Schiff base cyanoborohydride

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. 

Aldehyde-containing macromolecules will react spontaneously with hydrazide compounds to form hydrazone linkages. The hydrazone bond is a form of Schiff base that is more stable than the Schiff base formed from the interaction of an aldehyde and an amine. The hydrazone, however, may be reduced and further stabilized by the same reductants utilized for reductive amination purposes (Chapter 3, Section 4.8). The addition of sodium cyanoborohydride to a hydrazide-aldehyde reaction drives the equilibrium toward formation of a stable covalent complex. Mallia (1992) found that adipic acid dihydrazide derivatization of periodate-oxidized dextran (containing multiple formyl functionalities) proceeds with much greater yield when sodium cyanoborohydride is present. 

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... 

Derivatives of hydrazine, especially the hydrazide compounds formed from carboxylate groups, can react specifically with aldehyde or ketone functional groups in target molecules. Reaction with either group creates a hydrazone linkage (Reaction 44)—a type of Schiff base. This bond is relatively stable if it is formed with a ketone, but somewhat labile if the reaction is with an aldehyde group. However, the reaction rate of hydrazine derivatives with aldehydes typically is faster than the rate with ketones. Hydrazone formation with aldehydes, however, results in much more stable bonds than the easily reversible Schiff base interaction of an amine with an aldehyde. To further stabilize the bond between a hydrazide and an aldehyde, the hydrazone may be reacted with sodium cyanoborohydride to reduce the double bond and form a secure covalent linkage. 

Figure 3.14 Carbonyl groups can react with amine nucleophiles to form reversible Schiff base intermediates. In the presence of a suitable reductant, such as sodium cyanoborohydride, the Schiff base is stabilized to a secondary amine bond.

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... 

In a fume hood, add 10 pi of 5M sodium cyanoborohydride (Sigma) per ml of reaction solution. Caution cyanoborohydride is extremely toxic. All operations should be done with care in a fume hood. Also, avoid any contact with the reagent, as the 5M stock solution is dissolved in 1 N NaOH. If a higher pH buffer was used for the Schiff base formation, then adjust the solution to pH 7.5 before adding the cyanoborohydride. 

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). 

The conjugate may be stabilized by addition of a reductant such as sodium borohy-dride or sodium cyanoborohydride. Usually sodium cyanoborohydride is recommended for specific reduction of Schiff bases, but since the conjugate has already formed at this point, the use of sodium borohydride will both reduce the associated Schiff bases and eliminate any remaining aldehyde groups. Add sodium borohydride to a final concentration of lOmg/ml. Continue to react for 1 hour at 4°C. 

Hapten molecules containing aldehyde residues may be crosslinked to carrier molecules by use of reductive animation (Chapter 3, Section 4). At alkaline pH values, the aldehyde groups form intermediate Schiff bases with available amine groups on the carrier. Reduction of the resultant Schiff bases with sodium cyanoborohydride or sodium borohydride creates a stable conjugate held together by secondary amine bonds. 

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). 

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. 

In the reductive animation method, it is important to use reagents and reactants of high purity. The carbohydrate sample should not contain carbohydrate contaminants, as, if they were present, an adsorbent containing more than one type of ligand would be obtained. The sodium cyanoborohydride should be of high purity, because reduction of the Schiff base may not occur with impure preparations. Special procedures have been developed for purifying sodium cyanoborohydride, and these should be employed.25... 

Oxidation of 8-hydroxymethyl-2-isopropyl-l 1 //-pyrido[2,l-b]quinazolin-11-one with pyridinium chlorochromate in methylene chloride gave the 8-carboxaldehyde, which was converted into its 8-aminomethyl derivatives by reacting with amines followed by reduction of the Schiff bases with sodium cyanoborohydride in acetic acid (87JOC2469). 

Sodium cyanoborohydride, on the other hand, reduces the Schiff base much more rapidly than the carbonyl group88 at neutral pH. The... 

Anyway, MADAM-6 is not active. And the equally intriguing positional isomer, the easily made MADAM-2, will certainly contribute to these speculations. A quiz for the reader Will 2,N-dimethyl-3,4-methylenedioxyamphetamine (MADAM-2) be (1) Of much reduced activity, akin to MADAM-6, or (2) Of potency and action similar to that of MDMA, or (3) Something unexpected and unanticipated I know only one way of finding out. Make the Schiffs base between piperonal and cyclohexylamine, treat this with butyl lithium in hexane with some TMEDA present, add someN-methylformanilide, convert theformed benzaldehyde toanitrostyrenewith nitroethane, reduce this with elemental iron to thephenylacetone, reduce this in the presence of methylamine with sodium cyanoborohydride, then taste the result. 

Aldehyde groups can be converted into terminal amines by a reductive amination process with ammonia or a diamine compound. The reaction proceeds by initial formation of a Schiff base interaction—a dehydration step yielding an imine derivative. Reduction of the Schiff base with sodium cyanoborohydride or sodium bor-ohydride produces the primary amine (in the case of ammonia) or a secondary amine derivative terminating in a primary amine (for a diamine compound) (Fig. 88). 

Although both borohydride and cyanoborohydride have been used for reductive amination purposes, borohydride will reduce the reactive aldehyde groups to hydroxyls at the same time it converts any Schiff bases present to secondary amines. 

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