The effects of 5R-5,6-dihydro-5-hydroxythymidine on duplex DNA stability and structure.

An improved method for the chemical synthesis of oligodeoxynucleotides containing 5 R -5,6-dihydro-5-hydroxythymidine (1) at a defined site is reported. UV melting studies carried out on duplexes containing1synthesized in this manner correlate with previously reported enzyme inhibition experiments, as well as computational studies. The melting experiments suggest that1destabilizes duplex DNA, but that the lesion preferentially base pairs to deoxyadenosine. These experiments also suggest that the presence of1in a duplex disrupts base pairing at the 5'-adjacent nucleotide and results in the thermally preferred misincorporation of purines opposite the 5'-deoxyadenosine stacked above 1 at this position. Despite the disruptions in base stacking, the UV melting experiments and enzymatic ligation/electrophoretic migration assays are consistent with the predicted macroscopic duplex structure containing intrahelical nucleotides.

The reactivity of the 2-deoxyribonolactone lesion in single-stranded DNA and its implication in reaction mechanisms of DNA damage and repair.

The formal C1'-oxidation product, 2-deoxyribonolactone, is formed as a result of DNA damage induced via a variety of agents, including gamma-radiolysis and the enediyne antitumor antibiotics. This alkaline labile lesion may also be an intermediate during DNA damage induced by copper-phenanthroline. Oligo-nucleotides containing this lesion at a defined site were formed via aerobic photolysis of oligonucleotides containing a photolabile ketone, and were characterized by gel electrophoresis and electrospray mass spectrometry (ESI-MS). Treatment of oligo-nucleotides containing the lesion with secondary amines produces strand breaks consisting of 3'-phosphate termini, and products which migrate more slowly in polyacrylamide gels. MALDI-TOF mass spectrometry analysis indicates that the slower moving products are formal adducts of the beta-elimination product resulting from 2-deoxyribonolactone and one molecule of amine. The addition of beta-mercapto-ethanol to the reaction mixture produces thiol adducts as well. The stability of these adducts suggests that they cannot be the labile species characterized by gel electrophoresis in copper-phenanthroline-mediated strand scission. The characterization of these adducts by mass spectrometry also provides, by analogy, affirmation of proposals regarding the reactivity of nucleophiles with the beta-elimination product of abasic sites. Finally, the effects of this lesion and the various adducts on DNA repair enzymes are unknown, but their facile generation from oligonucleotides containing a photolabile ketone suggests that such issues could be addressed.

DNA damage induced via independent generation of the radical resulting from formal hydrogen atom abstraction from the C1'-position of a nucleotide.

BACKGROUND: Deoxyribonucleotide radicals resulting from formal C1'-hydrogen atom abstraction are important reactive intermediates in a variety of DNA-damage processes. The reactivity of these radicals can be affected by the agents that generate them and the environment in which they are produced. As an initial step in determining the factors that control the reactivity of these important radical species, we developed a mild method for their generation at a defined site within a biopolymer. RESULTS: Irradiation of oligonucleotides containing a photolabile nucleotide produced C1'-DNA radicals. In the absence of potential reactants other than O2, approximately 90% of the damage events involve formation of alkaline-labile lesions, with the remainder resulting in direct strand breaks. The ratio of alkaline-labile lesions to direct strand breaks (approximately 9:1) is independent of whether the radical is generated in single-stranded DNA or double-stranded DNA. Strand damage is almost completely quenched under anaerobic conditions in the presence of low thiol concentrations. Competition studies with O2 indicate that the trapping rate of C1'-DNA radicals by beta-mercaptoethanol is approximately 1.1 x 10(7) M-1s-1. CONCLUSIONS: The mild generation of the C1'-DNA radical in the absence of exogenous oxidants makes it possible to examine their intrinsic reactivity. In the absence of other reactants, the formation of direct strand breaks from C1'-radicals is, at most, a minor pathway. Competition studies between beta-mercaptoethanol and O2 indicate that significantly higher thiol concentrations than those in vivo or some means of increasing the effective thiol concentration near DNA are needed for these reagents to prevent the formation of DNA lesions arising from the C1'-radical under aerobic conditions.

