Bongsup P. Cho

Spectroscopic Properties of Polycyclic Aromatic Hydrocarbons: Effect of Solvent Polarity on the Fluorescence Emission Behavior of Select Fluoranthene, Fluorenochrysene, Indenochrysene, and Indenopyrene Derivatives

Fluorescence emission behavior is reported for benz[def]indeno-[1,2,3hi]chrysene, fluoreno[2,3,4,9defg]chrysene, benz[def]indeno-[l,2,3qr]chrysene, dibenzo[a,e]fluoranthene, indeno[1,2,3cd]pyrene, naphtho[1,2b]fluoranthene, benzo[b]fluoranthene, fluoranthene, benzo-[ghi]fluoranthene, naphtho[2,1a]fluoranthene, naphtho[2,3b]fiuoranthene, benzo[k]fluoranthene, and benzo[j]fluoranthene dissolved in organic nonelectrolyte solvents of varying polarity. Results of these measurements are used to classify the various solutes as either probe or nonprobe molecules, depending upon whether measured emission intensity ratios vary systematically with solvent polarity. Also discussed are primary and secondary inner-filtering artifacts associated with selective quenching agents used to help identify/analyze polycyclic aromatic hydrocarbons (PAHs) in unknown mixtures. Inner-filtering artifacts are illustrated by examining fluorescence emission intensities of 13 “nonalternant” PAHs at different nitromethane concentrations and solution absorbances.

Dynamic conformational heterogeneities of carcinogen-DNA adducts and their mutagenic relevance.

Abstract Arylamines and polycyclic aromatic hydrocarbons (PAHs), which are known as “bulky” carcinogens, have been studied extensively and upon activation in vivo, react with cellular DNA to form DNA-adducts. The available structure data accumulated thus far has revealed that conformational heterogeneity is a common theme among duplex DNA modified with these carcinogens. Several conformationally diverse structures have been elucidated and found to be in equilibrium in certain cases. The dynamics of the heterogeneity appear to be modulated by the nature of the adduct structure and the base sequences neighboring the lesion site. These can be termed as “adduct- and sequence-induced conformational heterogeneities,” respectively. Due to the small energy differences, the population levels of these conformers could readily be altered within the active sites of repair or replicate enzymes. Thus, the complex role of “enzyme-induced conformational heterogeneity” must also be taken into consideration for the establishment of a functional structure-mutation relationship. Ultimately, a major challenge in mutation structural biology is to carry out adduct- and site-specific experiments in a conformationally specific manner within biologically relevant environments. Results from such experiments should provide an accurate account of how a single chemically homogenous adduct gives rise to complex multiple mutations, the earliest step in the induction of cancer.

Inhibitory effects of polyphenol punicalagin on type-II collagen degradation in vitro and inflammation in vivo

Cartilage destruction is a crucial process in arthritis and is characterized by the degradation of cartilage proteins, proteoglycans, and type II collagen (CII), which are embedded within the extracellular matrix. While proteoglycan loss can be reversed, the degradation of CII is irreversible and has been correlated with an over-expression and over-activation of matrix metalloproteinases (MMPs). Among the various MMPs, the collagenase MMP-13 possesses the greatest catalytic activity for CII degradation. Here we show that the pomegranate-derived polyphenols, punicalagin (PA) and ellagic acid (EA), inhibit MMP-13-mediated degradation of CII in vitro. Surface plasmon resonance studies and molecular docking simulations suggested multiple binding interactions of PA and EA with CII. The effects of PA on bovine cartilage degradation (stimulated with IL-1β) were investigated by assaying proteoglycan and CII release into cartilage culture media. PA inhibited the degradation of both proteins in a concentration-dependent manner. Finally, the anti-inflammatory effects of PA (daily IP delivery at 10 and 50mg/kg for 14days) were tested in an adjuvant-induced arthritis rat model. Disease development was assessed by daily measurements of body weight and paw volume (using the water displacement method). PA had no effect on disease development at the lower dose but inhibited paw volume (P<0.05) at the higher dose.

Conformational and thermodynamic properties modulate the nucleotide excision repair of 2-aminofluorene and 2-acetylaminofluorene dG adducts in the NarI sequence

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 G1, G2 or G3 of NarI sequence (5′-CCG1G2CG3CC-3′). Our 19F-NMR/ICD results showed that FAAF at G1 and G3 prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G2. We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G3 and -G1 duplexes incised more efficiently than the B-type G2 duplex (G3∼G1 > G2). 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 G2∼G1 > G3, 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.

