Protein Chemistry. Structure, Properties, Research Methods - Shendryk A.N. 2022

Peptide Synthesis
Carboxyl Group Protection
Esterification

The primary purpose of carboxyl protection is not only to block the group, but also to disrupt the zwitterionic form of The amino acid. The simplest method for carboxyl protection is esterification, while alternative approaches include The formation of amides or hydrazides. The latter two Methods are used relatively rarely due to subsequent difficulties in removing the protecting group.

Methyl and ethyl esters. The standard Procedure for amino acid esterification involves treating them with an excess of the corresponding alcohol in the presence of an acid catalyst (such as HCl, thionyl chloride - SOCl₂, etc.). From the perspective of Peptide Synthesis, there are no significant differences between amino acid methyl and ethyl esters.

Methyl and ethyl esters of N-protected Amino Acids are prepared by introducing the N-protecting group into the already formed ester rather than the reverse.

Upon completion of peptide synthesis, the ester groups are cleaved via alkaline Hydrolysis. As the peptide chain lengthens, ester hydrolysis becomes increasingly difficult and requires more forcing reaction conditions, which in turn elevates the likelihood of racemization. Therefore, in such cases, alkaline hydrolysis is replaced by acidolysis.

Tert-butyl esters of Amino Acids and Peptides undergo exceptionally facile acid-catalyzed Cleavage, rendering them preferable to methyl and ethyl esters. Several methods for synthesizing amino acid tert-butyl esters are known.

> Via the esterification of free amino acids with isobutylene using sulfuric acid as a catalyst and dioxane as a solvent:

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> Via the transesterification of free amino acids with tert-butyl acetate using perchloric acid as a catalyst:

> Via the reaction of N-protected amino acids with tert-butanol and phosphorus oxychloride in the presence of excess pyridine.

Tert-butyl esters of Most amino acids are stable liquids that can be distilled without decomposition. They do not undergo self-Condensation upon storage and exhibit high stability toward Hydrazinolysis and aminolysis.

Tert-butyl esters are cleaved using hydrogen chloride in ethyl acetate or methylene chloride, toluenesulfonic acid in benzene, or trifluoroacetic acid. The latter is particularly common in the synthesis of biologically active peptide fragments.

Benzyl esters of amino acids are synthesized through the reaction of amino acid hydrochloride/acyl chloride derivatives with benzyl alcohol:

Reaction yields are generally modest.

When employing unprotected amino acids in this reaction, the process is facilitated by benzyl borate, which binds the Water formed during the reaction.

Benzyl esters can also be obtained by reacting amino acids with a 5–10-fold excess of dibenzyl sulfite in the presence of p-toluenesulfonic acid. This method is not applicable to Alanine, Lysine, and Proline.

Benzyl esters of amino acids and peptides are cleaved via catalytic hydrogenolysis in the presence of Pd. Selective hydrogenolysis of benzyl esters in the presence of a carbobenzoxy group is not feasible. Alternatively, benzyl esters can be cleaved using sodium in liquid ammonia. Alkaline hydrolysis of benzyl esters is utilized extremely rarely.

Modified Benzyl Esters

p-Nitrobenzyl esters of amino acids are prepared by refluxing carbobenzoxyamino acids with p-nitrobenzyl bromide or p-nitrobenzyl chloride in ethyl acetate using a tertiary amine as a catalyst (X denotes the carbobenzoxy group). The carbobenzoxy group is subsequently cleaved from the reaction product using hydrogen bromide in glacial acetic acid.

A second method for preparing p-nitrobenzyl esters involves the esterification of free amino acids with p-nitrobenzyl alcohol in the presence of toluenesulfonic acid in carbon tetrachloride.

A third approach is the reaction of sodium or trialkylammonium salts of carbobenzoxyamino acids with p-nitrobenzyl p-toluenesulfonate.

The cleavage of p-nitrobenzyl esters is typically achieved through alkaline hydrolysis or catalytic hydrogenolysis.