Conjugate sodium 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. 

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. 

Figure 7.11 Oxidation of glycoproteins with periodate, such as glycosylated antibodies, results in the formation of aldehyde groups that can be used for conjugation to dendrimers containing amine groups. Reductive amination with sodium cyanoborohydride results in coupling via secondary (or tertiary) amine bonds.

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). 

Figure 20.8 Enzymes that are glycoproteins like HRP may be oxidized with sodium periodate to produce reactive aldehyde residues. Conjugation with an antibody then may be done by reductive animation using sodium cyanoborohydride.

An interesting transformation was discovered by Sakai and Shinma (181) during the chemical investigation of corynanthe alkaloid (V-oxides. Polonovski reaction and sodium cyanoborohydride reduction of hirsuteine A-oxide (331) gave corynantheine (52) and 3-isocorynantheidine (65), the latter likely formed by reduction of the conjugated iminium intermediate 334. 

Reduction of unsaturated ketones to unsaturated alcohols is best carried out Nit v complex hydrides. a,/3-Unsaturated ketones may suifer reduction even at the conjugated double bond [764, 879]. Usually only the carbonyl group is reduced, especially if the inverse technique is applied. Such reductions are accomplished in high yields with lithium aluminum hydride [879, 880, 881, 882], with lithium trimethoxyaluminum hydride [764], with alane [879], with diisobutylalane [883], with lithium butylborohydride [884], with sodium boro-hydride [75/], with sodium cyanoborohydride [780, 885] with 9-borabicyclo [3.3.1]nonane (9-BBN) [764] and with isopropyl alcohol and aluminum isopro-... 

This step is optional. The biotin hydrazone-antibody conjugate formed in this reaction (steps 6 and 7) is considered by some researchers to be relatively unstable. To reduce the conjugate to a more stable, substituted hydrazide, treat the conjugate with sodium cyanoborohydride at a final concentration of 5 mA/by adding a 1/20 vol of a 100-mM stock solution. Incubate for 2 h at 4°C (see Note 5). 

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,... 

Desalt the biotinylated antibody by dialysis or gel filtration. Note that after hydrazide-based biotinylations, many researchers feel that it is necessary to stabilize the linkages between the hydrazides and aldehydes by treating the conjugate with sodium cyanoborohydride. 

With the procedure for constructing the quaternary carbon stereocenter in hand, the conversion of the ris-form to the trans form was explored in accordance with the synthetic plan shown in Scheme 9. The ketone moiety of the 1,4-conjugated adduct 61 was protected by an acetal group, followed by decarboxylation of compound 65 using sodium ethylthiolate to yield lactam trans-62 and cis-62 as an 8 1 diastereomixture [31]. The reason why the lactam trans-62 was obtained as a major product is that the subsequent protonation after decarboxylation proceeded kinetically. This assertion is supported by experimental results in which the trans- and cis-lactam diastereomixture (8 1) in ethanol was refluxed in the presence of potassium hydroxide to afford a 1 5 mixture [15,32,33]. The mixture of the lactam trans-62 and cis-62 was reduced with DIBALH, followed by treatment with sodium hydroxide to give bicyclic enamine 63. The kinetic iminium salt prepared from bicyclic enamine 63 with hydrochloric acid was reduced with sodium cyanoborohydride, leading to the frans-decahydroisoquinoline structure [22], The acetal moiety of the resultant 67 was removed to provide the objective ketones 68a and 2c. This method enabled the construction of the tra s-decahydroisoquinoline structure without an intermediate resembling the neurotoxic MPTP, and in fewer steps. 

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