The thermochemistry and transition states of the electrocyclic ring closures of the resonance-stabilized 1,4-pentadienyl radical to cyclopenten-3-yl, cyclobut-2-enylmethyl, and 2-vinylcyclopropyl are investigated at Hartree-Fock and coupled-cluster levels of theory. The CCSD(T)//QCISD/cc-pVDZ calculations predict activation barriers of 130, 169, and 236 kJ/mol, respectively, and DeltaH values of -60, 115, and 155 kJ/mol. Experimental evidence for the appearance of vinylcyclopropyl following photolytic generation of pentadienyl is more likely the result of a distinct electrocyclic reaction than quenching of a two-step mechanism for formation of cyclopentenyl. Higher energy pathways for formation of polycyclic structures are also briefly examined.
An allylic adenosine triphosphate analog (AATP) was tested as a substrate for commercially available DNA polymerases. All but one of the enzymes assayed incorporated AATP opposite thymidine (T) with concomitant termination of the elongation reaction. A concentration of only 1 microM was sufficient for complete termination of the polymerization reaction for a short template mediated by Ampli Taq DNA polymerase FS (Taq FS). This result suggests that AATP could be used as a 2',3'-dideoxyadenosine-5'-triphosphate (ddA) surrogate. Kinetics of incorporation revealed that AATP was 48 times less efficiently incorporated than ddA. Furthermore, AATP was used in dye-primer sequencing as a substitute for ddA.
Nucleoside triphosphates I with 3'-O-blocking groups that are both photolabile and fluorescent were required to investigate the viability of a strategy for sequencing DNA in a combinatorial fashion (see Figure 1). Four compounds were prepared to realize this goal. Two of them, 14 a and 14 t, had dansyl-functionalized, 3'-O-(2''-nitrobenzyl) ether groups, while the other two, 18 a and 18 t, had similar pendant carbonate groups. Tests for incorporation of these analogues were performed by using five different DNA replicating enzymes, but the analogues were not incorporated. These results were surprising in view of the fact that previous studies had shown that 3'-O-(2''-nitrobenzyl)adenosine triphosphate II was incorporated by Bst DNA polymerase I. However, molecular simulations with the coordinates of a T7 polymerase crystal structure as a model demonstrates that analogues 14 a, 14 t, 18 a and 18 t are too large to fit into the enzyme active site, whereas accommodation of the unsubstituted 2-nitrobenzyl compound II is much less demanding. We conclude that both the nucleoside triphosphates and the DNA polymerase enzyme must be modified if the proposed DNA sequencing scheme is to be viable.
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