Practical Protein Chemistry - A. Darbre 1989

Determination of C-terminal amino acid sequence
Determination of C-terminal groups
Determination of C-terminal amino acids as aldehydes

O-Pivaloylhydroxylamine reacts quantitatively with carbodiimide-activated carboxyl-containing acids to yield O-pivaloylpeptidohydroxamates (Fig. 18.7) [61].

Ionization of the N-H hydroxamate bond (pK_a 6.7–7.4) triggers the Lossen rearrangement in Peptides, leading to The formation of a peptidyl isocyanate, which subsequently converts into a mixture of peptidyl ureas. An aliquot is withdrawn from the solution, hydrolyzed with 6 M Hydrochloric acid, and the C-terminal amino acid is determined using a differential method; in the course of this Procedure, the C-terminal amino acid is converted into an aldehyde. The aldehyde can also be obtained by an alternative route: the pH is adjusted to 1–2, and the reaction mixture is heated for 2 h at 50 °C.

This procedure has been successfully applied to determine the C-terminal Amino Acids of eighteen di-, tri-, tetra-, and pentapeptides, as well as the A- and B-chains of Insulin. Here, the C-terminal amino acid was determined by a differential method, although in some cases the aldehyde was quantified by paper Chromatography as its 2,4-dinitrophenylhydrazone.

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FIG. 18.7. Reactions occurring during C-terminal analysis with aldehyde formation. O-Pivaloylhydroxylamine reacts quantitatively with the carbodiimide-activated C-terminal amino acid to yield a hydroxamate. Ionization of the hydroxamate N-H bond initiates the Lossen rearrangement, followed by The conversion of the C-terminal amino acid into an aldehyde [61].

Advantages of the described method include the feasibility of using aqueous solutions (and urea, if necessary), high yields (70–100% for Most amino acids), and the successful Determination of C-terminal Pro, Asn, and possibly Gln. However, low yields are observed for Asp and Glu (potentially due to the formation of cyclic anhydrides during carbodiimide activation), which leads to the regeneration of the starting amino acids upon Hydrolysis. Furthermore, to date, the method has been applied only to small peptides, for which performing a differential Amino acid analysis is actually meaningful.

18.3.4.1. O-Pivaloylhydroxylamine [61]. O-Pivaloylhydroxylamine hydrochloride (OPGA·HCl, 154 mg, 1 mmol) is dissolved in 4 mL of Water, and the pH is adjusted to 3.5 with 1 M sodium hydroxide. A solution of the peptide (1 μmol) in 2.0 mL of water is added, immediately followed by The addition of 0.4 mL of a 0.5 M aqueous EDC solution; the pH is maintained at 3.5 (pH-stat) by adding 0.1 M hydrochloric acid. The EDC solution is added in portions at 15-min intervals (three 0.4-mL portions). Excess EDC and OPGA·HCl are quenched with 0.5 mL of 5 M formate buffer (pH 3.5) with stirring for 20 min. The Lossen rearrangement is initiated by raising the pH to 8.5 and the Temperature to 50 °C. The pH is maintained at 8.5 for 20 h using 1 M NaOH. The process is monitored by the consumption of alkali required to maintain the pH (pH-stat). An aliquot is hydrolyzed with 6 M HCl and subjected to amino acid analysis; the C-terminal residue is determined by the differential method.