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The direct and unambiguous detection and identification of individual metabolite molecules present in complex biological mixtures constitute a major challenge in (bio)analytical research. In this context, nuclear magnetic resonance (NMR) spectroscopy has proven to be particularly powerful owing to its ability to provide both qualitative and quantitative atomic-level information on multiple analytes simultaneously in a noninvasive manner. Nevertheless, NMR suffers from a low inherent sensitivity and, moreover, lacks selectivity regarding the number of individual analytes to be studied in a mixture of a myriad of structurally and chemically very different molecules, e.g., metabolites in a biofluid. Here, we describe a method that circumvents these shortcomings via performing selective, photochemically induced dynamic nuclear polarization (photo-CIDNP) enhanced NMR spectroscopy on unmodified complex biological mixtures, i.e., human urine and serum, which yields a single, background-free one-dimensional NMR spectrum. In doing this, we demonstrate that photo-CIDNP experiments on unmodified complex mixtures of biological origin are feasible, can be performed straightforwardly in the native aqueous medium at physiological metabolite concentrations, and act as a spectral filter, facilitating the analysis of NMR spectra of complex biofluids. Due to its noninvasive nature, the method is fully compatible with state-of-the-art metabolomic protocols providing direct spectroscopic information on a small, carefully selected subset of clinically relevant metabolites. We anticipate that this approach, which, in addition, can be combined with existing high-throughput/high-sensitivity NMR methodology, holds great promise for further in-depth studies and development for use in metabolomics and many other areas of analytical research.

Herein, we perform for the first time a preliminary NMR and computational study of the spiroglycol structure. Spiroglycol is a highly symmetrical molecule, but it should be chiral due to the presence of a chiral axis. The presence of two enantiomers was demonstrated performing NMR enantiodifferentiation experiments using α,α′-bis(trifluoromethyl)-9,10-anthracenedimethanol (ABTE) as a chiral solvating agent (CSA). The addition of 0.6 equiv of ABTE allows the differentiation of several spiroglycol proton signals. The lack of resolution observed in the proton spectrum can be tackled through the corresponding 13C NMR spectrum where a significant enantiodifferentiation at the spirocarbon atom was observed. In order to physically separate both enantiomers, a SPG derivatization with camphorsulfonic acid and Mosher’s acid was performed affording the corresponding diastereoisomeric ester mixtures. Computations performed with the Gaussian16 package showed that the enantiodifferentiation is mainly due to the different compound thermodynamics stability.

The detection of ultra-long-range (4JCHand higher) heteronuclear connectivities can complement the conventional use of HMBC/HSQMBC data in structure elucidation NMR studies of proton-deficient natural products, where two-bond and three-bond correlations are usually observed. The performance of the selHSQMBC experiment with respect to its broadband HSQMBC counterpart is evaluated. Despite its frequency-selectivity nature, selHSQMBC efficiently prevents any unwanted signal phase and intensity modulations due to passive proton–proton coupling constants typically involved in HSQMBC. As a result, selHSQMBC offers a significant sensitivity enhancement and provides pure in-phase multiplets, improving the detection levels for short- and long-range cross-peaks corresponding to small heteronuclear coupling values. This is particularly relevant for experiments optimized to small nJCHvalues (2–3 Hz), referred to as LR-selHSQMBC, where key cross-peaks that are not visible in the equivalent broadband LR-HSQMBC spectrum can become observable in optimum conditions.

The congested nature of quaternary carbons hinders their preparation, most notably when stereocontrol is required. Here we report a biocatalytic method for the creation of quaternary carbon centers with broad substrate scope, leading to different compound classes bearing this structural feature. The key step comprises the aldol addition of 3,3-disubstituted 2-oxoacids to aldehydes catalyzed by metal dependent 3-methyl-2-oxobutanoate hydroxymethyltransferase from E. coli(KPHMT) and variants thereof. The 3,3,3-trisubstituted 2-oxoacids thus produced were converted into 2-oxolactones and 3-hydroxy acids and directly to ulosonic acid derivatives, all bearing gem-dialkyl, gem-cycloalkyl, and spirocyclic quaternary centers. In addition, some of these reactions use a single enantiomer from racemic nucleophiles to afford stereopure quaternary carbons. The notable substrate tolerance and stereocontrol of these enzymes are indicative of their potential for the synthesis of structurally intricate molecules.

The encapsulation of coordination complexes in a tetragonal prismatic nanocage (1·(BArF)8) built from Zn-porphyrin and macrocyclic Pd-clip-based synthons is described. The functional duality of the guest ligand L1allows for its encapsulation inside the cage 1·(BArF)8, along with the simultaneous coordination of ZnII, CuII, or FeIIImetal ions. Remarkably, the coordination chemistry inside the host–guest adduct L1⊂1·(BArF)8occurs in both solution solution and solid state. The resulting confined metallocomplexes have been characterized by means of UV-vis, ESI-HRMS, NMR, and EPR techniques. Furthermore, the emission of the Zn-porphyrin fluorophores of 1·(BArF)8is strongly quenched by the encapsulation of paramagnetic complexes, representing a remarkable example of guest-dependent tuning of the host fluorescence.

