Solution-phase chemical library synthesis using polymer-assisted purification techniques.

During the past few years, polymer-assisted solution-phase synthesis has become a prevalent method for the parallel synthesis of chemical libraries. This methodology allows for intermediate and final product purification by various resin-based sequestration techniques, which allow for the removal of excess reactants, by-products or side products from solution-phase reactions. The methodology has continued to expand, providing the practitioner with a broad range of ingenious purification methods, allowing single-step transformations as well as multistep syntheses to be performed in solution. The polymer-assisted solution-phase technology is currently being utilized for both the synthesis of lead generation and lead optimization libraries in the pharmaceutical arena and has also expanded into other disciplines.     

Recent applications of polymer-supported reagents and scavengers in combinatorial, parallel, or multistep synthesis

There has been a wealth of recent reports concerning support-bound reagents and scavengers in the solution-phase synthesis of compound libraries and natural products. Important advances in 1999 include the continued development and use of novel reagents for heterocycle synthesis, the increased use of catch-and-release purification, and the development of increasingly sophisticated techniques to allow sequestering of many types of impurities from desired compounds. These techniques have all been combined to enable the complicated multistep synthesis of natural products and of libraries of novel drug-like molecules, without conventional purification.     

Solid-phase/solution-phase combinatorial synthesis of neuroimmunophilin ligands

A novel solid-phase/solution-phase strategy for the synthesis of neuroimmunophilin ligands based on GPI 1046 was developed. The synthesis employs a solid-phase esterification strategy followed by a solution-phase pyruvic amide formation to produce multi-milligram quantities of discrete compounds for assay. The protocol was applied to a production library of 880 discrete compounds. A highlight of the strategy is an aqueous extractive purification of the final compounds using a novel liquid/ice extraction system developed for high throughput.     

Combinatorial carbohydrate chemistry

The application of combinatorial chemistry to the synthesis of carbohydrate-based compound collections has received increased attention in recent years. New strategies for the solution-phase synthesis of oligosaccharide libraries have been reported, and the use of monosaccharides as scaffolds in the generation of combinatorial libraries has been described. Novel approaches to the assembly of carbohydrate-based antibiotics, such as aminoglycoside analogs and vancomycin derivatives, have also been disclosed.     

Combinatorial chemistry in the agrosciences

Combinatorial chemistry and high throughput screening have had a profound effect upon the way in which agrochemical companies conduct their lead discovery research. The article reviews recent applications of combinatorial synthesis in the lead discovery process for new fungicides, herbicides and insecticides. The role and importance of bioavailability guidelines, natural products, privileged structures, virtual screening and X-ray crystallographic protein structures on the design of solid- and solution-phase compound libraries is discussed and illustrated.     

Efficient one-pot cyclization/folding of Rhesus θ-defensin-1 (RTD-1)

We report an efficient approach for the chemical synthesis of Rhesus θ-defensin-1 (RTD-1) using Fmoc-based solid-phase peptide synthesis in combination with an intramolecular version of native chemical ligation. The corresponding linear thioester precursor was cyclized and folded in a one-pot reaction using reduced glutathione. The reaction was extremely efficiently yielding natively folded RTD-1 with minimal or no purification at all. This approach is fully compatible with the high throughput production of chemical libraries using this peptide scaffold.     

Are porphyrin mixtures favorable photodynamic anticancer drugs? A model study with combinatorial libraries of tetraphenylporphyrins

