Epoxy Activation and Coupling

Modification of dextran polymers with 1,4-butanediol diglycidyl ether results in ether derivatives on the dextran hydroxyl groups containing hydrophilic spacers with terminal epoxy functions (Fig. 390). 

In a fume hood, mix 1 part 1,4-butanediol diglycidyl ether with 1 part 0.6 N NaOH containing 2 mg/ml sodium borohydride. 

With stirring, add 5 mg of dextran to each ml of the fcrs-epoxide solution. Mix well to dissolve. 

Extensively dialyze the solution against water to remove excess reactants. The activated dextran may be lyophilized for long-term storage. 

Noguchi et al. (1992) used an amine-terminal spacer arm derivative of dextran to react with SPDP (Chapter 5, Section 1.1) in the creation of a pyridyldisulfide-activated polymer (Fig. 391). Brunswick (1988) used a different amine-terminal spacer arm derivative of dextran and subsequently coupled iodoacetate to form a sulfhydryl-reactive polymer (Fig. 392). Heindel et al. (1991) used a unique approach. They 

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 

Porath, 1974). B/s-oxirane compounds also can be used to introduce epoxide groups into soluble dextran polymers in much the same manner (Boldicke et al., 1988 Bocher et al., 1992). The epoxide group reacts with nucleophiles in a ring-opening process to form a stable covalent linkage. The reaction can take place with primary amines, sulfhydryls, or hydroxyl groups to create secondary amine, thioether, or ether bonds, respectively (Chapter 2, Section 1.7). 

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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. 

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]. 

The coupling of enzymes to epoxy-activated carriers is commonly carried out at high ionic strength, because a salt-induced association between the macromolecule and the support surface increases the effective concenhation of nucleophilic groups on the protein close to the epoxide reactive sites, thus favoring the immobilization process [68, 69]. However, the salt concenhation needed for immobilizing an enzyme is highly dependent on the nature of the biocatalyst [69]. 

Bonn, Reiffenstuhl and Jandik were able to separate a number of divalent metal ions effectively in a single run using an IDA resin [17]. A silica based material (Nucleosil 300-7 of 7 pm diameter. 300 A average pore size) was derivatized with y-glycidoxypropyltrimethoxy silane, then iminodiacetic acid was covalently coupled to the epoxy activated surface. The final material was slurry packed into a 100 x 4.6 mm stainless steel column. A complexing eluent containing 10 mM citric acid plus... 

Epoxy-activated agarose has been coupled with iminodiacetic acid and converted into a zinc chelate form by equilibration with zinc chloride." The column has been successfully applied to the purification of human plasma a2-macroglobulin and tti-proteinase inhibitor. The column can be stripped with H4-edta and regenerated with zinc chloride. 

Genski and Taylor have employed sucx essfully epoxy-activated alkene 191 for coupling with paraformaldehyde in the presence of EtsAl/BusP to provide the MBH adduct 192, a highly functionalized molecule (containing epoxide and protected alcohol moieties in addition to the p-substituted enone), and transformed the adduct into the bioactive natural product ( )-c/)i-epoxydon (193) (Scheme 2.98). 

Enzymatic methods also yield a high conversion rate of L-lysine to AMV. Pukin et al. (2010) developed an enzyme (L-lysine a-oxidase enzyme from Trichoderma viride) immobilization system with an epoxy-activated solid support. They reported a 0.95 yield of 5-AMV acid. In this study, the enzyme showed an 8-fold lower activity as compared to the one determined in the standard assay under the experimental conditions, and the reason for the low enzymatic activity remained unclear. Liu et al. (2014) reported a simple composition of the two-enzyme coupled system and a 0.865 yield of 5-AMV. Considering all of the studies on AMV production, it seemed that L-lysine might be a suitable starting material for a higher production of AMV. 

Softening and cure is examined with the help of a torsional pendulum modified with a braid (65), which supports thermosets such as phenoHcs and epoxies that change from a Hquid to a soHd on curing. Another method uses vibrating arms coupled to a scrim-supported sample to measure storage and loss moduH as a function of time and temperature. An isothermal analytical method for phenoHc resins provides data regarding rate constants and activation energies and allows prediction of cure characteristics under conditions of commercial use (47). 

Other than the epoxy groups available on one Priostar dendrimer type and a methyl ester available on a PAMAM dendrimer, the commercial suppliers generally don t offer a selection of spontaneously reactive dendrimers for bioconjugation purposes. For this reason, most of the applications published for coupling biomolecules to dendrimers have used various modification or activation steps to create the appropriate reactive groups for conjugation (e.g., Leon etal., 1996). 

For protocol suggestions on conjugation to epoxy groups, see Chapter 2, Sections 1.7 and 4.1. Also, see Chapter 14, Section 4.11, Coupling to Epoxy Particles, for a method to attach affinity ligands to surfaces that are activated with epoxide groups. 

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