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The N-oxygenation of an amine group is one of the steps in the biosynthesis of the antibiotic chloramphenicol. The non-heme di-iron enzyme CmlI was identified as the enzyme catalyzing this reaction through bioinformatics studies and reconstitution of enzymatic activity. In vitro reconstitution was achieved using phenazine methosulfate and NADH as electron mediators, while in vivo activity was demonstrated in Escherichia coli using two substrates. Kinetic analysis showed a biphasic behavior of the enzyme. Oxidized hydroxylamine and nitroso compounds in the reaction were detected both in vitro and in vivo based on LC–MS. The active site metal was confirmed to be iron based on a ferrozine assay. These findings provide new insights into the biosynthesis of chloramphenicol and could lead to further development of CmlI as a useful biocatalyst.

Although aminoxyl radicals ( RR ′ N O ˙ ) are well-known as organic stable free radicals, the isomeric radicals, N-alkoxyaminyl radicals ( R N ˙ OR ′ ) , are scarcely studied in the area of theoretical study. We carried out the theoretical calculation of the isotropic 14N hyperfine coupling constants (aN), g-factors, electronic absorbances, and generation mechanisms of N-alkoxyaminyl radicals. For thirty one N-alkoxyaminyl radicals, the PCM-DFT calculation at B3LYP/6-31+G(d,p) could well reproduce the experimental data of aN and g-factors, where R2 were 0.9796 and 0.9439, respectively. The UV–vis spectra were moderately reproduced by PCM-TD-DFT (PBE0/6-31+G(d,p)), where R2 was 0.9205. Additionally, the use of a linear scaling procedure precisely reproduced the UV–vis spectrum. The activation Gibbs free energies for the generation of N-alkoxyaminyl radicals and aminoxyl radicals in the competing reactions of nitroso compounds with alkyl radicals were estimated by DFT and coupled cluster calculations. The activation free energies for the generation of N-alkoxyaminyl radicals were estimated to be 9–18 kcal/mol at B3LYP/6-31+G(d,p). The reported product ratios in the competing reactions could be reasonably explained by the differences in the activation free energies.

Tertiary aliphatic β- and γ-nitro nitriles and ketones deuterated in (several) selected positions had been synthesized. The deuterated nitro compounds served as a starting material for the corresponding deuterium labeled nitrones or hydroxylamines (reducing with aluminum amalgam). Further oxidation of the last two groups of compounds with sodium periodate or m-CPBA afforded the relevant deuterated tertiary C-nitroso compounds.

Aromatic nitroso compounds in the presence of base and 2-(α-hydroxyalkyl)-3,4-dimethylthiazolium trifluoromethanesulfonate and related salts furnish in variable yields O- and N-acyl-aryl hydroxylamines and 3,4-dimethylthiazolium trifluoromethanesulfonate. A primary kinetic isotope effect of 4.9, obtained for the appropriate 2α-deuterated thiazolium salt, points to the C2α–H bond cleavage as the rate determining step. Radical species detected by ESR were unambiguously identified as phenylhydronitroxide, but attempted trapping of the corresponding C–heterocyclic radicals by TEMPO was not successful, and substrates incorporating a potential cyclopropyl radical clock gave products with the cyclopropyl ring intact. Theoretical calculations revealed a large activation energy for such reaction, which thus cannot per se exclude the intervention of such radical species. Evidence for the likely operation of two concurrent mechanisms, a radical and a preponderant ionic pathway, involving the conjugate base of the thiazolium salt, as the chemical model for ‘active thiamine’, and ArNO is presented for the formation of the products of the reaction.

