Stephen P. Stanforth

Identification of volatile organic compounds produced by bacteria using HS-SPME-GC-MS.

The analysis of volatile organic compounds (VOCs) as a tool for bacterial identification is reported. Headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS) was applied to the analysis of bacterial VOCs with the aim of determining the impact of experimental parameters on the generated VOC profiles. The effect of culture medium, SPME fiber type and GC column were fully evaluated with the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae and the Gram-positive species Staphylococcus aureus. Multivariate analysis, including cluster analysis and principal component analysis, was applied to VOC data to determine whether the parameters under investigation significantly affected bacterial VOC profiles. Culture medium, and to a lesser extent, SPME fiber type, were found to significantly alter detected bacterial VOC profiles. The detected VOCs varied little with the polarity of the GC column. The results indicate that the generated bacterial VOC profiles need careful evaluation if they are to be used for clinical diagnostics. The whole process is limited by the need to grow the bacteria in broth (18 h) before extraction and analysis (63 min).

2-Arylbenzothiazole, benzoxazole and benzimidazole derivatives as fluorogenic substrates for the detection of nitroreductase and aminopeptidase activity in clinically important bacteria

A series of 2-(2-nitrophenyl)benzothiazole 7, 2-(2-nitrophenyl)benzoxazole 10 and 2-(2-nitrophenyl)benzimidazole 13 derivatives have been synthesised and assessed as indicators of nitroreductase activity across a range of clinically important Gram negative and Gram positive bacteria. The majority of Gram negative bacteria produced strongly fluorescent colonies with substrates 7 and 10 whereas fluorescence production in Gram positive bacteria was less widespread. The l-alanine 16 and 19 and β-alanine 21 and 23 derivatives have been prepared from 2-(2-aminophenyl)benzothiazole 14 and 2-(2-aminophenyl)benzoxazole 17. These four compounds have been evaluated as indicators of aminopeptidase activity. The growth of Gram positive bacteria was generally inhibited by these substrates but fluorescent colonies were produced with the majority of Gram negative bacteria tested.

Synthesis of pyridine derivatives using aza Diels–Alder methodology

Abstract Amidrazone 1 reacted with the unsymmetrical tricarbonyls 2a , 2c and 2d giving triazines 3a , 3c and 3d , respectively. These triazines were converted into their corresponding pyridine derivatives 6a , 6c and 6d in aza Diels–Alder reactions with 2,5-norbornadiene 5 . Triazines 3c and 3d gave the pyridolactones 9c and 9d with 2,3-dihydrofuran.

Desymmetrization of dichloroazaheterocycles

3,6-Dichloropyridizine 1a was converted in good yield into its mono-iodo derivative 1b when treated with a mixture of hydriodic acid and sodium iodide. Pure samples of the mono-iodo derivatives 2b, 3b and 4b could not be obtained from their corresponding dichlorinated precursors with these reagents. Compounds 1b and 4b underwent palladium catalysed Suzuki, Sonogashira and other coupling reactions.

Preparation and Cycloaddition Reactions of Novel Heterocyclic Mesomeric Betaines

Abstract The heterocyclic mesomeric betaines 6a–c reacted with dimethyl acetylenedicarboxylate and ethyl propiolate giving the 1,3-dipolar cycloaddition products 7a–c and 8a–c, respectively. With esters of maleic, fumaric, acrylic and methacrylic acids, mesomeric betaines 6a and 6b gave substituted tetralone derivatives.

Development of a novel method for detection of Clostridium difficile using HS‐SPME‐GC‐MS

A novel method has been developed that allows successful differentiation between Clostridium difficile culture‐positive and culture‐negative stool samples based on volatile organic compound (VOC) evolution and detection by headspace solid‐phase microextraction coupled with gas chromatography mass spectrometry (HS‐SPME‐GC‐MS).

Synthesis of 2,2′-bipyridyl derivatives using aza Diels-Alder methodology

Amidrazone 1 and the tricarbonyl derivatives 2a-c gave the triazines 3a-c, respectively, which reacted with 2,5-norbornadiene 4 in boiling ethanol yielding the corresponding novel 2,2′-bipyridines 5a-c in good yield. Triazine 6 gave the 2,2′-bipyridyl derivative 7 (65%) with compound 4 in 1,2-dichlorobenzene at 140°C.

Bacteria detection based on the evolution of enzyme-generated volatile organic compounds: Determination of Listeria monocytogenes in milk samples

The rapid detection of Listeria monocytogenes contamination in food is essential to prevent food-borne illness in humans. The aim of this study was to differentiate non-contaminated milk from milk contaminated with L. monocytogenes using enzyme substrates coupled with the analysis of volatile organic compounds (VOCs). The method is based on the activity of β-glucosidase and hippuricase enzymes and the detection of a specific VOC i.e. 2-nitrophenol and 3-fluoroaniline, respectively. VOCs were extracted, separated and detected by headspace-solid phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME GC-MS). This approach required the inclusion of the selective agents cycloheximide, nalidixic acid and acriflavine HCl in the growth medium to inhibit interfering bacteria. The VOCs were liberated by L. monocytogenes provided that samples contained at least 1-1.5×10(2) CFU ml(-1) of milk prior to overnight incubation. This approach shows potential for future development as a rapid method for the detection of L. monocytogenes contaminated milk.

The Wittig reaction of some fluorinated amide derivatives

Carboethoxymethylenetriphenylphosphorane 1 reacted with fluorinated amides 4, 6, 8 and 15 yielding products 12, 13, 14 and 3 respectively.

A convenient synthesis of substituted 2,2':6',2 -terpyridines

The 2,2′:6′,2″-terpyridines 7a–c were prepared in good yield by reacting α-acetoxy-α-chloro-β-keto-esters 3a–c with bis-amidrazone 4 and 2,5-norbornadiene 6 in ethanol at reflux. Compounds 3a and 3b gave the 2,2′:6′,2″-terpyridines 9a and 9b, respectively, in moderate yield when treated with compound 4 and enamine 8.