Christopher D. Maycock

Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge? Kinetics of NAD(+) and NADH pools determined in vivo by 13C NMR.

The involvement of nicotinamide adenine nucleotides (NAD+, NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using13C and 31P NMR to monitor in vivothe kinetics of the pools of NAD+, NADH, ATP, inorganic phosphate (Pi), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of13C-labeled NAD+ and NADH. The capacity ofL. lactis MG1363 to regenerate NAD+ was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH−deficient strain was constructed by double crossover. Upon supply of glucose, NAD+ was constant and maximal (∼5 mm) in the parent strain (MG1363) but decreased abruptly in the LDH− strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH− strain and NADH reached high levels (2.5 mm), reflecting severe limitation in regenerating NAD+. However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mm. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by NADH. The ATP/ADP/Pi content could play an important role.

Thermostabilization of Proteins by Diglycerol Phosphate, a New Compatible Solute from the Hyperthermophile Archaeoglobus fulgidus

ABSTRACT Diglycerol phosphate accumulates under salt stress in the archaeonArchaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, Appl. Environ. Microbiol. 63:896–902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, bakers yeast alcohol dehydrogenase, andThermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins fromDesulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianumrubredoxins. In contrast, the stability of D. desulfuricansrubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.

Copper-Catalyzed Regioselective Intramolecular Oxidative α-Functionalization of Tertiary Amines: An Efficient Synthesis of Dihydro-1,3-Oxazines†

Oxidative coupling reactions, especially through cross-dehydrogenative coupling (CDC) of C H bonds, which avoids the prefunctionalization of substrates, have recently been in focus for being more atom economical, directive, and environmentally benign than other cross-coupling reactions. Among the CDC reactions, the functionalization of sp C H bonds adjacent to a nitrogen atom has generally been achieved utilizing transition metal catalysts with co-oxidants, such as tert-butylhydroperoxide (TBHP), H2O2, molecular oxygen, and others, to generate iminium ion species, which in turn react with various nucleophiles. For this purpose, metals such as Ru, Fe, Rh, V, and others are often used. However, copper salts retain many advantages, such as ready availability, low cost, high efficiency and low toxicity, and have turned out to be the most efficient catalysts for the a-functionalization of tertiary amines. More recently, homogeneous copper catalysis have also been used to achieve the selective aerobic oxidative functionalization of C H bonds. However, such copper-catalyzed functionalization, whether under aerobic or anaerobic conditions, is mainly confined to the benzylic position of N-phenyltetrahydroisoquinoline. Examples relating to the activation of non-benzylic methyl/methylene C H, are scarce. Hence, despite significant development in recent years, still more research in this area is required to enhance the selectivity and substrate scope. Looking for synthetic advances in regiocontrolled C H functionalization, we became interested in the design of a synthetic strategy for a copper-catalyzed aerobic oxidative/ dehydrogenative a-functionalization of tertiary amines with subsequent intramolecular sp C O bond formation. Dihydro-1,3-oxazine derivatives are important heterocyclic molecules, which exhibit a wide range of pharmacological activity, including antibacterial, fungicidal, antitumor, antituberculosis, and anti-HIV. Naphthoxazine derivatives have therapeutic potential for the treatment of Parkinson s disease, and are also used as potent nonsteroidal progesterone receptor agonists. They can be used as intermediates in the synthesis of N-substituted aminoalcohols, bioactive natural products, or in asymmetric catalysis. Justifiably, the intrinsic versatility and synthetic utility of these heterocyclic systems has attracted great interest from chemists, who have developed several exquisite methods for their synthesis. However, except for a few examples, most of the reported strategies are primarily based on Mannich type condensations, which are restricted to the use of aliphatic alicyclic primary amines. Hence, regardless of these advances, investigations in search of more efficient routes for the synthesis of these compounds are still highly desirable for drug discovery and medicinal chemistry. Herein, we disclose an efficient, environmentally friendly, diastereoselective copper-catalyzed synthesis of naphtho and benzo2,3-dihydro-1,3-oxazines through regioselective C H bond activation and cyclization (Scheme 1).

Processing the interspecies quorum-sensing signal autoinducer-2 (AI-2): characterization of phospho-(S)-4,5-dihydroxy-2,3-pentanedione isomerization by LsrG protein.

The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is the precursor of the signal molecule autoinducer-2 (AI-2). AI-2 mediates interspecies communication and facilitates regulation of bacterial behaviors such as biofilm formation and virulence. A variety of bacterial species have the ability to sequester and process the AI-2 present in their environment, thereby interfering with the cell-cell communication of other bacteria. This process involves the AI-2-regulated lsr operon, comprised of the Lsr transport system that facilitates uptake of the signal, a kinase that phosphorylates the signal to phospho-DPD (P-DPD), and enzymes (like LsrG) that are responsible for processing the phosphorylated signal. Because P-DPD is the intracellular inducer of the lsr operon, enzymes involved in P-DPD processing impact the levels of Lsr expression. Here we show that LsrG catalyzes isomerization of P-DPD into 3,4,4-trihydroxy-2-pentanone-5-phosphate. We present the crystal structure of LsrG, identify potential catalytic residues, and determine which of these residues affects P-DPD processing in vivo and in vitro. We also show that an lsrG deletion mutant accumulates at least 10 times more P-DPD than wild type cells. Consistent with this result, we find that the lsrG mutant has increased expression of the lsr operon and an altered profile of AI-2 accumulation and removal. Understanding of the biochemical mechanisms employed by bacteria to quench signaling of other species can be of great utility in the development of therapies to control bacterial behavior.

