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Chemistry—Methods doi.org/10.1002/cmtd.202000047

1
2
3 Conceptual and Computational DFT-based In Silico
4
5
Fragmentation Method for the Identification of Metabolite
6 Mass Spectra
7
8 Emilie Cauët,*[a] Yannick J. Vanhaegenborgh,[a, b] Frank De Proft,[b] and Paul Geerlings[b]
9
10
11 Metabolomics is recognized as a crucial scientific domain, tandem MS data. Here, we propose a new innovative in silico
12 promising to advance our understanding of cell biology, approach employing quantum mechanical (QM) methods in
13 physiology and medicine. Tandem mass spectrometry (MS/MS) order to predict ion formation and subsequent fragmentation
14 has strong potential to elucidate the metabolites in complex patterns of arbitrary small molecules and validate putative
15 biological samples and to become a standard tool complement- annotations of tandem mass spectrometry (MS) data. The focus
16 ing established techniques. However, despite its potential for is on the evaluation of a new conceptual density functional
17 answering many key questions, a major challenge in the use of theory (CDFT) nuclear reactivity descriptor of the nuclear Fukui
18 tandem mass spectrometry for characterizing metabolites lies in function type, that characterizes the forces that the atomic
19 a lack of computational tools for accurate annotation and nuclei experience due to proton attachment and captures the
20 structure identification allowing us to turn complex data into onset of the change in the nuclear positions induced by it. A
21 molecular knowledge. Chemo-informatics and related machine- series of test compounds for which high quality experimental
22 learning in silico fragmentation tools have already been data exist and that were investigated before in a more
23 established and used for different classes of metabolites. For approximate theoretical framework have been examined. The
24 the classes of metabolites where existing chemo-informatics output of these calculations provides a list of the most probable
25 approaches produce insufficiently accurate predictions a super- molecular structures predicted to match the experimental
26 vised machine learning based strategy can be used to predict tandem MS spectrum (“de novo metabolite identification”).
27 possible molecular structures from “unassigned” experimental
28
29
30
1. Introduction ionization-mass spectrometry (GC-EI-MS) and liquid chromatog-
31
raphy-electrospray ionization-tandem mass spectrometry (LC-
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Metabolomics, the scientific study of metabolites (all small ESI-MS/MS). Recently, LC-ESI-MS/MS has become the analytical
33
molecules which are chemically transformed within cells of method of choice to elucidate the metabolites in complex
34
living organisms) has become recognized in recent years as a biological samples.[1d] This technique gives information beyond
35
crucial scientific domain, promising to advance our under- the compound mass by providing mass-to-charge (m/z) ratios
36
standing of cell biology, physiology and medicine.[1] It comple- and intensities of the measured intact metabolite and its
37
ments genomics, transcriptomics and proteomics by analysing fragments. However, a major challenge in the use of tandem
38
the final products of biochemical processes and by revealing mass spectrometry (MS/MS) for characterizing metabolites lies
39
the contributions of non-genetic factors such as the environ- in the difficulty for high-throughput annotation and structure
40
ment, diet and microbiome. identification of MS/MS signals.[2] The question is: Can we really
41
The metabolites are mainly detected and analysed using elucidate molecular structures of metabolites with unknown
42
several analytical technologies such as nuclear magnetic identity based on MS/MS data?
43
resonance (NMR) spectroscopy, gas chromatography-electron Reliable chemo-informatics methods and software tools
44
have been developed for analysing fragmentation mass spectra
45
of unknown metabolites.[3] This research field has been very
46
[a] Prof. E. Cauët, Y. J. Vanhaegenborgh active due to the structural diversity of metabolites and the
47 Spectroscopy, Quantum Chemistry and Atmospheric Remote Sensing (CP
complexity of their fragmentation patterns. Basically, the
48 160/09), Université libre de Bruxelles, 50 av. F. D. Roosevelt, B-1050 Brussels,
Belgium computational tools can be partitioned into two main types: (1)
49
E-mail: ecauet@ulb.ac.be in silico fragmentation methods (including in silico simulation
50 [b] Y. J. Vanhaegenborgh, Prof. F. De Proft, Prof. P. Geerlings
of tandem MS/MS spectra) and (2) fingerprint-based methods.
51 Algemene Chemie, Vrije Universiteit Brussel (VUB), Pleinlaan, 1050 Brussels,
Belgium The in silico fragmentation methods include tools such as
52
Supporting information for this article is available on the WWW under MetFrag,[4] CFM-ID,[5] MAGMa,[6] MIDAS/MIDAS-G,[7] MS-Finder,[8]
53 https://doi.org/10.1002/cmtd.202000047 Mass Frontier,[9] ACD/MS fragmenter[10] and MOLGEN/MS.[11] The
54 © 2020 The Authors. Published by Wiley-VCH GmbH. This is an open access tool MetFrag,[4] launched in 2010, was one of the first
55 article under the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided the combinatorial approaches developed. This tool selects molec-
56
original work is properly cited. ular candidates from compound databases (KEGG,[12]
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PubChem[13] or ChemSpider[14]) based on the neutral mass of more than 10 000 candidates, and, at the end, the correct
1
the parent ion. It generates all possible topological fragments molecular structure is still, in the best case, ranked at the top 10
2
of each individual candidate, using the bond dissociation position of the output list. For all the metabolites where above
3
approach, in order to match the theoretical mass of the approaches produce insufficiently accurate predictions, other
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generated fragments with the measured m/z peaks. A score prior information regarding the steps preceding the fragmenta-
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against each candidate is then calculated based on the position, tion might be beneficial such as the chances of a molecule to
6
intensity and bond dissociation energy for fragments that ionize and the likelihood that happens at a given site. One
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explain peaks. MetFrag generates molecular substructures in a alternative would be to supplement the existing methods by
8
combinatorial manner, which means it uses a systematic bond more refined quantum mechanical (QM) based calculations.
