Characterization of organic isomers: CID fragmentation technique on protonated hydroxybenzophenone isomers

We have studied the fragmentation dynamics of the protonated 2-, 3-and 4-hydroxybenzophenones ( 2 H + , 3 H + and 4 H + , 191 m/z ) obtained by CID (“Collision Induced Dissociation”) technique coupled to Mass Spectrometer with electrospray source (MS-ESI). The CID-fragmentation patterns involve the C 7 OH 5+ (105 m/z ) and C 7 O 2 H 5+ (121 m/z ) cations, for which the intensity ratio is typical to the isomer considered. 2 H + and 3 H + yield preferably C 7 O 2 H 5+ (121 m/z ) and C 7 OH 5+ (105 m/z ) respectively, while 4 H + yields both cations in similar quantity. The fragmentation pathways were analyzed and discussed, taking into account the “model of free - proton” and results from computational calculations at B3LYP/6-311++G(d,p) level of theory. CID/MS-ESI fragmentation technique employed to characterize hydroxybenzophenone isomers. Main structures in the spectrum: Parent cation (199 m/z ), C 7 O 2 H 5+ (121 m/z ), C 7 OH 5+ (105 m/z ).


Introduction
Protonation is a common occurring process within chemical and biological systems, 1 which usually results in the formation of hydrogen bonds. 2 Several studies have been carried out to determine the preferred site of protonation from small molecules, such as carbon monoxide, 3 up to large ones, such as proteins and peptides. 4In those systems, it has been found that the proton tends to attach to a thermodynamic favourable site that gives rise to a stable mono-charged cation (z = +1).However, in some cases, such as biological molecules, there might be different preferred sites of protonation (multi-protonated, z = +n) which indicates that protonation may be regarded as a competitive process between the thermodynamical and kinetical natures. 5Mass spectrometry is acknowledged as a powerful analytical technique which allows processes such as ionization/protonation/deprotonation/fragmentation in order to identify specific molecules and their products, and it may also be coupled with other complementary techniques, (e.g.CID) to conduct further structural analysis.
Typically, protonation takes place when the proton migrates from its preferred site to another region within the molecule.As an example, the protonation of the amide has been shown to occur at the oxygen located in the carbonyl group C=O, 6 but the fragmentation only takes place when the proton migrates to the nitrogen in the amide group, despite the fact that the N-protonated species might have higher energies than the O-protonates isomers. 7Since the previous process is a recurrent behaviour in many molecules, it has led to the proposition of the "mobile proton model" 8,9 to describe the mobility of the proton through protonated molecules.
In this research, it has been studied the fragmentation of protonated 2-, 3-and 4-hydroxybenzophenones (2H + , 3H + and 4H + ) using CID technique ("Collision Induced Dissociation") along with a triple quadrupole mass spectrometer (MS-TQ) coupled with electrospray source (MS-ESI).The experimental results have been analysed and rationalized by means of the density functional theory (DFT) computational calculations, performed at the B3LYP/6-311++G(d,p) level of theory.It is important to point out that CID fragmentation mass spectrometry experiments are very effective in the identification of chemical species.In this study, we have shown that through these techniques the isomer species could be unequivocally identified.

Results and Discussion
The Hydroxybenzophenones isomer species were protonated on their carbonyl groups C=O 10 and identified by the peak at 199.1 m/z (parent cation) in the mass spectrum.The CID fragmentation of these cations produced two fragments, identified as the 105 and 121 m/z peaks in the mass spectra (Figure 1), for which the relative intensities increase as the dissociation energy rises, while the intensity of parent cation decreases.
The previous result indicates the existence of two competitive channels of CID fragmentation, as described in the proposed mechanisms in Figure 2. In the first dissociation channel (I) it is produced a C7OH5 + cation (105 m/z) and a neutral phenol molecule; and in the second channel (II) it is produced a C7O2H5 + (121 m/z) cation and a neutral benzene molecule.The proportion of intensities of the peaks associated with these cations produce a CID pattern, which characterizes the fragmentation of these isomers (Figure 3).According to the proton mobile model, it is noticeable that depending on in which of the carbon atoms the fragmentation takes place, C1 or C2 on the benzene rings, the proton could migrate.It would lead to one of the two proposed fragmentation mechanisms.It is interesting to point out for the CID experiments involving the 2H + , 3H + and 4H + protonated-hydroxybenzophenones, that by varying the energy Ecm the formation of both fragments (at 105 and 121 m/z), with rates of formation (I105 + /I121 + ), were mostly independent of Ecm (Figure3).The fragmentation route (II) is preferable in the fragmentation of 2H + isomers, meanwhile the route (I) is followed by 3H + species.In the case of 4H + isomers, both routes have roughly the same probability.

