Derivatives of thymidine containing o-carboranylalkyl groups at the N-3 position and derivatives of 2'-deoxyuridine containing o-carboranylalkylmercapto groups at the C-5 position were synthesized. The alkyl spacers consist of 4-8 methylene units. The synthesis of the former compounds required 3-4 reaction steps in up to 75% overall yield and that of the latter 9-10 reaction steps with significantly lower overall yield. Derivatives of thymidine substituted with carboranylalkyl substituents at the N-3 position and short spacers were phosphorylated by both recombinant and purified cytosolic thymidine kinase (TK1) to a relatively high degree. None of the tested 2'-deoxyuridine derivatives possessing carboranyl substituents at the C-5 position were phosphorylated by either recombinant or purified TK1. The amounts of phosphorylation product detected for some of the C-5-substituted nucleosides with recombinant mitochondrial thymidine kinase (TK2) were low but significant and decreased with increasing lengths of the alkyl spacer. The data obtained in this study do not seem to support the tether concept applied in the synthesis of the new C-5- and N-3-substituted carboranyl nucleosides intended to reduce possible steric interference in the binding of carboranyl nucleosides with deoxynucleoside kinases. Instead, it appeared that a closer proximity of the bulky carborane moiety to the nucleoside scaffold resulted in better substrate characteristics.

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Examination of conserved motifs on the cloned subunits of the deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) of Lactobacillus acidophilus R-26 has begun with the Asp-Arg-Ser (DRS) motif. Replacement of Asp-78 of both subunits with Glu, Ala, or Asn reduced dGK and dAK activities to less than 0.2%, whereas replacement of Arg-79 with Lys, either on both subunits in tandem (R79K), or on the dGK subunit only (R79K:dGK), yielded active but kinetically modified enzymes. These were partially purified, and their kinetic and regulatory properties were analyzed. For dAK activity, the Vmax of the R79K:dGK enzyme was increased 28-fold, with no change in the limiting Km for dAdo, but with a slightly reduced Km for MgATP. The V/K efficiency ratio of dAK was also increased 29-fold, but that of dGK was decreased to 5-10% due to a 10-fold increase in Km for dGuo and a reduced Vmax. Therefore, the R79K substitution seems to have a greater effect on dGuo binding than on that of dAdo, but dGK modification appears to produce a stimulatory conformational effect on the opposite subunit, resembling the known unidirectional activation of dAK by either dGuo or dGTP.