NMR Conformational Analysis of DNA Duplexes Containing Diol Epoxide Adducts of Polycyclic Aromatic Hydrocarbons

The principal adducts formed between DNA and polycyclic aromatic hydrocarbon diol epoxides result from N-alkylation of the exocyclic amino groups of the purine bases by the benzylic carbon atom of the epoxide. To date, the solution conformations of more than a dozen alkylated DNA duplexes have been examined by 2D NMR spectroscopy. For trans opened diol epoxides, oligomer duplexes containing N²-adducts at deoxyguanosine have the hydrocarbon residue lying in the minor groove whereas those containing N⁶-adducts at deoxyadenosine have the hydrocarbon intercalated within the DNA helix. Absolute configuration at the site of attachment appears to be a major determinant in establishing whether the hydrocarbon lies to the 3′- or the 5′-side of the adducted base. For trans opened deoxyadenosine adducts with R-absolute configuration, the hydrocarbon residue is positioned toward the 5′-end of the adducted strand whereas trans opened deoxyguanosine adducts with R-absolute configuration have the hydrocarbon located toward the 3′-end of the adducted strand.     

Effect of Structure and Sequence on Mutations Induced by Diol Epoxide-DNA Adducts in an E. Coli−M13 Vector System

The effects of two DNA sequence contexts on the mutagenic response to dG and dA adducts derived from the optically active 7,8-diol 9,10-epoxides of benzo[a]pyrene (BaP DEs) and 3,4-diol 1,2-epoxides of benzo[c]phenanthrene (BcPh DEs) were examined. On replication of the adduct-containing, single-stranded vector M13mp 7L2 in E. coli SMH77, frequencies of substitution mutations ranging from 0.05% to 68% were observed. In general, the highest mutational frequencies were observed for BaP DE-dA adducts at the central position in a ～TAG～ sequence. The BaP DE adducts showed no relationship between stereochemistry and mutagenic response. In contrast, mutagenicity of the BcPh DE-dA adducts in the ～TAG～ sequence depended strongly on the configuration at C-1, the site of attachment of the hydrocarbon to the purine. In this sequence, BcPh DE-dA adducts with 1R configuration were 16-600 times less mutagenic than those with 1S configuration.     

Impact of Site-Specific Benzo[a]Pyrene Diol Epoxide-dG Lesions at or near Single/Double-Strand DNA Junctions on DNA Bending

Adducts derived from the binding of the (+)-7R,8S,9S,10R and (?)-7S,8R,9R,10S enantiomers of r7,t8-dihydrodiol-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) to 2′-deoxyguanosine residues in DNA are known to induce mutations due to error-prone DNA replication. Because the conformational properties of these lesions may be important in these phenomena, we have examined the effects of the stereoisomeric (+)- and (?)-trans-anti-[BP]-N ²-dG lesions positioned site-specifically at or near primer/template oligonucleotide junctions on DNA bending using high resolution gel electrophoresis. Remarkable differences in electrophoretic mobilities μ are observed in the two adducts derived from the tumorigenic (+)-anti-BPDE, and the non-tumorigenic (?)-anti-BPDE enantiomer. With the (+)-trans lesion positioned on the template strand adjacent to the 3′-end of the primer strand, a remarkable decrease in μ is observed. This suggests the existence of a bend at the single strand-double strand junction. Only modest decreases in μ are observed in the case of the (?)-trans lesion, or when the 3′-end is opposite to, or more distant from the lesion site. These observations are discussed in terms of the known NMR solution structures of these lesions in the same sequence context, and the properties of primer/template DNA in polymerases.     

siRNA Modified with 2′‐Deoxy‐2′‐C‐methylpyrimidine Nucleosides

(2′S)‐2′‐Deoxy‐2′‐C‐methyluridine and (2′R)‐2′‐deoxy‐2′‐C‐methyluridine were incorporated in the 3′‐overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2′S)‐2′‐deoxy‐2′‐C‐methylcytidine was incorporated in the 6‐position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC₅₀ values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3′‐overhang region by (2′S)‐ or (2′R)‐2′‐deoxy‐2′‐C‐methyluridine, reducing IC₅₀ to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.     

