Preparation of triphosphate
analogues:
ddTTP, or dideoxythymidine, had already been previous described, and first used in 1969, and therefore was available.
ddA had already been prepared by McCarthy
et al in
1966, and just needed to be converted into a triphospate.
This method was already in existence, and was being done by Tener
(1961), and Hoard and Ott (1965), and
therefore the
creation of ddATP wasn’t too complex.
Neither ddCTP not ddGTP have been performed previously. Sanger et al (1977), simply used the method described for ddATP to create ddGTP and ddCTP.
N-isobutyryl-5’-O-monomethoxytrityldeoxyguanosine was the starting material (this was prepared by F.E Barelle). Tosylation, or the addition of a toluene-4-sulphonyl group, of the 3’ OH group results in 2’3’-didehydro derivative after the removal of the group. If the isobutrylisobutryl group is not entirely removed, incubation of the molecule and NH3 ensure that it is.The didehydro derivative is reduced to a dideoxyderiative, and then converted to a triphospate. The product is purified by a DEAE-Sephadex column
ddCTP:
N-anisyl-5’-O-monomethoxytrityldeoxycytidine was used as the starting material, and the previous method was followed. The purification method was omitted due to low yield.
An attempt was made to prepare a solution from the intermediate: didehydrodideoxycytidine as Atkinson et al (1969) demonstrated its inhibitory ability, however this was unsuccessful. Therefore araCTP was obtained.