8,313 research outputs found
On the Michael addition of water to C = C bonds
No full text?-Hydroxy carbonyl compounds are an important class of compounds often found as a common structural motif in natural products. Although the molecules themselves look rather simple, their synthesis can be challenging. Water addition to conjugated C = C bonds opens up a straightforward route for the preparation of ?-hydroxy carbonyl compounds. Moreover, water addition to C = C bonds benefits a lot from its simplicity and excellent atom economy. However, the enantioselective addition of water to ?,?-unsaturated carbonyl (Michael) acceptors still represents a chemically very challenging reaction, due to the poor nucleophilicity of water and its small size, which make regio- and stereoinduction difficult. Equally, the often unfavorable equilibrium of water-addition reactions remains to be solved. In contrast, enzymes such as fumarase, malease, citraconase, aconitase, and enoyl-CoA hydratase have been successfully used on industrial scale, and their excellent (enantio-) selectivities are highly valued. Unfortunately, most hydratases are part of the primary metabolism where perfect substrate specificity is required. This very high substrate selectivity severely limits their practical applicability in organic synthesis. Thus, a straightforward approach with broad applicability still had not been described. The aim of the research presented in this thesis was to take up this challenge and dedicated to the search for a Michael hydratase with a more relaxed substrate specificity for the preparation of important ?-hydroxy carbonyl compounds. The stereospecificity of enzyme-catalysed reactions has been a fruitful source of information about the mechanisms of enzyme catalysis and vice versa; the application of stereospecifically labelled substrates allows for studying the course of the reaction. It offers a very promising opportunity to comprehensively understand the precise mechanistic and kinetic details of even the most complex enzymatic reactions. Thus Chapter 1 provides unifying ideas for stereochemistry of the enzymatic water addition to C = C bonds. This enhances our understanding of the chemistry of water addition to C = C bonds, and further allows us to find more hydratases from natural sources or obtained via protein engineering. In Chapter 2, a direct, enantioselective Michael addition of water in water to prepare important ?-hydroxy carbonyl compounds using whole cells of Rhodococcus strains is described. Good yields and excellent enantioselectivities were achieved with this method. This opens up an entirely new approach for the preparation of important ?-hydroxy carbonyl compounds. Deuterium labelling studies demonstrate that a Michael hydratase catalyzes the water addition exclusively with anti-stereochemistry, which belongs to the family members of hydratases: oleate hydratase, fumarase, malease, aconitase and type II dehydroquinase with a preference for the anti-addition; whereas, type I dehydroquinase, enoyl-CoA hydratase and artificial hydratase exclusive prefer for the syn-addition, as discussed in Chapter 1. The biocatalytic reaction system was carefully optimized for gram-scale synthesis, resulting in good conversions and excellent enantioselectivities. Under the optimized conditions, whole cells could be reused for 4 cycles without significant loss of activity while maintaining up to 90% ee. Since whole cells from Rhodococcus strains were used to catalyse the Michael addition of water in water to a series of ?,?-unsaturated carbonyl compounds, and when the work presented in Chapter 2 started, no genomic information of Rhodococcus strains was publically available, we sequenced and annotated the strain R. rhodochrous ATCC 17895. This is described in Chapter 3 together with features of the R. rhodochrous ATCC 17895. It is a Gram-positive aerobic bacterium with a rod-like morphology. The 6,869,887 bp long genome contains 6,609 protein-coding genes and 53 RNA genes. Our study suggests the Michael hydratase has not been described before. In the work presented in Chapter 2, we found that most ?-hydroxy ketones are not commercially available or commercially expensive as we mentioned in the first paragraph, which made the stereoselectivity determination of Michael addition products difficult. Indeed, many seemingly simple molecules have to be prepared via multi-step syntheses, in particular so if they are optically active. Therefore a straightforward approach to enantiomerically enriched (R)- and (S)-3-hydroxycyclopentanone was established by kinetic resolution in Chapter 4. This methodology allows us to prepare more ?-hydroxy carbonyl compounds structurally closely related to 3-hydroxycyclopentanone. The isolated chiral alcohols were used to determine the stereochemistry of the Michael addition of water in Chapter 2, saving us a lot of laboratory work. Moreover, unexpected stereoselective reduction of conjugated C = C bonds was discovered during studies on the enantioselective Michael addition of water. As mentioned in Chapter 2, the whole cells of R. rhodochrous ATCC 17895 reduced ?,?-unsaturated cyclic ketones into the corresponding ketones as initially undesired side reaction for the addition of water to C = C bonds. Therefore, ene-reductase activity was also investigated in Chapter 5. A series of substrates, including activated ketones, aldehydes, amines and nitro-compounds were screened for ene-reductase activity using whole cells of R. rhodochrous ATCC 17895. This showed that R. rhodochrous is a very promising catalyst for the reduction of C = C bonds and harbours ene-reductases. Indeed, looking for the annotated ene reductase from the genome of R. rhodochrous ATCC 17895 as described in Chapter 3, three candidates were observed and were classified as ene-reductases by amino acid sequence alignment with the known Old Yellow Enzymes (OYEs). Thus, the putative ene-reductase genes from R. rhodochrous ATCC 17895 were heterologously overexpressed in Escherichia coli and one of the encoded proteins was purified and characterized for their biocatalytic and biochemical properties. Based on these accomplishments it can be concluded that we have discovered a new Michael hydratase and three new ene reductases from Rhodococcus strains. Genome sequence and annotation of strain R. rhodochrous ATCC 17895 has been done, offering an excellent opportunity for the discovering novel enzymes, for instance, the Michael hydratase and S-selective ene reductase. The important chiral ?