Autoxidative self-condensation

Common impurities found in aldehydes are the corresponding alcohols, aldols and water from self-condensation, and the corresponding acids formed by autoxidation. Acids can be removed by shaking with aqueous 10% sodium bicarbonate solution. The organic liquid is then washed with water. It is dried with anhydrous sodium sulfate or magnesium sulfate and then fractionally distilled. Water soluble aldehydes must be dissolved in a suitable solvent such as diethyl ether before being washed in this way. Further purification can be effected via the bisulfite derivative (see above) or the Schiff base formed with aniline or benzidine. Solid aldehydes can be dissolved in diethyl ether and purified as above. Alternatively, they can be steam distilled, then sublimed and crystallised from toluene or petroleum ether. 

The kinetic instability of isoindoles dominates the chemistry of the simple systems and is principally to be ascribed to autoxidation and self-condensation reactions. The self-condensation reactions appear to be more important when both isoindole and isoindolenine tautomers can coexist (Eq. 6) hence, in general, /V-substituted isoindoles are more stable than /V-unsubstituted compounds. Although the autoxidation reactions have been worked out in several cases (Section V,G), the self-condensations are less well understood (but see Section V,F). Isoindole and C-alkylisoindoles become dark when kept at room temperature.6,78 C-Arylisoindoles are somewhat easier to handle, but 2-amino-1,3-diphenylisoindole decomposes rapidly on exposure to light and air.60 Operationally, unless strongly electron-withdrawing substituents are present at carbon, isoindoles must be kept in the freezer under nitrogen. 

AL has been synthesized by a wide variety of methods (SI, 62-68). It has been labeled with in positions 1 and 4 and in porition 5 (69). In general, 5-AL and the class of a-aminocarbonyl compounds, are stable in acid but rapidly and reversibly self-condense to dihydropyrazines (Fig. 11) in alkaline solution (pH > 8). These heterocycles autoxidize to the aromatic pyrazine. Methods for the isolation and characterization of 5-AL (70) have been given. It may be determined colorimetrically (70, 71). 

The formation of the GFP fluorophore involves self-processing reactions that bear similarity to the mechanism of formation of MIO discussed earlier (see Scheme 2). The proposed mechanism for the biosynthesis of the GFP fluorophore is shown in Scheme 16. It is proposed that after translation the protein (48) is folded in a way to encourage nucleophilic attack by the amide nitrogen of Gly67 on the amide carbon of Ser65 to form a cyclic intermediate (49), which loses water to yield an imidazolidin-5-one intermediate (50). This condensation reaction is followed by autoxidation of the a-/ bond of Tyr66 to yield the GFP fluorophore. It should be noted that the isolated tripeptide will not spontaneously undergo these reactions. For synthesis of the GFP cofactor to occur, the protein must be folded in such a manner to position the residues to facilitate the biosynthetic reactions. 

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