Novel Cβ-Cγ bond cleavages of tryptophan-containing peptide radical cations

Abstract

In this study, we observed unprecedented cleavages of the C(β)-C(γ) bonds of tryptophan residue side chains in a series of hydrogen-deficient tryptophan-containing peptide radical cations (M(•+)) during low-energy collision-induced dissociation (CID). We used CID experiments and theoretical density functional theory (DFT) calculations to study the mechanism of this bond cleavage, which forms [M - 116](+) ions. The formation of an α-carbon radical intermediate at the tryptophan residue for the subsequent C(β)-C(γ) bond cleavage is analogous to that occurring at leucine residues, producing the same product ions; this hypothesis was supported by the identical product ion spectra of [LGGGH - 43](+) and [WGGGH - 116](+), obtained from the CID of [LGGGH](•+) and [WGGGH](•+), respectively. Elimination of the neutral 116-Da radical requires inevitable dehydrogenation of the indole nitrogen atom, leaving the radical centered formally on the indole nitrogen atom ([Ind](•)-2), in agreement with the CID data for [WGGGH](•+) and [W(1-CH3)GGGH](•+); replacing the tryptophan residue with a 1-methyltryptophan residue results in a change of the base peak from that arising from a neutral radical loss (116 Da) to that arising from a molecule loss (131 Da), both originating from C(β)-C(γ) bond cleavage. Hydrogen atom transfer or proton transfer to the γ-carbon atom of the tryptophan residue weakens the C(β)-C(γ) bond and, therefore, decreases the dissociation energy barrier dramatically.

Fig. 1

CID spectra of (a) [GGGGW]^•+; (b) [GWGGG]^•+; (c) [WGGGG]^•+; (d) [GGGGW-OMe]^•+

Fig. 2

CID spectra of (a) [WGGGR]^•+; (b) [GWGGR]^•+; (c) [WGGGK]^•+; (d) [GWGGK]^•+; (e) [WGGGH]^•+; (f) [GWGGH]^•+

Fig. 3

CID spectrum of [W_α-CH3GGGH]^•+. “W_α-CH3” represents a modified tryptophan residue with the α-hydrogen substituted by a methyl group, which prevents the α-radical formation of tryptophan

Scheme 1

Possible mechanisms for the C_β–C_γ cleavage of tryptophan residue examined in this study

Fig. 4

CID spectra of (a) [M – 116]⁺ from [WGGGH]^•+; (b) [M – 43]⁺ from [LGGGH]^•+

Fig. 5

CID spectrum of [W_1-CH3GGGH]^•+. “W_1-CH3” represents a modified tryptophan residue with the 1-hydrogen of indole ring substituted by a methyl group, which prevents the dehydrogenation of the indole nitrogen of tryptophan

Fig. 6

Some critical conformers and transition structures of α-tryptophylmethylamino (a) radical and (b) radical cation involved in the elimination of neutral 116 Da radical via mechanisms shown in Scheme 1b and c, respectively. Relative enthalpies at 0 K are in kcal/mol. All related structures are shown in Figure S4