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Sorghum has gained popularity with consumers as a grain source with its gluten-free and high protein dietary characteristics. Acreage has increased recently, in part due to the demand for an alternative feed source for poultry and swine. New information is needed about the level of mycotoxin contamination in sorghum accessions, and accurate and affordable analytical methods need to be developed and tested to quantify mycotoxins in sorghum. A traditional method (solid-phase extraction chromatography with C18, followed by GC-MS) to quantify the mycotoxin deoxynivalenol (DON) produced inconsistent DON values following controlled spiking and recovery experiments with different sorghum accessions. Consequently, we incorporated a stable isotope (d1-DON) as an internal standard into a traditional GC-MS method. This method, stable isotope dilution assay (SIDA), was used to accurately determine DON levels in 196 sorghum samples representing 98 different accessions. Of the 98 accessions tested (two samples per accession), 76 of the accessions had DON levels that were greater than the limit of detection for both methods (0.20 μg g−1). For a USA regulatory threshold of 1 μg g−1, about one third of all the accessions (26/76) had at least 20% more DON using the SIDA method. For a regulatory threshold of 5 μg g−1, about 7% of all the accessions (5/76) had at least 20% more DON using the SIDA method. Using SIDA, the amount of DON in a sorghum sample can be accurately and reliably quantitated by basing calculations on the recovery of d1-DON, and this method may have future applications for quantifying DON from samples with complex matrices.

Amino acid homeostasis in plants is finely tuned to match developmental needs and response to adverse environments. Over-expression of the single-transmembrane domain protein GLUTAMINE DUMPER1 (GDU1) leads to increased amino acid export, reduced growth and constitutive induction of immune responses. We used an inducible gene expression system to tease apart the primary and secondary effects caused byGDU1, and demonstrated that the primary effect is increasing amino acid export, followed by increased amino acid content and abscisic acid (ABA) response, and a subsequent activation of defense responses. TheGDU1-mediated hypersensitivity to ABA partially depended on the E3 ubiquitin ligase LOSS-OF-GDU1 2 (LOG2), a known GDU1 interactor. More importantly, the lysine catabolite pipecolic acid played a pivotal role in theGDU1-induced defense responses. This work unravels a novel relationship between amino acid transport, ABA and defense responses, potentially mediated by the GDU1-LOG2 complex, critical for understanding how plants respond to amino acid imbalance.ONE SENTENCE SUMMARYGenetically induced disturbance of amino acid homeostasis sequentially triggers responses to abiotic stresses and plant defenses to pathogens in Arabidopsis through undefined sensing mechanisms

Plant hormones play an important role in plant adaptation to abiotic stress, but hormonal responses of cool-season turfgrass species to salt stress are not well documented. This study was carried out to investigate the responses of hormones to salt stress and examine if salt stress-induced injury was associated with hormonal alteration in kentucky bluegrass (KBG, Poa pratensis L.). The grass was grown in a growth chamber for 6 weeks and then subjected to salt stress (170 mm NaCl) for 28 days. Salt stress caused cell membrane damage, resulting in photosynthetic rate (Pn), chlorophyll (Chl), and turf quality decline in KBG. Salt stress increased leaf abscisic acid (ABA) and ABA/cytokinin (CK) ratio; reduced trans-zeatin riboside (ZR), isopentenyl adenosine (iPA), and indole-3-acetic acid (IAA), but did not affect gibberellin A4 (GA4). On average, salt stress reduced ZR by 67.4% and IAA by 58.6%, whereas it increased ABA by 398.5%. At the end of the experiment (day 28), turf quality, Pn, and stomatal conductance (gs) were negatively correlated with ABA and ABA/CK ratio, but positively correlated with ZR, iPA, and IAA. Electrolyte leakage (EL) was positively correlated with ABA and ABA/CK and negatively correlated with ZR, iPA, IAA, and GA4. GA4 was also positively correlated with turf quality and gs. The results of this study suggest that salt stress-induced injury of the cell membrane and photosynthetic function may be associated with hormonal alteration and imbalance in KBG.

The methionine salvage pathway (MSP) is critical for regeneration of S-adenosyl-l-methionine (SAM), a widely used cofactor involved in many essential metabolic reactions. The MSP has been completely elucidated in aerobic organisms, and found to rely on molecular oxygen. Since anaerobic organisms do not use O2, an alternative pathway(s) must be operating. We sought to evaluate whether the functions of two annotated MSP enzymes from Methanocaldococcus jannaschii, a methylthioinosine phosphorylase (MTIP) and a methylthioribose 1-phosphate isomerase (MTRI), are consistent with functioning in a modified anaerobic MSP (AnMSP). We show here that recombinant MTIP is active with six different purine nucleosides, consistent with its function as a general purine nucleoside phosphorylase for both AnMSP and purine salvage. Recombinant MTRI is active with both 5-methylthioribose 1-phosphate and 5-deoxyribose 1-phosphate as substrates, which are generated from phosphororolysis of 5'-methylthioinosine and 5'-deoxyinosine by MTIP, respectively. Together, these data suggest that MTIP and MTRI may function in a novel pathway for recycling the 5'-deoxyadenosine moiety of SAM in M. jannaschii. These enzymes may also enable biosynthesis of 6-deoxy-5-ketofructose 1-phosphate (DKFP), an essential intermediate in aromatic amino acid biosynthesis. Finally, we utilized a homocysteine auxotrophic strain of Methanosarcina acetivorans Δma1821-22Δoahs (HcyAux) to identify potential AnMSP intermediates in vivo. Growth recovery experiments of the M. acetivorans HcyAux were performed with known and proposed intermediates for the AnMSP. Only one metabolite, 2-keto-(4-methylthio)butyric acid, rescued growth of M. acetivorans HcyAux in the absence of homocysteine. This observation may indicate that AnMSP pathways substantially differ among methanogens from phylogenetically divergent genera.