Energetic analysis of fragmentation
The optimized geometries, obtained at B3LYP/6-311++G(d,p) level of theory, of the most stable hydroxybenzophenone-protonated rotamers, 2aH + , 3aH + and 4aH + are presented in Figure 4, for which the populations were 99.9%, 29% and 51%, respectively.It is important to notice that the proton in 2aH + forms a hydrogen bonding with the phenol part in its molecule, this feature seems to prevent the proton from behaving as a "free-proton" and thus influencing the fragmentation mechanism.On the contrary, for the 3H + and 4H + rotamers (and also other 2H + rotamers) that particularity was not observed.Fragmentation of 2H + : In Figure 5, distinct fragmentation pathways possible for the 2H + are shown.The proton on the most stable rotamer 2aH + is oriented towards C1 (phenolic group), but its fragmentation following the route (I) would have to overcome a high energy barrier with a transition state TS1a of 327.3 kJmol -1 .This barrier should be increased by the presence of a hydrogen bonding, as previously described.Consequently, the direct fragmentation of 2aH + is diminished in favour of the formation of the other rotamers such as 2bH + or 2fH + , overpassing much smaller barriers (TSi-k between 12 and 58 kJmol -1 ).Considering that 2aH + is subjected to this isomerization process with a population rebalancing, the rotamers with "free-protons" oriented towards C1, which will follow the fragmentation route (I), accounted for approximately 15% of the conformational population.The proportion of these species to those which will follow the route (II) has been found to be r = 0.18, a consistent value, comparable with the experiments (Figure 3).Fragmentation of 3H + (Figure 6): 3H + rotamers (including the most stable, 3aH + ) with the proton oriented against C1 represented 65.5% of the conformational population, and considering their "free-protons" character, these species tend to follow the fragmentation route (I) , overcoming energy barriers with TS1k between 172 and 177 kJmol -1 .

AUTHOR(S)
Rotamers with the proton oriented towards C2 (benzene group) represented a population of 34.5% and should follow the route (II) overpassing barriers with TS2k between 200 and 203 kJmol -1 kJmol -1 .It is easy to appreciate that the rotamers which fragment following the route (I) accounts for almost the double, proportionally, as compared to those which follow route (II), and they should be favoured by the fact that they have to overcome barriers of about 30 kJmol -1 lower in energy than by following route (I).This proportion, as described before, is in good agreement with the experimental results (Figure 3).
Fragmentation of 4H + (Figure 7): 4aH + and 4bH + rotamers, with the proton oriented towards C2, accounted for 91.5% of the conformational population.These species also behave as "free-protons" and should also tend to follow the fragmentation route (II), overcoming energy barriers with TS2k of approximately 207 kJmol -1 .4cH + and 4dH + rotamers, with the proton oriented towards C1, tend to follow route (I).Although these species only represent 8.5% of the population, their populations should increase at the expense of the most abundant ones (4aH + and 4bH + ).This is due to the barriers TS2k that they should surpass, which are almost 16 kJmol -1 less energetic than TS1k.

Conclusions
The analysis of the results of the CID fragmentation of protonated hydroxybenzophenone isomers, through the construction of characteristic fragmentation patterns, has allowed the unequivocal identification of these isomers, and to determine their relative populations.The fragmentation mechanisms were studied and analysed using DFT computational calculations, in conjunction with general scientific concepts, such as "mobile proton model", transition states, and conformational populations, which were useful for the prediction of the most favourable routes.From this approach, we have found that the 2H + isomers tend to fragment preferably following the route that leads to the formation of the C7O2H5 + (121 m/z) cation.On the contrary, the 3H + isomers preferably form the C7OH5 + cation (105 m/z), and the 4H + species fragment forming cations in similar proportions.The results obtained at B3LYP/6-311++G(d,p) level of theory were satisfactory to support the explanations of the fragmention pathways involving the hydroxybenzophenone isomers.