Novel structural templates for estrogen-receptor ligands and prospects for combinatorial synthesis of estrogens

BACKGROUND: The development of estrogen pharmaceutical agents with appropriate tissue-selectivity profiles has not yet benefited substantially from the application of combinatorial synthetic approaches to the preparation of structural classes that are known to be ligands for the estrogen receptor (ER). We have developed an estrogen pharmacophore that consists of a simple heterocyclic core scaffold, amenable to construction by combinatorial methods, onto which are appended 3-4 peripheral substituents that embody substructural motifs commonly found in nonsteroidal estrogens. The issue addressed here is whether these heterocyclic core structures can be used to prepare ligands with good affinity for the ER. RESULTS: We prepared representative members of various azole core structures. Although members of the imidazole, thiazole or isoxazole classes generally have weak binding for the ER, several members of the pyrazole class show good binding affinity. The high-affinity pyrazoles bear close conformational relationship to the nonsteroidal ligand raloxifene, and they can be fitted into the ligand-binding pocket of the ER-raloxifene X-ray structure. CONCLUSIONS: Compounds such as these pyrazoles, which are novel ER ligands, are well suited for combinatorial synthesis using solid-phase methods.     

Design and Synthesis of Carbohydrate Mimetics: A New Strategy for Tackling the Problem of Carbohydrate-Mediated Biological Recognition

Carbohydrates have been known as poor candidates for drug development. Recent studies have, however, shown that structurally simplified small molecules as mimics of complex carbohydrates recognized by receptors can be developed as inhibitors of carbohydrate-mediated biological recognition. In addition, small molecules with higher affinity and specificity than the parent ligands can be developed by incorporating additional hydrophobic or charged groups in the carbohydrate mimetic which contains essential functional groups for receptor binding. Representative examples are illustrated in the studies of sialyl Lewis x - selectin interactions, glycosidase and glycosyltransferase reactions and aminoglycoside antibiotic - RNA interactions.     

Combinatorial chemistry toward understanding the function(s) of carbohydrates and carbohydrate conjugates

Combinatorial chemistry has contributed significantly to understanding the structure-function relationships of biologically important molecules such as proteins and nucleic acids. However, carbohydrates and carbohydrate conjugates, which have been identified as key modulators of several biological functions have not enjoyed the same measure of success. The complexity and synthetic challenges of carbohydrate conjugates have resulted in a number of conceptual approaches to rapidly access sufficient quantities of these biomolecules. This article summarizes these combinatorial approaches and also highlights fully automated library synthesis of artificial glycopeptides with the goals of understanding their biological roles.     

Identification of alpha-galactosyl epitope mimetics through rapid generation and screening of C-linked glycopeptide library

A general methodology has been established for rapid generation and screening of combinatorial glycopeptide library and subsequent mass spectrometric sequencing to identify the mimetics of Galalpha(1,3)Gal epitopes. Using this approach, several active glycopeptide sequences were recognized and found to inhibit the binding of human natural anti-Gal antibodies with comparable IC(50)s to synthetic Galalpha(1,3)Gal oligosaccharides. The most active glycopeptides detected from the library included Gal-Tyr-Trp-Arg-Tyr, Gal-Thr-Trp-Arg-Tyr, and Gal-Arg-Trp-Arg-Tyr. These glycopeptides showed higher affinities to anti-Gal antibodies than known Galalpha(1,3)Gal peptide mimetics, such as DAHWESWL and SSLRGF. Our results suggest that, by combining a peptide sequence (the "functional" mimic part) with a terminal alpha-linked galactose moiety (the "structural" mimic part), the resulting glycopeptide could be a very good Galalpha(1,3)Gal mimetic. Analysis of these active glycopeptides provided first-hand information regarding the binding site of anti-Gal antibodies to facilitate the structurally based design of more potent and stable inhibitors.     

Combinatorial Synthesis and Multidimensional NMR Spectroscopy: An Approach to Understanding Protein–Ligand Interactions

Combinatorial chemistry is a laboratory emulation of natural recombination and selection processes. Strategies in this developing discipline involve the generation of diverse, molecular libraries through combinatorial synthesis and the selection of compounds that possess a desired property. Such approaches can facilitate the identification of ligands that bind to biological receptors, promoting our chemical understanding of cellular processes. This article illustrates that the coupling of combinatorial synthesis, multidimensional NMR spectroscopy, and biochemical methods has enhanced our understanding of a protein receptor used commonly in signal transduction, the Src Homology 3 (SH3) domain. This novel approach to studying molecular recognition has revealed a set of rules that govern SH3–ligand interactions, allowing models of receptor–ligand complexes to be constructed with only a knowledge of the polypeptide sequences. Combining combinatorial synthesis with structural methods provides a powerful new approach to understanding how proteins bind their ligands in general.     

