Synthesis, characterization and absolute configurations of methyl ladderanoates.

Methyl esters of [5]-ladderanoic acid and [3]-ladderanoic acid were prepared by esterification of the acids isolated from biomass at a wastewater treatment plant. Optical rotations at six different wavelengths (633, 589, 546, 436, 405 and 365 nm) and vibrational circular dichroism (VCD) spectra in the 1800-900 cm-1 region were measured in CDCl3 solvent and compared with quantum chemical (QC) predictions using B3LYP functional and 6-311++G(2d,2p) basis set with polarizing continuum model representing the solvent. QC predictions gave negative optical rotations at all six wavelengths for (R)-methyl [5]-ladderanoate and positive optical rotations for (R)-methyl [3]-ladderanoate, the same signs as previously reported for the corresponding acids. The crystal structure of (-)-methyl [5]-ladderanoate independently confirmed (R) configuration. The QC-predicted VCD spectra using Boltzmann population weighted spectra of individual conformers did not provide satisfactory quantitative agreement with the experimental VCD spectra. An improved quantitative agreement for VCD spectra could be obtained when conformer populations were optimized to maximize the similarity between experimental and predicted VCD spectra, but more improvements in VCD predictions are needed.

Alkali Metal- and Acid-Catalyzed Interconversion of Goniodomin A with Congeners B and C.

Goniodomin A (GDA, 1) is a phycotoxin produced by at least four species of Alexandrium dinoflagellates that are found globally in brackish estuaries and lagoons. It is a linear polyketide with six oxygen heterocyclic rings that is cyclized into a macrocyclic structure via lactone formation. Two of the oxygen heterocycles in 1 comprise a spiro-bis-pyran, whereas goniodomin B (GDB) contains a 2,7-dioxabicyclo[3.3.1]nonane ring system fused to a pyran. When H2O is present, 1 undergoes facile conversion to isomer GDB and to an α,ß-unsaturated ketone, goniodomin C (GDC, 7). GDB and GDC can be formed from GDA by cleavage of the spiro-bis-pyran ring system. GDA, but not GDB or GDC, forms a crown ether-type complex with K+. Equilibration of GDA with GDB and GDC is observed in the presence of H+ and of Na+, but the equilibrated mixtures revert to GDA upon addition of K+. Structural differences have been found between the K+ and Na+ complexes. The association of GDA with K+ is strong, while that with Na+ is weak. The K+ complex has a compact, well-defined structure, whereas Na+ complexes are an ill-defined mixture of species. Analyses of in vitro A. monilatum and A. hiranoi cultures indicate that only GDA is present in the cells; GDB and GDC appear to be postharvest transformation products.

Algal Toxin Goniodomin A Binds Potassium Ion Selectively to Yield a Conformationally Altered Complex with Potential Biological Consequences.

The marine toxin goniodomin A (GDA) is a polycyclic macrolide containing a spiroacetal and three cyclic ethers as part of the macrocycle backbone. GDA is produced by three species of the Alexandrium genus of dinoflagellates, blooms of which are associated with "red tides", which are widely dispersed and can cause significant harm to marine life. The toxicity of GDA has been attributed to stabilization of the filamentous form of the actin group of structural proteins, but the structural basis for its binding is not known. Japanese workers, capitalizing on the assumed rigidity of the heavily substituted macrolide ring, assigned the relative configuration and conformation by relying on NMR coupling constants and NOEs; the absolute configuration was assigned by degradation to a fragment that was compared with synthetic material. We have confirmed the absolute structure and broad features of the conformation by X-ray crystallography but have found GDA to complex with alkali metal ions in spite of two of the heterocyclic rings facing outward. Such an arrangement would have been expected to impair the ability of GDA to form a crown-ether-type multidentate complex. GDA shows preference for K+, Rb+, and Cs+ over Li+ and Na+ in determinations of relative affinities by TLC on metal-ion-impregnated silica gel plates and by electrospray mass spectrometry. NMR studies employing the K+ complex of GDA, formed from potassium tetrakis[pentafluorophenyl]borate (KBArF20), reveal a major alteration of the conformation of the macrolide ring. These observations argue against the prior assumption of rigidity of the ring. Alterations in chemical shifts, coupling constants, and NOEs indicate the involvement of most of the molecule other than ring F. Molecular mechanics simulations suggest K+ forms a heptacoordinate complex involving OA, OB, OC, OD, OE, and the C-26 and C-27 hydroxy groups. We speculate that complexation of K+ with GDA electrostatically stabilizes the complex of GDA with filamentous actin in marine animals due to the protein being negatively charged at physiological pH. GDA may also cause potassium leakage through cell membranes. This study provides insight into the structural features and chemistry of GDA that may be responsible for significant ecological damage associated with the GDA-producing algal blooms.