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disconnection to dissect a compound into all hypothetical Indeed, the QM calculations take the electron density effects
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fragments. MIDAS and MAGMa[15] are two other examples of throughout the entire metabolite into account which dictate
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available tools based on this algorithm solution. The focus here the ionization step and could significantly influence the
12
is more to identify the peaks in a measured fragmentation potential sites of bond cleavage. The question is how we do
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spectrum of a given species rather than to simulate its entire this without adding an even more laborious task.
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fragmentation spectrum. A major point of concern regarding Several attempts have been made to link the results of QM
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such approaches is that there is no attempt to create a calculations to mass spectra. Grimme[19] reported a QM-based
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mechanistically correct prediction of the fragmentation process approach named Quantum Chemistry Electron Ionization Mass
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resulting sometimes in ions that make little sense. Since the Spectrometry (QCEIMS) to predict complete Electron Ionization
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launch of MetFrag, statistical approaches have evolved, which mass spectra of organic molecules. However, this approach,
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are learning fragmentation processes on the basis of annotated based on Born-Oppenheimer Ab Initio Molecular Dynamics,
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experimental MS/MS data. For example, CFM-ID[5] uses Markov- requires huge computational resources and long simulation
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Chains to model the fragmentation process and machine times meaning that it can certainly not be a method of choice
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learning techniques to adapt the model parameters from to routinely validate or refine structural assignments.
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existing MS/MS data. This probabilistic generative model can be Cautereels et al.[20] have been using QM calculations (mostly
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used for both peaks prediction and assignment. Finally, Mass DFT) to elucidate fragmentation schemes in Electron Ionisation
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Frontier,[9] ACD/MS Fragmenter[10] and MOLGEN-MS[11] are rule- (EI) mass spectrometry for which they developed the innovative
26
based in silico fragmentation methods i. e. methods based on Quantum Chemical Mass Spectrometry for Materials Science
27
pre-determined fragmentation rules that are extracted from the (QCMS2) method. In order to reduce the number of QM
28
MS literature. They can be used for both the prediction and calculations, the QCMS2 method is based on three arbitrary
29
interpretation of tandem MS data. However, if we look at the rules regarding bond strength, bond cleavage and rearrange-
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existing spectral libraries, we realise that these are too small to ment, respectively. Despite of these rules, the number of QM
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cover all metabolites. Resulting rule-based approaches can only calculations remains very high given the fact that one
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thus cover a tiny part of the rules that should be known, while calculation on the radical cation is necessary to evaluate the
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novel compounds with potentially different fragmentation weakest bonds (i. e. bonds with the lowest Fractional Occupa-
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patterns are constantly being discovered. tion Hirshfeld Iterative (FOHI) bond order) and that, for each
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The second type of methods to deal with fragmentation bond considered for cleavage, the ΔE of three, four or five
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mass spectra of metabolites that cannot be found in spectral possible fragmentation pathways are evaluated and compared,
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libraries is the fingerprint-based methods such as Finger ID[16] the lowest-energy pathway being followed. Only rearrange-
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and CSI:Finger ID.[17] These methods predict structural features ments with hydrogen atoms are also considered. Non-hydrogen
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(or molecular fingerprints) of an unknown metabolite from its rearrangements are not taken into account in the QCMS2
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tandem mass spectrum using support vector machines (SVM). methodology. These above-mentioned QM studies have been
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The predicted molecular fingerprints are then used for match- investigating fragmentation rules in EI mass spectra which may
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ing and scoring against molecular structure databases. To this not be pertinent to the MS/MS fragments produced from an ion
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end, the “small” spectral libraries are replaced by the much (commonly the protonated parent molecule) derived from a
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larger structure databases. Recently, Gerlich and Neumann soft ionisation mechanism. In order to study the MS/MS
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introduced MetFusion[18] that combines the MetFrag approach fragments, Alex and coworkers[21] performed DFT QM-based
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with a similarity fingerprint to re-rank the molecular structures. calculations. They calculated the complete redistribution of
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Despite the success of the already established and probed electron density within a molecule as a result of protonation
48
chemo-informatics and related machine-learning methods such and investigated its effects of bond lengths. They then used
49
as those previously cited, it remains challenging to link the this information to help predict the cleavage sites of that
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large volume of known molecular structures to the data protonated molecule. Although this approach has led to some
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obtained with mass spectrometers. Although fragmentation interesting observations, this approach requires the perform-
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rules in MS/MS fragmentation have been largely investigated in ance of a series of iterative calculations on the protonated
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order to determine which chemical bonds are most likely to molecule, which seems unrealistic for large molecules. Also, as
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break separating a metabolite in small fragments, the resulting stated by the authors themselves, for a lot of classes of
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existing tools are only able to describe fragmentation pathways metabolites, other energetic forces must play a more significant
56
that are already known. Moreover, candidate lists may contain
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role in the fragmentation process and overpower the thermody- approach promises to be faster than conventional QM-based in
1
namic effects on which their calculations are based. silico fragmentation methods because only one single elec-
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In the present work, we will concentrate, for the first time, tronic structure calculation per metabolite (or fragment) is
3
on the ability to use DFT,[22] in particular Conceptual DFT performed.
