Conformation Of Adducts Research Articles

ChemInform Abstract: C‐Linked 8‐Aryl Guanine Nucleobase Adducts: Biological Outcomes and Utility as Fluorescent Probes

AbstractReview: 102 refs.

A Versatile Method for the Preparation of Ferroelectric Supramolecular Materials via Radical End-Functionalization of Vinylidene Fluoride Oligomers.

A synthetic method for the end-functionalization of vinylidene fluoride oligomers (OVDF) via a radical reaction between terminal olefins and I-OVDF is described. The method shows a wide substrate scope and excellent conversions, and permits the preparation of different disc-shaped cores such as benzene-1,3,5-tricarboxamides (BTAs), perylenes bisimide (PBI), and phthalocyanines (Pc) bearing three to eight ferroelectric oligomers at their periphery. The formation, purity, OVDF conformation, and morphology of the final adducts has been assessed by a combination of techniques, such as NMR, size exclusion chromatography, differential scanning calorimetry, polarized optical microscopy, and atomic force microscopy. Finally, PBI-OVDF and Pc-OVDF materials show ferroelectric hysteresis behavior together with high remnant polarizations, with values as high as Pr ≈ 37 mC/m(2) for Pc-OVDF. This work demonstrates the potential of preparing a new set of ferroelectric materials simply by attaching OVDF oligomers to different small molecules. The use of carefully chosen small molecules paves the way to new functional materials in which ferroelectricity and electrical conductivity or light-harvesting properties coexist in a single compound.

C-Linked 8-aryl guanine nucleobase adducts: biological outcomes and utility as fluorescent probes.

Aryl radical species derived from enzymatic transformations of aromatic mutagens preferentially react at the 8-site of the guanine (G) nucleobase to afford carbon-linked 8arylG adducts. The resulting lesions possess altered biophysical and genetic coding properties compared to the precursor G nucleoside in B-form DNA. Unlike other adducts, these lesions also possess useful fluorescent properties, since direct attachment of the 8aryl ring extends the purine π-system to afford G mimics with red-shifted excitation maxima and emission that can be sensitive to the microenvironment of the 8arylG base within nucleic acid structures. In B-form DNA, 8arylG adducts are disruptive to duplex formation because they prefer to adopt the syn-conformation about the bond connecting the nucleobase to the deoxyribose backbone, which perturbs Watson-Crick (WC) H-bonding with the opposing cytosine (C). Thus, in a B-form duplex, the emissive properties of 8arylG adducts can be employed as a tool to provide insight into adduct conformation, which can be related to their biological outcomes. However, since Gs preferentially adopt the syn-conformation in left-handed Z-DNA and antiparallel G-quadruplex (GQ) structures, 8arylG lesions can be inserted into syn-G positions without disrupting H-bonding interactions. In fact, 8arylG lesions can serve as ideal fluorescent probes in an antiparallel GQ because their emission is sensitive to GQ folding. This perspective outlines recent developments in the biological implications of 8arylG formation together with their utility as fluorescent G analogs for use in DNA-based diagnostic systems.

Chlorine Functionalization of a Model Phenolic C8-Guanine Adduct Increases Conformational Rigidity and Blocks Extension by a Y-Family DNA Polymerase

