Synthesis and aplication of linear and macrocyclic nitrogen ligand

Linear and macrocyclic nitrogen ligands have been found wide application during the years. Nitrogen has a much strong association with transition-metal ions because the electron pair is partucularly available for complexing purposes. We started our investigation with the synthesis of new chiral perazamacrocycles containing four pyrrole rings. This ligand was synthesized by the [2+2]condensation of (R,R)-diaminocyclohexane and dipirranedialdehydes and was tested, after a complexation with Cu(OAc)2, in Henry reactions. The best yields (96%) and higher ee’s (96%) were obtained when the meso-substituent on the dipyrrandialdehyde was a methyl group. The positive influence of the pyrrole-containing macrocyclic structure on the efficiency/enantioselectivity of the catalytic system was demonstrated by comparison with the Henry reactions performed using analogous ligands. Henry product was obtain in good yield but only 73% of ee, when the dialdehyde unit was replaced by a triheteroaromatic dialdehye (furan-pyrrol-furan). Another well known macrocyclic ligand is calix[4]pyrrole. We decided to investigate, in collaboration with Neier’s group, the metal-coordinating properties of calix[2]pyrrole[2]pyrrolidine compounds obtained by the reduction of calix[4]pyrrole. We focused our attention on the reduction conditions, and tested different Pd supported (charcoal, grafite) catalysts at different condition. Concerning the synthesis of linear polyamine ligands. We focused our attention to the synthesis of 2-heteroaryl- and 2,5-diheteroarylpyrrolidines. The reductive amination reaction of diarylketones and aryl-substitutedketo-aldehydes with different chiral amines was exploited to prepare a small library of diastereo-enriched substituted pyrrolidines. We have also described a new synthetic route to 1,2-disubstituted 1,2,3,4-tetrahydropyrrole[1,2-a]pyrazines, which involves the diastereoselective addition of Grignard reagents to chiral oxazolidines. The best diastereoselectivity (98:2) was dependent on the nature of both the chiral auxiliary, (S)-1-phenylglycinol, and the nature of the organometallic reagent (MeMgBr).

Effects of experimental nitrogen additions on plant diversity in an old-growth tropical forest

Response of plant biodiversity to increased availability of nitrogen (N) has been investigated in temperate and boreal forests, which are typically N-limited, but little is known in tropical forests. We examined the effects of artificial N additions on plant diversity (species richness, density and cover) of the understory layer in an N saturated old-growth tropical forest in southern China to test the following hypothesis: N additions decrease plant diversity in N saturated tropical forests primarily from N-mediated changes in soil properties. Experimental additions of N were administered at the following levels from July 2003 to July 2008: no addition (Control); 50 kg N ha−1 yr−1 (Low-N); 100 kg N ha−1 yr−1 (Medium-N), and 150 kg N ha−1 yr−1 (High-N). Results showed that no understory species exhibited positive growth response to any level of N addition during the study period. Although low-to-medium levels of N addition (≤100 kg N ha−1 yr−1) generally did not alter plant diversity through time, high levels of N addition significantly reduced species diversity. This decrease was most closely related to declines within tree seedling and fern functional groups, as well as to significant increases in soil acidity and Al mobility, and decreases in Ca availability and fine-root biomass. This mechanism for loss of biodiversity provides sharp contrast to competition-based mechanisms suggested in studies of understory communities in other forests. Our results suggest that high-N additions can decrease plant diversity in tropical forests, but that this response may vary with rate of N addition.

Evaluation of Proteferm® as a Nitrogen Supplement for Finishing Steers

Proteferm is a liquid by-product from the production of monosodium glutamate by fermentation that is 40% solids and contains 95% crude protein on a dry basis. Two trials were conducted with yearling steers to study the effects of different levels of Proteferm when added to 90% concentrate diets to replace corn and urea. Feeding Proteferm resulted in a decrease in urine pH, suggesting a metabolic acidosis that was probably caused by a negative cation–anion balance. Correcting the cation–anion imbalance resulted in an increase in feed intake and the performance of steers fed Proteferm as 1.5% of diet dry matter to be similar to the performance of control steers. Addition of sodium bicarbonate to the control diet or to the diet containing 1.5% Proteferm to produce a positive cation–anion balance did not improve performance. The results of these trials indicate that 1.5% Proteferm is the maximum that should be added to high concentrate diets fed to beef cattle without affecting performance or carcass value.