Synthesis of High‐Mannose Oligosaccharide Analogues through Click Chemistry: True Functional Mimics of Their Natural Counterparts Against Lectins?

Terminal “high‐mannose oligosaccharides” are involved in a broad range of biological and pathological processes, from sperm‐egg fusion to influenza and human immunodeficiency virus infections. In spite of many efforts, their synthesis continues to be very challenging and actually represents a major bottleneck in the field. Whereas multivalent presentation of mannopyranosyl motifs onto a variety of scaffolds has proven to be a successful way to interfere in recognition processes involving high‐mannose oligosaccharides, such constructs fail at reproducing the subtle differences in affinity towards the variety of protein receptors (lectins) and antibodies susceptible to binding to the natural ligands. Here we report a family of functional high‐mannose oligosaccharide mimics that reproduce not only the terminal mannopyranosyl display, but also the core structure and the branching pattern, by replacing some inner mannopyranosyl units with triazole rings. Such molecular design can be implemented by exploiting “click” ligation strategies, resulting in a substantial reduction of synthetic cost. The binding affinities of the new “click” high‐mannose oligosaccharide mimics towards two mannose specific lectins, namely the plant lectin concanavalin A (ConA) and the human macrophage mannose receptor (rhMMR), have been studied by enzyme‐linked lectin assays and found to follow identical trends to those observed for the natural oligosaccharide counterparts. Calorimetric determinations against ConA, and X‐ray structural data support the conclusion that these compounds are not just another family of multivalent mannosides, but real “structural mimics” of the high‐mannose oligosaccharides.     

β(1,3) Branched heptadeca- and linear hexadeca-saccharides possessing an aminoalkyl group as a strong ligand to dectin-1

In this report, we describe the convergent synthesis of β(1,3) oligosaccharides containing an aminoalkyl group. The branched heptadecasaccharide and linear hexadecasaccharide acted as ligands of dectin-1 whose binding affinity was only 10-fold weaker than that of natural SPG and exhibited dectin-1 agonist activity.     

Preparation of linear oligosaccharides by a simple monoprotective chemo-enzymatic approach

A monoprotective approach, involving acetyl ester as unique protective group in oligosaccharides synthesis, has been developed. Starting from peracetylated monosaccharides and glycals, by using an efficient and selective chemo-enzymatic ‘one-pot’ strategy (a regioselective hydrolysis catalyzed by immobilized lipases followed by a chemical acyl migration), different carbohydrate acceptors, only protected with acetyl ester, can be achieved. If combined with the use of an acetylated glycosyl donor, the glycosylation reaction with these glycosyl acceptors leads to peracetylated oligosaccharides. These compounds can be directly used as intermediates for the synthesis of glycopeptides used as antitumoral vaccines and, at the end of the process, can be easily fully deprotected in only one step. Thus, these key building blocks have been successfully used in glycosylation reactions for an efficient construction of peracetylated disaccharides, such as the biological relevant lactosamine, in multigram scale. Subsequently, glycosylation with the 3OH-tetraacetyl-α-d-galactose, used as carbohydrate acceptor, allowed the synthesis of a peracetylated N-trisaccharidic precursor of the lacto-N-neo-tetraose antigen. Extending this strategy to a 3OH-di-acetyl galactal, one peracetylated precursor of the T tumor-associated carbohydrate antigen has been synthesized.This efficient approach, characterized by the use of the acetyl ester as only protecting group during all the synthetical steps expected, represents an easy and efficient alternative to the classical synthetic methods in carbohydrate chemistry that involve several protecting group manipulation.     

Selective glycopeptide mapping of erythropoietin by on-line high-performance liquid chromatography--electrospray ionization mass spectrometry

Selective glycopeptide mapping of recombinant human erythropoietin (rhEPO) used as a model glycoprotein was successfully carried out by on-line high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) using a Vydac C18 column eluted in acetonitrile-1 mM ammonium acetate, pH 6.8. rhEPO expressed in a Chinese hamster ovary clone was exhaustively digested into four glycopeptides and nine peptides with endoproteinase Glu-C. Both glycopeptides and peptides were eluted with trifluoroacetic acid as the eluent, whereas only glycopeptides were eluted selectively with ammonium acetate in the following order: N38, N24, 0126, and N83. Furthermore, many glycoforms included in each glycopeptide were found to be separated by differences in the numbers of sialic acid and N-acetyllactosaminyl repeats. Twenty, 16 and 22 different N-linked oligosaccharides were determined at Asn24, 38, and 83, respectively, and two different O-linked oligosaccharides were observed at Ser126. Our method is simple, rapid, and useful for determining the carbohydrate structures at each glycosylation site and for elucidating the site-specific carbohydrate heterogeneity.     

