Split--pool synthesis of 1,3-dioxanes leading to arrayed stock solutions of single compounds sufficient for multiple phenotypic and protein-binding assays.

Diversity-oriented organic synthesis offers the promise of advancing chemical genetics, where small molecules are used to explore biology. While the split--pool synthetic method is theoretically the most effective approach for the production of large collections of small molecules, it has not been widely adopted due to numerous technical and analytical hurdles. We have developed a split--pool synthesis leading to an array of stock solutions of single 1,3-dioxanes. The quantities of compounds are sufficient for hundreds of phenotypic and protein-binding assays. The average concentration of these stock solutions derived from a single synthesis bead was determined to be 5.4 mM in 5 microL of DMSO. A mass spectrometric strategy to identify the structure of molecules from a split--pool synthesis was shown to be highly accurate. Individual members of the 1,3-dioxane library have activity in a variety of phenotypic and protein-binding assays. The procedure developed in this study allows many assays to be performed with compounds derived from individual synthesis beads. The synthetic compounds identified in these assays should serve as useful probes of cellular and organismal processes.     

A one-bead, one-stock solution approach to chemical genetics: part 1

BACKGROUND: In chemical genetics, small molecules instead of genetic mutations are used to modulate the functions of proteins rapidly and conditionally, thereby allowing many biological processes to be explored. This approach requires the identification of compounds that regulate pathways and bind to proteins with high specificity. Structurally complex and diverse small molecules can be prepared using diversity-oriented synthesis, and the split-pool strategy allows their spatial segregation on individual polymer beads, but typically in quantities that limit their usefulness. RESULTS: We report full details of the first phase of our platform development, including the synthesis of a high-capacity solid-phase bead/linker system, the development of a reliable library encoding strategy, and the design of compound decoding methods both from macrobeads and stock solutions. This phase was validated by the analysis of an enantioselective, diversity-oriented synthesis resulting in an encoded 4320-member library of structurally complex dihydropyrancarboxamides. CONCLUSIONS: An efficient and accessible approach to split-pool, diversity-oriented synthesis using high-capacity macrobeads as individual microreactors has been developed. Each macrobead contains sufficient compound to generate a stock solution amenable to many biological assays, and reliable library encoding allows for rapid compound structure elucidation post-synthesis. This 'one-bead, one-stock solution' strategy is a central element of a technology platform aimed at advancing chemical genetics.     

A one-bead, one-stock solution approach to chemical genetics: part 2

BACKGROUND: Chemical genetics provides a systematic means to study biology using small molecules to effect spatial and temporal control over protein function. As complementary approaches, phenotypic and proteomic screens of structurally diverse and complex small molecules may yield not only interesting individual probes of biological function, but also global information about small molecule collections and the interactions of their members with biological systems. RESULTS: We report a general high-throughput method for converting high-capacity beads into arrayed stock solutions amenable to both phenotypic and proteomic assays. Polystyrene beads from diversity-oriented syntheses were arrayed individually into wells. Bound compounds were cleaved, eluted, and resuspended to generate 'mother plates' of stock solutions. The second phase of development of our technology platform includes optimized cleavage and elution conditions, a novel bead arraying method, and robotic distribution of stock solutions of small molecules into 'daughter plates' for direct use in chemical genetic assays. This library formatting strategy enables what we refer to as annotation screening, in which every member of a library is annotated with biological assay data. This phase was validated by arraying and screening 708 members of an encoded 4320-member library of structurally diverse and complex dihydropyrancarboxamides. CONCLUSIONS: Our 'one-bead, multiple-stock solution' library formatting strategy is a central element of a technology platform aimed at advancing chemical genetics. Annotation screening provides a means for biology to inform chemistry, complementary to the way that chemistry can inform biology in conventional ('investigator-initiated') small molecule screens.     

