HAL Id: hal-03031677https://hal.archives-ouvertes.fr/hal-03031677

Submitted on 27 Nov 2021

HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.

Binding motifs in the naked complexes of target aminoacids with an excerpt of antitumor active biomolecule.

An ion vibrational spectroscopy assayBarbara Chiavarino, Rajeev Sinha, Maria Elisa Crestoni, Davide Corinti,Antonello Filippi, Caterina Fraschetti, Debora Scuderi, Philippe Maitre,

Simonetta Fornarini

To cite this version:Barbara Chiavarino, Rajeev Sinha, Maria Elisa Crestoni, Davide Corinti, Antonello Filippi, et al..Binding motifs in the naked complexes of target amino acids with an excerpt of antitumor activebiomolecule. An ion vibrational spectroscopy assay. Chemistry - A European Journal, Wiley-VCHVerlag, 2021, �10.1002/chem.202003555�. �hal-03031677�

Binding motifs in the naked complexes of target amino acids with an excerpt of antitumor active

biomolecule. An ion vibrational spectroscopy assay

Barbara Chiavarino,1 Rajeev K. Sinha,2 Maria Elisa Crestoni,1 Davide Corinti,1 Antonello Filippi,1 Caterina Fraschetti,1 Debora Scuderi,3 Philippe Maitre,3 Simonetta Fornarini1* 1Dipartimento di Chimica e Tecnologie del Farmaco, Università di Roma “La Sapienza”, P.le A. Moro 5, I-00185 Roma, Italy 2 Department of Atomic and Molecular Physics, Manipal University, Manipal-576104, Karnataka, India 3 Institut de Chimie Physique, UMR8000, CNRS, Université Paris-Saclay, F-91405, Orsay, France. Abstract. The structures of proton-bound complexes of 5,7-dimethoxy-4H-chromen-4-one (1) and basic

amino acids (AAs), namely histidine (His) and lysine (Lys) have been examined by mass spectrometry

coupled with IR ion spectroscopy and quantum chemical calculations. The choice is founded on the fact

that 1 represents a portion of Glabrescione B, a natural small molecule of promising antitumor activity,

while His and Lys are protein residues lining the cavity of the alleged binding site in the plausible receptor,

though obviously the isolated state of the present study bears little resemblance with the complex

biological environment. Common feature of [1+AA+H]+ complexes is the presence of a protonated AA

bound to neutral 1, in spite of the fact that the gas phase basicity of 1 is comparable to the one of Lys and

His. The carbonyl group of 1 acts as powerful hydrogen bond acceptor. Within [1+AA+H]+ the side chain

substituents (imidazole group for His and terminal amino group for Lys) present comparable basic

properties as the -amino group, taking part to a cooperative H-bond network. The structural assignment,

relying on the comparative analysis of the IRMPD spectrum and calculated IR spectra for the candidate

geometries, derives from an examination over two frequency ranges, 900-1800 cm-1 and 2900-3700 cm-1.

Information gained from the latter one proved especially valuable, for example pointing to the contribution

of species characterized by an unperturbed carboxylic OH- or imidazole NH-stretching mode.

Introduction

The subtle modulation of inter- and intramolecular binding motifs controls the role and function of

biomolecular host-guest adducts such as substrate-enzyme and drug-receptor complexes. Elucidating

conformational preferences and interaction energies is crucially important to understand their biological

activity although the structural characterization represents a challenging goal. The most usual methods are

based on X—ray crystallography and NMR spectroscopy, though each one presents known limitations. NMR

requires relatively major quantities of sample while X-ray analysis needs preliminary crystallization.

Therefore, the availability of alternative approaches is desirable and methods based on mass spectrometry

(MS) have a great potential because they are not exposed to these limitations.

Over the past couple of decades infrared multiple photon dissociation (IRMPD) spectroscopy combined

with mass spectrometry and theoretical calculations has emerged as a powerful tool to obtain structural

information about charged biomolecular complexes in an isolated state.1-5 The structure of naked

biomolecules in the gas phase may not reflect their native geometry in solution. However, it offers a useful

counterpart to the different perspective provided by solid state X-ray diffraction.

In the present contribution IRMPD spectroscopy is applied to inquire about structural features and non-

covalent interactions in ionic complexes of 5,7-dimethoxy-4H-chromen-4-one (1) (Scheme 1). This

molecule is an excerpt of Glabrescione B (Scheme 1), a natural small molecule found in the seeds of Derris

Glabrescens (Leguminosae). Glabrescione B is capable to inhibit glioma cell growth and represents a

promising candidate in the treatment of this lethal tumor.6-9 The basic unit chromone is an oxygen-

containing heterocyclic molecule forming the core of a large family of compounds widely spread in nature,

especially in the plant kingdom.10 With an aryl pendant in position 3 bearing two O-prenyl groups, the

isoflavone Glabrescione B has been suggested to operate by directly binding to the zinc finger domain of

Gli1 transcription factor and to inhibit its binding to DNA.7,8

Scheme 1. Structure of 5,7-dimethoxy-4H-chromen-4-one (1) and of Glabrescione B. Atom numbering is indicated on the chromone core of 1. The ionic complexes to be assayed by IRMPD spectroscopy are proton bound heterodimers formed by 1

and a basic amino acid, namely lysine and histidine. In a sequence of increasing molecular complexity the

earliest models to be sampled are protonated 1 and its adduct with 4(5)-Methylimidazole (MeIm, existing

as rapidly equilibrating tautomers). MeIm stands for the imidazole side chain substituent of histidine (His).11

The pKa near neutrality of this bifunctional group is responsible for the versatility of His residues in general

acid or base catalysis.12 Also due to this reason, His residues are frequently present in the active site of

protein enzymes. Lysine (Lys) is the second most basic (following after arginine and close to proline) among

the proteinogenic amino acids in water where the -amino group surpasses the -amino group in basicity.

A role has been ascribed to Lys residues in the interaction of Glabrescione B with Gli1.7

Proton bound complexes involving an amino acid (AA) partner have been profitably investigated by IRMPD

spectroscopy. IRMPD spectroscopy of proton-bound dimers consisting of valine and primary and secondary

amines of increasing basicity has revealed a transition from complexes holding protonated valine to

complexes of protonated base and neutral valine.13 Structural aspects of proton-bound homo and hetero

dimers composed of amino acids with aliphatic and aromatic side chains have been investigated by IRMPD

spectroscopy and electronic structure calculations.14-18 While isolated amino acids exist in the gas phase

exclusively in canonical form, in a complex with a charged partner the zwitterionic form can be stabilized

due to the presence of strong hydrogen bonds.18,19 IRMPD spectroscopy studies of proton bound

homodimers have shown a relationship between the stability of so-called salt bridge structures, holding a

zwitterionic AA, and the proton affinity of the AA.20-25 Gas-phase clusters of protonated methylamine and

several phenylalanine derivatives were shown by IRMPD spectroscopy to vary from charge solvated

structures to salt bridge structures, according to the protomeric form of the amino acid.26 Methylamine was

chosen to represent primary amino groups that are ubiquitous in biological system, in a similar perspective

as in the present study.

