A multidimensional overpressured layer chromatographic method for the characterization of tetrazine libraries

The recent combinatorial approach in synthetic organic chemistry started a new age in drug discovery. The generation of compound libraries in combination with high-throughput screening has become the method of choice for the production of new pharmacological leads for chemical optimization. Characterization and separation of such pool of compounds have been lagging behind the synthetic and screening methodologies. Overpressured layer chromatography (OPLC) is an instrumentalized planar liquid chromatographic technique associated with the use of optimized layers prepared from particles of narrow particle size distribution and small diameter. On one hand, uni-directional OPLC allows the simultaneous separation of large number of samples in minutes. On the other hand, two-dimensional OPLC offers multidimensional separation on a single layer. This paper shows the complete multidimensional separation of a tetrazine library prepared by parallel combinatorial synthesis. In general, this approach may become the method of choice for the characterization of compound libraries.     

Degradation of selected (bio-)surfactants by bacterial cultures monitored by calorimetric methods

The subjects of the article are investigations concerning the ability of both Rhodococcus opacus 1CP and mixed bacterial cultures to use selected surfactants as sole carbon and energy source. In a comparative manner the biosurfactants rhamnolipid, sophorolipid and trehalose tetraester, and the synthetic surfactant Tween 80 were examined. Particular emphasis was put on a combinatorial approach to determine quantitatively the degree of surfactant degradation by applying calorimetry, thermodynamic calculations and mass spectrometry, HPLC as well as determination of biomass. The pure bacterial strain R. opacus was only able to metabolize a part of the synthetic surfactant Tween 80, whereas the mixed bacterial cultures degraded all of the applied surfactants. Exclusive for the biosurfactant rhamnolipid a complete microbial degradation could be demonstrated. In the case of the other surfactants only primary degradation was observed.     

De novo synthetic route to a combinatorial library of peptidyl nucleosides

A stereoselective synthetic route has been developed for the combinatorial synthesis of a structurally unique class of C-4' side chain modified peptide-linked nucleosides. The synthetic strategy and approach involves initial synthesis of a strategically functionalized amino butenolide template, utilizing L-serine as a chiral starting material. Subsequent transformation of the above lactone to C4' aminoalkyl substituted nucleosides, followed by the peptidic coupling of the C4' side chain amine with various amino acids completed the syntheses of the target peptidyl nucleosides. Employing the above route, and utilizing a combination of easily available nucleobases (4) and amino acids (6) as the two diversity elements, synthesis of a 24-member combinatorial library of the title peptide-linked nucleosides has been accomplished.     

All D-amino acid hexapeptide inhibitors of melittin's cytolytic activity derived from synthetic combinatorial libraries

The identification of peptides that inhibit the biological functions of proteins was used as a means to explore protein/ligand interactions involved in molecular recognition processes. This approach is based on the use of synthetic combinatorial libraries (SCLs) for the rapid identification of individual peptides that block the interaction of proteins with their biological targets. Thus, each peptide mixture of an all-D -amino acid hexapeptide SCL in a positional scanning format was screened for its ability to inhibit the hemolytic activity of melittin, a model self-assembling protein. The potent inhibitory activity of the identified individual peptides suggests that protein-like complexes are able to specifically bind to peptides having an all-D configuration. These results also show that SCLs are useful for the identification of short, non-hydrolysable sequences having potential intracellular inhibitory activities.     

Synthesis of natural-product-based compound libraries

Natural products cover a diversity space not yet available from synthetic libraries, with an unrivalled success rate as drug leads. The combinatorial synthesis of non-oligomeric natural-product-based libraries, however, is still limited to few examples because access to easily modified units strongly depends on the availability of a core structure either from a natural source, or through a suitable synthetic route. Only a few resourceful groups have managed the latter approach for more demanding multifunctional natural drug leads, such as epothilones.     

Mesofluidic devices for DNA-programmed combinatorial chemistry

Hybrid combinatorial chemistry strategies that use DNA as an information-carrying medium are proving to be powerful tools for molecular discovery. In order to extend these efforts, we present a highly parallel format for DNA-programmed chemical library synthesis. The new format uses a standard microwell plate footprint and is compatible with commercially available automation technology. It can accommodate a wide variety of combinatorial synthetic schemes with up to 384 different building blocks per chemical step. We demonstrate that fluidic routing of DNA populations in the highly parallel format occurs with excellent specificity, and that chemistry on DNA arrayed into 384 well plates proceeds robustly, two requirements for the high-fidelity translation and efficient in vitro evolution of small molecules.     

Enzymes in the synthesis of bioactive compounds: the prodigious decades

The growing demand for enantiomerically pure pharmaceuticals has impelled research on enzymes as catalysts for asymmetric synthetic transformations. However, the use of enzymes for this purpose was rather limited until the discovery that enzymes can work in organic solvents. Since the advent of the PCR the number of available enzymes has been growing rapidly and the tailor-made biocatalysts are becoming a reality. Thus, it has been possible the use of enzymes for the synthesis of new innovative medicines such as carbohydrates and their incorporation to modern methods for drug development, such as combinatorial chemistry. Finally, the genomic research is allowing the manipulation of whole genomes opening the door to the combinatorial biosynthesis of compounds. In this review, our intention is to highlight the main landmarks that have led to transfer the chemical efficiency shown by the enzymes in the cell to the synthesis of bioactive molecules in the lab during the last 20 years.     

Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC

Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs.     

Comparison of mRNA-display-based selections using synthetic peptide and natural protein libraries

mRNA display is a genotype-phenotype conjugation method that allows the amplification-based, iterative rounds of in vitro selection to be applied to peptides and proteins. Compared to prior protein selection techniques, mRNA display can be used to select functional sequences from both long natural protein and short combinatorial peptide libraries with much higher complexities. To investigate the basic features and problems of using mRNA display in studying conditional protein-protein interactions, we compared the target-binding selections against calmodulin (CaM) using both a natural protein library and a combinatorial peptide library. The selections were efficient in both cases and required only two rounds to isolate numerous Ca2+/CaM-binding natural proteins and synthetic peptides with a wide range of affinities. Many known and novel CaM-binding proteins were identified from the natural human protein library. More than 2000 CaM-binding peptides were selected from the combinatorial peptide library. Unlike sequences from prior CaM-binding selections that correlated poorly with naturally occurring proteins, synthetic peptides homologous to the Ca2+/CaM-binding motifs in natural proteins were isolated. Interestingly, a large number of synthetic peptides that lack the conventional CaM-binding secondary structures bound to CaM tightly and specifically, suggesting the presence of other interaction modes between CaM and its downstream binding targets. Our results indicate that mRNA display is an ideal approach to the identification of Ca2+-dependent protein-protein interactions, which are important in the regulation of numerous signaling pathways.     

Using specificity to strategically target proteases

Proteases are a family of naturally occurring enzymes in the body whose dysregulation has been implicated in numerous diseases and cancers. Their ability to selectively and catalytically turnover substrate adds both signal amplification and functionality as parameters for the detection of disease. This review will focus on the development of activity-based methodologies to characterize proteases, and in particular, the use of positional scanning, synthetic combinatorial libraries (PS-SCL's), and substrate activity screening (SAS) assays. The use of these approaches to better understand a protease's natural substrate will be discussed as well as the technologies that emerged.     

Quarterly U.S. patent review: first quarter, 1999.

The following are among the U.S. patents, issued in the first quarter of 1999, directed to combinatorial chemistry and related technologies. Patent issuances in the field are growing in number. Additionally, patents claiming libraries themselves, as opposed to synthetic methodologies, are becoming more common. It is impossible to be comprehensive. The author would be pleased to recognize additional patents and invites the use of his e-mail address for this purpose.     

Solution-phase library generation: methods and applications in drug discovery

Solution-phase high throughput synthesis has emerged as a powerful method for the rapid generation of chemical libraries. The success of this approach is largely due to the development of novel synthetic methodologies that expedite the preparation of compounds. Several isolation/purification techniques have also been developed to eliminate the time-consuming purification procedures often associated with solution-phase chemistry. These methods are amenable to parallel synthesis and combinatorial strategies and can be fully automated. In addition, the compound libraries generated using solution-phase high throughput synthesis have been used to accelerate both lead identification and lead optimization programs at various companies.     

The recent impact of solid-phase synthesis on medicinally relevant benzoannelated nitrogen heterocycles

Benzoannelated heterocycles such as benzodiazepines and indoles can be prepared efficiently through cyclization on solid supports, although no single approach is currently universal for the preparation of all benzoannelated N-heterocycle chemistries. In this review, a number of synthetic strategies for the generation of benzoannelated nitrogen heterocycles using resin-bound substrates have been described. Classical heterocycle forming reactions such as the Fischer indole, the Bischler-Napieralski tetrahydroisoquinoline, the Pictet-Spengler tetrahydro-beta-carboline, the Tsuge, the Nenitzescu and the Richter cinnoline reaction are presented. In addition, the Heck, Sonogashira, Wittig, Diels-Alder, and olefin metathesis reactions have been also used. Multicomponent reactions such as the Grieco three-component assembly have been exploited for the synthesis of heterocycles. Cyclative cleavage from the solid support is particularly suitable for the synthesis of heterocycles while particular emphasis has been focused on the synthesis of libraries and the use of combinatorial chemistry techniques. In addition, the most relevant pharmacological properties of benzoannelated nitrogen heterocycles are included.     

羊毛硫肽的高通量工程改造方法新进展

羊毛硫肽(lanthipeptide)是由核糖体合成并经翻译后修饰产生的肽类天然产物,具有丰富的分子结构和多样的生物活性.新型羊毛硫肽是活性药物的重要来源,可以通过基因组挖掘和工程改造获得.羊毛硫肽前体肽由基因编码,同时其合成酶具有较高的底物杂泛性.基于这些特征,可以对羊毛硫肽的生物合成过程开展高通量工程改造,从而快速...     