Spin crossover (SCO) complexes, through their reversible spin transition under external stimuli, can work as switchable memory materials. Here, we present a protocol for the synthesis and characterization of a specific polyanionic iron SCO complex and its diluted systems. We describe steps for its synthesis and the determination of crystallographic structure of the SCO complex in diluted systems. We then detail a range of spectroscopic and magnetic techniques employed to monitor the spin state of the SCO complex in both diluted solid- and liquid-state systems.

Polyanionic and electroactive hybrids based on octasilsesquioxanes bearing metallacarborane units are developed. They show remarkable solubility in organic solvents and outstanding thermal stability. The metallacarboranes act as independent units simultaneously undergoing the reversible redox process.

Heteronuclear long-range NMR experiments are well established as essential NMR techniques for the structure elucidation of unknown natural products and small molecules. It is generally accepted that the absence of a given nJXHcorrelation in an HMBC or HSQMBC spectrum would automatically place the proton at least four bonds away from the carbon in question. This assumption can, however, be misleading in the case of a mismatch between the actual coupling constant and the delay used to optimize the experiment, which can lead to structural misassignments. Another scenario arises when an investigator, for whatever reason, needs to have access to very long-range correlations to confirm or refute a structure. In such cases, a conventional HMBC experiment will most likely fail to provide the requisite correlation, regardless of the delay optimization. Two recent methods for visualizing extremely weak or very long-range connectivities are the LR-HSQMBC and the HSQMBC-TOCSY experiments. Although they are intended to provide similar structural information, they utilize different transfer mechanisms, which differentiates the experiments, making each better suited for specific classes of compounds. In this report we have sought to examine the considerations implicit in choosing the best experiment to access weak or very long-range correlations for different types of molecules.

A recently developed NMR method to simultaneously obtain both long-range heteronuclear correlations and carbon multiplicity information in a single experiment, ME-selHSQMBC, is demonstrated as a potentially useful technique for chemical shift assignment and structure elucidation of natural products presenting complicated NMR spectra. Carbon multiplicities, even for C/CH2and odd for CH/CH3resonances, can be distinguished directly from the relative positive/negative phase of cross-peaks. In addition, connectivity networks can be further extended by incorporating a TOCSY propagation step. Staurosporine (1) and sungucine (2) are utilized as model compounds to demonstrate these techniques.

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Two silylated Pd–NHC complexes were immobilized on hybrid silicas by sol‐gel cocondensation with tetraethyl orthosilicate (TEOS) and performed well as recyclable catalysts towards the Heck, Suzuki, and Sonogashira coupling reactions. Remarkable conversion and recyclability were achieved in the Suzuki reaction with a challenging aryl chloride. No side products and no undesired homocoupling were observed in Suzuki or Heck reactions, which facilitates the final purification step for the cross‐coupling products. High turnover numbers and turnover frequencies were found for copper‐ and phosphane‐free Sonogashira reaction between p‐bromoacetophenone and phenylacetylene.

Simply successful: A proton‐selective HSQMBC‐TOCSY experiment can be used to measure small proton–carbon (nJCH; n>1) coupling constants on both protonated and non‐protonated carbon atoms (see spectrum). The method combines in a single pulse scheme all the benefits of the widely used HSQMBC and HSQC‐TOCSY experiments. The magnitude and the sign of nJCHcan be determined simply with excellent accuracy.

The first synthetic approach to the entire carbon skeleton of the sesquiterpenes dunniane and cumacrene has been developed. The sequence features a [2+2] photochemical reaction of a chiral 2(5H)‐furanone with ethylene to form the cyclobutane, a Meyer–Schuster‐type rearrangement of an α‐acetylenic alcohol to an α,β‐unsaturated aldehyde, and a microwave‐assisted Diels–Alder reaction of an elaborated dienamine with methyl acrylate to construct the six‐membered ring with the desired 1,4‐substitution pattern.

N‐Tosyl‐linked open‐chain yne‐ene‐yne enediynes 1and 2and yne‐yne‐ene enediynes 3and 4have been satisfactorily synthesised. The [2+2+2] cycloaddition process catalysed by the Wilkinson catalyst [RhCl(PPh3)3] was tested with the above‐mentioned substrates resulting in the production of high yields of the cycloadducts. Enediynes 1and 2gave standard [2+2+2] cycloaddition reactions whereas enediynes 3and 4suffered β‐hydride elimination followed by reductive elimination of the Wilkinson catalyst to give cycloadducts, which are isomers of those that would be obtained by standard [2+2+2] cycloaddition reactions. The different reactivities of these two types of enediyne have been rationalised by density functional theory calculations.

The palladium‐catalyzed Matsuda–Heck and Suzuki–Miyaura cross‐couplings of 2‐methoxycarbonylthiophene‐3‐diazonium tetrafluoroborate 1were performed to synthesize a series of thiophene derivatives functionalized in the β‐position. Good to excellent yields of the cross‐coupling products were obtained using palladium acetate [Pd(OAc)2], a ligandless palladium catalyst, without a base, at room temperature, under aerobic conditions and with short reaction times. A diazotization and cross‐coupling sequence can also be performed in a one‐pot process avoiding the isolation of the thiophenediazonium salt derivative. In addition, 2‐methoxycarbonylbenzo[b]thiophene‐3‐diazonium tetrafluoroborate 8was also efficiently arylated by applying the same Suzuki–Miyaura optimized reaction conditions.

