Targeted metabolic profiling of phenolics in urine and plasma after regular consumption of cocoa by liquid chromatography–tandem mass spectrometry

The biological properties of cocoa (Theobroma cacao L.) polyphenols are strictly dependent on their bioavailability. A long-term cocoa feeding trial was performed with subjects at high risk for cardiovascular disease. Subjects (n = 42) received two sachets of 20 g of cocoa powder/day with 250 mL of skimmed milk each, or only 500 mL/day of skimmed milk, both for two 4-week periods. The phenolic metabolic profile including phase II conjugated metabolites and phenolic acids derived from the intestinal microbiota was determined by LC-MS/MS in both 24-h urine and fasting plasma. The analysis of 24-h urine revealed significant increases of phase II metabolites, including glucuronides and sulfate conjugates of (−)-epicatechin, O-methyl-epicatechin, 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and 5-(3′-methoxy-4′-hydroxyphenyl)-γ-valerolactone, after regular cocoa intake. In the case of plasma, only glucuronide conjugates of dihydroxyphenylvalerolactones increased. Regular consumption of cocoa also resulted in a significant increase in the urinary excretion of colonic microbial-derived phenolic metabolites, including vanillic, 3,4-dihydroxyphenylacetic and 3-hydroxyphenylacetic acids, and particularly 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone, whereas only the two latter metabolites showed a significant increase in fasting plasma. The results found herein indicate that 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and hydroxyphenylacetic acids could be good biomarkers of the regular consumption of cocoa and therefore, of flavanol-rich foods.     

Highly enantioselective dynamic kinetic resolution and desymmetrization processes by cyclocondensation of chiral aminoalcohols with racemic or prochiral delta-oxoacid derivatives

Cyclocondensation reactions of aminoalcohols and with racemic or prochiral delta-oxoacid derivatives provide polysubstituted lactams with high enantioselectivity in a process that involves dynamic kinetic resolution and/or desymmetrization of enantiotopic or diastereotopic ester groups.     

Development of a new method for sulfide determination by vapor generator–inductively coupled plasma–mass spectrometry

A new sensitive methodology for the determination of total reduced sulfur species in natural waters and acid volatile sulfides in sediments at low levels (μg L^− 1) is described. Reduced sulfur species were separated from the water matrix in the form of H₂S after reaction with hydrochloric acid in a commercial vapor generator coupled to an inductively coupled plasma quadrupole mass spectrometer (VG–ICP–QMS) equipped with a reaction cell. The method avoided the effect of polyatomic isobaric interferences at m/z 32 caused by ¹⁶O¹⁶O⁺ and ¹⁴N¹⁸O⁺ through the elimination of the aqueous matrix, a source of oxygen. By introducing a mixture of helium and hydrogen gases into the octopole reaction cell, a series of ion-molecule reactions were induced to reduce the interfering polyatomic species. Operating conditions of the octopole reaction cell system and the analyzer were optimized to get the best signal to background ratio for ³²S; a full factorial 2³ experimental design was developed in order to evaluate which variables had a significant effect and a simplex methodology was applied to find the optimum conditions for the variables. The new method was evaluated by comparison to the standard potentiometric method. The analytical methodology developed was applied to the analysis of reduced sulfur species in natural waters and acid volatile sulfides in sea sediments.     

A Catalytic Asymmetric Synthesis ofN-Boc-beta-Methylphenylalanines

An efficient, stereodivergent, and enantioselective synthesis of the syn and anti diastereomers of N-Boc-beta-methylphenylalanine has been developed. Starting from enantiomerically pure (2S,3S)-2,3-epoxy-3-phenyl-1-propanol, a three-step sequence, consisting of the oxidation of the primary alcohol up to the carboxyl stage, ring opening of the epoxy acid with Me(2)CuCNLi(2), and esterification of the resulting hydroxy acid with methyl iodide, leads to the hydroxy ester anti-10, which has been converted in a stereodivergent manner into both the (2S,3R) and the (2R,3R) diastereomers of N-Boc-beta-methylphenylalanine, syn-1 and anti-1, respectively. Activation of the secondary hydroxy group in anti-10 as a mesylate, followed by nucleophilic displacement with sodium azide, hydrogenolysis with simultaneous protection of the amino group, and saponification with LiOH, affords syn-1. The same reaction sequence applied to syn-10, obtained in turn by Mitsunobu reaction of anti-10 with p-nitrobenzoic acid followed by the hydrolysis of the resulting p-nitrobenzoate, leads to anti-1. Both products have been obtained with >/=99% enantiomeric excess.     

New stereodivergent approach to 3-amino-2,3,6-trideoxysugars. Enantioselective synthesis of daunosamine,ristosamine, acosamine,and epi-daunosamine

[reaction: see text] An enantioselective preparation of the four diastereomeric 3-amino-2,3,6-trideoxy-hexoses, key components of anthracycline antibiotics, has been developed. Sharpless catalytic asymmetric epoxidation of the (2E)-2,5-hexadien-1-ol, regioselective ring opening with azide, followed by convenient functional group transformations, afforded the key aldehydes cis- or trans-6 in any configuration. The diastereoselective addition of methylmetal reagents to these aldehydes followed by ozonolysis gives access in a completely stereocontrolled manner to the four isomeric trideoxyaminosugars.     

