Solid-phase Support Research Articles

A novel anthracenyl tagged protecting group for "phase-switching" applications in parallel synthesis.

A new "phase-switching" protecting group 1 that facilitates the parallel synthesis of carboxylic acids, esters, and carboxamides is described. Its use permits chemistries to be performed in solution, which may be conveniently monitored with conventional analytical techniques, followed by subsequent immobilization onto a solid-phase support to aid compound purification. Carboxylic acids, esters, and carboxamides are then cleaved from the solid support following activation of the "safety-catch" and treatment with the desired nucleophile.

Characterization and optimization of a chromatographic process based on ethylenediamine- N, N, N′, N′-tetra(methylphosphonic) acid-modified zirconia particles

The primary objective of work was to characterize, optimize and model a chromatographic process based on ethylenediamine- N, N, N′, N′-tetra(methylphosphonic) acid (EDTPA)-modified zirconia particles. Zirconia particles were produced by spray-drying colloidal zirconia. Zirconia spheres produced were further classified, calcined and modified with EDTPA to yield a solid-phase support for use in bio-chromatography (r_PEZ). Specifically, the ability of r_PEZ to selectively bind and enrich IgG, IgA, and IgM from biological fluids was evaluated and demonstrated. To better understand the force of interaction between the IgG and the r_PEZ, the equilibrium disassociation constant ( K d) was determined by static binding isotherms, as a function of temperature and by frontal analysis at different linear velocities. The maximum static binding capacity ( Q max) was found to be in the range 55–65 mg IgG per ml of beads, and unaffected by temperature. The maximum dynamic binding capacity ( Q x ) was found to be in the range 20–12 mg IgG per ml of beads. The adsorption rate constant ( k a) was determined by a split-peak approach to be between 982 and 3242 l mol −1 s −1 depending on the linear velocity. The standard enthalpy and entropy values were estimated for this interaction of IgG with this novel support.

Two-photon microscopy to spatially resolve and quantify fluorophores in single-bead chemistry.

Solid-phase chemistry on polymer beads is widely used in the production and biological screening of combinatorial libraries of compounds.1 Since a lot of analytical methods used in solution chemistry cannot be applied to solid-phase chemistry, monitoring chemical processes on beads has been difficult. To understand solid-phase chemistry, it is desirable to describe the distribution of chemical groups within the solid-phase supports. Previous studies have described the use of autoradiography,2 scanning secondary ion mass spectrometry,3 fluorescence,4 and several microscopy methods, such as confocal fluorescence,5 confocal Raman,6,5d infrared,7 and more recently, two-photon microscopy (TPM).8 However, the spatial quantification of the chemical groups present has not been described before. Here, we show that TPM allows for the direct quantification of fluorophore distributions within single PEGA1900 (poly(ethylene glycol)acrylamide) beads. In TPM,9 the sample is irradiated with a laser with a wavelength approximately twice that of the normal excitation wavelength of the fluorophore. As a result, excitation can occur only when two photons are absorbed simultaneously. Such two-photon events occur at a very high photon density that is reached only at the focal point of the laser beam. Hence, the fluorescence detected originates only in the part of the sample that is in focus. Away from the focal point, there is essentially no absorption of the exciting beam, because there is no chromophore able to absorb single photons of this wavelength. Hence, TPM avoids the artifacts caused by excitation attenuation due to absorption that have been described for confocal fluorescence microscopy.7 In addition, the risk of photodamage to the sample is reduced by application of a pulsed laser permitting recovery of the fluorophore.

Frequent coinfection with hepatitis B virus strains of distinct genotypes detected by hybridization with type-specific probes immobilized on a solid-phase support

A genotype-specific probes assay (GSPA) was developed for distinguishing the seven genotypes (A–G) of hepatitis B virus (HBV). Nucleotide (nt) sequences corresponding to preS1 region were amplified by PCR with a primer labeled with biotin, and delivered to eight wells on which complementary sequences specific to one or other genotype had been immobilized. Thereafter, hybridization of HBV DNA sequences amplified from the test serum was detected by colorimetry. When 256 sera from HBV carriers in Bangladesh, Cameroon, Japan, South Africa, USA and Uzbekistan were subjected to GSPA, genotypes were concordant with those of ELISA with monoclonal antibodies to epitopes on preS2-region products in 242 (94.6%) of them; 8 sera (3.1%) were not genotypeable by either method. Cloning analysis confirmed the presence of two distinct HBV genotypes in the seven selected sera with coinfection. There were 7 (2.7%) sera with discordant genotyping results between GSPA and ELISA. When HBV DNA clones propagated from these sera were sequenced and analyzed phylogenetically, the genotypes determined by GSPA were verified. Coinfection with HBV strains of two distinct genotypes was identified by GSPA in 28 (10.9%) sera, while it was suggested by ELISA in only 2 (0.8%) sera. The GSPA method would be particularly useful for detecting the coinfection with distinct HBV genotypes of any clinical relevance, which seems to be more frequent than reported previously.