Enantiomerically enriched (4R,5S)- and (4S,5R)-indano[1,2-d]oxazolidinones were enzymatically prepared from (±)-1-amino-2-indanol. Racemic 1-(N′-chloroacetyl-N-carbamylamino)-2-indanol O-chloroacetate was hydrolyzed with immobilized Pseudomonas cepacia lipase in the presence of β-cyclodextrin in acetone-buffer solution, to afford (1S,2R)-1-(N′-chloroacetyl-N-carbamylamino)-2-indanol (90%e.e.) and the unreacted (1R,2S)-substrate (97%e.e.), in nearly quantitative yields. The deprotection provided enantiomers of 1-N-carbamylamino-2-indanol, the precursor of indanoxazolidinone, via nitrosation-deaminocyclization reaction.

2-Azadienes 1 bearing a trialkylsilyloxy group on position 3 can be regarded as carboxylic acid synthons. Their cycloadditions with several classes of nitroso compounds have been studied in details as well as the transformation of the adducts into alpha-amino acids. This study showed that arylnitroso compounds such as nitrosobenzene reacted with 2-azadienes to give adducts that are potential precursors of alpha-N-arylamino acids. We have outlined an important limitation to the use of alpha-chloronitroso compounds. They are not compatible with highly functionalized dienes like 2-azadienes. On the other hand alpha-cyanonitroso and acylnitroso compounds reacted with azadienes to give adducts which were readily converted into alpha-amino acid derivatives. Copyright (C) 1996 Elsevier Science Ltd

Reaction of Grignard reagents and organolithium compounds (RM) with the congested 1-chloro-1-nitroso-2,2,6,6-tetramethylcyclohexane 1 leads to the formation of significant amounts of the reduction product 2,2,6,6-tetramethylcyclohexanone oxime 3 (61–90%) together with the corresponding oxime O-R ether 4 (0–11%). Attack on nitrogen is unimportant as shown by very low yields of nitrone. Formation of the products is rationalised with a pathway involving transfer of an electron from RM to 1. This leads—after separation of MCI—to a radical pair consisting of R and the relatively stable iminoxy radical 2 (Schemes 1 and 2). Combination of these radicals explains formation of oxime ether 4 and nitrone 5, while reaction of iminoxy radical 2 with excess of RM can give oxime 3. Reactive radicals R (i.e. Me, Ph, and to a minor extent n-Bu) are furthermore capable of abstracting hydrogen from the solvent (diethyl ether, toluene, or cumene), and the solvent derived radicals can also combine with 2 on oxygen, under formation of oxime ether (26% of 6a). The corresponding benzyl- and cumyl ethers 6b and 6c are only formed in trace amounts because dimerisation of benzyl radicals (7%) and cumyl radicals (22%) is favoured.

Two model nitrosamides were used to illustrate the application of oxidative and non-oxidative nitrosamide photorearrangements. N-nitroso-N-acetyldehydroabietylamine photolytically rearranges to give stable 6α-nitroso and 6α-nitrato derivatives respectively. Similar photorearrangement of N-nitroso-N-methyl- o -toluamide gave good yields of the corresponding oximino and nitrate esters; they, however underwent facile photolytic and thermal reactions to give various products.

Regiospecificity in attack of the title- bis -diene by aromatic nitroso compounds as dienophilic components is lower than that of other dienophiles of the triazolinedione type.

The electronic absorption spectra of nitroso-methane, nitroso-ethylene and nitroso-benzene were investigated by the PPP and CNDO/2 method. The unusual energy level spacing may lead to an interesting photophysical behaviour making fluorescence from the second excited singlet state of aromatic nitroso-compounds conceivable. The source of intensity of the forbidden low-frequency transition at 13 kK in nitroso-benzene is also discussed.

α Chloronitrosoadamantane 1a gives upon reaction with 2-phenylpropene at room temperature virtually quantitatively a stable ketonitrone salt, the α,α-adamantylidene-N-(2-phenyl)prop-1-en-3-yl nitrone hydrochloride 2a . Evidence for the structure of the crystalline product is based on microanalytical data and spectroscopic properties, together with degradation studies. Similar aliphatic ketonitrone hydrochlorides have been obtained from reaction of 2-phenylpropene with other α-chloronitroso compounds 1b-1e (73–91%), and from α-chloroni-trosoadamantane . and a series of allylic olefins (76–95%). Rearrangement of an intermediary N-α-chloroalkyl-N-alkenylhydroxylamine, which has been initially formed by an ene-type process between the reactants, can explain formation of the product.

