31. SYNTHESIS OF POLYNUCLEOTIDES 117 



5). The method of phosphorylation using (8-cyanoethyl phosphate and 

 dicyclohexylcarbodiimide gives the /8-cyanoethyl phosphate' ester (XXI), 

 from which the cyanoethyl group is removed by mild alkaline treat- 

 ment. 



3. The Specific Synthesis of Interribonucleotide Bonds 



The problem of the specific synthesis of the C 3 '-C B ' or C 2 '-C 6 ' interribo- 

 nucleotide linkages is complicated by the presence of the vicinal m-hy- 

 droxyl group in ribonucleotides. It is the same factor which caused so much 

 difficulty in the elucidation of the nature of the internucleotide linkage in 

 the ribonucleic acids (Volume I, Chapter 12). The requirement is to prepare 

 appropriately blocked ribonucleoside derivatives, the protecting groups used 

 being such that they do not migrate to the adjacent hydroxyl group during 

 synthesis and can be removed at the end without damage to the phospho- 

 diester bonds synthesized. 



a. Ademjlyl-(2' -> 5')-uridine (XXVII) 22 



The synthesis of this substance in low (8%) yield was accomplished as 

 illustrated below (Scheme 6). 3',5'-Di-0-acetyladenosine (XXIII) was pre- 

 pared from adenosine and converted to the phosphite (XXIV). Chlorination 

 gave the corresponding phosphorochloridate (XXV) which served as the 

 phosphorylating agent for 2',3'-di-0-acetyluridine (XXVI). 



b. Uridylyl-(3' -+ 5')-uridine (XXXIII) 



Much greater interest clearly resides in the development of approaches 

 to the synthesis of ribo-oligonucleotides containing specifically the naturally 

 occurring C 3 '-C 5 < interribonucleotide linkages. A promising approach is 

 outlined in Scheme 7 and has been used in the synthesis of uridylyl-(3' — > 

 5')-uridine (XXXIII). 23 Uridine-5'-phosphate is converted in high yield to 

 uridine-3',5'-cyclic phosphate (XXVIII) in extremely dilute solution by 

 reaction with dicyclohexylcarbodiimide. Reaction of the cyclic phosphate, 

 as the free acid, with dihydropyran in dioxane gives the 2'-0-tetrahydro- 

 pyranyl ether (XXIX) quantitatively, which hydrolyses readily on being 

 heated in barium hydroxide, to a mixture of the 3'- and 5 '-phosphates 

 (XXX and XXXI) respectively, the former predominating. On treatment 

 of the mixture with triphenylmethyl chloride in pyridine, only the 3 '-phos- 

 phate derivative (XXX) reacts and the resulting (XXXII) can be readily 

 separated, by partition chromatography, from (XXXI), which itself is a 

 useful synthetic intermediate. The condensation of (XXXII) with 2',3'-di- 

 O-acetyluridine (XXVI) followed by mild alkaline and acidic treatments 

 gives uridylyl-(3' — » 5')-uridine (XXXIII) in high yield. 



22 A. M. Michelson, L. Szabo and A. R. Todd, J. Chem. Soc. p. 1546 (1956). 



23 M. Smith and H. G. Khorana, J. Am. Chem. Soc. 81, 2911 (1959). 