4
(CDFT),[23] in combination with computational DFT using Kohn
5
Sham equations,[24] to investigate the effect of the electronic
6
perturbations due to the protonation on bond strength and Computational Methods
7
hence to predict mass spectral fragmentation. This contribution
8 All DFT calculations were carried out at the B3LYP level[26] using the
can be seen as a counterpart to an earlier study by some of the 6–31G(d,p) basis set[27] with the Northwest Computational
9
present authors on electron-attachment induced DNA damage, Chemistry (NWChem 6.6) program package.[28] An optimization of
10
where instantaneous strand breaks were investigated with a the geometry for the neutral species was performed, and the
11 minimal energy conformation was verified by a frequency calcu-
similar methodology[25] (vide infra). Calculations will be reported
12 lation. For each molecule, the possible sites of protonation were
for the three test compounds: 2-(3-(diisopropylamino)-1-phenyl-
13 determined by investigating the molecular electrostatic potential
propyl)phenol), Fluconazole and Voriconazole (Figure 1), which (MEP) grid produced by NWChem 6.6. The possible protonation
14
were chosen to have varying number of functional groups. sites were determined by the most negative values on the MEP
15
These three test compounds have already been investigated in (indicating the most electron rich zones of the molecule most
16
detail in literature both experimentally and theoretically by Alex prone to interact with a proton).[29] If the molecule contained
17 multiple possible protonation sites, every site was considered and
et al..[21a] Their mass spectra, that will be used for comparison in
18 investigated independently. The molecule was then protonated by
this work, have been recorded using positive electrospray
19 placing a proton at 1 Å of each possible protonation site and a
ionization (ESI) on a LTQ Orbitrap XL Thermo Scientific with constrained geometry optimization for the protonated species was
20
source voltage 5 kV, entrance capillary voltage 35 V and performed where only the extra hydrogen was relaxed. No
21
resolution of 30 000 and present well-defined mass to charge symmetry constraints were imposed in the optimizations. Moreover,
22 we considered the well-known mobile proton model (MPM),[30] an
ratio peaks. Our calculations will define a uniform computa-
23 important concept that states that the proton added to the
tional approach, which will be usable for large databases of
24 molecule may transfer from an initial protonation site to another,
molecules. Our approach consists of using quantum mechanics
25 resulting in fragmentation when it moves over labile bonds. This
1) to identify the most probable protonation sites according to has been modeled as ‘consecutive’ fragmentation resulting from
26
the molecular electrostatic potential (evaluated at the DFT level) protonation on various protonation sites on the molecule as
27
that is created in the space around the molecule, 2) to evaluate opposed to focusing on one non-mobile proton attached to one
28 protonation site. The transfer of the hydrogen from the initial
a new CDFT reactivity descriptor investigating the forces
29 protonation site to another was elucidated by further analyzing the
(energy gradients) acting on the nuclei following the protona-
30 fragments, obtained from the first response to the initial proto-
tion, and 3) to estimate the kinetic energy delivered to all
31
bonds and hence to determine single-step fragments. This
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56 Figure 1. Optimized minimum energy geometries and structures of the three test compounds used in the study as a proof of concept.
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nation, but comprising an extra neutral hydrogen that has been Xa ¼ Fa ðM þ Hþ Þ Fa ðMÞ
1 (6)
transferred and attached to another site of protonation.
2
A new CDFT reactivity descriptor was used to investigate the In a first step, the magnitude of the change in force acting on the
3
coupling of the electronic density perturbation, induced by the nucleus α (a scalar) was investigated. Then, the projected
4 changes in forces along the bonds involving the nucleus α were
protonation, to the initial structural reorganization leading to
5 single-step fragmentation. In a general way, the electron density depicted as vectors with the origin located on the nucleus. Both
6 defines all ground state properties of a molecular system.[22,23c] quantities were visualized using VisIt2.13.2.[35] Let us note that, once
7 However, the response of the nuclei due to the perturbation of the the first-order fragments resulting from breaking a bond from the
8 electron density is still unknown.[31] Cohen et al.[32] introduced the parent molecular ion were obtained, higher-order fragmentation
nuclear Fukui function (Fa Þ that characterizes the changes in the has been investigated by calculating the remaining forces acting on
9
force (F) that the nucleus α experiences when the total number of the nuclei in product ions. In order to see if these forces were still
10 electrons N is changed at a constant external potential vðrÞ: relevant, the difference between the nuclear forces and the
11 corresponding nuclear forces in the parent ion has been calculated,
12 � � even though the number of electrons between the two species is
@Fa
13 Fa ¼ (1) different.
@N vðrÞ
14 The amount of kinetic energy (KE) delivered to bonds upon
15 Another interpretation was provided by Baekelandt[33] where the protonation as a function of time was estimated in analogy with
16 nuclear Fukui function can be viewed as the change in the chemical our previous work.[25] The KE was estimated as a sum of the KEa
17 potential (mÞ due to the conformational contribution (R). delivered to each nucleus involved in the bond:
18 � � � �
19 @Fa @m 1
Fa ¼ ¼ (2) KEa ¼ ma ðva Þ2 (7)
20 @N vðrÞ dRa N 2
21
For isolated molecules, nuclear Fukui function calculations can be where m and v are the nuclear mass and velocity, respectively.
22
performed in a finite difference method, approximating Fa , for a Assuming the initial velocity of the nucleus is zero, its v, after a
23 period of time ~t, is
nucleophilic or electrophilic reaction, as:
24
25 � �
@Fa þ va ¼ aa � Dt (8)
26 Fþa ¼ � Fa ðN þ 1Þ Fa ðNÞ (3)
@N vðrÞ
27 where aα is the nuclear acceleration, supposed here to be constant.
