Improved Strategies for Postoligomerization Synthesis of Oligodeoxynucleotides Bearing Structurally Defined Adducts at the <i>N</i><sup>2</sup> Position of Deoxyguanosine

DeCorte, Bart L.; Tsarouhtsis, Dimitrios; Kuchimanchi, Satyanarayan; Cooper, Monica; Horton, Pamela; Harris, Constance M.; Harris, Thomas M.

doi:10.1021/tx9501795

Cited by 75 publications

(96 citation statements)

References 31 publications

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“…Removal of the O 6 protecting group under acidic conditions yielded 29, which was oxidized to oligodeoxynucleotide 30. 47 Our post-synthetic modification strategy 48 allowed preparation of various dG enal adducts from a single modified phosphoramidite. A challenge was the preparation of stereochemically-defined amino diols for the crotonaldehyde (31,32) and 4-HNE adducts (33)(34)(35)(36) (Figure 2).…”

Section: Synthesis Of Oligodeoxynucleotides Containing 1n 2 -Dg Enalmentioning

confidence: 99%

Interstrand DNA Cross-Links Induced by α,β-Unsaturated Aldehydes Derived from Lipid Peroxidation and Environmental Sources

et al. 2008

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ConspectusSignificant levels of the 1,N 2 -γ-hydroxypropano-dG adducts of the α,β-unsaturated aldehydes acrolein, crotonaldehyde, and 4-hydroxy-2E-nonenal (HNE) have been identified in human DNA, arising from both exogenous and endogenous exposure. They yield interstrand DNA cross-links between guanines in the neighboring C•G and G•C base pairs located in 5′-CpG-3′ sequences, as a result of opening of the 1,N 2 -γ-hydroxypropano-dG adducts to form reactive aldehydes that are positioned within the minor groove of duplex DNA. Using a combination of chemical, spectroscopic, and computational methods, we have elucidated the chemistry of cross-link formation in duplex DNA. NMR spectroscopy revealed that, at equilibrium, the acrolein and crotonaldehyde cross-links consist primarily of interstrand carbinolamine linkages between the exocyclic amines of the two guanines located in the neighboring C•G and G•C base pairs located in 5′-CpG-3′ sequences, that maintain the Watson-Crick hydrogen bonding of the cross-linked base pairs. The ability of crotonaldehyde and HNE to form interstrand cross-links depends upon their common relative stereochemistry at the C6 position of the 1,N 2 -γ-hydroxypropano-dG adduct. The stereochemistry at this center modulates the orientation of the reactive aldehyde within the minor groove of the doublestranded DNA, either facilitating or hindering the cross-linking reactions; it also affects the stabilities of the resulting diastereoisomeric cross-links. The presence of these cross-links in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease. Reduced derivatives of these cross-links are useful tools for studying their biological processing. IntroductionThe α,β-unsaturated aldehydes (enals) acrolein, crotonaldehyde, and 4-hydroxynonenal (4-HNE) (Scheme 1) are endogenous byproducts of lipid peroxidation, arising as a consequence of oxidative stress. [1][2][3][4] Acrolein and crotonaldehyde exposures also occur from exogenous sources, e.g., cigarette smoke 5 and automobile exhaust. 6 Enals react with DNA nucleobases to give exocyclic adducts; they also react with proteins. 7 Addition of enals to dG involves Michael addition of the N 2 -amine to give N 2 -(3-oxopropyl)-dG adducts (1, 3-8), followed by * Michael P. Stone telephone, 615-322-2589; fax, 615-322-7591; michael.p The lipid peroxidation product 4-HNE afforded related dGadducts (13-16). 14 Identification of acrolein adducts of other nucleosides followed. 15,16 The principal acrolein adduct is γ-OH-PdG (9), 10,12 although the regioisomeric 6-hydroxypyrimido[1,2-a]purin-10(3H)-one (α-OH-PdG, 10) has also been observed. 12,17 The γ-OH-PdG adduct (9) exists as a mixture of C8-OH epimers. With crotonaldehyde, addition at N 2 -dG creates a stereocenter at C6. Of four possible products, the two with the trans relative configurations at C6 and C8 (11,12) are observed. 12,18 These are also formed through the reaction of dG with two equivalents of acetaldehyde. 5,19,20 The cor...

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Section: Synthesis Of Oligodeoxynucleotides Containing 1n 2 -Dg Enalmentioning

confidence: 99%

Interstrand DNA Cross-Links Induced by α,β-Unsaturated Aldehydes Derived from Lipid Peroxidation and Environmental Sources

et al. 2008

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“…(15) had observed more efficient coupling of 2-amino-2-phenylethanol with a fluorinated oligonucleotide after its cleavage from the support with NaOH as above, we subsequently observed that a separate NaOH cleavage step is not necessary with our less-hindered amine, because treatment (3 days at room temperature) of the glass beads with an aqueous solution of 0.9 M bis(3-aminopropyl)disulfide dihydrochloride and 4.5 M Et 3 N completely cleaves the oligonucleotide from the support concomitantly with replacement of the fluorine. The oligonucleotide, *20-Pri-C3, was purified by HPLC on the Hamilton column as above (t R , 15 min).…”

