Both organic carboxylic acid and inorganic acid can be used in the formation of acid anhydride. However, only a few have been widely applied, and in most cases, alkyl chloroformate is used. Ethyl chloroformate used in the past is mainly substituted by isobutyl chloride.
A mixed anhydride formed from carboxyl and chloroformate, the regioselectivity depends on the electrophilic and / or steric hindrance of the two competing carbonyl groups. In the case of the formation of mixed anhydrides in the presence of amino acid carboxylates (carboxyl groups) protected by N and alkyl chloroformate (active components, such as alkyl chloroformate), aucleophilic amines mainly attack the carboxyl groups of amino acid components, and then form the expected peptide derivatives, and release active components from the off acid form. When the alkyl chloroformate (R1 = isobutyl, ethyl, etc.) is used, the free alkyl carbonate is unstable and immediately decomposes into carbon dioxide and corresponding alcohols. However, there are also some contrary reports on the regioselectivity of nucleophilic attack, and the products are carbamates and the original N protected amino acid components.
In order to form mixed acid anhydride, the N-protected amino acids or peptides are dissolved in dichloromethane, tetrahydrofuran, dioxane, acetonitrile, ethyl acetate or DMF. Then with an equal amount of alkali (N-methyl piperidine, N-methyl morpholine, N-Ethyl morpholine) to treat. Then, an alkyl chloroformate was added at the temperature of - 15℃ - 5 ℃, stirring violently to form an asymmetric anhydride (activation). After a short time activation, nucleophilic amino acid components were added. If it is used as an ammonium salt (more base), you must avoid excessive use of alkali. If the above reaction conditions are strictly followed, the mixed acid anhydride method is easy to carry out and is one of the most effective condensation methods.
Benoiton and his colleagues conducted in-depth studies on the stability of mixed acid anhydride and the reduction of by-products, such as carbamate and racemes. Therefore, they further understand the mechanism of reaction and improve the efficiency of the method, which has been widely used. By studying the causes of excessive carbamate, especially in a different light ammonia acyl and valerian ammonia acyl, they found that the use of dichloromethane as solvent and N - methyl piperidine as alkali can prevent the main side effects. Mixed acid anhydride has high stability for hydrolysis, so it can be washed with water to purify mixed acid anhydride. The stability of mixed acid anhydride from alkyl chloroformate depends on the use of alkyl. The mixed acid anhydride produced from the protection of the isopropyl chloride of Boc, z-and Fmoc is more stable than the mixed acid anhydride obtained from ethyl chloroformate or isobutyl chloroformate. When there is no suitable nucleophilic reagent, the decomposition of mixed anhydride in organic solvent begins with cyclization, producing 2 alkoxy - 5 (4H) - oxazolone, releasing carbon dioxide and alcohol R2 - OH at the same time, and the by-product is symmetrical anhydride and ester.
In the practical application of mixed acid anhydride, the following aspects should be noted: although hydrous DMF is a good solvent for the formation of mixed anhydrides and subsequent condensation reactions, however, as in the reactions of Z-Gly-Xaa-OH (Xaa=Ala, Leu, Val, Phe) with H - Val-OEt, the degree of racemization promoted is much higher than that using tetrahydrofuran or halogenated reagents as solvents. Interestingly, in DMF or N- methylpyrrolidone, the activation of ethyl chloroformate was less than the activation of isobutyl chloroformate. However, triethylamine as the tertiary base of ethyl chloroformate has little practical value at present. The reaction of acid anhydride was first observed in amino acid activation of N alpha-methyl sulfonyl, N alpha-triphenylmethyl and N alpha-trifluoroacetyl.
Sometimes tevalic acid (2, 2-dimethylpropionic acid) is recommended as an activator for synthesis of mixed acid anhydride, especially for asparaginamide, a protective asparagine. Similarly, the asymmetric anhydride is made of N alpha-acyl amino acid and thopentyl chloride, and is high in production rate when it's reacted to the amino nucleophile. In terms of mechanism, the application of acyl phosphor salt as the active intermediate in peptide condensation is also mentioned.