Carbohydrate-based combinatorial libraries

Carbohydrate-based combinatorial libraries are tremendously valuable for studying the role of sugars in biology and for expanding accessible molecular diversity needed in broad-based drug screening programs. This review discusses the issues that are relevant to the successful implementation of comprehensive carbohydrate-based combinatorial library programs. In addition, details of oligosaccharide and glycoconjugate libraries constructed using both solid phase and solution phase strategies are presented. This revised version was published online in August 2006 with corrections to the Cover Date.     

Synthesis and diversity analysis of lead discovery piperazine-2-carboxamide libraries

A Lead Discovery Library ofpiperazine-2-carboxamide derivatives was produced forgeneral screening. This paper discloses two novelsolid phase synthetic routes used to produce 15 000single compounds via the Irori directed sortingtechnique. Computational methods such as reagentclustering and library profiling were used to maximizereagent diversity and optimize pharmacokineticparameters. The results of a four center pharmacophoreanalysis revealed the added diversity gained by usingtwo independent synthetic routes.     

Application of an almost traceless linker in the synthesis of 2-alkylthiobenzimidazole combinatorial libraries

Resin-bound triphenylphosphine was coupledto 4-fluoro-3-nitrobenzyl bromide, and2-alkylthiobenzimidazoles were synthesized onresin in 4 steps using standard chemistries. Cleavage of the compounds from the resin wasachieved with 10% NaOH in MeOH to leave amethyl group at the attachment point. A totalof 47 amines and 40 electrophiles wereevaluated, defining the scope of the reactions,culminating in the synthesis of an 80-membertest library of high purity as determined by HPLC.     

Solid phase synthesis of benzamidine and butylamine-derived hydantoin libraries

We have constructed a number of benzamidine- and butylamine-based hydantoin compounds by means of an efficient route using solid phase synthesis in which neat diisopropylamine was employed for a novel cyclization/traceless cleavage step. All library compounds were obtained in excellent yield and high purity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Getting more from IR-microscopy of resin-bound libraries

A linear pixel-array detector was employed to create spatially resolved multi-layered IR-images of a large collection of polymer beads supporting carbonyl and nitrile monomers. The feasibility of creating multi-layered IR-images with nitrile IR-band separation of 4 cm(-1) was demonstrated, an important issue when considering that many monomers used to develop combinatorial libraries are structurally analogous and therefore occupy very similar positions in the IR-spectrum. Strategies for obtaining high quality spectral data from both imaging and mapping IR-microscopes without compromising on sample area, analysis time, or spatial resolution are also described.     

Synthesis of hydantoins via N,N′-ureas derived from polymer-bound amino acids

Starting from carboxy-linked amino acids on trityl functionalized polystyrene resin a highly efficient solid-phase synthesis of hydantoins via N, N-ureas was elaborated. The polymer-bound hydantoins can be used as scaffolds for further combinatorial transformations, such as alkylation. Cleavage from the resins yielded the corresponding hydantoins in good yields and purities as shown by ESI-MS and HPLC.     

Analytical methods for quality control of combinatorial libraries

This literature review covers the applications of analytical techniques to solid phase organic chemistry and combinatorial chemistry published between June 96 and September 1997. Highlighted are mass spectrometry, NMR, IR and chromatographic analyses of solid phase synthesis reactions and combinatorial libraries.     

The solid-phase synthesis of novel 14-membered macrocycles for high throughput screening

A simple and efficient synthesis of novel 14-membered macrocycles from a resin-bound orthogonally protected lysine residue is described. Reductive alkylation of the lysine -nitrogen introduces the first diversity element. Acylation of the resultant secondary amine with an Fmoc-amino acid introduces the second diversity element providing a resin-bound protected di-peptide precursor. Removal of the Fmoc-group is followed by acylation with a succinic anhydride to introduce the final diversity elements. Removal of the methyltrityl-group from the amino group followed by macrocyclization provides the desired macrocycles, after TFA cleavage, in excellent yield and purity.     

Statistical molecular design of peptoid libraries

Statistical experimental design provides an efficient approach for selecting the building blocks to span the structural space and increase the information content in a combinatorial library. A set of renin-inhibitors, hexapeptoids, is used to illustrate the approach. Multivariate quantitative structure-activity relationships (MQSARs) were developed relating renin inhibition to the peptoid sequences variation, parametrized by the z-scales. By using the information from the models, the number of building block sets could be reduced from six to three. Using a statistical molecular design (SMD) reduces the number of compounds from more than 100 000 down to 90. A second SMD was used for comparison, based on less prior knowledge. This gave a reduction from over 2 billion to 120 compounds.     

