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Infrared multiple photon dissociation spectroscopy of cationized canavanine: Side-chain substitution influences gas-phase zwitterion formation

Dedicated to Professor Terry McMahon on the occasion of his 70th Birthday and in recognition of his outstanding contributions to gas-phase ion chemistry, thermochemistry, and spectroscopy.
https://doi.org/10.1016/j.ijms.2017.08.009Get rights and content

Highlights

  • We obtained IRMPD spectra for CavH+ and CavM+ (M = Li, Na, K, Cs).

  • Experimental spectra are compared with calculated spectra from DFT calculations.

  • CavH+, CavLi+ and CavNa+ were found to be charge-solvated structures in the gas phase.

  • CavK+ was found to exist in a mixture of charge-solvated and salt-bridge isomers.

  • CavCs+ was found to be a salt-bridge structure in the gas phase.

Abstract

Infrared multiple photon dissociation spectroscopy was performed on protonated and cationized canavanine (Cav), a non-protein amino acid oxy-analog of arginine. Infrared spectra in the XH stretching region (3000–4000 cm−1) were obtained at the Centre Laser Infrarouge d’Orsay (CLIO) facility. Comparison of the experimental infrared spectra with scaled harmonic frequencies at the B3LYP/6-31+G(d,p) level of theory indicates that canavanine is in a canonical neutral form in CavH+, CavLi+, and CavNa+; therefore, these cations are charge-solvated structures. The infrared spectrum of CavK+ is consistent with a mixture of Cav in canonical and zwitterionic forms leading to both charge-solvated and salt-bridged cationic structures. The Cav moiety in CavCs+ is shown to be zwitterionic, forming a salt-bridged structure for the cation. Infrared spectra in the fingerprint region (1000–2000 cm−1) obtained at the FELIX Laboratory in Nijmegen, Netherlands support these assignments. These results show that a single oxygen atom substitution in the side chain reduces the stability of the zwitterion compared to that of the protein amino acid arginine (Arg), which has been shown previously to adopt a zwitterionic structure in ArgNa+ and ArgK+. This difference can be explained in part due to the decreased basicity of Cav (PA = 1001 kJ/mol) as compared to arginine (PA = 1051 kJ/mol), but not entirely, as lysine, which has nearly the same proton affinity as Cav, (∼993 kJ/mol) forms only canonical structures with Na+, K +, and Cs+. A major difference between the zwitterionic forms of ArgM+ and CavM+ is that the protonation site is on the side chain for Arg and on the N-terminus for Cav. This results in systematically weaker salt bridges in the Cav zwitterions. In addition, the presence of another hydrogen-bonding acceptor atom in the side chain contributes to the stability of the canonical structures for the smaller alkali cations.

Introduction

As the building blocks of proteins and peptides, amino acids are fundamentally important biological species. In solution at physiological pH, amino acids occur naturally as zwitterions, with a protonated amino terminus and a deprotonated carboxylic acid group [1]. In contrast, the large energy cost to deprotonate amino acids in the gas phase (>1330 kJ/mol) [2], [3] is not fully compensated for by the energy savings from protonating the amino terminal group (<1050 kJ/mol) [4], [5], leading to canonical (COOH, NH2) structures for gas-phase amino acids. For the simplest amino acid, glycine, the zwitterionic form lies nearly 90 kJ/mol above the lowest energy canonical structure [6]. The stability of the zwitterionic form of isolated gas-phase amino acids is affected by the acidity of the COOH group and the basicity of the amino terminus/side chain. Arginine (Arg) is the most basic of the twenty protein amino acids (PAA) with a PA of 1051 kJ/mol [7], [8]. The increased basicity and the availability of multiple hydrogen- bonding sites in arginine helps to stabilize the zwitterionic form, and the difference in energy between the zwitterion and the canonical forms drops to around 15 kJ/mol [9]. Neutral arginine has been shown experimentally to be canonical through infrared cavity ring-down experiments [10].