5,7-dimethoxy-4H-chromen-4-one (1) Glabrescione B

OMe

MeO

OMe

MeO

OC5H9

OC5H9

1

2

3 6

5 4

8

7

Experimental section

Materials

5,7-dimethoxy-4H-chromen-4-one (1) is commercially available (Sigma-Aldrich/Merck) and was used as

received. The same supplier provided the other chemicals (4(5)-methylimidazole, L-lysine, L-histidine) and

research grade methanol used as solvent.

Mass spectrometry

Collision induced dissociation (CID) experiments were performed using a commercial hybrid triple-

quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source (2000 Q-TRAP Applied

Biosystems), with a Q1q2QLIT configuration (Q1 = mass analyzing quadrupole; q2 = N2-filled collision cell;

QLIT, linear ion trap). Electrosprayed ions obtained by infusion of a 10-5 M methanol solution of the selected

analytes were mass-selected (Q1) and allowed to collide with N2 in q2 at a nominal pressure of 1.1 × 10-5

mbar. Finally, the product ions were monitored scanning QLIT. The experimental parameters used were: ion

spray voltage at 5.5 kV, curtain gas at 20 psi, GS1 at 20 psi, declustering potential at 60 V and entrance

potential at 5 V.

IRMPD spectroscopy

Two distinct energy ranges were explored in IRMPD experiments. The fingerprint range (900-1900 cm-1)

was investigated using the beamline of the free electron laser (FEL) at the Centre Laser Infrarouge d’Orsay

(CLIO). In order to optimize the laser power in the frequency range of interest, the electron energy of the

FEL was set at 44,4 MeV. The FEL beamline is coupled with a hybrid FT-ICR tandem mass spectrometer

(APEX-Qe Bruker) equipped with a 7.0 T actively shielded magnet and a quadrupole-hexapole interface for

mass-filtering and ion accumulation, under control of the commercial software APEX 1.0 as described

previously.27 The ion of interest was selected in the quadrupole and then accumulated for 1 s in the

hexapole containing a buffer gas to allow collisional cooling before being transferred into the ICR cell. Ions

were then irradiated with the IR FEL light, after which the resulting mass spectra, recorded by averaging

four accumulations, were analyzed. The irradiation time was varied from 220 ms to 5 s depending on the

system and the use of up to 4 attenuators was necessary during the analysis of the proton bound

heterodimers to avoid saturated signals. Vibrational modes associated with the X-H (X = N, O or C) stretches

were investigated by recording IRMPD spectra in the 2900-3700 cm-1 frequency range. To this end, an

optical parametric oscillator/amplifier (OPO/OPA) (LaserVision) coupled to a Paul ion trap mass

spectrometer (Esquire 6000+, Bruker Daltonics), was used as previously illustrated.28 The average output

energy from the OPO/OPA laser (3-4 cm-1 bandwidth) was 17-20 mJ/pulse. In the ion trap, ions were first

accumulated for 10 ms and then mass-selected prior to IR irradiation. In the OPO/OPA experiments, the

irradiation time was varied from 0.3 to 1 s and each mass spectrum was obtained by the averaging of four

accumulations. IRMPD spectra were obtained by plotting the photofragmentation yield R (R = -

ln[Iparent/(Iparent + ΣIfragment)], where Iparent and Ifragment are the integrated intensities of the mass peaks of parent

and fragment ions, respectively) as a function of the wavenumber of the IR radiation.

Computational details

The optimized geometries and frequency analysis of [1+H]+ [1+MeIm+H]+ [1+His+H]+ ions [1+Lys+H]+ ions

reported in the following sections are the result of hybrid DFT calculations at B3LYP/6-311++G** level of

theory. A collection of initial trial structures for the sampled ions was obtained by performing a Monte

Carlo conformational survey employing the MMFF molecular mechanics model with the Spartan’16

program package as well as using chemical intuition. The plausible low energy structures were submitted

to hybrid DFT calculations at increasing level of theory, eventually yielding optimized structures

characterized by all positive frequency vibrational modes, thereby corresponding to local minima. In this

procedure binding motifs and low energy geometries already reported to describe the most stable

structures for protonated His and Lys were also obtained. Representative structures have been selected

from each set of closely related conformers as a basis for the results and discussion section. Calculated

vibrational frequencies were used to determine zero-point energies and to obtain relative enthalpies (H298)

and Gibbs free energies (G298) at 298 K. Harmonic frequencies are scaled with factors of 0.98 (0.96) in the

900-1900 cm-1 (2900-3700 cm-1) range, according to reported values that were found to properly account

for experimental frequencies in previous work using the same level of theory.29,30 Computed IR absorption

spectra are convoluted with a Lorentzian line shape with a full width at half maximum (fwhm) of 20 cm-1 (5

cm-1) in the 900-1900 cm-1 (2900-3700 cm-1) range for convenient comparison with the experimental IRMPD

spectra.

All DFT calculations were performed using the Gaussian09 software package.31

Results and Discussion

Formation of protonated species and proton-bound complexes. Sampling by activated dissociation

When a methanol solution of 1 is analyzed by ESI-MS in positive ion mode, the protonated species, [1+H]+,

is revealed at m/z 207 in significant abundance (Figure S1 in the Supporting Information, SI). The relative

ease of 1 to undergo protonation is ascribed to the stability and aromatic character of the carbonyl

protonated species which acquires the structure of a 4-hydroxybenzopyrylium ion.32 In the presence of

selected added solutes, namely MeIm and amino acids (AAs) endowed with basic groups in their side chain

(His, Lys), proton bound heterodimers are readily observed, namely [1+MeIm+H]+ and [1+AA+H]+ (Figures

S2-3 and S5). In contrast, no adducts are obtained in the presence of simple aliphatic AAs such as glycine or

valine, suggesting the requirement of a significantly basic functionality to provide adequate stabilization for

the complex to be observed. The isolated ionic species can be assayed either by collision induced

dissociation (CID) or by IR multiple photon dissociation. In both cases a stepwise activation process is

induced either by translational to internal energy conversion in a collision event or by direct absorption of

IR photons. The activation process triggers the fragmentation along the lowest energy dissociation channel.

[1+H]+ dissociates by loss of either 15u (likely a methyl group, 66%) or 44u (possibly CH3CHO, 33%) (Figure

S1). The dissociation of protonated substituted chromones has revealed fragmentation channels occurring

via ion-neutral complexes.33 However, the dissociation pathway of [1+H]+ is not the actual focus. The

dissociation of the proton bound heterodimers is more interesting because it releases the two components

either as neutral molecule or as protonated species. In doing so, it reveals the relative gas phase basicity

(GB) of the two partners.34-36

[1+MeIm+H]+ undergoes dissociation by exclusive loss of MeIm, indicating 1 to be more basic (Figure S2).

