Triphosphate preparation

The 31P NMR spectrum of a crude sample of nucleoside triphosphate prepared according to Protocol 10 is shown in Figure 9.7a (the sample shown is of TTP produced using a larger excess of phosphoryl chloride than normal to indicate likely minor impurities). After MPLC, the only usual contaminants are pyrophosphate... 

The 31P NMR spectrum after MPLC of a sample of (l-thio)triphosphate prepared according to Protocol 12 is shown in Figure 9.8a. Figure 9.8b shows the 31P NMR spectra of a pure sample of Sp (l-thio)triphosphate after MPLC and HPLC. 

P -[l-(2-Nitro)phenylethyl]adenosine-5 -triphosphate, prepared by treating ADP-morpholidate with l-(2-nitro)phenylethyl phosphate, has the useful property that upon illumination with laser light at 347 nm, the 2-nitrophenylethyl group is lost to afford ATP. Photolysis of muscle fibres perfused with this caged ATP thus removes limitations in resolving the time course of muscle fibre relaxation by creating an instantaneous concentration of ATP. ... 

Fig. 33. Stepwise preparative desorption chromatography of phosphoric esters of adenosine on CS-AV-17 cellosorbent 1) adenine, 2) adenosine, 3) adenosine monophosphate, 4) adenosine diphosphate, 5) adenosine triphosphate...

Luciferase assay. In this technique, firefly luciferase is used to measure small amounts of adenosine triphosphate (ATP) in a bacterial culture, ATP levels being reduced by the inhibitory action of aminoglycoside antibiotics. This method may find more application in the future as more active and reliable luciferase preparations become available. 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

Catalysis by flavoenzymes has been reviewed and various analogues of FAD have been prepared e.g. P -adenosine-P -riboflavin triphosphate and flavin-nicotinamide dinucleotide ) which show little enzymic activity. The kinetic constants of the interaction between nicotinamide-4-methyl-5-acetylimidazole dinucleotide (39) and lactic dehydrogenase suggest the presence of an anionic group near the adenine residue at the coenzyme binding site of the enzyme. ... 

Nucleoside triphosphates are prepared analogously to the diphosphates by the methods a, b, c, and d, representing conversion of a nucleoside phosphoric azolide with inorganic pyrophosphate (Method a), of a nucleoside diphosphoric azolide with inorganic phosphate (Method b), of a phosphoric bisazolide with a nucleoside diphosphate (Method c), and of a diphosphoric bisazolide (bisazolide of pyrophosphoric acid) with a nucleoside phosphate (Method d). 

By this method adenosine triphosphate (ATP) and inosine triphosphate (ITP) were prepared in 89% and 93% yield, respectively 51]... 

The following diphosphates and triphosphates can be prepared by reaction of nucleotide azolides with nucleotides, sugar phosphates, phosphopeptides, or aminohexyl phosphates, a) NAD-analogues and P1, / -dinucleoside-S -diphosphates 801... 

Prepare a 10 mM solution of K3P04 buffer. Use this solution as a blank and for diluting solutions of the standards. Prepare standard solutions of selected nucleotides and nucleoside triphosphates,... 

Dideoxyuridine (ddU) is an antiviral agent that proved ineffective at controlling human immunodeficiency virus type 1 (HIV-1) infection in human T-cells. This ineffectiveness was ascribed to a lack of substrate affinity of ddU for cellular nucleoside kinases, which prevent it from being metabolized to the active 5 -triphosphate. To overcome this problem, bis[(pivaloyloxy)methyl] 2, 3 -dideoxyuridine 5 -monophosphate (9.41) was prepared and shown to be a membrane-permeable prodrug of 2, 3 -di-deoxyuridine 5 -monophosphate (ddUMP, 9.42) [93]. Indeed, human T-cell lines exposed to 9.41 rapidly formed the mono-, di-, and triphosphate of ddU, and antiviral activity was observed. This example again documents... 

Before further testing and to confirm that the compounds are cytotoxic rather than merely interfering with the Alamar blue indicator dye, they are re-bioassayed using two other indicator dyes. Calcein-AM is a fluorescent dye that measures changes in cell mem brane permeability, an indicator for one of the penultimate steps of cell death, uci e e measures the amount of adenosine triphosphate (ATP) synthesis in a chemilurmne assay. For some compounds, cell death was also confirmed by microscopic exami Papanicolaou-stained cell preparations.11... 

The solution structures of 1 1 complex between paromomycin and gentamicin Cla and the A-site oligonucleotide have been solved at high resolution using heteronuclear NMR techniques These approaches require the preparation of uniformly labeled N- and C-RNA oligonucleotides via in vitro transcription with labeled nucleoside triphosphates. The use of RNAs labeled with NMR-active nuclei allows for the application of sophisticated heteronuclear NMR methods. These methods facilitate the assigmnent of NMR resonances and the acquisition of structural restraints for detailed structure determinations. 

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