Carbohydrate-naphthalene diimide conjugates as potential antiparasitic drugs: Synthesis, evaluation and structure-activity studies.

The neglected tropical diseases Human African Trypanosomiasis and leishmaniasis are caused by infection with trypanosomatid parasites Trypanosoma brucei and Leishmania spp, respectively. The genomes of these organisms contain multiple putative G-quadruplex (G4) forming sequences which have recently been proposed to mediate processes relevant for parasite survival. Therefore, G4 could be considered as potential targets for a novel approach towards the development of antiparasitic drugs. Recently, we have demonstrated that G4 ligands such as carbohydrate naphthalene diimide conjugates (carb-NDIs) possess notable antiparasitic activity. Herein, we have synthesized a new family of carb-NDIs, characterized by significant structural variability, and evaluated their anti-parasitic activity, with special focus on T. brucei. The interaction with relevant G4 sequences was evaluated in vitro through independent biophysical methods (FRET melting assays under competing conditions with double stranded DNA, circular dichroism and fluorescence titrations). Finally, flow cytometry and confocal microscopy experiments demonstrated that the conjugates exhibit excellent uptake into T. brucei parasites, localizing in the nuclei and kinetoplasts. Promising antiparasitic activity and selectivity against control mammalian cells, together with their peculiar mechanism of action, render the carb-NDI conjugates as suitable candidates for the development of an innovative treatment of trypanosomiasis.

A red-NIR fluorescent dye detecting nuclear DNA G-quadruplexes: in vitro analysis and cell imaging.

Aggregation, red-NIR emission and light-up upon nucleic acid G-quadruplex binding have been investigated for a prototype core-extended naphthalene diimide, which is capable of fast cellular entry and nucleolar localization. Both high-level colocalization with an anti-G-quadruplex antibody and nucleolin displacement reveal that the compound targets and thus makes visible nuclear DNA G-quadruplexes.

A naphthalene diimide dyad for fluorescence switch-on detection of G-quadruplexes.

A non-fluorescent naphthalene diimide (NDI) dimer, conjugating red and blue NDI dyes, becomes red/NIR emitting upon G-quadruplex binding. The fluorescence lifetime which is significantly different for the complexes, the G-quadruplex/dimer and the weakly emitting ds-DNA/dimer is the key feature for the development of new rationally engineered G-quadruplex sensors.

Photoinduced, ionic Meerwein arylation of olefins.

Irradiation of 4-chloroaniline or of its N,N-dimethyl derivative in polar solvents generates the corresponding triplet phenyl cations. These are trapped by alkenes yielding arylated products in medium to good yields. B3LYP calculations show that the triplet cation slides with negligible activation energy to a bonded adduct with ethylene, whereas it forms only a marginally stabilized CT complex with water (chosen as a representative sigma nucleophile). The structure of the final products depends on the preferred path from the adduct cation with the alkene. In the case of aryl olefins, this deprotonates to stilbene derivatives, while, from 2,3-dimethyl-2-butene and allytrimethylsilane, allylanilines are obtained by elimination of an electrofugal group in gamma. In the case of mono- and disubstituted alkenes the cation adds chloride rather than eliminating and beta-chloroalkylanilines are obtained. The regio- and sterochemistry of the addition across the alkene are best understood with a phenonium ion structure for the adduct. The nucleophile entering in beta can be varied under conditions in which the adduct cation is trapped more efficiently than the starting phenyl cation. Thus, beta-methoxyalkylanilines are formed when the irradiation is carried out in methanol. beta-Iodoalkylanilines are obtained in acetonitrile containing iodide and unsubstituted alkylanilines in the presence of sodium borohydride. A case of intramolecular nucleophilic trapping is found with 4-pentenoic acid. The reaction is a wide-scope ionic analogue of the radicalic Meerwin arylation of olefins.

Generation and reactivity of the 4-aminophenyl cation by photolysis of 4-chloroaniline.

4-Chloroaniline and its N,N-dimethyl derivative are photostable in cyclohexane but undergo efficient photoheterolysis in polar media via the triplet state and give the corresponding triplet phenyl cations. CASSCF and UB3LYP calculations show that the 4-aminophenyl triplet cation has a planar geometry and is stabilized by >10 kcal mol(-1) with respect to the slightly bent singlet. The triplet has a mixed carbene-diradical character at the divalent carbon. This species either adds to the starting substrate forming 5-chloro-2,4'-diaminodiphenyls (via an intermediate cyclohexadienyl cation) or is reduced to the aniline (via the aniline radical cation) in a ratio depending on the hydrogen-donating properties of the solvent. Transients attributable to the triplet aminophenyl cation as well as to the ensuing intermediates are detected. Chemical evidence for the generation of the phenyl cation is given by trapping via electrophilic substitution with benzene, mesitylene, and hexamethylbenzene (in the last case the main product is a 6-aryl-3-methylene-1,4-cyclohexadiene). Relative rates of electrophilic attack to benzene and to some alkenes and five-membered heterocycles are measured and span over a factor of 15 or 30 for the two cations. The triplet cation formed under these conditions is trapped by iodide more efficiently than by the best pi nucleophiles. However, in contrast to the singlet cation, it does not form ethers with alcohols, by which it is rather reduced.

o-Quinone methide as alkylating agent of nitrogen, oxygen, and sulfur nucleophiles. The role of H-bonding and solvent effects on the reactivity through a DFT computational study.