The GB of MeIm, equal to 921 kJ mol-1,37 is then a lower limit for the GB of 1.

The competitive dissociation of 1 or AA as neutral fragment from [1+AA+H]+ ions is more balanced (Eq. 1).

(1)

In the case of histidine, the ratio of 1 versus His loss is 45/55 under CID (Figure S3) and 25/75 under IRMPD

conditions (Figure S4). In the case of lysine, the 1 versus Lys loss ratio is 20/80 under CID (Figure S5) while

prevailing Lys departure (1 versus Lys loss ratio equal to ca. 10/90) is observed under IRMPD conditions

(Figure S6). The photofragmentation appears to be somewhat more selective than the CID process and

both activation methods indicate the GB of 1 to be close to the GB of the two amino acids. Reviewed values

are 949±4 kJ mol-1 for GB(His) and 947±4 kJ mol-1 for GB(Lys).38 These data, supporting a comparable GB of

the two AAs, slightly favoring His, are consistent with the bias for neutral loss of the less basic AA from the

sampled [1+AA+H]+ protonated heterodimers. Interestingly, the GB of 1 is exceptionally high when it is

compared with common GB values for compounds holding a carbonyl group as basic site.39 This outcome is

in line, though, with the expected high stability of the protonated form, the 4-hydroxybenzopyrylium ion.

Indeed, 1 approaches the basic properties of tropone, which upon protonation yields an aromatic

hydroxytropylium ion.40,41

Structural and vibrational features of [1+H]+ ions

The structure of [1+H]+ ions has been investigated by DFT calculations at B3LYP/6-311++G** level. The

results show the carbonyl protonated species to be characterized by the OH+ ···OCH3 hydrogen bond

interaction, yielding the optimized geometry Chrom_1 as the most stable one (Figure 1). The orientation of

the methoxy group in C7 has a minor effect, placing the relative energy of conformer Chrom_2 at 5.9 kJ

mol-1, while removal of the H-bond interaction in conformer Chrom_3 destabilizes protonated 1 by 31.0 kJ

mol-1. The IR spectra for the lowest energy candidates Chrom_1 and Chrom_2 are comparable and may

both well account for the experimental IRMPD spectrum of [1+H]+ (Figure 2).

Figure 1. Representative structures of [1+H]+ ions at B3LYP/6-311++G** level of theory. Relative enthalpy

and free energy (in italics) at 298 K are reported in brackets (kJ mol-1). Distances are in Å.

The IR spectra of the optimized structures plotted in Figure 2 and the vibrational mode assignments listed

in Table S1 allow to ascribe the most pronounced band at 1602 cm-1 in the IRMPD spectrum to C-C

[1+AA+H]+

[1+H]+ + AA

[AA+H]+ + 1

Chrom_2

(5.9; 5.9)

Chrom_1

(0.0; 0.0)

1.775 1.779

Chrom_3

(31.0; 31.2)

stretches of the ring frame calculated at 1645 and 1603 cm-1 in the IR spectrum of Chrom_1. A third

predicted highly active mode at 1569 cm-1, associated to the in plane COH bending, is apparently not

contributing in the experimental spectrum. This mode may be potentially diagnostic of the presence and

role of the OH+ ···OCH3 hydrogen bond because its frequency is blue-shifted in the IR spectrum of Chrom_1

when compared to the 1536 cm-1 wavenumber in the IR spectrum of Chrom_3. However, the in-plane COH

bending has been often found to be poorly active when the group is engaged in H-bonding.42-44 This missing

vibrational signature thus provides circumstantial evidence for the presence of H-bonded species

Chrom_1–2.

Figure 2. IRMPD spectrum of [1+H]+ ions (bottom panel, red and gray (x15) profiles), and calculated IR

spectra of Chrom_1-3 optimized structures.

Structural and vibrational features of [1+MeIm+H]+ ions

The proton-bound complex of 1 and 4(5)-methylimidazole (MeIm) is described by the lowest energy

geometries Chrom_MeIm_1-3 depicted in Figure 3. The noteworthy common feature is the proton residing

on the former aza group of MeIm that is engaged in a hydrogen bond with the carbonyl oxygen of 1. This

finding is unexpected because the relative GBs suggest protonation on 1 to be favored. In fact, as described

in the previous paragraph, the isolated molecules display thermodynamically favored protonation of 1 as

clearly evidenced by the dissociation products of the proton bound [1+MeIm+H]+ heterodimer, namely

Chrom_3

400

800

Chrom_2

400

800

Chrom_1

400

800

[1+H]+

1

2

1000 1200 1400 1600 1800

Wavenumber (cm-1)

1171 1223

1317 1422 1450

1602 R

IR in

ten

sit

ies (

km

mo

l-1)

[1+H]+ and neutral MeIm. The observed shift in protonation site is justified by the relative strength of the

C=O··· +HN(MeIm) hydrogen bond interaction when compared with the OH+··· N(MeIm) alternative H-bond

pattern,45 overlaying the intrinsic basicity properties of the isolated molecules.46

Figure 3. Representative structures of [1+MeIm+H]+ at B3LYP/6-311++G** level of theory. Relative enthalpy

and free energy (in italics) at 298 K are reported in brackets (kJ mol-1). Distances are in Å.

The lowest energy Chrom_MeIm_1 isomer is characterized by the favored orientation of the C7-methoxyl

group, already observed in the optimized structures of [1+H]+, while the different position of the methyl

group in the MeIm unit does not affect the energy of the [1+MeIm+H]+ heterodimer to any significant

extent (see the relative enthalpy of Chrom_MeIm_2 at 1 kJ mol-1). In view of the -electron rich character

of the imidazole ring, a specific search has been carried out for any -stacking interaction that may engage

MeIm and protonated 1 and thus stabilize the heterodimer in a proper arrangement. However, geometry

optimization from tentative starting structures did not yield any minimum, converging rather to

Chrom_MeIm_1.

A number of alternative geometries whereby protonated 1 interacts with neutral MeIm, oriented towards

different sites of the protonated partner, have been also investigated. However, their higher energy

content rather disproves their contribution, as further testified by their calculated IR spectra, meagerly

compatible with the sampled species (Figure S7).

In fact, the IRMPD spectrum of [1+MeIm+H]+ ions recorded both in the fingerprint range (900-1800 cm-1)

and in the X-H (X=O,N,C) stretching range (2900-3700 cm-1) is well accounted for by the IR spectra of the

low energy geometries Chrom_MeIm_1-2 (Figure 4). In fact both structures should contribute to the

thermal population at the room temperature of the experiment.

