Efficient and directed peptide bond formation in the gas phase via ion/ion reactions

Abstract

Amide linkages are among the most important chemical bonds in living systems, constituting the connections between amino acids in peptides and proteins. We demonstrate the controlled formation of amide bonds between amino acids or peptides in the gas phase using ion/ion reactions in a mass spectrometer. Individual amino acids or peptides can be prepared as reagents by (i) incorporating gas phaselabile protecting groups to silence otherwise reactive functional groups, such as the N terminus; (ii) converting the carboxyl groups to the active ester of N-hydroxysuccinimide; and (iii) incorporating a charge site. Protonation renders basic sites (nucleophiles) unreactive toward the N-hydroxysuccinimide ester reagents, resulting in sites with the greatest gas phase basicities being, in large part, unreactive. The N-terminal amines of most naturally occurring amino acids have lower gas phase basicities than the side chains of the basic amino acids (i.e., those of histidine, lysine, or arginine). Therefore, reagents may be directed to the N terminus of an existing "anchor" peptide to form an amide bond by protonating the anchor peptide's basic residues, while leaving the N-terminal amine unprotonated and therefore reactive. Reaction efficiencies of greater than 30% have been observed. We propose this method as a step toward the controlled synthesis of peptides in the gas phase.peptide ligation | ion chemistry T he formation of amide bonds between amino acids holds widespread interest as it is relevant to the origin of life. Although the process by which these early peptides were formed remains an open question, laboratory-based approaches for peptide synthesis have been in place for over a century. The first controlled synthesis of diglycine via hydrolysis of diketopiperazine by Fisher in 1901 (1) marked the beginning of a long path of development of peptide synthesis approaches. Since that time, peptide synthesis has undergone several developments, beginning as solution phase reactions and eventually evolving in 1963 with Merrifield's introduction of solid resins in peptide synthesis (2), more commonly referred to as solid phase peptide synthesis (SPPS). Although this technique has undergone a dynamic maturation over the past few decades, it is still regarded as expensive and inelegant (3, 4). Furthermore, there are still several challenges that are not likely to be overcome without a significant change to the synthesis approach, beyond inherent challenges such as, for example, solubility of reagents. SPPS requires significant use of protecting and deprotecting agents, cleaving agents, and other solvents for washing, drying, and dissolving the newly formed peptides. This method quickly becomes a rather wasteful approach when considering the sacrifices made to produce just a single amide bond (3). Alternative synthesis approaches, such as chemical ligation (5, 6) and molecular machine-mediated peptide synthesis (7), offer more elegant approaches despite maintaining several of the previously mentioned requirements.Amide b...

Efficient Covalent Bond Formation in Gas-Phase Peptide–Peptide Ion Complexes with the Photoleucine Stapler

“…Formation of covalent bonds in gas-phase ion complexes has been reported for several systems that relied on collision-induced chemical reactions between an anion and a cation in a complex that mimicked chemical methods used in solution [23,24]. In addition, covalent bond formation in gasphase ion complexes with 18-crown-6-ether has been accomplished by collision-induced dissociation of diazomalonate derivatives [25] or photodissociation of a diazirine labeled peptide [26] in which reactive carbenes played the role of reactive intermediates.…”

Selective Gas-Phase Oxidation and Localization of Alkylated Cysteine Residues in Polypeptide Ions via Ion/Ion Chemistry

“…35 Benefits of this approach include significantly shorter reaction times, reduced chemical noise via mass selection of reactants, and control over the extent of modification. 35 Recent examples of covalent modification ion/ion reactions include the synthesis of peptide bonds via N -hydroxysuccinimide 36 and Woodward’s Reagent K 37 conjugation chemistry, “Click” chemistry reactions between alkynes and azides, 38 and esterification of carboxylate groups via alkyl cation transfer from fixed charge cation reagents. 39 …”

Gas-Phase Oxidation of Neutral Basic Residues in Polypeptide Cations by Periodate

“…Recent examples of these modifications include the esterification of carboxylate groups via cation transfer [ 14 ], gas-phase conjugation of alkynes and azides via ‘Click’ chemistry reactions [15], the N- and C-terminal extension of peptides via conjugation with N-hydroxysuccinimide esters [ 16 ] and Woodward’s Reagent K [ 17 ], respectively, and the conjugation of aldehydes and amines via gas-phase Schiff base chemistry [18,19,20,21,22]. Gas-phase ion/ion reactions have several benefits over traditional solution-phase derivatizations including much shorter reaction times (<1 sec), reduction of side reactions via mass isolation of reactants, and control over the extent of reaction (viz., amount of product formed and number of modifications that occur) [1].…”