Secondary amines Schiffs base formation

Schiff base interactions between aldehydes and amines typically are not stable enough to form irreversible linkages. These bonds may be reduced with sodium cyanoborohydride or a number of other suitable reductants (Chapter 2, Section 5) to form permanent secondary amine bonds. However, proteins crosslinked by glutaraldehyde without reduction nevertheless show stabilities unexplainable by simple Schiff base formation. The stability of such unreduced glutaraldehyde conjugates has been postulated to be due to the vinyl addition mechanism, which doesn t depend on the creation of Schiff bases. 

Aldehydes and ketones can react with primary and secondary amines to form Schiff bases, a dehydration reaction yielding an imine (Reaction 45). However, Schiff base formation is a relatively labile, reversible interaction that is readily cleaved in aqueous solution by hydrolysis. The formation of Schiff bases is enhanced at alkaline pH values, but they are still not stable enough to use for crosslinking applications unless they are reduced by reductive amination (see below). 

Conjugation through Schiff base formation followed by reduction to secondary amine linkage. 

Neyroz et al. [97] have covalently linked 2NpOH to phos-phatidylethanolamine moiety by the Schiff-base formation between the NH2 of the phospholipid and the aldehyde moiety of 2-hydroxy-1-naphthaldehyde, followed by selective reduction of the imine to obtain a stable secondary amine. This fluorescent phospholipid easily incorporates into DML vesicle membrane and exhibits the typical behavior of ESPT probes. The emission spectrum of this probe inserted in the liposome is similar to that in ethanol medium and is affected by acetate used as a proton acceptor. 

Another unequivocal method of converting primary into secondary amines involves intermediate formation of Schiff bases and their quaternization. The quaternary salts are obtained almost quantitatively by heating the base with a lower alkyl halide in a sealed tube for several hours they are readily hydrolysed to the secondary amine. Benzaldehyde is usually taken to form the Schiff base. A number of A-alkyl-/ -methylphenethylamines have been prepared in excellent yield by this route.496 Also the allylmethylamine described above was obtained from allylamine in 71% yield by way of the anil.497... 

Tertiary amines. Using a secondary amine to decompose an ozonide derived from 1-alkene effects its alkylation. The amine initiates an eliminative fragmentation of the ozonide to generate an aldehyde and dialkylammonium formate. Schiff base formation from the aldehyde and another molecule of the amine is then followed by reduction by the formate ion. 

Pyridoxal Derivatives. Various aldehydes of pyridoxal (Table 3) react with hemoglobin at sites that can be somewhat controlled by the state of oxygenation (36,59). It is thereby possible to achieve derivatives having a wide range of functional properties. The reaction, shown for PLP in Figure 3, involves first the formation of a Schiff s base between the amino groups of hemoglobin and the aldehyde(s) of the pyridoxal compound, followed by reduction of the Schiff s base with sodium borohydride, to yield a covalendy-linked pyridoxyl derivative in the form of a secondary amine. 

It was reported only recently that A-methyl transfer from an oxaziridine to an amine occurs with formation of an N—N bond (79JA6671). N—N bond forming reactions with A-unsubstituted oxaziridines had been found immediately after discovery of this class of compound (64CB2521) and have led to simple hydrazine syntheses (79AHC(24)63). Secondary amines like diethylamine or morpholine are A-aminated by (52) in the course of some minutes at room temperature with yields exceeding 90% (77JPR195). Further examples are the amination of aniline to phenylhydrazine, and of the Schiff base (96) to the diaziridine (97). 

While enamines can usually be obtained directly from ketones and secondary amines their formation by an indirect route may bo advantageous. The previously mentioned condensation of rnethyl ketones during azeotropic enamine formation has prompted the alklyation (J) or acylation and reduction (59) of Schiff s bases. A parallel method uses the formation and desulfurization of N-acylthiazolines followed by hydride reduetion (60,61). 

The reductive alkylation of a primary amine with ketone leads to the formation of a stable imine. In the presence of hydrogen and a hydrogenation catalyst, the imine is reduced to a secondary amine. Similarly, a diamine reacts stepwise to form dialkylated secondary amines. However, several side reactions are possible for these reactions as outlined by Greenfield (12). The general scheme depicting the reaction between primary amine or diamine to yield secondary amine through a Schiff base is shown in Figure 17.1. 

Glutaraldehyde is the most popular b/s-aldchydc homobifunctional crosslinker in use today. Flowever, a glance at glutaraldehyde s structure is not indicative of the complexity of its possible reaction mechanisms. Reactions with proteins and other amine-containing molecules would be expected to proceed through the formation of Schiff bases. Subsequent reduction with sodium cyanoborohydride or another suitable reductant would yield stable secondary amine... 

Thus, glycoproteins such as HRP, GO, or most antibody molecules can be activated for conjugation by brief treatment with periodate. Crosslinking with an amine-containing protein takes place under alkaline pH conditions through the formation of Schiff base intermediates. These relatively labile intermediates can be stabilized by reduction to a secondary amine linkage with sodium cyanoborohydride (Figure 20.8). 

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