Interaction of transuranium elements with biologically important ligands: structural and spectroscopic evidence for nucleotide coordination to plutonium.

The first complex of a transuranium element (tetravalent plutonium) with nucleotide (deoxycytidinemonophosphate, dCMP) was synthesized and structurally characterized. The crystal structure of [Pu(4)(NO(3))(8)(HdCMP)(4)(H(2)O)(8)](NO(3))(4).2H(2)O consists of complex cations [Pu(4)(NO(3))(8)(HdCMP)(4)(H(2)O)(8)](4+), NO(3)(-) anions, and water molecules. There are two crystallographically independent Pu atoms in the structure, both having similar surroundings. Each of the Pu atoms is coordinated by three O atoms of phosphate groups belonging to three different (HdCMP)(-) anions, two bidentate nitrate anions, and two water molecules. The crystal structure is confirmed by IR and UV/vis/near-IR spectroscopic data.

5-Substituted N(4)-hydroxy-2'-deoxycytidines and their 5'-monophosphates: synthesis, conformation, interaction with tumor thymidylate synthase, and in vitro antitumor activity.

Convenient procedures are described for the synthesis of 5-substituted N(4)-hydroxy-2'-deoxycytidines 5a,b,d-h via transformation of the respective 5-substituted 3', 5'-di-O-acetyl-2'-deoxyuridines 1a-c,e-h. These procedures involved site-specific triazolation or N-methylimidazolation at position C(4), followed by hydroxylamination and deblocking with MeOH-NH(3). Nucleosides 5a,b,d-h were selectively converted to the corresponding 5'-monophosphates 6a,b,d-h with the aid of the wheat shoot phosphotransferase system. Conformation of each nucleoside in D(2)O solution, deduced from (1)H NMR spectra and confirmed by molecular mechanics calculations, showed the pentose ring to exist predominantly in the conformation S (C-2'-endo) and the N(4)-OH group as the cis rotamer. Cell growth inhibition was studied with two L5178Y murine leukemia cell lines, parental and 5-fluoro-2'-deoxyuridine (FdUrd)-resistant, the latter 70-fold less sensitive toward FdUrd than the former. With FdUrd-resistant L5178Y cells, 5-fluoro-N(4)-hydroxy-2'-deoxycytidine (5e) caused almost 3-fold stronger growth inhibition than FdUrd; 5e was only some 3-fold weaker growth inhibitor of the resistant cells than of the parental cells. Thymidylate synthase inhibition was studied with two forms of the enzyme differing in sensitivities toward 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), isolated from parental and FdUrd-resistant L1210 cell lines. All N(4)-hydroxy-dCMP (6a,b,d-h) and dUMP analogues studied were competitive vs dUMP inhibitors of the enzyme. Analogues 6b,d-h and 5-hydroxymethyl-dUMP, similar to N(4)-hydroxy-dCMP (6a) and FdUMP, were also N(5), N(10)-methylenetetrahydrofolate-dependent, hence mechanism-based, slow-binding inhibitors. 5-Chloro-dUMP, 5-bromo-dUMP, and 5-iodo-dUMP, similar to dTMP, did not cause a time-dependent inactivation of the enzyme. Instead, they behaved as classic inhibitors of tritium release from [5-(3)H]dUMP. 5-Bromo-dUMP and 5-iodo-dUMP showed substrate activity independent of N(5), N(10)-methylenetetrahydrofolate in the thymidylate synthase-catalyzed dehalogenation reaction. The =N-OH substituent of the pyrimidine C(4) prevented the enzyme-catalyzed release from the C(5) of Br(-) and I(-) (the same shown previously for H(+)). While FdUMP and 6a showed a higher affinity and greater inactivation power with the parental cell than FdUrd-resistant cell enzyme, an opposite relationship could be seen with 5-hydroxymethyl-dUMP.

Properties of an affinity-column-purified human deoxycytidylate deaminase.

Deoxycytidylate deaminase was purified about 7000-fold to homogeneity from a human source (HeLa cells). The final step in the purification employed an affinity column, which increased the specific activity of the enzyme from the previous step by 500-fold. Similar to most other dCMP deaminases, this enzyme is allosterically regulated by microM levels of dCTP and dTTP. However, unlike the other enzymes the most dramatic allosteric responses occur at substrate levels of 0.1 mM dCMP or less, where at least a 10-fold increase in activity is effected by dCTP. The enzyme is particularly sensitive to inhibition by dTTP with 50% inhibition being obtained at 1.5 x (10(-6) M in the absence of dCTP. Antibody to the human enzyme did not cross-react with a dCMP deaminase induced in Escherichia coli by T4-bacteriophage, nor did antibody to the phage-induced enzyme cross-react with the human deaminase. A potential transition-state analogue of the substrate, 2'-beta-D-deoxyribose-pyrimidin-2-one 5'-phosphate was prepared, and found to inhibit dCMP deaminase competitively with a Ki of 1.2 x 10(-8) M.

