Regioselective Rearrangement of Nitrogen- and Carbon-Centered Radical Intermediates in the Hofmann-Löffler-Freytag Reaction.

The Hofmann-Löffler-Freytag (HLF) reaction serves as a late-stage functionalization technique for generating pyrrolidine heterocyclic ring systems. Contemporary HLF protocols utilize in situ halogenated sulfonamides as precursors in the radical-mediated rearrangement cycle. Despite its well-established reaction mechanism, experiments toward the detection of radical intermediates using EPR techniques have only recently been attempted. However, the obtained spectra lack the distinct features of the N-centered radicals expected for the employed reactants. This paper presents phenylbutylnitrone spin-trapped C-centered and N-centered radicals, generated via light irradiation from N-halogen-tosyl-sulfonamide derivatives and detected using EPR spectroscopy. NMR spectroscopy and DFT calculations are used to explain the observed regioselectivity of the HLF reaction.

Regiodivergent and Enantioselective Synthesis of Cyclic Sulfones via Ligand-Controlled Nickel-Catalyzed Hydroalkylation.

Cyclic sulfones have demonstrated important applications in drug discovery. However, the catalytic and enantioselective synthesis of chiral cyclic sulfones remains challenging. Herein, we develop nickel-catalyzed regiodivergent and enantioselective hydroalkylation of sulfolenes to streamline the synthesis of chiral alkyl cyclic sulfones. The method has broad scope and high functional group tolerance. The regioselectivity can be controlled by ligands only. A neutral PYROX ligand favors C3-alkylation whereas an anionic BOX ligand favors C2-alkylation. This control is kinetic in origin as the C2-bound Ni intermediates are always thermodynamically more stable. Reactivity study of a wide range of relevant Ni intermediates reveal a NiI/NiIII catalytic cycle with a NiII-H species as the resting state. The regio- and enantio-determining step is the insertion of this NiII-H species into 2-sulfolene. This work provides an efficient catalytic method for the synthesis of an important class of organic compounds and enhances the mechanistic understanding of Ni-catalyzed stereoselective hydroalkylation.

Inner Filter Effect Correction for Fluorescence Measurements in Microplates Using Variable Vertical Axis Focus.

The inner filter effect (IFE) hinders fluorescence measurements, limiting linear dependence of fluorescence signals to low sample concentrations. Modern microplate readers allow movement of the optical element in the vertical axis, changing the relative position of the focus and thus the sample geometry. The proposed Z-position IFE correction method requires only two fluorescence measurements at different known vertical axis positions (z-positions) of the optical element for the same sample. Samples of quinine sulfate, both pure and in mixtures with potassium dichromate, showed a linear dependence of corrected fluorescence on fluorophore concentration (R2 > 0.999), up to Aex ≈ 2 and Aem ≈ 0.5. The correction extended linear fluorescence response over ≈98% of the concentration range with ≈1% deviation of the calibration slope, effectively eliminating the need for sample dilution or separate absorbance measurements to account for IFE. The companion numerical IFE correction method further eliminates the need for any geometric parameters with similar results. Both methods are available online at https://ninfe.science.

Ferrocenoyl-adenines: substituent effects on regioselective acylation.

A series of N 6-substituted adenine-ferrocene conjugates was prepared and the reaction mechanism underlying the synthesis was explored. The SN2-like reaction between ferrocenoyl chloride and adenine anions is a regioselective process in which the product ratio (N7/N9-ferrocenoyl isomers) is governed by the steric property of the substituent at the N 6-position. Steric effects were evaluated by using Charton (empirical) and Sterimol (computational) parameters. The bulky substituents may shield the proximal N7 region of space, which prevents the approach of an electrophile towards the N7 atom. As a consequence, the formation of N7-isomer is a kinetically less feasible process, i.e., the corresponding transition state structure increases in relative energy (compared to the formation of the N9-isomer). In cases where the steric hindrance is negligible, the electronic effect of the N 6-substituent is prevailing. That was supported by calculations of Fukui functions and molecular orbital coefficients. Both descriptors indicated that the N7 atom was more nucleophilic than its N9-counterpart in all adenine anion derivatives. We demonstrated that selected substituents may shift the acylation of purines from a regioselective to a regiospecific mode.