Synthesis of a 10,000-membered library of molecules resembling carpanone and discovery of vesicular traffic inhibitors

Split-and-pool synthesis of a 10,000-membered library of molecules resembling the natural product carpanone has been achieved. The synthesis features development of solid-phase multicomponent reactions between nitrogen nucleophiles, enones, and hydroxylamines, and a solid-phase application of the Huisgen cycloaddition affording substituted triazoles. The synthesis was performed in high-capacity (500 microm) polystyrene beads using a one bead-one stock solution strategy that enabled phenotypic screens of the resulting library. Using whole-cell fluorescence imaging, we discovered a series of molecules from the carpanone-based library that inhibit exocytosis from the Golgi apparatus. The most potent member of this series has an IC(50) of 14 microM. We also report structure-activity relationships for the molecules exhibiting this interesting phenotype. These inhibitors of exocytosis may be useful reagents for the study of vesicular traffic.     

Skeletal diversity via a branched pathway: efficient synthesis of 29 400 discrete,polycyclic compounds and their arraying into stock solutions

We report the synthesis and arraying of 29 400 structurally diverse and complex polycyclic carbocycles using diversity-oriented synthesis (DOS) and the "one bead-one stock solution" technology platform. Skeletal diversity, a difficult challenge in DOS, was achieved with a branching reaction pathway using one or two Diels-Alder reactions. This pathway yields small molecules having 10 different skeletons.     

Synthesis of fused multicyclic compounds containing macrocycles by dienyne ring-closing metathesis and Diels-Alder reactions

Fused bicyclic compounds comprising small and large rings were synthesised by dienyne ring-closing metathesis (RCM) using Grubbs' catalyst. By taking advantage of faster small ring cyclisation compared with macrocyclisation, single isomers were obtained rather than mixtures of two isomers with different ring sizes. Using this process, various fused bicyclic compounds comprising small rings (5-7- membered) and large rings (14-17- membered) were obtained. By increasing reaction temperature and catalyst loading, the product conversion was improved in a predicted manner. This method produced E-olefins on the macrocycles with high selectivity. Also, the selectivity issues of tandem RCM for the synthesis of fused bicyclic compounds comprising small and medium rings were investigated. Lastly, the prepared bicyclic compounds with small and large rings contained 1, 3-dienes that underwent a further modification reaction, such as Diels-Alder, to produce more complex compounds. These Diels-Alder reactions produced tri- and tetracyclic compounds containing a macrocycle with single diastereomers, suggesting that the methodology demonstrated here could be a powerful tool for rapid preparation of highly complex molecules.     

Edge spreading lithography and its application to the fabrication of mesoscopic gold and silver rings

A new form of edge lithography, edge spreading lithography (ESL), has been demonstrated and applied to the formation of coinage metal rings. In this process, alkanethiols are delivered from a flat PDMS stamp to the surface of a metal film through a two-dimensional array of spherical silica colloids. The thiols further spread on the metal surface, forming highly ordered SAMs in the form of a ring pattern. Following lift-off of beads, the pattern in the SAMs can be transferred into the metal film through wet chemical etching, with SAMs serving as the resist. The dimensions of the rings can be readily controlled by several parameters such as the beads diameter, the concentration of the thiol solution, and the contact time between the stamp and the silica beads.     

Pathway development and pilot library realization in diversity-oriented synthesis: exploring Ferrier and Pauson-Khand reactions on a glycal template

Through a correlation of the ability of small molecules to bind biological macromolecules and their ability to modulate cellular and organismal processes, chemistry can inform biology and vice versa. Diversity-oriented organic synthesis (DOS), which aims to provide structurally complex and diverse small molecules efficiently, can play a key role in such chemical genetic studies. Here we illustrate the trial-and-error experimentation that can refine an initial pathway-planning exercise and result eventually in an effective diversity pathway. By exploring Ferrier and Pauson-Khand reactions on a glycal template, we have developed efficient and stereoselective syntheses of tricyclic compounds. In this pathway, diversity results from the substituents and their spatial relationships about the tricyclic rings. A pilot split-pool library synthesis of 2500 tricyclic compounds highlights the use of planning considerations in DOS and a "one-bead, one-stock solution" technology platform. Additionally, it illustrates a promising synthetic pathway for future chemical genetic studies.     

