Tris phosphate preparation

The tetraphosphate anion is practically stable in neutral and alkaline solution at room temperature but at 65.9°C it is about half a power of ten less stable than the triphosphate anion over the whole pH range (61). Tetraphosphate has its maximum stability at 65.5°C at pH 10 and this is why it can be prepared from tetrametaphosphate at such temperatures. Its stability becomes smaller with decreasing pH. Apparently during the hydrolysis of an aqueous solution, which follows a first order law at constant pH, the tetraphosphate is cleaved initially exclusively at the end of the chain, to form mono- and tri-phosphate. 

Triethylaminoethyl cellulose powder (Serva) of capacity 0.71 m.equiv./gm is suspended in 2 M sodium chloride buffered with 0.1 M tris/phosphate pH 6.0 and the slurry is packed into a glass column 3.6 cm in diameter until the height of the packed material reaches 20 cm. The column is washed with a further 2 L of the solvent used for preparing the slurry and is then equilibrated with 0.01 M tris/phosphate buffer pH 8.5. Tris is an abbreviation for tris(hydroxymethyl)aminomethane. 330-360 mg of crude A. rhodostoma venom is dissolved in 20 ml of 0.01 M tris/phosphate buffer pH 8.5, centrifuged to remove insoluble material, and the clear supernatant is applied to the column. The fractionation is carried out at room temperature at a flow rate of 90-100 ml (35 ml/hour). The protein concentration in the eluate is estimated from the extinction of the solution at 280 m/t in 1 cm cells. The chromatogram is developed with the following buffers. In all cases the molarity of the buffers are with respect 40 to tris. 

Table 47 Preparations Di- and Tri-phosphate, Phosphate Ester, Phosphorate and Phosphonamate Complexes... 

A last problem in nucleotide mass spectrometry is related to the dinucleotides. Hunt and Hignite tried to prepare a matrix of several ribodinucleo-tides (silylated) for further application to the sequential analysis of oligonucleotides. The fact that common enzymatic and/or chemically induced hydrolyses do not stop at the nucleotide level, but rather continue until they become nucleosides, does not reduce the merit of their work. That work, as well as that of Biemann, enable one to identify any dinucleotide pair of the type B,pB2, where p stands for phosphate. This information is of very limited use, however, because from any given RNA, for instance, one obtains practically any possible B1PB2 combination of dinucleotides. 

Chemical Synthesis.—The phosphorimidazolidate method for the preparation of nucleoside di- and tri-phosphates has been re-examined and extended. For instance, ATP may be prepared either by activating ADP with carbonyl di-imidazole and treating the resulting phosphorimidazolidate with inorganic... 

A full paper has appeared on the tin-mediated allylation and benzylation of 1,2-0-isopropylidene-myo-inositol (see Vol. 27, p. 219, ref. 126 and Vol. 25, p. 215, ref. 108 for preliminary report) which resulted in the preparation of various protected myo-inositol 1,6-bis-and 1,5,6-tris-phosphates. ... 

Nucleoside bis-phosphates can be prepared (10.80). Some ribonucleoside tris-phosphates are known although their biochemical role appears to be limited. 

Radioiodination of lyt -octreotide using Chloramine-T (modified from Bakker et al 1990). Twenty-five microliters oflCT M Tyr -octreotide is mixed in a reaction vial with 10 MBq 10 pi of a freshly prepared solution oflCT M Chloramine-T in 0.25 M phosphate buffer pH 7.4 is added. After 30 s, 10 pi of a saturated solution of methionine in 0.25 M phosphate buffer pH 7.4 is added and the reaction mixture is vortexed. Purification is accomplished by reversed-phase HPLC. As Chloramine-T reveals a very similar retention time as the labeled Tyr -octreotide, the eluent system H20/acetonitrile containing TFA is not suited for this purpose. Using a gradient of0% B —> 100% B in 30 min (A = Tris-phosphate buffer pH 2.6 and B = methanol), flow rate = 0.7 ml mirT, the radiolabeled peptide can be purified. The 1-labeled peptide can be stored for up to 1 month at —20°C. 