-hydroxy carbonyl compounds can be prepared by kinetic resolution of racemic alcohols using lipases or the direct enantioselective Michael addition of water using whole cells of Rhodococcus strains. The isolated products from kinetic resolution were readily used for the stereochemistry determination of Michael addition of water in water, completes the story of water addition to C = C bonds.BiotechnologyApplied Science
Molecular basis of the fructose-2,6-bisphosphatase reaction of PFKFB3: Transition state and the C-terminal function
No full textThe molecular basis of fructose-2,6-bisphosphatase (F-2,6-P 2ase) of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) was investigated using the crystal structures of the human inducible form (PFKFB3) in a phospho-enzyme intermediate state (PFKFB3-P•F-6-P), in a transition state-analogous complex (PFKFB3•AlF 4), and in a complex with pyrophosphate (PFKFB3•PP i) at resolutions of 2.45, 2.2, and 2.3 Å, respectively. Trapping the PFKFB3-P•F-6-P intermediate was achieved by flash cooling the crystal during the reaction, and the PFKFB3•AlF 4 and PFKFB3•PP i complexes were obtained by soaking. The PFKFB3•AlF 4 and PFKFB3•PP i complexes resulted in removing F-6-P from the catalytic pocket. With these structures, the structures of the Michaelis complex and the transition state were extrapolated. For both the PFKFB3-P formation and break down, the phosphoryl donor and the acceptor are located within ∼5.1 Å, and the pivotal point 2-P is on the same line, suggesting an in-line transfer with a direct inversion of phosphate configuration. The geometry suggests that NE2 of His253 undergoes a nucleophilic attack to form a covalent N-P bond, breaking the 2O-P bond in the substrate. The resulting high reactivity of the leaving group, 2O of F-6-P, is neutralized by a proton donated by Glu322. Negative charges on the equatorial oxygen of the transient bipyramidal phosphorane formed during the transfer are stabilized by Arg252, His387, and Asn259. The C-terminal domain (residues 440-446) was rearranged in PFKFB3•PP i, implying that this domain plays a critical role in binding of substrate to and release of product from the F-2,6-P 2ase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction. © 2011 Wiley Periodicals, Inc
Michael Connelly Interview: 9/11, City of Bones, and Lost Light
No full textAn interview with American crime author Michael Connelly, exploring the role of crime narratives as outlets for critical engagement with the politics of the war on terror and the associated neoconservative narrativising of 9/11. The discussion principally delves into Connelly’s first two post 9/11 Harry Bosch novels; City of Bones (2002) and Lost Light (2003)
24th Annual African American Living Legends Series - Supervisor Burke and Michael Datcher
No full textCounty Supervisor Yvonne B. Burke (left) stands with event honoree author Michael Datcher (right)
24th Annual African American Living Legends Series - Michael Datcher and proclamation
No full textEvent honoree Michael Datcher signs the Living Legends proclamation at the 24th Annual African American Living Legends Series event honoring him and author Antwone Fisher
24th Annual African American Living Legends Series - Michael Datcher signs proclamation
No full textEvent honoree Michael Datcher signs the Living Legends proclamation at the 24th Annual African American Living Legends Series event honoring him and author Antwone Fisher
Postmodern Theory and the Choreography of Michael Clark
No full textThis study addresses two interrelated questions: Can postmodern ‘theory’ illuminate an understanding of Michael Clark’s work? and, a sub-question, In which ways, if at all, does Clark’s work demonstrate a postmodern sensibility? Chapter one, the introduction to this study, provides a ‘portrait’ of postmodernism, that is, it addresses the question What is postmodernism? Chapter two is a biography of Michael Clark. The seminal sections to this study, however, are chapters three and four. Here the author blends a discussion of a) subject matter, treatment and meanings in Clark’s choreography, b) journalistic criticisms of those features of his work, and c) postmodern theory. The outcome of these chapters is to demonstrate that Clark’s works do indeed require re-interpretation and re-evaluation, and to illustrate how these factors might be achieved
Tool for use in semi-automatic landslide mapping
No full textReport -- Maps.by Michael Bunn, Ben A. Leshchinsky, Michael J. Olsen, Nancy C. Calhoun, Jon J. Franczyk, and William J. Burns.This archived document is maintained by the State Library of Oregon as part of the Oregon Documents Depository Program. It is for informational purposes and may not be suitable for legal purposes.Includes bibliographical references (pages 39-41).Mode of access: Internet from the Oregon Government Publications Collection.Text in English
Interview with Dr. Michael C. Keith (Communication Department), author Of Night and Light: Stories (Blue Mustang Press, 2012)
No full textDr. Keith's latest book is a collection of shorts stories entitled Of Night and Light. This book collects more than 40 short stories covering all manner of speculative fiction, humor, and even a bit of romance. As the title notes, there are both light and dark themes within the book, though the author readily admits he leans a bit darker at times.Title supplied by cataloger
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