miR-375 Regulates Invasion-Related Proteins Vimentin and L-Plastin.

Invasion is a hallmark of advanced head and neck squamous cell carcinoma (HNSCC). We previously determined that low relative miR-375 expression was associated with poor patient prognosis. HNSCC cells with increased miR-375 expression have lower invasive properties and impaired invadopodium activity. Using stable isotope labeling with amino acids in cell culture and reverse-phase liquid chromatography mass spectrometry, we assessed the impact of miR-375 expression on protein levels in UM-SCC-1 cells. Increased miR-375 expression was associated with down-regulation of proteins involved in cellular assembly and organization, death and survival, and movement. Two invasion-associated proteins, vimentin and L-plastin, were strongly down-regulated by miR-375. Luciferase reporter assays demonstrated that high miR-375 expression reduced vimentin promoter activity, suggesting that vimentin is an indirect target of miR-375. Runt-related transcription factor 1 (RUNX1) is a potential miR-375 direct target, and its knockdown reduced vimentin and L-plastin expression. Data in The Cancer Genome Atlas HNSCC database showed a significant inverse correlation between miR-375 expression and RUNX1, vimentin, and L-plastin RNA expression. These clinical correlations validate our in vitro model findings and support a mechanism in which miR-375 suppresses RUNX1 levels, resulting in reduced vimentin and L-plastin expression. Furthermore, knockdown of RUNX1, L-plastin, and vimentin resulted in significant reductions in cell invasion in vitro, indicating the functional significance of miR-375 regulation of specific proteins involved in HNSCC invasion.

Apolipoprotein E Promotes Invasion in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) patients generally have a poor prognosis, because of the invasive nature of these tumors. In comparing transcription profiles between OSCC tumors with a more invasive (worst pattern of tumor invasion 5) versus a less invasive (worst pattern of tumor invasion 3) pattern of invasion, we identified a total of 97 genes that were overexpressed at least 1.5-fold in the more invasive tumor subtype. The most functionally relevant genes were assessed using in vitro invasion assays with an OSCC cell line (UM-SCC-1). Individual siRNA knockdown of 15 of these 45 genes resulted in significant reductions in tumor cell invasion compared to a nontargeting siRNA control. One gene whose knockdown had a strong effect on invasion corresponded to apolipoprotein E (APOE). Both matrix degradation and the number of mature invadopodia were significantly decreased with APOE knockdown. APOE knockdown also resulted in increased cellular cholesterol, consistent with APOE's role in regulating cholesterol efflux. APOE knockdown resulted in decreased levels of phospho-extracellular signal-regulated kinase 1/2, phospho-c-Jun N-terminal kinase, and phospho-cJun, as well as decreased activator protein 1 (AP-1) activity. Expression of matrix metalloproteinase 7 (MMP7), an AP-1 target, was also significantly decreased. Our findings suggest that APOE protein plays a significant role in OSCC tumor invasion because of its effects on cellular cholesterol and subsequent effects on cell signaling and AP-1 activity, leading to changes in the expression of invasion-related proteins, including MMP7.

