Carboxylates with Diamines

Raising the pi of macromolecules also can significantly alter the immune response toward them upon in vivo administration. Cationized proteins (those modified with diamines to increase their net charge or pi) are known to generate an increased immune response compared to their native forms (Muckerheide et al., 1987a, b Apple et al., 1988 Domen et al., 1987 Domen and Hermanson, 1992). The use of cationized BSA as a carrier protein for hapten conjugation can result in a dramatically higher antibody response toward a coupled hapten (Chapter 19). 

Protein containing positively Cationized protein containing 

Add the protein solution to an equal volume of diamine solution and mix. Alternatively, the solid protein can be dissolved directly in the diamine solution (after pH adjustment) 

Add EDC hydrochloride Thermo Fisher to a final concentration of 2mg/ml in the reaction solution. To aid in the addition of a small amount of EDC, a higher concentration stock solution may be prepared in water and an aliquot added to the reaction to give the proper concentration. Since EDC is labile in aqueous solutions, the stock solution must be made quickly and used immediately. 

Purify the modified protein by extensive dialysis against 0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4 (PBS) or another suitable buffer. 

Raising the pl of macromolecules also can significantly alter the immune response 

Dissolve the diamine chosen for modification at a concentration of 1 M made up in the coupling buffer. If a free-base form of diamine is used, then the solution will become highly alkaline on dissolution. This operation also will generate 

Protein Containing Positively-Charged Amines and Negatively-Charged Carboxylates 

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The following protocol using the carbodiimide EDC is an efficient way of modifying protein carboxylates with diamines to either increase the amount of amines present for further conjugation or to create a cationized protein having an increased net charge (Figure 1.90). Note that... 

Polyamides containing a-aminoacid units are readily obtained by reaction of bisazlactones (2-oxazolin-5-ones) with diamines. When polyamines such as diethylenetriamine (DETA) or triethylenetetramine (TETA) are used as the diamine component, the resultant polyamides readily cyclodehydrate above 200°C to produce polymers containing 2-imidazolin-5-one units in the backbone. Polyamides derived from simple diamines (e.g. 1,6-hexanedi amine) cyclodehydrate only in the presence of a suitable catalyst. Carboxylate salts and certain Lewis acids have been found to be efficient catalysts for this transformation. 

Modification of carboxylate groups with diamines also may be done in organic solvent for those molecules insoluble in aqueous buffers. Some peptides are quite soluble in solvents such as DMF and DMSO, but relatively insoluble in water. Such molecules may be reacted in these... 

Figure 1.95 Carboxylate-containing sugars may be modified with diamines using a carbodiimide-mediated reaction to create available amine groups for subsequent conjugation.

Diazepines 259 and 260 have been prepared282 by condensation of ethyl cyclohexanone-2-carboxylate with the appropriate diamine followed by cyclization of the initially formed imine. 

The effect of NaOH concentration on the ion transport and rheological properties of the Nafion ion exchange membranes may be attributable to some variation in the ionic domain structure in the presence of NaOH. Therefore, it is extremely Important to understand the ionic domain structure under these conditions. The anomalous behavior of Na" " ion transport as a function of NaOH concentration is seen more frequently in bilayer Nafion membranes in which one layer is treated with diamine and also in perfluorinated carboxylic ion exchange membranes. Several mechanisms have been proposed to explain their ion transport results including water absorption, transport of hydroxide ion tunneling, ion pairing mechanisms, etc. (54-56). As the ion transport properties are beyond the scope of this review, no detailed discussion will be presented. 

Polyamides (or nylons) are produced by heating diacids with diamines. The amine acts as the nucleophile and reacts with the carboxylic acid in a nucleophilic acyl substitution reaction (see Section 9.3). 

Since the reactions with phosphorus compounds give relatively high yields of carboxylic amides and esters and ureas, they have been extended to the direct polycondensations of dicarboxylic acids with diamines and bisphenols, of free a-amino acids or dipeptides, and of carbon dioxide and carbon disulfide with diamines under mild conditions to give polyamides, polyesters, polyureas, and polythioureas. 

Recently, the inverse electron demand Diels-Alder reaction of triethyl l,3,5-triazine-2,4,6-tri-carboxylate with in-situ generated alkene-1,1-diamines has been investigated in the preparation of suitably functionalized pyrimidines to be used as key intermediates in natural product synthesis.7-9... 

If less stringent requirements with regard to chemical resistance, abrasion, and hardness are placed on the cross-linking density, carboxyl-containing acrylates can also be cross-linked by salt formation (e.g., with diamines [2.44] or metal complexes [2.61]). This procedure is widely employed, particularly for aqueous dispersions. Cross-linking with bisoxazolines has also been reported [2.45]. Epoxy groups can be incorporated into the binder via glycidyl (meth)acrylate and cross-linked with dicar-boxylic acids [2.40]. 

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