Analysis of pathogenic bacteria using exogenous volatile organic compound metabolites and optical sensor detection

A novel, low-cost and simple method for the detection of pathogenic bacteria is proposed. The approach is based on the generation of an exogenous volatile organic compound (VOC) produced by the addition of an enzyme substrate to the bacterial sample. The generated VOC is then trapped in agarose gel allowing colour development to take place; visual detection is then possible by both the naked eye and by colorimetric analysis. Agarose gel has been evaluated as both a suitable VOC trapping matrix and host for the colour-generating reagents. This proof of concept method allowed for the discrimination between β-glucosidase and β-alanyl aminopeptidase producing bacteria. Enterococcus faecium and Klebsiella pneumoniae are both β-glucosidase producers and generated a yellow colour within agarose gels upon enzymatic hydrolysis of 2-nitrophenyl-β-D-glucoside. Pseudomonas aeruginosa is a known β-alanyl aminopeptidase producer and was shown to hydrolyse the trifluoroacetic acid (TFA) salt of 3-amino-N-phenylpropanamide resulting in the development of an orange colour within agarose gels spiked with the sodium salt of 1,2-naphthoquinone-4-sulfonic acid. 3-Amino-N-phenylpropanamide (as its TFA salt) and 2-nitrophenyl-β-D-glucoside concentrations of 20 μg mL−1 (or 72 μmol L−1) and 100 μg mL−1 (or 332 μmol L−1), respectively were the minimum quantities required for colour production following 18 h of incubation. The use of 3-amino-N-phenylpropanamide, TFA salt indicated that synthesised enzyme substrates can be tailor-made to liberate exogenous VOCs for colour generation

Addition of phenylisocyanate to 3,5-diphenyl-1,2-dithiolium-4-olate gives a thioketone

The Type B mesoionic heterocycle, 3,5-diphenyl-1,2-dithiolium-4-olate, reacts with phenylisocyanate producing a thioketone derivative rather than a pseudo-semi-conjugated heterocyclic mesomeric betaine. The structure of the thioketone product was confirmed by an X-ray crystallographic investigation

Detection of exogenous VOCs as a novel in vitro diagnostic technique for the detection of pathogenic bacteria

The evolution of volatile organic compounds (VOCs) provides an opportunistic approach for the detection of pathogenic bacteria. This approach can be enhanced by the application of VOC-labelled enzyme substrates that target specific enzyme activities of the bacteria under investigation. Detection of the VOCs provides a novel, specific and sensitive approach for the detection of pathogenic bacteria. This review highlights the importance of this approach alongside a range of alternative detection techniques for the identification of pathogenic bacteria

Development of novel routes to pyridines

Pyridines occupy a central part in modern day organic chemistry. Recent studies in various fields of chemistry, biology and physics have featured numerous examples and applications of these compounds. The purpose of this study was to produce a library of polysubstituted pyridines, 2,2'-bipyridines and 2,2':6',2"-terpyridines via pathways that allowed unusual or even unique substitution patterns. To achieve a generic pyridine synthesis that delivers a diversity of products tailored to different industrial needs, a strategy by which the target molecule is constructed in a [2+2+2]-manner was chosen, i.e. the six atoms of the pyridine ring and their pendant functionalities are traced back to three building blocks, each delivering two atoms to the pyridine ring. A range of a-acetoxy-a-chloro-P-keto esters were prepared in three steps from commercially available P-keto esters through a-chlorination with sulfuryl chloride, a-acetoxylation with acetic acid and triethylamine and a second a-chlorination in good overall yields (69 — 89 %) without the need for chromatographic purification. These a-acetoxy-a-chloro-j3-keto esters served as equivalents for a,[3-diketo esters (building block 1) in the synthesis of various 1,2,4- triazines through condensation with picolinohydrazonamides or thiosemicarbazides (building block 2). A subsequent aza Diels-Alder reaction of these 1,2,4-triazines with electron-rich dienophiles (building block 3) such as 2,5-norbornadiene, 1-pyrrolidino- 1 -cyclopentene and 2,3-dihydrofuran furnished an array of novel polysubstitued (bi)pyridines. The two-step sequence of condensation and aza Diels-Alder reaction could be advanced into a 'one-pot' synthesis on several occasions. Furthermore, we devised a feasible synthetic alternative towards a,(3-diketo esters. Alpha-picolinoyl-3-keto esters were prepared from the same starting materials as the a-acetoxy-a¬chloro-P-keto esters in a shortened two-step sequence of a-chlorination of P-keto esters with sulfuryl chloride and replacement of the chloro group by a picolinoyl group using picolinic acid and KHCO3. The overall yields of a-picolinoyl-f3-keto esters (55 — 91 %) were comparable to those of the a-acetoxy-a-chloro-P-keto esters. Copper(II) acetate-facilitated methanolysis of a-picolinoyl-P-keto esters and immediate oxidation of the in situ generated a-hydroxy-P-keto esters by excess copper(II) acetate afforded a,(3-diketo esters which reacted with hydrazonamides in the same manner as the a-chloro-a-acetoxy-P-keto esters. However, in terms of product purity and yield the `chloroacetate route' remains the superior strategy.EThOS - Electronic Theses Online ServiceVertellus Specialties UK LtdGBUnited Kingdo

