The gas-phase hydrogen/deuterium (HID) exchange kinetics of several protonated amino acids and dipeptides under a background pressure of CH3OD were determined in an external source Fourier transform mass spectrometer. H/D exchange reactions occur even when the gas-phase basicity of the compound is significantly larger (> 20 kcal/mol) than methanol. In addition; greater deuterium incorporation is observed for compounds that have multiple sites of similar basicities. A mechanism is proposed that involves a structurally specific intermediate with extensive interaction between the protonated compound and methanol.

H-D exchange reactions of methanol-d, with protonated amino acids were performed in an external-source Fourier transform mass spectrometer. Absolute rate constants were determined for the group which included glycine, alanine, valine, leucine, isoleucine and proline. By comparing reactivities with selected methyl esters, it was found that exchange on the carboxylic acid occurs 3-10 times faster than exchange on the amino group. No simple correlation is observed between the rates of H-D exchange on the acid group and the size of the alkyl group. However, the rates of exchange on the amine decrease with increasing gas-phase basicity. Glycine, the least basic amino acid, exchanges its amine hydrogens the fastest. These results are useful for determining the interaction of methanol with protonated amino acids and can provide insight into the H-D exchange reactions observed with polyprotonated proteins produced by electrospray ionization.

The probe of intramolecular interactions in gas-phase biomolecules using the combination of proton transfer reactions, hydrogeddeuterium exchange reactions, and molecular orbital calculations is illustrated by exploring the nearest-neighbor interactions in protonated peptides. The interactions, specifically -NHz***H+-*O=C and C=O**.H+***O=C, are investigated with peptides that model them. The compounds that include /?-Ala, p-Ala-Gly, and Gly-/?-Ala, Ala-Gly and Gly-Ala are used to evaluate the structural and electronic factors that are involved in the protonation of the terminal amine. Similarly, N-acetylglycine and N-acetylglycine amide are used to evaluate carbonyl group interactions in the peptide backbone. The /?-alanine residue on the terminal amine is found to increase the gas-phase basicity and decrease the WD exchange reactivity of the protonated compound relative to analogous compounds containing only a-amino acids. A p-alanine residue on the C-terminus produces compounds with similar gas-phase basicity and WD exchange behavior as those with a-amino acids. The gas-phase basicity and H/D exchange behavior of the acetylglycines point to stronger intramolecular hydrogen bonding in the amide derivative than in the acid. An amide carbonyl has a greater intrinsic basicity than a carboxylic carbonyl. An analysis proposed by MeotNer is used to separate electronic effects from structural effects in the two types of protonation sites.

The kinetics of gas-phase H-D exchange reactions of a series of protonated amino acids and peptides with deuterium-substituted alcohols (D,O, CH,OD, C,H,OD and 1-C,H,OD) were studied in an external source Fourier transform mass spectrometer. The number of exchanges observed on the time-scale of these experiments ranged from one to the total number of 'labile' substrate hydrogens, depending on the amino acid and the deuterating reagent. Exchange efficiencies, klk,,,, varied from <0.001 to 0.3. Within the series ROD, the reactivity increased with increasing size of the R group. For the amino acids with alkyl side-chains, a roughly linear correlation of log(k/k,,) with proton affinity difference (APA = PA of unprotonated substrate -P A of reagent) was observed. The amino acids lysine and histidine and the dipeptides alanylglycine and diglycine showed higher reactivity and greater tendency for multiple exchange, with a weaker dependence on APA. The ability of a peptide and an alcohol to exchange efficiently even when APA is larger is attributed to the occurrence of exchange within a cyclic hydrogen-bonded complex, in which the deuterating agent forms a bridge between the site of protonation and a basic site on the substrate.