Certain phenoxyl radicals can attach covalently to the C8-site of 2'-deoxyguanosine (dG) to afford oxygen-linked C8-dG adducts. Such O-linked adducts can be chemically synthesized through a nucleophilic displacement reaction between a phenolate and a suitably protected 8-Br-dG derivative. This permits the generation of model O-linked C8-dG adducts on scales suitable for insertion into oligonucleotide substrates using solid-phase DNA synthesis. Variation of the C8-aryl moiety provides an opportunity to derive structure-activity relationships on adduct conformation in duplex DNA and replication bypass by DNA polymerases. In the current study, the influence of chlorine C8-dG functionalization on in vitro DNA replication by Klenow fragment exo(-) (Kf(-)) and the Y-family polymerase (Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)) has been determined. Model O-linked C8-dG adducts derived from the pentachlorophenoxyl radical ([PCP]G) and 2,4,6-trichlorophenoxyl radical ([TCP]G) were inserted into the reiterated G3-position of the NarI sequence (12-mer, NarI(12); and 22-mer, NarI(22)), which is a known hotspot for frameshift mutations mediated by N-linked polycyclic C8-dG adducts in bacterial mutagenesis. Within the NarI(12) duplex, the unsubstituted C8-phenoxy-dG ([PhO]G) adduct adopts a minimally perturbed B-form helix. Chlorination of [PhO]G to afford [PCP]G does not significantly change the adduct conformation within the NarI(12) duplex, as predicted by molecular dynamics simulations. However, when using NarI(22) for DNA synthesis in vitro, the chlorinated [PCP]G and [TCP]G lesions significantly block DNA replication by Kf(-) and Dpo4, whereas [PhO]G is readily bypassed. These findings highlight the impact that chlorine substituents impart to bulky C8-dG lesions.

Conformations of adducts formed between the genotoxic benzo[a]pyrene-7,8-dione and 2′-deoxycytidine

Benzo[a]pyrene-7,8-dione (BPQ) is formed by the activation of benzo[a]pyrene(B[a]P), which is an environmental toxic substance that is easily exposed in daily life, due to P450/epoxide hydrolase, and is a substance that induces DNA deformation by forming adducts with DNA. In this study, to investigate the form of bonding between BPQ and DNA, the structures of adducts between BPQ and 2′-deoxycytidine were examined. To examine BPQ–dC adduct conformation, geometry optimization of a total of 16 structural isomers was performed using the density functional theory method. In the structures of BPQ–dC adducts, for the cis-form, the angle between BPQ and dC is nearly perpendicular; but for the trans-form, the bending angle is small. The trans-form had a larger energy gap between ground state and excited state than the cis-form, and had a smaller HOMO–LUMO gap than the cis-form. Therefore, it was found that the trans-form absorbs stronger light and has higher reactivity than the cis-form. Molecular electrostatic potential was calculated and analyzed. The calculated ESP contour map shows the electrophilic and nucleophilic regions of the molecule.

ChemInform Abstract: α‐Substituent Effect on Olefination of Ester Carbonyl Groups with Ynolates.

Abstract In the olefination reaction of ester carbonyl with ynolate, an α-hetero (N, O, S)-substituent on the substrates was found to markedly improve the yields of tetrasubstituted olefins, even though it was sterically hindered. The α-substituent effect may be due to several factors including the addition based on the polar Felkin–Anh model and restricted conformation of the initial adducts.

Nuclear Magnetic Resonance Studies of an N2-Guanine Adduct Derived from the Tumorigen Dibenzo[a,l]pyrene in DNA: Impact of Adduct Stereochemistry, Size, and Local DNA Sequence on Solution Conformations

The dimensions and arrangements of aromatic rings (topology) in adducts derived from the reactions of polycyclic aromatic hydrocarbon (PAH) diol epoxide metabolites with DNA influence the distortions and stabilities of double-stranded DNA, and hence their recognition and processing by the human nucleotide excision repair (NER) system. Dibenzo[a,l]pyrene (DB[a,l]P) is a highly tumorigenic six-ring PAH, which contains a nonplanar and aromatic fjord region that is absent in the structurally related bay region five-ring PAH benzo[a]pyrene (B[a]P). The PAH diol epoxide–DNA adducts formed include the stereoisomeric 14S and 14Rtrans-anti-DB[a,l]P-N2-dG and the stereochemically analogous 10S- and 10R-B[a]P-N2-dG (B[a]P-dG) guanine adducts. However, nuclear magnetic resonance (NMR) solution studies of the 14S-DB[a,l]P-N2-dG adduct in DNA have not yet been presented. Here we have investigated the 14S-DB[a,l]P-N2-dG adduct in two different sequence contexts using NMR methods with distance-restrained molecular dynamics simulations. In duplexes with dC opposite the adduct deleted, a well-resolved base-displaced intercalative adduct conformation can be observed. In full duplexes, in contrast to the intercalated 14R stereoisomeric adduct, the bulky DB[a,l]P residue in the 14S adduct is positioned in a greatly widened and distorted minor groove, with significant disruptions and distortions of base pairing at the lesion site and two 5′-side adjacent base pairs. These unique structural features are significantly different from those of the stereochemically analogous but smaller B[a]P-dG adduct. The greater size and different topology of the DB[a,l]P aromatic ring system lead to greater structurally destabilizing DNA distortions that are partially compensated by stabilizing DB[a,l]P-DNA van der Waals interactions, whose combined effects impact the NER response to the adduct. These structural results broaden our understanding of the structure–function relationship in NER.