Quantum dynamics of hydride transfer catalyzed by bimetallic electrophilic catalysis: synchronous motion of Mg(2+) and H(-) in xylose isomerase

Xylose isomerase exhibits a bridged-bimetallic active-site motif in which the substrate is bound to two metals connected by a glutamate bridge, and X-ray crystallographic studies suggest that metal movement is involved in the hydride transfer rate-controlling catalytic step. Here we report classical/quantal dynamical simulations of this step that provide new insight into the metal motion. The potential energy surface is calculated by treating xylose with semiempirical molecular orbital theory augmented by a simple valence bond potential and the rest of the system by molecular mechanics. The rate constant for the hydride-transfer step was calculated by ensemble-averaged dynamical simulations including both variational transition-state theory for determination of the statistically averaged dynamical bottleneck and optimized multidimensional tunneling calculations. The dynamics calculations include 25 317 atoms, with quantized vibrational free energy in 89 active-site degrees of freedom, and with 32 atoms moving through static secondary zone transition-state configurations in the quantum tunneling simulation. Our simulations show that the average Mg-Mg distance R increases monotonically as a function of the hydride-transfer progress variable z. The range of the average R along the reaction path is consistent with the X-ray structure, thus providing a dynamical demonstration of the postulated role of Mg in catalysis. We also predicted the primary deuterium kinetic isotope effect (KIE) for the chemical step. We calculated a KIE of 3.8 for xylose at 298 K, which is consistent with somewhat smaller experimentally observed KIEs for glucose substrate at higher temperatures. More than half of our KIE is due to tunneling; neglecting quantum effects on the reaction coordinate reduces the calculated KIE to 1.8.     

ENPDA: an evolutionary structure-based <Emphasis Type="Italic">de novo</Emphasis> peptide design algorithm

Summary One of the goals of computational chemists is to automate the de novo design of bioactive molecules. Despite significant advances in computational approaches to ligand design and binding energy evaluation, novel procedures for ligand design are required. Evolutionary computation provides a new approach to this design endeavor. We propose an evolutionary tool for de novo peptide design, based on the evaluation of energies for peptide binding to a user-defined protein surface patch. Special emphasis has been placed on the evaluation of the proposed peptides, leading to two different evaluation heuristics. The software developed was successfully tested on the design of ligands for the proteins prolyl oligopeptidase, p53, and DNA gyrase.     

Importance of the basis set for the spin-state energetics of iron complexes

We have performed a systematic investigation of the influence of the basis set on relative spin-state energies for a number of iron compounds. In principle, with an infinitely large basis set, both Slater-type orbital (STO) and Gaussian-type orbital (GTO) series should converge to the same final answer, which is indeed what we observe for both vertical and relaxed spin-state splittings. However, we see throughout the paper that the STO basis sets give consistent and rapidly converging results, while the convergence with respect to the basis set size is much slower for the GTO basis sets. For example, the large GTO basis sets that give good results for the vertical spin-state splittings of compounds 1-3 (6-311+G**, Ahlrichs VTZ2D2P) fail for the relaxed spin-state splittings of compound 4 (where 1 is Fe-(PyPepS)2 (PyPepSH 2 = N-(2-mercaptophenyl)-2-pyridinecarboxamide), 2 is Fe(tsalen)Cl (tsalen = N, N'-ethylenebis-(thiosalicylideneiminato)), 3 is Fe(N(CH2-o-C6H4S) 3)(1-Me-imidazole), and 4 is FeFHOH). Very demanding GTO basis sets like Dunning's correlation-consistent (cc-pVTZ, cc-pVQZ) basis sets are needed to achieve good results for these relaxed spin states. The use of popular (Pople-type) GTO, effective core potentials basis set (ECPB), or mixed ECPB(Fe):GTO(rest) basis sets is shown to lead to substantial deviations (2-10 kcal/mol, 14-24 kcal/mol for 3-21G), in particular for the high spin states that are typically placed at too low energy. Moreover, the use of an effective core potential in the ECPB basis sets results in spin-state splittings that are systematically different from the STO-GTO results.     

Assembling metals and clusters around an octaphosphine ligand based on N-substituted bis(diphenylphosphanyl)amines: structural characterization of dendrimer-like Co12 and Co16 branched clusters

A polypodal ligand based on an aromatic ring decorated by four dppa-type diphosphine moieties has been used to prepare Pt(II) complexes and to assemble four tricobalt or tetracobalt carbonyl clusters, leading to centrosymmetric Co12 and Co16 "clusters of clusters" which were characterized by X-ray diffraction.     

Profiles of paralytic shellfish poisoning toxins in shellfish from Portugal explained by carbamoylase activity

The presence of paralytic shellfish poisoning (PSP) toxins has not been recorded in the Portuguese coast since 1995. A bloom of Gymnodinium catenatum occurred in the NW coast of Portugal in the autumn of 2005, and PSP profiles were determined in several inshore and offshore shellfish species by HPLC after pre-column oxidation. Most of the species studied contained a complex toxin profile, typically representative of contamination by G. catenatum. However, clams such as Spisula solida contained mainly decarbamoyl toxins, while less extensive transformation was found in Scrobicularia plana. In vitro incubation of S. solida digestive glands with PSP standards revealed a rapid transformation of carbamate and N-sulfocarbamoyl toxins into their corresponding decarbamate analogues. After 24 h, less than 5% of the carbamate or N-sulfocarbamoyl toxins tested remained. After a 24 h in vitro incubation of S. plana digestive glands, no decarbamate analogues were detected. Artificial toxification of S. plana with cultures of G. catenatum revealed the conversion into decarbamoyl analogues progressed slowly: initially dcGTX2+3 and dcSTX accounted only for 5% of total non N-1 hydroxilated toxins, after 6 days these toxins accounted for 41% of the toxin composition. In vitro incubations of digestive glands from other commercial bivalves did not reveal production of decarbamoyl analogues over a 24 h period.