Small perturbations can affect the relative stability of the zwitterionic form of neutral amino acids relative to the canonical form. Addition of water molecules must lead to the zwitterionic forms of the amino acids becoming more stable, and many computational and experimental studies have been performed in order to determine the exact number of water molecules needed to cause the shift in stability [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]. A recent study by Perez de Tudela and Marx used spin- component scaled-MP2 to predict that the addition of nine water molecules in a specific bifurcated wire orientation is sufficient to cause the zwitterion form of glycine to be more stable than the canonical structure [22]. This number is between four and five for aliphatic amino acids, drops to around three waters for lysine, and it is predicted that a single water is sufficient to stabilize the zwitterionic form of arginine [23]. Bush et al. [24] showed that a single water molecule is sufficient to stabilize the salt-bridged structure of ArgLi+ relative to the charge-solvated isomer, which is the more-stable species in the gas-phase without the water molecule (see below) [25], [26].

Amino acids can form zwitterionic species when complexed to other molecules. The neutral dimer of arginine is predicted to contain one of the neutral arginine molecules in its zwitterionic state [27], and in the proton-bound dimer ion of Arg, hydrogen-deuterium exchange (HDX) [28] and Blackbody Infrared Dissociation (BIRD) [29] spectroscopy have been used to show that one of the neutrals is zwitterionic. In contrast, work by Wu et al. showed that the proton-bound dimer of lysine (less basic by ∼50 kJ/mol) is a charge-solvated structure [30].

The richest area of gas-phase amino acid zwitterion research centers on the ability of alkali and other metals to stabilize the zwitterionic form of amino acids through salt-bridged structures. Early ion mobility work from Bowers [31], a BIRD study by Jockush et al., [32] and a kinetic method study from Cerda and Wesdemiotis [33] established that coordination of arginine to increasingly larger alkali metals resulted in binding energies that were more consistent with arginine in a zwitterionic form than in a canonical structure. Early IRMPD studies from the Williams group established that arginine forms a salt-bridged structure when complexed with Na+, K+, and Cs+, but a charge-solvated structure with H+ and Li+ [25], [26].

IRMPD spectroscopy has become an invaluable tool for determining the structure of trapped gas-phase ions [34], [35], [36], [37]. Tunable infrared radiation from free-electron lasers [38], [39], [40] and more recently from bench-top OPO/OPA lasers [25], [41], [42], [43], [44], [45] has allowed vibrational spectroscopy to be applied to a variety of gas-phase cations and anions. Many recent studies of the structure of ionized amino acids and peptides using IRMPD have been carried out including investigations of their zwitterion/canonical forms [24], [25], [26], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], low-energy conformations of protonated amino acid clusters [30], [51], [74], [75], [76], [77], and hydration of ionized amino acids [41], [42], [78], [79], [80], [81], [82], [83], [84].

Of particular relevance to this work are recent studies on the structure of protonated and alkali-metallated arginine [25], [26] and on the structure of metallated lysine [52], [61]. In these studies, IRMPD spectroscopy was used to determine that 1) ArgLi+ is a charge-solvated structure with a canonical arginine moiety, 2) ArgNa+, ArgK+, ArgRb+, and ArCs + are salt-bridged structures containing a zwitterionic arginine moiety, 3) LysM+ forms exclusively charge-solvated structures, 4) N-methylation of lysine at either the amino terminus (α-N) or the side chain (ε-N) stabilizes the zwitterionic form of the lysine moiety such that MeLysNa+ and MeLysK+ are salt-bridged structures, and 5) dimethylation of the side chain results in exclusively salt-bridged structures for M = Li+, Na +, and K+. The PA of Lys has been measured to be between 988 and 1007 kJ/mol [85], [86], [87], [88], with recent high-level calculations centering in on a value of 993 kJ/mol [4], [5], [61]. Bush et al. calculated proton affinities for ε-NMeLys, α-NMeLys, and Me2Lys to be 997, 1008, and 1014 kJ/mol, respectively. The PA for arginine has recently been re-determined to be much higher than the lysine analogs (1051 kJ/mol) [8]. These studies reveal that the proton affinity of the isolated amino acids in and of itself is a poor predictor for whether they will adopt canonical or zwitterionic structures when complexed to alkali metals as the only salt-bridged cation containing lithium is the Me2LysLi+. Instead, one must consider the basicity in concert with the hydrogen-bonding ability of the side chain of the amino acid, the identity of the ionized groups, and the different metal binding motifs available to the amino acid. Given these results, we were inspired to investigate the structures of a series of metallated cations of canavanine, an oxy-analog of arginine with an oxyguanidino group as its side chain.