Cellular responses of BRCA1-defective and triple-negative breast cancer cells and in vitro BRCA1 interactions induced by metallo-intercalator ruthenium(II) complexes containing chloro-substituted phenylazopyridine

BackgroundTriple-negative breast cancer (TNBC) is defined by the absence of expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. Breast cancers with a BRCA1 mutation are also frequently triple-negative. Currently, there is a lack of effective therapies and known specific molecular targets for this aggressive breast cancer subtype. To address this concern, we have explored the cellular responses of BRCA1-defective and triple-negative breast cancer cells, and in vitro BRCA1 interactions induced by the ruthenium(II) complexes containing the bidentate ligand, 5-chloro-2-(phenylazo)pyridine.MethodsTriple-negative MDA-MB-231, BRCA1-defective HCC1937 and BRCA1-competent MCF-7 breast cancer cell lines were treated with ruthenium(II) complexes. The cytoxoxicity of ruthenium-induced breast cancer cells was evaluated by a real time cellular analyzer (RTCA). Cellular uptake of ruthenium complexes was determined by ICP-MS. Cell cycle progression and apoptosis were assessed using propidium iodide and Annexin V flow cytometry. The N-terminal BRCA1 RING protein was used for conformational and functional studies using circular dichroism and in vitro ubiquitination.ResultsHCC1937 cells were significantly more sensitive to the ruthenium complexes than the MDA-MB-231 and MCF-7 cells. Treatment demonstrated a higher degree of cytotoxicity than cisplatin against all three cell lines. Most ruthenium atoms were retained in the nuclear compartment, particularly in HCC1937 cells, after 24 h of incubation, and produced a significant block at the G2/M phase. An increased induction of apoptotic cells as well as an upregulation of p53 mRNA was observed in all tested breast cancer cells. It was of interest that BRCA1 mRNA and replication of BRCA1-defective cells were downregulated. Changes in the conformation and binding constants of ruthenium-BRCA1 adducts were observed, causing inactivation of the RING heterodimer BRCA1/BARD1-mediated E3 ubiquitin ligase activity.ConclusionsThis study has revealed the ability of ruthenium complexes to inhibit cell proliferation, induce cell cycle progression and apoptosis. Ruthenium treatment upregulated the marker genes involved in apoptosis and cell cycle progression while it downregulated BRCA1 mRNA and replication of HCC1937 cells. Our results could provide an alternative approach to finding effective therapeutic ruthenium-based agents with promising anticancer activity, and demonstrated that the BRCA1 RING domain protein was a promising therapeutic target for breast cancers.

Unusual enantioselectivities in heterogeneous organocatalyzed reactions: Reversal of direction using proline di- versus tri-peptides in the aldol addition

The heterogeneous asymmetric direct aldol reactions between aldehydes (2-nitrobenzaldehyde, 2-methylpropanal) and ketones (acetone, cyclohexanone) in the presence of polystyrene (PS) resin supported di- and tripeptides were studied under otherwise identical experimental conditions at room temperature in batch reactor. For all three asymmetric aldol reactions reversal of enantiomeric excess was observed on H-Pro-Pro-Glu(OH)-PS catalyst (ee 39–50%) as compared to the H-Pro-Glu(OH)-PS catalyst (ee 52–95%). The determinant role of the conformation of the intermediate adducts held responsible for chiral induction is interpreted by quantum chemical calculations.