Heterogeneous post-column immunoreaction detection using magnetized beads and a laboratory-constructed electromagnetic separator.

The nature of immune reactors allows development of quantitative analytical methods that are highly selective and can often be used directly with complex biological matrixes such as blood, plasma or urine. A major limitation of immunoassay is that antibodies are sometimes unable to discriminate structurally similar species such as drug metabolites and synthetic analogs. The problem associated with the lack of discrimination can be circumvented by coupling immunoassay with liquid chromatography post-column. The most commonly used separation method in post-column immunoreaction detection is the affinity column. Affinity columns may create undesired effects such as a compromise of the chromatographic separation efficiency, the requirement for an antibody with fast reaction kinetics and the need for flushing the column. This paper reports a post-column immunoreaction detection system coupled with a laboratory-constructed on-line magnetic separation flow chamber that is designed to overcome these problems. The system uses disposable magnetic beads as a solid-phase support for separation that can be easily removed from the system. The model analytes chosen for this study were digoxin and its metabolites due to the commercial availability of monoclonal antibodies for these compounds. Digoxin was separated using a chromatographic method prior to being interfaced through a liquid handler system to the immunoreactor. Compatibility of the HPLC mobile phase was determined to be acceptable with a mixing ratio of 1:3 between the LC fraction and immunoreagent solution. The dynamic range of the calibration curve in digoxin-spiked phosphate buffer was found to be 0.25-12 ng/ml and a quadratic fit was found to provide the best fit to the data with a correlation coefficient of 0.9974. The residual error for all standards was less than 15%. The percentage RSDs for the two controls, 2 and 10 ng/ml, were 6.88 and 4.82% (n = 6) and the percentage errors were 7.07 and -6.89% (n = 6), respectively.

Sequential Column Asymmetric Catalysis

AbstractFor Abstract see ChemInform Abstract in Full Text.

The Importance of Thermodynamic Equilibrium for High Throughput Gene Expression Arrays

We present an analysis of physical chemical constraints on the accuracy of DNA micro-arrays under equilibrium and nonequilibrium conditions. At the beginning of the article we describe an algorithm for choosing a probe set with high specificity for targeted genes under equilibrium conditions. The algorithm as well as existing methods is used to select probes from the full Saccharomyces cerevisiae genome, and these probe sets, along with a randomly selected set, are used to simulate array experiments and identify sources of error. Inasmuch as specificity and sensitivity are maximum at thermodynamic equilibrium, we are particularly interested in the factors that affect the approach to equilibrium. These are analyzed later in the article, where we develop and apply a rapidly executable method to simulate the kinetics of hybridization on a solid phase support. Although the difference between solution phase and solid phase hybridization is of little consequence for specificity and sensitivity when equilibrium is achieved, the kinetics of hybridization has a pronounced effect on both. We first use the model to estimate the effects of diffusion, crosshybridization, relaxation time, and target concentration on the hybridization kinetics, and then investigate the effects of the most important kinetic parameters on specificity. We find even when using probe sets that have high specificity at equilibrium that substantial crosshybridization is present under nonequilibrium conditions. Although those complexes that differ from perfect complementarity by more than a single base do not contribute to sources of error at equilibrium, they slow the approach to equilibrium dramatically and confound interpretation of the data when they dissociate on a time scale comparable to the time of the experiment. For the best probe set, our simulation shows that steady-state behavior is obtained in a relaxation time of ∼12–15 h for experimental target concentrations ∼(10 −13 - 10 −14) M, but the time is greater for lower target concentrations in the range (10 −15–10 −16) M. The result points to an asymmetry in the accuracy with which up- and downregulated genes are identified.

Sequential column asymmetric catalysis.

Since the introduction of catalysts and reagents on solid-support, researchers have developed new reaction systems to take advantage of their insoluble nature by designing multistep reaction sequences, high-throughput purification techniques, and combinatorial synthesis methods. The continuous flow system is one of these advancements and represents the foundation of a new technique termed sequential column asymmetric catalysis (CAC). In this strategy, reagents and catalysts are attached to a solid-phase support and loaded onto sequentially-linked columns. The substrates are present in the liquid phase that flows through the column. As a substrate encounters each successive column, it grows in complexity. Consequently, one can imagine a number of flow systems that consist of columns attached in series and/or in parallel that synthesize a fairly complex molecule. Herein, we discuss the development of the sequential CAC technique, beginning with the most relevant antecedents.