Solid phase synthesis of benzamidine-derived sulfonamide libraries

Using solid phase synthesis, a library has been constructed of benzamidine-derived sulfonamides which have strong inhibitory activity against blood coagulant thrombin. The library compounds were obtained in good yield and high purity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of novel antitumor agents from mixture-based synthetic combinatorial libraries using cell-based assays

A new strategy is presented here which integrates combinatorial library technology with the antitumor in vitro screening system at the National Cancer Institute in the search for novel antitumor agents. Mixture-based synthetic combinatorial libraries (SCLs) representing hundreds of thousands to millions of individual compounds were screened against the cell-based assay, which evaluates compounds for their ability to inhibit the growth of 60 different human tumor cell lines. Five different SCLs, composed of peptides, peptidomimetics, polyamines or small molecules were first tested against three cell lines to identify the most active SCLs. Two SCLs, namely the N-perbenzylated pentamine and the N-acylated permethylated triamine, were deconvoluted to yield individual compounds having significant activities against the 60 tumor cell lines. Active compounds were tested in mice to determine the maximum tolerated dose, followed by in vivo testing in a hollow fiber assay. Using this strategy, three different compounds identified directly from SCLs are currently being evaluated in human tumor xenografts. This study demonstrates for the first time the use of in vitro cell-based assays to identify antitumor lead compounds from mixture-based combinatorial libraries.     

Exploring privileged structures: The combinatorial synthesis of cyclic peptides

Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classesof receptor with high affinity. They may therefore be considered to beprivileged structures. This review outlines the strategies by which bothmacrocyclic cyclic peptides and cyclic dipeptides or diketopiperazines havebeen synthesised in combinatorial libraries. It also briefly outlines someof the biological applications of these molecules, thereby justifying theirinclusion as privileged structures.     

Six new photolabile linkers for solid phase synthesis. 2. Coupling of various building blocks and photolytic cleavage

Photolabile linkers are very useful in the generation of combinatorial libraries as they offer compound cleavage under mild conditions directly into a solvent suitable for biological testing. Six new photolabile linkers have been developed which allow coupling of building blocks with a carboxy, amino, hydroxy and sulfonyl group. Photolytic cleavage of these building blocks will give libraries with carboxy, amido, methylamido, amino, ureido, hydroxy, aminocarbonyloxy and aminosulfonyl terminal groups. Coupling conditions for these reactions were elucidated and the photolytic cleavage reaction was studied.     

1H NMR spectroscopy with internal and external standards for the quantification of libraries

A convenient and easy method based on 1H NMR spectroscopy with both external and internal standards is described for the quantification of members of libraries.     

Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: Deconvolution strategy

Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X₁–X₂–hinge–X₃–X₄–NH₂ (capped) and X₁–hinge–X₂–X₃ (uncapped), where X₁ through X₄ are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a central hinge residue in small peptides increases the opportunity for side chain interactions, providing a distinct advantage for hinge peptide libraries over conventional peptide libraries. Hinge peptide libraries are a rich source of novel ligands for modulation of biomechanisms. The library subsets uncovered in this study may possess peptides that will lead to effective therapies to neurotoxin poisoning.     

A new strategy for solid phase synthesis of a secondary amide library using sulfonamide linker via radical traceless cleavage

A new strategy for solid phase synthesis of a secondary amide library using sulfonamide linker via radical traceless cleavage is reported. Polystyrylsulfonyl chloride (1) reacted with primary amines to afford polystyryl-supported N-alkyl sulfonamides (2), which were acylated with acid chlorides and followed by radical cleavage with TiCl₄/Zn to afford secondary amides. It was interestingly found that the products released from acyl alkanesulfonamide resins are closely dependent on the substituents of benzene rings of alkyl or acyl groups on the resins. When the substituent on benzene ring of N-benzyl group of sulfonamides is an electron rich MeO-group, the products released from sulfonamide resins are dependent on the substitution position on benzene ring: para-MeO- to yield 1,2-bis (p-methoxylphenyl)ethane and N-p-methoxylbenzyl benzamide (30:1); ortho-MeO- to give 1,2-bis (o-methoxylphenyl)ethane and N-o-methoxylbenzyl benzamide (1:15); and meta-MeO- only to release N-m-methoxylbenzyl benzamide. Neither N-benzoyl sulfonamide resins on benzene ring with electron-drawing para-O₂N-, nor the one with electron-donating para-H₂N- could release any amide product, while the N-benzoyl sulfonamide resins on benzene ring with para-acetamido group released para-acetamidobenzamides. The conjugation effect to stabilize the radical groups in the radical cleaving process was observed.     