The reactivity of the alkylating agent o-quinone methide (o-QM) toward NH(3), H(2)O, and H(2)S, prototypes of nitrogen-, oxygen-, and sulfur-centered nucleophiles, has been studied by quantum chemical methods in the frame of DF theory (B3LYP) in reactions modeling its reactivity in water with biological nucleophiles. The computational analysis explores the reaction of NH(3), H(2)O, and H(2)S with o-QM, both free and H-bonded to a discrete water molecule, with the aim to rationalize the specific and general effect of the solvent on o-QM reactivity. Optimizations of stationary points were done at the B3LYP level using several basis sets [6-31G(d), 6-311+G(d,p), adding d and f functions to the S atom, 6-311+G(d,p),S(2df), and AUG-cc-pVTZ]. The activation energies calculated for the addition reactions were found to be reduced by the assistance of a water molecule, which makes easier the proton-transfer process in these alkylation reactions by at least 12.9, 10.5, and 6.0 kcal mol(-1) [at the B3LYP/AUG-cc-pVTZ//B3LYP/6-311+G(d,p) level], for ammonia, water, and hydrogen sulfide, respectively. A proper comparison of an uncatalyzed with a water-catalyzed reaction mechanism has been made on the basis of activation Gibbs free energies. In gas-phase alkylation of ammonia and water by o-QM, reactions assisted by an additional water molecule H-bonded to o-QM (water-catalyzed mechanism) are favored over their uncatalyzed counterparts by 5.6 and 4.0 kcal mol(-1) [at the B3LYP/6-311+G(d,p) level], respectively. In contrast, the hydrogen sulfide alkylation reaction in the gas phase shows a slight preference for a direct alkylation without water assistance, even though the free energy difference (DeltaDeltaG(#)) between the two reaction mechanisms is very small (by 1.0 kcal mol(-1) at the B3LYP/6-311+G(d,p),S(2df) level of theory). The bulk solvent effect, evaluated by the C-PCM model, significantly modifies the relative importance of the uncatalyzed and water-assisted alkylation mechanism by o-QM in comparison to the case in the gas phase. Unexpectedly, the uncatalyzed mechanism becomes highly favored over the catalyzed one in the alkylation reaction of ammonia (by 7.0 kcal mol(-1)) and hydrogen sulfide (by 4.0 kcal mol(-1)). In contrast, activation induced by water complexation still plays an important role in the o-QM hydration reaction in water as solvent.

Alkylation of amino acids and glutathione in water by o-quinone methide. Reactivity and selectivity.

o-Quinone methide (1) has been produced in water both thermally and photochemically from (2-hydroxybenzyl)trimethylammonium iodide (2). Michael addition reactions of 1 to various amines, and sulfides, including amino acids and glutathione have been carried out, obtaining alkylated adducts (3-16) in fairly good to quantitative yields. The reaction rate and selectivity of 1 toward nitrogen and sulfur nucleophiles, in competition with the hydration reaction, have been investigated at different pH by laser flash photolysis technique. The observed reactivity spans 7 orders of magnitude on passing from water (kNu = 5.8 M-1 s-1) to the most reactive nucleophile (2.8 x 10(8) M-1 s-1, 2-mercaptoethanol under alkaline conditions). These are the first direct reaction rate measurements of nucleophilic addition to the parent o-quinone methide (1). Competition experiments provided strong kinetic support to the involvement of free 1 as an intermediate in both thermal and photochemical reactions. Furthermore, several alkylation adducts regenerate 1 either by heating (9, 10, 13, and 14) or by irradiation (9, 11-13, 16). Such a thermal and photochemical reversibility of the alkylation process opens a new perspective for the use and application of such adducts as o-QM molecular carriers.