A New Class of 3′‐Sulfonyl BINAPHOS Ligands: Modulation of Activity and Selectivity in Asymmetric Palladium‐Catalysed Hydrophosphorylation of Styrene

Palladium‐catalysed monophosphorylation of (R)‐2,2′‐bisperfluoroalkanesulfonates of BINOL (R^F=CF₃ or C₄F₉) by a diaryl phosphinate [Ar₂P(O)H] followed by phosphine oxide reduction (Cl₃SiH) then lithium diisopropylamide‐mediated anionic thia‐Fries rearrangement furnishes enantiomerically‐pure (R)‐2′‐diarylphosphino‐2′‐hydroxy‐3′‐perfluoralkanesulfonyl‐1,1′‐binaphthalenes [(R)‐ 8ab and (R)‐ 8g–j ], which can be further diversified by Grignard reagent (RMgX)‐mediated CF₃‐displacement [→(R)‐ 8c–f ]. Coupling of (R)‐ 8a–j with (S)‐1,1′‐binaphthalene‐2,2′‐dioxychlorophosphine (S)‐ 9 generates 3′‐sulfonyl BINAPHOS ligands (R,S)‐ 10a–j in good yields (43–82%). These new ligands are of utlility in the asymmetric hydrophosphonylation of styrene ( 1 ) by 4,4,5,5‐tetramethyl‐1,3,2‐dioxaphospholane 2‐oxide ( 2 ), for which a combination of the chiral ligands with either [Pd(Cp)(allyl)] or [Pd(allyl)(MeCN)₂]⁺/NaCH(CO₂Me)₂ proves to be a convenient and active pre‐catalyst system. A combination of an electron‐rich phosphine moiety and an electron‐deficient 3′‐sulfone moiety provides the best enantioselectivity to date for this process, affording the branched 2‐phenethenephosphonate, (−)‐iso‐ 3 , in up to 74% ee with ligand (R,S)‐ 10i , where Ar=p‐anisyl and the 3′‐SO₂R group is triflone.     

Palladium(II) Complexes of C2‐Bridged Chiral Diphosphines: Application to Enantioselective Carbonyl‐Ene Reactions

(11bR,11′bR)‐4,4′‐(1,2‐Phenylene)bis[4,5‐dihydro‐3H‐dinaphtho[2,1‐c:1′,2′‐e]phosphepin] [abbreviated as (R)‐BINAPHANE], (3R,3′R,4S,4′S,11bS,11′bS)‐4,4′‐bis(1,1‐dimethylethyl)‐4,4′,5,5′‐tetrahydro‐3,3′‐bi‐3H‐dinaphtho[2,1‐c:1′,2′‐e]phosphepin [(S)‐BINAPINE], (1S,1′S,2R,2′R)‐1,1′‐bis(1,1‐dimethylethyl)‐2,2′‐biphospholane [(S,S,R,R)‐TANGPHOS] and (2R,2′R,5R,5′R)‐1,1′‐(1,2‐phenylene)bis[2,5‐bis(1‐methylethyl)phospholane] [(R,R)‐i‐Pr‐DUPHOS] are C₂‐bridged chiral diphosphines that form stable complexes with palladium(II) and platinum(II) containing a five‐membered chelate ring. The Pd(II)‐BINAPHANE catalyst displayed good to excellent enantioselectivities with ee values as high as 99.0% albeit in low yields for the carbonyl‐ene reaction between phenylglyoxal and alkenes. Its Pt(II) counterpart afforded improved yields while retaining satisfactory enantioselectivity. For the carbonyl‐ene reaction between ethyl trifluoropyruvate and alkenes, the Pd(II)‐BINAPHANE catalyst afforded both good yields and extremely high enantioselectivities with ees as high as 99.6%. A comparative study on the Pd(II) catalysts of the four C₂‐bridged chiral diphosphines revealed that Pd(II)‐BINAPHANE afforded the best enantioselectivity. The ee values derived from Pd(II)‐BINAPHANE are much higher than those derived from the other three Pd(II) catalysts. A comparison of the catalyst structures shows that the Pd(II)‐BINAPHANE catalyst is the only one that has two bulky (R)‐binaphthyl groups close to the reaction site. Hence it creates a deep chiral space that can efficiently control the reaction behavior in the carbonyl‐ene reactions resulting in excellent enantioselectivity.     

