Traceless synthesis of benzimidazoles on solid support

Traceless solid-phase syntheses of benzimidazoles and 5-(benzimidazol-2-yl)benzimidazoles on 2-(4-formyl-3-methoxyphenoxy)ethyl polystyrene are described. No auxiliary functional groups are left in the products after ultimate cleavage and cyclization.     

Reagents for the selective immobilization of oligonucleotides on solid supports

New reagents (CPGs and phosphoramidites) for automatic solid phase synthesis of modified oligonucleotides were designed. Three oligonucleotides carrying fluorescent label at the 5'-terminus and an anchor group at the 3'-terminus were prepared and their immobilization in orthogonal conditions on solid supports was studied.     

Quantitative techniques for the comparison of solid supports

The host of solid supports available to the synthetic chemist adds yet another level of complexity to solid-phase synthesis. Although the selection of the optimal solid support for a specific synthetic transformation is still empirically driven, significant progress has been made in the development of quantitative techniques to compare solid supports, providing new insight into the microenvironment created by the interaction of the solid support with solvent.     

Chemistry on solid supports: defining events and titers by use of cleavable, assayable linking molecules

The cleavable diamines cystamine, 5, and 1,6-diamino-3,4-dihydroxyhexane, 1, were bonded to solid supports and, with a simple, newly developed dinitrofluorobenzene-based assay, were used to define (a) titers of ligands and (b) chemistry distal to the support. Compound 1, which is cleavable with periodate, becomes a linking molecule which is stable to almost all conditions encountered in biochemistry and enjoys considerable hydrophilic character. Compound 5, which is cleavable with dithiothreitol, can be usefully applied to those systems which do not require reducing agents. These nucleophilic linking moieties were converted to cleavable electrophilic linkers by succinylation and p-nitrophenyl ester activation. The first preparation of a polysaccharide-linked support is described. The method also allows the chemical definition of ligands containing amino groups which are prepared by deblocking of protecting groups while on the support. The methodology should promote greater understanding of affinity chromatography materials and processes.     

Synthesis of hydrophilic and flexible linkers for peptide derivatization in solid phase

Four N-Fmoc protected polyoxyethylene-based amino acid type linkers were designed and synthesized for peptide derivatization in solid phase. Three of them were obtained in a crystalline form. The crystallized linkers can be stored at 4 degrees C for 2 years without significant decomposition. Protocols for biotinylation and fluorescent labeling of peptides in solid phase were developed. The linkers also provide good ionization ability for single-bead mass spectrometry analysis of peptides.     

Novel alpha-hydroxyethyl-polystyrene, alpha-chloroethyl-polystyrene and alpha-amino-oxyethyl-polystyrene linkers on the Multipin solid support for solid-phase organic synthesis

A simple method for the generation of three novel linkers, alpha-hydroxyethyl-polystyrene, alpha-chloroethyl-polystyrene and alpha-amino-oxyethyl-polystyrene on Multipin supports (SynPhase Crowns) has been developed. Applications of these linkers have been successfully demonstrated for solid-phase synthesis of dipeptide, oxime, and hydroxamic acid compounds in good yields and purities.     

Oxidiazole synthesis on solid supports.

Substituted 1,2,4-oxadiazoles were synthesized in good yields on solid supports under basic conditions at room temperature.     

Immobilization of plant protoplasts on solid supports

Brassica napus L. and Rosa sp. protoplasts were immobilized on variously treated Biosilon, Cytodex 1, 2 and 3, Separon, Sephadex, Sepharose and Sorfix micro carriers. Best anchorage of protoplasts of both species was obtained with Biosilon and Cytodex 3 coated with polylysine of high molecular weight (55 and 450 kDa), as well as with concanavalin A-coated Cytodex 1. Precoating of micro carriers with bovine serum albumin was generally without effect.     

Site-directed immobilization of glycoproteins on hydrazide-containing solid supports

Methods are described for the preparation and use of solid supports containing hydrazide functions for the immobilization of glycoproteins specifically through the oligosaccharide moieties. The solid supports are prepared from commercial "active ester" agarose by reaction with hydrazine hydrate. Glycoproteins are oxidized with sodium periodate, resulting in the production of aldehydes on the oligosaccharide moieties. Oxidized glycoprotein is then reacted with the hydrazide-derivatized solid support to produce stable hydrazone linkages. Data are presented for the optimization of binding of oxidized glycoprotein to hydrazide-derivatized agarose. Agarose hydrazide/glycoprotein gels were shown to be stable from pH 3 to 10 and activity studies using immobilized avidin show that this method of immobilization results in an increased "specific activity" of bound protein when compared with standard methods of immobilization.     

