Remote detection of hyperpolarized 129 Xe resonances via multiple distant dipolar field interactions with 1 H

A remote detection scheme utilizing the distant dipolar field interaction between two different spin species was proposed by Granwehr et al. [J. Magn. Reson. 176(2), 125 (2005)]. In that sequence 1H spins were detected indirectly via their dipolar field interaction with 129Xe spins, which served as the sensing spins. Here we propose a modification of the proposed detection scheme that takes advantage of the longer T1 relaxation time of xenon to create a long lasting dipolar field with which the fast relaxing 1H spins are allowed to interact many times during a single acquisition. This new acquisition scheme improves detection sensitivity, but it also presents some challenges

Identification and Characterization of 2′-Deoxyadenosine Adducts Formed by Isoprene Monoepoxides in Vitro

Isoprene, the 2-methyl analog of 1,3-butadiene, is ubiquitous in the environment, with major contributions to total isoprene emissions stemming from natural processes despite the compound being a bulk industrial chemical. Additionally, isoprene is a combustion product and a major component in cigarette smoke. Isoprene has been classified as possibly carcinogenic to humans (group 2B) by IARC and as reasonably anticipated to be a human carcinogen by the National Toxicology Program. Isoprene, like butadiene, requires metabolic activation to reactive epoxides to exhibit its carcinogenic properties. The mode of action has been postulated to be that of a genotoxic carcinogen, with formation of promutagenic DNA adducts being essential for mutagenesis and carcinogenesis. In rodents, isoprene-induced tumors show unique point mutations (A→T transversions) in the K-ras protooncogene at codon 61. Therefore, we investigated adducts formed after reaction of 2′-deoxyadenosine (dAdo1) with the two monoepoxides of isoprene, 2-ethenyl-2-methyloxirane (IP-1,2-O) and propen-2-yloxirane (IP-3,4-O), under physiological conditions. The formation of N1–2′-deoxyinosine (N1-dIno) due to deamination of N1-dAdo adducts was of particular interest, since N1-dIno adducts are suspected to have high mutagenic potential based on in vitro experiments. Major stable adducts were identified by HPLC, UV-Spectrometry and LC-MS/MS and characterized by 1H and 1H,13C HSQC and NMR experiments. Adducts of IP-1,2-O that were fully identified are: R,S-C1-N6-dAdo, R-C2-N6-dAdo, and S-C2-N6-dAdo; adducts of IP-3,4-O are: S-C3-N6-dAdo, R-C3-N6-dAdo, R,S-C4-N6-dAdo, S-C4-N1-dIno, R-C4-N1-dIno, R-C3-N1-dIno, S-C3-N1-dIno, and C3-N7-Ade. Both monoepoxides formed adducts on the external and internal oxirane carbons. This is the first study to describe adducts of isoprene monoepoxides with dAdo. Characterization of adducts formed by isoprene monoepoxides with deoxynucleosides and subsequently with DNA represent the first step toward evaluating their potential for being converted into a mutation, or as biomarkers of isoprene metabolism and exposure

Iminohydantoin Lesion Induced in DNA by Peracids and Other Epoxidizing Oxidants

The oxidation of guanine to 5-carboxamido-5-formamido-2-iminohydantoin (2-Ih) is shown to be a major transformation in the oxidation of the single-stranded DNA 5-mer d(TTGTT) by m-CPBA and DMDO as a model for peracid oxidants and in the oxidation of the 5-base pair duplex d[(TTGTT)·(AACAA)] with DMDO. 2-Ih has not been reported as an oxidative lesion at the level of single/double-stranded DNA or at the nucleoside/nucleotide level. The lesion is stable to DNA digestion and chromatographic purification suggesting that 2-Ih may be a stable biomarker in vivo. The oxidation products have been structurally characterized and the reaction mechanism probed by oxidation of the monomeric species dGuo, dGMP and dGTP. DMDO selectively oxidizes the guanine moiety of dGuo, dGMP and dGTP to 2-Ih, and both peracetic and m-chloroperbenzoic acids exhibit the same selectivity. The presence of the glycosidic bond results in the stereoselective induction of an asymmetric center at the spiro carbon to give a mixture of diastereomers, with each diastereomer in equilibrium with a minor conformer through rotation about the formamido C-N bond. Labeling studies with 18O2-m-CPBA and H218O to determine the source of the added oxygen atoms have established initial epoxidation of the guanine 4-5 bond with pyrimidine ring contraction by an acyl 1,2-migration of guanine carbonyl C6 to form a transient dehydrodeoxyspiroiminodihydantoin followed by hydrolytic ring opening of the imidazolone ring. Consistent with the proposed mechanism, no 8-oxoguanine was detected as a product of the oxidations of the oligonucleotides or monomeric species mediated by DMDO or the peracids. The 2-Ih base thus appears to be a pathway-specific lesion generated by peracids and possibly other epoxidizing agents and holds promise as a potential biomarker