A new ruthenium aquo catalyst with the formula trans‐[Ru(II)(CN‐Me)(trpy)OH2](PF6)2[where trpy=2,2′:6′,2′′‐terpyridine and CN‐Me=3‐methyl‐1‐(pyridin‐2‐yl)‐imidazolylidene] has been prepared and thoroughly characterized by spectroscopic and electrochemical techniques. The complex has been tested in epoxidation catalysis both in dichloromethane and dichloromethane:ionic liquid media, displaying excellent performances and selectivities. Reuse of the catalyst in ionic liquid:solvent media has been explored for the first time in ruthenium‐mediated epoxidation catalysis and its performance is fully maintained for up to ten runs.

The aldol addition of unphosphorylated dihydroxyacetone (DHA) to aldehydes catalyzed by L‐rhamnulose‐1‐phosphate aldolase (RhuA), a dihydroxyacetone phosphate‐dependent aldolase, is reported. Moreover, a single point mutation in the phosphate binding site of the RhuA wild type, that is, substitution of aspartate for asparagine at position N29, increased by 3‐fold the ${V{{app\hfill \atop {\rm max}\hfill}}}$of aldol addition reactions of DHA to other aldehyde acceptors rather than the natural L‐lactaldehyde. The RhuA N29D mutant modified the optimum enzyme design for the natural substrate and changed its catalytic properties making the aldolase more versatile to other aldol additions of DHA.

A minimalist active site redesign of the L‐fuculose‐1‐phosphate aldolase from E. coliFucA was envisaged, to extend its tolerance towards bulky and conformationally restricted N‐Cbz‐amino aldehyde acceptor substrates (Cbz=benzyloxycarbonyl). Various mutants at the active site of the FucA wild type were obtained and screened with seven sterically demanding N‐Cbz‐amino aldehydes including N‐Cbz‐prolinal derivatives. FucA F131A showed an aldol activity of 62 μmol h−1mg−1with (R)‐N‐Cbz‐prolinal, whereas no detectable activity was observed with the FucA wild type. For the other substrates, the F131A mutant gave aldol activities from 4 to about 25 times higher than those observed with the FucA wild type. With regard to the stereochemistry of the reactions, the (R)‐amino aldehydes gave exclusively the anticonfigured aldol adducts whereas their Scounterparts gave variable ratios of anti/syndiastereoisomers. Interestingly, the F131A mutant was highly stereoselective both with (R)‐ and with (S)‐N‐Cbz‐prolinal, exclusively producing the antiand synaldol adducts, respectively. Molecular models suggest that this improved activity towards bulky and more rigid substrates, such as N‐Cbz‐prolinal, could arise from a better fit of the substrate into the hydrophobic pocket created by the F131A mutation, due to an additional π–cation interaction with the residue K205′ and to efficient contact between the substrate and the mechanistically important Y113′ and Y209′ residues. An expedient synthesis of novel polyhydroxylated pyrrolizidines related to the hyacinthacine and alexine types was accomplished through aldol additions of dihydroxyacetone phosphate (DHAP) to hydroxyprolinal derivatives with the hyperactive FucA F131A as catalyst. The iminocyclitols obtained were fully characterised and found to be moderate to weak inhibitors (relative to 1,4‐dideoxy‐1,4‐imino‐L‐arabinitol (LAB) and 1,4‐dideoxy‐1,4‐imino‐D‐arabinitol (DAB)) against glycosidases and rat intestinal saccharidases.

RhodiumIcatalyzed [2+2+2] cycloaddition reactions of Ntosyl, carbon, and oxygentethered cyanodiynes in a completely intramolecular fashion have been optimized to afford highly functionalized pyridines by conventional andor microwave heating. Microwaves have been shown to enhance the process by allowing the reaction to be conducted effectively in short reaction times. The methodology has been extended for the synthesis of bipyridines, either by treating a cyanodiyne with an appended pyridine or by conducting a double [2+2+2] cycloaddition reaction on a dicyanotetrayne scaffold. The choice of the solvent in the microwave heating reaction has been shown to be crucial for the success of the process.

The synthesis, electrochemical, optical, and cation‐sensing properties of [2.2]ferrocenophanes, in which the two ferrocene subunits are linked through two aldiminic or iminophosphorane moieties, are reported. The new compounds show remarkably selective cation‐sensing properties due to the presence of redox‐active units (ferrocene) and aza‐unsaturated functionalities that are able to act as putative cation‐binding sites. In this structural motif, the aldimine groups act as a highly selective binding site for Zn2+cations, whereas the iminophosphorane bridges display an unusually strong binding affinity towards Li+cations, which could be explained by an additional Li⋅⋅⋅Fe interaction. The X‐ray structure of the complex 4⋅Li+as well as detailed NMR spectroscopic studies, both in solution and in the solid state, support this assessment. Experimental data and conclusions about the cation‐sensing capabilities of this family of compounds are supported by DFT calculations.

