Rosanna Mondelli

Dimers of nicotinamide adenine dinucleotide: New evidence for the structure and the involvement in an enzymatic redox process

Abstract The composition and the structure of the product from the known electrochemical dimerization of the NAD+ have been conclusively demonstrated. A detailed analysis of the 1H and 13C nmr spectra has in fact led to the conclusion that the product contains three diastereoisomeric dimers of the 4,4′-tetrahydrobipyridyl type. Furthermore, the cytoplasmic fraction obtained from a standard mitochondrial preparation of rat liver has been shown to catalyze the oxygen uptake by the dimers. A 1 : 1 molar ratio of the reagents in the redox process is indicated by manometric data on oxygen uptake complemented by spectrophotometric analysis of the oxidized substrates, suggesting that H2O2 is the reduction product. NAD+ was identified as the oxidation product by an enzymatic method.

13 C nuclear magnetic resonance spectroscopy of nitrogen heterocycles. Part 4. intra-extra Configuration of the N-acetyl group in phenothiazine and related systems with a ‘butterfly’ shape

Nitrogen lone-pair delocalization into aromatic rings has been studied for phenothiazine, N-acetylphenothiazine, and related tricyclic systems with a ‘butterfly’ conformation, by using 13C n.m.r. spectroscopy. Pyridazo[4,5-d][1,4]benzothiazines, phenoxazines, acridane, and simple model compounds such as diphenylamine, 2-methylthioaniline, and their corresponding N-acetyl derivatives were examined. The preferred configuration of the N-acetyl group has been determined for CDCl3 and Me2SO solutions. Nitrogen lone-pair delocalization of the N-acetyl group into the aromatic system is strongly hindered for all tricyclic compounds, and greatly reduced for N-acetyldiphenylamine and also for 2-methylthioacetanilide in Me2SO. For this last compound, the decrease of conjugation is due to a solvent-induced change of conformation; for tricyclic systems it is a consequence of the preferred extra-configuration of the N-acetyl group.

Interaction between double helix DNA fragments and a new topopyrone acting as human topoisomerase I poison

A water soluble derivative (2) of topopyrones was selected for NMR studies directed to elucidate the mode of binding with specific oligonucleotides. Topopyrone 2 can intercalate into the CG base pairs, but the residence time into the double helix is very short and a fast chemical exchange averaging occurs at room temperature between the free and bound species. The equilibria involved become slow below room temperature, thus allowing to measure a mean lifetime of the complex of ca. 7 ms at 15 degrees C. Structural models of the complex with d(CGTACG)(2) were developed on the basis of DOSY, 2D NOESY and (31)P NMR experiments. Topopyrone 2 presents a strong tendency to self-associate. In the presence of oligonucleotide a certain number of ligand molecules are found to externally stack to the double-helix, in addition to a small fraction of the same ligand intercalated. The external binding to the ionic surface of the phosphoribose chains may thus represents the first step of the intercalation process.

31P NMR studies on the interaction of morpholinyl anthracyclines and related compounds with d(CGTACG)2. Thermodynamic and kinetic parameters

Abstract31P NMR spectroscopy has been used to study the intercalation of the anthracyclines doxorubicin1, daunorubicin2, 4-demethoxydaunorubicin3, morpholinodoxorubicin4, methoxymorpholinodoxorubicin5 and 9-deoxydaunorubicin6 with the DNA fragment d(CGTACG)2. The individual phosphate resonances of the oligonucleotide were assigned in the free as well as in the intercalated species. The31P chemical shift variations allowed us to identify the intercalation sites, which resulted to be the same for all compoundsi.e. between the terminal CG base-pairs of the helix (two molecules of drugper duplex). The binding constants, the dissociation rate constants and ΔG# values have been determined in different conditions of ionic strength and temperature. The kinetic constant (koff) of the slow step of the anthracycline/duplex intercalation process has been directly measured by NOE exchange techniques. Binding constants depend on the ionic strength and on the self-association process so greatly, that their use to study by NMR anthracycline/DNA interactions is questionable. On the contrary, thekoff are not affected by these phenomena and present an interesting trend for1–6, thus showing that the average lifetime of the drug in the intercalation site appears to be important for determining the cytotoxicity and the antimitotic activity.