We have established the biosynthetic pathway and the associated genes for the biosynthesis of the 5-(aminomethyl)-3-furanmethanol (F1) moiety of methanofuran in the methanogenic archaeon Methanocaldococcus jannaschii. The recombinant enzyme, derived from the MJ1099 gene, was shown to readily condense glyceraldehyde 3-phosphate (Ga-3P) and dihydroxyacetone-P (DHAP) to form 4-(hydroxymethyl)-2-furancarboxaldehyde phosphate (4-HFC-P). The recombinant purified pyridoxal 5'-phosphate-dependent aminotransferase, derived from the MJ0684 gene, was found to be specific for catalyzing the transamination reaction between 4-HFC-P and [(15)N]alanine to produce [(15)N] 5-(aminomethyl)-3-furanmethanol-P (F1-P) and pyruvate. To confirm these results in cell extracts, we developed sensitive analytical methods for the liquid chromatography-ultraviolet-electrospray ionization mass spectrometry analysis of F1 as a 7-nitrobenzofurazan derivative. This method has allowed for the quantitation of trace amounts of F1 and F1-P in cell extracts and the measurement of the incorporation of stable isotopically labeled precursors into F1. After incubation of cell extracts with [1,2,3-(13)C3]pyruvate and DHAP, 4-([(2)H2]hydroxymethyl)-2-furancarboxylic acid phosphate (4-HFCA-P) or 4-([(2)H2]hydroxymethyl)-2-furancarboxaldehyde phosphate (4-HFC-P) was found to be incorporated into F1-P. 4-HFCA-P and 4-HFC-P were confirmed in cell extracts after removal of the phosphate. The low level of incorporation of [1,2,3-(13)C3]pyruvate into F1-P in these experiments is explained by the fact that the labeled pyruvate must first be converted into Ga-3-P through gluconeogenesis before being incorporated into 4-HFC-P. Cell extracts incubated with 4-HFC-P and a mixture of [(15)N]aspartate, [(15)N]glutamate, and [(15)N]alanine produced [(15)N]F1-P. We also demonstrated that aqueous solutions of methylglyoxal or pyruvate heated with dihydroxyacetone led to the formation of 4-HFC and 4-HFCA, suggesting a possible prebiotic route to this moiety of methanofuran.

Although fullerenes were discovered nearly three decades ago, the mechanism of their formation remains a mystery. Many versions of the classic 'bottom-up' formation mechanism have been advanced, starting with C2 units that build up to form chains and rings of carbon atoms and ultimately form those well-known isolated fullerenes (for example, I(h)-C60). In recent years, evidence from laboratory and interstellar observations has emerged to suggest a 'top-down' mechanism, whereby small isolated fullerenes are formed via shrinkage of giant fullerenes generated from graphene sheets. Here, we present molecular structural evidence for this top-down mechanism based on metal carbide metallofullerenes M2C2@C1(51383)-C84 (M = Y, Gd). We propose that the unique asymmetric C1(51383)-C84 cage with destabilizing fused pentagons is a preserved 'missing link' in the top-down mechanism, and in well-established rearrangement steps can form many well-known, high-symmetry fullerene structures that account for the majority of solvent-extractable metallofullerenes.

Structurally varied, carboxyl-containing cellulose derivatives were evaluated for their ability to form amorphous solid dispersions (ASD) with ellagic acid (EA), in order to improve the solubility of this high-melting, poorly bioavailable, but highly bioactive natural flavonoid compound. ASDs of EA with carboxymethylcellulose acetate butyrate (CMCAB), cellulose acetate adipate propionate (CAAdP), and hydroxypropylmethylcellulose acetate succinate (HPMCAS) were prepared, and EA dissolution from these ASDs was compared with that from pure crystalline EA and from EA/poly(vinylpyrrolidinone) (PVP) solid dispersions (SD). Polymer/drug mixtures were characterized by powder X-ray diffraction (XRPD), modulated differential scanning calorimetry (MDSC), nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FT-IR). The XRPD and FT-IR results indicated that EA was amorphous in solid dispersions with EA concentration up to 25 wt%. The stability against crystallization and solution concentrations of EA from these solid dispersions were significantly higher than those observed for physical mixtures and pure crystalline EA. HPMCAS stabilized EA most effectively, among the polymers tested, against both chemical degradation and recrystallization. The relative ability to solubilize EA from ASDs at pH 6.8 was PVP>>HPMCAS>>CMCAB. EA dissolves from ASD in PVP quickly and completely (maximum 92%) at pH 6.8, but EA is also released from PVP at pH 1.2, and then crystallizes rapidly. Therefore PVP is not a practical candidate for EA ASD. In contrast, the cellulose derivative ASDs show very slow EA release at pH 1.2 (

Amorphous solid dispersions (ASD) of quercetin (Que) in cellulose derivative matrices, carboxymethylcellulose acetate butyrate (CMCAB), hydroxypropylmethylcellulose acetate succinate (HPMCAS), and cellulose acetate adipate propionate (CAAdP) were prepared with the goal of identifying an ASD that effectively increased Que aqueous solution concentration. Crystalline quercetin and Que/poly(vinylpyrrolidinone) (PVP) ASD were evaluated for comparison. Powder X-ray diffraction (XRPD) and differential scanning calorimetry (DSC) were used to examine the crystallinity of ASDs, physical mixtures (PM) and quercetin. ASDs were amorphous up to 50 wt% Que. Que stability against crystallization and solution concentrations from these ASDs were significantly higher than those observed for physical mixtures and crystalline Que. PVP stabilizes against both Que degradation and recrystallization; in contrast, these carboxylated cellulose derivatives inhibit recrystallization but release Que slowly. PVP ASDs afforded fast and complete drug release, while ASDs using these three cellulose derivatives provide slow, incomplete, pH-triggered drug release.

