Scalable methods for the construction and analysis of virtual combinatorial libraries

One can distinguish between two kinds of virtual combinatorial libraries: viable and accessible . Viable libraries are relatively small in size, are assembled from readily available reagents that have been filtered by the medicinal chemist, and often have a physical counterpart. Conversely, accessible libraries can encompass millions or billions of structures, typically include all possible reagents that are in principle compatible with a particular reaction scheme, and they can never be physically synthesized in their entirety. Although the analysis of viable virtual libraries is relatively straightforward, the handling of large accessible libraries requires methods that scale well with respect to library size. In this work, we present novel, efficient and scalable techniques for the construction, analysis, and in silico screening of massive virtual combinatorial libraries.     

Chemoinformatics methods for systematic comparison of molecules from natural and synthetic sources and design of hybrid libraries

Until recently, the field of diversity and library design has more or less ignored natural products as a compound source. This is probably due to at least two reasons. First, combinatorial and reaction-based approaches have been major focal points in the early days of computational library design. In addition, a widespread view is that natural products are often highly complex and not amenable to medicinal chemistry efforts. This contribution introduces recent computational approaches to systematically analyze natural molecules and bridge the gap between natural products and synthetic chemistry programs. Large scale comparisons of natural and synthetic molecules are discussed as well as studies designed to identify `synthetic mimics' of natural products with specific activity. In addition, a concept for the design of natural/synthetic hybrid libraries is introduced. Although research in this area is still in its early stages, an important lesson to be learned from computational analyses is that there is no need to a priori `shy away' from natural products as a source for molecular design.     

Structural requirements for the biosynthesis of backbone cyclic peptide libraries

BACKGROUND: Combinatorial methods for the production of molecular libraries are an important source of ligand diversity for chemical biology. Synthetic methods focus on the production of small molecules that must traverse the cell membrane to elicit a response. Genetic methods enable intracellular ligand production, but products must typically be large molecules in order to withstand cellular catabolism. Here we describe an intein-based approach to biosynthesis of backbone cyclic peptide libraries that combines the strengths of synthetic and genetic methods. RESULTS: Through site-directed mutagenesis we show that the DnaE intein from Synechocystis sp. PCC6803 is very promiscuous with respect to peptide substrate composition, and can generate cyclic products ranging from four to nine amino acids. Libraries with five variable amino acids and either one or four fixed residues were prepared, yielding between 10(7) and 10(8) transformants. The majority of randomly selected clones from each library gave cyclic products. CONCLUSIONS: We have developed a versatile method for producing intracellular libraries of small, stable cyclic peptides. Genetic encoding enables facile manipulation of vast numbers of compounds, while low molecular weight ensures ready pharmacophore identification. The demonstrated flexibility of the method towards both peptide length and composition makes it a valuable addition to existing methods for generating ligand diversity.     

Design and screening of M13 phage display cDNA libraries

The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF) libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS) will be presented.     

Sustainable synthetic methods: domino construction of dihydropyridin-4-ones and beta-amino esters in aqueous ethanol

Domino reactions were designed to allow the byproduct of an upstream reaction to be internally recycled to catalyze a downstream reaction in a one-pot tandem sequence. Nitroarene reduction by In(0) generates an amine and In (III) byproducts. Addition of aldehyde followed by Danishefsky's diene or silyl ketene acetal provides access to dihydropyridin-4-ones or beta-amino esters, respectively, in yields that are comparable or superior to the reported stepwise reactions.     

Bicyclic homodetic peptide libraries: comparison of synthetic strategies for their solid-phase synthesis

Preliminary studies and synthesis development for the preparation of a bicyclic homodetic peptide library have been carried out using orthogonal protection schemes. The best results have been obtained using two Fmoc/tBu-based strategies, in which the first cycle is carried out in the solid phase through side chain functional groups previously protected with Aloc/Al groups. The second cycle is performed either in the solid phase, which requires side chain anchoring of a trifunctional amino acid and Dmb protection for the C-terminus carboxyl group, or in solution, which requires the use of highly labile resins, such as the 2-chlorotrityl (Barlos) resin. Only when the cycles are formed in a ziplike manner, that is, first the small cycle and then the larger ring, is the desired final product obtained.     

Random sequence libraries displayed on phage: identification of biologically important molecules

Phage display has become a widely used tool for the identification of proteins or peptides with affinity for a variety of biomolecules. The versatility, simplicity and cost effectiveness of this application has pervaded a wide variety of research areas. Although not without its limitations, phage display has provided a convenient methodology for obtaining ligands to study the function, structure and diagnostic or therapeutic potential of various macromolecules. This review highlights some recent research employing this technology that serves to illustrate its utility in various research and clinical applications.     