Section: Bis[(n-tert-butyloxycarbonyl)-3-aminopropyl]disulfide-mentioning

confidence: 99%

Trapping HIV-1 Reverse Transcriptase Before and After Translocation on DNA

Sarafianos

Clark

Tuske

et al. 2003

Journal of Biological Chemistry

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A disulfide cross-linking strategy was used to covalently trap as a stable complex (complex N) a shortlived, kinetic intermediate in DNA polymerization. This intermediate corresponds to the product of polymerization prior to translocation. We also prepared the trapped complex that corresponds to the product of polymerization after translocation (complex P). The crosslinking method that we used is a variation of a technique developed by the Verdine and Harrison laboratories. It involves disulfide interchange between an engineered sulfhydryl group of the protein (Q258C mutation) and a disulfide-containing tether attached at the N 2 amino group of a modified dG in either the template or the primer strand of the nucleic acid. We report here a highly efficient synthesis of the precursor, bis(3-aminopropyl)disulfide dihydrochloride, used to introduce this substituent into the oligonucleotide. Efficient cross-linking takes place when the base pair containing the substituent is positioned seven registers from the dNTP-binding site (N site) and the N site is occupied. Complex N, but not complex P, is a substrate for the ATP-based excision reaction that unblocks nucleoside reverse transcriptase inhibitor (NRTI)-terminated primers and causes resistance to several NRTIs, confirming predictions that the excision reaction takes place only when the 3-end of the primer is bound at the N site. These techniques can be used for biochemical and structural studies of the mechanism of DNA polymerization, translocation, and excision-based resistance of RT to NRTIs. They may also be useful in studying other DNA or RNA polymerases or other enzymes. HIV-11 reverse transcriptase (RT) is a complex molecular machine that uses several kinetically distinct steps to incorporate a nucleotide into a growing DNA strand. It is a heterodimer composed of a larger 560-residue subunit (p66) and a smaller subunit (p51) that contains the N-terminal 440 residues of p66. Both subunits contain subdomains that were named fingers, palm, thumb, and connection, because of the similarity of p66 to a right hand. The DNA polymerase active site is located in the p66 palm subdomain and the DNA binding cleft is formed primarily by the p66 fingers, palm, and thumb subdomains. The mechanism of polymerization by RT is similar to other polymerases and involves: 1) binding of the DNA substrate to the apo-enzyme; 2) binding of dNTP and divalent metal ions (required for catalysis) to the enzyme⅐DNA complex, followed by rate-limiting conformational changes; 3) formation of a phosphodiester bond between the 3Ј-OH primer terminus and the ␣-phosphate of dNTP, followed by release of the pyrophosphate product; 4) translocation of the elongated DNA primer (for processive synthesis) from the dNTP-binding site (N site) to the priming site (P site) or release of the nucleic acid (distributive synthesis) (Fig. 1).Extensive biochemical and crystallographic studies have enhanced our understanding of the details of the mechanism of DNA polymerization. However, the translocation step rem...

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“…Phosphoramidite reagents containing the required "activated" O 6 -(2-(trimethylsilylethyl)-2-fluorohypoxanthine (2) and 6-chloropurine (3) bases have been previously synthesized and incorporated in oligonucleotides via standard solid-phase methods (25,(34)(35)(36). The sitespecific synthesis of an M 1 dG-adducted oligonucleotide is shown in Scheme 2.…”

Section: Resultsmentioning

confidence: 99%

Site-Specific Synthesis of Oligonucleotides Containing Malondialdehyde Adducts of Deoxyguanosine and Deoxyadenosine via a Postsynthetic Modification Strategy

Wang

Kozekov

Kozekova

et al. 2006

Chem. Res. Toxicol.

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Malondialdehyde (MDA) and its reactive equivalent, base propenal, are products of oxidative damage to lipids and DNA, respectively; they are mutagenic in bacterial and mammalian systems and MDA is carcinogenic in rats. MDA adducts of deoxyguanosine (M 1 dG), deoxyadenosine (OPdA) and deoxycytidine (OPdC) have been characterized. We have developed site-specific syntheses of M 1 dG and OPdA adducted oligonucleotides that rely on a post-synthetic modification strategy. This work provides an alternative route to the M 1 dG adducted oligonucleotide and to date, the only viable strategy for the site-specific synthesis of OPdA modified oligonucleotides. The stability of the modified oligonucleotides was examined by UV thermal melting studies (T m ). In contrast to the M 1 dG adduct, OPdA caused very little change in the T m .

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Improved Strategies for Postoligomerization Synthesis of Oligodeoxynucleotides Bearing Structurally Defined Adducts at the N² Position of Deoxyguanosine

Cited by 75 publications

References 31 publications

Interstrand DNA Cross-Links Induced by α,β-Unsaturated Aldehydes Derived from Lipid Peroxidation and Environmental Sources

Interstrand DNA Cross-Links Induced by α,β-Unsaturated Aldehydes Derived from Lipid Peroxidation and Environmental Sources

Trapping HIV-1 Reverse Transcriptase Before and After Translocation on DNA

Site-Specific Synthesis of Oligonucleotides Containing Malondialdehyde Adducts of Deoxyguanosine and Deoxyadenosine via a Postsynthetic Modification Strategy

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Improved Strategies for Postoligomerization Synthesis of Oligodeoxynucleotides Bearing Structurally Defined Adducts at the N2 Position of Deoxyguanosine

Cited by 75 publications

References 31 publications

Interstrand DNA Cross-Links Induced by α,β-Unsaturated Aldehydes Derived from Lipid Peroxidation and Environmental Sources

Interstrand DNA Cross-Links Induced by α,β-Unsaturated Aldehydes Derived from Lipid Peroxidation and Environmental Sources

Trapping HIV-1 Reverse Transcriptase Before and After Translocation on DNA

Site-Specific Synthesis of Oligonucleotides Containing Malondialdehyde Adducts of Deoxyguanosine and Deoxyadenosine via a Postsynthetic Modification Strategy

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Improved Strategies for Postoligomerization Synthesis of Oligodeoxynucleotides Bearing Structurally Defined Adducts at the N² Position of Deoxyguanosine