TAC-scaffolded tripeptides as artificial hydrolytic receptors: a combinatorial approach toward esterase mimics

In this report, we present the first library of tripodal synthetic receptor molecules containing three different, temporarily N-terminal protected peptide arms capable of performing hydrolytic reactions. To construct this library, the orthogonally protected triazacyclophane (TAC)-scaffold was used in the preparation of a split-mix library of 19 683 resin bound tripodal receptor molecules. For the construction of the peptide arms, three different sets of amino acids were used, each focused on one part of the catalytic triad as found in several families of hydrolytic enzymes. Therefore, in the sets of amino acids used to assemble these tripeptides, basic (containing His and Lys), nucleophilic (containing Ser and Cys), or acidic (containing Asp and Glu) amino acid residues were present. In addition, nonfunctional hydrophobic amino acid residues were introduced. Possible unfavorable electrostatic interactions of charged N-termini or their acetylation during screening were circumvented by trifluoroacetylation of the N-terminal amines. Screening was performed with a known esterase substrate, 7-acetoxycoumarin, which upon hydrolysis gave the fluorescent 7-hydroxycoumarin, leading to fluorescence of beads containing a hydrolytically active synthetic receptor. Although many synthetic receptors contain catalytic triad combinations, apparently, only a few showed hydrolytic activity. Sequence analysis of the active receptors showed that carboxylate-containing amino acids are frequently found in the acidic arm and that substrate cleavage is mediated by lysine (noncatalytic) or histidine (catalytic) residues. Kinetic analysis of resynthesized receptors showed that catalysis depended on the number of histidine residues and was not assisted by significant substrate binding.     

The combinatorial approach to asymmetric hydrogenation: phosphoramidite libraries, ruthenacycles, and artificial enzymes

For a more general implementation of asymmetric catalysis in the production of fine chemicals, the screening for new catalysts and ligands must be dramatically accelerated. This is possible with a high-throughput experimentation (HTE) approach. However, implementation of this technology requires the rapid preparation of libraries of ligands/catalysts and consequently dictates the use of simple ligands that can be readily synthesised in a robot. In this concept article, we describe how the development of new ligands based on monodentate phosphoramidites enabled the development of an integral HTE protocol for asymmetric hydrogenation. This "instant ligand library" protocol makes it possible to synthesise 96 ligands in one day and screen them the next day. Further diversity is possible by using mixtures of monodentate ligands. This concept has already led to an industrial application. Other concepts, still under development, are based on chiral ruthenacycles as new transfer hydrogenation catalysts and the use of enzymes as ligands for transition-metal complexes.     

Formation of self-rolled polymer microtubes studied by combinatorial approach

This paper describes an experimental study of the polymer microtube formation in different fabrication conditions. A photolithography route is demonstrated for the fabrication of large amount of uniform and good quality polymer microtubes. TEM grids were used as mask and exposure of UV radiation was done in two steps to create asymmetric patterns. The diameter of tube was controlled by regulating the thickness of polymer layers, UV radiation dose and concentration of acidic solution. The results indicate that thickness of the bilayer film was the most dominating factor to determine the tube’s dimension, and diameter of the tubes increased with increase in the thickness of bilayer. The kinetics of the tube formation was studied with respect to acidity of the solution and UV dose. Rate of rolling increased with the acidity of the solution. Tube diameter and rate of rolling decreased with the increase of the UV exposure time. Films with two-dimensional gradients of layer thicknesses were prepared to study a broad range of parameters in a single experiment. The tubes were characterized in detail using optical microscopy and scanning electron microscopy. These polymer microtubes have potential applications in microfluidic devices and biotechnology field.     

Communication: efficient counterpoise corrections by a perturbative approach

We investigate the use of Hartree-Fock and density functional perturbative corrections for estimating the counterpoise correction (CPC) for interaction energies at the self-consistent field level. We test our approach using several popular basis sets on the S22 set of weakly bound systems, which can exhibit large basis set superposition errors. Our results show that the perturbative approaches typically recover over 95% of the CPC and can be up to twelve times faster to compute than the conventional methods and therefore provide an attractive alternative to calculating CPCs in the conventional way.     

Identification of protease substrates by combinatorial profiling on TentaGel beads

Reacting a 65,536 member combinatorial library of octapeptides on TentaGel beads with various proteases followed by selective staining of the free amino termini at the reacted bead surface and sequence determination by amino acid analysis allowed the rapid identification of protease substrates.     