An innovative approach for measuring phytase activity (PA) in surface water is presented. A substrate analog of -inositol hexakis(dihydrogen) phosphate (InsP), commonly referred to as phytic acid, 1--5--(1-oxo-1-(2' ,4,7,7' -tetrachloro-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)-5,8,11-trioxa-2-azatridecan-13-yl)-inositol 1,2,3,4,6-pentakis--(dihydrogen) phosphate, referred to as tetrachlorofluorescein (TET) tethered (T)InsP, has been developed that can be used to monitor the (phytase-catalyzed) phosphate ester bond-cleavage reaction. Test phytases, (wheat [4-] and [3-] phytase) sequentially remove phosphate groups from TET TInsP, producing dephosphorylated probe species that were readily separated by reversed-phase high-performance liquid chromatography (RP-HPLC). Because dephosphorylated probe species retain the TET group, highly sensitive quantification could be achieved using fluorescence detection (excitation/emission ' = 245/540 nm). Calibration curves for TET TInsP, which could be used as a standard for quantifying all probe species, were linear ( > 0.999) over the range of concentrations tested. Phytase-generated dephosphorylated probe species were characterized or identified using RP-HPLC with mass spectrometry. Results of mass spectrometry analysis show that the RP-HPLC system was capable of distinguishing between dephosphorylated probe species at the regioisomeric level. The TET TInsP molecular probe was used to successfully measure PA in pond water. We found that the PA associated with the particulate plus water-soluble fraction was greater than that observed for the water-soluble fraction alone. Moreover, it appeared that 4- and 3-phytase were active in pond water based on an analysis of the chromatographic profile (i.e., elution sequence) of dephosphorylated probe species produced. The advent of a fluorescent substrate analog of InsP affords environmental scientists with the means to unambiguously quantify an extremely small amount of phytase-generated dephosphorylated product(s), enabling the measurement of PA over a reasonably short time duration, in an environmental sample containing low concentrations of enzyme.

Aspergillus fumigatus is an opportunistic fungal pathogen and the most common causative agent of fatal invasive mycoses. The flavin-dependent monooxygenase siderophore A (SidA) catalyzes the oxygen and NADPH dependent hydroxylation of l-ornithine (l-Orn) to N5-l-hydroxyornithine in the biosynthetic pathway of hydroxamate-containing siderophores in A. fumigatus. Deletion of the gene that codes for SidA has shown that it is essential in establishing infection in mice models. Here, a fluorescence polarization high-throughput assay was used to screen a 2320 compound library for inhibitors of SidA. Celastrol, a natural quinone methide, was identified as a noncompetitive inhibitor of SidA with a MIC value of 2 μM. Docking experiments suggest that celastrol binds across the NADPH and l-Orn pocket. Celastrol prevents A. fumigatus growth in blood agar. The addition of purified ferric-siderophore abolished the inhibitory effect of celastrol. Thus, celastrol inhibits A. fumigatus growth by blocking siderophore biosy...

The first dual component pseudocryptand-type [2]pseudorotaxanes were designed and prepared via the self-assembly of synthetically easily accessible bis(meta-phenylene)-32-crown-10 pyridyl, quinolyl, and naphthyridyl derivatives with paraquat. The formation of the pseudocryptand structures in the complexes remarkably improved the association constant by forming the third pseudobridge via H-bonding with the guest and π-stacking of the heterocyclic units.

Multitopic dibenzylammonium derivatives (4) of C60 were prepared by Bingel reactions of C60 with a malonate diester (2) containing two t-BOC protected dibenzylamine moieties, followed by deprotection and protonation. Self-assembly of model pseudorotaxanes 5 from the multidibenzylammonium C60 derivatives with dibenzo-24-crown-8 was studied by 1H NMR spectroscopy and mass spectrometry. Self-assembly of linear and star-shaped pseudorotaxanes 8 with up to 12 arms based on polystyrenes bearing terminal DB24C8 host units (7) and the guest functionalized C60 salts was demonstrated by 1H NMR spectroscopy and solution phase viscometry. These studies provide further evidence of the potential of supramacromolecular chemistry in construction of complex polymeric architectures. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 6472–6495, 2009

Photochemically generated benzyl radicals react with Sc(3)N@C(80)-I(h) to produce a dibenzyl adduct [Sc(3)N@C(80)(CH(2)C(6)H(5))(2)] in 82% yield and high regioselectivity. The adduct's (1)H spectrum revealed high symmetry: only one AB pattern was observed for the methylene protons. The (13)C NMR spectrum suggested a C(2)-symmetrical structure. DFT calculations reveal that a 1,4-adduct is more favorable than a 1,2-adduct by10 kcal/mol. The 1,4-structure on [566] ring junctions was unambiguously confirmed by X-ray crystallographic analysis. UV-vis spectra revealed that the removal of two p orbitals from the pi system of the cage together with the benzylic substituents change the electronic properties of the metallofullerene in a manner similar to those reported for disilirane and trifluoromethyl moieties. Under the same conditions from Lu(3)N@C(80)-I(h) we prepared (63% yield) Lu(3)N@C(80)(CH(2)C(6)H(5))(2), which demonstrated properties similar to the 1,4-dibenzyl adduct of Sc(3)N@C(80)-I(h).

