Studies on the Oxidation of Aromatic Amines Catalyzed by Trametes versicolor Laccase.

The bio-oxidation of a series of aromatic amines catalyzed by T. versicolor laccase has been investigated exploiting either commercially available nitrogenous substrates [(E)-4-vinyl aniline and diphenyl amine] or ad hoc synthetized ones [(E)-4-styrylaniline, (E)-4-(prop-1-en-1-yl)aniline and (E)-4-(((4-methoxyphenyl)imino)methyl)phenol]. At variance to their phenolic equivalents, the investigated aromatic amines were not converted into the expected cyclic dimeric structures under T. versicolor catalysis. The formation of complex oligomeric/polymeric or decomposition by-products was mainly observed, with the exception of the isolation of two interesting but unexpected chemical skeletons. Specifically, the biooxidation of diphenylamine resulted in an oxygenated quinone-like product, while, to our surprise, in the presence of T. versicolor laccase (E)-4-vinyl aniline was converted into a 1,2-substited cyclobutane ring. To the best of our knowledge, this is the first example of an enzymatically triggered [2 + 2] olefin cycloaddition. Possible reaction mechanisms to explain the formation of these products are also reported.

Microbial transformations of the taxan ring of 10-DAB by some strains of the fungus Curvularia lunata: formation of the bis-abeotaxanes wallifoliol and 4-deacylwallifoliol.

The fermentation of 10-deacetylbaccatine III (10-DAB) (1) with Curvularia lunata afforded 4-deacylwallifoliol (4) and wallifoliol (5), the only natural taxoid with the unusual 5/6/6/6/4 ring system. The X-ray structure of compound 4 is reported. The skeletal rearrangements induced by the microbial enzymatic systems are also discussed.

Validating a strategy for molecular dynamics simulations of cyclodextrin inclusion complexes through single-crystal X-ray and NMR experimental data: a case study.

A theoretical and experimental study about the formation and structure of the inclusion complex (-)-menthyl-O-beta-D-glucopyranoside 1 with beta-cyclodextrin (beta-CD) 2 is presented as paradigmatic case study to test the results of molecular dynamics (MD) simulations. The customary methodological approach-the use of experimental geometrical parameters as restraints for MD runs-is logically reversed and the calculated structures are a posteriori compared with those obtained from NMR spectroscopy in D(2)O solution and single crystal X-ray diffraction so as to validate the simulation procedure. The guest molecule 1 allows for a broad repertoire of intermolecular interactions (dipolar, hydrophobic, hydrogen bonds) concurring to stabilize the host-guest complex, thus providing the general applicability of the simulation procedure to cyclodextrin physical chemistry. Many starting geometries of the host-guest association were chosen, not assuming any a priori inclusion. The simulation protocol, involving energy minimization and MD runs in explicit water, yielded four possible inclusion geometries, ruling out higher-energy outer adducts. By analysis of the average energy at room temperature, the most stable geometry in solution was eventually obtained, while the kinetics of formation showed that it is also kinetically favored. The reliability of such geometry was thoroughly checked against the NOE distances via the pair distribution functions, that is, the statistical distribution of intermolecular distances among selected diagnostic atoms calculated from the MD trajectories at room temperature. An analogous procedure was adopted both with implicit solvent and in vacuo. The most stable geometry matched that found with explicit solvent but major differences were observed in the relative stability of the metastable complexes as a consequence of the lack of hydration on the polar moiety of the guest. Finally, a control set of geometrical parameters of the thermodynamically favored complex matched the corresponding one obtained from the X-ray structure, while local conformational differences were indicative of packing effects.

Anomalous microbial transformations on the taxane ring of 10-DAB by a strain of the fungus Curvularia lunata: transbenzoylation, transacetylation, and opening of the oxetane ring.

The fermentation of 10-deacetylbaccatin III (10-DAB) (1) with Curvularia lunata afforded the taxane hemiacetals 2a and 3, characterized by extensive structural modification, including C-2 to C-1 transbenzoylation, oxidation of the C-2 hydroxyl, formation of a C-9/C13 hemiacetal, epimerization at C-10, and migration of the endocyclic double bond to an exocyclic location. In compound 3, an acetate-assisted opening of the oxetane ring was also observed.

Impurities of tazarotene: isolation and structural characterisation.

