Nitroxides as metabolic and EPR imaging probes in biological model systems

Nitroxides are unusually stable free radicals and sensitive “reporters” of the chemico-physical environment which surround them. The term “reporter group” was used to indicate this property, later the term spin label or spin probe was preferred. The facile synthesis and the multiplicity of compounds which have been coupled to the nitroxide moiety has allowed the development of a number of applications. Nitroxides have been used to study molecular mobility of proteins and lipids in membranes, enzyme active sites, DNA synthesis, and measure of electrical potential, pH, temperature as well as oxygen gradients across membranes; more recently, as contrast agents in magnetic resonance imaging and electron paramagnetic resonance imaging (EPRI). Aim of this work is to review and comment on the use of various nitroxides as metabolic probes, and in EPRI in studies of biomedical interest.     

Intermolecular nuclear relaxation in paramagnetic solutions: from free radicals to rare earths

The principles of the intermolecular relaxation of a nuclear spin by its fluctuating magnetic dipolar interactions with the electronic spins of the paramagnetic surrounding species in solution are briefly recalled. It is shown that a very high dynamic nuclear polarization (DNP) of solvent protons is obtained by saturating allowed transitions of free radicals with a hyperfine structure, and that this effect can be used in efficient Earth field magnetometers. Recent work on trivalent lanthanide Ln³⁺ aqua complexes in heavy water solutions is discussed, including paramagnetic shift and relaxation rate measurements of the ¹H NMR lines of probe solutes. This allows a determination of the effective electronic magnetic moments of the various Ln³⁺ ions in these complexes, and an estimation of their longitudinal and transverse electronic relaxation times T_1e and T_2e. Particular attention is given to Gd(III) hydrated chelates which can serve as contrast agents in magnetic resonance imaging (MRI). The full experimental electronic paramagnetic resonance (EPR) spectra of these complexes can be interpreted within the Redfield relaxation theory. Monte-Carlo simulations are used to explore situations beyond the validity of the Redfield approximation. For each Gd(III) complex, the EPR study leads to an accurate prediction of T_1e, which can be also derived from an independent relaxation dispersion study of the protons of the probe solutes.     

EPR studies of the hydroperoxy radical in x-irradiated single crystals of BaCl2·2H2O

Electron paramagnetic resonance studies of X-irradiated single crystals of BaCl₂-2H₂O have been made at room temperature. Four resonance lines are observed which can be grouped into two hyperfine doublets. Angular variation studies were made in the crystallographic ab, bc and ac planes. There are two magnetically equivalent but physically inequivalent magnetic complexes in the crystal lattice which are mutually perpendicular to each other. The paramagnetic species is identified as the hydroperoxy radical and a detailed discussion is made at the end.     

Use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide for rapid detection of methicillin-resistant Staphylococcus aureus by resonance light scattering

A rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) using resonance light scattering (RLS) on an ordinary fluorescence spectrometer was developed. The viable MRSA reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to produce insoluble particles which displayed intense resonance scattering light. It showed a linear relationship between the number of viable MRSA and the RLS intensity. Dead MRSA were unable to reduce MTT. MRSA exposed to flavonoids extracted from Marchantia convoluta (MCF) showed a MCF concentration-dependent inhibition of the ability to reduce MTT. The RLS could, in combination with the MTT assay, be a rapid and sensitive detection method for vitro-cultured MRSA.     

Spectral simplification in proton magnetic resonance spectrometry for 3-(4-aminophenyl)-3-ethyl-2,6-piperidinedione (aminoglutethimide) with achiral and chiral lanthanide shift reagents

The 60-MHz ¹H-NMR spectra of racemic aminoglutethimide (A; 3-(4-aminophenyl)-3- ethyl-2,6-piperidinedione) are studied with the achiral shift reagent, tris(6,6,7,7,8,8,8- heptafluoro-2,2-dimethyl-3,5-octanedionato)europiura(III) (B) and the chiral reagents, tris[3-(trifluororaethylhydroxyraethylene)-D-caraphorato]europium(III) (C) and tris[3- heptafluoropropylhydroxyraethylene-D-caraphorato]europiura(III) (D). Appreciable values of the enantioraeric shift difference, ΔΔδ, were observed for both sets of aryl protons and the methyl protons in CDCl₃ solutions ar 28°C with added C or D. Optical purity determinations were done with good reliability by using the methyl resonance and a D:A molar ratio near 0.5. Results are discussed in terms of possible coordination of the europiura at the amine or amide carbonyl groups. Some significant differences in results with B, C and D were noted.     