Low-molecular-weight liquid polybutadienes (1000-2 000 g mol -1 ) consisting of 60 mol-% poly(buta-1,2-diene) repeating units were synthesized via anionic telomerization. Maintaining the initiation and reaction temperature at less than 70 °C minimized chain transfer and enabled the polymerization to occur in a living fashion, which resulted in well-controlled molecular weights and narrow polydispersity indices. MALDI-TOF mass spectrometry confirmed that the end groups of liquid polybutadienes synthesized via anionic telomerization contained one benzyl end and one protonated end. In comparison, the end groups of liquid polybutadienes synthesized via living anionic polymerization contained one sec-butyl or butyl end and one protonated end.

Metallic nitride fullerenes (MNFs) are of interest to fundamental studies and applications. However, MNF sample availability for these studies has been limited. The lack of samples is related to the currently used chromatographic (HPLC) separation protocol, a tedious approach that is solvent intensive, expensive, and has a limited throughput. To address these drawbacks, we developed an alternative method (non-chromatographic) for removing contaminant C60 and C70 empty-cage fullerenes and other classical metallofullerenes (i.e. non-nitrogen containing endohedrals) from soot extracts. Specifically, we present a purification approach based on differences of cage reactivity of MNFs versus other fullerenes. Results indicate that reactive silica can preferentially bind to contaminant fullerenes and metallofullerenes using our Stir and Filter Approach (SAFA). With this process, fullerene contaminants are immobilized onto reactive silica, while unreacted MNFs remain in the filtrate. In this manner, purified samples of Sc3N@C80 are readily obtained.

Separation difficulties have led to a paucity of purified metallic nitride fullerenes (MNFs). Fundamental research and application development has been hampered with limited sample availability. Separation techniques designed to remove contaminant empty-cage fullerenes (e.g., C(60), C(70)...C(2)(n)) and classical metallofullerenes (e.g., non-MNFs) traditionally require expensive and tedious chromatographic methods. Our motivation is an alternative purification approach to minimize dependence on HPLC. Herein we report the use of cyclopentadienyl (CPD) and amino functionalized silica to selectively bind contaminant fullerenes. This "Stir and Filter Approach" (SAFA) provides purified MNF samples at ambient and reflux conditions. Under reflux conditions, purified MNFs (80% recovery, 41 h) are obtained using CPD silica. However, at room temperature, there is an equilibrium established between fullerenes and CPD silica, and no purified MNF samples are obtained using SAFA. In contrast, purified MNF samples (99+%) are readily obtained at room temperature using amino, diamino, and triamino silica at recoveries of 93% (11 h), 76% (9 h), and 50% (6 h), respectively.

The pure I(h) isomer of Sc3N@C80 was allowed to react with N-triphenylmethyl-5-oxazolidinone via the corresponding azomethine ylide. The reaction results in the formation of two monoadducts; one (1b) is the kinetic product, and the other (1a) is thermodynamically more stable. Small amounts of the bisadducts were also formed. The structure of the thermodynamic monoadduct 1a was shown conclusively by NMR spectroscopy and X-ray crystallography to result from addition across the 5,6-ring junction. The kinetic product 1b was demonstrated to be the 6,6-ring juncture adduct on the basis of NMR experiments and X-ray crystallography. In refluxing chlorobenzene pure 1b was converted to the more thermodynamically stable 1a isomer. These N-tritylpyrrolidino derivatives are potentially useful precursor compounds for further derivatization for various applications.

A crude extract of a marine alga showed activity against the enzyme Myt1 kinase. Bioassay-directed fractionation led to the isolation of two bioactive glycoglycerolipids. Lipid 1 was identified as sn-1,2-dipalmityl-3-(N-palmityl-6-deoxy-6-amino-a-D-glucosyl) glycerol and lipid2 as sn-1-palmityl-2-myristyl-3-(N-stearyl-6-deoxy-6-aminoglucosyl)glycerol. Compounds 1 and2 had IC50 values of 0.12 and 0.43 mg/mL, respectively, in the Myt1 kinase inhibitory bioassay, and were inactive against Akt and Chk1 kinases. q 2004 Elsevier Ltd. All rights reserved.

Methanofuran is the first in a series of coenzymes involved in the reduction of carbon dioxide to methane. All methanofuran structural variants contain a basic core structure of 4-[N-(γ-l-glutamyl-γ-l-glutamyl)-p-(β-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan (APMF-(Glu)2) with different attached side chains depending on the source organism. Recently, we discovered the biosynthetic route for the production of 5-(aminomethyl)-3-furanmethanol-phosphate (F1-P), a precursor to the furan moiety of methanofuran. However, how the γ-linked glutamates are incorporated into methanofuran's structure remains unknown. Here, we report the identification of an ATP-grasp enzyme encoded by the gene Mefer_1180 in Methanocaldococcus fervens (the homologue of MJ0815 in Methanocaldococcus jannaschii, annotated as MfnD) that catalyzes the ATP-dependent addition of one glutamate to tyramine via a γ-linked amide bond. The occurrence of this reaction is consistent with the presence of γ-glutamyltyramine in cell extracts of M. jannaschii. Our steady-state kinetic analysis of the recombinant enzyme showed that MfnD exhibits a catalytic ability comparable to other ATP-grasp enzymes such as the Escherichia coli glutathione synthetase (GS), with a similar apparent kcat and KM. In addition, its activity is divalent metal-dependent, with the highest activity observed with Mn(2+). The previously solved crystal structure of MfnD from Archaeoglobus fulgidus exhibits a classical ATP-grasp fold with three structural domains; the ATP-binding and metal-binding motifs are conserved in MfnD as seen in other ATP-grasp enzymes. We used site-directed mutagenesis and kinetic analysis to demonstrate that Arg251 is an important residue for both catalysis and glutamate binding. By comparing the active site of MfnD with GS and by molecular docking substrates to the MfnD active site, we predicted the possible glutamate- and tyramine-binding pocket. This is the first report describing the enzymology of the incorporation of the initial l-glutamate molecule into the methanofuran structure. It also provides the first example of an ATP-grasp enzyme activating the γ-carboxylate of glutamate as substrate.

