A parallel solution-phase synthesis of substituted 3,7-diazabicyclo[3.3.1]nonanes

The parallel solution-phase synthesis of a series of building blocks and combinatorial libraries based on natural bispidine scaffold has been accomplished. Key reactions include catalytic hydrogenation of the (-)-cytisine heterocyclic system, followed by alkali-mediated ring cleavage. Using this approach, a series of new bispidine core building blocks for combinatorial synthesis with three points of diversity were effectively synthesized. The libraries from libraries were then obtained in good yields and purities using solution-phase acylation reactions. Obtained combinatorial libraries of 3,4,7-trisubstituted bispidines are potentially useful in the discovery of novel physiologically active compounds.     

Library fingerprints: a novel approach to the screening of virtual libraries

We propose a novel method to prioritize libraries for combinatorial synthesis and high-throughput screening that assesses the viability of a particular library on the basis of the aggregate physical-chemical properties of the compounds using a na?ve Bayesian classifier. This approach prioritizes collections of related compounds according to the aggregate values of their physical-chemical parameters in contrast to single-compound screening. The method is also shown to be useful in screening existing noncombinatorial libraries when the compounds in these libraries have been previously clustered according to their molecular graphs. We show that the method used here is comparable or superior to the single-compound virtual screening of combinatorial libraries and noncombinatorial libraries and is superior to the pairwise Tanimoto similarity searching of a collection of combinatorial libraries.     

Solution-phase combinatorial libraries: modulating cellular signaling by targeting protein-protein or protein-DNA interactions

The high-throughput synthesis and screening of compound libraries hold tremendous promise for drug discovery and powerful methods for both solid-phase and solution-phase library preparation have been introduced. The question of which approach (solution-phase versus solid-phase) is best for the preparation of chemical libraries has been replaced by which approach is most appropriate for a particular target or screen. Herein we highlight distinctions in the two approaches that might serve as useful considerations at the onset of new programs. This is followed by a more personal account of our own focus on solution-phase techniques for the preparation of libraries designed to modulate cellular signaling by targeting protein-protein or protein-DNA interactions. The screening of our libraries against a prototypical set of extracellular and intracellular targets, using a wide range of assay formats, provided the first small-molecule modulators of the protein-protein interactions studied, and a generalized approach for conducting such studies.     

Three-component synthesis of 1,4-diazepin-5-ones and the construction of gamma-turn-like peptidomimetic libraries

The parallel synthesis of gamma-turn-inspired peptidomimetic libraries has been demonstrated through two approaches toward the preparation of 1,4-diazepin-5-ones. In the first approach, 1,4-diazepin-5-ones scaffolds were prepared on gram scale and subsequently diversified to produce libraries. In a second approach, libraries of 1,4-diazepin-5-ones were produced directly through a three-component strategy that maximizes the diversity obtained in a single step.     

Solid phase synthesis of structurally diverse tetra substituted pyrimidines for potential use in combinatorial chemistry

A new pyrimidine based scaffold has been identified for generation of combinatorial libraries using solid phase technique. The utility of the scaffolds was demonstrated by synthesizing small libraries of 12 substituted pyrimidines (4a-4l).     

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.     

An efficient solid phase synthesis of 5'-phosphodiester and phosphoramidate monoester nucleoside analogues

An easy and efficient strategy to obtain libraries of 5'-phosphodiester and 5'-phosphoramidate monoester nucleoside analogues in a highly pure form has been developed, starting from a new nucleoside based solid support. The nucleoside scaffold has been anchored through a 5'-phosphodiester linkage to Tentagel HL resin, functionalized with a 3-chloro-4-hydroxyphenylacetic linker. The solid phase synthesis of small libraries of 5'-phosphodiester and 5'-phosphoramidate monoester thymidine analogues is also reported.     

Synthesis of combinatorial libraries based on terpenoid scaffolds

Triterpenoid-based scaffolds betulinic acid (1a) and ursolic acid (1b), have been used for the generation of combinatorial libraries in parallel format using solid phase organic synthesis method. These templates have the potential for the synthesis and amplification of triterpenoid-based compounds with one and two-point diversity. This has been demonstrated by the synthesis of two small libraries comprising 18 derivatives each of betulinic acid and ursolic acid with structural diversity at C-3 and C-28 positions. The primary screening of antimalarial activity of these libraries against P. falciparum in vitro led to the identification of four compounds with 5 fold increase in the activity compared to betulinic and ursolic acids.     

Towards the systematic exploration of chemical space

The discovery of biologically active small molecules is shaped, in large part, by their synthetic (or biosynthetic accessibility). However, chemists' historical exploration of chemical space has been highly uneven and unsystematic. This article describes synthetic strategies that have emerged that may allow chemical space to be explored more systematically. Particular emphasis is placed on approaches that allow the scaffolds of small molecules to be varied combinatorially. In addition, some examples of bioactive small molecules that have been discovered by screening diverse small molecule libraries are highlighted. The authors comment on the likely scope of each of the strategies to deliver skeletally-diverse libraries. In addition, the authors highlight some key challenges for the future: the extension to libraries based on hundreds of distinct scaffolds; and the development of approaches that focus overtly on drug-relevant chemical space.     