Absolute Configurations of Naturally Occurring [5]- and [3]-Ladderanoic Acids: Isolation, Chiroptical Spectroscopy, and Crystallography.

We have isolated mixtures of [5]- and [3]-ladderanoic acids 1a and 2a from the biomass of an anammox bioreactor and have separated the acids and their phenacyl esters for the first time by HPLC. The absolute configurations of the naturally occurring acids and their phenacyl esters are assigned as R at the site of side-chain attachment by comparison of experimental specific rotations with corresponding values predicted using quantum chemical (QC) methods. The absolute configurations for 1a and 2a were independently verified by comparison of experimental Raman optical activity spectra with corresponding spectra predicted using QC methods. The configurational assignments of 1a and 2a and of the phenacyl ester of 1a were also confirmed by X-ray crystallography.

Mass spectrometric characterization of the seco acid formed by cleavage of the macrolide ring of the algal metabolite goniodomin A.

Goniodomin A (GDA) is a polyketide macrolide produced by multiple species of the marine dinoflagellate genus Alexandrium. GDA is unusual in that it undergoes cleavage of the ester linkage under mild conditions to give mixtures of seco acids (GDA-sa). Ring-opening occurs even in pure water although the rate of cleavage accelerates with increasing pH. The seco acids exist as a dynamic mixture of structural and stereo isomers which is only partially separable by chromatography. Freshly prepared seco acids show only end absorption in the UV spectrum but a gradual bathochromic change occurs, which is consistent with formation of α,ß-unsaturated ketones. Use of NMR and crystallography is precluded for structure elucidation. Nevertheless, structural assignments can be made by mass spectrometric techniques. Retro-Diels-Alder fragmentation has been of value for independently characterizing the head and tail regions of the seco acids. The chemical transformations of GDA revealed in the current studies help clarify observations made on laboratory cultures and in the natural environment. GDA has been found to reside mainly within the algal cells while the seco acids are mainly external with the transformation of GDA to the seco acids occurring largely outside the cells. This relationship, plus the fact that GDA is short-lived in growth medium whereas GDA-sa is long-lived, suggests that the toxicological properties of GDA-sa in its natural environment are more important for the survival of the Alexandrium spp. than those of GDA. The structural similarity of GDA-sa to that of monensin is noted. Monensin has strong antimicrobial properties, attributed to its ability to transport sodium ions across cell membranes. We propose that toxic properties of GDA may primarily be due to the ability of GDA-sa to mediate metal ion transport across cell membranes of predator organisms.

Thecal plate morphology, molecular phylogeny, and toxin analyses reveal two novel species of Alexandrium (Dinophyceae) and their potential for toxin production.

This study describes two novel species of marine dinophytes in the genus Alexandrium. Morphological characteristics and phylogenetic analyses support the placement of the new taxa, herein designated as Alexandrium limii sp. nov. and A. ogatae sp. nov. Alexandrium limii, a species closely related to A. taylorii, is distinguished by having a shorter 2'/4' suture length, narrower plates 1' and 6'', with larger length: width ratios, and by the position of the ventral pore (Vp). Alexandrium ogatae is distinguishable with its metasert plate 1' having almost parallel lateral margins, and by lacking a Vp. Production of paralytic shellfish toxins (PSTs), cycloimines, and goniodomins (GDs) in clonal cultures of A. ogatae, A. limii, and A. taylorii were examined analytically and the results showed that all strains contained GDs, with GDA as major variants (6-14 pg cell-1) for all strains except the Japanese strain of A. limii, which exclusively had a desmethyl variant of GDA (1.4-7.3 pg cell-1). None of the strains contained detectable levels of PSTs and cycloimines.

Hypermethylation of a cluster of Krüppel-type zinc finger protein genes on chromosome 19q13 in oropharyngeal squamous cell carcinoma.

