Solid-phase Support Research Articles

Polystyrene resins cross-linked with di- or tri(ethylene glycol) dimethacrylates as supports for solid-phase peptide synthesis

Polystyrene resins cross-linked with di(ethylene glycol) dimethacrylate (DEGDMA) and tri(ethylene glycol) dimethacrylate (TEGDMA), DEGDMA–PS and TEGDMA–PS, were synthesized by suspension copolymerization. Four functionalized resins, chloromethyl resin, 4-hydroxymethylphenoxymethyl resin (Wang resin), 4-methylbenzhydrylamine resin (MBHA resin) and 2-chlorotrityl chloride resin, were prepared from DEGDMA–PS and TEGDMA–PS. DEGDMA–PS and TEGDMA–PS showed high reactivity in the functionalization reactions in comparison with Merrifield resin (polystyrene cross-linked with divinylbenzene, DVB–PS). DEGDMA–PS–Wang resin and TEGDMA–PS–Wang resin were used as the solid-phase support for the synthesis of a difficult sequence, the fragment of acyl carrier protein 65–74. The yields of the crude peptide synthesized using DEGDMA–PS–Wang resin, TEGDMA–PS–Wang resin and DVB–PS–Wang resin were 92.3%, 91.6% and 78.8%, respectively. The purities of the crude peptides were 85.7%, 88.1% and 73.3%, respectively.

Polystyrene-graft-Polyglycerol Resins: A New Type of High-Loading Hybrid Support for Organic Synthesis

The preparation of a dendritic graft polymer by a very efficient synthesis of polyglycerol directly on a polystyrene resin is presented. This one-step process can be performed on a multigram scale to provide a chemically stable polymeric support. The resulting hybrid polymers were fully characterized by diverse analytical methods (NMR, IR, ESEM, UV detection of cleaved protecting groups, and mass-spectrometric methods). They combine a high loading capacity (up to 4.3 mmol g(-1)) with good swelling properties in a wide range of solvents (including water), which is the major drawback for many existing solid phase supports. In comparison to the widely employed PEGylated resins, these hybrid materials offer a 10-fold higher loading capacity. Their suitability as supports for organic synthesis and for the immobilization of reagents has been demonstrated. These materials also swell in water, and consequently, it should be possible to use these new hybrid materials for synthesis in protic solvents.

Clickity-click: highly functionalized peptoid oligomers generated by sequential conjugation reactions on solid-phase support

N-Substituted glycine peptoid oligomers were used as substrates for azide-alkyne [3 + 2] cycloaddition conjugation reactions and then elaborated through additional rounds of oligomerization and cycloaddition. This novel sequential conjugation technique allowed for the generation of complex peptidomimetic products in which multiple heterogeneous pendant groups were site-specifically positioned along the oligomer scaffold. Studies of a water-soluble estradiol-ferrocene peptoid conjugate demonstrated a potential application for the modular synthesis of biosensors.

Rapid Combinatorial Screening of Peptide Libraries for the Selection of Lanthanide-Binding Tags (LBTs)

Elucidating the structures and functions of novel proteins and their interaction partners is a ubiquitous challenge in laboratories worldwide, escalating the demand for tools for protein study. A small, versatile and multifaceted co-expression tag would therefore be extremely useful to protein scientists: Lanthanide-Binding Tags (LBTs) fulfill such criteria. These tags are remarkably short peptide sequences (15 to 20 residues) comprising only the encoded amino acids; post-expression addition of a lanthanide ion endows the LBTs with the abundant spectroscopic properties of lanthanides. Herein is reviewed a powerful combinatorial screen developed expressly for the synthetic evolution of short calcium-binding motifs into selective, potent binders of terbium and related lanthanide ions. A series of seven libraries resulted in peptides with dissociation constants nearly three orders of magnitude tighter than that of the parent sequence. These libraries utilized two orthogonal linkers on the solid phase support, enabling cleaved peptides to be screened off-resin, while leaving a portion of the peptide intact on the synthesis resin for later sequencing. Terbium luminescence from the bound LBTs provides the positive selection criterion, and mass spectroscopy using limited capping was implemented for sequence deconvolution. The roles and importance of various residues within the later-generation LBTs are also discussed.