Synthesis and Electron Paramagnetic Resonance Studies of Oligodeoxynucleotides Containing 2‐N‐tert‐Butylaminoxyl‐2′‐deoxyadenosines

Oligodeoxynucleotides (ODNs) containing 2‐N‐tert‐butylaminoxyl‐2′‐deoxyadenosine ( A* ) residues were synthesized to allow accurate monitoring of adenine motion by EPR spectroscopy through the agency of direct linkage of the acyclic aminoxyl group to the nucleobase, and EPR studies of the ODNs in single‐ and double‐stranded forms were performed. Upon duplex formation, peak broadening and decreases in peak height were observed in EPR spectra, and the synthesized ODNs were shown to be excellent monitors of hybridization. Comparison of peak height and the h₁/h₀ signal ratio provided information on the relative mobility of A* in duplexes with different stability. A second set of ODNs each containing two A* residues at different intervals and four dA residues were also synthesized. For these ODNs, correlations were observed between the EPR spectral shapes of the duplexes and the number of dA residues between A* residues, thus demonstrating the potential of A* residues in monitoring of the structures of nucleic acids.     

Metabolic Activation of Dibenzo[a,l]Pyrene by Cytochrome P450 Enzymes to Stable DNA Adducts Occurs Exclusively Through the Formation of the (−)-trans−(11R, 12R)-Diol

At low doses, the carcinogenic hexacyclic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) forms DNA adducts in human MCF-7 cells exclusively through formation of the (–)-anti?(11R, 12S)-diol (13S, 14R)-epoxide (DB[a,l]PDE) via its metabolic precursor, the (–)-(11R, 12R)-diol. The same result was obtained by exposure of V79 cells stably expressing human cytochrome P450 (P450) 1B1. Although several other metabolites such as the 7-phenol and a 11, 12-diol phenol are formed, no other DNA adducts are detectable after exposure to 0.1 μM DB[a,l]P. Exposure to 1 μM DB[a,l]P leads to the formation of low levels of (+)-syn?DB[a,l]P-DE-DNA adducts through intermediate generation of the (+)-(11S, 12S)-diol. In contrast, treatment of P450 1A1-expressing V79 cells results also in the formation of several polar DNA adducts. Incubation of the trans?8,9-diol and trans?11, 12-enantiomers of DB[a,l]P demonstrated that all polar DNA adducts detected in 1A1-expressing cells resulted from intermediate formation of the (–)-trans?11, 12-diol. Although the K-region trans?8,9-diol is metabolically converted to several diol phenols and bis?diols, this compound does not contribute to the strong DNA binding or cytotoxicity observed after exposure of P450 1A1- and 1B1-expressing cells to the parent hydrocarbon.     

New Rapid HPLC Method for Separation and Determination of Benzo[A]Pyrene Hydroxyderivatives

A new rapid, one-step semi-quantitative HPLC-MS method applicable for the separation and characterization of the group of total 10 BaP hydroxyderivatives (1-hydroxy benzo[a]pyrene, 2-hydroxybenzo[a]pyrene, 3-hydroxybenzo[a]pyrene, 4-hydroxybenzo [a]pyrene, 7-hydroxybenzo[a]pyrene, 8-hydroxybenzo[a]pyrene, 9-hydroxybenzo[a]py rene, 10-hydroxybenzo[a]pyrene, 12-hydroxybenzo[a]pyrene and benzo[a]pyrene-9,10-dihydrodiol) was developed, involving electrospray ionization technique (ESI) in positive mode. Its qualitative and quantitative characteristics were selected by the analysis of the model OH-BaP mixture consisted of compounds mentioned above. The model system offered the linear response in the whole concentration range used (50–1000 ng.mL^?1) as well as the resolution sufficient for semi-quantitative purposes (R_S > 0.5). Subsequently, the suitability of the developed method was tested on BaP decomposition products to be formed during photooxidation at λ = 365 nm. These analyses confirmed the applicability of the proposed method, succeeding in the identification of 5 BaP hydroxyderivatives formed upon the BaP photooxidation experiment.     