Identification of synthetic phosphatidylserine translocases from a combinatorial library prepared by directed split-and-pool synthesis

Simple sulfonamide and amide derivatives of tris(2-aminoethyl)amine (Tren) are known to promote the translocation or flip-flop of phosphatidylcholine, but not phosphatidylserine, across bilayer membranes. This paper describes the synthesis of a 300-member, spatially encoded library of Tren derivatives with appended peptide--sulfonamide and peptide--urea arms. The library was synthesized using the Encore method with SynPhase lanterns as the solid support. A high-throughput assay was developed to screen individual members of the library for an ability to translocate a fluorescent NBD derivative of phosphatidylserine across vesicle membranes. Several lead compounds were identified, and one was synthesized independently to confirm its high phosphatidylserine translocation activity.     

A combinatorial scaffold approach based upon a multicomponent reaction

A combinatorial scaffolding procedure for the synthesis and spatial arrangement of tripartite structures was developed. [reaction: see text]     

Diastereoselective Ru-catalyzed cross-metathesis-dihydroxylation sequence. an efficient approach toward enantiomerically enriched syn-diols

Sequential catalysis has evolved as a powerful concept within the past years and allows the more efficient use of catalytically active expensive transition metals in organic synthesis. In this paper we present the stereoselective cross-metathesis-dihydroxylation of various olefins with chiral auxiliary substituted acrylamides. The chiral information (i.e., the auxiliary) introduced in the metathesis reactions allows for a stereoselective subsequent RuO4-catalyzed dihydroxylation. The sequence is concluded by an unusual kinetic resolution of the diastereomeric diols obtained in the oxidation reaction. As a consequence a variety of structurally diverse enantiomerically enriched diols are obtained. To the best of our knowledge the results summarized in this paper represent the first highly efficient diastereoselective RuO4-catalyzed oxidation.     

Identification of novel functional mini-receptors by combinatorial screening of split-WW domains

β-Sheet motifs such as the WW domain are increasingly being explored as building blocks for synthetic biological applications. Since the sequence-structure relationships of β-sheet motifs are generally complex compared to the well-studied α-helical coiled coil (CC), other approaches such as combinatorial screening should be included to vary the function of the peptide. In this study, we present a combinatorial approach to identify novel functional mini-proteins based on the WW-domain scaffold, which takes advantage of the successful reconstitution of the fragmented WW domain of hPin1 (hPin1_WW) by CC association. Fragmentation of hPin1_WW was performed in both loop 1 (CC-hPin1_WW-L1) and loop 2 (CC-hPin1_WW-L2), and the respective fragments were linked to the strands of an antiparallel heterodimeric CC. Structural analysis by CD and NMR spectroscopy revealed structural reconstitution of the WW-domain scaffold only in CC-hPin1_WW-L1, but not in CC-hPin1_WW-L2. Furthermore, by using ¹H–¹⁵N HSQC NMR, fluorescence and CD spectroscopy, we demonstrated that binding properties of fragmented hPin1_WW in CC-hPin1_WW-L1 were fully restored by CC association. To demonstrate the power of this approach as a combinatorial screening platform, we synthesized a four-by-six library of N- and C-terminal hPin1_WW-CC peptide fragments that was screened for a WW domain that preferentially binds to ATP over cAMP, phophocholine, or IP6. Using this screening platform, we identified one WW domain, which specifically binds ATP, and a phosphorylcholine-specific WW-based mini-receptor, both having binding dissociation constants in the lower micromolar range.

A combinatorial approach toward novel functional WW domains based on coiled-coil-mediated reconstitution of split WW domains is presented. As such, an ATP-binding WW domain was found from a 4-by-6 library of N- and C-terminal WW domain fragments.     

Predicting sequences and structures of MHC-binding peptides: a computational combinatorial approach

Peptides bound to MHC molecules on the surface of cells convey critical information about the cellular milieu to immune system T cells. Predicting which peptides can bind an MHC molecule, and understanding their modes of binding, are important in order to design better diagnostic and therapeutic agents for infectious and autoimmune diseases. Due to the difficulty of obtaining sufficient experimental binding data for each human MHC molecule, computational modeling of MHC peptide-binding properties is necessary. This paper describes a computational combinatorial design approach to the prediction of peptides that bind an MHC molecule of known X-ray crystallographic or NMR-determined structure. The procedure uses chemical fragments as models for amino acid residues and produces a set of sequences for peptides predicted to bind in the MHC peptide-binding groove. The probabilities for specific amino acids occurring at each position of the peptide are calculated based on these sequences, and these probabilities show a good agreement with amino acid distributions derived from a MHC-binding peptide database. The method also enables prediction of the three-dimensional structure of MHC-peptide complexes. Docking, linking, and optimization procedures were performed with the XPLOR program [1].     