Details on the implementation of the simultaneous evolution of multiple frequencies into the same time period of a NMR pulse sequence are introduced. Timeshared TS versions of the most useful inverse 2D NMR experiments TSHMBC, TSHSQC, TSHSQCTOCSY, and TSHSQMBC are presented and illustrated for simultaneous acquisition of 1H13C and 1H15N NMR spectra. The major benefits associated to the parallel acquisition of multiple spectra from a single NMR experiment are spectrometer time savings and the achievement of multiple and complementary information with improved sensitivity gains per time unit. © 2010 Wiley Periodicals, Inc. Concepts Magn Reson Part A 36A: 1–23, 2010.

The chemoenzymatic synthesis of a collection of pyrrolidine‐type iminosugars generated by the aldol addition of dihydroxyacetone phosphate (DHAP) to C‐α‐substituted N‐Cbz‐2‐aminoaldehydes derivatives, catalyzed by DHAP aldolases is reported. L‐Fuculose‐1‐phosphate aldolase (FucA) and L‐rhamnulose‐1‐phosphate aldolase (RhuA) from E. coliwere used as biocatalysts to generate configurational diversity on the iminosugars. Alkyl linear substitutions at C‐α were well tolerated by FucA catalyst (i.e., 40–70 % conversions to aldol adduct), whereas no product was observed with C‐α‐alkyl branched substitutions, except for dimethyl and benzyl substitutions (20 %). RhuA was the most versatile biocatalyst: C‐α‐alkyl linear groups gave the highest conversions to aldol adducts (60–99 %), while the C‐α‐alkyl branched ones gave moderate to good conversions (50–80 %), with the exception of dimethyl and benzyl substituents (20 %). FucA was the most stereoselective biocatalyst (90–100 % anti(3R,4R) adduct). RhuA was highly stereoselective with (S)‐N‐Cbz‐2‐aminoaldehydes (90–100 % syn(i.e., 3R,4S) adduct), whereas those with Rconfiguration gave mixtures of anti/synadducts. For iPr and iBu substituents, RhuA furnished the antiadduct (i.e., FucA stereochemistry) with high stereoselectivity. Molecular models of aldol products with iPr and iBu substituents and as complexes with the RhuA active site suggest that the antiadducts could be kinetically preferred, while the synadducts would be the equilibrium products. The polyhydroxylated pyrrolidines generated were tested as inhibitors against seven glycosidases. Among them, good inhibitors of α‐L‐fucosidase (IC50=1–20 μM), moderate of α‐L‐rhamnosidase (IC50=7–150 μM), and weak of α‐D‐mannosidase (IC50=80–400 μM) were identified. The apparent inhibition constant values (Ki) were calculated for the most relevant inhibitors and computational docking studies were performed to understand both their binding capacity and the mode of interaction with the glycosidases.

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Number of members makes a difference: The [2+2+2] intramolecular cyclotrimerisation of a new series of 20‐ and 25‐membered azamacrocycles catalysed by the Wilkinson's catalyst are reported (see scheme). The 20‐ and 25‐membered azamacrocycles show different reactivity. Why? Theoretical calculations give insight into the reactivity differences observed for the 20‐ and 25‐membered macrocycles.

D‐Fructose‐6‐phosphate aldolase (FSA) is a key biocatalystfor the alternative synthetic construction of biologically active products with known therapeutic and research interest or novel structures relevant to drug discovery. Novel aldol addition reactions of dihydroxyacetone and hydroxyacetone to a variety of aldehydes catalyzed by FSA are presented (see scheme).

A novel serine hydroxymethyl transferase from Streptococcus thermophilus(SHMT) and a L‐threonine aldolase from Escherichia coli(LTA) were used as stereocomplementary biocatalysts for the aldol addition of glycine to N‐Cbz amino aldehydes and benzyloxyacetaldehyde (Cbz=benzyloxycarbonyl). Both threonine aldolases were classified as low‐specific L‐allo‐threonine aldolases, and by manipulating reaction parameters, such as temperature, glycine concentration, and reaction media, SHMT yielded exclusively L‐erythrodiastereomers in 34–60 % conversion, whereas LTA gave L‐threodiastereomers in 30:70 to 16:84 diastereomeric ratios and with 40–68 % conversion to product. SHMT is among the most stereoselective L‐threonine aldolases described. This is due, among other things, to its activity–temperature dependence: at 4 °C SHMT has high synthetic activity but negligible retroaldol activity on L‐threonine. Thus, the kinetic L‐erythroisomer was largely favored and the reactions were virtually irreversible, highly stereoselective, and in turn, gave excellent conversion. It was also found that treatment of the prepared N‐Cbz‐γ‐amino‐β‐hydroxy‐α‐amino acid derivatives with potassium hydroxide (1m) resulted in the spontaneous formation of 2‐oxazolidinone derivatives of the β‐hydroxyl and γ‐amino groups in quantitative yield. This reaction might be useful for further chemical manipulations of the products.