Nucleoside Phosphoramidites Containing Cleavable Linkers

Phosphoramidite reagents (linker phosphoramidites) containing a cleavable 3'-ester linkage between the nucleoside and the phosphoramidite group can be used to attach the first nucleoside to a solid-phase support. Inexpensive underivatized supports such as LCAA-CPG can then be used as universal supports for oligonucleotide synthesis. No modifications to synthesis coupling conditions and no 3'-dephosphorylation are required. Only oligonucleotides with terminal 3'-OH ends are produced. Phosphoramidites containing both a succinate and a sulfonyldiethanol linkage are particularly useful and create oligonucleotides with both a 3'-OH and 5'-phosphate. In addition, by using these reagents, one oligonucleotide sequence can be added onto the 5'-end of another (tandem synthesis) to produce a string of multiple oligonucleotides linked end-to-end. Deprotection releases the oligonucleotides from each other to yield a mixture of oligonucleotides. This approach is particularly useful for making pairs of PCR primers or both strands of a double-stranded sequence in a single operation.

Synthesis of 1-Alkyl-4-imidazolecarboxylates: A Catch and Release Strategy

The novel alkyl N-methyl-N-polystyreneamino-2-isocyanoacrylate was used for the synthesis of 1-substituted imidazole-4-carboxylates utilizing a "catch and release" methodology. The reactions were performed under microwave irradiation, affording the title compounds in both high yields and chemical purity directly to solution, from the solid phase support.

Evidence for the involvement of PECAM-1 in a receptor mediated signal-transduction pathway regulating capacitation-associated tyrosine phosphorylation in human spermatozoa

Mammalian spermatozoa must become ;capacitated' in the female reproductive tract before they gain the ability to fertilize the oocyte. The attainment of a capacitated state has been correlated with a number of biochemical changes, the most notable of which is a dramatic increase in the tyrosine phosphorylation status of these cells. Despite its biological importance, the mechanisms responsible for initiating this tyrosine phosphorylation cascade in vivo are unknown. Here, we report that this signalling pathway can be elicited in a rapid, dose-dependent and lectin-specific manner by wheat germ agglutinin (WGA), but none of 18 other lectins assessed. This response was abrogated by prior enzymatic cleavage of either sialic acid or GlcNAc residues from the sperm surface and by treatment with a range of pharmacological inhibitors directed against protein kinase A, protein tyrosine kinases and intermediates including Src. Proteomic analysis of the WGA-binding sites on the sperm surface identified the putative cognate receptor as platelet cell adhesion molecule 1 (PECAM-1/CD31). This conclusion was supported by the following evidence: (i) anti-PECAM-1 antibodies identified a molecule of the correct molecular mass in human spermatozoa, (ii) PECAM-1 could be isolated from a pool of sperm surface proteins using WGA immobilized on a solid phase support, (iii) PECAM-1 and WGA co-localized to the sperm surface and (iv) anti-PECAM-1 antibodies could completely block the ability of WGA to stimulate tyrosine phosphorylation in these cells. Collectively, these data provide the first evidence that a receptor-mediated signal transduction pathway triggers human sperm capacitation and identifies PECAM-1 as the probable initiator of this second messenger cascade.

Combinatorial Synthesis of MUC1 Glycopeptides: Polymer Blotting Facilitates Chemical and Enzymatic Synthesis of Highly Complicated Mucin Glycopeptides

The chemoselective polymer blotting method allows for rapid and efficient synthesis of glycopeptides based on a "catch and release" strategy between solid-phase and water-soluble polymer supports. We have developed a heterobifunctional linker sensitive to glutamic acid specific protease (BLase). The general procedure consists of five steps, namely (i) the solid-phase synthesis of glycopeptide containing BLase sensitive linker, (ii) subsequent deprotections and the release of the glycopeptide from the resin, (iii) chemoselective blotting of the glycopeptide intermediates in the presence of water-soluble polymers with oxylamino functional groups, (iv) sugar elongations using glycosyltransferases, and (v) the release of target glycopeptides from the polymer platform by selective BLase promoted hydrolysis. The combined use of the solid-phase chemical syntheses of peptides and the enzymatic syntheses of carbohydrates on water-soluble polymers would greatly contribute to the production of complicated glycopeptide libraries, thereby enhancing applicative research. We report here a high-throughput synthetic system for the various types of MUC1 glycopeptides exhibiting a variety of sugar moieties. It is our belief that this concept will become part of the entrenched repertoire for the synthesis of biologically important glycopeptides on the basis of glycosyltransferase reactions in automated and combinatorial syntheses.

Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes

Rapid, multiplexed, sensitive and specific molecular detection is of great demand in gene profiling, drug screening, clinical diagnostics and environmental analysis1,2,3. One of the major challenges in multiplexed analysis is to identify each specific reaction with a distinct label or 'code'4. Two encoding strategies are currently used: positional encoding, in which every potential reaction is preassigned a particular position on a solid-phase support such as a DNA microarray5,6,7,8, and reaction encoding, where every possible reaction is uniquely tagged with a code that is most often optical or particle based4,9,10,11,12,13. The micrometer size, polydispersity, complex fabrication process and nonbiocompatibility of current codes limit their usability1,4,12. Here we demonstrate the synthesis of dendrimer-like DNA-based, fluorescence-intensity-coded nanobarcodes, which contain a built-in code and a probe for molecular recognition. Their application to multiplexed detection of the DNA of several pathogens is first shown using fluorescence microscopy and dot blotting, and further demonstrated using flow cytometry that resulted in detection that was sensitive (attomole) and rapid.Supplementary informationThe online version of this article (doi:10.1038/nbt1106) contains supplementary material, which is available to authorized users.

Development of new procedures for the isolation of phytoplankton DNA from fixed samples

Phytoplankton samples collected for routine monitoring programmes have traditionally been preserved with fixatives before subsequent analytical procedures such as microscope-based identification, or simply to permit transport between laboratories. In recent years, to simplify identification and enumeration, the use of DNA or RNA probes coupled with the PCR assay has progressed and now represents a routine procedure for screening cultured and field samples. However, the phytoplankton cells have often still to be treated as fixed samples. The extraction of genomic DNA from fixed cultures of Alexandrium minutum cultures was compared using two new methods based on Magnetisable Solid Phase Support (MSPS) techniques with that using three commercial kits. Genomic DNA recovery and PCR amplification were observed and the results obtained from culture samples were validated using field samples. Among the DNA extraction techniques considered, the MSPS methods provided the best results.

Click to Fit: Versatile Polyvalent Display on a Peptidomimetic Scaffold

We describe an efficient protocol to effect multisite conjugation reactions to oligomers on solid-phase support. Sequence-specific N-substituted glycine "oligopeptoids" were utilized as substrates for azide-alkyne cycloaddition reactions. Diverse groups, including nucleobases and fluorophores, were conjugated at up to six positions on peptoid side chains with yields ranging from 88 to 96%. This strategy will be broadly applicable for generating polyvalent displays on peptides and other scaffolds, allowing precise control of spacing between the displayed groups.

Parallel synthesis and biological evaluation of different sizes of bicyclo[2,3]‐Leu‐enkephalin analogues

Parallel synthesis of peptides and peptidomimetics has been an important approach to search for biologically active ligands. A novel systematic synthesis of different size bicyclic dipeptide mimetics was developed on solid-phase supports. By taking advantage of the enantioselective synthesis of omega-unsaturated amino acids and their N-methylated derivatives, the hemiaminal problem was prevented in the pathway to thiazolidine formation. The bicyclic dipeptide was generated on the solid-phase support in three steps by an unconventional method. By inserting this bicyclic scaffold into the synthesis of a larger bioactive peptide, 11 different sizes of bicyclo[2,3]-Leu-enkephalin analogues were synthesized in a fast and efficient way. Modeling studies show that a reversed turn structure at positions 2-3 was favored when an L- and L-bicyclic scaffold was used, and that an extended conformation at the N-terminal was favored when a D- and L-bicyclic scaffold was inserted. Binding affinities and bioassay studies show ligands with micromolar binding affinities and antagonist bioactivities for the [6,5]- and [7,5]-bicyclo-Leu-enkephalin analogues.