Interaction of sulfate assimilation with carbon and nitrogen metabolism in Lemna minor

Cysteine synthesis from sulfide andO-acetyl-L-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. UsingLemna minor, we analyzed the effects of omission of CO2 from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5′-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO2 led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO2 on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO2, APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO2 also recovered both enzyme activities, with OAS again influenced only APR.35SO4 2− feeding showed that treatment in air without CO2 severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of 35S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of 35S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.

Regulation of Sulfate Assimilation by Nitrogen in Arabidopsis

Using Arabidopsis, we analyzed the effect of omission of a nitrogen source and of the addition of different nitrogen-containing compounds on the extractable activity and the enzyme and mRNA accumulation of adenosine 5′-phosphosulfate reductase (APR). During 72 h without a nitrogen source, the APR activity decreased to 70% and 50% of controls in leaves and roots, respectively, while cysteine (Cys) and glutathione contents were not affected. Northern and western analysis revealed that the decrease of APR activity was correlated with decreased mRNA and enzyme levels. The reduced APR activity in roots could be fully restored within 24 h by the addition of 4 mM each of NO3 −, NH4 +, or glutamine (Gln), or 1 mM O-acetylserine (OAS). 35SO4 2− feeding showed that after addition of NH4 +, Gln, or OAS to nitrogen-starved plants, incorporation of 35S into proteins significantly increased in roots; however, glutathione and Cys labeling was higher only with Gln and OAS or with OAS alone, respectively. OAS strongly increased mRNA levels of all three APR isoforms in roots and also those of sulfite reductase, Cys synthase, and serine acetyltransferase. Our data demonstrate that sulfate reduction is regulated by nitrogen nutrition at the transcriptional level and that OAS plays a major role in this regulation.

Molar gas ratios and stable oxygen and nitrogen isotopes of air entrapped in ice, North America

The molar ratios of atmospheric gases change during dissolution in water due to differences in their relative solubilities. We exploited this characteristic to develop a tool to clarify the origin of ice formations in permafrost regions. Extracted from ice, molar gas ratios can distinguish buried glacier ice from intrasedimental ground ice formed by freezing groundwaters. An extraction line was built to isolate gases from ice by melting and trapping with liquid He, followed by analysis of N2, O2, Ar, 18O-O2 and 15N-N2, by continuous flow mass spectrometry. The method was tested using glacier ice, aufeis ice (river icing) and intrasedimental ground ice from sites in the Canadian Arctic. O2/Ar and N2/Ar ratios clearly distinguish between atmospheric gas in glacial ice and gases from intrasedimental ground ice, which are exsolved from freezing water. 615NN2 and 618OO2 in glacier ice, aufeis ice and intrasedimental ground ice do not show clear distinguishing trends as they are affected by various physical processes during formation such as gravitational settling, excess air addition, mixing with snow pack, and respiration.

The nitrogen costs of photosynthesis in a diatom under current and future pCO2

With each cellular generation, oxygenic photoautotrophs must accumulate abundant protein complexes that mediate light capture, photosynthetic electron transport and carbon fixation. In addition to this net synthesis, oxygenic photoautotrophs must counter the light-dependent photoinactivation of Photosystem II (PSII), using metabolically expensive proteolysis, disassembly, resynthesis and re-assembly of protein subunits. We used growth rates, elemental analyses and protein quantitations to estimate the nitrogen (N) metabolism costs to both accumulate the photosynthetic system and to maintain PSII function in the diatom Thalassiosira pseudonana, growing at two pCO2 levels across a range of light levels. The photosynthetic system contains c. 15-25% of total cellular N. Under low growth light, N (re)cycling through PSII repair is only c. 1% of the cellular N assimilation rate. As growth light increases to inhibitory levels, N metabolite cycling through PSII repair increases to c. 14% of the cellular N assimilation rate. Cells growing under the assumed future 750 ppmv pCO2 show higher growth rates under optimal light, coinciding with a lowered N metabolic cost to maintain photosynthesis, but then suffer greater photoinhibition of growth under excess light, coincident with rising costs to maintain photosynthesis. We predict this quantitative trait response to light will vary across taxa.

