The Conversion of Starch and Sugars into Branched C10 and C11 Hydrocarbons.

Oligosaccharides, such as starch, cellulose, and hemicelluloses, are abundant and easily obtainable bio-derived materials that can potentially be used as precursors for fuels and chemical feedstocks. To access the pertinent molecular building blocks (i.e., 5- or 6-carbon containing sugar units) located within these biopolymers and transform them into useful fuel precursors, oligosaccharide depolymerization followed by chain extension is required. This chain extension can readily be performed via a Garcia-Gonzalez-like approach using ß-diketones under mild conditions to provide fuel precursors containing an increased carbon atom content that meets fuel requirements. In a subsequent step, ring opening and hydrodeoxygenation chemistry of these species allows for the preparation of branched alkanes under relatively mild conditions. This approach can be applied to monomeric sugars (glucose and xylose), oligosaccharides (starch), and potentially to hydrolyzed dedicated energy crops to allow the conversion of real biomass into fuel type molecules.

The hydrodeoxygenation of bioderived furans into alkanes.

The conversion of biomass into fuels and chemical feedstocks is one part of a drive to reduce the world's dependence on crude oil. For transportation fuels in particular, wholesale replacement of a fuel is logistically problematic, not least because of the infrastructure that is already in place. Here, we describe the catalytic defunctionalization of a series of biomass-derived molecules to provide linear alkanes suitable for use as transportation fuels. These biomass-derived molecules contain a variety of functional groups, including olefins, furan rings and carbonyl groups. We describe the removal of these in either a stepwise process or a one-pot process using common reagents and catalysts under mild reaction conditions to provide n-alkanes in good yields and with high selectivities. Our general synthetic approach is applicable to a range of precursors with different carbon content (chain length). This allows the selective generation of linear alkanes with carbon chain lengths between eight and sixteen carbons.

C-C or C-O bond cleavage in a phenolic lignin model compound: selectivity depends on vanadium catalyst.

The aerobic oxidation of a phenolic lignin model compound with a vanadium catalyst results in the oxidative cleavage of the C-C bond between the aryl ring and the adjacent hydroxy-substituted carbon atom. Labeling experiments indicate key mechanistic differences to a previously reported related C-O bond cleavage reaction. The selectivity in C-C versus C-O bond cleavage depends on the choice of the vanadium catalyst.

Design, synthesis, and a novel application of quorum-sensing agonists as potential drug-delivery vehicles.

Surface adhered bacterial colonies or biofilms are an important problem in medical and food industries. Bacteria use a chemical language to monitor their quorum and to express virulence factors, which eventually help them in colonization and manifestation of an infection. The LasR-LasI and RhlR-RhlI quorum-sensing (QS) systems of Pseudomonas aeruginosa control expression of virulence factors in a population density-dependent fashion. In this study we investigated the role of synthetic analogs to RhlR-RhlI system of P. aeruginosa strains (PAO-1; wild-type and mutants JP-1, PDO-100, and JP-2) responsible for production of acyl-homoserine lactones-2; butanol homoserine lactone (AHL-2; C(4)-HSL). We synthesized double (QS1207) and single (QS0108) sulfur analogs against (C(4)-HSL; AHL-2), an autoinducer of Pseudomonas QS system. Extensive biological investigation of these analogs suggested a growth promoting activity for these analogs in Pseudomonas controlling biofilm production and exo-protease secretion. We hypothesized that these thiolactone analogs could be potentially utilized as potent drug-delivery vehicles against biofilm-producing pathogens. As a proof of principle we conjugated the single sulfur analog QS0108 with the broad-spectrum antibiotic, ciprofloxacin (QS0108-Cip). The QS analog-antibiotic conjugate was significantly more effective at disrupting both the nascent and mature biofilms of P. aeruginosa than the free antibiotic.

Incorporation of tellurocysteine into glutathione transferase generates high glutathione peroxidase efficiency.

A rival to native peroxidase! An existing binding site for glutathione was combined with the catalytic residue tellurocysteine by using an auxotrophic expression system to create an engineered enzyme that functions as a glutathione peroxidase from the scaffold of a glutathione transferase (see picture). The catalytic activity of the telluroenzyme in the reduction of hydroperoxides by glutathione is comparable to that of native glutathione peroxidase.

Compensating bends in a 16-base-pair DNA oligomer containing a T(3)A(3) segment: A NMR study of global DNA curvature.