Reported here is the preparation of tetraphenylporphyrin libraries via efficient combinatorial solution-phase syntheses, their purification, and preliminary results from a bioorganic study on their uptake in liposome membranes. Libraries with up to 666 components were prepared with substituents including Br, CF3, Cl, CN, CO2Me, Et, F, OAc, and Ph. Further, a first example for the synthesis of more diverse libraries via a "latent libraries" approach is presented. This involves masking polar groups with lipophilic protecting groups. After purification of the latent library, the masking protecting groups are removed in a quantitative reaction that produces the library compounds as the only non-volatile components. Libraries were characterized by laser desorption time-of-flight mass spectrometry, NMR, and UV-vis spectroscopy. In vitro uptake into membranes of small sonicated liposomes was measured, both in terms of total porphyrin incorporation and in terms of structure-incorporation relationships. The latter were determined from isotopically-resolved laser-desorption mass spectra under conditions that yield quantitative results. Smaller libraries showed increased uptake of porphyrins bearing OH and CF3 substituents and lower uptake of ester-, alkyl-, and halide-bearing porphyrins. This structure-dependent selectivity disappears for larger libraries, however, where uniformly high uptake is observed, i.e., at a constant lipid:porphyrin ratio the total porphyrin incorporation is higher for libraries than for single compounds of similar polarity. We propose that the decreased concentration of individual compounds in large libraries is responsible for this effect. Membrane incorporation has previously been shown to correlate with photodynamic activity in vitro and in vivo.16 Therefore, these results may help to explain why photodynamic therapy of tumors, a modern anti-cancer treatment modality, is successfully performed with a complex mixture of porphyrins.     

Solution- and solid-phase oligosaccharide synthesis using glucosyl iodides: a comparative study

Glycosyl iodide donors have been used in both solid- and solution-phase syntheses yielding alpha-(1 --> 6)-linked glucosyl oligomers in highly efficient protocols. While the solid-phase strategy offers advantages in terms of ease of purification, it requires a total of 7.5 equiv of donor and approximately 12 h to complete the incorporation of one monosaccharide unit. In contrast, solution-phase methods require only 2.5 equiv of donor and 2-3 h reaction time per glycosylation. Moreover, since the reactions are virtually quantitative (> 90%) column chromatography of the material is facile. The overall advantages of solution-phase oligosaccharide synthesis were further illustrated in the convergent synthesis of a hexamer (methoxycarbonylmethyl 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl-(1 --> 6)-tetrakis-(2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl-(1 --> 6))-2,3,4-tri-O-benzyl-1-thio-alpha-D-glucopyranoside) that was constructed from dimer donor iodides in a two-plus-two and a two-plus-four fashion.     

Combinatorial search of substituted beta-cyclodextrins for phosphatase-like activity.

Thirteen per-6-akylamino-6-deoxy-beta-cyclodextrin libraries (beta-CD libraries) were generated by a solution-phase combinatorial synthesis starting from per-6-iodo-6-deoxy-beta-CD and different combinations of eleven individual amine nucleophiles. Certain libraries showed the ability to hydrolyzep-nitrophenyl phosphate in the presence of Zn2+.     

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity¹. Several biodegradable polymers have been studied as vaccine delivery vehicles¹; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses^2-12.The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses^10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation^14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods^16,17.Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described¹⁰. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs^10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).     

Chromosome specific c-DNA libraries: reduction of unspecific priming events by purification of heteronuclear RNA

Chromosome specific c-DNA libraries greatly facilitate the isolation of disease associated genes which have been previously linked to particular chromosomes. Recently, several methods have been developed and employed for the isolation of transcribed sequences from specific human chromosomes and chromosome regions. Heteronuclear (hn) RNA from somatic human/rodent cell hybrids has been used as starting material to selectively prime the synthesis of human specific c-DNAs. A drawback of this method is the high number of rodent clones found in these chromosome specific c-DNA libraries. Here, we provide direct evidence that unspecific priming events account for the majority of these rodent clones. Using an Alu consensus primer hn-RNA human specific c-DNA libraries have been established and the specificity of Alu-priming has been evaluated. Using a variety of purification schemes for isolating hn-RNA we have significantly reduced the percentage of unspecific priming events. We also included a comparison of the hn-RNA yield from different somatic hybrids prior and after purification.     

