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Peptide Synthesis - N-carboxylic Acid Anhydride Method

Hermann Leuchs found in 1906 that the carboxyl activation of amino acids and the acyl protection occurred in N-carboxyl anhydride (NCA) simultaneously. Therefore, in the German literature, it is also called Leuchs- anhydride. In principle, the derivatives should have the ideal precondition for the application of polypeptide synthesis.

The first N-carboxylic acid anhydride (1, 3- oxygen nitrogen-2, 5-dione) is derived from the elimination of chloroethane from N- (ethoxycarbonyl) amino acid acyl chloride. A fine method for preparing this kind of derivatives is free amino acid and photolysis reaction, and the corresponding amino formyl ammonia is intermediate. However, trace amounts of water can make N - carboxylic anhydride in polymerization occurs, because the initial carbamic acid is automatically removed from the carboxyl group to obtain free amine. This free amine is a nucleophile that has further opened loop reaction. Therefore, NCA method application in peptide synthesis has been restricted, it was not until 1966 to explore the right reaction conditions, which can be used for N - carboxylic acid in conditional peptide synthesis in aqueous medium use. The N-carboxyl anhydride can rapidly acylate amino acids and peptide under the conditions of low temperature and pH 10.2. When the pH increases to 10.2, the next N- carboxylic acid anhydride is added, and the next round of condensation is started. In order to reduce the exchange of carboxylate in intermediate peptide carbamate and amino components, the mixture must be agitated violently. Precise control of the pH is another prerequisite (the amino acid requires a pH of 10.2-10.5, and the peptide requires a pH of 10.2), because it produces a by-product of ethyl urea when the pH value is greater than 10.5.

The sulfur analogues of N-carboxyl acid anhydride (NTA) can also be used in peptide synthesis, because thiocarbamate has high stability. The acylation can be performed at a pH value of 9-9.5, thus preventing the possible hydrolysis of the hydrolysis into ethylcarbamide. NCA/NTA method is especially applicable to segment condensation that does not require separation of reaction intermediates. Three functional groups of amino acids (except lysine and cysteine) do not require side chain protection. Several fragments of ribonuclease S- protein have been combined with this method, and the complete s-protein can be obtained by means of azote method.

Recently, the NCA method has caused a great deal of attention again owing to the preparation of N-acetyl-semax acid anhydride (urethane -protected n-carboxy anhydrides, UNCA), which has been prepared to protect the amino acid ester, and it is used for peptide synthesis. In aprotic solvents and tertiary alkali condition, the use of appropriate reagent can acylate ring nitrogen atom of NCA acylation, and then introduce Boc, Z or Fmoc group and get the corresponding UNCA68 (Y = Boc, Z, Fmoc). UNCA showed high reaction activity to nucleophilic reagents, and in most anhydrous solvents (except alcohols) used in peptide solid phase and liquid phase synthesis, the peptide bonds were formed at high speed. UNCA crystals can be obtained from most amino acids and keep stable in the absence of water.

UNCA showed high reaction activity to nucleophilic reagents, and in most anhydrous solvents (except alcohols) used in peptide solid phase and liquid phase synthesis, the peptide bonds were formed at high speed. Carbon dioxide is the only byproduct, and there is no danger of oligomerization or polymerization because the growth of peptide chain amino end is still protected by urethan after condensation reaction.

The protective NCA of N-triphenylmethyl and N-phenyl group is also reported recently.