3,N(4)-ethano-2'-deoxycytidine: chemistry of incorporation into oligomeric DNA and reassessment of miscoding potential.

3,N(4)-Ethano-2'-deoxycytidine (ethano-dC) may be incorporated successfully into synthetic oligodeoxynucleotides by omitting the capping procedure used in the automated DNA synthetic protocols immediately after inserting the lesion and in all iterations thereafter. Ethano-dC is sensitive to acetic anhydride found in the capping reagent, and multiple oligomeric products are formed. These products were identified by examining the reaction of ethano-dC with the capping reagent, and several acetylated, ring-opened products were characterized by electrospray mass spectrometry and collision induced dissociation experiments on a tandem quadrupole mass spectrometer. A scheme for the formation of the acetylated products is proposed. In addition, the mutagenic profile of ethano-dC was re-examined and compared to that for etheno-dC. Ethano-dC is principally a blocking lesion; however, when encountered by the exo(-)Klenow fragment of DNA polymerase, dAMP (22%), TMP (16%), dGMP (5.3%) and dCMP (1.2%) were all incorporated opposite ethano-dC, along with an oligomer containing a one-base deletion (0.6%).

Synthesis of enzymatically noncleavable carbocyclic nucleosides for DNA-N-glycosylase studies.

Carbocyclic nucleosides have been of great interest as antiviral agents and in studies in the area of antisense technology. The recent finding that the replacement of a single 2'-deoxynucleoside in DNA by a carba analogue does not alter the Watson-Crick base pairing, yet at the same time provides a chemically and enzymatically stable "glycosidic" linkage, led us to examine this class of compound as enzyme inhibitors of the DNA-repair enzymes involved in oxidative damage. We now report the synthesis and incorporation into oligomeric DNA via suitable derivatives, the carbanucleosides 8-oxo-7,8-dihydro-2'-deoxycarbainosine, 8-oxo-7,8-dihydro-2'-deoxycarbaguanosine, and 2'-deoxyaristeromycin. Aristeromycin (1) was deoxygenated at the 2'-position as follows. Treatment of 1 with TPDSCl2 gave the 3',5'-protected derivative 3 (76%) which on phenylthiocarbonylation at the 2'-position gave 4 in 51% yield. The latter compound on reduction with Bu3SnH led to the 2'-deoxy derivative 5 (90%). Benzoylation followed by deprotection with TBAF in THF then gave the desired intermediate (6) in 65% yield. N2-Isobutyryl-8-oxo-7,8-dihydro-2'-deoxycarbaguanosine (16) was synthesized from 3-chloro-2'-deoxycarbainosine (9). Treatment of 9, either with hydrazine followed by catalytic reduction of the 2-hydrazino derivative or with 1-(2-nitrophenyl)ethylamine followed by photolysis of the resulting 2-substituted derivative, in both instances gave the desired 2'-deoxycarbaguanosine (12) in approximately 50% overall yield in each case. Bromination of 12 gave 13 (90%) which, when treated with BnONa in DMSO at 65 degrees C, led to the 8-benzyloxy derivative 14 (46%). Isobutyrylation of 14 followed by catalytic reduction then afforded 16. 8-Oxo-7,8-dihydro-2'-deoxycarbainosine (23) was prepared in four steps. Bromination of 2'-deoxyaristeromycin (19) at the 8-position gave 20 (> 95%) which was converted to the 8-benzyloxy derivative 21 (61%) using BnONa/DMSO at 80 degrees C. Reductive debenzylation of 21 then led to 8-oxo-7,8-dihydro-2'-deoxyaristeromycin (approximately 100%) which, when treated with adenosine deaminase, provided the desired carbainosine derivative 23 in quantitative yield. Compounds 6, 16, and 23 were converted to their respective 5'-O-DMT, 3'-O-[(2-cyanoethoxy)-(N,N-diisopropylamino)phosphine] derivatives (8, 18, and 25) in excellent overall yields. The latter were then used to synthesize a series of DNA oligomers by automated procedures.