α-Substituent effect on olefination of ester carbonyl groups with ynolates

In the olefination reaction of ester carbonyl with ynolate, an α-hetero (N, O, S)-substituent on the substrates was found to markedly improve the yields of tetrasubstituted olefins, even though it was sterically hindered. The α-substituent effect may be due to several factors including the addition based on the polar Felkin–Anh model and restricted conformation of the initial adducts.

Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo(-)) and DNA polymerase β (pol β) using (19)F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol β. The addition of a non-hydrolysable 2'-deoxycytosine-5'-[(α,β)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo(-) complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol β, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with (19)F NMR data. Surface plasmon resonance binding kinetics revealed that pol β binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential.

C8-Heteroaryl-2′-deoxyguanosine Adducts as Conformational Fluorescent Probes in the NarI Recognition Sequence

The optical, redox, and electronic properties of C(8)-heteroaryl-2'-deoxyguanosine (dG) adducts with C(8)-substituents consisting of furyl ((Fur)dG), pyrrolyl ((Pyr)dG), thienyl ((Th)dG), benzofuryl ((Bfur)dG), indolyl ((Ind)dG), and benzothienyl ((Bth)dG) are described. These adducts behave as fluorescent nucleobase probes with emission maxima from 379 to 419 nm and fluorescence quantum yields (Φ(fl)) in the 0.1-0.8 range in water at neutral pH. The probes exhibit quenched fluorescence with increased solvent viscosity and decreased solvent polarity. The (Fur)dG, (Bfur)dG, (Ind)dG, and (Bth)dG derivatives were incorporated into the G(3) position of the 12-mer oligonucleotide 5'-CTCG(1)G(2)CG(3)CCATC-3' that contains the recognition sequence of the NarI Type II restriction endonuclease. This sequence is widely used to study the biological activity (mutagenicity) of C(8)-arylamine-dG adducts with adduct conformation (anti vs syn) playing a critical role in the biological outcome. The modified NarI(X = (Fur)G, (Ind)G, (Bfur)G, or (Bth)G) oligonucleotides were hybridized to the complementary strand containing either C (NarI'(C)) or G (NarI'(G)) opposite the probe. The duplex structures were characterized by UV melting temperature analysis, fluorescence spectroscopy, collisional fluorescence quenching studies, and circular dichroism (CD). The emission of the probes showed sensitivity to the opposing base in the duplex, and suggested the utility of fluorescence spectroscopy to monitor probe conformation.

Nuclear Magnetic Resonance Solution Structure of an N2-Guanine DNA Adduct Derived from the Potent Tumorigen Dibenzo[a,l]pyrene: Intercalation from the Minor Groove with Ruptured Watson–Crick Base Pairing

The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the nonplanar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14 position with the exocyclic amino group of guanine. Here, we present the first nuclear magnetic resonance solution structure of a DB[a,l]P-derived adduct, the 14R-(+)-trans-anti-DB[a,l]P-N(2)-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N(2)-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3'-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3'-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE-DNA adduct conformation differs from (1) the classical intercalation motif in which Watson-Crick base pairing is intact at the lesion site and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix. The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed.

Structures of SHV-1 β-Lactamase with Penem and Penam Sulfone Inhibitors That Form Cyclic Intermediates Stabilized by Carbonyl Conjugation

Bacterial β-lactamase enzymes are in large part responsible for the decreased ability of β-lactam antibiotics to combat infections. The inability to overcome β-lactamase mediated resistance spurred the development of inhibitors with penems and penam sulfones being amongst the most potent and broad spectrum mechanism-based inactivators. These inhibitors form covalent, “suicide-type” inhibitory intermediates that are attached to the catalytic S70 residue. To further probe the details of the mechanism of β-lactamase inhibition by these novel compounds, we determined the crystal structures of SHV-1 bound with penem 1, and penam sulfones SA1-204 and SA3-53. Comparison with each other and with previously determined crystal structures of members of these classes of inhibitors suggests that the final conformation of the covalent adduct can vary greatly amongst the complex structures. In contrast, a common theme of carbonyl conjugation as a mechanism to avoid deacylation emerges despite that the penem and penam sulfone inhibitors form different types of intermediates. The detailed insights gained from this study could be used to further improve new mechanism-based inhibitors of these common class A serine β-lactamases.