Development of novel routes to pyridines

Pyridines occupy a central part in modern day organic chemistry. Recent studies in various fields of chemistry, biology and physics have featured numerous examples and applications of these compounds. The purpose of this study was to produce a library of polysubstituted pyridines, 2,2'-bipyridines and 2,2':6',2"-terpyridines via pathways that allowed unusual or even unique substitution patterns. To achieve a generic pyridine synthesis that delivers a diversity of products tailored to different industrial needs, a strategy by which the target molecule is constructed in a [2+2+2]-manner was chosen, i.e. the six atoms of the pyridine ring and their pendant functionalities are traced back to three building blocks, each delivering two atoms to the pyridine ring. A range of a-acetoxy-a-chloro-P-keto esters were prepared in three steps from commercially available P-keto esters through a-chlorination with sulfuryl chloride, a-acetoxylation with acetic acid and triethylamine and a second a-chlorination in good overall yields (69 — 89 %) without the need for chromatographic purification. These a-acetoxy-a-chloro-j3-keto esters served as equivalents for a,[3-diketo esters (building block 1) in the synthesis of various 1,2,4- triazines through condensation with picolinohydrazonamides or thiosemicarbazides (building block 2). A subsequent aza Diels-Alder reaction of these 1,2,4-triazines with electron-rich dienophiles (building block 3) such as 2,5-norbornadiene, 1-pyrrolidino- 1 -cyclopentene and 2,3-dihydrofuran furnished an array of novel polysubstitued (bi)pyridines. The two-step sequence of condensation and aza Diels-Alder reaction could be advanced into a 'one-pot' synthesis on several occasions. Furthermore, we devised a feasible synthetic alternative towards a,(3-diketo esters. Alpha-picolinoyl-3-keto esters were prepared from the same starting materials as the a-acetoxy-a¬chloro-P-keto esters in a shortened two-step sequence of a-chlorination of P-keto esters with sulfuryl chloride and replacement of the chloro group by a picolinoyl group using picolinic acid and KHCO3. The overall yields of a-picolinoyl-f3-keto esters (55 — 91 %) were comparable to those of the a-acetoxy-a-chloro-P-keto esters. Copper(II) acetate-facilitated methanolysis of a-picolinoyl-P-keto esters and immediate oxidation of the in situ generated a-hydroxy-P-keto esters by excess copper(II) acetate afforded a,(3-diketo esters which reacted with hydrazonamides in the same manner as the a-chloro-a-acetoxy-P-keto esters. However, in terms of product purity and yield the `chloroacetate route' remains the superior strategy.EThOS - Electronic Theses Online ServiceVertellus Specialties UK LtdGBUnited Kingdo