A topologically segregated one-bead-one-compound combinatorial glycopeptide library for identification of lectin ligands

A glycopeptide library containing more than 500,000 compounds has been constructed from a combination of Asn-linked carbohydrates using one-bead-one-compound combinatorial methodologies. The library was encoded with peptide markers that were topologically segregated on the interior of the solid support to negate interference with carbohydrate/protein recognition during lectin screening. Both the peptide backbone and carbohydrate components were randomized, but the glycosamine was limited to position 3 at the center of the pentapeptide to evaluate the influence of the peptide backbone in lectin recognition. Of the four lectins that were evaluated, remarkable selectivity was observed with wheat germ agglutinin (WGA), which recognizes N-acetyl glucosamine (GlcNAc). From more than 80,000 possible combinations, only six ligands were identified, all possessing GlcNAc. These compounds were independently synthesized, characterized, and evaluated in solution. All six of the glycopeptides showed higher affinity for WGA than GlcNAc, with one having a 4-fold increase. Modeling studies indicate that the peptide backbone is capable of interacting with amino acids in the active site of WGA, but these interactions are not strongly correlated with activity, suggesting that the primary role of the peptide is to properly orient the sugar in the recognition process.     

2-Oxabutane as a substitute for internal monomer units of oligosaccharides to create lectin ligands

The synthesis of bioactive oligosaccharides is too tedious to scale up for commercialization. However, structurally simplified glycomimetics are commercializable, if they can be synthesized much more easily than the oligosaccharides while having a comparable bioactivity. In this study, we propose a 2-oxabutane (OB) structure as an imitation of the internal monosaccharide units in oligosaccharides. Two trimannoside and three pentamannoside OB-glycomimics were synthesized in remarkably short steps. Among them, Manα1-OB-2Man 10, a trimannoside mimic, showed eight-fold affinity toward concanavalin A (ConA) relative to methyl mannoside in latex agglutination lectin assay and equilibrium dialysis assay (EDA), while the other mimics showed three- to four-fold affinities. EDA indicated that the bindings between each mimic molecule and a ConA subsite were all in one-to-one stoichiometry and thus these mimics were monovalent ligands, excluding multivalence effect for the high affinities. The strong affinity of 10 could be explained by the occupation of two mannose binding sites of a ConA subsite by its two mannose units. Mimic 10 proved to be even a better ligand for ConA than the natural disaccharide Manα1,2Man, while been much more easy to synthesize, thereby illustrating the potential of the approach here presented.     

Lock-and-key motif as a concept for designing affinity adsorbents for protein purification

The lock-and-key (LAK) motif, a common structural moiety found in subunit interfaces of glutathione S-transferases (GSTs), plays an important role in biomolecular recognition and quaternary structure integrity. Inspection of the key structural features of the LAK motif prompted the de novo design and combinatorial synthesis of a 13-membered solid-phase ligand library, employing as a lead ligand the Phe-Trz-X structure, mimicking the LAK motif. 1,3,5-Triazine (Trz) was used as the scaffold for assembly, substituted with different LAK-mimetic amino acids. De novo ligand design was effected using bioinformatics and molecular modeling and based on mimicking the interactions of the LAK motif. The library of affinity adsorbents was assessed for binding corn and human serum proteomes and purified proteins of different structure and ligand binding specificity. The results showed remarkable differences in the binding specificity of LAK-mimetic adsorbents for a wide range of proteins, as a consequence of minor changes in ligand structure. One LAK-mimetic adsorbent was integrated in a single-step purification protocol for human monoclonal anti-human immunodeficiency virus 2F5 antibody (mAb 2F5) from spiked corn extract, affording high recovery and purity. The results demonstrate that the principle of natural recognition found in the lock-and-key motif, in combination with de novo combinatorial design, may lead to synthetic affinity ligands, useful in downstream processing and proteomic research.     