� �
28 @Fa As described by Newton’s second law, the nucleus acceleration is
29 Fa ¼ � Fa ðNÞ Fa ðN 1Þ (4) parallel and directly proportional to the nucleus force Ξα projected
@N vðrÞ
30 here along the bond and inversely proportional to its mass m.
31 respectively, where Fa ðNÞ; Fa ðN þ 1Þ and Fa ðN 1Þ are the forces The KE delivered to a bond, as a function of time, is then finally
32 acting on the nucleus α of the neutral, anionic and cationic species, given by:
33 respectively (for an analytical approach see reference[34]).
34 The nuclear Fukui function was the key ingredient in our previously 1 X X 2a 2
KE ¼ t (9)
35 mentioned study[25] on the instantaneous strand breaks as occurring 2 a ma
36 in electron-attachment induced DNA damage, as bond breaking
37 events there take place under the influence of attachment of an This curve, in combination with the bond dissociation energy (BDE)
electron. Because we are interested, in this study, in the nuclear of the corresponding bond type[36] enables us to estimate the time
38
reactivity i. e. the forces (F) acting on the nuclei, not after electron necessary to reach bond breaking.
39 attachment (i. e. changing number of electrons) but following the
40 protonation, a new CDFT reactivity descriptor is called for. It should
41 characterize the changes in the force (F) (energy gradient) that a
42 nucleus α experiences when the external potential (v) is changed 2. Results and Discussion
43 through protonation of the molecule (at constant number of
electrons N). The required quantity, a new type of second order 2.1. Compound 1: 2-(3-diisopropylamino-1-phenylpropyl)
44 response function can then be written: phenol
45
46 � � � � � �
dFa d dE d2 E The first test system that was chosen to determine if the new
47 ¼ ¼ (5)
duðrÞ N duðrÞ dRa N dRa duðrÞ N CDFT computational approach could be applied successfully
48
was 2-(3-diisopropylamino-1-phenylpropyl)phenol (Figure 1).
49 For practical use we also turned to a finite difference type approach The experimental MS/MS spectrum reported by Alex et al.[21a] for
50 (cf. eqs (3) and (4)) by evaluating the difference in force on a
this compound at m/z 312.2316 is very simple (Figure 2). Only
51 nucleus α after and before protonation of the molecule M, i. e. Fα(M
+ H +) and Fα(M), respectively. The protonated species is calculated one product ion greater than a relative intensity of 15 % is
52
with the same geometry as the neutral system (except for the extra observed at m/z 270 while four much less intense peaks are
53
proton, which causes the change in external potential, which has present at m/z 228, 211, 183 and 133.
54 been optimized as described above). The MEP of 2-(3-diisopropylamino-1-phenylpropyl)phenol is
55
A vector quantity Ξα is then obtained obviously meeting the depicted in Figure 3. This figure shows how a proton is
56
constant N requirement: attracted to a particular location near the molecule. The red
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34 Figure 2. Positive ESI product ion spectrum of the [M + H] + ion of 2-(3-diisopropylamino-1-phenylpropyl)phenol (compound 1) at m/z 312.2316 reported by
Alex et al..[21a] Elucidation of the observed MS/MS product ion structures was conducted via the new CDFT computational approach and is presented as a
35 fragmentation tree. Parent ion structure is depicted in red while first-, second- and third-order fragment ion structures are presented in green, orange and
36 pink, respectively.
37
38
39
isosurface (negative value of the MEP) encloses the regions the projected reactivity descriptor for the heavy atoms are
40
favorable to proton attraction, while the blue isosurface shown in Figure 4(c-d). Only the forces corresponding to bond
41
(positive value of the MEP) encloses the regions where proton elongations are represented with the goal of identifying all
42
repulsion occurs. Large negative values of the MEP show up on possible bond dissociation reactions. Vector lengths (small to
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the nitrogen atom N19 ( 25.7 kcal/mol) and on the oxygen large) and colors reflect relative magnitudes of the projected
44
atom O51 ( 19.5 kcal/mol). These negative MEP values are forces. When 2-(3-diisopropylamino-1-phenylpropyl)phenol is
45
consistent with an accumulation of the electron density from protonated at N19 (D-1), large force responses occur on the
46
lone pairs, which is expected to attract a proton. These two alpha carbons i. e. C16 (16.6 kcal mol 1 · Å 1), C20
47
atoms N19 and O51 were therefore investigated as possible (17.9 kcal · mol · Å ) and C30 (17.8 kcal · mol 1 · Å 1). The com-
1 1
48
protonation sites. bined magnitude of the projected forces along the C N bonds
49
The magnitudes of the CDFT reactivity descriptor (Ξ), are 16.3 kcal · mol 1 · Å 1, 17.7 kcal · mol 1 · Å 1 and
50 1 1
characterizing the changes in the force that the atoms 17.6 kcal · mol · Å , for C16-N, C20-N and C30-N, respectively.
51
experience following the protonation of 2-(3-diisopropylamino- These forces are in agreement with the bond elongations
52
1-phenylpropyl)phenol at N19 (D-1) and O51 (D-2) are depicted in reported by Alex et al.[21a] and help us to envision a general
53
Figures 4a and 4b, respectively. Because the direction of the trend. However, given the small differences between the force
54
forces also influences their effects on the bonds in the responses, it is difficult to identify which bond is likely to break
55
molecule, we projected the atomic forces in the direction of all first. Thus, we performed an estimate of the amount of KE
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the bonds formed by the atom. The vector representations of delivered, as a function of time, to the C N bonds of the tertiary
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45 Figure 3. Molecular Electrostatic Potential (MEP) (in kcal/mol) for the three test compounds used in the study as a proof of concept with red indicating
regions favorable to proton attraction and blue indicating regions favorable to proton repulsion.
