Amines carbohydrate reactivity

The nature of the covalently bound nitrogen in ammonium lignin sulfonates or the associated carbohydrates has not yet been reported, but if it is present as a primary or secondary amine, the reactivity toward formaldehyde would likely be increased (5) and the crosslink density of the resultant adhesive thereby augmented. 

Carbohydrate molecules containing amine groups, such as D-glucosamine, easily may be conjugated to other macromolecules using a number of amine reactive chemical reactions and crosslinkers (Chapter 2, Section 1 and Chapter 3). Some polysaccharides containing acetylated... 

This same type of modification strategy also can be used to create highly reactive groups from functionalities of rather low reactivity. For instance, carbohydrate chains on glycoproteins can be modified with sodium periodate to transform their rather unreactive hydroxyl groups into highly reactive aldehydes. Similarly, cystine or disulfide residues in proteins can be selectively reduced to form active sulfhydryls, or 5 -phosphate groups of DNA can be transformed to yield modifiable amines. 

Note At a level of 50-pl probe addition, polyclonal human IgG will be modified at a level that gives an F/P ratio of about 0.113. Since the labeling occurs only at the oxidized carbohydrate sites, the fluorophore incorporation typically is less than that observed when using amine-reactive probes. 

The ethylene diamine-dextran derivative may be used for the coupling of carboxylate-contain-ing molecules by the carbodiimide reaction, for the coupling of amine-reactive probes, or to modify further using heterobifunctional crosslinkers. The hydrazide-dextran derivative may be used to crosslink aldehyde-containing molecules, such as oxidized carbohydrates or glycoproteins. 

Enzymes that contain carbohydrate, such as HRP or GO, may be oxidized with periodate to create reactive derivatives that subsequently can be used to label antibodies or other targeting molecules at their amine groups. The aldehyde-HRP intermediate may be stored for extended periods in a frozen or lyophilized state without loss of activity (either enzymatic or coupling potential). Avoid, however, storage in a liquid state, since polymerization may occur—resulting in precipitation and loss of activity. 

Hydroxylation may also take place at nitrogen atoms, resulting in hydroxyl-amines (e.g., acetaminophen). Benzene, polycyclic aromatic compounds (e.g., benzopyrene), and unsaturated cyclic carbohydrates can be converted by mono-oxygenases to epoxides, highly reactive electrophiles that are hepato-toxic and possibly carcinogenic. 

Phosphate-containing carbohydrates that are stable, such as the 5 -phosphate of the ribose derivatives of oligonucleotides, may be targeted for modification using a carbodiimide-facilitated reaction (Section 4.3). The water-soluble carbodiimide EDC can react with the phosphate groups to form highly reactive phospho-ester intermediates. These intermediates can react with amine- or hydrazide-containing molecules to form stable phosphoramidate bonds. 

At high pH (10), Traut s reagent also is reactive with aliphatic and aromatic hydroxyl groups, although the rate of reaction with these groups is only about 0.01 that of primary amines. In the absence of amines, however, carbohydrates such as agarose or cellulose membranes can be modified to contain sulfhydryl residues (Alagon and... 

Prepare the protein or macromolecule to be thiolated in a non-amine-containing buffer at pH 8.0. For the modification of ribosomal proteins (often cited in the literature) use 50 mM triethanolamine hydrochloride, 1 mM MgCl2, 50 mM KC1, pH 8. The magnesium and potassium salts are for stabilization of some ribosomal proteins. If other proteins are to be thiolated, the same buffer may be used without added salts for stabilization. Alternatively, 50 mM sodium phosphate, 0.15 M NaCl, pH 8, or 0.1 M sodium borate, pH 8.0 may be used. For the modification of polysaccharides, use 20 mM sodium borax, pH 10, to produce reactivity toward carbohydrate hydroxyl residues. Dissolve the protein to be modified at a concentration of 10 mg/ml in the reaction buffer of choice. Lower concentrations also may be used with a proportional scaling back of added 2-iminothiolane. 

---