Encoding method for OBOC small molecule libraries using a biphasic approach for ladder-synthesis of coding tags

In the "one-bead one-compound" (OBOC) combinatorial library method, each compound bead displays only one compound entity. Hundreds of thousands to millions of compound beads can be synthesized rapidly and screened simultaneously. Positive compound beads are then isolated for structural analysis. To fully exploit the power of OBOC combinatorial small molecule libraries, a robust and high throughput encoding method is needed to decode the positive compound beads. In this paper, we report on the development of a novel encoding strategy that combines the concepts of ladder-synthesis and chemical encoding on bilayer beads. In these encoded libraries, small molecule compounds are displayed on the bead surface, and cleavable coding tags consisting of a series of truncated molecules reside in the bead interior. Such a library can be easily constructed using the biphasic approach (J. Am. Chem. Soc.2002, 124, 7678) to topologically segregate the functionalities of the beads during library synthesis. The ladder members and coding tags are then released for MALDI-TOF-MS analysis. To simplify the interpretation of the mass spectra, we purposely add bromine into the cleavable linker so that the cleavage products generate a characteristic isotope fingerprint. The chemical structure of library compounds can be determined by analyzing the mass differences between adjacent peaks on the mass spectra. This encoding strategy also provides valuable information on the quality of the testing compound on the surface of the bead. To validate this methodology, a model OBOC small molecule library with 12,288 members was synthesized on TentaGel beads and screened against streptavidin. The chemical structures of the compound on each positive bead were unambiguously identified.     

NMR analysis of interaction between styrene-divinylbenzene gel beads and small molecules

The interaction of small molecules such as cyclohexane, tetrahydrofuran, and acetonitrile with styrene-divinylbenzene copolymer gel beads were investigated by ¹³C-NMR spectroscopy. When the gel content was 0.1–0.3 g/mL, the ¹³C-NMR spectrum of the solvent displayed two peaks. A sharp peak at lower magnetic field was assigned to the free solvent, and a broad one to the solvent affected by the gel. This signal splitting is attributed to the upfield shift caused by aromatic rings of styrene units in the polymer chain. The nitrile carbon of acetonitrile showed the largest upfield shift. The mobility of small molecules in gel beads was also investigated using the nuclear magnetic relaxation method. In the case of good solvents for gel beads, the mobility was affected by the cross-linking density, whereas poor solvents exhibited little dependence of the cross-linking density. © 1995 John Wiley & Sons, Inc.     

A library of spirooxindoles based on a stereoselective three-component coupling reaction

A collection of structurally complex and chemically diverse small molecules is a useful tool to explore cell circuitry. In this article, we report the split-pool synthesis of more than 3000 spirooxindoles on high capacity macrobeads. The key reaction to assemble the spirooxindole core stereoselectively is a Lewis acid variant of the Williams' three-component coupling. After formation, the skeleton was elaborated using Sonogashira couplings, amide forming reactions, and N-acylations of gamma-lactams. The final library was analyzed by sampling individual macrobeads and by using binomial confidence limits. It was determined that at least 82% of the library compounds should have better than 80% purity. To demonstrate the utility of our discovery process, a high-throughput chemical genetic modifier screen was performed using stock solutions of the resultant products. A number of positives were identified as enhancers of the cellular actions of latrunculin B, an actin polymerization inhibitor. Through resynthesis, we confirmed one of the positives and demonstrated that, in yeast cells, it has an EC50 in the sub-micromolar range.     