The theoretical IR spectra support the assignment of the major features in the IRMPD spectrum of

[1+MeIm+H]+ ions Table S2. The stretching of the free N-H at 3485 cm-1 accounts for the most prominent

band in the 2900-3700 cm-1 range, where a minor feature at 3167 cm-1 is assigned to CH stretches of the

imidazole ring. The band at 1586 cm-1 is due to the C=O stretching, a remarkably red shifted value for a

carbonyl group, reflecting its role as H-bond acceptor. The band at 1630 cm-1 encompasses CC stretching

modes of both chromone and imidazole ring frames while a distinct feature at 1300 cm-1 is associated to

ring breathing and CH in-plane bending modes.

Chrom-MeIm_1

(0.0; 0.0)

1.546 1.551

Chrom-MeIm _3

(6.9; 6.5)

Chrom-MeIm _2

(1.1; 1.6)

1.554

Figure 4. IRMPD spectrum of [1+MeIm+H]+ ions and calculated IR spectra of Chrom_MeIm_1-3.

Structural and vibrational features of [1+His+H]+ ions

The proton-bound complex of Chrom and His, [1+His+H]+, is characterized by several low lying structures,

displaying the common feature of being composed of protonated His associated with neutral 1. In other

words, in spite of the comparable GB of 1 and His emerging from the protonated heterodimer dissociation,

in the [1+His+H]+ complex the proton resides on the amino acid. However, two families of protonated His

have been recognized due to protonation on either one of the two competing basic sites, namely the amino

group and the imidazole substituent on the side chain. Both tautomers comprise a large array of different

conformers.47-53 The most stable structures result from protonation on the imidazole ring while the most

R

IR in

ten

sit

ies (

km

mo

l-1)

3485

3167

1630

1586

1300

1411 1166

Chrom-MeIm_1

Chrom-MeIm_2

Chrom-MeIm_3

400

800

50

100

150

50

100

150

400

800

400

800

50

100

150

Wavenumber (cm-1)

[1+MeIm+H]+

1000 1200 1400 1600 1800 3000 3200 3400 3600

0.5

1.0

1.5

1

2

3

4

favored NH2 protonated conformer is ca. 6 kJ mol-1 higher in energy relative to the imidazole protonated

absolute minimum.48

The IRMPD spectrum of protonated histidine has been reported both in the 600-1800 cm-1 fingerprint

range54 and in the 3200-3700 cm-1 range44 and ascribed to imidazole protonated species, the major

contribution due to a conformer stabilized by an imidazole-NHNH2O=C hydrogen bond network.54 Also

conformer selective IR spectra of protonated His using cold ion spectroscopy reveal structures where the

proton resides on the imidazole ring and is engaged in H-bonding with the amino group.55 In contrast, when

protonated His is complexed with 18-crown-6 ether (18C6) IRMPD spectroscopy and theoretical

calculations indicate that 18C6 binding shifts the competition towards amino group protonation.56,57 This

outcome is suggested to be due to the presence of three strong and nearly equivalent NH+···O interactions

occurring in the complexed -amino protonated species. This binding motif is found to play a role also in

the doubly charged complex of arginine with a 18C6 derivative, reported in an IRMPD spectroscopy study

of the binding of protonated arginine with the polyether macrocycle used as a model for the development

of arginine receptors.58

Within the proton bound [1+His+H]+ complex, a network of hydrogen bonding interactions characterizes

the conformational arrangement of the protonated (His+H+) and the neutral (1) partners. Three

representative low energy geometries are depicted in Figure 5 to stand for each of the two families of

proton bound heterodimers, hosting His protonated on either the imidazole (Chrom_His_1 and

Chrom_His_3) or the amino (Chrom_His_2) group.

Figure 5. Representative structures of [1+His+H]+ ions at B3LYP/6-311++G** level of theory. Relative

enthalpy and free energy (in italics) at 298 K are reported in brackets (kJ mol-1). Distances are in Å.

Chrom_His_1 and Chrom_His_2 lie at approximately the same energy level so that both tautomers are

expected to contribute to the sampled ion population and in fact the experimental IRMPD spectrum

supports the presence of both species when it is compared with the calculated IR spectra as displayed in

Figure 6.

Chrom_His_1

(0.0; 0.0)

Chrom_His_2

(2.3; 2.2)

Chrom_His_3

(2.6; 5.2)

1.579C

2.030

1.569

1.770

1.908

1.588

1.846

Figure 6. IRMPD spectrum of [1+His+H]+ ions and calculated IR spectra of representative isomers (Chrom_His_1-3). In this regard, the NH/OH stretching range is especially revealing. The most pronounced band at 3560 cm-1

is a common feature to all structures possessing a free OH in a syn carboxylic group (Chrom_His_1 and

Chrom_His_2), being calculated at 3597-3592 cm-1 Table S3. The second intense band at 3496 cm-1 is

instead characteristic of the NH stretching of imidazole when the group is not involved in H-bonding, as

found in Chrom_His_2. The minor feature at 3170 cm-1 is accounted for by the CH stretching of the

imidazole group in Chrom_His_1, properly matching the corresponding mode revealed in the IRMPD

spectrum of [1+MeIm+H]+ ions and supporting the presence of an imidazole protonated group. A broad

absorption extending through the 3090-3340 cm-1 range is associated to NH stretches engaged in H-

bonding. H-bonding is documented in IRMPD spectroscopy to result in broad structureless features to the

point of being poorly resolved from background signal.59-62 So these features do not convey any useful

structural information. The fingerprint range does not allow to discriminate the contribution of different

tautomers because of the comparable wavenumber and activity of the IR modes in the spectra of the

reported structures. The IRMPD spectrum is also similar here to the spectrum of [1+MeIm+H]+ ions. An

obvious addition is the band at 1786 cm-1 attributed to the C=O stretching of the carboxylic group. The

Wavenumber (cm-1)

IR in

ten

sit

ies (

km

mo

l-1)

3560

3496

1786

1636 1600

1416

1302

1161 3170

400

800

400

800

0.5

1.0

1.5

1000 1200 1400 1600 1800

[1+His+H]+

Chrom_His_1

400

800

Chrom_His_2

Chrom_His_3

1

2

3

4

3000 3200 3400 3600

50

100

150

50

100

150

50

100

150

R

dominant, partly merging bands at 1600 and 1636 cm-1 are due to ring CC stretching modes with additional

contributions of the umbrella mode of NH3 in Chrom_His_2 and the in-plane NH2 scissoring, respectively. In

the lower wavenumber range the particularly pronounced band at 1416 cm-1 suggests some contribution of

Chrom_His_3, characterized by a distinct band at 1392 associated to OH bending of the COOH group. To

summarize, protonation on both the amino group and the side imidazole group needs to be invoked to

account for the observed IRMPD spectra. Furthermore, different H-bonding motifs may be prevailing in the

sampled species. For example the NHs of protonated imidazole are H-bond donors to the carbonyl group of

1 and to the -amino group in Chrom_His_1 while the latter acceptor is replaced by the carbonyl group of

an anti carboxyl group in Chrom_His_3. Alternative conceivable structures whereby protonated 1 interacts

with neutral His placed along the border of the chromone ring are higher in energy (by ca. 40 kJ mol-1

relative to the global minimum) and their IR spectrum does not support any relevant contribution to the

sampled population.