The effects of secondary structure and O2 on the formation of direct strand breaks upon UV irradiation of 5-bromodeoxyuridine-containing oligonucleotides.

BACKGROUND: 5-Bromodeoxyuridine is a radiosensitizing agent that is currently being evaluated in clinical trials as an adjuvant in the treatment of a variety of cancers. gamma-Radiolysis and UV irradiation of oligonucleotides containing 5-bromodeoxyuridine result in the formation of direct strand breaks at the 5'-adjacent nucleotide by oxidation of the respective deoxyribose. We investigated the effects of DNA secondary structure and O2 on the induction of direct strand breaks in 5-bromodeoxyuridine-containing oligonucleotides. RESULTS: The efficiency of direct strand break formation in duplex DNA is dependent upon O2 and results in fragments containing 3'-phosphate and the labile 3'-ketodeoxyadenosine termini. The ratio of the 3'-termini is also dependent upon O2 and structure. Deuterium product isotope effects and tritium-transfer studies indicate that hydrogen-atom abstraction from the C1'- and C2'-positions occurs in an O2- and structure-dependent manner. CONCLUSIONS: The reaction mechanisms by which DNA containing 5-bromodeoxyuridine is sensitized to damage by UV irradiation are dependent upon whether the substrate is hybridized and upon the presence or absence of O2. Oxygen reduces the efficiency of direct strand break formation in duplex DNA, but does not affect the overall strand damage. It is proposed that the sigma radical abstracts hydrogen atoms from the C1'- and C2'-positions of the 5'-adjacent deoxyribose moiety, whereas the nucleobase peroxyl radical selectively abstracts the C1'-hydrogen atom from this site. This is the second example of DNA damage amplification by a nucleobase peroxyl radical, and might be indicative of a general reaction pattern for this family of reactive intermediates.

Synthesis of modified oligodeoxyribonucleotides on a solid-phase support via derivatization of a selectively revealed 2'-amino-2'-deoxyuridine.

[formula: see text] High yields of oligodeoxyribonucleotides modified at the C2'-position of site specifically incorporated 2'-amino-2'-deoxyuridine are obtained by photolytically unmasking the nucleophile in an otherwise protected solid-phase support-bound biopolymer.

Synthesis and deprotection of oligonucleotides under aprotic conditions.

Oligonucleotides containing an alkali-labile nucleotide are synthesized and deprotected using a synthetic method that eliminates the use of Bronsted acid and base. The development of a new family of exocyclic amine-protecting groups is an integral component of this approach.

Prevention of obesity in Avy/a mice by dehydroepiandrosterone.

Dehydroepiandrosterone, a mammalian glucose-6-phosphate dehydrogenase inhibitor, prevented Avy/a mice from becoming obese. Decreased accumulation of triacylglycerol accounted for a large portion of the weight difference between treated and control Avy/a mice. Hepatic lipogenesis as measured by 3H2O incorporation into total lipid was less in the dehydroepiandrosterone-treated mice. Dehydroepiandrosterone did not suppress appetite and had no apparent toxic effects at the doses used, and its weight controlling effects were reversible upon withdrawal of treatment.

Attachment of reporter and conjugate groups to the 3' termini of oligonucleotides.

Conjugation of oligonucleotides at the 3 terminus is less common because this site is used for covalent linkage to solid-phase oligonucleotide synthesis supports. However, 3-oligonucleotide conjugates have several valuable physicochemical properties, including their ability to stabilize nucleic acid hybridization complexes and to retard the activity of exonucleases. This unit discusses methods for preparing oligonucleotides conjugated at the 3 terminus.

The central nervous system and exposure to toluene: a risk characterization.