In-phase ligated DNA containing T(n)A(n) segments fail to exhibit the retarded polyacrylamide gel electrophoresis (PAGE) migration observed for in-phase ligated A(n)T(n) segments, a behavior thought to be correlated with macroscopic DNA curvature. The lack of macroscopic curvature in ligated T(n)A(n) segments is thought to be due to cancellation of bending in regions flanking the TpA steps. To address this issue, solution-state NMR, including residual dipolar coupling (RDC) restraints, was used to determine a high-resolution structure of [d(CGAGGTTTAAACCTCG)2], a DNA oligomer containing a T3A3 tract. The overall magnitude and direction of bending, including the regions flanking the central TpA step, was measured using a radius of curvature, Rc, analysis. The Rc for the overall molecule indicated a small magnitude of global bending (Rc = 138 +/- 23 nm) towards the major groove, whereas the Rc for the two halves (72 +/- 33 nm and 69 +/- 14 nm) indicated greater localized bending into the minor groove. The direction of bending in the regions flanking the TpA step is in partial opposition (109 degrees), contributing to cancellation of bending. The cancellation of bending did not correlate with a pattern of roll values at the TpA step, or at the 5' and 3' junctions, of the T3A3 segment, suggesting a simple junction/roll model is insufficient to predict cancellation of DNA bending in all T(n)A(n) junction sequence contexts. Importantly, Rc analysis of structures refined without RDC restraints lacked the precision and accuracy needed to reliably measure bending.

Analysis of GAA/TTC DNA triplexes using nuclear magnetic resonance and electrospray ionization mass spectrometry.

The formation of a GAA/TTC DNA triplex has been implicated in Friedreich's ataxia. The destabilization of GAA/TTC DNA triplexes either by pH or by binding to appropriate ligands was analyzed by nuclear magnetic resonance (NMR) and positive-ion electrospray mass spectrometry. The triplexes and duplexes were identified by changes in the NMR chemical shifts of H8, H1, H4, 15N7, and 15N4. The lowest pH at which the duplex is detectable depends upon the overall stability and the relative number of Hoogsteen C composite function G to T composite function A basepairs. A melting pH (pHm) of 7.6 was observed for the destabilization of the (GAA)2T4(TTC)2T4(CTT)2 triplex to the corresponding Watson-Crick duplex and the T4(CTT)2 overhang. The mass spectrometric analyses of (TTC)6.(GAA)6 composite function(TTC)6 triplex detected ions due to both triplex and single-stranded oligonucleotides under acidic conditions. The triplex ions disappeared completely at alkaline pH. Duplex and single strands were detectable only at neutral and alkaline pH values. Mass spectrometric analyses also showed that minor groove-binding ligands berenil, netropsin, and distamycin and the intercalating ligand acridine orange destabilize the (TTC)6.(GAA)6 composite function (TTC)6 triplex. These NMR and mass spectrometric methods may function as screening assays for the discovery of agents that destabilize GAA/TTC triplexes and as general methods for the characterization of structure, dynamics, and stability of DNA and DNA-ligand complexes.

An EPR, ESEEM, structural NMR, and DFT study of a synthetic model for the covalently ring-linked tyrosine-histidine structure in the heme-copper oxidases.

We report CW-EPR, ESEEM, and structural NMR results, as well as DFT calculations, on model compounds relevant to the unusual cross-linked Tyr-His (YH) moiety at the active site of the heme-copper oxidases. CW-EPR spectra of an (15)N isotopically labeled 4-methyl-2-(4-methyl-imidazole-1-yl)-phenol radical are nearly identical to those of the natural abundance (14)N compound. We obtain good simulations of these EPR spectra without including hyperfine couplings to the nitrogen nuclei. This implies that the electron distribution of the radical is largely localized on the phenol ring with only a small amount of spin delocalized onto the nitrogens of the imidazole. Using three-pulse ESEEM spectroscopy, we have successfully detected the two imidazole ring nitrogens, one near the "exact cancellation" ESEEM condition and the other more weakly coupled. We assign these to the imino and amino nitrogens, respectively, based on DFT calculations performed on this radical species. The experimental results and the supporting density functional calculations clearly show that the imidazole substituent has only a minor effect on the electronic structure of the substituted phenol radical.

Determining the solution state orientation of a Ti enolate via stable isotope labeling, NMR spectroscopy, and modeling studies.