Structural and vibrational features of [1+Lys+H]+ ions

As already found for [1+His+H]+, also the lowest energy structures of [1+Lys+H]+ complexes consistently

present protonation on the amino acid rather than on the chromone unit, in agreement with the somewhat

higher basic properties of Lys with respect to His (Figure 7). Between the two competing amino groups,

protonation of the NH2 side chain substituent is more favorable than protonation of the -amino group in

the isolated amino acid, just as in solution.38,49,50,52,63-66 The lowest energy forms of [Lys+H]+ are species

stabilized by a twofold hydrogen bond interaction in which the protonated amino group is hydrogen bond

donor to the N-terminus and to the carbonyl oxygen, as described by the NH3+ NH2 and NH3

+ O=C

network. The IR spectrum calculated for this molecular arrangement has been found to account well for the

major features in the IRMPD spectrum of [Lys+H]+ both in the fingerprint range67,68 and in the 2800-3700

cm-1 range.69

The NH3+ NH2 and NH3

+ O=C bonding scheme characterizes also the low energy geometries of

[1+Lys+H]+ complexes (a representative one is Chrom_Lys_2, shown in Figure 7). Here the third ammonium

hydrogen is engaged in H-bonding with the chromone carbonyl oxygen. In an alternative array of hydrogen

bonds (Chrom_Lys_1 in Figure 7) the -amino group does not interact with the protonated terminal but it

is rather H-bond acceptor towards a carboxylic group in anti conformation. The two isomers, Chrom_Lys_2

and Chrom_Lys_1 are comparable in energy whereas the latter H-bonding arrangement is destabilized by

ca. 8 kJ mol-1 in the free [Lys+H]+ ion.68 This bonding motif is reminiscent of the similar one in Chrom_His_3.

A third representative isomer (Chrom_Lys_3 in Figure 7) bears the additional proton on the -amino group

and lies 9 kJ mol-1 higher in energy relative to the most stable Chrom_Lys_1.

The experimental IRMPD spectrum results from the contribution of multiple structures although the

representative comparably stable Chrom_Lys_1 and Chrom_Lys_2 isomers of the terminal protonated

complex account well for the major features (Figure 8).

Figure 7. Representative structures of [1+Lys+H]+ ions at B3LYP/6-311++G** level of theory. Relative

enthalpy and free energy (in italics) at 298 K are reported in brackets (kJ mol-1). Distances are in Å.

Chrom_Lys_1

(0.0; 0.0)

Chrom_Lys_2

(1.4; 3.2)

Chrom_Lys_3

(7.6; 6.6)

1.874

1.849

1.598

1.629

2.192 1.940

1.643 1.768

Chrom_Lys_1

[1+Lys+H]+

IR in

ten

sit

ies (

km

mo

l-1)

Wavenumber (cm-1)

Chrom_Lys_2

Chrom_Lys_3

R

400

800

400

800

50

100

150

50

100

150

400

800

0.2

0.4

0.6

1000 1200 1400 1600 1800

0.2

0.6

1.0

3000 3200 3400 3600

50

100

150

3563

3433

3350 1750

1633

1590

1419

1300

1169

Figure 8. IRMPD spectrum of [1+Lys+H]+ ions and calculated IR spectra of representative isomers

(Chrom_Lys_1-3)

The free OH stretching at 3563 cm-1 is justified by the syn carboxylic group in Chrom_Lys_2 (and in

Chrom_Lys_3) whereas the OH is involved in H-bonding in Chrom_Lys_1 which contributes to a red shifted

and broadened absorption at 3060-3240 cm-1. The latter feature may encompass the NH stretching of

Chrom_Lys_1 also engaged in H-bonding while the free NH of the protonated amino group that

characterizes the IR spectra of Chrom_Lys_1 (at 3365 cm-1) and Chrom_Lys_2 (at 3353 cm-1) accounts for

the IRMPD band at 3350 cm-1. Other assignments in the OH/NH stretching range as well as in the

fingerprint region are described in Table S4. We rather tend to discard a contribution of Chrom_Lys_3

because the free NH of the protonated backbone amino group is expected to absorb at 3277 cm-1,

corresponding to a flat region in the experimental spectrum. One may also reason that this higher energy

isomer should be prone to rearrange to the more stable Chrom_Lys_2. This reaction would in fact involve a

proton transfer from the protonated backbone amino group to the terminal NH2 that is already engaged as

H-bond acceptor of the proton which is going to shift.

In the 900-1900 cm-1 wavenumber range the IRMPD spectrum of [1+Lys+H]+ resembles closely the one of

[1+His+H]+. The dominant bands at 1590 and 1633 cm-1 are mainly associated to highly coupled CC

stretching and NH bending vibrations and the third pronounced band at 1300 cm-1 is related to ring

breathing and in plane CH bending of the chromone unit. Overall, the most structurally characteristic and

informative portion of the inspected IR spectrum is rather the 2900-3700 cm-1 range.

Conclusions

The interaction between significant portions of biomolecules that are known to exert remarkable

biomolecular activity by mutual binding can be examined at a molecular level by IRMPD spectroscopy. The

two partners are isolated in the gas phase and made ionic by the addition of a proton. Also at physiological

pH the side chain substituent of the sampled amino acids, His and Lys, is at least partially (His) protonated.