NMR shielding in helium-3 atoms modified by gaseous nitrogen and oxygen

Helium-3 nuclear magnetic resonance (³He NMR) measurements were carried out for the gaseous mixtures of helium-3 with pure nitrogen and synthetic air as the solvents. It was found that ³He shielding is linearly dependent on solvent density up to approx. 6 mol/L. At higher density of the gaseous solvent, the change of ³He shielding is nonlinear and especially distinct when helium-3 atoms can interact with two O₂ molecules. The interaction with paramagnetic oxygen molecules can induce two kinds of ³He shielding changes: (1) due to the isotropic Fermi contact interaction and (2) from the dipolar magnetic interaction between unpaired O₂ electrons and ³He nuclear magnetic dipole moment. The two paramagnetic effects in helium-3 shielding cannot be experimentally separated, although for such small molecular objects, they could be presumably modeled by advanced theoretical calculations.     

Activatable 19F MRI Nanoparticle Probes for the Detection of Reducing Environments

¹⁹F magnetic resonance imaging (MRI) probes that can detect biological phenomena such as cell dynamics, ion concentrations, and enzymatic activity have attracted significant attention. Although perfluorocarbon (PFC) encapsulated nanoparticles are of interest in molecular imaging owing to their high sensitivity, activatable PFC nanoparticles have not been developed. In this study, we showed for the first time that the paramagnetic relaxation enhancement (PRE) effect can efficiently decrease the ¹⁹F NMR/MRI signals of PFCs in silica nanoparticles. On the basis of the PRE effect, we developed a reduction‐responsive PFC‐encapsulated nanoparticle probe, FLAME‐SS‐Gd³⁺ (FSG). This is the first example of an activatable PFC‐encapsulated nanoparticle that can be used for in vivo imaging. Calculations revealed that the ratio of fluorine atoms to Gd³⁺ complexes per nanoparticle was more than approximately 5.0×10², resulting in the high signal augmentation.     

Biohydrolysis of (S)-3-(thiophen-2-ylthio)butanenitrile

Whole-cell enzymatic hydrolysis was shown to be the choice in the preparation of (S)-3-(thiophen-2-ylthio)butanoic acid. While all chemical methods of hydrolysis failed, 12 bacterial strains expressing nitrile hydratase and amidase activities have been identified to hydrolyze (S)-3-(thiophen-2-ylthio)butanenitrile 1 directly into the corresponding acid. The substrate was also shown to be an efficient inducer of the enzymatic activity. However, it inhibited microbial growth. Acid 3 was prepared on a gram scale with the recombinant Rhodococcus erythropolis, formerly Brevibacterium sp. pYG811b as shown by sequencing of its 16S rRNA.     

Electron paramagnetic resonance of gamma-irradiated single crystals of 3-nitroacetanilide

The electron paramagnetic resonance of single crystals of 3-nitroacetanilide has been observed and analyzed for different orientations of the crystal in the magnetic field, after being damaged at 300 K by γ-irradiation. The crystals have been investigated between 123 and 300 K. The spectra were found to be temperature independent. The irradiation of 3-nitroacetanilide by γ-rays produces radicals at the nitrogen atoms in the molecule. The principal values of the hyperfine coupling tensor of the unpaired electron and the principal values of the g-tensor were determined.     

A scalable synthesis of (R)-3-(2-aminopropyl)-7-benzyloxyindole via resolution

(±)-3-(2-Aminopropyl)-7-benzyloxyindole 1, assembled from 7-benzyloxyindole 3 in 59% overall yield, is resolved with O,O′-di-p-toluoyl l-(2R,3R)-tartaric acid 7 into (R)-1, a key intermediate of AJ-9677 2 (selective adrenaline β₃-agonist) in 99.5% e.e. and 36% overall yield. The unwanted enantiomer (S)-1 (61.9% e.e.; recovered in 57% yield from the crystallization filtrate) can be reused in another round of resolution after its enantiomeric purity is lowered to 3.7% by Raney Co treatment under a hydrogen atmosphere.     

