Influence of “Alternative” C‐terminal amino acids on the formation of [b₃ + 17 + Cat]⁺ products from metal cationized synthetic tetrapeptides

Abstract

The aim of this study was to investigate the dissociation patterns, and in particular the relative abundance of [b(3) + 17 + Cat](+), for peptides with C-termini designed to allow transfer of the -OH required to generate the product ion, but not necessarily as the most favored pathway. Working with the hypothesis that formation of a five-membered ring intermediate, including intramolecular nucleophilic attack by a carbonyl oxygen atom, is an important mechanistic step, several model peptides with general sequence AcFGGX were synthesized, metal cationized by electrospray ionization and subjected to collision-induced dissociation (CID). The amino acid at position X was one that either required a larger ring intermediate (beta-alanine, gamma-aminobutyric acid and epsilon-amino-n-caproic acid to generate six-, seven- or nine- membered rings, respectively) to transfer -OH, lacked a structural element required for nucleophilic attack (aminoethanol) or prohibited cyclization because of the inclusion of a rigid ring (p- and m-aminobenzoic acid). For Ag(+), Li(+) and Na(+) cationized peptides, our results show that amino acids requiring the adoption of larger ring intermediates suppressed the formation of [b(3) + 17 + Cat](+), while amino acids that prohibit cyclization eliminated the reaction pathway completely. Formation of [b(3) - 1 + Cat](+) from the alkali metal cationized versions was not a favorable process upon suppression or elimination of the [b(3) + 17 + Cat](+) pathway: the loss of H(2)O to form [M - H(2)O + Cat](+) was instead the dominant dissociation reaction observed. Multiple-stage dissociation experiments suggest that [M - H(2)O + Cat](+) is not [b(4) - 1 + Cat](+) arising from the loss of H(2)O from the C-terminus, but may instead be a species that forms via a mechanism involving the elimination of an oxygen atom from an amide group.

Novel fragmentation pathway for CID of (bn−1+Cat)+ ions from model, metal cationized peptides

“…Recent investigations in our laboratory suggest that the reaction to eliminate H 2 O and combination of H 2 O and NH 3 are initiated by nucleophilic attack by the N-terminal amide carbonyl oxygen atom upon the carbon atom of the adjacent amide group. The absence of the two reaction products for the peptide containing ␤A adjacent to the N-terminus is consistent with the cyclization and intra-molecular attack involving a kinetically and entropically less favored 6-member ring and with our earlier study of the influence of amino acids on the generation of (b n ϩ17 ϩ Cat) ϩ°p roducts° [1].…”

“…Formation of ␣-lactones has been proposed to explain certain dissociation products°for°Ag ϩ -cationized°phenylalanine° [19,°20].°An alternative reaction pathway might involve another intramolecular nucleophilic attack upon the oxazolinone by the carbonyl group to the N-terminal side of the ring. However, we reason that such cyclization reactions might be less favored for the larger amino acids (i.e., because of kinetic and entropic factors associated with the larger cyclic intermediates, as demonstrated in our°earlier°study° [1])°such°as°␥Abu°and°Cap,°and impossible for 4AMBz because of the rigid aromatic ring.…”

“…The product ion is observed most prominently during the CID of Li ϩ and Na ϩ cationized peptides, only to a small extent for Ag ϩ cationized peptides, and not at all from protonated analogues. I n an attempt to enhance the understanding of how cation and sequence influence peptide fragmentation, we recently investigated the dissociation of model N-acetylated tetrapeptides with general sequence AcFGGX that featured C-termini designed to allow transfer of the ϪOH required to generate the (b 3 ϩ 17 ϩ Cat) ϩ product ion, but not necessarily as the most favored pathway [1]. The amino acid placed at position X either required a larger cyclic intermediate than the five-member ring presumably formed with ␣-amino acids (␤-alanine, ␥-aminobutyric acid, and -amino-n-caproic acid to generate 6-, 7-, or 9-member rings, respectively) or prohibited cyclization because of the inclusion of a rigid ring (para-and meta-aminobenzoic acid).…”

Disfavoring Macrocycle b Fragments by Constraining Torsional Freedom: The “Twisted” Case of QWFGLM b6

“…This is in marked contrast to previous studies, where mid-sized b fragments were found to be comprised of a mixture of oxazolone and macrocycle structures [18,21]. Sequence analogs of QWFGLMPG, incorporating proline and 4-aminomethylbenzoic acid (4AMBz) [42,43], were synthesized to investigate their effect on macrocycle formation in the corresponding b fragment.…”

Electrospray ionization mass spectral characteristics and fragmentation mechanisms of Angiotensin II and its analogues

“…The cyclic structure of Pro on the backbone of the peptide increases the rigidity of the C-terminal backbone, which makes the carbon atom more susceptible to nucleophilic attack [3,[27][28][29][30][31]. After a rearrangement of cyclic structure, the b 7 + 18 ion is generated with the transfer of -OH group to the Pro carbonyl carbon [32][33][34][35][36][37][38][39][40][41].…”