In the gas phase, which may be considered as a medium approaching the less polar environment prevailing

in the interior of a protein, the basicity of His and Lys becomes comparable and also similar to the one of

5,7-dimethoxy-4H-chromen-4-one. However, the role of non covalent interactions is already appreciable in

the [1+MeIm+H]+ complex. In fact, in spite of the higher GB of 1, within the complex the proton rather

resides on MeIm, as confirmed by the matching between the experimental IRMPD spectrum of the complex

and the calculated IR spectra of the lowest energy geometries. The basic character of the chromone

carbonyl oxygen appears in its role as powerful H-bond acceptor towards the protonated site of the partner

molecule. This role is in fact maintained in the [1+AA+H]+ complexes where AA is either His or Lys. The

experimental vibrational spectra interpreted by means of computed IR spectra for representative low

energy geometries assist in the structural assignment. In particular, the presence of a nearly unperturbed

NH stretching of the imidazole substituent supports the contribution of a [1+His+H]+ complex protonated

on the amino group together with an imidazole protonated tautomer. The latter isomer conforms to the

most stable structure depicting free protonated His. In contrast, the vibrational features of the [1+Lys+H]+

complex may be interpreted by the contribution of terminally protonated species, which may be a

reflection of both the slightly higher basicity of Lys relative to His and by the fact that in this case

protonation of either terminal or backbone NH2 yields an ammonium ion able to establish up to three H-

bond interactions. It may be underlined that solvation of ammonium hydrogens is responsible for the well

known discrepancy in the basicity order of (CH3)xNH3-x examined in water and in the gas phase. While in the

real case where the bioactive molecule is bound to a peptide or protein, only side chain substituents are

readily available for non covalent interactions, the results obtained in this study point out the subtle

balance governing the preferred coordination mode and proton location in the presence of competing

sites. Furthermore, IRMPD spectroscopy of ESI formed complexes is borne out to provide a suitable tool to

potentially investigate the variety of binding motifs underlying drug-receptor interactions.

References 1. Jasiková, L.; Roithová, J. Infrared Multiphoton Dissociation Spectroscopy with Free-Electron Lasers: On the Road from Small Molecules to Biomolecules. Chem. Eur. J. 2018, 24, 3374-3390. 2. Rijs, A. M.; Oomens, J. IR Spectroscopic Techniques to Study Isolated Biomolecules. In Gas Phase IR Spectroscopy and Structure of Biological Molecules; Rijs, A. M., Oomens, J., Eds.; Topics in Current Chemistry, Vol. 364, Springer: Berlin, 2015; pp. 1-42. 3. Polfer, N. C.; Stedwell, C. N. Infrared photodissociation of biomolecular ions. In Laser Photodissociation and Spectroscopy of Mass-separated Biomolecular Ions; Polfer, N.C., Dugourd, P., Eds.; Lecture Notes in Chemistry, Vol. 83, Springer: Switzerland, 2013; pp. 93-116. 4. Stedwell, C. N.; Galindo, J. F.; Roitberg, A. E.; Polfer, N. C. Structures of biomolecular ions in the gas phase probed by infrared light sources. Ann. Rev. Anal. Chem. 2013, 6, 267-285. 5. Polfer, N. C.; Oomens, J. Vibrational spectroscopy of bare and solvated ionic complexes of biological relevance. Mass Spectrom. Rev. 2009, 28, 468-494. 6. 1H-NMR metabolomics reveals the Glabrescione B exacerbation of glycolytic metabolism beside the cell growth inhibitory effect in glioma Giuseppina D’Alessandro, Deborah Quaglio , Lucia Monaco, Clotilde Lauro, Francesca Ghirga, Cinzia Ingallina, Michela De Martino, Sergio Fucile, Alessandra Porzia, Maria Amalia Di Castro, Federica Bellato, Francesca Mastrotto, Mattia Mori, Paola Infante, Paola Turano, Stefano Salmaso, Paolo Caliceti, Lucia Di Marcotullio, Bruno Botta, Veronica Ghini, Cristina Limatola Cell Commun. Signal. 2019, 17, 108 (1-12) 7. Gli1/DNA interaction is a druggable target for hedgehog-dependent tumors Paola Infante, Mattia Mori, Romina Alfonsi, Francesca Ghirga, Federica Aiello, Sara Toscano, Cinzia Ingallina, Mariangela Siler, Danilo Cucchi, Agnese Po, Evelina Miele, Davide D’Amico, Gianluca Canettieri, Enrico De Smaele, Elisabetta Ferretti, Isabella Screpanti, Gloria Uccello Barretta, Maurizio Botta, Bruno Botta, Alberto Gulino, Lucia Di Marcotullio EMBO J. 2015, 34, 200-217. 8. Current trends in hedgehog signaling pathway inhibition by small molecules. Ghirga F, Mori M, Infante P. Bioorg. Med. Chem. Lett. 2018, 28, 3131-3140. 9. Targeting GLI factors to inhibit the Hedgehog pathway Infante P, Alfonsi R, Botta B, Mori M, Di Marcotullio L. Trends Pharmacol. Sci. 2015, 36, 547-558. 10. Chromone: A Valid Scaffold in Medicinal Chemistry Alexandra Gaspar, Maria Joao Matos, Jorge Garrido, Eugenio Uriarte, Fernanda Borges Chem. Rev. 2014,

114, 4960−4992.

11. Cisplatin Binding to Biological Ligands Revealed at the Encounter Complex Level by IR Action Spectroscopy D. Corinti, C. Coletti, N. Re, B. Chiavarino, M. E. Crestoni, S. Fornarini Chem. Eur. J. 2016, 22, 3794-3803. 12.An amino acid as a cofactor for a catalytic polynucleotide Roth, A.; Breaker, R. R. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6027-6031. 13. Changes in Binding Motif of Protonated Heterodimers Containing Valine and Amines Investigated Using IRMPD Spectroscopy between 800 and 3700 cm-1 and Theory Jeremy T. O’Brien, James S. Prell, Jeffrey D. Steill, Jos Oomens,Evan R. Williams J. Am. Chem. Soc. 2009, 131, 3905–3912.

14. Structures of Aliphatic Amino Acid Proton-Bound Dimers by Infrared Multiple Photon Dissociation Spectroscopy in the 700−2000 cm-1 Region Rajabi, K.; Fridgen, T. D. J. Phys. Chem. A 2008, 112, 23–30. 15. Infrared Multiple Photon Dissociation Spectra of Proton- and Sodium Ion-Bound Glycine Dimers in the N−H and O−H Stretching Region Atkins, C. G.; Rajabi, K.; Gillis, E. A. L.; Fridgen, T. D. J. Phys. Chem. A 2008, 112, 10220–10225. 16. Experimental and Theoretical Investigation of the Proton-Bound Dimer of Lysine Ronghu Wu, Richard A. Marta, Jonathan K. Martens, Kris R. Eldridge, Terry B. McMahon J. Am. Soc. Mass Spectrom. (2011) 22, 1651-1659. 17. The structures of proton-bound dimers of glycine with phenylalanine and pentafluorophenylalanine Fu, W., Xiong, J., Lecours, M.J., Carr, P. J. J.; Marta, R. A.; Fillion, E.; McMahon, T.; Steinmetz, V., Hopkins, W.S. Journal of Molecular Spectroscopy 2016, 330, 194-199. 18. The structures and properties of proton- and alkali-bound cysteine dimers Ieritano, C., Carr, P.J.J., Hasan, M., Burt, M.; Marta, R. A.; Steinmetz, V.; Fillion, E.; McMahon, T. B.; Hopkins, W. S. Phys. Chem. Chem. Phys., 2016, 18, 4704. 18. Stabilization of the zwitterionic structure of proline by an alkylammonium ion in the gas phase Wu, R.H., McMahon, T.B.: Angew. Chem. Int. Edit. 46, 3668–3671 (2007) 19. Infrared multiple photon dissociation spectra of proline and glycine proton-bound homodimers. Evidence for zwitterionic structure. Wu, R.H., McMahon, T.B. J. Am. Chem. Soc. 129, 4864 (2007) 20. Structure of Proton-Bound Methionine and Tryptophan Dimers in the Gas Phase Investigated with IRMPD Spectroscopy and Quantum Chemical Calculations Åke Andersson, Mathias Poline, Meena Kodambattil, Oleksii Rebrov, Estelle Loire, Philippe Maître, Vitali Zhaunerchyk J. Phys. Chem. A 2020, 124, 12, 2408–2415.

