Palladium(II) acetate and palladium(II) chloride react with amino acids in acetone/water to yield cis or trans square planar bis-chelated palladium amino acid complexes. The naturally occurring amino acids and some N-alkylated and substituted derivatives and homologs were evaluated as ligands. Thirty-eight amino acids in total were investigated as ligands. The formation of aquo complexes in water was observed and studied by 13C NMR spectroscopy and modeled by DFT calculations. Each class of amino acid ligand is catalytically active with respect to the oxidative coupling of olefins and phenylboronic acids. Some enantioselectivity is observed and the formation of products not reported in other Pd(II) oxidative couplings is seen. Both activated and non-activated alkenes were oxidatively coupled to phenylboronic acids incorporating both electron-donating and electron-withdrawing groups. The crystal structures of nineteen catalyst complexes were obtained. The extended lattice structures arise from N-H..O or O..(HOH)..O hydrogen bonding. NMR, HRMS, FTIR, single crystal XRD, and powder XRD data are evaluated. / Ph. D.

Plant aromatic amino acid decarboxylases (AAADs) are a group of economically important enzymes categorically joined through their pyridoxal 5'-phosphate (PLP) dependence and sequence homology. Extensive evolutionary divergence of this enzyme family has resulted in a selection of enzymes with stringent aromatic amino acid substrate specificities. Variations in substrate specificities enable individual enzymes to catalyze key reactions in a diverse set of pathways impacting the synthesis of monoterpenoid indole alkaloids (including the pharmacologically active vinblastine and quinine), benzylisoquinoline alkaloids (including the pharmacologically active papaverine, codeine, morphine, and sanguinarine), and antioxidant and chemotherapeutic amides. Recent studies of plant AAAD proteins demonstrated that in addition to the typical decarboxylation enzymes, some annotated plant AAAD proteins are actually aromatic acetaldehyde synthases (AASs). These AASs catalyze a decarboxylation-oxidative deamination process of aromatic amino acids, leading to the production of aromatic acetaldehydes rather than the AAAD derived arylalkylamines. Research has implicated that plant AAS enzymes are involved in the production of volatile flower scents, floral attractants, and defensive phenolic acetaldehyde secondary metabolites. Historically, the structural elements responsible for differentiating plant AAAD substrate specificity and activity have been difficult to identify due to strong AAAD and AAS inter-enzyme homology. Through extensive bioinformatic analysis and experimental verification of plant AAADs, we have determined some structural elements unique to given types of AAADs. This document highlights structural components apparently responsible for the differentiation of activity and substrate specificity. In addition to producing primary sequence identifiers capable of AAAD activity and substrate specificity differentiation, this work has also demonstrated applications of AAAD enzyme engineering and novel activity identification. / Ph. D.

The oxidative addition of both monosubstituted and disubstituted a.-amino acids to [Ir(COD)(PMe3)3]Cl (COD = cyclooctadiene) was studied and the reactivity of the resulting complexes was examined. The reaction of [Ir(COD)(PMe3)3]CI with the disubstituted amino acids, diphenylglycine and methyl phenyl alanine, led to an almost exclusive facial product. Monosubstituted amino acid complexes were observed to be mixtures of the meridional and facial isomers with the meridional isomer largely predominating. The meridional isomer was found to convert to the facial isomer when heated for several days at 100°C. In fact, a predominantly meridional mixture was found to convert to a predominantly facial mixture upon heating. The facial isomer was therefore shown to be the thermodynamic product from the mixture. Small amounts of other isomers were observed in the hydride region of the proton NMR spectrum. One resonance at -23.75 ppm disappeared upon heating t-butyl acetylene with the amino acid complex. The disappearance of this hydride resonance may indicate the insertion of the unsaturate into the Ir-H bond, or alternatively, the conversion of this isomer to a more thermodynamically stable isomer. Reactions of these complexes with methylpropiolate and acrylamide were attempted but evidence of an insertion product was not found. / Master of Science

