Development of combinatorial chemistry methods for coatings: high-throughput weathering evaluation and scale-up of combinatorial leads

Combinatorial screening of materials formulations followed by the scale-up of combinatorial leads has been applied for the development of high-performance coating materials for automotive applications. We replaced labor-intensive coating formulation, testing, and measurement with a "combinatorial factory" that includes robotic formulation of coatings, their deposition as 48 coatings on a 9x12-cm plastic substrate, accelerated performance testing, and automated spectroscopic and image analysis of resulting performance. This high-throughput (HT) performance testing and measurement of the resulting properties provided a powerful set of tools for the 10-fold accelerated discovery of these coating materials. Performance of coatings is evaluated with respect to their weathering, because this parameter is one of the primary considerations in end-use automotive applications. Our HT screening strategy provides previously unavailable capabilities of (1) high speed and reproducibility of testing by using robotic automation and (2) improved quantification by using optical spectroscopic analysis of discoloration of coating-substrate structure and automatic imaging of the integrity loss of coatings. Upon testing, the coatings undergo changes that are impossible to quantitatively predict using existing knowledge. Using our HT methodology, we have developed several cost-competitive coatings leads that match the performance of more costly coatings. These HT screening results for the best coating compositions have been validated on the traditional scales of coating formulation and weathering testing. These validation results have confirmed the improved weathering performance of combinatorially developed coatings over conventional coatings on the traditional scale.     

London University Search Instrument: a combinatorial robot for high-throughput methods in ceramic science

This paper describes the design, construction, and operation of the London University Search Instrument (LUSI) which was recently commissioned to create and test combinatorial libraries of ceramic compositions. The instrument uses commercially available powders, milled as necessary to create thick-film libraries by ink-jet printing. Multicomponent mixtures are prepared by well plate reformatting of ceramic inks. The library tiles are robotically loaded into a flatbed furnace and, when fired, transferred to a 2-axis high-resolution measurement table fitted with a hot plate where measurements of, for example, optical or electrical properties can be made. Data are transferred to a dedicated high-performance computer. The possibilities for remote interrogation and search steering are discussed.     

Purification strategies for combinatorial and parallel chemistry

This review surveys the methods developed for the purification of intermediates and final compounds originating from parallel and combinatorial chemistry. Included will be reviews of polymer-assisted purification, liquid-phase combinatorial chemistry, fluorous synthesis, liquid-liquid and solid-phase extraction, reverse-phase HPLC and supercritical fluid chromatography. A critique of each method is given, highlighting the methodologies strengths and weaknesses.     

Surface-modified carbon felts: possible supports for combinatorial chemistry

It is possible to prepare carbon-based analogues of the Merrifield resin by electrochemical reduction of diazonium salts or oxidation of aryl acetates on high specific surface area carbon felts. These modified felts can undergo further reactions: nucleophilic substitution, Suzuki reaction, and finally reductive electrochemical cleavage, taking advantage of the conductivity of the carbon felt. This provides a simple example of the possible use of electrochemistry in combinatorial synthesis.     

Selection of new chromogenic substrates of serine proteinases using combinatorial chemistry methods

Chemical synthesis, physicochemical characterization and kinetic investigations of a tetrapeptide library of chromogenic substrates containing the amide of 5-amino-2-nitrobenzoic acid (Anb(5,2)-NH(2)) at their C-termini are reported. Anb(5,2)-NH(2) served as a chromophore released upon enzymatic action. The library consisting of 9567 peptides was synthesized using the portioning-mixing method and was screened against bovine a-chymotrypsin and human leukocyte elastase in solution applying an iterative approach. The selected chromogenic substrates were resynthesized and further modified at their N- and C-termini. Finally, two sequences, Z-Phe-Ala-Thr-Tyr-Anb(5,2)-NH(2) and Z-Phe-Phe-Pro-Val-Anb(5,2)-NH(2), were obtained as highly specific substrates for bovine alpha-chymotrypsin and human leukocyte elastase, respectively. The method of synthesis and selection of chromogenic substrates of serine proteinases described herein is straightforward and can be applied to design substrates for other proteases.     

Arrays for the combinatorial exploration of cell adhesion

A new method for the fabrication of arrays of self-assembled monolayers (SAMs) of alkane thiols (ATs) on gold to combinatorially assay surfaces for cell adhesion is reported. A fluorous SAM, which is both cytophobic and solvophobic, was used as the background between the array features. The resulting solvophobic background permits the application of an assembly after conjugation strategy for fabrication. SAMs containing mixtures of ATs and peptide-terminated ATs were generated. Multiple cell types demonstrated differential and specific binding to these surfaces. Additionally, pluripotent human embryonic stem cells proliferated on surfaces generated by this method.     

