A general method for the chemical synthesis of γ-32P-labeled or unlabeled nucleoside 5′-triphosphates and thiamine triphosphate

Bettendorff, Lucien; Nghiêm, Hoàng‐Oanh; Wins, Pierre; Lakaye, Bernard

doi:10.1016/j.ab.2003.08.013

Cited by 27 publications

(24 citation statements)

References 24 publications

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“…After unsatisfying phosphorylation attempts with some of these methods [21], [24], [25], we considered a more recent approach using organometallic synthesis [26]. The oxorhenium(VII)-catalyzed direct condensation of alcohols with phosphoric acid was, however, found to be inappropriate for the handling of radioactive compounds (reflux at temperatures above 140°C), and milder reaction conditions led to product mixtures [27] which were difficult to separate.…”

Section: Resultsmentioning

confidence: 99%

Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions

Nierth

Jäschke

2011

PLoS ONE

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Nature has efficiently adopted phosphorylation for numerous biological key processes, spanning from cell signaling to energy storage and transmission. For the bioorganic chemist the number of possible ways to attach a single phosphate for radioactive labeling is surprisingly small. Here we describe a very simple and fast one-pot synthesis to phosphorylate an alcohol with phosphoric acid using trichloroacetonitrile as activating agent. Using this procedure, we efficiently attached the radioactive phosphorus isotope 32P to an anthracene diene, which is a substrate for the Diels-Alderase ribozyme—an RNA sequence that catalyzes the eponymous reaction. We used the 32P-substrate for the measurement of RNA-catalyzed reaction kinetics of several dye-labeled ribozyme variants for which precise optical activity determination (UV/vis, fluorescence) failed due to interference of the attached dyes. The reaction kinetics were analyzed by thin-layer chromatographic separation of the 32P-labeled reaction components and densitometric analysis of the substrate and product radioactivities, thereby allowing iterative optimization of the dye positions for future single-molecule studies. The phosphorylation strategy with trichloroacetonitrile may be applicable for labeling numerous other compounds that contain alcoholic hydroxyl groups.

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Section: Resultsmentioning

confidence: 99%

Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions

Nierth

Jäschke

2011

PLoS ONE

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“…Thiamine, ThMP and ThDP were from Sigma-Aldrich NV/SA (Bornem, Belgium). ThTP and AThTP, which are not commercially available, were synthesized as described [42], [100]. All other chemicals were from Merck (Darmstadt, Germany) or from Sigma-Aldrich and of the highest purity available.…”

Section: Methodsmentioning

confidence: 99%

Thiamine Status in Humans and Content of Phosphorylated Thiamine Derivatives in Biopsies and Cultured Cells

et al. 2010

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BackgroundThiamine (vitamin B1) is an essential molecule for all life forms because thiamine diphosphate (ThDP) is an indispensable cofactor for oxidative energy metabolism. The less abundant thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and adenosine thiamine triphosphate (AThTP), present in many organisms, may have still unidentified physiological functions. Diseases linked to thiamine deficiency (polyneuritis, Wernicke-Korsakoff syndrome) remain frequent among alcohol abusers and other risk populations. This is the first comprehensive study on the distribution of thiamine derivatives in human biopsies, body fluids and cell lines.Methodology and Principal FindingsThiamine derivatives were determined by HPLC. In human tissues, the total thiamine content is lower than in other animal species. ThDP is the major thiamine compound and tissue levels decrease at high age. In semen, ThDP content correlates with the concentration of spermatozoa but not with their motility. The proportion of ThTP is higher in humans than in rodents, probably because of a lower 25-kDa ThTPase activity. The expression and activity of this enzyme seems to correlate with the degree of cell differentiation. ThTP was present in nearly all brain and muscle samples and in ∼60% of other tissue samples, in particular fetal tissue and cultured cells. A low ([ThTP]+[ThMP])/([Thiamine]+[ThMP]) ratio was found in cardiovascular tissues of patients with cardiac insufficiency. AThTP was detected only sporadically in adult tissues but was found more consistently in fetal tissues and cell lines.Conclusions and SignificanceThe high sensitivity of humans to thiamine deficiency is probably linked to low circulating thiamine concentrations and low ThDP tissue contents. ThTP levels are relatively high in many human tissues, as a result of low expression of the 25-kDa ThTPase. Another novel finding is the presence of ThTP and AThTP in poorly differentiated fast-growing cells, suggesting a hitherto unsuspected link between these compounds and cell division or differentiation.

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“…Thiamine triphosphate (ThTP) was synthesized and purified as previously described [35]. The CyaB expression plasmid pTRC99ACyaB was a gift of Drs Antoine Danchin and Agnieszka Sekowska (AMAbiotics SAS, Genopole Campus 1 - Genavenir 8, 5, rue Henri Desbruères, 91030 EVRY Cedex, France).…”

Section: Methodsmentioning

confidence: 99%

High Inorganic Triphosphatase Activities in Bacteria and Mammalian Cells: Identification of the Enzymes Involved

et al. 2012

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BackgroundWe recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities.Methodology/Principal FindingsRecombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPPi) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPPi but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility.Conclusions and General SignificanceWe show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPPi in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPPi, which could be cytotoxic because of its high affinity for Ca2+, thereby interfering with Ca2+ signaling.

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A general method for the chemical synthesis of γ-32P-labeled or unlabeled nucleoside 5′-triphosphates and thiamine triphosphate

Cited by 27 publications

References 24 publications

Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions

Radioactive Phosphorylation of Alcohols to Monitor Biocatalytic Diels-Alder Reactions

Thiamine Status in Humans and Content of Phosphorylated Thiamine Derivatives in Biopsies and Cultured Cells

High Inorganic Triphosphatase Activities in Bacteria and Mammalian Cells: Identification of the Enzymes Involved

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