Ligand design by a combinatorial approach based on modeling and experiment: application to HLA-DR4

Combinatorial synthesis and large scale screening methods are being used increasingly in drug discovery, particularly for finding novel lead compounds. Although these "random" methods sample larger areas of chemical space than traditional synthetic approaches, only a relatively small percentage of all possible compounds are practically accessible. It is therefore helpful to select regions of chemical space that have greater likelihood of yielding useful leads. When three-dimensional structural data are available for the target molecule this can be achieved by applying structure-based computational design methods to focus the combinatorial library. This is advantageous over the standard usage of computational methods to design a small number of specific novel ligands, because here computation is employed as part of the combinatorial design process and so is required only to determine a propensity for binding of certain chemical moieties in regions of the target molecule. This paper describes the application of the Multiple Copy Simultaneous Search (MCSS) method, an active site mapping and de novo structure-based design tool, to design a focused combinatorial library for the class II MHC protein HLA-DR4. Methods for the synthesizing and screening the computationally designed library are presented; evidence is provided to show that binding was achieved. Although the structure of the protein-ligand complex could not be determined, experimental results including cross-exclusion of a known HLA-DR4 peptide ligand (HA) by a compound from the library. Computational model building suggest that at least one of the ligands designed and identified by the methods described binds in a mode similar to that of native peptides.     

Kinetics of bimolecular chemical reaction by a modified diffusion equation

In order to describe the kinematical behavior of the bimolecular chmical reaction in a dilute solution of species characterized by a single diffusion coefficient and by some nonuniform spatial distribution of the active site, a diffusion equation with a simple sink term is derived by reducing the many-body problem to a one-body problem. The equation is normalized with the concentration of unreacted particles. The so-called second-order reaction rate constant can be calculated from the solution of the equation. The equation is applied to the intermolecular termination reaction of polymer radicals on the assumption of free draining. The reaction rate constant gradually decreases with time.     

Design and synthesis of artificial ribonucleases based on 1,4-diazabicyclo[2.2.2]octane and imidazole

The review surveys the results of our studies devoted to the design of highly efficient catalysts of hydrolysis of the phosphodiester bonds in RNA. These catalysts contain the imidazole residue in the catalytic domain, one or several bis-quaternized rings of 1,4-diazabicyclo[2.2.2]octane as a polycationic RNA-binding domain, and a lipophilic radical. A versatile approach to artificial ribonucleases of this type was proposed, which allows one to vary not only the number of positive charges in the RNA-binding domain, the structure of the catalytic site, and their mutual arrangement but also the domain structure of the molecule as a whole. Analysis of the catalytic properties of the synthesized constructs makes it possible to optimize the domain structure and the geometry of the molecule ensuring its maximum ribonuclease activity.     

Identification of a potent peptide deformylase inhibitor from a rationally designed combinatorial library

Peptide deformylase catalyzes the removal of the N-terminal formyl group from nascent polypeptides during prokaryotic protein synthesis and maturation and is essential for bacterial survival. Its apparent absence from mammalian organisms makes it an attractive target for designing novel antibacterial agents. Based on the substrate specificity of peptide deformylase from Escherichia coli, a focused library of peptide thiols was synthesized on TentaGel resin using a disulfide linkage. Screening of the library against the purified deformylase was carried out in solution phase after the inhibitors were released from the resin with a reducing agent. A potent deformylase inhibitor was obtained from a 750-member library and was further optimized through rational modification into a low nanomolar inhibitor (KI = 15 nM against E. coli deformylase).     

Fluorescent sensors for specific RNA: a general paradigm using chemistry and combinatorial biology

Here, we describe a new paradigm for the development of small molecule-based RNA sensors. We prepared a series of potential PET (photoinduced electron transfer) sensors on the basis of 2',7'-dichlorofluorescein (DCF) fluorophore conjugated with two aniline derivatives as electron donors (quenchers). NMR and fluorescent spectroscopic analyses of these DCF derivatives revealed the correlation between the conformations, the PET, and the fluorescent intensities of these DCF derivatives, enabling us to select a sensor candidate. RNA aptamers were raised against the aniline-based quencher via in vitro selection (SELEX). One of these aptamers enhanced the fluorescence intensity of the DCF-aniline conjugate in a concentration-dependent manner. To demonstrate the power and generality of this approach, additional in vitro selection was performed and aptamers from this selection were found to have similar activities. These results show that one can develop fluorescence-inducing reporter RNA and morph it into remotely related sequences without prior structural insight into RNA-ligand binding.