21. The Protonated and Sodiated dimers of Proline Studied by IRMPD Spectroscopy in the N−H and O−H

Stretching Region and Computational Methods.

Alahmadi, Y. J.; Gholami, A.; Fridgen, T. D. Phys. Chem. Chem. Phys. 2014, 16, 26855−26863.

22. Reinvestigation of the Structure of Protonated Lysine Dimer. Kong, X. J. Am. Soc. Mass Spectrom. 2014, 25, 422−426 23. Structure of Protonated Threonine Dimers in the Gas Phase: Salt-Bridged or Charge-Solvated? Yin, H.; Kong, X. J. Am. Soc. Mass Spectrom. 2015, 26, 1455−1461. 24. Side-chain Effects on the Structures of Protonated Amino Acid Dimers: A Gas-phase Infrared Spectroscopy Study. Seo, J.; Hoffmann, W.; Malerz, S.; Warnke, S.; Bowers, M. T.; Pagel, K.; von Helden, G. Int. J. Mass Spectrom.

2018, 429, 115−120

25. Probing Chirality Recognition of Protonated Glutamic Acid Dimers by Gas-phase Vibrational Spectroscopy and First-principles Simulations. Klyne, J.; Bouchet, A.; Ishiuchi, S.-i.; Fujii, M.; Schneider, M.; Baldauf, C.; Dopfer, O. Phys. Chem. Chem.

Phys. 2018, 20, 28452−28464.

26. Structures and Energetics of Protonated Clusters of Methylamine with Phenylalanine Analogs, Characterized by Infrared Multiple Photon Dissociation Spectroscopy and Electronic Structure Calculations

E. Kleisath, R. A. Marta, S. Martens, J. Martens, T. McMahon J. Phys. Chem. A 2015, 119, 6689−6702.

27. Gas-Phase Structure of a -Allyl-Palladium Complex: Efficient Infrared Spectroscopy in a 7 T Fourier Transform Mass Spectrometer J. M. Bakker, T. Besson, J. Lemaire, D. Scuderi, P. Maitre, J. Phys. Chem. A 2007, 111, 13415-13424 28. Vibrational Signatures of the Naked Aqua Complexes from Platinum(II) Anticancer Drugs A. De Petris, A. Ciavardini, C. Coletti, N. Re, B. Chiavarino, M. E. Crestoni, S. Fornarini, J. Phys. Chem. Lett. 2013, 4, 3631-3635. 29. An integrated approach to study novel properties of a MALDI matrix (4-maleicanhydridoproton sponge) for MS imaging analyses

D. Corinti, M. E.a Crestoni, S. Fornarini, M. Pieper, K. Niehaus, M. Giampà Anal. Bioanal. Chem. (2019) 411, 953–964 30. Complexation of halide ions to tyrosine: role of non-covalent interactions evidenced by IRMPD spectroscopy D. Corinti, B. Gregori, L. Guidoni, D. Scuderi, T. B. McMahon, B. Chiavarino, S. Fornarini, M. E. Crestoni Phys. Chem. Chem. Phys. 2018, 20, 4429-4441. 31. For the complete reference for Gaussian’09 program package see the Supporting Information. 32. J. D. Hepworth, C.r D. Gabbutt, B. M. Heron Pyrans and their Benzo Derivatives: Structure chapter 5.07 in Six-membered Rings with One Heteroatom and Fused Carbocyclic Derivatives Vol. 5 in Comprehensive Heterocyclic Chemistry II, 1996. Editors-in-Chief: Alan R. Katritzky, Charles W. Rees and Eric F.V. Scriven 33. Fragmentation of protonated 2-(2-phenylethyl)chromones from agarwood: The diagnostic role of ion/neutral complexes as reactive intermediates Yang, D., Xia, B., Jiang, Y., Mei, W., Kuck, D. Eur. J. Mass Spectrom. 2015, 21, 609-621. 34. Proton affinities from dissociations of proton-bound dimers S. A. McLuckey, D. Cameron, R. G. Cooks J. Am. Chem. Soc. 1981, 103, 1313–1317 35. Thermochemical determinations by the kinetic method R. G. Cooks, J. S. Patrick, T. Kotiaho, S. A. McLuckey Mass Spectrom. Rev. 1994, 13, 287–339. 36. Entropy considerations in kinetic method experiments C. Wesdemiotis J. Mass Spectrom. 2004, 39, 998–1003 37. E. P. Hunter, S. G. Lias "Proton Affinity Evaluation" in NIST Chemistry WebBook, NIST Standard Reference Database Number 69, Eds. P.J. Linstrom and W.G. Mallard, National Institute of Standards and Technology, Gaithersburg MD, 20899, https://doi.org/10.18434/T4D303, (retrieved March 24, 2020). 38. Gas Phase Basicities of Polyfunctional Molecules. Part 3: Amino Acids Bouchoux, G. Mass Spectrom Rev. 2012, 31, 391–435. 39.Gas-Phase Basicities of Polyfunctional Molecules. Part 4: Carbonyl Groups as Basic Sites Bouchoux, G. Mass Spectrom. Rev. 2015, 34, 493–534. 40. Heats of formation and protonation thermochemistry of gaseous benzaldehyde, tropone and quinine methides.

Bouchoux G. Chem Phys Lett. 2010, 495, 192–197.

41. IR spectroscopic feature of gaseous C7H7O+ ions: Benzylium versus tropylium ion structures

Chiavarino B, Crestoni ME, Fornarini S, Dopfer O, Lemaire J, Maitre P. J Phys Chem A 2006, 110, 9352–9360.