The principal health outcome of exposure to toluene is dysfunction of the central nervous system. Effects range from fatalities and severe neurological disorders in toluene abuse situations to deficits in neurobehavioral function in occupational populations. An Inhalation Reference Concentration (RfC) of 0.4 mg toluene/m3 or 0.1 ppm was developed by the U.S. EPA to protect general populations chronically exposed to toluene. The RfC was derived from results of an occupational study involving Asian workers who developed neurobehavioral deficits at a mean toluene exposure level at the time of the study of 88 ppm. The derivation incorporated several uncertainty factors, one of which was a factor of 10 to account for sensitive subpopulations. Recent evidence indicates that some Japanese and possibly other Asian populations harbor a defective gene for aldehyde dehydrogenase, and thus exhibit a decreased rate of toluene metabolism. Although it is not known if reduced metabolism by aldehyde dehydrogenase also was a factor in the occupational study, preshift blood levels of toluene were considerably higher than preshift levels from non-Asian workers exposed to similar air levels of toluene. The elevated blood levels are consistent with defective metabolism but remain to be confirmed. Inasmuch as air levels of toluene in urban environments are about 10-fold lower than the RfC, an adequate measure of protection is afforded by the RfC with or without an uncertainty factor for sensitive subgroups. However, the uncertainty factor for sensitive subgroups should be retained because there is no information regarding toluene metabolism in children.

In vitro and in vivo effects of oxidative damage to deoxyguanosine.

Biochemical, biophysical and biological studies of oligonucleotides containing lesions at defined sites provide a molecular basis for the effects of DNA lesions. dG (deoxyguanosine) is the most easily oxidized of the four native nucleotides. The chemical reactivity of dG correlates with compilations of mutations, which reveal that a significant fraction of transitions or transversions involve dG. OxodG (7,8-dihydro-8-hydroxy-2'-deoxyguanosine) is widely recognized as an important lesion derived from the oxidation of dG, and significant effort has been expended in studies of its effects on DNA structure and function. Recently, the properties of other lesions derived from dG and/or the oxidation of OxodG have been uncovered. Studies on these lesions reveal that they too are biologically significant.

Direct measurement of pyrimidine C6-hydrate stability.

Pyrimidine C6-hydrates are produced via UV-irradiation and undergo dehydration upon standing. The stability of these compounds has a direct bearing on their genotoxicity. The rate constants for elimination from 5'-benzyoylated derivatives of 5,6-dihydro-5-hydroxythymidine (6) and 5,6-dihydro-5-hydroxy-2'-deoxyuridine (9) were measured directly via HPLC. The rate constants for dehydration increase from pH 6.0 to 8.0. The half-lives for 6 and 9 at pH 7.4 and 37 degrees C are 46.5 and 24.4h, respectively. Deglycosylation is not observed, even upon heating at 90 degrees C. These observations reinforce proposals that pyrimidine hydrates are sufficiently long-lived that they can exert significant effects on biological systems.

Template-free segmental synthesis of oligonucleotides containing nonnative linkages.

Protected oligonucleotides containing 3'-alkyl carboxylic acids or 3'-alkylamines were obtained from photolabile solid-phase synthesis supports (1 and 4). Protected oligonucleotides containing 5'-alkylamines and 3'-hydroxyl groups were obtained using a photolabile solid-phase synthesis support (2) and a commercially available phosphoramidite reagent (3). Depending upon the source of alkylamine-containing oligonucleotide, the segments were coupled under mild conditions to form products containing either 5'-3' or 3'-3' linkages in good yield and high purity. Oligonucleotides as long as 40 nucleotides were prepared, and coupling yields of protected biopolymer segments were independent of length over the range examined. This method is particularly well suited for the convergent synthesis of oligonucleotides containing nonnative linkages and should be useful for the rapid assembly of modified biopolymers that are useful in biochemical studies.

Inhibition of klenow fragment (exo-) catalyzed DNA polymerization by (5R)-5,6-dihydro-5-hydroxythymidine and structural analogue 5,6-dihydro-5-methylthymidine.

