Schiff Base Formation and Reductive Amination

Conjugation reactions using CDI also may be done in organic solutions. This is a distinct advantage if the reactants are not very soluble in aqueous environments. In addition, organic coupling will not result in concomitant loss of activity due to hydrolysis as water-based reactions, thus nonaqueous reactions usually will provide greater yields. 

A protocol for the use of CDI in the activation of polyethylene glycol) is discussed in Chapter 25, Section 1.4, while CDI activation procedures for particles are described in Chapter 14. 

Add a quantity of the aldehyde-containing molecule to the solution in step 1 to obtain the desired molar ratio for conjugation. For instance, if the amine-containing protein is 

Add 10 pi of 5 M sodium cyanoborohydride in 1 N NaOH (Aldrich) per ml of the conjugation solution volume. Caution Highly toxic compound. Use a fume hood and be careful to avoid skin contact with this reagent. 

To block unreacted aldehyde sites, add 20 pi of 3 M ethanolamine (pH adjusted to desired value with HC1) per ml of the conjugation solution volume. React for 15 minutes at room temperature. 

Reductive amination (or alkylation) may be used to conjugate an aldehyde- or ketone-containing molecule with an amine-containing molecule. The reduction reaction is best facilitated by the use of a reducing agent such as sodium cyanoborohydride. 

Immobilization by reductive amination of amine-containing biological molecules onto aldehyde-containing solid supports has been used for quite some time (Sanderson and Wilson, 1971). The reaction proceeds with excellent efficiency (Domen et al., 1990). The optimum pH for the reaction is alkaline, although good yield can be realized from pH 7—10. At high pH (9—10) the formation of the Schiff bases is more efficient and the yield of conjugation or immobilization reactions can be dramatically increased (Hornsey et al., 1986). 

Dissolve the amine-containing protein to be conjugated at a concentration trf 1— 10 mg/ml in a buffer having a pH between 7 and 10. Higher pH reactions will result in greater yield of conjugate formation. Suitable buffers include 0.1 M sodium phosphate, 0.15 MNaCl, pH 7.2 0.1 M sodium borate, pH 9.5 or 0.05 M sodium carbonate, 0.1 M sodium citrate, pH 9.5. Avoid amine-containing buffers like Tris. 

---

Some workers avoid delay. Pai)adium-on-carbon was used effectively for the reductive amination of ethyl 2-oxo-4-phenyl butanoate with L-alanyl-L-proline in a synthesis of the antihyperlensive, enalapril maleate. SchifTs base formation and reduction were carried out in a single step as Schiff bases of a-amino acids and esters are known to be susceptible to racemization. To a solution of 4,54 g ethyl 2-oxO 4-phenylbutanoate and 1.86 g L-alanyl-L-proline was added 16 g 4A molecular sieve and 1.0 g 10% Pd-on-C The mixture was hydrogenated for 15 hr at room temperature and 40 psig H2. Excess a-keto ester was required as reduction to the a-hydroxy ester was a serious side reaction. The yield was 77% with a diastereomeric ratio of 62 38 (SSS RSS)((55). 

The synthesis pathway of quinolizidine alkaloids is based on lysine conversion by enzymatic activity to cadaverine in exactly the same way as in the case of piperidine alkaloids. Certainly, in the relatively rich literature which attempts to explain quinolizidine alkaloid synthesis °, there are different experimental variants of this conversion. According to new experimental data, the conversion is achieved by coenzyme PLP (pyridoxal phosphate) activity, when the lysine is CO2 reduced. From cadeverine, via the activity of the diamine oxidase, Schiff base formation and four minor reactions (Aldol-type reaction, hydrolysis of imine to aldehyde/amine, oxidative reaction and again Schiff base formation), the pathway is divided into two directions. The subway synthesizes (—)-lupinine by two reductive steps, and the main synthesis stream goes via the Schiff base formation and coupling to the compound substrate, from which again the synthetic pathway divides to form (+)-lupanine synthesis and (—)-sparteine synthesis. From (—)-sparteine, the route by conversion to (+)-cytisine synthesis is open (Figure 51). Cytisine is an alkaloid with the pyridone nucleus. 

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). 

Proteins may be modified with oxidized dextran polymers under mild conditions using sodium cyanoborohydride as the reducing agent. The reaction proceeds primarily through e-amino groups of lysine located at the surface of the protein molecules. The optimal pH for the reductive amination reaction is an alkaline environment between pH 7 and 10. The rate of reaction is greatest at pH 8-9 (Kobayashi and Ichishima, 1991), reflecting the efficiency of Schiff base formation at this pH. 

The rather labile Schiff base interaction can be chemically stabilized by reduction. The addition of sodium borohydride or sodium cyanoborohydride to a reaction medium containing an aldehyde compound and an amine-containing molecule will result in reduction of the Schiff base intermediate and covalent bond formation, creating a secondary amine linkage between the two molecules (Reaction 6). 

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. 

---