Chemistry of inorganic compounds – Phosphorus or compound thereof – Halogen containing
Patent
1993-08-25
1994-08-23
Langel, Wayne
Chemistry of inorganic compounds
Phosphorus or compound thereof
Halogen containing
423303, C01B 2510, C01B 2514
Patent
active
053405579
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a novel process for the preparation of .sup.32 P-thiophosphates.
Our published PCT Patent Application WO90/11289 describes the preparation of nucleoside thiotriphosphates carrying .sup.32 P in the gamma-thiophosphate group, which comprises reacting a nucleoside diphosphate with a .sup.32 P labelled thiophosphate salt. One problem is that the only methods available for the preparation of these .sup.32 P thiophosphates are time consuming and specific activities and yields are often low.
We have investigated the reaction of H.sub.3.sup.32 PO.sub.4 with thiophosphoryl halides and have discovered that .sup.32 P thiophosphates are obtained in high yield if the involatile portion of the reaction product is hydrolysed with an aqueous medium, preferably of pH at least 7.5.
Accordingly the present invention provides a novel process for the preparation of .sup.32 P thiophosphates of the general formula: ##STR2## wherein n =1, 2 or 3 and each M, which may be the same or different, is H or a cation, which comprises heating H.sub.3.sup.32 PO.sub.4 or a salt thereof with at least an equivalent amount of a thiophosphoryl halide and then treating the reaction product, after the removal of excess unreacted thiophosphoryl halide, with an aqueous medium, preferably of pH at least 7.5, to hydrolyse the reaction product and form the .sup.32 P thiophosphate.
The cation is usually an alkali metal or alkaline earth metal, but is preferably sodium. The thiophosphoryl halide is preferably chloride or bromide.
In the accompanying Drawing, FIG. 1 illustrates apparatus in which the reaction of the invention may take place.
It is preferred to react the H.sub.3.sup.32 PO.sub.4 in the form of a salt in which one or two but not all three of the acidic hydrogens are replaced by a cation, preferably an alkali metal cation or a quaternary ammonium cation. One particularly preferred reactant is LiH.sub.2.sup.32 PO.sub.4. The use of a salt of this type has the effect of reducing the extent of .sup.32 P exchange into reaction by-products e.g. .sup.32 PSCl.sub.3 when the intermediate reaction product is hydrolysed and so increases the specific activity of the desired .sup.32 P thiophosphate product. It is also desirable to wash the inside surfaces of the reactor first with acid e.g. HCl, and then with deionised water, before the condensation reaction commences, to remove surface bound cations.
The reaction between the thiophosphoryl halide and H.sub.3.sup.32 PO.sub.4 gives rise to a novel diphosphetane intermediate of the formula: ##STR3## wherein X is halogen, preferably chloro or bromo, and such diphosphetane intermediates form a further aspect of the invention.
The aqueous medium is preferably alkaline and has a pH usually in the range 7.5-14 and is preferably buffered at that pH. It is preferred to use a non-phosphate buffer, such as Tricine (N-tris[hydroxymethyl]methylglycine) or a borate buffer, to avoid dilution of the labelled thiophosphate with unlabelled orthophosphate at the end of the reaction. The hydrolysis of the intermediate diphosphetane reaction product can also be brought about using an aqueous medium of pH less than 7 and treatment of the diphosphetane with an aqueous medium of pH as low as 2.5 for several hours results in formation of the desired .sup.32 P thiophosphate.
The reaction may be carried out using a large excess of the thiophosphoryl halide in the presence of only a few picomoles of H.sub.3.sup.32 PO.sub.4, allowing the preparation of a high specific activity product on a very low scale. The reaction is normally carried out in the absence of solvent, preferably under anhydrous conditions, using temperatures between 100.degree.-200.degree. C., preferably between 150.degree.-180.degree. C. The reaction is carried out over preferably at least about 1 hour, but usually over 12 to 60 hours.
Where the reaction product is not a salt in which all 3M groups are cations, it is preferred to convert the reaction product to such a salt, each cation preferably being an alkali metal e.g., sodium,
REFERENCES:
patent: 3061405 (1962-10-01), Olah et al.
patent: 3399963 (1968-09-01), Brois
Chemical Abstracts, vol. 102 No. 27 (Jun. 17, 1987), (Columbus, Ohio, US), Abstract #206089u, p. 128.
Chemical Abstracts, vol. 88 No. 16 (Apr. 17, 1978), (Columbus, Ohio, US), Washburn et al., Abstract #114500k, p. 632.
Creighton Andrew M.
Jeffery William A.
British Technology Group Limited
Langel Wayne
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