Complete template-directed enzymatic synthesis of a potential antisense DNA containing 42 methylphosphonodiester bonds.

P alpha-Methyl thymidine triphosphate was prepared through the pyrophosphorolysis of P alpha-methyl thymidine diphosphate P beta-diphenyl ester and tested as an alternative substrate for E. coli DNA polymerase 1 (Klenow fragment) using several template-primer systems requiring the formation of 1 to 42 methylphosphono diester bonds. The enzyme catalyzes the incorporation of a P-methyl thymidylic residue with (Sp)-configuration at a single site in a recessive 3'-end as well as at multiple sites along a growing 167 nucleotide long chain. The synthesis of a full length product, containing 42 sites of methylphosphonate incorporation was observed.