Build-up of symbiotic methanogenic biofilms on solid supports

Support surfaces can have selective action which determines the relative quantities of acetogens (propionate and butyrate degraders) and methanogens (acetate degraders) immobilized in a symbiotic biofilm. The preference of the bacteria for hydrophilic substrata as their immobilization supports is in the order; butyrate degraders, acetate degraders followed by propionate degraders.     

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.     

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

Summary 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.     

Examining the influence of linkers and tertiary structure in the forced unfolding of multiple-repeat spectrin molecules

The unfolding pathways of multiple-repeat spectrin molecules were examined using steered molecular dynamics (SMD) simulations to forcibly unfold double- and triple-repeat spectrin molecules. Although SMD has previously been used to study other repeating-domain proteins, spectrin offers a unique challenge in that the linker connecting repeat units has a definite secondary structure, that of an alpha-helix. Therefore, the boundary conditions imposed on a double- or triple-repeat spectrin must be carefully considered if any relationship to the real system is to be deduced. This was accomplished by imposing additional forces on the system which ensure that the terminal alpha-helices behave as if there were no free noncontiguous helical ends. The results of the SMD simulations highlight the importance of the rupture of the alpha-helical linker on the subsequent unfolding events. Rupture of the linker propagates unfolding in the adjacent repeat units by destabilizing the tertiary structure, ultimately resulting in complete unfolding of the affected repeat unit. Two dominant classes of unfolding pathways are observed after the initial rupture of a linker which involve either rupture of another linker (possibly adjacent) or rupture of the basic tertiary structure of a repeat unit. The relationship between the force response observed on simulation timescales and those of experiment or physiological conditions is also discussed.     

Improved activity retention of enzymes deposited on solid supports

Enzymes deposited on solid support usually show good stability when operated in organic solvents. Decreased stability of the enzyme preparations was noticed when low enzyme loadings were used (e.g., with Celite as support; less than 1 mg enzyme/g). It was possible to avoid the activity loss by the addition of an additive which protects the enzyme during the immobilization. Proteins (such as albumin, gelatin, and casein) and poly(ethylene glycol) were effective additives whereas amino acids, monomeric carbohydrates, and polysaccharides had no effect. The amount of additive needed for stabilization was shown to depend on the structure of the support, more additive being required for a support with high porosity. The stabilizing effect was investigated in a series of glyceryl-controlled-pore glass (CPG) with varying specific surface areas (9.5-180 m(2)/g). The minimum addition of albumin, giving full stabilization, on the different supports correlated to a monolayer coverage of the surface, approximately 2-3 mg protein/m(2). The effect of the additive was less pronounced when increasing amounts of enzyme were immobilized (5-40 mg enzyme/g Celite). The effect of the additives was studied using mandelonitrile lyase, but alpha-chymotrypsin and lipase P were also shown to be stabilized. (c) 1993 John Wiley & Sons, Inc.     

Microwaves synthesis of solid supports for the synthesis of 3'-aminoalkyl oligodeoxynucleotides

Phthalimido-alkyl alcohol solid supports were rapidly prepared from solid supported phthalic anhydride and amino alcohol condensation induced by microwaves. These supports were used to synthesize 13-aminoalkyl oligodeoxynucleotides allowing a two step deprotection necessary to avoid aminolink alkylation.     

A new generation of cross‐linkers for selective detection by MALDI MS

We designed a new cross‐linker bearing a CHCA moiety. The use of the CHCA‐tagged cross‐linker JMV 3378 in conjunction with a neutral MALDI matrix α‐cyano‐4‐hydroxycinnamic methyl ester enabled specific signal enhancement in MALDI‐TOF MS of cross‐link containing peptides. Discrimination between modified and non‐modified peptides can be achieved by comparison of two spectra, one using CHCA and the other using the α‐cyano‐4‐hydroxycinnamic methyl ester matrix. The methodology was validated using cytochrome c and apo‐myoglobine as model proteins.