Role of substituents in the Hofmann-Löffler-Freytag reaction. A quantum-chemical case study on nicotine synthesis.

The Hofmann-Löffler-Freytag (HLF) reaction can be successfully used to synthesize saturated heterocyclic nitrogen-containing nature-derived pharmaceuticals such as nicotine and its derivatives. In this study the rate-determining hydrogen atom transfer (HAT) step in nicotine synthesis has been analyzed using quantum chemical methods. Through quantification of substituent effects in the HAT step of the reaction on both nitrogen and carbon atoms, optimized synthetic strategies are outlined for the racemic as well as the stereoselective synthesis of nicotine. This latter process can be achieved using common nitrogen protecting groups, such as Ac, TFAc, and Boc. The said protecting groups show superior nitrogen radical activation as compared to the commonly used Tosyl group. Computational results indicate that the 1,5-HAT step is in this case likely to work even for the reaction with primary unactivated carbon centers.

Transacylation in Ferrocenoyl-Purines. NMR and Computational Study of the Isomerization Mechanism.

In the reaction of purines with ferrocenoyl chloride in dimethylformamide (DMF), a regioselective acylation occurred. The two products have been isolated and, according to detailed NMR analysis, identified as N7- and N9-ferrocenoylated isomers. In a more polar solvent, for example, in dimethylsulfoxide (DMSO), the two isomers interconvert to each other. The N7/N9 isomerization was followed by 1H NMR spectroscopy, until dynamic equilibrium was reached. Both kinetics and thermodynamics of the transacylation process are governed by a C6-substituent on the purine ring (R = NH2, Me, NHBz, OBz). The observed rate constant for the N7/N9-isomerization in the adenine system (R = NH2) is kobs = 0.3668 h-1, whereas the corresponding process in the C6-benzyloxypurine is 56 times slower. By use of density functional theory calculations and molecular dynamics simulations, several reaction pathways were considered and explored. Only the reaction mechanism involving DMSO as a nucleophilic reactant is in harmony with the experimental kinetic data. The calculated barrier (ΔG⧧ = 107.9 kJ/mol; at the M06L/6-311+G(d,p)/SDD level of theory) for this SN2-like reaction in the adenine system agrees well with the experimental value of 102.7 kJ/mol. No isomerization was detected in other organic solvents, for example, acetonitrile, N,N-dimethylformamide, or acetone, which indicated the exceptional nucleophilicity of DMSO. Our results raise a warning when treating or dissolving acylated purines in DMSO as they are prone to isomerization. We observed that the N7/N9-group transfer was specific not only for the organometallic moiety only, but for other acyl groups in purines as well. The relevance of this isomerization may be expected for a series of nucleobases and heterocyclic systems in general.

Racemization of oxazepam and chiral 1,4-benzodiazepines. DFT study of the reaction mechanism in aqueous solution.

The tranquilizer and hypnotic drug oxazepam undergoes the racemization process in aqueous medium, which is relevant for its pharmacological profile. The experimental barrier value (ΔG‡298 ≈ 91 kJ mol-1) was determined earlier, but the exact mechanism of enantiomerization is not known. Four different mechanisms have been proposed in the literature: C3-H/H exchange reaction, keto-enol tautomerization, solvolytic identity reaction, and ring-chain tautomerization. However, none of the reported reactions has been confirmed as the main pathway for racemization. In this work, all these mechanisms were subjected to comprehensive analysis performed by high-level quantum-chemical models. Two density functionals (B3LYP and M062X) were employed for geometry optimization of all stationary points at the corresponding potential surfaces, and the double-hybrid model (B2PLYP) was used for improved energy calculations. Out of all the tested mechanisms, only the ring-chain tautomerism fits the two experimental targets: the measured energy barrier and the pH-rate profile of racemization. The latter reveals that no acid/base catalysis is required for racemization to occur. The ring-chain tautomerism is initiated by intramolecular proton transfer from the C3-hydroxyl group to the imine nitrogen, which triggers the benzodiazepine ring opening and the formation of the achiral aldehyde intermediate. The latter undergoes ring closure which results in the inverted configuration at the C3-chiral atom of oxazepam. Our computational results suggest that the same mechanism is operative in the fast racemization of different 1,4-benzodiazepines, which posses the hydroxyl group at the stereogenic C3-centre (e.g. lorazepam or temazepam). In other benzodiazepine members (e.g. cinazepam or camazepam) the keto-enol tautomerization and/or the C3-H/H exchange mechanism may become relevant for their much slower racemization. This computational study is not only revealing in terms of mechanistic details, but also has predictive power for optical stability estimates in the family of benzodiazepines and similar heterocycles.