Synthesis of lithium,sodium, cesium,and calcium salts of (E)-4- or (E,S)-3-methyl-2-(5-arylisoxazol-3-yl)-methyleneaminobutanoic, (E,S)-4-methyl-2- or (E,2S,3S)-3-methyl-2-(5-arylisoxazol-3-yl)methyleneaminopentanoic,and (E)-6-(5-arylisoxazol-3-yl)methyleneaminohexanoic acids

Preparative method for the synthesis of lithium, sodium, cesium, and calcium salts of (E)-4-(5-arylisoxazol-3-yl)methyleneaminobutanoic, (E)-6-(5-arylisoxazol-3-yl)methyleneaminohexanoic, (E,S)-3-methyl-2-(5-arylisoxazol-3-yl)methyleneaminobutanoic, (E,S)-4-methyl-2-(5-arylisoxazol-3-yl)methyleneaminopentanoic and (E,2S,3S)-3-methyl-2-(5-arylisoxazol-3-yl)methyleneaminopentanoic acids was developed by reacting 5-phenyl(4-tolyl)isoxazole-3-carbaldehydes with amino acids like 4-aminobutyric and 6-aminocaproic acids, L-valine, L-leucine or L-isoleucine in the presence of lithium hydride, sodium methoxide, cesium carbonate or calcium hydride in boiling methanol.     

Immunosensor Accuracy Improved by Coating Beads with Poly-2-[3-(Methacryloylamino)Propylammonio]Ethyl 3-Aminopropyl Phosphate

Poly-2-[3-(methacryloylamino)propylammonio]ethyl 3-aminopropyl phosphate (poly-3MAm3AP) is a polymer that contains phosphorylcholine groups that can attract water to form a water layer, and amino groups that can be used for the covalent attachment of receptors such as anti-LH (Luteinizing hormone) antibody and LH antigen. Poly-3MAm3AP was synthesized with the anticipation that it would aid the recognition of a large number of analytes and eliminate nonspecific adsorption. Poly-3MAm3AP was coated on beads and served as an antigen-antibody reaction interface. The specificity of this surface was evaluated using surface plasmon resonance (SPR) by comparing responses to specific anti-LH antibody and nonspecific anti-hemoglobin antibody.Poly-3MAm3AP did not adsorb nonspecific antibody due to the presence of free water associated with the phosphorylcholine moiety. An immunosensor system was fabricated by combining poly-3MAm3AP-coated beads containing immobilized anti-LH antibody with an optical detector. Poly-3MAm3AP not only reduced nonspecific adsorption, but also increased the amount of specific antibody-antigen interaction. These favorable characteristics are due to an increased amount of immobilized receptor and an increased amount of space for antigen-antibody interaction arising from electrostatic repulsion within the poly-3MAm3AP electrolyte.     

Isolation,Purification, and Characterization of Antimicrobial Compound 6-[1,2-dimethyl-6-(2-methyl-allyloxy)-hexyl]-3-(2-methoxy-phenyl)-chromen-4-one from Penicillium sp. HT-28

A fungal culture (Penicillium sp., HT-28), isolated from soil has been evaluated for its bioactivity, which showed broad spectrum antimicrobial activity and was effective against methicillin-resistant Staphylococcus aureus (MRSA) also. Statistical optimization of the medium by response surface methodology (RSM) enhanced the antimicrobial activity up to 1.8-fold. Column chromatography was used to isolate the active compound (A), which was characterized to be 6-[1,2-dimethyl-6-(2-methyl-allyloxy)-hexyl]-3-(2-methoxy-phenyl)-chromen-4-one by various spectroscopic techniques such as infrared (IR), ¹H and ¹³C nuclear magnetic resonance (NMR) spectra, and mass spectroscopy. Minimum inhibitory concentration (MIC) of the active compound (A) ranged from 0.5 to 15 μg/mL. Viable cell count studies of the active compound (A) showed S. aureus, Escherichia coli, Staphylococcus epidermidis, and Salmonella typhimurium 1 to be the most sensitive. The compound retained its bioactivity after treating it at 100 °C for 1 h. Furthermore, the compound (A) when tested for its biosafety was found neither to be cytotoxic nor mutagenic. The study demonstrated that an apparently novel compound isolated from Penicillium sp. (HT-28) seems to be a stable and potent antimicrobial.     