Directed development of high-performance membranes via high-throughput and combinatorial strategies

Combinatorial strategies are for the first time applied in membrane technology and prove to be a powerful new tool in the search for novel membrane materials. The selected system for this study is a polyimide solvent-resistant nanofiltration membrane prepared via phase inversion. The phase inversion process is a typical membrane synthesis procedure involving a large number of compositional components, which can each be varied in a wide concentration range. The optimization of the membrane dope composition was performed using evolutionary optimization via genetic algorithms. Compared with the best commercially available membranes, a substantially improved membrane performance could be realized, both on the level of membrane selectivity and on that of permeability. The miniaturized high-throughput synthesis procedure could be scaled up successfully when the polymer dope was sufficiently viscous. It can be anticipated that application of combinatorial techniques can potentially lead to major improvements in all fields of membrane technology, for example water treatment, gas separation, and dialysis, not only on the compositional level but also for instance on the level of membrane synthesis posttreatment and operational conditions.     

Preparation of polymer-bound pyrazolone active esters for combinatorial chemistry

The preparation of solid-phase active esters from a new pyrazolone linker resin is described. N-Acylation using this resin provides various amide products with a high conversion rate and good purity under mild conditions. The polymer-bound pyrazolone linkers are stable in the reaction conditions and are resistant to hydrolysis. Moreover, this resin can also be reused repeatedly without a loss of reactivity.     

Receptor-assisted combinatorial chemistry: thermodynamics and kinetics in drug discovery

Current drug discovery using combinatorial chemistry involves synthesis followed by screening, but emerging methods involve receptor-assistance to combine these steps. Adding stoichiometric amounts of receptor during library synthesis alters the kinetics or thermodynamics of the synthesis in a way that identifies the best-binding library members. Three main methods have emerged thus far in receptor-assisted combinatorial chemistry: dynamic combinatorial libraries, receptor-accelerated synthesis, and a new method, pseudo-dynamic libraries. Pseudo-dynamic libraries apply both thermodynamics and kinetics to amplify library members to easily observable levels, and attain selectivity heretofore unseen in receptor-assisted systems.     

Mass spectrometry and combinatorial chemistry: a short outline.

The rapid evolution of combinatorial chemistry in recent years has led to a dramatic improvement in synthetic capabilities. The goal is to accelerate the discovery of molecules showing affinity against a target, such as an enzyme or a receptor, through the simultaneous synthesis of a great number of structurally diverse compounds. This is done by generating combinatorial libraries containing as many as hundreds or thousands of compounds. The need to test all these compounds led to the development of high-throughput screening (HTS) techniques, and also high-throughput analytical techniques capable of assessing the occurrence, structure and purity of the products. In order to be applied effectively to the characterization of combinatorial libraries, an analytical technique must be adequately sensitive (to analyse samples which are typically produced in nanomole amounts or less), fast, affordable and easy to automate (to minimize analysis time and operator intervention). Although no method alone can meet all the analytical challenges underlying this task, the recent progress in mass spectrometric (MS) instrumentation renders this technique an essential tool for scientists working in this area. We describe here relevant aspects of the use of MS in combinatorial technologies, such as current methods of characterization, purification and screening of libraries. Some examples from our laboratory deal with the analysis of pooled oligomeric libraries containing n x 324(n = 1, 2) compounds, using both on-line high-performance liquid chromatography/MS with an ion trap mass spectrometer, and direct infusion into a triple quadrupole instrument. In the first approach, MS and product ion MS/MS with automatic selection of the precursor were performed in one run, allowing library confirmation and structural elucidation of unexpected by-products. The second approach used MS scans to characterize the entire library and also precursor ion and neutral loss scans to detect selectively components with given structural characteristics.     

Integrating virtual screening and combinatorial chemistry for accelerated drug discovery

Virtual screening is increasingly being used in drug discovery programs with a growing number of successful applications. Experimental methodologies developed to speed up the drug discovery processes include high-throughput screening and combinatorial chemistry. The complementarities between computational and experimental screenings have been recognized and reviewed in the literature. Computational methods have also been used in the combinatorial chemistry field, in particular in library design. However, the integration of computational and combinatorial chemistry screenings has been attempted only recently. Combinatorial libraries (experimental or virtual) represent a notable source of chemically related compounds. Advances in combinatorial chemistry and deconvolution strategies, have enabled the rapid exploration of novel and dense regions in the chemical space. The present review is focused on the integration of virtual and experimental screening of combinatorial libraries. Applications of virtual screening to discover novel anticancer agents and our ongoing efforts towards the integration of virtual screening and combinatorial chemistry are also discussed.     

