An unprecedented method of producing molecular radical cations of oligopeptides in the gas phase has been discovered. Electrospraying a methanolic mixture of a Cu(II)-amine complex, e.g., Cu II (dien)(NO 3 ) 2 (where dien ) diethylenetriamine), and an oligopeptide (M) yields the [Cu II (dien)M] •2+ ion, whose collision-induced dissociation (CID) produces [Cu I (dien)] + and M •+ , the molecular cation of the oligopeptide. Abundant M •+ is apparent when the oligopeptide contains both a tyrosyl and a basic residuesarginyl, lysyl, or histidyl. These structural requirements are similar to those in the metalloradical enzyme process in photosystem II. Tandem mass spectrometry of M •+ produces fragment ions that are both common to and also different from [M + H] + . The fragmentation chemistry of M •+ and of its products appear to be radical driven.Protein radicals have generated a lot of recent interest because of their unusual role in catalyzing a number of important reactions, 1 including the oxidation of water to oxygen for use in a photosynthesis system in plants and algae. 1a,b A common theme in these protein radicals is that they are synthesized posttranslationally and their formation involves metallo cofactors located either adjacent to the amino acid residue being oxidized or on a second subunit or activating enzyme that participates in the oxidation. 1 Frequent radical sites are located on the glycyl, tyrosyl, and tryptophanyl residues; the structure of the glycyl radical in pyruvate formate lyase has been found to be planar and maintain a gas-phase-like structure, despite being embedded in the protein. 1d Here we report results of a serendipitous discovery of an unprecedented route for generating molecular radical cations of oligopeptides in the gas phase. Some of the conditions under which these oligopeptide radical cations are generated bear a resemblance to those in vivo for protein radicals. It is noteworthy that this discovery centers on molecular radical cations as opposed to the more frequently encountered radical ions produced from protonated peptides capturing an electron 2 and metal-bearing peptide ternary complexes. 3 Although electron ionization (EI) is the most widely used method in mass spectrometry to generate radical cations, it is not amenable to oligopeptides because of their low volatility. Very few dipeptides and, as far as we know, only one tripeptide have been successfully ionized in this manner; their mass spectra were rich and contained a wealth of sequencing information. 4 We are reporting herein that electrospraying a methanolic mixture of a Cu(II)-amine complex, e.g., Cu II (dien)(NO 3 ) 2 (where dien ) diethylenetriamine), and an oligopeptide (M) yields the [Cu II -(dien)M] •2+ ion, whose collision-induced dissociation (CID) produces [Cu I (dien)] + and M •+ , the odd-electron (OE) molecular cation of the oligopeptide. Abundant M •+ is apparent only when the oligopeptide contains a tyrosyl and a basic residuesarginyl, lysyl, or histidyl. Figure 1 shows the product ion spectra of...

Molecular radical cations, M •+ , of amino acids and oligopeptides are produced by collision-induced dissociation of mixed complex ions, [Cu II (dien)M] •2+ , that contain Cu II , an amine, typically diethylenetriamine (dien), and the oligopeptide, M. With dien as the amine ligand, abundant M •+ formation is observed only for the amino acids tryptophan and tyrosine, and oligopeptides that contain either the tryptophanyl or tyrosyl residue. Dissociation of the M •+ ion is rich and differs considerably from that of protonated amino acids and peptides. Facile fragmentation occurs around the R-carbon of the tryptophanyl residue. Cleavage of the N-C R bond and proton transfer from the exocyclic methylene group in the side chain to the N-terminal residue results in formation of the [z n -H] •+ ion and elimination of the N-terminal fragment as ammonia or an amide, depending on the position of the tryptophanyl residue. Cleavage of the C R -C bond of an oligopeptide containing a C-terminal tryptophan residue and proton transfer from the carboxylic group to the N-terminal fragment (a carbonyl oxygen atom) results in formation of the [a n + H] •+ ion and elimination of carbon dioxide. Both types of fragmentation have no analogous reactions in protonated peptides. For the M •+ of tryptophanylglycylglycine, WGG, elimination of the tryptophanyl side chain results in GGG •+ . This radical cation fragments by eliminating its C-terminal glycine to give the [b 2 -H] •+ ion, which is an oxazolone and shares much of the structure and reactivity of the b 2 + ion from protonated triglycine. Density functional theory shows the mechanism of forming the [b 2 -H] •+ ion is similar to that of the b 2 + ion, although the free-energy barrier at 29.4 kcal/mol is lower. The [b 2 -H] •+ ion eliminates CO readily to give the [a 2 -H] •+ ion, which is an iminium radical ion.

The fragmentation mechanisms of protonated triglycine and its first-generation dissociation products have been investigated using a combination of density functional theory calculations and threshold collision-induced dissociation experiments. The activation barrier measured for the fragmentation of protonated triglycine to the b(2) ion and glycine is in good agreement with a calculated barrier at the B3LYP/6-31++G(d,p) level of theory reported earlier [Rodriquez, C. F. et al. J. Am. Chem. Soc. 2001, 123, 3006-3012]. The b(2) ion fragments to the a(2) ion via a transition state structure that is best described as acylium-like. Contrary to what is commonly assumed, the lowest energy structure of the a(2) ion is not an iminium ion, but a cyclic, protonated 4-imidazolidone. Furthermore, fragmentation of the b(2) to the a(1) ion proceeds not via a mechanism that results in HNCO and H(2)C=C=O as byproducts, as have been postulated, but via a transition state that contains an incipient a(1) ion and an incipient carbene. The fragmentation of a(2) to a(1) proceeds via a transition state structure that contains the a(1) ion, CO and an imine as incipient components.

Gas-phase dissociation reactions of protonated amino acidsphenylalanine, tyrosine, tryptophan, and histidineare rich and diverse. Considerable similarities exist among the four amino acids, but there are also significant differences. Facile reactions include the elimination of NH3, common to all aromatic amino acids except histidine, and the concomitant elimination of H2O and CO. Labeling experiments with deuteriums show considerable H/D scrambling prior to dissociation involving N−H, O−H, and C−H (both aliphatic and aromatic hydrogens). Mechanisms of this scrambling are proposed. At higher collision energies, eliminations of H2O, CO, CO2, and CH2CO occur after that of NH3. Similarly, eliminations of HCN, HCNH2, and NH3 occur after that of H2O and CO. The elimination of CH2CO is preceded by migration of the hydroxyl ion from the carboxylic group to the exocyclic carbon on the side chain. Aromatic amino acids, with the exception of tyrosine, were observed to yield cationic radical fragments by eliminating small radicals, including H•, CH3 •, and NHCH•.