We have been interested in the gas-phase chemistry of the so-called “non-protein” amino acids (NPAA), which are naturally occurring species that are not used for protein/peptide synthesis [89], [90]. These compounds are ubiquitous in nature and are often found as secondary products of plant metabolism. Many NPAAs are structurally similar to one or more of the 20 protein amino acids (PAA) and can compete with them in a variety of biochemical processes including mis-incorporation into proteins [91], [92], [93], [94], [95], [96], [97], [98], [99], [100], [101], [102], [103], [104], [105], [106]. In addition to their biological significance, these species are excellent models for examining the subtle interplay between structure and energetics in amino acids. We have measured gas-phase proton affinities [107] and acidities [108] for proline-analogs using the extended kinetic method and recently showed that the six-membered ring analog, pipecolic acid, leads to a selective fragmentation mechanism when inserted into peptides [109]. We have also determined proton affinities and gas-phase acidities of homologs of lysine, serine, and cysteine [87], [110].

In 2006, we measured the proton affinity of canavanine (Cav, Scheme 1), an oxy-analog of the PAA arginine, that results from oxygen atom substitution for the δ-CH2 group [111]. Using the extended kinetic method in a quadrupole ion trap instrument, we determined a PA for Cav of 1001 ± 9 kJ/mol. Thus, the single atom substitution in the side chain results in a nearly 50 kJ/mol reduction in proton affinity. This effect has been observed in solution, and the pKa of the oxyguanidino side-chain has been measured to be ∼7 units [93], [112]. Because of the similarity in structure between Cav and Arg, most organisms’ t-RNA synthetase molecules cannot differentiate between them, which allows for facile mis-incorporation of Cav into proteins [102], [106], [113], [114]. Cav is a potent natural insecticide [102], [106], [115], has been investigated for use as an anti-cancer drug [113], [116], and has been shown to increase the potency of other anti-cancer therapies [117], [118], [119]. The decrease in basicity of the side chain should result in less stable zwitterionic structures for Cav relative to Arg, and the addition of another hydrogen- bonding acceptor atom in the side chain should allow for different hydrogen-bonding schemes to be present in Cav. We present here a combined experimental/computational study of the structure of CavH+, CavLi+, CavNa+, CavK+, and CavCs+ and show that the behavior of the CavM+ ions is intermediate between that of ArgM+ (salt- bridged structure for Na+, K+, and Cs+) and that of LysM+ (charge-solvated structure for all cations).

Section snippets

CLIO

IRMPD spectra for several species were obtained using the CLIO free- electron laser (FEL) in Orsay, France. An infrared spectrum for CavH+ in the fingerprint region (∼1000–2000 cm−1) was obtained using the FEL in a modified Bruker Esquire quadrupole ion trap instrument [120], [121]. The laser light from the FEL comes in 8 μs-long macropulses at a repetition rate of 25 Hz. The average laser power was about 500 mW, which corresponds to a micropulse energy of 40 μJ, and a macropulse energy of 20 mJ. The

Materials

Canavanine, LiCl, NaCl, KCl, and CsCl were purchased from Sigma Aldrich (St. Louis, MO) and were used as provided with no further purification.

Canavanine and protonated canavanine

The oxyguanidino group in Cav has five tautomers of two general classes: can1 and can2. Four tautomers have the cav_can1 (a, Scheme 2) connectivity, which has a hydrogen atom on the ε-nitrogen (adjacent to the oxygen) and a formal double bond between the ζ-carbon and one of the η- nitrogen atoms (see Scheme 1 for lettering of Cav sites). These four tautomers comprise two sets of two cis/trans isomers that differ in terms of which η-nitrogen has the double bond. All four tautomers were

Conclusions

Infrared spectra in the Xsingle bondH and fingerprint regions of protonated and alkali-metallated canavanine were obtained using IRMPD. In contrast to arginine, which forms charge-solvated ions with Li+ and salt-bridged structures for Na+, K+, Rb+, and Cs+, and lysine which forms charge- solvated structures for all alkali metal ions, CavLi+ and CavNa+ were found to be charge-solvated, CavK+ was found to be a mixture of charge-solvated and salt-bridged isomers, and CavCs+ was found to be mostly salt

Acknowledgements

Funding for this project was generously provided by the National Science Foundation (CHE0911244 and CHEM:1464763), the National Institutes of Health, (1R15GM116180-01), and NWO Chemical Sciences under VICI project no. 724.011.002 (FELIX).

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