The Rh-catalysed hydroformylation of isopropylidenecyclohexane derivatives obtained by intramolecular diene cyclisations affords the corresponding aldehydes in good yields and in a completely chemo- and regioselective manner. Diastereoselectivities of ca. 90 % were achieved for all substrates when a bulky phosphite was used as the Rh ligand. Thestereochemical outcomes of the hydroformylation reactions were established by detailed NMR studies. The olfactory evaluation of the different aldehydes is also presented.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

The new chromophore-quencher tricarbonylrhenium(I) complexes [Re(4,4′-X2-bpy)(CO)3(PCA)]+, [(4,4′-X2-bpy)(CO)3Re(μ-PCA)Re(CO)3(4,4′-X2-bpy)]2+, and [(4,4′-X2-bpy)(CO)3ReI(μ-PCA)RuII(NH3)5]3+ (X = Ph or CO2Me and PCA = 4-pyridinealdazine) have been synthesized as their PF6– salts and characterized by spectroscopic, electrochemical, and photophysical techniques. In contrast to previously reported species with X = Me or H, these complexes emit at room temperature in CH3CN. The recovery of luminescence can thus be ascribed to the change of energy levels induced by adding electron-withdrawing substituents to the 2,2′-bipyridine ring, since the emissive ReII(X2bpy–) excited state becomes much lower in energy than the ReII(PCA–) non-emissive excited state. For the mononuclear and symmetric dinuclear rhenium(I) complexes with X = CO2Me, consecutive protonation of both the pyridinyl and imine N atoms of PCA in aqueous solution leads to unusual bell-shaped curves for both the absorption and the emission intensities vs. pH because of opposite effects on the electronic delocalization of PCA. This latter property can be employed to devise novel luminescent pH sensors of the on-off-on type. The corresponding unsymmetrical dinuclear complexes [(4,4′-X2-bpy)(CO)3ReI(μ-PCA)RuIII(NH3)5]4+ have been obtained in situ by oxidizing the [ReI,RuII] precursors in CH3CN solution. Spectral and electrochemical measurements with these complexes lead to the values of the metal-to-metal electronic coupling elements.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

A novel straightforward chemoenzymatic procedure for the synthesis of hyacinthacine stereoisomers based on the aldol addition of dihydroxyacetone phosphate (DHAP) to N-Cbz-prolinal under catalysis by L-rhamnulose 1-phosphate aldolase from E. coli is presented. The synthesis is complemented by a simple and effective purification protocol consisting of ion-exchange chromatography on CM-sepharose. As examples, (−)-hyacinthacine A2 [the enantiomer of (+)-hyacinthacine A2], 7-deoxy-2-epialexine (the enantiomer of 3-epihyacinthacine A2), ent-7-deoxyalexine (the enantiomer of 7-deoxyalexine) and 2-epihyacinthacine A2 were synthesized by these procedures and characterized for the first time. These new isomers were assayed as inhibitors of glycosidases. As a result, (−)-hyacinthacine A2 demonstrated to be a good inhibitor of α-D-glucosidase from rice whereas the natural enantiomer, hyacinthacine A2, was not. Moreover, a new family of inhibitors of α-L-rhamnosidase was uncovered.

Peptidic self-assembled nanostructures are said to have a wide range of applications in nanotechnology, yet the mechanistic details of hierarchical self-assembly are still poorly understood. The Phe-Phe recognition motif of the Alzheimer’s Aβ peptide is the smallest peptide able to assemble into higher-order structures. Here, we show that the Ile-Phe dipeptide analog is also able to self-associate in aqueous solution as a transparent, thermoreversible gel formed by a network of fibrillar nanostructures that exhibit strong birefringence upon Congo red binding. Besides, a second dipeptide Val-Phe, differing only in a methyl group from the former, is unable to self-assemble. The detailed analysis of the differential polymeric behavior of these closely related molecules provides insight into the forces triggering the first steps in self-assembly processes such as amyloid formation.

Heteronuclear coherence transfer in liquid-state NMR applications has been traditionally performed using pulse-interrupted delay schemes such as INEPT-type pulse trains. So far, the alternative use of heteronuclear crosspolarization (HCP) has only been limited to a few cases involving exclusively in-phase to in-phase transfers. In this revision work a theoretical description on the effect and the characteristic anisotropic features of HCP is introduced in terms of product operator formalism. A very intuitive and simple graphical black-box approach based on a pictorial nonclassical vector representation that only consider the available input/output magnetization is also presented to understand the general transformations that are undergoing under such rather complex HCP processes. The appropriate manipulation of magnetization components during the HCP block offers novel concepts in pulse sequence design. In this way, new liquid- state multidimensional NMR methods incorporating HCP-driven processes have recently been developed and successfully applied for both small-to-medium-sized molecules and large labeled bio-molecules, as will be discussed in this review.

The biocatalytic synthesis and purification of O-β-D-monoglucuronide conjugates of hydroxytyrosol, tyrosol, homovanillic alcohol, and 3-(4′-hydroxyphenyl)propanol, using porcine liver microsomes, are described here. The glucuronides were synthesized, analyzed and separated by HPLC-UV, identified by HPLC-MS, and their structures unequivocally established by NMR techniques. The outcome of the glucuronidation reaction depends on the structure of the phenolic compounds. Thus, the glucuronidation of hydroxytyrosol, biocatalyzed with porcine liver microsomes, proceeded exclusively on the phenolic hydroxy groups. The regioselectivity was similar to that observed for human and rat liver microsomes, the 4′-hydroxy position being more favorable than the 3′-hydroxy one. In the case of tyrosol, homovanillic alcohol, and hydroxyphenylpropanol, two products were formed during microsomal glucuronidation: a major one, the phenolic O-β-D-glucuronidated derivative and, a minor one, the O-β-D-glucuronidated aliphatic alcohol.