Rapid On-Chip Synthesis of Complex Glycomimetics from N-Glycan Scaffolds for Improved Lectin Targeting

C-type lectin receptor (CLR) carbohydrate binding proteins found on immune cells with important functions in pathogen recognition as well as self and non-self-differentiation are increasingly moving into the focus of drug developers as targets for the immune therapy of cancer autoimmune diseases and inflammation and to improve the efficacy of vaccines. The development of molecules with increased affinity and selectivity over the natural glycan binders has largely focused on the synthesis of mono and disaccharide mimetics but glycan array binding experiments have shown increased binding selectivity and affinity for selected larger oligosaccharides that are able to engage in additional favorable interactions beyond the primary binding site. Here, a platform for the rapid preparation and screening of N-glycan mimetics on microarrays is presented that turns a panel of complex glycan core structures into structurally diverse glycomimetics by a combination of enzymatic glycosylation with a nonnatural donor and subsequent cycloaddition with a collection of alkynes. All surface-based reactions were monitored by MALDI-TOF MS to assess conversion and purity of spot compositions. Screening the collection of 374 N-glycomimetics against the plant lectin WFA and the 2 human immune lectins MGL ECD and Langerin ECD produced a number of high affinity binders as lead structures for more selective lectin targeting probes.     

寡糖组合合成

寡糖是许多生物过程的重要协调物质,其结构与功能的关系正成为人们感兴趣的研究课题。然而传统的寡糖合成很复杂且费时费力,随着组合化学方法在寡糖合成领域的应用,方便快捷地制备各种各样的寡糖成为一种可能。本文从液相和固相反应两个方面综述了组合化学在寡糖合成领域的研究进展。     

Quality, not quantity: the role of natural products and chemical proteomics in modern drug discovery

Chemical genetics and reverse chemical genetics parallel classical genetics but target genes at the protein level and have proven useful in recent years for screening combinatorial libraries for compounds of biological interest. However, the performance of combinatorial chemistry in filling pharmaceutical pipelines has been lower than anticipated and the tide may be turning back to Nature in the search for new drug candidates. Even though diversity oriented synthesis is now producing molecules that are natural product-like in terms of size and complexity, these molecules have not evolved to interact with biomolecules. Natural products, on the other hand, have evolved to interact with biomolecules, which is why so many can be found in pharmacopoeias. However, the cellular targets and modes of action of these fascinating compounds are seldom known, hindering the drug development process. This review focuses on the emergence of chemical proteomics and reverse chemical proteomics as tools for the discovery of cellular receptors for natural products, thereby generating protein/ligand pairs that will prove useful in identifying new drug targets and new biologically active small molecule scaffolds. Such a system-wide approach to identifying new drugable targets and their small molecule ligands will help unblock the pharmaceutical product pipelines by speeding the process of target and lead identification.     

Divalent Sialylated Precision Glycooligomers Binding to Polyomaviruses and the Effect of Different Linkers

Divalent precision glycooligomers terminating in N‐acetylneuraminic acid (Neu5Ac) or 3′‐sialyllactose (3′‐SL) with varying linkers between scaffold and the glycan portions are synthesized via solid phase synthesis for co‐crystallization studies with the sialic acid‐binding major capsid protein VP1 of human Trichodysplasia spinulosa‐associated Polyomavirus. High‐resolution crystal structures of complexes demonstrate that the compounds bind to VP1 depending on the favorable combination of carbohydrate ligand and linker. It is found that artificial linkers can replace portions of natural carbohydrate linkers as long as they meet certain requirements such as size or flexibility to optimize contact area between ligand and receptor binding sites. The obtained results will influence the design of future high affinity ligands based on the structures presented here, and they can serve as a blueprint to develop multivalent glycooligomers as inhibitors of viral adhesion.     

Synthesis of 5-, 6- and 7-substituted-2-aminoquinolines as SH3 domain ligands

The Src homology 3 (SH3) domains are small protein-protein interaction domains that mediate a range of important biological processes and are considered valuable targets for the development of therapeutic agents. We have been developing 2-aminoquinolines as ligands for SH3 domains--so far the only reported examples of entirely small-molecule ligands for the SH3 domains. The highest affinity 2-aminoquinolines so far identified are 6-substituted compounds. In this article, the synthesis of several new 2-aminoquinolines, including 5-, 6- and 7-substituted compounds, for Tec SH3 domain ligand binding studies is presented. As a part of the synthetic investigation, the utility of different methods for the synthesis of 2-aminoquinolines was explored and potentially powerful methods were identified for the synthesis of 2-aminoquinolines with diverse functionality. Of the compounds prepared, the 5-substituted-2-aminoquinolines generally bound with similar affinities to unsubstituted 2-aminoquinoline, whilst the 7-substituted compounds generally bound with similar or lower affinity than unsubstituted 2-aminoquinoline. However, the 6-substituted-2-aminoquinolines generally bound with significantly higher affinity than unsubstituted 2-aminoquinoline. In addition, one 6-substituted-N-benzylated-2-aminoquinoline was also tested for SH3 binding and some evidence for the formation of additional contacts at other regions of the SH3 domain was found. These results provide new and useful SAR information that should greatly assist with the challenge of developing high affinity small-molecule ligands for the SH3 domains.     