Large antibody display libraries for isolation of high-affinity antibodies

Libraries of displayed antibodies have been increasingly used to select or screen antibodies. It is generally well recognized that large libraries provide high probability of finding a given antibody. In this review, the factors that limit the sizes of current in vivo and ribosome-based in vitro display libraries to be around 10(11) members are first discussed. An analysis resulting in a quantitative correlation between the library size and the antibody affinity is then presented. This analysis underscores the importance of large libraries in not only increasing the probability of finding a given antibody, but also enhancing the quality of a given antibody. Recent advances in preparing state-of-the-art large display libraries are then reviewed. Finally, potential improvements to current library technologies to generate libraries as large as 10(12) and their rationales are explored in length.     

Combinatorial library design using a multiobjective genetic algorithm

Early results from screening combinatorial libraries have been disappointing with libraries either failing to deliver the improved hit rates that were expected or resulting in hits with characteristics that make them undesirable as lead compounds. Consequently, the focus in library design has shifted toward designing libraries that are optimized on multiple properties simultaneously, for example, diversity and "druglike" physicochemical properties. Here we describe the program MoSELECT that is based on a multiobjective genetic algorithm and which is able to suggest a family of solutions to multiobjective library design where all the solutions are equally valid and each represents a different compromise between the objectives. MoSELECT also allows the relationships between the different objectives to be explored with competing objectives easily identified. The library designer can then make an informed choice on which solution(s) to explore. Various performance characteristics of MoSELECT are reported based on a number of different combinatorial libraries.     

Synthesis and mass spectrometry studies of branched oxime ether libraries. Mapping the substitution motif via linker stability and fragmentation pattern.

The oxime ether chemistry has recently been used as a convenient approach to preparing potentially highly diverse combinatorial libraries. The synthetically easiest way to form the libraries is convergent, i.e., via reaction of a branched scaffold containing two or more aminooxy linker groups, with a variety of carbonyl substituents. We show here that such reactions between aldehydes and ketones of different structure with the scaffolds containing different types of aminooxy groups can lead to the formation of virtually all expected components in the model mixtures 1-3 formed from three scaffolds (7-9) and eight substituents (R(1)-R(8)). One important problem with the branched libraries is that the libraries formed from the more complex scaffolds, such as 11, contain multiple regioisomers. The results of extensive analysis of a variety of library components by mass spectrometry presented here show that the differences in the MS-MS fragmentation energies for different linkers yield regiochemical information essential for identification of individual library components.     

Synthesis and evaluation of poly(oxyethylene glycol) polymer (POP) supports

Mono- and alpha,omega-bis-styryl-oligo(oxyethylene glycol) ethers have been constructed in an efficient two-step synthesis. From these precursors, poly(oxyethylene glycol) polymer (POP) supports of varying monomer and cross-linker composition have been produced. The swelling properties and mass-solvent uptake of these novel materials have been evaluated in a variety of solvents, demonstrating that POP supports exhibit enhanced solvent compatibilities over the commercial resins TENTA-GEL, ARGO-GEL, and Merrifield's resin. The utility of POP supports in solid-phase organic chemistry has also been demonstrated successfully. It is anticipated that these high-loading polymeric supports will have generic application in the solid-phase synthesis of combinatorial libraries and the in situ screening of these libraries in the aqueous environment of a bioassay.     

Rapid identification of immunostimulatory alpha-galactosylceramides using synthetic combinatorial libraries

Two 60+-membered libraries of alpha-galactosylceramides have been prepared by reactions between activated ester resins and two core, fully deprotected galactosylated sphingoid bases. The libraries were evaluated for their ability to stimulate CD1d-restricted NKT cells, using in vitro stimulation of a murine NKT cell hybridoma line and for their ability to induce the expansion of NKT cells from peripheral blood mononuclear cells (PBMC) of a normal human subject. Our results showed that many compounds constructed on a C18-phytosphingosine base had significant stimulatory activity in both assays. Because no product purification was required, this approach is particularly attractive as a method for rapid synthesis of large libraries of potential immunomodulatory glycosylceramides.     

Is nontarget screening of emerging contaminants by LC-HRMS successful? A plea for compound libraries and computer tools

This review focuses on the possibilities and limits of nontarget screening of emerging contaminants, with emphasis on recent applications and developments in data evaluation and compound identification by liquid chromatography-high-resolution mass spectrometry (HRMS). The general workflow includes determination of the elemental composition from accurate mass, a further search for the molecular formula in compound libraries or general chemical databases, and a ranking of the proposed structures using further information, e.g., from mass spectrometry (MS) fragmentation and retention times. The success of nontarget screening is in some way limited to the preselection of relevant compounds from a large data set. Recently developed approaches show that statistical analysis in combination with suspect and nontarget screening are useful methods to preselect relevant compounds. Currently, the unequivocal identification of unknowns still requires information from an authentic standard which has to be measured or is already available in user-defined MS/MS reference databases or libraries containing HRMS spectral information and retention times. In this context, we discuss the advantages and future needs of publicly available MS and MS/MS reference databases and libraries which have mostly been created for the metabolomic field. A big step forward has been achieved with computer-based tools when no MS library or MS database entry is found for a compound. The numerous search results from a large chemical database can be condensed to only a few by in silico fragmentation. This has been demonstrated for selected compounds and metabolites in recent publications. Still, only very few compounds have been identified or tentatively identified in environmental samples by nontarget screening. The availability of comprehensive MS libraries with a focus on environmental contaminants would tremendously improve the situation.     

