Enhanced Reactivity in Nucleophilic Acyl Substitution Ion/Ion Reactions Using Triazole-Ester Reagents
The acyl substitution reactions between 1-hydroxy-7-aza-benzotriazole (HOAt)/1-hydroxy-benzotriazole (HOBt) ester reagents and nucleophilic side chains on peptides have been demonstrated in the gas phase via ion/ion reactions. The HOAt/HOBt ester reagents were synthesized in solution and ionized via negative nano-electrospray ionization. The anionic reagents were then reacted with doubly protonated model peptides containing amines, guanidines, and imidazoles in the gas phase. The complexes formed in the reaction cell were further probed with ion trap collision induced dissociation (CID) yielding either a covalently modified analyte ion or a proton transfer product ion. The covalent reaction yield of HOAt/HOBt ester reagents was demonstrated to be higher than the yield with N-hydroxysuccinimide (NHS) ester reagents over a range of equivalent conditions. Density functional theory (DFT) calculations were performed with a primary amine model system for both triazole-ester and NHS-ester reactants, which indicated a lower transition state barrier for the former reagent, consistent with experiments. The work herein demonstrates that the triazole-ester reagents are more reactive, and therefore less selective, than the analogous NHS-ester reagent. As a consequence, the triazole-ester reagents are the first to show efficient reactivity with unprotonated histidine residues in the gas phase. For all nucleophilic sites and all reagents, covalent reactions are favored under long time, low amplitude activation conditions. This work presents a novel class of reagents capable of gas-phase conjugation to nucleophilic sites in analyte ions via ion/ion chemistry.
KeywordsIon/ion reactions HOAt/HOBt ester Nucleophilic attack
Tandem mass spectrometry is a well-established approach for obtaining structural information from ions derived from biomolecules. The structural information derived from a molecule of interest is highly related to the gas-phase ion type that is subjected to activation . Gas-phase ion/ion reactions have proven to be particularly useful for converting ions from one type to another. Examples include altering charge states and polarities with proton transfer reactions [2, 3, 4, 5], converting multiply protonated species to radical hypervalent species via electron transfer reactions [6, 7, 8], and removing or replacing metal ions [9, 10]. In addition to the small charged particle transfer ion/ion reactions just mentioned, ion/ion reactions that involve selective covalent chemistry have been developed, thereby enabling the chemical modification of analyte ions in the gas phase. Examples of gas-phase covalent modification reactions include Schiff base chemistry between amines and aldehydes , Click chemistry between azides and alkenes , and oxidation chemistry between periodate and various reductive groups . A reaction that is particularly useful for protein and peptide modification is the reaction between N-hydroxysuccinimide (NHS) esters and various nucleophiles commonly present in polypeptides such as neutral amines , neutral guanidines , and carboxylates . The reaction involves the covalent attachment of an acyl group to the nucleophile via the displacement of an NHS leaving group, resulting in the formation of an amide on the N-terminal amine or lysine side chain , an N-acyl guanidine on arginine side chains , or an anhydride on the C-terminus or side chain carboxylates .
The gas-phase ion/ion reactions that result in new covalent bond formation have been noted to proceed through a long-lived complex derived from the condensation of the oppositely charged reactant ions. Such complexes are usually sufficiently long-lived that they become stabilized by collisions in the relatively high pressure environment of an electrodynamic ion trap (e.g., 1–10 mTorr). Subsequent ion activation of the complex leads to observation of the products of the covalent reaction. However, charge transfer without covalent bond formation, usually in the form of proton transfer, is a competing reaction pathway in the break-up of an ion/ion complex. Simple charge transfer therefore constitutes a “side-reaction” when the selective formation of a covalent bond is the desired objective. The observed efficiency of the covalent reaction is governed by the energies and entropies of the barriers on the potential energy surface (PES) for the competing reactions (i.e., proton transfer versus covalent reaction) and by the activation conditions . While NHS esters have proven to be relatively efficient reagents for covalent bond formation, proton transfer is often highly competitive with covalent reaction. It is, therefore, of interest to explore the use of reagents that may be comprised of better leaving groups in order to increase the fraction of complexes that dissociate to yield the covalent reaction products. Triazoles, such as 1-hydroxy-benzotriazole (HOBt) and 1-hydroxy-7-aza-benzotriazole (HOAt), are a group of activating reagents for carboxylic acid functional groups and were originally used in solid-phase peptide synthesis . They are also commonly used as carboxylate activators  to facilitate amide bond formation between amines and carboxylates. Here, we describe a novel gas-phase ion/ion reaction between HOAt/HOBt ester reagents and nucleophiles. Similar to NHS ester reagents, HOAt/HOBt ester reagents react by covalently attaching an acyl group to certain nucleophiles with the loss of neutral HOAt/HOBt molecules (i.e., leaving groups). Density functional theory (DFT) calculations on model systems that have been conducted to compare reaction barrier heights for HOAt- and NHS-based reagents suggest that the former can be expected to react faster than the latter. As described herein, HOAt/HOBt ester reagents have also been demonstrated experimentally to be more reactive than NHS ester reagents, which leads to greater reaction efficiencies as well as reactivity with weaker nucleophiles, such as histidine side-chains.