Defining the antigenic structure of human GM-CSF and its implications for receptor interaction and therapeutic treatments

Three epitopes of human Granulocyte-Macrophage Colony-Stimulating Factor (hGM-CSF) recognised by neutralising and non-neutralising monoclonal antibodies (mAbs) were characterised using competitive binding assays, dissociation constant measurements with glycosylated and non-glycosylated rhGM-CSF, bioactivity inhibition studies, and synthetic peptide arrays. Based on the first approach, two different binding sites were identified: an area referred to as A, recognised by mAbs M1B8 and CC5B5, and an area referred to as B, recognised by mAbs CC1H7 and CC3C12. These binding sites on hGM-CSF were accurately delineated using cytokine-derived overlapping peptide scans and combinatorial hexapeptide libraries prepared by SPOT synthesis on cellulose membranes. We assigned the identical linear epitope A₁P₂A₃R₄ to both non-neutralising mAbs CC1H7 and CC3C12. The conformational epitopes A₁₈E₂₁R₂₃R₂₄F₁₁₉ and R₂₃E₂₁N₁₇W₁₃ recognised by mAb CC5B5, and P₁₁₈F₁₁₉EW₁₃E₁₄ recognised by mAb M1B8, were delineated by dual-positional scanning and subsequent iterative searches with two interrogating positions. Competitive binding assays with mAbs M1B8 and CC5B5 revealed the overlapping nature of the cytokine recognition. However, peptide library screening confirmed their binding to different epitopes of which the essential amino acids were found very closely located on the native protein surface. Inhibition of hGM-CSF biological activity by these mAbs demonstrated that these epitopes are located within or very near the receptor binding site of hGM-CSF. Finally, this work supports the importance of residues from helix A and residues from the C-terminal region of the cytokine, composing a common area that is indispensable for the cytokine's biological activity.     

Efficient library synthesis of imidazoles using a multicomponent reaction and microwave irradiation

Optimization of Radziszewski's four-component reaction employing a microwave-assisted protocol, led to a small library of 48 imidazoles with a success rate of 65% (conversion > 45%). All three diversity points of the four-component reaction were varied. Aromatic and aliphatic inputs were successfully implemented and mono-, di-, tri- and tetrasubstituted imidazoles with various substitution patterns were synthesized. Furthermore, unsymmetrical diketones could successfully be used which improved the intrinsic diversity of the method significantly. If the unsymmetrical diketone 1,2-phenylpropanedione (R¹ and R²) was used two regioisomers were formed. Depending on the type of amine (R⁴) and aldehyde (R³) applied, regioselectivity was modest to good. Based on these results, a reaction mechanism is proposed.     

高温超导体组合薄膜和相图表征高通量方法

铜氧化物超导体和铁基高温超导体是已知的两类高温超导体,研究高温超导机理是如今超导领域最具有挑战性的前沿课题.构建高温超导的高维精确相图、寻找决定超导转变温度的关键物理量可以为高温超导机理做好实验铺垫.对于铜氧化物高温超导体,多种自由度的相互关联与耦合使其相图呈现出复杂性与多样性.现有的研究方法在构建高维“全息”相图及获取定量化物理规律等方面面临着难以克服的困难,而材料的高通量制备与表征技术可以在相图空间实现参量的线扫描甚至面扫描,有望快速建立可靠的高温超导高维相图和高温超导关键参量数据库,并从中提取重要的统计物理规律.本文从阳离子掺杂、母体氧掺杂、双电层晶体管(静电场/电化学)、磁场等几个调控维度,回顾了主要基于输运手段获得的铜氧化物电子态相图,介绍了基于脉冲激光沉积技术和分子束外延技术的组合薄膜生长方法以及与之匹配的跨尺度选区输运测量技术,展示了高通量技术在高温超导研究中的初步应用.高通量实验技术与超导研究结合,逐步形成了新兴的高通量超导研究范式,将在构建高维精确相图、突破高温超导机理、推进超导材料实用化等方面发挥不可替代的作用.