Nucleoside analogues require phosphorylation to the 5 -triphosphate by cellular kinases prior to their inhibition of DNA synthesis. However, most studies have focused upon prodrugs of monophosphates, whereas for some nucleosides (for example, AZT) the formation of the diphosphate is rate limiting. Huynh-Dinh and coworkers [109, 110] have recently prepared P-acyl esters of 5 -di- (m = I) and tri-phosphates (m = 2) of both AZT and d4T (97, n = 6, 10, 12 or 14, Nu = 5 -AZT or 5 -d4T). The lipophilic acyl chain should facilitate passive diffusion across cellular membranes. Chemical hydrolyses at 37°C and physiological pH showed that the acyl phosphates were cleanly converted to the corresponding nucleoside di- and tri-phosphates. 

The conversion of glycerophosphoinositols to phosphoinositol has been optimised and the reaction has been exploited for the preparation of D-/nyo-inositol-l,4-bis- and 1,4,5-tris-phosphate in mg quantities from readily available preparations of mixed phosphoinositidesJ The pyridinium salt of the 1,4,5-trisphosphate, on treatment with DCC in aqueous pyridine, gave the 1,2-cyclic 4,5-trisphosphate7 ... 

Preparative separations of myo-inositol bis- and tris-phosphate isomers, yielding mg amounts of pure materials, were achieved by firactionations first on a cation- then on an anion-exchange resin. Stepwise elution of inositol mono- through hexa-phosphates from a strong-base anion-exchange resin in the formate form was demonstrated and used for the... 

Cyclic thiophosphates (35) of 1,2-O-isopropylidene-a-D-glucofuranose have been obtained from the corresponding 3,5,6-bicyclic thiophosphate by treatment with various nucleophiles. 6-Deoj -6-sulphono-o-D glucopyranosyl phosphate has been prepared as shown in Chapter 11, Scheme 5 the synthesis of individual enantiomers and racemates of mvo-inositol mono-, di-, and tri-phosphates is covered in Chapter 18, and the use of glycopyranosyl phosphates as gfyco I donors is referred to in Chapter 3. 

Several papers on the transformation of benzene and substituted benzenes to cis-diols using Pseudomonas putida have appeared. A summary on the preparation of cyclitols and natural products such as (+)-pinitol, (+)-conduritol F as well as D-(-)-myo-inositol-1- and l,4,S-tri-phosphate from benzene have been reported. Standard chemical transformations from benzene cu-diol involving resolution of an alcohol intermediate by way of (+)-sec phenylmethyl ethers led to a preparation of (+)-conduritol F and its enantiomei, whereas resolution of an alcohol intermediate using lipase-catalysed acetylation afforded (+)- and (-)-conduritol C. ... 

The above is a general procedure for preparing trialkyl orthophosphates. Similar yields are obtained for trimethyl phosphate, b.p. 62°/5 mm. triethyl phosphate, b.p. 75-5°/5 mm. tri-n-propyl phosphate, b.p. 107-5°/5 mm. tri-Mo-propyl phosphate, b.p. 83-5°/5 mm. tri-wo-butyl phosphate, b.p. 117°/5-5 mm. and tri- -amyl phosphate, b.p. 167-5°/5 mm. The alkyl phosphates are excellent alkylating agents for primary aromatic amines (see Section IV,41) they can also be ua for alkylating phenols (compare Sections IV,104-105). Trimethyl phosphate also finds application as a methylating agent for aliphatie alcohols (compare Section 111,58). 

Nickel Phosphate. Tri nickel orthophosphate [14396-43-17, Ni2(P0 2 7H20, exists as apple-green plates which decompose upon heating. It is prepared by the reaction of nickel carbonate and hot dilute phosphoric acid. Nickel phosphate is an additive to control the crystal size of ziac phosphate ia coaversioa coatiags which are appHed to steel prior to its being paiated (see Metal surface treatments). 

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