Phosphoromorpholidates

When AMP is heated under reflux in DMF, the 2, 3 -cyclic phosphate is formed, and cyclic phosphates can also be obtained from nucleosides and ortho-, pyro-, or poly-phosphoric acids under the same conditions. Promotion of phosphorylation by DMF is well known and the reaction with AMP is probably intermolecular as no 3, 5 -cyclic AMP can be detected. Minor products in the latter reaction are the 2, 3 -cyclic 5 -diphosphate and the 2 (30,5 -diphosphate. The synthesis of adenosine 2 (3 )-phosphate 5 -pyrophosphate has been achieved by the phosphoromorpholidate method used in a synthesis of Co A. ... 

Phosphoromorpholidate intermediates, initially developed by Khorana and Moffatt,19 have been widely used for the construction of nucleoside diphosphates. The construction of the diphosphate linkage typically involves exposure of a carbohydrate-derived phosphate nucleophile to phosphoromorpholidate electrophile in pyridine solvent. This protocol was attractive from the point of view that it can be executed on completely deprotected precursors. This method suffers, however, from the lengthy reaction times required for reasonable conversion, although a tetrazole modification, introduced by Wong, had been shown to shorten reaction times considerably. The major drawback of this method, for our own purposes, is that it would require construction of either a carbohydrate-derived phosphoromorpholidate or the preparation of a phosphoromorpholidate intermediate deriving from our rather expensive undecaprenyl monophosphate precursor. 

We have been unable to prepare the ATP analogue of PFA(II) by the phosphoromorpholidate route. When we attempt this preparation all we observe by TLC is the rapid formation of AMP. 

The synthesis of 2 -deoxyadenoslne nucleotides which contain an aminoalkyl chain at the 8-posltion covalently attached to fluoroscein and their subsequent incorporation into nucleic acids provides a route to fluorescent DNA probes. 8-(10-Aminodecyl)amino 5 -dAMP was treated with fluoroscein isothiocyanate in aqueous buffer at pH 9 to give the fluoroscelnylated monophosphate in good yield. The labelled triphosphate was most efficiently prepared by an analogous reaction of the phosphoromorpholidate with fluoroscein isothiocyanate... 

The first examples of supercharged nucleotide analogues (85-87) have been described, in which methylenebisphosphonic acid containing an additional ioni-sable acidic function has been incorporated into p,y-bridged derivatives of adenosine triphosphate. The compounds and their protected precursors were obtained following acid-catalysed reaction of the respective precursors (88-90) with adenosine 5 -phosphoromorpholidate in pyridine in yields of 80,75 and 25%. 

The chemical synthesis of uridine 5-(a-D-glucopyranosyl pyrophosphate) has been effected by condensing a-n-glucopyranosyl phosphate with uridine 5-phosphate in the presence of dicyclohexylcarbodiimide. The yield of glycosyl nucleotide in this reaction is very low, primarily because dicyclohexylcarbodiimide promotes the formation of n-glucose cyclic 1,2-phosphate in preference to the formation of the pyrophosphate bond. In an improved synthetic method, the nucleotide is first converted to a phosphoramidate (5) or phosphoromorpholidate (6), by condensation in the presence of dicyclohexylcarbodiimide with ammonia or morpholine, respectively. 

To prepare the [m-(acetylpyridinio)-n-alkyl]adenosine pyrophosphates, 10 mmoles of acetylpyridinio-n-alkylphosphoric acid and 10 mmoles of adenosine 5 -phosphoromorpholidate (as salt of 4-morpholine iV,iV -dicyclohexylcarboxamidine) are dissolved in 20 ml of freshly distilled o-chlorophenol. The mixture is stored at room temperature for 7 days. Progress of the condensation reaction is checked daily by paper electrophoresis (0.1 M Tris chloride at pH 8.1, 30 V/cm). After the reaction is completed, 60 ml of water are added to the mixture and the suspension is extracted 3 times, each with 200 ml of ethyl ether. The first ether extract obtained is reextracted with 30 ml of water. The combined aqueous phases are reduced to a volume of 5 ml under reduced pressure and are charged on to a Dowex 1X8 column (formate form, 2 X 50 cm, 100-200 mesh). The column is washed with 6.5 liters of water, and the coenzyme analogs are eluted from the resin with a convex gradient of formic acid ... 

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