Our group has used Ti-promoted aldol additions with an oxazolidineselone as the chiral auxiliary with much success. In these reactions, the Se atom in the auxiliary both promotes stereospecific addition as well as reports on, through the use of 77Se NMR spectroscopy, the ratio of diastereomers produced and the geometry of intermediates as the reaction proceeds. Through stable isotope labeling and NMR spectroscopy, we are able to experimentally observe a Ti enolate in solution and gain insight into its structure and reactivity. Results from molecular modeling calculations are also presented for comparison with NMR data.

Studies on the biosynthesis of asperparaline A: origin of the spirosuccinimde ring system.

The primary amino acid building blocks that constitute asperparaline A have been determined through feeding and incorporation of 13C-labeled intermediates. The beta-methyl proline residue is constituted from (S)-isoleucine, the novel spiro-succinime moiety is derived from the oxidative degradation of (S)-tryptophan, and (S)-adenosylmethionine contributes the two N-methyl residues. In addition, it was found that the incorporation of 13C-labeled acetate into the single isoprene unit clearly demonstrates that the isoprene moiety is derived from the mevalonate pathway.

Structural studies on the hairpins at the 3' untranslated region of an anthrax toxin gene.

Three proteins, namely, protective antigen (PA), edema factor (EF), and lethal factor (LF), encoded by the pX01 plasmid of Bacillus anthracis play a major role in the pathogenesis of target host cells. PA combines with EF and LF to form bipartite PA-EF and PA-LF toxins and facilitates intracellular delivery of EF and LF both of which cause cytotoxicity to the host. Since the level of PA is crucial to pathogenesis by anthrax toxins, it is important to understand how the host environment regulates the expression of the PA (or pagA) gene by utilizing the 5' and 3' untranslated regions (UTR). The 5' UTR sequence determines the initiation of transcription, whereas the 3' UTR sequence determines the efficient termination and stability of the transcript. Although, the role of the 5'UTR sequence of pagA has been investigated, little is known about the role of the 3' UTR. Since hairpin formation at the 3'UTR of a gene is an established mechanism for efficient termination and stability of the transcript, we carried out structural studies, including gel electrophoresis, circular dichroism, and two-dimensional nuclear magnetic resonance spectroscopy, to determine whether the 3' UTR sequences of pagA also form hairpin structures. Our results unequivocally demonstrate that both the coding and the noncoding 3' UTR sequences form stable hairpin structures. It is quite likely that the hairpins at the 3'UTR may contribute to efficient termination and stability of the pagA transcript.

Direct infrared detection of the covalently ring linked His-Tyr structure in the active site of the heme-copper oxidases.

Infrared spectroscopy, isotopic labeling ([(15)N(delta,epsilon)]histidine and ring-deuterated tyrosine), synthetic model studies, and normal mode calculations are employed to search for the spectroscopic signatures of the unique, covalently linked (His N(epsilon)-C(epsilon) Tyr) biring structure in the heme-copper oxidases. The specific enzyme examined is the cytochrome bo(3) quinol oxidase of E. coli. Infrared features of histidine and tyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm(-1)) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm(-1)). Two of these, at ca. 1480 and 1550 cm(-1), and their combination tones between 3010 and 3040 cm(-1), are definitively identified with the biring structure involving H284 and Y288 in the E. coli enzyme. Studies of a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show that a feature near 1540 cm(-1) is unique to the biring structure and is absent from the infrared spectrum of 4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques, and offers a direct spectroscopic probe for potential radical production involving the H-Y structure in the O(2) reduction cycle of the oxidases.

Synthesis and incorporation of [6,7]-selenatryptophan into dihydrofolate reductase.

Until recently, the only selenium containing amino acid which could be used to completely substitute for a wild type amino acid was selenomethionine (SeMet). In the last decade the preparation of SeMet containing proteins has proved to be valuable tools in the determination of three-dimensional structure by multiwavelength anomalous diffraction (MAD) techniques. The potential utility of a selenium containing tryptophan analog, beta-seleno[3,2-b]pyrrolyl-L-alanine ([4,5]selenatryptophan), has recently been demonstrated in the literature. This finding shows promise for the bioincorporation of its positional isomer, beta-selenolo[2,3-b]pyrrolyl-L-alanine ([6,7]selenatryptophan), thereby adding to the essential arsenal of selenium-containing amino acids for use in the characterization of proteins. The synthesis of [6,7]selenatryptophan by enzymatic biotransformation with tryptophan synthase from selenolo[2,3-b]pyrrole was carried out as well as its characterization by NMR spectroscopy and thin layer chromatography. Selenatryptophyl dihydrofolate reductase ([6,7]SeTrp-DHFR) was then synthesized in vivo, purified, and found to exhibit no perturbations to enzymatic activity.