Biomimetic synthesis and optimization of cyclic peptide antibiotics.

Molecules in nature are often brought to a bioactive conformation by ring formation (macrocyclization). A recurrent theme in the enzymatic synthesis of macrocyclic compounds by non-ribosomal and polyketide synthetases is the tethering of activated linear intermediates through thioester linkages to carrier proteins, in a natural analogy to solid-phase synthesis. A terminal thioesterase domain of the synthetase catalyses release from the tether and cyclization. Here we show that an isolated thioesterase can catalyse the cyclization of linear peptides immobilized on a solid-phase support modified with a biomimetic linker, offering the possibility of merging natural-product biosynthesis with combinatorial solid-phase chemistry. Starting from the cyclic decapeptide antibiotic tyrocidine A, this chemoenzymatic approach allows us to diversify the linear peptide both to probe the enzymology of the macrocyclizing enzyme, TycC thioesterase, and to create a library of cyclic peptide antibiotic products. We have used this method to reveal natural-product analogues of potential therapeutic utility; these compounds have an increased preference for bacterial over eukaryotic membranes and an improved spectrum of activity against some common bacterial pathogens.

An accurate method for the quantitation of Fmoc-derivatized solid phase supports

By performing the Fmoc resin loading determination with DBU instead of piperidine, highly reproducible results were obtained that showed excellent correlation with data obtained by independent analytical methods.

Fast [18F]FDG synthesis by alkaline hydrolysis on a low polarity solid phase support

AbstractThe synthesis of 2‐deoxy‐2‐[18F]fluoro‐D‐glucose ([18F]FDG) has been simplified by the use of a tC18 Sep Pak cartridge to effect purification and hydrolysis of the tetraacetylated [18F]fluoro‐glucose compound ([18F]TAG). After radiolabelling, this derivative was trapped on a solid phase extraction (SPE) cartridge and the residual reaction solvent (CH3CN), reagents (K222, K2CO3,…) and by‐products removed by washing the support with water. After this cleaning step, the acetyl groups were cleaved on the same tC18 column using 2N sodium hydroxide. This fast reaction proceeded near quantitatively (>98%) at room temperature in less than 2 min. The [18F]FDG was then recovered with a small amount of water, neutralized with a slight excess of 2N hydrochloric acid, buffered for pH with a citrate solution and finally purified on a neutral alumina oxide and a second tC18 column. After filtration, the radiochemical yield of this [18F]FDG isotonic solution after more than 100 production runs was found to be very reliable and reproducible (70±6% decay corrected). The synthesis time was about 22 min. Quality controls showed that the radiochemical purity was higher than 98% and in any case no [18F]FDM was detected. Only traces of 2‐chloro‐2‐deoxy‐glucose (ClDG) were found in the final sample (64±9 μg/ batch of 16 ml). [18F]FDG specific activity averaged between 1 and 20 Ci/µmol (EOS). No evaporation and use of ion retardation resin (AG11A8) are required. Moreover, this new approach is suitable for complete remote operation using available single use medical components. Copyright © 2002 John Wiley & Sons, Ltd.

Tandem oligonucleotide synthesis on solid-phase supports for the production of multiple oligonucleotides.

More than one oligonucleotide can be synthesized at a time by linking multiple oligonucleotides end-to-end in a tandem manner on the surface of a solid-phase support. The 5'-terminal hydroxyl position of one oligonucleotide serves as the starting point for the next oligonucleotide synthesis. The two oligonucleotides are linked via a cleavable 3'-O-hydroquinone-O,O'-diacetic acid linker arm (Q-linker). The Q-linker is rapidly and efficiently coupled to the 5'-OH position of immobilized oligonucleotides using HATU, HBTU, or HCTU in the presence of 1 equiv of DMAP. This protocol avoids introduction of phosphate linkages on either the 3'- or 5'-end of oligonucleotides. A single NH(4)OH cleavage step can simultaneously release the products from the surface of the support and each other to produce free 5'- and 3'-hydroxyl termini. Selective cleavage of one oligonucleotide out of two sequences has also been accomplished via a combination of succinyl and Q-linker linker arms. Tandem synthesis of multiple oligonucleotides is useful for producing sets of primers for PCR, DNA sequencing, and other diagnostic applications as well as double-stranded oligonucleotides. Tandem synthesis of the same sequence multiple times increases the yield of material from any single synthesis column for maximum economy in large-scale synthesis. This method can also be combined with reusable solid-phase supports to further reduce the cost of oligonucleotide production.

Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies.

The intracellular polymerization of cytoskeletal proteins into their supramolecular assemblies raises many questions regarding the regulatory patterns that control this process. Binding experiments using the ELISA solid phase system, together with protein assembly assays and electron microscopical studies provided clues on the protein-protein associations in the polymerization of tubulin and actin networks. In vitro reconstitution experiments of these cytoskeletal filaments using purified tau, tubulin, and actin proteins were carried out. Tau protein association with tubulin immobilized in a solid phase support system was inhibited by actin monomer, and a higher inhibition was attained in the presence of preassembled actin filaments. Conversely, tubulin and assembled microtubules strongly inhibited tau interaction with actin in the solid phase system. Actin filaments decreased the extent of in vitro tau-induced tubulin assembly. Studies on the morphological aspects of microtubules and actin filaments coexisting in vitro, revealed the association between both cytoskeletal filaments, and in some cases, the presence of fine filamentous structures bridging these polymers. Immunogold studies showed the association of tau along polymerized microtubules and actin filaments, even though a preferential localization of labeled tau with microtubules was revealed. The studies provide further evidence for the involvement of tau protein in modulating the interactions of microtubules and actin polymers in the organization of the cytsokeletal network.

Linker phosphoramidite reagents for oligonucleotide synthesis on underivatized solid-phase supports

Linker phosphoramidite reagents containing a protected nucleoside with a cleavable 3′-ester linkage to either succinic acid, diglycolic acid, or hydroquinone- O, O′-diacetic acid ( Q-Linker) allow the 3′-terminal nucleoside of an oligonucleotide sequence to be attached to underivatized ‘Universal’ amino or hydroxyl supports. After solid-phase oligonucleotide synthesis, treatment with NH 4OH cleaves the 3′-ester link to the support and produces oligonucleotides with only 3′-OH ends. No changes to existing phosphoramidite synthesis cycles, cleavage, or deprotection methods are required.

Reusable solid-phase supports for oligonucleotides and antisense therapeuticsElectronic supplementary information (ESI) available: CGE data for octathymidine phosphodiester sequences on supports P1–5, P7, P9, and P11–13 PAGE data for phosphorothioate products, dimethoxytrityl analysis and UV measurements on supports P1–7, P9 and P15, step listing for ABI394 DNA synthesizer and CGE and HPLC data for phosporothioate syntheses on support P3. See http://www.rsc.org/suppdata/p1/b1/b105089n/

A general method for oligonucleotide synthesis on reusable solid-phase supports has been developed which will significantly lower the cost of large-scale synthesis. It consists of five steps: 1, nucleoside attachment to an hydroxy derivatized support through a Q-linker (hydroquinone-O,O′-diacetic acid) linker arm; 2, chloro-acetylation of unreacted surface groups; 3, conventional phosphodiester or phosphorothioate oligonucleotide synthesis; 4, cleavage from the support with aqueous or anhydrous ammonia; and 5, support regeneration with methanolic potassium carbonate. The recycling process is fast, fully automatable, and does not require removal of the support from the synthesis column. Fifteen solid-phase supports were evaluated with glycerol-CPG providing the best results. Consecutive syntheses of ISIS 2302, a dGCCCAAGCTGGCATCCGTCA phosphorothioate sequence, on the same synthesis column were performed. A glycerol-CPG synthesis column was satisfactorily used six consecutive times when NH4OH was the cleavage reagent. However, anhydrous NH3 allowed twelve consecutive syntheses without any deterioration in support loading, product quality, or amount of product produced. An improved method for preparing the essential nucleoside-3′-hemiesters of the Q-Linker and an unexpectedly slower rate of cleavage for phosphorothioate DNA vs. phosphodiester DNA are also described.

A convenient and rapid method for the selective oxygen-17 enrichment of aspartyl peptides during solid-phase synthesis

In this work we describe, for the first time, a rapid and efficient method for 17O selective labeling on the β-carboxyl group of an aspartic acid residue already incorporated into a peptide sequence anchored on a solid-phase support. The β-O-benzyl ester of the Asp residue of the Ac-RGD-benzydrylamine resin was successfully saponified using Na17OH in a methanol/dichloromethane mixture. The 17O selective enriched peptide was then released from the solid support by acidic cleavage. The 17O NMR spectrum confirmed the 17O labeling of the Asp β-carboxylate.