The first synthesis of (-)-asperpentyn and efficient syntheses of (+)-harveynone, (+)-epiepoformin and (-)-theobroxide.

A generally applicable strategy for the synthesis of a range of polyoxygenated cyclohexane natural products has been developed. The enantioselective syntheses of (-)-theobroxide, a polyoxygenated cyclohexane natural compound with potent growth inducing properties in potato microtubers has been achieved via a 1,2 O-silyl migration between trans-hydroxyl groups and a remote hydroxyl directed epoxidation of an enone derived from quinic acid. A thus derived alpha-iodoenone was subjected to Stille coupling with tetramethylstannane to afford the first title compound. A similar strategy enabled a route to the complete asymmetric synthesis of the acetylenic phytotoxin (+)-harveynone. By selective reduction of (-)-theobroxide, (+)-epiepoformin was also prepared in enantiopure form and similarly, stereoselective reduction of (+)-harveynone completed the first enantioselective synthesis of (-)-asperpentyn, another natural compound with antimicrobial activity.

The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture

BackgroundNanotechnology has the potential to provide agriculture with new tools that may be used in the rapid detection and molecular treatment of diseases and enhancement of plant ability to absorb nutrients, among others. Data on nanoparticle toxicity in plants is largely heterogeneous with a diversity of physicochemical parameters reported, which difficult generalizations. Here a cell biology approach was used to evaluate the impact of Quantum Dots (QDs) nanocrystals on plant cells, including their effect on cell growth, cell viability, oxidative stress and ROS accumulation, besides their cytomobility.ResultsA plant cell suspension culture of Medicago sativa was settled for the assessment of the impact of the addition of mercaptopropanoic acid coated CdSe/ZnS QDs. Cell growth was significantly reduced when 100 mM of mercaptopropanoic acid -QDs was added during the exponential growth phase, with less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs addition. They were up taken by Medicago sativa cells and accumulated in the cytoplasm and nucleus as revealed by optical thin confocal imaging. As part of the cellular response to internalization, Medicago sativa cells were found to increase the production of Reactive Oxygen Species (ROS) in a dose and time dependent manner. Using the fluorescent dye H2DCFDA it was observable that mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive and linear increase of ROS accumulation.ConclusionsOur results showed that the extent of mercaptopropanoic acid coated CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors including QDs properties, dose and the environmental conditions of administration and that, for Medicago sativa cells, a safe range of 1-5 nM should not be exceeded for biological applications.

The preferential direction of enolization of some asymmetric 1,3-dicarbonyl compounds in solution: a study by multinuclear NMR spectroscopy

Abstract Previously described methods for determining the relative concentrations of the two cis -enol tautomers of asymmetric β-diketones using 13 C and 17 O NMR were compared and applied to a wider range of compounds. From the chemical shifts of the enol peaks and model shifts for the pure enol forms, the equilibrium constant for the enol—enol equilibrium was obtained. The small but significant effects of the β-substituents on the direction of enolization are in the following order of stabilization of the enol form at the adjacent carbonyl carbon: COOC 2 H 5 > CF 3 > C 6 H 5 > C(CH 3 ) 3 > CH 3 . This relative order does not totally coincide with the preferred location of the regioselective attack of those compounds to form monodithioacetals. This indicates that, besides electronic effects, steric effects may also be quite important in determining regioselectivity in this type of compound.

Synthesis of potassium (2R)-2-O-α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl-2,3-dihydroxypropanoate a natural compatible solute

Ethyl 6-O-acetyl-2,3,4-tribenzyl-1-thio-d-glucopyranoside, as a mixture of anomers, was employed for the stereoselective synthesis of the potassium salt of (2R)-2-O-alpha-d-glucopyranosyl-(1-->6)-alpha-d-glucopyranosyl-2,3-dihydroxypropanoic acid (alpha-d-glucosyl-(1-->6)-alpha-d-glucosyl-(1-->2)-d-glyceric acid, GGG), a recently isolated compatible solute. The alpha-anomer was by far the major product of both glycosylation reactions using NIS/TfOH as activator.

An efficient synthesis of the precursor of AI-2, the signalling molecule for inter-species quorum sensing.

Autoinducer-2 (AI-2) is a signalling molecule for bacterial inter-species communication. A synthesis of (S)-4,5-dihydroxypentane-2,3-dione (DPD), the precursor of AI-2, is described starting from methyl glycolate. The key step was an asymmetric reduction of a ketone with (S)-Alpine borane. This new method was highly reproducible affording DPD for biological tests without contaminants. The biological activity was tested with the previously available assays and compared with a new method using an Escherichia coli reporter strain thus avoiding the use of the pathogenic Salmonella reporter.

A synthesis of aziridines from α-iodoenones

Abstract Aziridines were prepared from α-iodocycloenones in very good yield, by a Michael addition/cyclisation (Gabriel–Cromwell) process employing a slight excess of primary amine and Cs 2 CO 3 as base at 95°C. Using chiral amines it was possible to prepare optically pure aziridines. The same method was also efficient for the synthesis of aziridines from acyclic α-halounsaturated compounds. 2-Oxoazabicycles reacted with several nucleophiles to afford α-heteroatom substituted cyclic enones in excellent yield.