Nonnatural deoxyribonucleoside D_3 incorporated in an intramolecular DNA triplex binds sequence-specifically by intercalation

Oligonucleotide-directed triple helix formation is one of the most powerful methods for the sequence-specific recognition of double-helical DNA. Pyrimidine oligonucleotides bind purine tracts in the major groove of DNA parallel to the purine Watson-Crick strand through the formation of specific Hoogsteen-type hydrogen bonds. Specificity is derived from thymine (T) recognition of adenine·thymine (A·T) base pairs (T·A·T triplet) and N3-protonated cytosine (C+) recognition of guanine-cytosine (G·C) base pairs (C + G·C triplets). The sequence-specific recognition of double-helical DNA by a third strand to form a triple helix is limited to mostly purine tracts. Although G in the third strand has been found to specifically bind to T·A, the lower stability of the G·T·A triplet and its dependence on the sequence of the neigh boring triplets reveals that this will have limitations. In an attempt to extend the recognition code to all four Watson-Crick base pairs, the nonnatural deoxyribonucleoside 1-(2-deoxy/ β-D-ribofuranosyl)-4-(3-benzamido)phenylimidazole [D_3] was synthesized and incorporated into pyrimidine DNA oligonucleotides (Figure 1a). It was found that D_3 selectively recognizes both T·A and C·G Watson-Crick base pairs within the pyrimidine·purine·pyrimidine triple-helix motif. This was also found to have a nearest neighbor dependence

Nonnatural deoxyribonucleoside D_3 incorporated in an intramolecular DNA triplex binds sequence-specifically by intercalation

Oligonucleotide-directed triple helix formation is one of the most powerful methods for the sequence-specific recognition of double-helical DNA. Pyrimidine oligonucleotides bind purine tracts in the major groove of DNA parallel to the purine Watson-Crick strand through the formation of specific Hoogsteen-type hydrogen bonds. Specificity is derived from thymine (T) recognition of adenine·thymine (A·T) base pairs (T·A·T triplet) and N3-protonated cytosine (C+) recognition of guanine-cytosine (G·C) base pairs (C + G·C triplets). The sequence-specific recognition of double-helical DNA by a third strand to form a triple helix is limited to mostly purine tracts. Although G in the third strand has been found to specifically bind to T·A, the lower stability of the G·T·A triplet and its dependence on the sequence of the neigh boring triplets reveals that this will have limitations. In an attempt to extend the recognition code to all four Watson-Crick base pairs, the nonnatural deoxyribonucleoside 1-(2-deoxy/ β-D-ribofuranosyl)-4-(3-benzamido)phenylimidazole [D_3] was synthesized and incorporated into pyrimidine DNA oligonucleotides (Figure 1a). It was found that D_3 selectively recognizes both T·A and C·G Watson-Crick base pairs within the pyrimidine·purine·pyrimidine triple-helix motif. This was also found to have a nearest neighbor dependence

Solution structure of a pyrimidine-purine-pyrimidine triplex containing the sequence-specific intercalating non-natural base D_3

