Precipitation-free high-affinity multivalent binding by inline lectin ligands

Multivalent ligand–protein interactions are a key concept in biology mediating, for example, signalling and adhesion. Multivalent ligands often have tremendously increased binding affinities. However, they also can cause crosslinking of receptor molecules leading to precipitation of ligand–receptor complexes. Plaque formation due to precipitation is a known characteristic of numerous fatal diseases limiting a potential medical application of multivalent ligands with a precipitating binding mode. Here, we present a new design of high-potency multivalent ligands featuring an inline arrangement of ligand epitopes with exceptionally high binding affinities in the low nanomolar range. At the same time, we show with a multi-methodological approach that precipitation of the receptor is prevented. We distinguish distinct binding modes of the ligands, in particular we elucidate a unique chelating binding mode, where four receptor binding sites are simultaneously bridged by one multivalent ligand molecule. The new design concept of inline multivalent ligands, which we established for the well-investigated model lectin wheat germ agglutinin, has great potential for the development of high-potency multivalent inhibitors as future therapeutics.

Integration of sugar epitopes into a backbone structure generates multivalent lectin ligands with a defined binding mode and high affinity without precipitating the protein.     

Solid-phase parallel synthesis of phosphite ligands

Various routes for the synthesis of polymer-bound phosphites and phosphoramidites have been investigated. In the presence of a suitable activator the supported phosphoramidites react cleanly with alcohols to give the corresponding monodentate phosphite ligands in solution. We have applied this novel solid-phase route in the parallel synthesis of several monodentate chiral and achiral phosphite ligands.     

Solid-phase synthesis of tetrasubstituted pyrazoles, novel ligands for the estrogen receptor

Most ligands for the estrogen receptor (ER) are not well suited for synthesis by combinatorial means, because their construction involves a series of carbon-carbon bond forming reactions that are not uniformly high yielding. In previous work directed to overcoming this limitation, we surveyed various phenol-substituted five-membered heterocycles, hoping to find a system that would afford both high ER binding affinity and whose synthesis could be adapted to solid-phase methods (Fink et al. Chem. Biol. 1999, 6, 206-219.) In this report, we have developed a reliable and efficient solid-phase method to prepare the best of these heterocycles, the tetrasubstituted pyrazoles, and we have used this methodology to produce small, discrete libraries of these novel ER ligands. We used a combination of FT-IR and nanoprobe (1)H NMR-MAS to characterize intermediates leading up to the final pyrazole products directly on the bead. We also developed a scavenging resin, which enabled us to obtain products free from inorganic contaminants. We prepared a 12-member test library, and then a 96-member library, and in both cases we determined product purity and ER binding affinity of all of the library members. Several interesting binding affinity patterns have emerged from these studies, and they have provided us with new directions for further exploration, which has led to pyrazoles having high affinity and potency as agonists and antagonists toward the ER alpha subtype.     

Chemoenzymatic synthesis and high-throughput screening of an aminoglycoside-polyamine library: identification of high-affinity displacers and DNA-binding ligands

Chemoenzymatic parallel synthesis and high-throughput screening were employed to develop a multivalent aminoglycoside-polyamine library for use as high-affinity cation-exchange displacers and DNA-binding ligands. Regioselective lipase-catalyzed acylation, followed by chemical aminolysis, was used to generate vinyl carbonate and vinyl carbamate linkers, respectively, of the aminoglycosidic cores. These were further derivatized with polyamines, leading to library generation. A parallel batch-displacement assay was employed to identify the efficacy of the library candidates as potential displacers for protein purification. Using this approach, low-molecular-mass displacers with affinities higher than those previously observed have been identified. The aminoglycoside-polyamine library was also screened for DNA binding efficacy using an ethidium bromide displacement assay. These highly cationic molecules exhibited strong DNA-binding properties and may have potential for enhanced gene delivery.     

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.     