Identification of epigenetically affected genes has become an important tool for understanding both normal and aberrant gene expression in cancer. Here we report a whole-genome analysis of DNA methylation profiles in fresh-frozen oropharyngeal squamous cell carcinoma (OPSCC) tissues and normal mucosa samples using microarray technology with patient genomic DNA. We initially compared whole-genome patterns of DNA methylation among 24 OPSCC primary tumors and 24 matched normal mucosal samples. From a survey of 27,578 CpG dinucleotide loci spanning more than 14,000 genes, we identified 958 CpG loci in which measurements of DNA methylation were altered in the primary tumors relative to the normal mucosal samples. These alterations were validated in an independent set of 21 OPSCC patients. A survey of these loci by chromosomal location revealed an abnormally high number of differentially methylated loci on chromosome 19. Many of the loci on chromosome 19 are associated with genes belonging to the Krüppel-type zinc finger protein genes. Hypermethylation was accompanied by a significant decrease in expression of these genes in OPSCC primary tumors relative to adjacent mucosa. This study reports the epigenetic silencing of Krüppel-type zinc finger protein genes on chromosome 19q13 in oropharyngeal cancer. The aberrant methylation of these genes represents a new avenue of exploration for pathways affected in this disease.

Novel monobactams utilizing a siderophore uptake mechanism for the treatment of gram-negative infections.

Novel siderophore-linked monobactams with in vitro and in vivo anti-microbial activity against MDR Gram-negative pathogens are described.

Deoxyguanosine forms a bis-adduct with E,E-muconaldehyde, an oxidative metabolite of benzene: implications for the carcinogenicity of benzene.

Benzene is employed in large quantities in the chemical industry and is an ubiquitous contaminant in the environment. There is strong epidemiological evidence that benzene exposure induces hematopoietic malignancies, especially acute myeloid leukemia, in humans, but the chemical mechanisms remain obscure. E,E-Muconaldehyde is one of the products of metabolic oxidation of benzene. This paper explores the proposition that E,E-muconaldehyde is capable of forming Gua-Gua cross-links. If formed in DNA, the replication and repair of such cross-links might introduce structural defects that could be the origin of the carcinogenicity. We have investigated the reaction of E,E-muconaldehyde with dGuo and found that the reaction yields two pairs of interconverting diastereomers of a novel heptacyclic bis-adduct having a spiro ring system linking the two Gua residues. The structures of the four diastereomers have been established by NMR spectroscopy and their absolute configurations by comparison of CD spectra with those of model compounds having known configurations. The final two steps in the formation of the bis-nucleoside (5-ring → 6-ring → 7-ring) have significant reversibility, which is the basis for the observed epimerization. The 6-ring precursor was trapped from the equilibrating mixture by reduction with NaBH(4). The anti relationship of the two Gua residues in the heptacyclic bis-adduct precludes it from being formed in B DNA, but the 6-ring precursor could readily be accommodated as an interchain or intrachain cross-link. It should be possible to form similar cross-links of dCyt, dAdo, the ε-amino group of lysine, the imidazole NH of histidine, and N termini of peptides with the dGuo-muconaldehyde monoadduct.

Formation of deoxyguanosine cross-links from calf thymus DNA treated with acrolein and 4-hydroxy-2-nonenal.

Acrolein (AC) and 4-hydroxy-2-nonenal (HNE) are endogenous bis-electrophiles that arise from the oxidation of polyunsaturated fatty acids. AC is also found in high concentrations in cigarette smoke and automobile exhaust. These reactive α,ß-unsaturated aldehyde (enal) covalently modify nucleic acids, to form exocyclic adducts, where the three-carbon hydroxypropano unit bridges the N1 and N(2) positions of deoxyguanosine (dG). The bifunctional nature of these enals allows them to undergo reaction with a second nucleophilic group and form DNA cross-links. These cross-linked enal adducts are likely to contribute to the genotoxic effects of both AC and HNE. We have developed a sensitive mass spectrometric method to detect cross-linked adducts of these enals in calf thymus DNA (CT DNA) treated with AC or HNE. The AC and HNE cross-linked adducts were measured by the stable isotope dilution method, employing a linear quadrupole ion trap mass spectrometer and consecutive reaction monitoring at the MS(3) or MS(4) scan stage. The lower limit of quantification of the cross-linked adducts is ∼1 adduct per 10(8) DNA bases, when 50 µg of DNA is assayed. The cross-linked adducts occur at levels that are ∼1-2% of the levels of the monomeric 1,N(2)-dG adducts in CT DNA treated with either enal.