N-hydroxysulfosuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) were purchased from Thermo Fisher Scientific (Rockford, IL, USA). 1-Hydroxybenzotriazole hydrate (HOBt·xH2O), acetic anhydride, and sodium 3-sulfobenzoate were purchased from Sigma Aldrich (St. Louis, MO, USA). 1-Hydroxy-7-azabenzotriazole (HOAt) was purchased from ABBLIS Chemicals LLC (Houston, TX, USA). The peptides KGAGGKGAGGKL, RGAGGRGAGGRL, and Ac-HGAGGHGAGGHL-OMe were custom synthesized by NeoBioLab (Cambridge, MA, USA). Methanol, dimethylformamide (DMF), and glacial acetic acid were purchased from Mallinckrodt (St. Louis, MO, USA). Water (HPLC grade), and acetonitrile were purchased from Fisher Scientific (Hampton, NH, USA).
Reagents sulfo-benzoyl-HOAt, sulfo-benzoyl-HOBt, and sulfo-benzoyl-NHS were prepared by combining 10 uL of 100 mM EDC, 100 mM sodium 3-sulfobenzoate, and 100 mM HOAt/HOBt/NHS in DMF. The solution was then diluted 100× with acetonitrile for nano electrospray ionization (nESI). Water is avoided as the reagents hydrolyze within a few hours at room temperature.
The N-terminus of the peptide RGAGGRGAGGRL was protected by combining the peptide with excessive acetic anhydride in pH 8 aqueous ammonium bicarbonate buffer. The solution was incubated for 2 h at room temperature. The reaction was quenched by the addition of glacial acetic acid to drop the pH to 2–3. All peptide solutions for positive nano-electrospray (nESI) were prepared in a 50/50 (vol/vol) solution of methanol/water at a concentration of ∼1 mM.
Experiments were performed using a QTRAP 4000 hybrid triple quadrupole/linear ion trap and a TripleTOF 5600 System (SCIEX, Concord, ON, Canada). Both instruments were modified for ion/ion reactions; modifications were similar to those published previously [20, 21]. Anions and cations were sequentially injected into the instruments via alternately pulsed nESI , isolated in transit through Q1, and transferred into the Q2/q2 reaction cell. The ions were mutually stored in the reaction cell for a defined period of time ranging from 10 to 500 ms. In the QTRAP 4000, reaction products were transferred to Q3, where they were further probed via resonance collisional induced dissociation (CID) and mass-analyzed using mass-selective axial ejection (MSAE) . In the TripleTOF 5600, the reaction products were probed directly in Q2 by either resonance CID or dipolar direct current (DDC) CID [24, 25] and then mass-analyzed using the orthogonal TOF.
The ion/ion reaction rate measured via DDC kinetic experiments has been described previously . Briefly, the dissociation of the precursor ion follows pseudo-first-order reaction kinetics. Therefore, dissociation rates can be determined from product ion spectra acquired as a function of activation time at various DDC amplitudes. Multiple pathways may exist in the dissociation process and compete with each other. The rate of appearance of each product can be calculated based on the partitioning between the two product peaks in the product spectrum and the overall dissociation rate.
DFT calculations were used to characterize reaction pathways. Optimizations and zero-point corrected energies (ZPE) were calculated using the Gaussian 09 package  at the unrestricted B3LYP/6-311G++(d,p) level of theory. Since the model molecules are small, all structures were directly optimized in Gaussian 09. Transition states were searched using the QST3 option and confirmed with intrinsic reaction coordinate (IRC) calculations . All stationary points have been subjected to frequency calculations and identified as local minima (zero imaginary frequencies) or transition states (one imaginary frequency). See Supplementary Material for the coordinates of all of the calculated structures.