Docking, synthesis, and NMR studies of mannosyl trisaccharide ligands for DC-SIGN lectin

DC-SIGN, a lectin, which presents at the surface of immature dendritic cells, constitutes nowadays a promising target for the design of new antiviral drugs. This lectin recognizes highly glycosylated proteins present at the surface of several pathogens such as HIV, Ebola virus, Candida albicans, Mycobacterium tuberculosis, etc. Understanding the binding mode of this lectin is a topic of tremendous interest and will permit a rational design of new and more selective ligands. Here, we present computational and experimental tools to study the interaction of di- and trisaccharides with DC-SIGN. Docking analysis of complexes involving mannosyl di- and trisaccharides and the carbohydrate recognition domain (CRD) of DC-SIGN have been performed. Trisaccharides Manalpha1,2[Manalpha1,6]Man 1 and Manalpha1,3[Manalpha1,6]Man 2 were synthesized from an orthogonally protected mannose as a common intermediate. Using these ligands and the soluble extracellular domain (ECD) of DC-SIGN, NMR experiments based on STD and transfer-NOE were performed providing additional information. Conformational analysis of the mannosyl ligands in the free and bound states was done. These studies have demonstrated that terminal mannoses at positions 2 or 3 in the trisaccharides are the most important moiety and present the strongest contact with the binding site of the lectin. Multiple binding modes could be proposed and therefore should be considered in the design of new ligands.     

Solid-phase methods for the synthesis of cyanine dyes

We report here a series of studies that explore solid-phase methodologies for the synthesis of various cyanine dyes. The scope of the previously reported catch-and-release method using sulfonyl chloride resin(1) has now been extended to include pentamethine and water-soluble cyanine dyes. We also report a new and chemically distinct synthetic strategy, employing the stepwise attack of heterocyclic carbon nucleophiles on immobilized polyene-chain precursors, allowing the clean synthesis of hydrophobic and hydrophilic trimethine and pentamethine dyes from more easily obtained starting materials. Overall, both approaches appear to be robust and versatile strategies to delivering a wide range of cyanine-based dyes in high purity.     

Mathematical model for determining the binding constants between immunoglobulins,bivalent ligands,and monovalent ligands

This paper analyzes the equilibria between immunoglobulins (R(2)), homo-bifunctional ligands (L(2)), monovalent ligands (I), and their complexes. We present a mathematical model that can be used to estimate the concentration of each species present in a mixture of R(2), L(2), and I, given the initial conditions defining the total concentration of R(2), L(2), I, and four dissociation constants (K(d)(inter), K(d)(intra), K(d)(mono), and α). This model is based on fewer assumptions than previous models and can be used to describe exactly a broad range of experimental conditions. A series of curves illustrates the dependence of the equilibria upon the total concentrations of receptors and ligands, and the dissociation constants. We provide a set of guidelines for the design and analysis of experiments with a focus on estimating the binding constants from experimental binding isotherms. Two analytical equations relate the conditions for maximum aggregation in this system to the binding constants. This model is a tool to quantify the binding of immunoglobulins to antigens and a guide to understanding and predicting the experimental data of assays and techniques that employ immunoglobulins.     

Solid-phase synthesis of beta-mannosides

The linkage of S-phenyl 2,3-di-O-benzyl-alpha-D-thiomannopyranoside to a cross-linked polystyrene support in the form of its 4,6-O-polystyrylborinate ester is described. The activation of this polymer-supported mannosyl donor is achieved at -60 degrees C in dichloromethane in the presence of 2,4,6-tri-tert-butylpyrimidine with the combination 1-benzenesulfinyl piperidine and trifluoromethanesulfonic anhydride. Addition of the donor alcohol at -60 degrees C followed by warming to room temperature and subsequent cleavage from the resin by gentle heating in aqueous acetone yields anomerically pure 2,3-di-O-benzyl-beta-D-mannopyranosides in excellent yield. Successful, diastereoselective coupling is demonstrated with a range of primary, secondary, and tertiary glycosyl acceptors, including typical carbohydrates and threonine derivatives.     

Solid-phase synthesis of dibenzoxazepinones

Two novel solid-phase routes to the pharmaceutically relevant dibenzoxazepinone nucleus are described. In one, a key cyclisation step involves intramolecular phenolate displacement of an activated aryl fluoride. In the second, the tricyclic nucleus is prepared in solution prior to derivatisation on resin.     