Mass spectrometry and combinatorial chemistry: new approaches for direct support-bound compound identification

Mass spectrometry is a powerful analytical tool allowing rapid and sensitive structural elucidation of a wide range of molecules issued from solution-, solid- and liquid-phase syntheses. Therefore, mass spectrometry has become the most widely used tool to probe combinatorial libraries. A significant portion of the reported combinatorial data are being produced using solid phase organic synthesis. In contrast to indirect strategies where the tethered structures were released from the support into solution to undergo standard mass spectrometric analyses, static - secondary ion mass spectrometry (S-SIMS) has enabled the identification of support-bound molecules without any chemical treatment of the resin bead. Such non-destructive characterization was applied at the bead level and facilitated the step-by-step monitoring of solid-phase peptide syntheses. Side-reactions were also detected. The relevance of S-SIMS in the rehearsal phase of combinatorial chemistry is demonstrated by comparison with infrared and nuclear magnetic resonance (NMR) spectroscopies, the two other techniques investigated in that field. An alternative to solid-phase synthesis consists of assembling molecules on a soluble polymer. This methodology is termed liquid-phase synthesis. Compound characterization is facilitated since the derivatized support is soluble in spectroscopic solvents used in NMR or in electrospray ionization mass spectrometry. The advantages and drawbacks of this approach will be discussed in terms of the direct monitoring of supported reactions during chemistry optimization and rehearsal library validation.     

Preparative high-performance liquid chromatography-mass spectrometry for the high-throughput purification of combinatorial libraries

Accurate results for the testing of combinatorial libraries necessitates high purity of the library members. Therefore, combinatorial libraries derived from a combinatorial solution or solid-phase synthesis often require the purification of compounds that do not achieve a certain purity threshold. This study describes that preparative high-performance liquid chromatography (HPLC)-mass spectrometry (MS) is the method of choice for the purification of large arrays of diverse compounds. The adoption of this technology to the workflow of a solution phase combinatorial chemistry laboratory producing more than 20,000 compounds per year is described. Furthermore, the setup and logistics are discussed as well as the purity achievable for large libraries. Efficiency, speed, quality, and universality of preparative HPLC-MS are presented in detail for a library of 140 compounds, including data logistics and downstream processes as well.     

Optimizing the reversibility of hydrazone formation for dynamic combinatorial chemistry

Hydrazones from hydrazines bearing electron withdrawing groups, and aromatic or aliphatic aldehydes form and hydrolyse rapidly in water at neutral pH.     

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.     

The teaching potential of evaluation and optimization methods in analytical chemistry

Summary The teaching potential for analytical chemistry of some mathematical and statistical tools is discussed. It is concluded that some of the so-called chemometric techniques should be taught and that they permit to develop a philosophy of problem solving in analytical chemistry.

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Unterrichtsmöglichkeiten für Bewertungs- und Optimierungsmethoden in der Analytischen Chemie

Zusammenfassung Die Notwendigkeit der Aufnahme solcher Methoden in die Unterrichtspläne für Analytische Chemie wird diskutiert und eine Reihe von statistischen und mathematischen Verfahren (sog. Chemometrie) wird beschrieben, die sich für die Aufnahme in die Lehrpläne eignen.

     

Combinatorial docking and combinatorial chemistry: Design of potent non-peptide thrombin inhibitors

A computational algorithm was used to design automatically novel thrombin inhibitors that are available from a single-step chemical reaction. The compounds do not contain amide bonds, are achiral and have a molecular weight below 400. Of the 10 compounds that were synthesized, five bind to thrombin with a Ki in the nanomolar range. Subsequent X-ray structure determination of the thrombin-inhibitor complex for the best compound (Ki=95 nM) confirms the predicted binding mode. The novel algorithm is applicable to a broad range of chemical reactions.     

The rise, fall and reinvention of combinatorial chemistry

Combinatorial chemistry provides a powerful tool for the rapid creation of large numbers of synthetic compounds. Ideally, these libraries should be a rich source of bioactive molecules, but there is the general feeling that the initial promise of combinatorial chemistry has not yet been realized. In particular, enthusiasm for conducting unbiased (non-structure-guided) screens of large libraries for protein or RNA ligands has waned. A central challenge in this area is to devise methods for the synthesis of chemically diverse, high-quality libraries of molecules with many of the desirable features of natural products. These include diverse functionality, a significant representation of chiral sp(3) centers that provide conformational bias to the molecule, significant skeletal diversity, and good pharmacokinetic properties. However, these libraries must be easy to make from cheap, readily available building blocks, ideally those that would support convenient hit optimization/structure reactivity relationship studies. Meeting these challenges will not be easy. Here I review some recent advances in this area and provide some thoughts on likely important developments in the next few years.     

Electrochemistry-enabled fabrication of orthogonal nanotopography and surface chemistry gradients for high-throughput screening

Gradient surfaces are emerging tools for investigating mammalian cell-surface interactions in high throughput. We demonstrate the electrochemical fabrication of an orthogonal gradient platform combining a porous silicon (pSi) pore size gradient with an orthogonal gradient of peptide ligand density. pSi gradients were fabricated via the anodic etching of a silicon wafer with pore sizes ranging from hundreds to tens of nanometers. A chemical gradient of ethyl-6-bromohexanoate was generated orthogonally to the pSi gradient via electrochemical attachment. Subsequent hydrolysis and activation of the chemical gradient allowed for the generation of a cyclic RGD gradient. Whilst mesenchymal stem cells (MSC) were shown to respond to both the topographical and chemical cues arising from the orthogonal gradient, the MSC's responded more strongly to changes in RGD density than to changes in pore size during short-term culture.