Epoxy activation

In a preliminary report, Ross et al. [40] used affinity chromatography to identify a putative bovine renal brush border Na /H exchanger. Brush border membranes were solubilized with Triton X-100 and chromatographed sequentially over lentil lectin Sepharose 4B and 5-(A-benzyl-iV-ethyl)amiloride coupled to epoxy-activated Sepharose 6B. The eluant contained 178- and 146-kDa proteins that were susceptible to Endo-F. Moreover, the eluants reacted on dot blot immunoassays with antisera to a 20-amino acid peptide of a human Na /H exchanger vide infra). The relationship between these proteins and the 66-kDa protein previously identified by the same investigators using amiloride photolabeling is presently unclear. 

Dilute the reaction mixture 10-fold with 50 percent methanol/water and purify the epoxy-activated dendrimer by repeated ultrafiltration using a membrane with a molecular... 

The epoxy-activated dendrimer may be conjugated to thiol-containing proteins by reaction in 50 mM sodium phosphate, pH 7.2. The reaction can be done at 4°C or at room temperature for 8-16 hours to form thioether linkages. 

Epoxy-activated bead containing extended spacer artn... 

Epoxy activation of hydroxylic polymers is commonly used as a means to immobilize molecules on solid phase chromatographic supports that contain hydroxyl groups (Sundberg and... 

Epoxy acrylates, 10 382-383 in varnish formulations, 10 442 Epoxy activation, in microarray fabrication, 16 385... 

Another factor that remarkably affects the enantioresolution of given enantiomeric pairs has been shown to be the binding chemistry used for the silica immobilization of glycopeptides (see Section 2.2). This was illustrated in the case of ristocetin A, which was covalently bonded to silica microparticles by immobilization onto epoxy-activated silica under mild conditions [22], and compared with the corresponding commercially available CSP, where the macrocycle was immobilized as previously reported for vancomycin, rifamycin B, thiostrepton [7], and teicoplanin [30]. The comparison proved that immobilization of ristocetin A onto epoxy-activated silica could significantly improve the enantioselectivity and the resolution of the corresponding CSP in the separation of a-amino acids under RP conditions [22]. 

Epoxy-activated gels are especially used for immobilization of carbohydrates. 

The structure of this gel is shown m Figure 3.19E. It provides for the attachment of ligands containing hydroxyl, thiol, or amino groups. The hydroxyl groups of mono-, oligo-, and polysaccharides can readily be attached to the gel. Epoxy-activated Sepharose 6B is available from Pharmacia. 

Epichlorohydrin (l-chloro-2,3-epoxypropane). Widely available. Used to epoxy activate the Sepharose CL-6B beads or other surfaces. The extent of epoxy activation of beads may be determined see Note 2). A high-purity (+99%) or equivalent should be used. It is a very unstable compound and must be stored at 0-4°C in an anhydrous environment. Hazards Flammable, poison, toxic by inhalation or contact with skin, and if swallowed may cause cancer. Toxicity data LD50 90 mg/kg oral, rat. Should be handled in a fume hood with safety glasses and gloves, and treated as a possible cancer hazard. 

Ammonia aqueous solution (35% v/v). Widely available chemical. Used to introduce free amino groups in the epoxy-activated beads, which can be quantified by the 2,4,6-trinitrobenzenesulphonic acid (TNBS) test (see Note 3). Hazards Poison,... 

Amination of agarose beads. The washed epoxy-activated gel is suspended in 1 mL of distilled water/g of gel in a 1-L conical flask. About 1.5 mL of ammonia/ g of gel are added and the gel incubated for 12 h at 30°C in a rotary shaker. Alternatively, 1,6-diaminohexanediamine (5 eq.) can be used to aminate the gel, yielding resins with a six-carbon spacer arm. The aminated gel is washed with 40 mL of distilled water/g of gel on a sinter funnel, and stored in 20% v/v ethanol at 0-4°C. The extent of amination is determined as described in Note 3. Aminated beads can also be purchased from Amersham Biosciences. 

Extent of epoxyactivation of agarose beads. Sodium thiosulphate (1.3 M, 3 mL) is added to 1 g of epoxy-activated gel and incubated at room temperature for 20 min. This mixture is neutralized with 0.1 MHC1 and the amount of HO used is recorded. The volume of 0.1 MHO added corresponds to the number of OH moles released (10 pmol for each 100 pL added), which equals the pmol of epoxy groups/g gel. Therefore, the extent of epoxy activation is expressed as pL HC1 used/10 pmol/g gel. The protocol usually results in 25 pmol epoxy groups/g moist weight gel. 

In 1995 Schwarz et al.159 described novel heterocyclic structures able to adsorb selectively immunoglobulins. The structures contained sulfur and nitrogen. Reported ligands were 2-mercapto-pyridine,2-mercapto-pyrimidine, and mercapto-thiazoline. These structures were chemically immobilized on silica and agarose beads using epoxy-activated matrices. Binding capacities for silica based material were about 25 mg of IgG per milliliter of resin, while for agarose beads it was about 18 mg/mL. 

Cellulose can be modified with organostannane chlorides, such as dibutyl or triphenyl derivatives [91,92], or with organotin halides in the presence of bisethylenediamine copper(II) hydroxide [93]. Epoxy-activated cellulose was prepared by reacting cellulose acetate fibers with sodium methoxide, followed by reacting it with epichlorohydrin in DMSO. This epoxy-activated cellulose has proved to be a useful intermediate to react with substances containing active hydrogen, such as amine, amino acid, or carboxylic acids [94], as shown in Fig. 3. Epoxidized cellulose has also been converted to a thiol derivative via reduction of a thiosulfate intermediate [95], and sulfoethylcellu-[ose has been obtained from sodium chloroethanesulfonate [96]. Cellulose... 

Figure 3 Reaction of epoxy-activated cellulose. R denotes cellulose.

When Candida rugosa lipase (formerly named Candida cylindracea lipase) was immobilized on an epoxy-activated resin it became resistant against acetaldehyde. Due to this immunization it could be repeatedly employed for the enantioselective acylation of secondary alcohols with vinyl acetate in dry organic solvents (Scheme 2.7) [78]. 

In a comparative study CALB was immobUized on epoxy-activated Sepabeads and amino Sepabeads with long and short spacers (glutaraldehyde was used as the coupling reagent) (see Figure 2.8). Lyophilized CALB and Novozym 435 were also included in the test The specific activity (U/g dry immobihzed CALB) of Novozym 435 was much lower than for CALB immobihzed on the different Sepabeads. In a thermal stabihty screening, Novozym 435 and CALB immobihzed on amino Sepabeads with short spacers displayed equal stabihty, while ah the other CALB preparations were less stable [32]. 

Scheme 2.7 Immobilization on an epoxy-activated resin protects C. rugosa lipase against acetaldehyde-induced deactivation.

Two epoxy-activated related supports, Sepabeads EC-EP3 and EC-EP5 (Figure 11.3), were assayed for the immobilization of A. aculeatus fructosyltransferase. By combination of nitrogen isotherms and mercury porosimetry analyses, the textural properties of both carriers were determined (Table 11.3). As shown, both samples... 

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