Concerted vs stepwise mechanism in 1,3-dipolar cycloaddition of nitrone to ethene, cyclobutadiene, and benzocyclobutadiene. A computational study

The problem of competition between concerted and stepwise diradical mechanisms in 1,3-dipolar cycloadditions was addressed by studying the reaction between nitrone and ethene with DFT (R(U)B3LYP/6-31G) and post HF methods. According to calculations this reaction should take place via the concerted cycloaddition path. The stepwise process is a viable but not competitive alternative. The R(U)B3LYP/6-31G study was extended to the reaction of the same 1, 3-dipole with cyclobutadiene and benzocyclobutadiene. The very reactive antiaromatic cyclobutadiene has an electronic structure that is particularly disposed to promote stepwise diradical pathways. Calculations suggest that its reaction with nitrone represents a borderline case in which the stepwise process can compete with the concerted one on similar footing. Attenuation of the antiaromatic character of the dipolarophile, i.e., on passing from cyclobutadiene to benzocyclobutadiene, causes the concerted 1,3-dipolar cycloaddition to become once again prevalent over the two-step path. Thus, our results suggest that, in 1,3-dipolar cycloadditions that involve normal dipolarophiles, the concerted path (Huisgen's mechanism) should clearly overwhelm its stepwise diradical (Firestone's mechanism) counterpart.

Epoxidation of acyclic chiral allylic alcohols with peroxy acids: spiro or planar butterfly transition structures? A computational DFT answer

The mechanism of the epoxidation of two chiral allylic alcohols, i.e., 3-methyl-3-buten-2-ol and (Z)-3-penten-2-ol, with peroxyformic acid has been investigated by locating 20 transition structures with the B3LYP/6-31G* method and by evaluating their electronic energy also at the B3LYP/6-311+G**@B3LYP/6-31G* theory level. Relative stability of TSs, as far as electronic energy is concerned, is basis set dependent; moreover, it also depends on entropy and solvent effects. Free enthalpies, calculated by using electronic energy at the higher theory level and with inclusion of solvent effects, indicates that syn, exo TSs, where the olefinic OH group hydrogen bonds the peroxy oxygens of the peroxy acid, outweigh syn, endo TSs, where the peroxy acid carbonyl oxygen is involved in hydrogen bonding. In the former TSs the peroxy acid moiety maintains its planar geometry while in the latter ones a strong out-of-plane distortion of peroxy acid is observed. This distortion makes it viable an unprecedented 1,2-H shift, as a possible alternative to the 1,4-H shift, for the peroxy acid hydrogen. In fact, for one syn, endo TS IRC analysis demonstrated that the 1,2-H shift mechanism is actually operative. The geometry of all TSs substantially conforms to a spiro (i.e., with the peroxy acid plane almost perpendicular to the C=C bond axis) butterfly orientation of the reactants while no TS resembles, even loosely, the planar butterfly structure. Theoretical threo/erythro epoxide ratios are in fair accord with experimental data. Calculations indicate that threo epoxides derive mostly from TSs in which the olefinic OH assumes an outside conformation while erythro epoxides originate from TSs with the OH group in an inside position. Computational findings do not support the qualitative TS models recently proposed for these reactions.

Facial selectivity in epoxidation of 2-cyclohexen-1-ol with peroxy acids. A computational DFT study

We addressed the mechanism of epoxidation of 2-cyclohexen-1-ol by locating all the transition structures (TSs) for the reaction of peroxyformic acid (PFA) with both pseudoequatorial and pseudoaxial cyclohexenol conformers (five TSs for each conformer) and, for purpose of comparison, also those for the PFA epoxidation of cyclohexene. Geometry optimizations were performed at the B3LYP/6-31G level, energies refined with single point B3LYP/6-311+G// B3LYP/6-31G calculations and solvent effects introduced with the CPCM method. Our results can be summarized as follows: (i) all TSs exhibit a spiro-like structure, that is, the dihedral angle between the peroxy acid plane and the forming oxirane plane is closer to 90 degrees than to 0 degrees (or 180 degrees ); (ii) there is a stabilizing hydrogen bonding interaction in syn TSs that, however, is partly counteracted by unfavorable entropic effects; (iii) syn,exo TSs with hydrogen bonding at the PFA peroxy oxygens are definitely more stable than syn,endo TSs hydrogen bonded at the PFA carbonyl oxygen; (iv) facial selectivity of epoxidation of both cyclohexenol conformers is mostly the result of competition between only two TSs, namely, an anti,exo TS and its syn,exo counterpart. The latter TS is more stable than the former one, as stabilization by hydrogen bonding overrides the unfavorable entropic and solvent effects; (v) calculations correctly predict both the experimental dominance of attack leading to syn epoxide for both cyclohexenol conformers and the higher syn selectivity observed for the pseudoequatorial as compared to the pseudoaxial derivative. Moreover, also the experimental relative and absolute epoxidation rates for cyclohexene and cyclohexenol as well as for pseudoaxial and pseudoequatorial cyclohexenol derivatives are fairly well reproduced by computational data.