Structure-activity relationships and hepatic safety risks of thiazole agonists of the thrombopoietin receptor.

5-F substitution of an aminothiazole moiety within a series of thrombopoietin receptor agonists leads to potent agents with an improved hepatic safety profile in rodent toxicology studies.

Revisiting the toxin profile of Alexandrium pseudogonyaulax; Formation of a desmethyl congener of goniodomin A.

During a survey of the production of goniodomin A (GDA) by Alexandrium pseudogonyaulax in Danish coastal waters, Krock et al. (2018) obtained mass spectral evidence for the presence of a truncated congener, herein termed GD754, having a molecular weight 14 Da lower than GDA and assigned it as goniodomin B (GDB). An erroneous structure of GDB involving deletion of a methylene group between rings B and D had previously been reported by Espiña et al. (2016) but without experimental details. HPLC properties reported by Krock for GD754 point to it being a homolog of GDA. Comparison of mass spectral fragmentation data reported for GD754 with fragmentation data for GDA, show it to be a truncated form of GDA with the deletion involving a CH2 group from ring F or one of the two methyl substituents on ring F, not elsewhere on the molecule. On biosynthetic grounds, the GD754 congener is proposed to be 34-desmethyl-GDA. Further experimental work will be required to confirm this hypothesis.

The toxin goniodomin, produced by Alexandrium spp., is identical to goniodomin A.

In 1968 Burkholder and associates (J. Antibiot. (Tokyo)1968, 21, 659-664) isolated the antifungal toxin goniodomin from an unidentified Puerto Rican dinoflagellate and partially characterized its structure. Subsequently, a metabolite of Alexandrium hiranoi was isolated by Murakami et al. from a bloom in Japan and its structure was established (Tetrahedron Lett.1988, 29, 1149-1152). The Japanese substance had strong similarities to Burkholder's but due to uncertainty as to whether it was identical or only similar, Murakami named his toxin goniodomin A. A detailed study of this question now provides compelling evidence that Burkholder's goniodomin is identical to goniodomin A. Morphological characterization of the dinoflagellate suggests that it was the genus Alexandrium but insufficient evidence is available to make a definite identification of the species. This is the only report of goniodomin in the Caribbean region.

Apoptosis signaling molecules as treatment targets in head and neck squamous cell carcinoma.

OBJECTIVES: To evaluate BCL-2 family signaling molecules in head and neck squamous cell carcinoma (HNSCC) and examine the ability of therapeutic agents with variable mechanisms of action to induce apoptosis in HNSCC cells. METHODS: messenger ribonculeic acid (mRNA) expression of BAK, BAX, B-cell lymphoma (Bcl-2), BCL2 Like 1 (BCL2L1), and MCL1 were measured in The Cancer Genome Atlas (TCGA) head and neck cancer dataset, as well as in a dataset from a cohort at Montefiore Medical Center (MMC). Protein expression was similarly evaluated in a panel of HNSCC cell lines (HN30, HN31, HN5, MDA686LN, UMSCC47). Cell viability and Annexin V assays were used to assess the efficacy and apoptotic potential of a variety of agents (ABT-263 [navitoclax], A-1210477, and bortezomib. RESULTS: Expression of BAK, BAX, BCL2L1, and MCL1 were each significantly higher than expression of BCL2 in the TCGA and MMC datasets. Protein expression demonstrated the same pattern of expression when examined in HNSCC cell lines. Treatment with combined ABT-263 (navitoclax)/A-1210477 or with bortezomib demonstrated apoptosis responses that approached or exceeded treatment with staurospaurine control. CONCLUSION: HNSCC cells rely on inhibition of apoptosis via BCL-xL and MCL-1 overexpression, and induction of apoptosis remains a potential therapeutic option as long as strategies overcome redundant anti-apoptotic signals. LEVEL OF EVIDENCE: NA Laryngoscope, 130:2643-2649, 2020.