Quantitative and kinetic examination of 32P-postlabeling of etheno-substituted nucleotides.

1,N6-ethenodeoxyadenosine-, 1,N2-ethenodeoxyguanosine- and 3,N4-ethenodeoxycytidine-3'-monophosphates were labeled by [gamma-32P] ATP using T4 polynucleotide kinase in conditions commonly used for the 32P-postlabeling assay. Kinetic studies showed that the reaction is fast reaching a plateau after 15-30 min. The efficiency of phosphorylation, as studied by substrate-product concentration dependency, was between 50-100% at the lower substrate concentrations. The adducts are labeled efficiently at sub-femtomole levels. All the adducts were sensitive to the 3'-dephosphorylation by P1 nuclease although the guanine derivative appeared to be more resistant than the two other adducts.

NMR study of stacking interactions and conformational adjustments in the dinucleotide-carcinogen adduct 2'-deoxycytidylyl-(3----5)-2'-deoxy-8-(N-fluoren-2-ylac etamido)guanosi ne.

The conformation and dynamics of the dinucleotide d-CpG modified at the C(8) position of the guanine ring by the carcinogen 2-(acetylamino)fluorene has been investigated by high-field 1H NMR spectroscopy. A two-state analysis of chemical shift data has enabled estimation of the extent of intramolecular stacking in aqueous solution as a function of temperature. The stacking, which is mostly fluorene-cytosine, is virtually complete in the low-temperature range. The 500-MHz 1H NMR spectrum consists of two subspectra near ambient temperatures due to a 14.3 +/- 0.3 kcal/mol barrier to internal rotation about the amide bond in the stacked form. A large barrier to internal rotation about the guanyl-nitrogen bond at C(8) has also been ascertained, but separate NMR subspectra were not detected due to the predominance of one of the torsional diastereomers (alpha' = 90 degrees) in the fully stacked state. Problems of self-association and chemical exchange were identified and overcome to enable analysis of the sugar-phosphate backbone conformation utilizing coupling constants. For the exocyclic C(4')-C(5') bond of the deoxyguanosine moiety, there is a high gauche+ (gamma = 60 degrees) conformer population, which is uncommon for a purine nucleotide with a syn orientation about the glycosyl bond. The gauche- conformation (gamma = 300 degrees), which is normally present in syn purine nucleotides in solution, was not detected. The exocyclic C(5')-O(5') torsion of the deoxy-guanosine moiety remains near the classical energy minimum (beta = 180 degrees) in the major stacked conformations. The sugar ring of the deoxycytidine moiety is predominantly in the C2'-endo conformation, while the deoxyguanosine ring is a mixture of conformations, one of which appears to be unusually puckered. The results support intercalation models of modified DNA and suggest a looped-out structure, with the modified guanine being the first base in the loop. Such structures could explain the relatively rapid rate of repair and the frame-shift mutations of this type of adduct.

Interaction of N4-hydroxy-2'-deoxycytidylic acid with thymidylate synthetase.

The interaction of dTMP synthetase with N4-hydroxy-2'-deoxycytidylate (N4-HOdCMP) has been investigated. With use of standard assay conditions, N4-HOd-CMP is a competitive inhibitor with an apparent Ki of 8.0 microM. Incubation of N4-HOdCMP with dTMP synthetase in the presence of 5,10-methylenetetrahydrofolate (CH2-H4folate) resulted in a rapid time-dependent inactivation of the enzyme which was not first order and the formation of complexes which could be isolated on nitrocellulose filter membranes. With use of radioactive ligands, the isolable native complex was shown to possess 2 mol of N4-HOdCMP and 2 mol of CH2-H4folate/mol of dimeric enzyme; the apparent dissociation constant of N4-HOdCMP was 1.0 microM. Ultraviolet difference spectroscopy of the ternary complex showed a loss of the pyrimidine chromophore which did not reappear upon denaturation with NaDodSO4. The rate of dissociation of N4-HOdCMP from the ternary complex was biphasic in which one-half of the initially bound ligand dissociated with t 1/2 congruent to 2.3 min and the remainder with t 1/2 congruent to 13 min. When the N4-HOdCMP-CH2-H4folate-enzyme complex was denatured, one-half of the CH2-H4folate dissociated whereas all of the N4-HOdCMP remained bound to the enzyme. Taken together, our results indicate that N4-HOdCMP forms a covalent bond with dTMP synthetase and reveal an unusual asymmetry in the two subunits of the N4-HOdCMP-CH2-H4folate-enzyme complex. It appears that one subunit is covalently bound to N4-HOdCMP, which, in turn, is covalently linked to CH2-H4folate whereas the other subunit is covalently bound to N4-HOdCMP but CH2-H4folate is bound by noncovalent interactions.