2- Cyanoethyl phosphate

Cyanoethyl phosphates are usually prepared by the phosphoramidite method using 2-cyanoethyl tyN-diisopropylchlorophosphoramidite,66 67 2-cyanoethyl N.tyN pN -tetraisopropylphosphordiamidite5 or bis(2-cyanoethyl) MN-diisopro-pylchlorophosphoramidite.71... 

Attempts to perform the phosphorylation step with either pyrophosphoryl chloride or 2-cyanoethyl phosphate failed. ... 

Protective group for nucleosides. Stepwise synthesis of oligonucleotides utilizing 2-cyanoethyl phosphate (1, 172-173) requires protection of the 3 -hydroxyl group which can be cleaved under essentially neutral conditions, since a methoxy-trityl ether is sensitive to acid and a /3-cyanoethyl phosphoric ester is sensitive to alkali. The /3-benzoylpropionyl group meets these requirements, since it is quantitatively cleaved by dilute solutions of hydrazine hydrate in pyridine-acetic acid. The esters are prepared by condensation with DCC (dicyclohexylcarbodiimide). Definitive paper R. L. Letsinger and P. S. Miller, Am. Soc., 91, 3356 (1969)... 

A most useful method of phosphorylation of nucleosides was developed by Gilham and Tener. - 2-Cyanoethyl phosphate (64) and an excess of... 

TV jTV -dicyclohexylcarbodiimide (65) reacted with 2, 3 -0-isopropyIidene acetals of nucleosides to fom 5 -(2-cyanoethyl phosphate) derivatives... 

In the iV,iV -dicyclohexylcarbodiimide-alkyl phosphate reaction described here, 2-cyanoethyl phosphate (64) offers an additional advantage, as the 2-cyanoethyl protecting group is readily eliminated by mild, alkaline hydrolysis, as shown in the conversion of (77) (see Scheme 3, R == nucleoside residue) into... 

Deoxycytidine was fully acylated with anisoyl chloride in pyridine to (117), and this was converted into A -anisoyl-2 -deoxycytidine (118). The mono-O-acyl derivative was treated with chloro-bis(p-methoxyphenyl)-phenylmethane (119) in pyridine, to yield 120, which was phosphorylated with 2-cyanoethyl phosphate and (65) in pyridine to the phospho-diester (121) in high yield. Removal of the protecting groups was achieved in two ways. One method involved the treatment of (121) with aqueous ammonia to give (122) which, on treatment with 80% acetic acid at room temperature for twenty minutes, afforded 2 -deoxycytidine 3 -phosphate... 

The procedures used for the synthesis of nucleotides of 2 -deoxycytidine were successfully applied to the preparation of their ptirine counterparts. 2 -Deoxyadenosine was fully benzoylated and then converted with alkali into the A -benzoyl derivative. Tritylation with (119) yielded (125), which was converted by the 2-cyanoethyl phosphate-iV,iV -dicyclohexyl-carbodiimide method, followed by removal of the protecting groups, into 2 -deoxyadenosine 3 -phosphate (126). With 2 -deoxyguanosine, direct tritylation with (119) gave (127), from which 2 -deoxyguanosine 3 -phos-phate (128) was obtained. ... 

Uridine 3 -phosphate (147) was prepared by Hall and Thedford by the phosphorylation of 2, 5 -di-0-trityluridine with 2-cyanoethyl phosphate and iVjiV -dicyclohexylcarbodiiinide to give the diester (146). This diester was de-esterified with alkali, and then hydrolyzed with 80% acetic acid at 100° for 90 minutes, to give (147). The structure of (14S) had been rigorously established by Yung and Fox, who methanesulfonated (145) to obtain (148). Treatment of (148) with sodium benzoate in N,N-di-methylformamide gave the 2,3 -anhydronucleoside (149). The anhydro... 

Treatment of (152) with 2-cyanoethyl phosphate and A, A -dicyclo-hexylcarbodiimide in pyridine yielded the diester (153) which was converted by 7 iV ammonium hydroxide (at 60° for 3 hours) into cytidine 2 -phosphate (154), exclusively. When this procedure was performed with the 2, 5 -dibenzoate (155), cytidine 3 -phosphate (157) was obtained, along with about 20% of the 2 -phosphate (154). It is clear that, in the reaction of (153) to give (154), 2-cyanoethyl elimination [see Scheme 3 (77)— (78)] preceded debenzoylation this accounts for the fact that (154) was obtained without contamination by cytidine 3 -phosphate. In the reaction (156)- (1S7), the authors suggested that some debenzoyla-... 

D-lyxofuranosyl)uracil (185) with 2-cyanoethyl phosphate and N,N -dicyclohexylcarbodiimide to give the 2 (3 )-phosphates (186), which were converted into l-/S-D-lyxofuranosyluracil 2 3 -cyclic phosphate (187). Conversion of (185) into (188) was accomplished by a five-step process (acetonation, debenzoylation, phosphorylation, deacetonation, and de-cyanoethylation). Treatment of (188) with J r,i T -dicyclohexylcarbodi-imide in pyridine gave a mixture of the 3 5 - and 2 5 -cycIic phosphates (189) and (190) of l-j3-D-lyxosyluracil, which differed in properties from the 2 3 -cyclic phosphate derivative (187) and did not consume metaperiodate. These cyclic phosphates (189) and (190) were separated. Only one of them was converted into the other by mild treatment with acid... 

The deprotection of crude oligodeoxynucleotides traditionally requires a basic treatment that allows the concomitant removal of the exocyclic acyl protecting groups, -elimination of the 2-cyanoethyl phosphate protecting group, and cleavage of the succinic ester bond that links the oligonucleotide to the solid support (Fig. 9). 

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