42. Gas-Phase IR Spectroscopy of Deprotonated Amino Acids Oomens, J.; Steill, J. D.; Redlich, B. J. Am. Chem. Soc. 2009, 131, 4310-4319. 43. Serine O-sulfation probed by IRMPD spectroscopy Paciotti, R.; Coletti, C.; Re, N.; Scuderi, D.; Chiavarino, B.; Fornarini, S.; Crestoni, M. E. Phys. Chem. Chem. Phys. 2015, 17, 25891-25904. 44. Cisplatin Primary Complex with L-Histidine Target Revealed by IRMPD Spectroscopy D. Corinti, A. De Petris, C. Coletti, N. Re, B. Chiavarino, M. E. Crestoni, S. Fornarini ChemPhysChem 2017, 18, 318-325. 45. The ionic hydrogen bond Meot-Ner (Mautner), M. Chem. Rev. 2005, 105, 213-284. 46. Large Proton Affinity Enhancements Triggered by Non-Covalent Interactions C. Martín-Fernández, M. M. Montero-Campillo, I. Alkorta, M. Yáñez, O. Mó, J. Elguero Chem. Eur. J. 2018, 24, 1971-1977. 47. Protonation Processes and Electronic Spectra of Histidine and Related Ions Z. Huang, Z. Lin and C. Song, J. Phys. Chem. A, 2007, 111, 4340–4352. 48. Gas-phase structures and thermochemistry of neutral histidine and its conjugated acid and base

V. Riffet, G. Bouchoux Phys. Chem. Chem. Phys. 2013, 15, 6097—6106.

49. Revising the Proton Affinity Scale of the Naturally Occurring -Amino Acids C. Bleiholder, S. Suhai and B. Paizs, J. Am. Soc. Mass Spectrom. 2006, 17, 1275–1281 50. Gas-phase structures and thermochemistry of neutral histidine and its conjugated acid and base

S. Gronert, D. C. Simpson, K. M. Conner J. Am. Soc. Mass Spectrom. 2009, 20, 2116–2123. 51. Comparisons of Computational and Experimental Thermochemical Properties of α-Amino Acids K. M. Uddin, P. L. Warburton, R. A. Poirier J. Phys. Chem. B, 2012, 116, 3220–3234. 52. Comprehensive theoretical study towards the accurate protonaffinity values of naturally occurring amino acids Dinadayalane, T. C.; Sastry, G. N.; Leszczynski, J. Int. J. Quant. Chem. 2006, 106, 2920–2933. 53. Gas phase structure of protonated histidine and histidine methyl ester: combined experimental mass spectrometry and theoretical ab initio study Kovacevic, B.; Rozman, M.; Klasinc, L. Maksic, Z. B.; Yanez, M. J. Phys. Chem. A 2005, 109, 8329-8335. 54. Infrared multiple photon dissociation spectroscopy of protonated histidine and 4-phenyl imidazole M. Citir, C. S. Hinton, J. Oomens, J. D. Steill, P. B. Armentrout Int. J. Mass Spectrom., 2012, 330–332, 6–15. 55. High Susceptibility of Histidine to Charge Solvation Revealed by Cold Ion Spectroscopy A. Y. Pereverzev, V. Kopysov, O. V. Boyarkin Angew. Chem. Int. Ed. 2017, 56, 15639 –15643. 56. Structural and Energetic Effects in the Molecular Recognition of Amino Acids by 18-Crown-6 Yu Chen and M. T. Rodgers J. Am. Chem. Soc. 2012, 134, 5863-5875. 57. Infrared multiple photon dissociation action spectroscopy of protonated glycine, histidine, lysine, and arginine complexed with 18-crown-6 ether McNary, C.P., Nei, Y.-W., Maitre, P., Rodgers, M.T., Armentrout, P.B. Physical Chemistry Chemical Physics 2019, 21, 12625-12639. 58. Guanidinium/ammonium competition and proton transfer in the interaction of the amino acid arginine with the tetracarboxylic 18-crown-6 ionophore J. R. Avilés-Moreno, G. Berden, J. Oomens, B. Martínez-Haya Phys. Chem. Chem. Phys., 2018, 20, 4067-4073. 59. Gas-Phase Infrared Multiple Photon Dissociation Spectroscopy of Mass-Selected Molecular Ions

Oomens, J.; Sartakov, B. G.; Meijer, G.; Von Helden, G. Int. J. Mass Spectrom. 2006, 254, 1−19.

60. Gas-Phase Structures of Phosphopeptide Ions: a Difficult Case Turecek, F.; Moss, C. L.; Pikalov, I.; Pepin, R.; Gulyuz, K.; Polfer, N. C.; Bush, M. F.; Brown, J.; Williams, J.;

Richardson, K. Int. J. Mass Spectrom. 2013, 354, 249−256.

61. Effect of Phenol and Acidic Side Chains on the Protonation Sites of b2 Ions Confirmed by IRMPD Spectroscopy

Wang, D.; Gulyuz, K.; Stedwell, C. N.; Yu, L.; Polfer, N. C. Int. J. Mass Spectrom. 2012, 330, 144−151.

62. Cation−π Interactions in Protonated Phenylalkylamines

B. Chiavarino, M. E. Crestoni, M. Schutz, A. Bouchet, S. Piccirillo, V. Steinmetz, O. Dopfer, S. Fornarini J.

Phys. Chem. A 2014, 118, 7130−7138.

63. Structures of lithiated lysine and structural analogues in the gas-phase: Effects of water and proton affinity on zwitterionic stability Lemoff, A.S.; Bush, M. F.; O’Brien, J. T.; Williams, E.R. J. Phys. Chem. A 2006, 110, 8433–8442. 64. Proton Affinity of Lysine Homologues from the Extended Kinetic Method Schroeder, O. E.; Andriole, E. J.; Carver, K. L.; Colyer, K. E.; Poutsma, J. C. J. Phys. Chem. A 2004, 108, 326-332. 65. Proton affinities of lysine and histidine: a theoretical consideration of the discrepancy between experimental results from the kinetic and bracketing methods Bliznyuk, A. A.; Schaefer, H. F.; Amster, I. J. J. Am. Chem. Soc. 1993, 115, 5149-5154 66. Gas phase basicities and proton affinities of lysine and histidine measured from the dissociation of proton bound dimers Wu, Z.; Fenselau, C. Rapid Commun. Mass Spectrom. 1994, 8, 777–780.

67. Infrared spectroscopy of cationized lysine and -N-methyllysine in the gas phase: Effects of alkali-metal ion size and proton affinity on zwitterion stability Bush, M. F.; Forbes, M. W.; Jockusch, R. A.; Oomens, J.; Polfer, N. C.; Saykally, R. J.; Williams, E, R, J Phys Chem A 2007, 111, 7753–7760.

68. An investigation of protonation sites and conformations of protonated aminoacids by IRMPD spectroscopy Wu, R.; McMahon, T. ChemPhysChem 2008, 9, 2826–2835. 69. Infrared spectroscopy and structure of photochemically protonated biomolecules in the gas phase: A noradrenaline analogue, lysine and alanyl alanine Vaden, T. D.; de Boer, T. S. J. A.; MacLeod, N. A.; Marzluff, E. M.; Simons, J. P.; Snoek, L. C. PhysChemChemPhys 2007, 9, 2549–2555.