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49
amine moiety and the surrounding C C bonds once the proton agreement with what is observed in the experimental MS/MS
50
has attached (Figure 5). According to the KE curves, the effects spectrum (Figure 2). Indeed, the most abundant product ion,
51
of the dynamic behaviors of the three C N bonds may be observed at m/z 270, corresponds to the loss of one of the
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significant. Although the time difference is very small, one of isopropyl groups (P1D in Figure 2). This result is in agreement
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the two C N isopropyl bonds (i. e. C20-N or C30-N) is expected to with the ‘even-electron rule’, a generalization often quoted to
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cleave first (after only ~ 30 fs). The fragmentation of one of two understand and predict the mass spectral behavior of organic
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C N isopropyl bonds (assuming that multiple bond breaking is compounds, which predicts that protonated molecules with an
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less likely to occur when there is no rearrangements[37]) is in
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28 Figure 4. (Top) Magnitude of the CDFT reactivity descriptor (Ξα) (in kcal/mol/Å) characterizing the changes in the force that the atoms experience following
29 the protonation of 2-(3-diisopropylamino-1-phenylpropyl)phenol (compound 1) at N19 (a) and O51 (b). (Bottom) Vector representations of the projected
30 reactivity descriptor for the heavy atoms following the protonation of 2-(3-diisopropylamino-1-phenylpropyl)phenol at N19 (c) and O51 (d). Only the forces
corresponding to bond elongations are represented with the goal of identifying all possible bond dissociation reactions. Vector lengths (small to large) and
31 colors reflect relative magnitudes of the projected forces within the molecule.
32
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46 Figure 5. Estimated amount of kinetic energy (KE) (in kcal/mol) delivered, as a function of time, to the selected (a) C N bonds and (b) C C bonds in 2-(3-
47 diisopropylamino-1-phenylpropyl)phenol (compound 1) once proton has attached at N19. For some bonds, KE raises very rapidly to values corresponding to
48 the bond dissociation energy (horizontal line) of the corresponding bond type.
49
50
51
even number of electrons would eliminate an even-electron the C4-O bond as a fragmentation site. However, the corre-
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neutral species (i. e. propene) rather than a radical.[37–38] sponding product ion at m/z 295 is not observed in the
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In order to elucidate the other product ions observed in the experimental MS/MS data.
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experimental spectrum, the single protonation of the oxygen The product ion observed at m/z 270 (P1D) can be
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atom was also investigated (D-2). Based on the results considered as a first-order fragment resulting from breaking a
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(Figures 4b-d), the calculated nuclear force responses predict bond (i. e. C20-N or C30-N) from the parent molecular ion. Second
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order fragmentation has been investigated by calculating the is identical as the one reported by Alex et al.[21a] Hence only the
1
remaining forces acting on the nuclei in cation P1D. If we look at product ions reported by Alex et al.[21a] are investigated in this
2
the results for the cation P1D resulting from breaking the C30-N work.
3
bond, the highest total forces are observed on the alpha The MEP of Fluconazole is depicted in Figure 3 that shows
4
carbons of the amine i. e. C16 (16.7 kcal · mol 1 · Å 1) and C20 large negative values on the trivalent nitrogen atoms of the
5
(18.8 kcal · mol 1 · Å 1) (see Figure S1 for details). The combined triazole moieties i. e. N5 ( 39.9 kcal/mol), N7 ( 30.7 kcal/mol),
6
magnitude of the projected forces along the C N bonds are N21 ( 33.8 kcal/mol) and N20 ( 26.7 kcal/mol). These trivalent
7
16.6 kcal · mol 1 · Å 1 and 18.6 kcal · mol 1 · Å 1, for C16-N and C20- nitrogen atoms are characterized by slightly different MEP
8
N, respectively. According to the KE distributions, both C16-N values and were therefore investigated independently as
9
and C20-N bonds are susceptible to break after ~ 30 fs. If possible protonation sites. Smaller negative MEP values occur
10
applying the even-electron rule, the product ion resulting from on the oxygen atom O22 ( 6.2 kcal/mol) as well as on the two
11
breaking the C20-N bond (P2D in Figure 2) elucidates the low tetravalent nitrogen atoms of the triazole moieties i. e. N6 and
12
intense peak observed at m/z 228 in the experimental MS/MS N19 ( 3.3 kcal/mol). Given the small values obtained for N6 and
13
spectrum, while the product ion resulting from breaking the N19, only the oxygen atom was investigated as an additional
14
C16-N bond (P3D in Figure 2) explains the peak at m/z 211. likely site of protonation.