Two impurities showing structures 5 and 6 were isolated and characterised by means of NMR analysis, during the optimisation of a synthetic procedure to tazarotene. Impurity 5, i.e. ethyl 6-((4,4-dimethyl-4H-thiochromen-6-yl)ethynyl)nicotinate, was a by-product of the reduction of the intermediate sulfoxide 7 with PCl(3). Impurity 6, i.e. 1,4-bis(4,4-dimethylthiochroman-6-yl)buta-1,3-diyne, was due to a side reaction of the Sonogashira coupling.

Isolation and characterisation of impurities in adapalene.

Three impurities of structure 2-4 were isolated and characterised during the optimisation of a synthetic procedure to adapalene. Impurity 1 was a by-product of the Friedel-Crafts reaction of adamantanol with 4-bromoanisole. Impurities 3 and 4 were due to side reactions of the final Negishi coupling.

Isolation and characterisation of a phenolic impurity in a commercial sample of duloxetine.

A phenolic impurity of duloxetine hydrochloride was isolated and characterised (MS, NMR, X-ray-analysis).

Microbial transformation of spirolaxine, a bioactive undecaketide fungal metabolite from the basidiomycete Sporotrichum laxum.

Incubation of (+)-spirolaxine (= (3R)-5-hydroxy-7-methoxy-3-{5-[(2R,5R,7R)-2-methyl-1,6-dioxaspiro[4.5]dec-7-yl]pentyl}-2-benzofuran-1(3H)-one; 1a) with Bacillus megaterium afforded two new mono- and one new dihydroxylated metabolite(s), all OH groups being introduced on the non-activated six-membered ring. In contrast, exposure of 1a to Cunninghamella echinulata gave rise to hydroxylation on the five-membered ring of the parent structure. The structures and absolute configurations of the new products 1b-e were deduced on the basis of MS and NMR data. The metabolite 1b was investigated, in comparison to 1a, for its cytotoxicity (sulforhodamin-B test) and for its antiproliferative activity towards bovine microvascular endothelial cells (BMEC).

The prediction of isotopic patterns in phenylpropanoids from their precursors and the mechanism of the NIH-shift: basis of the isotopic characteristics of natural aromatic compounds.

The theoretical 2H-distribution in the aromatic ring of phenylpropanoids can be predicted from that of their precursors--erythrose-4-phosphate, phosphoenolpyruvate and NADPH--and by invoking the mechanism of the NIH-shift and implied deuterium isotope effects. For each position in the non-oxygenated ring, the predicted natural 2H-abundance is in excellent agreement with experimental data obtained from quantitative 2H NMR-measurements on natural compounds, especially concerning the relative 2H-abundances p > o > or = m. For the p-hydroxylated derivatives, the experimentally determined 2H-abundance sequence order m > o can also be deduced, assuming an anisotropic migration (intramolecular isotope effect) of the p-hydrogen atom to the two differently 2H-substituted m-positions during the NIH-shift (intramolecular hydrogen transfer) and an in vivo deuterium kinetic isotope effect of approximately 1.20 on the final hydrogen elimination from the proposed ketodiene intermediate. The predicted 2H-distribution pattern of methyl salicylate 10, a representative of an o-hydroxylated natural compound, is in excellent agreement with that reported from 2H NMR analyses. However, for salicyl alcohol, minor differences between the theoretical and experimentally determined pattern are found that cannot yet be satisfactorily explained. On the other hand, a very good agreement is found between the theoretical and experimental pattern of coumarin, provided a deuterium kinetic isotope effect of approximately 1.30 is assumed for the elimination of the H-atoms from the ketodiene intermediate. The secondary m-hydroxylation of p-coumaric acid in the biosynthesis of vanillin seems to proceed without large isotope effects. Parallel differences are also observed for the 18O-kinetic isotope effects on the corresponding monooxygenase-catalysed reactions. The results demonstrate convincingly that the mechanisms of these general reactions of the phenylpropanoid biosynthetic pathway are identical and follow general principles. Small observed differences between the 2H-patterns of individual natural aromatic compounds originating from the same hydroxylation type can therefore be assigned to differences of the patterns of the precursors, the extent and the orientation of the hydrogen migration, and the kinetic isotope effect on the final hydrogen elimination. The evidence for the existence of general systematic rules governing isotopic patterns in the shikimic acid pathway and its subsequent reactions is further supported by the recently reported 13C-distribution pattern of vanillin, which is also in agreement with that predicted from the precursors. Hence, it is apparent that the systematics of the isotope patterns of phenylpropanoids are in line with the generally accepted biosynthetic reactions in the shikimic acid pathway and that this knowledge can strengthen their value as an essential support for the distinction of natural and synthetic aromatic compounds.

