Synthesis of 3'-Thioamido-Modified 3'-Deoxythymidine 5'-Triphosphates by Regioselective Thionation and Their Use as Chain Terminators in DNA Sequencing

Abstract

The thioamide derivatives 3′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-[(2-methyl-1-thioxopropyl)amino]thymidine (4a) and 3′-deoxy-5′-O-(4,4′-dimethoxytrityl)-3′-{{6-{[(9H-(fluoren-9-ylmethoxy)carbonyl]amino}-1-thioxohexyl}amino}thymidine (4b) were synthesized by regioselective thionation of the corresponding amides 3a and 3b with 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane 2,4-disulfide (Lawesson's reagent). The addition of exact amounts of pyridine to the reaction mixture proved to be essential for an efficient transformation. The thioamides were converted into the corresponding 5′-triphosphates 6a and 6b. Compound 6a was chosen for DNA sequencing experiments, and 6b was further labelled with fluorescein (8).