Synthesis of Duloxetine Intermediate (S)-3-Chloro-1-(2-thienyl)-1-propanol with Liquid-Core Immobilized Candida pseudotropicalis 104

(S) -3-Chloro-1-(2-thienyl)-1-propanol was synthesized by the asymmetric reduction of 3-chloro-1-(2-thienyl)propanone with liquid-core immobilized Candida pseudotropicalis 104. The optimum time was 28?h for the re-cultivation of immobilized cells. The optimum film solvent for the liquid-core capsule was 0.3?% chitosan (M _w 1.0?×?10⁵). Conversion decreased with the increase of the liquid-core capsule diameter and with the addition of more substrates at the same reduction time. The immobilized cells show good reduction ability in a potassium phosphate buffer (pH 6.6~7.2). The material outside the spread speed of immobilized cells was not restricted when the shaking speed was higher than 160?r/min. Liquid-core immobilized cells can be reused 11 times. Compared with the batch reduction, the continuous reduction of 3-chloro-1-(2-thienyl)propanone in the membrane reactor with liquid-core immobilized cells as catalyst can relieve the inhibition from a high-concentration substrate. Conversion and enantiometric excess of (S)-3-chloro-1-(2-thienyl)-1-propanol reached 100?% and >99?% in a continuous reduction of 12?g/L 3-chloro-1-(2-thienyl)propanone for 10?days.     

Structure,Z′ = 2 Crystal Packing Features of 3-(2-Chlorobenzylidene)-5-(p-tolyl)furan-2(3H)-one

3-(2-Chlorobenzylidene)-5-(p-tolyl)furan-2(3H)-one (1), C₁₈H₁₃ClO₂, crystallizes with Z = 8 and Z′ = 2, and the structure at 100 K has orthorhombic (Pna2₁) symmetry. Each kind of molecule takes part in π–π stacking interactions to form infinite chains parallel to the c axis. We believe that the existence of two forms can be explained by the probable rotation around a single C–C bond. The quantum chemical modeling reveals that these molecules are almost equivalent energetically, and they can be described as the two most stable conformers (rotamers) with a minor rotational barrier of about 0.67 kcal/mol.     

Novel (E)- and (Z)-3(5)-(2-hydroxyphenyl)-4-styrylpyrazoles from (E)- and (Z)-3-styrylchromones: the unexpected case of (E)-3(5)-(2-hydroxyphenyl)-4-(4-nitrostyryl)pyrazoles

An efficient synthetic method for the preparation of (E)- and (Z)-3(5)-(2-hydroxyphenyl)-4-styrylpyrazoles has been developed. The reaction of (E)- and (Z)-3-styrylchromones with hydrazine hydrate afforded the corresponding (E)- and (Z)-4-styrylpyrazoles, respectively, saved 4′-nitro-derivatives where both (E)- and (Z)-4′-nitro-3-styrylchromones afforded (E)-3(5)-(2-hydroxyphenyl)-4-(4-nitrostyryl)pyrazoles. The reaction mechanism for these transformations was discussed and the stereochemistry of all products was assigned by NMR experiments.     