1H-NMR study and structure determination of 4,4- and 4,6-dimers from electrochemical reduction of NADP+

The products arising from one-electron electrochemical reduction of the coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) have been studied by HPLC chromatography and 1H-NMR spectroscopy. HPLC and NMR analyses have shown seven dimeric species, the most abundant of which (40%) has been isolated and has resulted to be an NADP 4,4-linked dimer. The other two diastereoisomeric 4,4-dimers present for the 25% and 10%, respectively, have been detected in the crude reaction mixture, but have not been isolated. The 4,4-tetrahydrobipyridine structure and the stereochemistry at the ring-ring junction for these three isomers have been determined on the basis of their NMR parameters. Preparative HPLC chromatography also led to two fractions enriched in another four dimers, present in the crude mixture, which turned out to have a 4,6-tetrahydrobipyridine structure. All the chemical shifts and the H,H coupling constants of the 4,4- and 4,6-tetrahydrobipyridine systems have been obtained for the seven compounds. For the most abundant among the 4,4-dimers the NMR analysis also gave the coupling constant values of the ribose-diphosphate chain.

The interaction of nemorubicin metabolite PNU-159682 with DNA fragments d(CGTACG)(2), d(CGATCG)(2) and d(CGCGCG)(2) shows a strong but reversible binding to G:C base pairs.

The antitumor anthracycline nemorubicin is converted by human liver microsomes to a major metabolite, PNU-159682 (PNU), which was found to be much more potent than its parent drug toward cultured tumor cells and in vivo tumor models. The mechanism of action of nemorubicin appears different from other anthracyclines and until now is the object of studies. In fact PNU is deemed to play a dominant, but still unclear, role in the in vivo antitumor activity of nemorubicin. The interaction of PNU with the oligonucleotides d(CGTACG)(2), d(CGATCG)(2) and d(CGCGCG)(2) was studied with a combined use of (1)H and (31)P NMR spectroscopy and by ESI-mass experiments. The NMR studies allowed to establish that the intercalation between the base pairs of the duplex leads to very stable complexes and at the same time to exclude the formation of covalent bonds. Melting experiments monitored by NMR, allowed to observe with high accuracy the behaviour of the imine protons with temperature, and the results showed that the re-annealing occurs after melting. The formation of reversible complexes was confirmed by HPLC-tandem mass spectra, also combined with endonuclease P1digestion. The MS/MS spectra showed the loss of neutral PNU before breaking the double helix, a behaviour typical of intercalators. After digestion with the enzyme, the spectra did not show any compound with PNU bound to the bases. The evidence of a reversible process appears from both proton and phosphorus NOESY spectra of PNU bound to d(CGTACG)(2) and to d(CGATCG)(2). The dissociation rate constants (k(off)) of the slow step of the intercalation process, measured by (31)P NMR NOE-exchange experiments, showed that the kinetics of the process is slower for PNU than for doxorubicin and nemorubicin, leading to a 10- to 20-fold increase of the residence time of PNU into the intercalation sites, with respect to doxorubicin. A relevant number of NOE interactions allowed to derive a model of the complexes in solution from restrained MD calculations. The conformation of PNU bound to the oligonucleotides was also derived from the coupling constant values.

3D structure of microperoxidase-11 by NMR and molecular dynamic studies

Microperoxidase‐11 (MP11) is an undecapeptide, obtained from enzymatic cleavage of cytochrome c, which still exhibits peroxidase activity. It retains residues 11–21 of the protein, the heme‐c group and His18 as fifth ligand at the iron atom. We studied MP11 in the paramagnetic, Fe(III) low‐spin state by NMR and constrained molecular dynamics (MD). Well‐resolved 1H and 13C spectra were obtained in water–trifluoroethanol and water–methanol mixtures with different axial ligands to the iron, i.e. imidazole, NH3, OH, CN, at different temperatures from −15 to +25 °C. All the protons of the heme and the peptide moiety were assigned by using TOCSY and NOESY experiments. Some stereospecific assignments were also performed and eight coupling constants between the amide and the α‐protons were measured. Seventy‐one inter‐residue and heme peptide and 40 intra‐residue NOE interactions were translated into inter‐proton distances. Ten structures satisfying these distance constraints to within ±0.4 Å were obtained and showed that almost the whole peptide (segment 12–19) can form a right‐handed helix. The envelope structure of the peptide around the heme is well defined based on a number of NOE interactions between heme and peptide protons. Variable‐temperature experiments were performed in order to obtain information on the protons affected by the paramagnetic shift. An inverted temperature dependence (Curie law) was observed for the heme and meso protons, except for 3‐Me, when the sixth axial ligand is NH3 or imidazole. The pattern of 13C hyperfine shifts of heme methyl groups, similar in MP11 and cytochrome c, suggests that the orientation of His ring along the α–γ meso axis is conserved in MP11, which is consistent with MD calculations. Copyright

