A r&able prqxwation of S%yanm~y~4-th ad its incorporation into oligoribnucleoddes is reported.
Deproteciion qf oligo&otuuleotidks with DBU in acetonitrile followed by methanolic amraonio allmvs the use of standard N-benwyl and N-isoburyrylp7otectedp~~~r~t~. Cleavage of hammerhead rikqvmes using GCGCCGAAACACCGUGPUJCUCGAGC as the e&&d substrate and GGCUCGACUGAUGAGGCGC as rhe ribozyme resulted in a halving of the cleavage rate wher compared to the ummdifkd substrate, which is consistent with the proposal that the As-U1 7 base pair plays a key role in the active structure.
Syntktically.
sulphur has been incorporated into the phosphate backbonel2. sugars and basest of oligonucleotides. Two major approaches for incorpomting tbi&ases into oligodeoxynucleotides, using the standard phosphoramidlte approach, are currently in use. The first involves incmpomtion of either a S-cyanoethy14-6 or S-pivaloyloxymethy17.* protected thiopyrimidine or thiopurine into the growing oligonucleotide, followed by &protection. The other involves the incorpomdon of a suitably modified nucleotide which can be further altered after its incorporation into the oligonucleotide. Using this method a number of modified oligonucleotides can be pmpared from the same synthesis 9. 4-Thiopytimidlnes and 6-thiopurines have been used for post-synthetic modificatiot@ and photo cross-linkingttv*2.
The thione functionality also has different H-bonding properties to the carbonyl group and so can be used to p&e protein-nucleic acid and oligonucleotide-oligonucRot.ide interactions.
Most of the above moditications have heen on oligodeoxynucleotides, We report here the synthesis of S4-cyanoethyl-4thiouridlne and to our knowledge the first reported incorporation of Qthiourldine into a synthetic oligoribonucleotide, namely the 24-mer ribozyme substrate GCGCCGAAACACCGUG[~UJ-CUCGAGC, which is selectively cleaved at ~1*-~19 by a 19nucleotide ribozyme13 (Fig. ). Its rate of cleavage is compared with that of the wild-type strand In a preliminary experiment 4thiouridine was also * mcorpomted into the dirner [4SLJIG. Coleman and Siedlecki 14 have reported the synthesis of S4-cyanoethyl-protected 4-thiouridine and 2'-deoxy-dthio-