Mini-review: combinatorial approaches for the design of novel coating systems

Combinatorial and high throughput experimental methods are being applied to the design and development of novel polymers and coatings used in a number of application areas. Methods have been developed for polymer synthesis and screening and for the development of polymer thin film and coating libraries and the screening of these libraries for key properties such as surface energy and modulus. Combinatorial and high throughput methods enable the efficient exploration of a large number of compositional variables over a wide range. In the development of coatings for use in the marine environment, the key challenge is in the development of screening methods that can predict good performance. A number of assays are under development that will permit the rapid screening of the interaction of coatings with representative marine organisms.     

Oxidative folding and preparation of α‐conotoxins for use in high‐throughput structure–activity relationship studies

α‐Conotoxins are peptide neurotoxins that selectively inhibit various subtypes of nicotinic acetylcholine receptors. They are important research tools for studying numerous pharmacological disorders, with profound potential for developing drug leads for treating pain, tobacco addiction, and other conditions. They are characterized by the presence of two disulfide bonds connected in a globular arrangement, which stabilizes a bioactive helical conformation. Despite extensive structure–activity relationship studies that have produced α‐conotoxin analogs with increased potency and selectivity towards specific nicotinic acetylcholine receptor subtypes, the efficient production of diversity‐oriented α‐conotoxin combinatorial libraries has been limited by inefficient folding and purification procedures. We have investigated the optimized conditions for the reliable folding of α‐conotoxins using simplified oxidation procedures for use in the accelerated production of synthetic combinatorial libraries of α‐conotoxins. To this end, the effect of co‐solvent, redox reagents, pH, and temperature on the proportion of disulfide bond isomers was determined for α‐conotoxins exhibiting commonly known Cys loop spacing frameworks. In addition, we have developed high‐throughput ‘semi‐purification’ methods for the quick and efficient parallel preparation of α‐conotoxin libraries for use in accelerated structure–activity relationship studies. Our simplified procedures represent an effective strategy for the preparation of large arrays of correctly folded α‐conotoxin analogs and permit the rapid identification of active hits directly from high‐throughput pharmacological screening assays. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Mini-review: Combinatorial approaches for the design of novel coating systems

Abstract

Combinatorial and high throughput experimental methods are being applied to the design and development of novel polymers and coatings used in a number of application areas. Methods have been developed for polymer synthesis and screening and for the development of polymer thin film and coating libraries and the screening of these libraries for key properties such as surface energy and modulus. Combinatorial and high throughput methods enable the efficient exploration of a large number of compositional variables over a wide range. In the development of coatings for use in the marine environment, the key challenge is in the development of screening methods that can predict good performance. A number of assays are under development that will permit the rapid screening of the interaction of coatings with representative marine organisms.     

In vitro preclinical lead optimisation technologies (PLOTs) in pharmaceutical development

The explosion of genuine high throughput technologies has allowed large compound libraries to be screened with ever-increasing biological specificity, exacerbating the problem of lead candidate selection for subsequent drug development. To avoid creating a bottleneck, compounds identified from the high throughput screens undergo lead optimisation by employing medium-throughput screen which permits ranking in terms of their basic absorption, distribution, metabolism, excretion (ADME) and toxicological properties. The historical role of the CRO in the drug discovery/development continuum has been to perform efficacy and toxicology studies, simply to support the regulatory submission of lead candidates. This situation is, however, changing with the development of preclinical lead optimisation technologies facilitating the selection of leading candidates, thereby bridging the gap between high throughput efficacy screens and conventional safety assessment programmes.     