Synthesis and Properties of Cross-Linked 4-Vinylpyridine-Styrene-Halogen Complexes

Cross-linked 4-vinylpyridine-styrene beads (PVPS) containing various amounts of pyridine rings were synthesized and reaction with methyliodide and peroxyacetic acid gave corresponding N-methylpyridinium salts and N-oxides with more than 92% of the pyridine rings being transformed. PVPS formed stable complexes with bromine and chlorine in the ratio 1:1, and when a higher amount of halogen was used, complexes with two molecules of halogen on each pyridine were formed. Similar complexes were also formed with PVPS-N-oxides in the presence of bromine and chlorine, while the reaction of PVPS-hydrohalide with bromine and chlorine resulted in hydrobromide perbromide and hydrochloride perchloride resins. The chemical activity of halo-substituted resins was tested in the reaction with 1,1-diphenylethylene. Chi or o-substituted resins are very stable, while bromo-substituted beads gave bromoalkene, dibromide, and alkoxybromide, depending on the structure of the reagent, solvent, and reaction temperature.     

Organic synthesis in the solid state via hydrogen-bond-driven self-assembly

This Perspective describes how chemists can control intermolecular [2 + 2] photodimerizations in the solid state using small molecules as linear templates. The templates assemble olefins into positions for the reaction via hydrogen-bond-driven self-assembly. We attach functional groups to the olefins that complement hydrogen bond donor and acceptor groups of the templates. The resulting cyclobutane-based products form stereospecifically, quantitatively, and in gram amounts. The templates are used to direct the formation of a [2.2]paracyclophane and ladderanes. The organic solid state is an exciting medium within which to control chemical reactivity since it is possible to synthesize, or construct, molecules that may be, otherwise, unobtainable from solution. The products form with a high degree of stereocontrol provided by a crystal lattice. The critical covalent-bond-forming process also occurs in a solvent-free environment. That molecules are virtually frozen in position in a solid also means that this methodology enables chemists to employ principles of molecular recognition and self-assembly to direct and conduct organic synthesis.     

One-bead-one-compound library of end-capped dipeptides and deconvolution by microflow NMR

As part of our program to identify novel small molecules with interesting biological activity, we have designed and synthesized a library of end-capped dipeptides with an emphasis on compound diversity, complexity, and membrane permeability. An approximately 1500-member library was synthesized manually on large polystyrene beads using the mix-and-split method. The final compounds were cleaved into 384-well plates to generate individual stock solutions for input into high-throughput biological screens. Individual compounds were decoded using a combination of mass spectrometry and microflow NMR spectroscopy. In principle, this approach to deconvolution obviates the need for complicated binary encoding-decoding strategies for one-bead-one-compound libraries.     

One-pot preparation and uranyl adsorption properties of hierarchically porous zirconium titanium oxide beads using phase separation processes to vary macropore morphology

A simple and engineering friendly one-step process has been used to prepare zirconium titanium mixed oxide beads with porosity on multiple length scales. In this facile synthesis, the bead diameter and the macroporosity can be conveniently controlled through minor alterations in the synthesis conditions. The precursor solution consisted of poly(acrylonitrile) dissolved in dimethyl sulfoxide to which was added block copolymer Pluronic F127 and metal alkoxides. The millimeter-sized spheres were fabricated with differing macropore dimensions and morphology through dropwise addition of the precursor solution into a gelation bath consisting of water (H(2)O beads) or liquid nitrogen (LN(2) beads). The inorganic beads obtained after calcination (550 °C in air) had surface areas of 140 and 128 m(2) g(-1), respectively, and had varied pore architectures. The H(2)O-derived beads had much larger macropores (5.7 μm) and smaller mesopores (6.3 nm) compared with the LN(2)-derived beads (0.8 μm and 24 nm, respectively). Pluronic F127 was an important addition to the precursor solution, as it resulted in increased surface area, pore volume, and compressive yield point. From nonambient XRD analysis, it was concluded that the zirconium and titanium were homogeneously mixed within the oxide. The beads were analyzed for surface accessibility and adsorption rate by monitoring the uptake of uranyl species from solution. The macropore diameter and morphology greatly impacted surface accessibility. Beads with larger macropores reached adsorption equilibrium much faster than the beads with a more tortuous macropore network.     