13 C nuclear magnetic resonance of N-heterocycles. Part 2. Natural abundance carbon-13 and nitrogen-15 nuclear magnetic resonance studies of Δ3- and Δ4-pyrrolin-2-ones and model compounds

Carbon-13 and nitrogen-15 Fourier transform n.m.r. spectra of Δ3- and Δ4-pyrrolin-2-ones and their N-methyl and -acetyl derivatives, and those of succinimide, maleimide, pyrrolidin-2-one, piperidin-2-one, and 2-piperidone, have been measured and analysed. Carbon and nitrogen shifts are discussed in relation to charge density distributions and chemical reactivities for Δ3- and Δ4-pyrrolin-2-ones. Coupling constant values of these latter compounds are also reported.

Conformation and tautomerism of hypocrellins. Revised structure ofshiraiachrome A

Hypocrellins, natural perylenequinones from the fungi Hypocrella bambusae and Shiraia bambusicola, are being intensively studied for their photodynamic activity. As a recent paper reported the existence of a slow tautomeric equilibrium for hypocrellin A, the behaviour in solution of hypocrellin and hypocrellin A was reinvestigated by NMR. 1D and 2D ROESY spectra at different temperatures and in different solvents have been measured and quantitative NOE experiments have been performed to obtain the cross-relaxation rates and then the interproton distances, to be used for MM and MD calculations. This allowed us to confirm that tautomerism in these compounds is a fast process on the NMR time scale, and to establish the relative population of the two principal tautomers and the preferred conformation of hypocrellin, hypocrellin A and of their atropisomers. The atropisomeric interconversion process is fast enough to be studied by NMR; the rate constants, obtained by ROESY-exchange experiments, gave the activation parameters. The helix inversion also induces the inversion of the seven-membered ring, which adopts a twist–boat conformation in both atropisomers. The values of the dihedral angle C(1)–C(12b)–C(12a)–C(12), which shows the distortion of the perylenequinone system, have been obtained from energy minimisation of the structures derived from NOE data. They are in the range 25–29° for all stereoisomers. The structure of shiraiachrome A, another member of the series, has been revised to M(R),14S,16S. The conformation and the tautomeric equilibrium of this compound have been similarly determined.

Structure and dynamics of intercalation complexes of anthracyclines with d(CGATCG)2 and d(CGTACG)2. 2D-1H and 31P NMR investigations

The interaction of (2S)-2-methoxymorpholinodoxorubicin 4 and morpholinodoxorubicin 5 with the hexanucleotides d(CGATCG)2 and d(CGTACG)2 has been studied by a combined use of 2D-1H and 31P NMR techniques and molecular dynamics (MD) calculations, in comparison with doxorubicin 1, daunorubicin 2 and idarubicin 3. Both 1H and 31P chemical shifts of imino protons and phosphates respectively have been shown to be a sensitive probe for the intercalation sites (two anthracycline molecules at the CpG sites). A relevant number of NOE interactions allowed the structure of the complexes in solution to be derived through restrained MD calculations, which were compared with those obtained by X-ray analysis. In all the complexes the aglycone was shown to be located in the middle of the double helix, orthogonally oriented with respect to the base pairs, with ring D extending out of the helix on the major groove and ring A, with 9H8 conformation, between guanines G2 and G12. The daunosamine and morpholino moieties lie in the center of the minor groove, with slightly different positions than in the X-ray structures. In all the complexes the daunosamine ring is located at the A3 (T3) level, while the morpholino ring displays NOE interactions with the fourth unit T4 (A4). The deformations of the double helix are revealed by an increased distance between protons of the C5 and G6 units and by conformational changes at the level of the α, ζ, Iµ and δ angles of the phosphoribose backbone. The variation of the 31P chemical shifts is interpreted in terms of conformational equilibria leading to different populations of conformers. This is clearly shown from the values of the α and ζ torsion angles, monitored during the MD, which indicate a relevant population of trans forms for ζ and also for α angles of C5pG6 and G2pT3 (G2pA3) units, while the other phosphates exist entirely in the α, ζ, gauche,gauche conformation. The dissociation rate constants were measured by 2D 31P NOESY-exchange experiments for 1 and 4. The decrease of koff for 4, corresponding to a ten-fold increase of the residence time of the drug in the intercalation sites, is in line with the higher activity found for methoxymorpholinodoxorubicin 4.