Abstract 5469: Detection of deoxyguanosine adducts in the oral tissues of mice treated with the environmental carcinogen dibenzo[a, l]pyrene by LC-MS/MS

Abstract Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Our laboratory had developed a mouse model of oral cancer induced by DB[a, l]P. Our working hypothesis is that the stereochemical course of DB[a, l]P metabolism, the conformations of the DNA adducts formed and their removal by mammalian DNA repair enzymes, and their mutagenic properties if not removed in an error-free manner, all play critical roles during the induction of oral carcinogenesis. As an initial study to test our hypothesis, we have previously developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify DB[a, l]PDE-N6-dA adducts in oral tissues of mice treated with DB[a, l]P. We have shown that (-)-anti-cis- and (-)-anti-trans-DB[a, l]PDE-N6-dA adducts were detected from oral tissues of mice treated with DB[a, l]P. In the present study, to further test our hypothesis, we report on the development of a LC-MS/MS method to detect DB[a, l]PDE-N2-dG adducts in vivo. (±)-anti-[15N5]-DB[a, l]PDE-N2-dG adducts were synthesized as internal standards. The stereochemistry of adducts were characterized. Following the addition of internal standards, DNA isolated from oral tissues of mice treated with DB[a, l]P or DB[a, l]PDE was enzymatically hydrolyzed to 2′-deoxyribonucleosides and partially purified by solid-phase extraction. The LC-MS/MS analysis was carried out by monitoring transitions m/z 620 [M+H]+→ m/z 504 [(M+H)+−2′-deoxyribose] for DB[a, l]PDE-N2-dG adducts and m/z 625α m/z 509 for the internal standards. We have detected two N2-dG adducts from oral tissues of mice treated with DB[a, l]P, and four N2-dG adducts from oral tissues of mice treated with (±)-anti-DB[a, l]PDE. Collectively, the in vivo detection of dA and dG adducts indicated that DB[a, l]P is predominantly metabolized to (-)-anti-DB[a, l]PDE in oral tissues of mice. This sensitive LC-MS/MS method, is capable of simultaneously detecting both dA and dG adducts derived from anti-DB[a, l]PDE. Our results indicated that levels of dA adducts are significantly higher than dG adducts in vivo, which are consistent with those reported in literature in organs other than oral tissues, demonstrating that fjord region diol epoxide of DB[a, l]P predominantly form dA adducts than dG adducts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5469. doi:1538-7445.AM2012-5469

How Alkali Metal Ion Binding Alters the Conformation Preferences of Gramicidin A: A Molecular Dynamics and Ion Mobility Study

Here, we present a systematic study combing electrospray ionization-ion mobility experiments and an enhanced sampling molecular dynamics, specifically integrated tempering sampling molecular dynamics simulations (ITS-MDS), to explore the conformations of alkali metal ion (Na, K, and Cs) adducts of gramicidin A (GA) in vacuo. Folding simulation is performed to obtain inherent conformational preferences of neutral GA to provide insights about how the binding of metal ions influences the intrinsic conformations of GA. The comparison between conformations of neutral GA and alkali metal ion adducts reveals a high degree of structural similarity, especially between neutral GA and [GA + Na](+); however, the structural similarities decrease as ionic radius of the metal increases. Collision cross section (CCS) profiles for [GA + Na](+) and [GA + Cs](+) ions obtained from by ITS-MDS compare favorably with the experimental CCS, but there are significant differences from CCS profiles for [GA + K](+) ions. Such discrepancies between the calculated and measured CCS profiles for [GA + K](+) are discussed in terms of limitations in the simulation force field as well as possible size-dependent coordination of the [GA + K](+) ion complex.