Tailored polymer-supported templates in dynamic combinatorial libraries: simultaneous selection, amplification and isolation of synthetic receptors

The thermodynamically controlled synthesis and isolation of macrocyclic receptors from dynamic combinatorial libraries has been achieved in a single step using a polymer-supported template. The templates were cinchona alkaloids which show interesting enantio- and diastereoselective molecular recognition events in libraries based on pseudo-dipeptide building blocks. The synthetic routes used to derivatise the alkaloids and attach them to polymer supports minimised any influence of the tethering linkage on the templating activity. Systematic studies have been carried out to probe how the polymer morphology and the template loading affect the selectivity and isolation yield of the macrocyclic receptors. Molecular recognition between solid-phase bound templates and selected receptors also enabled their affinity-type chromatographic separation.     

Streamlined approach to high-quality purification and identification of compound series using high-resolution MS and NMR

Automated medicinal chemistry (parallel chemistry) has become an integral part of the drug-discovery process in almost every large pharmaceutical company. Parallel array synthesis of individual organic compounds has been used extensively to generate diverse structural libraries to support different phases of the drug-discovery process, such as hit-to-lead, lead finding, or lead optimization. In order to guarantee effective project support, efficiency in the production of compound libraries has been maximized. As a consequence, also throughput in chromatographic purification and analysis has been adapted. As a recent trend, more laboratories are preparing smaller, yet more focused libraries with even increasing demands towards quality, i.e. optimal purity and unambiguous confirmation of identity. This paper presents an automated approach how to combine effective purification and structural conformation of a lead optimization library created by microwave-assisted organic synthesis. The results of complementary analytical techniques such as UHPLC-HRMS and NMR are not only regarded but even merged for fast and easy decision making, providing optimal quality of compound stock. In comparison with the previous procedures, throughput times are at least four times faster, while compound consumption could be decreased more than threefold.     

Visual characterization and diversity quantification of chemical libraries: 2. Analysis and selection of size-independent, subspace-specific diversity indices

High Throughput Screening (HTS) is a standard technique widely used to find hit compounds in drug discovery projects. The high costs associated with such experiments have highlighted the need to carefully design screening libraries in order to avoid wasting resources. Molecular diversity is an established concept that has been used to this end for many years. In this article, a new approach to quantify the molecular diversity of screening libraries is presented. The approach is based on the Delimited Reference Chemical Subspace (DRCS) methodology, a new method that can be used to delimit the densest subspace spanned by a reference library in a reduced 2D continuous space. A total of 22 diversity indices were implemented or adapted to this methodology, which is used here to remove outliers and obtain a relevant cell-based partition of the subspace. The behavior of these indices was assessed and compared in various extreme situations and with respect to a set of theoretical rules that a diversity function should satisfy when libraries of different sizes have to be compared. Some gold standard indices are found inappropriate in such a context, while none of the tested indices behave perfectly in all cases. Five DRCS-based indices accounting for different aspects of diversity were finally selected, and a simple framework is proposed to use them effectively. Various libraries have been profiled with respect to more specific subspaces, which further illustrate the interest of the method.     

MS-Pep: a computer program for the interpretation of mass spectra of peptide libraries

Electrospray mass spectrometry (ESI-MS) is an established method for the qualitative analysis of synthetic peptide libraries and combinatorial mixtures or collections of small organic compounds. However, the calculation of the mass distribution of even small peptide mixtures is a time-consuming and error-proned task. Therefore, the computer program MS-Pep has been developed, which calculates the masses of expected peptides, byproducts and the mass distributions of peptide libraries.     

Synthesis, screening, and sequencing of cysteine-rich one-bead one-compound peptide libraries

Cysteine-rich peptides are valued as tags for biarsenical fluorophores and as environmentally important reagents for binding toxic heavy metals. Due to the inherent difficulties created by cysteine, the power of one-bead one-compound (OBOC) libraries has never been applied to the discovery of short cysteine-rich peptides. We have developed the first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries. First, we synthesized a heavily biased cysteine-rich OBOC library, incorporating 50% cysteine at each position (Ac-X8-KM-TentaGel). Then, we developed conditions for cysteine alkylation, cyanogen bromide cleavage, and direct MS/MS sequencing of that library at the single bead level. The sequencing efficiency of this library was comparable to a traditional cysteine-free library. To validate screening of cysteine-rich OBOC libraries, we reacted a library with the biarsenical FlAsH and identified beads bearing the known biarsenical-binding motif (CCXXCC). These results enable OBOC libraries to be used in high-throughput discovery of cysteine-rich peptides for protein tagging, environmental remediation of metal contaminants, or cysteine-rich pharmaceuticals.