An innovative approach for measuring phytase activity (PA) in surface water is presented. A substrate analog of -inositol hexakis(dihydrogen) phosphate (InsP), commonly referred to as phytic acid, 1--5--(1-oxo-1-(2' ,4,7,7' -tetrachloro-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)-5,8,11-trioxa-2-azatridecan-13-yl)-inositol 1,2,3,4,6-pentakis--(dihydrogen) phosphate, referred to as tetrachlorofluorescein (TET) tethered (T)InsP, has been developed that can be used to monitor the (phytase-catalyzed) phosphate ester bond-cleavage reaction. Test phytases, (wheat [4-] and [3-] phytase) sequentially remove phosphate groups from TET TInsP, producing dephosphorylated probe species that were readily separated by reversed-phase high-performance liquid chromatography (RP-HPLC). Because dephosphorylated probe species retain the TET group, highly sensitive quantification could be achieved using fluorescence detection (excitation/emission ' = 245/540 nm). Calibration curves for TET TInsP, which could be used as a standard for quantifying all probe species, were linear (0.999) over the range of concentrations tested. Phytase-generated dephosphorylated probe species were characterized or identified using RP-HPLC with mass spectrometry. Results of mass spectrometry analysis show that the RP-HPLC system was capable of distinguishing between dephosphorylated probe species at the regioisomeric level. The TET TInsP molecular probe was used to successfully measure PA in pond water. We found that the PA associated with the particulate plus water-soluble fraction was greater than that observed for the water-soluble fraction alone. Moreover, it appeared that 4- and 3-phytase were active in pond water based on an analysis of the chromatographic profile (i.e., elution sequence) of dephosphorylated probe species produced. The advent of a fluorescent substrate analog of InsP affords environmental scientists with the means to unambiguously quantify an extremely small amount of phytase-generated dephosphorylated product(s), enabling the measurement of PA over a reasonably short time duration, in an environmental sample containing low concentrations of enzyme.

Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site residues.

The nanoscale parameters of metal clusters and lattices have a crucial influence on the macroscopic properties of materials. Herein, we provide a detailed study on the size and shape of isolated yttrium carbide clusters in different fullerene cages. A family of diyttrium endohedral metallofullerenes with the general formula of Y(2)C(2n) (n = 40-59) are reported. The high field (13)C nuclear magnetic resonance (NMR) and density functional theory (DFT) methods are employed to examine this yttrium carbide cluster in certain family members, Y(2)C(2)@D(5)(450)-C(100), Y(2)C(2)@D(3)(85)-C(92), Y(2)C(2)@C(84), Y(2)C(2)@C(3v)(8)-C(82), and Y(2)C(2)@C(s)(6)-C(82). The results of this study suggest that decreasing the size of a fullerene cage with the same (Y(2)C(2))(4+) cluster results in nanoscale fullerene compression (NFC) from a nearly linear stretched geometry to a constrained "butterfly" structure. The (13)C NMR chemical shift and scalar (1)J(YC) coupling parameters provide a very sensitive measure of this NFC effect for the (Y(2)C(2))(4+) cluster. The crystal structural parameters of a previously reported metal carbide, Y(2)C(3) are directly compared to the (Y(2)C(2))(4+) cluster in the current metallofullerene study.

We describe an automated HPLC separation of the endohedral metallofullerenes such as SCC{sub 2n} and YC{sub 2n} from empty-cage fullerenes utilizing two polystyrene chromatographic columns (500 and 1000 A) in series. Rapid separation of the metallofullerene fraction from the empty-cage fullerenes (e.g., C{sub 60}) under anaerobic conditions is achieved. For the isolated SCC{sub 2n} fraction, all even-carbon-membered species from Sc{sub 2}C{sub 74} to Sc{sub 2}C{sub 104} were identified by negative-ion chemical ionization mass spectrometry. In addition, Sc{sub 3}C{sub 82} was a prominent component of this fraction. For the separated YC{sub 2n} sample, the mass spectral data indicate the presence of YC{sub 82} and all even-carbon-numbered diyttrium species from Y{sub 2}C{sub 82} to Y{sub 2}C{sub 104}. 14 refs., 7 figs.

IT was first proposed in 1985 that fullerenes can confine atoms in their interior because of their closed-cage structure. Attempts to verify this conjecture following the mass production of fullerenes have yielded metallofullerenes in bulk, and there is now good evidence that these compounds are endohedral—that is, that the metal atoms are inside. But direct confirmation in the form of structural data has been lacking, in part because of the difficulty of separating different metallofullerenes and obtaining pure crystals. Here we report the preparation of pure crystalline Sc_2@C_(84) and analyses of its structure by electron diffraction and high-resolution transmission electron microscopy. At room temperature the Sc_2@C_(84) molecules pack in a hexagonal-close-packed structure with a ratio of lattice constants c/ɑ = 1.63, the value expected for ideal-sphere packing. The molecular spacing of 11.2 A is the same as that found earlier in crystalline C_(84) (refs 5, 6). The match between our microscopic images and simulations is markedly better for endohedral models than for those in which the metal atoms reside in the lattice outside the C_(84) cages. We believe that this combination of observations points inevitably to the conclusion that the metal atoms are inside the fullerenes.

Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46 mM) and dopa (km 1.40 mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine.