α-Iodo-nitroalkanes decompose photochemically into α-nitroalkyl radicals and iodine. The α-nitroalkyl radical is detected by trapping with 2-methyl-2-nitrosopropane and identified by the hfs-constants of the resulting β-nitronitroxide observed with ESR. In the absence of nitroso compounds the α-nitroalkyl radicals can lose hydrogen to give nitro-olefins (70–80%). Minor products are nitroalkane (

Acryl radicals combine with nitroso compounds to give acyl-alkyl-nitroxides, which are identified by their ESR spectra. The acyl radicals are generated by oxidation of aldehydes with suitable hydrogen-acceptors such as t-butoxy radicals or by intermediates obtained int he photochemical and thermal decomposition of nitroso compounds. During photolysis of alkyl nitrites in hydrocarbon solvents the acyl-alkyl-nitroxides appear also and this is explained on the basis of known reactions of alkyl nitrites and their decomposition products.

The electronic structure of organic compounds containing NO bonds has been considered in relation to their observed lengths and characteristic frequencies. The relation between these properties and “bond-order” has been examined for a number of molecules and ions for which the electronic structures are well defined. These relationships are then used for the discussion of other compounds for which the electronic structures are less certain. It has been shown that many of the molecules contain bonds of non-integral order; structures in which the NO bonds contain an odd number of electrons have been proposed in certain cases, the double-quartet hypothesis being used to describe the electron distribution. It has been shown that the pattern of bond lenghts and characteristic frequencies can then be understood, as well as such unexplained observations as the dimerization of alkyl nitroso-compounds and the non-existence of dimers of nitrosyl halides.

Hydrogen peroxide reactions with ketones were studied by the nitroxide method. Fragmentation of ketones proceeded via hydrogen peroxide adducts. Radical substitution took place with boranes and plumbanes. Postulated radicals from photolysis of certain ketones have been trapped. Photolysis of aryliodides in the presence of t -nitrosobutane gave aromatic nitroxide radicals, the spectra of which were analyzed. Oxidative decarboxylation of carboxylic acid with thallic acetate proceeded via radicals. The splitting of the trichloromethyl nitroxide radicals is discussed. Some relationship between hyperfine Splittings and structure has been observed.

It is demonstrated that alkylnitroxides can be generated from nitrosoalkanes by thermal reduction with hydroxylamines or by photoreduction with thiophenol. The solvent dependence of the ratio of the nitrogen and hydrogen splitting constants in t-butylnitroxide is explained on the basis of mesomeric contributions to the structure of the radical.

The kinetics of Diels-Alder reactions with p -substituted nitroso benzenes and p -substituted 1-phenyl butadienes gives Hammett-reaction constants, clearly depending on the nature of the substituents. From this dependence a reaction scheme for diene synthesis with nitroso compounds is derived, describing the transition state of the reaction as an intermediate (quantitatively: as linear combination) between unpolar and zwitterionic structures. In our reactions it is an intermediate structure (depending on the substituents) between the transition states of a cyclo addition (concerted reaction) and an electrophilic addition.

Photolysis of the “pseudonitrole” 1-nitro-1-nitrosocyclohexane with red light in the absence of oxygen occurs by homolytic C-NO fission, and generates a radical pair: NO and the α-nitro-cyclohexyl radical. If one radical of this pair adds to the NO-group of unchanged starting material, there results a paramagnetic nitroxide, that can combine with the other radical of the pair to a diamagnetic intermediate, forming two isomeric intermediate adducts to the NO double bond. The ratio in which these isomers are formed depends on solvent. In methanol, solvolysis leads to equimolar amounts of cyclohexanone and cyclohexanone-oxime, together with methyl nitrite. In benzene, the major product is cyclohexanone. Small amounts are formed of 1,1-dinitrocyclohexane, 1-nitrocyclohexene and nitrocyclohexane; arising directly from the α-nitrocyclohexyl radical. The nitroxide derived from this radical and the starting material can be detected by ESR. The photolytic behaviour of 1-nitro-1-nitroso-1-cyclopropylethane illustrates the strong interaction between a nitrogroup and an adjacent C-radical centre: only ring-closed products are formed, showing considerable spin delocalisation away from the cyclopropyl ring.