Assessment of solution-phase positional scanning libraries based on distamycin A for the discovery of new DNA binding agents

The solution-phase synthesis of two 1000-membered positional scanning libraries of distamycin A analogues is described enlisting acid/base liquid-liquid extractions for isolation and purification of all intermediates and final products. The results of their screening for functional activity (L1210 cytotoxic potency) and DNA binding affinity were compared with those derived from libraries containing the same compound members but prepared in a smaller 10-compound mixture format. The positional scanning libraries, which are substantially less demanding to prepare, allowed the accurate detection of the global observations and the clearly more potent activities, but more subtle discoveries and less distinguishable activities were not detected. This is a natural consequence of testing the larger 100-compound mixtures and the relative insensitivity of the assays to the contribution of any single, uniquely acting compound in the mixture. Thus, the disadvantages associated with the loss of some information contained within the library must be balanced against the advantages of the ease of library synthesis and judged in light of the library screening objectives.     

Cocaine detoxification by combinatorially substituted beta-cyclodextrin libraries

Per-6-substituted- and A,C,E(F)-tri-6-substituted-6-deoxy-beta-cyclodextrin (beta-CD) libraries were generated using solution-phase combinatorial chemistry techniques starting from the corresponding iodo precursors and different combinations of individual amine nucleophiles. Using a high throughput electrospray mass spectrometry (ESMS) screen to monitor the hydrolysis of cocaine, certain libraries showed the ability to specifically hydrolyze the methyl ester of cocaine, with the most active per-6-substituted beta-CD library I producing complete hydrolysis in 24h. The cocaine hydrolytic activity in this series showed structure-activity relationships which appeared to involve specific interaction between the amine side chains and the cocaine molecule. Comparison of the composition of the most active per-6-substiuted beta-CD libraries and A,C,E(F)-tri-6-substituted-6-deoxy-beta-CD libraries (I and XV) showed three common side chains (3, 4, and 5), suggesting that active side chains in the tri-substituted beta-CD library might be predicted from evaluation of the more easily prepared per-6-substituted-6-deoxy-beta-CD series.     

Oligonucleotide synthesis using ionic liquids as soluble supports

The continuing evolution of the methodology for the solution-phase synthesis of oligonucleotides using soluble ionic tags as handles for easy purification is described. This methodology may provide a more cost efficient route for the large scale synthesis of oligonucleotides.     

Solution-phase synthesis of combinatorial libraries designed to modulate protein-protein or protein-DNA interactions

A short personal perspective on the development of an approach to the solution-phase synthesis of combinatorial libraries for modulating cellular signaling by inhibiting, promoting, or mimicking protein-protein or protein-DNA interactions is provided.     

Two-dimensional parallel array technology as a new approach to automated combinatorial solid-phase organic synthesis

An automated, 96-well parallel array synthesizer for solid-phase organic synthesis has been designed and constructed. The instrument employs a unique reagent array delivery format, in which each reagent utilized has a dedicated plumbing system. An inert atmosphere is maintained during all phases of a synthesis, and temperature can be controlled via a thermal transfer plate which holds the injection molded reaction block. The reaction plate assembly slides in the X-axis direction, while eight nozzle blocks holding the reagent lines slide in the Y-axis direction, allowing for the extremely rapid delivery of any of 64 reagents to 96 wells. In addition, there are six banks of fixed nozzle blocks, which deliver the same reagent or solvent to eight wells at once, for a total of 72 possible reagents. The instrument is controlled by software which allows the straightforward programming of the synthesis of a larger number of compounds. This is accomplished by supplying a general synthetic procedure in the form of a command file, which calls upon certain reagents to be added to specific wells via lookup in a sequence file. The bottle position, flow rate, and concentration of each reagent is stored in a separate reagent table file. To demonstrate the utility of the parallel array synthesizer, a small combinatorial library of hydroxamic acids was prepared in high throughput mode for biological screening. Approximately 1300 compounds were prepared on a 10 μmole scale (3-5 mg) in a few weeks. The resulting crude compounds were generally >80% pure, and were utilized directly for high throughput screening in antibacterial assays. Several active wells were found, and the activity was verified by solution-phase synthesis of analytically pure material, indicating that the system described herein is an efficient means for the parallel synthesis of compounds for lead discovery. Copyright 1998 John Wiley & Sons, Inc.