Lipid, carbon and nitrogen content and fatty acid composition of Temora longicornis females and different food species during the experiment

Temora longicornis, a dominant calanoid copepod species in the North Sea, is characterised by low lipid reserves and high biomass turnover rates. To survive and reproduce successfully, this species needs continuous food supply and thus requires a highly flexible digestive system to exploit various food sources. Information on the capacity of digestive enzymes is scarce and therefore the aim of our study was to investigate the enzymatic capability to respond to quickly changing nutritional conditions. We conducted two feeding experiments with female T. longicornis from the southern North Sea off Helgoland. In the first experiment in 2005, we tested how digestive enzyme activities and enzyme patterns as revealed by substrate SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) responded to changes in food composition. Females were incubated for three days fed ad libitum with either the heterotrophic dinoflagellate Oxyrrhis marina or the diatom Thalassiosira weissflogii. At the beginning and at the end of the experiment, copepods were deep-frozen for analyses. The lipolytic enzyme activity did not change over the course of the experiment but the enzyme patterns did, indicating a distinct diet-induced response. In a second experiment in 2008, we therefore focused on the enzyme patterns, testing how fast changes occur and whether feeding on the same algal species leads to similar patterns. In this experiment, we kept the females for 4 days at surplus food while changing the algal food species daily. At day 1, copepods were offered O. marina. On day 2, females received the cryptophycean Rhodomonas baltica followed by T. weissflogii on day 3. On day 4 copepods were again fed with O. marina. Each day, copepods were frozen for analysis by means of substrate SDS-PAGE. This showed that within 24 h new digestive enzymes appeared on the electrophoresis gels while others disappeared with the introduction of a new food species, and that the patterns were similar on day 1 and 4, when females were fed with O. marina. In addition, we monitored the fatty acid compositions of the copepods, and this indicated that specific algal fatty acids were quickly incorporated. With such short time lags between substrate availability and enzyme response, T. longicornis can successfully exploit short-term food sources and is thus well adapted to changes in food availability, as they often occur in its natural environment due seasonal variations in phyto- and microzooplankton distribution.

Mineral nitrogen from KCl extractions of soil from the Jena Experiment (Main Experiment up to 30cm depth, year 2002)

As an estimate of plant-available N, this data set contains measurements of inorganic nitrogen (NO3-N and NH4-N, the sum of which is termed mineral N or Nmin) determined by extraction with 1 M KCl solution of soil samples from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. Soil sampling and analysis: Five soil cores (diameter 0.01 m) were taken at a depth of 0 to 0.15 m and 0.15 to 0.3 m of the mineral soil from each of the experimental plots in September 2002. Samples of the soil cores per plot were pooled during each sampling campaign. NO3-N and NH4-N concentrations were determined by extraction of soil samples with 1 M KCl solution and were measured in the soil extract with a Continuous Flow Analyzer (CFA, Skalar, Breda, Netherlands).

Mineral nitrogen from KCl extractions of soil from the Jena Experiment (Main Experiment up to 30cm depth, year 2004)

As an estimate of plant-available N, this data set contains measurements of inorganic nitrogen (NO3-N and NH4-N, the sum of which is termed mineral N or Nmin) determined by extraction with 1 M KCl solution of soil samples from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. Soil sampling and analysis: Five soil cores (diameter 0.01 m) were taken at a depth of 0 to 0.15 m and 0.15 to 0.3 m of the mineral soil from each of the experimental plots in March and October 2004. Samples of the soil cores per plot were pooled during each sampling campaign. NO3-N and NH4-N concentrations were determined by extraction of soil samples with 1 M KCl solution and were measured in the soil extract with a Continuous Flow Analyzer (CFA, Skalar, Breda, Netherlands).

Dissolved nitrogen in soil solution of the Jena Experiment (Main Experiment, year 2004)

This data set contains measurements of dissolved nitrogen (total dissolved nitrogen: TDN, dissolved organic nitrogen: DON, dissolved ammonium: NH4+, and dissolved nitrate: NO3-) in samples of soil water collected from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. In April 2002 glass suction plates with a diameter of 12 cm, 1 cm thickness and a pore size of 1-1.6 µm (UMS GmbH, Munich, Germany) were installed in depths of 10, 20, 30 and 60 cm to collect soil solution. The sampling bottles were continuously evacuated to a negative pressure between 50 and 350 mbar, such that the suction pressure was about 50 mbar above the actual soil water tension. Thus, only the soil leachate was collected. Cumulative soil solution was sampled biweekly and analyzed for nitrate (NO3-) and ammonium (NH4+) concentrations with a continuous flow analyzer (CFA, Skalar, Breda, The Netherlands). Nitrate was analyzed photometrically after reduction to NO2- and reaction with sulfanilamide and naphthylethylenediamine-dihydrochloride to an azo-dye. Our NO3- concentrations contained an unknown contribution of NO2- that is expected to be small. Simultaneously to the NO3- analysis, NH4+ was determined photometrically as 5-aminosalicylate after a modified Berthelot reaction. The detection limits of NO3- and NH4+ were 0.02 and 0.03 mg N L-1, respectively. Total dissolved N in soil solution was analyzed by oxidation with K2S2O8 followed by reduction to NO2- as described above for NO3-. Dissolved organic N (DON) concentrations in soil solution were calculated as the difference between TDN and the sum of mineral N (NO3- + NH4+). In 5% of the samples, TDN was equal to or smaller than mineral N. In these cases, DON was assumed to be zero.