A quantum chemical study of HOCl-induced transformations of carbamazepine.

The antiepileptic drug carbamazepine (CBZ) is one of the most persistent pharmaceuticals in the environment. Its chemical fate is influenced by the type of wastewater treatment. This study sets out to determine the degradation mechanism and products in the reaction between CBZ and hypochlorous acid (HOCl), which is the main chlorinating species in water. In the search for the most feasible pathways of HOCl-induced transformations of CBZ, a quantum chemical approach was employed. Chlorination and epoxidation of CBZ are two initial, competitive processes that result in two key intermediates: N-chloramide and 10,11-epoxide. The calculated free energy barriers (ΔG) for these reactions are 105.7 and 95.7 kJ mol-1 resp., which is in agreement with the experimental energy barrier of 98.2 kJ mol-1. All transformation products detected in chlorination experiments were located by computational models, and the reaction mechanism underlying their formation was described in detail. Different computational methods (density functional and ab initio theory) were applied, and the double hybrid B2-PLYPD functional was found to be superior in terms of efficiency and accuracy. Of special interest are oxoiminostilbene and formylacridine, which are the final products in the degradation cascade. Their exceptional thermodynamic stability, as predicted by quantum chemical methods, suggests that these structures should be considered as recalcitrants in chlorinated waters. Fruitful interplay between computational models and experimental data proves that the quantum chemical approach can be used as a predictive tool in environmental degradation studies.

The stability of nitrogen-centered radicals.

Radical stabilization energies (RSEs) for a wide variety of nitrogen-centered radicals and their protonated counterparts have been calculated at G3(MP2)-RAD and G3B3 level. The calculated RSE values can be rationalized through the combined effects of resonance delocalization of the unpaired spin, electron donation through adjacent alkyl groups or lone pairs, and through inductive electron donation/electron withdrawal. The influence of ring strain effects as well as the synergistic combination of individual substituent effects (captodatively stabilized N-radicals) have also been explored. In symmetric N-radicals the substituents may also affect the relative ordering of electronic states. In most cases the π-type radical (unpaired spin distribution perpendicular to the plane of the N-radical) is found to be most stable. Closed shell precursors of biological and pharmaceutical relevance, for which neither experimental nor theoretical results on radical stabilities exist, have been included.

A computational study of the chlorination and hydroxylation of amines by hypochlorous acid.

The reactions of hypochlorous acid (HOCl) with ammonia, (di)methylamine, and heterocyclic amines have been studied computationally using double-hybrid DFT methods (B2PLYP-D and BK-PLYP) and a G3B3 composite scheme. In the gas phase the calculated energy barriers for N- and/or C-hydroxylation are ca. 100 kJ mol(-1) lower than the barrier for N-chlorination of amines. In the model solvent, however, the latter process becomes kinetically more favored. The explicit solvent effects are crucial for determination of the reaction mechanism. The N-chlorination is extremely susceptible to the presence of explicit water molecules, while no beneficial solvation effect has been found for the N- or C-hydroxylation of amines. The origin of the observed solvent effects arises from differential solvation of the respective transition states for chlorine- and oxygen-transfers, respectively. The nature of solvation of the transition state structures has been explored in more detail by classical molecular dynamics (MD) simulation. In agreement with the quantum mechanical approach, the most stable structural motif, which includes the amine, HOCl, and two reactive waters, has been identified during the MD simulation. The inclusion of 5 or 6 explicit water molecules is required to reproduce the experimental barriers for HOCl-induced formation of N-chloramines in an aqueous environment.