Oligonucleotides containing 5R-5,6-dihydro-5-hydroxythymidine (5R-3) and structural analogue 5,6-dihydro-5-methylthymidine (9) at defined sites were chemically synthesized via a method that obviates the use of NH4OH. Oligonucleotides prepared by this method were used to examine the effects of 5R-3 and 9 on the fidelity of Klenow (exo-) in vitro. The presence of lesions 5R-3 and 9 in DNA templates was shown to inhibit polymerization of primers hybridized to these templates. Inhibition was observed for both translesional synthesis and extension one nucleotide past the lesion, with the latter being more pronounced. The fidelity of Klenow (exo-) was reduced only slightly when utilizing substrates containing either dihyropyrimidine nucleotide. These results provide the first experimental verification of computational studies carried out on the effects of 3 on DNA templates, and are consistent with a structural model in which the C5-methyl group of 5R-3 adopts a pseudoaxial orientation resulting in a disruption in base stacking.

Oxygen-dependent DNA damage amplification involving 5,6-dihydrothymidin-5-yl in a structurally minimal system.

5,6-Dihydrothymidin-5-yl (1) was independently generated in a dinucleotide from a phenyl selenide precursor (4). Under free radical chain propagation conditions, the products resulting from hydrogen atom donation and radical-pair reaction are the major observed products in the absence of O(2). The stereoselectivity of the trapping process is dependent on the structure of the hydrogen atom donor. No evidence for internucleotidyl hydrogen atom abstraction by 1 was detected. The tandem lesion (17) resulting from hydrogen atom abstraction from the C1' position of the adjacent 2'-deoxyuridine by the peroxyl radical derived from 1 (3) is observed under aerobic conditions. The structure of this product is confirmed by independent synthesis and its transformation into a second independently synthesized product (24). Internucleotidyl hydrogen atom abstraction is effected selectively by the 5S-diastereomer of the peroxyl radical. The formation of dinucleotide 17 provides further support for the novel O(2)-dependent DNA damage amplification mechanism involving 1 reported previously (Greenberg, M. M.; et al. J. Am. Chem. Soc. 1997, 119, 1828).

Synthesis of 2'-modified oligodeoxynucleotides via on-column conjugation.

Oligodeoxynucleotides modified at the 2'-position of 2'-amino-2'-deoxyuridine or uridine were prepared in high yield and purity using phosphoramidites 2 and 3, respectively. Oligodeoxynucleotide conjugates were prepared on the solid-phase synthesis support following selective unmasking of the nucleophile incorporated in these phosphoramidites. Synthesis of oligodeoxynucleotides modified at the 2'-position of an internal nucleotide provides molecules that are complementary to those previously prepared via a similar approach using C5-substituted pyrimidines. The efficiency of functionalization of the 2'-O-alkylamino-uridine derived from 3 in a protected oligodeoxynucleotide was less susceptible to steric hindrance than the 2'-amino-2'-deoxyuridine in the same polymeric substrate. However, the greater reactivity of the 2'-O-alkylamine containing nucleotide gave rise to undesired acetamide formation resulting from nucleophilic attack on the 5'-terminal acetate in capped failure sequences. This problem was overcome by using 2,2,2-trimethylacetyl anhydride as a capping agent during the automated synthesis cycles. Finally, the efficiency of the photochemical unmasking of the support bound alkylamine on a 1 mumole scale was improved by using two 20 min photolysis cycles, coupled with removing reaction byproducts between cycles.

Convergent solution-phase synthesis of a nucleopeptide using a protected oligonucleotide.

A nucleopeptide was prepared in a convergent manner via segmental coupling of the protected biopolymers in solution. The resulting nucleopeptide (4b, 72%) containing the binding site of lambda repressor and a peptide containing the consensus sequence of the DNA binding helix of the helix turn-helix-proteins was obtained using only five equivalents of the peptide relative to the oligonucleotide. This demonstrates that the recently developed method for the solution phase coupling of protected oligonucleotides is amenable to the convergent synthesis of larger nucleopeptides that are potentially capable of adopting secondary structure.

