Coupling of thioamides with 4-bromocrotonate esters and subsequent conjugate addition for the rapid one-pot synthesis of functionalized thiazolines

An efficient one-pot synthesis of functionalized thiazolines via the coupling of thioamides with 4-bromocrotonate esters is described. A range of aryl- and alkyl-thioamides with various substitutions are well-tolerated. Additionally, the presence of the exocyclic ester functionality provides a convenient handle for the synthesis of more complex thiazoline-containing natural products and biologically-relevant compounds.     

Simulation of ultrasonic lamb wave generation, propagation and detection for a reconfigurable air coupled scanner

A computer simulator, to facilitate the design and assessment of a reconfigurable, air-coupled ultrasonic scanner is described and evaluated. The specific scanning system comprises a team of remote sensing agents, in the form of miniature robotic platforms that can reposition non-contact Lamb wave transducers over a plate type of structure, for the purpose of non-destructive evaluation (NDE). The overall objective is to implement reconfigurable array scanning, where transmission and reception are facilitated by different sensing agents which can be organised in a variety of pulse-echo and pitch-catch configurations, with guided waves used to generate data in the form of 2-D and 3-D images. The ability to reconfigure the scanner adaptively requires an understanding of the ultrasonic wave generation, its propagation and interaction with potential defects and boundaries. Transducer behaviour has been simulated using a linear systems approximation, with wave propagation in the structure modelled using the local interaction simulation approach (LISA). Integration of the linear systems and LISA approaches are validated for use in Lamb wave scanning by comparison with both analytic techniques and more computationally intensive commercial finite element/difference codes. Starting with fundamental dispersion data, the paper goes on to describe the simulation of wave propagation and the subsequent interaction with artificial defects and plate boundaries, before presenting a theoretical image obtained from a team of sensing agents based on the current generation of sensors and instrumentation.     

Nucleic acid sequence design via efficient ensemble defect optimization

We describe an algorithm for designing the sequence of one or more interacting nucleic acid strands intended to adopt a target secondary structure at equilibrium. Sequence design is formulated as an optimization problem with the goal of reducing the ensemble defect below a user‐specified stop condition. For a candidate sequence and a given target secondary structure, the ensemble defect is the average number of incorrectly paired nucleotides at equilibrium evaluated over the ensemble of unpseudoknotted secondary structures. To reduce the computational cost of accepting or rejecting mutations to a random initial sequence, candidate mutations are evaluated on the leaf nodes of a tree‐decomposition of the target structure. During leaf optimization, defect‐weighted mutation sampling is used to select each candidate mutation position with probability proportional to its contribution to the ensemble defect of the leaf. As subsequences are merged moving up the tree, emergent structural defects resulting from crosstalk between sibling sequences are eliminated via reoptimization within the defective subtree starting from new random subsequences. Using a Θ(N³) dynamic program to evaluate the ensemble defect of a target structure with N nucleotides, this hierarchical approach implies an asymptotic optimality bound on design time: for sufficiently large N, the cost of sequence design is bounded below by 4/3 the cost of a single evaluation of the ensemble defect for the full sequence. Hence, the design algorithm has time complexity Ω(N³). For target structures containing N ∈{100,200,400,800,1600,3200} nucleotides and duplex stems ranging from 1 to 30 base pairs, RNA sequence designs at 37°C typically succeed in satisfying a stop condition with ensemble defect less than N/100. Empirically, the sequence design algorithm exhibits asymptotic optimality and the exponent in the time complexity bound is sharp. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Synthetic, structural, and biosynthetic studies of an unusual phospho-glycopeptide derived from α-dystroglycan

Aberrant glycosylation of α-dystroglycan (α-DG) results in loss of interactions with the extracellular matrix and is central to the pathogenesis of several disorders. To examine protein glycosylation of α-DG, a facile synthetic approach has been developed for the preparation of unusual phosphorylated O-mannosyl glycopeptides derived from α-DG by a strategy in which properly protected phospho-mannosides are coupled with a Fmoc protected threonine derivative, followed by the use of the resulting derivatives in automated solid-phase glycopeptide synthesis using hyper-acid-sensitive Sieber amide resin. Synthetic efforts also provided a reduced phospho-trisaccharide, and the NMR data of this derivative confirmed the proper structural assignment of the unusual phospho-glycan structure. The glycopeptides made it possible to explore factors that regulate the elaboration of critical glycans. It was established that a glycopeptide having a 6-phospho-O-mannosyl residue is not an acceptor for action by the enzyme POMGnT1, which attaches β(1,2)-GlcNAc to O-mannosyl moietes, whereas the unphosphorylated derivate was readily extended by the enzyme. This finding implies a specific sequence of events in determining the structural fate of the O-glycan. It has also been found that the activity of POMGnT1 is dependent on the location of the acceptor site in the context of the underlying polypeptide/glycopeptide sequence. Conformational analysis by NMR has shown that the O-mannosyl modification does not exert major conformational effect on the peptide backbone. It is, however, proposed that these residues, introduced at the early stages of glycoprotein glycosylation, have an ability to regulate the loci of subsequent O-GalNAc additions, which do exert conformational effects. The studies show that through access to discrete glycopeptide structures, it is possible to reveal complex regulation of O-glycan processing on α-DG that has significant implications both for its normal post-translational maturation, and the mechanisms of the pathologies associated with hypoglycosylated α-DG.     

Identification of cyclized calmodulin antagonists from a phage display random peptide library

Summary To isolate peptide ligands that bound calmodulin (CaM) specifically, we screened an M13 phage library displaying cyclized octamer random peptides with immobilized bovine CaM. Isolates were recovered, sequenced, and deduced to express nine independent peptides, five of which contained the sequence Trp-Gly-Lys (WGK). Four of the nine peptide sequences were synthesized in cyclized, biotinylated form. All of the peptides required Ca²⁺ to bind CaM. The cyclized, disulfide-bonded form of one such peptide, SCLRWGKWSNCGS, bound CaM better than its reduced form or an analogue in which the cysteine residues were replaced by serine. The cyclized peptide also exhibited the ability to inhibit CaM-dependent kinase activity. Systematic alanine substitution of residues in this peptide sequence implicate the tryptophan residue as being critical for binding, with other residues contributing to binding to varying degrees. Cloning of ligand targets (COLT) confirmed the specificity of one of the cyclized peptides, yielding full-length and C-terminal CaM clones, in addition to a full-length clone of troponin C, a CaM-related protein. This study has demonstrated that conformationally constrained peptides isolated from a phage library acted as specific, Ca²⁺-dependent CaM ligands.