15
In order to elucidate higher order fragmentation, the The magnitudes of the CDFT reactivity descriptor (Ξ),
16
nuclear forces were calculated for cations P2D and P3D. From the characterizing the changes in the force that the atoms
17
results obtained for cation P2D (see Figure S2 for details), it can experience following the single protonation of Fluconazole at
18
be deduced that the C16-N bond would further break, and this N5 (F-1), N7 (F-2), N21 (F-3) and N20 (F-4), are depicted in
19
cleavage does lead to the already elucidated product ion (P3D) Figure 7(a–d). The vector representations of the projected
20
observed at m/z 211. The results obtained for cation P3D (see reactivity descriptor for the heavy atoms are shown in Fig-
21
Figure S3) help to explain one more product ion observed in ure 7(e-h). Generally, when Fluconazole is protonated at a
22
the MS/MS spectrum (Figure 2). Indeed, the nuclear forces trivalent nitrogen atom of a triazole ring (F-1 to F-4), large
23
support the third-order fragmentation of the C11-C13 bond dissociative nuclear forces occur on the atoms of the triazole
24
facilitating thus the loss of ethylene which accounts for the aromatic ring that was protonated. No forces appear on the rest
25
product ion (P4D in Figure 2) observed at m/z 183. According to of the molecule. As it is not likely that a triazole ring would
26
the remaining forces evaluated for cation P4D, a phenol cation fragment from soft ionization techniques due to the stability of
27
could be formed. However, these results cannot be confirmed the aromatic system,[21a] the only force response that matters
28
given the fact that these product ions are outside the mass occurs essentially between the bonded atoms C8-N6 and
29
range of the published MS/MS spectrum. between C12-N19, for cations F-1, F-2 and F-3, F-4, respectively.
30
Our findings are thus in complete agreement with what is The estimate of the amount of KE delivered, as a function of
31
observed experimentally. For this first test compound, we see time, to these bonds provides evidence that the cleavage of the
32
that all the product ions observed in the MS/MS spectrum, C N bond alpha to the triazole ring appears to be a likely
33
except one low intense peak at m/z 133, can be explained using fragmentation pathway for protonated Fluconazole (see Fig-
34
the new CDFT reactivity descriptor. We expect this low intense ure S4 for details). Following the ‘even-electron rule’, it is
35
peak at m/z 133 to result from a third-order fragmentation of expected that the protonated Fluconazole eliminates a neutral
36
the C11-C40 bond. Although the KE delivered to the C11-C40 bond triazole ring rather than a radical. Indeed, this fragmentation is
37
in cation P3D raises slowly (Figure S3), it does reach the bond consistent with the predictions previously reported in the
38
dissociation energy value of a C C bond type after ~ 100 fs literature and leads to the most abundant product ion observed
39
which could thus explain the last peak of the spectrum. at m/z 238 (P1F in Figure 6).
40
Nuclear force responses following protonation of Flucona-
41
zole at O22 (F-5) are depicted in Figure 8(a). A significantly large
42
2.2. Compound 2: Fluconazole force response appears on the central carbon atom C11
43
(27.4 kcal · mol 1 · Å 1). Projected force responses in Figure 8(b)
44
The second test system that was investigated was Fluconazole show that reactivity, as a result of protonation of Fluconazole at
45
(Figure 1). The experimental MS/MS spectrum reported by Alex the oxygen atom, affects both C11-O and C11-C (C11-C8, C11-C12
46
et al.[21a] for protonated Fluconazole at m/z 307.1111 is shown in and C11-C24) bonds. An estimate of the amount of KE delivered,
47
Figure 6. Three significant product ions (greater than a relative as a function of time, to the C11-O and C11-C bonds in cation F-5,
48
intensity of 15 %) are observed at m/z 289, 238 and 220. The once the proton is attached, is presented in Figures 8(c) and
49
spectrum also presents a much less intense peak at m/z 169. Let 8(d), respectively. The initial effects would likely occur on the
50
us note that a total of 17 ESI spectra for Fluconazole are C11-O bond for which the KE of the bond rises much more
51
reported in the MassBank database.[39] However, only two ESI rapidly. We see that the breakage of a C11-C bond is less likely
52
spectra with the same source voltage and entrance capillary to occur as initial step. The C11-O bond exhibits KE in the range
53
voltage are published. The first spectrum reported with the of 76.35 kcal/mol after only ~ 20 fs. This energy is of the same
54
same resolution[40] presents an additional peak at m/z 139 with order of magnitude as the bond dissociation energy (horizontal
55
a relative intensity of 2 % while the second spectrum[41] line) estimated for a C O bond in an aqueous solution.
56
measured in the same conditions but with a resolution of 7500 Following the ‘even-electron’ rule, the breakage of the C11-O
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33
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Figure 6. Positive ESI product ion spectrum of the [M + H] + ion of Fluconazole (compound 2) at m/z 307.1111 reported by Alex et al..[21a] Elucidation of the
35 observed MS/MS product ion structures was conducted via the new CDFT computational approach and is presented as a fragmentation tree. Parent ion
36 structure is depicted in red while first-order fragment ion structures are presented in green. The tentative fragment ion structures derived from modeling the
37 mobile proton model (MPM) are presented in blue.
38
39
40
bond thus leads to the loss of H2O (comprised of the hydroxyl proton response was expected. As explained earlier, when we
41
and the charge hydrogen) which results in the production of considered the single protonation of Fluconazole at N5 (F-1) or
42
the ion observed at m/z 289 (P2F in Figure 6). N7 (F-2), we saw that the first proton response was the cleavage
43
Second order fragmentation has been investigated by of the C8-N6 bond leading to the most abundant product ion
44
calculating the remaining forces acting on the nuclei in cations (P1F) observed at m/z 238 in the experimental MS/MS spectrum.
45
P1F and P2F. However, the remaining forces do not explain any However, because the neutral hydrogen may transfer from the
46
other ions observed in the MS/MS spectrum suggesting that initial protonation site (i. e. N5 or N7) to another, a second
47
the product ions P1F and P2F are not expected to break further. response may be expected. Thus, the nuclear force responses
48
From the MS/MS data, there are thus two other less abundant were calculated for the most abundant product ion (P1F)
49
product ions that cannot be explained by focusing on a single comprising a neutral hydrogen that has been transferred and
50
protonation site. Further calculations were then performed in attached to another site of protonation (i. e. N21, N20 and O22).
