Effects of basic site proximity on deprotonation and hydrogen/deuterium exchange reactions for model dodecapeptide ions containing lysine and glycine

Abstract

The effects of basic site proximity on gas-phase deprotonation and hydrogen/deuterium (H/D) exchange reactions were investigated for three model dodecapeptide ions in a Fourier transform ion cyclotron resonance mass spectrometer. Each peptide contained four high basicity lysine (K) residues and eight low basicity glycine (G) residues; however, the ordering of the residues differed. In the deprotonation studies, ‘fully protonated’ peptide ions, [M + 4H]⁴⁺, where M = (KGG)₄, (K₂G₄)₂, and K₄G₈, were reacted with reference compounds of known basicities.

Reaction efficiencies were in the order: [K₄G₈ + 4H]⁴⁺ > [(K₂G₄)₂ + 4H]⁴⁺ ∼ [(KGG)₄ + 4H]⁴⁺. The facile reaction of [K₄G₈ + 4H]⁴⁺ is consistent with this ion having the highest Coulomb energy. For gas-phase H/D exchange reactions with d₄-methanol, [K₄G₈ + 4H]⁴⁺ has the fastest exchange rate and undergoes the largest number of exchanges; 22 of the 26 labile hydrogens exchanged within the timescale studied. In contrast, [(KGG)₄ + 4H]⁴⁺ and [(K₂G₄)₂ + 4H]⁴⁺ reacted more slowly, but at similar rates, with a maximum of 14 observed exchanges for both ions. Molecular dynamics calculations were conducted to gain insights into conformations. In the lowest energy structures for [(KGG)₄ + 4H]⁴⁺ and [(K₂G₄)₂ + 4H]⁴⁺, the lysine n-butylamino chains stretch out to minimize Coulomb energy; there is little or no intramolecular hydrogen bonding involving the protonated amino groups. In contrast, for [K₄G₈ + 4H]⁴⁺, the proximity of the basicity residues makes minimization of the Coulomb energy difficult; instead, the structure becomes more compact with stabilization of the protonated amino groups by extensive intramolecular hydrogen bonding to heteroatoms in the peptide backbone. The calculated structures suggest that, in the H/D exchange reactions, the compact conformation of [K₄G₈ + 4H]⁴⁺ allows stabilization of the methanolpeptide intermediate by hydrogen bonding, thus lowering the barrier to proton transfer within the complex. The diffuse conformations of [(KGG)₄ + 4H]⁴⁺ and [(K₂G₄)₂ + 4H]⁴⁺ have lower Coulomb energies and fewer avenues for hydrogen bonding with methanol, which may limit their rate and extent of exchange.