Identification of amino acids in unknown dipeptides: A derivatization with 9-fluorenylmethyl chloroformate and HPLC

Abstract

This paper describes a biochemistry experiment in which students use HPLC to identify the amino acids in unknown dipeptides.

Advancement in the derivatizations of the amino groups with the o-phthaldehyde-thiol and with the 9-fluorenylmethyloxycarbonyl chloride reagents

“…This means, that out of 24 proposals [2, in 13 acetone [2,[75][76][77]79,81,84,[86][87][88]90,94,96], in 7 ACN [82,85,89,[91][92][93]95], in 2 acetone:ACN = (1:1, v/v) [74,80] were applied to dissolve FMOC. The advantage of acetone over ACN can be attributed to its higher polarity resulting in the increased reaction rate of the amine groups with the reagent: in particular in the cases of aspartic and glutamic acids.…”

“…Because of the considerable loss (25-50%) of the derivatized amino acids (histidine, ornithine, lysine being partly extracted into the organic phase), instead of C 5 H 12 , as extracting agents, also the mixture of C 5 H 12 /ethyl acetate = (9:1, v/v) [79], or C 6 H 14 [84], or C 7 H 16 [91] have been suggested. Although, quantitative separation of the amino acid derivatives, by extractions, could not be achieved.…”

A pH-dependent cyanate reactivity model: application to preparative N-carbamoylation of amino acids

“…(Adapted from Clapp et al 18 ): a 0.5 mL aliquot was taken, cooled and added to 0.5 mL of 0.2 M borate buffer (pH 7.7) containing 1 mM -alanine (internal reference). To half of this mixture 0.5 mL of a fluorenylmethyl chloroformate solution (15 mM) in acetone was added and the mixture was stirred for 30 s, then the solution was washed 3 times with 2 mL hexane.…”

N-Carbamoyl Derivatives and Their Nitrosation by Gaseous NOx − A New, Promising Tool in Stepwise Peptide Synthesis

“…To evaluate the extent of epimerisation during the coupling step, the enantiomeric excesses of peptide residues were measured after N ‐decarbamoylation (see next section) and then after peptide bond hydrolysis with boiling 6 n HCl 11. The enantiomeric excesses ( ee s) of the resulting amino acids were measured by GC analysis after derivatisation with (−)‐menthyl chloroformate in 2‐propanol 12.…”

“…Peptide Hydrolysis 11 and Derivatisation 12 for GC Analysis: A solution of ( N ‐deprotected) dipeptide ethyl ester (100 mg) in 6 n HCl (10 mL) was heated to 110−120 °C (oven) in a sealed Pyrex tube. After cooling and concentration to dryness in a rotary evaporator, the residue was dissolved in aqueous sodium borate (50 m m, 2 mL, pH = 9.0), and then diluted with sodium borate (0.2 m, 20 mL, pH = 7.7).…”