Abstract: We tested in the field the hypothesis that the specialist butterfly Euphydryas aurinia (Lepidoptera: Nymphalidae, Melitaeinae) lays eggs on leaves of Lonicera implexa (Caprifoliaceae) plants with greater iridoid concentrations. We conducted our investigations in a Mediterranean site by analyzing leaves with and without naturally laid egg clusters. There were no significant differences in iridoid glycoside concentrations between leaves from plants that did not receive eggs and the unused leaves from plants receiving eggs, a fact that would seem to indicate that E. aurinia butterflies do not choose plants for oviposition by their iridoid content. However, the leaves of L. implexa that bore egg clusters had dramatically greater (over 15-fold) concentrations of iridoid glycosides than the directly opposite leaves on the same plant. These huge foliar concentrations of iridoids (15% leaf dry weight) may provide specialist herbivores with compounds that they either sequester for their own defense or use as a means of avoiding competition for food from generalist herbivores. Nevertheless, it may still be possible that these high concentrations are detrimental to the herbivore, even if the herbivore is a specialist feeder on the plant.

Mycobacterium vaccaeis of major pharmaceutical interest as an immunotherapeutic agent. Although M. vaccae15483 ATCCTstrain displays smooth and rough colonial morphologies on solid culture media, it is not known in which conditions M. vaccaeswitches from one colonial morphotype to the other or whether there are biological differences, especially immunological, between them. We have found that the change from a smooth to rough stable variant occurs spontaneously at 30 °C. The analysis of the composition of the cell wall in both variants showed that the smooth morphotype presents an extracellular material that has never previously been described and was identified as a long-chain saturated polyester that, interestingly, is not produced by the rough morphotype. Our results also indicate that this compound could be implicated in the spreading ability of smooth colonies. Proliferation, IFN-$$\upgamma$$and IL-12(p40) production by splenocyte cultures was significantly higher in mice immunised with the rough variant compared with those immunised with the smooth one. This latter finding suggests that the different colonial morphology of M. vaccaemay affect the immunomodulatory effects induced from M. vaccaepreparations.Mycobacterium vaccaeis of major pharmaceutical interest as an immunotherapeutic agent. Although M. vaccae15483 ATCCTstrain displays smooth and rough colonial morphologies on solid culture media, it is not known in which conditions M. vaccaeswitches from one colonial morphotype to the other or whether there are biological differences, especially immunological, between them. We have found that the change from a smooth to rough stable variant occurs spontaneously at 30 °C. The analysis of the composition of the cell wall in both variants showed that the smooth morphotype presents an extracellular material that has never previously been described and was identified as a long-chain saturated polyester that, interestingly, is not produced by the rough morphotype. Our results also indicate that this compound could be implicated in the spreading ability of smooth colonies. Proliferation, IFN-$$\upgamma$$and IL-12(p40) production by splenocyte cultures was significantly higher in mice immunised with the rough variant compared with those immunised with the smooth one. This latter finding suggests that the different colonial morphology of M. vaccaemay affect the immunomodulatory effects induced from M. vaccaepreparations.

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A new family of RuIIcomplexes containing the tridentate meridional 2,2′:6′,2′′‐terpyridine (trpy) ligand, a C2‐symmetric didentate chiral oxazolinic ligand 1,2‐bis[4′‐alkyl‐4′,5′‐dihydro‐2′‐oxazolyl]benzene (Phbox‐R, R = Et or iPr), and a monodentate ligand, of general formula [Ru(Y)(trpy)(Phbox‐R)]n+(Y = Cl, H2O, py, MeCN, or 2‐OH‐py (2‐hydroxypyridine)) have been prepared and thoroughly characterized. In the solid state the complexes have been characterized by IR spectroscopy and by X‐ray diffraction analysis in two cases. In solution, UV/Vis, cyclic voltammetry (CV), and one‐dimensional (1D) and two‐dimensional (2D) NMR spectroscopy techniques have been used. We have also performed density functional theory (DFT) calculations with these complexes to interpret and complement experimental results. The oxazolinic ligand Phbox‐R exhibits free rotation along the phenyloxazoline axes. Upon coordination this rotation is restricted by an energy barrier of 26.0 kcal mol−1for the case of [Ru(trpy)(Phbox‐iPr)(MeCN)]2+thus preventing its potential interconversion. Furthermore due to steric effects the two atropisomers differ in energy by 5.7 kcal mol−1and as a consequence only one of them is obtained in the synthesis. Subtle but important structural effects occur upon changing the monodentate ligands that are detected by NMR spectroscopy in solution and interpreted by using their calculated DFT structures.