Structural perturbations induced by the alpha-anomer of the aflatoxin B(1) formamidopyrimidine adduct in duplex and single-strand DNA.

The guanine N7 adduct of aflatoxin B(1) exo-8,9-epoxide hydrolyzes to form the formamidopyrimidine (AFB-FAPY) adduct, which interconverts between alpha and beta anomers. The beta anomer is highly mutagenic in Escherichia coli, producing G --> T transversions; it thermally stabilizes the DNA duplex. The AFB-alpha-FAPY adduct blocks replication; it destabilizes the DNA duplex. Herein, the structure of the AFB-alpha-FAPY adduct has been elucidated in 5'-d(C(1)T(2)A(3)T(4)X(5)A(6)T(7)T(8)C(9)A(10))-3'.5'-d(T(11)G(12)A(13)A(14)T(15)C(16)A(17)T(18)A(19)G(20))-3' (X = AFB-alpha-FAPY) using molecular dynamics calculations restrained by NMR-derived distances and torsion angles. The AFB moiety intercalates on the 5' face of the pyrimidine moiety at the damaged nucleotide between base pairs T(4).A(17) and X(5).C(16), placing the FAPY C5-N(5) bond in the R(a) axial conformation. Large perturbations of the epsilon and zeta backbone torsion angles are observed, and the base stacking register of the duplex is perturbed. The deoxyribose orientation shifts to become parallel to the FAPY base and displaced toward the minor groove. Intrastrand stacking between the AFB moiety and the 5' neighbor thymine remains, but strong interstrand stacking is not observed. A hydrogen bond between the formyl group and the exocyclic amine of the 3'-neighbor adenine stabilizes the E conformation of the formamide moiety. NMR studies reveal a similar 5'-intercalation of the AFB moiety for the AFB-alpha-FAPY adduct in the tetramer 5'-d(C(1)T(2)X(3)A(4))-3', involving the R(a) axial conformation of the FAPY C5-N(5) bond and the E conformation of the formamide moiety. Since in duplex DNA the AFB moiety of the AFB-beta-FAPY adduct also intercalates on the 5' side of the pyrimidine moiety at the damaged nucleotide, we conclude that favorable 5'-stacking leads to the R(a) conformational preference about the C5-N(5) bond; the same conformational preference about this bond is also observed at the nucleoside and base levels. The structural distortions and the less favorable stacking interactions induced by the AFB-alpha-FAPY adduct explain its lower stability as compared to the AFB-beta-FAPY adduct in duplex DNA. In this DNA sequence, hydrogen bonding between the formyl oxygen and the exocyclic amine of the 3'-neighboring adenine stabilizing the E configuration of the formamide moiety is also observed for the AFB-beta-FAPY adduct, and suggests that the identity of the 3'-neighbor nucleotide modulates the stability and biological processing of AFB adducts.

Stereospecific formation of the (R)-gamma-hydroxytrimethylene interstrand N2-dG:N2-dG cross-link arising from the gamma-OH-1,N2-propano-2'-deoxyguanosine adduct in the 5'-CpG-3' DNA sequence.

