Nucleotide excision repair (NER) is a major repair pathway that recognizes and corrects various lesions in cellular DNA. We hypothesize that damage recognition is an initial step in NER that senses ...conformational anomalies in the DNA caused by lesions. We prepared three DNA duplexes containing the carcinogen adduct N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) at G(1), G(2) or G(3) of NarI sequence (5'-CCG(1)G(2)CG(3)CC-3'). Our (19)F-NMR/ICD results showed that FAAF at G(1) and G(3) prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G(2). We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G(3) and -G(1) duplexes incised more efficiently than the B-type G(2) duplex (G(3)∼G(1)> G(2)). The melting and thermodynamic data indicate that the S- and W-conformers produce greater DNA distortion and thermodynamic destabilization. The N-deacetylated N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF) adducts in the same NarI sequence are repaired 2- to 3-fold less than FAAF: however, the incision efficiency was in order of G(2)∼G(1)> G(3), a reverse trend of the FAAF case. We have envisioned the so-called N-acetyl factor as it could raise conformational barriers of FAAF versus FAF. The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts.
The metabolism of the antibacterial fluoroquinolone drug flumequine by
Cunninghamella elegans was investigated using cultures grown in Sabouraud dextrose broth with 308
μM flumequine. The cultures ...were extracted with ethyl acetate; metabolites were separated by high-performance liquid chromatography and identified by mass spectrometry and proton nuclear magnetic resonance spectroscopy. Flumequine was transformed to two diastereomers of 7-hydroxyflumequine (23 and 43% of the total chromatographic peak area at 280
nm) and 7-oxoflumequine (11% of the total peak area). This is the first time that the two 7-hydroxy diastereomers have been characterized structurally; the hydroxyflumequines are known to have less antimicrobial activity than flumequine.
Deeper exploration of uncharacterized Gcn5-related N-acetyltransferases has the potential to expand our knowledge of the types of molecules that can be acylated by this important superfamily of ...enzymes and may offer new opportunities for biotechnological applications. While determining native or biologically relevant in vivo functions of uncharacterized proteins is ideal, their alternative or promiscuous in vitro capabilities provide insight into key active site interactions. Additionally, this knowledge can be exploited to selectively modify complex molecules and reduce byproducts when synthetic routes become challenging. During our exploration of uncharacterized Gcn5-related N-acetyltransferases from Pseudomonas aeruginosa, we identified such an example. We found that the PA3944 enzyme acetylates both polymyxin B and colistin on a single diaminobutyric acid residue closest to the macrocyclic ring of the antimicrobial peptide and determined the PA3944 crystal structure. This finding is important for several reasons. (1) To the best of our knowledge, this is the first report of enzymatic acylation of polymyxins and thus reveals a new type of substrate that this enzyme family can use. (2) The enzymatic acetylation offers a controlled method for antibiotic modification compared to classical promiscuous chemical methods. (3) The site of acetylation would reduce the overall positive charge of the molecule, which is important for reducing nephrotoxic effects and may be a salvage strategy for this important class of antibiotics. While the physiological substrate for this enzyme remains unknown, our structural and functional characterization of PA3944 offers insight into its unique noncanonical substrate specificity.
A surface adlayer is generated on organic single crystals (tetracene and rubrene) using the site specific Diels-Alder reaction and a series of vapor phase dienophiles. X-ray photoelectron ...spectroscopy (XPS) confirms adsorption on the surfaces of tetracene and rubrene and mass spectrometry demonstrates the reaction's applicability to a range of dienophiles.
The most relevant human urinary metabolites of cocaine (nine metabolites) were measured in urban wastewater in Italy and USA. A novel analytical method based on liquid chromatography tandem mass ...spectrometry allowed the identification of ecgonine, ecgonine methyl ester and the pyrolytic derivatives of cocaine in untreated wastewater. The aim of this study was to verify whether the pattern of cocaine metabolites in wastewater reflected the human excretion profile in urine. The performance of the method was good, with recoveries higher than 60% and limits of quantifications in the low ng/L range. The stability in untreated wastewater was assessed for all metabolites and the best storage condition resulted freezing samples immediately after collection and keep them frozen until analysis. All the selected compounds were measured in wastewater at concentrations up to 1.5 μg/L and their weekly loads were calculated during a five weeks monitoring campaign in Milan (Italy). The profiles of cocaine metabolites in wastewater matched with those in human urine reported in the literature, suggesting that measures in wastewater reflect the real human excretion and that wastewater analysis is suitable for assessing drug consumption. Benzoylecgonine was confirmed as the best target for estimating cocaine use by wastewater analysis, while cocaine itself should not be considered because its amount in wastewater is affected by other environmental sources such as transport, handling and consumption. Results suggested that the measurement of other metabolites in combination with benzoylecgonine might reflect 60% of an administered dose of cocaine providing also information on different patterns of use.
► Cocaine and its most relevant urinary metabolites were measured in urban wastewater in Italy and USA. ► Weekly loads of the main metabolites of cocaine increased during the weekend. ► The pattern of cocaine metabolites in wastewater reflected the human excretion profile in urine. ► Wastewater analysis of drugs metabolites is suitable for assessing drug consumption in a population. ► Wastewater analysis can provide information on different pattern of use of a drug.
