Particles amine-containing

Catechin-immobilizing polymer particles were prepared by laccase-catalyzed oxidation of catechin in the presence of amine-containing porous polymer particles. The resulting particles showed good scavenging activity toward stable free l,l-diphenyl-2-picryl-hydrazyl radical and 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation. These particles may be applied for packed column systems to remove radical species such as reactive oxygen closely related to various diseases. 

DSC also is used to activate hydroxylic particles for coupling to amine-containing ligands (Miron and Wilchek, 1993). For methods involving particle conjugation using this homobifunctional compound, see Chapter 14. 

Figure 9.59 One of the first methods of preparing water-soluble QDs was to use thioacetic acid modification of the nanocrystal surface. This resulted in a negative charge on the surface of each dot that provides like charge repulsion of particles suspended in aqueous solution. The carboxylate group also could be used for conjugation with amine-containing molecules.

Carbodiimide coupling to carboxylate-containing QDs usually involves the use of EDC in a single-step or two-step process to form an amide bond. If a one-step reaction is done, the QD is activated with EDC in the presence of an amine-containing molecule, such as a protein. Many protocols use this method, but it can result in protein polymerization in addition to coupling, because proteins contain both carboxylates and amines. A two-step protocol results in better control of the reaction (Figure 9.61). In the first step, EDC is used in the presence of sulfo-NHS to activate the carboxylates on the particles to intermediate sulfo-NHS esters. After a quick separation step to remove excess reactants, the activated QDs are added to the protein solution to be coupled. This then results in amide bond formation without polymerization of the protein in solution. See Chapter 3, Section 1 and Chapter 14, Section 1 for additional information on this process. 

Figure 9.61 QDs containing carboxylate groups can be coupled to amine-containing proteins or other molecules using the EDC/sulfo-NHS reaction to form amide bond linkages. The intermediate sulfo-NHS ester is negatively charged and will help maintain particle stability due to like charge repulsion between particles.

Figure 14.5 A method of making particles biocompatible includes the use of PEG-based spacers. A lawn of mPEG molecules in interspersed with some longer PEG chains that terminate in carboxylate groups for coupling amine-containing molecules. The result is an extremely hydrophilic surface with low nonspecific binding.

Dissolve the amine-containing ligand to be coupled in 5 ml coupling buffer at a concentration sufficient to provide a 1- to 10-fold molar excess of ligand over the maximal calculated carboxylate group concentration for the amount and type of beads used. For particle manufacturers reporting a carboxylate concentration in meq/g, this is equivalent to pmol/mg. 

Primary amine-containing polymeric particles are available from a number of manufacturers and have either aliphatic or aryl amine groups on their surface. Occasionally, a particle type may have secondary or tertiary amines present, but these should be avoided for covalent coupling, as primary amines typically give better reaction yields than secondary amines and tertiary amines are unreactive. 

Figure 14.8 Amine-containing particles can be conjugated using alkylation or acylation reactions to result in secondary or tertiary amine linkages or amide bonds.

Figure 14.11 The modification of amine-containing particles with NHS-PEG4-maleimide produces hydrophilic PEG spacers containing terminal thiol-reactive groups. Coupling of thiol-containing proteins then results in the formation of thioether linkages.

Figure 14.14 Additional hydroxyl-particle activation methods include bis-epoxide modification, tosyl activation, and tresyl activation methods. The tosyl chloride and tresyl chloride activation procedures must be done in dry organic solvent, but the coupling of an amine-containing ligand can be done in either organic solvent or aqueous buffer.

Figure 14.15 CDI can be used to activate hydroxyl-particles in organic solvent and then the intermediate reactive imidazole carbamate brought into aqueous solution for coupling amine-containing ligands.

Figure 14.16 DSC can be used to activate hydroxyl-particles to a reactive NHS-carbonate derivative. The subsequent coupling of amine-containing ligands can be done in either organic solvent or aqueous conditions.

Add ethanolamine to the particle suspension at a final concentration of 0.1 M to quench any remaining active groups and react with mixing for 1 hour. Other amine-containing quenchers may be used, too, such as Tris buffer. Note DSC-activated sites on the particles that completely hydrolyze will revert back to the original hydroxyls. 

Figure 14.17 Cyanogen bromide can be used to activate a hydroxyl-particle to a reactive cyanate ester, which then can be used to couple amine-containing ligands.

Cyanogen bromide can be used to activate hydroxyl groups on particles to create reactive cyanate esters, which then can be coupled to amine-containing ligands to form an isourea bond (Figure 14.17). CNBr activation also can produce cyclic imidocarbonate groups, which are less reactive than the cyanate ester, but can form imidocarbonate bonds. The exact reactive species formed by the reaction is dependent on the structure of the hydroxylic support being activated (Kohn and Wilchek, 1982). 

Figure 14.21 Aldehyde-particles can be reacted with amine-containing proteins or other molecules to form intermediate Schiff bases, which can be stabilized by reduction with sodium cyanoborohydride.

Aldehyde particles are spontaneously reactive with hydrazine or hydrazide derivatives, forming hydrazone linkages upon Schiff base formation. Reactions with amine-containing molecules, such as proteins, can be done through a reductive amination process using sodium cyanoborohydride (Figure 14.21). 

Wash 10 mg of aldehyde particles 3 times with 10 mM sodium phosphate, pH 7.4 (coupling buffer). Buffers of higher pH value (i.e., carbonate buffer at pH 10) will result in more efficient Schiff base formation with amine-containing molecules than neutral pH conditions. 

Carboxylated silica particles may be coupled with amine-containing ligands, such as proteins, using a carbodiimide reaction with EDC. A similar protocol to that previously described for coupling to carboxylate polymer particles may be used. The following protocol is based on the method of Zhao et al. (2004), which was used for immobilizing monoclonal antibodies to E. coli 0157. 

Planar surfaces activated with the NHS-PEG groups may be sealed in a pouch and stored dry in the presence of a desiccant. Activated particles may be stored as a suspension in dry solvent under a head of nitrogen at 4°C until used for further conjugation. The addition of an amine-containing protein or other amine-molecule will cause covalent coupling to the surface NHS ester groups to form amide bonds. 

B.p. Enoksson (of Sweden), BritP 1147967(1969) CA 71, 23402n (1969) (AN with water-resistant and anticaking props was obtd by coating its particles with 0.05—0.1% of a mixt contg 2—10 moles of an alkylamine and 1 mole of an aliphatic acid. The acid and amine contained 12—20 carbon atoms. Stearylamine and stearic acid were used in one example)... 

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