The effects of the ring fragmentation product of thymidine C5-hydrate on phosphodiesterases and klenow (exo-) fragment.

N-(2-Deoxy-beta-D-erythro-pentofuranosyl)-N-3-(2R-hydroxyisobutyric acid)urea (alpha-R-hydroxy-beta-ureidoisobutyric acid, 8) was site specifically incorporated into a series of oligonucleotides via the ammonolysis of biopolymers containing 5R-thymidine C5-hydrate (3). alpha-R-hydroxy-beta-ureidoisobutyric acid (8) inhibits snake venom phosphodiesterase, lambda exonuclease and Klenow (exo-) fragment. Kinetic measurements for insertion of nucleotides opposite 8 by Klenow (exo-) fragment indicate that this lesion is instructive.

Observation and elimination of N-acetylation of oligonucleotides prepared using fast-deprotecting phosphoramidites and ultra-mild deprotection.

Commercially available 'fast-deprotecting' phosphoramidites are useful for synthesizing oligonucleotides containing alkali-sensitive nucleotides. However, N-acetylated oligonucleotides were observed during solid-phase synthesis using 'fast-deprotecting' phosphoramidites in conjunction with K2CO3/MeOH ('ultra-mild') deprotection. Transamidation was localized at deoxyguanosine, which is protected as its isopropylphenoxyacetyl amide. Substitution of trimethylacetic anhydride for acetic anhydride and appropriate modification of the automated synthesis cycles eliminated this problem.

Independent generation and reactivity of 2-deoxy-5-methyleneuridin-5-yl, a significant reactive intermediate produced from thymidine as a result of oxidative stress.

2'-Deoxy-5-methyleneuridin-5-yl (1) is produced in a variety of DNA damage processes and is believed to result in the formation of lesions that are mutagenic and refractory to enzymatic repair. 2'-Deoxy-5-methyleneuridin-5-yl (1) was independently generated under anaerobic conditions via Norrish Type I photocleavage during Pyrex filtered photolysis of the benzyl ketone 7. The radical (1) exhibits behavior consistent with that of a resonance-stabilized radical. The KIE for hydrogen atom transfer from t-BuSH was found to be 7.3 +/- 1.7. Competition studies between radical recombination and hydrogen atom donors (2,5-dimethyltetrahydrofuran, kTrap = 46.1 +/- 15.4 M(-1) s(-1); propan-2-ol, kTrap = 13.6 +/- 3.5 M(-1) s(-1)) chosen to mimic the carbohydrate components of 2'-deoxyribonucleotides suggest that 2'-deoxy-5-methyleneuridin-5-yl (1) may be able to transfer damage from the nucleobase to the deoxyribose of an adjacent nucleotide in DNA under hypoxic conditions.

An analysis of the relationships among obesity, plasma insulin and hepatic lipogenic enzymes in "viable yellow obese" mice (Avy/a).

The development of obesity, hyperinsulinemia and six hepatic lipogenic enzymes in Avy/a mice were compared to that in a/a mice. Correlation between body weight, liver weight, plasma insulin concentration and activities of hepatic enzymes was analyzed. In the Avy/a mice, body weight, liver weight and plasma insulin level increased steadily as the mice aged. In the a/a mice, the change of these three parameters was much slower. Plasma insulin concentration in a/a mice did not increase until eight months of age. Compared with a/a mice, Avy/a mice had higher 6-phosphogluconate dehydrogenase and fatty acid synthetase activities at two months of age; lower citrate cleavage enzyme, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities at three months of age; lower citrate cleavage enzyme and glucose-6-phosphate dehydrogenase and higher acetyl CoA carboxylase activities at five months of age; and higher malic enzyme, citrate cleavage enzyme and 6-phosphogluconate dehydrogenase activities at eight months of age. There were significant correlations between plasma insulin level and body weight and between plasma insulin level and the activities of malic enzyme and citrate cleavage enzyme in Avy/a mice. The correlation between body weight and malic enzyme and citrate cleavage enzyme activities disappeared after the analysis was adjusted for plasma insulin level.