NEW ALTERNATIVE TO BEAD SYNTHESIS

RESEARCHERS AT THE UNIVERSITY of Pittsburgh have developed a convenient new way to separate products from reaction mixtures produced in solution-phase organic synthesis. The method, in which reactants are drawn into and precipitated out of solution by attached auxiliary groups called precipitons, was devised to permit greater use of solution-phase synthesis as an alternative to solid-phase organic synthesis (SPOS). In SPOS, reactants are attached to solid-phase support beads to ease recovery of reaction products created from those reactants in subsequent chemical reactions. The SPOS approach is popular because an excess of reagents can be used to increase product yields and products can be isolated by simple filtration. But it has significant drawbacks—SPOS reactions are often slower than corresponding homogeneous solution reactions, development and optimization of reagent immobilization procedures can be time-consuming, and beads can interfere with reactions and reaction monitoring. As a result of thes...

Patient-tailored cloning of allergens by phage display: Peanut ( Arachis hypogaea) profilin, a food allergen derived from a rare mRNA

A peanut cDNA phage surface display library was constructed and screened for the presence of IgE-binding proteins. We used a serum from a peanut-sensitized individual with a low specific IgE level to peanut extract and suffering from mild symptoms after peanut ingestion. A total of 10 11 cDNA clones were screened by affinity selection towards serum IgE immobilized to solid-phase supports. After five rounds of selective enrichment, sequence determination of 25 inserts derived from different clones revealed presence of a single cDNA species. The cDNA-encoded gene product, formally termed Ara h 5, shows up to 80% amino acid sequence identity to the well-known plant allergen profilin, a 14 kD protein present only in low amount in peanut extracts. Immunoblot analysis of fifty sera from individuals sensitized to peanut showed that 16% had mounted a detectable IgE response to the newly identified peanut profilin. High-level expression as non-fusion protein in BL21 (DE3) was carried under control of the inducible T7 promoter. Peanut profilin was purified by affinity chromatography on poly-( l-proline)-Sepharose and yielded 30 mg l −1 culture of highly pure recombinant allergen. In spite of the high level of up to 80% amino acid identity to other plant profilins, inhibition experiments with recombinant profilins of peanut, cherry, pear, celery and birch revealed marked differences regarding their IgE-binding capacity.

(Immuno)affinity chromatography: a versatile tool for fast and selective purification, concentration, isolation and analysis.

Today, thanks to the availability of tailor made biomolecules with the desired biological functions, separations based upon (immuno)affinity techniques are more and more common in a large field of applications. By using the high selectivity of biomolecules (antibodies, receptors, specific proteins), this technique offers the possibility of isolating compounds from complex samples with a selectivity which cannot be achieved by other chromatographic methods. In order to succeed, however, the solid phase support for the immobilisation of the ligand of interest plays a prominent role. For this reason, numerous supports have already been introduced while research on new materials with additional advantages is continued. Here, a new solid phase support will be discussed for (immuno)affinity applications. This material demonstrates low non-specific adsorption and high ligand accessibility, which enables an enhanced selectivity and capacity. Because the material is available in large quantities and exhibits superb mechanical and physical stability, selective isolations have been performed on analytical as well as preparative scale. To demonstrate the potential of this new support, several applications will be presented. Based upon immunoaffinity, two applications for the determination of oestradiol in serum respectively vitamin B12 in fermentation broth will be presented. Regarding affinity chromatography, an enzyme reactor in which the enzyme glucose oxidase is immobilised on the new material, is made for the detection of glucose by Flow Injection Analysis and electrochemical detection. Next, to isolate, identify and test components on their xeno-oestrogenic activity, an affinity column is produced in which human oestrogen receptor is covalently coupled. Several components are screened on their biological activity and the results obtained will be presented here.

Immunological evidence for FGLamide‐ and W2W9‐allatostatins in the ovary of Gryllus bimaculatus (Ensifera, Gryllidae)

SummaryUsing polyclonal antisera to Gryllus bimaculatus allatostatin A1 (FGLamide) and B1 (W2W9‐amide), two sensitive and specific competitive enzyme‐linked immunosorbent assays (ELISAs) were developed, which allowed detection of A‐ and B‐allatostatin‐like immunoreactivity in extracts of ovaries and partially purified chromatography (HPLC) fractions. Immuno‐positive HPLC fractions inhibited biosynthesis of Juvenile Hormone in vitro in the rapid partition assay. Both antisera were also used to immunolocalize A‐ and B‐allatostatin epitopes in the ovary of larval and adult crickets. A‐ and B‐allatostatin immunoreactivity appeared in the cortical cytoplasm of the oocytes at the anterior cell poles. Controls treated with the pre‐immune sera or with antisera preadsorbed onto the respective peptides coupled to a solid phase support did not stain. There was no staining in any neural structures within the ovary.