Past and future perspectives of synthetic peptide libraries

Combinatorial preparation and HTS of arrays of compounds have increased the speed of drug discovery. A strong impulse in this field has come by the introduction of the solid phase synthesis method that, through automation and miniaturization, has paved the way to the preparation of large collections of compounds in compact and trackable formats. Due to the well established synthetic procedures, peptides have been largely used to develop the basic concepts of combinatorial chemistry and peptide libraries are still successfully employed in screening programs. However, peptides generally do not fulfil the requirements of low conformational flexibility, stability and bioavailability needed for good drug candidates and peptide leads with high potency and selectivity are often made "druggable" by conversion to more stable structures with improved pharmacological profiles. Such an approach makes the screening of peptide libraries still a valuable tool for drug discovery. We propose here a panoramic review of the most common methods for the preparation and screening of peptide libraries and the most interesting findings of the last decade. We also report on a new approach we follow in our laboratory that is based on the use of "simplified" libraries composed by a minimum number of non-redundant amino acids for the assembly of short peptides. The choice of amino acids is dictated by diversity in lipophilicity, MW, charge and polarity. Newly identified active sequences are then modified by preparing new variants containing analogous amino acids, so that the chemical space occupied by the excluded residues can be explored. This approach offers the advantage of simplifying the synthesis and deconvolution of libraries and provides new active compounds with a molecular size similar to that of small molecules, to which they can be easily converted.     

Agarose gel electrophoresis reveals structural fluidity of a phage T3 DNA packaging intermediate

We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (i) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for the stabilization of structure and then (ii) determining effective radius by two-dimensional agarose gel electrophoresis (2D-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase the production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2D-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when the ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging.     

A simple strategy for the construction of combinatorial cyclic peptoid libraries

Here we describe a simple method that allows for rapid and easy sequence determination of cyclic peptoids. The key idea in this strategy is a post-screening "ring-opening" reaction to convert cyclic peptoids selected from a high-throughput screen into linear peptoids, which can be sequenced by tandem mass spectrometry. Thus, there is no need for encoding.     

Uncommon multivariate statistical methods for environmental studies: A review

In this paper we describe the characteristics and the applications of the multivariate methods for spectroscopic and chromatographic techniques independent component analysis (ICA) and two-dimensional correlation spectroscopy (2DCOS) focused to their use in environmental studies. In our opinion, these methods are important because they allow to characterize environmental samples with different aims and scopes from those generally obtained by means of more common multivariate methods such as principal component analysis (PCA) and partial least squares (PLS). The new insights of these methods in recent environmental studies are reviewed and debated.     

Strategies for the construction and use of peptide and antibody libraries displayed on phages

Combinatorial chemistry and biology have become popular methods for the identification of bio-active molecules in drug discovery. A widely used technique in combinatorial biology is "phage display", by which peptides, antibody fragments and enzymes are displayed on the surface of bacteriophages, and can be selected by simple procedures of biopanning. The construction of phage libraries of peptides or antibody fragments provides a huge source of ligands and bio-active molecules that can be isolated from the library without laborious studies on antigen characteristics and prediction of ligand structure. This "irrational" approach for the construction of new drugs is extremely rapid and is now used by thousands of laboratories world-wide. The bottleneck in this procedure is the availability of large reliable libraries that can be used repeatedly over the years without loss of ligand expression and diversity. Construction of personalized libraries is therefore important for public and private laboratories engaged in the isolation of specific molecules for therapeutic or diagnostic use. Here we report the general strategies for constructing large phage peptide and antibody libraries, based on the experience of researchers who built the world's most widely used libraries. Particular attention is paid to advanced strategies for the construction, preservation and panning.     

General synthetic methods for the preparation of pinwheel receptors

The synthesis of bis-trityl diynes, used as cooperative fluorescent chemosensors, is described. The most convenient sequence for the preparation of the sensor framework was found to be intolerant of most functionality. Therefore, an unfunctionalized framework is constructed and recognition elements are selectively installed via electrophilic substitution on an electronically differentiated key intermediate. A divergent synthesis of a general class of chemical sensors emerges. This synthetic method is highlighted by its simplicity and modularity allowing for the introduction of various recognition elements and fluorophores.     

Spiroleucettadine: synthetic studies and investigations towards structural revision

Synthetic efforts towards spiroleucettadine are described, including the enantioselective synthesis of the presumed biosynthetic precursor. High level density functional theory calculations were used to predict the ¹³C NMR shifts of possible alternative structures and, along with a re-evaluation of the available NMR data, allow the proposal of revised structures for this spirocyclic alkaloid.     

LocaPep: localization of epitopes on protein surfaces using peptides from phage display libraries

The use of peptides from a phage display library selected by binding to a given antibody is a widespread technique to probe epitopes of antigenic proteins. However, the identification of interaction sites mimicked by these peptides on the antigen surface is a difficult task. LocaPep is a computer program developed to localize epitopes using a new clusters algorithm that focuses on protein surface properties. The program is constructed with the aim of providing a flexible computational tool for predicting the location of epitopes on protein structures. As a first set of testing results, the localization of epitope regions in eight different antigenic proteins for which experimental data on their antibody interactions exist is correctly identified by LocaPep. These results represent a disparate sample of biologically different systems. The program is freely available at http://atenea.montes.upm.es.