Acrolein reacts with dG to form hydroxylated 1,N(2)-propanodeoxyguanosine (OH-PdG) adducts. Most abundant are the epimeric 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2a] purin-10(3H)-ones, commonly referred to as the gamma-OH-PdG adducts. When placed complementary to deoxycytosine in duplex DNA, these undergo rearrangement to the N(2)-(3-oxopropyl)-dG aldehyde. The latter forms diastereomeric interstrand N(2)-dG:N(2)-dG cross-links in the 5'-CpG-3' sequence. Here we report the structure of the stereochemically favored (R)-gamma-hydroxytrimethylene N(2)-dG:N(2)-dG interstrand DNA cross-link in 5'-d(G(1)C(2)T(3)A(4)G(5)C(6)X(7)A(8)G(9)T(10)C(11)C(12))-3' x 5'-d(G(13)G(14)A(15)C(16)T(17)C(18)Y(19)C(20)T(21)A(22)G(23)C(24))-3' (X(7) is the dG linked to the alpha-carbon of the carbinolamine linkage, and Y(19) is the dG linked to the gamma-carbon of the carbinolamine linkage; the cross-link is in the 5'-CpG-3' sequence). The structure was characterized using isotope-edited (15)N nuclear Overhauser enhancement spectroscopy heteronuclear single quantum correlation (NOESY-HSQC) NMR, in which the exocyclic amines at X(7) or Y(19) were (15)N-labeled. Analyses of NOE intensities involving Y(19) N(2)H indicated that the (R)-gamma-hydroxytrimethylene linkage was the major cross-link species, constituting 80-90% of the cross-link. The X(7) and Y(19) imino resonances were observed at 65 degrees C. Additionally, for the 5'-neighbor base pair G(5) x C(20), the G(5) imino resonance remained sharp at 55 degrees C but broadened at 65 degrees C. In contrast, for the 3'-neighbor A(8) x T(17) base pair, the T(17) imino resonance was severely broadened at 55 degrees C. Structural refinement using NOE distance restraints obtained from isotope-edited (15)N NOESY-HSQC data indicated that the (R)-gamma-hydroxytrimethylene linkage maintained the C(6) x Y(19) and X(7) x C(18) base pairs with minimal structural perturbations. The (R)-gamma-hydroxytrimethylene linkage was located in the minor groove. The X(7) N(2) and Y(19) N(2) atoms were in the gauche conformation with respect to the linkage, which maintained Watson-Crick hydrogen bonding of the cross-linked base pairs. The anti conformation of the hydroxyl group with respect to C(alpha) of the tether minimized steric interaction and, more importantly, allowed the formation of a hydrogen bond between the hydroxyl group and C(20) O(2) located in the 5'-neighboring base pair G(5) x C(20). The formation of this hydrogen bond may, in part, explain the thermal stability of this carbinolamine interstrand cross-link and the stereochemical preference for the (R) configuration of the cross-link.

Interstrand DNA cross-links induced by alpha,beta-unsaturated aldehydes derived from lipid peroxidation and environmental sources.

Significant levels of the 1, N(2)-gamma-hydroxypropano-dG adducts of the alpha,beta-unsaturated aldehydes acrolein, crotonaldehyde, and 4-hydroxy-2E-nonenal (HNE) have been identified in human DNA, arising from both exogenous and endogenous exposures. 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)-gamma-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)-gamma-hydroxypropano-dG adduct. The stereochemistry at this center modulates the orientation of the reactive aldehyde within the minor groove of the double-stranded 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.

The development and evaluation of monoclonal antibodies for the detection of polycyclic aromatic hydrocarbons.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of 3- to 5-ring polycyclic aromatic hydrocarbons (PAHs) has been developed. A functional derivative of dibenzothiophene was synthesized and covalently linked to carrier proteins that were used to produce monoclonal antibodies (mAbs). During the conjugation step, the conjugation efficiency was improved by the presence of 25% N,N-dimethylformamide (DMF). Antibodies were selected based on a competitive inhibition assay to isolate those with the highest sensitivity for free PAHs. When using the mAb in an ELISA format, free PAHs were detected at a concentration as low as 0.1 microg/L (0.1 ppb) in aqueous samples.