Results and Discussion
DFT calculations using the Gaussian 09 package were performed on simple model systems to compare the energy requirements associated with the covalent reactions of HOAt ester and NHS ester reagents with primary amines. Here, neutral methyl amine was used to represent the analyte and a neutral acetyl HOAt/NHS ester was used to represent the reagent. The energy of the electrostatic complex was assigned 0 kcal/mol. The two reaction pathways are indicated on either side of the complex in Figure 1, although no modeling of the proton transfer process was performed, as this barrier is expected to be similar for all reagents with a given analyte ion. For reference, the proton transfer barrier between a doubly protonated amine system to a sulfonate has been reported previously as 28 kcal/mol . In the covalent modification pathway, to the right of the complex, the electrostatic complex overcomes a transition state barrier to yield the product complex, then breaks relatively weak van der Waals interactions between the modified peptide and the neutral HOAt/NHS leaving group to yield the covalently modified product. The transition state barrier of the HOAt ester reagent was determined to be 18 kcal/mol whereas that of the NHS ester pathway requires 21 kcal/mol of energy. Interestingly, while the calculations indicate that the NHS ester products are favored thermodynamically by 3 kcal/mol, they also suggest that the HOAt ester products should be expected to be favored on kinetic grounds. As CID conducted in tandem mass spectrometry is under kinetic control, this calculation suggests that HOAt esters should be more reactive than NHS ester reagents. Calculations were also conducted using benzoyl HOAt ester and sulfo-benzoyl HOAt ester reagents resulting in differences in transition state energies of ≤0.9 kcal/mol and product complex energies of ≤1.1 kcal/mol. All calculations are consistent with higher reactivity for HOAt esters. Results of the calculations are provided in SI Table 1 as well as listings of the coordinates for calculated structures.
We recently published an approach to characterizing the competition between proton transfer and covalent reaction based on the measurement of CID kinetics of relevant ion/ion complexes . It is based on measuring the overall dissociation rate, kdiss, as well as the rates for proton transfer, kPT, and covalent bond formation, kCov, where kdiss = kPT + kCov, as a function of ion trap collisional activation conditions. For each ion activation amplitude, CID spectra are collected as a function of time while monitoring the rate of loss of the precursor ion as well as the rates of appearance of the proton transfer and covalent reaction (i.e., the ion generated by loss of the leaving group) products. By attaching the strongly interacting sulfonate group to the portion of the reagent that remains bonded to the analyte ion, we generate the condition in which the rate-determining step for observation of the loss of the leaving group is the barrier for covalent reaction (i.e., the ‘case 1’ condition described in our earlier work ). The following sections relate our findings with respect to dissociation rates of ion/ion complexes comprised of peptide ions with the three different reagents, as a probe of relative reactivity. We employ DDC as the activation method for our detailed kinetics measurements because it is a broad-band activation approach and is, therefore, much less tuning-intensive relative to resonance ion acceleration based on the use of a supplementary AC signal. However, based on the finite time required to reach the full DC voltage in our DDC experiments, rates greater than roughly 30 s–1 could not be measured reliably because most, if not all, of the precursor ions were depleted before the final DC level was reached. It is straightforward to achieve higher dissociation rates using resonance AC CID. For this reason, a few spectra obtained using AC CID at relatively high dissociation rates are included here to illustrate the partitioning between proton transfer and covalent reaction under such conditions.
This work demonstrates gas-phase nucleophilic ion/ion reactions between triazole-ester reagents and various common neutral nucleophiles. Similar to other gas-phase ion/ion reactions that lead to selective covalent bond formation, this nucleophilic attack proceeds via a long-lived complex facilitated by the strong electrostatic interaction between anchoring groups on the analyte and the reagent (i.e., a sulfonate group on the reagent and a protonated site on the analyte). All of the experimental results in this study are consistent with triazole-ester reagents being more reactive than the analogous NHS-ester reagent, which is also consistent with a lower transition state barrier obtained via DFT calculations with a model primary amine. The triazole-ester reagents are the first to show efficient gas-phase reactivity with an imidazole, which makes these reagents less ‘selective’ than analogous NHS-ester reagents. In all cases, regardless of reagent or nucleophile, activation conditions can play a major role in the observed reaction efficiency. The main competing reaction, proton transfer, is increasingly favored as the activation conditions become more energetic. These nucleophilic displacement reactions are, therefore, most efficient using conditions that give rise to relatively low dissociation rates (e.g., low amplitudes and long times in ion trap CID).
This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award number FG02-00ER15105 and by the National Institutes of Health under Grant GM R37 45372. Frank Londry of Sciex is acknowledged for modifying the Sciex 5600 instrument to enable DDC experiments.
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