A stereselective synthesis of 3-oxabicyclo[3.2.0]heptane nucleoside analogues, which were designed as conformational mimics of the anti-HIV agents 2‘,3‘-didehydro-2‘,3‘-dideoxythimidine (stavudine, d4T) and 2‘,3‘-didehydro-2‘,3‘-dideoxyadenosine (d4A), is described. The target compounds were prepared by condensation of a common intermediate bicyclic acetate, derived from a homochiral 2(5H)-furanone, with pyrimidine and purine bases under modified Vorbrüggen conditions. The conformational behavior of the synthesized nucleoside analogues was studied by NMR spectroscopy and X-ray crystallography.

A family of highly stable organometallic CuIIIcomplexes with monoanionic triazamacrocyclic ligands (Li) with general formula [CuLi]+have been prepared and isolated, and their structural, spectroscopic, and redox properties thoroughly investigated. The HLiligands have been designed in order to understand and quantify the electronic effects exerted by electron donor and electron‐withdrawing groups on either the aromatic ring or the central secondary amine or on both. In the solid state the CuIIIcomplexes were mainly characterized by single‐crystal X‐ray diffraction analysis, whereas in solution their structural characterization was mainly based on 1H NMR spectroscopy given the diamagnetic nature of the d8square‐planar CuIIIcomplexes. Cyclic voltammetry together with 1H NMR and UV/Vis spectroscopy have allowed us to quantify the electronic effects exerted by the ligands on the CuIIImetal center. A theoretical analysis of this family of CuIIIcomplexes has also been undertaken by DFT calculations to gain a deeper insight into the electronic structure of these complexes, which has in turn allowed a greater understanding of the nature of the UV/Vis transitions as well as the molecular orbitals involved.

The complete structural analysis of the palladium complexes of the triolefinic macrocycles (E,E,E)‐1,6,11‐tris(arylsulfonyl)‐1,6,11‐triazacyclopentadeca‐3,8,13‐trienes, which featured from three identical to three different aryl groups, was achieved by performing X‐ray diffraction studies, NMR spectroscopy, and other calculations. The stereochemical complexity is determined by the different isomers formed through complexation of the metal to one or other face of each of the three olefins involved. The palladacyclopropane formulation of the palladium–olefin interaction offers a clear picture of the stereogenicity of the olefin carbon atoms that are complexed to the metal. The energetically favorable isomers were identified in the solid‐state and in solution by performing X‐ray diffraction and NMR spectroscopic analysis, respectively.

The potential of L‐fuculose‐1‐phosphate aldolase (FucA) as a catalyst for the asymmetric aldol addition of dihydroxyacetone phosphate (DHAP) to N‐protected amino aldehydes has been investigated. First, the reaction was studied in both emulsion systems and conventional dimethylformamide (DMF)/H2O (1:4 v/v) mixtures. At 100 mMDHAP, compared with the reactions in the DMF/H2O (1:4) mixture, the use of emulsion systems led to two‐ to three‐fold improvements in the conversions of the FucA‐catalyzed reactions. The N‐protected aminopolyols thus obtained were converted to iminocyclitols by reductive amination with Pd/C. This reaction was highly diastereoselective with the exception of the reaction of the aldol adduct formed from (S)‐N‐Cbz‐alaninal, which gave a 55:45 mixture of both epimers. From the stereochemical analysis of the resulting iminocyclitols, it was concluded that the stereoselectivity of the FucA‐catalyzed reaction depended upon the structure of the N‐Cbz‐amino aldehyde acceptor. Whereas the enzymatic aldol reaction with both enantiomers of N‐Cbz‐alaninal exclusively gave the expected 3R,4Rconfiguration, the stereochemistry at the C‐4 position of the major aldol adducts produced in the reactions with N‐Cbz‐glycinal and N‐Cbz‐3‐aminopropanal was inverted to the 3R,4Sconfiguration. The study of the FucA‐catalyzed addition of DHAP to phenylacetaldehyde and benzyloxyacetaldehyde revealed that the 4Rproduct was kinetically favored, but rapidly disappeared in favor of the 4Sdiastereoisomer. Computational models were generated for the situations before and after CC bond formation in the active site of FucA. Moreover, the lowest‐energy conformations of each pair of the resulting epimeric adducts were determined. The data show that the products with a 3R,4Sconfiguration were thermodynamically more stable and, therefore, the major products formed, in agreement with the experimental results.

The syntheses of new mononuclear ruthenium(II) complexes of the type: [Ru(bpy)(L)(tpm)](PF6)2 {tpm = tris(1-pyrazolyl)methane; bpy = 2,2′-bipyridine; L = pz (pyrazine; 1), 4,4′-bpy (4,4′-bipyridine; 2), and bpe [trans-1,2-bis(4-pyridyl)ethylene; 3]} are described, together with their spectroscopic, electrochemical, and photophysical properties. A complete assignment of the NMR resonances of the three species could be made in CD3CN by bidimensional techniques. A fine tuning of the energies of MLCT (metal-to-ligand charge transfer) states in these complexes is disclosed when comparing, in CH3CN, the values of their maximum absorption wavelengths for the most intense visible bands (λmax) and their redox potentials for the RuIII/RuII couples; this effect, relevant to the design of efficient photocatalysts, can be attributed to a decreasing order of dπ(Ru) → π*(2,2′-bpy) backbonding when decreasing the distance between both N atoms in the aromatic nitrogen heterocycle L that acts in a monodentate manner. Only the species with L = bpe emits at room temperature, pointing to the conclusion that MLCT excited states in this series become higher in energy than ddexcited states when the value of λmax is lower than 400 nm. These species are also useful building blocks for new dinuclear mixed-valent complexes. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