A general technique to rank protein-ligand binding affinities and determine allosteric versus direct binding site competition in compound mixtures

To realize the full potential of combinatorial chemistry-based drug discovery, generic and efficient tools must be developed that apply the strengths of diversity-oriented chemical synthesis to the identification and optimization of lead compounds for disease-associated protein targets. We report an affinity selection-mass spectrometry (AS-MS) method for protein-ligand affinity ranking and the classification of ligands by binding site. The method incorporates the following steps: (1) an affinity selection stage, where protein-binding compounds are selected from pools of ligands in the presence of varying concentrations of a competitor ligand, (2) a first chromatography stage to separate unbound ligands from protein-ligand complexes, and (3) a second chromatography stage to dissociate the ligands from the complexes for identification and quantification by MS. The ability of the competitor ligand to displace a target-bound library member, as measured by MS, reveals the binding site classification and affinity ranking of the mixture components. The technique requires no radiolabel incorporation or direct biochemical assay, no modification or immobilization of the compounds or target protein, and all reaction components, including any buffers or cofactors required for protein stability, are free in solution. We demonstrate the method for several compounds of wide structural variety against representatives of the most important protein classes in contemporary drug discovery, including novel ATP-competitive and allosteric inhibitors of the Akt-1 (PKB) and Zap-70 kinases, and previously undisclosed antagonists of the M(2) muscarinic acetylcholine receptor, a G-protein coupled receptor (GPCR). The theoretical basis of the technique is analyzed mathematically, allowing quantitative estimation of binding affinities and, in the case of allosteric interaction, absolute determination of binding cooperativity. The method is readily applicable to high-throughput screening hit triage, combinatorial library-based affinity optimization, and developing structure-activity relationships among multiple ligands to a given receptor.     

Oligosaccharide synthesis and library assembly by one-pot sequential glycosylation strategy

The oligosaccharide residue in glycoconjugates located in cell membranes is responsible for intercellular recognition and interaction: it acts as a receptor for proteins, hormones, and microorganisms and governs immune reactions. These significant activities have stimulated great interest in the field of oligosaccharides and glycoconjugates. Although many advances have been made in the synthesis of oligosaccharides, more convenient and efficient methods are still needed. This review describes one of these new methods-the one-pot sequential glycosylation approach as a potent tool for oligosaccharide assembly. The oligosaccharide library construction in a one-pot fashion is also summarized.     

Combined use of lectin affinity chromatography and endo-beta-galactosidase to study polylactosamine sequences isolated from haemopoietic cell surfaces

The trypsin-sensitive glycopeptides from cell surfaces of a multipotential murine haemopoietic cell line (DE) have been studied using serial lectin affinity chromatography on columns of immobilized lentil lectin (LCA), concanavalin A (Con A), and wheat-germ agglutinin (WGA). WGA-binding material consisted of glycopeptides that failed to bind to LCA and Con A. Step elution from the WGA-column with 0.01-, 0.1-, 0.5- and 1.0 M N-acetyl-D-glucosamine yielded four affinity classes of glycopeptide (WGA-W, WGA-I, WGA-S and WGA-SS respectively). WGA-W, WGA-I and WGA-S contained both alkali-stable (N-linked) and alkali-labile (O-linked) carbohydrate on high molecular weight glycopeptides. The WGA-SS fraction contained only N-linked carbohydrate. N-linked glycopeptides isolated from each WGA-binding class differed in molecular size, relative N-acetylneuraminic acid content and affinity for Ricinus communis 120 agglutinin. endo-beta-Galactosidase digestion showed that these glycopeptides contained polylactosamine-type glycans. Gel filtration profiles of the enzyme treated materials were different for each WGA-binding population suggesting variation in branching patterns and/or substitution with fucose residues. Affinity chromatography has shown that the WGA binding molecules are the major glycopeptide group at DE cell surfaces.