Solid-phase synthesis of DOTA-peptides

A general synthetic route to two DOTA-linked N-Fmoc amino acids (DOTA-F and DOTA-K) is described that allows insertion of DOTA at any endo-position within a peptide sequence. Three model pentapeptides were prepared to test the general utility of these derivatives in solid-phase peptide synthesis. Both DOTA derivatives reacted smoothly by means of standard HBTU activation chemistry to the point of insertion of the DOTA amino acid, but extension of the peptide chain beyond the DOTA-amino acid insertion required the use of pre-activated C-pentafluorophenyl ester N-alpha-Fmoc amino acids. Three Gal-80 binding peptides (12-mers) were then prepared by using this methodology with DOTA positioned either at the N terminus or at one of two different internal positions;the binding of the resulting GdDOTA-12-mers to Gal-80 were compared. The methodology described here allows versatile, controlled introduction of DOTA into any location within a peptide sequence. This provides a potential method for the screening of libraries of DOTA-linked peptides for optimal targeting properties.     

Solid-phase synthesis of solanesol

An efficient and convergent solid-phase strategy for the total synthesis of All- E solanesol is described. This method features avoidance of iterative and difficult purifications comparing with solution-phase synthesis and is suitable for the preparation of other oligoprenols.     

Solid-phase synthesis of fullerene-peptides

The solid-phase synthesis of peptides (SPPS) containing [60]fullerene-functionalized amino acids is reported. A new amino acid, fulleropyrrolidino-glutamic acid (Fgu), is used for the SPPS of a series of analogues of different length based on the natural Leu(5)-Enkephalin and on cationic antimicrobial peptides. These fullero-peptides were prepared on different solid supports to analyze the influence of the resin on the synthesis. Optimized protocols for the coupling and deprotection procedures were determined allowing the synthesis of highly pure peptides in sufficient quantities for evaluation of biological activities. In particular, to avoid side reactions of the fullerene moiety with bases and nucleophiles, the removal of the protecting groups was performed under inert conditions (nitrogen or argon in the dark). We have encountered serious problems with the recovery of the crude compounds, especially when Fgu was inserted in the proximity of the resin core as fullero-peptides tend to remain embedded inside the resin. Eventually, all of the fullero-peptides were easily purified, and the cationic peptides were tested for their antimicrobial activities. They displayed a specific activity against the Gram-positive bacterium S. aureus and also lysed erythrocytes. The availability of a fullero-amino acid easily useable in the SPPS of fullero-peptides may thus open the way to the synthesis of new types of biologically active oligomers.     

Solid-phase synthesis of isoxazolines

The polymer-supported synthesis of isoxazolines is described via nitrile oxide intermediates, starting from primary nitroalkanes in a one-pot process.     

Competition STD NMR for the detection of high-affinity ligands and NMR-based screening

The reported competition STD NMR method combines saturation transfer difference (STD) NMR with competition binding experiments to allow the detection of high-affinity ligands that undergo slow chemical exchange on the NMR time-scale. With this technique, the presence of a competing high-affinity ligand in the compound mixture can be detected by the disappearance or reduction of the STD signals of a low-affinity indicator ligand. This is demonstrated on a BACE1 (beta-site amyloid precursor protein cleaving enzyme 1) protein-inhibitor system. This method can also be used to derive an approximate value, or a lower limit, for the dissociation constant of the potential ligand based on the reduction of the signal intensity of the STD indicator, which is illustrated on an HSA (human serum albumin) model system. This leads to important applications of the competition STD NMR method for lead discovery: it can be used (i) for compound library screening against a broad range of drug targets to identify both high- and low-affinity ligands and (ii) to rank order analogs rapidly and derive structure-activity relationships, which are used to optimize these NMR hits into viable drug leads.     

Solid-phase synthesis of chiral N-acylethylenediamines and their use as ligands for the asymmetric addition of alkylzinc and alkenylzinc reagents to aldehydes

A solid-phase procedure has been developed for the synthesis of chiral N-acylethylenediamine ligands. The ligands are obtained in good yield and purity, without the need for chromatography or other purification methods. Several new and previously reported ligands were prepared using this procedure. These compounds were examined as catalysts for the enantioselective addition of alkylzinc reagents to aldehydes. In all cases, the crude ligands from the solid-phase syntheses catalyzed the reactions with similar yields and stereoselectivities when compared to reactions using ligands that had been purified by standard methods. Preliminary studies were also performed with ligands 3a and 3f as catalysts for the addition of alkenylzinc reagents to aldehydes to give chiral allylic alcohols. Ligand 3f was found to catalyze this addition reaction in up to 76% ee.