Flanking Bases Influence the Nature of DNA Distortion by Platinum 1,2-Intrastrand (GG) Cross-Links

The differences in efficacy and molecular mechanisms of platinum anti-cancer drugs cisplatin (CP) and oxaliplatin (OX) are thought to be partially due to the differences in the DNA conformations of the CP and OX adducts that form on adjacent guanines on DNA, which in turn influence the binding of damage-recognition proteins that control downstream effects of the adducts. Here we report a comprehensive comparison of the structural distortion of DNA caused by CP and OX adducts in the TGGT sequence context using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations. When compared to our previous studies in other sequence contexts, these structural studies help us understand the effect of the sequence context on the conformation of Pt-GG DNA adducts. We find that both the sequence context and the type of Pt-GG DNA adduct (CP vs. OX) play an important role in the conformation and the conformational dynamics of Pt-DNA adducts, possibly explaining their influence on the ability of many damage-recognition proteins to bind to Pt-DNA adducts.

Abstract 5550: Identification and quantification of DNA adducts in the oral tissues of mice treated with the environmental carcinogen dibenzo[a,l]pyrene by LC-MS/MS

Abstract Oral cancer is the major form of head and neck squamous cell carcinoma, which is the sixth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Our laboratory had developed a novel mouse model of oral cancer induced by DB[a,l]P. The metabolic activation of DB[a,l]P and genotoxic effect in rodent oral cavity have not been examined before. Our working hypothesis is that the stereochemical course of DB[a,l]P metabolism, the conformations of the DNA adducts formed, their removal by mammalian DNA repair enzymes, and their mutagenic properties if not removed in an error-free manner, all play critical roles during the induction of oral carcinogenesis. As an initial study to test our hypothesis, here we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify DB[a,l]PDE-N6-dA adducts in oral tissues of mice treated with DB[a,l]P. (±)-anti-[15N5]-DB[a,l]PDE-N6-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR and CD analysis. After addition of internal standards, DNA was enzymatically hydrolyzed to 2’-deoxyribonucleosides and partially purified by solid-phase extraction. The LC-MS/MS analysis was carried out by monitoring transitions m/z 604 [M+H]+ to m/z 335 [(M+H) +-dA-H2O] for DB[a,l]PDE-N6-dA adducts and m/z 609 to m/z 335 for the internal standards. The detection limit for DB[a,l]PDE-dA adduct is approximately 20 fmol in 100 ug DNA. We have compared the formation and removal of adducts as a function of time following direct application of DB[a,l]P into the oral cavity of B6C3F1 mice (24 nmol, 3 times per week, for 5 weeks). Adducts were measured at 48h, 1, 2 and 4 weeks after the last dose. Only (−)-anti-cis-and (−)-anti-trans- DB[a,l]PDE- N6-dA adducts were detected from oral tissues of DB[a,l]P treated mice, ranging from 1.9± 0.3 to 0.7± 0.1 and from 4.8 ± 0.2 to 2.3 ± 0.3 fmol/ug DNA, respectively. The levels of both adducts decreased in a time-dependent manner; approximately 50% of (−)-anti-cis- and (−)-anti-trans- DB[a,l]PDE- N6-dA adducts were removed within 4 weeks. The results suggest that DB[a,l]P is predominantly metabolized to (−)-anti-DB[a,l]PDE in oral tissues of mice, which is consistent with studies in other preclinical model systems. In this study we developed for the first time a sensitive LC-MS/MS method for the detection and quantification of DNA adducts derived from DB[a,l]P. The persistent (−)-anti-DB[a,l]PDE- N6-dA adducts may account for the initiation of carcinogenesis in the oral tissues of mice treated with DB[a,l]P. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5550. doi:10.1158/1538-7445.AM2011-5550