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The synthesis of a new Ru−H2O complex [Ru(phen)(H2O)([9]aneS3)](ClO4)2, 2, (phen = 1,10-phenanthroline, [9]aneS3 = 1,4,7-trithiacyclononane) is described. The complex is characterized by UV/Vis and 1-D and 2-D NMR spectroscopy in solution. The complex has been further characterized in the solid-state by X-ray diffraction analysis which displays a distorted octahedral coordination geometry around the Ru metal center. The cyclic voltammetric experiments of the complex show a pH-dependent chemically irreversible wave at Ep,a = 0.82 V, at pH = 7.0, indicating the low stability of the higher oxidation states. Nevertheless, before the complex decomposes, it is capable of electrocatalytically oxidizing benzyl alcohol (BzOH) to benzaldehyde with a second-order rate constant of 19.8 M⁻¹s⁻¹. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

The synthesis, structure and spectroscopic properties of a family of Zn complexes containing a dinucleating hexaazamacrocyclic ligand (L) and anionic ligands (X) of general formula [Zn2(X)4−n(L)]ⁿ⁺ (1−8; L = H2p, H3p, Me2p, Me2m, Me3p, Me3m; X = Cl, n = 0; X = NO3, n = 2) have been investigated. The solid-state structural characterisation has been performed by means of X-ray diffraction analysis for complexes 3, 5, 7·1/3CH2Cl2 and 8. The Zn complexes 5, 7·1/3CH2Cl2 and 8 containing chloride ligands present a distorted N2Cl2 tetrahedral coordination with two non-coordinated N atoms of the macrocyclic ligand. On the other hand, complex 3, which contains nitrate ligands, displays an N3O2 pentacoordination with all the N atoms of the macrocyclic ligand bonded to the metal centres. The different factors governing the relative Zn−Zn disposition are discussed. NMR spectroscopy in solution reveals the presence of fluxional behaviour and a completely different structural arrangement than in the solid state. In particular, evidence for the formation and breaking of Zn−N bonds involving all the macrocyclic ligand aminic N atoms is obtained for the chloride complexes 2 and 4−8. Finally, complexes 1, 3, 5 and 7 hydrolyse p-nitrophenyl acetate (NA) with second-order rate constants ranging from 0.045 to 0.193 M⁻¹s⁻¹, thus manifesting the influence of the ligands over the hydrolytic activity. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

The potential of dihydroxyacetone phosphate (DHAP)-dependent aldolases to catalyze stereoselective aldol additions is, in many instances, limited by the solubility of the acceptor aldehyde in aqueous/co-solvent mixtures. Herein, we demonstrate the efficiency of emulsion systems as reaction media for the class I fructose-1,6-bisphosphate aldolase (RAMA) and class II recombinant rhamnulose-1-phosphate aldolase from E. coli (RhuA)-catalyzed aldol addition between DHAP and N-benzyloxycarbonyl (N-Cbz) aminoaldehydes. The use of emulsions improved the RAMA-catalyzed aldol conversions by three to tenfold relative to those in conventional DMF/water mixtures. RhuA was more reactive than RAMA towards the N-Cbz aminoaldehydes regardless of the reaction medium. With (S)- or (R)-Cbz-alaninal, RAMA exhibited preference for the R enantiomer, while RhuA had no enantiomeric discrimination. The linear N-Cbz aminopolyols thus obtained were submitted to catalytic intramolecular reductive amination to afford the corresponding iminocyclitols. This reaction was diastereoselective in all cases examined; the face selectivity was controlled by the stereochemistry of the newly formed hydroxyl group originating from the aldehyde. Characterization of the resulting iminocyclitols allowed the assessment of the diastereoselectivity of the enzymatic aldol reactions with respect to the N-protected aminoaldehyde. RAMA formed single diastereoisomers from N-Cbz-glycinal and from both enantiomers of N-Cbz-alaninal, while 14 % of the epimeric product was observed from N-Cbz-3-aminopropanal. Diastereoselectivity from RhuA was lower than that observed from RAMA. Interestingly, a single diastereoisomer was formed from (S)-Cbz-alaninal, whereas only a 34 % diastereomeric excess was observed from its enantiomer (i.e., (R)-Cbz-alaninal).

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Non-selective and selective versions of several proton-detected 1D NMR experiments to be applied to ¹⁵N are proposed. Clean, artifact-free 1D spectra are easily obtained by the effective coherence selection by pulsed-field gradients and the attainable sensitivity is maximized using modern pulse schemes. Despite the low sensitivity inherent to ¹⁵N NMR spectroscopy, the successful application of these experiments is demonstrated for resonance assignments and accurate measurement of both one-bond and long-range proton–nitrogen coupling constants on a model tripeptide at natural abundance. Copyright © 2001 John Wiley & Sons, Ltd.