Crystal and molecular structure of methyl(±)-2-((1R,3R)-3-{ 2-[(3S)-1-ethyl-3-hydroxy-2-oxo-2,3-dihydro-1H-3-indolyl]acetyl}-2,2-dimethylcyclobutyl) acetate

The structure of methyl (±)-2-((1R,3R)-3-{ 2-[(3S)-1-ethyl-3-hydroxy-2-oxo-2,3-dihydro-1H-3-indolyl]acetyl}-2,2-dimethylcyclobutyl) acetate has been determined by single crystal X-ray diffraction. The crystal belongs to triclinic system; parameters of the unit cell are: a = 6.551(1) Å, b = 11.506(1) Å, c = 14.334(1) Å, α = 101.41(1)°, β = 97.57(1)°, γ = 104.72(1)°; space group P-1, Z = 2, composition C₂₁H₂₇NO₅. The structure of N-ethyloxindole fragment is usual for the present class of compounds. The configuration of the formed asymmetric carbon atom C(3) of the pyrrole ring along with the configuration of C(12) and C(14) atoms of 2,2-dimethylcyclobutane ring form the side chain of the molecule were determined. There is observed the generation of centrosymmetrical dimers in the crystal structure due to realized intermolecular hydrogen bond of O-H...O type, 2.808(2) Å.     

Preparation,molecular structures,and characteristic properties of (E)-1-(2-furyl)- and (E)-1-(2-thienyl)-2-(3-guaiazulenyl)ethylenes and (E)-1-(3-furyl)- and (E)-1-(3-thienyl)-2-(3-guaiazulenyl)ethylenes

Wittig reactions of 2-furaldehyde (20) [and thiophene-2-carbaldehyde (21)] with (3-guaiazulenylmethyl)triphenylphosphonium bromide (19) in ethanol containing NaOEt at 25 °C for 24 h under argon give (E)-1-(2-furyl)-2-(3-guaiazulenyl)ethylene (22E) and (E)-1-(2-thienyl)-2-(3-guaiazulenyl)ethylene (23E) in 53 and 36% yields. Similarly, Wittig reactions of 3-furaldehyde (29) [and thiophene-3-carbaldehyde (30)] with 19 under the same reaction conditions as for 20 and 21 afford (E)-1-(3-furyl)-2-(3-guaiazulenyl)ethylene (31E) and (E)-1-(3-thienyl)-2-(3-guaiazulenyl)ethylene (32E) in 32 and 46% yields. Molecular structures and characteristic properties as well as preparation of the title E (i.e., one of the geometrical isomers) forms, with a view to comparative study, are reported. Moreover, reactions of those conjugated π-electron systems with TCNE (=tetracyanoethylene) in benzene [and in DMF (=N,N-dimethylformamide)] at 25 °C for 24 h under argon yield unique products, possessing interesting molecular structures, respectively, whose characteristic properties and crystal structures are documented, also.     

Synthesis of methyl (E)-2-[(3S,4S)-4-hydroxy-3-(pent-3-yloxy)-pyrrolidin-2-ylidene]propanoate and its unusual recyclization

Methyl (2E,4S,5S)-5-hydroxy-6-mesyloxy-2-methyl-4-(pent-3-yloxy)hex-2-enoate was synthesized from l-tartaric acid. Attempted substitution of the mesyloxy group by the reaction with NaN₃ directly led to methyl (E)-2-[(3S,4S)-4-hydroxy-3-(pent-3-yloxy)pyrrolidin-2-ylidene]propanoate. The latter on treatment with CF₃COOH and then NaOH gave methyl (2E)-2-methyl-4-[(S)-oxiran-2-yl]-4-(pent-3-yloxy)but-2-enoate.     

Preparation of enantiomerically pure (3E)-alkyl-4-(hetero-2-yl)-2-hydroxybut-3-enoates by Candida parapsilosis ATCC 7330 mediated deracemisation and determination of the absolute configuration of (3E)-ethyl-4-(thiophene-2-yl)-2-hydroxybut-3-enoate

Deracemisation of racemic (3E)-alkyl-4-(hetero-2-yl)-2-hydroxybut-3-enoates using Candida parapsilosis ATCC 7330 resulted in the formation of one enantiomer in high enantiomeric excess [up to >99% ee] and isolated yields [up to 79%]. The absolute configuration of the enantiomerically pure (3E)-ethyl-4-(thiophene-2-yl)-2-hydroxybut-3-enoate as determined by ¹H NMR of the Mosher esters was found to be (S).