The condensation of rapanone1 (I) with various aldehydes and nitroso compounds yields the corresponding methylene-bis(2.5-dihydroxy-4-tridecyl-3,6-benzoquinones) (II) or their anhydro derivatives (III). The N-bis(anhydro-5-hydroxy-4-tridecyl-3,6-benzoquinones) (IV) have been synthesized and their properties recorded.

Unusual chemical shifts are reported for o -alkylnitrosobenzenes. The origin of these shifts is considered. The PMR spectrum of o -nitrosotoluene is found to contain signals attributed to both monomeric and dimeric forms in carbon tetrachloride and d -chloroform solution.

The photodecomposition of 2-nitro-2-nitrosopropane (2-propylpseudonitrole) with light of λ > 540 nm was studied. The product distribution changes drastically with the nature of the solvent but can be explained readily by homolytic cleavage of the CNO bond. No evidence of photoreduction by an excited state of the nitrosocompound was obtained. In aprotic solvents the major products were acetone, 2,2-dinitropropane, 2-nitropropene, nitrogen dioxide, nitric oxide and nitrogen. In methanol methyl nitrite, acetoxime, acetone dimethylketal and acetone together with dinitrogen monoxide and water were the main products. This pronounced solvent influence is shown to be caused by the solvolysis (in methanol) or secondary thermal decomposition (in aprotic solvents) of labile intermediates. The 2-nitropropyl radical formed in the primary dissociative step does not take part in any hydrogen abstraction reactions under the experimental conditions employed.

The isomeric structures of oximes, nitrones and nitroso compounds may be readily differentiated on the basis of their nitrogen chemical shifts. The tautomeric equilibria present in oximenitroso systems are easily observed by means of 14N NMR spectroscopy. An explanation of the range of chemical shifts found within the oxime and nitrone groups of compounds is presented, based upon the average excitation energy approximation to the paramagnetic term in the nuclear screening tensor. The increase in screening of the nitrogen found upon protonation of an oxime is analogous to that observed for pyridine-type structures and their N-oxides.

It is demonstrated that nitroxides can be generated by photochemical or thermal decomposition of C-nitroso compounds. The nitroxide-formation takes place in both cases via the nitroso monomer. The influence of the wavelength on the photochemical reaction is discussed.

Some paramagnetic species formed in the photoreactions of nitroalkanes with P-compounds have been characterized by ESR. The observed radicals are mostly nitroxides whose structures are consistent with the trapping of P-centred radicals by intermediate nitroso compounds.

Radical intermediates formed in reaction of substituted nitrobenzene compounds with nucleophilic reagents have been trapped by t-nitrosobutane to yield nitroxide radicals which have been characterized in situ by ESR. The formation of short-lived radicals originating from reagents and solvents has been shown.

By the condensation of embelin (I) with various nitroso compounds, the corresponding N-bis-anhydrobenzoquinones (II) have been synthesized and their properties recorded.

A general procedure has been devised for the preparation of α-nitrosulfonamides. Benzylic sulfonamides, and a wide variety of N,N-dialkylalkanesulfonamides may be converted to the corresponding nitrosulfonamides via displacement by a sulfonamide-stabilized carbanion on an alkyl nitrate.

By the condensation of 2,5-dihydroxyl-1,4-benzoquinone (I) with various nitroso compounds, the corresponding N-bis(anhydro-2,5-dihydroxy-3,6-benquinones) (II) were obtained and their properties recorded.