Enzyme-mediated preparation of the enantiomerically enriched isomers of the odorous tetrahydropyranyl acetates Jasmal and Jessemal, and their olfactory evaluation.

All four stereoisomers of the fragrance Jasmal of structure 3,4,5,6-tetrahydro-3-pentyl-2H-pyran-4-yl acetate were prepared by enzymatic resolutions of the corresponding alcohols. The absolute configurations were unambiguously determined by comparison with the enantiomer (3R,4S)-1 prepared from L-tartaric acid. The four stereoisomers of the fragrance Jessemal of structure 3-butyl-5-methyl-3,4,5,6-tetrahydro-2H-pyran-4-yl acetate were obtained starting from the epoxy alcohol 10, which was obtained in an optically pure state by enzymatic resolution of the racemic mixture. The olfactory evaluations of all stereoisomers are reported. (1)H-NMR Conformational analysis of diastereoisomers (3RS,4RS,5RS)-2 and (3RS,4SR,5RS)-2 is also reported.

The Co-identity of Lipiarmycin A3 and Tiacumicin B.

The co-identity of the antibiotics lipiarmycin A3 obtained from Actinoplanes deccanensis and tiacumicin B obtained from Dactylosporangium aurantiacum was unambiguously demonstrated through a number of experimental means. Spectroscopic analyses performed on both the antibiotics themselves and on their derivatives showed no difference between the two series of compounds. Moreover, unambiguous confirmation of the postulated identity of the two compounds was achieved by chemical degradation of lipiarmycin A3 and isolation of (3S,4R)-pentane-1,3,4-triol triacetate whose relative configuration was assigned by comparison with the authentic erythro and threo pentane-1,3,4-triol triacetates, obtained by chemical synthesis.

Stable isotope characterization of the ortho-oxygenated phenylpropanoids: coumarin and melilotol.

The natural abundance 2H NMR spectra of extractive coumarin 10 and of its dihydroderivative melilotol 11 produced by baker's yeast reduction has been compared with synthetic materials. Diagnostic for the differentiation of 10 are the (D/H)beta values, which are in the 128.1-133.6 ppm interval for the natural compounds but 258.5 and 189.8 ppm for the synthetic materials. Such a dramatic difference is also found for methyl cinnamate 12, which shows (D/H)beta values of 127.2 and 515.8 ppm, respectively. In extractive 10, the ratio (D/H)4para/(D/H)6ortho = 1.24 is similar to that observed in structurally related salicin and methyl salicylate. Coumarin 10 is transformed in salicyl alcohol 9, providing diacetate 14, showing in the natural series the trend (D/H)3meta > (D/H)4para > (D/H)5meta approximately (D/H)6ortho. A similar trend is shown also by the synthetic 10. A clear distinction between extractive and synthetic 10 is obtained through delta18O determinations on 10 and on chroman 13. The bulk delta18O values in the extractive series of 10 are 20.3, 23.6, and 22.6 per thousand, while those of the aromatic oxygen are 2.3, 0.5, and -0.5 per thousand. In the synthetic sample, the values are 12.6 and 5.6 per thousand, respectively. As a final product, the reduction of 10 leads to the dihydroderivative 11. Both the baker's yeast reduction and the catalytic hydrogenation lead to a marked decrease of the deuterium content of 11, which is stronger for the beta-position than for the alpha-position.

Investigation of the stereochemical course of ene reductase-catalysed reactions by deuterium labelling.

The stereoselective reduction of suitably substituted C═C bonds mediated by enzymes, called ene reductases, has received great attention in the last decade. Some successful applications of this biocatalysed procedure to the synthesis of chiral active pharmaceutical ingredients have been reported in the literature. The generation of suitable models to be used for predicting the stereochemical outcome of this kind of reductions is a challenging task. In the last years we have exploited deuterium labelling to investigate the stereochemical course of the enzyme-mediated reductions of a wide collection of substrates belonging to well-defined chemical classes. The results of this research have allowed us to draw conclusions on the relationship between the structural characteristics of the substrate and the binding mode it adopts in the enzyme active site. The collected data can be exploited to create an empirical model to rationalise and predict the stereoselectivity of old yellow enzyme (OYE)-catalysed reductions.

Penicillin Acylase-Mediated Synthesis of 2-Acetyl-1-pyrroline and of 2-Propionyl-1-pyrroline, Key Roast-Smelling Odorants in Food. Inclusion Complexes with beta-Cyclodextrin and Their NMR and MS Characterization.