Pathogens colonize their hosts by releasing molecules that can enter host cells. A biotrophic oomycete plant pathogen, Phytophthora sojae harbors a superfamily of effector genes whose protein products enter the cells of the host, soybean. Many of the effectors contain an RXLR-dEER motif in their N-terminus. More than 400 members belonging to this family have been previously identified using a Hidden Markov Model. Amino acids flanking the RXLR motif have been utilized to identify effector proteins from the P. sojae secretome, despite the high level of sequence divergence among the members of this protein family. I present here machine learning methods to identify protein candidates that belong to a particular class, such as the effector superfamily. Converting the flanking amino acid sequences of RXLR motifs (or other candidate motifs) into numeric values that reflect their physical properties enabled the protein sequences to be analyzed through these methods. The methods evaluated include Support Vector Machines and a related spherical classification method that I have developed. I also approached the effector prediction problem by building functional linkage networks and have produced lists of predicted P. sojae effector proteins. I tested the best candidate through gene gun bombardment assays using the beta-glucuronidase reporter system, which revealed that there is a high likelihood that the candidate can enter the soybean cells. / Ph. D.

No / A solid-phase Mitsunobu reaction between a resin-bound 1-thiosugar and an N-Fmoc protected amino alcohol was successfully employed for thio-linked glycopeptide synthesis. Facile cleavage and deprotection in one step afforded the target glycopeptide in good yield and purity.

No / The minerals involved in the formation of metazoan skeletons principally comprise glassy silica, calcium phosphate or carbonate. Because of their ancient heritage, glass sponges (Hexactinellida) may shed light on fundamental questions such as molecular evolution, the unique chemistry and formation of the first skeletal silica-based structures, and the origin of multicellular animals. We have studied anchoring spicules from the metre-long stalk of the glass rope sponge (Hyalonema sieboldi; Porifera, Class Hexactinellida), which are remarkable for their size, durability, flexibility and optical properties. Using slow-alkali etching of biosilica, we isolated the organic fraction, which was revealed to be dominated by a hydroxylated fibrillar collagen that contains an unusual [Gly-3Hyp-4Hyp] motif. We speculate that this motif is predisposed for silica precipitation, and provides a novel template for biosilicification in nature.

Yes / Studies on the substrate selectivity of recombinant ferrous-iron- and 2-oxoglutarate-dependent proline hydroxylases (PHs) reveal that they can catalyse the production of dihydroxylated 5-, 6-, and 7-membered ring products, and can accept bicyclic substrates. Ring-substituted substrate analogues (such hydroxylated and fluorinated prolines) are accepted in some cases. The results highlight the considerable, as yet largely untapped, potential for amino acid hydroxylases and other 2OG oxygenases in biocatalysis.

Glucosyltransferases (GTs) are enzymes which perform glucosylation reactions, which involve attaching a UDP-activated glucose molecule to acceptor molecules specifi c to the enzyme. The enzyme which our lab focuses its research on is a fl avonol-specifi c 3-OGT found in Citrus paradisi, or grapefruit (Cp3GT). This enzyme is part of the class of enzymes known as fl avonoid GTs, which are responsible for, among other things, the formation of compounds which can affect the taste of citrus. Our lab focuses its research on performing site-directed mutagenesis on Cp3GT in an attempt to discover the residues important for substrate and regiospecifi city. In this study, we are testing the basis of substrate septicity of Cp3GT. We hypothesize that incorporation of fi ve amino acids specifi c to Citrus sinensis GT (CsGT) into Cp3GT at 308th position may facilitate mCp3GT to use anthocyanidins as one of the substrates. We report our fi ndings thus far concerning the addition of specifi c residues to the Cp3GT’s amino acid sequence based on an alignment with the sequence of a putative fl avonoid GT found in Citrus sinensis.

Unnatural amino acids (uAA) expand the structural and functional possibilities of proteins. Numerous previous studies have demonstrated uAA as a powerful tool for protein engineering, but challenges also remain. Three notable such challenges include: (1) the fitness of uAA-incorporated proteins are difficult to predict and time-consuming to screen with conventional methods, (2) uAA incorporation in difficult-to-express proteins (e.g. membrane proteins such as G-protein coupled receptors) remain challenging, and (3) the incorporation of multiple types of uAA are still limited. In response, we pose cell-free protein synthesis (CFPS), a rapid and versatile in vitro expression system, as a platform to explore solutions to these challenges. The "cell-free" nature of CFPS enables it to accelerate protein expression and tolerate extensive modifications to its translational environment. In this work, these advantages were utilized to address the aforementioned challenges by: (1) rapidly expressing and screening uAA-containing proteins, (2) incorporating uAA in functional G-protein coupled receptor in the presence of membrane-mimicking lipid additives, and (3) engineer the translational environment extensively towards multiple uAA incorporation.

No description available.