Potato (Solanum tuberosum L.), a domesticated species that is the fourth most important world agricultural commodity, requires significant management to minimize the effects of herbivore and pathogen damage on crop yield. A wild relative, Solanum chacoense Bitt., has been of interest to plant breeders because it produces an abundance of novel steroidal glycoalkaloid compounds, leptines and leptinines, which are particularly effective deterrents of herbivory by the Colorado potato beetle (Leptinotarsa decemlineata Say). Biochemical approaches were used in this study to investigate the formation and accumulation of SGAs in S. chacoense. SGA contents were determined in various organs at different stages of organ maturity during a time course of plant development. Leptines and leptinines were the main contributors to the increased levels in SGA concentration measured in the aerial versus the subterranean organs of S. chacoense accession 8380-1. Leptines were not detected in aboveground stolons until the stage where shoots had formed mature chlorophyllous leaves. To gain insights into SGA biosynthesis, the abundance of SGAs and steady-state transcripts of genes coding for enzymes of the central terpene and SGA-specific pathways in various plant organs at anthesis were compared. For two genes of primary terpene metabolism, transcript and SGA abundances were correlated, although with some discrepancies. For genes associated with SGA biosynthesis, transcripts were not detected in some tissues containing SGAs; however these transcripts were detected in the progenitor tissues, indicating the possibility that under our standard growth conditions, SGA biosynthesis is largely limited to highly proliferative tissues such as shoot, root and floral meristems.

The dimetallic endohedral heterofullerene (EHF), Gd(2)@C(79)N, was prepared and isolated in a relatively high yield when compared with the earlier reported heterofullerene, Y(2)@C(79)N. Computational (DFT), chemical reactivity, Raman, and electrochemical studies all suggest that the purified Gd(2)@C(79)N, with the heterofullerene cage, (C(79)N)(5-) has comparable stability with other better known isoelectronic metallofullerene (C(80))(6-) cage species (e.g., Gd(3)N@C(80)). These results describe an exceptionally stable paramagnetic molecule with low chemical reactivity with the unpaired electron spin density localized on the internal diatomic gadolinium cluster and not on the heterofullerene cage. EPR studies confirm that the spin state of Gd(2)@C(79)N is characterized by a half-integer spin quantum number of S = 15/2. The spin (S = ½) on the N atom of the fullerene cage and two octet spins (S = 7/2) of two encapsulated gadoliniums are coupled with each other in a ferromagnetic manner with a small zero-field splitting parameter D. Because the central line of Gd(2)@C(79)N is due to the Kramer's doublet with a half-integer spin quantum number of S = 15/2, this relatively sharp line is prominent and the anisotropic nature of the line is weak. Interestingly, in contrast with most Gd(3+) ion environments, the central EPR line (g = 1.978) is observable even at room temperature in a toluene solution. Finally, we report the first EHF derivative, a diethyl bromomalonate monoadduct of Gd(2)@C(79)N, which was prepared and isolated via a modified Bingel-Hirsch reaction.

In this paper, we report the synthesis, purification, (13)C NMR, and other characterization studies of Y(3)N@C(88). The (13)C NMR, UV-vis, and chromatographic data suggest an Y(3)N@C(88) having an IPR-allowed cage with D(2)(35)-C(88) symmetry. In earlier density functional theory (DFT) computational and X-ray crystallographic studies, it was reported that lanthanide (A(3)N)(6+) clusters are stabilized in D(2)(35)-C(88) symmetry cages and have reduced HOMO-LUMO gaps relative to other trimetallic nitride endohedral metallofullerene cage systems, for example, A(3)N@C(80). In this paper, we report that the nonlanthanide (Y(3)N)(6+) cluster in the D(2)(35)-C(88) cage exhibits a HOMO-LUMO gap consistent with other lanthanide A(3)N@C(88) molecules based on electrochemical measurements and DFT computational studies. These results suggest that the reduced HOMO-LUMO gap of A(3)N@C(88) systems is a property dominated by the D(2)(35)-C(88) carbon cage and not f-orbital lanthanide electronic metal cluster (A(3)N)(6+) orbital participation.

The members of a new family of yttrium trimetallic nitride-templated (TNT) endohedral metallofullerenes (EMFs), Y(3)N@C(2n) (n = 40-43), have been synthesized and purified. On the basis of experimental and computational (13)C NMR studies, we propose cage structures for Y(3)N@I(h)-C(80) (IPR allowed), Y(3)N@D(5h)-C(80) (IPR allowed), Y(3)N@C(s)-C(82) (non-IPR), Y(3)N@C(s)-C(84) (non-IPR), and Y(3)N@D(3)-C(86) (IPR allowed). A significant result is the limited number of isomers found for each carbon cage. For example, there are 24 isolated pentagon rule (IPR) and 51 568 non-IPR structures possible for the C(84) cage, but only one major isomer of Y(3)N@C(s)-C(84) was found. The current study confirms the unique role of the trimetallic nitride (M(3)N)(6+) cluster template in the Kratschmer-Huffman electric-arc process for fullerene cage size and high symmetry isomer selectivity. This study reports the first (89)Y NMR results for Y(3)N@I(h)-C(80,) Y(3)N@C(s)(51365)-C(84), and Y(3)N@D(3)(19)-C(86), which reveal a progression from isotropic to restricted (Y(3)N)(6+) cluster motional processes. Even more surprising is the sensitivity of the (89)Y NMR chemical shift parameter to subtle changes in the electronic environment at each yttrium nuclide in the (Y(3)N)(6+) cluster (more than 200 ppm for these EMFs). This (89)Y NMR study suggests that (89)Y NMR will evolve as a powerful tool for cluster motional studies of EMFs.

Two new 6,6-open phenyl-C(81)-butyric acid methyl ester metallofulleroids, M(3)N@C(80)PCBM (M = Sc, Y), were synthesized by diazoalkane addition reactions and fully characterized. The results demonstrate that the reactive sites are the same for M(3)N@C(80) (M = Sc, Y) but dramatically different from that of C(60).