Unusual Totally Selective Cyclodimerization of Epoxides: Synthesis of a Pair of Diastereoisomers of Enantiopure 2,5‐Disubstituted‐1,4‐Dioxanes with C2 Symmetry

The synthesis of the C₂‐symmetrical (2R,5R)‐ and (2S,5S)‐2,5‐bis‐[(S)‐1‐(dibenzylaminoalkyl)]‐1,4‐dioxanes 1 or 2 in enantiopure form is reported. Compounds 1 and 2 were obtained by a completely selective and unusual cyclodimerization of chiral (2R,1′S)‐ or (2S,1′S)‐2‐(1‐aminoalkyl)epoxides 3 or 4 promoted by a mixture of diisopropylamine and boron trifluoride⋅diethyl etherate complex. The structure of the obtained dioxane was established by single‐crystal X‐ray diffraction analysis. A mechanism has been proposed to explain this transformation.     

Stereochemical inversion of pyrrolizidine alkaloids byMechanitis polymnia (Lepidoptera: Nymphalidae: Ithomiinae): Specificity and evolutionary significance

Pyrrolizidine alkaloids (PAs), acquired by adults or larvae of Danainae and Ithomiinae butterflies and Arctiidae moths from plants, protect these lepidopterans against predators and are biosynthetic precursors of male sex pheromones. The investigation of PAs in many species of wild-caught adults of Ithomiinae showed lycopsamine (1) [(7R)-OH, (2S)-OH, (3S)-OH] as the main alkaloid. In incorporation experiments, PA-free (freshly emerged) adults of the ithomiineMechanitis polymnia were fed seven PAs: lycopsamine and four of its known natural stereoisomers—indicine (2) [(7R)-OH, (2R)-OH, (3S)-OH], intermedine (3) [(7R)-OH, (2S)-OH, (3R)-OH], rinderine (4) [(7S)-OH, (2S)-OH, (3R)-OH], and echinatine (5) [(7S)-OH, (2S)-OH, (3S)-OH], and two PAs without the 7-OH: supinine (6) [(2S)-OH, (3R)-OH] and amabiline (7) [(2S)-OH, (3S)-OH]. Males epimerized PAs 3, 4, and 5 mainly to lycopsamine (1). Females fed these same three PAs changed a smaller proportion to lycopsamine; their lesser capacity to modify PAs corresponds to their normal acquisition of already transformed PAs from males during mating rather than through visits of adults to plant sources of PAs. The alkaloids1 and2, both 7R and 3S, were incorporated without or with minimum change by males and females. Feeding experiments with6 and7 (males only) showed an inversion at the 3 center of6 and no change in7. The inversion from 7S to 7R (probably via oxyreduction) may be closely related to the evolution of acquisition of PAs by butterflies and moths. Two hypotheses are discussed: (1) The ancestral butterflies are probably adapted to tolerate, assimilate, and use (7R)-PAs (most common in plants; all widespread 1,2-unsaturated macrocyclic PA diesters show this configuration). The development of (7R)-PA receptors in the butterflies could lead to a specialization on this configuration in two ways: to help find PA plants and to utilize these components in sexual chemical communication. A later appearance of (7S)-PAs in plants could have selected an enzymatic system for the inversion of this chiral center in order to continue producing (7R)-PA-derived pheromones. (2) The inversion would be due to the evolution of a enzyme system specialized in the transport of (7R)-PAs to the integument; the failure of this system to carry (7S)-PAs led to an enzymatic system to invert them to transportable (7R)-PAs. In this case, the 7R configuration is an effect and not a cause of (7R)-PA-derived pheromones. In both hypotheses, the partial inversion of the 3-asymmetric center, when the butterfly was fed intermedine (3), rinderine (4), and supinine (6), could be fortuitous due to the conformation of the molecule and/or the enzymatic system.This paper is part of the doctoral thesis of J.R.T.     

Computational Design of Enantiocomplementary Epoxide Hydrolases for Asymmetric Synthesis of Aliphatic and Aromatic Diols

The use of enzymes in preparative biocatalysis often requires tailoring enzyme selectivity by protein engineering. Herein we explore the use of computational library design and molecular dynamics simulations to create variants of limonene epoxide hydrolase that produce enantiomeric diols from meso-epoxides. Three substrates of different sizes were targeted: cis-2,3-butene oxide, cyclopentene oxide, and cis-stilbene oxide. Most of the 28 designs tested were active and showed the predicted enantioselectivity. Excellent enantioselectivities were obtained for the bulky substrate cis-stilbene oxide, and enantiocomplementary mutants produced (S,S)- and (R,R)-stilbene diol with >97 % enantiomeric excess. An (R,R)-selective mutant was used to prepare (R,R)-stilbene diol with high enantiopurity (98 % conversion into diol, >99 % ee). Some variants displayed higher catalytic rates (k_cat) than the original enzyme, but in most cases K_M values increased as well. The results demonstrate the feasibility of computational design and screening to engineer enantioselective epoxide hydrolase variants with very limited laboratory screening.     