51
order to simulate the electron density perturbation response Nuclear force responses following the H transfer from N5 (the
52
induced by the transfer of the mobile proton from one most likely protonation site) to N21 (the second most likely
53
protonation site to another (cf. MPM). All the possibilities protonation site) are depicted in Figure 9(a-b). A significantly
54
involving the most likely sites of protonation of Fluconazole large force response appears on the central carbon atom C11
55
(i. e. N5, N7, N20, N21 and O22) were considered. As we tried to (27.4 kcal · mol 1 · Å 1). Results show that reactivity as a result of
56
simulate the proton transfer between two sites, a two-step proton transfer from a trivalent nitrogen atom of one triazole
57

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19
20
21
22
Figure 7. (Top) Magnitude of the CDFT reactivity descriptor (Ξα) (in kcal/mol/Å) characterizing the changes in the force that the atoms experience following
23
the protonation of Fluconazole (compound 2) at N5 (a), N7 (b), N21 (c) and N20 (d). (Bottom) Vector representations of the projected reactivity descriptor for the
24 heavy atoms following the protonation of Fluconazole at N5 (e), N7 (f), N21 (g) and N20 (h). Only the forces corresponding to bond elongations are represented
25 with the goal of identifying all possible bond dissociation reactions. Vector lengths (small to large) and colors reflect relative magnitudes of the projected
forces within the molecule.
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52 Figure 8. (a) Magnitude of the CDFT reactivity descriptor (Ξα) (in kcal/mol/Å) and (b) vector representations of the projected reactivity descriptor for the heavy
atoms, following the protonation of Fluconazole (compound 2) at O22. Estimated amount of kinetic energy (KE) (in kcal/mol) delivered, as a function of time,
53
to the selected (c) C O bonds and (d) C C bonds in Fluconazole once proton has attached.
54
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22
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Figure 9. (a) Magnitude of the CDFT reactivity descriptor (Ξα) (in kcal/mol/Å) and (b) vector representations of the projected reactivity descriptor for the heavy
24
atoms, acting due the H-transfer from N5 to N21 in Fluconazole (compound 2). Estimated amount of kinetic energy (KE) (in kcal/mol) delivered, as a function of
25 time, to the selected (c) C N bonds and (d) C C bonds in Fluconazole once proton has transferred.
26
27
28
ring (here N5) to a trivalent nitrogen atom of the other triazole reported in the literature.[21a] The calculations described above
29
ring (N21) may affect both triazole moieties of the molecule. As appear to identify successfully all the product ions observed in
30
it is not likely that the triazole aromatic rings would fragment, the experimental MS/MS spectrum for protonated Fluconazole.
31
the expected response of this model is the loss of the first
32
protonated triazole ring followed by the loss of the other one.
33
This is clearly illustrated in Figure 9(c), which shows the amount 2.3. Compound 3: Voriconazole
34
of KE delivered, as a function of time, to the C12-N19 bond in P1F
35
due the H-transfer from N5 to N21. According to the KE Finally, in order to further validate our approach, we inves-
36
distributions, the effects of the dynamic behavior of the C12-N19 tigated a derivative of Fluconazole, namely Voriconazole (Fig-
37
bond are indeed significant, enabling cleavage of the C N bond ure 1). The experimental MS/MS spectrum for protonated
38
alpha to the triazole ring after only ~ 50 fs (much less than the Voriconazole at m/z 350 is shown in Figure 10. Three significant
39
vibrational time scale of C N). These fragmentations lead to a product ions (greater than a relative intensity of 15 %) are
40
product ion at m/z 169 observed in the mass spectrum, for observed at m/z 281, 224 and 127.
41
which a tentative fragment structure was assigned to (P3F in The MEP of Voriconazole is depicted in Figure 3. Five
42
Figure 6). Results involving the other coupled protonation sites possible sites of protonation, indicated by large negative MEP
43
comprising the nitrogen atoms of the triazole rings are values, appear on the trivalent nitrogen atoms of the triazole
44
presented in the Supporting Information (Figure S5). We see (i. e. N13 ( 36.3 kcal/mol), N12 ( 29.1 kcal/mol)) and pyrimidine
45
that they all lead to the production of the same ion at m/z 169 (i. e. N35 ( 30.4 kcal/mol) and N34 ( 27.1 kcal/mol)) moieties and
46
(P3F). Results concerning the H transfer from N5 (the most on the oxygen atom O14 ( 22.5 kcal/mol).
47
reactive site towards protonation) to the site of protonation O22 The magnitudes of the CDFT reactivity descriptor (Ξ),
48
are also presented in detail in the SI (Figure S6). They appear to showing the changes in the force that the atoms experience
49
show relevant nuclear force responses that can help to explain following the single protonation of Voriconazole at N13 (V-1)
50
the product ion observed at m/z 220, for which a tentative and N12 (V-2) are depicted in Figures 11a and 11b, respectively.