Chlorination of N-methylacetamide and amide-containing pharmaceuticals. Quantum-chemical study of the reaction mechanism.

Chlorination of amides is of utmost importance in biochemistry and environmental chemistry. Despite the huge body of data, the mechanism of reaction between amides and hypochlorous acid in aqueous environment remains unclear. In this work, the three different reaction pathways for chlorination of N-methylacetamide by HOCl have been considered: the one-step N-chlorination of the amide, the chlorination via O-chlorinated intermediate, and the N-chlorination of the iminol intermediate. The high-level quantum chemical G3B3 composite procedure, double-hybrid B2-PLYPD, B2K-PLYP methods, and global hybrid M06-2X and BMK methods have been employed. The calculated energy barriers have been compared to the experimental value of ΔG(#)298 ≈ 87 kJ/mol, which corresponds to reaction rate constant k(r) ≈ 0.0036 M(-1) s(-1). Only the mechanism in which the iminol form of N-methylacetamide reacts with HOCl is consistent (ΔG(#)298 = 87.3 kJ/mol at G3B3 level) with experimental results. The analogous reaction mechanism has been calculated as the most favorable pathway in the chlorination of small-sized amides and amide-containing pharmaceuticals: carbamazepine, acetaminophen, and phenytoin. We conclude that the formation of the iminol intermediate followed by its reaction with HOCl is the general mechanism of N-chlorination for a vast array of amides.

The chemical fate of paroxetine metabolites. Dehydration of radicals derived from 4-(4-fluorophenyl)-3-(hydroxymethyl)piperidine.

Quantum chemical calculations have been used to model reactions which are important for understanding the chemical fate of paroxetine-derived radicals in the environment. In order to explain the experimental observation that the loss of water occurs along the (photo)degradation pathway, four different mechanisms of radical-induced dehydrations have been considered. The elimination of water from the N-centered radical cation, which results in the formation of an imine intermediate, has been calculated as the most feasible process. The predicted energy barrier (ΔG = 98.5 kJ mol(-1)) is within the barrier limits set by experimental measurements. All reaction intermediates and transition state structures have been calculated using the G3(MP2)-RAD composite procedure, and solvent effects have been determined using a mixed (cluster/continuum) solvation model. Several new products, which comply with the available experimental data, have been proposed. These structures could be relevant for the chemical fate of antidepressant paroxetine, but also for biologically and environmentally related substrates.

Computational study of radicals derived from hydroxyurea and its methylated analogues.

Structural and electronic properties and chemical fate of free radicals generated from hydroxyurea (HU) and its methylated analogues N-methylhydroxyurea (NMHU) and O-methylhydroxyurea (OMHU) are of utmost importance for their biological and pharmacological effects. In this work the cis/trans conformational processes, tautomerizations, and intramolecular hydrogen and methyl migrations in hydroxyurea-derived radicals have been considered. Potential energy profiles for these reactions have been calculated using two DFT functionals (BP86 and B3LYP) and two composite models (G3(MP2)RAD and G3B3). Solvation effects have been included both implicitly (CPCM) and explicitly. It has been shown that calculated energy barriers for free radical rearrangements are significantly reduced when a single water molecule is included in calculations. In the case of HU-derived open-shell species, a number of oxygen-, nitrogen-, and carbon-centered radicals have been located, but only the O-centered radicals (e1 and z1) fit to experimental isomeric hyperfine coupling constants (hfccs) from EPR spectra. The reduction of NMHU and OMHU produces O-centered and N-centered radicals, respectively, with the former being more stable by ca. 60 kJ mol(-1). The NMHU-derived radical e4 undergoes rearrangements, which can result in formation of several conceivable products. The calculated hfccs have been successfully used to interpret the experimental EPR spectra of the most probable rearranged product 10. Reduction potentials of hydroxyureas, radical stabilization energy (RSE) and bond disocciation energy (BDE) values have been calculated to compare stabilities and reactivities of different subclasses of free radicals. It has been concluded, in agreement with experiment, that reductions of biologically relevant tyrosyl radicals by HU and NMHU are thermochemically favorable processes, and that the order of reactivity of hydroxyureas follows the experimentally observed trend NMHU > HU > OMHU.