A convenient synthesis of pyridine and 2,2′-bipyridine derivatives

α-Chloro-α-acetoxy-β-keto-esters 9 were readily prepared from β-keto-esters 6 in good overall yields. These compounds reacted as α,β-diketo-ester equivalents 2 with amidrazones 1 yielding triazines 3, generally in good yields. Picolinates 10 provided an alternative source of α,β-diketo-ester equivalents 2 when treated with copper(II) acetate. A ‘one-pot’ reaction of the α,β-diketo-ester equivalents 2 with amidrazones 1 in the presence of 2,5-norbornadiene 5 in boiling ethanol yielded the pyridines 4 and 2,2′-bipyridines 4 (R1=2-pyridyl) directly without the need to isolate the corresponding triazines 3. Triazine 3c reacted with the aza-dienophiles 13 and 17 affording the products 16 and 18, respectively, in good yields

Analysis of Listeria using exogenous volatile organic compound metabolites and their detection by static headspace–multi-capillary column–gas chromatography–ion mobility spectrometry (SHS–MCC–GC–IMS)

Listeria monocytogenes is a Gram-positive bacterium and an opportunistic food-borne pathogen which poses significant risk to the immune-compromised and pregnant due to the increased likelihood of acquiring infection and potential transmission of infection to the unborn child. Conventional methods of analysis suffer from either long turn-around times or lack the ability to discriminate between Listeria spp. reliably. This paper investigates an alternative method of detecting Listeria spp. using two novel enzyme substrates that liberate exogenous volatile organic compounds in the presence of α-mannosidase and d-alanyl aminopeptidase. The discriminating capabilities of this approach for identifying L. monocytogenes from other species of Listeria are investigated. The liberated volatile organic compounds (VOCs) are detected using an automated analytical technique based on static headspace–multi-capillary column–gas chromatography–ion mobility spectrometry (SHS–MCC–GC–IMS). The results obtained by SHS–MCC–GC–IMS are compared with those obtained by the more conventional analytical technique of headspace–solid phase microextraction–gas chromatography–mass spectrometry (HS–SPME–GC–MS). The results found that it was possible to differentiate between L. monocytogenes and L. ivanovii, based on their VOC response from α-mannosidase activity

New routes to functionalised pyridines

A novel method of preparing substituted pyridines has been developed. This method uses readily available [3-ketoesters and amidrazone as starting materials. The pyridines obtained do not require purification and different substitution patterns, not available by known methods, can be obtained. The formation of 1,2,3-tricarbonyl compounds was achieved by oxidation of the alcohol precursors, following two different methods. a-Chloro-ct-acetoxy-f3-dicarbonyls were prepared in excellent yields and were shown to react as tricarbonyl equivalents in the formation of 1,2,4-triazines. Regioselective condensation reactions were observed between different amidrazones with tricarbonyl and tricarbonyl equivalents to produce a series of novel 1,2,4-triazines in good yields with no contamination by any regioisomer. When 1,2,4-triazines were obtained from a-chloro-a-acetoxy-P-dicarbonyls, 2.5 equivalents of amidrazone were required. However, decomposition of a-chloro-a-acetoxy-P-dicarbonyls prior to reaction with 1 equivalent of amidrazone yielded the 1,2,4-triazines in good yields. These 1,2,4-triazines underwent aza Diels-Alder cycloaddition reactions with 2,5- norbornadiene to give a series of novel 2,3,6-trisubstituted pyridines. The pyridines bearing electron withdrawing groups as substituents could also be obtained in a 'one- pot' reaction from their corresponding tricarbonyls or tricarbonyl derivatives. The 1,2,4- triazines bearing electron donating groups could be converted to their corresponding pyridines either by changing the reaction conditions or, when possible, by conversion of the electron donating group into a more electron withdrawing substituent by oxidation (e.g. sulphoxide substituent). Pyridines bearing a sulphoxide substituent undergo nucleophilic substitutions, giving great scope to introduce different functionality in the C-6 of the pyridines.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Detection of l-alanylaminopeptidase activity in microorganisms using fluorogenic self-immolative enzyme substrates

A series of fluorogenic enzymatic substrates that incorporate a self-immolative spacer were synthesised for the purpose of identifying l-alanylaminopeptidase activity in microorganisms in agar media. These substrates resulted in the generation of fluorescent microorganism colonies with Gram-negative microorganisms