The synthesis of the strong natural roast odorants 1 and 2 is achieved from the C-6 isomeric alcohols 16 and 21 via the acetylenic C-6 and C-7 amines 8 and 9. Key step in the process is the hydrolysis of the N-phenylacetamides 12 and 13 by means of immobilized penicillin acylase, which affords the 1-amino-4,5-diketones 14 and 15, spontaneously ring closing to 1 and 2. These latter compounds form inclusion complexes with beta-cyclodextrin, as demonstrated by NMR measurements in deuterated water and FAB-MS spectra.

Preparation of (R)-Fluoropyruvaldehyde N,S-Ketals by Highly Stereospecific Tandem Pummerer Rearrangement/1,2-p-Tolylthio Group Migration of (R)-alpha-(Fluoroalkyl)-beta-sulfinylenamines.

Fluoropyruvaldehyde N,S-ketals (R)-2 have been prepared in good yields (up to 88%) and ee (up to 79%) from alpha-(fluoroalkyl)-beta-sulfinylenamines (R)-(Z)-1, through a new self-immolative tandem sequential process, consisting of a Pummerer reaction, promoted by trifluoroacetic anhydride, followed by a 1,2-migration of the p-tolylthio group, triggered by addition of silica gel or aqueous base. Each transfer of stereogenic center, from sulfur to the alpha-carbon and then to the beta-carbon, occurs with an average degree of enantioselectivity up to 94:6. Cis geometry between the sulfinyl and the amino groups of the starting enamine (R)-1 is necessary for achieving high level of stereocontrol, since neighboring group participation by the N-Cbz amino group prevents the sulfinyl center from racemization promoted by trifluoroacetic anhydride. NMR studies have shown that imines 3 are intermediate products of the Pummerer rearrangement, which are stable in the reaction environment.

Crystal architecture and conformational properties of the inclusion complex, neohesperidin dihydrochalcone-cyclomaltoheptaose (beta-cyclodextrin), by X-ray diffraction.

The crystal structure of the host-guest noncovalent complex of cyclomaltoheptaose (beta-cyclodextrin, betaCD) with the O-diglycosyl flavonoid neohesperidin dihydrochalcone [(3,5-dihydroxy-4-(3-hydroxy-4-methoxyhydrocinnamoyl)phenyl-2-O-(alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside, NDC] has been determined from single-crystal X-ray diffraction data collected at low temperature (130 K), using synchrotron radiation. The crystal data are as follows: a =15.125(5), b =30.523(5), c =41.332(5) Angstroms, orthorhombic, space group C222(1). The structure contains 19 molecules of water, of which 11 appeared well positioned, whereas 9 are disordered over 23-positions. The betaCD-NDC complex is characterized by one aromatic part of NDC deeply inserted into the hydrophobic cavity of the betaCD through the primary OH rim, and it is present in the crystal as a dimer. The dimeric units, formed by head-to-head assemblies of CD molecules, each with its guest, are self-assembled in columns. The stability of the columns is provided by host-guest and guest-guest attractive interactions, thus showing a key role of the guest molecules in the crystal architecture. The guest conformation in the complex is different from that reported in the literature for uncomplexed NDC. The host-induced conformational changes on NDC provide the optimum geometry requirements for the assembly of the dimeric units.

Differentiation of natural and synthetic phenylalanine and tyrosine through natural abundance 2H nuclear magnetic resonance.

The natural abundance deuterium NMR characterization of samples of the amino acids tyrosine (1) and phenylalanine (2), examined as the acetylated methyl esters 4 and 6, has been performed with the aim to identify by these means the contribution in animals of the hydroxylation of the diet l-phenylalanine (2) to the formation of l-tyrosine (1), a feature previously revealed on the same samples through the determination of the phenolic delta(18)O values. The study, which includes also the NMR examination of benzoic acid (5) from 2 and of tyrosol (7) from 1, substantially fails in providing the required information because the mode of deuterium labeling of tyrosine samples of different origins is quite similar but indicates a dramatic difference in the deuterium labeling pattern of the two amino acids 1 and 2. The most relevant variation is with regard to the deuterium enrichments at the CH(2) and CH positions, which are inverted in the two amino acids of natural derivation. Moreover, whereas the diastereotopic benzylic hydrogen atoms of l-tyrosine (1) appear to be equally deuterium enriched, in l-phenylalanine (2) the (D/H)(3)(R)() > (D/H)(3)(S)(). Similarly, benzoic acid (5) shows separate signals for the aromatic deuterium nuclei, which are quite indicative of the natural or synthetic derivation. The mode of deuterium labeling of the side chain of 1 and 2 is tentatively correlated to the different origins of the two amino acids, natural from animal sources for l-tyrosine and biotechnological probably from genetically modified microorganisms for l-phenylalanine.

