The unusual loss of an internal Val residue from the (M-H)- parent anions of the antimicrobial peptide citropin 1.1 and synthetically modified analogues -: Fragmentations which require a specific conformation of the decomposing anion

The electrospray negative ion mass spectra of citropin 1. 1 (GLFDVIKKVASVIGGL-NH2) and of synthetically modified analogues show loss of an internal Val residue from both the (M - H)(-) anion and certain fragment anions. The negative ion spectra of GLFDVIQQVASVIGGL-NH2 and of three synthetically modified versions where Val residues are successively replaced by Ile shows that it is Val 12 which is eliminated. A joint experimental and theoretical study indicates that this internal rearrangement requires the decomposing anion to have an alpha-helical structure in the reaction region. If the conformation of the anion is randomised, either by increasing the internal energy of the decomposing (M - H)anion, or by making the electrospray solution strongly acidic or strongly basic, the [(M - H) - Val] - fragmentation is either reduced or no longer observed. Using isopropanol (rather than water/acetonitrile) as the electrospay solvent enhances the abundances of [(M - H) - Val] peaks (compared with normal backbone cleavage peaks) up to 30 times. The process is initiated by the C-terminal CONH- group reacting at the carbonyl centre of the amide function situated between Ser11 and Val 12. The reacting groups can approach to within 2.5 Angstrom when the peptide has an alpha-helical structure in the reaction region. Nucleophilic substitution followed by decomposition of the tetrahedral intermediate gives a rearranged peptide with the original Val 12 now occupying the C-terminal position. Calculations at the HF/6-31G(d)//HF/6-31G(d) level of theory indicate that formation of the initial tetrahedral intermediate is exothermic (-11.9 kcal mol(-1)) with the rearrangement sequence being endothermic (17.1 kcal mol(-1)). The overall reaction requires the (M - H)(-) anion to have an excess energy of 40.8 kcal mol(-1) at the level of theory used in this study. The [Aspy-H2O-CH2O](-) fragment anion in the negative ion spectrum of citropin 1.1 rearranges to a similar tetrahedral intermediate which undergoes competitive loss of Val and cleavage of the peptide backbone. These reactions require the [Aspgamma-H2O-CH2O](-) decomposing anion to have a helical structure in the vicinity of the reaction centre. (C) 2004 Elsevier B.V. All rights reserved.