Effects of Polycyclic Aromatic Hydrocarbon Adducts with Deoxyguanosine and Deoxyadenosine in vivo and in vitro

Reactive metabolites from non-planar polycyclic aromatic hydrocarbon carcinogens react extensively with both deoxyadenosine and deoxyguanosine in DNA whereas those from planar molecules react predominantly only with deoxyguanosine. In vitro studies with single adducts in oligonucleotides showed that both types of adduct blocked primer extension and that the limited amount of nucleotide addition opposite the adduct varied with the polymerase, the sequence context of the adduct and the chemical structure of the adduct. When these same single adduct containing-oligonucleotides were introduced into a single-stranded vector that was allowed to replicate in Escherichia coli, the major events observed were blockage of replication, insertion of the correct nucleotide (i.e. T opposite an A adduct and C opposite a G adduct), and insertion of A opposite the adduct. In mouse skin, benzo[c]phenanthrene 4S,3R-dihydrodiol 2S,1R-epoxide initiated substantially more tumors per DNA adduct formed than did the other three isomers. This isomer generates mostly adenine adducts in DNA, suggesting that, in this case, adenine adducts are intrinsically more tumorigenic than guanine adducts.     

Two distinct pathways for the formation of hydroxy FA from linoleic acid by lactic acid bacteria

Twenty-three of 86 strains of lactic acid bacteria transformed linoleic acid into hydroxy FA. Two distinct conversion pathways were in operation. Two strains of Lactobacillus acidophilus and a strain of Pediococcus pentosaceus produced 13(S)-hydroxy-9-octadecenoic acid [13(S)-OH 18∶1] and 10,13-dihydroxyoctadecanoic acid (10,13-OH 18∶0) as main and minor products, respectively, whereas 13 strains, including L. casei subsp. casei, L. paracasei subsp, paracasei, L. rhamnosus, L. lactis subsp. cremoris, and Streptococcus salivarius subsp. thermophilus produced 10-hydroxy-12-octadecenoic acid (10-OH 18∶1). Seven strains of L. plantanum converted linoleic acid to 10-hydroxyoctadecanoic acid (10-OH 18∶0) through 10-OH 18∶1 Linoleic acid at 2 g/L was converted by L. acidophilus LFO13951^T to 1.3 g of 13(S)-OH 18∶1 and 0.09 g of 10,13-OH 18∶0 in 7 d. Lactobacillus paracasei subsp. paracasei JCM 1111 produced 10-OH 18∶1 in 91% yield, and L. plantarum JCM 8341, 10-OH 18∶0 in 59% yield from linoleic acid (2 g/L) under optimal conditions. To our knowledge, this is the first report on the production of 13(S)-OH 18∶1 by lactic acid bacteria other than ruminal bacteria, and of 10,13-OH 18∶0 by any bacteria.     

THE TOXICITY OF BENZO[a]PYRENE ON SOLE (SOLEA SOLEA) HEPATOCYTES: ASSESSMENT OF GENOTOXIC AND ENZYMATIC EFFECTS

The benzo[a]pyrene is a polycyclic aromatic hydrocarbon known to be genotoxic, mutagenic and carcinogenic in higher vertebrates. The aim of this study was to evaluate in vitro the enzymatic and genotoxic effects of BaP in a benthic fish species, Solea solea. Sole hepatocytes were exposed to BaP in order to measure the modulation of ethoxyresorufin-o-deethylase (EROD) activity and the DNA strand breaks induced by BaP metabolism. Exposures were performed in both culture flasks and microplate wells in order to check for the possible miniaturization of the exposure system. Moreover, sole liver microsomes were exposed to BaP in the presence of standard DNA in order to assess the potential formation of DNA adducts in sole. The results demonstrated the ability of sole hepatic enzymes to metabolize BaP into reactive species responsible for bulky DNA adducts and DNA strand breakage, whatever the tested exposure concentration and the mode of exposure.     