Sequence effects of aminofluorene-modified DNA duplexes: thermodynamic and circular dichroism properties

Circular dichroism (CD) and UV-melting experiments were conducted with 16 oligodeoxynucleotides modified by the carcinogen 2-aminofluorene, whose sequence around the lesion was varied systematically [d(CTTCTNG[AF]NCCTC), N = G, A, C, T], to gain insight into the factors that determine the equilibrium between base-displaced stacked (S) and external B-type (B) duplex conformers. Differing stabilities among the duplexes can be attributed to different populations of S and B conformers. The AF modification always resulted in sequence-dependent thermal (Tm) and thermodynamic (−ΔG°) destabilization. The population of B-type conformers derived from eight selected duplexes (i.e. -AG*N- and -CG*N-) was inversely proportional to the −ΔG° and Tm values, which highlights the importance of carcinogen/base stacking in duplex stabilization even in the face of disrupted Watson–Crick base pairing in S-conformation. CD studies showed that the extent of the adduct-induced negative ellipticities in the 290–350 nm range is correlated linearly with −ΔG° and Tm, but inversely with the population of B-type conformations. Taken together, these results revealed a unique interplay between the extent of carcinogenic interaction with neighboring base pairs and the thermodynamic properties of the AF-modified duplexes. The sequence-dependent S/B heterogeneities have important implications in understanding how arylamine–DNA adducts are recognized in nucleotide excision repair.

Structures of 2-Acetylaminofluorene Modified DNA Revisited: Insight into Conformational Heterogeneity

Despite the extensive data on dG-AAF, the major DNA adduct derived from the model carcinogen 2-acetylaminofluorene, little is known with respect to its solution structures. Here, we provide NMR/CD evidence for three conformers of dG-AAF in duplex DNA: major groove B-type (B), base-displaced stacked (S), and minor groove wedge (W). The S/B/W-conformational heterogeneities were found to be sensitive to the nature of the flanking DNA sequence contexts and pH.

A novel and practical synthesis of polycyclic fluoranthenes

Abstract A novel and general preparation of polycyclic fluoranthenes is described. The synthesis involves a Michael addition of silyl enol ethers to dibenzofulvene followed by cyclization and aromatization.

Influence of flanking sequence context on the conformational flexibility of aminofluorene-modified dG adduct in dA mismatch DNA duplexes

When positioned opposite to a dA in a DNA duplex, the prototype arylamine–DNA adduct [N-(2′-deoxyguanosin-yl)-7-fluoro-2-aminofluorene (FAF)] adopts the so-called ‘wedge’ (W) conformation, in which the carcinogen resides in the minor groove of the duplex. All 16 FAF-modified 12-mer NG*N/NAN dA mismatch duplexes (G* = FAF, N = G, A, C, T) exhibited strongly positive induced circular dichroism in the 290–360 nm range (ICD290–360 nm), which supports the W conformation. The ICD290–360 nm intensities were the greatest for duplexes with a 3′-flanking T. The AG*N duplex series showed little adduct-induced destabilization. An exception was the AG*T duplex, which displayed two well-resolved signals in the 19F NMR spectra. This was presumably due to a strong lesion-destabilizing effect of the 3′-T. The flanking T effect was substantiated further by findings with the TG*T duplex, which exhibited greater lesion flexibility and nucleotide excision repair recognition. Adduct conformational heterogeneity decreased in order of TG*T > AG*T > CG*T > AG*A > AG*G > AG*C. The dramatic flanking T effect on W-conformeric duplexes is consistent with the strong dependence of the ICD290-360 on both temperature and salt concentration and could be extended to the arylamine food mutagens that are biologically relevant in humans.

Sequence Verification of Oligonucleotides Containing Multiple Arylamine Modifications by Enzymatic Digestion and Liquid Chromatography Mass Spectrometry (LC/MS)

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.

Synthesis and Characterization of 4′-Amino and 4′-Nitro Derivatives of 4-N,N-Dimethylaminotriphenylmethane as Precursors for a Proximate Malachite Green Metabolite

Abstract This paper describes the preparation of 4′-amino ( 2 ) and 4′-nitro ( 3 ) derivatives of 4- N , N -dimethylaminotriphenylmethane as precursors for presumed DNA-binding metabolites of malachite green. The primary amine 2 was synthesized via a condensation of 4-lithiated bis-( N , N -trimethylsilyl)aniline and 4-(dimethylamino)benzophenone. A simple nitro-dediazoniation reaction of 2 failed to afford 3 , but gave exclusively a mixture derived from hydro-dediazoniation followed by nitration and N -dealkylative- N -nitrosation. Direct nitration of N , N -dimethylaminotriphenylmethane, however, afforded 3 as well as other nitro isomers.