A systematic spectroscopic and computational study was conducted in order to probe the influence of base sequences on stacked (S) versus B-type (B) conformational heterogeneity induced by the major ...dG adduct derived from the model carcinogen 7-fluoro-2-aminofluorene (FAF). We prepared and characterized eight 12-mer DNA duplexes (-AG*N- series, dCTTCTAG* N CCTC; -CG*N- series, dCTTCTCG* N CCTC), in which the central guanines (G*) were site-specifically modified with FAF with varying flanking bases (N = G, A, C, T). S/B heterogeneity was examined by CD, UV, and dynamic 19F NMR spectroscopy. All the modified duplexes studied followed a typical dynamic exchange between the S and B conformers in a sequence dependent manner. Specifically, purine bases at the 3‘-flanking site promoted the S conformation (G > A > C > T). Simulation analysis showed that the S/B energy barriers were in the 14−16 kcal/mol range. The correlation times (τ = 1/κ) were found to be in the millisecond range at 20 °C. The van der Waals energy force field calculations indicated the importance of the stacking interaction between the carcinogen and neighboring base pairs. Quantum mechanics calculations showed the existence of correlations between the total interaction energies (including electrostatic and solvation effects) and the S/B population ratios. The S/B equilibrium seems to modulate the efficiency of Escherichia coli UvrABC-based nucleotide excision repair in a conformation-specific manner: i.e., greater repair susceptibility for the S over B conformation and for the -AG*N- over the -CG*N- series. The results indicate a novel structure−function relationship, which provides insights into how bulky DNA adducts are accommodated by UvrABC proteins.
Product ion spectra from thirteen C8-substituted alkylaniline adducts of guanine and deoxyguanosine were generated using electrospray ionization and quadrupole ion trap mass spectrometry and studied ...to investigate the possibility of differentiating isomeric adduct structures based upon the relative abundances of fragment ions derived from the alkylaniline-modified guanine bases (BH
2
+ ions). The structural discrimination of the BH
2
+ ions formed by attachment of isomeric alkylanilines to the C8 position of guanine is a challenging problem because the ions tend to yield product ion spectra that are qualitatively identical upon collisional activation. In this study, a statistical method, referred to as a similarity index, was used to compare the product ion spectra of isomeric BH
2
+ ions and differentiate their structures. All the adducts investigated could be distinguished from SIs calculated using 5–6 product ions. These results suggest that a searchable database of product ion spectra may be created and used to characterize DNA adducts from aromatic amines whenever they are detected at levels amenable to mass spectral analysis.
We report synthesis and characterization of a complete set of α,β-methylene-2′-dNTPs (α,β-m-dNTP; N = A, C, T, G, 12−15) in which the α,β-oxygen linkage of natural dNTP was replaced by a methylene ...group. These nucleotides were designed to be noncleavable substrates for DNA polymerases. Synthesis entails preparation of 2′-deoxynucleoside 5′-diphosphate precursors, followed by an enzymatic γ-phosphorylation. All four synthesized α,β-m-dNTPs were found to be potent inhibitors of polymerase β, with K i values ranging 1−5 μM. During preparation of the dG and dT derivatives of α,β-methylene diphosphate, we also isolated significant amounts of 3,5′-cyclo-dG (16) and 2,5′-cyclo-dT (17), respectively. These novel 2′-deoxycyclonucleosides were formed via a base-catalyzed intramolecular cyclization (N3 → C5′ and O2 → C5′, respectively). In acidic solution, both 16 and 17 underwent glycolysis, followed by complete depurination. When exposed to alkaline conditions, 16 underwent an oxidative deamination to produce 3,5′-cyclo-2′-deoxyxanthosine (19), whereas 17 was hydrolyzed exclusively to dT.
An analytical method for the structure differentiation of arylamine modified oligonucleotides (ODNs) using on-line LC/MS analysis of raw exonuclease digests is described. Six different dodeca ODNs ...derived from the reaction of
N-acetoxy-
N-(trifluoroacetyl)-2-aminofluorene with the dodeca oligonucleotide 5′-CTCGGCGCCATC-3′ are isolated and sequenced with this LC/MS method using 3′- and 5′-exonucleases. When the three products modified by a single aminofluorene (AF) are subjected to 3′-exonuclease digestion, the exonuclease will cleave a modified nucleotide but when di-AF modified ODNs are analyzed the 3′-exonuclease ceases to cleave nucleotides when the first modification is exposed at the 3′-terminus. Small abundances of ODN fragments formed by the cleavage of an AF-modified nucleotide were observed when two of the three di-AF modified ODNs were subjected to 5′-exonuclease digestion. The results of the 5′-exonuclease studies of the three di-AF modified ODNs suggest that as the number of unmodified bases between two modifications in an ODN sequence increases, the easier it becomes to sequence beyond the modification closest to the 5′-terminus. The results of this study indicate that the LC/MS method described here would be useful in sequencing ODNs modified by multiple arylamines to be used as templates for site-specific mutagenesis studies.
PDF