Intercalative Conformations of the 14R (+)- and 14S (−)-trans-anti-DB[a,l]P-N6-dA Adducts: Molecular Modeling and MD Simulations

Among the polycyclic aromatic hydrocarbon class of chemical carcinogens, dibenzo[a,l]pyrene (DB[a,l]P) is the most potent tumorigen that has been identified to date. Structurally, it is bulky with six aromatic rings, and it contains the nonplanar fjord-region. The conformational properties of DB[a,l]P-derived DNA adducts responsible for its extraordinary carcinogenicity are hence of great interest. We have carried out molecular modeling and MD simulations for the 14R (+)- and 14S (-)-trans-anti-DB[a,l]P-N⁶-dA adducts derived from the reactions of the DB[a,l]P diol epoxides with adenine in double-stranded DNA. The structures are based on the classically intercalated NMR solution structures of the analogous fjord-region benzo[c]phenanthrene-derived-N⁶-dA adducts. One objective was to gain insight on the impact of the more bulky DB[a,l]P ring system on the structural characteristics of the intercalative adduct conformations. A further objective was to elucidate the effect of the flexible twist associated with the sterically hindered aromatic ring in the fjord-region on the intercalated conformations, for comparison with the intercalated but planar bay-region benzo[a]pyrene-derived-N⁶-dA adducts. For the DB[a,l]P-N⁶-dA adducts, our results show that the 14R (+)-adduct is more favorably intercalated on the 5'-side of the modified adenine than the stereoisomeric 14S (-)-adduct, intercalated on its 3'-side. The 14R (+)-adduct manifests better van der Waals stacking interactions with flanking base pairs, less perturbed Watson-Crick hydrogen bonding, less local groove enlargement, less unwinding, and a lower solvent exposure than the 14S (-)-adduct. These structural findings are consistent with observed thermodynamic melting data, UV absorption properties, and fluorescence quenching studies. By contrast, the NMR solution structures for the analogous but less bulky B[c]Ph-derived adducts reveal no such stereoisomeric effect, while the planar bay-region benzo[a]pyrene-derived-N⁶-dA adducts do. Differences in nucleotide excision repair susceptibilities of the fjord and bay region adducts stem from distinctions in their intercalative conformations, produced by the intrinsic topological variations in their polycyclic aromatic ring systems.

Conformation and Self-Assembly of the Transmembrane Peptide Gramicidin A: Insights from ion Mobility Spectrometry and Molecular Dynamics

Gramicidin A which is composed of alternating L- and D-amino acids is a naturally occurring pentadecapeptide from Bacillus brevis known to form monovalent metal ion channels in lipid membranes. The active form is a noncovalently bound dimer. The conformation and self-assembly behavior of gramicidin A highly depends on the solution environment. In this presentation, we report the use of electrospray-ion mobility-mass spectrometry to study the conformation of alkali metals adducts of gramicidin A monomer, as well as the monomerization and conformer interconversion equilibrium of gramicidin A dimer as a function of the solvent. The conformation of gramicidin A monomer vary significantly upon binding different metal adducts. Enhanced sampling molecular dynamics simulations are performed on alkali metals adducts to provide thermodynamics information of different conformers and gain insights of the interaction of different metal ions with the monomer. The kinetics of the monomerization and conformer interconversion processes of dimer in various alcohol solutions (Ethanol, 1-Propanol and, Isobutanol) are monitored by using the ion mobility profile of the monomer and the dimer. The rate constants and the temperature dependence of the rate constants of the monomer compare well with literature values which were obtained by using fluorescence. Furthermore, we found that the water content in the alcohol solution greatly influences the self-assembly process significantly. The role of water in catalyzing the conformer interconversion is being investigated further. Ion mobility spectrometry (IMS) combined with molecular dynamics simulations is a merging technique for conformational analysis of gas-phase low-lying energy level structures of biomolecules. In this study, we will demonstrate that this gas phase technique can also be of utility in studying a solution phase structural dynamics problem.