The idea1 that fullerenes might be able to encapsulate atoms and molecules has been verified by the successful synthesis of a range of endohedral fullerenes, in which metallic or non-metallic species are trapped inside the carbon cage2,3,4,5,6,7,8,9,10,11,12,13. Metal-containing endohedral fullerenes have attracted particular interest as they might exhibit unusual material properties associated with charge transfer from the metal to the carbon shell. However, current synthesis methods have typical yields of less than 0.5%, and produce multiple endohedral fullerene isomers, which makes it difficult to perform detailed studies of their properties. Here we show that the introduction of small amounts of nitrogen into an electric-arc reactor allows for the efficient production of a new family of stable endohedral fullerenes encapsulating trimetallic nitride clusters, ErxSc3-xN@C80 (x = 0–3). This ‘trimetallic nitride template’ process generates milligram quantities of product containing 3–5% Sc3N@C80, which allows us to isolate the material and determine its crystal structure, and its optical and electronic properties. We find that the Sc3N moiety is encapsulated in a highly symmetric, icosahedral C80 cage, which is stabilized as a result of charge transfer between the nitride cluster and the fullerene cage. We expect that our method will provide access to a range of small-bandgap fullerene materials, whose electronic properties can be tuned by encapsulating nitride clusters containing different metals and metal mixtures.

The first reactions of trimetallic nitride templated endohedral metallofullerenes (TNT EMFs) with carbon radicals generated from diethyl malonate catalyzed by manganese(III) acetate are reported. Two methano monoadducts, Sc3N@C80-A and Sc3N@C80-B, were isolated and characterized. Sc3N@C80-A contains two ester moieties, whereas Sc3N@C80-B contains only one ester group and a hydrogen atom on the central carbon of the addend. NMR spectroscopy of the two monoadducts suggests that the addition occurs regioselectively at a 6,6-ring juncture on the surface of the icosahedrally (Ih) symmetric Sc3N@C80, forming the first 6,6-ring-bridged methano Ih Sc3N@C80 derivatives. The measured 1J(C,H) = 147 Hz for the methano carbon with its hydrogen in monoadduct Sc3N@C80-B nearly perfectly matches the data for pi-homoaromatic systems, indicating an open [6,6]-methano structure. Geometry optimization also found that the "closed" [6,6]-methano structures were energetically unstable and always led to the open forms. Thus, an "open" [6,6]-methanofulleride structure is proposed, which was induced by the norcaradiene rearrangement, resulting in the cleavage of the cyclopropane ring and the formation of energetically stable open cage fullerene derivatives. These are the first examples of thermodynamically stable adducts of the "open" type at the 6,6-ring juncture of Ih Sc3N@C80, differing greatly from the "closed" 5,6-ring juncture adducts reported previously. In addition, bis-, tri-, and up to octaadducts of Sc3N@C80 were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry; this synthetic method was also applied to Lu3N@C80, producing adducts with up to 10 substituents on the carbon cage. These are the highest levels of substitution of TNT metallofullerenes reported so far.

We used Shiga-like toxin B subunit (SLTB) to deliver the photosensitizer, chlorin e6 (Ce6), to Vero cells expressing the Gb3 receptor. Our aim was to provide an example of carrier-enhanced photodynamic cell killing with which to start a systematic consideration of photosensitizer delivery at the subcellular level. SLTB, in contrast to many other potential protein carriers, is delivered intracellularly to the Golgi apparatus and endoplasmic reticulum (ER). Ce6 was chosen both for its phototoxic properties and its potential for covalent conjugation with SLTB. Ce6-SLTB after cleanup contained < or =10% noncovalently bound Ce6. The noncovalent binding of porphyrins and chlorins to protein conjugates has been well documented, and hence the effective cleanup procedure is a significant accomplishment. We demonstrate that Ce6-SLTB enhances delivery of Ce6 to target cells as compared to free Ce6. In Vero cells, Ce6-SLTB was over an order of magnitude more photodynamically toxic than free Ce6. Moreover, we show that in the case of Ce6-SLTB, photosensitizer accumulation is in a combination of subcellular sites including mitochondria, Golgi apparatus, ER and plasma membrane. The occurrence in nature of diverse B subunit binding sites and the possibilities of varied intracellular delivery make optimized use of B subunit carriers attractive.

In the evolving field of fullerenes, nanotubes and endohedral metallofullerenes, the isolated-pentagon rule (IPR)1 is sacrosanct — exceptions have been predicted2,3, but no bare carbon cages with adjacent pentagons have been characterized. Small organic molecules with metal-stabilized fused five-membered rings (pentalenes) have been created4, however, and here we describe a family of non-classical endohedral metallofullerenes with the general structure AxSc3−xN@C68 (where x = 0–2, A is a rare-earth metal, Sc is scandium and N is nitrogen) that has a non-IPR cage of only 68 carbon atoms containing annelated five-membered rings. This internal ring network is metal-stabilized and is accessible for external organic reaction chemistry.

The Kratschmer-Huffman electric-arc generator typically produces endohedral metallofullerenes in low yields with a wide array of different products, but the introduction of nitrogen leads to a new family of encapsulates. A family of endohedral metallofullerenes AnB3−nN@C2n (n=0–3, x=34, 39, and 40) where A and B are Group III and rare-earth metals is formed by a trimetallic nitride template (TNT) process in relatively high yields. The archetypal representative of this new class is the stable endohedral metallofullerene, Sc3N@C80 containing a triscandium nitride cluster encapsulated in an icosahedron (Ih), C80 cage. The Sc3N@C80 is formed in yields even exceeding empty-cage C84. Other prominent scandium TNT members are Sc3N@C68 and Sc3N@C78. The former Sc3N@C68 molecule represents an exception to the well known isolated pentagon rule (IPR). These new molecules were purified by chromatography with corresponding characterization by various spectroscopic approaches. In this paper we focus on the characterizat...

