIONALE: Tandem mass spectrometry (MS/MS) dissociation pathways can vary markedly between compound classes and can result in challenging and time-consuming interpretation of the data. Compound, class and substructure specific fragmentation rules for protonated molecules require refinement to aid the structural elucidation process. METHODS: The application of a predictive science approach using density functional theory (DFT) calculations has been investigated to estimate the abundances of first-generation product ions observed using an ion trap mass spectrometer. This has been achieved by application of Boltzmann population theory to electrospray ionisation (ESI)-MS and MS/MS data. RESULTS: Tandem ESI-MS data for this preliminary study were used to investigate the internal stabilities of protonated species and their product ions. The calculated relative abundances of 11.3%, 96.5%, and 1.1% for the product ion (m/z 192) of three quinazoline structural isomers are compared with the experimental values of 16%, 90% and 0% observed in the first-generation product ion mass spectra. CONCLUSIONS: Close correlation between calculated and experimental data has been demonstrated for these initial data. Applying this approach and establishing fragmentation rules, based on structure specific and common fragmentation behaviour, would improve and expedite the structural elucidation process.

Development of Simplified and Efficient Sample Preparation Methods for the Analysis of Problem Material within the Diesel Fuel Delivery System.

A new approach for the analysis of diesel engine fuel filters has been developed. This method involves minimal to no sample preparation, allowing rapid and unbiased analysis of diesel fuel filters. In recent years, diesel fuel filter plugging incidences have increased in parallel with changing emissions legislation. Fuel filter blockages can result in increased emissions, reduced efficiency, and engine failure. It is not fully understood why fuel filter blockages occur; as a result, there has been an international increase in research into the cause of fuel filter plugging. The method discussed in this paper utilizes a thermal desorption (TD) style sample introduction technique that can be used in conjunction with gas chromatography-mass spectrometry (GC-MS) and presents a fast, simple, and more sustainable approach to the analysis of fuel filters. When required, an efficient and straightforward sample cleanup process was developed and was used to simplify and improve confidence in the data identification and assignment; this method is up to three orders of magnitude faster than some procedures adopted in the literature. Further complementary analytical techniques, such as ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS) and high-resolution GC-MS, were used to access additional sample-specific information. This new approach has been successful in the identification of problematic materials deposited on blocked fuel filters, concurrent with recent research. This information can aid in the development of mitigation strategies to combat fuel filter plugging.

Self reporting RNA probes as an alternative to cleavable small molecule mass tags.

The large size of biological molecules such as proteins and oligonucleotides makes them inherently problematic to analyse and quantify directly by mass spectrometry. For these molecules, electrospray ionisation produces multiply charged species and associated alkali metal adducts which can reduce sensitivity and complicate quantification. Whereas time-of-flight mass analysers, often coupled to matrix-assisted laser desorption/ionisation, can have insufficient mass resolution to resolve these large molecules in the higher m/z range. This has led to the development of cleavable small molecule mass tag approaches for the indirect analysis of biomolecules such as proteins and oligonucleotides. Existing methodologies require the design and synthesis of a cleavable linker to join the biomolecule and the mass tag. Here, an alternative approach to small molecule mass tags is presented, which exploits the properties of the RNA molecule to afford self-reporting probes which can be easily synthesised using automated phosphoramidite chemistry. The sugar-phosphate backbone of RNA was used as a built-in enzyme cleavable linker and through the use of RNase digestion of bromine labelled oligonucleotides the observation of a range of small molecule mass tags by mass spectrometry is demonstrated. This study provides a proof-of-concept that RNase digestion can be used to produce labelled small molecule mass tags from oligonucleotide probes, thus eliminating the need for custom design and synthesis of a cleavable linker.

Synthesis of Ar@C60 using molecular surgery.

Synthesis of Ar@C60 is described, using a route in which high-pressure argon filling of an open-fullerene and photochemical desulfinylation are the key steps for >95% encapsulation of the noble gas. Enrichment by recycling HPLC leads to quantitative incorporation of argon in the product endofullerene, with a mass recovery of tens of milligrams, allowing the first characterisation of fine structure in the solution 13C NMR spectrum.

Total synthesis and stereochemical assignment of cis-uvariamicin I and cis-reticulatacin.

Diastereoisomeric mixtures of cis-uvariamicin I (15R,16R,19S,20S,36S and 15S,16S,19R,20R,36S) and cis-reticulatacin (17R,18R,21S,22S,36S and 17S,18S,21R,22R,36S) were synthesized to determine the stereochemistry of the natural products isolated from Annona muricata. It was not possible to resolve a mixture of the four synthetic isomers using chiral HPLC, but the mixed isomers could be distinguished using chiral HPLC EIMS with extracted fragment ion analysis. Comparison of synthetic standards with the natural isolate revealed that cis-uvariamicin I and cis-reticulatacin are present in nature as mixtures of threo-cis-threo diastereoisomers. It is suggested that the nomenclature for the natural products is amended as follows: (15R,16R,19S,20S,36S)-cis-uvariamicin I (cis-uvariamicin IA); (15S,16S,19R,20R,36S)-cis-uvariamicin I (cis-uvariamicin IB); (17R,18R,21S,22S,36S)-cis-reticulatacin (cis-reticulatacin A); (17S,18S,21R,22R,36S)-cis-reticulatacin (cis-reticulatacin B).

Small neutral molecular carriers for selective carboxylate transport.

A series of neutral thiourea receptors were found to mediate the antiport of chloride with a range of biologically relevant carboxylate anions across phospholipid bilayers. Simple structural modification of the carriers resulted in a change in the lactate/pyruvate transport selectivity.

Dietary Phytosterols Protective Against Peptic Ulceration.

BACKGROUND: In developing countries the prevalence of duodenal ulceration is related to the staple diet and not to the prevalence of Helicobacter pylori. Experiments using animal peptic ulcer models show that the lipid fraction in foods from the staple diets of low prevalence areas gives protection against ulceration, including ulceration due to non-steroidal anti-inflammatory drugs (NSAIDs), and also promotes healing of ulceration. The lipid from the pulse Dolichos biflorus (Horse gram) was highly active and used for further investigations. Further experiments showed the phospholipids, sterol esters and sterols present in Horse gram lipid were gastroprotective. Dietary phospholipids are known to be protective, but the nature of protective sterols in staple diets is not known. The present research investigates the nature of the protective phytosterols. METHODS: Sterol fractions were extracted from the lipid in Dolichos biflorus and tested for gastroprotection using the rat ethanol model. The fractions showing protective activity were isolated and identification of the components was investigated by Gas Chromatography-Mass Spectrometry (GC-MS). RESULTS: The protective phytosterol fraction was shown to consist of stigmasterol, ß-sitosterol and a third as yet unidentified sterol, isomeric with ß-sitosterol. CONCLUSIONS: Dietary changes, affecting the intake of protective phospholipids and phytosterols, may reduce the prevalence of duodenal ulceration in areas of high prevalence and may reduce the incidence of recurrent duodenal ulceration after healing and elimination of Helicobacter pylori infection. A combination of phospholipids and phytosterols, such as found in the lipid fraction of ulceroprotecive foods, may be of value in giving protection against the ulcerogenic effect of NSAIDs.