Prereactive complexes in chlorination of benzene, triazine, and tetrazine: a quantum chemical study.

In order to perform a complete search for prereactive complexes between arenes and chlorine, the stochastic search method was employed. Stationary points are optimized at B3LYP, M05-2X, and MP2 levels, while improved energetics are calculated using the B2PLYP-D method, which includes corrections important for accurate description of dispersion forces. New intermediates were located and their mechanistic relevance has been discussed. It has been suggested that, at least in the gas-phase, the T-shaped complex precedes the formation of classical benzene/chlorine π-complex. No σ-complex is found on the energy surface, unless explicit counterions are included in calculations. Neither π- nor σ-complexes were located on the reactant side of chlorination of triazine, but only linear and T-shaped complexes were identified as stable minima. These structures represent important prereactive complexes for chlorination of triazine. In the case of tetrazine, which is unlikely to undergo direct chlorination, only two complexes (resting and T-shaped) were located.

Base-catalyzed reactions of environmentally relevant N-chloro-piperidines. A quantum-chemical study.

Electronic structure methods have been applied to calculate the gas and aqueous phase reaction energies for base-induced rearrangements of N-chloropiperidine, N-chloro-3-(hydroxymethyl)piperidine, and N-chloro-4-(4-fluorophenyl)-3-(hydroxymethyl)piperidine. These derivatives have been selected as representative models for studying the chemical fate of environmentally relevant chloramines. The performance of different computational methods (MP2, MP4, QCISD, B3LYP and B2PLYP) for calculating the thermochemistry of rearrangement reactions was assessed. The latter method produces energies similar to those obtained at G3B3(+) level, which themselves have been tested against experimental results. Experimental energy barriers and enthalpies for ring inversion, nitrogen inversion and dehydrochlorination reactions in N-chloropiperidine have been accurately reproduced when solvent effects have been included. It was also found that the combined use of continuum solvation models (e.g. CPCM) and explicit consideration of a single water molecule is sufficient to properly describe the water-assisted rearrangement of N-chlorinated compounds in basic media. In the case of N-chloro-4-(4-fluorophenyl)-3-(hydroxymethyl)piperidine, which represents the chlorinated metabolite of the antidepressant paroxetine, several different reactions (intramolecular addition, substitution, and elimination reactions) have been investigated. Transition state structures for these processes have been located together with minimum energy structures of conceivable products. Imine 4A is predicted to be the most stable reaction product, closely followed by imine 4B and oxazinane 8, while formation of isoxazolidine 7 is much less favourable. Calculated reaction barriers in aqueous solution are quite similar for all four processes, the lowest barrier being predicted for the formation of imine 4A.

Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin.

Low-pressure pH gradient ion exchange separation provides a fast, simple and cost-effective method for preparative purification of native and desialylated apo-transferrin. The method enables easy monitoring of the extent of the desialylation reaction and also the efficient separation and purification of protein fractions after desialylation. The N-glycan analysis shows that the modified desialylation protocol successfully reduces the content of the sialylated fractions relative to the native apo-transferrin. In the optimized protocol, the desialylation capacity is increased by 150 %, compared to the original protocol provided by the manufacturer. The molar absorption coefficients in the near-UV region for the native and desialylated apo-transferrin differ by several percent, suggesting a subtle dependence of the glycoprotein absorbance on the variable sialic acid content. The method can easily be modified for other glycoproteins and is particularly appropriate for quick testing of sialic acid content in the protein glycosylation patterns prior to further verification by mass spectrometry.