We have used NMR spectroscopy to study a pyrimidine·purine·pyrimidine DNA triplex containing a non-natural base, 1-(2-deoxy-β-D-ribofuranosyl)- 4-(3-benzamido)phenylimidazole (D_3), in the third strand. The D_3base has been previously shown to specifically recognize T·A and C·G base-pairs via intercalation on the 3′ side (with respect to the purine strand) of the target base pair, instead of forming sequence-specific hydrogen bonds.1H resonance assignments have been made for the D_3base and most of the non-loop portion of the triplex. The solution structure of the triplex was calculated using restrained molecular dynamics and complete relaxation matrix refinement. The duplex portion of the triplex has an over-all helical structure that is more similar to B-DNA than to A-DNA. The three aromatic rings of the D_3base stack on the bases of all three strands and mimic a triplet. The conformation of the D_3base and its sequence specificity are discussed

Solution structure of a pyrimidine-purine-pyrimidine triplex containing the sequence-specific intercalating non-natural base D_3

We have used NMR spectroscopy to study a pyrimidine·purine·pyrimidine DNA triplex containing a non-natural base, 1-(2-deoxy-β-D-ribofuranosyl)- 4-(3-benzamido)phenylimidazole (D_3), in the third strand. The D_3base has been previously shown to specifically recognize T·A and C·G base-pairs via intercalation on the 3′ side (with respect to the purine strand) of the target base pair, instead of forming sequence-specific hydrogen bonds.1H resonance assignments have been made for the D_3base and most of the non-loop portion of the triplex. The solution structure of the triplex was calculated using restrained molecular dynamics and complete relaxation matrix refinement. The duplex portion of the triplex has an over-all helical structure that is more similar to B-DNA than to A-DNA. The three aromatic rings of the D_3base stack on the bases of all three strands and mimic a triplet. The conformation of the D_3base and its sequence specificity are discussed

Identification and Characterization of 2′-Deoxyadenosine Adducts Formed by Isoprene Monoepoxides in Vitro

Isoprene, the 2-methyl analog of 1,3-butadiene, is ubiquitous in the environment, with major contributions to total isoprene emissions stemming from natural processes despite the compound being a bulk industrial chemical. Additionally, isoprene is a combustion product and a major component in cigarette smoke. Isoprene has been classified as possibly carcinogenic to humans (group 2B) by IARC and as reasonably anticipated to be a human carcinogen by the National Toxicology Program. Isoprene, like butadiene, requires metabolic activation to reactive epoxides to exhibit its carcinogenic properties. The mode of action has been postulated to be that of a genotoxic carcinogen, with formation of promutagenic DNA adducts being essential for mutagenesis and carcinogenesis. In rodents, isoprene-induced tumors show unique point mutations (A→T transversions) in the K-ras protooncogene at codon 61. Therefore, we investigated adducts formed after reaction of 2′-deoxyadenosine (dAdo()) with the two monoepoxides of isoprene, 2-ethenyl-2-methyloxirane (IP-1,2-O) and propen-2-yloxirane (IP-3,4-O), under physiological conditions. The formation of N1–2′-deoxyinosine (N1-dIno) due to deamination of N1-dAdo adducts was of particular interest, since N1-dIno adducts are suspected to have high mutagenic potential based on in vitro experiments. Major stable adducts were identified by HPLC, UV-Spectrometry and LC-MS/MS and characterized by (1)H and (1)H,(13)C HSQC and NMR experiments. Adducts of IP-1,2-O that were fully identified are: R,S-C1-N(6)-dAdo, R-C2-N(6)-dAdo, and S-C2-N(6)-dAdo; adducts of IP-3,4-O are: S-C3-N(6)-dAdo, R-C3-N(6)-dAdo, R,S-C4-N(6)-dAdo, S-C4-N1-dIno, R-C4-N1-dIno, R-C3-N1-dIno, S-C3-N1-dIno, and C3-N7-Ade. Both monoepoxides formed adducts on the external and internal oxirane carbons. This is the first study to describe adducts of isoprene monoepoxides with dAdo. Characterization of adducts formed by isoprene monoepoxides with deoxynucleosides and subsequently with DNA represent the first step toward evaluating their potential for being converted into a mutation, or as biomarkers of isoprene metabolism and exposure