Synthesis of Polyaminooligonucleotides and Their Combinatorial Libraries

Abstract

A synthesis of phosphoramidites of 2′-deoxyadenosine and 2′-deoxyguanosine carrying a protected spermine moiety at N-6 and N-2 positions respectively is described. An approach to analyse properties of polyaminooligonucleotides using their synthetic combinatorial libraries is described and discussed. A synthesis of a polyaminooligonucleotide combinatorial library was carried out and the analysis of the library clearly showed that the presence of spermine moieties in oligodeoxyribonucleotides increases stability of their complexes.     

Mimotopes of the nicotinic receptor binding site selected by a combinatorial peptide library

Peptide libraries allow selecting new molecules, defined as mimotopes, which are able to mimic the structural and functional features of a native protein. This technology can be applied for the development of new reagents, which can interfere with the action of specific ligands on their target receptors. In the present study we used a combinatorial library approach to produce synthetic peptides mimicking the snake neurotoxin binding site of nicotinic receptors. On the basis of amino acid sequence comparison of different alpha-bungarotoxin binding receptors, we designed a 14 amino acid combinatorial synthetic peptide library with five invariant, four partially variant, and five totally variant positions. Peptides were synthesized using SPOT synthesis on cellulose membranes, and binding sequences were selected using biotinylated alpha-bungarotoxin. Each variant position was systematically identified, and all possible combinations of the best reacting amino acids in each variant position were tested. The best reactive sequences were identified, produced in soluble form, and tested in BIACORE to compare their kinetic constants. We identified several different peptides that can inhibit the binding of alpha-bungarotoxin to both muscle and neuronal nicotinic receptors. Peptide mimotopes have a toxin-binding affinity that is considerably higher than peptides reproducing native receptor sequences.     

Combinatorially-generated library of 6-fluoroquinolone analogs as potential novel antitubercular agents: a chemometric and molecular modeling assessment

The virtual combinatorial chemistry approach as a methodology for generating chemical libraries of structurally-similar analogs in a virtual environment was employed for building a general mixed virtual combinatorial library with a total of 53.871 6-FQ structural analogs, introducing the real synthetic pathways of three well known 6-FQ inhibitors. The druggability properties of the generated combinatorial 6-FQs were assessed using an in-house developed drug-likeness filter integrating the Lipinski/Veber rule-sets. The compounds recognized as drug-like were used as an external set for prediction of the biological activity values using a neural-networks (NN) model based on an experimentally-determined set of active 6-FQs. Furthermore, a subset of compounds was extracted from the pool of drug-like 6-FQs, with predicted biological activity, and subsequently used in virtual screening (VS) campaign combining pharmacophore modeling and molecular docking studies. This complex scheme, a powerful combination of chemometric and molecular modeling approaches provided novel QSAR guidelines that could aid in the further lead development of 6-FQs agents.     

Rational optimization of a short human P-selectin-binding peptide leads to nanomolar affinity antagonists

P-selectin plays an important role in the development of various diseases, including atherosclerosis and thrombosis. In our laboratory we recently identified a number of specific human P-selectin-binding peptides containing a Glu-Trp-Val-Asp-Val consensus motif, displaying a low micromolar affinity for P-selectin (IC(50) = 2 microm). In search of more potent antagonists for P-selectin, we have optimized the EWVDV pentapeptide core motif via a two-step combinatorial chemistry approach. A dedicated library of peptide derivatives was generated by introducing seven substituents at the N and C termini of the motif. In particular, pentapeptides with gallic acid or 1,3,5-benzenetricarboxylic acid substituents at the N terminus proved to be considerably more potent inhibitors of P-selectin binding than the parental peptide. After removal of the N-terminal glutamic acid from the core sequence, which appeared to be replaceable by a carboxamide function without loss of affinity, a second library was synthesized to map the chemical moieties within the gallic acid or 1,3,5-benzenetricarboxyl acid groups responsible for the enhanced P-selectin binding. Moreover, by varying the length and rigidity of the connective spacer, we have further optimized the spatial orientation of the N-terminal substituent. The combined use of phage display and subsequent combinatorial chemistry led to the design of a number of gallic acid- containing peptides with low nanomolar affinity for P-selectin both under static and dynamic conditions (IC(50) = 15.4 nm). These small synthetic antagonists, which are equally as potent as the natural ligand P-selectin glycoprotein ligand-1, are promising leads in anti-atherothrombotic therapy.     

Triazine-based condensing reagents

The synthesis, properties, and application of condensing reagents derived from 1,3,5-triazines are described. The mechanism of activation of carboxylic function, structure of reactive intermediates, and mechanism of acylation of nucleophiles are presented. The synthetic versatility of mono- and bifunctional reagents for syntheses in solution, triazine-based immobilized reagents, chiral triazines for enantiodifferentiating syntheses, are discussed. The scope and limitation of the synthetic utility of triazine reagents in the preparation of heterocyclic compounds, amides, esters, oligopeptides-including large-scale syntheses and use in the combinatorial chemistry-is demonstrated.