Solvent effects on the absorption and fluorescence spectra of Zaleplon: Determination of ground and excited state dipole moments.

Solvent effects on the absorption and fluorescence spectra of Zaleplon, a nonbenzodiazepine sedative/hypnotic drug that is mainly used for the short term treatment of insomnia, were investigated in 18 different solvents with diverse polarities. Dipole moments of the ground and excited state (µg and µe) were determined by Lippert-Mataga, Bakhshiev, Reichardt, McRae and Suppan solvatochromic methods. The dipole moment of Zaleplon ground state in the gas phase has been calculated as µg = 10.95 D (TD-DFT) with B3LYP/cc-pVTZ functional. There is a good agreement of theoretical data with Reichardt, McRae, and Suppan correlations, while some dissidence with Lippert-Mataga and Bakhshiev equations is suggesting the occurrence of specific solute-solvent interactions. Additionally, multiple linear regression analysis with Kamlet-Taft and Catalan solvatochromic models was applied to solute-solvent interactions. Dominant property of the solvent that affects the absorption band and Stokes shifts of Zaleplon is polarity of the solvent while the emission band is influenced mainly by solvent basicity.

Chlorination of 5-fluorouracil: Reaction mechanism and ecotoxicity assessment of chlorinated products.

What happens to drugs in the chlorinating environment? Degradation products may vary in pharmacological profiles and in ecotoxicity potentials compared to the parent compound. This study combines synthesis, NMR spectroscopy, quantum chemical calculations, and toxicity experiments on Daphnia magna to investigate chemical fate of antineoplastic drug 5-fluorouracil (5-FU) in chlorinated environment, which is common in waste-water treatment procedures, but also endogenous in activated neutrophils. A reduction of toxicity (EC50 after 48 h is 50% higher than for the parent 5-FU) was observed after the first chlorination step, in which a chlorohydrin 5-chloro-5-fluoro-6-hydroxy-5,6-dihydrouracil was formed. Further chlorination leads to N-chlorinated intermediate, that undergoes the pyrimidine ring opening reaction. The final product, 2-chloro-2-fluoro-3,3-dihydroxypropanoic acid was obtained after the loss of the chlorinated urea fragment. This is the most potent compound in the reaction sequence, with toxicity parameter EC50, after 48 h, more than twice lower compared to the parent 5-FU. Clearly, the contact time between chlorinating species and degradation products provide different ecotoxicological properties of reaction mixtures. Interplay between experimental and theoretical procedures, to properly describe reaction pathways and provide more information on toxicity profiles, is a way forward in environmental science research.

1,1'-Bis(thymine)ferrocene Nucleoside: Synthesis and Study of Its Stereoselective Formation.

The synthesis of 1,1'-bis(thymine)ferrocene nucleoside is reported. This nucleoside was obtained in a two-step synthetic methodology including a Michael addition reaction of 1,1'-bis(3-chloropropionyl)ferrocene with thymine to afford the bis(thymine) adduct in 44 % yield. In the second step, the two prochiral carbonyl functionalities in the Michael adduct were reduced to hydroxyl groups with sodium borohydride. This apparently straightforward reaction proceeds in a highly stereoselective fashion to yield the title ferrocenyl nucleoside as a racemic mixture that consists of the R,R and the S,S isomers. The absolute configuration of the chiral carbon atoms in the nucleoside was assigned on the basis of single-crystal X-ray diffraction analysis of the methyl derivative. Furthermore, the mechanism of reduction of the bis(thymine) adduct was investigated by using DFT calculations. The two critical minima, pre-reactive complex, and semi-reduced intermediate, as well as two corresponding transition states were located to support the observed stereoselectivity. The redox properties of 1,1'-bis(thymine)ferrocene nucleoside, its precursor, and congeners were investigated using cyclic voltammetry. For the title compound a reversible redox process was found at a low potential of -30 mV versus FcH/FcH+ (FcH=Fe(η5 -C5 H5 )2 ) as the reference redox couple.