51
fragment structure has again been assigned to (P4F in Figure 6). Projected force responses are shown in Figure 11(c-d). As with
52
Indeed, the estimate of the amount of KE delivered, as a Fluconazole, when a trivalent nitrogen atom of the triazole ring
53
function of time, to the bonds tend to demonstrate that the ion is protonated (V-1 and V-2), large dissociative nuclear forces
54
m/z 220 forms via a two-step process involving first the alpha occur on all the atoms of this aromatic ring. As it is not likely
55
cleavage of one of the triazole rings and second the loss of H2O. that an aromatic ring would fragment, the only force response
56
This finding is in agreement with the predictions previously that matters occurs between the bonded atoms C4-N11. This
57

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32 Figure 10. Positive ESI product ion spectrum of the [M + H] + ion of Voriconazole (compound 3) at m/z 350.1230 reported by Alex et al..[21a] Elucidation of the
observed MS/MS product ion structures was conducted via the new CDFT computational approach and is presented as a fragmentation tree. Parent ion
33
structure is depicted in red while first-order fragment ion structures are presented in green. The tentative fragment ion structure derived from modeling the
34 mobile proton model (MPM) is presented in blue.
35
36
37
provides evidence that the cleavage of the C N bond alpha to P1V and P2V. However, the results suggest that the product ions
38
the triazole ring appears to be a likely fragmentation pathway P1V and P2V are not expected to break further.
39
for protonated Voriconazole (see Figure S7 for details). Follow- Calculations were then performed in order to simulate the
40
ing the ‘even-electron rule’, this fragmentation leads to the electron density perturbation response induced by the transfer
41
most intense product ion observed at m/z 281 (P1V in Figure 10). of a proton from one protonation site to another. To not
42
Similarly, nuclear force responses following protonation of burden the reader, the results of only two coupled protonation
43
Voriconazole at a trivalent nitrogen atom of the pyrimidine ring, sites (N13 and O14) that predict a unique product ion observed in
44
N34 (V-3) or N35 (V-4), predict a fragmentation of the C1-N27 bond the experimental MS/MS spectrum, are discussed below. All the
45
(see Figure S8 for details). However, although the cleavage of rest of the results found in the SI (see Figure S10) do not explain
46
this bond seems favored, the corresponding product ion at m/z any observed peak. Following protonation of Voriconazole at
47
252 is not being observed in the experimental MS/MS spectrum N13, the first cleavage response would likely occur on the bond
48
suggesting that N34 and N35 are less likely to be protonated. C4-N11. If we suppose that the hydrogen atom transfers almost
49
Finally, single protonation of Voriconazole at O14 (V-5) immediately to the protonation site O14, we observe a
50
requires the elongation and possible fragmentation of the C3- subsequent proton response on the C3-O14 bond, leading to the
51
O14 bond. This event appears plausible given the nuclear force loss of H2O (comprised of the hydroxyl and the charge
52
magnitudes and directions for the C3 and O14 atoms and the KE hydrogen) which would result in the production of the ion
53
delivered to the C3-O14 bond (see Figure S9). The product ion observed at m/z 263 (P3V in Figure 10). A tentative product ion
54
resulting from this fragmentation (P2V in Figure 10) is indeed structure assigned to the peak is presented in the figure.
55
observed at m/z 332 in the experimental MS/MS spectrum. For the last test compound, all product ions observed in the
56
Second order fragmentation has been investigated for cations MS/MS spectrum for protonated Voriconazole are elucidated
57

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Figure 11. (Top) Magnitude of the CDFT reactivity descriptor (Ξα) (in kcal/mol/Å) characterizing the changes in the force that the atoms experience following
32
the protonation of Voriconazole (compound 3) at N13 (a) and N12 (b). (Bottom) Vector representations of the projected reactivity descriptor for the heavy
33 atoms following the protonation of Voriconazole at N13 (c) and N12 (d). Only the forces corresponding to bond elongations are represented with the goal of
34 identifying all possible bond dissociation reactions. Vector lengths (small to large) and colors reflect relative magnitudes of the projected forces within the
molecule.
35
36
37
38
except two. Those are expected to result from breaking the C1- kinetic energy supplied to the bonds, allows us to predict the
39
C3 bond. Although the KE delivered to the C1-C3 bond in cation motions of the nuclei required to accommodate the proton
40
V-3 or V-4 raises slowly, it does reach the bond dissociation attachment and hence the fragmentation patterns of molecules.
41
energy value of a C C bond type after ~ 200 fs which could The CDFT descriptor introduced in this work thus evaluates, in a
42
thus potentially explain the last peaks of the spectrum. single step, the strength of all bonds in a molecular structure,
43
providing a list of the most probable molecular structures
44
predicted to match the experimental MS/ MS spectrum.
45
3. Conclusions As a proof of concept, the MS/MS spectra of three test
46
compounds with high quality experimental data were inves-
47
The goal of our study has been to provide a new, generally tigated. For 2-(3-diisopropylamino-1-phenylpropyl)phenol (com-
48
applicable, QM-based approach to predict ion formation and pound 1), all the peaks except one in the MS/MS spectrum have
49
subsequent tandem mass spectrometric (MS/MS) fragmentation been identified by simulating first-, second- and third-order
50
of arbitrary molecules. Our approach relies on the development fragmentation of the protonated molecule. For Fluconazole
51
of a new conceptual density functional theory (CDFT) based (compound 2) and Voriconazole (compound 3), for which the
52
nuclear reactivity descriptor. This descriptor tends to show that number of potential sites of protonation reaches 7 and 6,
53
the forces on the atomic nuclei, which govern the changes in respectively, the fragmentation induced by non-mobile and
54
their position in response to protonation, become already mobile proton attachment have been modeled. For Flucona-
55
significant at the instant of proton attachment. The evaluation zole, all the peaks of the MS/MS spectrum have been elucidated
56
of the nuclear forces, at the nascent stage, as well as of the
57

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