Chemical Libraries Towards Protein Kinase Inhibitors

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis of Polymer‐bound Sensitizers and their Applications in the Preparation of Previtamin D3 from Tachysterol

AbstractTwo kinds of polymer‐bound photosensitizers were synthesized, in which anthracene acts as sensitizer moiety, polystyrene (PS) and poly(methyl methylacrylate) (PMMA) serve as solid phase supports, respectively. The photoisomerization of tachysterol to previtamin D3 was carried out with these polymer‐bound photosensitizers in ethanol and toluene solutions. The experiment results demonstrate that these solid phase photosensitizers are efficient for the photoisomerization with good recycle property and can be simply separated from the reaction mixtures by filtration.

B Cell Epitope Mapping Using Synthetic Peptides

Synthetic peptides are used for identification of functional B cell epitopes in antibody preparations. ELISA-type assays are used to identify sequences of proteins comprising antibody-binding regions. This unit describes three peptide display formats. Pepscan (pins) and SPOTs (cellulose membranes) may be used as solid-phase support media for peptide synthesis, followed by ELISA directly on the resulting peptide array. Alternatively, peptides may be cleaved from the array and tested in a standard microplate-based antibody capture ELISA format. The discussion includes choosing the peptide sequences by length and overlap, as well as determination of the minimum essential sequence for antibody binding. This method is highly effective for continuous epitopes and is often also useful for discontinuous epitopes.

Linker phosphoramidite reagents for the attachment of the first nucleoside to underivatized solid-phase supports.

New linker phosphoramidite reagents containing a cleavable 3'-ester linkage are used for attaching the first nucleoside to the surface of a solid- phase support. Inexpensive, underivatized amino supports, such as long chain alkylamine controlled-pore glass, can serve as universal supports. No modifications to phosphoramidite coupling conditions are required and, after synthesis, treatment with NH(4)OH releases the products with 3'-OH ends. No 3'-dephosphorylation is required. Phosphoramidite reagents containing a succinate and sulfonyl diethanol linkage between the nucleoside and phosphoramidite group are particularly advantageous and can be used to create both 3'-OH and 5'-phosphate ends on oligonucleotides. Reproducibility and quality of oligonucleotide synthesis is demonstrated for either column and 96-well plate formats on low-, medium- or high-loading CPG supports.

Convenient solid-phase synthesis of oligopeptides using pentacoordinated phosphoranes with amino acid residue as building blocks

The reactive intermediates of pentacoordinated phosphoranes with amino acids (P(5)-AA) as building blocks, which were obtained by the reaction of O-phenylene phosphorochloridate with N, O-bis(trimethylsilyl)amino acids, were linked to a solid-phase support containing a hydroxymethyl polystyrene functional group. The first amino acid residue was coupled to the solid-phase support after washing the resin with organic solvent. Repeating the procedure led to oligopeptides linked on the resin. A series of free oligopeptides including tetra-Gly, di-Val, tri-Val, di-Leu, di-Phe, and Phe–Leu were obtained after cleavage from solid-phase support. The structure of these oligopeptides were determined by IR, 1H NMR, FAB-MS, and HPLC.

Chemical libraries towards protein kinase inhibitors.

Over 500 human protein kinases identified to date are susceptible to play crucial roles in the regulation of many signal transduction pathways, making them significant drug discovery targets. However, their active sites share a high level of similarity, which constitutes a major challenge in the finding of selective and safe inhibitors. In order to meet this challenge, whether via traditional or alternative approaches, the use of chemical libraries to find either unknown natural ligands or specific inhibitors of particular kinases is more important than ever. This review briefly summarizes the recent literature on such libraries of peptides, natural product analogues, and small molecules. Significant chemical scaffolds, some synthetic routes particularly on solid-phase support, and computational tools employed for the efficient design of both selective and bioavailable inhibitors are highlighted.

Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures.

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.

Synthesis and application to asymmetric allylic amination of substituted monodonor diazaphospholidine ligands

The synthesis of a series of substituted monodonor diazaphospholidine ligands is described. A regioselective lithiation process is a key step in one of these syntheses. The compounds are designed to be incorporated into soluble polymer and other solid phase supports. Enantiomeric excesses of up to 88% were observed when these compounds were employed in palladium-catalysed asymmetric amination reactions.