Mineral nitrogen from KCl extractions of soil from the Jena Experiment (Main Experiment up to 15cm depth, year 2005)

As an estimate of plant-available N, this data set contains measurements of inorganic nitrogen (NO3-N and NH4-N, the sum of which is termed mineral N or Nmin) determined by extraction with 1 M KCl solution of soil samples from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. Soil sampling and analysis: Five soil cores (diameter 0.01 m) were taken at a depth of 0 to 0.15 m of the mineral soil from each of the experimental plots in April and September 2005. Samples of the soil cores per plot were pooled during each sampling campaign. NO3-N and NH4-N concentrations were determined by extraction of soil samples with 1 M KCl solution and were measured in the soil extract with a Continuous Flow Analyzer (CFA, Skalar, Breda, Netherlands).

Total nitrogen from solid phase in the Jena Experiment (Main Experiment up to 30cm depth, year 2008)

This data set contains measurements of total nitrogen from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. Soil sampling and analysis: Stratified soil sampling was performed in April 2008 to a depth of 30 cm. Three independent samples per plot were taken using a split tube sampler with an inner diameter of 4.8 cm (Eijkelkamp Agrisearch Equipment, Giesbeek, the Netherlands). Soil samples were segmented to a depth resolution of 5 cm in the field, giving six depth subsamples per core, and made into composite samples per depth. Sampling locations were less than 30 cm apart from sampling locations in other years. Samples were dried at 40°C. All soil samples were passed through a sieve with a mesh size of 2 mm. Because of much higher proportions of roots in the soil, the samples were further sieved to 1 mm according to common root removal methods. No additional mineral particles were removed by this procedure. Total nitrogen concentration was analyzed on ball-milled subsamples (time 4 min, frequency 30 s-1) by an elemental analyzer at 1150°C (Elementaranalysator vario Max CN; Elementar Analysensysteme GmbH, Hanau, Germany).

Total nitrogen from solid phase in the Jena Experiment (Block 2 Main Experiment up to 100cm depth, year 2002)

This data set contains measurements of total nitrogen from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. Soil sampling and analysis: Stratified soil sampling to a depth of 1m was performed before sowing in April 2002. Three independent samples per plot were taken of all plots in block 2 using a motor-driven soil column cylinder (Cobra, Eijkelkamp, 8.3 cm in diameter). Soil samples were dried at 40°C and segmented to a depth resolution of 5 cm giving 20 depth subsamples per core. All samples were analyzed independently. All soil samples were passed through a sieve with a mesh size of 2 mm. Rarely present visible plant remains were removed using tweezers. Total nitrogen concentration was analyzed on ball-milled subsamples (time 4 min, frequency 30 s-1) by an elemental analyzer at 1150°C (Elementaranalysator vario Max CN; Elementar Analysensysteme GmbH, Hanau, Germany).

Total nitrogen from solid phase in the Jena Experiment (Main Experiment up to 30cm depth, year 2004)

This data set contains measurements of total nitrogen from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below). In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. Soil sampling and analysis: Stratified soil sampling was performed in April 2004 to a depth of 30 cm. Three independent samples per plot were taken using a split tube sampler with an inner diameter of 4.8 cm (Eijkelkamp Agrisearch Equipment, Giesbeek, the Netherlands). Soil samples were segmented to a depth resolution of 5 cm in the field, giving six depth subsamples per core, and made into composite samples per depth. Sampling locations were less than 30 cm apart from sampling locations in other years. Samples were dried at 40°C. All soil samples were passed through a sieve with a mesh size of 2 mm. Because of much higher proportions of roots in the soil, the samples were further sieved to 1 mm according to common root removal methods. No additional mineral particles were removed by this procedure. Total nitrogen concentration was analyzed on ball-milled subsamples (time 4 min, frequency 30 s-1) by an elemental analyzer at 1150°C (Elementaranalysator vario Max CN; Elementar Analysensysteme GmbH, Hanau, Germany).