The biochemistry of the 13 steps involved in the conversion of alpha-ketoglutarate and acetylCoA to alpha-ketosuberate, a precursor to the coenzymes coenzyme B (7-mercapto heptanoylthreonine phosphate) and biotin, has been established in Methanosarcina thermophila. These series of reactions begin with the condensation of alpha-ketoglutarate and acetylCoA to form trans-homoaconitate. The trans-homoaconitate is then hydrated and dehydrated to cis-homoaconitate with (S)-homocitrate serving as an intermediate. Rehydration of the cis-homoaconitate produces (-)-threo-isohomocitrate [(2R,3S)-1-hydroxy-1,2, 4-butanetricarboxylic acid], which undergoes a NADP+-dependent oxidative decarboxylation to produce alpha-ketoadipate. The resulting alpha-ketoadipate then undergoes two consecutive sets of alpha-ketoacid chain elongation reactions to produce alpha-ketosuberate. In each of these sets of reactions, it has been shown that the homologues of cis-homoaconitate, homocitrate, and (-)-threo-isohomocitrate serve as intermediates. The protein product of the Methanococcus jannaschii MJ0503 gene aksA (AksA) was found to catalyze the condensation of alpha-ketoglutarate and acetylCoA to form trans-homoaconitate. This gene product also catalyzed the condensation of alpha-ketoadipate or alpha-ketopimelate with acetylCoA to form, respectively, the (R)-homocitrate homologues of (R)-2-hydroxy-1,2,5-pentanetricarboxylic acid and (R)-2-hydroxy-1,2, 6-hexanetricarboxylic acid. The alpha-ketosuberate resulting from this series of reactions then undergoes a nonoxidative decarboxylation to form 7-oxoheptanoic acid, a precursor to coenzyme B, and an oxidative decarboxylation to form pimelate, the precursor to biotin. Of the 13 intermediates in this pathway, eight have not previously been reported as occurring in biological systems.

The structure of isomer 2 of Tb3N@C84 has been determined through single-crystal X-ray diffraction on Tb3N@C84.NiII(OEP).2(C6H6). The carbon cage has a distinct egg shape due to the presence of a single pair of fused pentagons at one apex of the molecule. Thus, although 24 IPR structures are available to the C84 cage, Nature utilizes one of the 51 568 isomeric structures that do not conform to the IPR for this unusual molecule. The Tb3N portion of isomer 2 of Tb3N@C84 is strictly planar. One Tb atom is nestled within the fold of the fused pentagons, while the other Tb atoms are disordered over four pairs of sites.

Since the initial discovery of fullerenes nearly a decade ago [1], material scientists have focused attention on the possibility of encapsulating one or more metal atoms inside these spheroidal carbon frames. The experimental realization of macroscopic quantities of endohedral metallofullerenes (Am@C2n, n=30-55) in the early 1990's has heightened interest in developing this new class of tunable materials with possible electronic and/or optical applications [2,3]. They have been characterized by a number of spectroscopic techniques, for example, scanning tunneling microscope [4,5], EXAFS [6,7] and x-ray diffraction and electron microscopy [8]. However, low production yields and purification difficulties have hampered the development of this new class of materials. The soluble product distribution usually consists of high levels of the empty-caged fullerenes C60, C70, C84 and decreasing levels of the higher fullerenes, while the endohedral metallofullerene fraction usually constitutes less than 1% of the total soluble yield. Furthermore, the endohedral metallofullerene fraction consists of molecules with different numbers of metal atoms encapsulated (m=1-3), cage sizes (C2n) and isomers of the same mass (e.g., Er2@C82). The purification process is further complicated by the chemical reactivity of several endohedral metallofullerenes [9] in aerobic environments. For several years, we have been involved in a collaborative effort to develop methodology for detection, isolation, and characterization of endohedral metallofullerenes. The focus of the present study is on fullerenes encapsulating metals from Group II1b, (Sc@C2n, Y@C2n, and La@C2n) and the lanthanide series metal (Er@C2n).

The direct coupling of high-performance liquid chromatography (HPLC) with on-line electron paramagnetic resonance (EPR) detection is demonstrated for monitoring separations of endohedral metallofullerenes (MC{sub 2a}). The HPLC-EPR approach readily permits detection of the paramagnetic species, such as YC{sub 82} and Sc{sub 3}C{sub 82}, in the presence of the dominant empty-cage fullerenes (C{sub 60}, C{sub 70}) and diamagnetic metallofullerenes (e.g., M{sub 2}C{sub 2n}). The results indicate that on-line EPR provides a noninvasive, selective detector for HPLC metallofullerene separations that is readily adaptable to air-sensitive and/or labile compounds. Specifically, the `EPR-active` metallofullerenes, YC{sub 82} and Sc{sub 3}C{sub 82}, are selectively monitored on-line for an initial separation of the metallofullerene fraction from the dominant empty-cage fullerenes utilizing a combination of polystyrene columns. This preparative `cleanup` procedure is followed by HPLC-EPR separation and monitoring of YC{sub 82} and Sc{sub 3}C{sub 82} species using a selective tripodal {pi}-acidic-phase column (Trident-Tri-DNP) for the final stages of isolation. 30 refs., 8 figs.

The reaction of Sc3N@C80 with 6,7-dimethoxyisochroman-3-one (13C labeled) provides the first functionalized derivative of the trimetallic nitride template (TNT) endohedral metallofullerene family. The reaction mixture is dominated by a single 13C labeled monoadduct product that was purified by HPLC. The 13C labeled monoadduct was characterized by 1H NMR, 13C NMR, and MALDI-TOF mass spectrometry. The proposed structure for this novel symmetric monoadduct is consistent with derivatization at the [5,6] ring juncture on the Sc3N@C80 cage.

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