Self-aggregation of dendritic poly(benzyl ether)–poly(acrylic acid), an amphiphilic block copolymer, studied by 1H NMR

The dynamic properties of the micelles of a novel synthesized amphiphilic block copolymer, dendritic poly(benzyl ether)–poly(acrylic acid) (Dendr.PBE-PAA), formed in aqueous solutions were studied by the ¹H self-diffusion coefficient, relaxation measurements and 2D nuclear Overhauser enhancement spectroscopy. The experimental results show that Dendr.PBE-PAA molecules self-aggregate in aqueous solution. The dynamic properties of the Dendr.PBE-PAA micelles vary with their total concentration in the solution. The motion of the molecules in the micelles of a concentrated solution is more restricted than that in a less concentrated one. The main chains of PAA are densely packed in the surface layer of the hydrophobic core with the carboxyl side chain pointing to the aqueous medium and the hydrophobic phenoxy rings stay in the interior. The self-aggregate becomes larger as the degree of polymerization of PAA increases. However the phenoxy rings situated in the interior of the hydrophobic core become more loosely packed. n-Hexadecane is solubilized in the micelles. The optimal position of n-hexadecane is between the phenoxy rings next to the PAA chains. Received: 25 January 2001 Accepted: 18 July 2001     

An alkylsilyl-tethered, high-capacity solid support amenable to diversity-oriented synthesis for one-bead, one-stock solution chemical genetics

The synthesis and use of an alkylsilyl-tethered large (500-600 microm) polystyrene resin (1) are disclosed. An optimized Suzuki coupling of bromine-functionalized polystyrene and a silicon-functionalized alkylborane generates the silicon-substituted polystyrene 1 in large scale (>100 g). Resin loading is accomplished by activation as the silyl triflate, which can accommodate even sterically encumbered secondary alcohols and phenols. Treatment with HF/pyridine for linker cleavage is mild, efficient, and amenable to an automated, large-scale distribution system. This platform delivers, minimally, 50 nmol of each small molecule derived from a diversity-oriented, split-pool synthesis on a per bead basis for use in both forward and reverse chemical genetic assays. This technology satisfies many requirements of a one bead-one stock solution approach to chemical genetics.     

Site-site interactions within high-loading PAMAM dendrimer resin beads

Combinatorial chemistry and solid-phase synthesis have revolutionised the process of drug discovery in the last decade. Ever since the concept of split and mix synthesis was introduced in 1988, and in particular the concept of one bead one compound, this approach has been associated with the possibility of generating thousands or millions of compounds in only a relatively small number of synthetic steps. The demand for high loading resins has therefore increased over the last few years. Our research has focussed on bead-loading enhancement via a dendrimerisation process. Dendrimer resin beads have shown compatibility for many chemical conditions, however high functional group density could produce undesirable site-site interactions.     

New regioselective multicomponent reaction: one pot synthesis of spiro heterobicyclic aliphatic rings

In the context of our high-throughput organic synthesis program, we have studied the reactivity of special beta-keto esters toward the Biginelli reaction. We have found that a cyclic beta-keto ester reacts with one molecule of urea and two molecules of aldehyde to give a new family of spiro heterobicyclic aliphatic rings in good yields. Interestingly, the Biginelli product was not detected. After analysis of products using HPLC, 1H NMR, and 13C NMR, we have found that the reaction is driven by a regio-specific condensation of two molecules of aldehyde with the other reagents to afford only products harboring substituents exclusively in cis configuration. Monte Carlo minimization studies using MM2 force field suggest that cis products are energetically more stable than the trans counterparts. Together with previously reported data, these results suggest that the trans products were not obtained as result of steric hindrance produced by the equatorial position of one of the ring substituents. This new reaction is useful for high-throughput organic synthesis. Indeed, the new scaffold can be used to introduce additional groups in the molecules through remaining functional groups by a "domino strategy".