Stable isotope labeling pattern of resveratrol and related natural stilbenes.

The stable isotope characterization of resveratrol 1 from Polygonum cuspidatum and of related natural stilbenes 11 and 12 obtained by hydrolysis of the corresponding glucosides 2 and 3 from Rheum is reported. The C(6)-C(2)-C(6) framework of suitably protected derivatives of 1, 2, and 3 has been degraded with ozone to the C(6)-C(1) aldehydes 4, 5, 9, and 10, retaining all hydrogen atoms of the precursors. The natural and synthetic derivatives are characterized and distinguished by natural abundance deuterium nuclear magnetic resonance studies. In the case of anisaldehyde 4 the two series show, as expected, the characteristic difference of the aromatic labeling. The formyl deuterium contents of 4 and 5 from resveratrol are remarkably different, seemingly reflecting the different enrichments existing between positions 3 and 2, respectively, of the phenylpropanoid precursor. The positional delta(18)O values of the extractive materials 1-3 were also determined. In this instance a selective deoxygenation procedure was adopted, leading from 1 to the products 6, 7, and 8. The delta(18)O values of the latter compounds reveal, respectively, those at position 4' and positions 3 and 5 of 1. Similarly, the phenolic products 11 and 12 were converted into 13 and 14. From the delta(18)O values of the single components it is possible to design a detailed map of the oxygen fractionations which characterizes the stilbenes 1-3. In particular, the oxygen present at position 4' of the phenylpropanoid moiety of 1-3 shows delta(18)O values of +11.5, +1.8, and +6.7 per thousand, respectively. Moreover, the phenolic oxygen atom at position 3' of rhapontin 3 shows a value of +11.7 per thousand. The data are compared with those previously obtained on structurally related compounds. These results show the utility of simple chemical degradations in the stable isotope characterization of structurally complex food components.

Differentiation of extractive and synthetic salicin. The 2H aromatic pattern of natural 2-hydroxybenzyl alcohol.

The natural abundance deuterium NMR characterization of four commercially available samples (Kahlbaum, Aldrich, Fluka, and Extrasynthese) of salicin 1 in comparison with two extractive samples from Salix humboldiana and Salix purpurea L. and with a synthetic material, performed on the pentaacetate derivative 2 and on diacetyl salicyl alcohol 4, is reported. Product 2 from S. humboldiana and the sample from salicin Kahlbaum show mean (D/H)aromatic values of 117 and 121 ppm, whereas, for the remaining, values of 146, 154, 153, and 150 ppm are observed, thus suggesting that salicin Kahlbaum is from extractive origin. The (D/H) values at positions 5' and 6' of the sugar moiety suggest a hypothesis on the origin of the glucose residue discriminating between those deriving from C3 or C4 plants. The analysis of 4, obtained from 3, formed in the beta-glucosidase hydrolysis of salicin 1, reveals in the natural samples from S. purpurea and from Kahlbaum the trend (D/H)4(para) > (DH)3(meta) approximately (D/H)5(meta) > (D/H)6(ortho), the first example of deuterium pattern of an ortho-oxygen-substituted phenylpropanoid. The three samples derived from commercial 1 (Aldrich, Fluka, and Extrasynthese) and the synthetic sample show almost identical deuterium content at positions 4 and 6 (around 153 ppm), whereas for the two meta positions (D/H)3 > (D/H)5 (ca. 162 and 140 ppm, respectively). Product 4, obtained from 3 submitted to acid-catalyzed deuteration, shows different deuterium incorporations in the two meta positions (which are ortho/para to the activating phenolic hydroxyl group), suggesting that possibly the deuterium abundance at the two meta positions may be affected by exchange phenomena with the medium.

The co-identity of lipiarmycin A3 and tiacumicin B.

The co-identity of the antibiotics lipiarmycin A3 obtained from Actinoplanes deccanensis and tiacumicin B obtained from Dactylosporangium aurantiacum was unambiguously demonstrated through a number of experimental means. Spectroscopic analyses performed on both the antibiotics themselves and on their derivatives showed no difference between the two series of compounds. Moreover, unambiguous confirmation of the postulated identity of the two compounds was achieved by chemical degradation of lipiarmycin A3 and isolation of (3S,4R)-pentane-1,3,4-triol triacetate whose relative configuration was assigned by comparison with the authentic erythro and threo pentane-1,3,4-triol triacetates, obtained by chemical synthesis.