Highly Efficient and Enantioselective Iridium‐Catalyzed Asymmetric Hydrogenation of N‐Arylimines

A catalytic method employing the cationic iridium‐(S_c,R_p)‐DuanPhos [(1R,1′R,2S,2′S)‐2,2′‐di‐tert‐butyl‐2,2′,3,3′‐tetrahydro‐1H,1′H‐1,1′‐biisophosphindole] complex and BARF {tetrakis[3,5‐bis(trifluoromethyl)phenyl]borate} counterion effectively catalyzes the enantioselective hydrogenation of acyclic N‐arylimines with high turnover numbers (up to 10,000 TON) and excellent enantioselectivities (up to 98% ee), achieving the practical synthesis of chiral secondary amines.     

Enantioselective Synthesis of Chiral β‐Aryloxy Alcohols by Ruthenium‐Catalyzed Ketone Hydrogenation via Dynamic Kinetic Resolution (DKR)

A highly efficient enantioselective synthesis of chiral β‐aryloxy alcohols by the {RuCl₂[(S)‐SDP][(R,R)‐DPEN]} [(S_a,R,R)‐ 1a ; SDP=7,7′‐bis(diarylphosphino)‐1,1′‐spirobiindane; DPEN=trans‐1,2‐diphenylethylenediamine] complex‐catalyzed asymmetric hydrogenation of racemic α‐aryloxydialkyl ketones via dynamic kinetic resolution (DKR) has been developed. Enantioselectivities of up to 99% ee with good to high cis/anti‐selectivities (up to>99:1) were achieved.     

Synthesis of four chiral pharmaceutical intermediates by biocatalysis

Chiral intermediates were prepared by biocatalytic processes for the chemical synthesis of four pharmaceutical drug candidates. These include: (i) the microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic ethyl ester to (3S,5R)-dihydroxy-6-(benzyloxy) hexanoic acid ethyl ester, an intermediate for a new anticholesterol drug; (ii) synthesis of (2R,3S)-(-)-N-benzoyl-3-phenyl isoserine ethyl ester, a taxol side-chain synthon; (iii) the microbial oxygenation of 2,2-dimethyl-2H-1-benzopyran-6-carbonitrile to the corresponding (3S,4S) epoxide and (3S,4R)-trans diol, intermediates for synthesis of potassium channel opener; (iv) the biotransformation of (exo,exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimethanol to the corresponding chiral lactol and lactone, intermediates for thromboxane A₂ antagonist.     

Synthesis and Structure Determination of the Adducts of Dibenzo[a,l]pyrene Diol Epoxides and Deoxyadenosine or Deoxyguanosine

Abstract

(±)?Syn?dibenzo[a,l]pyrene diol epoxide (DB[a,l]PDE) and (±)?anti?DB[a,l]PDE were reacted with deoxyadenosine (dA) or deoxyguanosine (dG) in dimethylformamide at 100 °C for 30 min. The crude products were purified by reverse phase HPLC under gradient and isocratic conditions. The structure of each adduct was assigned by 1D and 2D NMR spectra and by fast atom bombardment mass spectrometry. Five adducts were isolated from the reaction of (±)?syn?DB[a,l]PDE and dA: syn?DB[a,l]PDE?N⁶dA?1, syn?DB[a,l]PDE?N⁶dA?2, syn?DB[a,l]PDE?N⁶dA?3, syn?DB[a,l]PDE?N⁶dA?4 and syn?DB[a,l]PDE?N7Ade. Four adducts were isolated from the reaction of (±)?anti?DB[a,l]PDE and dA: anti?DB[a,l]PDE?N⁶dA?1, anti?DB[a,l]PDE?N⁶dA?2, anti?DB[a,l]PDE?N⁶dA?3 and anti?DB[a,l]PDE?N⁶dA?4. Two adducts were isolated from the reaction of (±)?syn?DB[a,l]PDE and dG: (±)?11,12,13?trihydroxy?tetrahydroDB[a,l]P?14?N²dG and (±)?11,12,13?trihydroxy?tetrahydroDB[a,l]P?14?N7Gua. Two adducts were isolated from the reaction of (±)?anti?DB[a,l]PDE and dG: (±)?11,12,13?trihydroxy?tetrahydroDB[a,l]P?14?N²dG and (±)?11,12,13?trihydroxy?tetrahydroDB[a,l]P?14?N7Gua.